Medicine for treating HIV infection

Abstract

The purpose of the present invention is to provide a medicine useful for treating HIV infection by using compounds having different mechanisms in combination with kick and kill therapy. A medicine characterized by being a combination of (A) a polynucleotide or polypeptide that induces an anti-HIV Tat antibody that inhibits the activity of HIV Tat protein secreted from HIV-infected cells, and (B) a pattern recognition receptor agonist that induces type I IFN, is useful for the treatment, etc., of HIV infection.

Description

Medical drugs for the treatment of HIV infection

[0001] The present invention relates to a pharmaceutical agent for the treatment of human immunodeficiency virus (HIV) infection. More specifically, the present invention relates to a pharmaceutical agent for the treatment of HIV infection, characterized by combining (A) a polynucleotide or polypeptide that induces an anti-HIV Tat antibody that inhibits the activity of the HIV Tat protein secreted from HIV-infected cells, and (B) a pattern recognition receptor (PRR) agonist that induces type I interferon (IFN).

[0002] Since the development of combination anti-retroviral therapy (cART), the mortality rate from HIV infection has decreased significantly, and with continued cART, the average life expectancy is now almost the same as that of healthy individuals. However, a cure for HIV infection has not yet been established, and lifelong medication is necessary. For this reason, various approaches are being used in non-clinical and clinical research aimed at curing HIV infection, but to date, with the exception of bone marrow and umbilical cord blood transplants for patients with concomitant hematological cancers, no treatment has succeeded in curing HIV infection (Non-patent Literature 1). However, the "Kick (Shock) & Kill" therapy is the most promising treatment for curing HIV infection in patients who do not meet these prerequisites.

[0003] The biggest reason why a complete cure for HIV infection is difficult is that the HIV DNA genome can be inserted into human chromosomes, allowing the virus to lie dormant as a provirus without expressing viral proteins. "Kick & Kill" therapy is a method that reactivates (Kick) these HIV-infected cells to promote the expression of viral genes, and then eliminates (Kill) the viral protein-producing cells through the host immune system. The Kick agent (reactivator) in "Kick & Kill" therapy is called an LRA (Latency-Reversing Agent), and various drugs with different mechanisms of action have been reported, such as histone deacetylase (HDAC) inhibitors and protein kinase C (PKC) agonists. HDAC inhibitors, which had been developed earlier in the field of cancer, were the first to enter clinical trials in the field of HIV infection cure. While some clinical trials of HDAC inhibitors alone have observed reactivation of HIV-latent infected cells, they did not show a delay in viral rebound (generally meaning the detection of HIV RNA twice consecutively after virological suppression) or a significant reduction in the size (quantity) of the HIV reservoir (latent infected cells) (Non-Patent Documents 2, 3). As a LRA, AZD5582, an apoptosis inhibitor protein (IAPi) inhibitor, is expected to have a strong ability to reactivate HIV-latent infected cells, but it has not yet entered clinical trials (Non-Patent Documents 4, 5).

[0004] Among the drugs used in Kick & Kill therapy, various TLR (Toll-like receptor) agonists, which are pattern recognition receptor agonists, are considered to be the most promising. This is because TLR agonists are thought to indirectly possess Kick activity by producing IFNα (interferon α), and at the same time, they are thought to activate host immune cells and enhance Kill activity by inducing the production of various cytokines. Among TLR agonists, two drugs have progressed to clinical trials: the TLR7 agonist besatrimod (GS-9620) and the TLR9 agonist refitrimod. However, neither drug could suppress or delay viral rebound when used as a single agent (Non-Patent Literature 6, 7). Therefore, both drugs have been tested or are currently being tested in combination with Gag-conserved element (CE)-targeted DNA + IL-12 prime / MVA-boosted vaccines and broad-spectrum HIV neutralizing antibodies (bNAbs) that target HIV Env (envelope glycoprotein), with the expectation that they will enhance kill activity. However, to date, no cases of complete cure for HIV infection have been reported.

[0005] bNAb can inhibit new HIV infections by binding to HIV Env, and furthermore, it is expected to enhance kill activity by binding to Env presented or budding on MHC on the surface of infected cells, and by the binding of the Fc domain of bNAb to the Fc receptor on NK cells, thereby activating antibody-dependent cell-mediated cytotoxicity (ADCC). Non-patent document 8 describes that in a monkey model infected with SIV undergoing multidrug combination therapy, combination treatment with the TLR7 agonist besatrimod and the bNAb PGT121 maintained the blood viral load below the detection limit in some monkeys even after discontinuation of cART. This monkey model is an acute SIV infection model, which is a chimeric virus of HIV and SIV, and is a model in which the virus is easily eliminated from the body (Non-patent document 11), and is different from the chronic SIV infection model described in the present invention.

[0006] Non-Patent Document 10 and Patent Document 1 describe that administration of Tat peptide vaccine alone to patients during cART treatment has an effect of slowly reducing the size of the HIV reservoir. On the other hand, by estimating the attenuation rate of HIV-1 DNA using a random effects regression model, it is described that the time required to eradicate the HIV reservoir is estimated to be 83 years in the entire population administered with the Tat peptide vaccine, and the cure of HIV infection has not been achieved.

[0007] WO 2011 / 113618 WO 2017 / 057540 WO 2018 / 179172 WO 2025 / 063261 (Japanese Patent Application No. 2023-158238)

[0008] N Engl J Med. 2009 Feb 12;360(7):692-8 J Virus Erad 2020; 6:100004 Lancet HIV. 2014 Oct;1(1):e13-21 Nature. 2020 Feb;578(7793):160-165 Nat Med. 2023 Oct;29(10):2535-2546 Sci Transl Med 2021; 13:eabg3071 AIDS. 2019 Jul 1;33(8):1315-1325 Nature. 2018 Nov;563(7731):360-364 Nat Med. 2023 Oct;29(10):2547-2558 J Virus Erad. 2020 Sep; 6(3): 100004. PLoS One. 2011 Mar 25;6(3): e17965.

[0009] An object of the present invention is to provide a medicament useful for the treatment of HIV infection by combining a compound having a different mechanism with Kick&Kill therapy.

[0010] As a result of diligent research, the present inventors have found that administering (A) a polynucleotide or polypeptide that induces anti-HIV Tat antibodies that inhibit the activity of HIV Tat protein secreted from HIV-infected cells (hereinafter also referred to as "the HIV Tat activity inhibitor of the present invention"), in addition to (B) a compound such as a pattern recognition receptor agonist that induces type I IFN (hereinafter also referred to as "the pattern recognition receptor agonist of the present invention"), to HIV-infected patients may be useful in treating HIV infection. In other words, the present inventors have found that the pattern recognition receptor agonist of the present invention is useful as a kicker for latently infected HIV cells. Furthermore, we have found that the HIV Tat activity inhibitor of the present invention inhibits Tat-induced apoptosis in immune cells such as CTLs and NK cells activated by the pattern recognition receptor agonist of the present invention (hereinafter, this action will also be referred to as "Disarm"), thereby enabling immune cells to more effectively eliminate (Kill) HIV-infected cells and potentially leading to more effective treatment of HIV infection. This is the first instance suggesting that combining an HIV Tat activity inhibitor with "Kick & Kill" therapy alone can show superior therapeutic effects in the treatment of HIV infection where the therapeutic effect is insufficient.

[0011] Non-patent document 9 describes a study in which HIV-infected patients undergoing cART treatment were given lephyrimod, a TLR9 agonist, twice, followed by discontinuation of cART. The study then administered two types of bNAbs (3BNC117 and 10-1074) along with lephyrimod, and found that the delayed viral rebound caused by bNAb administration as a cART substitute was not enhanced by lephyrimod. In this document, lephyrimod is used to induce latent infected cells as LRAs and regrow the virus (Kick), while bNAbs are used to suppress new HIV infections and enhance or support the elimination of infected cells (Kill). In other words, the study describes the treatment of HIV infection using Kick & Kill therapy, but it neither suggests nor describes the administration of compounds with disarm activity in addition to Kick & Kill therapy.

[0012] Patent Documents 2 and 3 describe a "double-stranded oligonucleotide in which the first strand is a CpG oligonucleotide, the second strand is an oligonucleotide containing a sequence capable of hybridizing to the first strand, and a lipid is bound to the second strand", which is included in the pattern recognition receptor agonist of the present invention. It is described that when the double-stranded oligonucleotide is administered together with a cancer antigen peptide, the induction rate of antigen-specific CTL (cytotoxic T cells) by the vaccine is improved, showing strong antitumor activity. Furthermore, it is described that the double-stranded oligonucleotide exhibits immunostimulatory activity against B cells as an adjuvant for infectious disease vaccines. On the other hand, no use of the double-stranded oligonucleotide as a Kick agent is mentioned at all.

[0013] In other words, the present invention relates to the following: (1-1) A pharmaceutical for the treatment of HIV infection (hereinafter also referred to as "the pharmaceutical of the present invention") characterized by combining (A) a polynucleotide or polypeptide that induces an anti-HIV Tat antibody that inhibits the activity of the HIV Tat protein secreted from HIV-infected cells; and (B) a pattern recognition receptor agonist or an apoptosis inhibitor protein (IAPi) that induces type I IFNs. (1-2) The pharmaceutical according to (1-1), wherein (B) is a pattern recognition receptor agonist that induces type I IFNs. (2-1) The pharmaceutical according to (1-1) or (1-2), wherein (B) is (B-1) or (B-2). (B-1) A double-stranded oligonucleotide in which the first chain consists only of 8-50 base CpG oligonucleotides, the second chain is an oligonucleotide in which an 8-60 base DNA nucleoside containing a hybridizable sequence is bound to the first chain, the length of the second chain is 50% or more of the length of the first chain, and a lipid containing a hydrocarbon chain having 12-30 carbon atoms is bound to the second chain via or without a linker. (B-2) At least one compound selected from the group consisting of TLR3 agonists, TLR7 agonists, TLR9 agonists, and STING ligands. (2-2) The pharmaceutical product according to (2-1), wherein (B-2) is at least one compound selected from the group consisting of Poly I:C, PolyI:PolyC12U (Rintatolimod), Poly IC:LC (Oncovir), RGC100, ARNAX, ODN2006, lephytrimod, besatrimod, and tylsotrimod STING ligand. (2-3) The pharmaceutical product according to (2-1) or (2-2), wherein (B-2) is at least one compound selected from the group consisting of ODN2006, lephytrimod, besatrimod, tylsotrimod, and STING ligand. (2-4) The pharmaceutical product according to (1-1) or (1-2), wherein (B) is AZD5582. (3) (B) is (B-1), where (B-1) is a double-stranded oligonucleotide consisting of a first strand and a second strand, and the first strand is a strand represented by the following formula (an oligonucleotide consisting of the base sequence described in SEQ ID NO: 239): Formula: A pharmaceutical product according to any one of (2-1) to (2-4), wherein the second chain is a double-stranded oligonucleotide whose second chain is represented by the following formula (I) (an oligonucleotide consisting of the base sequence described in SEQ ID NO: 240, which includes a branched lipid at the 5' end). Formula (I): (4-1) The pharmaceutical product according to any one of (1-1) to (3), wherein (A) is a polynucleotide encoding HIV Tat protein or its antigenic fragment, or HIV Tat protein or its antigenic fragment. (4-2) The pharmaceutical product according to (4-1), wherein (A) is a polynucleotide encoding HIV Tat protein, or HIV Tat protein. (5) The pharmaceutical product according to (4-1) or (4-2), wherein a secretory signal peptide is further linked to the HIV Tat protein. (6-1) The pharmaceutical product according to (5), wherein the secretory signal peptide is a polypeptide consisting of the amino acid sequence described in any one of SEQ ID NOs: 161 to 167 and 183 to 187. (6-2) The pharmaceutical product according to any one of (4-1) to (6-1), wherein a functional sequence is further linked to the HIV Tat protein. (7-1) The pharmaceutical product according to any one of (4-1) to (6-2), wherein the HIV Tat protein is a polypeptide consisting of an amino acid sequence identical to or having 90% or more identity with the amino acid sequence described in any of SEQ ID NOs. 131 to 148 and 182. (7-2) The pharmaceutical product according to (7-1), wherein the HIV Tat protein is a polypeptide consisting of an amino acid sequence identical to or having 90% or more identity with the amino acid sequence described in SEQ ID NOs. 133 or 134.(8-1) (A) is mRNA containing an open reading frame encoding the HIV Tat protein or its antigenic fragment, and is one of the following: 5-methyluridine, pseudouridine, N1-methylpseudridine, 5-methoxyuridine, 2-thiouridine, 6-methyladenosine, 2-aminoadenosine, inosine, 5-methylcytidine, N1-ethylpseudridine, 4-thiouridine, 2-thio-1-methyl-1-deazapseudridine, 2-thio-1-methylpseudridine, 2-thio-5-a A pharmaceutical product according to any one of (1-1) to (7-2), wherein the mRNA contains at least one modified base selected from the group consisting of za-uridine, 2-thio-dihydropsuduridine, 2-thio-dihydropsuduridine, 2-thio-psuduridine, 4-methoxy-2-thio-psuduridine, 4-methoxypsuduridine, 4-thio-1-methylpsuduridine, 4-thio-psuduridine, 5-aza-uridine, dihydropsuduridine, and 2'-O-methyluridine. (8-2) A pharmaceutical product according to (8-1), wherein the modified base is at least one modified base selected from the group consisting of 5-methyluridine, N1-methylpsuduridine, and 5-methylcytidine. (8-3) The pharmaceutical product according to (8-1) or (8-2), wherein the mRNA comprises a 5' Cap structure, a 5' UTR, an open reading frame encoding the HIV Tat protein or an antigenic fragment thereof, a 3' UTR, and Poly(A). (9-1) The pharmaceutical product according to any one of (8-1) to (8-3), wherein the mRNA comprises a 5' Cap structure which is Cap1 or Cap2. (9-2) The pharmaceutical product according to (9-1), wherein the 5' Cap structure is Cap1. (10-1) The pharmaceutical product according to any one of (8-1) to (9-2), wherein the mRNA comprises 60 to 120 mers of Poly(A). (10-2) The pharmaceutical product according to (10-1), wherein the Poly(A) is 80 mers. (11-1) A pharmaceutical product according to any one of (8-1) to (10-2), wherein the mRNA contains a 5'UTR which is a polynucleotide consisting of a base sequence identical to or having 90% or more identity with the base sequence described in any of SEQ ID NOs: 1 to 63 and 211 to 218.(11-2) The pharmaceutical product according to (11-1), wherein the 5'UTR is a polynucleotide consisting of a base sequence identical to or having 90% or more identity with the base sequence described in any of SEQ ID NOs. 11-3) The pharmaceutical product according to (11-2), wherein the 5'UTR is a polynucleotide consisting of a base sequence identical to or having 90% or more identity with the base sequence described in any of SEQ ID NOs. 12-1) The pharmaceutical product according to any of (8-1) to (11-3), wherein the mRNA contains a 3'UTR which is a polynucleotide consisting of a base sequence identical to or having 90% or more identity with the base sequence described in any of SEQ ID NOs. 64 to 112. (12-2) The pharmaceutical product according to (12-1), wherein the 3'UTR is a polynucleotide consisting of a base sequence identical to or having 90% or more identity with the base sequence described in any of SEQ ID NOs. 64 to 69. (13-1) A pharmaceutical product according to any one of (8-1) to (12-2), wherein the open reading frame is a polynucleotide consisting of any of the base sequences described in SEQ ID NOs. 114-119, 121-126, and 198-210. (13-2) A pharmaceutical product according to (13-1), wherein the open reading frame is a polynucleotide consisting of any of the base sequences described in SEQ ID NOs. 121, 198, 200, and 202. (13-3) A pharmaceutical product according to any one of (8-1) to (13-2), wherein the mRNA is mRNA defined in any of SNG-299, 323, 325, and 327 (i.e., mRNA described in Table 1 of Example 2). (14) A pharmaceutical product according to any one of (1-1) to (13-3), wherein (A) and (B) are administered in combination. (15) A pharmaceutical product for use in combination with (B) according to (1-1), comprising (A) according to (1-1) as an active ingredient. (16) A pharmaceutical product containing (B) described in (1-1) as an active ingredient, for use in combination with (A) described in (1-1).

[0014] (17) A pharmaceutical agent for the treatment of HIV infection, characterized in that the target of administration is a patient in whom the viral load of HIV RNA in the blood or plasma is maintained below the detection limit, and comprising: (A) a polynucleotide or polypeptide that induces an anti-HIV Tat antibody that inhibits the activity of HIV Tat protein secreted from HIV-infected cells; and (B) a pattern recognition receptor agonist or IAPi that induces type I IFN; (18) The pharmaceutical agent according to (17), wherein the viral load of HIV RNA is less than 50 copies per 1 mL of blood or plasma. (19) The pharmaceutical agent according to (18), wherein the viral load of HIV RNA is less than 20 copies per 1 mL of blood or plasma.

[0015] (20) A pharmaceutical product according to any one of (1-1) to (19), wherein the target recipient is an HIV-infected patient taking an anti-HIV drug. (21) The pharmaceutical product according to (20), wherein the anti-HIV drug comprises one or more compounds selected from the group consisting of HIV integrase inhibitors, HIV reverse transcriptase nucleotype inhibitors, HIV reverse transcriptase non-nucleotype inhibitors, HIV protease inhibitors, HIV capsid inhibitors, HIV non-catalytic site integrase inhibitors, entry inhibitors, bNAbs, and pharmaceutically acceptable salts thereof. (That is, a pharmaceutical product, wherein the target recipient is an HIV-infected patient receiving combination therapy.) (22) The pharmaceutical product according to (21), wherein the anti-HIV drug comprises one or more compounds selected from the group consisting of HIV integrase inhibitors, HIV reverse transcriptase nucleotype inhibitors, and pharmaceutically acceptable salts thereof. (23) The pharmacopoeciliste according to (22), wherein the HIV integrase inhibitor is one or more compounds selected from the group consisting of dolutegravir, bictegravir, cabotegravir and pharmaceutically acceptable salts thereof. (24) The pharmacopoeciliste according to (22) or (23), wherein the nucleoside inhibitor of HIV reverse transcriptase is one or more compounds selected from the group consisting of tenofovir, abacavir, emtricitabine, lamivudine, islatravir and their prodrugs or pharmaceutically acceptable salts thereof. (25) The pharmacopoeciliste according to (24), wherein the nucleoside inhibitor of HIV reverse transcriptase is one or more compounds selected from the group consisting of tenofovir, abacavir and pharmaceutically acceptable salts thereof, and one or more compounds selected from the group consisting of emtricitabine, lamivudine and pharmaceutically acceptable salts thereof. (26) A pharmaceutical product according to any one of (21) to (25), wherein the anti-HIV drug is dolutegravir, tenofovir, and emtricitabine. (27) A pharmaceutical product according to any one of (21) to (24), wherein the anti-HIV drug is dolutegravir and lamivudine.

[0016] (28) The pharmaceutical product according to any one of (1-1) to (27), further characterized by combining (C) a broad-spectrum HIV neutralizing antibody (bNAb). (29) The pharmaceutical product according to (28), characterized in that the bNAb has ADCC activity and / or ADCP (antibody-dependent cell phagocytosis) activity.

[0017] (30) A method for treating HIV infection, characterized by combining (A) a polynucleotide or polypeptide that induces an anti-HIV Tat antibody that inhibits the activity of the HIV Tat protein secreted from HIV-infected cells; and (B) a pattern recognition receptor agonist or IAPi that induces type I IFNs.

[0018] (31) The pharmaceutical product according to any one of (1-1) to (29), characterized in that the measurement results of ADCP activity specific to HIV Tat protein in a biological sample taken from the administered subject suggest efficacy of (A) and (B) against the subject. (32) The pharmaceutical product according to (31), wherein the biological sample is a blood sample. (33) The pharmaceutical product according to (31) or (32), further characterized in that the administration of (A) and / or (B) is discontinued thereafter.

[0019] The pharmaceutical agent of the present invention exhibits remarkable effects, such as delayed HIV rebound and reduced reservoir size, compared to the effects of the HIV Tat activity inhibitor of the present invention alone and the effects of the pattern recognition receptor agonist of the present invention alone. Therefore, the pharmaceutical agent of the present invention is useful as a pharmaceutical, particularly as a pharmaceutical for the treatment of HIV infection.

[0020] Evaluation of the ability of Tat mRNA to induce cellular immunity in mice. Concept confirmation study in an SIV-infected cynomolgus monkey model. SIV Tat inhibition assay. Tat inhibitory activity during OLIGO administration. Evaluation of ADCC activity and ADCP activity.

[0021] The present invention will now be described with reference to embodiments. Throughout this specification, singular expressions should be understood to include the concept of their plural form unless otherwise specified. Therefore, singular articles (for example, "a," "an," "the," etc. in English) should be understood to include the concept of their plural form unless otherwise specified. Furthermore, terms used in this specification should be understood to have the meaning commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In case of any conflict, this specification (including definitions) shall prevail.

[0022] The term "consists of" means having only the constituent elements. The term "includes" means not being limited to the constituent elements and not excluding any elements not listed.

[0023] Terms used herein have the meaning commonly used in the art unless otherwise specified. In the present invention, methods of gene manipulation known in the art may be used. Examples include the methods described in *Molecular Cloning*, *A Laboratory Manual*, *Fourth Edition*, *Cold Spring Harbor Laboratory Press* (2012) and *Current Protocols Essential Laboratory Techniques*, *Current Protocols* (2012).

[0024] The HIV Tat protein is present in both HIV-1 and HIV-2 and plays a crucial role in regulating gene expression and replication in each. The HIV Tat protein has a proline-rich domain, a cysteine-rich domain, a core domain, an arginine-rich domain, and a glutamine-rich domain in exon 1, and an RGD motif in exon 2. The HIV-1 Tat protein consists of 99 to 106 residues, varying depending on the isolate, and is known to belong to the M, O, and N groups. Within the M group, subtypes A to D, CRF01 AE, F to H, J, and K are mainly known. On the other hand, the HIV-2 Tat protein consists of approximately 130 residues, varying depending on the isolate, and is mainly known to belong to subtypes A to G. Both the HIV-1 Tat gene and the HIV-2 Tat gene consist of two exons that encode the protein. Examples of amino acid sequences for HIV-1 Tat protein include SEQ ID NOs. 129-130 (A subtype), SEQ ID NOs. 131-148 (B subtype), SEQ ID NOs. 149-153 (C subtype), SEQ ID NOs. 154-156 (D subtype), and SEQ ID NOs. 157-158 (E subtype). Examples of amino acid sequences for HIV-2 Tat protein include SEQ ID NOs. 159. The HIV Tat protein is preferably HIV-1 Tat B subtype, and more preferably HIV-1 Tat Oyi or BH10. The amino acid sequence for HIV Tat protein is preferably the amino acid sequence described in any of SEQ ID NOs. 131-148, and more preferably the amino acid sequence described in SEQ ID NOs. 133 (HIV-1 Tat Oyi) or SEQ ID NOs. 134 (HIV-1 Tat BH10).

[0025] The "SIV Tat protein" is a protein found in the simian immunodeficiency virus that has a similar function to the HIV Tat protein. Examples of its amino acid sequences include SEQ ID NO: 160.

[0026] An "anti-HIV Tat antibody that inhibits the activity of HIV Tat protein secreted from HIV-infected cells" means an antibody that specifically binds to HIV Tat protein secreted from HIV-infected cells and inhibits the activity exerted by HIV Tat protein (also referred to herein as "the activity of HIV Tat protein secreted from HIV-infected cells"). The "activity exerted by HIV Tat protein" refers to the activity of invading HIV-uninfected cells and inducing apoptosis, the activity of suppressing the function of HIV Tat-specific CD8-positive T cells, and the activity of suppressing the phagocytic activity of macrophages. The inhibitory ability of the anti-HIV Tat antibody on the activity exerted by the HIV Tat protein can be measured, for example, using MT4 cells into which a luciferase gene has been incorporated downstream of the HIV LTR and HIV Tat peptide, in the same manner as in Example 7 (7-3) of this application. "Polynucleotides or polypeptides that induce anti-HIV Tat antibodies" means polynucleotides or polypeptides that directly or indirectly induce said antibodies. The ability of a polynucleotide or polypeptide to induce said antibodies can be measured by a method similar to that described in Example 5 of this application.

[0027] (A) A polynucleotide or polypeptide that induces an anti-HIV Tat antibody that inhibits the activity of the HIV Tat protein secreted from HIV-infected cells, that is, the HIV Tat activity inhibitor of the present invention, may, in one embodiment, be a polynucleotide encoding the HIV Tat protein or its antigenic fragment (the polynucleotide according to the present invention), or the HIV Tat protein or its antigenic fragment (the polypeptide according to the present invention).

[0028] The HIV Tat protein that is an HIV Tat activity inhibitor of the present invention may be a chimeric protein of HIV Tat, an HIV Tat protein having a mutation in the arginine-rich domain, an HIV Tat protein having a mutation in the RGD motif, a partial deletion mutant of the HIV Tat protein, or a mutant such as a linked HIV Tat protein with two or more valents.

[0029] "HIV Tat protein with mutations in the RGD motif" refers to HIV Tat protein in which a mutation has been introduced into the RGD motif located in exon 2 of the wild-type HIV Tat protein. HIV Tat protein has been reported to induce cell death in nerve cells, immune cells, and other cells via the RGD motif in exon 2. HIV Tat protein with specific mutations in the RGD motif is expected to have its cell death-inducing activity in nerve cells and immune cells eliminated or attenuated, and can induce immunity with neutralizing activity equivalent to that of unmutated HIV Tat protein. An example is the protein consisting of the amino acids in SEQ ID NO: 182.

[0030] The HIV Tat protein, which is an HIV Tat activity inhibitor of the present invention, is preferably a polypeptide consisting of an amino acid sequence that is identical to, or has 90% or more identity with, the amino acid sequence described in any of SEQ ID NOs. 131-148 and 182. Particularly preferably, it is a polypeptide consisting of an amino acid sequence that is identical to, or has 90% or more identity with, the amino acid sequence described in SEQ ID NOs. 133 or 134.

[0031] An "antigenic fragment of HIV Tat protein" refers to a fragment of HIV Tat protein that contains a portion of the HIV Tat protein and, like the full-length HIV Tat protein, is recognized by immune cells such as T cells and B cells, and can induce an immune response such as antibody production against HIV Tat protein. Examples of antigenic fragments of HIV Tat protein include fragments in which 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 4, or 1 to 2 amino acids are deleted from the N-terminus, middle, and / or C-terminus of the HIV Tat protein.

[0032] The HIV Tat activity inhibitor "HIV Tat protein" of the present invention may further have secretion signal peptides and / or functional sequences linked to it.

[0033] "Secretory signal peptides (SSPs)" refer to short peptides of about 15 to 30 amino acids that are located at the N-terminus, middle, or C-terminus of a protein or its peptide fragment, and that promote the transport of the protein or its peptide fragment into the endoplasmic reticulum and have a secretory function. Depending on the position of the secretory signal peptide within the protein or its peptide fragment, secretory signal peptides are classified as N-terminal secretory signal peptides, middle secretory signal peptides, and C-terminal secretory signal peptides, respectively. Secretory signal peptides may be operably linked (fused) to the protein or its peptide fragment, or they may be linked to a polypeptide from which methionine has been removed from the N-terminus of the protein or its peptide fragment.

[0034] Examples of amino acid sequences for secretory signal peptides include polypeptides consisting of amino acid sequences described in SEQ ID NOs. 1-1115 and 1728 of International Publication No. 2017 / 081082, and SEQ ID NOs. 161-167 and 183-187 of this specification, and polypeptides consisting of amino acid sequences in which one or more amino acids are deleted, substituted, inserted, or added, and which have secretory signal activity. Addition means that one or more amino acids are attached to the N-terminus or C-terminus of the amino acid sequence. "One or more" refers to 1 to 10 amino acids, preferably 1 to 5. Preferably, the secretory signal peptide is an N-terminal secretory signal peptide consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added, and which have secretory signal activity, as described in any of SEQ ID NOs. 161-167 or 183-187 of this specification. More preferably, IgE (SEQ ID NO: 161), Igk (SEQ ID NO: 162), JEV (SEQ ID NO: 163), SARS (SEQ ID NO: 164), IgG (SEQ ID NO: 167), tPA (SEQ ID NO: 165), albumin (SEQ ID NO: 166), Cystatin S (SEQ ID NO: 183), tPA (SEQ ID NO: 165), Mod-tPA (SEQ ID NO: 184), IL-6 (SEQ ID NO: 185), CTLA4 (SEQ ID NO: 186), or HSV / gDsec (SEQ ID NO: 187). More preferably, IgE, Igk, JEV, SARS, tPA, or IgG. Even more preferably, IgE, JEV, or SARS. Particularly preferably, IgE.

[0035] A "functional sequence" refers to a polypeptide of 3 to 60 amino acids located at the N-terminus, central, or C-terminus of a protein or its peptide fragment, which can determine the secretory pathway and localization of the protein or its peptide fragment. Functional sequences are synonymous with heterologous peptides. Functional sequences are distinguished as N-terminal functional sequences, central functional sequences, or C-terminal functional sequences depending on their position within the protein or its peptide fragment. Examples of functional sequences include signal peptides, linker peptides, immune-activating helper epitopes, TLR-activating peptides, and multimerization domain peptides such as SEQ ID NO: 189. Examples of signal peptides include secretory signal peptides, nuclear localization signal peptides, nuclear localization signal peptides, ubiquitinated signal peptides, endoplasmic reticulum-targeting signal peptides such as SEQ ID NO: 188, cell membrane localization signal peptides, Golgi apparatus localization signal peptides, endosome localization signal peptides, cytoskeleton localization signal peptides, and cytoplasmic localization signal peptides.

[0036] "Polynucleotide" refers to polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, their analogues, or mixtures thereof. This includes triple-stranded, double-stranded, and single-stranded deoxyribonucleic acid (DNA) and triple-stranded, double-stranded, and single-stranded ribonucleic acid (RNA). It also includes polynucleotides and cyclic polynucleotides in modified and unmodified forms, as well as those modified by alkylation and / or capping. Examples include polydeoxyribonucleotides containing 2-deoxy-D-ribose, polyribonucleotides such as tRNA, rRNA, hRNA, siRNA, and mRNA containing D-ribose, other types of polynucleotides that are N-glycosides or C-glycosides of purine or pyrimidine bases, and other polymers containing non-nucleotide backbones. Polymers include polyamides such as peptide nucleic acid PNA, polymorpholinopolymers, and other synthetic sequence-specific nucleic acid polymers. mRNA includes IVT mRNA, self-replicating RNA, and replicon RNA. Self-replicating RNA and replicon RNA include ssRNA viruses such as positive-strand ssRNA viruses.

[0037] The polynucleotide is preferably a ribonucleotide or a deoxyribonucleotide, and more preferably a non-natural mRNA. Non-natural mRNA means mRNA that differs from the wild-type sequence found in nature by at least one nucleotide. The mRNA is produced by existing gene modification technology. In one embodiment, the non-natural mRNA contains at least one modified base.

[0038] In one embodiment, the polynucleotide according to the present invention may be mRNA encoding the HIV Tat protein or its antigenic fragment (mRNA according to the present invention). The mRNA may be an mRNA comprising a 5' Cap structure, a 5' UTR, an open reading frame encoding a polypeptide containing the HIV Tat protein or its antigenic fragment, a 3' UTR, and Poly(A).

[0039] "5'UTR" is synonymous with "5'-untranslated region" and refers to the region of mRNA located between the 5'Cap structure and the start codon on the mRNA translated by the ribosome. Typically, it refers to the region from the nucleotide adjacent to the 3' end of the 5' Cap structure to the nucleotide adjacent to the 5' end of the start codon on the open reading frame (ORF). The 5'UTR may contain regulatory elements of gene expression. Examples of regulatory elements include ribosome binding sites or 5'-terminal oligopyrimidine tracts. Examples of the 5'UTR of mRNA according to the present invention include polynucleotides consisting of a nucleotide sequence that is identical to or has 80%, 85%, 90%, or 95% or more identity with the nucleotide sequences described in any of SEQ ID NOs: 1 to 63 and 211 to 218. Preferably, the polynucleotide is one that contains a base sequence identical to or having 90% or more identity with the base sequence described in any of SEQ ID NOs: 1 to 9, and more preferably, the polynucleotide is one that contains a base sequence identical to or having 90% or more identity with the base sequence described in any of SEQ ID NOs: 1 to 6. Particularly preferably, the polynucleotide is one consisting of a base sequence identical to the base sequence described in any of SEQ ID NOs: 1 to 3.

[0040] "3'UTR" is synonymous with 3'-untranslated region and refers to the region of mRNA located downstream (3' side) of the stop codon on mRNA translated by ribosomes. The 3'UTR may include gene expression regulatory elements, protein binding regions, or PolyA addition signal sequences. Examples of regulatory elements include ribosome binding sites and miRNA binding sites. The 3'UTR of mRNA according to the present invention includes polynucleotides consisting of a base sequence that is identical to or has 80%, 85%, 90%, or 95% or more identity with the base sequence described in any of SEQ ID NOs. 64 to 112. Preferably, it is a polynucleotide containing a base sequence that is identical to or has 90% or more identity with the base sequence described in any of SEQ ID NOs. 64 to 69, and more preferably, it is a polynucleotide containing a base sequence that is identical to or has 90% or more identity with the base sequence described in SEQ ID NOs. 64 to 66. Particularly preferably, it is a polynucleotide consisting of a base sequence identical to the base sequence described in SEQ ID NOs. 64 to 66.

[0041] "Identity" refers to the percentage of nucleotides or amino acids that match the same nucleotide or amino acid in the reference sequence at their respective sequence positions. Two or more sequences are considered identical if they have nucleotides or amino acids of the same length and order. When evaluating identity, the sequences being compared are assumed to have the same length, i.e., the length of the longest sequence among those being compared. This means that a first sequence consisting of 9 nucleotides or amino acids is 90% identical to a second sequence consisting of 10 nucleotides or amino acids that includes the first sequence. The identity of amino acid sequences or nucleotide sequences can be determined using the BLAST algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA (1993) 90:5873-7) or software such as CrystalW2 provided by the European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI).

[0042] "Poly(A)" is also called the PolyA tail or 3'-PolyA tail, and refers to a sequence of adenosine nucleotides of approximately 10 to 500 mere located directly downstream (i.e., on the 3' side) from the 3' UTR of the mRNA. In one embodiment, the mRNA according to the present invention may have the amino-terminal enhancer of split (AES) sequence of a split gene, the mitochondrial code 12S ribosomal RNA sequence, the A30LA70 (GCAUAUGACU) sequence, etc., inserted between the ORF and Poly(A). In another embodiment, the mRNA according to the present invention may have a mask structure of nucleotides other than adenyl(A) residues, INVERTED-dT, 2'-modified RNA, etc., inserted on the 3' side of the Poly(A) of the mRNA. The phosphate diester portion of Poly(A) may have a phosphorothioate, phosphorodithioate, phosphoroserenate, or phosphorodiserenate bond for the purpose of improving mRNA stability. In one embodiment, the mRNA according to the present invention may have Poly(A) and / or a histone stem loop at the 3' end, and may be present in combination with other elements such as a purine-rich region (hitone downstream element, or HDE).

[0043] The poly(A) of mRNA according to the present invention is preferably an adenosine nucleotide of 20 to 250 mer, more preferably 60 to 160 mer, 60 to 120 mer, and particularly preferably 75 to 85 mer or 80 mer.

[0044] An "Open Reading Frame (ORF)" refers to the nucleotide sequence from a start codon to a stop codon. The stop codon may contain one or more, and may contain any number of UAA, UAG, or UGA codons. The ORF may have all convertible codons converted and may be optimized using an optimization algorithm so that the mRNA forms a stable secondary structure. Codon optimization tools, algorithms, and services are known in the art and include GeneArt's services (Life Technologies), DNA2.0 (Menlo Park CA), ViennaRNA Package 2.0 (University of Vienna), and LinearDesign (Baidu).

[0045] Examples of ORF sequences for polynucleotides according to the present invention include sequences in which the expression level of the protein encoded by the ORF is increased by increasing the G / C content or optimizing the codons, without altering the amino acid sequence from the wild-type sequence. Preferably, the ORF sequence has a G / C content that is increased by 5%, 10%, 15%, or 20% or more compared to the ORF of wild-type Tat mRNA.

[0046] The ORF of the polynucleotide according to the present invention may encode a polypeptide containing HIV Tat protein and a secretory signal peptide. For example, Example 2 includes mRNA encoding a polypeptide in which a secretory signal peptide is linked to HIV-1 Tat protein. Examples of ORF sequences include the nucleotide sequences described in SEQ ID NOs: 114-119, 121-126, and 198-210. Preferably, it is the nucleotide sequence described in SEQ ID NOs: 121-126 and 198-210. More preferably, it is the nucleotide sequence described in any of SEQ ID NOs: 121, 198, 200, and 202.

[0047] 5'Cap refers to both native 5'Cap and modified 5'Cap. Native 5'Cap refers to the ribose-guanosine residue (m7G) with a methylated 7' position of the guanine base, which is present in all eukaryotes. m7G is linked to the 5'-terminal nucleotide of mRNA via a 5'-5'-triphosphate bond (hereinafter referred to as ppp). 5'Cap contributes to the efficient translation of mRNA-encoded proteins by protecting mRNA from degradation by exonucleases, promoting the transport of mRNA from the nucleus to the cytoplasm, and playing a crucial role in the assembly of the translation initiation complex. Modified 5'Cap refers to 5'Cap that has undergone some chemical modification to the native 5'Cap, resulting in a 5'Cap that has equivalent or superior function to the native 5'Cap in terms of protein translation.

[0048] Examples of modified 5'Cap include modified guanosine (hereinafter referred to as [G]), and structures described in International Publication Nos. 2017 / 066793, 2023 / 007019, CN116987137, and 2023 / 246860. Examples of nucleosides in [G] include guanosine with methylation at the 7th position of guanine and the 3' position of ribose (m7G-3'OMe), guanosine with methylation at the 7th position of the guanine residue and the 2' position of ribose (m7G-2'OMe), and other guanine modifications and sugar modifications. Examples of guanine modifications include hypoxanthine, N1-methyl-guanine, 7-deaza-guanine, and 8-oxo-guanine. Examples of sugar-modified riboses include riboses modified at the 2' and / or 3'1 positions, such as 2'-fluororibose, 2'-aminoribose, and 2'-azidribose; BNA (Bridged Nucleic Acid) with a cross-linking structure; and modified riboses described in International Publication No. 2014 / 081507.

[0049] The natural 5'Cap, m7G, and the modified 5'Caps, m7G-3'OMe and m7G-2'OMe, are specifically represented by the following structures. In this specification, the "5' Cap structure" refers to the 5'-[G]pppN structure located at the 5' end of mRNA. １N ２ N ３ (N １ 、N ２ 、N ３ represents any nucleoside.) By 5'-Cap structure, Cap0, Cap1 (N １ whose 2'-position is methylated, 5'-[m7G]-ppp-N １ mN ２ N ３ and is represented as.). Cap2 (N １ and N ２ whose 2'-position is methylated, 5'-[m7G]-ppp-N １ mN ２ mN ３ and is represented as.). Cap3 (N １ 、N ２ and N ３ whose 2'-position is methylated, 5'-[m7G]-ppp-N １ mN ２ mN ３ and is represented as m.). etc. are mentioned. Specifically, Cap1 is represented by the following structure. Base in the chemical formula refers to any base. For example, depending on the difference in capping methods, GGG, AGG, AUA, etc. can be taken. Note that for mRNA having a 5'-Cap structure represented by 5'-[m7G]-ppp-GmGG, 5'-[m7G]-ppp-AmGG or 5'-[m7G]-ppp-AmUA, it has been confirmed that the expression level in HEK293 cells and the expression level in mice are equivalent. Cap2 is represented by the following structure. Base in the chemical formula refers to any base. Cap3 is represented by the following structure. Base in the chemical formula refers to any base.

[0050] The 5'-5'-triphosphate bond (ppp) connecting the 5'-Cap and the nucleotide at the 5'-end of mRNA can use modified phosphate groups such as phosphorothioate, phosphorosenate, boranophosphate, boranophosphate ester, hydrogen phosphonate, phosphoramidate, alkyl or aryl phosphonate, and phosphotriester.

[0051] The 5' Cap structure of mRNA according to the present invention is preferably Cap1 or Cap2, and particularly preferably Cap1. The 5' Cap structure of mRNA according to the present invention is 5'-[G]pppN, depending on the reagent used for capping. １ N ２ N ３ N inside １ N ２ N ３ This can be GGG, AGG, AGA, AUG, GAA, or AUA. Therefore, in one embodiment, the mRNA base sequence according to the present invention begins with 5'-GGGG, 5'-GAGG, 5'-GAGA, 5'-GAUG, 5'-GGAA, or 5'-GAAUA, followed by a 5'UTR sequence.

[0052] The mRNA according to the present invention may have an internal ribosome entry site (IRES) instead of a 5' Cap structure. Preferred IRESs include those derived from picornavirus, plague virus, poliovirus, encephalomyocarditis virus, foot-and-mouth disease virus, hepatitis C virus, swine fever virus, mouse leukemia virus, simian immunodeficiency virus, and cricket paralysis virus.

[0053] The polynucleotide according to the present invention may contain at least one chemical modification. Any polynucleotide modification known in the art can be used in the polynucleotide according to the present invention, but examples include modified phosphate groups, modified bases, and sugar modifications.

[0054] Examples of modified phosphate groups include phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramides, alkyl or aryl phosphonates, and phosphotryesters. In phosphorothioates, both unbound oxygen atoms are substituted with sulfur. The phosphate linker may also be modified by substituting the bound oxygen atoms with nitrogen (bridged phosphoramide), sulfur (bridged phosphorothioate), and carbon (bridged methylene phosphonate).

[0055] Examples of modified bases include 5-methyluridine, pseudouridine (Ψ), N1-methylpseudridine (m1Ψ), 5-methoxyuridine, 2-thiouridine, 6-methyladenosine, 2-aminoadenosine, inosine, 5-methylcytidine, N1-ethylpseudridine, 4'-thiouridine, 2-thio-1-methyl-1-deazapseudridine, 2-thio-1-methylpseudridine, 2-thio-5-azapseudridine, 2-thio-dihydropseudridine, 2-thio-dihydropseudridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-1-methylpseudridine, 4-thiopseudridine, 5-azapseudridine, and dihydropseudridine. Furthermore, International Publication Nos. 2001 / 79502, 2002 / 98443, 2009 / 127230, US20100249219, 2012 / 019168, 2012 / 138453, 2013 / 143698, 2015 / 089511, US20150056253, and 2015 Modified bases described in publications such as 077123, 2016 / 209966, 2017 / 070613, US20180126003, 2020 / 243002, 2021 / 198258, 2021 / 251453, 2022 / 233880, and 2023 / 006999 are also available. The polynucleotide according to the present invention may contain multiple types of modified bases.Preferably, the mRNA modification base according to the present invention is at least one modification base selected from the group consisting of 5-methyluridine, pseudouridine, N1-methylpseudridine (m1Ψ), 5-methoxyuridine, 2-thiouridine, 6-methyladenosine, 2-aminoadenosine, inosine, 5-methylcytidine, N1-ethylpseudridine, 4-thiouridine, 2-thio-1-methyl-1-deazapseudridine, 2-thio-1-methylpseudridine, 2-thio-5-azapseudridine, 2-thio-dihydropseudridine, 2-thio-dihydrouridine, 2-thiopseudridine, 4-methoxy-2-thiopseudridine, 4-methoxypseudridine, 4-thio-1-methylpseudridine, 4-thiopseudridine, 5-azapseudridine, dihydropseudridine, and 2'-O-methyluridine. More preferably, it is at least one modified base selected from the group consisting of 5-methyluridine, pseudouridine, N1-methylpseudridine, 5-methoxyuridine, 2-thiouridine, 6-methyladenosine, inosine, and 5-methylcytidine. Even more preferably, it is at least one modified base selected from the group consisting of 5-methyluridine, N1-methylpseudridine, and 5-methylcytidine. Particularly preferred is N1-methylpseudridine.

[0056] Examples of sugar modifications include nucleosides having a substituent at the 2' position of the sugar and / or nucleosides having a crosslinking structure between the 4' and 2' positions of the sugar. Examples of substituents on the hydroxyl group at the 2' position of the sugar include oxy or deoxy substituents. Examples of "oxy" modifications include alkoxy groups or aryloxy (-OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), polyethylene glycol (PEG), and aminoalkoxy. Examples of "deoxy" modifications include hydrogen, amino, halogens (e.g., fluorine). Examples of aminos include alkylaminos, dialkylaminos, heterocyclyls, arylaminos, diarylaminos, heteroarylaminos, diheteroarylaminos, and amino acids. The amino may be bonded to the sugar via a linker, and the linker contains one or more atoms C, N, and O. Examples of the crosslinking structure include BNA (Bridged Nucleic Acid). BNAs include AmNA (Amido-bridged nucleic acid, see International Publication No. 2011 / 052436), TrNA (Triazole-Bridged Nucleic Acid, see International Publication No. 2014 / 126229), and 4'-(CH ２ ) ｎ -O-2' (n is an integer from 1 to 4), 4'-(CH ２ ) ｍ -C(=O)-NR １ -2' (where m is an integer from 0 to 4, R １ (wherein this is a hydrogen atom or an alkyl group). Specific examples and methods for their preparation are described in International Publication Nos. 98 / 39352, 2003 / 068795, 2005 / 021570, 2011 / 052436, and 2013 / 052523, etc.

[0057] Modifications of nucleotides known in this field and methods of modification are disclosed in the following patent documents, for example: International Publication No. 98 / 39352, International Publication No. 99 / 014226, International Publication No. 2000 / 056748, International Publication No. 2003 / 068795, International Publication No. 2004 / 016749, International Publication No. 2005 / 021570, International Publication No. 2005 / 083124, International Publication No. 2007 / 143315, International Publication No. 2009 / 071680, International Publication No. 2011 / 052436, International Publication No. 2014 / 112463, International Publication No. 2014 / 126229, etc.

[0058] The polynucleotides according to the present invention can be synthesized by conventional methods. For example, they can be synthesized by chemical synthesis, enzymatic synthesis generally referred to as in vitro transcription (IVT), or enzymatic or chemical cleavage of longer precursors, but are not limited to these methods. For example, this specification and Gait, M. J. (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, DC: IRL Press, 1984, and Herdewijn, P. This is disclosed in (ed.) Oligoncleotide synthesis: methods and applications, Methods in Molecular Biology, v. 288 (Clifton, N.J.; Totowa, N.J.; Humana Press, 2005), etc.

[0059] The mRNA according to the present invention can be synthesized by conventional methods. For example, the mRNA according to the present invention can be synthesized by performing IVT based on a template DNA encoding the mRNA sequence according to the present invention. When synthesizing mRNA by IVT, template DNA is required. Template DNA can be a plasmid linearized with restriction enzymes, or a DNA fragment produced by PCR. The template DNA for the mRNA according to the present invention includes, from the 5′ end, an RNA polymerase promoter sequence (e.g., T7 promoter sequence, SP6 promoter sequence, T3 promoter sequence, etc.), a 5′-UTR, an ORF region, and a 3′-UTR. The template DNA may further include a portion corresponding to Poly(A), and by including a portion corresponding to Poly(A) in the DNA template, the portion corresponding to Poly(A) is synthesized in the RNA synthesis reaction by RNA polymerase. Alternatively, as a method for adding Poly(A) to mRNA, the 3′ end of the mRNA can be polyadenylated after IVT using an enzyme such as E. coli Poly(A) polymerase.

[0060] Type I interferons (IFNs) are a family of cytokines, including IFNα and IFNβ.

[0061] A "pattern recognition receptor (PRR) agonist" refers to a molecule that binds to and activates one or more immune cell-associated receptors that recognize pathogen-associated molecular patterns (PAMPs) or injury-associated molecular patterns (DAMPs), thereby triggering immune cell activation and / or pathogen- or injury-induced inflammatory responses. Pattern recognition receptors are expressed by cells of the innate immune system, such as monocytes, macrophages, dendritic cells (DCs), neutrophils, and epithelial cells, as well as cells of the adaptive immune system. Examples of pattern recognition receptor agonists include Toll-like receptor (TLR) agonists, interferon-stimulating factor (STING) agonists, and RIG-I receptor agonists. Examples of TLR agonists include TLR1 / 2 agonists, TLR2 agonists, TLR3 agonists, TLR2 / 4 agonists, TLR4 agonists, TLR5 agonists, TLR7 agonists, TLR7 / 8 agonists, TLR8 agonists, and TLR9 agonists. Examples of STING agonists (ligands) include 2'3'-cGAMP, 3'3'-cGAMP, and c-di-GMP. Furthermore, there are double-stranded oligonucleotides in which the first strand consists only of 8-50 nucleotide CpG oligonucleotides, and the second strand is an oligonucleotide in which an 8-60 nucleotide DNA nucleoside containing a sequence that can hybridize to the first strand is bound.

[0062] "Type I interferon-inducing pattern recognition receptor agonists" refer to pattern recognition receptor agonists that have the ability to reactivate HIV-latently infected cells by inducing type I interferon. Preferred type I interferon-inducing pattern recognition receptor agonists are TLR3 agonists, TLR7 agonists, TLR9 agonists, and STING ligands. Examples of TLR3 agonists include Poly I:C, PolyI:PolyC12U (Rintatolimod), Poly IC:LC (Oncovir), RGC100, and ARNAX. Examples of TLR7 agonists include AL-034, DSP-0509, GS-9620 (Besatrimod), GS-986, LHC-165, TMX-101 (Imiquimod), GSK-2245035, Reshiquimod, DSR-6434, DSP-3025, IMO-4200, MCT-465, MEDI-9197, 3M-051, SB-9922, 3M-052, Limtop, TMX-30X, TMX-202, RG-7863, RG-7854, and RG-7795. Examples of TLR9 agonists include AST-008, CMP-001, IMO-2055, IMO-2125 (Tilsotrimod), Litenimod, MGN-1601, BB-001, BB-006, IMO-3100, IMO-8400, IR-103, IMO-9200, Agatrimod, DIMS-9054, DV-1079, DV-1179, AZD-1419, MGN-1703 (Refitrimod), CYT-003, CYT-003-QbG10, PUL-042, ODN2006, and OLIGO as described in this application. More preferably, as a pattern recognition receptor agonist that induces type I interferon, is Poly I:C, Poly I:PolyC12U (Rintatolimod), Poly IC:LC (Oncovir), RGC100, ARNAX, refitorimod, ODN2006, besatrimod, chilsotrimod, OLIGO as described in this application, 2'3'-cGAMP, c-di-GMP, or STING ligand. Even more preferably, is ODN2006, refitorimod, besatrimod, chilsotrimod, or STING ligand. Particularly preferred is OLIGO as described in this application.

[0063] "Reactivation of HIV latent infected cells" and "ability to reactivate HIV latent infected cells" refer to the re-induction of viral protein expression in latent infected cells that have HIV proviral DNA inserted into their chromosomes but do not express viral proteins, and the activity of such induction. The ability to reactivate HIV latent infected cells can be measured by a method similar to the method described in Example 4 of this application.

[0064] An "apoptosis inhibitor protein (IAP) inhibitor (IAPi)" is synonymous with an IAP antagonist and refers to a drug that inhibits (antagonizes) the inhibition of apoptosis by IAP in MDSCs / neutrophils. Examples include second mitochondrial caspase activator (SMAC) mimetic agents (SMACm, SM) such as LCL161, SM-164, SM-406, GDC-0152, ASTX660, AZD5582, and virinapant. Preferably, it is AZD5582.

[0065] An "oligonucleotide" refers to a nucleotide, which is a compound in which multiple identical or different nucleosides are linked together. A "nucleoside" refers to a compound in which a nucleic acid base and a sugar are linked by an N-glycosidic bond.

[0066] The bond between sugars in oligonucleotides (nucleoside bonds) may be a bond found in natural nucleic acids, a phosphodiester (D-oligo), an artificially modified bond, or a bond without a phosphorus atom. Any bond known in the art can be used. Examples of artificially modified bonds include phosphorothioates (S-oligo), methylphosphonates (M-oligo), and boranophosphonates. Bonds described in International Publications 2013 / 022966, 2011 / 005761, 2014 / 012081, and 2015 / 125845 can also be used. Examples of bonds without a phosphorus atom include divalent substituents derived from alkyl groups, non-aromatic carbocyclic groups, haloalkyl groups, and halogen-substituted non-aromatic carbocyclic groups. For example, divalent substituents derived from siloxanes, sulfides, sulfoxides, sulfones, acetyl, acetyl formate, acetyl thioformate, acetyl methyleneformate, acetyl thioformate, alkenyls, sulfamates, methyleneiminos, methylenehydrazinos, sulfonates, sulfonamides, amides, etc. The oligonucleotide may consist entirely of the same bond, or it may contain different bonds.

[0067] As one embodiment, the pattern recognition receptor agonist, etc. of the present invention may be a double-stranded oligonucleotide in which (B-1) the first chain consists only of an 8-50 base CpG oligonucleotide, the second chain is an oligonucleotide in which an 8-60 base DNA nucleoside containing a hybridizable sequence is bound to the first chain, the length of the second chain is 50% or more of the length of the first chain, and a lipid containing a hydrocarbon chain having 12-30 carbon atoms is bound to the second chain via / without a linker. Hereinafter, this will also be referred to as "double-stranded oligonucleotide according to the present invention".

[0068] A "CpG oligonucleotide (CpG ODN)" is a single-stranded oligonucleotide containing a non-methylated cytosine-guanine dinucleotide (5'-CpG-3') motif (CpG motif), and is known to be usable as a vaccine adjuvant because it induces an acquired immune response via TLR9 (Nat Rev Drug Discov, 2006, 5, 471-484; Expert Rev Vaccines, 2011, 10(4), 499-511). The CpG oligonucleotide used in the double-stranded oligonucleotide according to the present invention contains at least one CpG motif and may contain multiple CpG motifs.

[0069] The length of the CpG oligonucleotide used in the double-stranded oligonucleotide according to the present invention is 8 to 50 bases. For example, 8 to 50 bases, 8 to 40 bases, 8 to 30 bases, 10 to 25 bases, 15 to 25 bases, 18 to 25 bases, etc.

[0070] The term "CpG oligonucleotide" is not particularly limited to any known CpG oligonucleotide known to have immunostimulatory activity in the field. For example, synthesis methods and CpG oligonucleotides are cited in International Publication Nos. 2006 / 065751, 2007 / 092315, 2008 / 068638, 2010 / 067262, 2010 / 125480, 2014 / 047588, 2014 / 134698, 2015 / 041318, and U.S. Patent Application Publication No. 2011 / 0300163. These CpG oligonucleotides can be synthesized by referring to the methods described in the above-mentioned literature. CpG oligonucleotides are classified into classes A, B, C, P, and S based on their sequence, secondary structure, and effects on human peripheral blood mononuclear cells (PBMCs) (Advanced drug delivery reviews, 2009, 61(3), 195-204). Class A: ODN1585, ODN2216, ODN2336, etc.; Class B: ODNBW006, ODN D-SL01, ODN1668 (International Publication No. 2005 / 063264), ODN1826 (International Publication No. 2007 / 030580), ODN2006 (CpG7909, PF-3512676) (International Publication No. 98 / 18810), ODN2007, ODN684, etc.; Class C: ODN D-SL03, ODN 2395, ODN M362, etc. These can also be purchased from InvivoGen as research reagents.In addition, there are other examples such as CpG-28 (International Publication No. 2000 / 056342), CpG-685 (GNKG-168) (Blood, 2010, 115(24), 5041), CpG-ODN C274 (PLoS ONE, 2013, 8(4), e62373), KSK-13 (KSK-CpG) (U.S. Patent No. 7408050), CpG ODN 10104 (CpG-10104) (Drug Data Rep, 2006, 28(3), 258), and CpG ODN-1585 (International Publication No. 2001 / 022990). Examples include ODN-5890 (International Publication No. 2006 / 080946), 1018-ISS (International Publication No. 2008 / 073661), EMD-1201081 (HYB-2055, IMO-2055) (International Publication No. 2005 / 009355), D35-CpG, K3-CpG (Gene Design Co.), etc. Any class of CpG oligonucleotide can be used in the present invention, but preferably, a class A CpG oligonucleotide (e.g., ODN2216, ODN2336, D35-CpG, etc.), a class B CpG oligonucleotide (e.g., ODN1826, ODN2006, CpG-28, 1018-ISS, IMO2055, K3-CpG, ODN684, D-LS01, etc.), or a class C CpG oligonucleotide (e.g., D-LS03, ODN2395, ODN M362) is used. Particularly preferred are ODN1826 and ODN2006.

[0071] The second strand of the double-stranded oligonucleotide according to the present invention is an oligonucleotide of 8 to 60 bases (excluding RNA oligonucleotides) that contains a sequence capable of hybridizing to the first strand, which is a CpG oligonucleotide.

[0072] Preferably, the second chain is an 8-60 nucleotide oligonucleotide containing a sequence that can hybridize to the first chain CpG oligonucleotide under stringent conditions. The oligonucleotide of the second chain may have one or more mismatches at the hybridization site, as long as it can hybridize to the CpG oligonucleotide under stringent conditions. For example, an oligonucleotide whose hybridization site has at least 70%, preferably 80%, more preferably 90%, and most preferably 95% homology to the complementary sequence of the first chain CpG oligonucleotide is mentioned. Here, homology is shown by a score using the BLAST search program, which employs an algorithm developed by Altschul et al. (The Journal of Molecular Biology, 215, 403-410 (1990)).

[0073] "Stringent conditions" refer to conditions in which a certain base sequence forms a hybrid (so-called specific hybrid) with a specific sequence, and base sequences that do not have equivalent function do not form a hybrid (so-called nonspecific hybrid) with that specific sequence. Those skilled in the art can easily select such conditions by changing the temperature during the hybridization reaction and washing, and the salt concentrations of the hybridization reaction solution and washing solution. Specifically, 6×SSC (0.9M NaCl, 0.09M trisodium citrate) or 6×SSPE (3M NaCl, 0.2M NaH) ２ PO ４One example of stringent conditions in this invention is hybridizing in 20 mM EDTA-2Na (pH 7.4) at 42°C, followed by washing with 0.5 × SSC at 42°C, but this is not limited to this condition. As for the hybridization method, well-known and conventional methods in the art, such as Southern blot hybridization, can be used. Specifically, this can be carried out in accordance with the methods described in Molecular Cloning: A Laboratory Manual, Second Edition (1989) (Cold Spring Harbor Laboratory Press), Current Protocols in Molecular Biology (1994) (Wiley-Interscience), DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition (1995) (Oxford University Press), etc.

[0074] "One or several mismatches" means one to five mismatches, preferably one to three, and more preferably one or two.

[0075] The length of the second strand of the double-stranded oligonucleotide according to the present invention is 8 to 60 bases. For example, 8 to 60 bases, 8 to 50 bases, 8 to 40 bases, 8 to 30 bases, 10 to 25 bases, or 15 to 25 bases. The length of the second strand may be the same as the length of the first strand, which is a CpG oligonucleotide, or it may be one or several bases shorter than the length of the CpG oligonucleotide, insofar as it hybridizes with the CpG oligonucleotide. Alternatively, the length of the second strand may be longer than the length of the CpG oligonucleotide by adding one or several bases to one or both sides of the site that hybridizes with the CpG oligonucleotide. "One or several bases" means 1 to 10 bases, 1 to 5 bases, 1 to 3 bases, or 1 or 2 bases. The preferred length of the second strand depends on the length of the first strand, which is a CpG oligonucleotide. For example, the length of the first chain may be 50% or more, 60% or more, 70% or more, 50-100%, 60-100%, or 70-100% of the length of the first chain. Particularly preferred is a length of 50-100% of the length of the first chain.

[0076] "DNA nucleoside" refers to a naturally occurring DNA nucleoside, which is a part of a nucleotide, a unit that makes up an oligonucleotide. "Naturally occurring DNA nucleoside" means the following: (In the formula, B Ｘ１(These are adenine, guanine, cytosine, or thymine.) In the oligonucleotide of the second chain of the double-stranded oligonucleotide according to the present invention, any nucleoside bond known in the art can be used as the nucleoside bond, as exemplified above. All nucleoside bonds may be of the same type, or two or more types of bonds may be included. Preferably, D-oligo and / or S-oligo. When two or more types of nucleoside bonds are included, such as D-oligo and S-oligo, examples include an oligonucleotide that includes a central region and terminal regions on both sides of the central region, with at least one unnatural nucleoside bond (e.g., S-oligo) in the terminal regions on both sides, and a natural nucleoside bond (i.e., D-oligo) in the central region. For example, the 5' terminal region and / or the 3' terminal region may contain one or more, preferably 1 to 5, more preferably 2 to 3, unnatural nucleoside bonds. The type, number, and position of modifications within one terminal region may be the same as or different from those in the other terminal region. Another embodiment includes oligonucleotides containing randomly distributed non-natural nucleoside bonds.

[0077] The CpG oligonucleotide of the first strand and the oligonucleotide of the second strand in the double-stranded oligonucleotide according to the present invention can be synthesized by conventional methods in the art, and can be easily synthesized, for example, by commercially available automated nucleic acid synthesizers (e.g., Applied Biosystems, Dainippon Seiki Co., Ltd., etc.). Synthesis methods include solid-phase synthesis using phosphoramidite and solid-phase synthesis using hydrogen phosphonate. For example, these are disclosed in Example 1 of Patent Document 3, Tetrahedron Letters 22, 1859-1862 (1981), etc.

[0078] The synthesized first and second chains are hybridized by known methods to form a double-stranded oligonucleotide. For example, this is disclosed in Example 1 of Patent Document 3 and Example 1 of International Publication No. 2013 / 089283.

[0079] "Lipids" are hydrophobic compounds and are not particularly limited as long as they are known lipids, and may be linear, branched, or cyclic. Examples include fatty acids having an aliphatic chain of 8 to 30 carbon atoms (e.g., farnesol), diacyl lipids, cholesterol, cholesterol derivatives, steroid acids (e.g., bile acids), lipid A, tocopherol, or combinations thereof. Examples of fatty acids having an aliphatic chain include, but are not limited to, linear unsaturated fatty acids and saturated fatty acids, branched saturated fatty acids and unsaturated fatty acids, and fatty acid derivatives (e.g., fatty acid esters, fatty acid amides, fatty acid thioesters, etc.).

[0080] The lipids used in the double-stranded oligonucleotides according to the present invention are preferably lipids containing hydrocarbon chains. Preferably, lipids containing one or two hydrocarbon chains are included. The double-stranded oligonucleotides according to the present invention particularly preferably contain diacyl lipids. When lipids are bonded at two or more sites, if one lipid is a diacyl lipid, the other lipid may be a lipid other than a diacyl lipid. "Diacyl lipid" is a phosphate lipid, glycolipid, sphingolipid, or a combination thereof, and contains two hydrocarbon chains. Preferably, it is a group represented by (a) or (f) below. Each hydrocarbon chain in the lipid contains about 8 to 30 carbon atoms, is saturated, unsaturated, or a combination thereof, and may be branched. In the case of single-stranded lipids, the preferred chain length is 8 to 30 carbon atoms, more preferably 8 to 20. In the case of double-stranded lipids, the preferred chain length is 10 to 30 carbon atoms, more preferably 12 to 30, and even more preferably 14 to 24. In the case of double-stranded lipids, the two chains may be the same length or different. The chains in the lipid are bonded to sites containing phosphate, sugar, etc. (sites that bind to oligonucleotides) via ester bonds, amide bonds, thioester bonds, or combinations thereof. The double-stranded oligonucleotide according to the present invention has a lipid in which the second chain contains a hydrocarbon chain having 12 to 30 carbon atoms. If the lipid is bonded at two or more sites, if one lipid contains a hydrocarbon chain having 12 to 30 carbon atoms, the number of carbon atoms in the hydrocarbon chain contained in the other lipid may be less than 12.

[0081] Specifically, examples of "lipids" include the following: (In the formula, p and q are each an independent integer between 6 and 28, preferably each an independent integer between 8 and 28, more preferably each an independent integer between 10 and 28, and even more preferably each an independent integer between 10 and 18.)

[0082] In the double-stranded oligonucleotide according to the present invention, the lipid may be bound to any of the second chains. It may be bound to the 3' end, the 5' end, or within the second chain.

[0083] When a lipid binds to the 5' end of the second chain, it can bind in the following way, for example: (In the formula, B １ This is the second 3' terminal base, Y ａ (wherein is O or S, and p or q are each an independent integer between 6 and 28, preferably each an independent integer between 10 and 18.)

[0084] When a lipid binds to the 3' end of the second chain, it can bind in the following way, for example: (In the formula, B １ This is the second 5' terminal base, Y ａ (wherein is O or S, and p or q are each an independent integer between 6 and 28, preferably each an independent integer between 10 and 18.)

[0085] When lipids bind to the second chain, they can bind in the following ways, for example: or, (In the formula, B １ and B ２ is an adjacent base in the second chain, Y ａ or Y ｂ (Each is independently O or S, and p or q is independently an integer from 6 to 28, preferably independently 10 to 18.) Furthermore, it is preferable that the lipid is bound to one or two locations on the second chain. Preferably, the lipid is bound to the 3' end and / or the 5' end. More preferably, the lipid is bound to the 5' end.

[0086] Specific examples of lipids and their preparation methods can be synthesized by referring to techniques known in the field. For example, they are disclosed in Patent Document 3, etc.

[0087] In the double-stranded oligonucleotide according to the present invention, the lipid may be bonded to the second chain via a linker. Any linker used in the art can be used as the "linker". Examples include polar linkers, alkylene linkers, ethylene glycol linkers, ethylenediamine linkers, etc. When the lipid is a phosphate lipid, the linker has 4 to 26 atoms between the oxygen atom of the second chain and the phosphorus atom of the lipid. Specific examples include oligonucleotide linkers or the linkers described below.

[0088] (In the formula, Y' is O or S, and r or s is an integer from 1 to 10, preferably from 1 to 5, and more preferably from 1 to 3. t is an integer from 1 to 4, preferably from 1 to 3, and more preferably from 2 or 3.) The linker can be synthesized with reference to methods known in the art. The oligonucleotide linker can be synthesized in the same manner as the oligonucleotide synthesis method exemplified above.

[0089] Preferably, the linker is an oligonucleotide linker. The lengths of the oligonucleotide linkers are 2 to 10 bases, 2 to 5 bases, 2 bases, 3 bases, 4 bases, and 5 bases. For example, the linkers described below are included.

[0090] (In the formula, B １ and B ２ These are adenine (A), guanine (G), cytosine (C), 5-methylcytosine (5-Me-C), thymine (T), or uracil (U). Y' is independently either O or S. Z １ or Z ２ Each is independently H or OH, preferably H.) As an oligonucleotide linker, for example, a DNA linker, i.e., -(dX １ )u-(here, X１ Examples include A, G, C, or T, where u is an integer from 1 to 8. Specifically, examples include dG, dGdG, dGdGdGdG, dGdGdGdGdG, dT, dTdT, dTdTdTdT, dTdTdTdTdT, etc. Particularly preferred are dGdG, dGdGdGdGdG or dTdT. Phosphothioate bonds are preferred as nucleoside bonds in the DNA linker.

[0091] The lipid-unbound 3' or 5' end or linker of the double-stranded oligonucleotide according to the present invention may be further modified. Modification groups known in the art can be used to enable tracking of the oligonucleotide, to improve the pharmacokinetics or pharmacodynamics of the oligonucleotide, or to improve the stability or binding affinity of the oligonucleotide. Examples include hydroxyl group protecting groups, reporter molecules, cholesterol, phospholipids, dyes, fluorescent molecules, etc. Furthermore, the lipid-unbound 3' or 5' end of the double-stranded oligonucleotide according to the present invention may contain a phosphate ester moiety. "Phosphate ester moiety" means a terminal phosphate group that includes phosphate esters and modified phosphate esters. The phosphate ester moiety may be located at any end, but is preferably a 5'-terminal nucleoside. Specifically, it is a group represented by the formula: -O-P(=O)(OH)OH or a modified group thereof. That is, one or more of the O and OH may be substituted with H, O, OR', S, N(R') (where R' is H, an amino protecting group, or a substituted or unsubstituted alkyl group) or an alkyl group. The 5' or 3' terminus may independently contain substituted or unsubstituted phosphate ester moieties of 1 to 3.

[0092] The double-stranded oligonucleotide according to the present invention is preferably the OLIGO described in this application.

[0093] As one embodiment, the pharmaceutical product of the present invention is characterized by combining (A) a polynucleotide or polypeptide that induces an anti-HIV Tat antibody that inhibits the activity of the HIV Tat protein secreted from HIV-infected cells; and (B) a pattern recognition receptor agonist that induces type I IFNs.

[0094] The term "pharmaceuticals characterized by combination" includes pharmaceuticals containing each compound, forms in which each compound is used as a combination agent, forms in which each compound is used as a kit, forms in which each compound is used as a concomitant agent (i.e., forms in which they are administered simultaneously, forms in which they are administered at different times), and forms in which each compound is used in combination with other drugs. It is sometimes abbreviated as "combined," but these are all synonymous. Preferably, it is a concomitant agent.

[0095] When the pharmaceutical product of the present invention is administered as a combination agent, with the HIV Tat activity inhibitor and the pattern recognition receptor agonist of the present invention being administered as separate formulations, they can be administered simultaneously or with a time difference, but administration with a time difference is preferred. It is preferable to administer the HIV Tat activity inhibitor of the present invention first, and the pattern recognition receptor agonist of the present invention later. The administration methods for each may be the same or different.

[0096] The pharmaceutical product of the present invention exhibits a remarkable effect when used in combination with the HIV Tat activity inhibitor of the present invention, compared to the effect of using the pattern recognition receptor agonist, etc., of the present invention alone.

[0097] The pharmaceutical product of the present invention can be administered orally or parenterally. Parenteral administration methods include transdermal, subcutaneous, intravenous, intra-arterial, intramuscular, intraperitoneal, transmucosal, inhalation, nasal, ophthalmic, ophthalmic, ophthalmic, and vaginal administration.

[0098] For oral administration, the drug may be prepared and administered in any of the commonly used dosage forms, such as oral solid preparations (e.g., tablets, powders, granules, capsules, pills, films, etc.) or oral liquid preparations (e.g., suspensions, emulsions, elixirs, syrups, lemonades, alcoholic preparations, aromatic preparations, extracts, decoctions, tinctures, etc.), according to conventional methods. Tablets may be sugar-coated tablets, film-coated tablets, enteric-coated tablets, sustained-release tablets, lozenges, sublingual tablets, buccal tablets, chewable tablets, or orally disintegrating tablets; powders and granules may be dry syrups; and capsules may be soft capsules, microcapsules, or sustained-release capsules.

[0099] For parenteral administration, any commonly used dosage form such as injections, infusions, or topical preparations (e.g., eye drops, nasal drops, ear drops, aerosols, inhalants, lotions, injectables, ointments, gargles, enemas, ointments, plasters, jellies, creams, patches, poultices, topical powders, suppositories, etc.) can be suitably administered. Injectable preparations may also be emulsions of O / W, W / O, O / W / O, W / O / W type, etc.

[0100] The dosage and administration of the pharmaceutical product of the present invention can be appropriately selected based on clinically used dosages. For example, the HIV Tat activity inhibitor of the present invention can be administered once a week, once every two weeks, or at intervals of one, two, or three months following the initial dose. For example, the pattern recognition receptor agonist of the present invention can be administered at intervals of one, two, or three months following the initial dose. For example, in a human, the HIV Tat activity inhibitor of the present invention can be administered twice at monthly intervals, followed by ten doses of the pattern recognition receptor agonist of the present invention at monthly intervals. For example, in a human, the HIV Tat activity inhibitor of the present invention can be administered twice at monthly intervals, followed by ten or more doses of the pattern recognition receptor agonist of the present invention at monthly intervals. The dosage and administration of the pharmaceutical product of the present invention can also be determined using indicators such as HIV reservoir size, disappearance of anti-HIV antibodies, and ADCP activity. Furthermore, the efficacy of the pharmaceutical product of the present invention can be confirmed by measuring ADCP activity specific to HIV Tat protein in a biological sample collected from a subject (patient) who has been administered the pharmaceutical product of the present invention. "Biological sample" means a sample collected from a subject who has been administered the pharmaceutical product of the present invention, in which ADCP activity specific to HIV Tat protein can be detected. Examples of "biological samples" include blood samples and lymph nodes. Examples of "blood samples" include whole blood samples, peripheral blood samples, plasma samples, and serum samples.

[0101] ADCP activity can be evaluated using known measurement methods, such as the method described in Example 11 below, using antigen-presenting cells and effector cells, as well as plasma samples collected from subjects administered with the pharmaceutical of the present invention. Furthermore, it can also be evaluated using peripheral blood samples collected from subjects administered with the pharmaceutical of the present invention. By using the ADCP activity value as a biomarker that correlates with the rebound delay effect of the treatment according to the present invention, it is possible to adjust the dosage and administration of the pharmaceutical of the present invention, or to discontinue the administration of pharmaceuticals (A) and (B) of the present invention, or (A) or (B) in the pharmaceutical of the present invention, thereby providing more appropriate treatment to the patient.

[0102] (Embodiment 1) A pharmaceutical agent for the treatment of HIV infection, characterized by combining (A) an HIV Tat protein or its antigenic fragment that induces an anti-HIV Tat antibody that inhibits the activity of the HIV Tat protein secreted from HIV-infected cells; and (B) a pattern recognition receptor agonist that induces type I IFN.

[0103] (Embodiment 2) A pharmaceutical agent for treating HIV infection, characterized by combining: (A) an HIV Tat protein or its antigenic fragment that induces an anti-HIV Tat antibody that inhibits the activity of the HIV Tat protein secreted from HIV-infected cells; and (B) a double-stranded oligonucleotide in which the first chain consists only of an 8-50 base CpG oligonucleotide, and the second chain is a DNA nucleoside of 8-60 bases containing a hybridizable sequence to the first chain, the length of the second chain is 50% or more of the length of the first chain, and a lipid containing a hydrocarbon chain having 12-30 carbon atoms is bound to the second chain via / without a linker;

[0104] (Embodiment 3) A pharmaceutical agent for the treatment of HIV infection, characterized by combining (A) an HIV Tat protein or its antigenic fragment that induces an anti-HIV Tat antibody that inhibits the activity of the HIV Tat protein secreted from HIV-infected cells; and (B) a double-stranded oligonucleotide consisting of a first chain which is ODN2006 and a second chain which is represented by formula (I);

[0105] (Embodiment 4) A pharmaceutical agent for the treatment of HIV infection, characterized by combining (A) a polynucleotide encoding an HIV Tat protein or its antigenic fragment that induces an anti-HIV Tat antibody that inhibits the activity of the HIV Tat protein secreted from HIV-infected cells; and (B) a pattern recognition receptor agonist that induces type I IFNs.

[0106] (Embodiment 5) A pharmaceutical agent for treating HIV infection, characterized by combining: (A) mRNA encoding an HIV Tat protein or its antigenic fragment that induces an anti-HIV Tat antibody that inhibits the activity of the HIV Tat protein secreted from HIV-infected cells; and (B) a double-stranded oligonucleotide in which the first strand consists only of an 8-50 nucleotide CpG oligonucleotide, the second strand is an oligonucleotide in which an 8-60 nucleotide DNA nucleoside containing a hybridizable sequence is bound to the first strand, the length of the second strand is 50% or more of the length of the first strand, and a lipid containing a hydrocarbon chain having 12-30 carbon atoms is bound to the second strand via / without a linker;

[0107] (Embodiment 6) A pharmaceutical agent for the treatment of HIV infection, characterized by combining (A) mRNA encoding an HIV Tat protein or its antigenic fragment that induces an anti-HIV Tat antibody that inhibits the activity of the HIV Tat protein secreted from HIV-infected cells; and (B) a double-stranded oligonucleotide consisting of a first strand which is ODN2006 and a second strand which is represented by formula (I);

[0108] (Embodiment 7) A pharmaceutical agent for the treatment of HIV infection, characterized by combining (A) mRNA encoding an HIV Tat protein or its antigenic fragment, consisting of an amino acid sequence described in any of SEQ ID NOs. 131-148 and 182, which induces an anti-HIV Tat antibody that inhibits the activity of the HIV Tat protein secreted from HIV-infected cells; and (B) a pattern recognition receptor agonist that induces type I IFNs.

[0109] (Embodiment 8) A pharmaceutical agent for treating HIV infection, characterized by combining: (A) mRNA encoding an HIV Tat protein or an antigenic fragment thereof, consisting of an amino acid sequence described in any of SEQ ID NOs: 131-148 and 182, which induces an anti-HIV Tat antibody that inhibits the activity of the HIV Tat protein secreted from HIV-infected cells; and (B) a double-stranded oligonucleotide in which the first strand consists only of an 8-50 nucleotide CpG oligonucleotide, the second strand is an oligonucleotide in which an 8-60 nucleotide DNA nucleoside containing a hybridizable sequence is bound to the first strand, the length of the second strand is 50% or more of the length of the first strand, and a lipid containing a hydrocarbon chain having 12-30 carbon atoms is bound to the second strand via / without a linker;

[0110] (Embodiment 9) A pharmaceutical agent for the treatment of HIV infection, characterized by combining (A) mRNA encoding an HIV Tat protein or its antigenic fragment, which consists of an amino acid sequence described in any of SEQ ID NOs: 131-148 and 182, and (B) a double-stranded oligonucleotide consisting of a first strand which is ODN2006 and a second strand which is represented by formula (I); and (B)

[0111] (Embodiment 10) A pharmaceutical agent for the treatment of HIV infection, characterized by combining (A) mRNA comprising Cap1, a 5'UTR consisting of a nucleic acid sequence described in any of SEQ ID NOs. 1 to 63 and 211 to 218, an open reading frame encoding an HIV Tat protein consisting of an amino acid sequence described in any of SEQ ID NOs. 131 to 148 and 182, a 3'UTR consisting of a nucleic acid sequence described in any of SEQ ID NOs. 64 to 112, and Poly(A), which induces an anti-HIV Tat antibody that inhibits the activity of the HIV Tat protein secreted from HIV-infected cells; and (B) a pattern recognition receptor agonist that induces type I IFN.

[0112] (Embodiment 11) (A) mRNA comprising Cap1, a 5'UTR consisting of a nucleic acid sequence described in any of SEQ ID NOs: 1-63 and 211-218, an open reading frame encoding an HIV Tat protein consisting of an amino acid sequence described in any of SEQ ID NOs: 131-148 and 182, a 3'UTR consisting of a nucleic acid sequence described in any of SEQ ID NOs: 64-112, and Poly(A), which induces an anti-HIV Tat antibody that inhibits the activity of the HIV Tat protein secreted from HIV-infected cells; and (B) a double-stranded oligonucleotide in which the first strand consists only of an 8-50 nucleotide CpG oligonucleotide, the second strand is an oligonucleotide in which an 8-60 nucleotide DNA nucleoside containing a hybridizable sequence is bound to the first strand, the length of the second strand is 50% or more of the length of the first strand, and a lipid containing a hydrocarbon chain having 12-30 carbon atoms is bound to the second strand via / without a linker; A pharmaceutical drug for treating HIV infection, characterized by combining the following.

[0113] (Embodiment 12) A pharmaceutical agent for the treatment of HIV infection, characterized by combining (A) mRNA consisting of Cap1, a 5'UTR consisting of a nucleic acid sequence described in any of SEQ ID NOs: 1 to 63 and 211 to 218, an open reading frame encoding an HIV Tat protein consisting of an amino acid sequence described in any of SEQ ID NOs: 131 to 148 and 182, a 3'UTR consisting of a nucleic acid sequence described in any of SEQ ID NOs: 64 to 112, and Poly(A), which induces an anti-HIV Tat antibody that inhibits the activity of the HIV Tat protein secreted from HIV-infected cells; and (B) a double-stranded oligonucleotide consisting of a first strand which is ODN2006 and a second strand which is represented by formula (I);

[0114] (Embodiment 13) A pharmaceutical agent for the treatment of HIV infection, characterized by combining (A) an open reading frame which is a polynucleotide consisting of a nucleic acid sequence described in any of SEQ ID NOs: Cap1, 5'UTR consisting of a nucleic acid sequence described in any of SEQ ID NOs: 1 to 9, 114 to 119, 121 to 126, and 198 to 210, a 3'UTR consisting of a nucleic acid sequence described in any of SEQ ID NOs: 64 to 69, and 80 mer of Poly(A) mRNA; and (B) a double-stranded oligonucleotide consisting of a first strand which is ODN2006 and a second strand which is represented by formula (I);

[0115] (Embodiment 14) A pharmaceutical agent for the treatment of HIV infection, characterized by combining (A) an open reading frame which is a polynucleotide consisting of a nucleic acid sequence described in any of SEQ ID NOs: Cap1, 5'UTR consisting of a nucleic acid sequence described in any of SEQ ID NOs: 1 to 6, 114 to 119, 121 to 125 and 198 to 203, a 3'UTR consisting of a nucleic acid sequence described in any of SEQ ID NOs: 64 to 69 and 80 mer of Poly(A) mRNA; and (B) a double-stranded oligonucleotide consisting of a first strand which is ODN2006 and a second strand which is represented by formula (I);

[0116] (Embodiment 15) A pharmaceutical agent for the treatment of HIV infection, characterized by combining (A) mRNA consisting of Cap1, a 5'UTR consisting of the nucleic acid sequence described in SEQ ID NO: 1, an open reading frame which is a polynucleotide consisting of the base sequence described in any of SEQ ID NOs: 121, 198, 200, and 202, a 3'UTR consisting of the nucleic acid sequence described in SEQ ID NO: 64, and 80 mer of Poly(A); and (B) a double-stranded oligonucleotide consisting of a first strand which is ODN2006 and a second strand which is a strand represented by formula (I);

[0117] The pharmaceutical product of the present invention may further combine (C) a broad-spectrum HIV neutralizing antibody (bNAb) with each of the above embodiments.

[0118] bNAb (broad-spectrum HIV neutralizing antibody) refers to an antibody that has the function of binding to the Env of many HIV clinical strains and inhibiting infection. Examples include PGT121, VRC01, VRC07-523, N6-LS (VH3810109), Teropavimab (3BNC117, GS-5423), Zinlirvimab (10-1074, GS-2872), GS-8588, CAP256V2, PGDM1400, 8ANC195, 35O22, PGT151, 10E8V, DH511.2_K3, SF12, VRC-PG05, etc. The bNAb incorporated into the pharmaceutical of the present invention has ADCC activity and / or ADCP activity.

[0119] The pharmaceutical product of the present invention can be used as a pharmaceutical product for the treatment of HIV-related diseases such as HIV infection.

[0120] HIV-related diseases include HIV infection, candidiasis, coccidioidomycosis, cryptococcosis, cryptosporidiosis, cytomegalovirus, herpes simplex virus, shingles, histoplasmosis, isosporiasis, and Mycobacterium avium complex. Opportunistic infections selected from Pneumocystis pneumonia, progressive multifocal leukoencephalopathy, Salmonella, toxoplasmosis, tuberculosis, AIDS-related cancers selected from cervical cancer, Kaposi's sarcoma, and lymphoma, candidiasis of the esophagus, bronchi, trachea, or lung, invasive cervical cancer, disseminated or extrapulmonary coccidioidomycosis, extrapulmonary cryptococcosis, chronic enteric cryptosporidiosis, cytomegalovirus disease (other than liver, spleen, or nodule), cytomegalovirus retinitis with visual impairment, HIV-associated encephalopathy, herpes simplex (with chronic ulcers, bronchitis, interstitial pneumonia, or esophagitis), disseminated or extrapulmonary histoplasmosis, chronic enteric isosporiasis, Kaposi's sarcoma, Burkittli Examples include lymphoma, immunoblastic lymphoma, primary brain lymphoma, disseminated or extrapulmonary Mycobacterium avium complex or Kansasi, Mycobacterium tuberculosis in the lung or extrapulmonaryly, disseminated or extrapulmonary Mycobacterium species, Pneumocystis jirobesi pneumonia, recurrent pneumonia, progressive multifocal leukoencephalopathy, recurrent salmonella sepsis, cerebral toxoplasmosis, and HIV-induced wasting syndrome, AIDS-dementia complex, AIDS-induced encephalopathy, HIV encephalopathy, HIV-associated progressive encephalopathy, HIV-associated neurocognitive disorder (HAND), asymptomatic neurocognitive disorder (ANI), mild neurocognitive disorder (MND), HIV-associated dementia (HAD), mild cognitive motor disorder (MCMD), vacuolar myelopathy, peripheral neuropathy, and polymyositis. Particularly preferred is HIV infection.

[0121] "Treatment of HIV infection" means that the increase in the viral load of HIV in the subject's body is suppressed, stopped, paused, or terminated, and that the HIV reservoir or proviral load is reduced, eliminated, or removed from the body. For example, the viral load of HIV may be maintained below the detection limit or at a low level without therapeutic intervention such as anti-HIV drugs. The viral load of HIV in the subject's body can be measured by polymerase chain reaction (PCR) tests, bDNA (branched-chain DNA) tests, NASBA (nucleic acid sequence-based amplification) tests, HIV antigen detection tests (EIA, CLIA, FLIA, WB, etc.), etc. The HIV reservoir size and proviral load can be measured by complete proviral DNA assays (IPDA), quantitative viral outbreak assays (qVOA), etc.

[0122] In one embodiment, the pharmaceutical product of the present invention is administered to a patient whose viral load of HIV RNA in the blood or plasma is maintained below the detection limit. The controlled amount of HIV in the blood or plasma may be any HIV RNA below the detection limit, for example, less than 200 copies, less than 50 copies, or less than 20 copies of HIV RNA per mL. The prescribed period may be at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or 2 years.

[0123] In one embodiment, the viral load of HIV RNA in blood or plasma can be controlled by administering an anti-HIV drug. In other words, the target of administration of the pharmaceutical product of the present invention may have their HIV levels in blood or plasma controlled for a certain period of time by administering an anti-HIV drug.

[0124] Furthermore, upon administration of the pattern recognition receptor agonist of the present invention, a blip may be observed in which HIV is temporarily detected and then again falls below the detection limit. The amount of HIV that is temporarily detected may be any HIV RNA above the detection limit, for example, 200 copies or more, or 500 copies or more of HIV RNA per 1 mL.

[0125] "Anti-HIV drugs" refer to therapeutic drugs that have the activity to prevent HIV infection by inhibiting its adsorption to cells, inhibit the activity of viral enzymes necessary for HIV replication, or inhibit the maturation of HIV viral proteins. Examples include HIV protease inhibitors, HIV non-nucleoside inhibitors of reverse transcriptase, HIV nucleoside inhibitors of reverse transcriptase, HIV nucleotide inhibitors of reverse transcriptase, HIV integrase inhibitors, HIV non-catalytic site integrase inhibitors, gp41 inhibitors, CXCR4 inhibitors, entry inhibitors, CCR5 inhibitors, capsid inhibitors, pharmacokinetic enhancers, RNase H inhibitors, bNAbs, drugs containing one or more compounds selected from the group consisting of pharmaceutically acceptable salts thereof, and vaccines that induce bNAbs.

[0126] While a single anti-HIV drug may be used, it is preferable to select two or more drugs with different mechanisms of action and perform combination therapy (cART). Examples of anti-HIV drugs used in combination therapy include those listed in the Anti-HIV Treatment Guidelines ([Accessed August 9, 2024], Internet).<URL:https: / / hiv-guidelines.jp / 2024 / part01.htm> (Reference), Guidelines for the Use of Antiretroviral Agents in Adults and Adolescents with HIV ([Accessed August 9, 2024], Internet, <URL:https: / / clinicalinfo.hiv.gov / sites / default / files / guidelines / documents / adult-adolescent-arv / recommendations-adult-adolescent-arv.pdf> (Reference), European AIDS Clinical Society's EACS Guidelines ([Accessed August 9, 2024], Internet,<URL:https: / / www.eacsociety.org / media / guidelines-12.0.pdf> Examples of treatments recommended in reference, etc. are listed. Preferably, the anti-HIV drug is one or more compounds selected from the group consisting of HIV integrase inhibitors, HIV reverse transcriptase nucleotype inhibitors, HIV reverse transcriptase non-nucleotype inhibitors, HIV protease inhibitors, HIV capsid inhibitors, HIV non-catalytic site integrase inhibitors, entry inhibitors, bNAbs, and pharmaceutically acceptable salts thereof. More preferably, it is two or more drugs that combine one or more drugs selected from HIV integrase inhibitors and one or more drugs selected from HIV reverse transcriptase nucleotype inhibitors.More preferably, the combination is one or more drugs selected from the group consisting of dolutegravir, bictegravir and pharmaceutically acceptable salts thereof, which are HIV integrase inhibitors; one or more drugs selected from the group consisting of tenofovir, abacavir and pharmaceutically acceptable salts thereof, which are nucleoside inhibitors of HIV reverse transcriptase; and one or more drugs selected from the group consisting of emtricitabine, lamivudine and pharmaceutically acceptable salts thereof, which are nucleoside inhibitors of HIV reverse transcriptase. More preferably, the combination is one or more drugs selected from the group consisting of dolutegravir, lamivudine and pharmaceutically acceptable salts thereof; one or more drugs selected from the group consisting of dolutegravir, lamivudine, abacavir and pharmaceutically acceptable salts thereof; or one or more drugs selected from the group consisting of bictegravir, tenofovir, emtricitabine and pharmaceutically acceptable salts thereof. If there are two or more anti-HIV drugs, the combination may include each drug or use each drug as a combination agent. Anti-HIV drugs can be administered orally or parenterally. They may also be injectable, including sustained-release injectable formulations such as cabotegravir, rilpivirine, and lenacapavir.

[0127] The pharmaceuticals of the present invention can also be used in combination with other pharmaceuticals. Examples include LRAs and bNAbs with different mechanisms of action than pattern recognition receptor agonists, HIV infection treatment vaccines, and CAR-T. LRAs include IAPi, which stimulate the non-classical NF-κB pathway, thereby expressing cytokines and causing reactivation of proviruses inserted nearby (i.e., reactivation of HIV-latent infected cells). However, further administration of bNAbs is effective for HIV-infected patients who are taking anti-HIV drugs other than bNAbs.

[0128] The pharmaceutical product of the present invention may also be used in combination with pharmaceutical products for the treatment or prevention of other diseases other than HIV infection (e.g., bacterial or fungal infections, other viral infections such as hepatitis B or C, cancer). Additional therapeutic agents may be compounded with the pharmaceutical product of the present invention.

[0129] The pharmaceutical product of the present invention has any or all of the following excellent features: a) Induces anti-HIV Tat antibodies with high neutralizing activity. b) Induces HIV Tat-specific CD8-positive T cells and CD4-positive T cells. c) Induces HIV Tat-specific antibodies with ADCC activity and ADCP activity. d) When administered to HIV-infected patients taking anti-HIV drugs, it causes a decrease in the reservoir size of HIV virus in the blood. e) When administered to HIV-infected patients taking anti-HIV drugs, it causes a delay in the rebound of HIV virus in the blood.

[0130] The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto.

[0131] Example 1 Synthesis of lipid-binding double-stranded oligonucleotide (OLIGO) A double-stranded oligonucleotide (OLIGO) was synthesized according to the method described in Example 1 of Patent Document 3, in which the branched lipid-binding oligonucleotide is "a double-stranded oligonucleotide consisting of a first chain which is ODN2006 and a second chain which is represented by formula (I)". The activity of OLIGO was evaluated by a reporter assay according to the method described in Example 2 of Patent Document 3. 50% activation concentration (EC) against the human TLR9 gene ５０ The value was 436.7 (nM).

[0132] Example 2 Synthesis of Tat mRNA HIV Tat mRNA with 5'UTR, ORF, 3'UTR, Poly(A), and modified bases as described in Table 1 was synthesized. The 5'Cap structure of each Tat mRNA is Cap1, and is represented as 5'-[m7G]-ppp-GmGG. Here, Gm represents guanosine with methylated 2' position. Specifically, it is represented by the following structure. In all Tat mRNAs, all uridine bases in the sequence are replaced with N1-methylpseudridine (m1Ψ) bases. In the table, SNG-327 represents mRNA encoding IgE HIV-Tat Oyi protein (SEQ ID NO: 174), in which secretory signal peptide IgE (SEQ ID NO: 161) is ligated to the N-terminus of HIV-Tat Oyi protein (SEQ ID NO: 133). SNG-327 is mRNA consisting of a 5' Cap structure consisting of 5'-[m7G]-ppp-GmGG in the 5'-3' direction, a 5' UTR represented by SEQ ID NO: 1, an ORF represented by SEQ ID NO: 121, a 3' UTR represented by SEQ ID NO: 64, and approximately 80 mer of Poly(A). The other mRNAs in Tables 1 and 2 are similar. Note that Mod-Oyi refers to the Tat Oyi protein with a mutation in the RGD motif, while Oyi refers to the wild-type Tat Oyi protein.

[0133] Furthermore, we purchased SIV Tat mRNA from Trilink, which has the structure described in Table 2 and in which all uridine bases in the sequence are replaced with N1-methylpseudridine (m1Ψ) bases.

[0134]

[0135] Example 3 Preparation of mRNA-encapsulated nucleic acid lipid particles using Tat mRNA Cationic lipids described in International Publication No. 2022 / 168884 (hereinafter referred to as Lipid), distearoylphosphatidylcholine (1,2-Distearoyl-sn-glycero-3-phosphacholine: hereinafter referred to as DSPC, NOF CORPORATION), Cholesterol (hereinafter referred to as Chol, NIPPON FINE CHEMICAL CO., LTD.), and polyethylene glycol with a molecular weight of approximately 2000, 1,2-Dimyristoyl-sn-Glycero-3-Methoxypolyethylene Glycol (hereinafter referred to as PEG-DMG, NOF A lipid solution was obtained by dissolving CORPORATION) in ethanol in a molar ratio of DSPC:Chol:Lipid:PEG-DMG = 8.4:36:54:1.6 to a total lipid concentration of 33 mM. On the other hand, the Tat mRNA synthesized in Example 2 was prepared to 292 μg / mL in citrate buffer (pH 4.0) to obtain an mRNA solution. The above lipid solution and mRNA solution were mixed in a volume ratio of 1:3 using NanoAssemblr® IGNITE. ＴＭ A crude dispersion of nucleic acid lipid particles was obtained by mixing in a microfluidic channel using Precision Nanosystems Inc. The crude dispersion of nucleic acid lipid particles and phosphate buffer (pH 7.5) were mixed in a volume ratio of 2:1 using in-line dilution (using a microfluidic cartridge IGNITE NxGen mixer with dilution function) to obtain a dispersion of nucleic acid lipid particles. The dispersion of nucleic acid lipid particles was dialyzed with approximately 100 times the volume of 20 mM Tris-HCl buffer for 3 hours, and then dialyzed again with approximately 100 times the volume of 20 mM Tris-HCl solution for 12 to 18 hours (Slide-A-Lyzer). ＴＭEthanol was removed by Dialesis Cassette G2, MWCO: 10 kD, Spectra / Po, and the solution was concentrated using Amicon (Amicon® Ultra-15 PLHK Ultracel-PL membrane, MWCO 100 kDa, Merck). After adjusting the concentration with 20 mM Tris-HCl buffer, it was mixed with an additive stock (dispersion: 20 mM Tris-HCl buffer and 1.05 mM Sucross mixed in a volume ratio of 1:2) to obtain a dispersion of purified mRNA-encapsulated nucleic acid lipid particles (mRNA vaccine) in 20 mM Tris-HCl buffer and 350 mM Sucross solution.

[0136] Example 4 Reactivation test of HIV latent infected cells by OLIGO in a SIV chronically infected cynomolgus monkey model (4-1) Preparation of SIV chronically infected cynomolgus monkeys When infecting cynomolgus monkeys with SIVmac239, general anesthesia was administered by intramuscular injection of Ketalar (registered trademark) (Daiichi Sankyo Propharma Co., Ltd.) (5-10 mg as ketamine), and 5 x 10 ４ SIVmac239 at TCID50 (Median tissue culture infectious dose, 50% infectious dose) was administered intravenously via the saphenous vein. All monkeys used in the infection experiments tested negative for simian immunodeficiency virus, simian retrovirus type D, simian T-cell leukemia virus, sulfomyvirus, Epstein-Barr virus, cytomegalovirus, and B virus. Clinical arterial therapy (CART) was initiated 23 to 63 weeks after infection, with subcutaneous administration of 10 mg / kg dolutegravir, 60 mg / kg tenofovir, and 120 mg / kg emtricitabine in a 6-day continuous administration followed by a 1-day break.

[0137] (4-2) Reactivation test of HIV latent infected cells using OLIGO Cynomolgus monkeys in which the blood viral load was suppressed to below the detection limit by cART using the method in (4-1) were subcutaneously administered 1 mg / kg of OLIGO once a week for a total of 10 times. On the day after each OLIGO administration, blood was collected from the femoral vein in the inguinal region into a Venojet® II vacuum blood collection tube (TERUMO) containing EDTA-2Na. The blood collection tube was centrifuged (780 g, 25 minutes, 25°C) to separate the plasma and cells. The plasma was dispensed into screw tubes and stored at -80°C. The amount of viral RNA in the plasma of SIV-infected cynomolgus monkeys was measured by real-time qRT-PCR. Viral RNA was purified from plasma using the MagNA PureCompact Nucleic Acid Isolation Kit (Roche Diagnostics). Real-time qRT-PCR was performed using the QuantiTec Probe RT-PCR Kit (Qiagen) and the LightCycle® 480 thermocycle (Roche Diagnostics). The gag gene of SIVmac239 was amplified using primer 1: 5'-GCAGAGGAGGGAAAAATTACCCAGTAC-3' (SEQ ID NO: 219) and primer 2: 5'-CAATTTTAACCCAGGCATTTTAATGTT-3' (SEQ ID NO: 220), and 5'-FAM-TGTCCCACCTGCCCATTAAGTCCCGA-TAMRA-3' (SEQ ID NO: 221) was used as the probe. Here, FAM is 6-carboxyfluorescein and TAMRA is 6-carboxytetramethylrhodamine. The copy number of viral RNA was 3 x 10⁻¹⁶, using SIV gag RNA as the standard sample. ５ The calculation was performed using a calibration curve obtained from a 10-fold dilution series starting from copies / μL. The detection limit was set to 45 copies / mL.

[0138] When a viral load was detected, it was determined that reactivation of HIV-latent infected cells had occurred, and the number of times this occurred was evaluated. The results are summarized in Table 3. In the table, % (N) for cynomolgus monkeys means the number of individuals in which blip was observed at least once, and % for blip count means the number of blips observed relative to the total number of OLIGO doses administered per monkey (10 doses). The ability of OLIGO, a double-stranded oligonucleotide according to the present invention, to reactivate HIV-latent infected cells was equivalent to or better than the excellent ability of AZD5582 to reactivate HIV-latent infected cells, as shown in Non-Patent Literature 4.

[0139]

[0140] Example 5 Comparative study of antibody induction ability of Tat peptide vaccine and Tat mRNA using mice Six mice were intramuscularly injected with either 30 μg of Tat BH10 peptide (SEQ ID NO: 134) or Tat Oyi peptide (SEQ ID NO: 133), dissolved in PBS, into the hind limb thigh muscle three times at 4-week intervals. For comparison, six mice were administered an mRNA vaccine containing 25 μg of SNG-327. Two weeks after the third immunization, total blood was collected from the abdominal vena cava using a syringe with a needle containing 10 μL of heparin, and the blood was centrifuged in a refrigerated centrifuge (TOMY MX-305) (20,400 g, 5 minutes, 4°C) to obtain plasma. The anti-Tat antibody titer in the plasma was measured by ELISA. Tat Oyi peptide (SEQ ID NO: 133) was prepared at a concentration of 1 μg / mL using PBS (Gibco) at pH 7.4. 25 μL of the prepared solution was dispensed into 384-well plates, the plates were sealed, and left to stand overnight at 4°C. The following day, the antigen solution was removed from the plates, and 90 μL of washing buffer (PBS-T (TAKARA)) was added per well, followed by two washes. 50 μL of blocking buffer (1% BSA (SIGMA) / PBS) was added per well to the washed plates, and they were left to stand at room temperature. After one hour, the blocking buffer was removed from the plates, and 90 μL of washing solution was added per well, followed by three washes. Plasma was diluted with dilution buffer (1% BSA / PBS) and added at a rate of 25 μL per well to the washed plates, and the same conditions were applied to 2 wells of each plate. The plates were then left to stand at room temperature. After 2 hours, the plasma sample was removed from the plate, and the plate was washed three times with 90 μL of washing solution per well. Diluted HRP-labeled goat anti-mouse IgG Fc antibody (Jackson Immuno Research) was added to the washed plate as a secondary antibody at a rate of 25 μL per well, and the plate was left to stand at room temperature. After 1 hour, the secondary antibody solution was removed from the plate, and the plate was washed three times with 90 μL of washing solution per well. TMB solution (Thermo Scientific) was added to the washed plate at a rate of 25 μL per well, and the plate was left to stand at room temperature. The reaction was stopped by adding 25 μL of 2N sulfuric acid (Fujifilm Wako) per well for 10 minutes. The absorbance at 450 nm was measured using a spectrophotometer. The anti-Tat antibody titer was calculated using the following method.The absorbance of the well sample with only dilution buffer added was used as the blank, and the cutoff value was set by adding 0.5 to the average value of the blank. In the dilution series of plasma samples, the dilution factor at which the average absorbance of N=2 corresponds to the cutoff value was calculated as the anti-Tat antibody titer, and the GMT (Geometric Mean Titer) for each group was calculated. The results are shown in Table 4.

[0141] mRNA vaccines provide immunity with two doses. ５ from 10 ６ While antibodies with a titer of 1 could be induced, in the case of Tat BH10 peptide or Oyi peptide, even after 3 immunizations, the titer was only 10. ５ The antibody induction ability was limited. Therefore, it was shown that Tat mRNA has superior antibody induction ability compared to Tat peptide.

[0142]

[0143] Example 6 Evaluation of cellular immunity induction ability by Tat mRNA in mice ELISPOT plates (MABTECH: IFNγMulti, IL-4, IL-5 ELISPOT plates) were washed four times with 200 μL of PBS(-), then 200 μL of Complete Culture Medium was added and the plates were allowed to stand at room temperature for at least 30 minutes. DAPI staining solution was added to spleen cell suspension collected from vaccinated mice, and the number of cells was counted. The spleen cell suspension was centrifuged (470 g, 5 min), and 3 x 10⁶ cells were collected in Complete Culture Medium. ６ The solution was prepared to cells / mL. Complete Culture Medium containing Tat Oyi peptide was added at 100 μL / well after removing the Complete Culture Medium from the above-mentioned ELISPOT plate. Furthermore, 100 μL / well of a separately prepared spleen cell suspension was seeded and left to stand at room temperature for 30 minutes. After that, it was incubated in a carbon dioxide incubator at 37°C and 5% CO2. ２The cells were cultured for two days under the specified conditions. The ELISPOT plates were washed five times with 300 μL of PBS using Multidrop 384 (Thermo Scientific). Then, 100 μL / well of antibodies to detect IFNγ (Anti-mouse IFN-γ mAb (R4-6A2), biotin (Mabtec)), IL-4 (Anti-mouse IL-4 mAb (BVD6-24G2), biotin (Mabtec)), and IL-5 (Anti-mouse IL-5 mAb (TRFK4), biotin (Mabtec)) were added to each ELISPOT plate. After incubating at room temperature for 2 hours, the ELISPOT plate was washed five times with 300 μL of PBS using Multidrop 384 (Thermo Scientific), then 100 μL / well of Streptabidin-ALP solution was added, and the reaction was allowed to proceed at room temperature for 1 hour. After washing the ELISPOT plate five times with 300 μL of PBS using Multidrop 384 (Thermo Scientific), 100 μL / well of BCIP / NBT substrate solution was added, and the reaction was stopped by adding water after 4 minutes. The results were measured using a Novosysteme Advanced Flow Cytometer (AGILENT) and analyzed.

[0144] Compared to SNG-299 and SNG-323, which encode Tat Oyi proteins with mutations in the RGD motif, SNG-325 and SNG-327, which encode wild-type Tat Oyi proteins, showed higher activation ability in both TH1 cells (IFN-γ) and TH2 cells (IL-4 and IL-5) (Figure 1).

[0145] Example 7 Combined administration study of TLR9 agonist and Tat mRNA vaccine in an SIV-infected cynomolgus monkey model (7-1) Concept confirmation study in an SIV-infected cynomolgus monkey model Cynomolgus monkeys were infected with SIV according to the protocol described in (4-1) of Example 4, and the first dose of 100 μg of SNG-3 was administered 15-29 weeks after the start of cART. The control group was administered the same amount of PBS. General anesthesia was administered by intramuscular injection of Ketalar® (Daiichi Sankyo Propharma Co., Ltd.) (5-10 mg as ketamine) at the time of vaccination. The second dose was administered 4 weeks later. From 2 weeks thereafter, a total of 10 doses of OLIGO or PBS were administered by subcutaneous injection once a week, and cART was discontinued 1 week after the final dose (Figure 2). The treatment from the administration of the Tat mRNA vaccine to the final administration of OLIGO is referred to as "the treatment according to the present invention." Cynomolgus monkeys infected with SIV were divided into four groups: a control group of 2 monkeys administered only PBS, an OLIGO-administered group of 7 monkeys administered only OLIGO, a SIV Tat mRNA vaccine group of 5 monkeys administered SIV Tat mRNA vaccine and PBS, and a combined administration group of 7 monkeys administered SIV Tat mRNA vaccine and OLIGO. Blood was collected from the cynomolgus monkeys at one or two-week intervals from the femoral vein in the inguinal region, and collected in Venojet II vacuum blood collection tubes (TERUMO) containing EDTA-2Na. The blood collection tubes were centrifuged using a centrifuge (TOMY AX-310) (780g, 25 minutes, 25°C) to separate plasma and cells. Plasma was dispensed into screw tubes and stored at -80°C. The leukocyte fraction was collected in a 15 mL centrifuge tube, and erythrocytes were lysed with BD Pharma Lyse (Becton Dickinson). The cells were then centrifuged using a TOMY AX-310 centrifuge (780 g, 5 minutes, 25°C), and the resulting cells were suspended in 3.5 mL of RPMI-1640 medium containing 10% FBS and penicillin streptomycin. The suspension was overlaid on 6.5 mL of Ficol-Paque PLUS (Cytiva) and centrifuged using a TOMY AX-310 centrifuge (1200 g, 15 minutes, 25°C).Subsequently, peripheral blood mononuclear cells were suspended in FBS containing 10% DMSO, frozen to -80°C using a programmable freezer (Strex Corporation), and immediately stored in a liquid nitrogen tank for use in various evaluations.

[0146] (7-2) Measurement of Antibody Titer of Anti-SIV Tat Antibody To confirm that anti-Tat antibodies were induced by the administration of SNG-3, analysis was performed using the ELISA method. Chemically synthesized SIV Tat protein (PeptiStar Co., Ltd.) was immobilized on a 96-well plate as the antigen, and serially diluted cynomolgus monkey plasma was bound to it to measure the antibody titer. The titer was measured using an absorbance meter, and the dilution ratio showing 0.1 OD was defined as the antibody titer.

[0147] (7-3) Assay to inhibit Tat function by anti-Tat antibodies The Tat function inhibition assay was used to evaluate whether the induced anti-Tat antibodies could inhibit Tat function. It is known that Tat protein secreted from infected cells is taken up by uninfected cells and induces apoptosis (EMBO J. 2002 Dec 16; 21(24): 6801-6810. etc.). In the Tat inhibition assay, when chemically synthesized SIV Tat protein is added to a culture medium of MT4 cells in which the luciferase gene is incorporated downstream of the SIV LTR, transcriptional activity is enhanced by Tat and downstream luciferase is expressed. When purified anti-Tat antibody is added to this culture system, the uptake of Tat into cells is inhibited and luciferase activity is reduced or eliminated. The Tat function inhibitory activity of the anti-Tat antibody was evaluated by utilizing this property (Figure 3). 1 x 10⁶ cells in 384 wells ４MT-4 / LTR-Luc cells were seeded at 40 μL / well. Plasma from multiple individuals assigned to either the SIV Tat mRNA vaccine group or the combination therapy group was mixed and purified using a column (Thermo Fisher Scientific, NAb® Protein A / G Spin Kit, 0.2 mL) for evaluation. 4 μL / well of serially diluted plasma samples from each group were added to the cell seeded wells and mixed to prepare final concentrations of 180, 60, 20, 6.7, 2.2, and 0.74 μg / mL. 0.8 μL of Tat Oyi S22C solution (Peptide Laboratory, Lot No. 992-10651, 0.175 μg / μL) was added and stirred, and the mixture was incubated at 37°C for 5 to 7 hours. 10 μL of ONE-Glo Reagent (ONE-Glo Luciferase Assay System (Promega)) was added and stirred, and the mixture was allowed to stand at room temperature for 10 minutes. The activity of luciferase induced by Tat was measured using Enspire (PerkinElmer), and the inhibition rate at each test sample concentration was determined by substituting the measured values ​​into the following formula to find the percentage of activity reduction due to the addition of anti-Tat antibody. IC50 (μg / mL) was then determined using ExcelFit (XLfit® 5.3.1.3). Inhibition rate = (Value when plasma sample is added) / (Value of positive control (with Tat added, without plasma sample)) × 100 The neutralizing activity of purified IgG antibodies was investigated immediately before the first administration of OLIGO (OLIGO_Shot01), immediately before the fifth administration (OLIGO_Shot05), and immediately before the tenth administration (OLIGO_Shot10), which are the peaks of anti-Tat antibody induction by the SIV Tat mRNA vaccine. As a result, neutralizing activity was confirmed in all individuals administered the SIV Tat mRNA vaccine. Figure 4 shows the neutralizing activity data (IC50 (μg / mL)) for monkey individuals Mon#001, Mon#002, and Mon#003. Furthermore, it was confirmed that the neutralizing activity correlates with the antibody titer, and a similar correlation was confirmed for the mRNA according to the present invention. In this test protocol, the antibody titer was 10 ３ Based on the above, we were able to determine the IC50 of the Tat inhibitory activity.

[0148] Example 8 Evaluation of Rebound Delay Effect After cART Interruption After treatment according to the invention described in Example 7 (7-1), blood samples were taken weekly or bi-weekly, the amount of virus in the plasma was measured, and the rebound delay effect was analyzed. Rebound was determined if the virus was detected in the plasma in two consecutive tests. The detection limit here was 45 copies / mL. The results are summarized in Table 5. In the control group, rebound occurred on average in 22 days (15-29 days), while in the OLIGO administration group, rebound occurred on average in 25 days (15-29 days), and no delay effect was observed. On the other hand, in the Tat mRNA vaccine administration group, the average was 46.8 days (21-141 days), showing a delay effect of about twice as long. Furthermore, in the group receiving OLIGO and Tat mRNA vaccine in combination, a delay effect of approximately three times was observed, with an average of 60.9 days (7 to 141 days, i.e., 7 days to 141 days or more), and no rebound was observed in 2 out of 7 dogs for 141 days or more after cART discontinuation (Table 5). In the table, the number in parentheses in the column for "No rebound for 141 days" represents the percentage of individuals assigned to each group that did not rebound for 141 days. The number in parentheses in the column for "Average time to rebound" represents the range between the minimum and maximum time to rebound for individuals assigned to each group.

[0149]

[0150] Example 9 Measurement of Intact Provirus SIV DNA in Peripheral Blood Mononuclear Cells (PBMCs) To estimate the reduction in SIV reservoir size in the blood due to the treatment according to the present invention, blood was collected from each individual at two or three time points (before treatment according to the present invention) 17 to 22 weeks prior to the treatment interruption point corresponding to the administration of the Tat mRNA vaccine, and at one time point (after treatment according to the present invention) at the time of cART treatment discontinuation. DNA was separated from each PBMC sample using a commercially available nucleic acid extraction kit (QIAGEN). The amount of intact provirus SIV DNA in the extracted DNA that was free from fatal deletions or mutations and capable of viral replication was measured as the reservoir size using Intact Provirus DNA Assay (IPDA). IPDA is a measurement method that uses three types of duplex droplet digital PCR (ddPCR). The first type is a primer / probe set specific to two regions in the ribbonuclease P / MRP subunit p30 (RPP30) gene (RPP30-1F: 5'-AGGATGCTCCGGGAGTATGTA-3' (SEQ ID NO: 222), RPP30-1R: 5'-CCTGCTTTGTCAACCTATATAACAT-3' (SEQ ID NO: 223), RPP30-1 ddPCR was performed using prove:5'-FAM-TCAAGCTGGGAGAACGGGAAGAGTCCAGT-ZEN / IABkFQ-3' (SEQ ID NO: 224), RPP30-2P:5'-ACAGACTCAACACAATTTTAGG-3' (SEQ ID NO: 225), RPP30-2R:5'-ACATTCATGCCAACTGCACTC-3' (SEQ ID NO: 226), and RPP30-2 prove:5'-HEX-ACAGGGTCTCCAACTTTTTGTTCCA-ZEN / IABkFQ-3' (SEQ ID NO: 227). The measured values ​​were used for cell count and correction calculations for genomic DNA breaks that occur during DNA extraction.The second type is a primer / probe set specific to the wild-type sequences of the SIV pol gene and SIV env gene (polF: 5'-GCAGGGGATAGAGCACACCTTTG-3' (SEQ ID NO: 228), pollR: 5'-CTATGGTTTTCTACTGAATTTGCTTTTTC-3' (SEQ ID NO: 229), poll intact probe: 5'-FAM-TTTCCAGGTGGTGATTCA-MGB-3' (SEQ ID NO: 230), poll hyper probe: 5'-TAGGTGGTGATTTTATTT-MGB-3' (SEQ ID NO: 231), env F: 5'-CCTCCAATAAAGCCTTGTGTTAAAAATTATC-3' (SEQ ID NO: 232), env In ddPCR using R:GTGTTTATTTGATTTTGTCAATCCC-3' (SEQ ID NO: 233), env intact probe:5'-VIC-TGCATTACTATGAGAATGC-MGB-3' (SEQ ID NO: 234), and env hyper probe:5'-TGCATTACTATAAAATGC-MGB-3' (SEQ ID NO: 235), the double-positive measurement where both sequences are detected was defined as the amount of SIV DNA without mutations or deletions. The third type is a primer / probe set specific to the wild-type sequence of the SIV 2LTR region and the SIV env gene (2LTR_F: 5'-GCAGGGGATAGAGCACACCTTTG-3' (SEQ ID NO: 236), 2LTR_R: 5'-CTATGGTTTTCTACTGAATTTGGCTTTTC-3' (SEQ ID NO: 237), 2LTR_probe: 5'-FAM-CCCTGGCTGTTTAGGAACCCTTTTTCTGCTTTTG-ZEN / IABkFQ-3' (SEQ ID NO: 238), env F (SEQ ID NO: 232), env R (SEQ ID NO: 233), env intact probe (SEQ ID NO: 234), env hyper Using ddPCR with probe (SEQ ID NO: 235), the amount of SIV 2LTR circle present in a state that is not inserted into chromosomes and does not produce virus was measured and subtracted from the results of the second measurement. From these three ddPCR measurements, the amount of intact provirus SIV DNA per 1 million PBMCs was calculated.Measurements at two or three time points before Tat mRNA vaccine administration were calculated as geometric mean values ​​for each individual and used for comparative analysis. The change in reservoir size from before Tat mRNA vaccine administration to the time of treatment discontinuation was calculated as log reduction, and the reduction effect of Tat mRNA vaccine and OLIGO added to cART was evaluated by subtracting the median value of the control group from each individual's value. The degree of reservoir size reduction in each group was greatest in the combination therapy group, with a median reduction of -0.53 log (Table 6). On the other hand, the OLIGO monotherapy group showed a median reduction of -0.16 log, and the vaccine monotherapy group showed a median reduction of -0.23 log. These results confirm that combination therapy with vaccine and OLIGO is the most effective in reducing SIV reservoir size.

[0151]

[0152] Example 10 Clinical Trial Protocol The safety and immunogenicity of the pharmaceutical product of the present invention can be evaluated by the following clinical trial protocol. The trial will be conducted as a placebo-controlled, double-blind study in subjects infected with HIV and undergoing cART treatment. Subjects will have maintained an HIV RNA concentration of 50 copies per mL or less in their blood or plasma for at least 6 months. The study will consist of three cohorts (Cohorts A to C). Cohort A will receive two intramuscular administrations of SNG-327 once a month, followed by ten subcutaneous administrations of OLIGO once a month. Cohort B will receive two intramuscular administrations of placebo (saline) once a month, followed by ten subcutaneous administrations of OLIGO once a month. Cohort C will receive two intramuscular administrations of SNG-327 once a month, followed by ten subcutaneous administrations of placebo (saline). The primary endpoint of the study is the change in HIV reservoir size in blood or plasma at the start of administration and at day 366. If a decrease in reservoir size is observed, cART treatment will be discontinued, and the time until the amount of HIV in blood or plasma rebounds will be measured. HIV reservoir size can be measured by extracting DNA from PBMCs isolated from each subject's blood and performing IPDA using an appropriate primer-probe set.

[0153] Example 11 Evaluation of ADCC activity and ADCP activity In order to search for biomarkers that correlate with the rebound delay effect of the treatment according to the present invention, ADCC activity and ADCP activity were evaluated using plasma samples from groups 3 and 4 used in the test of Example 8, according to the following protocol.

[0154] The ADCC assay and ADCP assay are based on Jurkat-Lucia ＴＭ NFAT-CD16 cells and THP-1-Lucia ＴＭ The procedure was performed using NKκB cells (InvivoGen). First, a plasmid expressing SIV Tat protein was introduced into HEK239A cells using 293fectin. ＴＭ SIV Tat-expressing cells were obtained by transfection using a transfection reagent (Thermo Fisher Scientific). Subsequently, 2.0 x 10⁶ cells per well were transferred to a flat-bottom 96-well cell culture plate. ４ Seed at cell density, 37°C, 5% CO2 ２ The cells were incubated overnight under the specified conditions. The following day, diluted plasma samples from groups 3 and 4 used in Example 8 were added, and the cells were incubated at 37°C in 5% CO2. ２ The cells were cultured for 1 hour under the specified conditions. Afterward, they were washed twice with PBS to remove the effector cells (Jurkat-Lucia). ＴＭ NFAT-CD16 cells or THP-1-Lucia ＴＭ NKκB cells) 1.0 x 10 ５ Individual seeds were sown. After 12 hours of incubation, the culture supernatant and QUANTI-Luc were used. ＴＭLucia / Gaussia (InvivoGen) was mixed, and luminescence was measured using an EnSpire Multimode Plate Reader (PerkinElmer). Tat-specific ADCC activity was similar in the group that rebounded immediately after cART interruption (8 individuals) and the group that rebounded late (4 individuals) (4463 vs. 4623). In contrast, Tat-specific ADCP activity was significantly higher in the group that rebounded late compared to the group that rebounded immediately after cART interruption (19207 vs. 53647, p-value = 0.0485) (Figure 5). This result suggests that ADCP activity correlates with rebound delay.

[0155] The pharmaceutical product of the present invention may be useful for treating HIV infection.

Claims

1. A pharmaceutical agent for the treatment of HIV infection, characterized by combining (A) a polynucleotide or polypeptide that induces an anti-HIV Tat antibody that inhibits the activity of the HIV Tat protein secreted from HIV-infected cells; and (B) a pattern recognition receptor agonist that induces type I interferon.

2. The pharmaceutical product according to claim 1, wherein (B) is (B-1) or (B-2). (B-1) A double-stranded oligonucleotide in which the first chain consists only of an 8-50 base CpG oligonucleotide, the second chain is an oligonucleotide in which an 8-60 base DNA nucleoside containing a hybridizable sequence is bound to the first chain, the length of the second chain is 50% or more of the length of the first chain, and a lipid containing a hydrocarbon chain having 12-30 carbon atoms is bound to the second chain via or without a linker. (B-2) At least one compound selected from the group consisting of ODN2006, lephytrimod, besatrimod, tylsotrimod, and STING ligand.

3. (B) is (B-1), where (B-1) is a double-stranded oligonucleotide consisting of a first strand and a second strand, and the first strand is represented by the following formula: Formula: The pharmaceutical product according to claim 2, wherein the second chain is a double-stranded oligonucleotide represented by the following formula (I). Formula (I):

4. The pharmaceutical product according to any one of claims 1 to 3, wherein (A) is a polynucleotide encoding the HIV Tat protein or an antigenic fragment thereof, or the HIV Tat protein or an antigenic fragment thereof.

5. The pharmaceutical product according to claim 4, wherein a secretion signal peptide is further linked to the HIV Tat protein.

6. The pharmaceutical product according to claim 5, wherein the secretory signaling peptide is a polypeptide comprising the amino acid sequence described in any of SEQ ID NOs: 161-167 and 183-187.

7. The pharmaceutical product according to any one of claims 4 to 6, wherein the HIV Tat protein is a polypeptide consisting of an amino acid sequence that is identical to, or has 90% or more identity with, the amino acid sequence described in any of SEQ ID NOs. 131 to 148 and 182.

8. The pharmaceutical product according to any one of claims 1 to 7, wherein (A) is mRNA containing an open reading frame encoding the HIV Tat protein or an antigenic fragment thereof, and comprises at least one modified base selected from the group consisting of 5-methyluridine, pseudouridine, N1-methylpseudridine, 5-methoxyuridine, 2-thiouridine, 6-methyladenosine, inosine, and 5-methylcytidine.

9. The pharmaceutical product according to claim 8, comprising a 5'Cap structure in which the mRNA is Cap1 or Cap2.

10. The pharmaceutical product according to claim 8 or 9, wherein the mRNA contains 60 to 120 mer of Poly(A).

11. The pharmaceutical product according to any one of claims 8 to 10, wherein the mRNA comprises a 5'UTR which is a polynucleotide consisting of a base sequence identical to or having 90% or more identity with any of the base sequences described in SEQ ID NOs: 1 to 63 and 211 to 218.

12. The pharmaceutical product according to any one of claims 8 to 11, wherein the mRNA comprises a 3'UTR which is a polynucleotide consisting of a base sequence identical to or having 90% or more identity with the base sequence described in any of sequence numbers 64 to 112.

13. The pharmaceutical product according to any one of claims 8 to 12, wherein the open reading frame is a polynucleotide consisting of any of the base sequences described in SEQ ID NOs: 114-119, 121-126, and 198-210.

14. A pharmaceutical product according to any one of claims 1 to 13, wherein (A) and (B) are administered in combination.

15. A pharmaceutical product for use in combination with (B) according to claim 1, comprising (A) according to claim 1 as an active ingredient.

16. A pharmaceutical product for use in combination with (A) according to claim 1, comprising (B) according to claim 1 as an active ingredient.

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