CD155 binding molecule and application thereof

Abstract

The invention belongs to the field of immunology, and relates to a CD155 binding molecule and application thereof. The CD155 binding molecule is composed of heavy chains, and the heavy chains comprise a heavy chain CDR1 as shown in any one of SEQ ID NO: 23 to SEQ ID NO: 26, a heavy chain CDR2 as shown in SEQ ID NO: 27 and a heavy chain CDR3 as shown in any one of SEQ ID NO: 28 to SEQ ID NO: 31. Compared with the prior art, the invention has the beneficial effects that the specific single-domain antibody aiming at CD155 is screened by using a biological genetic engineering technology, and the affinity of the antibody is better.

Description

TECHNICAL FIELD

[0001] The present application relates to a molecule capable of binding to CD155, and a pharmaceutical composition containing the CD155 binding molecule as an active ingredient, and a pharmaceutical therapeutic use thereof. BACKGROUND

[0002] Cluster of differentiation 155 (CD155) is a single transmembrane cell surface protein, which is rarely expressed or not expressed in normal tissues, but is highly expressed in various malignant tumors including skin adenocarcinoma, cholangiocarcinoma, colorectal cancer, non-small cell lung cancer, bladder cancer, breast cancer, making it an ideal target for anti-tumor drugs. CD155, as a cell adhesion molecule, is involved in tumor cell adhesion, migration and polarization, and also acts as an immune regulatory molecule to interact with co-stimulatory or co-inhibitory receptors including TIGIT, DNA-1, CD96 on T cells or NK cells, to exert immune regulation and affect the immune microenvironment.

[0003] The earliest drug development for targeting CD155 to treat tumors is to directly target tumor cells overexpressing CD155 by recombinant oncolytic poliovirus effectively infecting tumor cells, leading to lysis and cell death of tumor cells, and further exerting immune regulation. With the gradual deepening of the function of CD155, the role of CD155 as an immune checkpoint inhibitor is targeted to block, and the inhibitory interaction of immune cells in the tumor microenvironment is blocked by antibodies to reverse immune suppression, increase tumor infiltrating lymphocyte (TIL) activation and cytotoxicity, and ultimately lead to tumor cell death. Or use monoclonal antibodies to block tumor cells overexpressing CD155 to exert an anti-tumor effect by blocking the development and metastasis of tumor cells. In addition, the immune checkpoint inhibition pathway can be blocked by targeting CD155 receptors.

[0004] Nanobodies are the new darling of the antibody field, and can obtain bivalent, trivalent or bispecific antibodies through simple molecular cloning technology due to their small molecular weight. Nanobodies can achieve high yield in both prokaryotic expression systems (E. coli) and eukaryotic expression systems (CHO cells, 293 cells, etc.) due to their small molecular size. The rapid development of nanobodies has become a potential force in the development of antibody drugs, representing an important development direction for antibody drugs in the future. SUMMARY

[0005] The purpose of the present application is to provide a CD155 binding molecule and its use. In this specification, the CD155 binding molecule is a single-domain antibody against CD155.

[0006] The first aspect of the present application provides a CD155 binding molecule, which is composed of a heavy chain, the heavy chain comprising a heavy chain CDR1 as shown in any one of SEQ ID NO: 23-SEQ ID NO: 26, a heavy chain CDR2 as shown in SEQ ID NO: 27, and a heavy chain CDR3 as shown in any one of SEQ ID NO: 28-SEQ ID NO: 31.

[0007] Preferably, the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 are one of the following (1)-(5):

[0008] (1) CDR1 as shown in SEQ ID NO: 23, CDR2 as shown in SEQ ID NO: 27, and CDR3 as shown in SEQ ID NO: 31;

[0009] (2) CDR1 as shown in SEQ ID NO: 23, CDR2 as shown in SEQ ID NO: 27, and CDR3 as shown in SEQ ID NO: 29;

[0010] (3) CDR1 as shown in SEQ ID NO: 24, CDR2 as shown in SEQ ID NO: 27, and CDR3 as shown in SEQ ID NO: 31;

[0011] (4) CDR1 as shown in SEQ ID NO: 25, CDR2 as shown in SEQ ID NO: 27, and CDR3 as shown in SEQ ID NO: 30;

[0012] (5) CDR1 as shown in SEQ ID NO: 26, CDR2 as shown in SEQ ID NO: 27, and CDR3 as shown in SEQ ID NO: 28.

[0013] All of the above sequences can be replaced by a sequence having "at least 80% homology" with the sequence or a sequence with only one or a few amino acid substitutions; preferably "at least 85% homology", more preferably "at least 90% homology", more preferably "at least 95% homology", and most preferably "at least 98% homology".

[0014] In one embodiment, one to five arbitrary amino acid residues in any one or more of the heavy chain CDR1, CDR2, and CDR3 can be replaced by a conservative amino acid substitution, respectively. Specifically, 1 to 5 amino acid residues in the heavy chain CDR1 can be replaced by a conservative amino acid substitution; 1 to 5 amino acid residues in the heavy chain CDR2 can be replaced by a conservative amino acid substitution; and 1 to 5 amino acid residues in the heavy chain CDR3 can be replaced by a conservative amino acid substitution.

[0015] As used herein, the term "sequence homology" denotes the extent to which two (nucleotide or amino acid) sequences have the same residues at the same positions in an alignment, and is usually expressed as a percentage. Preferably, homology is determined over the entire length of the sequences being compared. Thus, two copies having exactly the same sequence have 100% homology.

[0016] In some embodiments, sequences that differ from the foregoing sequences by only one or a small number of amino acid substitutions, e.g., comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, can also achieve the objects of the invention. Such variants include, but are not limited to, deletions from, and / or insertions into, and / or substitutions of, one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and the addition of one or more (usually 20 or fewer, preferably 10 or fewer, more preferably 5 or fewer) amino acids to the C-terminus and / or the N-terminus of the single-domain antibody. Indeed, when determining the degree of sequence identity between two amino acid sequences or when determining the CDR1, CDR2, and CDR3 combination in a single-domain antibody, the skilled artisan can take into account so-called "conservative" amino acid substitutions, which, in the case of a substitution, will preferably be conservative amino acid substitutions. Such conservative amino acids, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue having similar chemical structure, and which have little or no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, e.g., conservative amino acid substitutions are one or a small number of amino acids within the following groups (a)-(d) are replaced with another or a small number of amino acids within the same group: (a) polar, negatively charged residues and their uncharged amides: Asp, Asn, Glu, Gin; (b) polar, positively charged residues: His, Arg, Lys; (c) aromatic residues: Phe, Trp, Tyr; (d) aliphatic, nonpolar or weakly polar residues: Ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val, Cys. Particularly preferred conservative amino acid substitutions are as follows: Asp for Glu; Asn for Gin or His; Glu for Asp; Gin for Asn; His for Asn or Gin; Arg for Lys; Lys for Arg, Gin; Phe for Met, Leu, Tyr; Trp for Tyr; Tyr for Phe, Trp; Ala for Gly or Ser; Ser for Thr; Thr for Ser; Gly for Ala or Pro; Met for Leu, Tyr or Ile; Leu for Ile or Val; Ile for Leu or Val; Val for Ile or Leu; Cys for Ser. In addition, the skilled artisan will appreciate that the inventive single-domain antibody is inventive in the CDR1-3 regions, and that the framework region sequences FR1-4 are not immutable, and that the sequences of FR1-4 can take conservative sequence variants of the sequences disclosed herein.

[0017] The meaning of "a single-domain antibody against CD155" in the present application includes not only the intact single-domain antibody, but also fragments, derivatives and analogs of the single-domain antibody against CD155. As used herein, the terms "fragment", "derivative" and "analog" have the same meaning, and refer to a polypeptide that substantially maintains the same biological function or activity of the antibody of the present application. The polypeptide fragment, derivative or analog of the present application can be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues can or can not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of the mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, for example, polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or a secretion sequence or a sequence for purification of the polypeptide or a proteinogen sequence, or a fusion protein with an Fc tag). These fragments, derivatives and analogs are within the scope known to those skilled in the art according to the teachings herein.

[0018] In a preferred embodiment, the sequence of the CD155 binding molecule further comprises a framework region FR; the framework region FR comprises the amino acid sequences of FR1, FR2, FR3 and FR4; the amino acid sequences of the framework region FR are respectively:

[0019] FR1 or a variant of FR1 as shown in any one of SEQ ID NOs: 11-12, the variant of FR1 comprising up to 5 substitutions of amino acids in the FR1;

[0020] FR2 or a variant of FR2 as shown in any one of SEQ ID NOs: 13-15, the variant of FR2 comprising up to 5 substitutions of amino acids in the FR2;

[0021] FR3 or a variant of FR3 as shown in any one of SEQ ID NOs: 16-20, the variant of FR3 comprising up to 5 substitutions of amino acids in the FR3;

[0022] FR4 or a variant of FR4 as shown in any one of SEQ ID NOs: 21-22, the variant of FR4 comprising up to 5 substitutions of amino acids in the FR4.

[0023] The second aspect of the present application provides an amino acid sequence of the CD155 binding molecule, which is shown in SEQ ID NO. 1-5 respectively, or the single domain antibody has at least 80% sequence homology with the amino acid sequence of SEQ ID NO. 1-5, and can specifically bind to the CD155 protein.

[0024] In a preferred embodiment, the amino acid sequence of the CD155 binding molecule has at least 1 amino acid residue in FR1, FR2, FR3 or FR4 sequence substituted by a conservative amino acid compared with any one of SEQ ID NO: 1-5.

[0025] In an embodiment, the CD155 binding molecule has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology with the amino acid sequence selected from SEQ ID NO: 1-5, and can specifically bind to the CD155 protein.

[0026] The third aspect of the present application provides the Fc fusion antibody or humanized antibody of the CD155 binding molecule of any of the foregoing.

[0027] The fourth aspect of the present application provides a nucleotide molecule encoding the CD155 binding molecule of the foregoing or the Fc fusion antibody of the foregoing or the humanized antibody of the foregoing, the nucleotide sequence of which is shown in SEQ ID NO: 6-10 respectively, or the amino acid sequence encoded by the nucleotide sequence is the same as the amino acid sequence encoded by any one of SEQ ID NO: 6-10, or has at least 95% sequence homology with any one of SEQ ID NO: 6-10.

[0028] In an embodiment, the nucleic acid molecule encoding the CD155 binding molecule has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology with the nucleotide sequence selected from SEQ ID NO: 6-10, and the CD155 binding molecule encoded thereby can specifically bind to the CD155 protein.

[0029] The fifth aspect of the present application provides an expression vector comprising a nucleotide molecule encoding the CD155 binding molecule or the Fc fusion antibody or the humanized antibody, the nucleotide sequence of which is shown in SEQ ID NO: 6-10 respectively.

[0030] In a preferred embodiment, the expression vector used is RJK-V4-hFC (a nucleotide molecule encoding a CD155 binding molecule or an Fc fusion antibody or a humanized antibody thereof is integrated into RJK-V4-hFC by genetic engineering). Other general expression vectors can also be selected as needed.

[0031] A sixth aspect of the present invention is to provide a host cell capable of expressing the aforementioned CD155 binding molecule, Fc fusion antibody or humanized antibody, or an expression vector comprising the aforementioned host cell. Preferably, the host cell is a bacterial cell, a fungal cell or a mammalian cell.

[0032] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell, including bacteria and fungi.

[0033] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or a combination thereof.

[0034] In another preferred embodiment, the prokaryotic cell is selected from the group consisting of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, or a combination thereof.

[0035] In another preferred embodiment, the eukaryotic cell is selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, or a combination thereof.

[0036] In another preferred embodiment, the eukaryotic cells are selected from the group consisting of insect cells such as fall armyworm, plant cells such as tobacco, BHK cells, CHO cells, COS cells, myeloma cells, or a combination thereof.

[0037] In another preferred embodiment, the host cell is a suspension ExpiCHO-S cell.

[0038] In another preferred embodiment, the host cell is a suspension 293F cell.

[0039] A seventh aspect of the present application provides a recombinant protein comprising the aforementioned CD155 binding molecule. The recombinant protein can be the single domain antibody shown in SEQ ID NO. 1-5, or a single domain antibody having at least 80% homology with SEQ ID NO. 1-5, or a multi-epitope antibody, a bispecific antibody, a multi-specific antibody, and a multivalent antibody; for example, the multi-epitope antibody can be composed of more than one sequence in SEQ ID NO. 1-5; the multivalent antibody can be composed of a sequence in SEQ ID NO. 1-5 repeated several times; the multi-specific antibody includes, but is not limited to, a tri-specific antibody, a tetra-specific antibody; in addition, the recombinant protein can be a fragment, derivative, and analog of the aforementioned antibody.

[0040] An eighth aspect of the present application provides a pharmaceutical composition comprising the aforementioned CD155 binding molecule and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally determined according to the isoelectric point of the antibody (the pH of the aqueous carrier medium needs to deviate from the isoelectric point of the antibody, and differ from the isoelectric point of the antibody by about 2). The prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): intravenous, transdermal (directly applying or pasting ointment on the affected area).

[0041] The pharmaceutical composition of the present application contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the aforementioned single domain antibody and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be matched with the administration method. The pharmaceutical composition of the present application can be prepared in the form of a needle, for example, using normal saline or an aqueous solution containing glucose and other adjuvants by conventional methods. The pharmaceutical composition such as needle and solution should be manufactured under sterile conditions.

[0042] A ninth aspect of the present application provides a medicament for treating diseases, comprising the aforementioned CD155 binding molecule as an active ingredient.

[0043] A tenth aspect of the present application provides a kit for detecting the level of CD155, comprising the aforementioned CD155 binding molecule. In a preferred embodiment of the present application, the kit further comprises a container, instructions for use, a buffer, etc.

[0044] In a preferred embodiment, the kit comprises an antibody recognizing the CD155 protein, a lysis medium for lysing the sample, and general reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc. The detection kit can be an in vitro diagnostic device.

[0045] In a preferred embodiment, the kit further comprises a second antibody and an enzyme or fluorescent or radioactive label for detection, and buffers.

[0046] In a preferred embodiment, the second antibody of the kit can be an antibody (as an anti-antibody) of the aforementioned CD155 binding molecule, and can be a single domain antibody, a monoclonal antibody, a polyclonal antibody, or any other form of antibody.

[0047] In an eleventh aspect of the present application, a method for producing a CD155 binding molecule (a single domain antibody against CD155) is provided, comprising the steps of:

[0048] (a) culturing the host cell of the sixth aspect of the present application under conditions suitable for production of a single domain antibody, thereby

[0049] obtaining a culture comprising the single domain antibody against CD155; and

[0050] (b) isolating or recovering the single domain antibody against CD155 from the culture; and

[0051] (c) optionally, purifying and / or modifying the single domain antibody against CD155 obtained in step (b).

[0052] In a twelfth aspect of the present application, use of the aforementioned CD155 binding molecule or the aforementioned pharmaceutical composition in the manufacture of a medicament for treating a disease is provided.

[0053] In a preferred embodiment, the disease comprises a solid tumor, bladder cancer, brain tumor, head and neck cancer, breast cancer, rectal cancer, prostate cancer, glioblastoma, melanoma, head and neck squamous cell carcinoma, diabetic nephropathy, diabetic retinopathy, and non-alcoholic steatohepatitis.

[0054] Advantages

[0055] Advantages of the present application over the prior art are:

[0056] (1) The single domain antibody of the present application is specific to the CD155 protein having a correct spatial structure.

[0057] (2) The single domain antibody obtained by the application has flexible expression system selection, can be expressed in a prokaryotic system or a eukaryotic system of yeast cells or mammalian cells, and has low expression cost in the prokaryotic expression system, thereby reducing the production cost in the later stage.

[0058] (3) The single domain antibody obtained by the application has simple modification of multi-combination form of the antibody, and the multi-valent and multi-specific antibody can be obtained by simple concatenation through genetic engineering, and the immunological heterogeneity is low, and a strong immune response is not generated without humanization modification.

[0059] (4) The single domain antibody obtained by the application has a wider affinity range, and the affinity range can be from nM level to pM level before affinity maturation, thereby providing multiple choices for the antibody for different uses in the later stage. BRIEF DESCRIPTION OF DRAWINGS

[0060] In order to more clearly illustrate the technical solutions of the present application, the drawings needed in the embodiments will be briefly introduced below. Obviously, other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0061] Figure 1 Assay for antibody blocking CD155 binding to CD226 (1B3);

[0062] Figure 2 Assay for antibody blocking CD155 binding to CD226 (3B4);

[0063] Figure 3 Assay for antibody blocking CD155 binding to CD226 (2C10);

[0064] Figure 4 Assay for antibody blocking CD155 binding to CD226 (4F3);

[0065] Figure 5 Assay for antibody blocking CD155 binding to CD226 (4C8);

[0066] Figure 6 Assay for antibody blocking CD155 binding to CD226 (Tab1, hIgG);

[0067] Figure 7 Assay for antibody blocking CD155 binding to TIGIT (1B3);

[0068] Figure 8 Assay for antibody blocking CD155 binding to TIGIT (2C10);

[0069] Figure 9 Assay to determine if an antibody blocks CD155 binding to TIGIT (3B4);

[0070] Figure 10 Assay to determine if an antibody blocks CD155 binding to TIGIT (4F3);

[0071] Figure 11 Assay to determine if an antibody blocks CD155 binding to TIGIT (4C8);

[0072] Figure 12 Assay to determine if an antibody blocks CD155 binding to TIGIT (Tab2, hIgG). DETAILED DESCRIPTION

[0073] The application will now be further described by way of example with reference to the accompanying drawings wherein:

[0074] As used herein, a "single domain antibody" (sdAb, also referred to by the developers Ablynx as a Nanobody or VHH) is well known to those skilled in the art. A single domain antibody is an antibody whose complementarity determining regions are part of a single polypeptide. Thus, a single domain antibody comprises a single complementarity determining region (a single CDR1, a single CDR2 and a single CDR3). Examples of single domain antibodies are heavy chain only antibodies (which naturally do not comprise a light chain), single domain antibodies derived from conventional antibodies and engineered antibodies.

[0075] Single domain antibodies can be derived from any species, including mouse, human, camel, llama, goat, rabbit and bovine. For example, naturally occurring VHH molecules can be derived from antibodies provided by camelids (e.g. camels, dromedaries, llamas and guanacos). Like intact antibodies, single domain antibodies are able to selectively bind to a specific antigen. Single domain antibodies can contain only the variable domain of an immunoglobulin chain, which domain has a CDR1, a CDR2 and a CDR3 as well as framework regions.

[0076] As used herein, the term "sequence homology" denotes the extent to which two (nucleotide or amino acid) sequences have the same residues at the same positions in an alignment, and is usually expressed as a percentage. Preferably, the homology is determined over the entire length of the sequences being compared. Thus, two copies of a sequence that have exactly the same sequence have 100% homology.

[0077] As used herein, the term "Fc-fusion antibody" refers to a novel protein produced by fusing the Fc fragment of a target antibody with a certain functional protein molecule having a biological activity using genetic engineering technology.

[0078] The term "humanized antibody" refers to an antibody obtained by fusing the heavy chain variable region of a target antibody (such as an animal antibody) with the constant region of a human antibody, or an antibody obtained by grafting the complementarity determining region (CDR1-3 sequence) of a target antibody into the variable region of a human antibody, or an antibody obtained by mutating the amino acids of a target antibody according to the characteristics of the human antibody framework region (FR1-4). The humanized antibody can be obtained by synthetic or site-directed mutagenesis.

[0079] In the present application, sequences with high sequence homology to the CDR1-3 sequences disclosed in the present application can also obtain single-domain antibodies against CD155. In some embodiments, sequences with "at least 80% homology" to the sequences in SEQ ID NO. 1-5, or sequences with "at least 85% homology", "at least 90% homology", "at least 95% homology", "at least 98% homology" can achieve the purpose of the application.

[0080] In some embodiments, sequences that only replace one or a small number of amino acids compared to the sequences of SEQ ID NOs: 1-5, e.g., comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, can also achieve the objects of the invention. Indeed, in determining the degree of sequence homology between two amino acid sequences or in determining the CDR1, CDR2, and CDR3 combinations in a single domain antibody, the skilled artisan can take into account so-called "conservative" amino acid substitutions, which, in the case of a substitution, will preferably be a conservative amino acid substitution, which can generally be described as an amino acid substitution in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, and which substitution has little or substantially no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, e.g., conservative amino acid substitutions are one or a small number of amino acids within the following groups (a)-(d) are replaced by another amino acid within the same group: (a) polar, negatively charged residues and their uncharged amides: Asp, Asn, Glu, Gin; (b) polar, positively charged residues: His, Arg, Lys; (c) aromatic residues: Phe, Trp, Tyr; (d) aliphatic, nonpolar or weakly polar residues: Ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val, Cys. Particularly preferred conservative amino acid substitutions are as follows: Asp for Glu; Asn for Gin or His; Glu for Asp; Gin for Asn; His for Asn or Gin; Arg for Lys; Lys for Arg, Gin; Phe for Met, Leu, Tyr; Trp for Tyr; Tyr for Phe, Trp; Ala for Gly or Ser; Ser for Thr; Thr for Ser; Gly for Ala or Pro; Met for Leu, Tyr or Ile; Leu for Ile or Val; Ile for Leu or Val; Val for Ile or Leu; Cys for Ser. In addition, the skilled artisan is aware that the inventive single domain antibody is creative in the CDR1-3 regions, and that the framework region sequences FR1-4 are not immutable, and that the sequences of FR1-4 can take conservative sequence variants of the sequences disclosed herein.

[0081] Preferred host cells of the present invention are bacterial cells, fungal cells, or mammalian cells.

[0082] The patent is prepared by gene engineering technology to prepare target protein and truncated form of target protein, and then the obtained antigen protein is immunized with Inner Mongolia Alashan Bactrian camel, and after multiple immunization, the peripheral blood lymphocytes or spleen cells of the camel are obtained, the camel antibody variable region coding sequence is recombined into a phage display vector by gene engineering method, the specific antibodies against the antigen protein are screened by phage display technology, and the binding ability of the antibodies with the antigen is further detected.

[0083] The above technical solutions will be split and explained in detail, which will be described in the form of specific examples:

[0084] Example 1: Preparation of human CD155 recombinant extracellular domain protein:

[0085] The human recombinant extracellular domain protein used in the patent is obtained by the company itself, and the expression vector design scheme of the human recombinant CD155 protein is as follows:

[0086] (1) The coding sequence of CD155 is obtained by searching in NCBI, and the accession number is NM_006505.4. The amino acid sequence generated by the sequence has the accession number NP_006496.4.

[0087] (2) The nucleotide sequence encoding the 1st-343rd amino acid of the extracellular end of CD155 is cloned into the vector pcDNA3.4 by gene synthesis. After Sanger sequencing of the constructed vector, the original sequence is compared and confirmed to be correct, and the recombinant plasmid is extracted in large quantities to remove endotoxin, and transfected into suspended 293F cells for expression and purification of the target protein. The purity is more than 90%, which meets the requirements of animal immunization.

[0088] Example 2: Construction of single-domain antibody library against CD155 protein:

[0089] 1 mg of human recombinant CD155 protein purified in Example 1 is mixed with an equal volume of Freund's complete adjuvant to immunize one Inner Mongolia Alashan Bactrian camel, and the immunization is performed once a week for a total of 7 times. Except for the first immunization, the remaining six times are mixed with 1 mg of CD155 protein and an equal volume of Freund's incomplete adjuvant for animal immunization. This immunization process is to stimulate the camel to produce antibodies against CD155 protein.

[0090] After the animal immunization is completed, 150 mL of peripheral blood lymphocytes of the camel are extracted, and the RNA of the cells is extracted. The total RNA is used to synthesize cDNA, and the VHH (antibody heavy chain variable region) is amplified by nested PCR reaction with cDNA as template.

[0091] Then the pMECS vector and VHH fragments were digested by restriction enzymes respectively, and then the digested fragments and the vector were linked. The linked fragments were electroporated into competent cells TG1 to construct a phage display library of CD155 protein and determine the library capacity, and the library capacity of the library was about 1 x 10 9 At the same time, the correct insertion rate of the library in the target fragment was detected by colony PCR identification.

[0092] The results showed that after PCR amplification of 30 colonies randomly selected from the library, 29 clones could amplify a band with a size of the predicted size, and one clone amplified an incorrect band, so the correct insertion rate was 29 ÷ 30 x 100% ≈ 97%.

[0093] Example 3: Screening of single-domain antibody against CD155 protein

[0094] 200 μL of recombinant TG1 cells in Example 2 were taken into 2 x TY medium for culture, 40 μL of helper phage VCSM13 was added to infect the TG1 cells during the culture, and the phage was amplified overnight. The next day, the phage was precipitated by PEG / NaCl, and the amplified phage was collected by centrifugation.

[0095] 500 μg of CD155 protein diluted in 100 mM pH 8.3 NaHCO3 was coupled to an enzyme-labeled plate, and was placed at 4°C overnight, while a negative control well (medium control) was set up; the next day, 200 μL of 3% skim milk was added, and was blocked at room temperature for 2 h; after the blocking was completed, 100 μl of the amplified phage library (about 2 x 10 11 phage particles) was added, and was reacted at room temperature for 1 h; after 1 h of reaction, PBS + 0.05% Tween-20 was used to wash 15 times to wash away the unbound phage.

[0096] The phage specifically bound to CD155 protein was dissociated with trypsin at a final concentration of 25 mg / mL, and was used to infect E. coli TG1 cells in the logarithmic growth phase, which were cultured at 37°C for 1 h to produce and collect phage for the next round of screening. The same screening process was repeated for 1 round, and was gradually enriched.

[0097] Example 4: Screening of specific positive clones against CD155 by phage enzyme-linked immunosorbent assay (ELISA)

[0098] According to the screening method in the above embodiment 3, the anti-CD155 protein single domain antibody was screened for 2 rounds, the enrichment factor of the anti-CD155 protein phage reached more than 10, and after the screening was completed, 384 single colonies were selected from the positive clones obtained in the screening and inoculated in a 96 deep well plate containing 2xTY medium containing 100 μg / mL ampicillin, and a blank control was set. After being cultured at 37°C to the logarithmic phase, 1 mM IPTG was added, and the culture was incubated at 28°C overnight.

[0099] The crude antibody was obtained by osmotic rupture method; the CD155 recombinant protein was released into 100 mM pH 8.3 NaHCO3, and 100 μg of protein was coated in an enzyme-labeled plate (ELISA plate) at 4°C overnight. 100 μL of the obtained antibody crude extract was transferred to the ELISA plate with antigen, and incubated at room temperature for 1 h; the unbound antibody was washed with PBST, 100 μl of Mouse Anti-HA tag Antibody (HRP) diluted 1:2000 was added, and incubated at room temperature for 1 h; the unbound antibody was washed with PBST, and horseradish peroxidase color developing solution was added. After reaction at 37°C for 15 min, the stop solution was added, and the absorbance value was read on an enzyme-labeled instrument at 450 nm wavelength.

[0100] When the OD value of the sample well was more than 5 times that of the control well, it was determined to be a positive clone well; the bacteria in the positive clone well were transferred to LB medium containing 100 μg / mL ampicillin for plasmid extraction and sequencing.

[0101] According to the sequence alignment software VectorNTI, the gene sequences of each clone were analyzed, the strains with the same CDR1, CDR2 and CDR3 sequences were regarded as the same clone, and the strains with different sequences were regarded as different clones, and finally the single domain antibody specific to CD155 protein was obtained.

[0102] The amino acid sequence of the antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 structure, which constitutes the whole VHH. The obtained single domain antibody recombinant plasmid can be expressed in a prokaryotic system or a eukaryotic system, and finally the single domain antibody protein is obtained.

[0103] The CDR and FR sequences of the 5 single domain antibodies are shown in Tables 1-7, and the amino acid sequences and nucleotide sequences of the 5 single domain antibodies are shown in Tables 8 and 9, respectively.

[0104] Table 1 CDR1 sequence of 5 antibodies

[0105]

[0106]

[0107] Table 2 CDR2 sequences of 5 antibodies

[0108]

[0109] Table 3 CDR3 sequences of 5 antibodies

[0110]

[0111] Table 4 FR1 sequences of 5 antibodies

[0112]

[0113] Table 5 FR2 sequences of 5 antibodies

[0114]

[0115] Table 6 FR3 sequences of 5 antibodies

[0116]

[0117]

[0118] Table 7 FR4 sequences of 5 antibodies

[0119]

[0120] Table 8 Amino acid sequences of 5 antibodies

[0121]

[0122] Table 9 Nucleic acid sequences of 5 antibodies

[0123]

[0124]

[0125] Example 5: Purification and expression of specific single-domain antibody of CD155 protein in host bacteria Escherichia coli

[0126] The plasmids (pMECS-VHH) of different clones obtained by sequencing analysis in Example 4 were electrotransformed into Escherichia coli HB2151, and were coated on LB+amp+glucose culture plates containing ampicillin and glucose, and incubated at 37°C overnight; single colonies were selected and inoculated in 5 mL LB culture solution containing ampicillin, and incubated at 37°C overnight.

[0127] Inoculate 1 mL of overnight culture into 330 mL of TB medium and culture at 37°C with a shaker. When the OD600nm value reaches 0.6-0.9, add 1 M IPTG and culture overnight at 28°C with a shaker. Centrifuge to collect the E. coli and obtain a crude antibody extract using the osmotic swelling method.

[0128] The antibody was purified by nickel affinity chromatography.

[0129] Example 6: Construction of a eukaryotic expression vector for an anti-CD155 single-domain antibody Fc fusion antibody

[0130] (1) Subcloning the target sequence obtained in Example 4 into a eukaryotic expression vector: The antibodies screened in Example 4 were subjected to Sanger sequencing to obtain their nucleotide sequences;

[0131] (2) The above nucleotide sequence was synthesized into the vector RJK-V4-hFC designed and modified by our company to obtain a recombinant eukaryotic expression vector. The modification method of the vector is as described in Example 10;

[0132] (3) transforming the recombinant eukaryotic expression vector constructed in step (2) into DH5α Escherichia coli, culturing and performing plasmid extraction to remove endotoxins;

[0133] (4) Sequencing the extracted plasmid;

[0134] (5) The confirmed recombinant vector is prepared for subsequent eukaryotic cell transfection and expression, and the Fc protein of VHH is expressed by the method of Example 7 or 8, and the above-mentioned antibody is purified by the method of Example 9.

[0135] Example 7: Expression of single domain antibodies against CD155 protein in suspension ExpiCHO-S cells

[0136] (1) 3 days before transfection, 2.5×10 5 / mL cell passaging and expansion culture of ExpiCHO-S TM The calculated volume of cells was transferred to a fresh, pre-warmed 120 mL (final volume) of ExpiCHO TM The cells were cultured in a 500 mL shake flask containing expression medium; the cell concentration reached approximately 4 × 10 6 -6×10 6 Viable cells / mL;

[0137] (2) One day before transfection, TM The cells were diluted to a concentration of 3.5 × 10 6 viable cells / mL, and cells were cultured overnight;

[0138] (3) On the day of transfection, measure the cell density and percentage of viable cells. The cell density should reach about 7×10 6 -10×10 6 Viable cells / mL;

[0139] (4) Use fresh ExpiCHO preheated to 37℃ TM Dilute the cells to 6 × 10 6 The calculated required volume of cells was transferred to a fresh, pre-warmed 100 mL (final volume) of ExpiCHO TM in a 500 mL shake flask containing expression medium;

[0140] (5) Gently invert to mix ExpiFectamine TM CHO reagent, use 3.7 mL OptiPRO TM Dilute ExpiFectamine in culture medium TM CHO reagent, swirl or mix;

[0141] (6) Use 4 mL of refrigerated OptiPRO TM Dilute the plasmid DNA in the culture medium and vortex to mix;

[0142] (7) Incubate the ExpiFectamine CHO / plasmid DNA (the plasmid DNA is the eukaryotic expression vector for the Fc fusion antibody of the anti-CD155 single-domain antibody prepared in Example 6) complex at room temperature for 1-5 minutes, then gently add it to the prepared cell suspension, gently shaking the flask during the addition process;

[0143] (8) The cells were cultured with shaking at 37°C, 8% CO2, and humidified air;

[0144] (9) On the first day after transfection (18-22 hours later), add 600ul of ExpiFectamine TM CHO Enhancer and 24mLExpiCHO feed.

[0145] (10) Collect the supernatant approximately 8 days after transfection (cell viability is less than 70%).

[0146] Example 8: Expression of single domain antibodies against CD155 protein in suspension 293F cells

[0147] Recombinant single domain antibody expression experimental process (taking 500mL shake flask as an example):

[0148] (1) 3 days before transfection, 2.5×10 5Passage and expansion of cells 293F cells were passaged and expanded to the calculated required cell volume was transferred to a 500 mL shake flask containing fresh pre-warmed 120 mL (final volume) of OPM-293CD05 Medium. The cell concentration was brought to about 2 x 10 6 -3 x 10 6 viable cells / mL.

[0149] (2) On the day of transfection, the cell density and percentage of viable cells were determined. The cell density should be brought to about 2 x 10 6 -3 x 10 6 viable cells / mL.

[0150] (3) The cells were diluted to 1 x 10 6 viable cells / mL with pre-warmed OPM-293CD05 Medium. The calculated required cell volume was transferred to a 500 mL shake flask containing fresh pre-warmed 100 mL (final volume) of medium.

[0151] (4) The PEI (1 mg / mL) reagent was diluted with 4 mL Opti-MEM medium, mixed by inverting or flicking the tube; the plasmid DNA (the Fc fusion antibody eukaryotic expression vector of the single-domain antibody against CD155 prepared in Example 6) was diluted with 4 mL Opti-MEM medium, mixed by inverting, and filtered with a 0.22-μm filter. Incubation was performed at room temperature for 5 min.

[0152] (5) The diluted PEI reagent was added to the diluted DNA, mixed by inverting. The PEI / plasmid DNA complex was incubated at room temperature for 15-20 min, and then added to the prepared cell suspension gently, inverting the shake flask during the addition.

[0153] (6) The cells were cultured at 37°C, 5% CO2, 120 rpm shaking.

[0154] (7) 5 mL of OPM-CHO PFF05 feed was added at 24 h and 72 h after transfection.

[0155] (8) The supernatant was collected about 7 days after transfection (when the cell viability was less than 70%).

[0156] Example 9: Purification of the single-domain antibody of the anti-CD155 protein

[0157] (1) The protein expression supernatant obtained in Example 7 or 8 was filtered with a 0.45-μm disposable filter to remove insoluble impurities;

[0158] (2) The filtrate is purified by affinity chromatography using a protein purification system, which utilizes the ability of human Fc to bind to Protein A, and a Protein A-coupled agarose filler is used for purification;

[0159] (3) The filtrate is passed through a Protein A pre-packed column at a flow rate of 1 mL / min, and the target protein in the filtrate binds to the filler in this step;

[0160] (4) The bound impurity proteins on the column are washed with low-salt and high-salt buffers;

[0161] (5) The bound target protein on the column is eluted with a low-pH buffer;

[0162] (6) The eluate is quickly added to a Tris-HCl solution at pH 9.0 for neutralization;

[0163] (7) After dialysis of the neutralized protein solution, SDS-PAGE analysis is performed to determine that the protein purity is above 95% and the concentration is above 0.5 mg / mL, and the protein is stored at low temperature for future use.

[0164] Example 10: Construction of a single-domain antibody eukaryotic expression vector RJK-V4-hFC

[0165] The general target vector RJK-V4-hFC mentioned for nanobodies is a vector that is modified based on the human IgG1 heavy chain coding sequence Fc segment of our company's commercialized vector pCDNA3.4 (vector data link: https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / pcdna3_4_topo_ta_cloning_kit_man.pdf), i.e., the vector contains the hinge region (Hinge) CH2 and CH3 region of the IgG1 heavy chain. The specific modification scheme is as follows:

[0166] (1) Select the restriction enzyme cutting sites XbaI and AgeI on pcDNA3.4;

[0167] (2) Introduce a multiple cloning site (MCS, Multiple Cloning Site) and a 6×His tag at the 5' and 3' ends of the Fc fragment coding sequence by overlap PCR;

[0168] (3) Use a pair of primers with XbaI and AgeI enzyme cutting sites to amplify the above-mentioned fragment by PCR;

[0169] (4) Using restriction enzymes Xbal and Agel to respectively digest pcDNA3.4 and the recombinant DNA fragment in (3);

[0170] (5) The digested vector and insert were ligated under the action of T4 ligase, and then the ligation product was transformed into E. coli, amplified, sequenced and verified to obtain the recombinant plasmid.

[0171] Example 11: Expression and purification of tool antibodies (Tab) targeting human CD155

[0172] Herein, Tab1 is 4E5 in CN109071666A, and Tab2 is 7D4 in CN109071666A. The searched sequences were entrusted to General Biosystems (Anhui) Co., Ltd. for codon optimization in a mammalian cell expression system and cloned into a pcDNA3.1 vector. After resistance screening, plasmid-positive bacteria were selected for amplification, and the plasmid was extracted using a plasmid extraction kit (Macherey Nagel, Cat# 740412.50). According to the addition of 100 μg of plasmid (40 μg of heavy chain + 60 μg of light chain) per 100 mL of cells, PEI was used for transient expression in 293F cells (culture medium: FreeStyle 293 Expression medium, Thermo, Cat# 12338026 + F-68, Thermo, Cat# 24040032); 5% volume of 10% Peptone (Sigma, Cat# P0521-100G) was added after 6-24 h of transfection, and the cells were cultured at 8% CO2 130 rpm for about 7-8 days; the expression supernatant was collected when the cell viability decreased to 50%, and the Protein A (GE, Cat# 17-5438-02) gravity column was used for purification; after PBS dialysis, the concentration was determined using a Nanodrop, the purity was identified by SEC, and the binding capacity was verified by indirect ELISA;

[0173] The Tab obtained by the method has a concentration of not less than 2 mg / ml and a purity of greater than 95%.

[0174] Example 12: Determination of antigen binding dose-response curve of antibody

[0175] This example adopts a standard enzyme-linked immunosorbent assay (ELISA) operation process.

[0176] (1) Coat 50 μL of 1 μg / mL human CD155 protein at 4°C overnight.

[0177] (2) Wash the plate; add 200 μL of 5% milk and block at 37°C for 2 h.

[0178] (3) Dilute VHH-hFc to 2 μg / mL, then 5-fold gradient dilute the antibody to 8 concentration gradients. Here, VHH-hFc refers to the Fc fusion antibody of the single-domain antibody of the anti-CD155 protein prepared in Example 8 (expressed in 293F cells) purified in Example 9. In addition, hIgG, Tab control are also set up respectively; Tab1, Tab2 are prepared in Example 11;

[0179] (4) Wash the plate; add 50 μL of the single-domain antibody diluted in step (3), two duplicate wells, 37°C for 1 h.

[0180] (5) Wash the plate; add 50 μL of HRP-Goat anti hIgG secondary antibody, 37°C for 30 min.

[0181] (6) Wash the plate (several times); add 50 μL of pre-warmed TMB, avoid light, room temperature for 15 min.

[0182] (7) Add 50 μL of stop solution (1N HCl), read the plate on the microplate reader and save.

[0183] (8) Draw the curve and calculate the EC50, where hIgG refers to the isotype control, an immunoglobulin molecule that does not bind to any target, obtained by commercial purchase. The results obtained are shown in Table 10:

[0184] Table 10 EC50 values of antibody antigen binding

[0185]

[0186] Compared with hIgG and Tab control, single-domain antibodies 1B3, 2C10, 3B4, 4F3 and 4C8 all exhibit excellent binding performance to CD155 antigen, with better affinity and specificity.

[0187] Example 13: RBA effect detection of specific single-domain antibodies of CD155 protein - determination of antibody blocking CD155 binding to CD226

[0188] This example uses standard enzyme-linked immunosorbent assay (ELISA) operating procedures.

[0189] (1) Coat 50 μL, 1 μg / mL CD226 (Acro, Cat: DN1-H5257), 4°C overnight.

[0190] (2) Wash the plate; add 200 μL of 5% milk, 37°C for 2 h.

[0191] (3) Mix Biotin-CD155 (Acro, Cat: CD5-H82F6) with gradient-diluted VHH-hFc to obtain a mixture (the concentration of Biotin-CD155 in the mixture is 19.8 ng / ml, and the concentration of VHH-hFc in the mixture is 5-fold dilution from 10 μg / mL, with 7 concentrations). Here, VHH-hFc refers to the Fc fusion antibody of the single-domain antibody against CD155 protein prepared in Example 8 (expressed in 293F cells) purified in Example 9. In addition, hIgG and Tab controls are set; hIgG refers to a homologous control, an immunoglobulin molecule that does not bind to any target, which is obtained by commercial purchase;

[0192] (4) Wash the plate; add 50 μL of the above mixture to two duplicate wells, and incubate at 37°C for 1 h.

[0193] (5) Wash the plate; add 50 μL of Streptavidin-HRP, and incubate at 37°C for 30 min.

[0194] (6) Wash the plate (several times); add 50 μL of pre-warmed TMB, and react at room temperature for 15 min in the dark.

[0195] (7) Add 50 μL of stop solution (1N HCl), and read the result with a microplate reader.

[0196] (8) Draw a curve, and calculate the EC50. The results are shown in Figures 1-6 . Figures 1-6 The abscissa represents the specific concentration of the gradient-diluted VHH-hFc. It can be seen that the single-domain antibody effectively blocks the binding of CD155 to CD226.

[0197] Example 14: RBA effect detection of specific single-domain antibody of CD155 protein - determination of antibody blocking CD155 binding to TIGIT

[0198] This example uses a standard enzyme-linked immunosorbent assay (ELISA) operating procedure.

[0199] (1) Coat 50 μL of 1 μg / mL TIGIT protein (Acro, Cat: DN1-H5257) at 4°C overnight.

[0200] (2) Wash the plate; add 200 μL of 5% milk, and block at 37°C for 2 h.

[0201] (3) Mix Biotin-CD155 (Acro, Cat: CD5-H82F6) with gradient-diluted VHH-hFc to obtain a mixture (the concentration of Biotin-CD155 in the mixture is 101.985 ng / ml, and the concentration of VHH-hFc in the mixture is 5-fold dilution from 10 μg / mL, with 7 concentrations). Here, VHH-hFc refers to the Fc fusion antibody of the single-domain antibody of the anti-CD155 protein prepared in Example 8 (expressed in 293F cells) purified in Example 9. In addition, hIgG and Tab controls are also set; hIgG refers to a homologous control, an immunoglobulin molecule that does not bind to any target, which is obtained by commercial purchase;

[0202] (4) Wash the plate; add 50 μL of the above mixture to two duplicate wells, and incubate at 37°C for 1 h.

[0203] (5) Wash the plate; add 50 μL of Streptavidin-HRP, and incubate at 37°C for 30 min.

[0204] (6) Wash the plate (several times); add 50 μL of pre-warmed TMB, and react at room temperature for 15 min in the dark.

[0205] (7) Add 50 μL of stop solution (1N HCl), and read the reading of the microplate reader.

[0206] (8) Draw a curve, and calculate the EC50. The results are shown in Figures 7-12 Figures 7-12 The horizontal axis represents the specific concentration of the gradient-diluted VHH-hFc. It can be seen that the single-domain antibody effectively blocks the binding of CD155 to TIGIT.

[0207] The basic principles, main features and advantages of the present application are shown and described above. It should be understood by those skilled in the art that the above examples do not limit the present application in any form, and any technical solution obtained by equivalent substitution or equivalent transformation falls within the protection scope of the present application.​

Claims

1. A CD 155 binding molecule characterized in that: The CD155 binding molecule is composed of a heavy chain, and the heavy chain comprises a heavy chain CDR1 shown in any one of SEQ ID NOs: 23-26, a heavy chain CDR2 shown in SEQ ID NO: 27, and a heavy chain CDR3 shown in any one of SEQ ID NOs: 28-31. The amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 are one of the following (1)-(5): (1) the CDR1 shown in SEQ ID NO: 23, the CDR2 shown in SEQ ID NO: 27, and the CDR3 shown in SEQ ID NO: 31; 2. The CD155 binding molecule of claim 1, wherein: (2) the CDR1 shown in SEQ ID NO: 23, the CDR2 shown in SEQ ID NO: 27, and the CDR3 shown in SEQ ID NO: 29; (3) the CDR1 shown in SEQ ID NO: 24, the CDR2 shown in SEQ ID NO: 27, and the CDR3 shown in SEQ ID NO: 31; (4) the CDR1 shown in SEQ ID NO: 25, the CDR2 shown in SEQ ID NO: 27, and the CDR3 shown in SEQ ID NO: 30; (5) the CDR1 shown in SEQ ID NO: 26, the CDR2 shown in SEQ ID NO: 27, and the CDR3 shown in SEQ ID NO:

28. The single-domain antibody further comprises a framework region FR; the framework region FR comprises the amino acid sequences of FR1, FR2, FR3, and FR4; and the amino acid sequences of the framework region FR are respectively: the FR1 shown in any one of SEQ ID NOs: 11-12 or a variant of the FR1, wherein the variant of the FR1 comprises at most 5 substitutions of amino acids in the FR1; 3. The CD155 binding molecule of claim 1, wherein: the FR2 shown in any one of SEQ ID NOs: 13-15 or a variant of the FR2, wherein the variant of the FR2 comprises at most 5 substitutions of amino acids in the FR2; the FR3 shown in any one of SEQ ID NOs: 16-20 or a variant of the FR3, wherein the variant of the FR3 comprises at most 5 substitutions of amino acids in the FR3; the FR4 shown in any one of SEQ ID NOs: 21-22 or a variant of the FR4, wherein the variant of the FR4 comprises at most 5 substitutions of amino acids in the FR4. The amino acid sequences of the CD155 binding molecule are shown in any one of SEQ ID NOs: 1-5, respectively; or the amino acid sequence of the single-domain antibody is compared with any one of SEQ ID NOs: 1-5, at least one amino acid residue in the FR1, the FR2, the FR3, or the FR4 sequence is substituted by a conservative amino acid.

5. An Fc fusion antibody or a humanized antibody of the CD155 binding molecule according to any one of claims 1-4.

4. A CD 155 binding molecule characterized in that: The recombinant protein comprises the CD155 binding molecule according to any one of claims 1-4. The recombinant protein comprises the CD155 binding molecule according to any one of claims 1-4.

6. A recombinant protein, characterized in that, ​ 7. A nucleotide molecule encoding the CD 155 binding molecule of any one of claims 1 to 4. a nucleotide sequence as shown in any one of SEQ ID NOs: 6-10, or an amino acid sequence encoded by the nucleotide sequence is identical to an amino acid sequence encoded by any one of SEQ ID NOs: 6-10.

8. An expression vector, characterized by: a nucleotide molecule encoding the CD155 binding molecule according to any one of claims 1 to 4 or the Fc fusion antibody or humanized antibody according to claim 5, or the nucleotide molecule according to claim 7.

9. A host cell, characterized in that: an expression vector according to claim 8, which can express the CD155 binding molecule according to any one of claims 1 to 4 or the Fc fusion antibody or humanized antibody according to claim 5.

10. A pharmaceutical composition, characterized by: the pharmaceutical composition comprises the CD155 binding molecule according to any one of claims 1 to 4, and a pharmaceutically acceptable carrier.

11. A medicament for the treatment of a disease, characterized in that: the pharmaceutical composition comprises the CD155 binding molecule according to any one of claims 1 to 4 as an active ingredient.

12. Use of the CD155 binding molecule according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 10 in the manufacture of a medicament for treating a disease.

13. Use according to claim 12, characterized in that: the disease includes a solid tumor, bladder cancer, brain tumor, head and neck cancer, breast cancer, rectal cancer, prostate cancer, glioblastoma, melanoma, head and neck squamous cell carcinoma, diabetic nephropathy, diabetic retinopathy, and non-alcoholic steatohepatitis.

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