Anti-CD155 antibody and its applications

Abstract

The present invention provides anti-CD155 antibodies and their applications, and provides anti-CD155 chimeric antibodies, humanized antibodies, and affinity-matured humanized antibodies, which specifically target and bind to the CD155 receptor, inhibit the binding of CD155 to its receptors TIGIT, CD96, and CD226, and have the characteristic of not inducing apoptosis of CD155 cells.

Description

[Technical Field]

[0001] The present invention relates to the biomedicine field, specifically to anti-CD155 antibodies and their applications. [Background technology]

[0002] CD155, also known as NECL-5 or PVR, is a type I transmembrane protein whose extracellular region contains one IgV domain and two IgC domains, and whose intracellular region contains an ITIM motif. Studies have shown that CD155 can be shed from the cell surface to form soluble CD155 protein. CD155 is expressed on the surface of tissue cells, such as monocytes and dendritic cells, and its expression is elevated in various tumor tissues. Studies have shown that CD155 promotes tumor invasion and metastasis, and high CD155 expression is associated with poor tumor prognosis.

[0003] NK cells and T cells express multiple CD155 receptors on their surfaces, including TIGIT, CD96, and CD226. TIGIT and CD96 are inhibitory receptors, while CD226 is an activating receptor. Immune checkpoint antibodies targeting TIGIT and CD96 have already entered clinical trials, with Roche's TIGIT monoclonal antibody showing the most rapid progress, currently in Phase III clinical trials. CD155 shed from the cell surface can bind to CD226, downregulating CD226 and weakening the anti-cancer functions of NK cells and T cells. Eli Lilly is currently developing an agonist CD226 antibody.

[0004] Studies have shown that patients with non-small cell lung cancer and melanoma with high CD155 expression have a poorer prognosis and a lower response to PD-1 / L1 therapy. Therefore, the development of antibody drugs targeting CD155 is crucial, as they can be used in PD-L1-negative patients while improving their response to PD-1 / L1 therapy. To date, the only CD155 antibody in clinical trials is Nectin's NTX-1088 antibody (named NB1088 in early development). This antibody is an IgG4 subtype and can block the binding of CD155 to its receptors TIGIT, CD96, and CD226. Our research has shown that this antibody strongly induces apoptosis in CD155-positive cells. However, CD155 is also expressed on human peripheral blood mononuclear cells and various normal tissue cells, posing safety risks for its clinical application. Another CD155 antibody commonly used in scientific research, SKII.4, is also an inhibitory antibody that can block the binding of CD155 to its receptor, and in our research, this antibody also strongly induces apoptosis of CD155-positive cells.

[0005] Therefore, research is needed to develop antibody drugs that target CD155 and are highly safe. Summary of the Invention [Problem to be solved by the invention]

[0006] The present application has been filed based on the inventor's discovery of the following problems and facts.

[0007] CD155 is expressed on the surface of tissue cells such as monocytes and dendritic cells, and its expression is elevated in several tumor tissues. CD155 promotes tumor invasion and metastasis, and high CD155 expression is associated with poor tumor prognosis. However, because CD155 binds to three receptors, TIGIT, CD96, and CD226, screening for inhibitory anti-CD155 monoclonal antibodies is challenging. Furthermore, existing antibodies that block the CD155 receptor pose a safety risk by inducing apoptosis of CD155-positive normal tissue cells, making the design and development of anti-CD155 monoclonal antibodies challenging.

[0008] The present inventors have successfully screened for an inhibitory CD155 antibody that does not induce cell apoptosis. Specifically, the inventors used hybridoma technology to screen for a series of high-affinity anti-CD155 monoclonal antibodies that inhibit the binding of CD155 to its receptors, such as TIGIT, CD96, and CD226. They then humanized the constant regions of the mouse-derived anti-CD155 monoclonal antibodies and retained the variable regions of the mouse-derived anti-CD155 monoclonal antibodies to obtain a series of anti-CD155 chimeric monoclonal antibodies. Based on this, the inventors humanized the framework regions of the mouse-derived variable regions to obtain humanized CD155 antibodies. Furthermore, the inventors obtained affinity-matured humanized CD155 antibodies by mutating the CDRs of the humanized CD155 antibodies. The inventors discovered that the chimeric antibodies, humanized antibodies, and affinity-matured humanized antibodies obtained in this application specifically target and bind to the CD155 receptor, inhibiting the binding of CD155 to its receptors TIGIT, CD96, CD226, etc., and not only effectively treating and / or preventing CD155-mediated related diseases, such as tumors, but also have the characteristic of not inducing apoptosis of CD155 cells. [Means for solving the problem]

[0009] Thus, in a first aspect, the present invention provides an antibody or antigen-binding fragment. According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises a CDR sequence selected from at least one of the heavy chain variable region CDR sequences of SEQ ID NOs: 1-3, 7-9, 13-15, and 19-21 and the light chain variable region CDR sequences of SEQ ID NOs: 4-6, 10-12, 16-18, and 22-24, or an amino acid sequence having at least 80% identity thereto. The antibody or antigen-binding fragment according to an embodiment of the present invention is an affinity-matured humanized antibody, which has stronger specificity, a longer half-life, and stronger potency, and can bind to human or monkey CD155 protein and inhibit the binding of CD155 to its receptors, such as TIGIT, CD96, and CD226, effectively treating or preventing CD155-mediated related diseases at a relatively low dosage, without inducing apoptosis of CD155-positive cells, resulting in fewer toxic side effects and greater safety.

[0010] In a second aspect, the present invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the antibody or antigen-binding fragment described in the first aspect. The antibody or antigen-binding fragment encoded by the nucleic acid molecule according to this embodiment of the present invention has stronger specificity, longer half-life, and stronger potency, and can bind to human or monkey CD155 protein and inhibit the binding of CD155 to its receptors, such as TIGIT, CD96, and CD226. It effectively treats or prevents CD155-mediated related diseases at a relatively low dosage, does not induce apoptosis of CD155-positive cells, and has low toxic side effects and is therefore safer.

[0011] In a third aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the nucleic acid molecule described in the second aspect. The expression vector may include a selectable regulatory sequence, which is operably linked to the nucleic acid molecule. The regulatory sequence may be one or more regulatory sequences capable of directing expression of the nucleic acid molecule in a host. The expression vector according to an embodiment of the present invention can efficiently express the antibody or antigen-binding fragment in a suitable host cell. The antibody or antigen-binding fragment has stronger specificity, a longer half-life, and stronger potency, and can bind to human or monkey CD155 protein and inhibit the binding of CD155 to its receptors, such as TIGIT, CD96, and CD226. This effectively treats or prevents CD155-mediated related diseases at a relatively low dosage, without inducing apoptosis of CD155-positive cells, resulting in fewer toxic side effects and greater safety.

[0012] In a fourth aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment described in the first aspect. According to an embodiment of the present invention, the method includes introducing the expression vector described in the third aspect into cells and culturing the cells under conditions suitable for protein expression and secretion, to obtain the antibody or antigen-binding fragment. This method according to an embodiment of the present invention can effectively obtain the antibody or antigen-binding fragment, which has stronger specificity, a longer half-life, and stronger potency, and is capable of binding to human or monkey CD155 protein and inhibiting the binding of CD155 to its receptors, such as TIGIT, CD96, and CD226. This method effectively treats or prevents CD155-mediated diseases at a relatively low dosage, without inducing apoptosis of CD155-positive cells, resulting in fewer toxic side effects and greater safety.

[0013] In a fifth aspect, the present invention provides a recombinant cell. According to some embodiments of the present invention, the recombinant cell carries the nucleic acid described in the second aspect or the expression vector described in the third aspect, or is capable of expressing the antibody or antigen-binding fragment described in the first aspect. The recombinant cell is obtained by transfection or transformation with the expression vector. According to some specific embodiments of the present invention, the recombinant cell can efficiently express large amounts of the antibody or antigen-binding fragment under appropriate conditions. The antibody or antigen-binding fragment has stronger specificity, longer half-life, and stronger potency, binds to human or monkey CD155 protein, and inhibits the binding of CD155 to its receptors, such as TIGIT, CD96, and CD226. This antibody or antigen-binding fragment can effectively treat or prevent CD155-mediated related diseases at a relatively low dosage, without inducing apoptosis of CD155-positive cells, resulting in fewer toxic side effects and greater safety.

[0014] In a sixth aspect, the present invention provides an immunoconjugate. According to an embodiment of the present invention, the immunoconjugate comprises the antibody or antigen-binding fragment described in the first aspect and a therapeutic agent. As described above, the antibody or antigen-binding fragment according to an embodiment of the present invention can effectively bind to CD155 protein and inhibit the binding of CD155 protein to its receptor. Therefore, an immunoconjugate comprising the antibody or antigen-binding fragment can similarly bind to human or monkey CD155 protein. The immunoconjugate has a good effect in preventing and / or treating CD155-mediated diseases, does not induce apoptosis of CD155-positive cells, has low toxic side effects, and is highly safe.

[0015] In a seventh aspect, the present invention provides compositions, which, according to embodiments of the present invention, comprise the aforementioned antibodies, nucleic acid molecules, expression vectors, or recombinant cells. As described above, the aforementioned antibodies or antigen-binding fragments according to embodiments of the present invention can effectively bind to CD155 protein and inhibit the binding of CD155 protein to its receptor, without inducing apoptosis of CD155-positive cells. Therefore, compositions containing the aforementioned substances can also effectively inhibit the binding of CD155 protein to its receptor, have a good effect on preventing and / or treating CD155-mediated diseases, and do not induce apoptosis of CD155-positive cells.

[0016] In an eighth aspect, the present invention provides use of the aforementioned antibodies or antigen-binding fragments thereof, nucleic acid molecules, expression vectors, recombinant cells, or compositions in the preparation of a medicament for preventing and / or treating CD155-related diseases. As described above, the aforementioned antibodies or antigen-binding fragments according to embodiments of the present invention can effectively bind to CD155 protein and inhibit the binding of CD155 protein to its receptor without inducing apoptosis of CD155-positive cells. Therefore, drugs containing the aforementioned substances can also effectively inhibit the binding of CD155 protein to its receptor, have a good effect on preventing and / or treating CD155-mediated diseases, and do not induce apoptosis of CD155-positive cells.

[0017] In a ninth aspect, the present invention provides a drug. According to an embodiment of the present invention, the drug comprises the antibody nucleic acid molecule, expression vector, recombinant cell, or composition described above. As described above, the antibody or antigen-binding fragment according to an embodiment of the present invention can effectively bind to CD155 protein and inhibit the binding of CD155 protein to its receptor, without inducing apoptosis of CD155-positive cells. Therefore, drugs containing the above substances can also effectively inhibit the binding of CD155 protein to its receptor, have good effects in preventing and / or treating CD155-mediated diseases, and do not induce apoptosis of CD155-positive cells.

[0018] In a tenth aspect, the present invention provides use of the aforementioned antibodies or antigen-binding fragments in the preparation of a kit. According to an embodiment of the present invention, the kit is used to detect CD155. As described above, the antibodies or antigen-binding fragments according to embodiments of the present invention can effectively bind to human or monkey CD155 protein and inhibit the binding of CD155 protein to its receptor. Therefore, the aforementioned antibodies or antigen-binding fragments can be used to prepare a kit for detecting CD155 protein, which can effectively detect CD155 protein qualitatively or quantitatively.

[0019] In an eleventh aspect, the present invention provides a kit for detecting CD155. According to an embodiment of the present invention, the kit contains the above-described antibody or antigen-binding fragment thereof. As described above, the antibody or antigen-binding fragment according to an embodiment of the present invention can effectively bind to human or monkey CD155 protein and inhibit the binding of the CD155 protein to its receptor. Therefore, the kit containing the above-described antibody or antigen-binding fragment can effectively detect CD155 protein qualitatively or quantitatively.

[0020] In a twelfth aspect, the present invention provides a method for preventing and / or treating a CD155-related disease, according to an embodiment of the present invention, the method comprising administering to a subject at least one of the above-described antibody or antigen-binding fragment, the above-described nucleic acid molecule, the above-described expression vector, the above-described recombinant cell, the above-described immunoconjugate, or the above-described composition.

[0021] Additional aspects and advantages of the present invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the present invention. [Brief explanation of the drawings]

[0022] The above and / or additional aspects and advantages of the present invention will become apparent and easier to understand from the following description of the embodiments taken in conjunction with the drawings. [Figure 1] This is a flow ceteometry result diagram showing the binding of different concentrations of mouse-derived CD155 antibodies according to a specific embodiment of the present invention to CHO-K1-human CD155 cells expressing human CD155 protein and CHO-K1-Rhesus CD155 cells expressing monkey CD155 protein. [Figure 2] FIG. 10 shows the results of the inhibition of binding of TIGIT-Fc, CD96-Fc, and CD226-Fc to CHO-K1-human CD155 cells expressing human CD155 protein by mouse-derived CD155 antibodies at different concentrations according to a specific embodiment of the present invention. [Figure 3] FIG. 1 shows the ELISA results of mouse-derived CD155 antibodies according to a specific embodiment of the present invention at different concentrations inhibiting the binding of TIGIT, CD96, and CD266 to recombinantly expressed human CD155 protein. [Figure 4] FIG. 1 shows flow ceteometry results of binding of different concentrations of a human-mouse chimeric CD155 antibody according to a specific embodiment of the present invention to CHO-K1-huamn CD155 and CHO-K1-Rhesus CD155 cells. [Figure 5] FIG. 1 shows ELISA results of different concentrations of affinity-matured humanized CD155 antibodies according to specific embodiments of the present invention binding to human and monkey CD155 proteins. [Figure 6] This figure shows the ELISA results of the inhibition of the binding of TIGIT, CD96, and CD226 to human or monkey CD155 proteins expressed in CHO-K1-huamn CD155 and CHO-K1-Rhesus CD155 cells at different concentrations of affinity-matured humanized CD155 antibodies according to a specific embodiment of the present invention. [Figure 7] FIG. 1 shows flow ceftometry results of the binding of affinity-matured humanized CD155 antibodies according to specific embodiments of the present invention at different concentrations to CHO-K1-human CD155 and CHO-K1-Rhesus CD155, respectively. [Figure 8] FIG. 1 shows the results of humanized CD155 antibodies according to specific embodiments of the present invention promoting apoptosis of U-937-CD155 cells. [Figure 9] FIG. 1 shows the results of detecting that a humanized CD155 antibody according to a specific embodiment of the present invention promotes immune reconstitution in mice against human tumors. DETAILED DESCRIPTION OF THE INVENTION

[0023] Hereinafter, examples of the present invention will be described in detail. The examples described below are illustrative and are used only to explain the present invention, and should not be understood as limitations on the present invention.

[0024] It should be noted that the terms "first," "second," etc. are for descriptive purposes only and cannot be considered to indicate or imply relative importance or the express number of technical features. Thus, a feature qualified as "first" or "second" may expressly or imply the inclusion of one or more of the feature. Furthermore, in the present description, unless specifically stated otherwise, the meaning of "plurality" refers to two or more than two.

[0025] Hereinafter, specific embodiments of the present invention will be described in detail. It should be understood that the specific embodiments described herein are only intended to illustrate the present invention and are not intended to limit the present invention.

[0026] The endpoints of ranges and any value invented herein should be understood to be not limited to such exact ranges or values, but to include values ​​close to these ranges or values. In the case of ranges of numerical values, the range endpoints, the range endpoints and any single point value, and the single point values ​​may be combined with each other to form one or more new numerical ranges, and these numerical ranges are considered to be specifically invented in the specification.

[0027] To facilitate understanding of the present invention, certain technical and scientific terms are defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the meaning commonly understood by those of ordinary skill in the art. Abbreviations for amino acid residues refer to the standard three-letter and / or one-letter codes used in the art to represent one of the 20 commonly used L-amino acids.

[0028] In the present invention, unless otherwise specified, the term antigen-binding fragment used is "antibody fragment", and antibody fragment generally refers to an antigen-binding antibody fragment, which can include a portion of an intact antibody, generally the antigen-binding region or variable region, including, for example, Fab, Fab', F(ab')2, Fv or scFv, diabodies, linear antibodies, single-chain antibody molecules, etc.

[0029] The term "complementarity determining region" or "CDR" or "CDR sequence" refers to the amino acid sequence responsible for antigen binding in an antibody, typically consisting of amino acid residues 23-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region, and amino acid residues 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable region (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)), and / or "high-variability loops" (e.g., amino acid residues 26-32 (LI), 50-52 (L2), and 91-96 (L3) in the light chain variable region, and amino acid residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable region (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987))

[0030] The term "conservatively modified amino acid sequence" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence, and includes amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present invention by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions refer to replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions of antibodies of the invention can be replaced with other amino acid residues from the same side chain family, and the resulting antibody is tested for retention of function using the functional assays described herein. Preferably, the number of conservative modifications does not exceed one or two.

[0031] With respect to polypeptides, the term "substantial homology" means that when two polypeptides or their designated sequences are optimally aligned and compared (with appropriate insertions or deletions of nucleotides), at least about 80% of the amino acids are similar, usually at least about 90% to 95%, and more preferably at least about 98% to 99.5% of the amino acids are similar.

[0032] The percent identity between two sequences varies depending on the number of identical positions shared by the sequences when the sequences are optimally aligned (i.e., % homology = number of identical positions / total number of positions x 100), and optimal alignment is determined by considering the number of gaps and the length of each gap that need to be introduced to achieve optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be completed using a mathematical algorithm, as described in the non-limiting examples below.

[0033] Those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acids in the sequences of the present invention, obtaining variants of the antibody or functional fragment thereof, provided that the antibody activity is not substantially affected (retaining at least 95% activity). These variants are considered to be within the scope of the present invention. For example, amino acids with similar properties can be substituted in the variable region. The variant sequences described in the present invention have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence. Sequence identity described in the present invention can be measured using sequence analysis software, such as the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. All amino acid sequences referred to in the present invention are presented in N- to C-terminal order.

[0034] As described above, the antibodies of the present invention may be full-length antibodies (e.g., IgG1 or IgG4 antibodies), may comprise only the antigen-binding portion (e.g., Fab, F(ab')2, or scFv fragments), or may be modified to affect function. The present invention includes anti-CD155 antibodies with modified glycosylation patterns. For some applications, it may be useful to perform modifications to remove undesired glycosylation sites, or antibodies lacking fucose moieties on the oligosaccharide chains, for example, to enhance antibody-dependent cellular cytotoxicity (ADCC) function. For other applications, galactosylation modifications can be performed to alter complement-dependent cytotoxicity (CDC).

[0035] The term "functional fragment" as used herein refers in particular to antibody fragments, such as Fv, scFv (sc refers to single chain), Fab, F(ab')2, Fab', scFv-Fc fragments or diabodies, or any fragment whose half-life can be extended by chemical modification or liposome encapsulation, e.g., the addition of a poly(alkylene)diol, such as polyethylene glycol ("pegylated, PEGylated") (referred to as pegylated fragments of Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG or Fab'-PEG), where "PEG" is polyethylene glycol), and which fragments retain CD155-binding activity. Preferably, the functional fragment consists of or comprises a subsequence of the heavy or light chain variable region of the antibody from which it is derived, said subsequence being sufficient to retain the same binding specificity and sufficient affinity for CD155 as the antibody from which it is derived, preferably at least 1 / 100 of the affinity of the antibody from which it is derived, and in a more preferred embodiment at least 1 / 10. Such functional fragments comprise at least 5 amino acids, preferably 10, 15, 25, 50 and 100 consecutive amino acids of the antibody sequence from which it is derived.

[0036] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof, comprising at least one CDR sequence selected from heavy chain variable region CDR sequences: SEQ ID NOs: 1-3, 7-9, 13-15, and 19-21, and light chain variable region CDR sequences: SEQ ID NOs: 4-6, 10-12, 16-18, and 22-24, or an amino acid sequence having at least 80% identity thereto. The antibody or antigen-binding fragment according to embodiments of the present invention is an affinity-matured humanized antibody, which has stronger specificity, a longer half-life, and stronger potency, and can bind to human or monkey CD155 protein and inhibit the binding of CD155 to its receptors, such as TIGIT, CD96, and CD226, effectively treating or preventing CD155-mediated related diseases at a relatively low dosage without inducing apoptosis of CD155-positive cells, resulting in fewer toxic side effects and greater safety.

[0037] In a preferred embodiment of the present invention, antibodies are humanized to improve their biological acceptability, specificity, and efficacy. Furthermore, the inventors perform affinity maturation on the humanized antibodies, for example, by mutating the CDRs to obtain affinity-matured antibodies. The term "chimeric antibody" refers to a recombinant antibody obtained by using recombinant DNA technology to replace the amino acid sequences of the constant region of a monoclonal antibody derived from one species (e.g., mouse) with the constant region of an antibody derived from another species (e.g., human). The term "humanized antibody" refers to a recombinant antibody obtained by using recombinant DNA technology to replace all amino acid sequences of the constant region and non-CDRs of the variable region (Fv framework region (FR)) of a monoclonal antibody derived from one species (e.g., mouse) with the amino acid sequences of the constant region and non-CDRs of the variable region of an antibody derived from another species (e.g., human). In other words, an antibody in which the constant region of one antibody is humanized is called a chimeric antibody, and an antibody in which all non-CDR amino acid sequences of the constant region and variable region are humanized is called a humanized antibody. Humanization methods can be performed using conventional antibody engineering techniques, and will not be repeated here. "Antibody affinity maturation" refers to the process of randomly mutating amino acids in the CDR regions of an antibody and screening the resulting mutant library to obtain an antibody with higher corresponding antigen affinity.

[0038] According to some specific embodiments of the present invention, the antibody or antigen-binding fragment further comprises at least one of the following additional technical features:

[0039] According to some specific embodiments of the present invention, the antibody or antigen-binding fragment comprises at least one heavy chain variable region CDR1 selected from the amino acid sequences set forth in SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:19, and conservatively modified versions thereof; at least one heavy chain variable region CDR2 selected from the amino acid sequences set forth in SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, and conservatively modified versions thereof; and at least one heavy chain variable region CDR3 selected from the amino acid sequences set forth in SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:21, and conservatively modified versions thereof.

[0040] According to some specific embodiments of the present invention, the antibody or antigen-binding fragment may further comprise at least one light chain variable region CDR1 selected from the amino acid sequences set forth in SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:22, and conservatively modified versions thereof; at least one light chain variable region CDR2 selected from the amino acid sequences set forth in SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:23, and conservatively modified versions thereof; and at least one light chain variable region CDR3 selected from the amino acid sequences set forth in SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:24, and conservatively modified versions thereof.

[0041] According to some specific embodiments of the present invention, the antibody or antigen-binding fragment comprises: 1) a heavy chain variable region CDR1 shown in SEQ ID NO:1, a heavy chain variable region CDR2 shown in SEQ ID NO:2, a heavy chain variable region CDR3 shown in SEQ ID NO:3, a light chain variable region CDR1 shown in SEQ ID NO:4, a light chain variable region CDR2 shown in SEQ ID NO:5, and a light chain variable region CDR3 shown in SEQ ID NO:6; or 2) a heavy chain variable region CDR1 shown in SEQ ID NO:7, a heavy chain variable region CDR2 shown in SEQ ID NO:8, a heavy chain variable region CDR3 shown in SEQ ID NO:9, a light chain variable region CDR1 shown in SEQ ID NO:10, a light chain variable region CDR2 shown in SEQ ID NO:11, and a light chain variable region CDR3 shown in SEQ ID NO:12; or 3) a heavy chain variable region CDR1 shown in SEQ ID NO:13, a light chain variable region CDR2 shown in SEQ ID NO:14, and a light chain variable region CDR3 shown in SEQ ID NO:15. 3) a heavy chain variable region CDR1 shown in SEQ ID NO: 19, a heavy chain variable region CDR2 shown in SEQ ID NO: 20, a heavy chain variable region CDR3 shown in SEQ ID NO: 21, a light chain variable region CDR1 shown in SEQ ID NO: 22, a light chain variable region CDR2 shown in SEQ ID NO: 23, and a light chain variable region CDR3 shown in SEQ ID NO: 24.

[0042] According to some specific embodiments of the invention, the antibody or antigen-binding fragment comprises i) a heavy chain variable region having an amino acid sequence at least 80% identical to at least one of the amino acid sequences set forth in SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, and conservatively modified versions thereof, and / or ii) a light chain variable region having an amino acid sequence at least 80% identical to at least one of the amino acid sequences set forth in SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, and conservatively modified versions thereof.

[0043] According to some specific embodiments of the invention, the heavy chain variable region comprises an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a heavy chain variable region selected from i), and the light chain variable region comprises an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a light chain variable region selected from ii).

[0044] According to some specific embodiments of the invention, the antibody or antigen-binding fragment comprises 1) a heavy chain variable region set forth in SEQ ID NO:25 and a light chain variable region set forth in SEQ ID NO:26, or 2) a heavy chain variable region set forth in SEQ ID NO:27 and a light chain variable region set forth in SEQ ID NO:28, or 3) a heavy chain variable region set forth in SEQ ID NO:29 and a light chain variable region set forth in SEQ ID NO:30, or 4) a heavy chain variable region set forth in SEQ ID NO:31 and a light chain variable region set forth in SEQ ID NO:32.

[0045] According to some specific embodiments of the present invention, the antibody comprises at least one of a heavy chain constant region and a light chain constant region, and at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a human-derived antibody, a primate-derived antibody, or a variant thereof.

[0046] According to some specific embodiments of the invention, both the light and heavy chain constant regions of the antibody are derived from a human IgG antibody or a variant thereof.

[0047] According to some specific embodiments of the present invention, both the light chain constant region and the heavy chain constant region of the antibody are derived from a human IgG1 antibody, a human IgG4 antibody, or a variant thereof.

[0048] According to some specific embodiments of the invention, the antibody has a heavy chain constant region of the amino acid sequence set forth in SEQ ID NO:41, 42 or 43 and / or a light chain constant region of the amino acid sequence set forth in SEQ ID NO:44.

[0049] According to some specific embodiments of the invention, the antibody comprises a heavy chain having the amino acid sequence set forth in any one of SEQ ID NOs: 33, 35, 37, and 39, and a light chain having the amino acid sequence set forth in any one of SEQ ID NOs: 34, 36, 38, and 40.

[0050] According to some specific embodiments of the invention, the antibody is a monoclonal antibody, a murine antibody, a chimeric antibody, a humanized antibody, a human antibody, an Fv, a single chain antibody (scFv), Fab, Fab', Fab'-SH, or F(ab')2.

[0051] According to some specific embodiments of the invention, the antibody or antigen-binding fragment thereof is capable of binding to the amino acid sequence shown in SEQ ID NO:73 and / or 74.

[0052] Nucleic acids encoding the heavy and / or light chains of the antibodies of the present invention are within the scope of the present invention, and the corresponding nucleic acid sequences can be easily obtained by those skilled in the art based on the amino acid sequences of the heavy and / or light chains.

[0053] Thus, in another aspect, the present invention provides a nucleic acid molecule encoding the antibody or antigen-binding fragment of aspect 1. In some specific embodiments of the present invention, the antibody or antigen-binding fragment encoded by the nucleic acid molecule has stronger specificity, longer half-life, and stronger potency, and can bind to human or monkey CD155 protein and inhibit the binding of CD155 to its receptors, such as TIGIT, CD96, and CD226. It effectively treats or prevents CD155-mediated related diseases at a relatively low dosage, does not induce apoptosis of CD155-positive cells, and has low toxic side effects and is therefore safer.

[0054] According to some specific embodiments of the present invention, the nucleic acid molecule may further include at least one of the following additional technical features:

[0055] According to some specific embodiments of the invention, the nucleic acid molecule is DNA.

[0056] It should be understood by those skilled in the art that the nucleic acid molecules referred to herein actually include either or both of the complementary strands. For convenience, in many cases, only one strand is shown in this specification and claims, but the complementary strand is also disclosed. Furthermore, references to nucleic acid sequences in this application include DNA and RNA forms, and disclosure of one means disclosure of the other.

[0057] In another aspect, the present invention provides an expression vector carrying the aforementioned nucleic acid molecule. The expression vector may include a selectable control sequence, which is operably linked to the nucleic acid molecule. The control sequence is one or more control sequences capable of directing expression of the nucleic acid molecule in a host. Expression vectors according to embodiments of the present invention can efficiently express antibodies or antigen-binding fragments in suitable host cells, and the antibodies or antigen-binding fragments have stronger specificity, longer half-life, and stronger potency. They can bind to human or monkey CD155 protein and inhibit the binding of CD155 to its receptors, such as TIGIT, CD96, and CD226. They can effectively treat or prevent CD155-mediated related diseases at relatively low dosages, without inducing apoptosis of CD155-positive cells, and have low toxic side effects and higher safety. When the nucleic acid molecule is linked to a vector, the nucleic acid molecule can be linked directly or indirectly to control elements on the vector, as long as these control elements are capable of controlling the translation and expression of the nucleic acid molecule. Of course, these control elements may be derived directly from the vector itself, or may be exogenous, i.e., not derived from the vector itself. Of course, the nucleic acid molecule may be operably linked to the control elements. As used herein, "operably linked" refers to the connection of an exogenous gene to a vector, allowing the control elements within the vector, such as transcriptional control sequences and translational control sequences, to function in regulating the transcription and translation of the intended exogenous gene. Of course, nucleic acid molecules encoding antibodies or antigen-binding fragments can be inserted individually into different vectors, but are often inserted into the same vector. Commonly used vectors include plasmids and bacteriophages, such as Plasmid-X plasmids.

[0058] In one aspect, the present invention provides a method for preparing the aforementioned antibody or antigen-binding fragment, comprising introducing the aforementioned expression vector into cells and culturing the cells under conditions suitable for protein expression and secretion to obtain the antibody or antigen-binding fragment. The method according to some specific embodiments of the present invention can effectively obtain an antibody or antigen-binding fragment with stronger specificity, longer half-life, and stronger potency, which can bind to human or monkey CD155 protein and inhibit the binding of CD155 to its receptors, such as TIGIT, CD96, and CD226, and can effectively treat or prevent CD155-mediated related diseases at a relatively low dosage without inducing apoptosis of CD155-positive cells, resulting in low toxic side effects and greater safety.

[0059] According to some specific embodiments of the present invention, the above-mentioned method for preparing an antibody or antigen-binding fragment may further include at least one of the following additional technical features:

[0060] According to some specific embodiments of the present invention, the cells are not particularly limited, and either prokaryotic or eukaryotic cells can be used, and when the cells are eukaryotic cells, for example, mammalian cells, the expression efficiency of recombinant antibodies is high.

[0061] According to some specific embodiments of the invention, the cell is a eukaryotic cell.

[0062] According to some specific embodiments of the present invention, the eukaryotic cell is a mammalian cell. According to some specific examples of the present invention, when the cell is a eukaryotic cell, for example a mammalian cell, the expression efficiency of the recombinant antibody is high.

[0063] According to some specific embodiments of the invention, eukaryotic cells do not include animal germ cells, fertilized eggs or embryonic stem cells.

[0064] In another aspect of the present invention, the present invention provides recombinant cells, which carry the aforementioned nucleic acid or expression vector or are capable of expressing the aforementioned antibody or antigen-binding fragment. The recombinant cells are obtained by transfecting or transforming the expression vector. According to some specific embodiments of the present invention, the recombinant cells efficiently express large amounts of the aforementioned antibody or antigen-binding fragment under appropriate conditions. The antibody or antigen-binding fragment has stronger specificity, longer half-life, and stronger potency, and can bind to human or monkey CD155 protein and inhibit the binding of CD155 to its receptors, such as TIGIT, CD96, and CD226. This effectively treats or prevents CD155-mediated related diseases at a relatively low dosage, without inducing apoptosis of CD155-positive cells, resulting in low toxic side effects and greater safety.

[0065] The recombinant cells of the present invention are not particularly limited and may be prokaryotic cells, eukaryotic cells, or bacteriophages. Prokaryotic cells may be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis. Eukaryotic cells may be fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Trichoderma; insect cells such as grass rotifers; plant cells such as tobacco; or mammalian cells such as BHK cells, CHO cells, COS cells, or myeloma cells. In some embodiments, the recombinant cells of the present invention are preferably mammalian cells, including BHK cells, CHO cells, NSO cells, or COS cells, but do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0066] The term "appropriate conditions" as used herein refers to conditions suitable for the expression of the antibody or antigen-binding fragment of the present application. Those skilled in the art should understand that suitable conditions for the expression of an antibody or antigen-binding fragment include, but are not limited to, an appropriate transformation or transfection method, appropriate transformation or transfection conditions, a healthy host cell state, an appropriate host cell density, an appropriate cell culture environment, and an appropriate cell culture time. The term "appropriate conditions" is not particularly limited, and those skilled in the art will optimize the optimal conditions for the expression of an antibody or antigen-binding fragment according to the specific environment of their laboratory.

[0067] In a further aspect, the present invention provides an immunoconjugate, comprising the aforementioned antibody or antigen-binding fragment and a therapeutic agent. As described above, the antibodies or antigen-binding fragments according to embodiments of the present invention can effectively bind to CD155 protein and inhibit the binding of CD155 protein to its receptor. Therefore, immunoconjugates comprising the antibodies or antigen-binding fragments can similarly bind to human or simian CD155 protein. The immunoconjugates have good efficacy in preventing and / or treating CD155-mediated diseases, do not induce apoptosis of CD155-positive cells, have low toxic side effects, and are safer.

[0068] In one aspect, the present invention provides a composition comprising the above-described antibody, nucleic acid molecule, expression vector, or recombinant cell. As described above, the antibody or antigen-binding fragment according to some specific embodiments of the present invention can effectively bind to CD155 protein and inhibit the binding of CD155 protein to its receptor, without inducing apoptosis of CD155-positive cells. Therefore, a composition comprising the above substance can also effectively inhibit the binding of CD155 protein to its receptor, has a good effect in preventing and / or treating CD155-mediated diseases, and does not induce apoptosis of CD155-positive cells. The type of composition is not particularly limited, and may be a food composition or a pharmaceutical composition.

[0069] The compositions of the present invention can be administered in combination with each other or with one or more other therapeutic compounds, for example, in combination with a chemotherapeutic agent. Thus, the compositions may further comprise a chemotherapeutic agent. The antibodies or antigen-binding fragments thereof, or immunoconjugates of the present invention may be combined with a second therapeutic agent, examples of which include, but are not limited to, other agents that inhibit CD155 (such as other antibodies or antigen-binding fragments thereof, peptide inhibitors, small molecule antagonists, etc.) and / or agents that interfere with signal transduction upstream or downstream of CD155.

[0070] The term "composition" includes combinations of components separated in time and / or space, as long as they can act cooperatively to achieve the objectives of the present invention. For example, the components contained in the composition can be administered to a subject as a whole, or separately. When the components contained in the composition are administered to a subject separately, the components can be administered to the subject simultaneously or sequentially.

[0071] In another aspect, the present invention provides use of the aforementioned antibodies or antigen-binding fragments thereof, nucleic acid molecules, expression vectors, recombinant cells, or compositions in the preparation of a medicament for preventing and / or treating CD155-related diseases. As described above, the antibodies or antigen-binding fragments according to some specific embodiments of the present invention can effectively bind to CD155 protein and inhibit the binding of CD155 protein to its receptor, without inducing apoptosis of CD155-positive cells. Therefore, drugs containing the above substances can also effectively inhibit the binding of CD155 protein to its receptor, have good effects on preventing and / or treating CD155-mediated diseases, and do not induce apoptosis of CD155-positive cells.

[0072] According to some specific embodiments of the present invention, the use in preparing the above-mentioned drug may further include at least one of the following additional technical features:

[0073] According to some specific embodiments of the invention, the CD155-associated disease is cancer, an autoimmune disease, transplant rejection or an infectious disease.

[0074] According to some specific embodiments of the present invention, the cancer comprises at least one of malignant melanoma, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0075] According to some specific embodiments of the present invention, the infectious disease includes, but is not limited to, HIV viral infection and / or hepatitis b viral infection.

[0076] In a further aspect, the present invention provides a drug comprising the above-mentioned antibody nucleic acid molecule, expression vector, recombinant cell, or composition. As described above, the antibody or antigen-binding fragment according to some specific embodiments of the present invention can effectively bind to CD155 protein and inhibit the binding of CD155 protein to its receptor, without inducing apoptosis of CD155-positive cells. Therefore, a drug comprising an effective amount of an antibody active ingredient or a series of substances thereof can also effectively inhibit the binding of CD155 protein to its receptor, and the antibody or antigen-binding fragment has a good effect in preventing and / or treating CD155-mediated diseases without inducing apoptosis of CD155-positive cells.

[0077] According to some specific embodiments of the present invention, the drug may further include at least one of the following additional technical features:

[0078] According to some specific embodiments of the present invention, the medicament is used to treat or prevent a CD155-associated disease.

[0079] According to some specific embodiments of the invention, the CD155-associated disease is cancer, an autoimmune disease, transplant rejection or an infectious disease.

[0080] According to some specific embodiments of the present invention, the cancer comprises at least one of malignant melanoma, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0081] According to some specific embodiments of the present invention, the infectious disease includes, but is not limited to, HIV viral infection and / or hepatitis b viral infection.

[0082] According to some specific embodiments of the present invention, it may further comprise a pharmaceutically acceptable vector.

[0083] As used herein, the term "effective amount" or "effective dose" means an amount that produces a function or activity in humans and / or animals and is acceptable to humans and / or animals.

[0084] As used herein, a "pharmaceutically acceptable" component is a substance that can be administered to humans and / or mammals without undue side effects (e.g., toxicity, irritation, allergies, etc.), i.e., a substance that has a reasonable benefit / risk ratio. The term "pharmaceutically acceptable vector" refers to a vector used to administer a therapeutic agent, including various excipients and diluents.

[0085] The medicament of the present invention comprises a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable vector. Such vectors include, but are not limited to, saline, buffer, glucose, water, glycerin, ethanol, and combinations thereof. Typically, the drug formulation is tailored to the mode of administration, which may be oral, nasal, intradermal, subcutaneous, intramuscular, intravenous, or intraperitoneal. The dosage form of the medicament of the present invention may be an injection, oral preparation (tablet, capsule, oral liquid), transdermal preparation, or sustained-release preparation. For example, it may be prepared by a conventional method using saline or an aqueous solution containing glucose and other excipients. Preferably, the medicament is prepared under aseptic conditions.

[0086] The effective amount of the active ingredient of the present invention can vary depending on the mode of administration and the severity of the disease being treated. The selection of a preferred effective amount can be determined by one skilled in the art based on various factors (e.g., clinical trials). These factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life, the severity of the disease being treated in the patient, the patient's body weight, the patient's immune status, and the route of administration. For example, depending on the exigencies of the therapeutic situation, the active ingredient can be administered in divided doses per day or the dose can be proportionally reduced.

[0087] Of course, the anti-CD155 antibodies herein may be prepared as part of a kit or other diagnostic reagent, if desired.

[0088] In one aspect, the present invention includes the use of the aforementioned antibodies or antigen-binding fragments in the preparation of a kit for detecting CD155. As described above, the antibodies or antigen-binding fragments according to some specific embodiments of the present invention can effectively bind to human or monkey CD155 protein and inhibit the binding of CD155 protein to its receptor. Therefore, the antibodies or antigen-binding fragments can be used to prepare a kit for detecting CD155 protein, and the kit can effectively detect CD155 protein qualitatively or quantitatively.

[0089] In another aspect, the present invention provides a kit for detecting CD155, which includes the aforementioned antibody or antigen-binding fragment thereof. As described above, antibodies or antigen-binding fragments according to some specific embodiments of the present invention can effectively bind to human or monkey CD155 protein and inhibit the binding of CD155 protein to its receptor. Therefore, kits containing the antibody or antigen-binding fragment can effectively detect CD155 protein qualitatively or quantitatively. Kits embodying the present invention may be kits used for detection utilizing the specific binding ability of antibodies to the CD155 antigen, such as immunoblotting or immunoprecipitation. These kits may also include one or more of the following: antagonists, anti-CD155 antibody or drug reference materials, protein purification columns, immunosphere protein affinity purification buffers, cell assay diluents, instructions, literature, etc. Anti-CD155 antibodies can be used in various types of diagnostic tests, for example, to detect the presence of various diseases or drugs, toxins, or other proteins in vitro or in vivo, for example, by detecting them in a subject's serum or blood to test for related diseases. Such related diseases may include CD155-related diseases, such as cancer, autoimmune diseases, transplant rejection, or infectious diseases, including at least one of malignant melanoma, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer. Of course, the antibodies provided herein can also be used for radioimmunodetection and radioimmunotherapy of the above diseases.

[0090] According to some specific embodiments of the present invention, the kit may further comprise reagents commonly used to detect CD155, such as a coating solution.

[0091] The present invention further relates to a method for preventing and / or treating a CD155-associated disease, the method comprising the step of administering to a patient an effective amount of at least one of an antibody or antigen-binding fragment thereof, a nucleic acid molecule, an expression vector, a recombinant cell, an immunoconjugate, and a composition of the present invention.

[0092] A "patient" or "subject" in the context of the present invention generally refers to a mammal, such as a primate and / or rodent, particularly a human or a mouse.

[0093] The sequence descriptions relevant to the present invention are detailed in Table 1.

[0094] [Table 1(1)] [Table 1(2)] [Table 1(3)] [Table 1(4)] [Table 1(5)] [Table 1(6)] [Table 1(7)] [Table 1(8)] [Table 1(9)] [Table 1 (10)] [Table 1 (11)] [Table 1 (12)] [Table 1(13)] [Table 1(14)]

Table 1(15)

Table 1(16)

Table 1(18)

Table 1(19)

Table 1(20)

Table 1(21)

Table 1(22)

Table 1(23)

Table 1(24)

Table 1(25)

Table 1(26)

Table 1(27)

Table 1(28)

Table 1(29)

Table 1(30)

Table 1(32)

Table 1(33)

Table 1(34)

Table 1(35)

Table 1(37)

Table 1(38)

Table 1(39)

Table 1(40)

Table 1(42)

Table 1(43)

Table 1(44)

Table 1(45)

Table 1(46)

Table 1(47)

[0095] The present invention will be described in detail with reference to the following examples. In the examples or test examples, unless specific experimental conditions are specified, the experimental methods are carried out under ordinary conditions.

[0096] The following examples are used in combination to illustrate the present invention. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If specific techniques or conditions are not specified in the examples, they should be carried out according to the techniques or conditions described in the literature in the field or according to the product specifications. If the manufacturer of the reagents or equipment used is not specified, they are conventional products that can be purchased commercially.

[0097] Example 1: Preparation of antibodies

[0098] This example was used to obtain a mouse-derived monoclonal antibody against human CD155. Balb / c mice (9 weeks old, purchased from Shanghai Resc, weighing approximately 20 g) were immunized with purified recombinant CD155 extracellular region Fc fusion protein (CD155-Fc) (a fusion protein of the recombinant CD155 extracellular region and hIgG1-Fc, the amino acid sequence of which is shown in SEQ ID NO: 75) as an antigen.

[0099] The immunized mice were intraperitoneally immunized three times using purified antigen and complete Freund's adjuvant, and immune responses were detected after blood collection from the tail vein. Serum screening was performed using ELISA and flow cytometry according to the usual procedure to obtain mice with anti-human CD155 immunoglobulin protein. Splenocytes were collected from the mice with the highest levels of anti-CD155 immunoglobulin protein and fused with mouse myeloma cells SP2 / 0 (ATCC number CRL-1581). Antibody screening was performed on the fused hybridoma cells to obtain mouse monoclonal antibodies. The specific procedure is as follows: The total number of candidate hybridoma cells was increased to 10 6The hybridoma cells were cultured at 100°C for 10 minutes, and then centrifuged at 800 rpm to harvest the cells. Total RNA from the hybridoma cells was extracted using a Trizol kit (Invitrogen). A cDNA library (Invitrogen) was then synthesized by reverse transcription using the total RNA as a template. The cDNA was then used as a template to amplify the variable region nucleic acid sequences corresponding to the hybridoma cells using PCR. The primer sequences used in the PCR amplification reaction were complementary to the first framework region or signal peptide region and constant region of the antibody variable region (see Larrick, JW, et al. (1990) Scand. J. Immunol., 32, 121-128 and Coloma, JJ, et al. (1991) BioTechniques, 11, 152-156 for details). The PCR amplification reaction system and process are as follows: A 50 μL reaction mixture was prepared by adding 2 μL of cDNA, 5 μL of 10× PCR buffer, 2 μL (5 μmol) of upstream and downstream primers, 2 μL of dNTPs, 1 μL of Taq enzyme (Takara, Ex Taq), and 38 μL of HO. The mixture was pre-denatured at 95°C for 5 minutes, followed by temperature cycling for PCR amplification. The reaction conditions were 30 s denaturation at 94°C, 45 s annealing at 58°C, and 50 s elongation at 72°C for a total of 32 cycles, followed by 7 min of elongation at 72°C. After sequencing the amplified product, the sequences of the heavy and light chain variable regions of the mouse monoclonal antibody (the amino acid sequences of which are shown in SEQ ID NOs: 53-54) were obtained.

[0100] Example 2: Detection of binding activity of mouse-derived CD155 antibody

[0101] Flow cerebrospinal fluid (FCE) experiments were performed to detect the binding properties of CD155 antibodies. Human CD155 protein (CHO-K1-human CD155) and monkey CD155 protein (CHO-K1-Rhesus CD155) were overexpressed in CHO-K1 cells (ATCC number 10111). The strength of the detection signal after the addition of the mouse-derived antibody obtained in Example 1 was used to determine the binding properties of the antibody to CD155. The specific experimental procedures were as follows: 5 × 10 HEK293T cells5 Cells were plated at 1000 μL per well in a 6-well plate and cultured overnight in DMEM medium without the double antibody. Before transfection, discard the medium and add 1 mL of fresh DMEM medium without the double antibody. pLVX-EF1a-CD155-IRES-puro (inserted with the nucleotide sequence SEQ ID NO: 45 encoding the human CD155 protein) or pLVX-EF1a-cynomolgus CD155-IRES-puro (inserted with the nucleotide sequence SEQ ID NO: 46 encoding the Rhesus CD155 protein) was added to 200 μL of serum-free DMEM medium at a 2:1:1 ratio with pMD2G or psPAX2 vector (3 μg total). Next, 12 μg of polyetherimide (PEI, Polysciences) was added to cleave the pLVX-EF1a-IRES-puro vector between EcoRI and BamHI. After uniform mixing, the mixture was left to stand for 16 minutes, and then the entire liquid was added to a 6-well plate containing HEK293T cells. After 6 hours of incubation, the medium was discarded and fresh complete DMEM medium was added and incubated. 48 hours after transfection, the cell culture supernatant was collected and passed through a 0.45 μm filter (Millipore), which is the viral supernatant. The different viral supernatants obtained were all diluted to 1 × 10 4 The cells were added to a 6-well plate containing 1000 CHO-K1 cells, and polybrene (Sigma) was added at a final concentration of 4 μg / mL. The cells were then cultured for 12 hours. The supernatant was then completely removed, and fresh complete DMEM medium was added. The resulting cells were CHO-K1-CD155 or CHO-K1-Rhesus CD155 cells.

[0102] 2 x 10 CHO-K1-human CD155 or CHO-K1-Rhesus CD155 cells in PBS 6The cells were diluted to 100 μL / tube and added to 1.5 mL EP tubes. 10 μL / tube of goat serum (purchased from Shanghai Biotechnology) was added and blocked for 30 min at 4°C. Different concentrations of CD155 antibody were added and incubated for 30 min at 4°C. 1 mL of PBS was added to the EP tubes, and the cells were centrifuged at 3500 rpm for 5 min at 4°C. The supernatant was completely removed, and the cells were washed once with PBS and pelleted. After centrifugation, the supernatant was completely removed, and the cells were resuspended in 100 μL / tube of PBS. 0.1 μL / tube of Alexa-647-conjugated goat anti-mouse secondary antibody (Biolegend) was added and incubated for 30 min at 4°C, protected from light. The cells were washed twice with PBS, centrifuged, and the supernatant was completely removed. The cells were resuspended in 200 μL / tube of PBS and detected by flow cytometry. IgG (purchased from Biolegend) was used as a control. Figure 1 shows that the mouse-derived antibody of the present invention (mutgi1005) can bind to human and monkey CD155 proteins.

[0103] Example 3: Detection of inhibition of activity of mouse-derived CD155 antibody

[0104] CD155 antibodies bind to the extracellular domain of CD155, inhibiting the signaling pathway between CD155 and its receptors TIGIT, CD96, and CD226. Flow cerebrospinal fluid (FCE) experiments were used to detect the inhibition of CD155 binding to its receptors by CD155 antibodies. The specific experimental procedures are as follows: 2 × 10 CHO-K1-human CD155 cells (same as above) obtained in Example 2 were diluted with PBS. 6The cells were diluted to 1 / mL and placed in 1.5 mL EP tubes at a volume of 100 μL per tube. 10 μL of mouse serum was added per tube and blocked at 4°C for 30 minutes. After blocking, different concentrations of CD155 antibodies were added and incubated at 4°C for 30 minutes. Next, 2 μg / tube of TIGIT extracellular domain Fc fusion protein (TIGIT-Fc, SEQ ID NO: 76, expressed in our laboratory), 4 μg / tube of CD96 extracellular domain Fc fusion protein (CD96-Fc, SEQ ID NO: 77, expressed in our laboratory), or 2 μg / tube of CD226 extracellular domain Fc fusion protein (CD226-Fc, SEQ ID NO: 78, expressed in our laboratory) were added and incubated at 4°C for 30 minutes. 1 mL of PBS was added to the EP tubes at 4°C. The cells were centrifuged at 3500 rpm for 5 min, the supernatant completely removed, and washed once with PBS. After centrifugation, the supernatant was completely removed and the cells were resuspended in 100 μL of PBS per tube. Then, 1 μL of 647-labeled mouse anti-human secondary antibody (HP6017, Biolegend) was added per tube and incubated at 4°C for 30 min, protected from light. The cells were washed twice with PBS, centrifuged, and the supernatant completely removed. The cells were resuspended in 200 μL of PBS per tube and detected by flow cytometry, using IgG (purchased from Biolegend) as a control antibody. As shown in Figure 2, the mouse-derived CD155 antibody (mutgi1005) of the present invention was able to inhibit the binding of CD155 to TIGIT, CD96, and CD226.

[0105] Example 4: Experiment to detect inhibition of activity of mouse-derived CD155 antibody

[0106] ELISA experiments were used to detect the effects of antibody binding to CD155 and its receptors TIGIT, CD96, and CD226. The specific experiments are as follows:

[0107] CD155-His tag fusion protein (purchased from Acro) was diluted to 5 μg / mL in PBS buffer and added to a 96-well plate at a volume of 100 μL per well and incubated overnight at 4°C. The PBS buffer in the 96-well plate was then aspirated and the plate was washed six times with PBST (pH 7.2, 0.1% Tween 20). 200 μL of PBS / 10% BSA was added per well and incubated at 37°C for 2 hours for blocking. The blocking solution was then removed and the plate was washed six times with PBST. The appropriate concentrations of the target mouse CD155 antibodies (5 μg / mL TIGIT-Avi tag (purchased from Acro), 3.2 μg / mL CD96-Avi tag (purchased from Acro), or 10 μg / mL CD226-Avi tag (purchased from Acro) were added to the plate at 100 μL per well and incubated at 37°C for 1 hour. The reaction mixture was removed, and the plate was washed six times with PBST. 100 μL / well of HRP (horse radish peroxidase)-conjugated streptavidin secondary antibody (purchased from Southern Biotech) was diluted in PBST / 0.05% BSA and incubated at 37°C for 1 hour. After washing the plate six times with PBST, 80 μL / well of TMB (tetramethylbenzidine) was added and incubated at room temperature for 3 minutes. 80 μL / well of 4M sulfuric acid was added to stop the reaction. Absorbance readings were taken at 450 nm using a microplate reader with mIgG (purchased from Biolegend) and SKII4 (purchased from Biolegend) as controls. As shown in Figure 3, the mouse CD155 antibody (Mutgi1005) of the present invention inhibits the binding of CD155 to its receptors TIGIT, CD96, and CD226.

[0108] Example 5: Flow cetocemometry binding experiments of chimeric CD155 antibodies

[0109] 2 × 10 CHO-K1-human CD155 and CHO-K1-Rhesus CD155 cells obtained in Example 2 were cultured in PBS. 6The cells were diluted to 1 mL / mL and added to 1.5 mL EP tubes at a volume of 100 μL / tube, to which 10 μL / tube of mouse serum was added, followed by blocking at 4°C for 30 minutes. Different concentrations of mutgi1005-hIgG1 or mutgi1005-hIgG4 CD155 chimeric antibodies (constructed by fusing the light chain variable region and heavy chain variable region of the mouse-derived antibody obtained in Example 1 with human wild-type IgG1 or IgG4, respectively, and represented by SEQ ID NOs: 69-72) were added and incubated at 4°C for 30 minutes. 1 mL of PBS was added to the EP tubes, and the mixture was centrifuged at 3,500 rpm at 4°C for 5 minutes. The supernatant was completely removed, and the cells were washed once again with PBS. After centrifugation, the supernatant was completely removed, and the cells were resuspended in 100 μL / tube of PBS. 1 μL / tube of Alexa-647-labeled mouse anti-human secondary antibody (HP6017, Biolegend) was added and incubated at 4°C for 30 minutes, protected from light. The cells were washed twice with PBS, centrifuged, and the supernatant was completely removed. The cells were resuspended in 200 μL / tube of PBS and analyzed by flow cytometry. The results, shown in Figure 4, further demonstrate that the chimeric antibodies of the present invention can bind to human or monkey CD155 proteins.

[0110] Example 6: Antibody humanization and affinity maturation experiments

[0111] Using the CD155 antibody heavy chain variable region sequence (shown in SEQ ID NO: 53) and light chain variable region sequence (shown in SEQ ID NO: 54) obtained in Example 1, a humanization template that best matched the non-CDR regions was designed and selected using MOE software. The CDR regions of the mouse-derived antibody were grafted onto the selected humanization template to replace the CDR regions of the human-derived template, resulting in a humanized antibody. Next, based on the three-dimensional structure of the mouse-derived antibody, restoration mutations were made to buried residues, residues that directly interact with the CDRs, and residues in the FR region that have a significant impact on the conformation of the VL and VH, to obtain a humanized antibody. The sequence of the heavy chain variable region of the humanized CD155 antibody (tgi1005 V0-hIgG1 / 4) is shown in SEQ ID NO: 63, and the sequence of the light chain variable region is shown in SEQ ID NO: 64.

[0112] An affinity-matured antibody was obtained by randomly mutating the CDR regions of the CD155 antibody. The sequence of the heavy chain variable region of the affinity-matured CD155 antibody is shown in SEQ ID NO:25, and the sequence of the light chain variable region is shown in SEQ ID NO:26; alternatively, the sequence of the heavy chain variable region of the affinity-matured CD155 antibody is shown in SEQ ID NO:27, and the sequence of the light chain variable region is shown in SEQ ID NO:28; alternatively, the sequence of the heavy chain variable region of the affinity-matured CD155 antibody is shown in SEQ ID NO:29, and the sequence of the light chain variable region is shown in SEQ ID NO:30; or the sequence of the heavy chain variable region of the affinity-matured CD155 antibody is shown in SEQ ID NO:31, and the sequence of the light chain variable region is shown in SEQ ID NO:32. Specific sequences are shown in Tables 1 and 2.

[0113] [Table 2]

[0114] Example 7: Binding activity detection experiment of affinity-matured humanized CD155 antibodies

[0115] ELISA experiments were performed to detect the binding properties of the affinity-matured humanized CD155 antibodies obtained in Example 6. CD155-His tagged fusion protein (purchased from Acro) and Rhesus CD155-His tagged fusion protein (purchased from Acro) were coated onto a 96-well plate, and the intensity of the detection signal after addition of the antibody was used to determine the binding properties of the antibody to human and monkey CD155.

[0116] CD155-His tagged fusion protein and Rhesus CD155-His tagged fusion protein were diluted to 2 μg / mL in PBS buffer and added to a 96-well plate at a volume of 100 μL / well and incubated at 4°C overnight. The PBS buffer in the 96-well plate was then aspirated and the plate was washed six times with PBST (pH 7.2, 0.1% Tween 20). 200 μL / well of PBS / 10% BSA was added and incubated at 37°C for 2 hours for blocking. After removing the blocking solution and washing six times with PBST, 100 μL / well of the affinity-matured humanized CD155 antibody to be measured, diluted to the appropriate concentration in PBST / 0.05% BSA, was added and incubated at 37°C for 1 hour. After removing the reaction mixture and washing the plate six times with PBST, 100 μL / well of HRP (horse radish peroxidase)-conjugated mouse anti-human IgG (Fab specific) secondary antibody (Sigma) was diluted in PBST / 0.05% BSA and incubated at 37°C for 1 h. After washing the plate six times with PBST, 80 μL / well of TMB (tetramethylbenzidine) was added and incubated at room temperature for 3 min. 80 μL / well of 4M sulfuric acid was added to stop the reaction. hIgG1 (purchased from Biolegend and proprietary NB1088-hIgG1 (SEQ ID NOs: 79 and 81) and NB1088-hIgG4 (SEQ ID NOs: 80 and 81) antibodies expressed in this laboratory) were used as control antibodies. Absorbance readings were taken at 450 nm using a microplate reader. The results are shown in Figure 5. LALA refers to the L234A and L235A mutations in the hIgG1 constant region. The affinity-matured humanized CD155 antibodies of the present invention can bind to human and monkey CD155, and the binding activity of the affinity-matured antibodies is higher than that of the non-affinity-matured antibodies.

[0117] Example 8: Experiments to detect inhibition of the activity of affinity-matured humanized CD155 antibodies

[0118] The ELISA experiment is used to detect the effect of the affinity-matured humanized CD155 antibody obtained in Example 6 on binding between CD155 and its receptor.

[0119] CD155-His tag fusion protein (purchased from Acro) was diluted to 5 μg / mL in PBS buffer, added to a 96-well plate at a volume of 100 μL / well, and left overnight at 4°C. The PBS buffer in the 96-well plate was then aspirated and the plate was washed six times with PBST (pH 7.2 PBS containing 0.1% Tween 20) buffer. 200 μL / well of PBS / 10% BSA was then added and incubated at 37°C for 2 hours for blocking. After removing the blocking solution and washing six times with PBST, 100 μL / well of the affinity-matured humanized CD155 antibody of interest was added at the appropriate concentration in PBST / 0.05% BSA. The antibody was then incubated at 37°C for 1 hour. The reaction mixture was removed, the plate was washed six times with PBST, and 100 μL / well of HRP-conjugated Avidin secondary antibody was added at 37°C for 1 hour. After washing the plate six times with PBST, 80 μl / well of TMB (tetramethylbenzidine) was added and incubated at room temperature for 3 minutes. The reaction was stopped by adding 80 μl / well of 4 M sulfuric acid. Absorbance readings were taken at 450 nm using a microplate reader. The results, shown in Figure 7, demonstrate that the affinity-matured humanized CD155 antibody of the present invention can inhibit the binding of CD155 to TIGIT, CD96, and CD226.

[0120] Example 9: Flow cerebrospinal fluid binding experiments of affinity-matured humanized CD155 antibodies

[0121] 2×10 CHO-K1-human CD155 and CHO-K1-Rhesus CD155 cells obtained in Example 2 were diluted in PBS. 6The cells were diluted to 1 mL / mL and added to 1.5 mL EP tubes at a volume of 100 μL per tube. 10 μL of rat serum was added per tube and blocked at 4°C for 30 minutes. The affinity-matured humanized CD155 antibody obtained in Example 6 was added at different concentrations and incubated at 4°C for 30 minutes. 1 mL of PBS was added to the EP tube, and the mixture was centrifuged at 3500 rpm for 5 minutes at 4°C. The supernatant was completely removed. The cells were washed once again with PBS, and the supernatant was completely removed after centrifugation. The cells were resuspended in 100 μL per tube of PBS, and 1 μL of Alexa-647-labeled rat anti-human secondary antibody (Biolegend) was added per tube. The mixture was incubated at 4°C for 30 minutes, protected from light. The cells were washed twice with PBS, and the supernatant was completely removed after centrifugation. The cells were resuspended in 200 μL / tube of PBS and detected by flow cytometry. hIgG1 was used as a control antibody. The experimental results, as shown in Figure 6, demonstrated that the affinity-matured humanized antibody of the present invention can bind to CD155.

[0122] Example 10: Affinity-matured humanized CD155 antibodies affect apoptosis in U-937-CD155 cells

[0123] Flow cerebrospinal fluid (FPE) experiments were performed to detect the functional properties of the affinity-matured humanized CD155 antibody obtained in Example 6. Human CD155 protein (U-937-CD155) was overexpressed in U937 cells (ATCC No. CRL-1593.2) using the pLVX-EF1a-IRES-puro plasmid. The antibody was added and incubated for 4 hours. 7-AAD was then added. The intensity of the 7-AAD signal was used to determine the effect of the CD155 antibody on the functional properties of apoptosis in U-937-CD155 cells.

[0124] 5 × 10 HEK293T cells 5Cells were plated at 1000 cells / well in a 6-well plate and cultured overnight in DMEM medium without the double antibody. Before transfection, the medium was discarded and 1 mL of fresh DMEM medium without the double antibody was added. pLVX-EF1a-CD155-IRES-pur (the coding sequence of human CD155 protein inserted between the EcoRI and BamHI enzyme cleavage sites of the pLVX-EF1a-IRES-puro vector (SEQ ID NO: 45)), pMD2G, and psPAX2 vectors (3 μg in total) were added to 200 μL of serum-free DMEM medium at a 2:1:1 ratio, and 12 μg of polyetherimide (PEI, Polysciences) was added. After uniform mixing, the mixture was left to stand for 16 minutes, and then the entire liquid was added to the HEK293T cells in a 6-well plate. After 6 hours of incubation, the medium was discarded and fresh complete DMEM medium was added and incubated. 48 hours after transfection, the cell culture supernatant was collected and passed through a 0.45 μm filter (Millipore) to obtain the viral supernatant. The viral supernatant was then diluted to 1 × 10 4 A 6-well plate of U-937 cells was cultured with 4 μg / mL polybrene (Sigma) at a final concentration of 4 μg / mL for 12 hours. The supernatant was then completely removed and fresh complete IMDM medium was added. The resulting cells are U-937-CD155 cells.

[0125] 2 × 10 U-937-CD155 cells in complete IMDM 5 The affinity-matured humanized CD155 antibodies were diluted to 1 / mL and added to a 96-well round-bottom plate at a volume of 100 μL per tube. The affinity-matured humanized CD155 antibodies were then added and incubated at 37°C, 5% CO for 4 hours. 1 μg / tube of 7-AAD was added, and the cell suspension was transferred to a flow tube for detection by flow cytometry. The results, as shown in Figure 8, further demonstrate that the affinity-matured humanized CD155 antibodies of the present invention have distinct functional properties: v0 can promote apoptosis of U-937-CD155 cells, while v20, v28, and v40 do not induce apoptosis.

[0126] Example 11: Anti-cancer effects in mice with affinity-matured humanized CD155 antibodies

[0127] In vivo efficacy experiments were used to detect the enhanced anti-cancer function of the affinity-matured humanized CD155 antibody obtained in Example 6 in immune reconstituted mice. (1) On day 0, human PBMCs were administered via the tail vein into NCG mice (purchased from Chubu Pharmaceutical Co., Ltd.) at a dose of 1 × 10 7 / animals, (2) On day 4, tumors developed subcutaneously on the right flank of NCG mice, with 1 × 10 per mouse. 6 tumor cells are injected, (3) on days 7, 10, 13, and 16, mice are intraperitoneally injected with the affinity-matured humanized antibody at 100 μg or 200 μg per mouse; (4) After the injection of the above antibodies, the tumor volume was measured once every three days (days 7, 10, 13, 16, 19, and 22).

[0128] The results, as shown in Figure 9, demonstrate that the affinity-matured humanized CD155 antibody of the present invention can effectively inhibit tumor growth and has anti-cancer function.

[0129] Combined with the experimental results of Examples 1-11 above, the antibodies obtained in the present invention can bind to human and monkey CD155 proteins, effectively inhibit the interaction between CD155 and its receptors TIGIT, CD96, and CD226, and promote immune reconstitution anti-cancer in mice.

[0130] Although the preferred embodiments of the present invention have been described in detail above, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical means of the present invention, and each technical feature can be combined in any other suitable manner, and these simple modifications and combinations should also be considered as the inventive content of the present invention, and all fall within the protection scope of the present invention.

[0131] In the description herein, a statement referring to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples" means that a particular feature, structure, material, or characteristic described with reference to that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, general references to such terms do not necessarily refer to the same embodiment or example. In addition, a particular feature, structure, material, or characteristic described may be incorporated in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art can combine and combine the various embodiments or examples described herein and the features of the various embodiments or examples without mutual contradiction.

[0132] Although the embodiments of the present invention have been shown and described, the above embodiments are illustrative and should not be construed as limiting the present invention. Those skilled in the art will understand that various changes, modifications, substitutions and variations can be made to the above embodiments within the scope of the present invention.

Claims

1. An antibody or antigen-binding fragment, Heavy chain variable region CDR sequences having SEQ ID NOs: 1-3, 7-9, 13-15 and 19-21; and An antibody or antigen-binding fragment thereof, comprising at least one CDR sequence selected from the light chain variable region CDR sequences of SEQ ID NOs: 4-6, 10-12, 16-18 and 22-24, or an amino acid sequence having at least 80% identity thereto.

2. At least one heavy chain variable region CDR1 selected from the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 19 and conservatively modified amino acid sequences thereof; At least one heavy chain variable region CDR2 selected from the amino acid sequences set forth in SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 14, and SEQ ID NO: 20, and conservatively modified amino acid sequences thereof; and The antibody or antigen-binding fragment of claim 1, characterized in that it comprises at least one heavy chain variable region CDR3 selected from the amino acid sequences shown in SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 21, and conservatively modified amino acid sequences thereof.

3. At least one light chain variable region CDR1 selected from the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 16, and SEQ ID NO: 22, and conservatively modified amino acid sequences thereof; At least one light chain variable region CDR2 selected from the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 17, and SEQ ID NO: 23 and conservatively modified amino acid sequences thereof; and The antibody or antigen-binding fragment of claim 1, characterized in that it contains at least one light chain variable region CDR3 selected from the amino acid sequences shown in SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 24, and conservatively modified amino acid sequences thereof.

4. 1) a heavy chain variable region CDR1 shown in SEQ ID NO: 1, a heavy chain variable region CDR2 shown in SEQ ID NO: 2, a heavy chain variable region CDR3 shown in SEQ ID NO: 3, a light chain variable region CDR1 shown in SEQ ID NO: 4, a light chain variable region CDR2 shown in SEQ ID NO: 5, and a light chain variable region CDR3 shown in SEQ ID NO: 6; or 2) a heavy chain variable region CDR1 shown in SEQ ID NO: 7, a heavy chain variable region CDR2 shown in SEQ ID NO: 8, a heavy chain variable region CDR3 shown in SEQ ID NO: 9, a light chain variable region CDR1 shown in SEQ ID NO: 10, a light chain variable region CDR2 shown in SEQ ID NO: 11, and a light chain variable region CDR3 shown in SEQ ID NO: 12; or 3) a heavy chain variable region CDR1 shown in SEQ ID NO: 13, a heavy chain variable region CDR2 shown in SEQ ID NO: 14, a heavy chain variable region CDR3 shown in SEQ ID NO: 15, a light chain variable region CDR1 shown in SEQ ID NO: 16, a light chain variable region CDR2 shown in SEQ ID NO: 17, and a light chain variable region CDR3 shown in SEQ ID NO: 18; or 4) The antibody or antigen-binding fragment according to any one of claims 2 to 3, comprising a heavy chain variable region CDR1 shown in SEQ ID NO: 19, a heavy chain variable region CDR2 shown in SEQ ID NO: 20, a heavy chain variable region CDR3 shown in SEQ ID NO: 21, a light chain variable region CDR1 shown in SEQ ID NO: 22, a light chain variable region CDR2 shown in SEQ ID NO: 23, and a light chain variable region CDR3 shown in SEQ ID NO:

24.

5. i) a heavy chain variable region comprising an amino acid sequence having at least 80% identity with at least one of the amino acid sequences set forth in SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, and conservatively modified versions thereof; and / or ii) The antibody or antigen-binding fragment thereof according to claim 4, characterized in that it comprises a light chain variable region comprising an amino acid sequence having at least 80% homology with at least one of the amino acid sequences set forth in SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, and conservatively modified forms thereof.

6. 6. The antibody or antigen-binding fragment thereof according to claim 5, wherein the heavy chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a heavy chain variable region selected from i), and the light chain variable region comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a light chain variable region selected from ii).

7. 1) a heavy chain variable region set forth in SEQ ID NO: 25 and a light chain variable region set forth in SEQ ID NO: 26, or 2) a heavy chain variable region set forth in SEQ ID NO: 27 and a light chain variable region set forth in SEQ ID NO: 28; or 3) a heavy chain variable region set forth in SEQ ID NO: 29 and a light chain variable region set forth in SEQ ID NO: 30; or 4) The antibody or antigen-binding fragment thereof according to claim 6, characterized in that it comprises a heavy chain variable region shown in SEQ ID NO: 31 and a light chain variable region shown in SEQ ID NO:

32.

8. The antibody or antigen-binding fragment of claim 1, wherein the antibody comprises at least one of a heavy chain constant region and a light chain constant region, and at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from a human antibody, a primate antibody, or at least one of their variants.

9. The antibody or antigen-binding fragment of claim 8, wherein both the light chain constant region and the heavy chain constant region of the antibody are derived from a human IgG antibody or a mutant thereof.

10. The antibody or antigen-binding fragment of claim 8, wherein both the light chain constant region and the heavy chain constant region of the antibody are derived from a human IgG1 antibody, a human IgG4 antibody, or a mutant thereof.

11. The antibody or antigen-binding fragment of claim 8, wherein the antibody has a heavy chain constant region of the amino acid sequence shown in SEQ ID NO: 41, 42 or 43 and / or a light chain constant region of the amino acid sequence shown in SEQ ID NO:

44.

12. The antibody or antigen-binding fragment of claim 8, characterized in that it has a heavy chain having an amino acid sequence set forth in any one of SEQ ID NOs: 33, 35, 37 and 39, and a light chain having an amino acid sequence set forth in any one of SEQ ID NOs: 34, 36, 38 and 40.

13. The antibody may be a monoclonal antibody, a murine antibody, a chimeric antibody, a humanized antibody, a human antibody, an Fv, a single chain antibody (scFv), an Fab, an Fab', an Fab'-SH or an F(ab'). ２ The antibody or antigen-binding fragment thereof according to claim 1,

14. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that the antibody or antigen-binding fragment thereof is capable of binding to the amino acid sequence shown in SEQ ID NO: 73 and / or 74.

15. A nucleic acid molecule encoding the antibody or antigen-binding fragment of any one of claims 1 to 14.

16. The nucleic acid molecule of claim 15, wherein the nucleic acid molecule is DNA.

17. An expression vector comprising the nucleic acid molecule according to any one of claims 15 to 16.

18. A recombinant cell characterized in that it carries a nucleic acid molecule according to any one of claims 5 to 16, an expression vector according to claim 17, or is capable of expressing an antibody or antigen-binding fragment according to any one of claims 1 to 14.

19. The recombinant cell according to claim 18, characterized in that the recombinant cell is obtained by introducing the expression vector according to claim 17 into a host cell.

20. 20. The recombinant cell of claim 19, wherein the expression vector is introduced into the host cell by electrotransfer.

21. The recombinant cell of claim 19, wherein the recombinant cell is a eukaryotic cell.

22. The recombinant cell of claim 19, wherein the recombinant cell is a mammalian cell.

23. An immunoconjugate comprising the antibody or antigen-binding fragment of any one of claims 1 to 14 and a therapeutic agent.

24. A composition comprising an antibody according to any one of claims 1 to 14, a nucleic acid molecule according to any one of claims 15 to 16, an expression vector according to claim 17, or a recombinant cell according to any one of claims 18 to 22.

25. Use of an antibody or antigen-binding fragment thereof described in any one of claims 1 to 14, a nucleic acid molecule described in any one of claims 15 to 16, an expression vector described in claim 17, a recombinant cell described in any one of claims 18 to 22, or a composition described in claim 24 in the preparation of a drug for preventing and / or treating a CD155-related disease.

26. The use according to claim 25, characterized in that the CD155-associated disease is cancer, an autoimmune disease, transplant rejection or an infectious disease.

27. The use of claim 26, characterized in that the cancer includes at least one of malignant melanoma, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

28. A drug comprising an antibody according to any one of claims 1 to 14, a nucleic acid molecule according to any one of claims 15 to 16, an expression vector according to claim 17, a recombinant cell according to any one of claims 18 to 22, or a composition according to claim 24.

29. Use of the antibody or antigen-binding fragment of any one of claims 1 to 14 in the preparation of a kit for detecting CD155.

30. A kit for detecting CD155, characterized in that the kit comprises an antibody or antigen-binding fragment thereof according to any one of claims 1 to 14.

31. A method for preventing and / or treating a CD155-associated disease, comprising the step of administering to a subject at least one of the antibody or antigen-binding fragment of any one of claims 1 to 14, the nucleic acid molecule of any one of claims 15 to 16, the expression vector of claim 17, the recombinant cell of any one of claims 18 to 22, the immunoconjugate of claim 23, or the composition of claim 24.

32. The method of claim 31, wherein the CD155-associated disease is cancer, an autoimmune disease, transplant rejection or an infectious disease.

33. 33. The method of claim 32, wherein the cancer comprises at least one of malignant melanoma, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

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