A rabbit-derived monoclonal antibody against human NPTXR protein

Abstract

The disclosure provides a rabbit-derived monoclonal antibody against human NPTXR protein, and belongs to the field of biological detection and bioengineering technology. The rabbit-derived monoclonal antibody against human NPTXR protein comprises HZK26-B0008. The monoclonal antibody provided by the disclosure can highly specifically recognize NPTXR protein, and compared with mouse or rat antibodies, the rabbit-derived monoclonal antibody is more prone to humanization due to the characteristics of large genetic diversity, simple immunoglobulin structure and high affinity.

Description

Technical Field

[0001] This disclosure relates to the fields of biological detection and bioengineering, and in particular to a rabbit-derived monoclonal antibody against human NPTXR protein. Background Technology

[0002] Alzheimer's disease (AD) is a prevalent degenerative disease of the central nervous system. Its important pathological features include β-amyloid (Aβ) plaque deposition, neurofibrillary tangles (NFTs) formed by phosphorylated tau protein, and neuronal damage and loss.

[0003] Related technologies indicate that, in addition to classic pathological molecules such as β-amyloid and tau protein, synaptic-associated proteins also play a crucial role in the development and progression of neurodegenerative diseases. The expression level or distribution of the neuronal pentraxin receptor (NPTXR) is abnormal in various neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and frontotemporal degeneration, and this abnormal change may precede the appearance of typical pathological features.

[0004] However, existing detection methods for NPTXR proteins still have limitations. Reported antibodies vary in specificity, affinity, and stability, making it difficult to meet the demands for high sensitivity and reproducibility. Furthermore, antibody resources targeting different epitopes are relatively scarce, limiting their application in detection and diagnostic products. Therefore, there is a need to develop highly specific monoclonal antibodies against human NPTXR proteins and related detection technologies for the detection, auxiliary diagnosis, and disease progression assessment of neurodegenerative diseases. Summary of the Invention

[0005] In view of this, in order to at least partially solve at least one of the aforementioned technical problems, this disclosure provides a rabbit-derived monoclonal antibody against human NPTXR protein.

[0006] According to one embodiment of this disclosure, a rabbit-derived monoclonal antibody against human NPTXR protein is provided. The monoclonal antibody includes a light chain variable region and a heavy chain variable region. The monoclonal antibody includes HZK26-B0008, wherein the heavy chain variable region of HZK26-B0008 includes the CDR sequences shown in SEQ ID No. 67 to SEQ ID No. 69, and the light chain variable region includes the CDR sequences shown in SEQ ID No. 70 to SEQ ID No. 72.

[0007] According to another embodiment of this disclosure, a nucleic acid molecule is provided that encodes the aforementioned monoclonal antibody.

[0008] According to another aspect of the present disclosure, a recombinant expression vector is provided, comprising the aforementioned nucleic acid molecules.

[0009] According to another embodiment of this disclosure, a host cell is provided, comprising the above-described recombinant expression vector.

[0010] According to another aspect of this disclosure, a composition is provided comprising at least one of the above-described monoclonal antibody, the above-described nucleic acid molecule, the above-described recombinant expression vector, and the above-described host cell.

[0011] According to another embodiment of this disclosure, the use of the above-described monoclonal antibody, the above-described nucleic acid molecule, the above-described recombinant expression vector, the above-described host cell, or the above-described composition in the preparation of a kit for detecting NPTXR protein is provided.

[0012] According to another embodiment of this disclosure, a method for preparing a monoclonal antibody is provided, including the step of culturing the host cells described above.

[0013] According to the embodiments of this disclosure, a rabbit monoclonal antibody HZK26-B0008 that recognizes human NPTXR protein has been developed. It does not cross-react with other homologous sites of human NPTXR protein or related proteins at other sites (such as NP1 and NP2). Compared to mouse or rat antibodies, the rabbit monoclonal antibody provided in this disclosure has greater genetic diversity and a simpler immunoglobulin structure, making it easier to humanize for subsequent clinical applications and reducing potential immunogenicity. The HZK26 series of monoclonal antibodies prepared in this disclosure can bind to human NPTXR protein with high specificity, and more specific detection kits for related humoral markers can be developed based on this. Attached Figure Description

[0014] The above and other objects, features, and advantages of this disclosure will become clearer from the following description of embodiments of the present disclosure with reference to the accompanying drawings, in which:

[0015] Figure 1 The diagram below illustrates the flow cytometry process for preparing monoclonal antibodies to sort B cells according to an embodiment of this disclosure. In the diagram, A is a forward scattering light-area map, B is a 7-aminoactinomycin D screening map, C is an immunoglobulin G signal map, and D is a phycocyanin-area map.

[0016] Figure 2 This is a schematic diagram illustrating the principle of obtaining variable region fragments of antibody heavy and light chains by nested PCR amplification as disclosed in this invention.

[0017] Figure 3This is a Western blot result of the HZK26-B0008 antibody binding to a human brain tissue sample, as described in an embodiment of this disclosure. Detailed Implementation

[0018] The embodiments of the present disclosure will now be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of the disclosure. In the following detailed description, numerous specific details are set forth to provide a thorough understanding of the embodiments of the present disclosure for ease of explanation. However, it will be apparent that one or more embodiments may be practiced without these specific details. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of the present disclosure.

[0019] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. The term "comprising" as used herein indicates the presence of features, steps, or operations, but does not exclude the presence or addition of one or more other features.

[0020] When using expressions such as "at least one of A, B, and C," the expression should generally be interpreted in accordance with the meaning commonly understood by a person skilled in the art (e.g., "a system having at least one of A, B, and C" should include, but is not limited to, a system having A alone, a system having B alone, a system having C alone, a system having A and B, a system having A and C, a system having B and C, and / or a system having A, B, and C, etc.). When using expressions such as "at least one of A, B, or C," the expression should generally be interpreted in accordance with the meaning commonly understood by a person skilled in the art.

[0021] The term "homology" refers to the level of similarity or percentage identity between polynucleotide sequences in terms of percentage nucleotide positional similarity (i.e., sequence similarity or identity). As used here, homology also refers to the concept of similar functional properties between different polynucleotide molecules; for example, promoters with similar functions may have homologous cis elements. Polynucleotide molecules are homologous when they specifically hybridize under certain conditions to form a double-stranded molecule. Under these conditions (called stringent hybridization conditions), one polynucleotide molecule can be used as a probe or primer to identify another polynucleotide molecule sharing homology.

[0022] The term "promoter" refers to a polynucleotide molecule that, in its native state, is located upstream of or 5' of the translation start codon in the reading frame (or protein-coding region) and participates in the recognition and binding of RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription.

[0023] The term "operably linked" refers to the linkage of a first polynucleotide molecule (e.g., a promoter) to a second transcribed polynucleotide molecule (e.g., a target gene), wherein the polynucleotide molecules are arranged such that the first polynucleotide molecule influences the function of the second polynucleotide molecule. Preferably, the two polynucleotide molecules are portions of a single, consecutive polynucleotide molecule, and more preferably, they are adjacent. For example, if a promoter regulates or mediates the transcription of a target gene within the cell, then the promoter is operably linked to the target gene.

[0024] The term "conversion" refers to the method of introducing a heterologous DNA sequence into a host cell or organism.

[0025] The term "expression" refers to the transcription and / or translation of endogenous or exogenous genes in host cells.

[0026] The term "host cell" refers to a cell containing the polynucleotides of this disclosure, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, pairing, or other methods known in the art. The exogenous polynucleotides may remain as, for example, non-integrating vectors of plasmids or may be integrated into the host genome.

[0027] The term "monoclonal antibody," unless otherwise specified, generally refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one "light" (L) chain and one "heavy" (H) chain). In a general sense, the heavy chain can be understood as the larger polypeptide chain in the antibody, and the light chain as the smaller polypeptide chain. Light chains can be classified as κ and λ light chains. Heavy chains are typically classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The constant region of the light chain consists of a single CL domain. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antibody binding sites.

[0028] The aforementioned CDR is a sequence defined according to the Kabat numbering system. Within the scope of this disclosure, the antibody may be in various forms, such as a full-length antibody, a Fab fragment, an F(ab')2 fragment, or a single-chain Fv fragment.

[0029] In realizing the concept disclosed herein, it was discovered that during the occurrence and development of neurodegenerative diseases such as Alzheimer's disease (AD), abnormal changes occur in the expression level, spatial distribution, or conformational state of human neuronal synapse-associated protein X receptor (NPTXR), thereby disrupting synaptic structural stability and leading to synaptic dysfunction. This, in turn, causes pathological changes such as impaired information transmission between neurons and gradual neuronal loss. Studies have shown that human NPTXR protein in pathological states can coexist in soluble and abnormally aggregated forms, and its distribution characteristics on the neuronal membrane surface and synaptic region are closely related to the occurrence and development of neurodegenerative diseases.

[0030] Abnormal changes in human NPTXR protein levels or related molecular characteristics can be detected in cerebrospinal fluid and peripheral body fluid samples from AD patients. Since obtaining cerebrospinal fluid samples requires invasive procedures such as lumbar puncture, it is difficult to promote its application in large-scale population screening. Related technologies indicate that NPTXR-related indicators in peripheral blood and other body fluid samples also have the potential for disease prediction and assessment. Therefore, developing highly specific antibodies against human NPTXR protein holds significant promise for constructing a fluid-based biomarker-based detection method for neurodegenerative diseases.

[0031] Specifically, according to one aspect of this disclosure, a rabbit-derived monoclonal antibody against human NPTXR protein is provided. The monoclonal antibody includes a light chain variable region and a heavy chain variable region, and the monoclonal antibody includes HZK26-B0008. The respective heavy chain and light chain variable regions are shown below.

[0032] The heavy chain variable region of the HZK26-B0008 monoclonal antibody contains the following CDR sequence:

[0033] (i) GFTLSSYY (as shown in SEQ ID No. 67);

[0034] (ii) IYIDSSGI (as shown in SEQ ID No. 68);

[0035] (iii) AREGYGTGGDYGYDL (as shown in SEQ ID No. 69);

[0036] The light chain variable region of the HZK26-B0008 monoclonal antibody contains the following CDR sequence:

[0037] (i) QSTASY (as shown in SEQ ID No. 70);

[0038] (ii) QAS (as shown in SEQ ID No. 71);

[0039] (iii) HAYYGISYTA (as shown in SEQ ID No. 72).

[0040] According to the embodiments of this disclosure, a rabbit monoclonal antibody HZK26-B0008 that recognizes human NPTXR protein has been developed. It does not cross-react with other homologous sites of human NPTXR protein or related proteins at other sites (such as NP1 and NP2). Compared to mouse or rat antibodies, the rabbit monoclonal antibody provided in this disclosure has greater genetic diversity and a simpler immunoglobulin structure, making it easier to humanize for subsequent clinical applications and reducing potential immunogenicity. The HZK26 series of monoclonal antibodies prepared in this disclosure can bind to human NPTXR protein with high specificity, and more specific detection kits for related humoral markers can be developed based on this.

[0041] According to embodiments of this disclosure, the heavy chain and light chain variable region sequences of the monoclonal antibody HZK26-B0008 are shown below.

[0042] The amino acid sequence of the B0008-H variable region (rabbit IgG1) (SEQ ID No. 124):

[0043] QSVKESGGDLVKPGASLTLTCTASGFTLSSYYMCWVRQAPGKGLKWIGCIYIDSSGIWYASWAKGRFTISKTSSTTVTLQMTSLTVADTATYFCAREGYGTGGDYGYDLWGPGTLVTVSS.

[0044] B0008-L variable region amino acid sequence (rabbit IgG1) (SEQ ID No. 136):

[0045] DVVMTQTPASVSEPVGGTVTIKCQASQSTASYLAWYQQKPGQPPKRLIYQASKVASGVPSRFKGSGSGTEYTLTISDLECADAATYYCHAYYGISYTAFGGGSEVVVK.

[0046] According to embodiments of this disclosure, the heavy chain sequence of HZK26-B0008 is shown in SEQ ID No. 4, and the light chain sequence is shown in SEQ ID No. 16.

[0047] B0008-H full-length amino acid sequence (rabbit IgG1) (SEQ ID No.4):

[0048] MGWSLILLFLVAVATRVLSQSVKESGGDLVKPGASLTLTCTASGFTLSSYYMCWVRQAPGKGLKWIGCIYIDSSGIWYASWAKGRFTISKTSSTTVTLQMTSLTVADTATYFCAREGYGTGGDYGYDLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK .

[0049] B0008-L full-length amino acid sequence (rabbit kappa chain) (SEQ ID No.16):

[0050] MGWSCIILFLVATATGVHSDVVMTQTPASVSEPVGGTVTIKCQASQSTASYLAWYQQKPGQPPKRLIYQASKVASGVPSRFKGSGSGTEYTLTISDLECADAATYYCHAYYGISYTAFGGGSEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC.

[0051] According to an embodiment of another aspect of this disclosure, nucleotides encoding any of the above-described monoclonal antibodies are provided. The nucleic acid molecule comprises the base sequence shown in any one of SEQ ID No. 28, SEQ ID No. 40, SEQ ID No. 148, and SEQ ID No. 160.

[0052] B0008-H full-length nucleotide sequence (rabbit IgG1) (SEQ ID No. 28):

[0053]

[0054] Full-length nucleotide sequence of B0008-L (rabbit kappa chain) (SEQ ID No.40):

[0055] ATGGGCTGGTCCTGTATCATCCTGTTCCTGGTGGCTACAGCCACAGGAGTGCATAGTGATGTCGTGATGACCCAGACTCCAGCCTCCGTGTCTGAACCTGTGGGAGGCACAGTCACCATCAAGTGTCAGGCCAGTCAGAGCACTGCTAGCTACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCGCCTGATCTACCAGGCATCCAAAGTGGCATCTGGGGTCCCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTACACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCACGCCTATTATGGTATTAGTTATACTGCTTTCGGCGGAGGGTCCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAA。

[0056] B0008-H variable region nucleotide sequence (rabbit IgG1) (SEQ ID No.148): CAGTCGGTGAAGGAGTCCGGGGGAGACCTGGTCAAGCCTGGGGCATCCCTGACACTCACCTGCACAGCTTCTGGATTCACCCTCAGTAGCTACTACATGTGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGAAATGGATCGGATGCATTTATATTGATAGTAGTGGCATATGGTA CGCGAGCTGGGCGAAAGGCCGGTTCACCATCTCCAAAACCTCGTCGACCACGGTGACTCTGCAAATGACCAGTCTGACAGTCGCGGATACGGCCACCTATTTCTGTGCGAGAGAGGGATATGGTACTGGTGGTGATTATGGTTATGACTTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA.

[0057] B0008-L variable region nucleotide sequence (rabbit kappa chain) (SEQ ID No.160): TGTCGTGATGACCCAGACTCCAGCCTCCGTGTCTGAACCTGTGGGAGGCACAGTCACCATCAAGTGTCAGGCCAGTCAGAGCACTGCTAGCTACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCGCCTGATCTACCAGGCATCCAAA GTGGCATCTGGGGTCCCATCCGGTTCAAAGGCAGTGGATCTGGGACAGAGTACACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCACGCCTATTATGGTATTAGTTATACTGCTTTCGGCGGAGGGTCCGAGGTGGTGGTCAAA.

[0058] According to an embodiment of another aspect of this disclosure, a recombinant expression vector is provided, comprising a nucleic acid molecule with a base sequence as shown in any one of SEQ ID No. 28, SEQ ID No. 40, SEQ ID No. 148, and SEQ ID No. 160 above.

[0059] Specifically, this disclosure does not limit the types of recombinant expression vectors. For example, expression vectors can be constructed using isolated mammalian cells, and corresponding recombinant expression vectors can be obtained through adaptive design.

[0060] According to embodiments of this disclosure, a vector refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell through transformation, transduction, or transfection, allowing the genetic material elements it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage, and animal viruses. A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may also contain a replication initiation site.

[0061] According to another aspect of the present disclosure, a host cell comprising the above-described nucleotide sequence or the above-described expression vector is provided.

[0062] For example, genes encoding antibody heavy and light chains (whether the complete sequence of the variable region fused with the constant region, or the variable region fused with the vector's own constant region sequence) are inserted into the multiple cloning site of such vectors to form heavy chain expression plasmids and light chain expression plasmids, and then co-transfected into HEK293 cells for expression to obtain the corresponding host cells.

[0063] According to another aspect of this disclosure, a composition is provided comprising at least one of the above-described monoclonal antibody, the above-described nucleic acid molecule, the above-described recombinant expression vector, and the above-described host cell.

[0064] According to embodiments of this disclosure, the composition further includes a pharmaceutically acceptable carrier.

[0065] According to embodiments of this disclosure, the above composition may include pharmaceutically acceptable excipients, diluents or carriers, etc.; it may also include essential components as a detection kit.

[0066] Specifically, pharmaceutically acceptable excipients refer to substances added to pharmaceutical formulations other than the active ingredient, used to improve the physical properties, stability, or bioavailability of the drug. They can be classified into various types according to their function, such as disintegrants, binders, and lubricants. They must meet conditions such as being non-toxic, non-irritating, and not chemically reacting with the active ingredient to ensure the safety and efficacy of the drug. Diluents are mainly used to adjust drug concentrations to achieve appropriate dosage specifications. Diluents need to be chemically stable, not affect the activity of antibody or antigen-binding fragments, and have good flowability and compressibility to facilitate formulation molding.

[0067] Specifically, the essential components of a test kit may include solid supports, such as ELISA plates, nitrocellulose membranes, and chips, for coating antigens or antibodies; calibrators and quality controls, including a series of standards containing known concentrations of NPTXR protein, for plotting standard curves and monitoring the effectiveness of the detection process; labeled secondary antibodies, such as horseradish peroxidase-labeled goat anti-rabbit IgG, for signal amplification and detection; substrate chromogenic solutions / chemiluminescent substrates, which react with labeled enzymes to generate detectable signals (color or light); and washing, diluents, and blocking solutions, for sample dilution, blocking of non-specific binding sites, and washing between steps.

[0068] In some specific embodiments of this disclosure, the monoclonal antibody composition may exist in various forms, such as liquid, lyophilized powder, and other formulations.

[0069] According to another aspect of this disclosure, a composition is provided comprising the above-described monoclonal antibody, the above-described nucleic acid molecule, the above-described recombinant expression vector, the above-described host cell, or the above-described composition, for use in the preparation of a kit for detecting NPTXR protein.

[0070] Specifically, nucleic acid molecules may also include all regulatory sequences necessary to initiate the expression of nucleic acid molecules encoding antibody or antigen-binding fragments. These regulatory sequences, under compatible conditions, guide the expression of the coding sequence in suitable host cells. Regulatory sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequence must include a promoter and termination signals for transcription and translation. To introduce specific restriction enzyme sites into the vector for linking the regulatory sequence to the coding region of the nucleic acid sequence encoding the protein, a regulator-linked regulatory sequence may be provided. The regulatory sequence may be a suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains transcriptional regulatory sequences that mediate protein expression. The promoter may be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutated, truncated, and heterozygous promoters, and may be derived from genes encoding extracellular or intracellular proteins that are homologous or heterologous to those of the host cell. The regulatory sequence may also be a suitable transcription terminator sequence, i.e., a sequence that can be recognized by the host cell to terminate transcription. The termination sequence is operatively linked to the 3' end of a nucleic acid sequence encoding an antibody or antigen-binding fragment. Any terminator that functions in a selected host cell may be used in this disclosure. Any leader sequence that functions in a selected host cell may be used in this disclosure. The regulatory sequence may also be a signal peptide coding region encoding an amino acid sequence linked to the amino terminus of a protein (i.e., an antibody or antigen-binding fragment) that guides the encoded protein (i.e., the antibody or antigen-binding fragment) into the cellular secretory pathway. Signal peptide coding regions that guide the expressed protein (i.e., the antibody or antigen-binding fragment) into the secretory pathway of the host cell used may be used in this disclosure. Adding a regulatory sequence that can regulate protein (i.e., antibody or antigen-binding fragment) expression according to the growth status of the host cell may also be necessary. Examples of regulatory sequences are those that respond to chemical or physical stimuli (including in the presence of regulatory compounds), thereby opening or closing gene expression. Other examples of regulatory sequences are those that enable gene amplification. In these examples, the nucleic acid sequence encoding the protein (i.e., the antibody or antigen-binding fragment) should be operatively linked to the regulatory sequence.

[0071] According to embodiments of this disclosure, the kit is used for one or more of the following: treating or preventing neurodegenerative diseases, diagnosing or assisting in the diagnosis of neurodegenerative diseases, monitoring NPTXR levels, differentiating neurodegenerative diseases from other forms of disease, and assessing the effectiveness of treatment and disease progression in neurodegenerative diseases.

[0072] According to another aspect of the embodiments of this disclosure, neurodegenerative diseases include Alzheimer's disease, Pick's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, or spinocerebellar ataxia, and other forms of disease include Lewy body dementia and frontotemporal degeneration.

[0073] In some specific embodiments of this application, the above-described kit can be used to detect NPTXR levels in peripheral blood and other body fluids to differentiate and diagnose neurodegenerative diseases and other forms of diseases, and to assess the treatment efficacy and disease progression of neurodegenerative diseases.

[0074] According to another embodiment of this disclosure, a method for preparing a monoclonal antibody is provided, including the step of culturing the host cells described above.

[0075] Specifically, an expression vector containing the heavy and light chain nucleotide sequences encoding the monoclonal antibody disclosed herein can be introduced into a suitable mammalian host cell (e.g., but not limited to, HEK293 cells, CHO cells, or SP2 / 0 cells) to obtain a recombinant host cell capable of expressing the antibody.

[0076] The technical solutions of this disclosure will be further explained and described below with reference to specific embodiments and accompanying drawings. Unless otherwise specified, all reagents used are commercially available reagents or can be prepared by known methods, and all test / experiment methods used are conventional methods in the art.

[0077] Example 1: Preparation of NPTXR monoclonal antibody

[0078] 1.1 Immunoassay and Serum Titer Detection

[0079] Immunological and serum titer assay reagents: complete Freund's adjuvant (Sigma), incomplete Freund's adjuvant (BD), horseradish enzyme-labeled goat anti-rabbit IgG (H+L) (IR), coating solution (Yiqiao Shenzhou), washing solution (Yiqiao Shenzhou), blocking solution (Yiqiao Shenzhou), secondary antibody diluent (Yiqiao Shenzhou), sample diluent (Yiqiao Shenzhou), chromogenic solution (Yiqiao Shenzhou), and stop solution (Yiqiao Shenzhou).

[0080] Immunization and Serum Titer Detection Methods: Two healthy New Zealand white rabbits were used in this experiment. For the initial immunization, 500 μg of antigen was emulsified with an equal volume of complete Freund's adjuvant and injected subcutaneously into the rabbits at multiple sites on the back. Subsequently, booster immunizations were performed every two weeks, using the same dose of antigen emulsified with an equal volume of incomplete Freund's adjuvant. After the fourth immunization, blood was collected from the marginal ear vein, and serum titer was measured using an indirect ELISA method to determine if a follow-up immunization was necessary. Once the serum titer reached the acceptable standard, peripheral blood was collected for B cell sorting.

[0081] 1.2 Antigen labeling and flow cytometry sorting of B cells

[0082] Antigen labeling and flow cytometry reagents for B cell sorting: 2-Iminothiolactone (2-IT, SIGMA), phycoerythrin (PE, Agilent), allophycocyanin (APC, Agilent), succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylate (SMCC, Thermo), N-ethylmaleimide (NEM, SIGMA), protective agent (Sino-American), phosphate-buffered saline (PBS, Sino-American), rabbit IgG antibody (Anti-rabbit IgG), rabbit IgM antibody (Anti-rabbit IgM), S1h-3C-crosslinker-APC fluorescent labeling complex (S1h-3C-NPTXR-16070H1-APC), S1h-3C-crosslinker-PE fluorescent labeling complex (S1h-3C-NPTXR-16070H1-PE), 7-aminoactinomycin (D7-AAD).

[0083] PE marking method:

[0084] (1) Concentrate PE to approximately 3 mg / mL and determine its accurate concentration using ultraviolet spectrophotometry. Subsequently, add crosslinking agent SMCC at a certain molar ratio and react at room temperature in the dark. The N-hydroxysuccinimide (NHS) end of SMCC will react with the primary amino group on the surface of PE protein, thereby introducing maleimide (MAL) active groups onto PE.

[0085] (2) Mix the activated PE with an appropriate amount of peptide SinoA12331 dissolved in PBS and mix thoroughly. Place the mixture at 4°C and react overnight in the dark.

[0086] (3) After the reaction was completed, the conjugated product was subjected to multiple buffer changes (replaced with the required storage buffer PBS) using ultrafiltration centrifuge tubes to thoroughly remove unreacted peptides, free dyes, and small molecule byproducts. Finally, the concentration of the purified product was determined again by ultraviolet spectrophotometry, and the yield of the final PE-peptide conjugate was calculated.

[0087] APC tagging method:

[0088] (1) APC was concentrated to about 3 mg / mL. After the accurate concentration was determined by ultraviolet spectrophotometry, SMCC was added and the reaction was carried out at room temperature in the dark. The N-hydroxysuccinimide (NHS) end of SMCC was bound to the primary amino group on the surface of APC, thereby introducing maleimide (MAL) active groups onto APC.

[0089] (2) Mix the NPTXR antigen peptide 16070-H07H (Yiqiao Shenzhou) dissolved in PBS with the activated APC, mix thoroughly, and react overnight at 4°C in the dark.

[0090] (3) After the reaction was completed, the conjugated product was replaced with an ultrafiltration centrifuge tube with a suitable buffer such as PBS to remove unreacted peptides and small molecule impurities. Finally, the concentration of the purified product was determined by ultraviolet spectrophotometry, and the final yield of the APC-peptide conjugate was calculated.

[0091] Flow cytometry sorting of B cells:

[0092] (1) Add EDTA-anticoagulated rabbit whole blood to a centrifuge tube containing gradient separation medium (Ficoll) and centrifuge. After centrifugation, aspirate the white, cloudy cell layer (i.e., the white membrane layer rich in lymphocytes and monocytes) located between the separation medium interfaces to a new centrifuge tube. Add sufficient phosphate buffer to wash the cells, centrifuge, and discard the supernatant to obtain relatively pure peripheral blood mononuclear cells (PBMCs).

[0093] (2) Add IgG, IgM antibodies and fluorescently labeled proteins to the isolated PBMC cells and incubate them. After incubation, wash the cells with PBS 2-3 times to completely remove unbound free antibodies. Finally, resuspend the cells with an appropriate amount of PBS.

[0094] (3) The cells were detected by flow cytometry. The flow cytometry results are as follows: Figure 1 As shown.

[0095] Figure 1 The diagram below illustrates the flow cytometry process for preparing monoclonal antibodies to sort B cells according to an embodiment of this disclosure. In the diagram, A is a forward scattering area map, B is a 7-aminoactinomycin D screening map, C is an immunoglobulin G signal map, and D is an allophycocyanin area map.

[0096] pass Figure 1 The sorting strategy can gradually screen out target B cells from mixed cells, namely antigen-specific B cells that can bind to NPTXR.

[0097] 1.3 Expansion of positive rabbit B cell clones

[0098] Rabbit B cell positive clone amplification reagents: 3T3-mouse CD40 ligand feeder cells (3T3-msCD40L feeder cells, CellRON), mitomycin C (MCE), modified Durbeco medium (IMDM medium, GIBCO), phytohemagglutinin-M (PHA-M, Roche), fetal bovine serum (GIBCO), interleukin-2, interleukin-21 (IL2, IL21 cytokines, Sinocare).

[0099] One day before B cell sorting, prepare mitomycin C-treated feeder cells and seed them into 96-well cell culture plates to allow them to adhere. On the day of sorting, inject single B cells that meet the screening criteria into wells containing feeder cells. After sorting, add culture medium containing mitogens and cytokines and incubate in a CO2 incubator for about one week. Take a portion of the culture supernatant for ELISA detection.

[0100] The reagents used in ELISA are shown in Table 1:

[0101] Table 1

[0102]

[0103] ELISA detection method:

[0104] (1) Dilute the project peptide and cross-peptide to working concentrations (0.5 μg / mL and 5 μg / mL, respectively) using coating buffer. Add 100 μL of the diluted antigen solution to each well of the ELISA plate, seal and coat overnight at 4°C.

[0105] (2) Discard the coating solution inside the plate and pat dry on absorbent paper. Add 300 μL of blocking solution to each well, seal and incubate at room temperature (25℃) for 1 hour.

[0106] (3) Add 300 μL of washing solution to each well, let stand for about 30 seconds and then discard the liquid. Repeat twice and drain on absorbent paper.

[0107] (4) Use sample diluent to dilute the cell culture supernatant by a ratio of 5, and vortex thoroughly to mix well, and set aside.

[0108] (5) Add 100 μL of diluted sample supernatant to each well of the sealed and washed ELISA plate. Place the ELISA plate on a horizontal shaker and mix at low speed. Incubate at room temperature for 2 hours.

[0109] (6) Wash the plate 3 times with 300 μL / well washing solution, and pat dry the plate after the last wash.

[0110] (7) Dilute the horseradish peroxidase-labeled Goat Anti-Rabbit IgG Fc / HRP to the working concentration, mix well, add at 100 μL / well, and incubate at room temperature for 2 h.

[0111] (8) Same as step (3), but increase the number of washes to three to thoroughly remove unbound components.

[0112] (9) Mix solution A and solution B at a ratio of 1:1, add 200 μL to each well, and incubate at room temperature in the dark for 3 min.

[0113] (10) Add 50 μL of stop solution to each well and immediately measure the absorbance (OD value) of each well at a wavelength of 450 nm using an ELISA reader.

[0114] The results of the first sorting of B cell culture supernatant by ELISA are shown in Table 2.

[0115] Table 2

[0116]

[0117] Based on the data in the table above, positive B cell clones capable of secreting specific antibodies could be screened by detecting the antibody binding signal to NPTXR protein in the culture supernatant of each well. The results showed that all B cell culture supernatants exhibited higher OD values ​​for NPTXR protein than the blank control, indicating that these B cells could secrete antibodies that specifically bind to NPTXR. These positive clones were further used for subsequent recombinant antibody expression.

[0118] 1.4 Recombinant Antibody Expression

[0119] Recombinant antibody expression reagents: cell lysis buffer (Lysis Buffer, E-Tech), PCR kit (E-Tech), reverse transcription kit (SRT-200T, E-Tech), RNase inhibitor (Thermo), dithiothreitol (DTT, Thermo), deoxyribonucleoside triphosphate mixture (dNTPs, Thermo), one-step rapid cloning kit (Shanghai Yisheng).

[0120] Recombinant antibody expression method: After lysing qualified B cells from culture supernatant, cDNA was obtained by reverse transcription using the Sinocare Reverse Transcription Kit. The antibody heavy and light chain variable region sequences were then obtained through single B cell amplification technology (the amplification principle is as follows). Figure 2 As shown in the figure, after constructing the variable region fragments of the heavy and light chains into expression vectors, the correctly sequenced heavy and light chain expression plasmids were obtained, which were then transduced into HEK 293 for transient expression and secreted into complete IgG antibodies.

[0121] Figure 2 This is a schematic diagram illustrating the principle of nested PCR amplification to obtain variable region fragments of the heavy and light chains of the antibody. Primers are designed from the stable region of the antibody gene to amplify the target gene DNA. Subsequently, random primers are designed to shorten the amplification range and clone the VDJ fragment.

[0122] 1.5 Antibody Purification

[0123] The reagents used for antibody purification are shown in Table 3:

[0124] Table 3

[0125]

[0126] Antibody purification methods:

[0127] (1) Sample preparation. The collected cell supernatant was centrifuged using a benchtop centrifuge at 4000g for 30 min. The cell supernatant was collected after centrifugation and filtered through a 0.45 μM filter membrane.

[0128] (2) Select a protein A column of appropriate specifications according to the expression level and connect it to the purification system; wash with ultrapure water for 3 column volumes to replace the 25% ethanol stored in the column, and then equilibrate with binding buffer (AC Binding + 1 / 5 stock buffer) for at least 3 column volumes until the UV absorption baseline is stable.

[0129] (3) Load the pretreated sample at an appropriate flow rate to ensure that the antibody and protein A filler are fully combined.

[0130] (4) After loading the sample, use the rinsing buffer to rinse the chromatography column for 5-10 column volumes at the same flow rate until the UV absorption signal returns to the baseline level to remove non-specifically bound proteins.

[0131] (5) Elute with elution buffer (AC Elution) and collect the elution peaks according to the real-time UV monitoring spectrum. Immediately add 2 M Tris-HCl buffer (pH 8.0) to the collected eluent to neutralize it and prevent the antibody from being inactivated under acidic conditions.

[0132] (6) Add 2M Tris, pH 8.0 to neutralize and elute the antibody.

[0133] (7) Use binding buffer (AC Binding) to equilibrate 3 column volumes to neutral.

[0134] (8) In-situ cleaning (CIP) of 5 column volumes or more.

[0135] (9) Use AC Binding to flush with alkali until the pH of the effluent returns to neutral.

[0136] (10) Use 25% ethanol to balance the two column volumes, and seal and store the fully balanced chromatography column.

[0137] The above steps allow for the capture of antibodies from the cell culture supernatant. After elution and neutralization, high-purity antibody solutions B0005, B0006, B0007, B0008, B0010, B0012, B0013, B0014, B0016, B0017, B0026, and B0027 are obtained.

[0138] Example 2: Identification of Monoclonal Antibodies

[0139] 2.1 ELISA detection and identification

[0140] The ELISA test reagents are the same as those in Table 1 of Example 1.

[0141] ELISA detection and identification method:

[0142] (1) Dilute the NPTXR antigen peptide 16070-H07H to 0.1 μg / mL and 1 μg / mL respectively with coating buffer, add 100 μL of the corresponding concentration of antigen solution to each well of the microplate, seal and coat overnight at 4°C.

[0143] (2) Discard the coating solution in the plate and pat dry on absorbent paper. Add 300 μL of blocking solution to each well, seal and incubate at room temperature for 1 hour to cover the non-specific binding sites in the well.

[0144] (3) Discard the sealing solution, add 300 μL of washing solution to each well, wash the plate twice, and pat dry the plate on the last wash.

[0145] (4) Dilute antibodies HZK26-B0005, HZK26-B0006, HZK26-B0007, HZK26-B0008, HZK26-B0010, HZK26-B0012, HZK26-B0013, HZK26-B0014, HZK26-B0016, HZK26-B0017, HZK26-B0026, and HZK26-B0027 (primary antibody) to 0.1 μg / mL, add 100 μL of each to the corresponding well plate, mix well, and incubate at room temperature for 2 h.

[0146] (5) Discard the primary antibody and wash the plate three times with washing solution as in step 3. Pat the plate dry on the last wash to completely remove the unbound primary antibody.

[0147] (6) Dilute the horseradish peroxidase-labeled goat anti-rabbit IgG (Goat Anti-Rabbit IgGFc / HRP) secondary antibody to the recommended working concentration using antibody dilution buffer. Add 100 μL of the diluted secondary antibody to each well and incubate at room temperature for 1 hour.

[0148] (7) Discard the secondary antibody and wash the plate three times with washing solution as in step 3 to completely remove the unbound secondary antibody.

[0149] (8) Mix color development solution A and color development solution B at a ratio of 1:1, add 200 μL of the mixture to each well, and incubate at room temperature in the dark for 20 minutes.

[0150] (9) Add 50 μL of stop solution to each well, mix gently, and immediately use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0151] The ELISA test results are shown in Table 4.

[0152] Table 4. ELISA Detection Results of Purified Antibodies

[0153]

[0154] 2.2 Identification by immunoblotting

[0155] Immunoblotting assay

[0156] (1) Take a human brain tissue sample, add pre-cooled protein lysis buffer (containing protease inhibitor) for homogenization, and lyse on ice for 30 minutes. Then centrifuge at 4℃ and 12000rpm for 20 minutes, and collect the supernatant, which is the total protein sample. Determine the protein concentration using the BCA method, etc., and mix the sample with 5× loading buffer and boil at 100℃ for 5 minutes to denature the protein.

[0157] (2) Prepare a 16% SDS-PAGE gel. Add the prepared protein sample to the well according to the predetermined loading amount. Add electrophoresis buffer to the electrophoresis tank, set the voltage to 120V, and electrophore for about 1 hour until the bromophenol blue indicator reaches the bottom of the gel.

[0158] (3) After electrophoresis, remove the gel and cut 0.22 μm PVDF membranes and filter paper according to the gel size. The PVDF membrane needs to be activated by soaking in methanol for 5-10 seconds. Then, place the sponge, filter paper, gel, PVDF membrane, filter paper, and sponge in the transfer clamp in sequence, ensuring that there are no air bubbles between the layers. Place the transfer clamp into the transfer tank and add pre-cooled transfer buffer. Transfer the membrane at a constant current of 240 mA for 30 minutes under ice bath conditions.

[0159] (5) Use blocking buffer to block the membrane for 1 hour at room temperature.

[0160] (6) Incubate the membrane with the appropriate diluted primary antibody at 4°C overnight; incubate the membrane with the conjugated secondary antibody dilution in the blocking buffer for 1 hour at room temperature.

[0161] (7) Mix the peroxide solution and enhancer in the ECL chemiluminescent substrate in equal volumes (500 μL each) to prepare a reaction solution. Add the reaction solution evenly to the surface of the PVDF film, ensuring the liquid covers all areas. Then, perform exposure detection in a chemiluminescence imager or dark chamber. Adjust the exposure time according to the signal intensity to obtain a clear band image, as shown in the figure. Figure 3 As shown.

[0162] Figure 3 This is a Western blot result of the HZK26-B0008 antibody binding to a human brain tissue sample, as described in an embodiment of this disclosure.

[0163] according to Figure 3 It can be seen that rabbit B cell supernatant and the purified HZK26-B0008 antibody clone can effectively distinguish NPTXR.

[0164] The specific embodiments described above further illustrate the purpose, technical solutions, and beneficial effects of this disclosure. It should be understood that the above descriptions are merely specific embodiments of this disclosure and are not intended to limit this disclosure. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the protection scope of this disclosure.

Claims

1. A rabbit-derived monoclonal antibody against human NPTXR protein, characterized in that, The monoclonal antibody includes a light chain variable region and a heavy chain variable region, and the monoclonal antibody includes HZK26-B0008; wherein... The heavy chain variable regions CDR1, CDR2, and CDR3 sequences of HZK26-B0008 are shown in SEQ ID No. 67 to SEQ ID No. 69, and the light chain variable regions CDR1, CDR2, and CDR3 sequences are shown in SEQ ID No. 70 to SEQ ID No.

72.

2. The monoclonal antibody according to claim 1, characterized in that, The heavy chain variable region sequence of HZK26-B0008 is shown in SEQ ID No. 124, and the light chain variable region sequence is shown in SEQ ID No.

136.

3. The monoclonal antibody according to claim 1 or 2, characterized in that, The heavy chain sequence of HZK26-B0008 is shown in SEQ ID No. 4, and the light chain sequence is shown in SEQ ID No.

16.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the monoclonal antibody as described in any one of claims 1 to 3.

5. The nucleic acid molecule according to claim 4, characterized in that, The nucleic acid molecule includes the nucleotide sequence shown in any one of SEQ ID No. 28, SEQ ID No. 40, SEQ ID No. 148, and SEQ ID No.

160.

6. A recombinant expression vector, characterized in that, Includes the nucleic acid molecules described in claim 4 or 5.

7. A host cell, characterized in that, Includes the recombinant expression vector as described in claim 6.

8. A composition, characterized in that, The composition comprises the monoclonal antibody according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4 or 5, the recombinant expression vector according to claim 6, or the host cell according to claim 7.

9. The composition according to claim 8, characterized in that, It also includes pharmaceutically acceptable carriers.

10. The use of a monoclonal antibody according to any one of claims 1 to 3, a nucleic acid molecule according to claim 4 or 5, a recombinant expression vector according to claim 6, a host cell according to claim 7, or a composition according to claim 8 or 9 in the preparation of a kit for detecting NPTXR protein.

11. The application according to claim 10, characterized in that, The kit is used for one or more of the following: diagnosing or assisting in the diagnosis of neurodegenerative diseases, monitoring NPTXR protein levels in cerebrospinal fluid, differentiating neurodegenerative diseases from other forms of disease, and evaluating the treatment efficacy and disease progression of neurodegenerative diseases; The neurodegenerative diseases include Alzheimer's disease, Pick's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, or spinocerebellar ataxia, and the other forms of disease are Lewy body dementia and frontotemporal degeneration.

12. A method for preparing a monoclonal antibody, comprising the step of culturing the host cells of claim 7.

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