Anti-LY6G6D single-domain antibody, its preparation method and uses

Abstract

This invention belongs to the field of biotechnology and provides an anti-LY6G6D single-domain antibody, its preparation method, and its uses. The antibody comprises a heavy chain variable region, which includes three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained in the sequences shown in SEQ ID NO:1, 5, or 7. The antibody or its antigen-binding fragment provided by this invention can bind to human LY6G6D and exhibits many superior properties.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to anti-LY6G6D antibodies and their uses, particularly anti-LY6G6D single-domain antibodies. Background Technology

[0002] Colorectal cancer (CRC) ranks second in cancer mortality worldwide. However, first-line targeted therapies for metastatic CRC (mCRC) are scarce and suffer from low efficacy and drug resistance. Chemotherapy drugs also present clinical challenges such as low efficacy, significant side effects, and resistance reversal, leading to poor prognosis for CRC patients. Current targeted therapies are primarily immune checkpoint inhibitors (ICIs), but due to their limited availability, the vast majority of colorectal cancer patients do not benefit from ICI treatment.

[0003] Lymphocyte antigen 6 complex locus G6D (LY6G6D), also known as LY6-D, LY6-G, G6D, NG25, MEGT1, and C6orf23, is a member of the Lymphocyte antigen 6 superfamily (Ly6SF). It is a phosphatidylinositol that can attach to the cell membrane via glycosylphosphatidylinositol (GPI) anchorage. LY6G6D is a dual-mechanism target: it exhibits TSA-specific expression in CRC, with minimal expression in normal tissues; simultaneously, it can regulate immunity. JAK / STAT inhibitors can increase CRC cell death by targeting the STAT5 / LY6G6D axis.

[0004] Compared to normal colorectal tissue, LY6G6D is highly expressed in both primary and metastatic colorectal tumors. High LY6G6D expression is associated with significant infiltration of immunosuppressive cells; in colon cancer with reduced CD8+ T lymphocytes, LY6G6D expression and STAT5 activation are enhanced. STAT5 knockout significantly reduces LY6G6D levels, indicating that STAT5 regulates LY6G6D expression. Furthermore, the proliferation of LY6G6D-positive MSS cells (which can be transmitted through myeloid-derived suppressor cells, MDSCs) inhibits T cell proliferation. Similarly, in CRC, inhibitors of p38 α MAPK and knockdown of the DNA methyltransferase DNMT1 lead to decreased LY6G6D expression. In metastatic CRC, LY6G6D hypermethylation predicts resistance to first-line FOLFOX (the drugs leucovorin, fluorouracil (5-FU), and oxaliplatin). Therefore, differentially high expression of LY6G6D in colorectal cancer is closely related to cancer development and is expected to serve as a potential predictive indicator or therapeutic target for colorectal cancer. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an isolated antibody or antigen-binding fragment thereof that binds to the human lymphocyte antigen 6 complex locus G6D (LY6G6D).

[0006] In a first aspect, the present invention provides a single-domain antibody or antigen-binding fragment thereof having binding specificity to human lymphocyte antigen 6 complex site G6D (LY6G6D), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region, the heavy chain variable region comprising three heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3 contained in the sequence shown in SEQ ID NO: 1, 5 or 7.

[0007] Those skilled in the art can determine the CDR sequence based on the illustrated heavy chain and light chain variable region sequences using methods known in the art. These known methods may include the Kabat scheme, the AbM scheme, the Chothia scheme, or the Contact scheme. It is well known in the art that CDR sequences obtained using different schemes for the same variable region sequence will differ. Furthermore, those skilled in the art will understand that CDR regions obtained using different schemes are all within the scope of protection of this invention.

[0008] In some implementations, the Kabat scheme is used to determine the CDR region sequence.

[0009] In some embodiments, the amino acid sequences of HCDR1, HCDR2, and HCDR3 contained in the antibody or its antigen-binding fragment are shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; or,

[0010] The amino acid sequences of HCDR1, HCDR2, and HCDR3 contained in the antibody or its antigen-binding fragment are shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 6, respectively; or,

[0011] The amino acid sequences of HCDR1, HCDR2 and HCDR3 contained in the antibody or its antigen-binding fragment are shown in SEQ ID NO: 2, SEQ ID NO: 8 and SEQ ID NO: 9, respectively.

[0012] In some embodiments, the antibody or its antigen-binding fragment comprises a heavy chain variable region, the amino acid sequence of which is as shown in SEQ ID NO: 1, 5 or 7, or has at least 90% sequence identity with the sequence shown in SEQ ID NO: 1, 5 or 7, such as 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% sequence identity.

[0013] In some embodiments, the antibody or its antigen-binding fragment further comprises a heavy chain constant region, an Fc region, or a combination thereof.

[0014] In some implementations, the heavy chain constant region is selected from the IgG, IgM, IgA, IgE, or IgD class.

[0015] In some implementations, the heavy chain constant region is a heavy chain constant region selected from the IgG1, IgG2, IgG3 or IgG4 subclasses.

[0016] In some embodiments, the antibody or its antigen-binding fragment further comprises a human IgG1 heavy chain constant region or a variant thereof.

[0017] In some embodiments, the sequence of the human IgG1 heavy chain constant region is shown in SEQ ID NO: 10.

[0018] In some implementations, the antibody or its antigen-binding fragment is a chimeric antibody or a humanized antibody.

[0019] In some embodiments, the antibody or its antigen-binding fragment is VHH.

[0020] In some embodiments, the antibody or its antigen-binding fragment is a single-domain antibody or a nanobody.

[0021] In some embodiments, the sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 15, 16 or 17, or has at least 90% sequence identity with the sequence shown in SEQ ID NO: 15, 16 or 17, such as 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% sequence identity.

[0022] In some embodiments, the antibody or its antigen-binding fragment is a monoclonal antibody, a polyclonal antibody, or a multispecific antibody (e.g., a bispecific antibody).

[0023] In some embodiments, the antibody is a tandem antibody, comprising two or more antibodies or antigen-binding fragments thereof linked in tandem.

[0024] In some embodiments, two or more antibodies or their antigen-binding fragments are linked by a linker. The linker may be a peptide linker conventionally used in the art, such as (GGGGS)n, where n is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. In some preferred embodiments, the linker is GGGGSGGGGSGGGGS (SEQ ID NO:27).

[0025] In some embodiments, the tandem antibody comprises a first antibody or an antigen-binding fragment thereof and a second antibody or an antibody-binding fragment thereof, wherein,

[0026] The first antibody or its antigen-binding fragment includes three heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3 contained in the sequence shown in SEQ ID NO: 5;

[0027] The second antibody or its antigen-binding fragment includes three heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3 contained in the sequence shown in SEQ ID NO: 7.

[0028] In some preferred embodiments, the amino acid sequence of the first antibody or its antigen-binding fragment is as shown in SEQ ID NO: 5, or has at least 90% sequence identity with the sequence shown in SEQ ID NO: 5, such as 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% sequence identity. The amino acid sequence of the second antibody or its antigen-binding fragment is as shown in SEQ ID NO: 7, or has at least 90% sequence identity with the sequence shown in SEQ ID NO: 7, such as 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% sequence identity.

[0029] In some implementations, the tandem antibody further includes an Fc segment.

[0030] In some embodiments, the amino acid sequence of the tandem antibody is as shown in SEQ ID NO: 18, or has at least 90% sequence identity with the sequence shown in SEQ ID NO: 18, such as 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% sequence identity.

[0031] In some embodiments, the antibody further includes a CD8α signal peptide, a CD8α hinge, a CD8α transmembrane region, a 4-1BB intracellular domain, and a partial CD3 intracellular domain.

[0032] In some embodiments, from the N-terminus to the C-terminus, the antibody comprises, in sequence, a CD8α signal peptide, a single-domain antibody of the present invention or an antigen-binding fragment thereof or a tandem antibody of the present invention, a CD8α hinge, a CD8α transmembrane region, a 4-1BB intracellular domain, and a partial intracellular domain of CD3.

[0033] In some embodiments, the amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO: 19, the amino acid sequence of the CD8α hinge and CD8α transmembrane region is shown in SEQ ID NO: 21, the amino acid sequence of the 4-1BB intracellular domain is shown in SEQ ID NO: 23, and the amino acid sequence of the CD3 partial intracellular domain is shown in SEQ ID NO: 25.

[0034] In a preferred embodiment, from the N-terminus to the C-terminus, the antibody comprises, in sequence, a CD8α signal peptide, a tandem antibody, a CD8α hinge, a CD8α transmembrane region, a 4-1BB intracellular domain, and a partial CD3 intracellular domain. The amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO: 19, the amino acid sequence of the tandem antibody is shown in SEQ ID NO: 18, the amino acid sequences of the CD8α hinge and the CD8α transmembrane region are shown in SEQ ID NO: 21, the amino acid sequence of the 4-1BB intracellular domain is shown in SEQ ID NO: 23, and the amino acid sequence of the partial CD3 intracellular domain is shown in SEQ ID NO: 25.

[0035] In the second aspect, a biomaterial is provided, selected from the following a) to c)

[0036] a) A nucleic acid molecule encoding the antibody or its antigen-binding fragment as described in this invention;

[0037] In some embodiments, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA. In other embodiments, the nucleic acid molecule may be RNA, such as mRNA or hnRNA.

[0038] b) A recombinant vector containing the aforementioned nucleic acid molecules;

[0039] The vector can be an expression vector; in some embodiments, the vector is a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a bacteriophage vector, etc.

[0040] c) Recombinant cells containing the above-mentioned nucleic acid molecules and / or the above-mentioned recombinant vectors.

[0041] The recombinant cells are obtained by transforming or infecting host cells with the nucleic acid molecules or recombinant vectors of the present invention. In some embodiments, the host cell can be any prokaryotic or eukaryotic cell, such as bacterial or insect, fungal, plant, or animal cells. In some embodiments, the host cell is prokaryotic, such as *Escherichia coli*. In other embodiments, the host cell is eukaryotic, such as 293 cells, CHO cells, yeast cells, or plant cells. In some embodiments, the host cell is another cell suitable for preparing antibodies or their antigen-binding fragments.

[0042] In a third aspect, a method for preparing the antibody or antigen-binding fragment thereof described in this invention is provided, the method comprising culturing recombinant cells containing a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of this invention, in a manner suitable for antibody expression.

[0043] In some embodiments, the method further includes: recovering the antibody or its antigen-binding fragment from the recombinant cells or culture medium.

[0044] In a fourth aspect, a composition is provided comprising the antibody or antigen-binding fragment thereof or biological material as described in this invention, and a pharmaceutically acceptable carrier.

[0045] In some embodiments, the above composition is a pharmaceutical composition.

[0046] Fifthly, the use of any of the above-described antibodies or their antigen-binding fragments, biological materials, and / or compositions in the preparation of any of the following products is provided:

[0047] (a) Products tested for LY6G6D;

[0048] (b) Products that stimulate or enhance the immune response;

[0049] (c) Products for the prevention and / or treatment of diseases associated with abnormal LY6G6D expression;

[0050] (d) Products that kill or inhibit the growth of cells expressing LY6G6D in vitro or in vivo.

[0051] In some preferred embodiments, the disease is selected from the group consisting of colorectal cancer.

[0052] The antibody or its antigen-binding fragment provided by this invention can bind with high affinity to human LY6G6D, triggering a tumor-specific immune response and providing a new direction for the treatment of colorectal cancer. Attached Figure Description

[0053] Figure 1 To assess the binding activity of chimeric nanobodies against human LY6G6D to colorectal cancer HT-55 cells.

[0054] Figure 2 To assess the binding activity of the chimeric nanobody against human LY6G6D to the HEK293 cell line overexpressing human LY6G6D.

[0055] Figure 3 To assess the binding activity of chimeric nanobodies against human LY6G6D to the HEK293 cell line.

[0056] Figure 4 To assess the affinity of the chimeric nanobody 00FY9P001 against human LY6G6D to human LY6G6D protein.

[0057] Figure 5 To assess the affinity of the chimeric nanobody 00FY9P002 against human LY6G6D protein.

[0058] Figure 6 To assess the affinity of the chimeric nanobody 00FY9P003 against human LY6G6D protein.

[0059] Figure 7 The affinity of the positive control antibody HZ6E10 against human LY6G6D for human LY6G6D protein.

[0060] Figure 8 To investigate the binding activity of the anti-human LY6G6D tandem chimeric nanobody 00FY9P002+003-hFc to HEK293 cell lines overexpressing human LY6G6D.

[0061] Figure 9 To assess the binding activity of the anti-human LY6G6D tandem chimeric nanobody 00FY9P002+003-hFc to the HEK293 cell line.

[0062] Figure 10 To enhance the affinity of the anti-human LY6G6D tandem chimeric nanobody 00FY9P002+003-hFc for human LY6G6D protein.

[0063] Figure 11 To enhance the affinity of the anti-human LY6G6D tandem chimeric nanobody 00FY9P002 for human LY6G6D protein.

[0064] Figure 12 To enhance the affinity of the anti-human LY6G6D tandem chimeric nanobody 00FY9P003 for human LY6G6D protein.

[0065] Figure 13 To investigate the in vitro killing activity of the anti-human LY6G6D tandem chimeric nanobody 00FY9P002+003-hFc against LS1034 target cells endogenously expressing LY6G6D.

[0066] Figure 14 To investigate the in vitro killing activity of the anti-human LY6G6D tandem chimeric nanobody 00FY9P002+003-hFc against HT-55 target cells endogenously expressing LY6G6D. Detailed Implementation

[0067] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the techniques used in the embodiments are conventional practices in the art, or experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, the reagents and biological materials used in the embodiments are commercially available.

[0068] Abbreviations and Definitions

[0069] Unless otherwise stated, the following terms shall have the meanings described below. Other terms or abbreviations shall have meanings known in the art.

[0070] "Antibody" refers to any form of antibody that exhibits a desired biological activity (e.g., inhibiting the binding of a ligand to its receptor or by inhibiting receptor signal transduction induced by a ligand). Therefore, "antibody" is used in its broadest sense and explicitly includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies and multispecific antibodies (e.g., bispecific antibodies), fully human, humanized, primate-derived, chimeric antibodies, single-chain antibodies, etc.

[0071] "Antigen-binding fragment" refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of its structure, the antibody fragment binds to the same antigen recognized by the intact antibody. The term "antigen-binding fragment" includes aptamers, mirror isoforms, and bivalent antibodies. The term "antigen-binding fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by binding to a specific antigen to form a complex.

[0072] A "Fab fragment" consists of a light chain, a heavy chain (CH1), and a variable region. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0073] The “Fc region” contains two heavy chain segments, including the CH2 and CH3 domains of the antibody. The two heavy chain segments are held together by two or more disulfide bonds and through the hydrophobic interaction of the CH3 domain.

[0074] The “Fv region” contains variable regions from both the heavy and light chains, but lacks constant regions.

[0075] A "single-chain Fv antibody" (or "scFv antibody") is an antibody fragment containing both VH and VL domains of the antibody, wherein these domains are contained within a single polypeptide chain. Generally, Fv polypeptides also contain a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding. For a review of scFv, see U.S. Patent No. 6,423,538.

[0076] A "single-domain antibody" is an antigen-binding fragment containing only a single antibody variable region. Single-domain antibodies can bind to antigens without pairing with another corresponding polypeptide containing a different CDR (e.g., VL).

[0077] In this paper, a single-domain antibody containing a variable domain of the heavy chain is referred to as a "VHH (variable domain of heavy chain antibody)," also known as a nanobody (Nb). The VHH used in this invention is preferably derived from camel species, such as alpacas, or from their humanized or sequence-optimized forms. The VHH has the following structure from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0078] Those skilled in the art will understand that antibody heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses (e.g., γ1-γ4). The properties of this chain determine the "type" of the antibody, namely IgG, IgM, IgA, IgD, or IgE. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, etc., have been well characterized and their assigned functional specificities are known. All immunoglobulin types are within the scope of protection disclosed in this invention. In some embodiments, the immunoglobulin molecule is of the IgG type. IgG typically comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons and two identical heavy chain polypeptides with a molecular weight of approximately 53,000-70,000. These four chains are linked by disulfide bonds in a "Y" configuration, wherein the light chain begins at the "Y" port and continues to surround the heavy chain through a variable region. Antibodies in the form of IgG1 are a subclass of IgG, with their heavy chain being the γ1 subtype. In some embodiments, the antibody disclosed in this invention is IgG1.

[0079] As used herein, the term "heavy chain constant region" includes an amino acid sequence derived from the immunoglobulin heavy chain. A polypeptide containing a heavy chain constant region comprises at least one of the following: a CH1 domain, a hinge (e.g., an upper hinge region, a middle hinge region, and / or a lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide used in this invention may comprise: a polypeptide chain containing a CH1 domain; a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, and a CH2 domain; a polypeptide chain containing both a CH1 domain and a CH3 domain; a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, and a CH3 domain; or a polypeptide chain containing at least a portion of a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide of this invention comprises a polypeptide chain containing a CH3 domain. Furthermore, antibodies used in this invention may lack at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). As described above, those skilled in the art will understand that the heavy chain constant region can be modified to differ in amino acid sequence from naturally occurring immunoglobulin molecules. In some cases, the single-domain antibody is derived from cartilaginous fish, such as sharks, in which case the antibody comprises one variable region and five constant regions (C1-C5).

[0080] The "hypervariant region" refers to the antibody amino acid residues responsible for antigen binding. The hypervariant region contains the following amino acid residues: amino acid residues from the "complementarity-determining region" or "CDR" defined by sequence alignment. "Framework" residues or "FR" residues are variable domain residues other than those defined in the hypervariant region.

[0081] "Isolated antibody" is an antibody separated from all or part of its natural environmental components. The contaminating components of its natural environment are substances that could interfere with the diagnostic or therapeutic application of the antibody and may include enzymes, hormones, and other proteolysinic or non-proteolysinic substances. In some embodiments, the antibody is purified to the extent that: (1) more than 95% by weight, such as more than 99% by weight, as determined by the Lowry method; (2) sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a twist-cup sequencer; or (3) determined to be homogeneous by SDS-PAGE stained with Coomassie blue or silver under reducing or non-reducing conditions. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the antibody's natural environment will be absent. Isolated antibodies are typically prepared by at least one purification step. In some embodiments, the purity of the isolated antibody is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or a range between any two of these values ​​(including the endpoint) or any value therein.

[0082] "Nucleic acid" or "polynucleotide" refers to a polymer molecule composed of a single nucleotide: adenine (a), cytosine (c), guanine (g), thymine (t) (or uracil (u) in RNA), such as DNA, RNA, or modifications thereof. Nucleic acid molecules can be natural or synthetic nucleic acid molecules, or a combination of one or more natural nucleic acid molecules with one or more synthetic nucleic acid molecules. Examples of nucleic acids include, but are not limited to: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribonuclease, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.

[0083] "Isolated nucleic acid molecules" are nucleic acid molecules that have been identified and separated from at least one contaminating nucleic acid molecule. Isolated nucleic acid molecules differ from their naturally occurring form or environment. Therefore, isolated nucleic acid molecules are distinct from nucleic acid molecules present in their natural cells. However, isolated nucleic acid molecules include nucleic acid molecules contained in cells that normally express antibodies, for example, where the chromosomal location of the nucleic acid molecule differs from its chromosomal location in natural cells.

[0084] "Monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies, the individual antibodies constituting the group being identical. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, unlike conventional (polyclonal) antibody preparations, which typically include multiple different antibodies targeting multiple different determinants (epitopes), each monoclonal antibody targets only a single determinant on the antigen.

[0085] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain originates from one source or species, while the remainder of the heavy chain and / or light chain originates from a different source or species.

[0086] "Immune cells" include cells that have a hematopoietic origin and play a role in the immune response. Immune cells include: B lymphocytes, T lymphocytes, natural killer cells, monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0087] The sequence “variant” used in this article refers to a sequence that differs from the sequence shown at one or more amino acid residues but retains the biological activity of the resulting molecule.

[0088] Amino acids are organic compounds that contain both amino and carboxyl groups, such as α-amino acids, which can be encoded by nucleic acids directly or in their precursor form. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). The fact that the same amino acid can be encoded by different codons is called "degeneracy of the genetic code." Amino acids include both natural and non-natural 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).

[0089] "Variations of conserved substitutions" or "conserved amino acid substitutions" refer to amino acid substitutions known to those skilled in the art that such substitutions generally do not alter the biological activity of the resulting molecule. Generally, it is generally accepted by those skilled in the art that a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter its biological activity. Conserved substitutions can be made by amino acid substitutions with chemically similar side chains, such as: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid.

[0090] As used herein, the term "about" means a numerical value within an acceptable margin of error for a specific value determined by a person skilled in the art, the numerical value depending in part on how it is measured or determined (i.e., the limits of the measurement system). Alternatively, "about" may mean a range of up to ±20%, such as ±10%, ±5%, or ±1%. Unless otherwise stated, when a specific value appears in this application and claims, the meaning of "about" should be assumed to be within an acceptable margin of error for that specific value.

[0091] The term "identity" as used in this article can be evaluated by the naked eye or by computer software (such as the software program described in Current Protocols in Molecular Biology by Ausubel et al. eds. (2007)). When the positions in the compared sequences are occupied by the same bases or amino acids, the molecules are identical at that position. Identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. "Sequence identity" of a polynucleotide or amino acid sequence with another sequence at a certain percentage (e.g., 90%, 95%, 98%, or 99%) means that when the sequences are aligned, that percentage of bases or amino acids are the same in the two compared sequences.

[0092] When referring to ligand / receptor, antibody / antigen, or other binding pairs, "specific" binding means determining the presence of a binding reaction of the protein, such as LY6G6D, within a heterogeneous population of proteins and / or other biological reagents. Therefore, under specified conditions, a particular ligand / antigen binds to a specific receptor / antibody and does not bind in significant amounts to other proteins present in the sample.

[0093] When applied to polynucleotides, the term "encoding" refers to a polynucleotide that, if in its natural state or when manipulated by methods known to those skilled in the art, can be transcribed and / or translated to produce mRNA containing a polypeptide and / or fragments thereof, is called "encoding" a polypeptide. The antisense strand is the complement of this nucleic acid, and the coding sequence can be deduced from it.

[0094] When "giving" and "treating" are used to refer to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, it means contacting an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Giving" and "treating" can refer to, for example, methods of treatment, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating cells includes contacting a reagent with cells and contacting a reagent with a fluid, wherein the fluid contacts the cells. "Giving" and "treating" also mean, for example, in vitro and ex vivo treatment of cells by means of a reagent, diagnostic agent, conjugated composition, or other cells.

[0095] The term "treatment" refers to the improvement or cessation of a condition or its symptoms. Treatment includes suppression, such as reducing the overall frequency of attacks of a condition or its symptoms.

[0096] The term "prevention" includes avoiding the initial stages of a disease or its symptoms.

[0097] As used herein, the term "therapeutic effective dose" or "effective dose" refers to the amount of anti-LY6G6D single-domain antibody or its antigen-binding fragment that, when administered alone or in combination with another therapeutic agent to cells, tissues, or a treated individual, effectively prevents or alleviates the disease or condition to be treated. Therapeutic effective dose further refers to the amount of the antibody or its antigen-binding fragment sufficient to result in symptom relief, such relief being, for example, treatment, cure, prevention, or alleviation of an associated medical condition, or an increase in the rate of treatment, cure, prevention, or alleviation of the symptom. The effective dose for a specific treated individual can vary depending on various factors, such as the disease to be treated, the patient's overall health condition, the route and dosage of administration, and the severity of side effects. The effective dose may be the maximum dose or administration regimen that avoids significant side effects or toxicity. When administered to an individual as a single active ingredient, the therapeutic effective dose refers to that single ingredient. When administered in combination, the therapeutic effective dose refers to the combined amount of active ingredients that produce a therapeutic effect, regardless of whether they are administered in combination, continuously, or simultaneously.

[0098] Pharmaceutical Composition

[0099] The present invention also provides pharmaceutical compositions. Such compositions comprise an effective dose of an antibody or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.

[0100] In some implementations, the term "pharmaceuticalally acceptable carrier" refers to a substance approved by a government regulatory agency or listed in another recognized pharmacopoeia for use in animals (particularly for humans). Furthermore, "pharmaceuticalally acceptable carrier" will generally be any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation adjuvant.

[0101] The term "carrier" refers to a diluent, adjuvant, excipient, or carrier used in conjunction with an active ingredient for therapeutic purposes. Such drug carriers can be sterile liquids, such as water and oils, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In some embodiments, the carrier can be water when the drug composition is administered intravenously. Saline solutions, glucose solutions, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Examples of suitable drug carriers are described in EW Martin's Remington's Pharmaceutical Sciences, which are incorporated herein by reference. Such compositions will contain a clinically effective dose of an antibody or antibody fragment, along with a suitable carrier, to provide a dosage form suitable for the patient. The formulation should be suitable for the mode of administration. The formulation can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0102] In some embodiments, the pharmaceutical compositions of the present invention may be administered by any suitable route known in the art, including but not limited to: oral, nasal, intradermal, subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, and / or cerebrospinal fluid administration.

[0103] In some embodiments, the composition is formulated into a pharmaceutical composition suitable for intravenous injection into the human body according to conventional procedures. Compositions for intravenous administration are typically solutions in sterile isotonic buffer solutions. The pharmaceutical composition may also contain a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site. Generally, the active ingredient is supplied individually or in combination in unit doses, such as as a dry lyophilized powder or anhydrous concentrate in a sealed container (such as an ampoule or sachet) indicating the amount of active agent. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, ampoules of sterile water or saline for injection can be used, allowing the active ingredient to be mixed before administration.

[0104] The antibodies or antigen-binding fragments thereof of the present invention include their salt forms. Pharmaceutically acceptable salts include those derived from anions such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those derived from cations such as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0105] Example 1: Animal Immunization and Library Construction

[0106] 1. Obtaining single-domain antibodies

[0107] Two alpacas were immunized with LY6G6D protein. Peripheral blood was collected after immunization to produce V. H H phage libraries were subjected to affinity panning to obtain single-domain antibodies. The specific method is as follows:

[0108] Alpaca Immunization: Human LY6G6D Protein, C-term Fc Tag (Baiying Biotechnology, catalog number: B22256102) and Human LY6G6D Protein, N-term Fc Tag (ACRO, catalog number: LYD-H5265) were used as immunogens and diluted to 1 mg / mL with physiological saline. Then, an equal volume of adjuvant CFA (Sigma, F5881) or IFA (Sigma, F5506) was mixed (CFA was used for the initial immunization, and IFA for subsequent immunizations) and administered subcutaneously to two alpacas (400 μg / alpaca). Protein immunization was performed at least three times at two-week intervals. Seven days after the last immunization, 50 mL of peripheral blood was collected, and PBMCs were isolated. Total RNA was extracted from the PBMCs using TRIZOL reagent according to the manufacturer's protocol. cDNA was synthesized using an RNA template, oligo(dT)20 primers, and the PrimeScript™ II Reverse Transcriptase (Takara, 2690A) first-strand cDNA synthesis kit, following the manufacturer's instructions. Alpaca cDNA amplification V H H, to produce V H H phage library.

[0109] 100 μL of the phage display library was taken from 2 mL of solution, serially diluted, and 4 μL was inoculated onto a plate containing carbenicillin. The library volume was estimated by counting colonies. The phage display library (pfu) was approximately 5E+08. Single clones were randomly selected for sequencing. Sequencing results showed that the effective insertion rate of the phage display library was 100%.

[0110] Affinity panning: The constructed immune library was panned multiple times for biotinylated LY6G6D protein (Kaikai Biotechnology, catalog number: LYD-HM4GDB) and HEK293 overexpressing LY6G6D (Jiman Biotechnology Co., Ltd., catalog number: GM-C27573). After each round of panning, the panned library was amplified. From the eluent titer plate, 96 clones were randomly selected with a sterile toothpick and inoculated into 1 mL of 2×YT-A sterile liquid medium (prepared and provided by Baiying Biotechnology). The culture was incubated at 37°C with shaking at 230 r / min for 8 h. 200 µL of the above culture was taken and M13K07 helper phage (NEB, catalog number: N0315S) was added at a cell:phage ratio of 1:20. The culture was incubated at 37°C for 15 min, then shaken at 220 r / min for 45 min. Add 800 µL of 2×YT-AK (provided by Baiying Biotechnology), incubate overnight at 30°C with vigorous shaking. The next day, centrifuge at 12000 rpm for 2 min, collect the supernatant, and use it for cell binding FACS identification.

[0111] Cellular FACS screening for positive clones: HEK293 cells overexpressing human LY6G6D (Jiman Biotechnology Co., Ltd., catalog number: GM-C27573) or empty HEK293 cells were adjusted to a concentration of 1×10⁻⁶ cells using FACS buffer (PBS solution containing 1% FBS). 6Cells / mL were collected at a rate of 100 μL / well in a 96-well U-bottom plate. After centrifugation, the supernatant was discarded. 50 μL of phage supernatant, positive control antibody HZ20A12 (the humanized 20A12 sequence from patent WO2021119505A1, see Table 3, synthesized by Hongcheng Pharmaceutical), and negative control antibody (Baiying Biotechnology, catalog number: 00K0W3003) were added, and the cells were incubated at 4°C for 1 hour. After incubation, the cells were centrifuged and washed three times with FACS buffer. PE-labeled Anti-M13 Mouse Antibody (SinoBiological, 11973-MM05T-P) was diluted 100-fold with FACS buffer (PBS solution containing 1% FBS) or Alexa Fluor® 647 AffiniPure Goat Anti-Human IgG (Jacksons Lab, catalog number: 109-605-190) with FACS buffer (PBS solution containing 1% FBS). 100 μL of secondary antibody dilution buffer was added to each well to resuspend the cell clumps, and the mixture was pipetted and incubated at 4°C for approximately 30 minutes. After incubation, the cells were centrifuged, washed three times with FACS buffer, and then resuspended in FACS buffer at 100 μL / well. The mean fluorescence intensity (MFI) was read using a flow cytometer (Agilent, NovoCyte 2060R), and the results are shown in Table 1. The screening results yielded 189 positive clones. Phylogenetic sequencing, combined with grouping of CDR3 sequences by different lengths, and phylogenetic tree analysis were performed. Regions with significant differences in CDR3 were selected, ultimately resulting in six recombinant single-domain antibodies. All six different sequences were validated using colorectal cancer HT55 cells.

[0112] Table 1. Phage supernatant screening results

[0113]

[0114] HT-55 cell binding validation for colorectal cancer: The concentration of HT-55 cells (Nanjing Kebai, catalog number: CBP60012) was adjusted to 1×10⁻⁶ using FACS buffer (PBS solution containing 1% FBS). 6Cells / mL were collected and injected into 96-well U-bottom plates at a rate of 100 μL / well. After centrifugation, the supernatant was discarded. 50 μL of phage supernatant, positive control antibody hz20A12, and negative control antibody (Baiying Biotechnology, catalog number: 00K0W3003) were added to each well, and the plates were incubated at 4°C for 1 hour. After incubation, the cells were centrifuged and washed three times with FACS buffer. PE-labeled Anti-M13 Mouse Antibody (Sino Biological, 11973-MM05T-P) was diluted 100-fold with FACS buffer (PBS solution containing 1% FBS) or Alexa Fluor® 647 AffiniPure Goat Anti-Human IgG (Jacksons Lab, catalog number: 109-605-190) with FACS buffer (PBS solution containing 1% FBS). 100 μL of secondary antibody dilution buffer was added to each well to resuspend the cell clumps, and the mixture was pipetted and incubated at 4°C for approximately 30 minutes. After incubation, the cells were centrifuged, washed three times with FACS buffer, and then resuspended in FACS buffer at 100 μL / well. The mean fluorescence intensity (MFI) was read using a flow cytometer (Agilent, NovoCyte 2060R).

[0115] The results are as follows Figure 1 As shown in the figure. Through HT-55 binding experiments, three of them (CP1R3-25, CP1R3-31, and CP1R3-40, with sequences detailed in Table 2) bound to HT-55. These were selected for recombinant nanobody expression and further characterized.

[0116] 2. Preparation of chimeric nanobodies against human LY6G6D

[0117] The CP1R3-25, CP1R3-31, and CP1R3-40 sequences selected above are shown in Table 2.

[0118] Table 2: CDR sequence and variable region sequence of chimeric nanobodies against human LY6G6D (determined according to the Kabat protocol)

[0119]

[0120] The heavy chain variable region (Table 2) was constructed onto the human constant region (IgG1, Table 3) to construct the corresponding chimeric antibodies (Table 4), and the sequences were verified by sequencing.

[0121] The corresponding nucleic acid encoding the chimeric antibody was expressed in Expi293 cells and purified using a Protein A column, as follows:

[0122] Expi293 cells expressing chimeric antibodies: One day before transfection, Expi293 cells (Thermo, catalog number: A14635CN) were diluted to a density of 1.5 × 10⁻⁶. 6 Cells / mL were cultured at 37°C in an 8% CO2 shaker at 120 rpm. On the second day, viable cell density and survival rate were measured; the cell transfection density should be 3 × 10⁻⁶ cells / mL. 6 Cells / mL, cell viability >95%. Preparation of the PEI / plasmid complex: Mix PEI (1 mg / mL, Polysciences, catalog number: 24765-1) by inverting. Dilute the expression plasmid (Invitrogen pCDNA3.4 vector, catalog number: A14697) with OPM-293CD05 medium (Shanghai Aopomai Biotechnology Co., Ltd., catalog number: 81075-001), to a total plasmid volume of 1 μg / mL. The volume of the medium used to dilute the plasmid should be 1 / 20 of the transfection volume. Mix gently, ensuring a light to heavy chain plasmid ratio of 1:1.5. Dilute the PEI reagent with OPM-293CD05 medium, to a volume 1 / 20 of the transfection volume. Mix gently by inverting and incubate at room temperature for 5 minutes. Add the diluted PEI reagent to the diluted plasmid and mix gently by inverting. Incubate the PEI / plasmid complex at room temperature for 15 minutes, then slowly add the solution dropwise to a transfer flask, gently rotating the flask during the addition. After transfection, incubate the flask at 37°C and 8% CO2 on a shaker at 120 rpm. On the second day after transfection (24 hours post-transfection), add 10% OPM-293 ProFeed (Shanghai Aopumai Biotechnology Co., Ltd., catalog number: F081918) to the flask, gently rotating the flask during the addition. Then, return the flask to the shaker and continue incubating for 5-7 days, harvesting the supernatant.

[0123] Protein A column purification of antibodies: Prepare a gravity chromatography column. Open the column cap and place the gasket at the bottom of the column, pressing it firmly. Prepare the packing material, Protein A (Cytiva, catalog number: 17549801). Calculate the required packing suspension volume precisely based on the target packing volume and the packing suspension ratio: Required packing suspension volume = Target packing volume / Packing suspension ratio. Vortex the packing material thoroughly to ensure complete suspension. Add the packing suspension to the bottom of the gravity chromatography column. Add at least 10 CV of equilibration buffer (PBS) to the column. After equilibration, check the outlet pH. If the target pH of 7.4 is not reached, continue adding equilibration buffer until the target pH of 7.4 is reached. Slowly add a certain volume of sample to the column. Add at least 10 CV of eluent to the column. Slowly add 5CV of elution buffer (10-50 mM NaAc, pH 3.0-3.5) to the gravity chromatography column and incubate for 3-5 minutes. Collect the eluent. Repeat the elution step as needed. Neutralization: Adjust the pH to the target pH of 7.4 with neutralization buffer (1M Tris). Determine the protein concentration using Nanodrop. Replace the antibody-containing buffer with PBS via ultrafiltration.

[0124] Table 3: Constant region sequences and positive control antibody sequences

[0125]

[0126] Table 4: Full-length sequence of anti-human LY6G6D chimeric nanobody

[0127]

[0128] Example 2: Functional identification of chimeric nanobodies against human LY6G6D

[0129] 1. Binding activity of anti-human LY6G6D chimeric nanobody to HEK293 cell line overexpressing human LY6G6D

[0130] The concentration of HEK293 cells expressing human LY6G6D (Jiman Biotechnology Co., Ltd., catalog number: GM-C27573) or empty HEK293 cell lines was adjusted to 1×10⁻⁶ using FACS buffer (PBS solution containing 1% FBS). 6Cells / mL were collected and placed at 100 μL / well in a 96-well U-bottom plate. After centrifugation, the supernatant was discarded. The chimeric nanobody against human LY6G6D, the positive control antibody HZ20A12, and the negative control antibody (Baiying Biotechnology, catalog number: 00K0W3003) were diluted to an initial working concentration of 100 nM in FACS buffer (PBS solution containing 1% FBS). Then, serial dilutions were performed using FACS buffer (4-fold dilution, 8 concentration points in total). The serially diluted antibody was added at 100 μL / well to resuspend the cells, mixed by pipetting, and incubated at 4°C for 1 hour. After incubation, the cells were centrifuged and washed three times with FACS buffer. The Alexa Flour-647-labeled anti-human secondary antibody (Invitrogen, A-21445) was diluted 1:1000 with FACS buffer (PBS solution containing 1% FBS). 100 μL of the secondary antibody dilution was added to each well to resuspend cell clumps, and the mixture was pipetted and incubated at 4°C for approximately 45 minutes. After incubation, the cells were centrifuged, washed three times with FACS buffer, and then resuspended in 100 μL of FACS buffer per well. Mean fluorescence intensity (MFI) was read using flow cytometry. The experimental data were analyzed using Graphpad Prism 8.0 software, with the logarithm of antibody concentration on the x-axis and the corresponding MFI value on the y-axis. A four-parameter regression model was used to fit the antibody dose-response curve and calculate the EC50. 50 The result is as follows Figure 2 , 3 As shown in Table 5.

[0131] Table 5: Binding activity of anti-human LY6G6D chimeric nanobodies to HEK293 cell lines overexpressing human LY6G6D

[0132]

[0133] The results showed that the anti-human LY6G6D chimeric nanobody had EC50 (nM) binding activity comparable to the positive control antibody against HEK293 cells overexpressing human LY6G6D, but had no binding activity against HEK293 blank cells.

[0134] 2. Antibody affinity determination based on surface plasmon resonance (SPR)

[0135] The affinity of the anti-human LY6G6D chimeric nanobody was determined using the Biacore 8K SPR system (Cytiva, catalog number: 29277881) based on surface plasmon resonance technology. The anti-LY6G6D chimeric nanobody and HZ6E10 (the humanized 6E10 sequence in patent WO2021119505A1, see Table 3, synthesized by Hongcheng Pharmaceutical) were immobilized on the surface of a Protein A chip (Cytiva; catalog number: Biacore Sensor Chip Protein A). The human LY6G6D (C-His) protein (Kaikai Biotechnology, catalog number: LYD-HM1GD) was then diluted to a concentration of 200 nM and flowed through the chip surface at a flow rate of 30 μL / min. The binding time was 180 s, and the dissociation time was 1200 s. After each cycle, the chip surface was regenerated with 10 M, pH 1.5 Glycine. The kinetic rate constant was adjusted by subtracting the blank control, and data were fitted using a 1:1 global fit analysis model. The dissociation equilibrium rate constant (KD) was calculated using the following formula: K D =kd / ka. The result is as follows: Figure 4 , 5 As shown in 6, 7 and Table 6.

[0136] Table 6: Affinity of anti-human LY6G6D chimeric nanobodies

[0137]

[0138] The results showed that the anti-human LY6G6D chimeric nanobody had binding activity to the recombinant human LY6G6D (C-His) protein, with an affinity range of 1.11E-07 M to 3.54E-08 M.

[0139] Example 3: Functional Identification of Tandem Chimeric Nanobodies

[0140] To enhance antibody affinity, two nanobodies with higher affinity (00FY9P002 and 00FY9P003) were expressed as tandem nanobodies with Fc tags (00FY9P002+003-hFc), the sequences of which are shown in Table 7. Tandem chimeric nanobodies were prepared according to the method for preparing the anti-human LY6G6D chimeric nanobodies in Example 1.

[0141] Table 7. Sequences of anti-human LY6G6D tandem chimeric nanobodies

[0142]

[0143] 1. Binding activity of anti-human LY6G6D tandem chimeric nanobody to HEK293 cell line overexpressing human LY6G6D

[0144] The concentration of HEK293 cells overexpressing human LY6G6D (Jiman Biotechnology Co., Ltd., catalog number: GM-C27573) or empty HEK293 cell lines was adjusted to 1×10⁻⁶ using FACS buffer (PBS solution containing 1% FBS). 6 Cells / mL were collected and placed at 100 μL / well in a 96-well U-bottom plate. After centrifugation, the supernatant was discarded. The anti-human LY6G6D tandem chimeric antibody, positive control antibody HZ20A12, and negative control antibody (Baiying Biotechnology, catalog number: 00K0W3003) were diluted to an initial working concentration of 100 nM using FACS buffer (PBS solution containing 1% FBS). Then, a serial dilution (4-fold dilution, 8 concentration points) was performed using FACS buffer. Cells were resuspended in the serially diluted antibody at 100 μL / well, mixed by pipetting, and incubated at 4°C for 1 hour. After incubation, the cells were centrifuged and washed three times with FACS buffer. The Alexa Flour-647-labeled anti-human secondary antibody (Invitrogen, A-21445) was diluted 1:1000 with FACS buffer (PBS solution containing 1% FBS). 100 μL of the secondary antibody dilution was added to each well to resuspend cell clumps, and the mixture was pipetted and incubated at 4°C for approximately 45 minutes. After incubation, the cells were centrifuged, washed three times with FACS buffer, and then resuspended in 100 μL of FACS buffer per well. Mean fluorescence intensity (MFI) was read using flow cytometry. The experimental data were analyzed using Graphpad Prism 8.0 software, with the logarithm of antibody concentration on the x-axis and the corresponding MFI value on the y-axis. A four-parameter regression model was used to fit the antibody dose-response curve and calculate the EC50. 50 The result is as follows Figure 8 , 9 As shown in Table 8.

[0145] Table 8 Cell line binding activity of anti-human LY6G6D tandem chimeric nanobodies

[0146]

[0147] The results showed that the anti-human LY6G6D tandem chimeric nanobody had binding activity comparable to the positive control antibody HZ20A12 in HEK293 cells overexpressing human LY6G6D, but had no binding activity in HEK293 blank cells.

[0148] 2. Affinity determination of anti-human LY6G6D tandem chimeric nanobodies

[0149] The affinity of anti-human LY6G6D chimeric nanobodies was determined using a Biacore 8K SPR system (Cytiva, catalog number 29277881) based on surface plasmon resonance (SPR) technology. Human antigen LY6G6D (LY6G6D(C-His)) was immobilized on the surface of a CM5 chip (Cytiva, catalog number Biacore Sensor Chip CM5). The antibody to be tested was then diluted to 200 nM and flowed through the chip surface at a flow rate of 30 μl / min. The binding time was 180 s, and the dissociation time was 1200 s. After each cycle, the chip surface was regenerated with 10 M, pH 1.5 Glycine. The kinetic rate constant was calculated by subtracting the blank control and fitting the data using a 1:1 global fit analysis. The dissociation equilibrium rate constant (KD) was calculated using the following formula: K D =kd / ka.

[0150] The results are as follows Figure 10 , Figure 11 , Figure 12 As shown in Table 9, the results indicate that the anti-human LY6G6D tandem chimeric nanobody (00FY9P002+003-hFc) significantly improved the affinity for the recombinant human LY6G6D (C-His) protein.

[0151] Table 9: Affinity of anti-human LY6G6D tandem chimeric nanobodies

[0152]

[0153] Example 4: In vitro cell killing experiment of CAR-T cells

[0154] (1) Preparation of anti-LY6G6D CAR-T

[0155] A CAR structure was constructed using an anti-LY6G6D tandem chimeric nanobody (00FY9P002+003-hFc) and a positive control HZ20A12 (the heavy chain variable region was linked to the light chain variable region via 3×G4S to construct an scFV). The full-length CAR from the N-terminus to the C-terminus consists of: CD8α signal peptide, LY6G6D binding domain (nanobody or positive control antibody), CD8α hinge, CD8α transmembrane region, 4-1BB intracellular domain, and a partial intracellular domain of CD3. The amino acid and nucleotide sequences of each element of the CAR are shown in Table 10.

[0156] Table 10. Amino acid and nucleotide sequences of each CAR element

[0157]

[0158] (2) Lentiviral preparation

[0159] On day 0, HEK-293T cells (ATCC) were injected at a rate of 25,000 cells / cm³. 3 Inoculate into 10-layer cell factories, adding 1000 mL of DMEM medium containing 10% FBS to each factory; the next day, mix 200 μg PMD2.G, 200 μg PREV, 400 μg PRRE, and 800 μg PEV of the lentiviral packaging vector in 50 mL of DMEM (DNA mixture) and incubate at room temperature for 5 minutes; add 1.6 mL of PEI to 50 mL of DMEM, incubate at room temperature for 5 minutes, then add to the DNA mixture, incubate at room temperature for 15 minutes, and then add to the cell factories. Change the medium 6-8 hours after transfection. 48 hours after transfection, collect the viral supernatant, centrifuge to remove debris (4000g, 15 min), then centrifuge to concentrate (16000g, 4 h), remove the supernatant, resuspend in viral preservation solution, and aliquot.

[0160] (3) CAR-T preparation

[0161] On day 0, PBMCs (Shanghai Miaoshun, catalog number P122051102C) were resuscitated. T cells were sorted using DYNABEADs CD3 / CD28 (Gibco, 40203D) and activated in X-VIVO 15 (Lonza, BEBP02-054Q) supplemented with 5% FBS and 100 IU / mL IL-2 (RD, BT-002-GMP-050). T cells were re-seeded at 2E+05 cells / mL. Lentiviral virus was added to the T cells on day 2. On day 3, the culture medium was replaced with T cell expansion medium, i.e., X-VIVO 15 supplemented with 5% AB (GEMINI, 100-512) and 100 IU / mL IL-2 GMP. On day 5, DYNABEADs CD3 / CD28 were removed from the cell suspension using a magnetic pole, and the T cell expansion medium was replaced. On days 9 and 13, transduction efficiency was determined by detecting the percentage of T cells recognizing recombinant LY6G6D using flow cytometry. Cells were expanded using T cell expansion medium in larger culture vessels as needed. LY6G6D CAR-T cells were cryopreserved on day 14.

[0162] (4) CAR-T in vitro functional testing

[0163] HT-55 and LS1034 (ATCC, catalog number CRL2158ATC) luciferase-expressing target cells in good growth condition (1E+04 cells / well, 100 μL / well) were seeded into 96-well white opaque cell culture plates with 3-5 replicates. Then, the target-effect ratio was calculated based on the CAR positivity rate, and effector cells (100 μL / well) were added at target-effect ratios of 4:1, 2:1, 1:1, 1:2, 1:4, and 1:8, respectively. Culture medium was added as a negative control, and 2% Triton X-100 (BioFroxx, catalog number 1139ML100) was added as a maximum release control (MAX). After cell adhesion, effector cells were added. After co-incubation for 24h±2h, centrifuge at 125g for 5min, and aspirate 130μl of cell supernatant for cytokine detection. Add 70μL (1:1 with the liquid volume in the well plate) of pre-mixed glow-type firefly luciferase (Shanghai Yisheng, 11404ES80) to each well of the plate, shake well in the dark for 5-10min, detect the luciferescence signal, and calculate the CAR-T killing rate according to the following formula.

[0164] CAR-T kill rate (%) = (RLU_NC - RLU_(CAR-T)) / (RLU_NC - RLU_MAX) × 100%

[0165] The results of the in vitro killing ability test of LS1034 and HT-55 target cells endogenously expressing LY6G6D are as follows: Figure 13 , Figure 14 As shown, the CAR-T constructed from the tandem chimeric nanobody (00FY9P002+003-hFc) achieved in vitro killing activity comparable to the positive control in LS1034 and HT-55 cells.

Claims

1. A single-domain antibody against LY6G6D, wherein the antibody comprises a heavy chain variable region, the heavy chain variable region comprising three heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3; wherein, The amino acid sequences of HCDR1, HCDR2, and HCDR3 contained in the antibody are shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively; or, The amino acid sequences of HCDR1, HCDR2, and HCDR3 contained in the antibody are shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 6, respectively; or, The amino acid sequences of HCDR1, HCDR2 and HCDR3 contained in the antibody are shown in SEQ ID NO: 2, SEQ ID NO: 8 and SEQ ID NO: 9, respectively.

2. The anti-LY6G6D single-domain antibody according to claim 1, comprising a heavy chain variable region, wherein the amino acid sequence of the heavy chain variable region has at least 90% sequence identity with the sequence shown in SEQ ID NO: 1, 5 or 7.

3. The anti-LY6G6D single-domain antibody according to claim 1, comprising a heavy chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 1, 5 or 7.

4. An antibody comprising the anti-LY6G6D single-domain antibody according to any one of claims 1 to 3, further comprising a heavy chain constant region or an Fc region.

5. The antibody according to claim 4, wherein, The sequence of the Fc region is shown in SEQ ID NO:

10.

6. The antibody according to claim 5, wherein its sequence has at least 90% sequence identity with the sequence shown in SEQ ID NO: 15, 16 or 17.

7. The antibody according to claim 5, wherein the sequence is shown in SEQ ID NO: 15, 16 or 17.

8. A tandem antibody comprising two anti-LY6G6D single-domain antibodies as described in any one of claims 1 to 3, connected in tandem, wherein, Two anti-LY6G6D single-domain antibodies are linked by a linker. The amino acid sequence of the first antibody is shown in SEQ ID NO: 5, and the amino acid sequence of the second antibody is shown in SEQ ID NO:

7. The linker is (GGGGS)n, where n is an integer from 1 to 10.

9. A tandem antibody, the amino acid sequence of which is shown in SEQ ID NO:

18.

10. A CAR comprising the tandem antibody of claim 8 or 9, further comprising a CD8α signal peptide, a CD8α hinge, a CD8α transmembrane region, a 4-1BB intracellular domain, and a partial CD3 intracellular domain, wherein, From the N-terminus to the C-terminus, the CAR comprises, in sequence, a CD8α signal peptide, the tandem antibody as described in claim 8 or 9, a CD8α hinge, a CD8α transmembrane region, a 4-1BB intracellular domain, and a partial CD3 intracellular domain; the amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO: 19, the amino acid sequences of the CD8α hinge and the CD8α transmembrane region are shown in SEQ ID NO: 21, the amino acid sequence of the 4-1BB intracellular domain is shown in SEQ ID NO: 23, and the amino acid sequence of the partial CD3 intracellular domain is shown in SEQ ID NO:

25.

11. A biological material selected from the following a) to c) a) A nucleic acid molecule encoding the anti-LY6G6D single-domain antibody as described in any one of claims 1 to 3, the antibody as described in any one of claims 4 to 7, the tandem antibody as described in any one of claims 8 to 9, or the CAR as described in claim 10; b) A recombinant vector comprising the nucleic acid molecule described in a); c) Recombinant cells comprising the nucleic acid molecules described in a) and / or the recombinant vector described in b).

12. A method for preparing the anti-LY6G6D single-domain antibody according to any one of claims 1 to 3, the antibody according to any one of claims 4 to 7, the tandem antibody according to any one of claims 8 to 9, or the CAR according to claim 10, comprising, when suitable for antibody expression, culturing recombinant cells comprising a nucleic acid molecule encoding the anti-LY6G6D single-domain antibody according to any one of claims 1 to 3, the antibody according to any one of claims 4 to 7, the tandem antibody according to any one of claims 8 to 9, or the CAR according to claim 10.

13. A composition comprising the anti-LY6G6D single-domain antibody of any one of claims 1 to 3, the antibody of any one of claims 4 to 7, the tandem antibody of any one of claims 8 to 9, the CAR of claim 10, or the biomaterial of claim 11, and a pharmaceutically acceptable carrier.

14. The use of the anti-LY6G6D single-domain antibody according to any one of claims 1 to 3, the antibody according to any one of claims 4 to 7, the tandem antibody according to any one of claims 8 to 9, or the CAR according to claim 10, the biomaterial according to claim 11, and / or the composition according to claim 13 in the preparation of a reagent for detecting LY6G6D.

15. The use of the antibody according to any one of claims 4 to 7, the tandem antibody according to claim 9, or the CAR according to claim 10 in the preparation of a medicament for treating a disease associated with abnormal LY6G6D expression; wherein the disease is colorectal cancer.

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