Test products and methods for measuring botulinum toxin activity or synaptosome-related proteins

Abstract

The present invention discloses a detection product and method for determining botulinum toxin activity or synaptosome-related proteins. The detection product includes a kit, a test strip or a protein chip, which is used to detect SNAP25 or to determine botulinum toxin activity, wherein the detection product comprises an antibody or an antigen-binding fragment thereof that binds to SNAP25. The present invention achieves complete control of the experiment at each stage through phage display technology, the experimental cycle is relatively short, a large-capacity antibody library is generated, and different types of antibodies can be recombinantly produced. The antibody prepared by the present invention has good specificity and an affinity that is superior to commercial animal-derived SNAP25 polyclonal antibodies, and can be used for the detection of botulinum toxin endopeptidase activity, as well as the detection of SNAP25 in samples such as blood, cerebrospinal fluid, and exosomes.

Description

Technical Field

[0001] The present invention relates to the field of synaptosome-related protein detection, and in particular to a detection product and method for determining botulinum toxin activity or synaptosome-related protein. Background Art

[0002] Synaptosome-associated protein of 25 kDa (SNAP25) is a small 25 kDa (206 amino acids) helical protein first identified by Oyler et al. It consists of two isoforms, SNAP25a and SNAP25b, which appear at different stages of neural development. Structurally, SNAP25 is a helical protein. Two α-helical SNAP25s and two other helical vesicle-associated membrane proteins (VAMP) and syntaxin (STX) together form a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Functionally, SNAP25 is involved in the release of presynaptic neurotransmitters. Moreover, residues 180-197 play a key role in the regulation of calcium dynamics. In addition, the C-terminal zipper of SNAP25 has also been found to trigger the opening of the fusion pore and conformational changes of the SNARE complex.

[0003] Botulinum toxins (BoNTs) are the most toxic natural protein toxins known to humans, with a median lethal dose of 0.1-1.0 ng / kg. After entering cells, BoNTs block the release of neurotransmitters at the neuromuscular junction by cleaving SNARE proteins, ultimately leading to flaccid nerve paralysis. Therefore, BoNTs are widely used to treat nervous system-related diseases and the beauty industry, such as chronic migraine, trigeminal neuralgia, hemifacial spasm, dystonia, overactive bladder, frown lines, etc.

[0004] Currently, there is still a need to develop an anti-hSNAP25 humanized single-chain antibody and establish a method for detecting botulinum toxin activity in different sera.

[0005] The information in the background technology is only for illustrating the general background of the present invention and should not be regarded as admitting or suggesting in any form that such information constitutes the prior art known to a person skilled in the art. Summary of the invention

[0006] In order to solve at least some of the technical problems in the prior art, the present invention screens anti-hSNAP25 humanized single-chain antibodies from a human natural single-chain antibody library to ensure full humanization of the antibody, and then constructs a eukaryotic expression vector with the help of genetic engineering technology, and expresses it in eukaryotic cells 293F. In addition, the present invention converts the screened anti-hSNAP25 fully humanized single-chain antibody into a specific fully humanized complete hSNAP25-IgG1 antibody, and then separates and purifies it, and evaluates its biological properties such as specificity and affinity, thereby establishing a BoNT / A, BoNT / C and BoNT / E activity detection method. Specifically, the present invention includes the following contents.

[0007] In a first aspect of the present invention, a method for determining the activity of botulinum toxin is provided, wherein the method for determining the activity of botulinum toxin comprises the following steps:

[0008] (a) contacting the botulinum toxin with the SNAP25 protein for a time and under conditions sufficient to exert its biological activity, thereby obtaining a botulinum toxin-cleaved SNAP25;

[0009] (b) contacting the antibody or antigen-binding fragment thereof with the SNAP25 cleaved by botulinum toxin under conditions suitable for binding of the antibody or antigen-binding fragment thereof to the SNAP25 cleaved by botulinum toxin to obtain a first complex, wherein the antibody or antigen-binding fragment thereof comprises heavy chain CDR1-3 as shown in the amino acid sequences of SEQ ID NOs. 1-3 and / or light chain CDR1-3 as shown in the amino acid sequences of SEQ ID NOs. 4-6;

[0010] (c) contacting the detection antibody with the first complex under conditions suitable for the detection antibody to bind to the first complex, thereby obtaining a second complex;

[0011] (d) measuring the amount of the second complex and determining the botulinum toxin activity.

[0012] In certain embodiments, according to the method for determining the activity of botulinum toxin of the present invention, the detection antibody is an alkaline phosphatase-conjugated antibody, a horseradish peroxidase-conjugated antibody or a fluorescent dye-conjugated antibody.

[0013] A second aspect of the present invention provides a method for detecting SNAP25, the method comprising the following steps:

[0014] (1) contacting the sample with an antibody or antigen-binding fragment thereof for a time and under conditions sufficient to form an antibody or antigen-binding fragment thereof / antigen complex, wherein the antibody or antigen-binding fragment thereof binds to SNAP25 in the sample, and the antibody or antigen-binding fragment thereof comprises heavy chain CDR1-3 with amino acid sequences as shown in SEQ ID NOs. 1-3 and / or light chain CDR1-3 with amino acid sequences as shown in SEQ ID NOs. 4-6;

[0015] (2) determining the binding of the sample to the antibody or antigen-binding fragment thereof, or determining the presence of the antibody or antigen-binding fragment thereof / antigen complex, to determine the presence of SNAP25 in the sample.

[0016] In a third aspect of the present invention, an antibody or an antigen-binding fragment thereof is provided, wherein the antibody or the antigen-binding fragment thereof is capable of targeting SNAP25, and the antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1-3 whose amino acid sequence is shown in SEQ ID NO.1-3 and / or a light chain CDR1-3 whose amino acid sequence is shown in SEQ ID NO.4-6.

[0017] In certain embodiments, the antibody or antigen-binding fragment thereof according to the present invention has any one of the amino acid sequences shown in (I)-(III):

[0018] (I) comprising the heavy chain amino acid sequence shown in SEQ ID NO.7 and / or the light chain amino acid sequence shown in SEQ ID NO.8;

[0019] (II) an amino acid sequence that has at least 90% homology to the amino acid sequence shown in (I) and has the same function;

[0020] (III) an amino acid sequence obtained by modifying, replacing, deleting or adding one or more amino acids to the amino acid sequence shown in (I) or (II) and having the same function.

[0021] In certain embodiments, according to the antibody or antigen-binding fragment thereof described in the present invention, the antibody includes a monoclonal antibody, a chimeric antibody or a humanized antibody; the antigen-binding fragment includes Fab (a fragment comprising the entire variable region (VH and VL) and the constant region CH1), Fab' (a fragment removing the crystallizable region), F(ab)2 (a fragment connected by a disulfide bond), F(ab')2 (a fragment connected by a disulfide bond removing the crystallizable region) or scFv (single-chain variable fragment).

[0022] The fourth aspect of the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof described in the present invention.

[0023] The fifth aspect of the present invention provides a vector molecule, wherein the vector molecule comprises the nucleic acid molecule described in the present invention.

[0024] The sixth aspect of the present invention provides a host cell, wherein the host cell comprises the nucleic acid molecule or the vector molecule described in the present invention.

[0025] In a seventh aspect, the present invention provides a method for preparing an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof is prepared by artificial synthesis or genetic engineering.

[0026] In an eighth aspect of the present invention, a detection product is provided, wherein the detection product comprises a kit, a test strip or a protein chip, the detection product is used to detect SNAP25 or to determine the activity of botulinum toxin, and the detection product comprises the antibody or antigen-binding fragment thereof described in the present invention.

[0027] Although commercial SNAP25 antibodies are often used to detect the biological activity of botulinum toxin, most SNAP25s are polyclonal or monoclonal antibodies of animal origin (including mouse and rabbit sources). Animal-derived monoclonal antibodies are produced by hybridoma technology, which requires animal immunization, complex hybridoma fusion and screening, etc. The entire cycle takes a long time and has poor repetitive production performance. The present invention uses phage display technology to achieve complete control of the experiment at each stage, with a relatively short experimental cycle, a large-capacity antibody library, and can recombinantly produce different types of antibodies, which is suitable for industrial preparation. The antibody prepared by the present invention has good specificity and better affinity than commercial animal-derived SNAP25 polyclonal antibodies, and can be used for the detection of botulinum toxin endopeptidase activity, as well as the detection of SNAP25 in samples such as blood, cerebrospinal fluid, and exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the result of the recombinant human SNAP25 protein conjugated with biotin (B-hSNAP25), verified by 10% SDS-PAGE under reducing conditions.

[0029] Figure 2 The SDS-PAGE verification results of SNAP25H05 under reducing (lane 1) and non-reducing (lane 2) conditions are shown.

[0030] Figure 3 The purity of the SNAP25H05 antibody was determined using HPLC.

[0031] Figure 4This is the affinity test result of SNAP25H05 antibody. The grey curves from top to bottom in the figure represent 50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.125 nM respectively, and the black curve is the corresponding fitting curve.

[0032] Figure 5 Comparison of SNAP25H05 and commercially available S9684 antibody in detecting BoNT / A endopeptidase activity. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0036] Testing products

[0037] In one aspect of the present invention, a detection product is provided, which includes a kit, a test strip or a protein chip. The detection product is used to detect (or quantify) SNAP25 or to determine the activity of botulinum toxin, and the detection product comprises the antibody or antigen-binding fragment thereof described in the present invention and an optional detection antibody, as well as instructions for performing the detection of SNAP25 and botulinum toxin activity according to the present invention.

[0038] In addition to the above components, the kit of the present invention may also include precautions related to regulating the manufacture, use or sale of diagnostic kits. In addition, the kit of the present invention may also be provided with detailed instructions for storage and troubleshooting. The kit may also be optionally arranged in a suitable robotically operated device preferably for high throughput settings.

[0039] In certain embodiments, the components of the kit of the present invention may be arranged in a container. The container will typically include at least one vial, test tube, flask, bottle, syringe and / or other container means, in which the solvent is optionally placed in equal parts. The kit may also include means for a second container containing a sterile, pharmaceutically acceptable buffer and / or other solvent.

[0040] In certain embodiments, the components of the kit of the present invention can be provided in the form of a solution, such as an aqueous solution. In the case of being in the form of an aqueous solution, the concentration or content of these components can be conveniently determined by those skilled in the art according to different requirements.

[0041] The kit of the present invention may further comprise other reagents or components. For example, reagents or components for conducting experiments such as enzyme-linked immunosorbent assay, protein extraction and purification. These other reagents or components are known to those skilled in the art and can be easily obtained through public publications such as the fourth edition of Molecular Cloning Laboratory Manual.

[0042] The kit of the present invention may optionally contain additional antibodies, which may be detection antibodies. The detection antibodies may be any antibodies known in the art that are capable of specifically binding to cleaved SNAP25, such as antibodies derived from mouse, rabbit or humanized sources. Different detection antibodies may be selected according to different types of botulinum toxins to be detected. For example, when the botulinum toxin to be detected is BoNT / A, any suitable anti-SNAP25 antibody that is capable of specifically binding to SNAP25 with 197 amino acids may be selected, such as SNAP25 197 Mouse monoclonal antibody, those skilled in the art may also simultaneously or use other antibodies such as SNAP25 198 SNAP25 180 In a preferred embodiment, the antibody is a human SNAP25H05 antibody, and the detection antibody is a SNAP25 197 -HRP mouse monoclonal antibody.

[0043] Method for determining botulinum toxin activity

[0044] In one aspect of the present invention, a method for determining the activity of botulinum toxin is provided, wherein the BoNTs used for determination include at least one of BoNT / A, BoNT / E and BoNT / C, wherein the above serotypes of BoNTs have unique specificity for the target protein and the cleaved peptide bond, wherein BoNT / A and BoNT / E cleave SNAP25 at the carboxyl terminus to release a 9 or 26 amino acid fragment, respectively, and BoNT / C cleaves SNAP25 near the carboxyl terminus to release an 8 amino acid fragment. Therefore, the SNAP25 antibody or antigen-binding fragment thereof of the present invention can be used to establish a method for detecting the activity of BoNT / A, BoNT / C and BoNT / E.

[0045] The method for determining the activity of botulinum toxin of the present invention comprises steps (a) to (d), which are described in detail below.

[0046] Step (a) of the present invention is a step of contacting the botulinum toxin with the SNAP25 protein for a time and under conditions sufficient to exert its biological activity to obtain SNAP25 cleaved by the botulinum toxin. In one embodiment of step (a), the reaction conditions are incubation at 30-45°C for 5-30 min, preferably incubation at 35-40°C for 8-15 min.

[0047] Step (b) of the present invention is to contact the antibody or its antigen-binding fragment with the SNAP25 cleaved by the botulinum toxin under conditions suitable for the antibody or its antigen-binding fragment to bind to the SNAP25 cleaved by the botulinum toxin to obtain a first complex, wherein the antibody or its antigen-binding fragment comprises heavy chain CDR1-3 as shown in amino acid sequences of SEQ ID NO.1-3 and / or light chain CDR1-3 as shown in amino acid sequences of SEQ ID NO.4-6. In one embodiment of step (b), the reaction conditions are incubation at 0-10°C for 5-24 h, preferably incubation at 0-4°C for 7-12 h. The antibody or its antigen-binding fragment is preferably immobilized on a solid phase carrier, and the method of connecting the antibody or its antigen-binding fragment to a solid phase carrier is known in the art and is not particularly limited thereto.

[0048] Step (c) of the present invention is a step of contacting the detection antibody with the first complex under conditions suitable for the detection antibody to bind to the first complex to obtain a second complex. The detection antibody can be any antibody known in the art that can specifically bind to the cleaved SNAP25, such as an antibody from mouse, rabbit or humanized. Different detection antibodies can be selected according to different types of botulinum toxin to be detected. As an example, when the botulinum toxin to be detected is BoNT / A, any suitable anti-SNAP25 antibody that can specifically bind to SNAP25 with 197 amino acids can be selected, such as SNAP25 197Mouse monoclonal antibody, those skilled in the art may also simultaneously or use other antibodies such as SNAP25 198 SNAP25 180 The combined determination of different types of botulinum toxin activities can be achieved. In the present invention, the detection antibody or other antibody is an antibody containing a detectable label, preferably an alkaline phosphatase-conjugated antibody, a horseradish peroxidase-conjugated antibody or an antibody conjugated with a fluorescent dye.

[0049] Step (d) of the present invention is a step of measuring the amount of the second complex and determining the activity of the botulinum toxin. A person skilled in the art can select a suitable detection method to determine the amount (or concentration) of the second complex based on different detectable groups, thereby determining the activity of the botulinum toxin, and there is no particular limitation on this.

[0050] Methods for detecting SNAP25

[0051] In one aspect of the present invention, a method for detecting SNAP25 is provided, wherein the detection comprises determining the presence or absence and / or quantifying the amount of SNAP25 in a sample, wherein SNAP25 comprises its full-length form and fragments of the full-length SNAP25 after cleavage.

[0052] In one embodiment of the present invention, the method for detecting SNAP25 is not used for prediction or diagnosis of a disease, but is used to determine whether SNAP25 is present in a sample, or to perform quantitative detection of SNAP25, and the result is used to assist standard diagnostic techniques or provide intermediate information related to neuropathological diseases of confirmed or suspected subjects to treating physicians, or for scientific research, drug screening, etc. It is currently known that SNAP25 can be used as a potential biomarker and therapeutic target for diseases. Therefore, in another embodiment of the present invention, the detection of SNAP25 can be used for prediction or diagnosis of diseases, such as for prediction, progression, or even prognosis of neuropathological diseases. Based on this, the method of the present invention is also referred to as "the use of a reagent in the preparation of a diagnostic product (e.g., a diagnostic kit) for prediction, progression, or prognosis of a neuropathological disease", and wherein the reagent comprises an antibody or an antigen-binding fragment thereof, and the antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1-3 having an amino acid sequence as shown in SEQ ID NO.1-3 and / or a light chain CDR1-3 having an amino acid sequence as shown in SEQ ID NO.4-6. In the present invention, neuropathological diseases include but are not limited to Creutzfeldt-Jakob disease, neurodegenerative diseases (such as Alzheimer's disease), neuropathic pain, schizophrenia, epilepsy, ataxia and speech disorders.

[0053] In the present invention, the samples to be tested may include skin, muscle (including but not limited to skeletal muscle and smooth muscle), bladder, glandular tissue (including but not limited to prostate, lacrimal gland, endocrine gland and exocrine gland), blood vessels, spinal cord and brain (including nerve fibers in any or all of these tissues).

[0054] In one embodiment of the method for detecting SNAP25 of the present invention, the antibody or antigen-binding fragment thereof of the present invention may carry any detectable label. Examples of labels that may be used include, but are not limited to, radioactive substances, fluorescent groups, chemical labels, biological agents (such as, but not limited to, biotin / streptavidin detection), or enzyme substrate labels. In other embodiments, a detection antibody that carries a detectable label may be used in the present invention, and the use of the detection antibody may enhance the signal of the antibody of the present invention in some cases.

[0055] In the present invention, "time and conditions sufficient to form an antibody or antigen-binding fragment thereof / antigen complex" are not particularly limited, as long as SNAP25 in the sample binds to and forms a stable antibody or antigen-binding fragment thereof / antigen complex or conjugate after contact with the antibody or antigen-binding fragment thereof of the present invention.

[0056] Those skilled in the art are familiar with how to detect the binding of SNAP25 to an antibody or an antigen-binding fragment thereof in the sample, or detect the presence of an antibody or an antigen-binding fragment thereof / antigen complex, so as to determine whether SNAP25 is present in the sample or the amount of SNAP25. Such detection techniques include, but are not limited to, immunofluorescence, radioimmunoassay, enzyme-linked immunosorbent assay, agglutination test, immunoadsorption, immunoelectrophoresis, immunoelectron microscopy, and the like.

[0057] Antibodies or antigen-binding fragments thereof

[0058] In one aspect of the present invention, an antibody or an antigen-binding fragment thereof is provided, which is capable of binding to full-length SNAP25 and / or a fragment of SNAP25 with high affinity and targeting, and the antibody or the antigen-binding fragment thereof has the heavy chain CDR1-3 shown in SEQ ID NO.1-3 and / or the light chain CDR1-3 shown in SEQ ID NO.4-6.

[0059] As used herein, the term "antibody" refers to an immunoglobulin molecule that has the ability to specifically bind to a specific antigen. Antibodies typically contain a variable region and a constant region in each heavy chain and light chain. The variable regions of the antibody heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors. Therefore, most antibodies have a heavy chain variable region (VH) and a light chain variable region (VL), which together form the antibody portion that binds to the antigen.

[0060] The "light chain variable region (VL)" or "heavy chain variable region (VH)" of the present invention consists of a "framework" region intercalated with three "complementarity determining regions (CDRs)". The framework region is used to adjust the CDRs for specific binding to antigenic epitopes. The CDRs contain the amino acid residues in the antibody that are primarily responsible for antigen binding. From the amino terminus to the carboxyl terminus, both the VL and VH domains contain the following framework (FR) regions and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0061] In the present invention, the antibody or antigen-binding fragment thereof has any one of the amino acid sequences shown in (I)-(III):

[0062] (I) comprising the heavy chain amino acid sequence shown in SEQ ID NO.7 and / or the light chain amino acid sequence shown in SEQ ID NO.8;

[0063] (II) an amino acid sequence that has at least 90% homology to the amino acid sequence shown in (I) and has the same function;

[0064] (III) an amino acid sequence obtained by modifying, replacing, deleting or adding one or more amino acids to the amino acid sequence shown in (I) or (II) and having the same function.

[0065] As used herein, the terms "homology" and "identity" are used interchangeably. Homologous sequences include amino acid sequences that are at least 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the sequences of the present invention. In order to determine sequence identity, sequence alignment can be performed, which can be performed in a variety of ways known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software, etc. Those skilled in the art can determine appropriate parameters for alignment, including any algorithm required for achieving optimal alignment in the full-length sequence being compared.

[0066] Herein, variant antibody sequences obtained by modification, substitution, deletion or addition of one or more amino acids also fall within the scope of protection of the present invention. The term "modification" refers to any chemical modification of an amino acid sequence. The term "substitution" refers to the replacement of one or more amino acids by different amino acids. "Deletion" refers to the reduction of one or more amino acids in an amino acid sequence. "Insertion" or "addition" refers to an increase in one or more amino acids compared to a naturally occurring molecule resulting from a change in an amino acid sequence. It should be noted that, in the above-mentioned variant antibodies provided by the present invention, the modification, substitution, deletion or addition preferably occurs in a region outside the variable region, such as in the framework region or constant region of the antibody, and the modified, substituted, deleted or added antibody still retains the desired functional properties of the antibody of the present invention or its antigen-binding fragment, or has improved antigen-binding properties.

[0067] In the present invention, antibodies include monoclonal antibodies, chimeric antibodies or humanized antibodies.

[0068] The term "monoclonal antibody" as used herein, sometimes also referred to as "monoclonal antibody" or Ab, refers to an immunoglobulin obtained from a pure line of cells, having the same structure and chemical properties, and being specific to a single antigenic determinant. Monoclonal antibodies are different from conventional polyclonal antibody preparations (usually having different antibodies for different determinants), in that each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the benefit of monoclonal antibodies is that they are obtained by hybridoma or recombinant engineering cell culture and are not mixed with other immunoglobulins. This characteristic is contrasted with polyclonal antibody preparations, which generally include antibodies for different antigenic determinants. The modifier "monoclonal" indicates the characteristic of the antibody, which is obtained from a homogeneous antibody population, but this should not be interpreted as requiring any special method to produce the antibody.

[0069] Herein, the term "chimeric antibody" generally refers to an antibody in which a portion of each heavy or light chain amino acid sequence is homologous to the corresponding amino acid sequence in an antibody from a particular species, or belongs to a particular class, while the remaining segments of the chain are homologous to the corresponding sequence in another species. For example, the variable regions of the light and heavy chains are both from the variable regions of an antibody from one animal species, while the constant portions are homologous to the antibody sequences from another species. For example, to obtain a chimeric antibody, a non-human B cell or hybridoma cell can be used to produce the variable region, while the constant region combined therewith is from a human. The variable region has the advantage of being easy to prepare, and its specificity is not affected by the source of the constant region combined therewith.

[0070] Herein, the term "humanized antibody" generally refers to a chimeric antibody that contains fewer sequences from non-human immunoglobulins, thereby reducing the immunogenicity of xenogeneic antibodies when introduced into humans, while maintaining the antibody's full antigen binding affinity and specificity.

[0071] Herein, the term "antigen-binding fragment" generally refers to one or more fragments of an antibody that specifically bind to an antigen. It is understood that the antigen-binding function of an antibody can be achieved by a full-length fragment of the antibody, or by a heavy chain comprising Fab, Fab', F(ab)2, F(ab')2, or scFv, or a light chain comprising Fab, Fab', F(ab)2, F(ab')2, or scFv.

[0072] In the present invention, the terms "binding", "specific binding", "directed against" and "targeting" are used interchangeably and generally refer to the non-covalent interaction that occurs between an immunoglobulin molecule and an antigen that is specific to the immunoglobulin. The strength or affinity of an immunological binding interaction can be expressed as a dissociation constant (Kd), where a smaller Kd represents a higher affinity. "Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, when used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of a binding dissociation equilibrium constant. Affinity can be measured by conventional methods well known in the art, including those known in the prior art and described herein.

[0073] Nucleic acid molecules

[0074] In one aspect of the present invention, a nucleic acid molecule is provided, which comprises a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of the present invention.

[0075] The term "nucleic acid" as used herein is intended to include polymeric forms of nucleotides of any length, containing deoxyribonucleotides, ribonucleotides and / or their analogs, including DNA, RNA and DNA / RNA hybrids, including DNA or RNA analogs, such as those containing modified backbones (e.g., peptide nucleic acids (PNA) or thiophosphates) or modified bases. Therefore, nucleic acids of the present invention include DNA, cDNA, mRNA, recombinant nucleic acids, etc.

[0076] Once the coding sequence of the antibody or antigen-binding fragment thereof of the present invention is obtained, the antibody or antigen-binding fragment thereof can be obtained in large quantities by recombinant technology. An exemplary method is to clone the coding gene into a vector, transfer it into cells, and then separate it from the propagated host cells by conventional methods.

[0077] Carrier molecules

[0078] One aspect of the present invention provides a vector molecule comprising the nucleic acid molecule of the present invention.

[0079] The vector of the present invention refers to an artificial construct that can deliver and preferably express one or more target genes or sequences in a host cell. The vector of the present invention is not limited and can be an expression vector, a viral vector, etc. In certain embodiments, the vector comprises a target gene, a promoter, a terminator encoding an antibody or an antigen-binding fragment thereof of the present invention, or optionally further comprises a marker gene. The vector can use a known vector or a self-constructed vector. Known vectors include phage vectors, plasmid vectors, lentiviral vectors, adenoviral vectors, AAV viral vectors, etc.

[0080] Host cells

[0081] In one aspect of the present invention, a host cell is provided, which comprises the nucleic acid molecule or the vector molecule described in the present invention.

[0082] The host cell of the present invention refers to any cell type suitable for transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a nucleic acid molecule of the present invention. A host cell includes any progeny of a parent cell that differs from the parent cell due to mutations that occur during replication.

[0083] Preparation method

[0084] In one aspect of the present invention, a method for preparing the antibody or antigen-binding fragment thereof of the present invention is provided. The preparation method is not particularly limited, and includes preparation by artificial synthesis or genetic engineering.

[0085] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention is obtained by artificial synthesis. Methods for artificially synthesizing antibodies or antigen-binding fragments thereof are known in the art, for example, the antibody or antigen-binding fragment thereof of the present invention is obtained by direct amino acid synthesis.

[0086] In certain embodiments, the antibody or antigen-binding fragment thereof of the present invention is obtained by genetic engineering expression. Genetic engineering expression systems include prokaryotic cell expression systems, eukaryotic cell expression systems, and cell-free expression systems. Examples of prokaryotic cell expression systems include Escherichia coli expression systems. Eukaryotic cell expression systems include zymocyte expression systems, insect cell expression systems, and mammalian cell expression systems.

[0087] In a specific embodiment, the method for preparing an antibody of the present invention comprises:

[0088] (1) constructing a recombinant vector for the antibody heavy chain expressing the amino acid sequence shown in SEQ ID NO.7 and a recombinant vector for the antibody light chain expressing the amino acid sequence shown in SEQ ID NO.8;

[0089] (2) transforming the vector into host cells and culturing the cells under conditions suitable for antibody expression;

[0090] (3) Collect and purify antibodies.

[0091] Example 1

[0092] This example shows a method for coupling recombinant human SNAP25 protein (hSNAP25) expressed in E. coli with biotin to obtain conjugate B-hSNAP25. The specific steps are as follows:

[0093] 1) Dissolve biotin in DMSO to 10 mg / mL and add to the protein at a mass ratio of 10:1.

[0094] 2) Seal with sealing film and fix on a shaker at 37°C for 2 hours;

[0095] 3) Wash the G25 column with PBS for 30 minutes. After PBS flows downward, transfer the well-reacted protein to the G25 column with a pipette on a 37°C shaker. Add 1 ml of PBS for elution. Do not transfer the first drop and transfer the remaining bound protein to a 2 ml tube until the protein is completely eluted to obtain B-hSNAP25. The SDS-PAGE verification result is as follows: Figure 1 shown.

[0096] Example 2

[0097] This example illustrates the preparation of antibodies using a phage library preparation method as an example.

[0098] Typical biopanning may require 3-5 rounds of screening to achieve acceptable enrichment. In this example, 4 rounds of screening were performed, with pre-absorption performed before each round of panning to minimize non-specific binders.

[0099] The experimental steps are as follows:

[0100] 1) Incubate the ELISA plate coated with streptavidin-coupled magnetic beads M-280 for 2 h;

[0101] 2) Coat the ELISA plate with B-hSNAP25 and incubate overnight;

[0102] 3) Block the ELISA plate with blocking buffer and incubate overnight;

[0103] 4) Add the phage library to each well and incubate at 37°C for 2 h;

[0104] 5) Wash 5 times with an ELISA plate washer to remove unbound phages;

[0105] 6) Add elution buffer and incubate at 37°C for 10 min. Pipette up and down vigorously 10 times to elute the positive phages from the target antigen. Then, transfer the filtrate to a microcentrifuge tube containing neutralization buffer.

[0106] 7) The partially eluted phages were -2 and 10 -10 Serial dilutions were performed between cfu, and the infection logarithmic phase E.coil TG1 (OD600≈0.5), infected E.coil TG1 was spread on 2×YT-A (2×YT medium supplemented with ampicillin) agar plates, grown overnight at 37°C, and the number of recovered phages was calculated;

[0107] 8) Infect partially eluted phage E.coil After TG1, 2×YT-AG medium (2×YT medium supplemented with ampicillin and glucose) was added, grown overnight at 37°C, stirred at 5000 rpm for 15 min at 4°C, and the pellet was resuspended in 2× yeast extract tryptone medium (2×YT medium) containing glycerol and stored at −80°C;

[0108] 9) Inoculate part of the recovered glycerol stock solution into 2×YT-AG medium and grow to the logarithmic phase (OD 600 ≈ 0.5). Add 1×10 11 pfu of M13K07 helper phage, incubated in a 37°C water bath for 30 min, centrifuged at 5000 rpm for 15 min, resuspended the pellet in 2×YT-AK medium (2×YT medium supplemented with ampicillin and kanamycin), and grown overnight at 30°C with shaking (225 rpm);

[0109] 10) Centrifuge the culture at 4000 g for 20 min in a 50 mL centrifuge tube to precipitate the bacteria and collect the supernatant (containing the phage library). To precipitate the phage, add 1 / 5 volume of 5× PEG / NaCl to the supernatant and place on ice for at least 1 h (PEG will promote the precipitation of the phage complex from the solution). Remove the supernatant thoroughly by centrifugation at 12000 g for 15 min at 4°C. Resuspend the pellet in 1 mL of sterile PBS. To eliminate bacterial contamination of the phage in the suspension, centrifuge in a microcentrifuge (2 min). Transfer the supernatant to a new tube for the next round of panning.

[0110] 11) Repeat steps 1)-10) 4 times.

[0111] Table 1 Monitoring of phage antibody library screening process

[0112]

[0113] Enrichment factor = number of input phages / number of eluted phages.

[0114] Example 3

[0115] This example shows an ELISA experiment of monoclonal phage.

[0116] 1) Coat a 96-well plate with streptavidin-conjugated B-hSNAP25 (SA+B-hSNAP25) and use PBS as a negative control at 4°C overnight;

[0117] 2) Wash the plate, add blocking buffer, incubate at 37°C for 1 hour, and then wash the plate;

[0118] 3) Single colonies picked from the plates were cultured overnight at 37°C, 2-5 μL / well was inoculated from the overnight plate, and grown to exponential phase (OD 600 ≈ 0.5), and a mixture was prepared by adding M13KO7 helper phage, with a phage to bacteria ratio of about 20:1, and incubated with shaking for several hours;

[0119] 4) Combine the recovered monoclonal phage supernatant (25 μL / well) with 75 μL of blocking buffer and add to the antigen-coated 96-well plate and incubate at 37°C for 1 h;

[0120] 5) After washing the plate, add 100 μL / well of 1:5000 diluted anti-M13-HRP and incubate at 37°C for 1 hour;

[0121] 6) After washing the plate, add 100 μL / well TMB colorimetric solution and incubate at room temperature in the dark for 5-30 min;

[0122] 7) Add 50 μL / well 1 M H2SO4 to terminate the reaction and read the absorbance of each well at 450 nm in a microplate reader.

[0123] An antibody that binds to the antigen was screened and named Clone159-scFv, and sequenced.

[0124] After sequencing, the amino acid sequence of Clone159-scFv antibody is shown in SEQ ID No. 9. The amino acid sequence of heavy chain CDR1-3 is shown in SEQ ID No. 1-3, the amino acid sequence of light chain CDR1-3 is shown in SEQ ID No. 4-6, the amino acid sequence of heavy chain variable region VH is shown in SEQ ID No. 10, and the amino acid sequence of light chain variable region VL is shown in SEQ ID No. 11.

[0125] Table 2 Amino acid sequences of antibodies

[0126]

[0127] Example 4

[0128] This example shows an ELISA experiment of soluble phage.

[0129] The identified positive clones were subjected to soluble phage ELISA.

[0130] 1) Place it in E.coil In TG1, expression was induced with IPTG at 30°C and 37°C;

[0131] 2) After expression is complete, the bacteria are collected by centrifugation for periplasmic extraction;

[0132] 3) The periplasmic extract was diluted 1:10 and added to the pre-coated SA+B-hSNAP25 ELISA plate, and PBS was used as a control;

[0133] 4) The soluble expression of Clone159-scFv was detected using mouse anti-E-tag monoclonal antibody at a dilution of 1:2000 and HRP goat anti-mouse polyclonal antibody at a dilution of 1:5000 as the secondary antibody and the tertiary antibody. As shown in Table 3, Clone159-scFv was soluble and positively bound to B-hSNAP25.

[0134] Table 3 Soluble phage ELISA

[0135]

[0136] Example 5

[0137] This example shows the expression of a fully human SNAP25-IgG1 (SNAP25H05) antibody.

[0138] Based on the above research, a specific anti-hSNAP25 humanized single-chain antibody, namely Clone159-scFv, was screened from a large-capacity phage antibody library. Since the scFv single-chain antibody has no Fc segment, its affinity is not as good as that of a complete IgG1. Therefore, in this example, the anti-Clone159-scFv single-chain antibody was converted into a complete IgG1 antibody, named SNAP25H05. The amino acid sequence of the SNAP25H05 heavy chain is shown in SEQ ID No.7, and the amino acid sequence of the SNAP25H05 light chain is shown in SEQ ID No.8.

[0139] Using genetic engineering technology, the human antibody heavy chain constant region and the heavy chain variable region screened from the phage library, the human antibody light chain constant region and the light chain variable region screened from the phage library were amplified to the eukaryotic cell expression plasmid pcDNA3.4 to construct the light chain expression plasmid and the heavy chain expression plasmid respectively.

[0140] 1) Plasmid preparation: Transform the SNAP25H05 antibody light chain plasmid and the SNAP25H05 antibody heavy chain plasmid into DH5α competent bacteria respectively, and then inoculate them into LB agar plates containing Amp antibiotics for resistance screening. Pick positive clones and inoculate them into 150 ml LB medium containing 100X Amp antibiotics, shake well, incubate overnight, and extract the plasmid according to the instructions of the plasmid extraction kit;

[0141] 2) Transfection of 293F cells: 293F cells were cultured in suspension at 37°C, 5% CO2 and 120 rpm, maintaining a cell density of 2×10 5 Cells / ml were cultured every 4 days, and the cell confluence was checked to be between 70-80%. The 293F cell density was adjusted to about 1.1×10 6 cells / mL, dilute the heavy chain and light chain plasmids and polyethyleneimine (PEI) separately in Opi-MEM, and then mix them together (light chain: heavy chain = 1:1; PEI: plasmid = 2.7:1), add the mixture to the flask containing 293F cells at one time, shake gently and move crosswise to ensure sufficient mixing. If the cells are in suspension, transfection is more efficient. Place the flask in a CO2 incubator at 37°C and 5% CO2 for 6 days;

[0142] 3) SNAP25H05 antibody purification: The 293F cell suspension was centrifuged at 8000 rpm for 30 min at 4°C, and the cell supernatant was filtered through a 0.22 μm filter membrane. The antibody was purified with protein A, and the filtrate was transferred to a dialysis bag and stirred overnight at 4°C. The dialysate was removed, and the final antibody concentration was detected by Nanodrop. The sample was filtered through a 0.22 μm filter membrane, and 0.1 mg of protein was taken for concentration determination, SDS-PAGE, and HPLC. Figure 2 SDS-PAGE analysis of SNAP25H05 under reducing (lane 1) and non-reducing (lane 2) conditions. The gel was stained with Coomassie Brilliant Blue for 30 min. Under reducing conditions, the light and heavy chains migrated at approximately 50 kDa and 25 kDa, respectively, while under non-reducing conditions, the protein migrated at 130-180 kDa. Figure 3 The purity of SNAP25H05 antibody was tested by high performance liquid chromatography, and the results showed that the purity of SNAP25H05 was 99.5%.

[0143] Example 6

[0144] This example shows the affinity determination of the SNAP25H05 antibody.

[0145] 1) Anti-human IgG (Fc) immobilization: dilute anti-human IgG (Fc) to 25 μg / mL in the immobilization buffer and immobilize the antibody onto the surface of the CM5 sensor chip at 10 μL / min for 420 s. The amount of anti-human IgG (Fc) antibody coupled to the CM5 sensor chip is approximately 7000-14000 RU.

[0146] 2) SNAP25H05 antibody dilution: dilute SNAP25H05 antibody to 2.5 μg / mL with running buffer and inject it into the sample channel at 10 μL / min to achieve a capture level of approximately 100-130 RU;

[0147] 3) Sample dilution: Different concentrations of SNAP25 protein (3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM) were diluted in running buffer for affinity and kinetic measurements. The 1:1 binding model was used to measure binding affinity and / or kinetics. The specific binding affinity results are shown in Figure 4 And as shown in Table 4.

[0148] Table 4 SNAP25H05 antibody affinity

[0149]

[0150] Example 7

[0151] This example shows the application of human SNAP25H05 antibody in the detection of botulinum toxin endopeptidase activity.

[0152] 1) Primary antibody incubation: dilute the human SNAP25H05 antibody and the commercially available SNAP25 rabbit polyclonal antibody S9684 with 1× PBS buffer at 1:1000, 100 μL / well, coat with shaking at 4°C overnight, wash three times with a plate washer (PBST), pat dry, block with 2% BSA solution at room temperature for 1.5 h, discard, and pat dry;

[0153] 2) Add the sample to be tested: dilute the BoNT / A to be tested with diluent (50 mmol / L HEPES, 20 μmol / L ZnCl2, 10 mmol / L DTT, 1 mg / ml BSA, 0.5% Tween20; pH 7.0) from 100 U / mL in 2-fold gradients for 10 times; add 50 μL / well to a 96-well transparent plate, and activate it in a constant temperature shaker at 37°C, 400 rpm for 30 min; dilute the h-SNAP25 protein to 0.25 μg / ml with the prepared diluent, add 50 μL / well to the activated toxin well, place it in a constant temperature shaker at 37°C, 400 rpm, incubate and cut for 10 min, remove it from the constant temperature shaker after 10 min, add 90 μL / well to the blocked ELISA enzyme-labeled plate, shake it at 4°C overnight, wash it 3 times with a plate washer, and pat it dry;

[0154] 3) Secondary antibody incubation: SNAP25 197 -HRP mouse monoclonal antibody (50 μg / ml) was diluted 1:10000 with 2% BSA solution, 100 μL / well, shaken at room temperature for 1.5 h, washed three times with a plate washer, and patted dry;

[0155] 4) ELISA color development: Mix the color development solution in a 1:1 ratio, add 100 μL / well of color development solution, and detect the optical density value at D450 on an ELISA reader.

[0156] The results were analyzed by four-parameter fitting. Figure 5 The results showed that the EC50 of SNAP25H05 for detecting BoNT / A was 6.08 U / ml, and the EC50 of commercially available SNAP25 rabbit polyclonal antibody S9684 for detecting BoNT / A was 25.19 U / ml, indicating that the SNAP25H05 antibody of the present invention has significantly higher affinity than S9684.

[0157] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims should be based on the broadest interpretation to cover all modifications and equivalent structures and functions.

Claims

1. A kit, characterized in that: The kit is used for detecting synaptosome-associated proteins or for determining botulinum toxin activity, and comprises an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises heavy chain CDR1-3 whose amino acid sequences are respectively shown in SEQ ID NO.1-3 and light chain CDR1-3 whose amino acid sequences are respectively shown in SEQ ID NO.4-6.

2. A method for determining the activity of botulinum toxin, characterized in that: The method for determining the activity of botulinum toxin comprises the following steps: (a) contacting the botulinum toxin with a synaptosome-associated protein for a time and under conditions sufficient for the botulinum toxin to exert its biological activity, thereby obtaining a synaptosome-associated protein cleaved by the botulinum toxin; (b) contacting the antibody or antigen-binding fragment thereof with the synaptosome-associated protein cleaved by botulinum toxin under conditions suitable for binding of the antibody or antigen-binding fragment thereof to the synaptosome-associated protein cleaved by botulinum toxin to obtain a first complex, wherein the antibody or antigen-binding fragment thereof comprises heavy chain CDR1-3 as shown in amino acid sequences of SEQ ID NOs. 1-3, respectively, and light chain CDR1-3 as shown in amino acid sequences of SEQ ID NOs. 4-6, respectively; (c) contacting the detection antibody with the first complex under conditions suitable for the detection antibody to bind to the first complex to obtain a second complex; (d) measuring the amount of the second complex and determining the botulinum toxin activity; Wherein, the detection antibody is an alkaline phosphatase-conjugated antibody, a horseradish peroxidase-conjugated antibody or a fluorescent dye-conjugated antibody.

3. Use of the reagent in preparing a kit for detecting synaptosome-related proteins, characterized in that: The reagent comprises an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises heavy chain CDR1-3 whose amino acid sequences are respectively shown in SEQ ID NO.1-3 and light chain CDR1-3 whose amino acid sequences are respectively shown in SEQ ID NO.4-6; wherein, The detection of synaptosome-associated proteins comprises the following steps: (1) contacting a sample with an antibody or antigen-binding fragment thereof for a time and under conditions sufficient to form an antibody or antigen-binding fragment thereof / antigen complex, wherein the antibody or antigen-binding fragment thereof binds to a synaptosome-associated protein in the sample; (2) determining the binding of the sample to the antibody or its antigen-binding fragment, or determining the presence of the antibody or its antigen-binding fragment / antigen complex, so as to determine the presence of synaptosome-associated protein in the sample or determine the amount of synaptosome-associated protein in the sample.

4. An antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof can target synaptosome-associated proteins, and the antibody or antigen-binding fragment thereof comprises heavy chain CDR1-3 whose amino acid sequences are respectively shown in SEQ ID NO.1-3 and light chain CDR1-3 whose amino acid sequences are respectively shown in SEQ ID NO.4-6.

5. The antibody or antigen-binding fragment thereof according to claim 4, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain amino acid sequence shown in SEQ ID NO.7 and a light chain amino acid sequence shown in SEQ ID NO.

8.

6. The antibody or antigen-binding fragment thereof according to claim 4, characterized in that: The antibody includes a monoclonal antibody, a chimeric antibody or a humanized antibody; the antigen-binding fragment includes Fab, Fab', F(ab)2, F(ab')2 or scFv.

7. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 4 to 6.

8. A carrier molecule, characterized in that The vector molecule comprises the nucleic acid molecule according to claim 7.

9. A host cell, characterized in that The host cell comprises the nucleic acid molecule according to claim 7 or the vector molecule according to claim 8.

10. The method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 4 to 6, characterized in that: The antibody or antigen-binding fragment thereof is prepared by artificial synthesis or genetic engineering.

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