JP6501247B2 - Immunostimulatory polynucleotide / schizophyllan complex and pharmaceutical composition containing the same - Google Patents

Description

The present invention relates to novel immunostimulatory polynucleotide / schizophyllan complexes and pharmaceutical compositions comprising the same.

  The basic principle of preventing infection by a vaccine is to induce acquired immunity by artificial artificial infection, and to induce antibody production and cellular immunity against specific pathogens. In acquired immunity, T cells and B cells responsible for immune "memory" play a central role, and the diversity of antibody variable regions due to DNA rearrangement causes specific immune responses to numerous antigens. It is known to make it possible. On the other hand, innate immunity in which phagocytes such as white blood cells, macrophages and dendritic cells play a central role has hitherto been a nonspecific process of phagocytosing pathogens and foreign substances, and as "temporary" until acquired immunity is established. It is thought that it plays only a role, but progress in research on the molecular mechanism of innate immunity leads to specific recognition of self / non-self and innate immunity acquired even in innate immunity. It was clarified that it is essential for the establishment of immunity. More specifically, Toll-like receptor (TLR) family present in antigen-presenting cells such as dendritic cells, macrophages and B cells react with various pathogens to induce the production of cytokines, and naive T cells are Recent studies have revealed that induced immunity can be induced through promotion of differentiation into Th1 cells, activation of killer T cells, and the like.

  The components of pathogens recognized by a series of TLR families are diverse, one of which is DNA with CpG sequences (CpG DNA), which is a ligand for TLR9. The CpG sequence is a sequence based on six bases in which cytosine (C) and guanine (G) are aligned at the central portion, and is a nucleotide sequence which is small in mammals and frequently in microorganisms. Also, in mammals, most of the few CpG sequences are methylated. Unmethylated CpG sequences, which are scarcely present in mammals, have potent immunostimulatory activity (see, for example, non-patent documents 1 to 3). CpG DNA taken up into cells by endocytosis is recognized by TLR9 present in phagosome-like endoplasmic reticulum and strongly induces a Th1 response. The Th1 response has strong antitumor activity, while suppressing the allergic response in which the Th2 response is dominant. In addition, CpG DNA does not have side effects such as toxicity as seen with some adjuvants. Therefore, in addition to infection prevention, CpG DNA is expected as an adjuvant for allergic diseases and neoplastic diseases (see, for example, Non-Patent Document 4).

  However, when CpG DNA is used as an adjuvant for immunotherapy, how to allow CpG DNA to reach the inside of target cells while avoiding degradation due to nucleases in cytoplasm or plasma and nonspecific binding with proteins is considered. It becomes a problem.

  The present inventors focused on schizophyllan (hereinafter sometimes abbreviated as "SPG") as a novel gene carrier, and so far, schizophyllan includes nucleic acid drugs (antisense DNA, CpG DNA) It has been found to form new types of complexes with various nucleic acids (see, for example, Patent Documents 1 and 2 and Non-patent Documents 5 to 7).

  A single-stranded nucleic acid after dissolving Schizophyllan, which is naturally present in a triple helix, in an aprotic polar organic solvent such as dimethyl sulfoxide (DMSO) or an alkaline solution of 0.1 N or more and dissociating it into a single strand. Were added, and the solvent was returned to water or neutral, and it was found that a triple helical complex consisting of one nucleic acid molecule and two schizophyllan molecules was formed. In this case, it is considered that the schizophyllan molecule and the nucleic acid molecule in the triple helix complex mainly form hydrogen bonding and hydrophobic interaction to form an intermolecular bond (see Non-Patent Document 8).

  As mentioned above, undesired interactions between nucleic acid molecules and in vivo proteins, such as hydrolysis of the nucleic acid molecules by nucleases and nonspecific binding of plasma proteins to the nucleic acids by complexing the nucleic acid with Schizophyllan It has become possible to allow the nucleic acid to reach the inside of the cell while suppressing Successful delivery of CpG DNA into cells using a complex of schizophyllan and a nucleic acid, and a ternary complex containing a protein having antigenicity (for example, Patent Documents 3 and 4, Non-patent Document 9) ~ 11).

WO 01/34207 WO 02/072152 JP 2007-174107 A JP, 2010-70307, A

Bacterial CpG DNA Activates Immune Cells to Signal Infectious Danger, H. Wagner, Adv. Immunol., 73, 329-368 (1999). CpG Motifs in Bacterial DNA and Their Immune Effects, M. Krieg, Annu. Rev. Immunol., 20, 709-760 (2002). The discovery of immunostimulatory DNA sequence, S. Yamamoto, T. Yamamoto, and T. Tokunaga, Springer Seminars in Immunopathology, 22, 11-19 (2000). Standard Immunology, 2nd Edition, School of Medicine, 2002, p. 333 Molecular Recognition of Adenine, Cytosine, and Uracil in a Single-Stranded RNA by a Natural Polysaccharide: Schizophylla. K. Sakurai and S. Shinkai, J. Am. Chem. Soc., 122, 4520-4521 (2000). Polysaccharide-Polynucleotide Complexes. 2. Complementary Polynucleotide Mimic Behavior of the Natural Polysaccharide Complex of the Macromolecular Complex with Single-Stranded RNA and DNA. K. Sakurai, M. Mizu and S. Shinkai, Biomacromolecules, 2, 641-650 (2001) . Sect. Mochizuki and K. Sakurai, J. Control. Release, 151 (2011) 155-161. Dectin-1 targeting delivery of TNF-α anti-sense ODNs complexed with β-1,3- glucan protectors mice from LPS-induced hepatitis. Structural Analysis of the Curdlan / Poly (cytidylic acid) Complex with Semiempirical Molecular Orbital Calculations. K. Miyoshi, K. Uezu, K. Sakurai and S. Shinkai, Biomacromolecules, 6, 1540-1546 (2005). A. Polysaccharide Carrier for Immunostimulatory CpG DNAs to Enhance Cytokine Secretion, M. Mizu, K. Koumoto, T. Anada, T. Matsumoto, M. Numata, S. Shinkai, T. Nagasaki and K. Sakurai, J. Am. Soc., 126, 8372-8373 (2004). Protection of Polynucleotides against Nuclease-Mediated Hydrolysis by Complexation with Schizophyllan, M. Mizu, K. Koumoto, T. Kimura, K. Sakurai and S. Shinkai, Biomaterials, 25, 15, 3109-3116 (2004). Synthesis and in Vitro Characterization of Antigen-Conjugated Polysaccharide as a CpG DNA Carrier, N. Shimada, KJ Ishii, Y. Takeda, C. Coban, Y. Torii, S. Shinkai, S. Akira and K. Sakurai, Bioconjugate Chem. , 17 1136-1140 (2006).

  However, the above-described conventional techniques have the following problems. For example, in the method for producing a ternary complex of schizophyllan / protein having antigenicity / CpG DNA described in Non-Patent Document 11, a formyl group is generated on the glucose residue of the side chain of schizophyllan by periodic acid oxidation. The Schizophyllan and the antigenic peptide are covalently bonded by reacting a formyl group with the amino group of a peptide having antigenicity (hereinafter sometimes abbreviated as "antigenic peptide") by reductive amination. Although forming a complex, it had the subject that the yield was very low. In view of such circumstances, for example, in the method of producing a ternary complex of schizophyllan / antigenic protein (antigenic peptide) / CpG DNA described in Patent Document 4, schizophyllan having a formyl group in the side chain and antigenic peptide Is reacted in an aqueous alkaline solution and at the same time neutralized, or reacted in an aqueous alkaline solution and sequentially neutralized to react the formyl group on the side chain of schizophyllan with the amino group of the antigenic peptide. And improve the yield. However, since there are a plurality of amino groups in the peptide, control of the reaction point is difficult. Therefore, there are concerns about problems such as differences in immunogenicity depending on the reaction position of the antigenic peptide, and difficulties in separation and purification due to the reaction product with schizophyllan becoming a complicated mixture. In addition, the formation of a complex based on the formation of a covalent bond between schizophyllan and an antigenic peptide requires a complicated step, as compared to the formation of a complex of schizophyllan and DNA using complex formation by hydrogen bonding. In these respects, the method for producing a ternary complex of schizophyllan / antigenic peptide / CpG DNA described in Patent Document 4 still has problems in terms of productivity and the like.

The present invention has been made in view of such circumstances, and it is an object of the present invention to provide an immunostimulatory polynucleotide / schizophyllan complex having excellent productivity and high immunostimulatory activity, and a pharmaceutical composition comprising the same.

なお、式（Ｉ）において、θはＸ線の散乱角を表し、λはＸ線の波長を表す。
本発明の第２の態様は、シゾフィランと、
前記シゾフィランと水素結合を介して結合し、ＣｐＧ配列を有するポリデオキシリボヌクレオチド又は該ポリデオキシリボヌクレオチドにおいてホスホジエステル結合の一部又は全部がホスホロチオエート結合もしくはホスホロジチオエート結合で置換されたポリデオキシリボヌクレオチド誘導体とを含み、
前記ポリデオキシリボヌクレオチド又は前記ポリデオキシリボヌクレオチド誘導体の分子鎖１本と前記シゾフィランの分子鎖２本とからなる三重螺旋構造を有する複合体を形成しており、
多角度光散乱測定又はＸ線小角散乱による慣性半径の測定値が２０ｎｍ以上２００ｎｍ以下であり、
Ｘ線小角散乱を用いて測定したＸ線の散乱強度Ｉを、上記の式（Ｉ）で定義されるｑ値の対数に対しプロットした場合、ｑが１０ ^−１ ｎｍ ^−１ から１ｎｍ ^−１ の範囲における傾きａが−１．５以上−０．５以下であり、
前記ポリデオキシリボヌクレオチド又は前記ポリデオキシリボヌクレオチドの誘導体のうち、前記シゾフィランと水素結合を介して結合する部分の重合度が１０以上であり、且つポリデオキシリボヌクレオチドにおいてホスホジエステル結合がホスホロジチオエート結合に置換されたポリデオキシリボヌクレオチドの誘導体であることを特徴とする免疫賦活用ポリヌクレオチド／シゾフィラン複合体を提供することにより上記課題を解決するものである。 According to a first aspect of the present invention, which meets the above object, there is provided schizophyllan,
The schizophyllan and linked via hydrogen bonds, poly deoxyribonucleic polynucleotide or the polypeptide deoxyribonucleic nucleotides some phosphodiester bonds or in the whole of poly deoxyribonucleic nucleotide derivative substituted with phosphorothioate bonds or phosphorodithioate bonds having a CpG sequence ( Hereinafter, the present invention includes “the above-mentioned polydeoxyribonucleotide derivative” or “ polydeoxyribonucleotide derivative” for short.
Forms a complex with the polypeptide deoxyribonucleic polynucleotide or triple helix structure composed of the polypeptide deoxyribonucleic nucleotide derivative chains one with a molecular chain two of said schizophyllan,
The measurement value of the radius of inertia by multi-angle light scattering measurement or small-angle X-ray scattering is 20 nm or more and 200 nm or less,
When the scattered intensity I of X-rays measured using small-angle X-ray scattering is plotted against the logarithm of q value defined by the following formula (I), q is 10 ⁻¹ nm ⁻¹ to 1 nm ⁻¹ slope a is -1.5 or more in the range -0.5 Ri der below,
There is provided an immunostimulatory polynucleotide / schizophyllan complex , wherein the degree of polymerization of a portion of the polydeoxyribonucleotide or the derivative of the polydeoxyribonucleotide bound to the schizophyllan via a hydrogen bond is 60 or more. It solves the above-mentioned subject by doing.

In equation (I), θ represents the scattering angle of X-rays, and λ represents the wavelength of X-rays.
According to a second aspect of the present invention there is provided:
A polydeoxyribonucleotide having a CpG sequence and a polydeoxyribonucleotide having a CpG sequence or a polydeoxyribonucleotide derivative in which part or all of phosphodiester bonds in the polydeoxyribonucleotide is substituted with a phosphorothioate bond or a phosphorodithioate bond, which is bound to the schizophyllan via hydrogen bond Including
Forming a complex having a triple helical structure comprising one molecular chain of the polydeoxyribonucleotide or the polydeoxyribonucleotide derivative and two molecular chains of the schizophyllan,
The measurement value of the radius of inertia by multi-angle light scattering measurement or small-angle X-ray scattering is 20 nm or more and 200 nm or less,
The scattering intensity I of X-rays was measured using an X-ray small angle scattering, when plotted against the logarithm of q values defined by the above formula (I), q is ^{1 nm -1} from ¹⁰ -1 ^{nm -1} The slope a in the range is -1.5 or more and -0.5 or less,
In the polydeoxyribonucleotide or the derivative of the polydeoxyribonucleotide, the degree of polymerization of the moiety bonded to the schizophyllan via hydrogen bond is 10 or more, and in the polydeoxyribonucleotide, the phosphodiester bond is substituted to the phosphorodithioate bond The object of the present invention is to solve the above-mentioned problems by providing an immunostimulatory polynucleotide / schizophyllan complex characterized by being a derivative of the polydeoxyribonucleotide.

In the immunostimulatory polynucleotide / schizophyllan complex according to the first or second aspect of the present invention, the molecular weight may be 1 × 10 ⁵ or more and 3 × 10 ⁶ or less.

The third aspect of the present invention solves the above-mentioned problems by providing a pharmaceutical composition comprising the immunostimulatory polynucleotide / schizophyllan complex according to the first or second aspect of the present invention.

According to the present invention, the following advantageous effects are obtained.
(1) Regardless of the type, molecular weight, combination, etc. of schizophyllan and polynucleotides having CpG sequences, a polynucleotide / schizophyllan complex can be formed via hydrogen bonding between schizophyllan and the polynucleotide, so An immunostimulatory polynucleotide / schizophyllan complex can be obtained by a single operation for combinations of polynucleotides having CpG sequences.

(2) Hydrogen bonding between schizophyllan and a polynucleotide having a CpG sequence can rapidly and efficiently proceed the reaction by changing the pH of the solvent. Therefore, the polynucleotide / schizophyllan complex can be obtained in a high yield in a short time, and the time required for isolation and purification can also be reduced. Therefore, the immunostimulatory polynucleotide / schizophyllan complex of the present invention is excellent in productivity and can be produced at low cost.

(3) The use of the immunostimulatory polynucleotide / schizophyllan complex of the present invention induces induction of an immune response specific to a polynucleotide having a CpG sequence, as compared to when the polynucleotide having a CpG sequence is used alone. It can be done more effectively.

(4) immunostimulatory polynucleotide / schizophyllan pharmaceutical composition comprising a conjugate of the present invention, than with a polynucleotide having a CpG sequence alone, specific immune for polynucleotides having a broad CpG sequence The response can be induced more effectively. Therefore, application as a vaccine and an immunostimulant can be expected.

It is a graph which shows the molecular weight of a Sd (A) _40- D35 complex, and the relation of a radius of inertia.

  Subsequently, an embodiment of the present invention will be described to provide an understanding of the present invention.

[1] first according to the embodiment immunostimulatory polynucleotide / schizophyllan complex of immunostimulatory polynucleotides / schizophyllan complexes present invention (hereinafter, sometimes simply referred to as "polynucleotide / schizophyllan complex" ) Is bound to schizophyllan via hydrogen bond with schizophyllan, and a polynucleotide or polynucleotide derivative having a CpG sequence (a part or all of phosphodiester bonds in the polynucleotide is substituted by phosphorothioate bond or phosphorodithioate bond) (Polynucleotide derivatives) and

  In the polynucleotide / schizophyllan complex, the polynucleotide or the polynucleotide derivative and the schizophyllan form a complex having a triple helical structure consisting of one molecular chain of the polynucleotide or the polynucleotide derivative and two molecular chains of the schizophyllan. doing.

  The polynucleotide or polynucleotide derivative used for production of the polynucleotide / schizophyllan complex is capable of binding to schizophyllan via hydrogen bond and has a CpG sequence.

  Oligonucleotides having an immune response stimulating activity were discovered in 1984 by Tokunaga et al. In the process of searching for an antitumor component of BCG. Then, it was revealed that the activation action is attributable to a specific base sequence containing cytosine guanine dinucleotide (5′-CpG-3 ′: so-called CpG sequence) (eg, Tokunaga, T. et al. , et al., J. Natl. Cancer Inst., 72, 955 (1984) and Tokunaga, T., et al., J. Natl. Cancer Res., 79, 682 (1988)).

  Similar activity is also observed in genomic DNA having CpG sequences other than vertebrates or plants. Sequences before and after the CpG core are also considered to be important for the immunostimulatory activity, and in particular, 5'-PuPuCpGPyPy having non-methylated CpGs and in which substituted purines (Pu) and substituted pyrimidines (Py) are arranged. The consensus is -3 'as a representative unmethylated CpG motif (see, eg, Krieg, A., et al., Nature, 374, 576 (1995)). Here, the unmethylated CpG motif is, as is well known, a short nucleotide sequence (generally 4 to 10 nucleotides) containing at least one cytosine (C) -guanine (G) sequence Sequence), wherein the 5-position of cytosine in the cytosine-guanine sequence is not methylated. In the following description, CpG means unmethylated CpG unless otherwise specified.

  Examples of useful CpG motifs (hexamers) are described below (where A: adenine, G: guanine, C: cytosine, T: thymine, U: uracil).

  AACGTT, AGCGTT, GACGTT, GGCGTT, AACGTC, AGCGTC, GACGTC, AGCGTC, AACGCC, AGCGCC, GACGCC, GAGGCCC, AACGCT, AGCGCT, GACGCT, and GGGCCT

  Oligonucleotides composed of about 8 to 100 including these sequences are those having immunostimulatory activity.

  The following sequences are examples of immunostimulatory oligonucleotides containing CpG motifs that have been reported to be effective for NK cell activation (underlined portions indicate CpG motifs and capital letters indicate thiolated DNA) (eg S. Iho, T. Yamamoto, T. Takahashi and S. Yamamoto, J. immunol., 1999, 163, 3642-3652.).

accgat accggt gccggt gacgg caccacg
accgat agcgct gccggtgacggcaccacg
accgat gacgtc gccggtgacggcaccacg
accgat tcgcga gccggtgacggcaccacg
gggggggggggg cgatcg gggggggggggg
gggggggggg gacgatcgtc gggggggggg
gggggggggggg aacgtt gggggggggggg
GAG AACGCT CGACCTTCGAT
TCCAT GACGTT CCTGATGCT
TCTCCCAG CG TG CG CCAT
GGggt caacgtt gaGGGGGg

  The polynucleotide (for example, RNA or DNA) containing the CpG sequence described above may be hybridized with a polynucleotide or polynucleotide derivative having a base sequence that binds to Schizophyllan via a hydrogen bond, and such a hybrid is It can be obtained or synthesized using any known method. When the polynucleotide or the polynucleotide derivative is a chimeric nucleic acid in which the 5 'end of RNA is bound to the 3' end of DNA, the phosphodiester bond between RNA and DNA is particularly susceptible to degradation. The hydroxyl group at the 2 'position of the 5' terminal nucleotide of RNA bound to DNA is substituted with a methoxy group or a fluoro group and / or the 3 'position of the first ribonucleotide bound to DNA and the adjacent RNA Preferably, derivatization such as substitution of a phosphodiester group between the 5'-positions with a phosphorothioate group is carried out to improve the resistance to hydrolysis.

  Since polynucleotides are susceptible to degradation by nucleases in vivo, polynucleotide derivatives may be used instead of polynucleotides to improve their stability in vivo. Examples of polynucleotide derivatives include those in which all or part of the hydroxyl group at the 2 'position of ribonucleotide is substituted with a fluorine or methoxy group, phosphodiester of polyribonucleotide (RNA) or polydeoxyribonucleotide (DNA) Those in which all or part of the groups are substituted with phosphorothioate groups are exemplified. When part of the phosphodiester group of the polyribonucleotide or polydeoxyribonucleotide is substituted with a phosphorothioate group, it is preferable that 50% or more of the phosphodiester bond is substituted with a phosphorothioate group. The position of the phosphodiester group substituted by the phosphorothioate group is not particularly limited, and a plurality of consecutive phosphodiester groups may be substituted or the phosphorothioate groups may be substituted so as not to be adjacent to each other.

  Specific examples of the polynucleotide or polynucleotide derivative having a partial base sequence linked via a hydrogen bond include polyadenosine (polyadenylate, polyriboadenylate) (polyA), polycytidine (polycytidylate), which has a high binding ability to schizophyllan. Polyribocytidylic acid (polyC), polydeoxyadenosine (polydeoxyadenylate, polydeoxyriboadenylate) (poly (dA)), polydeoxythymidine (polydeoxythymidylate, polydeoxyribotimidylate) (poly (dT)) Be The number of bases of the polynucleotide is not particularly limited as long as it can form a complex having a triple helix structure with Schizophyllan as described above, but in order to improve the complexing ability, the polynucleotide binds to Schizophyllan It is preferable to have a repeating sequence of any of polyadenosine (polyA), polycytidine (polyC), polydeoxyadenosine (poly (dA)), and polydeoxythymidine (poly (dT)) having high performance. The type of base and nucleotide or nucleotide derivative constituting the preferable repeating sequence and the number of bases are appropriately determined according to the length of the polynucleotide or polynucleotide derivative having the CpG sequence, the molecular weight of the schizophyllan to be used, and the like. If the number of bases is small, it is difficult to form a triple helix structure by hydrogen bonding with schizophyllan, so the number of bases needs to be 10 or more, and preferably 20 to 80. And 30 to 80 are more preferable.

The molecular weight of schizophyllan is appropriately adjusted according to the base sequence and base length of the polynucleotide or polynucleotide derivative contained in the polynucleotide / schizophyllan complex. However, if the molecular weight is small, it is not preferable because the so-called cluster effect (cooperative phenomenon of polymer systems) hardly occurs. Usually, the weight-average molecular weight (per molecule) of schizophyllan capable of forming a complex with a nucleic acid varies depending on the type and higher-order structure of the nucleic acid base, but is preferably at least 20,000, more preferably at least 40,000. And more preferably 60,000 or more. In addition, the number of hydroxyl groups that form hydrogen bonds with nucleic acid bases on the polynucleotide is usually 5 or more, preferably 8 or more, and more preferably 10 or more.
The weight average molecular weight of schizophyllan can be determined using any known method such as light scattering method, sedimentation velocity method (ultracentrifugation method) and the like.

  Since schizophyllan is generally produced by fungi and eubacteria, it is obtained by culturing these microorganisms, homogenizing the cells, and isolating them from impurities such as cell elution and insoluble residue by a method such as ultracentrifugation. be able to. In general, the schizophyllan thus obtained has a triple helical structure with a high molecular weight (weight average molecular weight of several hundreds of thousands). If necessary, the molecular weight may be reduced. Molecular weight reduction is carried out by appropriately selecting an appropriate method and conditions from enzymatic degradation, acid hydrolysis and the like.

(3) Preparation of polynucleotide / schizophyllan complex Schizophyllan usually exhibits a triple helical structure consisting of three molecular chains of schizophyllan in water. Therefore, in order to form a complex with a polynucleotide or a polynucleotide derivative, Schizophyllan is dissolved in a solvent such as DMSO to break the association state due to intermolecular hydrogen bonding and hydrophobic interaction into a single strand. An aqueous solution containing a polynucleotide or a polynucleotide derivative (or a solution of a polar solvent such as alcohol) is added to the solution of single-stranded schizophyllan obtained in this manner, and the solvent is added by the addition of water. As the polarity increases, the hydrophobic interaction causes the polynucleotide and the schizophyllan to associate, and while the molecular chain of the polynucleotide or the polynucleotide derivative is incorporated, a complex of the polynucleotide or the polynucleotide derivative and the schizophyllan is formed. As a result, a complex having a triple helical structure consisting of one molecular chain of the polynucleotide or polynucleotide derivative and two molecular chains of schizophyllan is formed. The formation of the complex can be confirmed by examining the conformational change of the polynucleotide or the polynucleotide derivative, for example, by measuring a CD (circular dichroism) spectrum.

The weight average molecular weight of the polynucleotide / schizophyllan complex can be measured using any known method such as gel permeation chromatography (GPC) and viscosity measurement. The molecular weight of the polynucleotide / schizophyllan complex is preferably 1 × 10 ⁵ or more and 3 × 10 ⁶ or less.

  In the polynucleotide / schizophyllan complex, the measurement value of the radius of inertia by multi-angle light scattering measurement or small-angle X-ray scattering is preferably 20 nm or more and 200 nm or less. When the radius of inertia of the polynucleotide / schizophyllan complex is less than 20 nm or more than 200 nm, it is considered that the receptor of Schizophyllan present on the cell surface is less likely to be recognized, and therefore, it is difficult for cellular uptake to occur.

  Although any known method can be used to measure the radius of inertia of the polynucleotide / schizophyllan complex, it is described, for example, in the literature (The Journal of Physical Chemistry B 116 (1), 87-94 (2011)). Methods, and more specifically, from the molecular weight of the polynucleotide / schizophyllan complex, it is expected to be less than 10 nm for the light scattering method for those for which the radius of inertia is expected to be 10 nm or more Small-angle X-ray scattering is preferably used.

In the polynucleotide / schizophyllan complex, X-ray scattering intensity I measured using small-angle X-ray scattering was plotted against the logarithm of q value (absolute value of scattering vector) defined by the following formula (I) In the case where q is in the range of 10 ⁻¹ nm ⁻¹ to 1 nm ⁻¹ , the slope a is preferably −1.5 or more and −0.5 or less.

  In equation (I), θ represents the scattering angle of X-rays, and λ represents the wavelength of X-rays. Therefore, q value is a function of X-ray scattering angle θ and X-ray wavelength λ, and the relationship between X-ray scattering intensity I and q value indicates the angle and wavelength dependence of X-ray scattering intensity I .

The relationship I∝q ^a is established between the X-ray scattering intensity I and the q value. Therefore, the a value corresponds to the slope a when the scattered intensity I of the X-ray is plotted against the logarithm of the q value. The a value reflects the shape of the polymer chain, and when a = -1, it indicates that it is a rod-like shape without branching. When | a | <1, it indicates that it is a bent rod-like or soft rod-like, and when | a |> 1, it indicates that it has a branched structure. The larger the value of | a |, the higher the degree of branching. In the polynucleotide / schizophyllan complex, the slope a in the q range of 10 ⁻¹ nm to 1 nm ⁻¹ is preferably −1.5 or more and −0.5 or less.

  Even when a is out of the above range, the receptor for Schizophyllan present on the cell surface is less likely to be recognized, so that it is considered that cellular uptake is less likely to occur.

  The pharmaceutical composition according to the second embodiment of the present invention (hereinafter referred to as "pharmaceutical composition") comprises the polynucleotide / schizophyllan complex according to the first embodiment of the present invention. .

  For the preparation of the pharmaceutical composition, in addition to the polynucleotide / schizophyllan complex and the polynucleotide / schizophyllan complex as active ingredients, any known components (optional carriers, excipients and additives acceptable for pharmaceutical use) And formulation methods can be used. For example, the pharmaceutical composition can take the form of tablets, suppositories, capsules, microcapsules such as syrups and nanogels, sterile solutions, injections such as suspensions, aerosols, sprays and the like.

  The pharmaceutical composition can be administered to human or warm-blooded animal (mouse, rat, rabbit, sheep, pig, cow, horse, chicken, cat, dog, monkey, etc.) by any of oral and parenteral routes. . The parenteral administration route includes subcutaneous, intradermal and intramuscular injection, intraperitoneal administration, drip, spray on nasal mucosa and pharynx, and the like.

  The dose of the active ingredient polynucleotide / schizophyllan complex varies depending on the activity, the disease to be treated, the type of animal to be administered, body weight, sex, age, severity of disease, administration method and the like. When taking an adult weighing 60 kg as an example, for oral administration, the daily dose is usually about 0.1 to about 100 mg, preferably about 1.0 to about 50 mg, more preferably about 1.0 to about 20 mg For parenteral administration, the daily dose is usually about 0.01 to about 30 mg, preferably about 0.1 to about 20 mg, more preferably about 0.1 to about 10 mg. When administered to another animal, the dose is converted to the dose per unit body weight, and then the dose obtained by multiplying the weight of the animal to be administered is used.

  The pharmaceutical composition can be used as a vaccine for treatment and prevention of infections caused by infection of pathogens such as bacteria and viruses by activating immunity, and vaccines, immunostimulants and the like for tumors such as cancers.

Example 1 Preparation of Schizophyllan of Different Molecular Weights In this Example, Schizophyllum commune Fries was cultured, and Schizophyllan produced in the culture solution was separated and purified to obtain high molecular weight Schizophyllan. The polymer Schizophyllan was sonicated to physically decompose Schizophyllan. Moreover, the molecular weight of schizophyllan obtained by changing the time of ultrasonication was able to be changed.

  In order to obtain an even lower molecular weight schizophyllan, it was treated with formic acid. The obtained Schizophyllan of different molecular weight is described in the literature (Norisuye et al., J. Polym. Sci., Polym. Phys. Ed. 1980, 18, 547, Yanaki et al., Macromolecules 1980, 13, 1462, Kashiwagi et al., Macromolecules 1981, 14 Water was fractionally purified using a good solvent, acetone, or methanol as a poor solvent, as described in U.S. Pat.

The obtained sample names and the obtained molecular weights are shown in Table 1. For each sample, ¹ H NMR measurement is performed at 70 ° C. in DMSO, and the β-1,3-glucan skeleton is retained, in particular from the peak ratios of the chemical shifts of 4.1 ppm and 4.5 ppm. It was confirmed that the side chains of 1,6-glucan bonds were maintained in the range of 2 to 5 to glucose 10 of the main chain. In addition, the chemical structure is described on pages 87-90 of Toshio Yanagi's thesis (Osaka University Faculty of Science 1984), (1) Hydrolysis with sulfuric acid is followed by appropriate processing and analysis by gas chromatogram Method, (2) The box guard method (literature: Bulletin of Yamagata University (Agriculture) Vol. 11, No. 4: 907 to 956, separate volume, January, 1993) was confirmed. The molecular weight was measured by scattering method using a light scattering photometer, centrifugal equilibrium method using ultracentrifugation, and scattering method using small-angle X-ray scattering. The light scattering method is suitable for determining the molecular weight of a relatively high molecular weight sample, the centrifugal equilibrium method is a relatively medium to low molecular weight sample, and the small angle X-ray scattering is a relatively low molecular weight sample. Since molecular weight determination in aqueous systems is subject to errors, it was determined in two or more independent ways. In DMSO, the viscosity of the solvent is high, the time to reach sedimentation equilibrium is long by centrifugal equilibrium, and measurement is difficult for samples of high molecular weight, and X-ray small-angle scattering makes X-ray transmission poor and difficult to measure Met.

  Table 1 shows the results. Phys. Ed. 1980, 18, 547. Yanaki et al., Macromolecules 1980, 13, 1462, Kashiwagi et al., Macromolecules 1981, 14, 1220. as indicated by the literature. Is molecularly dispersed as one polymer chain in DMSO. Therefore, if the ratio of the molecular weight in DMSO to the molecular weight in water is 3, it is judged that schizophyllan takes a triple helical structure in water. From the results shown in the table, when the weight average molecular weight is 20,000 or more, preferably 30,000 or more as one polymer chain of Schizophyllan, the majority of Schizophyllan has a triple helical structure in water. In addition, when the weight average molecular weight is 10,000 or less, it can be determined that it is present as one polymer chain in water.

Note 1: LS indicates light scattering, UCF indicates ultracentrifugation, and SAXS indicates small-angle X-ray scattering. The molecular weight distribution is the ratio of weight-average molecular weight to number-average molecular weight determined from the chromatogram of GPC for light scattering, and for ultracentrifugation, the data obtained at the time of centrifugal equilibria (Fujita, H. Foundations of Ultracentrifugal Analysis; Wiley: New York, 1975 ) Determined according to the method described.
Note 2: In order to make the measurement of light scattering accurate, it was measured in 0.01 N NaOH aqueous solution which is known to maintain the same conformation of Schizophyllan as neutral water.

Example 2: at different molecular weights schizophyllan and nucleic poly _{(dA) X} of the complexed known nucleic acid solid phase synthesis, was synthesized poly _{(dA) X} is a polymer of deoxyadenosine monophosphate. Here, X indicates the degree of polymerization. That is, (dA) _X is an X-mer of deoxyadenosine. As this analogue, S-poly (dA) _X , which is a phosphorothioate derivative in which one of the oxygen atoms of phosphodiester-linked phosphate is substituted with sulfur, and two of the oxygen atoms of phosphoric acid are substituted with sulfur A phosphorodithioate derivative D-poly (dA) _X was also synthesized. All were purified using HPLC to obtain a purified product having a purity of 99% or more.

As described in Bioorganic Chemistry Vol. 38. P260-264 (2010), gelation electrophoresis, gel permeation chromatography (GPC), and circular dichroism spectra are used to determine the degree of conjugation with schizophyllan. When it is 95% or more (○), 50% or more (Δ), and 10% or less (×), the samples in Table 1 were classified. The results are shown in Tables 2 to 4. Table 2 shows the results of phosphodiester bonds, Table 3 shows the results of S-poly (dA) _X in which one of the oxygens is substituted by sulfur, and Table 4 shows the results of D-poly (dA) _X in which two of the oxygens are substituted by sulfur. It is.

For preparation of the complex, Schizophyllan is dissolved in 0.25 N NaOH solution (15 mg / mL) and left for 2 days or more to completely dissociate Schizophyllan into a single strand, and then poly (deoxyribonucleotide / schizophyllan complex Solution) and phosphate buffer (330 mM NaH ₂ PO ₄ , pH 4.5), so that the pH is in the range of 6 to 7, and the mixing ratio of schizophyllan to deoxyribonucleotide is 3 in molar ratio: A basic aqueous solution of schizophyllan was added and stirred to be 1. After the obtained solution was allowed to stand overnight at 4 ° C., various measurements were performed. Although the prior patent discloses a method of adding an appropriate concentration of Schizophyllan / DMSO solution to a DNA solution, it is not preferable because DMSO remains when it is used in biological experiments.

From the results shown in Tables 2 to 4, in the phosphodiester bond, the polymerization X of deoxyadenosine monophosphate is preferably 20 or more, more preferably 40 or more, and 60 to make the yield of the complex close to 100%. The above is necessary. In the phosphorothioate type S-poly (dA) _X , preferably 10 or more, more preferably 20 or more, and 40 or more are required to make the yield of the complex close to 100%. In the case of phosphorodithioate type D-poly (dA) _X , preferably 10 or more and 20 or more are required to make the yield of the complex close to 100%.

Example 3 Complexation of Schizophyllan of Different Molecular Weights with CpG DNA with Poly (dA) _X Tail Added CpG DNA added poly dA tail: (dA) ₄₀ at the 5'-end as a K-type CpG DNA the _{5 '- (dA) 40 -ATCGACTCTCGAGCGTTCTC} -3'; 5 ' (SEQ ID NO: 1 _(dA) 40 k3 hereinafter), and as D- type _{CpG DNA - (dA) 40 -GGTGCATCGATGCAGGGGGG} ( SEQ ID NO: 1 (DA) ₄₀ -D35 for short) was used. Both phosphate backbones of DNA are phosphorothioate type, and both samples are synthetic products of Hokkaido System Science, and are purified by high performance liquid chromatography. In addition, K3- (dA) ₄₀ and D35- (dA) ₄₀ to which a poly dA tail was added at the 3'-end were similarly synthesized. The complexation of these samples was evaluated in the same manner as in Example 2. The results are shown in Table 5.

  The basic aqueous solution of Schizophyllan was added and stirred so that the mixing ratio of Schizophyllan and dA was 3: 1 in molar ratio. From this result, it can be seen that when CpG is added to dA, although it is slight, complexation is difficult.

Example 4 Purification of Complex and Measurement of Molecular Weight, Spread and Cell Stimulation Unreacted DNA can be separated by GPC because its molecular weight is much smaller than that of the complex. The solution flowing out of the detector was separated while confirming the complex fraction. Moreover, unreacted schizophyllan was removed by the method using the anion exchange column described in Unexamined-Japanese-Patent No. 2011-178707. From the mixed solution having a complexing ratio of 50% or less, it was possible to make the complexing ratio 90% or more using these methods. The purified complex did not change its conjugation rate even when left as an aqueous solution at room temperature for 10 days.

  The weight average absolute molecular weight Mw and the sample having a complexing ratio of 90% or more obtained in this manner are described by the method described in the literature (The Journal of Physical Chemistry B 116 (1), 87-94 (2011)). The inertial radius Rg was measured. For the measurement of the radius of inertia Rg, the light scattering method was used for 10 nm or more, and the small-angle X-ray scattering method was used for less than 10 nm. Since the distribution of molecular weight and radius of inertia increases in the complex, the minimum value and the maximum value of both measured values were recorded.

  Moreover, the enhancement effect (hereinafter referred to as "physiological activity") of the cytokine IL-12 production amount from mouse-derived peritoneal macrophages by stimulation of CpG DNA was examined by the following method. That is, isolation of mouse-derived peritoneal macrophages was carried out by a standard method described in the literature. That is, the carotid artery of an eight-week-old female Balb / c mouse was cut and decapitated, disinfected with 70% ethanol, cut in the abdominal skin, exposed the outer skin and exposed the peritoneum. Cold PBS (phosphate buffered saline) was injected into 5 mL of the abdomen, massaged well, and then the fluid was recovered. Using a polypropylene centrifuge tube, centrifugation was performed at 1,000 rpm for 10 minutes at 4 ° C. The supernatant was removed and suspended in RPMI 1640 medium containing 10% fetal calf serum (Nippon Biochemical Society, edited by NeoBiochem. 12 Molecular Immunology I Immunocytes / Cytokine, Tokyo Kagaku Dojin (1989)).

Seed 2 × 10 ⁵ mouse-derived peritoneal macrophages suspended in RPMI 1640 medium containing 100 μL of 10% fetal calf serum in a 96-well plate, and incubate at 37 ° C. in 5% CO ₂ for 2 hours. After adhesion, CpG DNA and a complex of CpG DNA and Schizophyllan were added, and the culture supernatant was recovered after culturing for 24 hours at 37 ° C. under 5% CO ₂ . Measurement of the total amount of IL-12 in mice contained in the culture supernatant was performed using Mouse Interleukin-12 Total ELISA (manufactured by ENDOGEN) according to the attached protocol. The total amount of IL-12 contained in the culture supernatant was higher in the immunostimulant of the present invention in the complex than administration of CpG DNA alone. The results are shown as ○ when the difference is more than twice as high as the physiological activity, and × when the difference is about the same. The table shows the results for complexes containing D-type CpG DNA, but the results were similar for complexes containing K-type CpG DNA.

In the composite of Sd (A) _40- D35 using S1, S2, and S3, the graph which plotted the molecular weight computed from the multi-angle light scattering measurement with respect to the radius of inertia is shown in FIG. It was found that the molecular weight and the radius of inertia of the complex formed with the increase of the molecular weight of schizophyllan were increased. When the physiological activity of the prepared complex was evaluated, the complex having a large molecular weight and radius of inertia exhibited high activity, but the activity of a complex having a small radius of inertia was very small. This is considered to be due to the fact that the smaller molecule of schizophyllan has a lower conjugation ratio and the smaller abundance ratio of the complex, the smaller molecules are less likely to be taken up into cells (less recognized by the receptors of schizophyllan) (see Tables 6 and 7). ).

Example 5 Preparation of Polynucleotide / Schizofiran Complex and Relationship Between Concentration and Branching Degree and Physiological Activity In the complex of Sd (A) ₄₀ -D35 using S3, in Example 2, the preparation of the complex The solution is to dissolve schizophyllan in 0.25N NaOH solution to a concentration of 15 mg / mL to dissociate schizophyllan into single strands, and then use poly (deoxyribonucleotide / schizophyllan complex) solution and phosphate buffer (330 mM NaH). ₂ PO ₄ (pH 4.5) was mixed. As the concentration of schizophyllan and poly (deoxyribonucleotide / schizophyllan complex) is increased to 5, 10 and 30 times, the molecular weight of the complex increases and the scattering angle of the scattering intensity measured by small-angle X-ray scattering The dependence (denoted as I∝q ^a , where q is the absolute value of the scattering vector) is a when q is in the range of 10 ⁻¹ to 1 nm ⁻¹ and the concentration of the polynucleotide / schizophyllan complex is low. Although the sign was always negative as the concentration increased, the absolute value | a | of a increased. Similar measurements were also performed on Sd (A) ₄₀ -D35 complexes using S4. In the case of S4, the concentration was prepared from 1/100 of the standard.

A complex (| a |> 1) having a branched structure is formed as the concentration of Schizophyllan is increased in the complex of Sd (A) ₂₀ -D 35 and SD 35-d (A) ₄₀ using S3. It was found that the physiological activity decreased. It is considered that the branched structure of the complex affects cellular uptake, that is, the ability to recognize the receptor is reduced. Also in the complex using S4, it is understood that the physiological activity is lost by having the branched structure as well. Furthermore, it is considered that the physiological activity is also greatly reduced as compared to S3 due to the reduction of the radius of inertia accompanying the reduction of the molecular weight of schizophyllan (see Table 8).

Claims (4)

With sizofilan,
Linked via the schizophyllan hydrogen bonds, and a poly deoxyribonucleic nucleotide derivative some or all of the phosphodiester bonds in the poly deoxyribonucleic polynucleotide or the polypeptide deoxyribonucleic nucleotides were replaced by phosphorothioate bonds or phosphorodithioate bonds having a CpG sequence Including
Forms a complex with the polypeptide deoxyribonucleic polynucleotide or triple helix structure composed of the polypeptide deoxyribonucleic nucleotide derivative chains one with a molecular chain two of said schizophyllan,
The measurement value of the radius of inertia by multi-angle light scattering measurement or small-angle X-ray scattering is 20 nm or more and 200 nm or less,
When the scattered intensity I of X-rays measured using small-angle X-ray scattering is plotted against the logarithm of q value defined by the following formula (I), q is 10 ⁻¹ nm ⁻¹ to 1 nm ⁻¹ slope a is -1.5 or more in the range -0.5 Ri der below,
An immunostimulatory polynucleotide / schizophyllan complex , wherein a polymerization degree of a portion of the polydeoxyribonucleotide or the derivative of the polydeoxyribonucleotide bound to the schizophyllan via a hydrogen bond is 60 or more .

In equation (I), θ represents the scattering angle of X-rays, and λ represents the wavelength of X-rays.   With sizofilan,
  A polydeoxyribonucleotide having a CpG sequence and a polydeoxyribonucleotide having a CpG sequence or a polydeoxyribonucleotide derivative in which part or all of phosphodiester bonds in the polydeoxyribonucleotide is substituted with a phosphorothioate bond or a phosphorodithioate bond, which is bound to the schizophyllan via hydrogen bond Including
  Forming a complex having a triple helical structure comprising one molecular chain of the polydeoxyribonucleotide or the polydeoxyribonucleotide derivative and two molecular chains of the schizophyllan,
  The measurement value of the radius of inertia by multi-angle light scattering measurement or small-angle X-ray scattering is 20 nm or more and 200 nm or less,
  When the scattered intensity I of the X-ray measured using small-angle X-ray scattering is plotted against the logarithm of the q value defined by the following formula (I), q is 10 ^-1 nm ^-1 To 1 nm ^-1 In the range of −1.5 to −0.5,
  In the polydeoxyribonucleotide or the derivative of the polydeoxyribonucleotide, the degree of polymerization of the moiety bonded to the schizophyllan via hydrogen bond is 10 or more, and in the polydeoxyribonucleotide, the phosphodiester bond is substituted to the phosphorodithioate bond An immunostimulatory polynucleotide / schizophyllan complex, characterized in that it is a derivative of the polydeoxyribonucleotide.

  In equation (I), θ represents the scattering angle of X-rays, and λ represents the wavelength of X-rays. The immunostimulatory polynucleotide / schizophyllan complex according to claim 1 or 2, wherein the molecular weight is 1 × 10 ⁵ or more and 3 × 10 ⁶ or less. A pharmaceutical composition comprising the immunostimulatory polynucleotide / schizophyllan complex according to any one of claims 1 to 3 .

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