CD155-targeted antigen-binding protein and use thereof

Abstract

A CD155-targeted antigen-binding protein and the use thereof. The present application specifically relates to an isolated antigen-binding protein capable of binding to CD155, the antigen-binding protein comprising at least one CDR in a heavy chain variable region of an antibody. Provided are a chimeric antigen receptor comprising the antigen-binding protein, an immunoconjugate, a pharmaceutical composition, a pharmaceutical combination, a nucleic acid encoding the antigen-binding protein, a vector comprising the nucleic acid molecule, a cell comprising the vector, and the use of the antigen-binding protein in the prevention and / or treatment of diseases.

Description

Antigen binding proteins targeting CD155 and uses thereof TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to an antigen binding protein targeting CD155 and uses thereof. BACKGROUND

[0002] CD155, also known as PVR (Poliovirus Receptor) or Necl-5 (Nectin-like molecule 5), is a transmembrane glycoprotein belonging to the immunoglobulin superfamily (IgSF), composed of two immunoglobulin-like C2-type domains and one immunoglobulin-like V-type domain, and is a protein required for poliovirus binding and uptake. It is mainly expressed in various cell types in the human body, including immune cells, neural cells and tumor cells, etc. CD155 has diverse functions, it is one of the receptors for viral entry into host cells, and also participates in various physiological and pathological processes.

[0003] In the field of immunology, CD155 was found to be overexpressed in the tumor microenvironment, especially on the surface of tumor cells. This overexpression is related to the immune escape of tumors, because after CD155 binds to the inhibitory receptors (immune checkpoints) of T cells such as TIGIT (T cell immunoreceptor with Ig and ITIM domains), it inhibits the activation and killing effect of T cells, thereby protecting tumor cells from the attack of the immune system. Tumors can be treated by inhibiting the interaction between CD155 and T cell inhibitory receptors.

[0004] Therefore, as a highly potential target for tumor treatment, CD155 needs to develop more effective therapeutic drugs that can effectively bind to it to meet the treatment needs of the majority of diseases. SUMMARY

[0005] The present application provides an antigen binding protein targeting CD155. In the present application, the antigen binding protein has one or more of the following properties: (1) capable of binding CD155 and having good binding activity; (2) capable of binding CD155 expressed on the surface of a target cell; (3) capable of killing tumors; (4) capable of blocking the binding of CD155 to its receptor. The present application also provides a chimeric antigen receptor comprising the antigen binding protein, and a cell comprising and / or expressing the chimeric antigen receptor, which has one or more of the following properties: (1) capable of specifically binding CD155 and having good binding activity; (2) capable of specifically binding CD155 expressed on the surface of a target cell; (3) capable of killing tumors; (4) capable of blocking the binding of CD155 to its receptor; (5) capable of secreting more cytokines under the stimulation of target cells; (6) capable of targeting proliferation.

[0006] In one aspect, the present application provides an isolated antigen binding protein capable of binding CD155, wherein the antigen binding protein comprises an antibody heavy chain variable region VH, which comprises heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 59 or SEQ ID NO: 15.

[0007] In certain embodiments, the amino acid sequence of the HCDR3 is shown in any one of SEQ ID NOs: 14-19.

[0008] In certain embodiments, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 58 or SEQ ID NO: 9.

[0009] In certain embodiments, the amino acid sequence of the HCDR2 is shown in any one of SEQ ID NOs: 7-13.

[0010] In certain embodiments, the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 57 or SEQ ID NO: 3.

[0011] In certain embodiments, the amino acid sequence of the HCDR1 is shown in any one of SEQ ID NOs: 1-6.

[0012] In certain embodiments, the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 57, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 58, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 59; or the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 3, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 9, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 15.

[0013] In certain embodiments, the amino acid sequences of the HCDR1, HCDR2, and HCDR3 are selected from any one of the following combinations:

[0014] (1) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 7, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 14;

[0015] (2) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 8, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 14;

[0016] (3) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 3, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 9, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 15;

[0017] (4) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 4, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 10, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 16;

[0018] (5) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 4, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 13, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 19;

[0019] (6) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 5, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 11, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 17; and

[0020] (7) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 6, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 12, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 18.

[0021] In certain embodiments, the VH comprises H-FR1, H-FR2, H-FR3, and H-FR4, the amino acid sequence of the H-FR1 is set forth in any one of SEQ ID NOs: 20-24, the amino acid sequence of the H-FR2 is set forth in any one of SEQ ID NOs: 25-30, the amino acid sequence of the H-FR3 is set forth in any one of SEQ ID NOs: 31-37, and the amino acid sequence of the H-FR4 is set forth in any one of SEQ ID NOs: 38-39.

[0022] In certain embodiments, the VH comprises H-FR1, H-FR2, H-FR3, and H-FR4, the amino acid sequence of the H-FR1, H-FR2, H-FR3, and H-FR4 is selected from any one of the following combinations:

[0023] (1) the amino acid sequence of the H-FR1 is set forth in SEQ ID NO: 20, the amino acid sequence of the H-FR2 is set forth in SEQ ID NO: 25, the amino acid sequence of the H-FR3 is set forth in SEQ ID NO: 31, and the amino acid sequence of the H-FR4 is set forth in SEQ ID NO: 38;

[0024] (2) the amino acid sequence of the H-FR1 is set forth in SEQ ID NO: 21, the amino acid sequence of the H-FR2 is set forth in SEQ ID NO: 26, the amino acid sequence of the H-FR3 is set forth in SEQ ID NO: 31, and the amino acid sequence of the H-FR4 is set forth in SEQ ID NO: 38;

[0025] (3) the amino acid sequence of the H-FR1 is set forth in SEQ ID NO: 23, the amino acid sequence of the H-FR2 is set forth in SEQ ID NO: 25, the amino acid sequence of the H-FR3 is set forth in SEQ ID NO: 31, and the amino acid sequence of the H-FR4 is set forth in SEQ ID NO: 38;

[0026] (4) the amino acid sequence of the H-FR1 is set forth in SEQ ID NO: 20, the amino acid sequence of the H-FR2 is set forth in SEQ ID NO: 25, the amino acid sequence of the H-FR3 is set forth in SEQ ID NO: 32, and the amino acid sequence of the H-FR4 is set forth in SEQ ID NO: 38;

[0027] (5) the amino acid sequence of the H-FR1 is shown as SEQ ID NO:21, the amino acid sequence of the H-FR2 is shown as SEQ ID NO:25, the amino acid sequence of the H-FR3 is shown as SEQ ID NO:32, and the amino acid sequence of the H-FR4 is shown as SEQ ID NO:38;

[0028] (6) the amino acid sequence of the H-FR1 is shown as SEQ ID NO:20, the amino acid sequence of the H-FR2 is shown as SEQ ID NO:29, the amino acid sequence of the H-FR3 is shown as SEQ ID NO:32, and the amino acid sequence of the H-FR4 is shown as SEQ ID NO:38;

[0029] (7) the amino acid sequence of the H-FR1 is shown as SEQ ID NO:22, the amino acid sequence of the H-FR2 is shown as SEQ ID NO:27, the amino acid sequence of the H-FR3 is shown as SEQ ID NO:33, and the amino acid sequence of the H-FR4 is shown as SEQ ID NO:38;

[0030] (8) the amino acid sequence of the H-FR1 is shown as SEQ ID NO:20, the amino acid sequence of the H-FR2 is shown as SEQ ID NO:28, the amino acid sequence of the H-FR3 is shown as SEQ ID NO:34, and the amino acid sequence of the H-FR4 is shown as SEQ ID NO:38;

[0031] (9) the amino acid sequence of the H-FR1 is shown as SEQ ID NO:24, the amino acid sequence of the H-FR2 is shown as SEQ ID NO:27, the amino acid sequence of the H-FR3 is shown as SEQ ID NO:35, and the amino acid sequence of the H-FR4 is shown as SEQ ID NO:38;

[0032] (10) the amino acid sequence of the H-FR1 is shown as SEQ ID NO:20, the amino acid sequence of the H-FR2 is shown as SEQ ID NO:30, the amino acid sequence of the H-FR3 is shown as SEQ ID NO:36, and the amino acid sequence of the H-FR4 is shown as SEQ ID NO:39; and

[0033] (11) the amino acid sequence of the H-FR1 is set forth in SEQ ID NO: 20, the amino acid sequence of the H-FR2 is set forth in SEQ ID NO: 30, the amino acid sequence of the H-FR3 is set forth in SEQ ID NO: 37, and the amino acid sequence of the H-FR4 is set forth in SEQ ID NO: 39.

[0034] In certain embodiments, the amino acid sequence of the VH is set forth in any one of SEQ ID NOs: 43-54.

[0035] In certain embodiments, the antigen binding protein further comprises an immunoglobulin constant region.

[0036] In certain embodiments, the immunoglobulin constant region is an immunoglobulin Fc region.

[0037] In certain embodiments, the immunoglobulin constant region is an IgG Fc region.

[0038] In certain embodiments, the antigen binding protein is an antibody or an antigen binding fragment thereof.

[0039] In certain embodiments, the antigen binding fragment is a Fab, (Fab)2, F(ab’)2, scFv, di-scFv, Fv, VHH, or dAb fragment of the antibody.

[0040] In certain embodiments, the antigen binding protein is a VHH.

[0041] In certain embodiments, the antigen binding protein is a chimeric antibody, a humanized antibody, or a fully human antibody.

[0042] In certain embodiments, the antigen binding protein is a monovalent antibody, a bivalent antibody, or a multivalent antibody.

[0043] In certain embodiments, the antigen binding protein is a monospecific antibody, a bispecific antibody, or a multispecific antibody.

[0044] In certain embodiments, the antigen binding protein is a bispecific antibody, the bispecific antibody comprising a first targeting moiety and a second targeting moiety, the first targeting moiety comprising the antigen binding protein, the antigen binding protein being capable of binding CD155.

[0045] In certain embodiments, the first targeting moiety comprises a VHH capable of binding CD155, the amino acid sequence of the VHH being set forth in any one of SEQ ID NOs: 43-54.

[0046] In certain embodiments, the second targeting moiety is capable of binding an immune checkpoint target.

[0047] In some embodiments, the second targeting moiety is capable of binding PD-L1.

[0048] In some embodiments, the second targeting moiety comprises an antigen binding protein capable of binding PD-L1.

[0049] In some embodiments, the second targeting moiety comprises an antibody or antigen binding fragment thereof capable of binding PD-L1.

[0050] In some embodiments, the second targeting moiety comprises HCDR1-3 and LCDR1-3, the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 64, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 66, the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 68, the amino acid sequence of the LCDR1 is set forth in SEQ ID NO: 71, the amino acid sequence of the LCDR2 is set forth in SEQ ID NO: 73, and the amino acid sequence of the LCDR3 is set forth in SEQ ID NO: 75.

[0051] In some embodiments, the second targeting moiety comprises a VH and a VL, the amino acid sequence of the VH is set forth in SEQ ID NO: 42, and the amino acid sequence of the VL is set forth in SEQ ID NO: 55.

[0052] In some embodiments, the second targeting moiety comprises a heavy chain constant region and a light chain constant region.

[0053] In some embodiments, the second targeting moiety comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is set forth in SEQ ID NO: 69, and the amino acid sequence of the light chain is set forth in SEQ ID NO: 77.

[0054] In some embodiments, the second targeting moiety is located at the C-terminus of the first targeting moiety.

[0055] In some embodiments, the first targeting moiety and the second targeting moiety are connected by a linker.

[0056] In some embodiments, the bispecific antibody comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the heavy chain variable region VH capable of binding PD-L1 and a heavy chain constant region CH, and the VHH capable of binding CD155; wherein the second polypeptide chain comprises the light chain variable region VL capable of binding PD-L1 and a light chain constant region CL.

[0057] In some embodiments, the heavy chain constant region CH and the VHH capable of binding CD155 are connected by a linker.

[0058] In some embodiments, the amino acid sequence of the first polypeptide chain is set forth in SEQ ID NO: 78, and the amino acid sequence of the second polypeptide chain is set forth in SEQ ID NO: 77.

[0059] In another aspect, the present application also provides a chimeric antigen receptor comprising the antigen binding protein.

[0060] In some embodiments, the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain.

[0061] In some embodiments, the antigen binding domain comprises the antigen binding protein.

[0062] In some embodiments, the transmembrane domain comprises a transmembrane domain derived from any one of the following proteins: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 epsilon, CD3 zeta, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc epsilon RI gamma, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

[0063] In some embodiments, the transmembrane domain is derived from a transmembrane domain of CD8 alpha.

[0064] In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain derived from any one of the following proteins: CD3 zeta, CD3 delta, CD3 gamma, CD3 epsilon, CD79a, CD79b, Fc epsilon RI gamma, Fc epsilon RI beta, Fc gamma RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM.

[0065] In some embodiments, the intracellular signaling domain is derived from an intracellular signaling domain of CD3 zeta.

[0066] In some embodiments, the chimeric antigen receptor further comprises a costimulatory domain.

[0067] In certain embodiments, the co-stimulatory domain comprises a co-stimulatory domain derived from one or more of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc epsilon RIGAM, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand for CD83, CD40, and MyD88.

[0068] In certain embodiments, the co-stimulatory domain is derived from a co-stimulatory domain of 4-1BB.

[0069] In certain embodiments, the chimeric antigen receptor further comprises a hinge region.

[0070] In certain embodiments, the hinge region comprises a hinge region derived from any one of the following proteins: CD28, IgGl, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc epsilon RIGAM, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

[0071] In certain embodiments, the hinge region is derived from a hinge region of CD8a.

[0072] In another aspect, the present application also provides a modified immune cell, wherein the immune cell comprises the chimeric antigen receptor.

[0073] In certain embodiments, the immune cell is a T cell, an NK cell, an iNKT cell, a dendritic cell, and / or a macrophage.

[0074] In certain embodiments, the modified immune cell further comprises and / or expresses a low density lipoprotein receptor-related protein or a fragment thereof.

[0075] In certain embodiments, the low density lipoprotein receptor-related protein or a fragment thereof comprises one or more selected from the group consisting of low density lipoprotein receptor-related proteins 1-12 and functional fragments thereof.

[0076] In certain embodiments, the low density lipoprotein receptor-related protein or a fragment thereof is low density lipoprotein receptor-related protein 5 and / or 6 or a fragment thereof.

[0077] In some embodiments, the low density lipoprotein receptor-related protein or fragment thereof comprises an amino acid sequence as set forth in SEQ ID NO: 60.

[0078] In some embodiments, the low density lipoprotein receptor-related protein or fragment thereof is an exogenous low density lipoprotein receptor-related protein or fragment thereof.

[0079] In some embodiments, the antigen binding protein is an antibody.

[0080] In some embodiments, the antigen binding protein is an antibody.

[0081] In some embodiments, the antigen binding protein is an antibody.

[0082] In some embodiments, the antigen binding protein is an antibody.

[0083] In some embodiments, the antigen binding protein is an antibody.

[0084] In some embodiments, the antigen binding protein is an antibody.

[0085] In some embodiments, the antigen binding protein is an antibody.

[0086] In some embodiments, the antigen binding protein is an antibody.

[0087] In some embodiments, the antigen binding protein is an antibody.

[0088] In some embodiments, the antigen binding protein is an antibody.

[0089] In another aspect, the present application also provides use of the antigen binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition in the manufacture of a medicament for preventing and / or treating a tumor.

[0090] In certain embodiments, the tumor is a solid tumor and / or a hematological tumor.

[0091] In certain embodiments, the tumor is positive for CD155 expression.

[0092] In certain embodiments, the tumor is a melanoma, a lung cancer, a liver cancer, an ovarian cancer, a cervical cancer, a skin cancer, a bladder cancer, a colon cancer, a breast cancer, a glioma, a kidney cancer, a gastric cancer, an esophageal cancer, an oral squamous cell carcinoma, a head and neck cancer, a lymphoma, a multiple myeloma and / or a leukemia.

[0093] In another aspect, the present application also provides a method for preventing and / or treating a tumor, comprising administering the antigen binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition to a subject in need thereof.

[0094] In certain embodiments, the tumor is a solid tumor and / or a hematological tumor.

[0095] In certain embodiments, the tumor is positive for CD155 expression.

[0096] In certain embodiments, the tumor is a melanoma, a lung cancer, a liver cancer, an ovarian cancer, a cervical cancer, a skin cancer, a bladder cancer, a colon cancer, a breast cancer, a glioma, a kidney cancer, a gastric cancer, an esophageal cancer, an oral squamous cell carcinoma, a head and neck cancer, a lymphoma, a multiple myeloma and / or a leukemia.

[0097] In another aspect, the present application also provides the antigen binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition for use in preventing and / or treating a tumor.

[0098] In certain embodiments, the tumor is a solid tumor and / or a hematological tumor.

[0099] In certain embodiments, the tumor is positive for CD155 expression.

[0100] In some embodiments, the tumor is melanoma, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, head and neck cancer, lymphoma, multiple myeloma, and / or leukemia.

[0101] Other aspects and advantages of the present application will become apparent from the following detailed description, taken in conjunction with the accompanying drawings. Only illustrative embodiments of the present application are described below. As will be realized by one of ordinary skill in the art, the application is capable of modifications in various obvious aspects, all without departing from the spirit and scope of the underlying inventive concept. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not restrictive. BRIEF DESCRIPTION OF DRAWINGS

[0102] The specific features of the application are set forth in the appended claims. A better understanding of the features and advantages of the present application will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the application are utilized, and the accompanying drawings of which:

[0103] FIGS. 1A-1B show the results of Pool ELISA verification of the CD155 antibody described herein.

[0104] FIG. 2 shows the results of the binding activity of the CD155 antibody described herein to human CD155 protein.

[0105] FIG. 3 shows the results of the binding activity of the CD155 antibody described herein to human CD155 transfected CHO cells.

[0106] FIG. 4 shows the results of the blocking ability of the CD155 antibody described herein on the interaction between TIGIT and CD155.

[0107] FIG. 5 shows the results of the binding activity of the humanized CD155 antibody described herein to human CD155 transfected CHO cells.

[0108] FIG. 6 shows the results of the blocking ability of the humanized CD155 antibody described herein on the interaction between TIGIT and CD155.

[0109] FIG. 7 shows the results of the in vitro expansion of the CD155 CAR-T cells described herein.

[0110] FIG. 8 shows the results of the in vitro targeting killing effect of the CD155 CAR-T cells described herein.

[0111] Figure 9 shows the results of IL-2 secretion from co-culture of CD155 CAR-T cells and target cells according to the present application.

[0112] Figure 10 shows the results of IFN-γ secretion from co-culture of CD155 CAR-T cells and target cells according to the present application.

[0113] Figure 11A shows the structure of CD155-PD-L1 bispecific antibody according to the present application; Figure 11B shows the results of anti-tumor effect of CD155 antibody and CD155-PD-L1 bispecific antibody according to the present application; and Figure 11C shows the results of up-regulation of CD226 expression level by CD155 antibody according to the present application. DETAILED DESCRIPTION

[0114] The following specific examples illustrate the embodiments of the present application, and other advantages and effects of the present application can be easily understood by those skilled in the art from the disclosure of the present specification.

[0115] Definitions of terms

[0116] In the present application, the terms "CD155", "PVR" and "Necl-5" can be used interchangeably, and generally refer to a transmembrane glycoprotein belonging to the immunoglobulin superfamily, consisting of two immunoglobulin-like C2-type domains and one immunoglobulin-like V-type domain, which is a protein required for poliovirus binding and uptake. In the present application, the CD155 receptor can recognize and / or bind to CD155. For example, the CD155 receptor can be an immunoglobulin superfamily receptor. For example, the CD155 receptor can be TIGIT, CD226 (DNAM-1) or CD96. In the present application, the CD155 can be intact CD155 and functionally active fragments, homologues, analogues, variants or derivatives thereof. For example, the CD155 can be full-length CD155 or truncated CD155 retaining functional activity. In the present application, the CD155 can be of any species origin. For example, the CD155 can be human CD155. In the present application, the CD155 can be wild type or artificially modified. For example, the CD155 can be modified CD155.

[0117] In the present application, the term "antigen binding protein" generally refers to a protein having the ability to bind an antigen. In the present application, the antigen binding protein can comprise an antigen binding moiety and optionally, a scaffold or framework moiety that allows the antigen binding moiety to adopt a conformation that facilitates the antigen binding moiety binding to an antigen. In the present application, the antigen binding protein can be wild type or artificially engineered. In the present application, the antigen binding protein can comprise a protein scaffold of antibody origin or an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives. In the present application, the CDRs can be determined by various encoding systems. For example, the CDRs can be determined by CCG, Kabat, Chothia, IMGT, AbM, integrated consideration of Kabat / Chothia, etc. For example, the CDRs can be determined by the Kabat encoding system. For example, the CDRs can be determined by the IMGT encoding system. In the present application, the CDRs encompass CDR sequences determined according to any CDR partitioning scheme. In the present application, the CDRs can encompass variants thereof. For example, the CDRs can have one or more amino acids, e.g., 1-30, 1-20, or 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions. For example, the CDRs encompass homologues. For example, the homologues can be amino acid sequences having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more) sequence homology to the amino acid sequence of the CDRs. In the present application, the antigen binding protein can be secreted by an immune cell or artificially synthesized. For example, the antigen binding protein can be secreted by an immune cell, e.g., an effector B cell. For example, the antigen binding protein can be derived from plasma, a hybridoma, or a recombinant cell line. In the present application, the antigen binding protein can be of animal origin or non-animal origin. For example, the antigen binding protein can be of camelid origin. For example, the antigen binding protein can be of human origin. In the present application, the antigen binding protein can be an antibody or an antigen binding fragment thereof, as well as variants, homologues, derivatives, or analogues thereof. In the present application, antigen binding proteins can include, but are not limited to, antibodies, antigen binding fragments (Fabs, Fab’s, F(ab)2s, Fv fragments, F(ab’)2s, scFvs, di-scFvs, VHHs, and / or dAbs), immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogues, or fusion proteins, etc., as long as they exhibit the desired antigen binding activity. In the present application, the antibody can be a chimeric antibody, a humanized antibody, or a fully human antibody.In the present application, the antibody can be a recombinant, hybrid, mutated or grafted antibody. In the present application, the antigen binding protein can be a VHH. In the present application, the antigen binding protein can specifically bind to CD155. In the present application, the antigen binding protein can block the binding of CD155 to its receptor.

[0118] In the present application, the terms "VHH", "nanobody" and "single domain antibody" can be used interchangeably, and generally refer to an antibody structure consisting of a heavy chain variable region. In the present application, the VHH can consist of VH. In the present application, the VHH can comprise complementarity determining regions (CDRs) and framework regions (FRs). In the present application, the VHH comprises three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. In the present application, the VHH can be secreted by immune cells or artificially synthesized. In the present application, the VHH can be of animal origin or non-animal origin. In the present application, the VHH can be a chimeric antibody, a humanized antibody or a fully human antibody.

[0119] In the present application, the terms "immunoglobulin constant region" and "constant region" can be used interchangeably, and the constant region refers to the region of the antibody non-variable region. In the present application, the constant region is not directly involved in binding to the antigen, but shows various effector functions. In the present application, the constant region can be all or part of the antibody non-variable region. For example, the constant region can be a heavy chain constant region. For example, the constant region can be an Fc region. In the present application, the constant region can be derived from animals. For example, the constant region can be derived from humans, goats, rabbits, rats or guinea pigs. For example, the Fc region can be a human Fc region.

[0120] In the present application, the terms "bispecific antibody" and "BsAb" are generally used interchangeably and generally refer to an antibody or antibody construct having dual specificity in its binding arms. In the present application, the bispecific antibody can be obtained from two different sources and constructed by recombinant DNA or cell fusion technology. In the present application, the bispecific antibody can have two different antigen binding sites. In the present application, the bispecific antibody can interact with the same or two cell surface antigens. In the present application, the bispecific antibody can simultaneously block two different mediators / pathways that play unique or overlapping functions in pathogenesis. In the present application, the bispecific antibody can comprise a first targeting moiety and a second targeting moiety. In the present application, the first targeting moiety and / or the second targeting moiety can be an antigen binding protein. In the present application, the first targeting moiety and / or the second targeting moiety can comprise an antibody or an antigen binding fragment thereof. For example, the first targeting moiety can comprise a VHH. For example, the second targeting moiety can comprise an antibody. In the present application, the bispecific antibody can comprise a first polypeptide chain and a second polypeptide chain. In the present application, the first polypeptide chain can be a heavy chain and the second polypeptide chain can be a light chain. In the present application, the bispecific antibody can further comprise a constant region. For example, the bispecific antibody can further comprise a human IgG constant region.

[0121] The proteins and / or amino acid sequences referred to in the present application are also understood to encompass variants or homologues of the recited proteins having the same or similar function. In the present application, the variants can be proteins or polypeptides in which one or more amino acids have been substituted, deleted or added to the amino acid sequence of the recited protein (e.g., an antigen binding protein recited in the present application). For example, the functional variants can comprise proteins or polypeptides that have been altered by at least 1, e.g., 1-30, 1-20, or 1-10, and for example, 1, 2, 3, 4, or 5 amino acid substitutions, deletions and / or insertions. The functional variants can substantially retain the biological properties of the recited protein or the recited polypeptide prior to the alterations (e.g., substitutions, deletions or additions). For example, the functional variants can retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding ability) of the recited protein or the recited polypeptide prior to the alterations. For example, the substitutions can be conservative substitutions. In the present application, a portion of the amino acid sequence of the antigen binding protein can be homologous to the corresponding amino acid sequence in an antibody from a particular species or belonging to a particular class. For example, both the variable region and the constant portion of the antibody can be from the variable region and the constant region of an antibody of an animal species (e.g., human). In the present application, the homologues can be proteins or polypeptides having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more) sequence homology to the amino acid sequence of the recited protein and / or the recited polypeptide (e.g., an antigen binding protein recited in the present application).

[0122] In the present application, the terms "chimeric antigen receptor" and "CAR" are generally used interchangeably and generally refer to a recombinant polypeptide comprising at least an extracellular domain that specifically binds to an antigen or target, a transmembrane domain, and an intracellular domain. In the present application, the extracellular domain can comprise an antigen binding domain and a hinge region. In the present application, the extracellular domain can further comprise a signal peptide. In the present application, the intracellular domain can comprise an intracellular signaling domain. In the present application, the intracellular domain can further comprise one or more costimulatory domains. In the present application, the CAR can comprise, in order, an optional antigen binding domain, a hinge region, a transmembrane domain region, and / or a costimulatory domain. In the present application, the CAR can comprise, in order, an optional signal peptide, an antigen binding domain, a hinge region, a transmembrane domain region, a costimulatory domain, and / or an intracellular signaling domain. In the present application, the CAR can be secreted by a cell or artificially synthesized. In the present application, the chimeric antigen receptor can further comprise a low-density lipoprotein receptor-related protein or a fragment thereof.

[0123] In the present application, the term "signal peptide" generally refers to a leader sequence at the amino terminal (N-terminal) end of a nascent CAR, which can direct the nascent protein to the endoplasmic reticulum and express at the time of translation or post-translation. In the present application, the signal peptide can comprise a signal peptide derived from CD8.

[0124] In the present application, the term "hinge region" generally refers to a peptide, polypeptide, or protein molecule. In the present application, the hinge region can be a polypeptide molecule. In the present application, the hinge region can be flexible, allowing independent movement of the antigen binding domain. In the present application, the hinge region can be subdivided into upper, middle, and lower hinge domains. In the present application, the hinge region can comprise a hinge region derived from any one of the following proteins: CD28, IgGl, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc epsilon R gamma, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT. In the present application, the hinge region can comprise a hinge region derived from CD8a.

[0125] In the present application, the term "transmembrane domain" generally refers to a domain of a peptide, polypeptide, or protein that is capable of spanning the plasma membrane of a cell. These domains can be employed to anchor an extracellular domain to the cell membrane. For example, the transmembrane region can comprise a transmembrane domain derived from any one of the following proteins: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 epsilon, CD3 zeta, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc epsilon R gamma, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM. For example, the transmembrane domain can comprise a transmembrane domain derived from CD8a.

[0126] In the present application, the term "co-stimulatory domain" generally refers to a portion of a CAR that is capable of transducing an effector signal in the intracellular signaling region. For example, the co-stimulatory domain can include a co-stimulatory domain derived from one or more proteins selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc epsilon R gamma, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, ligand for CD83, CD40, and MyD88. For example, the co-stimulatory domain can be a co-stimulatory domain derived from 4-1BB.

[0127] In the present application, the term "intracellular signaling domain" generally refers to an intracellular region that can generate a signal that promotes immune effector function of a CAR-containing cell (e.g., a CAR-T cell or a CAR-iNKT cell). For example, the intracellular signaling region can be derived from the intracellular signaling region of any one of the following proteins: CD3 zeta, CD3 delta, CD3 gamma, CD3 epsilon, CD79a, CD79b, Fc epsilon R gamma, Fc epsilon R beta, Fc gamma RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM. For example, the intracellular signaling region can be a signaling domain derived from CD3 zeta.

[0128] In the present application, the term "immune cell" generally refers to a cell that participates in an immune response. For example, the immune cell can be an immune cell that exerts an effector function. For example, the exerting an effector function can include clearing a foreign antigen or promoting an immune effector response, etc. In the present application, the immune effector cell can include a T cell, a B cell, a natural killer cell (NK cell), a macrophage, an NKT cell, an iNKT cell, a dendritic cell, a granulocyte, a lymphocyte, a leukocyte, a peripheral blood mononuclear cell, an embryonic stem cell, a lymphoid progenitor cell, and / or a pluripotent stem cell. For example, the immune effector cell can be a T cell.

[0129] In the present application, the term "low-density lipoprotein receptor-related protein" refers to a cell surface protein belonging to endocytic receptors, which is widely distributed in the body and has great inter-tissue differences, and the main function is to uptake cholesterol into cells for cell proliferation and synthesis of sterol hormones and bile acid salts. For example, the low-density lipoprotein receptor-related protein can be from any vertebrate. For example, the low-density lipoprotein receptor-related protein or its fragment can be located at the C-terminal of the intracellular signal region. For example, the low-density lipoprotein receptor-related protein or its fragment can comprise one or more selected from the group consisting of low-density lipoprotein receptor-related proteins 1-12 and functional fragments thereof. For example, the low-density lipoprotein receptor-related protein or its fragment can be low-density lipoprotein receptor-related protein 6 or a fragment thereof.

[0130] In the present application, the term "immunoconjugate" generally refers to a conjugate formed by conjugating other active ingredients with the antigen-binding protein. In the present application, the active ingredients can be covalently linked to the antigen-binding protein through a linker molecule. In the present application, the immunoconjugate can be an antigen-binding protein conjugated with a payload. For example, the payload can be a toxin, a polymer, a protein, a drug, a radioisotope, a nucleic acid compound, or a glucocorticoid. In the present application, the immunoconjugate can be an antibody drug conjugate (ADC), a PROTAC-antibody conjugate (DAC), or a targeted radionuclide (RDC). In the present application, the conjugate can deliver the payload to the target cell through specific binding of the antigen-binding protein to the antigen on the target cell. For example, the target cell can be a tumor cell.

[0131] In the present application, the term "pharmaceutically acceptable carrier" generally refers to a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. For example, the pharmaceutically acceptable carrier includes a pharmaceutically acceptable carrier, excipient or stabilizer, which is non-toxic to cells or mammals exposed to it at the dose and concentration used. For example, the physiologically acceptable carrier can be water, salt, protein, polysaccharide, lipid or inactive virus particle.

[0132] In the present application, the term "preventing and / or treating" generally refers to preventing and / or treating a disease. For example, the prevention and / or treatment can be preventing the onset of the disease, slowing or reversing the disease progression, preventing or slowing the onset of one or more symptoms associated with the disease, reducing or alleviating one or more symptoms associated with the disease, reducing the severity and duration of the disease and any symptoms associated therewith, or preventing further increase in the severity of the disease and any symptoms associated therewith. In the present application, the disease can be a tumor disease. For example, preventing or alleviating the onset of one or more symptoms associated with a tumor, reducing the severity and duration of a tumor and symptoms associated therewith.

[0133] In the present application, the term "tumor" generally refers to any new pathological tissue proliferation in which tumor antigens recognizable by the immune system are present. In the present application, the tumor can include benign or malignant tumors (cancers). In the present application, the cancer can be metastatic and non-metastatic cancers. In the present application, the tumor can include solid tumors and blood tumors. In the present application, the solid tumor generally refers to a tangible tumor that can be detected by clinical examination means. For example, the solid tumor can include a neoplasm or a solid lesion formed by abnormal cell growth. In the present application, the blood tumor generally refers to a type of hematopoietic system disease. In the present application, the blood tumor can include various types of leukemia, multiple myeloma, or malignant lymphoma. In the present application, the tumor can be a CD155-expressing tumor. For example, the tumor can be melanoma, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, head and neck cancer, lymphoma, multiple myeloma, and / or leukemia.

[0134] In the present application, the term "subject" generally refers to a human or a non-human animal, including but not limited to a cat, a dog, a horse, a pig, a cow, a sheep, a rabbit, a mouse, a rat, or a monkey.

[0135] In the present application, the term "comprising" generally means including the recited features but not excluding other elements.

[0136] In the present application, the term "about" generally means a variation within a range of 0.5-10% above or below the specified numerical value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified numerical value.

[0137] DETAILED DESCRIPTION

[0138] Antigen-binding protein

[0139] The CDRs of an antibody, also known as complementarity determining regions, are part of the variable region. The amino acid residues of this region can contact the antigen or epitope. Antibody CDRs can be determined by various numbering systems, such as CCG, Kabat, Chothia, IMGT, AbM, integrated consideration of Kabat / Chothia, etc. These numbering systems are known in the art, and can be found, for example, at http: / / www.bioinf.org.uk / abs / index.html#kabatnum. One of skill in the art can determine the CDR regions from the sequence and structure of an antibody using different numbering systems. The CDR regions can differ using different numbering systems. In the present application, the CDRs encompass CDR sequences determined according to any CDR partitioning scheme; also encompass variants thereof, which include substitution, deletion, and / or addition of one or more amino acids of the CDR amino acid sequence. For example, 1-30, 1-20, or 1-10 amino acid substitutions, deletions, and / or insertions; also encompass homologues thereof, which can be an amino acid sequence having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more) sequence homology to the CDR amino acid sequence. For example, the CDRs of the isolated antigen binding proteins described in the Sequence Listing of the present application can be determined using IMGT.

[0140] In one aspect, the present application provides an isolated antigen binding protein that is capable of binding to CD155, wherein the antigen binding protein comprises at least one CDR in an antibody heavy chain variable region.

[0141] In the present application, the antigen binding protein can comprise an antibody heavy chain variable region VH, which can comprise heavy chain complementarity determining regions HCDR1, HCDR2, and / or HCDR3.

[0142] In the present application, the antigen binding protein has one or more of the following properties: (1) capable of binding to CD155 and has good binding activity; (2) capable of binding to CD155 expressed on the surface of target cells; (3) capable of killing tumors; (4) capable of blocking the binding of CD155 to its receptor. In the present application, the antigen binding protein can bind to CD155 with higher affinity / binding activity than the control antibody NTX-1088. For example, the antigen binding protein can bind to CD155 with a higher KD value than the control antibody NTX-1088. For example, the antigen binding protein can bind to CD155 with a similar or lower EC50 value than the control antibody NTX-1088. In the present application, the antigen binding protein can have a similar ability to block the binding of TIGIT to CD155 as the control antibody NTX-1088. In the present application, the EC50 value, KD value or blocking ability can be determined by routine techniques in the art. In the present application, the KD value can be determined by Octet, SPR, ELISA, competitive ELISA or BIACORE or KINEXA. In the present application, the isolated antigen binding protein can specifically bind to CD155. In the present application, the specific binding can be determined by FACS.

[0143] In the present application, the VH can comprise a HCDR3, the amino acid sequence of which is set forth in SEQ ID NO: 59 or SEQ ID NO: 15.

[0144] In the present application, the VH can comprise a HCDR3, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 14-19.

[0145] In the present application, the VH can comprise a HCDR2, the amino acid sequence of which is set forth in SEQ ID NO: 58 or SEQ ID NO: 9.

[0146] In the present application, the VH can comprise a HCDR2, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 7-13.

[0147] In the present application, the VH can comprise a HCDR1, the amino acid sequence of which is set forth in SEQ ID NO: 57 or SEQ ID NO: 3.

[0148] In the present application, the VH can comprise a HCDR1, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 1-6.

[0149] In the present application, the antigen binding protein can comprise an antibody heavy chain variable region VH, which can comprise heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 can be as shown in SEQ ID NO: 57 or SEQ ID NO: 3, the amino acid sequence of the HCDR2 can be as shown in SEQ ID NO: 58 or SEQ ID NO: 9, and the amino acid sequence of the HCDR3 can be as shown in SEQ ID NO: 59 or SEQ ID NO: 15.

[0150] In the present application, the antigen binding protein can comprise an antibody heavy chain variable region VH, which can comprise heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 can be as shown in SEQ ID NO: 57, the amino acid sequence of the HCDR2 can be as shown in SEQ ID NO: 58, and the amino acid sequence of the HCDR3 can be as shown in SEQ ID NO: 59. In the present application, the antigen binding protein can comprise an antibody heavy chain variable region VH, which can comprise heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 can be as shown in SEQ ID NO: 3, the amino acid sequence of the HCDR2 can be as shown in SEQ ID NO: 9, and the amino acid sequence of the HCDR3 can be as shown in SEQ ID NO: 15.

[0151] In the present application, GSIX1X2IWV (SEQ ID NO: 57), wherein X1may be A, F or S, and X2may be D or N.

[0152] In the present application, X1X2VX3GGX4TD (SEQ ID NO: 58), wherein X1may be A or V, X2may be D, I or L, X3may be N or S, and X4may be S or Y.

[0153] In the present application, X1X2KPX3GRPX4I (SEQ ID NO: 59), wherein X1may be K or N, X2may be A, I or L, X3may be A or V, and X4may be E or G.

[0154] For example, the amino acid sequences of the HCDR1, HCDR2 and HCDR3 can be selected from any one of the following combinations:

[0155] (1) the amino acid sequence of the HCDR1 is as shown in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO: 7, and the amino acid sequence of the HCDR3 is as shown in SEQ ID NO: 14;

[0156] (2) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 8, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 14;

[0157] (3) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 3, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 9, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 15;

[0158] (4) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 4, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 10, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 16;

[0159] (5) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 4, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 13, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 19;

[0160] (6) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 5, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 11, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 17; and

[0161] (7) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 6, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 12, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 18.

[0162] In the present application, the VH can comprise a H-FR1, a H-FR2, a H-FR3, and / or a H-FR4.

[0163] In the present application, the VH can comprise a H-FR1, the amino acid sequence of the H-FR1 is set forth in any one of SEQ ID NOs: 20-24.

[0164] In the present application, the VH can comprise a H-FR2, the amino acid sequence of the H-FR2 is set forth in any one of SEQ ID NOs: 25-30.

[0165] In the present application, the VH can comprise a H-FR3, the amino acid sequence of the H-FR3 is set forth in any one of SEQ ID NOs: 31-37.

[0166] In the present application, the VH can comprise H-FR4, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 38-39.

[0167] In the present application, the VH comprises H-FR1, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 20-24, H-FR2, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 25-30, H-FR3, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 31-37, and H-FR4, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 38-39.

[0168] For example, the VH can comprise H-FR1, H-FR2, H-FR3 and H-FR4, which can be selected from any one of the following combinations:

[0169] (1) the amino acid sequence of H-FR1 is set forth in SEQ ID NO: 20, the amino acid sequence of H-FR2 is set forth in SEQ ID NO: 25, the amino acid sequence of H-FR3 is set forth in SEQ ID NO: 31, and the amino acid sequence of H-FR4 is set forth in SEQ ID NO: 38;

[0170] (2) the amino acid sequence of H-FR1 is set forth in SEQ ID NO: 21, the amino acid sequence of H-FR2 is set forth in SEQ ID NO: 26, the amino acid sequence of H-FR3 is set forth in SEQ ID NO: 31, and the amino acid sequence of H-FR4 is set forth in SEQ ID NO: 38;

[0171] (3) the amino acid sequence of H-FR1 is set forth in SEQ ID NO: 23, the amino acid sequence of H-FR2 is set forth in SEQ ID NO: 25, the amino acid sequence of H-FR3 is set forth in SEQ ID NO: 31, and the amino acid sequence of H-FR4 is set forth in SEQ ID NO: 38;

[0172] (4) the amino acid sequence of H-FR1 is set forth in SEQ ID NO: 20, the amino acid sequence of H-FR2 is set forth in SEQ ID NO: 25, the amino acid sequence of H-FR3 is set forth in SEQ ID NO: 32, and the amino acid sequence of H-FR4 is set forth in SEQ ID NO: 38;

[0173] (5) the amino acid sequence of the H-FR1 is shown as SEQ ID NO:21, the amino acid sequence of the H-FR2 is shown as SEQ ID NO:25, the amino acid sequence of the H-FR3 is shown as SEQ ID NO:32, and the amino acid sequence of the H-FR4 is shown as SEQ ID NO:38;

[0174] (6) the amino acid sequence of the H-FR1 is shown as SEQ ID NO:20, the amino acid sequence of the H-FR2 is shown as SEQ ID NO:29, the amino acid sequence of the H-FR3 is shown as SEQ ID NO:32, and the amino acid sequence of the H-FR4 is shown as SEQ ID NO:38;

[0175] (7) the amino acid sequence of the H-FR1 is shown as SEQ ID NO:22, the amino acid sequence of the H-FR2 is shown as SEQ ID NO:27, the amino acid sequence of the H-FR3 is shown as SEQ ID NO:33, and the amino acid sequence of the H-FR4 is shown as SEQ ID NO:38;

[0176] (8) the amino acid sequence of the H-FR1 is shown as SEQ ID NO:20, the amino acid sequence of the H-FR2 is shown as SEQ ID NO:28, the amino acid sequence of the H-FR3 is shown as SEQ ID NO:34, and the amino acid sequence of the H-FR4 is shown as SEQ ID NO:38;

[0177] (9) the amino acid sequence of the H-FR1 is shown as SEQ ID NO:24, the amino acid sequence of the H-FR2 is shown as SEQ ID NO:27, the amino acid sequence of the H-FR3 is shown as SEQ ID NO:35, and the amino acid sequence of the H-FR4 is shown as SEQ ID NO:38;

[0178] (10) the amino acid sequence of the H-FR1 is shown as SEQ ID NO:20, the amino acid sequence of the H-FR2 is shown as SEQ ID NO:30, the amino acid sequence of the H-FR3 is shown as SEQ ID NO:36, and the amino acid sequence of the H-FR4 is shown as SEQ ID NO:39; and

[0179] (11) the amino acid sequence of the H-FR1 is shown as SEQ ID NO:20, the amino acid sequence of the H-FR2 is shown as SEQ ID NO:30, the amino acid sequence of the H-FR3 is shown as SEQ ID NO:37, and the amino acid sequence of the H-FR4 is shown as SEQ ID NO:39.

[0180] In the present application, the C-terminus of the H-FR1 can be directly or indirectly linked to the N-terminus of the HCDR1. In the present application, the H-FR2 can be located between the HCDR1 and the HCDR2. In the present application, the H-FR3 can be located between the HCDR2 and the HCDR3. In the present application, the N-terminus of the H-FR4 can be directly or indirectly linked to the C-terminus of the HCDR3.

[0181] In the present application, the antigen binding protein can comprise a HCDR1, a HCDR2, a HCDR3, a H-FR1, a H-FR2, a H-FR3, and a H-FR4, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, the H-FR2 is located between the HCDR1 and the HCDR2, the H-FR3 is located between the HCDR2 and the HCDR3, and the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3.

[0182] In the present application, the amino acid sequence of the FR can be of any species origin. For example, the FR can be of murine, rabbit, goat, llama, or human origin. For example, the FR can be of llama origin. For example, the FR can be of human origin.

[0183] In the present application, the amino acid sequence of the FR can be adjusted as needed. For example, the FR can be of wild-type sequence. For example, during the process of humanization of the antibody, the amino acid sequence of the FR can be changed without reducing the binding activity / affinity of the antigen binding protein to CD155. For example, during the process of humanization of the antibody, one or more amino acid sequences in the FR can be mutated or optimized without reducing the binding activity / affinity of the antigen binding protein to CD155. For example, the FR can be a variant thereof, which comprises one or more amino acids substituted, deleted, and / or added to the amino acid sequence of the FR. For example, 1-30, 1-20, or 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions. For example, the FR can be a homolog, which can be an amino acid sequence having at least about 85% (for example, having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more) sequence homology to the amino acid sequence of the FR.

[0184] In the present application, the direct or indirect linkage can be linked by intermolecular forces, or can be linked by a linker.

[0185] In the present application, the amino acid sequence of the VH can be as set forth in any one of SEQ ID NOs: 43-54. For example, the VH can be a wild-type sequence. For example, one or more amino acid sequences in the VH can be mutated or optimized without reducing the binding activity / affinity of the antigen binding protein to CD155. For example, the VH can be a variant thereof, which includes the amino acid sequence of the VH being substituted, deleted, and / or added with one or more amino acids. For example, the VH can also comprise a sequence having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more homology to the amino acid sequence as set forth in any one of SEQ ID NOs: 43-54.

[0186] In the present application, the antigen binding protein can also comprise an immunoglobulin constant region. In the present application, the immunoglobulin constant region can be an immunoglobulin Fc region. In the present application, the Fc region can be a wild-type sequence, or can be mutated or optimized. In the present application, the Fc region can be an Fc region of any species origin. For example, the immunoglobulin constant region can be an IgG Fc region. For example, the immunoglobulin constant region can be an IgG1 Fc region. For example, the Fc region can be derived from a murine, rabbit, goat, llama, or human Fc region. For example, the immunoglobulin constant region can be a human IgG Fc region.

[0187] In the present application, the antigen binding protein can be an antibody or an antigen binding fragment thereof. For example, the isolated antigen binding protein described in the present application can include, but is not limited to, an antibody, an antigen binding fragment, an immunoconjugate, a multispecific antibody, an antibody fragment, an antibody derivative, an antibody analog, or a fusion protein, etc.

[0188] In the present application, the antigen binding fragment can be a Fab, (Fab)2, F(ab’)2, scFv, di-scFv, Fv, VHH, or dAb fragment of the antibody. For example, the antigen binding fragment can be a VHH, the amino acid sequence of which is as set forth in any one of SEQ ID NOs: 43-54.

[0189] In the present application, the antigen binding protein can be a chimeric antibody, a humanized antibody, or a fully human antibody. For example, the antigen binding protein is mutated / optimized for FR and constant region without reducing the binding activity / affinity of the antigen binding protein to CD155. For example, the antigen binding protein has lower immunogenicity without reducing the binding activity / affinity of the antigen binding protein to CD155.

[0190] In the present application, the antigen binding protein can be a monovalent antibody, a bivalent antibody, or a multivalent antibody. For example, the antigen binding protein can have one, two, or more antigen binding sites.

[0191] In the present application, the antigen binding protein can be a monospecific antibody, a bispecific antibody, or a multispecific antibody. For example, the antigen binding protein can comprise one, two, or more targeting moieties. In the present application, the antigen binding protein can be a monospecific antibody. For example, the antigen binding protein can comprise only a first targeting moiety.

[0192] In the present application, the antigen binding protein can be a bispecific antibody. For example, the antigen binding protein can comprise a first targeting moiety and a second targeting moiety. In the present application, the first targeting moiety can target a tumor antigen. For example, the first targeting moiety can target a tumor-associated antigen or a tumor-specific antigen. For example, the first targeting moiety can target a solid tumor or / a hematological tumor. For example, the first targeting moiety can target CD155. For example, the first targeting moiety can bind to a CD155-positive tumor. For example, the first targeting moiety can block the binding of CD155 to its receptor. For example, the first targeting moiety can block the binding of CD155 to TIGIT or CD226 (DNAM-1). In the present application, the first targeting moiety can comprise an antigen binding fragment capable of binding to CD155. In the present application, the first targeting moiety can comprise an antibody or an antigen binding fragment thereof capable of binding to CD155. For example, the antigen binding fragment in the first targeting moiety can be a Fab, a (Fab)2, a F(ab’)2, a scFv, a di-scFv, a Fv, a VHH, or a dAb fragment of the antibody. For example, the first targeting moiety can comprise a VHH capable of binding to CD155. For example, the first targeting moiety can comprise a VHH, which can comprise a HCDR1, a HCDR2, and a HCDR3, the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 57 or SEQ ID NO: 3, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 58 or SEQ ID NO: 9, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 59 or SEQ ID NO: 15. For example, the first targeting moiety can comprise a VHH, which can comprise a HCDR1, a HCDR2, and a HCDR3, the amino acid sequence of the HCDR1 is set forth in any one of SEQ ID NOs: 1-6, the amino acid sequence of the HCDR2 is set forth in any one of SEQ ID NOs: 7-13, and the amino acid sequence of the HCDR3 is set forth in any one of SEQ ID NOs: 14-19.

[0193] For example, the first targeting moiety can comprise a VHH, which can comprise HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are selected from any one of the following combinations:

[0194] (1) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 7, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 14;

[0195] (2) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 8, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 14;

[0196] (3) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 3, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 9, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 15;

[0197] (4) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 4, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 10, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 16;

[0198] (5) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 4, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 13, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 19;

[0199] (6) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 5, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 11, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 17; and

[0200] (7) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 6, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 12, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 18.

[0201] For example, the first targeting moiety can comprise a VHH, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 43-54. For example, the first targeting moiety can comprise a VHH, the amino acid sequence of which is set forth in SEQ ID NO: 53. In the present application, the antigen binding protein in the first targeting moiety can further comprise a constant region. For example, the constant region can be a human constant region. For example, the constant region can comprise a heavy chain constant region and / or a light chain constant region.

[0202] In the present application, the second targeting moiety can target a tumor antigen. For example, the second targeting moiety can target a tumor-associated antigen or a tumor-specific antigen. For example, the second targeting moiety can target a solid tumor or / a hematological tumor. For example, the second targeting moiety can target an immune checkpoint target. For example, the second targeting moiety can target PD-L1. For example, the second targeting moiety can bind to a PD-L1 positive tumor. In the present application, the second targeting moiety can comprise an antigen binding protein capable of binding to PD-L1. In the present application, the second targeting moiety can comprise an antibody or an antigen binding fragment thereof capable of binding to PD-L1. For example, the antigen binding fragment in the second targeting moiety can be a Fab, a (Fab)2, a F(ab’)2, a scFv, a di-scFv, a Fv, a VHH or a dAb fragment of the antibody. For example, the second targeting moiety can comprise an antigen binding protein capable of binding to PD-L1. For example, the second targeting moiety can comprise an antigen binding protein capable of binding to PD-L1, which comprises an antibody or an antigen binding fragment thereof capable of binding to PD-L1. For example, the second targeting moiety can comprise an antigen binding protein capable of binding to PD-L1, which comprises an antibody heavy chain variable region VH and an antibody light chain variable region VL. For example, the second targeting moiety can comprise an antigen binding protein capable of binding to PD-L1, which comprises a VH comprising a HCDR1, a HCDR2 and a HCDR3, an amino acid sequence of the HCDR1 is shown as SEQ ID NO: 64, an amino acid sequence of the HCDR2 is shown as SEQ ID NO: 66, and an amino acid sequence of the HCDR3 is shown as SEQ ID NO: 68. For example, the second targeting moiety can comprise an antigen binding protein capable of binding to PD-L1, which comprises a VL comprising a LCDR1, a LCDR2 and a LCDR3, an amino acid sequence of the LCDR1 is shown as SEQ ID NO: 71, an amino acid sequence of the LCDR2 is shown as SEQ ID NO: 73, and an amino acid sequence of the LCDR3 is shown as SEQ ID NO: 75.For example, the second targeting moiety can comprise an antigen binding protein capable of binding PD-L1, the antigen binding protein comprising a VH, which can comprise a HCDR1, a HCDR2, and a HCDR3, the HCDR1 having the amino acid sequence of SEQ ID NO: 64, the HCDR2 having the amino acid sequence of SEQ ID NO: 66, the HCDR3 having the amino acid sequence of SEQ ID NO: 68; and a VL, which can comprise a LCDR1, a LCDR2, and a LCDR3, the LCDR1 having the amino acid sequence of SEQ ID NO: 71, the LCDR2 having the amino acid sequence of SEQ ID NO: 73, the LCDR3 having the amino acid sequence of SEQ ID NO: 75. For example, the VH can have the amino acid sequence of SEQ ID NO: 42. For example, the VL can have the amino acid sequence of SEQ ID NO: 55. For example, the second targeting moiety can further comprise a constant region. For example, the constant region can be a human constant region. For example, the second targeting moiety can comprise a heavy chain constant region CH. For example, the second targeting moiety can comprise a light chain variable region CL. For example, the second targeting moiety can comprise CH and CL. For example, the second targeting moiety can comprise a heavy chain. For example, the heavy chain can have the amino acid sequence of SEQ ID NO: 69. For example, the second targeting moiety can comprise a light chain. For example, the light chain can have the amino acid sequence of SEQ ID NO: 77. For example, the second targeting moiety can comprise a heavy chain and a light chain, the heavy chain having the amino acid sequence of SEQ ID NO: 69, and the light chain having the amino acid sequence of SEQ ID NO: 77. For example, the second targeting moiety can be located at the N-terminus of the first targeting moiety. For example, the second targeting moiety can be located at the C-terminus of the first targeting moiety. For example, the first targeting moiety and the second targeting moiety can be directly or indirectly connected. For example, the bispecific antibody can further comprise a linker. For example, the first targeting moiety and the second moiety can be indirectly connected by a linker.

[0203] In the present application, the antigen binding protein can be a bispecific antibody. For example, the antigen binding protein can comprise a first polypeptide chain and a second polypeptide chain. In the present application, the first polypeptide chain can comprise an antibody heavy chain variable region VH capable of binding to PD-L1 and a VHH capable of binding to CD155; and the second polypeptide chain can comprise an antibody light chain variable region VL capable of binding to PD-L1. In the present application, in the first polypeptide chain, the heavy chain variable region VH capable of binding to PD-L1 is located at the N-terminus of the VHH capable of binding to CD155. In the present application, in the first polypeptide chain, the heavy chain variable region VH capable of binding to PD-L1 is located at the C-terminus of the VHH capable of binding to CD155. In the present application, the first polypeptide chain can further comprise a constant region. For example, the first polypeptide chain can further comprise a heavy chain constant region CH. For example, the heavy chain constant region can be an IgG constant region. For example, the IgG constant region can be a human IgG constant region. For example, the IgG constant region can be a human IgG1 constant region. For example, the CH can be located at the C-terminus of the heavy chain variable region VH capable of binding to PD-L1 and at the N-terminus of the VHH capable of binding to CD155. For example, the CH can be located at the C-terminus of the heavy chain variable region VH capable of binding to PD-L1, which is located at the C-terminus of the VHH capable of binding to CD155. For example, the first polypeptide chain can further comprise a linker. For example, the heavy chain constant region CH and the VHH capable of binding to CD155 are connected by a linker. In the present application, the amino acid sequence of the first polypeptide chain can be as shown in SEQ ID NO: 78.

[0204] In the present application, the second polypeptide chain can further comprise a light chain constant region CL. For example, the light chain constant region can be an Igλ constant region. For example, the light chain constant region can be a human Igλ constant region. For example, the CL can be located at the C-terminus of the light chain variable region VL capable of binding to PD-L1. In the present application, the amino acid sequence of the second polypeptide chain can be as shown in SEQ ID NO: 77. In the present application, the bispecific antibody can comprise a first polypeptide chain and a second polypeptide chain, the amino acid sequence of the first polypeptide chain is as shown in SEQ ID NO: 78, and the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO: 77.

[0205] In the present application, the antigen binding protein can be expressed in the form of a fusion protein. For example, the antigen binding protein can be fused to a peptide, a polypeptide, an amino acid or a protein. In the present application, the fusion protein can be a Fab fusion protein, an Fc fusion protein or a single-chain antibody fusion protein. For example, the antigen binding protein can be fused to one or more functional molecules. For example, the functional molecule can be one or more antigen binding proteins. For example, the functional molecule can be one or more Fc regions.

[0206] In the present application, the antigen binding protein can be wild type. For example, the antigen binding protein can be produced by an immune cell, such as a B cell. For example, the antigen binding protein can comprise a wild type sequence. In the present application, the antigen binding protein can be recombinant. For example, the antigen binding protein can be produced using recombinant DNA technology. In the present application, the antigen binding protein can be modified. For example, the antigen binding protein can be mutated or optimized. For example, the antigen binding protein can have one or more amino acids substituted, deleted and / or added. For example, 1-30, 1-20 or 1-10, for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and / or insertions.

[0207] Antigen recognizing receptor

[0208] In another aspect, the present application provides an antigen recognizing receptor comprising the antigen binding protein.

[0209] In the present application, the antigen recognizing receptor can be a chimeric antigen receptor (CAR). In the present application, the antigen recognizing receptor can be a T cell receptor (TCR).

[0210] In the present application, the chimeric antigen receptor can comprise an extracellular domain, a transmembrane domain and an intracellular domain. In the present application, the extracellular domain can comprise an antigen binding domain and a hinge region. In the present application, the extracellular domain can further comprise a signal peptide. In the present application, the intracellular domain can comprise an intracellular signaling domain. In the present application, the intracellular domain can further comprise one or more costimulatory domains. For example, the chimeric antigen receptor can comprise, in order, an optional antigen binding domain, a transmembrane domain region and / or an intracellular signaling domain. For example, the chimeric antigen receptor can comprise, in order, an optional antigen binding domain, a hinge region, a transmembrane domain region, a costimulatory domain and / or an intracellular signaling domain.

[0211] In the present application, the chimeric antigen receptor can comprise an antigen binding domain. In the present application, the antigen binding domain can comprise the antigen binding protein. For example, the antigen binding domain can be an antibody or an antigen binding fragment thereof. For example, the antigen binding fragment can be a Fab, a (Fab)2, a F(ab’)2, a scFv, a di-scFv, a Fv, a VHH or a dAb fragment. For example, the antigen binding fragment can be a VHH. For example, the VHH can comprise a HCDR1, a HCDR2 and a HCDR3. In the present application, the VHH can comprise a FR1, a HCDR1, a FR2, a HCDR2, a FR3, a HCDR3 and a FR4.

[0212] In the present application, the antigen binding domain can comprise an antigen binding protein comprising an antibody heavy chain variable region VH, which can comprise a heavy chain complementarity determining region HCDR1, HCDR2 and / or HCDR3. For example, the antigen binding domain can comprise an antigen binding protein, which can comprise an antibody heavy chain variable region VH, which can comprise a heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR3 being set forth in SEQ ID NO: 57 or SEQ ID NO: 3, the amino acid sequence of the HCDR2 being set forth in SEQ ID NO: 58 or SEQ ID NO: 9, the amino acid sequence of the HCDR3 being set forth in SEQ ID NO: 59 or SEQ ID NO: 15. For example, the antigen binding domain can comprise an antigen binding protein, which can comprise an antibody heavy chain variable region VH, which can comprise a heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 being set forth in any one of SEQ ID NOs: 1 -6, the amino acid sequence of the HCDR2 being set forth in any one of SEQ ID NOs: 7-13, the amino acid sequence of the HCDR3 being set forth in any one of SEQ ID NOs: 14-19.

[0213] For example, the antigen binding domain can comprise an antigen binding protein, which can comprise an antibody heavy chain variable region VH, which can comprise a heavy chain complementarity determining region HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1, HCDR2 and HCDR3 being selected from any one of the following combinations:

[0214] (1 ) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 7, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 14;

[0215] (2) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 2, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 8, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 14;

[0216] (3) the amino acid sequence of the HCDR1 is set forth in SEQ ID NO: 3, the amino acid sequence of the HCDR2 is set forth in SEQ ID NO: 9, and the amino acid sequence of the HCDR3 is set forth in SEQ ID NO: 15;

[0217] (4) the amino acid sequence of the HCDR1 is shown in SEQ ID NO:4, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 10, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 16;

[0218] (5) the amino acid sequence of the HCDR1 is shown in SEQ ID NO:4, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 13, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 19;

[0219] (6) the amino acid sequence of the HCDR1 is shown in SEQ ID NO:5, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 11, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 17; and

[0220] (7) the amino acid sequence of the HCDR1 is shown in SEQ ID NO:6, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 12, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 18.

[0221] For example, the antigen binding domain can comprise an antigen binding protein, which can comprise an antibody heavy chain variable region VH, the amino acid sequence of which is shown in any one of SEQ ID NOs:43-54.

[0222] In the present application, the hinge region can comprise a hinge region derived from CD28, IgGl, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and LIGHT. For example, the hinge region is derived from the hinge region of CD8a.

[0223] In the present application, the transmembrane domain comprises a transmembrane domain derived from any one of the following proteins: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 epsilon, CD3 zeta, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc epsilon RI gamma, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM. For example, the transmembrane domain can be derived from the transmembrane domain of CD8 alpha.

[0224] In the present application, the costimulatory domain can comprise a costimulatory domain derived from one or more of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc epsilon RI gamma, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, ligand of CD83, CD40, and MyD88. For example, the costimulatory domain can be derived from the costimulatory domain of 4-1BB.

[0225] In the present application, the intracellular signaling domain can comprise an intracellular signaling domain derived from the following proteins: CD3 zeta, CD3 delta, CD3 gamma, CD3 epsilon, CD79a, CD79b, Fc epsilon RI gamma, Fc epsilon RI beta, Fc gamma RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12, or a domain comprising at least one ITAM. For example, the intracellular signaling domain is derived from the intracellular signaling domain of CD3 zeta.

[0226] In the present application, the CAR can further comprise a low-density lipoprotein receptor-related protein or a fragment thereof. For example, the low-density lipoprotein receptor-related protein or the fragment thereof can be located at the C-terminus of the CAR. For example, the low-density lipoprotein receptor-related protein or the fragment thereof can comprise low-density lipoprotein receptor-related proteins 1-12 and functional fragments thereof. For example, the low-density lipoprotein receptor-related protein or the fragment thereof can be low-density lipoprotein receptor-related proteins 5 and / or 6 or a fragment thereof. For example, the low-density lipoprotein receptor-related protein or the fragment thereof can be low-density lipoprotein receptor-related protein 6 or a fragment thereof. In the present application, the low-density lipoprotein receptor-related protein or the fragment thereof can comprise the amino acid sequence shown in SEQ ID NO: 60. In the present application, the low-density lipoprotein receptor-related protein or the fragment thereof can be an exogenous low-density lipoprotein receptor-related protein or a fragment thereof.

[0227] In the present application, the sequence of the low-density lipoprotein receptor-related protein or the fragment thereof in the CAR can be linked to the C-terminal sequence of the CAR through a self-cleaving peptide (e.g., T2A, P2A, E2A, etc. 2A peptide). For example, the low-density lipoprotein receptor-related protein or the fragment thereof can be linked to the C-terminus of the intracellular signaling region through T2A.

[0228] In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting CD155, a transmembrane domain, and an intracellular signaling domain. In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting CD155, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain. In the present application, the chimeric antigen receptor can comprise an antigen binding domain targeting CD155, a hinge region, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain. In the present application, the chimeric antigen receptor can comprise a signal peptide, an antigen binding domain targeting CD155, a hinge region, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain. In the present application, the chimeric antigen receptor can comprise a signal peptide, an antigen binding domain targeting CD155, a hinge region, a transmembrane domain, a costimulatory domain, an intracellular signaling domain, and a low-density lipoprotein receptor-related protein or a fragment thereof. For example, the chimeric antigen receptor can comprise a VHH targeting CD155, a CD8a transmembrane domain, and a CD3 zeta intracellular signaling domain. For example, the chimeric antigen receptor can comprise a VHH targeting CD155, a CD8a transmembrane domain, and a CD3 zeta intracellular signaling domain, the amino acid sequence of the VHH being represented by any one of SEQ ID NOs: 43-54. For example, the chimeric antigen receptor can comprise a VHH targeting CD155, a CD8a transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain. For example, the chimeric antigen receptor can comprise a VHH targeting CD155, a hinge region, a CD8a transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the amino acid sequence of the VHH being represented by any one of SEQ ID NOs: 43-54. For example, the chimeric antigen receptor can comprise a VHH targeting CD155, a CD8a hinge region, a CD8a transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain. For example, the chimeric antigen receptor can comprise a VHH targeting CD155, a CD8a hinge region, a CD8a transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain, the amino acid sequence of the VHH being represented by any one of SEQ ID NOs: 43-54. For example, the chimeric antigen receptor can comprise a VHH targeting CD155, a CD8a hinge region, a CD8a transmembrane domain, a 4-1BB costimulatory domain, a CD3 zeta intracellular signaling domain, and a low-density lipoprotein receptor-related protein or a fragment thereof comprising the amino acid sequence represented by SEQ ID NO: 60, the amino acid sequence of the VHH being represented by any one of SEQ ID NOs: 43-54.

[0229] In the present application, the chimeric antigen receptor can further comprise a signal peptide. For example, the signal peptide can be a CD8 signal peptide.

[0230] In the present application, the TCR can comprise the antigen binding protein.

[0231] In the present application, the TCR can specifically bind to CD155.

[0232] In the present application, the TCR can comprise an alpha chain comprising an alpha chain variable region (TRAV) and an alpha chain constant region (TRAC), and a beta chain comprising a beta chain variable region (TRBV) and a beta chain constant region (TRBC).

[0233] For example, the TRAV and / or TRBV can comprise three hypervariable regions CDR1, CDR2 and CDR3.

[0234] In another aspect, the present application provides a modified immune cell, wherein the immune cell can comprise the chimeric antigen receptor and / or the T cell receptor.

[0235] In the present application, the immune cell can be a T cell, a B cell, a natural killer cell (NK cell), a macrophage, an NKT cell, an iNKT cell, a TIL cell, a CIK cell, a gd T cell, a DNT cell, a dendritic cell, a granulocyte, a lymphocyte, a leukocyte, a peripheral blood mononuclear cell, an embryonic stem cell, a lymphoid progenitor cell, and / or a pluripotent stem cell. For example, the immune cell can be an immune effector cell. For example, the immune cell can be a T cell. For example, the immune cell can be a mixture, which can comprise different immune cell species, for example, the mixture can comprise one or more immune cells.

[0236] In the present application, the immune cell can comprise and / or express one or more antigen recognition receptors. In the present application, the immune cell can comprise and / or express one or more antigen recognition receptors.

[0237] In the present application, the immune cell can comprise and / or express one or more chimeric antigen receptors. In the present application, the immune cell can comprise and / or express one or more chimeric antigen receptors.

[0238] In the present application, the immune cell can comprise and / or express the CAR and the low density lipoprotein receptor-related protein or a fragment thereof.

[0239] Immunoconjugate

[0240] In another aspect, the present application also provides an immunoconjugate, wherein the immunoconjugate can comprise the antigen binding protein. In the present application, the immunoconjugate can specifically bind to CD155.

[0241] In the present application, the immunoconjugate can comprise an antigen binding protein, a linker, and a payload. In the present application, the parts of the immunoconjugate are directly or indirectly linked. For example, the parts of the immunoconjugate are indirectly linked. For example, the parts of the immunoconjugate are linked by a linker.

[0242] In the present application, the payload can be a toxin, a polymer, a protein, a drug, a radioisotope, a nucleic acid compound, or a glucocorticoid.

[0243] In the present application, the immunoconjugate can be an antibody drug conjugate (ADC), a PROTAC-antibody conjugate (DAC), or a targeted radionuclide (RDC).

[0244] In the present application, the antibody drug conjugate can comprise the antigen binding protein, a linker, and a cytotoxic drug. In the present application, the cytotoxic drug can be a chemotherapeutic drug, a tubulin inhibitor, a DNA damaging agent, or a topoisomerase I inhibitor. In the present application, the chemotherapeutic drug can be vinblastine or doxorubicin. In the present application, the tubulin inhibitor can be auristatin, eribulin, or maytansine. In the present application, the DNA damaging agent can be a calicheamicin, a duocarmycin, or an ansamycin derivative PBD. In the present application, the topoisomerase I inhibitor can be camptothecin, topotecan, irinotecan, or irinotecan.

[0245] In the present application, the PROTAC-antibody conjugate can comprise the antigen binding protein, a linker, and a PROTAC. In the present application, the PROTAC can comprise a target protein ligand, a linker, an E3 ligase ligand. In the present application, the E3 ligase ligand can be CRBN, VHL, IAP, MDM2, DCF15, RNF114, DCAF16, KEAP1, or FEM1B.

[0246] In the present application, the targeted radionuclide can comprise the antigen binding protein, a linker, and a radioisotope. In the present application, the radioisotope can be iodine-131, radium-223, thallium-201, or arsenic-211.

[0247] Nucleic acid molecules, vectors, and cells

[0248] In another aspect, the present application also provides an isolated nucleic acid molecule, which can encode the antigen binding protein and / or the chimeric antigen receptor.

[0249] In the present application, the nucleic acid molecule can further comprise a sequence encoding a signal peptide.

[0250] In the present application, the nucleic acid molecule can further comprise a sequence encoding the low density lipoprotein receptor-related protein or a fragment thereof. For example, the nucleic acid molecule comprises a sequence capable of expressing the chimeric antigen receptor and the low density lipoprotein receptor-related protein or a fragment thereof in a cell. In the present application, the nucleic acid sequence encoding the chimeric antigen receptor can be linked to the nucleic acid sequence encoding the low density lipoprotein receptor-related protein or a fragment thereof by a cleavable peptide. For example, the nucleic acid molecule encoding the low density lipoprotein receptor-related protein or a fragment thereof can comprise the nucleotide sequence set forth in SEQ ID NO: 60.

[0251] In the present application, the nucleic acid molecule can be produced or synthesized by (i) amplification in vitro, for example, produced by polymerase chain reaction (PCR) amplification, (ii) produced by recombinant cloning, (iii) purified, for example, fractionated by enzyme digestion and gel electrophoresis, or (iv) synthesized, for example, by chemical synthesis.

[0252] In the present application, the nucleic acid molecule can be DNA and / or RNA. In the present application, the nucleic acid molecule can be an artificially synthesized nucleic acid analog.

[0253] In the present application, the nucleic acid molecule can be a modified nucleic acid molecule.

[0254] In another aspect, the present application provides a vector comprising the nucleic acid molecule.

[0255] In the present application, the vector can comprise one or more of the nucleic acid molecules. In the present application, the vector can comprise one or more of the nucleic acid molecules.

[0256] In the present application, the vector can be an expression vector or a cloning vector. In the present application, the vector can be a viral vector or a non-viral vector. In the present application, the vector can be a viral vector, a plasmid vector, a phage vector, or other vectors commonly used in, for example, genetic engineering. For example, the viral vector can be an adenovirus, an adeno-associated virus, a retrovirus (including lentivirus). In the present application, the vector can be a fusion type vector or a non-fusion type vector.

[0257] In the present application, the vector can further comprise other genes. For example, the other genes can be marker genes.

[0258] In the present application, the vector can contain various elements that control expression. For example, the vector can comprise a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and / or a reporter gene. For example, the vector can also contain a replication initiation site. For example, the vector can comprise components that assist in entry into a cell. To allow the nucleic acid molecule to replicate in the vector, the 5' end and 3' end of the nucleic acid molecule can also comprise long terminal repeat sequences.

[0259] In another aspect, the present application provides a cell comprising the nucleic acid molecule and / or the vector.

[0260] In the present application, the cell can comprise progeny of a single cell. The progeny can not necessarily be identical to the original parent cell (in the morphology of the total DNA complement or on the genome) due to natural, accidental or intentional mutations.

[0261] In the present application, the cell can be a prokaryotic cell (e.g., a bacterial cell), a CHO cell, an NS / 0 cell, a HEK293T cell, or a HEK293A cell, or other eukaryotic cell, such as a fungal or yeast cell, etc.

[0262] In the present application, the cell can comprise one or more of the nucleic acid molecules and / or one or more vectors. In the present application, the vector can comprise one or more of the nucleic acid molecules and / or one or more vectors.

[0263] In the present application, the vector can be introduced into the cell by methods known in the art. For example, the method can be electroporation, Lipofectine transfection, or Lipofectamin transfection.

[0264] In another aspect, the present application provides a method of producing the antigen binding protein and / or chimeric antigen receptor, the method comprising culturing the cell under conditions such that the antigen binding protein and / or chimeric antigen receptor is expressed.

[0265] Pharmaceutical composition

[0266] In another aspect, the present application also provides a pharmaceutical composition, which can comprise the antigen binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0267] In the present application, the pharmaceutical composition can comprise a suitable formulation of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. Acceptable ingredients of the composition are preferably non-toxic to the recipient at the doses and concentrations employed. The pharmaceutical composition of the present application can include liquid, frozen, and lyophilized compositions.

[0268] In the present application, the pharmaceutically acceptable carrier can comprise any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, which are compatible with pharmaceutical administration, are generally safe, non-toxic and neither biologically nor otherwise undesirable.

[0269] In the present application, the pharmaceutical composition can comprise parenteral, transdermal, intracavitary, intraarterial, intrathecal and / or intranasal administration or direct injection into a tissue. For example, the pharmaceutical composition can be administered to a patient or subject by infusion or injection. In certain embodiments, administration of the pharmaceutical composition can be performed in different ways, for example, intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

[0270] Pharmaceutical composition

[0271] In another aspect, the present application provides a pharmaceutical combination comprising the antigen binding protein, which can further comprise one or more active ingredients other than the antigen binding protein.

[0272] For example, the pharmaceutical combination can further comprise a substance related to an immune response. For example, the pharmaceutical combination can further comprise a drug related to an immune response.

[0273] In another aspect, the present application provides a pharmaceutical combination comprising the antigen binding protein, the nucleic acid molecule and / or the cell and a therapeutic agent. In the present application, the therapeutic agent can be selected from one or more of the following group: an antitumor drug, a chemotherapeutic agent, a radioisotope or an immune checkpoint inhibitor.

[0274] In another aspect, the present application also provides a regimen of the antigen binding protein in combination with one or more additional active ingredients. For example, the antigen binding protein is used in combination with one or more therapeutic agents. In the present application, the drug combination can be administered separately, simultaneously or sequentially. In the present application, the drug combination can be administered at the same or different dose or administration route. For example, the active ingredients in the drug combination are administered to the patient as separate entities at the same / different dose, administration route. In the present application, the components in the drug combination can be administered to the patient simultaneously as a single entity or dose. For example, the components in the drug combination are administered to the patient as separate entities simultaneously, concurrently or sequentially. In the present application, the specific administration route can be determined according to the class of the active ingredient, and the specific administration dose can be adjusted according to the severity of the condition, the physical condition of the subject, etc.

[0275] In the present application, the different active ingredients in the drug combination can be mixed or placed separately. For example, the active ingredients can be placed in the same container. For example, the active ingredients can be placed in different containers.

[0276] Preparation method

[0277] In another aspect, the present application provides a method of preparing the antigen binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition. For example, the method can comprise culturing the cell under conditions such that the antigen binding protein and / or the chimeric antigen receptor is expressed. For example, the method can comprise introducing the vector into an immune cell.

[0278] Use

[0279] In another aspect, the present application also provides the use of the antigen binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition in the preparation of a medicament, which can be used for preventing, diagnosing and / or treating a disease and / or a disorder.

[0280] In another aspect, the present application also provides a method of preventing, diagnosing and / or treating a disease and / or a disorder, which can comprise administering the antigen binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition to a subject in need.

[0281] In another aspect, the present application also provides the antigen binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition for use in preventing, diagnosing and / or treating a disease and / or a disorder.

[0282] In the present application, the disease and / or disorder can be a CD155 related disease and / or disorder.

[0283] In the present application, the disease and / or disorder can be a tumor. In the present application, the tumor can be a solid tumor and / or a blood tumor. In the present application, the tumor can be a CD155 positive tumor. In the present application, the blood tumor can include various types of leukemia, multiple myeloma or malignant lymphoma. In the present application, the solid tumor can be melanoma, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, head and neck cancer, lymphoma, multiple myeloma and / or leukemia.

[0284] In the present application, the prevention, diagnosis and / or treatment can be preventing the onset of the disease, slowing down or reversing the disease progression, preventing or slowing down the onset of one or more symptoms associated with the disease, reducing or alleviating one or more symptoms associated with the disease, reducing the severity and duration of the disease and any symptoms associated therewith or preventing further increase in the severity of the disease and any symptoms associated therewith.

[0285] In another aspect, the present application provides the use of the antigen binding protein for the preparation of a diagnostic agent for diagnosing a disease and / or a disorder associated with the expression of CD155 protein.

[0286] In another aspect, the present application provides the antigen binding protein for use in diagnosing a disease and / or a disorder associated with the expression of CD155 protein.

[0287] In the present application, the diagnostic agent can be used alone or in combination with an instrument, appliance, device or system. The diagnostic agent can be used for in vitro detection of human samples (e.g., various body fluids, cells, tissue samples, etc.) in the process of prevention, diagnosis, treatment monitoring, prognosis observation, health status evaluation and prediction of genetic diseases.

[0288] In the present application, the diagnostic agent can be selected from the group consisting of reagents, kits, calibrators and quality controls.

[0289] In the present application, the method of in vitro detection can be selected from the group consisting of Western Blot, ELISA and immunohistochemical method.

[0290] For example, the reagent can include a reagent capable of measuring the expression amount of the CD155 protein.

[0291] For example, the reagent can be selected from the group consisting of a reagent to perform Western Blot, a reagent to perform ELISA, and a reagent to perform immunohistochemistry.

[0292] In another aspect, the present application also provides a detection kit, which can include the antigen binding protein, for detecting the presence and / or amount of CD155 in a sample or subject. For example, the detection kit can be used for preventing, diagnosing and / or treating a disease and / or disorder.

[0293] For example, the present application relates to an immunodetection kit for use in immunodetection methods such as ELISA, immunohistochemistry, Western Blot, flow cytometry, etc. using the antibodies of the present application.

[0294] In the present application, the antigen binding protein of the present application is included in the kit for detecting CD155-related cancer cells, and specifically, the immunodetection kit will contain the antibody of the present application as a first antibody binding to CD155, and optionally an immunodetection reagent, in a suitable container member.

[0295] For example, the antibody can be pre-bound to a solid support, such as a column matrix and / or a microtiter plate well.

[0296] In the present application, the immunodetection reagent in the kit can be in any of a variety of forms, including those detectable labels bound or linked to a given antibody. Detectable labels bound or linked to secondary binding ligands can also be included. Exemplary secondary ligands are those secondary antibodies having binding affinity for the first antibody.

[0297] In the present application, other immunodetection reagents suitable for use in the kits of the present application include two-component reagents comprising a secondary antibody having binding affinity for the first antibody, and a third antibody having binding affinity for the second antibody, the third antibody being linked to a detectable label. As noted above, a variety of exemplary labels are known in the art and all such labels can be used in conjunction with the present application.

[0298] In another aspect, the antigen binding protein, the chimeric antigen receptor, the modified immune cell, the immunoconjugate, the nucleic acid molecule, the vector, the cell and / or the pharmaceutical composition of the present application are used for the manufacture of a diagnostic test kit.

[0299] In the present application, the kit can further comprise a suitable aliquot of the CD155 composition, whether labeled or unlabeled, which can be used to prepare a standard curve for the detection assay. The kit can contain the antibody-label conjugate in fully conjugated form, in intermediate form, or separate parts to be conjugated by the user of the kit. The components of the kit can be packaged in an aqueous medium or in lyophilized form.

[0300] In the present application, the container means of the kit will generally include at least one vial, test tube, flask, bottle, syringe, or other container means, into which the antibody or preferably a suitable aliquot of the antibody can be contained. The kits of the present application will also typically include a container means for containing the antibody, antigen, and any other reagents containers in sealed form, ready for use.

[0301] In another aspect, the present application also provides a method of detecting the presence and / or amount of CD155, which can comprise using the antigen binding protein.

[0302] In another aspect, the present application provides a method of diagnosing a disease and / or disorder associated with the expression of CD155 protein in a subject, which comprises contacting a sample derived from the subject with the antigen binding protein, and determining the presence and / or amount of a substance capable of specifically binding to the antigen binding protein in the sample.

[0303] In another aspect, the present application provides a method of detecting CD155 in a sample or a subject, which comprises administering the antigen binding protein. In the present application, the administration can be performed in different ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. Without wishing to be bound by any theory, the examples below are merely intended to illustrate the antigen binding protein, the method of preparation and use of the present application, and are not intended to limit the scope of the present application.

[0304] Without wishing to be bound by any theory, the examples below are merely intended to illustrate the antigen binding protein, the method of preparation and use of the present application, and are not intended to limit the scope of the present application.

[0305] Examples

[0306] Example 1 Screening of Nanobodies Targeting CD155-his

[0307] 1.1 Construction of Phage Display Immunonanobody Library

[0308] First, the extracellular domain (ECD) of recombinant human CD155 was expressed and the llama was immunized, which was fused with 6xHis tag for subsequent protein purification. According to the known scheme in the art, the llama was immunized with CD155-His antigen, and the animal immunization was outsourced to Chengdu Apaek Biotechnology Co., Ltd., with a single immunization dose of 1-2 mg of protein. After the third and fourth immunization, 5 mL of peripheral blood was collected, the serum was separated, and the immune effect was detected by ELISA. The ELISA titer reached more than 1:16000 (coating antigen 5 μg / mL, OD value greater than 2.0). After the impact immunization, 150 mL of peripheral blood was collected. Then the PBMC was separated and the total RNA was extracted using the RNA extraction kit provided by QIAGEN. Finally, the extracted RNA was reverse transcribed into cDNA using the X kit (Super-Script III FIRST STRAND SUPERMI).

[0309] Under the known scheme in the art, the variable region (VHH) of the heavy chain antibody was amplified by two rounds of nested PCR, the target fragment was gel recovered, and the restriction enzyme (from Thermo) Sfil was used to clone it into the phage display vector pComb3XSS. After desalting the plasmid, it was electroporated into electrocompetent E. coli TG1 to construct the phage display nanobody library NanoCD155, and the library was evaluated for diversity.

[0310] 1.2 Nanobody panning targeting CD155-his

[0311] His-control and CD155-His protein 10 μg / ml were used to coat the plate and placed at 4°C overnight. The next day, after washing 3 times with 1xPBST (containing 0.05% Tween-20 in PBS), 0.5% BSA was used for room temperature blocking for 2 hours, and 1xPBST was used for washing 3 times. 100 μl of llama immunized phage library was added to the His-control well for negative screening, and after 1 hour, the phage in the His-control well was transferred to the CD155-His well for positive screening. After 1.5 hours, 1xPBST was used for washing 10 times to wash away the phage that did not bind to the antigen. Finally, glycine-HCl with pH=2.2 was used for elution, 100 μl / well, and then neutralized with Tris-HCl with pH=8.0. Half of the eluted phage was used to infect TG1 in the logarithmic growth phase, and after half an hour, M13KO7 was superinfected and cultured overnight. The next day, the phage was precipitated for the next round of screening. Similar screening process was repeated for 4 rounds. After the second round of positive screening, 1xPBST was used for washing 20 times, 1xPBST was used for washing 30 times in the third round, and 1xPBST was used for washing 40 times in the fourth round.

[0312] 1.3 Pool ELISA

[0313] One day in advance, 96-well plates were coated with 1 μg / ml of CD155-His, His-control, murine CD155-His and Rhesus CD155-His proteins, respectively, and left overnight. The next day, the plates were washed three times with 1x PBST, and then blocked with 0.5% BSA for 2 hours. After blocking, the plates were washed three times with 1x PBST, and 30 μl of each round of phage library was added, and incubated at room temperature for 1 hour with shaking. After washing three times with 1x PBST, secondary antibody Anti-M13-HRP (Sino Biological, Cat. No. 11973-MM05T-H) was added, and incubated at room temperature for 30-60 minutes. After washing seven times with 1x PBST, TMB color developing solution was added, and after color development, 2M phosphoric acid was added to stop the reaction. The absorbance value at 450 nm was read.

[0314] The ELISA results are shown in FIGS. 1A-1B, and the experimental results show that specific antibodies targeting CD155-His and Rhesus CD155-His were significantly enriched from the third and fourth rounds of screening, and there was no enrichment of antibodies targeting murine CD155-His, and the non-specificity was good.

[0315] 1.4 Identification of specific positive monoclonal antibodies by phage enzyme-linked immunoassay (ELISA)

[0316] After 4 rounds of panning, the reserved products of the fourth round were plated on 2YT / carb, and the next day single colonies were randomly picked into 800 μl of 2YT / carb / M13KO7, and incubated overnight with shaking to produce phage. One day in advance, 384-well plates were coated with 1 μg / ml of CD155-His and His-control proteins, and left overnight. The next day, the plates were washed three times with 1x PBST, and then blocked with 0.5% BSA for 2 hours, while the phage supernatant was collected by centrifugation. After blocking, the plates were washed three times with 1x PBST, and 30 μl of phage supernatant was added, and incubated at room temperature for 1 hour with shaking. After washing three times with 1x PBST, secondary antibody Anti-M13-HRP was added, and incubated at room temperature for 30 minutes. After washing seven times with 1x PBST, TMB color developing solution was added, and after color development, 2M phosphoric acid was added to stop the reaction. The absorbance value at 450 nm was read. When the OD value of the sample well was more than 5 times that of the control well, it was determined to be positive. Finally, the positive phage was re-infected into TG1, and sequencing was performed. According to the analysis of the amino acid sequences of each clone by sequence alignment software BioEdit, clones with the same CDR1, CDR2 and CDR3 sequences were considered as the same antibody strain. The obtained antibodies and their sequences are shown in Table 1.

[0317] Table 1: Schematic table of antibody sequences targeting CD155

[0318] Example 2: Identification of the binding activity and function of nanobodies targeting CD155

[0319] 2.1 Construction of CD155 VHH-Fc fusion protein and expression and purification in eukaryotic cells

[0320] Specific antibody sequences were selected from the sequencing results, cloned into the vector pcDNA3.4-Fc vector (inserted into pcDNA3.4 with a human IgG1-Fc fragment), the recombinant plasmid was transformed into E. coli DH5a (Tiangen Biotech Co., Ltd., Catalog No: CB101-02), and then the bacterial solution was uniformly coated on 2YT solid medium containing 100 μg / ml ampicillin and incubated at 37°C overnight. The next day, single colonies were picked and incubated at 37°C for about 6 hours, half of the bacterial solution was sent for sequencing, and the other half was stored at 4°C. The correctly sequenced clones were cultured and the plasmids were extracted. The expression plasmid was transfected into Expi293F cells using the PEI transfection method, and expressed in a 37°C cell incubator for 5 days. The cell supernatant was then collected and purified using a Protein A affinity chromatography column. Finally, CD155 VHH-Fc antibody protein with a purity of more than 90% was obtained.

[0321] 2.2 Enzyme-linked immunosorbent assay for detecting specific binding of CD155 VHH-Fc to human CD155 protein

[0322] A high adsorption 96-well plate was coated with 1 μg / ml of CD155-His and His-control protein, incubated at 4°C overnight, washed 3 times with 1×PBST, blocked with 0.5% BSA at room temperature for 2 hours, washed 3 times with 1×PBST, added 100 nM of VHH-Fc, incubated at room temperature for 1 hour, washed 3 times with 1×PBST, added secondary antibody anti-human IgG Fc antibody HRP (abcam, Catalog No: ab99759), incubated at room temperature for 30-60 minutes, washed 7 times with 1×PBST, added TMB developing solution, added 2M phosphoric acid to stop the reaction after color development, and read the absorbance value at 450 nm wavelength. After the nanobody sequence was expressed and purified in the form of VHH-Fc fusion protein, ELISA was performed, and NTX-1088 (WO 2021 / 070181) was used as a positive control.

[0323] The results of the binding activity to human CD155 protein are shown in Figure 2. The EC50 values of all sequences (23, 25, 27, 28, 32, 35, 36, 38, 49, 54) are similar to or lower than those of the positive control antibody NTX-1088, i.e., the binding activity is similar to or higher than that of the positive control antibody NTX-1088. The experimental results show that the CD155 antibody described in the present application has a higher or similar binding ability to CD155 than the positive control, and has a higher binding affinity.

[0324] 2.3 Flow cytometry to identify the binding activity of CD155 VHH-Fc to human CD155 transfected CHO

[0325] Recover CHO-CD155 cells and passage, harvest cells on the experimental day, adjust the cell density to 1 x 10 6 / ml, 30 μl / well (3 x 10 4 / well) after counting. Representative CD155 nanobodies and positive antibody NTX-1088 were set at 100 nM as the highest concentration, 3-fold ratio, 7 gradients, and PBS control, 30 μl / well, mixed and incubated at 4°C for 1 hour. Wash twice with PBS containing 0.1% BSA, 500g, 5 min, 4°C, and spin dry. Dilute fluorescent goat F(ab')2 anti-human IgG-Fc (DyLight 650, Goat pAb to Hu IgG, abcam, item number: ab98593) 1:200, 30 μl / well, mix and incubate at 4°C for 30 minutes. Wash twice with PBS containing 0.1% BSA, 500g, 5 min, 4°C, spin dry, resuspend 30 μl / well, and read on a high-throughput flow detector (IQue). Collect the data and use Graphpad to make a three-parameter fitting curve.

[0326] The results of the binding activity to human CD155 transfected CHO are shown in Figure 3. The MFI values of all sequences are higher than the positive control antibody NTX-1088. The experimental results show that the CD155 antibody described in the application has a binding ability to CHO cells overexpressing human CD155 much higher than the positive control, and has a higher binding affinity.

[0327] 2.4 Flow cytometry to identify the blocking function of CD155 VHH-Fc on the interaction of cell surface CD155 and TIGIT Recover CHO-CD155 cells and passage, harvest cells, adjust the cell density to 1 x 10 6 / ml, 30 μl / well (3 x 10 4 / well) after counting. The concentration gradient of CD155 nanobodies and positive antibody NTX-1088 is shown in the abscissa of Figure 4, 15 μl / well, and then add 15 μl of 3.2 μg / ml TIGIT-His protein, mix and incubate at 4°C for 1 hour. Wash twice with PBS containing 0.1% BSA, 500g, 5 min, 4°C, and spin dry. Dilute 650 Anti-6X His Ab (Abeam, Cat# ab117504), 30 μl / well, mix well and incubate at 4°C for 30 min. Wash twice with PBS containing 0.1% BSA, 500g, 5 min, 4°C, and spin dry. Resuspend 30 μl / well and read on a high-throughput flow cytometer (IQue).

[0328] The blocking results are shown in Figure 4, all sequences can block the binding of TIGIT to CHO cells overexpressing human CD155. The experimental results show that the CD155 antibody described in the application has good ability to block the binding of TIGIT to CD155, has the effect of inhibiting the binding of CD155 to the inhibitory receptor of T cells, and is beneficial to the treatment of tumors.

[0329] 2.5 Humanization and identification of CD155 nanobodies

[0330] The framework regions of CD155 nanobodies No. 38 and No. 54 were humanized. First, the sequence of human antibody DP-47 was obtained from the literature (Tomlinson et al., 1992, https: / / doi.org / 10.1016 / 0022-2836(92)90223-7), and DP-47 was numbered on the IMGT website (http: / / www.imgt.org / ) to determine the framework region and CDR, and then the amino acids on the framework of No. 38 and No. 54 sequences were replaced with the corresponding amino acids on the framework region of DP-47. Since FR2 plays an important role in the stability of nanobodies, it is left unchanged. The corresponding sequences of No. 38 and No. 54 CD155 antibodies after humanization are No. 55 and No. 56 sequences, respectively.

[0331] The VHH amino acid sequence of No. 55 is shown as SEQ ID NO: 53, the CDR1 amino acid sequence is shown as SEQ ID NO: 5, the CDR2 amino acid sequence is shown as SEQ ID NO: 11, and the CDR3 amino acid sequence is shown as SEQ ID NO: 17.

[0332] The VHH amino acid sequence of No. 56 is shown as SEQ ID NO: 54, the CDR1 amino acid sequence is shown as SEQ ID NO: 4, the CDR2 amino acid sequence is shown as SEQ ID NO: 13, and the CDR3 amino acid sequence is shown as SEQ ID NO: 19.

[0333] The humanized sequence was cloned into the expression vector pcDNA3.4, and the antibody protein was produced by transient transfection of EXPI293, and purified by protein A. Then the binding ability and function of the humanized CD155 nanobody were identified according to the methods of Examples 2.3 and 2.4.

[0334] The binding activity results of CHO transfected with human CD155 are shown in Figure 5. The MFI values of the antibodies after humanization are comparable to those before humanization. The experimental results show that the CD155 antibodies described in the present application still have high binding affinity after humanization.

[0335] The blocking results are shown in Figure 6. The antibodies after humanization can block the binding of TIGIT to CHO cells overexpressing human CD155. The experimental results show that the CD155 antibodies described in the present application still have good ability to block the binding of TIGIT to CD155 after humanization, have the effect of inhibiting the binding of CD155 to the inhibitory receptor of T cells, and are beneficial to the treatment of tumors.

[0336] Example 3 CAR-T lentivirus packaging and T cell infection and expansion

[0337] 3.1 Lentivirus packaging

[0338] Select a general lentivirus vector (Beijing Yiqiao God Science and Technology Co., Ltd., Catalog No. LVCV-01) as the CAR lentivirus core empty vector (hereinafter referred to as CAR lentivirus empty vector), construct the core sequence and Ori element to the expression region of the CAR lentivirus empty vector to form a new vector sequence pCore vector (originally constructed by Original Biology Co., Ltd.). The second-generation CAR structure composed of CD8 signal peptide, CD155 antibody sequence, CD8 alpha hinge region, CD8 alpha transmembrane domain, 4-1BB costimulatory domain and CD3 zeta intracellular signaling domain is constructed into the CAR lentivirus empty vector. The four-plasmid system is used for lentivirus packaging in the present application. The virus packaging process is as follows: 1 x 10 6293FT cells were suspended in 2 ml DMEM (10% FBS) medium, and were plated in a 6-well plate. The cells were fully dispersed and uniformly distributed, and were cultured at 37°C in 5% CO2 for about 24 h. The plasmids and reagents were equilibrated at room temperature. 350 μl Opti-MEM medium contained 7 μg (PRH1 1.17 μg: PMH2 1.17 μg: PVH3 2.33 μg: core plasmid 2.33 μg = 1:1:2:2) and 21 μl (plasmid: FuGENE HD = 1:3) transfection reagent (Promega, item number: E2311). The packaging plasmids were fully mixed, and then the transfection reagent was slowly added dropwise. After the addition was completed, the mixture was fully mixed and was left to stand at room temperature for 15 min. The original 400 μl DMEM medium (containing 10% FBS) was removed, and the liposome-cation complex was added dropwise in portions. The cells were cultured at 37°C in 5% CO2 for about 17 h. The medium containing the plasmids was removed (about 200 μl remained), and 2.5 ml of DMEM medium containing 5% FBS (preheated at 37°C for 30 min) was added. The cells were cultured at 37°C in 5% CO2 for about 24-26 h. The virus supernatant was collected, centrifuged at 3000 rpm for 10 min, and was aliquoted at about 720 μl in cryotubes and stored at -80°C. An appropriate amount was taken for determination of the lentivirus titer.

[0339] 3.2 Determination of lentivirus titer

[0340] The 293T cells in good condition were washed with 1xPBS, and were digested with 1.5 ml 0.25% trypsin at 37°C. The digestion was terminated with 11 ml of medium. The cell system was adjusted to 2x10 5 The cell line medium was added with 10 μg / ml polybrene at 1:1000, and was fully mixed. 1 ml of the cell suspension was added to each well of a 12-well plate. 5 μl of the obtained virus stock solution was added dropwise in portions, and the volume in each well was made up to 2.5 ml. The cell suspension without virus was set as a blank control for flow detection. The cells were cultured at 37°C in 5% CO2 for about 42-48 h. The supernatant was removed, and the cells were washed with PBS. The cells were digested with 400 μl 0.25% trypsin (gibco, item number: 25200072) at 37°C for about 1 min, and the digestion was terminated with 2.5 ml complete medium. The cells were fully dispersed by gently blowing with a 1 ml pipette, and were transferred to a 1.5 ml EP tube. The cells were counted. 5x10 5 cells were taken for flow detection.

[0341] 3.3 Infection and expansion of T cells

[0342] The PBMC cells were resuscitated, and were added with 11 ml X-VIVO. The cells were centrifuged at 500 g for 5 min, were added with 12 ml X-VIVO, were centrifuged at 400 g for 5 min, were added with 12 ml X-VIVO, and were centrifuged at 300 g for 5 min. The cells were suspended in 1 ml X-VIVO, and the cell density was adjusted to 1x106 Cells / ml, CD3 / CD28 (4 x 10 8 / ml) magnetic beads (Thermo, Cat: 11131D) were added to T cells at a ratio of 1:3 (cells:magnetic beads), mixed well, and 700 μl of cell suspension (7 x 10 5 Cells / well / 700 μl) was added to each well of a 12-well plate. The activated PBMCs were incubated at 37°C in a CO2incubator for 18 h. Virus supernatant was added at a MOI of 4-5, polybrene was added to a final concentration of 5 μg / ml (1:2000, by volume), the appropriate medium was added, the cells were mixed, a blank control was set up, and the cells were centrifuged at 1200 rpm for 1 h. The cells were incubated at 37°C in a 5% CO2incubator for 24 h. The cells were mixed gently, collected in 1.5 ml EP tubes, centrifuged at 300 g for 5 min, the supernatant was removed (about 50-100 μl of medium remained), 1 ml of X-VIVO medium was added for resuspension, and the cells were transferred to a 12-well plate, 500 μl of medium was added, and the cells were incubated at 37°C in a 5% CO2incubator for 48 h. The medium was observed, an appropriate amount of X-VIVO medium (800 μl-1 ml) was added, and the cells were incubated at 37°C in a 5% CO2incubator. The cells were counted every 2 days, and the cell density was adjusted to 7 x 10 5 / ml, 6-well plates / 3 ml, and the total cell number was 2.1 x 10 6 The cells were incubated at 37°C in a 5% CO2incubator for 24 h. An appropriate amount of medium (1 ml) was added, and the cells were incubated for 24 h. The cells were mixed gently, counted, and the total cell number was calculated by dividing the counted number by the previous total cell number 2.1 x 10 6 , which was the expansion fold T2. The cell density was adjusted to 7 x 10 5 / ml, 6-well plates / 3 ml, and the total cell number was 2.1 x 10 6 Passage. 6 x 10 5 The cells were incubated at 37°C in a 5% CO2incubator for 24 h. An appropriate amount of medium (1 ml) was added, and the cells were incubated for 24 h. The cells were mixed gently, counted, and the total cell number was calculated by dividing the counted number by the previous total cell number 2.1 x 10 6 , which was the expansion fold T3. The cell density was adjusted to 7 x 10 5 / ml, 6-well plates / 3 ml, and the total cell number was 2.1 x 10 6 Passage. 6 x 10 5 The cells were incubated at 37°C in a 5% CO2incubator for 24 h. An appropriate amount of medium (1 ml) was added, and the cells were incubated for 24 h. The cells were mixed gently, counted, and the total cell number was calculated by dividing the counted number by the previous total cell number 2.1 x 10 6, which is the expansion fold T4. Take the corresponding cells for CAR-T repeated stimulation experiment. Take an appropriate amount of cells for CAR-T positive rate, CD25 / CD69, PD-1 / TIM3 / LAG3 CAR detection (remove magnetic beads for 1 min). Expansion fold: T1*T2*T3*T4.

[0343] The in vitro expansion results of CD155 CAR-T cells are shown in Figure 7. All sequences are well expanded, and the expansion fold is higher than the positive control antibody NTX-1088 after 11 days, with higher expansion capacity. Among them, 25 and 32 have the highest expansion fold.

[0344] Example 4: Evaluation of CD155 CAR-T in vitro tumor killing activity experiment

[0345] 4.1 Cytotoxicity detection

[0346] The luciferase reporter gene-based cytotoxicity detection method was used, and the SK-OV3 target cell strain stably transfected with luciferase was used for the experiment. First, the CAR-T positive rate was detected to adjust the proportion of the infected CAR group to be consistent, and the total number of cells in each group was consistent. The number of target cells in the 96-well plate was 2x10 4 cells / well, and the effector cells: target cells (E:T) were 1:1, 1:3, and 1:9. A well containing only the same amount of target cells as the experimental group was set as a control group in which the target cells were not killed. The corresponding effector cells and target cells were incubated in 200 μl X-VIVO (without FBS) medium, and incubated in a 37°C, 5% CO2 incubator for 20-24 h. The appropriate amount of target cells was counted and resuspended in a non-transparent 96-well plate with 100 μl X-VIVO, and the appropriate amount of effector cells was taken according to the effector-to-target ratio, and the appropriate amount of blank T cells was added to make up 500g for 5 min. 100 μl X-VIVO was resuspended with target cells in a 96-well plate, and incubated at 37°C, 5% CO2, 20-24 h. After incubation, 100 μl of cell culture supernatant was carefully removed, and then 100 μl of ONE-Glo luciferase reporter gene detection system (ONE-Glo Luciferase Assay System, Promega, Cat: E6120) was added to each well, incubated for 5 min, and then read using a microplate reader to calculate the cell killing ratio.

[0347] The cytotoxicity detection results are shown in Figure 8. All sequences have specific killing ratios comparable to the positive control NTX-1088 at different effector-to-target ratios. The experimental results show that the CD155 antibodies described in the present application have high in vitro killing function of target cells after being prepared as CAR-T, and have good anti-tumor ability.

[0348] 4.2 Cytokine detection

[0349] CAR-T was prepared according to the T cell infection and expansion method in Example 3. The supernatant was co-incubated for IL-2, IFN-γ, and other cytokine detection. The CAR-T positive rate was detected, and the proportion of the infected CAR group was adjusted to be consistent with the uninfected blank T cells. The total number of cells in each group was consistent (the required number of CAR-T cells, the minimum CAR positive proportion was the base), the total number of T cells was consistent, VT (T+CELL group) = CAR T+T. The blank T cell wells equivalent to the experimental group were set as the T cell alone group, and the effector cell wells equivalent to the experimental group were set as the effector cell alone group. The number of target cells was 1×10 4 cells / well, and the effector cells (containing CAR-T positive cells 1×10 4 ) and target cells (1×10 4 ) were mixed, and the final volume was 200 uL. Lightly mix, 37°C, 5% CO2 incubator, 24h, 400xg, 5min centrifugation, take 50ul cell supernatant for cytokine detection.

[0350] The results of cytokine detection are shown in Figures 9-10. When CAR-T cells were co-cultured with SK-OV3 cells, CAR-T cells were activated and produced a large amount of cytokines IL-2 and IFN-γ, both of which were higher than the group with only CAR-T cells. The experimental results show that the CAR-T prepared by the CD155 antibody described in the application is activated after co-cultured with target cells, which can promote the release of cytokines and better kill target cells.

[0351] Example 5 CD155 monoclonal antibody and CD155-PD-L1 double antibody show excellent anti-tumor effect in preclinical animal models

[0352] We further constructed CD155-PD-L1 double antibody on the basis of the CD155 antibody with immune checkpoint blockade effect. The CD155 antibody sequence No. 55 was inserted into the PD-L1-H (PCT / CN2018 / 102584) vector digested by Age I and Apa I by homologous recombination, and the structure is shown in Figure 11A. In order to evaluate the anti-tumor effect of CD155 monoclonal antibody No. 55, PD-L1 monoclonal antibody (PCT / CN2018 / 102584, YN035), and CD155-PD-L1 double antibody, we constructed a tumor model of humanized immune system. 7.5×10 6 A549 tumor cells, 2.5×10 6Human PBMCs and an equal volume of basement membrane matrix (Matrigel) were mixed thoroughly and inoculated subcutaneously into B-NDG mice at the corresponding dose, and then the tumor growth was observed. On the third day after tumor inoculation, the mice were divided into four groups according to the tumor volume (the average volume at the time of grouping was 53 mm 3 ) and intraperitoneally injected with IgG1-Fc (cIg, 10 mg / kg, once every three days, for a total of 6 times), CD155 Ab (10 mg / kg, once every three days, for a total of 6 times), PD-L1 Ab (YN035, 7.5 mg / kg, once every three days, for a total of 6 times), and CD155-PD-L1 bispecific antibody (YN035-55, 9 mg / kg, once every three days, for a total of 6 times). During the period, the body weight and tumor volume of the mice were measured and recorded two to three times a week, and the mice were continuously observed for more than 3 weeks.

[0353] The results of the anti-tumor effect are shown in FIG. 11B. The tumor volume of the mice in the IgG1-Fc group gradually increased after inoculation. The tumor of the mice in the CD155 Ab group, the PD-L1 Ab group, and the CD155-PD-L1 bispecific antibody group showed an obvious inflection point on the 9th to 12th day after inoculation, and the volume began to gradually decrease. No obvious change in the body weight of the mice was observed during the entire experiment. The experimental results show that the CD155 antibody and the bispecific antibody comprising the CD155 antibody described in the present application have excellent anti-tumor effects and can inhibit the growth of tumor cells.

[0354] To understand the anti-tumor related mechanism of the CD155 antibody, the tumor tissues of the mice in different groups were taken on the 18th day, cut into pieces, and then digested with 1 mg / ml collagenase IV and 1 mg / ml DNAase I at 37 degrees on a shaking table at 150 rpm for 1 hour. After passing through a 70 μm filter membrane, the cells were collected by centrifugation for flow cytometry detection of tumor microenvironment infiltrating T cells. The changes in the surface activation molecule CD226 (DNAM-1) of the T cells in the tumor microenvironment of the cIg treatment group and the CD155 antibody treatment group were analyzed.

[0355] The results of the changes in CD226 are shown in FIG. 11C. The CD226 molecule in the CD155 antibody treatment group was significantly higher than that in the cIg group. The experimental results show that the CD155 antibody described in the present application can up-regulate the expression level of the activation receptor CD226 in the tumor microenvironment, thereby exerting an obvious anti-tumor effect.

[0356] The above results show that the CD155 antibody and the CD155-PD-L1 bispecific antibody, which block the interaction between CD155 / CD226 and CD155 / TIGIT, have good anti-tumor effects, and the CD155 antibody has the potential to exert high anti-tumor ability with other immune checkpoint bispecific antibodies.

Claims

1. An isolated antigen-binding protein capable of binding CD155, wherein the antigen-binding protein comprises an antibody heavy chain variable region VH, the VH comprising heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR3 being shown in SEQ ID NO:59 or SEQ ID NO:

15.

2. The antigen-binding protein according to claim 1, wherein the amino acid sequence of HCDR3 is shown in any one of SEQ ID NOs:14-19.

3. The antigen-binding protein according to any one of claims 1-2, wherein the amino acid sequence of HCDR2 is as shown in SEQ ID NO:58 or SEQ ID NO:

9.

4. The antigen-binding protein according to any one of claims 1-3, wherein the amino acid sequence of HCDR2 is shown in any one of SEQ ID NOs:7-13.

5. The antigen-binding protein according to any one of claims 1-4, wherein the amino acid sequence of HCDR1 is as shown in SEQ ID NO:57 or SEQ ID NO:

3.

6. The antigen-binding protein according to any one of claims 1-5, wherein the amino acid sequence of HCDR1 is shown in any one of SEQ ID NOs:1-6.

7. The antigen-binding protein according to any one of claims 1-6, wherein the amino acid sequence of HCDR1 is as shown in SEQ ID NO:57, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:58, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:59; or the amino acid sequence of HCDR1 is as shown in SEQ ID NO:3, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:9, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:

15.

8. The antigen-binding protein according to any one of claims 1-7, wherein the amino acid sequences of said HCDR1, HCDR2, and HCDR3 are selected from any group of the following combinations: (1) 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:7, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

14. (2) The amino acid sequence of HCDR1 is shown in SEQ ID NO:2, the amino acid sequence of HCDR2 is shown in SEQ ID NO:8, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

14. (3) The amino acid sequence of HCDR1 is shown in SEQ ID NO:3, the amino acid sequence of HCDR2 is shown in SEQ ID NO:9, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

15. (4) The amino acid sequence of HCDR1 is shown in SEQ ID NO:4, the amino acid sequence of HCDR2 is shown in SEQ ID NO:10, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

16. (5) The amino acid sequence of HCDR1 is shown in SEQ ID NO:4, the amino acid sequence of HCDR2 is shown in SEQ ID NO:13, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

19. (6) The amino acid sequence of HCDR1 is shown in SEQ ID NO:5, the amino acid sequence of HCDR2 is shown in SEQ ID NO:11, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:17; and (7) The amino acid sequence of HCDR1 is shown in SEQ ID NO:6, the amino acid sequence of HCDR2 is shown in SEQ ID NO:12, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

18.

9. The antigen-binding protein according to any one of claims 1-8, wherein the VH comprises H-FR1, H-FR2, H-FR3 and H-FR4, wherein the amino acid sequence of H-FR1 is shown in any one of SEQ ID NOs:20-24, the amino acid sequence of H-FR2 is shown in any one of SEQ ID NOs:25-30, the amino acid sequence of H-FR3 is shown in any one of SEQ ID NOs:31-37, and the amino acid sequence of H-FR4 is shown in any one of SEQ ID NOs:38-39.

10. The antigen-binding protein according to any one of claims 1-9, wherein the VH comprises H-FR1, H-FR2, H-FR3, and H-FR4, and the amino acid sequences of said H-FR1, H-FR2, H-FR3, and H-FR4 are selected from any group of the following combinations: (1) The amino acid sequence of H-FR1 is shown in SEQ ID NO:20, the amino acid sequence of H-FR2 is shown in SEQ ID NO:25, the amino acid sequence of H-FR3 is shown in SEQ ID NO:31, and the amino acid sequence of H-FR4 is shown in SEQ ID NO:38; (2) The amino acid sequence of H-FR1 is shown in SEQ ID NO:21, the amino acid sequence of H-FR2 is shown in SEQ ID NO:26, the amino acid sequence of H-FR3 is shown in SEQ ID NO:31, and the amino acid sequence of H-FR4 is shown in SEQ ID NO:38; (3) The amino acid sequence of H-FR1 is shown in SEQ ID NO:23, the amino acid sequence of H-FR2 is shown in SEQ ID NO:25, the amino acid sequence of H-FR3 is shown in SEQ ID NO:31, and the amino acid sequence of H-FR4 is shown in SEQ ID NO:38; (4) The amino acid sequence of H-FR1 is shown in SEQ ID NO:20, the amino acid sequence of H-FR2 is shown in SEQ ID NO:25, the amino acid sequence of H-FR3 is shown in SEQ ID NO:32, and the amino acid sequence of H-FR4 is shown in SEQ ID NO:

38. (5) The amino acid sequence of H-FR1 is shown in SEQ ID NO:21, the amino acid sequence of H-FR2 is shown in SEQ ID NO:25, the amino acid sequence of H-FR3 is shown in SEQ ID NO:32, and the amino acid sequence of H-FR4 is shown in SEQ ID NO:

38. (6) The amino acid sequence of H-FR1 is shown in SEQ ID NO:20, the amino acid sequence of H-FR2 is shown in SEQ ID NO:29, the amino acid sequence of H-FR3 is shown in SEQ ID NO:32, and the amino acid sequence of H-FR4 is shown in SEQ ID NO:38; (7) The amino acid sequence of H-FR1 is shown in SEQ ID NO:22, the amino acid sequence of H-FR2 is shown in SEQ ID NO:27, the amino acid sequence of H-FR3 is shown in SEQ ID NO:33, and the amino acid sequence of H-FR4 is shown in SEQ ID NO:38; (8) The amino acid sequence of H-FR1 is shown in SEQ ID NO:20, the amino acid sequence of H-FR2 is shown in SEQ ID NO:28, the amino acid sequence of H-FR3 is shown in SEQ ID NO:34, and the amino acid sequence of H-FR4 is shown in SEQ ID NO:

38. (9) The amino acid sequence of H-FR1 is shown in SEQ ID NO:24, the amino acid sequence of H-FR2 is shown in SEQ ID NO:27, the amino acid sequence of H-FR3 is shown in SEQ ID NO:35, and the amino acid sequence of H-FR4 is shown in SEQ ID NO:

38. (10) The amino acid sequence of H-FR1 is shown in SEQ ID NO:20, the amino acid sequence of H-FR2 is shown in SEQ ID NO:30, the amino acid sequence of H-FR3 is shown in SEQ ID NO:36, and the amino acid sequence of H-FR4 is shown in SEQ ID NO:39; and (11) The amino acid sequence of H-FR1 is shown in SEQ ID NO:20, the amino acid sequence of H-FR2 is shown in SEQ ID NO:30, the amino acid sequence of H-FR3 is shown in SEQ ID NO:37, and the amino acid sequence of H-FR4 is shown in SEQ ID NO:

39.

11. The antigen-binding protein according to any one of claims 1-10, wherein the amino acid sequence of the VH is shown in any one of SEQ ID NOs:43-54.

12. The antigen-binding protein according to any one of claims 1-11, wherein the antigen-binding protein further comprises an immunoglobulin constant region.

13. The antigen-binding protein of claim 12, wherein the immunoglobulin constant region is the immunoglobulin Fc region.

14. The antigen-binding protein according to any one of claims 12-13, wherein the immunoglobulin constant region is the IgG Fc region.

15. The antigen-binding protein according to any one of claims 1-14, wherein the antigen-binding protein is an antibody or an antigen-binding fragment thereof.

16. The antigen-binding protein according to claim 15, wherein the antigen-binding fragment is a Fab, (Fab)2, F(ab')2, scFv, di-scFv, Fv, VHH, or dAb fragment of the antibody.

17. The antigen-binding protein according to any one of claims 1-16, wherein the antigen-binding protein is VHH.

18. The antigen-binding protein according to any one of claims 1-17, wherein the antigen-binding protein is a chimeric antibody, a humanized antibody, or a fully human antibody.

19. The antigen-binding protein according to any one of claims 1-18, wherein the antigen-binding protein is a monovalent antibody, a bivalent antibody, or a multivalent antibody.

20. The antigen-binding protein according to any one of claims 1-19, wherein the antigen-binding protein is a monospecific antibody, a bispecific antibody, or a multispecific antibody.

21. The antigen-binding protein according to any one of claims 1-20, wherein the antigen-binding protein is a bispecific antibody, the bispecific antibody comprising a first targeting portion and a second targeting portion, the first targeting portion comprising the antigen-binding protein according to any one of claims 1-20, the antigen-binding protein being capable of binding CD155.

22. The antigen-binding protein of claim 21, wherein the first targeting portion comprises a VHH capable of binding CD155, the amino acid sequence of said VHH being shown in any one of SEQ ID NOs:43-54.

23. The antigen-binding protein according to any one of claims 21-22, wherein the second targeting portion is capable of binding to an immune checkpoint target.

24. The antigen-binding protein according to any one of claims 21-23, wherein the second targeting portion is capable of binding PD-L1.

25. The antigen-binding protein according to any one of claims 21-24, wherein the second targeting portion comprises an antigen-binding protein capable of binding PD-L1.

26. The antigen-binding protein according to any one of claims 21-25, wherein the second targeting portion comprises an antibody or an antigen-binding fragment thereof capable of binding to PD-L1.

27. The antigen-binding protein according to any one of claims 21-26, wherein the second targeting portion comprises HCDR1-3 and LCDR1-3, the amino acid sequence of HCDR1 is shown in SEQ ID NO:64, the amino acid sequence of HCDR2 is shown in SEQ ID NO:66, the amino acid sequence of HCDR3 is shown in SEQ ID NO:68, the amino acid sequence of LCDR1 is shown in SEQ ID NO:71, the amino acid sequence of LCDR2 is shown in SEQ ID NO:73, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

75.

28. The antigen-binding protein according to any one of claims 21-27, wherein the second targeting portion comprises VH and VL, the amino acid sequence of VH being as shown in SEQ ID NO:42, and the amino acid sequence of VL being as shown in SEQ ID NO:

55.

29. The antigen-binding protein according to any one of claims 21-28, wherein the second targeting portion comprises a heavy chain constant region and a light chain constant region.

30. The antigen-binding protein according to any one of claims 21-29, wherein the second targeting portion comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain being as shown in SEQ ID NO:69, and the amino acid sequence of the light chain being as shown in SEQ ID NO:

77.

31. The antigen-binding protein according to any one of claims 21-30, wherein the second targeting portion is located at the C-terminus of the first targeting portion.

32. The antigen-binding protein according to any one of claims 21-31, wherein the first targeting portion and the second targeting portion are connected by a linker.

33. The antigen-binding protein according to any one of claims 1-32, wherein the antigen-binding protein is a bispecific antibody, wherein the bispecific antibody comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the heavy chain variable region VH and the heavy chain constant region CH capable of binding PD-L1, and the VHH capable of binding CD155; wherein the second polypeptide chain comprises the light chain variable region VL and the light chain constant region CL capable of binding PD-L1.

34. The antigen-binding protein of claim 33, wherein the heavy chain constant region CH and the antigen-binding protein are... The VHH of CD155 is connected via a connector.

35. The antigen-binding protein according to any one of claims 33-34, wherein the amino acid sequence of the first polypeptide chain is as shown in SEQ ID NO:78, and the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO:

77.

36. A chimeric antigen receptor comprising the antigen-binding protein of any one of claims 1-35.

37. The chimeric antigen receptor of claim 36, wherein the chimeric antigen receptor comprises an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain.

38. The chimeric antigen receptor of claim 37, wherein the antigen-binding domain comprises the antigen-binding protein of any one of claims 1-35.

39. The chimeric antigen receptor according to any one of claims 37-38, wherein the transmembrane domain comprises a transmembrane domain derived from any of the following proteins: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

40. The chimeric antigen receptor according to any one of claims 37-39, wherein the transmembrane domain is derived from the transmembrane domain of CD8α.

41. The chimeric antigen receptor according to any one of claims 37-40, wherein the intracellular signal transduction domain comprises an intracellular signal transduction domain derived from any of the following proteins: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM.

42. The chimeric antigen receptor according to any one of claims 37-41, wherein the intracellular signal transduction domain is derived from the intracellular signal transduction domain of CD3ζ.

43. The chimeric antigen receptor according to any one of claims 37-42, wherein the chimeric antigen receptor further comprises a co-stimulatory domain.

44. The chimeric antigen receptor of claim 43, wherein the co-stimulatory domain comprises a co-stimulatory domain derived from one or more of the following proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88.

45. The chimeric antigen receptor according to any one of claims 43-44, wherein the co-stimulatory domain is derived from the co-stimulatory domain of 4-1BB.

46. ​​The chimeric antigen receptor according to any one of claims 37-45, wherein the chimeric antigen receptor further comprises a hinge region.

47. The chimeric antigen receptor of claim 46, wherein the hinge region comprises a hinge region derived from any of the following proteins: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

48. The chimeric antigen receptor according to any one of claims 46-47, wherein the hinge region is derived from the hinge region of CD8α.

49. A modified immune cell, wherein the immune cell comprises a chimeric antigen receptor according to any one of claims 36-48.

50. The modified immune cell according to claim 49, wherein the immune cell is a T cell, NK cell, iNKT cell, dendritic cell and / or macrophage.

51. The modified immune cell according to any one of claims 49-50, further comprising and / or expressing low-density lipoprotein receptor-associated protein or a fragment thereof.

52. The modified immune cell of claim 51, wherein the low-density lipoprotein receptor-associated protein or a fragment thereof comprises one or more selected from the group consisting of low-density lipoprotein receptor-associated proteins 1-12 and functional fragments thereof.

53. The modified immune cell according to any one of claims 51-52, wherein the low-density lipoprotein receptor-associated protein or a fragment thereof is low-density lipoprotein receptor-associated protein 5 and / or 6 or a fragment thereof.

54. The modified immune cell according to any one of claims 51-53, wherein the low-density lipoprotein receptor-associated protein or a fragment thereof comprises the amino acid sequence shown in SEQ ID NO:

60.

55. The modified immune cell according to any one of claims 51-54, wherein the low-density lipoprotein receptor-associated protein or a fragment thereof is an exogenous low-density lipoprotein receptor-associated protein or a fragment thereof.

56. An immunoconjugate comprising the antigen-binding protein of any one of claims 1-35.

57. The immunoconjugate of claim 56, wherein the immunoconjugate comprises an antigen-binding protein, a linker, and a payload.

58. The immunoconjugate of claim 57, wherein the payload is a toxin, polymer, protein, drug, radioisotope, nucleic acid compound, or glucocorticoid.

59. The immunoconjugate according to any one of claims 56-58, wherein the immunoconjugate is an antibody-drug conjugate (ADC), a PROTAC-antibody conjugate (DAC), or a targeted radiopharmaceutical (RDC).

60. An isolated nucleic acid molecule encoding an antigen-binding protein according to any one of claims 1-35 and / or a chimeric antigen receptor according to any one of claims 36-48.

61. A vector comprising the nucleic acid molecule of claim 60.

62. A cell comprising the nucleic acid molecule of claim 60 and / or the vector of claim 61.

63. A pharmaceutical composition comprising an antigen-binding protein according to any one of claims 1-35, a chimeric antigen receptor according to any one of claims 36-48, a modified immune cell according to any one of claims 49-55, an immune conjugate according to any one of claims 56-59, a nucleic acid molecule according to claim 60, a carrier according to claim 61, and / or a cell according to claim 62, and optionally a pharmaceutically acceptable carrier.

64. A detection kit comprising an antigen-binding protein according to any one of claims 1-35, a chimeric antigen receptor according to any one of claims 36-48, a modified immune cell according to any one of claims 49-55, an immunoconjugate according to any one of claims 56-59, a nucleic acid molecule according to claim 60, a carrier according to claim 61, a cell according to claim 62, and / or a pharmaceutical composition according to claim 63, the detection kit being used to detect the presence and / or content of CD155 in a sample or subject.

65. A method for detecting the presence and / or content of CD155, comprising using an antigen-binding protein according to any one of claims 1-35, a chimeric antigen receptor according to any one of claims 36-48, a modified immune cell according to any one of claims 49-55, an immunoconjugate according to any one of claims 56-59, a nucleic acid molecule according to claim 60, a carrier according to claim 61, a cell according to claim 62, and / or a pharmaceutical composition according to claim 63.

66. Use of the antigen-binding protein of any one of claims 1-35, the chimeric antigen receptor of any one of claims 36-48, the modified immune cell of any one of claims 49-55, the immune conjugate of any one of claims 56-59, the nucleic acid molecule of claim 60, the carrier of claim 61, the cell of claim 62, and / or the pharmaceutical composition of claim 63 in the preparation of a medicament for the prevention and / or treatment of tumors.

67. The use according to claim 66, wherein the tumor is a solid tumor and / or a hematoma.

68. The use according to any one of claims 66-67, wherein the tumor is positively expressed for CD155.

69. The use according to any one of claims 66-68, wherein the tumor is melanoma, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, head and neck cancer, lymphoma, multiple myeloma and / or leukemia.

70. A method for preventing and / or treating tumors, comprising administering to a subject in need an antigen-binding protein of any one of claims 1-35, a chimeric antigen receptor of any one of claims 36-48, a modified immune cell of any one of claims 49-55, an immune conjugate of any one of claims 56-59, a nucleic acid molecule of claim 60, a carrier of claim 61, a cell of claim 62, and / or a pharmaceutical composition of claim 63.

71. The method of claim 70, wherein the tumor is a solid tumor and / or a hematoma.

72. The method according to any one of claims 70-71, wherein the tumor is positively expressed for CD155.

73. The method according to any one of claims 70-72, wherein the tumor is melanoma, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma, head and neck cancer, lymphoma, multiple myeloma and / or leukemia.

74. The antigen-binding protein of any one of claims 1-35, the chimeric antigen receptor of any one of claims 36-48, the modified immune cell of any one of claims 49-55, the immune conjugate of any one of claims 56-59, the nucleic acid molecule of claim 60, the carrier of claim 61, the cell of claim 62, and / or the pharmaceutical composition of claim 63, for the prevention and / or treatment of tumors.

75. The antigen-binding protein, chimeric antigen receptor, modified immune cell, immune conjugate, nucleic acid molecule, carrier, cell and / or pharmaceutical composition according to claim 74, wherein the tumor is a solid tumor and / or hematologic malignancy.

76. The antigen-binding protein, chimeric antigen receptor, modified immune cell, immune conjugate, nucleic acid molecule, carrier, cell and / or pharmaceutical composition according to any one of claims 74-75, wherein the tumor CD155 is positively expressed.

77. The antigen-binding protein, chimeric antigen receptor, modified immune cell, immune conjugate, nucleic acid molecule, carrier, cell and / or pharmaceutical composition according to any one of claims 74-76, wherein the tumor is melanoma, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, head and neck cancer, lymphoma, multiple myeloma and / or leukemia.

Images