Nanometer antibody targeting Ly6G6D and application thereof

Abstract

The invention provides a nano antibody targeting Ly6G6D and application of the nano antibody, particularly provides a nano antibody resisting Ly6G6D (a lymphocyte antigen 6 compound and a gene locus G61) and an antigen binding fragment, and further provides application of the nano antibody or the antigen binding fragment in preparation of drugs for treating Ly6G6D positive cancers.

Description

Technical Field

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

[0002] Nanobodies are currently the smallest antibody molecules, initially discovered by Belgian scientist Hamers in camel blood, and they are a class of highly concerned engineered antibody products. The main advantages of nanobodies are as follows: First, their volume is small, only one-tenth of that of ordinary antibodies. Due to their small volume, they have stronger penetration in animal tissues. For example, they can pass through the human brain tissue and reach the inside of tumors with high density, which ordinary antibodies cannot do. The small volume advantage of nanobodies allows people to use them to treat certain tumors or brain diseases. Second, their efficacy is stable. They have a wide temperature adaptation range and can still function at temperatures as high as 90 °C, while traditional antibodies will be inactivated under such conditions. Even under extreme pH values, they still perform very stably and can even pass through the human stomach while maintaining effectiveness. Third, their antigen specificity is good and they are easy to genetically modify. Nanobodies can recognize unique antigenic structural epitopes, have a wider antigen-binding ability than ordinary antibodies, and are convenient for artificial modification to obtain antibodies against different pathogens. Nanobodies can be easily synthesized in microorganisms and can be highly expressed in microorganisms such as phages, Escherichia coli, and yeasts, making them easy to produce on a large scale.

[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, encoding a protein of 133 amino acid residues with a molecular weight of approximately 13.7 kDa. Like most family members, Ly6G6D is attached to the cell membrane by glycosylphosphatidylinositol (GPI) anchoring. Multiple studies have shown that lymphocyte antigen 6 family members are very important for regulating the functions of the immune system, nervous system, and complement. Especially in cancer, numerous lymphocyte antigen 6 family members play key roles in many types of cancers, such as gastric cancer, cervical cancer, breast cancer, ovarian cancer, lung cancer, and bladder cancer. Therefore, the lymphocyte antigen 6 gene family may play an important role in clinical practice, not only as a marker for disease prognosis but also as an important target for developing new drugs. In-depth study of their biological functions will be of great significance for elucidating the functions of the lymphocyte antigen 6 family, analyzing protein-protein interactions in their structures, exploring disease pathogenesis, and discovering new therapeutic targets. As a new member of the lymphocyte antigen 6 gene family, Ly6G6D has been successively reported to play an important role in the occurrence of diseases such as colorectal cancer. Currently, a bispecific antibody based on Ly6G6D is used for the treatment of colorectal cancer, indicating that Ly6G6D has the potential to become a promising target for antibody-based therapy.

[0004] In the prior art, ScFv, Fab, or full IgG class anti-Ly6G6D antibody molecules have complex structures and large molecular sizes. Although they can connect active molecules to Ly6G6D, they affect the functions of active molecules, have complex methods, and low loading efficiencies; nanobody molecules are smaller, easier to operate, and have stable physicochemical properties and low immunogenicity. Summary of the Invention

[0005] The present invention provides a Ly6G6D nanobody, which has a high affinity for 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. The nanobody can specifically bind to Ly6G6D, and the heavy chain variable region in the nanobody contains complementary determining regions: CDR1, CDR2, and CDR3;

[0007] Among them, 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, 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;

[0018] The 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] The 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, the CDR1 has the amino acid sequence shown in SEQ ID NO: 1; the CDR2 has the amino acid sequence shown in SEQ ID NO: 3; the CDR3 has the amino acid sequence shown in SEQ ID NO: 5.

[0021] In some embodiments of the present invention, the CDR1 has the amino acid sequence shown in SEQ ID NO: 8; the CDR2 has the amino acid sequence shown in SEQ ID NO: 11; the 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; 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; CDR3 has the amino acid sequence shown in SEQ ID NO:7.

[0024] YYAIG (SEQ ID NO:1).

[0025] SYAME (SEQ ID NO:8).

[0026] SYMME (SEQ ID NO:9).

[0027] SYAMG (SEQ ID NO:10).

[0028] CISSSGGSTVYADTVKG (SEQ ID NO:3).

[0029] VISWTGGSTYYADSVKG (SEQ ID NO:11).

[0030] VHSWTGGVTYYADSVKG (SEQ ID NO:12).

[0031] AVSWYGGSTYYADSAKG (SEQ ID NO:13).

[0032] GPECSRWYKDS (SEQ ID NO:5).

[0033] ESRGFTHYNGNYYSRPDY (SEQ ID NO:6).

[0034] RAPVKGDITPSNYYGLDY (SEQ ID NO:7).

[0035] 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 alternately in sequence.

[0036] Specifically, the alternate arrangement of the framework regions and the complementarity determining regions is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0037] In some embodiments of the present invention, FR1 has the amino acid sequence shown in SEQ ID NO: 14;

[0038] 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 is selected from F or R; X 11 is selected from L or F; X 12 is selected from D, E or S;

[0039] FR2 has the amino acid sequence shown in SEQ ID NO: 15;

[0040] 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;

[0041] FR3 has the amino acid sequence shown in SEQ ID NO: 16;

[0042] 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;

[0043] FR4 has the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO: 18;

[0044] WGQGTQVTVSS(SEQ ID NO: 17);

[0045] SGKGTLVIVSS(SEQ ID NO: 18).

[0046] 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;

[0047] FR2 has the amino acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 24;

[0048] FR3 has the amino acid sequence shown in SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27;

[0049] FR4 has the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO: 18.

[0050] 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; FR4 has the amino acid sequence shown in SEQ ID NO: 17.

[0051] 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; FR4 has the amino acid sequence shown in SEQ ID NO: 17.

[0052] 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; FR4 has the amino acid sequence shown in SEQ ID NO: 17.

[0053] 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; FR4 has the amino acid sequence shown in SEQ ID NO: 18.

[0054] QLQLVESGGGLVQPGGSLRLSCAASGFTLD (SEQ ID NO: 19).

[0055] QVQLVESGGGLVQAGGSLRLSCAASGRTFS (SEQ ID NO: 20).

[0056] QVQLVESGGGLVQAGGSLRLSCAASGRTFE (SEQ ID NO: 21).

[0057] QLQLVESGGGLVQAGGSLRLSCAASGRTFS (SEQ ID NO:22).

[0058] WFRQAPGKEREGVS (SEQ ID NO:23).

[0059] WFRQAPGKEREFVA (SEQ ID NO:24).

[0060] RFTISRDNTKNTVYLQMDSLKPEDTAVYYCAA (SEQ ID NO:25).

[0061] RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA (SEQ ID NO:26).

[0062] RFTISRDNAKNALYLQMDSLKPEDTAVYYCAA (SEQ ID NO:27).

[0063] In some embodiments of the present invention, the present invention provides a nanobody, and the variable region of the heavy chain of the nanobody has the 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.

[0064] In some embodiments of the present invention, the present invention provides a nanobody, and the variable region of the heavy chain of the nanobody has the amino acid sequence shown in any one of SEQ ID NO:28 - 31 or an amino acid sequence having 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% identity with any one of SEQ ID NO:28 - 31.

[0065] QLQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSGGSTV YADTVKGRFTISRDNTKNTVYLQMDSLKPEDTAVYYCAAGPECSRWYKDSWGQGTQVTV SS (SEQ ID NO:28).

[0066] In the present invention, the antibody containing the variable region of the heavy chain shown in SEQ ID NO:28 is numbered: 1A4.

[0067] QVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMEWFRQAPGKEREFVAVISWTGGST YYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAESRGFTHYNGNYYSRPDYW GQGTQVTVSS (SEQ ID NO:29).

[0068] In the present invention, the antibody containing the heavy chain variable region shown in SEQ ID NO:29 is numbered: 1D5.

[0069] QVQLVESGGGLVQAGGSLRLSCAASGRTFESYMMEWFRQAPGKEREFVAVHSWTGGV TYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAESRGFTHYNGNYYSRPDY WGQGTQVTVSS (SEQ ID NO:30).

[0070] In the present invention, the antibody containing the heavy chain variable region shown in SEQ ID NO:30 is numbered: 1D5G8.

[0071] QLQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAVSWYGGST YYADSAKGRFTISRDNAKNALYLQMDSLKPEDTAVYYCAARAPVKGDITPSNYYGLDYSG KGTLVIVSS (SEQ ID NO:31).

[0072] In the present invention, the antibody containing the heavy chain variable region shown in SEQ ID NO:31 is numbered: 2G5.

[0073] In some embodiments of the present invention, the nanobody of the present invention is camelized.

[0074] On the other hand, the present invention provides a fusion protein, which contains a functional domain capable of specifically binding to Ly6G6D; wherein, the functional domain is composed of the nanobody of the present invention that specifically binds to Ly6G6D.

[0075] In some embodiments of the present invention, the fusion protein of the present invention further contains a constant region.

[0076] Specifically, the constant region is the constant region of human IgG or its mutant.

[0077] Specifically, the constant region of the human IgG is the constant region of human IgG1 or a mutant thereof.

[0078] 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.

[0079] EPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 32).

[0080] In some embodiments of the present invention, the fusion protein of the present invention further comprises a linker peptide Linker.

[0081] Specifically, the linker peptide Linker of the fusion protein 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 through the linker peptide Linker. An example of the amino acid sequence of the linker peptide Linker is: (GnS)m, where n is a natural number from 3 to 5, n is preferably 4, and m is a natural number from 1 to 5, and m is preferably 3.

[0082] GGGGSGGGGSGGGGS (SEQ ID NO: 33).

[0083] On the other hand, the present invention provides an anti-Ly6G6D antibody, which is a conventional antibody or a functional fragment thereof, and the heavy chain variable region of the conventional antibody or its functional fragment is composed of the nanobody specifically binding to Ly6G6D of the present invention.

[0084] In some embodiments of the present invention, the antibody functional fragment is a Fab, Fab’, (Fab’)2, Fv, scFv or sdFv structure of a conventional antibody.

[0085] Conventional antibodies are structurally composed of two identical heavy chains and two identical light chains. The light chain has a variable light chain region (VL) and a constant light chain region (CL); the heavy chain has a variable heavy chain region (VH) and constant heavy chain regions (CH1, CH2, CH3, and / or CH4). On the premise that the present invention discloses the structure of a nanobody capable of specifically binding to Ly6G6D, those skilled in the art can easily think of using the nanobody of the present invention to modify conventional antibodies. For example, applying the CDR region structure of the nanobody of the present invention to conventional antibodies to obtain conventional antibodies that can specifically bind to Ly6G6D. Such conventional antibodies also fall within the protection scope of the present invention; further, based on the structure of conventional antibodies, some of their structures, such as Fab, Fab’, (Fab’)2, Fv, scFv, or sdFv structures, etc., also have Ly6G6D binding specificity, which also belongs to the protection scope of the present invention.

[0086] Among them, Fab is composed of a complete light chain, the variable domain of the heavy chain (VH), and the first constant domain of one heavy chain (CH1). Fab can be the product generated by digesting an antibody with papain; the difference between Fab’ and Fab is that the Fab’ fragment adds some residues at the carboxyl terminus of the CH1 domain, and these residues contain one or more cysteines missing from the antibody hinge region; (Fab’)2 is equivalent to two Fab’ fragments connected by a disulfide bond, having bivalent antigen-binding activity and still being able to cross-link antigens. (Fab’)2 can be the product of pepsin digestion of an antibody; Fv is composed of a tight, non-covalent dimer of a variable heavy chain domain and a variable light chain domain; scFv (single-chain antibody) contains an antibody fragment in which the VH and VL antibody domains are linked to a single polypeptide chain. Preferably, the scFv further contains a polypeptide linker between the VH and VL domains to enable the scFv to form the required antigen-binding structure; sdFv refers to an Fv fragment linked by a disulfide bond.

[0087] On the other hand, the present invention provides a bispecific or multispecific antibody that contains the nanobody specifically binding to Ly6G6D described in the present invention. To produce the bispecific or multispecific antibody, the nanobody described in the present invention can be linked (for example, by chemical coupling, gene fusion, non-covalent association, or other means) to one or more other binding molecules (such as another antibody, antibody fragment, peptide, or binding mimetic).

[0088] In some embodiments, the bispecific or multispecific antibody specifically binds to Ly6G6D and additionally specifically binds to one or more other targets.

[0089] In some embodiments, the bispecific or multispecific antibody further comprises at least one second antibody having a second binding specificity for a second target.

[0090] In yet another aspect, the present invention also provides a conjugate comprising a nanobody specifically binding to Ly6G6D or a bispecific or multispecific antibody according to the present invention and a coupling moiety.

[0091] In some embodiments, the nanobody specifically binding to Ly6G6D according to the present invention is optionally conjugated to the coupling moiety via a linker.

[0092] In some embodiments, the coupling moiety is selected from protein tags. 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 a suitable protein tag according to the desired purpose (e.g., purification, detection, or tracing).

[0093] In some embodiments, a His tag (e.g., 6×His) is linked to the C-terminus of the nanobody specifically binding to Ly6G6D according to the present invention.

[0094] In some embodiments, the coupling moiety is selected from detectable labels, such as enzymes (e.g., horseradish peroxidase), radionuclides, fluorescent dyes, luminescent substances (such as chemiluminescent substances), or biotin. The detectable label according to the present invention can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrochemistry, optics, or chemical means. Such labels are well known in the art, and examples thereof 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 markers 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 markers.

[0095] In some embodiments, such labels can be applicable to immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescent immunoassay, etc.).

[0096] In some embodiments, the detectable label as described above can be linked to the nanobody or its antigen-binding fragment of the present invention via linkers of different lengths to reduce potential steric hindrance.

[0097] In some embodiments, the coupling moiety is selected from therapeutic agents, such as anti-inflammatory drugs or immunosuppressive agents.

[0098] In some embodiments, the coupling moiety is selected from additional bioactive polypeptides.

[0099] On the other hand, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the nanobody, the antibody, or the fusion protein of the present invention.

[0100] In some embodiments of the present invention, the nucleic acid molecule is DNA.

[0101] In some embodiments of the present invention, the present invention further provides a nucleic acid construct comprising the nucleic acid molecule of the present invention; further, the nucleic acid construct comprises one or more control sequences operably linked to the nucleic acid molecule, and the one or more control sequences direct the production of the antibody or its antigen-binding fragment of the present invention in a suitable host cell, and the one or more control sequences are selected from: promoters, enhancers, stop signals, signal peptides, leading sequences, transcription terminators, and any combination thereof.

[0102] On the other hand, the present invention provides an expression vector carrying the nucleic acid molecule of the present invention.

[0103] In some embodiments of the present invention, the expression vector of the present invention is selected from: plasmids, cosmids, viruses, minichromosomes, and artificial chromosomes.

[0104] In some embodiments of the present invention, the expression vector is a eukaryotic expression vector.

[0105] In some embodiments of the present invention, the eukaryotic expression vector is selected from: pCRII, pCR3, and pcDNA3.4, pBSII, pET 15, pGEX, pEGFP-N1, pETL, pDSR-α, and pFastBacDual, etc.

[0106] On the other hand, the present invention provides a host cell comprising the nucleic acid molecule or the expression vector of the present invention.

[0107] In some embodiments of the present invention, the host cell is Escherichia coli, yeast or a eukaryotic cell.

[0108] In some embodiments of the present invention, the host cell is a eukaryotic host cell.

[0109] In some embodiments of the present invention, the host cell is a mammalian host cell.

[0110] On the other hand, the present invention provides a pharmaceutical composition comprising 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.

[0111] In some embodiments of the present invention, the pharmaceutical composition of the present invention further comprises a pharmaceutically acceptable carrier.

[0112] The pharmaceutical composition of the present invention may comprise 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.

[0113] A "prophylactically effective amount" means an amount sufficient to prevent, arrest, or delay the onset of a disease. A "therapeutically effective amount" means 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 to be treated, the overall status of the patient's own immune system, the general condition of the patient such as age, weight, and gender, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0114] 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 mode of administration and therapeutic use.

[0115] A preferred dosage form is an injection. Such an injection can be a sterile injection solution. For example, a sterile injection solution can be prepared by incorporating the required dose of the antibody or its antigen-binding fragment of the present invention in a suitable solvent, and optionally, simultaneously incorporating other desired components (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, the sterile injection solution can be prepared as a sterile lyophilized powder (for example, by vacuum drying or freeze drying) for ease of storage and use.

[0116] The nanobody, antibody, fusion protein, nucleic acid molecule, expression vector, host cell or composition of the present 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, intracisternal, inguinal, intravesical, local (such as powder, ointment or drops), or nasal routes. However, for many therapeutic uses, the preferred route of administration / mode is parenteral administration (such as intravenous injection or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art should understand that the route of administration and / or mode will vary according to the intended purpose.

[0117] In some embodiments, the nanobody, antibody, fusion protein, nucleic acid molecule, expression vector, host cell or composition of the present invention is administered by intravenous injection or bolus injection.

[0118] On the other hand, the present invention provides the use of the nanobody, antibody, fusion protein, nucleic acid molecule, expression vector, host cell or 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.

[0119] 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.

[0120] On the other hand, the present invention provides a kit for detecting Ly6G6D, which kit contains the nanobody, antibody or fusion protein of the present invention and an instruction manual.

[0121] On the other hand, the present invention provides a method for detecting the presence or amount of Ly6G6D in a sample, which includes using the nanobody of the present invention.

[0122] In some embodiments, the method is an immunological detection, such as immunoblotting, enzyme immunoassay (such as ELISA), chemiluminescent immunoassay, fluorescence immunoassay or radioimmunoassay.

[0123] In some embodiments, the conjugate for the method contains the nanobody of the present invention and a detectable label.

[0124] In some embodiments, the nanobody used in the method is labeled with a detectable label.

[0125] In some embodiments, the nanobody used in the method is not labeled with a detectable label. Thus, the method may further include using other reagents (such as a second antibody) labeled with a detectable label to detect the nanobody of the present invention.

[0126] In some embodiments, the method includes the following steps:

[0127] (1) Contacting the sample with the nanobody of the present invention;

[0128] (2) Detecting the formation of a complex between the nanobody and Ly6G6D or detecting the amount of the complex.

[0129] The formation of the complex indicates the presence of Ly6G6D or cells expressing Ly6G6D.

[0130] The method can be used for diagnostic purposes or non-diagnostic purposes (for example, the sample is a cell sample rather than a sample from a patient).

[0131] In some embodiments, the method is used to diagnose whether a subject has a disease related to Ly6G6D. In such embodiments, the method may further include: 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 known not to have a disease related to Ly6G6D (such as a healthy control) (also referred to as a "negative reference value"). For example, if the amount of Ly6G6D in a sample from the subject is elevated relative to the negative reference value, it indicates that the subject has a disease related to Ly6G6D.

[0132] In some embodiments, the sample may be selected from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue-associated cells (i.e., free cells), tissues (such as surgically removed tumor tissues, biopsy specimens, or fine needle aspiration tissues), histological preparations, etc.

[0133] Term Definitions

[0134] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Also, the operation steps such as cell culture, molecular biology, biochemistry, nucleic acid chemistry, immunology, etc. used herein are all conventional steps widely used in the corresponding fields. At the same time, for a better understanding of the present invention, the definitions and explanations of related terms are provided below.

[0135] When the present invention uses terms such as "for example", "such as", "including", "comprising" or variants thereof, these terms will not be considered restrictive terms, but will be construed to mean "but not limited to" or "not limited to".

[0136] Unless the present invention otherwise indicates or is clearly contradictory according to the context, the terms "a", "an" and "the" and similar referents in the context of describing the present invention (especially in the context of the following claims) shall be construed to cover the singular and the plural.

[0137] As used in the present invention, the term "camel-derived antibody" refers to an antibody against an antigen produced by a Camelidae animal (including camels, alpacas and llamas) that has been immunized or invaded by an antigen. As is known to those skilled in the art, there is a "heavy chain antibody" (Camelid heavy chain antibody, HCAb) lacking a light chain among the antibodies produced by Camelidae animals. This antibody only contains a single variable domain of the heavy chain (variable domain of heavy chain of HCAb, VHH) and two conventional CH2 and CH3 regions, and the separately cloned and expressed VHH region has good structural stability and antigen-binding activity. VHH is the smallest unit known to be able to bind to a target antigen.

[0138] As used in the present invention, the term "nanobody" has the meaning commonly understood by those skilled in the art, which refers to an antibody fragment composed of a single monomeric variable antibody domain (such as a single variable domain of the heavy chain), usually derived from the variable region of a heavy chain antibody (such as a Camelidae animal antibody or a shark antibody). Typically, a nanobody consists of 4 framework regions and 3 complementarity-determining regions, having the structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The nanobody can be truncated at the N-terminus or C-terminus so that it only contains 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. Nanobodies are also referred to as single-domain antibodies (single domain antibody, sdAb), and the two can be used interchangeably.

[0139] As used herein, 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 the same antigen to which the nanobody binds, and / or competes with the nanobody for specific binding to the antigen, which is also referred to as the "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of the antibodies of the invention can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of the nanobodies of the invention. In some embodiments, the "antigen-binding fragment" of the nanobody can be truncated at the N-terminus or C-terminus compared to the full-length nanobody such that it contains only a portion of FR1 and / or FR4, or lacks one or both of those framework regions, so long as it substantially retains antigen binding and specificity.

[0140] Antigen-binding fragments of nanobodies can be obtained from a given nanobody (such as a nanobody provided by the invention) using conventional techniques known to those of skill in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and antigen-binding fragments of nanobodies can be screened for specificity in the same manner as for intact nanobodies.

[0141] As used herein, unless the context clearly dictates otherwise, when referring to the term "nanobody", it includes not only the intact nanobody, but also antigen-binding fragments of the nanobody.

[0142] As used herein, the term "complementary determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. Nanobodies contain 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, such as 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, those skilled in the art will readily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0143] 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.

[0144] As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (KD) of that 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 an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0145] The specific binding properties between two molecules can be determined using methods well-known in the art. One method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentrations and the actual rates 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). The KD, kon, and kdis values can be measured using any valid method. In certain embodiments, surface plasmon resonance (SPR) in a Biacore can be used to measure the dissociation constant. In addition, bioluminescence interferometry or Kinexa can also be used to measure the dissociation constant.

[0146] The term "amino acid" refers to a compound containing two functional groups, an amino group and a carboxyl group, such as an α-amino acid. Two or more amino acids can form a polypeptide through an amide bond (also called a peptide bond). A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The encoding of the same amino acid 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).

[0147] As used herein, the term "expression vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector enables the expression of a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction, or transfection, so that the genetic material elements carried by it can be 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); phages such as lambda phage or M13 phage, and animal viruses, etc. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, polyomaviruses (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. In addition, the vector can also contain an origin of replication.

[0148] As used herein, 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 cDNA.

[0149] 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 Drosophila S2 cells or Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells. The host cell can include a single cell or a cell population.

[0150] As used herein, the term "identity" is used to refer to the sequence match between two polypeptides or between two nucleic acids. When a position in both of the two sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of 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 × 100. For example, if 6 of 10 positions in two sequences match, then the two sequences have 60% identity. Generally, comparison is made when the two sequences are aligned to yield maximum identity. Such alignment can be accomplished by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed by computer programs such as the Align program (DNAstar, Inc.). The algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), incorporated into the ALIGN program (version 2.0), can also be used, with a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 to determine the percent identity between two amino acid sequences. In addition, the algorithm of Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) incorporated into the GAP program in the GCG software package (available at www.gcg.com) can be used, with a Blossum 62 matrix or 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 to determine the percent identity between two amino acid sequences.

[0151] As used in the present invention, 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, e.g., 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 for maintaining osmotic pressure, agents for delaying absorption, and preservatives. For example, pH regulators include, but are not limited to, phosphate buffer solutions. 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, etc. Agents for maintaining osmotic pressure include, but are not limited to, sugars, NaCl, and their analogs. Agents for delaying absorption include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffer solutions (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 commonly 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, sugars (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 includes 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 solutions (such as 0.9% (w / v) NaCl), glucose solutions (such as 5% glucose), solutions containing surfactants (such as 0.01% polysorbate 20), pH buffer solutions (such as phosphate buffer solutions), Ringer's solution, and any combination thereof.

[0152] As used in the present invention, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease or disorder or symptom (e.g., a disease associated with Ly6G6D) in a subject. As used herein, the term "treatment" refers to a method implemented 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., not worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether in part or in whole), whether detectable or not. In addition, "treatment" may also refer to extending the survival period compared to the expected survival period (if untreated).

[0153] 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 (e.g., a human) has a disease associated with Ly6G6D.

[0154] The embodiments of the present invention will be described in detail below in conjunction with the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention and not for limiting the scope of the present invention. According to the following detailed description of the drawings and preferred embodiments, various objects and advantageous aspects of the present invention will become practicable to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0155] Figure 1 It is a graph showing the binding result of the antibody to the hLY6G6D antigen confirmed by ELISA;

[0156] Figure 2 It is a graph showing the binding result of the antibody to the cynoLY6G6D antigen confirmed by ELISA;

[0157] Figure 3 It is a graph showing the binding result of the antibody to HEK23T cells highly expressing hLY6G6D detected by flow cytometry;

[0158] Figure 4 It is a graph showing the binding result of the antibody to HEK23T cells highly expressing cynoLY6G6D detected by flow cytometry;

[0159] Figure 5 It is a graph showing the result of evaluating the in vitro ADCC activity using the Jurkat-NFAT-Luc2-CD16 cell model. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0160] The present invention will now be described with reference to the following examples which are intended to illustrate the present invention (but not to limit the present invention).

[0161] Unless otherwise specified, the molecular biology experimental methods and immunoassay methods used in the present invention are basically carried out according to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Current Protocols in Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995. Those skilled in the art know that the examples describe the present invention by way of illustration and are not intended to limit the scope of the present invention claimed.

[0162] Example 1: Screening of LY6G6D nanobodies by phage display technology and flow cytometry

[0163] By phage-displaying the VH form of nanobodies, panning was carried out using phage library screening technology, and monoclonal antibodies that could produce high binding to Human LY6G6D at the molecular level were screened by ELISA method. The extracellular region-encoding genes of Human LY6G6D and cynoLY6G6D proteins were designed using Uniprot and NCBI databases and constructed into a mammalian eukaryotic expression system. The extracellular regions of Human LY6G6D and cynoLY6G6D proteins were expressed and used for screening the immune nanobody library. The extracellular region of the expressed Human LY6G6D protein was used to immunize alpacas 6 times until the titer reached 10 4 , PBMC was isolated from the alpaca blood, and the VH sequences of nanobodies were amplified therefrom to establish an immune nanobody phage library. The immune nanobody phage library was panned with the Human LY6G6D protein. After 3 rounds of panning, monoclonal phages that could bind to Human LY6G6D were screened by the ELSIA method. The binding results of 4 candidate phages to Human LY6G6D are shown in Table 1 below:

[0164] Table 1

[0165] Number 1A4 1D5 1D5G8 2G5 OD450 1.312 0.613 1.558 1.429

[0166] Example 2: Expression and purification of antibodies

[0167] Sequenced the 4 candidate phages obtained in Example 1 to obtain the sequences encoding the heavy chain variable regions of the antibody candidates. Used gene synthesis to synthesize the gene encoding sequence of the heavy chain IgG antibody (VH-Linker-Fc, where VH has the amino acid sequence shown in any one of SEQ ID NOs: 28 to 31, Fc has the amino acid sequence shown in SEQ ID NO: 32, and Linker has the amino acid sequence shown in SEQ ID NO: 33). Constructed a series of antibody expression vectors by molecular cloning and carried out recombinant expression in the 293F expression system. After resuscitating and culturing the 293F host cells with OPMI medium, when the cell density was about 3*10 6 cell / mL, collected the cells and transfected them with the PEI reagent. On the second day of culture, fed-batch and added glucose to a culture concentration of 8 g / L. On the 6th day of culture, collected the cell culture supernatant.

[0168] Detected a series of candidate antibodies at the translational level. Purified the collected cell culture supernatant using a Protein A chromatography column, collected the absorption peak, and performed mass spectrometry detection. The mass spectrometry detected a series of candidate antibodies. The molecular weight of the candidate antibody VHH-Fc was 75 KDa, which was consistent with the theoretical molecular weight and was in the form of a dimer. At the same time, the collected samples were detected by 10% SDS-PAGE electrophoresis after reduction and non-reduction. The reduced SDS-PAGE electrophoresis pattern of the candidate antibody VH-Fc showed around 37 KDa, and the non-reduced SDS-PAGE electrophoresis pattern showed a single band at around 75 KDa. The size of the electrophoresis pattern bands was consistent with the theory. The candidate antibody VHH-Fc with a smaller volume had better tumor penetration. The purified sample was dialyzed overnight at 4°C with 0.02 M PBS buffer at pH 7.4.

[0169] Example 3: Detection of the binding characteristics between the antibody and the antigen

[0170] Confirmed the binding of the expressed antibody to the hLY6G6D antigen by ELISA. The positive control antibody 20A12 was from patent CN202080086360.1. Coated an ELISA plate with 100 ng / well of hLY6G6D and placed it in a 37°C electrothermal constant temperature incubator for 2 h. Then blocked the non-specific binding sites overnight at 4°C with 5% skim milk powder. Incubated with different concentrations of VHH for 1 h, washed 3 times with 0.1% PBST, added HRP-mouse anti-human IgG1 Fc (SouthernBiotech) to detect the bound VHH, then washed 5 times with 0.1% PBST, added the TMB substrate (Yunqiao Biotech), terminated the reaction after color development, and read the absorbance at 450 nm. The results were as Figure 1As shown in Table 2, the affinity of the candidate antibodies is comparable to that of the positive antibody 20A12, and the antibody affinities of 1A4 and 2G5 are even better than that of the positive antibody 20A12; the heavy chain of the positive antibody 20A12 contains the amino acid sequence shown in SEQ ID NO:34, and the light chain contains the amino acid sequence shown in SEQ ID NO:35;

[0171] EVQLLESGGGLVQPGGSLRLSCAASGFDFVNNAMIWVRQAPGKGLEWVSALSFADNTAYYATWASGRFTISRDSSKTTVYLQMNSLRAEDTAVYYCMRGDLWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:34);

[0172] DIQMTQSPSTLSASVGDRVTITCQASESITRYLNWYQQKPGKAPKLLIYDASKLPSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQSTSFRGRSYQNTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:35).

[0173] Table 2

[0174] Number 1A4 1D5 1D5G8 2G5 20A12 EC50 (nM) 5.03 7.329 8.756 4.917 7.89

[0175] Example 4: Detection of the binding characteristics between the antibody and the antigen

[0176] Cross-reactivity detection with monkeys: The binding of the expressed antibody to cynoLY6G6D antigen was confirmed by ELISA. The positive control antibody 20A12 was sourced from patent CN202080086360.1. An ELISA plate was coated with 100 ng / well of cynoLY6G6D and placed in an electrothermal incubator at 37 °C for 2 h. Then, non-specific binding sites were blocked overnight at 4 °C with 5% skim milk powder. Different concentrations of VHH were incubated for 1 h, washed 3 times with 0.1% PBST, HRP-mouse anti-human IgG1Fc (SouthernBiotech) was added to detect the bound VHH, then washed 5 times with 0.1% PBST, TMB substrate (Yunqiao Biotech) was added, the reaction was terminated after color development, and the absorbance was read at 450 nm. The results are as Figure 2 shown in Figure Figure 2 and Table 3, both the candidate antibody and the positive antibody bind to cynoLY6G6D.

[0177] Table 3

[0178]

[0179]

[0180] Example 5: Detection of the binding characteristics between the antibody and the antigen

[0181] Flow cytometry was used to detect the binding between HEK23T cells highly expressing hLY6G6D and the antibody. The positive control antibody 20A12 was sourced from patent CN202080086360.1. 2×10 5 cells were resuspended in a 96-well plate, incubated with different concentrations of VHH for 1 h, then the cells were washed 2 times in PBS. PE-Goat anti-human IgG Fc (Jackson ImmunoResearch) was diluted 1:500 and incubated at 4 °C for 30 min to detect the bound antibody. Then the cells were washed 2 times in PBS and analyzed on a FACS (beckman). The results are as Figure 3 shown in Figure Figure 3 and Table 4, the affinity of the candidate antibody is comparable to that of the positive antibody 20A12.

[0182] Table 4

[0183] Number 1A4 1D5 1D5G8 2G5 20A12 EC50 (nM) 4.569 6.313 7.137 7.938 4.565

[0184] Example 6: Detection of the binding characteristics between the antibody and the antigen

[0185] Cross-reactivity detection with monkeys: Flow cytometry was used to detect the binding between HEK23T cells highly expressing cynoLY6G6D and the antibody. The positive control antibody 20A12 was sourced from patent CN202080086360.1. 2×105 Cells were resuspended in a 96-well plate, incubated with different concentrations of VHH for 1 h, then the cells were washed twice with PBS. PE-Goat anti-human IgG Fc (Jackson ImmunoResearch) was diluted 1:500 and incubated at 4 °C for 30 min to detect the bound antibody. Then the cells were washed twice with PBS and analyzed on a FACS (beckman). The results are as Figure 4 shown in Table 5. Both the candidate antibody and the positive antibody bound to cynoLY6G6D.

[0186] Table 5

[0187] Number 1A4 1D5 1D5G8 2G5 20A12 EC50 (nM) 3.483 4.309 5.299 5.957 4.072

[0188] Example 7: Evaluation of in vitro ADCC activity using the Jurkat-NFAT-Luc2-CD16 cell model

[0189] A Jurkat-NFAT-Luc2-CD16 reporter gene cell model stably transfected with the FcγRIIIa receptor and NFAT (nuclear factor of activated T cells) was constructed using jurkat cells to evaluate the ADCC activity of candidate molecules.

[0190] Using the constructed hLY6G6D-HEK293T cells as target cells and Jurkat-NFAT-Luc2-CD16 cells as effector cells, the effector cells and target cells were seeded in a 384-well plate, 5000 cells per well. The positive control antibody 20A12 was from patent CN202080086360.1. Diluted antibody solutions at different concentrations were added to the 384-well plate, and the effector cells and target cells were co-cultured in an incubator at 37 °C and 5% carbon dioxide for 14 h. The antibody could specifically bind to hLY6G6D on the surface of the target cells, and the Fc end of the antibody could bind to the FcγRIIIa receptor on the surface of the effector cells, thus forming a target cell-antibody-effector cell complex, activating the NFAT-Luc signaling pathway in the effector cells, so that the effector cells produced luciferase. Luciferase detection substrate was added and reacted for 3 min, and the luminescence values of each well were read using a multifunctional microplate reader. The results are as Figure 5 shown in Table 6. The ADCC activities of the candidate antibody and the positive antibody 20A12 were comparable.

[0191] Table 6

[0192] Number 1A4 1D5 1D5G8 2G5 20A12 EC50 (nM) 0.02771 0.02655 0.03815 0.01326 0.01213

[0193] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the teachings that have been published, and such changes are within the scope of protection of the present invention. The entire scope of the present 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 comprises complementary determining regions: CDR1, CDR2 and CDR3; Wherein, the CDR1 has an amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; YYAIG (SEQ ID NO: 1); SYX1MX2 (SEQ ID NO: 2); wherein X1 is selected from A or M, and X2 is selected from E or G; The CDR2 has an amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4; CISSSGGSTVYADTVKG(SEQ ID NO:3); 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; The CDR3 has an 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, characterized in that Said 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 includes framework regions: FR1, FR2, FR3 and FR4; wherein the framework regions and the complementarity determining regions are arranged alternately in sequence.

4. The Nanobody according to claim 3, characterized in that in, FR1 has the amino acid sequence shown in SEQ ID NO:14; 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; 10 Selected from F or R; X 11 Select from L or F; X 12 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 Select from G or F; X 14 Select from S or A; FR3 has the amino acid sequence shown in SEQ ID NO: 16; RFTISRDNX 15 KNX 16 X 17 YQ 18 SLKPEDTAVYYCAA (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 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, characterized in that Said 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 the amino acid sequence shown in SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27; FR4 has the amino acid sequence shown in SEQ ID NO:17 or SEQ ID NO:

18.

6. The Nanobody according to any one of claims 1 to 5, characterized in that The Nanobody heavy chain variable region has 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.

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

8. The anti-Ly6G6D antibody according to claim 7, characterized in that The antibody functional fragment is a traditional antibody Fab, Fab', (Fab')2, Fv, scFv or sdFv structure.

9. A fusion protein, characterized in that The fusion protein comprises a functional domain capable of specifically binding to Ly6G6D; wherein the functional domain is composed of the 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 comprises a nucleotide sequence encoding the Nanobody according to any one of claims 1 to 6, the antibody according to claim 7 or 8, or the fusion protein according to claim 9; Preferably, the nucleic acid molecule is DNA.

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

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

14. Use of the Nanobody of any one of claims 1 to 6, the antibody of claim 7 or 8, the fusion protein of claim 9, the nucleic acid molecule of claim 10, the expression vector of claim 11, the host cell of claim 12 or the composition of claim 13 in the preparation of a medicament 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, colon cancer or adenocarcinoma, etc.

16. A kit for detecting Ly6G6D, characterized in that: The kit comprises the Nanobody according to any one of claims 1 to 6, the antibody according to claim 7 or 8, the fusion protein according to claim 9, and instructions for use.

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