Binding molecules targeting AAV8 and AAV9 and uses thereof

Abstract

The present invention provides binding molecules targeting AAV8 and AAV9 and uses thereof. Specifically, the binding molecule provided by the invention is a nano antibody, and the nano antibody can bind to AAV of AAV8 and AAV9 serotypes with high affinity, binds to AAV under a neutral pH condition and does not bind to AAV under an acidic pH condition, and can be applied to purification, production, actual development and other application prospects related to recombinant AAV.

Description

[0001] This application is a divisional application of the invention patent application filed on October 21, 2024, with application number 202411470499.2 and invention title "A binding molecule targeting AAV8 and AAV9 and its use therein". Technical Field

[0002] This invention relates to the field of biomedicine, and more specifically, to binding molecules that target AAV8 and AAV9 and their uses. Background Technology

[0003] Adeno-associated virus (AAV) is the simplest structurally simple, non-enveloped, single-stranded DNA virus discovered to date, with a viral genome length of approximately 4.7 kb. It belongs to the parvovirus family. Parvoviruses, belonging to the Parvoviridae family, have viral particles approximately 20-25 nm in size, making them the smallest known animal viruses. AAV is a replication-defective virus, requiring the helper virus (adenovirus or herpes simplex virus) for replication. In the absence of helper viruses, AAV can remain latent for extended periods in many cell types. Although AAV infection is common in humans, it is not associated with any known diseases. Its ability to transduce dividing and quiescent cells, very low immunogenicity, and long-term transgenic expression (unlike wild-type AAV virus, which integrates into the host cell DNA after infection, recombinant AAV vectors primarily exist as extrachromosomal free organisms) contribute to its high stability, broad cellular infectivity, and long-term cellular persistence. It expresses exogenous genes in target cells through gene transcription and replication. These characteristics make AAV a highly efficient and safe transgenic vector, which is widely used in gene therapy, neural circuit labeling, in vivo imaging, gene editing, and the preparation of animal models of nervous system diseases.

[0004] In AAV products, residual host cell proteins, bovine serum albumin, nucleases, or nucleic acids can not only affect transfection efficiency but also trigger immune responses. To obtain high-purity products, a combination of ultracentrifugation, liquid chromatography, chemical reagent evaporation, and ultrafiltration is typically required. However, traditional preparation processes are complex, with each step increasing yield loss and the risk of product instability. Therefore, a more efficient AAV purification protocol is urgently needed. Key factors for efficient AAV purification are high recovery rates and minimizing empty capsid product impurities. Affinity capture is a highly efficient method that can specifically bind to and concentrate the target protein while simultaneously achieving very high impurity removal rates.

[0005] AAV8 has attracted widespread attention from researchers due to its unique advantages in the field of gene therapy. AAV8 is one of many AAV serotypes, and it is particularly well-suited for targeting liver cells, thus showing great potential in gene therapy for treating liver diseases. Furthermore, AAV8 can also effectively infect a variety of other tissues, including the heart, skeletal muscle, and various regions of the central nervous system.

[0006] AAV9's ability to cross the blood-brain barrier makes it a strong candidate for treating neurodegenerative diseases. Furthermore, AAV9 exhibits relatively high transduction efficiency in cardiomyocytes, suggesting its potential role in heart disease treatment. In the treatment of cardiomyopathy and heart failure, researchers are exploring AAV9-mediated gene therapy to express beneficial therapeutic proteins or correct disease-causing genes, thereby improving myocardial function and patients' quality of life. Beyond its potential in disease treatment, AAV9 also has important applications in basic scientific research. For example, in the creation of animal models, AAV9 can be used for the efficient transfer of target genes, elucidating gene function and disease pathogenesis. Due to its good tissue specificity and low immunogenicity, AAV9 offers unique advantages as a long-term expression vector in the construction of chronic disease models.

[0007] There are currently no reports of AAV8 and AAV9 specific serotype nanobodies. The development of AAV8 and AAV9 nanobodies and their affinity packing materials that can be used for affinity purification may have good application prospects. Summary of the Invention

[0008] The purpose of this invention is to provide nanobodies that can bind to AAV8 and AAV9 serotypes with high affinity and specificity, and the application of the nanobodies used in AAV detection and AAV separation and purification.

[0009] In a first aspect of the invention, an anti-adeno-associated virus (AAV) nanobody is provided, wherein the complementarity-determining region (CDR) of the VHH chain of the anti-AAV nanobody is selected from the group consisting of: (1) CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 2, and CDR3 shown in SEQ ID NO: 3; (2) CDR1 shown in SEQ ID NO: 6, CDR2 shown in SEQ ID NO: 7, and CDR3 shown in SEQ ID NO: 8; or (3) CDR1 shown in SEQ ID NO: 11, CDR2 shown in SEQ ID NO: 12, and CDR3 shown in SEQ ID NO: 13.

[0010] In another preferred embodiment, any of the amino acid sequences described above further includes a derived sequence which has optionally been added, deleted, modified and / or substituted at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and retains the ability to specifically bind to AAV.

[0011] In another preferred embodiment, the anti-AAV nanobody is capable of specifically binding to AAV8 and / or AAV9 serotypes.

[0012] In another preferred embodiment, the anti-AAV nanobody further includes a framework region (FR).

[0013] In another preferred embodiment, CDR1, CDR2 and CDR3 in the VHH chain are separated by the frame regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0014] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-AAV nanobody is selected from the sequence shown in SEQ ID NO:4, 9 or 14, or has at least 80% (preferably at least 90%, for example 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with the sequence shown in SEQ ID NO:4, 9 or 14.

[0015] In another preferred embodiment, the anti-AAV nanobody includes humanized antibodies, camel-derived antibodies, and chimeric antibodies.

[0016] In another preferred embodiment, the affinity of the anti-AAV nanobody for AAV8 is EC50 ≤ 500 nM, preferably ≤ 300 nM, and more preferably ≤ 200 nM.

[0017] In another preferred embodiment, the affinity of the anti-AAV nanobody for AAV9 is EC50 ≤ 800 nM, preferably ≤ 600 nM, and more preferably ≤ 300 nM.

[0018] In a second aspect of the invention, an anti-AAV antibody is provided, the antibody comprising one or more VHH chains of an anti-AAV nanobody as described in the first aspect of the invention.

[0019] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-AAV nanobody is selected from the sequence shown in SEQ ID NO:4, 9 or 14.

[0020] In another preferred embodiment, the anti-AAV antibody may be a monomer, a bivalent antibody, and / or a multivalent antibody.

[0021] In a third aspect of the invention, a recombinant protein is provided, said recombinant protein having: (i) anti-AAV nanobodies as described in the first aspect of the present invention, or anti-AAV antibodies as described in the second aspect of the present invention; and (ii) Optional tag sequences to assist in expression and / or purification.

[0022] In another preferred embodiment, the tag sequence includes an Fc tag, an HA tag, and a His tag.

[0023] In another preferred embodiment, the recombinant protein specifically binds to AAV8 and / or AAV9.

[0024] In a fourth aspect of the invention, a polynucleotide is provided that encodes a protein selected from the group consisting of: an anti-AAV nanobody as described in the first aspect of the invention, an anti-AAV antibody as described in the second aspect of the invention, or a recombinant protein as described in the third aspect of the invention.

[0025] In another preferred embodiment, the polynucleotide includes RNA, DNA, or cDNA.

[0026] In another preferred embodiment, the polynucleotide encodes a VHH chain of an anti-AAV nanobody as described in the first aspect of the invention; preferably, the sequence of the polynucleotide is as shown in SEQ ID NO: 5, 10 or 15.

[0027] In a fifth aspect of the invention, an expression vector is provided, the expression vector containing the polynucleotide as described in the fourth aspect of the invention.

[0028] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof; preferably, the expression vector includes viral vectors such as lentiviruses, adenoviruses, AAV viruses, retroviruses, or combinations thereof.

[0029] In another preferred embodiment, the expression vector is the pPICZαA plasmid.

[0030] In a sixth aspect of the invention, a host cell is provided, the host cell containing an expression vector as described in the fifth aspect of the invention, or having a genome containing polynucleotides as described in the fourth aspect of the invention.

[0031] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0032] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0033] In another preferred embodiment, the host cell is a Pichia pastoris cell.

[0034] In a seventh aspect of the invention, a method for generating anti-AAV nanobodies as described in the first aspect of the invention is provided, comprising the steps of: (a) Under conditions suitable for the production of nanobodies, host cells as described in the sixth aspect of the present invention are cultured to obtain a culture containing the anti-AAV nanobodies. (b) Isolate and / or recover the anti-AAV nanobody from the culture.

[0035] In another preferred embodiment, the method further includes step (c): purifying and / or modifying the anti-AAV nanobody obtained in step (b).

[0036] In an eighth aspect of the invention, the use of an anti-AAV nanobody as described in the first aspect of the invention, an anti-AAV antibody as described in the second aspect of the invention, or a recombinant protein as described in the third aspect of the invention, for the preparation of: i) Reagents used for the separation and purification of AAV; or ii) Reagents used to detect AAV.

[0037] In another preferred embodiment, the reagents for separating and purifying AAV can be used for laboratory preparation or industrial production of AAV.

[0038] In another preferred embodiment, the reagent shown for detecting AAV is a diagnostic reagent, preferably a test strip or test plate.

[0039] In another preferred embodiment, the diagnostic reagent is used to detect AAV or fragments thereof in a sample.

[0040] In another preferred embodiment, the reagent for detecting AAV is a quality control reagent used to detect the quality of the generated recombinant AAV product.

[0041] In another preferred embodiment, the detection includes flow cytometry, immunofluorescence assay, and ELISA.

[0042] In another preferred embodiment, the serotypes of the AAV include AAV8 and AAV9.

[0043] In a ninth aspect of the present invention, a reagent for isolating and purifying AAV is provided, comprising an anti-AAV nanobody as described in the first aspect of the present invention, an anti-AAV antibody as described in the second aspect of the present invention, or a recombinant protein as described in the third aspect of the present invention.

[0044] In a tenth aspect of the invention, an affinity chromatography packing material for separating and purifying AAV is provided, comprising a resin coupled with an AAV-binding molecule, wherein the AAV-binding molecule is selected from anti-AAV nanobodies as described in the first aspect of the invention, anti-AAV antibodies as described in the second aspect of the invention, or recombinant proteins as described in the third aspect of the invention.

[0045] In another preferred embodiment, the resin is a porous cross-linked microbead agarose that has been activated by periodoacetic acid groups.

[0046] In another preferred embodiment, the resin is selected from Epoxy activated Bestarose 6B or PabPurSulfoLink Beads 4FF.

[0047] In an eleventh aspect of the present invention, an AAV detection reagent is provided, the detection reagent containing an anti-AAV nanobody as described in the first aspect of the present invention, an anti-AAV antibody as described in the second aspect of the present invention, or a recombinant protein as described in the third aspect of the present invention.

[0048] In another preferred embodiment, the detection reagent is used for flow cytometry, cell immunofluorescence assay, or ELISA assay.

[0049] In another preferred embodiment, the detection reagent is a diagnostic reagent.

[0050] In another preferred embodiment, the diagnostic reagent is used to detect AAV or fragments thereof in a sample.

[0051] In another preferred embodiment, the detection reagent is a quality control reagent used to detect the quality of the generated recombinant AAV product.

[0052] In a twelfth aspect of the present invention, an AAV detection plate is provided, the detection plate comprising: a substrate and a test strip, the test strip containing an anti-AAV nanobody as described in the first aspect of the present invention, an anti-AAV antibody as described in the second aspect of the present invention, or a recombinant protein as described in the third aspect of the present invention.

[0053] In a thirteenth aspect of the invention, a kit is provided comprising an anti-AAV nanobody as described in the first aspect of the invention, an anti-AAV antibody as described in the second aspect of the invention, or a recombinant protein as described in the third aspect of the invention.

[0054] In another preferred embodiment, the kit further includes an instruction manual indicating that the kit is used for detecting AAV or fragments thereof in a sample.

[0055] In a fourteenth aspect of the present invention, a method for in vitro detection of AAV or fragments thereof in a sample is provided, the method comprising the steps of: (1) In vitro, the sample is contacted with an anti-AAV nanobody as described in the first aspect of the present invention, an anti-AAV antibody as described in the second aspect of the present invention, or a recombinant protein as described in the third aspect of the present invention; (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of AAV or its fragments in the sample.

[0056] In another preferred embodiment, the detection includes diagnostic or non-diagnostic methods.

[0057] In a fifteenth aspect of the present invention, a method for detecting AAV is provided, comprising the steps of: (i) Obtain a sample from the diagnostic subject and contact the sample with an anti-AAV nanobody as described in the first aspect of the present invention, an anti-AAV antibody as described in the second aspect of the present invention, or a recombinant protein as described in the third aspect of the present invention; (ii) Detect whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of AAV in the subject.

[0058] In another preferred embodiment, the subject is a patient receiving AAV vector gene therapy.

[0059] In another preferred embodiment, in step (i), the sample is contacted with an immunoconjugate as described in the eighth aspect of the invention, wherein the conjugated portion is a quantitatively detectable marker, and the method may optionally further include the step of: (iii) Quantitatively detect the content of the marker to obtain the content of AAV in the subject of diagnosis.

[0060] In another preferred embodiment, the sample includes: blood, body fluid, tissue sample, or a combination thereof.

[0061] In a sixteenth aspect of the invention, a method for purifying AAV is provided, using a reagent for separating and purifying AAV as described in a ninth aspect of the invention, or an affinity chromatography packing material as described in a tenth aspect of the invention.

[0062] In another preferred embodiment, the aforementioned AAV serotypes include AAV8 and AAV9.

[0063] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0064] The following figures are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the invention as defined by the claims.

[0065] Figure 1 This is an SDS-PAGE gel image of AAV8 and AAV9 specific nanobodies.

[0066] Figure 2 The results are obtained by ELISA detection of the binding activity of candidate nanobodies with different subtypes of AAV.

[0067] Figure 3 This is the result of ELISA detection of the binding activity of candidate nanobodies under different reaction conditions.

[0068] Figure 4 This is the SDS-PAGE result of the expression supernatant of the candidate nanobody in Pichia pastoris.

[0069] Figure 5 This is a graph showing the expression yield of the candidate nanobody in a 7L fermenter.

[0070] Figure 6 These are the SDS-PAGE results of the candidate nanobody at different time points in a 7L fermenter.

[0071] Figure 7 This is an SDS-PAGE gel image of a sample obtained during the purification of AAV8 adenovirus using candidate nanobody fillers. Detailed Implementation

[0072] Through extensive and in-depth research, the inventors have, for the first time, screened and obtained nanobodies capable of specifically binding to AAV8 and AAV9 serotypes of adeno-associated virus (AAV). Experiments have demonstrated that the nanobodies of this invention can bind to AAV8 and AAV9 serotypes with high affinity. Furthermore, the nanobodies of this invention bind to AAV under neutral pH conditions but not under acidic pH conditions, making them suitable as ligands for affinity chromatography. This facilitates elution under relatively mild conditions during AAV purification, maintaining the good biological activity of AAV. In addition, the nanobodies of this invention exhibit good tolerance in 20% ethanol and completely dissociate from AAV under 0.1M NaOH conditions, which is beneficial for the CIP process of the packing material, making them highly suitable for the industrial production of AAV.

[0073] Based on this, the present invention was completed.

[0074] the term As used herein, the terms “nanobody of the present invention”, “anti-AAV nanobody of the present invention”, and “anti-AAV nanobody” are used interchangeably and all refer to single-domain antibodies that specifically recognize and bind to AAV8 and AAV9 serotypes of AAV.

[0075] The amino acid sequence, base sequence, and CDR region sequence of the VHH chain of the nanobody of the present invention, as well as the CDR region sequence obtained according to the CDR calculation algorithm (the present invention uses the IMGT partitioning method), are shown in Table A below.

[0076] Table A. Sequence List of Nanobodies of the Present Invention As used herein, the terms "single domain antibody (sdAb)," "nanobody," and "VHH" have the same meaning and are used interchangeably. They refer to the cloning of the variable region of an antibody heavy chain to construct a single domain antibody consisting of only one heavy chain variable region. It is the smallest antigen-binding fragment with complete function. Typically, antibodies lacking both the light chain and the heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody consisting of only one heavy chain variable region.

[0077] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0078] As used herein, the term "variable" refers to the fact that certain portions of the variable region in an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire antibody variable region. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable regions are called framework regions (FRs). The variable regions of both the natural heavy and light chains each contain four FR regions, which are roughly arranged in a 360-460-460 pattern. - A folded configuration, formed by three CDRs connecting to form a connecting loop, which in some cases can form a partial... Folded structure. The CDRs in each chain are tightly packed together via FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0079] As used in this article, the terms “variable region” and “complementarity determining region (CDR)” are used interchangeably.

[0080] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity-determining regions CDR1, CDR2, and CDR3.

[0081] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the aforementioned heavy chain variable region and heavy chain constant region.

[0082] In this invention, the terms "antibody of the invention," "protein of the invention," or "peptide of the invention" are used interchangeably and all refer to peptides that specifically bind to AAV proteins, such as proteins or peptides having a heavy chain variable region. They may or may not contain initiating methionine.

[0083] This invention also provides other proteins or fusion expression products having the antibodies of this invention. Specifically, this invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, provided that the variable region is the same as or at least 90% homologous to the heavy chain variable region of the antibody of this invention, preferably at least 95% homologous.

[0084] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called the variable region (CDR). This segment is divided into four frame regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, and are spatially close to each other through the β-sheets formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0085] The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because at least a portion of them are involved in binding antigens. Therefore, the present invention includes molecules having variable regions of antibody heavy chains with CDRs, provided that their CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology with the CDRs identified herein.

[0086] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0087] As used herein, the terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0088] The term "antibody of the present invention" refers to a polypeptide containing the aforementioned CDR region that has AAV protein-binding activity. This term also includes variants of polypeptides containing the aforementioned CDR region that have the same function as the antibodies of the present invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids, and the addition of one or more amino acids at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition of one or more amino acids at the C-terminus and / or N-terminus generally does not alter the function of the protein. This term also includes active fragments and active derivatives of the antibodies of the present invention.

[0089] The variant forms of the polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0090] The present invention also provides other polypeptides, such as fusion proteins comprising a single-domain antibody or a fragment thereof. In addition to nearly full-length polypeptides, the present invention also includes fragments of the single-domain antibody of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0091] In this invention, "a conserved variant of the antibody of the present invention" refers to a polypeptide formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table B.

[0092] Table B The present invention also provides a polynucleotide molecule encoding the aforementioned antibody or a fragment thereof or a fusion protein thereof. The polynucleotide of the present invention may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0093] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0094] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0095] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60ºC; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0096] The full-length nucleotide sequence or fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein.

[0097] Once the relevant sequence is obtained, it can be obtained in large quantities using recombinant methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms.

[0098] Currently, the DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0099] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0100] The host cell can be a prokaryotic cell, such as a bacterial cell, preferably Bacillus subtilis cells; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; Salmonella typhimurium bacterial cells; fungal cells such as yeast; Drosophila S2 or Sf9 insect cells; and animal cells such as CHO, COS7, and 293 cells.

[0101] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0102] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0103] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0104] The antibodies of the present invention can be used alone or in combination or conjugated with detectable markers (for diagnostic purposes), PK (protein kinase) modified portions, or any combination of the above substances.

[0105] Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes that can produce detectable products.

[0106] AAV and anti-AAV nanobodies As used in this article, the term "adeno-associated virus (AAV)" is a replication-defective virus whose genome is a single-stranded DNA of approximately 4.7 kb, with 145-nucleotide terminal inverted repeats (ITRs) at both ends. There are numerous AAV serotypes, each with different capsid protein amino acid sequences and structures. These differences, along with their varying interactions with host cytokines, lead to different serotypes of AAV exhibiting varying infectivity in different tissues and cells. Currently, 12 human AAV serotypes (AAV1 to AAV12) and over 100 non-human primate AAV serotypes have been identified.

[0107] This invention provides nanobodies capable of binding serum AAV8 and AAV9, wherein the antibody comprises the CDR shown in Table A above, or a VHH chain having an amino acid sequence selected from SEQ ID NO: 4, 9, or 14. The nanobodies of this invention exhibit an affinity EC50 of ≤500 nM for AAV8, preferably ≤300 nM, more preferably ≤200 nM; and an affinity EC50 of ≤800 nM for AAV9, preferably ≤600 nM, more preferably ≤300 nM.

[0108] In this invention, an anti-AAV antibody is also provided, which includes a monomer, a bivalent (bivalent antibody), a tetravalent (tetravalent antibody), and / or a multivalent (multivalent antibody).

[0109] In a preferred embodiment of the present invention, the anti-AAV antibody has one or more VHH chains selected from the amino acid sequences shown in SEQ ID NO:4, 9 or 14.

[0110] In one embodiment of the invention, the nanobody or antibody carries a detectable marker. More preferably, the marker is selected from the group consisting of isotopes, colloidal gold markers, colored markers, or fluorescent markers.

[0111] Preferably, the nanobody carries a quantitatively detectable marker.

[0112] The anti-AAV nanobody of the present invention has excellent specificity and can be used for the localization, tracing and detection of AAV.

[0113] The labeled anti-AAV single-domain antibody of the present invention can be used to detect the presence of AAV in a subject (e.g., a patient receiving AAV vector gene therapy), preferably to detect the AAV content in the subject, thereby assessing the efficacy of AAV gene therapy and the risk of subsequent side effects and complications.

[0114] Test reagents, test plates and kits This invention provides a detection reagent for AAV containing the antibody or a fragment thereof or an immunoconjugate thereof. The detection reagent includes diagnostic reagents for detecting AAV or fragments thereof in a sample, and also includes quality control reagents for detecting the quality of recombinant AAV products produced in industrial processes.

[0115] The present invention also provides a detection plate comprising a substrate (or support plate) and a test strip, wherein the test strip contains an antibody or fragment thereof or an immunoconjugate thereof as a detection reagent.

[0116] The present invention also provides a kit containing the antibody or fragment thereof or the immunoconjugate thereof of the present invention, or the above-mentioned detection plate. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, buffer, etc.

[0117] In one embodiment of the present invention, the kit is a detection kit for detecting AAV, comprising an antibody that recognizes AAV, a lysis medium for dissolving samples, and universal reagents and buffers required for detection, such as various buffers, detection labels, and detection substrates. The detection kit can be an in vitro detection device.

[0118] Detection methods The present invention also relates to a method for detecting AAV. The method comprises the following steps: obtaining cell and / or tissue samples; dissolving the samples in a medium; and detecting the level of AAV in the dissolved samples.

[0119] In the detection method of the present invention, there are no particular limitations on the samples used. Representative examples are peripheral blood samples or cell-containing samples present in cell preservation solutions.

[0120] The detection methods of the present invention include non-diagnostic in vitro methods, as well as methods for detecting the presence of AAV in a diagnostic subject.

[0121] Reagents for separating and purifying AAV This invention provides a reagent for separation and purification containing the antibody of this invention. In a preferred embodiment of this invention, an affinity chromatography packing material for separating and purifying AAV is provided, comprising a resin coupled with the antibody of this invention. The affinity chromatography packing material is prepared by: activating the resin with an iodoacetic acid group; adding a free cysteine ​​residue to the C-terminus of the antibody of this invention; and forming an irreversible thioether bond through a specific reaction between the iodoacetic acid group and a thiol group, thereby covalently immobilizing the cysteine ​​peptide and other thiol molecules to form an antibody-coupled resin.

[0122] Methods for purifying AAV The present invention also provides a method for purifying AAV, namely, using the separation and purification reagents described in the present invention to separate and purify the produced AAV.

[0123] In one embodiment of the present invention, AAV purification is performed using affinity chromatography, wherein the packing material for affinity chromatography is a resin coupled with the antibody of the present invention. Furthermore, neutral loading and acidic elution chromatography conditions are used during affinity chromatography.

[0124] application As described above, the nanobody of the present invention has broad industrial and biomedical application value, and its applications involve multiple fields such as basic medical research and biological research related to AAV. Specifically, the nanobody of the present invention has the following uses: i) Used for the production, preparation, separation, purification, or combination thereof of AAV; ii) For in vivo and in vitro localization, tracing, detection, or combinations thereof for AAV.

[0125] iii) Used for the diagnosis of AAV-related diseases.

[0126] In a preferred embodiment of the present invention, the nanobody of the present invention is used to detect the AAV content in the body of a patient receiving AAV gene therapy, thereby assessing the efficacy of AAV gene therapy and the risk of causing side effects and complications.

[0127] In a preferred embodiment of the present invention, the nanobody of the present invention is used for the separation and purification of AAV production.

[0128] The main advantages of this invention include: (1) This invention is the first to develop nanobodies specifically targeting AAV8 and AAV9 serum types; (2) The nanobody of the present invention has a high affinity for binding to AAV8 and AAV9; (3) The nanobody of the present invention binds to AAV under neutral pH conditions and does not bind to AAV under acidic pH conditions. It can be used as a packing material ligand for affinity chromatography, which is convenient to be eluted under relatively mild conditions during the AAV purification process and maintains the good biological activity of AAV. In addition, the nanobody of the present invention has good tolerance in 20% ethanol and completely dissociates from AAV under 0.1M NaOH conditions, which is beneficial to the CIP process of the packing material. Therefore, it is very suitable for industrial production of AAV. (4) The nanobody preparation method of the present invention is simple and easy to mass-produce.

[0129] The results of the present invention are further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the following embodiments are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommended conditions. Unless otherwise stated, percentages and parts are weight percentages and parts by weight results.

[0130] Example 1: Construction of AAV8 and AAV9 nanobody display libraries and antibody screening One 2-3 year old Bactrian camel was immunized with high-purity AAV8 and AAV9 recombinant adeno-associated virus (purchased from Shanghai Weizhen Biotechnology Co., Ltd.). Immunization was performed weekly, with each dose containing 1 mg of recombinant adeno-associated virus. After seven immunizations, peripheral blood was collected to detect serum antibody titers. Lymphocytes were isolated and a phage nanobody display library was constructed upon confirmation of high serum titers. The VHH target fragment was amplified using two rounds of PCR and cloned into the pMECS vector, then electroporated into TG1 competent cells to construct an AAV8 and AAV9 specific nanobody library. The quality of the library, such as its volume and insertion rate, was then assessed.

[0131] Nanobodies specifically recognizing AAV8 and AAV9 were obtained using phage display panning technology. AAV8 and AAV9 viruses were separately coated onto ELISA plates, and phage display libraries containing nanobodies were added for 2-3 rounds of panning. From the enriched pool, 300 clones were randomly selected for PE-ELISA to verify binding to AAV8 and AAV9 adenovirus particles. Positive clones were sequenced and classified based on the diversity of amino acid sequences in the CDR3 region of the nanobodies.

[0132] Example 2: Purification of candidate nanobodies Candidate nanobodies were expressed using *E. coli*. Plasmids from different clones obtained from sequencing analysis in Example 1 were electroporated into the expression strains and plated on LA+glucose (containing ampicillin and glucose) plates, incubated overnight at 37°C. Single clones were randomly picked from the plates and inoculated into LA liquid medium for expansion to the logarithmic growth phase, followed by IPTG induction and overnight incubation for 16 hours. Cells were then collected and lysed using a high-glucose solution. The lysate was incubated with a Ni column, and single-domain antibody proteins were prepared by one-step Ni column affinity chromatography. Nine candidate nanobodies were simultaneously prepared using this method.

[0133] SDS-PAGE analysis showed that the purity of all nine candidate nanobodies exceeded 95%, as indicated by the results. Figure 1 As shown.

[0134] Example 3: ELISA identification of the binding activity of candidate nanobodies to AAV8 and AAV9 antigens The binding activity of the nine candidate nanobodies described in Example 2 to AAV8 and AAV9 was detected using ELISA.

[0135] AAV8 and AAV9 antigens were coated onto 96-well ELISA plates at 37°C for 2 hours. After washing with PBST, BSA blocking buffer was added and the plates were blocked at 37°C for 2 hours. After washing with PBST, serially diluted candidate nanobodies were added and incubated at 37°C for 1 hour. After washing with PBST, diluted biotinylated anti-his antibody was added and incubated, followed by diluted SA-HRP. After washing with PBST, chromogenic solution and stop solution were added, and the absorbance was measured using an ELISA reader.

[0136] The results are as follows Figure 2 As shown, all nine candidate nanobodies can effectively bind to AA8 and AAV9.

[0137] Example 4: Identification of the binding activity of candidate nanobodies under different solvent conditions The purpose of this invention is to screen for antibodies that bind under neutral pH conditions and dissociate under low pH conditions, in order to mimic the characteristic of ligands capturing antigens during affinity purification.

[0138] Candidate antibodies were replaced with buffers under different conditions (including pH 3.0, pH 3.5, pH 4.0, pH 7.0, 20% ethanol, and 0.1M NaOH). AAV8 antigen was coated onto an ELISA plate, washed with PBST, and then blocked at 37°C for 2 hours with BSA blocking buffer. After washing with PBST, candidate nanobodies diluted in different solvents were added and incubated at 37°C for 1 hour. After washing with PBST, diluted biotinylated anti-his antibody was added and incubated, followed by diluted SA-HRP. After washing with PBST, chromogenic and stop solutions were added, and absorbance was measured using an ELISA reader.

[0139] The results are as follows Figure 3 As shown, candidate nanobodies Nb4, Nb5, and Nb9 (amino acid sequences shown in SEQ ID NO: 9, 14, and 4, respectively) bind to the antigen under neutral conditions (pH 7.0), but do not bind to AAV8 under low pH conditions. This indicates that these three candidate nanobodies, as ligands, can be easily eluted under relatively mild conditions during AAV8 virus purification, which is beneficial for maintaining the good biological characteristics of AAV8. Furthermore, the candidate nanobodies exhibit good tolerance in 20% ethanol and completely dissociate from the antigen under 0.1M NaOH conditions, which is beneficial for the subsequent CIP process of the packing material.

[0140] Example 5: Expression of candidate nanobodies in Pichia pastoris Based on the above screening results, Nb4, Nb5, and Nb9 were selected for the construction and expression of Pichia pastoris strains, with a free cysteine ​​residue added to their C-terminus for directional coupling with the media material. The base sequences of the above candidate nanobodies (as shown in SEQ ID NO: 10, 15, and 5, respectively) were constructed into the pPICZαA vector, linearized by enzyme digestion, and then electroporated into Pichia pastoris X-33 host. Clones were screened by plating on Zeocin-resistant plates, and expression verification was performed in well plates.

[0141] The expression of candidate nanobodies in Pichia pastoris supernatant is shown in the figure. Figure 4 The SDS-PAGE detection results (NR in the figure represents non-reduced, and R represents reduced)

[0142] The candidate nanobody was then cultured in a 7L fermentation tank. Wet weight and Titer curves during fermentation are shown in [Figure number missing]. Figure 5 The SDS-PAGE results of the supernatant during fermentation are shown in the figure. Figure 6The results showed that the yield of Nb9 nanobody steadily increased during the fermentation process in a 7L tank, reaching a high of 7g / L at the end of fermentation.

[0143] Example 6: Conjugation test of candidate nanobodies with different media The candidate Nb9 nanobody was designed to contain free cysteine ​​residues. Under normal conditions, antibodies readily form dimers; therefore, before coupling with the medium, they were treated with TCEP or dithiothreitol to form monomers, reducing and exposing all free cysteine ​​residues to facilitate coupling with the groups on the medium microspheres. For AAV affinity purification testing of the nanobody, two different resins were selected for coupling: Epoxy-activated Bestarose 6B and PabPurSulfoLink Beads 4FF. The coupling resin is a porous, cross-linked agarose bead activated with periodoacetic acid groups. Iodoacetic acid groups specifically react with thiol groups to form irreversible thioether bonds, achieving covalent fixation of the cysteine ​​peptide and other thiol molecules. The coupling loading of the candidate Nb9 nanobody with the coupling resin is shown in Table 1. The results in Table 1 indicate that the candidate Nb9 nanobody has a higher coupling capacity and better coupling effect with Epoxy-activated Bestarose 6B.

[0144] Table 1. Conjugation effects of candidate nanobodies with different matrix materials Example 7: Purification effect test of candidate nanobody fillers Affinity chromatography media for AAV8 were tested using Epoxy-activated Bestarose 6B resin conjugated with candidate Nb9 nanobodies as the packing material.

[0145] The purification efficiency of this resin for AA8 samples was evaluated using an AKTA Explorer device (1 mL pre-packed column). The retention time was 5 min, and the column was equilibrated with a pH-neutral buffer of 20 mmol / L PB and 150 mmol / L NaCl. Fermentation supernatant mixed with AAV8 standards was loaded onto the column in 10 mL volumes. Elution was then performed under neutral conditions with 20 mmol / L PB and 150 mmol / L NaCl for the first elution, followed by 20 mmol / L PB and 1 mol / L NaCl for the second elution, and finally 20 mmol / L PB for the third elution under neutral conditions. The tested AAV8 binding capacity was 2.0 × 10⁻⁶. 13The recovery rate was 90.5% (vg / ml). Subsequently, the purity of each component tested for AAV8 was determined by SDS-PAGE. The results showed that the nanobody and chromatographic packing material could effectively capture AAV8 from the supernatant, and a highly purified AAV8 adenovirus was obtained. Figure 7 ).

[0146] The above results demonstrate that the nanobody and chromatography packing material have high specificity and good potential for development into AAV affinity chromatography packing material.

[0147] All references to this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A nanobody against adeno-associated virus (AAV), characterized in that, The complementarity-determining region (CDR) of the VHH chain of the anti-AAV nanobody is shown below: CDR1 shown in SEQ ID NO: 6, CDR2 shown in SEQ ID NO: 7, and CDR3 shown in SEQ ID NO:

8.

2. The anti-AAV nanobody as described in claim 1, characterized in that, The anti-AAV nanobody can specifically bind to AAV8 and / or AAV9 serotypes.

3. The anti-AAV nanobody as described in claim 1, characterized in that, The amino acid sequence of the VHH chain of the anti-AAV nanobody is shown in SEQ ID NO: 9, or has at least 80% sequence identity with the sequence shown in SEQ ID NO:

9.

4. An antibody against AAV, characterized in that, The antibody comprises one or more VHH chains of anti-AAV nanobodies as described in claim 1.

5. A recombinant protein, characterized in that, The recombinant protein has the following characteristics: (i) anti-AAV nanobodies as described in the first aspect of the present invention, or anti-AAV antibodies as described in the second aspect of the present invention; and (ii) Optional tag sequences to assist in expression and / or purification.

6. A polynucleotide, characterized in that, The polynucleotide encodes a protein selected from the group consisting of: the anti-AAV nanobody as described in claim 1, the anti-AAV antibody as described in claim 4, or the recombinant protein as described in claim 5.

7. The use of the anti-AAV nanobody as described in claim 1, the anti-AAV antibody as described in claim 4, or the recombinant protein as described in claim 5, for the preparation of: i) Reagents used for the separation and purification of AAV; or ii) Reagents used to detect AAV.

8. A reagent for isolating and purifying AAV, comprising the anti-AAV nanobody as described in claim 1, the anti-AAV antibody as described in claim 4, or the recombinant protein as described in claim 5.

9. An affinity chromatography packing material for separating and purifying AAV, comprising a resin coupled with an AAV-binding molecule, wherein the AAV-binding molecule is selected from the anti-AAV nanobody of claim 1, the anti-AAV antibody of claim 4, or the recombinant protein of claim 5.

10. A method for purifying AAV, characterized in that, AAV is purified using the reagent for separating and purifying AAV as described in claim 8, or the affinity chromatography packing material as described in claim 9.

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