CN121108346A - Anti-LY 6G6D single domain antibody, and preparation method and application thereof - Google Patents

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

Anti-LY 6G6D single domain antibody, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to an anti-LY 6G6D antibody and application thereof, in particular to an anti-LY 6G6D single domain antibody.

Background

Colorectal cancer (colorectal cancer, CRC) ranks second in global cancer mortality, but mCRC first-line targeted drugs are scarce and have problems of low efficacy, resistance. The chemotherapy drugs have clinical difficulties such as low curative effect, large toxic and side effects, reversion of drug resistance and the like, so that the prognosis of a CRC patient is poor. The existing targeted drugs are mainly immune checkpoint inhibitors ICI, but most colorectal cancer patients do not benefit from ICI treatment due to the proportion of problems.

The lymphocyte antigen 6 complex site G6D (Lymphocyte antigen 6 complex locus G6D, LY G6D), also known as LY6-D, LY6-G, G6D, NG, MEGT1, C6orf23, is one of the members of the lymphocyte antigen 6 superfamily (Ly 6 SF), is a phosphatidylinositol, and can be anchored to the cell membrane by Glycosyl Phosphatidylinositol (GPI). LY6G6D is a dual mechanism target, has TSA characteristics, CRC specific expression and very little normal tissue expression, can regulate immune JAK/STAT inhibitor, and can increase CRC cell death by targeting STAT5/LY6G6D axis.

LY6G6D is highly expressed in primary and metastatic colorectal tumor tissue compared to normal colorectal tissue. High expression of LY6G6D is associated with high infiltration of immunosuppressive cells, and increased LY6G6D expression and STAT5 activation in colon cancer with reduced cd8+ T lymphocytes. The LY6G6D level was significantly reduced after STAT5 knockout, indicating that STAT5 was able to regulate LY6G6D expression. In addition, LY6G6D positive MSS cells (which may be myeloid derived suppressor cells, MDSCs) expand, thereby suppressing T cell proliferation. Also in CRC, inhibitors of p38α MAPK and DNA methyltransferase DNMT1 knockdown may result in reduced LY6G6D expression. In the metastatic CRC group LY6G6D hypermethylation is indicative of resistance to FOLFOX (the drugs folinic acid, fluorouracil (5 FU) and oxaliplatin) first-line therapy. Therefore, in colorectal cancer, LY6G6D differential high expression is closely related to cancer occurrence, and is expected to be used as a potential prediction index or treatment target of colorectal cancer.

Disclosure of Invention

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 recombination site G6D (Lymphocyte antigen 6 complex locus G6D, LY G6D).

In a first aspect, the invention provides a single domain antibody or antigen binding fragment thereof having binding specificity to human lymphocyte antigen 6 recombination site G6D (LY 6G 6D), wherein said antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising three heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 comprising the sequences shown in SEQ ID NOs 1, 5 or 7.

The sequences of CDRs can be determined by one skilled in the art using methods well known in the art from the heavy chain variable region sequences and light chain variable region sequences shown. The methods known in the art may be Kabat protocol, abM protocol, chothia protocol or Contact protocol. CDR region sequences obtained using different schemes for the same variable region sequence will vary, as is well known in the art. And those skilled in the art will appreciate that CDR regions obtained in different schemes are within the scope of the present invention.

In some embodiments, the CDR region sequences are determined using the Kabat protocol.

In some embodiments, the antibody or antigen binding fragment thereof comprises the amino acid sequences of HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4, respectively, or

The antibody or antigen binding fragment thereof comprises the amino acid sequences of HCDR1, HCDR2 and HCDR3 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 or antigen binding fragment thereof are shown as SEQ ID NO. 2, SEQ ID NO.8 and SEQ ID NO. 9 respectively.

In some embodiments, the antibody or antigen binding fragment thereof comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID No. 1, 5 or 7, or having at least 90% sequence identity, 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%, at least 99% sequence identity, to the sequence set forth in SEQ ID No. 1, 5 or 7.

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

In some embodiments, the heavy chain constant region is selected from IgG, igM, igA, igE or IgD classes.

In some embodiments, the heavy chain constant region is a heavy chain constant region selected from the subclasses IgG1, igG2, igG3, or IgG 4.

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

In some embodiments, the sequence of the human IgG1 heavy chain constant region is set forth in SEQ ID NO. 10.

In some embodiments, the antibody or antigen binding fragment thereof is a chimeric, humanized antibody.

In some embodiments, the antibody or antigen binding fragment thereof is a VHH.

In some embodiments, the antibody or antigen binding fragment thereof is a single domain antibody or nanobody.

In some embodiments, the antibody or antigen binding fragment thereof has a sequence as set forth in SEQ ID NO. 15, 16 or 17, or has at least 90% sequence identity, 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%, at least 99% sequence identity, to the sequence set forth in SEQ ID NO. 15, 16 or 17.

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

In some embodiments, the antibody is a tandem antibody comprising two or more antibodies or antigen binding fragments thereof described above connected in tandem.

In some embodiments, two or more antibodies or antigen binding fragments thereof are linked by a linker. The linker may be a polypeptide linker conventionally used in the art, such as (GGGGS) n, where n is an integer of 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).

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

The first antibody or antigen binding fragment thereof comprises three heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 comprised by the sequence shown as SEQ ID No. 5;

The second antibody or antigen binding fragment thereof comprises three heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 comprised by the sequence shown as SEQ ID No. 7.

In some preferred embodiments, the amino acid sequence of the first antibody or antigen binding fragment thereof is as set forth in SEQ ID NO. 5, or has at least 90% sequence identity, 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%, at least 99% sequence identity, to the sequence set forth in SEQ ID NO. 5. The amino acid sequence of the second antibody or antigen binding fragment thereof is as shown in SEQ ID NO. 7, or has at least 90% sequence identity, 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%, at least 99% sequence identity, to the sequence shown in SEQ ID NO. 7.

In some embodiments, the tandem antibody further comprises an Fc fragment.

In some embodiments, the amino acid sequence of the tandem antibody is as set forth in SEQ ID NO. 18, or has at least 90% sequence identity, 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%, at least 99% sequence identity, to the sequence set forth in SEQ ID NO. 18.

In some embodiments, the antibody further comprises a CD8 alpha signal peptide, a CD8 alpha hinge, a CD8 alpha transmembrane region, a 4-1BB intracellular domain, a CD3 partial intracellular domain.

In some embodiments, the antibody comprises, from N-terminus to C-terminus, a CD8 a signal peptide, a single domain antibody of the invention or antigen binding fragment thereof, or a tandem antibody of the invention, a CD8 a hinge, a CD8 a transmembrane region, a 4-1BB intracellular domain, a CD3 moiety intracellular domain, linked in sequence.

In some embodiments, the amino acid sequence of the CD 8. Alpha. Signal peptide is shown in SEQ ID NO. 19, the amino acid sequences of the CD 8. Alpha. Hinge and CD 8. Alpha. 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 CD3 partial intracellular domain is shown in SEQ ID NO. 25.

In a preferred embodiment, the antibody comprises, from N-terminus to C-terminus, a CD8 alpha signal peptide, a tandem antibody, a CD8 alpha hinge, a CD8 alpha transmembrane region, a 4-1BB intracellular domain, and a CD3 portion of the intracellular domain, wherein the amino acid sequence of the CD8 alpha 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 CD8 alpha hinge and CD8 alpha 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 CD3 portion of the intracellular domain is shown in SEQ ID NO. 25, which are sequentially linked.

In a second aspect, a biomaterial is provided, selected from a) to c) below

A) A nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of the invention;

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, or the like.

B) A recombinant vector comprising the above nucleic acid molecule;

The vector may be an expression vector, in some embodiments, a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector, and the like.

C) A recombinant cell comprising the above nucleic acid molecule and/or the above recombinant vector.

The recombinant cells are obtained by transforming or infecting the nucleic acid molecules or recombinant vectors of the invention with host cells. In some embodiments, the host cell may be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal, plant or animal cell. In some embodiments, the host cell is prokaryotic, such as e.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 the production of antibodies or antigen-binding fragments thereof.

In a third aspect, there is provided a method of making an antibody or antigen-binding fragment thereof of the invention, the method comprising culturing a recombinant cell comprising a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of the invention, under conditions suitable for expression of the antibody.

In some embodiments, the method further comprises recovering the antibody or antigen binding fragment thereof from the recombinant cell or culture medium.

In a fourth aspect, there is provided a composition comprising an antibody or antigen-binding fragment thereof or a biological material according to the invention, and a pharmaceutically acceptable carrier.

In some embodiments, the above composition is a pharmaceutical composition.

In a fifth aspect, there is provided the use of an antibody or antigen-binding fragment thereof, a biological material and/or a composition as described in any one of the preceding claims for the preparation of a product as described in any one of the following:

(a) Detecting a product of LY6G 6D;

(b) A product that stimulates or enhances an immune response;

(c) A product for preventing and/or treating a disease associated with abnormal expression of LY6G 6D;

(d) A product that kills cells expressing LY6G6D or inhibits growth of cells expressing LY6G6D in vitro or in vivo.

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

The antibody or antigen binding fragment thereof provided by the invention can be combined with human LY6G6D with high affinity, can trigger anti-tumor specific immune response, and provides a new direction for colorectal cancer treatment.

Drawings

FIG. 1 shows the binding activity of chimeric nanobodies against human LY6G6D to colorectal cancer HT-55 cells.

FIG. 2 is the binding activity of chimeric nanobodies against human LY6G6D to HEK293 cell lines overexpressing human LY6G 6D.

FIG. 3 is a graph showing the binding activity of chimeric nanobodies against human LY6G6D to HEK293 cell lines.

FIG. 4 is a graph showing the affinity of chimeric nanobody 00FY9P001 against human LY6G6D with human LY6G6D protein.

FIG. 5 is a graph showing the affinity of chimeric nanobody 00FY9P002 against human LY6G6D with human LY6G6D protein.

FIG. 6 is a graph showing the affinity of chimeric nanobody 00FY9P003 against human LY6G6D with human LY6G6D protein.

FIG. 7 is the affinity of the positive control antibody HZ6E10 against human LY6G6D for human LY6G6D protein.

FIG. 8 is the binding activity of the tandem chimeric nanobody 00FY9P002+003-hFc against human LY6G6D to HEK293 cell line overexpressing human LY6G 6D.

FIG. 9 is a graph showing the binding activity of the tandem chimeric nanobody 00FY9P002+003-hFc against human LY6G6D to HEK293 cell line.

FIG. 10 shows the affinity of the anti-human LY6G6D tandem chimeric nanobody 00FY9P002+003-hFc to human LY6G6D protein.

FIG. 11 is an illustration of the affinity of anti-human LY6G6D tandem chimeric nanobody 00FY9P002 to human LY6G6D protein.

FIG. 12 is an affinity of anti-human LY6G6D tandem chimeric nanobody 00FY9P003 with human LY6G6D protein.

FIG. 13 is an in vitro killing activity of anti-human LY6G6D tandem chimeric nanobody 00FY9P002+003-hFc against LS1034 target cells endogenously expressing LY6G 6D.

FIG. 14 is an in vitro killing activity of anti-human LY6G6D tandem chimeric nanobody 00FY9P002+003-hFc against HT-55 target cells endogenously expressing LY6G 6D.

Detailed Description

The invention will be further illustrated with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Unless otherwise indicated, all technical means used in the examples are routine in the art or according to the experimental methods suggested by the manufacturers of the kits and instruments. Reagents and biological materials used in the examples were obtained commercially unless otherwise specified.

Abbreviations and definitions

Unless otherwise indicated, the following terms shall have the meanings set forth below. Other terms or abbreviations have the meaning well known in the art.

An "antibody" refers to any form of antibody that exhibits a desired biological activity (e.g., inhibits ligand binding to its receptor or receptor signaling induced by inhibition of ligand). Thus, "antibody" is used in its broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies), fully humanized, primatized, chimeric antibodies, single chain antibodies, and the like.

An "antigen binding fragment" refers to a portion of an antibody, such as F (ab ') 2, F (ab) 2, fab', fab, fv, scFv, and the like. Regardless of its structure, the antibody fragment binds to the same antigen that is recognized by the intact antibody. The term "antigen binding fragment" includes aptamers, stereoisomers, and diabodies. 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.

"Fab fragment" consists of a light chain and a heavy chain CH1 and variable domains. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

The "Fc region" contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic effect of the CH3 domain.

The "Fv region" comprises variable regions from both the heavy and light chains, but lacks constant regions.

"Single chain Fv antibody" (or "scFv antibody") refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, fv polypeptides additionally comprise a polypeptide linker between the VH and VL domains that allows the scFv to form the desired structure for antigen binding. For an overview of scFv, see U.S. Pat. No. 6423538.

"Single domain antibody" refers to an antigen binding fragment comprising only a single antibody variable region. The single domain antibody is capable of binding to an antigen without the need to pair with a corresponding other polypeptide (e.g., VL) comprising a different CDR.

Herein, a single domain antibody comprising a heavy chain variable domain is referred to as a "VHH (variable domain of HEAVY CHAIN of heavy-chain antibody)", also referred to as Nanobody (Nb). The VHH used in the present invention is preferably derived from a camelid, such as alpaca, or a humanized or sequence optimised version thereof. VHH has the structure from N-terminal to C-terminal FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

Those skilled in the art will appreciate that classes of antibody heavy chains include gamma, mu, alpha, delta or epsilon (γ, μ, α, δ, ε), some of which are also subclasses (e.g., γ1- γ4). The nature of this chain determines the "class" of antibody as IgG, igM, igA, igD or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., igG1, igG2, igG3, igG4, etc., have been well characterized and the functional specificities conferred are also known. All immunoglobulin classes are within the scope of the present disclosure. In some embodiments, the immunoglobulin molecule is an IgG class. IgG typically comprises two identical light chain polypeptides having a molecular weight of about 23,000 daltons and two identical heavy chain polypeptides having a molecular weight of about 53,000-70,000. The four chains are linked by disulfide bonds in a "Y" configuration, wherein the light chain starts at the "Y" mouth and continues through the variable region surrounding the heavy chain. Antibodies in the form of IgG1 are a subclass of IgG, with the heavy chain being the gamma 1 subclass. In some embodiments, the presently disclosed antibodies are IgG1.

As used herein, the term "heavy chain constant region" includes amino acid sequences derived from an immunoglobulin heavy chain. The polypeptide comprising a heavy chain constant region comprises at least one of a CH1 domain, a hinge (e.g., upper hinge region, middle hinge region, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen binding polypeptide for use in the present invention may comprise a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH2 domain, a polypeptide chain comprising a CH1 domain and a CH3 domain, a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, the polypeptide of the invention comprises a polypeptide chain comprising a CH3 domain. Furthermore, antibodies for use in the invention may lack at least a portion of a CH2 domain (e.g., all or a portion of a CH2 domain). As described above, one of ordinary skill in the art will appreciate that the heavy chain constant region can be modified such that it differs in amino acid sequence from a naturally occurring immunoglobulin molecule. In some cases, the single domain antibody is derived from a cartilaginous fish, such as a shark, where the antibody comprises 1 variable region and 5 constant regions (C1-C5).

"Hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region comprises amino acid residues from a "complementarity determining region" or "CDR" defined by the sequence alignment. "framework" residues or "FR" residues are variable domain residues other than the hypervariable region residues defined herein.

An "isolated antibody" is an antibody that is separated from all or part of a natural environmental component. The contaminating components of its natural environment are substances that interfere with the diagnostic or therapeutic use of the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the antibodies are purified to a degree (1) of more than 95% by weight of the antibody, such as more than 99% by weight, as determined by the Lowry method, (2) of a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by use of a rotary cup sequencer, or (3) as homogeneous by SDS-PAGE stained with coomassie blue or silver under reducing or non-reducing conditions. The isolated antibody includes an antibody in situ within the recombinant cell because at least one component of the antibody's natural environment will not be present. The 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 (inclusive) or any value therein.

"Nucleic acid" or "polynucleotide" refers to a polymer molecule composed of the individual nucleotides adenine (a), cytosine (c), guanine (g), thymine (t) (or uracil (u) in RNA), such as DNA, RNA, or modifications thereof. The nucleic acid molecule may be a natural nucleic acid molecule or a synthetic nucleic acid molecule or a combination of one or more natural nucleic acid molecules and 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, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, 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.

An "isolated nucleic acid molecule" is a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule. The isolated nucleic acid molecule differs from its naturally occurring form or environment. Thus, an isolated nucleic acid molecule is distinguished from a nucleic acid molecule that is present in its natural cell. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in a cell, such as a cell that normally expresses an antibody, e.g., the nucleic acid molecule is located at a chromosomal location different from that of a native cell.

"Monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, each antibody comprising the population being identical. Monoclonal antibodies are highly specific and can be directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include a plurality of different antibodies directed against a plurality of different determinants (epitopes), each monoclonal antibody is directed against only a single determinant on the antigen.

The term "chimeric antibody" refers to an antibody in which a portion of the heavy and/or light chains are derived from one source or species, while the remainder of the heavy and/or light chains are derived from a different source or species.

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

As used herein, a sequence "variant" 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.

"Amino acid" refers to an organic compound containing both amino and carboxyl groups, such as an alpha-amino acid, which may be encoded by a nucleic acid directly or in precursor form. A single amino acid is encoded by a nucleic acid consisting of three nucleotides, a so-called codon or base triplet. The same amino acid may be encoded by different codons 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).

"Conservatively substituted variant" or "conservative amino acid substitution" refers to an amino acid substitution known to those of skill in the art that is made without normally altering the biological activity of the resulting molecule. In general, it is well recognized by those skilled in the art that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity. Conservative substitutions may be made by amino acids containing chemically similar side chains, such as 1) aliphatic side chains of glycine, alanine, valine, leucine and isoleucine, 2) aliphatic hydroxyl side chains of serine and threonine, 3) amide-containing side chains of asparagine and glutamine, 4) aromatic side chains of phenylalanine, tyrosine and tryptophan, 5) basic side chains of lysine, arginine and histidine, 6) acidic side chains of aspartic acid and glutamic acid.

The term "about" as used herein means that the value is within an acceptable error range for the particular value being determined by one of ordinary skill in the art, which value depends in part on how the measurement or determination is made (i.e., the limits of the measurement system). Or "about" may mean a range of up to ±20%, such as ±10%, 5% or ±1% range. Unless otherwise indicated, when a particular value is found in the present disclosure and claims, the meaning of "about" should be assumed to be within an acceptable error range for that particular value.

The term "identity" as used herein may be assessed with the naked eye or in computer software such as the software program described in Ausubel et al eds. (2007) at Current Protocols in Molecular Biology. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position. The identity between two or more sequences may be expressed in percent (%), which may be used to evaluate the identity between related sequences. A polynucleotide sequence or amino acid sequence has a percentage (e.g., 90%, 95%, 98%, or 99%) of "sequence identity" with another sequence, meaning that when the sequences are aligned, the percentage of bases or amino acids in the two sequences that are compared are identical.

When referring to ligand/receptor, antibody/antigen or other binding pair, "specific" binding refers to determining the presence or absence of a binding reaction of a protein, such as LY6G6D, in a heterogeneous population of proteins and/or other biological agents. Thus, under the specified conditions, a particular ligand/antigen binds to a particular receptor/antibody and does not bind in significant amounts to other proteins present in the sample.

The term "encoding" when applied to a polynucleotide refers to a polynucleotide that is said to "encode" a polypeptide if it can be transcribed and/or translated to produce an mRNA of the polypeptide and/or fragment thereof in its native state or when manipulated by methods well known to those of skill in the art. The antisense strand is the complement of such a nucleic acid and from which the coding sequence can be deduced.

When referring to an animal, human, subject, cell, tissue, organ or biological fluid with "administration" and "treatment" it is meant that the exogenous drug, therapeutic, diagnostic agent or composition is contacted with the animal, human, subject, cell, tissue, organ or biological fluid. "administration" and "treatment" may refer to, for example, therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating the cell includes contacting the agent with the cell and contacting the agent with a fluid, wherein the fluid is in contact with the cell. "administration" and "treatment" also mean in vitro and ex vivo treatment of cells, e.g., by agents, diagnostic agents, binding compositions, or by other cells.

The term "treatment" includes amelioration or cessation of a disorder or symptoms thereof. Treatment includes inhibition, e.g., reducing the overall frequency of onset of the disorder or symptoms thereof.

The term "preventing" includes avoiding the onset of a disorder or symptoms thereof.

The term "therapeutically effective amount" or "effective amount" as used herein refers to an amount effective to prevent or slow down a disease or disorder to be treated when an anti-LY 6G6D single domain antibody or antigen-binding fragment thereof is administered alone or in combination with another therapeutic agent to a cell, tissue or subject. A therapeutically effective dose further refers to an amount of the antibody or antigen binding fragment thereof sufficient to cause a alleviation of symptoms, such as treatment, cure, prevention, or alleviation of a relevant medical condition, or an increase in the rate of treatment, cure, prevention, or alleviation of the condition. The effective amount for a particular subject can vary depending upon a variety of factors, such as the disease to be treated, the overall health of the patient, the route of administration, and the dosage and severity of the side effects. An effective amount may be the maximum dose or regimen that avoids significant side effects or toxic effects. When administered to an individual an active ingredient administered alone, a therapeutically effective amount refers to the individual ingredient. When a combination is administered, a therapeutically effective amount refers to the amount of the combination of active ingredients that produces a therapeutic effect, whether administered in combination, serially or simultaneously.

Pharmaceutical composition

The invention also provides a pharmaceutical composition. Such compositions comprise an effective amount of an antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

In some embodiments, the term "pharmaceutically acceptable carrier" refers to a substance listed in the pharmacopoeia approved by a regulatory agency of the government or otherwise generally recognized for use in animals, and particularly in humans. Furthermore, a "pharmaceutically acceptable carrier" will generally be any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid.

The term "carrier" refers to a diluent, adjuvant, excipient, or carrier used with the active ingredient for treatment. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal or vegetable origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. In some embodiments, when the pharmaceutical composition is administered intravenously, the carrier may be water. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences of e.w. Martin, incorporated herein by reference. Such compositions will contain a clinically effective dose of the antibody or antibody fragment, along with a suitable carrier, to provide a form of administration suitable for the patient. The formulation should be suitable for the mode of administration. The formulations may be packaged in ampules, disposable syringes or multiple dose vials made of glass or plastic.

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.

In some embodiments, the composition is formulated according to conventional procedures into a pharmaceutical composition suitable for intravenous injection into the human body. Compositions for intravenous administration are typically solutions in sterile isotonic aqueous buffers. The pharmaceutical composition may also contain a solubilizing agent and a local anesthetic such as lidocaine, thereby alleviating pain at the injection site. In general, the active ingredients are supplied individually or in admixture in unit dosage form, such as in the form of a dry lyophilized powder or dry concentrate, in a sealed container (e.g., ampoule or pouch) that is indicative of the amount of active agent. In the case of administration of the composition by infusion, the composition may be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline. In the case of administering the composition by injection, an ampoule of sterile water for injection or saline may be used so that the active ingredients may be mixed prior to administration.

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

Example 1 animal immunization and library construction

1. Obtaining of Single-Domain antibodies

Two alpacas are immunized by LY6G6D protein, peripheral blood is collected after immunization is finished, and a V _H H phage library is generated for affinity panning to obtain a single-domain antibody. The specific method comprises the following steps:

Alpaca immunization two alpacas were subcutaneously immunized (400 μg/alpaca) with Protein (CFA) by mixing Human LY6G6D Protein, C-TERM FC TAG (Baiying organism, cat No. B22256102) with Human LY6G6D Protein, N-TERM FC TAG (ACRO, cat No. LYD-H5265) as immunogen, diluted to 1 mg/mL with physiological saline, and then with an equal volume adjuvant CFA (Sigma, F5881) or IFA (Sigma, F5506) (CFA for the initial immunization and IFA for the subsequent immunization). Protein immunization was performed 3 or more times in succession at 2 week intervals, and after 7 days of the last immunization, 50mL peripheral blood was taken, mononuclear cells (PBMCs) were isolated, and total RNA was extracted from PBMCs using TRIZOL reagent according to the manufacturer's protocol. The cDNA was synthesized based on RNA template using oligo (dT) 20 primer using PRIMESCRIPTTMII REVERSE TRANSCRIPTASE (Takara, 2690A) first strand cDNA synthesis kit according to the manufacturer's protocol, alpaca cDNA was amplified for V _H H to generate a V _H H phage library.

100. Mu.L of phage display library solution was removed from 2 mL, serially diluted, and 4. Mu.L was inoculated onto plates containing carbenicillin. The library capacity of phage display libraries was estimated by counting colonies. Phage display library (p.f.u.) is approximately 5E+08. The single clones were randomly picked for sequencing. Sequencing results showed that the phage display library had an effective insertion rate of 100%.

Affinity panning the constructed immune library was subjected to multiple rounds of panning against biotinylated LY6G6D protein (kai cuo organisms, cat# LYD-HM4 GDB) and HEK293 overexpressing LY6G6D (Ji-Mang Biotechnology Co., ltd., cat# GM-C27573). After each round of panning, the post-panning library was amplified, 96 clones were randomly selected from plates of panning eluate titer using sterile toothpicks, inoculated into 1ml of 2 XYT-A sterile liquid medium (supplied by Baiying Biopreparation), shake-cultured at 37℃at 230 r/min for 8 h, 200 μl of the above culture was added to M13K07 helper phage (NEB, cat# N0315S) at a ratio of cell: phage=1:20, at 37℃and allowed to stand for 15min, and shake-cultured at 220 r/min for 45 min. A volume of 800. Mu.L of 2 XYT-AK (supplied by Baiying Biochemical Co., ltd.) was added, and the culture was vigorously shaken overnight at 30 ℃. The next day 12000 rpm was centrifuged for 2min and the supernatant was taken for cell binding FACS identification.

Cell binding FACS screening of positive clones HEK293 cells (Ji Man Biotechnology Co., ltd., cat# GM-C27573) overexpressing human LY6G6D or empty HEK293 cells were adjusted to 1X 10 ⁶ cells/mL with FACS buffer (PBS solution containing 1% FBS), placed in 96-well U-bottom plates at 100. Mu.L/well and the supernatant was discarded after centrifugation. 50. Mu.L of phage supernatant samples, positive control antibody HZ20A12 (humanized 20A12 sequence in patent WO2021119505A1, see Table 3, macropharmaceutical synthesis), negative control antibody (Baiying organism, cat# 00K0W 3003) were added and incubated at 4℃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-MM 05T-P) was diluted 100-fold with FACS buffer (PBS solution containing 1% FBS) or Alexa Fluor 647 AffiniPure Goat Anti-Human IgG (Jacksons Lab, cat# 109-605-190) with FACS buffer (PBS solution containing 1% FBS), 100. Mu.L of secondary Anti-dilution was added per well, cell pellet was resuspended, and air-blown and mixed, and incubated at 4℃for about 30 minutes. After incubation, the cells were centrifuged and washed three times with FACS buffer, and the cells were resuspended by adding FACS buffer to the well plate at 100 μl/well. The results of flow cytometry (Agilent, novoCyte 2060R) reading the average fluorescence intensities (Median Fluorescence Intensity, MFI) are shown in table 1. Screening results show that 189 positive clones are obtained, evolutionary sequencing is performed, the CDR3 groups with different lengths are synthesized, the analysis results of the evolutionary tree are performed, CDR3 regions with larger differences are selected, and finally 6 recombinant single-domain antibodies are obtained. All 6 different sequences were verified using colorectal cancer HT55 cells.

TABLE 1 phage supernatant screening results

Colorectal cancer HT-55 cell binding assay HT-55 cells (CBP 60012) were adjusted to 1X 10 ⁶ cells/mL using FACS buffer (PBS solution with 1% FBS), the supernatant was discarded after centrifugation at 100. Mu.L/well in 96-well U-bottom plates. 50. Mu.L of phage supernatant sample, positive control antibody hz20A12, negative control antibody (Baiying organism, cat# 00K0W 3003) were added, respectively, and incubated at 4℃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-MM 05T-P) was diluted 100-fold with FACS buffer (PBS solution containing 1% FBS) or Alexa Fluor 647-Human IgG (Jacksons Lab, cat. No. 109-605-190) with FACS buffer (PBS solution containing 1% FBS), 100. Mu.L of secondary Antibody dilution was added to each well, the cell pellet was resuspended, and air-blown and mixed, and incubated at 4℃for about 30 minutes.

The results are shown in FIG. 1. By HT-55 binding experiments, 3 of which (CP 1R3-25, CP1R3-31, CP1R3-40, see Table 2 for sequence details) bind HT-55, they were selected for recombinant nanobody expression and further characterized.

2. Preparation of chimeric nanobody against human LY6G6D

The sequences of the above-screened CP1R3-25, CP1R3-31 and CP1R3-40 are shown in Table 2.

TABLE 2 CDR sequences and variable region sequences of chimeric nanobodies against human LY6G6D (determined according to Kabat protocol)

Heavy chain variable regions (table 2) were each constructed onto human constant regions (IgG 1, table 3), corresponding chimeric antibodies (table 4) were constructed, and the sequences were verified by sequencing.

The corresponding nucleic acid encoding the chimeric antibody was expressed using an Expi293 cell and purified using a Protein a column as follows:

Expi293 cells express chimeric antibodies Expi293 cells (Thermo, cat. No. A14635 CN) were density diluted to 1.5X10- ⁶ cells/mL and incubated in a 37℃8% CO ₂ shaker at 120 rpm day prior to transfection. The next day, viable cell density and viability were measured, cell transfection density should be 3×10 ⁶ cells/mL, cell viability was greater than 95%. PEI/plasmid complexes were prepared by mixing PEI (1 mg/mL, polysciences, cat. No. 24765-1) upside down. The expression plasmid (Invitrogen pCDNA3.4 vector, cat# A14697) was diluted with OPM-293 CD05 medium (Shanghai A Pu Mai Biotechnology Co., ltd., cat# 81075-001) to a total of 1. Mu.g/mL, the volume of the medium diluted with the plasmid was 1/20 of the transfection volume, gently mixed, and the plasmid ratio for expressing light and heavy chains was 1:1.5. PEI reagent was diluted with OPM-293 CD05 medium, the volume of the diluted PEI medium was 1/20 of the transfection volume, mixed gently upside down, and incubated at room temperature for 5 minutes. Diluted PEI reagent was added to the diluted plasmid and mixed gently upside down. PEI/plasmid complex was incubated at room temperature for 15 minutes, then the solution was slowly added dropwise to the transfer flask, which was gently swirled during the addition. After transfection, shake flasks were incubated at 37℃in a shaker with 8% CO ₂ at 120 rpm. On the next day after transfection (24 hours after transfection), 10% OPM-293 ProFeed (Shanghai Ao Pu Mai Biotech Co., ltd., cat# F081918) was added to the shake flask, the shake flask was gently rotated during the addition, and then returned to the shaker for further cultivation for 5-7 days, and the supernatant was harvested.

The Protein A column is used for purifying the antibody, namely, a gravity chromatographic column is prepared, an upper cover of the gravity chromatographic column is opened, and a gasket is arranged at the bottom of the gravity column and is pressed. Filler Protein a (Cytiva, cat No. 17549801) was prepared, and the required filler suspension volume was precisely calculated from the target filler volume and the filler suspension ratio, and the required filler suspension volume=target filler volume/filler suspension ratio. And (5) fully vortex oscillating the filler to ensure that the filler is completely suspended. And adding the filler suspension into the bottom of the gravity chromatographic column. At least 10 CV of equilibration buffer PBS was added to the gravity chromatography column and after equilibration, the exit pH was measured. If it is not at the target pH7.4, the addition of equilibration buffer is continued until it equilibrates to the target pH7.4. A volume of sample was slowly added to the gravity column. At least 10 CV of the elution buffer is added to the gravity chromatography column. The eluate was collected by slowly adding 5CV of elution buffer (10-50 mM NaAc, pH 3.0-pH 3.5) to the gravity column and incubating for 3-5 minutes. The elution step was repeated as necessary. Neutralization, the pH was adjusted to the target pH7.4 with a neutralization buffer (1M Tris). Protein concentration was determined using Nanodrop. The buffer holding the antibody was replaced with PBS by ultrafiltration.

TABLE 3 constant region sequences and positive control antibody sequences

TABLE 4 full-length sequence of anti-human LY6G6D chimeric nanobody

Example 2 functional identification of chimeric nanobodies against human LY6G6D

1. Binding Activity of chimeric nanobody against human LY6G6D and HEK293 cell line overexpressing human LY6G6D

HEK293 cells expressing human LY6G6D (Ji Man Biotechnology Co., ltd., cat# GM-C27573) or empty HEK293 cell line concentration was adjusted to 1X 10 ⁶ cells/mL with FACS buffer (PBS solution containing 1% FBS), and the supernatant was discarded after centrifugation at 100. Mu.L/well in 96-well U-well bottom plates. The chimeric nanobody against human LY6G6D, the positive control antibody HZ20A12, and the negative control antibody (Baiying organism, cat# 00K0W 3003) were diluted to an initial working concentration of 100 nM by FACS buffer (PBS solution containing 1% FBS), and then subjected to gradient dilution (4-fold dilution, total of 8 concentration points) by FACS buffer. The antibody was added to the well plate at 100. Mu.L/well to resuspend the cells, blow mixed well and incubated at 4℃for 1 hour. After incubation, the cells were centrifuged and washed three times with FACS buffer. AlexaFlour-647 labeled anti-human secondary antibody (Invitrogen, A-21445) was diluted 1:1000 with FACS buffer (PBS solution with 1% FBS), 100. Mu.L of secondary antibody dilution was added to each well, the cell pellet was resuspended, air-blown, mixed, and incubated at 4℃for about 45 minutes. After incubation, the cells were centrifuged and washed three times with FACS buffer, and the cells were resuspended by adding FACS buffer to the well plate at 100 μl/well. The flow cytometer reads the average fluorescence intensity (Median Fluorescence Intensity, MFI), analyzes the test data by using GRAPHPAD PRISM 8.0 software, takes the logarithm of the antibody concentration as the x axis, takes the corresponding MFI value as the y axis, selects a four-parameter equation regression model, fits an antibody dose response curve, and calculates EC ₅₀. The results are shown in FIGS. 2,3 and Table 5.

TABLE 5 HEK293 cell line binding Activity of chimeric nanobodies against human LY6G6D and over-expression of human LY6G6D

The results show that the anti-human LY6G6D chimeric nanobody has an EC50 (nM) binding activity on HEK293 cells overexpressing human LY6G6D, comparable to that of a positive control antibody, but has no binding activity on HEK293 blank cells.

2. Antibody affinity assay based on Surface Plasmon Resonance (SPR)

The affinity against human LY6G6D chimeric nanobody was determined using Biacore 8K SPR system based on surface plasmon resonance (Cytiva Situo, cat# 29277881). anti-LY 6G6D chimeric nanobody, HZ6E10 (humanized 6E10 sequence in patent WO2021119505A1, see Table 3, macropharmaceutical synthesis) was immobilized on the surface of Protein A chip (Cytiva Situo; cat# Biacore Sensor Chip Protein A). Human LY6G6D (C-His) protein (kai cuo organisms, cat# LYD-HM1 GD) was then diluted to 200 nM concentration and flowed over the chip surface at 30. Mu.L/min each, binding time 180s, dissociation time 1200s after each cycle was completed, the chip surface was regenerated with 10M, pH1.5 Glycine kinetic rate constants were subjected to subtraction of blank controls, data fitting was performed using global fit analysis method 1:1 binding model, dissociation equilibrium rate constants (KD) were calculated using the following formula K _D =kd/ka. results are shown in FIGS. 4, 5, 6,7 and Table 6.

TABLE 6 affinity of anti-human LY6G6D chimeric nanobodies

The results show that the anti-human LY6G6D chimeric nanobody has binding activity to recombinant protein human LY6G6D (C-His) with an affinity ranging from 1.11E-07M to 3.54E-08M.

Example 3 functional identification of tandem chimeric nanobodies

To increase the affinity of the antibodies, two nanobodies with higher affinity (00 FY9P002, 00FY9P 003) were expressed as tandem nanobody Fc tags (00fy 9P 002+003-hFc) with the sequences shown in table 7. Tandem chimeric nanobodies were prepared according to the method for preparing chimeric nanobodies against human LY6G6D in example 1.

TABLE 7 anti-human LY6G6D series chimeric nanobody sequences

1. Binding Activity of anti-human LY6G6D series chimeric nanobody and HEK293 cell line overexpressing human LY6G6D

HEK293 cells overexpressing human LY6G6D (Ji Man Biotechnology Co., ltd., cat# GM-C27573) or empty HEK293 cell line concentration was adjusted to 1X 10 ⁶ cells/mL with FACS buffer (PBS solution containing 1% FBS), placed in 96-well U-bottom plates at 100. Mu.L/well, and the supernatant was discarded after centrifugation. The serial chimeric antibody against human LY6G6D, the positive control antibody HZ20A12, and the negative control antibody (Baiying organism, cat# 00K0W 3003) were diluted to an initial working concentration of 100 nM with FACS buffer (1% FBS in PBS), and then subjected to gradient (4-fold dilution, total of 8 concentration points) dilution with FACS buffer. The antibody was added to the well plate at 100. Mu.L/well to resuspend the cells, blow mixed well and incubated at 4℃for 1 hour. After incubation, the cells were centrifuged and washed three times with FACS buffer. AlexaFlour-647 labeled anti-human secondary antibodies (Invitrogen, A-21445) were diluted 1:1000 with FACS buffer (PBS containing 1% FBS), 100. Mu.L of secondary antibody dilution was added to each well, the cell pellet was resuspended, air-blown, and mixed well, incubated at 4℃for about 45 minutes, cells after incubation were centrifuged, washed three times with FACS buffer, and the FACS buffer was added to the well plate to resuspend cells at 100. Mu.L/well, flow cytometry read the mean fluorescence intensity (Median Fluorescence Intensity, MFI), test data were analyzed using GRAPHPAD PRISM 8.0.0 software, logarithmic antibody concentration was x-axis, corresponding MFI value was y-axis, a four parameter equation regression model was selected, and antibody dose-response curves were fitted, and EC ₅₀ was calculated as shown in FIGS. 8,9 and Table 8.

TABLE 8 anti-human LY6G6D series chimeric nanobody cell line binding Activity

The results show that the anti-human LY6G6D tandem chimeric nanobody has equivalent binding activity to HEK293 cells over-expressing human LY6G6D as the positive control antibody HZ20A12, but has no binding activity to HEK293 blank cells.

2. Affinity assay for anti-human LY6G6D tandem chimeric nanobody

The affinity against human LY6G6D chimeric nanobody was determined using Biacore 8K SPR system based on surface plasmon resonance (Cytiva Situo, cat. No. 29277881). Human antigen LY6G6D (LY 6G6D (C-His)) was immobilized on the CM5 chip surface (Cytiva Situo, cat. No. Biacore Sensor Chip CM 5), and then the antibody to be tested was diluted to 200nM and flowed over the chip surface at a flow rate of 30. Mu.l/min, respectively. Binding time 180s and dissociation time 1200s. After each cycle, the chip surface was regenerated with 10M Glycine at pH 1.5. The kinetic rate constant was subtracted from the blank control and data fitting was performed using global fit analysis method 1:1 binding model. The dissociation equilibrium rate constant (KD) is calculated using the formula K _D =kd/ka.

The results are shown in fig. 10, 11, 12 and table 9. The results show that the anti-human LY6G6D tandem chimeric nanobody (00FY9P002+003-hFc) significantly improved the affinity for recombinant protein human LY6G6D (C-His).

TABLE 9 affinity of anti-human LY6G6D tandem chimeric nanobodies

Example 4 in vitro cell killing experiments with CAR-T cells

(1) Preparation of anti-LY 6G6D CAR-T

The CAR structure was constructed by anti-LY 6G6D tandem chimeric nanobody (00fy9p002+003-hFc) and positive control HZ20a12 (heavy chain variable region was constructed as scFV by 3×g ₄ S joining light chain variable region), full length CAR from N-terminus to C-terminus: CD8 a signal peptide, LY6G6D binding domain (nanobody or positive control antibody), CD8 a hinge, CD8 a transmembrane region, 4-1BB intracellular domain, CD3 part intracellular domain, the amino acid and nucleotide sequences of the CAR elements are shown in table 10.

Amino acid and nucleotide sequences of elements of Table 10 CAR

(2) Lentivirus preparation

HEK-293T cells (ATCC) were inoculated at 25000 cells/cm ³ into 10-layer cell factories each containing 1000mL of DMEM medium containing 10% FBS on day 0, 200. Mu.g/PREV 400. Mu. g PRRE/800. Mu.g PEV of lentiviral packaging vector was mixed in 50 mL of DMEM (DNA mixture) the following day, incubated at room temperature for 5 minutes, 1.6 mL PEI was added to 50 mL of DMEM, incubated at room temperature for 5 minutes, added to DNA mixture, and incubated at room temperature for 15 minutes, and then added to cell factories. Changing the liquid 6-8h after transfection, collecting virus supernatant 48h after transfection, centrifuging to remove fragments (4000 g,15 min), centrifuging to concentrate (16000 g,4 h), removing supernatant, re-suspending with virus preservation liquid, and packaging.

(3) CAR-T preparation

On day 0 PBMC (Shanghai Miaoshun, cat. P122051102C), T cells were sorted using DYNABEADs CD/CD 28 (Gibco, 40203D) and purified T cells were activated in X-VIVO 15 (Lonza, BEBP 02-054Q) supplemented with 5% FBS and 100IU/ml IL-2 (RD, BT-002-GMP-050). T cells were seeded with 2e+05 cells/mL reselection, and lentiviruses were added to T cells on day 2. On day 3, the medium was replaced with T cell expansion medium, X-VIVO 15 supplemented with 5% AB (GEMINI, 100-512) and 100IU/mL IL-2 GMP. On day 5, DYNABEADs CD/CD 28 was removed from the cell suspension using a magnetic pole and the T cell expansion medium was changed. Transduction efficiencies were determined by detecting the percentage of T cells that recognize recombinant LY6G6D using flow cytometry on days 9 and 13. If necessary, cells are expanded in a larger culture vessel using a T cell expansion medium. LY6G6D CAR-T cells were cryopreserved on day 14.

(4) CAR-T in vitro functional test

And (3) plating luciferase expression target cells HT-55 and LS1034 (ATCC, product number CRL2158 ATC) with good growth state (1 E+04 cells/hole, 100 mu L/hole) into a 96-hole white opaque cell culture plate, and setting 3-5 compound holes. Subsequently, the target ratio was calculated according to the CAR positive rate, effector cells (100 μl/well) were added at target ratios of 4:1, 2:1, 1:1, 1:2, 1:4, 1:8, respectively, medium was added as negative control group, 2% triton X-100 (BioFroxx, cat No. 1139ML 100) was used as maximum release control (MAX), and effector cells were added after plating after cell attachment. After 24 h+ -2 h of co-incubation, 125g is centrifuged for 5min, 130 μl of cell supernatant is extracted for cytokine detection, 70 μl (1:1 of liquid volume in the well plate) of mixed glow type firefly luciferase (Shanghai next holt, 11404ES 80) is added to each well of the plate, shaking and mixing are conducted for 5-10 min in a dark place, a Lumineancence signal is detected, and the CAR-T killing rate is calculated according to the following formula.

CAR-T killing ratio (%) = (RLU_NC-RLU_ (CAR-T))/(RLU_NC-RLU_MAX) ×100%

Results of in vitro killing ability tests on LS1034 and HT-55 target cells endogenously expressing LY6G6D are shown in FIG. 13 and FIG. 14, and CAR-T constructed by the serial chimeric nanobody (00FY9P002+003-hFc) realizes in vitro killing activity on LS1034 and HT-55 cells comparable to positive control.

Claims (16)

1. An anti-LY 6G6D single domain antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3 comprised by a sequence as set forth in SEQ ID No. 1,5, or 7, wherein the HCDR1, HCDR2, and HCDR3 are determined by a Kabat protocol, an AbM protocol, a Chothia protocol, or a Contact protocol.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises the amino acid sequences of HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.2, SEQ ID NO. 3 and SEQ ID NO. 4, respectively, or

The antibody or antigen binding fragment thereof comprises the amino acid sequences of HCDR1, HCDR2 and HCDR3 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 or antigen binding fragment thereof are shown as SEQ ID NO. 2, SEQ ID NO.8 and SEQ ID NO. 9 respectively.

3. The antibody or antigen binding fragment thereof of claim 1, comprising a heavy chain variable region having an amino acid sequence as set forth in SEQ ID No.1, 5 or 7, or having at least 90% sequence identity to the sequence set forth in SEQ ID No.1, 5 or 7.

4. The antibody or antigen-binding fragment thereof of claim 1, further comprising a heavy chain constant region, an Fc region, or a combination thereof.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein the heavy chain constant region has the sequence shown in SEQ ID No. 10.

6. The antibody or antigen binding fragment thereof of claim 5, having a sequence as set forth in SEQ ID No. 15, 16 or 17, or having at least 90% sequence identity to the sequence set forth in SEQ ID No. 15, 16 or 17.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which is a tandem antibody comprising two or more antibodies or antigen-binding fragments thereof according to any one of claims 1 to 4 connected in a tandem manner, wherein the two or more antibodies or antigen-binding fragments thereof are connected by a linker, wherein the linker is (GGGGS) n, wherein n is an integer of 1 to 10.

8. The antibody or antigen-binding fragment thereof of claim 7, wherein the linker is GGGGSGGGGSGGGGS.

9. The antibody or antigen-binding fragment thereof according to claim 8, wherein the tandem antibody comprises a first antibody or antigen-binding fragment thereof and a second antibody or antibody-binding fragment thereof,

The first antibody or antigen binding fragment thereof comprises three heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 comprised by the sequence shown as SEQ ID No. 5;

The second antibody or antigen binding fragment thereof comprises three heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 comprised by the sequence shown as SEQ ID No. 7.

10. The antibody or antigen-binding fragment thereof according to claim 9, wherein the amino acid sequence of the first antibody or antigen-binding fragment thereof is as shown in SEQ ID No. 5 or has at least 90% sequence identity to the sequence shown in SEQ ID No. 5 and the amino acid sequence of the second antibody or antigen-binding fragment thereof is as shown in SEQ ID No. 7 or has at least 90% sequence identity to the sequence shown in SEQ ID No. 7.

11. The antibody or antigen binding fragment thereof of claim 10, wherein the amino acid sequence of the tandem antibody is as set forth in SEQ ID No. 18 or has at least 90% sequence identity to the sequence set forth in SEQ ID No. 18.

12. The antibody or antigen binding fragment thereof of claim 11, further comprising a CD8 a signal peptide, a CD8 a hinge, a CD8 a transmembrane region, a 4-1BB intracellular domain, a CD3 portion of an intracellular domain, wherein, from N-terminus to C-terminus, the antibody or antigen binding fragment thereof comprises, in sequence, a CD8 a signal peptide, the antibody or antigen binding fragment thereof of claim 11, a CD8 a hinge, a CD8 a transmembrane region, a 4-1BB intracellular domain, a CD3 portion of an intracellular domain, the amino acid sequence of the CD8 a signal peptide is shown in SEQ ID No. 19, the amino acid sequence of the antibody is shown in SEQ ID No. 18, the amino acid sequences of the CD8 a hinge and CD8 a 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 CD3 portion of an intracellular domain is shown in SEQ ID No. 25.

13. A biomaterial selected from a) to c) below

A) A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-12;

b) A recombinant vector comprising the nucleic acid molecule of a);

c) A recombinant cell comprising a) said nucleic acid molecule and/or b) said recombinant vector.

14. A method of preparing the antibody or antigen-binding fragment thereof of any one of claims 1-12, comprising culturing a recombinant cell comprising a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-12 under conditions suitable for expression of the antibody.

15. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-12 or the biological material of claim 13, and a pharmaceutically acceptable carrier.

16. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, a biomaterial according to claim 13 and/or a composition according to claim 15 for the preparation of a product as shown in any one of the following:

(a) Detecting a product of LY6G 6D;

(b) A product that stimulates or enhances an immune response;

(c) A product for preventing and/or treating a disease associated with abnormal expression of LY6G6D, wherein the disease is colorectal cancer;

(d) A product that kills cells expressing LY6G6D or inhibits growth of cells expressing LY6G6D in vitro or in vivo.