Antibody targeting ATRN protein or antigen-binding fragment thereof, and use thereof

Abstract

An antibody targeting an ATRN protein or an antigen-binding fragment thereof, and the use thereof, and belongs to the field of antibodies. The antibody or the antigen-binding fragment thereof comprises a heavy chain complementarity determining region HCDR, wherein the HCDR comprises at least one selected from amino acid sequences of SEQ ID NOs: 1-3 or an amino acid sequence having at least 80% homology thereto. The antibody or the antigen-binding fragment thereof is capable of binding to a human ATRN protein with high specificity and affinity, blocking the interaction thereof with receptors, inhibiting the signal transduction process, and restoring cell activity, thereby regulating obesity, melanin deposition, neural cell function and anti-tumor function of immune cells in organisms.

Description

Antibodies targeting ATRN protein or their antigen-binding fragments and their applications

[0001] Priority information:

[0002] This disclosure claims priority and benefits to patent application (202411024067.9) filed with the China National Intellectual Property Administration on July 26, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application belongs to the field of antibodies, specifically relating to antibodies against the ATRN protein or antigen-binding fragments thereof and their applications. Background Technology

[0004] ATRN (Attractin) is a membrane-associated molecule that is rapidly upregulated on activated T cells and possesses CD26 protease activity. It is encoded by the ATRN gene on human chromosome 20. ATRN is expressed in a variety of tissues, including skin, brain, liver, and adipose tissue, indicating that it plays an important role in a variety of physiological processes.

[0005] ATRN performs immunomodulatory functions by capturing and proteolytically modifying the N-termini of various cytokines and chemokines. This regulatory activity allows immune cells to interact and form immunomodulatory clusters, and helps downregulate chemokine and cytokine activity after the initiation of the response. Furthermore, ATRN plays an important role in adipocyte differentiation, neural function, skin pigmentation, and extracellular matrix interactions. High expression of ATRN is associated with metabolic disorders such as obesity, and in the nervous system, ATRN participates in neuronal development, synaptic function, and neuroprotection, making it a hot topic in neurological disease research.

[0006] ATRN also interacts with a variety of proteins, including LRP1, Syndecans, HSPGs, Neogenin, and NCAM, and is involved in cell signaling, adhesion, and neural development. Therefore, blocking the interaction of ATRN with other proteins may have potential therapeutic value for related diseases. However, its specific mechanism of action and its role in disease states remain not fully understood, and there is a lack of specific antibodies against ATRN.

[0007] Therefore, there is an urgent need to develop a therapeutic anti-ATRN antibody with high affinity. Summary of the Invention

[0008] This application aims to solve at least one of the aforementioned technical problems. Therefore, one objective of this application is to provide an antibody with high affinity and high specificity that binds to human ATRN protein.

[0009] Specifically, this application provides the following technical solution:

[0010] In a first aspect of this application, an antibody or its antigen-binding fragment is provided. According to embodiments of this application, it includes a heavy chain complementarity-determining region (HCDR), said HCDR comprising at least one of SEQ ID NO: 1-3 or an amino acid sequence having at least 80% homology with it. In some examples of this application, the aforementioned antibody or its antigen-binding fragment can bind to the extracellular segment of ATRN protein with high specificity and affinity, blocking its interaction with the receptor, inhibiting signal transduction processes, restoring cell activity, thereby regulating obesity, melanin deposition, nerve cell function, and the anti-tumor function of immune cells.

[0011] It should be noted that the aforementioned term "receptor" includes the melanocortin receptor, melanocortin / adrenocorticotropic hormone (ACTH) receptor, melanocortin receptor, melanocortin receptor, or other receptors that interact with ATRN. In some examples of this application, the aforementioned other receptors that interact with ATRN have biological functions.

[0012] In a second aspect of this application, a nucleic acid molecule is provided. According to embodiments of this application, the nucleic acid molecule encodes the antibody or its antigen-binding fragment described in the first aspect of this application. In some examples of this application, based on the aforementioned nucleic acid molecule, the aforementioned antibody or its antigen-binding fragment can be efficiently expressed in vitro. This nucleic acid molecule enables the large-scale in vitro expression of the aforementioned antibody or its antigen-binding fragment.

[0013] In a third aspect, this application provides an expression vector. According to embodiments of this application, the expression vector carries the nucleic acid molecule described in the second aspect of this application. In some examples of this application, after the expression vector is introduced into suitable recipient cells, the expression of the aforementioned antibody or its antigen-binding fragment can be effectively achieved under the mediation of a regulatory system, thereby enabling the large-scale in vitro preparation of the antibody or its antigen-binding fragment.

[0014] In a fourth aspect of this application, a method for preparing the antibody or antigen-binding fragment thereof described in the first aspect is provided. According to embodiments of this application, the method includes: introducing the expression vector described in the third aspect of this application into cells; culturing the cells under conditions suitable for protein expression and secretion to obtain the antibody or antigen-binding fragment thereof. In some examples of this application, based on the above method, antibodies or antigen-binding fragments thereof capable of binding to ATRN proteins with high specificity and affinity can be efficiently prepared in vitro.

[0015] In a fifth aspect of this application, a recombinant cell is provided. According to embodiments of this application, the recombinant cell carries the antibody or its antigen-binding fragment described in the first aspect, the nucleic acid molecule described in the second aspect, or the expression vector described in the third aspect. The recombinant cell is obtained by transfecting or transforming the expression vector. In some examples of this application, the recombinant cell can efficiently express the aforementioned antibody or its antigen-binding fragment under suitable conditions, and the obtained antibody or its antigen-binding fragment can bind to the ATRN protein with high specificity and affinity.

[0016] In a sixth aspect of this application, a recombinant protein is proposed. According to embodiments of this application, the recombinant protein comprises: the antibody or its antigen-binding fragment described in the first aspect of this application. In some examples of this application, the aforementioned engineered protein can effectively target the ATRN protein, block its interaction with the receptor, inhibit signal transduction processes, restore cell activity, thereby regulating obesity, melanin deposition, nerve cell function, and the anti-tumor function of immune cells in organisms.

[0017] In a seventh aspect of this application, a composition is proposed. According to embodiments of this application, the composition comprises: the antibody or its antigen-binding fragment described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fifth aspect, or the recombinant protein described in the sixth aspect. As previously mentioned, the antibody or its antigen-binding fragment can bind to the ATRN protein with high specificity and affinity. Therefore, compositions containing the antibody or its antigen-binding fragment, such as food compositions and pharmaceutical compositions, can also effectively target the ATRN protein, block its interaction with the receptor, inhibit signal transduction processes, restore cell activity, and thereby regulate obesity, melanin deposition, nerve cell function, and the anti-tumor function of immune cells in organisms.

[0018] In an eighth aspect of this application, the use of the antibody or antigen-binding fragment thereof described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fifth aspect, the recombinant protein described in the sixth aspect, or the composition described in the seventh aspect in the preparation of a medicament for the treatment or prevention of ATRN-mediated diseases. In some examples of this application, medicaments prepared based on antibodies or antigen-binding fragments thereof, recombinant proteins, nucleic acid molecules, expression vectors, recombinant cells, or pharmaceutical compositions, wherein the aforementioned antibodies or antigen-binding fragments thereof can bind to ATRN proteins with high specificity and affinity, blocking their interaction with receptors, inhibiting signal transduction processes, restoring cell activity, thereby regulating obesity, melanin deposition, nerve cell function, and the anti-tumor function of immune cells in organisms.

[0019] In a ninth aspect of this application, a medicament is proposed. According to embodiments of this application, the medicament comprises: an antibody or antigen-binding fragment thereof as described in the first aspect, a nucleic acid molecule as described in the second aspect, an expression vector as described in the third aspect, recombinant cells as described in the fifth aspect, a recombinant protein as described in the sixth aspect, or a composition as described in the seventh aspect. The medicament is used to treat or prevent ATRN-mediated diseases. In some examples of this application, medicaments prepared based on antibodies or antigen-binding fragments thereof, recombinant proteins, nucleic acid molecules, expression vectors, recombinant cells, or medicament compositions can effectively target the ATRN protein, block its interaction with its receptor, inhibit signal transduction processes, and restore cell activity, thereby regulating obesity, melanin deposition, nerve cell function, and the anti-tumor function of immune cells in organisms.

[0020] In a tenth aspect of this application, the use of the antibody or antigen-binding fragment thereof described in the first aspect in the preparation of a kit for detecting ATRN is proposed. In some examples of the application, kits prepared based on antibodies or antigen-binding fragments thereof, recombinant proteins, nucleic acid molecules, expression vectors, or recombinant cells can be used for portable detection of ATRN protein.

[0021] In the eleventh aspect of this application, a kit is provided. According to an embodiment of this application, the kit comprises the antibody or antigen-binding fragment thereof described in the first aspect. As previously mentioned, the antibody or antigen-binding fragment thereof can bind to ATRN protein with high specificity and affinity; therefore, the kit comprising the antibody or antigen-binding fragment thereof can be used for efficient and portable detection of ATRN. In some examples of this application, the kit can be used for scientific research, such as for qualitative or quantitative detection of ATRN protein in biological samples, and can also be used to assess an individual's condition, such as determining whether the individual's ATRN level is higher or lower than normal after obtaining the individual's ATRN level.

[0022] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 is a schematic diagram of the chromatographic results of the purified ATRN extracellular fragment protein (antigen) provided in the embodiments of this application;

[0025] Figure 2 is a schematic diagram of the SDS-PAGE characterization results of ATRN protein provided in the embodiments of this application;

[0026] Figure 3 is a schematic diagram of antibody chromatography detection and purification results provided in the embodiments of this application;

[0027] Figure 4 is a schematic diagram of the chromatographic results of the removal of the FC segment and TEV protease by enzymatic cleavage of nanobodies provided in the embodiments of this application;

[0028] Figure 5 is an electrophoresis diagram of the protein SDS-PAGE gel provided in the embodiments of this application; wherein, Nb(UD) represents the nanobody containing the FC fragment; Nb(Dig) represents the nanobody after enzyme digestion and separation by nickel column and Protein A column; FC represents the FC fragment separated from the nanobody after enzyme digestion; TEV represents the tobacco etched virus protease separated from the nanobody after enzyme digestion.

[0029] Figure 6 is a schematic diagram of the verification results of the binding ability of ATRN antigen and nanobody by ELISA provided in the embodiments of this application;

[0030] Figure 7 is a schematic diagram of flow cytometry detection of the ability of nanobody-stimulated human peripheral blood mononuclear cells (PBMCs) to secrete cytokines, according to the embodiments of this application. In Figure 7(A), ISO represents the isotype control flow cytometry antibody sample detected by ATRN flow cytometry after 96 hours of PHA stimulation, Ctrl represents the sample detected by ATRN flow cytometry without PHA stimulation, and PHA represents the sample detected by ATRN flow cytometry after 96 hours of PHA stimulation. The vertical axis of the flow cytometry plot represents the cell number, and the horizontal axis represents the AF647 fluorescence intensity. Figure 7(B) shows the gates representing the flow cytometry detection of NK cell cytokine secretion ability. Figure 7(C) shows the gate diagram and statistical graph for the flow cytometry detection of CD8+ T cell cytokine secretion ability. FC represents the control group of PBMCs stimulated by FC protein, and Anti-ATRN represents the experimental group of PBMCs stimulated by nanobodies. The vertical axis of the flow cytometry graph represents the fluorescence intensity of the BV421 fluorescence channel, and the horizontal axis represents the fluorescence intensity of the BV605 fluorescence channel.

[0031] Figure 8 is a schematic diagram of the inhibition of HepG2 subcutaneous tumor growth by the anti-ATRN nanobody provided in the embodiments of this application. Detailed Implementation

[0032] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present application.

[0033] In this application, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0034] In this application, the term "antibody" refers to an immunoglobulin, which is composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain differ, thus their antigenicity also differs. Immunoglobulins can be classified into five classes: IgM, IgD, IgG, IgA, and IgE. The same class of immunoglobulins can also be divided into different subclasses based on their amino acid composition, such as IgG1, IgG2, IgG3, and IgG4. The immunoglobulin light chain is divided into κ chains or λ chains based on the difference in its constant region.

[0035] The sequence of approximately 110 amino acids near the N-terminus of both the antibody heavy and light chains varies considerably and is known as the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are called the constant region. The variable region includes three hypervariable regions (HVRs) and four relatively conserved backbone regions (FRs). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0036] In this application, the term "monoclonal antibody (abbreviated as MABS)" refers to an antibody that can recognize only one specific antigenic epitope. Common MABS consist of two lighter light chains and two heavier heavy chains, linked by disulfide bonds to form a tetrapeptide molecule. The amino-terminal (N-terminus) amino acid sequence of this heavy or light chain varies considerably and is called the variable region (V-terminus), while the carboxyl-terminus (C-terminus) of the heavy or light chain is relatively stable and changes very little, and is called the constant region (C-terminus). The V-termini of the L-chain and H-chain are called VL and VH, respectively. MABS can also be small molecule antibodies, which mainly include Fab antibodies, Fv antibodies, single-chain antibodies, single-domain antibodies, and the smallest recognition unit.

[0037] In this application, the term "polyclonal antibody (abbreviated as polyclonal antibody)" refers to an antibody that can recognize multiple antigenic epitopes, such as an antibody that can recognize two antigenic epitopes (abbreviated as biclonal antibody), an antibody that can recognize three antigenic epitopes, or an antibody that can recognize four antigenic epitopes. It is interpreted in a broad sense, and the specific structure is not limited, as long as it can recognize multiple antigenic epitopes.

[0038] In this application, the term "nanobody" refers only to the heavy chain variable region (VH) and the conventional CH2 and CH3 regions, which bind specifically to the antigen through the heavy chain variable region.

[0039] In this application, the term "antigen-binding fragment" refers to a Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, or ScFv fragment having antigen-binding activity; which contains one or more CDR regions selected from SEQ ID NO:1 to 3 of the antibody described in this application.

[0040] In this application, the amino acid sequences of the CDRs listed above are all as shown in the IMGT definition rules. However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as the Kabat rule, the Chothia rule, etc. Those skilled in the art should understand that, unless otherwise specified, the terms "CDR" and "complementarity-determining region" for a given antibody or its region (e.g., variable region) should be understood to encompass complementarity-determining regions defined as described in any of the known schemes above. Although the scope of protection claimed in this application is based on the sequences shown in the IMGT definition rules, amino acid sequences corresponding to other CDR definition rules should also be included in the scope of protection of this application.

[0041] In this application, the term "amino acid" is represented by a single-letter or three-letter code, with the following meanings: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Aspartic acid (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamic acid); G: Gly (glycine); H: Histidine; I: Ile (isoleucine); L: Leu (leucine); K: Lysine (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Proline (proline); S: Serine (serine); T: Threonine (threonine); W: Tryptophan (tryptophan); Y: Tyrosine (tyrosine); V: Valine (valine).

[0042] For nucleotides, the terms "homology," "identity," or "similarity" are used to describe or compare the degree of nucleotide similarity between two or more nucleotide sequences. The percentage of "sequence homology" between a first and a second sequence can be calculated by dividing the number of nucleotides in the first sequence that are identical to those at the corresponding positions by the number of nucleotides in the second sequence. This is calculated by subtracting the total number of nucleotides in the first sequence from the number of nucleotides in the second sequence and then multiplying by 100%, where each deletion, insertion, substitution, or addition of a nucleotide in the second sequence—relative to the first sequence—is considered a difference at a single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using standard settings and known computer algorithms for sequence alignment, such as NCBI Blast v2.0. Other techniques, computer algorithms, and settings used to determine the degree of sequence identity include, for example, those in WO 04 / 037999, EP 0 967 284, EP 1 085 089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185, and GB 2357768-A.

[0043] For peptides, the terms "(substantial) homology," "identity," or "similarity" are used to describe or compare the degree of amino acid similarity between two or more peptides or their designated sequences at optimal alignment and comparison (where appropriate insertions or deletions of nucleotides are made). The percentage of homology between two sequences varies with the number of identical positions shared by these sequences at optimal alignment (i.e., homology % = number of identical positions / total number of positions × 100), where optimal alignment is determined taking into account the number of vacancies introduced to achieve optimal alignment of the two sequences and the length of each vacancy. Sequence comparison and identity percentage determination between two sequences can be performed using mathematical algorithms, as described in the non-limiting examples below.

[0044] In this application, without substantially affecting antibody activity (retaining at least 95% of the activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids to obtain sequences of the antibody or its functional fragments. These are all considered to be included within the scope of protection of this invention. For example, amino acids with similar properties can be substituted in the variable region. The variant sequences described in this application can have at least 80% identity (or homology) with the reference sequences, meaning at least 80%, which can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with each reference sequence. The sequence consistency described in this application can be measured using sequence analysis software. For example, using the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The amino acid sequences described in this invention are shown from the N-terminus to the C-terminus.

[0045] In this document, the term "vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule into host cells and / or between host cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having multiple of the aforementioned functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the vector, the vector can produce the desired expression product.

[0046] In this application, the term "composition" generally refers to a unit dosage form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining an active ingredient with a carrier constituting one or more adjunct components. Typically, compositions are prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a finely pulverized solid carrier, or both.

[0047] In this application, the term "pharmaceuticalally acceptable" means a substance that is suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio.

[0048] In this application, the term "pharmaceuticalally acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a particular target dosage form. The use of any conventional excipients that are incompatible with the compounds of this invention, such as any adverse biological effects they produce or interactions that occur harmfully with any other component of the pharmaceutically acceptable composition, is also within the scope of this invention.

[0049] In this application, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The antibodies or antigen-binding fragments, recombinant proteins, multispecific antibodies, conjugates, or pharmaceutical compositions of the present invention can be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified routes of administration. Preferably, the compositions of the present invention are administered via intravenous or subcutaneous injection.

[0050] In this application, the term "treatment" is used to refer to achieving a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, "treatment" covers diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of a condition in individuals susceptible to disease but not yet diagnosed with it; (b) suppression of disease, such as inhibiting disease progression; or (c) alleviating disease, such as reducing symptoms associated with the disease. As used herein, "treatment" covers any administration of a drug or compound to an individual to treat, cure, alleviate, improve, reduce, or suppress the individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0051] In this application, the terms "effective amount" or "effective dose" refer to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0052] The amino acid sequences involved in this application are shown in Table 1.

[0053] This application discloses an antibody or antigen-binding fragment thereof targeting the ATRN protein, a method for its preparation, a nucleic acid molecule, an expression vector, a recombinant cell, a recombinant protein, a composition, a pharmaceutical use, a drug, a use in a preparation kit, a kit, a method for treating a disease, and a use for treating a disease, which will be described in detail below.

[0054] Antibody or its antigen-binding fragment

[0055] In one aspect of this application, an antibody or antigen-binding fragment thereof is provided, comprising a heavy chain complementarity-determining region (HCDR), said HCDR containing an amino acid sequence selected from at least one of SEQ ID NO: 1 to 3 or having at least 80% homology with it. In some examples of this application, antibodies or antigen-binding fragments thereof having the aforementioned HCDR amino acid sequence are capable of binding ATRN proteins with high affinity and specificity.

[0056] In some examples of this application, the aforementioned HCDRs include: HCDR1, HCDR2, and HCDR3, wherein HCDR1 contains an amino acid sequence as shown in SEQ ID NO:1 or having at least 80% homology with SEQ ID NO:1, HCDR2 contains an amino acid sequence as shown in SEQ ID NO:2 or having at least 80% homology with SEQ ID NO:2, and HCDR3 contains an amino acid sequence as shown in SEQ ID NO:3 or having at least 80% homology with SEQ ID NO:3. Antibodies based on the aforementioned HCDR sequences or their antigen-binding fragments exhibit high binding affinity and specificity to ATRN proteins.

[0057] In some preferred embodiments of this application, the aforementioned HCDR includes HCDR1, HCDR2, and HCDR3, respectively, with amino acid sequences as shown in SEQ ID NO:1–3. Experimental verification has shown that antibodies or their antigen-binding fragments having amino acid sequences as shown in SEQ ID NO:1–3 exhibit high binding affinity and specificity to ATRN proteins.

[0058] In some examples of this application, the aforementioned antibody or its antigen-binding fragment further includes a light chain complementarity-determining region (LCDR), wherein the LCDR includes LCDR1, LCDR2, and LCDR3.

[0059] It should be noted that one or more amino acid residues in the aforementioned HCDR targeting the ATRN protein can be replaced by other amino acid residues from the same side chain family, and the retention function of the modified antibody can be tested using the functional assay methods described herein.

[0060] In some examples of this application, the aforementioned antibody or its antigen-binding fragment further includes a heavy chain framework region.

[0061] In some examples of this application, at least a portion of the aforementioned heavy chain framework region is independently derived from at least one of alpaca-derived antibodies, shark-derived antibodies, mouse-derived antibodies, rabbit-derived antibodies, primate-derived antibodies, or mutants thereof. In a preferred example of this application, the aforementioned heavy chain framework region is selected from alpaca-derived antibodies.

[0062] In some preferred embodiments of this application, the heavy chain variable region of the aforementioned antibody or its antigen-binding fragment comprises an amino acid sequence selected from or having at least 80% homology with SEQ ID NO:4. In some more preferred embodiments of this application, the amino acid sequence of the heavy chain variable region of the aforementioned antibody or its antigen-binding fragment is as shown in SEQ ID NO:4.

[0063] In some examples of this application, the aforementioned antibody or its antigen-binding fragment further includes a light chain framework region.

[0064] In some examples of this application, at least a portion of the light chain framework region is independently derived from at least one of a mouse antibody, a rabbit antibody, a primate antibody, or a mutant thereof.

[0065] Antibodies or antigen-binding fragments based on the aforementioned heavy chain variable region sequence exhibit high affinity and specificity for ATRN protein binding.

[0066] In some examples of this application, the aforementioned antibody or its antigen-binding fragment further includes a heavy chain constant region.

[0067] In some examples of this application, the aforementioned antibody or its antigen-binding fragment further includes a light chain constant region.

[0068] In some examples of this application, at least a portion of the aforementioned heavy chain constant region and the aforementioned light chain constant region are independently derived from at least one of rabbit-derived antibodies, mouse-derived antibodies, sheep-derived antibodies, goat-derived antibodies, primate-derived antibodies, or mutants thereof. In some preferred examples of this application, at least a portion of the aforementioned heavy chain constant region and the aforementioned light chain constant region are derived from mouse-derived antibodies.

[0069] In some examples of this application, the aforementioned antibody includes at least one selected from polyclonal antibodies and monoclonal antibodies; or the aforementioned antigen-binding fragment includes at least one selected from F(ab')2 fragment, Fab' fragment, Fab fragment, Fv fragment, scFv fragment, scFv-Fv fusion protein and minimum recognition unit.

[0070] In some preferred embodiments of this application, the aforementioned antibody is a nanobody. This nanobody was first obtained by the inventors through mutation of the ATRN nanosequence using an AI model (HIE BL, SHANKER VR, XU D, et al. Efficient evolution of human antibodies from general protein language models[J]. Nature Biotechnology, 2024, 42(2):275-283.), and it has shown excellent tumor suppression effects in the HepG2 humanized tumor model.

[0071] Those skilled in the art will understand that the aforementioned antibody or antigen-binding fragments are applicable to a variety of animal tumor models, including but not limited to mouse tumor models.

[0072] In some examples of this application, the aforementioned nanobodies have an amino acid sequence as shown in SEQ ID NO:4 or with at least 80% homology to SEQ ID NO:4. In some examples of this application, sufficient experimental verification has shown that nanobodies with an amino acid sequence as shown in SEQ ID NO:4 can bind to ATRN proteins with high affinity and high specificity.

[0073] According to the embodiments of this application, the aforementioned antibody or antigen-binding fragment can bind to the extracellular domain of human ATRN protein, blocking the inhibitory signal transduction of ATRN on NK cells and CD8+ T cells, promoting the ability of human peripheral blood NK cells to secrete perforin, and CD8+ T cells to secrete interferon, tumor necrosis factor, and perforin, thereby enhancing the killing function of NK cells and CD8+ T cells.

[0074] Nucleic acid molecules

[0075] In another aspect of this application, a nucleic acid molecule is proposed that encodes an antibody or an antigen-binding fragment thereof as described above. In some examples of this application, antibodies or antigen-binding fragments thereof can be efficiently expressed in vitro based on the aforementioned nucleic acid molecule.

[0076] In some examples of this application, the nucleic acid molecule is DNA.

[0077] It should be noted that those skilled in the art will understand that the nucleic acid molecules mentioned in this specification and claims actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases in this specification and claims, the other complementary strand is actually disclosed as well. In addition, the nucleic acid sequences in this application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0078] expression carrier

[0079] In another aspect of this application, an expression vector is proposed that carries the nucleic acid molecule as described above. In some examples of this application, the aforementioned expression vector enables the efficient in vitro preparation of antibodies or antigen-binding fragments.

[0080] It should be noted that when linking the aforementioned nucleic acid molecules to the vector, the nucleic acid molecules can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecules. These control elements can originate directly from the vector itself or be exogenous, i.e., not derived from the vector itself. Of course, the connection between the nucleic acid molecules and the control elements must be operably established.

[0081] According to embodiments of the present invention, the vector may refer to a cloning vector or an expression vector, and can be obtained by operatively ligating the nucleic acid to a commercially available vector (such as a plasmid or viral vector). The vector in this invention is not particularly limited; commonly used plasmids such as pSeTag2, PEE14, and pMH3 can be used.

[0082] In this document, the term "operably ligated" refers to ligating a foreign gene to a vector such that the control elements within the vector, such as amino acid sequences controlling transcription and amino acid sequences controlling translation, can perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used vectors include viral vectors, plasmids, bacteriophages, etc. After the expression vector according to some specific embodiments of the present invention is introduced into suitable recipient cells, the expression of the aforementioned nucleic acid molecules can be effectively achieved under the mediation of a regulatory system, thereby enabling the large-scale in vitro production of the proteins encoded by the nucleic acid molecules.

[0083] In some examples of this application, the vector is a eukaryotic vector or a prokaryotic vector.

[0084] In some examples of this application, the vector includes at least one selected from plasmid vectors, adenovirus vectors, lentivirus vectors, and adeno-associated virus vectors.

[0085] Preparation method of antibody or its antigen-binding fragment

[0086] In another aspect of this application, a method for preparing the aforementioned antibody or its antigen-binding fragment is provided, comprising: introducing the aforementioned expression vector into cells; and culturing the cells under conditions suitable for protein expression and secretion in order to obtain the aforementioned antibody or its antigen-binding fragment.

[0087] In some examples of this application, the cells are eukaryotic cells.

[0088] In some specific examples of this application, the eukaryotic cells are mammalian cells. When the cells are mammalian cells, the expression efficiency of the antibody or its antigen-binding fragment is higher.

[0089] It should be noted that the eukaryotic cells mentioned do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0090] Recombinant cells

[0091] In another aspect of this application, a recombinant cell is provided, carrying the aforementioned antibody or its antigen-binding fragment, the aforementioned nucleic acid molecule, or the aforementioned expression vector. In some examples of this application, the recombinant cell is obtained by transfecting or transforming the expression vector, and the aforementioned recombinant cell can efficiently express the aforementioned antibody or antigen-binding fragment under suitable conditions.

[0092] It should be noted that the recombinant cells described in this application are not particularly limited and can be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, etc. The eukaryotic cells can be fungi including Pichia pastoris, Saccharomyces cerevisiae, Schizosoma, Trichoderma, etc.; insect cells such as armyworms; plant cells such as tobacco; and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells. In some embodiments, the recombinant cells described in this application are preferably mammalian cells, including BHK cells, CHO cells, NSO cells, or COS cells, and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0093] The aforementioned “transformation” or “transfection” refers to the introduction of nucleic acids (e.g., vectors) into cells using various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of this application and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in this invention include immortalized hybridoma cells, NS / O myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0094] It should be noted that the "suitable conditions" mentioned in this application refer to conditions suitable for antibody expression. Those skilled in the art will readily understand that suitable conditions for antibody expression include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the optimal conditions for antibody expression based on the specific environment of their laboratory.

[0095] Recombinant protein

[0096] In another aspect of this application, a recombinant protein is proposed, comprising: the aforementioned antibody or its antigen-binding fragment. In some examples of this application, the recombinant protein is capable of binding to ATRN protein with high affinity and high specificity.

[0097] In some examples of this application, the aforementioned recombinant protein may further include at least one selected from bioactive proteins or fragments thereof, bioactive peptides or fragments thereof.

[0098] In some examples of this application, the bioactive protein or fragment thereof includes at least one selected from protein tags, protein toxins or fragments thereof, tumor necrosis factor or fragments thereof, interferon or fragments thereof, biological response regulators or fragments thereof, and Fc fragments.

[0099] The term "protein tag" typically refers to a polypeptide or protein fused together with a target protein (antibody or antigen-binding fragment) for expression. It can be used for the expression, detection, detection, or purification of the target protein. Examples include, but are not limited to, His tags (also known as His-Tag, sequence HHHHHH), Flag tags (also known as Flag-Tag, sequence DYKDDDDK), GST tags (also known as GST-Tag, glutathione thiotransferase tag), SUMO tags, and C-Myc tags.

[0100] The term "toxin" generally refers to substances toxic to the host, including protein toxins and non-protein toxins. Protein toxins include, but are not limited to, abrin, ricin A, Pseudomonas exotoxin, and diphtheria toxin. In this application, the protein toxin is preferably an enzymatic protein toxin.

[0101] The term "tumor necrosis factor" usually refers to substances that can cause hemorrhagic necrosis in various tumors, including but not limited to TNF-α and TNF-β.

[0102] The term "interferon" typically refers to a glycoprotein that can directly kill or inhibit viruses. This includes, but is not limited to, IFN-α, IFN-β, and IFN-γ.

[0103] The term "biological response modulators" generally refers to a class of protein substances that enhance the body's anti-tumor effects directly or indirectly through the immune system. These include, but are not limited to, lymphokines, IL-2, IL-6, IL-10, and GM-CSF.

[0104] The term "Fc fragment" typically refers to the Fc region of IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM, including CH2, CH3 regions and optionally a hinge region. Preferably, the IgG, IgA1, IgA2, IgD, IgE, or IgM is derived from alpaca, mouse, or primate sources.

[0105] Composition

[0106] In another aspect of this application, a composition is provided comprising: the aforementioned antibody or its antigen-binding fragment, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, or the aforementioned recombinant protein. In some examples of this application, the aforementioned composition can be used to prepare related products targeting the ATRN protein, such as drugs, reagent kits, etc.

[0107] It should be noted that the composition includes combinations that are separate in time and / or space, as long as they can work together to achieve the purpose of this application. For example, the components contained in the composition may be administered to the subject as a whole or separately. When the components contained in the composition are administered to the subject separately, the individual components may be administered to the subject simultaneously or sequentially.

[0108] Pharmaceutical Uses

[0109] In another aspect of this application, the application proposes the use of the aforementioned antibody or its antigen-binding fragment, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, the aforementioned recombinant protein, or the aforementioned composition in the preparation of a medicament for the treatment or prevention of ATRN-mediated diseases.

[0110] In some examples of this application, the ATRN-mediated related diseases include neurological disorders, autism spectrum disorder (ASD), Alzheimer's disease, schizophrenia, Parkinson's disease, bipolar disorder, epilepsy, diabetic nephropathy, glomerulonephritis, chronic kidney disease, polycystic kidney disease, nephrotic syndrome, renal cell carcinoma, obesity, type 2 diabetes, non-alcoholic fatty liver disease, metabolic syndrome, hypothyroidism, Cushing's syndrome, hyperlipidemia, coronary artery disease, heart failure, stroke, peripheral artery disease, atrial fibrillation, atherosclerosis, hypertension, coronary artery disease, heart failure, arrhythmia, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, psoriasis, melanoma, breast cancer, lung cancer, colorectal cancer, prostate cancer, ovarian cancer, renal cell carcinoma, liver cancer, lung cancer, stomach cancer, colorectal cancer, breast cancer, esophageal cancer, thyroid cancer, skin cancer, prostate cancer, kidney cancer, endometrial cancer, cervical cancer, and hematologic malignancies.

[0111] drug

[0112] In another aspect of this application, a medicament is provided, comprising: the aforementioned antibody or its antigen-binding fragment, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, the aforementioned recombinant protein, or the aforementioned composition, said medicament for treating or preventing ATRN-mediated diseases.

[0113] The ATRN-mediated diseases include neurological disorders, autism spectrum disorder (ASD), Alzheimer's disease, schizophrenia, Parkinson's disease, bipolar disorder, epilepsy, diabetic nephropathy, glomerulonephritis, chronic kidney disease, polycystic kidney disease, nephrotic syndrome, renal cell carcinoma, obesity, type II diabetes, non-alcoholic fatty liver disease, metabolic syndrome, hypothyroidism, Cushing's syndrome, hyperlipidemia, coronary artery disease, heart failure, stroke, peripheral artery disease, atrial fibrillation, atherosclerosis, hypertension, coronary artery disease, heart failure, arrhythmia, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, psoriasis, melanoma, breast cancer, lung cancer, colorectal cancer, prostate cancer, ovarian cancer, renal cell carcinoma, liver cancer, lung cancer, stomach cancer, colorectal cancer, breast cancer, esophageal cancer, thyroid cancer, skin cancer, prostate cancer, kidney cancer, endometrial cancer, cervical cancer, and hematological malignancies.

[0114] In some examples of this application, pharmaceutically acceptable excipients are further included.

[0115] In some examples of this application, the excipients include one or more pharmaceutically acceptable excipients, diluents, stabilizers, or carriers.

[0116] In some examples of this application, the pharmaceutical composition is an injectable formulation.

[0117] It should be noted that the pharmaceutical composition includes combinations that are separate in time and / or space, as long as they can work together to achieve the objectives of the present invention. For example, the components contained in the pharmaceutical composition may be administered to the subject as a whole or separately. When the components contained in the pharmaceutical composition are administered to the subject separately, the individual components may be administered to the subject simultaneously or sequentially.

[0118] The drug of this application contains a safe and effective amount of the active ingredient (antibody) of this application and pharmaceutically acceptable excipients. Such excipients include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical formulation should be matched to the route of administration; the dosage form of the drug of this application is an injection, oral formulation (tablets, capsules, oral liquid), transdermal formulation, or sustained-release formulation. For example, it is prepared using physiological saline or an aqueous solution containing glucose and other excipients by conventional methods. The drug is preferably manufactured under aseptic conditions.

[0119] The effective amount of the active ingredient described in this application may vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0120] Pharmaceutically acceptable excipients described in this application include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched to the route of administration, as is well known to those skilled in the art.

[0121] Uses in the preparation kit

[0122] In another aspect of this application, the use of the aforementioned antibody or its antigen-binding fragment in the preparation of a kit for detecting ATRN is provided.

[0123] As previously described, the antibodies or antigen-binding fragments of this application can specifically bind to ATRN; therefore, the antibodies or antigen-binding fragments can be used to detect ATRN proteins. Furthermore, they can be used to prepare ATRN-related kits for scientific research, such as qualitative or quantitative detection of ATRN protein molecules in biological samples. More specifically, they can be used in kits involving the specific binding properties of ATRN and antibodies, such as immunoblotting and immunoprecipitation. These kits may contain any one or more of the following: antagonists, the antibodies of this application, or pharmaceutical reference materials; protein purification columns; immunoglobulin affinity purification buffers; and cell assay diluents. The antibodies of this application can be used in different types of diagnostic tests, such as detecting the presence of various diseases, drugs, toxins, or other proteins in vitro or in vivo. For example, they can be used to test for ATRN-mediated diseases (such as liver cancer) by detecting the serum or blood of a subject.

[0124] Reagent test kit

[0125] In another aspect of this application, a kit is provided comprising the antibody or its antigen-binding fragment described in the first aspect. The antibody or its antigen-binding fragment in the kit provided by this application can effectively bind to the ATRN protein. Furthermore, under suitable conditions, the nucleic acid molecule, expression vector, or recombinant cell can express the antibody or its antigen-binding fragment. Further, the kit containing the above substances can bind to ATRN with high specificity and can be used for the effective detection of ATRN. The kit can be used for scientific research, such as for qualitative or quantitative detection of ATRN in biological samples, and can also be used to assess an individual's condition, such as determining whether the individual's ATRN level is too high or too low than normal after obtaining the individual's ATRN level. The biological sample can be cells, tissues, etc.

[0126] In some examples of this application, the kit is used to detect ATRN.

[0127] Disease treatment methods

[0128] In another aspect of this application, a method for preventing and / or treating ATRN-mediated diseases is provided. According to embodiments of this application, the method includes administering to a subject a pharmaceutically acceptable amount of an antibody or its antigen-binding fragment, expression vector, recombinant cells, recombinant protein, composition, or drug.

[0129] It should be noted that the terms "subject," "individual," and "patient" are used interchangeably herein and refer to a mammal being evaluated for treatment and / or being treated. In one implementation, the mammal is a human. The terms "subject," "individual," and "patient" include, but are not limited to, individuals with cancer, individuals with autoimmune diseases, individuals with pathogen infections, etc. Subjects can be humans, but also include other mammals, particularly mammals that can be used as laboratory models of human diseases, such as mice, rats, etc.

[0130] The effective amount of the antibody or its antigen-binding fragment, nucleic acid molecule, expression vector, recombinant protein, composition, or drug described in this application may vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0131] In some examples of this application, the ATRN-mediated related diseases include neurological disorders, autism spectrum disorder (ASD), Alzheimer's disease, schizophrenia, Parkinson's disease, bipolar disorder, epilepsy, diabetic nephropathy, glomerulonephritis, chronic kidney disease, polycystic kidney disease, nephrotic syndrome, renal cell carcinoma, obesity, type II diabetes, non-alcoholic fatty liver disease, metabolic syndrome, hypothyroidism, Cushing's syndrome, hyperlipidemia, coronary artery disease, heart failure, stroke, peripheral artery disease, atrial fibrillation, atherosclerosis, hypertension, coronary artery disease, heart failure, arrhythmia, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, psoriasis, melanoma, breast cancer, lung cancer, colorectal cancer, prostate cancer, ovarian cancer, renal cell carcinoma, liver cancer, lung cancer, stomach cancer, colorectal cancer, breast cancer, esophageal cancer, thyroid cancer, skin cancer, prostate cancer, kidney cancer, endometrial cancer, cervical cancer, and hematologic malignancies.

[0132] Uses in disease treatment

[0133] In another aspect of this application, the use of the aforementioned antibodies or antigen-binding fragments thereof, nucleic acid molecules, expression vectors, recombinant proteins, compositions, or drugs in the treatment or prevention of ATRN-mediated diseases is proposed. In some examples of this application, administration of an effective dose of the antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, recombinant protein, composition, or drug to a subject can effectively treat ATRN-mediated diseases.

[0134] In some examples of this application, the ATRN-mediated related diseases include neurological disorders, autism spectrum disorder (ASD), Alzheimer's disease, schizophrenia, Parkinson's disease, bipolar disorder, epilepsy, diabetic nephropathy, glomerulonephritis, chronic kidney disease, polycystic kidney disease, nephrotic syndrome, renal cell carcinoma, obesity, type II diabetes, non-alcoholic fatty liver disease, metabolic syndrome, hypothyroidism, Cushing's syndrome, hyperlipidemia, coronary artery disease, heart failure, stroke, peripheral artery disease, atrial fibrillation, atherosclerosis, hypertension, coronary artery disease, heart failure, arrhythmia, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, psoriasis, melanoma, breast cancer, lung cancer, colorectal cancer, prostate cancer, ovarian cancer, renal cell carcinoma, liver cancer, lung cancer, stomach cancer, colorectal cancer, breast cancer, esophageal cancer, thyroid cancer, skin cancer, prostate cancer, kidney cancer, endometrial cancer, cervical cancer, and hematologic malignancies.

[0135] Table 1

[0136] The present application's solution will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0137] Example 1: Expression, purification, and elution of ATRN extracellular fragment protein

[0138] In this embodiment, the specific steps for expressing, purifying, and eluting the extracellular domain of ATRN protein are as follows:

[0139] Primers were designed to amplify the extracellular segment of ATRN using the full-length ATRN gene sequence as a template. The vector was selected from pTT5. The pTT5-ATRN plasmid was constructed using Gibson assembly.

[0140] The extracted plasmids were transfected into HEK293F cells using polyethyleneimine (PEI) as the transfection reagent. The reagents and dosages are shown in Table 2. The mammalian expression system was transfected using the pTT5-ATRN plasmid.

[0141] Table 2. Reagents and dosages for the transfection system

[0142] Preheat DMEM to 37°C in a water bath before transfection. Place 15 ml of DMEM in two 50 ml tubes, add the plasmid to one tube and PEI to the other. Incubate both tubes at room temperature for 10 minutes. Then transfer the PEI diluted with DMEM to the plasmid tube and incubate at room temperature for 15 minutes. HEK293F cells were grown at a count of 2 million / ml and a total volume of 350 ml. After plasmid-PEI incubation, transfer it dropwise to 400 ml of cells and incubate at 37°C for 20 minutes without shaking. Subsequently, culture the cells in a shaker at 120 rpm and 5% CO2 for 72 to 96 hours. Centrifuge the cells at 4347 rcf for 30 minutes, and then pass the supernatant through 0.45 μm and 0.22 μm filters, respectively. Load the filtered sample onto a peristaltic pump and purify the protein using affinity chromatography (protein A column). The pTT5 vector carries an Fc(IgG) tag, which can specifically bind to the resin of the Protein A column, thereby enriching the target protein in the supernatant onto the column.

[0143] After loading, the protein column was eluted using the AKTA Prime Plus purification system. 0.2 M Tris-HCl (pH = 7) was added to the protein collection tube, and the program was run to elute the protein from 0% B elution buffer to 100% B protocol. Liquid A used was PBS, and liquid B was elution buffer (0.1 M acetic acid solution) used to elute the bound protein. The harvested protein peak pattern is shown in Figure 1, indicating the acquisition of high-purity ATRN extracellular fragment protein.

[0144] Example 2: Detection of ATRN extracellular protein

[0145] The ATRN extracellular fragment protein sample obtained in Example 1 was added to the staining agent and denatured at 95°C for 3 minutes to denature the protein. A 12.5% ​​SDS-gel electrophoresis gel was prepared using the Omni-Easy One-Step SDS-Gel Pretreatment Kit according to standard instructions. The gel was transferred to an electrophoresis tank containing running buffer (Tris-glycine buffer), and the sample and standard label were loaded into the corresponding wells. The power was turned on, and the voltage was set to 90V for 20 minutes, then increased to 130V until the dye reached the bottom. The gel was stained with cresol blue solution to visualize the protein bands, and then placed in the staining solution for appropriate staining. The gel was rinsed with water and incubated at 70°C (changing the water after 10 minutes until the background became clear and the protein bands became visible) to remove excess dye. The gel was imaged using a gel imaging system, as shown in Figure 2, indicating that the ATRN extracellular fragment protein of the correct size was obtained.

[0146] Example 3: Mutation and purification elution of anti-ATRN nanobodies

[0147] The existing ATRN nanobody sequences were analyzed using an AI model (HIE BL, SHANKER VR, XU D, et al. Efficient evolution of human antibodies from general protein language models[J]. Nature Biotechnology, 2024, 42(2):275-283.), and related amino acid mutations were performed. The corresponding nucleotide sequences were synthesized by the company and cloned into the pTT5 vector, and purified using the method in Example 1.

[0148] The antibody chromatographic detection results are shown in Figure 3, indicating that high-purity antibodies were obtained.

[0149] Example 4: TEV digestion of anti-ATRN nanobodies with FC fragment

[0150] To detect the binding affinity of specific nanobodies to ATRN antigens with FC fragments using ELISA, the FC fragments of the nanobodies were further enzymatically digested and the VHH fragments of the anti-ATRN antibody were purified. For this purpose, the purified nanobodies were enzymatically digested with Tobacco Etch Virus protease (TEV protease). The TEV protease was added to the collected proteins and stored overnight at 4°C. Subsequently, the digested proteins were passed through a protein A column and a nickel (Ni-NTA) column to completely remove undigested proteins, FC fragments, and TEV protease, respectively.

[0151] Protein A and the nickel column were then eluted to verify the successful removal of the FC tag and TEV protease. The results are shown in Figure 4, demonstrating the successful removal of the FC tag and TEV protease from the mixture.

[0152] The undigested nanobody protein, the nanobody protein after digestion with the FC fragment removed, the FC fragment, and the TEV protease were subjected to SDS-PAGE electrophoresis. The results are shown in Figure 5, indicating that the obtained proteins are of the correct size.

[0153] Example 5: Detection and verification of the binding affinity between nanobodies and ATRN by ELISA

[0154] Add 0.2 μg of enzyme-digested antibody to each well, add 100 μL of PBS, and incubate overnight at 4°C. Wash the immunoplate three times with PBS, then add 300 μL of MPBS (containing BSA to block non-specific binding sites) to each well and block for 3 hours at room temperature. Wash the immunoplate three times with PBST, add 1000 nM of Fc-tagged antigen (dissolved in MPBS) to the starting well, and then sequentially dilute 10-fold for each subsequent row, adding 100 μL to each well. Incubate with shaking at room temperature for one hour. Wash the immunoplate three times with PBST, then add HRP-conjugated anti-IgG1 Fc antibody (Beijing Yiqiao Shenzhou) to detect the antigen-bound nanobody-Fc fusion protein. Incubate with shaking at room temperature for 1 hour. Wash the immunoplate five times with PBST, then add 100 μL of TMB (chromogenic substrate) to each well. Incubate at room temperature in the dark for 10 minutes, then add 50 μL of 1M H2SO4 to each well to stop the reaction and start the instrument. As shown in Figure 6, the OD450 value gradually increased with the increase of antigen concentration. When the antigen concentration was 1000 nM, the OD450 reached a maximum value of 1.081, indicating that the nanobody can specifically bind to the extracellular segment of ATRN and has high affinity.

[0155] Example 6: Flow cytometry detection of the ability of nanobody-stimulated human peripheral blood mononuclear cells (PBMCs) to secrete cytokines

[0156] PBMCs were cultured in 96-well plates in 1640 medium supplemented with PHA (5 μg / ml), 1% penicillin antibiotics, and 10% FBS at 37°C and 5% CO2 for 96 h, resulting in a cell concentration of 2 × 10⁻⁶ cells / well. 6 The control group received culture medium without PHA. PBMCs were then blocked with PBS containing 10% mouse serum for 15 minutes, followed by labeling with flow cytometry antibodies against CD3-BV786 (Biolegend), CD8-PC7 (Biolegend), and CD56-BV605 (Biolegend). After incubation at 4°C for 30 minutes, cells were washed three times with PBS, then fixed with fixative for 1 hour (Invitrogen eBioscience), labeled with ATRN-AF647 flow cytometry antibody (Santa Cruz) and its isotype control antibody (Biolegend), incubated at 4°C for 30 minutes, and washed three times with permeation buffer (Invitrogen eBioscience). Finally, flow cytometry analysis was performed. The results are shown in Figure 7(A). Analysis using FlowJO software showed that, compared to the control group, CD8+ in PBMCs stimulated with PHA for 96 hours was significantly higher. + T cells and NK cells highly express ATRN.

[0157] Anti-ATRN nanobodies and FC protein were added to PBMCs stimulated with PHA (5 μg / ml) for 96 h, and the antibody concentration was 200 nmol. The cells were cultured at 37 °C and 5% CO2. PBMCs were then stimulated for 4 h with PMA (30 ng / ml), iomycin (1 μg / ml), and monensin (2.5 μg / ml) for 48 h and 96 h, respectively, followed by cell collection by centrifugation. Blocking and labeling with external standard flow cytometry antibodies CD3-BV786 (Biolegend), CD8-PC7 (Biolegend), and CD56-BV605 (Biolegend), followed by membrane perforation and fixation, and labeling with internal standard flow cytometry antibodies IFN-γ-FITC (Biolegend), Perforin-BV421 (Biolegend), Granzyme B-ECD (Biolegend), and TNF-α-APC (Biolegend), as well as their isotype control antibodies (Biolegend), were performed. Flow cytometry was used for detection, and the results were analyzed using FlowJO software. The results are shown in Figure 7 (BC). Compared to the FC group, co-incubation with anti-ATRN nanobody for 48 h significantly enhanced the ability of NK cells to secrete perforin, and co-incubation with anti-ATRN nanobody for 96 h significantly enhanced the ability of CD8 cells to secrete perforin. + The ability of T cells to secrete interferon, tumor necrosis factor, and perforin.

[0158] Example 7: Anti-ATRN nanobody inhibits the growth of HepG2 subcutaneous tumors

[0159] To verify the therapeutic potential of the obtained nanobodies, 5 × 10⁶ nanobodies were suspended in PBS and matrix gel (2:1). 6 HepG2 cells were injected subcutaneously into age-appropriate NSG mice (6-8 weeks) to establish a tumor model. On day 12 post-injection, 5 × 10⁵ cells were infused into each mouse via the tail vein. 6 For PBMCs, when the tumor volume reached at least approximately 100 cubic millimeters (day 15 after tumor injection), randomly assigned mice were intraperitoneally injected with nanobodies. Each dose contained approximately 200 μg of nanobodies (10 mg / kg), with doses administered 2-3 days apart, and each mouse received 5 treatments. Control mice received PBS containing the same amount of the drug solution. Tumor growth was monitored every 2-3 days for 24 days after tumor cell injection, with volume measured as length × width. 2 The result, calculated using ×0.52, is shown in Figure 8. After the fifth treatment, tumor growth in the experimental group mice was significantly slower compared to the control group. This indicates that the anti-ATRN nanobody has good therapeutic potential against HepG2 tumors.

[0160] The preferred embodiments of this application have been described in detail above; however, this application is not limited thereto. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solution of this application, including combining various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in this application and are all within the protection scope of this application.

[0161] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0162] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. An antibody or its antigen-binding fragment, characterized in that, include: The heavy chain complementarity-determining region (HCDR) comprises an amino acid sequence selected from at least one of SEQ ID NO:1 to 3 or having at least 80% homology with it.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The HCDRs include: HCDR1, HCDR2, and HCDR3, wherein: The HCDR1 contains an amino acid sequence as shown in SEQ ID NO:1 or having at least 80% homology with SEQ ID NO:

1. The HCDR2 comprises an amino acid sequence as shown in SEQ ID NO:2 or having at least 80% homology with SEQ ID NO:2, and the HCDR3 comprises an amino acid sequence selected from the amino acid sequence shown in SEQ ID NO:3 or having at least 80% homology with SEQ ID NO:3; Preferably, the HCDR includes HCDR1, HCDR2 and HCDR3, respectively, with amino acid sequences as shown in SEQ ID NO:1 to 3.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, Further includes: The light chain complementarity determination region LCDR includes LCDR1, LCDR2 and LCDR3.

4. The antibody or its antigen-binding fragment according to claim 2 or 3, characterized in that, The antibody or its antigen-binding fragment further includes: a heavy chain framework region; Optionally, at least a portion of the heavy chain framework region is independently derived from at least one of alpaca-derived antibodies, shark-derived antibodies, mouse-derived antibodies, rabbit-derived antibodies, primate-derived antibodies, or mutants thereof, preferably alpaca-derived antibodies; Preferably, the heavy chain variable region of the antibody or its antigen-binding fragment comprises an amino acid sequence selected from or having at least 80% homology with SEQ ID NO:4; preferably, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is as shown in SEQ ID NO:4; Optionally, the antibody or its antigen-binding fragment further includes a light chain framework region; Optionally, at least a portion of the light chain framework region is independently derived from at least one of a mouse antibody, a rabbit antibody, a primate antibody, or a mutant thereof.

5. The antibody or its antigen-binding fragment according to claim 4, characterized in that, The antibody or its antigen-binding fragment further includes: a heavy chain constant region; Optionally, the antibody or its antigen-binding fragment further includes a light chain constant region; Optionally, at least a portion of the heavy chain constant region and the light chain constant region are independently derived from at least one of rabbit-derived antibodies, mouse-derived antibodies, sheep-derived antibodies, goat-derived antibodies, primate-derived antibodies, or mutants thereof.

6. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody includes at least one selected from polyclonal antibodies and monoclonal antibodies; or The antigen-binding fragment includes at least one selected from the F(ab')2 fragment, Fab' fragment, Fab fragment, Fv fragment, scFv fragment, scFv-Fv fusion protein, and the smallest recognition unit; Preferably, the antibody is a nanobody.

7. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or its antigen-binding fragment as described in any one of claims 1 to 6; Optionally, the nucleic acid molecule is DNA.

8. An expression carrier, characterized in that, Carrying the nucleic acid molecule as described in claim 7.

9. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, characterized in that, include: The expression vector according to claim 8 is introduced into cells; The cells are cultured under conditions suitable for protein expression and secretion in order to obtain the antibody or its antigen-binding fragment; Optionally, the cells are eukaryotic cells.

10. A recombinant cell, characterized in that, The recombinant cells carry the nucleic acid molecule of claim 7 or the expression vector of claim 8.

11. A recombinant protein, characterized in that, include: The antibody or antigen-binding fragment thereof as described in any one of claims 1 to 6; Optionally, it further includes at least one selected from bioactive proteins or fragments thereof, bioactive peptides or fragments thereof; Optionally, the bioactive protein or fragment thereof includes at least one selected from protein tags, protein toxins or fragments thereof, tumor necrosis factor or fragments thereof, interferon or fragments thereof, biological response regulators or fragments thereof, and Fc fragments.

12. A composition, characterized in that, include: The antibody or its antigen-binding fragment according to any one of claims 1 to 6, the nucleic acid molecule carrying the nucleic acid molecule according to claim 7, the expression vector according to claim 8, the recombinant cell according to claim 10, or the recombinant protein according to claim 11.

13. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, carrying the nucleic acid molecule according to claim 7, the expression vector according to claim 8, the recombinant cell according to claim 10, the recombinant protein according to claim 11, or the composition according to claim 12 in the preparation of a medicament for the treatment or prevention of ATRN-mediated diseases.

14. The use according to claim 13, characterized in that, The ATRN-mediated diseases include neurological disorders, autism spectrum disorder (ASD), Alzheimer's disease, schizophrenia, Parkinson's disease, bipolar disorder, epilepsy, diabetic nephropathy, glomerulonephritis, chronic kidney disease, polycystic kidney disease, nephrotic syndrome, renal cell carcinoma, obesity, type II diabetes, non-alcoholic fatty liver disease, metabolic syndrome, hypothyroidism, Cushing's syndrome, hyperlipidemia, coronary artery disease, heart failure, stroke, peripheral artery disease, atrial fibrillation, atherosclerosis, hypertension, coronary artery disease, heart failure, arrhythmia, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, psoriasis, melanoma, breast cancer, lung cancer, colorectal cancer, prostate cancer, ovarian cancer, renal cell carcinoma, liver cancer, lung cancer, stomach cancer, colorectal cancer, breast cancer, esophageal cancer, thyroid cancer, skin cancer, prostate cancer, kidney cancer, endometrial cancer, cervical cancer, and hematological malignancies.

15. A drug, characterized in that, include: The drug is used to treat or prevent ATRN-mediated diseases and comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, the expression vector according to claim 8, the recombinant cell according to claim 10, the recombinant protein according to claim 11, or the composition according to claim 12.

16. The medicament according to claim 15, characterized in that, The ATRN-mediated diseases include neurological disorders, autism spectrum disorder (ASD), Alzheimer's disease, schizophrenia, Parkinson's disease, bipolar disorder, epilepsy, diabetic nephropathy, glomerulonephritis, chronic kidney disease, polycystic kidney disease, nephrotic syndrome, renal cell carcinoma, obesity, type II diabetes, non-alcoholic fatty liver disease, metabolic syndrome, hypothyroidism, Cushing's syndrome, hyperlipidemia, coronary artery disease, heart failure, stroke, peripheral artery disease, atrial fibrillation, atherosclerosis, hypertension, coronary artery disease, heart failure, arrhythmia, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, psoriasis, melanoma, breast cancer, lung cancer, colorectal cancer, prostate cancer, ovarian cancer, renal cell carcinoma, liver cancer, lung cancer, stomach cancer, colorectal cancer, breast cancer, esophageal cancer, thyroid cancer, skin cancer, prostate cancer, kidney cancer, endometrial cancer, cervical cancer, and hematological malignancies. Optionally, the drug further includes pharmaceutically acceptable excipients; Optionally, the excipients include: One or more pharmaceutically acceptable excipients, diluents, stabilizers, or carriers; Optionally, the drug is an injectable form.

17. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 in the preparation of a kit for detecting ATRN.

18. A reagent kit, characterized in that, The kit contains: The antibody or antigen-binding fragment thereof as described in any one of claims 1 to 6.

19. The reagent kit according to claim 18, characterized in that, The kit is used to detect ATRN.

20. A method for preventing and / or treating ATRN-mediated diseases, characterized in that, include: Administering a pharmaceutically acceptable amount of an antibody or its antigen-binding fragment, expression vector, recombinant cell, recombinant protein, composition or drug to a subject.

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