CN118909108A

CN118909108A - Antibody targeting ATRN protein or antigen binding fragment thereof and application thereof

Info

Publication number: CN118909108A
Application number: CN202411024067.9A
Authority: CN
Inventors: 孙成; 高爽; 穆罕默德·伊姆兰·汗; 阿萨德·汗; 孙鹏; 阿希特沙姆·乌马尔
Original assignee: University of Science and Technology of China USTC
Current assignee: University of Science and Technology of China USTC
Priority date: 2024-07-26
Filing date: 2024-07-26
Publication date: 2024-11-08

Abstract

The application relates to an antibody targeting ATRN proteins or antigen binding fragments thereof and application thereof, belonging to the field of antibodies. Wherein the antibody or antigen binding fragment thereof comprises: a heavy chain complementarity determining region HCDR comprising an amino acid sequence selected from at least one of SEQ ID NOs 1 to 3 or having at least 80% homology therewith. The antibody or the antigen binding fragment thereof can be combined with human ATRN protein with high specificity and affinity, block the interaction of the human ATRN protein and a receptor, inhibit the signal transduction process and restore the cell activity, thereby regulating the obesity, the melanin deposition, the nerve cell function and the anti-tumor function of immune cells of organisms.

Description

Antibody targeting ATRN protein or antigen binding fragment thereof and application thereof

Technical Field

The application belongs to the field of antibodies, and particularly relates to an antibody of ATRN proteins or antigen-binding fragments thereof and application thereof.

Background

ATRN (Attractin) is a membrane-associated molecule that is rapidly upregulated on activated T cells, has CD26 protease activity, and is encoded by the ATRN gene on chromosome 20 in humans. ATRN are expressed in a variety of tissues, including skin, brain, liver, and adipose tissue, indicating that they have important functions in a variety of physiological processes.

ATRN perform immunomodulatory functions by capturing and proteolytically modifying the N-terminus of a variety of cytokines and chemokines. This regulatory activity allows immune cells to interact and form immunoregulatory clusters and helps down-regulate chemokines and cytokine activity after initiation of the response. In addition ATRN plays an important role in adipocyte differentiation, neural function, skin pigmentation, and extracellular matrix interactions, etc. High expression ATRN is related to metabolic disorders such as obesity, and ATRN is involved in neuronal development, synaptic function and neuroprotection in the nervous system, and becomes a hot spot for research of nervous system diseases.

ATRN also interact with a variety of proteins, including LRP1, SYNDECANS, HSPGS, NEOGENIN, and NCAM, involved in cell signaling, adhesion, and neural development. Thus, blocking ATRN interactions with other proteins may be potentially valuable for the treatment of related diseases. However, its specific mechanism of action and its role in the disease state is still not completely understood, while the lack of specific antibodies to ATRN is at the same time lacking.

For this reason, there is a need to develop a therapeutic anti-ATRN antibody with high affinity.

Disclosure of Invention

The present application aims to solve at least one of the above technical problems. To this end, it is an object of the present application to provide a high affinity, high specificity antibody that binds human ATRN protein.

Specifically, the application provides the following technical scheme:

In a first aspect of the application, the application provides an antibody or antigen binding fragment thereof. According to an embodiment of the application, it comprises: a heavy chain complementarity determining region HCDR comprising an amino acid sequence selected from at least one of SEQ ID NOs 1 to 3 or having at least 80% homology therewith. In some examples of the application, the aforementioned antibodies or antigen binding fragments thereof are capable of binding to ATRN extracellular proteins with high specificity and affinity, and blocking their interaction with a receptor, inhibiting the signal transduction process, restoring cellular activity, and thereby modulating obesity, melanin deposition, neuronal cell function, and anti-tumor function of immune cells of an organism.

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

In a second aspect of the application, the application provides a nucleic acid molecule. According to an embodiment of the application, the nucleic acid molecule encodes an antibody or antigen binding fragment thereof according to the first aspect of the application. In some examples of the application, the above-described antibodies or antigen-binding fragments thereof may be efficiently expressed in vitro based on the above-described nucleic acid molecules. Based on which the in vitro high-volume expression of the aforementioned antibodies or antigen-binding fragments thereof can be achieved.

In a third aspect of the application, the application provides an expression vector. According to an embodiment of the application, the expression vector carries a nucleic acid molecule according to the second aspect of the application. In some examples of the application, the expression vector is introduced into a suitable recipient cell, and the expression of the antibody or antigen-binding fragment thereof is effectively achieved under the mediation of a regulatory system, so that the in vitro mass production of the antibody or antigen-binding fragment thereof is achieved.

In a fourth aspect of the application, the application provides a method of preparing an antibody or antigen binding fragment thereof according to the first aspect. According to an embodiment of the application, the method comprises: introducing an expression vector according to the third aspect of the application into a cell; culturing the cells under conditions suitable for protein expression and secretion to obtain the antibody or antigen-binding fragment thereof. In some examples of the application, antibodies or antigen-binding fragments thereof capable of binding ATRN proteins with high specificity and affinity can be prepared efficiently in vitro based on the present methods described above.

In a fifth aspect of the application, the application provides a recombinant cell. According to an embodiment of the application, the recombinant cell carries an antibody or antigen-binding fragment thereof according to the first aspect of the application, a nucleic acid molecule according to the second aspect or an expression vector according to the third aspect. Wherein the aforementioned recombinant cells are obtained by transfecting or transforming the expression vector. In some examples of the application, the recombinant cells can efficiently express the above antibodies or antigen-binding fragments thereof under suitable conditions, and the resulting antibodies or antigen-binding fragments thereof are capable of binding ATRN proteins with high specificity and affinity.

In a sixth aspect of the application, the application provides a recombinant protein. According to an embodiment of the application, the recombinant protein comprises: the antibody or antigen-binding fragment thereof of the first aspect of the application. In some examples of the application, the foregoing engineered proteins are capable of effectively targeting ATRN proteins, blocking their interaction with receptors, inhibiting signal transduction processes, restoring cellular activity, and thereby modulating obesity, melanin deposition, neuronal cell function, and anti-tumor function of immune cells of an organism.

In a seventh aspect of the application, the application provides a composition. According to an embodiment of the application, the composition comprises: the antibody or antigen binding fragment thereof of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fifth aspect, or the recombinant protein of the sixth aspect are claimed. As described above, the antibody or antigen-binding fragment thereof can bind to ATRN proteins with high specificity and affinity, and thus, compositions, such as food compositions, pharmaceutical compositions, and the like, comprising the antibody or antigen-binding fragment thereof can also be effective in targeting ATRN proteins, blocking their interaction with receptors, inhibiting signal transduction processes, restoring cellular activity, and thus modulating obesity, melanin deposition, neuronal cell function, and anti-tumor function of immune cells of an organism.

In an eighth aspect of the application, the application provides the use of an antibody or antigen binding fragment thereof according to the first aspect, a nucleic acid molecule according to the second aspect, an expression vector according to the third aspect, a recombinant cell according to the fifth aspect, a recombinant protein according to the sixth aspect or a composition according to the seventh aspect for the manufacture of a medicament for the treatment or prevention of ATRN-mediated related diseases. In some examples of the application, a drug prepared based on an antibody or antigen-binding fragment thereof, a recombinant protein, a nucleic acid molecule, an expression vector, a recombinant cell, or a pharmaceutical composition, is capable of binding to ATRN protein with high specificity and affinity, blocking its interaction with a receptor, inhibiting a signal transduction process, restoring cellular activity, and thereby modulating obesity, melanin deposition, neuronal cell function, and anti-tumor function of immune cells of an organism.

In a ninth aspect of the application, the application provides a medicament. According to an embodiment of the application, the medicament comprises: an antibody or antigen binding fragment thereof according to the first aspect, a nucleic acid molecule according to the second aspect, an expression vector according to the third aspect, a recombinant cell according to the fifth aspect, a recombinant protein according to the sixth aspect or a composition according to the seventh aspect, for use in the treatment or prevention of ATRN-mediated related diseases. In some examples of the application, a drug prepared based on an antibody or antigen binding fragment thereof, a recombinant protein, a nucleic acid molecule, an expression vector, a recombinant cell, or a pharmaceutical composition is capable of effectively targeting ATRN a protein, blocking its interaction with a receptor, inhibiting a signal transduction process, restoring cellular activity, and thereby modulating obesity, melanin deposition, neuronal cell function, and anti-tumor function of immune cells of an organism.

In a tenth aspect of the application, the application provides the use of an antibody or antigen binding fragment thereof according to the first aspect for the preparation of a kit for detection ATRN. 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 may be used for portable detection of ATRN proteins.

In an eleventh aspect of the application, the application provides a kit. According to an embodiment of the application, the kit comprises an antibody or antigen binding fragment thereof according to the first aspect. As previously described, the antibody or antigen binding fragment thereof is capable of binding to ATRN proteins with high specificity and affinity, and therefore, kits comprising the antibody or antigen binding fragment thereof can be used for efficient portable assays ATRN. In some examples of the application, the kit may be used in scientific research, such as qualitative or quantitative detection of ATRN protein in a biological sample, and may also be used to determine the status of an individual, such as whether the level of ATRN is above or below normal after the level of ATRN in the individual has been obtained.

Additional aspects and advantages of the 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 the application.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graphical representation of the results of chromatography of purified ATRN extracellular portion protein (antigen) provided in an example of the present application;

FIG. 2 is a schematic diagram showing the results of SDS-PAGE characterization ATRN of the protein provided in the examples of the present application;

FIG. 3 is a schematic diagram of the results of antibody chromatography detection and purification provided in the examples of the present application;

FIG. 4 is a schematic diagram showing the chromatographic results of the enzyme-cleaved nanobody provided by the embodiment of the application for removing the FC segment and the TEV protease;

FIG. 5 is an electrophoretogram of a protein SDS-PAGE gel provided in an embodiment of the present application; wherein Nb (UD) represents a nanobody containing an FC fragment; nb (Dig) represents a nano antibody separated by a nickel column and a Protein A column after enzyme digestion; FC refers to the FC fragment separated after cleavage in the nanobody; TEV represents tobacco etching virus protease separated after enzyme cutting in the nano antibody;

FIG. 6 is a schematic diagram of the results of verification of the binding capacity of the antigen ATRN antigen and the nanobody of the Elisa assay provided by the example of the present application;

FIG. 7 is a schematic diagram of flow cytometry detection of the ability of nanobodies to stimulate secretion of cytokines by human Peripheral Blood Mononuclear Cells (PBMC) provided by an embodiment of the application; wherein, ISO in fig. 7 (a) represents a sample of isotype control flow antibody detected using ATRN flow antibody after 96h of PHA stimulation, ctrl represents a sample detected using ATRN flow antibody without PHA stimulation, PHA represents a sample detected using ATRN flow antibody after 96h of PHA stimulation, the vertical axis of the flow chart is cell number, and the horizontal axis is AF647 fluorescence intensity; FIG. 7 (B) is a graph showing a gate representation and a statistical graph of the ability of flow-through NK cells to secrete cytokines, FC is a control group of FC protein stimulated PBMC, anti-ATRN is an experimental group of nanobody stimulated PBMC, the vertical axis of the flow-through graph is the fluorescence intensity of BV421 fluorescence channel, and the horizontal axis is the fluorescence intensity of BV605 fluorescence channel; FIG. 7 (C) is a graph showing a gate representation and a statistical graph of the ability of flow-type CD8+ T cells to secrete cytokines, FC is a control group of FC protein stimulated PBMC, anti-ATRN is an experimental group of nanobody stimulated PBMC, the vertical axis of the flow-type graph is FITC, BV421 and the fluorescence intensity of the APC fluorescence channel, respectively, and the horizontal axis is the fluorescence intensity of the PC7 fluorescence channel;

Fig. 8 is a schematic diagram of inhibition of growth of HepG2 subcutaneous tumor by anti-ATRN nano-antibody provided in the example of the present application.

Detailed Description

Reference will now be made in detail to embodiments of the present application, examples of which are illustrated in the accompanying drawings. The embodiments described below by referring to the drawings are illustrative and intended to explain the present application and should not be construed as limiting the application.

In the present disclosure, the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the present application, the meaning of "plurality" means at least two, for example, two, three, etc., unless specifically defined otherwise.

In the present application, the term "antibody" refers to an immunoglobulin formed by two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Immunoglobulins can be divided into five classes, igM, igD, igG, igA, igE. The same class of immunoglobulins can also be of different subclasses, e.g., igG1, igG2, igG3, igG4, depending on the amino acid composition. Immunoglobulin light chains are classified as either kappa chains or lambda chains depending on the constant region.

The sequences of the heavy and light chains of antibodies, near the N-terminus, vary widely, being the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region. The variable region includes 3 hypervariable regions (HVRs) and 4 Framework Regions (FR) that are relatively conserved in sequence. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each Light Chain Variable Region (LCVR) and Heavy Chain Variable Region (HCVR) consists of 3 CDR regions and 4 FR regions, arranged in the order from amino-to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR3.

In the present application, the term "monoclonal antibody (abbreviated as" monoclonal antibody ") refers to an antibody recognizing only one specific epitope. Wherein, the common monoclonal antibody comprises two light chains with lighter molecular weight and two heavy chains with heavier molecular weight, the heavy chains (H chains) and the light chains (L chains) are connected by disulfide bonds to form a tetrapeptide chain molecule, the amino terminal (N-terminal) amino acid sequence of the heavy chains or the light chains is greatly changed and is called a variable region (V region), and the carboxyl terminal (C terminal) of the heavy chains or the light chains is relatively stable and has little change and is called a constant region (C region); the V chains of the L chain and H chain are referred to as VL and VH, respectively. The monoclonal antibodies can also be small molecule antibodies, which mainly comprise: fab antibodies, fv antibodies, single chain antibodies, single domain antibodies, and minimal recognition units.

In the present application, the term "polyclonal antibody (abbreviated as polyclonal antibody)" refers to an antibody capable of recognizing various epitopes, for example, an antibody capable of recognizing two epitopes (abbreviated as diabody), an antibody of three epitopes or an antibody of four epitopes, which is broadly understood, and the specific structure is not limited, and may recognize various epitopes.

In the present application, the term "nanobody" comprises only the heavy chain variable region (VH) and the conventional CH2 and CH3 regions, which specifically bind to an antigen through the heavy chain variable region.

In the present 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; comprising one or more CDR regions selected from SEQ ID NOS 1-3 of the antibodies of the present application.

In the present application, the amino acid sequences of the CDRs listed above are all shown according to IMGT definition rules. However, it is well known to those skilled in the art that the CDRs of an antibody can be defined by a variety of methods, such as Kabat's rule, chothia's rule, etc. It will be appreciated by those skilled in the art that unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) are to be understood as encompassing complementarity determining regions defined in any of the above known schemes as described by the present application. Although the claimed scope of the present application is based on the sequences shown by IMGT definition rules, amino acid sequences corresponding to definition rules according to other CDRs should also be included in the scope of the present application.

In the present application, the term "amino acid" is represented by a single letter or three letter code, having the following meaning: a: ala (alanine); r: arg (arginine); n: asn (asparagine); d: asp (aspartic acid); c: cys (cysteine); q: gln (glutamine); e: glu (glutamic acid); g: gly (glycine); h: his (histidine); i: ile (isoleucine); l: leu (leucine); k: lys (lysine); m: met (methionine); f: phe (phenylalanine); p: pro (proline); s: ser (serine); t: thr (threonine); w: trp (tryptophan); y: tyr (tyrosine); v: val (valine).

For nucleotides, the terms "homology", "identity" or "similarity" are used to describe or compare the degree of nucleotide similarity of two or more nucleotide sequences. The percentage of "sequence homology" between a first sequence and a second sequence can be calculated by dividing [ the number of nucleotides in the first sequence that are identical to the nucleotides at the corresponding positions ]. The nucleotides in the second sequence are subtracted by [ the total number of nucleotides in the first sequence ], then multiplied by [100% ], wherein the deletion, insertion, substitution or addition of each nucleotide in the second nucleotide sequence-relative to the first nucleotide sequence-is considered as a difference in single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences may be calculated using standard settings using known computer algorithms for sequence alignment, such as NCBI Blast v 2.0. Some other techniques for determining the degree of sequence identity, computer algorithms and settings are described, for example, 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.

For polypeptides, the terms "(substantial) homology", "identity" or "similarity" are used to describe or compare the degree of amino acid similarity of two or more polypeptides or specified sequences thereof when optimally aligned and compared (with appropriate insertions or deletions of nucleotides). The% homology between two sequences varies with the number of identical positions shared by the sequences when optimally aligned (i.e.,% homology =number of identical positions/total number of positions x 100), where the optimal alignment is determined by taking into account the number of gaps that need to be introduced to achieve optimal alignment of the two sequences and the length of each gap. Sequence comparison and percent identity determination between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

In the present application, one skilled in the art may replace, 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 the sequences of the present application to obtain variants of the sequences of the antibodies or functional fragments thereof without substantially affecting the activity of the antibodies (retaining at least 95% of the activity). They are all considered to be included within the scope of the present application. Such as substitution of amino acids with similar properties in the variable region. The variant sequences of the application may have at least 80% identity (or homology) to a reference sequence, meaning at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity to each reference sequence. Sequence identity as described herein can be measured using sequence analysis software. Such as computer programs BLAST, in particular BLASTP or TBLASTN, using default parameters. The amino acid sequences mentioned in the present application are all shown in N-terminal to C-terminal fashion.

In this context, the term "vector" generally refers to a nucleic acid molecule capable of insertion into a suitable host for self-replication, which transfers the inserted nucleic acid molecule into and/or between host cells. The vector may include a vector mainly used for inserting DNA or RNA into a cell, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of transcription and/or translation of DNA or RNA. The carrier also includes a carrier having a plurality of functions as described above. The vector may be a polynucleotide capable of transcription and translation into a polypeptide when introduced into a suitable host cell. Typically, the vector will produce the desired expression product by culturing a suitable host cell comprising the vector.

In the present application, the term "composition" generally refers to a unit dosage form and may be prepared by any of the methods well known in the pharmaceutical arts. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. Generally, the compositions are prepared by uniformly and intimately bringing into association the active compound with liquid carriers, finely divided solid carriers or both.

In the present application, the term "pharmaceutically acceptable" ingredients are substances which are suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation and allergic response), i.e. a reasonable benefit/risk ratio.

In the present application, the term "pharmaceutically acceptable excipients" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for the particular target dosage form. In addition to the extent to which any conventional adjuvant is incompatible with the compounds of the present application, such as any adverse biological effects produced or interactions with any other component of the pharmaceutically acceptable composition in a deleterious manner, their use is also contemplated by the present application.

In the present application, the term "administering" refers to introducing a predetermined amount of a substance into a patient by some suitable means. The antibody or antigen binding fragment, recombinant protein, multispecific antibody, conjugate, or pharmaceutical composition of the application may be administered by any common route so long as it reaches the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the application is not limited to these illustrated modes of administration. Preferably, the compositions of the present application are administered by intravenous or subcutaneous injection.

In the present application, the term "treatment" is intended to mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or symptoms thereof, and/or may be therapeutic in terms of partially or completely curing the disease and/or adverse effects caused by the disease. As used herein, "treating" encompasses diseases in mammals, particularly humans, including: (a) Preventing the occurrence of a disease or disorder in an individual susceptible to the disease but not yet diagnosed with the disease; (b) inhibiting disease, e.g., arresting disease progression; or (c) alleviating a disease, e.g., alleviating symptoms associated with a disease. As used herein, "treating" or "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, reduce or inhibit a disease in the individual, including, but not limited to, administration of a drug comprising a compound described herein to an individual in need thereof.

In the present application, the term "effective amount" or "effective dose" refers to an amount that is functionally or actively administered to and acceptable to humans and/or animals.

In the present application, the amino acid sequences are shown in Table 1.

The application provides an antibody or antigen binding fragment thereof targeting ATRN proteins, a preparation method thereof, a nucleic acid molecule, an expression vector, a recombinant cell, a recombinant protein, a composition, pharmaceutical use, a medicament, use in preparation of a kit, a disease treatment method and disease treatment use, and the detailed description of the antibody or antigen binding fragment is provided.

Antibodies or antigen binding fragments thereof

In one aspect of the application, the application provides an antibody or antigen binding fragment thereof comprising: a heavy chain complementarity determining region HCDR comprising an amino acid sequence selected from at least one of SEQ ID NOs 1 to 3 or having at least 80% homology therewith. In some examples of the application, antibodies or antigen binding fragments thereof having the aforementioned HCDR amino acid sequences are capable of binding ATRN proteins with high affinity and specificity.

In some examples of the application, the HCDR includes: HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises 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 as shown in SEQ ID NO. 3 or having at least 80% homology with SEQ ID NO. 3. Antibodies or antigen binding fragments thereof based on the aforementioned HCDR sequences have higher binding affinity and specificity to ATRN proteins.

In some preferred examples of the application, the aforementioned HCDR comprises: HCDR1, HCDR2 and HCDR3 with the amino acid sequences shown in SEQ ID NO 1-3. Experiments prove that the antibody or antigen binding fragment thereof with the amino acid sequence shown in SEQ ID NO. 1-3 has higher binding affinity and specificity with ATRN protein.

In some examples of the application, the foregoing antibodies or antigen binding fragments thereof further comprise: light chain complementarity determining region LCDR, said LCDR comprising: LCDR1, LCDR2, and LCDR3.

It is noted that one or more amino acid residues in the HCDR of the foregoing targeting ATRN protein may be replaced with other amino acid residues from the same side chain family, and that it may be tested for the retention function of the altered antibodies using the functional assay methods described herein.

In some examples of the application, the foregoing antibodies or antigen binding fragments thereof further comprise: heavy chain framework regions.

In some examples of the application, at least a portion of the aforementioned heavy chain framework regions are independently derived from at least one of alpaca-derived antibodies, shark-derived antibodies, murine-derived antibodies, rabbit-derived antibodies, primates-derived antibodies, or mutants thereof. In a preferred embodiment of the present application, the heavy chain framework region is selected from alpaca-derived antibodies.

In some preferred examples of the application, the heavy chain variable region of the foregoing antibodies or antigen binding fragments thereof comprises an amino acid sequence selected from the group consisting of those shown in SEQ ID NO. 4 or having at least 80% homology with SEQ ID NO. 4. In some more preferred examples of the application, the amino acid sequence of the heavy chain variable region of the aforementioned antibody or antigen binding fragment thereof is shown in SEQ ID NO. 4.

In some examples of the application, the foregoing antibodies or antigen binding fragments thereof further comprise: light chain framework regions.

In some examples of the application, at least a portion of the light chain framework region is independently derived from at least one of a murine antibody, a rabbit antibody, a primate-source antibody, or a mutant thereof.

The antibody or antigen binding fragment thereof based on the heavy chain variable region sequence has higher binding affinity and specificity to ATRN protein.

In some examples of the application, the foregoing antibodies or antigen binding fragments thereof further comprise: heavy chain constant region.

In some examples of the application, the foregoing antibodies or antigen binding fragments thereof further comprise: a light chain constant region.

In some examples of the application, at least a portion of the foregoing heavy chain constant region and the foregoing light chain constant region are independently derived from at least one of a rabbit-derived antibody, a murine-derived antibody, a sheep-derived antibody, a goat-derived antibody, a primate-derived antibody, or a mutant thereof. In some preferred examples of the application, at least a portion of the foregoing heavy chain constant region and the foregoing light chain constant region are derived from a murine antibody.

In some examples of the application, the aforementioned antibodies include at least one selected from the group consisting of polyclonal antibodies and monoclonal antibodies; or the aforementioned antigen binding fragment comprises at least one selected from the group consisting of a F (ab ') 2 fragment, a Fab' fragment, a Fab fragment, an Fv fragment, an scFv-Fv fusion protein and a minimal recognition unit.

In some preferred examples of the application, the aforementioned antibodies are nanobodies. The nano antibody is obtained by the inventor through the mutation of an AI model (HIE BL,SHANKER V R,XU D,et al.Efficient evolution of human antibodies from general protein languagemodels[J].Nature Biotechnology,2024,42(2):275-283.) to ATRN nano sequence for the first time, and shows excellent tumor inhibition effect in a HepG2 humanized tumor model.

It will be appreciated by those skilled in the art that the foregoing antibodies or antigen binding fragments are suitable for use in a variety of animal tumor models, including but not limited to mouse tumor models.

In some examples of the application, the foregoing nanobodies have an amino acid sequence as shown in SEQ ID NO. 4 or have at least 80% homology with SEQ ID NO. 4. In some examples of the application, it was found through sufficient experimental verification that nanobodies having the amino acid sequence shown in SEQ ID NO. 4 are capable of binding ATRN proteins with high affinity and high specificity.

According to the embodiment of the application, the antibody or the antigen binding fragment can be combined with the extracellular segment of human ATRN protein, so that the inhibiting signal transmission of ATRN to NK cells and CD8+T is blocked, the capacity of NK cells in human peripheral blood to secrete perforin and CD8+T cells to secrete interferon, tumor necrosis factor and perforin is promoted, and the killing function of NK cells and CD8+T cells is further enhanced.

Nucleic acid molecules

In another aspect of the application, the application provides a nucleic acid molecule encoding an antibody or antigen binding fragment thereof as described above. In some examples of the application, antibodies or antigen binding fragments thereof may be efficiently expressed in vitro based on the aforementioned nucleic acid molecules.

In some examples of the application, the nucleic acid molecule is DNA.

It is to be noted that, for the nucleic acid molecules mentioned in the description and claims of the present application, a person skilled in the art will understand that either one or both of the complementary double strands are actually included. For convenience, in the present description and claims, although only one strand is shown in most cases, the other strand complementary thereto is actually disclosed. In addition, the nucleic acid sequences of the present application include DNA forms or RNA forms, one of which is disclosed, meaning the other is also disclosed.

Expression vector

In another aspect of the application, the application provides an expression vector carrying a nucleic acid molecule as described above. In some examples of the application, efficient in vitro preparation of antibodies or antigen binding fragments can be achieved based on the aforementioned expression vectors.

In the case of attaching the above-mentioned nucleic acid molecule to a vector, the nucleic acid molecule may be directly or indirectly attached to a control element on the vector, so long as the control element is capable of controlling translation, expression, etc. of the nucleic acid molecule. Of course, these control elements may be directly from the carrier itself or may be exogenous, i.e., not from the carrier itself. Of course, the nucleic acid molecule may be operably linked to a control element.

According to embodiments of the invention, the vector may be referred to as a cloning vector or as an expression vector, and may be obtained by operably linking the nucleic acid to a commercially available vector, such as a plasmid or viral vector. The vector of the present invention is not particularly limited, and commonly used plasmids such as pSeTag, PEE14, pMH3, etc. can be used.

As used herein, the term "operably linked" refers to the linkage of a foreign gene to a vector such that control elements within the vector, e.g., transcription-controlled amino acid sequences such as those that are translation-controlled, and the like, are capable of performing their intended functions of regulating transcription and translation of the foreign gene. The usual vectors may be, for example, viral vectors, plasmids, phages and the like. After the expression vector according to some embodiments of the present invention is introduced into a suitable recipient cell, the expression of the nucleic acid molecule described above can be effectively achieved under the mediation of a regulatory system, thereby achieving in vitro mass-production of the protein encoded by the nucleic acid molecule.

In some examples of the application, the vector is a eukaryotic vector or a prokaryotic vector.

In some examples of the application, the vector comprises at least one selected from the group consisting of a plasmid vector, an adenovirus vector, a lentiviral vector, and an adeno-associated virus vector.

Method for preparing antibody or antigen binding fragment thereof

In another aspect of the application, the application provides a method of preparing the aforementioned antibody or antigen binding fragment thereof, comprising: introducing the aforementioned expression vector into a cell; the cells are cultured under conditions suitable for protein expression and secretion to obtain the aforementioned antibodies or antigen-binding fragments thereof.

In some examples of the application, the cell is a eukaryotic cell.

In some specific examples of the application, the eukaryotic cell is a mammalian cell. The antibody or antigen binding fragment thereof is expressed more efficiently when the cell is a mammalian cell.

The eukaryotic cell does not include an animal germ cell, a fertilized egg, or an embryonic stem cell.

Recombinant cells

In another aspect of the application, the application provides a recombinant cell carrying the aforementioned antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule or the aforementioned expression vector. In some examples of the application, the recombinant cells are obtained by transfecting or transforming the expression vector, and the recombinant cells can efficiently express the antibodies or antigen-binding fragments under suitable conditions.

It should be noted that the recombinant cells of the present application are not particularly limited, and may be prokaryotic cells, eukaryotic cells, or phage. The prokaryotic cell can be escherichia coli, bacillus subtilis, streptomycete or proteus mirabilis and the like. The eukaryotic cells can be fungi such as pichia pastoris, saccharomyces cerevisiae, schizosaccharomyces, trichoderma and the like, insect cells such as armyworm and the like, plant cells such as tobacco and the like, and mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells and the like. In some embodiments, the recombinant cells of the 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.

The foregoing "transformation" or "transfection" refers to the introduction of a nucleic acid (e.g., vector) into a cell by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of the application and can be used for expression and/or secretion of a target protein. Suitable host cells can be transformed or transfected with the DNA sequences of the application and can be used for expression and/or secretion of a target protein. Examples of suitable host cells that can be used in the present application include immortalized hybridoma cells, NS/0 myeloma cells, 293 cells, chinese Hamster Ovary (CHO) cells, heLa cells, cap cells (human amniotic fluid derived cells) and CoS cells.

The term "suitable conditions" as used herein refers to conditions suitable for expression of the antibody of the present application. Those skilled in the art will readily appreciate that conditions suitable for antibody expression include, but are not limited to, suitable transformation or transfection means, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, suitable cell culture environment, suitable cell culture time. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the conditions for optimal antibody expression according to the specific environment of the laboratory.

Recombinant proteins

In another aspect of the application, the application provides a recombinant protein comprising: the aforementioned antibody or antigen-binding fragment thereof. In some examples of the application, the recombinant protein is capable of binding ATRN proteins with high affinity and specificity.

In some examples of the present application, the aforementioned recombinant protein may further include at least one selected from a biologically active protein or a fragment thereof, a biologically active polypeptide or a fragment thereof.

In some examples of the application, the biologically active protein or fragment thereof comprises at least one selected from the group consisting of a protein tag, a protein toxin or fragment thereof, a tumor necrosis factor or fragment thereof, an interferon or fragment thereof, a biological response modifier or fragment thereof, and an Fc fragment.

Wherein the term "protein tag" generally refers to a polypeptide or protein that is expressed in fusion with a protein of interest (antibody or antigen binding fragment), which can be used for expression, detection, missing or purification of the protein of interest, etc. Including but 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 sulfhydryl transferase tags), SUMO tags, C-Myc tags, and the like.

By "toxin" is generally meant a substance that is toxic to the host, including protein toxins and non-protein toxins. Among these, protein toxins include, but are not limited to, abrin, ricin a, pseudomonas exotoxin, diphtheria toxin, and the like. In the present application, the protein toxin is preferably a protein toxin having enzymatic activity.

By "tumor necrosis factor" is generally meant a substance that causes hemorrhagic necrosis of a variety of tumors, including but not limited to TNF- α and TNF- β.

The term "interferon" generally refers to a glycoprotein that has direct killing or inhibition of a virus. Including but not limited to IFN- α, INF- β and IFN- γ.

By "biological response modifier" is generally meant a class of proteinaceous substances that directly or indirectly enhance the antitumor effect of the body through the immune system. Including but not limited to lymphokines, IL-2, IL-6, IL-10, and GM-CSF, etc.

By "Fc fragment" is generally meant an Fc region from IgG (e.g., igG1, igG2, igG3, or IgG4 subtype), igA1, igA2, igD, igE, or IgM, including CH2, CH3 regions, and optionally hinge regions. Preferably, igG, igA1, igA2, igD, igE or IgM is derived from alpaca, murine or primate sources.

Composition and method for producing the same

In another aspect of the application, the application provides a composition comprising: the antibody or antigen binding fragment thereof, the nucleic acid molecule, the expression vector, the recombinant cell or the recombinant protein. In some examples of the application, the foregoing compositions can be used to prepare related products, such as pharmaceuticals, kits, etc., that target ATRN proteins.

It is noted that the compositions include combinations that are separated in time and/or space, so long as they are capable of co-acting to achieve the objects of the present application. For example, the ingredients contained in the composition may be administered to the subject in whole or separately. When the components contained in the composition are separately administered to a subject, the individual components may be administered to the subject simultaneously or sequentially.

Pharmaceutical use

In a further aspect of the application, the application provides the use of the aforementioned antibody or antigen binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, the aforementioned recombinant protein or the aforementioned composition for the preparation of a medicament for the treatment or prevention of ATRN-mediated related diseases.

In some examples of the application, the ATRN-mediated related disease includes neurological diseases, autism Spectrum Disorders (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, nonalcoholic fatty liver, metabolic syndrome, hypothyroidism, cushing's syndrome, hyperlipidemia, coronary artery disease, heart failure, stroke, peripheral arterial disease, atrial fibrillation, atherosclerosis, hypertension, coronary artery disease, heart failure, cardiac arrhythmias, 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, gastric cancer, colorectal cancer, breast cancer, esophageal cancer, thyroid cancer, skin cancer, prostate cancer, renal cancer, endometrial cancer, cervical cancer, and hematological tumors.

Medicament

In yet another aspect of the application, the application provides a medicament comprising: the aforementioned antibodies or antigen binding fragments thereof, the aforementioned nucleic acid molecules, the aforementioned expression vectors, the aforementioned recombinant cells, the aforementioned recombinant proteins or the aforementioned compositions, which are useful for the treatment or prevention of ATRN-mediated related diseases.

The ATRN-mediated related diseases include neurological diseases, 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, nonalcoholic fatty liver, metabolic syndrome, hypothyroidism, cushing's syndrome, hyperlipidemia, coronary artery disease, heart failure, stroke, peripheral arterial disease, atrial fibrillation, atherosclerosis, hypertension, coronary artery disease, heart failure, cardiac arrhythmias, 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, gastric cancer, colorectal cancer, breast cancer, esophageal cancer, thyroid cancer, skin cancer, prostate cancer, renal cancer, endometrial cancer, cervical cancer, and hematological tumors.

In some examples of the application, further comprising a pharmaceutically acceptable excipient.

In some examples of the application, the adjunct comprises: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers.

In some examples of the application, the pharmaceutical composition is an injection.

It is noted that the pharmaceutical composition includes combinations that are separated in time and/or space, as long as they can co-act to achieve the object of the present invention. For example, the ingredients 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 separately administered to a subject, the individual components may be administered to the subject simultaneously or sequentially.

The medicine of the application contains safe and effective amount of the active ingredient (antibody) of the application and pharmaceutically acceptable auxiliary materials. Such excipients include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation is matched with the administration mode, and the dosage forms of the medicine are injection, oral preparation (tablet, capsule and oral liquid), transdermal agent and sustained release agent. For example, by using physiological saline or an aqueous solution containing glucose and other auxiliary agents by conventional methods. The medicament is preferably manufactured under aseptic conditions.

The effective amount of the active ingredient described herein may vary depending upon the mode of administration, the severity of the condition being treated, and the like. The selection of the preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). The foregoing factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life etc.; the severity of the disease to be treated in the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. For example, separate doses may be administered several times per day, or the dose may be proportionally reduced, as dictated by the urgent need for the treatment of the condition.

Pharmaceutically acceptable excipients described herein 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 compatible with the mode of administration and will be well known to those of ordinary skill in the art.

Use in the preparation of a kit

In a further aspect of the application, the application provides the use of the aforementioned antibody or antigen binding fragment thereof in the preparation of a kit for detection ATRN.

As previously described, the antibodies or antigen-binding fragments thereof of embodiments of the application are capable of specifically binding to ATRN, and thus, the antibodies or antigen-binding fragments thereof can be used to detect ATRN protein. Further, it can be used for preparing ATRN related kits and for scientific research, such as qualitative or quantitative detection of ATRN protein molecules in biological samples. More specifically, it can be used for immunoblotting, immunoprecipitation, etc. involving a kit for detection using the specific binding properties of ATRN and an antibody, etc. These kits may comprise any one or more of the following: antagonists, antibodies of the application, or pharmaceutical reference materials; a protein purification column; immunoglobulin affinity purification buffers; assay of cells diluent. The antibodies of the application may be used in different types of diagnostic tests, for example, to detect the presence of a wide variety of diseases or drugs, toxins or other proteins, etc., in vitro or in vivo. For example, the kit can be used for testing ATRN for related diseases (such as liver cancer, etc.) mediated by serum or blood of a subject.

Kit for detecting a substance in a sample

In a further aspect of the application, the application provides a kit comprising an antibody or antigen binding fragment thereof according to the first aspect. The antibody or antigen binding fragment thereof in the kit provided by the application can be effectively combined with ATRN protein, in addition, under proper conditions, the nucleic acid molecule, the expression vector or the recombinant cell can express the antibody or antigen binding fragment thereof, and further, the kit containing the antibody or antigen binding fragment thereof can be combined with ATRN with high specificity, so that the kit can be used for effectively detecting ATRN. The kit can be used for scientific research, such as qualitative or quantitative detection of ATRN in a biological sample, and also can be used for judging the state of an individual, such as judging whether the ATRN level of the individual is higher or lower than the normal level after the ATRN level of the individual is obtained, wherein the biological sample can be cells, tissues and the like.

In some examples of the application, the kit is used for detection ATRN.

Method for treating disease

In yet another aspect of the application, the application provides a method of preventing and/or treating ATRN-mediated diseases. According to an embodiment of the application, the method comprises: administering a pharmaceutically acceptable amount of an antibody or antigen-binding fragment thereof, an expression vector, a recombinant cell, a recombinant protein, a composition, or a medicament to a subject.

It is noted that the terms "subject," "individual," and "patient" are used interchangeably herein to refer to a mammal being evaluated for treatment and/or being treated. In one embodiment, 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 infection, and the like. The subject may be a human, but also includes other mammals, particularly mammals that may be used as laboratory models of human diseases, such as mice, rats, and the like.

The effective amount of the antibodies or antigen binding fragments thereof, nucleic acid molecules, expression vectors, recombinant proteins, compositions or medicaments of the present application may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life etc.; the severity of the disease to be treated in the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. For example, separate doses may be administered several times per day, or the dose may be proportionally reduced, as dictated by the urgent need for the treatment of the condition.

In some examples of the application, the ATRN-mediated related disease includes neurological diseases, autism Spectrum Disorders (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, nonalcoholic fatty liver, metabolic syndrome, hypothyroidism, cushing's syndrome, hyperlipidemia, coronary artery disease, heart failure, stroke, peripheral arterial disease, atrial fibrillation, atherosclerosis, hypertension, coronary artery disease, heart failure, cardiac arrhythmias, 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, gastric cancer, colorectal cancer, breast cancer, esophageal cancer, thyroid cancer, skin cancer, prostate cancer, renal cancer, endometrial cancer, cervical cancer, and hematological tumors.

Use for the treatment of diseases

In a further aspect of the application, the application provides the use of the aforementioned antibody or antigen binding fragment thereof, nucleic acid molecule, expression vector, recombinant protein, composition or medicament for the treatment or prevention of ATRN-mediated related diseases. In some examples of the application, administering an effective dose of an antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, recombinant protein, composition or drug to a subject is effective to treat ATRN-mediated disease.

TABLE 1

The scheme of the present application will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present application and should not be construed as limiting the scope of the application. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1: ATRN expression, purification and elution of extracellular portion proteins

In this example, the expression, purification and elution of ATRN extracellular portion proteins were performed as follows:

The primer is designed to amplify ATRN extracellular gene fragment by taking ATRN full-length gene sequence as a template, and the vector is selected from pTT5. pTT5-ATRN plasmid was constructed using Gibson assembly.

The extracted plasmid was transfected into HEK293F cells using the transfection reagent Polyethylenimine (PEI). The reagents and amounts are shown in Table 2, and mammalian expression systems were transfected with pTT5-ATRN plasmid.

TABLE 2 reagents and amounts of transfection System

DMEM was preheated in a 37 ℃ water bath prior to transfection. Two 50ml tubes were used to hold 15ml DMEM, respectively, and then the plasmid was added to one of the tubes and PEI was added to the other tube. The two tubes were left to stand at room temperature for 10 minutes. PEI diluted with DMEM was then transferred to plasmid tubes and incubated for 15 minutes at room temperature. HEK293F cells were grown at 200 ten thousand/ml counts and a total volume of 350 ml. After incubation of plasmid-PEI, it was transferred drop-wise into 400ml cells and then allowed to stand in an incubator at 37℃for 20 minutes without shaking. Subsequently, the cells were cultured in a shaker incubator at 120rpm, 5% CO ₂ for 72 to 96 hours. The cells were centrifuged at 4347rcf for 30min and the supernatants were then passed through 0.45 μm and 0.22 μm filters, respectively. The filtered sample was loaded onto a peristaltic pump and the protein was purified using affinity chromatography (protein a column). The pTT5 vector carries an Fc (IgG) tag and can specifically bind to the resin of the Protein A column, thereby enriching the target Protein in the supernatant onto the column.

After loading, the protein column was eluted on AKTA PRIME plus purification system, 0.2M Tris-HCl (ph=7) was added to the protein collection tube and the procedure was run to pass the protein from 0% b elution buffer to 100% b protocol. The liquid a used was PBS and the liquid B was elution buffer (0.1M acetic acid solution) for eluting the bound protein. The peak diagram of the protein obtained is shown in FIG. 1, which shows that the high-purity ATRN extracellular portion protein is obtained.

Example 2: ATRN extracellular domain protein detection

The ATRN extracellular portion protein sample obtained in example 1 was added with a stain and denatured at 95℃for 3 minutes to denature the protein. A12.5% SDS-gel electrophoresis gel was prepared by using a support (Omni-Easy one-step SDS-gel pretreatment kit) according to the standard instructions, transferred to an electrophoresis tank filled with running buffer (Tris-glycine buffer), filled with a sample and a standard marker in the corresponding well, powered on, set at 90 volts for 20 minutes, and then raised to 130 volts until the dye reached the bottom. The gel was stained with a cresol blue solution to visualize the protein bands, and the gel was placed in the staining solution for appropriate staining. The gel was rinsed with water and incubated at 70 degrees celsius (water was changed after 10 minutes until the background became clear and then the protein bands became visible) to remove excess dye from the gel. Imaging of the gel with a gel imaging system, as shown in fig. 2, indicated that the correct size ATRN extracellular portion protein was obtained.

Example 3: mutation and purification elution of anti-ATRN nanometer antibody

The existing ATRN nm antibody sequence was analyzed by AI model (HIE B L,SHANKER V R,XU D,et al.Efficient evolution of human antibodies fromgeneral protein language models[J].Nature Biotechnology,2024,42(2):275-283.) and subjected to related amino acid mutations, and the corresponding nucleotide sequence was synthesized by company and cloned into the pTT5 vector, which was purified by the method of example 1.

The results of the antibody chromatography are shown in FIG. 3, indicating that high purity antibodies were obtained.

Example 4: anti ATRN nanometer antibody with FC segment by TEV enzyme digestion

To detect the binding capacity of a particular nanobody to ATRN antigen with FC using ELISA, the FC fragment of the nanobody was further digested and the VHH fragment of the anti-ATRN antibody was purified. To this end, tobacco etch virus protease (TobaccoEtchVirus protease, also known as TEV protease) digested the purified nanobody. TEV protease was added to the collected protein and stored overnight at 4 ℃. Subsequently, the digested proteins were passed through a protein column (protein A) and a nickel (Ni-NTA) column to completely remove undigested protein, FC and TEV enzymes, respectively.

Protein a and nickel columns were then eluted, verifying successful removal of FC tag and TEV protease. The results are shown in FIG. 4, where the FC tag and TEV protease were successfully removed from the mixture.

The results of SDS-PAGE electrophoresis of the nanobody protein without cleavage, the nanobody protein from which the FC fragment was removed after cleavage, the FC fragment and the TEV protease are shown in FIG. 5, and the obtained proteins were correctly sized.

Example 5: detection and validation of nanobody binding affinity to ATRN by ELISA

Mu.g of the digested antibody was used per well, 100. Mu.L of PBS was added thereto, and the mixture was cooled overnight at 4 ℃. The immune plates were washed three times with PBS, then 300 μl of MPBS (containing BSA for blocking non-specific binding sites) was added to each well and blocked for 3 hours at room temperature. The immunoplates were washed three times with PBST, 1000nM of Fc-tagged antigen (dissolved in MPBS) was added from the initial well, followed by ten-fold dilutions per row, 100 μl per well, and incubation with shaking at room temperature for one hour. The immune plates were washed 3 times with PBST, then HRP-conjugated anti-IgG 1 Fc antibody (beggar, sykeda) was added for detection of the nanobody-Fc fusion protein bound to antigen and incubated with shaking at room temperature for 1 hour. The immunoplates were washed five times with PBST, then 100. Mu.L TMB (chromogenic substrate) was added to each well, incubated at room temperature for 10 minutes in the dark, and 50. Mu.L 1M H ₂SO₄ was added to each well to terminate the reaction. The results are shown in FIG. 6, where the OD450 value gradually increases with increasing antigen concentration, and at an antigen concentration of 1000nM, OD450 reaches a maximum of 1.081, indicating that nanobody is able to specifically bind to the extracellular segment of ATRN with higher affinity.

Example 6: flow cytometry detects the ability of nanobodies to stimulate secretion of cytokines by human Peripheral Blood Mononuclear Cells (PBMCs)

PBMC were incubated with PHA (5. Mu.g/ml), 1% diabody and 10% FBS in 1640 medium in 96-well plates at 37℃in 5% CO2, cell concentration 2X 10 ⁶/ml, control medium without PHA, PBMC were blocked with PBS containing 10% mouse serum for 15min, then labeled CD3-BV786 (bioleged), CD8-PC7 (bioleged), CD56-BV605 (bioleged) flow antibody, 3 times with PBS after incubation in a refrigerator at 4℃for 30min, PBMC were fixed with fixative for 1 hour (Invitrogen eBioscience), labeled ATRN-AF647 flow antibody (Stokes) and isotype control antibody (bioleged) thereof, incubated for 30min at 4℃in refrigerator for 3 times with permeabilization buffer (Invitrogen eBioscience), and finally examined using a flow cytometer. The results are shown in fig. 7 (a), analyzed by flowjo software, with high expression of ATRN by CD8 ⁺ T cells and NK cells in PBMC stimulated with PHA for 96h compared to control.

Anti-ATRN nanobody and FC protein were added to PBMC stimulated with PHA (5. Mu.g/ml) for 96h at 200nmol and incubated in an environment of 5% CO2 at 37 ℃. PBMC were stimulated with PMA (30 ng/ml), ionomycin (1. Mu.g/ml) and monensin (2.5. Mu.g/ml) for 4 hours at nanobody stimulation for 48h and 96h, respectively, after which cells were harvested by centrifugation. Blocking, labeling of external standard flow antibody CD3-BV786 (bioleged), CD8-PC7 (bioleged), CD56-BV605 (bioleged), membrane disruption immobilization, labeling of internal standard flow antibody IFN-. Gamma. -FITC (Biolegend), perforin-BV421 (bioleged), granzyme B-ECD (Biolegend), TNF-. Alpha. -APC (Biolegend), and isotype control antibodies thereof (bioleged) were performed and detected using a flow cytometer, and finally analyzed with flowjo software. The results are shown in FIG. 7 (B-C), which shows that compared with the FC group, the ability of NK cells to secrete perforin is obviously enhanced after the FC group is incubated for 48 hours with the anti-ATRN nanometer antibody, and the ability of CD8 ⁺ T cells to secrete interferon, tumor necrosis factor and perforin is obviously enhanced after the FC group is incubated for 96 hours with the anti-ATRN nanometer antibody.

Example 7: anti-ATRN nanometer antibody inhibits growth of HepG2 subcutaneous tumor

To verify the therapeutic potential of the nanobodies obtained, 5×10 ⁶ HepG2 cells suspended in PBS and matrigel (2:1) were injected subcutaneously into mice of the age-appropriate NSG mice (6-8 weeks) to establish tumor models. On day 12 post injection, 5X 10 ⁶ PBMC were returned from the tail vein to each mouse, and when the tumor volume reached at least about 100 cubic millimeters (day 15 of tumor injection), randomly grouped mice were given 5 treatments per mouse by intraperitoneal injection of nanobody, each dose containing about 200 μg nanobody (10 mg/kg), at 2-3 days intervals. In addition, control mice received an equivalent amount of PBS as the drug-containing solution. Tumor growth was monitored every 2-3 days for 24 days after tumor cell injection, and the volume was calculated as length x width ² x 0.52, as shown in figure 8, and after treatment 5, tumor growth was significantly slowed in mice in the experimental group compared to the control group. The anti-ATRN nanometer antibody has good therapeutic potential on HepG2 tumor.

The preferred embodiments of the present application have been described in detail above, but the present application is not limited thereto. Within the scope of the technical idea of the application, a number of simple variants of the technical solution of the application are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the application, all falling within the scope of protection of the application.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

While embodiments of the present application have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the application, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the application.

Claims

1. An antibody or antigen-binding fragment thereof, comprising: a heavy chain complementarity determining region HCDR comprising an amino acid sequence selected from at least one of SEQ ID NOs 1 to 3 or having at least 80% homology therewith.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the HCDR comprises: HCDR1, HCDR2 and HCDR3, wherein:

The HCDR1 comprises an amino acid sequence as set forth 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 group consisting of those 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 with the amino acid sequences shown in SEQ ID NO 1-3.

3. The antibody or antigen-binding fragment thereof of claim 1, further comprising: a light chain complementarity determining region LCDR comprising: LCDR1, LCDR2, and LCDR3.

4. The antibody or antigen-binding fragment thereof of claim 2 or 3, wherein the antibody or antigen-binding fragment thereof further comprises: heavy chain framework regions;

Optionally, at least a portion of the heavy chain framework region is independently derived from at least one of an alpaca-derived antibody, a shark-derived antibody, a murine antibody, a rabbit-derived antibody, a primates-derived antibody or a mutant thereof, preferably an alpaca-derived antibody;

preferably, the heavy chain variable region of the antibody or antigen binding fragment thereof comprises an amino acid sequence selected from the group consisting of those shown in SEQ ID No. 4 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 antigen binding fragment thereof is shown in SEQ ID NO. 4;

optionally, the antibody or antigen binding fragment thereof further comprises: 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 murine antibody, a rabbit antibody, a primate antibody, or a mutant thereof.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein the antibody or antigen-binding fragment thereof further comprises: a heavy chain constant region;

Optionally, the antibody or antigen binding fragment thereof further comprises: 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 a rabbit-derived antibody, a murine-derived antibody, a sheep-derived antibody, a goat-derived antibody, a primate-derived antibody, or a mutant thereof.

6. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody comprises at least one selected from the group consisting of a polyclonal antibody and a monoclonal antibody; or (b)

The antigen binding fragment comprises at least one selected from the group consisting of a F (ab ') 2 fragment, a Fab' fragment, a Fab fragment, an Fv fragment, an scFv-Fv fusion protein, and a minimal recognition unit;

preferably, the antibody is a nanobody.

7. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-6;

optionally, the nucleic acid molecule is DNA.

8. An expression vector carrying the nucleic acid molecule of claim 7.

9. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1-6, comprising:

introducing the expression vector of claim 8 into a cell;

culturing the cells under conditions suitable for protein expression and secretion to obtain the antibody or antigen-binding fragment thereof;

Optionally, the cell is a eukaryotic cell.

10. A recombinant cell carrying the nucleic acid molecule of claim 7 or the expression vector of claim 8.

11. A recombinant protein which is prepared from the recombinant protein, characterized by comprising the following steps:

The antibody or antigen-binding fragment thereof of any one of claims 1-6;

Optionally, further comprising at least one member selected from the group consisting of a biologically active protein or fragment thereof, a biologically active polypeptide or fragment thereof;

Optionally, the biologically active protein or fragment thereof comprises at least one selected from the group consisting of a protein tag, a protein toxin or fragment thereof, a tumor necrosis factor or fragment thereof, an interferon or fragment thereof, a biological response modifier or fragment thereof, and an Fc fragment.

12. A composition of matter comprising a blend of two or more of the above, characterized by comprising the following steps:

the antibody or antigen-binding fragment thereof of any one of claims 1-6, carrying the nucleic acid molecule of claim 7, the expression vector of claim 8, the recombinant cell of claim 10, or the recombinant protein of claim 11.

13. Use of an 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 for the preparation of a medicament for the treatment or prevention of ATRN-mediated related diseases.

14. The use according to claim 13, wherein the ATRN-mediated related diseases include neurological diseases, autism Spectrum Disorders (ASD), alzheimer's disease, schizophrenia, parkinson's disease, bipolar disorders, epilepsy, diabetic nephropathy, glomerulonephritis, chronic kidney disease, polycystic kidney disease, nephrotic syndrome, renal cell carcinoma, obesity, type II diabetes, nonalcoholic fatty liver, metabolic syndrome, hypothyroidism, cushing's syndrome, hyperlipidemia, coronary artery disease, heart failure, stroke, peripheral arterial disease, atrial fibrillation, atherosclerosis, hypertension, coronary artery disease, heart failure, cardiac arrhythmias, 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, gastric cancer, colorectal cancer, breast cancer, esophageal cancer, thyroid cancer, skin cancer, prostate cancer, renal cancer, endometrial cancer, cervical cancer, and hematological tumors.

15. A medicament, comprising: the antibody or antigen-binding fragment thereof of any one of claims 1-6, carrying the nucleic acid molecule of claim 7, the expression vector of claim 8, the recombinant cell of claim 10, the recombinant protein of claim 11, or the composition of claim 12 for use in the treatment or prevention of ATRN-mediated related diseases.

16. The medicament of claim 15, wherein the ATRN-mediated related diseases include neurological diseases, autism Spectrum Disorders (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, nonalcoholic fatty liver, metabolic syndrome, hypothyroidism, cushing's syndrome, hyperlipidemia, coronary artery disease, heart failure, stroke, peripheral arterial disease, atrial fibrillation, atherosclerosis, hypertension, coronary artery disease, heart failure, cardiac arrhythmias, 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, renal cancer, endometrial cancer, cervical cancer, and hematological tumors;

optionally, the medicament further comprises pharmaceutically acceptable excipients;

Optionally, the auxiliary materials comprise: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers;

optionally, the medicament is an injection.

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

18. A kit is characterized in that, the kit comprises:

The antibody or antigen-binding fragment thereof of any one of claims 1-6.

19. The kit of claim 18, wherein the kit is for use in detecting ATRN.