Anti-lc-1 antibodies or antigen-binding fragments thereof, methods of making and uses

Abstract

The application provides an anti-LC-1 antibody or antigen binding fragment thereof, a preparation method and application, and relates to the technical field of biotechnology.The anti-LC-1 antibody or antigen binding fragment thereof contains a complementarity determining region of a heavy chain variable region and a complementarity determining region of a light chain variable region, the complementarity determining region of the heavy chain variable region includes an amino acid sequence identical to VH-CDR1, VH-CDR2 and VH-CDR3 of the heavy chain variable region shown in SEQ ID NO.1; the complementarity determining region of the light chain variable region includes an amino acid sequence identical to VL-CDR1, VL-CDR2 and VL-CDR3 of the light chain variable region shown in SEQ ID NO.2.The anti-LC-1 antibody or antigen binding fragment thereof has good specific binding capacity with the LC-1 antigen, and can be used for identification of the anti-LC-1 antibody or the LC-1 antigen, and auxiliary diagnosis and detection of diseases positive to the anti-LC-1 antibody.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and in particular to an anti-LC-1 antibody or its antigen-binding fragment, preparation method and application. Background Technology

[0002] The following statements are provided only as background information in relation to the present invention and do not necessarily constitute prior art.

[0003] Anti-hepatocyte cytoplasmic type I antibody (anti-LC-1 antibody) is considered another marker antibody for type II AIH (autoimmune hepatitis). In 1988, Martini et al. first confirmed the presence of anti-LC-1 antibody in the serum of six adult patients with autoimmune hepatitis using the IIF and ID methods. The target antigen is present in the cytoplasm of hepatocytes, and its components are imine-methyltetrahydrofolate cyclodeaminase and argininosuccinate lyase. The IB method detects it as a 58-62 kDa hepatocyte cytoplasmic polypeptide protein. Detection of anti-LC-1 antibody is of great significance for the diagnosis and classification of AIH.

[0004] Anti-LC-1 antibody is a serum-specific antibody for type II autoimmune hepatitis, with a positive rate of 56%–72%. Clinically, anti-LC-1 antibodies are more common in young AIH patients under 20 years of age, and less common in AIH patients over 40 years of age. Anti-LC-1 antibodies often coexist with anti-LKM-1 antibodies. Among patients positive for anti-LC-1 antibodies, 32%–67% can also detect anti-liver and kidney microsomal antibodies (anti-LKM-1 antibodies); among patients positive for anti-LKM-1 antibodies, 25%–50% can detect anti-LC-1 antibodies. Therefore, anti-LC-1 antibodies and anti-LKM-1 antibodies are closely related. Hepatitis C virus (HCV) infection is not related to LC-1, and there is no cross-reaction between anti-HCV and anti-LC-1 antibodies. Therefore, anti-LC-1 antibodies are more specific for AIH than anti-LKM-1 antibodies. Anti-LC-1 antibodies are associated with the disease activity of AIH-II and serve as a marker of disease activity and prognostic indicators for AIH.

[0005] Therefore, the development of high-affinity anti-LC-1 recombinant monoclonal antibodies is of great significance and value for the development of clinical diagnostic kits.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The purpose of this invention is to provide an anti-LC-1 antibody or its antigen-binding fragment, which exhibits good specific binding ability to the LC-1 antigen. Based on the anti-LC-1 antibody or its antigen-binding fragment provided by this invention, another objective is to provide its applications. A further objective of this invention is to develop an anti-LC-1 antibody or its antigen-binding fragment with good binding activity using a simpler method, which can be used for the identification of the LC-1 antigen and to assist in the diagnosis or auxiliary diagnosis of diseases positive for anti-LC-1 antibodies.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0009] Definition of noun:

[0010] In this article, "LC-1 antigen" refers to the antigen that can be bound by natural anti-LC-1 antibodies (anti-hepatocyte cytoplasmic type I antibodies). These antigens are usually present in the cytoplasm of hepatocytes and their components are imine methyltetrahydrofolate cyclodeaminase and argininosuccinate lyase, which appear as 58-62kD hepatocyte cytoplasmic polypeptide proteins as detected by the IB method.

[0011] In this article, "anti-LC-1 antibody or its antigen-binding fragment" refers to a molecule that can specifically bind to the above definition of "LC-1 antigen".

[0012] In this document, the term "antibody or antigen-binding fragment thereof" refers to all proteins and protein fragments containing a complementarity-determining region (CDR). Proteins and protein fragments can be antibodies; the terms "antibody" and "full-length antibody" include both polyclonal and monoclonal antibodies. Furthermore, the term "antibody" includes both naturally occurring and non-naturally occurring antibodies, including, for example, chimeric, bifunctional, and humanized antibodies, as well as related synthetic isoforms. Non-naturally occurring antibodies are also referred to herein as "recombinant antibodies," and the term "antibody" is used interchangeably with "immunoglobulin." Proteins and protein fragments can be antigen-binding fragments containing part or all of the antibody's CDR, lacking at least some amino acids present in the full-length antibody chain but still capable of specifically binding to antigens. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Such fragments are selected from, but are not limited to, any one of F(ab')2, Fab', Fab, Fv (composed of VH and VL), ScFv (single-chain antibody, where VH and VL are linked by a linker peptide), dsFv (disulfide-stabilized Fv fragments, dsFv)), bispecific antibodies, nanobodies, and the smallest recognition unit of an antibody. In addition to the functional fragments mentioned above, any fragment with an extended half-life is also included.

[0013] The term "variable region" or "variable domain" refers to the amino-terminal domain of an antibody's heavy or light chain that recognizes and binds to antigens. The composition and arrangement of the amino acids in this region determine the antibody's specificity in recognizing antigens. The heavy chain variable domain can be referred to as "VH," and the light chain variable domain as "VL." These domains are typically the most variable parts of the antibody and contain antigen-binding sites. Both the heavy and light chain variable regions consist of three complementarity-determining regions (CDRs) (also known as hypervariable regions) connected by four framework regions (FRs). The extent of the backbone region and CDRs has been precisely defined, for example, in Kabat (see Sequences of Proteins of Immunological Interest, E. Kabat et al.) and Chothia. Any CDR determination method well-known in the art, including combinations of methods, can identify CDRs of variable domains. CDRs in each chain are held together closely by FRs to form variable regions. Typically, the variable regions VL / VH of the heavy and light chains can be obtained by linking the following numbered CDRs with FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0014] The term "constant region" or "constant domain" refers to the constant region of a single antibody light chain or a constant region of an antibody heavy chain. An antibody heavy chain has a variable domain (VH) followed by one or more constant domains or regions, such as a hinge, CH1, CH2, CH3, and CH4. The CH1 domain is adjacent to the VH domain and is located at the amino terminus of the hinge region of the antibody heavy chain, and does not form a portion of the Fc region of the antibody. The hinge region includes the portion of the heavy chain molecule that links the CH1 domain to the CH2 domain. The N-terminus of CH2 is typically a CH3 domain, which usually forms the C-terminal portion of the antibody. In some antibody types, such as IgM and IgE, the constant region also includes a CH4 domain. The constant region of an antibody can originate from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD, as well as the constant regions of their subclasses and mutant forms.

[0015] This invention does not limit the method of obtaining the anti-LC-1 antibody or its antigen-binding fragment. In some alternative embodiments, the corresponding antibody can be obtained by linking a polynucleotide encoding the anti-LC-1 antibody or its antigen-binding fragment to a vector and expressing it in cells. The above-mentioned vector can be introduced into eukaryotic cells, especially mammalian cells, to construct a structure capable of expressing the anti-LC-1 antibody or its antigen-binding fragment. In other alternative embodiments, the anti-LC-1 antibody or its antigen-binding fragment can also be obtained by recombinant genetic techniques known to those skilled in the art or by peptide synthesis, such as automated peptide synthesizers (e.g., automated peptide synthesizers sold by Applied BioSystems, etc.); the antigen-binding fragment can also optionally be generated by enzymatic cleavage of antigen-binding molecules (including intact antibodies), such as pepsin or papain cleavage; or by chemical cleavage, such as by chemical reduction of disulfide bonds to obtain the above-mentioned antigen-binding fragment.

[0016] The terms "specific recognition," "selective binding," "selective binding," and "specific binding," or similar expressions, refer to the binding of an antibody or its antigen-binding fragment to a pre-defined epitope on an antigen. Typically, antibodies or their antigen-binding fragments bind at a rate of approximately less than 10... -5 M, for example, approximately less than 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller K d Value binding. The K value of the antibody can be determined using methods well-established in the art. d Values. Other standard assays for evaluating the binding ability of ligands, such as antibodies, to targets are known in the art, including, for example, ELISA, Western blotting, RIA, and flow cytometry.

[0017] The binding affinity of anti-LC-1 antibodies or their antigen-binding fragments to the target antigen can also be expressed as the "half-maximum effective concentration (EC50)". EC50 refers to the concentration of drug or antibody that achieves 50% of the maximum biological effect after a specific exposure time. Generally, a lower EC50 indicates better affinity, meaning that it can bind to the target protein at a lower concentration. The EC50 value can be determined using binding detection methods known in the art, such as direct or indirect binding detection methods (e.g., enzyme-linked immunosorbent assay (ELISA), flow cytometry, and other binding detection methods).

[0018] In this document, the term "signaling substance" refers to a substance that can provide a detectable signal, which can be directly observed by the naked eye or detected by conventional instruments acceptable in the art. The signaling substance can provide a signal directly, such as color (e.g., colloidal gold, colored microspheres), fluorescence (fluorescent molecules), magnetism, radiation, or luminescence; or it can provide a signal indirectly through a reaction in which the signaling substance participates, such as catalyzing a specific substrate reaction to produce any of the above signals.

[0019] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of polynucleotides include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derived nucleotide bases. The polynucleotide encodes the aforementioned anti-LC-1 antibody or its antigen-binding fragment, optionally encoding either the sense or antisense strand. Polynucleotides can be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.

[0020] In this article, the term "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, it is called an expression vector. Vectors can be introduced into cells through transformation, transduction, or transfection, allowing the genetic material they carry to be expressed in the cells.

[0021] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids, episome plasmids, microcircular DNA, phage particles, and Cosmids; 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. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses. In some embodiments, the vectors of this invention contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0022] The terms “cell,” “cell line,” and “cell culture” used herein are used interchangeably, and all such names include progeny. Progeny may not be identical to primary cells due to natural, accidental, or intentional mutations, and may differ from primary cells morphologically and / or in genomic DNA. “Transformation” and “transformed cell” include primary test cells and cultures derived from them.

[0023] In this document, the terms “purified” or “isolated” associated with peptides or nucleic acids mean that the peptide or nucleic acid is not in its native medium or in its native form. Therefore, the term “isolated” includes peptides or nucleic acids removed from their original environment, such as if they are naturally occurring. For example, isolated peptides typically do not contain at least some proteins or other cellular components that are normally bound to or mixed with or in solution with them. Isolated peptides include naturally produced peptides contained in cell lysates, peptides in purified or partially purified forms, recombinant peptides, peptides expressed or secreted by cells, and peptides in heterologous cells or cultures. As associated with nucleic acids, the terms “isolated” or “purified” indicate, for example, that the nucleic acid is not in its native genomic background (e.g., in a vector, as an expression cassette, linked to a promoter, or artificially introduced into heterologous cells).

[0024] In this article, "anti-LC-1 antibody positive disease" refers to a disease that can cause a patient's anti-LC-1 antibody level to be higher than that before the disease. Exemplary anti-LC-1 antibody positive diseases include, but are not limited to, type II autoimmune hepatitis.

[0025] In this document, unless otherwise stated, any numbering is used to distinguish one entity or behavior from another, and is not required to require or imply any actual relationship, order or importance between these entities or behaviors, such as numbering (I), (II)...(V).

[0026] In this document, unless otherwise stated, “optional,” “optional,” “optional,” or “optional” means that the event or situation described below may, but does not have to, occur, including the circumstances in which the event or situation may or may not occur.

[0027] In this document, the terms “comprising” or “including” mean that the stated elements, integers or steps are included, but do not exclude any other elements, integers or steps.

[0028] In a first aspect, an anti-LC-1 antibody or an antigen-binding fragment thereof is provided, comprising a heavy chain variable region and a light chain variable region.

[0029] The heavy chain variable region includes complementarity-determining regions VH-CDR1, VH-CDR2, and VH-CDR3; the light chain variable region includes complementarity-determining regions VL-CDR1, VL-CDR2, and VL-CDR3.

[0030] The VH-CDR1 comprises an amino acid sequence consistent with the VH-CDR1 of the heavy chain variable region shown in SEQ ID NO.1; the VH-CDR2 comprises an amino acid sequence consistent with the VH-CDR2 of the heavy chain variable region shown in SEQ ID NO.1; and the VH-CDR3 comprises an amino acid sequence consistent with the VH-CDR3 of the heavy chain variable region shown in SEQ ID NO.1; and the VL-CDR1 comprises an amino acid sequence consistent with the VL-CDR1 of the light chain variable region shown in SEQ ID NO.2; the VL-CDR2 comprises an amino acid sequence consistent with the VL-CDR2 of the light chain variable region shown in SEQ ID NO.2; and the VL-CDR3 comprises an amino acid sequence consistent with the VL-CDR3 of the light chain variable region shown in SEQ ID NO.2.

[0031] It is understood that the amino acid sequences of the variable regions shown in SEQ ID NO. 1 or 2, excluding the CDR region, are not intended to limit the anti-LC-1 antibody or its antigen-binding fragment provided by this invention. For example, if the anti-LC-1 antibody or its antigen-binding fragment provided by this application contains a backbone region, it may differ from the backbone region in the variable region shown in SEQ ID NO. 1 or 2. The CDR region in the variable region shown in SEQ ID NO. 1 or 2 may be divided according to any optional manner known in the art. Optionally, the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 of the variable region may be defined by any one or a combination of multiple definition systems such as Kabat, Chothia, IMGT, ABM, or Contact. Taking Kabat, Chothia, IMGT, ABM, or Contact as examples, the amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 in the heavy chain variable region of SEQ ID NO.1 and the amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 in the light chain variable region of SEQ ID NO.2 are shown in Table 1:

[0032] Table 1

[0033]

[0034] In an optional embodiment, the anti-LC-1 antibody or its antigen-binding fragment has VH-CDR1, VH-CDR2, and VH-CDR3, the heavy chain variable regions defined in Table 1, and VL-CDR1, VL-CDR2, and VL-CDR3, the light chain variable regions defined in Table 1. Taking the IMGT definition as an example: the amino acid sequence of VH-CDR1 is shown in SEQ ID NO.11, the amino acid sequence of VH-CDR2 is shown in SEQ ID NO.16, and the amino acid sequence of VH-CDR3 is shown in SEQ ID NO.19; the amino acid sequence of VL-CDR1 is shown in SEQ ID NO.22, the amino acid sequence of VL-CDR2 is QMS, and the amino acid sequence of VL-CDR3 is shown in SEQ ID NO.25.

[0035] In an optional implementation, the heavy chain variable region further includes at least one skeleton region, such as one skeleton region, two skeleton regions, three skeleton regions, or four skeleton regions.

[0036] In an optional implementation, the light chain variable region further includes at least one skeleton region, such as one skeleton region, two skeleton regions, three skeleton regions, or four skeleton regions.

[0037] In optional embodiments, the species origin of each skeletal region is independent, including but not limited to one or more of rabbits, cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, camels, donkeys, deer, mink, chickens, ducks, geese, turkeys, fighting cocks, humans, and their mutants.

[0038] In an optional embodiment, the backbone region VH-FR1 of the heavy chain variable region includes an amino acid sequence consistent with that of the heavy chain variable region VH-FR1 shown in SEQ ID NO.1; and / or the backbone region VH-FR2 includes an amino acid sequence consistent with that of the heavy chain variable region VH-FR2 shown in SEQ ID NO.1; and / or the backbone region VH-FR3 includes an amino acid sequence consistent with that of the heavy chain variable region VH-FR3 shown in SEQ ID NO.1; and / or the backbone region VH-FR4 includes an amino acid sequence consistent with that of the heavy chain variable region VH-FR4 shown in SEQ ID NO.1.

[0039] In an optional embodiment, the backbone region VL-FR1 of the light chain variable region includes an amino acid sequence consistent with that of the light chain variable region VL-FR1 shown in SEQ ID NO.2; and / or the backbone region VL-FR2 includes an amino acid sequence consistent with that of the light chain variable region VL-FR2 shown in SEQ ID NO.2; and / or the backbone region VL-FR3 includes an amino acid sequence consistent with that of the light chain variable region VL-FR3 shown in SEQ ID NO.2; and / or the backbone region VL-FR4 includes an amino acid sequence consistent with that of the light chain variable region VL-FR4 shown in SEQ ID NO.2.

[0040] In an optional embodiment, the amino acid sequence of the heavy chain variable region of the anti-LC-1 antibody or its antigen-binding fragment is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2.

[0041] In an optional embodiment, the anti-LC-1 antibody or its antigen-binding fragment is an antibody or antigen-binding fragment that includes a constant region.

[0042] In an optional embodiment, at least a portion of the constant region sequence of the anti-LC-1 antibody or its antigen-binding fragment is a human constant region sequence.

[0043] In an optional embodiment, the constant region sequence of the anti-LC-1 antibody or its antigen-binding fragment is selected from the sequence of part or all of the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD, wherein IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD include their subclasses and mutant forms.

[0044] In an optional embodiment, the anti-LC-1 antibody or its antigen-binding fragment contains a heavy chain constant region.

[0045] In an optional embodiment, the heavy chain constant region sequence of the anti-LC-1 antibody or its antigen-binding fragment is selected from part or all of the constant region sequence of human IgG1, preferably including CH1, CH2 and CH3 of the constant region of human IgG1.

[0046] In an optional embodiment, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.3.

[0047] In an optional embodiment, the anti-LC-1 antibody or its antigen-binding fragment contains a light chain constant region.

[0048] In an optional implementation, the light chain constant region sequence is selected from the light chain constant region of a mouse.

[0049] In an optional embodiment, the amino acid sequence of the light chain constant region is shown in SEQ ID NO.4.

[0050] In an optional embodiment, the anti-LC-1 antibody or its antigen-binding fragment is a human-mouse chimeric antibody, wherein the heavy chain amino acid sequence of the anti-LC-1 antibody or its antigen-binding fragment is as shown in SEQ ID NO.5, and the light chain amino acid sequence is as shown in SEQ ID NO.6.

[0051] In a second aspect, a biological material is also provided, comprising a polynucleotide, a carrier, or a cell; wherein the polynucleotide encodes the aforementioned anti-LC-1 antibody or its antigen-binding fragment; the carrier carries the polynucleotide; and the cell carries the polynucleotide, or contains the carrier, or is capable of expressing the anti-LC-1 antibody or its antigen-binding fragment.

[0052] By linking a vector with a polynucleotide encoding an anti-LC-1 antibody or its antigen-binding fragment, the vector can be introduced into eukaryotic cells, especially mammalian cells, to construct a cell line that can express the anti-LC-1 antibody or its antigen-binding fragment, and the corresponding protein can be obtained through cell expression.

[0053] In an optional embodiment, the cells used to express the anti-LC-1 antibody or its antigen-binding fragment are 293 cells (human kidney epithelial cell line), preferably 293F cells.

[0054] In an optional embodiment, the cells used to express the anti-LC-1 antibody or its antigen-binding fragment are CHO cells (Chinese hamster ovary cells).

[0055] Thirdly, a method for preparing the anti-LC-1 antibody or its antigen-binding fragment as described in the first aspect is also provided, comprising culturing cells capable of expressing the anti-LC-1 antibody or its antigen-binding fragment.

[0056] In an optional embodiment, the preparation method further includes converting and expressing a polynucleotide encoding the anti-LC-1 antibody or its antigen-binding fragment into cells, and obtaining the anti-LC-1 antibody or its antigen-binding fragment by purification.

[0057] In an optional embodiment, the preparation method further includes, as needed, synthesizing a polynucleotide containing a gene encoding the anti-LC-1 antibody or its antigen-binding fragment, and / or, as needed, preparing a suitable expression vector, transforming the expression vector into the desired cells and expressing it, and obtaining the anti-LC-1 antibody or its antigen-binding fragment through purification.

[0058] In an optional embodiment, the cells are prepared by converting cells with a polynucleotide encoding an anti-LC-1 antibody or an antigen-binding fragment thereof as described in the first aspect, the polynucleotide including a heavy chain expression plasmid and a light chain expression plasmid, and the conversion includes co-converting the heavy chain expression plasmid and the light chain expression plasmid into the cells.

[0059] In an optional implementation, the C-terminus of the heavy chain variable region is fused with a constant region fragment to construct a complete heavy chain expression plasmid.

[0060] In an optional embodiment, the constant region segment includes one or more of CH1, CH2 and CH3, preferably including CH1, CH2 and CH3.

[0061] In an optional embodiment, the cell is a eukaryotic cell, preferably a mammalian cell.

[0062] In an optional embodiment, the mammalian cells include 293 cells or CHO cells, preferably 293F cells.

[0063] Fourthly, the application of the anti-LC-1 antibody or its antigen-binding fragment from the first aspect, or the biological material from the second aspect, in any of the following (I) to (VII) is also provided:

[0064] (I) Non-diagnostic and therapeutic target testing for anti-LC-1 antibodies;

[0065] (II) Preparation of products for detecting anti-LC-1 antibodies;

[0066] (III) Prepare products for the diagnosis and / or auxiliary diagnosis of diseases with positive anti-LC-1 antibodies;

[0067] (IV) Non-diagnostic and treatment-oriented detection of LC-1 antigen;

[0068] (V) Prepare products for detecting LC-1 antigen;

[0069] (VI) Used for the isolation, enrichment and / or purification of LC-1 antigen;

[0070] (VII) Prepare products for the isolation, enrichment and / or purification of LC-1 antigen;

[0071] In the applications described in (I) to (III) above, anti-LC-1 antibodies or their antigen-binding fragments can be used as standards and / or quality control materials for detection, providing a reliable reference for the test results, and can also be used to construct standard curves.

[0072] In an optional implementation, the anti-LC-1 antibody positive disease includes type II autoimmune hepatitis.

[0073] The applications described in aspects (IV) to (VII) above can utilize the ability of anti-LC-1 antibodies or their antigen-binding fragments to specifically target and bind to LC-1 antigens, thereby enabling the detection, separation, enrichment, and / or purification of LC-1 antigens. In an optional embodiment, when the anti-LC-1 antibody or its antigen-binding fragment is an immunoconjugate, for example, linked to a signal, the localization or real-time detection of the LC-1 antigen can be achieved by detecting the signal. In an optional embodiment, the detection, separation, enrichment, and / or purification of LC-1 antigens can be achieved by separating the anti-LC-1 antibody-LC-1 antigen immune complex.

[0074] In optional embodiments, in aspects (II), (III), (V) or (VII) above, those skilled in the art can prepare corresponding products (such as the immunoconjugates mentioned above) according to actual uses, and select other reagent components in the product, including but not limited to one or more of the following: signaling agents, solid-phase carriers, buffer reagents, salts, secondary antibodies, chromogenic substrates, blocking solutions, washing solutions, solvents, elution solutions, conjugates, negative controls, positive controls, standards, quality control products, and markers.

[0075] Fifthly, a reagent or kit is also provided, the reagent or kit comprising the anti-LC-1 antibody of the first aspect or its antigen-binding fragment, or the biological material of the second aspect.

[0076] In an optional embodiment, the kit is used to detect anti-LC-1 antibody or anti-LC-1 antibody-positive diseases; the kit includes standards and / or quality control products, the standards and / or quality control products containing the anti-LC-1 antibody or its antigen-binding fragment.

[0077] In an optional implementation, the kit is used to detect type II autoimmune hepatitis.

[0078] In an optional embodiment, the kit further includes a detection reagent, which includes an anti-LC-1 antibody detection reagent.

[0079] In an optional embodiment, the kit may further include a detection reagent for detecting anti-LKM-1 antibodies.

[0080] In an optional embodiment, the kit further includes a solid support.

[0081] In optional embodiments, the anti-LC-1 antibody or its antigen-binding fragment in the reagent or kit is coupled to a solid-phase carrier; or the anti-LC-1 antibody or its antigen-binding fragment in the reagent or kit is packaged separately from the solid-phase carrier. By coupling the anti-LC-1 antibody or its antigen-binding fragment to the solid-phase carrier, it can be used to capture LC-1 antigen in the test sample. Alternatively, by coupling the anti-LC-1 antibody or its antigen-binding fragment to the solid-phase carrier, it can be used to purify LC-1 antigen.

[0082] In an optional embodiment, the anti-LC-1 antibody detection reagent includes a solid-phase carrier conjugated with the LC-1 antigen.

[0083] In an optional implementation, the kit may further include a signaling agent.

[0084] In optional embodiments, the anti-LC-1 antibody or its antigen-binding fragment in the reagent or kit is conjugated with a signaling agent; or the anti-LC-1 antibody or its antigen-binding fragment in the reagent or kit is packaged separately from the signaling agent. By conjugating the anti-LC-1 antibody or its antigen-binding fragment with the signaling agent, it can be used to locate and detect LC-1 antigen or to detect LC-1 antigen in a sample by Western blotting.

[0085] The reagents or kits described above may also optionally include reagents and / or consumables well known to those skilled in the art for use in detecting reactions or purifying proteins, including but not limited to one or more of buffers, salts, secondary antibodies, chromogenic substrates, blocking solutions, washing solutions, solvents, elution solutions, coupling agents, negative controls, positive controls, standards, quality controls, and markers.

[0086] The above-described reagents or kits can be used in general immunoassay methods acceptable in the art, including but not limited to immunofluorescence staining, flow cytometry, immunoblotting, immunohistochemistry, ELISA, immunochromatography, or immunomagnetic beads. Those skilled in the art can formulate other reagents in the reagents or kits according to the corresponding detection methods, and the present invention does not limit this.

[0087] The signaling agents in any of the above embodiments include, but are not limited to, one or more of the following: enzymes, luminescent labels, fluorescent microspheres, colored microspheres, latex microspheres, colloidal gold, quantum dots, biotin, streptavidin, radionuclides, radioactive contrast agents, paramagnetic ions, metals, and photosensitizers.

[0088] Examples of enzymes include, but are not limited to, alkaline phosphatase or horseradish peroxidase. Luminescent labels include, but are not limited to, fluorescent proteins, synthetic small molecules, or polymer dyes. Specific examples include, but are not limited to, Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminoacridine, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethylrhodamine, and Cascade. Blue, Cy2, Cy3, Cy5, Cy7, 6-FAM, Dansyl chloride, Fluorescein, HEX, 6-JOE, NBD (7-nitrobenzo-2-oxa-1,3-diazole), Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, Phthalic acid, Terephthalic acid, Isophthalic acid, Cresol Violet, Cresol Blue Violet, Brilliant Cresol Blue, p-Aminobenzoic acid, Erythrosine, Phthalocyanine, Azocyanine, Anthocyanin, Xanthine, Succinyl fluorescein, Rare earth metal cavitation compounds, Tribispyridyldiamine europium, europium cavitation compounds or chelates, Diamine, Dianthocyanin, La Jolla Blue dye, Allococyanin B. Phycocyanin C, Phycocyanin R, Thiamine, Phycoerythrin, Phycoerythrin R, REG, Rhodamine Green, Rhodamine Isothiocyanate, Rhodamine Red, ROX, TAMRA, TET, TRIT (tetramethylrhodamine isothiol), tetramethylrhodamine, and Texas Red, one or more of these. Fluorescent microspheres, colored microspheres, and latex microspheres are each independently selected from products acceptable in the art, such as those derived from commercially available products. Radionuclides include, but are not limited to, those derived from... 110 In、 111 In、 177 Lu、 18 F, 52 Fe、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 86 Y、 90 Y、 89 Zr、 94 mTc, 94 Tc, 99 mTc, 120 I,123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C 13 N、 15 O、 186 Re、 188 Re、 51 Mn, 52 mMn, 55 Co、 72 As、 75 Br、 76 Br、 82 mRb and 83 One or more of Sr. Paramagnetic ions include, but are not limited to, one or more of chromium (III), manganese (II), iron (III), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (III).

[0089] The solid-phase support in any of the above embodiments includes, but is not limited to, microtubes, columns, microparticles, nitrocellulose membranes, chromatography matrices, or side-flow devices; more specifically, it can be, but is not limited to, enzyme-labeled wells, immunochromatographic test strips, or magnetic beads. The chromatography matrix can be any known chromatography matrix acceptable in the art, including but not limited to polystyrene, polysaccharide polymers, or silica gel. In optional embodiments, the chromatography matrix includes gel particles.

[0090] This invention discloses an anti-LC-1 antibody or its antigen-binding fragment, which is further used to prepare a human-mouse chimeric recombinant monoclonal antibody. This invention has the following beneficial effects:

[0091] (1) This method prepares anti-LC-1 antibodies or their antigen-binding fragments with known sequences, which can be recombinantly expressed in vitro. The operation is simple, time-consuming, and the production process is controllable with small batch-to-batch differences. It also has the advantages of good stability. After treatment at 4℃ for 7 days, the retention rate is greater than 95%, and after treatment at 37℃ for 7 days, the retention rate is greater than 85%. It has good application prospects.

[0092] (2) The antibody of the present invention has good affinity, with EC50 values ​​of 1.42 nM and 2.63 nM, respectively, and can specifically bind to LC-1 antigen. It can be used for LC-1 antigen detection, including but not limited to using liquid chromatography chips, immunofluorescence staining, flow cytometry, fluorescent microspheres and other techniques to identify LC-1 antigen; the anti-LC-1 antibody or its antigen-binding fragment can also be coupled to a chromatography matrix for immunoaffinity chromatography purification of LC-1 antigen, thereby improving antigen purity.

[0093] (3) The anti-LC-1 antibody or its antigen-binding fragment can be used for anti-LC-1 antibody detection, anti-LC1 antibody positive disease diagnostic products (such as auxiliary diagnostic products for autoimmune hepatitis type II), for example, as a quality control and / or standard for anti-LC-1 antibody detection products; when the anti-LC-1 antibody or its antigen-binding fragment is used as a quality control in the test kit, it can alleviate the problems of complicated operation of polyclonal antibodies and low subsequent conjugation efficiency; it can reduce production costs, stabilize product quality, and significantly improve reaction values; on the other hand, compared with directly using human serum, it can also avoid the problems of difficult sample sources and high costs. Attached Figure Description

[0094] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0095] Figure 1 The results of the ELISA assay in Example 2 show the binding activity of recombinant monoclonal antibody Ab01-LC-1 to LC-1 antigen. Ab01-LC-1 represents the binding activity of recombinant monoclonal antibody Ab01-LC-1 to LC-1 antigen, and Ab-BSA represents the non-specific binding activity of recombinant monoclonal antibody Ab01-LC-1 to control protein BSA.

[0096] Figure 2 The results of the ELISA assay in Example 2 show the binding activity of recombinant monoclonal antibody Ab02-LC-1 to LC-1 antigen. Ab02-LC-1 represents the binding activity of recombinant monoclonal antibody Ab02-LC-1 to LC-1 antigen, and Ab-BSA represents the non-specific binding activity of recombinant monoclonal antibody Ab02-LC-1 to control protein BSA.

[0097] Figure 3 This is an SDS-PAGE protein electrophoresis image of recombinant monoclonal antibody Ab01-LC-1. M represents the marker. "Reduced" indicates the reduced SDS-PAGE electrophoresis result of recombinant monoclonal antibody Ab01-LC-1, with heavy and light chains of 50 kDa and 25 kDa, respectively. "Non-reduced" indicates the non-reduced SDS-PAGE electrophoresis result of recombinant monoclonal antibody Ab01-LC-1, with a size of approximately 150 kDa. Detailed Implementation

[0098] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0099] Example 1: Preparation of LC-1 chimeric antibody

[0100] Phage display technology was used to screen and obtain LC-1-specific high-affinity monoclonal strains. After sequencing, the Fab region sequence was obtained and the genes were synthesized. The synthesized light chain gene and heavy chain gene were constructed into the vector PTT5, respectively.

[0101] The light chain gene was a complete mouse light chain, with the constant region of the light chain shown in SEQ ID NO.4. The heavy chain gene was constructed by fusing the C-terminus of the variable region (VH) of the heavy chain with a constant region fragment of human IgG1 (including CH1-CH2-CH3), thus creating a complete IgG heavy chain, with the constant region of the heavy chain shown in SEQ ID NO.3. Two clones with better screening results were selected for sequencing, yielding two different LC-1 antibody sequences, Ab01-LC-1 and Ab02-LC-1.

[0102] The constructed heavy chain gene expression plasmid and light chain gene expression plasmid were mixed with PEI at a 1:2 molar ratio and transfected into 293F suspension cells in logarithmic growth phase. The cells were cultured in a 37°C shaking incubator at 120 rpm. After 5 days, the cell supernatant was collected, and the antibody was purified with Protein A, eluted with 0.1M Glycine (pH 3.0), and neutralized with 1M Tris (pH 8.0). After elution, the ultrafiltration centrifuge tubes were replaced with PBS buffer and concentrated to determine protein concentration.

[0103] Example 2: ELISA determination of the binding activity of recombinant monoclonal antibody to LC-1 antigen.

[0104] (1) The LC-1 protein (reference sequence NP_996848.1, 2 μg / mL) was coated on an ELISA plate, and BSA was coated as a non-specific binding control. The plates were incubated overnight at 4°C.

[0105] (2) Discard the coating solution, wash the plate with PBST, pat dry, add 3% milk, and block at 37°C for 2 hours.

[0106] (3) Discard the blocking solution, wash the plate with PBST, pat dry, add 1 μM of antibody in a 3-fold serial dilution, and incubate at 37°C for 1.5 h.

[0107] (4) Discard the primary antibody, wash the plate with PBST, pat dry, add horseradish peroxidase (HRP) labeled mouse anti-human IgG secondary antibody, and incubate at 37°C for 1 h.

[0108] (5) Discard the secondary antibody, wash the plate with PBST, pat dry, add the chromogenic substrate for color development, add the stop solution after 15 min to stop the reaction, and measure the OD value with an ELISA reader.

[0109] (6) Plot a nonlinear fit graph with OD value as the vertical axis and the logarithm of antibody molar concentration as the horizontal axis.

[0110] like Figure 1 and Figure 2 As shown, the EC50 values ​​of the two antibodies are 1.42 nM (Ab01-LC-1) and 2.63 nM (Ab02-LC-1), respectively.

[0111] Example 3: Stability of the LC-1 recombinant monoclonal antibody

[0112] The LC-1 humanized antibody prepared in Example 1 was diluted to 2 mg / mL, and then diluted 1000 times using this concentration as the initial concentration. The solutions were then placed at -80℃, 4℃, and 37℃ for 7 days, respectively. The magnetic barcodes conjugated with the LC-1 antigen were then detected using a fully automated multiplex immunoassay analyzer from Zhuhai Lizhu Reagent Co., Ltd. The test results are shown in Table 2.

[0113] Table 2. Signal retention rate after antibody thermal acceleration

[0114]

[0115] As shown in Table 2, after the antibody was diluted 1000-fold and treated at 4℃ for 7 days, the signal retention rates were 97.8% and 93%, respectively. After treatment at 37℃ for 7 days, the signal retention rates were 88.2% and 85.9%, respectively. Overall, the Ab01-LC-1 antibody exhibits better stability.

[0116] Example 4: Sequence information of the recombinant monoclonal antibody Ab01-LC-1 (LC-1)

[0117] SDS-PAGE image of recombinant monoclonal antibody Ab01-LC-1 is shown below. Figure 3 As shown in SEQ ID NO.1, the variable region sequence of the heavy chain is shown in SEQ ID NO.2; the constant region sequence of the heavy chain is shown in SEQ ID NO.3 (human-derived sequence), and the constant region sequence of the light chain is shown in SEQ ID NO.4 (mouse-derived sequence). The complete heavy chain sequence is shown in SEQ ID NO.5, and the complete light chain sequence is shown in SEQ ID NO.6. CDR sequence information is shown in Table 1.

[0118] Heavy chain variable region (SEQ ID NO.1)

[0119] QVQLQQSGNELVKPGSSVILSCKASGFTFTNYDINWVRQRPEQGLEWIGWIFPGDGHTRYNEK FKGKATLTMDKSSNTAYMQLSRLASEDSAVYFCAREYDHFDYWGQGTTLTVSS

[0120] Light chain variable region (SEQ ID NO.2)

[0121] DIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVP DRFSGSGSGTEFTLRISRVEAEDVGVYYCGQNLEVPLTFGAGTKLELK

[0122] Table 1

[0123]

[0124] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An anti-LC-1 antibody or its antigen-binding fragment, characterized in that, It includes variable regions for heavy chains and variable regions for light chains; The heavy chain variable region includes complementarity-determining regions VH-CDR1, VH-CDR2, and VH-CDR3; the light chain variable region includes complementarity-determining regions VL-CDR1, VL-CDR2, and VL-CDR3. The VH-CDR1, VH-CDR2, and VH-CDR3 are amino acid sequences identical to those of the VH-CDR1, VH-CDR2, and VH-CDR3 in the heavy chain variable region shown in SEQ ID NO.1; the VL-CDR1, VL-CDR2, and VL-CDR3 are amino acid sequences identical to those of the VL-CDR1, VL-CDR2, and VL-CDR3 in the light chain variable region shown in SEQ ID NO.

2. According to the IMGT definition: the amino acid sequence of VH-CDR1 is shown in SEQ ID NO.11, the amino acid sequence of VH-CDR2 is shown in SEQ ID NO.16, and the amino acid sequence of VH-CDR3 is shown in SEQ ID NO.19; the amino acid sequence of VL-CDR1 is shown in SEQ ID NO.22, the amino acid sequence of VL-CDR2 is QMS, and the amino acid sequence of VL-CDR3 is shown in SEQ ID NO.

25.

2. The anti-LC-1 antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region further includes a backbone region, and / or the light chain variable region further includes a backbone region.

3. The anti-LC-1 antibody or its antigen-binding fragment according to claim 2, characterized in that, Each of the aforementioned skeletal regions is independently derived from one of the following species: rabbit, cattle, horse, pig, sheep, goat, rat, mouse, dog, cat, camel, donkey, deer, mink, chicken, duck, goose, turkey, human, or mutants thereof.

4. The anti-LC-1 antibody or its antigen-binding fragment according to claim 3, characterized in that, The cow in question is a dairy cow.

5. The anti-LC-1 antibody or its antigen-binding fragment according to claim 2, characterized in that, At least one backbone region of the heavy chain variable region includes an amino acid sequence consistent with the backbone region of the heavy chain variable region shown in SEQ ID NO.

1.

6. The anti-LC-1 antibody or its antigen-binding fragment according to claim 2, characterized in that, At least one backbone region of the light chain variable region includes an amino acid sequence consistent with the backbone region of the light chain variable region shown in SEQ ID NO.

2.

7. The anti-LC-1 antibody or its antigen-binding fragment according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the anti-LC-1 antibody or its antigen-binding fragment is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

2.

8. The anti-LC-1 antibody or its antigen-binding fragment according to claim 1, characterized in that, The anti-LC-1 antibody or its antigen-binding fragment is a complete antibody, F(ab')2, Fab', Fab, Fv, scFv, dsFv, or a bispecific antibody.

9. The anti-LC-1 antibody or its antigen-binding fragment according to claim 8, characterized in that, The antibody sequence, excluding the CDR region, is derived from one of the following species: mouse, rat, guinea pig, hamster, rabbit, ferret, cat, dog, goat, sheep, cow, pig, horse, monkey, and human.

10. The anti-LC-1 antibody or its antigen-binding fragment according to any one of claims 1 to 9, characterized in that, It also includes constant regions.

11. The anti-LC-1 antibody or its antigen-binding fragment according to claim 10, characterized in that, At least a portion of the constant region sequence is a human constant region sequence.

12. The anti-LC-1 antibody or its antigen-binding fragment according to claim 11, characterized in that, The constant region sequence is selected from a portion or all of the constant region sequence of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

13. The anti-LC-1 antibody or its antigen-binding fragment according to claim 10, characterized in that, The anti-LC-1 antibody or its antigen-binding fragment contains a heavy chain constant region, and the heavy chain constant region sequence is selected from the human heavy chain constant region.

14. The anti-LC-1 antibody or its antigen-binding fragment according to claim 13, characterized in that, The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.

3.

15. The anti-LC-1 antibody or its antigen-binding fragment according to claim 10, characterized in that, The anti-LC-1 antibody or its antigen-binding fragment contains a light chain constant region.

16. The anti-LC-1 antibody or its antigen-binding fragment according to claim 15, characterized in that, The light chain constant region sequence is selected from the light chain constant region of mice, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO.

4.

17. The anti-LC-1 antibody or its antigen-binding fragment according to claim 8, characterized in that... The anti-LC-1 antibody or its antigen-binding fragment is a complete antibody, with the heavy chain amino acid sequence shown in SEQ ID NO.5 and the light chain amino acid sequence shown in SEQ ID NO.

6.

18. A biomaterial, characterized in that, Including polynucleotides, carriers, or cells; A 1. The polynucleotide encodes the anti-LC-1 antibody or its antigen-binding fragment as described in any one of claims 1 to 17; A2. The vector carries the polynucleotide described in A1; A3. The cell carries the polynucleotide described in A1, or contains the carrier described in A2, or is capable of expressing the anti-LC-1 antibody or its antigen-binding fragment as described in any one of claims 1 to 17.

19. A method for preparing the anti-LC-1 antibody or its antigen-binding fragment according to any one of claims 1 to 17, characterized in that, This includes culturing the cells as described in claim 18.

20. The preparation method according to claim 19, characterized in that, The cells are prepared by converting cells with a polynucleotide encoding an anti-LC-1 antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 16, wherein the polynucleotide includes a heavy chain expression plasmid and a light chain expression plasmid, and the conversion includes co-converting the heavy chain expression plasmid and the light chain expression plasmid into the cells.

21. The preparation method according to claim 19, characterized in that, The cells in question are eukaryotic cells.

22. The preparation method according to claim 21, characterized in that, The eukaryotic cells are mammalian cells.

23. The preparation method according to claim 22, characterized in that, The mammalian cells are 293 cells or CHO cells.

24. The preparation method according to claim 23, characterized in that, The cells in question are 293F cells.

25. The use of the anti-LC-1 antibody or its antigen-binding fragment according to any one of claims 1 to 17, or the biological material according to claim 18, in any one of the following (I) to (VII): (I) Detection of anti-LC-1 antibodies for non-diagnostic and non-therapeutic purposes; (II) Preparation of products for detecting anti-LC-1 antibodies; (III) To prepare products for the diagnosis and / or auxiliary diagnosis of diseases positive for anti-LC-1 antibodies; (IV) Non-diagnostic and non-therapeutic target testing of LC-1 antigen; (V) Prepare products for detecting LC-1 antigen; (VI) Used for the isolation, enrichment and / or purification of LC-1 antigen; (VII) Prepare products for the isolation, enrichment and / or purification of LC-1 antigen; In any of (I) to (III), the anti-LC-1 antibody or its antigen-binding fragment is used as a standard or quality control for LC-1 antibody detection; The disease described as positive for anti-LC-1 antibodies is type II autoimmune hepatitis.

26. A reagent or kit, characterized in that, The reagent or kit comprises the anti-LC-1 antibody or its antigen-binding fragment as described in any one of claims 1 to 17, or the biological material as described in claim 18.

27. The reagent or kit according to claim 26, characterized in that, The kit is used to detect anti-LC-1 antibody or anti-LC-1 antibody-positive diseases; the kit includes standards and / or quality control products, the standards and / or quality control products containing the anti-LC-1 antibody or its antigen-binding fragment; The disease described as positive for anti-LC-1 antibodies is type II autoimmune hepatitis.

28. The reagent or kit according to claim 27, characterized in that, The kit also includes an anti-LC-1 antibody detection reagent.

29. The reagent or kit according to claim 27, characterized in that, The kit also includes a detection reagent for detecting anti-LKM-1 antibodies.

Images