CN118085066B - AAV-resistant monoclonal antibody and application thereof - Google Patents
AAV-resistant monoclonal antibody and application thereof Download PDFInfo
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Abstract
The application provides an AAV-resistant monoclonal antibody and application thereof. The application also provides an ELISA kit containing the antibody and a detection method thereof. Specifically, the application provides antibodies against AAV RC-C07V5, and ELISA kits comprising the antibodies. The kit provided by the application can finish quantitative detection of AAV RC-C07V5, and has a wider linear range. The antibodies of the application are also useful as AAV RC-C07V5 virus neutralizing antibodies.
Description
Technical Field
The present invention relates to the field of biological detection. In particular, the present invention relates to an anti-AAV monoclonal antibody and uses thereof.
Background
Adeno-associated virus (AAV) is the major gene delivery platform for the treatment of a variety of human diseases. AAV belongs to the genus Paramyxoviridae, dependovirus, is non-enveloped, and consists of an icosahedral protein capsid of about 22nm diameter, which encapsulates the DNA genome, and a linear single-stranded DNA genome of about 4.7 kb. The genome consists of inverted terminal repeats (INVERTED TERMINAL REPEATS, ITR) on both sides and the middle Rep gene, which encodes four replication proteins, designated by their molecular weights: rep78, rep68, rep52 and Rep40, the Cap gene encodes three capsid proteins, VP1, VP2 and VP3, with different start codons, these three structural proteins having different amino-termini but sharing one carboxy-terminus, the three capsid proteins VP1, VP2 and VP3 being present as a whole at 1:1:10 to form an icosahedral viral capsid.
The total number of classes of currently registered AAV serotypes has exceeded 200, with the major difference between AAV serotypes being their capsid proteins, which also results in differences in the infectivity of different AAV's for different tissues and cell types. The Shanghai Langmuir Biotechnology Co.Ltd.obtains a series of AAV9 capsid variants with obviously improved properties based on AAV9 capsids through rational design (see CN 117247434A), wherein AAV RC-C07V5 capsids are obtained by replacing 15 functional amino acid (HQSAQAQAQTGWVQN, SEQ ID NO. 41) mutations of variable region VIII (584-598 aa) of AAV9 capsid VP1 with 25 specific amino acids (LQRGNLALGDVTRPARQAATADVNT, SEQ ID NO. 42), LALGDVTRPA (SEQ ID NO. 43) is a functional short peptide with retina tissues, and the variable region VIII region mutations weaken the binding capacity of AAV9 capsids to cell surface Glu (galactose), so that RC-C07V5 new capsids obtain more efficient inner limiting membrane penetration capacity (through intravitreal administration), can be distributed on the whole retina, especially photosensitive cells capable of reaching external membranous tissues, and stabilize continuously expressed supplementary proteins, and have the same capsid production capacity and stability as AAV 9.
After AAV RC-C07V5 is prepared, QC is needed to be carried out on viruses, wherein the particle number of the viruses in the product can be measured by quantifying capsids, and the ratio of VP/VG is an important index reflecting the real heart rate of the viruses. After in vivo administration, the number of viral particles in the targeted organs and tissues also needs to be detected, and the escape of viruses in the non-targeted organs and tissues is monitored. The ELISA method is an efficient AAV capsid detection method, however, commercial ELISA detection kits only can aim at a few wild AAV types such as AAV1, AAV2, AAV3, AAV5, AAV6, AAV8, AAV9, AAVrh10 and the like, and no ELISA detection kit and antibody suitable for AAV RC-C07V5 exist.
Therefore, there is a need in the art to develop a monoclonal antibody that specifically recognizes the spatial conformational epitope of AAV RC-C07V5 capsid protein and a kit that can quantitatively detect AAV RC-C07V5 solid and/or empty capsids using ELISA, thereby providing the possibility and convenience for AAV RC-C07V5 detection.
Disclosure of Invention
The invention aims to provide a detection method of AAV RC-C07V 5.
The invention provides an anti-AAV RC-C07V5 antibody.
The invention provides a detection kit containing an anti-AAV RC-C07V5 antibody and application thereof.
In a first aspect of the invention, there is provided an antibody against AAV RC-C07V5, said antibody comprising a heavy chain variable region and a light chain comprising a light chain variable region, wherein said antibody comprises complementarity determining region CDRs as set forth in (a), (b), (C) or (d):
(a) Defined according to the Kabat numbering system:
the amino acid sequence is shown as a light chain CDR1 shown in SEQ ID NO.3,
Light chain CDR2 with the amino acid sequence shown as SEQ ID NO.4,
Light chain CDR3 with the amino acid sequence shown in SEQ ID NO.5,
The amino acid sequence is shown as a heavy chain CDR1 shown in SEQ ID NO.6,
A heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO.7, and
A heavy chain CDR3 with an amino acid sequence shown as SEQ ID NO. 8;
(b) Defined according to IMGT numbering system:
the amino acid sequence is shown as a light chain CDR1 shown in SEQ ID NO.9,
Light chain CDR2 with amino acid sequence as shown in RAN,
Light chain CDR3 with the amino acid sequence shown in SEQ ID NO.5,
The amino acid sequence is shown as a heavy chain CDR1 shown in SEQ ID NO.10,
A heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO.11, and
A heavy chain CDR3 with an amino acid sequence shown as SEQ ID NO. 12;
(c) Defined according to the Chothia numbering system:
the amino acid sequence is shown as a light chain CDR1 shown in SEQ ID NO.3,
Light chain CDR2 with the amino acid sequence shown as SEQ ID NO.4,
Light chain CDR3 with the amino acid sequence shown in SEQ ID NO.5,
The amino acid sequence is shown as a heavy chain CDR1 shown in SEQ ID NO.13,
A heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO.14, and
A heavy chain CDR3 with an amino acid sequence shown as SEQ ID NO. 8; and
(D) Defined according to the Contact numbering system:
The amino acid sequence is shown as a light chain CDR1 shown in SEQ ID NO.15,
Light chain CDR2 with the amino acid sequence shown in SEQ ID NO.16,
Light chain CDR3 with the amino acid sequence shown in SEQ ID NO.17,
The amino acid sequence is shown as a heavy chain CDR1 shown in SEQ ID NO.18,
A heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO.19, and
The amino acid sequence is shown as heavy chain CDR3 of SEQ ID NO. 20.
In another preferred embodiment, the heavy chain variable region and the light chain variable region each further comprise a Framework Region (FR).
In another preferred embodiment, CDR1, CDR2 and CDR3 of the light or heavy chain variable region are separated by framework regions FR1, FR2, FR3 and FR4, respectively.
In another preferred embodiment, FR1, FR2, FR3 and FR4 of the light chain variable region have the amino acid sequences shown in SEQ ID nos. 23, 24, 25, 26, respectively; and/or the heavy chain variable regions FR1, FR2, FR3 and FR4 have the amino acid sequences shown in SEQ ID nos. 27, 28, 29, 30, respectively.
In another preferred embodiment, FR1, FR2, FR3 and FR4 of the light chain variable region have the amino acid sequences shown in SEQ ID nos. 31, 32, 33, 26, respectively; and/or the heavy chain variable regions FR1, FR2, FR3 and FR4 have the amino acid sequences shown in SEQ ID nos. 34, 35, 36, 30, respectively.
In another preferred embodiment, the heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO.39, or has a sequence identity of ≡85%,. Gtoreq.90%,. Gtoreq.95%,. Gtoreq.96%,. Gtoreq.97%,. Gtoreq.98% or 99% thereto.
In another preferred embodiment, the light chain variable region amino acid sequence of the antibody is shown as SEQ ID NO.40, or has a sequence identity of ≡85%,. Gtoreq.90%,. Gtoreq.95%,. Gtoreq.96%,. Gtoreq.97%,. Gtoreq.98% or 99% thereto.
In another preferred embodiment, the heavy chain variable region of the antibody has an amino acid sequence as shown in SEQ ID No. 39 and/or the light chain variable region has an amino acid sequence as shown in SEQ ID No. 40.
In another preferred embodiment, the light chain and/or heavy chain of the antibody further comprises a constant region.
In another preferred embodiment, the constant region is a human constant region.
In another preferred example, the heavy chain amino acid sequence of the antibody is shown as SEQ ID NO.1, or has more than or equal to 85%,. Gtoreq.90%,. Gtoreq.95%,. Gtoreq.96%,. Gtoreq.97%,. Gtoreq.98%, or more than or equal to 99% sequence identity thereto.
In another preferred example, the light chain amino acid sequence of the antibody is as shown in SEQ ID NO.2 or has more than or equal to 85%, more than or equal to 90%, more than or equal to 95%, more than or equal to 96%, more than or equal to 97%, more than or equal to 98% or more than or equal to 99% sequence identity with the light chain amino acid sequence.
In another preferred embodiment, the heavy chain of the antibody has the amino acid sequence shown in SEQ ID NO.1 and/or the light chain has the amino acid sequence shown in SEQ ID NO. 2.
In another preferred embodiment, any of the above amino acid sequences further comprises a derivative sequence, optionally with at least one amino acid added, deleted, modified and/or substituted, that is capable of retaining AAV RC-C07V5 binding affinity.
In another preferred embodiment, the antibody does not bind or does not substantially bind to wild-type AAV capsids.
In another preferred embodiment, the wild-type AAV is selected from the group consisting of: AAV2, AAV5, AAV6, AAV8, AAV9, or a combination thereof.
In another preferred embodiment, the number of amino acids added, deleted, modified and/or substituted does not exceed 30%, preferably 20%, more preferably 10% of the total number of amino acids of the original amino acid sequence.
In another preferred embodiment, the antibody is an animal-derived antibody, a chimeric antibody or a humanized antibody.
In another preferred embodiment, the antibody is a single chain antibody, a diabody, or an antigen-binding fragment.
In a second aspect of the present invention, there is provided a fusion protein comprising:
(1) An antibody according to the first aspect of the invention; and
(2) Optionally a tag sequence to assist expression and/or purification.
In another preferred embodiment, the tag comprises an Fc tag, a FLAG tag, a 6His tag, or a combination thereof.
In a third aspect of the invention there is provided a polynucleotide encoding an antibody according to the first aspect of the invention or a fusion protein according to the second aspect of the invention.
In another preferred embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:21.
In another preferred embodiment, the polynucleotide comprises the nucleotide sequence of SEQ ID NO:22.
In a fourth aspect of the invention there is provided a vector comprising a polynucleotide according to the third aspect of the invention.
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.
In a fifth aspect of the invention there is provided a genetically engineered host cell comprising a vector according to the fourth aspect of the invention or a polynucleotide according to the third aspect of the invention integrated into the genome.
In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell.
In another preferred embodiment, the host cell is selected from the group consisting of: coli, yeast cells, mammalian cells.
In a sixth aspect of the invention, there is provided an antibody conjugate comprising:
(a) An antibody moiety which is an antibody according to the first aspect of the invention; and
(B) A coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, a drug, or a combination thereof.
In another preferred embodiment, the antibody moiety is coupled to the coupling moiety by a chemical bond or linker.
In another preferred embodiment, the detectable label is selected from the group consisting of: fluorescent or luminescent labels, biotin, radioactive labels, MRI (magnetic resonance imaging) or CT (electronic computer tomography) contrast agents, or enzymes, gold nanoparticles/nanorods, nanomagnetic particles, or nanoparticles of any form capable of producing a detectable product.
In another preferred embodiment, the detectable label is biotin.
In another preferred embodiment, the agent is a small molecule drug, a biological agent, or a combination thereof.
In another preferred embodiment, the drug is an antiviral drug.
In a seventh aspect of the invention, there is provided the use of an antibody according to the first aspect of the invention or an antibody conjugate according to the sixth aspect of the invention in the preparation of a reagent or kit for detecting AAV RC-C07V 5.
In an eighth aspect of the invention there is provided the use of an antibody according to the first aspect of the invention, a polynucleotide according to the third aspect of the invention, a vector according to the fourth aspect of the invention, a host cell according to the fifth aspect of the invention or an antibody conjugate according to the sixth aspect of the invention in the preparation of an AAV RC-C07V5 virus neutralising formulation or medicament.
In another preferred embodiment, the neutralizing agent neutralizes the capsid epitope of AAV RC-C07V5 that binds to a target cell surface receptor.
In a ninth aspect of the invention, there is provided an AAV RC-C07V5 detection kit comprising an antibody according to the first aspect of the invention and/or an antibody conjugate according to the sixth aspect of the invention.
In another preferred embodiment, the kit is an ELISA kit.
In another preferred embodiment, the ELISA kit comprises a capture antibody and a detection antibody.
In another preferred embodiment, the capture antibody and the detection antibody are the antibodies of the first aspect of the invention.
In another preferred embodiment, the capture antibody is bound to a solid support.
In another preferred embodiment, the detection antibody carries a detectable label, preferably the detectable label is biotin.
In another preferred embodiment, the assay kit further comprises a component selected from the group consisting of: labeled (e.g., HRP-labeled) streptavidin, AAV RC-C07V5 standard, wash solution, sample dilution, chromogenic solution, stop solution, or a combination thereof.
In a tenth aspect of the invention, there is provided a method of detecting AAV RC-C07V5 in a sample using an antibody according to the first aspect of the invention, an antibody conjugate according to the sixth aspect of the invention, or a detection kit according to the ninth aspect of the invention.
In another preferred embodiment, the method further comprises the steps of: and detecting the AAV RC-C07V5 standard substance to obtain a standard curve, and calculating the AAV RC-C07V5 content in the tested sample according to the standard curve.
In another preferred embodiment, the sample to be tested is selected from the group consisting of: cell culture fluid, whole blood, serum, plasma, body fluids, or combinations thereof.
In an eleventh aspect of the invention, there is provided a formulation or pharmaceutical composition comprising:
(i) An antibody according to the first aspect of the invention, a polynucleotide according to the third aspect of the invention, a vector according to the fourth aspect of the invention, a host cell according to the fifth aspect of the invention, an antibody conjugate according to the sixth aspect of the invention, or a combination thereof; and
(Ii) A pharmaceutically acceptable carrier.
In another preferred embodiment, the formulation or pharmaceutical composition is in the form of an injection.
In another preferred embodiment, the formulation or pharmaceutical composition is used to neutralize a capsid epitope of AAV RC-C07V5 that binds to a target cell surface receptor.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
The following drawings are illustrative of particular embodiments of the invention and are not intended to limit the scope of the invention as defined by the claims.
FIG. 1 shows the results of specific detection of anti-RC-C07V 5 recombinant antibodies.
FIG. 2 shows that the anti-RC-C07V 5 recombinant antibodies bind to AAV RC-C07V5 without binding to denatured AAV RC-C07V5.
FIG. 3 shows the detection range of the double antibody sandwich ELISA kit.
FIG. 4 shows that high concentrations of anti-RC-C07V 5 recombinant antibodies were found to bind denatured AAV RC-C07V5 and AAV RC-C07V5 by Western Blot analysis.
FIG. 5 shows the neutralizing capacity of an anti-RC-C07V 5 recombinant antibody against AAV RC-C07V 5.
FIG. 6 shows the IC50 values for neutralizing AAV RC-C07V5 with anti-RC-C07V 5 recombinant antibodies.
Detailed Description
The present inventors have conducted extensive and intensive studies and have developed, for the first time, an anti-AAV monoclonal antibody and uses thereof. Specifically, the invention provides a monoclonal antibody specifically recognizing a novel antigen AAV RC-C07V5 capsid protein space conformational epitope and a kit capable of quantitatively detecting AAV RC-C07V5 solid and/or empty capsids by ELISA, thereby providing possibility and convenience for AAV RC-C07V5 detection. On this basis, the present invention has been completed.
Terminology
In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Before describing the present invention, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.
As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
As used herein, the terms "comprising," "including," and "containing" are used interchangeably, and include not only closed-form definitions, but also semi-closed-form and open-form definitions. In other words, the term includes "consisting of … …", "consisting essentially of … …".
As used herein, the term "pharmaceutically acceptable carrier" component refers to a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio.
"Sequence identity" as used herein refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate substitutions, insertions, or deletions of mutations. The sequence identity between the sequences described in the present invention and sequences with which it has identity may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.
Antibodies to
As used herein, the term "antibody" or "immunoglobulin" is an iso-tetralin protein of about 150000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H), having identical structural features. Each light chain is linked to the heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of 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 a plurality of constant regions. One end of each light chain is provided with a variable region (VL) and the other end is provided with a constant region; the constant region of the light chain is opposite the first constant region of the heavy chain and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.
As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the light and heavy chain variable regions called Complementarity Determining Regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are in a substantially b-folded configuration, joined by three CDRs forming a linker loop, which in some cases may form part of a b-folded structure. The CDRs in each chain are held closely together by 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 public No. 91-3242, vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.
The "light chain" of a vertebrate antibody (immunoglobulin) can be classified into one of two distinct classes (called k and l) according to the amino acid sequence of its constant region. Immunoglobulins can be assigned to different classes based on the amino acid sequence of their heavy chain constant region. There are mainly 5 classes of immunoglobulins: igA, igD, igE, igG and IgM, some of which can be further divided into subclasses (isotypes) such as IgG1, igG2, igG3, igG4, igA and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are designated a, d, e, g, and m, respectively. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (typically having different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma culture and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring any particular method for producing the antibody.
In general, the antigen binding properties of antibodies can be described by 3 specific regions located in the heavy and light chain variable regions, called variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, the β -sheets formed by the FR therebetween are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of the same type of antibody.
The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. Fragments of full length antibodies have been shown to be useful for performing the antigen binding function of antibodies. Examples of binding fragments included in the term "antigen-binding fragment of an antibody" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) A F (ab') 2 fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bridge over the longer chain region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VH and VL domains of a single arm of an antibody. Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and have a minimal antibody fragment of the entire antigen binding site. Generally, fv antibodies also comprise a polypeptide linker between the VH and VL domains, and are capable of forming the structures required for antigen binding.
The invention includes not only intact monoclonal antibodies but also immunologically active antibody fragments or fusion proteins of antibodies with other sequences, such as Fab or (Fab') 2 fragments; antibody heavy chain; an antibody light chain. Thus, the invention also includes fragments, derivatives and analogues of said antibodies.
The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes generally comprise at least 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 contiguous or non-contiguous amino acids in a unique spatial conformation. Epitopes may be discrete on an antigen, three-dimensional spatial sites recognized by an antibody or antigen binding fragment of the invention.
The terms "specific binding," "selective binding," "selectively binding," and "specifically binding" refer to binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody binds with an affinity (KD) of about less than 10 -7 M, e.g., about less than 10 -8M、10-9 M or l0 -10 M or less.
In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be obtained by standard DNA recombination techniques, all of which are useful antibodies. Chimeric antibodies are a molecule in which different portions are derived from different animal species, e.g., chimeric antibodies having variable regions from murine monoclonal antibodies, and constant regions from human immunoglobulins (see, e.g., U.S. Pat. No. 4,816,567 and U.S. Pat. No. 4,816,397, incorporated herein by reference in their entirety). Humanized antibodies refer to antibody molecules derived from non-human species having one or more Complementarity Determining Regions (CDRs) derived from the non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Pat. No. 5,585,089, incorporated herein by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombination techniques well known in the art.
In the present invention, antibodies may be monospecific, bispecific, trispecific, or more multispecific.
As used herein, the term "heavy chain variable region" is used interchangeably with "VH".
As used herein, the term "light chain variable region" is used interchangeably with "VL".
As used herein, the term "complementarity determining regions (complementarity determining region, CDRs)" refers to hypervariable regions within the variable domains of an antibody that contribute primarily to antigen binding. One of the most commonly used definitions of the CDRs is provided by Kabat E.A et al, (1991) Sequences of proteins of immunological interface. Furthermore, IMGT (Lefranc, 2003), chothia (Al-Lazikani, 1997), etc. all provide CDR definition rules, which are well known to those skilled in the art.
In a preferred embodiment of the invention, the heavy chain of the antibody comprises a heavy chain variable region and a heavy chain constant region, which may be murine or human in origin.
In a preferred embodiment of the invention, the light chain of the antibody comprises a light chain variable region and a light chain constant region, which may be murine or human in origin.
In the present invention, the antibodies of the invention also include conservative variants thereof, meaning that up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are replaced by amino acids of similar or similar nature to the amino acid sequence of the antibodies of the invention to form a polypeptide. These conservatively mutated polypeptides are preferably produced by amino acid substitution according to Table 1.
TABLE 1
Anti-AAV RC-C07V5 antibodies
As used herein, the terms "antibody of the invention", and "AAV RC-C07V5 antibody of the invention" are used interchangeably, and each refer to an antibody targeting AAV RC-C07V5 as described in the first aspect of the invention.
The term "AAV (Adeno-associated virus)", i.e. Adeno-associated virus, belongs to the family picoviridae. The diameter is about 20-25nM, the genome is single-stranded DNA, the length is about 4.7kb, and the genome is composed of two ITR (inverted terminal repeat) ends.
The term "AAV RC-C07V5" refers to the AAV RC-C07V5 capsid disclosed in CN 117247434A. The AAV RC-C07V5 capsid is an engineered AAV capsid obtained by replacing the 15 functional amino acid (HQSAQAQAQTGWVQN, SEQ ID No. 41) mutation of the variable region VIII (584-598 aa) of the AAV9 capsid VP1 with 25 specific amino acids (LQRGNLALGDVTRPARQAATADVNT, SEQ ID No. 42).
The function of the antibodies of the invention is determined by the antibody light and heavy chain variable region gene-specific gene sequences. The antibody of the invention can specifically bind to AAV RC-C07V5, has high affinity and high specificity, does not bind to AAV2, AAV5, AAV6 and AAV8, and has weak binding activity to AAV 9. The AAV RC-C07V5 antibody of the present invention can also be used as a neutralizing antibody for neutralizing the capsid epitope of AAV RC-C07V5 binding to a target cell surface receptor.
The AAV RC-C07V5 antibodies of the invention may be intact immunoglobulins, antigen binding fragments thereof, or antigen binding molecules. Antibodies of the invention may be of any isotype or class (e.g., igM, igD, igG, igE and IgA) or any subclass (e.g., igGl-4, igAl-2) or any type of subclass (e.g., igG2a, igG2 b), and may have a kappa or lambda light chain. Antigen binding fragments of antibodies include, for example, fab, fv, scFv and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies (scabs), single domain antibodies (dabs), single domain heavy chain antibodies, single domain light chain antibodies, bispecific antibodies, multispecific antibodies, and fusion proteins comprising the antigen-binding portion of an antibody and a non-antibody protein.
Polynucleotide
The present invention also provides a polynucleotide encoding the above-described antibody of the present invention or a fusion protein comprising the antibody of the present invention.
The preparation method of the polynucleotide is a preparation method conventional in the art, and preferably comprises the following steps: the nucleic acid molecules encoding the above proteins are obtained by gene cloning techniques or by artificial total sequence synthesis.
It is known to those skilled in the art that a nucleotide sequence encoding the amino acid sequence of the above protein may be appropriately introduced into a substitution, deletion, alteration, insertion or addition to provide a homolog of a polynucleotide. Homologs of the polynucleotides of the invention may be obtained by substitution, deletion or addition of one or more bases of the gene encoding the protein sequence within a range that retains antibody activity.
Carrier body
The invention also provides a recombinant expression vector comprising the polynucleotide.
Wherein said recombinant expression vector is obtainable by methods conventional in the art, namely: the polynucleotide molecule is constructed by connecting the polynucleotide molecule to various expression vectors. The expression vector is a variety of vectors conventional in the art, as long as it is capable of harboring the aforementioned polynucleotide molecule. The carrier preferably comprises: various plasmids, cosmids, phage or viral vectors, and the like.
The invention also provides a recombinant expression transformant containing the recombinant expression vector.
Wherein, the preparation method of the recombinant expression transformant is a preparation method conventional in the field, preferably: the recombinant expression vector is transformed into a host cell. The host cell is a variety of host cells conventional in the art, so long as the recombinant expression vector can stably replicate itself and the polynucleotide carried can be expressed effectively. Preferably, the host cell is an E.coli TG1 or E.coli BL21 cell (expressing a single chain antibody or Fab antibody), or HEK293 or CHO cell (expressing a full length IgG antibody). The recombinant expression plasmid is transformed into a host cell, so that the preferred recombinant expression transformant of the invention can be obtained. Wherein the conversion process is conventional in the art, preferably chemical, heat shock or electrotransformation.
Preparation of antibodies
The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique such as amplification by PCR or screening of a genomic library. In addition, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.
Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.
At present, it is already possible to obtain the DNA sequences encoding the antibodies of the invention (or fragments or derivatives thereof) described, entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.
The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein.
The host cell may be a prokaryotic cell; or lower eukaryotic cells; or higher eukaryotic cells, such as mammalian cells.
Typically, the transformed host cell is cultured under conditions suitable for expression of the antibodies of the invention. The antibodies of the invention are then purified by conventional isolation and purification means well known to those skilled in the art.
The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined using immunoprecipitation or in vitro binding assays, such as Radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA). The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis by Munson et al, anal biochem.,107:220 (1980).
The antibodies of the invention may be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art.
Detection application and kit
The invention also provides the use of the antibodies, fusion proteins and/or antibody conjugates of the invention, e.g. for the preparation of diagnostic formulations or for the preparation of medicaments.
The antibodies of the invention are useful in detection applications, e.g., for quality control of AAV RC-C07V5 products.
The invention provides a kit for quantitatively detecting AAV RC-C07V5 solid and/or empty capsids by ELISA, wherein the kit is coated and detected by the antibody. The anti-AAV RC-C07V5 antibody has weak binding activity with linear epitopes of AAV9 and RC-C07V5, so that the antibody can be also used for WB detection of AAV9 and RC-C07V 5. In a preferred embodiment of the invention, the kit further comprises a container, instructions for use, buffers, etc.
When used in detection applications, the antibodies of the invention may be labeled with, for example, a radioactive, enzymatic or fluorescent group. The antibodies of the invention may also be conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (a member of a biotin-avidin specific binding pair). The antibodies of the invention may also be bound to a solid support, such as polystyrene plates or beads, etc.
Pharmaceutical composition
The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising an antibody or active fragment thereof or fusion protein thereof or corresponding immune cell as described above, and a pharmaceutically acceptable carrier. The pharmaceutical compositions of the invention can be used to neutralize capsid epitopes of AAV RC-C07V5 binding to target cell surface receptors.
The pharmaceutical composition of the present invention contains a safe and effective amount of the monoclonal antibody of the present invention as described above and a pharmaceutically acceptable carrier or excipient. The pharmaceutical formulation should be compatible with the mode of administration. The amount of active ingredient administered is a therapeutically effective amount.
In the present invention, the pharmaceutical composition of the present invention preferably further comprises one or more pharmaceutically acceptable carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the field, and can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipients. The pharmaceutical excipients are conventional pharmaceutical excipients in the field, and preferably comprise pharmaceutically acceptable excipients, fillers or diluents and the like.
In the present invention, the pharmaceutical composition is preferably administered in an amount effective to reduce or delay the progression of the disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be based in part on the symptoms to be treated and the consideration of the results sought. The skilled artisan can determine the effective amount by using the factors described above on an individual basis and the like and using no more than routine experimentation.
The main advantages of the invention include:
1) The invention provides antibodies specifically targeting AAV RC-C07V5, which possess affinity and specificity. The antibody of the invention has good ELISA detection sensitivity for AAV RC-C07V5, has wider linear range and meets the use requirement.
2) The antibody of the present invention can neutralize a capsid epitope bound to a target cell surface receptor of AAV RC-C07V5, and can be used as a neutralizing antibody for AAV RC-C07V 5.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.
Example 1 AAV9 antibody preparation and ELISA kit construction
1. AAV RC-C07V5 packaging
The rep-cap plasmid RC-C07V5, the GOI plasmid GOI-E04V8 (GOI-E04V 8 is the GOI plasmid with EGFP gene deleted in the complete expression frame, EGFP is deleted to avoid the influence of foreign protein when immunizing mice) and phelper are taken, and AAV packaging is performed by referring to the conventional three-plasmid packaging method.
2. AAV purification
Affinity chromatography was performed using POROS ™ Captureselect ™ AAVX AFFINITY RESIN (thermo, A36741), and detection of viral genome vg was performed using 100kD super-isolation tube (MILLIPORE, UFC 910096) with buffer replaced with PBS, qPCR and ddPCR.
3. AAV animal immunization
C57BL/6 mice were immunized by muscle immunization with about 10 11 vg of RC-C07V5 virus. Subsequently, the immunization was repeated every 2to 3 weeks, thereby boosting the experimental mice 3 times. When the serum titer of the mice reaches more than 10 5, the spleens of the mice are taken for subsequent antibody discovery work.
4. Acquisition of AAV antibody hybridoma cell strain and preparation of monoclonal antibody
A mouse spleen single cell suspension and a myeloma cell (SP 2/0) single cell suspension were prepared, respectively, and hybridoma cells were prepared by fusing spleen cells with SP2/0 mouse myeloma cells using electrofusion. After 7 days of incubation, the presence of anti-RC-C07V 5 antibodies was tested using an indirect ELISA method. Subcloning was performed using limiting dilution, and monoclonal cells were selected while the supernatant was subjected to the ELISA binding described above, and the results showed that monoclonal antibody LX-mAb01 reacted only with RC-C07V5, but not with AAV9, indicating that the monoclonal antibody had good specificity.
Indirect ELISA specific method: 100ul of 7E+08 vg/ml AAV9 and RC-C07V5 were coated on microplates, respectively, overnight at 4℃and the coating was removed, plates were washed with PBST (PBS containing 0.05% Tween 20), and 150ul of PBST containing 2% BSA was added to each well after drying, and blocked at 37℃for 1 hour. The blocking solution was discarded, the plate was washed once with PBST after the beating, 100ul of hybridoma cell culture supernatant was added to each well after the beating, and then incubated at 37 ℃ for 1 hour. The supernatant was discarded, the plate was washed three times with PBST, and 100ul of PBS 1 was added to each well after drying: 1000 dilution of HRP (horseradish peroxidase) -labeled goat anti-mouse (bi yun day, a 0216) was incubated at 37 ℃ for 1 hour. The plates were washed five times with PBST, after which 50ul of TMB color development was added and incubated in the dark for 10 minutes at room temperature. Finally, 50ul of 1M HCl stop solution (Guozhen, 10011018) was added to stop the reaction. Plates were read at 450nm using an enzyme-labeled instrument and the positive wells with the highest OD values were selected for subsequent experiments.
5. Variable region sequencing of monoclonal antibodies and monoclonal antibody production
After expansion of the above LX-mAb01 cell culture, total RNA was extracted from about 1×10 6 hybridoma cells using TRIzol (Life Technology, 15596-026) and reverse transcribed into cDNA using antibody subtype specific primers and universal primers (PRIME SCRIPTTM 1stStrand cDNA Synthesis Kit,Takara), followed by amplification of murine immunoglobulin heavy and light chain V-region fragments by RACE PCR (GenScript) and subcloning of the resulting PCR fragments into the pEASY-Blunt Cloning Kit vector system (full gold, CB 101-02) and sequencing of the insert using vector specific primers. Finally, the unique V-region protein amino acid sequence is obtained: KC225 heavy chain variable region amino acid sequence (SEQ ID NO. 39) and KC224 light chain variable region amino acid sequence (SEQ ID NO. 40). The sequence is added with signal peptide genes to synthesize the gene sequences of the heavy chain and light chain variable regions and the constant regions of the antibody, and the gene sequences are constructed into a mammalian cell expression vector. Recombinant plasmids were used to transfect the Expi293 mammalian cells to secrete expressed antibodies. And (3) carrying out affinity purification on cell expression supernatant by protein A to obtain the RC-C07V5 specific recombinant antibody.
6. ELISA detection of monoclonal antibodies
100Ul 7E+08 vg/ml AAV2/AAV5/AAV6/AAV8/AAV 9/RC-C07V 5 were coated on microplates, respectively, and the specificity of the anti-RC-C07V 5 antibody was again tested using the indirect ELISA method described above. The results are shown in FIG. 1. It can be seen that the anti-RC-C07V 5 antibody specifically binds only RC-C07V5 and does not react with AAV2/AAV5/AAV6/AAV8/AAV 9.
The results are shown in FIG. 1, where the anti-RC-C07V 5 antibody has good specificity for RC-C07V 5.
To test whether the anti-RC-C07V 5 antibody binds to denatured RC-C07V5, 100ul 7E+08 vg/ml of RC-C07V5 was denatured at 95℃for 5min and then naturally cooled to room temperature, the specificity of the anti-RC-C07V 5 antibody was also tested by the indirect ELISA method described above, and the control was undenatured RC-C07V5. The results are shown in FIG. 2, where the anti-RC-C07V 5 antibody binds only RC-C07V5, and not to denatured RC-C07V5.
From the above results, it was found that the anti-RC-C07V 5 antibody specifically binds only RC-C07V5 having the epitope in the complete spatial conformation.
7. Preparation of double antibody sandwich ELISA kit
7.1 Principle of: the capture antibody is pre-coated in a 96-well reaction plate and packaged to ensure the activity. After adding standard substances or samples to be tested with different dilution concentrations, the capsids in the standard substances or samples can be specifically bound to the capture antibodies of the reaction plate due to the special space conformational epitopes, then the detection antibodies and the HRP labels are added in steps, and finally the antibody-antigen-detection antibody-HRP label compound is formed, and the superfluous detection antibodies and the HRP labels are removed through washing operation. After adding the chromogenic solution, HRP catalyzes the color development, and the color development intensity is in direct proportion to the capsid titer of RC-C07V5 in the sample. After terminating the reaction by using a termination solution, reading an absorption value at a wavelength of 450 nm on an enzyme-labeled instrument, preparing a standard curve by using the capsid concentration of the standard substance and the corresponding OD450 value, and calculating the titer of the RC-C07V5 capsid in the sample to be detected by using the standard curve.
Configuration of ELISA kit components:
standard substance: the solid rate of AAV RC-C07V5 was measured by AUC, then the total number of virus particles vp=vg/real heart rate was obtained;
capture antibody: anti-RC-C07V 5 antibody, PBST diluted to 5ug/ml;
Detection of antibodies: biotin (Biotin) -labeled anti-RC-C07V 5 antibody, PBST was diluted to 0.1ug-0.3ug/ml;
HRP label: HRP-labeled strepavidin (streptavidin), PBST diluted to 0.1ug/ml;
Color development liquid: TMB;
stop solution: 1M HCl.
7.2 Detection range and sensitivity analysis:
100ul 5ug/ml capture antibody was coated on microplates, respectively, overnight at 4℃and the coating was discarded, plates were washed with 300ul PBST, and 150ul of 2% BSA in PBST was added to each well after drying and blocked at 37℃for 1 hour. The blocking solution was discarded, washed 3 times with 300ul of PBST, and after drying by pipetting 100ul of standard virus diluted 3-fold from 1E+11 vp/ml was added and incubated for 1 hour at 37 ℃. The supernatant was discarded, the plate was washed 3 times with 300ul of PBST, 100ul of detection antibody was added to each well after pipetting dry, and incubation was performed for 1 hour at 37 ℃. The supernatant was discarded, the plate was washed 3 times with 300ul of PBST, 100ul of HRP-tag was added to each well after pipetting dry and incubated for 1 hour at 37 ℃. Plates were washed 4 times with 300ul of PBST, after which 50ul of TMB chromogenic solution was added and incubated at room temperature in the dark for 10 minutes. Finally, 50ul of 1M HCl stop solution (Guozhen, 10011018) was added to stop the reaction. Plates were read at 450nm using an microplate reader. The results are shown in FIG. 3. As can be seen from the results of FIG. 3, the kit exhibited a good linear detection range from 4.3E+07vp/ml to 2.75E+09 vp/ml.
EXAMPLE 2 detection of AAV RC-C07V5 by the anti-RC-C07V 5 antibody WB
Mu.L of 2E+09vg AAV9 and RC-C07V5 virus were placed in a 1.5mL EP tube, 5. Mu.L of 4 Xsample buffer (Thermo, NP 0007) was added, and heated in a metal bath at 95℃for 10min.12000rpm, centrifugation for 10min, SDS-PAGE of 10ul supernatant, transfer, and blocking with 5% mill-PBST at room temperature for 60min after transfer. The blocking solution was decanted, primary antibody diluted with 5% mill-PBST was added and incubated overnight at 4 ℃. The antibody was decanted, washed three times with PBST, the wash was decanted, 10mL of secondary antibody diluted with 5% milk-PBST was added, and incubated for 60min at room temperature. The secondary antibody was decanted, washed three times with PBST, and developed and photographed.
The results are shown in FIG. 4. As can be seen from the results of FIG. 4, the anti-RC-C07V 5 antibody bound to both AAV9 and RC-C07V5 at a concentration of 8. Mu.g/mL, but bound to RC-C07V5 much more strongly than AAV9.
EXAMPLE 3 determination of neutralizing Activity of anti-RC-C07V 5 antibodies
1. And (3) paving: 96-well plate HEK-293T cells are plated, counted by a cell counter, 5E3 cells are plated in each well, and virus infection is carried out when the cell confluence reaches 30% -40%.
2. Virus dilution: counting the number of single Kong Xibao before infection, calculating the virus volume required by each well according to the formula V (μl) =moi cell number/virus titer 1000, and diluting the virus with DMEM complete medium as diluent; dilution of anti-RC-C07V 5 antibody: antibody dilution was performed by 4-fold gradient dilution method using DMEM complete medium as diluent, resulting in dilutions of 1 (antibody stock solution), 1: 4. 1: 16. 1: 64. 1: 256. 1: 1024. 1:4096 and 0 (DMEM complete medium); positive control antibody (intravenous human immunoglobulin PH4, shandongtaibang, concentration 5%) was diluted: similarly, the dilution of the antibody was performed by 4-fold gradient dilution method using DMEM complete medium as a diluent, to obtain dilutions of 1 (stock antibody solution), 1: 4. 1: 16. 1: 64. 1: 256. 1: 1024. 1:4096 and 0 (DMEM complete medium).
3. Viral and antibody co-incubation: antibodies and viruses were mixed in a volume ratio of 1:1 were mixed and incubated overnight in a 37℃incubator.
4. Viral infection: the actual MOI was calculated and recorded by counting single Kong Xibao prior to infection, adding 100 μl of virus and antibody mixture to each well, 2 replicate wells per gradient for each sample, and incubating the well plates in a 37℃carbon dioxide incubator after infection.
5. Streaming detection (LSRFortessa ™ X-20, BD): 72h after infection, cell supernatants were removed, cells were washed once with PBS, pancreatin was fully digested, DMEM containing 10% FBS was added to stop the reaction, followed by flow-through detection, and the output results were analyzed for percentage of fluorescent cells by FlowJo software.
6. As shown in fig. 5 and 6, the anti-RC-C07V 5 antibody was effective in neutralizing RC-C07V5 virus, with ic50=16427, which was stronger than the neutralizing effect of human immunoglobulin on RC-C07V 5. The anti-RC-C07V 5 antibody was unable to neutralize AAV9.
Antibody heavy chain (SEQ ID NO. 1):
MGWSWIFLFFLSGTAGVLSEVLLQQSGPELVKPGASVKIPCKASGYSFTDYNMDWVKQSHGKSLEWIGDINPNNGDTFYNQKFKGKATLTVDKSSSTAYMELRSLTSEDTAVYYCARWDSRYDGSMDYWGQGTSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK
antibody light chain (SEQ ID No. 2):
MRTPAQFLGILLLGFPGIKCDIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGGGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC
CDR and FR amino acid sequences defined according to the Kabat numbering system:
CDR-L1: KASQDINSYLS (SEQ ID NO.3)
CDR-L2: RANRLVD (SEQ ID NO.4)
CDR-L3: LQYDEFPLT (SEQ ID NO.5)
CDR-H1: DYNMD (SEQ ID NO.6)
CDR-H2: DINPNNGDTFYNQKFKG (SEQ ID NO.7)
CDR-H3: WDSRYDGSMDY (SEQ ID NO.8)
FR-L1: DIKMTQSPSSMYASLGERVTITC (SEQ ID NO.23)
FR-L2: WFQQKPGKSPKTLIY (SEQ ID NO.24)
FR-L3: GVPSRFSGSGSGQDYSLTISSLEYEDMGIYYC (SEQ ID NO.25)
FR-L4: FGGGTKLELK (SEQ ID NO.26)
FR-H1: EVLLQQSGPELVKPGASVKIPCKASGYSFT (SEQ ID NO.27)
FR-H2: WVKQSHGKSLEWIG (SEQ ID NO.28)
FR-H3: KATLTVDKSSSTAYMELRSLTSEDTAVYYCAR (SEQ ID NO.29)
FR-H4: WGQGTSVTVSS (SEQ ID NO.30)
CDR and FR amino acid sequences defined according to IMGT numbering system:
CDR-L1: QDINSY (SEQ ID NO.9)
CDR-L2: RAN
CDR-L3: LQYDEFPLT (SEQ ID NO.5)
CDR-H1: GYSFTDYN (SEQ ID NO.10)
CDR-H2: INPNNGDT (SEQ ID NO.11)
CDR-H3: ARWDSRYDGSMDY (SEQ ID NO.12)
FR-L1: DIKMTQSPSSMYASLGERVTITCKAS (SEQ ID NO.31)
FR-L2: LSWFQQKPGKSPKTLIY (SEQ ID NO.32)
FR-L3: RLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYC (SEQ ID NO.33)
FR-L4: FGGGTKLELK (SEQ ID NO.26)
FR-H1: EVLLQQSGPELVKPGASVKIPCKAS (SEQ ID NO.34)
FR-H2: MDWVKQSHGKSLEWIGD (SEQ ID NO.35)
FR-H3: FYNQKFKGKATLTVDKSSSTAYMELRSLTSEDTAVYYC (SEQ ID NO.36)
FR-H4: WGQGTSVTVSS (SEQ ID NO.30)
CDR amino acid sequence defined according to Chothia numbering system:
CDR-L1: KASQDINSYLS (SEQ ID NO.3)
CDR-L2: RANRLVD (SEQ ID NO.4)
CDR-L3: LQYDEFPLT (SEQ ID NO.5)
CDR-H1: GYSFTDY (SEQ ID NO.13)
CDR-H2: NPNNGD (SEQ ID NO.14)
CDR-H3: WDSRYDGSMDY (SEQ ID NO.8)
CDR amino acid sequences defined according to the Contact numbering system
CDR-L1: NSYLSWF (SEQ ID NO.15)
CDR-L2: TLIYRANRLV (SEQ ID NO.16)
CDR-L3: LQYDEFPL (SEQ ID NO.17)
CDR-H1: TDYNMD (SEQ ID NO.18)
CDR-H2: WIGDINPNNGDTF (SEQ ID NO.19)
CDR-H3: ARWDSRYDGSMD (SEQ ID NO.20)
Heavy chain nucleotide sequence (SEQ ID NO. 21) of AAV monoclonal antibody
ATGGGGTGGTCTTGGATATTTTTGTTTTTCCTTAGTGGCACAGCGGGTGTTCTTTCTGAAGTGCTCTTGCAACAGTCAGGTCCAGAACTTGTGAAACCAGGAGCCAGCGTTAAAATCCCTTGTAAGGCCAGCGGGTACTCATTCACCGACTATAACATGGACTGGGTCAAACAAAGCCACGGTAAATCACTGGAGTGGATTGGCGACATCAATCCGAACAACGGCGACACTTTTTACAACCAGAAATTCAAGGGGAAAGCTACACTGACCGTGGACAAGTCTTCAAGTACTGCGTACATGGAGCTGCGCAGTCTCACAAGCGAGGACACAGCCGTGTATTATTGTGCAAGATGGGACTCCCGATACGACGGCTCCATGGATTACTGGGGACAGGGAACCTCCGTGACCGTTAGCTCAGCAAAGACAACCGCTCCGAGTGTCTACCCATTGGCACCTGTTTGTGGTGACACAACTGGCAGCAGCGTAACACTCGGTTGTTTGGTGAAAGGCTACTTCCCGGAGCCAGTGACCCTTACCTGGAACAGCGGAAGCCTGTCAAGTGGAGTCCACACCTTCCCAGCAGTTCTCCAATCTGACCTTTACACTCTTTCCAGCTCTGTGACCGTAACTAGCAGCACATGGCCATCCCAATCCATCACATGTAATGTTGCTCATCCGGCTTCTTCTACCAAGGTGGACAAGAAGATAGAACCCCGCGGGCCTACTATTAAGCCTTGTCCACCGTGCAAGTGCCCCGCACCTAACCTCTTGGGTGGCCCCTCCGTGTTCATCTTTCCTCCCAAGATCAAGGATGTGCTGATGATCAGCCTTAGCCCTATCGTGACATGCGTGGTGGTGGACGTGTCCGAGGACGACCCCGACGTGCAGATTTCTTGGTTCGTGAACAACGTGGAGGTGCACACCGCTCAGACCCAGACCCACAGGGAGGACTACAACTCTACACTGCGGGTGGTGTCCGCCCTGCCTATTCAGCACCAGGACTGGATGTCCGGAAAGGAGTTCAAGTGCAAGGTGAACAACAAAGACCTGCCCGCACCAATAGAGAGAACCATCAGCAAACCCAAAGGCTCTGTCAGGGCTCCTCAGGTGTATGTCTTGCCACCACCAGAGGAAGAAATGACAAAAAAACAGGTCACTCTTACATGTATGGTGACAGATTTCATGCCCGAGGATATATACGTGGAGTGGACCAATAACGGGAAGACTGAACTCAACTACAAAAATACTGAGCCAGTTCTGGATAGTGATGGCTCCTACTTTATGTACAGTAAGTTGCGAGTGGAGAAAAAGAACTGGGTTGAACGCAACAGCTACAGTTGCAGTGTCGTGCATGAAGGACTGCACAATCACCATACAACCAAGAGTTTTTCACGAACGCCAGGTAAAtaa
Anti-AAV monoclonal antibody light chain nucleotide sequence (SEQ ID NO. 22)
ATGCGAACCCCTGCTCAGTTCCTTGGTATCCTTCTTCTCGGATTTCCAGGGATCAAGTGTGACATTAAGATGACCCAGTCCCCCAGCTCTATGTATGCTAGCCTTGGCGAGAGGGTCACAATTACCTGTAAAGCCTCCCAGGATATTAACTCCTATCTGAGCTGGTTTCAACAGAAACCTGGCAAATCCCCTAAGACGTTGATCTACAGGGCTAACAGACTCGTTGACGGAGTTCCTAGCAGGTTTTCTGGGAGTGGAAGTGGCCAAGATTACAGTCTTACAATCTCCTCCTTGGAATATGAAGACATGGGCATCTATTACTGCTTGCAGTATGATGAATTTCCACTGACATTCGGTGGGGGAACCAAATTGGAATTGAAAAGGGCCGATGCCGCTCCTACAGTGAGCATCTTTCCTCCTTCCTCCGAGCAGCTGACAAGCGGCGGCGCCAGCGTGGTGTGTTTCCTGAACAACTTCTATCCTAAGGACATCAATGTGAAGTGGAAGATCGACGGCAGCGAGAGACAGAACGGCGTGCTGAACTCCTGGACCGACCAGGATTCCAAGGACTCCACCTACTCCATGTCCTCCACACTGACCCTGACCAAGGATGAGTACGAGAGGCACAACAGCTACACATGCGAGGCCACACACAAGACCTCCACCAGCCCTATCGTGAAGAGCTTCAATAGAAACGAGTGCtaa
Light chain signal peptide sequence: MRTPAQFLGILLLGFPGIKC (SEQ ID NO. 37)
Heavy chain signal peptide sequence: MGWSWIFLFFLSGTAGVLS (SEQ ID NO. 38)
Antibody heavy chain variable region (SEQ ID NO. 39):
EVLLQQSGPELVKPGASVKIPCKASGYSFTDYNMDWVKQSHGKSLEWIGDINPNNGDTFYNQKFKGKATLTVDKSSSTAYMELRSLTSEDTAVYYCARWDSRYDGSMDYWGQGTSVTVSS
Antibody light chain variable region (SEQ ID NO. 40):
DIKMTQSPSSMYASLGERVTITCKASQDINSYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLT。
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
Claims (12)
1. An antibody against AAV RC-C07V5, wherein said antibody comprises a heavy chain comprising a heavy chain variable region and a light chain comprising a light chain variable region, wherein said antibody comprises complementarity determining region CDRs as set forth in (a), (b), (C), or (d):
(a) Defined according to the Kabat numbering system:
the amino acid sequence is shown as a light chain CDR1 shown in SEQ ID NO.3,
Light chain CDR2 with the amino acid sequence shown as SEQ ID NO.4,
Light chain CDR3 with the amino acid sequence shown in SEQ ID NO.5,
The amino acid sequence is shown as a heavy chain CDR1 shown in SEQ ID NO.6,
A heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO.7, and
A heavy chain CDR3 with an amino acid sequence shown as SEQ ID NO. 8;
(b) Defined according to IMGT numbering system:
the amino acid sequence is shown as a light chain CDR1 shown in SEQ ID NO.9,
Light chain CDR2 with amino acid sequence as shown in RAN,
Light chain CDR3 with the amino acid sequence shown in SEQ ID NO.5,
The amino acid sequence is shown as a heavy chain CDR1 shown in SEQ ID NO.10,
A heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO.11, and
A heavy chain CDR3 with an amino acid sequence shown as SEQ ID NO. 12;
(c) Defined according to the Chothia numbering system:
the amino acid sequence is shown as a light chain CDR1 shown in SEQ ID NO.3,
Light chain CDR2 with the amino acid sequence shown as SEQ ID NO.4,
Light chain CDR3 with the amino acid sequence shown in SEQ ID NO.5,
The amino acid sequence is shown as a heavy chain CDR1 shown in SEQ ID NO.13,
A heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO.14, and
A heavy chain CDR3 with an amino acid sequence shown as SEQ ID NO. 8; and
(D) Defined according to the Contact numbering system:
The amino acid sequence is shown as a light chain CDR1 shown in SEQ ID NO.15,
Light chain CDR2 with the amino acid sequence shown in SEQ ID NO.16,
Light chain CDR3 with the amino acid sequence shown in SEQ ID NO.17,
The amino acid sequence is shown as a heavy chain CDR1 shown in SEQ ID NO.18,
A heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO.19, and
The amino acid sequence is shown as heavy chain CDR3 of SEQ ID NO. 20.
2. The antibody of claim 1, wherein the heavy chain variable region of said antibody has the amino acid sequence set forth in SEQ ID No.39, and
The amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 40.
3. The antibody of claim 1, wherein the heavy chain of the antibody has the amino acid sequence shown in SEQ ID No.1, and
The amino acid sequence of the light chain of the antibody is shown as SEQ ID NO. 2.
4. A fusion protein, the fusion protein comprising:
(1) The antibody of claim 1; and
(2) Tag sequences that facilitate expression and/or purification.
5. A polynucleotide encoding the antibody of claim 1 or the fusion protein of claim 4.
6. A vector comprising the polynucleotide of claim 5.
7. A genetically engineered host cell comprising the vector of claim 6 or the polynucleotide of claim 5 integrated into the genome.
8. An antibody conjugate, comprising:
(a) An antibody moiety which is an antibody according to claim 1; and
(B) A coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, a drug, or a combination thereof.
9. Use of the antibody of claim 1, the polynucleotide of claim 5, the vector of claim 6, the host cell of claim 7, or the antibody conjugate of claim 8 in the preparation of an AAV RC-C07V5 virus neutralization formulation or medicament.
10. An AAV RC-C07V5 detection kit comprising the antibody of claim 1 and/or the antibody conjugate of claim 8.
11. A method of detecting AAV RC-C07V5 in a sample for non-diagnostic purposes, wherein the method is detected using the antibody of claim 1, the antibody conjugate of claim 8, or the detection kit of claim 10.
12. A formulation or pharmaceutical composition, characterized in that it comprises:
(i) The antibody of claim 1, the polynucleotide of claim 5, the vector of claim 6, the host cell of claim 7, the antibody conjugate of claim 8, or a combination thereof; and
(Ii) A pharmaceutically acceptable carrier.
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