IL268983B1 - Nucleic acid derivative having immunostimulatory activity - Google Patents

Description

Description Title of the Invention:NUCLEIC ACID DERIVATIVE HAVING IMMUNOSTIMULATORY ACTIVITY Field of the Invention [0001]The present invention relates to a nucleic acid derivative having immunostimulatory activity. In more detail, it relates to a double-stranded oligonucleotide wherein the first strand is a CpG oligonucleotide, and the second strand binds to a lipid.

Background Art [0002]A vaccine which is a medicine for infectious disease or cancer utilizes an antigen-specific immune response. An adjuvant is a compound having an immunostimulatory activity which is used to enhance efficacy or durability of a vaccine, and various kinds of adjuvants such as aluminum salt, emulsion and liposome have been researched or developed (Non-patent Document 1 or the like).[0003]A single-strand oligodeoxynucleotide comprising a dinucleotide motif of unmethylated cytosine guanine (5’-CpG-3’) (ssCpG ODN) is known as a one of the adjuvants. ssCpG ODNs are ligands of TLR9 (Toll-like receptor 9), and extremely efficient inducers of Th1 immunity or cytotoxic T-lymphocyte (CTL) responses thorough TLR9 to stimulate the immune system (Non-patent Document 1). However, there are problems related to in vivo stability, toxicity, pharmacokinetics or the like of ssCpG ODNs to use alone. A method that ssCpG ODN is encapsulated in a nanoparticle made of lipid bilayer (Non­patent Document 2), a method that lipid binds to 5’ end of ssCpG ODN (Non­patent Document 3 or Patent Document 1) or the like is known as means for solving these problems.[0004]Furthermore, it is known that the charactericity as an adjuvant was1 vanished when ssCpG ODN was administered as a double-stranded DNA (dsCpG ODN) by annealing the first strand and the second strand (Non-patent Document 4). Non-patent Document 5 discloses that only dsCpG ODN did not show the immunostimulatory activity, but when dsCpG ODN was encapsulated in a lipofectin particle, the dsCpG ODN comprising either CpG motif or GpC motif showed the immunostimulatory activity.

Prior Art Document Patent Document [0005]Patent Document 1: WO2013/1517Non-patent Document [0006]Non-patent Document 1: Trends in immunology, 2009, 30(1), 23-Non-patent Document 2: Advanced Drug Delivery Review, 2009, 61(3), 233­242Non-patent Document 3: Nature, 2014, 507, 519-5Non-patent Document 4: Eur. J. Immunol., 2003, 33, 1382-13Non-patent Document 5: BMB reports, 2010, 43(3), 164-169 Summary of the inventionProblems to be solved by the Invention[0007]The purpose of the present invention is to provide new nucleic acid derivatives having immunostimulatory activity which are useful as an adjuvant of a vaccine and/or a vaccine itself.

Means for Solving the Problem [0008]Non-patent Documents 4 and 5 disclose that administering dsCpG ODN did not show the immunostimulatory activity. Furthermore, Non-patent Document 5 discloses that when dsCpG ODN was encapsulated in a lipofectin particle, the dsCpG ODN comprising either CpG motif (ODN4531) or GpC motif (ODN4531GC) induced IL-8 and HLR-DRA expression, that is, by a method2 which is independent of a CG sequence. It suggested based on the fact that dsCpG ODN was rapidly degraded in cells compared to ssCpG ODN that the reason why dsCpG ODN encapsulated in a lipofectin particle showed immunostimulatory activity is that encapsulation of dsCpG ODN may protect against rapid degradation (page 165, the right column, lines 14 to 16).[0009]The present inventors have intensively studied to synthesize a double- stranded oligonucleotide (a lipid binding double-stranded oligonucleotide of the present invention) wherein a first strand is a CpG oligonucleotide, and a second strand is an oligonucleotide comprising a sequence capable of hybridizing with the first strand and binds to a lipid. They found that the inductivity of antigen-specific CTL by a vaccine was enhanced and showed the anti-tumor effect by administering a lipid binding double-stranded oligonucleotide of the present invention as an adjuvant with a tumor antigen peptide (when a lipid binding double-stranded oligonucleotide of the present invention is used as an adjuvant, it is also referred as "an adjuvant of the present invention"). In addition, they found that there is a lipid binding double-stranded oligonucleotide in the present invention which shows strong anti-tumor effect by itself, that is, which is useful as a cancer vaccine (when a lipid binding double-stranded oligonucleotide of the present invention is used as a vaccine, it is also referred as "a vaccine of the present invention"). That is, a lipid binding double-stranded oligonucleotide of the present invention has the immunostimulatory activity. Furthermore, lipid binding double-stranded oligonucleotides of the present invention have high metabolic stability, water solubility and less toxicity to be safe enough as a medicine.[0010]For example, when an adjuvant of the present invention wherein the second strand is a DNA oligonucleotide, an oligonucleotide consisting of nucleoside derivatives having OMe at the 2’ position of the sugar or an oligonucleotide consisting of nucleoside derivatives having F at the 2’ position of the sugar was administered with a tumor antigen peptide, they showed the enhanced CTL inductivity or the strong anti-tumor effect compared to ssCpG ODN (e.g., Result 1-A, B, Result 7-A, B and Result 9-A, B in Example 3). On the other hand, the lipid binding double-stranded oligonucleotide wherein the3 second strand is a RNA oligonucleotide (SEQ-40) did not show the anti-tumor effect (Result 7-B).When an adjuvant of the present invention wherein a lipid comprising two of the acyl chains having 14 to 24 carbon atoms binds at 3’ or 5’ end of the second strand was administered with a tumor antigen peptide, it showed the strong anti-tumor effect compared to ssCpG ODN (e.g., Result 2-A, B in Example 3). On the other hand, when a double-stranded oligonucleotide wherein a lipid comprising two acyl chains having 10 carbon atoms binds at 5’ end of the second strand (SEQ-12) was administered with a tumor antigen peptide, it did not show the enhanced anti-tumor effect compared to ssCpG ODN. In addition, when an adjuvant of the present invention wherein a lipid comprising two of the acyl chains having 12 to 20 carbon atoms binds at 3’ or 5’ end of the second strand, and furthermore a lipid binds at the other end was administered with a tumor antigen peptide, it showed the enhanced CTL inductivity or the strong anti-tumor effect compared to ssCpG ODN (e.g.,Result 3-A, B in Example 3).When an adjuvant of the present invention wherein the length of the second strand is 50 % to 100 % of that of the first strand (the CpG oligonucleotide) was administered with a tumor antigen peptide, it showed the enhanced CTL inductivity or the strong anti-tumor effect compared to ssCpG ODN (e.g., Result 4-A, B, Result 5-A, Result 8-A, B, and Result 9-A, B in Example 3).When an adjuvant of the present invention wherein a lipid binds to a second strand through an oligonucleotide linker (e.g., dGdG, dTdT and dAdA) was administered with a tumor antigen peptide, it showed the further enhanced CTL inductivity or the stronger anti-tumor effect compared to an adjuvant of the present invention without a linker (e.g., Result 8-A, B in Example 3).[0011]In addition, lipid binding double-stranded oligonucleotides of the present invention have no systemic toxicity, and it suggested the high safety (Example 5).[0012]Furthermore, the result of immunization with an adjuvant of the4 present invention and PCRV protein derived from Pseudomonas aeruginosa which is a cause of an infectious disease also suggested the immunostimulatory activity as an adjuvant of a vaccine for an infectious disease (Example 6).[0013]When the adjuvant of the present invention wherein lipid binds to a second strand through an oligonucleotide linker (dGdGdGdGdG) (SEQ-121) was administered alone, it showed the strong anti-tumor effect compared to ssCpG ODN (Example 4).[0014]That is, the present invention is related to the followings.(A1) A double-stranded oligonucleotide, whereina first strand is a CpG oligonucleotide consisting of 8 to 50 nucleotides, a second strand is an oligonucleotide consisting of 8 to 60 nucleotides and comprising a sequence capable of hybridizing with the first strand, but excluding a RNA oligonucleotide,the length of the second strand is 50 % or more of that of the first strand, and a lipid comprising C12 to C30 hydrocarbon chain(s) binds to the second strand through a linker.(A2) The double-stranded oligonucleotide of (A1), wherein the oligonucleotide of the second strand is an oligonucleotide consisting of DNA nucleosides and/or nucleoside derivatives.(A3) The double-stranded oligonucleotide of (A2), wherein the nucleoside derivative is a nucleoside having a substituent at the 2’ position of the sugar and/or a nucleoside having a bridge structure between the 4’ and 2’ positions of the sugar.(A4) The double-stranded oligonucleotide of (A3), wherein the bridge structure between the 4’ and 2’ positions of the sugar is 4’-(CH2)m-O-2’, wherein m is an integer of 1 to 4.(A5) The double-stranded oligonucleotide of any one of (A1) to (A4), wherein the lipid is a diacyl lipid.(A6) The double-stranded oligonucleotide of (A1) to (A5), wherein the lipid binds at the 3’ end and/or 5’ end of the second strand.(A7) The double-stranded oligonucleotide of any one of (A1) to (A6), wherein the linker is an oligonucleotide linker.

(A8) The double-stranded oligonucleotide of (A7), wherein the linker is -(dXOu- , wherein X1 is each independently, A, G, C or T, and u is an integer of 1 to 8. (A9) The double-stranded oligonucleotide of (A1) selected from the group of consisting of SEQ-61, SEQ-119, SEQ-121, SEQ-170 and SEQ-192.(A10) The double-stranded oligonucleotide of (A1) selected from the group of consisting of SEQ-59, SEQ-166, SEQ-168, SEQ-216, SEQ-272, SEQ-280, SEQ- 290, SEQ-294, SEQ-310, SEQ-373 and SEQ-384.(A11) A pharmaceutical composition comprising the double-stranded oligonucleotide of any one of (A1) to (A10).(A12) The pharmaceutical composition of (A11), further comprising an antigen. (A13) A method for treating or preventing a cancer or an infectious disease, comprising administering the double-stranded oligonucleotide of any one of (A1) to (A10).(A14) Use of the double-stranded oligonucleotide of any one of (A1) to (A10) for the manufacture of an agent for treating or preventing a cancer or an infectious disease.(A15) The double-stranded oligonucleotide of any one of (A1) to (A10) for treating or preventing a cancer or an infectious disease.[0015](A16) The pharmaceutical composition of (A12), wherein the antigen is a microbial antigen, a self-antigen or an addictive substance.(A18) The pharmaceutical composition of (A16), wherein the microbial antigen is a bacterial antigen, a viral antigen or a parasitic antigen.(A18) The pharmaceutical composition of (A16), wherein the self-antigen is a tumor antigen, an antigen associated with Alzheimer's Disease, an antigen against a human antibody, or an antigen that is expressed from human endogenous retroviral elements.(A19) The pharmaceutical composition of (A16), the addictive substance is nicotine or cocaine.(A20) A method for increasing an immune response in a subject comprising administering to the subject an effective amount of the pharmaceutical composition of any one of (A12), (A16) to (A19).(A21) The method of (A20), wherein the immune response is an increase in inductivity of specific cytotoxic T-lymphocyte compared to a control.6 (A22) The method of (A20) or (A21), wherein the subject has a cancer or an infectious disease.[0016]In addition, the present invention includes the followings.(B1) An adjuvant consisting of a double-stranded oligonucleotide, wherein a first strand is a CpG oligonucleotide consisting of 8 to 50 nucleotides, a second strand is an oligonucleotide consisting of 8 to 60 nucleotides and comprising a sequence capable of hybridizing with the first strand, and a lipid binds to the second strand through a linker.(B2) The adjuvant of (B1), wherein the oligonucleotide of the second strand is an oligonucleotide consisting of DNA nucleosides and/or nucleoside derivatives. (B3) The adjuvant of (B2), wherein the nucleoside derivative is a nucleoside having a substituent at the 2’ position of the sugar and/or a nucleoside having a bridge structure between the 4’ and 2’ positions of the sugar.(B4) The adjuvant of (B3), wherein the substituent is OCH3.(B5) The adjuvant of any one of (B1) to (B4), wherein the lipid is a diacyl lipid. (B6) The adjuvant of (B5), wherein the acyl chain of the diacyl lipid has 14 to carbon atoms.(B7) The adjuvant of (B5) or (B6), wherein the lipid binds at the 5’ the end of the second strand.(B8) The adjuvant of any one of (B1) to (B7), wherein the length of the second strand is 50 % or more of that of the first strand.(B9) The adjuvant of any one of (B1) to (B9), wherein the linker is an oligonucleotide linker.(B10) The adjuvant of (B9), wherein the linker is dX:dX2, wherein X1 or X2 is A, G, C or T.(B11) A vaccine composition comprising an antigen and the adjuvant of any one of (B1) to (B10).[0017](B12) The vaccine composition of (B11) wherein the antigen is a microbial antigen, a self-antigen or an addictive substance.(B13) The vaccine composition of (B12) wherein the microbial antigen is a bacterial antigen, a viral antigen or a parasitic antigen.(B14) The vaccine composition of (B12) wherein the self-antigen is a tumor antigen, an antigen associated with Alzheimer's Disease, an antigen against a human antibody, or an antigen that is expressed from human endogenous retroviral elements.(B15) The vaccine composition of (B12) wherein the addictive substance is nicotine or cocaine.(B16) A method of increasing an immune response in a subject comprising administering to the subject an effective amount of the vaccine composition of any one of (B11) to (B15).(B17) The method of (B16), wherein the immune response is an increase in inductivity of induced specific cytotoxic T-lymphocyte compared to a control. (B18) The method of (B16) or (B17), wherein the subject has a cancer or an infectious disease.(B19) A method of treating a cancer or an infectious disease comprising administering to the subject an effective amount of the vaccine composition of any of (B11) to (B14) to reduce one or more symptoms of the cancer or infectious disease compared to a control.

Effect of the Invention [0018]Lipid binding double-stranded oligonucleotides of the present invention show the superior immunostimulatory activity against a target antigen. No systemic toxicity was detected, and therefore they are expected to apply to a medicine as an adjuvant of a vaccine and/or a vaccine itself.

Brief Description of the Drawings [0019][Fig. 1] The anti-tumor effect when ssCpG ODN (ODN1826, SEQ-1), dsCpG ODN (SEQ-4) or the adjuvant of the present invention (SEQ-16) was administered with a tumor antigen peptide (TRP2 peptide).[Fig. 2] The anti-tumor effect when ssCpG ODN (ODN1826, SEQ-1) or the adjuvant of the present invention having lipids at the both ends (SEQ-46 or SEQ-48) was administered with a tumor antigen peptide (TRP2 peptide).[Fig. 3] The anti-tumor effect when ssCpG ODN (ODN1826, SEQ-1), ssCpG ODN introducing a lipid ligand (SEQ-2) or the adjuvant of the present8 invention which the length of the complementary strand is different (SEQ-8 or SEQ-10) was administered with a tumor antigen peptide (TRP2 peptide).[Fig. 4] The anti-tumor effect when ssCpG ODN (ODN1826, SEQ-1), ssCpG ODN introducing a lipid ligand (SEQ-2), the adjuvant of the present invention (SEQ-26) or Montanide was administered with a tumor antigen peptide (TRPpeptide).[Fig. 5] The anti-tumor effect when ssCpG ODN (ODN1826, SEQ-1), ssCpG ODN introducing a lipid ligand (SEQ-2) or the adjuvant of the present invention (SEQ-38) was administered with a tumor antigen peptide (TRPpeptide).[Fig. 6] The anti-tumor effect when ssCpG ODN (ODN2006, SEQ-49), the adjuvant of the present invention which the length of the complementary strand is different (SEQ-51 or SEQ-61) or the adjuvant of the present invention having a linker (SEQ-63 or SEQ-65) was administered with a tumor antigen peptide (TRP2 peptide).[Fig. 7] The anti-tumor effect when ssCpG ODN (ODN2006, SEQ-49) or the adjuvant of the present invention wherein the nucleic monomer of the complementary strand is 2’-OMe-RNA (SEQ-67 or SEQ-69) was administered with a tumor antigen peptide (TRP2 peptide).[Fig. 8] The anti-tumor effect when ssCpG ODN (ODN2006, SEQ-49) or the adjuvant of the present invention having an oligonucleotide linker (SEQ-119 or SEQ-192) was administered with a tumor antigen peptide (TRP2 peptide).[Fig. 9] The anti-tumor effect when ssCpG ODN (ODN2006, SEQ-49) or the adjuvant of the present invention having an oligonucleotide linker (SEQ-121) was administered with a tumor antigen peptide (TRP2 peptide).[Fig. 10] The anti-tumor effect when ssCpG ODN (ODN2006, SEQ-49) or the adjuvant of the present invention having an oligonucleotide linker (SEQ-152 or SEQ-158) was administered with a tumor antigen peptide (TRP2 peptide).[Fig. 11] The anti-tumor effect when ssCpG ODN (ODN2006, SEQ-49) or the adjuvant of the present invention wherein a lipid binds at the 3’ end and/or 5’ end (SEQ-188, SEQ-192 or SEQ-194) was administered with a tumor antigen peptide (TRP2 peptide).[Fig. 12] The anti-tumor effect when ssCpG ODN (ODN2006, SEQ-49) or the adjuvant of the present invention having an oligonucleotide linker (SEQ-164)9 was administered with a tumor antigen peptide (TRP2 peptide).[Fig. 13] The anti-tumor effect when ssCpG ODN (ODN2006, SEQ-49) or the adjuvant of the present invention wherein a lipid binds only at the 3’ end (SEQ-170) was administered with a tumor antigen peptide (TRP2 peptide). [Fig. 14] The anti-tumor effect of ssCpG ODN (ODN2006, SEQ-49) or the vaccine of the present invention (SEQ-121) under the absence of a tumor antigen peptide.[Fig. 15] Antibody titer after subcutaneous administration of PCRV antigen vaccine with ssCpG ODN (ODN2006, SEQ-49), the adjuvant of the present invention (SEQ-61 or SEQ-121) or Freund’s adjuvant.

Embodiments for Carrying out the invention [0020]Terms used herein, unless otherwise indicated, are used in a sense normally used in the art.In the present invention, a genetic manipulation method which is well known in the art can be used. For example, it is a method described in Molecular Cloning, A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press (2012), or Current Protocols Essential Laboratory Techniques, Current Protocols (2012).[0021]Terms used in this description are explained below. Each term, unless otherwise indicated, has the same meaning when it is used alone or together with other terms.[0022]In this description, "adjuvant" means a compound having the immunostimulatory activity which is utilized to enhance efficacy or durability of immune response of a vaccine antigen.[0023]A "nucleoside" means a compound that a nucleic acid base and a sugar are bonded by an N-glycoside bond.An "oligonucleotide" means nucleotides that some of same or different kinds of nucleotide are bonded by an internucleoside linkage such as a phosphodiester bond. id="p-24" id="p-24"

[0024]A linkage between a sugar and a sugar in an oligonucleotide (internucleoside linkage) may be a linkage having a natural nucleic acid, phosphodiester (D-oligo), an artificially modified linkage or a linkage without phosphorus atom. Any linkage which is well-known in this field can be used. Examples of an artificially modified linkage are phosphorothioate (S-oligo), methylphosphonate (M-oligo) and boranophosphate. Furthermore, a linkage described in WO2013/022966, WO2011/005761, WO2014/012081, WO2015/125845 or the like can be used. An example of a linkage without phosphorus atom is a bivalent substituent deriving from non-aromatic carbocyclyl or the like substituted with alkyl, non-aromatic carbocyclyl, haloalkyl or halogen. Example is a bivalent substituent deriving from siloxane, sulfide, sulfoxide, sulfone, acetyl, acetyl formate, acetyl thioformate, acetyl methylene formate, acetyl thioformate, alkenyl, sulfamate,methyleneimino, methylenehydrazino, sulfonate, sulfonamide, amide or the like. In an oligonucleotide, linkages may be same or different.[0025]In this description, a "DNA nucleoside" or "RNA nucleoside" means natural DNA nucleoside or natural RNA nucleoside, and a part of nucleotide, which is 1 unit for a component of an oligonucleotide. A "natural DNA nucleoside" is as below. wherein Bx1 is adenine, guanine, cytosine or thymine. A "natural RNA nucleoside" is as below. wherein Bx2 is adenine, guanine, cytosine or uracil. [0026] A "DNA oligonucleotide" means an oligonucleotide which some DNA nucleosides are bounded, and an "RNA oligonucleotide" means an oligonucleotide which some RNA nucleosides are bounded.[0027]In this description, a "nucleoside derivative" means a nucleoside whose nucleic base and/or sugar part of DNA nucleoside or RNA nucleoside was artificially modified. Any well-known modification for a nucleoside in this field can be used.[0028]Examples of modification for a nucleic base are 5-methylcytosine, 5- hydroxy methylcytosine and 5-propynylcytosine.[0029]An example of modification for a sugar part is a substituent at the 2’ position of a sugar. Examples are 2’-F, 2’-OCH3 (2’־OMe) and 2’- OCH2CH2OCH3 (2’-MOE).The other example is the following bridge structure between the 4’ and 2’ positions of a sugar. 4’-(CR1R2)m-O-2’, 4’-(CR1 R2)m-S-2’, 4’-(CR1 R2)m-O-C(=O)-2’, 4’- (CR1 R2 )m-NR3 -O-(CR1 R2 )m1 -2’, 4’-(CR1 R2 )m1 -C(=O)-NR3 -2’, 4’-(CR1 R2 )m2 - C(=O)-NR3 -Y4 -2’, 4’-(CR1 R2 )m1 -SO2 -NR3 -2’, or Y4 is O, S, NH or CH2,R1 is each independently, hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl or substituted or unsubstituted alkynyl,R2 is each independently, hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl or substituted or unsubstituted alkynyl, R3 is hydrogen atom, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aromatic carbocyclyl, substituted or unsubstituted non-aromatic carbocyclyl, substituted or unsubstituted aromatic heterocyclyl, substituted or unsubstituted non-aromatic heterocyclyl, substituted or unsubstituted aromatic carbocyclylalky, substituted or unsubstituted non-aromatic carbocyclylalky, substituted or unsubstituted aromatic heterocyclylalkyl or substituted or unsubstituted non-aromatic heterocyclylalkyl,Y1 is CR4 or N,Y2 is CR5 or N,Y3 is CR6 or N,R4, R5 and R6 are each independently, hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted amino, substituted or unsubstituted alkyloxy, substituted or unsubstituted alkylcarbonylamino, substituted or unsubstituted alkenylcarbonylamino, substituted or unsubstituted alkynylcarbonylamino, substituted or unsubstituted alkylcarbamoyl, substituted or unsubstituted alkenylcarbamoyl, or substituted or unsubstituted alkynylcarbamoyl, m is an integer of 1 to 4, m1 is an integer of 0 to 3, and m2 is 0 or 1.[0030]R1 and R2 is preferably hydrogen atom.[0031]R3 is preferably hydrogen atom, alkyl, alkenyl, alkynyl, aromatic carbocyclyl, non-aromatic carbocyclyl, aromatic heterocyclyl, non-aromatic heterocyclyl, aromatic carbocyclylalky, non-aromatic carbocyclylalky, aromatic heterocyclylalkyl or non-aromatic heterocyclylalkyl, and may have one or more substituent(s) selected from Group a.Group a: a hydroxyl group, alkyl, alkyloxy, mercapto, alkylthio, amino, alkylamino and halogen.[0032]The bridge structure is preferably 4’-(CR1 R2 )m-O-2’ or 4’-(CR1 R2)m! -13 C(=O)-NR3 -2’ (AmNA, Bridged nucleic acid), wherein,R1 is each independently, hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl,R2 is each independently, hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl,R3 is hydrogen atom, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl, m is an integer of 1 to 4, and m1 is an integer of 0 to 2.[0033]The bridge structure is more preferably 4’-(CH2)m-O-2’, wherein m is an integer of 1 to 4, or 4’-C(=O)-NR3 -2’, wherein R3 is hydrogen atom or alkyl.4’-(CH2 )m-O-2’, wherein m is an integer of 1 to 4, is more preferably 4’- CH2 -O-2’ (LNA, Locked nucleic acid). Examples and the methods for preparation are described in WO98/39352, WO2003/068795, WO2005/021570 or the like.4’-C(=O)-NR3 -2’, wherein R3 is hydrogen atom or alkyl, is more preferably 4’-C(=O)-NCH3 -2’. Examples and the methods for preparation are described in WO2011/052436.[0034]Examples of the well-known modification of nucleotide and the method for modification in this field are described in the following patent documents. WO98/39352, WO99/014226, WO2000/056748, WO2005/021570, WO2003/068795, WO2011/052436, WO2004/016749, WO2005/083124, WO2007/143315, WO2009/071680, WO2014/112463, WO2014/126229 and the like.[0035]"Halogen" includes a fluorine atom, a chlorine atom, a bromine atom and an iodine atom. A fluorine atom and a chlorine atom are especially preferable.[0036]14 "Alkyl" includes a C1 to C15, preferably a C1 to C10, more preferably a C1 to C6 and even more preferably a C1 to C4 linear or branched hydrocarbon group. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl, isooctyl, n-nonyl and n-decyl.A preferred embodiment of "alkyl" is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl or n-pentyl. A more preferred embodiment is methyl, ethyl, n-propyl, isopropyl or tert-butyl.[0037]"Alkenyl" includes a C2 to C15, preferably a C2 to C10, more preferably a C2 to C6 and further preferably a C2 to C4 linear or branched hydrocarbon group having one or more double bond(s) at any position(s). Examples include vinyl, allyl, propenyl, isopropenyl, butenyl, isobutenyl, prenyl, butadienyl, pentenyl, isopentenyl, pentadienyl, hexenyl, isohexenyl, hexadienyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl and pentadecenyl.A preferred embodiment of "alkenyl" is vinyl, allyl, propenyl, isopropenyl or butenyl.[0038]"Alkynyl" includes a C2 to C10, preferably a C2 to C8, more preferably a C2 to C6 and further preferably a C2 to C4 linear or branched hydrocarbon group having one or more triple bond(s) at any position(s). Examples include ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl and decynyl. Furthermore, it may have double bond(s) at any position(s).A preferred embodiment of "alkynyl" is ethynyl, propynyl, butynyl or pentynyl.[0039]"Aromatic carbocyclyl" means a cyclic aromatic hydrocarbon group which is monocyclic or polycyclic having two or more rings. Examples include phenyl, naphthyl, anthryl and phenanthryl.A preferred embodiment of "aromatic carbocyclyl" is phenyl.[0040]"Non-aromatic carbocyclyl" means a cyclic saturated hydrocarbon group or cyclic unsaturated non-aromatic hydrocarbon group, which is monocyclic or15 polycyclic having two or more rings. Examples of the non-aromatic carbocyclyl, which is polycyclic having two or more rings, include a fused ring group wherein a non-aromatic carbocyclyl, which is monocyclic or polycyclic having two or more rings, is fused with a ring of the above "aromatic carbocyclyl".In addition, examples of the "non-aromatic carbocyclyl" also include a group having a bridge and a group to form a spiro ring as follows: The non-aromatic carbocyclyl, which is monocyclic, is preferably C3 to C16, more preferably C3 to C12 and further preferably C4 to C8 carbocyclyl. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl and cyclohexadienyl.Examples of non-aromatic carbocyclyl, which is polycyclic having two or more rings, include indanyl, indenyl, acenaphthyl, tetrahydronaphthyl and fluorenyl.[0041]"Aromatic heterocyclyl" means an aromatic cyclyl, which is monocyclic or polycyclic having two or more rings, containing one or more of heteroatom(s) selected independently from O, S and N.Examples of aromatic heterocyclyl, which is polycyclic having two or more rings, include a fused ring group wherein an aromatic heterocyclyl, which is monocyclic or polycyclic having two or more rings, is fused with a ring of the above "aromatic carbocyclyl" and/or "non-aromatic carbocyclyl".The aromatic heterocyclyl, which is monocyclic, is preferably a 5- to 8- membered and more preferably 5- to 6- membered ring. Examples include pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazolyl, triazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, oxazolyl, oxadiazolyl, isothiazolyl, thiazolyl and thiadiazolyl.Examples of aromatic heterocyclyl, which is bicyclic, include indolyl,16 isoindolyl, indazolyl, indolizinyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, naphthyridinyl, quinoxalinyl, purinyl, pteridinyl, benzimidazolyl, benzisoxazolyl, benzoxazolyl, benzoxadiazolyl, benzisothiazolyl, benzothiazolyl, benzothiadiazolyl, benzofuryl, isobenzofuryl, benzothienyl, benzotriazolyl, imidazopyridyl, triazolopyridyl, imidazothiazolyl, pyrazinopyridazinyl, oxazolopyridyl and thiazolopyridyl.Examples of aromatic heterocyclyl, which is polycyclic having three or more rings, include carbazolyl, acridinyl, xanthenyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl and dibenzofuryl.[0042]"Non-aromatic heterocyclyl" means a non-aromatic cyclyl, which is monocyclic or polycyclic having two or more rings, containing one or more heteroatom(s) selected independently from O, S and N.Examples of non-aromatic heterocyclyl, which is polycyclic having two or more rings, include a fused ring group wherein a non-aromatic heterocycle, which is monocyclic or polycyclic having two or more rings, is fused with a ring of "aromatic carbocyclyl", "non-aromatic carbocyclyl" and/or "aromatic heterocyclyl".In addition, examples of the "non-aromatic heterocyclyl" also include a group having a bridge and a group to form a spiro ring as follows: O The non-aromatic heterocyclyl, which is monocyclic, is preferably a 3- to 8-membered and more preferably 5- to 6- membered ring. Examples include dioxanyl, thiiranyl, oxiranyl, oxetanyl, oxathiolanyl, azetidinyl, thianyl, thiazolidinyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperidyl, piperazinyl, morpholinyl, morpholino, thiomorpholinyl, thiomorpholino, dihydropyridyl, tetrahydropyridyl, tetrahydrofuryl, tetrahydropyranyl, dihydrothiazolyl, tetrahydrothiazolyl, tetrahydroisothiazolyl, dihydrooxazinyl, hexahydroazepinyl,tetrahydrodiazepinyl, tetrahydropyridazinyl, hexahydropyrimidinyl,17 dioxolanyl, dioxazinyl, aziridinyl, dioxolinyl, oxepanyl, thiolanyl, thiinyl and thiazinyl.Examples of non-aromatic heterocyclyl, which is polycyclic having two or more rings, include indolinyl, isoindolinyl, chromanyl and isochromanyl.[0043]"Alkyloxy" means a group wherein "alkyl" binds to an oxygen atom. Examples include methyloxy, ethyloxy, n-propyloxy, isopropyloxy, n-butyloxy, tert-butyloxy, isobutyloxy, sec-butyloxy, pentyloxy, isopentyloxy and hexyloxy.A preferred embodiment of "alkyloxy" is methyloxy, ethyloxy, n- propyloxy, isopropyloxy or tert-butyloxy.[0044]"Haloalkyl" means a group wherein one or more "halogen" binds to "alkyl". Examples include monofluoromethyl, monofluoroethyl, monofluoropropyl, 2,2,3,3,3-pentafluoropropyl, monochloromethyl, trifluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 2,2,2-trichloroethyl, 1,2- dibromoethyl, and 1,1,1-trifluoropropane-2-yl.A preferred embodiment of "haloalkyl" is trifluoromethyl or trichloromethyl.[0045]"Alkylthio" means a group wherein "alkyl" binds to sulfur atom.[0046]"Alkylamino" includes monoalkylamino and dialkylamino."Monoalkylamino" means a group wherein a hydrogen atom attached to a nitrogen atom of an amino group is replaced with "alkyl". Examples include methylamino, ethylamino and isopropylamino. Preferably, it is methylamino or ethylamino."Dialkylamino" means a group wherein two hydrogen atoms attached to a nitrogen atom of an amino group are replaced with two "alkyl". These two alkyl groups may be the same or different. Examples include dimethylamino, diethylamino, N, N-diisopropylamino, N-methyl-N-ethylamino and N-isopropyl- N-ethylamino. Preferably, it is dimethylamino or diethylamino.[0047]"Alkylcarbonylamino" means a group wherein one or two hydrogen atoms attached to a nitrogen atom of an amino group are replaced with one or18 two alkylcarbonyl. The two alkylcarbonyl groups may be the same or different. Examples include methylcarbonylamino, ethylcarbonylamino, propylcarbonylamino, isopropylcarbonylamino, tert-butylcarbonylamino, isobutylcarbonylamino, sec-butylcarbonylamino, dimethylcarbonylamino, diethylcarbonylamino and N, N-diisopropylcarbonylamino.A preferred embodiment of "alkylcarbonylamino" is methylcarbonylamino and ethylcarbonylamino.[0048]"Alkenylcarbonylamino" means a group wherein one or two hydrogen atoms attached to a nitrogen atom of an amino group are replaced with one or two alkenylcarbonyl. The two alkenylcarbonyl groups may be the same or different. Examples include vinylcarbonylamino and propenylcarbonylamino. [0049]"Alkynylcarbonylamino" means a group wherein one or two hydrogen atoms attached to a nitrogen atom of an amino group are replaced with one or two alkynylcarbonyl. The two alkynylcarbonyl groups may be the same or different. Examples include ethynylcarbonylamino and propynylcarbonylamino.[0050]"Alkylcarbamoyl" means a group wherein one or two hydrogen atoms attached to a nitrogen atom of a carbamoyl group are replaced with one or two "alkyl". These two alkyl groups may be the same or different. Examples include methylcarbamoyl, ethylcarbamoyl, dimethylcarbamoyl and diethylcarbamoyl.[0051]"Alkenylcarbamoyl" means a group wherein one or two hydrogen atoms attached to a nitrogen atom of a carbamoyl group are replaced with one or two "alkenyl". These two alkenyl groups may be the same or different. Examples include vinylcarbamoyl and propenylcarbamoyl.[0052]"Alkynylcarbamoyl" means a group wherein one or two hydrogen atoms attached to a nitrogen atom of a carbamoyl group are replaced with one or two "alkynyl". These two alkynyl groups may be the same or different. Examples include ethynylcarbamoyl and propynylcarbamoyl.19 id="p-53" id="p-53"

[0053]Alkyl part of "aromatic carbocyclylalky", "non-aromatic carbocyclylalky", "aromatic heterocyclylalkyl" or "non-aromatic heterocyclylalkyl" is the same as "alkyl".[0054]"Aromatic carbocyclylalkyl" means an alkyl substituted with one or more "aromatic carbocyclyl". Examples include benzyl, phenethyl, phenylpropyl, benzhydryl, trityl, naphthylmethyl and a group of the formula of A preferred embodiment of "aromatic carbocyclylalkyl" is benzyl, phenethyl or benzhydryl.[0055]"Non-aromatic carbocyclylalkyl" means an alkyl substituted with one or more "non-aromatic carbocyclyl". The "non-aromatic carbocyclylalkyl" also includes "non-aromatic carbocyclylalkyl" wherein the alkyl part is substituted with "aromatic carbocyclyl". Examples include cyclopropylmethyl, cyclobutylmethyl, cyclopenthylmethyl, cyclohexylmethyl and a group of the formula ofJW id="p-56" id="p-56"

[0056]"Aromatic heterocyclylalkyl" means an alkyl substituted with one or more "aromatic heterocyclyl". The "aromatic heterocyclylalkyl" also includes "aromatic heterocyclylalkyl" wherein the alkyl part is substituted with the above "aromatic carbocyclyl" and/or "non-aromatic carbocyclyl". Examples include pyridylmethyl, furanylmethyl, imidazolylmethyl, indolylmethyl, benzothiophenylmethyl, oxazolylmethyl, isoxazolylmethyl, thiazolylmethyl, isothiazolylmethyl, pyrazolylmethyl, isopyrazolylmethyl, pyrrolidinylmethyl,benzoxazolylmethyl and groups of the formula of v/W oj id="p-57" id="p-57"

[0057]"Non-aromatic heterocyclylalkyl" means an alkyl substituted with one or more "non-aromatic heterocyclyl". The "non-aromatic heterocyclylalkyl" also includes "non-aromatic heterocyclylalkyl" wherein the alkyl part is substituted with "aromatic carbocyclyl", "non-aromatic carbocyclyl" and/or "aromatic heterocyclyl". Examples include tetrahydropyranylmethyl, morpholinylethyl, piperidinylmethyl, piperazinylmethyl and groups of the formula ofjxpjx *sru id="p-58" id="p-58"

[0058]Examples of the substituents for "substituted or unsubstituted alkyl", "substituted or unsubstituted alkenyl", "substituted or unsubstituted alkynyl", "substituted or unsubstituted alkyloxy", "substituted or unsubstituted alkylcarbonylamino", "substituted or unsubstituted alkenylcarbonylamino", "substituted or unsubstituted alkynylcarbonylamino", "substituted or unsubstituted alkylcarbamoyl", "substituted or unsubstituted alkenylcarbamoyl "or "substituted or unsubstituted alkynylcarbamoyl" include the following substituents. A carbon atom at any position(s) may be bonded to one or more group(s) selected from the following substituents.Substituent: halogen, hydroxy, carboxy, amino, imino, hydroxyamino, hydroxyimino, formyl, formyloxy, carbamoyl, sulfamoyl, sulfanyl, sulfino, sulfo, thioformyl, thiocarboxy, dithiocarboxy, thiocarbamoyl, cyano, nitro, nitroso, azide, hydrazino, ureide, amidino, guanidino, trialkylsilyl, alkyloxy, alkenyloxy, alkynyloxy, haloalkyloxy, alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, monoalkylamino, dialkylamino, alkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, monoalkylcarbonylamino, dialkylcarbonylamino, monoalkylsulfonylamino, dialkylsulfonylamino, alkylimino, alkenylimino, alkynylimino, alkylcarbonylimino,21 alkenylcarbonylimino, alkynylcarbonylimino, alkyloxyimino, alkenyloxyimino, alkynyloxyimino, alkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy, alkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, alkylsulfanyl, alkenylsulfanyl, alkynylsulfanyl, alkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, monoalkylcarbamoyl, dialkylcarbamoyl, monoalkylsulfamoyl, dialkylsulfamoyl, aromatic carbocyclyl, non-aromatic carbocyclyl, aromatic heterocyclyl, non-aromatic heterocyclyl, aromatic carbocyclyloxy, non-aromatic carbocyclyloxy, aromatic heterocyclyloxy, non-aromatic heterocyclyloxy, aromatic carbocyclylcarbonyl, non-aromatic carbocyclylcarbonyl, aromatic heterocyclylcarbonyl, non-aromatic heterocyclylcarbonyl, aromatic carbocyclyloxycarbonyl, non-aromatic carbocyclyloxycarbonyl, aromatic heterocyclyloxycarbonyl, non-aromatic heterocyclyloxycarbonyl, aromatic carbocyclylalkyoxy, non-aromatic carbocyclylalkyoxy, aromatic heterocyclylalkyloxy, non-aromatic heterocyclylalkyloxy, aromatic carbocyclylalkyoxycarbonyl, non-aromatic carbocyclylalkyoxycarbonyl, aromatic heterocyclylalkyloxycarbonyl, non-aromaticheterocyclylalkyloxycarbonyl, aromatic carbocyclylalkyamino, non-aromatic carbocyclylalkyamino, aromatic heterocyclylalkylamino, non-aromatic heterocyclylalkylamino, aromatic carbocyclylsulfanyl, non-aromatic carbocyclylsulfanyl, aromatic heterocyclylsulfanyl, non-aromatic heterocyclylsulfanyl, non-aromatic carbocyclylsulfonyl, aromatic carbocyclylsulfonyl, aromatic heterocyclylsulfonyl, and non-aromatic heterocyclylsulfonyl. Furthermore, the substituent may have one or more substituent(s) selected from the above Group a.[0059]Examples of the substituents on the ring of "aromatic carbocyclyl", "non­aromatic carbocyclyl", "aromatic heterocyclyl" or "non-aromatic heterocyclyl" of "substituted or unsubstituted aromatic carbocyclyl", "substituted or unsubstituted non-aromatic carbocyclyl", "substituted or unsubstituted aromatic heterocyclyl", and "substituted or unsubstituted non-aromatic heterocyclyl" include the following substituents. An atom at any position(s) on the ring may be bonded to one or more group(s) selected from the following substituents.Substituent: halogen, hydroxy, carboxy, amino, imino, hydroxyamino,22 hydroxyimino, formyl, formyloxy, carbamoyl, sulfamoyl, sulfanyl, sulfino, sulfo, thioformyl, thiocarboxy, dithiocarboxy, thiocarbamoyl, cyano, nitro, nitroso, azide, hydrazino, ureide, amidino, guanidino, trialkylsilyl, alkyl, alkenyl, alkynyl, haloalkyl, alkyloxy, alkenyloxy, alkynyloxy, haloalkyloxy, alkyloxyalkyl, alkyloxyalkyloxy, alkylcarbonyl, alkenylcarbonyl, alkynylcarbonyl, monoalkylamino, dialkylamino, alkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, monoalkylcarbonylamino, dialkylcarbonylamino, monoalkylsulfonylamino, dialkylsulfonylamino, alkylimino, alkenylimino, alkynylimino, alkylcarbonylimino, alkenylcarbonylimino, alkynylcarbonylimino, alkyloxyimino, alkenyloxyimino, alkynyloxyimino, alkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy, alkyloxycarbonyl, alkenyloxycarbonyl, alkynyloxycarbonyl, alkylsulfanyl, alkenylsulfanyl, alkynylsulfanyl, alkylsulfinyl, alkenylsulfinyl, alkynylsulfinyl, monoalkylcarbamoyl, dialkylcarbamoyl, monoalkylsulfamoyl, dialkylsulfamoyl, aromatic carbocyclyl, non-aromatic carbocyclyl, aromatic heterocyclyl, non-aromatic heterocyclyl, aromatic carbocyclyloxy, non-aromatic carbocyclyloxy, aromatic heterocyclyloxy, non-aromatic heterocyclyloxy, aromatic carbocyclylcarbonyl, non-aromatic carbocyclylcarbonyl, aromatic heterocyclylcarbonyl, non-aromatic heterocyclylcarbonyl, aromatic carbocyclyloxycarbonyl, non-aromatic carbocyclyloxycarbonyl, aromatic heterocyclyloxycarbonyl, non-aromatic heterocyclyloxycarbonyl, aromatic carbocyclylalky, non-aromatic carbocyclylalky, aromatic heterocyclylalkyl, non­aromatic heterocyclylalkyl, aromatic carbocyclylalkyoxy, non-aromatic carbocyclylalkyoxy, aromatic heterocyclylalkyloxy, non-aromatic heterocyclylalkyloxy, aromatic carbocyclylalkyoxycarbonyl, non-aromatic carbocyclylalkyoxycarbonyl, aromatic heterocyclylalkyloxycarbonyl, non­aromatic heterocyclylalkyloxycarbonyl, aromatic carbocyclylalkyoxyalkyl, non­aromatic carbocyclylalkyoxyalkyl, aromatic heterocyclylalkyloxyalkyl, non­aromatic heterocyclylalkyloxyalkyl, aromatic carbocyclylalkyamino, non­aromatic carbocyclylalkyamino, aromatic heterocyclylalkylamino, non-aromatic heterocyclylalkylamino, aromatic carbocyclylsulfanyl, non-aromatic carbocyclylsulfanyl, aromatic heterocyclylsulfanyl, non-aromatic heterocyclylsulfanyl, non-aromatic carbocyclylsulfonyl, aromatic carbocyclylsulfonyl, aromatic heterocyclylsulfonyl, and non-aromatic heterocyclylsulfonyl. Furthermore, the substituent may have one or more substituent(s) selected from the above Group a.[0060]Additionally, "substituted or unsubstituted non-aromatic carbocyclyl" and "substituted or unsubstituted non-aromatic heterocyclyl" may be substituted with "oxo". In this case, it means a group wherein two hydrogen atoms on a carbon atom are substituted as below.vA/WAAA id="p-61" id="p-61"

[0061]Here, the present invention is explained in detail.A lipid binding double-stranded oligonucleotide of the present invention consists of a double-stranded oligonucleotide, wherein a first strand is a CpG oligonucleotide consisting of 8 to 50 nucleotides, a second strand is an oligonucleotide consisting of 8 to 60 nucleotides and comprising a sequence capable of hybridizing with the first strand, and a lipid binds to the second strand through a linker.More preferably, it is a double-stranded oligonucleotide, wherein a first strand is a CpG oligonucleotide consisting of 8 to 50 nucleotides, a second strand is an oligonucleotide consisting of 8 to 60 nucleotides and comprising a sequence capable of hybridizing with the first strand, but excluding a RNA oligonucleotide,the length of the second strand is 50 % or more of that of the first strand, and a lipid comprising C12 to C30 hydrocarbon chain(s) binds to the second strand through a linker.[0062]A first strand of a lipid binding double-stranded oligonucleotide of the present invention is a CpG oligonucleotide consisting of 8 to 50 nucleotides. [0063]The "CpG oligonucleotide (CpG ODN)" means a single-stranded oligonucleotide comprising dinucleotide of non-methylated cytosine guanine (5’-24 CpG-3’) motif (CpG motif) and it is well known that it can be useful as a vaccine adjuvant because it induces acquired immune response through TLR(Nat Rev Drug Discov, 2006, 5, 471-484, Expert Rev Vaccines., 2011, 10(4), 499-511). The CpG oligonucleotide used for the present invention comprises at least one CpG motif and may comprise several CpG motifs.[0064]The length of the CpG oligonucleotide used for the present invention is to 50 nucleotides. For example, it is 8 to 50 nucleotides, 8 to 40 nucleotides, to 30 nucleotides, 10 to 25 nucleotides, 15 to 25 nucleotides or 18 to nucleotides.[0065]As "the CpG oligonucleotide", it is not especially limited to the CpG oligonucleotide, which is well known for the immunostimulatory activity in this field can be used. For example, the CpG oligonucleotide or the method for preparing is described in WO2006/065751, WO2007/092315, WO2008/068638, WO2010/067262, WO2010/125480, WO2014/047588, WO2014/134698, WO2015/041318, US2011/0300163 or the like. These CpG oligonucleotides can be synthesized in reference to methods described in the above documents.The CpG oligonucleotides are classified based on the sequence, secondary structure and effect on human peripheral blood mononuclear cells (PBMC), into class A, class B, class C, class P or class S (Advanced drug delivery reviews, 2009, 61(3),195-204).Class A: ODN1585, ODN2216, ODN2336 or the like;Class B: ODNBW006, ODN D-SL01, ODN1668 (WO2005/063264), OND18(WO2007/030580), OND2006 (CpG7909, PF-3512676) (WO98/18810), ODN2007, ODN684 or the like; andClass C: ODN D-SL03, ODN 2395, ODN M362 or the like. These can be purchased from InvivoGen as a research reagent. In addition,CpG-28 (WO2000/056342),CpG-685(GNKG-168) (Blood, 2010, 115(24), 5041),CpG-ODN C274 (PLoS ONE, 2013, 8(4), e62373),KSK-13 (KSK-CpG) (US7408050),CpG ODN 10104 (CpG-10104) (Drug Data Rep, 2006, 28(3), 258),CpG ODN-1585 (WO2001/022990),25 ODN-5890 (WO2006/080946),1018-ISS (WO2008/073661),EMD-1201081 (HYB-2055, IMO-2055) (WO2005/009355),D35-CpG, K3-CpG (GeneDesign, Inc.),or the like. For the present invention, the CpG oligonucleotides of any class can be used. The CpG oligonucleotides of class A (e.g., ODN2216, ODN23and D35-CpG), the CpG oligonucleotides of class B (e.g., ODN1826, ODN2006, CpG-28, 1018-ISS, IMO2055, K3-CpG, ODN684 and D-LS01) or the CpG oligonucleotides of class C (e.g., D-LS03, ODN2395 and ODN M362) is preferable. ODN1826 or ODN2006 is especially preferable.[0066]A second strand of a lipid binding double-stranded oligonucleotide of the present invention is an oligonucleotide consisting of 8 to 60 nucleotides and comprising a sequence capable of hybridizing with the CpG oligonucleotide which is the first strand, but excluding a RNA oligonucleotide,[0067]Preferably, a second strand is an oligonucleotide consisting of 8 to nucleotides and comprising a sequence capable of hybridizing with the CpG oligonucleotide which is a first strand under a stringent condition.Any oligonucleotide can be used for a second strand as long as it can be hybridized with the CpG oligonucleotide under a stringent condition, and the one comprising 1 or several mismatch(es) in a part of hybridization can be exemplified.For example, it is the oligonucleotide whose part for hybridization has at least 70 % or more, preferably 80 % or more, more preferably 90 % or more, and most preferably 95 % or more homology to a complementary sequence of the CpG oligonucleotide of a first strand.The homology shows the similarity as a score for example, by BLAST, a search program using algorithm discovered by Altschul et al. (The Journal of Molecular Biology, 215, 403-410 (1990).)[0068]The "stringent conditions" mean conditions under which a base sequence forms hybrid (so-called specific hybrid) with a specific sequence but any base sequence without the equivalent function does not form hybrid (so-called non­26 specific hybrid) with the specific sequences. People skilled in the art can easily select the conditions by changing a temperature during hybridization reaction or washing, salt concentration in hybridization or washing buffer, or the like. In detail, an example of the stringent conditions of this invention is, but not limited to the condition, which the oligonucleotide is hybridized in 6xSSC (0.9 M NaCl, 0.09 M trisodium citrate) or 6xSSPE (3 M NaCl, 0.2 M NaH2PO4, 20 mM EDTA-2Na, pH 7.4) at 42 °C and washed with 0.5xSSC at 42 °C. As a hybridization method, well-known methods in the art, for example, southern blot hybridization or the like can be used. In detail, it can be performed according to a method disclosed in Molecular Cloning: A Laboratory Manual, Second Edition (1989) (Cold Spring Harbor Laboratory Press), Current Protocols in Molecular Biology (1994) (Wiley-Interscience), DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition (1995) (Oxford University Press) or the like.[0069]"1 or several mismatch(es)" means 1 to 5, preferably 1 to 3, and more preferably 1 or 2 mismatches.[0070]The length of a second strand of a lipid binding double-stranded oligonucleotide of the present invention is 8 to 60 nucleotides. For example, it is 8 to 60 nucleotides, 8 to 50 nucleotides, 8 to 40 nucleotides, 8 to nucleotides, 10 to 25 nucleotides or 15 to 25 nucleotides. The length of the second strand can be same with that of the CpG oligonucleotide which is a first strand, and 1 or several nucleotide(s) shorter than that of the CpG oligonucleotide as long as it can be hybridized with the CpG oligonucleotide. Furthermore, the length of a second strand can be longer than that of the CpG oligonucleotide by adding 1 or several nucleotide(s) at one or both sides of a part hybridizing with the CpG oligonucleotide."1 or several nucleotide(s)" means 1 to 10, 1 to 5, 1 to 3, or 1 or nucleotide(s).A preferable length of a second strand depends on the length of the CpG oligonucleotide of a first strand. For example, it is a length of 50 % or more, % or more, 70 % or more, 50 to 100 %, 60 to 100 % or 70 to 100 % of the length of a first strand. 50 to 100% of the length of a first strand is especially27 preferable.[0071]The oligonucleotide of a second strand of a lipid binding double-stranded oligonucleotide of the present invention is an oligonucleotide which nucleosides selected from the group consisting of DNA nucleosides, RNA nucleosides and nucleoside derivatives are bounded. All nucleosides can be same, or two or more kinds of nucleoside. However, an RNA oligonucleotide which all nucleosides are a RNA nucleoside is excluded. As a nucleoside comprised in a second strand, an oligonucleotide which DNA nucleosides and/or nucleoside derivatives are bounded is preferable. All nucleosides can be a DNA nucleoside, nucleoside derivative, or both of them.When an oligonucleotide comprises DNA nucleosides and nucleoside derivatives, example is an oligonucleotide comprising a center region and end regions at the both sides of the center region and comprising at least one nucleoside derivative in the end region at the both sides. In detail, 5’ and/or 3’ end region(s) comprises 1 or more, preferably 1 to 5, and more preferably to 3 nucleoside derivatives. The kind, number and position of modification(s) in one end region may be same or different from those in the other end region. In the other embodiment, it is an oligonucleotide randomly comprising nucleoside derivatives.[0072]As a nucleoside derivative in a second strand of a lipid binding double- stranded oligonucleotide of the present invention, any modification(s) for a nucleoside which is well-known in this field such as the above examples can be used.A nucleoside having a substituent at the 2’ position of sugar and/or a nucleoside having a bridge structure between at the 4’ and 2’ positions of sugar are preferable.As a substituent at the 2’ position of a sugar, F, OCH3 or OCH2CH2OCH3 is preferable. OCH3 is especially preferable.As a bridge structure between the 4’ and 2’ positions of a sugar, 4’- (CH2)m-O-2’, wherein m is an integer of 1 to 4, or 4’-C(=O)-NR3-2’, wherein Ris hydrogen atom or alkyl, is preferable.[0073]28 As an internucleoside linkage in the oligonucleotide of a second strand of a lipid binding double-stranded oligonucleotide of the present invention, any well-known internucleoside linkage such as the above examples in this field can be used. All internucleoside linkages can be same, or two or more kinds of linkage. D-oligo and/or S-oligo is preferable.When 2 or more kinds of internucleoside linkages are comprised such as D-oligo and S-oligo, example is an oligonucleotide comprising a center region and end regions at the both sides of the center region, and comprising at least one unnatural internucleoside linkage (e.g., S-oligo) in the end region at the both sides and natural internucleoside linkage (e.g., D-oligo) in the center region. For example, 5’ and/or 3’ end region(s) comprises 1 or more, preferably 1 to 5, and more preferably 2 to 3 unnatural internucleoside linkages. The kind, number and position of modification(s) in one end region may be same or different from those in the other end region. In the other embodiment, it is an oligonucleotide randomly comprising unnatural internucleoside linkages.[0074]The CpG oligonucleotide of a first strand and oligonucleotide of a second strand in a lipid binding double-stranded oligonucleotide of the present invention can be synthesized according to the usual methods in this field. For example, they can be easily synthesized by an automated nucleic acid synthesizer which is commercially available (e.g., the synthesizer by AppliedBiosystems, Dainippon Seiki). A method for synthesizing is solid- phase synthesis using phosphoramidite, solid-phase synthesis using hydrogen phosphonate or the like. Examples are disclosed in the following Example 1, Tetrahedron Letters 22, 1859-1862 (1981) and the like.[0075]The synthesized first and second strands form a double-stranded oligonucleotide by hybridizing according to the well-known method. Examples are disclosed in the following Example 1, Example 1 in WO2013/089283 and the like.[0076]In the lipid binding double-stranded oligonucleotide of the present invention, a lipid binds to the second strand through a linker. id="p-77" id="p-77"

[0077]"Lipid" means a hydrophobic compound. It is not limited as long as it is a well-known lipid, and may be a straight, branched or cyclic form. Examples are fatty acid with a C8 to C30 aliphatic chain(s) (e.g., farnesol), diacyl lipid, cholesterol, cholesterol derivatives, steroid acid (e.g., bile acid), lipid A, tocopherol and combination thereof. Examples of fatty acid with an aliphatic chain(s) are, but not limited to, unsaturated or saturated fatty acid in a straight or branched form, and fatty acid derivatives (e.g., fatty acid esters, fatty acid amides and fatty acid thioesters).[0078]As a lipid used for the present invention, a lipid comprising a hydrocarbon chain(s) is preferable. Preferably, it is a lipid comprising 1 or hydrocarbon chain(s) or the like. Especially preferably, a lipid binding double-stranded oligonucleotide of the present invention has a diacyl lipid. When lipids bind at two or more positions and one lipid is a diacyl lipid, the other lipid can be a lipid except for a diacyl lipid."Diacyl lipid" is phospholipid, glycolipid, sphingolipid or combination thereof, which comprising two hydrocarbon chains. Preferably, it is the group of the following (a) or (f).Hydrocarbon chains in the lipid independently comprise about C8 to Ccarbon atoms. They can be saturated, unsaturated, or combination thereof, and branched. When the lipid has one chain, the length of the chain is preferably C8 to C30, and more preferably C8 to C20. When the lipid has two chains, the length of the chains is preferably C10 to C30, more preferably Cto C30, and most preferably C14 to C24. When the lipid has two chains, the length of two chains may be same or different. Chains in a lipid bind a part comprising phosphoric acid, sugar or the like (a position binding with an oligonucleotide) through ester bond, amide bond, thioester bond, combination thereof or the like.A lipid binding double-stranded oligonucleotide of the present invention has a lipid comprising a C12 to C30 hydrocarbon chain(s) in a second strand. When lipids bind at 2 or more positions and one lipid is a lipid comprising a C12 to C30 hydrocarbon chain(s), the number of carbon atoms of a hydrocarbon chain(s) in the other lipid can be less than 12.30 id="p-79" id="p-79"

[0079]Examples of "lipid" are the followings. wherein p and q are each independently an integer of 6 to 28, preferably 8 to 28, more preferably 10 to 28, and most preferably 10 to 18.[0080]In a lipid binding double-stranded oligonucleotide of the present invention, a lipid can bind at any position of the second strand. It can bind at the 3’ end, the 5’ end or in the second strand.[0081]When the lipid binds at the 5’ end, for example, it can bind as below.

Oligonucleotide B1O^ O O Linker Ya-P­IOH LipidONH OO wherein B1 is a base at the 3’ end of the second strand, Ya is O or S, and p or q is each independently an integer of 6 to 28, and preferably 10 to 18.[0082]When the lipid binds at the 3’ end, for example, it can bind as below. wherein B1 is a base at the 5’ end of the second strand, Ya is O or S, and p or q is each independently an integer of 6 to 28, and preferably 10 to 18. [0083]When the lipid binds in a second strand, for example, it can bind asbelow.

A ס / V H3C or A wherein B1 and B2 are the neighboring bases in the second strand, Ya or Yb is each independently O or S, and p and q is each independently an integer of 6 to 28, and preferably 10 to 18.Furthermore, the lipid preferably binds at one or two position(s) in the second strand. It preferably binds at 3’ and/or 5’ end. It more preferably binds at 5’ end.[0084]The lipid can be synthesized in reference to well-known methods in this field. Examples of the lipid or the preparation methods are disclosed in the following Example 1, Patent Document 1 and the like.[0085]In a lipid binding double-stranded oligonucleotide of the present invention, the lipid binds to a second strand through a linker. As a "linker", any linker used in this field can be used. Examples are polar linker, alkylene linker, ethylene glycol linker and ethylenediamine linker. When a lipid is a phospholipid, it is a linker which is 4 to 26 atoms between oxygen atom of a second strand and phosphorus atom of a lipid. Examples are an oligonucleotide linker or the following linkers.

Lipid id="p-86" id="p-86"

[0086] Oligonucleotide- Lipid NH Lipid Lipid Oligonucleotide -P-0 Lipid Oligonucleotide- Lipid Oligonucleotide Lipid '0OH׳ 0 Oligonucleotide Oligonucleotide Oligonucleotide------ P^q wherein Y’ is O or S, and r or s is each independently an integer of 1 to 10, preferably 1 to 5, and more preferably 1 to 3. t is an integer of 1 to 4,preferably 1 to 3, and more preferably 2 or 3.The linker can be synthesized in reference to the well-known method in this field. The oligonucleotide linker can be synthesized by the method similar to the method for synthesizing the oligonucleotides in the above example.[0087]The linker is preferably an oligonucleotide linker. The length of an oligonucleotide linker is 2 to 10, 2 to 5, 2, 3, 4 or 5 nucleotides. An example is the following.[0088] -°S Oligonucleotide Lipid—Linker wherein B1 or B2is adenine(A), guanine(G), cytosine (C), 5-methylcytosine (5- Me-C), thymine (T) or uracil(U). Y’ is each independently O or S. Z1 or Z2 is each independently H or OH, and preferably H.An example of an oligonucleotide linker is a DNA linker, that is, - (dX!)u-, wherein X1 is each independently A, G, C or T, and u is an integer of to 8. In detail, it is dG, dGdG, dGdGdGdG, dGdGdGdGdG, dT, dTdT, dTdTdTdT, dTdTdTdTdT or the like. dGdG, dGdGdGdGdG or dTdT is especially preferable. The internucleoside linkage in the DNA linker is preferably phosphorothioate linkage.[0089]3’ end, 5’ end or linker without binding lipid of a lipid binding double- stranded oligonucleotide of the present invention can be further modified. To be capable of tracking of the oligonucleotide, to improve pharmacokinetics or pharmacodynamics of the oligonucleotide, or to enhance the stability or binding affinity of the oligonucleotide, the well-known modified group in this field can be used. Examples are a protecting group of a hydroxyl group, reporter molecule, cholesterol, phospholipid, pigment, fluorescent molecule and the like.Furthermore, 3’ or 5’ end without binding the lipid of a lipid binding double-stranded oligonucleotide of the present invention may comprise a phosphate ester moiety. The "phosphate ester moiety" means a phosphate group at the end comprising phosphate ester or modified phosphate ester. Although the phosphate ester moiety may be at the either end, it is preferably 5’ end nucleoside. In detail, it is a group of the formula: -O-P(=O)(OH)OH or the modified group. That is, one or more of O or OH is optionally substituted with H, O, OR’, S, N(R’), wherein R’ is H, amino-protecting group, or substituted or unsubstituted alkyl, or alkyl. 5’ or 3’ end may each independently comprise substituted or unsubstituted 1 to 3 phosphate ester moiety.[0090]The present invention encompasses a pharmaceutical composition (a pharmaceutical composition of the present invention) or a vaccine composition (a vaccine composition of the present invention) comprising a lipid binding double-stranded oligonucleotide of the present invention (a vaccine or adjuvant of the present invention of the present invention).In addition, the present invention encompasses a pharmaceutical or vaccine composition comprising an antigen and a lipid binding double-stranded oligonucleotide of the present invention (an adjuvant of the present invention). [0091]An "antigen" is a molecule that is capable of inducing an immune response. Examples include, but are not limited to, cells, cell extracts, proteins, recombinant proteins, purified proteins, polypeptides, peptides, polysaccharides, polysaccharide conjugates, peptide or non-peptide mimics of polysaccharides, other molecules encoded by plasmid DNA, haptens, small molecules, lipids, glycolipids, carbohydrates, whole killed pathogens, viruses, viral extracts, live attenuated virus, viral vector, live attenuated bacteria, bacterial vectors, multicellular organisms such as parasites and allergens.The antigen may be provided as a single antigen or combination of antigens. The antigen may be provided as a complex mixture of polypeptide or oligonucleotide.Antigens include, but are not limited to, microbial antigens, self­antigens and addictive substances.[0092]A "microbial antigen" means an antigen of a microorganism, and the microorganism includes, but is not limited to, a bacterium, virus, parasite and fungus.A "bacterium" is not especially limited as long as it is a bacterium which causes a disease in human, pet, livestock or the like. In detail, it is Streptococcus (Streptococcus pyogens, Streptococcus pneumoniae or the like), Staphylococcus aureus (MSSA, MRSA or the like), Staphylococcus epidermidis, Enterococcus, bacteria belonging to the genus Listeria, bacteria causing36 meningitis, Gonococcus, pathogenic Escherichia coli, Friedlander bacilli, Proteus bacilli, Bordetella pertussis, Pseudomonas aeruginosa, Serratia marcescens, Citrobacter, Acinetobacter, Enterobacter, Mycoplasma, Clostridia, Tubercle bacilli, Cholera bacilli, Yersinia pestis, Corynebacterium diphtheriae, Dysentery bacillus, Bacillus anthracis, Treponema pallidum, Tetanus bacillus, Mycobacterium leprae, Legionella pneumophila, Leptospira, Borrelia, Francisella, Coxiella, Rickettsia, Chlamydia, Burkholderia mallei, Helicobacter pylori or the like.A "virus" is not especially limited as long as it is a virus which causes a disease in human, pet, livestock or the like. Examples are influenza virus, respiratory syncytial virus (RSV), papilloma virus, hepatitis virus (type A, B,C, D, E, F, G, TT or the like), rhinovirus, variola virus, morbillivirus, rubella virus, poliovirus, varicella zoster virus, norovirus, Norwalk virus, sapovirus, Sapporo virus, mumps virus, adenovirus, enterovirus, rotavirus, human immunodeficiency virus such as HIV-1 and HIV-2, rabies virus, T-lymphotropic virus, yellow fever virus, cytomegalovirus, SARS-CoV, coronavirus such as MERS-CoV, ebola virus, polyomavirus, JC virus, BK virus, herpes virus such as herpes simplex virus 1 (HSV1) and herpes simplex virus 2 (HSV2), lymphocryptovirus , roseolovirus, Japanese encephalitis virus, Coxsackie virus, dengue virus, West Nile virus, coronavirus, parvovirus, Epstein-Barr virus, Marburg virus, hantavirus, Lassa virus, Chikungunya virus, Hantaan virus, louping ill virus, lymphocytic choriomeningitis virus, bornavirus, Rift Valley fever, Thogoto virus, Dhori virus, foot-and-mouth disease virus, Newcastle disease virus, bovine papular stomatitis virus, rinderpest virus, swine vesicular disease virus, calicivirus, torovirus, African horse sickness virus, arterivirus, sheep pox virus, capripoxvirus, sheep-associated malignant catarrhal fever virus, viral hemorrhagic septicemia virus and vesicular stomatitis virus.A "parasite" is not especially limited as long as it is a parasite which causes a disease in human, pet, livestock or the like. Examples are entamoeba histolytica, malaria, toxoplasma, leishmania, cryptosporidium, trypanosoma, echinococcus, schistosoma japonicum, filaria, roundworm and diphyllobothrium.A "Fungus" is not especially limited as long as it is a fungus which causes a disease in human, pet, livestock or the like. In detail, it is aspergilloma, Candida, cryptococcus, trichophyton, histoplasma, pneumocystis or the like.[0093]A "self-antigen" means a tumor antigen, an antigen associated with Alzheimer’s disease, an antigen against a human antibody, an antigen expressed from human endogenous retroviral elements or the like.An example of a "tumor antigen" is an antigen specifically expressed in cancer cells. Examples include protein, peptide and fusion peptide comprising them. Examples are peptides described in WO2006/090810, WO2007/145318, WO2008/047473, WO2008/102557, WO2009/025117, WO2009/025196, WO2009/1539992, WO2010/013485, WO2010/021112, WO2010/073551, WO2010/095428, WO2010/131452, WO2010/137295, WO2011/067920, WO2011/074236, WO2011/089921, WO20U/1U392, WO2012/053200, WO2012/053206, WO2012/169200, WO2013/024582, WO2013/061594, WO2014/041784, WO2014/087626 and the like.An example of an antigen associated with Alzheimer's disease is tau, 6- amyloid or the like.An example of an antigen against a human antibody is IgE.[0094]An "addictive substance" means nicotine, cocaine or the like. An example of a nicotine antigen is a nicotine hapten conjugated to a carrier (e.g., diphtheria toxin).[0095]A pharmaceutical or vaccine composition of the present invention can further comprise a well-known adjuvant(s) as long as the effect of the lipid binding double-stranded oligonucleotide of the present invention is maintained. For example, it is cholera toxin, salmonella toxin, alum or an agonist for a Toll-like receptor (TLR) that is not TLR9. Examples of agonists for TLR are an agonist for TLR3 such as stabilized poly(l:C); an agonist for TLR4 such as a derivative of lipopolysaccharide (LPS) (e.g., MPL and GLA); an agonist for TLR5 such as flagellin; an agonist for TLR7; and an agonist for TLR8. Examples include aluminum salt such as aluminum hydroxide, an immune stimulatory complex (ISCOM), oil-in-water or water-in-oil emulsion, liposome and a delivery system such as nanoparticle and microparticle. [0096]As the following examples, a pharmaceutical or vaccine composition of the present invention comprising a tumor antigen has any or all of the following excellent characteristics:a) 1 % or more CTL inductivity.b) Inhibition on tumor implantation.c) Effect of tumor regressiond) Good pharmacokinetics such as a high bioavailability, moderate clearance and the like. Especially, efficient delivery to lymph-nodes.e) High metabolic stability.f) No cytokine release syndrome.g) Weak local irritation.h) No mutagenicity.i) Low cardiovascular risk.j) Low acute toxicity risk.[0097]Any administration method and formulation for the pharmaceutical or vaccine composition of the present invention can be used if it is a well-known administration method and formulation in this field.[0098]A pharmaceutical or vaccine composition of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Examples of an administration method include topical (including ophthalmic, intravaginal, intrarectal, intranasal and transdermal), oral and parenteral. Examples of parenteral administration include intravenous injection or drip, subdermal, intraperitoneal or intramuscular injection, lung administration by aspiration or inhalation, intrathecal administration and intraventricular administration. Intravenous injection or subcutaneous administration is preferable.[0099]When the pharmaceutical or vaccine composition of the present invention is topically administered, a formulation such as a transdermal patch, ointment, lotion, cream, gel, drop, suppository, spray, liquid and powder can be39 used.Examples of the composition for oral administration include powder, granule, suspension or solution dissolved in water or non-aqueous vehicle, capsule, powder and tablet.Examples of the composition for parenteral, intrathecal or intraventricular administration include sterile aqueous solutions which contain buffers, diluents and other suitable additives.[0100]A pharmaceutical or vaccine composition of the present invention can be obtained by mixing an effective amount with various pharmaceutical additives suitable for the administration form, such as excipients, binders, moistening agents, disintegrants, lubricants and diluents as needed. When the composition is an injection, it together with a suitable carrier can be sterilized to obtain a composition.[0101]Examples of the excipients include lactose, saccharose, glucose, starch, calcium carbonate and crystalline cellulose.Examples of the binders include methylcellulose, carboxymethylcellulose, hydroxypropylcellulose, gelatin and p olyvinylpyrrolidone.Examples of the disintegrants include carboxymethylcellulose, sodium carboxymethylcellulose, starch, sodium alginate, agar and sodium lauryl sulfate.Examples of the lubricants include talc, magnesium stearate and macrogol. Cacao oil, macrogol, methylcellulose or the like may be used as base materials of suppositories.When the composition is prepared as solutions, emulsified injections or suspended injections, solubilizing agents, suspending agents, emulsifiers, stabilizers, preservatives, isotonic agents and the like which are usually used may be added as needed. For oral administration, sweetening agents, flavors or the like may be added.[0102]Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several40 months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of a pharmaceutical or vaccine composition accumulation in the body. Persons of ordinary skill in the art can determine optimal dosages, dosing methodologies and repetition rates.[0103]The content rate of a lipid binding double-stranded oligonucleotide of the present invention of a pharmaceutical or vaccine composition of the present invention is, but not especially limited to, usually about 0.01 to 99.99 wt% against 100 wt% of a pharmaceutical or vaccine composition.When an antigen was comprised, considering the content rate of the antigen and an adjuvant of the present invention, the adjuvant is usually about 10 to 1000 parts by weight against 1 part by weight of the antigen.The dosage of a pharmaceutical or vaccine composition is diversified depending on the desired degree of immune stimulation, age of the subject of administration, sex or the like, so it can be appropriately set, e.g., 0.001 to mg/kg weight/day, to which it is not especially limited.In addition, according to the degree of effectiveness of the desired pharmaceutical or vaccine composition, age of the subject of administration, sex or the like, a pharmaceutical or vaccine composition of the present invention can be administered to the same subject at several times, e.g., up to about 2 to 4 times. When a pharmaceutical or vaccine composition of the present invention is administered to the same subject at several times, the interval can be appropriately set, e.g., about 14 to 30 days.[0104]A pharmaceutical or vaccine composition of the present invention can be administered in combination of 1 or more kind(s) of medicines. The therapeutic agent known as a therapeutic agent for the target disease in this field can be used as the medicines. For example, when the disease is cancer, a pharmaceutical or vaccine composition of the present invention can be co­administered with a chemotherapeutic agent(s). When the disease is bacterial infections, a pharmaceutical or vaccine composition of the present invention can be co-administered with an antibiotic(s). A pharmaceutical or vaccine composition of the present invention may be simultaneously or41 separately administered with the agent(s). In addition, a pharmaceutical or vaccine composition of the present invention and the therapeutic agent(s) may be administered as the different formulations independently comprising each one or a single formulation comprising the pharmaceutical or vaccine composition and the therapeutic agent(s). Furthermore, when the disease is cancer, a pharmaceutical or vaccine composition of the present invention can be administered in combination of the surgical treatment which is known in this field.[0105]Moreover, the present invention encompasses a method for increasing an immune response in a subject, comprising administering to the subject an effective amount of a pharmaceutical or vaccine composition of the present invention. An example of "immune response" is an increase in inductivity of specific cytotoxic T-lymphocyte (CTL) compared to a control. A specific method for measuring CTL inductivity is disclosed in, e.g., the following Example 3 and WO2013/024582.[0106]Furthermore, the present invention encompassed a method for treating a cancer or infectious disease, comprising administering to a subject an effective amount of a pharmaceutical or vaccine composition of the present invention to reduce one or more symptoms of the cancer or infectious disease compared to a control.[0107]"The subject" means any individual which is a target for a treatment with a pharmaceutical or vaccine composition of the present invention. Examples include mammals such as humans, laboratory animals (e.g., mice and rats), pets (e.g., dogs, cats, ferrets and birds) and livestock (e.g., cattle, pigs, chickens, goats, ostriches, sheep and horses).[0108]"An effective amount" means enough dosage to induce or enhance the immune response, provide the desired pharmacological and/or physiological effect, or provide a treatment for the disorder, disease or status. The exact dosage may be changeable depending on various factors such as the subject- dependent variable (e.g., age and health condition of immune system), disease,42 stage of disease and the provided treatment.[0109]"Cancer" is not especially limited as long as it is a cancer which a human, pet, livestock or the like may contract. Examples are chronic myeloid leukemia (CML), acute myeloid leukemia (AML), lymphoma, Hodgkin's disease, Non-Hodgkin lymphoma, multiple myeloma, brain tumor, breast cancer, endometrial carcinoma, cervical cancer, ovarian cancer, esophageal cancer, stomach cancer such as diffuse gastric cancer, appendiceal cancer, colorectal cancer, liver cancer, hepatocellular carcinoma, gallbladder cancer, cholangiocarcinoma, pancreatic cancer, adrenal cancer, gastrointestinal stromal tumor, mesothelioma, head and neck carcinoma, laryngeal cancer, oral cancer, gingival cancer, tongue cancer, buccal mucosa cancer, salivary gland cancer, paranasal cancer, carcinoma of maxillary sinus, carcinoma of the frontal sinus, ethmoid sinus cancer, sphenoid sinus cancer, thyroid cancer, renal cancer, lung cancer such as Non-Small Cell Lung Cancer (NSCLC) and small cell lung cancer (SCLC), osteosarcoma, prostate cancer, testicular cancer, bladder cancer, rhabdomyosarcoma, skin cancer, anal carcinoma, chondrosarcoma, synovial sarcoma, endometriosis, soft tissue tumor and osteoblastoma.[0110]An "infectious disease" means acute or chronic infectious disease, especially viral infectious disease. Examples include, but not limited to, local or systemic viral infections such as immunodeficiency by HIV or the like, papilloma by HPV or the like, herpes by HSV or the like, encephalitis, influenza by human influenza virus A or the like and cold by human rhinovirus.[0111]In this description, meaning of each abbreviation is as follows:Ac: acetylCPG: Controlled Pore Glass DIEA: N,N-diisopropylethylamine DMAP: 4-dimethylaminopyridine DMTr: dimethoxytritylDMT-MM: 4-(4,6-dimethoxy1,3,5-triazine2-yl)-4-methylmorpholinium chloride DMF: N,N’-dimethylformamide Et: ethylFmoc: 9-fluorenylmethyloxycarbonylHBTU: O-benzotriazole-N,N,N’,N’-tetramethyluronium-hexafluoro-phosphate Me: methylMMTr: 4-methoxyphenyldiphenylmethylMMTrCl: 4-methoxytrityl chloridePBS: phosphate buffered salineTBS: tert-butyldimethylsilylTBAF: tetrabutylammonium fluorideTFA: trifluoroacetic acidTHF: tetrahydrofuran Examples[0112]The present invention will be described in more detail with reference to, but not limited to, the following Examples, Reference Examples and Test Examples.NMR analysis of each compound obtained in Examples was performed by 300 MHz or 400 MHz using CD3OD, CDCl3 or DMSO-d6.[0113]UPLC analysis was performed under the following conditions.Mobile phases: [A] is 0.1 % aqueous formic solution, [B] is acetonitrile solution containing 0.1 % aqueous formic acidGradient: linear gradient of 5 % to 100 % solvent [B] for 3.5 minutes was performed, and 100 % solvent [B] was maintained for 0.5 minute.Column: ACQUITY UPLC (Registered trademark) BEH C18 (1.7 pm, i.d. 2.1 x mm) (Waters)Flow rate: 0.8 mL/minPDA detection wavelength: 254 nm (Detection range 210-500 nm)[0114]Example 1 Synthesis of a lipid binding double-stranded oligonucleotide of the present invention A) Synthesis of lipids44 id="p-115" id="p-115"

[0115]1-1) Synthesis of 4-n wherein n is an integer of 6 to 28. [0116]1-1-1) Synthesis of Compound 3-Step 1 Compound 2-6 (3.20 g, 22.2 mmol, Tokyo Chemical Industry Co., Ltd.)was dissolved in THF-DMF (5:1, 60 mL), and DIEA (4.84 mL, 27.7 mmol) and HBTU (8.84 g, 23.3 mmol) were added thereto. The mixture was stirred at room temperature for 30 minutes. Compound 1 (2.2 g, 24.41 mmol) in DMF solution (5 mL) was added dropwise over 10 minutes and stirred for 3 hours.The amount of the reaction mixture was concentrated to the half underreduced pressure, and it was added dropwise to water (100 mL). The mixture was stirred for 10 minutes, and the resulting solid was collected by filtration. The solid was washed with water (50 mL) and acetonitrile (150 mL) to obtain Compound 3-6 (3.2 g, 9.34 mmol) as white solid.1H-NMR (CDCl3) 8: 6.45 (2H, t, J = 6.0 Hz), 4.39 (1H, s), 3.78-3.73 (1H, m),3.33 (4H, t, J = 5.6 Hz), 2.22 (4H, t, J = 7.7 Hz), 1.67 (4H, dt, J = 31.0, 14.Hz), 1.30-1.27 (16H, m), 0.88 (6H, t, J = 6.8 Hz).[0117]1-1-2) Synthesis of Compound 4-25 Step 1Compound 2-8 (7.65 g, 44.40 mmol, Wako Pure Chemical Industries, Ltd.) was dissolved in DMF (50.0 mL) and dichloromethane (100.0 mL). DIEA (8.72 mL, 66.6 mmol) and HBTU (18.52 g, 48.8 mmol) were added thereto, and the mixture was vigorously stirred at room temperature for 30 minutes. To the resulting suspended solution, Compound 1 (2.0 g, 22.2 mmol) was added at room temperature, and the mixture was vigorously stirred for 3 hours. To the reaction mixture was added aqueous saturated sodium bicarbonate solution (mL), the resulting white solid was collected by filtration. The resulting solid was washed with water (100 mL) and acetonitrile (100 mL) and dried to obtain Compound 3-8 (6.6 g, 16.6 mmol) as white solid.1H-NMR(CDCla)8:6.29 (brs, 2H), 4.12 (s, 1H), 3.76 (dd, 1H, J = 4.5, 4.5 Hz), 3.42-3.35 (m, 2H), 3.31-3.25 (m, 2H), 2.22 (t, 4H, J = 7.5 Hz), 1.65-1.60 (m, 4H), 1.29-1.26 (m, 24H), 0.88 (t, 6H, J = 6.5 Hz).ESI-MS(m/z): 340 (M+1).

Step 2Compound 3-8 (2.88 g, 7.22 mmol) was suspended in dichloromethane (60 mL), and DIEA (5.30 mL, 30.3 mmol) was added thereto. Then, 2- cyanoethyl N,N-diisopropylchlorophosphoramidite (3.23 mL, 14.5 mmol) was added at room temperature, and the mixture was heated under reflux for hours. After cooling to room temperature, the reaction mixture was partitioned by the separatory funnnel, and the organic layer diluted with dichloromethane (80 mL) was washed twice with aqueous saturated sodium bicarbonate solution (20 mL), twice with water (20 mL), and once with brine (20 mL). After the resulting organic layer was dried over magnesium sulfate, the solvent was concentrated under reduced pressure. The resulting brown oil, Compound 4-8 (2.88 g, 4.81 mmol) was obtained as a crude product. Formation of the compound was determined based on introduction of trivalent phosphorus by 31P-NMR.31P-NMR(CDCl3)8:148.2 (s)[0118]1-1-3) Synthesis of Compound 4-Step 1Compound 2-10 (9.78 g, 48.8 mmol, Tokyo Chemical Industry Co., Ltd.) was dissolved in DMF (150 mL) and dichloromethane (75 mL). DIEA (12.mL, 73.2 mmol) and HBTU (20.37 g, 53.7 mmol) were added thereto, and the mixture was vigorously stirred at room temperature for 45 minutes. To the resulting suspended solution, Compound 1 (2.2 g, 24.41 mmol) was added at46 room temperature, and the mixture was vigorously stirred. Then, the mixture was heated to 80 °C, and then stirred for 4 hours. To the reaction mixture was added aqueous saturated sodium bicarbonate solution (200 mL) and water (50 mL), the resulting white solid was collected by filtration. The resulting solid was washed with water (200 mL) and acetonitrile (400 mL) to obtain Compound 3-10 (9.95 g, 21.88 mmol) as white solid.1H-NMR (CDCla)8: 6.25 (2H, t, J = 5.8 Hz), 4.07 (1H, s), 3.75 (1H, s), 3.44-3.(2H, m), 3.29-3.23 (2H, m), 2.22 (4H, t, J = 7.6 Hz), 1.67-1.59 (4H, m), 1.30-1.(64H, m), 0.88 (6H, t, J = 6.8 Hz).

Step 2Compound 3-10 (230 mg, 0.506 mmol) was suspended in dichloromethane (11 mL), and DIEA (0.353 mL, 2.023 mmol) was added thereto. Then, 2- cyanoethyl N,N-diisopropylchlorophosphoramidite (0.226mL, 1.012mmol) was added at room temperature, and the mixture was heated under reflux for hours. After cooling to room temperature, the reaction mixture was partitioned by the separatory funnnel, and the organic layer was washed twice with aqueous saturated sodium bicarbonate solution (10 mL), five times with water (10 mL) and once with brine (10 mL). After the resulting organic layer was dried over magnesium sulfate, the solvent was concentrated under reduced pressure. The resulting brown oil, Compound 4-10 (365 mg) was obtained as a crude product. Formation of the compound was determined based on introduction of trivalent phosphorus by 31P-NMR.31P-NMR(CDCl3 )8:148.2 (s)[0119]1-1-4) Synthesis of Compound 4-Step 1Compound 2-12 (5.07 g, 22.19 mmol, Tokyo Chemical Industry Co., Ltd.) was dissolved in DMF (51.8 mL) and dichloromethane (28.6 mL). DIEA (5.mL, 33.3 mmol) and HBTU (9.26 g, 24.4 mmol) were added thereto, and the mixture was vigorously stirred at room temperature for 30 minutes. To the resulting suspended solution, Compound 1 (1.0 g, 11.1 mmol) was added at room temperature, and the mixture was vigorously stirred. Then, the mixture was heated to 40 °C, and then stirred for 2 hours. To the reaction mixture47 was added aqueous saturated sodium bicarbonate solution (10 mL), the resulting white solid was collected by filtration. The resulting solid was washed with water (50 mL), acetonitrile (50 mL) and dichloromethane (50 mL) to obtain Compound 3-12 (4.8 g, 9.4 mmol) as white solid. 1H-NMR(CDCla)8:6.20 (brs, 2H), 3.96 (d, 1H, J = 4.0 Hz, 1H), 3.75 (m, 1H),3.40 (dd, 2H, J=4.0, 12.0 Hz), 3.25 (dd, 2H, J = 4.0, 12.0 Hz), 2.22 (t, 4H, J = 12.0 Hz, 2H), 1.62 (d, 4H, J = 8.0 Hz), 1.29-1.25 (m, 40H), 0.90-0.86 (m, 6H) ESI-MS(m/z): 512 (M+1).

Step 2Compound 3-12 (5.10 g, 9.98 mmol) was suspended in dichloromethane (257 mL), and DIEA (6.97 mL, 39.9 mmol) was added thereto. Then, 2- cyanoethyl N,N-diisopropylchlorophosphoramidite (4.46mL, 20.0mmol) was added at room temperature, and the mixture was heated under reflux for hours. After cooling to room temperature, the reaction mixture was partitioned by the separatory funnnel, and the organic layer was washed twice with aqueous saturated sodium bicarbonate solution (100 mL), twice with water (100 mL) and once with brine (100 mL). After the resulting organic layer was dried over magnesium sulfate, the solvent was concentrated under reduced pressure. The resulting brown oil, Compound 4-12 (4.80 g, 6.mmol) was obtained as a crude product. Formation of the compound was determined based on introduction of trivalent phosphorus by 31P-NMR. 31P-NMR(CDCl3)8:148.2 (s)[0120]1-1-5) Synthesis of Compound 4-14Compound 2-14 (12.52 g, 48.8 mmol, Tokyo Chemical Industry Co., Ltd.) was dissolved in DMF (150 mL) and dichloromethane (75 mL). DIEA (12.mL, 73.2 mmol) and HBTU (20.37 g, 53.7 mmol) were added thereto, and the mixture was vigorously stirred at room temperature for 2.5 hours. To the resulting suspended solution, Compound 1 (2.2 g, 24.41 mmol) was added at room temperature, and the mixture was vigorously stirred. Then, the mixture was heated to 80 °C, and then stirred for 5 hours. To the reaction mixture was added aqueous saturated sodium bicarbonate solution (800 mL) and water (50 mL), the resulting white solid was collected by filtration. The resulting48 solid was washed with water (500 mL), acetonitrile (300 mL) and dichloromethane (100 mL) to obtain Compound 3-14 (10.9 g, 19.23 mmol) as white solid.1H-NMR (CDCla)8: 6.21 (2H, s), 3.97 (1H, d, J = 4.0 Hz), 3.77-3.74 (1H, m), 3.45-3.38 (2H, m), 3.28-3.22 (2H, m), 2.22 (4H, t, J = 7.6 Hz), 1.65-1.61 (4H, m), 1.29-1.25 (48H, m), 0.88 (6H, t, J = 6.8 Hz).

Step 2Compound 3-14 (1.00 g, 1.76 mmol) was suspended in dichloromethane (50 mL), and DIEA (0.924 mL, 5.29 mmol) was added thereto. Then, 2- cyanoethyl N,N-diisopropylchlorophosphoramidite (0.870mL, 3.53mmol) was added at room temperature, and the mixture was heated under reflux for hours. After cooling to room temperature, the reaction mixture was diluted with dichloromethane (50 mL). The solution was partitioned by the separatory funnnel, and then the organic layer was washed twice with aqueous saturated sodium bicarbonate solution (100 mL), twice with water (100 mL) and once with brine (100 mL). After the resulting organic layer was dried over magnesium sulfate, the solvent was concentrated under reduced pressure. The resulting white amorphous, Compound 4-14 (1.00 g, 1.30 mmol) was obtained as a crude product. Formation of the compound was determined based on introduction of trivalent phosphorus by 31P-NMR.31P-NMR(CDCl3 )8:148.2 (s)[0121]1-1-6) Synthesis of Compound 4-16According to methods described in Non-patent Document 3, Compound 3­was synthesized from Compound 2-16, and then Compound 4-16 was synthesized.Compound 3-16: 1 H-NMR(CDCl3) 8:6.20 (brs, 2H), 3.95 (m, 1H), 3.76 (m, 1H), 3.40 (m, 2H), 3.25 (m, 2H), 2.24 (m, 4H), 1.68-1.20 (m, 60H), 0.88 (t, 6H, J=8.Hz)Compound 4-16: 31 P-NMR(CDCl3) 8:148.2 (s)[0122]1-1-7) Synthesis of Compound 4-Step 149 Compound 2-18 (13.9 g, 44.4 mmol, Tokyo Chemical Industry Co., Ltd.) was dissolved in DMF (207 mL) and dichloromethane (214 mL). DIEA (16.mL, 93 mmol) and HBTU (18.5 g, 48.8 mmol) were added thereto, and the mixture was vigorously stirred at room temperature for 30 minutes. To the resulting suspended solution, Compound 1 (2.0 g, 22.2 mmol) was added at room temperature, and the mixture was vigorously stirred. Then, the mixture was heated to 40 °C, and then stirred for 2 hours. To the reaction mixture was added aqueous saturated sodium bicarbonate solution (10 mL), the resulting white solid was collected by filtration. The resulting solid was washed with water (100 mL), acetonitrile (100 mL) and dichloromethane (1mL) to obtain Compound 3-18 (10.0 g, 14.7 mmol) as white solid. 1H-NMR(CDCl3)8:6.20 (brs, 2H), 3.96 (m, 1H), 3.75 (m, 1H), 3.42 (m, 2H), 3.(m, 2H), 2.22 (m, 4H), 1.68-1.20 (m, 68H), 0.88 (m, 6H) Step 2Compound 3-18 (3.8 g, 5.60 mmol) was suspended in dichloromethane (230 mL), DIEA (5.86 mL, 33.6 mmol) was added thereto. Then, 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (3.74mL, 16.79mmol) was added at room temperature, and the mixture was heated under reflux for 2 hours.After cooling to room temperature, the reaction mixture was partitioned by the separatory funnnel, and the organic layer was washed twice with aqueous saturated sodium bicarbonate solution (100 mL), once with water (50 mL) and once with brine (50 mL). After the resulting organic layer was dried over magnesium sulfate, the solvent was concentrated under reduced pressure.After adding acetonitrile to the residue, white solid was collected by filtration. The resulting solid was washed with aqueous saturated sodium bicarbonate solution, water and acetonitrile to obtain Compound 4-18 (4.13 g, 4.70 mmol) as white solid. Formation of the compound was determined based on introduction of trivalent phosphorus by 31P-NMR.31P-NMR(CDCl3 )8:148.2 (s)[0123]1-1-8) Synthesis of Compound 4-Step 1Compound 2-20 (10.2 g, 30.0 mmol, Tokyo Chemical Industry Co., Ltd.)50 was dissolved in DMF (250 mL) and dichloromethane (250 mL). DIEA (7.mL, 45 mmol) and HBTU (12.52 g, 33.0 mmol) were added thereto, and the mixture was vigorously stirred at room temperature for 30 minutes. To the resulting suspended solution, Compound 1 (1.35 g, 15.0 mmol) was added at room temperature, and the mixture was vigorously stirred. Then, the mixture was heated to 40 °C, and then stirred for 2 hours. To the reaction mixture was added aqueous saturated sodium bicarbonate solution (50 mL), the resulting white solid was collected by filtration. The resulting solid was washed with water (50 mL), acetonitrile (50 mL) and dichloromethane (50 mL) to obtain Compound 3-20 (7.20 g, 9.79 mmol) as white solid.1H-NMR(CDCl3)8:6.18 (brs, 2H), 3.75 (m, 1H), 3.41 (m, 2H), 3.27 (m, 2H), 2.(m, 4H), 1.58-1.25 (m, 76H), 0.89-0.86 (m, 6H) Step 2Compound 3-20 (1.0 g, 1.36 mmol) was suspended in chloroform (50 mL), and DIEA (0.713 mL, 4.08 mmol) was added thereto. Then, 2-cyanoethyl N,N- diisopropylchlorophosphoramidite (0.607mL, 2.72mmol) was added at room temperature, and the mixture was heated under reflux for 2 hours. After cooling to room temperature, the reaction mixture was added dropwise to acetonitrile (300 mL) under vigorously stirring. The precipitated solid was collected by filtration, and the solid was washed twice with aqueous saturated sodium bicarbonate solution (20 mL), twice with water (20 mL) and twice with acetonitrile (20 mL). The resulting solid was dried under reduced pressure to obtain Compound 4-20 (843 mg, 0.901 mmol) as white solid. Formation of the compound was determined based on introduction of trivalent phosphorus by 31P-NMR.31P-NMR(CDCl3)8:148.2 (s)[0124]1-1-9) Synthesis of Compound 4-Step 1Compound 2-22 (8.79 g, 23.8 mmol, Wako Pure Chemical Industries, Ltd.) was dissolved in DMF (250 mL) and dichloromethane (250 mL). DIEA (6.25 mL, 35.8 mmol) and HBTU (9.95 g, 26.2 mmol) were added thereto, and the mixture was vigorously stirred at room temperature for 30 minutes. To the51 resulting suspended solution, Compound 1 (1.07 g, 11.9 mmol) was added at room temperature, and the mixture was vigorously stirred. Then, the mixture was heated to 40 °C, and then stirred for 2 hours. To the reaction mixture was added aqueous saturated sodium bicarbonate solution (50 mL), the resulting white solid was collected by filtration. The resulting solid was washed with water (50 mL), acetonitrile (50 mL) and dichloromethane (50 mL) to obtain Compound 3-22 (8.10 g, 8.17 mmol) as white solid.1H-NMR(CDCl3)8:6.06 (brs, 2H), 3.73 (m, 1H), 3.36 (dd, 2H, J = 6.0, 14.4 Hz), 3.22 (dd, 2H, J = 5.2, 14.4 Hz), 2.17 (m, 4H), 1.61 (m, 4H), 1.59-1.24 (m, 80H), 0.87-0.84 (m, 6H) Step 2Compound 3-22 (1.0 g, 1.36 mmol) was suspended in chloroform (50 mL), and DIEA (0.713 mL, 4.08 mmol) was added thereto. Then, 2-cyanoethyl N,N- diisopropylchlorophosphoramidite (0.607mL, 2.72mmol) was added at room temperature, and the mixture was heated under reflux for 2 hours. After cooling to room temperature, the reaction mixture was added dropwise to acetonitrile (300 mL) under vigorously stirring. The precipitated solid was collected by filtration, and the solid was washed twice with aqueous saturated sodium bicarbonate solution (20 mL), twice with water (20 mL) and twice with acetonitrile (20 mL). The resulting solid was dried under reduced pressure to obtain Compound 4-22 (814 mg, 0.821 mmol) as white solid. Formation of the compound was determined based on introduction of trivalent phosphorus by 31P-NMR.31P-NMR(CDCl3)8:148.2 (s)[0125]1-2) Synthesis of Compound 4-n,o wherein n or o is an integer of 6 to 28.[0126]1-2-1) Synthesis of Compound 4-8,Step 1Compound 2-8 is dissolved in DMF and solution. DIEA and HBTU are added thereto, and the mixture is stirred at room temperature. To the resulting suspended solution, Compound 1 is added at room temperature, and the mixture was stirred. The activated solution of Compound 2-18 which is separately prepared [Compound 2-18 is dissolved in DMF and solution. DIEA and HBTU are added thereto, and the mixture is stirred at room temperature] was added to the reaction vessel, and the mixture is stirred at room temperature. The mixture is heated to 40 °C and then stirred. To thereaction mixture is added aqueous saturated sodium bicarbonate solution, the resulting solid is collected by filtration. The resulting solid is washed with water, acetonitrile and dichloromethane to obtain Compound 6-8,18. 20Step 2Compound 6-8,18 is suspended in chloroform, and DIEA is added thereto. Then, 2-cyanoethyl N,N-diisopropylchlorophosphoramidite is addedat room temperature, and the mixture is heated under reflux. After cooling to53 room temperature, the reaction mixture is added dropwise to acetonitrile under stirring. The precipitated solid is collected by filtration, and the solid is washed twice with saturated sodium bicarbonate solution, twice with water and twice with acetonitrile. The resulting solid is dried under reduced pressure to obtain Compound 4-8,18. Formation of the compound is determined based on introduction of trivalent phosphorus by 31P-NMR.[0127]2) Synthesis of Compound 8-n wherein n is an integer of 6 to 28.[0128]2-1) Synthesis of Compound 8-6Compound 7-6 (1.00 g, 7.68 mmol, Tokyo Chemical Industry Co., Ltd.) was dissolved in dichloromethane (15 mL), and triethylamine (2.13 mL, 15.mmol) was added thereto. Then, 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (1.71mL, 7.68mmol) was added at room temperature, and the mixture was stirred at room temperature for 3 hours.The reaction was stopped by aqueous saturated sodium bicarbonate solution, and the mixture was extracted twice with ethyl acetate (50 mL). The organic layer was washed once with aqueous saturated sodium bicarbonate solution (mL), three times with water (10 mL) and once with brine (10 mL), and then dried over magnesium sulfate. The organic layer was concentrated under reduced pressure to obtain brown oil, Compound 8-6 (2.60 g) as a crude product. Formation of the compound was determined based on introduction of trivalent phosphorus by 31P-NMR.31P-NMR(CDCl3)8:147.2 (s)[0129]2-2) Synthesis of Compound 8-10Compound 7-10 (1.00 g, 5 .37 mmol, Tokyo Chemical Industry Co., Ltd.)54 was dissolved in dichloromethane (15 mL), and triethylamine (1.49 mL, 10.mmol) was added thereto. Then, 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (1.20 mL, 5.37 mmol) was added at room temperature, and the mixture was stirred at room temperature for 3 hours.The reaction was stopped by aqueous saturated sodium bicarbonate solution, and the mixture was extracted twice with ethyl acetate (50 mL). The organic layer was washed once with aqueous saturated sodium bicarbonate solution (mL), three times with water (10 mL) and once with brine (10 mL), and then dried over magnesium sulfate. The organic layer was concentrated under reduced pressure to obtain brown oil, Compound 8-10 (2.09 g) as a crude product. Formation of the compound was determined based on introduction of trivalent phosphorus by 31P-NMR.31P-NMR(CDCl3)8:147.2 (s)[0130]2-3) Synthesis of Compound 8-12Compound 7-12 (4.29 g, 20.0 mmol, Tokyo Chemical Industry Co., Ltd.) was dissolved in dichloromethane (52 mL), and DIEA (10.5 mL, 60.0 mmol) was added thereto. Then, 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (5.36 mL, 24.00 mmol) was added at room temperature, and the mixture was stirred at room temperature for 12 hours. The reaction was stopped by aqueous saturated sodium bicarbonate solution (20 mL), and then the mixture was partitioned by the separatory funnnel. After the organic layer was washed once with water (100 mL), the organic layer was washed once with aqueous saturated sodium bicarbonate solution (100 mL) and twice with water (100 mL), and then dried over magnesium sulfate. The organic layer was concentrated under reduced pressure to obtain brown oil, Compound 8-12 (4.g, 11.6 mmol) as a crude product. Formation of the compound was determined based on introduction of trivalent phosphorus by 31P-NMR. 31P-NMR(CDCl3)8:147.3 (s)[0131]2-4) Synthesis of Compound 8-14Compound 7-14 (1.00 g, 4.12 mmol, NACALAI TESQUE, INC.) was dissolved in dichloromethane (15 mL), and triethylamine (1.14 mL, 8.25 mmol) was added thereto. Then, 2-cyanoethyl N,N-diisopropylchlorophosphoramidite55 (0.92 mL, 4.12 mmol) was added at room temperature, and the mixture was stirred at room temperature for 3 hours. The reaction was stopped by aqueous saturated sodium bicarbonate solution, and the mixture was extracted twice with ethyl acetate (50 mL). The organic layer was washed once with aqueous saturated sodium bicarbonate solution (10 mL), three times with water (10 mL) and once with brine (10 mL), and then dried over magnesium sulfate. The organic layer was concentrated under reduced pressure to obtain brown oil, Compound 8-14 (1.87 g). Formation of the compound was determined based on introduction of trivalent phosphorus by 31P-NMR. 31P-NMR(CDCl3)8:147.2 (s)[0132]2-5) Synthesis of Compound 8-16Compound 7-16 (5.41 g, 20.0 mmol, Tokyo Chemical Industry Co., Ltd.) was dissolved in dichloromethane (52 mL), and DIEA (10.5 mL, 60.0 mmol) was added thereto. Then, 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (4.91mL, 22.00mmol) was added at room temperature, and the mixture was stirred at room temperature for 12 hours. The reaction was stopped with aqueous saturated sodium bicarbonate solution (20 mL), and then the mixture was partitioned by the separatory funnnel. After the organic layer was washed with water (100 mL), the organic layer was washed once with aqueous saturated sodium bicarbonate solution (100 mL) and twice with water (1mL), and then dried over magnesium sulfate. The organic layer was concentrated under reduced pressure to obtain brown oil, Compound 8-16 (4.g, 9.77 mmol) as a crude product. Formation of the compound was determined based on introduction of trivalent phosphorus by 31P-NMR. 31P-NMR(CDCl3)8:147.3 (s)[0133]2-6) Synthesis of Compound 8-18Compound 7-18 (2.99 g, 10.0 mmol, Tokyo Chemical Industry Co., Ltd.) was dissolved in chloroform (81 mL), and DIEA (3.67 mL, 21.0 mmol) was added thereto. Then, 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (2.34 mL, 10.5 mmol) was added at room temperature, and the mixture was stirred at room temperature for 12 hours. The reaction mixture was concentrated, and the residue was washed with acetonitrile (50 mL). The56 precipitated solid was collected by filtration and washed with acetonitrile (mL). Then, yellow solid was dried under reduced pressure to obtain Compound 8-18 (1.22 g, 2.45 mmol). Formation of the compound was determined based on introduction of trivalent phosphorus by 31P-NMR. 31P-NMR(CDCl3)8:147.2 (s)[0134]2-7) Synthesis of Compound 8-20Compound 7-20 (3.27 g, 10.0 mmol, Tokyo Chemical Industry Co., Ltd.) was dissolved in chloroform (81 mL), and DIEA (3.67 mL, 21.0 mmol) was added thereto. Then, 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (2.34 mL, 10.5 mmol) was added at room temperature, and the mixture was stirred at room temperature for 12 hours. The reaction mixture was concentrated, and the residue was washed with acetonitrile (50 mL). The precipitated solid was collected by filtration and washed with acetonitrile (mL). Then, yellow solid was dried under reduced pressure to obtain Compound 8-20 (3.19 g, 6.06 mmol). Formation of the compound was determined based on introduction of trivalent phosphorus by 31P-NMR. 31P-NMR(CDCl3)8:147.2 (s)[0135]2-8) Synthesis of Compound 8-22Compound 7-22 (3.55 g, 10.0 mmol, Tokyo Chemical Industry Co., Ltd.) was dissolved in chloroform (81 mL), and DIEA (3.67 mL, 21.0 mmol) was added thereto. Then, 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (2.34 mL, 10.5 mmol) was added at room temperature, and the mixture was stirred at room temperature for 12 hours. The reaction mixture was concentrated, and the residue was washed with acetonitrile (50 mL). The precipitated solid was collected by filtration and washed with acetonitrile (mL). Then, yellow solid was dried under reduced pressure to obtain Compound 8-22 (4.97 g, 8.96 mmol). Formation of the compound was determined based on introduction of trivalent phosphorus by 31P־NMR. 31P-NMR(CDCl3)8:147.2 (s)[0136]3) Synthesis of Compound 10-n -n N.p.O. וO NC DIEA wherein n is an integer of 6 to 28.[0137]3-1) Synthesis of Compound 10-16Step 1Under nitrogen atmosphere, to Compound 7-16 (12.1 g, 44.4 mmol) in DMF (104 mL)-dichloromethane (57.1 mL) solution, bis-(p-nitrophenyl) carbonate (13.5 g, 44.4 mmol) and DIEA (11.6 mL, 66.6 mmol) were added, and then the mixture was stirred at room temperature for 8 hours. Next,Compound 1 (2.0 g, 22.2 mmol) was added thereto, and the mixture was heated under reflux at 60 °C for 2 hours. The resulting solid was collected by filtration and washed with dichloromethane (100 mL), water (100 mL) and acetonitrile (100 mL), and then dried under reduced pressure to obtain Compound 9-16 (15.44 g, 22.6 mmol) as white solid.1H-NMR(CDCla)8:5.20 (brs, 2H), 4.05 (m, 4H), 3.79 (m, 1H), 3.24 (m, 4H), 1.55­1.21 (m, 68H), 0.88 (m, 6H) Step 2Under nitrogen atmosphere, to Compound 9-16 suspended indichloromethane (582 mL), DIEA (15.8 mL, 90.0 mmol) and 2-cyanoethyl N,N- diisopropylchlorophosphoramidite (10.1 mL, 45.2 mmol) were added, and then the mixture was heated under reflux at 50 °C for 2 hours. After cooling the reaction mixture to room temperature, the organic layer was washed twice with aqueous saturated sodium bicarbonate solution (300 mL), once with water (300 mL) and once with brine (300 mL). The organic layer was dried over magnesium sulfate, and then concentrated under reduced pressure. The residue was dissolved in dichloromethane (50 mL) and added dropwise to acetonitrile (150 mL) to powderize. The resulting solid was dried under reduced pressure to obtain Compound 10-16 (14.2 g, 16.1 mmol). 31P-NMR(CDCla)8:149.1 (s)[0138]3-2) Synthesis of Compound 10-Step 1Under nitrogen atmosphere, to Compound 7-18 (4 g, 13.4 mmol) in DMF (60 mL)-dichloromethane (40 mL) solution, bis-(p-nitrophenyl) carbonate (4.g, 13.4 mmol) and DIEA (3.5 mL, 20.1 mmol) were added, and then the mixture was stirred at room temperature for 5 hours. Next, Compound 1 (0.6 g, 6.mmol) in DMF solution (5 mL) was added thereto, and the mixture was stirred overnight. The resulting solid was collected by filtration and washed with dichloromethane, water and acetonitrile, and then dried under reduced pressure to obtain Compound 9-18 (4.1 g, 5.55 mmol) as white solid. 1H-NMR(CDCl3)8:5.20 (brs, 2H), 4.05 (t, 4H, J = 8Hz), 3.79 (s, 1H), 3.32 (m, 2H), 3.23 (m, 2H), 1.25 (s, 72H), 0.88 (t, 6H, J = 8Hz) Step 2Under nitrogen atmosphere, to Compound 9-18 (1.0 g, 1.35 mmol) suspended in dichloromethane (60 mL), DIEA (1.2 mL, 6.8 mmol) and 2- cyanoethyl N,N-diisopropylchlorophosphoramidite (0.75 mL, 3.4 mmol) were added, and the mixture was stirred at 50°C for 1.5 hours. The reaction mixture was cooled to room temperature, diluted with dichloromethane (mL), and washed with aqueous saturated sodium bicarbonate solution (40 mL x 2), water (40 mL) and brine (40 mL). The organic layer was dried over magnesium sulfate, and then concentrated under reduced pressure. The residue was dissolved in dichloromethane (15 mL) and added dropwise to acetonitrile (150 mL) to powderize. The resulting solid was dried under59 reduced pressure to obtain Compound 10-18 (0.98 g, 1.04 mmol) as white solid.31P-NMR(CDCl3)8:149.1 (s) [0139]4) Synthesis of Compound 15-n 5 wherein n is an integer of 6 to 28.[0140]4-1) Synthesis of Compound 15-6Steps 1 and 2To Compound 11 (US2014/0142253) (722 mg, 1.075 mmol) in dichloromethane (5.6 mL), diethylamine (1.4 mL, 13.40 mmol) was added, and the mixture was stirred at room temperature for 17 hours. After ethanol was added to the reaction mixture, and the mixture was stirred, the solvent wasconcentrated under reduced pressure. The resulting residue was coevaporated twice with ethanol to obtain the crude product of Compound 12.To Compound 2-6 (239 mg, 1.505 mmol) in ethanol solution (5.0 mL), DMT-MM (476 mg, 1.720 mmol) was added, the mixture was stirred at room temperature for 15 minutes. The resulting reaction mixture was added to thecrude product of Compound 12 in ethanol solution (2.5 mL), and the mixture60 was stirred at room temperature for 4.5 hours. After the solvent was concentrated under reduced pressure, aqueous saturated sodium bicarbonate solution and water were added to the resulting residue, and extracted with ethyl acetate. The organic layer was washed with water and brine, and then dried over sodium sulfate. The solvent was concentrated under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (n-hexane: ethyl acetate =70:30^20:80) to obtain Compound13-6 (320 mg, Yield 52 %) as colorless oil.1H-NMR (CDCla)8: 7.42-7.40 (2H, m), 7.32-7.26 (6H, m), 7.21 (1H, t, J = 7.Hz), 6.83 (4H, d, J = 8.8 Hz), 5.37 (1H, s), 3.79 (6H, s), 3.71-3.65 (1H, m), 3.63­3.57 (1H, m), 3.27 (1H, dd, J = 9.2, 4.0 Hz), 3.25-3.15 (2H, m), 3.07 (1H, dd, J = 9.2, 7.2 Hz), 2.45 (1H, t, J = 5.6 Hz), 2.12 (2H, t, J = 7.6 Hz), 1.78 (1H, s), 1.62­1.58 (2H, m), 1.47-1.40 (2H, m), 1.36-1.20 (12H, m), 0.87 (3H, t, J = 6.8 Hz).

Step 3To Compound 13-6 (310 mg, 0.538 mmol) in dichloromethane (3.0 mL), DMAP (6.6 mg, 0.054 mmol), DIEA (0.282 mL, 1.614 mmol) and succinic anhydride (81 mg, 0.807 mmol) were added, and the mixture was stirred at room temperature for 2 days. The solvent was concentrated under reduced pressure, and then the resulting crude product was purified by silica gel column chromatography (chloroform: methanol = 100:0^90:10) to obtain Compound 14-6 (360 mg, Yield 99 %) as colorless oil.1H-NMR (CDCl3)8: 7.42-7.40 (2H, m), 7.31-7.25 (6H, m), 7.19 (1H, t, J = 7.Hz), 6.82 (4H, d, J = 8.8 Hz), 5.74 (1H, t, J = 5.6 Hz), 4.24 (1H, dd, J = 10.8, 5.Hz), 4.12 (1H, dd, J = 10.8, 6.0 Hz), 3.79 (6H, s), 3.52-3.45 (1H, m), 3.24-2.(4H, m), 2.90 (1H, q, J = 7.2 Hz), 2.59-2.50 (3H, m), 2.14 (2H, t, J = 7.6 Hz), 1.90-1.85 (1H, m), 1.61-1.58 (2H, m), 1.48-1.18 (14H, m), 0.87 (3H, t, J = 6.Hz).

Step 4To Compound 14-6 (216 mg, 0.320 mmol) in acetonitrile solution (mL), DIEA (0.186 mL, 1.065 mmol) and HBTU (89 mg, 0.234 mmol) were added, and the mixture was shaken at room temperature for 15 minutes. To the reaction mixture, Native Amino lcaa CPG 1000A (ChemGenes Corporation) (4.2 g) was added, and the mixture was shaken for 24 hours. After the reaction mixture was filtered, CPG resin was washed three times with acetonitrile and three times with diethyl ether, and dried under reduced pressure. To the dried CPG, a mixture of CapA (PROLIGO, L840045-06) and CapB (PROLIGO, L850045-06) (1:1, 42 mL) was added, and the mixture was shaken for 1.5 hours. After filtrating the reaction mixture, CPG resin was washed twice with pyridine, twice with isopropanol and twice with diethyl ether, and dried under reduced pressure. The supported amount of Compound14- 6 was calculated by colorimetric assay of the DMTr cation, and Compound15- 6 whose supported amount is 53 pmol/g was obtained.[0141]4-2) Synthesis of Compound 15-Steps 1 and 2In a similar method to Step 1 of 4-1), the crude product of Compound (392 mg) was obtained.To Compound 2-10 (155 mg, 0.772 mmol) in ethanol solution (2.6 mL), DMT-MM (214 mg, 0.772 mmol) was added, the mixture was stirred at room temperature for 30 minutes. The resulting reaction mixture was added to the crude product of Compound 12 (392 mg) in ethanol solution (1.3 mL), and the mixture was stirred at room temperature for 4 hours. To Compound 2-10 (mg, 0.356 mmol) in ethanol solution (1.3 mL), DMT-MM (99 mg, 0.356 mmol) was added, and the mixture was stirred at room temperature for 15 minutes. The mixture was combined with the other reaction mixture and stirred at room temperature for 1.5 hours. After the solvent was concentrated under reduced pressure, aqueous saturated sodium bicarbonate solution and water were added to the resulting residue, and extracted with ethyl acetate. The organic layer was washed with water and brine, and dried over sodium sulfate. The solvent was concentrated under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (n-hexane: ethyl acetate =80:20^30:70) to obtain Compound 13-10 (165 mg, 44 %) as colorless oil.1H-NMR (CDCl3)8: 7.41 (2H, d, J = 8.0 Hz), 7.31-7.26 (6H, m), 7.21 (1H, t, J = 6.6 Hz), 6.83 (4H, d, J = 8.4 Hz), 5.35 (1H, s), 3.79 (6H, s), 3.70-3.57 (2H, m), 3.28-3.16 (3H, m), 3.07 (1H, t, J = 8.0 Hz), 2.43 (1H, s), 2.12 (2H, t, J = 7.2 Hz),62 1.78 (1H, s), 1.61-1.58 (2H, m), 1.46-1.39 (2H, m), 1.25 (20H, s), 0.87 (3H, t, J = 6.0 Hz).

Step 3To Compound 13-10 (222 mg, 0.352 mmol) in dichloromethane (2.2 mL), DMAP (4.3 mg, 0.035 mmol), DIEA (0.184 mL, 1.055 mmol) and succinic anhydride (53 mg, 0.528 mmol) were added, and the mixture was stirred at room temperature for 2 days. The solvent was concentrated under reduced pressure, and then the resulting crude product was purified by silica gel column chromatography (chloroform: methanol = 100:0^90:10) to obtain Compound 14-10 (243 mg, 94 %) as colorless oil.1H-NMR (CDCl3)8: 7.41 (2H, d, J = 6.0 Hz), 7.31-7.26 (6H, m), 7.20 (1H, d, J =7.2 Hz), 6.82 (4H, d, J = 7.6 Hz), 5.59 (1H, s), 4.29 (1H, d, J = 10.4 Hz), 4.17­4.13 (1H, m), 3.79 (6H, s), 3.23-2.98 (4H, m), 2.58 (4H, s), 2.16 (2H, t, J = 8.Hz), 1.90 (1H, s), 1.60 (2H, s), 1.42-1.19 (22H, m), 0.88 (3H, t, J = 6.8 Hz).

Step 4To Compound 14-10 (209 mg, 0.285 mmol) in acetonitrile solution (mL), DIEA (0.166 mL, 0.950 mmol) and HBTU (79 mg, 0.209 mmol) were added, and the mixture was shaken at room temperature for 15 minutes. To the reaction mixture, Native Amino lcaa CPG 1000A (ChemGenes Corporation) (3.8 g) was added, and the mixture was shaken for 23 hours. After the reaction mixture was filtered, CPG resin was washed three times with acetonitrile and three times diethyl ether, and dried under reduced pressure. To the dried CPG, a mixture of CapA (PROLIGO, L840045-06) and CapB (PROLIGO, L850045-06) (1:1, 38 mL) was added, and the mixture was shaken for 1.5 hours. After filtrating the reaction mixture, CPG resin was washed twice with pyridine, twice with isopropanol and twice with diethyl ether, and dried under reduced pressure. The supported amount of Compound 14-10 was calculated by colorimetric assay of the DMTr cation, and Compound 15-whose supported amount is 40 pmol/g was obtained.[0142]4-3) Synthesis of Compound 15-Steps 1 and 263 In a similar method to Step 1 of 4-1), the crude product of Compound (392 mg) was obtained.To Compound 2-14 (345 mg, 1.346 mmol) in ethanol solution (4.0 mL), DMT-MM (372 mg, 1.346 mmol) was added, and the mixture was stirred at room temperature for 15 minutes. The resulting reaction mixture was added to crude product of Compound 12 in ethanol solution (2.0 mL), and the mixture was stirred at room temperature for 28 hours. After the solvent was concentrated under reduced pressure, aqueous saturated sodium bicarbonate solution and water were added to the resulting residue, and extracted with ethyl acetate. The organic layer was washed with water and brine, and dried over sodium sulfate. The solvent was concentrated under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (n-hexane: ethyl acetate =70:30^20:80) to obtain Compound13-14 (228 mg, Yield 37 %) as colorless oil.1H-NMR (CDCl3)8: 7.40 (2H, d, J = 6.8 Hz), 7.31-7.21 (7H, m), 6.83 (4H, d, J = 6.4 Hz), 5.35 (1H, s), 3.79 (6H, s), 3.67 (1H, s), 3.61 (1H, s), 3.28-3.19 (3H, m), 3.07 (1H, t, J = 7.2 Hz), 2.43 (1H, s), 2.12 (2H, t, J = 6.0 Hz), 1.77 (1H, s), 1.(2H, s), 1.43 (2H, s), 1.25 (28H, s), 0.88 (3H, s).

Step 3To Compound 13-14 (224 mg, 0.326 mmol) in dichloromethane (2.2 mL), DMAP (4.0 mg, 0.033 mmol), DIEA (0.171 mL, 0.977 mmol) and succinic anhydride (49 mg, 0.488 mmol) were added, and the mixture was stirred at room temperature for 2 days. The solvent was concentrated under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (chloroform: methanol = 100:0^90:10) to obtain Compound 14­(163 mg, Yield 64 %) as colorless oil.1H-NMR (CDCl3)8: 7.40 (2H, d, J = 6.4 Hz), 7.30-7.26 (6H, m), 7.19 (1H, t, J =7.2 Hz), 6.81 (4H, d, J = 7.2 Hz), 5.57 (1H, s), 4.29 (1H, d, J = 10.8 Hz), 4.17­4.13 (1H, m), 3.79 (6H, s), 3.23-2.98 (4H, m), 2.58 (4H, s), 2.15 (2H, t, J = 7.Hz), 1.90 (1H, s), 1.59 (4H, s), 1.46-1.25 (28H, m), 0.87 (3H, t, J = 5.6 Hz).

Step 4To Compound 14-14 (162 mg, 0.206 mmol) in acetonitrile solution (2864 mL), DIEA (0.122 mL, 0.700 mmol) and HBTU (58 mg, 0.154 mmol) were added, and the mixture was shaken at room temperature for 15 minutes. To the reaction mixture, Native Amino lcaa CPG 1000A (ChemGenes Corporation) (2.8 g) was added, and the mixture was shaken for 24 hours. After the reaction mixture was filtered, CPG resin was washed three times with acetonitrile and three times with diethyl ether, and dried under reduced pressure. To the dried CPG, a mixture of CapA (PROLIGO, L840045-06) and CapB (PROLIGO, L850045-06) (1:1, 28 mL) was added, and the mixture was shaken for 1.5 hours. After filtrating the reaction mixture, CPG resin was washed twice with pyridine, twice with isopropanol and twice with diethyl ether, and dried under reduced pressure. The supported amount of Compound14- 14 was calculated by colorimetric assay of the DMTr cation, and Compound15- 14 whose supported amount is 42 pmol/g was obtained.[0143]4-4) Synthesis of Compound 15-Steps 1 and 2In a similar method to Step 1 of 4-1), the crude product of Compound (392 mg) was obtained.To Compound 2-18 (466 mg, 1.490 mmol) in ethanol solution (5.0 mL), DMT-MM (471 mg, 1.702 mmol) was added, the mixture was stirred at room temperature for 15 minutes. The resulting reaction mixture was added to the crude product of Compound 12 in ethanol solution (2.5 mL), the mixture was stirred at room temperature for 4.5 hours. After the solvent was concentrated under reduced pressure, aqueous saturated sodium bicarbonate solution and water were added to the resulting residue, and extracted with ethyl acetate. The organic layer was washed with water and brine, and dried over sodium sulfate. The solvent was concentrated under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (n- hexane: ethyl acetate =70:30^20:80) to obtain Compound 13-18 (494 mg, Yield %) as colorless oil.1H-NMR (CDCl3)8: 7.41 (2H, d, J = 7.2 Hz), 7.32-7.26 (6H, m), 7.21 (1H, t, J =7.2 Hz), 6.83 (4H, d, J = 8.8 Hz), 5.36 (1H, s), 3.79 (6H, s), 3.70-3.65 (1H, m), 3.63-3.57 (1H, m), 3.27 (1H, dd, J = 9.2, 4.0 Hz), 3.24-3.13 (2H, m), 3.07 (1H, dd, J = 9.2, 7.2 Hz), 2.45 (1H, t, J = 5.6 Hz), 2.12 (2H, t, J = 7.6 Hz), 1.78 (1H, s), 1.63-1.25 (40H, m), 0.88 (3H, t, J = 6.8 Hz).

Step 3To Compound 13-18 (352 mg, 0.473 mmol) in dichloromethane (3.5 mL), DMAP (5.8 mg, 0.047 mmol), DIEA (0.248 mL, 1.419 mmol) and succinic anhydride (71 mg, 0.709 mmol) were added, and the mixture was stirred at room temperature for 2 days. The solvent was concentrated under reduced pressure, and then the resulting crude product was purified by silica gel column chromatography (chloroform: methanol = 100:0^90:10) to obtain Compound 14-18 (225 mg, 56 %) as colorless oil.1H-NMR (CDCl3)8: 7.41 (2H, d, J = 7.2 Hz), 7.31-7.26 (6H, m), 7.20 (1H, t, J = 6.4 Hz), 6.82 (4H, d, J = 7.6 Hz), 5.57 (1H, s), 4.30 (1H, dd, J = 10.4, 2.4 Hz),4.15 (1H, dd, J = 10.4, 6.4 Hz), 3.79 (6H, s), 3.24-2.98 (4H, m), 2.59 (4H, s),2.16 (2H, t, J = 7.6 Hz), 1.90 (1H, s), 1.59 (4H, s), 1.44-1.21 (36H, m), 0.88 (3H, t, J = 5.6 Hz).

Step 4To Compound 14-18 (223 mg, 0.264 mmol) in acetonitrile solution (mL), DIEA (0.154 mL, 0.880 mmol) and HBTU (73 mg, 0.194 mmol) were added, and the mixture was shaken at room temperature for 15 minutes. To the reaction mixture, Native Amino lcaa CPG 1000A (ChemGenes Corporation) (3.5 g) was added, and the mixture was shaken for 24 hours. After the reaction mixture was filtered, CPG resin was washed three times with acetonitrile and three times diethyl ether, and dried under reduced pressure. To the dried CPG, a mixture of CapA (PROLIGO, L840045-06) and CapB (PROLIGO, L850045-06) (1:1, 35mL) was added, and the mixture was shaken for 1.5 hours. After filtrating the reaction mixture, CPG resin was washed twice with pyridine, twice with isopropanol and twice with diethyl ether, and dried under reduced pressure. The supported amount of Compound 14-18 was calculated by colorimetric assay of the DMTr cation, and Compound 15-whose supported amount is 56 pmol/g was obtained.[0144]5) Synthesis of Compound 17 ODMTr ODMTrOTBSFmocHNOHFmocHN Steps 1 and 2To Compound 11 (US2014/0142253, 292 mg, 0.435 mmol) in DMF solution (2.0 mL), imidazole (71 mg, 1.044 mmol) and t-butylchlorodimethylsilane (79 mg, 0.522 mmol) were added, and the mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with water and extracted with cyclopentyl methyl ether. The organic layer was washed with water and brine, and dried over sodium sulfate. The solvent was concentrated under reduced pressure to obtain the crude product of Compound 16 (352 mg).To the crude product of Compound 16 (352 mg) in dichloromethane (2.mL), diethylamine (0.6 mL, 5.74 mmol) was added, and the mixture was stirred at room temperature for 16 hours. After ethanol was added to the reaction mixture, the solvent was concentrated under reduced pressure. The residue was coevaporated twice with ethanol, and the resulting crude product was purified by amino silica gel column chromatography (chloroform) to obtain Compound 17 (190 mg, 78 %) as colorless oil.1H-NMR (CDCla)8: 7.45-7.43 (2H, m), 7.32 (4H, d, J = 8.8 Hz), 7.29-7.25 (2H,m), 7.22-7.18 (1H, m), 6.81 (4H, d, J = 8.8 Hz), 3.79 (6H, s), 3.68-3.61 (2H, m),3.08-3.02 (2H, m), 2.63 (2H, t, J = 7.2 Hz), 1.75-1.69 (1H, m), 1.41-1.30 (6H, m), 1.27-1.15 (2H, m), 0.84 (9H, s), 0.01 (6H, s).[0145]6) Synthesis of Compound 22-n wherein n is an integer of 6 to 28.[0146]6-1) Synthesis of Compound 22-6Step 1To Compound 3-6 (1.0 g, 2.92 mmol) in THF (20 mL)-chloroform (20 mL) solution, DIEA (1.53 mL, 8.76 mmol), bis(nitrophenyl) carbonate (1.33 g, 4.mmol) and DMAP (178 mg, 1.46 mmol) were added, and the mixture was stirred at 60 °C for 1 hour. The reaction mixture was filtered. After the mother liquid was concentrated under reduced pressure, the resulting crude product was purified by silica gel column chromatography (hexane: ethyl acetate= 60:40^20:80) to obtain Compound 18-6 (982 mg, 66 %) as white solid.68 1H-NMR (CDC13) 8: 8.32-8.26 (2H, m), 7.42 (2H, dt, J = 9.9, 2.5 Hz), 6.36 (2H, t, J = 6.4 Hz), 4.80 (1H, ddd, J = 10.7, 5.6, 3.3 Hz), 3.65-3.50 (4H, m), 2.26 (4H, t, J = 7.6 Hz), 1.69-1.62 (4H, m), 1.28 (16H, dt, J = 19.1, 4.7 Hz), 0.87 (6H, t, J = 6.8 Hz).

Step 2To Compound 17 (500 mg, 0.89 mmol) in dichloromethane (10.0 mL), Compound 18-6 (450 mg, 0.89 mmol) was added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure and purified by amino silica gel column chromatography (hexane: ethyl acetate =65:35^10:90) to obtain Compound 19-6 (625 mg, 76 %) as colorless oil.1H-NMR (CDCl3) 8: 7.42 (2H, d, J = 7.4 Hz), 7.31 (4H, t, J = 6.2 Hz), 7.26 (3H, t, J = 3.9 Hz), 7.19 (1H, t, J = 7.2 Hz), 6.82 (4H, t, J = 6.0 Hz), 6.25 (2H, t, J =5.8 Hz), 4.70 (2H, dd, J = 10.3, 5.3 Hz), 3.79 (6H, d, J = 4.4 Hz), 3.62 (2H, dd, J = 10.1, 5.1 Hz), 3.51 (2H, dd, J = 13.3, 6.4 Hz), 3.32-3.26 (2H, m), 3.08 (4H, dt,J = 20.2, 6.6 Hz), 2.19 (4H, t, J = 7.7 Hz), 1.70 (1H, t, J = 5.7 Hz), 1.61 (8H, t, J = 9.3 Hz), 1.42 (2H, t, J = 7.3 Hz), 1.26 (20H, tt, J = 26.0, 10.5 Hz), 0.88 (6H, dd, J = 12.0, 5.3 Hz), 0.83 (9H, s).

Step 3To Compound 19-6 (625 mg, 0.67 mmol) in THF (10 mL), TBAF (1M THF, 1.34 mL, 1.34 mmol) was added, and the mixture was stirred at room temperature for 24 hours. The reaction mixture was concentrated under reduced pressure, and the resulting crude product was purified by diol silica gel column chromatography (hexane: ethyl acetate= 50:50^10:90) to obtain Compound 20-6 (541 mg, 99 %) as colorless liquid.1H-NMR (CDCl3) 8: 7.41 (2H, t, J = 4.3 Hz), 7.26 (9H, ddt, J = 31.6, 12.0, 4.Hz), 6.83 (4H, d, J = 8.8 Hz), 6.38 (2H, q, J = 6.1 Hz), 4.88 (1H, t, J = 5.6 Hz), 4.67 (1H, t, J = 5.0 Hz), 3.79 (6H, t, J = 7.5 Hz), 3.69-3.61 (2H, m), 3.50-3.(2H, m), 3.30 (3H, tt, J = 20.6, 6.5 Hz), 3.15-3.06 (3H, m), 2.63 (1H, s), 2.21­2.17 (4H, m), 1.78 (1H, s), 1.62 (4H, t, J = 6.9 Hz), 1.43 (2H, t, J = 5.4 Hz), 1.(20H, dt, J = 29.2, 11.0 Hz), 0.87 (6H, t, J = 6.9 Hz).[0147]69 Step 4To Compound 20-6 (541 mg, 0.66 mmol) in dichloromethane (2 mL), DIEA (0.35 mL, 1.98 mmol), DMAP (8.0 mg, 0.066 mmol) and succinic anhydride (132 mg, 1.32 mmol) were added, and the mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (chloroform: methanol =40:1^10:1) to obtain Compound 21-6 (591 mg, 97 %) as colorless liquid.1H-NMR (CDCl3) 8: 7.41 (2H, d, J = 7.5 Hz), 7.31-7.25 (8H, m), 7.20 (1H, t, J =7.2 Hz), 6.82 (4H, d, J = 8.5 Hz), 6.62 (1H, t, J = 6.3 Hz), 6.48 (1H, t, J = 6.Hz), 5.91 (1H, t, J = 5.5 Hz), 4.71 (1H, t, J = 5.3 Hz), 4.42 (1H, dd, J = 11.0, 3.Hz), 4.14 (1H, dd, J = 10.9, 5.9 Hz), 3.79 (6H, s), 3.40 (4H, tt, J = 20.4, 7.0 Hz), 3.08 (4H, dq, J = 33.3, 8.0 Hz), 2.69-2.49 (4H, m), 2.20 (4H, dd, J = 15.6, 8.Hz), 1.95 (1H, s), 1.61 (4H, d, J = 7.0 Hz), 1.27 (22H, d, J = 5.0 Hz), 0.87 (6H, dd, J = 6.8, 5.1 Hz).

Step 5To Compound 21-6 (312 mg, 0.34 mmol) in a mixture of acetonitrile/dichloromethane (4:1, 25 mL), DIEA (0.30 mL, 1.70 mmol) and HBTU (142 mg, 0.37 mmol) were added, and the mixture was shaken at room temperature for 15 minutes. To the reaction mixture, HybridCPG amino form 2000A (Prime Synthesis, Inc.) (2.8 g) was added, and the mixture was shaken for 24 hours. After the reaction mixture was filtered, HybridCPG resin was washed three times with acetonitrile and three times with diethyl ether, and dried under reduced pressure. To the dried HybridCPG, a mixture of THF/acetic anhydride/pyridine (8:1:1, 30 mL) was added, and the mixture was shaken for 3 hours. After the reaction mixture was filtered, HybridCPG resin was washed twice with pyridine, twice with isopropanol and twice with diethyl ether, and dried under reduced pressure. The supported amount of Compound21- 6 was calculated by colorimetric assay of the DMTr cation, and Compound22- 6 whose supported amount is 114 pmol/g was obtained.[0148]6-2) Synthesis of Compound 22-Step 170 To Compound 3-8 (1 g, 2.5 mmol) in THF (20 mL) and chloroform (mL), bis(nitrophenyl) carbonate (1.14 g, 3.76 mmol), DIEA (1.3 mL, 7.5 mmol) and DMAP (0.15 g, 1.25 mmol) were added, ant the mixture was stirred at °C for 1 hour. The reaction mixture was filtered. After the mother liquid was concentrated under reduced pressure, the resulting crude product was purified by silica gel column chromatography (hexane: ethyl acetate =60:40^20:80) to obtain Compound 18-8 (1.17 g, 83 %) as white solid.1H-NMR (CDCl3) 8: 8.27 (2H, dt, J = 9.9, 2.5 Hz), 7.41 (2H, dt, J = 9.9, 2.5 Hz),6.42 (2H, t, J = 6.5 Hz), 4.81-4.78 (1H, m), 3.65-3.50 (4H, m), 2.26 (4H, t, J =7.6 Hz), 1.68-1.62 (4H, m), 1.28 (24H, t, J = 9.5 Hz), 0.87 (6H, t, J = 6.8 Hz).

Step 2To Compound 17 (500 mg, 0.89 mmol) in dichloromethane (10.0 mL), Compound 18-8 (500 mg, 0.89 mmol) and DIEA (0.23 mL, 1.33 mmol) were added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, and then purified by amino silica gel column chromatography (hexane: ethyl acetate =50:50^10:90) to obtain Compound 19-8 (661 mg, 75 %) as pale yellow solid. 1H-NMR (CDCl3) 8: 7.42 (2H, d, J = 7.4 Hz), 7.31 (4H, t, J = 6.3 Hz), 7.26 (3H, t, J = 3.9 Hz), 7.19 (1H, t, J = 7.2 Hz), 6.81 (4H, d, J = 8.8 Hz), 6.25 (2H, t, J = 6.0 Hz), 4.70 (2H, q, J = 5.4 Hz), 3.79 (6H, s), 3.63 (2H, t, J = 5.3 Hz), 3.51 (2H, dd, J = 13.1, 6.5 Hz), 3.29 (2H, dd, J = 12.9, 7.0 Hz), 3.08 (4H, dt, J = 20.0, 6.Hz), 2.18 (4H, t, J = 7.7 Hz), 1.70 (1H, t, J = 5.8 Hz), 1.62 (6H, d, J = 11.2 Hz),1.42 (2H, t, J = 7.2 Hz), 1.27 (28H, dt, J = 37.9, 14.6 Hz), 0.87 (6H, t, J = 6.Hz), 0.83 (9H, s).

Step 3To Compound 19-8 (661 mg, 0.669 mmol) in THF (10 mL), TBAF (1 M THF, 1.34 mL, 1.34 mmol) was added, and the mixture was stirred at room temperature for 25 hours. The reaction mixture was concentrated under reduced pressure, and then purified by diol silica gel column chromatography (hexane: ethyl acetate= 50:50^10:90) to obtain Compound 20-8 (549 mg, 94 %) as colorless oil..1H-NMR (CDCl3) 8: 7.41 (2H, t, J = 4.4 Hz), 7.32-7.19 (9H, m), 6.84 (4H, d, J =71 8.8 Hz), 6.35 (2H, q, J = 6.1 Hz), 4.85 (1H, t, J = 5.8 Hz), 4.67 (1H, t, J = 5.Hz), 3.79 (6H, s), 3.64 (2H, dd, J = 14.3, 7.4 Hz), 3.47 (2H, dt, J = 14.1, 5.8 Hz), 3.30 (3H, tt, J = 19.5, 6.2 Hz), 3.15-3.06 (3H, m), 2.60 (1H, s), 2.21-2.17 (4H, m), 1.78 (1H, s), 1.62 (4H, s), 1.44 (2H, d, J = 5.1 Hz), 1.27 (28H, dd, J = 11.0, 3.7 Hz), 0.87 (6H, t, J = 6.8 Hz).[0149]Step 4To Compound 20-8 (549 mg, 0.63 mmol) in dichloromethane (10 mL), DMAP (7.7 mg, 0.063 mmol), succinic anhydride (126 mg, 1.25 mmol) and DIEA (0.32 mL, 1.88 mmol) were added, and the mixture was stirred at room temperature for 4 hours. After the reaction mixture was concentrated under reduced pressure, the resulting crude product was purified by silica gel column chromatography (chloroform :methanol= 20:1^10:1) to obtain Compound 21-(582 mg, 95 %) as colorless liquid.1H-NMR (CDCl3) 8: 7.41 (2H, d, J = 7.5 Hz), 7.31-7.25 (8H, m), 7.20 (1H, t, J =7.2 Hz), 6.82 (4H, d, J = 8.7 Hz), 6.61 (1H, t, J = 6.2 Hz), 6.47 (1H, t, J = 6.Hz), 5.91 (1H, t, J = 5.6 Hz), 4.71 (1H, t, J = 5.1 Hz), 4.42 (1H, dd, J = 11.0, 3.Hz), 4.14 (1H, dd, J = 10.9, 5.8 Hz), 3.80 (6H, d, J = 6.1 Hz), 3.47-3.33 (4H, m), 3.08 (4H, ddd, J = 33.6, 15.6, 8.5 Hz), 2.69-2.49 (4H, m), 2.20 (4H, dd, J = 15.6,8.2 Hz), 1.95 (1H, s), 1.55 (4H, dt, J = 34.3, 6.5 Hz), 1.27 (30H, t, J = 7.2 Hz), 0.87 (6H, t, J = 6.8 Hz).

Step 5To Compound 21-8 (300 mg, 0.31 mmol) in a mixture of acetonitrile/dichloromethane (4:1, 25mL), DIEA (0.27 mL, 1.54 mmol) and HBTU (128 mg, 0.34 mmol) were added, and the mixture was shaken at room temperature for 15 minutes. HybridCPG amino form 2000A (Prime Synthesis, Inc.)(2.5g) was added to the reaction mixture, and the mixture was shaken for hours. After the reaction mixture was filtered, HybridCPG resin was washed three times with acetonitrile and three times with diethyl ether, and dried under reduced pressure. To the dried HybridCPG, a mixture of THF/acetic anhydride /pyridine (8:1:1, 30 mL) was added, and the mixture was shaken for 3 hours. After the reaction mixture was filtered, HybridCPG resin was washed twice with pyridine, twice with isopropanol, and twice with diethyl72 ether, and dried under reduced pressure. The supported amount of Compound21- 8 was calculated by colorimetric assay of the DMTr cation, and Compound22- 8 whose supported amount is 107 pmol/g was obtained.[0150]6-3) Synthesis of Compound 22-Step 1To Compound 3-10 (2.0 g, 3.92 mmol) in THF (50 mL), pyridine (0.3mL, 4.70 mmol) and 4-nitrophenyl chloroformate (947 mg, 4.70 mmol) were added, and the mixture was stirred at 60 °C for 1 hour. The reaction mixture was filtered. After the mother liquid was concentrated under reduced pressure, the resulting crude product was purified by silica gel column chromatography (chloroform: methanol = 100:0^90:10) to obtain Compound 18­(1.1 g, 42 %) as white solid.1H-NMR (CDCl3)8: 8.28 (2H, d, J = 8.8 Hz) , 7.42 (2H, d, J = 8.8 Hz),6.91 (2H, d, J = 9.1 Hz),4.80 (1H, s),3.57 (4H, m),2.26 (4H, t, J = 7.6 Hz),1.66 (4H, t, J =6.9 Hz),1.27 (40H, d, J = 20.2 Hz),0.88 (6H, t, J = 6.7 Hz).

Step 2To Compound 17 (41 mg, 0.074 mmol) in dichloromethane (5.0 mL), Compound 18-10 (50 mg, 0.074 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was purified by silica gel column chromatography (chloroform: methanol=100:0^90:10) to obtain Compound 19-10 (80 mg, 98 %) as yellow liquid.1H-NMR (CDCl3)8: 7.42 (2H, d, J = 7.5 Hz),7.31 (7H, t, J = 6.4 Hz),6.81 (4H, d, J = 8.8 Hz),6.23 (2H, d, J = 5.5 Hz),4.68 (1H, s),3.79 (6H, s),3.63 (2H, t, J = 5.Hz),3.52 (2H, t, J = 6.8 Hz),3.28 (2H, t, J = 7.3 Hz),3.11-3.04 (4H, m),2.18 (4H, t, J = 7.5 Hz),1.62 (6H, t, J = 7.2 Hz),1.25 (40H, s),0.88 (6H, t, J = 6.8 Hz),0.(9H, s), 0.01 (6H, s) Step 3To Compound 19-10 (559.2 mg, 0.535 mmol) in THF (5 mL), triethylamine (4.4 mL, 7.21 mmol) and TBAF (1 M THF, 0.4 mL, 0.40 mmol) were added, and the mixture was stirred at room temperature for 12 hours.The reaction mixture was diluted with chloroform (10 mL), and then washed with aqueous saturated sodium bicarbonate solution (10 mL). After the resulting organic layer was concentrated under reduced pressure, the resulting crude product was purified by silica gel column chromatography (chloroform :methanol = 100:0^90:10) to obtain Compound 20-10 (384 mg, 77 %) as colorless liquid.1H-NMR (CDCla)8: 7.41 (2H, t, J = 4.3 Hz),7.31 (7H, d, J = 8.5 Hz),6.84 (4H, d, J = 8.8 Hz),6.32 (2H, t, J = 6.3 Hz),4.83 (1H, t, J = 6.0 Hz),4.67 (1H, s, J = 5.Hz),3.79 (6H, s),3.68-3.63 (2H, m),3.47 (2H, dd, J = 12.9, 6.1 Hz),3.34-3.24 (3H, m),3.10 (3H, dt, J = 17.5, 5.6 Hz),2.59 (1H, s),2.21-2.17 (4H, m),1.71 (1H, m),1.62 (4H, t, J = 7.2 Hz),1.25 (36H, s),0.88 (6H, t, J = 6.8 Hz).[0151]Step 4To Compound 20-10 (384 mg, 0.431 mmol) in dichloromethane (10.1 mL), DMAP (5.04 mg, 0.041 mmol) and succinic anhydride (62.0 mg, 0.619 mmol) were added, and the mixture was stirred at room temperature for 1 day. After the reaction mixture was concentrated under reduced pressure, the resulting crude product was purified by diol silica gel column chromatography (chloroform :methanol = 100:0^90:10) to obtain Compound 21-10 (308 mg, %) as colorless liquid.1H-NMR (CDCl3)8: 7.41 (2H, t, J = 4.3 Hz),7.31 (6H, d, J = 8.5 Hz),6.84 (4H, d, J = 8.8 Hz), 6.81 (1H, br s),6.61 (1H, br s),5.83 (1H, br s),4.70 (1H, s),4.38 (1H, m),4.13 (1H, d, J = 10.4 Hz),3.79 (6H, s),3.78-3.41 (6H, m),3.05 (6H, m),2.(6H, m),2.62-2.55 (6H, m),2.20 (4H, d, J = 7.2 Hz),1.94 (1H, s),1.62 (4H, t, J =7.2 Hz),1.25 (36H, s),0.88 (6H, t, J = 7.2 Hz).

Step 5To Compound 21-10 (247 mg, 0.240 mmol) in a mixture of acetonitrile/dichloromethane (1:1, 20 mL), DIEA (0.168 mL, 0.982 mmol) and HBTU (100 mg, 0.264 mmol) were added, and the mixture was shaken at room temperature for 20 minutes. To the reaction mixture, Native Amino lcaa CPG 1000 A (ChemGenes Corporation)(2.0 g) was added, and the mixture was shaken for 12 hours. After the reaction mixture was filtered, CPG resin was washed three times with dichloromethane and three times with diethyl ether, and dried under reduced pressure. To the dried HybridCPG, a mixture of74 CapA (PROLIGO, L840045-06) and CapB (PROLIGO, L850045-06) (1:1, 20 mL) was added, and the mixture was shaken for 30 minutes. After the reaction mixture was filtered, CPG resin was washed twice with dichloromethane and twice with diethyl ether, and dried under reduced pressure. The supported amount of Compound 21-10 was calculated by colorimetric assay of the DMTr cation, and Compound 22-10 whose supported amount is 69 pmol/g was obtained.[0152]6-4) Synthesis of Compound 22-Step 1To Compound 3-12 (2.0 g, 3.92 mmol) in THF (50 mL), pyridine (0.3mL, 4.70 mmol) and 4-nitrophenyl chloroformate (947 mg, 4.70 mmol) were added, and the mixture was stirred at 60 °C for 1 hour. The reaction mixture was filtered. After the mother liquid was concentrated under reduced pressure, the resulting crude product was purified by silica gel column chromatography (chloroform: methanol = 100:0^90:10) to obtain Compound 18­(1.1 g, 42 %) as white solid.1H-NMR (CDCl3)8: 8.28 (2H, d, J = 8.8 Hz) , 7.42 (2H, d, J = 8.8 Hz),6.91 (2H, d, J = 9.1 Hz),4.80 (1H, s),3.57 (4H, m),2.26 (4H, t, J = 7.6 Hz),1.66 (4H, t, J =6.9 Hz),1.27 (40H, d, J = 20.2 Hz),0.88 (6H, t, J = 6.7 Hz).

Step 2To Compound 17 (41 mg, 0.074 mmol) in dichloromethane (5.0 mL), Compound 18-12 (50 mg, 0.074 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was purified by silica gel column chromatography (chloroform: methanol = 100:0^90:10) to obtain Compound 19-12 (80 mg, 98 %) as yellow liquid.1H-NMR (CDCl3)8: 7.42 (2H, d, J = 7.5 Hz),7.31 (6H, t, J = 6.4 Hz),6.81 (4H, d, J = 8.8 Hz),6.23 (2H, d, J = 5.5 Hz),4.68 (1H, s),3.79 (6H, s),3.63 (2H, t, J = 5.Hz),3.52 (2H, t, J = 6.8 Hz),3.28 (2H, t, J = 7.3 Hz),3.11-3.04 (4H, m),2.18 (4H, t, J = 7.5 Hz),1.62 (6H, t, J = 7.2 Hz),1.25 (40H, s),0.88 (6H, t, J = 6.8 Hz),0.(6H, s), 0.01 (6H, s) Step 375 To Compound 19-12 (90 mg, 0.082 mmol) in THF (5 mL), triethylamine (1.0 mL, 7.21 mmol) and TBAF (1 M THF, 0.4 mL, 0.40 mmol) were added, and the mixture was stirred at room temperature for 12 hours. The reaction mixture was diluted with chloroform (10 mL), and then washed with aqueous saturated sodium bicarbonate solution (10 mL). After the resulting organic layer was concentrated under reduced pressure, the resulting crude product was purified by silica gel column chromatography (chloroform: methanol = 100:0^95:5) to obtain Compound 20-12 (84 mg, 100 %) as colorless liquid.1H-NMR (CDCl3)8: 7.41 (2H, t, J = 4.3 Hz),7.31 (6H, d, J = 8.5 Hz),6.84 (4H, d, J = 8.8 Hz),6.34 (2H, t, J = 6.3 Hz),4.83 (1H, s),4.67 (1H, s),3.79 (6H, s),3.68- 3.63 (2H, m),3.47 (2H, dd, J = 12.9, 6.1 Hz),3.34-3.24 (3H, m),3.10 (3H, dt, J = 17.5, 5.6 Hz),2.60 (1H, s),2.21-2.17 (4H, m),1.62 (4H, t, J = 6.9 Hz),1.25 (40H, s),0.88 (6H, t, J = 6.8 Hz).[0153]Step 4To Compound 20-12 (60 mg, 0.061 mmol) in pyridine solution (2 mL), DMAP (0.7 mg, 0.006 mmol) and succinic anhydride (7.3 mg, 0.073 mmol) were added, and the mixture was stirred at room temperature for 4 days. After the reaction mixture was concentrated under reduced pressure, the resulting crude product was purified by silica gel column chromatography (chloroform: methanol = 100:0^95:5) to obtain Compound 21-12 (56 mg, 85 %) as colorless liquid.ESI-MS (m/z) : 1085 (M-H).

Step 5To Compound 21-12 (52 mg, 0.048 mmol) in a mixture of acetonitrile/chloroform (1:1, 10 mL), DIEA (0.043 mL, 0.248 mmol) and HBTU (31 mg, 0.083 mmol) were added, and the mixture was shaken at room temperature for 15 minutes. To the reaction mixture, Native Amino lcaa CPG 1000A (ChemGenes Corporation) (0.5 g) was added, and the mixture was shaken for 23 hours. After the reaction mixture was filtered, CPG resin was washed three times with acetonitrile and three times with diethyl ether, and dried under reduced pressure. To the dried CPG, a mixture of CapA (PROLIGO, L840045-06) and CapB(PROLIGO, L850045-06) (1:1, 5 mL) was76 added, and the mixture was shaken for 1.5 hours. After the reaction mixture was filtered, CPG resin was washed twice with pyridine, twice with isopropanol and twice with diethyl ether, and then dried under reduced pressure. The supported amount of Compound 21-12 was calculated by colorimetric assay of the DMTr cation, and Compound 22-12 whose supported amount is 31 pmol/g was obtained.[0154]6-5) Synthesis of Compound 22-Step 1To Compound 3-14 (1.5 g, 3.92 mmol) in THF (60 mL), pyridine (0.3mL, 3.97 mmol) and 4-nitrophenyl chloroformate (800 mg, 3.97 mmol) were added, and the mixture was heated under reflux for 3 hours. The reaction mixture was filtered. After the mother liquid was concentrated under reduced pressure, the resulting crude product was purified by silica gel column chromatography (hexane :ethyl acetate =66:34^45:55) to obtain Compound 18­(1.56 g, 81 %) as white solid.1H-NMR (CDCl3) 8: 8.29 (2H, d, J = 10.0 Hz), 7.43 (2H, d, J = 10.0 Hz), 6.(2H, t, J = 6.4 Hz), 4.81-4.76 (1H, m), 3.65-3.58 (2H, m), 3.55-3.48 (2H, m), 2.(4H, t, J = 7.6 Hz), 1.67-1.61 (4H, m), 1.25 (48H, s), 0.88 (6H, t, J = 6.8 Hz).

Step 2To Compound 17 (1.06 g, 1.88 mmol) in dichloromethane (30 mL), Compound 18-14 (1.38 g, 1.88 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After the solvent was concentrated under reduced pressure, the resulting crude product was purified by silica gel column chromatography (hexane: ethyl acetate =66:34^45:55) to obtain Compound 19­(1.47 g, 68 %) as white solid.ESI-MS (m/z) : 1155 (M-H).

Step 3To Compound 19-14 (1.47 g, 1.27 mmol) in THF (30 mL), TBAF (1 M THF, 3.81 mL, 3.81 mmol) was added, and the mixture was stirred at room temperature for 18 hours. After the reaction mixture was diluted with ethyl acetate, the solvent was concentrated under reduced pressure. The resulting77 crude product was purified by silica gel column chromatography (hexane: ethyl acetate =50:50^5:95) to obtain Compound 20-14 (1.04 g, 79 %) as white solid.1H-NMR (CDCl3) 8: 7.41 (2H, d, J = 7.6 Hz), 7.32-7.27 (6H, m), 7.21 (1H, t, J =7.2 Hz), 6.83 (4H, d, J = 8.8 Hz), 6.37-6.35 (2H, m), 4.86 (1H, t, J = 5.6 Hz), 4.69-4.64 (1H, m), 3.79 (6H, s), 3.69-3.61 (2H, m), 3.50-3.24 (6H, m), 3.15-3.(3H, m), 2.61 (1H, s), 2.21-2.17 (4H, m), 1.25 (58H, s), 0.88 (6H, t, J = 6.8 Hz). [0155]Step 4To Compound 20-14 (1.04 g, 0.998 mmol) in dichloromethane (20 mL), DMAP (12 mg, 0.10 mmol), DIEA (0.523 mL, 2.99 mmol) and succinic anhydride (170 mg, 1.70 mmol) were added, and the mixture was stirred at room temperature for 16 hours. After the reaction mixture was concentrated under reduced pressure, the resulting crude product was purified by diol silica gel column chromatography (chloroform) to obtain Compound 21-14 (1.17 g) as white solid.ESI-MS (m/z) : 1141 (M-H).

Step 5To Compound 21-14 (585 mg, 0.512 mmol) in a mixture of acetonitrile/dichloromethane (1:1, 40 mL), DIEA (0.447 mL, 2.56 mmol) and HBTU (214 mg, 0.563 mmol) were added, and the mixture was shaken at room temperature for 15 minutes. To the reaction mixture, Native Amino lcaa CPG 1000A (ChemGenes Corporation) (4.0 g) was added, and the mixture was shaken for 21 hours. After the reaction mixture was filtered, CPG resin was washed with a mixture of acetonitrile/dichloromethane (1:1, 120 mL) and diethyl ether (60 mL), and then dried under reduced pressure. To the dried CPG, a mixture of CapA (PROLIGO, L840045-06) and CapB (PROLIGO, L850045-06) (1:1, 40 mL) was added, and the mixture was shaken for 1.hours. After the reaction mixture was filtered, CPG resin was washed with pyridine (40 mL), isopropanol (60 mL) and diethyl ether (60 mL), and then dried under reduced pressure. The supported amount of Compound 21-14 was calculated by colorimetric assay of the DMTr cation, and Compound 22-whose supported amount is 40 pmol/g was obtained.[0156]78 6-6) Synthesis of Compound 22-Step 1To Compound 3-18 (1.0 g, 1.47 mmol) in THF (40 mL), pyridine (0.1mL, 1.76 mmol) and 4-nitrophenyl chloroformate (356 mg, 1.77 mmol) were added, and the mixture was stirred at 60 °C for 1 hour. After the reaction mixture was filtered, the mother liquid was concentrated under reduced pressure. The crude product of resulting solid was washed with ethyl acetate to obtain Compound 18-18 (508 mg, 41 %) as white solid.1H-NMR (CDCla)8: 8.29 (2H, d, J = 8.8 Hz),7.43 (2H, d, J = 9.1 Hz),6.29 (2H, s),3.60-3.50 (4H, m),2.25 (4H, t, J = 7.6 Hz),1.64 (4H, d, J = 6.6 Hz),1.25 (64H, s),0.88 (6H, t, J = 6.4 Hz).

Step 2To Compound 17 (62 mg, 0.110 mmol) in dichloromethane (4.0 mL), Compound 18-18 (93 mg, 0.110 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was purified by silica gel column chromatography (chloroform: methanol = 100:0^90:10) to obtain Compound 19-18 (113 mg, 81 %) as yellow liquid.1H-NMR (CDCl3)8: 7.42 (2H, d, J = 13.4 Hz),7.31 (7H, d, J = 9.3 Hz),6.81 (4H, d, J = 8.8 Hz),6.22 (2H, s),4.69 (1H, s),3.78 (6H, s),3.62 (2H, t, J = 5.6 Hz),3.(2H, dd, J = 10.9, 6.1 Hz),3.32-3.27 (2H, m),3.11-3.04 (4H, m),2.17 (4H, t, J = 3.7 Hz),1.62 (6H, dd, J = 10.5, 4.7 Hz),1.25 (64H, s),0.88 (6H, t, J = 6.8 Hz),0.83 (9H, s), 0.01 (6H, s).

Step 3To Compound 19-18 (100 mg, 0.079 mmol) in THF (4 mL), triethylamine (0.1 mL, 0.79 mmol) and TBAF (1 M THF, 0.32 mL, 0.32 mmol) were added, and the mixture was stirred at room temperature for 24 hours. The reaction mixture was diluted with chloroform (10 mL), and then washed with aqueous saturated sodium bicarbonate solution (10 mL). After the resulting organic layer was dried over magnesium sulfate and concentrated under reduced pressure, the resulting crude product was purified by silica gel column chromatography (chloroform: methanol = 100:0^95:5) to obtain Compound 20­(90 mg, 99 %) as colorless liquid.79 1H-NMR (CDCl3)8: 7.41 (2H, d, J = 8.6 Hz),7.31 (7H, d, J = 8.8 Hz),6.84 (4H, d, J = 8.6 Hz),6.31 (2H, s),4.81 (1H, s),4.67 (1H, s),3.79 (6H, s),3.71-3.63 (2H, m),3.49 (2H, dd, J = 14.7, 11.1 Hz),3.30 (3H, tt, J = 18.8, 7.4 Hz),3.13-3.07 (3H, m),2.58 (1H, s),2.18 (4H, d, J = 7.6 Hz),1.60 (6H, dd, J = 9.2, 4.4 Hz)1.25 (64H, s),0.88 (6H, t, J = 6.3 Hz).[0157]Step 4To Compound 20-18 (87 mg, 0.075 mmol) in pyridine solution (2 mL), DMAP (0.9 mg, 0.007 mmol) and succinic anhydride (15.8 mg, 0.151 mmol) were added, and the mixture was stirred at room temperature for 7 days.After the reaction mixture was concentrated under reduced pressure, the resulting crude product was purified by silica gel column chromatography (chloroform: methanol = 100:0^95:5) to obtain Compound 21-18 (85 mg, 90 %) as colorless liquid.ESI-MS (m/z) : 1253 (M-H).

Step 5To Compound 21-18 (85 mg, 0.068 mmol) in a mixture of acetonitrile/chloroform (1:1, 10 mL), DIEA (0.043 mL, 0.248 mmol) and HBTU (38 mg, 0.10 mmol) were added, and the mixture was stirred at room temperature for 15 minutes. To the reaction mixture, Native Amino lcaa CPG 1000A (ChemGenes Corporation) (0.5 g) was added, and the mixture was shaken at room temperature for 23 hours. After the reaction mixture was filtered, CPG resin was washed three times with acetonitrile and three times with diethyl ether, and dried under reduced pressure. To the dried CPG, a mixture of CapA (PROLIGO, L840045-06) and CapB (PROLIGO, L850045-06) (1:1, 5 mL) was added, and the mixture was shaken for 1.5 hours. After the reaction mixture was filtered, CPG resin was washed twice with pyridine, twice with isopropanol and twice with diethyl ether, and dried under reduced pressure. The supported amount of Compound 21-18 was calculated by colorimetric assay of the DMTr cation, and Compound 22-18 whose supported amount is 15 pmol/g was obtained.[0158]6-7) Synthesis of Compound 22-2080 Step 1To Compound 3-20 (1.0 g, 1.36 mmol) in THF (35 mL), bis(4-nitrophenyl) carbonate (1.241 mL, 4.08 mmol) and DMAP (498 mg, 4.08 mmol) were added, and the mixture was stirred at 55 °C for 1 hour. The reaction mixture was concentrated under reduced pressure. To the residue, acetonitrile (50 mL) was added and the mixture was stirred until the solid was precipitated. To the suspended solution, water (20 mL) was added and the mixture was vigorously stirred. The resulting solid was collected by filtration and washed with water (50 mL) and acetonitrile (50 mL) in order to obtain Compound 18­(1.1 g, 92 %) as white solid.1H-NMR (CDCla)8: 8.28 (2H, dd, J = 2.0, 6.8 Hz) , 7.42 (2H, dd, J = 2.4, 7.Hz),6.30 (2H, s),4.79 (1H, s),3.61 (2H, m),3.52 (2H, m),2.20 (4H, m),1.66 (4H, m),1.25 (72H, m),0.88 (6H, t, J = 6.8 Hz).

Step 2To Compound 17 (219 mg, 0.388 mmol) in THF (6.5 mL), Compound 18­(350 mg, 0.388 mmol) and DMAP (47.5 mg, 0.388 mmol) were added, and the mixture was stirred at 65 °C for 2 hours. To the reaction mixture, 10 % hydrous acetonitrile solution (70 ml) was added, and the mixture was stirred for a while. The precipitated solid was collected by filtration to obtain Compound 19-20 (420 mg, 82 %) as white solid.1H-NMR (CDCl3)8: 7.43 (2H, d, J = 7.6 Hz),7.31 (7H, m),6.81 (4H, d, J = 8.Hz),6.26 (2H, br s),4.73 (1H, br s),4.69 (1H, br s),3.79 (6H, s),3.79 (6H, s),3.(2H, m),3.49 (2H, m),3.29 (2H, d, J = 14.0 Hz),3.10-3.06 (4H, m),2.18 (4H, t, J = 7.6 Hz),1.68 (4H, m),1.42-1.25 (m, 72H),0.88 (6H, t, J = 6.4 Hz),0.83 (s,9H),0.00 (s, 6H).

Step 3To Compound 19-20 (559 mg, 0.422 mmol) in THF (5 mL), TBAF (1 M THF, 0.506 mL, 0.506 mmol) was added, and the mixture was stirred at 50 °C for 5 hours. The reaction mixture was added dropwise to 10 % hydrous acetonitrile solution (100 mL), and then the precipitated solid was collected by filtration to obtain Compound 20-20 (344 mg, 67 %) as white solid.1H-NMR (CDCl3)8: 7.41 (2H, t, J = 4.3 Hz),7.31 (7H, d, J = 8.5 Hz),6.84 (4H, d,81 J = 8.8 Hz),6.35 (2H, br s),4.85 (1H, s),4.67 (1H, s),3.79 (6H, s),3.68 (2H, m),3.47 (2H, dd, J = 12.9, 6.1 Hz),3.34-3.24 (3H, m),3.10 (3H, dt, J = 17.5, 5.Hz),2.60 (1H, s),2.21-2.17 (4H, m),1.62 (4H, m), 1.60-1.45 (m, 4H),1.25-1.(72H, m),0.88 (6H, t, J = 6.8 Hz).[0159]Step 4To Compound 20-20 (344 mg, 0.284 mmol) in dichloromethane (10 mL), DMAP (3.5 mg, 0.0284 mmol) and succinic anhydride (42.6 mg, 0.426 mmol) were added, and the mixture was stirred at room temperature overnight. The reaction mixture was added dropwise to 10 % hydrous acetonitrile solution (100 mL), and the precipitated solid was collected by filtration to obtain Compound 21-20 (361 mg, 97 %) as white solid.1H-NMR (CDCla)8: 7.42 (2H, d, J = 7.6 Hz),7.31-7.26 (7H, m),6.83 (4H, t, J = 8.4 Hz),6.56 (1H, br s),6.40 (1H, br s),5.89 (1H, br s),4.71 (1H, m),4.41 (1H, d, J = 8.0 Hz),4.14 (1H, dd, J = 6.0,11.2 Hz),3.79 (6H, s),3.65 (1H, m), 3.43-3.(4H, m),3.05-3.02 (4H, m),2.19-2.17 (4H, m),1.61-1.25 (76H, m),0.88 (6H, t, J =6.8 Hz).

Step 5To Compound 21-20 (190 mg, 0.145 mmol) in a mixture of acetonitrile/chloroform (1:3, 10 mL), DIEA (0.127 mL, 0.725 mmol) and HBTU (60.5 mg, 0.159 mmol) were added, and the mixture was shaken at 40 °C for 15ominutes. To the reaction mixture, Native Amino lcaa CPG 1000A (ChemGenes Corporation) (3.0 g) was added, and the mixture was shaken for hours. After the reaction mixture was filtered, CPG resin was washed three times with chloroform, once with ethanol and three times with acetonitrile, and dried under reduced pressure. To the dried CPG, a mixture of CapA (PROLIGO, L840045-06) and CapB (PROLIGO, L850045-06) (1:1, 20 mL) was added, and the mixture was shaken for 1.5 hours. After the reaction mixture was filtered, CPG resin was washed three times with chloroform and three times with acetonitrile, and then dried under reduced pressure. The supported amount of Compound 21-20 was calculated by colorimetric assay of the DMTr cation, and Compound 22-20 whose supported amount is 48 pmol/g was obtained.82 id="p-160" id="p-160"

[0160]6-8) Synthesis of Compound 22-Step 1To Compound 3-22 (2.0 g, 2.53 mmol) in THF (35 mL), bis-(p- nitrophenyl) carbonate (1.54 g, 5.05 mmol) and DMAP (618 mg, 5.05 mmol) were added, and the mixture was stirred at 65 °C for 2 hours. The reaction mixture was concentrated under reduced pressure. To the residue, acetonitrile (100 mL) was added, and the mixture was stirred until the solid was precipitated. To the suspended solution, water (20 mL) was added, and the mixture was vigorously stirred. The resulting solid was collected by filtration and washed in order with water (100 mL) and acetonitrile (100 mL) to obtain Compound 18-22 (2.27 g, 89 %) as white solid.1H-NMR (CDCla)8: 8.28 (2H, d, J = 8.8 Hz) , 7.43 (2H, d, J = 8.82 Hz),6.29 (2H, br t, J = 6.4 Hz),4.79 (1H, br s),3.61 (2H, m),3.52 (2H, m),2.25 (4H, t, J = 7.Hz),1.64 (4H, m),1.26 (84H, m),0.88 (6H, t, J = 6.0 Hz) Step 2To Compound 17 (666 mg, 1.81 mmol) in THF (10.0 mL), DMAP (144 mg, 1.81 mmol) and Compound 18-22 (1.13 g, 1.18 mmol) were added, and the mixture was stirred at 65 °C for 2 hours. To the reaction mixture, acetonitrile (150 mL) was added slowly. The resulting precipitate was collected by filtration and washed three times with acetonitrile to obtain Compound 19-(1.5 g, 92 %) as white solid.1H-NMR (CDCl3)8: 7.42 (2H, d, J = 6.8 Hz),7.32-7.20 (7H, m),6.81 (4H, d, J =6.8 Hz),6.26 (2H, br m),4.70 (2H, s),3.63 (2H, m),3.49 (2H, m),3.31 (2H, m),3.09-3.04 (4H, m),2.18 (4H, m),1.60 4H, m),1.25 (84H, m),0.86 (6H, t, J = 6.Hz),0.84 (9H, s), 0.01 (6H, s) Step 3To Compound 19-22 (1.5 g, 1.07 mmol) in THF (8.9 mL), TBAF (1 M THF, 1.64 mL, 1.69 mmol) was added, and the mixture was stirred at 65 °C for hours. To the reaction mixture, acetonitrile (150 mL) was added slowly.The resulting precipitate was collected by filtration and washed three times with acetonitrile to obtain Compound 20-22 (1.2 g, 87 %) as white solid.83 1H-NMR (CDCl3)8: 7.41 (2H, t, J = 7.2 Hz),7.31-7.21 (7H, m),6.84 (4H, d, J = 8.0 Hz),6.34 (2H, m),4.83 (1H, s),4.67 (1H, s),3.79 (6H, s),3.67 (2H, m),3.46 (2H, m),3.33-3.24 (3H, m),3.10 (3H, m),2.17 (4H, t, J = 7.6 Hz),1.78 (4H, m),1.44- 1.25 (84H, m),0.88 (6H, t, J = 6.0 Hz).[0161]Step 4To Compound 20-22 (100 mg, 0.079 mmol) in dichloromethane (3 mL), DMAP (1 mg, 0.008 mmol) and succinic anhydride (11.9 mg, 0.118 mmol) were added, and the mixture was stirred at 45 °C for 4 hours. To the reaction mixture, acetonitrile (10 mL) was added dropwise, and the precipitated solid was collected by filtration. The resulting solid was washed three times with acetonitrile to obtain Compound 21-22 (361 mg, 97 %) as white solid.1H-NMR (CDCl3)8: 7.42 (2H, d, J = 7.6 Hz),7.31-7.26 (7H, m),6.81 (4H, t, J =8.8 Hz),6.58 (1H, br m),6.43 (1H, br m),5.88 (1H, br m),4.71 (1H, m),4.40 (1H, d, J = 8.0 Hz),4.14 (1H, dd, J = 6.0,10.8 Hz),3.79 (6H, s),3.65 (1H, m), 3.43-3.(4H, m),3.13-3.02 (4H, m),2.63-2.53 (4H, m),2.23-2.18 (4H, dd, J = 7.2, 14.Hz),2.00-1.25 (4H, m) 1.25-1.11(84H, m),0.88 (6H, t, J = 6.8 Hz).

Step 5To Compound 21-22 (92 mg, 0.067 mmol) in a mixture of acetonitrile/dichloromethane/chloroform (1:2:2, 10 mL), DIEA (0.059 mL, 0.3mmol) and HBTU (28 mg, 0.074 mmol) were added, and the mixture was shaken at 40 °C for 15 minutes. To the reaction mixture, Native Amino lcaa CPG 1000A (ChemGenes Corporation) (0.9 g) was added, and the mixture was shaken at 40 °C for 3 hours. After the reaction mixture was filtered, CPG resin was washed three times with chloroform, three times with acetonitrile and three times with ethanol, and dried under reduced pressure. To the dried CPG, a mixture of CapA (PROLIGO, L840045-06) and CapB (PROLIGO, L850045-06) (1:1, 60 mL) was added, and the mixture was shaken for 1.hours. After the reaction mixture was filtered, CPG resin was washed three times with acetonitrile, and dried under reduced pressure. The supported amount of Compound 21-22 was calculated by colorimetric assay of the DMTr cation, and Compound 22-22 whose supported amount is 47 pmol/g was obtained.84 id="p-162" id="p-162"

[0162]8-1) Synthesis of Compound 26 Under nitrogen atmosphere, to cholesterol (Compound 25, 1.00 g, 2.mmol, Wako Pure Chemical Industries, Ltd.) in dichloromethane (10 mL),DIEA (0.9 mL, 5.17 mmol) was added, and the mixture was cooled with ice-cold water. Methyl N,N-diisopropylchlorophosphoramidite (0.86 mL, 3.85 mmol) was added thereto and the mixture was stirred under ice cooling for minutes. The reaction mixture was diluted with dichloromethane and ethyl acetate (100 mL), and then washed with aqueous saturated sodium bicarbonate solution (100 mL) and brine (100 mL). The organic layer was dried over magnesium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (SiO2:20 g, n-hexane: ethyl acetate =50:50^0:100) to obtain Compound (1.45 g, Yield 95 %) as colorless foam. Formation of the compound was determined based on introduction of trivalent phosphorus by 31P־NMR. 31P-NMR(CDCla)8:145.46 (d)[0163]8-2) Synthesis of Compound 5 Farnesol (Compound 52, 1.0 mL, 3.99 mmol, Junsei Chemical Co., Ltd.) was dissolved in dichloromethane (8.9 mL), and DIEA (1.53 mL, 8.78 mmol) was added thereto. Then, 2-cyanoethyl N, N-diisopropylchlorophosphoramidite (0.98 mL, 4.39 mmol) was added thereto at room temperature, and the mixture was stirred at room temperature for minutes. Dichloromethane (90 mL) and aqueous saturated sodium bicarbonate solution (100 mL) were added to the reaction solvent to stop the reaction, and then the mixture was partitioned by the separatory funnnel.The organic layer was washed with brine (100 mL), and then dried over magnesium sulfate. The organic layer was concentrated under reduced pressure to obtain Compound 53 (1.20 g, 2.84 mmol) as pale yellow oil as a crude product. Formation of the compound was determined based on introduction of trivalent phosphorus by 31P-NMR.1H-NMR (CDCl3) 8: 5.37 (1H, t, J = 6.6 Hz), 5.10-5.08 (2H, m), 4.19-4.13 (2H, m), 3.90-3.77 (2H, m), 3.66-3.54 (2H, m), 2.64 (2H, t, J = 6.6 Hz), 2.08-1.99 (8H, m), 1.68-1.67 (6H, m), 1.61-1.60 (6H, m), 1.20-1.17 (12H, m).31P-NMR (CDCl3) 8: 147.92 (1H, s).[0164]9) Synthesis of Compound 36-n ,NFmoc DMTrO^ 29 HOh^/NFmoc O^OH OHODMTrO' HN O OMe 1sfp"cr 33-n n O 3-n wherein n is an integer of 6 to 28.[0165]9-1) Synthesis of Compound 36-16Step 1According to methods described in Nucleic Acids Research, 42, 8796­8807 (2014), Compound 28 was synthesized from Compound 27 in two steps.

Step 2To Compound 28 (5.0 g, 7.79 mmol) in DMF solution (20 mL), imidazole (690mg, 10.1mmol, 1.3eq.) and tert-butyldimethylsilyl chloride (1.29 g, 8.mmol, 1.1eq.) were added in order at room temperature, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with ethyl acetate, washed with brine, and then dried over magnesium sulfate. The solvent was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (SiO2 :120 g, n-hexane: ethyl acetate: triethylamine =90:10:1^65:35:1) to obtain Compound 29 (5.50 g, Yield%) as colorless foam. By 1H-NMR, it was observed a mixture of rotamers,87 which is 1:1.ESI-MS (m/z) : 778 (M+H).H-NMR(CDCl3)8: 7.68 (d, J=7.5Hz, 1H), 7.62 (d, J=7.5Hz, 1H), 7.58-7.51 (m, 1H), 7.40-7.05 (m, 14H), 6.74-6.63 (m, 5H), 4.61-4.49 (m, 1H), 4.33-4.15 (2m, 1H), 4.12-4.03 (m, 1H), 3.93-3.85 (m, 1H), 3.62 (s, 6H), 3.56 (dd, J=10.7, 5.4Hz, 0.5H), 3.41-3.31 (m, 1H), 3.16 (dd, J=9.0, 4.3Hz, 0.5H), 3.00 (m, 0.5H), 2.90 (m, 0.5H), 2.13-2.02 (m, 1H), 1.97-1.82 (m, 1H), 0.82 (s, 1.5H), 0.81 (s, 3H), 0.7(s, 3H), 0.787 (s, 1.5H), 0.007 (s, 1.5H), 0.000 (s, 1.5H), -0.015 (s, 1.5H), -0.0(s, 1.5H). it 3 To Compound 29 (2.63 g, 3.48 mmol) in DMF solution (10 ml), piperidine (0.379 ml, 3.83 mmol, 1.1 eq.) was added. The mixture was stirred at room temperature for 2 hours, and then concentrated under reduced pressure. The residue was diluted with ethyl acetate, washed with brine, and then dried over magnesium sulfate. The solvent was concentrated under reduced pressure. Methanol (10 ml) was added thereto, and the resulting precipitate was filtered. The filtrate was concentrated to obtain the crude product of Compound 30(2.20 g).ESI-MS (m/z) : 524 (M+H). HPLC Peak RT = 3.29 min.

Step 4To Compound 30 (2.20 g, crude) in dichloromethane (15 ml), triethylamine (3.65 ml, 36.9 mmol) and acetoxyacetyl chloride (3.65 ml, 36.mmol) were added in order at room temperature, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with ethyl acetate, washed with brine, and then dried over magnesium sulfate. The solvent was concentrated under reduced pressure to obtain the crude product of Compound 31.ESI-MS (m/z) : 634 (M+H). HPLC Peak RT = 3.40 min.

Step 5After Compound 31 obtained in Step 4 was dissolved in methanol (ml), 28 % sodium methoxide-methanol solution (0.60ml) was added thereto,88 and the mixture was stirred at room temperature for 1 hour. The reaction mixture was diluted with ethyl acetate, washed with brine, and then dried over magnesium sulfate. The solvent was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (SiO2:80 g, n-hexane :ethyl acetate :triethylamine =80:20:1^65:35:1) to obtain Compound 32 (1.45 g, Yield from Compound 30: 71 %) as colorless foam. By 1H-NMR, it was observed a mixture of rotamers, which is 78:22.ESI-MS (m/z) : 592 (M+H). HPLC Peak RT = 3.36 min.1H-NMR(CDCla)8: (Major) 7.39-7.32 (m, 2H), 7.32-7.18 (m, 7H), 6.86-6.79 (m, 4H), 4.73 (m, 1H), 4.35 (m, 1H), 4.06 (dd, J=15.1, 4.4Hz, 1H), 3.98 (dd, J=15.1, 4.4Hz, 1H), 3.79 (s, 6H), 3.57 (dd, J=10.0, 4.0Hz, 1H), 3.52 (dd, J=10.0, 6.0Hz, 1H), 3.47 (dd, J=4.4, 4.4Hz, 1H), 3.13 (dd, J=10.0, 2.5Hz, 1H), 3.08 (dd, J=10.0, 5.0Hz, 1H), 2.19 (m, 1H), 1.93 (ddd, J=13.0, 8.8, 6.0Hz, 1H), 0.87 (s, 9H), 0.(s, 3H), 0.07 (s, 3H). (Minor) 7.39-7.32 (m, 2H), 7.32-7.18 (m, 7H), 6.86-6.79 (m, 4H), 4.54 (m, 1H), 4.06-3.98 (m, 1H), 3.92 (dd, J=14.6, 4.3Hz, 1H), 3.79 (s, 6H), 3.68 (dd, J=12.0, 4.0Hz, 1H), 3.62-3.42 (m, 2H), 3.16-3.02 (m, 2H), 2.11 (ddd, J=13.0, 5.8, 4.0Hz, 1H), 2.02 (m, 1H), 0.86 (s, 9H), 0.051 (s, 3H), 0.049 (s, 3H). [0166]Step 6To Compound 3-16 (2.00 g, 3.21 mmol), which was synthesized in a similar method to 1-1-6), suspended in dichloromethane (60 ml), DIEA (3.mL, 19.3 mmol, 4.0 eq.) was added, and the solution was added to methyl N,N- diisopropylchlorophosphoramidite (2.54 g, 12.8 mmol, 2.0 eq.) in dichloromethane (10 ml). The mixture was stirred at 45 °C for 10 minutes.After cooling to room temperature, the mixture was poured to aqueous saturated sodium bicarbonate solution, extracted with chloroform, and then washed with brine. After drying over magnesium sulfate, the solvent was concentrated under reduced pressure. Over stirring, acetonitrile (20 ml) was added to the resulting residue. The precipitated object was collected by filtration to obtain Compound 33-16 (2.36 g, Yield 94 %) as white solid.H-NMR(CDCl3)8:6.39 (t, J = 5.5 Hz, 1H, -NH), 6.18 (t, J = 5.5 Hz, 1H, - NH),3.95 (m, 0.5H), 3.67-3.51 (m, 4.5H), 3.42 (s, 1.5H), 3.39 (s, 1.5H), 3.19-3.(m, 1H), 3.05-2.97 (m, 1H), 2.30-2.15 (m, 4H), 1.70-1.57 (m, 4H), 1.36-1.23 (m, 56H), 1.23-1.14 (m, 12H), 0.88 (t, J=6.5Hz, 6H). 31 P-NMR(CDCl3 )8: 149.0689 Step 7To Compound 32 (70 0mg, 1.18 mmol) and Compound 33-16 (1.87 g, 2.mmol, 2.0 eq.) dissolved in dichloromethane (14 ml), 1H-tetrazole (124 mg, 1.mmol, 1.5 eq.) was added, and the mixture was stirred at room temperature for hours. 0.5 M DDTT solution ([(dimethylamino-methylidene)amino]-3H- 1,2,4-dithiazaolin-3-thion, 4.73 ml, dissolved in 3-picoline:acetonitrile =1:1) was added thereto, and the mixture was stirred at same temperature for hour. The reaction mixture was diluted with ethyl acetate, washed with 10 % citric acid solution, aqueous saturated sodium bicarbonate solution and brine in order, and then dried over magnesium sulfate. The solvent was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (SiO2 :45 g, n-hexane :ethyl acetate :triethylamine =75:25:1^50:50:1) to obtain Compound 34-16 (1.19 g, Yield %) as colorless foam.1H-NMR(CDCl3)8:7.40-7.11 (m, 9H), 6.87-6.78 (m, 4H), 4.94-4.84 (m, 1H), 4.82­4.72 (m, 1H), 4.58-4.43 (m, 2H), 4.38-4.31 (m, 1H), 3.86-3.53 (m, 4H), 3.80 (s, 3H), 3.79 (s, 6H), 3.33-2.99 (m, 4H), 2.30-1.88 (m, 6H), 1.65-1.55 (m, 4H), 1.35­1.20 (m, 56H), 0.91-0.84 (m, 15H), 0.08 (s, 3H), 0.01 (s, 3H).

Step 8To Compound 34-16 (1.02 g, 0.780 mmol) in THF (10 ml),mol/l TBAF-THF (0.937 ml, 1.2 eq.) was added at room temperature, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (SiO2 :30 g, chloroform :methanol :triethylamine =97.5:2.5:1^90:10:1) to obtain Compound 35-16 (608 mg, Yield %).1H-NMR(CDCl3)8:7.40-7.17 (m, 9H), 6.91 (m, 1H, -NH), 6.88-6.78 (m, 5H),5.00- 4.35 (m, 5H), 3.85-3.44 (m, 4H), 3.79 (s, 3H), 3.78 (s, 6H), 3.35-3.06 (m, 4H), 2.31-2.09 (m, 5H), 2.06-1.94 (m, 1H), 1.68-1.52 (m, 4H), 1.36-1.18 (m, 56H),0.88 (t, J=7.0Hz, 6H).

Step 990 To Compound 35-16 (210 mg, 0.176 mmol) in dichloromethane (3 ml), triethylamine (0.073ml, 0.528mmol, 3.0eq.), succinic anhydride (35 mg, 0.3mmol, 2.0 eq.) and DMAP (4 mg, 0.033 mmol) was added in order at room temperature, and the mixture was stirred at room temperature for 12 hours.The reaction mixture was concentrated under reduced pressure. Theresulting residue was purified by silica gel column chromatography (SiO2 :24 g, chloroform :methanol :triethylamine = 100:0:1^95:5:1) to obtain Compound 36­(172 mg, Yield 76 %) as colorless solid.1H-NMR(CDCl3)8:7.38-7.32 (m, 2H), 7.38-7.32 (m, 7H), 6.90-6.76 (m, 4H), 5.10 (br.s, 1H), 5.04-4.39 (m, 4H), 3.87-3.55 (m, 4H), 3.79 (s, 3H), 3.78 (s, 6H), 3.31­3.07 (m, 4H), 2.65-2.43 (m, 4H), 2.38-2.28 (m, 1H), 2.28-2.08 (m, 5H), 1.67-1.(m, 4H), 1.35-1.19 (m, 56H), 0.88 (t, J=6.5Hz, 6H).[0167]10) Synthesis of Compound 46-nHD HO HD wherein n is an integer of 6 to 28.[0168]10-1) Synthesis of Compound 46-16Step 1According to methods described in Nucleic Acids Research, 42, 8796­8807 (2014), Compound 37 was synthesized from Compound 28.

Step 2To Compound 37 (3.00 g, 7.15 mmol) in dichloromethane (15 ml), triethylamine (1.98 ml, 14.3 mmol, 2.0 eq.) and succinic anhydride (751 mg, 7.51 mmol, 1.05 eq.) were added at room temperature, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (SiO2 :120g, chloroform: methanol:92 triethylamine =95:5:1^75:25:1) to obtain Compound 38 (3.37 g, Yield 91 %) as colorless powder. By 1H-NMR, it was observed a mixture of rotamers, which is 63:37.ESI-MS (m/z) : 530 (M+H). HPLC Peak RT = 1.86 min.1H-NMR(CDCl3)8:(Major) 7.39-7.33 (m, 2H), 7.30-7.22 (m, 6H), 7.22-7.16 (m, 1H), 6.85-6.77 (m, 4H), 4.50 (br.s, 1H), 4.41 (m, 1H), 3.88 (d, J=11.0Hz, 1H), 3.775 (s, 6H), 3.65 (dd, J=11.0, 4.0Hz, 1H), 3.43 (dd, J=9.2, 4.5Hz, 1H),3.14 (dd, J=9.2, 2.7Hz, 1H), 2.85-1.97 (m, 6H). (Minor) 7.39-7.33 (m, 2H), 7.30-7.22 (m, 6H), 7.22-7.16 (m, 1H), 6.85-6.77 (m, 4H), 4.41 (m, 1H), 4.31 (br.s, 1H), 4.11 (d, J=12.3Hz, 1H), 3.783 (s, 6H), 3.25 (dd, J=12.3, 3.5Hz, 1H), 3.18 (dd, J=9.5, 4.8Hz, 1H),3.10 (dd, J=9.5, 4.8Hz, 1H), 2.85-1.97 (m, 6H).

Step 3According to methods described in Journal of Medicinal Chemistry, 48, 7781 (2005), Compound 39 was synthesized from Compound 1.

Step 4To Compound 39 (3.00 g, 7.15 mmol) in a mixture of THF-water (9:1) (30ml), triphenylphosphine (1.98 ml, 14.3 mmol, 2.0 eq.) was added at room temperature, and the mixture was stirred at room temperature for 1 hour.The temperature was raised up to 70 °C, and the mixture was stirred for hours. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure to the crude product of obtain Compound as colorless oil.

Step 5To Compound 40 (7.15 mmol) in dichloromethane (30 ml), triethylamine (2.10 ml, 15.1 mmol, 1.2 eq.) and Fmoc-Cl (3.59 g, 13.9 mmol, 1.1 eq.) were added at room temperature, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was diluted with chloroform, washed with brine, and then dried over magnesium sulfate. The solvent was concentrated under reduced pressure. The resulting solid was washed with n-hexane and a little of chloroform, and then purified by silica gel column chromatography (SiO2:120g, n-hexane :ethyl acetate =75:25^0:100) to obtain Compound 4193 (4.18 g, Yield from Compound 39: 65 %) as colorless foam.ESI-MS (m/z) : 512 (M+H). HPLC Peak RT = 2.71 min.H-NMR(CDCl3)8:7.80 (d, J=7.5Hz, 2H), 7.60 (d, J=7.5Hz, 2H), 7.40 (dd, J=7.5, 7.5Hz, 2H), 7.30 (dd, J=7.5, 7.5Hz, 2H), 6.02 (br.s, 1H), 5.21 (br.s, 2H), 4.46­4.27 (m, 2H), 4.21 (m, 1H), 3.59 (m, 1H), 3.45-3.29 (m, 2H), 3.27-3.13 (m, 2H), 1.46 (s, 18H).[0169]Step 6To Compound 41 (3.00 g, 7.15 mmol), TFA (30 ml) was added, and the mixture was stirred at room temperature for 1 hour. The mixture was concetrated under reduced pressure to obtain the crude product of Compound 42.ESI-MS (m/z) : 312 (M+H). HPLC Peak RT = 1.00 min.

Step 7To Compound 42 in dichloromethane (10 ml), triethylamine (1.17 ml, 8.44 mmol, 6.0 eq.) and stearoyl chloride (938 mg, 3.10 mmol, 2.2 eq.) was added at room temperature, and the mixture was stirred at room temperature for 1 hour. The resulting white precipitate was collected by filtration, washed with a little of chloroform, water and n-hexane, and then dried under reduced pressure to obtain Compound 43-16 (888 mg, Yield from Compound 40: 75 %) as colorless solid.H-NMR(CDCl3)8:7.76 (d, J=7.5Hz, 2H), 7.60 (d, J=7.5Hz, 2H), 7.34 (d, J=7.5, 7.5Hz, 2H), 7.31 (d, J=7.5, 7.5Hz, 2H), 6.51 (br.s, 2H), 6.39 (br.s, 1H), 4.39-4.(m, 2H), 4.21 (m, 1H), 3.68-3.54 (m, 2H), 3.26-3.07 (m, 2H), 2.29-2.18 (m, 4H), 1.70-1.58 (m, 4H), 1.37-1.17 (m, 56H), 0.88 (t, J=7.0Hz, 6H).

Step 8To Compound 43-16 (860 mg, 1.02 mmol) in DMF solution (5 ml), piperidine (0.111 ml, 1.12 mmol, 1.1 eq.) was added, and the mixture was stirred at 80 °C for 1.5 hours. After standing still at room temperature for hours, the resulting precipitate was collected by filtration, washed with n- hexane, and dried under reduced pressure. The resulting solid was purified by silica gel column chromatography (SiO2 :24g, chloroform :methanol94 =98:2^75:25) to obtain Compound 44-16 (198 mg, Yield 31 %) as colorless solid.H-NMR(CDCl3)8:6.34 (br.s, 2H), 3.48-3.36 (m, 2H), 3.06-2.98 (m, 2H), 2.99 (s, 1H), 2.25-2.18 (m, 4H), 1.70-1.58 (m, 4H), 1.37-1.18 (m, 56H), 0.88 (t, J=7.0Hz, 6H).

Step 9To Compound 38 (134 mg, 0.257 mmol) derived from Step 2 in DMF solution (2 mL), DIEA(225 pi, 1.29 mmol) and HBTU (127 mg, 0.334 mmol) were added at room temperature, and the mixture was stirred at room temperature for 10 minutes. The mixture was added to Compound 44-16 (2mg, 17.1 mmol) in dichloromethane (3 mL) at 45 °C, and then the mixture was stirred for 30 minutes at 45 °C. The reaction mixture was washed with brine. After drying over magnesium sulfate, the solvent was concentrated under reduced pressure. The resulting residue was dissolved in ethyl acetate, and n- hexane was added thereto at 80 °C. The precipitated solid was collected by filtration to obtain Compound 45-16 (244 mg, Yield 85 %) as colorless solid.By 1H-NMR, it was observed a mixture of rotamers, which is 63:37. 1H-NMR(CDCl3)8:(Major) 7.35-7.32 (m, 2H), 7.31-7.16 (m, 7H), 6.85-6.77 (m, 4H), 6.46 (br.s, 1H, -NH), 4.38 (br.s, 1H), 3.781 (s, 6H), 3.72-3.58 (m, 2H), 3.51­3.36 (m, 2H), 3.28-3.08 (m, 3H), 2.78-2.60 (m, 2H), 2.48-1.98 (m, 7H), 1.64-1.(m, 4H), 1.35-1.19 (m, 56H), 0.88 (t, J=7.0Hz, 6H). (Minor) 7.35-7.32 (m, 2H), 7.31-7.16 (m, 7H), 6.85-6.77 (m, 4H), 6.46 (br.s, 1H, -NH), 4.53 (br.s, 1H), 3.7(s, 6H), 3.72-3.58 (m, 2H), 3.51-3.36 (m, 2H), 3.28-3.08 (m, 3H), 2.48-1.98 (m, 7H), 1.64-1.56 (m, 4H), 1.35-1.19 (m, 56H), 0.88 (t, J=7.0Hz, 6H).

Step 10To Compound 45-16 (340 mg, 0.303 mmol) in dichloromethane (3 ml), triethylamine (0.237 ml, 1.71 mmol, 5.6 eq.), succinic anhydride (85 mg, 0.8mmol, 2.8 eq.) and DMAP (4 mg) were added in order at room temperature. After the mixture was stirred at 45 °C for 2 hours, the reaction mixture was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (SiO2 :12 g, chloroform :methanol :triethylamine = 100:0:1^80:20:1), and then washed with n-hexane to obtain95 Compound 46-16 (246 mg, Yield 66 %) as colorless solid. By 1H-NMR, it was observed a mixture of rotamers, which is 77:23.1H-NMR(CDCl3)8:(Major) 7.38-7.31 (m, 2H), 7.31-7.12 (m, 6H), 7.17-7.11 (m, 1H), 7.00 (br.s, 1H), 6.85-6.77 (m, 4H), 6.74 (br.s, 1H, -NH), 5.33 (br.s, 1H),4.34 (br.s, 1H), 3.90-3.74 (m, 2H), 3.788 (s, 6H), 3.64-3.37 (m, 2H), 3.35-3.11(m, 2H), 3.11-2.92 (m, 2H), 2.68-2.07 (m, 11H), 1.68-1.51 (m, 4H), 1.37-1.16 (m, 56H), 0.88 (t, J=6.6Hz, 6H). (Minor) 7.38-7.31 (m, 2H), 7.31-7.12 (m, 6H), 7.17­7.11 (m, 1H), 7.00 (br.s, 1H), 6.85-6.77 (m, 4H), 6.74 (br.s, 1H, -NH), 5.20 (br.s,IH) , 4.17 (br.s, 1H), 3.90-3.74 (m, 2H), 3.678 (s, 6H), 3.64-3.37 (m, 2H), 3.35­3.11 (m, 2H), 3.11-2.92 (m, 2H), 2.68-2.07 (m, 11H), 1.68-1.51 (m, 4H), 1.37­1.16 (m, 56H), 0.88 (t, J=6.6Hz, 6H).[0170]II) Synthesis of Compound 49-n wherein n is an integer of 6 to 28.[0171]11-1) Synthesis of Compound 49-20 Step 1In a similar method to synthesis of Compound 34-16 described in 9-1), Compound 47-16 was obtained as colorless foam (Yield 78 %). 1H-NMR(CDCl3)8:7.34 (d, J=8.0Hz, 2H), 7.31-7.21 (m, 6H), 7.18 (m, 1H), 6.87­6.79 (m, 4H), 3.78 (s, 6H), 2.25-2.02 (m, 5H), 1.94-1.84 (m, 1H), 1.64-1.49 (m,4H), 1.33-1.16 (m, 56H), 0.88 (t, J=7.0Hz, 6H), 0.87 (s, 9H), 0.06 (s, 6H).

Step 2In a similar method to synthesis of Compound 35-16 described in 9-1), Compound 48-16 was obtained as colorless foam (Yield 61 %).ESI-MS (m/z) : 1177 (M+). HPLC Peak RT = 3.64 min.1H-NMR(CDCla)8:7.35 (d, J=7.8Hz, 2H), 7.31-7.16 (m, 7H), 6.87-6.78 (m, 4H), 4.98-4.49 (m, 3H), 4.48-4.31 (m, 2H), 3.91-3.44 (m, 5H), 3.78 (s, 6H), 3.37-3.(m, 4H), 2.27-2.11 (m, 5H), 2.08-1.94 (m, 1H), 1.66-1.52 (m, 4H), 1.34-1.21 (m, 56H), 0.88 (t, J=6.7Hz, 6H).

Step 3To Compound 48-16 (624 mg, 0.529 mmol) in dichloromethane (5 ml), triethylamine (0.330 ml, 2.39 mmol, 4.5 eq.), succinic anhydride (106 mg, 0.1mmol, 3.0 eq.) and DMAP (13 mg, 10.6 pmol, 0.2 eq.) were added in order at room temperature, and the mixture was stirred at room temperature for hours. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (SiO2 :24g, chloroform :methanol :triethylamine =97.5:2.5:1^80:20:1) to obtain Compound 49-16 (482 mg, Yield 71 %) as colorless solid.[0172]Although Compound 49-16 was a mixture of 2 kinds of isomers, a part of the mixture was separated by silica gel column chromatography and the various equipment data were measured.

Isomer 1 of Compound 49-16 (isomer which has higher Rf value based on TLC deploying with the solvent of chloroform : methanol = 5 : 1) : By 1H-NMR, it was observed a mixture of rotamers, which is 65:35.ESI-MS (m/z) : 1277 (M+). HPLC Peak RT = 3.72 min.1H-NMR(CDCl3)8:(Major) 7.34 (d, J=7.5Hz, 2H), 7.31-7.14 (m, 7H), 6.83 (d, J=8.3Hz, 4H), 5.45 (br.s, 1H), 4.75 (dd, J=14.1, 8.3Hz, 1H), 4.55-4.38 (m, 2H), 4.33 (m, 1H), 3.79 (s, 6H), 3.72-3.48 (m, 4H), 3.44-3.08 (m, 4H), 2.70-2.44 (m, 4H), 2.38-2.14 (m, 6H), 1.68-1.51 (m, 4H), 1.34-1.21 (m, 56H), 0.88 (t, J=6.7Hz, 6H). (Minor) 7.34 (d, J=7.5Hz, 2H), 7.31-7.14 (m, 7H), 6.83 (d, J=8.3Hz, 4H), 5.20 (br.s, 1H), 4.84 (m, 1H), 4.55-4.38 (m, 2H), 4.33 (m, 1H), 3.79 (s, 6H), 3.72­3.48 (m, 4H), 3.44-3.08 (m, 4H), 2.70-2.44 (m, 4H), 2.38-2.14 (m, 6H), 1.68-1.5197 (m, 4H), 1.34-1.21 (m, 56H), 0.88 (t, J=6.7Hz, 6H). 31 P-NMR(CDCla)8: 58.1 Isomer 2 of Compound 49-16 (isomer which has lower Rf value based on TLC deploying with the solvent of chloroform :methanol = 5 : 1)ESI-MS (m/z) : 1277 (M+). HPLC Peak RT = 3.72 min.1H-NMR(CDCl3)8:7.83 (br.s, 1H), 7.37-7.10 (m, 9H), 6.74 (d, J=8.6Hz, 4H), 5.30 (br.s, 1H), 4.62 (dd, J=14.0, 9.2Hz, 1H), 4.42-4.33 (m, 2H), 4.30 (dd, J=14.0, 9.2Hz, 1H), 3.71 (s, 6H), 2.63-2.34 (m, 4H),2.31-2.21 (m, 1H), 2.16-2.(m, 5H), 1.59-1.42 (m, 4H), 1.24-1.09 (m, 56H), 0.81 (t, J=6.7Hz, 6H). 31P- NMR(CDCl3)8: 57.[0173]12) Synthesis of Compound 50-n wherein n is an integer of 6 to 28.[0174]12-1) Synthesis of Compound 50-16Compound 50-16 was obtained by subjecting Compound 36-16 obtained from 9-1) to the similar condition of a reaction to support on solid resin in 4). The supported amount of Compound 36-16 was calculated by colorimetric assay of the DMTr cation, and Compound 50-16 whose supported amount is umol/g was obtained.In a similar method, Compound 49-n can be supported on solid resin.[0175]13) Synthesis of Compound 51-n wherein n is an integer of 6 to 28.[0176]13-1) Synthesis of compound 51-16Compound 51-16 was obtained by subjecting Compound 46-16 obtained from 10-1) to the similar condition of a reaction to support on solid resin in 4). The supported amount of Compound 46-16 was calculated by colorimetric assay of the DMTr cation, and Compound 51-16 whose supported amount is 10 umol/g was obtained.[0177]14) Synthesis of compound 55 Compound 55 was obtained by subjecting Compound 54 described in US2008/0085869 to the similar condition of a reaction to support on solid resin in 4). The supported amount of Compound 54 was calculated by colorimetric assay of the DMTr cation, and Compound 55 whose supported amount is umol/g was obtained.[0178]15) Synthesis of compound 63-n 100 OH O FmocHN' ""0 '0' ׳׳' OTBS "ODMTr O"O' 58 FmocHN "OTBS "ODMTr O OTBS "ODMTr O"O' OH "ODMTr O0' OH O "O' 0' 60-n 0' 61-n O"O' HoN ^ 0' O!f'HH׳S 0 n NA A , O H O %' HN H r, , 0 n N A A . O H "O' ׳nr0™י O O H 62-n 63-n wherein n is an integer of 6 to 28.[0179]15-1) Synthesis of Compound 63-Step 1it 1 To Compound 57 (Sigma-Aldrich, 0.517 g, 0.952 mmol) in DMF solution 101 (4.9 mL), DIEA (0.447 mL, 3.46 mmol) and HBTU (349 mg, 1.04 mmol) were added, and the mixture was stirred at room temperature for 30 minutes.Then, Compound 17 (0.488 g, 0.865 mmol) in DMF solution was added thereto, and the mixture was stirred overnight. After separating and extracting with hexane/ethyl acetate =1:1 (2x50 mL) and aqueous saturated sodium bicarbonate solution (50 mL), the organic layers were combined, washed with brine (50 mL), and dried over magnesium sulfate. After filtrating, it was concentrated by rotary evaporator under reduced pressure, and purified by silica gel column chromatography (chloroform: methanol = 100:0^70:30) to give Compound 58 (0.837 g, 85 %) as colorless solid.1H-NMR (CDCl3) 8: 7.75 (2H, d, J = 7.5 Hz), 7.60 (2H, d, J = 7.4 Hz), 7.42-7.(4H, m), 7.32-7.28 (7H, m), 7.24 (1H, s), 7.18 (1H, t, J = 7.3 Hz), 6.81 (4H, d, J = 8.9 Hz), 6.49 (1H, s), 6.08 (1H, s), 5.56 (1H, s), 4.39 (2H, d, J = 6.9 Hz), 4.(1H, t, J = 6.7 Hz), 3.79 (6H, s), 3.63-3.52 (15H, m), 3.34-3.28 (4H, m), 3.(2H, dd, J = 13.9, 6.4 Hz), 3.03 (2H, d, J = 5.6 Hz), 1.80-1.65 (5H, m), 1.63 (4H, s), 1.43-1.36 (2H, m), 1.31-1.26 (2H, m), 1.18-1.12 (2H, m), 0.84 (9H, s), 0.(6H, s).

Step 2To Compound 58 (800 mg, 0.735 mmol) in DMF solution (8 mL), piperidine (80 uL) was added, and the mixture was stirred at room temperature for 2 hours. After separating and extracting with hexane/ethyl acetate =1:4 and water, it was dried over anhydrous sodium sulfate overnight. After filtrating, it was concentrated by rotary evaporator under reduced pressure, and purified by amino silica gel column chromatography (chloroform: methanol = 100:0^97:3) to give Compound 59 (0.178 g, 0.205 mmol) as colorless oil.1H-NMR (CDCl3) 8: 7.37 (8H, dt, J = 50.4, 5.2 Hz), 7.26-7.18 (2H, m), 6.89 (1H, s), 6.82 (4H, t, J = 5.9 Hz), 6.25 (1H, t, J = 5.1 Hz), 3.81 (6H, s), 3.66-3.53 (15H, m), 3.35 (2H, q, J = 6.0 Hz), 3.16 (2H, dd, J = 13.9, 6.5 Hz), 3.04 (2H, d, J = 5.Hz), 2.80 (2H, t, J = 6.7 Hz), 1.80-1.17 (17H, m), 0.85 (9H, s), 0.03-0.01 (6H, m).

Step 3After Compound 3-18 (140 mg, 0.205 mmol) and DMAP (63 mg, 0.514102 mmol) were broken into small pieces in a flask, it dissolved in THF (1.8 mL). bis(4-nitrophenyl) carbonate (156 mg, 0.514 mmol) was added thereto, the mixture was stirred at 55 °C for 30 minutes. After cooling to room temperature, THF was concentrated under reduced pressure, and the residue was suspended in acetonitrile (10 mL) again. After heating to be almost solution, it cooled to room temperature, and the solid was precipitated. Then, the precipitation is promoted by ultrasonic breaking. The precipitated solid was collected by filtration, washed with acetonitrile (10 mL), water (10 mL) and acetonitrile (10 mL) in order, and then dried under vacuum to give Compound 18-18 (196 mg, 0.232 mmol) as pale yellow solid. Compound 18-(196 mg) was dissolved in THF (1.8 mL). Compound 59 (178 mg, 0.205 mmol) and DMAP (25 mg, 0.205 mmol) were added thereto, and the mixture was stirred at 55 °C for 2 hours. After cooling to room temperature, the solid was precipitated, and acetonitrile (18 mL) was added thereto. After heating and ultrasonic breaking, water (1.8 mL) was added thereto, and then the object, Compound 60-18 (251 mg, 0.160 mmol) as pale yellow solid, was obtained by filtration with a Kyriyama funnel.[0180]Step 4After Compound 60-18 (246 mg, 0.157 mmol) in THF (4.9 mL) was cooled by ice-water bath, TBAF solution (1 M THF, 495 uL, 0.495 mmol) was added thereto, and the mixture was stirred and warmed to room temperature for minutes. After the solvent was concentrated under reduced pressure, the resulting crude product was purified by amino silica gel column chromatography (only chloroform) to give Compound 61-18 (192 mg, 0.1mmol) as colorless solid.1H-NMR (CDCl3) 8: 7.41 (2H, d, J = 7.3 Hz), 7.30 (5H, dd, J = 8.9, 2.1 Hz), 7.(1H, t, J = 7.2 Hz), 6.83 (5H, d, J = 8.7 Hz), 6.76 (1H, t, J = 6.2 Hz), 6.44 (1H, t, J = 6.6 Hz), 5.63 (1H, t, J = 5.7 Hz), 4.72-4.66 (1H, m), 3.79 (6H, s), 3.76-3.(1H, m), 3.64-3.48 (17H, m), 3.39-3.14 (6H, m), 3.06 (2H, dd, J = 9.0, 7.6 Hz), 2.76 (1H, t, J = 5.8 Hz), 2.50-2.46 (6H, m), 2.19 (6H, t, J = 7.6 Hz), 1.79-1.(7H, m), 1.49-1.42 (6H, m), 1.24 (68H, d, J = 11.8 Hz), 0.89-0.87 (6H, t, J = 6.Hz). 103 Step 5To Compound 61-18 (191 mg, 0.131 mmol) in dichloromethane (1.9 mL), DIEA (0.069 mL, 0.393 mmol), DMAP (1.6 mg, 0.013 mmol) and succinic anhydride (20 mg, 0.197 mmol) were added, and the mixture was stirred under reflux for 3 hours. The solvent was concentrated under reduced pressure, and the residue was purified by diol silica gel column chromatography (only chloroform) to give Compound 62-18 (201 mg, 0.129 mmol) as colorless solid. 1H-NMR (CDCl3) 8: 7.42 (2H, d, J = 7.4 Hz), 7.30 (5H, d, J = 8.7 Hz), 7.19 (1H, t, J = 7.2 Hz), 6.81 (5H, d, J = 8.7 Hz), 6.74 (1H, t, J = 5.8 Hz), 6.26 (1H, t, J = 5.9 Hz), 5.55 (1H, t, J = 5.9 Hz), 4.72-4.67 (1H, m), 3.79 (6H, s), 3.58-3.49 (17H, m), 3.32-3.27 (6H, m), 3.14 (2H, q, J = 6.5 Hz), 3.04 (2H, d, J = 5.4 Hz), 2.49­2.46 (4H, m), 2.20 (5H, t, J = 7.5 Hz), 1.76-1.72 (4H, m), 1.65-1.62 (10H, m), 1.42-1.41 (3H, m), 1.25 (68H, s), 0.88 (6H, t, J = 6.6 Hz).

Step 6After Compound 62-18 (153 mg, 0.098 mmol) was dissolved in a mixture of acetonitrile/dichloromethane (1:1, 10 mL), DIEA (0.067 mL, 0.392 mmol) and HBTU (41 mg, 0.108 mmol) were added, and the mixture was shaken at room temperature for 20 minutes. To the reaction mixture, Native Amino lcaa CPG 1000A (ChemGenes Corporation) (1.0 g) was added, and the mixture was shaken for 24 hours. After the reaction mixture was filtered, CPG resin was washed twice with acetonitrile, twice with dichloromethane and twice with diethyl ether, and dried under reduced pressure. To the dried CPG, a mixture of CapA (PROLIGO, L840045-06) and CapB (PROLIGO, L850045-06) (1:1, 20mL) was added, and the mixture was shaken for 30 minutes. After the reaction mixture was filtered, CPG resin was washed twice with acetonitrile, twice with dichloromethane and twice with diethyl ether, and dried under reduced pressure. The supported amount of Compound 62-18 was calculated by colorimetric assay of the DMTr cation, and Compound 63-18 whose supported amount is 56 umol/g was obtained.[0181]B) Synthesis of oligonucleotidesOligonucleotides used in examples of this description were synthesized using phosphoramidite method by AKTA Oligopilot10 (GE Healthcare), NS-8-I104 (Dainippon Seiki co., ltd.) or NS-8-II (Dainippon Seiki co., ltd.). A monomer was prepared in 0.1 M acetonitrile solution using the amidite derived from the above amidite synthesis. The coupling time was 32 seconds to 10 minutes, and 8 to 10 equivalents of the amidite unit were used to condense with one monomer. 0.02 M Oxidizer (Sigma-Aldrich) andiodine/pyridine/water/=12.7/9/1 (w/v/v) were used for PO oxidation. 50 mM DDTT ((dimethylamino-methylidyne) amino-3H-1,2,4-dithiazoline-3-thion) in acetonitrile/3-picoline 1/1(v/v) or 1/4(v/v) and acetonitrile/pyridine 1/4 (v/v) solution were used for PS oxidation. ETT activator (5-ethylthio)-1H-tetrazole) (Sigma-Aldrich) was used as an activator, CapA and CapB (Sigma-Aldrich) was used as a capping reagent. Deb (3 w/v% TCA CH2 Cl2 solution) (Wako Pure Chemical Industries, Ltd.) or Deb (3 w/v% Dichloroacetin acid, Toluene Solution) was used as a detritylation reagent.SEQ-321, SEQ-323, SEQ-340, SEQ-343, SEQ-346, SEQ-349, SEQ-352, SEQ-355 and SEQ-358 were derived by providing Compound 22-18 to GeneDesign Inc. to consign nucleic synthesis and purification of oligonucleotides. DMT-butanediol phosphoramidite used for synthesis of SEQ-316 were purchased from ChemGenes Corporation.[0182]C) Synthesis of lipid-binding oligonucleotides 1) Synthesis from a synthesis amidite unitA stirrer, Molecular Sieves 4A 1/16 and the amidite synthesized in the above A) (e.g., Compound 4-n, Compound 4-n,o, Compound 8-n and Compound 10-n. 10 to 100 equivalents of an oligonucleotide) were put in a microwavetube (2-5 ml, 10-20 ml) made by Biotage, and the solution was adjusted to 0.M with chloroform (added 2-methyl-2-butane as a stabilizer). After drying for hours, a oligonucleotides supported to solid phase (CPG resin or polystyrene resin) and 0.25 M ETT activator, which is ((5-ethylthio)-1H-tetrazole) dichloromethane (the same amount of chloroform) were added, sealed and heated at 40 °C for 10 minutes to 1 hour. After cooling to room temperature, the reaction mixture was diluted twice with chloroform, and the resin was collected by filtration. The resulting resin was used in PS oxidization in NS- 8-I (Dainippon Seiki co., ltd.) or NS-8-II (Dainippon Seiki co., ltd.). Then the dried resin was subject to the following deprotection condition I or II to 105 synthesize the target lipid-binding oligonucleotide.2) Synthesis from lipid-supported resinUsing a resin supported lipid synthesized in the above A) (e.g., Compound 15-n, Compound 22-n, Compound 50-n, Compound 51-n, Compound and Compound 63-n), the target lipid-binding oligonucleotides were synthesized in a similar method to the above B).[0183]D) Deprotection1) Cleavage from the resin, and phosphate deprotection and base deprotectionFor cutting out DNA oligonucleotide, 28 % ammonia water (SEQ-1, 3 or 49) or 28 % ammonia water/EtOH4/1(v/v) (Single strand oligonucleotides described in Examples except for SEQ-1, 3 and 49) was used and the solution was shaken at room temperature for 1 hour and at 55 °C for 5 hours. 1 ml, ml or 10 ml of ammonia solution was used for 1 pmol, 5 pmol or 10 pmol synthesis, respectively, for cutting-out reaction. After the resin was washed with 50 % ethanol water, the filtrate was concentrated under reduced pressure to about 1 to 5 mL.When the sequence contains RNA, the resulting solution was lyophilized to obtain white powder, and then the following deprotection reaction of the 2’- TBS group was performed.[0184]2) Deprotection of 2’-TBS groupTo the resulting white powder, N-methylpyrrolidone/triethylamine/triethylamine trihydrofluoride=6/1/2(v/v) was added and the solution was stirred at 65 °C for 1.5 hours. To the reaction mixture was added the same amount of ethoxytrimethylsilane, and the solution was vigorously stirred at room temperature for 10 minutes to obtain the precipitate. After centrifugation at 2500 x g (2 minutes), the organic solvent layer was carefully removed. To the resulting precipitate, diethyl ether was added, and the solution was vigorously stirred. Then, in a similar way, the centrifugation was carried out and removed the organic solvent to obtain crude RNA unit (white solid).[0185]E) Purification 106 Oligonucleotides without a lipid ligand such as SEQ-1, 3 and 49 were purified by reversed phase HPLC in Condition 1.

Condition for reversed phase HPLC Condition Mobile phasesBuffer A: 100 mM TEAA (triethylammoniumacetate, pH 7.0) aqueous solution or 100 mM AcONa aqueous solution (pH5.4)Buffer B: acetonitrile B concentration gradient: 10-30 %(Condition 1-1)Column: Hydrosphere C18 (YMC co., ltd.) 100x20mm I.D., S-5 pm, 12 nmFlow rate: 10 mL/minColumn temperature: room temperatureDetection UV: 260 nm (Condition 1-2)Column: Hydrosphere C18 (YMC co., ltd.) 150x10mm I.D., S-5 pm, 12 nmFlow rate: 4 mL/minColumn temperature: room temperatureDetection UV: 260 nm Single strand oligonucleotides with a lipid ligand(s) (Single strand oligonucleotides described in Examples except for SEQ-1, 3 and 49) was purified by reversed phase HPLC in Condition 2.Condition 2Condition for reversed phase HPLCAccording to lipid solubility of the compound, B concentration at the beginning was adjusted from 20 % to 50 %.Mobile phasesBuffer A: 100 mM TEAA (Triethylammonium acetate pH7.0) aqueous solution or 100 mM AcONa aqueous solution (pH5.4)Buffer B: acetonitrileB concentration gradient: 20-80 % (compounds having L4 - 8 or L4-10),107 -60 % (compounds having Ltoc or Lchoi),30-80 % (compounds having L4 -12, L4 -1 4 , L4 -16 , L4 -18 , M2 2-12 or M51-16),40-80 % (compounds having L4 - 2 0 or L4 - 2 2),5080־ % (compounds having M2 2-18) (Condition 2-1)Column: YMC-Pack C4 (YMC co., ltd.) 100x20mm I.D., S-5 pm, 12 nmFlow rate: 10 mL/minColumn temperature: room temperatureDetection UV: 260 nm (Condition 2-2)Column: YMC-Pack C4 (YMC co., ltd.)150x10mm I.D., S-5 pm, 12 nm Flow rate: 4 mL/min Column temperature: room temperature Detection UV: 260 nm [0186]F) Desalting and freeze-drying of the purified oligonucleotideUsing VivaSpin20 (MWCO 3000) (Sartorius) and Amicon Ultra-Centrifugal Filter Units-3K, ultrafiltration was repeated for the resulting oligonucleotide to remove salt component from the fraction. Then, it was lyophilized to obtain the target oligonucleotide as powder. For the oligonucleotides purified using TEAA solvent, the desalting procedure was carried out after transforming the salt form with 100 mM sodium acetate solution (20 mL).[0187]G) Purity analysis of oligonucleotidesThe resulting oligonucleotide was confirmed as the target sequence by matching the found molecular weights determined by UPLC/MS measurement and the calculated molecular weights.Condition 1 (SEQ-1, 3 or 49)Xevo G2 Tof System (Waters)Column: Aquity OST C18 (2.1x50 mm) (Waters)Mobile phases108 Buffer A: 200 mM 1,1,1,3,3,3-hexafluoro-2-propanol/8mM triethylamine aqueous solutionBuffer B: methanolB concentration gradient: 10-30 % (10 min)Temperature: 50 °C Flow rate: 0.2 mL/min Condition 2 (Single strand oligonucleotides described in Examples except for SEQ-1, 3 and 49)Xevo G2 T of System (Waters)Column: ACQUITY UPLC Protein BEH C4 Column, 300A, 1.7pm, 2.1mmx100mm, 1/pkg (Waters)Mobile phasesBuffer A: 200 mM 1,1,1,3,3,3-hexafluoro-2-propanol/8mM triethylamine aqueous solutionBuffer B: methanolB concentration gradient: 10-95 % (10 min)Temperature: 50 °C Flow rate: 0.2 mL/min [0188]The results were shown in Tables 1 to 3.[0189] 109 [Table 1] No. Theoretical Mw [M H] FoundMw[Ml No. Theoretical Mw [M H] FoundMw[M]SEQ 1 6363 6365 SEQ 76 5117 5116SEQ 2 7064 7065 SEQ 78 5554 5554SEQ 3 6254 6256 SEQ 80 5610 5610SEQ 5 6955 6955 SEQ 82 5666 5666SEQ 7 3842 3842 SEQ S4 6798 6799SEQ 9 5367 5367 SEQ S6 6854 6855SEQ 11 6731 6731 SEQ 88 6910 6911SEQ 13 6843 6843 SEQ 90 8451 8452SEQ 15 7012 7011 SEQ 92 8451 8452SEQ 17 6698 6699 SEQ 94 8451 8452SEQ 19 6369 6370 SEQ 96 8563 8564SEQ 21 6040 6040 SEQ 98 8731 8733SEQ 23 5736 5736 SEQ 100 8675 8677SEQ 25 5423 5423 SEQ 102 8731 8733SEQ 27 5133 5134 SEQ 104 8395 8396SEQ 29 4829 4829 SEQ 106 8507 8508SEQ 31 4500 4500 SEQ 108 8563 8564SEQ 33 4211 4211 SEQ 110 6042 6042SEQ 35 3898 3898 SEQ 112 6362 6363SEQ 37 7570 7570 SEQ 114 7323 7323SEQ 39 7291 7291 SEQ 116 6067 6067SEQ 41 7330 7331 SEQ 118 6412 6413SEQ 43 5844 5845 SEQ 120 7448 7447SEQ 45 7363 7363 SEQ 122 7287 7287SEQ 47 7419 7419 SEQ 124 7607 7607SEQ 49 7697 7698 SEQ 126 8568 8569SEQ 50 8211 8212 SEQ 128 7312 7312SEQ 52 7569 7568 SEQ 130 7657 7658SEQ 04 6967 6966 SEQ 132 8693 8693SEQ 56 6348 6348 SEQ 134 8385 8386SEQ 58 5722 5722 SEQ 136 5598 5599SEQ 60 5409 5409 SEQ 138 8216 8218SEQ 62 6049 6050 SEQ 140 5430 5431SEQ 64 6099 6099 SEQ 142 8399 839SSEQ 66 8932 8933 SEQ 143 9089 9090SEQ 68 5859 5860 SEQ 144 8652 8651SEQ 70 5816 5817 SEQ 145 5426 5427SEQ 72 8451 8452 SEQ 147 5410 5410SEQ 74 5161 5160 SEQ 149 5470 5470 110 id="p-190" id="p-190"

[0190][Table 2] No. Theoretical Mw [M H] FoundMw[M] No. Theoretical Mw [M H] FoundMw[M]SEQ 151 6758 6758 SEQ 229 5992 5992SEQ 153 7102 7102 SEQ 231 6048 6049SEQ 155 7793 7793 SEQ 233 5987 5987SEQ 157 8139 8139 SEQ 235 8370 8370SEQ 159 8484 8484 SEQ 237 6700 6701SEQ 161 7761 7761 SEQ 239 7390 7392SEQ 163 8074 S075 SEQ 241 6756 6757SEQ 165 5912 5912 SEQ 243 7446 7448SEQ 167 6602 6603 SEQ 245 6644 6645SEQ 169 7638 7638 SEQ 247 7334 7336SEQ 171 6380 6380 SEQ 249 6700 6701SEQ 173 736S 7368 SEQ 251 7390 7391SEQ 175 5782 5785 SEQ 253 6348 6349SEQ 177 6408 6408 SEQ 254 5793 5792SEQ 179 7509 7508 SEQ 256 6326 6328SEQ 181 8481 8482 SEQ 257 5615 5615SEQ 1S3 5694 5695 SEQ 259 5687 5687SEQ 1S5 6073 6075 SEQ 261 5743 5743SEQ 187 6764 6765 SEQ 263 5967 5968SEQ 189 7800 7801 SEQ 265 6023 6024SEQ 191 6129 6130 SEQ 267 5951 5952SEQ 193 6820 6819 SEQ 269 6007 6010SEQ 195 7856 7856 SEQ 271 6063 6064SEQ 197 6017 6018 SEQ 273 6642 6642SEQ 199 6707 6709 SEQ 275 6698 6699SEQ 201 6073 6074 SEQ 277 6754 6754SEQ 203 6764 6766 SEQ 279 6468 6469SEQ 205 6007 6008 SEQ 2S1 6524 6525SEQ 207 6063 6063 SEQ 283 6552 6553SEQ 209 6119 6120 SEQ 285 7609 7608SEQ 211 6698 6698 SEQ 287 7196 7196SEQ 213 6754 6754 SEQ 289 8441 8442SEQ 215 6810 6812 SEQ 291 7252 7252SEQ 217 6119 6119 SEQ 293 8498 8499SEQ 219 6175 6177 SEQ 295 7180 7184SEQ 221 6810 6811 SEQ 297 7236 7240SEQ 223 6867 6869 SEQ 299 7292 7296SEQ 225 6192 6192 SEQ 301 7870 7873SEQ 227 6028 6027 SEQ 303 7926 7929 111 id="p-191" id="p-191"

[0191][Table 3] No. Theoretical Found No. Theoretical FoundMw Mw Mw Mw[M H] [M] [M H] [M]SEQ 305 7983 7986 SEQ 346 5944 5942SEQ 307 8425 8426 SEQ 348 9346 9345SEQ 309 8537 8541 SEQ 349 5919 5916SEQ 311 9115 9118 SEQ 351 7048 7046SEQ 313 9171 9175 SEQ 352 5921 5920SEQ 315 9227 9230 SEQ 354 8050 8049SEQ 317 6247 6248 SEQ 355 5927 5926SEQ 319 6213 6214 SEQ 357 6778 6777SEQ 321 6109 6107 SEQ 358 5775 5771SEQ 323 4248 4249 SEQ 360 5574 5575SEQ 325 7236 7238 SEQ 362 5839 5840SEQ 327 7292 7293 SEQ 364 5895 5897SEQ 329 7348 7350 SEQ 366 5631 5631SEQ 331 7926 7929 SEQ 368 5895 5896SEQ 333 7983 7985 SEQ 370 5951 5953SEQ 335 8039 8041 SEQ 372 8481 8483SEQ 337 5619 5619 SEQ 374 8537 8539SEQ 339 6433 6434 SEQ 376 8593 8595SEQ 340 5775 5773 SEQ 378 9171 9173SEQ 342 7342 7341 SEQ 380 7151 7154SEQ 343 5936 5934 SEQ 3S1 5860 5861SEQ 345 8341 8339 SEQ 383 8833 8835 id="p-192" id="p-192"

[0192]I) Preparation of the double-stranded oligonucleotideLipid binding double-stranded oligonucleotides of the present invention were prepared as below. After mixing the equimolecular amount of 100 pM solution of each oligonucleotide, the solution was heated at 75 °C for 10 minutes, and naturally cooled to room temperature to obtain the double-stranded nucleic acids. Conformation of the double-stranded formation was carried out with size exclusion chromatography.Column: YMC-PAC Diol-120 (4.6 x 300 mm) (YMC co., ltd.)Mobile phases: 40 % acetonitrile in 1 x PBS solution Flow rate: 0.5 mL/min 112 Temperature: room temperature [0193] The synthesized oligonucleotides are shown in Tables 4 to 27.[0194][Table 4] No. ID (SEQ ID) Sequence: CpG(5'=>3) Complementary strand (3’=>5)SEQ-1 ODN1826 (1) 5'־tAcAcAaAtAgAaAcAgAtAtAcAcAtAgAaAcAgAtAt*3'SEQ-2 Amphl826 (1) 5'־L416AtAcAcAaAtAgAaAcAgAtAtAcAcAtAgAaAcAgAtAt3־'SEQ-3 S2) 1־) 3'־aAgAgtactgcaaggactgAcAaAa-5'SEQ-4 ODN1826 (1)S2) 1־)5'-tAcAcAaAtAgAaAcAgAtAtAcAcAtAgAaAcAgAtAt-3'3’-aAgAgtactgcaaggactgAcAaAa5־’SEQ-5 S-2 (2) 3'-aAgAgtactgcaaggactgAcAaAaAL416*5’SEQ-6 ODN1826 (1)S-2 (2)5'־tAcAcAaAtAgAaAcAgAtAtAcAcAtAgAaAcAgAtAt3־’3'־a A g A gt act gcaaggact g Ac A a A a A L4165 ־'SEQ-7 S-3 (3) 3'־aAgAgactgAcAaAaAL4165־'SEQ-S ODN1826 (1)S-3 (3)5'-tAcAcAaAtAgAaAcAgAtAtAcAcAtAgAaAcAgAtAt3־’3’־aAgAgactgAcAaAaAL4165־'SEQ-9 S-4 (4) 3'־cAtAgcaaggactgAcAaAaAL4165־'SEQ-10 ODN1826 (1)S*4 (4)5'-tAcAcAaAtAgAaAcAgAtAtAcAcAtAgAaAcAgAtAt-3' 3'־cAtAgcaaggactgAcAaAaAL4165־'SEQ-11 S2) 5־) 3'־aAgAgtactgcaaggactgAcAaAaAL4 85־’SEQ-12 ODN1826 (1)S-5 (2)5'-tAcAcAaAtAgAaAcAgAtAtAcAcAtAgAaAcAgAtAt-3’ 3'-aAgAgtactgcaaggactgAcAaAaAL4 8*5’SEQ-13 S-6 (2) 3׳־aAgAgtactgcaaggactgAcAaAaAL4125־'SEQ-14 ODN 1826(1)S-6 (2)5'-tAcAcAaAtAgAaAcAgAtAtAcAcAtAgAaAcAgAtAt-3’ 3'־aAgAgtactgcaaggactgAcAaAaAL4 125־’SEQ*15 S2) 7־) 3'־aAgAgtactgcaaggactgAcAaAaAL4185־’SEQ-16 ODN1826 (1)S2) 7־)5'־tAcAcAaAtAgAaAcAgAtAtAcAcAtAgAaAcAgAtAt3־' 3’־aAgAgtactgcaaggactgAcAaAaAL4185־’SEQ-17 S5) 8־) 3'־gAgAtactgcaaggactgAcAaAaAL4185־'SEQ-18 ODN1826 (1)S5) 8־)5’־tAcAcAaAtAgAaAcAgAtAtAcAcAtAgAaAcAgAtAt-3' 3'־gAgAtactgcaaggactgAcAaAaAL4185־’SEQ-19 S-9 (6) 3׳־gAtAactgcaaggactgAcAaAaAL4185־’SEQ-20 ODN 1826 (1)S-9 (6)5'־tAcAcAaAtAgAaAcAgAtAtAcAcAtAgAaAcAgAtAt3־'3’-gAtAactgCaaggactgACAaAaAL4-18*5’SEQ-21 S-10 (7) 3'־t Aa Actgcaaggactg Ac Aa A a A L4185־’SEQ-22 ODN 1826 (1) S-10 (7)5'־tAcAcAaAtAgAaAcAgAtAtAcAcAtAgAaAcAgAtAt3־' 3'־tAaActgcaaggactgAcAaAaAL4185־'SEQ-23 S-ll (8) 3'־aAcAtgcaaggactgAcAaAaAL4185־'SEQ-24 ODN1826 (1) S-ll (8)5'*tAcAcAaAtAgAaAcAgAtAtAcAcAtAgAaAcAgAtAt3־'3’־aAcAtgcaaggactgAcAaAaAL4185־’SEQ-25 S-12 (4) 3’־cAtAgcaaggactgAcAaAaAL4185־'SEQ-26 ODN1826 (1) S-12 (4)5'־tAcAcAaAtAgAaAcAgAtAtAcAcAtAgAaAcAgAtAt3־' 3'־cAtAgcaaggactgAcAaAaAL4185־'SEQ-27 S-13 (9) 3׳-tAgAcaaggactgAcAaAaAL4185־’SEQ-28 ODN 1826 (1) S-13 (9)5'־tAcAcAaAtAgAaAcAgAtAtAcAcAtAgAaAcAgAtAt3־' 3'־tAgAcaaggactgAcAaAaAL4185־' id="p-195" id="p-195"

[0195] 113 [Table 5] No. ID (SEQ ID) Sequence: CpG(5‘=>3') Complementary strand (3‘=>5') SEQ 29 S 14(10) 3' gAcAaaggactgAcAaAaAL4-1s 5' SEQ 30 ODN1S26Q) S 14(10) 5' tAcAcAaAtAgAaAcAgAtAtAcAcAtAgAaAcAgAtAt 3' 3׳ gAcAaaggactgAcAaAaAL4-1s 5’ SEQ 31 S 15(11) 3' cAaAaggactgAcAaAaAL4- 1 s 3’ SEQ 32 ODN1826(l) S 15(11) 5'tAcAcAaAtAgAaAcAgAtAtAcAcAtAgAaAcAgAtAt 3’ 3' cAaAaggactgAcAa aAL4-1s 5' SEQ 33 S 16(12) 3' aAaAggactgAcAaAaAL4-1s 5' SEQ 34 ODN1826(l) S 16(12) 5־ tAcAcAaAtAgAaAcAgAtAtAcAcAtAgAaAcAgAtAt 3־ 3־ aAaAggactgAcAaAaAL4- 1 s 5' SEQ 35 S 17(3) 3' aAgAgactgAcAaAaAL4-1s 5' SEQ 36 ODN1826Q) S 17(3) 5־ tAcAcAaAtAgAaAcAgAtAtAcAcAtAgAaAcAgAtAt 3' 3׳ aAgAgactgAcAaAaAL4-!s 5’ SEQ 37 S 18(2) 3' a0MeAgOMeAgOMeU 0 MeU 0 Mea 0 MeC 0 MeU 0 MeU 0 MegOMeC 0 Mea 0 Mea OMegOMe g0Mea0MeC0MeU0MeU0Meg0MeAC0MeAa0MeAa0Me' L4-1S 5' SEQ 38 ODN1826Q) S 18(2) 5־ tAcAcAaAtAgAaAcAgAtAtAcAcAtAgAaAcAgAtAt 3־ 3' a0MeAgOMeAgOMeU 0 MeU 0 Mea 0 MeC 0 MeU 0 MeU 0 MegOMeC 0 Mea 0 Mea OMegOMe g0Mea0MeC0MeU0MeU0Meg0MeAC0MeAa0MeAa0Me ' L4-1S 5' SEQ 39 S 19(2) 3־ AaGaGTACTGCAAGGACTGaCaAaAaL4-18 5־ SEQ 40 ODN1826Q) S 19(2) otccatgacgttcctgacgt to 3־ AAGAGTACTGCAAGGACTGACAAAAAL4-1S 5׳ SEQ 41 S 20(2) 3־ Af aGf aGfUfAfCfUfGfCfAfAfGfGfAfCfUfG f aCf a AfaAfaL4-1s 3־ SEQ 42 ODN1826Q) S 20(2) 5' tAcAcAaAtAgAaAcAgAtAtAcAcAtAgAaAcAgAtAt 3' 3־ Af aGf aGfUfAfC fUfGfCfAfAfGfG fAf C fUfGfaCf a AfaAfaL43 18־’ SEQ 43 S 21(4) 3' C0MeAU0MeU0MeAg0MeC0Mea0Mea0Meg0Meg0Mea0MeC0MeU0MeU OMegOMe A COMe A aOMe ׳' aOMe' L4-1S 3' SEQ 44 ODN1826(l) S 21(4) 5'tAcAcAaAtAgAaAcAgAtAtAcAcAtAgAaAcAgAtAt 3־ 3' C0MeAU0MeU0MeAg0MeC0Mea0Mea0Meg0Meg0Mea0MeC0MeU0MeO OMegOMe A COMeA aOMe ' aOMe' L4-1S 3' SEQ 45 S 22(2) 3' M15-6AaAgAgtactgcaaggactgAcAaAaAL4-1s 5' SEQ 46 ODN1826Q) S 22(2) 5'־tAcAcAaAtAgAaAcAgAtAtAcAcAtAgAaAcAgAtAt 3־ 3' M15-6AaAgAgtactgcaaggactgAcAaAaAL4-1s 5' SEQ 47 S 23(2) 3' M15-10AaAgAgtactgcaaggactgAcAaAaAL4-1s3' SEQ 48 ODN1826Q) S 23(2) 5־ tAcAcAaAtAgAaAcAgAtAtAcAcAtAgAaAcAgAtAt 3׳ 3' M15-10AaAgAgtactgcaaggactgAcAaAaAL4-1s3' SEQ 49 QDN2006Q3) 5־ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3־ id="p-196" id="p-196"

[0196] 114 [Table 6] No. ID (SEQ ID) Sequence: CpG(5'=>3 ) Complementary strand (3'=>5')SEQ 50 S 24(14) 3' aAgAcagcaaaacagcaaaacagAcAaAaAL4-is 5'SEQ 51 ODN2006(13)S 24(14)5־ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3'3' aAgAcagcaaaacagcaaaacagAcAaAaAL4-1s 5'SEQ 52 S 25(15) 3' cAaAgcaaaacagcaaaacagAcAaAaAL4-1s 5'SEQ 53 ODN2006(13)S 25(15)5־ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3׳3' cAaAgcaaaacagcaaaacagAcAaAaAL4-1s 5'SEQ 54 S 26(16) 3' gAcAaaaacagcaaaacagAcAaAaAL4-1s 5'SEQ 55 ODN2006(13)S 26(16)5־ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3־3' gAcAaaaacagcaaaacagAcAaAaAL4-1s 5'SEQ 56 S 27(17) 3' aAaAaacagcaaaacagAcAaAaAL4-1s 5'SEQ 57 ODN2006(13)S 27(17)5’ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3'3' aAaAaacagcaaaacagAcAaAaAL4-1s 5’SEQ 58 S 28(18) 3' aAaAcagcaaaacagAcAaAaAL4-1s 5’SEQ 59 ODN2006(13)S 28(18)5־ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3’3' aAaAcagcaaaacagAcAaAaAL4-1s 5’SEQ 60 S 29(19) 3' aAcAagcaaaacagAcAaAaAL4-1$ 5'SEQ 61 ODN2006(13)S 29(19)5־ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3׳3' aAcAagcaaaacagAcAaAaAL4-1s 5'SEQ 62 S 30(19) 3' aAcAagcaaaacagAcAaAaAtAt ’ L4-185'SEQ 63 ODN2006U3)S 30(19)5' tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3'3' aAcAagcaaaacagAcAaAaAtAt' L41s5'SEQ 64 S 31(19) 3' aAcAagcaaaacagAcAaAaAgAgAL4-1s 5'SEQ 65 ODN2006Q3)S 31(19)5' tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3'3' aAcAagcaaaacagAcAaAa gAgAL4-1s 5'SEQ 66 S 32(14) 3' a0MeAg0MeAC0Mea0Meg0MeC0Mea0Mea0Mea0Mea0MeC0Mea0Me g0MeC0Mea0Mea0Mea0Mea0MeC0Mea0Meg0MeAC0MeA a0MeA a0Me A L4-185’SEQ 67 ODN2006(13)S 32(14)5־ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3׳3' aoMe" g0MeAC0Mea0Meg0MeC0Mea0Mea0Mea0Mea0MeC0Mea0Me g0MeC0Mea0Mea0Mea0Mea0MeC0Mea0Meg0MeAC0Me a0Me a0Me L4-1S5־ id="p-197" id="p-197"

[0197] 115 [Table 7] No. ID (SEQ ID) Sequence: CpG(5'=>3 ) Complementary sti'and (3'=>5*)SEQ 68 S 33(19) 3' a0MeAC0MeAa0Meg0MeC0Mea0Mea0Mea0Mea0MeC0Mea0Meg0Me ACOMe Aa0Me Aa0Me׳ L41S 5' SEQ 69 ODN2006Q3)S 33(19) 5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3' 3' a0MeACOMe a0MegOMeC0Mea0Mea0Mea0Mea0.MeC0Mea0MegOMe AC0MeAa0MeAa0MeAL41S 5' SEQ 70 S 34(19) 3’ M15-10AaAcAagcaaaacagAcAaAaAL4-1s 5' SEQ 71 ODN2006(13)S 34(19) 5’ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3' 3' M!5-10AaAcAagcaaaacagAcAaAaAL4-1s 5' SEQ 72 S 35(14) 3' M15-10AaAgAcagcaaaacagcaaaacagAcAaAaAL4-12 5' SEQ 73 ODN2006Q3)S 35(14) 5' tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3' 3'M15-l0AaAgAcagcaaaacagcaaaacagAcAaAaAL4-12 5’ SEQ 74 S 36(19) 3' aAcAagcaaaacagAcAaAaALToo 5' SEQ 75 ODN2006Q3)S 36(19) 5־ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3' 3' aAcAagcaaaacagAc aAaAL 10 c 5’ SEQ 76 S 37(19) 3' aAcAagcaaaacagAcAaAaALchol 5' SEQ 77 ODN2006Q3)S 37(19) 5 t c g t c g t t t t g t c g t t t t g t c g t t־o 3' aAcAagcaaaacagAcAaAaALch 01 5' SEQ 78 S 38(18) 3’ aAaAcagcaaaacagAcAaAaAL4-12 5' SEQ 79 ODN2006Q3)S 38(18) 5' tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3' 3' aAaAcagcaaaacagAcAaAaAL4-12 5’ SEQ 80 S 39(18) 3' aAaAcagcaaaacagAcAaAaAL 4-14 5' SEQ 81 ODN2006Q3)S 39(18) 5' tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3' 3' aAaAcagcaaaacagAcAaAaAL4-14 5' SEQ 82 S 40(18) 3’ aAaAcagcaaaacagAcAaAaAL4-16 5־’ SEQ 83 ODN2006Q3)S 40(18) 5־ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3׳ 3' aAaAcagcaaaacagAcAaAaAL4-!6 •5' id="p-198" id="p-198"

[0198] 116 [Table 8] No. ID (SEQ ID) Sequence: CpG(5'=>3') Complementary strand (3‘=>5’)SEQ 84 S 41(16) 3' gAcAaaaacagcaaaacagAcAaAaAL4-12 5'SEQ 85 ODN2006Q3)S 41(16)5' tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3׳3' gAcAaaaacagcaaaacagAcAaAaAL4-12 5'SEQ 86 S 42(16) 3' gAcAaaaacagcaaaacagAcAaAaAL4-14 5'SEQ 87 ODN2006Q3)S 42(16)5' tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3׳3' gAcAaaaacagcaaaacagAcAaAaAL4-14 5'SEQ 88 S 43(16) 3' gAcAaaaacagcaaaacagAcAaAaAL4-16 5'SEQ 89 ODN2006Q3)S 43(16)5־ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3־3' gAcAaaaacagcaaaacagAcAaAaAL4-16 5'SEQ 90 S 44(14) 3' M!5-6AaAgAcagcaaaacagcaaaacagAcAaAaAL4-14 5'SEQ 91 ODN2006G3)S 44(14)5־ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3׳3' M15-6AaAgAcagcaaaacagcaaaacagAcAaAaAL4-14 5'SEQ 92 S 45(14) 3' M1514AaAgAcagcaaaacagcaaaacagAcAaAaAL410 5'SEQ 93 ODN2006Q3)S 45(14)5' tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3׳3' M!5-14AaAgAcagcaaaacagcaaaacagAcAaAaAL4-10 5'SEQ 94 S 46(14) 3' M15-1sAaAgAcagcaaaacagcaaaacagAcAaAaAL4-s 5'SEQ 95 ODN2006Q3)S 46(14)5' tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3'3' M15-1sAaAgAcagcaaaacagcaaaacagAcAaAaAL4-s 5'SEQ 96 S 47(14) 3' M15-6AaAgAcagcaaaacagcaaaacagAcAaAaAL4-!s 5'SEQ 97 ODN2006Q3)S 47(14)5’ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3׳3' M15-6AaAgAcagcaaaacagcaaaacagAcAaAaAL4-1s 5' id="p-199" id="p-199"

[0199] [Table 9] No. ID (SEQ ID) Sequence: CpG(5'=>3) Complementary strand (3’=>5)SEQ 98 S 48(14) 3' M15-10AaAgAcagcaaaacagcaaaacagAcAaAaAL4-1s 5'SEQ 99 ODN2006(13)S 48(14)5' tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3'3' M15-10AaAgAcagcaaaacagcaaaacagAcAaAaAL4-1s 5'SEQ 100 S 49(14) 3' M15-14AaAgAcagcaaaacagcaaaacagAcAaAaAL4-1s 5'SEQ 101 ODN2006G3)S 49(14)5־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3־3' M15-14AaAgAcagcaaaacagcaaaacagAcAaAaAL4-1s 5'SEQ 102 S 50(14) 3' M15-1sAaAgAcagcaaaacagcaaaacagAcAaAaAL4-1s5־'SEQ 103 ODN2006Q3)S 50(14)^ ' *.A.A ״AiA.A״AiAlAiAlA״A4A. A״AiAiAi AiA-AiA.A-AiAi Q •Otcgtcgttttgtcgttttgtcgtto3' M15-1sAaAgAcagcaaaacagcaaaacagAc aAaAL4-1s 5'SEQ 104 S 51(14) 3' M15-6AaAgAcagcaaaacagcaaaacagAcAaAaAL412 5'SEQ 105 ODN2006Q3)S 51(14)5־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3׳3' M15-6AaAgAcagcaaaacagcaaaacagAcAaAaAL4-12 5'SEQ 106 S 52(14) 3' M15-!4AaAgAcagcaaaacagcaaaacagAcAaAaAL412 5'SEQ 107 ODN2006Q3)S 52(14)5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3־3' M15-!4AaAgAcagcaaaacagcaaaacagAcAaAaAL412 5'SEQ 108 S 53(14) 3' M15-1sAaAgAcagcaaaacagcaaaacagAcAaAaAL4-12 5'SEQ 109 ODN2006(13)S 53(14)5’tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3־3' M!5-1sAaAgAcagcaaaacagcaaaacagAcAaAaAL4-12 5’SEQ 110 S 54(18) 3' aAaAcagcaaaacagAcAaAaAtAL4-18 5'SEQ 111 ODN2006Q3)S 54(18)5־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3־3' aAaAcagcaaaacagAcAaAaAtAL4-!s 5' id="p-200" id="p-200"

[0200][Table 10] No. ID (SEQ ID) Sequence: CpG(5’=^3) Complementary strand (3‘=>5')SEQ 112 S 55(18) 3' aAaAcagcaaaacagAcAaAaAtAt/L4-1s 5'SEQ 113 ODN2006Q3) 5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3'S 55(18) 3' aAaAcagcaaaacagAcAaAaAtAtAL4-1s 5'SEQ 114 S 56(18) 3' aAaAcagcaaaacagAcAaAaAtAtAtAtAtAL4-1s 5’SEQ 115 ODN2006Q3) 5־־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3'S 56(18) 3' aAaAcagcaaaacagAcAaAaAtAtAtAtAtAL4-1s 5'SEQ 116 S 57(18) 3' aAaAcagcaaaacagAcAaAaAgAL4-1s 5'SEQ 117 ODN2006<13) 5־ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3־S 57(18)3' aAaAcagcaaaacagAcAaAaAgAL4-18 5’SEQ 118 S 58(18) 3' aAaAcagcaaaacagAcAaAaAgAgAL4-18 5'SEQ 119 ODN2006(13) 5’ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3'S 58(18) 3' aAaAcagcaaaacagAcAaAaAgAgAL41s 5'SEQ 120 S 59(18) 3’ aAaAcagcaaaacagAcAaAaAgAgAgAgAgAL4-1s 5׳SEQ 121 ODN2006Q3) 5־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3־S 59(18) 3' aAaAcagcaaaacagAcAaAaAgAgAgAgAgAL4-18 5’SEQ 122 S 60(16) 3' gAcAaaaacagcaaaacagAcAaAaAtAL4-1s 5'SEQ 123 ODN2006Q3) 5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3־S 60(16) 3' gAcAaaaacagcaaaacagAcAaAaAtAL4-1s 5'SEQ 124 S 61(16) 3' gAcAaaaacagcaaaacagAcAaAaAtAtAL4-!s 5'SEQ 125 ODN2006(13) 5־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3'S 61(16) 3' gAcAaaaacagcaaaacagAcAaAaAtAtAL45 18־’SEQ 126 S 62(16) 3' gAcAaaaacagcaaaacagAcAaAaAtAtAtAtAtAL4-1S 5’SEQ 127 ODN2006Q3) 5' tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3’S 62(16) 3' gAcAaaaacagcaaaacagAcAaAaAtAtAtAtAt aL41s 5'SEQ 128 S 63(16) 3' gAcAaaaacagcaaaacagAcAaAaAgAL4-18 5’SEQ 129 ODN2006Q3) 5’־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3־S 63(16) 3' gAcAaaaacagcaaaacagAcAaAaAgAL4-!s 5' id="p-201" id="p-201"

[0201] 118 [Table 11] No. ID (SEQ ID) Sequence: CpG(5’=>3) Complementary strand (3'=>5)SEQ 130 S 64(16) 3' gAcAaaaacagcaaaacagAcAaAaAgAgAL4-1s 5'SEQ 131 ODN2006Q3) S 64(16) ' tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtA cAgAtAt 3'3' gAcAaaaacagcaaaacagAcAaAaAgAgAL4-1s 5'SEQ 132 S 65(16) 3' gAcAaaaacagcaaaacagAcAaAaAgAgAgAgAgAL4-1S 5’SEQ 133 ODN2006(13) S 65(16) ' tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3י3' gAcAaaaacagcaaaacagAcAaAaAgAgAgAgAgAL4-1s 5'SEQ 134 S 66(14) 3' M22-l8AaAgAcagcaaaacagcaaaacagAcAaAa 5'SEQ 135 ODN2006Q3) S 66(14) ־ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtA cAgAtAt 3’3’ M22-1sAaAgAcagcaaaacagcaaaacagAcAaAa 5'SEQ 136 S 67(20) 3' M22-18AaAgAcagcaaaacaAgAcAa 5'SEQ 137 ODN2006Q3) S 67(20) ־ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtA cAgAtAt 3’3' M22-1sAaAgAcagcaaaacaAgAcAa 5'SEQ 138 S 68(14) 3' M22-12AaAgAcagcaaaacagcaaaacagAcAaAa 5'SEQ 139 ODN2006Q3) S 68(14) ־ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtA cAgAtAt 3’3' M2212AaAgAcagcaaaacagcaaaacagAcAaAa 5'SEQ 140 S 69(20) 3' M22-12AaAgAcagcaaaacaAgAcAa 5'SEQ 141 ODN2006Q3) S 69(20) ־ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtA cAgAtAt 3־3' M22-12AaAgAcagcaaaacaAgAcAa 5'SEQ 142 Amph2006(13) 5' L4-16AtAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAg AtAt 3־SEQ 143 Amph2006GG(13)5' L4-16AgAgAtAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAt AcAgAtAt 3־SEQ 144 3' Amph20(13)5־ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtA cAgAtAtAM51-16 3' id="p-202" id="p-202"

[0202] [Table 12] No. ID (SEQ ID) Sequence: CpG(5‘=^3‘) Complementary strand (3'=>5')SEQ 145 S 70(20)3' aAgAcagcaaaacaAgAcAa L4-1s 5'SEQ 146 ODN2006G3) S 70(20) ־ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtA cAgAtAt 3’3' aAgAcagcaaaacaAgAcAa/ L4-1S 5’SEQ 147 S 71(21) 3' gAcAaaaacagcaaaAaAcAL4-!s 5'SEQ 148 ODN2006G3) S 71(21) ’ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtA cAgAtAt 3’3' gAcAaaaacagcaaaAaAc' L4-1s 5’SEQ 149 S 72(19) 3' aAcAagcaaaacagAcAaAaAL10-1S 5'SEQ 150 ODN2006G3) S 72(19) ־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtA cAgAtAt 3'3' aAcAagcaaaacagAcAaAaAL!0-18 5' 119 id="p-203" id="p-203"

[0203][Table 13] No. ID (SEQ ID) Sequence: CpG(5'=>3) Complementary strand (3'=>5‘)SEQ 151 S 73(18) 3׳ aAaAcagcaaaacagAcAaAaAgAgAgAL4-1s 5'SEQ 152ODN2006(13)S 73(18)5’ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3־3’ aAaAcagcaaaacagAcAaAaAgAgAgAL4-1s 5'SEQ 153 S 74(18) 3' aAaAcagcaaaacagAcAaAaAgAgAgAgAL41s5־'SEQ 154ODN2006(13)S 74(18)5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3'3' aAaAcagcaaaacagAcAaAaAgAgAgAgAL4-1s 5'SEQ 155 S 75(18)3־ aAaAcagcaaaacagAcAaAaAgAgAgAgAgAgAL4-1s •5'SEQ 156ODN2006(13)S 75(18)5' tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3־3' aAaAcagcaaaacagAcAaAaAgAgAgAgAgAgAL4-1s 5'SEQ 157 S 76(18) 3׳ aAaAcagcaaaacagAcAaAaAgAgAgAgAgAgAgAL4-1s 5'SEQ 158ODN2006Q3)S 76(18)5' tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3'3' aAaAcagcaaaacagAcAaAaAgAgAgAgAgAgAgAL4-1s 5' id="p-204" id="p-204"

[0204][Table 14] No. ID (SEQ ID) Sequence: CpG(5'=>3‘) Complementary strand (3'=>5)SEQ 159S 77(18) 3' aAaAcagcaaaacagAcAaAaAgAgAgAgAgAgAgAgAL4-1S 5'SEQ 160ODN2006G3)S 77(18)5׳־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3־3’ aAaAcagcaaaacagAcAaAaAgAgAgAgAgAgAgAgAL41s 5'SEQ 161 S 78(24) 3’ aAaAacagcaaaacagAcAaAaAgAgAgAgAgAL41s 0' SEQ 162ODN2006G3)S 78(24)5' tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3'3' aAaAacagcaaaacagAcAaAaAgAgAgAgAg׳ L4-1S 5'SEQ 163 S 79(17)3׳ aAaAaacagcaaaacagAcAaAaAgAgAgAgAg׳ L4-1s 5’SEQ 164ODN2006G3)S 79(17)5׳ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3־3׳ aAaAaacagcaaaacagAcAaAaAgAgAgAgAgAL4-1s 5׳SEQ 165 S 80(25) 3' M22-18AaAgAcagcaaaacagAcAaAa 5‘SEQ 166ODN2006G3)S 80(25)5׳tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3־3' M22-18AaAgAcagcaaaacagAcAaAa 5’SEQ 167 S 81(25) 3' M22-1sAgAgAaAgAcagcaaaacagAcAaAa 5'SEQ 168ODN2006G3)S 81(25)5׳tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3־3' M22-1sAgAgA aAgAcagcaaaacagAcAaAa 5’SEQ 169 S 82(25) 3' M22-1sAgAgAgAgAgAaAgAcagcaaaacagAcAaAa 5'SEQ 170ODN2006Q3)S 82(25)5׳tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3־3' M22-1sAgAgAgAgAgAaAgAcagcaaaacagAcAaAa 5'SEQ 171 S 83(IS) 3' aAaAcagcaaaacagAcAaAaAaAaAL4-1s 5'SEQ 172ODN2006Q3)S 83(18)5’ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3'3' aAaAcagcaaaacagAcAaAaAaAaAL4-1s 5'SEQ 173 S 84(18) 3’ aAaAcagcaaaacagAcAaAa AaAaAaAaAaAL4-is 5'SEQ 174ODN2006G3)S 84(18)5׳tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3־3’ aAaAcagcaaaacagAcAaAaAaAaAaAaAaAL4-!8 5' 120 id="p-205" id="p-205"

[0205][Table 15] No. ID (SEQ ID) Sequence: CpG(5'=>3) Complementary strand (3=^5) SEQ 175 S 85(25) 3' aAgAcagcaaaacagAcAaAaAL10-1s 5' ODN2006Q3) 5'tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt־ SEQ 176 ־ 3 S 85(25) 3' aAgAcagcaaaacagAcAaAaAL!0-1s 6' SEQ 177 S 86(17) 3' aAaAaacagcaaaacagAcAaAaAL10-18 5' ODN2006(13) 5־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt־ SEQ 178 ־ 3 S 86(17) 3' aAaAaacagcaaaacagAcAaAaAL10-1s 5' SEQ 179 S 87(25) 3' aAgAcagcaaaacagAcAaAaAgAgAgAgAgAL!0-1s 5' ODN2006(13) 5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt SEQ ISO 3' S 87(25) 3' aAgAcagcaaaacagAcAaAaAgAgAgAgAgAL10-18 5' SEQ 1S1 S 88(14) 3' M22-12AaAgAcagcaaaacagcaaaacagAcAaAa/ Ls-10 5' ODN2006Q3) 5’ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt־ SEQ 182 ־ 3 S 88(14) 3' M22-12AaAgAcagcaaaacagcaaaacagAcAaAaALs-10 5' SEQ 183 S 89(19) 3' M22-12AaAcAagcaaaacagAcAaAaAL810 6' ODN2006(13) 5'-tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt־ SEQ 184 3* S 89(19) 3' M22-12AaAcAagcaaaacagAcAaAaAL8-10 5' SEQ 185 S 90(18) 3' M15-6AaAaAcagcaaaacagAcAaAaAL418 5’ ODN2006Q3) 5' tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt־ SEQ 1S6 ־ 3 S 90(18) 3' M15-6AaAaAcagcaaaacagAcAaAaAL4-1s 5' SEQ 187 S 91(18) 3'M15-6AaAaAcagcaaaacagAcAaAaAgAgAL4-18 5' ODN2006Q3) 5' tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt SEQ 188 3' S 91(18) 3'M15-6AaAaAcagcaaaacagAcAaAaAgAgAL4-18 5' SEQ 189 S 92(18) 3' M15-6AaAaAcagcaaaacagAcAaAaAgAgAgAgAgAL418 5' ODN2006(13) 5'tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt־ SEQ 190 3' S 92(18) 3' M15-6AaAaAcagcaaaacagAcAaAaAg׳־ gAgAgAgAL4-18 0' id="p-206" id="p-206"

[0206] 121 [Table 16] No. ID (SEQ ID) Sequence: CpG(5‘=>3) Complementary strand (3'=>5') SEQ 191 S 93(18) 3' M15-10AaAaAcagcaaaacagAcAaAaAL4-18 5' SEQ 192 ODN2006Q3) S 93(18) 5'-tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3’ 3' M15-10AaAaAcagcaaaacagAcAaAaAL4-1s 5' SEQ 193 S 94(18) 3' M15-10AaAaAcagcaaaacagAcAaAaAgAgAL4-1s 5' SEQ 194 ODN2006Q3) S 94(18) 5׳ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3־ 3' M15-10AaAaAcagcaaaacagAcAaAaAgAgAL4-18 5' SEQ 190 S 95(18) 3' M15-10AaAaAcagcaaaacagAcAaAaAgAgAgAgAgAL4-18 5' SEQ 196 ODN2006(13) S 95(18) 5׳ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3־ 3' M15-10AaAaAcagcaaaacagAcAaAaAgAgAgAgAgAL4-1s 5' SEQ 197 S 96(18) 3’ M15-6AaAaAcagcaaaacagAcAaAaAL4-16 5' SEQ 198 ODN2006Q3) S 96(18) 5׳ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3’ 3' M15-6AaAaAcagcaaaacagAcAaAaAL4 !6 5' SEQ 199 S 97(18) 3’ M15-6AaAaAcagcaaaacagAcAaAaAgAgAL4-16 5' SEQ 200 ODN2006<13) S 97(18) 5' tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3־ 3' M15-6AaAaAcagcaaaacagAcAaAaAgAgAL4-16 5' SEQ 201 S 98(18) 3' M!5-10AaAaAcagcaaaacagAcAaAaAL4-16 5' SEQ 202 ODN2006<13) S 98(18) 5׳tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3־ 3' M15-10AaAaAcagcaaaacagAcAaAaAL4-16 5' SEQ 203 S 99(18) 3' M!510AaAaAcagcaaaacagAcAaAaAgAgAL4-16 5' SEQ 204 ODN2006(13) S 99(18) 5' tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3’ 3' M15-10AaAaAcagcaaaacagAcAaAaAgAgAL4-16 5' SEQ 205 S 100(25) 3' M22-14AaAgAcagcaaaacagAcAaAaAL8-6 3־' SEQ 206 ODN2006Q3) S 100(25) 5׳ tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3־ 3' M22-14AaAgAcagcaaaacagAcAaAaAL8-6 •3' SEQ 207 S 101(25) 3' M22-14AaAgAcagcaaaacagAcAaAaALs-10 5' SEQ 208 ODN2006Q3) S 101(25) 5' tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt 3' 3’ M22-14AaAgAcagcaaaacagAcAaAaAL8-10 5' id="p-207" id="p-207"

[0207] 122 [Table 17] No. ID (SEQ ID) Sequence: CpG(5’=>3’) Complementary strand (3’=>5’) SEQ-209 S25)102־) 3’־M2214AaAgAcagcaaaacagAcAaAaALs145־' SEQ-210 ODN2006(13) S25)102־) 5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־' 3’־M22-14AaAgAcagcaaaacagAcAaAaAL8-145־' SEQ-211 S25)103 ־) 3’־M2214AgAgAaAgAcagcaaaacagAcAaAaALs-65־' SEQ-212 ODN2006(13) S25)103־) 5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־' 3’־M22-14AgAgAaAgAcagcaaaacagAcAaAaAL8-65־' SEQ-213 S25)104־) 3’־M22-14AgAgAaAgAcagcaaaacagAcAaAaALs-105־' SEQ-214 ODN2006(13) S25)104־) 5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־' 3’־M22-14AgAgAaAgAcagcaaaacagAcAaAaALs-105־' SEQ-215 S25)105־) 3’־M22-14AgAgAaAgAcagcaaaacagAcAaAaAL8-145־' SEQ-216 ODN2006(13) S25)105־) 5,־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־' 3’־M22-14AgAgAaAgAcagcaaaacagAcAaAaALs-145־' SEQ-217 S25)106־) 3’־M22-18AaAgAcagcaaaacagAcAaAaAL8-6'5' SEQ-218 ODN2006(13) S25)106־) 5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־' 3’־M22-18AaAgAcagcaaaacagAcAaAaALs-65־' SEQ-219 S25)107־) 3’־M22-18AaAgAcagcaaaacagAcAaAaAL8-105־' SEQ-220 ODN2006(13) S25)107־) 5'-tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־' 3’־M22-18AaAgAcagcaaaacagAcAaAaALs-105־' SEQ-221 S25)108־) 3’־M22-18AgAgAaAgAcagcaaaacagAcAaAaAL8-6'5' SEQ-222 ODN2006(13) S25)108 ־) 5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־' 3’־M22-18AgAgAaAgAcagcaaaacagAcAaAaAL8-6'0' SEQ-223 S25)109־) 3’־M22-18AgAgAaAgAcagcaaaacagAcAaAaAL8-105־' SEQ-224 ODN2006(13) S25)109 ־) 5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־' 3’־M22-18AgAgAaAgAcagcaaaacagAcAaAaAL8-105־' SEQ-225 S18)110־) 3’־M22-12AaAaAcagcaaaacagAcAaAaALchor5' SEQ-226 ODN2006(13) S18)110־) 5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־’ 3’־M22-12AaAaAcagcaaaacagAcAaAaALehor5' SEQ-227 S־lll(18) 3’־M22-12AaAaAcagcaaaacagAcAaAaALfarr5' SEQ-228 ODN2006(13) S18)111 ־) 5’־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcA°־AtAt3־’ 3’־M22-12AaAaAcagcaaaacagAcAaAaALfarr5' id="p-208" id="p-208"

[0208] 123 [Table 18] No. ID (SEQ ID) Sequence: CpG(5’=>3’) Complementary strand (3’=>5’)SEQ-229 S18)112־) 3’־M22-12AaAaAcagcaaaacagAcAaAaALs-105־'SEQ-230ODN2006(13)S18)112־)5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־'3’־M22-12AaAaAcagcaaaacagAcAaAaALs-105־'SEQ-231 S18)113־) 3’־M22-12AaAaAcagcaaaacagAcAaAaALs-145־'SEQ-232ODN2006(13)S-113(18)5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־'־ י M22-12AaAaAcagcaaaacagAcAaAaALs-145־,SEQ-233 S18)114־) 3’־Mch01AaAaAcagcaaaacagAcAaAaALfarr5'SEQ-234ODN2006(13)S18)114־)5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־'3’־Mcho1AaAaAcagcaaaacagAcAaAaALfar15־'SEQ235־ S14)115־) 3'־aAgAcagcaaaacaK22-1sgcaaaacagAcAaAa5־'SEQ-236ODN2006(13)S14)115־)5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt ׳ 3 ־3'־aAgACagcaaaaCaK22-1sgcaaaacagAcAaAa5־'SEQ-237 S17)116־) 3’־M15-6AaAaAaacagcaaaacagAcAaAaAL4185־'SEQ-238ODN2006(13)S17)116־)5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־’3’־M15-6AaAaAaacagcaaaacagACAaAaAL4-185־'SEQ-239 S17)117־) 3’־M15-6AaAaAaacagcaaaacagAcAaAaAgAgAL4-185־'SEQ-240ODN2006(13)S17)117־)5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־’3’־M1s-6AaAaAaacagcaaaacagAcAaAaAgAgAL4-185־'SEQ-241 S17)118־) 3’־M15-10AaAaAaacagcaaaacagAcAaAaAL4-185־'SEQ-242ODN2006(13)S17)118־)5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־’3’־M15-10AaAaAaacagcaaaacagAcAaAaAL4-185־'SEQ-243 S17)119־) 3’־M15-10AaAaAaacagcaaaacagAcAaAaAgAgAL4-185־'SEQ-244ODN2006(13)S17)119־)5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־'3’־M15-10AaAaAaacagcaaaacagAcAaAaAgAgAL4-185־'SEQ245־ S17)120־) 3’־M15-6AaAaAaacagcaaaacagACAaAaAL4165־'SEQ-246ODN2006(13)S17)120־)5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־'3’־M15-6AaAaAaacagcaaaacagACAaAaAL4-165־' id="p-209" id="p-209"

[0209] 124 [Table 19] No. ID (SEQ ID) Sequence: CpG(5’=>3’) Complementary strand (3’=>5י) SEQ-247 S17)121־) 3’־M15-6AaAaAaacagcaaaacagAcAaAaAgAgAL4-165־, SEQ-248 ODN2006(13) S17)121־) 5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־' 3’־M15-6AaAaAaacagcaaaacagAcAaAaAgAgAL4-165־' SEQ-249 S17)122־) 3’־M15-10AaAaAaacagcaaaacagAcAaAaAL4-16-5' SEQ250־ ODN2006(13) S17)122־) 3’־M15-10AaAaAaacagcaaaacagAcAaAaAL4-165־' SEQ-251 S17)123־) 3’־M15-10AaAaAaacagcaaaacagAcAaAaAgAgAL4 165־' SEQ-252 ODN2006(13) S17)123־) 5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־' 3’־M15-10AaAaAaacagcaaaacagAcAaAaAgAgAL4-165־' SEQ-253 K3־CpG(26) 5'־aAtAcAgAaAcAtAcAtAcAgAaAgAcAgAtAtAcAtAc3־' SEQ254־ S27)124־) 3’־tAgAagagctcgcaaAgAaAgAL41s5־' SEQ-255 K3־CpG(26) S27)124־) 5'־aAtAcAgAaAcAtAcAtAcAgAaAgAcAgAtAtAcAtAc3־' 3’־tAgAagagctcgcaaAgAaAgAL4-1s5־' SEQ256־ D35־CpG(28) 5'־gAgtgcatcgatgcaggggAgAg3־' SEQ257־ S29)125־) 3'־gAtAagctacgtcccAcAcAcAL4-1s5־' SEQ-258 D35־CpG(28) S29)125־) 5'־gAgtgcatcgatgcaggggAgAg3־' 3'־gAtAagctacgtcccAcAcAcAL4-185־' SEQ-259 S25)126־) 3’־M22-10AaAgAcagcaaaacagAcAaAa5־' SEQ-260 ODN2006G3) S25)126־) 5,־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt-3' 3’־M22-10AaAgAcagcaaaacagAcAaAa5־' SEQ-261 S25)127־) 3’־M22-12AaAgAcagcaaaacagAcAaAa5־' SEQ-262 ODN2006G3) S25)127־) 5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־' 3’־M22-12AaAgAcagcaaaacagAcAaAa5־' SEQ-261 S25)127־) 3’־M22-12AaAgAcagcaaaacagAcAaAa5־' SEQ-262 ODN2006G3) S25)127־) 5’־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־' 3’־M22-12AaAgAcagcaaaacagAcAaAa5־' id="p-210" id="p-210"

[0210] 125 [Table 20] No. ID (SEQ ID) Sequence: CpG(5’=>3’) Complementary strand (3’=45’) SEQ-263 S25)128־) 3’־M22-20AaAgAcagcaaaacagAcAaAa5־' SEQ-264 ODN2006(13) S25)128־) Ac'^0,^^^^A^AgA^AcAgA^A|.A^A^.AgA|.AcAQ.A^ 3’־M22-20 AaAgAcagcaaaacagAcAaAa5־' SEQ265־ S25)129־) 3’־M22-22AaAgAcagcaaaacagAcAaAa5־' SEQ-266 ODN2006(13) S25)129־) 5'"t AcA£yA^AcAgAt-A|A^A^AgA^AcAgA^At-At׳A^AgA^AcA0.A^ 3’־M22-22AaAgAcagcaaaacagAcAaAa5־' SEQ-267 S-130(25) 3’־M22-12AaAgAcagcaaaacagAcAaAaAL8-6'5' SEQ-268 ODN2006(13) S25)130־) S^tAcA^A^Ac^gA^A^A^A^AgAt-AcAgA^Ai■ A^A^AgA^AcAQ-At-A^>3• 3’־M22-12AaAgAcagcaaaacagAcAaAaALs 65־' SEQ-269 S25)131־) 3’־M22-12AaAgAcagcaaaacagAcAaAaAL8-105־' SEQ-270 ODN2006(13) S25)131 ־) 5l’tAcA(yAt)AcA0‘A^A^.A^A^AgA^AcAgA^A|. A^A^AgA^AcAQ-A^ A^«3> 3’־M22-12AaAgAcagcaaaacagAcAaAaAL8-105־' SEQ-271 S25)132־) 3’־M22-12AaAgAcagcaaaacagAcAaAaAL8-145־' SEQ-272 ODN2006C13) S25)132־) S1*! AcA0‘AtjAcA0'A^A׳|׳A^A^AgA^AcAgA|.At׳A^A^AgA^AcAQ.A^A|.3> 3’־M22-12AaAgAcagcaaaacagAcAaAaALs-145־' SEQ-273 S25)133־) 3’־M22-12AgAgAaAgAcagcaaaacagAcAaAaAL8-65־' SEQ-274 ODN2006(13) S25)133־) 5,’tAcA(yAt|AcA0־A^A^.A^A^AgA^AcAgA^A|.A^A^AgA^AcA0.A^ A^«3> 3’־M22-12AgAgAaAgAcagcaaaacagAcAaAaALs-65־' SEQ275־ S25)134־) 3’־M22-12AaAgAcagcaaaacagAcAaAaAgAgAL8105־' SEQ-276 ODN2006(13) S25)134־) -..^AcA^Ai-AcAgA^A^A^A^AgA^AcAgAi-At.A^A^AgA^AcA^A^A^.31 3’־M22-12AgAgAaAgAcagcaaaacagAcAaAaALs-105־' SEQ277־ S25)135 ־) 3’־M22-12AgAgAaAgAcagcaaaacagAcAaAaAL8-145־' SEQ-278 ODN2006(13) S25)135־) 5'"t AcA^A^AcA(yA|.A^A^A^AgA^AcAgA|.A^.A^ A^AgA|.AcAgA^A^.3> 3’־M22-12AgAgAaAgAcagcaaaacagAcAaAaAL8-145־' SEQ-279 S18)136־) 3'־aAaAcagcaaaacagAcAALNAAALNA־AgAgAL4-185־' SEQ-280 ODN2006(13) S18)136 ־) 5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־' 3'־aAaAcagcaaaacagAcAALNAAALNA־AgAgAL4-185־' id="p-211" id="p-211"

[0211] 126 [Table 21] No. ID (SEQ ID) Sequence: CpG(5’=>3’) Complementary strand (3’=>50 SEQ-281 S18)137־) 3'־aAaAcagcaALNAALNAacagAcAALNAAALNAAgAgAL4-185־' SEQ-282 ODN2006CL3) S18)137־) '־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־' 3,־aAaAcagcaALNAALNAacagAcAAtNAAALNAAgAgAL4-185־' SEQ-283 S18)138־) 3'־aAALNAACagCaALNAALNAacagACAALNAAALNAAgAgAL4-185־' SEQ-284 ODN2006(13) S18)138־) 5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־' 3'־aAALNAAcagcaALNAALNAacagAcAALNAAALNAAgAgAL41s5־' SEQ285־ S18)139־) 3'־aAaAcAaAgAcAaAaAaAaAcAaAgAcAaAaAgAgAgAgAgA L4-185־' SEQ-286 ODN2006(13) S18)139־) ^!.^A^AQ.A^AcAQ.A^A^A^A^AgA^AcAgA^A^A^A^AgA^AQAgA^A|..3' 3'־aAaAcAaAgAcAaAaAaAaAcAaAgAcAaAaAgAgAgAgAgA L4-1s5־' SEQ-287 S30)140־) 3’־M22-20AaAgAcagcaaaacagcaaaAaAcAa5־' SEQ-288 ODN2006(13) S30)140־) 5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־, 3’־M22-20AaAgAcagcaaaacagcaaaAaAcAa5־' SEQ-289 S-14K14) 3’־M22-20AaAgAcagcaaaacagcaaaacagAcAaAa5־' SEQ-290 ODN2006I13) S14)141־) 5**tAc^^A(Ac^^A^A^A^A|.AgA^AcAgA|.A|.A^A^AgA^AcAgA^A^.3' 3’־M22-20AaAgAcagcaaaacagcaaaacagAcAaAa5־' SEQ-291 S30)142־) 3’־M22-22AaAgAcagcaaaacagcaaaAaAcAa5־' SEQ-292 ODN2006(13) S30)142־) 5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־' 3’־M22-22AaAgAcagcaaaacagcaaaAaAcAa5־' SEQ-293 S14)143 ־) 3’־M22-22AaAgAcagcaaaacagcaaaacagAcAaAa5־' SEQ-294 ODN2006(13) S14)143־) 3’־M22-22AaAgAcagcaaaacagcaaaacagAcAaAa5־' SEQ-295 S30)144־) 3’־M22-12AaAgAcagcaaaacagcaaaAaAcAaAL8-65־' SEQ-296 ODN2006(13) S30)144־) S^tAcA^A^AcAo-A^A^ A^A^.AgA^AcAgAt.A^A^A^AgA^AcAgA^A^.3• 3’־M22-12AaAgAcagcaaaacagcaaaAaAcAaAL8-6'5' id="p-212" id="p-212"

[0212] 127 [Table 22] No. ID (SEQ ID) Sequence: CpG(5’=>3’) Complementaiy strand (3’=>5’) SEQ-297 S30)145־) 3’־M22-12AaAgAcagcaaaacagcaaaAaAcAaAL8-105־' SEQ-298 ODN2006(13) S30)145־) ^ t _ ^ 0■ ^ Q Ql ^ ^ ^ ^ 0. ^ Q ^ ^ ^ ^ ^ Q ^ ^ a ^ 1 3’־M22-12AaAgAcagcaaaacagcaaaAaAcAaAL810־o' SEQ-299 S30)146־) 3’־M22-12AaAgAcagcaaaacagcaaaAaAcAaAL8-145־' SEQ-300 ODN2006(13) S30)146־) ^ A 0 ^y ^ 0 ^y ^ ^ ^ ^y ^ 0 ^y A ^ ^ ^ ^y ^ 0 0 ^ A ^ a ^ t 3’־M22-12AaAgAcagcaaaacagcaaaAaAcAaALs14־o' SEQ-301 S30)147 ־) 3’־M22-12AgAgAaAgAcagcaaaacagcaaaAaAcAaALs-65־' SEQ-302 ODN2006(13) S30)147־) ^ ^ 0 A ^y ^ 0 ^y ^ ^ ^ ^y ^ 0 ^y A ^ ^ A ^ ^y ^ 0 ^y ^ ^ M ^ ! 3’־M22-12AgAgAaAgAcagcaaaacagcaaaAaAcAaALs-6־o' SEQ-303 S30)148־) 3’־M22-12AgAgAaAgAcagcaaaacagcaaaAaAcAaALs-105־' SEQ-304 ODN2006(13) S30)148־) 5l"tAc^^^tAc^^^t^tAtAtA^A|Ac^O,^^A^A|A^AgA|.AcA0A^A|..3l 3’־M22-12AgAgAaAgAcagcaaaacagcaaaAaAcAaALs105־' SEQ-305 S30)149־) 3’־M22-12AgAgAaAgAcagcaaaacagcaaaAaAcAaALs-145־' SEQ-306 ODN2006(13) S30)149־) 5’־tAcA o־AtAcAo־AtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־’ 3’־M22-12AgAgAaAgAcagcaaaacagcaaaAaAcAaAL8-145־' SEQ-307 S14)150־) 3’־M22-12AaAgAcagcaaaacagcaaaacagAcAaAaALs-65־' SEQ-308 ODN2006(13) S14)150־) 5’״t AcAgAt AcAgAt At AtAt AgAt AcAgAt AtAtAt AgAt AcAgAt At3־f 3’־M22-12AaAgAcagcaaaacagcaaaacagAcAaAaALs-65־' SEQ-309 S14)151־) 3’־M22-12AaAgAcagcaaaacagcaaaacagAcAaAaALs-145־' SEQ-310 ODN2006(13) S-15K14) 5HAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־' 3’־M22-12AaAgAcagcaaaacagcaaaacagAcAaAaAL8-140־' id="p-213" id="p-213"

[0213] [Table 23] No. ID (SEQ ID) Sequence: CpG(5’=^3’) Complementary strand (3’=^5’) SEQ-311 S14)152־) 3’־M22-12AgAgAaAgAcagcaaaacagcaaaacagAcAaAaALs-65־' SEQ-312 ODN2006(13) S-152G4) 5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־' 3’־M22-12AgAgAaAgAcagcaaaacagcaaaacagAcAaAaAL8-65־' SEQ-313 S14)153־) 3’־M22-12AgAgAaAgAcagcaaaacagcaaaacagAcAaAaAL8-105־' SEQ-314 ODN2006(13) S14)153־) ^ ^ ^ 0 0 0 ^y ^ ^ ^ ^y ^ 0 ^y ^ ^ ^ ^y A ^ 0 ^y ^ ^ a ^ 1 3’־M22-12AgAgAaAgAcagcaaaacagcaaaacagAcAaAaALs-105־' SEQ-315 S14)154־) 3’־M22-12AgAgAaAgAcagcaaaacagcaaaacagAcAaAaAL8-145־' SEQ-316 ODN2006(13) S14)154־) 5 ־ ז tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt י 3 ־ 3’־M22-12AgAgAaAgAcagcaaaacagcaaaacagAcAaAaALs-145־' SEQ-317 S18)155־) 3'־M22-1sABuABuAaAaAcagcaaaacagAcAaAa-5’ SEQ-318 ODN2006(13) S18)155־) 5׳-tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt ׳ 3 ־ 3'־M22-18ABuABuAaAaAcagcaaaacagAcAaAa5־’ SEQ-319 S18)156־) 3'־Mteg -18AaAaAcagcaaaacagAcAaAa5־’ SEQ-320 C)DN2006(13) S18)156־) 5'-tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־' 3'־MTEG-1sAaAaAcagcaaaacagAcAaAa5־’ SEQ-321 S25)157־) 3'־M22-1sAALNAAGLNAA5mCLNAagcaaaacagA5111CLNAA AlnaaAlna5־’ SEQ-322 ODN2006(13) S25)157־) '־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־' 3'־M22-18AALNAAGLNAA5mCLNAagcaaaacagA5mCLNAAAlnaaAlna5־’ SEQ-323 S31)158־) 3'־M22-18AALNAAGLNAA5mCLNAagCaAALNAAALNAAALNA5־’ SEQ-324 ODN2006(13) S31)158־) 5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־' 3'־M22-18AALNAAGLNAA5mCLNAagCaAALNAAALNAAALNA5־’ 128 id="p-214" id="p-214"

[0214] [Table 24] No. ID (SEQ ID) Sequence: CpG(5’=^3’) Complementaiy strand (3’=^5’)SEQ-325 S30)159־) 3’־M22-14AaAgAcagcaaaacagcaaaAaAcAaAL8-65־'SEQ-326ODN2006(13)S30)159־)5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־'3’־M22-14AaAgAcagcaaaacagcaaaAaAcAaAL8-65־'SEQ-327 S30)160־) 3’־M22-14AaAgAcagcaaaacagcaaaAaAcAaAL8-105־'SEQ-328ODN2006(13)S30)160־)5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־'3’־M22-14AaAgAcagcaaaacagcaaaAaAcAaAL8-105־'SEQ-329 S-16K30) 3’־M22-14AaAgAcagcaaaacagcaaaAaAcAaALs-145־'SEQ-330ODN2006(13)S-16K30)5’־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־’ 3’־M22-14AaAgAcagcaaaacagcaaaAaAcAaALs 145־'SEQ-331S30)162 ־)3’־M22-14AgAgAaAgAcagcaaaacagcaaaAaAcAaAL8-6‘5'SEQ-332ODN2006(13)S-162(30)5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־'3’־M22-14AgAgAaAgAcagcaaaacagcaaaAaAcAaAL8-65־'SEQ-333 S30)163־) 3’־M22-14AgAgAaAgAcagcaaaacagcaaaAaAcAaALs-105־'SEQ-334ODN2006(13)S-163(30)5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־'3,־M22-14AgAgAaAgAcagcaaaacagcaaaAaAcAaAL8-105־'SEQ-335 S-164(30) 3’־M22-14AgAgAaAgAcagcaaaacagcaaaAaAcAaALs-145־'SEQ-336ODN2006(13)S30)164־)5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־'3’־M22-14AgAgAaAgAcagcaaaacagcaaaAaAcAaAL8-145־'SEQ-337 S25)165־) 3'־aAC0MeAag0MeCa0Meaa0MeaC0Meag0MeACAa0MeAaAL4-185־' SEQ-338ODN2006(13)S25)165־)5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־' 3'־aAC0MeAag0MeCa0Meaa0MeaC0Meag0MeACAa0MeAaAL4-185־' id="p-215" id="p-215"

[0215] 129 [Table 25] No. ID (SEQ ID) Sequence: CpG(5'=>3') Complementary strand (3'=)5)SEQ 339 ODN2216(32) 5' gAgAgggacgatcgtcgAgAgAgAgAg 3'SEQ 340 S 166(33) 3' M22-1sAcAcAccctgctagcaAgAcAc 5'SEQ 341ODN2216(32)S 166(33)5' gAgAgggacgatcgtcgAgAgAgAgAg 3’3' M22-1sAcAcAccctgctagcaAgAcAc 5'SEQ 342 ODN6S4<34) 5־ tAcAgAaAcAgAtAtAcAgAtAcAgAtAtAcAgAtAcAgAtAtAc 3־SEQ 343 S 167(35) 3’ M22-18AaAgActgcaagcagcAaAaAg 5‘SEQ 344ODN6S4<34)S 167(35)5־ tAcAgAaAcAgAtAtAcAgAtAcAgAtAtAcAgAtAcAgAtAtAc 3־3' M22-1sAaAgActgcaagcagcAaAaAg 5'SEQ 340 D LS0K36)5-־tAcAgAcAgAaAcAgAtAtAcAgAcAcAcAgAaAcAgAtAtAcAgAg AtAa 3־SEQ 346 S 168(37) 3' M22-18AaAgAcgctgcaagcgAgAgAc 5' SEQ 347D LS0K36)S 168(37)5־ tAcAgAcAgAaAcAgAtAtAcAgAcAcAcAgAaAcAgAtAtAcAgAg AtAa 3־3' M22-18AaAgAcgctgcaagcgAgAgAc 5'SEQ 348 D LS03(38)5־ tAcAgAcAgAaAaAcAgAtAtAcAgAcAcAgAcAgAtAtAcAgAaA aAcAgAcAgAg 3'SEQ 349 S 169(39) 3’ M22-1sAaAgAcgcttgcaagcAgAgAc 5' SEQ 350D LS03(38)S 169(39)5־־tAcAgAcAgAaAaAcAgAtAtAcAgAcAcAgAcAgAtAtAcAgAaA aAcAgAcAgAg 3’3' M22-1sAaAgAcgcttgcaagcAgAgAc 5'SEQ 351 ODN2395(40) 5־ tAcAgAtAcAgAtAtAtAtAcAgAgAcAgAcAgAcAgAcAcAg 3־SEQ 352 S 170(41) 3' M22-18 aAgAcagcaaaagccAgAc g 5'SEQ 353ODN2395(40)S 170(41)5־ tAcAgAtAcAgAtAtAtAtAcAgAgAcAgAcAgAcAgAcAcAg 3־3' M22-18AaAgAcagcaaaagccAgAcAg 5'SEQ 354 ODNM362(42)5־tAcAgAtAcAgAtAcAgAtAtAcAgAaAaAcAgAaAcAgAtAtAgAa At 3'SEQ 355 S 171(43) 3' M22-1sAaAgAcagcagcaagcAtAtAg 5' SEQ 356ODNM362(42)S 171(43)5'־tAcAgAtAcAgAtAcAgAtAtAcAgAaAaAcAgAaAcAgAtAtAgAa At 3־3' M22-l8AaAgAcagcagcaagcAtAtAg 5'SEQ 357 ODN2336(44) 5' gAgAggacgacgtcgtggAgAgAgAgAg 3’SEQ 358 S 172(45) 3' M22-1sAcAcAcctgctgcagcAaAcAc 5'SEQ 359ODN2336(44)S 172(45)5' gAgAggacgacgtcgtggAgAgAgAgAg 3'3' M22-1sAcAcAcctgctgcagcAaAcAc 5' id="p-216" id="p-216"

[0216] 130 [Table 26] No. ID (SEQ ID) Sequence: CpG(5’=>3’) Complementary strand (3’=>5’) SEQ-360 S25)173־) 3’־M22-6AaAgAcagcaaaacagAcAaAa5־' SEQ-361 ODN2006(13) S25)173־) 5,־tAcAgAtAcA°־AtAtAtAtAgAtAcAgAtAtAtAtA0־AtAcA0־AtAt3־' 3’־M22-6AaAgAcagcaaaacagAcAaAa5־' SEQ-362 S25)174־) 3’־M22-6AaAgAcagcaaaacagAcAaAaALs-105־' SEQ-363 ODN2006(13) S25)174־) ״t.^AcA0.AtAcAa־AtAtAtAtAgAtACAgAtAtAtAtA0־AtACAgAtAt3־' 3’־M22-6AaAgAcagcaaaacagAcAaAaAL8-105־' SEQ-364 S25)175 ־) 3’־M22-6AaAgAcagcaaaacagAcAaAaAL8-145־' SEQ365־ ODN2006(13) S25)175־) 5,־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־’ 3’־M22-6AaAgAcagcaaaacagAcAaAaAL8-145־' SEQ-366 S25)176 ־) 3’־M22-8AaAgAcagcaaaacagAcAaAa5־' SEQ367־ ODN2006(13) S25)176־) 5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־' 3’־M22-8AaAgAcagcaaaacagAcAaAa5־’ SEQ-368 S25)177־) 3’־M22-8AaAgAcagcaaaacagAcAaAaAL8-105־' SEQ-369 ODN2006(13) S25)177 ־) 5,־tAcA0־AtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־' 3’־M22-8AaAgAcagcaaaacagAcAaAaAL8-10*5' SEQ-370 S25)178־) 3’־M22-8AaAgAcagcaaaacagAcAaAaAL8-145־' SEQ-371 ODN2006(13) S25)178־) 5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־' 3,־M22-8AaAgAcagcaaaacagAcAaAaAL8-14־o' SEQ-372 S14)179־) 3’־M22-14AaAgAcagcaaaacagcaaaacagAcAaAaAL8-65־' SEQ-373 ODN2006(13) S14)179־) 5 ־ י tAcAgAtAcAcrAtAtAtAtAgAtAcAgAtAtAtAtA0־AtAcA0■AtAt3־, 3’־M22-14AaAgAcagcaaaacagcaaaacagAcAaAaALs-6'5' SEQ374־ S14)180־) 3’־M22-14AaAgAcagcaaaacagcaaaacagAcAaAaAL8-105־' SEQ375־ ODN2006(13) S-180(14) ~»_j.AcAa.Aj.AcAa.A.£;A£AtA.|.AgA£AcAgA.j.AtA.kA£AgAj:AcAgA£A.|:_g» 3’־M22-14AaAgAcagcaaaacagcaaaacagAcAaAaAL8-105־' id="p-217" id="p-217"

[0217] [Table 27] No. ID (SEQ ID) Sequence: CpG(5’=>3’) Complementary strand (3’=>5י) SEQ376־ S-18K14) 3’־M22-14AaAgAcagcaaaacagcaaaacagAcAaAaAL8145־' SEQ377־ ODN2006(13) S-18K14) 5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־' 3’־M22-14AaAgAcagcaaaacagcaaaacagAcAaAaAL8-145־' SEQ-378 S14)182־) 3’־M22-14AgAgAaAgAcagcaaaacagcaaaacagAcAaAaAL8-6'5' SEQ-379 ODN2006(13) S-182(14) 5*.tAcA g g A tAt־AtAtA g A tA 0 A g A tAtA^A^A g A tA 0 A tA t ~ 3^ 3’־M22-14AgAgAaAgAcagcaaaacagcaaaacagAcAaAaAL8-6'5' SEQ-380 1018ISS(46) 5'־tAgAaAcAtAgAtAgAaAaAcAgAtAtAcAgAaAgAaAtAgAa3־' SEQ-381 S-183(47) 3’־M22-18AaAcAtgacacttgcaAaAgAc5־' SEQ-382 1018ISS(46) S47)183־) 5'־tAgAaAcAtAgAtAgAaAaAcAgAtAtAcAgAaAgAaAtAgAa3־' 3’־M22-18AaAcAtgacacttgcaAaAgAc5־' SEQ-383 S14)184־) 3’־M22-22ABuABuAaAgAcagcaaaacagcaaaacagAcAaAa5־' SEQ-384 ODN2006(13) S14)184־) 5'־tAcAgAtAcAgAtAtAtAtA0־AtAcAgAtAtAtAtAgAtAcA0־AtAt3־' 3’־M22-22ABuABuAaAgAcagcaaaacagcaaaacagAcAaAa5־' SEQ-385 S14)185־) 3’־M22-22AgAgAgAgAgAaAgAcagcaaaacagcaaaacagAcAaAa5־' SEQ-386 ODN2006(13) S-185(14) 5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcA°־AtAt3־' 3’־M22-22AgAgAgAgAgAaAgAcagcaaaacagcaaaacagAcAaAa5־' SEQ-387 S14)186־) 3’־M22-22ABuABuAgAgAgAgAgAaAgAcagcaaaacagcaaaacag AcAaAa5־' SEQ-388 ODN2006(13) S14)186־) 5'־tAcAgAtAcAgAtAtAtAtAgAtAcAgAtAtAtAtAgAtAcAgAtAt3־' 3’־M22-22ABuABuAgAgAgAgAgAaAgAcagcaaaacagcaaaacag AcAaAa5־' id="p-218" id="p-218"

[0218]In Tables 4 to 27, n (small letter) is DNA, no Me is 2’-OMe-RNA, N (capital letter) is RNA, NF is 2’-deoxy-2’-F-RNA, Nlna is LNA and 5mC is 5- methylcytosine. A is -P(S)OH-, and the bond without any symbol is -P(O)OH-.

No symbol: L is a compound introduced at the 5’ end of oligonucleotide possibly comprising an oligonucleotide linker, and covalently binds with a hydroxyl group at the 5’ end of oligonucleotide via the described bond. M is a compound introduced at the 3’ end of oligonucleotide possibly comprising an oligonucleotide linker, and covalently binds with a hydroxyl group at the 3’ end of oligonucleotide via the described bond. In Lx - n , x is a figure corresponding to the structure of Compound 4-n or Compound 10-n which is double strand or Compound 8-n which is single strand, and n is an integer of 6 to corresponding to the carbon chain(s). In Mx - n , x is a figure corresponding to the structure of Compound 22-n, Compound 49-n or Compound 51-n which is double strand of Compound 15-n which is single strand, and n is an integer of to 28 corresponding to the carbon chain(s).[0219]In detail, Lx - n is a group described below.O O■8-n Lb°L4-n O-10-n 132 wherein n is an integer of 6 to 28.L4-n is a group derived from Compound 4־n which is synthesized in 11־) of the above A). L8 ־ n is a group derived from Compound 8־n which is synthesized in 2) of the above A). Li 0 - n is a group derived from Compound 10־n which is synthesized in 3) of the above A).[0220] Lchoi or Ltoc in Table is a group described below. -chol Lchoi is a group derived from Compound 26 which is synthesized in 81־) of the above A). Ltoc is a group derived from Compound 5’־Tocopherol־CE־ Phosphoramidite which is purchased from Link Technologies Ltd.[0221] Lfarl in Table is a group described below.

Lfarl is a group deriving from Compound 53 synthesized in 82־) of the above A).[0222] Mx־n is a group described below. 133 O A.OOH NH 122-nM OHv/ NnH15-nM 51-nM49-nM 'O-t OH OO O ,ס- OH ON O wherein n is an integer of 6 to 28.M1 5 - n is a group deriving from Compound 15־n synthesized in 4) of the above A), M2 2 ־ n is a group deriving from Compound 22־n synthesized in 6) of the above A), M4 9 ־ n is a group deriving from Compound 49־n synthesized in 11) of the above A), and M5 1 - n is a group deriving from Compound 51־n synthesized in 13) of the above A).[0223]Mchoi in Table is a group described below.

Mchoi is a group deriving from Compound 55 synthesized in 14) of the above A).[0224]Mt e g - n in Table is a group described below. 134 wherein n is an integer of 6 to 28.MTEG-n is a group deriving from Compound 63־n synthesized in 15) of the above A).[0225]K2 2 ־ n in Table is a group described below. wherein n is an integer of 6 to 28.K22 -n is a group deriving from Compound 22־n synthesized in 6) of the above A).[0226]The following linker (ABuABuA in Table) is used for S155־, S184־ or S־186. id="p-227" id="p-227"

[0227]Example 2 Activity evaluation of lipid binding double־stranded oligonucleotides of the present invention using the reporter assay (Materials and Methods)HEK־BlueTM hTLR9 cells (Invivogen) used for the reporter assay were made by introducing human TLR9 gene and a reporter gene that secretory alkaline phosphatase gene bound to NF־kB• AP1־ binding region sequence in HEK293 cells, and are the cells stably expressing the both genes. 3.6x!04 of 135 the cells suspended in HEK-BlueTM Detection (Invitrogen) comprising substrates against the alkaline phosphatase were seeded on the 96 well plate, and 3 nM to 30 pM of a lipid binding double-stranded oligonucleotide of the present invention, the known CpG oligonucleotides (ODN2006, SEQ-49), or adjuvant based on design in Patent Document 1, i.e., ssCpG ODN introducing a lipid ligand (SEQ-142, 143 or 144) was added. After culturing in a 37 °C, 5 % CO2 incubator for 16 hours, absorbency at 620 nm was measured using the culture supernatant developed a color. The result value was analyzed with TIBCO Spotfire software and calculated the 50 % effective concentration (EC5 0) of each oligonucleotide.[0228](Result)As previously described, it is known that the charactericity as an adjuvant was vanished when ssCpG ODN was administered as a double- stranded DNA (dsCpG ODN) by annealing the first and second strand (Non­patent Document 4). To examine that the adjuvant design of double-stranded nucleic acid shows an activity as a TLR9 agonist, activity evaluation of a double-stranded oligonucleotide was carried out by a reporter assay.As shown in Table 28 and 29, ODN2006 (SEQ-49) which is a ssCpG ODN showed the activity as a TLR9 agonist, and the EC5 0 value was about 467 nM (Experiment 1, Table 28) and about 245 nM (Experiment 2, Table 29). On the other hand, a lipid binding double-stranded oligonucleotide of the present invention comprising ODN2006 as a CpG oligonucleotide showed TLR9 agonist activity at a range of several tens to one thousand and several hundreds nM. Most adjuvants of the present invention described in Table 28 and 29 had a tendency to enhance the activity compared to ODN2006. In addition, sequences of SEQ-57, SEQ-59, SEQ-61, SEQ-63, SEQ-65, SEQ-111, SEQ-117, SEQ-135, SEQ-146, SEQ-226, SEQ-282, SEQ-284, SEQ-294, SEQ-324, SEQ-3and SEQ-363 had a tendency to slightly lower the activity compared to ODN2006, but the difference was about 3 times. Therefore, it became clear that all lipid binding double-stranded oligonucleotides of the present invention have activity as a TLR9 agonist.Furthermore, to examine that the modification form of SEQ-2 disclosed in Patent Document 1 applied for ODN2006 which is the human type CpG 136 oligonucleotide sequence shows an activity as TLR9 agonist, activity evaluation was carried out in the same manner (SEQ-141, 142 or 143). SEQ- 142, 143 or 144 could not induce the activation of TLR9 in the measured concentration range, and it was impossible to calculate the EC5 0 values (n.d.in Table 28). That is, it is suggested that design (ssCpG ODN introducing a lipid ligand) disclosed in Patent Document 1 cannot be useful as an adjuvant of vaccine and/or vaccine itself because it showed the activity for ODN18(mouse type) but did not for ODN2006 (human type) as described in the example.In contrast, it is suggested that a lipid binding double-strandedoligonucleotide of the present invention can be useful as adjuvant of vaccine and/or vaccine itself regardless of the sequence of the CpG oligonucleotides because it showed the activity for ODN2006 as well as ODN1826.[0229]Experiment [Table 28] No. EC50(nM) No. EC50(nM) No. EC50(nM)SEQ-49 466.8 SEQ-89 300.9 SEQ-125 305.6SEQ-51 405.9 SEQ-91 68.4 SEQ-127 299.9SEQ-55 377.9 SEQ-93 106.4 SEQ-129 459.0SEQ-57 976.8 SEQ-95 33.4 SEQ-131 312.2SEQ-59 542.5 SEQ-97 ' 308.9 SEQ-133 169.7SEQ-61 608.1 SEQ-99 311.3 SEQ-135 509.8SEQ-63 598.9 SEQ-101 33.5 SEQ-137 254.7SEQ-65 500.9 SEQ-105 96.9 SEQ-139 58.4SEQ-75 300.6 SEQ-111 490.1 SEQ-141 31.0SEQ-77 296.8 SEQ-113 449.5 SEQ-146 1128.2SEQ-79 162.9 SEQ-115 303.2 SEQ-148 376.6SEQ-81 225.9 SEQ-117 616.5 SEQ-150 309.9SEQ-83 324.9 SEQ-119 436.7 SEQ-142 n.d.SEQ-85 72.7 SEQ-121 282.3 SEQ-143 n.d.SEQ-87 102.8 SEQ-123 326.0 SEQ-144 n.d. id="p-230" id="p-230"

[0230] 137 Experiment [Table 29] No. EC50(nM) No. EC50(nM) No. EC50(nM)SEQ-49 245.3 SEQ-218 59.8 SEQ-292 58.9SEQ-152 86.4 SEQ-220 57.2 SEQ-294 262.4SEQ-154 94.5 SEQ-222 31.9 SEQ-296 10.1SEQ-156 90.0 SEQ-224 12.4 SEQ-298 10.2SEQ-158 56.6 SEQ-226 362.0 SEQ-300 11.4SEQ-160 65.1 SEQ-228 21.0 SEQ-302 10.2SEQ-162 89.7 SEQ-230 39.8 SEQ-304 11.6SEQ-164 64.2 SEQ-232 72.7 SEQ-306 11.3SEQ-166 90.4 SEQ-234 27.0 SEQ-308 8.4SEQ-168 62.8 SEQ-236 203.1 SEQ-310 10.7SEQ-170--- ~v — - - 46.0 SEQ-238 131.9 SEQ-312 19.1SEQ-172 161.9 SEQ-240 62.0 SEQ-314 10.8SEQ-174 101.0 SEQ-242 102.4 SEQ-316 10.9SEQ-176 241.2 SEQ-244 101.8 SEQ-318 81.3SEQ-178 210.1 SEQ-246 102.6 SEQ-320 68.4SEQ-180 46.9 SEQ-248 108.3 SEQ-322 50.4SEQ-182 11.2 SEQ-250 92.4 SEQ-324 261.1SEQ-184 34.2 SEQ-252 87.4 SEQ-326 24.6SEQ-186 127.7 SEQ-260 39.9 SEQ-328 24.1SEQ-188 67.7 SEQ-262 25.7 SEQ-330 33.7SEQ-190 46.9 SEQ-264 86.3 SEQ-332 24.8SEQ-192 123.9 SEQ-266 58.1 SEQ-334 25.3SEQ-194 99.8 SEQ-268 11.5 SEQ-336 31.4SEQ-196 33.2 SEQ-270 16.3 SEQ-338 188.1SEQ-198 114.9 SEQ-272 33.3 SEQ-361 771.8SEQ-200 96.3 SEQ-274 29.7 SEQ-363 290.2SEQ-202 89.5 SEQ-276 30.9 SEQ-365 177.4SEQ-204 83.4 SEQ-278 33.3 SEQ-367 178.2SEQ-206 40.7 SEQ-280 168.3 SEQ-369 135.9SEQ-208 31.3 SEQ-282 275.1 SEQ-371 170.0SEQ-210 56.7 SEQ-284 255.8 SEQ-373 21.5SEQ-212 46.9 SEQ-286 45.0 SEQ-375 21.9SEQ-214 47.4 SEQ-288 65.4 SEQ-377 29.8SEQ-216 99.5 SEQ-290 151.7 SEQ-379 20.3 id="p-231" id="p-231"

[0231] 138 As shown in Table 30, even when the CpG oligonucleotides except for ODN2006 were used, most adjuvants of the present invention showed the tendency of activity enhancement compared to ssCpG ODN. It became clear that every lipid binding double-stranded oligonucleotide of the present invention shows activity as TLR9 agonist.[0232][Table 30] No. EC50(nM)SEQ-253 (K3־CpG) 2870.2SEQ-255 173.4SEQ-256 (D35־CpG) 3421046.2SEQ-258 16328.8SEQ-339 (ODN2216) 7244.7SEQ-341 7915.8. SEQ-342 (ODN684) 2339.9SEQ-344 111.4SEQ-345 (D-LS01) 6280.7SEQ-347 10.7SEQ-351 (ODN2395) 11569.8SEQ-353 1982.2SEQ-354 (ODN M362) 16893838.1SEQ-356 292.6 id="p-233" id="p-233"

[0233]Example 3 In vivo evaluation of adjuvants of the present inventionA) Evaluation of CTL inductivity(Animals)C57BL/6JJcl mice (6 to 8 weeks old) were purchased from CLEA Japan,Inc.

(Components of Vaccine 1)•OVA2 5 7-2 6 4 peptide (SIINFEKL, SEQ ID NO: 22) which synthesized and purified by reversed phase HPLC and purchased from Sigma-Aldrich.•an adjuvant of the present invention or the well-known CpG ODN 139 (Components of Vaccine 2)•TRP21 8 0-1 8 8 peptide (CSVYDFFVWL, SEQ ID NO: 23) which synthesized and purified by reversed phase HPLC and purchased from Sigma-Aldrich.•an adjuvant of the present invention or the well-known CpG ODN (Components of Vaccine 3)•OVA2 5 7-2 6 4 peptide or TRP21 80-1 8 8 peptide •Montanide ISA51 (SEPPIC) id="p-234" id="p-234"

[0234](Preparation of Vaccine 1)The specific immunity was made by vaccinating a mouse twice at 7 days intervals. Each vaccine was that 100 pg of OVA peptide, and 1.57 or 4.nmol of an adjuvant of the present invention or well-known CpG ODN, were dissolved in 1xPBS. 100 pL of the vaccine was administered subcutaneous injection on the flank of a mouse.

(Preparation of Vaccine 2)The specific immunity was induced by vaccinating a mouse twice at days intervals. Each vaccine was that 100 pg of TRP2 peptide, and 1.57 or 4.71 nmol of an adjuvant of the present invention or well-known CpG ODN, were dissolved in 1xPBS. 100 pL of the vaccine was administered subcutaneous injection on the flank of a mouse.

(Preparation of Vaccine 3)1:1 of 2 mg/ml of TRP2 peptide in PBS and Montanide (the above vaccine component 3) were mixed using a cylinder and pumping connector to form emulsion. 100 pL of the emulsion vaccine was administered subcutaneous injection on the flank of a mouse twice at 7 days intervals.[0235](Tetramer staining)On 7 days after second vaccination, the blood was collected, red blood cells were removed by BD Pharm Lyse (BD pharmingen), and the cells were suspended in FACS buffer (1% fetal bovine serum, 2 mM PBS with EDTA).Anti-mouse CD16/32 monoclonal antibody (BioXcell) was added thereto, and Fc140 receptor blocking was carried out at 4 °C for 30 minutes. Next, PE labeled H- 2Kb tetramer corresponding to the vaccine antigen (T-Select H-2Kb OVA Tetramer-SIINFEKL or T-Select H-2Kb TRP-2 Tetramer-SVYDFFVWL; Both were purchased from Medical & Biological Laboratories Co., Ltd.) and Alexa 647 labeled anti-CD8 antibody (Medical & Biological Laboratories Co., Ltd.) were added thereto, and staining was carried out at room temperature for minutes. Then, staining was carried out with DAPI (Invitrogen) and the cells were washed twice with FACS buffer. It was resuspended in FACS buffer and analyzed by FACS verse Flow Cytometer (BD bioscience) and FAC Suite software (BD bioscience). For analysis, after DAPI negative fraction was gated, white blood cells fraction was provided using forward and side-way scattering as indicators. In the white blood cells fraction, both positive cells of Alexa 647-CD8 and PE-H-2Kb Tetramer were defined as CTLs. CTL ratio in white blood cells was used for evaluation.

Calculation method of the indicator of CTL inductivityAfter calculation of the average CTL inductivity of each group per experiment, the ratio against average value of CTL inductivity of ssCpG ODN, ODN1826 (SEQ-1) or ODN2006 (SEQ-49) as a control was calculated.

In the case of an adjuvant comprising ODN1826(Average of CTL inductivity of the adjuvant group)/ (Average of CTL inductivity of the ODN1826 group) = the ratio against average value In the case of an adjuvant comprising ODN2006(Average of CTL inductivity of the adjuvant group)/ (Average of CTL inductivity of the ODN2006 group) = the ratio against average value CTL inductivity of each adjuvant was shown when CTL inductivity of ssCpG ODN was taken as 1, so the bigger value means the higher CTL inductivity.[0236]B) Evaluation of anti-tumor effect of TRP2 peptide vaccine using an adjuvant of the present invention141 After immunization by TRP2 peptide vaccine, B16F10 cells (ATTC) which is mouse melanoma expressing TRP2 protein was subcutaneously transplanted to evaluate anti-tumor effect. In a similar method to Example A), on the next day that tetramer-positive ratio in peripheral blood of mouse vaccinated twice every 7 days was observed, B16F10 cells (1x105 cells/mouse) were subcutaneously transplanted in the right shoulder. The major and minor axes of the engrafted tumor were measured every 2 to 3 days, and the tumor volume was calculated by the formula:(major axis x minor axis2)/2 Calculation method for indicators of anti-tumor effect For the adjuvant comprising ODN1826100-(average tumor volume of adjuvant group)/ (average tumor volume of ODN1826 group) For the adjuvant comprising ODN2006100-(average tumor volume of adjuvant group)/ (average tumor volume of ODN2006 group) The compound with bigger value has the stronger anti-tumor activity.[0237](Result using ODN1826 as a CpG oligonucleotide) Result 1-A: Comparison of CTL inductivity between an adjuvant of the present invention and the known adjuvantsWhen mice were immunized with TRP2 peptide and ODN1826 (SEQ-1), TRP2-specific CTL was induced. When dsCpG ODN (SEQ-4) was used, TRP2- specific CTL inductivity was lowered at 0.46 times compared to ODN1826.That is, double strands led to lower the CTL inductivity compared to a single­strand adjuvant. This suggested that when the CpG oligonucleotide is just made as a double-stranded form, the immunostimulatory activity is lowered as disclosed in Non-patent Document 4 and the like. On the other hand, when an adjuvant of the present invention, i.e., lipid binding double-stranded oligonucleotide adjuvant (SEQ-16) was used for immunization, the CTL inductivity was enhanced at 1.46 times compared to ODN1826. Therefore, it 142 suggested that an adjuvant of the present invention has unexpected enhancement of activity. The results were shown in Table 31. [0238][Table 31] Peptide Adjuvant dose/head nmol Ratio of average value of antigen־specific CTL TRP2 SEQ-1 1.57 1.00 TRP2 SEQ-4 1.57 0.46 TRP2 SEQ-16 1.57 1.46 id="p-239" id="p-239"

[0239]Result 1-B: Comparison of anti-tumor effect between an adjuvant of the present invention and the known adjuvantsWhen mice were immunized with TRP2 peptide and ODN1826 (SEQ-1), they had little effect of inhibition on tumor progression of B16F10 cells. Furthermore, dsCpG ODN (SEQ-4) had little effect of inhibition on tumor progression of B16F10 cells. On the other hand, an adjuvant of the present invention (SEQ-16) showed the strong effect of inhibition on tumor progression of B16F10 cells, 43% compared to ODN1826 on Day 14 after transplantation. The results were shown in Figure 1.[0240]Result 2-A, B: CTL inductivity and anti-tumor effect of adjuvants of the present invention comprising a lipid ligand with different number of carbon atomsThe activity of an adjuvant of the present invention having a lipid comprising two acyl chains comprising 10 to 20 carbon atoms as a ligand was examined with OVA peptide. CTL inductivity and tumor inhibition rate of SEQ-12 (10 carbon atoms) was lowered compared to ODN 1826 (SEQ-1). On the other hand, there is tendency to enhance CTL inductivity as the chain length is longer like SEQ-14 (14 carbon atoms), SEQ-6 (18 carbon atoms) and SEQ-16 (20 carbon atoms). In addition, tumor inhibition rate of SEQ-showed about 94 % inhibition of the tumor growth compared to ODN1826 on Day 14 after transplantation, and SEQ-6 and SEQ-16 perfectly inhibited the tumor growth on Day 14 after transplantation. The results were shown in Table 32.143 id="p-241" id="p-241"

[0241] [Table 32] Peptide Adjuvant dose/head nmol Ratio of average value of antigen־specific CTL Tumor inhibition rate (Day 14 after transplantation) OVA SEQ-1 1.57 1.00 0.00 OVA SEQ-12 1.57 0.42 -62.88 OVA SEQ-14 1.57 0.85 93.94 OVA SEQ-6 1.57 0.77 100.00 OVA SEQ-16 1.57 2.32 100.00 id="p-242" id="p-242"

[0242]Result 3-A: CTL inductivity of adjuvants of the present invention introduced lipids at the both endsThe activity of an adjuvant of the present invention with a lipid comprising two acyl chains comprising 20 carbon atoms as a ligand and further comprising a single-strand lipid comprising 8 or 12 carbon atoms at 3’ end of the CpG oligonucleotide (SEQ-46 or SEQ-48) was examined with TRP2 peptide. The both CTL inductivities were enhanced at about 2.8 times compared to ODN1826 (SEQ-1). The results were shown in Table 33.[0243][Table 33] Peptide Adjuvant dose/head nmol Ratio of average value of antigen-specific CTL TRP2 SEQ-1 1.57 1.00 TRP2 SEQ-46 1.57 2.81 TRP2 SEQ-48 1.57 2.84 id="p-244" id="p-244"

[0244]Result 3-B: Anticancer effect of adjuvants of the present invention introduced lipids at the both endsWhen mice were immunized with TRP2 peptide and an adjuvant of the present invention, SEQ-46 or SEQ-48, it showed the strong effect of inhibition on tumor progression of B16F10 cells, respectively 52% or 43% on Day 13 after transplantation compared to ODN1826 (SEQ-1). The results were shown in Figure 2. 144 id="p-245" id="p-245"

[0245]Result 4-A: CTL inductivity of adjuvants of the present invention with the different lengths of the complementary strandIn a double-stranded oligonucleotide adjuvant design, it is necessary to release the CpG oligonucleotide, which is an active ingredient, in the lymph nodes. The speed to dissociate a double strand is proportionate to the heat stability, and the more complementary sites in the double strand structure are, the more stably the double strand exists. Then, the activity of adjuvants of the present invention whose chain length of complementary strand is different was examined with TRP2 peptide.The adjuvant whose complementary strand is 10 mer for ODN1826 (20 mer), i.e., length of the complementary strand for the CpG oligonucleotide is 50 % (SEQ-8) and the adjuvant whose complementary strand if 15 mer, i.e., length is % (SEQ-10) were used. Both SEQ-8 and SEQ-10 dominantly enhanced CTL inductivity compared to ODN1826 (SEQ-1) at about 1.2 and 2.5 times, respectively. The results were shown in Table 34.[0246][Table 34] Peptide Adjuvant dose/head nmol Ratio of average value of antigen-specific CTL TRP2 SEQ-1 1.57 1.00 TRP2 SEQ-2 1.57 1.68 TRP2 SEQ-8 1.57 1.15 TRP2 SEQ-10 1.57 2.50 id="p-247" id="p-247"

[0247]Result 4-B: Anticancer effect of adjuvants of the present invention with the different lengths of the complementary strandWhen mice were immunized with TRP2 peptide and ssCpG ODN introducing a lipid ligand in Patent Document 1 (SEQ-2), the tumor inhibition rate was about 3.5 % on Day 12 after transplantation compared to ODN18(SEQ-1). On the other hand, SEQ-8 which is an adjuvant of the present invention with 10 mer of complementary strand showed about 50 % of the tumor inhibition rate compared to ODN1826 on Day 12 after transplantation. Furthermore, SEQ-10 which has 15 mer of complementary strand showed the 145 strong effect of inhibition on tumor progression of B16F10 cells, about 76 % compared to ODN1826 on Day 12 after transplantation. The results were shown in Figure 3.[0248]Result 5-A: CTL inductivity of adjuvants of the present invention with the different chain length of complementary strandThe activity of an adjuvant of the present invention whose chain length of complementary strand is from 10 to 20 mer against ODN1826 (20 mer), i.e., complementary strand is 50 to 100 % against the CpG oligonucleotide was examined with TRP2 peptide. The adjuvant whose complementary strand is mer (SEQ-28), 15 mer (SEQ-26), 17 mer (SEQ-22), 19 mer (SEQ-18) and mer (SEQ-16) showed enhancement of CTL inductivity compared to ODN18(SEQ-1). Especially, adjuvant with 19 mer (SEQ-18), 17 mer (SEQ-22) and 15mer (SEQ-26) showed CTL inductivity at about 3 times compared to ODN1826 (SEQ-1). The results were shown in Table 35.[0249][Table 35] Peptide Adjuvant dose/head nmol Ratio of average value of antigen־ specific CTL TRP2 SEQ-1 1.57 1.00 TRP2 SEQ-16 1.57 1.63 TRP2 SEQ-18 1.57 3.40 TRP2 SEQ-22 1.57 2.69 TRP2 SEQ-26 1.57 2.82 TRP2 SEQ-28 1.57 2.21 TRP2 SEQ-32 1.57 1.10 TRP2 SEQ-36 1.57 1.07 id="p-250" id="p-250"

[0250]Result 6-A: Comparison of CTL inductivity with MontanideThe activities of ODN1826 (SEQ-1), ssCpG ODN introducing a lipid ligand (SEQ-2), an adjuvant of the present invention (SEQ-26) and Montanide which is an adjuvant used a lot in the clinical trials as an adjuvant of a peptide vaccine were compared with TRP2 peptide as antigen. SEQ-2 and SEQ-showed higher CTL inductivity than ODN1826. On the other hand, CTL inductivity of Montanide was lowered to one-seventh compared to ODN1826. 146 The results were shown in Table 36. [0251][Table 36] Peptide Adjuvant dose/head nmol Ratio of average value of antigen-specific CTL TRP2 SEQ-1 1.57 1.00 TRP2 SEQ-2 1.57 3.23 TRP2 SEQ-26 1.57 2.82 TRP2 Mondanide 100 pL 0.13 id="p-252" id="p-252"

[0252]Result 6-B: Comparison of the anti-tumor effect against ssCpG ODN introducing a lipid ligandWhen mice were immunized with TRP2 peptide and an adjuvant of the present invention, SEQ-26, it showed the very strong effect of inhibition on tumor progression of B16F10 cells, 76.8% on Day 12 after transplantation compared to ODN1826 (SEQ-1). The tumor inhibition rate of ssCpG ODN introducing a lipid ligand (SEQ-2) was 64.3% on Day 12 after transplantation compared to ODN1826. That is, an adjuvant of the present invention showed the stronger effect of inhibition on tumor progression of B16F10 cells than the adjuvant of SEQ-2. On the other hand, Montanide used as an adjuvant has no anti-tumor effect. The results were shown in Figure 4.[0253]Result 7-A: CTL inductivity of adjuvants of the present invention with the different kinds of nucleic acid monomer in a complementary strandActivity of an adjuvant of the present invention whose nucleic acid monomers in the complementary strand were RNA, 2’-OMe-RNA or 2’-F-RNA was examined with TRP2 peptide. The adjuvant that all of the nucleic acid monomers were RNA (SEQ-40) or 2’-F-RNA (SEQ-42) did not show big enhancement of CTL inductivity compared to ODN1826 (SEQ-1). On the other hand, the adjuvant with 2’-OMe-RNA as a nucleic acid monomer (SEQ-38 and SEQ-44) showed enhancement of CTL inductivity at 2.9 or 2.4 times respectively compared to ODN1826. These results suggested that 2’־OMe־ RNA is useful as nucleic acid monomer of an adjuvant of the present invention as well as DNA. The results were shown in Table 37. 147 id="p-254" id="p-254"

[0254] [Table 37] Peptide Adjuvant dose/head nmol Ratio of average value of antigen-specific CTL Tumor inhibition rate (Day 14 after transplantation) TRP2 SEQ-1 1.57 1.00 0.00 TRP2 SEQ-38 1.57 2.85 86.19 TRP2 SEQ-40 1.57 1.24 3.73 TRP2 SEQ-42 1.57 0.99 57.33 TRP2 SEQ-44 1.57 2.39 57.51 TRP2 SEQ-2 1.57 3.11 72.64 id="p-255" id="p-255"

[0255]Result 7-B: Anti-tumor effect of adjuvants of the present invention with the different kinds of nucleic acid monomer in a complementary strandWhen mice were immunized with TRP2 peptide and an adjuvant of the present invention with 2’-OMe-RNA as nucleic acid monomers (SEQ-38), it showed the very strong effect of inhibition on tumor progression of B16Fcells, about 86.2 % on Day 14 after transplantation compared to ODN18(SEQ-1). The tumor inhibition rate of ssCpG ODN introducing a lipid ligand (SEQ-2) was about 72.6 % on Day 14 after transplantation compared to ODN1826. That is, an adjuvant of the present invention showed the stronger effect of inhibition on tumor progression of B16F10 cells than the adjuvant of SEQ-2. In addition, the adjuvant whose chain length of SEQ-38 was shorten (SEQ-44) and the adjuvant with 2’-F-RNA as a nucleic acid monomer (SEQ-42) showed the effect of inhibition on tumor progression in B16F10 cells. On the other hand, the adjuvant with RNA as a nucleic acid monomer (SEQ-40) showed no effect of inhibition on tumor progression of B16F10 cells. The results were shown in Table 37 and Figure 5.[0256](Result using ODN2006 as a CpG oligonucleotide)Result 8-A: CTL inductivity of adjuvants of the present invention with the different chain length of the complementary strand or a linkerThe activities of ODN2006 (SEQ-49, 24 mer) and adjuvants of the present invention were compared with TRP2 peptide as antigen. The adjuvant whose chain length of the complementary strand is 24 mer, i.e., the 148 length of the complementary strand against the CpG oligonucleotide is 100 % (SEQ-51) and the adjuvant whose chain length of the complementary strand is mer, i.e., the length of the complementary strand against the CpG oligonucleotide is 62. 5 % (SEQ-61) were used. Both of SEQ-51 and SEQ-showed high CTL inductivity at more than 5 times compared to ODN2006. Furthermore, the adjuvant with dTdT as a linker (SEQ-63) or dGdG (SEQ-65) was used. Both SEQ-63 and SEQ-65 showed very high CTL inductivity at about 10 times compared to ODN2006. The results were shown in Table 38. [0257][Table 38] Peptide Adjuvant dose/head nmol Ratio of average value of antigen-specific CTL TRP2 SEQ-49 4.71 1.00 TRP2 SEQ51־ 4.71 5.82 TRP2 SEQ-61 4.71 5.73 TRP2 SEQ-63 4.71 11.73 TRP2 SEQ-65 4.71 9.21 id="p-258" id="p-258"

[0258]Result 8-B: Anti-tumor effect of an adjuvant of the present invention with the different chain length of a complementary strand or a linkerWhen mice were immunized with TRP2 peptide and ODN2006 (SEQ-49), it showed little effect of inhibition on tumor progression of B16F10 cells. On the other hand, an adjuvant of the present invention whose length of the complementary strand is 100 % against the CpG oligonucleotide (SEQ-51) showed 20 % of the effect of inhibition on tumor progression of B16F10 cells on Day 10 after transplantation compared to ODN2006. In addition, each the adjuvant whose length of the complementary strand is 62.5 % against the CpG oligonucleotide (SEQ-61), and the adjuvant with dTdT as a linker (SEQ-63) or dGdG (SEQ-65) showed about 50 % of the effect of inhibition on tumor progression of B16F10 cells on Day 10 after transplantation compared to ODN2006. The results were shown in Figure 6.[0259]Result 9-A: CTL inductivity of adjuvants of the present invention whose nucleic acid monomer in the complementary strand is 2’-OMe-RNA 149 Activity of an adjuvant of the present invention whose nucleic acid monomer in the complementary strand is 2’-OMe-RNA was examined with TRP2. The chain length of the complementary strand is 24 mer, i.e., the adjuvant whose length of the complementary strand against the CpG oligonucleotides was 100 % (SEQ-67) and, the adjuvant whose length of the complementary strand is 15 mer, i.e., the adjuvant whose length of the complementary strand against the CpG oligonucleotide is 62.5 % (SEQ-69) were used. SEQ-67 and SEQ-69 showed CTL inductivity at 1.4 or 1.2 times respectively compared to ODN2006 (SEQ-49). The results were shown in Table 39.[0260][Table 39] Peptide Adjuvant dose/head nmol Ratio of average value of antigen־specific CTL TRP2 SEQ-49 4.71 1.00 TRP2 SEQ-67 4.71 1.39 TRP2 SEQ-69 4.71 1.21 id="p-261" id="p-261"

[0261]Result 9-B: Anti-tumor effect of adjuvants of the present invention whose nucleic acid monomer in the complementary strand is 2’-OMe-RNAWhen mice were immunized with TRP2 peptide and an adjuvant of the present invention which has 2’-OMe-RNA as a nucleic acid monomer, adjuvant whose length of complementary strand against the CpG oligonucleotides is 1% (SEQ-67) showed very strong effect of inhibition on tumor progression of B16F10 cells, about 75 % on Day 10 after transplantation compared to ODN2006 (SEQ-49). The adjuvant whose length of the complementary strand against CpG oligonucleotides is 62. 5 % (SEQ-69) showed about 20 % of the effect of inhibition on tumor progression of B16F10 cells on Day 10 after transplantation compared to ODN2006. The results were shown in Figure 7.These suggested that 2’-OMe-RNA was useful also for ODN2006 as nucleic acid monomer for an adjuvant of the present invention as well as DNA. [0262]In similar methods, CTL inductivity or anti-tumor effect of adjuvants of 150 the present invention was measured. Results are shown in Tables 40 to and Figures 8 to 13 separately by experiments. For these experiments, TRPpeptide was used and the dosage of the adjuvant of the present invention was 4.71 nmol. NT in tables means "not tested".[0263][Table 40] AdjuvantRatio of average value of antigen־specific CTLSEQ-49 1.00SEQ-59 5.34SEQ-57 4.28SEQ55־ 3.02SEQ-53 2.86 id="p-264" id="p-264"

[0264][Table 41] AdjuvantRatio of average value of antigen-specific CTL Tumor inhibition rate (Day 12 after transplantation)SEQ-49 1.00 0.00SEQ-119 2.05 55.1SEQ-192 1.74 41.5 id="p-265" id="p-265"

[0265] [Table 42] AdjuvantRatio of average value of antigen-specific CTL Tumor inhibition rate (Day 12 after transplantation)SEQ-49 1.00 0.00SEQ-121 3.99 56.8 id="p-266" id="p-266"

[0266] 151 [Table 43] Adjuvant Ratio of average value of antigen-specific CTL Tumor inhibition rate (Day 14 after transplantation) SEQ-49 1.00 0.00 SEQ-152 1.17 80.45 SEQ-154 2.47 NT SEQ-158 3.06 100 id="p-267" id="p-267"

[0267][Table 44] Adjuvant Ratio of average value of antigen־specific CTL SEQ-49 1.00 SEQ-182 2.24 SEQ-184 2.38 id="p-268" id="p-268"

[0268] [Table 45] Adjuvant Ratio of average value of antigen־specific CTL Tumor inhibition rate (Day 14 after transplantation) SEQ-49 1.00 0.00 SEQ-186 2.37 NT SEQ-188 1.49 51.03 SEQ-190 1.95 NT SEQ-192 3.61 46.16 SEQ-194 2.48 55.02 SEQ-196 3.26 NT id="p-269" id="p-269"

[0269] 152 [Table 46] Adjuvant Ratio of average value of antigen־specific CTL Tumor inhibition rate (Day 14 after transplantation) SEQ-49 1.00 0.00 SEQ-156 1.70 NT SEQ-160 3.65 NT SEQ-162 3.44 NT SEQ-164 4.88 53.69 id="p-270" id="p-270"

[0270][Table 47] Adjuvant Ratio of average value of antigen־specific CTL SEQ-49 1.00 SEQ-166 2.57 SEQ-168 1.96 id="p-271" id="p-271"

[0271] [Table 48] Adjuvant Ratio of average value of antigen־specific CTL Tumor inhibition rate (Day 14 after transplantation) SEQ-49 1.00 0.00 SEQ-170 3.10 79.09 SEQ-172 1.24 NT SEQ-174 1.78 NT id="p-272" id="p-272"

[0272] 153 [Table 49] AdjuvantRatio of average value of antigen־specific CTLSEQ-49 1.00SEQ-176 1.97SEQ-178 5.44SEQ-180 3.59SEQ-206 1.07SEQ-208 1.28SEQ-214 1.38SEQ-216 1.95 id="p-273" id="p-273"

[0273][Table 50] AdjuvantRatio of average value of antigen־ specific CTLSEQ-49 1.00SEQ-198 3.12SEQ-200 3.24SEQ-204 3.98 id="p-274" id="p-274"

[0274] [Table 51] AdjuvantRatio of average value of antigen־specific CTLSEQ-49 1.00SEQ-226 1.84SEQ-228 2.10SEQ-230 3.10SEQ-232 1.28SEQ-234 1.96 id="p-275" id="p-275"

[0275] 154 [Table 52] AdjuvantRatio of average value of antigen־specific CTLSEQ-49 1.00SEQ-218 2.87SEQ-220 1.68SEQ-222 3.11SEQ-224 2.25 id="p-276" id="p-276"

[0276][Table 53] AdjuvantRatio of average value of antigen־specific CTLSEQ-49 1.00SEQ-236 2.66 id="p-277" id="p-277"

[0277] [Table 54] AdjuvantRatio of average value of antigen-specific CTLSEQ-49 1.00SEQ-238 1.09SEQ-240 1.45SEQ-242 1.62SEQ-244 3.24 id="p-278" id="p-278"

[0278] [Table 55] AdjuvantRatio of average value of antigen־specific CTLSEQ-49 1.00SEQ-246 2.51SEQ-248 1.67SEQ-250 1.86SEQ-252 1.14 155 id="p-279" id="p-279"

[0279] [Table 56] AdjuvantRatio of average value of antigen־ specific CTLSEQ-256 (D35־CpG)1.00SEQ-258 2.46 id="p-280" id="p-280"

[0280][Table 57] AdjuvantRatio of average value of antigen-specific CTLSEQ-49 1.00SEQ-260 1.89SEQ-262 1.45SEQ-264 3.44SEQ-266 2.51 id="p-281" id="p-281"

[0281][Table 58] AdjuvantRatio of average value of antigen־specific CTLSEQ-49 1.00SEQ-268 2.09SEQ-270 2.31SEQ-272 1.77SEQ-274 4.03SEQ-276 3.77SEQ-278 2.58 id="p-282" id="p-282"

[0282] 156 [Table 59] AdjuvantRatio of average value of antigen־specific CTLSEQ-49 1.00SEQ-280 5.27SEQ-282 2.82SEQ-284 5.57SEQ-286 2.40 id="p-283" id="p-283"

[0283][Table 60] AdjuvantRatio of average value of antigen־specific CTL SEQ-49 1.00SEQ-288 4.22SEQ-290 2.80SEQ-292 4.63SEQ-294 5.30 id="p-284" id="p-284"

[0284] [Table 61] AdjuvantRatio of average value of antigen־specific CTLSEQ-49 1.00SEQ-296 2.43SEQ-298 2.08SEQ-300 3.48SEQ-302 2.58SEQ-304 2.42SEQ-306 3.04 id="p-285" id="p-285"

[0285] 157 [Table 62] AdjuvantRatio of average value of antigen־specific CTLSEQ-49 1.00SEQ-308 2.52SEQ-310 4.91SEQ-312 1.75SEQ-314 2.58SEQ-316 1.30 id="p-286" id="p-286"

[0286][Table 63] AdjuvantRatio of average value of antigen־specific CTLSEQ-49 1.00SEQ-318 4.19SEQ-320 3.81SEQ-322 2.15SEQ-324 1.92 id="p-287" id="p-287"

[0287] [Table 64] AdjuvantRatio of average value of antigen-specific CTLSEQ-49 1.00SEQ-326 4.27SEQ-328 2.93SEQ-330 2.53SEQ-332 2.98SEQ-334 1.66SEQ-336 3.62SEQ-338 1.72 id="p-288" id="p-288"

[0288] 158 [Table 65] AdjuvantRatio of average value of antigen־specific CTLSEQ339־(ODN2216)1.00SEQ-341 1.25SEQ342־(ODN684)1.00SEQ-344 1.20SEQ345־(D־LS01)1.00SEQ-347 2.73 id="p-289" id="p-289"

[0289][Table 66] AdjuvantRatio of average value of antigen־specific CTLSEQ354־(ODN M362)1.00SEQ-356 1.91 id="p-290" id="p-290"

[0290][Table 67] AdjuvantRatio of average value of antigen־specific CTLSEQ-49 1.00SEQ-373 2.21SEQ-375 2.58SEQ-377 1.57 id="p-291" id="p-291"

[0291] [Table 68] AdjuvantRatio of average value of antigen-specific CTLSEQ-49 1.00SEQ-361 1.09SEQ-365 2.04SEQ-369 1.70SEQ-371 1.11 15159 id="p-292" id="p-292"

[0292]Example 4Antigen-specific CTL inductivity and anti-tumor effect of ODN20(SEQ-49) and SEQ-121 in the absence of a tumor antigen peptide were evaluated. In similar methods of A) and B) of Example 3, the effect of vaccine without a tumor antigen peptide was verified. Both of the antigen-specific CTL values of SEQ-49 and SEQ-121 were undetectable. On the other hand, SEQ-121 showed 85.3 % of the effect of inhibition on tumor progression of B16F10 cells on Day 12 after transplantation compared to ODN2006. From these results, SEQ-121 was expected to apply to therapeutic vaccine with anti­tumor effect even it is a single agent (Table 69, Figure 14).[0293][Table 69] AdjuvantRatio of average value of antigen־ specific CTL Tumor inhibition rate (Day 12 after transplantation)SEQ-49 Undetectable 0.00SEQ-121 Undetectable 85.3 id="p-294" id="p-294"

[0294]Example 5 Irritation test in a single dose of adjuvants of the present invention A) Subcutaneous irritation test in a single doseSubcutaneous irritation of a compound of the present invention was evaluated by gross examination and histopathological examination about the skin at necropsy one day and one or four week(s) after subcutaneous administration in a single dose.The compound of the present invention was subcutaneously administered to the center of back between the shoulder blades of rats. At necropsy one day, one or four week(s) after administration (isoflurane anesthesia, euthanasia by bleeding), skin at the administration site was collected, grossly observed and fixed in 10 % neutral buffered formalin solution. The fixed skin was cut out, embedded and put in a thin slice by the usual methods, and Hematoxylin-eosin (HE) stained sections were made. The pathological observers were observed the HE stained sections by an optical 160 microscope and recorded the histopathological findings. The changes in pathological findings were evaluated in four grades of Minimal; ±, Mild; +, Moderate; 2+ and Marked; 3+. When any grade was not appropriate for the finding, it was recorded as Positive.[0295](Result)To verify the safety of adjuvants of the present invention as a medicine, the subcutaneous irritation was compared to that of Montanide used in clinical trials as an adjuvant, Amph1826 (SEQ-2) or ODN2006 (SEQ-49) which is the well-known adjuvant.Montanide is an adjuvant consisting of mineral oil and surfactant, and used in clinical trials as an adjuvant of peptide vaccine by preparation of emulsion with aqueous formulation. Although it is an adjuvant with excellent safety profile about systemic safety concerns, there is a problem about the occurrence of skin induration caused by remaining emulsion in the administration site. In this experiment, the administration site reaction of the double-stranded oligonucleotide was compared to that of Montanide, and it was examined whether it could be an adjuvant with week local irritation.Rat subcutaneous irritation tests in a single dose were carried out for Montanide, SEQ-2, SEQ-49 or an adjuvant of the present invention (SEQ-61, SEQ-119, SEQ-121, SEQ-170, SEQ-192 or SEQ-216). Montanide was administered at 1 mL/site in emulsion of Montanide: saline (1:1). SEQ-2, SEQ-49 or an adjuvant of the present invention was dissolved in phosphate buffered saline and administered at 4.71, 15.7 or 47.1 nmol/1mL/site, respectively.As a result, it became clear that Montanide was not absorpted/decomposed/eliminated because white accumulation or white nodules was grossly observed under the skin and cyst-like structures was histologically confirmed one day, one and four week(s) after administration. Montanide remaining under the skin continued eliciting inflammation from one day to four weeks after administration. One day after administration, neutrophil infiltration and edema were observed. One week after administration, neutrophil infiltration and edema continued, but the inflammation became slightly chronic to observe as granulomatous inflammation. Four weeks after161 administration, thick fibrous capsule encapsulated Montanide was formed. Inflammation was slightly reduced compared to one week after administration, but even four weeks after administration, the animals with continuous neutrophil infiltration or edema were observed.In the case of SEQ-2, inflammation cellular infiltration, edema and crust were observed one day and one week after administration. Even four weeks after administration, skin inflammation was not recovered, and granulomatous inflammation by continuous activation of monocyte/macrophage system was observed.In the case of SEQ-49, and SEQ-61, SEQ-119, SEQ-121, SEQ-170, SEQ- 192 and SEQ-216, which is an adjuvant of the present invention, acute inflammation, mainly neutrophil infiltration, was observed under the skin one day after administration. One week after administration, inflammation, mainly lymphocyte and macrophage, was observed from subcutaneous to dermis tissue, and four weeks after administration, inflammation was almost recovered. Regarding SEQ-119, SEQ-121 and SEQ-170, subcutaneous irritation evaluation four weeks after administration was not carried out, but when their subcutaneous irritation one day and one week after administration were compared to those of SEQ-119, SEQ-121, SEQ-170 and SEQ-61, the irritation were equal to or weaker than SEQ-61. These suggested that regarding SEQ-119, SEQ-121 and SEQ-170, inflammation was almost completely recovered four weeks after administration.Tables 70 to 72 showed the results one day after administration, Tables to 75 showed the results one week after administration, and Tables 76 and showed the results four weeks after administration. The notes in the tables are as below.1) Values after findings are numbers of the observed animals.2) Values after findings are scores. The scores were calculated the total, provided that grades of each individual were put as Minimal; 1, Mild; 2 and Moderate; 3.[0296]The above showed that compared between adjuvants of the present invention and Montanide, adjuvants of the present invention did not cause the accumulation of the compounds under the skin (low risk of induration),162 necrosis or neutrophil infiltration was not occurred even one week after administration, and inflammation was almost completely recovered four weeks after administration. Therefore, it was concluded that subcutaneous irritation of the adjuvants of the present invention was weaker than that of Montanide. Also, compared between adjuvants of the present invention and Amph1826 (SEQ-2), inflammation caused by adjuvants of the present invention was almost completely recovered four weeks after administration, but on the other hand skin inflammation caused by SEQ-2 was not recovered, and granulomatous inflammation by continuous activation of monocyte/macrophage system was observed even four weeks after administration. Therefore, it was concluded that subcutaneous irritation of the adjuvants of the present invention was weaker than that of SEQ-2. Subcutaneous irritation of adjuvants of the present invention was no significant difference from that of ODN2006 (SEQ-49).[0297] 163 [Table 70] 47.1CO/ 0o o CO in-o in CO SEQ-6115.7CO/ 0o o CD CO o o in CM 4.71CO/ 0o o ID CsJ-o CO SEQ-4947.1CO/ 0ok kCD>.inkCMkokinkok .7CO/ 0o o CO in o CO 4.71CO/ 0o o CO CM o CM MontanideCO/ 0CO k o CO L CO k CNJ k CO k o k o k Compound nameDose (nmol/site)Number of animalsNumber of deaths/imminent dissectionsSubcutaneous:White accumulationSubcutaneous:Red spots. ErythemaSubcutaneous:• • • 2 )Neutrophilic infiltration2)Subcutaneous: Edema2)Subcutaneous:Bleeding 'Subcutaneous:Cvst-like structure1Muscle layer:2)Neutrophilic infiltration '2)Muscle laver: Edema ^(/)Ok.O Histopathological AdministrationsitePathology 164[0298] [Table 71] SEQ-2 1 47.1 1CO| 0 /0 cokcokCOk k kcokcokי^־kOh 15.7 CO 0 /0 COkinkcok kCMk סל kח־־יkcokOk 4.71 CO 0 /0 חייkcokok k kin י*k kOk S E Q -216 47.1 CO 0 /0 ink־י*־k kokokCOkCMk k k 15.7 CO 0 /0 CO o CO CO CM o 4.71 CO 0 /0 CO in COk k.CM CO CM o SEQ-192 47.1 CO 0 /0 in CM co ־ M ־ CO o .7 CO 0 /0 in coי- oי-co CO CM o 4.71 CO 0 /0 CO r^־ CMי-CM co CM-o Compound name Dose (nmol/site) Number of animals Number of deaths/imminent dissections Inflammatory cell • • 2) infiltration 'CO E o דכLLI egUCס 1<ע ס םס eg.<׳>(/> o V­oa>Z Cyst-like structure1 Inflammatory cell • . 2) infiltration ' Inflammatory cell • 2) infiltration 'egCO E eg(/>'(/>OV­O o Z Subcutaneous tissue Dermis Muscle layer 2 ) Histopathological Administration site Pathology id="p-299" id="p-299"

[0299] 165 [Table 72] T— o r*•' CO CM o CO CO CO יגיג־ o סנ— וז־ o in CO O O יג סר CM CM o LUk k k k kC0y— o CO 1— o CO ao T— T—יג■ o k k k k k k— ד o ‘ ח*• CO o CM CO r* in COיג־ o ס L k b. k kr-ך־ r־^ o ססר CO T— CM CO in o 00 o LUk k k k k kC0 o CO T— CO CO T— coיג־ o k k b. k k— ד o r-' CO o o CO in CO COיג־ o T"CMי־־ r- o uo CO o o in CO יג inס o JJk k(/)y— o CO o o uo סנ T— CMיג■ o "© "© "©o o o S'- L S'״O eg o O eg c +־׳ C -׳ 4 4-* CO _ © o © oיה £O'!© E £ '•£ -׳ 4 £ E 2 E 2 £ 2 03 © © ±i־סC C LU c c c c (0כ V.©©£©ם ­! >>£ס ­.סcD c<סי ­ ו ­© כ כ ס w •ם 0 < כ £c© _©©o(0כH LU C0 ■£ a 2eg©Oס) ­CO ס c _o o -C-׳ 4o ©© a /י 1co .■eE coס/—s 03 -C— E o ■t־׳E -׳ 4 £־ס©Cco c ©־ס<ס ­ o a- ־ 4 >ם£O oכ k_ o ס © o a £ Si -QF.0FJO■*־׳ס o 3 ־ 3 ־ ©ס o z z Q. id="p-300" id="p-300"

[0300] 166 [Table 73] y—oCO o o o O r- ס CD O־ 3 יok k. k k. k k k k CDIr־*oo in CO o o o o CO ס in oUJ— ייoCOk. k- koCO o V— o o CM ס oי^־o k k k- k. koco o o o o 00 ס uo oo».k k kTj־ooUJin CO soo- י o o CD ס CD oCO k- k- k- koco o T— o o CO ס o־*r oL. h k. k Lk4>דכ c o1 CO CO O CM CD o in coco o L 1. k k kCMcס-׳ 4 S CMc E מCOok- o -׳ 4 'מ c E 03 E~ o a>B o pPcסג־סk.-׳ 4ZUJ .. .. ..i3* • co.. c a 2 CO co .E ־= ״> co כ z כ i 3 a. o ״ כ ^ כ ס כ 3 o — o o ° o .E ס o oV סג CO סג — סג ~ סג <ס סנ סג סג 2 C c S c J= 2 § c ® 5 > 2 1 c c & 2 * 3 * -Q - סג ס ­ כ o JO 3 s O jo כ 3 § JO c 3 § -Q03 O jQ 3i _Q - כ 3 3 > co 3 UJ ט z ט 2 ט ט ט ט c ט «5 o CO 00 c o o o a o CO-5 0) (0 o Q. CO סג דכ ט X — -י 4 c סג C ס c -י 4 b סג b E ti .־ co CO ״י c סג E N ✓־־ סג CO E CO 03 E דכ ־ 1 < oa c סג c 03 ־ס ס ­ o > ם E c O k_ ס k_ םס ס o סג סג ס a. E סג CO _Q E _Q E o o 3 3Q_ O O z z 167 id="p-301" id="p-301"

[0301] [Table 74] oCO o ס r— o o 00 CO o co a> COok k k k k. k k k k. kCMוייי* oLOCO o ס T—o O 00 O T— co ־^־ID oס/נk k k k k k k k k k k— זoCO o ס O o o CO o ook k k k k k k k k ky—oCO o T— o CM o LO CM CMT—COocok k k k L k. k k k k kCMooLO CO oo o" י " יO CO CM o CM LOLUk k k k L k. k k k ky—or^■CO o o o o o COy— O CM o ook k k k k k k k k k ky—oCO o o oy—o r- CM co CM CO ־ 1 ־* oCM k k k k L י■ ויייי oLO CO s o o o T— o CO COy— T—LO LO O o ID k k k k k T—o r- CO o o o O o Tf CO o o CM o ־י*־ o k k k k k "׳־י* Gk_ ״G ■*־׳ ־G ״G CO O o O O c 5CMo c .2 O <י* כ V. L O 1 .= 1 !ש !ש (שס O .2 £.2 LD z o •S .2 c c ס V. G ע 0 ( ■*־׳ o & 0 ( כ o G k. G > <ס E ־ס G •״_ <ס V, C (ס ■*־׳ כ coJ2GGe .־ b ס E Ok. G D .ס k_-C o ־ס k- כ G DQ LD o 00 Q 2o« גCO ה ­cס _o O־׳ 4O-C■*־׳G סרCO(0CO<0כס ­י ­ ■*cG o k.o■*־׳CO if S rc E .■*־׳ E <סG t i _CO(0joס•. -- ׳ <ס JZ c E ס E ■*־׳Q E CTJco C G ־סC Q ס ­ 168 id="p-302" id="p-302"

[0302] 169 [Table 75] SEQ -119 47.1 CO/ 0CSJkokCOkCO Tfk. .7 CO/ 0o o ־י!־ CM CM 4.71 CO/ 0kokk k. or־~■ T LUסר 47.1 CO/ 0o CM 00 in CO .7 CO/ 0 CM CO o in 4.71 CO/ 0k kk kk SEQ-121 47.1 CO/ 0o o CO CO CO .7 CO/ 0okokin TT in 4.71 CO/ 0o o in - CM Compound name Dose (nm ol/site) Number of animals Number of deaths/imminent dissections Turbidity Erythema Inflammatory cell infiltration2 > Inflammatory cell infiltration2 Inflammatory cell • 2) infiltration Subcutaneous Dermis Muscle layer (/) o k. O Histopathological2 Administration site Pathology 170 id="p-303" id="p-303"

[0303] [Table 76] 171 id="p-304" id="p-304"

[0304] 172 [Table 77] o r-’ CO o o o o o o tj־ o k k k. k k CM 1 r־» o O LO CO o o LO o o o o ־ 3 ^ LU o CO k k k. k k k o CO o o ־ 3 י* o o o o tj־ o k k k k k k o CO o ▼— o o o o o o ־ 3 ־* o co k k k k k k k CM r* o LO CO o o " י o o o " י o י־ LU CO k k L k L k k k o CO o o O o o o o o ־ 3 ־* o k k k k k k. o r-’ CO o o o o o o o o o CM k O י־ o JL LO CO o o o o o o o O o LU k k k — י o CO o o o o o o o o ■ 3 ־* o k k h "0 "0 y) o o c O C £־״ <0 ■° O 3 •*־ E ~ p i e E 2 y> O ±J o E SE ס ס ra ii <ע k_ y> 3 i ± מ £ d C ix ־ס XI ?כ > C iX o a .e c c UJ Li- O to £ .E co y> c. 0 O -» 4 ־* 4 (0 c E *J c/> <ס -׳ O (/> ’e .if 3 _Q k. -C k_ 3 0 $ o CO o "o o y> '50 c o 4-» o o JZ -י 4 cz y) y> y> a מ o u O k_ *♦־» w c c o X ej c E O •*-׳ 0 /־S J2 o E <0 -C o k. -4-» y> ‘c s ite E .*e E ■*־׳ <ס E <ס y) C 0 דכ c o ס ­ < c E o ס o c k_ 0) c. _o a סכ CO JD _Q U E E E -C «*־» o o 3 3 o O o z: -z. CL 173 id="p-305" id="p-305"

[0305]B) Intradermal irritation test in a single doseSubcutaneous irritation of adjuvants of the present invention of the present invention is evaluated by gross examination and histopathological examination about the skin at necropsy one day, one and four week(s) after subcutaneous administration in a single dose.An adjuvant of the present invention is subcutaneously administered to skin of back of rats. The following procedure is carried out in a similar method to A) of Example 5.[0306]Example 6 Evaluation of inducibility of antibody production of the adjuvant of the present invention in immunization by Pseudomonas aeruginosa PCRV antigen vaccineWhen antigen and adjuvant are administered as an infectious disease vaccine and induced antibody production from B lymphocyte in the immunized animal, effect of prevention and treatment for infectious disease is expected. Therefore, in expectation of apply for an infectious disease vaccine of this adjuvant, and antibody production from B lymphocyte was evaluated. PCRV antigen which is Pseudomonas aeruginosa was used for the examination as an antigen. It has been already reported that Pseudomonas aeruginosa PCRV antigen have a vaccine effect (Moriyama et al., Infect. Immun., Sep. 2001, vol. 69, no. 9, 5908-5910), and therefore effect of inducibility of antibody production by the adjuvant was evaluated compared to PCRV antigen administeration group. Freund’s adjuvant was used for a control group. Freund’s adjuvant is an adjuvant that tubercle bacillus killed by heating is mixed in oil consisting of paraffin oil and mannide monooleate, and known as an adjuvant strongly inducing antibody production in non-clinical studies. Freund’s adjuvant shows strong effect in non-clinical studies, but the use in clinical trials has been prohibited because of concern of side effects (The European Agency for the Evaluation of Medical Products Evaluation of Medicines for Human Use, March 2004, Internet (URL:http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/11/WC500015469.pdf)).[0307]174 Pcrv nucleotide sequence (SEQ ID NO: 48) derived from Pseudomonas Aeruginosa Strain PAO1 was cloned at NdeI-XhoI site in pET21a vector, and Escherichia coli strain BL21 (DE3) was transformed with the prepared plasmid. Strain pcrV-BL21 (DE3) was cultured in 500 mL of LB/Ampicillin liquid medium at 37 °C, and 200 pL of 0.1 M IPTG was added when OD6became 0.5 to induce expression of recombinant PCRV protein. After culturing at 16 °C for 18 hours, bacteria were separated by a centrifuge, and ml of Buffer A (50 mM Tris-HCl (pH8.0), 0.2M NaCl) comprising 0.5% lysozyme (SIGMA Corporation) was added. After putting at 4 °C for 30 minutes, ultrasonic treatment was carried out. After centrifugation at 12,000 rpm for minutes, soluble fraction was obtained. 1 mL of Ni-NTA agarose gel (QIAGEN) was added to Polyprep column (BIO-RAD Laboratories, Inc) and equilibrated with 10mL of Buffer A. Soluble fraction was added to the column and passed. The column was washed with 20 mL of Buffer A + 20 mM imidazole and eluted with 1 mL of Buffer A + 250 mM imidazole. Superdex 200 16/60 GL column was connected to AKTA Explorer (GE Healthcare), and equilibrated with 1.5 CV of Buffer A. The fraction eluted with imidazole was purified by gel filtration and recombinant PCRV protein C-end HIS (SEQ ID NO: 49) was purified. The resulting recombinant PCRV protein was measured the concentration with BCA assay kit (PIERCE) and kept at -80°C.[0308]pg of recombinant PCRV protein and 33 nmol of an adjuvant of the present invention were mixed with saline to be 0.7 mL. To each 5 mice of weeks old female A/J mice (Japan SLC, Inc.), 0.1 mL of immunogen was intradermaly injected on Day 0, 7 and 27. As a control, only recombinant PCRV protein was used for immunization in a similar method. Regarding Freund’s adjuvant, 100 pg of recombinant PCRV protein was mixed with saline to be 0.5 mL, and emulsion was prepared with the same amount of Complete Freund’s adjuvant (Difco Laboratories). 0.1 mL was intradermaly injected on Day 7 as initial immunization. On Day 27, Incomplete Freund’s adjuvant (Difco Laboratories) was used for immunization in a similar method. On Day 33, blood was collect using a heparin from tail vein of mice, and centrifuged at 6,000 rpm for 15 minutes. The supernatant was collected as an antiserum and kept at -40 °C.175 id="p-309" id="p-309"

[0309]Antibody titer was measured as blow. Recombinant PCRV protein was mixed with PBS (pH7.4) (Invitrogen) to be 1 pg/mL, and 20 pL/well of the solution was added to Nunc Maxisorp 384 well plate (Thermo). The plate was sealed with a plate seal and incubation was carried out at 4 °C over night. After washing twice with 90 pL/well of Washing Buffer (9 g/L NaCl, 0.5 g/L Proclin 150, 0.1 g/L Tween-20), 60 pL/well of 1xAssay Buffer (Invitrogen) was added. The plate was sealed with a plate seal and blocking was carried out at room temperature for 2 hours. After removing the liquid by tapping, antiserum was diluted with lxAssay Buffer at 103 to 107 times and 20 pL/well of the solution was added. The plate was sealed with a plate seal and incubation was carried out at 4 °C over night. After washing three times with Washing Buffer, GOAT Anti-mouse IgG Fc-HRP (JacksonImmunoResearch) was diluted at 20,000 with lxAssay Buffer and 20 pL/well of the solution was added. The plate was sealed with a plate seal and incubation was carried out at room temperature for 2 hours. After washing three times with Washing Buffer, 20 pL/well of TMB substrate (Dako) was added and incubation was carried out at room temperature for 30 minutes. 20 pL/well of 0.5 N sulfuric acid (Nacalai) was added, and absorbance at 450 nm was measured with a plate reader. The dilution ratio when absorbance at 450 nm is 1.0 was calculated and put as an antibody titer. The results were shown in Figure 15.[0310]The antibody titers were 3.0x106 for SEQ-121 which is the highest, 2.5x106 for Freund’s adjuvant, 7.1x105 for SEQ-61 and 5.3x105 for ODN20(SEQ-49) in order. The antibody titer for only PCRV protein was 1.3x105 . These results suggested that the adjuvants of the present invention have strong immunostimulatory activity against B cells. Especially, antibody titer for SEQ-121 showed higher inducibility of antibody production than ODN20and almost same with antibody titer for Freund’s adjuvant. Then, it was suggested that adjuvants of the present invention are excellent as an adjuvant for vaccine against infectious diseases. From these results, the adjuvants of the present invention are expected to apply for a vaccine against infectious diseases. 176 Industrial Applicability [0311]As it is clear from the above examples, lipid binding double-stranded oligonucleotides of the present invention show the excellent immunostimulatory activity. Therefore, they are very useful especially as an adjuvant to enhance the effect of vaccine. 177

Claims (9)

1./ 1 Claims [Claim 1] A pharmaceutical composition comprising a double-stranded oligonucleotide, wherein a first strand consists only of a CpG oligonucleotide consisting of 8 to nucleotides, a second strand is an oligonucleotide consisting of 8 to 60 nucleotides and comprising a sequence capable of hybridizing with the first strand, but excluding an RNA oligonucleotide, the length of the second strand is 50 % or more of that of the first strand, and a lipid comprising C12 to C30 hydrocarbon chain(s) binds to the second strand through a linker.

2.[Claim 2] The pharmaceutical composition comprising a double-stranded oligonucleotide of claim 1, wherein the oligonucleotide of the second strand is an oligonucleotide consisting of DNA nucleosides and/or nucleoside derivatives.

3.[Claim 3] The pharmaceutical composition comprising a double-stranded oligonucleotide of claim 2, wherein the nucleoside derivative is a nucleoside having a substituent at the 2’ position of the sugar and/or a nucleoside having a bridge structure between the 4’ and 2’ positions of the sugar.

4.[Claim 4] The pharmaceutical composition comprising a double-stranded oligonucleotide of claim 3, wherein the bridge structure between the 4’ and 2’ positions of the sugar is 4’-(CH2 )m-O-2’, wherein m is an integer of 1 to 4.

5.[Claim 5] The pharmaceutical composition comprising a double-stranded oligonucleotide 268983/ 1 of any one of claims 1 to 4, wherein the lipid is a diacyl lipid.

6.[Claim 6] The pharmaceutical composition comprising a double-stranded oligonucleotide of claim 5, wherein the diacyl lipid is wherein p and q are each independently an integer of 10 to 28.

7.[Claim 7] The pharmaceutical composition comprising a double-stranded oligonucleotide of any one of claims 1 to 6, wherein the lipid binds at the 3’ end and/or 5’ end of the second strand.

8.[Claim 8] The pharmaceutical composition comprising a double-stranded oligonucleotide of any one of claims 1 to 7, wherein the linker is an oligonucleotide linker.

9.[Claim 9] The pharmaceutical composition comprising a double-stranded oligonucleotide of claim 8, wherein the linker is -(dX)u-, wherein X is each independently, A, G, C or T, and u is an integer of 1 to 8. 268983/ 1 [Claim 10] The pharmaceutical composition comprising a double-stranded oligonucleotide of any one of claims 1 to 9, wherein the CpG oligonucleotide is an oligonucleotide consisting of the sequence of SEQ ID NO: 13. [Claim 11] A pharmaceutical composition comprising a double-stranded oligonucleotide consisting of a first strand and a second strand, wherein the first strand is a strand of the following formula: and the second strand is a strand represented by the formula selected from the 268983/ 1 followings, the formula ： the formula ： 268983/ 1 the formula ： 268983/ 1 the formula ： 268983/ 1 the formula ： 268983/ 1 the formula ： 268983/ 1 the formula ： 268983/ 1 the formula ： 268983/ 1 the formula ： 268983/ 1 the formula ： 268983/ 1 the formula ： 268983/ 1 and the formula ： 268983/ 1 . [Claim 12] The pharmaceutical composition of any one of claims 1 to 11, further comprising an antigen. 268983/ 1 [Claim 13] The pharmaceutical composition of claim 12, wherein the antigen is a microbial antigen, a self-antigen or an addictive substance. [Claim 14] The pharmaceutical composition of Claim 13, wherein the antigen is a microbial antigen, and the microbial antigen is a bacterial antigen, a viral antigen or a parasitic antigen. [Claim 15] The pharmaceutical composition of Claim 13, wherein the antigen is a self-antigen, and the self-antigen is a tumor antigen, an antigen associated with Alzheimer's Disease, an antigen against a human antibody, or an antigen that is expressed from human endogenous retroviral elements. [Claim 16] The pharmaceutical composition of Claim 13, wherein the antigen is an addictive substance, and the addictive substance is nicotine or cocaine.