Anti-klhl11 antibodies and uses thereof

Abstract

The present application relates to an anti-KLHL11 antibody and its application, and belongs to the technical field of antibodies, wherein the anti-KLHL11 antibody comprises a VHH, and the VHH comprises CDR1, CDR2 and CDR3 in the amino acid sequence shown as SEQ ID NO: 2. The present application first develops an anti-KLHL11 antibody with high specificity and high affinity, which can be used as a positive reference for an anti-KLHL11 autoantibody kit, thereby solving the problems of difficult development and quality control of a diagnostic kit caused by the difficulty in obtaining positive serum and the complexity of components in the prior art, and the anti-KLHL11 antibody is particularly suitable for a diagnostic kit for autoimmune diseases of the nervous system (such as autoimmune encephalitis).

Description

Technical Field

[0001] This invention belongs to the field of antibodies, specifically relating to an anti-KLHL11 antibody and its applications. Background Technology

[0002] Autoimmune diseases of the nervous system are a group of diseases caused by inflammation and damage resulting from the recognition of autoantigens (self-antigens) by autoimmune molecules and immune cells of the nervous system's own tissue cells. These diseases can occur in the central nervous system, peripheral nervous system, and neuromuscular junction, and include autoimmune encephalitis (AE), central demyelinating diseases, and autoimmune peripheral neuropathy. AE is the most common, clinically presenting with acute or subacute onset (<3 months) and one or more neurological and psychiatric symptoms or clinical syndromes: 1) Limbic symptoms: recent memory loss, seizures, and abnormal mental and behavioral patterns; 2) Encephalitis syndrome: clinical manifestations of diffuse or multifocal brain damage; 3) Clinical manifestations involving the basal ganglia and / or diencephalon / hypothalamus; 4) Mental disorders, but not considered by psychiatric specialists to be non-organic diseases. These diseases primarily affect young and middle-aged adults and have high mortality and disability rates.

[0003] Anti-KLHL11 (Kelch-like protein 11) autoantibodies have become increasingly important biomarkers in neurological disease research in recent years, particularly in the diagnosis of autoimmune neurological disorders.

[0004] Currently, there are detection kits for anti-KLHL11 (Kelch-like protein 11) autoantibodies. However, regardless of whether the kits are prepared using various methods such as cell-based immunofluorescence, immunospot assays, chemiluminescence, colloidal gold assays, or ELISA, numerous difficulties exist in the product design and development stage. Furthermore, there are currently no commercially available national or industry standards for reference. Companies must establish their own reference standards using positive and negative sera for performance verification and quality control studies. However, due to the complex composition of serum and the low incidence of this disease, positive sera are scarce and difficult to collect, which cannot meet the needs of early-stage research and long-term product quality control.

[0005] Therefore, there is an urgent need in this field to develop humanized chimeric antibodies that bind to KLHL11 to meet the requirements for positive reference materials in diagnostic kits. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the present invention aims to provide an anti-KLHL11 antibody and its application, wherein the antibody has high specificity and high sensitivity, can meet the requirements of positive reference materials for diagnostic kits, is easy to store and modify, and wherein the antibody has the ability to recognize the target antigen KLHL11.

[0007] In a first aspect, the present invention provides an anti-KLHL11 antibody comprising VHH, wherein the VHH comprises CDR1, CDR2 and CDR3 in the amino acid sequence shown in SEQ ID NO:2.

[0008] Optionally, CDR1 to CDR3 are determined according to the Kabat numbering system, AbM numbering system, IMGT numbering system, Chothia numbering system, or Contact numbering system.

[0009] Optionally, the amino acid sequence of CDR1 is shown in SEQ ID NO:3, the amino acid sequence of CDR2 is shown in SEQ ID NO:4, and the amino acid sequence of CDR3 is shown in SEQ ID NO:5.

[0010] Optionally, the VHH comprises an amino acid sequence with more than 90% homology obtained by substituting, deleting, and / or adding one or more amino acids and / or terminal modification of any one or more amino acids in the amino acid sequence shown in SEQ ID NO:2.

[0011] Optionally, the VHH comprises an amino acid sequence as shown in SEQ ID NO:2.

[0012] Optionally, the anti-KLHL11 antibody is a humanized chimeric antibody.

[0013] Optionally, the VHH is linked to the human IgG1 Fc segment.

[0014] In a second aspect, the present invention provides the use of the anti-KLHL11 antibody as described in the first aspect in the preparation of a kit for detecting anti-KLHL11 autoantibodies.

[0015] Optionally, the method for detecting anti-KLHL11 autoantibodies is selected from at least one of CBA, immunoblotting, membrane strip method, ELISA, chemiluminescence, radioimmunoassay, liquid phase chip method, lateral chromatography, electrochemistry, and flow cytometry.

[0016] Thirdly, the present invention provides a kit for detecting anti-KLHL11 autoantibodies, comprising a positive reference, wherein the positive reference is the anti-KLHL11 antibody.

[0017] Optionally, the kit is used for autoimmune diseases of the nervous system, such as autoimmune encephalitis. Compared with the prior art, the present invention has the following advantages:

[0018] The anti-KLHL11 antibody screened in this invention has high specificity and affinity. The chimeric antibody Nb-30 binds to HEK293 cells overexpressing human KLHL11 (hKLHL11-HEK293) via ECMO. 50 It reached 3.9 nM and did not bind to empty host HEK293 cells;

[0019] The anti-KLHL11 antibody screened by this invention can meet the requirements of high-sensitivity detection, which makes it suitable as a positive reference (or positive control) for the kit, providing a quantitative and traceable standard.

[0020] The antibodies screened in this invention employ a humanized chimeric design (such as the VHH-Fc form) and are efficiently produced using the ExpiCHO transient expression system. The purification process is simple, and compared to serum reference standards, these antibodies have clearly defined components, high stability, and are easy to store and control for long periods, avoiding the problems of serum variability and scarcity. Addressing the current lack of national standards for anti-KLHL11 autoantibody detection kits, this invention provides standardized positive reference standards, aiding product development and quality control, and possessing significant research and clinical value. Attached Figure Description

[0021] Figure 1 The binding activity of the anti-KLHL11 chimeric antibody to hKLHL11-HEK293 cells was demonstrated.

[0022] Figure 2 The binding activity of the anti-KLHL11 chimeric antibody to HEK293 cells was demonstrated. Detailed Implementation

[0023] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures for cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are all standard procedures widely used in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0024] The term “antibody” is used in the broadest sense in this article to refer to a protein that contains an antigen-binding site, encompassing natural and artificial antibodies of various structures, including but not limited to polyclonal antibodies, monoclonal antibodies, single-chain antibodies, intact antibodies, and antibody fragments.

[0025] As used herein, “sdAb” refers to a single monomeric variable antibody domain capable of binding to an antigen (e.g., a single-domain antibody that binds to brain-specific protein product 9.5). Single-domain antibodies include the VHH domain described herein. Examples of single-domain antibodies include, but are not limited to, antibodies naturally lacking the light chain, such as antibodies from camelid species (e.g., camels), single-domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single-domain antibodies other than single-domain scaffolds derived from antibodies. Single-domain antibodies can be derived from any species, including but not limited to mice, humans, camels, goats, rabbits, and cattle. For example, single-domain antibodies can be derived from antibodies produced in camelid species (e.g., camels, dromedary camels, alpacas, and guanacos), as described herein. Other species besides camelids may also produce heavy-chain antibodies naturally lacking the light chain; VHHs derived from these other species are also within the scope of this disclosure. The single-domain antibody (e.g., VHH) provided in this article can have the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0026] The term "complementarity-determining region" or "CDR region" or "CDR" refers to a region within the variable domain of an antibody that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). CDRs are primarily responsible for binding to antigen epitopes and are sequentially numbered from the N-terminus as CDR1, CDR2, and CDR3. In a given variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any of a number of known antibody CDR assignment systems or combinations thereof, including, for example: Chothia (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)) based on antibody sequence variability; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics database (IMGT). (http: / / imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering utilizing a large number of crystal structures. Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the foregoing methods. A CDR may also be determined based on having the same AbM numbering position as a reference CDR sequence (e.g., any of the CDR sequences in the examples of this invention). In one embodiment, the CDR of the single-domain antibody of this invention is positioned according to the AbM numbering scheme. Unless otherwise stated, in this invention, when referring to the positions of residues (including heavy chain variable region residues) in the antibody variable region and CDR, it refers to the numbering position according to the AbM numbering system.

[0027] A "chimeric antibody" is an antibody molecule created by splicing the V region gene of another non-human antibody with the C region gene of a human antibody to form a chimeric gene, which is then inserted into an expression vector for expression. For example, camel antibodies can be modified by replacing their constant region with a constant region derived from human immunoglobulins. By replacing it with a human constant region, the chimeric antibody can retain its specificity in recognizing antigens, while exhibiting reduced antigenicity in humans compared to the original camel antibody.

[0028] The term "anti-Kelch-like protein 11 (KLHL11) antibody-associated encephalitis" refers to a type of paraneoplastic encephalitis associated with testicular seminoma, with a minority of cases presenting with symptoms of limbic encephalitis. Vertigo, hearing loss, and tinnitus are common and distinctive clinical symptoms in patients with this disease.

[0029] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, conditions described in a laboratory manual, or conditions recommended by the manufacturer.

[0030] Example 1: Preparation of overexpression cell lines

[0031] Construction of the HEK293 cell line overexpressing human KLHL11 (hereinafter referred to as hKLHL11-HEK293): The full-length human KLHL11 (UniProt:Q9NVR0) nucleic acid sequence was constructed into the pLVX-puro plasmid. The resulting plasmid was then electroporated into HEK293 cells (ATCC® CRL-1573™). After electroporation, the cells were transferred to DMEM medium containing 10% FBS (v / v) and free of antibiotics. The cells were then seeded in 10×10cm cell culture dishes and cultured for 48 hours. Subsequently, the cells were aliquoted into 96-well cell culture plates at an average density of 0.5 cells / well. Puromycin at a final concentration of 2 µg / mL was added as a selection pressure. Cell clonal growth was observed after approximately 2 weeks, and cell lines that formed clones were selected for identification. Flow cytometry analysis showed that hKLHL11-HEK293 highly expressed human KLHL11 on its surface.

[0032] Example 2 Screening of anti-human KLHL11 nanobodies

[0033] A human Fc tag linked to the C-terminus of the full-length human KLHL11 (UniProt:Q9NVR0) gene was used as an antigen to immunize alpacas, obtaining high-titer antiserum. After animal immunization, 50 mL of fresh alpaca blood was collected, and peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque density gradient separation medium. RNA was extracted, reverse transcribed, amplified using universal primers, cloned into phage particles, transformed into TG1 strain, and a phage library was established for screening monoclonal antibodies. Nanobodies with high specificity and affinity were obtained and named Nb-30 and Nb-28.

[0034] Example 3: Generation and expression of anti-human KLHL11 chimeric antibody

[0035] The anti-human KLHL11 nanobody (hereinafter referred to as VHH) obtained in Example 2 was fused with the human IgG1 Fc fragment (SEQ ID NO:1), wherein the C-terminus of the VHH gene sequence was linked to the N-terminus of the human IgG1 Fc fragment gene sequence to construct the expression vector pcDNA3.4-TOPO for the VHH-Fc chimeric antibody. Expression was performed using the ExpiCHO transient transduction expression system. The cell culture supernatant expressing the target protein was centrifuged at 15000 g for 10 min. The obtained supernatant was purified by affinity using MabSelect SuRe LX, then eluted with 100 mM sodium acetate (pH 3.0), neutralized with 1 M Tris-HCl, and finally the obtained protein was transferred to PBS buffer via ultrafiltration to obtain the VHH-Fc chimeric antibody.

[0036] Example 4: Cell-specific binding assay of anti-human KLHL11 chimeric antibody

[0037] hKLHL11-HEK293 and HEK293 cells in good growth condition were digested with Trypsin containing 0.25% EDTA. 1×10⁵ cells were incubated with a 3-fold serially diluted chimeric antibody for 1 hour. An hIgG1 isotype-independent antibody was used as a negative control. Cells were washed twice with FACS buffer and then incubated with 0.5 µg of PE-labeled goat anti-human IgG Fc secondary antibody at 4°C for 1 hour. Afterward, the cells were washed three times with FACS buffer, and the binding of the candidate antibody to the cells was detected by flow cytometry. The flow cytometry results are as follows: Figure 1 The results showed that the chimeric antibody Nb-30 was superior to Nb-28 in binding to hKLHL11-HEK293 cells, with Nb-30 having an EC50 of 3.9 nM and Nb-28 having an EC50 of 6.4 nM. Figure 2This indicates that the screened chimeric antibody does not bind to HEK293. The results show that the chimeric antibody Nb-30 has a better affinity than Nb-28 and a lower non-specific binding rate than Nb-28. Therefore, chimeric antibody Nb-30 was ultimately selected (the amino acid sequence of chimeric antibody Nb-30 VHH is shown in SEQ ID NO:2, and the amino acid sequences of CDR1, CDR2, and CDR3 as defined by Kabat are shown in SEQ ID NO:3-5, respectively).

[0038] Example 5: KLHL11 Antibody Detection Method Based on Enzyme-Linked Immunosorbent Assay (ELISA)

[0039] The purified human KLHL11 protein was coated as the detection antigen using carbonate buffer (pH 9.6) at a concentration of 100 ng / well overnight at 4°C (coating buffer was 25 mL carbonate buffer, pH 9.6). The next day, the wells were washed three times with PBST solution for 5 min each time. 2% BSA was added to fill the wells for blocking, and the wells were incubated at 37°C for 1 h. After incubation, the wells were washed three times with PBST for 5 min each time. The chimeric antibody Nb-30 prepared in Example 3, prepared in serially diluted form (cerebrospinal fluid from patients with Kelch-like protein 11 (KLHL11) antibody-associated encephalitis was used as a positive control, and cerebrospinal fluid from healthy individuals was used as a negative control) was incubated separately. 100 μL of the above dilution was added to each well, and the wells were incubated at 37°C for 1 h. The wells were washed three times with PBST for 3 min each time. Goat anti-human IgG-HRP secondary antibody was diluted 1:5000 and added to the plate. The plate was incubated at 37°C for 30 min. After incubation, the wells were washed three times with PBST, and TMB was added for color development for 3 min. 2M... The assay was terminated with H2SO4, and the absorbance was measured at 450 nm. The experimental results are shown in Table 1. The chimeric antibody Nb-30 specifically recognizes the human KLHL11 antigen, and at an antigen coating amount of 100 ng / well, the titer of Nb-30 can reach ≥1:256K, making it a viable serum alternative to meet the needs of long-term diagnostic reagents. Humanized chimeric antibodies retain the ability of parental antibodies to specifically bind antigens while significantly reducing the immunogenicity of heterologous antibodies. Because humanized chimeric antibodies can specifically recognize corresponding antigens, they can, to some extent, replace the function of positive serum. Concentration can be determined through protein quantification, and titer can be determined through antibody titer testing, thus providing a quantifiable and traceable enterprise reference material.

[0040] Table 1

[0041]

Claims

1. An anti-KLHL11 antibody, wherein the anti-KLHL11 antibody is a humanized chimeric antibody containing VHH, wherein VHH contains the amino acid sequence shown in SEQ ID NO:2, and the VHH in the humanized chimeric antibody is linked to the human IgG1 Fc fragment.

2. A kit for detecting anti-KLHL11 autoantibodies, characterized in that, It includes a positive reference, wherein the positive reference is the anti-KLHL11 antibody as described in claim 1.

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