Antibody specifically binding to CD155 and uses thereof

Abstract

The present application provides: an anti-CD155 antibody or an antigen-binding fragment thereof; a conjugate of the antibody or antigen-binding fragment thereof and a drug; a composition for detecting CD155, a composition for diagnosing cancer, and a pharmaceutical composition for preventing, ameliorating, or treating cancer, each comprising the antibody or antigen-binding fragment thereof; and a pharmaceutical composition for preventing, ameliorating, or treating cancer comprising the conjugate, and thus achieves a better anticancer effect.

Description

Antibody that specifically binds to CD155 and its uses

[0001] The present application relates to an antibody that specifically binds to CD155 and its uses.

[0002] Cross-citation with related application(s)

[0003] This application claims the benefit of priority based on Korean Patent Application No. 10-2025-0003342 dated January 9, 2025, and all contents disclosed in the document of said Korean patent application are incorporated herein as part of this specification.

[0004] The poliovirus receptor (PVR), also known as CD155, is a transmembrane glycoprotein involved in mediating cell adhesion to extracellular matrix molecules. CD155 is known as a tumor antigen associated with neuroectodermal cancers, including glioblastoma, medulloblastoma, and colorectal cancer, as well as pancreatic cancer. CD155 is known to shorten the GO / GI phase of the cell cycle through sero-induced activation via Ras-Raf-MEK-ERK signaling, the upregulation of cyclins D2 and E, and the downregulation of p27Kip1, which inhibits cell cycle progression. For this reason, it is anticipated that blocking CD155 in tumor cells may induce apoptosis.

[0005] Furthermore, CD155 is known to be an important immune checkpoint ligand. As a cell surface marker expressed in various cancer cells, CD155 can regulate anti-cancer immune responses by binding to immune checkpoint receptors, such as TIGIT, expressed in immune cells.

[0006] Therefore, there is a need to develop anti-CD155 antibodies that can further promote the death of cancer cells.

[0007]

[0008] One example provides an anti-CD155 antibody or an antigen-binding fragment thereof.

[0009] Another example provides a polynucleotide encoding the above anti-CD155 antibody or its antigen-binding fragment.

[0010] Another example provides a vector comprising a polynucleotide (nucleic acid molecule) encoding the above-mentioned anti-CD155 antibody or its antigen-binding fragment. The vector may be a recombinant vector or an expression vector.

[0011] Another example provides a recombinant cell containing the above recombinant vector.

[0012] Another example provides a method for producing an anti-CD155 antibody or an antigen-binding fragment thereof, comprising the step of expressing the above polynucleotide in a host cell.

[0013] Another example provides a composition for detecting CD155 comprising the above-mentioned anti-CD155 antibody or an antigen-binding fragment thereof.

[0014] Another example provides a method for detecting CD155 in a biological sample, comprising the step of contacting the above anti-CD155 antibody or its antigen-binding fragment with a biological sample for which CD155 expression detection is required.

[0015] Another example provides a composition for diagnosing cancer comprising the above-mentioned anti-CD155 antibody or an antigen-binding fragment thereof. Herein, the cancer may be a cancer expressing CD155.

[0016] Another example provides a method for diagnosing cancer of a biological sample or a subject from which the biological sample is derived, or a method for providing information for cancer diagnosis, comprising the step of contacting the anti-CD155 antibody or an antigen-binding fragment thereof with a biological sample. The biological sample may be isolated from the subject.

[0017] Another example provides a conjugate comprising (1) the anti-CD155 antibody or its antigen-binding fragment and (2) one or more selected from the group consisting of useful polymers, labeling substances, drugs, proteins, and peptides. For example, the conjugate may be an antibody-drug conjugate comprising the anti-CD155 antibody or its antigen-binding fragment and a drug. In one example, the drug may be a cytotoxic drug, for example, an anticancer drug.

[0018] Another example provides a pharmaceutical composition comprising the anti-CD155 antibody or its antigen-binding fragment; and / or the conjugate. The pharmaceutical composition may further comprise a pharmaceutically acceptable excipient. The pharmaceutical composition may have anticancer activity. Here, the pharmaceutical composition may comprise the anti-CD155 antibody or its antigen-binding fragment; and / or the conjugate as an active ingredient.

[0019] Another example provides a pharmaceutical composition for the prevention, improvement, and / or treatment of cancer comprising the above-mentioned anti-CD155 antibody or its antigen-binding fragment; and / or the above-mentioned conjugate. The cancer may be a cancer expressing CD155. Here, the pharmaceutical composition may comprise the above-mentioned anti-CD155 antibody or its antigen-binding fragment; and / or the above-mentioned conjugate as an active ingredient.

[0020] Another example provides a method for the prevention, improvement, and / or treatment of cancer, comprising the step of administering a pharmaceutically effective amount of the anti-CD155 antibody or its antigen-binding fragment; and / or the conjugate, to a subject requiring prevention, improvement, and / or treatment of cancer. The method may further comprise, prior to the step of administering, a step of identifying (diagnosing, determining) the subject requiring prevention, improvement, and / or treatment of cancer.

[0021] Another example provides the use of the above anti-CD155 antibody or its antigen-binding fragment; and / or the above conjugate for the prevention, improvement, and / or treatment of cancer.

[0022] Another example provides a use for the preparation of a pharmaceutical composition for the prevention, improvement, and / or treatment of cancer, of the above anti-CD155 antibody or its antigen-binding fragment; and / or the conjugate.

[0023] The present disclosure is summarized as follows:

[0024] 1. H-CDR1 containing the amino acid sequence of SEQ ID NO. 1;

[0025] H-CDR2 containing the amino acid sequence of SEQ ID NO. 2;

[0026] H-CDR3 containing the amino acid sequence of SEQ ID NO. 3;

[0027] L-CDR1 containing the amino acid sequence of SEQ ID NO. 4;

[0028] L-CDR2 comprising the amino acid sequence of SEQ ID NO. 5; and

[0029] L-CDR3 containing the amino acid sequence of SEQ ID NO. 6

[0030] An anti-CD155 antibody or an antigen-binding fragment thereof comprising

[0031] 2. The above-mentioned heavy chain variable region comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs 7 to 11 or said amino acid sequence; and

[0032] An anti-CD155 antibody or an antigen-binding fragment thereof comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs 12 to 17, or comprising a light chain variable region consisting of said amino acid sequence.

[0033] 3. In any one of the preceding claims, a heavy chain comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs 46 to 66 or said amino acid sequence; and

[0034] An anti-CD155 antibody or an antigen-binding fragment thereof comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs 67 to 72, or comprising a light chain consisting of said amino acid sequence.

[0035] 4. In any one of the preceding claims, the anti-CD155 antibody or the antigen-binding fragment thereof is an anti-CD155 antibody or the antigen-binding fragment thereof that recognizes CD155 domain 1 as an epitope.

[0036] 5. In any one of the preceding claims, the anti-CD155 antibody or its antigen-binding fragment has one or more features selected from the group consisting of:

[0037] (i) The above anti-CD155 antibody or its antigen-binding fragment does not bind to peripheral blood cells;

[0038] (ii) The above anti-CD155 antibody or its antigen-binding fragment inhibits the binding of TIGIT and CD155;

[0039] (iii) The above anti-CD155 antibody or its antigen-binding fragment inhibits the binding of CD226 and CD155; and

[0040] (iv) The above anti-CD155 antibody or its antigen-binding fragment inhibits the binding of CD96 and CD155.

[0041] 6. In any one of the preceding paragraphs, the anti-CD155 antibody is an anti-CD155 antibody or an antigen-binding fragment thereof that is an animal antibody, a chimeric antibody, or a humanized antibody.

[0042] 7. In any one of the preceding claims, the antigen-binding fragment is an anti-CD155 antibody or an antigen-binding fragment thereof that is a scFv, scFv-Fc, (scFv)2, Fab, Fab', F(ab')2, diabody, or minibody of the anti-CD155 antibody.

[0043] 8. An anti-CD155 antibody of any one of the preceding paragraphs or a polynucleotide encoding an antigen-binding fragment thereof.

[0044] 9. A recombinant vector comprising a polynucleotide encoding the anti-CD155 antibody of any one of the preceding claims or an antigen-binding fragment thereof.

[0045] 10. A polynucleotide encoding an anti-CD155 antibody of any one of the preceding claims or an antigen-binding fragment thereof; and / or a recombinant cell comprising a recombinant vector containing the same.

[0046] 11. A composition for detecting CD155 comprising an anti-CD155 antibody of any one of the preceding claims or an antigen-binding fragment thereof.

[0047] 12. A composition for diagnosing cancer comprising an anti-CD155 antibody or an antigen-binding fragment thereof according to any one of the preceding claims.

[0048] 13. A pharmaceutical composition for the prevention, improvement, or treatment of cancer, comprising an anti-CD155 antibody or an antigen-binding fragment thereof according to any one of the preceding paragraphs.

[0049] 14. A pharmaceutical composition in which, in any one of the compositions of the preceding paragraph, the cancer is a cancer expressing CD155.

[0050] 15. (1) The anti-CD155 antibody of claim 1 or an antigen-binding fragment thereof; and

[0051] (2) A conjugate comprising one or more selected from the group consisting of polymers, labeling substances, drugs, proteins, and peptides.

[0052] 16. The conjugate according to claim 15 above, wherein the conjugate comprises a drug and has an antibody-linker-drug structure.

[0053] 17. A conjugate of any one of the preceding paragraphs, wherein the drug is an anticancer agent.

[0054] 18. A pharmaceutical composition for the prevention, improvement, or treatment of cancer, comprising a conjugate of any one of the preceding paragraphs.

[0055] 19. A pharmaceutical composition in which, in any one of the compositions of the preceding paragraph, the cancer is a cancer expressing CD155.

[0056] 20. A method for the prevention, improvement, and / or treatment of cancer, comprising the step of administering to an individual the anti-CD155 antibody of any one of the preceding claims or an antigen-binding fragment thereof; and / or the conjugate of any one of the preceding claims.

[0057] 21. Use of the anti-CD155 antibody of any one of the preceding claims or its antigen-binding fragment for the prevention, improvement, and / or treatment of cancer; and / or the conjugate of any one of the preceding claims.

[0058] 22. Use of the anti-CD155 antibody of any one of the preceding claims or its antigen-binding fragment for the manufacture of a drug for the prevention, improvement, and / or treatment of cancer; and / or use of the conjugate of any one of the preceding claims.

[0059] This is explained in detail as follows. Meanwhile, each description and embodiment of the present disclosure may be applied to other descriptions and embodiments. That is, all combinations of the various elements of the present disclosure fall within the scope of the present disclosure. Furthermore, the scope of the present disclosure is not to be considered limited by the specific descriptions provided below. Additionally, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated by reference into this specification in their entirety to more clearly explain the level of the art to which the present invention pertains and the content of the present invention. Furthermore, a person skilled in the art can recognize or identify numerous equivalents for the specific embodiments of the present application described in this application using only ordinary experiments. Moreover, such equivalents are intended to be included in the present application.

[0060]

[0061] Definition of Terms

[0062] In this specification, "consisting of a sequence," "essentially consisting of a sequence," or "comprising a sequence" may mean all cases comprising said sequence, but are not intended to exclude cases comprising other sequences other than said sequence.

[0063] In this specification, the terms “protein or polypeptide comprising or comprising an amino acid sequence recognized by a sequence number” and “gene or polynucleotide comprising or comprising a nucleic acid sequence recognized by a sequence number (which may also be used interchangeably with “nucleic acid molecule”) are essentially composed of said amino acid sequence or nucleic acid sequence, or while maintaining inherent and / or intended activity and / or function, said amino acid sequence or nucleic acid sequence and at least 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5% or more, 93% or more, 93.5% or more, 94% or more, 94.5% or more, 95% or more, 95.5% or more, 96% or more, 96.5% or more, 97% or more, 97.5% or more, 97.6% or more, 97.7% or more, 97.8% or more, 97.9% or more, 98% or more, 98.1% or more, 98.2% or more, 98.3% or more, 98.4% or more, 98.5% or more, 98.6% or more, 98.7% or more, 98.8% or more, 98.9% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, It may refer to a protein (or polypeptide) or gene (or polynucleotide) having 99.7% or more, 99.8% or more, or 99.9% or more of sequence homology or sequence identity.

[0064] Due to the degeneracy of codons, the nucleic acid sequences described herein may undergo various modifications to the coding region within a range that does not alter the amino acid sequence and / or function of the protein expressed from the coding region, taking into account the codons preferred by the microorganism intended to express the protein.

[0065] In this specification, the term "homology" refers to the degree of correspondence with a given nucleic acid sequence or amino acid sequence and may be expressed as a percentage (%). Homology to a nucleic acid sequence can be determined, for example, using well-known algorithms such as BLAST or FASTA, and based on these BLAST algorithms, programs called BLASTN or BLASTX have been developed (see: http: / www.ncbi.nlm.nih.gov).

[0066] In this specification, the term "antibody" refers collectively to proteins that specifically bind to a particular antigen. These may be proteins produced within the immune system in response to antigenic stimulation, or proteins produced chemically or recombinantly, and their types are not particularly limited. An antibody may mean an intact immunoglobulin of any isotype, or an antigen-binding fragment capable of competing with an intact antibody for binding to a target antigen. An antibody is also a type of antigen-binding protein itself. An intact antibody may generally comprise at least two full-length heavy chains and two full-length light chains. In this specification, an antibody or its antigen-binding fragment may be unnaturally generated, e.g., recombinant or synthetic. For example, an antibody or its antigen-binding fragment may be produced by hybridomas, recombinant DNA technology, or enzymatic or chemical cleavage of an intact antibody. In this specification, unless otherwise noted, the term antibody may include antibodies comprising two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments, and / or mutants thereof. The antibody may be an animal antibody (e.g., mouse antibody, etc.), a chimeric antibody (e.g., mouse-human antibody), a humanized antibody, or a human antibody. The antibody may be a monoclonal antibody (or monoclonal antibody) or a polyclonal antibody (or polyclonal antibody).

[0067] As used herein, the term "antibody" may encompass a wide range of biochemically distinguishable polypeptides. Those skilled in the art will understand that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε) and have some subclasses (e.g., γ1-γ4), and that light chains are classified as kappa or lambda (K, λ). It is the characteristics of these chains that determine the "class" of the antibody as IgG, IgM, IgA, IgD, or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and are known to confer functional specialization.

[0068] A whole antibody comprises two full-length light chains and two full-length heavy chains, each light chain being connected to a heavy chain by a disulfide bond. The antibody has a heavy chain constant region and a light chain constant region. The heavy chain constant region is of the gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) type, which can be further classified into gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), or alpha 2 (α2). The light chain constant region may be of the kappa (κ) or lambda (λ) type.

[0069] The term "heavy chain" refers to a variable region V containing an amino acid sequence sufficient to provide specificity to the antigen. H , and the three invariant regions C H1 , C H2 and C H3 It may mean a full-length heavy chain or a fragment thereof including , and a hinge region. The term "light chain" refers to a variable region V containing an amino acid sequence sufficient to provide specificity to the antigen. L- and invariant region C L It may refer to a full-length light chain or a fragment thereof containing...

[0070] The term "complementary determining region (CDR)" refers to an amino acid sequence found in the hypervariable region of the heavy or light chain of an immunoglobulin. The heavy and light chains may each contain three CDRs (H-CDR1, H-CDR2, and H-CDR3; and L-CDR1, L-CDR2, and L-CDR3). CDRs may provide residues that play a crucial role in the antibody binding to an antigen or epitope. The terms "specifically binding" or "specifically recognized" are well known to those skilled in the art and indicate that the antibody and the antigen interact specifically to induce immunological activity.

[0071] In this specification, antibodies may comprise, but are not limited to, polyclonal antibodies or monoclonal antibodies; and / or antibodies derived from humans, humanized animals (e.g., mice, rabbits, etc.), or chimeric antibodies (e.g., mouse-human chimeric antibodies).

[0072] Animal-derived antibodies, produced by immunizing animals with a desired antigen, can generally induce an immune rejection reaction when administered to humans for therapeutic purposes; to suppress this rejection, chimeric antibodies were developed. Chimeric antibodies are formed by using genetic engineering methods to replace the constant region of animal-derived antibodies—which causes anti-isotyping reactions—with the constant region of human antibodies. Although chimeric antibodies show significantly improved anti-homologous reactions compared to animal-derived antibodies, potential side effects due to anti-iotyping reactions still exist because animal-derived amino acids remain in the variable region. Humanized antibodies were developed to address these side effects. They are manufactured by transplanting the Complementary Determining Region (CDR), which plays a crucial role in antigen binding within the variable region of chimeric antibodies, into a human antibody framework.

[0073] In this specification, antibodies are understood to include antigen-binding fragments of antibodies having antigen-binding ability, unless otherwise specifically stated.

[0074] The term “antigen-binding fragment” means any form of polypeptide comprising a portion to which an antigen can bind (e.g., the six CDRs defined herein). For example, it may be one or more selected from the group consisting of, but not limited to, scFv, scFv-Fc, scFv-Fc-scFv, (scFv)2, Fab, Fab', Fab-Fc, Fab'-Fc, F(ab')2, diabody, and minibody of the antibody.

[0075] Among the above antigen-binding fragments, Fab has a structure having a variable region of the light chain and heavy chain, a constant region of the light chain and a first constant region (CH1) of the heavy chain, and has one antigen-binding site.

[0076] Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain.

[0077] F(ab')2 antibodies are produced when cysteine ​​residues in the hinge region of Fab' form disulfide bonds.

[0078] Recombinant techniques for generating Fv fragments, which are minimal antibody fragments possessing only a heavy chain variable region and a light chain variable region, are widely known in the art. Two-chain Fvs are formed in which the heavy chain variable region and the light chain variable region are connected by non-covalent bonds, while single-chain Fvs (scFvs) can form single-chain dimer structures in which the heavy chain variable region and the short chain variable region are connected by covalent bonds (peptide bonds) through a common peptide linker or directly (without a linker). scFv-Fc is formed when Fc is linked to scFv. scFv-Fc-scFv is formed in which scFv is linked to each end (N-terminus and C-terminus) of Fc. A minibody is formed in which a CH3 group is linked to scFv. A diabody contains two scFv molecules.

[0079] The above antigen-binding fragment can be produced by chemically cutting an intact antibody or enzymatically cutting it using a proteolytic enzyme (for example, if the whole antibody is restricted to papain, Fab can be obtained, and if it is cut with pepsin, the F(ab')2 fragment can be obtained), or through genetic recombination technology.

[0080] The term "hinge region" refers to a region within the heavy chain of an antibody that exists between the CH1 and CH2 regions and functions to provide flexibility to the antigen-binding site within the antibody.

[0081] In this specification, the “Fc” region refers to a heavy chain fragment comprising CH2 and CH3 domains of an antibody, and may, in some cases, be in a form including a hinge region at the N-terminus of the CH2 and CH3 domains (i.e., the N-terminus of the CH2 domain). Two Fc regions may be joined to each other by two or more disulfide bonds and / or hydrophobic interactions of the CH3 domain.

[0082] In this specification, a “bivalent antibody or bivalent antigen-binding fragment” comprises two antigen-binding sites comprising six CDRs (H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3). The two antigen-binding sites included in the bivalent antibody or bivalent antigen-binding fragment may have the same antigen specificity or have bispecificity, each binding to a different antigen. In this specification, a “monovalent antibody or monovalent antigen-binding fragment” comprises one antigen-binding site comprising six CDRs (H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3).

[0083] In this specification, a bispecific antibody or its antigen-binding fragment refers to an antibody or its antigen-binding fragment that targets two antigens, and is a hybrid antibody that binds to two different epitopes. The two epitopes of the bispecific antibody or antigen-binding fragment may be located on the same or different target proteins (antigens).

[0084] The antibody provided in this specification may be a monoclonal antibody. The monoclonal antibody may be manufactured according to methods widely known in the art. For example, it may be manufactured using a phage display technique. Alternatively, the antibody may be manufactured as a monoclonal antibody derived from an animal (e.g., mouse, rat, rabbit, etc.) by conventional methods, or as a humanized antibody by conventional methods.

[0085] In this specification, “antigen” or “immunogen” means a molecule or part of a molecule to which, for example, an antigen-binding protein (e.g., an antibody or an immunologically functional antigen-binding fragment thereof) can bind, and may be used in the production of antibodies capable of binding to an antigen in animals. The antigen may include one or more epitopes capable of interacting with other antibodies or fragments thereof.

[0086] In this specification, “epitope” is a part of a molecule to which an antibody or its antigen-binding fragment is bound or recognized by them, and comprises any determinant capable of specifically binding to an antigen-binding protein, such as an antibody or a T cell receptor, for example. The epitope may be continuous or discontinuous, and may be amino acid residues located at positions that are not continuous with each other in the polypeptide sequence but are bound by a single antigen-binding protein in terms of the molecule, such as a conformational epitope. The epitope determinant may be a chemically active group formed on the surface of a molecule, such as an amino acid, a sugar side chain, a phosphoryl group, and / or a sulfonyl group, or may have specific three-dimensional structural features and / or specific charge features.

[0087] In this specification, the statement that an antibody (e.g., CDR, variable region, or heavy / light chain, antigen-binding fragment, etc.) “comprising a specific amino acid sequence, or composed of or expressed by a specific amino acid sequence” may mean both cases where the antibody essentially comprises said amino acid sequence and cases where insignificant variations (e.g., substitution, deletion, and / or addition of amino acid residues) that do not have a significant effect on antibody activity (e.g., antigen affinity, pharmacological activity, etc.) are introduced into said amino acid sequence.

[0088]

[0089] Anti-CD155 antibody or its antigen-binding fragment

[0090] Specific examples of the present application provide a novel anti-CD155 antibody or an antigen-binding fragment thereof, and in detail, said anti-CD155 antibody or the antigen-binding fragment thereof may comprise the following:

[0091] H-CDR1 containing the amino acid sequence of SEQ ID NO. 1;

[0092] H-CDR2 containing the amino acid sequence of SEQ ID NO. 2;

[0093] H-CDR3 containing the amino acid sequence of SEQ ID NO. 3;

[0094] L-CDR1 containing the amino acid sequence of SEQ ID NO. 4;

[0095] L-CDR2 comprising the amino acid sequence of SEQ ID NO. 5; and

[0096] L-CDR3 containing the amino acid sequence of SEQ ID NO. 6.

[0097] The amino acid sequence of the CDR of the above anti-CD155 antibody or its antigen-binding fragment is exemplified in Table 1 below.

[0098] Sequence No. Sequence 1H-CDR1SSYIS2H-CDR2WIYAGTGDTNYNQNFTG3H-CDR3HDYGNNYDWYFDV4L-CDR1RSSTGAVTTSNYAN5L-CDR2GTNNRAP6L-CDR3ALWYSNHWV

[0099] The heavy chain variable region and light chain variable region of the above anti-CD155 antibody or its antigen-binding fragment may be an antibody or antigen-binding fragment derived from IgG, and may be an antibody or antigen-binding fragment in which a mutation has been introduced to a wild-type sequence, e.g., a framework region (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, and / or L-FR4). In one example, the mutation may be for stabilization and / or the formation of a disulfide bridge. For example, the antigen-binding fragment may have one or more (e.g., one) G(Gly)→C(Cys) mutations (G replaced by C) introduced to the framework region of the antibody's variable region, but is not limited thereto. In one embodiment, if the CD155 antibody is a complete IgG antibody or a part thereof (e.g., Fab or Fab-Fc), the heavy chain variable region and light chain variable region included herein may be of wild-type IgG origin. In another embodiment, if the anti-CD155 antibody is an antigen-binding fragment of the anti-CD155 antibody (e.g., scFv, etc.), the heavy chain variable region and light chain variable region included herein may have a G→C mutation introduced into the framework region. In some cases (e.g., when recombinantly produced), the heavy chain variable region and / or light chain variable region may additionally include an appropriate signal sequence at the N-terminus.

[0100] In one example, the above anti-CD155 antibody or its antigen-binding fragment is,

[0101] A heavy chain variable region comprising H-CDR1 having the amino acid sequence of SEQ ID NO. 1; H-CDR2 having the amino acid sequence of SEQ ID NO. 2; and H-CDR3 having the amino acid sequence of SEQ ID NO. 3; and / or

[0102] It may include a light chain variable region comprising L-CDR1 having the amino acid sequence of SEQ ID NO. 4; L-CDR2 having the amino acid sequence of SEQ ID NO. 5; and L-CDR3 having the amino acid sequence of SEQ ID NO. 6.

[0103] In one example, the above anti-CD155 antibody or its antigen-binding fragment is,

[0104] A heavy chain variable region comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs. 7 to 11 or said amino acid sequence; and / or

[0105] It may include any one amino acid sequence selected from the group consisting of SEQ ID NOs 12 to 17, or a light chain variable region consisting of said amino acid sequence.

[0106] In one example, the anti-CD155 antibody or its antigen-binding fragment may include a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 7; and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 12, 13, 14, 15, 16, or 17.

[0107] In one example, the anti-CD155 antibody or its antigen-binding fragment may include a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 8; and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 12, 13, 14, 15, 16, or 17.

[0108] In one example, the anti-CD155 antibody or its antigen-binding fragment may include a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 9; and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 12, 13, 14, 15, 16, or 17.

[0109] In one example, the anti-CD155 antibody or its antigen-binding fragment may include a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 10; and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 12, 13, 14, 15, 16, or 17.

[0110] In one example, the anti-CD155 antibody or its antigen-binding fragment may include a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 11; and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 12, 13, 14, 15, 16, or 17.

[0111] The amino acid sequences of the heavy chain variable region and the light chain variable region of the above anti-CD155 antibody or its antigen-binding fragment are exemplified in Table 2 below.

[0112] 서열번호명명서열7mVHEVQLQESGAELVKPGASVKLSCKTSGFTFSSSYISWLKQKPGQSLEWIAWIYAGTGDTNYNQNFTGKAQLTVDTSSSTAYMQFSSLTTEDSAIYYCARHDYGNNYDWYFDVWGAGTTVTVSS8VH1 (HU1186-hVH1)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSSYISWVRQAPGQRLEWMGWIYAGTGDTNYNQNFTGRVTITVDTSASTAYMELSSLRSEDTAVYYCARHDYGNNYDWYFDVWGQGTTVTVSS9VH2 (HU1186-hVH2-1)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSSYISWVRQAPGQRLEWMGWIYAGTGDTNYNQNFTGRVTITRDTSASTAYMELSSLRSEDTAVYYCARHDYGNNYDWYFDVWGRGTLVTVSS10VH3 (HU1186-hVH3)QVQLVQSGAEVVKPGASVKVSCKASGFTFSSSYISWVRQAPGQRLEWMGWIYAGTGDTNYNQNFTGRVTITVDTSASTAYMELSSLRSEDTAIYYCARHDYGNNYDWYFDVWGQGTTVTVSS11VH4 (HU1186-hVH4)QVQLVQSGAEVKKPGASVKVSCKASGFTFSSSYISWLKQKPGQRLEWMGWIYAGTGDTNYNQNFTGRVTITRDTSASTAYMELSSLRSEDTAVYYCARHDYGNNYDWYFDVWGQGTTVTVSS12mVLQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVL13VL1 (HU1186-hVL1)QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNNRAPWTPARFSGSLLGGKAALTLLGAQPEDEAEYYCALWYSNHWVFGGGTKLTVL14VL2(HU1186-hVL2)QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNNRAPGTPARFSGSLLGDKAALTLLGAQPEDEAEYYCALWYSNHWVFGGGTKLTVL15VL3 (HU1186-hVL3)QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQLFRGLIGGTNNRAPGTPARFSGSLLGDKAALTLLGAQPEDEAEYFCALWYSNHWVFGGGTKLTVL16VL4 (HU1186-hVL4)QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWFQQKPGHAPRGLIYGTNNRAPWTPARFSGSLLGDKAALTISGAQPEDEAEYFCALWYSNHWVFGGGTKLTVL17VL5 (HU1186-hVL5-1)QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWFQQKPGQAPRTLIYGTNNRAPWTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNHWVFGGGTKLTVL

[0113] The amino acid sequence of the framework of the variable region of the above anti-CD155 antibody or its antigen-binding fragment is exemplified in Table 3 below.

[0114] Sequence Number H-FR1 Sequence Number H-FR2 Sequence Number H-FR3 Sequence Number H-FR418EVQLQESGAELVKPGASVKLSCKTSGFTFS22WLKQKPGQSLEWIA25KAQLTVDTSSSTAYMQFSSLTTEDSAIYYCAR29WGAGTTVTVSS19QVQLVQSGAEVKKPGASVKVSCKASGYTFT23WVRQAPGQRLEWMG26RVTITVDTSAST AYMELSSLRSEDTAVYYCAR30WGQGTTVTVSS20QVQLVQSGAEVVKPGASVKVSCKAGSGTFFS24WLKQKPGQRLEWMG27RVTITRDTSASTA YMELSSLRSEDTAVYYCAR31WGRGTLVTVSS21QVQLVQSGAEVKKPGASVKVSCKASGFTFS28RVTITVDTSASTAYMELSSLRSEDTAIYYCA R Sequence Number L-FR1 Sequence Number L-FR2 Sequence Number L-FR3 Sequence Number L-FR432QAVVTQESALTTSPGETVTLTC34WVQEKPDHLFTGLIG39GVPARFSGSLIGDKAALTITGAQTEDEAIYFC45FGGGTKLTVL33QAVVTQEPSLTVSPGGTVTLTC35WVQQKPGQAPRGLIG40WTPARFSGSLLGGKAALTLLGAQPED EAEYYC36WVQQKPGQLFRGLIG41GTPARFSGSLLGDKAALTLLGAQPEDEAEYYC37WFQQKPGHAPRGLIY42GTPARFSGSLLGDKAALTLL GAQPEDEAEYFC38WFQQKPGQAPRTLIY43WTPARFSGSLLGDKAALTISGAQPEDEAEYFC44WTPARFSGSLLGGKAALTLSGAQPEDEAEYYC

[0115] In one example, the above anti-CD155 antibody or its antigen-binding fragment is,

[0116] A heavy chain comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs 46 to 66 or said amino acid sequence; and / or

[0117] It may include any one amino acid sequence selected from the group consisting of SEQ ID NOs 67 to 72, or a light chain consisting of said amino acid sequence.

[0118] In one example, the above anti-CD155 antibody or its antigen-binding fragment is,

[0119] A heavy chain comprising the amino acid sequence of SEQ ID NO. 46; and

[0120] It may include a light chain containing the amino acid sequence of SEQ ID NO. 67.

[0121] In one example, the above anti-CD155 antibody or its antigen-binding fragment is,

[0122] A heavy chain comprising the amino acid sequence of SEQ ID NO. 47, 48, 49, or 50; and

[0123] It may include a light chain containing the amino acid sequence of SEQ ID NO. 68.

[0124] In one example, the above anti-CD155 antibody or its antigen-binding fragment is,

[0125] A heavy chain comprising the amino acid sequence of SEQ ID NO. 51, 52, 53, or 54; and

[0126] It may include a light chain containing the amino acid sequence of SEQ ID NO. 69.

[0127] In one example, the above anti-CD155 antibody or its antigen-binding fragment is,

[0128] A heavy chain comprising the amino acid sequence of SEQ ID NO. 55, 56, 57, or 58; and

[0129] It may include a light chain containing the amino acid sequence of SEQ ID NO. 70.

[0130] In one example, the above anti-CD155 antibody or its antigen-binding fragment is,

[0131] A heavy chain comprising the amino acid sequence of SEQ ID NO. 59, 60, 61, or 62; and

[0132] It may include a light chain containing the amino acid sequence of SEQ ID NO. 71.

[0133] In one example, the above anti-CD155 antibody or its antigen-binding fragment is,

[0134] A heavy chain comprising the amino acid sequence of SEQ ID NO. 63, 64, 65, or 66; and

[0135] It may include a light chain containing the amino acid sequence of SEQ ID NO. 72.

[0136] In one example, the above anti-CD155 antibody or its antigen-binding fragment may be an anti-CD155 scFv (single-chain variable fragment) comprising the following:

[0137] A heavy chain variable region comprising H-CDR1 having the amino acid sequence of SEQ ID NO. 1; H-CDR2 having the amino acid sequence of SEQ ID NO. 2; and H-CDR3 having the amino acid sequence of SEQ ID NO. 3; and

[0138] A light chain variable region comprising L-CDR1 having the amino acid sequence of SEQ ID NO. 4; L-CDR2 having the amino acid sequence of SEQ ID NO. 5; and L-CDR3 having the amino acid sequence of SEQ ID NO. 6.

[0139] At this time, the heavy chain variable region and the light chain variable region can be connected to each other in any order directly (e.g., without a linker) or through a peptide linker.

[0140] In one example, the above-mentioned claim-CD155 scFv may include the following:

[0141] A heavy chain variable region comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs 7 to 11 or said amino acid sequence; and

[0142] A light chain variable region comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs 12 to 17, or said amino acid sequence.

[0143] At this time, the heavy chain variable region and the light chain variable region can be connected to each other in any order, either directly or through a peptide linker.

[0144] In this specification, the anti-CD155 scFv comprises a heavy chain variable region and a light chain variable region in any order. For example, the anti-CD155 scFv may comprise a light chain variable region and a heavy chain variable region in the direction from the N-terminus to the C-terminus. Alternatively, the anti-CD155 scFv may comprise a heavy chain variable region and a light chain variable region in the direction from the N-terminus to the C-terminus. In one example, the anti-CD155 scFv may comprise a light chain variable region, a peptide linker, and a heavy chain variable region in the direction from the N-terminus to the C-terminus. In another example, the anti-CD155 scFv may comprise a heavy chain variable region, a peptide linker, and a light chain variable region in the direction from the N-terminus to the C-terminus.

[0145] In this specification, the term “peptide linker” may mean an oligopeptide comprising 1 to 100 amino acids, particularly 2 to 50 amino acids, each of which may be any type of amino acid without limitation. Any conventional peptide linker may be used with or without appropriate modifications to suit a particular purpose. In certain embodiments, the peptide linker may comprise, for example, Gly, Asn, and / or Ser residues, and / or neutral amino acids such as Thr and / or Ala. Amino acid sequences suitable for the peptide linker may be known in the relevant art. The length of the peptide linker may be appropriately determined to the extent that the function of the polypeptide and / or scFv is not affected. For example, the peptide linker may be formed of a total of about 1 to about 100 amino acids, about 2 to about 50 amino acids, or about 5 to about 25 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25), each of which may be independently selected from the group consisting of Gly, Asn, Ser, Thr, and Ala. In one embodiment, the peptide linker is (G m S l ) n (m, l, and n are "G", "S" and "(G" respectively) m S l It can be expressed as the number of ”, each independently selected from about 1 to about 10, particularly, integers 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0146]

[0147] The antibody or antigen-binding fragment provided herein has a binding affinity to an antigen (e.g., human antigen protein) measured, for example, by a binding affinity (K) measured by surface plasmon resonance (SPR). D Based on EC50 measured by ELISA or ) it may be 5 nM or less, 1 nM or less, 0.5 nM or less, 0.2 nM or less, 0.1 nM or less, 0.05 nM or less, 0.01 nM or less, 0.005 nM or less, or 0.001 nM or less, for example, 0.001 nM to 5 nM, 0.001 nM to 1 nM, but is not limited thereto.

[0148] In one example, the anti-CD155 antibody or its antigen-binding fragment is 10 nM or less, 7 nM or less, 5 nM or less, 3 nM or less, 2 nM or less, 1.5 nM or less, or 1.0 nM or less EC 50It may have a value. For example, the antibody of the present invention or its antigen-binding fragment is 1.4 nM or less, 1.3 nM or less, 1.2 nM or less, 1.1 nM or less, 1.0 nM or less, 0.97 nM or less, 0.95 nM or less, 0.93 nM or less, 0.90 nM or less, 0.87 nM or less, 0.85 nM or less, 0.83 nM or less, 0.80 nM or less, 0.77 nM or less, 0.75 nM or less, 0.73 nM or less, 0.70 nM or less, 0.67 nM or less, 0.65 nM or less, 0.63 nM or less, 0.60 nM or less, 0.57 nM or less, 0.55 nM or less, 0.53 nM or less, 0.50 nM or less, 0.47 nM or less, 0.45nM or less, 0.43nM or less, 0.40nM or less, 0.37nM or less, 0.35nM or less, 0.33nM or less, 0.30nM or less, 0.27nM or less, 0.25nM or less, 0.23nM or less, 0.20nM or less, 0.17nM or less, 0.15nM or less, 0.13nM or less, 0.10nM or less, 0.07nM or less, 0.05nM or less, 0.03nM or less, 0.02nM or less, 0.01nM or less, 0.009nM or less, 0.008nM or less, 0.007nM or less, 0.006nM or less, 0.005nM or less, 0.004nM or less, EC of 0.003nM or less, 0.002nM or less, or 0.001nM or less 50 It can be combined with CD155 as a value.

[0149] In one example, the anti-CD155 antibody may be an anti-CD155 antibody or an antigen-binding fragment thereof, which is an animal antibody, a chimeric antibody, or a humanized antibody.

[0150] In one example, the antigen-binding fragment may be a scFv, scFv-Fc, (scFv)2, Fab, Fab', F(ab')2, diabody, or minibody of the anti-CD155 antibody.

[0151] The anti-CD155 antibody or its antigen-binding fragment provided herein has functions such as CD155 inhibition (e.g., inhibition of CD155 expression, inhibition of activity, and / or inhibition of binding with other proteins (TIGIT, CD226, CD96, etc.), targeting of cancer cells by specific binding to cancer cells (e.g., CD155-expressing cancer cells), and anticancer activity (e.g., cancer cell death, inhibition of cancer cell proliferation, etc.). Accordingly, the anti-CD155 antibody or its antigen-binding fragment can be usefully applied to the prevention, improvement, and / or treatment of cancer.

[0152] The above anti-CD155 antibody or its antigen-binding fragment may have specificity for CD155 and a suitable affinity for use as an antibody therapeutic or diagnostic agent.

[0153] The above anti-CD155 antibody or its antigen-binding fragment may have cross-reactivity with human and monkey CD155.

[0154] Since the above anti-CD155 antibody or its antigen-binding fragment is cross-reactive with monkey CD155, it has the advantage of allowing for better evaluation of anticancer effects in animal studies prior to clinical trials and can increase the likelihood of success in human trials.

[0155] The above anti-CD155 antibody or its antigen-binding fragment specifically binds to CD155 and can prevent the binding of CD155 to at least one of a T cell immune receptor (TIGIT) having a ligand, Ig, and ITIM domain, CD96, and CD226 (DNAM-1).

[0156] In one example, the anti-CD155 antibody or its antigen-binding fragment may recognize CD155 domain 1 as an epitope. More specifically, the anti-CD155 antibody or its antigen-binding fragment may recognize the C-terminus of CD155 domain 1 as an epitope, but is not limited thereto. The CD155 domain 1 may include, for example, one or more amino acid sequences selected from the group consisting of SEQ ID NOs 111 to 113, or may consist of said amino acid sequences, but is not limited thereto.

[0157] In one example, the anti-CD155 antibody or its antigen-binding fragment may have one or more features selected from the group consisting of the following:

[0158] (i) The anti-CD155 antibody or its antigen-binding fragment does not bind to peripheral blood cells (e.g., granulocytes, lymphocytes, monocytes, and / or red blood cells);

[0159] (ii) The above anti-CD155 antibody or its antigen-binding fragment inhibits the binding of TIGIT and CD155;

[0160] (iii) The above anti-CD155 antibody or its antigen-binding fragment inhibits the binding of CD226 and CD155; and

[0161] (iv) The above anti-CD155 antibody or its antigen-binding fragment inhibits (prevents, blocks) the binding of CD96 and CD155.

[0162] In one example, the anti-CD155 antibody or its antigen-binding fragment can inhibit the binding of CD155 to at least one of TIGIT, CD226, and CD96.

[0163] FIG. 1 of this specification shows a schematic diagram illustrating receptors expressed in immune cells and tumor cells. TIGIT is known as an inhibitory receptor expressed in immune cells such as T cells. DNAM-1 (also named CD226) is also a receptor expressed in immune cells such as T cells and is known to act as an activating receptor. PVR (CD155) is a ligand for TIGIT, DNAM-1, and CD96 expressed in many tumor cells; it acts as an inhibitory ligand by binding strongly to TIGIT, and binds more weakly to DNAM-1 and CD96. Nectin-2 (CD112) is an activating ligand for DNAM-1 expressed in many tumor cells and exhibits marginal recognition of TIGIT. The anti-CD155 antibody of the present application can exhibit the effect of increasing the activity of immune cells by specifically binding to PVR (CD155) and blocking the interaction between CD155 and TIGIT. Meanwhile, PVRIG is known to be an activating ligand for DNAM-1, which binds to the CD112 receptor overexpressed in tumor cells, and to bind weakly to TIGIT.

[0164]

[0165] Method for preparing polynucleotides, vectors, cells, and antibodies

[0166] Another example provides a polynucleotide (or nucleic acid molecule) encoding (coding for) the above anti-CD155 antibody or its antigen-binding fragment.

[0167] In one example, the polynucleotide is the heavy chain complementarity determining site, heavy chain variable region, or heavy chain of the anti-CD155 antibody; and / or

[0168] It may be a light chain complementarity determining site, light chain variable region, or a light chain encoding of the anti-CD155 antibody.

[0169] In one example, the polynucleotide encoding the heavy chain variable region of the anti-CD155 antibody may comprise any one nucleic acid sequence selected from the group consisting of SEQ ID NOs 73 to 77, or may be composed of said nucleic acid sequence.

[0170] In one example, the polynucleotide encoding the light chain variable region of the anti-CD155 antibody may comprise any one nucleic acid sequence selected from the group consisting of SEQ ID NOs 78 to 83, or may be composed of said nucleic acid sequence.

[0171] In one example, the polynucleotide encoding the heavy chain of the anti-CD155 antibody may comprise any one nucleic acid sequence selected from the group consisting of SEQ ID NOs 84 to 104, or may be composed of said nucleic acid sequence.

[0172] In one example, the polynucleotide encoding the light chain of the anti-CD155 antibody may comprise any one nucleic acid sequence selected from the group consisting of SEQ ID NOs 105 to 110, or may be composed of said nucleic acid sequence.

[0173] Another example provides a vector comprising a polynucleotide encoding the anti-CD155 antibody or its antigen-binding fragment. The vector may be a recombinant vector or an expression vector.

[0174] Another example provides a recombinant cell containing the above recombinant vector.

[0175] In one example, the recombinant cell may be a hybridomy.

[0176] In this application, the term "vector" refers to a means for expressing a target gene in a host cell. Examples include viral vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors, lentivirus vectors, adenovirus vectors, retrovirus vectors and adeno-associated virus vectors. The vectors that can be used as the above-mentioned recombinant vectors are plasmids commonly used in the art (e.g., pBR series, pUC series, pBluescriptII series, pGEM series, pGEX series, pTZ series, pCL, pcDNA series, pET series, etc.; more specifically, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pcDNA3.1, pcDNA3.3, etc.), phages (e.g., λgt4λB, λ-Charon, λΔz1, and M13, etc.) or viruses (e.g. For example, it can be manufactured by manipulating the SV40, but is not limited to this.

[0177] In the above recombinant vector, the polynucleotide may be operatively linked to a promoter. The term "operatively linked" means a functional linkage between a nucleotide expression regulatory sequence (e.g., a promoter sequence) and another nucleotide sequence. The regulatory sequence may regulate the transcription and / or decoding of another nucleotide sequence by being "operatively linked."

[0178] The recombinant vector can typically be constructed as a vector for cloning or as a vector for expression. The expression vector may be a conventional one used in the art to express foreign proteins in plants, animals, or microorganisms. The recombinant vector can be constructed through various methods known in the art.

[0179] The above recombinant vector can be constructed using prokaryotic or eukaryotic cells as hosts. For example, if the vector used is an expression vector and the host is a prokaryotic cell, a potent promoter capable of advancing transcription (e.g., pL λ It is common for the vector to include a promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.), a ribosome binding site for initiating translation, and a transcription / translation termination sequence. When eukaryotic cells are used as the host, the replication origins that operate in eukaryotic cells included in the vector include, but are not limited to, f1 replication origins, SV40 replication origins, pMB1 replication origins, adeno replication origins, AAV replication origins, and BBV replication origins. Additionally, promoters derived from the genome of mammalian cells (e.g., metallothionein promoters) or promoters derived from mammalian viruses (e.g., adenovirus late promoters, vaccinia virus 7.5K promoters, SV40 promoters, cytomegalovirus promoters, and HSV tk promoters) may be used, and generally have a polyadenylation sequence as the transcription termination sequence.

[0180] The recombinant cell may be obtained by introducing the recombinant vector into a suitable host cell. Any host cell known in the art that is capable of stably and continuously cloning or expressing the recombinant vector may be used as the host cell, and prokaryotic cells include, for example, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. Bacillus strains such as coliW3110, Bacillus subtilis, and Bacillus churingensis, as well as intestinal bacteria and strains such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species, may be used as host cells when transforming into eukaryotic cells, such as yeast (Saccharomyce cerevisiae), insect cells, plant cells, and animal cells, for example, Sp2 / 0, CHO (Chinese hamster ovary) K1, CHO DG44, CHO S, CHO DXB11, CHO GS-KO, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, MDCK cell lines, etc., but are not limited thereto.

[0181] The delivery (introduction) of the above nucleic acid molecule or a recombinant vector containing it into a host cell may be carried out using a delivery method widely known in the art. For example, if the host cell is a prokaryotic cell, the delivery method may be the CaCl2 method or the electroporation method, and if the host cell is a eukaryotic cell, the delivery method may be the microinjection method, calcium phosphate precipitation method, electroporation method, liposome-mediated transfection method, and gene bombardment method, but is not limited thereto.

[0182] The method for selecting the above-mentioned transformed host cells can be easily carried out according to methods widely known in the art by utilizing the phenotype expressed by the selection marker. For example, if the selection marker is a specific antibiotic resistance gene, the transformed cells can be easily selected by culturing the transformed cells in a medium containing the antibiotic.

[0183] Another example provides a method for producing an anti-CD155 antibody or an antigen-binding fragment thereof, comprising the step of expressing the above-mentioned polynucleotide in a host cell. The step of expressing the above-mentioned nucleic acid molecule in a host cell may include the step of culturing a cell containing the above-mentioned nucleic acid molecule or a recombinant vector containing the same. The manufacturing method may optionally further include the step of isolating and / or purifying the antibody or antigen-binding fragment from a culture medium after the culturing step.

[0184] When the antibody or its antigen-binding fragment provided in this specification is produced recombinantly, it may be in a form bound to a conventional signal peptide, cleavage site, tag, etc. for purification. Accordingly, in a non-limiting example, the antibody or its antigen-binding fragment provided in this specification may be in a form additionally comprising one or more selected from the group consisting of a signal peptide, cleavage site, tag (e.g., His tag, GST (glutathione-s-transferase) tag, MBP (maltose binding protein) tag, etc.) that are typically used in the process of recombinant production of proteins, or in a purified form from which these are removed.

[0185]

[0186] CD155 detection and cancer diagnosis applications

[0187] Another example provides a composition for CD155 detection or a kit for CD155 detection comprising the above anti-CD155 antibody or its antigen-binding fragment, and / or a polynucleotide encoding the above anti-CD155 antibody or its antigen-binding fragment.

[0188] Another example provides a method for detecting CD155 in a biological sample, comprising the step of contacting a biological sample in which CD155 expression detection is required with the anti-CD155 antibody or its antigen-binding fragment, and / or a polynucleotide encoding the anti-CD155 antibody or its antigen-binding fragment. The method may further comprise, after the contacting step, the step of measuring an antibody-antigen reaction (binding) in the biological sample treated (contacted) with the anti-CD155 antibody or its antigen-binding fragment, and / or the polynucleotide encoding the anti-CD155 antibody or its antigen-binding fragment. In one embodiment, the method may be performed in vitro or in vivo.

[0189] Another example provides a use for the detection of CD155 of the above anti-CD155 antibody or its antigen-binding fragment, and / or a polynucleotide encoding the above anti-CD155 antibody or its antigen-binding fragment.

[0190] Another example provides a cancer diagnostic composition or a cancer diagnostic kit comprising the above-mentioned anti-CD155 antibody or its antigen-binding fragment, and / or a polynucleotide encoding the above-mentioned anti-CD155 antibody or its antigen-binding fragment. The cancer may be a cancer that expresses CD155.

[0191] Another example provides a method for diagnosing cancer of a biological sample or a subject from which the biological sample is derived, or a method for providing information for cancer diagnosis, comprising the step of contacting a biological sample with the anti-CD155 antibody or its antigen-binding fragment, and / or a polynucleotide encoding the anti-CD155 antibody or its antigen-binding fragment. The biological sample may be isolated from the subject. The method may further comprise, after the contacting step, the step of measuring an antibody-antigen reaction (binding) in the biological sample treated (contacted) with the anti-CD155 antibody or its antigen-binding fragment, and / or the polynucleotide encoding the anti-CD155 antibody or its antigen-binding fragment, and / or, if binding is detected in the treated (contacted) biological sample, the step of determining (identifying) the biological sample or the subject from which the biological sample is derived (isolated) as a cancer patient, or further determining that such a subject will respond to treatment with the anti-CD155 antibody or its antigen-binding fragment. In one embodiment, the method may be performed in vitro or in vivo. The cancer may be a cancer expressing CD155.

[0192] Another example provides a cancer diagnostic use of the above anti-CD155 antibody or its antigen-binding fragment, and / or a polynucleotide encoding the above anti-CD155 antibody or its antigen-binding fragment.

[0193] In the composition and / or method for CD155 detection and / or cancer diagnosis provided herein, the biological sample may be a cell, tissue, body fluid (e.g., serum), transformed cell, or culture thereof derived (isolated) from any animal, e.g., mammals including humans, primates such as monkeys, rats, rodents such as mice, etc., and may be, e.g., a human or a cell, tissue, or body fluid (e.g., serum) isolated from a human.

[0194]

[0195] Conjugate

[0196] Another example provides a conjugate comprising (1) the above anti-CD155 antibody or its antigen-binding fragment and (2) one or more selected from the group consisting of useful polymers, labeling substances, drugs, proteins, and peptides.

[0197] In one example, the conjugate may contain a drug and have an antibody-linker-drug structure. In one example, the conjugate may be an antibody-drug conjugate. That is, one example of the present invention may provide an antibody-drug conjugate in which (1) the anti-CD155 antibody or its antigen-binding fragment and (2) a drug are linked.

[0198]

[0199] In this specification, "conjugate" refers to a cell binder that is covalently bonded to one or more molecules of a cytotoxic compound. Here, the "cell binder" is a molecule having an affinity for a biological target, which may be, for example, a ligand, a protein, an antibody, or an antigen-binding fragment thereof, and the binder functions to guide the biologically active compound to the biological target. In one embodiment of the present invention, the conjugate may be designed to target cancer cells via a cell surface antigen. The antigen may be a cell surface antigen that is overexpressed or expressed in abnormal cell types. Specifically, the target antigen may be one that is primarily expressed on proliferating cells (e.g., cancer cells). The target antigen may typically be selected based on different expression between proliferating tissues and normal tissues.

[0200] The anti-CD155 antibody or its antigen-binding fragment provided in this specification may be used in the form of a conjugate conjugated with one or more selected from the group consisting of useful polymers, labeling substances, drugs, proteins, and peptides.

[0201] The above useful polymer may be, for example, a polymer that increases the half-life of polypeptides, antibodies, and / or antigen-binding fragments in the body, and may be exemplified by one or more hydrophilic polymers selected from the group consisting of polyethylene glycol (PEG) (e.g., PEG having a molecular weight of 2 kDa, 5 kDa, 10 kDa, 12 kDa, 20 kDa, 30 kDa, or 40 kDa), dextran, monomethoxypolyethylene glycol (mPEG), etc., but is not limited thereto.

[0202] The above labeling material may be selected from fluorescent or chemiluminescent small molecule compounds, radioisotopes, etc., commonly used in the industry, but is not limited thereto.

[0203] The above drugs may be one or more selected from the group consisting of cytotoxic drugs (e.g., anticancer agents, antibiotics, etc.) and contrast agents. The above cytotoxic drugs may be any drugs having cytotoxic activity, particularly cytotoxic activity on cancer cells or bacteriological / viral toxicity, and may be, for example, tubulin inhibitors, DNA alkylating agents, topoisomerase inhibitors, RNA polymerase inhibitors, ribosome inactivating proteins, NAMPT inhibitors, immunomodulators, etc., but are not limited thereto, and may be included without limitation as long as they are capable of inhibiting the growth, proliferation, and / or progression of cancer cells or tumors.

[0204] The above drugs are, for example, deruxtecan, maytansine class drugs (e.g., Mertansine (DM1), Ravtansine (DM4)), auristatin class drugs (e.g., Monomethylauristatin E (MMAE), Monomethylauristatin F (MMAF), etc.), calicheamicin class drugs, pyrrobenzodiazepine class drugs, duocarmycin, docetaxel, doxorubicin, carboplatin (paraplatin), cyclophosphamide, ifosfamide, nidran, nitrogen mustard, and mechlorethamine hydrochloride. It may be HCL), bleomycin, mitomycin C, cytarabine, flurouracil, gemcitabine, trimetrexate, methotrexate, etoposide, vinblastine, vinorelbine, Alimta, Altretamine, procarbazine, paclitaxel (Taxol), taxotere, topotecan, irinotecan, exatecan, or analogs thereof.

[0205] The CD155 antibody of the present invention or its antigen-binding fragment may be directly or indirectly linked to the drug and used in the form of an antibody-drug conjugate (ADC), and the drug may be a metansin-class drug, an auristatin-class drug, a calicemycin-class drug, a pyrrolobenzodiazepine-class drug, duocamycin, irinotecan, or exatecan.

[0206] In the above conjugate, the antibody or its antigen-binding fragment and the cytotoxic drug (anticancer agent, etc.) may be connected through chemical bonding, e.g., covalent bonding. For example, the antibody and the cytotoxic drug may be connected to each other through thiol coupling (SH coupling) or amine coupling (NH2 coupling). To this end, the antibody may be modified or substituted (or mutated) with cysteine ​​so that any amino acid residue of the heavy chain and / or light chain (e.g., within the constant region) has a functional group capable of thiol coupling or amine coupling, or the drug may be derivatized to have a functional group capable of thiol coupling or amine coupling. Alternatively, the antibody or antigen-binding fragment and the cytotoxic drug may be connected to each other through glycosylation of the asparagine (N) residue in the Fc of the antibody or antigen-binding fragment. That is, an antibody-drug conjugate according to one example of the present invention may be conjugated via the N-glycan of the anti-CD155 antibody or its antigen-binding fragment.

[0207] In one example, the antibody or its antigen-binding fragment and a cytotoxic drug (such as an anticancer agent) may be conjugated through immunochemical bonding. For example, an antibody-drug conjugate according to one example of the present invention comprises F of the anti-CD155 antibody or its antigen-binding fragment. CA secondary antibody that recognizes the site and has a drug (e.g., MMAE) attached to it can be prepared by reacting the anti-CD155 antibody or its antigen-binding fragment with the secondary antibody. Accordingly, the antibody-drug conjugate formed through the immunochemical bonding may have the form of the anti-CD155 antibody or its antigen-binding fragment-secondary antibody-drug.

[0208] In the present invention, the term "drug-to-antibody ratio" (DAR) refers to the average number of therapeutic moieties, e.g., drugs, bound to the conjugate according to the present invention. The drug-to-antibody ratio may be measured by a liquid chromatography-mass spectrometry method, etc. The drug-to-antibody ratio of the conjugate according to the present invention may be 1 to 10, specifically 2 to 8, but is not limited thereto and may be appropriately adjusted depending on the type and characteristics of the drug or linker used.

[0209] In this specification, “Antibody-Drug Conjugate” (ADC) is a conjugate for delivering a drug to a cell to which the antibody can specifically bind, and, for example, can selectively deliver a drug to a cell expressing CD155 by conjugating the drug with the antibody of the present invention or its antigen-binding fragment that can specifically bind to CD155.

[0210] In the present invention, the conjugation between the antibody and the drug may be a direct chemical bond, or it may be an indirect bond by a linker or a secondary antibody bound to the antibody, but is not limited thereto.

[0211] The above antibody-drug conjugate may include a linker, and the linker refers to a compound that covalently binds an activator to a ligand. For example, the linker may be cleavable, non-cleavable, hydrophilic, or hydrophobic, but is not limited thereto. For example, the linker sequence may be attached to the Fc sequence of the antibody or its antigen-binding fragment according to the present invention, and the linker sequence may be linked with a drug to be prepared and used in the form of an antibody-linker-drug.

[0212]

[0213] medicinal use

[0214] Another example provides a pharmaceutical composition comprising: the anti-CD155 antibody or its antigen-binding fragment; a polynucleotide encoding the antibody or its antigen-binding fragment; a recombinant vector comprising the polynucleotide; a recombinant cell comprising the polynucleotide or the recombinant vector; and / or the conjugate. In one example, the antibody or its antigen-binding fragment, etc., may be included as an active ingredient in the pharmaceutical composition. In one example, the pharmaceutical composition may further comprise a pharmaceutically acceptable excipient. In one example, the pharmaceutical composition may have anticancer activity. That is, in one example, the pharmaceutical composition may be a pharmaceutical composition for the prevention, improvement, and / or treatment of cancer.

[0215] Another example provides a pharmaceutical composition for the prevention, improvement, and / or treatment of cancer comprising the above anti-CD155 antibody or its antigen-binding fragment; the above polynucleotide; the above recombinant vector; the above recombinant cell; and / or the above conjugate. In one example, the antibody or its antigen-binding fragment, etc., may be included as an active ingredient in the pharmaceutical composition. In one example, the cancer may be a cancer expressing CD155.

[0216] Another example provides a method for the prevention, improvement, and / or treatment of cancer, comprising the step of administering a pharmaceutically effective amount of the above-mentioned anti-CD155 antibody or its antigen-binding fragment; the above-mentioned polynucleotide; the above-mentioned recombinant vector; the above-mentioned recombinant cell; and / or the above-mentioned conjugate to a subject (individual, subject) who requires prevention, improvement, and / or treatment of cancer. The method may further comprise, prior to the step of administering, a step of identifying (diagnosing, determining) the subject who requires prevention, improvement, and / or treatment of cancer.

[0217] Other examples provide the use of the above anti-CD155 antibody or its antigen-binding fragment; the above polynucleotide; the above recombinant vector; the above recombinant cell; and / or the above conjugate for the prevention, improvement, and / or treatment of cancer.

[0218] Another example provides a use for the preparation of a pharmaceutical composition (drug) for the prevention, improvement, and / or treatment of cancer of the above anti-CD155 antibody or its antigen-binding fragment; the above polynucleotide; the above recombinant vector; the above recombinant cell; and / or the above conjugate.

[0219] The conjugate included in the pharmaceutical composition described above may include a cytotoxic drug (e.g., an anticancer agent). In one example, the conjugate may be an antibody-drug conjugate.

[0220] The above cancer may be a solid tumor or a blood cancer, and may be, for example, a cancer having the characteristic of expressing CD155 (e.g., expression (or overexpression) on the cell surface), but is not limited thereto. For example, the above cancers include, but are not limited to, breast cancer, lung cancer, prostate cancer, ovarian cancer, brain cancer, liver cancer, colorectal cancer, colon cancer, colorectal cancer, rectal cancer, cervical cancer, endometrial cancer, uterine cancer, kidney cancer, nephroblastoma, skin cancer, oral squamous cell carcinoma, squamous cell carcinoma, epidermal carcinoma, nasopharyngeal cancer, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, lymphangial cancer (e.g., Hodgkin lymphoma or non-Hodgkin lymphoma), gastric cancer, pancreatic cancer, testicular cancer, thyroid cancer, thyrofollicular carcinoma, hepatocellular carcinoma, oral cancer, renal cell carcinoma, adrenal cancer, melanoma, myeloma, multiple myeloma, mesothelioma, osteosarcoma, myelodysplastic syndrome, tumors of mesenchymal origin, soft tissue sarcoma, liposarcoma, gastrointestinal stromal sarcoma, malignant peripheral nerve sheath tumor (MPNST), Ewing sarcoma, leiomyosarcoma, mesenchymal Examples may include chondrosarcoma, lymphosarcoma, fibrosarcoma, rhabdomyosarcoma, teratoma, neuroblastoma, medulloblastoma, glioma, benign tumors of the skin, leukemia, Burkitt lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, etc. Lung cancer may be, for example, small cell lung carcinoma (SCLC) or non-small cell lung carcinoma (NSCLC). Leukemia may be, for example, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), or chronic lymphoblastic leukemia (CLL). The cancer may be primary or metastatic cancer.

[0221] In one example, the above cancer may be T-cell cancer, pancreatic cancer, colorectal cancer, stomach cancer, liver cancer, lung cancer and / or prostate cancer.

[0222] The above cancer may be a cancer that overexpresses CD155 (e.g., a cancer with a high expression level of CD155 compared to normal cells or normal tissues).

[0223] The above anticancer activity or preventive and / or therapeutic effect of cancer may refer to any anticancer and / or antitumor action that eliminates (kills) cancer cells, inhibits the occurrence and / or growth of cancer cells, suppresses the worsening of cancer due to migration, invasion, metastasis, etc., alleviates or improves symptoms, and / or partially or completely eliminates cancer.

[0224] The target of administration of the anti-CD155 antibody or its antigen-binding fragment, the polynucleotide, the recombinant vector, the recombinant cell, the conjugate, and / or the pharmaceutical composition comprising the same provided in this specification may be any animal or cell, for example, an animal selected from mammals including humans, primates such as monkeys, rats, mice, etc., or cells, tissues, body fluids (e.g., serum) derived from (isolated from) said animals, or cultures thereof, for example, a human or cells, tissues, body fluids (e.g., serum) isolated from a human.

[0225] The above pharmaceutical composition may further comprise, in addition to the anti-CD155 antibody or its antigen-binding fragment as an active ingredient, the polynucleotide, the recombinant vector, the recombinant cell, and the conjugate, a pharmaceutically acceptable excipient and / or carrier, and one or more of the excipients and / or carriers commonly used in the formulation of protein drugs may be appropriately selected and used by a person skilled in the art.

[0226] The content of the active ingredient, which is the anti-CD155 antibody or its antigen-binding fragment, polynucleotide, recombinant vector, recombinant cell, or conjugate in the above pharmaceutical composition, can be varied depending on factors such as the formulation method, mode of administration, patient's age, body weight, sex, pathological condition, food, time of administration, interval of administration, route of administration, excretion rate, and response responsiveness.

[0227] In the above pharmaceutical composition or method, the pharmaceutically effective amount of the anti-CD155 antibody or its antigen-binding fragment, the polynucleotide, the recombinant vector, the recombinant cell, and / or the conjugate may be provided together with one or more additives selected from the group consisting of pharmaceutically acceptable carriers, diluents, and excipients.

[0228] The above-mentioned pharmaceutically acceptable carrier is one that is commonly used in the formulation of antibodies and may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc., but is not limited thereto. In addition to the above components, the above-mentioned pharmaceutical composition may further include one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc., which are commonly used in the manufacture of pharmaceutical compositions.

[0229] The above anti-CD155 antibody, its antigen-binding fragment, the above polynucleotide, the above recombinant vector, the above recombinant cell, the above conjugate, and / or pharmaceutical composition may be administered orally or parenterally. In the case of parenteral administration, it may be administered via intravenous infusion, subcutaneous infusion, intramuscular infusion, intraperitoneal infusion, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration. When administered orally, since proteins or peptides are digested, the oral composition must be formulated to coat the active agent or protect it from degradation in the stomach. Additionally, the composition may be administered by any device capable of delivering the active substance to target cells.

[0230] Appropriate dosages of the above anti-CD155 antibody, its antigen-binding fragment, the polynucleotide, the recombinant vector, the recombinant cell, the conjugate, and / or pharmaceutical composition may be prescribed in various ways depending on factors such as the method of formulation, the mode of administration, the patient's age, body weight, sex, pathological condition, food, time of administration, route of administration, excretion rate, and response responsiveness. For example, the daily dosage of the above anti-CD155 antibody, its antigen-binding fragment, conjugate, and / or pharmaceutical composition may be in the range of 0.001 to 1000 mg / kg, specifically 0.01 to 100 mg / kg, more specifically 0.1 to 50 mg / kg, but is not limited thereto. The daily dosage may be formulated as a single preparation in the form of a unit dose, formulated in appropriately divided quantities, or prepared by being contained in a multi-dose container. The term "pharmaceutical effective dose" refers to an amount of the above-mentioned active ingredient capable of producing the desired effect, namely, the effect of preventing and / or treating cancer, and may be prescribed in various ways depending on factors such as the formulation method, mode of administration, patient's age, weight, sex, pathological condition, food, time of administration, route of administration, excretion rate, and response responsiveness.

[0231] The above pharmaceutical composition may be prepared in a unit dose form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier and / or excipient, according to a method that can be easily carried out by a person skilled in the art. In this case, the formulation may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0232] In addition, the above pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

[0233] Meanwhile, the above pharmaceutical composition may include an antibody or an antigen-binding fragment, in which case the pharmaceutical composition may be formulated into an immunoliposome. The liposome containing the antibody may be prepared according to methods widely known in the art. The immunoliposome may be prepared by reverse-phase evaporation as a lipid composition comprising phosphatidylcholine, cholesterol, and polyethylene glycol-derived phosphatidylethanolamine. For example, the Fab' fragment of the antibody may be conjugated to the liposome via a disulfide-exchange reaction. A chemotherapeutic agent such as doxorubicin may additionally be included within the liposome.

[0234] The patient to whom the above pharmaceutical composition is administered or the patient to whom the above preventive and / or therapeutic method is administered may be a mammal, such as humans, primates such as monkeys, or rodents such as rats and mice, but is not limited thereto, and may be a cancer patient who has developed resistance to existing anticancer agents, such as antagonists for the above target cell membrane protein (e.g., CD155).

[0235]

[0236] The present application provides a novel anti-CD155 antibody or an antigen-binding fragment thereof; a conjugate of said antibody or antigen-binding fragment and a drug; and a use thereof, thereby enabling a superior anticancer effect.

[0237]

[0238] Figure 1 is a schematic diagram showing receptors expressed in immune cells and tumor cells.

[0239] Figure 2 shows the results of confirming the degree of binding of the selected clones by performing fluorescence staining on the CD155-expressing CHO-K1 cell line transformed in Examples 1-2 and the SNU-601 cell line known as a CD155-expressing positive gastric cancer cell line and analyzing them with a flow cytometer.

[0240] Figure 3 is a result showing that the mouse anti-CD155 antibody prepared according to one example of the present invention showed a negative reaction to granulocytes, lymphocytes, monocytes, and red blood cells (RBCs) in peripheral blood.

[0241] Figure 4 is the result of measuring the protein size of an antigen recognized by a mouse anti-CD155 antibody prepared according to one example of the present invention.

[0242] Figure 5 is the result of comparing the affinity of a mouse anti-CD155 antibody and a chimeric anti-CD155 antibody prepared according to one example of the present invention in the SNU-601 cell line.

[0243] Figure 6 is a result showing that the chimeric anti-CD155 antibody prepared according to one example of the present invention exhibits similar reactivity to CD155 domain full-hFc, CD155 domain mutant (CG)-hFc, and CD155 domain mutant (EG)-hFc, indicating that the epitope of the antibody recognizes the CD155 domain 1 C-term.

[0244] Figure 7 shows the results of measuring the affinity of humanized anti-CD155 antibodies to an antigen prepared according to one example of the present invention.

[0245] Figure 8 shows the results of confirming the antigen binding of chimeric and humanized anti-CD155 antibodies prepared according to an example of the present invention using an ELISA analysis method.

[0246] Figure 9 shows the results of confirming the antigen binding of chimeric and humanized anti-CD155 antibodies prepared according to an example of the present invention using the FACS analysis method.

[0247] Figure 10 is the result of confirming cell surface binding affinity in SNU-601 cell lines by applying a concentration gradient of chimeric and humanized anti-CD155 antibodies prepared according to one example of the present invention.

[0248] Figure 11 is a result confirming that a mouse anti-CD155 antibody prepared according to one example of the present invention inhibits TIGIT / CD155 binding.

[0249] Figure 12 is a result confirming that a humanized anti-CD155 antibody prepared according to an example of the present invention inhibits TIGIT / CD155 binding.

[0250] Figure 13 is a result confirming that a mouse anti-CD155 antibody prepared according to one example of the present invention inhibits CD226 / CD155 binding.

[0251] Figure 14 is a result confirming that a humanized anti-CD155 antibody prepared according to one example of the present invention inhibits CD226 / CD155 binding.

[0252] Figure 15 is a result confirming that a humanized anti-CD155 antibody prepared according to one example of the present invention inhibits CD96 / CD155 binding.

[0253] Figure 16 is a result showing that a humanized anti-CD155 antibody prepared according to one example of the present invention has cross-reactivity with human CD155 and monkey CD155, but does not cross-reactivity with mouse CD155.

[0254] Figure 17 is a result confirming that a humanized anti-CD155 antibody prepared according to one example of the present invention can specifically bind to the surface of tumor cells and induce the internalization of the antibody into the cell.

[0255] Figure 18 is a result showing that the chimeric anti-CD155 antibody and the humanized anti-CD155 antibody prepared according to one example of the present invention showed a negative reaction to granulocytes, lymphocytes, monocytes, and red blood cells of peripheral blood.

[0256] Figure 19 is a result showing that the cell death effect was significantly increased when an antibody-drug conjugate comprising a humanized anti-CD155 antibody and a drug (MMAE or Deruxtecan) prepared according to one example of the present invention was utilized.

[0257] FIG. 20 shows the results of observing that an antibody-drug conjugate comprising a humanized anti-CD155 antibody and a drug (MMAE) prepared according to an example of the present invention inhibited tumor growth in a gastric cancer mouse model (PBS, PBS administration group; 5D1-MMAE_3 mg / kg_single, 5D1-MMAE conjugate (3 mg / kg) single administration group; 5D1-MMAE_3 mg / kg, 5D1-MMAE conjugate (3 mg / kg) triple administration group; 5D1-MMAE_0.3 mg / kg, 5D1-MMAE conjugate (0.3 mg / kg) triple administration group).

[0258] FIG. 21 shows the results of observing that an antibody-drug conjugate comprising a humanized anti-CD155 antibody and a drug (deruxtecan; Dxd) prepared according to an example of the present invention inhibited tumor growth in a breast cancer mouse model (Vehicle; PBS administration group; Hu IgG-Dxd, antibody subtype-Dxd conjugate administration group; 5D1-Dxd_single, 5D1-Dxd conjugate (5 mg / kg) single administration group; 5D1-Dxd, 5D1-Dxd conjugate (5 mg / kg) triple administration group).

[0259] The present disclosure is to be explained more specifically below by the following examples. However, these are merely illustrative of the present disclosure, and the scope of the present disclosure is not limited by these examples.

[0260]

[0261] Example 1. Preparation and Analysis of 5D1 Antibody

[0262] To develop a new antibody specific to CD155, the following experiments were conducted, and the developed antibody was named "5D1".

[0263]

[0264] Example 1-1. Method for designing and selecting a target site for the production of 5D1 antibodies.

[0265] To develop a monoclonal antibody that specifically recognizes CD155, the monoclonal antibody against hCD155-Fc (Sino Biological, Cat #. 10109H02H) was established by immunizing the cells and then using a cell fusion method. Subsequently, during the hybridoma screening process, antibodies that were positive for the recombinant antigen hCD155-Fc, negative for human Fc, and positive for the gastric cancer cell line SNU-601 (KCLB, Cat #. 00601) were selected.

[0266]

[0267] Example 1-2. Establishment of a transformed cell line for screening

[0268] Cell lines expressing recombinant antigens were developed by transforming CHO-K1 cells (ATCC, CRL-9618) with commercially available TIGIT DNA (Sino Biological, Cat #. HG10917-UT) and CD155 DNA (Sino Biological, Cat #. HG10109-UT), respectively. The detailed experimental methods are as follows.

[0269] First, transfer 1 x 10⁶ CHO-K1 cells to a 6-well plate the day before transformation. 5Cells were inoculated at a concentration of cells / well and 3 ml of DMEM medium (Dulbecco's modified Eagle's medium, Biowest, USA) containing 10% Fetal bovine serum (ATLAS, USA) was added. The cells were cultured for 18 hours at 37°C under 5% CO2 conditions. The prepared DNA was then transfected into CHO-K1 cells using Lipofectamine (Invitrogen, USA). Three days after transfection, cells were harvested, and CD155 expression was confirmed by FACS using a control antibody. To establish a stable cell line, a screening process was conducted using 100 μg / ml Hygromycin (Gibco, USA). After the screening process, a limiting dilution was performed to secure a single colony and establish the final cell line.

[0270]

[0271] Examples 1-3. Screening of antibodies for hybridoma preparation

[0272] To develop CD155-specific antibodies, 100 μl of hCD155-Fc (Sino Biological, Cat #. 10109H02H) and Addavax (InvivoGen, Cat #. vac-adx-10) were mixed and 1 x 10⁶ were administered per 6-week-old female Balb / c mouse. 7 Immunization was performed by injecting the number of mice into the intraperitoneal cavity (IP) three times at 3-week intervals.

[0273] As described above, the spleen of an immunized mouse was resected to obtain a single-cell suspension, which was washed twice with RPMI1640. Then, 0.4% trypan blue (Sigma) was mixed in a 1:1 (v / v) ratio, and the number of cells was counted using the trypan blue staining method, which measures unstained cells under a microscope. X63 mouse myeloma cell line (ATCC, CRL-1580) was used as the cell fusion partner cell, and the number of cells was counted after washing in the same manner as the spleen cells.

[0274] The above myeloma cells and splenocytes were mixed, and then PEG (polyethylene glycol) 1500 was added to fuse the cells, after which the cells were centrifuged. The fused hybridoma cells were suspended in RPMI (20% FBS, hypoxanthine-aminopterin-thymidine) containing 1xHAT, 150 μl was dispensed into a 96-well plate, and cultured in a 37°C 5% CO2 incubator. After the above fusion, a screening process was carried out in a medium containing HAT for a certain period, and when wells forming colonies were observed, the medium was replaced with HT medium and cultured in a 37 ℃ 5% CO2 incubator for 48 hours. Then, the reactivity to the same recombinant proteins used for immunization, hCD155-Fc (Sino Biological, Cat #. 10109H02H), human Fc (Jackson immunoresearch, Cat #. 109-035-098), and Nectin-2 (Sino Biological, Cat #. 10005-HCCH), was observed using the ELISA method. For the ELISA, 100 ng of recombinant protein hCD155-Fc (Sino Biological, Cat #. 10109H02H) was added to a Maxisorp ELISA plate per well and incubated at 37°C for one hour to coat the antigen, and then 200 μl of 1x blocking solution (T&I, Cat #. BEB-1100) was added per well and incubated at 37°C for one hour to block. To the prepared plate, a CD155-specific antibody, a control antibody 5B9 (WO 2017-149538 A1 (Publication Date: Sep. 8, 2017)), known as an antibody against CD155, and 100 μl of PBS were added respectively, incubated at 37°C for one hour, and washed with PBS-T to remove unbound antibodies.Subsequently, diluted Goat anti-Mouse IgG-HRP (Jackson ImmunoResearch, USA) was added and reacted for 30 minutes. After washing with PBST, 50 μl of TMB solution was added to each well and reacted for 10 minutes. Then, 50 μl of sulfuric acid was added to stop the reaction, and the absorbance was measured at 450 nm. The CD155 antibody used as a positive control was produced as a mouse recombinant antibody by adapting the sequence of the 5B9 antibody disclosed in International Publication WO 2017-149538 A1 (Publication Date: Sep. 8, 2017). The measured absorbance results are shown in Table 4 below.

[0275]

[0276] In the above method, clones that are positive for CD155-hFc and negative for CD112 (Nectin-2), a common ligand for human Fc and DNAM-1, were selected. The selected clones were then fluorescently stained as follows on the CD155-expressing CHO-K1 cell line transformed in Examples 1-2 and the SNU-601 cell line, known as a CD155-expressing gastric cancer cell line, respectively, and analyzed using a flow cytometer to confirm the degree of binding. Similarly, the CD155 antibody used as a positive control was produced as a mouse recombinant antibody by applying the sequence of the above 5B9 antibody.

[0277] Specifically, 100 μl of hybridoma culture supernatant was added to each prepared cell and incubated at 4°C for 30 minutes. Then, 3 ml of PBS was added and the cells were centrifuged at 1700 rpm for 3 minutes to wash away unbound antibodies. To identify bound antibodies, a secondary antibody, goat anti-Mouse Ig-FITC (Jackson ImmunoResearch, USA), was diluted 200-fold and added. The cells were incubated at 4°C for 15 minutes, washed with 3 ml of PBS in the same manner as above, and then measured using a flow cytometer. The results of the flow cytometry analysis are shown in Figure 2.

[0278] As shown in Table 4 and Figure 2 above, in the above method, an antibody that is positive for hCD155-Fc and CD155 / CHO-K1 and negative for human Fc and Nectin-2 (hereinafter referred to as "5D1 mouse antibody") was selected, and finally, 5D1 hybridoma cells that produce a single colony of 5D1 mouse antibody through limiting dilution were obtained.

[0279]

[0280] Examples 1-4. Analysis of antibody expression in various cell lines

[0281] (1) Check the degree of binding to peripheral blood

[0282] The degree of binding to peripheral blood and red blood cells was determined using Whole Blood (Zenbio, USA).

[0283] Specifically, to determine the degree of binding to peripheral blood, an equal amount of RBC lysis buffer (Invirtogen, Cat #. 00-4300-54) was added to peripheral blood and left at room temperature for 10 minutes, after which peripheral blood cells were separated by centrifugation at 1700 rpm for 5 minutes. For red blood cells, only the RBC cells located at the bottom of the whole blood sample were collected and used after centrifugation at 1700 rpm for 5 minutes.

[0284] 100 μl of hybridoma supernatant was added to each of the prepared peripheral blood cells and RBCs and incubated at 4°C for 30 minutes. Then, 3 ml of PBS was added and the cells were centrifuged at 1700 rpm for 3 minutes to wash away unbound antibodies. To identify bound antibodies, a secondary antibody, goat anti-Mouse Ig-FITC (Jackson ImmunoResearch, USA) or goat anti-Human Ig-FITC (Jackson ImmunoResearch, USA), was diluted 200-fold and added. After incubating at 4°C for 15 minutes, the cells were washed with 3 ml of PBS in the same manner as above, and the results were measured using a flow cytometer and recorded (Fig. 3).

[0285] Figure 3 shows the results of the reactivity test of 5D1 mouse antibodies with peripheral blood and RBCs. As shown in the figure above, 5D1 mouse antibodies showed a negative reaction to granulocytes, lymphocytes, monocytes, and red blood cells (RBCs) in peripheral blood.

[0286]

[0287] (2) Antibody expression in various cell lines

[0288] The binding of 5D1 mouse antibodies to various cancer cell lines obtained from KCLB (Korean Cell Line Bank) and ATCC (American Type Culture Collection) was confirmed using flow cytometry.

[0289] Specifically, cancer cell lines purchased and used were SNU-601 (KCLB #. 00601), Huh-7 (KCLB #. 60104), HepG2 (KCLB #. 88065), NCI-H460 (KCLB #. 30177), and PC-3 (KCLB #. 21435) from KCLB (Korean Cell Line Bank), and H9 (ATCC #. HTB-176), Jurkat (ATCC #. TIB-152), Capan-1 (ATCC #. HTB-79), Capan-2 (ATCC #. HTB-80), DLD-1 (ATCC #. CCL-221), AGS (ATCC #. CRL-1739), and Hep 3B2. 1-7 (ATCC #. HB-8064) from ATCC (American Type Culture Collection).

[0290] Cancer cell lines were cultured in RPMI1640 medium supplemented with 10% heat-inactivated FBS or DMEM medium according to the culture media and methods presented in the KCLB (Korean Cell Line Bank) and ATCC (American Type Culture Collection), using an incubator at 37°C under 5% CO2 conditions. The 5D1 mouse antibody was added to each of the cultured cancer cells and incubated at 4°C for 30 minutes, followed by washing with PBS. Then, FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, USA) was added and incubated at 4°C for 15 minutes. After washing again with PBS, the reactivity of the 5D1 mouse antibody in various solid tumor and hematological cancer cell lines was confirmed by analysis using a FACS caliber (Becton Dickinson, USA) (Table 5).

[0291] Analysis of 5D1 Mouse Antibody Expression in Various Cell Lines Origin Cell line 5D1PBMC lymphocyte-monocyte-granulocyte-T cell H9++PancreasCapan-1++Capan-2++ColonDLD-1++StomachAGS+++SNU-601+++LiverHep 3B2. 1-7+Huh-7++HepG2+++LungNCI-H460++ProstatePC-3+++

[0292] The proportion of 5D1 positive cells was calculated through FACS analysis.

[0293] -: Positive cells less than 20%,

[0294] +: 20-30%,

[0295] ++: 40-70%,

[0296] +++: 60-100%.

[0297] As described in Table 5 above, it was confirmed that the 5D1 mouse antibody binds to a wide variety of solid tumors, particularly pancreatic cancer, liver cancer, gastric cancer, and prostate cancer. This indicates that the 5D1 mouse antibody can be used as a treatment for solid tumors, as it shows a positive reaction in various solid tumor cells and a negative reaction in normal blood cells.

[0298]

[0299] Examples 1-5. Analysis of Antigens and Epitopes of 5D1 Antibodies

[0300] Western blot was performed to determine the protein size of the antigen recognized by the 5D1 mouse antibody and to examine epitope characteristics.

[0301] Specifically, HepG2 liver cancer cell lines with high antigen expression for the 5D1 mouse antibody were lysed in a cell lysis solution (RIPA cell lysis buffer, Thermo Fisher Scientific) containing a protease inhibitor cocktail (Sigma, Cat. #. 1136170001), and then loaded under reducing (R) and non-reducing (NR) conditions using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis on a polyacrylamide gel, the cells were transferred to an Immobilon-P membrane (Millipore Corporation, Billerica, MA, USA). Subsequently, the purified 5D1 mouse antibody was applied to the membrane, and the bands were visualized by color development using an ECL system (GE Healthcare, Buckinghamshire, UK) (Fig. 4).

[0302] As shown in Figure 4, a band of approximately 70 kDa was detected under non-reduced and reduced conditions, indicating that the 5D1 mouse antibody recognizes linear epitopes.

[0303]

[0304] Examples 1-6. Determination of the isotype of the 5D1 antibody

[0305] To determine the isotype of the 5D1 mouse antibody prepared in Example 1 above, it was analyzed using a mouse immunoglobulin isotyping ELISA kit (BD Biosciences, USA). As a result, it was confirmed that the 5D1 antibody possesses a mouse IgG1 / lambda light chain. (Result not specified).

[0306]

[0307] Examples 1-7. 5D1 antibody variable region sequence analysis

[0308] The 5D1 mouse antibody gene was cloned using the Mouse Ig-Primer Set (Merck Millipore, Cat. #.69831). PCR was performed using the Mouse Ig-Primer Set on RNA isolated from 5D1 hybridoma cells producing 5D1 mouse antibodies. After insertion into the pGEM-T vector (Promega, Cat. #. A3600), the DNA base sequence was confirmed via sequencing, and the 5D1 mouse antibody gene was identified via the IMGT site (www.imgt.org). Table 6 shows the sequences in the heavy and light chain variable regions of the analyzed 5D1 mouse antibody. In the amino acid sequences listed below, the parts underlined in bold indicate the complementarity determining region. The sequence positions in Table 6 below are based on the Kabat numbering system.

[0309] Determination of Heavy and Light Chain Complementarity of 5D1 Mouse Antibody Site Nomenclature Sequence Sequence Number Sequence Position Length 5D1 VH-FR1EVQLQESGAELVKPGASVKLSCKTSGFTFS181-30305D1 VH-CDR1SSYIS131-3555D1 VH-FR2WLKQKPGQSLEWIA2236-49145D1 VH-CDR2WIYAGTGDTNYNQNFTG250-66175D1 VH-FR3KAQLTVDTSSSTAYMQFSSLTTEDSAIYYCAR2567-98325D1 VH-CDR3HDYGNNYDWYFDV399-111135D1 VH-FR4WGAGTTVTVSS29112-122115D1 Ab VHEVQLQESGAELVKPGASVKLSCKTSGFTFSSSYISWLKQKPGQSLEWIAWIYAGTGDTNYNQNFTGKAQLTVDTSSSTAYMQFSSLTTEDSAIYYCARHDYGNNYDWYFDVWGAGTTVTVSS71-1221225D1 VL-FR1QAVVTQESALTTSPGETVTLTC321-22225D1 VL-CDR1RSSTGAVTTSNYAN423-36145D1 VL-FR2WVQEKPDHLFTGLIG3437-51155D1 VL-CDR2GTNNRAP552-5875D1 VL-FR3GVPARFSGSLIGDKAALTITGAQTEDEAIYFC3959-90325D1 VL-CDR3ALWYSNHWV691-9995D1 VL-FR4FGGGTKLTVL45100-109105D1 Ab VLQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVL121-109109

[0310] Example 2. Development of 5D1 Chimeric Antibody

[0311] Example 2-1. Preparation of 5D1 Chimeric Antibody

[0312] Based on the amino acid sequence of the 5D1 mouse antibody obtained in Examples 1-7, an anti-CD155 chimeric antibody, the 5D1 chimeric antibody, was constructed. Specifically, for the expression of the 5D1 chimeric antibody, a plasmid for heavy chain expression and a plasmid for light chain expression were constructed, respectively. In order to express the variable region coding cDNA and constant region coding cDNA of each antibody as a continuous amino acid sequence without the insertion of additional amino acids, gene fragments were synthesized (Biointron) by linking the cloned variable region coding sequence with the known human IgGl constant region (heavy chain) and kappa constant region (light chain) coding sequences, and then ligated into the pcDNA3.4 vector to construct the complete antibody expression plasmids. The above-described pcDNA3.4-anti-CD155 heavy chain expression plasmid and pcDNA3.4-anti-CD155 light chain expression plasmid were inserted into ExpiCHO-S cells using the ExpiCHO Expression system (Gibco, USA), which is derived from CHO cells, and the supernatant was harvested and purified after 5 days of culture.

[0313] Figure 5 shows the results of comparing the affinity in the CD155 antigen-positive SNU-601 cell line after cloning the antibody gene from the 5D1 mouse antibody and expressing it as a 5D1 chimeric antibody. As can be seen in Figure 5, the 5D1 mouse antibody and the 5D1 chimeric antibody showed similar affinities.

[0314]

[0315] Example 2-2. Domain Mapping Analysis by ELISA Binding Assay

[0316] To verify the domain recognized by the 5D1 antibody, recombinant antigens containing or not containing epitope sites were prepared as described in Table 7 below, and an immune response was performed against the 5D1 chimeric antibody of Example 2-1, and the results are shown in Figure 6.

[0317]

[0318] (1) Preparation and production of CD155 Domain-1 mutant recombinant gene

[0319] Recombinant CD155 mutant genes were obtained by inserting into pcDNA3.4 genes to which hFc was attached to the whole nucleotide sequence of the CD155-domain1 antigen (human CD155; see Uniprot ID P15151) and two CD155 domain1 mutant sequences obtained by cleaving the CD155-domain1 antigen differently. The sequences of the CD155 mutants prepared as described above are shown in Table 7 below.

[0320] CD155 Domain-1 mutant 재조합 단백질 서열name서열서열번호Cy155D1-Fc (Domain 1 fμll)VVQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLTWARHGESGSMAVFHQTQGPSYSESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVDIWLRVLAGGGGSGGGGSGGGGSEPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK111Cy155D2-Fc (domain 1 C-G)VTHVSQLTWARHGESGSMAVFHQTQGPSYSESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVDIWLRVLAGGGGSGGGGSGGGGSEPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK112Cy155D3-Fc (domain 1EG)LGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVDIWLRVLAGGGGSGGGGSGGGGSEPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK113

[0321] The pcDNA 3.4 vector containing the above CD155 domain full-hFc, CD155 domain mutant (CG)-hFc, and CD155 domain mutant (EG)-hFc was inserted into ExpiCHO-S cells using the ExpiCHO Expression system (Gibco, USA), and the supernatant was harvested and purified after culturing for 5 days.

[0322]

[0323] (2) Verification of the CD155 domain 1 binding site of the 5D1 antibody

[0324] To verify the epitope for CD155 domain 1 of the 5D1 antibody, 50 ng of Goat-anti-Human Fc specific IgG (Jackson Immuniresearch, USA) was added per well and reacted at 37°C for one hour to coat, and 200 μl of 1x blocking solution (T&I, Korea) was added per well and reacted at 37°C for 20 minutes to block. To the prepared plate, 200 ng each of the CD155 domain 1 full-hFc, CD155 domain mutant (CG)-hFc, and CD155 domain mutant (EG)-hFc prepared in Example 2-2-(1) were added per well and reacted at 37°C for one hour to coat with the capture antigen. Subsequently, 200 ng of the 5D1 chimeric antibody prepared in Example 2-1 and the control antibody (5B9, International Publication WO 2017-149538 A1 (Publication date: Sep. 8, 2017)) were added per well, reacted at 37°C for one hour, and washed with PBS to remove unbound antibodies. As a secondary antibody, Goat-anti-Human (Fab')2 specific HRP (Jackson Immuniresearch, USA) was diluted in 1x blocking solution and added, reacted at 37°C for 30 minutes, then washed with PBS. Afterward, 50 μl of TMB solution was added per well and reacted for 5 minutes, then 50 μl of sulfuric acid was added to stop the reaction, and the absorbance was measured at 450 nm.

[0325] As described in Figure 6, the 5D1 chimeric antibody showed similar reactivity to CD155 domain full-hFc, CD155 domain mutant (CG)-hFc, and CD155 domain mutant (EG)-hFc, which indicates that the epitope of this antibody recognizes the C-terminal region (C-term) of CD155 domain 1.

[0326]

[0327] Example 3. Development of a humanized antibody of 5D1 antibody

[0328] To reduce the immunogenicity caused by the human anti-mouse antibody (HAMA) reaction upon human administration of mouse-derived antibodies and to secure an antibody with high draggability, a humanized antibody was constructed by replacing the variable region and the remaining part of the 5D1 mouse antibody with human Fc. It was confirmed that the selected 5D1 humanized antibody had antigen specificity and affinity similar to the original 5D1 mouse antibody.

[0329]

[0330] Example 3-1. Selection of Recombinant Antibody Sequence by In Silico Humanization

[0331] The variable region, CDR, and framework sequences of the 5D1 mouse antibody are as shown in Table 6 above. Humanized 5D1 antibodies with equivalent or superior antigen binding affinity were selected by recombining the sequence region of the Framework region in silico based on the germline sequence encoding the human antibody gene, while maintaining the heavy and light chain amino acid sequences of the 5D1 mouse antibody as similar as possible. The human antibody germline gene used as the backbone for the humanized recombinant antibody sequence, which has the highest similarity to the heavy and light chain sequences of the 5D1 mouse antibody, is shown in Table 8 below.

[0332] Human Ab GermlineHeavy chainLight chainIGHV3-3*01IGLV7-46*02IGLV7-46*01

[0333] The amino acid sequences of the heavy chain variable region and the light chain variable region of the above-selected 5D1 humanized antibody are shown in Table 9 below. In the amino acid sequences described below, the parts underlined in bold indicate the complementarity determining region.

[0334] 중쇄 가변영역아미노산 서열서열번호VH1 (HU1186-hVH1)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSSYISWVRQAPGQRLEWMGWIYAGTGDTNYNQNFTGRVTITVDTSASTAYMELSSLRSEDTAVYYCARHDYGNNYDWYFDVWGQGTTVTVSS8VH2 (HU1186-hVH2-1)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSSYISWVRQAPGQRLEWMGWIYAGTGDTNYNQNFTGRVTITRDTSASTAYMELSSLRSEDTAVYYCARHDYGNNYDWYFDVWGRGTLVTVSS9VH3 (HU1186-hVH3)QVQLVQSGAEVVKPGASVKVSCKASGFTFSSSYISWVRQAPGQRLEWMGWIYAGTGDTNYNQNFTGRVTITVDTSASTAYMELSSLRSEDTAIYYCARHDYGNNYDWYFDVWGQGTTVTVSS10VH4 (HU1186-hVH4)QVQLVQSGAEVKKPGASVKVSCKASGFTFSSSYISWLKQKPGQRLEWMGWIYAGTGDTNYNQNFTGRVTITRDTSASTAYMELSSLRSEDTAVYYCARHDYGNNYDWYFDVWGQGTTVTVSS11경쇄 가변영역아미노산 서열서열번호VL1 (HU1186-hVL1)QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNNRAPWTPARFSGSLLGGKAALTLLGAQPEDEAEYYCALWYSNHWVFGGGTKLTVL13VL2 (HU1186-hVL2)QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNNRAPGTPARFSGSLLGDKAALTLLGAQPEDEAEYYCALWYSNHWVFGGGTKLTVL14VL3 (HU1186-hVL3)QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQLFRGLIGGTNNRAPGTPARFSGSLLGDKAALTLLGAQPEDEAEYFCALWYSNHWVFGGGTKLTVL15VL4(HU1186-hVL4)QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWFQQKPGHAPRGLIYGTNNRAPWTPARFSGSLLGDKAALTISGAQPEDEAEYFCALWYSNHWVFGGGTKLTVL16VL5 (HU1186-hVL5-1)QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWFQQKPGQAPRTLIYGTNNRAPWTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNHWVFGGGTKLTVL17

[0335] Example 3-2. Expression and production of 5D1 humanized antibody.

[0336] DNA sequences were synthesized by codon-optimizing the amino acid sequences corresponding to the expression genes of the four heavy chain types (including VH1 to VH4) and five light chain types (including VL1 to VL5) in Table 9 above. The synthesized DNA was then linked to the human Fc region gene (the heavy chain was the human IgG1 Fc gene, and the light chain was the human Ig lambda constant gene) via overlap PCR and cloned into the pcDNA3.4 vector included in the ExpiCHO-S Expression system kit (Gibco, USA). The light and heavy chain expression plasmids of the constructed 5D1 humanized antibodies were transfected into the ExpiCHO-S cell line using the ExpiCHO-S expression system kit (Invitrogen / Life technologies) to produce a total of 20 types of 5D1 humanized antibodies. The culture medium of the expressed 5D1 humanized antibody was purified using proteinA affinity chromatography, and the purity of the purified 5D1 humanized antibodies was analyzed by size-exclusion chromatography (SEC) and SDS-PAGE (Table 10).

[0337] Antibody (VH# / VL#)Expression yield(mg / L)Antibody concentration(mg / ml)Purity(SEC, 280nm)Purity(SDS-PAGE)# of back-mutation(H+L)VH1 / VL12061.4799.35%95%1+3VH1 / VL22231.35100%95%1+5VH1 / VL32491.51100%95%1+8VH1 / VL42191.3799.45%95%1+5VH1 / VL5199.51.2199. 52%95%1+0VH2 / VL12361.4399.67%95%0+3VH2 / VL22111.28100%95%0+5VH2 / VL3246 1.49100%95%0+8VH2 / VL42061.4299.78%95%0+5VH2 / VL5204.51.2499.16%95%0+0VH 3 / VL12051.4499.51%95%5+3VH3 / VL2204.51.41100%95%5+5VH3 / VL3218.51.56100 %95%5+8VH3 / VL4204.51.24100%95%5+5VH3 / VL5176.51.0799.55%95%5+0VH4 / VL118 01.2100%95%5+3VH4 / VL22241.6100%95%5+5VH4 / VL32431.52100%95%5+8VH4 / VL42321.4598.64%95%5+5VH4 / VL52061.4799.14%95%5+0mVH / mVL206.51.2599.40%95%* # of back-mutation: The number of back-mutations in the heavy and light chains is indicated in the form of #(heavy chain)+#(light chain).

[0338] Example 3-3. Antigen binding test of 5D1 humanized antibody using ViaCore analysis method

[0339] As shown in Table 10 above, the binding affinity of the purified 5D1 humanized antibodies to the CD155 antigen was tested. To confirm CD155 antigen binding and affinity, SPR was performed using the CD155 antigen (Sino Biological, Cat #10109-H08H). SPR was performed using Biacore 8K (Cytiva), and antibody immobilization was performed using a proteinA chip (Cytiva; Cat #. 29127556). The conditions for SPR use are as follows (Table 11).

[0340] namedtailsCapture ligand2 / 4 μg / ml antibody in running buffer,10 μl / min, capture about 295~596 RURunning bufferHBS-EP+ bufferAssociation / dissociation flow rate30 μl / min, association 120 s, dissociation 200 sRegeneration buffer10 mM pH 1.5 Gly-HClRegeneration flow rate30 μl / min, 30s

[0341] SPR was first performed by fixing the 21 types of antibodies of Table 10, which were purified, to a single antigen concentration. The affinity values ​​of two types of 5D1 humanized antibodies (VH3 / VL3, VH4 / VL3) among the antibodies are shown in Tables 12 and 13 below, and Figure 7.

[0342] Antibody (VH# / VL#)Analyteconcentration1:1 bindingKa (1 / Ms)Kd (1 / s)KD (M)mVH / mVL (chimeric Ab.)200 nM1.30E+056.87E-025.28E-07VH3 / VL3200 nM1.76E+058.77E-024.99E-07VH4 / VL3200 nM1.67E+055.97E-023.58E-07

[0343] Antibody (VH# / VL#)Analyte concentration1:1 bindingKa (1 / Ms)Kd (1 / s)KD (M)mVH / mVL (chimeric Ab.)6.25 ~ 200 nM(2-fold)9.07E+045.73E-026.32E-07VH3 / VL36.25 ~ 200 nM(2-fold)1.77E+058.50E-024.80E-07VH4 / VL36.25 ~ 200 nM(2-fold)1.72E+056.00E-023.49E-07

[0344] As can be seen in Tables 12 and 13 and Figure 7 above, the 5D1 humanized antibody (VH3 / VL3, VH4 / VL3) had a lower KD value compared to the control (5D1 chimeric antibody of Example 2), and thus had excellent antigen affinity.

[0345]

[0346] Example 3-4. Antigen binding test of 5D1 humanized antibody using ELISA method

[0347] The 5D1 humanized antibodies, whose antigen binding was confirmed via SPR, were reconfirmed by performing ELISA with the CD155 antigen (Sino Biological, Cat #. 10109-H08H). As a conventional ELISA method, the CD155 antigen was coated (100 ng / well), and the 5D1 humanized antibody and chimeric antibody were conjugated as primary antibodies by serially diluting them threefold starting from 2000 ng / ml. Goat anti-humanIg-HRP (Jackson ImmunoResearch Laboratories; Cat # 109-035-003) was used as the secondary antibody.

[0348]

[0349] The results of the above ELISA are shown in Table 14 and Figure 8 below. As can be seen in Table 14 and Figure 8, the 5D1 humanized antibody (VH3 / VL3, VH4 / VL3) showed high efficacy (binding power) with a lower EC50 value compared to the control (5D1 chimeric antibody of Example 2).

[0350] Antibody concentration (ng / ml)humanized 5D1VH3 / VL3humanized 5D1VH4 / VL3chimeric 5D120001.07631.41230.9654666.71.13321.36680.7598222.21.55581.40690.60774.10.48951.04210.2 40424.70.22220.60530.11798.20.11190.31180.08282.70.08250.15690.07240.90.070.09780.0675EC50 (ng / ml)80.220935.3159198.483

[0351] Examples 3-5. Antigen binding test of 5D1 humanized antibody using FACS analysis method

[0352] The cell surface binding affinity of the 5D1 humanized antibody was compared using CD155 antigen-positive cancer cells. After staining the cell surface with human stomach cancer cell lines AGS and SNU-601, the results were analyzed using a flow cytometer.

[0353] As shown in Fig. 9, both of the 5D1 humanized antibodies of the present invention (VH3 / VL3 and VH4 / VL3) effectively bound to the cell surface, and in particular, the 5D1 humanized antibody (VH4 / VL3) showed a higher cell surface binding affinity than the control (5D1 chimeric antibody of Example 2).

[0354] Using the SNU-601 cell line, cell surface binding of the antibodies was confirmed by applying a concentration gradient for each antibody. It was reconfirmed through FACS staining with a concentration gradient that the 5D1 humanized antibody (VH4 / VL3) showed higher cell surface binding affinity than the control (5D1 chimeric antibody of Example 2) (Fig. 10).

[0355]

[0356] Example 4. TIGIT / CD155 binding inhibition test of 5D1 antibody

[0357] To confirm the inhibitory effect of the 5D1 mouse antibody on the binding of CD155 molecules to TIGIT, a TIGIT-CD155 (PVR) blocking assay was performed. Specifically, 2 μg each of recombinant CD155-hFc (Sino Biological, Cat #10109-H02H) and the 5D1 mouse antibody were mixed and added to the TIGIT-expressing CHO cell line established in Examples 1-2 above, and reacted at 4°C for 30 minutes. Then, 3 ml of PBS was added and the mixture was centrifuged at 1700 rpm for 3 minutes to wash away unbound antigens and antibodies. Subsequently, the secondary antibody Goat Anti-Human IgG, Fcγ fragment specific FITC (Jackson ImmunoResearch Laboratories; Cat # 109-095-190) was diluted 200-fold and added, and the mixture was reacted at 4°C for 15 minutes. After washing with 3 ml of PBS in the same manner as above, the results were measured using a flow cytometer and recorded (Fig. 11; top).

[0358] As a result, it was confirmed that the degree of binding was significantly lower when the 5D1 mouse antibody was added (b) compared to the control group (a) in which TIGIT and CD155-hFc were bound.

[0359]

[0360] To confirm the above experiment, a TIGIT-CD155 blocking assay was performed using the SNU-601 gastric cancer cell line, which is known to express CD155. Specifically, 2 μg each of recombinant TIGIT-hFc (sinobiological; Cat #. 10917-H02H) and 5D1 mouse antibody were mixed and added to the SNU-601 cell line and reacted at 4°C for 30 minutes. Then, 3 ml of PBS was added and the mixture was centrifuged at 1700 rpm for 3 minutes to wash away unbound antigens and antibodies. Subsequently, the secondary antibody Goat Anti-Human IgG, Fcγ fragment specific FITC (Jackson ImmunoResearch Laboratories; Cat #. 109-095-190) was diluted 200-fold and added, and the mixture was reacted at 4°C for 15 minutes. After washing with 3 ml of PBS in the same manner as above, the results were measured using a flow cytometer and recorded (Fig. 11; bottom).

[0361] As a result, it was confirmed that the degree of binding was significantly lower when the 5D1 mouse antibody was added (b) compared to the control group (a) in which TIGIT-hFc and CD155 were bound. Based on these results, it was confirmed that the 5D1 antibody, which is specific to CD155, has the function of inhibiting TIGIT / CD155 binding with CD155, which is a ligand for TIGIT.

[0362]

[0363] The degree of inhibition of TIGIT / CD155 binding using the 5D1 humanized antibody (VH4 / VL3) prepared in Example 3 above was reconfirmed by ELISA using recombinant antigens. Specifically, 50 ng of TIGIT-HisTag (Sinobiological, Cat #. 10917-H08C) was added per well and reacted at 37°C for one hour to coat the plate, and 200 μl of 1x blocking solution (T&I, Korea) was added per well and reacted at 37°C for 20 minutes to block the plate. To the prepared plate, 200 ng each of the 5D1 humanized antibody (VH4 / VL3) and recombinant CD155-hFc (Sino Biological, Cat #. 10109-H02H) were added per well and reacted at 37°C for one hour. Goat-anti-Human (Fab')2 specific HRP (Jackson Immunoresearch, USA) was diluted in 1x blocking solution and added as a secondary antibody, and the reaction was carried out at 37°C for 30 minutes. For the CD155-hFc single well used as a positive control, Goat Anti-Human IgG, Fcγ fragment specific IgG (Jackson Immunoresearch Laboratories, USA) was used as the secondary antibody. After 30 minutes of adding the secondary antibody, the wells were washed with PBS, then 50 μl of TMB solution was added to each well and the reaction was carried out for 5 minutes. The reaction was then stopped by adding 50 μl of sulfuric acid, and the absorbance was measured at 450 nm. The results are shown in Figure 12.

[0364] As can be seen in Figure 12, it was confirmed that the 5D1 humanized antibody also has the function of inhibiting TIGIT / CD155 binding with CD155, which is a ligand for TIGIT.

[0365]

[0366] Example 5. CD226 / CD155 binding inhibition test of 5D1 antibody

[0367] To confirm the inhibitory effect of the 5D1 mouse antibody on the binding of CD155 molecules to CD226 (DNAM-1, DNAX Accessory Molecule-1), a CD226-CD155 (PVR) blocking assay was performed. Specifically, the CD226-CD155 blocking assay was conducted using the SNU-601 gastric cancer cell line, which is known to express CD155. 2 μg each of recombinant CD226-hFc (Sino Biological, Cat #. 10565-H02H) and the 5D1 mouse antibody were mixed and added to the SNU-601 cell line. After incubation at 4°C for 30 minutes, 3 ml of PBS was added, and the mixture was centrifuged at 1700 rpm for 3 minutes to wash away unbound antigens and antibodies. Afterwards, the secondary antibody Goat Anti-Human IgG, Fcγ fragment specific FITC (Jackson ImmunoResearch Laboratories, Cat #. 109-095-190) was diluted 200-fold and added, and the mixture was reacted at 4°C for 15 minutes. After washing with 3 ml of PBS in the same manner as above, the results were measured using a flow cytometer and recorded (Fig. 13).

[0368] As a result, it was confirmed that the degree of binding was slightly lower when the 5D1 mouse antibody was added (b) compared to the control group (a) in which CD226-hFc and CD155 were bound. As a result, it was confirmed that the CD155-specific 5D1 antibody has the function of inhibiting CD226 / CD155 binding with CD155, which is a ligand for CD226.

[0369]

[0370] The degree of CD226 / CD155 binding inhibition by the 5D1 humanized antibody (VH4 / VL3) prepared in Example 3 above was reconfirmed using ELISA with recombinant antigens. Specifically, 50 ng of CD226-HisTag (Sinobiological, Cat #. 10565-H08H) was added per well and reacted at 37°C for one hour to coat the plate, and 200 μl of 1x blocking solution (T&I, Korea) was added per well and reacted at 37°C for 20 minutes to block the plate. To the prepared plate, 200 ng each of the 5D1 humanized antibody (VH4 / VL3) and recombinant CD155-hFc (Sino Biological, Cat #. 10109-H02H) were added per well and reacted at 37°C for one hour. Goat-anti-Human (Fab')2 specific HRP (Jackson Immunoresearch, USA) was diluted in 1x blocking solution and added as a secondary antibody, and the mixture was reacted at 37°C for 30 minutes. For the CD155-hFc single well used as a positive control, Goat Anti-Human IgG, Fcγ fragment specific IgG (Jackson Immunoresearch Laboratories, USA) was used as the secondary antibody. After 30 minutes of adding the secondary antibody, the wells were washed with PBS, then 50 μl of TMB solution was added to each well and reacted for 5 minutes. The reaction was stopped by adding 50 μl of sulfuric acid, and the absorbance was measured at 450 nm and the results were recorded (Fig. 14).

[0371] As can be seen in Figure 14, it was confirmed that the 5D1 humanized antibody also has the function of inhibiting CD226 / CD155 binding with CD155, which is a ligand for CD226.

[0372]

[0373] Example 6. CD96 / CD155 binding inhibition test of 5D1 antibody

[0374] To confirm the inhibitory effect of the 5D1 humanized antibody (VH4 / VL3) on the binding of CD155 molecules to CD96 (TACTILE: T cell activation, increased late expression), a CD96-CD155 (PVR) blocking assay was performed. Specifically, 50 ng of CD96 HisTag (Acrobiosystems, Cat #. TAE-H52H0) was added per well and reacted at 37°C for one hour to coat the plate, and 200 μl of 1x blocking solution (T&I, Korea) was added per well and reacted at 37°C for 20 minutes to block the plate. To the prepared plate, 200 ng each of the 5D1 humanized antibody (VH4 / VL3) and recombinant CD155-hFc (Sino Biological, Cat #. 10109-H02H) were added and reacted at 37°C for one hour. Goat-anti-Human (Fab')2 specific HRP (Jackson Immunoresearch, USA) was diluted in 1x blocking solution and added as a secondary antibody, and the reaction was carried out at 37°C for 30 minutes. For the CD155-hFc single well used as a positive control, Goat Anti-Human IgG, Fcγ fragment specific IgG (Jackson Immunoresearch Laboratories, USA) was used as the secondary antibody. After 30 minutes of adding the secondary antibody, the wells were washed with PBS, then 50 μl of TMB solution was added to each well and the reaction was carried out for 5 minutes. The reaction was then stopped by adding 50 μl of sulfuric acid, and the absorbance was measured at 450 nm. The results are shown in Figure 15.

[0375] As a result, as shown in Figure 15, it was confirmed that the 5D1 antibody specific to CD155 has the function of inhibiting CD96 / CD155 binding with CD155, which is a ligand for CD96.

[0376]

[0377] Example 7. Confirmation of interspecies cross-reactivity of 5D1 antibody

[0378] To confirm the species cross-reactivity of the 5D1 humanized antibody (VH4 / VL3), binding to Human CD155 (Sino Biological, Cat #. 10109-H08H), mouse CD155 (Sino Biological, Cat #. 50259-M08H), and rhesus CD155 (Sino Biological, Cat #. 90005-C08H) antigens was confirmed by ELISA. Specifically, 100 μL of each of the three CD155 antigens, diluted to a concentration of 2 μg / mL in PBS, was added to each well of a 96-well plate and coated by overnight incubation at 4°C. Then, 200 μL of 1x blocking solution (T&I, Korea) was added to each well and reacted at 37°C for 20 minutes to block the antibodies. 5D1 humanized antibody (VH4 / VL3) was diluted to a concentration of 2 μg / mL, 100 μl was added per well, and the mixture was incubated at 37°C for 1 hour. After washing three times with PBST, Goat Anti-Human IgG, Fcγ fragment specific IgG (Jackson ImmunoResearch Laboratories, USA) was added as a secondary antibody, and the mixture was incubated at 37°C for 30 minutes. 50 μl of TMB solution was added per well, and color development was observed. The reaction was stopped with 1 N H2SO4, and the color intensity was checked at an absorbance of 450 nm. Cross-reactivity with human, rhesus, and mouse CD155 antigens was confirmed, and the results are shown in Figure 16.

[0379] Figure 16 shows the results of measuring the binding of the 5D1 humanized antibody (VH4 / VL3) to human, mouse, and rhesus CD155 antigens using ELISA to confirm species cross-reactivity. As a result, it was confirmed that the 5D1 humanized antibody (VH4 / VL3) has cross-reactivity with human CD155 and rhesus CD155, but does not cross-react with mouse CD155 antigen.

[0380]

[0381] Example 8. Confirmation of endocellular integration of 5D1 antibody

[0382] The endocellular uptake of CD155-specific 5D1 antibodies bound to CD155-positive cells was confirmed using a high-content screening (HCS) instrument (Molecular devices, ImageXpress® Micro Confocal system). Specifically, the SNU-601 cell line was suspended in growth medium (RPMI + 10% FBS) at a rate of 1 x 10⁶ 4Cells were plated in a 96-well plate at cells / well. After incubating cells at 37°C and 5% CO2 for 18 hours, the supernatant was carefully removed to prevent attached cells from detaching. 5D1 mouse antibody was diluted to 1 mg / ml, 100 μl was added to each well, and the mixture was incubated at 4°C for 30 minutes. Subsequently, 3 ml of PBS was added, and the mixture was centrifuged at 1700 rpm for 3 minutes to wash away unbound antigens and antibodies. Afterward, the secondary antibody, Goat Anti-Mouse IgG, Fcγ fragment specific-Alexa Fluor 488 (Jackson ImmunoResearch Laboratories; Cat #. 115-545-071), was diluted 200-fold and added. The mixture was incubated at 4°C for 15 minutes, followed by washing with 3 ml of PBS in the same manner as above. Finally, real-time images were measured using an HCS instrument at 10, 30, 60 minutes, and 1-24 hours.

[0383] Figure 17 shows the degree of internalization of the 5D1 mouse antibody measured at 30 minutes and 2 hours after attachment of the 5D1 mouse antibody. Consequently, as shown in the figure above, the 5D1 mouse antibody specifically bound to the surface of tumor cells and induced the internalization of the antibody into the cells.

[0384] The endocellular uptake of the humanized 5D1 antibody was confirmed using an internalization reagent (Acro Biosystems, Cat #.IGG-PZF2001). The humanized 5D1 antibody (VH4 / VL3) from Example 3 was diluted to 8 μg / ml, mixed with the working solution and internalization solution, and reacted at room temperature for 10 minutes to form a complex of the humanized 5D1 antibody and pHrodo-dye (humanized 5D1 antibody / pHrodo-dye complex). 2×10 SNU601 cells were used. 6After preparing the cells / ml, 25 μl of the humanized 5D1 antibody / pHrodo-dye complex was added and reacted for the time specified in Table 15 below. Subsequently, 100 μl of 4% paraformaldehyde / PBS was added to each sample for fixation, followed by analysis using a flow cytometer. The analysis was conducted utilizing the characteristic of pH-rodo dye to exhibit fluorescence under weakly acidic conditions, such as pH 6.0, similar to that of lysomes, and the results are shown in Table 15.

[0385] time (hr)% (+)mean fluorescence intensity0.10.252440.538909163.51,283278.51,671488.42,229887.22,24324812,369

[0386] As shown in the table above, it was confirmed that cells treated with the humanized 5D1 antibody / pHrodo-dye complex emitted fluorescence after the complex was internalized into the cell. The above results demonstrate that the 5D1 humanized antibody of the present invention can reach the lysosome after specifically binding to tumor cells and then being internalized into the cell (antibody internalization).

[0387]

[0388] Example 9. Confirmation of the binding degree of chimeric antibodies and humanized antibodies to peripheral blood

[0389] The degree of binding of the 5D1 chimeric antibody (the chimeric antibody of Example 2) and the 5D1 humanized antibody (VH4 / VL3) to peripheral blood and red blood cells was measured. Peripheral blood cells and red blood cells were prepared using the same procedure as in Example 1-4-(1), and 100 μl of the hybridoma supernatant was added to each of the prepared peripheral blood cells and RBCs, and the cells were reacted with the 5D1 chimeric antibody and the 5D1 humanized antibody, respectively, at 4°C for 30 minutes. After the reaction was completed, 3 ml of PBS was added and the mixture was centrifuged at 1700 rpm for 3 minutes to wash away unbound antibodies. To identify bound antibodies, a secondary antibody, goat anti-Mouse Ig-FITC (Jackson ImmunoResearch, USA) or goat anti-Human Ig-FITC (Jackson ImmunoResearch, USA), was added at a 200-fold dilution and reacted at 4°C for 15 minutes. Afterward, the mixture was washed with 3 ml of PBS in the same manner as above and analyzed using a flow cytometer.

[0390] Figure 18 shows the results of the reactivity tests between peripheral blood and RBCs for each of the 5D1 chimeric antibody and the 5D1 humanized antibody. As shown in Figure 18, both the 5D1 chimeric antibody and the 5D1 humanized antibody showed negative reactions to granulocytes, lymphocytes, monocytes, and red blood cells in peripheral blood.

[0391]

[0392] Example 10. Preparation of 5D1 Antibody-Drug Conjugate and Measurement of Its In Vitro Cytotoxicity

[0393] Example 10-1. Preparation of a 5D1 antibody linker-drug solution

[0394] A 5D1 humanized antibody (VH4 / VL3) was conjugated to a linker-drug of the chemical formula to obtain a mixture of cysteine-linked antibody-drug conjugates of the chemical formula. The antibody-linker-drug conjugates were prepared using the commercially available AGLink ADC conjugation kit (ACRO Biosystems, Cat #. ADC-P009) purchased according to the manufacturer's instructions. Specifically, for the preparation of the antibody-drug conjugates, the buffer (PBS) was replaced with a buffer (25 mM Tris-HCl buffer, pH 7.5) using a desalting spin column. The drug conjugation method involved passing through the N-glycan of the antibody's Fc domain; the enzyme required for the glycan conjugation reaction and the substrate containing the drug (MMAE, Monomethyl Auristatin E, ACRO Biosystems) were mixed in the amounts specified in the kit and incubated at 30°C for 24 hours. During this process, the enzyme reaction was adjusted so that the DAR (drug antibody ration) was 4. A spin column with protein A resin was used to separate the antibody-drug conjugate from the unconjugated drug, and the antibody-drug conjugate was separated and purified according to the method presented in the kit.

[0395] An antibody-drug conjugate with another isolated and purified drug, deruxtecan (Dxd, CellMosaic), was prepared using the antibody-deruxtecan conjugation kit (CellMosaic, Cat #. CM11431). The antibody-deruxtecan conjugate was prepared using the method described in the kit, similar to the method described above, and after isolation and purification of the antibody-deruxtecan conjugate, the antibody was quantified using a quantification calibration curve.

[0396]

[0397] Example 10-2. Evaluation of the In Vitro Cell Proliferation Inhibitory Activity of 5D1 Antibody-Drug Conjugate

[0398] Cytotoxicity assays were performed on SNU-601 (gastric cancer cell line), cell line A, and PC-3 (prostate cancer cell line), which are positive for the CD155 antigen, against the 5D1 humanized antibody (VH4 / VL3)-drug conjugate prepared in Example 10-1 to confirm the in vitro cytotoxic effect.

[0399] Specifically, SNU-601 cell line and PC-3 cell line were each suspended in growth medium (RPMI + 10% FBS) at a rate of 5 x 10 3Cells were plated in a 96-well plate at cells / well. After incubating the cells at 37°C and 5% CO2 for 18 hours, the supernatant was removed carefully to prevent the attached cells from detaching. An antibody-drug conjugate containing the 5D1 humanized antibody (VH4 / VL3) and MMAE was prepared in the same manner as in Example 10-1. Additionally, as another 5D1 antibody-MMAE conjugate, the 5D1 humanized antibody (VH4 / VL3) was prepared by diluting it 2X to the concentration to be used in the experiment, and IgGs Anti-Human IgG Fc-MMAE Antibody with Cleavable Linker (MORADEC, Cat #. AH-102AE), a kit that indirectly links the 5D1 humanized antibody (VH4 / VL3) with the chemotoxic substance MMAE, was prepared by diluting it to 2 μg / ml in cell growth medium (RPMI + 10% FBS). An antibody-drug conjugate containing the 5D1 humanized antibody (VH4 / VL3) and Deruxtecan (Dxd) was prepared by the same method as described in Example 10-1. 50 μl each of the prepared antibody and drug were added to a 96-well plate with attached cells, and then incubated for 72 hours at 37°C and 5% CO2. After 72 hours, 10 μl of EZ-Cytox (DOGEN, Cat #. EZ-1000), a kit capable of measuring cell cytotoxicity, was dispensed onto the plate, and the degree of color development was checked at an absorbance of 450 nm after 1 hour. The results of cytotoxicity are described in Fig. 19, specifically, the treatment results of the 5D1 humanized antibody-MMAE drug conjugate prepared in Example 10-1 are shown in the upper left of Fig. 19, and the treatment results of the 5D1 humanized antibody-MMAE drug conjugate prepared with the MORADEC kit are shown in the upper right of Fig. 19.

[0400] As shown in Figure 19, both the antibody-drug conjugate containing the 5D1 humanized antibody and MMAE and the antibody-drug conjugate containing the 5D1 humanized antibody and deruxtecan (Dxd) were observed to have very high apoptotic effects.

[0401]

[0402] Example 11. Evaluation of In Vivo Anticancer Efficacy of 5D1 Antibody-Drug Conjugate

[0403] To confirm the substantial in vivo anticancer effect of the 5D1 humanized antibody-drug conjugate, the reduction in tumor size by administering the 5D1 humanized antibody-MMAE conjugate was evaluated in an NSG mouse animal model prepared by transplanting SUN-601, a human-derived gastric cancer cell line.

[0404] The 5D1 humanized antibody (VH4 / VL3)-drug (MMAE) conjugate was produced through the Osong Advanced Medical Industry Promotion Foundation (KBio Health), a specialized antibody-drug conjugate manufacturer.

[0405] The cancer animal model is 5x10 6 The gastric cancer cell line SNU-601 was mixed with Matrigel in a 1:1 ratio and prepared by intraperitoneal injection (IP) into the flanks of 8-week-old NSG mice. After 1–2 weeks had elapsed following the injection of the gastric cancer cell line, 100 to 150 mm 3Five mice with formed tumor volumes were selected for each group and used in the experiment. The tumor-forming NSG mice were divided into four groups; one group was administered PBS, and two groups were administered intravenously (IV) a total of three times on days 0, 7, and 14 at 0.3 mg / kg and 3 mg / kg, respectively, of the 5D1 humanized antibody (VH4 / VL3)-MMAE conjugate. The remaining group was administered the 5D1 humanized antibody-MMAE conjugate once on day 0 at 3 mg / kg, and subsequently, the size of the cancer cells injected subcutaneously was observed and measured.

[0406] The in vivo anticancer efficacy of the 5D1 humanized antibody-drug conjugate of the present invention in a gastric cancer mouse model is shown in Fig. 20. No significant anticancer effect was observed in the group administered a low dose of 0.3 mg / kg of the 5D1 humanized antibody-drug conjugate (5D1-MMAE_0.3 mg / kg), but the group administered a dose of 3 mg / kg of the 5D1 humanized antibody-drug conjugate (5D1-MMAE_3 mg / kg) showed a strong anticancer effect (tumor growth inhibition; TGI) compared to the group injected only with PBS (PBS). The above results support the dose-dependent anticancer effect of the antibody-drug conjugate of the present invention. In addition, the group administered a single dose of the 5D1 humanized antibody-drug conjugate (5D1-MMAE_3mg / kg_single) also showed a potent anticancer effect compared to the PBS group, and it was observed that the anticancer effect was maintained for more than 3 weeks. These results demonstrate the excellent anticancer effect of the 5D1 antibody-drug conjugate of the present invention.

[0407]

[0408] Furthermore, to verify that the 5D1 humanized antibody of the present invention can exert an anticancer effect when conjugated with various drugs, a 5D1 humanized antibody-Deruxtecan conjugate was additionally manufactured and the in vivo anticancer effect was confirmed. The 5D1 humanized antibody (VH4 / VL3)-deruxtecan conjugate was manufactured by Wuxi (China), a specialized antibody-drug conjugate manufacturer.

[0409] The anticancer effect of the above antibody-Deruxtecan conjugate was evaluated using a Balb / c-nude mouse model transplanted with JIMT-1, a human breast cancer cell line expressing intermediate levels of CD155. The mouse model was prepared using the same method as the gastric cancer mouse model described above. Experiments were conducted on a total of four groups, with five mice per group; the group injected with PBS (Vehicle) and the group injected with the antibody subtype-deruxtecan conjugate (Hu IgG-Dxd) were designated as controls. The group administered the 5D1 humanized antibody (VH4 / VL3)-deruxtecan conjugate received a total of three intravenous injections at a dose of 5 mg / kg on days 0, 7, and 14, while the other group received a single injection at a dose of 5 mg / kg on day 0 (5D1-Dxd_single).

[0410] The in vivo anticancer efficacy of the 5D1 humanized antibody-deruxtecan conjugate in a breast cancer mouse model is shown in Fig. 21. The group administered the antibody-drug conjugate of the present invention a total of three times showed a high TGI, confirming a potent anticancer effect, and a significant anticancer effect was observed even in the group injected with the antibody-drug conjugate only once. The above results support the fact that the 5D1 antibody according to the present invention can exert a potent anticancer effect when conjugated with various anticancer agents.

[0411]

[0412] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present disclosure may be implemented in other specific forms without altering the technical concept or essential features thereof. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present disclosure should be interpreted as including all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the detailed description above.

Claims

1. H-CDR1 containing the amino acid sequence of SEQ ID NO. 1; H-CDR2 containing the amino acid sequence of SEQ ID NO. 2; H-CDR3 containing the amino acid sequence of SEQ ID NO. 3; L-CDR1 containing the amino acid sequence of SEQ ID NO. 4; L-CDR2 comprising the amino acid sequence of SEQ ID NO. 5; and L-CDR3 containing the amino acid sequence of SEQ ID NO. 6 An anti-CD155 antibody or an antigen-binding fragment thereof comprising 2. In claim 1, a heavy chain variable region comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs 7 to 11 or said amino acid sequence; and An anti-CD155 antibody or an antigen-binding fragment thereof comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs 12 to 17, or comprising a light chain variable region consisting of said amino acid sequence.

3. In claim 1, a heavy chain comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs 46 to 66 or said amino acid sequence; and An anti-CD155 antibody or an antigen-binding fragment thereof comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs 67 to 72, or comprising a light chain consisting of said amino acid sequence.

4. In claim 1, the anti-CD155 antibody or its antigen-binding fragment is an anti-CD155 antibody or its antigen-binding fragment that recognizes CD155 domain 1 as an epitope.

5. The anti-CD155 antibody or its antigen-binding fragment according to claim 1, wherein the anti-CD155 antibody or its antigen-binding fragment has one or more features selected from the group consisting of: (i) The above anti-CD155 antibody or its antigen-binding fragment does not bind to peripheral blood cells; (ii) The above anti-CD155 antibody or its antigen-binding fragment inhibits the binding of TIGIT and CD155; (iii) The above anti-CD155 antibody or its antigen-binding fragment inhibits the binding of CD226 and CD155; and (iv) The above anti-CD155 antibody or its antigen-binding fragment inhibits the binding of CD96 and CD155.

6. In paragraph 1, the anti-CD155 antibody is an anti-CD155 antibody or an antigen-binding fragment thereof, which is an animal antibody, a chimeric antibody, or a humanized antibody.

7. In paragraph 1, the antigen-binding fragment is an anti-CD155 antibody or an antigen-binding fragment thereof that is a scFv, scFv-Fc, (scFv)2, Fab, Fab', F(ab')2, diabody, or minibody of the anti-CD155 antibody.

8. An anti-CD155 antibody of any one of claims 1 to 7 or a polynucleotide encoding an antigen-binding fragment thereof.

9. A recombinant vector comprising the polynucleotide of claim 8.

10. A recombinant cell comprising the polynucleotide of claim 8 or a recombinant vector containing the same.

11. A composition for detecting CD155 comprising an anti-CD155 antibody of any one of claims 1 to 7 or an antigen-binding fragment thereof.

12. A composition for diagnosing cancer comprising an anti-CD155 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7.

13. A pharmaceutical composition for the prevention, improvement, or treatment of cancer, comprising an anti-CD155 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7.

14. A pharmaceutical composition according to claim 13, wherein the cancer is a cancer expressing CD155. 15.(1) The anti-CD155 antibody of claim 1 or an antigen-binding fragment thereof; and (2) A conjugate comprising one or more selected from the group consisting of polymers, labeling substances, drugs, proteins, and peptides.

16. In paragraph 15, the conjugate comprises a drug and has an antibody-linker-drug structure.

17. In paragraph 16, the above drug is a conjugate that is an anticancer agent.

18. A pharmaceutical composition for the prevention, improvement, or treatment of cancer, comprising a conjugate of any one of claims 15 to 17.

19. A pharmaceutical composition according to claim 18, wherein the cancer is a cancer expressing CD155.

20. A method for the prevention, improvement, or treatment of cancer, comprising the step of administering to an individual an anti-CD155 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7; or a conjugate according to any one of claims 15 to 17.

21. Use of an anti-CD155 antibody of any one of claims 1 to 7 or an antigen-binding fragment thereof for the prevention, improvement, or treatment of cancer; or a conjugate of any one of claims 15 to 17.

22. Use of an anti-CD155 antibody of any one of claims 1 to 7 or an antigen-binding fragment thereof for the manufacture of a drug for the prevention, improvement, or treatment of cancer; or a conjugate of any one of claims 15 to 17.

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