Ly6g6d-targeted nanobody and use thereof

Abstract

Provided are a Ly6G6D-targeted nanobody and a use thereof. Specifically, provided are an anti-Ly6G6D (lymphocyte antigen 6 complex, locus G6D) nanobody and an antigen-binding fragment. Also provided is a use of the nanobody or the antigen-binding fragment in the preparation of a drug for treating Ly6G6D-positive cancer.

Description

A nanobody targeting Ly6G6D and its application Technical Field

[0001] The present invention belongs to the field of antibody engineering. Specifically, the present invention relates to a single-domain antibody, in particular to a nanobody targeting Ly6G6D and its use in preparing drugs. Background Art

[0002] Nanobodies are the smallest antibody molecules currently available. Originally discovered in camel blood by Belgian scientist Hamers, they have attracted considerable attention as a class of engineered antibody products. Their key advantages include: first, their small size, only one-tenth the size of a typical antibody. This small size allows them to penetrate animal tissue more effectively. For example, they can penetrate human brain tissue and reach high-density tumors, something that typical antibodies cannot achieve. The small size of nanobodies makes them suitable for treating certain tumors or brain diseases. Second, their efficacy is stable, adapting to a wide temperature range and remaining effective at temperatures up to 90°C, whereas traditional antibodies become inactive at such temperatures. They are also highly stable even at extreme pH values ​​and can even pass through the human stomach without losing effectiveness. Third, they possess excellent antigen specificity and are amenable to genetic modification. Nanobodies can recognize unique antigenic epitopes, possessing a broader antigen-binding capacity than typical antibodies, and can be easily engineered to produce antibodies against different pathogens. Nanobodies can be easily synthesized in microorganisms and expressed in large quantities in microorganisms such as bacteriophages, E. coli, and yeast, making them amenable to large-scale production.

[0003] Lymphocyte antigen 6 family member G6D (Ly6G6D) is a leukocyte antigen cluster located in the major histocompatibility complex (MHC) class III region of chromosome 6. It encodes a 133-amino acid protein with a molecular weight of approximately 13.7 kDa. Like most family members, Ly6G6D is attached to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor. Numerous studies have demonstrated that members of the LAG6 family are crucial for regulating immune, nervous, and complement functions. In cancer, many LAG6 family members play a key role in numerous cancer types, including gastric, cervical, breast, ovarian, lung, and bladder cancers. Therefore, the LAG6 gene family has the potential to play a significant role in clinical practice, not only as a biomarker for disease prognosis but also as a key target for new drug development. In-depth understanding of their biological functions will be crucial for elucidating the functions of the LAG6 family, analyzing protein interactions within their structure, exploring disease pathogenesis, and discovering new therapeutic targets. Ly6G6D is a new member of the lymphocyte antigen 6 gene family. Studies have shown that Ly6G6D plays an important role in the development of diseases such as colorectal cancer. Currently, there is a Ly6G6D-based bispecific antibody for the treatment of colorectal cancer, which indicates that Ly6G6D is expected to become a potential target for antibody-based therapy.

[0004] The ScFv, Fab or full IgG anti-Ly6G6D antibody molecules in the existing technology have complex structures and large molecules. Although they can connect active molecules to Ly6G6D, they affect the function of the active molecules, the methods are complex, and the loading efficiency is low. Nanoantibody molecules are smaller, easy to operate, and have stable physical and chemical properties and low immunogenicity. Summary of the Invention

[0005] The present invention provides a Ly6G6D nanobody, which has high affinity to human recombinant Ly6G6D protein and has good application prospects.

[0006] Specifically, on the one hand, the present invention provides a nanobody that specifically binds to Ly6G6D, wherein the nanobody can specifically bind to Ly6G6D, and the heavy chain variable region in the nanobody comprises complementary determining regions: CDR1, CDR2 and CDR3;

[0007] wherein the CDR1 has the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2;

[0008] YYAIG (SEQ ID NO: 1);

[0009] SYX1MX2 (SEQ ID NO: 2); wherein X1 is selected from A or M, and X2 is selected from E or G;

[0010] The CDR2 has the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4;

[0011] CISSSGGSTVYADTVKG(SEQ ID NO:3);

[0012] X3X4SWX5GGX6TYYADSX7KG (SEQ ID NO: 4); wherein X3 is selected from V or A; X4 is selected from I, H or V; X5 is selected from T or Y; X6 is selected from V or S; X7 is selected from V or A;

[0013] The CDR3 has the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7;

[0014] GPECSRWYKDS (SEQ ID NO: 5);

[0015] ESRGFTHYNGNYYSRPDY(SEQ ID NO:6);

[0016] RAPVKGDITPSNYYGLDY (SEQ ID NO:7).

[0017] In some embodiments of the present invention, CDR1 has the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10;

[0018] CDR2 has the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 13;

[0019] CDR3 has the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.

[0020] In some embodiments of the present invention, CDR1 has the amino acid sequence shown in SEQ ID NO: 1; CDR2 has the amino acid sequence shown in SEQ ID NO: 3; and CDR3 has the amino acid sequence shown in SEQ ID NO: 5.

[0021] In some embodiments of the present invention, CDR1 has the amino acid sequence shown in SEQ ID NO: 8; CDR2 has the amino acid sequence shown in SEQ ID NO: 11; and CDR3 has the amino acid sequence shown in SEQ ID NO: 6.

[0022] In some embodiments of the present invention, CDR1 has the amino acid sequence shown in SEQ ID NO: 9; CDR2 has the amino acid sequence shown in SEQ ID NO: 12; and CDR3 has the amino acid sequence shown in SEQ ID NO: 6.

[0023] In some embodiments of the present invention, CDR1 has the amino acid sequence shown in SEQ ID NO: 10; CDR2 has the amino acid sequence shown in SEQ ID NO: 13; and CDR3 has the amino acid sequence shown in SEQ ID NO: 7.

[0024] In some embodiments of the present invention, the heavy chain variable region further comprises framework regions: FR1, FR2, FR3 and FR4; wherein the framework regions and the complementarity determining regions are arranged in a staggered order.

[0025] Specifically, the framework regions and complementarity determining regions are arranged in a staggered manner as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0026] In some embodiments of the present invention, FR1 has the amino acid sequence shown in SEQ ID NO: 14;

[0027] QX8QLVESGGGLVQX9GGSLRLSCAASGX 10 TX 11 X 12 (SEQ ID NO: 14); wherein X8 is selected from V or L; X9 is selected from P or A; X 10 Selected from F or R; X 11 Select from L or F; X 12 Selected from D, E or S;

[0028] FR2 has the amino acid sequence shown in SEQ ID NO: 15;

[0029] WFRQAPGKEREX 13 VX 14 (SEQ ID NO: 15); wherein X 13 Select from G or F; X 14 Select from S or A;

[0030] FR3 has the amino acid sequence shown in SEQ ID NO: 16;

[0031] RFTISRDNX 15 KNX 16 X 17 YQ 18SLKPEDTAVYYCAA (SEQ ID NO: 16); wherein X 15 Select from T or A; X 16 Select from T or A; X 17 Selected from V or L; X 18 Select N or D;

[0032] FR4 has the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO: 18;

[0033] In some embodiments of the present invention, FR1 has the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22;

[0034] FR2 has the amino acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 24;

[0035] FR3 has the amino acid sequence shown in SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27;

[0036] FR4 has the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO: 18.

[0037] In some embodiments of the present invention, FR1 has the amino acid sequence shown in SEQ ID NO: 19; FR2 has the amino acid sequence shown in SEQ ID NO: 23; FR3 has the amino acid sequence shown in SEQ ID NO: 25; and FR4 has the amino acid sequence shown in SEQ ID NO: 17.

[0038] In some embodiments of the present invention, FR1 has the amino acid sequence shown in SEQ ID NO:20; FR2 has the amino acid sequence shown in SEQ ID NO:24; FR3 has the amino acid sequence shown in SEQ ID NO:26; and FR4 has the amino acid sequence shown in SEQ ID NO:17.

[0039] In some embodiments of the present invention, FR1 has the amino acid sequence shown in SEQ ID NO:21; FR2 has the amino acid sequence shown in SEQ ID NO:24; FR3 has the amino acid sequence shown in SEQ ID NO:26; and FR4 has the amino acid sequence shown in SEQ ID NO:17.

[0040] In some embodiments of the present invention, FR1 has the amino acid sequence shown in SEQ ID NO: 22; FR2 has the amino acid sequence shown in SEQ ID NO: 24; FR3 has the amino acid sequence shown in SEQ ID NO: 27; and FR4 has the amino acid sequence shown in SEQ ID NO: 18.

[0041] In some embodiments of the present invention, the present invention provides a Nanobody, the Nanobody heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 28 to 31, or an amino acid sequence that is at least 85%, at least 90%, at least 95% or at least 99% identical to any one of SEQ ID NOs: 28 to 31.

[0042] In some embodiments of the invention, the invention provides a Nanobody, the Nanobody heavy chain variable region having the amino acid sequence shown in any one of SEQ ID NOs: 28 to 31, or an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any one of SEQ ID NOs: 28 to 31.

[0043] In the present invention, the antibody comprising the heavy chain variable region shown in SEQ ID NO: 28 is numbered as 1A4.

[0044] In the present invention, the antibody comprising the heavy chain variable region shown in SEQ ID NO: 29 is numbered as 1D5.

[0045] In the present invention, the antibody comprising the heavy chain variable region shown in SEQ ID NO: 30 is numbered as 1D5G8.

[0046] In the present invention, the antibody comprising the heavy chain variable region shown in SEQ ID NO: 31 is numbered as 2G5.

[0047] In some embodiments of the present invention, the Nanobodies of the present invention are alpaca-derived.

[0048] On the other hand, the present invention provides a fusion protein comprising a functional domain capable of specifically binding to Ly6G6D; wherein the functional domain is composed of the nanobody that specifically binds to Ly6G6D according to the present invention.

[0049] In some embodiments of the present invention, the fusion protein of the present invention further comprises a constant region.

[0050] Specifically, the constant region is the constant region of human IgG or a mutant thereof.

[0051] Specifically, the constant region of human IgG is the constant region of human IgG1 or a mutant thereof.

[0052] In some embodiments of the present invention, the constant region of the IgG1 of the present invention comprises an Fc segment, and the Fc segment comprises the amino acid sequence shown in SEQ ID NO:32.

[0053] In some embodiments of the present invention, the fusion protein of the present invention further comprises a linker peptide.

[0054] Specifically, the fusion protein connecting peptide linker is used to connect the heavy chain variable region and the constant region, wherein the C-terminus of the heavy chain variable region is connected to the N-terminus of the constant region via the connecting peptide linker. An example of the amino acid sequence of the connecting peptide linker is: (GnS)m, where n is a natural number of 3-5, preferably 4, and m is a natural number of 1-5, preferably 3.

[0055] In yet another aspect, the present invention provides an anti-Ly6G6D antibody, which is a traditional antibody or a functional fragment thereof, wherein the heavy chain variable region of the traditional antibody or the functional fragment thereof is composed of the nanobody that specifically binds to Ly6G6D according to the present invention.

[0056] In some embodiments of the present invention, the functional antibody fragment is a Fab, Fab', (Fab')2, Fv, scFv or sdFv structure of a traditional antibody.

[0057] Traditional antibodies are structurally composed of two identical heavy chains and two identical light chains, the light chain having a light chain variable region (VL) and a light chain constant region (CL); the heavy chain having a heavy chain variable region (VH) and a heavy chain constant region (CH1, CH2, CH3 and / or CH4). Under the premise that the present invention discloses the structure of a nanobody capable of specifically binding to Ly6G6D, it is easy for those skilled in the art to think of using the nanobody of the present invention to modify traditional antibodies, for example, applying the CDR region structure of the nanobody of the present invention to a traditional antibody to obtain a traditional antibody that can specifically bind to Ly6G6D. Such traditional antibodies also fall within the scope of protection of the present invention. Further, based on the structure of traditional antibodies, some of their structures, such as Fab, Fab', (Fab')2, Fv, scFv or sdFv structures, also have Ly6G6D binding specificity, which also falls within the scope of protection of the present invention.

[0058] Among them, Fab is composed of a complete light chain, a heavy chain variable region domain (VH) and the first constant domain (CH1) of a heavy chain, and Fab can be a product produced by papain digestion of an antibody; Fab' differs from Fab in that the Fab' fragment has some residues added to the carboxyl terminus of the CH1 domain, and these residues contain one or more missing cysteines from the antibody hinge region; (Fab')2 is equivalent to two Fab' fragments connected by disulfide bonds with divalent antigen-binding activity and still capable of cross-linking antigens, and (Fab')2 can be a product of pepsin cleavage of the antibody; Fv is composed of a tight, non-covalently associated dimer of a heavy chain variable region domain and a light chain variable region domain; scFv (single-chain antibody) comprises an antibody fragment comprising the VH and VL antibody domains connected to a single polypeptide chain, preferably, the scFv further comprises a polypeptide linker between the VH and VL domains so that the scFv forms the desired antigen-binding structure; sdFv refers to an Fv fragment connected by a disulfide bond.

[0059] In yet another aspect, the present invention provides a bispecific or multispecific antibody comprising a Nanobody that specifically binds to Ly6G6D as described herein. To produce such bispecific or multispecific antibodies, the Nanobody described herein can be linked (e.g., by chemical coupling, genetic fusion, non-covalent association or other means) to one or more other binding molecules (e.g., additional antibodies, antibody fragments, peptides or binding mimetics).

[0060] In some embodiments, the bispecific or multispecific antibody specifically binds Ly6G6D and additionally specifically binds one or more other targets.

[0061] In some embodiments, the bispecific or multispecific antibody further comprises at least one second antibody with a second binding specificity for a second target.

[0062] In yet another aspect, the present invention also provides a conjugate comprising the Nanobody or bispecific or multispecific antibody that specifically binds to Ly6G6D as described in the present invention and a coupling portion.

[0063] In some embodiments, the Nanobody that specifically binds to Ly6G6D described in the present invention is optionally conjugated to the coupling portion via a linker.

[0064] In some embodiments, the coupling moiety is selected from a protein tag. Such protein tags are well known in the art, and examples thereof include but are not limited to His, Flag, GST, MBP, HA, Myc, GFP, or biotin, and those skilled in the art know how to select an appropriate protein tag according to the desired purpose (e.g., purification, detection, or tracing).

[0065] In some embodiments, the C-terminus of the Nanobody that specifically binds to Ly6G6D of the present invention is connected to a His tag (eg, 6×His).

[0066] In some embodiments, the coupling moiety is selected from a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin. The detectable label of the present invention can be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical, or chemical means. Such labels are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3 H. 125 I. 35 S. 14 C or 32 P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent substances such as acridinium ester compounds), magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above labels.

[0067] In some embodiments, such labels can be suitable for use in immunological detection (eg, enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.).

[0068] In some embodiments, the detectable labels described above can be linked to the Nanobodies or antigen-binding fragments thereof of the invention via linkers of varying lengths to reduce potential steric hindrance.

[0069] In some embodiments, the conjugated moiety is selected from a therapeutic agent, such as an anti-inflammatory drug or an immunosuppressant.

[0070] In some embodiments, the conjugation moiety is selected from additional biologically active polypeptides.

[0071] In yet another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the Nanobody of the invention, the antibody of the invention or the fusion protein of the invention.

[0072] In some embodiments of the present invention, the nucleic acid molecule is DNA.

[0073] In some embodiments of the present invention, the present invention also provides a nucleic acid construct, which comprises the nucleic acid molecule described in the present invention; further, the nucleic acid construct comprises one or more control sequences operably linked to the nucleic acid molecule, wherein the one or more control sequences direct the production of the antibody or antigen-binding fragment thereof described in the present invention in an appropriate host cell, and the one or more control sequences are selected from: a promoter, an enhancer, a stop signal, a signal peptide, a leader sequence, a transcription terminator, and any group thereof.

[0074] In yet another aspect, the present invention provides an expression vector carrying the nucleic acid molecule of the present invention.

[0075] In some embodiments of the present invention, the expression vector of the present invention is selected from the group consisting of: plasmid, cosmid, virus, minichromosome and artificial chromosome.

[0076] In some embodiments of the present invention, the expression vector is a eukaryotic expression vector.

[0077] In some embodiments of the present invention, the eukaryotic expression vector is selected from the group consisting of: pCRII, pCR3, pcDNA3.4, pBSII, pET15, pGEX, pEGFP-N1, pETL, pDSR-α, and pFastBacDual.

[0078] In yet another aspect, the present invention provides a host cell comprising the nucleic acid molecule of the present invention or the expression vector of the present invention.

[0079] In some embodiments of the present invention, the host cell is Escherichia coli, yeast or a eukaryotic cell.

[0080] In some embodiments of the present invention, the host cell is a eukaryotic host cell.

[0081] In some embodiments of the present invention, the host cell is a mammalian host cell.

[0082] In yet another aspect, the present invention provides a pharmaceutical composition comprising the Nanobody described in the present invention, the antibody described in the present invention, the fusion protein described in the present invention, the nucleic acid molecule described in the present invention, the expression vector described in the present invention, or the host cell described in the present invention.

[0083] In some embodiments of the present invention, the pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier.

[0084] The pharmaceutical composition of the present invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of the Nanobody of the present invention, the antibody of the present invention, the fusion protein of the present invention, the nucleic acid molecule of the present invention, the expression vector of the present invention or the host cell of the present invention.

[0085] A "prophylactically effective amount" is an amount sufficient to prevent, arrest, or delay the onset of a disease. A "therapeutically effective amount" is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount may vary depending on factors such as the severity of the disease being treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and other concurrently administered therapies.

[0086] The pharmaceutical composition of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended route of administration and therapeutic use.

[0087] A preferred dosage form is an injection. Such injections can be sterile injection solutions. For example, sterile injection solutions can be prepared by the following method: incorporating the necessary dose of the antibody of the present invention or its antigen-binding fragment in an appropriate solvent, and optionally, simultaneously incorporating other desired ingredients (including but not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration sterilization. In addition, sterile injection solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze drying) for storage and use.

[0088] Nanobodies according to the invention, antibodies according to the invention, fusion proteins according to the invention, nucleic acid molecules according to the invention, expression vectors according to the invention, host cells according to the invention, or compositions according to the invention can be administered by any suitable method known in the art, including but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (e.g., powders, ointments, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / mode of administration is parenteral administration (e.g., intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). It will be understood by the skilled artisan that the route and / or mode of administration will vary depending on the intended purpose.

[0089] In some embodiments, the Nanobodies of the invention, the antibodies of the invention, the fusion proteins of the invention, the nucleic acid molecules of the invention, the expression vectors of the invention, the host cells of the invention or the compositions of the invention are administered by intravenous injection or bolus injection.

[0090] On the other hand, the present invention provides the use of the Nanobody of the present invention, the antibody of the present invention, the fusion protein of the present invention, the nucleic acid molecule of the present invention, the expression vector of the present invention, the host cell of the present invention or the pharmaceutical composition of the present invention in the preparation of a drug for treating or alleviating Ly6G6D-positive cancer in a subject in need thereof.

[0091] In some embodiments of the present invention, the Ly6G6D-positive cancer is colorectal cancer, head and neck cancer, ovarian cancer, cervical cancer, melanoma, gastric cancer, esophageal cancer, small intestine cancer, large intestine cancer or adenocarcinoma, etc.

[0092] In yet another aspect, the present invention provides a kit for detecting Ly6G6D, the kit comprising the Nanobody of the present invention, the antibody of the present invention or the fusion protein of the present invention and instructions for use.

[0093] In yet another aspect, the invention provides a method for detecting the presence or amount of Ly6G6D in a sample, comprising the use of the Nanobody of the invention.

[0094] In some embodiments, the method is an immunological assay, such as immunoblotting, enzyme immunoassay (eg, ELISA), chemiluminescent immunoassay, fluorescent immunoassay, or radioimmunoassay.

[0095] In some embodiments, the conjugate for use in the methods comprises a Nanobody of the invention and a detectable label.

[0096] In some embodiments, the Nanobodies used in the methods are detectably labeled.

[0097] In some embodiments, the Nanobodies used in the methods do not carry a detectable label. Thus, the methods may further comprise detecting the Nanobodies of the invention using other reagents (such as a second antibody) that carry a detectable label.

[0098] In some embodiments, the method comprises the following steps:

[0099] (1) contacting the sample with the Nanobody of the present invention;

[0100] (2) Detecting the formation of a complex between the Nanobody and Ly6G6D or detecting the amount of the complex.

[0101] The formation of the complex indicates the presence of Ly6G6D or cells expressing Ly6G6D.

[0102] The methods may be used for diagnostic purposes, or for non-diagnostic purposes (eg, the sample is a cell sample rather than a sample from a patient).

[0103] In some embodiments, the method is used to diagnose whether a subject suffers from a disease associated with Ly6G6D. In such embodiments, the method may further comprise a step of comparing the amount of Ly6G6D in a sample from the subject with a reference value. The reference value may be the level of Ly6G6D in a sample from a subject (e.g., a healthy control) known not to have a disease associated with Ly6G6D (also referred to as a "negative reference value"). For example, if the amount of Ly6G6D in a sample from the subject increases relative to a negative reference value, it indicates that the subject suffers from a disease associated with Ly6G6D.

[0104] In some embodiments, the sample can be selected from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue associated cells (i.e., free cells), tissue (e.g., surgically resected tumor tissue, biopsy tissue, or fine needle aspirate tissue), histological preparations, etc.

[0105] Definition of terms

[0106] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, procedures in cell culture, molecular biology, biochemistry, nucleic acid chemistry, immunology, and the like used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0107] When the present invention uses the terms "for example", "such as", "including", "comprising" or their variations, these terms will not be considered as limiting terms, but will be interpreted as meaning "but not limited to" or "not limited to".

[0108] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0109] As used herein, the term "camel-derived antibody" refers to antibodies produced by Camelidae animals (including camels, alpacas, and llamas) against an antigen following immunization or antigen invasion. It is known to those skilled in the art that camelid antibodies contain light chain-deficient "heavy chain antibodies" (HCAbs). These antibodies consist of only a single heavy chain variable domain (VHH) and two conventional CH2 and CH3 regions. The separately cloned and expressed VHH region exhibits excellent structural stability and antigen-binding activity. VHH is the smallest unit currently known to bind to a target antigen.

[0110] As used in the present invention, the term "nanobody" has the meaning generally understood by those skilled in the art and refers to an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region), typically derived from the variable region of a heavy chain antibody (e.g., a camelid antibody or a shark antibody). Typically, a nanobody consists of four framework regions and three complementarity determining regions, with a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. A nanobody may be truncated at the N-terminus or C-terminus so that it contains only part of FR1 and / or FR4, or lacks one or two of those framework regions, as long as it substantially maintains antigen binding and specificity. Nanobodies are also referred to as single domain antibodies (sdAbs), and the two are used interchangeably.

[0111] As used in the present invention, the term "antigen-binding fragment" of a Nanobody refers to a polypeptide comprising a fragment of a Nanobody that retains the ability to specifically bind to the same antigen bound by the Nanobody and / or competes with the Nanobody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of the antibodies of the invention can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the Nanobodies of the invention. In some embodiments, the "antigen-binding fragment" of such a Nanobody may be truncated at the N-terminus or C-terminus compared to the full-length Nanobody so that it only comprises part of FR1 and / or FR4, or lacks one or both of those framework regions, as long as it substantially retains antigen binding and specificity.

[0112] Antigen-binding fragments of Nanobodies can be obtained from a given Nanobody (such as the Nanobodies provided herein) using conventional techniques known to those skilled in the art (for example, recombinant DNA techniques or enzymatic or chemical cleavage methods), and screened for specificity in the same manner as for intact Nanobodies.

[0113] In this document, unless the context clearly indicates otherwise, when referring to the term "Nanobody" it includes not only intact Nanobodies but also antigen-binding fragments of Nanobodies.

[0114] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. In nanobodies, there are three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, for example, according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given Nanobody, a person skilled in the art will easily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (for example, see Lefrance et al., Dev. Comparat. Immunol. 27: 55-77, 2003).

[0115] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0116] As used in the present invention, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its targeted antigen. The strength or affinity of a specific binding interaction can be expressed as the equilibrium dissociation constant (KD) of the interaction. In the present invention, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0117] The specific binding properties between two molecules can be measured using methods well known in the art. One method involves measuring the speed of formation and dissociation of antigen binding sites / antigen complexes. Both "association rate constant" (ka or kon) and "dissociation rate constant" (kdis or koff) can be calculated by concentration and the actual rate of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of kdis / kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473). KD, kon and kdis values ​​can be measured by any effective method. In certain embodiments, the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR). In addition, the dissociation constant can also be measured using bioluminescence interferometry or Kinexa.

[0118] The term "amino acid" refers to compounds containing both amino and carboxyl functional groups, such as α-amino acids. Two or more amino acids can form polypeptides via amide bonds (also known as peptide bonds). Individual amino acids are encoded by nucleic acids consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called "degeneracy of the genetic code." Amino acids include natural amino acids and unnatural amino acids. Natural amino acids include alanine (three-letter code: Ala, one-letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).

[0119] As used herein, the term "expression vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. A vector is called an expression vector when it can express the protein encoded by the inserted polynucleotide. A vector can be introduced into a host cell via transformation, transduction, or transfection, so that the genetic material it carries is expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0120] As used in the present invention, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. The nucleic acid molecule can be single-stranded or double-stranded and can be a cDNA.

[0121] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells. A host cell may include a single cell or a cell population.

[0122] As used herein, the term "identity" refers to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. Typically, two sequences are compared when they are aligned for maximum identity. Such an alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0123] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH regulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, carbohydrates (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In some exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0124] As used in the present invention, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or condition or symptom (e.g., a disease associated with Ly6G6D) in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or completely), whether detectable or undetectable. In addition, "treatment" can also refer to extending survival compared to the expected survival (if not receiving treatment).

[0125] As used in the present invention, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In some embodiments, the subject (eg, human) suffers from a disease associated with Ly6G6D.

[0126] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only intended to illustrate the present invention and are not intended to limit the scope of the invention. Based on the following detailed description of the drawings and preferred embodiments, the various objects and advantages of the present invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0127] FIG1 is a diagram showing the results of confirming the binding of antibodies to hLY6G6D antigen by ELISA;

[0128] FIG2 is a diagram showing the results of confirming the binding of the antibody to the cynoLY6G6D antigen by ELISA;

[0129] FIG3 is a graph showing the binding results of HEK23T cells highly expressing hLY6G6D and antibodies detected by flow cytometry;

[0130] FIG4 is a graph showing the results of flow cytometry detection of the binding between HEK23T cells that highly express cynoLY6G6D and antibodies;

[0131] FIG5 is a graph showing the results of evaluating in vitro ADCC activity using the Jurkat-NFAT-Luc2-CD16 cell model. DETAILED DESCRIPTION

[0132] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0133] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., Molecular Biology: A Compendium of Laboratory Manuals, 3rd edition, John Wiley & Sons, Inc., 1995. It will be appreciated by those skilled in the art that the examples are provided to illustrate the present invention and are not intended to limit the scope of the invention.

[0134] Example 1: Phage display technology and flow cytometry screening of LY6G6D nanobodies

[0135] By displaying the nanoantibody VH format on phage, panning was performed using phage library screening technology, and ELISA was used to screen for monoclonal antibodies that highly bind to HumanLY6G6D at the molecular level. The genes encoding the extracellular regions of the HumanLY6G6D and cynoLY6G6D proteins were designed using the Uniprot and NCBI databases, and constructed into a mammalian eukaryotic expression system to express the extracellular regions of the HumanLY6G6D and cynoLY6G6D proteins for screening of the immune nanoantibody library. The expressed extracellular regions of the HumanLY6G6D protein were used to immunize alpacas six times, and the titer reached 10 4 PBMC were isolated from alpaca blood, and the VH sequence of the nanobody was amplified from it. An immune nanobody phage library was established, and the immune nanobody phage library was panned with HumanLY6G6D protein. After three rounds of panning, the ELSIA method was used to screen monoclonal phages that could bind to HumanLY6G6D. The binding results of the four candidate phages to HumanLY6G6D are shown in Table 1 below:

[0136] Table 1

[0137] Example 2: Expression and purification of antibodies

[0138] The four candidate phages obtained in Example 1 were sequenced to obtain sequences encoding heavy chain variable regions of candidate antibodies. The heavy chain IgG antibody (VH-Linker-Fc, wherein VH has an amino acid sequence as shown in any one of SEQ ID NOs: 28 to 31, Fc has an amino acid sequence as shown in SEQ ID NO: 32, and Linker has an amino acid sequence as shown in SEQ ID NO: 33) gene coding sequences were synthesized using whole gene synthesis. A series of antibody expression vectors were constructed using molecular cloning methods and recombinant expression was performed in the 293F expression system. After the 293F host cells were revived and cultured with Aupromycin medium, the cells were cultured at a density of approximately 3*10 6When the cell count reached 100 cells / mL, cells were harvested and transfected with PEI reagent. On the second day of culture, feed was added and glucose was added to the culture concentration to 8 g / L. On the sixth day of culture, the cell culture medium was harvested.

[0139] A series of candidate antibodies were tested at the translational level. The collected cell culture fluid was purified using a Protein A column, and the absorption peaks were collected for mass spectrometry analysis. Mass spectrometry analysis of a series of candidate antibodies revealed a molecular weight of 75 kDa for the candidate antibody VHH-Fc, consistent with the theoretical molecular weight, indicating a dimer. The collected samples were also analyzed by 10% SDS-PAGE electrophoresis after both reduction and non-reduction conditions. The reduced SDS-PAGE electrophoresis pattern of the candidate antibody VH-Fc showed a band around 37 kDa, while the non-reduced SDS-PAGE electrophoresis pattern revealed a single band around 75 kDa, consistent with the theoretical band size. Candidate antibody VHH-Fc with a smaller size exhibited improved tumor penetration. The purified samples were dialyzed overnight at 4°C using 0.02 M PBS buffer (pH 7.4).

[0140] Example 3: Detection of binding properties between antibodies and antigens

[0141] Binding of the expressed antibodies to the hLY6G6D antigen was confirmed by ELISA. The positive control antibody 20A12 was obtained from patent CN202080086360.1. ELISA plates were coated with 100 ng / well of hLY6G6D and placed in a 37°C incubator for 2 hours. Nonspecific binding sites were then blocked with 5% skim milk powder overnight at 4°C. The plates were incubated with various concentrations of VHHs for 1 hour, washed three times with 0.1% PBST, and bound VHHs were detected with HRP-mouse anti-human IgG1Fc (SouthernBiotech). The plates were then washed five times with 0.1% PBST, and TMB substrate (Yunqiao Biotechnology) was added. The reaction was terminated after color development and read at 450 nm. The results are shown in Figure 1 and Table 2. The candidate antibodies had comparable affinities to the positive antibody 20A12, with 1A4 and 2G5 having even better affinities than the positive antibody 20A12. The heavy chain of the positive antibody 20A12 comprised the amino acid sequence set forth in SEQ ID NO:34, and the light chain comprised the amino acid sequence set forth in SEQ ID NO:35.

[0142] Table 2

[0143] Example 4: Detection of binding properties between antibodies and antigens

[0144] Cross-reactivity with monkeys: Binding of the expressed antibodies to the cynoLY6G6D antigen was confirmed by ELISA. The positive control antibody 20A12 was obtained from patent CN202080086360.1. ELISA plates were coated with 100 ng / well of cynoLY6G6D and placed in a 37°C incubator for 2 hours. Nonspecific binding sites were then blocked with 5% skim milk powder overnight at 4°C. The plates were incubated with various concentrations of VHHs for 1 hour, washed three times with 0.1% PBST, and bound VHHs were detected with HRP-mouse anti-human IgG1Fc (SouthernBiotech). The plates were then washed five times with 0.1% PBST, and TMB substrate (Yunqiao Bio) was added. The reaction was terminated after color development and read at 450 nm. The results, as shown in Figure 2 and Table 3, show that both candidate and positive antibodies bound to cynoLY6G6D.

[0145] Table 3

[0146] Example 5: Detection of binding properties between antibodies and antigens

[0147] Flow cytometry was used to detect the binding of HEK23T cells with high expression of hLY6G6D to the antibody. The positive control antibody 20A12 was obtained from patent CN202080086360.1. 5 The cells were resuspended in a 96-well plate and incubated with different concentrations of VHH for 1 hour. The cells were then washed twice in PBS. PE-Goat anti-human IgG Fc (Jackson ImmunoResearch) was diluted 1:500 and incubated at 4°C for 30 minutes to detect bound antibodies. The cells were then washed twice in PBS and analyzed on FACS (Beckman). The results are shown in Figure 3 and Table 4. The affinity of the candidate antibodies was comparable to that of the positive antibody 20A12.

[0148] Table 4

[0149] Example 6: Detection of binding properties between antibodies and antigens

[0150] Cross-reaction detection with monkeys: Flow cytometry was used to detect the binding of HEK23T cells that highly expressed cynoLY6G6D to the antibody. The positive control antibody 20A12 was derived from patent CN202080086360.1. 5The cells were resuspended in a 96-well plate and incubated with different concentrations of VHH for 1 hour. The cells were then washed twice in PBS. PE-Goat anti-human IgG Fc (Jackson ImmunoResearch) was diluted 1:500 and incubated at 4°C for 30 minutes to detect bound antibodies. The cells were then washed twice in PBS and analyzed on a FACS (Beckman). The results are shown in Figure 4 and Table 5. Both the candidate antibodies and the positive antibodies bound to cynoLY6G6D.

[0151] Table 5

[0152] Example 7: Evaluation of in vitro ADCC activity using the Jurkat-NFAT-Luc2-CD16 cell model

[0153] A Jurkat-NFAT-Luc2-CD16 reporter gene cell model was constructed using Jurkat cells stably transfected with FcγRIIIa receptor and NFAT (nuclear factor of activated T cells) to evaluate the ADCC activity of candidate molecules.

[0154] hLY6G6D-HEK293T cells were constructed as target cells, and Jurkat-NFAT-Luc2-CD16 cells were used as effector cells. Both effector and target cells were plated in 384-well plates, with 5,000 cells per well. The positive control antibody, 20A12, was derived from patent CN202080086360.1. Different concentrations of antibody dilutions were added to the 384-well plates, and the effector and target cells were incubated in a 37°C, 5% CO2 incubator for 14 hours. The antibody specifically binds to hLY6G6D on the target cell surface, and the Fc terminus of the antibody binds to the FcγRIIIa receptor on the effector cell surface, forming a target cell-antibody-effector cell complex. This activates the NFAT-Luc signaling pathway within the effector cells, leading to luciferase production. Luciferase detection substrate was added, and the reaction was allowed to proceed for 3 minutes. Luminescence readings for each well were obtained using a multi-functional microplate reader. The results, as shown in Figure 5 and Table 6, show that the candidate antibodies exhibited comparable ADCC activity to the positive antibody, 20A12.

[0155] Table 6

[0156] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. A nanobody that specifically binds to Ly6G6D, characterized in that, The nanobody can specifically bind to Ly6G6D, and the heavy chain variable region in the nanobody contains complementary determining regions: CDR1, CDR2, and CDR3; Among them, the CDR1 has the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2; YYAIG (SEQ ID NO:1); SYX1MX2 (SEQ ID NO:2); where X1 is selected from A or M, and X2 is selected from E or G; The CDR2 has the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:4; CISSSGGSTVYADTVKG (SEQ ID NO:3); X3X4SWX5GGX6TYYADSX7KG (SEQ ID NO:4); where X3 is selected from V or A; X4 is selected from I, H, or V; X5 is selected from T or Y; X6 is selected from V or S; X7 is selected from V or A; The CDR3 has the amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7; GPECSRWYKDS (SEQ ID NO:5); ESRGFTHYNGNYYSRPDY (SEQ ID NO:6); RAPVKGDITPSNYYGLDY (SEQ ID NO:7).

2. The nanobody according to claim 1, wherein The CDR1 has the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10; CDR2 has the amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13; CDR3 has the amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:

7.

3. The nanobody according to claim 1 or 2, characterized in that, The heavy chain variable region also contains framework regions: FR1, FR2, FR3, and FR4; among them, the framework regions and the complementary determining regions are arranged alternately in sequence.

4. The nanobody according to claim 3, characterized in that, Among them, FR1 has the amino acid sequence shown in SEQ ID NO:14; QX8QLVESGGGLVQX9GGSLRLSCAASGX 10 TX 11 X 12 (SEQ ID NO:14); Among them, X8 is selected from V or L; X9 is selected from P or A; X 10 is selected from F or R; X 11 is selected from L or F; X 12 is selected from D, E or S; FR2 has the amino acid sequence shown in SEQ ID NO:15; WFRQAPGKEREX 13 VX 14 (SEQ ID NO:15); wherein, X 13 is selected from G or F; X 14 is selected from S or A; FR3 has the amino acid sequence shown in SEQ ID NO:16; RFTISRDNX 15 KNX 16 X 17 YLQMX 18 SLKPEDTAVYYCAA(SEQ ID NO:16); wherein, X 15 is selected from T or A; X 16 is selected from T or A; X 17 is selected from V or L; X 18 is selected from N or D; FR4 has the amino acid sequence shown in SEQ ID NO:17 or SEQ ID NO:18; WGQGTQVTVSS (SEQ ID NO:17); SGKGTLVIVSS (SEQ ID NO:18).

5. The nanobody according to claim 4, wherein The FR1 has the amino acid sequence shown in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22; FR2 has the amino acid sequence shown in SEQ ID NO:23 or SEQ ID NO:24; FR3 has an amino acid sequence shown in SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27; FR4 has an amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO:

18.

6. The nanobody according to any one of claims 1-5, characterized in that, The variable region of the nanobody heavy chain has an amino acid sequence shown in any one of SEQ ID NO: 28 - 31 or an amino acid sequence having at least 85%, at least 90%, at least 95% or at least 99% identity with any one of SEQ ID NO: 28 - 31.

7. An anti-Ly6G6D antibody, characterized in that, The antibody is a conventional antibody or a functional fragment thereof, and the variable region of the heavy chain of the conventional antibody or its functional fragment is composed of a nanobody that specifically binds to Ly6G6D as described in any one of claims 1 - 6.

8. The anti-Ly6G6D antibody according to claim 7, wherein The functional fragment of the antibody is in the structure of Fab, Fab’, (Fab’)2, Fv, scFv or sdFv of a conventional antibody.

9. A fusion protein, characterized in that, The fusion protein contains a functional domain that can specifically bind to Ly6G6D; wherein, the functional domain is composed of a nanobody that specifically binds to Ly6G6D as described in any one of claims 1 - 6.

10. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule contains a nucleotide sequence encoding the nanobody as described in any one of claims 1 - 6, the antibody as described in claim 7 or 8, or the fusion protein as described in claim 9; Preferably, the nucleic acid molecule is DNA.

11. An expression vector, characterized in that, The expression vector carries the nucleic acid molecule as described in claim 10; Preferably, the expression vector is a eukaryotic expression vector.

12. A host cell, characterized in that, The host cell contains the nucleic acid molecule as described in claim 10 or the expression vector as described in claim 11; Preferably, the host cell is a eukaryotic host cell; More preferably, the host cell is a mammalian host cell.

13. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the nanobody as described in any one of claims 1 - 6, the antibody as described in claim 7 or 8, the fusion protein as described in claim 9, the nucleic acid molecule as described in claim 10, the expression vector as described in claim 11 or the host cell as described in claim 12; Preferably, the pharmaceutical composition further contains a pharmaceutically acceptable carrier.

14. Use of the nanobody as described in any one of claims 1 - 6, the antibody as described in claim 7 or 8, the fusion protein as described in claim 9, the nucleic acid molecule as described in claim 10, the expression vector as described in claim 11, the host cell as described in claim 12 or the composition as described in claim 13 in the preparation of a drug for treating or alleviating Ly6G6D - positive cancer in a subject in need thereof.

15. The use according to claim 14, characterized in that, The Ly6G6D - positive cancer is colorectal cancer, head and neck cancer, ovarian cancer, cervical cancer, melanoma, gastric cancer, esophageal cancer, small intestine cancer, large intestine cancer, adenocarcinoma, etc.

16. A kit for detecting Ly6G6D, characterized in that, The kit contains the nanobody as described in any one of claims 1 - 6, the antibody as described in claim 7 or 8, the fusion protein as described in claim 9 and an instruction manual.

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