A kit, antibody and method for measuring botulinum toxin activity or SNAP25

Abstract

The present invention discloses a kit, an antibody and a method for determining botulinum toxin activity or 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. In addition, the antibody prepared by the present invention has a higher affinity and is higher than the commercial animal-derived SNAP25 polyclonal antibody, and has a wide range of application prospects in SNAP25-related detection.

Description

Technical Field

[0001] The present invention relates to the field of synaptosome-related protein detection, and in particular to a kit, an antibody and a method for determining botulinum toxin activity or SNAP25. Background Art

[0002] Synaptosome-associated protein of 25 kDa (SNAP25) was first identified by Oyler et al. It is a small 25 kDa (206 amino acids) helical protein. 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. The screened anti-hSNAP25 fully humanized single-chain antibody is converted into a specific fully humanized complete hSNAP25-IgG1 antibody, which is then separated and purified, and its biological properties such as specificity and affinity are evaluated, 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 detecting SNAP25 is provided, wherein the method for detecting SNAP25 comprises the following steps:

[0008] (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;

[0009] (2) detecting the binding of the sample to the antibody or antigen-binding fragment thereof, or detecting the presence of the antibody or antigen-binding fragment thereof / antigen complex, to determine the presence of SNAP25 in the sample.

[0010] In a second 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:

[0011] (a) contacting the botulinum toxin with the SNAP25 protein for a time and under conditions sufficient to exert its biological activity, thereby obtaining SNAP25 cleaved by the botulinum toxin;

[0012] (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;

[0013] (c) contacting the additional antibody with the first complex under conditions suitable for binding of the first complex to the additional antibody to obtain a second complex;

[0014] (d) detecting the amount of the second complex and determining the botulinum toxin activity.

[0015] In certain embodiments, according to the method for determining botulinum toxin activity of the present invention, the additional antibody is an alkaline phosphatase-conjugated antibody, a horseradish peroxidase-conjugated antibody, or a fluorescent dye-conjugated antibody.

[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 kit is provided, wherein the kit is used to detect SNAP25 or to determine the activity of botulinum toxin, and the kit comprises an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1-3 having an amino acid sequence as shown in SEQ ID NOs. 1-3 and / or a light chain CDR1-3 having an amino acid sequence as shown in SEQ ID NOs. 4-6.

[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, for example, mouse and rabbit origin). 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, the experimental cycle is relatively short, a large-capacity antibody library is generated, and different types of antibodies can be recombinantly produced. In addition, the antibodies prepared by the present invention have higher affinity and are higher than commercial animal-derived SNAP25 polyclonal antibodies, and have broad application prospects in SNAP25-related detection. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0029] Figure 2 SDS-PAGE analysis of SNAP25H03 under reducing (lane 1) and non-reducing (lane 2) conditions is shown.

[0030] Figure 3 The results of SNAP25H03 antibody purity assayed using HPLC are shown.

[0031] Figure 4 The affinity test results of SNAP25H03 antibody detected by Biacore are shown. The gray 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 The comparison of SNAP25H03 and commercially available S9684 antibody in detecting BoNT / A endopeptidase activity is shown. 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] Methods for detecting SNAP25

[0037] 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.

[0038] 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 quantitatively detect SNAP25, and the result can be used to assist standard diagnostic techniques or provide intermediate information related to neuropathological diseases for treating physicians in subjects diagnosed or suspected of having the disease. Considering that it is currently known that SNAP25 can be used as a potential biomarker and therapeutic target for a disease, therefore, in another embodiment of the present invention, the detection of SNAP25 can be used for prediction or diagnosis of a disease, such as for prediction, progression, or even prognosis of a neuropathological disease. 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, Alzheimer's disease, neuropathic pain, schizophrenia, epilepsy, ataxia and speech disorders.

[0039] In the present invention, the sample to be tested may include at least one of the following: 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).

[0040] 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), or enzyme substrate labels. In other embodiments, additional antibodies that may carry a detectable label may be used in the present invention, and the use of the additional antibodies may in some cases enhance the signal of the antibodies of the present invention.

[0041] 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.

[0042] 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 the 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.

[0043] Method for determining botulinum toxin activity

[0044] In one aspect of the present invention, a method for determining the activity of botulinum toxin (BoNTs) 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 binding of the antibody or its antigen-binding fragment with 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 additional antibody with the first complex under conditions suitable for the binding of the first complex with the additional antibody to obtain a second complex. The additional antibody can be any antibody known in the art that can specifically bind to cleaved SNAP25, such as an antibody from mouse, rabbit or humanized. Different additional antibodies can be selected according to different types of botulinum toxin to be tested. For example, when the botulinum toxin to be tested is BoNT / A, any suitable anti-SNAP25 antibody that can specifically bind to SNAP25 with 197 amino acids can 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 The combined determination of different types of botulinum toxin activities can be achieved. In the present invention, the additional 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 detecting 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 of the second complex and thereby determine the activity of the botulinum toxin according to different detectable groups, and this is not particularly limited.

[0050] Antibodies or antigen-binding fragments thereof

[0051] 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.

[0052] 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.

[0053] 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.

[0054] In the present invention, the antibody or antigen-binding fragment thereof has any one of the amino acid sequences shown in (I)-(III):

[0055] (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;

[0056] (II) an amino acid sequence that has at least 90% homology to the amino acid sequence shown in (I) and has the same function;

[0057] (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.

[0058] 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.

[0059] 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.

[0060] In the present invention, antibodies include monoclonal antibodies, chimeric antibodies or humanized antibodies.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] Unless otherwise indicated, the antibodies or antigen-binding fragments thereof described herein are isolated antibodies or antigen-binding fragments thereof. The term "isolated" as used herein refers to an antibody or antigen-binding fragment thereof that has been extracted from its natural environment. An antibody or antigen-binding fragment thereof that has been "isolated" thus includes an antibody or antigen-binding fragment thereof that has been purified by standard purification methods. The term also includes antibodies or antigen-binding fragments thereof prepared by recombinant expression in a host cell and chemically synthesized antibodies or antigen-binding fragments thereof.

[0067] Nucleic acid molecules

[0068] 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.

[0069] 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.

[0070] 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 isolate it from the propagated host cells by conventional methods.

[0071] Carrier molecules

[0072] One aspect of the present invention provides a vector molecule comprising the nucleic acid molecule of the present invention.

[0073] 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 plasmid vectors, lentiviral vectors, adenoviral vectors, AAV viral vectors, etc.

[0074] Host cells

[0075] 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.

[0076] 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.

[0077] Preparation method

[0078] 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.

[0079] 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.

[0080] 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.

[0081] In a specific embodiment, the method for preparing an antibody of the present invention comprises:

[0082] (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;

[0083] (2) transforming the vector into host cells and culturing the cells under conditions suitable for antibody expression;

[0084] (3) Collect and purify antibodies.

[0085] Reagent test kit

[0086] The present invention further provides a kit, wherein the kit is used to detect SNAP25 or to determine the activity of botulinum toxin, and the kit comprises an antibody or an antigen-binding fragment thereof, wherein 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. The kit may also comprise optional additional antibodies, and instructions for how to perform the detection of SNAP25 and botulinum toxin activity according to the present invention.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[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 protein:biotin.

[0094] 2) Seal with sealing film and fix on a shaker at 37°C for 2 hours. Wash the G25 column with PBS for 30 minutes. After PBS flows down, use a pipette to transfer the well-reacted protein from the 37°C shaker to the G25 column. Add 1 ml 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.

[0095] Example 2

[0096] This example illustrates the preparation of antibodies using a phage library preparation method as an example.

[0097] 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.

[0098] The experimental steps are as follows:

[0099] 1) Coat the ELISA plate with streptavidin-coupled magnetic beads M-280 and incubate for 2 h.

[0100] 2) Coat ELISA plates with B-hSNAP25 and incubate overnight.

[0101] 3) Block the ELISA plate with blocking buffer and incubate overnight.

[0102] 4) Add the phage library to each well and incubate at 37°C for 2 hours.

[0103] 5) Wash 5 times with an ELISA plate washer to remove unbound phages.

[0104] 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.

[0105] 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 counted.

[0106] 8) Infect partially eluted phage E.coil After TG1, add 2×YT-AG medium (2×YT medium supplemented with ampicillin and glucose), grow overnight at 37°C, stir at 5000 rpm for 15 min at 4°C, resuspend the pellet in 2× yeast extract tryptone medium (2×YT medium) containing glycerol, and store at −80°C.

[0107] 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 M13K07 helper phage, incubate in a 37°C water bath for 30 min, centrifuge at 5 000 rpm for 15 min, resuspend the pellet in 2×YT-AK medium (2×YT medium supplemented with ampicillin and kanamycin), and grow overnight at 30°C with shaking (225 rpm).

[0108] 10) Centrifuge the culture at 4000 g for 20 min in a 50 mL centrifuge tube to pellet 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). Completely remove the supernatant by centrifugation at 12,000 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) and transfer the supernatant to a new tube for the next round of panning.

[0109] 11) Repeat steps 1)-10) 4 times.

[0110] Table 1 Monitoring of phage antibody library screening process

[0111]

[0112] Enrichment factor = number of input phages / number of eluted phages.

[0113] Example 3

[0114] This example shows an ELISA experiment of monoclonal phage.

[0115] 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.

[0116] 2) Wash the plate, add blocking buffer, incubate at 37°C for 1 hour, and then wash the plate.

[0117] 3) Culture a single colony picked from the plate overnight at 37°C, inoculate 2-5 μL / well from the overnight plate, grow to exponential phase (OD600≈0.5), prepare a mixture by adding M13KO7 helper phage, with a phage to bacteria ratio of approximately 20:1, and incubate with shaking for several hours.

[0118] 4) Combine the recovered monoclonal phage supernatant (25 μL / well) with 75 μL of blocking buffer and add it to the antigen-coated 96-well plate and incubate at 37°C for 1 h.

[0119] 5) After washing, add 100 μL / well of 1:5000 diluted anti-M13-HRP and incubate at 37°C for 1 hour.

[0120] 6) After washing, add 100 μL / well TMB colorimetric solution and incubate at room temperature in the dark for 5-30 min.

[0121] 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.

[0122] An antibody that binds to the antigen was screened and named Clone100-scFv antibody, and then sequenced.

[0123] After sequencing, the amino acid sequence of the Clone100-scFv antibody is shown as SEQ ID No.9, wherein the amino acid sequence of the heavy chain CDR1-3 is shown as SEQ ID No.1-3, and the amino acid sequence of the light chain CDR13 is shown as SEQ ID No.4-6.

[0124] Table 2 Amino acid sequences of antibodies or antigen-binding fragments thereof

[0125]

[0126] Example 4

[0127] This example shows an ELISA experiment of soluble phage.

[0128] The identified positive clones were subjected to soluble phage ELISA.

[0129] 1) Place it in E.coil The expression was induced by isopropylthio-β-galactoside (IPTG) at 30°C and 37°C in TG1.

[0130] 2) After expression is complete, the bacteria are collected by centrifugation for periplasmic extraction.

[0131] 3) Dilute the periplasmic extract 1:10 and add it to the pre-coated SA+B-hSNAP25 ELISA plate, and use PBS as a control.

[0132] 4) 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 were used as the secondary and tertiary antibodies to detect the soluble expression of Clone100-scFv. As shown in Table 3, Clone100-scFv was soluble and positively bound to B-hSNAP25.

[0133] Table 3 Soluble phage ELISA

[0134]

[0135] Example 5

[0136] This example shows the expression of intact hSNAP25-IgG1 (SNAP25H03) antibody.

[0137] Based on the above research, a specific anti-hSNAP25 humanized single-chain antibody, namely Clone100-scFv, was screened from a large-capacity phage antibody library. Since the 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-Clone100-scFv single-chain antibody was converted into a complete IgG1 antibody, named SNAP25H03. The amino acid sequence of the SNAP25H03 heavy chain is shown in SEQ ID No.7, and the amino acid sequence of the SNAP25H03 light chain is shown in SEQ ID No.8.

[0138] 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.

[0139] 1) Plasmid preparation: Transform SNAP25H03 antibody light chain plasmid and SNAP25H03 antibody heavy chain plasmid into DH5α competent bacteria respectively, and then inoculate them into LB agar culture plates containing Amp antibiotics for resistance screening and culture. Pick positive clones and inoculate them into 150 ml LB medium containing 150 μl 100X Amp antibiotics, shake well, and incubate overnight. Extract the plasmid according to the instructions of the plasmid extraction kit.

[0140] 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. Check that the cell confluence reached 70-80%. The 293F cell density was adjusted to about 1.1×10 6 cells / mL. Dilute the heavy and light chain plasmids and polyethyleneimine (PEI) separately in Opi-MEM and mix them together (light chain: heavy chain = 1:1; PEI: plasmid = 2.7:1). Add the mixture to the flask containing 293F cells at once and shake gently, moving crosswise to ensure thorough mixing. Transfection is more efficient if the cells are in suspension. Place the flask in a CO2 incubator at 37°C and 5% CO2 for 6 days.

[0141] 3) SNAP25H03 antibody purification: Centrifuge the 293F cell suspension at 8000 rpm for 30 minutes at 4°C, and filter the cell supernatant with a 0.22 μm filter. Purify the antibody with protein A, transfer the filtrate to a dialysis bag, and stir overnight at 4°C. Remove the dialysate, detect the final antibody concentration with Nanodrop, filter with a 0.22 μm filter, and take a 0.1 mg protein sample for concentration determination, SDS-PAGE, and HPLC.

[0142] like Figure 2 As shown, 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 proteins migrated at 130-180 kDa. Figure 3 The results of SNAP25H03 antibody purity determination using HPLC are shown, and the results show that the purity of SNAP25H03 is 99.3%.

[0143] Example 6

[0144] This example shows the affinity determination of the SNAP25H03 antibody.

[0145] 1) Immobilization of anti-human IgG (Fc): Anti-human IgG (Fc) was diluted to 25 μg / mL in the immobilization buffer and immobilized on the surface of the carboxymethyl dextran (CM5) sensor chip (BIAcore), 10 μL / min, 420 s. The amount of anti-human IgG (Fc) antibody coupled to the CM5 sensor chip was approximately 7000-14000 RU.

[0146] 2) SNAP25H03 antibody dilution: Dilute SNAP25H03 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.

[0148] The specific binding affinity results are shown in Table 4.

[0149] Table 4 SNAP25H03 antibody affinity

[0150]

[0151] Example 7

[0152] This example shows the application of human SNAP25H03 antibody in the detection of botulinum toxin endopeptidase activity.

[0153] 1) Primary antibody incubation: Dilute the human SNAP25H03 antibody and the commercially available SNAP25 rabbit polyclonal antibody S9684 with 1×PBS buffer at 1:1000, 100 μL / well, coat with shaking overnight at 4°C, wash three times with a plate washer (PBST), and pat dry. Block with 2% BSA solution at room temperature for 1.5 h, discard, and pat dry.

[0154] 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; pH7.0) 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 hSNAP25 protein to 0.25 μg / ml with the prepared diluent, and add 50 μL / well to the activated toxin well. Place in a constant temperature shaker at 37°C, shake at 400 rpm, and incubate and cut for 10 min. After 10 min, remove from the constant temperature shaker, add 90 μL / well to the blocked ELISA enzyme-labeled plate, shake overnight at 4°C, wash three times with a plate washer, and pat dry.

[0155] 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.

[0156] 4) ELISA color development: Mix the color development solution in a 1:1 ratio, add 100 μL / well of color development solution, and measure the optical density value at D450 on an ELISA reader.

[0157] Figure 5 The results of the detection of BoNT / A endopeptidase activity by SNAP25H03 and the commercially available S9684 antibody are shown. The data were analyzed by four-parameter fitting using GraphPad Prism 9.4.1. The results show that the EC value of SNAP25H03 for detecting BoNT / A is 50 The EC value of S9684 antibody for detecting BoNT / A is 8.47 U / ml. 50 It is 25.19 U / ml.

[0158] 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 SNAP25 or for determining the activity of botulinum toxin, and comprises an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof is capable of targeting full-length SNAP25 and / or a fragment of SNAP25, and the antibody or the antigen-binding fragment thereof comprises heavy chain CDR1-3 having an amino acid sequence as shown in SEQ ID NOs.1-3 and light chain CDR1-3 having an amino acid sequence as shown in SEQ ID NOs.4-6.

2. Use of the reagent in preparing a kit for determining SNAP25, characterized in that: The reagent comprises an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof is capable of targeting the full-length SNAP25 and / or the fragment of SNAP25, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1-3 as shown in the amino acid sequence of SEQ ID NO.1-3 and a light chain CDR1-3 as shown in the amino acid sequence of SEQ ID NO.4-6, and the determination of SNAP25 comprises the following steps: (1) contacting the sample with the antibody or antigen-binding fragment thereof for a time and under conditions sufficient to form an antibody or antigen-binding fragment thereof / antigen complex; (2) detecting the binding of the sample to the antibody or antigen-binding fragment thereof, or detecting the presence of the antibody or antigen-binding fragment thereof / antigen complex, so as to determine the presence of SNAP25 in the sample or the amount of SNAP25 in the sample.

3. Use of the reagent in preparing a kit for determining the activity of botulinum toxin, characterized in that: The reagent comprises an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof is capable of targeting the full-length SNAP25 and / or the fragment of SNAP25, wherein 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 a light chain CDR1-3 having an amino acid sequence as shown in SEQ ID NO.4-6, and the determination of the botulinum toxin activity comprises the following steps: (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; (b) contacting the antibody or antigen-binding fragment thereof with the SNAP25 cleaved by the botulinum toxin under conditions suitable for binding of the antibody or antigen-binding fragment thereof to the SNAP25 cleaved by the botulinum toxin to obtain a first complex; (c) contacting the additional antibody with the first complex under conditions suitable for binding of the first complex to the additional antibody to obtain a second complex; (d) detecting the amount of the second complex and determining the botulinum toxin activity; Wherein, the additional antibody is an alkaline phosphatase-conjugated antibody, a horseradish peroxidase-conjugated antibody or a fluorescent dye-conjugated antibody.

4. An antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof can target SNAP25, and the antibody or antigen-binding fragment thereof comprises heavy chain CDR1-3 as shown in amino acid sequences of SEQ ID NO.1-3 and light chain CDR1-3 as shown in amino acid sequences of 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 has any one of the amino acid sequences shown in (I)-(III): (I) comprising the heavy chain amino acid sequence shown in SEQ ID NO.7 and the light chain amino acid sequence shown in SEQ ID NO.8; (II) an amino acid sequence that has at least 90% homology to the amino acid sequence shown in (I) and has the same function; (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.

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 of claim 7.

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

Images