Antibodies or antigen-binding fragments thereof against slc6a8 and compositions and uses thereof

Abstract

The present application provides an antibody or antigen-binding fragment thereof against SLC6A8, and compositions and uses thereof. Specifically, an antibody or antigen-binding fragment thereof against SLC6A8 is provided, which can effectively bind to human SLC6A8 transmembrane protein. The present application also provides an antibody drug conjugate comprising the antibody or antigen-binding fragment, and the like, which can be applied to the preparation of a medicament for treating SLC6A8 overexpression disease, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to antibodies against SLC6A8 or their antigen-binding fragments, their compositions, and their applications. Background Technology

[0002] SLC6A8 is significantly upregulated in various tumors, and its expression is associated with the survival of cancer patients and the infiltration of immune cells. Studies have found that SLC6A8 inhibitors have anti-tumor activity against various primary and metastatic colorectal cancers. The SLC6A8 transporter small molecule inhibitor RGX-202 can inhibit creatine infusion into tissues, reduce phosphocreatine and ATP levels in cells and tumors, inhibit tumor cell proliferation, induce tumor cell apoptosis, and increase serum and urinary creatine excretion in cancer patients.

[0003] However, small molecule inhibitors have short half-lives, low target specificity, and are prone to adverse reactions, necessitating the development of new SLC6A8 drugs. Antibody drugs, with their long half-lives and high specificity, can compensate for the shortcomings of small molecule drugs. In SLC6A8 research, in addition to developing inhibitory antibodies, there is also a need for antibodies capable of binding to SLC6A8 to detect the SLC6A8 protein in scientific research, diagnostics, and detection technologies. However, the limited number of existing SLC6A8 detection / labeling antibodies hinders research progress.

[0004] Therefore, there is an urgent need in this field to develop a series of detection antibodies or inhibitory antibodies targeting SLC6A8. Summary of the Invention

[0005] This invention provides antibodies against SLC6A8 or antigen-binding fragments thereof, compositions thereof, and applications.

[0006] In a first aspect of the invention, an anti-SLC6A8 antibody or an antigen-binding fragment thereof is provided, said antibody or antigen-binding fragment having three complementarity-determining regions (CDRs) of the heavy chain variable region and three complementarity-determining regions (CDRs) of the light chain variable region selected from the group consisting of: (1) HCDR1 shown in SEQ ID NO: 55, HCDR2, as shown in SEQ ID NO: 56, HCDR3, as shown in SEQ ID NO: 71, LCDR1 shown in SEQ ID NO: 73, LCDR2 shown in SEQ ID NO: 74 LCDR3 as shown in SEQ ID NO: 75; (2) HCDR1 shown in SEQ ID NO: 55, HCDR2, as shown in SEQ ID NO: 56, HCDR3, as shown in SEQ ID NO: 91, LCDR1 shown in SEQ ID NO: 93, LCDR2 shown in SEQ ID NO: 94 LCDR3 as shown in SEQ ID NO: 95; (3) HCDR1 shown in SEQ ID NO: 55, HCDR2, as shown in SEQ ID NO: 56, HCDR3, as shown in SEQ ID NO: 132, LCDR1 shown in SEQ ID NO: 134 LCDR2 shown in SEQ ID NO: 135, LCDR3 as shown in SEQ ID NO: 136; (4) HCDR1 shown in SEQ ID NO: 55, HCDR2, as shown in SEQ ID NO: 56, HCDR3, as shown in SEQ ID NO: 140, LCDR1 shown in SEQ ID NO: 117 LCDR2 shown in SEQ ID NO: 118 LCDR3 as shown in SEQ ID NO: 119; (5) HCDR1 shown in SEQ ID NO: 55, HCDR2, as shown in SEQ ID NO: 56, HCDR3, as shown in SEQ ID NO: 145, LCDR1 shown in SEQ ID NO: 147 LCDR2 shown in SEQ ID NO: 74 LCDR3 as shown in SEQ ID NO: 148.

[0007] In another preferred embodiment, the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

[0008] In another preferred embodiment, the antigen-binding fragment includes the Fab fragment, the F(ab')2 fragment, and the Fv fragment.

[0009] In another preferred embodiment, the heavy chain variable region and light chain variable region of the anti-SLC6A8 antibody or its antigen-binding fragment are selected from the group consisting of: (a) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 70, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO: 72.

[0010] (b) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 90, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO: 92.

[0011] (c) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 131, and the light chain variable region with the amino acid sequence shown in SEQ ID NO: 133.

[0012] (d) The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 139, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO: 141.

[0013] (e) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 144, and the light chain variable region with the amino acid sequence shown in SEQ ID NO: 146.

[0014] In another preferred embodiment, the heavy chain of the antibody or its antigen-binding fragment further includes a heavy chain constant region; the light chain of the antibody or its antigen-binding fragment further includes a light chain constant region.

[0015] In another preferred embodiment, the antibody is a single-chain antibody, a double-chain antibody, or an antigen-binding fragment.

[0016] In another preferred embodiment, the antibody is a humanized antibody, a murine antibody, or a chimeric antibody.

[0017] In another preferred embodiment, the heavy chain constant region is of human or mouse origin.

[0018] In another preferred embodiment, the light chain constant region is of human or mouse origin.

[0019] In another preferred embodiment, the antibody is a full-length antibody protein or an antigen-binding fragment.

[0020] In another preferred embodiment, the antibody is a monoclonal antibody.

[0021] In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.

[0022] In another preferred embodiment, the antibody further comprises a linker peptide located between the heavy chain variable region and the light chain variable region.

[0023] In a second aspect of the invention, a recombinant protein is provided, the recombinant protein having: (i) the anti-SLC6A8 antibody or its antigen-binding fragment as described in the first aspect of the present invention; and (ii) Tag sequences that optionally assist in expression and / or purification.

[0024] In another preferred embodiment, the label includes an Fc label, a FLAG label, a 6His label, a Strep label, an HA label, or a combination thereof.

[0025] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0026] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0027] In a third aspect of the invention, an isolated nucleic acid molecule is provided, said nucleic acid molecule encoding the anti-SLC6A8 antibody or its antigen-binding fragment as described in the first aspect of the invention.

[0028] In another preferred embodiment, the nucleotide molecule is DNA, RNA, or cDNA.

[0029] In a fourth aspect of the invention, a carrier is provided, the carrier containing the nucleic acid molecule described in the third aspect of the invention.

[0030] In another preferred embodiment, the vector includes: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0031] In another preferred embodiment, the vector is a eukaryotic expression vector.

[0032] In a fifth aspect of the invention, a host cell is provided, the host cell containing the vector described in the fourth aspect of the invention, or having the nucleic acid molecule described in the third aspect of the invention integrated into its genome.

[0033] In another preferred embodiment, the cell is a eukaryotic cell or a prokaryotic cell.

[0034] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0035] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0036] In another preferred embodiment, the prokaryotic cell is Escherichia coli.

[0037] In another preferred embodiment, the cell is an immune cell, and its surface simultaneously expresses a chimeric antigen receptor.

[0038] In another preferred embodiment, the immune cells are T cells, NK cells, or a combination thereof.

[0039] In another preferred embodiment, the immune cells are chimeric antigen receptor T cells (CAR-T cells).

[0040] In another preferred embodiment, the chimeric antigen receptor is an anti-SLC6A8 antibody or its antigen-binding fragment.

[0041] In a sixth aspect of the invention, an antibody-drug conjugate is provided, the antibody-drug conjugate comprising: (a) An antibody portion comprising the anti-SLC6A8 antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention; and (b) A conjugation portion coupled to the antibody or its antigen-binding fragment, the conjugation portion being selected from the group consisting of detectable markers, drugs, or combinations thereof.

[0042] In another preferred embodiment, the detectable marker is selected from the group consisting of: (A) Radioactive isotopes, such as 35 S, 14 C 125 I, 3 H and 131 I. Antibodies can be labeled with radioactive isotopes, and radioactivity can be measured using scintillation counting.

[0043] (B) Fluorescent markers, such as rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, erythroline, phycoerythrin, Texas red, and Brilliant Violet. TM Yes, it is available. Fluorescent labels can be conjugated to antibodies using techniques disclosed, for example, in Current Protocols in Immunology (ibid.). Fluorescence can be quantified using flow cytometry, imaging microscopy, or a fluorometer.

[0044] (C) Various enzyme-substrate labels are available. Enzymes typically catalyze chemical changes in chromogenic substrates, which can be measured using various techniques. For example, an enzyme can catalyze a color change in a substrate, which can be measured spectrophotometrically. Alternatively, an enzyme can alter the fluorescence or chemiluminescence of a substrate. Techniques for quantifying fluorescence changes are described above. Chemiluminescent substrates become electronically excited through a chemical reaction and then emit light that can be measured (e.g., using a chemiluminometer) or supply energy to a fluorescent acceptor. Examples of enzyme labels include luciferases (e.g., firefly luciferase and bacterial luciferase; luciferin, 2,3-dihydrophthalazinedione, malate dehydrogenase, urease, peroxidases such as horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, etc.

[0045] The indirect conjugation of labels to antibodies is a technique known to those skilled in the art. For example, antibodies can be conjugated to biotin, and any of the three broad categories of labels mentioned above can be conjugated to avidin, or vice versa. Biotin selectively binds to avidin, and therefore labels can be conjugated to antibodies in this indirect manner.

[0046] In another preferred embodiment, the expression for the antibody-drug conjugate is: mAb-(XY)n; in, mAb is the anti-SLC6A8 antibody or its antigen-binding fragment; X is a connector; Y represents the coupling portion, which is a drug; n is a positive integer ≤ 8; The conjugation portion is conjugated to the anti-SLC6A8 antibody or its antigen-binding fragment via a linker.

[0047] In a seventh aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising: (i) an antibody or antigen-binding fragment thereof as described in the first aspect of the invention, a recombinant protein as described in the second aspect of the invention, a nucleic acid molecule as described in the third aspect of the invention, a vector as described in the fourth aspect of the invention, a host cell as described in the fifth aspect of the invention, or an antibody-drug conjugate as described in the sixth aspect of the invention; and (ii) Pharmaceutically acceptable carriers, diluents or excipients.

[0048] In another preferred embodiment, the pharmaceutical composition is an injectable dosage form.

[0049] In another preferred embodiment, the pharmaceutical composition is used to prepare a medicament for treating diseases caused by SLC6A8 overexpression.

[0050] In another preferred embodiment, the disease is a tumor.

[0051] In another preferred embodiment, the tumor includes colorectal cancer.

[0052] In an eighth aspect of the invention, the use of an antibody or antigen-binding fragment thereof as described in the first aspect of the invention, a recombinant protein as described in the second aspect of the invention, and an antibody-drug conjugate as described in the sixth aspect of the invention are provided for the preparation of pharmaceuticals, reagents, detection plates or kits. The reagents, detection plates, or kits are used to detect SLC6A8 protein in samples; The agent is used to treat or prevent diseases that overexpress the SLC6A8 protein.

[0053] In another preferred embodiment, the SLC6A8 protein is a multi-transmembrane protein.

[0054] In a ninth aspect of the present invention, a method for preparing the anti-SLC6A8 antibody or its antigen-binding fragment as described in the first aspect of the present invention is provided, the method comprising the following steps: (a) Under expression conditions, host cells as described in the fifth aspect of the present invention are cultured to express the anti-SLC6A8 antibody or its antigen-binding fragment; (b) Isolate and purify the anti-SLC6A8 antibody or its antigen-binding fragment described in (a).

[0055] In a tenth aspect of the present invention, a method for detecting SLC6A8 protein in a sample is provided, the method comprising the steps of: (S1) Contact the sample with an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention; (S2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of SLC6A8 protein in the sample.

[0056] In another preferred embodiment, the sample includes: human or animal tissue samples, or exfoliated cell samples.

[0057] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0058] In another preferred embodiment, the method is an in vitro method.

[0059] In another preferred embodiment, the method further includes step (3) analyzing the affinity between the antibody and the antigen.

[0060] In an eleventh aspect of the present invention, a detection plate is provided, the detection plate comprising a substrate (support plate) and a test strip, the test strip containing an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, a recombinant protein as described in the second aspect of the present invention, or an antibody-drug conjugate as described in the sixth aspect of the present invention.

[0061] In another preferred embodiment, the test strip also contains an antigen spotting area.

[0062] In another preferred embodiment, the test strip is composed of filter paper, chromatography material, nitrocellulose membrane and absorbent paper stacked in sequence.

[0063] In a twelfth aspect of the invention, a kit is provided, the kit comprising: (1) A first container containing an antibody or an antigen-binding fragment thereof as described in the first aspect of the invention; and / or (2) A second container containing a secondary antibody against an antibody or an antigen-binding fragment thereof as described in the first aspect of the invention; and / or (3) A third container containing a cell lysis reagent; or, The kit contains a detection plate as described in the eleventh aspect of the present invention.

[0064] In another preferred embodiment, the antibody in the first container is labeled with a detectable tag.

[0065] In another preferred embodiment, the antibody in the second container is labeled with a detectable tag.

[0066] In a thirteenth aspect of the invention, a method for treating a disease associated with SLC6A8 overexpression is provided, comprising the steps of administering to a subject requiring treatment a therapeutically effective amount of the anti-SLC6A8 antibody or its antigen-binding fragment as described in the first aspect of the invention, the recombinant protein as described in the second aspect of the invention, the nucleic acid molecule as described in the third aspect of the invention, the expression vector as described in the fourth aspect of the invention, the antibody-drug conjugate as described in the sixth aspect of the invention, or a pharmaceutical composition as described in the seventh aspect of the invention.

[0067] In another preferred example, the disease associated with SLC6A8 overexpression is tumors.

[0068] In another preferred embodiment, the tumor includes colorectal cancer.

[0069] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0070] Figure 1 The construction of the purified SLC6A8 protein for expression is shown.

[0071] Figure 2 The results of the flow sorting example in Example 2 are shown.

[0072] Figure 3 The heavy chain vector of human IgG used to construct the antibody is shown.

[0073] Figure 4 The light chain vector of human IgG used to construct the antibody is shown.

[0074] Figure 5 The statistical plot of MFI values ​​for antibody gradient and CAC protein flow cytometry detection is shown.

[0075] Figure 6A and Figure 6B The results of flow cytometry analysis are shown (including negative and positive controls). Detailed Implementation

[0076] Through extensive and in-depth research and numerous screenings, the inventors unexpectedly obtained a class of antibodies against SLC6A8. Experimental results show that the anti-SLC6A8 antibody of this invention possesses high affinity and good biological activity, and can be used for antigen detection of SLC6A8 and as an inhibitor for the treatment of SLC6A8-related diseases. Based on this, the present invention was completed.

[0077] Specifically, SLC6A8 is a multi-transmembrane protein, and technically, it is difficult to obtain high-affinity specific antibodies for multi-transmembrane proteins. However, in the experiments of this invention, 14 high-affinity SLC6A8 monoclonal antibodies were successfully obtained, including antibodies numbered SJ12, SJ24, SJ25, SJ26, SJ28, SJ30, SJ32, SJ36, SJ38, SJ39, SJ40, SJ41, SJ42, and SJ43.

[0078] the term To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0079] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101.

[0080] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0081] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0082] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0083] As used herein, the term “pharmaceuticalally acceptable carrier” refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio.

[0084] As used herein, the term "therapeutic effective amount" refers to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals. Those skilled in the art will understand that the "therapeutic effective amount" can vary depending on the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and whether it is used in combination with other drugs.

[0085] SLC6A8 SLC6A8 Solute Carrier Family 6 Member 8 (CT1, Creatine Transporter 1) encodes a multi-transmembrane protein on the cell membrane. It is a member of the solute carrier family 6, specifically the GABA transporter subfamily. This protein relies on sodium and chloride ions to transport creatine across the cell membrane. Slc6a8 is highly expressed in tissues that require creatine, such as the brain and skeletal muscle, accumulating high concentrations of creatine intracellularly and creating a sharp concentration gradient. Adequate creatine transport is crucial for maintaining the normal physiological functions of high-energy-consuming organs.

[0086] Antibody In this invention, the terms "antibody (Ab)" and "immunoglobulin G (IgG)" refer to heterotetraglycoproteins with the same structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by a constant region, which consists of three domains: CH1, CH2, and CH3. Each light chain has a variable region (VL) at one end and a constant region at the other end, with the light chain constant region including a domain CL; the light chain constant region pairs with the CH1 domain of the heavy chain constant region, and the light chain variable region pairs with the heavy chain variable region. Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC). Heavy chain constant regions include IgG1, IgG2, IgG3, and IgG4 isotypes; light chain constant regions include κ (Kappa) or λ (Lambda). The heavy and light chains of an antibody are covalently linked by disulfide bonds between the CH1 domain of the heavy chain and the CL domain of the light chain. The two heavy chains of an antibody are covalently linked by interpeptide disulfide bonds formed between their hinge regions.

[0087] In this invention, the terms "Fab" and "Fc" refer to the ability of papain to cleave an antibody into two identical Fab fragments and one Fc fragment. The Fab fragment consists of the VH and CH1 domains of the antibody's heavy chain and the VL and CL domains of its light chain. The Fc fragment, or crystallizable fragment, consists of the antibody's CH2 and CH3 domains. The Fc fragment lacks antigen-binding activity and is the site of interaction between the antibody and effector molecules or cells.

[0088] In this invention, the term "scFv" refers to a single-chain antibody fragment (scFv), which is composed of the variable regions of the antibody heavy chain and the variable regions of the light chain, typically linked by a short peptide (linker) of 15 to 25 amino acids.

[0089] In this invention, the term "variable" refers to the fact that certain portions of the variable region in an antibody differ in sequence, resulting in the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of the antibody. It is concentrated in three segments within the variable regions of the heavy and light chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable regions are called frame regions (FRs). The variable regions of the natural heavy and light chains each contain four FR regions, which are generally β-sheet configurations, linked by three CDRs forming a linking loop, and in some cases may form a partial β-sheet structure. The CDRs in each chain are closely packed together through the FR regions and together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)).

[0090] As used herein, the term "frame region" (FR) refers to the amino acid sequence inserted between CDRs, that is, those portions of the variable regions of the light and heavy chains of immunoglobulins that are relatively conserved among different immunoglobulins within a single species. Each of the light and heavy chains of an immunoglobulin has four FRs, designated L-FR1, L-FR2, L-FR3, L-FR4 and H-FR1, H-FR2, H-FR3, H-FR4, respectively. Accordingly, the light chain variable domain can therefore be designated (L-FR1)-(LCDR1)-(L-FR2)-(LCDR2)-(L-FR3)-(LCDR3)-(L-FR4), and the heavy chain variable domain can therefore be represented as (H-FR1)-(HCDR1)-(H-FR2)-(HCDR2)-(H-FR3)-(HCDR3)-(H-FR4). Preferably, the FR of the present invention is a human antibody FR or a derivative thereof, wherein the derivative of the human antibody FR is substantially the same as the naturally occurring human antibody FR, that is, the sequence identity reaches 85%, 90%, 95%, 96%, 97%, 98% or 99%.

[0091] Knowing the amino acid sequence of the CDR, those skilled in the art can easily determine the framework regions L-FR1, L-FR2, L-FR3, L-FR4 and / or H-FR1, H-FR2, H-FR3, H-FR4.

[0092] As used herein, the term "human frame region" is a frame region that is substantially identical (approximately 85% or more, specifically 90%, 95%, 97%, 99%, or 100%) to the frame region of a naturally occurring human antibody.

[0093] As used herein, the term "linker" refers to an insertion into an immunoglobulin domain that provides sufficient mobility for the light and heavy chains to fold into one or more amino acid residues of an exchangeable dual variable region immunoglobulin. In this invention, preferred linkers are Linker1 and Linker2, wherein Linker1 links the VH and VL of a single-chain antibody (scFv), while Linker2 is used to link the scFv to the heavy chain of another antibody.

[0094] Suitable examples of linkers include monoglycine (Gly) or serine (Ser) residues, and the identification and sequence of amino acid residues in the linker can vary depending on the type of secondary structural element that needs to be achieved in the linker.

[0095] In this invention, the antibody also includes its conserved variants, which are polypeptides formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the specific antibody of this invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0096] Table A In this invention, the terms "antibody," "binding," and "specific binding" refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and its targeted antigen. Typically, antibodies bind at a rate of less than approximately 10... -7 M, for example, less than approximately 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 The antibody binds to the antigen with an equilibrium dissociation constant (KD) of M or smaller. In this invention, the term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. For example, the binding affinity between the antibody and the antigen can be determined using surface plasmon resonance (SPR) in a BIACORE instrument or using ELISA to determine the relative affinity of antibody-antigen binding.

[0097] In this invention, the term "epitope" refers to a polypeptide determinant that specifically binds to an antibody. The epitopes of this invention are regions of an antigen that are bound to antibodies.

[0098] Polynucleotides, vectors and host cells The present invention also provides a polynucleotide molecule encoding the above-described antibody or a fragment thereof or a fusion protein thereof. The polynucleotide of the present invention may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0099] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0100] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0101] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0102] The full-length nucleotide sequence or fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein.

[0103] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms.

[0104] Currently, the DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0105] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0106] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[0107] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0108] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0109] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0110] The antibodies of the present invention can be used alone or in combination or conjugated with detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modified parts, or any combination of the above substances.

[0111] Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes that can produce detectable products.

[0112] Therapeutic agents that can bind to or conjugate with the antibodies of this invention include, but are not limited to: 1. radionuclides; 2. biotoxicants; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorobars; 5. viral particles; 6. liposomes; 7. magnetic nanoparticles; 8. prodrug-activating enzymes (e.g., DT-cardiac flavinase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0113] Pharmaceutical Compositions and Applications This invention also provides a composition. Preferably, the composition is a pharmaceutical composition containing the aforementioned antibody or its active fragment or fusion protein, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intravenous injection, intravenous infusion, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (e.g., intraperitoneal), intracranial injection, or intracavitary injection. In this invention, the term "pharmaceutical composition" refers to a pharmaceutical formulation composition in which the specific antibody of this invention can be combined with a pharmaceutically acceptable carrier to exert its therapeutic effect more stably. These formulations ensure the conformational integrity of the amino acid core sequence of the specific antibody disclosed in this invention, while also protecting the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamination, or oxidation). The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described specific antibody (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. Furthermore, the specific antibody of the present invention can also be used with other therapeutic agents.

[0114] When using a pharmaceutical composition, a safe and effective amount of the specific antibody or its immunoconjugate is administered to a mammal. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is between about 10 micrograms per kilogram of body weight and about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0115] Antibody-drug conjugates (ADCs) The present invention also provides antibody-drug conjugates (ADCs) based on the antibodies of the present invention.

[0116] Typically, the antibody-drug conjugate comprises an antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a drug with therapeutic activity. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic agent, a small molecule drug, or a radionuclide.

[0117] The antibody and the effector molecule of the present invention can be coupled via a coupling agent. Examples of the coupling agent include any one or more of non-selective coupling agents, carboxyl-based coupling agents, peptide chains, and disulfide bonds. The non-selective coupling agent refers to a compound that covalently links the effector molecule and the antibody, such as glutaraldehyde. The carboxyl-based coupling agent can be any one or more of maleic aconitine-based coupling agents (e.g., maleic aconitine) and acylhydrazone-based coupling agents (with an acylhydrazone as the coupling site).

[0118] Certain residues on antibodies (such as Cys or Lys) are used to link to a variety of functional groups, including imaging reagents (e.g., chromophores and fluorophores), diagnostic reagents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. Antibodies can be conjugated to functional agents to form antibody-functional agent conjugates. Functional agents (e.g., drugs, detection reagents, stabilizers) are conjugated (covalently linked) to antibodies. Functional agents can be directly attached to antibodies or indirectly through linkers.

[0119] Antibodies can be conjugated to drugs to form antibody-drug conjugates (ADCs). Typically, an ADC contains a linker between the drug and the antibody. The linker can be degradable or non-degradable. Degradable linkers are typically readily degraded in intracellular environments, such as at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptide-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronidase-containing linkers. Peptide linkers can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0120] Prior to attachment to the antibody, the linker has a reactive group capable of reacting with certain amino acid residues, and the attachment is achieved through the reactive group. Thiol-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethyl ketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones; pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, with the counter ion being acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, a maleimide attached to the antibody via a thiosuccinimide.

[0121] The drug can be any cytotoxic, cell growth-inhibiting, or immunosuppressive drug. In one embodiment, the linker connects the antibody and the drug, and the drug has a functional group that can bond with the linker. For example, the drug may have an amino, carboxyl, thiol, hydroxyl, or ketone group that can bond with the linker. In the case where the drug is directly linked to the linker, the drug has a reactive group before being linked to the antibody.

[0122] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, etc. In this invention, the drug-linker can be used to form an ADC in a single, simple step. In other embodiments, bifunctional linker compounds can be used to form an ADC in two or more steps. For example, cysteine ​​residues react with the reactive portion of the linker in a first step, and in subsequent steps, functional groups on the linker react with the drug to form an ADC.

[0123] Typically, functional groups on the linker are selected to facilitate specific reaction with suitable reactive groups on the drug moiety. As a non-limiting example, azide-based moieties can be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently bound to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphine (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other linking strategies, such as those described in Bioconjugation Techniques, Second Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will understand that for selective reaction between the drug moiety and the linker, when a complementary pair of reactive functional groups is selected, each member of that complementary pair can be used for either the linker or the drug.

[0124] The present invention also provides a method for preparing an ADC, which may further include: binding an antibody to a drug-adaptor compound under conditions sufficient to form an antibody-drug conjugate (ADC).

[0125] In some embodiments, the method of the present invention includes binding an antibody to a bifunctional adapter compound under conditions sufficient to form an antibody-adaptor conjugate. In these embodiments, the method of the present invention further includes binding the antibody-adaptor conjugate to a drug moiety under conditions sufficient to covalently link a drug moiety to the antibody via the adapter.

[0126] In some implementations, the antibody-drug conjugate (ADC) has the following molecular formula: in: Ab is an antibody. LU stands for connector; D is a drug; Furthermore, the subscript p is a value selected from 1 to 8.

[0127] Detection uses and kits The antibodies of this invention can be used in detection applications, such as for testing samples, to provide diagnostic information.

[0128] In this invention, the samples used include cells, tissue samples, and biopsy specimens. The term "biopsy" as used in this invention should include all types of biopsies known to those skilled in the art. Therefore, biopsies used in this invention can include tissue samples prepared, for example, by endoscopic methods or by puncture or needle biopsy of organs.

[0129] The samples used in this invention include fixed or preserved cell or tissue samples.

[0130] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be immobilized on a detection plate.

[0131] application This invention provides the use of the antibody of this invention, for example, for the preparation of diagnostic agents, or for the preparation of drugs for the prevention and / or treatment of SLC6A8-related diseases.

[0132] In a preferred embodiment, the SLC6A8-related disease is a disease characterized by SLC6A8 overexpression. In a preferred embodiment, the disease is a tumor. In a preferred embodiment, the disease includes colorectal cancer.

[0133] The main advantages of this invention include: (a) Compared with SLC6A8 small molecule inhibitors, the antibody of the present invention has a longer half-life and higher specificity.

[0134] (b) The antibodies prepared in this invention not only provide new tools for scientific research and disease diagnosis of SLC6A8, but also open up new avenues for drug development. With further research, anti-SLC6A8 monoclonal antibodies are expected to demonstrate their unique value and broad prospects in more fields.

[0135] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0136] Example 1: Obtaining the native conformation of human SLC6A8 protein In this embodiment, conventional techniques in the art were used to construct the full-length human SLC6A8 gene sequence (Bgee:ENSG00000130821) into a mammalian cell expression vector, such as the pUC57 vector, and HEK293 cells were used for exogenous expression in order to obtain a large number of SLC6A8 protein samples.

[0137] The expressed protein will be purified using conventional biochemical techniques in the field, with purification conditions optimized to preserve the protein's native conformation as much as possible. The purified SLC6A8 protein will then be packaged using Amphipol reagent to maximize the mimicry of the native SLC6A8 protein conformation on the cell membrane surface. Alternatively, natural human full-length SLC6A8 protein can be purchased. Figure 1 To construct and express the purified SLC6A8 protein.

[0138] Example 2: Mouse immunization, mouse B cell sorting and culture The SLC6A8 protein obtained in Example 1 was used to immunize mice using conventional and known techniques. The immunization strategy was as follows: 10-12 week old C57BL / 6 mice were selected and divided into an immunization group and a control group. Each mouse in the immunization group was injected intraperitoneally and tarsal joint with 30 µg of the SLC6A8 protein (antigen) prepared in Example 1 and 20 µg of CpG OND 2395 (5'-tcgtcgttttcggcgcgcgccg-3') (SEQ ID NO: 153) and an equal volume of AddaVax™ (InvivoGen). The control group was injected with 20 μg of CpG OND 2395 and an equal volume of AddaVax™. The mice were immunized once every 3-4 days for 4-6 times.

[0139] Sorting of mouse B cells: 3-7 days after the last immunization, mice in both the experimental and control groups were sacrificed, and spleen and lymph node tissues were obtained. Single-cell suspensions were prepared from the spleen and lymph nodes (popliteal fossa and groin), and then red blood cells were removed using erythrocyte lysis buffer (Sangon Biotech Cat.b541001-0100). After washing twice with 2% FBS in PBS (FACS buffer), the cells were prepared for staining. A Cytoflex SRT (Beckman Coulter) cell sorting system was then used to sort the cells to obtain the target cells. The staining gating strategy was as follows: DAPI-CD19+CD38+GL7-IgG1+; The following reagents were used: DAPI (live / dead; Invitrogen Cat. D1306), anti-mouse CD19 (APC / Cyanine7; Biolegend Cat.115530), anti-mouse CD38 (PE / Cyanine7; Biolegend Cat.102718), anti-mouse / human GL7 Antigen (T and B cell Activation Marker), AlexaFluor® 488; Biolegend Cat.144612); anti-mouse IgG1 (APC; Biolegend Cat.406610).

[0140] Following immunization, mouse IgG1-positive memory B cells were sorted by flow cytometry.

[0141] Example results of flow sorting are as follows Figure 2 As shown in the figure. Group A is the control group, and group B is the experimental group. Figure 2 It can be seen that the control group obtained very few IgG1+ positive B cells (0.20%), while the experimental group obtained a large number of IgG1+ positive B cells (1.15%) after immunization. Compared with the control group, the antigen immunization group showed a significant immune response. Based on the above results, this immunization protocol can effectively induce a sufficient number of antigen-specific memory B cells and germinal center B cells, and the sorting strategy can efficiently enrich antigen-specific memory B cells.

[0142] Culture of mouse B cells: The IgG1-positive B cells obtained above were cultured, and the culture method can be referred to Hanida and Kitamura, (2019). Induced Germinal Center B CellCulture System, Bio-protocol 9 (4): e3163. DOI: 10.21769 / BioProtoc.3163. The feeding medium was: RPMI-1640, (Gibco, Cat.11875-093), 10% Fetal Bovine Serum (FBS, VivaCell, Cat. C04002-500), 55 μM 2-mercury ethanol (Thermo Fisher, Cat.21985), 100 units / ml Penicillin (Biosharp), 100 μg / ml Streptomycin (Biosharp), 10 mM HEPES (Thermo Fisher, The medium contained 1 mM Sodium Pyruvate (Thermo Fisher, Cat.11360-070) and 0.1 mM MEM nonessential amino acid (Thermo Fisher, Cat.11140-050). One day before sorting, feeder cells expressing CD40L and BAFF were seeded into 96-well plates at 800-1200 cells per well. Single IgG1-positive memory B cells were sorted into each well, and 2-10 ng / ml IL-4 was added to the feeder medium and cultured for two days. Then, 4-10 ng / ml IL-21 was added and cultured for another 6-8 days, changing the medium daily. On day 10, the supernatant was collected and stored at 4°C. The cells in the 96-well plates were stored at -80°C for subsequent lysis to obtain RNA from single B cells.

[0143] In the experiment, sorting was performed three times, with 100 plates sorted each time for culture. Flow cytometry data showed that the target memory B cell population accounted for approximately 87.7%, with an IgG1 positivity rate of approximately 1.15%, indicating that the sorting strategy can efficiently sort memory B cells. PBS with 20 μg of CpG OND 2395 and an equal volume of AddaVax™ served as the immunization control group. Subsequently, approximately 60 pairs of monoclonal antibody VDJ / VJ sequences were obtained using molecular cloning methods. However, after further flow cytometry verification of bead antigen and antibody surface binding, only 14 highly specific monoclonal antibodies were obtained. SLC6A8 is a multi-transmembrane protein, and technically, it is difficult to obtain high-affinity specific antibodies for multi-transmembrane proteins. In this experiment, through extensive work and technical expertise, 14 high-affinity SLC6A8 monoclonal antibodies were successfully obtained.

[0144] Example 3: ELISA Experiment, Results, and Analysis The SLC6A8 antigen protein prepared in Example 1 was coated onto a 96-well ELISA plate and incubated overnight at 4°C under humid conditions. After discarding the coating buffer, 100-120 μL of blocking buffer (4% BSA in 1×PBS) was added to the plate and incubated at room temperature for 2 hours. After removing the blocking buffer, 20-60 μL of single-cell culture supernatant was added to each well and incubated overnight at 4°C under humid conditions. After washing three times, 20-30 μL of secondary antibody (AKP goat anti-mouse IgG1, Southern Biotech, cat. 1030-04) was added and incubated at room temperature for 2 hours. After washing four times, 20-30 μL of chromogenic solution containing disodium 4-nitrophenyl phosphate hexahydrate (CSNpharm, Cat. CSN66207) was added to each well. OD405 was measured using an MD SpectraMax iD3.

[0145] The results are shown in Tables 1-2 below: Partial detection data of ELISA assay using 96-well plates.

[0146] Table 1. OD405 results of IgG1 positive control in plate 1 Table 2. OD405 results of antigen-antibody specificity detection on plate 1 ELISA results showed that the supernatant of the cultured IgG1+ positive B cells contained antibodies that specifically bind to the SLC6A8 antigen. The color development varied depending on the antibody binding strength or concentration in the culture supernatant, with antibodies showing specific antigen-antibody binding displaying higher values. After multiple batches of ELISA testing, approximately 60-80 highly specific antigen-antibody binding antibodies were obtained across all batches.

[0147] Example 4 Molecular cloning method and obtaining the monoclonal antibody VDJ / VJ sequence Based on the ELISA test results analysis of Example 3, cells from the positive wells (antigen-antibody binding) of a 96-well cell culture plate that were pre-frozen at -80℃ were selected for RNA extraction from the single B cells, and subsequent molecular cloning was performed to obtain the VDJ / VJ sequence.

[0148] Total RNA was extracted from B cells in 96-well cell culture plates that tested positive (antigen-antibody binding) using TRIzol Reagent (Thermo Fisher). Reverse transcription and PCR were performed according to the paper (Cloning and expression of murine Iggenes from single B cells, Thomas Tiller et al., Journal of Immunological Methods, 2009). In short, cDNA synthesis was performed using Maxima H Minus reverse transcriptase (Thermo Fisher) at 42°C, 5 min, 25°C, 10 min, 50°C, 60 min, and 94°C. Two rounds of semi-nested PCR were then performed using HotStar DNA polymerase (Qiagen) to enrich heavy and light chains. The PCR products were purified and sequenced. Sequencing results were analyzed using IgBlast and the IMGT database. The VDJ / VJ fragment was amplified using gene-specific primers (Table 12). The cloned VDJ fragment heavy chain and / or VJ fragment light chain were ligated into a vector using homologous recombination or T4 ligase. The vector can be mouse-derived, human-derived, etc. It can be in the form of a full-length antibody, or in Fab or scFV form, and can carry His, Strep, Flag, Fc tags, etc., with the antibody form selected according to experimental requirements.

[0149] The primers used for the two-round semi-nested PCR are shown in Table 11. The PCR program was as follows: 95℃, 15 minutes; 95℃, 30 seconds; 50-65℃, 30 seconds; 72℃, 5 minutes.

[0150] Reverse transcription to cDNA: Add the following reagents sequentially to Mix1: 1.4 µL RNase-free water; 1 µL 10 mM dNTP; 1 µL 100 µM Random Primers; 1 µL template RNA. Incubate at 65 °C for 5 min. Add the following reagents sequentially to Mix2: 4 µL 5×RT buffer; 0.1 µL RNase inhibitor; 0.25 µL reverse transcriptase; 11.25 µL RNase-free water. Mix1 and Mix2 are then combined and subjected to the following reverse transcription PCR program: 42 °C for 5 min; 25 °C for 10 min; 50 °C for 60 min; 85 °C for 5 min; store at 4 °C.

[0151] The reaction system for the first round of heavy chain PCR is shown in Table 3 below: (or the reaction system can be scaled up or down as needed). Table 3 The reaction system for the second round of heavy chain PCR is shown in Table 4 below: (or the reaction system can be scaled up or down as needed).

[0152] Table 4 The first round PCR reaction system for Kappa light chain is shown in Table 5 below: (or the reaction system can be scaled up or down as needed).

[0153] Table 5 The second round PCR reaction system for Kappa light chain is shown in Table 6 below: (or the reaction system can be scaled up or down as needed).

[0154] Table 6 The PCR reaction system for the first round of lambda light chain is shown in Table 7 below: Table 7 The second round PCR reaction system for lambda light chain is shown in Table 8 below: Table 8 The primer mix in the heavy chain, Kappa chain, and lambda light chain mentioned above refers to the mixture of the corresponding primers in List 9 below in the first and second rounds of semi-nested PCR in the corresponding heavy chain, Kappa chain, and lambda light chain. For example, in the IgK 1st PCR, 5′ L-Vk mix Fw is a mixture of 5L-Vκ_3, 5L-Vκ_4, 5L-Vκ_5, 5L-Vκ_6, 5L-Vκ_6-8-9, 5L-Vκ_14, 5L-Vκ_19, and 5L-Vκ_20, with each primer at 10 μM. The mix in other PCR systems is explained accordingly.

[0155] Table 9 Semi-nested PCR primers Table 10 Gene-specific primers for amplifying VDJ / VJ The VDJ / VJ-specific primers in this embodiment include homologous arms of the heavy / light chain vectors, as well as specific portions of the VDJ / VJ fragments. Generally, the homologous arms of these specific primers will vary depending on the cloning vector used. This invention is not limited to the heavy and light chain vectors and specific primers used in this embodiment.

[0156] After reverse transcription and two rounds of semi-nested PCR, the nucleotide and amino acid sequences of the VDJ / VJ variable regions of the antibodies were sequenced, resulting in antibodies numbered SJ12, SJ24, SJ25, SJ26, SJ28, SJ30, SJ32, SJ36, SJ38, SJ39, SJ40, SJ41, SJ42, and SJ43, whose sequences are shown in Table 11.

[0157] Table 11 In this embodiment, the vector map of the antibody against SLC6A8 prepared using the heavy chain constant region and light chain constant region vector of human IgG1 is as follows. Figure 3 and Figure 4 As shown.

[0158] The amino acid sequence of the heavy chain constant region of the humanized antibody is shown in SEQ ID NO: 151, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 152.

[0159] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 151) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 152) Example 5: Detection of SLC6A8 antigen and antibody bead binding In this embodiment, the successfully paired antibody heavy-light chain plasmid was transfected into 293F cells for expression and purification using conventional techniques in the art, for subsequent experimental use. In the experiment, polystyrene Protein A (Spherotech) microspheres were resuspended in 1x PBS at room temperature, centrifuged, and then resuspended in 10 μg / ml human anti-flag antibody protein. The mixture was stirred and incubated for 15 minutes, then washed twice with 1x PBS. Each time, 2 μg / ml SL6A8-2% FACS solution was added and incubated for 1-2 hours (SLC6A8 antigen has a flag tag). The mixture was then washed twice with 2% FACS solution, followed by the addition of 50 μl of Fab antibody (antibody vector with a Strep tag) diluted in 2% FACS solution, and incubated for 1 hour. After washing twice with 1x PBS, 50 μl of secondary antibody (FITC Strep Tag II Antibody, LSBio, Cat. LS-C203631) was added, and the mixture was incubated on ice for 15 minutes. All procedures were performed on ice. Flow cytometry was performed using CytoFLEX LX (Beckman) and MFI was analyzed. Positive controls were mouse serum immunized with SLC6A8 antigen.

[0160] The results are as follows Figure 5 , Figure 6A , Figure 6B Table 12 shows the specific data for the flow cytometry detection of antibody binding to SLC6A7 protein at eight serial dilution points.

[0161] Table 12 MFI values ​​of SLC6A8 The results above show that antibodies SJ25, SJ32, SJ41, SJ42, and SJ43 all have very good antigen-binding activity.

[0162] In addition, the EC50 values ​​of antibody binding to SLC6A8 protein by flow cytometry are shown in Table 13 below.

[0163] Table 13 All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An anti-SLC6A8 antibody or its antigen-binding fragment, characterized in that, The antibody or its antigen-binding fragment has the following three complementarity-determining regions (CDRs) of the heavy chain variable region and three complementarity-determining regions (CDRs) of the light chain variable region: HCDR1, as shown in SEQ ID NO: 55, HCDR2, as shown in SEQ ID NO: 56, HCDR3, as shown in SEQ ID NO: 71, LCDR1 shown in SEQ ID NO: 73, LCDR2 shown in SEQ ID NO: 74 LCDR3 as shown in SEQ ID NO: 75; The amino acid sequence of LCDR2 is YTS.

2. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The heavy chain variable region and light chain variable region of the anti-SLC6A8 antibody or its antigen-binding fragment are: The heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 70, and the light chain variable region with an amino acid sequence as shown in SEQ ID NO:

72.

3. A recombinant protein, characterized in that, The recombinant protein consists of the following: (i) the anti-SLC6A8 antibody of claim 1 or its antigen-binding fragment; and (ii) Tag sequences that assist in expression and / or purification.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-SLC6A8 antibody of claim 1 or its antigen-binding fragment.

5. A carrier, characterized in that, The carrier contains the nucleic acid molecule as described in claim 4.

6. A host cell, characterized in that, The host cell contains the vector of claim 5, or has the nucleic acid molecule of claim 4 integrated into its genome.

7. The use of the antibody or its antigen-binding fragment as described in claim 1 or the recombinant protein as described in claim 3, characterized in that, Used for preparing reagents, detection plates, or kits; The reagents, detection plates, or kits are used to detect SLC6A8 protein in samples.

8. A method for preparing the anti-SLC6A8 antibody or its antigen-binding fragment as described in claim 1, characterized in that, The method includes the following steps: (a) Under expression conditions, host cells as described in claim 6 are cultured to express the anti-SLC6A8 antibody or its antigen-binding fragment; (b) Isolate and purify the anti-SLC6A8 antibody or its antigen-binding fragment described in (a).

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