Nanometer antibody, fusion protein containing nanometer antibody and application of nanometer antibody to purification of multiple serotypes AAV

Abstract

The invention discloses a nano antibody, a fusion protein containing the nano antibody and application of the nano antibody to purification of various serotypes AAV. The nano antibody comprises a heavy chain variable region containing three complementary determining regions, namely CDR1, CDR2 and CDR3; wherein the CDR1 comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 1, the CDR2 comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 2, and the CDR3 comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 3. The nano antibody can target multiple serotypes of AAV, and can be used for detection and purification of AAV; multi-step purification can be replaced by a single-step purification step, so that the time cost is reduced, and meanwhile, the yield of AAV is improved.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to nanoantibodies, fusion proteins containing the nanoantibodies, and their use in purifying various serotypes of AAV. Background Art

[0002] Adeno-associated virus (AAV) belongs to the Parvoviridae family and the genus Parvoviridae. Its life cycle depends on the presence of a helper virus (e.g., AdV). AAV consists of an icosahedral protein capsid with a diameter of 26 nm and a 4.7 kb single-stranded DNA genome. The capsid is composed of three subunits: VP1, VP2, and VP3, with a ratio of VP1:VP2:VP3 = 1:1:10. Two T-shaped inverted repeats (ITRs) are located at either end of the genome, primarily serving as viral replication initiation and packaging signals. Rep encodes four proteins required for viral replication: Rep78, Rep68, Rep52, and Rep40, while cap encodes three protein subunits. The AAV genome can integrate into the AAVS1 locus in the human genome, during which AAV enters a latent phase. Recombinant AAV has been successfully used as a viral delivery system for therapeutic genes in multiple human clinical trials.

[0003] At present, the purification of AAV mainly includes CsCl density gradient centrifugation or iodixanol density gradient centrifugation, ion exchange chromatography, affinity chromatography and size exclusion chromatography. Density gradient centrifugation is only suitable for small-scale production in the laboratory and is not realistic for large-scale application. Among them, the disadvantage of ion exchange chromatography is at least that it is sensitive to the conditions. Slight changes in pH or salt concentration may significantly affect the binding efficiency, and the process development is complicated. The disadvantage of affinity chromatography is at least that there are serotype restrictions and high costs. In addition, the above-mentioned partial purification methods often require multiple steps to be combined with each other. Although AAV with higher purity can be obtained in the end, it is time-consuming and has low yields.

[0004] Therefore, there is an urgent need in the art to develop new methods for purifying AAV. Summary of the Invention

[0005] Based on this, it is necessary to provide at least one nanobody, a fusion protein containing the nanobody, and its use in purifying multiple serotypes of AAV.

[0006] In the first aspect of the present application, a nanobody or an antigen-binding fragment thereof targeting different serotypes of adeno-associated viruses (AAV) is provided, wherein the nanobody or the antigen-binding fragment thereof comprises a heavy chain variable region comprising three complementarity determining regions CDR1, CDR2 and CDR3; wherein the CDR1 comprises an amino acid fragment whose sequence is shown in SEQ ID NO: 1, CDR2 comprises an amino acid fragment whose sequence is shown in SEQ ID NO: 2, and CDR3 comprises an amino acid fragment whose sequence is shown in SEQ ID NO: 3.

[0007] In the second aspect of the present application, a fusion protein is provided, which comprises the Nanobody or antigen-binding fragment thereof described in the first aspect.

[0008] In the third aspect of the present application, a nucleic acid is provided, which comprises a nucleotide sequence encoding the Nanobody or antigen-binding fragment thereof as described in the first aspect or the fusion protein as described in the second aspect.

[0009] In the fourth aspect of the present application, a recombinant expression vector is provided, which includes the nucleic acid as described in the third aspect.

[0010] In the fifth aspect of the present application, a host cell is provided, which expresses the Nanobody or antigen-binding fragment thereof as described in the first aspect or the fusion protein as described in the second aspect.

[0011] In the sixth aspect of the present application, a method for preparing the Nanobody or antigen-binding fragment thereof as described in the first aspect or the fusion protein as described in the second aspect is provided.

[0012] In the seventh aspect of the present application, a solid phase carrier is provided, the surface of which is coupled with the nanobody or antigen-binding fragment thereof as described in the first aspect or the fusion protein as described in the second aspect.

[0013] In an eighth aspect of the present application, a method for purifying AAV is provided, comprising the following steps:

[0014] contacting the solid phase carrier described in the seventh aspect with a sample containing AAV;

[0015] separating the solid phase carrier and the sample;

[0016] The AAV bound to the solid phase carrier is eluted using a solution to obtain an eluate.

[0017] In the ninth aspect of the present application, a kit is provided, comprising one or more of the nanobody or antigen-binding fragment thereof as described in the first aspect, the fusion protein as described in the second aspect, and the solid phase carrier as described in the seventh aspect.

[0018] In a tenth aspect of the present application, a method for detecting AAV or its content in a sample for non-diagnostic purposes is provided, comprising:

[0019] contacting the Nanobody or antigen-binding fragment thereof as described in the first aspect or the fusion protein as described in the second aspect with the sample to obtain an eluate containing AAV;

[0020] The amount of AAV in the eluate was detected.

[0021] The present application provides nanobodies or antigen-binding fragments thereof that can target multiple serotypes of AAV, which can be used for the detection and purification of AAV; a single-step purification step can be used instead of a multi-step purification step, thereby reducing time costs while increasing the yield of AAV. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the implementation methods and examples of this application and to more completely understand the application and its beneficial effects, the following briefly introduces the drawings required for the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in explaining the present application.

[0023] Figure 1 This is a diagram verifying the expression effect of nano antibodies in one embodiment of the present application.

[0024] Figure 2 This is a diagram verifying the purification effect of nano antibodies in one embodiment of the present application.

[0025] Figure 3 This is a diagram verifying the purification effect of AAV of different serotypes in one embodiment of the present application.

[0026] Figure 4 This is a diagram verifying the purification effect of AAV of different serotypes in one embodiment of the present application. DETAILED DESCRIPTION

[0027] To facilitate understanding of the present application, a more comprehensive description of the present application will be provided below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present application. However, the present application may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present application.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0029] In this application, unless otherwise specified, "one or more" refers to any one of the listed items or any combination of the listed items. Similarly, "one or more" and other similar expressions that refer to "one or more" are also understood in the same way unless otherwise specified.

[0030] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0031] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method", etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0032] In this application, the terms "further," "further," "particularly," "for example," "such as," "example," and "for example" are used for descriptive purposes to indicate that the preceding and following technical solutions are related in terms of the content covered, but should not be construed as limiting the preceding technical solution or the scope of protection of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it is understood that A is not limited to B.

[0033] In this application, the terms "optionally," "optional," and "optional" mean optional, that is, they refer to either option selected from the two parallel options of "yes" or "no." If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "optional" is independent. Unless otherwise specified, the descriptions "optionally include," "optionally include," etc. in this application, taking "optionally include" as an example, mean "may include or not include."

[0034] As used herein, the terms "comprising," "including," and "include" are synonymous and are inclusive or open-ended, not excluding additional, unrecited members or features. Examples of members or features include materials or components, structures, elements, and instruments. Non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, and states.

[0035] In this application, the technical features or technical solutions described in open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0036] In this application, exemplary descriptions such as "in some embodiments (or examples)" and "in one embodiment (or example)" may include but are not limited to the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0037] In this application, the terms "first," "second," "third," "fourth," etc. in "the first aspect," "the second aspect," "the third aspect," "the fourth aspect," etc. are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor should they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc. serve only for the purpose of non-exhaustive enumeration and description, and should be understood not to constitute a closed-ended limitation on quantity.

[0038] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values in the numerical interval is considered to be continuous and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval only refers to an integer in the numerical interval, including the two endpoint integers of the numerical range and each integer between the two endpoints, is equivalent to directly enumerating each integer. When multiple numerical ranges are provided to describe a feature or characteristic, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0039] In this application, if a method flow involves multiple steps, unless otherwise specified herein, there is no strict order restriction on the execution of these steps, and they can be executed in an order other than the order described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages do not necessarily need to be completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating, or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0040] the term

[0041] In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning given below.

[0042] The terms "nanoantibody", "single domain antibody", "antibody of the present application", "nanoantibody of the present application", etc. are used interchangeably and refer to the nanoantibody as described in the first aspect of the present application that specifically recognizes and binds to AAV.

[0043] As used herein, the terms "single-domain antibody," "VHH," "Nanobody," and "single-domain antibody (sdAb, or nanobody)" 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 (VHH) consisting solely of a single heavy chain variable region. This is the smallest fully functional antigen-binding fragment. Typically, an antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting solely of a single heavy chain variable region.

[0044] As used herein, the term "variable" refers to certain portions of the variable region in an antibody that differ in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of native heavy and light chains each contain four FR regions, which are generally arranged in a β-sheet configuration and are connected by three CDRs that form a connecting loop, and in some cases can form a partial β-sheet structure. The CDRs in each chain are closely together through the 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. 1, pp. 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cytotoxicity of the antibody.

[0045] As used herein, the terms “hypervariable region”, “hypervariable region”, “complementarity determining region” and “complementarity determining region (CDR)” are used interchangeably.

[0046] As used herein, the terms "fragment," "derivative," and "analog" 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 conservative or non-conservative amino acid residues (preferably conservative 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 a substituent group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein formed with a 6×His tag). According to the teachings of the present application, these fragments, derivatives, and analogs are within the scope known to those skilled in the art.

[0047] The present application provides a nanobody or an antigen-binding fragment thereof targeting different serotypes of adeno-associated viruses (AAV).

[0048] Generally, an antibody's antigen-binding properties are described by three specific regions within the variable region of the heavy chain, known as the variable regions (CDRs). This region is divided into four framework 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, spatially close to each other through the β-sheet formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antibody's antigen-binding site. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0049] In some embodiments, the heavy chain variable region of a Nanobody or antigen-binding fragment thereof comprises three complementarity determining regions: CDR1, CDR2, and CDR3.

[0050] In some embodiments, CDR1 comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 1, CDR2 comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 2, and CDR3 comprises an amino acid fragment with a sequence as shown in SEQ ID NO: 3.

[0051] Illustratively, the heavy chain variable region comprises, in order, FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0052] The nanoantibody of the present application refers to a polypeptide having AAV binding activity and comprising the above-mentioned CDR region. The term also includes variant forms of polypeptides comprising the above-mentioned CDR region and having the same function as the antibody of the present application. These variant forms include (but are not limited to): one or more (usually 1-50, optionally 1-30, further optionally 1-20, and further optionally 1-10) amino acid deletions, insertions and / or substitutions, and addition of one or several (usually within 20, optionally within 10, further optionally within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is generally not changed. For another example, adding one or several amino acids to the C-terminus and / or N-terminus generally does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present application.

[0053] Variant forms of the polypeptide include: homologous sequences, conservative 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 stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0054] The heavy chain variable regions of the nanobodies of the present application are of particular interest because at least part of them is involved in binding to the antigen. Therefore, the present application includes molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (optionally more than 95%, further optionally more than 98%) homology with the CDRs identified herein.

[0055] In some embodiments, the Nanobody or its antigen-binding fragment comprises an amino acid fragment whose sequence is shown in SEQ ID NO: 4.

[0056] In some embodiments, the heavy chain of the Nanobody or antibody comprises the above-mentioned heavy chain variable region and heavy chain constant region.

[0057] In some embodiments, the Nanobody or antigen-binding fragment thereof further comprises an Fc fragment.

[0058] The present application also provides other proteins or fusion expression products having the nanobodies or antigen-binding fragments thereof of the present application. Specifically, the present application 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, as long as the variable region is identical to or at least 90% homologous to the heavy chain variable region of the antibody of the present application, for example, at least 95% homologous.

[0059] The present application includes not only nanobodies, but also fragments of nanobodies with immunological activity or fusion proteins formed by nanobodies and other sequences. Therefore, the present application also includes fragments, derivatives and analogs of the nanobodies.

[0060] In some embodiments, in the fusion protein, the N-terminus and / or C-terminus of the Nanobody or its antigen-binding fragment carries a purification tag and / or a fluorescent tag.

[0061] Exemplarily, the purification tag may be selected from the group consisting of 6×His, Flag, GST, Myc, MBP and Strep.

[0062] The above-mentioned tag and Nanobody or antigen-binding fragment thereof are operably linked, for example, via a linker (such as a flexible linker).

[0063] The present application also provides polynucleotide molecules (or nucleic acids) encoding the aforementioned Nanobodies, fragments thereof, or fusion proteins thereof. The polynucleotides of the present application may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0064] The polynucleotide encoding the mature polypeptide of the present application includes: a coding sequence encoding only a mature polypeptide; a coding sequence of a mature polypeptide and various additional coding sequences; a coding sequence of a mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0065] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and / or non-coding sequences.

[0066] The present application also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, optionally at least 70%, and further optionally at least 80% identity between the two sequences. The present application particularly relates to polynucleotides that can hybridize to the polynucleotides described in the present application under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf 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%. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0067] The full-length nucleotide sequence of the antibody of the present application or its fragments can generally be obtained by PCR amplification, recombination, or artificial synthesis. One feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Generally, by first synthesizing multiple small fragments and then ligating them, very long fragments can be obtained. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6×His) to form a fusion protein.

[0068] Once the relevant sequence is obtained, recombinant methods can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) referred to in this application include biomolecules in isolated form.

[0069] Currently, DNA sequences encoding proteins of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the present application by chemical synthesis.

[0070] The present application also relates to recombinant expression vectors comprising the above nucleic acid and appropriate promoter or control sequence. These recombinant expression vectors can be used to transform appropriate host cells to enable them to express proteins.

[0071] The backbone of a recombinant expression vector includes, for example, an origin of replication (ori), a selectable marker, a multiple cloning site, and corresponding expression regulatory elements (e.g., a promoter, RBS, terminator, etc.). In some embodiments, the backbone of a recombinant expression vector is a plasmid, cosmid, phage, or viral vector. The backbone of a plasmid can be, for example, pET28a.

[0072] The present application also provides a host cell, which expresses the nanobody or antigen-binding fragment thereof as described above or the fusion protein as described above.

[0073] In some embodiments, it comprises one or more of the nucleic acid described above and the recombinant expression vector described above.

[0074] 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 prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2, using procedures 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 eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0075] The transformant obtained can be cultivated with conventional methods to express the polypeptide encoded by the gene of the present application. Depending on the host cell used, the culture medium used in the cultivation can be selected from various conventional culture media. Cultivate under conditions suitable for host cell growth. After the host cells grow to an appropriate cell density, induce the promoter of choice with a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0076] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.

[0077] Examples of prokaryotic cells include Escherichia coli and Bacillus subtilis.

[0078] The eukaryotic cells are, for example, yeast cells or fungal cells, wherein the fungal cells may be derived from the genus Aspergillus or Trichoderma.

[0079] The present application also provides a method for preparing the above-mentioned Nanobody or its antigen-binding fragment or fusion protein, which comprises:

[0080] culturing the above host cells to obtain an intracellular soluble fraction;

[0081] The intracellular soluble portion is purified to obtain a nanobody or an antigen-binding fragment or fusion protein thereof.

[0082] In some embodiments, the host cells are induced during culture.

[0083] Exemplarily, IPTG (isopropyl-β-D-thiogalactopyranoside) induction is used.

[0084] In some embodiments, IPTG is used for induction at 30°C for 12 h.

[0085] The present application also provides a solid phase carrier, the surface of which is coupled with the above-mentioned nanobody or antigen-binding fragment or fusion protein. Exemplarily, the coupling is performed via a spacer arm with an active group.

[0086] The term "solid-phase carrier" refers to any conventional solid-phase carrier in the art. Its materials include, but are not limited to, polymer compounds, glass, metals, magnetic materials (e.g., magnetic particles), resin compositions containing magnetic materials, and combinations thereof. Examples of polymer compounds include polystyrenes, polyethylenes, polypropylenes, polyesters, poly(meth)acrylonitrile, styrene-butadiene copolymers, poly(meth)acrylates, fluororesins, cross-linked dextran, and polysaccharides. The shape of the solid-phase carrier is not particularly limited; examples include trays, spheres (e.g., large-diameter silica spheres), particles, fibers, rods, disks, containers, microchannels, and plates. The magnetic particles may be magnetic microparticles formed from metals such as ferroferric oxide (Fe₃O₄), ferrites (γ-Fe₂O₃), various ferrites, iron, manganese, nickel, cobalt, and chromium; magnetic microparticles formed from alloys of these metals; and magnetic particles containing these magnetic microparticles in a resin. Examples of the resin include hydrophobic polymers and hydrophilic polymers.

[0087] In some embodiments, the solid phase carrier is, for example, a filler, magnetic microspheres, polymer microspheres, or a membrane for separation.

[0088] Unless otherwise specified, the term "separation membrane" in the present application refers to membranes formed of organic materials such as cellulose acetate, polysulfone, and polyethylene, or membranes formed of inorganic materials such as alumina and zirconia, as needed.

[0089] Exemplary fillers may be agarose pre-activated fillers (Epoxy, SulfoLink, NHS, etc.).

[0090] The present application also provides a method for purifying AAV, which comprises the following steps:

[0091] S100: contacting the solid phase carrier with a sample containing AAV;

[0092] S200: separation of solid phase carrier and sample;

[0093] S300: Using a solution to elute the AAV bound to the solid phase carrier to obtain an eluate.

[0094] The AAV in the present application may optionally include one or more of serotypes AAV1 to AAV9.

[0095] The pH of the solution used in step S300 may be 2.0-4.0, for example, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, or a value or range between any two values.

[0096] In some embodiments, the pH of the solution used in step S300 is 3.0.

[0097] The present application also provides a kit, which comprises one or more of the Nanobodies or antigen-binding fragments thereof, the fusion protein and the solid phase carrier as described above; and optionally, instructions for the kit.

[0098] The present application also provides a method for detecting AAV or its content in a sample for non-diagnostic purposes, comprising:

[0099] contacting the above-mentioned Nanobody or antigen-binding fragment or fusion protein with the sample to obtain an eluate containing AAV;

[0100] The amount of AAV in the eluate was detected.

[0101] In some embodiments, the method comprises obtaining an eluate using the above-described method for purifying AAV;

[0102] Analyze the components in the eluate by gel electrophoresis;

[0103] Silver stain the gel after electrophoresis.

[0104] In some embodiments, the method comprises obtaining an eluate using the method for purifying AAV as described above;

[0105] The viral genome in the eluate is extracted and subjected to real-time fluorescent quantitative polymerase chain reaction to obtain the genome titer.

[0106] In some embodiments, the method further comprises determining the content of full capsids by measuring the genome titer.

[0107] Some examples are provided below.

[0108] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. For experimental methods in the following examples where the conditions are not specified, reference is made to the guidance provided in the present application. The methods may also be performed according to experimental manuals in the art (e.g., Antibody Engineering (Springer, 3rd Ed.); Protein Purification: Principles and Practice (Scopes, Springer); Bioconjugation Technology Manual (Invitrogen); Molecular Cloning: A Laboratory Manual (Sambrook & Russell), etc.) or conventional conditions, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0109] Example 1 Nanobody Sequence

[0110] The sequence of the designed nanobody is:

[0111] MQVQLQESGGGLVQAGGSLLRLSCAASGRTH GYTFSSYAMG WFRQAPGKEREFVA VQSSDGADTHY VKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA GRIIMSGSYNAADDYDY WGQGTQVTVSSACPGCA (SEQ ID NO: 4; the bold and underlined parts are CDR1, CDR2, and CDR3, and the sequences are SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively).

[0112] Example 2 Expression and purification of nanobodies

[0113] 1. Expression of Nanobodies

[0114] To facilitate subsequent nanobody purification and conjugation, a 6×his tag and Met (methionine) were added to the N-terminus of the nanobody sequence to generate a new fusion protein. This fusion protein was ligated to pET28a, and the vector containing the fusion protein was transformed into competent Escherichia coli. IPTG was added to a final concentration of 0.1 mM and induced at 30°C for 12 hours. The cells were lysed and the supernatant was collected to obtain a sample containing the fusion protein. The intracellular soluble and insoluble fractions were subjected to SDS-PAGE. Figure 1 Shown: Compared with the empty pET28a, the supernatant of the sample with fusion protein has obvious target bands. Figure 1From left to right in the figure: control intracellular soluble, control intracellular insoluble, Marker, intracellular soluble sample linked to nanobody, intracellular insoluble sample linked to nanobody.

[0115] 2. Purification of Nanobodies

[0116] The above sample was centrifuged at 8000 rpm for 20 min and the supernatant was discarded. PBS was added to resuspend the cells and then ultrasonically disrupted. The supernatant was collected by centrifugation at 9000 rpm for 30 min. The supernatant was filtered through a 0.45 μm filter and then affinity chromatographed using Ni Purose 6 FF (Qianchun Bio, A46302). The flow-through, wash, and elution samples were collected and subjected to SDS-PAGE. Figure 2 It shows that a single target band of the correct size can be obtained by one-step purification. Figure 2 From left to right in the figure: intracellular soluble, flow-through, washing, elution and marker of the connected nanoantibody sample.

[0117] Example 3: Purification of AAV using nanobody-bonded fillers

[0118] 1. Binding of Nanobodies

[0119] Add the epoxy pre-activated filler into the bonding reactor and cross-link it with the purified nanoantibody to obtain a filler that can be used for affinity AAV. Other types of pre-activated fillers can also refer to the above method.

[0120] 2. AAV purification

[0121] The prepared affinity matrix was loaded into a 1 mL empty column. After equilibration with 5 CV of equilibration buffer, the AAV-containing sample was allowed to fully contact the affinity matrix. The equilibration buffer was then applied for an additional 10 CV to separate the unbound sample from the matrix. Finally, the bound AAV was eluted from the matrix using a pH 3.0 eluent. AAV is easily inactivated at low pH, so 1 / 10 volume of 0.2 MBTP was added to the collection tube for neutralization. The flow-through and eluate were collected separately during this process.

[0122] 3. Silver staining

[0123] The collected solution was subjected to SDS-PAGE. After the protein gel was removed, silver staining was performed using an external kit (Biyuntian, P0017S). The staining was completed through the following steps: "fixation-washing-water washing-sensitization-water washing-silver staining-water washing-color development-stopping". Figure 3 and Figure 4 It can be seen that there are obvious target bands in the eluate. Figure 3From left to right: Marker, sample containing AAV1, flowthrough, eluate, sample containing AAV2, flowthrough, eluate, sample containing AAV3, flowthrough, eluate, sample containing AAV4, flowthrough, eluate, sample containing AAV5, flowthrough, eluate. Figure 4 From left to right in the figure: Marker, sample containing AAV6, flowthrough, eluate, sample containing AAV7, flowthrough, eluate, sample containing AAV8, flowthrough, eluate, sample containing AAV9, flowthrough, eluate.

[0124] 4. AAV full capsid assay

[0125] The collected fluid was pre-treated (viral genome extraction) and then subjected to qPCR to obtain the corresponding Ct value, which was then used to calculate the initial template content. The results are shown in Table 1.

[0126] Table 1

[0127]

[0128] The above results show that the nanobodies in this application can be used as ligands with affinity for different serotypes of AAV for AAV purification research. It should be noted that the elution titers of the above serotypes are only a verification of the filler's adsorption effect on AAV and are not used as the maximum loading capacity of the filler.

[0129] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0130] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the patent in this application shall be determined by the appended claims, and the specification and drawings shall serve to interpret the claims.

Claims

1. Nanobodies or antigen-binding fragments thereof targeting different serotypes of adeno-associated viruses, characterized in that: The Nanobody or antigen-binding fragment thereof comprises a heavy chain variable region comprising three complementarity determining regions CDR1, CDR2 and CDR3; wherein the CDR1 comprises an amino acid fragment whose sequence is shown in SEQ ID NO: 1, the CDR2 comprises an amino acid fragment whose sequence is shown in SEQ ID NO: 2, and the CDR3 comprises an amino acid fragment whose sequence is shown in SEQ ID NO:

3.

2. The Nanobody or antigen-binding fragment thereof according to claim 1, wherein It includes an amino acid fragment whose sequence is shown in SEQ ID NO: 4; Further optionally, the Nanobody or antigen-binding fragment thereof also includes an Fc fragment.

3. A fusion protein, characterized in that It includes the Nanobody or antigen-binding fragment thereof as described in claim 1 or 2; Optionally, in the fusion protein, the N-terminus and / or C-terminus of the Nanobody or its antigen-binding fragment carries a purification tag and / or a fluorescent tag; the purification tag is optionally selected from the group consisting of 6×His, Flag, GST, Myc, MBP and Strep; the tag and the Nanobody or its antigen-binding fragment are operably connected, optionally via a linker.

4. A nucleic acid, characterized in that It comprises a nucleotide sequence encoding the Nanobody or antigen-binding fragment thereof as claimed in claim 1 or 2 or the fusion protein as claimed in claim 3.

5. A recombinant expression vector, characterized in that: It comprises the nucleic acid according to claim 4; Optionally, the backbone of the recombinant expression vector includes a replication origin, a selection marker, a multiple cloning site and corresponding expression regulatory elements; Optionally, the backbone of the recombinant expression vector is a plasmid, cosmid, phage or viral vector; the backbone of the plasmid is optionally pET28a.

6. A host cell, characterized in that It expresses the Nanobody or antigen-binding fragment thereof as described in claim 1 or 2 or the fusion protein as described in claim 3; Alternatively, it comprises one or more of the nucleic acid according to claim 4 and the recombinant expression vector according to claim 5; Optionally, the host cell is selected from the group consisting of prokaryotic cells and eukaryotic cells; the prokaryotic cell is optionally Escherichia coli or Bacillus subtilis, and the eukaryotic cell is optionally a yeast cell or a fungal cell; the fungal cell is optionally derived from the genus Mold or Trichoderma.

7. A method for preparing the Nanobody or antigen-binding fragment thereof according to claim 1 or 2 or the fusion protein according to claim 3, characterized in that: It includes: Cultivating the host cell according to claim 6 to obtain an intracellular soluble fraction; Purifying the intracellular soluble portion to obtain the nanobody or antigen-binding fragment thereof or the fusion protein; Optionally, the host cells are induced during the culture process.

8. A solid phase carrier, characterized in that Its surface is coupled with the Nanobody or antigen-binding fragment thereof as claimed in claim 1 or 2 or the fusion protein as claimed in claim 3; optionally, the coupling is performed through a spacer arm with an active group; Optionally, the solid phase carrier is selected from the group consisting of fillers, magnetic microspheres, polymer microspheres and separation membranes; the filler optionally includes agarose pre-activated filler.

9. A method for purifying adeno-associated virus, characterized in that: It includes the following steps: contacting the solid phase carrier according to claim 8 with a sample containing an adeno-associated virus; separating the solid phase carrier and the sample; Using a solution to elute the adeno-associated virus bound to the solid phase carrier to obtain an eluate; Wherein, the adeno-associated virus optionally includes one or more of serotype adeno-associated virus 1 to serotype adeno-associated virus 9; optionally, the adeno-associated virus is serotype adeno-associated virus 5; The pH of the solution is optionally 2.0-4.

0.

10. A kit, characterized in that It comprises one or more of the nanobody or antigen-binding fragment thereof as described in claim 1 or 2, the fusion protein as described in claim 3, and the solid phase carrier as described in claim 8.

11. A method for detecting adeno-associated virus or its content in a sample for non-diagnostic purposes, characterized in that: It includes: contacting the Nanobody or antigen-binding fragment thereof as described in claim 1 or 2 or the fusion protein as described in claim 3 with the sample to obtain an eluate containing adeno-associated virus; detecting the amount of adeno-associated virus in the eluate; Optionally, the method comprises obtaining an eluate using the method for purifying adeno-associated virus according to claim 9; Analyze the components in the eluate by gel electrophoresis; Silver stain the gel after electrophoresis; Optionally, the method comprises obtaining an eluate using the method for purifying adeno-associated virus according to claim 9; extracting the viral genome from the eluate and performing real-time fluorescent quantitative polymerase chain reaction to obtain the genome titer; Optionally, the method further comprises determining the content of full capsids by measuring the genome titer.

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