Bispecific antibodies that alter the targeting of adeno-associated viral vectors

Abstract

To provide a targeting molecule capable of specifically transducing a gene into a target by modifying the targeting property of an AAV vector in a gene therapy using the AAV vector.SOLUTION: A bispecific antibody comprising a first single chain antibody that specifically binds to a capsid protein of AAV and a second single chain antibody that specifically binds to a target protein expressed in a cell targeted by gene therapy, and a pharmaceutical composition for administering an AAV vector and a bispecific antibody comprising a first single chain antibody that specifically binds to a capsid protein of AAV and a second single chain antibody that specifically binds to a target protein expressed in a cell targeted by gene therapy simultaneously or separately in gene therapy using an AAV vector.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a bispecific antibody that alters the targeting properties of an adeno-associated virus (AAV) vector, enabling specific gene transfer into target cells or tissues. [Background technology]

[0002] Viral vectors can express specific genes of interest in target cells or tissues by infecting cells. Viral vectors utilize the infectious properties of viruses, resulting in high gene transfer and expression efficiencies. This allows them to transfer genes into hematopoietic cells and primary cultured cells, which are difficult to transduce using other gene transfer methods. They are also capable of in vivo gene transfer into many animals, including humans, rats, and mice. Among viral vectors, adeno-associated viral vectors (AAV vectors) are nonpathogenic, can transfer genes into terminally differentiated, non-dividing cells such as neurons, and produce physicochemically stable viral particles that can be purified and concentrated. These advantages make them the most widely used in clinical trials, with an increasing number of vector formulations being approved and marketed.

[0003] However, while the clinical application of AAV vectors in gene therapy has progressed, AAV vectors have the drawback of having broad tropism for multiple tissues and lacking specificity. For example, there have been cases where AAV vector-based gene therapy caused severe liver dysfunction, forcing the discontinuation of development. This is an off-target effect caused by AAV vectors' high tropism for the liver. Such off-target effects can cause side effects in organs other than the target and result in a relative decrease in gene transfer efficiency in the organ originally targeted by the therapy. Furthermore, it is difficult to target organs with low tropism for AAV, such as hematopoietic cells, which is a barrier to expanding the range of diseases for which gene therapy is applicable.

[0004] To address these issues, directed evolution has been used to modify AAV capsids by mimicking natural evolution and altering AAV tropism. In directed evolution, wild-type AAV capsid genes are mutated using techniques such as error-prone PCR, random peptide display, and DNA family shuffling to construct an AAV capsid library containing a variety of mutant AAV capsid genes. After packaging the libraries into viruses, the libraries are screened in vitro or in vivo to identify novel AAV capsids with desired functions (Non-Patent Document 1). Furthermore, a capsid selection method called Cre recombination-based AAV targeted evolution (CREATE) has been reported to produce AAV variants with high central nervous system (CNS) transduction efficiency (Non-Patent Document 2) and AAV variants with high muscle transduction efficiency (Non-Patent Document 3). However, these methods for modifying capsids in a directed manner based on directed evolution are limited to "modifying" the directionality and cannot eliminate uptake into non-target tissues.

[0005] On the other hand, another approach to solving the above problems is to modify the targeting of AAV vectors by inserting a targeting molecule into the AAV capsid. For example, there is a method of modifying targeting by inserting an artificial protein (DARPin) with high affinity for a surface antigen (HER2) into a part of the AAV capsid protein (Non-Patent Document 4), and a method of using an antibody as a targeting molecule (Patent Document 1). However, it is known that incorporating a long peptide such as an antibody into the AAV capsid interferes with capsid assembly, significantly reducing the efficiency of vector particle production. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO2020 / 232292 [Non-patent literature]

[0007] [Non-Patent Document 1] Kotterman, Melissa A, Schaffer, David V, Engineering adeno-associated viruses for clinical gene therapy. Nat. Rev. Genet. 2014 July;15(7):445-451 [Non-patent document 2] Benjamin E. Deverman et al., Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain, Nat Biotechnol 2016 Feb;34(2):204-9 [Non-patent document 3] Mohammadsharif Tabebordbar et al, Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species, Cell 2021 Sep 16;184(19):4919-4938 [Non-patent document 4] Robert C Munch et al. DARPins: an efficient targeting domain for lentiviral vectors, Mol Ther 2011 Apr;19(4):686-93. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention aims to provide a targeting molecule that can modify the targeting properties of an AAV vector in gene therapy using an AAV vector and enable specific gene transfer to a target. [Means for solving the problem]

[0009] As a result of extensive research to solve the above problems, the present inventors discovered that when an AAV vector is used to transfer a gene into a target, it is possible to transfer a gene specifically into a target by using a bispecific antibody, which is formed by linking a single-chain antibody (scFv) against the AAV capsid and a single-chain antibody (scFv) against a surface antigen of the target cell, as a targeting molecule, and thus completed the present invention.

[0010] That is, the present invention includes the following inventions. (1) A bispecific antibody comprising a first single-chain antibody that specifically binds to an AAV capsid protein and a second single-chain antibody that specifically binds to a target protein expressed in cells targeted for gene therapy. (2) The bispecific antibody according to (1), wherein the first single-chain antibody that specifically binds to an AAV capsid protein is a single-chain antibody having a heavy chain variable region and a light chain variable region described in any one of the following (a) to (c): (a) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2 (b) a heavy chain variable region consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2 (c) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1 with one or more amino acid substitutions, deletions, insertions, and / or additions, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2 with one or more amino acid substitutions, deletions, insertions, and / or additions. (3) The bispecific antibody according to (1), wherein the second single-chain antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody against a target protein. (4) The bispecific antibody according to any one of (1) to (3), wherein the heavy chain variable region and light chain variable region of the first single-chain antibody, the first single-chain antibody and second single-chain antibody, and the heavy chain variable region and light chain variable region of the second single-chain antibody are linked via peptide linkers, respectively. (5) The bispecific antibody according to any one of (1) to (3), wherein the bispecific antibody is dimerized using the Fc region of IgG as a dimerization domain. (6) A pharmaceutical composition for simultaneous or separate administration of an AAV vector and a bispecific antibody comprising a first single-chain antibody that specifically binds to an AAV capsid protein and a second single-chain antibody that specifically binds to a target protein expressed in cells targeted by the gene therapy in gene therapy using an AAV vector. [Effects of the Invention]

[0011] The bispecific antibodies of the present invention, when used together with an AAV vector, enable specific gene transfer to specific target tissues, cells, or organs, thereby enabling efficient and safe gene therapy using an AAV vector. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows a schematic diagram of gene transfer of an AAV vector into target cells using the bispecific antibody (bispecific scFv) of the present invention. [Figure 2] FIG. 2 shows one embodiment of a method for constructing an AAV vector. [Figure 3] FIG. 3 shows the structure of a vector for expressing a bispecific antibody (monomer) of the present invention. [Figure 4] Figure 4 shows the results of a gene (GFP) transduction test into HER2-high expressing cells (SK-BR-3) using an AAV vector and a bispecific antibody (a construct linking an anti-HER2 antibody (ML39) and an anti-capsid antibody (A20, 100E4)). [Figure 5]Figure 5 shows the results of a gene (GFP) transduction test into HER2-high expressing cells (SK-BR-3) using an AAV vector and a bispecific antibody (a construct linking an anti-HER2 antibody (ML39) and an anti-capsid antibody (A20, 100E4)). (Left panel: wild-type AAV vector (rAAV2-WT) used; right panel: mutant AAV vector (rAAV2-m) used.) [Figure 6] FIG. 6 shows the results of a gene (GFP) transfection test into HER2-highly expressing cells (SK-BR-3) using a wild-type AAV vector and various concentrations of a bispecific antibody (ML39-A20HL). [Figure 7] FIG. 7 shows the results of a gene (GFP) transfection test into HER2-high expressing cells (SK-BR-3) using a wild-type AAV vector and a bispecific antibody (ML39-A20HL) at various multiplicities of infection (MOI). [Figure 8] FIG. 8 shows the results of a gene (GFP) transfection test into HER2-highly expressing cells (SK-BR-3) using a mutant AAV vector and various concentrations of a bispecific antibody (ML39-A20HL). [Figure 9] FIG. 9 shows the results of a gene (GFP) transduction test into HER2-high expressing cells (SK-BR-3) using mutant AAV vectors at various multiplicities of infection (MOI) and a bispecific antibody (ML39-A20HL). [Figure 10] Figure 10 shows the procedure for generating cells co-expressing HER2 and mCherry. [Figure 11] FIG. 11 shows a comparison of the relative expression levels of HER2 in HER2-expressing cell lines (HEK293, SK-BR-3, HEK293-HER2-low, and HEK293-HER2-high). [Figure 12] Figure 12 shows the results of a gene (GFP) transfection test using a mutant AAV vector and a bispecific antibody (ML39-A20HL) in each cell line (HEK293, HEK293-ΔHER2, HEK293-HER2-low, HEK293-HER2-high). [Figure 13] FIG. 13 shows a schematic diagram of cell transduction with a mutant AAV vector and a bispecific antibody (ML39-A20HL). [Figure 14] Figure 14A shows flow cytometry data for cells (HEK293-HER2-high+HEK293) transfected with a mutant AAV vector and a bispecific antibody (ML39-A20HL). Figure 14B shows flow cytometry data for cells (HEK293-HER2-low+HEK293) transfected with a mutant AAV vector and a bispecific antibody (ML39-A20HL). [Figure 15] FIG. 15 shows a schematic diagram of dimerization of a bispecific antibody of the invention. [Figure 16] FIG. 16 shows the structure of a vector for expressing a bispecific antibody (dimer) of the present invention. [Figure 17] FIG. 17 shows the differences in bispecific antibody yield and dimerization efficiency depending on the type of dimerization domain and the presence or absence of a structure-stabilizing mutation (CC mutant) in the anti-capsid antibody. [Figure 18] FIG. 18 shows a comparison of gene transfer efficiency between the monomer and dimer of the bispecific antibody of the present invention. [Figure 19] Figure 19 shows the results of a gene (GFP) transfection test into highly EGFR-expressing cells (A-431) using a mutant AAV vector and a bispecific antibody (a construct linking an anti-EGFR antibody (C225) and an anti-capsid antibody (A20)). [Figure 20] Figure 20 shows the results of a gene (GFP) transfection test into highly EGFR-expressing cells (A-431) using a mutant AAV vector and a bispecific antibody (a construct linking an anti-EGFR antibody (2224) and an anti-capsid antibody (A20)). [Figure 21] Figure 21 shows the results of a test to transduce a gene (GFP) into highly CD19-expressing cells (Daudi) using a mutant AAV vector and a bispecific antibody (a construct linking an anti-CD19 antibody and an anti-capsid antibody (A20)). [Figure 22] Figure 22 shows the results of a gene (GFP) transfection test into CD22-high-expressing cells (Daudi) using a mutant AAV vector and a bispecific antibody (a construct linking an anti-CD22 antibody and an anti-capsid antibody (A20)). DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention will be described in detail below. 1. Bispecific antibodies The bispecific antibody (BsAb) of the present invention comprises a first single-chain antibody that specifically binds to an adeno-associated virus (AAV) capsid protein and a second single-chain antibody that specifically binds to a target protein expressed in cells targeted for gene therapy. When used together with an AAV vector carrying a gene of interest, the bispecific antibody of the present invention can specifically introduce the gene of interest into target cells and suppress uptake into non-target cells (Figure 1).

[0014] As used herein, the term "bispecific antibody" refers to an antibody that possesses binding specificities for two different antigen molecules or epitopes in a single molecule. Specifically, bispecific antibodies of the present invention are capable of specifically binding to an AAV capsid protein and also to a target protein expressed in cells targeted by gene therapy. Here, "specifically binds" means that the binding activity of an antibody for a certain target (which may be an epitope) is, for example, preferably at least 2-fold, 3-fold, 5-fold, more preferably at least 10-fold, 20-fold, 30-fold, and even more preferably at least 50-fold higher than its binding activity for another target.

[0015] Cells targeted by gene therapy include, but are not limited to, cancer cells, blood cells, leukocytes, lymphocytes, T cells, B cells, hematopoietic stem cells, etc. The type of cancer is also not limited to, and includes, but is not limited to, gastric cancer, breast cancer, lung cancer, esophageal cancer, prostate cancer, liver cancer, colon cancer, kidney cancer, pharyngeal cancer, skin cancer, uterine cancer, testicular cancer, ovarian cancer, bladder cancer, brain tumor, osteosarcoma, bone marrow tumor, leukemia, malignant lymphoma, glioma, etc. The target protein is an antigen expressed in abnormal cells causing the target disease, and is preferably a membrane protein.

[0016] In the present invention, the first single-chain antibody consists of a single-chain variable fragment (scFv) in which the heavy chain variable region (VH) and light chain variable region (VL) of an antibody that specifically binds to an AAV capsid protein are linked via a peptide linker, and the second single-chain antibody consists of a single-chain variable fragment (scFv) in which the heavy chain variable region (VH) and light chain variable region (VL) of an antibody that specifically binds to a target protein expressed in cells targeted by gene therapy (sometimes referred to herein as a "targeting antibody") are linked via a peptide linker. The bispecific antibody of the present invention is a single-chain polypeptide in which these two scFvs are further linked via a peptide linker, and is a tandem bispecific single-chain antibody (taFv), which is one form of bispecific antibody.

[0017] In the present invention, the first single-chain antibody that specifically binds to the AAV capsid protein has a heavy chain variable region and a light chain variable region described in any one of (a) to (c) below. (a) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2 (b) a heavy chain variable region consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2 (c) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1 with one or more amino acid substitutions, deletions, insertions, and / or additions, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2 with one or more amino acid substitutions, deletions, insertions, and / or additions.

[0018] The single-chain antibodies (b) and (c) above are antibodies that substantially retain the same functions as single-chain antibodies having a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2, for example, the antigen specificity of the variable regions.

[0019] In (b) above, "an amino acid sequence having 90% or more sequence identity" refers to an amino acid sequence having a sequence identity of at least 90% or more, preferably 95% or more, more preferably 97% or more, and most preferably 98% or more. Amino acid sequence identity can be determined using methods well known to those skilled in the art, sequence analysis software, etc. Examples include the blastp program of the BLAST algorithm and the fasta program of the FASTA algorithm. Herein, the sequence identity of an amino acid sequence is a value expressed as a percentage, obtained by comparing the amino acid sequence to be evaluated with the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, and determining the frequency at which identical amino acids appear at the same positions. Hereinafter, the term "sequence identity" of an amino acid sequence will be used in the same sense in this specification.

[0020] In the above (c), "multiple" refers to, for example, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. Furthermore, the amino acid substitution is preferably a conservative amino acid substitution. Here, conservative amino acid substitution refers to substitution between amino acids with similar properties, such as polarity, electrical properties, and structural properties, such as hydrophobic amino acids, polar amino acids, acidic amino acids, basic amino acids, amino acids with branched side chains, and aromatic amino acids. Examples of hydrophobic (nonpolar) amino acids include glycine, alanine, valine, leucine, isoleucine, and proline; examples of polar amino acids include serine, threonine, cysteine, methionine, asparagine, and glutamine; examples of acidic amino acids include aspartic acid and glutamic acid; examples of basic amino acids include lysine, arginine, and histidine; examples of branched side chain amino acids include valine, isoleucine, and leucine; and examples of aromatic amino acids include phenylalanine, tyrosine, tryptophan, and histidine. Preferred conservative amino acid substitutions include substitutions between amino acids selected from valine, leucine, and isoleucine, phenylalanine, and tyrosine, lysine and arginine, alanine and valine, and asparagine and glutamine.

[0021] Heavy chain or light chain variable regions consisting of an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, and / or added can be prepared using known methods such as site-directed mutagenesis, random mutagenesis, chain shuffling, and CDR walking.

[0022] In the present invention, the nucleotide sequence encoding the single-chain polypeptide constituting the bispecific antibody of the present invention can be modified so that it uses codons (optimal codons) suited to the host cell, without changing the amino acids themselves. By modifying the codons to be optimal in this way, the expression efficiency of the single-chain polypeptide in the host cell can be improved.

[0023] Furthermore, the first single-chain antibody that specifically binds to the capsid protein of AAV has the amino acid sequence of SEQ ID NO: 1 (SDVQLQESGPDLVKPSQSLSLTC TVTGYSITSGYTWH WIRQFPGNKQEWMG YIHFSGYTN YNPSLKSRVSITRDTSKNQFFLHLNSVTTEDTATYYC ARGDYGYEWFTY a heavy chain variable region having CDR1 consisting of TVTGYSITSGYTWH (SEQ ID NO: 37) corresponding to the underlined portion of (WGQGTLVTVSA), CDR2 consisting of YIHFSGYTN (SEQ ID NO: 38), and CDR3 consisting of ARGDYGYEWFTY (SEQ ID NO: 39); and an amino acid sequence of SEQ ID NO: 2 (DIQMTQSSSSFSVSLGDRVTITC KASEDIHNRLA WYKQKPGNAPRLLI SGATSLET GVPSRFSGSGSGKDYTLSITSLQNEDVATYYC QQYWIGPFT The antibody may have a light chain variable region having CDR1 consisting of KASEDIHNRLA (SEQ ID NO: 40), which corresponds to the underlined portion of FGSGTNLEIK, CDR2 consisting of SGATSLET (SEQ ID NO: 41), and CDR3 consisting of QQYWIGPFT (SEQ ID NO: 42).

[0024] The heavy chain variable region (VH) and light chain variable region (VL) of the first single-chain antibody and the heavy chain variable region (VH) and light chain variable region (VL) of the second single-chain antibody each consist of approximately 100 to 120 amino acid residues known as the immunoglobulin fold, share the same overall structure, and are primarily composed of β-sheets. In each single-chain antibody, the VL and VH may be arranged either as a construct in which the VL is at the N-terminus, followed by a peptide linker and VH (VL-peptide linker-VH construct), or as a construct in which the VH is at the N-terminus, followed by a peptide linker and VL (VH-peptide linker-VL construct).

[0025] In the bispecific antibodies of the present invention, the peptide linker between the heavy chain variable region and light chain variable region in the first single-chain antibody (first peptide linker) and the peptide linker between the heavy chain variable region and light chain variable region in the second single-chain antibody (second peptide linker) are not particularly limited in length, as long as they allow the heavy chain variable region and light chain variable region to associate and react specifically with their respective antigens to form antigen-binding sites, and examples of the length include 1 or more, 5 or more, 10 or more, 15 or more, and 30 or less, 25 or less, and 20 or less. The amino acids constituting the peptide linkers are not particularly limited, as long as they are natural amino acids. Examples of such amino acids include glycine, alanine, valine, isoleucine, leucine, serine, threonine, cysteine, methionine, phenylalanine, tryptophan, tyrosine, proline, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, and histidine, with glycine, serine, and proline being preferred.

[0026] Furthermore, in the bispecific antibodies of the present invention, the length and amino acid types of the peptide linker between the first single-chain antibody and the second single-chain antibody are the same as those described above.

[0027] The bispecific antibodies of the present invention can be produced using various means known to those skilled in the art, such as genetic engineering techniques or chemical synthesis. Examples of genetic engineering techniques include constructing a vector into which the nucleotide sequences encoding the first single-chain antibody, a peptide linker, and the second single-chain antibody are ligated and inserted, transforming host cells with this vector, culturing the transformed host cells to express the nucleic acids in the host cells, and recovering and purifying the nucleic acids.

[0028] The vector used in the present invention is not particularly limited as long as it is replicable in a host or capable of integrating a nucleic acid of interest into the host genome, and examples thereof include plasmid DNA and phage DNA.

[0029] Examples of plasmid DNA include plasmids derived from Escherichia coli (e.g., pBR322, pBR325, pUC118, pUC119, pUC18, etc.), plasmids derived from Bacillus subtilis (e.g., pUB110, pTP5, etc.), and plasmids derived from yeast (e.g., YEp13, YEp24, YCp50, etc.), and examples of phage DNA include λ phage (λgt10, λgt11, λZAP, etc.).

[0030] To insert a nucleotide sequence encoding a single-chain polypeptide comprising a first single-chain antibody, a peptide linker, and a second single-chain antibody into a vector, a method may be employed in which the purified DNA is first cleaved with an appropriate restriction enzyme, inserted into a restriction enzyme site or multicloning site of an appropriate vector DNA, and then ligated to the vector.

[0031] In addition to the nucleotide sequence encoding the single-chain polypeptide, other cis elements such as a promoter, an enhancer if desired, a splicing signal, a poly(A) addition signal, a selection marker, a ribosome binding sequence (SD sequence), a start codon, and a stop codon can be ligated to the vector of the present invention. A tag sequence can also be ligated to facilitate the production of the bispecific antibody. Examples of tag sequences that can be used include nucleotide sequences encoding known tags such as His tags, GST tags, and MBP tags.

[0032] Whether or not the desired nucleotide sequence has been inserted into a vector can be confirmed using known genetic engineering techniques. For example, in the case of a plasmid vector, the vector can be subcloned using competent cells, the DNA extracted, and the nucleotide sequence identified using a DNA sequencer. Similar techniques can be used for other vectors that can be subcloned using bacteria or other hosts. Vector selection using a selection marker such as a drug resistance gene is also effective.

[0033] Any cells known to those skilled in the art can be used as host cells. Representative host cells include prokaryotic cells such as Escherichia coli (E. coli), and eukaryotic cells such as mammalian cells such as Chinese hamster ovary cells (CHO cells) and human-derived cells, yeast, and insect cells.

[0034] The single-chain polypeptide obtained by expression in such host cells is generally recovered from the culture medium as a secreted polypeptide, but if it is directly produced without a secretory signal, it can be recovered from the host cell lysate.

[0035] The purification procedure can be carried out by appropriately combining any method known to those skilled in the art, such as centrifugation, hydroxylapatite chromatography, gel electrophoresis, dialysis, fractionation on an ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin Sepharose, anionic or cationic resin chromatography (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and affinity chromatography.

[0036] Furthermore, the bispecific antibodies of the present invention can be dimerized via a dimerization domain, which can be an Fc region derived from an antibody, or a variant or fragment thereof, and is preferably an Fc region derived from an IgG.

[0037] 2. AAV Vector Adeno-associated virus (AAV) is a single-stranded DNA virus of approximately 4.7 kb belonging to the Parvoviridae family. It has an icosahedral capsid with a diameter of 20-30 nm and infects a wide host range because it recognizes heparan sulfate proteoglycan, a universal component of cell membranes. The AAV genome structure contains inverted terminal repeats (ITRs) at both ends, Rep (a regulatory protein that controls replication and transcription), and Cap (three capsid proteins: VP1, VP2, and VP3).

[0038] Known adeno-associated viruses include AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), and AAV type 10 (AAV10), and any of these can be used to produce the AAV vector used in the present invention. Artificially produced AAV vectors such as AAV-DJ and AAV-PHP.B may also be used.

[0039] AAV vectors are produced by co-transfecting three plasmids into virus-producing cells: a vector plasmid (transfer plasmid) containing an expression cassette for the gene of interest between the ITRs at both ends of the adeno-associated virus genome; a plasmid (packaging plasmid) containing the AAV Rep and Cap genes; and a helper plasmid expressing E2A, E4, and VA-RNA; to generate cells capable of producing virus, and then collecting the AAV vector of interest produced in the culture supernatant or intracellularly.

[0040] The virus-producing cells are cells (packaging cells) that have the ability to produce viruses and into which elements necessary for forming virus particles have been introduced in such a way that virus particles are formed and produced intracellularly. The virus-producing cells used in the present invention include all mammalian cells, including humans, such as somatic cells constituting a living body, precursor cells, and cancer cells, cells (cell lines) that have been isolated from a living body, acquired immortalization ability, and stably maintained ex vivo, and cells that have been isolated from a living body and artificially genetically modified. The origin of the cells is not particularly limited, and examples thereof include humans, mice, rats, guinea pigs, hamsters, rabbits, dogs, cats, pigs, cows, and horses.

[0041] Specifically, human cells include HEK293 cells, HEK293T cells derived from HEK293 cells, HEK293S cells, HEK293F cells, HEK293FT cells, HEK293FTM cells, HEK293SG cells, HEK293SGGD cells, HEK293H cells, HEK293E cells, HEK293MSR cells, and VPC2.0 cells. Commercially available cells developed for adenovirus (e.g., Adeno-X 293 Cell Line) and commercially available cells developed for AAV (e.g., AAVpro 293T Cell Line) can also be used. Examples of cells include G3T-hi cells (Takara Bio), HeLa cells (ATCC CCL-2), MOLT-4 cells (ATCC CRL-1582), human lung cancer-derived A549 cells, human fibrosarcoma HT-1080 cells, human retinal tissue-derived cells such as PER.C6 cells, human tissue-derived mesenchymal stem cells, cells derived from human uterine contents, placenta, and fetal tissue, and human liver-derived cells. Non-human primate cells include Vero cells, COS-1 cells (ATCC CRL-1650), and COS-7 cells (ATCC CRL-1651). Rodent cells include BHK cells, CHO cells (ATCC CCL-61), and HePa1-6 cells (ATCC CRL-1830).

[0042] Furthermore, the "gene of interest" is typically a therapeutic gene, but may also include marker genes for evaluating the efficiency of gene introduction or expression stability, such as genes encoding GFP (Green Fluorescent Protein), β-galactosidase, luciferase (Luc), etc.

[0043] The above-mentioned plasmid can be transfected into cells for producing an AAV vector by any method for introducing DNA into animal cells, such as electroporation, calcium phosphate, lipofection, or DEAE-dextran, which are well known to those skilled in the art.

[0044] In the present invention, the medium used for culturing virus-producing cells may be a medium commonly used for culturing animal cells. Specifically, a basal medium containing components necessary for cell survival and proliferation (e.g., inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins) may be used, such as Dulbecco's Modified Eagle's Medium (D-MEM), Dulbecco's Modified Eagle's Medium:Nutrient Mixture F-12 (D-MEM / F-12), Glasgow MEM (G-MEM), Basal Medium Eagle (BME), Minimum Essential Medium (MEM), Eagle's minimal essential medium (EMEM), Iscove's Modified Dulbecco's Medium (IMDM), RPMI 1640, Medium 199, αMEM, Ham's medium, Fischer's medium, and mixtures thereof. The medium may also contain growth factors (FGF, EGF, etc.), interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27 supplements, N2 supplements, antibiotics (penicillin, streptomycin, etc.), etc., as needed. The medium may be serum-containing or serum-free. From the viewpoint of preventing contamination with components derived from different animal species, it is preferable to use serum-free medium or serum derived from the same animal species as the cells to be cultured. Serum substitutes such as albumin may also be used.

[0045] Virus-producing cells into which a plasmid has been introduced can be cultured under standard culture conditions. The culture temperature is not particularly limited, but is, for example, 30 to 40°C, preferably 35 to 37°C. The CO2 concentration is, for example, 1 to 10%, preferably 5 to 6%. The culture time is not particularly limited, but is, for example, 24 to 120 hours, preferably 48 to 96 hours.

[0046] After culturing, the culture supernatant or cells are collected to obtain the AAV vector. In the present invention, the AAV vector is produced in the form of the supernatant, a filtrate obtained by filtering the supernatant, an extract obtained by disrupting the cells, or a concentrate or purified product concentrated or purified by a known method, and is stored by an appropriate method, for example, by freezing, until use.

[0047] 3. Pharmaceutical Compositions The pharmaceutical composition of the present invention is a pharmaceutical composition for simultaneous or separate administration of an AAV vector and a bispecific antibody comprising a first single-chain antibody that specifically binds to an AAV capsid protein and a second single-chain antibody that specifically binds to a target protein expressed in cells that are the target of the gene therapy, in gene therapy using an AAV vector.

[0048] The administration form of the pharmaceutical composition of the present invention is preferably the most effective for treatment, and examples thereof include intravenous, intraarterial, intramuscular, intraperitoneal, subcutaneous, intratumoral, oral, transdermal, rectal, oral, intratracheal, and intranasal administration, with intravenous administration being preferred. For example, in the case of gene therapy for cancer, for solid tumors in various organs that are easily accessible by surgical procedures, administration can be carried out by local injection into or near the tumor using a stereotactic needle or the like, while for non-solid tumors such as leukemia, cancers in sites that are difficult to access by surgical procedures, such as brain tumors, and metastatic cancers, administration can be carried out by intravenous injection. In addition, the above-mentioned administration methods can be appropriately selected and used depending on the type and site of cancer.

[0049] Gene therapy can be performed in two ways: ex vivo, in which target cells are removed from the body and a gene is introduced into them, and in vivo, in which a gene is introduced into the body. The pharmaceutical composition of the present invention can be used in both of these treatment methods. In ex vivo methods, patient-derived cells are cultured outside the body and then treated with the pharmaceutical composition of the present invention for gene introduction before being administered to the patient. In in vivo methods, the pharmaceutical composition of the present invention can be administered directly into the patient's body (organ tissue, skin, muscle, etc.).

[0050] The method of administering the pharmaceutical composition of the present invention may be any method in which an AAV vector and a bispecific antibody (targeting antibody) comprising a first single-chain antibody that specifically binds to an AAV capsid protein and a second single-chain antibody that specifically binds to a target protein expressed in cells targeted by gene therapy are administered substantially simultaneously. For example, a formulation comprising the AAV vector and a formulation comprising the targeting antibody may be administered to a subject completely simultaneously, or may be administered consecutively within a short period of time (preferably within a few minutes). Alternatively, a formulation in which the AAV vector and the targeting antibody are thoroughly mixed in advance may be administered.

[0051] The pharmaceutical compositions of the present invention can be formulated into various dosage forms, such as tablets, powders, granules, fine granules, capsules, oral liquids (suspensions, syrups, emulsions, etc.), topical liquids (infusions, sprays / aerosols, inhalants, liniments, etc.), injections, infusions, and suppositories. Pharmacologically and pharmaceutically acceptable additives may be added, depending on the dosage form and intended use, such as formulation bases, carriers, excipients, diluents, binders, lubricants, coating agents, disintegrants or disintegration aids, stabilizers, preservatives, antiseptics, bulking agents, dispersants, wetting agents, buffers, solubilizers or solubilizers, isotonicity agents, pH adjusters, and colorants. These additives may be added as appropriate, depending on the dosage form and intended use, and formulated into various dosage forms that can be administered orally or parenterally, systemically, or locally, by various known methods. The pharmaceutical compositions of the present invention formulated into various dosage forms can be administered orally or parenterally, systemically, or locally. When the pharmaceutical composition of the present invention is administered orally, it may be formulated into tablets, capsules, granules, powders, pills, oral solutions, suspensions, emulsions, syrups, etc., or may be made into a dry product to be redissolved when used. When the pharmaceutical composition of the present invention is administered parenterally, it may be formulated into intravenous injections (including drip infusions), intramuscular injections, intraperitoneal injections, intrathecal injections, subcutaneous injections, suppositories, etc., and injectable preparations are provided in the form of unit-dose ampoules or multi-dose containers.

[0052] There are no particular limitations on the diseases to which the pharmaceutical composition of the present invention can be applied, and examples include cancer (solid cancer, blood cancer, etc.), infectious diseases, genetic diseases, inflammatory diseases, cardiovascular diseases, etc. There are also no particular limitations on the subjects to which the pharmaceutical composition of the present invention can be applied (such as the age or symptoms of the patient).

[0053] The dosage of the pharmaceutical composition of the present invention is not particularly limited, and may be in accordance with the dosage used clinically, and may be appropriately selected depending on the age, weight, type and symptoms of the disease, administration method, administration period, etc. For example, when vg (vector genome) is used as the administration unit for an AAV vector, 1 to 10 6 vg, preferably 10 2 ~10 6 vg, more preferably 10 4 ~10 6 Doses in the vg range are used for bispecific antibodies, with a dosage of 10 mol per target cell. -22 ~10 -15 mol, preferably 10 -20 ~10 -15 mol, more preferably 10 -17 ~10 -15 The dosage can be selected from, but is not limited to, the range of 100 to 150 mol. [Example]

[0054] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0055] Example 1: Construction of AAV vectors (1) Construction of AAV vector (AcGFP gene expression) AAV vectors were produced using the triple transfection method (Figure 2). First, HEK293 cells (HEK293EB cells) stably expressing the E1 and Bcl-xL genes were seeded onto 245 mm square dishes (Corning) for AAV vector production. After three days, the cells were confirmed to have grown to approximately 90% confluence. Three plasmids were then transfected into the 293EB cells using Polyethylenimine Max: a helper plasmid (pHelper) containing the adenovirus-derived E2A, E4, and VA genes; a packaging plasmid (pRC2) containing the rep and cap genes of serotype 2 AAV; and a vector plasmid containing the AcGFP gene flanked by terminal inverted repeats. After three days, the cells were harvested, and the AAV vectors were recovered and purified using the AAVpro® Purification Kit (Takara Bio Inc.) according to the manufacturer's protocol. The titer of the resulting vector was measured by real-time quantitative PCR using primers designed for the AcGFP gene.

[0056] (2) Construction of mutant AAV vectors To eliminate the infectivity of AAV vectors to cells, mutations were introduced into the capsid protein. Specifically, mutations were introduced into the serotype 2 AAV packaging plasmid (pRC2) using the PrimeSTAR® Mutagenesis Basal Kit (Takara Bio Inc.) to replace the arginine residues at positions 585 and 588 of the amino acid sequence (SEQ ID NO: 43) encoded by the cap gene with alanine (SEQ ID NO: 44). The arginine residues at positions 585 and 588 are the primary binding sites for heparan sulfate proteoglycan, the primary cellular receptor for AAV. The resulting mutant plasmid was designated pRC2-m.

[0057] Next, mutant AAV vectors were generated using the triple transfection method, similar to the AAV vectors described above. Instead of pRC2, pRC2-m was used as the packaging plasmid. This was mixed with the helper and vector plasmids and transfected into 293EB cells using Polyethylenimine Max. After three days, the cells were harvested, and the AAV vectors were collected and purified using the AAVpro® Purification Kit (Takara Bio Inc.) according to the manufacturer's protocol. The titer of the resulting vectors was measured by real-time quantitative PCR using primers designed for the AcGFP gene. When the resulting mutant AAV vectors were added to HEK293 cells, no gene transfer was observed, confirming that infectivity of the cells had almost completely disappeared.

[0058] Example 2: Comparison of anti-AAV capsid antibodies To search for anti-AAV capsid antibodies, three clones (A20, 100E4, and 46D10) were compared. 1. Method (1) Sequence information The sequences of the heavy and light chain variable regions of the anti-AAV capsid antibodies (A20, 100E4, 46D10) used in this example (Table 1), the sequences of the heavy and light chain variable regions of the anti-HER2 antibody (ML39) (Table 2), and the sequences of the linkers (between the anti-HER2 antibody and anti-AAV capsid antibody, and between the heavy and light chain variable regions of the anti-HER2 antibody and anti-AAV capsid antibody) (Table 3) are shown below.

[0059] [Table 1]

[0060] [Table 2]

[0061] [Table 3]

[0062] (2) Construction of bispecific antibodies (monomers) Using HER2 as a model target antigen, a bispecific antibody construct linking the anti-HER2 antibody ML39 and an anti-AAV capsid antibody was prepared as follows.

[0063] First, the amino acid sequence of the anti-HER2 antibody ML39 was obtained from the sequence of the Addgene plasmid pACgp67B-Her2. The sequence of its high-affinity variant, ML39-B1D2, was obtained from a paper (J Mol Biol. 1996 Nov 8;263(4):551-67). Next, the amino acid sequences of the anti-AAV capsid antibodies were obtained. The sequences of 46D10 and 100E4 were obtained from a patent (WO2016 / 176212A1), and the sequence of A20 was obtained from a paper (Virology. 2012 Sep;431(1-2):40-9). Nucleotide sequences were created from these amino acid sequences. The codons were optimized for hamster hamsters using the Codon Optimization Tool on the IDT website. Plasmids containing the obtained sequences were then artificially synthesized by Thermo Fisher Scientific.

[0064] Next, the nucleotide sequence of each antibody was amplified by PCR. The intervening sequence (linker) between the antibodies was obtained by oligo synthesis at Eurofins Genomics. The backbone pSecTag2A plasmid was digested with restriction enzymes and then subcloned using the In-Fusion HD Cloning Kit (Takara Bio Inc.) in the order of anti-HER2 antibody, linker, and anti-AAV capsid antibody. This resulted in the expression plasmid for the bispecific antibody (Figure 3). The anti-AAV capsid antibody was used as a single-chain antibody by tandemly linking the heavy and light chain variable regions. Two linking orders were tested: heavy and light chain (HL) and the reverse, light and heavy chain (LH). Thus, a total of six constructs were produced, with two different heavy and light chain orders for each of the three clones (46D10, 100E4, and A20).

[0065] Bispecific antibodies were produced using the ExpiCHO Expression System Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. First, Expi-CHO cells cultured at 37°C, 8% CO2, and 125 rpm were transfected with the expression plasmids for the bispecific antibodies prepared above using ExpiFectamine CHO Reagent. The next day, ExpiFectamine CHO Enhancer and ExpiCHO Feed were added. After further culture at 32°C, 5% CO2, and 125 rpm for one week, culture media containing each bispecific antibody were obtained. The culture media were centrifuged twice at 4,000 × g for 30 minutes at 4°C or purified by Sartoclear Dynamics. (R) Cells or cell debris in the culture medium were removed by adding Filter Aid (Sartorius), and the mixture was further filtered through a PES membrane filter to obtain a clear supernatant.

[0066] Next, ProBond TM The bispecific antibodies were purified from the supernatant using the Thermo Fisher Purification System according to the manufacturer's instructions. The purified antibodies were further concentrated using Amicon Ultra-15 centrifugal filter units, 10k (Merck) by replacing the buffer with 0.1% Tween 20 / PBS.

[0067] (3) Gene transfer into HER2-expressing cell lines The bispecific antibody prepared in (2) was added to the culture medium of the breast cancer cell line SK-BR-3 together with the serotype 2 AAV vector (wild type: rAAV2-WT) expressing the AcGFP gene prepared in Example 1 or a mutant AAV vector (rAAV2-m, an AAV vector prepared from a capsid protein in which the arginine residues at positions 585 and 588 of the serotype 2 AAV capsid protein were substituted with alanine). The antibody concentration was 100 nM, and the multiplicity of infection of the AAV vector was 10 5 The AcGFP positive rate was measured after 3 days.

[0068] 2.Results When AAV vectors (wild-type) were added to the culture medium of the breast cancer cell line SK-BR-3 together with bispecific antibodies, the yield of bispecific antibodies was significantly low for clone 46D10, but sufficient amounts of bispecific antibodies were obtained for both HL and LH for clones 100E4 and A20. Of the four clones tested, the highest gene transfer efficiency was obtained with 20HL (Figure 4, Figure 5, left).

[0069] On the other hand, when the mutant AAV vector (rAAV2-m) was added to the culture medium of the breast cancer cell line SK-BR-3 together with the bispecific antibody, gene transfer was not possible when 100E4 was used as the anti-AAV capsid antibody, whereas green AcGFP fluorescence was observed in almost all cells when A20, especially A20HL, was used (Figure 5, right).

[0070] Based on the above results, it was decided to use A20HL as the anti-AAV capsid antibody in the following examples.

[0071] Example 3: Targeting of wild-type AAV vectors by bispecific antibodies A bispecific antibody (ML39-A20HL) linking an anti-HER2 antibody (ML39) and A20HL was added to the culture medium of the breast cancer cell line SK-BR-3 at concentrations of 0.1 to 100 nM, along with a wild-type AAV vector of serotype 2 expressing the AcGFP gene. The multiplicity of infection of the AAV vector was 10 5 As a result, a significant increase in gene transfer efficiency was observed from 10 nM (Figure 6).

[0072] Subsequently, the cells were incubated with saline (PBS) or 100 nM ML39-A20HL at a multiplicity of infection of 10. 3 ~10 5 A wild-type AAV vector was added at 2 × 10 vg / cell. 4A significant increase in gene transfer efficiency was observed at a multiplicity of infection of 1000 vg / cell or more. Thus, the bispecific antibody enhanced targeting to SK-BR-3 cells, which strongly express HER2 (Figure 7).

[0073] Example 4: Targeting of mutant AAV vectors by bispecific antibodies ML39-A20HL was added to the culture medium of the breast cancer cell line SK-BR-3 at concentrations of 0.1 to 100 nM together with a mutant AAV vector expressing the AcGFP gene. The multiplicity of infection of the AAV vector was 10 5 As a result, a significant increase in gene transfer efficiency was observed from 20 nM (Fig. 8).

[0074] Subsequently, the cells were incubated with 100 nM ML39-A20HL at a multiplicity of infection of 10 3 ~10 5 The mutant AAV vector was added at 5 × 10 vg / cell. 3 A clear increase in gene transfer efficiency was observed at a multiplicity of infection of 1000 vg / cell or higher (Fig. 9). Thus, gene transfer to SK-BR-3 cells, which strongly express HER2, was possible using a bispecific antibody, even when a mutant AAV vector that cannot bind to heparan sulfate was used.

[0075] (Example 5) Gene transfer into HER2-expressing cell lines Using a lentiviral vector, we generated HEK293 cells stably expressing HER2 and the red fluorescent dye mCherry (Figure 10). Two cell lines were obtained: one with high HER2 expression (HEK293-HER2-high) and one with low HER2 expression (HEK293-HER2-low) (Figure 11). As a control, we also generated a cell line (HEK293-ΔHER2) expressing HER2 with a transmembrane domain deleted to prevent cell surface expression. ML39-A20HL was added to HEK293 and these cell lines, along with a mutant AAV vector expressing the AcGFP gene. The multiplicity of infection of the AAV vector was 10 5The concentration of the bispecific antibody was 100 nM. While green AcGFP fluorescence was barely observed in HEK293 and HEK293-ΔHER2, it was observed in approximately 50% of HEK293-HER2-low cells and in almost all HEK293-HER2-high cells (Figure 12).

[0076] Next, HEK293-HER2-high or HEK293-HER2-low cells were mixed with HEK293 cells, and ML39-A20 HL was added along with a mutant AAV vector expressing the AcGFP gene (Figure 13). HER2 was expressed only in HEK293-HER2-high or -low cells, which exhibited red fluorescence due to mCherry. Three days after gene transfection, cells were harvested and analyzed by flow cytometry. Triplicate samples were analyzed for each. In both HEK293-HER2-high and HEK293-HER2-low cells, green fluorescence due to AcGFP was observed almost exclusively in mCherry-positive cells (Figure 14). This confirms that gene transfection can be performed specifically in HER2-positive cells.

[0077] Example 6: Dimerization of bispecific antibodies using immunoglobulin constant domains To improve the performance of the bispecific antibody, we attempted to dimerize it (Figure 15). We used the constant regions of three immunoglobulins, IgG, IgM, and IgE, as the domains (dimerization domains) that allow the bispecific antibody to form homodimers. Specifically, we created a construct in which this dimerization domain was sandwiched between an anti-HER2 antibody and an anti-AAV capsid antibody (Figure 16).

[0078] 1. Method (1) Sequence information The sequences of the dimerization domains and linkers (between the dimerization domain and the targeting antibody, and between the dimerization domain and the anti-AAV capsid antibody) used in this example are shown in Table 4 below.

[0079] [Table 4]

[0080] (2) Construction of bispecific antibodies (dimers) First, the amino acid sequence of the dimerization domain was obtained. The IgG constant region was obtained from a paper (mAbs. 2009;1(6):572-9), the IgM constant region from a patent (WO2013 / 156148A1), and the IgE constant region from a patent (WO2013 / 156148A1). For the IgG constant region, the sequence obtained from the paper was modified with four mutations (L234A, L235A, G237A, and K322A) to inhibit complement and Fc receptor binding. Linkers were added before and after these amino acid sequences to create the nucleotide sequence. The codons were optimized for hamster hamsters using the Codon Optimization Tool on the IDT website. A plasmid containing the obtained sequence was then synthesized by Thermo Fisher Scientific.

[0081] Next, the nucleotide sequences of the dimerization domain and linker were amplified by PCR. Expression plasmids for the ML39-B1D2 and A20 bispecific antibodies (monomers) were amplified by inverse PCR and used as the backbone. These resulting DNA fragments were ligated using the In-Fusion HD Cloning Kit (Takara Bio Inc.) to obtain the expression plasmid for the dimerized bispecific antibody (Figure 16).

[0082] To stabilize the protein structure of the anti-AAV capsid antibody A20 during dimerization of the bispecific antibody, mutations were introduced to allow intermolecular cross-linking via disulfide bonds between the heavy and light chain variable regions of A20. Specifically, a plasmid containing a base sequence in which lysine residue 46 and serine residue 235 were replaced with cysteine ​​(CC mutant) was artificially synthesized by Thermo Fisher Scientific. Using this as a template, a DNA fragment amplified by PCR was used as the insert. The expression plasmid for the bispecific antibody (dimer) consisting of the ML39-B1D2 backbone and A20 was digested with restriction enzymes and then subcloned using the In-Fusion HD Cloning Kit (Takara Bio).

[0083] Bispecific antibodies (dimers) were produced in the same manner as in Example 2(1) using the expression plasmids for the bispecific antibodies prepared above (with and without mutations introduced into the anti-AAV capsid antibody portion (CC mutant)).

[0084] (3) Gene transfer into HER2-expressing cells The dimerized bispecific antibody prepared in (2) was added to the culture medium of the breast cancer cell line SK-BR-3 together with the mutant AAV vector prepared in Example 1 (an AAV vector prepared from a capsid protein in which the arginine residues at positions 585 and 588 of the serotype 2 AAV capsid protein were substituted with alanine). The antibody concentration was 100 nM, and the multiplicity of infection of the AAV vector was 10 5 The AcGFP-positive rate was measured after 3 days.

[0085] 2.Results When bispecific antibodies (without mutations in the anti-AAV capsid antibody portion) were used, dimers were hardly obtained, and the yield was extremely low. This was thought to be due to the fact that the addition of the dimerization domain interfered with protein folding. On the other hand, when bispecific antibodies were prepared from bispecific antibodies (with mutations in the anti-AAV capsid antibody portion), the yield was significantly improved and dimer formation was observed. In particular, the use of the IgG constant region showed high dimerization efficiency (Figure 17).

[0086] Example 7 Comparison of dimerized bispecific antibodies with monomers A dimerized bispecific antibody consisting of an anti-HER2 antibody (ML39-B1D2) and A20HL was added to the culture medium of the breast cancer cell line SK-BR-3 at concentrations ranging from 1 fM to 10 pM, along with a mutant AAV vector expressing the AcGFP gene. The multiplicity of infection of the AAV vector was 10 5 The transfection efficiency was increased from 20 fM to 2 pM, peaking at 2 pM. The transfection efficiency of the anti-HER2 antibody (ML39-B1D2) and A20HL monomeric bispecific antibodies showed a clear increase at 200 pM, indicating that dimerization of the bispecific antibodies enabled transfection at much lower concentrations (Figure 18).

[0087] (Example 8) Gene transfer using EGFR as a target antigen Two anti-EGFR antibodies were tested: C225 (Cetuximab) and 2224. Constructs were prepared by linking these antibodies to an IgG constant region and an anti-AAV capsid antibody (A20 HL), and dimerized bispecific antibodies were prepared in the same manner as in Example 6.

[0088] The anti-EGFR antibodies (C225, 2224) used in this example, the sequences of the heavy and light chain variable regions of the anti-EGFR antibodies, and the sequence of the linker between the heavy and light chain variable regions of the anti-EGFR antibodies are shown in Table 5 below.

[0089] [Table 5]

[0090] The two bispecific antibodies obtained were added to A-431, a cell line that highly expresses EGFR, together with a mutant AAV vector expressing the AcGFP gene. The antibody concentrations were 10 pM to 10 nM, and the multiplicity of infection of the AAV vector was 10 5 The transfection efficiency was measured at a concentration of 1000 μg / cell. When C225 was used, the transfection efficiency increased from 50 pM and peaked at 2 nM (Figure 19). When 2224 was used, the transfection efficiency increased from 20 pM and peaked at 2 nM (Figure 20).

[0091] (Example 9) Gene transfer using CD19 as a target antigen A construct was prepared by linking an anti-CD19 antibody called HD37 (Blinatumomab), an IgG constant region, and an anti-AAV capsid antibody (A20 HL), and a dimerized bispecific antibody was prepared in the same manner as in Example 6.

[0092] The anti-CD19 antibody (HD37) used in this example, the sequences of the heavy and light chain variable regions of the anti-CD19 antibody, and the sequence of the linker between the heavy and light chain variable regions of the anti-CD19 antibody are shown in Table 6 below.

[0093] [Table 6]

[0094] The obtained bispecific antibody was added to Daudi, a cell line that highly expresses CD19, together with a mutant AAV vector expressing the AcGFP gene. The antibody concentration was 10 pM to 10 nM, and the multiplicity of infection of the AAV vector was 10 5The transfection efficiency increased from 10 pM and peaked at 100 pM (Figure 21). When transfected with a wild-type AAV vector of serotype 2 (rAAV2-WT) into Daudi, the AcGFP positivity rate was approximately 25%, and a similar transfection efficiency was obtained.

[0095] (Example 10) Gene transfer using CD22 as a target antigen A construct was prepared by linking an anti-CD22 antibody, designated RPE_E6, with an IgG constant region and an anti-AAV capsid antibody (A20HL), and a dimerized bispecific antibody was prepared in the same manner as in Example 6.

[0096] The anti-CD22 antibody (RPE_E6) used in this example, the sequences of the heavy and light chain variable regions of the anti-CD22 antibody, and the sequence of the linker between the heavy and light chain variable regions of the anti-CD22 antibody are shown in Table 7 below.

[0097] [Table 7]

[0098] The obtained bispecific antibody was added to Daudi, a cell line that highly expresses CD22, together with a mutant AAV vector expressing the AcGFP gene. The antibody concentration was 10 pM to 10 nM, and the multiplicity of infection of the AAV vector was 10 5 The transfection efficiency increased from 10 pM and peaked at 5 nM (Fig. 22). When transfected with a wild-type AAV vector of serotype 2 (rAAV2-WT) into Daudi cells, the AcGFP positivity rate was approximately 25%, whereas a transfection efficiency of approximately 80% was achieved. [Industrial Applicability]

[0099] The present invention can be used in the field of producing AAV vectors, which are tools for gene therapy.

Claims

1. A bispecific antibody comprising a first single-chain antibody that specifically binds to an AAV capsid protein and a second single-chain antibody that specifically binds to a target protein expressed in cells targeted for gene therapy.

2. The bispecific antibody of claim 1, wherein the first single-chain antibody that specifically binds to an AAV capsid protein is a single-chain antibody having a heavy chain variable region and a light chain variable region described in any one of (a) to (c) below: (a) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2 (b) a heavy chain variable region consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2 (c) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 1 in which one or more amino acids have been substituted, deleted, inserted, and / or added; and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 2 in which one or more amino acids have been substituted, deleted, inserted, and / or added.

3. 2. The bispecific antibody of claim 1 , wherein the second single-chain antibody comprises the heavy chain variable region (VH) and light chain variable region (VL) of an antibody against a target protein.

4. The bispecific antibody according to any one of claims 1 to 3, wherein the heavy chain variable region and light chain variable region of the first single-chain antibody, the first single-chain antibody and second single-chain antibody, and the heavy chain variable region and light chain variable region of the second single-chain antibody are each linked via a peptide linker.

5. The bispecific antibody according to any one of claims 1 to 3, wherein the bispecific antibody is dimerized using the Fc region of IgG as a dimerization domain.

6. A pharmaceutical composition for simultaneous or separate administration of an AAV vector and a bispecific antibody comprising a first single-chain antibody that specifically binds to an AAV capsid protein and a second single-chain antibody that specifically binds to a target protein expressed in cells targeted by gene therapy in gene therapy using an AAV vector.

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