Polypeptides with binding ability to antibodies and genes

Abstract

To provide polypeptides with binding ability to both a binding antibody between an amino acid sequence and nucleic acid, which enables selective delivery of nucleic acid (gene), and a nucleic acid; to provide complexes comprising the polypeptide, antibody and nucleic acid; and to provide methods of manufacturing the same.SOLUTION: The present invention relates to a polypeptide represented by an amino acid sequence comprising an amino acid sequence (region 1) that specifically binds to a constant site of an antibody and an amino acid sequence (region 2) that binds to a nucleic acid, to a complex comprising the polypeptide, an antibody and a nucleic acid, and to a method of manufacturing the complex, comprising mixing the polypeptide, antibody and nucleic acid in a solution.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a polypeptide capable of binding to both an antibody and a gene; a complex comprising said polypeptide, an antibody that binds to said polypeptide, and a nucleic acid that binds to said polypeptide; a method for producing the same; and a method for introducing nucleic acid or a gene into a target cell using the complex. [Background technology]

[0002] Methods for introducing nucleic acids or genes into cells, particularly animal cells, can be broadly divided into two categories: methods using viral vectors and non-viral vector methods. Viral vector methods use viral vectors such as lentivirus, adeno-associated virus, retrovirus, and adenovirus. Non-viral vector methods include chemical introduction methods (e.g., cationic liposomes, cationic polymers, cell-penetrating peptides (CPPs)) and physical methods (e.g., electroporation and sonoporation).

[0003] Antibody-drug conjugates (ADCs) are known as a method for selectively delivering small molecular weight compounds or proteins, instead of nucleic acids, as a payload to cells. ADCs are antibodies to which small molecular weight drugs are attached via a linker. After binding to an antigen on the surface of a target cell, they are taken up into lysosomes through endocytosis or transport processes, and the small molecular weight drug is released into the target cell following cleavage of the antibody and linker (Non-Patent Document 1). In recent years, CPPs and ADCs in particular have attracted attention as effective options for cancer treatment strategies.

[0004] CPPs are known to have the function of transporting complexes containing the peptide and other substances (such as proteins and nucleic acids) through biological membranes in mammalian and human cell lines (Non-Patent Documents 2 to 5). However, this method results in nonspecific uptake by a wide variety of cells, making it impossible to deliver genes only to target cells. For example, the cell membrane-permeable peptide described in Patent Document 1 can efficiently transport hydrophilic physiologically active substances into cells, but does not have cell selectivity. Furthermore, Patent Document 2 describes a cell membrane-permeable peptide rich in basic amino acids such as arginine and lysine, but does not have cell selectivity. Attempts have been made to modify peptide sequences to confer affinity to specific cell types, but this has been tedious, as it requires the design and synthesis of peptides individually for each cell type.

[0005] Furthermore, although ADCs have superior cell selectivity compared to CPPs, there is no established technology for delivering nucleic acids as a payload, and there are major challenges in terms of flexibility in payload design. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] US2012 / 065124 [Patent Document 2] US2013 / 129726 [Non-patent literature]

[0007] [Non-Patent Document 1] Drago, JZ, et al., (2021) Nature Reviews, 18, 327-344. [Non-patent document 2] Kurrikoff, K., et al., (2016) Expert Opin. Drug Deliv., 13, 373-387. [Non-patent document 3] Boisguerin, P., et al., (2015) Adv. Drug Deliv. Rev., 87, 52-67. [Non-patent document 4] Lonn, P. & Dowdy, SF (2015) Expert Opin. Drug Deliv., 12, 1627-1636. [Non-Patent Document 5] Stanzl, EG, et al., (2013) Acc. Chem. Res., 46, 2944-2954. Summary of the Invention [Problem to be solved by the invention]

[0008] An objective of the present invention is to provide a polypeptide capable of binding to both an antibody and a nucleic acid, which enables cell-selective delivery of a nucleic acid (e.g., a gene); a complex comprising the polypeptide, an antibody, and a nucleic acid; a method for producing the complex; and a method for introducing a nucleic acid or a gene using the complex. [Means for solving the problem]

[0009] The present inventors constructed a carrier peptide as a fusion peptide that combines an amino acid sequence that specifically binds to a constant site of an antibody with an amino acid sequence that binds to a nucleic acid, and discovered that by mixing the carrier peptide with an antibody and a nucleic acid to form a complex, it is possible to introduce a gene only into specific cells that have affinity for the antibody, thereby completing the present invention. That is, the present invention is as follows.

[0010] [1] A polypeptide comprising an amino acid sequence (region 1) that specifically binds to a constant region of an antibody and an amino acid sequence (region 2) that specifically binds to a nucleic acid. [2] The amino acid sequence of the region 1 has a Kd value of 1×10 for binding to the constant site of an antibody. -3 The polypeptide according to [1], having a binding affinity of M or less. [3] The polypeptide according to [1] or [2], wherein the amino acid sequence of region 1 is selected from the group consisting of antibody binding sites of protein A, protein G, protein L, M protein family, Fc alpha receptor, Fc gamma receptor, Fc epsilon receptor, Fc alpha / mu receptor, rheumatoid factor, recombinant derivatives thereof, and polypeptides derived from a library of random polypeptides that specifically bind to the constant site of an antibody. [4] The amino acid sequence of region 1 is NKFRGKYK (SEQ ID NO: 1), FYWHCLDE (SEQ ID NO: 2), (RTY)4K2KG (SEQ ID NOs: 71 and 72), NARKFYKG (SEQ ID NO: 7), FYCHWALE (SEQ ID NO: 8), FYCHTIDE (SEQ ID NO: 9), TWKTSRISIF (SEQ ID NO: 10), FGRLVSSIRY (SEQ ID NO: 11), DCAWHLGELVWCT (SEQ ID NO: 12), PAWHLGELVWP (SEQ ID NO: 13), PDCAWHLGELVWCP (SEQ ID NO: 14), CDCAWHLGELVWCTC (SEQ ID NO: 15), EPIHRSTLTALL (SEQ ID NO: 16), (CFHH)2KG (SEQ ID NOs: 73 and 74), HWRGWV (SEQ ID NO: 17), HYFKFD (SEQ ID NO: 18), HFRRHL (SEQ ID NO: 19), HWCitGWV (SEQ ID NO: 75), RWHYFK (SEQ ID NO: 20), MWFRHYK (SEQ ID NO: 21), RRGW (SEQ ID NO: 22), KHRFNKD (SEQ ID NO: 23), GSYWYDVWF (SEQ ID NO: 24), CPSTHWK (SEQ ID NO: 25), NVQYFAV (SEQ ID NO: 26), ASHTQKS (SEQ ID NO: 27), QPQMSHM (SEQ ID NO: 28), TNIESLK (SEQ ID NO: 29), NCHKCWN (SEQ ID NO: 30), SHLSKNF (SEQ ID NO: 31), CVFYRNGKSFQFS (SEQ ID NO: 32), HKRSFWADN (SEQ ID NO: 33), RTQFRPNQT (SEQ ID NO: 34), QLCDFWRTR (SEQ ID NO: 35), FEDFNEQRT (SEQ ID NO: 36), LAKFLKGKD (SEQ ID NO: 37), WHRRTHKTF (SEQ ID NO: 38), RTIQTRSHW (SEQ ID NO: 39), IKLAQLHSV (SEQ ID NO: 40), WRHRNATEW (SEQ ID NO: 41), QNWIKDVHK (SEQ ID NO: 42), WKDKLVYNVL (SEQ ID NO: 43), WKDKPLVKVT (SEQ ID NO: 44), WKNTALHKVT (SEQ ID NO: 45), WRNWDVYKVI (SEQ ID NO: 46), HMVCLAYRGRPVCFAL (SEQ ID NO: 47), HMVCLSYRGRPVCFSL (SEQ ID NO: 48), KEQQERQKNLEELERQSQREVEKRYQEQLQKQQQL (SEQ ID NO: 49), KLEKKSEDVERHYLRQLDQEYKEQQERQ (SEQ ID NO: 50), YYALSDAKEEEPRYKALRGENQDLREKERKYQDKIKKLEEKEKNLEKKSC (SEQ ID NO: 51), DPQYRALMGENQDLRKREGQYQDKIEELE (SEQ ID NO: 52), AVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 53), IDEILAALPKTDTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE (SEQ ID NO: 54), The polypeptide according to any one of [1] to [3], which is a polypeptide of the present invention or a derivative thereof. [5] The polypeptide according to any one of [1] to [4], wherein the amino acid sequence of region 2 contains 40 mol % or more cationic amino acid residues. [6] The polypeptide according to any one of [1] to [5], wherein the amino acid sequence of region 2 is RRRRRRRR (SEQ ID NO: 3), KWKWKKA (SEQ ID NO: 4), KKKKKKKK (SEQ ID NO: 5), or RRRRRRWR (SEQ ID NO: 6). [7] A complex comprising the polypeptide according to any one of [1] to [6], an antibody that binds to the polypeptide, and a nucleic acid that binds to the polypeptide. [8] The complex according to [7], wherein the binding between the polypeptide and the antibody and the binding between the polypeptide and the nucleic acid are due to intermolecular interactions. [9] The complex according to [7] or [8], which has an average particle size of 1 to 300 nm.

[10] A method for producing a complex comprising the polypeptide according to any one of [1] to [6], an antibody that binds to the polypeptide, and a nucleic acid that binds to the polypeptide, the method comprising mixing the polypeptide, the antibody, and the nucleic acid in a solution.

[11] A method for selectively introducing nucleic acid into target cells, comprising the steps of contacting the polypeptide according to any one of [1] to [6] with an antibody and nucleic acid to form a complex, and contacting the resulting complex with target cells.

[12] A method for selectively introducing nucleic acid into target cells, the method comprising the step of contacting the complex according to any one of [7] to [9] with the target cells. [Effects of the Invention]

[0011] Generally, when nucleic acids (genes) are directly administered into the body, they cannot penetrate cell membranes and are rapidly degraded by nucleases without exerting their efficacy. Furthermore, they cannot be specifically introduced into target cells. However, in the present invention, the nucleic acid (gene) to be functional in the body is coated with the peptide of the present invention, and the outer layer is further coated with an antibody to form a complex, thereby forming a complex resistant to intracellular nucleases and proteases. This complex retains its ability to introduce genes into cells and remains highly stable in the body. As a result, the retention of the nucleic acid, which is the active ingredient, in the body can be increased. In addition, because the outermost layer contains an antibody that specifically binds to a specific antigen, the complex accumulates in target tissues, actively binding to and being introduced into target cells. On the other hand, the outermost layer of the antibody prevents contact, binding, and introduction of the complex into cells in non-target tissues. As a result, simple, safe, and efficient selective nucleic acid (gene) introduction into target cells is possible while suppressing non-specific gene introduction into non-target tissues. Therefore, the present invention may be extremely effective in treating genetic diseases and non-genetic diseases such as malignant tumors. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows the results of EMSA in Example 2, which confirmed the ability of the polypeptide of the present invention to bind to nucleic acids. [Figure 2] FIG. 2 is a graph showing the results of confirming the binding ability of the polypeptide of the present invention to an antibody using ELISA in Example 3. [Figure 3] FIG. 3 shows the results of observing particles of a complex of the polypeptide, antibody, and nucleic acid of the present invention in Example 4. [Figure 4] FIG. 4 is a graph showing the results of measuring the particle size and particle number of the complex of the polypeptide, antibody, and nucleic acid of the present invention in Example 4. [Figure 5]FIG. 5 is a graph showing the results of nucleic acid transfer into cells using the complex of the present invention in which SpA1-R8 was used as the polypeptide in Example 5. [Figure 6] FIG. 6 is a graph showing the results of nucleic acid transfer into cells using the complex of the present invention in which Fcγ1-R8 is used as the polypeptide in Example 6. [Figure 7] FIG. 7 is a graph showing the results of nucleic acid transfer into cells using the complex of the present invention in which SpA1-K8 was used as the polypeptide in Example 7. [Figure 8] 8 is a schematic diagram showing the structure of a complex of the present invention in one embodiment of the present invention, in which 1 represents a gene, 2 represents a polypeptide, and 3 represents an antibody. [Figure 9] FIG. 9 is a graph showing the results of nucleic acid transfer into cells using the complex of the present invention, which uses SpA1-R8 as the polypeptide and anti-EAAT2 antibody clone E-1 as the antibody in Example 8. [Figure 10] FIG. 10 shows in Example 9 the transfer of nucleic acid into mouse skin tissue using the complex of the present invention. [Figure 11] FIG. 11 is a graph showing the quantitative measurement results of the luminescence intensity of luciferase derived from nucleic acid introduced into mouse skin tissue by the complex of the present invention in Example 9. [Figure 12] FIG. 12 shows, in Example 10, the transfer of nucleic acid into mouse skin tissue using the complex of the present invention. [Figure 13] FIG. 13 is a graph showing in Example 11 that the nucleic acid encapsulated in the complex of the present invention is resistant to DNase. [Figure 14] FIG. 14 is a graph showing that in Example 12, nucleic acid transfer efficiency and selectivity can be improved by changing the preparation process of the complex of the present invention. [Figure 15] FIG. 15 shows, in Example 13, the transfer of nucleic acid into mouse central nervous tissue using the complex of the present invention. [Figure 16]FIG. 16 is a graph showing the results of measuring the amount of nucleic acid transferred into mouse central nervous tissue by the complex of the present invention in Example 13. [Figure 17] FIG. 17 is a graph showing the results of measuring the particle size and particle number of the complex of the present invention after freezing and thawing in Example 14. [Figure 18] FIG. 18 is a graph showing the results of measuring the particle size and particle number of the complex of the present invention after freezing, vacuum freeze-drying, and dissolution in Example 14. [Figure 19] FIG. 19 is a graph showing the resistance of the complex of the present invention to proteases in Example 15. [Figure 20] FIG. 20 is a graph showing the antitumor effect of the complex of the present invention in Example 16. [Figure 21] FIG. 21 is a diagram showing the structure of the complex of the present invention confirmed using an electron microscope in Example 17. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention will now be described. Terms used in this specification have the meanings commonly used in the art unless otherwise specified. When amino acids and the like are represented by abbreviations in this specification, they are based on the abbreviations specified by the IUPAC-IUB Commission on Biochemical Nomenclature or common abbreviations used in the art.

[0014] 1. Polypeptides capable of binding to both antibodies and nucleic acids The present invention provides a polypeptide capable of binding to both an antibody and a nucleic acid, specifically a polypeptide comprising an amino acid sequence (region 1) that specifically binds to a constant region of an antibody and an amino acid sequence (region 2) that binds to a nucleic acid (hereinafter also referred to as the "polypeptide of the present invention"). The amino acid sequence of region 1 is not particularly limited as long as it specifically binds to the constant region of an antibody and does not inhibit the binding of the antibody to a target antigen. The antibody to which the amino acid sequence of region 1 specifically binds may be any of IgA, IgD, IgE, IgG, and IgM. IgG is preferred. If the antibody has subclasses, any subclass may be used as long as the desired effect of the present invention is obtained. For example, five subclasses of human IgG are known: IgG1, IgG2a, IgG2b, IgG3, and IgG4. However, any IgG subclass may be used as long as the desired effect of the present invention is obtained. A low-molecular-weight antibody such as a single chain Fv or VHH may also be used. In this specification, the "constant region" of an antibody refers to a region that changes relatively little in the amino acid sequence of the antibody. Examples of the constant region of an antibody include regions corresponding to the constant regions (C regions) of each class of antibody (e.g., C L area, C H Area (C H 1. C H 2. C H Examples of such regions include the Fc region (V region, Fc region, etc.) and constant regions in the variable region (V region). The amino acid sequence of Region 1 of the present invention may bind to any constant region of any isoform, as long as it does not inhibit antibody binding to the target antigen, but is preferably one that binds to the Fc region of IgG1. Examples of amino acid sequences in region 1 include those derived from protein A, protein G, protein L, the M protein family, rheumatoid factor, and Fc receptors. Protein A is a 46.7 kDa protein present in the cell wall of Staphylococcus aureus. Protein A specifically binds to the constant site of immunoglobulins (especially IgG). It has an affinity binding site for IgG (Ka=10 -8Protein G is a protein present in the cell wall of group G streptococci. Protein G specifically binds to the constant site of immunoglobulins (especially IgG). It has four affinity binding sites for IgG (Ka=10 -8 Two regions of the Fc receptor (M) are known. Protein L is a 35.8 kDa protein derived from Peptostreptococcus magnus that specifically binds to the constant region of the kappa light chain of immunoglobulins (especially IgG). The M protein family, which comprises group A streptococci, includes several proteins that bind to the constant region of immunoglobulins. Rheumatoid factor (RF) is an autoantibody against the Fc region of IgG observed in rheumatoid arthritis, other autoimmune diseases, and chronic hepatitis. Fc receptors are receptor proteins that bind to the Fc region of immunoglobulin molecules and are present on cell surfaces. Receptors for the immunoglobulin molecules IgG, IgA, IgE, and IgM are called FcγR, FcαR, FcεR, and FcμR, respectively. The sequence derived from which receptor is used depends on the subclass of antibody to which the amino acid sequence in region 1 binds. For example, if the antibody is intended to be IgG, an amino acid sequence derived from FcγR is preferred.

[0015] Methods for determining peptide sequences capable of binding to antibodies include, for example, constructing a polypeptide library from the amino acid sequences of these antibody-binding proteins using physical or virtual techniques, and selecting and modifying amino acid sequences that specifically bind to constant regions of antibodies (Biochemical Engineering Journal, Volume 79, October 15, 2013, Pages 33-40; Biochemical Engineering Journal, Volume 88, July 15, 2014, Pages 1-11). Another method involves selecting and modifying amino acid sequences that specifically bind to constant regions of antibodies from a polypeptide library constructed with random amino acid sequences.

[0016] In one embodiment of the present invention, the amino acid sequence of region 1 is preferably FYWHCLDE (SEQ ID NO: 2), a sequence derived from protein A, or NKFRGKYK (SEQ ID NO: 1), a sequence derived from FcγR, and more preferably the amino acid sequence of SEQ ID NO: 2. In another embodiment of the present invention, the amino acid sequence of region 1 may be: (RTY)4K2KG (also referred to as "TG19320", SEQ ID NOs: 71 and 72); NARKFYKG (SEQ ID NO: 7), FYCHWALE (SEQ ID NO: 8), FYCHTIDE (SEQ ID NO: 9), TWKTSRISIF (SEQ ID NO: 10), FGRLVSSIRY (SEQ ID NO: 11), DCAWHLGELVWCT (SEQ ID NO: 12), PAWHLGELVWP (SEQ ID NO: 13), PDCAWHLGELVWCP (SEQ ID NO: 14), CDCAWHLGELVWCTC (SEQ ID NO: 15), EPIHRSTLTALL (SEQ ID NO: 16), (CFHH)2KG (SEQ ID NOs: 73 and 74), HWRGWV (SEQ ID NO: 17), HYFKFD (SEQ ID NO: 18), HFRRHL (SEQ ID NO: 19), HWCitGWV ("Cit" is citrullinated arginine) (SEQ ID NO: 75); RWHYFK (SEQ ID NO: 20), MWFRHYK (SEQ ID NO: 21), RRGW (SEQ ID NO: 22), KHRFNKD (SEQ ID NO: 23), GSYWYDVWF (SEQ ID NO: 24), CPSTHWK (SEQ ID NO: 25), NVQYFAV (SEQ ID NO: 26), ASHTQKS (SEQ ID NO: 27), QPQMSHM (SEQ ID NO: 28), TNIESLK (SEQ ID NO: 29), NCHKCWN (SEQ ID NO: 30), SHLSKNF (SEQ ID NO: 31), CVFYRNGKSFQFS (SEQ ID NO: 32), HKRSFWADN (SEQ ID NO: 33), RTQFRPNQT (SEQ ID NO: 34), QLCDFWRTR (SEQ ID NO: 35), FEDFNEQRT (SEQ ID NO: 36), LAKFLKGKD (SEQ ID NO: 37), WHRRTHKTF (SEQ ID NO: 38), RTIQTRSHW (SEQ ID NO: 39), IKLAQLHSV (SEQ ID NO: 40), WRHRNATEW (SEQ ID NO: 41), QNWIKDVHK (SEQ ID NO: 42), WKDKLVYNVL (SEQ ID NO: 43), WKDKPLVKVT (SEQ ID NO: 44), WKNTALHKVT (SEQ ID NO: 45), WRNWDVYKVI (SEQ ID NO: 46), HMVCLAYRGRPVCFAL (SEQ ID NO: 47), HMVCLSYRGRPVCFSL (SEQ ID NO: 48), KEQQERQKNLEELERQSQREVEKRYQEQLQKQQQL (SEQ ID NO: 49), KLEKKSEDVERHYLRQLDQEYKEQQERQ (SEQ ID NO: 50), YYALSDAKEEEPRYKALRGENQDLREKERKYQDKIKKLEEKEKNLEKKSC (SEQ ID NO: 51), DPQYRALMGENQDLRKREGQYQDKIEELE (SEQ ID NO: 52), AVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 53) or IDEILAALPKTDTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE (SEQ ID NO: 54).

[0017] In one embodiment of the present invention, the amino acid sequence of region 1 may be a derivative of the amino acid sequence of SEQ ID NO: 1, 2, or 7 to 54, etc., above.

[0018] As used herein, the phrase "region 1 specifically binds to the constant region of an antibody" means that the Kd value of the binding affinity of region 1 to the constant region of an antibody is 1×10 -3 M or less (preferably 2 × 10 -4 M or less, 1×10 -4 M or less, 1×10 -5 M or less, 1×10 -6 M or less, 1×10 -7 M or less, 1×10 -8 M or less, 1×10 -9 M or less, 1×10 -10 M or less). NKFRGKYK (SEQ ID NO: 1) has a Kd of 1.7x10 -7 M, FYWHCLDE (SEQ ID NO: 2) has a Kd of 1.5x10 -6 M, HWRGWV (SEQ ID NO: 17) Kd = 1.0 x 10 -5 M, HWCitGWV is Kd=1.1x10 -4 It's M.

[0019] The amino acid sequence of region 2 is not particularly limited as long as it is an amino acid sequence that binds to nucleic acids. The binding mode between region 2 and nucleic acids may be covalent or non-covalent (e.g., electrostatic interaction, hydrogen bonding, van der Waals forces, etc.). However, because nucleic acids are negatively charged regardless of the genetic information they contain, electrostatic interaction is preferred. More specifically, the phosphate groups of the ribose-phosphate backbone of nucleic acids are negatively charged in the neutral to basic pH range. Therefore, by using a positively charged peptide as the amino acid sequence of region 2, the two can be electrostatically bound. Therefore, in a preferred embodiment of the present invention, the amino acid sequence of region 2 of the present invention has a polycationic sequence, contains at least three amino acid residues selected from lysine (K), arginine (R), and histidine (H), and forms a stable bond with nucleic acids under physiological conditions. In addition to the positively charged amino acid residues (cationic amino acid residues) lysine, arginine, and histidine, the polycation component may also contain neutral amino acids, provided that its overall nature is sufficiently cationic and that it forms stable bonds with nucleic acids under physiological conditions. The length of the amino acid sequence in region 2 is preferably 5 to 100 amino acid residues, more preferably 5 to 50, even more preferably 7 to 20, and even more preferably 7 to 10 amino acid residues. The proportion of cationic amino acid residues in the amino acid sequence is preferably 40 mol% or more, more preferably 60 mol% or more, even more preferably 80 mol% or more, and most preferably 90 mol% or more. A polycation sequence consisting solely of cationic amino acid residues is most preferably used.

[0020] Furthermore, when electrostatic interaction is adopted as the binding mode between the amino acid sequence of region 2 and the nucleic acid, the binding between the amino acid sequence of region 2 and the nucleic acid relies on the positive charge of region 2 and the negative charge of the phosphate group in the ribose-phosphate backbone of the nucleic acid, and the content of the genetic information of the nucleic acid does not impose any limitations on the implementation of the present invention, which is extremely preferable in that the polypeptide of the present invention can be used for an extremely wide range of purposes.

[0021] In one embodiment of the present invention, in addition to employing electrostatic interaction as the binding mode between the amino acid sequence of region 2 and nucleic acid, a binding mode other than electrostatic interaction can also be used in combination. For example, aromatic amino acids such as phenylalanine (F), tryptophan (W), and tyrosine (T) are known to form π-π interactions or CH-π interactions between nucleic acid bases and cyclic sugar chains (Nucleic Acids Res. 2014 Jun;42(10):6726-41). Therefore, the binding strength between the amino acid sequence of region 2 and nucleic acid may be strengthened or stabilized by including an aromatic amino acid in the amino acid sequence of region 2. In one embodiment of the present invention, the amino acid sequence of region 2 is preferably RRRRRRRR (SEQ ID NO: 3), KWKWKKA (SEQ ID NO: 4), KKKKKKKK (SEQ ID NO: 5) or RRRRRRWR (SEQ ID NO: 6), and more preferably the amino acid sequence of SEQ ID NO: 3.

[0022] The polypeptide of the present invention is a polypeptide represented by an amino acid sequence containing an amino acid sequence (region 1) that specifically binds to a constant region of an antibody and an amino acid sequence (region 2) that binds to a nucleic acid.

[0023] The amino acids constituting the polypeptide may be either L- or D-amino acids, natural or unnatural amino acids, or may be chemically modified as appropriate. The polypeptide may be cyclic, linear, or branched.

[0024] The order of Region 1 and Region 2 within the polypeptide is not particularly limited as long as they maintain their ability to bind to antibodies and nucleic acids, respectively, and may be Region 1 → Region 2 from the N-terminus, or Region 2 → Region 1. Preferably, Region 1 is on the N-terminus and Region 2 is on the C-terminus. In the polypeptides of the present invention, the polypeptide having the amino acid sequence of region 1 and the polypeptide having the amino acid sequence of region 2 can be bonded chemically by a conventional peptide bonding reaction, or biologically using an enzyme such as ligase. For example, this can be done according to a general peptide synthesis method such as solid-phase synthesis (e.g., Fmoc solid-phase synthesis). An appropriate amino acid sequence can be added to the N-terminus or C-terminus of region 1 and region 2, or between the two regions. For example, a linker consisting of one to several amino acids can be inserted between the two regions, and the amino acid residues constituting the linker can be selected appropriately. A group capable of forming an intrinsically unfolded structure that can freely change its shape to bind to other proteins, and more preferably composed of glycine (G) and serine (S), is preferred. Furthermore, functional amino acid sequences such as an enzymatic degradation sequence or a nuclear localization signal can be added or inserted, for example, at the end of each region or between the two regions.

[0025] The polypeptides of the present invention can also be obtained by recombinant DNA techniques. For example, a DNA fragment encoding the amino acid sequence of region 1 can be ligated to one or both ends of a DNA fragment encoding the amino acid sequence of region 2 by ligation with an appropriate DNA adapter or by in vitro mutagenesis. Such genetic engineering methods are well known to those skilled in the art of molecular biology.

[0026] In one embodiment, a polypeptide of the invention can be: FYWHCLDERRRRRRRR (SEQ ID NO: 55), FYWHCLDEKKKKKKKK (SEQ ID NO: 56), FYWHCLDEKWKWKKA (SEQ ID NO: 57), FYWHCLDERRRRRRWR (SEQ ID NO: 58), NKFRGKYKRRRRRRRR (SEQ ID NO: 59), NKFRGKYKKKKKKKKK (SEQ ID NO: 60), NKFRGKYKKWKWKKA (SEQ ID NO: 61), NKFRGKYKRRRRRRWR (SEQ ID NO: 62), HWRGWVRRRRRRRR (SEQ ID NO: 63), HWRGWVKKKKKKKK (SEQ ID NO: 64), HWRGWVKWKWKKA (SEQ ID NO: 65), HWRGWVRRRRRRWR (SEQ ID NO: 66), KHRFNKDRRRRRRRR (SEQ ID NO: 67) KHRFNKDKKKKKKKK (SEQ ID NO: 68) KHRFNKDKWKWKKA (SEQ ID NO: 69), KHRFNKDRRRRRRWR (SEQ ID NO: 70), HWCitGWVRRRRRRRR (SEQ ID NO: 76), HWCitGWVKKKKKKKK (SEQ ID NO: 77), HWCitGWVKWKWKKA (SEQ ID NO: 78), or HWCitGWVRRRRRRWR (SEQ ID NO: 79).

[0027] 2. Complex containing polypeptide, antibody and nucleic acid and method for producing the same The present invention provides a complex comprising the polypeptide described in 1 above, an antibody that binds to the polypeptide, and a nucleic acid that binds to the polypeptide (hereinafter also referred to as the complex of the present invention).

[0028] The nucleic acid constituting the complex of the present invention is not particularly limited, and can be any nucleic acid whose introduction into target cells is desired. The nucleic acid may be linear or circular. It may also be single-stranded or double-stranded. It may also be DNA, RNA, or a hybrid of DNA and RNA. DNA encompasses DNA molecules of all types and sizes, including cDNA, plasmids, genomic DNA, and derivatives thereof. Furthermore, such nucleic acids can be chemically modified as long as their binding to the amino acid sequence (polycation sequence) of region 2 of the polypeptide of the present invention is preserved. Examples of suitable modified nucleic acids include thioates and dithioates. Furthermore, nucleic acids with chemically modified nucleotide bases can also be used. For example, RNA molecules in which the 2'-OH groups of one to several nucleotides are replaced with O-alkyl groups, halogens, or other modifying groups can also be used. The nucleic acid to be introduced into target cells is preferably DNA or RNA, modified as desired. For example, the nucleic acid to be introduced into target cells can contain genetic information to be expressed in the target cells. This method can, for example, remove gene-dependent defects. On the other hand, to suppress the expression of a specific gene in the target cell, the nucleic acid introduced into the target cell may have antisense properties (i.e., the nucleic acid is complementary to the mRNA expressed in the target cell). As such a nucleic acid, the nucleic acid of interest may have ribozyme properties, i.e., the ability to cleave a specific RNA molecule in the target cell.

[0029] In one embodiment of the present invention, the nucleic acid introduced into the target cell may be a nucleic acid drug, i.e., an antisense oligonucleotide (ASO, antisense nucleic acid), RNAi (siRNA), microRNA (miRNA), aptamer, or decoy.

[0030] The size of the nucleic acid contained in the complex of the present invention is not particularly limited as long as it can be introduced into the target cell, and short-chain RNA of about 20 base pairs to double-stranded DNA of about several hundred kilobase pairs can be used. For example, in the case of double-stranded DNA, the size of the nucleic acid to be introduced is usually 20 base pairs to 20 kilobase pairs, preferably about 50 base pairs to 10 kilobase pairs.

[0031] The antibody constituting the complex of the present invention is not particularly limited, and is an antibody that enables delivery to target cells and is directed against a marker molecule that is specifically expressed on the cell surface of the target cells.

[0032] In one embodiment, the antibody may be an antibody that can be internalized into cells in an endocytic manner through interaction with an antigen (antigen-antibody internalization pathway). Without wishing to be bound by any theory, this is because it suggests that the excellent nucleic acid transfer efficiency of the complex of the present invention into target cells is largely due to the internalization ability of the antibody rather than the cell permeability of the polypeptide. The internalization ability of the antibody can be confirmed, for example, by contacting a pH-responsive fluorescently labeled antibody alone with target cells and detecting intracellular fluorescence.

[0033] Both the nucleic acids and the antibodies are commercially available or can be prepared by methods known per se.

[0034] The mode of binding between a polypeptide and an antibody or between a polypeptide and a nucleic acid is not particularly limited, but binding via intermolecular interactions such as electrostatic interaction, van der Waals force, dipole-dipole interaction, dispersion force, hydrogen bond, charge transfer, and hydrophobic interaction is preferred, and binding via electrostatic interaction is particularly preferred. For example, the nucleic acid binds to the polypeptide of the present invention via electrostatic interaction between the negative charge of the nucleic acid and the positive charge of the amino acid sequence in region 2.

[0035] The complex of the present invention can be produced by mixing the polypeptide of the present invention described in 1 above, a nucleic acid that binds to the polypeptide, and an antibody that binds to the polypeptide in a solution. The order of mixing is not particularly limited, but in a preferred embodiment, the complex is produced by first mixing the nucleic acid and the antibody, and then adding the peptide to the mixture. Furthermore, when mixing the components, the solution may be stirred using pipetting, a vortex, or the like. In another preferred embodiment, the complex can be prepared by mixing a solution containing the nucleic acid and a solution containing the antibody by pipetting, and then adding the peptide solution to the mixture while vortexing.

[0036] Furthermore, in the step of mixing the polypeptide of the present invention, antibody, and nucleic acid to form a complex, the respective mixing ratios are set appropriately depending on the nucleic acid, polypeptide of the present invention, and antibody used. However, when the nucleic acid is a plasmid gene composed of one molecule of DNA bases with a molecular weight of 330, the polypeptide of the present invention is approximately 2,000, and the antibody is an IgG antibody with an molecular weight of approximately 150,000, the "molar ratio of nucleic acid:polypeptide of the present invention:antibody" is preferably 6,000-10,000:250-200,000:50-1,600, and more preferably 6,000-10,000:500-100,000:100-800. The step of forming a complex comprising the polypeptide of the present invention, nucleic acid, and antibody can be carried out, for example, by mixing the polypeptide of the present invention, nucleic acid, and antibody in a solution. In this case, the concentration of the polypeptide of the present invention is usually 0.01 to 50 mg / mL, preferably 0.1 to 5 mg / mL, the concentration of the nucleic acid solution is usually 1 to 2000 μg / mL, preferably 10 to 200 μg / mL, and the concentration of the antibody is usually 0.01 to 1000 mg / mL, preferably 0.1 to 1000 mg / mL.

[0037] Although there are no particular limitations on the form of the complex of the present invention formed as described above, in embodiments in which the introduced nucleic acid is expressed in target cells, it is generally a circular or linear nucleic acid incorporating a promoter region and the like in addition to the desired gene sequence, and in such embodiments, the complex of the present invention forms a unique particulate structure in which the peptide of the present invention is arranged around the nucleic acid and the antibody is arranged further outside of that, as shown in Figure 8. The average particle diameter of the complex forming the particulate structure in such embodiments depends on the size of the circular nucleic acid used, but may typically be 1 nm or more, preferably 5 nm or more, 10 nm or more, 15 nm or more, 20 nm or more, 25 nm or more, or 30 nm or more, and may typically be 300 nm or less, preferably 200 nm or less, 150 nm or less, 100 nm or less, 80 nm or less, 60 nm or less, or 40 nm or less. The average particle size may be in the range of 1 to 300 nm, preferably 5 to 200 nm, 10 to 150 nm, 15 to 100 nm, 20 to 80 nm, 25 to 60 nm, or 30 to 40 nm. The average particle size can be measured by dynamic light scattering (DLS) or nanoparticle tracking analysis (NTA), which measures the properties of both scattered light and Brownian motion.

[0038] 3. Method for selectively introducing nucleic acid into target cells The present invention provides a method for selectively introducing nucleic acid into target cells. One embodiment of the method comprises the step of contacting the polypeptide of the present invention with an antibody and nucleic acid to form a complex as described above in 1, and contacting the resulting complex with target cells. Alternatively, another embodiment comprises the step of contacting the complex of the present invention with target cells as described above in 2. The "contacting step" specifically refers to a mixing step (in solution). For example, the method can be carried out by adding a solution of the complex of the present invention to the target cells and incubating them at room temperature in an incubator. The incubation time is preferably 5 to 150 hours, more preferably 10 to 30 hours.

[0039] Here, the nucleic acids and antibodies that can be used are similar to those described in 2 above, and are appropriately selected depending on the intended use (eg, gene therapy use and research use).

[0040] For example, the present invention can be used in a combination therapy of antibody drug therapy and gene therapy for cancer. For example, in oral squamous cell carcinoma, overexpression of EGFR (epidermal growth factor receptor) is observed on the surface of cancer cells in over 90% of cases. EGFR acts to promote cancer cell proliferation by activating the signaling pathway downstream of EGFR. Antibody drugs that bind to and inhibit EGFR (e.g., cetuximab) are commercially available. Nucleic acid drugs that inhibit its downstream signaling pathway are also considered promising. Therefore, for example, the present invention can be used to form and administer a complex consisting of an EGFR-inhibiting antibody and an shRNA that blocks downstream signaling of EGFR, or a plasmid vector gene medicine encoding the shRNA. The complex accumulates in cancer lesions expressing EGFR due to the action of the antibody, enabling selective gene transfer to cancer cells. In one embodiment of the present invention, a cancer therapeutic antibody drug and a cancer therapeutic gene medicine can be combined via the peptide of the present invention to form a complex of the present invention that exerts a synergistic antitumor effect.

[0041] Furthermore, for example, the present invention can be used as a method for selective gene delivery to cells in central nervous system tissues for the purpose of research or disease treatment. Central nervous system tissues are composed of multiple types of cells, including not only neurons but also astrocytes, oligodendrocytes, and microglia.

[0042] Astrocyte-specific surface antigens AQP4 and EAAT2 are known to be internalized upon antibody binding. By utilizing this mechanism and the present invention, it is possible to selectively transfer genes into astrocytes.

[0043] Furthermore, for example, the present invention can be used as a selective gene delivery method for cells of skin and mucosal tissues for the purpose of research or disease treatment. Desmoglein 1 (Dsg1) and desmoglein 3 (Dsg3) are known as cell surface antigens specific to skin and mucosal cells. Dsg1 and Dsg3 have different expression patterns in skin and mucosal tissues. In skin, Dsg3 is strongly expressed in cells of the basal and parabasal layers of the lower epidermis, while Dsg1 expression increases from the lower to upper layers of the epidermis. In contrast, in mucosa, Dsg3 is strongly expressed in cells of all layers of the mucosal epithelium, whereas Dsg1 expression is weaker than Dsg3.

[0044] Desmoglein 3 (Dsg3) is known to be internalized upon antibody binding, and by utilizing this mechanism and the present invention, it is possible to selectively transfer genes to specific cells in the skin and mucosa.

[0045] The present invention can also be made into a kit for introducing nucleic acids into target cells. The kit of the present invention is characterized by comprising the polypeptide of the present invention described in 1. The kit of the present invention may further comprise an antibody or nucleic acid that binds to the polypeptide. The kit may also include an instruction manual, reagents and tools for complex formation and cell introduction, etc.

[0046] The present invention will be described in detail below using examples, but the present invention is not limited in any way. Unless otherwise specified, the reagents and materials used are commercially available or can be prepared according to known literature. Furthermore, those skilled in the art will understand that any substance having the same effect or action can be substituted. [Example]

[0047] Example 1: Polypeptide Production A fusion peptide (NKFRGKYKRRRRRRRR; SEQ ID NO: 59) having the amino acid sequence of SEQ ID NO: 1 as the amino acid sequence of region 1 and the amino acid sequence of SEQ ID NO: 3 as the amino acid sequence of region 2 was produced by Fmoc solid-phase synthesis and named Fcγ1-R8 peptide. Chemical Formula: C 96 H 173 N 47 O 19 Molecular Weight:2289.79 g / mol

[0048] A fusion peptide (FYWHCLDERRRRRRRR; SEQ ID NO: 55) having the amino acid sequence of SEQ ID NO: 2 as the amino acid sequence of region 1 and the amino acid sequence of SEQ ID NO: 3 as the amino acid sequence of region 2 was produced by Fmoc solid-phase synthesis and named SpA1-R8 peptide. Chemical Formula: C 101 H 161 N 43 O 22 Molecular weight: 2361.78 g / mol

[0049] A fusion peptide (FYWHCLDEKKKKKKKK; SEQ ID NO: 56) having the amino acid sequence of SEQ ID NO: 1 as the amino acid sequence of region 1 and the amino acid sequence of SEQ ID NO: 5 as the amino acid sequence of region 2 was produced by Fmoc solid-phase synthesis and named SpA1-K8 peptide. Chemical Formula: C 101 H 161 N 27 O 22 Molecular weight: 2137.7 g / mol

[0050] Example 2: Confirmation of binding between polypeptide and nucleic acid 1. Materials and Methods (1) Modified gel shift assay (EMSA) (2) DNA probe A 104-bp double-stranded DNA derived from pNL1.3.CMV[secNluc / CMV] (Promega Corporation) was used. The DNA was amplified by PCR using primers labeled with IRDye 700 (synthesized by IDT) and purified. 10 ng was used per lane (equivalent to 15.15 pmol, assuming one DNA base pair is one molecule and a molecular weight of 660 g / mol). Diluted with 10 mM HEPES (pH 7.4) to a volume of 2 μl. (3) Polypeptide Fcγ1-R8 (molecular weight 2289.79 g / mol) was used. -Synthesized and purified by Fmoc solid phase synthesis (synthesis commissioned to Biologica, synthesis by GL Biochem). Dissolved in 10mM HEPES (pH 7.4). Polypeptide in amounts of 0 (no polypeptide), 2, 4, 8, 16, 32, and 64 times the molar ratio of DNA base pairs was dissolved and adjusted to a liquid volume of 2 μl. (4) Mixing DNA probes and polypeptides (2) and (3) were mixed to make a total of 4 μl of solution, and the solution was left to stand at room temperature for 20 minutes. (5) Electrophoresis The Odyssey® EMSA Kit (IL-COR) was prepared by mixing 2 μl of 10X Binding Buffer (100 mM Tris, 500 mM KCl, 10 mM DTT, pH 7.5), 2 μl of 25 mM DTT, 2.5% Tween® 203, 1 μl of Poly(dI·dC) (1 μg / μL in 10 mM Tris, 1 mM EDTA, pH 7.5), and 11 μl of ultrapure water. Then, 4 μl of (4) was mixed. Mixed with 2 μl of 10X Orange Loading Dye from Odyssey® EMSA Kit (IL-COR). · 20 μl of the mixture was injected into a lane of a 6% TBE gel. Electrophoresis was carried out at 70 V for 90 minutes (running buffer was 0.5X TBE). (6) Shooting After electrophoresis, the gel was photographed directly using an Odyssey CLx (LI-COR). Excitation light source: 685 nm semiconductor laser, detection wavelength: 700 ch: 710-730 nm

[0051] 2. Results and Discussion The results are shown in Figure 1. As the molar ratio of polypeptide to DNA probe increased, the mobility of the probe in electrophoresis decreased, and when the molar ratio became too high, the DNA probe could no longer enter the gel. This is thought to be the result of the molecular weight of the DNA probe increasing due to the binding of the polypeptide to the nucleic acid through intermolecular interactions.

[0052] Example 3: Confirmation of binding between polypeptide and antibody 1. Materials and Methods (1) Modified ELISA method (2) Polypeptide Fcγ1-R8 (molecular weight 2289.79 g / mol) was used. Dissolved in 10mM HEPES (pH 7.4). A Peptide Coating Kit (Takara Bio Inc.) was used, which includes a Reaction Plate, Coupling Reagent, Reaction Buffer, and Blocking Solution. The polypeptide dissolved in HEPES was serially diluted with Reaction Buffer to 4.2 μg, 8.4 μg, 16.9 μg, 33.8 μg, and 67.5 μg per well, with a total volume of 50 μl. (3) Immobilization of polypeptides (2) was poured into a Reaction Plate, and Coupling Reagent was added to each well at 100 μg. Allow to stand at room temperature for 2 hours. Washed three times with ultrapure water. (4) Blocking 200 μl of blocking solution was added per well and incubated at 37°C for 1 hour. (5) Primary antibody binding reaction Normal human IgG, whole molecule (Fujifilm Wako Pure Chemical Industries, Ltd.) was used. The antibody molecular weight was calculated as 150,000 g / mol. 172.8 ng of antibody was dissolved in 200 μl of PBS-T per well and injected. Incubated at 37°C for 1 hour. Washed with PBS-T. (6) Secondary antibody binding reaction Peroxidase-conjugated AffiniPure Goat Anti-Human IgG (H+L) (Proteintech) was diluted 1:1000 in PBS-T and 200 μl was injected per well. Incubated at 37°C for 1 hour. Washed with PBS-T. (7) Color reaction 100 μl of TMB solution (1 reagent type) (Beacon) was injected per well. After leaving the plate at room temperature for 30 minutes, 50 μl of 2N H2SO4 was added per well to stop the reaction. (8) Absorbance measurement The absorbance was measured at a wavelength of 450 nm using a microplate reader (Thermo Fisher Scientific Multiskan Go).

[0053] 2. Results and Discussion The results are shown in Figure 2. The absorbance increased with increasing amounts of peptide coated on the plate. This is thought to be the result of human IgG antibody binding to the polypeptide, followed by binding of the peroxidase-modified secondary antibody to the antibody.

[0054] Example 4: Confirmation of complex formation between polypeptide, antibody and nucleic acid (1) Genes The plasmid vector pNL1.3.CMV[secNluc / CMV] (Promega Corporation), 3945 bp, encoding secreted luciferase for mammalian cell expression was used. The molecular weight of 1 bp was calculated as 660 g / mol, and the molecular weight per vector (3945 bp) was calculated as 2603700 g / mol. (2) Polypeptide SpA1-R8 (molecular weight 2361.78 g / mol) and Fcγ1-R8 (molecular weight 2289.79 g / mol) were used. (3) Antibodies Normal human IgG, whole molecule (Fujifilm Wako Pure Chemical Industries, Ltd.) was used. The antibody molecular weight was calculated as 150,000 g / mol. (4) Antibody + Polypeptide + Gene Complex Formation 2500ng of gene was diluted with HEPES to a solution volume of 50μl and filtered through a 0.1μm PVDF filter. SpA1-R8 and Fcγ1-R8 polypeptides were diluted in HEPES at a molar ratio of 96,000 times the gene content to make a solution volume of 50 μl, and filtered through a 0.1 μm PVDF filter. The antibody was diluted in HEPES at a molar ratio of 200 times that of the gene to make a solution volume of 50 μl, and filtered through a 0.1 μm PVDF filter. 50 μl each of the gene, polypeptide, and antibody were mixed to a total volume of 150 μl and allowed to stand for 20 minutes to form antibody + SpA1-R8+ gene and antibody + Fcγ1-R8+ gene complexes. 50 μl of each of the solutions of gene only, antibody only, SpA1-R8 only, and Fcγ1-R8 only, which had been diluted and filtered in the same manner, was diluted with 100 μl of HEPES to a total of 150 μl. (5) Observation of particles and measurement of particle size and particle number The solutions of antibody + SpA1-R8 + gene and antibody + Fcγ1-R8 + gene complexes prepared in (4) were diluted 15-fold with HEPES to a solution volume of 2250 μl. The gene alone, antibody alone, SpA1-R8 alone, and Fcγ1-R8 alone were each diluted 15-fold with HEPES to a solution volume of 2250 μl. Using a NanoSight (LM10-SHR, NTA software v3.2, Japan Quantum Design), the scattered light from each particle was observed using a camera mounted on a microscope equipped with a 20x objective lens, and particle size and particle number concentration were measured in the range of 0.5 nm to 999.5 nm by nanoparticle tracking (trajectory) analysis. The particle number concentration of the original solution was calculated by multiplying the measured value by 15. The particle number concentration for each particle size was integrated to calculate the total particle number concentration in the solution. 2. Results and Discussion The results of observing scattered light using the NanoSite are shown in Figure 3. Particles of antibody + SpA1-R8 + gene complex and antibody + Fcγ1-R8 + gene complex were observed. The results of measuring the particle count and particle size using NanoSite are shown in Figure 4. The antibody + SpA1-R8 + gene complex had a mean value of 169.2 nm and a mode value of 85.4 nm, totaling 7.68 × 10 particles. 9 The antibody + Fcγ1-R8 + gene complex was observed at a concentration of 5.37 × 10 particles with a mean value of 190.9 nm and a mode value of 98.0 nm. 9 / ml concentration was observed. The particle concentrations of the gene only, antibody only, SpA1-R8 only, and Fcγ1-R8 only solutions were 0 / ml and 2.29×10 9 / ml, 1.39 × 10 9 / ml, and 0 / ml. - It was confirmed that a large amount of complex particles with an average size of less than 200 nm and a mode of less than 100 nm were formed in a mixture with a molar ratio of gene:polypeptide:antibody of 1:96000:200.

[0055] Example 5: Introduction of nucleic acid into cells using a complex of polypeptide, antibody and nucleic acid - 1 1. Materials and Methods (1) Polypeptide SpA1-R8 (molecular weight 2361.78 g / mol) (synthesis requested from Biologica, synthesis by GL Biochem) was used. (2) Antibodies Cetuximab (Erbitux®: Bristol-Myers Squibb, Merck Serono, ImClone) was used as an anti-EGFR antibody. Normal human IgG, whole molecule (Fujifilm Wako Pure Chemical Industries, Ltd.) was used as a negative control. The antibody molecular weight was calculated as 150,000 g / mol. (3) Cell line The HSC2 cell line (oral epithelial carcinoma cell line) was used. The expression of EGFR on the cell membrane of HSC2 has been previously confirmed by immunohistochemistry (preliminary study, data not shown). Internalization of anti-EGFR antibody (cetuximab) by HSC2 has been confirmed using pHAb-Reactive-Dyes (Promega) (preliminary study, data not shown). The day before the experiment, plate 1 x 10 cells in a cell culture plate. 4 The cells were seeded at 10 cells / well and cultured at 37°C in the presence of 5% CO2. (4) Genes The plasmid vector pNL1.3.CMV[secNluc / CMV] (Promega Corporation), 3945 bp, encoding secreted luciferase for mammalian cell expression was used. The molecular weight of 1 bp was calculated as 660 g / mol, and the molecular weight per vector (3945 bp) was calculated as 2603700 g / mol. (5) Formation of antibody + polypeptide + gene complex and administration to cells 100 ng of gene (0.0384 pmol of vector as one molecule) per well was mixed with polypeptide at molar ratios of 0 (no polypeptide), 12,000, 24,000, 48,000, and 96,000. Furthermore, 100 ng of the gene was mixed with antibody at a molar ratio of 200 times. The mixture was left at room temperature for 20 minutes to allow the antibody, polypeptide, and gene complexes to form. A solution containing the formed complex was administered to wells where cells were cultured, and the gene was introduced. (6) Gene expression analysis 24 hours after transfection, 10 μl of cell culture medium (culture supernatant) was collected from each well. The plate was reacted with Coelenterazine 400 a (GoldBio) dissolved in 0.01% Tween / 0.3% EDTA / PBS at 1 μg / 100 μl per well. The luminescence intensity for 10 seconds starting from 2 seconds after the reaction was measured using a multiplate reader (Spark 10M SparkControl Magellan v1.2) (TECAN) and integrated.

[0056] 2. Results and Discussion The results are shown in Figure 5. The horizontal axis shows the molar ratio of polypeptide / gene, with 0 (no polypeptide), 24,000, 48,000, and 96,000 times indicated as 0k, 24k, 48k, and 96k, respectively. Increasing the amount of polypeptide increased the gene transfer efficiency. When cetuximab was conjugated (Cmab in the figure), transfection efficiency was significantly enhanced. On the other hand, when normal IgG was conjugated (IgG in the figure), almost no increase in transfection efficiency was observed. It was thought that increasing the amount of polypeptide and increasing the binding amount of cetuximab + polypeptide + gene would improve the efficiency of gene transfer. In complexes formed using normal IgG that does not bind to EGFR on the cell surface, even when the amount of polypeptide was increased, active uptake by cells did not occur. This suggests that by binding to the negatively charged gene, the positive charge of the polypeptide's gene-binding region was neutralized, weakening its ability to permeate the cell membrane. While not wishing to be bound by theory, taking into consideration the results of preliminary experiments (EGFR expression on the cell surface, cellular internalization of cetuximab), it is believed that the enhancement of gene transfer efficiency by the complex of the present invention is due more to the effect of the complex being taken up by target cells via the antibody's internalization pathway than to the cell membrane permeation effect of polycations.

[0057] Example 6: Introduction of nucleic acid into cells using a complex of polypeptide, antibody and nucleic acid - 2 A complex was prepared in the same manner as in Example 5, except that Fcγ1-R8 was used as the polypeptide, and administered to cultured HSC2 cells. The luminescence intensity was measured 24 hours after administration (after gene transfer) and gene expression analysis was performed. The results are shown in Figure 6. As in the case of using SpA1-R8, gene transfer was significantly promoted when cetuximab was bound as the amount of polypeptide increased.

[0058] Example 7: Introduction of nucleic acid into cells using a complex of polypeptide, antibody and nucleic acid - 3 A complex was prepared in the same manner as in Example 5, except that SpA1-K8 was used as the polypeptide, and administered to cultured HSC2 cells. The luminescence intensity was measured 24 hours after administration (after gene transfer) and gene expression analysis was performed. The results are shown in Figure 7. When cetuximab was bound, the incorporation was significantly promoted.

[0059] Example 8: Introduction of nucleic acid into cells using a complex of polypeptide, antibody and nucleic acid - 3 1. Materials and Methods (1) Polypeptide SpA1-R8 (molecular weight 2361.78 g / mol) (synthesis requested from Biologica, synthesis by GL Biochem) was used. (2) Antibodies Anti-EAAT2 antibody clone E-1 (mouse monoclonal IgG2bEAAT2 antibody) (Santa Cruz Biotechnology) was used. Normal human IgG whole molecule (Fujifilm Wako Pure Chemical Industries) was used as a negative control. The antibody molecular weight was calculated as 150,000 g / mol. (3) Cell line The C8-D1A cell line (a spontaneously immortalized astrocyte cell line derived from mice) was used. The expression of EAAT2 on the cell membrane of C8-D1A cells has been previously confirmed by immunohistochemistry (preliminary study, data not shown). The internalization of anti-EAAT2 antibody (E-1) by C8-D1A has been confirmed using pHAb-Reactive-Dyes (Promega) (preliminary test, data not shown). The day before the experiment, plate 1 x 10 cells in a cell culture plate. 4 The cells were seeded at 10 cells / well and cultured at 37°C in the presence of 5% CO2. (4) Genes The plasmid vector pNL1.3.CMV[secNluc / CMV] (Promega Corporation), 3945 bp, encoding secreted luciferase for mammalian cell expression was used. The molecular weight of 1 bp was calculated as 660 g / mol, and the molecular weight per vector (3945 bp) was calculated as 2603700 g / mol. (5) Formation of antibody + polypeptide + gene complex and administration to cells 100 ng of gene (0.0384 pmol of vector as one molecule) per well was mixed with polypeptide at molar ratios of 0 (no polypeptide), 24,000, 48,000, 96,000, and 192,000. Furthermore, 100 ng of the gene was mixed with antibody at a molar ratio of 100 times. The mixture was left at room temperature for 20 minutes to allow the antibody, polypeptide, and gene complexes to form. A solution containing the formed complex was administered to wells where cells were cultured, and the gene was introduced. (6) Gene expression analysis 24 hours after transfection, 10 μl of cell culture medium (culture supernatant) was collected from each well. The plate was reacted with Coelenterazine 400 a (GoldBio) dissolved in 0.01% Tween / 0.3% EDTA / PBS at 1 μg / 100 μl per well. The luminescence intensity for 10 seconds starting from 2 seconds after the reaction was measured using a multiplate reader (Spark 10M SparkControl Magellan v1.2) (TECAN) and integrated.

[0060] 2. Results and Discussion The results are shown in Figure 9. The horizontal axis shows the molar ratio of polypeptide / gene, with 0 (no polypeptide), 24,000, 48,000, 96,000, and 192,000 times indicated as 0k, 24k, 48k, 96k, and 192k, respectively. Increasing the amount of polypeptide increased the gene transfer efficiency. The transduction efficiency was significantly enhanced when EAAT2 antibody was bound (EAAT2), whereas the transduction efficiency was barely increased when normal IgG was bound (IgG). It was thought that increasing the amount of polypeptide and increasing the binding amount of EAAT2 + polypeptide + gene would improve the efficiency of gene introduction. In complexes formed using normal IgG that does not bind to EAAT2 on the cell surface, even when the amount of polypeptide was increased, active uptake by cells did not occur. This suggests that by binding to the negatively charged gene, the positive charge of the gene-binding region of the polypeptide was neutralized, thereby weakening its ability to permeate the cell membrane. While not wishing to be bound by theory, taking into consideration the results of preliminary experiments (expression of EAAT2 on the cell surface and internalization of EAAT2 in cells), it appears that the enhancement of gene transfer efficiency by the complex of the present invention is due more to the effect of the complex being taken up by target cells via the antibody's internalization pathway than to the cell membrane permeation effect of polycations.

[0061] Example 9: Introduction of nucleic acid into mouse skin tissue using a complex of polypeptide, antibody, and nucleic acid - 1 1. Materials and Methods (1) Polypeptide SpA1-K8 (molecular weight 2137.7 g / mol), SpA1-KW1 (molecular weight 2068.5 g / mol, SEQ ID NO: 57), and Fcγ1-K8 (molecular weight 2065.71 g / mol, SEQ ID NO: 60) (synthesis requested from Biologica and synthesis by GL Biochem) were used. (2) Antibodies The anti-desmoglein 3 antibody clone AK18 (mouse monoclonal IgG1) (MBL Life Sciences) was used. Desmoglein 3 is a cell surface antigen specifically expressed in mucous membranes and skin. Normal human IgG, whole molecule (Fujifilm Wako Pure Chemical Industries, Ltd.) was used as a negative control. The antibody molecular weight was calculated as 150,000 g / mol. (3)Animals Eight-week-old male BALB / c mice were used. The day before the experiment, the hair on the back of the mice was removed. (4) Genes The mammalian cell-expressing luciferase plasmid vector pGL4.51[luc2 / CMV / Neo] (Promega Corporation), 6358 bp, was used. The molecular weight of one DNA base was calculated as 330 g / mol. (5) Formation of antibody + polypeptide + gene complex and administration to tissue 500 ng of gene was used per injection, and the molar ratio was 200 antibodies per 8,000 bases of DNA. The polypeptide was then mixed with the DNA at a molar ratio of 500 molecules per 8,000 bases to form a complex of antibody, polypeptide, and gene. The solution containing the formed complex was injected intradermally into the back of mice under isoflurane inhalation anesthesia. (6) Gene expression analysis After 48 hours of breeding, the mice were intraperitoneally injected with 100 μL of 30 mg / mL D-luciferin under inhalation anesthesia. The mice were placed in a supine position inside an IVIS Lumina II (Xenogen), and images were taken using Living Image version 3.2 (Xenogen) by integrating the luminescence intensity for 1 minute starting 15 minutes after injection. Using the same software, an ROI was set at the injection site and the luminescence intensity was quantified.

[0062] 2. Results and Discussion The photographed images are shown in Figure 10. The images were taken by injecting complexes of the polypeptides SpA1-K8, SpA1-KW1, and Fcγ1-K8 with the luciferase gene, each modified with normal human IgG (IgG), into the left side of the mouse's back from the cranial to the caudal side, and complexes modified with anti-desmoglein 3 antibody (Dsg3) into the right side. When the desmoglein 3 antibody-modified complex was injected, luminescence due to gene expression was confirmed whether the polypeptides SpA1-K8, SpA1-KW1, or Fcγ1-K8 were used. When normal human IgG was modified with the conjugate, no luminescence due to gene expression was observed, regardless of whether the polypeptides SpA1-K8, SpA1-KW1, or Fcγ1-K8 were used. The quantitative measurement results of the luminescence intensity are shown in Figure 11. The horizontal axis shows the conditions under which complexes of the polypeptides SpA1-K8, SpA1-KW1, and Fcγ1-K8 with the luciferase gene, each modified with normal human IgG (IgG) or anti-desmoglein 3 antibody (Dsg3), were injected, and the vertical axis shows the relative light units (RLU) under each condition. When the desmoglein 3 antibody-modified complex was injected, luminescence due to gene expression was confirmed whether the polypeptides SpA1-K8, SpA1-KW1, or Fcγ1-K8 were used. When normal human IgG modified with the conjugate was injected, no luminescence due to gene expression was observed, regardless of whether the polypeptides SpA1-K8, SpA1-KW1, or Fcγ1-K8 were used, and the values ​​were negative and below the measurement reference value.

[0063] Example 10: Introduction of nucleic acid into mouse skin tissue using a complex of polypeptide, antibody, and nucleic acid - 2 The complex formation conditions were either no antibody modification or modification with normal human IgG, and the complex preparation and intradermal injection experiments into mice were carried out in the same manner as in Example 9, except that imaging using an IVIS Lumina II (Xenogen Corporation) was performed 25 minutes after substrate injection.

[0064] The photographed images are shown in Figure 12. The images were taken by injecting the SpA1-K8, SpA1-KW1, or Fcγ1-K8 polypeptide complex with the luciferase gene, without modification with antibody (without IgG), and the right side of the mouse, from the cranial to the caudal side of the back. To clearly indicate the area where luminescence was measured, the ROI and its measurement values ​​were shown in the photograph. When the conjugates without antibody modification were injected, weak luminescence due to gene expression was confirmed whether the polypeptides pA1-K8, SpA1-KW1, or Fcγ1-K8 were used. When normal human IgG modified with the conjugate was injected, no luminescence due to gene expression was observed, regardless of whether polypeptides pA1-K8, SpA1-KW1, or Fcγ1-K8 were used, and the values ​​were negative and below the measurement reference value. By further coating the surface of the gene-peptide complex with an antibody, a new effect was achieved that was not achieved with existing peptide-based gene transfer methods: non-specific gene transfer to organs other than the target organ was blocked.

[0065] Example 11: Evaluation of DNase resistance by a complex of a polypeptide, an antibody, and a nucleic acid - 1 1. Materials and Methods (1) Polypeptide SpA1-R8 (molecular weight 2361.78 g / mol) and Fcγ1-K8 (molecular weight 2065.71 g / mol) (synthesis requested from Biologica, synthesis by GL Biochem) were used. (2) Antibodies Mouse IgG2b isotype control clone MPC-11 (BioLegend) was used. The antibody molecular weight was calculated as 150,000 g / mol. (3) Genes The mammalian cell-expressing luciferase plasmid vector pNL1.3.CMV[secNluc / CMV] (Promega Corporation) 3945 bp was used. The molecular weight of one DNA base was calculated as 330 g / mol. (4) Antibody + Polypeptide + Gene Complex Formation 5000ng of gene was used per sample, and the molar ratio was mixed so that there were 400 antibodies per 8000 bases of DNA. The polypeptide was then mixed with the DNA at a molar ratio of 1,000 molecules per 8,000 bases to form a complex of antibody, polypeptide, and gene. As controls, a sample containing only the gene but not the antibody or polypeptide, and a sample containing only the polypeptide and gene but not the antibody were also prepared. (5) DNA decomposition enzyme treatment DNase I (RNase-free) (Nippon Gene Co., Ltd.) was used as the DNA degrading enzyme. One unit of this DNA enzyme is defined as the enzyme activity that increases the absorbance at 260 nm of the reaction solution by 0.001 per minute at 25°C and pH 7.9 using calf thymus DNA as a substrate. Five units of DNA enzyme per sample was mixed with the solution containing the complex and allowed to stand at 25°C for 20 minutes. (6) Extraction of remaining DNA After DNase treatment, the remaining DNA was purified and extracted from the solution containing the complex using the alkali-SDS method. Purification was performed using the FastGene Plasmid Mini Kit (Nippon Genetics Co., Ltd.). (7) Measurement of the concentration of remaining DNA The absorbance of the extracted solution at 260 nm was measured using a NanoDrop 2000c (Thermo Scientific) to calculate the DNA concentration. The DNA concentration of the solution extracted from a sample that did not contain antibodies or polypeptides, but contained only genes and was not treated with DNase, was set at 100%. -Compared with this, the percentage of remaining DNA concentration in each sample after DNase treatment was calculated.

[0066] 2. Results and Discussion The results are shown in Figure 13. The horizontal axis shows samples containing only the gene but no antibody or polypeptide, treated with DNase (Naked), and samples containing the gene and polypeptides SpA1-R8 and Fcγ1-K8, but without antibody (IgG -) or with antibody (IgG +), both of which were treated with DNase. The vertical axis shows the residual DNA rate for each sample, with the DNA concentration of the sample that did not contain antibodies or polypeptides, but contained only genes and was not treated with DNase set at 100%. When a complex was formed between the gene and either the polypeptide SpA1-R8 or Fcγ1-K8, the survival rate of DNA after DNase treatment was improved compared to the gene alone. When a complex was formed between the gene and either the polypeptide SpA1R8 or Fcγ1-K8, the survival rate of DNA after DNase treatment was improved when the complex was modified with antibody compared to when it was not modified with antibody. It is thought that by forming a complex in which the gene is covered with polypeptide, contact with DNase is avoided, resulting in DNase resistance. This example provided the novel insight that DNase resistance can be improved by further coating the outer periphery of nucleic acids and polypeptides with antibodies.

[0067] Example 12: Introduction of nucleic acid into cells using a complex of polypeptide, antibody and nucleic acid - 4 1. Materials and Methods (1) Polypeptide SpA1-R8 (molecular weight 2361.78 g / mol) (synthesized by Biologica and synthesized by GL Biochem) was used. (2) Antibodies Cetuximab (Erbitux®: Bristol-Myers Squibb, Merck Serono, ImClone) was used as an anti-EGFR antibody. Normal human IgG, whole molecule (Fujifilm Wako Pure Chemical Industries, Ltd.) was used as a negative control. The antibody molecular weight was calculated as 150,000 g / mol. (3) Cell line The HSC2 cell line (oral epithelial carcinoma cell line) was used. (4) Genes The mammalian cell-expressing luciferase plasmid vector pNL1.3.CMV[secNluc / CMV] (Promega Corporation) 3945 bp was used. The molecular weight of 1 bp was calculated as 660 g / mol, and the molecular weight per vector (3945 bp) was calculated as 2603700 g / mol. (5) Antibody + Polypeptide + Gene Complex Formation 100 ng of gene was used per sample, and the molar ratio was 48,000 or 96,000 polypeptides per vector, and 200 antibodies. The mixing order and mixing method of genes, polypeptides, and antibodies were divided into two patterns. Pattern A: Mix by pipetting in the order of gene, peptide, and antibody. Pattern B: After mixing the gene and antibody by pipetting, add the peptide while vortexing the mixture and mix. The luciferase gene was introduced under all other conditions identical to those in Example 5.

[0068] 2. Results and Discussion The results are shown in Figure 14. The horizontal axis shows the molar ratio of polypeptide / gene in pattern A or pattern B, 48,000 and 96,000 times, as 48k and 96k, respectively. - The cases where cetuximab and normal IgG were used are indicated as Cmab and IgG, respectively. The vertical axis shows the relative light units (RLU) due to the expression of the luciferase gene. In both Pattern A and Pattern B, the gene transfer rate was improved when cetuximab was used to modify the antibody compared to when normal IgG was used to modify the antibody. When mixed with pattern B, the transfection efficiency when modifying normal IgG decreased, and the transfection efficiency when modifying cetuximab increased, compared to when mixed with pattern A. While not wishing to be bound by theory, it is possible that adding peptide to a solution that already contained genes and antibodies while mixing with a vortex resulted in the simultaneous and uniform formation of antibody + peptide + gene complexes, improving gene transfer efficiency and selectivity.

[0069] Example 13: Introduction of nucleic acid into mouse central nervous tissue using a complex of polypeptide, antibody, and nucleic acid - 1 1. Materials and Methods (1) Polypeptide SpA1-K8 (molecular weight 2137.7 g / mol) (synthesis requested from Biologica, synthesis by GL Biochem) was used. (2) Antibodies Anti-EAAT2 antibody clone E-1 (mouse monoclonal IgG2b) (Santa Cruz Biotechnology) and anti-AQP4 antibody clone 4 / 18 (mouse monoclonal IgG3) were used. Both EAAT2 and AQP4 are surface antigens specific to astrocytes, which are constituent cells of the central nervous system. Normal human IgG, whole molecule (Fujifilm Wako Pure Chemical Industries, Ltd.) was used as a negative control. The antibody molecular weight was calculated as 150,000 g / mol. (3)Animals Sixteen-week-old male BALB / c mice were used. The day before the experiment, the hair on the back of the mice was removed. (4) Genes The mammalian cell-expressing luciferase plasmid vector pGL4.51[luc2 / CMV / Neo] (Promega Corporation), 6358 bp, was used. The molecular weight of one DNA base was calculated as 330 g / mol. (5) Formation of antibody + polypeptide + gene complex and administration to animals 2000ng of gene was used per injection, and the molar ratio was 100 antibodies per 8000 bases of DNA. The polypeptide was then mixed with the DNA at a molar ratio of 2,000 molecules per 8,000 bases to form a complex of antibody, polypeptide, and gene. 100 μL of the solution containing the formed complex was injected into the cerebrospinal cavity of mice via the 5th / 6th lumbar intervertebral space under isoflurane inhalation anesthesia. (6) Gene expression analysis After the transfection, the mice were raised for 48 hours and then injected intraperitoneally with 100 μL of 30 mg / mL D-luciferin under inhalation anesthesia. The mice were placed in a supine position inside an IVIS Lumina II (Xenogen), and images were taken using Living Image version 3.2 (Xenogen) by integrating the relative luminescence intensity for 1 minute starting 15 minutes after injection. Using this software, an ROI was set that included the entire brain and spinal cord, and the relative luminescence intensity was quantified.

[0070] 2. Results and Discussion The photographed images are shown in Figure 15. These images show an example of an individual injected with a complex modified with whole normal human IgG molecules (IgG), an individual injected with a complex modified with anti-EAAT2 antibody (EAAT2), and an individual injected with a complex modified with anti-AQP4 antibody (AQP4). Each ROI and its RUI measurement are shown in the figure. The results of similar experiments performed on three animals each are shown in Figure 16. The horizontal axis shows the antibody conditions used to modify the complex, and the vertical axis shows the relative light units (RLU) within the ROI. The complex modified with normal human IgG barely transferred genes into mouse central nervous tissue. On the other hand, the complex modified with either anti-EAAT2 or anti-AQP4 antibodies efficiently transferred genes into mouse central nervous tissue, with the complex modified with anti-AQP4 antibody showing particularly high gene transfer efficiency. It is thought that by modifying the surface of the complex with astrocyte-specific surface antigens, astrocyte-specific gene transfer occurred.

[0071] Example 14: Evaluation of the stability of complexes of polypeptides, antibodies, and nucleic acids 1. Materials and Methods (1) Polypeptide SpA1-K8 (molecular weight 2137.7 g / mol) (synthesized by Biologica and synthesized by GL Biochem) was used. (2) Antibodies Normal human IgG, whole molecule (Fujifilm Wako Pure Chemical Industries, Ltd.) was used. (3) Genes The mammalian cell-expressing luciferase plasmid vector pNL1.3.CMV[secNluc / CMV] (Promega Corporation) 3945 bp was used. The molecular weight of one DNA base was calculated as 330 g / mol. (4) Antibody + Polypeptide + Gene Complex Formation and Dilution The antibodies, peptides, and genes used were each filtered through a 0.1 μm PVDF filter. 10,000 ng of gene was used per sample, and the molar ratio was 200 antibodies per 8,000 bases of DNA. The polypeptide was then mixed with the DNA at a molar ratio of 2,000 molecules per 8,000 bases to form a complex of antibody, polypeptide, and gene. Using the same method, two antibody + polypeptide + gene complexes were prepared (Samples A and B). The solutions containing the two antibody + polypeptide + gene complexes were placed in 2 mL tubes, capped, and flash-frozen in liquid nitrogen for at least 1 minute. Sample A was stored at -20°C for 48 hours and then rethawed. For sample B, a hole was pierced in the cap of a 2 mL tube with an 18G syringe needle, and the tube was vacuum freeze-dried for 24 hours at a cold trap temperature of -50°C using a vacuum freeze-dryer, FreeZone 4.5 Liter Benchtop Freeze Dry System (LABCONCO). The 2 mL tube containing sample B was again replaced with a standard cap and stored at 4°C for 24 hours. After filtering through a 0.22 μm PVDF filter, the sample was reconstituted using an amount of 10 mM HEPES equal to the volume of the liquid before drying. The solutions of samples A and B were each diluted 10-fold with 10 mM HEPES filtered through a 0.22 μm PVDF filter. (5) Observation of particles and measurement of particle size and particle number The solutions containing the diluted antibody + peptide + gene complexes formed in (4) were analyzed using a NanoSight (LM10-SHR, NTA software v3.2, Japan Quantum Design) with a camera mounted on a microscope equipped with a 20x magnification objective lens to observe the scattered light from each particle, and particle diameter and particle number concentration were measured in the range of 0.5 nm to 999.5 nm by nanoparticle tracking (trajectory) analysis. The particle number concentration of the original solution was calculated by multiplying the measured value by 10. The particle number concentration for each particle size was integrated to calculate the total particle number concentration in the solution.

[0072] 2. Results and Discussion Figure 17 shows the particle count and particle size measurement results at Nanosite for the solution diluted after remelting sample A, converted into the original solution concentration. The antibody + SpA1-K8 + gene complex had a mean particle size of 91.5 ± 8.4 nm and a mode of 83.7 ± 20.0 nm, with a mean particle size of 2.0 ± 1.11 × 10 9 / ml concentration was observed. Figure 18 shows the particle count and particle size measurement results at Nanosite for the solution diluted after redissolving sample B, converted into the original solution concentration. The antibody + SpA1-K8 + gene complex had a mean particle size of 134.6 ± 8.2 nm and a mode of 100.6 ± 6.9 nm, with a mean particle size of 2.13 ± 0.20 × 10 9 / ml concentration was observed. The antibody + peptide + gene complex formed by mixing can be frozen and stored, and it was found that the particles remain intact even after re-thawing. Furthermore, it was found that the antibody + peptide + gene complex formed by mixing retained its particle structure even after vacuum freeze-drying.

[0073] Example 15: Evaluation of protease resistance of complexes of polypeptides, antibodies, and nucleic acids (1) Polypeptide A polypeptide (molecular weight 2864.24 g / mol) in which the N-terminus of SpA1-R8 was modified with FITC (synthesis commissioned from Biologica, synthesis by GL Biochem) was used. (2) Antibodies Normal mouse IgG, whole molecule (Fujifilm Wako Pure Chemical Industries, Ltd.) was used. The antibody molecular weight was calculated as 150,000 g / mol. (3) Genes The mammalian cell-expressing luciferase plasmid vector pNL1.3.CMV[secNluc / CMV] (Promega Corporation) 3945 bp was used. The molecular weight of one DNA base was calculated as 330 g / mol. (4) Antibody + Polypeptide + Gene Complex Formation The experimental sample used was 1600 ng of gene per sample, and the mixture was mixed at a molar ratio of 100 antibodies per 8000 bases of DNA. The polypeptide was then mixed with the DNA at a molar ratio of 2,000 molecules per 8,000 bases to form a complex of antibody, polypeptide, and gene. As controls, a sample containing the antibody and polypeptide but not the gene, a sample containing the gene and polypeptide but not the antibody, and a sample containing only the polypeptide but not the antibody or gene were prepared. To ensure that the combined protein and polypeptide concentrations in all samples were the same, a control without antibody was spiked with the same mass of bovine serum albumin as the antibody. (5) Proteolytic enzyme treatment Proteinase K (Nacalai Tesque) was used as the proteolytic enzyme. Proteinase K nonspecifically cleaves peptide bonds, degrading proteins and peptides. Proteinase K was mixed with all samples to a final concentration of 100 μg / mL and left on ice for 30 minutes. Phenylmethylsulfonyl fluoride (Fujifilm Wako Pure Chemical Industries, Ltd.) was added as a proteinase K inhibitor to a final concentration of 2 mM, and the mixture was allowed to stand for 10 minutes. (6) Preparation of calibration curve samples Standard curve samples were prepared using the same gene amount, antibody amount, and mixing method as the experimental samples, with the following polypeptides per 8000 bases of DNA: 2000 (100% peptide amount), 1000 (50% peptide amount), 500 (peptide amount), and 250 (12.5% ​​peptide amount). (7) Electrophoresis Peptide-PAGE mini precast gel 1mm 10well (TEFCO) and Tricine buffer kit (TEFCO) were used. All samples were mixed with equal volumes of Tricine sample buffer and heated at 95°C for 5 minutes. The electrophoresis tank containing the precast gel was filled with a 10-fold diluted electrophoresis buffer kit, and 10 μL of the heat-treated sample was injected. The electrophoresis was carried out at 125V for 60 minutes. (8) Gel photography and analysis Images were captured and analyzed using an Amersham Imager 680 (software version 2.0) (Cytiva). The gel containing the post-electrophoresis samples was removed from the gel plate and placed on the black tray of the device. The sample was irradiated with blue LED (wavelength 460 nm) and the excited fluorescence band was photographed. The fluorescent bands of the FITC-modified SpA1-R8 peptide were identified, and the band volumes of all samples were quantitatively measured. Using Microsoft Excel version 2205, a simple regression analysis was performed based on the band volumes of the calibration curve samples to create a calibration curve. The residual rate (%) of the peptide in the experimental sample was calculated based on the created calibration curve.

[0074] 2. Results and Discussion The results are shown in Figure 19. In samples containing neither antibody nor gene, the residual peptide rate decreased to 44.3% (Gene -, IgG - ). When the gene and peptide were mixed, the survival rate increased to 65.2% (Gene +, IgG - ). When the antibody and peptide were mixed, the survival rate increased to 61.6% (Gene -, IgG + ). When both the gene and antibody were mixed with the peptide, the survival rate increased to 81.5% (Gene +, IgG + ). - By mixing the gene or antibody with the peptide, a bond is formed between each, avoiding contact with proteolytic enzymes, which is thought to make the peptide less susceptible to degradation. By forming a complex between the gene and peptide and covering its surface with antibodies, contact with proteolytic enzymes is further avoided, making the peptide less susceptible to degradation. By forming a complex between a gene and a peptide and then coating the surface with an antibody, a new function was obtained: improving retention as a gene delivery carrier.

[0075] Example 16: Experiment to confirm the antitumor effects of polypeptide, antibody drug, and gene therapy drug 1. Materials and Methods (1) Polypeptide SpA1-R8 or SpA1-K8 (both synthesized by Biologica and GL Biochem) were used. (2) Antibodies Cetuximab (Erbitux®: Bristol-Myers Squibb, Merck Serono, ImClone) was used as an anti-EGFR antibody. The antibody molecular weight was calculated as 150,000 g / mol. (3) Cell line The HSC2 cell line (human oral epithelial carcinoma cell line) was used. As described in Example 5, the expression of EGFR on the cell membrane of HSC2 has been previously confirmed by immunohistochemistry (preliminary test, data not shown). As described in Example 5, it has been confirmed that anti-EGFR antibody (cetuximab) is internalized by HSC2 using pHAb-Reactive-Dyes (Promega) (preliminary test, data not shown). The day before the experiment, 1 x 10 cells were plated in a 96-well multiplate. 4 Cells were seeded at 100 cells / well. (4) Genes Akt1 shRNA Plasmid (h) (Santa Cruz) encoding shRNA for human Akt1 was used. Akt1 is expressed downstream of the EGFR signaling pathway and promotes tumor cell proliferation. · shRNA transcribed from the Akt1 shRNA plasmid (h) introduced into the cell nucleus is processed into siRNA, which degrades the Akt1 gene mRNA and suppresses tumor growth. The molecular weight of one DNA base was calculated as 330 g / mol. (5) Formation of antibody + polypeptide + gene complex and administration to cells 400 ng of gene was used per well, and the molar ratio was mixed to give 400 antibodies per 8000 bases of DNA. The polypeptide was then mixed with the DNA at a molar ratio of 12,500 molecules per 8,000 bases to form a complex of antibody, polypeptide, and gene. The solution containing the formed complex was then administered to wells where the cells were cultured. As controls, solutions containing only the same amount of polypeptide, only the same amount of gene, and only the same amount of antibody were prepared and administered to wells in which cells were cultured. (6) Antitumor effect analysis Forty-eight hours after administration, 20 μL of CellTiter 96 AQueous One Solution Reagent (Promega Corporation) was added per well to wells containing cells and wells containing medium alone, and the cells were cultured for an additional 2 hours. The absorbance of the wells was measured at 490 nm using a Multiskan GO microplate reader (Thermo Scientific). The absorbance of the well containing only the medium was used as the blank value and was subtracted from the absorbance of all wells. The absorbance of the well showing the highest absorbance was set as 100%, and the ratio of the absorbance of the wells to which other antibodies, polypeptides, genes, and their complexes were administered was calculated to represent the cell viability.

[0076] 2. Results and Discussion The results are shown in Figure 20. Administration of the complex of antibody + polypeptide + gene is indicated as Complex, administration of polypeptide alone as Pep. alone, administration of gene alone as DNA alone, and administration of antibody alone as Cmab alone. The vertical axis shows the cell viability under each condition. The absorbance of the wells to which the polypeptide SpA1-K8 was administered alone was the highest and was set at 100%. Administration of the polypeptide alone or the gene alone did not result in a decrease in cell viability below 90%. Administration of the antibody alone reduced cell viability by 82.5%. Administration of antibody, gene complex with polypeptide SpA1-K8, or SpA1-R8 reduced cell viability by 76.2% and 69.2%, respectively. By administering the antibody drug cetuximab and the gene therapy drug Akt1 shRNA plasmid as a complex linked via a peptide, selective gene transfer to EGFR-expressing cells was achieved using cetuximab as a delivery site, and the anti-tumor effect was thought to be enhanced by the synergistic effect of the antibody drug and the gene therapy drug.

[0077] Example 17: Structural identification of complexes of polypeptides, antibodies and nucleic acids by electron microscopy 1. Materials and Methods (1) Polypeptide SpA1-K8 (molecular weight 2137.7 g / mol) (synthesized by Biologica and synthesized by GL Biochem) was used. (2) Antibodies Normal human IgG, whole molecule (Fujifilm Wako Pure Chemical Industries, Ltd.) was used. The antibody molecular weight was calculated as 150,000 g / mol. (3) Genes The mammalian cell-expressing luciferase plasmid vector pNL1.3.CMV[secNluc / CMV] (Promega Corporation) 3945 bp was used. The molecular weight of one DNA base was calculated as 330 g / mol. (4) Formation and preservation of antibody + polypeptide + gene complexes. The antibodies, peptides, and genes used were each filtered through a 0.1 μm PVDF filter. 10,000 ng of gene was used, and the molar ratio was 200 antibodies per 8,000 bases of DNA. The polypeptide was then mixed with the DNA at a molar ratio of 2,000 molecules per 8,000 bases to form a complex of antibody, polypeptide, and gene. The antibody + polypeptide + gene complexes were frozen and stored at -20°C. (5) Preparation and photography of specimens The frozen complex solution formed in (4) was thawed and diluted 100 times with distilled water, and the resulting droplet was placed on an EM fine grid support membrane made of 400 mesh Cu (Nissin EM). The grid was placed in distilled water and washed twice. After washing, the grid was placed on a drop of 2% uranyl acetate solution for negative staining. The negatively stained grid was inserted into a transmission electron microscope JEM-1400Flash (JEOL) and photographed.

[0078] 2. Results and Discussion The photograph taken at a magnification of 50,000 times is shown in Figure 21. Negative staining revealed an image of a complex nanoparticle with the plasmid gene at the center and the surface covered with a shell of antibodies bound to peptides. The structure of the complex of the present invention, which exhibits the distinctive functions of promoting selective gene transfer, inhibiting non-selective gene transfer, and improving the retention of nucleic acids and peptides, has been elucidated. [Industrial Applicability]

[0079] According to the present invention, it is possible to conveniently and safely transfer a gene (nucleic acid) selectively into target cells.

[0080] This application is based on patent application No. 2021-115889 filed in Japan (filing date: July 13, 2021), the contents of which are incorporated in their entirety into this specification.

Claims

1. A polypeptide comprising an amino acid sequence (Region 1) that specifically binds to the constant region of an antibody and an amino acid sequence (Region 2) that specifically binds to a nucleic acid.

2. The amino acid sequence of the said region 1 has a binding affinity with a constant site of an antibody and a Kd value of 1 × 10 －３ M or less, and the polypeptide according to claim 1.

3. The polypeptide according to claim 1 or 2, wherein the amino acid sequence of Region 1 is selected from the group consisting of polypeptides derived from Protein A, Protein G, Protein L, M protein family, Fc alpha receptor, Fc gamma receptor, Fc epsilon receptor, Fc alpha / mu receptor, antibody-binding site of rheumatoid factor, recombinant derivatives thereof, and libraries of random polypeptides, which specifically bind to the constant region of an antibody.

4. The amino acid sequence of Region 1 is NKFRGKYK (SEQ ID NO: 1), FYWHCLDE (SEQ ID NO: 2), (RTY) 4 K 2 KG (sequence numbers 71 and 72), NARKFYKG (SEQ ID NO: 7), FYCHWALE (SEQ ID NO: 8), FYCHTIDE (SEQ ID NO: 9), TWKTSRISIF (SEQ ID NO: 10), FGRLVSSIRY (SEQ ID NO: 11), DCAWHLGELVWCT (SEQ ID NO: 12), PAWHLGELVWP (SEQ ID NO: 13), PDCAWHLGELVWCP (SEQ ID NO: 14), CDCAWHLGELVWCTC (SEQ ID NO: 15), EPIHRSTLTALL (SEQ ID NO: 16), (CFHH) 2 KG (SEQ ID NOs: 73 and 74), HWRGWV (SEQ ID NO: 17), HYFKFD (SEQ ID NO: 18), HFRRHL (SEQ ID NO: 19), HWCitGWV (SEQ ID NO: 75), RWHYFK (SEQ ID NO: 20), MWFRHYK (SEQ ID NO: 21), RRGW (SEQ ID NO: 22), KHRFNKD (SEQ ID NO: 23), GSYWYDVWF (SEQ ID NO: 24), CPSTHWK (SEQ ID NO: 25), NVQYFAV (SEQ ID NO: 26), ASHTQKS (SEQ ID NO: 27), QPQMSHM (SEQ ID NO: 28), TNIESLK (SEQ ID NO: 29), NCHKCWN (SEQ ID NO: 30), SHLSKNF (SEQ ID NO: 31), CVFYRNGKSFQFS (SEQ ID NO: 32), HKRSFWADN (SEQ ID NO: 33), RTQFRPNQT (SEQ ID NO: 34), QLCDFWRTR (SEQ ID NO: 35), FEDFNEQRT (SEQ ID NO: 36), LAKFLKGKD (SEQ ID NO: 37), WHRRTHKTF (SEQ ID NO: 38), RTIQTRSHW (SEQ ID NO: 39), IKLAQLHSV (SEQ ID NO: 40), WRHRNATEW (SEQ ID NO: 41), QNWIKDVHK (SEQ ID NO: 42), WKDKLVYNVL (SEQ ID NO: 43), WKDKPLVKVT (SEQ ID NO: 44), WKNTALHKVT (SEQ ID NO: 45), WRNWDVYKVI (SEQ ID NO: 46), HMVCLAYRGRPVCFAL (SEQ ID NO: 47), HMVCLSYRGRPVCFSL (SEQ ID NO: 48), KEQQERQKNLEELERQSQREVEKRYQEQLQKQQQL (SEQ ID NO: 49), KLEKKSEDVERHYLRQLDQEYKEQQERQ (SEQ ID NO: 50), YYALSDAKEEEPRYKALRGENQDLREKERKYQDKIKKLEEKEKNLEKKSC (SEQ ID NO: 51), DPQYRALMGENQDLRKREGQYQDKIEELE (SEQ ID NO: 52), AVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 53), IDEILAALPKTDTYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE (SEQ ID NO: 54), or a derivative thereof, the polypeptide according to claim 1.

5. The polypeptide according to claim 1, wherein the amino acid sequence of region 2 contains 40 mol% or more of cationic amino acid residues.

6. The polypeptide according to claim 1, wherein the amino acid sequence of region 2 is RRRRRRRR (SEQ ID NO: 3), KWKWKKA (SEQ ID NO: 4), KKKKKKKK (SEQ ID NO: 5) or RRRRRRWR (SEQ ID NO: 6).

7. A complex comprising the polypeptide according to claim 1, an antibody that binds to the polypeptide, and a nucleic acid that binds to the polypeptide.

8. The complex according to claim 7, wherein the binding between the polypeptide and the antibody and the binding between the polypeptide and the nucleic acid are due to intermolecular interactions.

9. The complex according to claim 7 or 8, having an average particle diameter of 1 to 300 nm.

10. A method for producing a complex comprising the polypeptide according to claim 1, an antibody that binds to the polypeptide, and a nucleic acid that binds to the polypeptide, the method comprising mixing the polypeptide, the antibody, and the nucleic acid in a solution.

11. A method for selectively introducing a nucleic acid into a target cell, the method comprising the steps of contacting the polypeptide according to claim 1 with an antibody and a nucleic acid to form a complex, and contacting the resulting complex with the target cell.

12. A method for selectively introducing a nucleic acid into a target cell, the method comprising the step of contacting the complex according to claim 7 with the target cell.

Images