Antibodies or antigen-binding fragments targeting CD155 and their applications

Abstract

The present invention belongs to the field of immunoengineering technology, and specifically relates to an antibody or antigen-binding fragment targeting CD155 and its application. The antibody includes a heavy chain variable region having at least 70% identity with the sequence shown in SEQ ID NO: 23 and a light chain variable region having at least 70% identity with the sequence shown in SEQ ID NO: 24, or a heavy chain variable region having at least 70% identity with the sequence shown in SEQ ID NO: 25 and a light chain variable region having at least 70% identity with the sequence shown in SEQ ID NO: 26. The antibody has excellent binding ability to CD155, and the CAR-T constructed based on the antibody has significant killing ability against tumor cells expressing CD155, as well as obvious cell internalization, promotion of NK cell killing toxicity and promotion of macrophage phagocytosis, showing excellent anti-tumor effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunological engineering, and in particular relates to an antibody or antigen-binding fragment targeting CD155 and applications thereof. Background Art

[0002] CD155 (also known as PVR, NECTIN-2, or Necl-5) is a multifunctional single-pass transmembrane protein that plays a key role in a variety of biological processes. It plays a dual role in immune regulation. On the one hand, as a ligand for DNAM-1, CD155 can activate NK cells and T cells, promoting the killing effect of immune cells; on the other hand, by binding to immune checkpoint receptors such as TIGIT and CD96, CD155 can inhibit the activation and function of immune cells, forming an immunosuppressive tumor microenvironment.

[0003] Existing studies have shown that in many types of tumors, the expression of CD155 is often significantly upregulated, becoming one of the common characteristics of many types of tumors. High expression of CD155 regulates tumor progression by affecting processes such as cell proliferation, migration, and adhesion; specifically, it can promote cell proliferation by activating specific signaling pathways (such as the Ras-Raf-MEK-ERK signaling pathway); at the same time, it can also interact with growth factor receptors to further affect the cell proliferation process; in terms of affecting cell migration, CD155 can induce epithelial-mesenchymal transition in tumor cells and promote tumor cell migration by participating in multiple tumor-related signaling pathways. In addition, CD155 inhibits the activation and function of NK cells and T cells by binding to immune checkpoint receptors such as TIGIT and CD96, thereby reducing the ability of immune cells to kill tumor cells. In addition, it can also promote the recruitment and activation of immunosuppressive cells (such as MDSCs, Tregs, etc.), further exacerbating tumor immune escape.

[0004] Given its important role in tumor immune escape, CD155 has become one of the important targets for tumor immunotherapy. Currently, one of the immunotherapy strategies targeting CD155 and its receptor is to develop antibodies specific for CD155 to block its binding to immune checkpoint receptors and restore the activity and function of immune cells. Summary of the Invention

[0005] The present invention has developed an antibody or antigen-binding fragment targeting CD155. The antibody includes a humanized antibody that has a strong binding ability to CD155. CAT-T cells constructed based on the antibody have a significant cell-killing ability against some tumor cells.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] On the one hand, the present invention provides an antibody or antigen-binding fragment targeting CD155, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, and the light chain variable region comprising LCDR1, LCDR2 and LCDR3; the amino acid sequence of HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 3; the amino acid sequence of LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 6.

[0008] Preferably, in the above-mentioned antibody or antigen-binding fragment targeting CD155, the heavy chain variable region further includes: HFR1 having at least 80% identity with the sequence shown in SEQ ID NO:7 and / or HFR2 having at least 80% identity with the sequence shown in SEQ ID NO:8 and / or HFR3 having at least 80% identity with the sequence shown in SEQ ID NO:9 and / or HFR4 having at least 80% identity with the sequence shown in SEQ ID NO:10; and / or the light chain variable region further includes: LFR1 having at least 80% identity with the sequence shown in SEQ ID NO:11 and / or LFR2 having at least 80% identity with the sequence shown in SEQ ID NO:12 and / or LFR3 having at least 80% identity with the sequence shown in SEQ ID NO:13 and / or LFR4 having at least 80% identity with the sequence shown in SEQ ID NO:14.

[0009] Preferably, in the above-mentioned antibody or antigen-binding fragment targeting CD155, the heavy chain variable region further includes: HFR1 having at least 80% identity with the sequence shown in SEQ ID NO: 15 and / or HFR2 having at least 80% identity with the sequence shown in SEQ ID NO: 16 and / or HFR3 having at least 80% identity with the sequence shown in SEQ ID NO: 17 and / or HFR4 having at least 80% identity with the sequence shown in SEQ ID NO: 18; and / or the light chain variable region further includes: LFR1 having at least 80% identity with the sequence shown in SEQ ID NO: 19 and / or LFR2 having at least 80% identity with the sequence shown in SEQ ID NO: 20 and / or LFR3 having at least 80% identity with the sequence shown in SEQ ID NO: 21 and / or LFR4 having at least 80% identity with the sequence shown in SEQ ID NO: 22.

[0010] Preferably, the above-mentioned antibody or antigen-binding fragment targeting CD155 is selected from any one of the following antibodies: (a) the antibody or antigen-binding fragment targeting CD155 comprises: a heavy chain variable region having at least 70% identity with the sequence shown in SEQ ID NO: 23 and a light chain variable region having at least 70% identity with the sequence shown in SEQ ID NO: 24; (b) the antibody targeting CD155 comprises: a heavy chain variable region having at least 70% identity with the sequence shown in SEQ ID NO: 25 and a light chain variable region having at least 70% identity with the sequence shown in SEQ ID NO: 26.

[0011] Preferably, the above-mentioned antibody or antigen-binding fragment targeting CD155 further comprises a heavy chain constant region and / or a light chain constant region, and at least a portion of the heavy chain constant region and / or the light chain constant region is derived from at least one of a human antibody, a primate antibody or a mutant thereof.

[0012] Preferably, the above-mentioned heavy chain constant region and light chain constant region are both derived from human IgG antibodies or mutants thereof.

[0013] Preferably, the above-mentioned heavy chain constant region and light chain constant region are both derived from human IgG1 antibody, human IgG4 antibody or mutants thereof.

[0014] Preferably, the heavy chain constant region comprises a sequence that is at least 70% identical to the sequence shown in SEQ ID NO: 34, and the light chain constant region comprises a sequence that is at least 70% identical to the sequence shown in SEQ ID NO: 35.

[0015] In another aspect, the present invention provides a CAR structure comprising the antibody or antigen-binding fragment targeting CD155 of the present invention.

[0016] Preferably, the above-mentioned CAR structure further includes: a hinge region having at least 80% identity with the amino acid sequence encoded by the sequence shown in SEQ ID NO: 27 and / or a transmembrane region and / or an intracellular signaling domain having at least 80% identity with the amino acid sequence encoded by the sequence shown in SEQ ID NO: 28; the intracellular signaling domain includes an intracellular costimulatory domain having at least 80% identity with the amino acid sequence encoded by the sequence shown in SEQ ID NO: 29 and an intracellular signaling domain having at least 80% identity with the amino acid sequence encoded by the sequence shown in SEQ ID NO: 30.

[0017] In another aspect, the present invention provides any of the following substances:

[0018] (i) a nucleic acid molecule encoding an antibody or antigen-binding fragment targeting CD155 of the present invention or encoding a CAR structure of the present invention;

[0019] (ii) an expression vector comprising the nucleic acid molecule of (i);

[0020] (iii) an engineered cell comprising the nucleic acid molecule of (i) and / or the expression vector of (ii);

[0021] (iv) a product for detecting CD155, comprising the antibody or antigen-binding fragment targeting CD155 of the present invention;

[0022] (v) a pharmaceutical composition comprising the antibody or antigen-binding fragment targeting CD155 of the present invention and / or the CAR structure of the present invention and / or the nucleic acid molecule of (i) and / or the expression vector of (ii) and / or the engineered cell of (iii);

[0023] (vi) a pharmaceutical preparation comprising an antibody targeting CD155 of the present invention and / or an antigen-binding fragment of the present invention and / or a CAR structure of the present invention and / or the nucleic acid molecule of (i) and / or the expression vector of (ii) and / or the engineered cell of (iii) and / or the pharmaceutical composition of (v).

[0024] Preferably, the pharmaceutical composition is a bispecific antibody, a polyspecific antibody, an ADC or a fusion protein.

[0025] In another aspect, the present invention provides a use of the antibody or antigen-binding fragment targeting CD155 of the present invention in the preparation of a product for detecting CD155 levels.

[0026] On the other hand, the present invention provides an antibody or antigen-binding fragment targeting CD155 of the present invention and / or the CAR structure of the present invention and / or the nucleic acid molecule and / or expression vector and / or engineered cell of the present invention for use in the preparation of a drug, which is used to prevent and / or treat CD155-related diseases.

[0027] Preferably, the CD155-related disease is cancer, autoimmune disease, transplant rejection or infectious disease.

[0028] Preferably, the cancer includes at least one of acute myeloid leukemia, monocytic leukemia, melanoma, lung cancer, liver cancer, ovarian cancer, cervical cancer, pancreatic cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer.

[0029] Preferably, the above applications include one or more combinations of the following applications:

[0030] (a) Use of the CD155-targeting antibody or antigen-binding fragment of the present invention in mediating cellular internalization;

[0031] (b) Use of the CD155-targeting antibody or antigen-binding fragment of the present invention in promoting the killing toxicity of NK cells;

[0032] (c) Use of the antibody or antigen-binding fragment targeting CD155 of the present invention in promoting phagocytosis of macrophages.

[0033] The beneficial effects of the present invention include at least:

[0034] (1) The mouse-derived antibody targeting CD155 provided by the present invention has excellent binding ability to CD155, with an EC50 value of 0.969 nM for binding to CD155 recombinant protein and 21.51 nM for binding to CD155 overexpressing cell lines.

[0035] (2) The humanized modified antibody targeting CD155 provided by the present invention has excellent binding ability to CD155, with an EC50 value of 0.315 nM for binding to CD155 recombinant protein and 12.20 nM for binding to CD155 overexpressing cell lines.

[0036] (3) The humanized modified antibody targeting CD155 provided by the present invention has obvious cell internalization effect, promotes the killing toxicity of NK cells and promotes the phagocytosis of macrophages.

[0037] (4) CAT-T cells constructed based on the humanized modified antibody targeting CD155 provided by the present invention have significant killing ability against some tumor cells, and the killing ability is stronger than that of T cells; for example, the killing efficiency of CAT-T cells against human acute myeloid leukemia cell lines can reach 62.50%, the killing efficiency against human monocytic leukemia can reach 59.32%, the killing efficiency against human liver cancer cells can reach 49.10%, the killing efficiency against human glioma cells can reach 33.52%, the killing efficiency against human breast cancer cells can reach 46.30%, the killing efficiency against human colon cancer cells can reach 41.52%, the killing efficiency against human non-small cell lung cancer cells can reach 16.58%, and the killing efficiency against human pancreatic cancer cells can reach 11.25%. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 CD155 expression in various tumor tissues and normal tissues;

[0039] Figure 2 The expression values ​​of CD155 in each cell subset of normal tissues are compared with those of EGFR\ERBB2\ROR1;

[0040] Figure 3ELISA analysis of the binding of mouse antibody B03 to human CD155 protein;

[0041] Figure 4 Binding analysis of mouse antibody B03 and cell lines overexpressing human CD155;

[0042] Figure 5 ELISA analysis of the binding of humanized modified antibody B03-1 to human CD155 protein;

[0043] Figure 6 Binding analysis of humanized antibody B03-1 and cell lines overexpressing human CD155;

[0044] Figure 7 The humanized antibody B03-1 specifically binds to a monkey CD155-overexpressing cell line;

[0045] Figure 8 Analysis of the internalization ability of the humanized antibody B03-1;

[0046] Figure 9 To investigate the effect of humanized antibody B03-1 on enhancing antibody-mediated NK cell killing;

[0047] Figure 10 To analyze the effect of humanized antibody B03-1 on enhancing antibody-mediated macrophage phagocytosis by flow cytometry;

[0048] Figure 11 This is a bar graph showing the enhancement of antibody-mediated macrophage phagocytosis by the humanized antibody B03-1;

[0049] Figure 12 This is the CD155 CAR plasmid vector map. DETAILED DESCRIPTION

[0050] The examples are provided to better illustrate the present invention, but are not intended to limit the present invention to the examples. Therefore, non-essential improvements and adjustments to the embodiments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection of the present invention.

[0051] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context clearly has a different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "comprise" and the like are intended to indicate the presence of features, numbers, operations, materials or combinations. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0052] As used herein, the term "antigen-binding fragment" refers to antigen-binding fragments of antibodies and antibody analogs, which generally include at least a portion of the antigen-binding region or variable region of the parent antibody, such as one or more CDRs; antibody fragments retain at least some of the binding specificity of the parent antibody.

[0053] An embodiment of the present invention provides an antibody or antigen-binding fragment targeting CD155, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, and the light chain variable region comprising LCDR1, LCDR2, and LCDR3; the amino acid sequence of HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 3; the amino acid sequence of LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 6.

[0054] It should be noted that the CD155-targeting antibody in the present invention can be any antibody form such as a monoclonal antibody or a recombinant antibody; in terms of source, it can be a humanized antibody or an animal-derived antibody, such as a mouse-derived, rabbit-derived or camel-derived antibody; in addition, HCDR3 and LCDR3 in the above-mentioned antibodies belong to the hypervariable regions of the heavy chain variable region and the light chain variable region, and are less stable than HCDR1 and HCDR2 and LCDR1 and LCDR2.

[0055] In some specific examples, in the above-mentioned antibodies or antigen-binding fragments targeting CD155, the heavy chain variable region also includes: HFR1 having at least 80% identity with the sequence shown in SEQ ID NO:7 and / or HFR2 having at least 80% identity with the sequence shown in SEQ ID NO:8 and / or HFR3 having at least 80% identity with the sequence shown in SEQ ID NO:9 and / or HFR4 having at least 80% identity with the sequence shown in SEQ ID NO:10; and / or the light chain variable region also includes: LFR1 having at least 80% identity with the sequence shown in SEQ ID NO:11 and / or LFR2 having at least 80% identity with the sequence shown in SEQ ID NO:12 and / or LFR3 having at least 80% identity with the sequence shown in SEQ ID NO:13 and / or LFR4 having at least 80% identity with the sequence shown in SEQ ID NO:14.

[0056] It should be noted that the FRs (HFR1, HFR2, HFR3, HFR4 and LFR1, LFR2, LFR3, LFR4) in the above-mentioned antibodies are framework regions used to connect the CDR regions and are relatively stable. In addition, the sequences of the FR regions and the CDR regions of the above-mentioned heavy and light chains can be arranged according to FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 to constitute the heavy chain variable region and light chain variable region of the above-mentioned two antibodies, respectively. In addition, the at least 80% identity in the present invention includes ≥80%, ≥85%, ≥90%, ≥95% or 100% identity, etc. In addition, the above-mentioned FR sequences are derived from mice and can be combined with the CDRs in the present invention to form antibodies with strong affinity and high specificity for the target CD155.

[0057] In some specific examples, in the above-mentioned antibodies or antigen-binding fragments targeting CD155, the heavy chain variable region also includes: HFR1 with at least 80% identity to the sequence shown in SEQ ID NO: 15 and / or HFR2 with at least 80% identity to the sequence shown in SEQ ID NO: 16 and / or HFR3 with at least 80% identity to the sequence shown in SEQ ID NO: 17 and / or HFR4 with at least 80% identity to the sequence shown in SEQ ID NO: 18; and / or the light chain variable region also includes: LFR1 with at least 80% identity to the sequence shown in SEQ ID NO: 19 and / or LFR2 with at least 80% identity to the sequence shown in SEQ ID NO: 20 and / or LFR3 with at least 80% identity to the sequence shown in SEQ ID NO: 21 and / or LFR4 with at least 80% identity to the sequence shown in SEQ ID NO: 22.

[0058] It should be noted that the above-mentioned FR sequence is a humanized modified sequence, which can be combined with the CDR region of the present invention to form an antibody for recognizing CD155, and has a strong binding ability to monkey CD155; similarly, the FR (HFR1, HFR2, HFR3, HFR4 and LFR1, LFR2, LFR3, LFR4) in the above-mentioned antibody is a framework region used to connect the CDR region and is relatively stable; in addition, the sequences of the FR regions of the above-mentioned heavy chain and light chain and the sequences of the CDR regions can be arranged according to FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0059] In some specific examples, the antibody or antigen-binding fragment targeting CD155 is selected from any one of the following antibodies:

[0060] (a) an antibody or antigen-binding fragment targeting CD155 comprising: a heavy chain variable region having at least 70% identity to the sequence shown in SEQ ID NO: 23 and a light chain variable region having at least 70% identity to the sequence shown in SEQ ID NO: 24;

[0061] (b) An antibody or antigen-binding fragment targeting CD155 comprises a heavy chain variable region that is at least 70% identical to the sequence shown in SEQ ID NO: 25 and a light chain variable region that is at least 70% identical to the sequence shown in SEQ ID NO: 26.

[0062] It should be noted that the antibody or antigen-binding fragment targeting CD155 in the present invention may preferably be an antibody consisting of a heavy chain variable region composed of the above-mentioned HCDR1 to HCDR3 and murine HFR1 to HFR4, and a light chain variable region composed of the above-mentioned LCDR1 to LCDR3 and murine LFR1 to LFR4; similarly, an antibody may also be preferably composed of a heavy chain variable region composed of the above-mentioned HCDR1 to HCDR3 and humanized HFR1 to HFR4, and a light chain variable region composed of the above-mentioned LCDR1 to LCDR3 and murine LFR1 to LFR4.

[0063] In some specific examples, the above-mentioned antibody or antigen-binding fragment targeting CD155 further comprises a heavy chain constant region and / or a light chain constant region, and at least a portion of the heavy chain constant region and / or the light chain constant region is derived from at least one of a human antibody, a primate antibody or a mutant thereof.

[0064] In some specific examples, the heavy chain constant region and the light chain constant region are both derived from human IgG antibodies or mutants thereof.

[0065] In some specific examples, the heavy chain constant region and the light chain constant region are both derived from human IgG1 antibody, human IgG4 antibody or mutants thereof.

[0066] In some specific examples, the heavy chain constant region comprises a sequence that is at least 70% identical to the sequence shown in SEQ ID NO:34, and the light chain constant region comprises a sequence that is at least 70% identical to the sequence shown in SEQ ID NO:35.

[0067] It should be noted that the CD155-targeting antibodies or antigen-binding fragments of the present invention, in addition to the aforementioned heavy chain variable region and light chain variable region, also include a heavy chain constant region and a light chain constant region. The heavy chain constant region and light chain constant region can be derived from human or other animal sources (e.g., rabbit, porcine, etc.), and are regions that are virtually immune to mutation. Furthermore, the heavy and light chains of the antibodies can also include signal peptides, which can facilitate antibody membrane penetration; the signal peptides can be any known signal peptide in the art.

[0068] An embodiment of the present invention further provides a CAR structure, which includes the antibody or antigen-binding fragment targeting CD155 of the present invention.

[0069] It should be noted that the variable region fragment of the antibody targeting CD155 in the present invention can be used as the antigen recognition region of the CAR structure and other structures (such as the transmembrane region, hinge region or intracellular signal transduction region) to form a CAR structure; it is more conducive to the targeted killing of CD155.

[0070] In some specific examples, the above-mentioned CAR structure also includes: a hinge region having at least 80% identity with the amino acid sequence encoded by the sequence shown in SEQ ID NO: 27 and / or a transmembrane region and / or an intracellular signaling domain having at least 80% identity with the amino acid sequence encoded by the sequence shown in SEQ ID NO: 28; the intracellular signaling domain includes an intracellular co-stimulatory domain having at least 80% identity with the amino acid sequence encoded by the sequence shown in SEQ ID NO: 29 and an intracellular signaling domain having at least 80% identity with the amino acid sequence encoded by the sequence shown in SEQ ID NO: 30.

[0071] It should be noted that, as described above, the transmembrane region, hinge region, or intracellular signaling domain in the present invention may preferably be the above-mentioned sequences. Similarly, at least 80% identity includes identity ≥80%, identity ≥85%, identity ≥90%, identity ≥95%, or 100% sequence identity; it should be understood that 100% identity means that the amino acid sequence of the hinge region is the amino acid sequence encoded by the sequence shown in SEQ ID NO: 27, the sequence of the transmembrane region is the amino acid sequence encoded by the sequence shown in SEQ ID NO: 28, and the sequence of the intracellular signaling domain is the amino acid sequence encoded by the sequences shown in SEQ ID NO: 29 and SEQ ID NO: 30.

[0072] The present invention also provides any of the following substances:

[0073] (i) a nucleic acid molecule encoding an antibody or antigen-binding fragment targeting CD155 of the present invention or encoding a CAR structure of the present invention; specifically, the nucleic acid molecule of the present invention is obtained by translating the above-mentioned antibody or antigen-binding fragment targeting CD155 or CAR structure according to conventional methods; in addition, it can also be a nucleotide sequence obtained by further modifying the sequence obtained by translating the above-mentioned amino acid sequence; the modification method is a nucleotide modification method known in the art to increase expression efficiency or other purposes;

[0074] (ii) an expression vector comprising the nucleic acid molecule of (i); specifically, the expression vector of the present invention can be selected from any one of a lentiviral expression vector, a retroviral expression vector, an adenoviral expression vector, an adeno-associated viral expression vector, a DNA vector, an RNA vector, and a plasmid. The lentiviral vector can be selected from the following group: human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), visna-maedivirus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV);

[0075] (iii) an engineered cell, comprising the nucleic acid molecule of (i) and / or the expression vector of (ii); specifically, the engineered cell of the present invention may be a host cell, and the expression vector may be introduced into the host cell to encode the antibody of the present invention; when the engineered cell comprises a CAR structure, the engineered cell may be a T cell or a B cell, and the CAR structure may be transduced into the T cell or B cell to form a CAR-T cell or CAR-B cell, which can achieve precise killing of cells secreting CD155;

[0076] (iv) a product for detecting CD155, comprising the antibody or antigen-binding fragment targeting CD155 of the present invention; specifically, the antibody or antigen-binding fragment targeting CD155 can specifically bind to CD155 and can be prepared into a product for detecting CD155, such as a detection reagent, a kit, or a microfluidic chip;

[0077] (v) a pharmaceutical composition comprising the antibody or antigen-binding fragment targeting CD155 of the present invention and / or the CAR structure of the present invention and / or the nucleic acid molecule of (i) and / or the expression vector of (ii) and / or the engineered cell of (iii); specifically, the antibody or antigen-binding fragment targeting CD155 of the present invention and / or the CAR structure or nucleic acid molecule or expression vector or engineered cell of the present invention can be combined with other active ingredients to form a pharmaceutical composition to improve the therapeutic effect; the other active ingredients can be small chemical molecules or polypeptides, etc.;

[0078] (vi) A pharmaceutical preparation comprising an antibody targeting CD155 of the present invention and / or an antigen-binding fragment of the present invention and / or a CAR structure of the present invention and / or a nucleic acid molecule of (i) and / or an expression vector of (ii) and / or an engineered cell of (iii) and / or a pharmaceutical composition of (v); specifically, the above-mentioned antibody and / or antigen-binding fragment and / or CAR structure and / or nucleic acid molecule and / or expression vector and / or engineered cell and / or pharmaceutical composition can be added with a pharmaceutically acceptable carrier to prepare pharmaceutical preparations of different dosage forms, and the pharmaceutically acceptable carrier is selected according to different dosage forms according to methods known in the art; dosage forms include injections, iron preparations, ointments, tablets or powders, etc.; the specific dosage form can be selected according to clinical conditions.

[0079] In some specific examples, the pharmaceutical composition is a bispecific antibody, a polyspecific antibody, an ADC, or a fusion protein.

[0080] It should be noted that the CD155-targeting antibody or antigen-binding fragment of the present invention can be combined with other antibodies to form a bispecific antibody or polyspecific antibody to increase the target of action or therapeutic effect; in addition, the CD155-targeting antibody or antigen-binding fragment can be linked to a small molecule with biological activity to form an ADC drug; in addition, the antibody or antigen-binding fragment of the present invention and the bispecific antibody and monoclonal antibody composed thereof can also be fused with a protein to form a fusion protein drug; the specific composition method is well known in the art.

[0081] An embodiment of the present invention further provides a use of the antibody or antigen-binding fragment targeting CD155 in the present invention in the preparation of a product for detecting CD155 levels.

[0082] The embodiments of the present invention also provide an antibody or antigen-binding fragment targeting CD155 of the present invention and / or the CAR structure of the present invention and / or the nucleic acid molecule and / or expression vector and / or engineered cell of the present invention for use in the preparation of a drug, which is used to prevent and / or treat CD155-related diseases.

[0083] In some embodiments, the CD155-associated disease is cancer, autoimmune disease, transplant rejection, or infectious disease.

[0084] In some specific examples, the cancer includes at least one of acute myeloid leukemia, monocytic leukemia, melanoma, lung cancer, liver cancer, ovarian cancer, cervical cancer, pancreatic cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, and head and neck cancer.

[0085] In some specific examples, the above applications include one or more combinations of the following applications:

[0086] (a) Use of the CD155-targeting antibody or antigen-binding fragment of the present invention in mediating cellular internalization;

[0087] (b) Use of the CD155-targeting antibody or antigen-binding fragment of the present invention in promoting the killing toxicity of NK cells;

[0088] (c) Use of the antibody or antigen-binding fragment targeting CD155 of the present invention in promoting phagocytosis of macrophages.

[0089] It should be noted that cellular internalization refers to the potential for ADC drug development; the three major mechanisms of action of antibodies—ADCC, ADCP, and CDC—are key to the therapeutic effects of antibody drugs. The ADCC mechanism involves antibody-dependent cell-mediated cytotoxicity, which indirectly leads to target cell destruction (i.e., the cytotoxic effect of NK cells) through the binding of the antibody Fc segment to the FcR on the surface of killer cells; the ADCP mechanism involves phagocytosis mediated by monocytes, macrophages, neutrophils, and dendritic cells through the expression of specific FcγRs (the phagocytic effect of macrophages).

[0090] In order to better understand the present invention, the content of the present invention is further explained below with reference to specific examples, but the content of the present invention is not limited to the following examples.

[0091] 1. CD155 expression in different tumors and flow cytometry detection of tumor cells

[0092] The expression of CD155 in various tumor tissues and normal tissues was analyzed through the website http: / / gepia.cancer-pku.cn / index.html. Figure 1As shown, CD155 is significantly expressed in various tumor tissues. CD155 expression in normal tissues was subsequently analyzed and compared with that in EGFR, ERBB2, and ROR1. Forty-nine single-cell RNA sequencing (scRNA-seq) datasets from nine healthy tissues were combined. scRNA-seq data were analyzed using the R package Seurat (version 4.2.0). Strict quality control was performed based on three metrics: total UMI count, number of detected genes, and the proportion of mitochondrial gene UMI counts, to screen out low-quality cells. The following steps were then performed: Normalization: The NormalizeData function was used for library size correction and logarithmic transformation; Variable feature selection: The FindVariableFeatures function selected the top 2000 highly variable genes (hvg); Scaling: The HVG expression matrix was scaled using the ScaleData function; Principal Component Analysis (PCA): The scaled HVG expression matrix was subjected to PCA using RunPCA, retaining the top 30 components for further analysis; Dimensionality reduction: The RunUMAP function was used for dimensionality reduction, and cells were embedded in coordinates for visualization. Cell clusters were annotated based on the expression of classic cell markers, identifying major cell types such as T cells, B cells, NK cells, and epithelial cells. A single gene count matrix was created and used to plot the average expression values ​​of different cell types. All preprocessing and analysis steps were run in Python 3.9 using RStudio v4.2.0, and visualization was performed using the pheatmap function. The results are shown in Figure 2. Figure 2 As shown, the expression value of CD155 in various normal tissues is significantly lower than that of EGFR, ERBB2 (HER2), and ROR1. EGFR, ERBB2 (HER2), and ROR1 are now being widely developed in clinical practice as targets for anti-tumor drugs.

[0093] 2. CD155 Antibody Screening

[0094] Balb / c mice (8 weeks old, purchased from Jiangsu Jicui Pharmaceutical, weighing approximately 20 g) were immunized with hCD155 protein (10109-H08H, Sino Biological) as an antigen. The immunized mice were immunized three times with purified antigen and complete Freund's adjuvant. Serum titers were tested by ELISA to obtain mice with the highest serum titer of anti-human CD155 immunoglobulin.

[0095] The spleen and bone marrow of mice with the highest serum titer of anti-human CD155 antibodies were harvested, plasma cells were enriched using magnetic beads, and the cells were analyzed using Bruker's single-cell photoconductive platform. Screening plasma cells that secrete CD155 antigen positive, deriving single cells, sequencing by single cell PCR to obtain BCR, and finally obtaining antibody sequences; specifically including:

[0096] (1) The spleen was ground and resuspended in 20 ml of PBS buffer. The suspension was added to a 50 ml centrifuge tube containing 10 ml of Ficoll solution (Cytiva, 17144003). The tube was centrifuged at 800 g for 30 min, with the 1:1 ratio followed by the 0:0 ratio. The lymphocyte layer was removed and mixed with bone marrow-derived cells.

[0097] (2) Plasma cells were isolated according to the instructions of Miltenyi CD138 Microbeads (130-098-257); the enriched plasma cells were introduced into the single cell light-guided platform of Bruker. Antigen-positive plasma cells were screened according to the manufacturer's instructions, and the variable region sequence of the B03 antibody was obtained; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 23, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 24, and the specific sequences of the CDR and FR regions are shown in Table 1 below.

[0098] Table 1B03 variable region sequences

[0099]

[0100]

[0101] 2. Expression and purification of full-length chimeric antibodies

[0102] The B03 variable region sequence was combined with the human antibody heavy chain and light chain constant regions to construct the full-length B03 antibody (the amino acid sequence of the human antibody heavy chain constant region is shown in SEQ ID NO: 34, and the amino acid sequence of the human antibody light chain constant region is shown in SEQ ID NO: 35). The antibody was expressed and purified according to the method described in patent CN117264050A (performed according to the method described in paragraphs

[0130] to

[0136] of the specification). After purification, the antibody was added with 5% mannitol and 5% trehalose, aliquoted, and stored at -20°C for long-term storage.

[0103] 3. Analysis of Binding Ability of Mouse Chimeric Antibodies

[0104] (1) Antibody-antigen protein binding analysis

[0105] For antibody-antigen protein binding analysis, an ELISA experiment was performed using purified B03 antibody (full length) and hCD155-his (10109-H08H, Sino Biological) recombinant protein. The specific experimental steps are as follows:

[0106] (1) Dilute hCD155-his to 2 μg / mL with PBS and add 100 μL to each well of the ELISA plate for coating at 4°C overnight;

[0107] (2) Discard the supernatant coating solution, wash with PBST, and block with PBST containing 1% BSA at 37°C for 2 h;

[0108] (3) Discard the blocking solution in the wells and wash with PBST;

[0109] (4) Add different concentrations of B03 antibody (full length) diluted 1:3 in 1% BSA solution and incubate at 37°C for 1 h;

[0110] (5) Discard the liquid and wash with PBST; add diluted HRP secondary antibody to each well and incubate at 37°C for 1 h; discard the liquid and wash with PBST;

[0111] (6) TMB colorimetric solution was added to each well and the reaction was carried out at room temperature for 10-15 minutes. The reaction was terminated by adding the stop solution. The OD value at 450 nm was read, a graph was drawn, and the analysis was performed using GraphPad Prism 9. The EC50 value was calculated (the concentration of the antibody when the OD450 value was half of the maximum value was the EC50 value of the antibody. The smaller the EC50 value, the stronger the affinity of the antibody and the higher the binding ability to the hCD155-his protein). The results are shown in Figure 5. Figure 3 As shown, B03 bound to hCD155-his in a concentration-dependent manner, with an EC50 value of 0.969 nM.

[0112] (II) Analysis of the ability of antibodies to bind to cell lines overexpressing CD155

[0113] To analyze the ability of the antibody to bind to cell lines overexpressing CD155, flow cytometry was used for detection. The specific steps are as follows:

[0114] (1) Virus packaging and cell line construction: HEK293T cells were cultured at a rate of 5×10 5 Cells / well were plated in six-well plates and cultured overnight in DMEM medium without double antibody;

[0115] (2) Before transfection, discard the culture medium and add 1 mL of fresh DMEM culture medium without double antibody. PCDH-CMV-human CD155-EF1-RFP-T2A-Puro (hCD155 was constructed into the vector PCDH-CMV-MCS-EF1-RFP-T2A-Puro provided by Qingke Biotechnology and sent to Qingke Biotechnology for synthesis, and the CD155 sequence was synthesized using uniprot accession: P15151, 1W-417R), PCDH-CMV-Rhesus CD155-EF1-RFP-T2A-Puro (RCD155 was constructed into the vector PCDH-CMV-MCS-EF1-RFP-T2A-Puro provided by Qingke Biotechnology and sent to Qingke Biotechnology for synthesis, and the CD155 sequence was synthesized using NCBI accession: NP_001036851.1, 1M-407R), pMD2G, and psPAX2 vectors (3 μg in total) were added to 200 μL serum-free DMEM medium at a mass ratio of 2:1:1;

[0116] (3) After mixing, let it stand for 16 minutes, and then add all the liquid to the six-well plate containing HEK293T cells;

[0117] (4) After 6 h of culture, the culture medium was discarded and fresh complete DMEM medium was added;

[0118] (5) 48 h after transfection, the cell culture supernatant was collected and filtered through a 0.45 μm filter (Millipore) to obtain the viral supernatant;

[0119] (6) All the obtained different virus supernatants were added to the 5×10 5 To a 6-well plate of HEK293T cells, polybrene (Sigma) was added at a final concentration of 4 μg / mL and cultured for 12 h. The supernatant was then discarded and fresh complete DMEM medium was added; the resulting cells were HEK293T-hCD155 and HEK293T-rCD155.

[0120] (7) Dilute the HEK293T-hCD155 cells to 2×10 6 pcs / mL, added to 1.5mL EP tubes at a volume of 100μL / tube, added with different concentrations of B03 antibody (full length), and incubated at 4℃ for 60min;

[0121] (8) Add 1 mL of PBS to the EP tube, centrifuge at 3500 rpm for 5 min at 4°C, discard the supernatant, and wash the pellet again with PBS;

[0122] (9) After centrifugation, discard the supernatant, resuspend the cells with 100 μL / tube PBS, add 0.1 μL / tube AF647 secondary antibody, and incubate at 4°C in the dark for 30 min; wash twice with PBS, centrifuge, and discard the supernatant;

[0123] (10) Resuspend the cells in 200 μL / tube of PBS, detect them by flow cytometry, and analyze them by graphing using GraphPad Prism 9. Figure 4 As shown, the B03 antibody was able to bind to the cell line overexpressing CD155 in a concentration-dependent manner, with an EC50 of 21.51 nM.

[0124] IV. Construction and Verification of Humanized Antibodies

[0125] (1) Humanization of mouse antibodies

[0126] The amino acid sequences of the heavy and light chain variable regions of the mouse B03 antibody were entered into the Discovery Studio modeling environment (DS, version 19.1.0.18287), and the framework regions (FRs) and complementarity-determining regions (CDRs) were annotated. The antibody's 3D structure was modeled using templates of the FR and CDR regions. The optimal model was selected based on sequence similarity and energy minimization. DS identified three typical structure-determining residues and confirmed their role in maintaining conformation through 3D visualization. Human FRs with high homology were then selected from the Fab sequence database, and the mouse CDRs were grafted onto these human FRs. Finally, based on the typical structure-determining residues and the predicted impact on structural stability and binding affinity, the key residues for backmutation were identified to obtain the variable region sequence of the humanized antibody B03-1. The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:25, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:26. The specific sequences of its CDR and FR regions are shown in Table 2 below.

[0127] Table 2B03-1 Variable region sequences

[0128] Name (kabat annotation) Amino acid sequence B03-1HFR1 QVQLVESGGGLVKPGGSLRLSCAASGFTFS(SEQ ID NO:15) B03-1HCDR1 SYAMS (SEQ ID NO: 1) B03-1HFR2 WVRQAPGKGLEIVA (SEQ ID NO: 16) B03-1HCDR2 TISSIGFTYYPDSVKG (SEQ ID NO: 2) B03-1HFR3 RFTISRDTSKNTLYLQMSSLRAEDTAVYYCAR(SEQ ID NO:17) B03-1HCDR3 PSYYGNYGWYFDV (SEQ ID NO: 3) B03-1HFR4 WGQGTLVTVSS (SEQ ID NO: 18) B03-1LFR1 DIVMTQSPSSLSASPGDRVTISC(SEQ ID NO:19) B03-1LCDR1 RASQDIRNYLN (SEQ ID NO: 4) B03-1LFR2 WYQQKPGQAPKLLIY (SEQ ID NO: 20) B03-1LCDR2 YTSRLHS (SEQ ID NO: 5) B03-1LFR3 GVPSRFSGSGSGTDFTLTISSLQPEDEATYYC(SEQ ID NO:21) B03-1LCDR3 QQGNTFPLT (SEQ ID NO: 6) B03-1LFR4 FGGGTKVEIK (SEQ ID NO: 22)

[0129] (II) Analysis of humanized antibody binding ability

[0130] (1) Analysis of humanized antibody-enhanced antibody binding ability

[0131] The variable region sequence of the humanized antibody B03-1 was combined with the heavy chain and light chain constant regions of the human antibody to construct the full-length B03-1 antibody (the amino acid sequence of the human antibody heavy chain constant region is shown in SEQ ID NO: 34, and the amino acid sequence of the human antibody light chain constant region is shown in SEQ ID NO: 35). The antibody was purified according to the expression and purification method described in patent CN117264050A (according to the method described in paragraphs

[0130] to

[0136] of the specification). After purification, the antibody was analyzed for its binding ability to CD155 recombinant protein and CD155 overexpressing cell lines (according to the above-mentioned analysis method for the B03 antibody). Figure 5 and Figure 6 As shown, the humanized B03-1 antibody significantly enhanced its binding ability to CD155 protein and overexpressing cell lines, with the EC50 value for binding to CD155 recombinant protein being 0.315 nM and the EC50 value for binding to CD155 overexpressing cell lines being 12.20 nM.

[0132] (2) Analysis of the binding ability of humanized antibodies to monkey CD155

[0133] Referring to the flow cytometry analysis method in the analysis of the ability of antibodies to bind to cell lines overexpressing CD155 in step 3 (2), 293T-rCD155 cells were subjected to flow cytometry analysis using B03-1 antibody at concentrations of 1ug / ml, 5ug / ml, and 10ug / ml, respectively. Figure 7 As shown, B03-1 antibody can significantly bind to 293T cells overexpressing monkey CD155 compared to the isotype control.

[0134] (III) Verification of the different effects of humanized antibody B03-1

[0135] (1) B03-1-mediated cellular internalization

[0136] Logarithmic phase THP-1 cells were resuspended and incubated with 1 μg / ml B03-1 full-length antibody for 1 hour, and then incubated at 37°C for 0 hour, 1 hour, 3 hours, and 6 hours. After internalization, the cells were fixed and human IgG (Jackson, 109-606-170) 2 μg / ml was added and incubated for 1 hour. The cells were washed twice with PBS and subjected to flow cytometry. The results are shown in the figure. Figure 8 As shown, the cell surface fluorescence gradually decreased with time, and the surface antigen-antibody complex was gradually internalized.

[0137] (2) B03-1-mediated ADCC

[0138] NK cells were isolated from the peripheral blood of healthy blood donors using a human NK cell enrichment kit (Miltenyi, 130092657) and incubated overnight in a culture medium containing 500 IU / ml human recombinant IL-2 (Peprotech, 200-02). THP-1 cells in the logarithmic growth phase were stained and washed with CTV (invitrogen, C34557). NK cells and THP-1 cells were mixed at an effector-target ratio of 4:1, 2:1, and 1:1, and cultured at 37°C for 6 hours. The killing efficiency was detected by flow cytometry using Annexin V-APC. The percentage of CTV+Annexin V-APC+ positive cells was the cell killing efficiency. Figure 9 As shown, at different concentrations, the B03-1 full-length antibody significantly promoted the cytotoxic effect of NK cells compared with the isotype control antibody.

[0139] (3) B03-1-mediated ADCP effect

[0140] M2 macrophages were differentiated in vitro according to the method described in Example 11 of patent CN 114423787A. B03-1 antibody was diluted to a final concentration of 1 μg / ml and 5 μg / ml in DMEM + 10% FBS + 1% double-antibody culture medium. A549 cells in the logarithmic growth phase were collected and labeled with CTV dye, washed with PBS, and resuspended in antibody solution for counting 1*10 5 cells , the final volume was 50ul, and 5*10 macrophages were added 4 cells The final volume was 100ul, and the cells were cultured in a 37℃ incubator for 4 hours. The cells were resuspended and labeled with APC-labeled anti-human CD11b antibody for flow cytometry analysis. The total macrophages were APC-CD11b+ positive; the macrophages undergoing phagocytosis were APC-CD11b+CTV+ double positive; the antibody-dependent cellular phagocytosis efficiency (%) = the number of macrophages undergoing phagocytosis / the total number of macrophages*100%. Figure 10 and Figure 11 As shown, at different concentrations, B03-1 antibody significantly promoted the phagocytosis of macrophages.

[0141] 5. Construction and verification of CAR plasmid based on humanized B03-1

[0142] (I) Construction of CAR plasmid targeting CD155

[0143] The B03-1 scFv antibody sequence was sent to Yunzhou Biovector for CAR structure plasmid construction. The plasmid sequence is shown in Table 3 below, and the vector map is shown in Figure 12As shown, the punctured bacteria were obtained from the company, and then the plasmid was extracted by streaking and shaking according to the general molecular biology method; the virus packaging method (three (two)) described above was used to obtain the virus supernatant.

[0144] Table 3 CAR structure vector sequence

[0145]

[0146] Note: In 6272B03-1, the heavy chain variable region and the light chain variable region are connected by a (G4S) 3-link sequence.

[0147] (2) CAR-T cell construction

[0148] Lymphocytes were separated by gradient centrifugation. After centrifugation, the second white lymphocyte layer was collected, washed with saline, and cultured in RPMI 1640 complete medium containing 10% FBS to obtain human PBMCs. The obtained PBMCs were activated with anti-CD3.CD28 monoclonal antibodies for 24 hours, and the viral supernatant obtained above was added to the activated cells for infection. The CAR-T positivity rate was detected on the third day of viral infection (the detection method was flow cytometry, and the detection protein was biotinylated human CD155 protein and streptavidin-APC fluorescent secondary antibody (biolegend, 405207). The ScFv sequence in the CAR antigen recognition region can recognize biotinylated human CD155 protein, and biotin can bind to the streptavidin-APC fluorescent secondary antibody. Therefore, the APC fluorescent signal can be used to detect the CAR positivity rate and CAR expression intensity). Three days after viral transduction, the CAR-T positivity rate was 65.2%.

[0149] (III) In vitro killing ability test of different tumor cells

[0150] Tumor cell lines MOLM13 (human acute myeloid leukemia cell line), THP-1 (human monocytic leukemia), HepG2 (human liver cancer cells), U251 (human glioma cells), MDA-MB-231 (human breast cancer cells), HT29 (human colon cancer cells), A549 (human non-small cell lung cancer cells), and Capan-1 (human pancreatic cancer cells) were used as positive target cells. CAR-T cells or control T cells were plated on the target cells at an effector-target ratio of 1:2. After 4 hours, the killing efficiency was detected by flow cytometry staining of DAPI and Annexin V-APC. The Annexin V-APC positive percentage was the cell killing efficiency. The killing efficiency of CAR-T cells against various tumor cell lines is shown in Table 4 below.

[0151] Table 4 Killing effect of CAR-T cells on different tumor cells

[0152] Tumor cell lines CD155CAR-T killing efficiency (%) Killing efficiency of controlT (%) MOLM13 62.50 14.61 THP-1 59.32 14.21 HepG2 49.10 13.60 U251 33.52 4.26 MDA-MB-231 46.30 8.52 HT29 41.52 12.86 A549 16.58 3.63 Capan-1 11.25 2.58

[0153] As can be seen from Table 1 above, the constructed targeted CD155 CAR-T cells have a significant killing effect on MOLM13 (human acute myeloid leukemia cell line), U937 (human histiocytic lymphoma cells), THP-1 (human monocytic leukemia), HepG2 (human liver cancer cells), U251 (human glioma cells), MDA-MB-231 (human breast cancer cells), HT29 (human colon cancer cells), A549 (human non-small cell lung cancer cells), and Capan-1 (human pancreatic cancer cells).

[0154] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be covered by the scope of the claims of the present invention.

Claims

1. An antibody or antigen-binding fragment targeting CD155, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3; characterized in that: The amino acid sequence of HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 3; the amino acid sequence of LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

6.

2. The antibody or antigen-binding fragment targeting CD155 according to claim 1, characterized in that The heavy chain variable region further comprises: an HFR1 having at least 80% identity to the sequence shown in SEQ ID NO: 7 and / or an HFR2 having at least 80% identity to the sequence shown in SEQ ID NO: 8 and / or an HFR3 having at least 80% identity to the sequence shown in SEQ ID NO: 9 and / or an HFR4 having at least 80% identity to the sequence shown in SEQ ID NO: 10; and / or The light chain variable region also includes: LFR1 that is at least 80% identical to the sequence shown in SEQ ID NO:11 and / or LFR2 that is at least 80% identical to the sequence shown in SEQ ID NO:12 and / or LFR3 that is at least 80% identical to the sequence shown in SEQ ID NO:13 and / or LFR4 that is at least 80% identical to the sequence shown in SEQ ID NO:

14.

3. The antibody or antigen-binding fragment targeting CD155 according to claim 1, characterized in that The heavy chain variable region further comprises: an HFR1 having at least 80% identity to the sequence shown in SEQ ID NO: 15 and / or an HFR2 having at least 80% identity to the sequence shown in SEQ ID NO: 16 and / or an HFR3 having at least 80% identity to the sequence shown in SEQ ID NO: 17 and / or an HFR4 having at least 80% identity to the sequence shown in SEQ ID NO: 18; and / or The light chain variable region also includes: LFR1 that is at least 80% identical to the sequence shown in SEQ ID NO:19 and / or LFR2 that is at least 80% identical to the sequence shown in SEQ ID NO:20 and / or LFR3 that is at least 80% identical to the sequence shown in SEQ ID NO:21 and / or LFR4 that is at least 80% identical to the sequence shown in SEQ ID NO:

22.

4. The antibody or antigen-binding fragment targeting CD155 according to claim 1, wherein The antibody or antigen-binding fragment targeting CD155 is selected from any one of the following antibodies: (a) an antibody targeting CD155 comprising: a heavy chain variable region having at least 70% identity to the sequence shown in SEQ ID NO: 23 and a light chain variable region having at least 70% identity to the sequence shown in SEQ ID NO: 24; (b) An antibody targeting CD155 comprises a heavy chain variable region that is at least 70% identical to the sequence shown in SEQ ID NO: 25 and a light chain variable region that is at least 70% identical to the sequence shown in SEQ ID NO:

26.

5. The antibody or antigen-binding fragment targeting CD155 according to any one of claims 1 to 4, characterized in that The antibody or antigen-binding fragment targeting CD155 further comprises a heavy chain constant region and / or a light chain constant region, at least a portion of which is derived from at least one of a human antibody, a primate antibody, or a mutant thereof.

6. The antibody or antigen-binding fragment targeting CD155 according to claim 5, characterized in that The heavy chain constant region and the light chain constant region are both derived from human IgG antibodies or their mutants.

7. The antibody or antigen-binding fragment targeting CD155 according to claim 6, characterized in that The heavy chain constant region and the light chain constant region are both derived from human IgG1 antibody, human IgG4 antibody or mutants thereof.

8. The antibody or antigen-binding fragment targeting CD155 according to claim 5, characterized in that The heavy chain constant region comprises a sequence at least 70% identical to SEQ ID NO:34, and the light chain constant region comprises a sequence at least 70% identical to SEQ ID NO:

35. 9.CAR structure, characterized in that The invention comprises the antibody or antigen-binding fragment targeting CD155 according to any one of claims 1 to 8.

10. The CAR structure according to claim 9, characterized in that: The CAR structure also includes: a hinge region having at least 80% identity to the amino acid sequence encoded by the sequence shown in SEQ ID NO: 27 and / or a transmembrane region and / or an intracellular signaling domain having at least 80% identity to the amino acid sequence encoded by the sequence shown in SEQ ID NO: 28; the intracellular signaling domain includes an intracellular costimulatory domain having at least 80% identity to the amino acid sequence encoded by the sequence shown in SEQ ID NO: 29 and an intracellular signaling domain having at least 80% identity to the amino acid sequence encoded by the sequence shown in SEQ ID NO:

30.

11. Any of the following substances: (i) a nucleic acid molecule encoding the antibody or antigen-binding fragment targeting CD155 according to any one of claims 1 to 8 or encoding the CAR structure according to any one of claims 9 to 10; (ii) an expression vector comprising the nucleic acid molecule of (i); (iii) an engineered cell comprising the nucleic acid molecule of (i) and / or the expression vector of (ii); (iv) a product for detecting CD155, comprising the antibody or antigen-binding fragment targeting CD155 according to any one of claims 1 to 8; (v) a pharmaceutical composition comprising the antibody or antigen-binding fragment targeting CD155 according to any one of claims 1 to 8 and / or the CAR structure according to any one of claims 9 to 10 and / or the nucleic acid molecule in (i) and / or the expression vector in (ii) and / or the engineered cell in (iii); (vi) A pharmaceutical preparation comprising an antibody or antigen-binding fragment targeting CD155 according to any one of claims 1 to 8 and / or a CAR structure according to any one of claims 9 to 10 and / or the nucleic acid molecule in (i) and / or the expression vector in (ii) and / or the engineered cell in (iii) and / or the pharmaceutical composition in (v).

12. Use of the antibody or antigen-binding fragment targeting CD155 according to any one of claims 1 to 8 in the preparation of a product for detecting CD155 levels.

13. Use of an antibody or antigen-binding fragment targeting CD155 as described in any one of claims 1 to 8 and / or a CAR structure as described in any one of claims 9 to 10 and / or a nucleic acid molecule and / or an expression vector and / or an engineered cell as described in claim 11 in the preparation of a drug, the drug being used to prevent and / or treat a CD155-related disease, wherein the CD155-related disease is at least one of acute myeloid leukemia, histiocytic lymphoma, monocytic leukemia, non-small cell lung cancer, liver cancer, pancreatic cancer, colon cancer, breast cancer, and glioma.

14. The application according to claim 13, wherein the application comprises one or more combinations of the following applications: (a) Use of the antibody or antigen-binding fragment targeting CD155 according to any one of claims 1 to 8 in mediating cellular internalization; (b) Use of the antibody or antigen-binding fragment targeting CD155 according to any one of claims 1 to 8 in promoting the killing and toxic effects of NK cells; (c) Use of the antibody or antigen-binding fragment targeting CD155 according to any one of claims 1 to 8 in promoting phagocytosis of macrophages.

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