BR112019019177A2 - pharmaceutical composition comprising nucleic acid derivatives having immunostimulatory activity - Google Patents

Abstract

the present invention relates to double-stranded oligonucleotides comprising the cpg oligonucleotide mentioned below, as a nucleic acid derivative having an immunostimulatory activity. an adjuvant comprising 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 to the first strand, and a lipid attaches to the second strand through a binder.

Description

Descriptive Report of the Invention Patent for PHARMACEUTICAL COMPOSITION UNDERSTANDING NUCLEIC ACID DERIVATIVES HAVING IMMUNOSIMULATORY ACTIVITY.

Field of the invention [001] The present invention relates to a nucleic acid derivative having immunostimulatory activity. In more detail, it refers to a double-stranded oligonucleotide in which the first strand is a CpG oligonucleotide and the second strand binds to a lipid.

Background art [002] A vaccine that is a drug for infectious disease or cancer uses a specific immune response to the antigen. An adjuvant is a compound with immunostimulatory activity that is used to enhance the effectiveness or durability of a vaccine, and various types of adjuvants, such as ammonium salt, emulsion and liposome, have been researched or developed (Non-Patent Document 1 or similar).

[003] A single-stranded oligodeoxynucleotide comprising a non-methylated cytosine-guanine (5'-CpG-3 ') (ssCpG ODN) dinucleotide motif is known as one of the adjuvants. ssCpG ODNs are TLR9 ligands (Toll-like receptor 9) and extremely efficient inducers of Th1 immune responses or cytotoxic T lymphocytes (CTL) through TLR9 to stimulate the immune system (Non-Patent Document 1). However, there are problems related to the in vivo stability, toxicity, pharmacokinetics or similar of ssCpG ODNs for use alone. A method in which ssCpG ODN is encapsulated in a nanoparticle made of lipid bilayer (Non-Patent Document 2), a method in which a lipid attaches to the 5 'end of ssCpG ODN (Non-Patent Document 3 or Patent Document 1) , or similar methods, are known means for solving these problems.

2/187 [004] Furthermore, it is known that characterization as an adjuvant disappeared when ssCpG ODN was administered as double-stranded DNA (dsCpG ODN) by annealing the first strand with the second strand (Non-Patent Document 4). Non-Patent Document 5 describes that only dsCpG ODN did not exhibit immunostimulatory activity, but when dsCpG ODN was encapsulated in a lipofectin particle, dsCpG ODN comprising the CpG motif or the GpC motif showed immunostimulatory activity. Prior Art Documents Patent Document [005] Patent Document 1: WO2013 / 151771 Non-Patent Document [006] Non-Patent Document 1: Trends in immunology, 2009, 30 (1), 23-32;

[007] Non-Patent Document 2: Advanced Drug Delivery Review, 2009, 61 (3), 233-242;

[008] Non-Patent Document 3: Nature, 2014, 507, 519-522;

[009] Non-Patent Document 4: Eur. J. Immunol., 2003, 33,

1382-1392;

[0010] Non-Patent Document 5: BMB reports, 2010, 43 (3), 164-169.

Summary of the invention

Problems to be solved by the invention [0011] The purpose of the present invention is to provide new nucleic acid derivatives having immunostimulatory activity that are useful as an adjuvant to a vaccine and / or the vaccine itself.

Means to solve the problem [0012] Non-Patent Documents 4 and 5 describe that the administration of dsCpG ODN did not show immunostimulatory activity. Furthermore, Non-Patent Document 5 reveals that,

IBWaS

3/187 when dsCpG ODN was encapsulated in a lipofectin particle, the dsCpG ODN comprising the CpG motif (ODN4531) or the GpC motif (ODN4531GC) induced the expression of IL-8 and HLR-DRA, that is, by a method that is independent of a CG sequence. It has been suggested, based on the fact that dsCpG ODN was rapidly degraded in cells, when compared to ssCpG ODN, that the reason why dsCpG ODN encapsulated in a lipofectin particle showed immunostimulatory activity is that the encapsulation of dsCpG ODN may protect against rapid degradation (page 165, the right column, lines 14 to 16).

[0013] The present inventors have intensively studied the synthesis of a double-stranded oligonucleotide (a lipid linking the 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 able to hybridize with the first strand and binds to a lipid. They found that the ability to induce antigen-specific CTL by a vaccine was improved, in addition to showing the antitumor effect by administering a lipid-linked double-stranded oligonucleotide of the present invention, as an adjuvant, with a tumor antigenic peptide (when a lipid-linked double-stranded oligonucleotide of the present invention is used as an adjuvant, it is also called an adjuvant of the present invention). In addition, they found that a lipid-linked double-stranded oligonucleotide in the present invention exhibits, in itself, a strong antitumor effect, that is, it is useful as a cancer vaccine (when a lipid-bound double-stranded oligonucleotide of the present invention). invention is used as a vaccine, it is also called a vaccine of the present invention). That is, a lipid-linked double-stranded oligonucleotide of the present invention has immunostimulatory activity. In addition, double-stranded oligonucleotides

4/187 the lipid of the present invention has high metabolic stability, water solubility and less toxicity to be safe enough as a medicine.

[0014] For example, when an adjuvant of the present invention, wherein the second strand is a DNA oligonucleotide, an oligonucleotide consisting of nucleoside derivatives with OMe in the 2 'position of the sugar or an oligonucleotide consisting of nucleoside derivatives with F at the 2 'position of the sugar, was administered with a tumor antigenic peptide, the improved ability to induce CTL or the strong antitumor effect compared to ssCpG ODN (for example, Result 1-A, B, Result 7- A, B and Result 9-A, B in Example 3). On the other hand, the lipid-linked double-stranded oligonucleotide in which the second strand is an RNA oligonucleotide (SEQ-40) did not show the antitumor effect (Result 7-B).

[0015] When an adjuvant of the present invention, in which a lipid comprising two of the acyl chains with 14 to 24 carbon atoms attaches to the 3 'or 5' end of the second strand, was administered with a tumor antigen peptide, it revealed the strong antitumor effect compared to ssCpG ODN (for example, Result 2A, B in Example 3). On the other hand, when a double-stranded oligonucleotide in which a lipid comprising two acyl chains with 10 carbon atoms bonds to the 5 'end of the second strand (SEQ-12) was administered with a tumor antigen peptide, it did not show the enhanced antitumor effect compared to ssCpG ODN. In addition, when an adjuvant of the present invention, in which a lipid comprising two of the acyl chains with 12 to 20 carbon atoms binds to the 3 'or 5' end of the second strand and, in addition, a lipid binds to the other end, it was administered with a tumor antigenic peptide, it showed increased capacity for CTL induction or the strong antitumor effect compared to

5/187 ssCpG ODN (for example, Result 3-A, B in Example 3).

[0016] When an adjuvant of the present invention, in which 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 increased ability to induce CTL or the strong antitumor effect compared to ssCpG ODN (for example, Result 4-A, B, Result 5-A, Result 8-A, B, and Result 9-A, B in Example 3).

[0017] When an adjuvant of the present invention, in which a lipid attaches to a second strand through an oligonucleotide ligand (connector) (e.g., dGdG, dTdT and dAdA), was administered with a tumor antigenic peptide, it showed the increased ability for CTL induction to have the strongest antitumor effect when compared to an adjuvant of the present invention without a linker (for example, Result 8-A, B in Example 3).

[0018] Furthermore, double-stranded lipid-linked oligonucleotides of the present invention have no systemic toxicity, which suggested the high level of safety (Example 5).

[0019] Furthermore, the result of immunization with an adjuvant of the present invention and PCRV protein derived from Pseudomonas aeruginosa, which is a cause of infectious disease, also suggested immunostimulatory activity as an adjuvant to a vaccine for an infectious disease (Example 6).

[0020] When the adjuvant of the present invention, in which the lipid binds to a second strand through an oligonucleotide ligand (dGdGdGdGdG) (SEQ-121), was administered alone, it showed the strong antitumor effect compared to ssCpG ODN (Example 4).

[0021] That is, the present invention is related to the following.

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6/187 [0022] (A1) A double-stranded oligonucleotide, in which:

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 to the first strand, but excluding an RNA oligonucleotide, the length the second strand is 50% or more than that of the first strand and a lipid comprising C12 to C30 hydrocarbon chain (s) is attached to the second strand via a binder.

[0023] (A2) The (A1) double-stranded oligonucleotide, wherein the second-stranded oligonucleotide is an oligonucleotide consisting of DNA nucleosides and / or nucleoside derivatives.

[0024] (A3) The double-stranded oligonucleotide of (A2), wherein the nucleoside derivative is a nucleoside with a substituent in the 2 'position of the sugar and / or a nucleoside with a bridge structure between the 4' and 2 'of the sugar.

[0025] (A4) The double-stranded oligonucleotide of (A3), in which the bridge structure between the 4 'and 2' positions of the sugar is 4 '- (CH2) mO-2', where m is a number integer from 1 to 4.

[0026] (A5) The double-stranded oligonucleotide of any of (A1) to (A4), in which the lipid is a diacyl-lipid.

[0027] (A6) The double-stranded oligonucleotide from (A1) to (A5), wherein the lipid binds to the 3 'end and / or the 5' end of the second strand. [0028] (A7) The double-stranded oligonucleotide of any one of (A1) to (A6), wherein the linker is an oligonucleotide linker.

[0029] (A8) The double-stranded oligonucleotide of (A7), where the linker is - (dX ¹ ) u-, where X ¹ is, each independently A, G, C or T, eu is a number integer from 1 to 8.

[0030] (A9) The double-stranded oligonucleotide from (A1) selected at

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7/187 from the group consisting of SEQ-61, SEQ-119, SEQ-121, SEQ170eSEQ-192.

[0031] (A10) The double-stranded oligonucleotide from (A1) selected from the group consisting of SEQ-59, SEQ-166, SEQ-168, SEQ216, SEQ-272, SEQ-280, SEQ-290, SEQ -294, SEQ-310, SEQ-373 and SEQ-384.

[0032] (A11) A pharmaceutical composition comprising the double-stranded oligonucleotide of any one of (A1) to (A10).

[0033] (A12) The pharmaceutical composition of (A11), further comprising an antigen.

[0034] (A13) A method for treating or preventing cancer or an infectious disease, comprising administering the double-stranded oligonucleotide from either (A1) to (A10).

[0035] (A14) Use of double-stranded oligonucleotide from any of (A1) to (A10) in the production of an agent to treat or prevent cancer or an infectious disease.

[0036] (A15) The double-stranded oligonucleotide of any of (A1) to (A10) to treat or prevent cancer or an infectious disease.

[0037] (A16) A pharmaceutical composition of (A12), in which the antigen is a microbial antigen, a self-antigen or an addictive substance.

[0038] (A17) The pharmaceutical composition of (A16), in which the microbial antigen is a bacterial antigen, a viral antigen or a parasitic antigen.

[0039] (A18) The pharmaceutical composition of (A16), in which the autoantigen is a tumor antigen, an antigen associated with Alzheimer's disease, an antigen against a human antibody or an antigen that is expressed from endogenous retroviral elements humans.

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8/187 [0040] (A19) The pharmaceutical composition of (A16), the addictive substance is nicotine or cocaine.

[0041] (A20) A method of enhancing an immune response in an individual comprising administering to the individual an effective amount of the pharmaceutical composition from either (A12), (A16) to (A19).

[0042] (A21) The (A20) method, in which the immune response is an increase in the ability to induce specific cytotoxic T lymphocytes compared to a control.

[0043] (A22) The method of (A20) or (A21), in which the individual has a cancer or an infectious disease.

[0044] In addition, the present invention includes the following.

[0045] (B1) An adjuvant consisting of a double-stranded oligonucleotide, in which:

a first strand is a CpG oligonucleotide that consists of 8 to 50 nucleotides, a second strand is an oligonucleotide that consists of 8 to 60 nucleotides and that comprises a sequence capable of hybridizing to the first strand and a lipid attaches to the second strand through of a binder.

[0046] (B2) The adjuvant of (B1), wherein the oligonucleotide of the second strand is an oligonucleotide consisting of DNA nucleosides and / or nucleoside derivatives.

[0047] (B3) The adjuvant of (B2), wherein the nucleoside derivative is a nucleoside with a substituent at the 2 'position of the sugar and / or a nucleoside with a bridge structure between the 4' and 2 'positions of the sugar.

[0048] (B4) The adjuvant of (B3), where the substituent is OCH3.

[0049] (B5) The adjuvant of any of (B1) to (B4), wherein the lipid is a diacyl-lipid.

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9/187 [0050] (B6) The adjuvant of (B5), in which the diacyl-lipid acyl chain has 14 to 30 carbon atoms.

[0051] (B7) The adjuvant of (B5) or (B6), wherein the lipid attaches to the 5 'end of the second strip.

[0052] (B8) The adjunct to any of (B1) to (B7), where ο the length of the second tape is 50% or more than that of the first tape.

[0053] (B9) The adjuvant of any one of (B1) to (B9), wherein the linker is an oligonucleotide linker.

[0054] (B10) The adjuvant of (B9), where the linker is dX ¹ dX ² , where X ¹ or X ² is A, G, C or T.

[0055] (B11) A vaccine composition comprising an antigen and the adjuvant of any one of (B1) to (B10).

[0056] (B12) The vaccine composition of (B11), in which the antigen is a microbial antigen, a self-antigen or an addictive substance. [0057] (B13) The vaccine composition of (B12), in which the microbial antigen is a bacterial antigen, a viral antigen or a parasitic antigen.

[0058] (B14) The vaccine composition of (B12), in which the autoantigen is a tumor antigen, an antigen associated with Alzheimer's disease, an antigen against a human antibody or an antigen that is expressed from endogenous retroviral elements humans.

[0059] (B15) The vaccine composition of (B12), in which the addictive substance is nicotine or cocaine.

[0060] (B16) A method of enhancing an immune response in an individual, comprising administering to the individual an effective amount of the vaccine composition from either (B11) to (B15).

[0061] (B17) The (B16) method, in which the immune response is an increase in the ability to induce specific cytotoxic T lymphocytes induced compared to a control.

[0062] (B18) The method of (B16) or (B17), in which the individual has

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10/187 a cancer or an infectious disease.

[0063] (B19) A method of treating a cancer or an infectious disease, comprising administering to the individual an effective amount of the vaccine composition from either (B11) to (B14) to reduce one or more symptoms of the cancer or disease infectious compared to a control.

Effect of the invention [0064] The lipid-linked double-stranded oligonucleotides of the present invention show superior immunostimulatory activity against a target antigen. No systemic toxicity has been detected and, therefore, its application to a drug as an adjuvant to a vaccine and / or as the vaccine itself is expected.

Brief description of the drawings [0065] Figure 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).

[0066] Figure 2. The antitumor effect when ssCpG ODN (ODN1826, SEQ-1) or the adjuvant of the present invention having lipids at both ends (SEQ-46 or SEQ-48) was administered with a tumor antigen peptide (peptide TRP2 ).

[0067] Figure 3. The antitumor effect when ssCpG ODN (ODN1826, SEQ-1), ssCpG ODN with an introduced lipid ligand (SEQ-2) or the adjuvant of the present invention in which the complementary strip length is different (SEQ- 8 or SEQ-10) was administered with a tumor antigenic peptide (TRP2 peptide).

[0068] Figure 4. The antitumor effect when ssCpG ODN (ODN1826, SEQ-1), ssCpG ODN with an introduced lipid linker (SEQ-2), the adjuvant of the present invention (SEQ-26) or Montanide was administered with a peptide tumor antigen (TRP2 peptide).

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11/187 [0069] Figure 5. The antitumor effect when ssCpG ODN (ODN1826, SEQ-1), ssCpG ODN with an introduced lipid ligand (SEQ-2) or the adjuvant of the present invention (SEQ-38) was administered with a tumor antigenic peptide (TRP2 peptide).

[0070] Figure 6. The anti-tumor effect when ssCpG ODN (ODN2006, SEQ-49), the adjuvant of the present invention in which the length of the complementary tape is different (SEQ-51 or SEQ-61) or the adjuvant of the present invention with a linker (SEQ-63 or SEQ65), was administered with a tumor antigenic peptide (TRP2 peptide).

[0071] Figure 7. The anti-tumor effect when ssCpG ODN (ODN2006, SEQ-49) or the adjuvant of the present invention wherein the complementary strand nucleic acid monomer is 2'-OMe-RNA (SEQ-67 or SEQ-69 ) was administered with a tumor antigenic peptide (TRP2 peptide).

[0072] Figure 8. The antitumor effect when ssCpG ODN (ODN2006, SEQ-49) or the adjuvant of the present invention with an oligonucleotide linker (SEQ-119 or SEQ-192) was administered with a tumor antigenic peptide (TRP2 peptide ).

[0073] Figure 9. The antitumor effect when ssCpG ODN (ODN2006, SEQ-49) or the adjuvant of the present invention with an oligonucleotide linker (SEQ-121) was administered with a tumor antigenic peptide (TRP2 peptide).

[0074] Figure 10. The antitumor effect when ssCpG ODN (ODN2006, SEQ-49) or the adjuvant of the present invention with an oligonucleotide ligand (SEQ-152 or SEQ-158) was administered with a tumor antigenic peptide (TRP2 peptide ).

[0075] Figure 11. The antitumor effect when ssCpG ODN (ODN2006, SEQ-49) or the adjuvant of the present invention in which a lipid binds to the 3 'end and / or the 5' end (SEQ-188, SEQPetition 870290039082, 10 / Φ3 / 2Ο29, page 2S / 22S

12/187

192 or SEQ-194) was administered with a tumor antigenic peptide (TRP2 peptide).

[0076] Figure 12. The antitumor effect when ssCpG ODN (ODN2006, SEQ-49) or the adjuvant of the present invention with an oligonucleotide linker (SEQ-164) was administered with a tumor antigenic peptide (TRP2 peptide).

[0077] Figure 13. The antitumor effect when ssCpG ODN (ODN2006, SEQ-49) or the adjuvant of the present invention in which a lipid binds only to the 3 'end (SEQ-170) was administered with a tumor antigen peptide ( TRP2 peptide).

[0078] Figure 14. The anti-tumor effect of ssCpG ODN (QDN2006, SEQ-49) or the vaccine of the present invention (SEQ-121) in the absence of a tumor antigenic peptide.

[0079] Figure 15. Antibody titer after subcutaneous administration of the PCRV antigen vaccine with ssCpG ODN (QDN2006, SEQ-49), the adjuvant of the present invention (SEQ-61 or SEQ-121) or Freund's adjuvant.

Modalities for carrying out the invention [0080] The terms used in this specification, unless otherwise indicated, are used in the sense normally used in the art.

[0081] In the present invention, a method of genetic manipulation well known in the field can be used. For example, this 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).

[0082] The terms used in this description are explained below.

Each term, unless otherwise indicated, has the same meaning when used alone or in conjunction with other terms.

[0083] In this description, adjuvant means a compound with the

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13/187 immunostimulatory activity that is used to reinforce the effectiveness or durability of the immune response of a vaccine antigen.

[0084] A nucleoside means a compound in which a nucleic acid base and a sugar are joined by a Nglycoside bond.

[0085] An oligonucleotide means nucleotides in which the same or different types of nucleotide are joined by an internucleoside bond like a phosphodiester bond.

[0086] A bond between a sugar and a sugar in an oligonucleotide (internucleoside bond) can be a bond with a natural nucleic acid, phosphodiester (D-oligo), an artificially modified bond or a bond without a phosphorus atom. Any link that is well known in this field can be used. Examples of an artificially modified bond are phosphorothioate (S-oligo), methylphosphonate (M-oligo) and boranophosphate. In addition, a connection described in WQ2013 / 022966, WQ2011 / 005761, WQ2014 / 012081, WQ2015 / 125845 or the like can be used. An example of a bond without a phosphorus atom is a bivalent substituent derived from non-aromatic carbocyclyl or the like substituted with alkyl, non-aromatic carbocyclyl, haloalkyl or halogen. The example is a bivalent substituent derived from siloxane, sulfide, sulfoxide, sulfone, acetyl, acetyl formate, acetyl thioformate, methylene acetyl formate, acetyl thioformide, alkenyl, sulfamate, methyleneimino, methylene hydrazine, sulfonate, sulfonamide, amide or the like. In an oligonucleotide, the bonds can be the same or different.

[0087] In this description, a DNA nucleoside or RNA nucleoside means a natural DNA nucleoside or natural RNA nucleoside and a nucleotide part, which is 1 unit for an oligonucleotide component. A natural nucleoside from

DNA is as below:

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where Βχι is adenine, guanine, cytosine or thymine.

[0088] A natural RNA nucleoside is as below:

OH i ww where Bx2 is adenine, guanine, cytosine or uracil.

[0089] A DNA oligonucleotide means an oligonucleotide in which some DNA nucleosides are joined, and an RNA oligonucleotide means an oligonucleotide in which some RNA nucleosides are joined.

[0090] In this description, a nucleoside derivative means a nucleoside whose nucleic base and / or part of the DNA nucleoside or RNA nucleoside sugar has been artificially modified. Any modification well known in this field can be used.

[0091] Examples of modification to a nucleic base are

5-methylcytosine, 5-hydroxy-methylcytosine and 5-propynylcytosine.

[0092] An example of modification for a sugar part is a substituent at the 2 'position of a sugar. Examples are 2'-F, 2'OCH ₃ (2'-OMe) and 2'-OCH ₂ CH ₂ OCH3 (2'-MOE).

[0093] The other example is the bridge structure between positions 4 'and 2' following a sugar:

[0094] 4 '- (CR ¹ R ² ) mO-2', 4 '- (CR ¹ R ² ) mS-2', 4 '- (CR ¹ R ² ) mOC (= O) 2', 4 ' - (CR ¹ R ² ) m-NR ³ -O- (CR ¹ R ² ) mi-2 ', 4' - (CR ¹ R ² ) mi-C (= O) -NR ³ -2 ', 4' (CR ¹ R ² ) m2-C (= O) -NR ³ -Y ⁴ -2 ', 4' - (CR ¹ R ² ) mi-SO2-NR ³ -2 ', or

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on what:

[0095] Y ⁴ is Ο, S, ΝΗ or CH ₂ , [0096] R ¹ is each independently hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl or substituted or unsubstituted alkynyl substituted, [0097] R ² is each independently hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl or substituted or unsubstituted alkynyl, [0098] R ³ is hydrogen atom, alkyl substituted or unsubstituted, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aromatic carbocyclyl, substituted or unsubstituted aromatic carbocyclyl, substituted or unsubstituted aromatic heterocyclyl, substituted or unsubstituted aromatic heterocyclyl, aromatic substituted carbocycloalkyl or unsubstituted, non-aromatic carbocycloalkyl substituted substituted or unsubstituted aromatic heterocycloalkyl or unsubstituted or unsubstituted heterocycloalkyl, [0099] Y ¹ éCR ⁴ ouN, [00100] Y ² éCR ⁵ ouN, [00101] Y ³ éCR ⁶ ouN, [00102] R ⁴ , R ⁵ and R ⁶ 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, substituted or unsubstituted alkylcarbonylamino, substituted or unsubstituted alkenylcarbonylamino, substituted or unsubstituted alkynylcarbonylamino, substituted alkylcarbamoyl or

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16/187 unsubstituted, substituted or unsubstituted alkenyl carbamoyl or substituted or unsubstituted alkynyl carbamoyl, [00103] m is an integer from 1 to 4, [00104] mi is an integer from 0 to 3 and [00105] iri2é0ou1.

[00106] R ¹ and R ² are preferably y hydrogen atom.

[00107] R ³ is preferably hydrogen atom, alkyl, alkenyl, alkynyl, aromatic carbocyclyl, non-aromatic carbocyclyl, aromatic heterocyclyl, non-aromatic heterocyclyl, aromatic carbocycloalkyl, non-aromatic carbocycloalkyl, aromatic heterocycloalkyl or non-aromatic heterocycloalkyl and non-heterocycloalkyl and non-aromatic heterocycloalkyl. have one or more substituents selected from Group a.

[00108] Group a: hydroxyl, alkyl, alkyloxy, mercapto, alkylthio, amino, alkylamino and halogen group.

[00109] The bridged structure is preferably 4 '- (CR ¹ R ² ) mO-2' or 4 '- (CR ¹ R ² ) mi-C (= O) -NR ³ -2' (AmNA, Acid bridged nucleic acid), [00110] where, [00111] R ¹ is each independently hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl, [ 00112] R ² is each independently hydrogen atom, halogen, cyano, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl, [00113] R ³ is hydrogen atom, substituted alkyl or unsubstituted, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl, [00114] m is an integer from 1 to 4 and [00115] rm is an integer from 0 to 2.

[00116] The bridged structure is more preferably 4 '- (CH2) m-O2', where m is an integer from 1 to 4, or 4'-C (= O) -NR ³ -2 ', in

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17/187 that R ³ is hydrogen atom or alkyl.

[00117] 4 '- (CH2) m-O-2', where m is an integer from 1 to 4, is most preferably 4'-CH2-O-2 '(LNA, blocked nucleic acid). Examples and methods of preparation are described in WO98 / 39352, W02003 / 068795, W02005 / 021570 or the like.

[00118] 4'-C (= O) -NR ³ -2 ', where R ³ is hydrogen atom or alkyl, more preferably 4'-C (= O) -NCH3-2'. Examples and methods for preparation are described in WO2011 / 052436.

[00119] Examples of the well-known modification of nucleotides and the method for modification in this field are described in the following Patent Documents:

[00120] WO98 / 39352, WO99 / 014226, W02000 / 056748, W02005 / 021570,

W02003 / 068795, WO2011 / 052436, W02004 / 016749, W02005 / 083124, WO2007 / 143315, W02009 / 071680, WO2014 / 112463, WO2014 / 126229 and the like.

[00121] 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.

[00122] Alkyl includes a straight or branched C1 to C15 hydrocarbon group, preferably C1 to C10, more preferably C1 to C6 and even more preferably C1 to C4. Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, npentyl, isopentyl, neopentyl, n-hexyl, isohexyl, n-heptyl, isoheptyl, n-octyl, isooctyl , n-nonila and n-decila.

[00123] A preferred embodiment of alkyl is methyl, ethyl, npropyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl or n-pentyl.

A more preferred embodiment is methyl, ethyl, n-propyl, isopropyl or tert-butyl.

[00124] Alkenyl includes a straight or branched C2 to C15 hydrocarbon group, preferably C2 to C10, more preferably C2 to C6

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18/187 and even more preferably C2 to C4 with one or more double bonds in 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, dodecenyl, dodecenyl and

[00125] A preferred embodiment of alkenyl is vinyl, allyl, propenyl, isopropenyl or butenyl.

[00126] Alquinyl includes a straight or branched C2 to C10 hydrocarbon group, preferably C2 to 08, more preferably 02 to 06 and even more preferably 02 to 04 with one or more triple bonds in any position (s). Examples include ethinyl, propynyl, butynyl, pentynyl, hexynyl, heptinyl, octinyl, noninyl and decynyl. Furthermore, they can have double bond (s) in any position (s).

[00127] A preferred mode of alkynyl is ethynyl, propynyl, butynyl and pentynyl.

[00128] Aromatic carbocyclyl means a cyclic aromatic hydrocarbon group that is monocyclic or polycyclic with two or more rings. Examples include phenyl, naphthyl, anthryl and phenanthryl.

[00129] A preferred embodiment of aromatic carbocyclyl is phenyl.

[00130] Non-aromatic carbocyclyl means a saturated non-aromatic cyclic hydrocarbon group or unsaturated non-aromatic cyclic hydrocarbon group, which is monocyclic or polycyclic with two or more rings. Examples of non-aromatic carbocyclyl, which is polycyclic with two or more rings, include a fused ring group in which a non-aromatic carbocyclyl, which is monocyclic or polycyclic with two or more rings, is fused with an aromatic carbocyclyl ring above.

[00131] In addition, examples of non-aromatic carbocyclyl size 870290039082, from 10 / Φ3 / 2Ο29, p. 29/220

19/187 also include a group with a bridge and a group forming a spiro ring as follows:

[00132] Non-aromatic carbocyclyl, which is monocyclic, is preferably C3 to C16 carbocyclyl, more preferably C3 to C12 and even more preferably C4 to C8. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl and cyclohexadienyl.

[00133] Examples of non-aromatic carbocyclyl, which is polycyclic with two or more rings, include indanila, indenila, acenaftila, tetrahydronaftila and fluorenila.

[00134] Aromatic heterocyclyl means an aromatic cyclyl, which is monocyclic or polycyclic with two or more rings, containing one or more of heteroatoms independently selected from O, S and N.

[00135] Examples of aromatic heterocyclyl, which is polycyclic with two or more rings, include a fused ring group, wherein an aromatic heterocyclyl, which is monocyclic or polycyclic with two or more rings, is fused with an aromatic carbocyclyl ring and / or non-aromatic carbocyclyl above.

[00136] Aromatic heterocyclyl, which is monocyclic, is preferably a 5 to 8 membered ring and more preferably 5 to 6 membered.

Examples include pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazolyl, triazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, oxazolyl, oxadiazolyl, isothiazolyl, thiazolyl and thiad.

[00137] Examples of aromatic heterocyclyl, which is bicyclic, inPetição 870290039082, from 10 / Φ3 / 2Ο29, p. 30/220

20/187 include indolyl, isoindolyl, indazolyl, indolizinyl, quinolinyl, isoquinolinyl, cinolinyl, phthalazinyl, quinazolinyl, naphthyridinyl, quinoxalinyl, purinyl, pteridinyl, benzimidazolyl, benzisoxazolyl, benzoxazolyl, benzoxazolol, benzoxazolol, benzoxazole benzotriazolyl, imidazopyridyl, triazolopyridyl, imidazothiazolyl, pyrazinopyridazinyl, oxazolopyridyl and thiazolopyridyl.

[00138] Examples of aromatic heterocyclyl, which is polycyclic with three or more rings, include carbazolyl, acridinyl, xanthenyl, phenothiazinyl, phenoxathinyl, phenoxazinyl and dibenzofuryl.

[00139] Non-aromatic heterocyclyl means a non-aromatic cyclyl, which is monocyclic or polycyclic with two or more rings, containing one or more heteroatoms independently selected from O, S and N.

[00140] Examples of non-aromatic heterocyclyl, which is polycyclic with two or more rings, include a fused ring group, in which a non-aromatic heterocycle, which is monocyclic or polycyclic with two or more rings, is fused with a ring of aromatic carbocyclyl, non-aromatic carbocyclyl and / or aromatic heterocyclyl.

[00141] In addition, examples of non-aromatic heterocyclyl also include a group with a bridge and a group forming a spiro ring as follows:

[00142] Non-aromatic heterocyclyl, which is monocyclic, is preferably a 3 to 8 membered ring and more preferably 5 to 6 membered. Examples include dioxanil, tyranyl, oxiranyl, oxetanil, oxathiolanil, azetidinyl, thianyl, thiazolidinyl, pyrrolidinyl, pyrrolinyl, imiPetition 87Ο29ΟΦ3Θ082, from 10 / Φ3 / 2Ο29, p. 3Í / 22S

21/187 dazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperidyl, piperazinyl, morpholinyl, morpholino, thiomorpholinyl, thiomorpholino, dihydropyridyl, tetrahydropyridyl, tetrahydrofuryl, tetrahydropyril, tetrahydro, hydrofluid, tetrahydro, hydrofluid -hydroazepinyl, tetrahydrodiazepinyl, tetrahydropyridazinyl, hexahydropyrimidinyl, dioxolanyl, dioxazinyl, aziridinyl, dioxolinyl, oxepanyl, thiolanil, thiinyl and thiazinyl.

[00143] Examples of non-aromatic heterocyclyl, which is polycyclic with two or more rings, include indolinyl, isoindolinyl, chromanyl and isochromanyl.

[00144] Alkyloxy means a group in which alkyl is attached to an oxygen atom. Examples include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, isobutoxy, sec-butoxy, pentoxy, isopentoxy and hexoxy.

[00145] A preferred alkyloxy modality is methoxy, ethoxy, npropoxy, isopropoxy or tert-butoxy.

[00146] Haloalkyl means a group in which one or more halogens bond to alkyl. Examples include monofluoromethyl, monofluoroethyl, monofluoropropyl, 2,2,3,3,3-pentafluoropropyl, monochloromethyl, trifluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 2,2,2trichloroethyl, 1,2-dibromoethyl, and 1,1 , 1-trifluoropropane-2-yl.

[00147] A preferred embodiment of haloalkyl is trifluoromethyl or trichloromethyl.

[00148] Alkylthio means a group in which alkyl is attached to a sulfur atom.

[00149] Alkylamino includes monoalkylamino and dialkylamino.

[00150] Monoalkylamino means a group in which a hydrogen atom attached to a nitrogen atom of an amino lump is replaced by alkyl. Examples include methylamino, ethylamino and isopropylamino. Preferably, it is methylamino or ethylamino.

Petition 870290039082, of 10 / (03/2029, page 3S / 22S

22/187 [00151] Dialkylamino means a group in which two hydrogen atoms attached to a nitrogen atom of an amino group are replaced by two alkyls. These two alkyl groups can 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.

[00152] Alkylcarbonylamino means a group in which one or two hydrogen atoms attached to a nitrogen atom of an amino group are replaced by one or two alkylcarbonyl. The two alkylcarbonyl groups can be the same or different. Examples include methylcarbonylamino, ethylcarbonylamino, propylcarbonylamino, isopropylcarbonylamino, tert-butylcarbonylamino, isobutylcarbonylamino, sec-butylcarbonylamino, dimethylcarbonylamino, diethylcarbonylamino and N, N-diisopropylcarbonylamino.

[00153] A preferred embodiment of alkylcarbonylamino is methylcarbonylamino and ethylcarbonylamino.

[00154] Alkenylcarbonylamino means a group in which one or two hydrogen atoms attached to a nitrogen atom of an amino group are replaced by one or two alkenylcarbonyl. The two alkenylcarbonyl groups can be the same or different. Examples include vinylcarbonylamino and propenylcarbonylamino.

[00155] Alquinylcarbonylamino means a group in which one or two hydrogen atoms attached to a nitrogen atom of an amino group are replaced by one or two alkynylcarbonyl. The two alkynylcarbonyl groups can be the same or different. Examples include ethinylcarbonylamino and propynylcarbonylamino.

[00156] Alkylcarbamoyl means a group in which one or two hydrogen atoms attached to a nitrogen atom of a carbamoyl group are replaced by one or two alkyl. These two alkyl groups can be the same or different. Examples include mePetition 870290 (039082, from 10 / Φ3 / 2Ο29, pg. 33 / 22S

23/187 tilcarbamoyl, ethylcarbamoyl, dimethylcarbamoyl and diethylcarbamoyl.

[00157] Alkenylcarbamoyl means a group in which one or two hydrogen atoms attached to a nitrogen atom in a carbamoyl group are replaced by one or two alkenyl. These two alkenyl groups can be the same or different. Examples include vinylcarbamoyl and propenylcarbamoyl.

[00158] Alquinylcarbamoyl means a group in which one or two hydrogen atoms attached to a nitrogen atom of a carbamoyl group are replaced by one or two alkynyl. These two alkynyl groups can be the same or different. Examples include ethinylcarbamoyl and propynylcarbamoyl.

[00159] Alkyl portion of aromatic carbocycloalkyl, non-aromatic carbocycloalkyl, aromatic heterocycloalkyl or non-aromatic heterocycloalkyl is the same as alkyl.

[00160] Aromatic carbocyclylalkyl means alkyl substituted with one or more aromatic carbocyclyl. Examples include benzyl, phenethyl, phenylpropyl, benzhydryl, trityl, naphthylmethyl and a group of the formula:

[00161] A preferred modality of aromatic carbocyclylalkyl is benzyl, phenethyl or benzhydryl.

[00162] Non-aromatic carbocyclylalkyl means alkyl substituted with one or more non-aromatic carbocyclyls. Non-aromatic carbocycloalkyl also includes non-aromatic carbocycloalkyl in which the alkyl part is replaced with aromatic carbocyclyl. Examples include cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl and a group of the formula:

Petition 870290039082, of 10 / (03/2029, page 38 / 22S

24/187 σνυχ

[00163] Aromatic heterocycloalkyl means alkyl substituted with one or more aromatic heterocyclyl. Aromatic heterocycloalkyl also includes aromatic heterocycloalkyl in which the alkyl part is replaced with the aromatic carbocyclyl and / or the non-aromatic carbocyclyl above. Examples include pyridylmethyl, furanylmethyl, imidazolylmethyl, indolylmethyl, benzothiophenylmethyl, oxazolylmethyl, isoxazolylmethyl, thiazolylmethyl, isothiazolylmethyl, pyrazolylmethyl, isopyrazolylmethyl, pyrrolidinylmethyl, and pyrrolidinylmethyl;

[00164] Non-aromatic heterocycloalkyl means alkyl substituted with one or more non-aromatic heterocyclyl. Non-aromatic heterocycloalkyl also includes non-aromatic heterocycloalkyl in which the alkyl part is replaced with aromatic carbocyclyl, non-aromatic carbocyclyl and / or aromatic heterocyclyl. Examples include tetrahydropyranylmethyl, morpholinylethyl, piperidinylmethyl, piperazinylmethyl and groups of the formulas:

[00165] Examples of 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

Petition 870290 (039082, from 10 / Φ3 / 2Ο29, page 39 / 22S

25/187 substituted or unsubstituted, substituted or unsubstituted alkylcarbamoyl, substituted or unsubstituted alkenylcarbamoyl or substituted or unsubstituted alkynylcarbamoyl include the following substituents. A carbon atom in any position (s) can be attached to one or more groups selected from the following substituents.

[00166] Substituent: halogen, hydroxy, carboxy, amino, imino, hydroxyamino, hydroxyimino, formyl, formyloxy, carbamoyl, sulfamoyl, sulfanyl, sulfine, sulfo, thioformyl, thiocarboxy, dithiocarbon, thiocarbamoyl, cyano, azide, nitro, nitro , hydrazino, ureido, amidino, guanidino, trialquilsilila, alkyloxy, alkenyloxy, alkynyloxy, haloalkoxy, alkylcarbonyl, alquenilcarbonila, alquinilcarbonila, monoalkylamino, dialkylamino, alquilsulfonila, alquenilsulfonila, alquinilsulfonila, monoalquilcarbonilamino, dialquilcarbonilamino, monoalquilsulfonilamino, dialkylsulfonylamino, alkylimino, alquenilimino, alquinilimino, alquilcarbonilimino , alkenylcarbonylimino, alkynylcarbonylimino, alkoxyimino, alkenyloxyimino, alkynyloxyimino, alkylcarbonyloxy, alkenylcarbonyloxy, alkynylcarbonyloxy, alkoxycarbonyl, alkenyloxycarbonyl, alkenyloxycarbonyl, alkenylsulfanyl, alkylsulfanyl ila, monoalkylcarbamoyl, dialkylcarbamoyl, monoalkylsulfamoyl, dialkylsulfamoyl, aromatic carbocyclyl, non-aromatic carbocyclyl, aromatic heterocyclyl, non-aromatic heterocyclyl, aromatic carbocyclyloxy, non-aromatic carbocyclyl, carbocyclyl, carbocyclic, aryl, aryl, aryl aromatic, aromatic carbocyclyloxycarbonyl, non-aromatic carbocyclyloxycarbonyl, aromatic heterocyclyloxycarbonyl, non-aromatic heterocyclyloxycarbonyl, aromatic carbocycloalkoxy, non-aromatic carbocycloalkoxy, aromatic heterocycloalkoxy / non-aromatic, carbocyclicΘΟPomom carb 30/220

26/187 cloalcoxicarbonila non-aromatic, heterocicloalcoxicarbonila aromatic, non-aromatic heterocicloalcoxicarbonila, carbocicloalquilamino aromatic, non-aromatic carbocicloalquilamino, heterocycloalkylamino aromatic, non-aromatic heterocycloalkylamino, carbociclossulfanila aromatic, non-aromatic carbocarbociclossulfanila, heterocarbociclossulfanila aromatic, non-aromatic heterocarbociclossulfanila, carbocarbociclossulfonila non-aromatic, aromatic carbocarbociclossulfonila, heterocarbociclossulfonila aromatic and non-aromatic heterocarbocyclosulfonyl. In addition, the substituent may have one or more substituents selected from Group α above.

[00167] Examples of the substituents on the aromatic carbocyclyl ring, non-aromatic carbocyclyl, aromatic heterocyclyl or substituted or unsubstituted aromatic heterocyclyl, substituted or unsubstituted non-aromatic carbocyclyl, substituted or unsubstituted aromatic heterocyclyl and unsubstituted heterocyclyl or unsubstituted include the following substituents. An atom at any position (s) in the ring can be attached to one or more groups selected from the following substituents.

[00168] Substituent: halogen, hydroxy, carboxy, amino, imino, hydroxyamino, hydroxyimino, formyl, formyloxy, carbamoyl, sulfamoyl, sulfanyl, sulfine, sulfo, thioformil, thiocarboxy, dithiocarbon, thiocarbamoyl, cyano, azide, nitro, nitro , hydrazino, ureido, amidino, guanidino, trialquilsilila, alkyl, alkenyl, alkynyl, haloalkyl, alkyloxy, alkenyloxy, alkynyloxy, haloalkoxy, alkoxyalkyl, alkoxyalkoxy, alkylcarbonyl, alquenilcarbonila, alquinilcarbonila, monoalkylamino, dialkylamino, alquilsulfonila, alquenilsulfonila, alquinilsulfonila, monoalquilcarbonilamino, dialquilcarbonilamino , monoalkylsulfonylamino, dialkylsulfonylamino, alkylimino, alkenylimino, alkynylimino, alkylcarbonylimino, alkenylcarbonylimino, alkynylcarbonylimino, alkoxyimino, alkenyloxyPetition 87Ο29ΟΦ3Θ082, from 10 / Φ3 / 2 3T / 22S

27/187 imino, imino-alkynyloxy, alkylcarbonyloxy, alkenylcarbonyloxy, alquinilcarbonilóxi, alkoxycarbonyl, alqueniloxicarbonila, alquiniloxicarbonila, alquilsulfanila, alquenilsulfanila, alquinilsulfanila, alquilsulfinila, alquenilsulfinila, alquinilsulfinila, monoalquilcarbamoíla, dialquilcarbamoíla, monoalquilsulfamoíla, dialquilsulfamoíla, carbociclila aromatic, non-aromatic carbociclila, aromatic heterocyclyl , non-aromatic heterocyclyl, aromatic carbocyclyloxy, non-aromatic carbocyclyloxy, aromatic heterocyclyloxy, non-aromatic heterocyclyloxy, aromatic carbocyclylcarbonyl, non-aromatic heterocyclyl, aromatic heterocyclyl, carbocyclycylcyclohexylcarbonyl, aromatic, carbocyclohexylcarbonyl non-aromatic carbocycloalkyl, aromatic heterocycloalkyl, heterocycle alkyl non-aromatic, aromatic carbocicloalcóxi, carbocicloalcóxi non-aromatic, aromatic heterocycloalkoxy, heterocycloalkoxy non-aromatic, aromatic carbociclicoalcoxicarbonila, non-aromatic carbocicloalcoxicarbonila, aromatic heterocicloalcoxicarbonila, non-aromatic heterocicloalcoxicarbonila, aromatic carbocicloalcoxialquila, non-aromatic carbocicloalcoxialquila, aromatic heterocicloalcoxialquila, non-aromatic heterocicloalcoxialquila aromatic carbocicloalquilamino, carbocicloalquilamino non-aromatic, aromatic heterocycloalkylamino, non-aromatic heterocycloalkylamino, aromatic carbocarbocyclosulfanyl, non-aromatic carbocarbocyclosulfanyl, aromatic heterocarbocyclosulfanyl, non-aromatic heterocarbocyclosulfanyl, aromatic carbocarbocyclobromoscarbonate, aromatic carbocarbocyclic and aromatic. Furthermore, the substituent may have one or more substituents selected from Group α above.

[00169] Additionally, substituted or unsubstituted non-aromatic carbocyclyl and unsubstituted or unsubstituted heterocyclyl

Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 38/220

28/187 can be replaced with oxo. In this case, it means a group in which two hydrogen atoms in a carbon atom are replaced as below

[00170] Here, the present invention is explained in detail.

[00171] A lipid-linked double-stranded oligonucleotide of the present invention consists of a double-stranded oligonucleotide, in which:

[00172] a first strand is a CpG oligonucleotide consisting of 8 to 50 nucleotides, [00173] a second strand is an oligonucleotide consisting of 8 to 60 nucleotides and comprising a sequence capable of hybridizing to the first strand and [00174] a lipid attaches to the second strand through a linker.

[00175] More preferably, it is a double-stranded oligonucleotide, in which:

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 to the first strand, but excluding an RNA oligonucleotide, the length the second strand is 50% or more than that of the first strand and a lipid comprising hydrocarbon chain (s) C12 to C30 is attached to the second strand via a binder.

[00176] The first strand of a lipid-linked double-stranded oligonucleotide of the present invention is a CpG oligonucleotide consisting of 8 to 50 nucleotides.

Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 39/220

29/187 [00177] The CpG oligonucleotide (CpG ODN) means a single-stranded oligonucleotide comprising a dinucleotide with unmethylated cytosine-guanine motif (5'-CpG-3 ') (CpG motif), and is well known that it may be useful as a vaccine adjuvant because it induces an immune response acquired through TLR9 (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 can comprise several CpG motifs.

[00178] The length of the CpG oligonucleotide used for the present invention is 8 to 50 nucleotides. For example, it is 8 to 50 nucleotides, 8 to 40 nucleotides, 8 to 30 nucleotides, 10 to 25 nucleotides, 15 to 25 nucleotides or 18 to 25 nucleotides.

[00179] Regarding the CpG oligonucleotide, it is not limited to the CpG oligonucleotide, which is well known for the immunostimulatory activity in this field and can be used. For example, the CpG oligonucleotide or the preparation method is described in W02006 / 065751, W02007 / 092315, W02008 / 068638, WO2010 / 067262, WO2010 / 125480, WO2014 / 047588, WO2014 / 134698, W02015 / 041318, US2011 / 030016 similar. These CpG oligonucleotides can be synthesized with reference to the methods described in the documents above.

[00180] CpG oligonucleotides are classified based on the sequence, secondary structure and effect on human peripheral blood mononuclear cells (PBMC), in class A, class B, class C, class P or class S (Advanced drug delivery reviews , 2009, 61 (3), 195-204).

[00181] Class A: ODN1585, ODN2216, ODN2336 or the like;

[00182] Class B: ODNBW006, ODN D-SL01, ODN1668 (W02005 / 063264), OND1826 (W02007 / 030580), OND2006 (CpG7909, PF-3512676) (WO98 / 18810), ODN2007, ODN684 or simNPet / Φ3 / 2Ο29, p. âê / 220

30/187 homes; and [00183] Class C: ODN D-SL03, ODN 2395, ODN M362 or the like. These can be purchased from InvivoGen as a research reagent. In addition, [00184] CpG-28 (W02000 / 056342), [00185] CpG-685 (GNKG-168) (Blood, 2010, 115 (24), 5041), [00186] CpG-ODN C274 (PLoS ONE, 2013, 8 (4), e62373), [00187] KSK-13 (KSK-CpG) (US7408050), [00188] CpG ODN 10104 (CpG-10104) (Drug Data Rep, 2006, 28 (3), 258) , [00189] CpG ODN-1585 (WO2001 / 022990), [00190] ODN-5890 (W02006 / 080946), [00191] 1018-ISS (W02008 / 073661), [00192] EMD-1201081 (HYB-2055, IMO -2055) (W02005 / 009355), [00193] D35-CpG, K3-CpG (GeneDesign, Inc.), [00194] or the like. For the present invention, CpG oligonucleotides of any class can be used. Class A CpG oligonucleotides (eg ODN2216, ODN2336 and D35CpG), class B CpG oligonucleotides (eg ODN1826, ODN2006, CpG-28, 1018-ISS, IMO2055, K3-CpG, ODN684 and D-LS01 ) or CpG class C oligonucleotides (for example, D-LS03, ODN2395 and ODN M362) are preferable. ODN 1826 or ODN2006 is especially preferable.

[00195] A second strand of a lipid-linked double-stranded oligonucleotide of the present invention is an oligonucleotide consisting of 8 to 60 nucleotides and comprising a sequence capable of hybridizing to the CpG oligonucleotide which is the first strand, but excluding an oligonucleotide of RNA.

[00196] Preferably, a second strand is an oligonucleotide consisting of 8 to 60 nucleotides and comprising a sequence

Petition 870290 (039082, of 10 / Φ3 / 2Ο29, page ií / 220

31/187 capable of hybridizing to the CpG oligonucleotide which is a first strand under strict conditions.

[00197] Any oligonucleotide can be used for a second strand as long as it can hybridize with the CpG oligonucleotide under strict conditions, and one that comprises 1 or several mismatches in a hybridization part can be exemplified.

[00198] 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 even more preferably 95% or more homology with a complementary sequence of the oligonucleotide CpG of a first tape.

[00199] The homology shows similarity as a score, for example, by BLAST, a search program using an algorithm discovered by Altschul et al. (The Journal of Molecular Biology, 215, 403-410 (1990)) [00200] Strict conditions mean conditions under which a sequence of bases forms a hybrid (the so-called specific hybrid) with a specific sequence, but any sequence of bases without the equivalent function it does not form a hybrid (the so-called non-specific hybrid) with the specific sequences. Those skilled in the art can easily select conditions by changing a temperature during the hybridization or wash reaction, the concentration of salt in the hybridization or wash buffer, or the like. In detail, an example of the stringent conditions of this invention is, but not limited to, the oligonucleotide is hybridized in 6xSSC (NaCI 0.9, trisodium citrate 0.09 M) or 6 * SSPE (NaCI 3 M, NaH2PO4 0.2 M , 20 mM EDTA-2N, pH 7.4) at 42 ° C and washed with 0.5xSSC at 42 ° C. As a method of hybridization, methods well known in the art, for example, hybridization by Southern Blot or simi

Petition 87Ο29ΟΦ3Θ082, of 10 / (03/2029, page iS / 220

32/187 homes can be used. In detail, hybridization can be performed according to a method described in Molecular Cloning: A Laboratory Manual, second edition (1989) (Cold Spring Harbor Laboratory Press), Current Protocols in Molecular Biology (1994) (WileyInterscience), DNA Cloning 1: Core Techniques, A Practical Approach, second edition (1995) (Oxford University Press) or similar.

[00201] 1 or several pairing errors means 1 to 5, preferably 1 to 3 and more preferably 1 or 2 pairing errors.

[00202] The length of a second strand of a lipid-linked 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 30 nucleotides, 10 to 25 nucleotides or 15 to 25 nucleotides. The length of the second strand can be equal to that of the CpG oligonucleotide which is a first strand, and 1 or several nucleotides shorter than that of the CpG oligonucleotide as long as it can be hybridized with the CpG oligonucleotide. In addition, the length of a second strand can be longer than that of the CpG oligonucleotide, adding 1 or several nucleotides to one or both sides of a part that hybridizes to the CpG oligonucleotide.

[00203] 1 or several nucleotides means 1 to 10, 1 to 5, 1 to 3 or 1 or 2 nucleotides.

[00204] A preferable length of a second strand depends on the length of the CpG oligonucleotide of a first strand. For example, its length is 50% or more, 60% or more, 70% or more, 50 to 100%, 60 to 100% or 70 to 100% the length of a first tape. 50 to 100% of the length of a first tape is especially preferable.

[00205] The second-stranded oligonucleotide of a lipid-linked double-stranded oligonucleotide of the present invention is an oligonuPetition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. Í3 / 22S

33/187 cleotides to which nucleosides selected from what consists of DNA nucleosides, nucleosides of RNAs and nucleoside derivatives are linked. All nucleosides can be the same, or two or more nucleosides. However, an RNA oligonucleotide is excluded in which all nucleosides are RNA nucleosides. As a nucleoside comprised on a second strand, an oligonucleotide to which DNA nucleosides and / or nucleoside derivatives are linked is preferable. All nucleosides can be DNA nucleosides, derived from nucleosides, or both.

[00206] When an oligonucleotide comprises DNA nucleosides and nucleoside derivatives, the example is an oligonucleotide comprising a central region and end regions on both sides of the central region and comprising at least one nucleoside derivative in the end region on both sides . In detail, the region (s) of the 5 'and / or 3' ends comprise (m) 1 or more, preferably 1 to 5 and more preferably 2 to 3 nucleoside derivatives. The type, number and position of the modification (s) in one region at the end can be the same or different from those in the region at the other end. In another embodiment, it is an oligonucleotide that comprises nucleoside derivatives at random.

[00207] As a nucleoside derivative in a second strand of a lipid-linked double-stranded oligonucleotide of the present invention, any modification to a nucleoside that is well known in this field, such as the examples above, can be used.

[00208] A nucleoside with a substituent at the 2 'position of the sugar and / or a nucleoside with a bridge structure between the 4' and 2 'positions of the sugar are preferable.

[00209] As a substituent in the 2 'position of a sugar, it is preferable

F, OCHs or OCH2CH2OCH3. OCH3 is especially preferable.

[00210] As a bridge structure between the 4 'and 2' positions of a

Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. ^ 8/220

34/187 sugar, 4 '- (CH2) mO-2' is preferred, where m is an integer from 1 to 4, or 4'-C (= O) -NR ³ -2 ', where R ³ it is hydrogen atom or alkyl. [00211] As an internucleoside linkage in the oligonucleotide of a second strand of a lipid-linked double-stranded oligonucleotide of the present invention, any well-known internucleoside linkage, as the examples above, in this field can be used. All internucleoside bonds can be the same, or two or more types of bonds. D-oligo and / or S-oligo are preferable.

[00212] When 2 or more types of internucleoside linkages are comprised, such as D-oligo and S-oligo, the example is an oligonucleotide comprising a central region and final regions on both sides of the central region, and comprising at least one unnatural internucleoside bond (eg, S-oligo) in the end region on both sides and a natural internucleoside bond (eg, D-oligo) in the center region. For example, the region (s) of the 5 'and / or 3' ends comprises (m) 1 or more, preferably 1 to 5 and more preferably 2 to 3 unnatural internucleoside bonds. The type, number and position of the modification (s) in one end region can be the same or different from those in the other end region. In another embodiment, it is an oligonucleotide that comprises randomly unnatural internucleoside bonds.

The first-stranded CpG oligonucleotide and the second-stranded oligonucleotide in a lipid-linked 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 a commercially available automatic nucleic acid synthesizer (for example, the AppliedBiosystems synthesizer, Dainippon Seiki). One method for synthesis is solid phase synthesis using phosphoramidite, solid phase synthesis

Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. Í0 / 22S

35/187 using hydrogen phosphonate or the like. Examples are described in Example 1 below, in Tetrahedron Letters 22, 1859-1862 (1981) and the like.

[00214] The first and second synthesized strands form a double stranded oligonucleotide by hybridization according to the well-known method. Examples are described in Example 1 below, in Example 1 in WO2013 / 089283 and the like.

[00215] In the lipid-linked double-stranded oligonucleotide of the present invention, a lipid attaches to the second strand through a linker.

[00216] Lipid means a hybrophobic compound. It is not limited as long as it is a well-known lipid, and can be in linear, branched or cyclic form. Examples are fatty acids with aliphatic chain (s) C8 to C30 (eg farnesol), diacyl-lipid, cholesterol, cholesterol derivatives, steroid acids (eg bile acid), lipid A, tocopherol and a combination thereof. Examples of fatty acids with aliphatic chain (s) are, inter alia, unsaturated or saturated fatty acid in linear or branched form and derivatives of fatty acids (for example, fatty acid esters, fatty acid amides and thioesters fatty acids).

[00217] As a lipid used for the present invention, a lipid comprising hydrocarbon chain (s) is preferred. Preferably, it is a lipid comprising 1 or 2 hydrocarbon chains or the like. A lipid-linked double-stranded oligonucleotide of the present invention with a diacyl-lipid is especially preferable. When lipids bond in two or more positions and one lipid is a diacyl-lipid, the other lipid can be any lipid except a diacyl-lipid.

[00218] Diacil-lipid is a phospholipid, glycolipid, sphingolipid or a combination thereof that comprises two hydrocarbon chains. Preferably, it is the group of (a) or (f) following.

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36/187 [00219] The hydrocarbon chains in the lipid independently comprise approximately C8 to C30 carbon atoms. They can be saturated, unsaturated or a combination of them and branched. When the lipid has a chain, the chain length is preferably C8 to C30 and more preferably C8 to C20. When the lipid has two chains, the length of the chains is preferably from C10 to C30, more preferably C12 to C30 and even more preferably C14 to C24. When the lipid has two chains, the length of the chains can be the same or different. The chains on a lipid attach to a moiety comprising phosphoric acid, sugar or the like (a binding position with an oligonucleotide) via ester bond, amide bond, thioester bond, a combination thereof or the like.

[00220] A lipid-linked double-stranded oligonucleotide of the present invention has a lipid comprising hydrocarbon chain (s) C12 to C30 on a second strand. When lipids bond at 2 or more positions and a lipid is a lipid comprising C12 to C30 hydrocarbon chain (s), the number of carbon atoms in one or more hydrocarbon chains in the other lipid may be below 12.

[00221] Examples of lipid are as follows:

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[00222] where p and q are each independently an integer from 6 to 28, preferably 8 to 28, more preferably 10 to 28 and even more preferably 10 to 18.

[00223] In a lipid-linked double-stranded oligonucleotide of the present invention, a lipid can attach at any position on the second strand. It can attach to the 3 'end, the 5' end or the second ribbon.

[00224] When the lipid binds at the 5 'end, for example, it can bind as below.

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where B ¹ is a base at the 3 'end of the second ribbon, Y ^a is O or S ep or q is each independently an integer from 6 to 28, preferably 10 to 18.

[00225] When the lipid attaches to the 3 'end, for example, it can attach as below.

where B ¹ is a base at the 5 'end of the second ribbon, Y ^a is O or S ep or q is each independently an integer from 6 to 28, preferably 10 to 18.

[00226] When the lipid binds on a second ribbon, for example, it can bind as below.

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or

where B ¹ and B ² are the neighboring bases on the second strip, Y ^a or Y ^b is each independently O or S epeq is each independently an integer from 6 to 28, preferably 10 to 18.

[00227] Furthermore, the bound lipid may preferably be in one or two positions on the second strip. It preferably binds at the 3 'and / or 5' end. It most preferably binds at the 5 'end.

[00228] The lipid can be synthesized with reference to methods well known in this field. Examples of the lipid or preparation methods are described in Example 1 below, in Patent Document 1 and the like.

[00229] In a lipid-linked double-stranded oligonucleotide of the present invention, the lipid attaches to a second strand through a linker. As a binder, any binder used in this field may have 870290 (039082, from 10 / Φ3 / 2Ο29, page 50/220

40/187 to be used. Examples are polar linker, alkylene linker, ethylene glycol linker and ethylenediamine linker. When a lipid is a phospholipid, it is a linker with 4 to 26 atoms between the oxygen atom of a second ribbon and the phosphor atom of a lipid. Examples are an oligonucleotide linker or the following linkers.

Y '

Oligonucleotide - p ------- l Lipid

OH

Oligonucleotide

Oligonucleotide

H where Y 'is O or S and r or s is each independently an integer from 1 to 10, preferably 1 to 5 and more preferably 1 to

3. t is an integer from 1 to 4, preferably 1 to 3 and more preferably 2 or 3.

[00230] The ligand can be synthesized with reference to the method well known in this field. The oligonucleotide linker can be synthesized by the method similar to the method for synthesizing the oligonucleotides in the example above.

[00231] 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 as follows.

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Lipid

Oligonucleotide in which B ¹ or B ² is adenine (A), guanine (G), cytosine (C), 5-methylcytosine (5-Me-C), thymine (T) or uracil (U). Y 'is each independently O or S. Z ¹ or Z ² is each independently H or OH, preferably H.

[00232] An example of an oligonucleotide ligand is a DNA ligand, that is, - (dX ¹ ) u-, where X ¹ is each independently A, G, C or T eu is an integer of 1 to 8. In detail, it is dG, dGdG, dGdGdGdG, dGdGdGdGdG, dT, dTdT, dTdTdTdT, dTdTdTdTdT or similar. dGdG, dGdGdGdGdG or dTdT is especially preferable. The internucleoside bond in the DNA ligand is preferably a phosphorothioate bond.

The 3 'end, the 5' end or the linker without the lipid-binding lipid-bound oligonucleotide of the present invention can be further modified. In order to be able to trace the oligonucleotide, to improve the pharmacokinetics or pharmacodynamics of the oligonucleotide or to increase the stability or binding affinity of the oligonucleotide, the modified group well known in this field can be used. Examples are a hydroxyl protecting group, a reporter molecule, cholesterol, phospholipid, a pigment, a fluorescent molecule and the like.

[00234] In addition, the lipid-free 3 'or 5' end of a lipid-linked 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 portion can be at either end, it is preferably used in 10 / Φ3 / 2Ο29, p. êS / 220

42/187 nucleoside 5 'end. 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'), where R 'is H, substituted or unsubstituted alkyl or alkyl protecting group or alkyl. The 5 'or 3' end may each independently comprise 1 to 3 substituted or unsubstituted phosphate ester moieties.

[00235] 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-linked double-stranded oligonucleotide of the present invention (a vaccine or adjuvant of the present invention) invention of the present invention).

[00236] Furthermore, the present invention encompasses a pharmaceutical or vaccine composition comprising an antigen and a lipid-bound double-stranded oligonucleotide of the present invention (an adjuvant to the present invention).

[00237] 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, peptides, polysaccharides, polysaccharide conjugates, peptide or non-peptide polysaccharide mimetics, other molecules encoded by plasmid DNA, haptens, small molecules, lipids, glycolipids, carbohydrates, hydrated pathogens, whole dead pathogens, viruses, viral extracts, live attenuated viruses, viral vectors, live attenuated bacteria, bacterial vectors, multicellular organisms such as parasites and allergens.

[00238] The antigen can be provided as a single antigen or a combination of antigens. The antigen can be provided as a complex mixture of polypeptides or oligonucleotides.

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43/187 [00239] Antigens include, but are not limited to, microbial antigens, autoantigens and addictive substances.

[00240] A microbial antigen means an antigen of a microorganism, and the microorganism includes, among others, bacteria, viruses, parasites and fungi.

[00241] There are no special restrictions on bacteria as long as a disease-causing bacteria in humans, pets, farm animals or other animals. In detail, it is Streptococcus (Streptococcus pyogens, Streptococcus pneumoniae or similar), Staphylococcus aureus (MSSA, MRSA or similar), Staphylococcus epidermidis, Enterococcus, bacteria belonging to the genus Listeria, bacteria causing meningitis, Gonococcus, Escherichia, Escherichia Proteus bacillus, Bordetella pertussis, Pseudomonas aeruginosa, Serratia marcescens, Citrobacter, Acinetobacter, Enterobacter, Mycoplasma, Clostridia, tuberculosis bacillus, cholera bacillus, Yersinia pestis, Corynebacterium difteriae, dysentery bacillus, bacillus de treacone, bacillus anthrone , Mycobacterium leprae, Legionella pneumophila, Leptospira, Borrelia, Francisella, Coxiella, Rickettsia, Chlamydia, Burkholdeha mallei, Helicobacter pylori or the like.

[00242] There are no special restrictions regarding the virus as long as it is a disease-causing virus in humans, pets, livestock or similar. Examples are Influenza virus, respiratory syncytial virus (RSV), papillomavirus, hepatitis virus (type A, B, C, D, E, F, G, TT or similar), rhinovirus, smallpox virus, morbilivirus, 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-cell lymphotropic virus , yellow fever virus, cytomegalovirus, SARS-CoV, coronavirus as

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MERS-CoV, ebola virus, polyomavirus, JC virus, BK virus, herpes virus such as herpes simplex virus 1 (HSV1) and herpes simplex virus 2 (HSV2), lymphocriptovirus, roseola virus, Japanese encephalitis virus, virus Coxsackie, dengue virus, West Nile virus, coronavirus, parvovirus, Epstein-Barr virus, Marburg virus, hantavirus, Lassa fever virus, chicungunha virus, Hantaan virus, ovine encephalomyelitis virus (louping ill), choriomeningitis virus lymphocyte, bornavirus, Rift valley fever, Thogoto virus, Dhori virus, foot and mouth disease virus, Newcastle disease virus, bovine papular stomatitis virus, bovine plague virus, swine vesicular disease virus, calicivirus, torovirus, plague virus African horse, arterivirus, smallpox virus, capripoxvirus, sheep bluetongue virus, viral hemorrhagic septicemia virus and vesicular stomatitis virus.

[00243] There are no special restrictions on the parasite as long as it is a parasite that causes disease in humans, pets, livestock or similar. Examples are Entamoeba histolytica, which causes malaria, toxoplasmosis, leischmaniasis, Cryptosporidium, Trypanosoma, Echinococcus, Schistosoma japonicum, filariasis, ascaris and Diphyllobothrium.

[00244] There are no special restrictions on the fungus as long as it is a disease-causing fungus in humans, pets, farm animals or the like. In detail, it is Aspergilloma, Candida, Cryptococcus, Trichophyton, Histoplasma, Pneumocystis or similar.

[00245] An autoantigen 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.

[00246] An example of tumor antigen is a specific antigen 870290 (039082, from 10 / Φ3 / 2Ο29, p. 49/220

45/187 expressed in tumor cells. Examples include protein, peptide and fusion peptide that comprises them. Examples are peptides described in W02006 / 090810, WO2007 / 145318,

W02008 / 047473,

W02009 / 025196,

WO2010 / 021112,

W02010 / 131452,

WO2011 / 074236,

WO2012 / 053200,

WO2013 / 024582,

W02008 / 102557, W02009 / 1539992, WO2010 / 073551, WO2010 / 137295, WO2011 / 089921, WO2012 / 053206, WO2013 / 061594,

W02009 / 025117, WO2010 / 013485, WO2010 / 095428, WO2011 / 067920, WO2011 / 111392, WO2012 / 169200, WO2014 / 041784,

WO2014 / 087626 and the like.

[00247] An example of an antigen associated with Alzheimer's disease is tau, β-amyloid or the like.

[00248] An example of an antigen against a human antibody is igE.

[00249] An addictive substance means nicotine, cocaine or similar. An example of a nicotine antigen is a nicotine hapten conjugated to a vehicle (for example, diphtheria toxin).

[00250] A pharmaceutical or vaccine composition of the present invention can further comprise one or more well-known adjuvants as long as the effect of the lipid-linked double-stranded oligonucleotide of the present invention is maintained. For example, it is choleric toxin, Salmonella toxin, aluminum or a non-TLR9 agonist of a Toll-like receptor (TLR). Examples of TLR agonists are a TLR3 agonist as a stabilized poly (1: C); a TLR4 agonist as a lipopolysaccharide (LPS) derivative (for example, MPL and GLA); a TLR5 agonist like flagellin; a TLR7 agonist; and a TLR8 agonist. Examples include aluminum salt such as aluminum hydroxide, an immunostimulating complex (ISCOM), oil in water and water in oil emulsion, liposome and a

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46/187 release as nanoparticle and microparticle.

[00251] Like 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: [00252] 1% or more of CTL-inducing capacity.

[00253] Inhibition of tumor implantation.

Tumor regression effect [00254] Good pharmacokinetics as high bioavailability, moderate clearance similar. Especially, efficient delivery to lymph nodes.

[00255] High metabolic stability.

[00256] Absence of cytokine release syndrome.

[00257] Local irritation of little intensity.

[00258] Absence of mutagenicity.

[00259] Low cardiovascular risk.

[00260] Low risk of acute toxicity.

[00261] Any mode of administration and formulation for the pharmaceutical or vaccine composition of the present invention can be employed if for a mode of administration and formulation well known in the field.

[00262] A pharmaceutical or vaccine composition of the present invention can be administered in several ways depending on whether local or systemic treatment and the area to be treated are desired. Examples of mode of administration include topical (including ophthalmic, intravaginal, intrarectal, intranasal and transdermal), oral and parenteral. Examples of parenteral administration include injection or in drops, subdermal, intraperitoneal or intramuscular injection, pulmonary administration by aspiration or inhalation, intrathecal administration and intraventricular administration. Intravenous injection or subcutaneous administration is preferable.

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47/187 [00263] When the pharmaceutical or vaccine composition of the present invention is administered topically, a formulation such as transdermal patch, ointment, lotion, cream, gel, drops, suppository, spray, liquid and powder can be used.

[00264] Examples of the composition for oral administration include powder, granule, suspension or solution dissolved in water or non-aqueous vehicle, capsule, powder and tablet.

[00265] Examples of the composition for parenteral, intrathecal or intraventricular administration include sterile aqueous solutions containing buffers, diluents and other suitable additives.

[00266] A pharmaceutical or vaccine composition of the present invention can be obtained by mixing an effective amount with various pharmaceutical additives suitable for the form of administration, such as excipients, binders, wetting agents, disintegrants, lubricants and diluents as needed. When it is an injection, the composition, together with a suitable vehicle, can be sterilized to obtain a composition.

[00267] Examples of excipients include lactose, sucrose, glucose, starch, calcium carbonate and crystalline cellulose.

[00268] Examples of binders include methylcellulose, carboxymethylcellulose, hydroxypropylcellulose, gelatin and polyvinylpyrrolidone.

[00269] Examples of disintegrants include carboxymethylcellulose, sodium carboxymethylcellulose, starch, sodium alginate, agar and sodium lauryl sulfate.

[00270] Examples of lubricants include talc, magnesium stearate and macrogol. Cocoa oil, macrogol, methylcellulose or similar can be used as base materials for suppositories.

[00271] When the composition is prepared as a solution, emulsified injection or suspended injection, solubilizing agents, suspending agents, emulsifiers, stabilizers,

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48/187 preservatives, isotonic agents and the like commonly used, as needed. For oral administration, sweetening, flavoring or similar agents can be added.

[00272] The dosage is dependent on the severity and responsiveness of the disease state to be treated, with the duration of the treatment cycle from several days to several months, or until a cure or reduction of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of a pharmaceutical composition or the accumulation of the vaccine in the body. Any person skilled in the art can determine ideal doses, dosing methodologies and repetition frequencies.

[00273] The content of a lipid-linked double-stranded oligonucleotide of the present invention in a pharmaceutical or vaccine composition of the present invention is, among others, normally between 0.01 and 99.99% by weight versus 100% by weight of a pharmaceutical or vaccine composition.

[00274] When an antigen is understood, considering the content of the antigen and an adjuvant of the present invention, the adjuvant is present between about 10 and 1000 parts by weight against 1 part by weight of the antigen.

[00275] The dose of a pharmaceutical or vaccine composition is diversified depending on the desired degree of immune stimulation, the age of the individual to be administered, the sex or similar factors, so that it can be properly fixed at, for example, 0.001 and 10 mg / kg of weight / day, range to which it is not particularly limited.

[00276] In addition, according to the degree of effectiveness of the desired pharmaceutical or vaccine composition, the age of the individual to be administered, sex or similar factors, a pharmaceutical compositionPetition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 59/220

49/187 of the present invention may be administered to the same individual several times, for example, up to 2 to 4 times. When a pharmaceutical or vaccine composition of the present invention is administered to the same individual several times, the interval can be suitably fixed, for example, between approximately 14 and 30 days.

[00277] A pharmaceutical or vaccine composition of the present invention can be administered in combination with 1 or more types of drugs. The therapeutic agent, known as the therapeutic agent for the target disease in this field, can be used as the drugs. For example, when the disease is cancer, a pharmaceutical or vaccine composition of the present invention can be co-administered with one or more chemotherapeutic agents. When the disease is bacterial infection, a pharmaceutical or vaccine composition of the present invention can be co-administered with one or more antibiotics. A pharmaceutical or vaccine composition of the present invention can be administered simultaneously or separately from the agent (s). In addition, a pharmaceutical or vaccine composition of the present invention and the therapeutic agent (s) can be administered as different formulations, each independently, or as a single formulation comprising the pharmaceutical or vaccine and the therapeutic agent (s) ( s). Furthermore, when the disease is cancer, a pharmaceutical or vaccine composition of the present invention can be administered in combination with the surgical treatment known in the field.

[00278] Furthermore, the present invention encompasses a method for enhancing an immune response in an individual, comprising administering to the individual an effective amount of a pharmaceutical or vaccine composition of the present invention. An example of an immune response is an increase in the ability to induce cytotoxic T lymphocytesPetition 87Ο29ΟΦ3Θ082, 10 / Φ3 / 2Ο29, p. 60/220

50/187 specific (CTL) compared to a control. A specific method for measuring CTL induction capacity is described in, for example, Example 3 below and in WO2013 / 024582.

[00279] Furthermore, the present invention encompasses a method for treating cancer or an infectious disease, comprising administering to an individual an effective amount of a pharmaceutical or vaccine composition of the present invention to reduce one or more symptoms of cancer or infectious disease compared to a control.

[00280] The individual means any individual who is a target for treatment with a pharmaceutical or vaccine composition of the present invention. Examples include mammals such as humans, laboratory animals (for example, mice and rats), pets (for example, dogs, cats, ferrets and birds) and farmed animals (for example, bovine, swine, chickens, goats, ostriches, sheep and horses).

[00281] An effective amount means a dose sufficient to induce or increase the immune response, provide the desired pharmacological and / or physiological effect or provide a treatment for the disorder, disease or condition. The exact dose can be changed depending on several factors such as the individual's dependent variable (eg age and health status of the immune system), disease, stage of the disease and the treatment provided.

[00282] There are no special restrictions on cancer as long as it is a cancer that humans, pets, farm animals or the like can contract. Examples are chronic myeloid leukemia (CML), acute myeloid leukemia (AML), lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, brain tumor, breast cancer, endometrial carcinoma, cervical cancer, ovarian cancer, esophageal cancer , stomach cancer as canPetition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 6Í / 22S

51/187 diffuse gastric cer, appendix cancer, colorectal cancer, liver cancer, hepatocellular carcinoma, gallbladder cancer, cholangiocarcinoma, pancreatic cancer, adrenal cancer, gastrointestinal stromal tumor, mesothelioma, head and neck carcinoma, cancer of larynx, oral cancer, gingival cancer, tongue cancer, oral mucosa cancer, salivary gland cancer, paranasal cancer, maxillary sinus carcinoma, frontal sinus carcinoma, ethmoidal sinus cancer, sphenoid sinus cancer, thyroid cancer, cancer kidney, lung cancer such as non-small cell lung cancer (CPNPC) and small cell lung cancer (CPPC), osteosarcoma, prostate cancer, testicular cancer, bladder cancer, rhabdomyosarcoma, skin cancer, anal carcinoma, chondrosarcoma, synovial sarcoma, endometriosis, soft tissue tumor and osteoblastoma.

[00283] An infectious disease means acute or chronic infectious disease, especially viral infectious disease. Examples include, but are not limited to, local or systemic viral infections such as immunodeficiency by HIV or others, papilloma by HPV or others, herpes by HSV or others, encephalitis, influenza by human Influenza A virus or others, and cold by human rhinovirus.

[00284] In this description, the meaning of each abbreviation is as follows:

Ac: acetyl

CPG: Controlled pore glass DIEA: N, N-diisopropylethylamine DMAP: 4-dimethylaminopyridine DMTr: dimethoxytrityl

DMT-MM: 4- (4,6-dimethoxy1,3,5-triazine-2-yl) -4-methylmorpholine chloride

DMF: N, N'-dimethylformamide

Et: ethyl

Fmoc: 9-fluorenylmethoxycarbonyl

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HBTU: O-benzotriazole hexafluorophosphate-N, N, N ', N'-tetramethyluronium Me: methyl

MMTr: 4-methoxyphenyldiphenylmethyl

MMTrCI: 4-methoxytrityl chloride

PBS: saline solution with phosphate buffer

TBS: tert-butyldimethylsilyl

TBAF: tetrabutylammonium fluoride

TFA: trifluoroacetic acid

THF: tetrahydrofuran

Examples [00285] The present invention will be described in more detail with reference, among others, to the Examples, Reference Examples and Test Examples below.

[00286] The NMR analysis of each compound obtained in the Examples was performed with CD3OD, CDCh or DMSO-d6 at 300 MHz or 400 MHz.

[00287] UPLC analysis was performed under the following conditions:

[00288] Mobile phases: [A] is 0.1% aqueous formic acid solution, [B] is acetonitrile solution containing 0.1% aqueous formic acid [00289] Gradient: linear gradient from 5% to 100% solvent [B] performed for 3.5 minutes, and 100% solvent [B] was maintained for 0.5 minutes.

[00290] Column: ACQUITY UPLC (Trademark) BEH C18 (1.7 pm, internal diameter 2.1 x 50 mm) (Waters) [00291] Flow rate: 0.8 mL / minute [00292] Detection wavelength PDA: 254 nm (Detection range: 210-500 nm) [00293] Example 1. Synthesis of lipid-linked double-stranded oligonucleotide of the present invention

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Synthesis of lipids

Synthesis of 4-n

where n is an integer from 6 to 28.

Synthesis of Compound 3-6

Step 1 [00294] 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 half under reduced pressure and was added dropwise to the 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 a white solid.

[00295] ¹ H NMR (CDCh) δ: 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; 14.1 Hz ), 1.30-1.27 (16H, m), 0.88 (6H, t, J = 6.8 Hz).

Synthesis of Compound 4-8

Step 1 [00296] Compound 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 stirred vigorously at room temperature for 30 minutes. Compound 1 (2.0

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54/187 g, 22.2 mmol) to the resulting suspended solution at room temperature and the mixture was stirred vigorously for 3 hours. Saturated aqueous sodium bicarbonate solution (20 mL) was added to the reaction mixture and 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 a white solid.

[00297] ¹ H NMR (CDCh) δ: 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).

[00298] ESI-MS (m / z): 340 (M + 1).

Step 2 [00299] Compound 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 2 hours. After cooling to room temperature, the reaction mixture was partitioned through the separating funnel, and the organic layer diluted with dichloromethane (80 mL) was washed twice with saturated aqueous 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. The formation of the compound was determined based on the introduction of trivalent phosphorus by ³¹ P.

[00300] ³¹ P NMR (CDCh) δ: 148.2 (s)

Synthesis of Compound 4-10

Step 1 [00301] Compound 2-10 (9.78 g, 48.8 mmol, Tokyo Chemical InPetition 87Ο29ΟΦ3Θ082, from 10 / Φ3 / 2Ο29, page 60/220

55/187 dustry Co., Ltd.) was dissolved in DMF (150 ml) and dichloromethane (75 ml). DIEA (12.79 mL, 73.2 mmol) and HBTU (20.37 g, 53.7 mmol) were added thereto, and the mixture was stirred vigorously at room temperature for 45 minutes. Compound 1 (2.2 g, 24.41 mmol) was added to the resulting suspended solution at room temperature and the mixture was stirred vigorously. Then, the mixture was heated to 80 ° C and then stirred for 4 hours. Saturated aqueous sodium bicarbonate solution (200 ml) and water (50 ml) were added to the reaction mixture and 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 a white solid.

[00302] ¹ H NMR (CDCb) δ: 6.25 (2H, t, J = 5.8 Hz), 4.07 (1H, s), 3.75 (1H, s), 3.44- 3.38 (2H, m), 3.29-3.23 (2H, m), 2.22 (4H, t, J = 7.6 Hz),

1.67-1.59 (4H, m), 1.30-1.25 (64H, m), 0.88 (6H, t, J = 6.8 Hz).

Step 2 [00303] Compound 3-10 (230 mg, 0.556 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 2 hours. After cooling to room temperature, the reaction mixture was partitioned through the separating funnel, and the organic layer was washed twice with saturated aqueous 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. The formation of the compound was determined based on the introduction of trivalent phosphorus by NMR31 p

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56/187 [00304] ³¹ P NMR (CDCb) δ: 148.2 (s)

Synthesis of Compound 4-12

Step 1 [00305] Compound 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 (5.81 mL, 33.3 mmol) and HBTU (9.26 g, 24.4 mmol) were added thereto, and the mixture was stirred vigorously at room temperature for 30 minutes. Compound 1 (1.0 g, 11.1 mmol) was added to the resulting suspended solution at room temperature and the mixture was stirred vigorously. Then, the mixture was heated to 40 ° C and then stirred for 2 hours. Saturated aqueous sodium bicarbonate solution (10 mL) was added to the reaction mixture and 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 a white solid.

[00306] ¹ H NMR (CDCb) δ: 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) [00307] ESI-MS (m / z): 512 (M + 1).

Step 2 [00308] Compound 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 (4.46 mL, 20.0 mmol) 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 through the separating funnel, and the organic layer was washed twice with saturated aqueous sodium bicarbonate solution (100 mL), twice with water (100 mL) and

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57/187 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, 6.75 mmol), was obtained as a crude product. The formation of the compound was determined based on the introduction of trivalent phosphorus by NMR31 p [00309] NMR- ³¹ P (CDCh) δ: 148.2 (s)

Synthesis of Compound 4-14 [00310] Compound 2-14 (12.52 g, 48.8 mmol, Tokyo Chemical Industry Co., Ltd.) was dissolved in DMF (150 ml) and dichloromethane (75 ml). DIEA (12.79 mL, 73.2 mmol) and HBTU (20.37 g, 53.7 mmol) were added thereto, and the mixture was stirred vigorously at room temperature for 2.5 hours. Compound 1 (2.2 g, 24.41 mmol) was added to the resulting suspended solution at room temperature and the mixture was stirred vigorously. Then, the mixture was heated to 80 ° C and then stirred for 5 hours. Saturated aqueous sodium bicarbonate solution (800 ml) and water (50 ml) were added to the reaction mixture and the resulting white solid was collected by filtration. The resulting 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 a white solid.

[00311] ¹ H NMR (CDCh) δ: 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 2 [00312] Compound 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 (0.870 mL, 3.53 mmol) was added at room temperature and the mixture

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58/187 was heated under reflux for 2 hours. After cooling to room temperature, the reaction mixture was diluted with dichloromethane (50 ml). The solution was partitioned through the separating funnel, and then the organic layer was washed twice with saturated aqueous 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. Compound formation was determined based on the introduction of trivalent phosphorus by ³¹ P NMR. [00313] ³¹ P NMR (CDCh) δ: 148.2 (s)

Synthesis of Compound 4-16 [00314] According to the methods described in Non-Patent Document 3, Compound 3-16 was synthesized from Compound 216 and then Compound 4-16 was synthesized.

[00315] Compound 3-16: ¹ H NMR (CDCh) δ: 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 = 8.0 Hz) [ 00316] Compound 4-16: ³¹ P NMR (CDCh) δ: 148.2 (s)

Synthesis of Compound 4-18

Step 1 [00317] 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 (16.3 mL, 93 mmol) and HBTU (18.5 g, 48.8 mmol) were added thereto, and the mixture was stirred vigorously at room temperature for 30 minutes. Compound 1 (2.0 g, 22.2 mmol) was added to the resulting suspended solution at room temperature and the mixture was stirred vigorously. Then, the mixture was heated to 40 ° C and then stirred for 2 hours. Saturated aqueous sodium bicarbonate solution (10 mL) was added to the reaction mixture and the solid

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The resulting white 59/187 was collected by filtration. The resulting solid was washed with water (100 ml), acetonitrile (100 ml) and dichloromethane (100 ml) to obtain Compound 3-18 (10.0 g, 14.7 mmol) as a white solid.

[00318] ¹ H NMR (CDCh) δ: 6.20 (brs, 2H), 3.96 (m, 1H), 3.75 (m, 1H), 3.42 (m, 2H), 3, 23 (m, 2H), 2.22 (m, 4H), 1.68-1.20 (m, 68H), 0.88 (m, 6H) Step 2 [00319] Compound 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 (3.74 mL, 16.79 mmol) 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 through the separating funnel and the organic layer was washed twice with saturated aqueous 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 acetonitrile was added to the residue, the white solid was collected by filtration. The resulting solid was washed with saturated aqueous sodium bicarbonate solution, water and acetonitrile to obtain Compound 4-18 (4.13 g, 4.70 mmol) as a white solid. The formation of the compound was determined based on the introduction of trivalent phosphorus by ³¹ P NMR.

[00320] ³¹ P NMR (CDCh) õ: 148.2 (s)

Synthesis of Compound 4-20

Step 1 [00321] Compound 2-20 (10.2 g, 30.0 mmol, Tokyo Chemical Industry Co., Ltd.) was dissolved in DMF (250 ml) and dichloromethane (250 ml). DIEA (7.86 mL, 45 mmol) and HBTU (12.52 g, 33.0 mmol) were added thereto, and the mixture was stirred vigorously at time 870290 (039082, 10 / Φ3 / 2Ο29, pg. 00 / 220

60/187 ambient temperature for 30 minutes. Compound 1 (1.35 g, 15.0 mmol) was added to the resulting suspended solution at room temperature and the mixture was stirred vigorously. Then, the mixture was heated to 40 ° C and then stirred for 2 hours. Saturated aqueous sodium bicarbonate solution (50 ml) was added to the reaction mixture and 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 a white solid.

[00322] ¹ H NMR (CDCh) δ: 6.18 (brs, 2H), 3.75 (m, 1H), 3.41 (m, 2H), 3.27 (m, 2H), 2, 22 (m, 4H), 1.58-1.25 (m, 76H), 0.89-0.86 (m, 6H) Step 2 [00323] Compound 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 vigorous stirring. The precipitated solid was collected by filtration, being washed twice with saturated aqueous 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 a white solid. The formation of the compound was determined based on the introduction of trivalent phosphorus by ³¹ P NMR.

[00324] ³¹ P NMR (CDCh) δ: 148.2 (s)

Synthesis of Compound 4-22

Step 1 [00325] Compound 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

Petition 870290 (039082, of 10 / Φ3 / 2Ο29, p. 05/220

61/187 added to it, and the mixture was stirred vigorously at room temperature for 30 minutes. Compound 1 (1.07 g, 11.9 mmol) was added to the resulting suspended solution at room temperature and the mixture was stirred vigorously. Then, the mixture was heated to 40 ° C and then stirred for 2 hours. Saturated aqueous sodium bicarbonate solution (50 ml) was added to the reaction mixture and 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 a white solid. [00326] ¹ H NMR (CDCh) δ: 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 2 [00327] Compound 3-22 (1.0 g, 1.36 mmol) was suspended in chloroform (50 mL), and DIEA (0.713 mL, 4 , 08 mmol) was added to it. Then, 2-cyanoethyl N, N-diisopropylchlorophosphoramidite (0.607 mL, 2.72 mmol) 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 vigorous stirring. The precipitated solid was collected by filtration, being washed twice with saturated aqueous 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 a white solid. The formation of the compound was determined based on the introduction of trivalent phosphorus by ³¹ P.

[00328] ³¹ P NMR (CDCh) δ: 148.2 (s)

Synthesis of Compound 4-n, the

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62/187

ο

4-η, ο where η or ο is an integer from 6 to 28.

1-2-1) Synthesis of Compound 4-8.18

Step 1 [00329] Compound 2-8 is dissolved in DMF and solution. Dl EA and HBTU are added thereto, and the mixture is stirred at room temperature. Compound 1 is added to the resulting suspended solution at room temperature and the mixture is stirred. The activated solution of Compound 2-18, which is prepared separately [Compound 2-18 is dissolved in DMF and solution. DIEA and HBTU are added to it and the mixture is stirred at room temperature], it is added to the reaction vessel and the mixture is stirred at room temperature. The mixture is heated to 40 ° C and then stirred. Saturated aqueous sodium bicarbonate solution is added to the reaction mixture and the resulting solid is collected by filtration. The resulting solid is washed with water, acetonitrile and dichloromethane to obtain Compound 6-8,18.

Step 2 [00330] Compound 6-8.18 is suspended in chloroform, and DIEA is added to it. Then, 2-cyanoethyl N, N-diisopropylchlorophosphoramidite is added at room temperature and the mixture is heated under reflux. After cooling to room temperature, the reaction mixture is added dropwise to the acetonitrile under stirring. The precipitated solid is collected by filtration, being washed twice with SoPetition 87ΟΦ29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, pg. 03/220

63/187 saturated solution of sodium bicarbonate, twice with water and twice with acetonitrile. The resulting solid is dried under reduced pressure to obtain Compound 4-8.18. Compound formation is determined based on the introduction of trivalent phosphorus by ³¹ P NMR.

2) Synthesis of Compound 8-n

where n is an integer from 6 to 28.

2-1) Synthesis of Compound 8-6 [00331] Compound 7-6 (1.00 g, 7.68 mmol, Tokyo Chemical Industry Co., Ltd.) was dissolved in dichloromethane (15 mL), and triethylamine ( 2.13 mL, 15.4 mmol) was added thereto. Then, 2-cyanoethyl N, N-diisopropylchlorophosphoramidite (1.71 ml, 7.68 mmol) was added at room temperature and the mixture was stirred at room temperature for 3 hours. The reaction was stopped by a saturated aqueous solution of sodium bicarbonate and the mixture was extracted twice with ethyl acetate (50 ml). The organic layer was washed once with saturated aqueous sodium bicarbonate solution (10 ml), three times with water (10 ml) and once with brine (10 ml) and then dried with magnesium sulfate. The organic layer was concentrated under reduced pressure to obtain a brown oil, Compound 8-6 (2.60 g) as a crude product. The formation of the compound was determined based on the introduction of trivalent phosphorus by ³¹ P NMR.

[00332] ³¹ P NMR (CDCh) δ: 147.2 (s)

2-2) Synthesis of Compound 8-10 [00333] Compound 7-10 (1.00 g, 5.37 mmol, Tokyo Chemical Industry Co., Ltd.) was dissolved in dichloromethane (15 mL), and triethylamine ( 1.49 mL, 10.7 mmol) was added thereto. Then, add Petition 870290 (039082, from 10 / Φ3 / 2Ο29, page 08/220

64/187 2-cyanoethyl Ν, Ν-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 a saturated aqueous solution of sodium bicarbonate and the mixture was extracted twice with ethyl acetate (50 ml). The organic layer was washed once with saturated aqueous sodium bicarbonate solution (10 ml), three times with water (10 ml) and once with brine (10 ml) and then dried with magnesium sulfate. The organic layer was concentrated under reduced pressure to obtain a brown oil, Compound 8-10 (2.09 g) as a crude product. The formation of the compound was determined based on the introduction of trivalent phosphorus by ³¹ P NMR.

[00334] ³¹ P NMR (CDCh) δ: 147.2 (s)

2-3) Synthesis of Compound 8-12 [00335] Compound 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 a saturated aqueous solution of sodium bicarbonate (20 mL) and then the mixture was partitioned through the separating funnel. After being washed once with water (100 ml), the organic layer was washed once with saturated aqueous sodium bicarbonate solution (100 ml) and twice with water (100 ml) and then dried with magnesium sulfate . The organic layer was concentrated under reduced pressure to obtain a brown oil, Compound 8-12 (4.80 g, 11.6 mmol) as a crude product. The formation of the compound was determined based on the introduction of trivalent phosphorus by ³¹ P NMR.

[00336] ³¹ P NMR (CDCh) δ: 147.3 (s)

2-4) Synthesis of Compound 8-14

Petition 870290 (039082, dated 10 / Φ3 / 2Ο29, page 09/220

65/187 [00337] Compound 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-diisopropylchlorophosphoramidite (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 a saturated aqueous solution of sodium bicarbonate and the mixture was extracted twice with ethyl acetate (50 ml). The organic layer was washed once with saturated aqueous sodium bicarbonate solution (10 ml), three times with water (10 ml) and once with brine (10 ml) and then dried with magnesium sulfate. The organic layer was concentrated under reduced pressure to obtain a brown oil, Compound 8-14 (1.87 g). The formation of the compound was determined based on the introduction of trivalent phosphorus by ³¹ P NMR.

[00338] ³¹ P NMR (CDCh) δ: 147.2 (s)

2-5) Synthesis of Compound 8-16 [00339] Compound 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 (4.91 mL, 22.00 mmol) was added at room temperature and the mixture was stirred at room temperature for 12 hours. The reaction was stopped with saturated aqueous sodium bicarbonate solution (20 mL) and the mixture was then partitioned through the separating funnel. After being washed with water (100 ml), the organic layer was washed once with saturated aqueous sodium bicarbonate solution (100 ml) and twice with water (100 ml) and then dried with magnesium sulfate. The organic layer was concentrated under reduced pressure to obtain a brown oil, Compound 8-16 (4.60 g, 9.77 mmol) as a crude product. The formation of the compound was determined based on the introduction of trivalent phosphorus by ³¹ P NMR.

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66/187 [00340] ³¹ P NMR (CDCh) δ: 147.3 (s)

2-6) Synthesis of Compound 8-18 [00341] Compound 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, washed with acetonitrile (50 ml). The precipitated solid was collected by filtration and washed with acetonitrile (50 ml). Then, the yellow solid was dried under reduced pressure to obtain Compound 8-18 (1.22 g, 2.45 mmol). The formation of the compound was determined based on the introduction of trivalent phosphorus by ³¹ P NMR.

[00342] ³¹ P NMR (CDCh) õ: 147.2 (s)

2-7) Synthesis of Compound 8-20 [00343] Compound 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, washed with acetonitrile (50 ml). The precipitated solid was collected by filtration and washed with acetonitrile (50 ml). Then, the yellow solid was dried under reduced pressure to obtain Compound 8-20 (3.19 g, 6.06 mmol). The formation of the compound was determined based on the introduction of trivalent phosphorus by ³¹ P NMR.

[00344] ³¹ P NMR (CDCh) õ: 147.2 (s)

2-8) Synthesis of Compound 8-22 [00345] Compound 7-22 (3.55 g, 10.0 mmol, Tokyo Chemical Industry Co., Ltd.) was dissolved in chloroform (81 mL), and DIEA ( 3.67

Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 7T / 22S

67/187 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, washed with acetonitrile (50 ml). The precipitated solid was collected by filtration and washed with acetonitrile (50 ml). Then, the yellow solid was dried under reduced pressure to obtain Compound 8-22 (4.97 g, 8.96 mmol). The formation of the compound was determined based on the introduction of trivalent phosphorus by ³¹ P NMR.

[00346] 31P NMR (CDCb) δ: 147.2 (s)

3) Synthesis of Compound 10-n

OH

9-n

DIEA

10-n where n is an integer from 6 to 28.

3-1) Synthesis of Compound 10-16

Step 1 [00347] Under a nitrogen atmosphere, bis- (p-nitrophenyl) carbonate (13.5 g, 44.4 mmol) and DIEA (11.6 mL, 66.6 mmol) were added to Compound 7-16 ( 12.1 g, 44.4 mmol) in DMF solution (104 mL) dichloromethane (57.1 mL) and then the mixture was stirred at room temperature for 8 hours. Then, Compound 1 (2.0 g,

Petition 870290 (039082, of 10 / Φ3 / 2Ο29, page 78/220

68/187

22.2 mmol) 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 a white solid.

[00348] ¹ H NMR (CDCh) δ: 5.20 (brs, 2H), 4.05 (m, 4H), 3.79 (m, 1H), 3.24 (m, 4H), 1, 55-1.21 (m, 68H), 0.88 (m, 6H) Step 2 [00349] Under nitrogen atmosphere, DIEA (15.8 mL, 90.0 mmol) and

2-cyanoethyl N, N-diisopropylchlorophosphoramidite (10.1 ml, 45.2 mmol) were added to Compound 9-16 suspended in dichloromethane (582 ml) 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 saturated aqueous 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 convert to a powder. The resulting solid was dried under reduced pressure to obtain Compound 10-16 (14.2 g, 16.1 mmol).

[00350] ³¹ P NMR (CDCh) δ: 149.1 (s)

3- 2) Synthesis of Compound 10-18

Step 1 [00351] Under a nitrogen atmosphere, bis- (p-nitrophenyl) carbonate (4.1 g, 13.4 mmol) and DIEA (3.5 mL, 20.1 mmol) were added to Compound 7-18 ( 4 g, 13.4 mmol) in DMF solution (60 mL) dichloromethane (40 mL) and then the mixture was stirred at room temperature for 5 hours. Then, Compound 1 (0.6 g,

6.7 mmol) in DMF solution (5 mL) and the mixture was stirred

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69/187 at night. 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 a white solid.

[00352] ¹ H NMR (CDCh) δ: 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 2 [00353] Under nitrogen atmosphere, DIEA (1.2 mL, 6 , 8 mmol) and 2-cyanoethyl N, N-diisopropylchlorophosphoramidite (0.75 mL, 3.4 mmol) were added to Compound 9-18 (1.0 g, 1.35 mmol) suspended in dichloromethane (60 mL) and the mixture it was stirred at 50 ° C for 1.5 hours. The reaction mixture was cooled to room temperature, diluted with dichloromethane (40 ml) and washed with saturated aqueous 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 make a powder. The resulting solid was dried under reduced pressure to obtain Compound 10-18 (0.98 g, 1.04 mmol) as a white solid.

[00354] ³¹ P NMR (CDCh) õ: 149.1 (s)

4) Synthesis of Compound 15-n

Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 80/220

70/187

where n is an integer from 6 to 28.

4-1) Synthesis of Compound 15-6

Steps 1 and 2 [00355] Diethylamine (1.4 mL, 13.40 mmol) was added to Compound 11 (US2014 / 0142253) (722 mg, 1.075 mmol) in dichloromethane (5.6 mL) and the mixture was stirred at room temperature for 17 hours. After ethanol was added to the reaction mixture, the mixture was stirred and the solvent, concentrated under reduced pressure. The resulting residue was coevaporated twice with ethanol to obtain the crude product of Compound 12.

[00356] D MT-MM (476 mg, 1.720 mmol) was added to Compound 2-6 (239 mg, 1.505 mmol) in ethanol solution (5.0 mL) and 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), and the mixture was stirred at room temperature for 4.5 hours. After the solvent was concentrated under reduced pressure, saturated aqueous sodium bicarbonate solution and water were added to the resulting residue and extracted with acetate

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71/187 of ethyl. The organic layer was washed with water and brine, and then dried with sodium sulfate. The solvent was concentrated under reduced pressure and the resulting crude product purified by silica gel column chromatography (n-hexane: ethyl acetate = 70: 30 ^ 20: 80) to obtain Compound 13-6 (320 mg, yield: 52%) as colorless oil.

[00357] 1H NMR (CDCb) δ: 7.42-7.40 (2H, m), 7.32-7.26 (6H, m), 7.21 (1H, t, J = 7.2 Hz), 6.83 (4H, d, J = 8.8 Hz), 5.37 (1H, s), 3.79 (6H, s), 3.71-3.65 (1H, m), 3.63-3.57 (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), 1.62-1.58 (2H, m), 1.47-1.40 (2H, m), 1.36-1.20 (12H, m), 0.87 ( 3H, t, J = 6.8 Hz).

Step 3 [00358] DMAP (6.6 mg, 0.054 mmol), DIEA (0.282 mL, 1.614 mmol) and succinic anhydride (81 mg, 0.807 mmol) were added to Compound 13-6 (310 mg, 0.538 mmol) in dichloromethane (3.0 mL) 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.

[00359] ¹ H NMR (CDCb) δ: 7.42-7.40 (2H, m), 7.31-7.25 (6H, m),

7.19 (1H, t, J = 7.2 Hz), 6.82 (4H, d, J = 8.8 Hz), 5.74 (1H, t, J = 5.6 Hz),

4.24 (1H, dd, J = 10.8; 5.2 Hz), 4.12 (1H, dd, J = 10.8; 6.0 Hz), 3.79 (6H, s), 3 , 52-3.45 (1H, m), 3.24-2.98 (4H, m), 2.90 (1H, q, J = 7.2 Hz), 2.592.50 (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 = 6.8 Hz).

Step 4 [00360] DIEA (0.186 mL, 1.065 mmol) and HBTU (89 mg, 0.234 mmol) were added to Compound 14-6 (216 mg, 0.320 mmol) in acetonitrile solution (42 mL) and the mixture was stirred at Ambient temperature 87Ο29ΟΦ3Θ082, from 10 / Φ3 / 2Ο29, p. 80/220

72/187 for 15 minutes. Native Amino Icaa CPG 1000Â (ChemGenes Corporation) (4.2 g) was added to the reaction mixture, and the mixture was stirred for 24 hours. After the reaction mixture was filtered, the CPG resin was washed three times with acetonitrile and three times with diethyl ether and dried under reduced pressure. A mixture of CapA (PROLIGO, L840045-06) and CapB (PROLIGO, L850045-06) (1: 1, 42 mL) was added to the dry CPG resin, and the mixture was stirred for 1.5 hours. After filtering the reaction mixture, the CPG resin was washed twice with pyridine, twice with isopropanol and twice with diethyl ether and was dried under reduced pressure. The amount in the support of Compound 14-6 was calculated by colorimetric assay of the DMTR cation, having been obtained Compound 15-6 whose amount in the support is 53 pmol / g.

4-2) Synthesis of Compound 15-10

Steps 1 and 2 [00361] In a method similar to Step 1 of 4-1), the crude product of Compound 12 (392 mg) was obtained.

[00362] D MT-MM (214 mg, 0.772 mmol) was added to Compound 2-10 (155 mg, 0.772 mmol) in ethanol solution (2.6 mL) and 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. DMT-MM (99 mg, 0.356 mmol) was added to Compound 2-10 (71 mg, 0.356 mmol) in ethanol solution (1.3 mL) 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, saturated aqueous 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

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73/187 reduced pressure and the resulting crude product, 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.

[00363] ¹ H NMR (CDCh) δ: 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), 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 3 [00364] DMAP (4.3 mg, 0.035 mmol), DIEA (0.184 mL, 1.055 mmol) and succinic anhydride (53 mg, 0.528 mmol) Compound 13-10 (222 mg, 0.352 mmol) in dichloromethane was added (2.2 mL) 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.

[00365] ¹ H NMR (CDCh) δ: 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, 17-4.13 (1H, m), 3.79 (6H, s), 3.23-2.98 (4H, m),

2.58 (4H, s), 2.16 (2H, t, J = 8.0 Hz), 1.90 (1H, s), 1.60 (2H, s), 1.42-

1.19 (22H, m), 0.88 (3H, t, J = 6.8 Hz).

Step 4 [00366] DIEA (0.166 mL, 0.950 mmol) and HBTU (79 mg, 0.209 mmol) were added to Compound 14-10 (209 mg, 0.285 mmol) in acetonitrile solution (38 mL) and the mixture was stirred at room temperature for 15 minutes. Native Amino Icaa CPG 1000A (ChemGenes Corporation) (3.8 g) was added to the reaction mixture, and the mixture was stirred for 23 hours. After the reaction mixture was filtered, the CPG resin was washed three times with acetonitrile and three times with ether

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74/187 diethyl and was dried under reduced pressure. A mixture of CapA (PROLIGO, L840045-06) and CapB (PROLIGO, L850045-06) (1: 1, 38 mL) was added to the dry CPG resin and the mixture was stirred for 1.5 hours. After filtering the reaction mixture, the CPG resin was washed twice with pyridine, twice with isopropanol and twice with diethyl ether and was dried under reduced pressure. The amount in the support of Compound 14-10 was calculated by colorimetric assay of the DMTr cation, having obtained Compound 15-10 whose amount in the support is 40 pmol / g.

[00367] 4-3) Synthesis of Compound 15-14

Steps 1 and 2 [00368] In a method similar to Step 1 of 4-1), the crude product of Compound 12 (392 mg) was obtained.

[00369] D MT-MM (372 mg, 1.346 mmol) was added to Compound 2-14 (345 mg, 1.346 mmol) in ethanol solution (4.0 mL) and 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 (2.0 ml) and the mixture was stirred at room temperature for 28 hours. After the solvent was concentrated under reduced pressure, saturated aqueous 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 purified by silica gel column chromatography (n-hexane: ethyl acetate = 70: 30 ^ 20: 80) to obtain Compound 13-14 (228 mg, yield: 37%) as colorless oil.

[00370] ¹ H NMR (CDCh) δ: 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), 1.59 (2H, s), 1.43 (2H, s), 1.25 (28H, s),

0.88 (3H, s).

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Step 3 [00371] DMAP (4.0 mg, 0.033 mmol), DIEA (0.171 mL, 0.977 mmol) and succinic anhydride (49 mg, 0.488 mmol) were added to Compound 13-14 (224 mg, 0.326 mmol) in dichloromethane (2.2 mL) and the mixture was stirred at room temperature for 2 days. The solvent was concentrated under reduced pressure and the resulting crude product, purified by silica gel column chromatography (chloroform: methanol = 100: 0 ^ 90: 10) to obtain Compound 14-14 (163 mg, yield: 64% ) as colorless oil.

[00372] ¹ H NMR (CDCb) δ: 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, 17-4.13 (1H, m), 3.79 (6H, s), 3.23-2.98 (4H, m),

2.58 (4H, s), 2.15 (2H, t, J = 7.2 Hz), 1.90 (1H, s), 1.59 (4H, s), 1.46-

1.25 (28H, m), 0.87 (3H, t, J = 5.6 Hz).

Step 4 [00373] DIEA (0.122 mL, 0.700 mmol) and HBTU (58 mg, 0.154 mmol) were added to Compound 14-14 (162 mg, 0.206 mmol) in acetonitrile solution (28 mL) and the mixture was stirred at room temperature for 15 minutes. Native Amino Icaa CPG 1000Â (ChemGenes Corporation) (2.8 g) was added to the reaction mixture, and the mixture was stirred for 24 hours. After the reaction mixture was filtered, the CPG resin was washed three times with acetonitrile and three times with diethyl ether and was dried under reduced pressure. A mixture of CapA (PROLIGO, L840045-06) and CapB (PROLIGO, L850045-06) (1: 1, 28 mL) was added to the dry CPG resin, and the mixture was stirred for 1.5 hours. After filtering the reaction mixture, the CPG resin was washed twice with pyridine, twice with isopropanol and twice with diethyl ether and was dried under reduced pressure. The amount in the support of Compound 14-14 was calculated by colorimetric assay of the DMTr cation, having obtained Compound 15-14 whose amount in the support is 42 pmol / g.

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4-4) Synthesis of Compound 15-18

Steps 1 and 2 [00374] In a method similar to Step 1 of 4-1), the crude product of Compound 12 (392 mg) was obtained.

[00375] D MT-MM (471 mg, 1.702 mmol) was added to Compound 2-18 (466 mg, 1.490 mmol) in ethanol solution (5.0 mL) and 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) and the mixture was stirred at room temperature for 4.5 hours. After the solvent was concentrated under reduced pressure, saturated aqueous 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 purified by silica gel column chromatography (n-hexane: ethyl acetate = 70: 30 ^ 20: 80) to obtain Compound 13-18 (494 mg, yield: 62%) as colorless oil.

[00376] ¹ H NMR (CDCh) δ: 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 3 [00377] DMAP (5.8 mg, 0.047 mmol), DIEA (0.248 mL, 1.419 mmol) and succinic anhydride (71 mg, 0.709 mmol) were added to Compound 13-18 (352 mg, 0.473 mmol) in dichloromethane (3.5 mL) 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 (chloroforPetition 87Ο29ΟΦ3Θ082, 10 / Φ3 / 2Ο29, page 8T / 22S

77/187 medium: methanol = 100: 0 ^ 90: 10) to obtain Compound 14-18 (225 mg, 56%) as a colorless oil.

[00378] ¹ H NMR (CDCh) ó: 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 4 [00379] DIEA (0.154 ml, 0.880 mmol) and HBTU (73 mg, 0.194 mmol) were added to Compound 14-18 (223 mg, 0.264 mmol) in acetonitrile solution (35 ml) and the mixture was stirred at room temperature for 15 minutes. Native Amino Icaa CPG 1000Â (ChemGenes Corporation) (3.5 g) was added to the reaction mixture, and the mixture was stirred for 24 hours. After the reaction mixture was filtered, the CPG resin was washed three times with acetonitrile and three times with diethyl ether and was dried under reduced pressure. A mixture of CapA (PROLIGO, L840045-06) and CapB (PROLIGO, L850045-06) (1: 1, 35mL) was added to the dry CPG resin, and the mixture was stirred for 1.5 hours. After filtering the reaction mixture, the CPG resin was washed twice with pyridine, twice with isopropanol and twice with diethyl ether and was dried under reduced pressure. The amount in the support of Compound 14-18 was calculated by colorimetric assay of the DMTr cation, having obtained Compound 15-18 whose amount in the support is 56 pmol / g.

5) Synthesis of Compound 17

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, ODMTr , ODMTr  ^ OH --- -k ^ OTBS FmocHN FmocHN 11 16 JDDMTr A, OTBS

Steps 1 and 2 [00380] Imidazole (71 mg, 1.044 mmol) and t-butylchlorodimethylsilane (79 mg, 0.522 mmol) were added to Compound 11 (US2014 / 0142253, 292 mg, 0.435 mmol) in DMF solution (2.0 ml) 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).

[00381] Diethylamine (0.6 ml, 5.74 mmol) was added to the crude product of Compound 16 (352 mg) in dichloromethane (2.4 ml) 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 column chromatography on amino-silica gel (chloroform) to obtain Compound 17 (190 mg, 78%) as a colorless oil.

[00382] ¹ H NMR (CDCh) ó: 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).

6) Synthesis of Compound 22-n

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where n is an integer from 6 to 28.

6-1) Synthesis of Compound 22-6

Step 1 [00383] DIEA (1.53 mL, 8.76 mmol), bis (nitrophenyl) carbonate (1.33 g, 4.38 mmol) and DMAP (178 mg, 1.46 mmol) were added to Compound 3 -6 (1.0 g, 2.92 mmol) in THF solution (20 mL) chloroform (20 mL) and the mixture was stirred at 60 ° C for 1 hour. The reaction mixture was filtered. After the mother liquor 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 a white solid.

[00384] ¹ H NMR (CDCh) δ: 8.32-8.26 (2H, m), 7.42 (2H, dt, J = 9.9;

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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 2 [00385] Compound 18-6 (450 mg, 0.89 mmol) was added to Compound 17 (500 mg, 0.89 mmol) in dichloromethane (10.0 mL) and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure and purified by column chromatography on amino-silica gel (hexane: ethyl acetate = 65: 35 ^ 10: 90) to obtain Compound 19-6 (625 mg, 76%) colorless oil.

[00386] ¹ H NMR (CDCh) δ: 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 3 [00387] TBAF (1M THF, 1.34 mL, 1.34 mmol) was added to Compound 19-6 (625 mg, 0.67 mmol) in THF (10 mL) and the mixture was stirred at room temperature for 24 hours. The reaction mixture was concentrated under reduced pressure and the resulting crude product, purified by column chromatography on diol-silica gel (hexane: ethyl acetate = 50: 50 ^ 10: 90) to obtain Compound 20-6 (541 mg, 99%) as a colorless liquid.

[00388] ¹ H NMR (CDCh) δ: 7.41 (2H, t, J = 4.3 Hz), 7.26 (9H, ddt,

J = 31.6; 12.0; 4.9 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),

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3.69- 3.61 (2H, m), 3.50-3.44 (2H, m), 3.30 (3H, tt, J = 20.6; 6.5 Hz), 3.15-3, 06 (3H, m), 2.63 (1H, s), 2.21 - 2.17 (4H, m), 1.78 (1H, s), 1.62 (4H, t, J = 6, 9 Hz), 1.43 (2H, t, J = 5.4 Hz), 1.30 (20H, dt, J = 29.2; 11.0 Hz), 0.87 (6H, t, J = 6.9 Hz).

Step 4 [00389] DIEA (0.35 mL, 1.98 mmol), DMAP (8.0 mg, 0.066 mmol) and succinic anhydride (132 mg, 1.32 mmol) were added to Compound 20-6 (541 mg , 0.66 mmol) in dichloromethane (2 mL) and the mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated under reduced pressure and the resulting crude product, purified by silica gel column chromatography (chloroform: methanol = 40: 1 ^ 10: 1) to obtain Compound 21-6 (591 mg, 97% ) as a colorless liquid.

[00390] ¹ H NMR (CDCh) δ: 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 , 5 Hz), 5.91 (1H, t, J = 5.5 Hz), 4.71 (1H, t, J = 5.3 Hz), 4.42 (1H, dd, J = 11.0 ; 3.2 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; 8.2 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 5 [00391] DIEA (0.30 mL, 1.70 mmol) and HBTU (142 mg, 0.37 mmol) were added to Compound 21-6 (312 mg, 0.34 mmol) in a mixture of acetonitrile / dichloromethane (4: 1, 25 mL) and the mixture was stirred at room temperature for 15 minutes. HybridCPG amino form 2000A (Prime Synthesis, Inc.) (2.8 g) was added to the reaction mixture, and the mixture was stirred for 24 hours. After the reaction mixture was filtered, the HybridCPG resin was washed three times with acetonitrile and three times with diethyl ether and was dried under reduced pressure. AdditionPetition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 80/220

82/187 a THF / acetic anhydride / pyridine mixture (8: 1: 1, 30 mL) was added to the dry HybridCPG resin, and the mixture was stirred for 3 hours. After the reaction mixture was filtered, the HybridCPG resin was washed twice with pyridine, twice with isopropanol and twice with diethyl ether and was dried under reduced pressure. The amount in the support of Compound 21-6 was calculated by colorimetric assay of the DMTR cation, having obtained Compound 22-6 whose amount in the support is 114 pmol / g.

6-2) Synthesis of Compound 22-8

Step 1 [00392] 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 to Compound 3-8 (1 g, 2.5 mmol) in THF (20 mL) and chloroform (20 mL) and the mixture was stirred at 60 ° C for 1 hour. The reaction mixture was filtered. After the mother liquor 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 a white solid.

[00393] ¹ H NMR (CDCh) δ: 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 2 [00394] Compound 18-8 (500 mg, 0.89 mmol) and DIEA (0.23 mL, 1.33 mmol) were added to Compound 17 (500 mg, 0.89 mmol) in dichloromethane (10 , 0 mL) and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure and then purified by column chromatography on aminosilica gel (hexane: ethyl acetate = 50: 50A 0:90) to obtain Compound 19-8 (661 mg, 75%) as a light yellow solid.

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83/187 [00395] ¹ H NMR (CDCb) δ: 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; 6.5 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 = 6.8 Hz), 0.83 (9H, s). Step 3 [00396] TBAF (1M THF, 1.34 mL, 1.34 mmol) was added to Compound 19-8 (661 mg, 0.699 mmol) in THF (10 mL) and the mixture was stirred at room temperature for 25 minutes. hours. The reaction mixture was concentrated under reduced pressure and then purified by column chromatography on diol-silica gel (hexane: ethyl acetate = 50: 50 ^ 10: 90) to obtain Compound 20-8 (549 mg, 94%) as colorless oil.

[00397] ¹ H NMR (CDCb) δ: 7.41 (2H, t, J = 4.4 Hz), 7.32-7.19 (9H, m), 6.84 (4H, d, J = 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 , 1 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).

Step 4 [00398] DMAP (7.7 mg, 0.063 mmol), succinic anhydride (126 mg,

1.25 mmol) and DIEA (0.32 mL, 1.88 mmol) were added to Compound 20-8 (549 mg, 0.63 mmol) in dichloromethane (10 mL) 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 21-8 (582 mg, 95%) as a colorless liquid.

Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 88/220

84/187 [00399] ¹ H NMR (CDCh) δ: 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 = 6.4 Hz), 5.91 (1H, t, J = 5.6 Hz), 4.71 (1H, t, J = 5.1 Hz), 4.42 (1H, dd, J = 11.0; 3.2 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 5 [00400] DIEA (0.27 mL, 1.54 mmol) and HBTU (128 mg, 0.34 mmol) were added to Compound 21-8 (300 mg, 0.31 mmol) in a mixture of acetonitrile / dichloromethane (4: 1, 25 mL) and the mixture was stirred at room temperature for 15 minutes. HybridCPG amino form 2000A (Prime Synthesis, lnc.) (2.5g) was added to the reaction mixture, and the mixture was stirred for 24 hours. After the reaction mixture was filtered, the HybridCPG resin was washed three times with acetonitrile and three times with diethyl ether and was dried under reduced pressure. To the dry HybridCPG resin, a mixture of THF / acetic anhydride / pyridine (8: 1: 1, 30 mL) was added and the mixture was stirred for 3 hours. After the reaction mixture was filtered, the HybridCPG resin was washed twice with pyridine, twice with isopropanol and twice with diethyl ether and was dried under reduced pressure. The amount in the support of Compound 21-8 was calculated by colorimetric assay of the DMTR cation, having obtained Compound 22-8 whose amount in the support is 107 pmol / g.

6-3) Synthesis of Compound 22-10

Step 1 [00401] Pyridine (0.379 mL, 4.70 mmol) and 4-nitrophenyl chloroformate (947 mg, 4.70 mmol) were added to Compound 3-10 (2.0 g, 3.92 mmol) in THF (50 ml) and the mixture was stirred at 60 ° C for

Petition 870290 (039082, 10 / Φ3 / 2Ο29, p. 89/220

85/187 hour. The reaction mixture was filtered. After the mother liquor 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 18-10 (1.1 g, 42%) as a white solid.

[00402] ¹ H NMR (CDCh) δ: 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 2 [00403] Compound 18-10 (50 mg, 0.074 mmol) was added to Compound 17 (41 mg, 0.074 mmol) in dichloromethane (5.0 mL) 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 a yellow liquid.

[00404] ¹ H NMR (CDCh) δ: 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 = 5.4 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), 0.84 (9H, s), 0.01 (6H, s) Step 3 [00405] Triethylamine (4.4 mL, 7.21 mmol) and TBAF (1M THF, 0.4 mL, 0.40 mmol) were added to Compound 19-10 (559.2 mg, 0.535 mmol) in THF (5 mL) and the mixture was stirred at room temperature for 12 hours. The reaction mixture was diluted with chloroform (10 ml) and then washed with saturated aqueous 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

Petition 87Ο29ΟΦ3Θ0Η2, of 10 / Φ3 / 2Ο29, p. 90/220

86/187 = 100: 0 ^ 90: 10) to obtain Compound 20-10 (384 mg, 77%) as a colorless liquid.

[00406] ¹ H NMR (CDCh) δ: 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 = 5.2 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).

Step 4 [00407] DMAP (5.04 mg, 0.041 mmol) and succinic anhydride (62.0 mg, 0.619 mmol) were added to Compound 20-10 (384 mg, 0.431 mmol) in dichloromethane (10.1 mL) 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 column chromatography on diol-silica gel (chloroform: methanol = 100: 0 ^ 90: 10) to obtain Compound 21-10 (308 mg, 71%) as a colorless liquid.

[00408] ¹ H NMR (CDCh) δ: 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), 2.89 (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 5 [00409] DIEA (0.168 mL, 0.982 mmol) and HBTU (100 mg, 0.264 mmol) were added to Compound 21-10 (247 mg, 0.240 mmol) in an acetonitrile / dichloromethane mixture (1: 1, 20 mL ) and the mixture was stirred at room temperature for 20 minutes. Native Amino Icaa CPG 1000 A (ChemGenes Corporation) (2.0 g) was added to the reaction mixture, and the mixture was stirred for 12 hours. After the reaction mixture 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 9T / 22S

87/187 was filtered, the CPG resin was washed three times with dichloromethane and three times with diethyl ether and was dried under reduced pressure. A mixture of CapA (PROLIGO, L840045-06) and CapB (PROLIGO, L850045-06) (1: 1, 20 mL) was added to the dry HybridCPG resin, and the mixture was stirred for 30 minutes. After the reaction mixture was filtered, the CPG resin was washed twice with dichloromethane and twice with diethyl ether and was dried under reduced pressure. The amount in the support of Compound 21-10 was calculated by colorimetric assay of the DMTr cation, having obtained Compound 22-10 whose amount in the support is 69 pmol / g.

6-4) Synthesis of Compound 22-12

Step 1 [00410] Pyridine (0.379 mL, 4.70 mmol) and 4-nitrophenyl chloroformate (947 mg, 4.70 mmol) were added to Compound 3-12 (2.0 g, 3.92 mmol) in THF (50 ml) and the mixture was stirred at 60 ° C for 1 hour. The reaction mixture was filtered. After the mother liquor 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 18-12 (1.1 g, 42%) as a white solid.

[00411] ¹ H NMR (CDCh) δ: 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 2 [00412] Compound 18-12 (50 mg, 0.074 mmol) was added to the

Compound 17 (41 mg, 0.074 mmol) in dichloromethane (5.0 ml) 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,

Petition 870290 (039082, of 10 / Φ3 / 2Ο29, page 98/220

88/187

98%) as a yellow liquid.

[00413] ¹ H NMR (CDCb) δ: 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 = 5.4 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), 0.84 (6H, s), 0.01 (6H, s) Step 3 [00414] Triethylamine (1.0 mL, 7.21 mmol) and TBAF (1M THF, 0.4 mL, 0.40 mmol) were added to Compound 19-12 (90 mg, 0.082 mmol) in THF (5 mL) and the mixture was stirred at room temperature for 12 hours. The reaction mixture was diluted with chloroform (10 ml) and then washed with saturated aqueous 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 a colorless liquid.

[00415] ¹ H NMR (CDCb) δ: 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).

Step 4 [00416] DMAP (0.7 mg, 0.006 mmol) and succinic anhydride (7.3 mg, 0.073 mmol) were added to Compound 20-12 (60 mg, 0.061 mmol) in pyridine solution (2 mL) 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

Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 99/220

89/187 = 100: 0 ^ 95: 5) to obtain Compound 21-12 (56 mg, 85%) as a colorless liquid.

[00417] ESI-MS (m / z): 1085 (M-H).

Step 5 [00418] DIEA (0.043 mL, 0.248 mmol) and HBTU (31 mg, 0.083 mmol) were added to Compound 21-12 (52 mg, 0.048 mmol) in an acetonitrile / chloroform mixture (1: 1, 10 mL ) and the mixture was stirred at room temperature for 15 minutes. Native Amino Icaa CPG 1000A (ChemGenes Corporation) (0.5 g) was added to the reaction mixture, and the mixture was stirred for 23 hours. After the reaction mixture was filtered, the CPG resin was washed three times with acetonitrile and three times with diethyl ether and was dried under reduced pressure. A mixture of CapA (PROLIGO, L840045-06) and CapB (PROLIGO, L850045-06) (1: 1.5 mL) was added to the dry CPG resin, and the mixture was stirred for

1.5 hours. After the reaction mixture was filtered, the CPG resin was washed twice with pyridine, twice with isopropanol and twice with diethyl ether and then dried under reduced pressure. The amount in the support of Compound 21-12 was calculated by colorimetric assay of the DMTr cation, having obtained Compound 22-12 whose amount in the support is 31 pmol / g.

[00419] 6-5) Synthesis of Compound 22-14

Step 1 [00420] Pyridine (0.320 mL, 3.97 mmol) and 4-nitrophenyl chloroformate (800 mg, 3.97 mmol) were added to Compound 3-14 (1.5 g, 3.92 mmol) in THF (60 ml) and the mixture was heated under reflux for 3 hours. The reaction mixture was filtered. After the mother liquor 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 18-14 (1 , 56 g, 81%) as a white solid.

ΙΙ7 «ΙΏΖ2 (Ε«) 1. ipijg. HWEZ®

90/187 [00421] ¹ H NMR (CDCh) δ: 8.29 (2H, d, J = 10.0 Hz), 7.43 (2H, d, J = 10.0 Hz), 6.31 (2H, t, J = 6.4 Hz), 4.81 - 4.76 (1H, m), 3.65-3.58 (2H, m), 3.55-3.48 (2H, m ), 2.25 (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 2 [00422] Compound 18-14 (1.38 g, 1.88 mmol) was added to Compound 17 (1.06 g, 1.88 mmol) in dichloromethane (30 mL) and the mixture was stirred at temperature room 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: 34A5: 55) to obtain Compound 19-14 (1.47 g, 68%) as a white solid.

[00423] ESI-MS (m / z): 1155 (M-H).

Step 3 [00424] TBAF (1M THF, 3.81 mL, 3.81 mmol) was added to Compound 19-14 (1.47 g, 1.27 mmol) in THF (30 mL) 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 resulting 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 a white solid.

[00425] ¹ H NMR (CDCh) δ: 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), 3.15-3.06 (3H, m), 2.61 (1H, s), 2.21-2.17 (4H, m), 1.25 (58H, s), 0.88 (6H, t, J = 6.8 Hz).

Step 4 [00426] DMAP (12 mg, 0.10 mmol), DIEA (0.523 mL, 2.99 mmol) and succinic anhydride (170 mg, 1.70 mmol) were added to the Com

ΙΙ7 «ΙΏΖ2 (Ε«) 1. ipijg. NWffiTb

91/187 set 20-14 (1.04 g, 0.998 mmol) in dichloromethane (20 mL) 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 column chromatography on diol-silica gel (chloroform) to obtain Compound 21-14 (1.17 g) as a white solid.

[00427] ESI-MS (m / z): 1141 (M-H).

Step 5 [00428] DIEA (0.447 mL, 2.56 mmol) and HBTU (214 mg, 0.563 mmol) were added to Compound 21-14 (585 mg, 0.512 mmol) in an acetonitrile / dichloromethane (1: 1, 40 ml) and the mixture was stirred at room temperature for 15 minutes. Native Amino Icaa CPG 1000A (ChemGenes Corporation) (4.0 g) was added to the reaction mixture, and the mixture was stirred for 21 hours. After the reaction mixture was filtered, the CPG resin was washed with a mixture of acetonitrile / dichloromethane (1: 1, 120 ml) and diethyl ether (60 ml) and then was dried under reduced pressure. A mixture of CapA (PROLIGO, L840045-06) and CapB (PROLIGO, L850045-06) (1: 1, 40 mL) was added to the dry CPG resin, and the mixture was stirred for 1.5 hours. After the reaction mixture was filtered, the CPG resin was washed with pyridine (40 ml), isopropanol (60 ml) and diethyl ether (60 ml) and then dried under reduced pressure. The amount in the support of Compound 21-14 was calculated by colorimetric assay of the DMTr cation, having obtained Compound 22-14 whose amount in the support is 40 pmol / g.

6-6) Synthesis of Compound 22-18

Step 1 [00429] Pyridine (0.143 mL, 1.76 mmol) and 4-nitrophenyl chloroformate (356 mg, 1.77 mmol) were added to Compound 3-18 (1.0 g, 1.47 mmol) in THF (40 ml) and the mixture was stirred at 60 ° C for 1 hour. After the reaction mixture was filtered, the mother liquor was

ΙΙ7 «ΙΏΖ2 (Ε«) 1. ipijg. HHZ2ZB

92/187 concentrated under reduced pressure. The crude product of the resulting solid was washed with ethyl acetate to obtain Compound 18-18 (508 mg, 41%) as a white solid.

[00430] ¹ H NMR (CDCb) δ: 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 2 [00431] Compound 18-18 (93 mg, 0.110 mmol) was added to Compound 17 (62 mg, 0.110 mmol) in dichloromethane (4.0 mL) 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 a yellow liquid.

[00432] ¹ H NMR (CDCb) δ: 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), 3.51 (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 3 [00433] Triethylamine (0.1 mL, 0.79 mmol) and TBAF (1M THF, 0.32 mL, 0.32 mmol) were added to Compound 19-18 (100 mg, 0.079 mmol) in THF ( 4 mL) and the mixture was stirred at room temperature for 24 hours. The reaction mixture was diluted with chloroform (10 ml) and then washed with saturated aqueous 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 -18 (90 mg, 99%) as a colorless liquid.

[00434] ¹ H NMR (CDCb) δ: 7.41 (2H, d, J = 8.6 Hz), 7.31 (7H, d,

93/187

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).

Step 4 [00435] DMAP (0.9 mg, 0.007 mmol) and succinic anhydride (15.8 mg, 0.151 mmol) were added to Compound 20-18 (87 mg, 0.075 mmol) in pyridine solution (2 mL) 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 a colorless liquid.

[00436] ESI-MS (m / z): 1253 (M-H).

Step 5 [00437] DIEA (0.043 mL, 0.248 mmol) and HBTU (38 mg, 0.10 mmol) were added to Compound 21-18 (85 mg, 0.068 mmol) in an acetonitrile / chloroform (1: 1, 10 mL) and the mixture was stirred at room temperature for 15 minutes. Native Amino Icaa CPG 1000Â (ChemGenes Corporation) (0.5 g) was added to the reaction mixture, and the mixture was stirred at room temperature for 23 hours. After the reaction mixture was filtered, the CPG resin was washed three times with acetonitrile and three times with diethyl ether and was dried under reduced pressure. A mixture of CapA (PROLIGO, L840045-06) and CapB (PROLIGO, L850045-06) (1: 1, 5 mL) was added to the dry CPG resin, and the mixture was stirred for 1.5 hours. After the reaction mixture was filtered, the CPG resin was washed twice with pyridine, twice with isopropanol and twice with diethyl ether and was dried under reduced pressure. The amount in the support of Compound 2194/187 was calculated by colorimetric assay of the DMTR cation, having obtained Compound 22-18 whose amount in the support is 15 pmol / g.

[00438] 6-7) Synthesis of Compound 22-20

Step 1 [00439] bis (4-nitrophenyl) carbonate (1.241 mL, 4.08 mmol) and DMAP (498 mg, 4.08 mmol) were added to Compound 3-20 (1.0 g, 1.36 mmol) in THF (35 ml) and the mixture was stirred at 55 ° C for 1 hour. The reaction mixture was concentrated under reduced pressure. Acetonitrile (50 ml) was added to the residue, and the mixture was stirred until the solid precipitated. Water (20 ml) was added to the suspended solution, and the mixture was stirred vigorously. The resulting solid was collected by filtration and washed with water (50 ml) and acetonitrile (50 ml) to obtain Compound 18-20 (1.1 g, 92%) as a white solid.

[00440] ¹ H NMR (CDCb) δ: 8.28 (2H, dd, J = 2.0; 6.8 Hz), 7.42 (2H, dd, J = 2.4; 7.2 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 2 [00441] Compound 18-20 (350 mg, 0.388 mmol) and DMAP (47.5 mg, 0.388 mmol) were added to Compound 17 (219 mg, 0.388 mmol) in THF (6.5 mL) and the The mixture was stirred at 65 ° C for 2 hours. Aqueous 10% acetonitrile solution (70 ml) was added to the reaction mixture, and the mixture was stirred for a while. The precipitated solid was collected by filtration to obtain Compound 19-20 (420 mg, 82%) as a white solid.

[00442] ¹ H NMR (CDCb) δ: 7.43 (2H, d, J = 7.6 Hz), 7.31 (7H, m),

6.81 (4H, d, J = 8.4 Hz), 6.26 (2H, br s), 4.73 (1H, br s), 4.69 (1H, br s),

3.79 (6H, s), 3.79 (6H, s), 3.63 (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 3

95/187 [00443] TBAF (1M THF, 0.556 mL, 0.556 mmol) was added to Compound 19-20 (559 mg, 0.422 mmol) in THF (5 mL) and the mixture was stirred at 50 ° C for 5 hours. The reaction mixture was added dropwise to a 10% aqueous solution of acetonitrile (100 ml) and then the precipitated solid was collected by filtration to obtain Compound 2020 (344 mg, 67%) as a white solid.

[00444] ¹ H NMR (CDCh) δ: 7.41 (2H, t, J = 4.3 Hz), 7.31 (7H, d, J = 8.5 Hz), 6.84 (4H, d, J = 8.8 Hz), 6.35 (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; 5.6 Hz), 2.60 (1H, s), 2.21-2.17 (4H, m) , 1.62 (4H, m), 1.60-1.45 (m, 4H), 1.25-1.01 (72H, m), 0.88 (6H, t, J = 6.8 Hz ).

Step 4 [00445] DMAP (3.5 mg, 0.0284 mmol) and succinic anhydride (42.6 mg, 0.426 mmol) were added to Compound 20-20 (344 mg, 0.284 mmol) in dichloromethane (10 mL) and the mixture was stirred at room temperature overnight. The reaction mixture was added dropwise to a 10% aqueous solution of acetonitrile (100 ml), and the precipitated solid was collected by filtration to obtain Compound 2120 (361 mg, 97%) as a white solid.

[00446] ¹ H NMR (CDCh) δ: 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), 3.43-3.37 (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 5 [00447] DIEA (0.127 ml, 0.725 mmol) and HBTU (60.5 mg, 0.159 mmol) were added to Compound 21-20 (190 mg, 0.155 mmol) in an acetonitrile / chloroform mixture (1: 3, 10 mL) and the mixture was stirred at 40 ° C for 15 minutes. Native Amino Icaa CPG has been added

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1000Â (ChemGenes Corporation) (3.0 g) to the reaction mixture, and the mixture was stirred for 2 hours. After the reaction mixture was filtered, the CPG resin was washed three times with chloroform, once with ethanol and three times with acetonitrile and was dried under reduced pressure. A mixture of CapA (PROLIGO, L840045-06) and CapB (PROLIGO, L850045-06) (1: 1, 20 mL) was added to the dry CPG resin, and the mixture was stirred for 1.5 hours. After the reaction mixture was filtered, the CPG resin was washed three times with chloroform and three times with acetonitrile and then dried under reduced pressure. The amount in the support of Compound 21-20 was calculated by colorimetric assay of the DMTr cation, having obtained Compound 22-20 whose amount in the support is 48 pmol / g.

6-8) Synthesis of Compound 22-22

Step 1 [00448] bis- (p-nitrophenyl) carbonate (1.54 g, 5.05 mmol) and DMAP (618 mg, 5.05 mmol) were added to Compound 3-22 (2.0 g, 2, 53 mmol) in THF (35 mL) and the mixture was stirred at 65 ° C for 2 hours. The reaction mixture was concentrated under reduced pressure. Acetonitrile (100 ml) was added to the residue, and the mixture was stirred until the solid precipitated. Water (20 ml) was added to the suspended solution, and the mixture was stirred vigorously. The resulting solid was collected by filtration and washed in this order with water (100 ml) and acetonitrile (100 ml) to obtain Compound 18-22 (2.27 g, 89%) as a white solid.

[00449] ¹ H NMR (CDCh) δ: 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 , 6 Hz), 1.64 (4H, m), 1.26 (84H, m), 0.88 (6H, t, J = 6.0 Hz) Step 2 [00450] DMAP (144 mg, 1, 81 mmol) and Compound 18-22 (1.13 g,

1.18 mmol) were added to Compound 17 (666 mg, 1.81 mmol)

Petition 870290 (039082, of 10 / Φ3 / 2Ο29, page 10T / 22S

97/187 in THF (10.0 mL) and the mixture was stirred at 65 ° C for 2 hours. Acetonitrile (150 ml) was added to the reaction mixture slowly. The resulting precipitate was collected by filtration and washed three times with acetonitrile to obtain Compound 19-22 (1.5 g, 92%) as a white solid. [00451] ¹ H NMR (CDCh) δ: 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 = 6.8 Hz), 0.84 (9H, s), 0.01 (6H, s). Step 3 [00452] TBAF (1M THF, 1.64 mL, 1.69 mmol) was added to Compound 19-22 (1.5 g, 1.07 mmol) in THF (8.9 mL) and the mixture was stirred at 65 ° C for 2 hours. Acetonitrile (150 ml) was added to the reaction mixture slowly. The resulting precipitate was collected by filtration and washed three times with acetonitrile to obtain Compound 20-22 (1.2 g, 87%) as a white solid.

[00453] ¹ H NMR (CDCh) δ: 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).

Step 4 [00454] DMAP (1 mg, 0.008 mmol) and succinic anhydride (11.9 mg, 0.118 mmol) were added to Compound 20-22 (100 mg, 0.079 mmol) in dichloromethane (3 mL) and the mixture was stirred at 45 ° C for 4 hours. Acetonitrile (10 ml) was added dropwise to the reaction mixture, 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 a white solid.

[00455] ¹ H NMR (CDCh) δ: 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,

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98/187 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), 3.43-3.37 (4H, m), 3.13-3.02 (4H, m), 2.63- 2.53 (4H, m), 2.23-2.18 (4H, dd, J = 7.2; 14.8 Hz), 2.00-1.25 (4H, m), 1.25- 1.11 (84H, m), 0.88 (6H, t, J = 6.8 Hz).

Step 5 [00456] DIEA (0.059 mL, 0.336 mmol) and HBTU (28 mg, 0.074 mmol) were added to Compound 21-22 (92 mg, 0.067 mmol) in an acetonitrile / dichloromethane / chloroform mixture (1: 2: 2, 10 mL) and the mixture was stirred at 40 ° C for 15 minutes. Native Amino Icaa CPG 1000Â (ChemGenes Corporation) (0.9 g) was added to the reaction mixture, and the mixture was stirred at 40 ° C for 3 hours. After the reaction mixture was filtered, the CPG resin was washed three times with chloroform, three times with acetonitrile and three times with ethanol and was dried under reduced pressure. A mixture of CapA (PROLIGO, L840045-06) and CapB (PROLIGO, L850045-06) (1: 1, 60 mL) was added to the dry CPG resin, and the mixture was stirred for 1.5 hours. After the reaction mixture was filtered, the CPG resin was washed three times with acetonitrile and was dried under reduced pressure. The amount in the support of Compound 21-22 was calculated by colorimetric assay of the DMTR cation, having obtained Compound 22-22 whose amount in the support is 47 pmol / g.

8-1) Synthesis of Compound 26

[00457] Under a nitrogen atmosphere, DIEA (0.9 mL, 5.17 mmol) was added to cholesterol (Compound 25, 1.00 g, 2.59 mmol, Wako Pure

Chemical Industries, Ltd.) in dichloromethane (10 m), and the mixture was resPetition 87Ο29ΟΦ3Θ082, 10 / Φ3 / 2Ο29, p. 109/220

99/187 chilled with ice water. Methyl N, N-diisopropylchlorophosphoramidite (0.86 mL, 3.85 mmol) was added thereto and the mixture was stirred under ice cooling for 40 minutes. The reaction mixture was diluted with dichloromethane and ethyl acetate (100 ml) and then washed with saturated aqueous 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 26 (1.45 g, yield: 95% ) as a colorless foam. The formation of the compound was determined based on the introduction of trivalent phosphorus by ³¹ P NMR.

[00458] ³¹ P NMR (CDCh) δ: 145.46 (d)

8-2) Synthesis of Compound 5

[00459] 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) has been added to it. 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 40 minutes. Dichloromethane (90 mL) and saturated aqueous sodium bicarbonate solution (100 mL) were added to the solvent to stop the reaction, and then the mixture was partitioned through the separating funnel. 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) in light yellow oil as a crude product. Compound formation was determined based on the introduction of trivalent phosphorus

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100/187 by ³¹ P NMR.

[00460] ¹ H NMR (CDCl 3) δ: 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).

[00461] ³¹ P NMR (CDCl 3) δ: 147.92 (1H, s).

9) Synthesis of Compound 36-n

where n is an integer from 6 to 28.

9-1) Synthesis of Compound 36-16

Step 1 [00462] According to the methods described in Nucleic Acids RePetition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. ΙΦί / 220

101/187 search, 42, 8796-8807 (2014), Compound 28 was synthesized from Compound 27 in two stages.

Step 2 [00463] Imidazole (690 mg, 10.1 mmol, 1.3 eq.) And tertbutyldimethylsilyl chloride (1.29 g, 8.57 mmol, 1.1 eq.) Were added, in that order, to Compound 28 (5.0 g, 7.79 mmol) in DMF solution (20 mL) 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 with magnesium sulfate. The solvent was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (SiO2: 120 g, nhexane: ethyl acetate: triethylamine = 90: 10: 1 ^ 65: 35: 1) to obtain Compound 29 (5.50 g, yield: 93%) as a colorless foam. By ¹ H NMR, a mixture of rotamers, which is 1: 1, was observed.

[00464] ESI-MS (m / z): 778 (M + H).

[00465] ¹ H NMR (CDCh) δ: 7.68 (d, J = 7.5 Hz, 1H), 7.62 (d, J = 7.5 Hz, 1H), 7.58-7.51 ( m, 1H), 7.40-7.05 (m, 14H), 6.74-6.63 (m, 5H), 4,614.49 (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), 0.779 (s, 3H), 0.777 (s, 1.5H), 0.007 (s, 1.5H), 0.000 (s, 1.5H), -0.015 (s, 1.5H), 0.026 (s, 1.5H).

Step 3 [00466] Piperidine (0.379 mL, 3.83 mmol, 1.1 eq.) Was added to Compound 29 (2.63 g, 3.48 mmol) in DMF solution (10 mL). 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 with magnesium sulfate. The solvent was concentrated under reduced pressure. Methanol was added

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102/187 (10 mL) and the resulting precipitate was filtered. The filtrate was concentrated to obtain the crude product of Compound 30 (2.20 g).

[00467] ESI-MS (m / z): 524 (M + H). Peak HPLC RT = 3.29 min. Step 4 [00468] Triethylamine (3.65 ml, 36.9 mmol) and acetoxyacetyl chloride (3.65 ml, 36.9 mmol) were added in this order to Compound 30 (2.20 g, crude) in dichloromethane ( 15 mL) 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 with magnesium sulfate. The solvent was concentrated under reduced pressure to obtain the crude product of Compound 31.

[00469] ESI-MS (m / z): 634 (M + H). Peak HPLC RT = 3.40 min. Step 5 [00470] After Compound 31 obtained in Step 4 was dissolved in methanol (10 ml), a 28% sodium methoxide-methanol solution (0.60 ml) was added thereto, 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 with 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 a colorless foam. By ¹ H NMR, a mixture of rotamers, which is 78:22, was observed.

[00471] ESI-MS (m / z): 592 (M + H). Peak HPLC RT = 3.36 min.

[00472] ¹ H NMR (CDCh) δ: (Majority) 7.39-7.32 (m, 2H), 7,327.18 (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; 6.0 Hz, 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),

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2.19 (m, 1H), 1.93 (ddd, J = 13.0; 8.8; 6.0Hz, 1H), 0.87 (s, 9H), 0.08 (s, 3H), 0.07 (s, 3H). (Minority) 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).

Step 6 [00473] DIEA (3.36 mL, 19.3 mmol, 4.0 eq.) Was added to Compound 3-16 (2.00 g, 3.21 mmol), which was synthesized in a method similar to 1-1-6), suspended in dichloromethane (60 mL), 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.

[00474] After cooling to room temperature, the mixture was poured into a saturated aqueous solution of sodium bicarbonate, extracted with chloroform and then washed with brine. After drying with magnesium sulfate, the solvent was concentrated under reduced pressure. During 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 a white solid.

[00475] ¹ H NMR (CDCh) δ: 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, 19-3.11 (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 (CDCh) δ: 149.06 Step 7 [00476] 1H-tetrazole (124 mg, 1.77 mmol, 1.5 eq.) Was added to the

Compound 32 (70.0 mg, 1.18 mmol) and Compound 33-16 (1.87 g, 2.37 mmol, 2.0 eq.) Dissolved in dichloromethane (14 mL), and the mixture was stirred at room temperature for 2 hours. A DDTT solution

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0.5 M ([(dimethylamino-methylidene) amino] -3H-1,2,4-d itiazaolin-3-thione, 4.73 mL, dissolved in 3-picoline: acetonitrile = 1: 1) was added to it , and the mixture was stirred at the same temperature for 1 hour. The reaction mixture was diluted with ethyl acetate, washed with 10% citric acid solution, saturated aqueous sodium bicarbonate solution and brine, in that order, and then dried with 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: 79%) as a colorless foam.

[00477] ¹ H NMR (CDCh) δ: 7.40-7.11 (m, 9H), 6.87-6.78 (m, 4H), 4.94-4.84 (m, 1H) , 4.82-4.72 (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), 1.35-1.20 (m, 56H), 0.91-0.84 (m, 15H), 0.08 (s, 3H), 0.01 (s , 3H).

Step 8 [00478] 1 mol / L TBAF-THF (0.937 mL, 1.2 eq.) Was added to Compound 34-16 (1.02 g, 0.780 mmol) in THF (10 mL) 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: 65%).

[00479] ¹ H NMR (CDCh) δ: 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 = 7.0 Hz, 6H).

Step 9 [00480] Triethylamine (0.073 mL, 0.528 mmol, 3.0 eq.), SuePetition 87Ο29ΟΦ3Θ0Η2, of 10 / Φ3 / 2Ο29, pg. 1Φ0 / 220

105/187 cynical (35 mg, 0.352 mmol, 2.0 eq.) And DMAP (4 mg, 0.033 mmol) were added, in that order, to Compound 35-16 (210 mg, 0.176 mmol) in dichloromethane (3 mL) at room temperature, and the mixture was stirred at room temperature for 12 hours. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (SiO2: 24 g, chloroform: methanol: triethylamine = 100: 0: 1 ^ 95: 5: 1) to obtain Compound 3616 (172 mg, yield: 76% ) as a colorless solid.

[00481] ¹ H NMR (CDCb) δ: 7.38-7.32 (m, 2H), 7.38-7.32 (m, 7H), 6.90-6.76 (m, 4H) , 5.42 (br.s, 1H), 5.04-4.39 (m, 4H), 3.87-3.55 (m, 4H), 3.79 (s, 3H), 3.78 (s, 6H), 3.31-3.07 (m, 4H), 2.65-2.43 (m, 4H), 2.38-2.28 (m, 1H), 2.28-2 .08 (m, 5H), 1.67-1.53 (m, 4H), 1.35-1.19 (m, 56H), 0.88 (t, J = 6.5Hz, 6H).

10) Synthesis of Compound 46-n

FmocNH

Petition 870190Φ3ΘΒ82, of 10 / Φ3 / 2Ο1Θ, p. 11Θ / 22Β

106/187 where n is an integer from 6 to 28.

10-1) Synthesis of Compound 46-16

Step 1 [00482] According to the methods described in Nucleic Acids Research, 42, 8796-8807 (2014), Compound 37 was synthesized from Compound 28.

Step 2 [00483] Triethylamine (1.98 mL, 14.3 mmol, 2.0 eq.) And succinic anhydride (751 mg, 7.51 mmol, 1.05 eq.) Were added to Compound 37 (3.00 g, 7.15 mmol) in dichloromethane (15 mL) 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 (S1O2: 120g, chloroform: methanol: triethylamine = 95: 5: 1 ^ 75: 25: 1) to obtain Compound 38 (3.37 g, yield: 91 %) as colorless powder. By ¹ H NMR, a mixture of rotamers, which is 63:37, was observed.

[00484] ESI-MS (m / z): 530 (M + H). Peak HPLC RT = 1.86 min. [00485] ¹ H NMR (CDCh) δ: (Majority) 7.39-7.33 (m, 2H), 7.307.22 (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; 2.7 Hz, 1H), 2.85-1.97 (m, 6H). (Minority) 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 3 [00486] According to the methods described in the Journal of Medicinal Chemistry, 48, 7781 (2005), Compound 39 was synthesized from Compound 1.

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Step 4 [00487] Triphenylphosphine (1.98 mL, 14.3 mmol, 2.0 eq.) Was added to Compound 39 (3.00 g, 7.15 mmol) in a THF-water mixture (9: 1 ) (30 mL) at room temperature, and the mixture was stirred at room temperature for 1 hour. The temperature was raised to 70 ° C and the mixture was stirred for 4 hours. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure to the crude product to obtain Compound 40 as a colorless oil.

Step 5 [00488] Triethylamine (2.10 mL, 15.1 mmol, 1.2 eq.) And Fmoc-CI (3.59 g, 13.9 mmol, 1.1 eq.) Were added to Compound 40 ( 7.15 mmol) in dichloromethane (30 mL) 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 with magnesium sulfate. The solvent was concentrated under reduced pressure. The resulting solid was washed with n-hexane and a little chloroform and then purified by silica gel column chromatography (SiO2: 120 g, n-hexane: ethyl acetate = 75: 25 ^ 0: 100) to obtain Compound 41 (4.18 g, yield: from Compound 39: 65%) as a colorless foam.

[00489] ESI-MS (m / z): 512 (M + H). Peak HPLC RT = 2.71 min. [00490] ¹ H NMR (CDCh) δ: 7.80 (d, J = 7.5 Hz, 2H), 7.60 (d, J = 7.5 Hz, 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 ), 4.46-4.27 (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).

Step 6 [00491] TFA (30 ml) was added to Compound 41 (3.00 g, 7.15 mmol) and the mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure to obtain the crude product of Compound 42.

Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 118/220

108/187 [00492] ESI-MS (m / z): 312 (M + H). Peak HPLC RT = 1.00 min. Step 7 [00493] Triethylamine (1.17 mL, 8.44 mmol, 6.0 eq.) And stearoyl chloride (938 mg, 3.10 mmol, 2.2 eq.) Were added to Compound 42 in dichloromethane ( 10 ml) 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 chloroform, water and n-hexane, and then dried under reduced pressure to obtain Compound 43-16 (888 mg, yield: from Compound 40: 75 %) as a colorless solid.

[00494] ¹ H NMR (CDCh) δ: 7.76 (d, J = 7.5 Hz, 2H), 7.60 (d, J = 7.5 Hz, 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 ), 4.39-4.26 (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 8 [00495] Piperidine (0.111 mL, 1.12 mmol, 1.1 eq.) Was added to Compound 43-16 (860 mg, 1.02 mmol) in DMF solution (5 mL) and the mixture was stirred at 80 ° C for 1.5 hours. After standing at room temperature for 2 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: 24 g, chloroform: methanol = 98: 2 ^ 75: 25) to obtain Compound 44-16 (198 mg, yield: 31%) as a colorless solid .

[00496] ¹ H NMR (CDCh) δ: 6.34 (br.s, 2H), 3.48-3.36 (m, 2H), 3.062.98 (m, 2H), 2.99 (s , 1H), 2.25-2.18 (m, 4H), 1.70-1.58 (m, 4H), 1,371.18 (m, 56H), 0.88 (t, J = 7.0Hz , 6H).

Step 9 [00497] DIEA (225 pL, 1.29 mmol) and HBTU (127 mg, 0.334 mmol) were added to Compound 38 (134 mg, 0.257 mmol), derived

Petition 8701Θ0Φ3ΘΘ82, of 10 / Φ3 / 2Ο29, p. 119/220

109/187 in Step 2, in DMF solution (2 mL) at room temperature, and the mixture was stirred at room temperature for 10 minutes. The mixture was added to Compound 44-16 (218 mg, 17.1 mmol) in dichloromethane (3 ml) at 45 ° C and then it was stirred for 30 minutes at 45 ° C. The reaction mixture was washed with brine. After drying with 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 a colorless solid. By ¹ H NMR, a mixture of rotamers, which is 63:37, was observed.

[00498] 1H NMR (CDCh) õ: (Majority) 7.35-7.32 (m, 2H), 7,317.16 (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), 3.51-3, 36 (m, 2H), 3.28-3.08 (m, 3H), 2.78-2.60 (m, 2H), 2.48-1.98 (m, 7H), 1.64- 1.56 (m, 4H), 1.35-1.19 (m, 56H), 0.88 (t, J = 7.0 Hz, 6H). (Minority) 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), 3.783 (s, 6H), 3.72-3.58 (m, 2H), 3.51-3.36 (m, 2H), 3.28-3.08 (m, 3H), 2,481.98 (m, 7H), 1.64-1.56 (m, 4H), 1.35-1.19 (m, 56H), 0, 88 (t, J = 7.0 Hz, 6H).

Step 10 [00499] Triethylamine (0.237 mL, 1.71 mmol, 5.6 eq.), Succinic anhydride (85 mg, 0.844 mmol, 2.8 eq.) And DMAP (4 mg) were added, in that order, to the Compound 45-16 (340 mg, 0.303 mmol) in dichloromethane (3 ml) 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 obtain the Compound 46-16 (246 mg, yield: 66%) as a colorless solid. By ¹ H NMR, it was

Petition 870290039082, of 10 / Φ3 / 2Ο29, p. 120/220

110/187 observed a mixture of rotamers, which is 77:23.

[00500] ¹ H-NMR (CDCH) δ: (Majority) 7.38-7.31 (m, 2H), 7,317,12 (m, 6H), 7.17 to 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). (Minority) 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),

5.20 (br.s, 1H), 4.17 (br.s, 1H), 3.90-3.74 (m, 2H), 3.688 (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 = 6.6 Hz, 6H).

11) Synthesis of Compound 49-n

where n is an integer from 6 to 28.

11-1) Synthesis of Compound 49-16

Step 1 [00501] In a method similar to the synthesis of Compound 34-16 described in 9-1), Compound 47-16 was obtained as a colorless foam (Yield: 78%).

[00502] ¹ H NMR (CDCh) δ: 7.34 (d, J = 8.0 Hz, 2H), 7.31-7.21 (m,

6H), 7.18 (m, 1H), 6.87-6.79 (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 = 7.0 Hz, 6H), 0.87 (s, 9H), 0.06 (s, 6H).

Step 2

Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 125/220

111/187 [00503] In a method similar to the synthesis of Compound 35-16 described in 9-1), Compound 48-16 was obtained as a colorless foam (Yield: 61%).

[00504] ESI-MS (m / z): 1177 (M +). Peak HPLC RT = 3.64 min. [00505] ¹ H NMR (CDCh) δ: 7.35 (d, J = 7.8 Hz, 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) , 3.37-3.08 (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 = 6.7 Hz, 6H). Step 3 [00506] Triethylamine (0.330 mL, 2.39 mmol, 4.5 eq.), Succinic anhydride (106 mg, 0.159 mmol, 3.0 eq.) And DMAP (13 mg, 10.6 pmol, 0, 2 eq.) Were added, in that order, to Compound 48-16 (624 mg, 0.529 mmol) in dichloromethane (5 ml) at room temperature, and the mixture was stirred at room temperature for 12 hours. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (SiO2: 24 g, chloroform: methanol: triethylamine = 97.5: 2.5: 1 ^ 80: 20: 1) to obtain Compound 49-16 (482 mg, yield: 71%) as a colorless solid.

[00507] Although Compound 49-16 was a mixture of 2 types of isomers, a part of the mixture was separated by silica gel column chromatography and the various equipment data were measured.

[00508] Isomer 1 of Compound 49-16 (isomer with higher Rf value based on TLC implemented with the chloroform solvent: methanol = 5: 1): By ¹ H NMR, a mixture of rotamers, which is 65 : 35.

[00509] ESI-MS (m / z): 1277 (M +). Peak HPLC RT = 3.72 min.

[00510] ¹ H NMR (CDCh) δ: (Majority) 7.34 (d, J = 7.5 Hz, 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), 3,723.48 (m, 4H), 3.44 -3.08 (m, 4H), 2.70-2.44 (m, 4H), 2.38-2.14 (m, 6H),

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112/187

1.68-1.51 (m, 4H), 1.34-1.21 (m, 56H), 0.88 (t, J = 6.7Hz, 6H). (Minority) 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) , 3.72-3.48 (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). ³¹ P NMR (CDCh) δ: 58.1.

[00511] Isomer 2 of Compound 49-16 (isomer with a lower Rf value based on TLC implemented with the chloroform solvent: methanol = 5: 1) [00512] ESI-MS (m / z): 1277 (M +). Peak HPLC RT = 3.72 min. [00513] ¹ H NMR (CDCh) δ: 7.83 (br.s, 1H), 7.37-7.10 (m, 9H), 6.74 (d, J = 8.6 Hz, 4H) , 5.30 (br.s, 1H), 4.62 (dd, J = 14.0; 9.2Hz, 1H), 4,424.33 (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, 03 (m, 5H), 1.59-1.42 (m, 4H), 1.24-1.09 (m, 56H), 0.81 (t, J = 6.7Hz, 6H). ³¹ P NMR (CDCh) δ: 57.3.

12) Synthesis of Compound 50-n

where n is an integer from 6 to 28.

12-1) Synthesis of Compound 50-16 [00514] Compound 50-16 was obtained by subjecting Compound 3616 obtained in 9-1) to the similar condition of a reaction supported on a solid resin in 4). The amount in the support of Compound 36-16 was calculated by colorimetric assay of the DMTr cation, having obtained Compound 50-16 whose amount in the support is 32 pmol / g.

Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 123/220

113/187 [00515] In a similar method, Compound 49-n can be supported on solid resin.

13) Synthesis of Compound 51-n

where n is an integer from 6 to 28.

13-1) Synthesis of Compound 51-16 [00516] Compound 51-16 was obtained by subjecting Compound 4616 obtained in 10-1) to the similar condition of a reaction for support in solid resin in 4). The amount in the support of Compound 4616 was calculated by colorimetric assay of the DMTr cation, having obtained Compound 51-16 whose amount in the support is 33 pmol / g.

14) Synthesis of Compound 55

[00517] Compound 55 was obtained by submitting Compound 54,

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114/187 described in US2008 / 0085869, to the similar condition of a reaction for support in solid resin in 4). The amount in the support of Compound 54 was calculated by colorimetric assay of the DMTR cation, having obtained Compound 55 whose amount in the support is 90 pmol / g.

15) Synthesis of Compound 63-n

63-n where n is an integer from 6 to 28.

15-1) Synthesis of Compound 63-18

Step 1 [00518] DIEA (0.447 mL, 3.46 mmol) and HBTU (349 mg, 1.04 mmol)

Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 129/220

115/187 was added to Compound 57 (Sigma-Aldrich, 0.517 g, 0.952 mmol) in DMF solution (4.9 ml) 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 separation and extraction with hexane / ethyl acetate = 1: 1 (2x50 mL) and saturated aqueous sodium bicarbonate solution (50 mL), the organic layers were combined, washed with brine (50 mL) and dried with magnesium sulfate . After being filtered, it was concentrated by rotary evaporator under reduced pressure and purified by silica gel column chromatography (chloroform: methanol = 100: 0 ^ 70: 30) giving Compound 58 (0.837 g, 85%) as a colorless solid.

[00519] ¹ H NMR (CDCl 3) o: 7.75 (2H, d, J = 7.5 Hz), 7.60 (2H, d, J = 7.4 Hz), 7.42-7, 38 (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), 4.21 (1H, t, J = 6.7 Hz), 3.79 (6H, s), 3.63-3.52 (15H, m), 3.34-3.28 ( 4H, m), 3.14 (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), 0.01 (6H, s). Step 2 [00520] Piperidine (80 µL) was added to Compound 58 (800 mg, 0.735 mmol) in DMF solution (8 mL) and the mixture was stirred at room temperature for 2 hours. After separation and extraction with hexane / ethyl acetate = 1: 4 and water, it was dried over anhydrous sodium sulfate overnight. After being filtered, it was concentrated by rotary evaporator under reduced pressure and purified by column chromatography on amino-silica gel (chloroform: methanol = 100: 0 ^ 97: 3) providing Compound 59 (0.178 g, 0.205 mmol) as oil colorless.

[00521] ¹ H NMR (CDCl 3) δ: 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),

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116/187

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 = 5.5 Hz), 2.80 (2H, t, J = 6.7 Hz), 1,801.17 (17H, m), 0 , 85 (9H, s), 0.03-0.01 (6H, m).

Step 3 [00522] After being broken into small pieces in a flask, Compound 3-18 (140 mg, 0.205 mmol) and DMAP (63 mg, 0.514 mmol) were dissolved in THF (1.8 mL). Bis (4-nitrophenyl) carbonate (156 mg, 0.514 mmol) was added and 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 being heated until it almost became a solution, the suspension was cooled to room temperature and the solid was precipitated. Then, precipitation is promoted by ultrasonic breakage. The precipitated solid was collected by filtration, washed with acetonitrile (10 ml), water (10 ml) and acetonitrile (10 ml), in that order, and then dried under vacuum to provide Compound 18-18 (196 mg, 0.232 mmol) as a light yellow solid. Compound 18-18 (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) added to it. After heating and ultrasonic breaking, water (1.8 mL) was added to it and then the object, Compound 60-18 (251 mg, 0.160 mmol), was obtained as a light yellow solid by filtration with a funnel of Kyriyama.

Step 4 [00523] After Compound 60-18 (246 mg, 0.157 mmol) in THF (4.9 mL) was cooled with an ice water bath, TBAF in solution (1M THF, 495 pL, 0.495 mmol) was added at the same time, and the mixture was stirred and heated to room temperature for 30 minutes.

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117/187

After the solvent was concentrated under reduced pressure, the resulting crude product was purified by column chromatography on aminosilica gel (chloroform only) providing Compound 61-18 (192 mg, 0.132 mmol) as a colorless solid.

[00524] ¹ H NMR (CDCl 3) δ: 7.41 (2H, d, J = 7.3 Hz), 7.30 (5H, dd, J = 8.9; 2.1 Hz), 7, 21 (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), 3.76-3.74 (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), 1.79-1.73 (7H, m), 1.49-1.42 (6H, m),

1.24 (68H, d, J = 11.8 Hz), 0.89-0.87 (6H, t, J = 6.3 Hz).

Step 5 [00525] DIEA (0.069 mL, 0.393 mmol), DMAP (1.6 mg, 0.013 mmol) and succinic anhydride (20 mg, 0.197 mmol) were added to Compound 61-18 (191 mg, 0.131 mmol) in dichloromethane (1.9 mL) and the mixture was stirred at reflux for 3 hours. The solvent was concentrated under reduced pressure and the residue purified by silica gel column chromatography (chloroform only) providing Compound 62-18 (201 mg, 0.129 mmol) as a colorless solid.

[00526] ¹ H NMR (CDCl 3) δ: 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), 2.49-2.46 (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 6 [00527] After Compound 62-18 (153 mg, 0.098 mmol) has been dissolved in an acetonitrile / dichloromethane (1: 1, 10 mL), DIEA (0.067 mL, 0.392 mmol) and HBTU (41 mg , 0.108 mmol) were postponed 870290039082, of 10 / Φ3 / 2Ο29, p. 128/220

118/187 and the mixture was stirred at room temperature for 20 minutes. Native Amino Icaa CPG 1000Â (ChemGenes Corporation) (1.0 g) was added to the reaction mixture, and the mixture was stirred for 24 hours. After the reaction mixture was filtered, the CPG resin was washed twice with acetonitrile, twice with dichloromethane and twice with diethyl ether and was dried under reduced pressure. A mixture of CapA (PROLIGO, L840045-06) and CapB (PROLIGO, L850045-06) (1: 1, 20mL) was added to the dry CPG resin, and the mixture was stirred for 30 minutes. After the reaction mixture was filtered, the CPG resin was washed twice with acetonitrile, twice with dichloromethane and twice with diethyl ether and dried under reduced pressure. The amount in the support of Compound 62-18 was calculated by colorimetric assay of the DMTr cation, having obtained Compound 63-18 whose amount in the support is 56 pmol / g.

B) Synthesis of oligonucleotides [00528] The oligonucleotides used in the Examples of this description were synthesized using the phosphoramidite method with AKTA Oligopilot10 (GE Healthcare), NS-8-1 (Dainippon Seiki co., Ltd.) Or NS-8-II (Dainippon Seiki co., Ltd.). A monomer was prepared in a 0.1 M acetonitrile solution using the amidite derived in the above amidite synthesis. The coupling time was 32 seconds to 10 minutes, and 8 to 10 equivalents of the amidite unit were used for condensation with a monomer. Oxidizer 0.02 M (Sigma-Aldrich) and iodine / pyridine / water / = 12.7 / 9/1 (w / v / v) were used for oxidation of PO. 50 mM DDTT ((dimethylamino-methylidino) amino-3H-1,2,4-dithiazoline-3-thione) in 1/1 (v / v) or 1/4 (v / v) acetonitrile / 3-picoline solution and acetonitrile / pyridine 1/4 (v / v) were used for oxidation of PS. The activator ETT ((5-ethylthio) -1 H-tetrazole) (Sigma-Aldrich) was used as an activator and CapA and CapB (Sigma-Aldrich) used as a capping reagent. Deb (TCA-CH2CI2 solution 3% w / v) (Wako Pure Chemical IndusPetition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 129/220

119/187 tries, Ltd.) or Deb (dichloroacetic acid solution, 3% w / v toluene) was used as a destritylation reagent.

[00529] SEQ-321, SEQ-323, SEQ-340, SEQ-343, SEQ-346, SEQ349, SEQ-352, SEQ-355 and SEQ-358 were derived by supplying Compound 22-18 to GeneDesign Inc. to consign nucleic acid synthesis and oligonucleotide purification. DMT-butanediol phosphoramidite used for the synthesis of SEQ-316 were purchased from ChemGenes Corporation.

C) Synthesis of lipid-linked oligonucleotides Synthesis from a synthetic amidite unit [00530] An agitator, Molecular Sieves 4A 1/16 and the amidite synthesized in A) above (for example, Compound 4-n, Compound 4-n, o, Compound 8-neo Compound 10-No. 10 to 100 equivalents of an oligonucleotide) were placed in a microwave tube (2-5 mL, 1020 mL) made by Biotage, and the solution was adjusted to 0.2 M with chloroform (2-methyl-2-butane added as stabilizer). After drying for 5 hours, oligonucleotides supported in solid phase (CPG resin or polystyrene resin) and the activator ETT 0.25 Μ, which is ((5ethylthio) -1H-tetrazole) dichloromethane (the same amount of chloroform) , sealed and heated to 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 to oxidize PS to NS-8-I (Dainippon Seiki co., Ltd.) Or NS-8-II (Dainippon Seiki co., Ltd.). Then, the dry resin was subjected to the deprotection I or II condition for synthesis of the oligonucleotide bound to the target lipid.

2) Synthesis from resin supported lipid [00531] Using a resin supported lipid, synthesized in A) above (for example, Compound 15-n, Compound 22-n, Compound 50-n,

Compound 51-n, Compound 55 and Compound 63-n), oligonucleotidesPetition 870290 (039082, 10 / Φ3 / 2Ο29, p. 130/220

120/187 deos linked to target lipids were synthesized in a method similar to B) above.

D) Deprotection

Cleavage of resin and deprotection of phosphate and base [00532] To cut the DNA oligonucleotide, 28% water ammonia (SEQ-1, 3 or 49) or 28% ammonia / EtOH 4/1 (v / v) was used ) (single-stranded oligonucleotides described in the Examples, except for SEQ1, 3 and 49) and the solution was stirred at room temperature for 1 hour and at 55 ° C for 5 hours. 1 ml, 5 ml or 10 ml of ammonia solution were used for the synthesis of 1 pmol, 5 pmol or 10 pmol, respectively, in the clipping reaction. After the resin was washed with water and 50% ethanol, the filtrate was concentrated under reduced pressure to 1 to 5 ml.

[00533] When the sequence contained RNA, the resulting solution was lyophilized to obtain a white powder and then the next deprotection reaction of the 2'-TBS group was carried out.

2) Deprotection of the 2'-TBS group [00534] N-methylpyrrolidone / triethylamine / triethylamine trifluorohydrate = 6/1/2 (v / v) was added to the resulting white powder and the solution was stirred at 65 ° C for 1, 5 hours. The same amount of ethoxytrimethylsilane was added to the reaction mixture, and the solution was stirred vigorously 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. Diethyl ether was added to the resulting precipitate, and the solution was stirred vigorously. Then, similarly, centrifugation was performed and the organic solvent was removed to obtain the crude unit of RNA (white solid).

E) Purification [00535] Oligonucleotides without a lipid linker, such as SEQ-1, 3 and

49, were purified by reverse phase HPLC in Condition 1.

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Condition for reverse phase HPLC

Condition 1

Mobile phases:

Buffer A: 100 mM TEAA aqueous solution (triethylammonium acetate, pH 7.0) or 100 mM AcONa aqueous solution (pH 5.4)

Buffer B: acetonitrile

B concentration gradient: 10-30%

Condition 1-1

Column: Hydrosphere C18 (YMC co., Ltd.) 100x20 mm D.I.,

S-5 pm, 12 nm

Flow rate: 10 mL / min

Column temperature: room temperature

UV detection: 260 nm

Condition 1-2

Column: Hydrosphere C18 (YMC co., Ltd.) 150x10 mm D.I.,

S-5 pm, 12 nm

Flow rate: 4 mL / min

Column temperature: room temperature UV detection: 260 nm [00536] Single-stranded oligonucleotides with or more lipid ligands (single-stranded oligonucleotides described in the Examples, except for SEQ-1, 3 and 49) were purified by reverse phase HPLC in Condition 2 .

Condition 2

Condition for reverse phase HPLC [00537] According to the compound's lipid solubility, the concentration of B was adjusted at the beginning of 20% to 50%.

Mobile phases:

Buffer A: 100 mM TEAA aqueous solution (Triethylammonium acetate, pH 7.0) or 100 mM AcONa aqueous solution (pH5.4)

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Buffer B: acetonitrile

B concentration gradient: 20-80% (compounds with L4-8 or L4-10),

30-60% (compounds with Ltocou Lchoi),

30-80% (compounds with L4-12, L4-14, L4-16, L4-18, M22-12 or M51-16),

40-80% (compounds with L4-20 or L4-22),

50-80% (compounds with M22-18)

Condition 2-1

Column: YMC-Pack C4 (YMC co., Ltd.) 100x20 mm D.I., S-5 pm, 12 nm

Flow rate: 10 mL / min

Column temperature: room temperature UV detection: 260 nm

Condition 2-2

Column: YMC-Pack C4 (YMC co., Itd.) 150x10 mm D.I., S-5 pm, 12 nm

Flow rate: 4 mL / min

Column temperature: room temperature UV detection: 260 nm

F) Desalination and lyophilization of the purified oligonucleotide [00538] Using VivaSpin20 (MWCO 3000) (Sartorius) and Amicon Ultra-4 Centrifugal Filter Units-3K, ultrafiltration was repeated on the resulting oligonucleotide to remove the saline component from the fraction. Then, lyophilization was performed to obtain the target powder oligonucleotide. For oligonucleotides purified using the TEAA solvent, the desalination procedure was performed after transformation of the salt form with 100 mM sodium acetate solution (20 ml).

G) Purity analysis of oligonucleotides

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123/187 [00539] The resulting oligonucleotide was confirmed as the target sequence, comparing the molecular masses (Mw) found, determined by measurement with UPLC / MS, and the calculated molecular masses.

Condition 1 (SEQ-1.3 or 49)

Xevo G2 Tof System (Waters)

Column: Aquity OST C18 (2.1x50 mm) (Waters)

Mobile phases:

Buffer A: 200 mM 1,1,1,3,3,3-hexafluoro-2propanol / 8 mM triethylamine aqueous solution

Buffer B: methanol

B concentration gradient: 10-30% (10 min) Temperature: 50 ° C Flow rate: 0.2 mL / min

Condition 2 (Single-stranded oliqonucleotides described in the Examples, except for SEQ-1, 3 and 49)

Xevo G2 Tof System (Waters)

Column: ACQUITY UPLC Protein BEH C4 Column, 300Â,

1.7 pm, 2.1 mmx100 mm, 1 / pkg (Waters)

Mobile phases:

Buffer A: 200 mM 1,1,1,3,3,3-hexafluoro-2propanol / 8 mM triethylamine aqueous solution

Buffer B: methanol

B concentration gradient: 10-95% (10 min) Temperature: 50 ° C Flow rate: 0.2 mL / min [00540] The results were shown in Tables 1 to 3.

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Table 1

No. Theoretical Mw [M-H] - Mw found [M] No. Theoretical Mw [M-H] - Mw found [M] SEQ 1 6363 6365 SEQ'76 5117 5116 SEQ-2 7064 7065 SEQ-78 5554 5554 SEQ- 3 6254 6256 SEQ 80 561.0 5610 SEQ 5 6955 6955 SEQ 82 5666 5666 SEQ * - 7 8842 3842 SEQ 84 6798 6799 SEQ9 5367 5367 SEQ-86 6854 6855 SEQ 11 6731 6731 SEQ 88 6910 6911 SEQ-13 6843 6843 SEQ-90 8451 8452 SEQ 15 7012 7011 SEQ-92 8451 8452 SEQ 17 6698 6699 SEQ 94 8451 8452 SEQ-19 6369 6370 SEQ -96 8563 8564 SEQ21 6040 6040 SEQ-98 .8731 8733 SEQ 23 5736 5736 SEQ 100 8675 8677 SEQ'25 5423 5423 SEQ-102 .8731 8733 SEQ-27 5133 5134 SEQ 104 8395 8396 SEQ-29 4829 4829 SEQ-106 8507 8508 SEQ-31 4500 4500 SEQ-108 8563 8564 SEQ-S3 4211 4211 SEQ-110 6042 6042 SEQ-35 3898 3898 SEQ-112 6362 6363 SEQ-37 7570 7570 SEQ 114 7323 7323 SEQ 39 7291 7291 SEQ-116 6067 6067 SEQ-41 7330 7331 SEQ-118 6412 6413 SEQ 43 »844 5845 SEQ 120 * 448 <44 i SEQ 45 7363 7363 SEQ 122 7287 7287 SEQ-4 7 7419 7419 SEQ 124 7607 7607 SEQ49 7697 7698 SEQ-126 8568 8569 SEQ-ÃO 8211 8212 SEQ T 28 7312 7312 SEQ 52 7569 7568 SEQ 130 7657 7658 SEQ'54 6967 6966 SEQ-132 8693 8693 SEQ 56 6348 6348 SEQ 134 8385 8386 SEQ - 58 5722 5722 SEQ-136 5598 5599 SEQ 60 5409 5409 SEQ138 8216 8218 SEQ-62 6049 6050 SEQ-140 5430 · 5431 SEQ-64 6099 6099 SEQ 142 8399 8398 SEQ; -66 8932 8933 SEQT43 9089 9090 SEQ 68 5859 5860 SEQ-144 8652 8651 SEQ-70 5816 5817 SEQ-145 5426 5427 SEQ-72 8451 8452 SEQ 147 5410 5410 SEQ-74 5161 5160 SEQ-149 5470 5470

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Table 2

No. Theoretical Mw [M-H] - Mw found [M] No. Theoretical Mw [M-H] - Mw found [M] SEQ 151 6758 6758 SEQ-229 5992 5992 SEQ 153 7102 7102 SEQ-231 6048 6049 SEQ-155 7793 7793 SEQ-233 5987 5987 SEQ 157 8139 8139 SEQ-235 8370 8370 SEQ-159 8484 8484 SEQ-23 7 6700 6701 SEQ-161 7761 7761 SEQ-239 7390 7392 SEQ 163 8074 8075 SEQ-241 6756 6757 SEQ-165 5912 5912 SEQ-243 744-6 7448 SEQ167 6662 6603 SEQ-245 6644 6645 SEQ 169 7638 7638 SEQ-247 7334 7336 SEQ 171 6386 6380 SEQ-249 6700 6701 SEQ · 173 7368 7368 SEQ-2Ô1 7390 7391 SEQ 175 5782 5785 SEQ-253 6348 6349 SEQ177 6468 6408 SEQ-254 5793 5792 SEQ-179 7569 7508 SEQ-256 6326 6328 SEQ-181 8481 8482 SEQ-257 5615 5615 SEQ-183 5694 5695 SEQ-2Ô9 5687 5687 SEQ-185 6073 6075 SEQ-261 5743 5743 SEQ 187 6764 6765 SEQ-263 5967 5968 SEQ-189 7800 7801 SEQ-265 6023 6024 SEQ 191 6129 6130 SEQ 267 5951 5952 SEQ-195 6820 6819 SEQ-269 6007 6010 SEQ -195 7 856 / 856 SEQ-271 6063 6064 SEQ 197 6017 6618 SEQ-273 6642 6642 SEQ-199 6767 6709 SEQ-275 6698 6699 SEQ 201 6073 6074 SEQ-277 6754 6/54 SEQ 203 6764 6766 SEQ-279 6-468 6469 SEQ-20Õ 6067 6008 SEQ-281 6324 6525 SEQ-207 6063 6063 SEQ-283 6552 6553 SEQ 209 6119 6126 SEQ-285 7609 7608 SEQ21 6698 6698 SEQ-287 719-6 7196 SEQ-213 6754 6754 SEQ- 289 S441 8442 SEQ-215 6816 6812 SEQ-291 7252 7252 SEQ-217 6119 6119 SEQ-293 8498 8499 SEQ-219 6175 6177 SEQ-295 7180 7184 SEQ221 6810 6811 SEQ-297 7236 7240 SEQ -223 6867 6869 SEQ-299 7292 7296 SEQ-225 6192 6192 SEQ-301 7870 7873 SEQ-227 6028 6027 SEQ-303 7926 7929

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Table 3

No. Theoretical Mw [M-H] - Mw found [M] No. Theoretical Mw [M-H] - Mw found [M] SEQ-305 7983 7986 SEQ 346 5944 5942 SEQ-307 8425 8426 SEQ 348 9346 9345 SEQ 309 8537 8541 SEQ 349 5919 5916 SEQ-311 9115 9118 SEQ351 7048 7046 SEQ 313 9171 9175 SEQ 352 5921 5920 SEQ 315 9227 9230 SEQ-354 8050 8049 SEQ317 6247 6248 SEQ 355 5927 5926 SEQ 319 6213 6214 SEQ-357 6 778 6777 SEQ 321 6109 6107 SEQ-358 5775 5771 SEQ-323 4248 4249 SEQ-360 5574 5575 SEQ 325 7236 7238 SEQ-362 5839 5840 SEQ327 7292 7293 SEQ-364 5895 5897 SEQ-329 7348 7350 SEQ 366 5631 5631 SEQ 331 7926 7929 SEQ- 368 5895 5896 SEQ> 7983 7985 SEQ-370 5951 5953 SEQ. U 8039 8041 SEQ-372 8481 8483 SEQ · 337 5619 5619 SEQ-374 8537 8539 SEQ 339 6433 6434 SEQ-376 8593 8595 SEQ 340 5775 5773 SEQ 378 9171 9173 SEQ 342 7342 7341 SEQ 380 7151 7154 SEQ-343 5936 5934 SEQ 381 5860 5861 SEQ 345 8341 8339 SEQ 383 8833 8835

I) Preparation of the double-stranded oligonucleotide [00541] The lipid-bound double-stranded oligonucleotides of the present invention were prepared as follows. After mixing the equimolecular amount of a 100 μΜ solution of each oligonucleotide, the solution was heated to 75 ° C for 5 minutes and cooled naturally to room temperature to obtain double-stranded nucleic acids. The formation of the double stripe was performed 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

Temperature: room temperature [00542] The synthesized oligonucleotides are shown in Tables 4 to 27.

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Table 4

10 ΈΟ JJ> fe String; <Complementary tape: 'Ütotolto 0 tos ;; <· to't''g'a''e''g ^: 'to''to'e <' r ''gtoV'e ^? 'g't't'to ^: \ na ¹ 'νΛ' I 5 ^ to ^A c ^A and ^A ftoto g 'a' toto to toto 'togrto ^Λ g' t ^A 1 to S-5 <> toto U ^A gwsgeaa ^ í>cto''íto »to · -'EQ-4 | OWlSSfí (1) . UI U> 'to toe a í *>> t t- ς <to a to e £ to to>' â to 'to tototogeaa ^ gaetototo to' 5 ‘ '5 ÈQ'5 S- to ' : V’U g 'toaefsva: xggas? Í g' <'' íí 'a L * í <eto sfou | ÜBMSÚS W Sto to ’ toítoto-to ’tog '3' to; ú 5’ toe'-to to «’ <· '»' <to '2' -V-.5 g 'gtaeígeaaggatfg' <'tf’U ««' 5 ' Lm w to'3''g'gatog ·;. '' o''3'1 ... s 5 5 ' SEQ-5 ODtoHStoto <; s §Htoto'to ^A t- ^; ^ ^A to ^A e. ^ TotoFto ^z e ^A t ^A g.to ^A e. ^x g ^A l ^A t-rS: · ' sfq-5 to'4 w ____________________________________________________________________ toto t 'geaaggaetgr' cVtoW » ¹ SEQ-to Ui u> §To to '''toa' rg to to 'g to' to etoto'g ss ^{x [} to g to. to-to àto 'ϊ' gcôa g ga toto 'c''to ^Λ aL ». ^> - 5 ' toEQ-U 'í-õ to * 3to g gtaet-reAaggactg to to- a 'L; ? -5 ’ stvyvj toON Etoto iO S-é to ⁵ .y-fv <-to'to'to ^{, A} at toto toto 'to' g a'c'to torsi 'toa'to'gtaftos: r & agg »<tg ^A <? · 'toto L. ^ ·?' S E <) '; 4 toof. to> Stotoçtotacs gcaa ^ garrto 'e'; to ^A L <$ zV * EQ-H the [i \ '-totoi * f <. > “>! “Etoto 'Hto''#to?''' To toto to ^A a * 'f? G ^A t' t * 3 ^! V \ sto Ui. u ^ EQ't . '' to to} to-a 'ii. 'gl; <etuí' «s <g«; w3g to · ís a L, f ^ ‘ '(WW Ü) V? \ '' 3 i g a <£ 5 i e '<': g n> 'g t to-a' £ 'gtaetgcaa ^ íxetg' ’to 'e'a' ívõ’ 3to ‘g 'Utt ^ cas ^ aetitoctototoLi;« - â' SEO'Lx πηχ ^: u) - '' * <v 5 ^s ' t '(?' E * a ^A t to ^A ac to t ^A í ^Λ « ^Λ '-''''''a<''&' ^{r Λ} 1 'to 3to to' í ací gs.3 aggaeíg 'eaa' U · ιη'λ ' -EQ-ie Ul> ’G> to '' aefst'-a iggaíg v e 3 Ϊ ,. vU YOUR 'òD'XEto6 to' _s U9 bj> to- {'' 3 i I. '(. g 5 i <\' 'g' «'t'> ' 3'-g 'í artgcaagg'aet g' and ‘aV Ls-vto ' -to? to · T 'í' a ^; € tgvaaggatog> :: 'aa to- s s- &' 'FO ‘2- ’UOMtoU tos - 1? to ' 5 '-í e' e ^A a ^Λ t to ^A ac ^Λ g · 't to to'<'t * ga' e * g r '' t to 4'-tto '«g ^f Si.aggaeíg'to' 3 'to U s§-.5' ’? 5. < 3'-3 ·: '' Igeaaggi-ijg 'to'a' ii 'Ϊ3;>.'? * ' i W-4 ’> DM? Í <u -Ui 5to 'c' ea ^A t ^A g ^A to '''g ^A t''t * <e ^Λ t ^A g' a ^Λ c 'g ^A t' to · to: l'-a '<? · 'g <8agto ^: tofg'e ·· ato' U x-rto i -eu - i? '< 3 to- 'r' geaaggngí í ''? ' a a 'to {. $ - to toi <to 12 to ./ 5'-i '<· asga <g 5 5 and a. ga <gt. · <________________________>'·''' to << ³ í. & · „?: ______________________________ hE ^ -27 («» _: to 1 'g toVgg.v ¢ 4 ^f 3 W l><7 51-ÀJ-2E ÕDÜISÍU d) to-t «\»> t 2 3 «\ toto x '< ⁵ e'a ’To evgganj. '«' To Lm ^ S’

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Table 5

At the. NEQ ID) 8 Sequence: pG * ik Complementary tape SEQ'29 8 14 «10) 3 ^: gNVaaggaety and .- to 1 ..-. + + SEQ 30 ODN 1826 (11 & 14 <10) b + t '· cN + a + t + g? tr ^! * t ⁷ T; . ena LüôÕ SEQ 31 5 15HD S + c + a + aggacig <· a to Lj .V SEQ 32 ODNlxlh *! ' 8 15) 1B 5'-t''c + c + a ^A i ^A ga ^ 'etEc.'tgaogttT í ta ot ^ a ·' · - '.1 to 7> SEQ-33 S 16112, S + a + a + ggact ^ 3 c '3 L _; .. 1 SEQ 34 ODN1826 (1} S 16112) õ + t ^A s ^% c ^A a ^? t / guckttc ”ga <· wrt 3 * 3 '-><a - L ^ ift s SEQ 36 8 17’3) 2 1 = g ·> tg <'> - L- * · - SEQ 30 ODN 1826 (11 8 17 <3> r <- · 'tt _t ''gitvr? 1 <gtt 3 »· g - gsetg <to 5» L4i £ -: 5 ' SEy 3'í 8 18i2) 3 'aoiíe + gWe'gOMfUasjeUeSieP-'ilde ~ f iltcv-u cxs ^ eawea 03>, fegos & 5ΟΜΒ &<.<Μϊ0Ο1χΣϊί-ί05ΐϊ«31ί; ίΜ _β ^ Οΐ &' • ÇOlfe ^ aOMe SOMe * 'L ^ -ls SEQ 38 ÔDN1826 (1) 8 + 18 (2} .δ + f'e '''c''''a''t' g 'a'c'g'g'a'N' gt ”t 8 '3' SOM * '· gOJíe + gWeCSOMíUOKçaOMíéOiís ^ j ^ WO ^ ígOMeC.pJsííaoJáea SXfegfcMe gOMeaOM »COMeU0i £ eUOM» §OS & ^A CGM: »'' aCS & a <fcfe 'Li-ts &' SEq íh 8 11-2) 3'-A · G- GTACTGCAVIGAC'TG 'C' A · A L41S 8 ' SEQ 4Í> ODN1A2H <1? 8 1912 ' á ^: (<-: <- · -p 't'·.> - 's / ΰ V' t 'r' · c W § ++ '++'g'N't 3 '..1 AG ^Z GTACTGCAAGGAC'TG CA Λ L; is 3 ' SEQ-41 8 2 (-2 = ί A _t G.UiA L UrG _s .Cj.A; .Aj..G _? Gj. A ç l fG _} . '' Cp '·' A _t H. lj ' SEQ-42. ODN1826 (1} 3-20 (2} 5 t. . 3 r 1? . ttk> ⁺ 1 '_& * y 2 A _t <GG'«'t'FG _í ..C _5; A _i .: A _í G _í G _f A _i; C _{: f} .UtG _f / ^: Cp + A ₍ À, Uís V SEQ-43. ·. 8-21 (4) <. <£,! · WJI »3> Je Lí 1 = 5 ' SEQ-44 ιΊΆΙΆβ *!) 8-21 (4.) t · .. <~ ar »0 A = 'G έ' + tg + 3 gt + t 'S' ³ '1? #. 1> \ íe> ifeÇ <'VtOUeC ^ é ^ iíWeU QMWÍfSi * <!' 3 <'? Of Lh 7 SEQ 45 ’. ° * í '<' 'v. ^ O’33.' etc SEQ 46 ÜDNU26ÍD 't ^ .i-'arsa ^ = ££'. ' + _the <<'tt Ί 2'J>. .. g =. ^Γ '>' Τ- 'Ί to 7 <' SEQ-47 S 2> M2? Μ. K> 1 S yt + - ayg r _t ^ _4ί 1 SEQ 48 ODNls26tl> 8 2 ’« 2 / · .. ”. »Tg> _c rt <_ = <'' - Mi'1> 3 g gíaotg '^ +«; g'7i'íg cg -1 .-. +. + SEQ - 49 DD \ ’j <> .36 * 13» V t, s ^T <stttt £ ⁺ <gttttgtg'-f '· 1-3'

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Table 6

N- II) .SEtf 1D- Rí Sequence: · λ · α5 '-? 3') Cs · Complementary tape S ') SEQ 5-0 S J4Í14 ’ λ, ·. - 3t. 'a to Lj o' <Ες .1 ODN> 06 ’i + '+ 2104 = 5 'tcarcj.ttttg'l _β t »tt _& r ^A c: ^A g ^A t ^A t-3' 3 3 - '..íi.iji / í'- -I .-, ..: 4 ΐ 4. 3 3 Lj v 7 7EQ · .: 3 <-.t> 3, u, Lí _s cr SEQ Ί G »E» N2 (> oGl · · £ ^ 13 tc /: c _T · * - ^r gr, jr '* · ₃ t í í:: .3' 3? α V- 3 3 Ll 2S <:>' + EO '.4 S> /! * '· 3 '* <J υ' G * W ίβ .oDN2ooéila> S- 2 6'16) 5 t, tt. t3 « ^A g ^A t ^A t.-§ '9'><\ 333 <Ί -.«. 3ΛΊ33 ^ 3 ^ vu ·. L ·. Λ ' SEQ õ> 7 S 27117) (as P3tá: '3-f' _C , 't 3 1.4 1; 7 SEQ .-. 7 ODN2Ô06Í13) S 27 (171 Stp ^ rc ^ rttt ^ t '* t - tr _& x- ^s Ω. <Í '? <J. J. 2j4-£ -? 8EC - S 38 «18) 3 4 ’1 3’ ^^ í 'U'4 „- ϊ’ L, 1 ;? SEQ> 9 GDN2O067.1..3Í 3 28 (18) > t <gtc »r - r 1 gt <. gt i-3 '' -í - _H cg ^ aaoscfig run 'L $ I3 -> 8EQ-60 S 29 ·)! + 3 -4 <'bl3.iil <· Λ 3 E; SEQ & l 1> DI- 2í>'! ^} 1 · S 29'19 ' '7 ttt <- tt ΐ tt xtt 3' 3 C Λ »<3 saw \>'A _P 0 to 3 Lí li 5' SEQ b2 S 30 (19; '(υ 4 3 -. <’-í 1’ ί is, 3 t t L'. · <· ' SEQ G3 ODN2006Ú.3) S 30 (19) S'r'arcgtrttat *, rr t '''t''' g ^A t ^A .c ^A g ^A t ^A t-3 '' 7 a <atccu-iji wag <aatt L, st SEQ G-t S 31'19 ' 3 \>'4 ^ 1 <3 <- 3' _c \ -. 'to 4' s 'T.-í'. SEy> '! GDN20Ü6 <13) S 3K19) .3 t <· λ rr --rtairjE »· t ^A t ^A g 'í' ¹ is ^A g ^A t''t'3 'p 3 3 í-. g 3 Lji _t 7 ' SEQS6 S 32 (14) 3 'auv · ÃOVe «\ V ^ OSt-fc-'Gi.M.O''í» arai * 3>. ^ TeaoJ> íeáOS &' € ÓMeaCi & fc - .. 'λ. ac's-Ai-k '' W-ic-Wd. <'; ^ 3c? .I>3Q4' _F 'cuii _i ^A aoi £ i,'^; isc! ji _S · ' SEQ G7 ODNJOOGÚSí S ._ \ 14) 3 '£ ·; regr: i 't -'. ι - ·. · -. =. ·. ϊ ^Α ί ^Α 8 · '·''ί: ^Α σ ^Α ® ^Α 1 ^Α 1; · 3' 3 'sSOLIi SUS &''cM ^ i'2de-leps,> y _f >K> _ls to ^ t> Sí 'COME ^ ^' ^ and the IOI & CQjvaoi ^ <^^ a ^ and Q ^ <^ the ®í-OM ^'the GIS & çcssí ^{"the a} to aííMe <Mé ···. U i- Yes .____________

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Table 7

Μώ, ID (SEQ ID) S String:) ptí (o ^ 3'í C <Complementary tape -> 3 '« SEQ 68 S-33 (19) -i <-Ο'Λ c <Μ * Μ- a -> \ íe Ε, λ ' a ^ eâÔJí & a <: e & aoMÍCOSE ₅ ÃMEGO ^ SEQ 69 ODN2606Í13) S 33 «19.’ ώ - - i * r 'r' Cw ,. aiCiie ^ OV. Pií, acsíe-: aoMe 'Lá-ÍS' · »* t Λ ftfeS OMeaoSfe SüMs ^ OStóSO & SígOW 8EQ S 34 (195 - Μι Pd C c to ΐ L «Ρ V ~ -EQ 71 ODN200í> (W- S 34 «19? τ · ' Μ ^ § t <t t t £ p ã < ί <; c ^s ad L 'ttt _έ 't'-e- · 4-13 · - * g- «-i- 3‘ j 2 S 35 «14? 7 Μι ρ a c ^ l’aa ^ ea ^ aa ^ -H <a. L4 :. 3Fq> 7 ODN2006Ú3) S 35 (14) 5 'τ < 'Ml l, i 8 g cjycss ^ acaií 38.w .u t t t s r »'  . <Lí 12 . t.M <3 ’ BE-Q 74 s-36 «ias 2 '1 κ 3tC3i <\ '· 3 j £> · <χ 3 SEQ 75 ODN-2006Ó3) S 36 <19? ^λ : <”/ = ¾ L ' j 'tl t' t'i Λ ^ ’Γΐιί Γ Si.-Í L V ΐ r r 3 't’V WK ’ SEQ 76 S 37 «19? 2 Ί 3 νδδ ’r to 3 I. <hJ '< SEQ-77 ODN2006 (13) 8-37 «19? :> · R · ν » ·> T. · .. κ 1 ' =. ΐ - '<? ·· - r -r t Ά ’ 3-, 'iío' d 3 3 t <= - Le> 4 3 r t τ g 't' '' c · ' 'rT-t ·! 3EQ 7 S 38113) 1 i <. ( 3 L-t1; ' <EQ 73 ODND’OCnU S 38 «IS? 3 ’Λ -¾ <a t? t .. l _1} · y rMM ^A g ^: M ^A e ' SEQSO S 39 (18) • J ·? 'Τ ?? ca $? c333d ^ 3 _C · <, a ' to L ₄ 14 .- SÈQ 31 ODN2096 (13) S 39 «13) 3 '. <. < »T <ct ^{r T} ts'.« - _β <.J t C í ’ 3 Li 14 --- r 'i''t ^/ - g -:''t: ^/ ' c ·· 'gWtrf 8FQ a? S _; 3 · '} ^Γ } · ί 2a ^ ra333ca £ u ί • L _T if ' S Eq »3 ODX2uf 'K <13? 3 40-1 ^ 3 V κ .. 5 1 C t ΐ t < ⁽ v3 ^ <333d ^ 3 ^ <. A ' t L> t Li Li. ' t '+ hqri'k'

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131/187

Table 8

At the. Ilí sSEQ ILH ; String: CpGiS '^ S') C Complementary tape ίδ’ΌΝ) SEQ 8 · s 4iac. ’ 3'N ‘''«: + '3s: gacàereaààacs «' 'W al'E-i-js S' SEQS5 GDN? 0060P 3 41 * 16 * 3 't. irt ΐ l _and 'ir. *> rt 3 ^f 7 = i <aa Ls »2 y SE.Q 86 S 42 * 16) 2 u <a ·. t d- t t> SEQ-S7 G £ »N2 () õ6G5; S 42 * 16 ' ^ tcíT r r - 2t t 4. t t t t κ t ’Γ 3 ' 3 12 '1 ΐ x,> SEQ 88 3,4306; 2 l λ3 <<3 L. </ ODNUOCiíi 3 » 3 il ·. 1 * 0 y :: vu,. <ittcg * ii - *> $ i 1 ^f <<a '^^'Ρι'Ίί<. ax '4'> V SEQ-90 S 44 * 14 · ’ 5i -, ΐ =. - ·? .3; · <. · -,. 3 j L „i4 5 SEQ 91 ODN2006il: U S 44 * 14J ⁷ c t. tt * ⁺ t ⁺ t, t ΐ trt? í <et 't 3' -? 4 ia - .. '' thereKPi,. <> L „ii 5 SEQ 92 3 45 * 14) '51,., Ί =, ---. 4,3.-51 at,'ij.ii.:'=í r 3 -i L, i _t 3 SEQ 93 ODN '<“' ó ¹ 13 * E-45> i4 ' Ή3 ' 3rd:; ¢ 5 5 .--- ι, π, .3.: -, .., 5, - c Λ to 1.11. Λ SEQ ^ Í S ’μ>: 14 ' ί? C a - u> i s cEQ '? ₃ ί> ΠΧ2ΓΌ (>, 1 O S 4 »” 14 ’ 'ΐ, - ^' - ' _Ε .ΐί ^ί · ^< · _ί . + gtttfgtkg * ·' t 3 '3 .'O: <· a «-, o <. í>'? õ ⁽ . íao-hi ·. -., 1,: S F j Mb + EQ <4 <· 14 'UDN2V ‘? B' 1 j) + 4, -14) 2_ο Γ3 ^ ΜΆ313? <. 33ΜΑΓ 'to Γ 1 to L, l _s 5________ L · tt 7 2 Γ <J Ul G _f A γ Z''x ^λ

Table 9

Ng. I.D * SEQ ID) S Sequence: EpGf.S ’^ S '? C Complementary tape '^ SO SÉ0 98 S 4S »14> 3 'Mis-ieG · ’g' cagcaaaacagcaaaacag 'c'’ 'a' to L .; is 5 ' 3EQ 99 00X2006 * 13 · » S 43 * 14! 5 't V'g + 1/0' £ 'th ⁷ r' rg 't'-cg ^s t't''t''t'•^' r- 'c + g' 0 t 3 '3' M-sj-io 'a' g · 'cagcaaaacageaaaacag'>? ' aa 'Li-is:?' SEQ 100 S 49 * 14 '* 3 'Mu.-iOa' g 'cageaaaacagcaaaacag + c + a ··' a ’'Li-is» ’ 8EQ191 Ο.Γ'.Μ2ν06 * L3 ^! S 190 0 yt ^{, x} £ § rr§ ·· '' rryyg ^ tr · gvryrrg / ye ^A g 'r ΐ 3 * U yijJ-14 íl £ C & Ã .L>4-it'. Q 8r. ^ * 2 8 50 * 14 » 3 ^: Msí.-ís's''g'cAgcaaaacagcaaaacag' c 'aO' L-iís 5 ' '' tlAj _ * V: *! PN2 *) 06 * 1 3> g. 50 * 14! 5 't 5- g' t-s'Vr't rtg f-c 'gt ·' rh ^ - c'g- rr 3 '3' Mj = -js 'ag ·' cagcaaasi-agcaaascag · ''? · · A 'a-'Li-is 5' XF cr? b 51 * 14 » 3 ^: Miâ-s '' a 'g Osgcaaaacageaaaacag' · ca O L <-i; 5 ' 8EQ 101 *> L) N10 Y * 1P S ‘1 * 14’ 5 '1' g 1 'and' g 't' f 't /' f 'g ·' t 'ç' § Ό 't' t 't g'tc' · gt t 3 '3' Mi = -e '' a ''g'-cagcaaaacagcaaaaeag ^- ca 's. Lj-is Õ ' -'EQ lÍ ** t S 12 * 11 » 3 ^: Mis-iá 'a' g 'cagcaaaacae <aaaacag' and a 'a' Lj- ·; 5 ' 8EQ 10. ’ (Jl’N-4 »CO J.i 8 52Ί4 '» 5 're 2 rrrregt á tt 1'S'-Mis-u'a' g '''<? Ageaaaacapcaaaaca? R ^z e' ^- a '· a' L4-12 5 ' SEQ-108 3 ãi * 14 » 3 'Sliá-is' a 'go cagcaaaaeageaaascag <?' s' a 'Lí-í: ã' SEQ-109 OD \ '_ o Ι-Ό3) b 58 * 14 » ã'íc-'gf 'cg' Ό t / t + t + g 'È''c''g' 1 'r' rt g't / c gt't 3 '3' Aíij-is '3' g '' cag'caasaca «caaaaí.'ag” · 'c' a 'a' L4-12 5 ' SEQ1110 3 54 * 1 '» 3 '3.' a 'cagca & ase ag ’' c '' a · 'a tLi-1» · 5' SEq-lll ÍDNJ0Í4 03? 5 ^: t ^Λ ο 'g ^x tc' g “t 't' t 't' .i 'tt' g 't' t '* t''t' g 't' c 'g' t '4 3' 3 ^: -a- '.a''cageasaacagl' c 'a''s' t ·· L4.13- 5 ^:

Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 100/220

132/187

Table 10

Ng, ÍL> «EQ ID) S String: j6? V ^ -í ¹ C Complementary tape '= ^ §'í 8EQT12- SEQ113 ΟΓ \ 2 * 106 (13) _3 '<' + í '· ..- SJA-CW-! y <3 \ l''i '+ L.' If - / ______________________ V tc - r <- s + tttgtcstrr fg- 't ^A c «. ϊ. E j '> + · 3 1 to T t 1.4 - V SEQ 114 tf Γ · ό 'ItfJ 3 't Ά <! $' Ϊ́c a 3 · '’r t t L & ig S' 2 cr w < tf 36 ' ·>'Ί ·. * · Í 'i' τ rttgtk s- i »'t ^A _: g-' t ·· '' _ç ..XgA |., Vj..g ·· '>' 3 3 HH jue.i · Λ {· 's ί T ttt ΐ Li-is ã' SEQ 11 « 4 .., already. 5 3 * · aa τγ \ v · 5 ^Λ SEQ U7 nnxvoG 13í tf 37 'l · > 'r c t j t t t t g t c j? r t t t * a ~. cagcaaaac ^^ tf'c- SEQ 1H The otf x lò / 3 ’3. <.i '.-Igcaaaacag' <? ' a. · ’a’ g 'g 1.4 ·> t>' SEQ 119- <'DX Ojv 1.31 8? + ^: 15' 't <. tV'g ' ^x t' t 'rv g- ^ v' c ^A g ^z r''t ^z 't- ^A í: - ^x K ^A t ^Á t í tfl tf%> a <3 L <^ gcaaaÃÇ < Ac <λ ά S r_ + ’Jí ' S 69Ί8 ’ 3 'ί, t 3 - - g 4 1,412 3 ‘ SEQ 121 OPX2COG + 3 < 8? 69 (18) τ v t 4 'ς i t t g t r ΐ t ΐ r t V a f <.3geaaaA-'A £ e> a 4 L-iistftf SF 0 11 2 s eoiiG » 3 4 ·? M-lCAAJtill’dl: 3 <'c t L'. > - Q 12? OPN.v-Oó 13 » tf 'i-Hj'. · 11? V tc = r <z. - rtt _& tctrrt - 't' · '''gesaaaeagcaaaaeag' c ^A a''a ^A t * L4-i8 3 ' SEQ 124- tf í 1 t> > · 'Laaaea ^ caaaaesg' c a f't r £ 4-3.5 5 ' SEQ-125- <-ΓΧ '<106 (13) V tl ís t ''? 'g'X' t 't' t 'g'f c-' g · 't ^z t ·' t ^A έ'Ά t 'VV i 1,1 - .. ·' .., · -. .ill 'X>,' it L. · .1>' SEQ-126 tf 2 ’-G« 3 'TA -'> «ü C 1 t t t Wia 8E4T27 ODN2006Í13.S S 62ÍS) ¢ .- + -. +> _O ·. a. <v '· »_c taaeiíi <aorrr <''Lí-isS' S.EQ12.S. 8 63% () 3 'g' € '' aa.aacageaaaaeag ^ c ^ a: · '- »: ·' g ^? L4-is õ ' 8EQ-1M ODK200tV13> S -63 (16) »1 - t _ ··: r 1 - r - 'ttt>' + - '· 3 ^: -g ^A <-.' aaaacagcaaaaeag - c - '3 -';; § 1 ..: 3> .. 5 '

Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 143/220

133/187

Table 11

TD SEQ ID.) Sequence Complementary tape (3 '= +> §') SEQ 1 30 S <? 4 b '» 3 3 g g I. SEQ 131 <· »ΙΡ: 2οθδ <13ί) 3 t c, t <? t - - ’, r t- t t 4 t S -6: 4 (16) T g 'xf <1 \ ~ x ° ^Λ a g u 2; - -ΓΥ SEQ 132 S-SSÍIS) ’H t. U v The I. : èy- σ £ SEQ 133 ODN2Ô06Ü.-3) > tl í i ( _& i 't <_& t V g ΐ t t r gt <'Οί Α ^Λ ΐ · - 3 S> ’ 3 '1 <Í3Í? G 3 t 'and ê Las < £ EQ : 5-1 S -1614 ' > There ”R A - ΙΛ-Ι, ί-Τ. '!. ·.:; c a <3Se SEQ 1 • ΑΓΑ '<Ob <'> '· 5 rc à tot ♦ t - _e + s ώΐ r t r 4 t S 66! 14 * C · δγχ · -Τ3  3L'2B 3 Wp '- ’. AW just go Γ ’· SEQ 136 S 67-120S > \ I t - t t: SEQ £ 2> í vDivfOOS * 1? ¹ ~ r ^x t <'gtttt - <g t r t r g r S 67! 29í T AÍ22 15 3 a vat, ·, 3 A’‘l _ t-5 * SEQ 138 S 62> 14> _k - C '' \ V a · a ’ SEQ 139 QDNSut® 13.! '<r>'_& tcg ΐ - - tg - c t 't 't ’• ΐ- £ ί X S -’χ.1 Γ : <11! - _& <3U <. .. i V '>' A3. c a. & 5 ‘ SEQ 140 S 'There ^! ·> M ’i As • 'A ·” l .. ’s> A SEQ. 14.1 <> E> 2 <2í tíoilâ) : t and _ t <= · - · >> 2 c e 'r t 'l. t S 646269 1 M „2! 2 3 uViSidi-M j.  3? SEQ 1.42: ' AEnph20Õ6 * 13 ^; > Ls c '' t- ^T c - tt .Γ r s t + c- ·· ' g- · £ ' SEQ 143 Aniy h20U§GGÍ13) ^: '.>: -2 *' 2 _o T- - · £ ,. C e; TT ·. · ?! t ! túg · f C ' gE ^: Ua''úa ^A g '''c SEQ: 144 Amph2 * 306 't''L' =. tttt 2 - 31 _if 3 ΐ <íA-rt t t s' t

Table 12

ID <SEQ ID) CpG Sequence> 7 ^ 3> Complementary tape (3 ^ 57 SEQ145 S . (· <. '<> s ’3 g C12.338K3 g c'a Lj is 5 SEQ 146 ODNAitWS) 8-70 (20) 5'r> 'g t ε' = 't ΐ t t g t c g tUh'T'g’t * -' 't t. 7 'a g>' ia '«Aaíj ^ A« λ Lj í- 5' SEQ-147 8-71 (21) X g x J: L.j _ = ' SEQ 14§ ODNA.tO '> la < 8 TblD ííç i ιυ Cj ¢ 7 ^ - ία ç. <+ · 3 r-, naked r'f- 1 SEQ-149 S 7V] <3 7 a r A-, sa ΑΛ '>> - ’La - 3 SEQ: 1 & 0 ODX'2'A ^! A Í2 ¹ S 72 (10) Atcg ^T Agt * tt rgttt ^A t ^ Great ^A C -fe ΐ.- ί? ΐ>·>'8<' aiejg3Svag ^: ' ^: -è ^A á ^A to ^A LiG-is · S'

Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 138 / 22S

134/187

Table 13

Naked.. 1D (SEQ ID.) Sequence: t: pGts- ~> 3 '..> Complementary tape SEQ 151 S 73! IS) '> 3 3 <ukJj-íI-í-' A 1 =. fe fc Li SEQ-L53- ODN2006Ü5) S-73118) 5 't? è t <gtttt £ t <, sr ^T ttgte â ϊ ϊ V 1 3 3 <\ 2κ33Α 1'a = A 3 = 'g i. I read '*>' S.EQ453 S 74'1 S · ¹ '> Ί 1 i333 ¢ .- 1 3 0 3 ¢ - _c ' t L43 * - 3 SEQÍ54 ODN2006U3) S 74 <IS- ¹ 'J' go »'«. tttt «X · gti ΐ t X xg f-'t” 3 ^? > ·· ... '. ₍ .n, 3. · 3 0 ΐ _r - Lil „Ã SE ^ 155 8 75 «1 ·. * Λ <>'í-> aaed.v. ? di _ · -><>! .- =. Li _{ir r} 5 SEQASS ODN2Ui) <- '}.' 7 8 75Ί + ti τ · ^Λ £ ο Τ ⁽ a Λ 'Λ> _ <1 1 3 ° - - L .; It> SEQ / IS7 S 76'18) > * a ι-i ax - j; · y a g ->? i L-i-> SEQ15Ô OD.x-Woti '.;' 8 76’18) 5't <“tetrt 1 tt <, ft -r + t \ t <, 2 r 't 3' -3 -.-- 3; _: a -cageaasa-cag ·. ç; a;. »· g- g -g-'g- · g · g: g Li-ig-õ

Table 14

At the. ID ‘SE'Q ID) String: CpGiSM-SÒ Complementary tape 3M57 SEQ 159 8-77 (18) ; 8) -a5'a Li-is-5 'c-Aé «áááacág' '<r.a' 'a v? ·. & -. SEQ 160 ODN2006 <13> S 77 (18) t < Í and J'd ΐ ’a.Jk, Í 3 3 * Γ> t t & ϊ: - £ t T r . .g ^ :. to 1 It t r 5 SEQ 161 8 78134) i 3 c a 3 * S to S - SEQ 162 ODN 20060 31 S 7St24) V t C * i 3 Í IL L T t L £ * T ~ r g - < Li k ’ £ r r r SEQ-168 8 79’17) and d • t 1 t \ U 'i' -_ _c . 't t _v _ a s. g to 1-4 Is- SEQ 164 <’DX 'uuC.q-D S 79’17) Ϊ ^! a ⁺ ttt _t . 4 1 - <* u -. dt * <t t t t a - S) a t to <<'L < to t · t 7 SEQ185 S 80135) 'V ·' U t SEQ 166 ÜDN21W13) S 80) 351 3 t < L 3L? Ϊ Is 3 i. .UlCv T c r- t V case 1 4 ^r C I'm t 1 5 ^! 8EQ '167 3 81’25) M_ K ° t g <a SEQ 168 ODN2006O3 » 3 81'25 ») > XL -. i eg ⁺ tlt ^ V 2 9 1 „<2 _t t <gttt 'í < _e tt * 4 t < t t T SEQ-169 $ 82’25.) ? 2Ί> 1 'L * - ³ ° '3 > G -3 ·; < SEQ 170 ODN2006H3) S 82’35) 5 t i. 5 M J Ϊ ' _Λ - 1 1 \ $. 4 5 j / r c r t ϊ t £ 7 <3 d χ ' t y SEQ 171 3 83 (18) rj <í 4Í v <? g «? yx vua *. «A u a to L '.? SEQ 175 GDN2OO ‘? 13) 8 S’M ^ ' 3 '· 3/3. gtf ΐ ttr * £. ^ · ΑΛ. ' _{Λ | ί} <1 3 ΐ <I t t to 3. Li-iK 5 ' t g Í <· <f · t-3 · SEQ173 S 84 «18) 3 '-a' a 'You? 3 x u · .; to 3 3 d e Li> y SEQ 174 ODN20G6tl3j S 84’18) 5 't-'c 3 'a'‘a g ' ^x f Γ £ f TT it £ Cag'NAd33t3g <3 a r r g ΐ t ΐ <'el 4 t 3 t 8 tLi ,. 5 gN- tL

Petition 8701Θ0Φ3ΘΒ82, of 10 / Φ3 / 2Ο29, p. 149/223

135/187

Table 15

ID 'SEQ ID). Sequence: CpG> 5 * -à39 (3 ’” 4 complementary tape S')' 8EQ-17S S ·>: · ΓΥ5 '·? i. J <s g - '' csgca & 33c ag a LiC-ls S ' ODN2COG · 13 ' r- <d: CIO ν'-fCform O Yfe .0¾ o 8 8 3 C) 3 '- &-' ^x g ^A cagcaaaa <3í> to Li; 1,- SEQ 177 s _>) Ta- ^A to ^A aac ^ caa8.x.7 “3 I.; a SEQ 178 0 ^TV \ '' ^, H 96 (13) 8 86 (17) & * - t '·' · g + ttt: gt <'t ⁺ t.- r'-g-'t - <' gt '3' S 'a' a ^Λ áacagcaaaacag- 'o'U'- a - 'Έιο-β;The' SEQ .-- 1.79 8 87 <25) SF.Q 180 ODN20Q6 (13) 5 'íc' g ^A i8é? ^v g + 1 '·' s ^Λ IFt * g ^A t / e ^A -g8-1'- · £ + - (8ΐ ^Λ g ^Λ ί / ' t ' '- X -Çs- · <- ^r 8 $ 7'F’’i SEQ 181 Oftâ 8 'M: l a g eegcaa & aeageaaaacag + c +' a ^ a + Lg-ig 5 ' SEQ-182 ODN2096Ü.3) S-88 (14) 5’t ε 'g t <? 3 ’ + Μ · _ ·. .: Λ g f.lgCX.tfV’A.'tAlt »'- ·. K a rL> S-19 --- 5 ’ AND ' SEQT83 8-89 (19) 3 M __ <_ 3 C <. i L- ' SEQ 18 i ODN20G6 (13) 8 89 (19) 5'tcgt <'gt t' gt? 6 tttt g + t + e '' § '£' 3 'δ-ΛΓ. a '· -i Víu «Λ Η ^Λ Ε84ό; 5' T SEQ.W 8 90 (18) .S -. Ms-t ’a -i m> a ma <? 'A'-L ^ ss &’ SE% 18Ô ODN2006 (13) 8 90 (18 » '5 ^< -t- ⁷ : e -'- g + 4: ^A ® ^v g + t: + t +: t ^A t-g' + t ^A ç ^íV § ⁷ -Í _; + t + t + t + ^< g- ^f; 't ^? 'ç Ί _t ·. .. cageaaaaoag <- 'a' a Lz is 3 ' AND SEQ187 8-91 (18) *? 11.bk <> cageaaaacag '<? ^A to ^A to ^z 'g' g 'L ^ -is -5' SEQ-188. ODXWlF 8 91 * 18) '<t <g t <' g + tt '·' (g'-f-e'g + f t ' : V 31 «t> <> 'cagcaaaacag'’ <· a 'a' ’’ g '’g' 'La s 5’ X ' SEQ 18-9 8 92 (18) 8 31 \ 3 3 a -cageaaaacag 'c' a. '' A 'g' g 'g g’’g'-Ei-Ls 3' 8EQ1.90 GDN2ffi6 (l: 3) ’ 8--92318) 1't _<t at 'g *> Tn TGH ^- c GT Vg ^ tT tVYH' · the 'S · ^Λ 6 to MS5 will "cagcaaáacág ^Λ C - ^{Λ z>} g ^Λ g''g ⁷ 'g + g': Lé-iá · a T- '

Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 140/220

136/187

Table 16

Ní: ID (SEQ ED> Sequence: 5> C <.5 =? 3) Complementary tape <3M5 '.) SEQ 191 SA3Í1SÍ 3 Mio 11 '3 t' .tp rê.lVXV <o · »1-4 Ib 5 SEQ 192 ODN200 <> (13) S 93 (181 '’-Cgtcgtrrtx * par'’rt« rii7è'V’V'i3 ’ -> Mi. ii a ΐ * «caaaa <-ag · 'a a Lus’' SEQT93 S 94Í1 3'Mis It a 3 C3v '* aajeaá c a. <C- Li *' ' SEQ-194 <; DN2 (’0Gi13í S 94 (185 '• tegtrgj-rttuxratxttgx ’' β 'V t-3’ _ · 2> 1' i, a t v i3 '- <ay' -.as. , - 1, ^ 7 SÉQ119S S 95C18> 'Mt it * a (3¾. Trucks''3 g £ g _; s L4 £ § &' SEQ-196 ODN2006 <13> S-SailS · ¹ r 1 * t «. gtr 'r _e r ♦ ^r t Micic ft 1' ie <.j? at · aw <· ae ώ gg L-sis U SEQI97 S 96 <13; ? Mts. ' 3rd street ^ aejiat-at '' si SEQ-1'38 ODN2006QS) S 90 (184 5 j-'tt'rrjft '4''tM-3' Vic to 3 <3-3 cs Luc! ' SEQ-l® S 97 (185 Mi · & a · -. '* íva.>avaí' λ * s _& L-iir 5 ' SEQ-20Õ 0DX2 ¹ 'S 97Í1SÍ 5 g '+ MS' 3 Mio j 1 to c3g- _is i33 <3g as g. and L, go SEQ-201 S-OStlS! • Mi iv 3 * c, -. LjiÈÍ ’ SEQ-202 ODX2006; 13) S 98ilS> 'rgrtg''t + t-3 * 3 Muh a 3 3 _b .'iaaa.' «aca 3 I.-j is 1 ' SEQ-203 S 99 (18. ' 1 Ml if · 3 * -4 ^ 1.33.13>>? X s 4 β X L * ie 5 ’ SEQ ζ · »4- ODN2OOG; 13> S 99ί13> - t t t t «r <3’ 3 Mi · is. * 3 csgi'taaacag - a · 3 g- ¢ -I 4 is ã ' SE ·? V S 100 (25) 1 MD_1. 3 g 'agt.33 4ai.3si. '. L '> 5' SEQ?> <' <) D? Í2 (»OO <i3> S 100'23) '• ts'gtcgttrrgTi'irrrt g ^A t ^A c'-g-'t- ⁱ t - 8'? M; _ 14 aa ^ asfsanaas 37 · '1 a L- < SEQ -207 S 101 (25! 3 ΆΙ2214 a í veraadft'a ^ <. at 0 Lsi 5 ' SEQ 208 > <’. 1 '> S 101''25) 3 'M „. 1, a g ^ a> <yyyy <. 'G' i a Lti ’>

Petition 870190Φ3ΘΒ82, of 10 / Φ3 / 2Ο29, p. 14T / 22B

137/187

Table 17

At the. ID (SEQ ID) Sequence: CpG (5M3 ') complementary tape. (3M5 ’) SEQ-209 S-102 (25.) 3'-M22-i4 ^A a''g ^A cagcaaaacag ^A c ^A a ^A a ^A Le-ir5 ' SEQ-210 ODN2006Ú3) S-102 (25) õ ^, -V'c ' ^, g ^A V'cg- ^- 'V't' ^- t- ^A r'g ^A t ^A c'-g ^A t ^A t ^A V't''g-'t ^A c ^A g ^A t ^A t-3 '3'-M22-i4 ^A to ^A g ^A cagcaaaacag ^A c ^A to ^A to ^A Ls -'. 4'5 ' SEQ-211 S-103 (25) 3 '- M22-14 ^{Λ Λ} g ^A g A ^to G ^The aca aa cagca ^g? c ^Λ a ^Λ a ^Λ Ls-g - 5 ' SEQ-212 ODN2006 (13) 8-103 (25) 5 ^, -t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t -3 '3 ^f -M22-i4 ^A g ^A g ^A to ^A g ^A cagcaaaacag ^A c ^A to ^A to ^A Ls-g-ã' SEQ-213 8-104 (25) 3 '- M22-14 ^A g ^A g' ^Λ a ^Λ g ^A ca gcaaaaeag ^A and ^A a ^A to ^A Le-w 5 ' SEQ-214 ODN2006 (13) S-104 (25) 5'-1 ^A c ^A g ^Λ t. · ^Λ c ^Λ 'gtΐ.' t ^A tg''t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t · ^Λ c ^A g ^A t ^A t-3 '3'-M22-i4' * g ^A g ^A a ^A g ^A cagcaaaacag ^? c ^A to ^A to ^A Ls - o-5 ' SEQ-215 8-105) 25.) 3'-M22-i4 ^A g ^A g 'a ^A g ^A eagcaaaacag ^A c ^A a ^A to ^A Ls-i4-5' SEQ-216 ODN2006Ü3) 8-105) 25) õ'-t ^A c''g ^A t ^A c '''' g ^A t- ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^a t ^a t-3 '3' - M22-14 ^a g ^a g ^'the g * ^Λ to ca gcaaaac AG ^Λ c a ^a a ^a LS go 5' SEQ-217 S-106 (25) 3'-i8-M22 ^{a a} g ^The cagcaaaaeag ^{Λ A} and ^A to the LS-8'5 ' SEQ-218 ODN2006 (13) 8-106 (25) 5 '-1c ^A g ^Λ t ^A c ^Λ g ^A t ^Λ t ^A t ^A t ^A g' t ^ft c ^Λ gt 't ^A t ^A tg ^A t' c ^A g '* t' t - 3 '3' -M22-i8 ^A to ^A g ^A cagcaaaaeag ^A c ^A to ^A to ^A Ls - «- 5 ' SEQ-219 8-107 (25) ^The 3'-M22- is ^to G ^The cage aaa acag ^{Λ A} and ^{Λ A} to the LG- yo '5' SEQ-220 ODN2006Ü3) 8-107 (25) 5'-t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t -3 ', 3'-M22 is' a ^to g ^the cagcaaaacag ^{Λ a} and ^a to the LS-io * 5' 8EQ-221 8-108) 25) 3 'M22-1 s ^Λ g · ^Λ g''ag ^A ca gcaaa ac ag ^Λ c' ^L a ^A to ^A Ls - s ^- 5 ' SEQ-222 ODN2006 (13) 8-108 (25) 5'-t ^A c ^A g ^A t ^A c ^A g ^A t. ^At t. ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t-3 '3'-M22-is ^A g ^A g ^A a ^A g ^A ca gcaaaaeag ^A and ^A to ^A to ^A L <8-6-5 ' ¢ 1 & Kl rfí. 8-109 (25) 3'-M22-i8 ^λ g ^A g ^A a ^A g ^A cagcaaaacag ^A c ^A a ^A a ^A Ls jo'5 ' SEQ-224 ODN2006 (13) 8-109 (25) 5 '-1 ^Λ T C ^{A A} g ^A and g ^{Λ Λ} T T ^{A A} T ^A T ^A T g ^{Λ Λ Λ} c t g ^A T ^A T ^{Λ Λ} TG ^A T ^A C ^A t g ^Λ' t - 3 '3'-M22-i8 ^A g ^/ ' g ^A to ^A g ^A cagcaaaacag ^A c ^A to ^A to ^A Ls it>'5' SEQ-225 8-110 (18) 3'- M22-12 ^A a ^A a ^A shit a y g ^A c ^A a ^A a ^A Lehe '5' SEQ-226 ODN2006Ü3) 8-110 (18) 5'-t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ' ^A t ^A c ^A g ^A fc ^A t ^A t ^A r ^A g ^A t- ^A c ^A g'''t ^A t-3' 3 M22-12 “a ^A a ^A ca gca aa acag ^A c ^Λ a '· a ^A L ^ r 5' SEQ-227 8-111 (18) 3'-M22-i2 ^A a''a''cagc.aaaacag ^A c ^A a''a ^A Lf _S rr5 ' SEQ-228 ODN2006 (13) 8-111) 18) ã'-t ^A C ^A g ^A t ^A C ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A C ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A C ^A g ^A t ^A t -3 '3' - M22-12 ^Λ a ^Λ a ^Λ cagca aaa ca g ^A c ^A a ^Λ a 'Ε ^ γ 5'

Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 148/220

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Table 18

At the. ID ISSEQ ID) S <String: pG (5M> 3 ’) Co Complementary tape +5’) SEQ-229 8-112 (18) 3'-M22-12 ^A to ^A to ^A CagCaaaacag ^A C ^A to ^A to ^A Ls-10-5 ' SEQ-230 ODN2006 (13) 8-112 (18) õ1 ^A c ^A g ^A t ^Λ c ^A g ^A t ^Λ t tt'g ^A t ^A c ^A gr · '' t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t - 3 * 3 '' -M22-i2 ^/ 'a ^A to ^A cagcaaaacag ^/ ' c ^A a ^{/ 1} a ^/ 'L8-iQ-5' fa cn 8-113 (18} 3'-M22-i2 ^A to ^A to ^A cagcaaaacag ^A c ^A to ^A to ^A Lsi4-5 ' SEQ-232 ODN2C »06 (13) 8-113 (18} 5 ^! -t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t. ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t. ^A t-3 '3'- M22-12' a ^A to ^A ca gca aa acag ^A c ^A to ^A to ^A Lg -14 - 5 ' SEQ-233 8-114 (18) ^The -a 3 to ^the cagcaaa ^The fitness g c ^a a ^a a & ^{Α Ε} τ - 5 ' SEQ-234 ODN2006 (13) 8-114 (18) 5'-t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t''t ^A t ^A g ^A t ^A c ^A g ^A t ^A t-3 '3 -Metei aa ^A cagcaaaaca g ^Λ c ^A a ^Λ a ^A L & xr 5' SEQ / 235 8-115 (14) 3'-a ^A g ^A cagcaaaaeaK22isgcaaaacag ^A c '' a ^A -5 ' SEQ-236 ODN2006 (13) 8-115 (14) 5 ^! -t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t. ^A g ^A t ^A c ^A g ^A t ^A t-3 '3'-a ^A g ^A cagcaaaacaK22 isgcaaaacag' * c ^A to ^A a-5 ' SEQ-237 8-116 (17) 3 -Mi5- and ^A aa ^Λ a acagcaa a aca ^A ca ^A L 4-1 g- 5 ' SEQ-238 ODN2006 (13.) 8-116 (17} 5'-t ^A and ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t -3 * 3 '-M1 ã-e ^A a ^Λ a ^Λ a ac ag ca aaa cag- ^A c ^A a ^A a ^Λ I4-13'5' SEQ-239 8-117 (17) 3 ^, -Mi3-g ^A to ^A to ^A aacagcaaaacag ^A c ^A to ^A to ^A g ^/ g ^A L4-i8-5 ' SEQ-240 ODN2006 (13) 3-117 (17) õ'-t ^A c ^A g ^A t'c ^A g ^A t ^A t ^A t ^A t ^A g ^A t'c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t -3 '3'-Mi5-e ^A to ^A to ^A aacagcaaaacag ^A c ^A to ^A to ^A g ^A g ^A L4-i85' SEQ-241 8-118 (17) ^The aacageaaaacag the third ^{Λ A} and ^{Λ A} at the L4-i8-5 ' SEQ-242 ODN2006Í13) 8-118 (17} 5 ^! -t ^A c ^A g ^A t- ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t- -3 '3'-Mi $ - 13 ^a to ^A to ^A aacagcaaaacag ^A c ^A to ^A to ^A L4- is * 5' SEQ-243 8-119 (17) 3 '-M10- ies ^A a ^A to ^A aac agcaa a ac g ^A c ^A to ^A a ^A g ^A g ^A Lj-1 s 5' SEQ-244 ODN2006 (13) 8-119 (17) 5 -1 * ^A C ^{A A} T g ^'The g c ^{Λ Λ} T T ^A T ^A r T ^Λ g ^The C ^to G ^A T ^A T ^A T ^A T ^A T ^A g ^a c ^a t g ^A T ^A - 3 '3' -Mia- · io * a ^a a ^a Agca aac aca aa ^a and ^Λ g a ^a a ^{a a a} g g s L4-1 - 5 ' SEQ-245 8-120 (17} 3'-Miã-e ^A to ^A to ^A aacagcaaaacag ^A c ^A to ^A to ^A L4-t8-5 ' SEQ-246 ODN2C »06 (13) 8-120 (17} 5 ^! -t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t. ^A g ^A t ^A c ^A g ^A t ^A t. ^A t ^A t ^A g ^A t ^A c ^A g ^A t. ^A t-3 '3'-Miõ-e ^A to ^A to ^A aacagcaaaacag ^A c ^A to ^A to ^A L4-te-5'

Petition 870190Φ3ΘΒ82, of 10 / Φ3 / 2Ο29, p. 149 / 22B

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Table 19

At the. ID (SEQ ID} Sequence: CpG (5M3j complementary tape (3M5 ') SEQ-247 S-121Ü7) 3'-Miõ- « ^Λ a ^Λ a ^A aaca gcaaaaca g ^A c ^A aa ^A g ^A g ' ^- ' 1.» - 1 g- 5 ' SEQ-248 ODN2006Í13) S-121Ü7) 5'-t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A (· ' 4-3 * 3'-Mis-e ^A to ^A to ^A aacagcaaaacag ^A c ^A to ^A to ^A g''g ^A L4-ie-5 ' SEQ-249 S-122Ü7) 3'-M · 5- is ^A a ^A to ^A aacagcaaaacag ^A c ^A a ^Λ a ^Λ Li-, c> - 5 ' SEQ-250 ODN2006ÍI3) 8’122 (17) 5'-t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t -3 '3' -Mis-ίο 'a *' a ^A aacagcaaaacag ^A c ^A a ^Λ a Li- ig- 5 ' SEQ-251 8-123 (17) 3'-Mi5-io ^A to ^A to ^A aacagcaaaacag ^A c ^A a''a ^A g ^A g ^A L4-ig-5 ^! SEQ-252 ODN2006 (13) 8-123 (17) 5'-t ^A c ^A g ^A t ^A c ^A g ^A i ^A t. ^A t ^A t '''g ^A t ^A c ^A g''t. ^At t. ^A t ^A t ^A g ^A t ^A c ^A g ^A t · t-3 '3'-Mis-io ^A a ^A a ^A aacagcaaaacag ^A c ^A a''a ^A g ^A g''' Li ιβ'5 ' SEQ-253 K3-CpG (26) 5'-a ^A t ^A c ^A g ^A a ^A c ^A t ^A c ^A t · * e ^A g ^A a ^A g ^A and ^A g ^A t ^A t ^{A A} c-3 ' SEQ-254 8-124 (27) 3 '-t ^A g ^{, A} aga gct-cgcaa ^A g ^A to ^A g ^A Lís - 5' SEQ-255 K3-CpG (26) 8-124 (27) 5'-a ^A t ^A c ^A g ^A a ^A c ^A t ^A c ^A t ^A c ^A g ^A a ^A g ^A c ^A g ^z t ^A t ^A c ^A t ^A c-3 '3'-t ^A g ^A agagetcgcaa ^A g ^A to ^A g ' ^J -L-is-ã' SEQ-256 D35-CpG (28) 5 ^; -g ^A gtgcaícgatgcagggg ^A g '''' g-3 ' SEQ-257 8-125 (29.) 3 '- g ^A t ^A a gctaegtcccc c ^A c ^A Lí-ir- 5' SEQ-2S8 D35-CpG (28) 8-125 (29) 5'-g ^A gt-gcaícgatgcagggg · 'g ^A g-3'3'-g ^A t ^A agctacgtccc ^A c ^A c ^A c ^A 14-ia- 5 ' SEQ-259 S-126 (25) 3'-Μ22 · ιο ^λ a ^A g ^Λ ea gcaaa ac ag ^A c ^A a ^A a - 5 ' SEQ-260 ODN2006 (13) S-126 (25) 5 ^; -t ^A 'c ^A g ^A t ^A c ^A g''' t ^A t ^A t ^A t ^/ '' g ^A t ^A c ^A g '''t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t-3 '?' -M 22- ir · ^A to ^A g ^A cagcaaaacag ^A <? ^A to ^A a-5 ' SEQ-261 8-127 (25) 3'-i'eÍ22-i2 ^A a 'g ^A cagcaaaaeag ^A c ^A a ^A a-5 * SEQ-262 ODN2006G3.) 8-127 (25) 5'-T ^A and T g ^{A A} C ^A T ^A g ^a i:. '''T ^A T ^A G -''' T ^A C ^{A A} T ^A T g ^/ T - ^'A T ^A g t ^/ 'c.''' g ^A t ^A t-3 ^! 3 '-M22-12 A ^to G ^{Λ Λ} c ^{z The} cagcaaaacag to ^A' -5 ' SEQ-261 8-127 (25) 3'-M22-12 ^A to ^A g ^A ca gcaaa ac ag ^A c ^A to ^A a - 5 ' SEQ-262 ODN2006Ú3) 8-127 (25) 5 ^í -t: ^A ₍ 2 - '' g ^A t ^A C ^A 'g''' t ^A t ^A t ^A t ^A g ^A t ^A C ^A g''t '''t ^A t ^A t ^A g ^A t ^A C ^A g ^A r '''t-3 * 3' -M22-12 ^A to ^A g '' cagcaaaacag ^A c ^A to ^A at -5 '

Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. readsO / 220

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Table 20

At the. ID (SEQ ID) String: jpG (5 ^: ^> 3 ') Complementary tape (3Ά5Ί SEQ-263 8-128 (25) 3 '-M22- 20 ^{A to} G ^The cage yyyy ca ca ^Λ to ^Λ g - 5' SEQ-264 ODN2006 (13) S-128 (25) õ * -t- ^A c ^A g · 'V'e ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A and ^A g ^A t ^The tS ^! 3 '-M22-2ü ^A a ^A g ^A cageaaaacag ^A c ^Λ a ^Λ a - 5 ^! SEQ-265 8-129 (25) 3'-M22-22 ^A a ^A g ^A cagcaaaaeag ^A c ^A a ^A a-õ ' SEQ-266 ODN20Q6 (13) 8-129 (25.) 5 '-1 ^A c ^A g ^A t ^A c' g ^A t ^A t ^A t 't ^A g ^A t ^A eg * t ^Λ t ^Λ t ^Λ t ^Λ g' t 'c ^A g ^A t ^A t - 3 '3 ^? -M> 2-22 ^a a ^Λ g 'cage aaa aca g ^A c ^A a ^A a õ' SEQ-267 S 130 (25) 3 -M22-12 ^A to ^A g ^A cageaaaa ca g ^A c ^A to ^A to ^A Ls-e ^- 5 ' SEQ-268 ODN2006Ü3) S-130 (25) 5 * -t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t -3 ^! 3 - M ·> 12 ^A to ^Λ g ^A cageaaaacag ^A c ^A to ^A to ^A L = .5-5 ' SEQ-269 S-131 (25.) 3'-M22-i2 ^A to ^A g ^A cagcaaaacag ^A c ^A to ^A to ^A Ls io ~ 5 ^! SEQ-270 ODN2C> 06 (13) 8-131 (25) õ'-t ^A c ^A g ^A t ^A c ^A g ^A t- ^A t ^A t ^A t- ^A g ^A t- ^A c''g ^A t ^A t ^A t ^A t ^A g ^A t '“c ^A g ^A t ^A t-3 '3'-M22-i2 ^A to ^A g ^A cageaaaacag ^A c ^A to ^A to ^A L8-io-5 ⁽ SEQ-271 8-132 (25) 3-M22i2 ^A to ^A g ^A cagcaaaacag ^A c ^A to ^A to ^A L8; 4'5 ^! SEQ-272 ODN2006Ü3) 8-132 (25) õ'-t ^A c ^A g ^A t ^A c ^A g''t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t-3 ^! 3'-M22-i2 ^A to ^A g ^A cagcaaaacag ^A c ^A to ^A to ^A Ls-i4'5 ' SEQ-27 3 8-133 (25) 3 ^'The -M22-12 g g ^{Λ Λ Λ} the GCA geaaaacag ^{A A} A ^A A c. L ^ - 5 ' SEQ-27 4 ODN2006Í13) 8-133 (25) 5 ^! -t ^A c ^A g ^A t. ^A c ^A g ^A t ^A t''t ^/ t ^A g ^A t ^A c ^A g''t ' ^A t ^A ' t ^A t ^A g ^A t ^A c ^A g ^A t ^A t-3 '3'- M22-i2 ^A g ^A g ^A a ^A g ^A cagcaaaacag ^A c ^A a ^A a ^A Ls ^ -5 ' SEQ-275 8-134 (25.) 3'-M22-i2 ^A a ^A g ^A cageaaaacag ^A c ^A a ^A 'a ^A, g ^A g ^A L8-i <? - 5 * SEQ-276 ODN2006 (13) 8-134 (25) õ ^, -t. ^A c ^A g ^A t ^A 'c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t-3 ¹ 3 '-M22- 12 ^a g ^A g ^A a ^A g ^A shit a ag ^A and ^A a ^A a ^A Ls- w 5' SEQ-277 8-135 (25) 3'-M22-i2 ^A g ^A g ^A to ^A g ^/ cageaaaacag 'c ^A to ^A to ^A Ls 14'5' SEQ-278 ODN2006 (13) 8-135 (25) 5 ^! -t ^A c ^A g ^A t ^A c ^A g ^A t. ^At t. ^A t ^A t ^A g ^A t. ^A and ^A g ' ^A t' ^A t ^A t ^A t ^A g ^A t ' ^A c ^A g ^A t''t-3' 3 ^a M22- 12 ^A g ^Λ g ^Λ a ^Λ g ^A ca geaaaacag ^A c ^A the ALS-u'5 ' SEQ-279 8-136 (18) 3 ^! -a ^A a ^A cagcaaaacag ^A c ^A ALNA''ALNA ' ^A g''g ^A L4-i8-5' SEQ-280 ODN2006 (13) 8-13 (5 (18) 5'-t · ^A c '·' g ^A f.- ^A c ^A g ^A r ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A and ^A g ^A t ^A t-3 ^! 3'-a ^A a ^- 'cageaaaacag ^A and ^A ALNA ^LA ' ALNA '''' g ^A g ^A L4-i8-5 '

Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. read / 220

141/187

Table 21

At the. ID (SEQ: ID) Sequence: CpGÍõMSj Complementary tape (3 ++ 5j SEQ-2S1 8-137 (18? ύ -a cagcaALNAÀLNAaoag c Alna Alna g g ’Lí-is-õ SEQ.-282 ODN2006ÍI3) 8-137 (18) 5'-t ^A c ^A g ^A t ^A c ^A g ^A t. ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t-3 '3'-a ^A a ^A cagcaAu4AALNAacag ^A c ^A ALNA ^A ALNA ^A g ^A g ^A Li-i8-5 ' SEQ-283 8-138 (18) 3 '* a ^Λ Alna ^a cageaALNAÂLNAacag ^A c ^A Alna Aln a ^a g ^A g ^A L + 8'5' SEQ-284 ODN2006 (13) 8-138 (18? 5'-t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A i ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t -3 '3'-a ^Λ ALNA ^A cageaALNAALNAacag ^A c ·''ÂLNA ^A ALNA ^A g ^A g' ^A l4-i8'5 * SEQ-285 8-139 (18) 3'-a ^A a ^A c ^A a ^A g ^A and ^A a ^A a ^A a ^A a ^A c ^A a ^A g ^A c ^A a ^A a ^A g ^A g ^A g ^A g ^A g ^A Lib 18-5 ' SEQ-286 ODN2006Ü3) 8-139 (18) 5H ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t: ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t-3 '3'-a ^A a ^A c ^A a ^A g ^A and ^A a ^A a ^A a ^A a ^A c ^A a ^A g ^A c ^A a ^A a ^A g ^A g ^A g ^A g ^A g ^A Lib-is' 5 ' SEQ-287 8-140 (30? 3'- M22-28 ^A ag ^A cagcaaaacagcaaa ^A to ^A c ^A to -5 ' SEQ.-288 ODN2006Ü3) 8-140 (30) ã'-t. ^A c ^A g ^A t ^A c ^A g ^A t ^A t. ^A t- ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t- ^A t-3 '3'-M22-2Q ^A a ^A g ^A cagcaaaacagcaaa ^A a ^A c ^A a-5 ' SEQ-289 8-1411.14) 3'-M22-2s ^A to ^A £ ^A cageaaaaeageaaaacag ^A c ^A to ^A a -> ^! SEQ-290 ODN2006 (13) 8-141 (14) 5'-t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^' g ^A t ^A c ^A g ^A t''t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t-3 '3'-M22-28 ^A a ^A g ^A cagcaaaacagcaaaacag ^A c ^A a ^A a-5 ^! SEQ-291 8-142 (30) 3'-M22-22 ^A a ^A g ^A cagcaaaacagcaaa ^? a ^A and ^A a-5 ' SEQ-292 ODN2006 (13.) 8-142 (30? 5'-t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t -3 '3' -M22 22 ^A to ^A g ^A ca gcaaaaca gca aa ^A a ^Λ e ^A a - 5 ' SEQ-293 8-143 (14) 3'-M22-22 ^A a ^A g ^A cagcaaaacagcaaaacag ^A c ^A a ^A -5 ^! SEQ-294 ODN2006 (13) 8-143 (14? 5 ^! -t ^A c ^A g ^A t ^A and ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^/ 'g ^A t'' ^, t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t-3 '3'-M-22-22''a''g''cagcaaaacagcaaaacag ^A c ^A to ^A a-5' SEQ-295 8-144 (30) 3'-Maa-12a ^Λ g ^A ca gcaaaaca gca aa ^A a'c ^A a ^Λ Lg-s · 5 * SEQ-296 ODN2006ÍI3) S-144.30) 5'-t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t -3 '3' -M22-12 ^{a to} g ^a CA EASO the acageaaa ^a to ^a and ^Λ the Lg-s'

Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. reads / 220

142/187

Table 22

At the. ID (SEQ ID) String: CpGíã ^ S ') Complementary tape (3' ^ 5 ') SEQ-297 8-145 (30) 3'-M22-i2 ^A a ^A g ^A cagcaaaacag «®aa ^A a ^A c ^A a ^A Lô-ic-S ' SEQ-298 ODN2006 (13) 8-145 (30) 5Tc ^A g'4'xYtYt / V-g''t * c ^A g'h.Wt''g'-t ^A c ^A g ^A t ^A t-3 '3 ^f -M22-i2 ^A a ^A g ^A cagcaaaacagcaaa ^A to ^A c ^A to ^A Ls-io-5 ' SEQ-299 8-146 (30) 3'-M22-i2 ^A to ^A g ^A cagcaaaacagcaaa ^A to ^A c ^A to ^A Ls-i4-5 ^! SEQ-300 ODN2006 (13) 8-146 (30) 5 - t ''' _C - <gA _t A _c ^A g ^A t ^A t ^A t ^A t''g''' fc ^A g ^A t ^A t ^A t ^A t ^A g ^A t''c ^A g '''t ^A t-3 ^! 3'-M22-i2 ^A to ^A g ^A cagcaaaacagcaaa ^A to ^A c. ^A to ^A Ls-i4ã ' SEQ-301 8-147 (30) 3 '-M22 -12 ^A g' ^A g ^{, A} to ^A g ^{, A} shit aaa <agca aa ^A ac- ^A to ^A Lg g-5 ^: SEQ-302 ODN2006 (13) 8-147 (30) ^T 5 -t. ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t-3 '3 '-M22-i2 ^λ g ^A g ^A to ^A g ^A cagcaaaacagcaaa ^A to ^A c ^A to ^A Ls g- 5' SEQ-30 3 8-148 (30) 3'-M22-i2 ^A g ^A g ^A to ^A g ^A cagcaaaacagcaaa ^A to ^A € ^A to ^A L8.ío'5 ' SEQ-304 ODN2006 (13) 8-148 (30) 5H ^A c ^A g ^A t ^A c ^A g ^A t. ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t'H-'t ^A t ^A g ^A t ^A c ^A g ^A t. ^The t-3 ^! 3'-M22-i2 ^A g ^A g ^A to ^A g ^A eagcaaaaeagcaaa ^A to ^A c ^A to ^A L6-ic-5 ' SEQ-305 8-149 (30) 3'-M22-i2 ^A g ^A g ^A a ^A g ^A cagcaaaacagcaaa ^A a''c ^A to ^A L8.i4-5 ' SEQ-306 ODN2006 (13) 8-149 (30) 5 '-1' ^Λ c t g ^A T ^A CG ^{Λ Λ Λ} T ^Λ t t g ^A T ^{A A} C ^A t g ^{Λ Λ} T ^A T ^A T ^A T ^A g ^A and g ^{Λ 'Λ} t t - 3 '3'-M22-i2 ^A g' ^A g ^A a ^A g ^A cageaaaaeagcaaa''a ''<?' ^{A A} Lg.i4-5' SEQ'307 8-150 (14) 3'-M22-i2 ^A to ^A g ^A cagcaaaacagcaaaacag ^A c ^A to ^A to ^A L8 -g-5 ' SEQ-308 ODN2006C13) 8-150 (14) 5'-t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t. ^A g ^A t ^A c ^A g ^A t ^A t-3 '3'-M22-i2 ^A a ^A g ^A cagcaaaaeagcaaaacag ^A c ^A a ^A a ^A Le-8-5' SEQ-309 8-151 (14) 3'-M22-i2 ^A to ^A g ^A cagcaaaacagcaaaacag ^A c ^A to ^A to ^A L8-i4-5 ' SEQ-310 ODN2006CL3) 8-151 (14) 5'-t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t. ^A c ^A g ^A t ^A t ^A t ^A t. ^A g ^A t- ^A c ^A g ^A t ^A t-3 * S'-Mss-i2 ^A a ^A g ^A cagcaaaacagcaaaacag ^A c ^A a ^A a ^A Le · ιγ5 *

Table 23

At the ID (SEQ ID) Sequence CpGKS '^ S') Complementary tape (3 '^ 5') SEQ-311 8-152 (14) 3'-M22-í2 ^A g ^A g ^A a''g ^A cagcaaaacagcaaaacag ^A c ^A to ^A to ^A L8S'5 ' SEQ-312 ODN2006 (13) 8 * 152 (14) 5'-t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t -3 '3'-M22-i2 ^A g ^A g ^A to ^A g ^A cagcaaaacagcaaaacag ^A c ^A to ^A to ^A Ls -s-5' SEQ-313 8-153 (14) 3 ^, -M22-i2 ^A g ^A g ^A to ^A g '''' cagcaaaacagc3.aaacag ^A c ^A to ^A to ^A L8io-5 ' SEQ-314 C®N2006 (13) 8-153 (14) 5'-t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t -3 'S ^ Mss.ia ^ g ^ g ^^ a ^ g ^ cagõaaaacagcaaaacag ^ c ^ a ^^ a ^ Ls-io-õ' SEQ-315 8-154 (14) 3'-M22-s2 ^A g ^A g ^A a ^A g ^A cagcaaaaeagcaaaacag ^A c ^A a / 'a ^A Ls-u' 5 ' SEQ-316 ODN2006 (13) 8-154 (14) 5'-t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t- ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t-3 '3'-M22-S2 ^A g ^A g ^A to ^A g ^A cagcaaaacagcaaaaeag ^A c ^A to ^A to ^A L8-i4-5' SEQ-317 S155 (18) 3'-M22-i8 ^A Bu ^A Bu ^A a ^A a ^A cagcaaaacag ^A c ^A a ^A a-5 ^! SEQ-318 ODN2006Q3) S-155Ü8) 5'-t ^Λ c ^A g ^ft i · 'c ^A g' t ^A r ^A t ^A t ^A gt ^Λ e ^A g 'tt ·' tr ^Λ g ^A t. ^'The g c' * TT 3 '3' s ^The -M22- Bu u ^A B A ^A A ^A AA ca gca aca g ^{Λ A} is the ^A to-5 ' SEQ-319 8-156 (13) 3 'Mteg- 18 ^A a ^A a' ¹ cagcaaaaca g ^A c ^Λ a 'a - 5' SEQ-320 ODN2006Ü3) S-156 (18) 5'-t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t. ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t.-3 '3'-Mteo-: s ^A a ^A a''cagcaaaacag''c ^Λ a ^A a - 5 ' SEQ-321 S-157 (25) 3'-M22-í8 ^a Alna ^a Glsa 5mCLNAagcaaaacag ^A 5mCiNA ^A Alna 'Alna'5 SEQ-322 ODN2006Q3) S-15’7 (25) 5 't ^Λ c ^A g ^A t ^Λ c ^A gt' t ^A t ^Λ t ^A g ^A t ^A c ^A g ^Λ t 't ^Λ t ^A t ^A g ^A t' c ^A g ^Λ t ^Λ t - 3 '3'-M22-i8 ^The ALNA ^The glnA ^The 5mCLiíAagcaaaacag ^The 5mCLNA ^The Alna 'Alna 5 SEQ-323 8-158 (31) 3'-M-22-ís ^to Alna ^a GlnaSmCbNAagca '' Alna Alna ^a Alna'5 ' SEQ-324 ODN2006Í13) S-158 (31) 5'-t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t -3 '3'? Vl ^A Alka ^a Glna ^a 5iiíClnAagca ^A Alna ^a Alna ^a Alna'5

Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 143/220

143/187

Table 24

At the. ID (SEQ ID) Sequence: CpGíõ ^ S) OlM-õO complementary tape SEQ-325 8-159 (30) 3 ^! · Μ · 22-ΐ4 ^Λ 8 ^A g ^A cageaaaacagcaaa ^A a ^A ca ^Λ Ls- <3-.5 ' SEQ-326 ODN2006Í13) S-159 (30) 5'-t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A i ^A g ^A t ^A c ^A g ^A t- ^A t ^A t ^A t ^A g ^A t- ^A c ^A g ^A t ^A t-3 'S'-Msa i4 ^A to ^A g ^A cagcaaaacagcaaa ^A to ^A c ^A to ^A le-e-5' SEQ-327 S-160 (30) 3'-M22-i4 ^A to ^A g ^A eagcaaaacagcaaa ^A to ^A c ^A to ^A L8-i <> - 5 ^! SEQ-328 ODN2006 (13) 8-160 (30) S'-T ^A C ^{A A} T ^A g ^Λ c t g ^A T ^{A A A ί Λ ΐ-} g ^A T ^A C ^{A A} g ^A t T t ^A t ^A t ^A g ^A · ^Λ C ^to G ^z 't ^A t-3'3'-M22-i4 ^A to ^A g ^A cagcaaaacagcaaa''a ^A c ^A to ^A L8-io-5 ^! SEQ-329 S-161 (30) 3 - l ..: 14 to 'g cagca ^The acagca aa a aa ^A C ^A · 14-5 AL® SEQ-330 ODN2006 (13) 3-161 (30) 5'-t ^A c ^A g ^/, t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A and ^A g ^A t- ^A t ^A t · '· t ^A g ^A t ^A c ^A g ^A t ^A t-3 '3 ^> -M22-i4 ^A to ^A g ^A cagcaaaacagcaaa ^A to ^A c ^A to ^A L8-i4'5' SEQ-331 S-162 (30) 3l; 1 ^Λ g g g ^A to ^{A A} to ^A ac gcaaaacagcaaa ^A and ALG-s 5 ' SEQ-332 ODN2006 (13) 8-162 (30) 5'-t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t- ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t-3 '3'-M22-i4 ^A g ^A g' * a ^A g ^A cagcaaaacagcaaa ^A a ^A c ^A a ^A L8 - «- 5 ' SEQ-333 8-163 (30) 3'-M22-i4 ^A g ^A g ^A a ^A g ^A cagcaaaacagcaaa ^A a ^A c ^A a ^z 'L8-ií>-5' SEQ-334 ODN2006 (13) 8-163 (30) 5'-t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t -3 '3'-M22-i4 ^A g''g' * 'a ^A g ^A cagcaaaacagcaaa ^A a ^A c ^A a ^A L8-iQ-5' SEQ-335 8-164 (30) 3'-M22-i4 ^A g ^A g ^z a ^A g ^A cagcaaaacagcaaa ^A to ^A c ^A to ^A L8-i4'5 ' SEQ-336 ODN2006 (13) S-164 (30) 5 ^, -t ^A and ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t -3 '3 ^A M22-i4 ^A g ^A g ^A to ^z ' g ^A cagcaaaticagcaaa ^A to ^A c ^A to ^A Ls-i4-5 ^! SEQ-337 8-165 (25.) ^The 3'-eoMe the 'agGifecaosieaaoMeacoM' agoM 'aome ^A C ^{A A} A ^A * 18'5 L4- SEQ-338 ODN2006 (13) 8-165 (25) 5'-t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t. ^ft C ^to G ^A T ^A T ^A T ^A T ^A T ^A g ^The C ^to G ^A T ^A T-3 '3'coife to ^A * ^A and ^A agoàfecaoMeaaoMeacoMeagoM AOM''in ^{Λ Τι-} it is'

Petition 87Ο29ΟΦ3Θ0Η2, of 10 / Φ3 / 2Ο29, p. readsH / 220

144/187

Table 25

At the. ID (SEQ ID) S Sequence: ..- pG (õ = ^ 3- /. C <Complementary tape 057 SEQ 339 ODX ^ IS '^ S h ¹ <> DX2 ^''--- 1 (- .. » ⁼ e- Ϊ gfcêJ ^ 'tCgt <g _& 2 _{S &} g> SEQ 34Θ. ^Λ XL-ii ens SEQ: .341 5 ° gAf-ACgvOgb g ^J a 7 3 21 ... 1- '<-, · a A r' SEQ-342 ' »3 5 tek ί ' _e ttikt <gtt (. Í ^A c-3' SEQ 3 13 S] (ϊ » · - Xk_ Is 1 SS 1 | -, S13. yes, ή SEQ-344 S lf 5 t - A ag ^: ''^< t '''C''g''' t: - 'Í --'- C-3' 3 \ k „i„ ⁵ a otAr'tat <3g '3 3 - ³ SEX ·) .. '' ΐ.θ D LS0V3SÍ te i. v - 3 'A ^A e -''g-''t ^A t ^A is''' g- ^A g t 3 5 SEQ 346 S 168'37) 3 There „'j is 3 i''cgctgeaagcg-'- g- ·' g ^; ç&' SEQ-347 I »LS> '\ S-168 (37) õ rg <'a. ^A c ^A g ^A t ^A t ^A <- -x - <<'g ^A s.' ^A c ^A g ^A t ^A 't ^A í / g''- g -' 6 'to 3 Ό 2 <: g tg -...- 1 Í.1 Aí'A g CA SEQ-848 D -LS03 (3S) 'tl: «í -' 3 3:. gi - CC · '^; ^ ^Λ ί5-§- · 'ϊ:'Ό'ί ^Λ § '''' · 3 '''· 3. L g- 5 SEQ '549 S 169 (39.1 rs <CJ • n 16 n ° rirç ^f o ^ t- > EQ 7 Ml D LSU3 '38i S 169139.1 0 t <. g- · <. k. 3 3 and 2 ttck < ^r a Og2ΐ'- · ΐ ^; + “§'5 & ^Α aagcg: g · -? ^; M- -a- • ^ 'egettgcaagc ^ g ^ g ^ ç-S ·' > 3M EQ. ODN2j9Q40 ’ 7 r gteg ^A O + ^A g-3 ' SEQ s: -2 S 3 70’41.1 9 λΐ ^λ ι ^ λ £ nwuydr-t · '·' SEQ -> 3rd ODN2395UO) S 170 (411 5 rcr V gt I ttegg C gcg <g <eg 3 '3 M22 ia 3. · · «·' ca ^ caaaa.gtsc7 ^> 8r '''c ····· ^ · &' SEQ-354 ODN’M362'.42; rt>'av -cg -'- t ^A c'-g ^A t ^A t ^: -c ^A g''''a''' a '''-cg/' a ^ c- g ^ tO-g ' - '''»t>' SEQ- 353 S 171U3) 3 t r 0 5 SEQ - 356 ηΠΝΑΙ'.υ- 'Í2> S 1 ’7- 13 ' 5 're _e rcreg: τ ·' gia ^v e - '' g ' ^A to ^A c: -' g ^A -t '''t ^A g- ^A to ^A Ê-3' 2 XhiiS · g <<íq .'Vs ftg Λ SEQ 357 QDN2s36'44 ‘ b A '-'r- ^ rio ^c y V SEQ 3 · - > 1 72-.45 ’ 2 '6221 = <· .t> v 5 SEQ 35? 01’232 · S ’-> '44 · S 172'451 • 1 g <£ -j £ & g £ O gg A 2 2'JLc í-- <. ^r g'.tg '---.' -t - c>

Petition 8702Θ0Φ3ΘΒ82, of 10 / Φ3 / 2Ο29, p. 159/220

145/187

Table 26

At the. ID (SEQ ID) Sequence: CpG (5 '= + 3') Complementary tape (3 ^: ==? 5 \ í SEQ-360 3-173 (25) 3 'M22 g and ^{Λ A} to ^A ca ca ^The gcaaaacag the ^Λ - 5' SEQ-361 ODN2006Í13) 8-173 (25} 5 ^, -t- ^, 7 :: ^A g ^A t / '' 7? '' ^, G ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g '''t- ^A c ^A g ^A t ^A t-3' 3 ^! -M22-e ^A to ^A g ^A cagcaaaacag ^A c ^A to ^A a-5 ' SEQ-362 8-174 (25) 3 -M22 g ^A to ^A g ^A cagcaaaacag ^A c ^A to ^A to ^A Ls · <) - 5 ' 8EQ-363 ODN2006Í13) 8-174 (25, · 5'-t ^A c ^A g ^A t- ^A c ^A g ^A t ^A t ^A t ^A t''g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t'3 ^< 3'-M22-e ^A to ^A g ^A cagcaaaacag ^A c ^A to ^A to ^A Ls-ie'5 ' SEQ-364 8-175 (25) 3 'NI22 6 ^A to ^A g''CagcaaaílCag'C''a ^A a ^/ Έ $ -ι4 “· Ϋ 8EQ-365 ODN2006Í13) í úCZü.í 5 ^, -t ^A c ^A g ^A t. ^A c ^A g ^A t ^A t ^A t ^A t''g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t-3 ^< 3'-M22-e ^A a ^A g ^A cagcaaaacag ^A c ^A a ^A a ^A Ls-i4'5 * SEQ-366 8-176 (25) 3 '· M ·? 2-8 ^A a ^Λ g ^A ca gc aaaacag ^A c ^A a ^A a- 5' SEQ'367 ODN2006Ú3) S'176 (25? 5'-t ^A c ^A g ^A t. ^A c ^A g ^A t ^A t ^A t ^A t''g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t-3 ^< 3'-M22-8 ^z 'a ^A g ^A cagcaaaacag ^A c ^A a ^A a-5 * SEQ-368 8-177 (25) 3 '-M22-8 ^A a ^A g ^A ca gc aaaacag ^A c ^A a ^A a ^A Ls- · cr 5' SEQ-369 ODN2006Í13) 8-177 (25) 5'-tg ^A t. ^A c ^A g ^A t ^A t ^A t ^A t''g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t'3 ^< 3'-M22-8 ^A a ^A g ^A cagcaaaacag ^A c ^A a ^A a ^A Ls-ie-5 * SEQ-370 8-178 (25) 3'-M22-s ^aa 'g ^A cagcaaaacag ^A c ^A a ^A to ^A Ls: i-5' SEQ-371 ODN2006Ü3) 8-178 (25) 5'-t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A cg ^A t ^A t-3 '3'-M22-8 ^A to ^A g ^A cagcaaaacag ^A c ^A to ^A to ^A Ls-i4'5 * SEQ-372 8-179 (14) 3'-M22-i4 ^A a''g ^A cagcaaaacagcaaaacag ^A c ^A a ^A to ^A Ls-e-ã ' SEQ-373 ODN2006 (13) 8-179 (14) 5'-t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A tg ^A t ^A t-3 '3 '-M32i4 ^A to ^A g ^A cagcaaaacagcaaaacag ^A c ^A to ^A to ^A Ls-6-5' SEQ-374 8-180 (14) 3'-M22 i4 ^A to ^A g ^A cagcaaaaeagcaaaacag ^A c ^A to ^A to ^A L8-io-5 ' SEQ-375 GDN2006 (13) 8-180 (14) 5'-t ^A c ^A g ^A t / 'c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t'<! g ^A t ^A t-3 '3'-M22i4 ^A to ^A g ^A cagcaaaacagcaaaacag ^A c ^A to ^A a''Ls-io-5'

Table 27

At the. ID (SEQ ID) S String:! PG (5M3 ') C <Complementary tape · = Φ0'.) SEQ-376 8-181 (14} 3 -M22-i4 ^A to ^A g ^A cagcaaaacagcaaaacag ^A c ^A to ^A to ^A Ls-i4'5 * SEQ-3 f7 ODN2006 (13) 8-181 (14) 5 ^! -t ^A c ^A g ^A t ^A _c ^A g ^A t ^A t ^A t ^A t '''g ^A t'' ⁱ c ^A g ^A t ^A t ^A t''' t ^A g ^A t ^A c ^A g ^A t ^A t-3 '3'-M22-i4 ^A to ^A g ^A eagcaaaacageaaaacag ^A c ^A to ^A a ^/ ' Ls-i4 * 5 ' SEQ-378 8-182 (14) 3 '-M22- μ ^A g ^A ga ^Λ g ^A ca gcaaaac ag ca aaa cag ^A c ^A a ^A to ^A Ls «5' SEQ-379 ODN2006 (13) 8-182 (14) 5 ^, -t ^A c ^A g ^A t ^A c ^A g ^A t ^A t. ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t-3 '3' -M22-14g ^A g ^A a ^Λ g ^A cagcaaaaca gca a aa cag ^A c ^A a ^A a ^A Ls -g - 5 ' SEQ-380 1018188 (46) 5 ^[ -t ^A g ^A a ^A c ^A t ^A g ^A t. '''G' ^A a ^A a ^A c ^A g ^A t ^A t ^A c ^A g ^A a ^A g ^A a ^A t- ^A g ^A a-3 ' SEQ-381 8-183 (47) 3 '-M22-1 s ^A to ^A c ^A tg acac ttgca ^A to ^A g ^A c 5 ^: SEQ-882 1018188 (46) 8-183 (47} 5 ^A t ^A g '''a ^A c ^A t ^A g ^A t. ^A g ^A A ^A A ^A C ^A t g ^A T ^{A A} C ^{A A} g A ^to G ^A a ^A t ^A g '' to 3 '3 1 -M22 · s at'<T TG acact gca ^A to ^A g ^A c 5 ' SEQ-383 8-184 (14) 3'-M22-22 ^A Bu ^A Bu ^A a ^A g ^A cagcaaaacagcaaaacag ^A and ^A a ^A a-5 * SEQ-384 ODN2006 (13) 8-184 (14) 5 ^A t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A s; ^A g ^A t ^A t ^A t ^A t ^A g ' ^A t ^A c ^A g ^A t ^A t-3'3'-M22-22 ^A Bu ^A Bu ^A a ^A g ^A cagcaaaacagcaaaacag ^A c ^A a ^A a-5 ' SEQ-385 S-185I14) 3'-M22-22 ^A g ^A g ^A g ^A g ^A g ^A a ^A g ^A cagcaaaacagcaaaacag ^A w ^/ a ^A -5 ' SEQ-386 ODN2006Í13) 8-185 (14) 5 ^! -1 ^A c ^A g ^A t ^A c ^A g ^A t ^Λ t ^A t ^A t ^A g ^A t ^A c ^A g ^{, A} i ^A t ^A t ^A t ^A g ^A t ^A and ^A g ^A t ^A t 3 '3'-M22-22 ^A g ^A g ^A g ^A g ^A g ^A a ^A g ^A cagcaaaacagcaaaacag ^A c ^A to ^A a-5' SEQ-387 8-186 (14) 3 ^"The M22-22 bu bu g ^A g ^A g ^A g ^A g ^A A ^A g ^A • cagcaaaacagcaaaacag ^A and ^A to ^{A f} a'5 SEQ-388 ODN2006Ú3) 8-186 (14} 5 ^> -t ^A c ^A g ^A t ^A c ^A g ^A t ^A t ^A È ^A t ^A g ^A t ^A c ^A g ^A t ^A t ^A t ^A t ^A g ^A t ^A c ^A g ^A t ^A t -3 '3'-M22-22 ^A Bu ^A Bu ^A g ^A g ^A g ^A g ^A g ^A a ^A g ^A cagcaaaacagcaaaacag ^A c ^A a ^A a-5'

[00543] In Tables 4 to 27, n (lowercase) is DNA, noMe is 2'Petition 87Ο29ΟΦ3Θ0Η2, from 10 / Φ3 / 2Ο29, p. 15Θ / 220

146/187

OMe-RNA, N (capital letter) is RNA, Nfó 2'-deoxy-2'-F-RNA, Nlna is LNA and 5mC is 5-methylcytosine. ^Λ is -P (S) OH- and the connection without any symbol is -P (O) OH-,

, A

OH

No symbol:

[00544] L is a compound introduced at the 5 'end of the oligonucleotide possibly comprising an oligonucleotide linker and which covalently bonds to a hydroxyl group at the 5' end of the oligonucleotide via the described link. M is a compound introduced at the 3 'end of the oligonucleotide possibly comprising an oligonucleotide linker and which covalently bonds to a hydroxyl group at the 3' end of the oligonucleotide through the described link. In L _x . _n , x is a number that corresponds to the structure of Compound 4-n or Compound 10-n, which is double-stranded, or Compound 8-n which is single-stranded, and n is an integer from 6 to 28 corresponding to ( s) carbon chain (s). In M _x . _n , x is a number that corresponds to the structure of Compound 22-n, Compound 49-n or Compound 51-n which is double-stranded from Compound 15-n which is single-stranded, and n is a corresponding integer from 6 to 28 to the carbon chain (s).

[00545] In detail, L _x . _n is a group described below.

O

Petition 870190Φ3ΘΒ82, of 10 / Φ3 / 2Ο29, p. 15T / 22B

147/187 where n is an integer from 6 to 28.

[00546] l_4-n is a group derived from Compound 4-n which is synthesized in 1-1) of A) above. Ls- _n is a group derived from Compound 8-n that is synthesized in 2) from A) above. Lw-n is a group derived from Compound 10-n that is synthesized in 3) from A) above.

[00547] Lchoi or Ltoc in the Table is a group described below.

[00548] Lchoi is a group derived from Compound 26 which is synthesized in 8-1) of A) above. Ltoc is a group derived from Compound 5'Tocopherol-CE-Phosphoramidite which is purchased from Link Technologies Ltd. [00549] Lfari in the Table is a group described below.

[00550] Lfari is a group that is derived from Compound 53 synthesized in 8-2) of A) above.

[00551] Mx-n is a group described below.

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148/187 where n is an integer from 6 to 28.

[00552] Mis-η θ a group that is derived from Compound 15-n synthesized in 4) of A) above, M ₂₂ . _n is a group that is derived from Compound 22-n synthesized in 6) of A) above, M49- _n is a group that is derived from Compound 49-n synthesized in 11) of A) above, and Msi- _n is a group derived from Compound 51-n synthesized in 13) of A) above.

[00553] Mchoi in the Table is a group described below.

[00554] Mchoi is a group that is derived from Compound 55 synthesized in 14) of A) above.

[00555] Mteg-π in the Table is a group described below.

O

where n is an integer from 6 to 28.

[00556] Mteg-π θ a group that is derived from Compound 63-n synthesized in 15) of A) above.

[00557] K ₂₂ -n in the Table is a group described below.

O

where n is an integer from 6 to 28.

[00558] K ₂₂ -n is a group derived from Compound 22-n synthesizesPetition 8701Θ0Φ3ΘΘ82, of 10 / Φ3 / 2Ο29, p. 159 / 22B

149/187 of in 6) of A) above.

[00559] The following linker ( ^A Bu ^A Bu ^A in the Table) is used for S155, S-184 or S-186.

Example 2. Evaluation of the activity of double-stranded oligonucleotides linked to lipids of the present invention using the reporter assay Materials and methods [00560] HEK-Blue ™ hTLR9 cells (Invivogen), used for the reporter assay, were produced by introducing the gene Human TLR9 and a reporter gene which is the alkaline phosphatase secreting gene linked to the NF-kB-AP-1 binding region sequence in HEK293 cells, and the cells stably expressing the two genes. 3.6x10 ⁴ of the cells suspended in HEK-Blue ™ Detection (Invitrogen), which comprises substrates against alkaline phosphatase, were seeded in the 96-well plate, a lipid-linked double-stranded oligonucleotide of the present invention, 3 nM at 30 μΜ , the known CpG oligonucleotides (ODN2006, SEQ-49) or adjuvant, based on the design in Patent Document 1, that is, ssCpG ODN with an introduced lipid linker (SEQ-142, 143 or 144) was added. After culture in an incubator at 37 ° C, CO25% for 16 hours, the absorbance at 620 nm was measured using the culture supernatant that developed color. The result value was analyzed with the TIBCO Spotfire software and the 50% effective concentration (ECso) of each oligonucleotide was calculated.

Result [00561] As previously described, it is known that the characterization as an adjuvant disappeared when ssCpG ODN was administered as double-stranded DNA (dsCpG ODN) by annealing the first with the second strand (Non-Patent Document 4). To examine whether

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150/187 the design of the double-stranded nucleic acid adjuvant shows activity as a TLR9 agonist, the evaluation of the activity of a double-stranded oligonucleotide was performed by a reporter assay.

[00562] As shown in Tables 28 and 29, ODN2006 (SEQ-49), which is a ssCpG ODN, revealed the activity as a TLR9 agonist, and the ECso value was approximately 467 nM (Experiment 1, Table 28) and approximately 245 nM (Experiment 2, Table 29). On the other hand, a lipid-linked double-stranded oligonucleotide of the present invention, comprising ODN2006 as a CpG oligonucleotide, revealed TLR9 agonist activity in the nM range between several tens and a thousand and many hundreds. Most of the adjuvants of the present invention, described in Tables 28 and 29, had the tendency to increase activity when compared to ODN2006. In addition, 16 sequences, SEQ-57, SEQ-59, SEQ-61, SEQ-63, SEQ-65, SEQ-111, SEQ-117, SEQ-135, SEQ-146, SEQ-226, SEQ-282, SEQ-284, SEQ294, SEQ-324, SEQ-361 and SEQ-363, tended to decrease activity slightly compared to ODN2006, but the difference was approximately 3 times. Therefore, it was clear that all lipid-linked double-stranded oligonucleotides of the present invention have TLR9 agonist activity.

[00563] In addition, to examine whether the modified form of SEQ2, described in Patent Document 1, applied for ODN2006, which is the sequence of the human type CpG oligonucleotide, shows activity as a TLR9 agonist, the activity evaluation has been carried out in the same way (SEQ-141, 142 or 143). SEQ-142, 143 or 144 could not induce TLR9 activation in the measured concentration range, and it was impossible to calculate the ECso values (n.d. in Table 28). That is, it is suggested that the design (ssCpG ODN with an inserted lipid ligand) described in Patent Document 1 cannot be useful as a vaccine adjuvant and / or the vaccine itself, as it revealed the

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151/187 activity for ODN1826 (mouse type), but not for ODN2006 (human type) as described in the example.

In contrast, it is suggested that a lipid-linked double-stranded oligonucleotide of the present invention may be useful as a vaccine adjuvant and / or the vaccine itself, regardless of the sequence of the CpG oligonucleotides, since it has revealed the activity for ODN2006 as well as for ODN1826.

Experiment 1

Table 28

No. EC50 (nM) No. EC50 (nM) No. EC50 (nM) SEQ-49 466.8 SEQ-89 300.9 SEQ-125 305.6 SEQ-51 405.9 SEQ-91 68.4 SEQ-127 299.9 SEQ-55 377.9 SEQ-93 106.4 SEQ-129 459.0 SEQ-57 976.8 SEQ-95 33.4 SEQ-131 312.2 SEQ-59 542.5 SEQ-97 308.9 SEQ-133 169.7 SEQ-61 608.1 SEQ-99 311.3 SEQ-135 509.8 SEQ-63 598.9 SEQ-101 33.5 SEQ-137 254.7 SEQ-65 500.9 SEQ-105 96.9 SEQ-139 58.4 SEQ-75 300.6 SEQ-111 490.1 SEQ-141 31.0 SEQ-77 296.8 SEQ-113 449.5 SEQ-146 1128.2 SEQ-79 162.9 SEQ-115 303.2 SEQ-148 376.6 SEQ-81 225.9 SEQ-117 616.5 SEQ-150 309.9 SEQ-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.

Experiment 2

Table 29

No. EC50 (nM) No. EC50 (nM) No. EC50 (nM) SEQ-49 245.3 SEQ-218 59.8 SEQ-292 58.9 SEQ-152 86.4 SEQ-220 57.2 SEQ-294 262.4 SEQ-154 94.5 SEQ-222 31.9 SEQ-296 10.1 SEQ-156 90.0 SEQ-224 12.4 SEQ-298 10.2 SEQ-158 56.6 SEQ-226 362.0 SEQ-300 11.4 SEQ-160 65.1 SEQ-228 21.03 SEQ-302 10.2 SEQ-162 89.7 SEQ-230 39.8 SEQ-304 11.6 SEQ-164 64.2 SEQ-232 72.7 SEQ-306 11.3 SEQ-166 90.4 SEQ-234 27.0 SEQ-308 8.4 SEQ-168 62.8 SEQ-236 203.1 SEQ-310 10.7 SEQ-170 46.0 SEQ-238 131.9 SEQ-312 19.1 SEQ-172 161.9 SEQ-240 62.0 SEQ-314 10.8 SEQ-174 101.0 SEQ-242 102.4 SEQ-316 10.9 SEQ-176 241.2 SEQ-244 101.8 SEQ-318 81.3 SEQ-178 210.1 SEQ-246 102.6 SEQ-320 68.4 SEQ-180 46.9 SEQ-248 108.3 SEQ-322 50.4 SEQ-182 11.2 SEQ-250 92.4 SEQ-324 261.1

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No. EC50 (nM) No. EC50 (nM) No. EC50 (nM) SEQ-184 34.2 SEQ-252 87.4 SEQ-326 24.6 SEQ-186 127.7 SEQ-260 39.9 SEQ-328 24.1 SEQ-188 67.7 SEQ-262 25.7 SEQ-330 33.7 SEQ-190 46.9 SEQ-264 86.3 SEQ-332 24.8 SEQ-192 123.9 SEQ-266 58.1 SEQ-334 25.3 SEQ-194 99.8 SEQ-268 11.5 SEQ-336 31.4 SEQ-196 33.2 SEQ-270 16.3 SEQ-338 188.1 SEQ-198 114.9 SEQ-272 33.3 SEQ-361 771.8 SEQ-200 96.3 SEQ-274 29.7 SEQ-363 290.2 SEQ-202 89.5 SEQ-276 30.9 SEQ-365 177.4 SEQ-204 83.4 SEQ-278 33.3 SEQ-367 178.2 SEQ-206 40.7 SEQ-280 168.3 SEQ-369 135.9 SEQ-208 31.3 SEQ-282 275.1 SEQ-371 170.0 SEQ-210 56.7 SEQ-284 255.8 SEQ-373 21.5 SEQ-212 46.9 SEQ-286 45.0 SEQ-375 21.9 SEQ-214 47.4 SEQ-288 65.4 SEQ-377 29.8 SEQ-216 99.5 SEQ-290 151.7 SEQ-379 20.3

[00565] As shown in Table 30, even when CpG oligonucleotides, except for ODN2006, were used, most of the adjuvants of the present invention showed a tendency to improve activity compared to ssCpG ODN. It was clear that all lipid-linked double-stranded oligonucleotides of the present invention show activity as a TLR9 agonist.

Table 30

At the. EC50 i SEQ-2Õ3 tKH'CpU) SEQ-255 287Q.3 173.4 SEQ-25h (DSfrCpG) 3421046.2 16328.8 SEQ-3H9 (ODN22 1Φ 7244.7 SEQ-341 . SEQ-342 CODN684) 7915.8 233S.9 SEQ-314 111.4 SEQ'347 Φ-LSOn 6283.7 SEQ-SU7 10.7 SEQ'351 11569.8 SEQ 873 1982.2 16893838.1 292.8

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Example 3: In vivo evaluation of adjuvants of the present invention

A) Evaluation of CTL induction capacity Animals [00566] C57BL / 6JJcl mice (6 to 8 weeks old) were purchased from CLEA Japan, Inc.

Vaccine components 1

Peptide OVA257-264 (SIINFEKL, SEQ ID NO: 22), synthesized and purified by reverse phase HPLC and purchased from SigmaAldrich.

an adjuvant to the present invention or the well-known CpG ODN

Components of the vaccine 2

TRP2i ₈ o-i8 ₈ peptide (CSVYDFFVWL, SEQ ID NO: 23), synthesized and purified by reverse phase HPLC and purchased from SigmaAldrich.

an adjuvant to the present invention or the well-known CpG ODN

Vaccine components 3

OVA257-264OU peptide TRP2iso-i ₈₈ Montanide ISA51 (SEPPIC)

Preparation of vaccine 1 [00567] Specific immunity was produced by vaccinating a mouse twice at 7-day intervals. Each vaccine consisted of 100 pg of OVA peptide and 1.57 or 4.71 nmol of an adjuvant of the present invention or well-known CpG ODN, dissolved in 1 * PBS. 100 pL of the vaccine was administered by subcutaneous injection in the flank of a mouse.

Preparation of vaccine 2 [00568] Specific immunity was induced by vaccinating a mouse twice at 7-day intervals. Each vaccine consisted of

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100 pg of TRP2 peptide and 1.57 or 4.71 nmol of an adjuvant of the present invention or well-known CpG ODN, dissolved in 1xPBS. 100 μΙ_ of the vaccine was administered by subcutaneous injection in the flank of a mouse.

Preparation of vaccine 3 [00569] The 2 mg / mL TRP2 peptide in PBS and Montanide (the component of vaccine 3) were mixed 1: 1 using a cylinder and pumping connector to form an emulsion. 100 μΙ_ of the vaccine emulsion was administered by subcutaneous injection in the flank of a mouse twice at 7-day intervals.

Tetramer staining [00570] On the 7th day after the second vaccination, blood was collected, the red cells removed by BD Pharm Lyse (BD pharmingen) and the cells were suspended in FACS buffer (1% fetal bovine serum, 2 mM PBS with EDTA). The mouse anti-CD16 / 32 monoclonal antibody (BioXcell) was added to it, and the blocking of Fc receptors was performed at 4 ° C for 30 minutes. Then, the PE-labeled H-2Kb tetrameter, corresponding to the vaccine antigen (T-Select H2Kb OVA Tetramer-SIINFEKL or T-Select H-2Kb TRP-2 TetramerSVYDFFVWL; both 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 performed at room temperature for 45 minutes. Then, staining was performed with DAPI (Invitrogen) and the cells were washed twice with FACS buffer. The suspension in FACS buffer was repeated and staining was analyzed with the FACS Verse flow cytometer (BD bioscience) and the FAC Suite software (BD bioscience). For analysis, after creating a gate to delimit the negative fraction by DAPI, the light scattered in the same direction (forward) and perpendicularly (side-way), as indicators, provided the fraction. In the fraction

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155/187 leukocytes, cells positive for Alexa 647-CD8 and PE-HT 2Kb diameter were defined as CTLs. The CTL ratio in the leukocytes was used for evaluation.

Calculation method of the CTL induction capacity indicator [00571] After calculating the average CTL induction capacity of each group per experiment, the ratio to the mean value of the CTL induction capacity of ssCpG ODN, ODN1826 (SEQ -1) or ODN2006 (SEQ-49), as a control, was calculated.

[00572] In the case of ODN 1826 comprising adjuvant:

(Average of the CTL induction capacity of the group with adjuvant) / (Average of the CTL induction capacity of the group ODN 1826) = the ratio to the average value [00573] In the case of ODN2006 comprising adjuvant:

(Average of the CTL induction capacity of the group with adjuvant) / (Average of the CTL induction capacity of the ODN2006 group) = the ratio to the average value [00574] The CTL induction capacity of each adjuvant was shown, when the CTL induction capacity of ssCpG ODN was considered 1, so that the higher value means the greater CTL induction capacity.

B) Evaluation of the antitumor effect of the vaccine with TRP2 peptide using an adjuvant of the present invention [00575] After immunization by the vaccine with the TRP2 peptide, B16F10 cells (ATTC), which are from mouse melanoma expressing the TRP2 protein, were transplanted via subcutaneous to assess the antitumor effect. In a method similar to Example 3 A), the next day when a positive tetrimeter ratio was observed in the peripheral blood of mice vaccinated twice every 7 days, B16F10 cells (1x10 ⁵ cells / mouse) were transplanted subcutaneously in the shoulder right. The major and minor axis of the

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156/187 grafted tumors were measured every 2 to 3 days, and the tumor volume was calculated using the formula:

(major axis χ minor axis ² ) / 2

Calculation method for indicators of antitumor effect [00576] For ODN 1826 comprising adjuvant

100- (average tumor volume of the group with adjuvant) / (average tumor volume of the group ODN1826) [00577] For ODN2006 comprising the adjuvant

100- (average tumor volume in the group with adjuvant) / (average tumor volume in the ODN2006 group) [00578] The Compound with the highest value has the strongest antitumor activity.

Result using ODN1826 as CpG oligonucleotide Result 1-A: Comparison of the CTL-inducing ability between an adjuvant of the present invention and known adjuvants [00579] When mice were immunized with TRP2 and ODN 1826 (SEQ-1) peptide, CTL specific for TRP2 were induced. When dsCpG ODN (SEQ-4) was used, the induction capacity of TRL2-specific CTL was 0.46 times lower compared to ODN 1826. That is, a double-stranded adjuvant led to less CTL induction capacity when compared to a single tape. This suggested that when the CpG oligonucleotide is produced only in the form of a double strand, the immunostimulatory activity is decreased as described in Non-Patent Document 4 and the like. On the other hand, when an adjuvant of the present invention, that is, a lipid-bound double-stranded oligonucleotide adjuvant (SEQ-16) for immunization, was used, the CTL-inducing ability improved 1.46 times more compared to ODN 1826. Therefore, it has been suggested that an adjuvant of the present invention exhibits unexpected improvement in

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157/187 activity. The results were shown in Table 31.

Table 31

Peptide Adjuvant Nmol dose / head Ratio of mean antigen-specific CTL value TRP2 SEQ-1 1.57 1.00 TRP2 SEQ-4 1.57 0.46 TRP2 SEQ-16 1.57 1.46

Result 1-B: Comparison of the antitumor effect between an adjuvant of the present invention and known adjuvants [00580] When mice were immunized with peptide TRP2 and ODN 1826 (SEQ-1), they obtained little inhibitory effect on tumor progression of B16F10 cells . Furthermore, dsCpG ODN (SEQ-4) exhibited little inhibitory effect on tumor progression of B16F10 cells. On the other hand, an adjuvant of the present invention (SEQ-16) showed the inhibitory effect on tumor progression of B16F10 cells, 43% stronger compared to ODN1826 on Day 14 after transplantation. The results were shown in Figure 1.

Result 2-A, B: CTL-inducing ability and anti-tumor effect of adjuvants of the present invention comprising lipid ligand with different number of carbon atoms [00581] The activity of an adjuvant of the present invention, having a lipid comprising two acyl chains including 10 to 20 carbon atoms as a linker, was examined with OVA peptide. The CTL induction capacity and tumor inhibition rate of SEQ-12 (10 carbon atoms) was lower when compared to ODN 1826 (SEQ-1). On the other hand, there is a tendency to improve the CTL induction capacity as the chain length gets longer, as in SEQ-14 (14 carbon atoms), SEQ-6 (18 carbon atoms) and SEQ- 16 (20 carbon atoms). In addition, the tumor inhibition rate of SEQ-14 was approximately 94% inhibition of tumor growth when compared to ODN 1826 on Day 14 after transplantation, and SEQ-6 and SEQ-16 perfectly inhibited the

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158/187 tumor growth on Day 14 after transplantation. The results were shown in Table 32.

Table 32

Peptide Adjuvant Nmol dose / head Ratio of mean antigen-specific CTL value Tumor inhibition rate (Day 14 after transplant) 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

Result 3-A: Ability to induce CTL DE adjuvants of the present invention with lipids introduced at both ends [00582] The activity of an adjuvant of the present invention with a lipid comprising two acyl chains including 20 carbon atoms as a linker and further comprising a single-stranded lipid including 8 or 12 carbon atoms at the 3 'end of the CpG oligonucleotide (SEQ-46 or SEQ-48) was examined with TRP2 peptide. Both CTL induction capabilities have been improved approximately 2.8 times more compared to ODN1826 (SEQ-1). The results were shown in Table 33.

Table 33

Peptide Adjuvant Nmol dose / head Ratio of mean antigen-specific CTL value TRP2 SEQ-1 1.57 1.00 TRP2 SEQ-46 1.57 2.84 TRP2 SEQ-48 1.57 2.84

Result 3-B: Effect against cancer of adjuvants of the present invention with lipids introduced at both ends [00583] When mice were immunized with peptide

TRP2 and an adjuvant of the present invention, SEQ-46 or SEQ-48, it revealed the inhibitory effect on tumor progression of cells

B16F10, respectively, 52% or 43% stronger on Day 13 after transplantation, compared to ODN 1826 (SEQ-1). The results were obtained from 870290 (039082, from 10 / Φ3 / 2Ο29, p. 169/220

159/187 ram shown in Figure 2.

Result 4-A: CTL-inducing ability of adjuvants of the present invention with different lengths of the complementary strip [00584] In 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 tape is proportional to the thermal stability and the more complementary sites there are in the double tape structure, the more stable the double tape is. Then, the adjuvant activity of the present invention whose complementary strand chain length is different was examined with TRP2 peptide.

[00585] The adjuvant whose complementary strand is 10 mer for ODN1826 (20 mer), that is, the length of the complementary strand in relation to the CpG oligonucleotide is 50% (SEQ-8) and the adjuvant whose complementary strand is 15 mer , that is, the length is 75% (SEQ10). Both SEQ-8 and SEQ-10 dominantly improved CTL induction capacity compared to ODN 1826 (SEQ-1) approximately 1.2 and 2.5 times more, respectively. The results were shown in Table 34.

Table 34

Peptide Adjuvant Nmol dose / head Ratio of mean antigen-specific CTL value 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

Result 4-B: Cancer effect of adjuvants of the present invention with different lengths of complementary strand [00586] When mice were immunized with peptide

TRP2 and ssCpG ODN with a lipid ligand introduced in Patent Document 1 (SEQ-2), the tumor inhibition rate was approximately 3.5% on Day 12 after transplant compared to ODN 1826

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160/187 (SEQ-1). On the other hand, SEQ-8, which is an adjuvant of the present invention with 10 mer of complementary tape, revealed approximately 50% of the tumor inhibition rate compared to ODN 1826 on Day 12 after transplantation. In addition, SEQ-10, which has 15 mer of complementary tape, exhibited the inhibitory effect on tumor progression of B16F10 cells, approximately 76% stronger compared to ODN 1826 on Day 12 after transplantation. The results were shown in Figure 3.

Result 5-A: CTL induction capacity of adjuvants of the present invention with a different chain length than the complementary strip [00587] The activity of an adjuvant of the present invention, whose complementary strip chain length is 10 to 20 mer in Regarding ODN 1826 (20 mer), that is, the complementary strand is 50 to 100% in relation to the CpG oligonucleotide, it was examined with TRP2 peptide. Adjuvant whose complementary stripe is 14 mer (SEQ-28), 15 mer (SEQ-26), 17 mer (SEQ-22), 19 mer (SEQ-18) and 20 mer (SEQ-16) showed improved ability to CTL induction compared to ODN 1826 (SEQ-1). Especially, the 19 mer (SEQ-18), 17 mer (SEQ-22) and 15mer (SEQ-26) adjuvant revealed a CTL induction capacity approximately 3 times greater when compared to ODN 1826 (SEQ-1). The results were shown in Table 35.

Table 35

Peptide Adjuvant Nmol dose / head Ratio of mean antigen-specific CTL value 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

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Result 6-A: Comparison of CTL induction capacity with Montanide [00588] The activities of ODN1826 (SEQ-1), ssCpG ODN with an introduced lipid ligand (SEQ-2), an adjuvant of the present invention (SEQ-26) and Montanide, which is an adjuvant used extensively in studies as an adjunct to a peptide vaccine, were compared with TRP2 peptide as an antigen. SEQ-2 and SEQ-26 showed greater CTL induction capacity than ODN1826. On the other hand, Montanide's CTL induction capacity was a seventh lower compared to ODN 1826. The results are shown in Table 36.

Table 36

Peptide Adjuvant Nmol dose / head Ratio of mean antigen-specific CTL value 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

Result 6-B: Comparison of the anti-tumor effect against ssCpG ODN with an introduced lipid ligand [00589] When mice were immunized with TRP2 peptide and an adjuvant of the present invention, SEQ-26, it showed a very strong inhibiting effect on tumor progression of cells B16F10, 76.8% on Day 12 after transplant compared to ODN1826 (SEQ-1). The tumor inhibition rate of ssCpG ODN with an introduced lipid ligand (SEQ-2) was 64.3% on Day 12 after transplant compared to ODN 1826. That is, an adjuvant of the present invention exhibited the greatest inhibitory effect on tumor progression of B16F10 cells than the SEQ-2 adjuvant. On the other hand, Montanide used as an adjuvant had no antitumor effect. The results were shown in Figure 4.

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162/187 feels an invention with the different types of nucleic acid monomer in a complementary strand [00590] The activity 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 in which all the nucleic acid monomers were RNA (SEQ-40) or 2'-F-RNA (SEQ-42) did not show much improvement in the CTL-inducing capacity compared to ODN 1826 (SEQ-1). On the other hand, the adjuvant with 2'-OMe-RNA as a nucleic acid monomer (SEQ-38 and SEQ-44) exhibited 2.9 or 2.4 times better CTL induction capacity, respectively, compared to ODN 1826. These results suggested that 2'-OMe-RNA is useful as a nucleic acid monomer for an adjuvant of the present invention as well as DNA. The results were shown in Table 37.

Table 37

Peptide Adjuvant Nmol dose / head Ratio of mean antigen-specific CTL value Tumor inhibition rate (Day 14 after transplant) 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

Result 7-B: Antitumor effect of adjuvants of the present invention with the different types of nucleic acid monomer in a complementary strand [00591] When mice were immunized with TRP2 peptide and an adjuvant of the present invention as 2'-OMe-RNA as a monomer nucleic acid (SEQ-38), it exhibited the very strong inhibiting effect on tumor progression of B16F10 cells, approximately 86.2% on Day 14 after transplantation compared to ODN1826 (SEQ-1). The rate of tumor inhibition of ssCpG ODN with an introduced lipid ligand (SEQ-2) was approximately 72.6% on Day 14 after transplantation compared to ODN 1826. That is, an 87Ο29ΟΦ3Θ082 adjudication, of 10 / Φ3 / 2Ο29, p. 103/220

163/187 ahead of the present invention revealed a stronger inhibitory effect on tumor progression of B16F10 cells than the adjuvant of SEQ-

2. In addition, the adjuvant whose chain length of SEQ-38 has been shortened (SEQ-44) and the adjuvant with 2'-F-RNA as nucleic acid monomer (SEQ-42) exhibited the inhibitory effect on tumor progression in B16F10 cells. On the other hand, the RNA adjuvant as nucleic acid monomer (SEQ-40) did not show an inhibitory effect on tumor progression of B16F10 cells. The results were shown in Table 37 and Figure 5.

Result using QDN2006 as CpG oligonucleotide

Result 8-A: CTL-inducing ability of adjuvants of the present invention with a different chain length than that of the complementary strip or a binder [00592] The activities of ODN2006 (SEQ-49, 24 mer) and adjuvants of the present invention were compared, with TRP2 peptide as antigen. The adjuvant whose complementary strand chain length is 24 mer was used, that is, the complementary strand length in relation to the CpG oligonucleotide is 100% (SEQ-51) and the adjuvant whose complementary strand chain length is 15 mer, that is, the length of the complementary strip in relation to the CpG oligonucleotide is 62.5% (SEQ-61). Both SEQ-51 and SEQ-61 showed a high CTL induction capacity more than 5 times when compared to QDN2006. In addition, the adjuvant with dTdT was used as a binder (SEQ-63) or dGdG (SEQ-65). Both SEQ-63 and SEQ65 showed very high CTL induction capacity, about 10 times when compared to QDN2006. The results were shown in Table 38.

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Table 38

Peptide Adjuvant Nmol dose / head Ratio of mean antigen-specific CTL value TRP2 SEQ-49 4.71 1.00 TRP2 SEQ-51 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

Result 8-B: Antitumor effect of an adjuvant of the present invention with the different chain length of a complementary strand or a ligand [00593] When mice were immunized with peptide TRP2 and QDN2006 (SEQ-49), it showed little inhibition effect on tumor progression of B16F10 cells. On the other hand, an adjuvant of the present invention, whose complementary strand length is 100% in relation to the CpG oligonucleotide (SEQ-51), exhibited 20% of the inhibitory effect on tumor progression of B16F10 cells on Day 10 after transplantation in comparison the QDN2006. In addition, the adjuvant whose complementary strand length is 62.5% in relation to the CpG oligonucleotide (SEQ-61) and the adjuvant with dTdT as ligand (SEQ-63) or dGdG (SEQ-65) show about 50% of the inhibitory effect on tumor progression of B16F10 cells on Day 10 after transplant compared to QDN2006. The results were shown in Figure 6.

Result 9-A: CTL-inducing ability of adjuvants of the present invention whose nucleic acid monomer in the complementary strand is 2'-OMe-RNA [00594] The 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 length of the complementary strand chain is 24 mer, that is, we used the adjuvant whose complementary strand length in relation to the CpG oligonucleotide was 100% (SEQ67) and the adjuvant whose complementary strand length is 15 mer, or

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165/187 that is, the adjuvant whose complementary strip length in relation to the CpG oligonucleotide is 62.5% (SEQ-69) was used. SEQ-67 and SEQ-69 showed 1.4 or 1.2 times more, respectively, CTL induction capacity compared to ODN2006 (SEQ-49). The results were shown in Table 39.

Table 39

Peptide Adjuvant Nmol dose / head Ratio of mean antigen-specific CTL value TRP2 SEQ-49 4.71 1.00 TRP2 SEQ-51 4.71 1.39 TRP2 SEQ-61 4.71 1.21

Result 9-B: Antitumor effect of adjuvants of the present invention whose nucleic acid monomer in the complementary strand is 2'-0Me-RNA [00595] When mice were immunized with TRP2 peptide and an adjuvant of the present invention that has 2'-OMe- RNA as a nucleic acid monomer, the adjuvant whose complementary strand length relative to the CpG oligonucleotide is 100% (SEQ67) exhibited very strong inhibiting effect on tumor progression of B16F10 cells, approximately 75% on Day 10 after transplantation QDN2006 (SEQ-49). The adjuvant whose complementary strand length relative to CpG oligonucleotides is 62.5% (SEQ-69) showed about 20% of the inhibitory effect on tumor progression of B16F10 cells on Day 10 after transplant compared to QDN2006. The results were shown in Figure 7.

[00596] These suggested that 2'-OMe-RNA was also useful for QDN2006 as the nucleic acid monomer of an adjuvant of the present invention as well as DNA.

[00597] In similar methods, the ability to induce

CTL or the anti-tumor effect of adjuvants of the present invention were measured. The results are shown in Tables 40 to 68 and Figures 8 to 13, separately by experiments. For these experiments, the TRP2 peptide and the dose of the adjuvant used in this study were used.

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166/187 invention was 4.71 nmol. NT in the Tables means not tested.

Table 40

Adjuvant Ratio of mean antigen-specific CTL value SEQ-49 1.00 SEQ-59 5.34 SEQ-57 4.28 SEQ-55 3.02 SEQ-53 2.86

Table 41

Adjuvant Ratio of mean antigen-specific CTL value Tumor inhibition rate (Day 12 after transplant) SEQ-49 1.00 0.00 SEQ-119 2.05 55.1 SEQ-192 1.74 41.5

Table 42

Adjuvant Ratio of mean antigen-specific CTL value Tumor inhibition rate (Day 12 after transplant) SEQ-49 1.00 0.00 SEQ-121 3.99 56.8

Table 43

Adjuvant Ratio of mean antigen-specific CTL value Tumor inhibition rate (Day 14 after transplant) SEQ-49 1.00 0.00 SEQ-152 1.17 80.45 SEQ-154 2.47 NT SEQ-158 3.06 100

Table 44

Adjuvant Ratio of mean antigen-specific CTL value SEQ-49 1.00 SEQ-182 2.24 SEQ-184 2.38

Table 45

Adjuvant Ratio of mean antigen-specific CTL value Tumor inhibition rate (Day 14 after transplant) 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 3.29 NT

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Table 46

Adjuvant Ratio of mean antigen-specific CTL value Tumor inhibition rate (Day 14 after transplant) 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

Table 47

Adjuvant Ratio of mean antigen-specific CTL value SEQ-49 1.00 SEQ-166 2.57 SEQ-168 1.96

Table 48

Adjuvant Ratio of mean antigen-specific CTL value Tumor inhibition rate (Day 14 after transplant) SEQ-49 1.00 0.00 SEQ-170 3.10 79.09 SEQ-172 1.24 NT SEQ-174 1.78 NT

Table 49

Adjuvant Ratio of mean antigen-specific CTL value SEQ-49 1.00 SEQ-176 1.97 SEQ-178 5.44 SEQ-180 3.59 SEQ-206 1.07 SEQ-208 1.28 SEQ-214 1.38 SEQ-216 1.95

Table 50

Adjuvant Ratio of mean antigen-specific CTL value SEQ-49 1.00 SEQ-198 3.12 SEQ-200 3.24 SEQ-204 3.98

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Table 51

Adjuvant Ratio of mean antigen-specific CTL value SEQ-49 1.00 SEQ-226 1.84 SEQ-228 2.10 SEQ-230 3.10 SEQ-232 1.28 SEQ-234 1.96

Table 52

Adjuvant Ratio of mean antigen-specific CTL value SEQ-49 1.00 SEQ-218 2.87 SEQ-220 1.68 SEQ-222 3.11 SEQ-224 2.25

Table 53

Adjuvant Ratio of mean antigen-specific CTL value SEQ-49 1.00 SEQ-236 2.66

Table 54

Adjuvant Ratio of mean antigen-specific CTL value SEQ-49 1.00 SEQ-238 1.09 SEQ-240 1.45 SEQ-242 1.62 SEQ-244 3.24

Table 55

Adjuvant Ratio of mean antigen-specific CTL value SEQ-49 1.00 SEQ-256 2.51 SEQ-248 1.67 SEQ-250 1.86 SEQ-252 1.14

Table 56

Adjuvant Ratio of mean antigen-specific CTL value SEQ-256 (D35-CpG) 1.00 SEQ-258 2.46

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Table 57

Adjuvant Ratio of mean antigen-specific CTL value SEQ-49 1.00 SEQ-260 1.89 SEQ-262 1.45 SEQ-264 3.44 SEQ-266 2.51

Table 58

Adjuvant Ratio of mean antigen-specific CTL value SEQ-49 1.00 SEQ-268 2.09 SEQ-270 2.31 SEQ-272 1.77 SEQ-274 4.03 SEQ-276 3.77 SEQ-278 2.58

Table 59

Adjuvant Ratio of mean antigen-specific CTL value SEQ-49 1.00 SEQ-280 5.27 SEQ-282 2.82 SEQ-284 5.57 SEQ-286 2.40

Table 60

Adjuvant Ratio of mean antigen-specific CTL value SEQ-49 1.00 SEQ-288 4.22 SEQ-290 2.80 SEQ-292 4.63 SEQ-294 5.30

Table 61

Adjuvant Ratio of mean antigen-specific CTL value SEQ-49 1.00 SEQ-296 2.43 SEQ-298 2.08 SEQ-300 3.48 SEQ-302 2.58 SEQ-304 2.42 SEQ-306 3.04

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Table 62

Adjuvant Ratio of mean antigen-specific CTL value SEQ-49 1.00 SEQ-308 2.52 SEQ-310 4.91 SEQ-312 1.75 SEQ-314 2.58 SEQ-316 1.30

Table 63

Adjuvant Ratio of mean antigen-specific CTL value SEQ-49 1.00 SEQ-318 4.19 SEQ-320 3.81 SEQ-322 2.15 SEQ-324 1.92

Table 64

Adjuvant Ratio of mean antigen-specific CTL value SEQ-49 1.00 SEQ-326 4.27 SEQ-328 2.93 SEQ-330 2.53 SEQ-332 2.98 SEQ-334 1.66 SEQ-336 3.62 SEQ-338 1.72

Table 65

Adjuvant Ratio of mean antigen-specific CTL value SEQ-339 (ODN2216) 1.00 SEQ-341 1.25 SEQ-342 (ODN684) 1.00 SEQ-344 1.20 SEQ-345 (D-LS01) 1.00 SEQ-347 2.73

Table 66

Adjuvant Ratio of mean antigen-specific CTL value SEQ-354 (ODN M362) 1.00 SEQ-356 1.91

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Table 67

Adjuvant Ratio of mean antigen-specific CTL value SEQ-49 1.00 SEQ-373 2.21 SEQ-375 2.58 SEQ-377 1.57

Table 68

Adjuvant Ratio of mean antigen-specific CTL value SEQ-49 1.00 SEQ-361 1.09 SEQ-365 2.04 SEQ-369 1.70 SEQ-371 1.11

Example 4 [00598] The ability to induce antigen-specific CTL and the antitumor effect of ODN2006 (SEQ-49) and SEQ-121 in the absence of a tumor antigenic peptide were evaluated. In methods similar to A) and B) of Example 3, the effect of the vaccine without a tumor antigenic peptide was verified. Both values for antigen-specific CTL of SEQ-49 and SEQ-121 were undetectable. On the other hand, SEQ-121 showed 85.3% of the inhibitory effect on tumor progression of B16F10 cells on Day 12 after transplantation compared to QDN2006. From these results, it was predicted that SEQ-121 would apply to a therapeutic vaccine with antitumor effect even if it were an isolated agent (Table 69, Figure 14).

Table 69

Adjuvant Ratio of mean antigen-specific CTL value Tumor inhibition rate (Day 12 after transplant) SEQ-49 Undetectable 0.00 SEQ-121 Undetectable 85.3

Example 5: Irritation test with a single dose of adjuvants of the present invention

A) Subcutaneous irritation test with a single dose [00599] Subcutaneous irritation of a compound of the present invention

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172/187 tion was assessed by macroscopic and histopathological examination of the skin at necropsy, one day and one or four weeks after subcutaneous administration of a single dose.

[00600] The Compound of the present invention was administered subcutaneously to the center of the back in the interscapular region of rats. At necropsy, one day, one or four weeks after administration (isoflurane anesthesia, sacrifice by exsanguination), the skin at the site of administration was collected, observed macroscopically and fixed in a neutral 10% formaldehyde buffered solution. The fixed skin was cut and inserted into a thin slice by the usual methods, and thin cuts were stained with hematoxylin-eosin (HE). The slices stained with HE were observed under an optical microscope, and histopathological findings were recorded. Changes in pathological findings were assessed in four degrees: Minimal, ±, Mild, +, Moderate, 2+ and Marked, 3+. If no grade was considered for the finding, it was recorded as Positive.

Result [00601] To verify the safety of adjuvants of the present invention as a medicine, subcutaneous irritation was compared to that of Montanide used in clinical studies as an adjuvant, Amph1826 (SEQ-2) or ODN2006 (SEQ-49), which are well adjuvant known.

[00602] Montanide is an adjuvant consisting of mineral oil and surfactant, and used in clinical studies as an adjunct to peptide vaccine, preparing an emulsion with aqueous formulation. Although it is an adjuvant with an excellent safety profile with respect to issues related to systemic safety, there is a problem with the occurrence of skin hardening caused by the remaining emulsion at the administration site. In this experiment, the reaction at the site of administration of the double-stranded oligonucleotide was compared to that

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173/187 de Montanide, and it was examined whether this could be an adjunct with weekly local irritation.

[00603] Skin irritation tests with a single dose on rats were performed 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 with Montanide emulsion: saline (1: 1). SEQ-2, SEQ-49 or an adjuvant of the present invention was dissolved in saline with phosphate buffer was administered at 4.71, 15.7 or 47.1 nmol / 1 ml / site, respectively.

[00604] As a result, it became evident that Montanide was not absorbed / decomposed / eliminated by the white accumulation or white nodules seen macroscopically under the skin, and cysts-like structures were confirmed histologically one day, one and four weeks after administration. The Montanide remaining under the skin continued to cause inflammation from the first day to four weeks after administration. One day after administration, neutrophilic inflammation and edema were observed. One week after administration, neutrophilic inflammation and edema continued, the inflammation became slightly chronic and was seen as granulomatous inflammation. Four weeks after the administration, a thick fibrous capsule, encapsulating, Montanide had formed. Inflammation had reduced slightly compared to one week after administration, however, even four weeks after administration, animals were observed that continued with neutrophilic inflammation or edema.

[00605] In the case of SEQ-2, inflammation with cell infiltration, edema and crust was observed one day and one week after administration. Even four weeks after administration, the skin inflammation had not yet recovered, and granulomatous inflammation

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174/187 by continuous monocyte / macrophage activation was observed.

[00606] In the case of SEQ-49, and SEQ-61, SEQ-119, SEQ-121, SEQ170, SEQ-192 and SEQ-216, which are an adjunct to the present invention, acute inflammation, mainly neutrophilic infiltration, was observed under the skin one day after administration. One week after the administration, inflammation, mainly with lymphocytes and macrophages, was observed from the subcutaneous tissue to the dermis and, four weeks after the administration, the inflammation had almost recovered. Regarding SEQ-119, SEQ-121 and SEQ-170, the assessment of subcutaneous irritation four weeks after administration was not performed, however when their assessments of 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 was the same or weaker than that caused by SEQ-61. This suggested that, in relation to SEQ-119, SEQ-121 and SEQ-170, the inflammation was almost completely recovered four weeks after administration.

[00607] Tables 70 to 72 showed results from one day after administration, Tables 73 to 75 showed results from one week after administration and Tables 76 and 77 showed results from four weeks after administration. The observations in the tables are as follows.

1) The values in the findings are numbers of the animals observed.

2) The values in the findings are scores. The scores were calculated in total, provided that the degrees of each individual had been Minimum: 1, Light: 2 and Moderate: 3.

[00608] The precedent showed that, in the comparison between adjuvants of the present invention and Montanide, the adjuvants of the present invention did not cause the accumulation of Compounds under the skin (low

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175/187 risk of hardening), necrosis or neutrophilic infiltration did not occur even one week after administration and the inflammation had recovered almost completely four weeks after administration. Therefore, it is concluded that the subcutaneous irritation of the adjuvants of the present invention was weaker than that of Montanide. Furthermore, in the comparison between adjuvants of the present invention and Amph1826 (SEQ-2), the inflammation caused by the adjuvants of the present invention had recovered almost completely four weeks after administration, but, on the other hand, the skin inflammation caused by SEQ -2 had not recovered, and granulomatous inflammation by continuous activation of the monocyte / macrophage system was observed even four weeks after administration. Therefore, it is concluded that the subcutaneous irritation of the adjuvants of the present invention was weaker than that of SEQ-2. Subcutaneous irritation of adjuvants of the present invention showed no significant difference from that of QDN2006 (SEQ-49).

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Table 70

Compound name Montanide SEQ-49 SEQ-61 Dose (nmol / local) - 4.71 15.7 47.1 4.71 15.7 47.1 Number of animals 3 3 3 3 3 3 3 Number of imminent deaths / dissections 0/0 0/0 0/0 0/0 0/0 0/0 0/0 Pathology Administration location Macroscopy ¹ * Subcutaneous: White buildup 3 0 0 0 0 0 0 Subcutaneous: Red spots, erythema 0 0 0 1 0 0 0 Histopathology Subcutaneous: Neutrophilic infiltration ² * 3 6 6 6 5 6 6 Subcutaneous: Edema ² * 3 4 5 5 2 3 5 Subcutaneous: Bleeding ² * 2 2 1 2 1 0 1 Subcutaneous: cyst-like structure ¹ * 3 0 0 0 0 0 0 Muscle layer: Neutrophilic infiltration ² * 0 4 6 5 4 5 5 Muscle layer: Edema ² * 0 2 1 0 3 2 3

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Table 71

Compound name SEQ-192 SEQ-216 SEQ-2 Dose (nmol / local) 4.71 15.7 47.1 4.71 15.7 47.1 4.71 15.7 47.1 Number of animals 3 3 3 3 3 3 3 3 3 Number of imminent deaths / dissections 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 Pathology Administration location Histopathology ² ' Subcutaneous tissue Infiltration by inflammatory cells ² ' 6 5 5 6 6 5 7 6 6 Edema ² ' 7 6 4 5 7 4 3 5 6 Bleeding ² ' 2 1 1 3 1 1 0 3 3 Necrosis ² ' 1 0 1 1 0 0 1 1 1 Cyst-like structure ¹ ' 2 1 2 2 3 0 1 2 1 Dermis Infiltration by inflammatory cells ² ' 6 6 6 7 7 6 5 9 6 Muscle layer Infiltration by inflammatory cells ² ' 2 3 4 3 6 2 4 7 6 Edema ² ' 1 2 3 2 2 1 1 3 4 Necrosis ² ' 0 0 0 0 0 1 0 0 0

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Table 72

Compound name SEQ-121 SEQ-170 SEQ-119 Dose (nmol / local) 4.71 15.7 47.1 4.71 15.7 47.1 4.71 15.7 47.1 Number of animals 3 3 3 3 3 3 3 3 3 Number of imminent deaths / dissections 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 Pathology Administration location Macroscopy ¹⁾ Turbidity 0 0 0 1 1 0 1 0 2 And rite ma 0 0 0 1 2 2 0 0 0 Histopathology ²⁾ Subcutaneous tissue Infiltration by inflammatory cells ²⁾ 5 5 6 3 6 8 3 4 6 Edema ²⁾ 9 8 5 6 5 7 5 5 6 Dermis Infiltration by inflammatory cells ²⁾ 1 4 6 1 0 5 1 2 6 Muscle layer Infiltration by inflammatory cells ² ) 2 5 6 3 5 6 1 2 4

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Table 73

Compound name Montanide SEQ-49 SEQ-61 Dose (nmol / local) - 4.71 15.7 47.1 4.71 15.7 47.1 Number of animals 3 3 3 3 3 3 3 Number of imminent deaths / dissections 0/0 0/0 0/0 0/0 0/0 0/0 0/0 Pathology Administration location Macroscopy ¹⁾ Subcutaneous: White nodules 3 0 0 0 0 0 0 Epidermis: Crust 0 1 1 0 1 0 0 Histopathology Subcutaneous: Necrosis ² ' 2 0 0 0 0 0 0 Subcutaneous: ² 'neutrophilic infiltration 6 0 0 0 0 0 0 Subcutaneous: Infiltration by monocytes ² ' 0 3 6 8 2 3 7 Subcutaneous: Granulomatous inflammation ² ' 7 0 0 0 0 0 0 Subcutaneous: Edema ² ' 5 4 6 5 4 5 6 Subcutaneous: Cyst-like structure ¹ ' 3 0 0 0 0 0 0

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Petition 87Ο29ΟΦ3Θ0Η2, of 10 / Φ3 / 2Ο29, p. 180/220

Table 74

Compound name SEQ-192 SEQ-216 SEQ-2 Dose (nmol / local) 4.71 15.7 47.1 4.71 15.7 47.1 4.71 15.7 47.1 Number of animals 3 3 3 3 3 3 3 3 3 Number of imminent deaths / dissections 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 Pathology Administration location Macroscopy ¹⁾ White matter accumulation 0 0 0 0 0 0 0 0 0 Dark red dots 0 0 0 0 0 1 0 0 0 Edema 0 0 0 0 1 0 0 1 1 Crust 0 1 1 0 1 2 0 0 0 Histopathology ² ) Subcutaneous Granulomatous inflammation ²⁾ 0 0 0 0 0 0 0 0 0 Infiltration by inflammatory cells ² ) 4 6 7 3 6 5 8 8 Edema ²⁾ 3 3 2 1 2 2 3 4 3 Necrosis ² ) 0 1 3 0 0 2 0 0 0 Cyst-like structure ¹ ) 0 1 2 2 2 1 1 1 3 Dermis Infiltration by inflammatory cells ² ) 2 5 7 0 5 8 4 6 9 Muscle layer Infiltration by inflammatory cells ² ) 0 5 3 0 4 4 0 4 6

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Petition 870290039082, of 10 / Φ3 / 2Ο29, p. 185/220

Table 75

Compound name SEQ-121 SEQ-170 SEQ-119 Dose (nmol / local) 4.71 15.7 47.1 4.71 15.7 47.1 4.71 15.7 47.1 Number of animals 3 3 3 3 3 3 3 3 3 Number of imminent deaths / dissections 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 Pathology Administration location Macroscopy ¹⁾ Turbidity 0 0 0 1 1 0 1 0 2 And rite ma 0 0 0 1 2 2 0 0 0 Histopathology ²⁾ Subcutaneous Infiltration by inflammatory cells ²⁾ 5 5 6 3 6 8 3 4 6 Dermis Infiltration by inflammatory cells ²⁾ 1 4 6 1 0 5 1 2 6 Muscle layer Infiltration by inflammatory cells ² ) 2 5 6 3 5 6 1 2 4

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Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 180/220

Table 76

Compound name Montanide SEQ-49 SEQ-61 Dose (nmol / local) - 4.71 15.7 47.1 4.71 15.7 47.1 Number of animals 3 3 3 3 3 3 3 Number of imminent deaths / dissections 0/0 0/0 0/0 0/0 0/0 0/0 0/0 Pathology Administration location Macroscopy ¹ ' Subcutaneous: White nodules 3 0 0 0 0 0 0 Histopathology Muscle layer: monocyte infiltration ² ' 0 0 0 0 0 0 1 Subcutaneous: ² 'neutrophilic infiltration 3 0 0 0 0 0 0 Subcutaneous: ² 'granulomatous infiltration 6 0 0 0 0 0 0 Subcutaneous: Edema ² ' 1 0 0 0 0 0 0 Subcutaneous: Cyst-like structure accompanied by formation of fibrous capsule ¹ ' 3 0 0 0 0 0 0

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Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 183/220

Table 77

Compound name SEQ-192 SEQ-216 SEQ-2 Dose (nmol / local) 4.71 15.7 47.1 4.71 15.7 47.1 4.71 15.7 47.1 Number of animals 3 3 3 3 3 3 3 3 3 Number of imminent deaths / dissections 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 Pathology Administration location Macroscopy ¹⁾ White matter accumulation 0 0 0 0 0 0 0 0 0 Crust 0 0 0 0 1 1 0 0 0 Histopathology ²⁾ Subcutaneous Granulomatous inflammation ² ) 0 0 0 0 0 0 4 5 7 Infiltration by inflammatory cells ² ) 0 0 0 0 0 0 0 0 0 Edema ²⁾ 0 0 0 0 0 0 0 0 0 Fibrosis ² ) 0 0 0 0 1 0 0 0 Cyst-like structure ¹ ) 0 0 0 0 0 0 0 0 0 Dermis Infiltration by inflammatory cells ² ) 0 1 0 0 1 0 0 4 7

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B) Intradermal irritation test with a single dose [00609] Subcutaneous irritation by adjuvants of the present invention of the present invention is assessed by microscopic and histopathological examination of the skin, at necropsy, one day, one and four weeks after subcutaneous administration of a Single dose.

[00610] An adjuvant of the present invention is administered subcutaneously to the skin of rats. The following procedure is carried out in a method similar to that of Example 5).

Example 6 Evaluation of the ability to induce antibody production of the adjuvant of the present invention in immunization by vaccine with PCRV antigen from Pseudomonas aeruginosa [00611] When the antigen and adjuvant are administered, as in a vaccine for infectious disease, and induced production of antibodies by B lymphocytes in the immunized animal, the prevention and treatment effect for the disease is expected. Therefore, in the expectation of applying this adjuvant for a vaccine against an infectious disease, production by B lymphocytes was evaluated. The PCRV antigen, which is from Pseudomonas aeruginosa, was used for the examination as an antigen. There are already reports that the PCRV antigen of Pseudomonas aeruginosa has a vaccine effect (Moriyama et al., Infect. Immun., Sep. 2001, vol. 69, no. 9, 5908-5910) and, therefore, the induction effect of antibody production by the adjuvant was evaluated in comparison to the group to which the PCRV antigen was administered. Freund's adjuvant was used for a control group. Freund's adjuvant is an adjuvant that eliminated the tuberculosis bacillus by heat treatment, is mixed in oil that consists of paraffin oil and mannan monooleate and is known to strongly induce the production of antibodies in non-clinical studies. Freund's adjuvant has a strong effect in non-clinical studies, but its use in clinical studies has been banned due to concerns about side effects (The European Agency for

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Evaluation of Medicinal Products for Human Use, 25 March 2004, Internet (URL: http://www.ema.europa.eu/docs/en__GB/ documentjibrary / Scientific_guideline / 2009/11 / WC500015469.pdf)).

The Pcrv nucleotide sequence (SEQ ID NO: 48), derived from the PAO1 strain of Pseudomonas Aeruginosa was cloned at the Ndel-Xhol site in the vector pET21a, and the BL21 strain of Escherichia coli (DE3) was transformed with the prepared plasmid . The pcrV-BL21 (DE3) strain was grown in 500 mL of LB / Ampicillin liquid medium at 37 ° C, and 200 µL of 0.1M IPTG was added when OD600 reached 0.5 to induce the expression of the recombinant PCRV protein. After culture at 16 ° C for 18 hours, the bacteria were separated by centrifugation, and 15 mL of Buffer A (50 mM Tris-HCI (pH8.0), 0.2 M NaCI) comprising 0.5% lysozyme (SIGMA Corporation) have been added. After 30 minutes at 4 ° C, ultrasonic treatment was performed. After centrifugation at 12,000 rpm for 10 minutes, the soluble fraction was obtained. 1 ml of Ni-NTA agarose gel (QIAGEN) was added to the Poliprep column (BIO-RAD Laboratories, Inc) which was equilibrated with 10 ml of Buffer A. The 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. The 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 the recombinant PCRV protein with HIS at the C end. (SEQ ID NO: 49) was purified. The resulting recombinant PCRV protein had its concentration measured with the BCA assay kit (PIERCE) and was maintained at -80 ° C.

[00613] 70 pg of recombinant PCRV protein and 33 nmol of an adjuvant of the present invention were mixed with saline to obtain 0.7 ml. 0.1 ml of the immunogen was injected intradermally on Day 0, 7 and 27 in each of the 5 mice A / J fePetição 870290 (030082, 10 / Φ3 / 2Ο29, p. 190/220

186/187 5 week old females (Japan SLC, Inc.). As a control, only the 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 obtain 0.5 ml, and the emulsion was prepared with the same amount of complete Freund's adjuvant (Difco Laboratories). 0.1 mL was injected intradermally on Day 7 as an initial immunization. On Day 27, Freund's incomplete adjuvant (Difco Laboratories) was used for immunization in a similar method. On Day 33, blood was collected with heparin from a vein in the mice's tail, which was centrifuged at 6,000 rpm for 15 minutes. The supernatant was collected as an antiserum and kept at -40 ° C.

[00614] The antibody titer was measured as a stroke. Recombinant PCRV protein was mixed with PBS (pH7.4) (Invitrogen) until it reached 1 pg / mL, and 20 pL / well of the solution was added to the 384-well Nunc Maxisorp plate (Thermo). The plate was closed with a plate seal and incubated at 4 ° C overnight. After washing twice with 90 pL / well of Wash Buffer (NaCI 9 g / L, Proclin 1500.5 g / L Tween-20 0.1 g / L), 60 pL / well of 1x Assay Buffer (Invitrogen) have been added. The plate was closed with a plate seal and the block was performed at room temperature for 2 hours. After removing the liquid by tapping, the antiserum was diluted with 1x Assay Buffer 10 ³ to 10 ⁷ times and 20 µl / well solution was added. The plate was closed with a plate seal and incubated at 4 ° C overnight. After washing three times with Wash Buffer, the goat anti-IgG-Fc-HRP mouse antibody (JacksonlmmunoResearch) was diluted 20,000 with 1x Assay Buffer and 20 µl / well solution was added. The plate was closed with a plate seal and incubated at room temperature for 2 hours. After washing three times with Wash Buffer,

Petition 870190Φ3ΘΒ82, of 10 / Φ3 / 2Ο29, p. 19T / 22B

187/187 pL / well of TMB substrate (Dako) were 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 the absorbance at 450 nm was measured with a plate reader. The dilution ratio when the absorbance at 450 nm is 1.0 was calculated and considered as the antibody titer. The results were shown in Figure 15.

[00615] The antibody titers were 3.0x10 ⁶ for SEQ-121, which is the highest, 2.5 * 10 ⁶ for Freund's adjuvant, 7.1 × 10 ⁵ for SEQ61 and 5.3x10 ⁵ for ODN2006 (SEQ -49) in that order. The antibody titer for PCRV protein alone was 1.3 x 10 ⁵ . These results suggested that the adjuvants of the present invention have strong immunostimulatory activity against B cells. In particular, the antibody titer for SEQ-121 revealed a greater ability to induce antibody production than QDN2006 and almost the same antibody titer as the Freund. Therefore, it has been suggested that the adjuvants of the present invention are excellent as an adjuvant for infectious disease vaccine. From these results, adjuvants of the present invention are expected to apply to a vaccine against infectious disease.

Industrial applicability [00616] As was clear from the examples above, the lipid-bound double-stranded oligonucleotides of the present invention show excellent immunostimulatory activity. Therefore, they are very useful especially as an adjuvant to increase the vaccine effect.

Claims (16)

1. Pharmaceutical composition, characterized by the fact that it comprises a double-stranded oligonucleotide in which a first strand consists only of a CpG oligonucleotide consisting of 8 to 50 nucleotides, a second strand is an oligonucleotide consisting of 8 to 60 nucleotides and which comprises a sequence capable of hybridizing to the first strand and 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) attaches to the second tape through a binder. 2. Pharmaceutical composition comprising a double-stranded oligonucleotide according to claim 1, characterized in that the second-stranded oligonucleotide is an oligonucleotide consisting of DNA nucleosides and / or nucleoside derivatives. 3. Pharmaceutical composition comprising a double-stranded oligonucleotide according to claim 2, characterized by the fact that: the nucleoside derivative is a nucleoside that has a substituent at the 2 'position of the sugar and / or a nucleoside that has a bridge structure between the 4' and 2 'positions of the sugar. 4. Pharmaceutical composition comprising a double-stranded oligonucleotide according to claim 3, characterized in that the bridge structure between the 4 'and 2' positions of the sugar is 4 '- (CH2) mO-2', where m is an integer from 1 to 4. Pharmaceutical composition comprising a double-stranded oligonucleotide according to any one of claims 1 to 4, characterized in that the lipid is a diacyl-lipid. Petition 870190Φ3ΘΒ82, of 10 / Φ3 / 2Ο29, p. 199 / 22B 2/16 6. Pharmaceutical composition comprising a double-stranded oligonucleotide according to claim 5, characterized by the fact that the diacyl-lipid is

where p and q are each independently an integer from 10 to 28. Pharmaceutical composition comprising a double-stranded oligonucleotide according to any one of claims 1 to 6, characterized in that the lipid binds to the 3 'end and / or the 5' end of the second strand. Pharmaceutical composition comprising a double-stranded oligonucleotide according to any one of claims 1 to 7, characterized in that the linker is an oligonucleotide linker. Pharmaceutical composition comprising a double-stranded oligonucleotide according to claim 8, characterized in that the linker is - (dX ¹ ) u-, where X ¹ is each independently A, G, C or T, eu is an integer from 1 to 8. Pharmaceutical composition comprising a double-stranded oligonucleotide according to any one of claims 1 to 9, characterized in that the CpG oligonucleotide is Petition 87Ο29ΟΦ3Θ0Η2, of 10 / Φ3 / 2Ο29, p. 290/220 3/16 is an oligonucleotide consisting of the sequence of SEQ ID NO: 13. 11. Pharmaceutical composition, characterized by the fact that it comprises a double-stranded oligonucleotide consisting of a first strand and a second strand, in which

and the second ribbon is a ribbon represented by the formula selected from the following: the formula: Petition 870290039082, of 10 / Φ3 / 2Ο29, p. 295/220 4/16 OH -------- -------------- P = S

(S-28) the formula: Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 290/220 5/16 HO I OH I P = S

(S-29) the formula: Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 293/220 6/16 OH

(S-58) the formula: Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 298/220 7/16

the formula: Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 299/220 8/16

(S-80) the formula: Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 200/220 9/16 ο

the formula: Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 20T / 22S 10/16

HO

(S-93) the formula: Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 208/220 11/16 HO I P = S

(S-136) the formula: Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 209/220 12/16 OH

the formula: Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 2/220 13/16

the formula: Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 2/5/220 14/16

and the formula: Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 2Φ0 / 220 15/16

Pharmaceutical composition according to any one of claims 1 to 11, characterized in that it further comprises an antigen. 13. Pharmaceutical composition according to claim Petition 87Ο29ΟΦ3Θ082, of 10 / Φ3 / 2Ο29, p. 2/3/220 16/16 12, characterized by the fact that the antigen is a microbial antigen, a self-antigen or an addictive substance. 14. Pharmaceutical composition according to claim 13, characterized by the fact that the microbial antigen is a bacterial antigen, a viral antigen or a parasitic antigen. 15. Pharmaceutical composition according to claim 13, characterized by the fact that the autoantigen is a tumor antigen, an antigen associated with Alzheimer's disease, an antigen against a human antibody or an antigen that is expressed from endogenous retroviral elements humans. 16. Pharmaceutical composition according to claim 13, characterized by the fact that the addictive substance is nicotine or cocaine.