WO2025188136A1

WO2025188136A1 - Anti-tigit/anti-4-1bb bispecific antibodies and uses thereof

Info

Publication number: WO2025188136A1
Application number: PCT/KR2025/099526
Authority: WO
Inventors: Wonjun Son; Ye Lim Park; Kyeongsu PARK; Sora Kim; Hyunseong YOUN; Gihoon YOU; Jaehyoung JEON; Arim SEO; Byungje Sung; Minji PARK; Hyeran Lee; Kyeong Nang MOON; Han Byul Lee; Yangsoon Lee; Hyejin Chung
Original assignee: ABL Bio Inc
Current assignee: ABL Bio Inc
Priority date: 2024-03-04
Filing date: 2025-03-04
Publication date: 2025-09-12
Anticipated expiration: 2026-09-04
Also published as: WO2025188136A8

Abstract

The invention relates to a bispecific antibody or an antigen-binding fragment thereof comprising a first antigen binding site that can bind to TIGIT and a second antigen binding site that can bind to 4-1BB; pharmaceutical compositions comprising such bispecific antibodies and uses thereof. In particular, the antibody or the antigen-binding fragment thereof or the bispecific antibody or an antigen-binding fragment thereof can cause activation of T cells and then it can effectively prevent or treat the cancer.

Description

ANTI-TIGIT/ANTI-4-1BB BISPECIFIC ANTIBODIES AND USES THEREOF

The present invention pertains to antibodies, compositions comprising such antibodies and uses of such antibodies and compositions. More specifically, provided anti-TIGIT/anti-4-1BB bispecific antibodies and uses thereof.

Co-inhibitory or immune checkpoint receptors play a critical role in the maintenance of immune homeostasis: their expression on effector T cells ensures the proper contraction of effector T cell responses, while their expression on regulatory T (Treg) cells guarantees the proper functioning of Treg cells to control effector T cells. Accordingly, their function in regulating pro-inflammatory T cell responses and maintaining self-tolerance has been most widely studied in this context. More recently, the role of co-inhibitory receptors has come to the forefront particularly in cancer, where these receptors are highly expressed and are being targeted clinically to improve anti-tumor capability. While current immunotherapies directed against the coinhibitory receptors CTLA-4 and PD(L)-1 are exhibiting unprecedented efficacy in several cancer indications and chronic viral infections, there are still many patients that do not respond to these therapeutic approaches and some tumor types that remain largely refractory to such therapies.

In an aspect, the present disclosure provides an anti-TIGIT/anti-4-1BB bispecific antibody or an antigen-binding fragment thereof.

In another aspect, the present disclosure provides an isolated nucleic acid encoding the anti-TIGIT/anti-4-1BB bispecific antibody or an antigen-binding fragment thereof; a vector comprising the isolated nucleic acid; and a host cell comprising the vector.

In another aspect, the present disclosure provides a pharmaceutical composition of the anti-TIGIT/anti-4-1BB bispecific antibody or an antigen-binding fragment thereof.

In another aspect, the present disclosure provides a method for treating or preventing cancer in a patient in need thereof, comprised of administering to the patient an effective amount of the anti-TIGIT/anti-4-1BB bispecific antibody or an antigen-binding fragment thereof, or the use of the anti-TIGIT/anti-4-1BB bispecific antibody or an antigen-binding fragment thereof in the manufacture of a medicament for treating or preventing cancer.

An aspect of the present disclosure provides an anti-TIGIT/anti-4-1BB bispecific antibody or an antigen-binding fragment thereof.

In an embodiment, the bispecific antibody or antigen-binding fragment thereof comprising: (i) a first antigen binding site that binds to TIGIT (T-cell immunoreceptor with immunoglobulin and ITIM domain) and (ii) a second antigen binding site that binds to 4-1BB.

In an embodiment, the bispecific antibody or antigen-binding fragment thereof may comprise the first antigen binding site comprising any one of the following: (a) a heavy chain CDR1 of SEQ ID NO: 2; a heavy chain CDR2 of SEQ ID NO: 4; and a heavy chain CDR3 of SEQ ID NO: 6; (b) a heavy chain CDR1 of SEQ ID NO: 37; a heavy chain CDR2 of SEQ ID NO: 38; a heavy chain CDR3 of SEQ ID NO: 39; a light chain CDR1 of SEQ ID NO: 31; a light chain CDR2 of SEQ ID NO: 32; a light chain CDR3 of SEQ ID NO: 33; (c) a heavy chain CDR1 of SEQ ID NO: 47; a heavy chain CDR2 of SEQ ID NO: 48; a heavy chain CDR3 of SEQ ID NO: 49; a light chain CDR1 of SEQ ID NO: 42; a light chain CDR2 of SEQ ID NO: 43; a light chain CDR3 of SEQ ID NO: 44; (d) a heavy chain CDR1 of SEQ ID NO: 57; a heavy chain CDR2 of SEQ ID NO: 58; a heavy chain CDR3 of SEQ ID NO: 59; a light chain CDR1 of SEQ ID NO: 52; a light chain CDR2 of SEQ ID NO: 53; a light chain CDR3 of SEQ ID NO: 54; and (e) a heavy chain CDR1 of SEQ ID NO: 67; a heavy chain CDR2 of SEQ ID NO: 68; a heavy chain CDR3 of SEQ ID NO: 69; a light chain CDR1 of SEQ ID NO: 62; a light chain CDR2 of SEQ ID NO: 63; a light chain CDR3 of SEQ ID NO: 64.

In an embodiment, a bispecific antibody or antigen-binding fragment thereof may comprise the first antigen binding site thereof comprising: a heavy chain framework 1 (H-FR1) comprising an amino acid sequence of SEQ ID NO: 1 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 1; a heavy chain framework 2 (H-FR2) comprising an amino acid sequence of SEQ ID NO: 3 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 3; a heavy chain framework 3 (H-FR3) comprising an amino acid sequence of SEQ ID NO: 5 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 5; and a heavy chain framework 4 (H-FR4) comprising an amino acid sequence of SEQ ID NOs: 7 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO:7.

In an embodiment, the bispecific antibody or antigen-binding fragment thereof may comprise the first antigen binding site comprising any one of the following: (a) a single-domain antibody having a heavy chain CDR1 of SEQ ID NO: 2; a heavy chain CDR2 of SEQ ID NO: 4; and a heavy chain CDR3 of SEQ ID NO: 6; (b) a Fv fragment or scFv having a heavy chain CDR1 of SEQ ID NO: 37; a heavy chain CDR2 of SEQ ID NO: 38; a heavy chain CDR3 of SEQ ID NO: 39; a light chain CDR1 of SEQ ID NO: 31; a light chain CDR2 of SEQ ID NO: 32; a light chain CDR3 of SEQ ID NO: 33; (c) a Fv fragment or scFv having a heavy chain CDR1 of SEQ ID NO: 47; a heavy chain CDR2 of SEQ ID NO: 48; a heavy chain CDR3 of SEQ ID NO: 49; a light chain CDR1 of SEQ ID NO: 42; a light chain CDR2 of SEQ ID NO: 43; a light chain CDR3 of SEQ ID NO: 44; (d) a Fv fragment or scFv having a heavy chain CDR1 of SEQ ID NO: 57; a heavy chain CDR2 of SEQ ID NO: 58; a heavy chain CDR3 of SEQ ID NO: 59; a light chain CDR1 of SEQ ID NO: 52; a light chain CDR2 of SEQ ID NO: 53; a light chain CDR3 of SEQ ID NO: 54; and (e) a Fv fragment or scFv having a heavy chain CDR1 of SEQ ID NO: 67; a heavy chain CDR2 of SEQ ID NO: 68; a heavy chain CDR3 of SEQ ID NO: 69; a light chain CDR1 of SEQ ID NO: 62; a light chain CDR2 of SEQ ID NO: 63; a light chain CDR3 of SEQ ID NO: 64.

In an embodiment, the bispecific antibody or antigen-binding fragment thereof may comprise the first antigen binding site comprising any one of the following: (a) a heavy chain variable region of SEQ ID NO: 8; (b) a heavy chain variable region of SEQ ID NO: 36 and a light chain variable region of SEQ ID NO: 30; (c) a heavy chain variable region of SEQ ID NO: 46 and a light chain variable region of SEQ ID NO: 41; (d) a heavy chain variable region of SEQ ID NO: 56 and a light chain variable region of SEQ ID NO: 51; and (e) a heavy chain variable region of SEQ ID NO: 66 and a light chain variable region of SEQ ID NO: 61.

In an embodiment, the bispecific antibody or antigen-binding fragment thereof may comprise the second antigen binding site comprising: (a) a light chain CDR1 of SEQ ID NO: 10; (b) a light chain CDR2 of SEQ ID NO: 12; (c) a light chain CDR3 of SEQ ID NO: 14; (d) a heavy chain CDR1 of SEQ ID NO: 18; (e) a heavy chain CDR2 of SEQ ID NO: 20; and (f) a heavy chain CDR3 of SEQ ID NO: 22.

In an embodiment, the bispecific antibody or antigen-binding fragment thereof may comprise the second antigen binding site comprising a light chain variable region of SEQ ID NO: 16.

In an embodiment, the bispecific antibody or antigen-binding fragment thereof may comprise the second antigen binding site comprising a heavy chain variable region of SEQ ID NO: 24.

In an embodiment, the bispecific antibody or antigen-binding fragment thereof may be a mouse antibody, a chimeric antibody, a humanized antibody or a fully human antibody.

In an embodiment, the bispecific antibody or antigen-binding fragment thereof may be independently selected from a group consisting of a whole IgG, sdAb (single-domain antibody), Fab, Fab', F(ab')2, xFab, scFab, dsFv, Fv, scFv, IgG-scFv, sdAb-Fc, sdAb-Fc-scFv, scFv-Fc, scFab-Fc, diabody, minibody, scAb, dAb, half-IgG and combinations thereof.

In an embodiment, the bispecific antibody or antigen-binding fragment thereof may comprise an Fc region of an IgG1, IgG2, IgG3, or IgG4 antibody, or a hybrid Fc region or a constant region.

In an embodiment, the bispecific antibody or antigen-binding fragment thereof may be covalently linked with the second antigen binding site.

In an embodiment, the bispecific antibody or antigen-binding fragment thereof may comprise the first antigen binding site or the second antigen binding site, which are linked with a peptide linker.

In an embodiment, the bispecific antibody or antigen-binding fragment thereof may comprise the peptide linker of SEQ ID NOs: 25 or 26.

In an embodiment, the bispecific antibody or antigen-binding fragment thereof may comprise hinge region of SEQ ID NO: 27.

In an embodiment, a pharmaceutical formulation comprising the bispecific antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

In an embodiment, a pharmaceutical composition for use in a method of preventing or treating cancer, comprising the bispecific antibody or antigen-binding fragment thereof.

In an embodiment, the cancer may be immune checkpoint inhibitor-resistant cancer.

In an embodiment, the cancer may be TIGIT-positive cancer.

In an embodiment, wherein the cancer is selected from the group consisting of the cancer may be selected from the group consisting of leukemia, rectal cancer, endometrial cancer, nephroblastoma, basal cell carcinoma, nasopharyngeal cancer, bone tumor, esophageal cancer, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, follicular thyroid cancer, hepatocellular carcinoma, oral cancer, renal cell carcinoma, multiple myeloma, mesothelioma, osteosarcoma, myelodysplastic syndrome, mesenchymal tumor, soft tissue sarcoma, liposarcoma, gastrointestinal stromal tumor, malignant peripheral nerve sheath tumor (MPNST), Ewing sarcoma, leiomyosarcoma, mesenchymal chondrosarcoma, lymphosarcoma, fibrosarcoma, rhabdomyosarcoma, teratoma, neuroblastoma, medulloblastoma, glioma, benign skin tumor, Burkitt's lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, marginal zone lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy-cell leukemia, myelodysplastic syndrome (MDS), chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML), neuroectodermal tumor, epithelial tumor, cutaneous T-cell lymphoma (CTCL), peripheral T cell lymphoma (PTCL), pancreatic cancer, hematological malignancies, kidney cancer, tumor vasculature, breast cancer, renal cancer, ovarian cancer, epithelial ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), Head and neck squamous cell carcinoma (HNSCC), glioblastoma multiforme (GBM), brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer and adrenal cancer.

In an embodiment, the present invention provides a method for preventing or treating a cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the bispecific antibody or antigen-binding fragment.

In an embodiment, the present invention provides a use of the bispecific antibody or antigen-binding fragment thereof in the manufacture of medicament for treating or preventing cancer.

In an embodiment, the present invention provides a method for restoring T cells in a patient in need thereof, comprising administering to the patient an effective amount of the bispecific antibody or antigen-binding fragment.

In an embodiment, the present invention provides an isolated nucleic acid encoding the bispecific antibody or antigen-binding fragment thereof.

In an embodiment, the present invention provides a vector comprising the isolated nucleic acid.

In an embodiment, the present invention provides a host cell comprising the vector.

In an embodiment, the present invention provides combination therapy comprising an anti-TIGIT/anti-4-1BB bispecific antibody or an antigen-binding fragment thereof and an immune checkpoint inhibitor.

According to one of the embodiments, the bispecific antibodies or antigen binding fragments thereof may activate cytotoxic T cells and block immunosuppressive regulatory T cells by disrupting TIGIT and activating 4-1BB and thus provide synergistic therapeutic tumor treatment. Through multiple functions, the bispecific antibodies or an antigen binding fragments thereof exhibited potent tumor-killing activity and memory response, therefore can be a promising immunotherapy with the combination of anti-PD-(L)1 therapies for cancer patients.

FIG.1(a) and Fig.1(b) are graphs showing antigen binding activity of the bispecific antibody to TIGIT and 4-1BB Protein by SACE.

FIG.2 is a graph showing antigen binding activity of the bispecific antibody to TIGIT and 4-1BB Protein by DACE.

FIG.3(a) and Fig.3(b) are graphs showing cross-reactivity of anti-TIGIT/anti-4-1BB bispecific antibody to TIGIT from various species by SACE.

FIG.4(a) and Fig.4(b) are graphs showing binding activity of anti-TIGIT/anti-4-1BB bispecific antibody on the cell surface by FACS.

FIG.5 is a graph showing binding activity of anti-TIGIT/anti-4-1BB bispecific antibody to Treg cells by FACS

FIG.6 is a graph showing 4-1BB signal activation depending on TIGIT expression.

FIG.7 is a graph showing evaluation of the potency and stability of anti-TIGIT/anti-4-1BB bispecific antibody by in vitro TIGIT/CD155 blockade bioassay.

FIG.8(a) to Fig.8(c) are graphs showing 4-1BB signal activation dependent on FcγRI engagement.

FIG.9(a) to Fig.9(d) are graphs showing efficacy evaluation of anti-TIGIT/anti-4-1BB bispecific antibody in the treatment of the CT26 mouse colon cancer model in BALB/c-h4-1BB mice.

FIG.10(a) to Fig.10(d) are graphs showing efficacy evaluation of anti-TIGIT/anti-4-1BB bispecific antibody in the treatment of the H22 mouse liver cancer model in BALB/c-h4-1BB mice.

FIG.11(a) to Fig.11(d) are graphs showing efficacy evaluation of anti-TIGIT/anti-4-1BB bispecific antibody in the treatment of the MC38 mouse colon cancer model in C57BL/6-h4-1BB mice.

FIG.12(a) and Fig.12(b) are graphs showing antigen binding activity by SACE.

FIG.13 is a graph showing antigen binding activity by DACE.

FIG.14(a) and Fig.14(b) are graphs showing binding activity on the cell surface.

FIG.15 is a graph showing binding activity to Treg cells.

FIG.16(a) and Fig.16(b) are graphs showing 4-1BB signal activation depending on TIGIT expression.

FIG.17 is a graph showing evaluation of the potency and stability of anti-TIGIT/anti-4-1BB bispecific antibody by in vitro TIGIT/CD155 blockade bioassay.

FIG.18 is a graph showing in vivo efficacy study of anti-TIGIT/anti-4-1BB bispecific antibodies in CT26 model.

FIG.19(a) to Fig.20(b) are graphs showing in vivo TIL analysis in H22 model.

FIG.21(a) to Fig.21(h) are graphs showing efficacy evaluation of anti-TIGIT/anti-4-1BB bispecific antibody and combination therapy of TIGIT/4-1BB bispecific antibody with anti-PD-1 antibody in the treatment of the CT26-hPD-L1 mouse colon cancer Model in BALB/c-hPD-1/hPD-L1/h4-1BB mice.

FIG.22(a) to Fig.22(i) are graphs showing efficacy evaluation of anti-TIGIT/anti-4-1BB bispecific antibody and combination therapy of TIGIT/4-1BB bispecific antibody with anti-PD-L1 antibody in the treatment of the MC38 mouse colon cancer model in C57BL/6-h4-1BB/hTIGIT mice.

Definitions

Unless defined otherwise, technical and scientific terms used herein have the same meaning as generally those used in the art to which this invention belongs. For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa.

The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific and multi-specific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding affinity.

The term "monospecific" antibody as used herein denotes an antibody that has one or more binding sites each of which bind to the same epitope of the same antigen.

The term "bispecific" means that the antibody is able to specifically bind to at least two distinct antigenic determinants. For example, two binding sites each formed by any one of an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL) or both, binding to different antigens or to different epitopes on the same antigen.

The antibody provided herein is a multispecific antibody, e.g. a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. Provided herein is a bispecific antibody, with binding specificities for TIGIT and 4-1BB. Bispecific antibodies of the invention include, for example, multivalent single chain antibodies, diabodies and triabodies, as well as antibodies having the constant domain structure of full length antibodies to which further antigen-binding sites (e.g., single chain Fv, Fv, a VH domain and/or a VL domain, Fab, or (Fab)2) are linked via one or more peptide-linkers. The antibodies can be full length from a single species, or be chimerized or humanized. For another example, the multispecific antibody may include a multiparatopic antibody.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities, and "knob-in-hole" engineering. Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules; cross-linking two or more antibodies or fragments; using leucine zippers to produce bi-specific antibodies; using "diabody" technology for making bispecific antibody fragments; and using single-chain Fv (scFv) dimers; and preparing trispecific antibodies as described. The term "valent" as used within the current application denotes the presence of a specified number of binding domains in an antibody or antibody fragment. As such, the terms "monovalent", "bivalent", "trivalent", "tetravalent", "pentavalent", and "hexavalent" denote the presence of one binding domain, two binding domains, three binding domains, four binding domains, five binding domains, and six binding domains, respectively, in an antibody. The bispecific antibodies according to the invention are at least "bivalent" and may be "trivalent" or "multivalent" (e.g., "tetravalent", "pentavalent" or "hexavalent"). In a particular aspect, the antibodies of the present invention have two or more binding sites and are bispecific. That is, the antibodies may be bispecific even in cases where there are more than two binding sites (i.e., that the antibody is trivalent or multivalent).

The complementarity determining regions (CDRs) in the variable region allow the antibody to selectively recognize and specifically bind epitopes on antigens. More specifically, in a conventional antibody such as IgG, the antigen-binding site is defined by three CDRs on each of the VH and VL chains (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3). In some instances, e.g., certain immunoglobulin molecules derived from camelid species or engineered based on camelid immunoglobulins, a complete immunoglobulin molecule may consist solely of heavy chains, with no light chains.

The terms "CDR-H", "HCDR" and "CDRH" herein are used interchangeably to refer to a VH chain of the CDR (e.g., CDR-H1, HCDR1 and CDRH1 are refer to a VH1 of the CDR). The terms "CDR-L", "LCDR" and "CDRL" herein are used interchangeably to refer to a VL chain of the CDR (e.g., CDR-L1, LCDR1 and CDRL1 are a refer to a VL1 of the CDR).

In naturally occurring antibodies, the "complementarity determining regions" or "CDRs" present in each antigen-binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain as the antibody assumes its three-dimensional configuration in an aqueous environment. The remainder of the amino acids in the antigen-binding domains, referred to as "framework" regions, show less inter-molecular variability. The CDRs and the framework regions included in an antibody, respectively, can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been precisely defined.

In the case where there are two or more definitions of a term which is used and/or accepted within the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated otherwise. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the noncontiguous antigen combining sites found within the variable region of heavy and/or light chain polypeptides. This particular region has been described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) or by Chothia et al., J. MoI. Biol. 196: 901-917 (1987). In addition, the numbering scheme of the international ImMunoGeneTics information system (IMGT), the AbM definition (see bioinfo.org.uk/abs/), Martin Antibody Numbering (Enhanced Chothia or AbM) or Honneger's numbering scheme (AHo) can be used for the identification of variable regions and CDRs. The definitions of CDR according to such multiple numbering schemes include overlaps or subsets of amino acid residues when compared against each other. Nevertheless, application of any definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.

The definitions of CDR according to Kabat and Chothia include overlaps or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.

Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this "Kabat numbering" system to any variable domain sequence, without reliance on any experimental data beyond the sequence itself.

Antibodies disclosed herein may be from any animal origin, including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies.

As used herein, the term "heavy chain constant region" includes amino acid sequences derived from an immunoglobulin heavy chain. As set forth above, it will be understood by one of ordinary skill in the art that the heavy chain constant region may be modified such that they vary in amino acid sequence from the naturally occurring immunoglobulin molecule.

The heavy chain constant region of an antibody disclosed herein may be derived from different immunoglobulin molecules. For example, a heavy chain constant region of a polypeptide may comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, a heavy chain constant region can comprise a hinge region derived, in part, from an IgG1 molecule and, in part, from an IgG3 molecule. In another example, a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgG1 molecule and, in part, from an IgG4 molecule. In another example, a heavy chain constant region can comprise a CH2 domain derived from an IgG1 molecule and a hinge region derived from an IgG1 molecule.

An "antibody fragment" or "antigen-binding fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. An immunologically functional immunoglobulin fragment includes sdAb (single-domain antibody), Fab, Fab', F(ab')₂, xFab, scFab, dsFv, Fv, scFv, IgG-scFv, sdAb-Fc, sdAb-Fc-scFv, scFv-Fc, scFab-Fc, diabody, minibody, scAb, dAb, half-IgG or combinations thereof, but not limited thereto. The term "Fab" used in Fab, Fab', F(ab')₂, xFab and scFab may include a traditional Fab fragment and the chimeric Fab-like domain. In addition, it may be derived from any mammal including human, mouse, rat, camelid or rabbit, but not limited thereto. The functional part of the antibody such as one or more CDRs described herein may be linked with a secondary protein or small molecular compound by a covalent bond, thereby being used as a target therapeutic agent to a specific target. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g., E. coli or phage), as described herein.

The term "single-domain antibody" or "sdAb" refers to a single antigen-binding polypeptide having three complementary determining regions (CDRs). The sdAb alone is capable of binding to the antigen without pairing with a corresponding CDR-containing polypeptide. In some cases, single-domain antibodies are engineered from camelid HCAbs, and their heavy chain variable domains may be referred as "VHHs" (Variable domain of the heavy chain of the Heavy chain antibody). For example, Camelid sdAb is one of the smallest known antigen-binding antibody fragments. A basic VHH has the following structure from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which FR1 to FR4 refer to framework regions 1 to 4, respectively. The term "Fv fragment (Fv)" refers to a covalently or noncovalently associated heterodimer of a VH domain and a VL domain that specifically binds to an antigen, forming the Fv region. Each VH and VL domain contains three complementary determining regions (CDRs).

Antibodies that mediate cytotoxicity by recruiting and activating endogenous immune cells are an emerging class of next generation antibody therapeutics. According to the invention, bispecific antibodies are provided wherein both the TIGIT-binding site and the 4-1BB-binding site bind to the antigens on T cells, antigen presenting cells, natural killer cells, etc.―i.e., tumor microenvironment.

As used herein, the term "antigen binding domain" or "antigen binding site" refers to the part of the antibody or antibody fragment that specifically binds to an antigenic determinant. More particularly, the term "antigen binding domain" refers to the part of an antibody that comprises the area which specifically binds to and is complementary to part or all of an antigen. In one aspect, the antigen binding domain is able to bind to its antigen and block or partly block its function. Antigen binding domains that specifically bind to TIGIT and/or to 4-1BB include antibodies and fragments thereof as further defined herein. In addition, antigen binding domains may include scaffold antigen binding proteins, e.g., binding domains which are based on designed repeat proteins or designed repeat domains.

As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site (e.g., a contiguous stretch of amino acids or a conformational configuration made up of different regions of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen binding moiety binds, forming an antigen binding moiety-antigen complex. "Specific binding" means that the binding is selective for the antigen and can be discriminated from unwanted or non-specific interactions. The ability of an antibody or antibody fragment to bind to a specific antigen can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g., Surface Plasmon Resonance (SPR) technique (analyzed on a BIAcore쪠 instrument), and traditional binding assays.

As used herein, "Affinity" or "binding affinity" refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen).

The term "TIGIT/4-1BB bispecific antibody", "anti-TIGIT/4-1BB bispecific antibody" "TIGIT/4-1BB", "TIGITx4-1BB" or an "anti-TIGIT/anti-4-1BB antibody" are used interchangeably herein and refer to an antibody that can bind to both TIGIT and 4-1BB with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting TIGIT and 4-1BB.

As used herein, the terms "TIGIT protein" or "TIGIT antigen" or "TIGIT" are used interchangeably and refer to TIGIT (T cell immunoreceptor with Ig and ITIM domains) that binds the poliovirus receptor (PVR-also known as CD155). TIGIT is also known as VSIG9, VSTM3, or WUCAM. Reference to TIGIT includes the native human TIGIT protein naturally expressed in the human host and/or on the surface of human cultured cell lines, as well as recombinant forms and fragments thereof and also naturally occurring mutant forms.

TIGIT is an inhibitory immune checkpoint receptor suppressing T cell activation by interacting with the poliovirus receptor (also known as CD155) expressed in dendritic cells, macrophages, and tumor cells. TIGIT expression is increased on tumor infiltrating lymphocytes (TILs) and in disease settings such as HIV infection. TIGIT expression marks exhausted T cells that have lower effector function as compared to TIGIT negative counterparts. Conversely, Treg cells that express TIGIT show enhanced immunosuppressive activity as compared to TIGIT negative Treg population. Like CTLA4 which competes with CD28 for CD80/86 binding, TIGIT competes with its counter-receptor CD226, a costimulatory receptor, for binding to CD155. TIGIT expression is induced after T cell activation and regulates T cell activity by disrupting CD226-CD155 interactions and triggering T cell inhibitory signaling. In the CITYSCAPE phase II study of advanced non-small cell lung cancer (NSCLC), the combination of tiragolumab (600 mg) plus atezolizumab (1200 mg) showed increased efficacy compared to placebo plus atezolizumab [ORR (31.3% versus 16.2%; median progression free survival (PFS): 5.4 versus 3.6 months. However, in the phase III SKYSCRAPER-01 study of PD-L1^high metastatic NSCLC, the combination of tiragolumab and atezolizumab did not meet the primary endpoint of PFS and awaits further clinical assessment.

Antibodies targeting TIGIT have different mechanisms with either an "enabled" Fc function (able to interact with various Fc receptors) or "silent" Fc function (mutated to prevent interaction with Fc receptors).

TIGIT was highlighted as one of key immune checkpoints as the next promising immunotherapy overcoming resistance to PD-(L)1 blocking antibodies. However, clinical efficacies of TIGIT antibodies were moderate in monotherapy and combination efficacy with PD-(L)1 antibodies resulted in somewhat mixed clinical outcome.

The terms "anti-TIGIT antibody", "an antibody that binds to TIGIT" and "an antibody comprising an antigen binding domain that binds to TIGIT" refer to an antibody or antigen binding fragment that is capable of binding TIGIT, especially a TIGIT polypeptide expressed on a cell surface, with sufficient affinity. Especially, the anti-TIGIT antibody VH28 may recognize an epitope of TIGIT which is different from those of known anti-TIGIT antibodies. Thus, VH28 may have superior results in this regard compared to other commercial anti-TIGIT antibodies. VH28 may enable a higher level of immune activation compared to others and overcome resistance to PD-(L)1 blocking antibodies or other commercial anti-TIGIT antibodies by binding to the unique epitope of TIGIT. As used herein, the term "4-1BB" is a member of TNF-receptor superfamily (TNFRSF) and is a co-stimulatory molecule which is expressed following the activation of immune cells, both innate and adaptive immune cells. 4-1BB plays important role in modulate the activity of various immune cells. 4-1BB agonists enhance immune cell proliferation, survival, secretion of cytokines and cytolytic activity CD8 T cells. Many other studies showed that activation of 4-1BB enhances immune response to eliminate tumors in mice. Therefore, it suggests that 4-1BB is a promising target molecule in cancer immunology. 4-1BB, as a strong, inducible costimulatory receptor, also has been an attractive target for antitumor therapeutics but development of 4-1BB agonists was hampered by unexpected challenges such as liver toxicity or low efficacy. In addition, increased expression of 4-1BB within intra-tumoral Tregs was reported.

The term "4-1BB" refers to CD137, or TNFRSF9 (TNF Receptor 25 Superfamily Member 9), is a member of TNF-receptor superfamily (TNFRSF) and is a co-stimulatory molecule which is expressed following the activation of immune cells, both innate and adaptive immune cells. As used herein, 4-1BB may be originated from a mammal, for example, Homo sapiens (human) (NCBI Accession No. NP_001552.2).

As described herein, the term "4-1BB" includes variants, isoforms, homologs, orthologs, and paralogs. For example, antibodies specific for a human 4-1BB protein may, in certain cases, cross-react with a 4-1BB protein from a species other than human. In other embodiments, the antibodies specific for a human 4-1BB protein may be completely specific for the human 4-1BB protein and may exhibit species or other types of cross-reactivity, or may cross-react with 4-1BB from certain other species but not all other species (e.g., cross-react with monkey 4-1BB, but not mouse 4-1BB). The term "human 4-1BB" refers to human sequence 4-1BB, such as the complete amino acid sequence of human 4-1BB having NCBI Accession No. NP_001552.2. The term "mouse 4-1BB" refers to mouse sequence 4-1BB, such as the complete amino acid sequence of mouse 4-1BB having NCBI Accession No. NP 033430.1. 4-1BB also can be known in the art as, for example, CD137. The human 4-1BB sequence in the disclosure may differ from human 4-1BB of NCBI Accession No. NP_001552.2 by having, e.g., conserved mutations or mutations in non-conserved regions and the 4-1BB in the disclosure has substantially the same biological function as the human 4-1BB of NCBI Accession No. NP_001552.2.

The terms "anti-4-1BB antibody", "an antibody that binds to 4-1BB" and "an antibody comprising an antigen binding domain that binds to 4-1BB" refer to an antibody or antigen binding fragment that can bind to 4-1BB, especially a 4-1BB polypeptide expressed on a cell surface, with sufficient affinity.

The term "restoration" refers to repairing a function of effector T cell (cytotoxic T cell) which was affected by cancer cells or other cells. For example, inhibition of TIGIT, an immune checkpoint inhibitor expressed by effector T cells, binding using antagonistic antibodies has shown potential to restore T-cell function.

The term "Immune　checkpoint" refers to inhibitory pathways hardwired into the　immune　system that are crucial for maintaining self-tolerance and modulating the duration and amplitude of physiological　immune　responses in peripheral tissues in order to minimize collateral tissue damage.　Immune　checkpoint molecules can be stimulatory or inhibitory to an　immune　checkpoint. The present disclosure and claims refer to inhibitory molecules of　immune　checkpoints as "immune　checkpoint molecules". Preliminary clinical findings with agents that block　immune　checkpoint molecules, suggest opportunities to enhance antitumor immunity with the potential to produce effective clinical responses. An　immune　checkpoint inhibitor is a type of drug that blocks the signaling of　immune　checkpoint molecule(s) made by some types of　immune　system cells, such as T cells and some cancer cells.　Immune　checkpoint inhibitors therefore can cause　immune　checkpoint blockade.　Immune　checkpoint molecules help keep　immune　responses in check and can keep T cells from killing cancer cells. When these molecules are blocked, the "brakes" on the　immune　system are released (inhibition of the　immune　system is reduced or blocked) and T cells are able to kill cancer cells better. In some embodiments,　immune　checkpoint molecules are proteins. In some embodiments,　immune　checkpoint molecules are nucleic acids that encode the proteins. In some embodiments,　immune　checkpoint inhibitors bind to and/or antagonize　immune　checkpoint molecules.

The term "peptide linker" refers to a peptide comprising one or more amino acids, typically about 2 to 30 amino acids. Peptide linkers are known in the art or are described herein. Suitable, non-immunogenic linker peptides are, for example, (GS)_n, (G₄S)_n, (SG₄)_n or G₄(SG₄)_n pep peptide linkers, wherein "n" is generally a number between 1 and 10, i.e. the peptides selected from the group consisting of (GS)₉: SEQ ID NO: 26 and (G₄S)_4": SEQ ID NO: 25.

As used herein, the terms "engineer, engineered, engineering" are considered to include any manipulation of the peptide backbone or the post-translational modifications of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modifications of the amino acid sequence, the glycosylation pattern, or the side chain group of individual amino acids, as well as combinations of these approaches.

The term "mutation" as used herein in relation to amino acid, protein or antibody is meant to encompass amino acid substitutions, deletions, insertions, addition, and modifications. Any combination of substitution, deletion, insertion, addition, and modification can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., reduced binding to an Fc receptor, or increased association with another peptide. As used herein, the term "polypeptide" is intended to encompass a singular "polypeptide" as well as plural "polypeptides," and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" may be used instead of, or interchangeably with, any of these terms. Considering the purpose of the present disclosure, the useful polypeptide fragment includes an immunological functional fragment of an antibody comprising an antigen-binding domain. In the case of TIGIT or 4-1BB binding antibody, such a useful fragment includes a CDR sequence comprising 1, 2, or 3 of heavy chains and/or light chains, or all or a portion of the antibody chain comprising a variable region or constant region of a heavy chain or light chain, but not limited thereto.

As used herein, "variant" of a polypeptide such as, for example, an antigen-binding fragment, a protein or an antibody, is a polypeptide in which one or more amino acid residues are inserted, deleted, added and/or substituted, as compared to another polypeptide sequence, and includes a fusion polypeptide. In addition, a protein variant includes one modified by protein enzyme cutting, phosphorylation or other posttranslational modification, but maintains biological activity of the antibody disclosed herein, for example, specific binding to TIGIT and/or 4-1BB and biological activity.

As used herein, "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences.

As used herein, "percent (%) sequence identity" and "homology" with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

A "therapeutically effective amount" of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent, for example, eliminates, decreases, delays, minimizes or prevents adverse effects of a disease.

The term "pharmaceutical composition" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the composition would be administered.

A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is considered to be nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, "treatment" (and grammatical variations thereof such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the antibody of the present disclosure may be used to delay development of a disease or to slow the progression of a disease.

Anti-TIGIT antibody

An anti-TIGIT antibody may comprise an anti-TIGIT antibody or an antigen-binding fragment thereof as a TIGIT targeting moiety. The anti-TIGIT antibody or antigen binding fragment thereof may exhibit potent binding and inhibitory activities to TIGIT and be useful for therapeutic uses.

In one embodiment, the anti-TIGIT antibody or fragment thereof may be capable of specificity to a mouse, cynomolgus and/or human TIGIT protein.

In an embodiment, the anti-TIGIT antibody or the antigen-binding fragment thereof may comprise any one of the following: (a) a heavy chain CDR1 of SEQ ID NO: 2; a heavy chain CDR2 of SEQ ID NO: 4; and a heavy chain CDR3 of SEQ ID NO: 6; (b) a heavy chain CDR1 of SEQ ID NO: 37; a heavy chain CDR2 of SEQ ID NO: 38; a heavy chain CDR3 of SEQ ID NO: 39; a light chain CDR1 of SEQ ID NO: 31; a light chain CDR2 of SEQ ID NO: 32; a light chain CDR3 of SEQ ID NO: 33; (c) a heavy chain CDR1 of SEQ ID NO: 47; a heavy chain CDR2 of SEQ ID NO: 48; a heavy chain CDR3 of SEQ ID NO: 49; a light chain CDR1 of SEQ ID NO: 42; a light chain CDR2 of SEQ ID NO: 43; a light chain CDR3 of SEQ ID NO: 44; (d) a heavy chain CDR1 of SEQ ID NO: 57; a heavy chain CDR2 of SEQ ID NO: 58; a heavy chain CDR3 of SEQ ID NO: 59; a light chain CDR1 of SEQ ID NO: 52; a light chain CDR2 of SEQ ID NO: 53; a light chain CDR3 of SEQ ID NO: 54; and (e) a heavy chain CDR1 of SEQ ID NO: 67; a heavy chain CDR2 of SEQ ID NO: 68; a heavy chain CDR3 of SEQ ID NO: 69; a light chain CDR1 of SEQ ID NO: 62; a light chain CDR2 of SEQ ID NO: 63; a light chain CDR3 of SEQ ID NO: 64.

The CDR sequences of anti-TIGIT (VH28) antibody or the antigen-binding fragment to be comprised in heavy chain variable regions of the antibody or antigen-binding fragment according to one embodiment of the present invention are shown in table 1 below.

Region	Sequence	SEQ ID NO:
HFR1	EVQLVESGGGLVQPGGSLRLSCAASGYKYG	1
VH CDR1	VYSMG	2
HFR2	WFRQAPGKGLEGVS	3
VH CDR2	AICSGGRTTYSDSVKG	4
HFR3	RFTISRDNSNQILYLQMNSLRAEDTAVYYCAA	5
VH CDR3	RPLWTGDCDLSSSWYKT	6
HFR4	WGQGTLVTVSS	7
TIGIT single domain	EVQLVESGGGLVQPGGSLRLSCAASGYKYGVYSMGWFRQAPGKGLEGVSAICSGGRTTYSDSVKGRFTISRDNSNQILYLQMNSLRAEDTAVYYCAARPLWTGDCDLSSSWYKTWGQGTLVTVSS	8

In an embodiment, the antibody or antigen-binding fragment thereof may comprise a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 41, 51, 61 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 41, 51, 61.

In an embodiment, the antibody or antigen-binding fragment thereof may comprise a heavy chain framework 1 (H-FR1) comprising an amino acid sequence of SEQ ID NO: 1 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 1; a heavy chain framework 2 (H-FR2) comprising an amino acid sequence of SEQ ID NO: 3 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 3; a heavy chain framework 3 (H-FR3) comprising an amino acid sequence of SEQ ID NO: 5 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 5; a heavy chain framework 4 (H-FR4) comprising an amino acid sequence of SEQ ID NOs: 7 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO:7.

In an embodiment, the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 46, 56, 66 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 46, 56, 66.

In an embodiment, the antibody or antigen-binding fragment thereof may comprise a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 70, 71, 72, 73 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 70, 71, 72, 73.

In an embodiment, the antibody or antigen-binding fragment thereof may comprise a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 40, 50, 60 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 40, 50, 60.

Anti-4-1BB antibody

An anti-4-1BB antibody may comprise an anti-4-1BB antibody or an antigen-binding fragment thereof as a 4-1BB targeting moiety. The anti-4-1BB antibody or antigen-binding fragment thereof may exhibit potent binding and inhibitory activities to 4-1BB and be useful for therapeutic uses.

In an embodiment, the anti-4-1BB antibody or fragment thereof can specifically bind to 4-1BB (e.g., human 4-1BB) protein.

In an embodiment, the anti-4-1BB antibody or the antigen-binding fragment thereof may comprise (a) a VL CDR1 comprising an amino acid sequence of SEQ ID NO: 10; (b) a VL CDR2 comprising an amino acid sequence of SEQ ID NO: 12; (c) a VL CDR3 comprising an amino acid sequence of SEQ ID NO: 14; (d) a VH CDR1 comprising an amino acid sequence of SEQ ID NO: 18; (e) a VH CDR2 comprising an amino acid sequence of SEQ ID NO: 20; and (f) a VH CDR3 comprising an amino acid sequence of SEQ ID NO: 22.

The CDR sequences of anti-4-1BB (1A10M12) antibody or the antigen-binding fragment to be comprised in heavy chain variable regions of the antibody or antigen-binding fragment according to one embodiment of the present invention are shown in table 2 below.

In an embodiment, the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 24 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 24.

In an embodiment, the antibody or antigen-binding fragment thereof may comprise a light chain variable region comprising an amino acid sequence of SEQ ID NO: 16 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NOs: 16.

In an embodiment, the antibody or antigen-binding fragment thereof may comprise a heavy chain framework 1 (H-FR1) comprising an amino acid sequence of SEQ ID NO: 17 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 17; a heavy chain framework 2 (H-FR2) comprising an amino acid sequence of SEQ ID NO: 19 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 19; a heavy chain framework 3 (H-FR3) comprising an amino acid sequence of SEQ ID NO: 21 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 21; a heavy chain framework 4 (H-FR4) comprising an amino acid sequence of SEQ ID NO: 23 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 23; a light chain framework 1 (L-FR1) comprising an amino acid sequence of SEQ ID NO: 9 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 9; a light chain framework 2 (L-FR2) comprising an amino acid sequence of SEQ ID NO: 11 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 11; a light chain framework 3 (L-FR3) comprising an amino acid sequence of SEQ ID NO: 13 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 13; and a light chain framework 4 (L-FR4) comprising an amino acid sequence of SEQ ID NO: 15 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 15.

Anti-TIGIT/anti-4-1BB bispecific antibody

An anti-TIGIT/anti-4-1BB bispecific antibody may comprise the anti-TIGIT antibody or antigen-binding fragment thereof; and anti-4-1BB antibody or antigen binding fragment thereof. The anti-TIGIT/anti-4-1BB bispecific antibody may be useful for therapeutic uses.

Thus, to overcome the limitations of combined therapies, anti-TIGIT/anti-4-1BB bispecific antibody, which has restored T cell activity by abolishing TIGIT-CD155 interactions and activating 4-1BB in a TIGIT- and CD64-dependent manner and the activation of 4-1BB induces an immune memory response that leads to anticipated long-lasting anti-tumor activity, is expected to exhibit superior efficacy compared to conventional combined therapies.

By implementing additional functional activities including TIGIT-dependent 4-1BB activation and FcγR binding activity, TIGIT antibody was potentiated to stimulate innate as well as adaptive immune responses. Outstanding antitumor efficacy as well as combination efficacy with PD(L)-1 antibodies were observed arising from T cell activation and tumor microenvironment (TME) change involving Treg depletion and myeloid cell activation.

In one embodiment, the bispecific antibody comprises the TIGIT antigen binding site and the 4-1BB antigen binding site.

In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof and the anti-4-1BB antibody or antigen-binding fragment thereof may be fused, directly or via a peptide linker.

In one embodiment, each of the anti-TIGIT antibody or antigen-binding fragment thereof and the anti-4-1BB antibody or antigen-binding fragment thereof may independently be a sdAb, Fv, scFv or a Fab molecule.

In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof is a single-domain antibody (sdAb), and the anti-4-1BB antibody or antigen-binding fragment thereof is a single-chain fragment variables (scFvs); the anti-TIGIT antibody or antigen-binding fragment thereof is fused, at the C-terminus of the sdAb of the anti-TIGIT antibody or antigen-binding fragment thereof, to the C-terminus of the scFvs of the anti-4-1BB antibody or antigen-binding fragment thereof and the anti-4-1BB antibody or antigen-binding fragment thereof is fused, at the C-terminus of the scFvs of the anti-4-1BB antibody or antigen-binding fragment thereof, to the N-terminus of the Fc domain, in a symmetric structure.

The amino acid sequences of anti-TIGIT/anti-4-1BB bispecific antibody or antigen-binding fragment according to one embodiment of the present invention are shown in table 3 below.

Region	Sequence	SEQ ID NO:
TIGIT single domain	EVQLVESGGGLVQPGGSLRLSCAASGYKYGVYSMGWFRQAPGKGLEGVSAICSGGRTTYSDSVKGRFTISRDNSNQILYLQMNSLRAEDTAVYYCAARPLWTGDCDLSSSWYKTWGQGTLVTVSS	8
Hinge region	DKTHTCPPCPAPELLGGP	27
IgG1	SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK	28
GS9 linker	GSGSGSGSGSGSGSGSGS	26
4-1BB light chain variable region	QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVTWYQQLPGTAPKLLIYADSHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVL	16
G4S linker	GGGGSGGGGSGGGGSGGGGS	25
4-1BB heavy chain variable region	EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKCLEWVSWISYSGGSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRNSMREFDYWGQGTLVTVSS	24

Fc Domain

The Fc domain confers to the antibody or bispecific antibody favorable pharmacokinetic properties, including a long serum half-life which contributes to good accumulation in the target tissue and a favorable tissue-blood distribution ratio. Wild-type Fc domain is involved in antibody dependent cellular cytotoxicity (ADCC) which is an Fc-dependent effector function of IgG important for anti-viral immunity and anti-tumor therapies. On the other hand, it may lead to undesirable targeting of the antibody or bispecific antibody to cells expressing Fc receptors rather than to the preferred antigen-bearing cells.

In one embodiment, the Fc domain of the present invention is wild-type Fc domain. In another embodiment, the amino acid mutation that reduces the binding affinity of the Fc domain to a Fc receptor and/or effector function may be introduced to the antibody. For example, the Fc domain may comprise an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329 (numberings according to Kabat EU index). For example, the further amino acid substitution may be E233P, L234A, L235A, L235E, N297A, N297D or P331S.

Therapeutic Methods and Compositions

Any of the antibodies or bispecific antibodies provided herein may be used in therapeutic methods. Antibodies or bispecific antibodies of the invention may be used as immunotherapeutic agents, for example in the treatment of cancers.

In one aspect, antibodies or bispecific antibodies of the invention for use as a medicament are provided. In further aspects, antibodies or bispecific antibodies of the invention for use in treating a disease are provided. In certain embodiments, antibodies or bispecific antibodies of the invention for use in a method of treatment are provided.

In one embodiment, the invention provides bispecific antibodies as described herein for use in the treatment of a cancer in an individual in need thereof.

In one embodiment, the cancer may be a solid cancer or a blood cancer.

In one embodiment, the cancer may be selected from the group consisting of leukemia, rectal cancer, endometrial cancer, nephroblastoma, basal cell carcinoma, nasopharyngeal cancer, bone tumor, esophageal cancer, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, follicular thyroid cancer, hepatocellular carcinoma, oral cancer, renal cell carcinoma, multiple myeloma, mesothelioma, osteosarcoma, myelodysplastic syndrome, mesenchymal tumor, soft tissue sarcoma, liposarcoma, gastrointestinal stromal tumor, malignant peripheral nerve sheath tumor (MPNST), Ewing sarcoma, leiomyosarcoma, mesenchymal chondrosarcoma, lymphosarcoma, fibrosarcoma, rhabdomyosarcoma, teratoma, neuroblastoma, medulloblastoma, glioma, benign skin tumor, Burkitt's lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, marginal zone lymphoma, neuroectodermal tumor, epithelial tumor, cutaneous T-cell lymphoma (CTCL), peripheral T cell lymphoma (PTCL), pancreatic cancer, haematological malignancies, kidney cancer, tumor vasculature, breast cancer, renal cancer, ovarian cancer, epithelial ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), Head and neck squamous cell carcinoma (HNSCC), glioblastoma multiforme (GBM), brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer and adrenal cancer.

Hereinafter, the present invention will be described in more detail by way of examples. These examples are only for describing the present invention in more detail, and it will be apparent to those skilled in the art that according to the gist of the present invention, the scope of the present invention is not limited to these examples.

Combination Therapy in Cancer

The present disclosure also provides methods for combination therapies in which an agent known to modulate other pathways, or other components of the same pathway, or even overlapping sets of target enzymes are used in combination with a compound of the present disclosure, or a pharmaceutically acceptable salt thereof. In one aspect, such therapy includes but is not limited to the combination of one or more compounds of the disclosure with chemotherapeutic agents, therapeutic antibodies, and radiation treatment, to provide a synergistic or additive therapeutic effect.

A further embodiment of the invention includes a method of using a combination comprising the TIGIT/4-1BB bispecific antibody with another therapeutic agent selected from a chemotherapeutic agent, a PD-1 inhibitor, or a PD-L1 inhibitor for the treatment of cancer.

Administered "in combination", as used herein, means that two (or more) different treatments are delivered to the patient during the course of the patient's affliction with the cancer, e.g., the two or more treatments are delivered after the patient has been diagnosed with the cancer and before the cancer has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as "simultaneous" or "concurrent delivery". In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration.

Examples

The disclosures of each of the below-referenced applications are incorporated by reference herein in their entirety.

Example 1. Production of anti-TIGIT/anti-4-1BB bispecific antibody

1-1. Generation of anti-TIGIT antibody

1-1-1. Generation of anti-TIGIT sdAb

(1) Immunization

Camels were immunized with recombinant TIGIT-Fc (ACROBiosystems) protein under all current animal welfare regulations. For immunization, the antigen was formulated as an emulsion with CFA (Complete Freund's adjuvant; primary immunization) or IFA (incomplete Freund's adjuvant; boost immunizations). The antigen was administered subcutaneously at the neck. Each animal received 5 injections of the emulsion, containing 100 pg of TIGIT-Fc in CFA emulsion and 4 subsequent injections of TIGIT-Fc in IFA emulsion at two-week intervals. At different time points during immunization, 10 mL blood samples were collected from the animal and sera were prepared. The induction of an antigen specific humoral immune response was verified using the serum samples in an ELISA-based experiment with immobilized TIGIT-His protein. Five days after the last immunization, a blood sample of 300 mL was collected. Peripheral blood lymphocytes (PBLs), as the genetic source of the camel HCAbs, were isolated from the 300 mL blood sample using a FicolL-Paque쪠 gradient (Amersham Biosciences).

(2) Library construction

RNA extracted from PBLs was used as starting material for RT-PCR to amplify sdAb encoding gene fragments. These fragments were cloned into an in-house phagemid vector. In frame with the sdAb coding sequence, the vector coded for a C-terminal His-Tag. The library phage was prepared according to a standard protocol and stored after filter sterilization at 4°C for further use.

(3) Selections and high-throughput screening

Selections were carried out with the above libraries using solid panning as well as cell-based panning. Each selection output was analyzed for enrichment factor (# of phage present in eluate relative to control), diversity and percentage of TIGIT positive clones (ELISA). Based on these parameters the best selections were chosen for further analysis. To this end, the output from each selection was recloned as a pool into a soluble expression vector for high-throughput screening. In frame with the sdAb coding sequence, the vector coded for a C terminal His-Tag. Colonies were picked and grown in 96 deep well plates (1 mL volume) and induced by adding IPTG and 0.1% Triton for sdAb expression in the supernatant. The supernatant was analyzed for their ability to bind to TIGIT protein (by ELISA) and TIGIT expressing CHO-K1 stable cell line (by FACS). The positive binders were sequenced and the unique clones were selected for further characterization. The unique clones were grown in 2XYT medium and induced by IPTG for sdAb expression in the supernatant. The supernatants of unique binders were analyzed for their ability to inhibit the interaction between CD155 and TIGIT. To this end, TIGIT-expressing stable CHO cells were incubated with the sdAb-containing supernatant first, then with CD155-Fc (ACROBiosystems) followed by fluorophore labelled secondary antibody against human Fc. Shift in mean fluorescent intensity (MFI) as compared with samples without anti-TIGIT sdAb blocking represents the blockade of CD155/TIGIT binding. All potential inhibitors were selected for KD analysis by surface plasmon resonance (SPR) on a BIAcore쪠 T200 instrument. The dissociation phase was used to calculate the K_off values for each individual sdAb.

(4) Humanization, production and characterization of anti-TIGIT sdAb-Fc fusion proteins

Among humanized variants, (AS19584) VH28 was selected for production and characterization according to affinity and small-scale production level. The humanized anti-TIGIT sdAb-Fc fusion protein constructs were generated by fusing humanized anti-TIGIT sdAbs with human IgG1 Fc region. The maxiprep of the constructs were prepared for HEK293 cell transient expression and purification. The expressed humanized anti-TIGIT sdAb-Fc fusion proteins were purified by chromatography through a column containing Protein A agarose resin. Protein purity was determined by SEC-HPLC.

1-1-2. Generation of anti-TIGIT IgG antibody

Anti-TIGIT clones COM-902 (Compugen's anti-TIGIT antibody, Patent No. US 10,751,415), F04 (Yuhan's anti-TIGIT antibody, Patent No. KR 2019-0103996), Tiragolumab (Roche's anti-TIGIT antibody, Patent No. US 10,017,572), and Vibostolimab (Merck's anti-TIGIT antibody, Patent No. US 10,618,958) were prepared in IgG format as disclosed in U.S. Patent No. 10,751,415, Republic of Korea Patent No. 10-2019-0103996, U.S. Patent No. 10,017,572, and U.S. Patent No. 10,618,958, respectively. Specifically, two types of were generated using the IgG1 and IgG4 backbones.

1-2. Generation of anti-4-1BB Antibody

1-2-1. Preparation of full human anti-4-1BB monoclonal antibodies

Full human anti-4-1BB monoclonal antibodies were prepared as disclosed in WO2020/111913. Specifically, for panning of a phage library (obtained from Kbio Health) against target molecules, A total of four rounds of panning were carried out using 4-1BB (NCBI Accession No. NP_001552.2) coated immunotubes. Bacterial colonies from the 3 rounds of panning output were grown in SB-Carbenicillin in 96 deep well plate until turbid, at which point 10¹¹ pfu of VCSM13 helper phage was added to each well. After 1 hour infection at 37℃ with gentle shaking (80 rpm), 70 μg/mL of kanamycin was added, and the cells were cultured overnight at 30℃ with shaking at 200 rpm. Next day, the plates were centrifuged and the supernatants containing the phages were added to 4-1BB antigen-coated ELISA plates blocked with 3% BSA in PBST. After 1 h incubation at room temperature, the plates were washed three times with PBST and anti M13 antibody was added. The plates were incubated for 1 hour, washed three times with PBST, and the binding affinity was measured using tetramethylbenzidine (TMB).

The 4-1BB specific binders were amplified for plasmid DNA sequencing. Ig light chain variable (VL) genes and Ig heavy chain (VH) genes were analyzed to identify unique sequences and determine sequence diversity.

1-2-2. Preparation of anti-4-1BB scFv antibodies

Anti-4-1BB scFv antibodies with a structure of (N')-VL-linker [(GGGGS)₄ SEQ ID NO:25] VH-(C') were prepared using the variable regions of the full human monoclonal antibodies against 4-1BB obtained in Example 1-2-1 above, wherein the amino acid residue "G" at the position 44 of a heavy chain variable region was substituted with "C", and the amino acid residue "G" at the position 103 of a light chain variable region was substituted with "C". Such amino acid substitution from "G" to "C" in scFv can contribute to increase in stabilities of bispecific antibodies comprising the scFv as one target specific moiety.

1-3. Generation of anti-TIGIT/anti-4-1BB Bispecific antibody

The anti-TIGIT clones and the anti-4-1BB clone mentioned above were selected to prepare anti-TIGIT/anti-4-1BB bispecific antibodies. The anti-TIGIT/anti-4-1BB bispecific antibodies were generated with the sequences shown in Table 6. In these constructs, an scFv antibody fragment specific to one antigen is fused to the C-terminal of IgG Fc region, while an antibody fragment specific to the other antigen is fused to the N-terminal of the Fc region. When TIGIT is placed in full IgG part or TIGIT sdAb is linked to Fc region, wild type of IgG1 backbone were used. The CDR sequences of each anti-TIGIT/anti-4-1BB bispecific antibody are also bolded and underlined in Table 6.

For anti-TIGIT/anti-4-1BB bispecific antibodies having full IgG TIGIT antibody, cloning of the bispecific antibody was performed as follows: A DNA segment 1 having a nucleotide sequence encoding a heavy chain of an IgG antibody of the anti-TIGIT/anti-4-1BB bispecific antibody was inserted into pcDNA 3.4 (Invitrogen, A14697; plasmid 1), and a DNA segment 2 having a nucleotide sequence encoding a light chain of an IgG antibody of the anti- TIGIT/anti-4-1BB bispecific antibody was inserted into pcDNA 3.4 (Invitrogen, A14697; plasmid 2). Thereafter, a DNA segment 3 encoding a scFv was fused at a part of the DNA segment 1 corresponding to the c-terminus of the Fc region of the IgG antibody inserted into the plasmid 1, using a DNA segment 4 encoding a linker peptide having 16 amino acid lengths consisting of (GGGGS)₄: SEQ ID NO: 25 or using a DNA segment 5 encoding a linker peptide having 18 amino acid lengths consisting of (GS)₉: SEQ ID NO: 26 to construct vectors for the expression of bispecific antibodies. In case of anti-TIGIT/anti-4-1BB bispecific antibodies having anti-TIGIT sdAb, the same cloning method was used except that DNA segment 2―encoding a light chain―was not used. TIGIT-binding single domain antibody (sdAb) was cloned to the N-terminal hinge region of CH2 of IgG1 Fc region. Anti-4-1BB scFvs were cloned into the C-terminals of CH3 through (GS)₉ linkers: SEQ ID NO: 26.

The constructed vectors were transiently expressed in ExpiCHO-S™ cells (Thermo Fisher, A29127) using ExpiFectamine쪠 CHO Kit (ThermoFisher, A29129), cultured in ExpiCHO-S™ Expression medium (ThermoFisher, A2910002) under the conditions of 37℃ for 7 days in a CO₂ incubator equipped with rotating shaker. Plasmid DNA (250 μg) and ExpiFectamin™ CHO Reagent (800 μL) were mixed with Opti-MEM® I medium (20 mL final volume) and allowed to stand at room temperature for 5 minutes. The mixed solution was added to 6x10⁶ ExpiCHO™ cells cultured in ExpiCHO™ Expression Medium and gently mixed in a shaker incubator at 37℃ with a humidified atmosphere of 8% CO₂ in air. At 18 hours post-transfection, 1.5 mL of ExpiFectamin™ CHO Transfection Enhancer and 60 mL of ExpiFectamin™ CHO Transfection Feed were added to each flask. After transfection for 7 days, cells were harvested, and the supernatant was used for purification.

Each bispecific antibody was purified from the cell culture supernatant by recombinant Protein A affinity chromatography (MabSelect™ SuRe™, Cytiva, 17-5438-03) and gel filtration chromatography with a HiLoad 26/600 Superdex-200 prep grade column (Cytiva, 28-9893-36). SDS-PAGE (NuPAGE 4-12% Bis-Tris gel, NP0321) and size exclusion HPLC (Agilent, 1200 series) analysis with SE-HPLC column (TSKgel G3000SWXL ID 7.8 mm, 30 cm, 5 μm, TOSOH, 0008541) were performed to detect and confirm the size and purity of each bispecific antibody. The purified proteins were buffer-exchanged and concentrated with 20 mM Histidine-HCl, pH 6.0, 8% (w/v) Sucrose buffer using an Amicon Ultra -15, 30K centrifugal filter unit (Millipore, UFC903096) and then formulation was performed by spiking the 20 mM Histidine-HCl, pH 6.0, 8% (w/v) Sucrose, 2% (w/v) Polysorbate 80 stock solution to become the final 20 mM Histidine-HCl, pH 6.0, 8%(w/v) Sucrose, 0.02% (w/v) Polysorbate 80. And then, protein concentrations were estimated using a Nanodrop One (ThermoFIsher).

For anti-TIGIT/anti-4-1BB bispecific antibodies having full IgG TIGIT antibody, a two-vector system was applied and the ratio between light to heavy chain was 1:2 by weight. Alternatively, for anti-TIGIT/anti-4-1BB bispecific antibodies having anti-TIGIT sdAb, a one-vector system was used as described above.

Example 2. Evaluation of anti-TIGIT/anti-4-1BB bispecific antibody

2-1. Antigen binding activity

2-1-1. Antigen binding activity of the bispecific antibody to TIGIT and 4-1BB Protein by SACE

To evaluate the binding activity to TIGIT and 4-1BB, tested antibodies were subjected to SACE (Single antigen captured ELISA). Anti-TIGIT/anti-4-1BB bispecific antibody (VH28/1A10M12), TIGIT single domain antibody (VH28), Tiragolumab, 1A10M12 and Urelumab (BMS's anti-4-1BB antibody) were tested.

Briefly, a micro plate was coated with 100 ng/well of human TIGIT-His protein (Sinobiological, 10917-H08H), and human 4-1BB-His protein (Sinobiological, 10041-H08H) in PBS at 4℃ overnight. After removing the human TIGIT protein, the human 4-1BB protein plate was incubated with 200 μl/well of 1% BSA buffer (1X PBS, 1% BSA) for 2 hours at 37℃. Four-fold dilutions of each tested antibody, starting from 100 nM, were added to each well. And the plate was incubated for 1 hour at 37℃ and then washed with PBS-T (1X PBS/0.05% Tween-20). Then, the plate was washed with PBS-T and then incubated with HRP (Horse Radish Peroxidase) conjugated Anti-Fc antibody (Pierce, 31413) for 1 hour at 37℃. After washing, the plate was developed as a TMB substrate, and the reaction was stopped using sulfuric acid. And the plate was analyzed by spectrophotometer at OD 450-650nm. A Four-parameter logistic curve analysis was performed with GraphPad software.

The results are shown in the figures 1(a) and 1(b). hIgG1 was used as a negative control. As shown in the figures, the anti-TIGIT/anti-4-1BB bispecific antibody showed comparable binding activity to both human TIGIT and 4-1BB compared to anti-TIGIT or anti-4-1BB monospecific antibodies (mAb). This suggests that the anti-TIGIT antibody and the anti-4-1BB antibody, even in the form of bispecific antibody, retain their binding activity to their respective targets without substantial interference.

2-1-2. Antigen binding activity of the bispecific antibody to TIGIT and 4-1BB Protein by DACE

To evaluate the two-way binding activity to TIGIT and 4-1BB, anti-TIGIT/anti-4-1BB bispecific antibody (VH28/1A10M12) was subjected to DACE (Dual antigen captured ELISA).

Briefly, a micro plate was coated with 100 ng/well of human TIGIT-Fc protein (Sinobiological, 10917-H02H) in PBS at 4℃ overnight. After removing the human TIGIT protein, the plate was incubated with 200 μl/well of 1% BSA buffer (1X PBS, 1% BSA) for 2 hours at 37℃. Four-fold dilutions of each the tested antibody, starting from 100 nM, were added to each well. And the plate was incubated for 2 hours at 37℃ and then washed with PBS-T (1X PBS/0.05% Tween-20). And then 100 ng/well of human 4-1BB-His protein (Sinobiological, 10041-H08H) in 1% BSA was added to each well and incubated for 1 hour at 37℃. The plate was washed with PBS-T and then incubated with Anti-His HRP (Roche, Cat: 11965085001) for 1 hour at 37℃. After washing, the plate was developed as a TMB substrate, and the reaction was stopped using sulfuric acid. And the plate was analyzed by a spectrophotometer at OD 450-650nm. Four-parameter logistic curve analysis was performed with GraphPad software.

The results are shown in the figure 2. As shown in the figure, the anti-TIGIT/anti-4-1BB bispecific antibody showed binding activity to both human TIGIT and 4-1BB.

2-2. Cross-reactivity of anti-TIGIT/anti-4-1BB bispecific antibody to TIGIT from various species by SACE

To evaluate the cross-reactivity of the VH28/1A10M12 bispecific antibody to TIGIT of mouse and cynomolgus monkey, VH28/1A10M12 and the TIGIT antibodies were subjected to an ELISA test. As TIGIT monospecific antibodies, COM-902 (IgG4), Tiragolumab, and Vibostolimab were used.

Briefly, a micro plate was coated with 100 ng/well of mouse TIGIT-His protein (Sinobiological, 50939-M08H), cynomolgus TIGIT-His protein (Abcam, ab223112) in PBS at 4℃ overnight. After removing the mouse TIGIT protein, the cynomolgus TIGIT protein plate was incubated with 200 μl/well of 1% BSA buffer (1X PBS, 1% BSA) for 2 hours at 37℃. Six-fold dilutions of each tested antibody, starting from 100 nM, were added to each well. And the plate was incubated for 1 hour at 37℃ and then washed with PBS-T (1X PBS/0.05% Tween-20). The plate was washed with PBS-T and then incubated with HRP (Horse Radish Peroxidase) conjugated Anti-Fc antibody (Pierce, 31413) for 1 hour at 37℃. After washing, the plate was developed as a TMB substrate, and the reaction was stopped using sulfuric acid. And the plate was analyzed by a spectrophotometer at OD 450-650nm. Four-parameter logistic curve analysis was performed with GraphPad software.

The results are shown in the figures 3(a) and 3(b). As shown in the figures, VH28/1A10M12 bispecific antibody and COM-902 bound to both mouse TIGIT and cynomolgus TIGIT while Tiragolumab and Vibostolimab only bound to cynomolgus TIGIT.

2-3. Binding activity

2-3-1. Binding activity of anti-TIGIT/anti-4-1BB bispecific antibody on the cell surface (FACS)

To evaluate the binding activity of anti-TIGIT/anti-4-1BB bispecific antibody VH28/1A10M12 to human TIGIT and human 4-1BB expressed on the cell surface, mean fluorescence intensity (MFI) was evaluated by flow cytometry (BD bioscience, LSRFortessa X-20). MFI intensity for cells indicated the ability of tested antibodies to bind to cells.

In brief, human 4-1BB overexpressed Jurkat (Promega, J2332) and human TIGIT overexpressed CHO-K1 cells (GenScript, M00542) were placed in 96 well plate, then washed with 200 μl/well of 1% BSA buffer. Four-fold dilutions of each tested antibody, starting from 100 nM, were added to each well and incubated for 1 hour at 4℃. The plates were washed with PBS-T 1% BSA buffer. And then FITC-conjugated anti-human IgG (Fc specific) (sigma, F9512) in 1% BSA buffer was added to each well and incubated for 1 hour at 4℃. The plates were washed with PBS-T 1% BSA buffer. The MFI of FITC was evaluated by flow cytometry. The obtained MFI values were subjected to a four-parameter logistic curve analysis with GraphPad software.

The results are shown in the figures 4(a) and 4(b). In dual antigen captured ELISA (DACE), anti-TIGIT/anti-4-1BB bispecific antibody bound to TIGIT and 4-1BB simultaneously with EC₅₀ of 0.3 nM (Fig 2). The anti-TIGIT/anti-4-1BB bispecific antibody bound to CHO-K1 and Jurkat T cells expressing TIGIT and 4-1BB with EC₅₀ of 0.2 and 3.3 nM, respectively (Figs 4(a) and 4(b)). VH28/1A10M12 showed lower 4-1BB binding activity than urelumab (EC₅₀: 0.3 nM) but showed comparable binding activity to its parent anti-4-1BB antibody, 1A10M12 (EC₅₀: 2.6 nM, Fig 4(b)). Anti-TIGIT/anti-4-1BB bispecific antibody bound to CHO-K1 expressing hTIGIT (EC₅₀: 0.2 nM) with comparable affinity to that of the parent anti-TIGIT antibody (EC₅₀: 0.1 nM, indicated as TIGIT sdAb, Fig. 4(a)). As shown below regarding Fig. 5, the binding of the bispecific antibody to expanded Treg cells (EC₅₀: 0.2 nM, Fig. 5) was also comparable to that of parent anti-TIGIT antibody. The analysis of Tiragolumab (EC₅₀: 0.7 nM), parent anti-TIGIT antibody (EC₅₀: 0.1 nM), and the anti-TIGIT/anti-4-1BB bispecific antibody showed that Tiragolumab had lower binding activity than the anti-TIGIT/anti-4-1BB bispecific antibody. In surface plasmon resonance (SPR) analysis (the results are shown in the table 4 below), it was shown that the anti-TIGIT/anti-4-1BB bispecific antibody bound to TIGIT and 4-1BB with a KD of 0.5x10^-9 M and 4.5x10^-9 M, respectively. Interestingly, the anti-TIGIT/anti-4-1BB bispecific antibody bound to mouse TIGIT with a KD of 0.5 Х 10^-9 M, supporting the successful establishment of the syngeneic mouse model for in vivo analysis.

2-3-2. Binding activity of anti-TIGIT/anti-4-1BB bispecific antibody to Treg cells (FACS)

To evaluate the binding activity of anti-TIGIT/anti-4-1BB bispecific antibody VH28/1A10M12 to human Treg cells (Regulatory T cells), which are naturally expressing TIGIT and 4-1BB, mean fluorescence intensity (MFI) was evaluated by flow cytometry (BD bioscience, LSRFortessa X-20). MFI intensity for cells indicates the ability of tested antibody to bind to cells.

In brief, Primary human CD4+ T cells were isolated from cryopreserved peripheral blood mononuclear cells (CTL, CTL-UP1) using a Miltenyi CD4+ T cell isolation kit (Miltenyi Biotec, 130-096-533), according to the manufacturer's instructions. For the expansion of Treg cells, human CD4+ T cells were plated at 5x10⁵cells/mL in Human T cell Expansion Media containing 20 ng/mL recombinant human IL-2 (Peprotech, 200-02-100UG). Then, 75 μL of Cloudz™ Treg CD3/CD28 was added per 2mL of cells to each well containing cells by using Cloudz™ Human Treg Expansion Kit, and the cells were cultured for 9 days. The rhIL-2 media was changed on days 2 and 3. To generate Treg cells, these Treg cells were placed in a 96-well plate, then washed with 200 μl/well of 1% BSA buffer. 5 μl of Human TruStain FcX™ (Biolegend, 422302) was added per million cells in a 100 μl staining volume and incubated at room temperature for 5-10 minutes. Six-fold dilutions of each tested antibody, starting from 100 nM, were added to each well and incubated for 1 hour at 4℃. The plates were washed with PBS-T 1% BSA buffer. And then AF647-conjugated 'F(ab')₂ Fragment anti-human IgG (Jackson Immunoresearch, 109-606-098) in 1% BSA buffer was added to each well and incubated for 1 hour at 4℃. The plates were washed with PBS-T 1% BSA buffer. The MFI of AF647 was evaluated by flow cytometry. The obtained MFI values were subjected to a four-parameter logistic curve analysis with GraphPad software.

The results are shown in the figure 5. As shown in the figure, the anti-TIGIT/anti-4-1BB bispecific antibody bound to Treg cells with comparable binding activity to parental TIGIT sdAb and Tiragolumab. Such results may be due to the potential of the anti-TIGIT/anti-4-1BB bispecific antibody to suppress tumor growth by depleting immunosuppressive Treg cells within the tumor.

2-4. Binding activity analysis using Surface Plasmon Resonance (SPR)

SPR was used to measure the binding activity of an anti-TIGIT/anti-4-1BB bispecific antibody VH28/1A10M12, with human / mouse TIGIT and human 4-1BB. The antigens used in here are as follows: TIGIT (Human: R&D systems, Cat.9525-TG-050, Mouse: Sino Biological / Cat.50939-M08H), 4-1BB (Human: R&D systems, Cat. 9220-4B-100). All binding activities were measured using Biacore™ T200 (Cytiva). To evaluate the binding activity of the anti-TIGIT/anti-4-1BB bispecific antibody, human / mouse TIGIT were diluted to 0, 6.25, 12.5, 25, 50, and 100 nM, and human 4-1BB was diluted to 0, 15.625, 31.25, 62.5, 125, and 250 nM using HBS-EP (1X). And then the anti-TIGIT/anti-4-1BB bispecific antibody was captured on the surface of Protein A chip (Cytiva, Cat.29127556) at about 200 RU. Diluted analytes were injected at a flow rate of 30 μL/minute for 60 seconds over the surface that the anti-TIGIT/anti-4-1BB bispecific antibody was captured. To monitor complex dissociation, HBS-EP (1X) was flowed at 30 μL/minute for 180 seconds after the association phase. To recover the chip surface, 10 mM Glycine-HCl pH 1.5 (Cytiva, Cat.BR100354) was injected at 30 μL/minute for 30 seconds. All measurements were independently performed twice at 25℃. The kinetic parameters of the anti-TIGIT/anti-4-1BB bispecific antibody to human/mouse TIGIT and human 4-1BB were calculated using a 1:1 binding model (Biacore™ T200 evaluation software ver.1.0).

The embodiments are shown in the Table 5 below. The anti-TIGIT/anti-4-1BB bispecific antibody has binding activity to both human and mouse TIGIT, and human 4-1BB

Antigen	Ka (1/Ms, x10 ⁵ )	Kd (1/s, x10 ^-4 )	K _D (M, x10 ^-9 )	Rmax (RU)
Human TIGIT	3.655 ± 0.375	1.756 ± 0.431	0.489 ± 0.168	37.40 ± 0.71
Mouse TIGIT	6.275 ± 0.100	3.122 ± 0.054	0.498 ± 0.016	43.29 ± 0.13
Human 4-1BB	2.095 ± 0.012	9.482 ± 0.112	4.527 ± 0.028	56.51 ± 0.90

Example 3. Activity and efficacy of anti-TIGIT/anti-4-1BB bispecific antibody

3-1. 4-1BB signal activation depending on TIGIT expression

To measure 4-1BB signal activation in anti-TIGIT/anti-4-1BB bispecific antibody, a cell-based 4-1BB NF-kB luciferase reporter assay (4-1BB activation assay) was performed. For the test antibodies, VH28/1A10M12 was used as an anti-TIGIT/anti-4-1BB bispecific antibody, and VH28 (TIGIT sdAb) and Tiragolumab were used as anti-TIGIT monospecific antibody. In the 4-1BB activation assay, the activity of tested antibodies in the presence of human TIGIT antigens was evaluated using human TIGIT overexpressing CHO-K1 cells. GloResponse™ NFκB-luc2/4-1BB Jurkat cell line (Promega, cat# CS196004) was used as effector cells. GloResponse™ NFκB-luc2/4-1BB Jurkat cell line was genetically modified to stably express 4-1BB and luciferase downstream of a response element. Luciferase expression is induced upon antibody binding to the 4-1BB receptor.

In brief, CHOK1-hTIGIT (TIGIT positive hamster ovarian epithelial cells, 2.5x10⁴) were added on a white 96-well plate containing 100 μl culture medium, and the plate was incubated overnight at 37℃ in a 5% CO₂ humidification incubator. After incubation, 100 μl of culture medium was removed, and 25 μl of analysis medium (RPMI1640 containing 1% (v/v) FBS) was added to pre-attached target cells. 25 μl of tested antibody (5-fold dilution starting from 40 nM) was added to the plate. GloResponse™ NFκB-luc2/4-1BB Jurkat cell lines were recovered and re-entered on an analysis medium. 25 μl of GloResponse™ NFκB-luc2/4-1BB Jurkat cells were added to the plate to have 2.5x10⁴ cells per well, and the plate was incubated at 37℃ for 6 hours in a 5% CO₂ humidification incubator. During incubation time, the BIO-GLO™ reagent was reconstructed according to the manufacturer's manual. After 6 hours of incubation, 75 μl of BIO-GLO™ Reagent per well was added to the plate. It waited for 5 minutes and measured light emission using a microplate reader. The four-parameter logistic curve was evaluated using GraphPad software.

The results using CHOK1-hTIGIT cells are shown in the figure 6. As shown in the figure 6, the anti-TIGIT/anti-4-1BB bispecific antibody activated 4-1BB in a TIGIT-dependent manner with EC₅₀ of 0.1 nM (Fig 6), but not in the presence of mock CHO-K1 cells (data not shown). However, either anti-TIGIT mAb alone or a combination of anti-TIGIT monospecific antibody and anti-4-1BB monospecific antibody did not induce 4-1BB activation. These results indicated that the crosslinking of TIGIT and 4-1BB as a bispecific antibody is required for T cell activation (Fig 6). In other words, an anti-TIGIT/anti-4-1BB bispecific antibody may trigger a highly potent 4-1BB activation. Such results were raised due to the capability of an anti-TIGIT/anti-4-1BB bispecific antibody to induce robust TIGIT-dependent 4-1BB clustering when TIGIT exists, thereby facilitating 4-1BB activation.

3-2. Evaluation of the potency and stability of anti-TIGIT/anti-4-1BB bispecific antibody by in vitro TIGIT/CD155 blockade bioassay

To compare the TIGIT blocking activity of VH28/1A10M12 with the previous anti-TIGIT monospecific antibodies, VH28/1A10M12 was analyzed for its ligand blocking activity (in vitro TIGIT Bioassay) by using Promega kit system (Promega, J2092) with anti-TIGIT antibodies from Roche, and TIGIT single domain antibody.

In brief, CD155 aAPC/CHO-K1 cells (4x10⁴ cells/well) were incubated in a white 96-well assay plate in 100 μL culture medium for overnight at 37℃ in a 5% CO₂ humidified incubator. After the overnight culture, 100 μl of culture medium was removed, and 25 μl of analysis medium (RPMI1640 containing 1% (v/v) FBS) was added to the pre-attached target cells. 25 μL of TIGIT blocking antibodies (3X) were indicated: VH28/1A10M12, TIGIT sdAb, Tiragolumab (Roche). 25 μL of TIGIT effector cells (1.5x10⁵ cells/well) were dispensed for each assay well and then cultured for 6 hours at 37℃ in a 5% CO₂ humidified incubator. During the incubation time, BIO-GLO™ reagent was reconstituted according to the manufacturer's instruction. After 6 hours of incubation, 75 μL of BIO-GLO™ reagent was added per well to the assay plate. After 5 minutes waiting period, luminescence was quantified using a microplate reader (PHERAstar FS, BMG LABTECH, Ortenberg, Germany). The four-parameter logistic curve analysis was performed using GraphPad Prism® software v10.0.1 (GraphPad Software Inc., San Diego, CA, USA).

The results are shown in figure 7. As shown in the figure, VH28/1A10M12 strongly inhibited TIGIT-CD155 interactions, restoring T cell activity with EC₅₀ of 2.8 nM compared to Tiragolumab treatment with EC₅₀ of 8.1 nM. It was found that VH28/1A10M12 exhibited relatively stronger blocking activity among the tested antibodies, even though VH28/1A10M12 is in a bispecific antibody format. In other words, VH28/1A10M12 showed better TIGIT blocking effect than anti-TIGIT monospecific antibodies.

3-3. 4-1BB signal activation dependent on FcγRI engagement

The anti-TIGIT/anti-4-1BB bispecific antibody was designed to retain FcγR binding activity to maximize the efficacy of the anti-TIGITx4-1BB BsAb. It was assumed that Fc-competent TIGITx4-1BB could induce both FcγR-dependent 4-1BB activation and TIGIT-dependent 4-1BB activation. To identify the main FcγR isotype interacting with the Fc region of TIGITx4-1BB antibody VH28/1A10M12 leading to 4-1BB activation, CHO-K1 cells expressing FcγRI (CD64, Genscript, M00597), FcγRIIb (CD32, Promega, JA2255), or FcγRIIIa (CD16, Genscript, M00588) were incubated with the luciferase reporter-expressing NF-kB-Luc2/4-1BB Jurkat T cells. Four-parameter logistic curve analysis was performed with GraphPad Prism® software. The anti-TIGIT/anti-4-1BB bispecific antibody strongly induced 4-1BB activation only in the presence of FcγRI-expressing CHO-K1 cells (Fig 8(a) to Fig 8(c)).

The results are shown in figures 8(a) to 8(c). As shown in the figures, the anti-TIGIT/anti-4-1BB bispecific antibody activated T cells in TIGIT and FcγRI expression-dependent manner in addition to blocking TIGIT-mediated T cell suppression. In other words, the anti-TIGIT/anti-4-1BB bispecific antibody showed activation of 4-1BB via FcγRI-dependent cross-linking. It has been confirmed that by binding exclusively to FcγRI and not to FcγRIIb and FcγRIIIa, the risk of nonspecific immune responses of the bispecific antibody is reduced, thus minimizing potential side effects.

Example 4. In vivo efficacy study of anti-TIGIT/anti-4-1BB bispecific antibody

4-1. Efficacy Evaluation of anti-TIGIT/anti-4-1BB bispecific antibody in the Treatment of the CT26 mouse colon cancer Model in BALB/c-h4-1BB Mice

Tumor inhibitory activity of anti-TIGIT/anti-4-1BB bispecific antibody was compared with that of Human IgG1 and parent TIGIT single domain Ab (TIGIT sdAb). 24 BALB/c-h4-1BB mice were subcutaneously injected with CT26 tumor cells (5Х10⁵/0.1 mL/mouse) (ATCC (Manassas, VA, USA), Cat No. CRL-2638) in the right behind flank for tumor development. 13 days post inoculation, the 24 tumor-bearing animals were randomly enrolled into three study groups when the mean tumor size reached 81.45 mm³. Each group consisted of 8 mice. The three groups were G1: Human IgG1 (3 mg/kg), G2: TIGIT sdAb (1.6 mg/kg) and G3: anti-TIGIT/anti-4-1BB bispecific antibody VH28/1A10M12 (2.6 mg/kg). Treatment began on the day of grouping (Day 0). All test antibodies were intraperitoneally administered to tumor-bearing mice twice weekly for a total of six times. The tumor volume and body weight were measured and recorded twice weekly.

The results are shown in figure 9(a). As shown in the figure, while the anti-TIGIT/anti-4-1BB bispecific antibody exerted strong tumor growth inhibition, TIGIT sdAb only showed moderate tumor growth inhibition. This suggests that TIGIT blocking alone is not enough for complete tumor regression. The anti-TIGIT/anti-4-1BB bispecific antibody treatment resulted in complete tumor regression in 6 out of 8 mice and its activity lasted for 100 days even after discontinuation of drug treatment.

After the first CT26 transplantation, the anti-TIGIT/anti-4-1BB bispecific antibody was administered to confirm the memory response using mice that survived by immunotherapy. After administration of the bispecific antibody, CT26 was transplanted into the mice in which the tumor completely disappeared 100 days later.

The results are shown in figure 9(b). As shown in the figure, tumors of naive mice used as control grew, but mice that survived after administration of the anti-TIGIT/anti-4-1BB bispecific antibody did not grow tumors. The anti-TIGIT/anti-4-1BB bispecific antibody showing complete regression (CR) in the primary tumor challenge were protected from further tumor re-challenge (Fig 9(b)).

100 μL of peripheral blood was collected from naive mice and the anti-TIGIT/anti-4-1BB bispecific antibody-treated mice with complete regression (CR) at days -7, 7, 14, and 28 after tumor re-challenge and analyzed for memory T cell levels via Flow Cytometry (panel: L/D, mCD45, mCD3, mCD8 (clone KT15), mCD4, mCD44, mCD62L, AH1 (gp70) tetramer). Recovered mice from the previous challenge showed increased proportions of memory CD4+ (CD4+CD44+) and CD8+ T cells (CD8+CD44+) at day 7~28 (Fig 9(c) and 9(d)). This suggests that immune memory is established by the anti-TIGIT/anti-4-1BB bispecific antibody.

Compared with naive mice, the anti-TIGIT/anti-4-1BB bispecific antibody treatment showed significantly increased the proportion of tumor-specific memory T cells in the blood, suggesting long-term immune memory had been established. As a result, mice that survived after receiving the anti-TIGIT/anti-4-1BB bispecific antibody had specific memory immunity against CT26. In other words, the anti-TIGIT/anti-4-1BB bispecific antibody showed inhibition of tumor growth of CT26 in h4-1BB knock-in mice and protection of mice from tumor re-challenge.

4-2. Efficacy evaluation of anti-TIGIT/anti-4-1BB bispecific antibody in the Treatment of the H22 mouse liver cancer Model in BALB/c-h4-1BB Mice

15 Female BALB/c-h4-1BB mice (6-8 weeks old) were subcutaneously injected with H22 hepatoma cells (1Х10⁶/0.1 mL/mouse) (CCTCC (Wuhan, China)) in the upper right flank for tumor development. 5 days post inoculation, the 15 tumor-bearing animals were randomly enrolled into three study groups when the mean tumor size reached 77.37 mm³. Each group consisted of 5 mice. The three groups were G1: Human IgG1 (7.5 mg/kg), G2: parent anti-TIGIT sdAb (4 mg/kg) and G3: anti-TIGIT/anti-4-1BB bispecific antibody VH28/1A10M12 (6.65 mg/kg). Treatment began on the day of grouping (Day 0). All test antibodies were intraperitoneally administered to tumor-bearing mice once every three days for a total of four times. The tumor volume and body weight were measured and recorded twice weekly.

As shown in the figures 10(b) to 10(d), in another mouse model with murine H22 hepatoma expressing high levels of endogenous CD155, both the anti-TIGIT mAb and the anti-TIGIT/anti-4-1BB bispecific antibody exerted robust antitumor activities in the beginning, however, tumors relapsed shortly in anti-TIGIT mAb-treated group around day 30 after treatment. This indicated that, although anti-TIGIT mAb monotherapy showed marked inhibition of tumor growth, its antitumor effects were ephemeral. This tumor inhibitory efficacy of anti-TIGIT mAbs was not observed in other tumor models, including the CT26 and MC38 (Fig 9(a)). Because H22 overexpresses CD155, it is likely that the blockade of TIGIT-CD155 interactions initially promoted tumor regression, but TIGIT blockade and Fc-mediated NK cell activation might not sustain long-term effects. On the contrary to TIGIT mAb, the anti-TIGITx4-1BB BsAb strongly inhibited tumor growth with complete regression in all mice for up to 100 days.

For the re-challenge study, mice with complete regression (CR, cured mice) were monitored for ~3 months without antibody treatment. Mice were re-challenged with H22 cells (1 Х 10⁶). Five naive mice were used as controls. In detail, after the first H22 transplantation, anti-TIGIT/anti-4-1BB bispecific antibody was administered to confirm the memory response using mice that survived by immunotherapy. After administration of the bispecific antibody, H22 was transplanted into the mice in which the tumor completely disappeared 100 days later. Tumors of naive mice used as control grew, but mice that survived after administration of the double antibody did not grow tumors.

As observed in the CT26 model (Fig 9(b)), cured H22 model mice from primary tumor challenge were also protected from secondary tumor development (Fig 10(a)). This suggests that mice that survived after receiving the TIGIT/4-1BB bispecific antibody had specific memory immunity against H22, and in other words, the TIGIT/4-1BB bispecific antibody showed sustained tumor-suppressive activity and protection from repeated tumor rechallenge in the H22 syngeneic model.

4-3. Efficacy evaluation of anti-TIGIT/anti-4-1BB bispecific antibody in the treatment of the MC38 mouse colon cancer model in C57BL/6-h4-1BB mice

15 C57BL/6-h4-1BB mice were subcutaneously injected with MC38 tumor cells (5Х10⁵/0.1 mL/mouse) (Shunran Shanghai Biological Technology (Shanghai, China)) in the right behind flank for tumor development. 7 days post inoculation, 15 tumor-bearing animals were randomly enrolled into three study groups when the mean tumor size reached 84 mm³. Each group consisted of 5 mice. The three groups were G1: Human IgG1 (1.5 mg/kg), G2: TIGIT sdAb (0.8 mg/kg) and G3: anti-TIGIT/anti-4-1BB bispecific antibody VH28/1A10M12 (1.3 mg/kg). Treatment began on the day of grouping (Day 0). All test antibodies were intraperitoneally administered to tumor-bearing mice once every three days for a total of six times. The tumor volume and body weight were measured and recorded twice weekly.

The obtained results are shown in figures 11(a) to 11(d), especially figure 11(a) provides the summary of the results shown in figures 11(b) to 11(d). As shown in the figures, anti-TIGIT/anti-4-1BB bispecific antibodies showed superior anti-tumor efficacy in MC38 tumor compared to TIGIT sdAb. In other words, the anti-TIGIT/anti-4-1BB bispecific antibody showed sustained tumor suppressive activity in MC38 model.

Example 5. Confirmation using various anti-TIGIT/anti-4-1BB bispecific antibodies

5-1. Antigen binding activity

5-1-1. Antigen binding activity by SACE

To evaluate the binding activity of various anti-TIGIT/anti-4-1BB bispecific antibodies to TIGIT and 4-1BB, tested antibodies were subjected to SACE (Single antigen captured ELISA). VH28/1A10M12, COM-902x1A10M12 and F04x1A10M12 bispecific antibodies were tested.

Briefly, a micro plate was coated with 100 ng/well of human TIGIT-His protein (Sinobiological, 10917-H08H), and human 4-1BB-His protein (Sinobiological, 10041-H08H) in PBS at 4℃ overnight. After removing the human TIGIT protein, the human 4-1BB protein plate was incubated with 200 μl/well of 1% BSA buffer (1X PBS, 1% BSA) for 2 hours at 37℃. Four-fold dilutions of each tested antibody, starting from 100 nM, were added to each well. And the plate was incubated for 1 hour at 37℃ and then washed with PBS-T (1xPBS/0.05% Tween-20). Then, the plate was washed with PBS-T and then incubated with HRP (Horse Radish Peroxidase) conjugated Anti-Fc antibody (Pierce, 31413) for 1 hour at 37℃. After washing, the plate was developed as a TMB substrate, and the reaction was stopped using sulfuric acid. And the plate was analyzed by spectrophotometer at OD 450-650nm. A Four-parameter logistic curve analysis was performed with GraphPad software.

The results are shown in the figures 12(a) and 12(b). As shown in the figures 12(a) and 12(b), all the tested anti-TIGIT/anti-4-1BB bispecific antibodies showed binding activity to both human TIGIT and 4-1BB.

5-1-2. Antigen binding activity by DACE

To evaluate the two-way binding activity of various TIGIT/4-1BB bispecific antibodies to TIGIT and 4-1BB, tested antibodies were subjected to DACE (Dual antigen captured ELISA). VH28/1A10M12, COM-902x1A10M12, F04x1A10M12, Tiragolumabx1A10M12 and Vibostolimabx1A10M12 bispecific antibodies were tested.

Briefly, a micro plate was coated with 100 ng/well of human TIGIT-Fc protein (Sinobiological, 10917-H02H) in PBS at 4℃ overnight. After removing the human TIGIT protein, the plate was incubated with 200 μl/well of 1% BSA buffer (1X PBS, 1% BSA) for 2 hours at 37℃. Four-fold dilutions of each the tested antibody, starting from 100 nM, were added to each well. And the plate was incubated for 2 hours at 37℃ and then washed with PBS-T (1xPBS/0.05% Tween-20). And then 100 ng/well of human 4-1BB-His protein (Sinobiological, 10041-H08H) in 1% BSA was added to each well and incubated for 1 hour at 37℃. The plate was washed with PBS-T and then incubated with Anti-His HRP (Roche, Cat: 11965085001) for 1 hour at 37℃. After washing, the plate was developed as a TMB substrate, and the reaction was stopped using sulfuric acid. And the plate was analyzed by a spectrophotometer at OD 450-650nm. Four-parameter logistic curve analysis was performed with GraphPad software.

The results are shown in the figure 13. As shown in the figure 13, all the tested TIGIT/4-1BB bispecific antibodies showed binding activity to both human TIGIT and 4-1BB.

The amino acid sequences of the tested antibody were illustrated in Table 6, with the variable regions underlined and the CDRs bolded and underlined.

Tested antibodies	Region	Sequence	SEQ ID NO:
COM-902x1A10M12 (IgG4 backbone)	Light chain	QSALTQPRSASGNPGQRVTISC SGSSSNMGRRPVN WYQQIPGTAPKLLIY SQNQRPS GVPDRFSGSQSGTSASLTISGLQSEDEAEYFC AVWDDIGRVLQ LGGGTQLAVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS	29
COM-902x1A10M12 (IgG4 backbone)	Heavy chain_IgG4	EVQLVETGGGLIQPGRSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVA VISYAGEVKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DPLPLHYYGMDV WGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK	35
COM-902x1A10M12 (IgG1 backbone)	Heavy chain_IgG1	EVQLVETGGGLIQPGRSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVA VISYAGEVKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DPLPLHYYGMDV WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK	36
COM-902x1A10M12 (IgG1 backbone)	Heavy Chain_COM-902x1A10M12(hIgG1)	EVQLVETGGGLIQPGRSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVA VISYAGEVKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DPLPLHYYGMDV WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSGSGSGSGSQSVLTQPPSASGTPGQRVTISC SGSSSNIGNNYVT WYQQLPGTAPKLLIY ADSHRPS GVPDRFSGSKSGTSASLAISGLRSEDEADYYC ATWDYSLSGYV FGCGTKLTVLGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFS SYDMS WVRQAPGKCLEWVS WISYSGGSIYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DAQRNSMREFDY WGQGTLVTVSS	70
F04x1A10M12 (IgG1 backbone)	Light chain	EIVLTQSPGTLSLSPGERATLSC RASQSVSSSYLA WYQQKPGQAPRLLIY GASSRAT GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQGYHRYAT FGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	40
	Heavy chain	EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYYMS WVRQAPGKGLEWVS SIGSGSPSSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR SSYSGGNGYYYYAYAFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK	45
	Heavy Chain_F04x1A10M12	EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYYMS WVRQAPGKGLEWVS SIGSGSPSSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR SSYSGGNGYYYYAYAFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSGSGSGSGSQSVLTQPPSASGTPGQRVTISC SGSSSNIGNNYVT WYQQLPGTAPKLLIY ADSHRPS GVPDRFSGSKSGTSASLAISGLRSEDEADYYC ATWDYSLSGYV FGCGTKLTVLGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFS SYDMS WVRQAPGKCLEWVS WISYSGGSIYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DAQRNSMREFDY WGQGTLVTVSS	71
Tiragolumabx1A10M12 (IgG1 backbone)	Light chain	DIVMTQSPDSLAVSLGERATINC KSSQTVLYSSNNKKYLA WYQQKPGQPPNLLIY WASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQYYSTPFT FGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	50
	Heavy chain	EVQLQQSGPGLVKPSQTLSLTCAISGDSVS SNSAAWN WIRQSPSRGLEWLG KTYYRFKWYSDYAVSVKG RITINPDTSKNQFSLQLNSVTPEDTAVFYCTR ESTTYDLLAGPFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK	55
	Heavy Chain_Tiragolumabx1A10M12	EVQLQQSGPGLVKPSQTLSLTCAISGDSVS SNSAAWN WIRQSPSRGLEWLG KTYYRFKWYSDYAVSVKG RITINPDTSKNQFSLQLNSVTPEDTAVFYCTR ESTTYDLLAGPFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSGSGSGSGSQSVLTQPPSASGTPGQRVTISC SGSSSNIGNNYVT WYQQLPGTAPKLLIY ADSHRPS GVPDRFSGSKSGTSASLAISGLRSEDEADYYC ATWDYSLSGYV FGCGTKLTVLGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFS SYDMS WVRQAPGKCLEWVS WISYSGGSIYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAQRNSMREFDY WGQGTLVTVSS	72
Vibostolimabx1A10M12 (IgG1 backbone)	Light chain	DIQMTQSPSSLSASVGDRVTITC RASEHIYSYLS WYQQKPGKVPKLLIY NAKTLAE GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC QHHFGSPLT FGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC	60
	Heavy chain	EVQLVQSGAEVKKPGSSVKVSCKASGYTFS SYVMH WVRQAPGQGLEWIG YIDPYNDGAKYAQKFQG RVTLTSDKSTSTAYMELSSLRSEDTAVYYCAR GGPYGWYFDV WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK	65
	Heavy Chain_Vibostolimabx1A10M12	EVQLVQSGAEVKKPGSSVKVSCKASGYTFS SYVMH WVRQAPGQGLEWIG YIDPYNDGAKYAQKFQG RVTLTSDKSTSTAYMELSSLRSEDTAVYYCAR GGPYGWYFDV WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSGSGSGSGSGSGSGSGSQSVLTQPPSASGTPGQRVTISC SGSSSNIGNNYVT WYQQLPGTAPKLLIY ADSHRPS GVPDRFSGSKSGTSASLAISGLRSEDEADYYC ATWDYSLSGYV FGCGTKLTVLGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFS SYDMS WVRQAPGKCLEWVS WISYSGGSIYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DAQRNSMREFDY WGQGTLVTVSS	73

5-2. Cell binding activity

5-2-1. Binding activity on the cell surface

To evaluate the binding activity of various anti-TIGIT/anti-4-1BB bispecific antibodies to human TIGIT and human 4-1BB expressed on the cell surface, mean fluorescence intensity (MFI) was evaluated by flow cytometry. MFI intensity for cells indicated the ability of tested antibodies to bind to cells. VH28/1A10M12, COM-902x1A10M12, F04x1A10M12, Tiragolumabx1A10M12 and Vibostolimabx1A10M12 bispecific antibodies were tested.

In brief, human 4-1BB overexpressed Jurkat and human TIGIT overexpressed CHO-K1 cells (GenScript (Piscataway, NJ, USA), Cat No. M00542) were placed in 96 well plate, then washed with 200 μl/well of 1% BSA buffer. Four-fold dilutions of each tested antibody, starting from 100 nM, were added to each well and incubated for 1 hour at 4℃. The plates were washed with PBS-T 1% BSA buffer. And then FITC-conjugated anti-human IgG (Fc specific) (sigma, F9512) in 1% BSA buffer was added to each well and incubated for 1 hour at 4℃. The plates were washed with PBS-T 1% BSA buffer. The MFI of FITC was evaluated by flow cytometry. The obtained MFI values were subjected to a four-parameter logistic curve analysis with GraphPad software.

The results are shown in figures 14(a) and 14(b). As shown in Figure 14(a), all the tested antibodies, except for urelumab, showed binding activity to human TIGIT. Figure 14(b) shows that all the tested antibodies presented binding activity to human 4-1BB. As a result, all the tested TIGIT/4-1BB bispecific antibodies showed binding activity to both human TIGIT- and 4-1BB-expressing cells.

5-2-2. Binding activity to Treg cells

To evaluate the binding activity of various anti-TIGIT/anti-4-1BB bispecific antibodies to human Treg cells, which are naturally expressing TIGIT and 4-1BB, mean fluorescence intensity (MFI) was evaluated by flow cytometry. MFI intensity for cells indicates the ability of tested antibody to bind to cells. VH28/1A10M12, COM-902x1A10M12, F04x1A10M12 and Tiragolumabx1A10M12 bispecific antibodies were tested.

In brief, Primary human CD4+ T cells were isolated from cryopreserved peripheral blood mononuclear cells using a Miltenyi CD4+ T cell isolation kit (Miltenyi Biotec, 130-096-533), according to the manufacturer's instructions. For the expansion of Treg cells, human CD4+ T cells were plated at 5x10⁵ cells/mL in Human T cell Expansion Media containing 20 ng/mL recombinant human IL-2 (Peprotech, 200-02-100UG). Then, 75 μL of Cloudz™ Treg CD3/CD28 was added per 2mL of cells to each well containing cells by using Cloudz™ Human Treg Expansion Kit, and the cells were cultured for 9 days. The rhIL-2 media was changed on days 2 and 3. To generate Treg cells, these Treg cells were placed in a 96-well plate, then washed with 200 μl/well of 1% BSA buffer. 5 μl of Human TruStain FcX™ (Biolegend, 422302) was added per million cells in a 100 μl staining volume and incubated at room temperature for 5-10 minutes. Six-fold dilutions of each tested antibody, starting from 100 nM, were added to each well and incubated for 1 hour at 4℃. The plates were washed with PBS-T 1% BSA buffer. And then AF647-conjugated'F(ab')₂ Fragment anti-human IgG (Jackson Immunoresearch, 109-606-098) in 1% BSA buffer was added to each well and incubated for 1 hour at 4℃. The plates were washed with PBS-T 1% BSA buffer. The MFI of AF647 was evaluated by flow cytometry. The obtained MFI values were subjected to a four-parameter logistic curve analysis with GraphPad software.

The results are shown in figure 15. As shown in the figure, all the tested anti-TIGIT/anti-4-1BB bispecific antibodies bound to Treg cells with comparable binding activity, while the IgG1 antibody showed no binding activity. Such results arose due to the potential of the anti-TIGIT/anti-4-1BB bispecific antibodies to suppress tumor growth by depleting immunosuppressive Treg cells within the tumor.

5-3. Activity and efficacy

5-3-1. 4-1BB signal activation depending on TIGIT expression

To measure 4-1BB signal activation of various anti-TIGIT/anti-4-1BB bispecific antibodies, a cell-based 4-1BB NF-kB luciferase reporter assay (4-1BB assay) was performed. VH28/1A10M12, COM-902x1A10M12, F04x1A10M12, Tiragolumabx1A10M12 and Vibostolimabx1A10M12 bispecific antibodies were tested.

In the 4-1BB assay, the activity of tested antibodies in the presence of human TIGIT antigens was evaluated using human TIGIT overexpressing CHO-K1 cells. GloResponse™ NFkB-luc2/4-1BB Jurkat cell line (Promega, Cat No. CS196004) was used as effector cells. GloResponse™ NFkB-luc2/4-1BB Jurkat cell line was genetically modified to stably express 4-1BB and luciferase downstream of a response element. Luciferase expression is induced upon antibody binding to the 4-1BB receptor.

In brief, CHOK1-hTIGIT (TIGIT positive hamster ovarian epithelial cells, 2.5x10⁴) were added on a white 96-well plate containing 100 μl culture medium, and the plate was incubated overnight at 37℃ in a 5% CO₂ humidification incubator. After incubation, 100 μl of culture medium was removed, and 25 μl of analysis medium (RPMI1640 containing 1% (v/v) FBS) was added to pre-attached target cells. 25 μl of tested antibody (5-fold dilution starting from 40 nM) was added to the plate. GloResponse™ NFkB-luc2/4-1BB Jurkat cell lines were recovered and re-entered on an analysis medium. 25 μl of GloResponse™ NFkB-luc2/4-1BB Jurkat cells were added to the plate to have 2.5x10⁴ cells per well, and the plate was incubated at 37℃ for 6 hours in a 5% CO₂ humidification incubator. During incubation time, the BIO-GLO™ reagent was reconstructed according to the manufacturer's manual. After 6 hours of incubation, 75 μl of BIO-GLO™ Reagent per well was added to the plate. It waited for 5 minutes and measured light emission using a microplate reader. The four-parameter logistic curve was evaluated using GraphPad software.

The results using CHOK1-hTIGIT cells are shown in figures 16(a) and 16(b). As shown in the figures, all the tested anti-TIGIT/anti-4-1BB bispecific antibodies triggered 4-1BB activation.

5-3-2. Evaluation of the potency and stability of anti-TIGIT/anti-4-1BB bispecific antibody by in vitro TIGIT/CD155 blockade bioassay

To confirm the TIGIT blocking activity of various anti-TIGIT/anti-4-1BB bispecific antibodies, VH28/1A10M12, COM-902x1A10M12, F04x1A10M12, Tiragolumabx1A10M12 and Vibostolimabx1A10M12 bispecific antibodies were analyzed for their ligand blocking activity (in vitro TIGIT Bioassay) by using Promega kit system (Promega, J2092).

In brief, TIGIT Effector cells (Promega, Cat No. J1695) were incubated in a white 96-well assay plate in 80 μL culture medium for overnight at 37℃ in a 5% CO₂ humidified incubator. After the overnight culture, 20 μL of TIGIT blocking antibodies (6X) were indicated: VH28/1A10M12, COM-902x1A10M12 (COM-902(Compugen)), F04x1A10M12 (F04(Yuhan)), Tiragolumabx1A10M12 (Tiragolumab(Roche)) and Vibostolimabx1A10M12 (Vibostolimab(Merck)). 20 μL of CD155 aAPC/CHO-K1 cells (aAPC/CHO-K1 cells (Promega, Cat No. J1805) were dispensed for each assay well and then cultured for 6 hours at 37℃ in a 5% CO₂ humidified incubator. During the incubation time, BIO-GLO™ reagent was reconstituted according to the manufacturer's instruction. After 6 hours of incubation, 120 μL of BIO-GLO™ reagent was added to each well on the assay plate. After 5 minutes waiting period, luminescence was quantified using a microplate reader. Four-parameter logistic curve analysis was performed with GraphPad software. Data were generated using thaw-and-use cells.

The results are shown in figure 17. As shown in the figure, all the tested anti-TIGIT/anti-4-1BB bispecific antibodies inhibited the interaction between TIGIT and CD155 in a dose-dependent manner.

5-4. In vivo efficacy study of anti-TIGIT/anti-4-1BB bispecific antibodies in CT26 model

In vivo efficacy of anti-TIGIT/anti-4-1BB bispecific antibodies was evaluated. 64 BALB/c-h4-1BB mice were subcutaneously injected with CT26 tumor cells (5Х10⁵/0.1 mL/mouse) in the right behind flank for tumor development. 11 days post-inoculation, the 64 tumor-bearing animals were randomly enrolled into eight study groups when the mean tumor size reached 79.5 mm³. Each group consisted of 8 mice. The eight groups were G1: Human IgG1 (3 mg/kg), G2: TIGIT sdAb (1.6 mg/kg), G3: combination of TIGIT sdAb and 1A10M12 (1.6 mg/kg + 3 mg/kg), G4: VH28/1A10M12 (2.6 mg/kg), G5: COM-902(hIgG1) (3 mg/kg), G6: COM-902(hIgG1)x1A10M12 (4 mg/kg), G7: F04 (3 mg/kg), G8: F04x1A10M12 (4 mg/kg).

Treatment began on the day of grouping (Day 0). All test antibodies were intraperitoneally administered to tumor-bearing mice twice weekly for a total of four times. The tumor volume and body weight were measured and recorded twice weekly.

The results are shown in figure 18. As shown in the figure, all the tested anti-TIGIT/anti-4-1BB bispecific antibodies showed superior anti-tumor efficacy in CT26 tumor when compared to monospecific anti-TIGIT antibody or combination therapy of anti-TIGIT monospecific antibody with anti-4-1BB monospecific antibody (1A10M12).

5-5. In vivo TIL analysis in H22 model

To understand the mechanisms of action by anti-TIGIT/anti-4-1BB bispecific antibody, 21 female BALB/c-h4-1BB mice (6-8 weeks old, supplied by GemPharmatech) were subcutaneously injected with H22 hepatoma cells (1Х10⁶ cells/0.1 mL/mouse) in the upper right flank for tumor development. 10 days post inoculation, 21 tumor-bearing animals were randomly enrolled into three study groups when the mean tumor size reached 157 mm³. Each group consisted of 7 mice. The three groups were as follows: G1: Human IgG1 (7.5 mg/kg), G2: parent anti-TIGIT sdAb (4 mg/kg), and G3: anti-TIGIT/anti-4-1BB bispecific antibody VH28/1A10M12 (6.65 mg/kg). Treatment began on the day of grouping (Day 0). All test antibodies were intraperitoneally administered to tumor-bearing mice on Days 0 and 3. Four days after the second administration, the mice were assessed for the analysis of immune cells and myeloid cells by flow cytometry (panel: L/D, mCD45, mCD3, mCD8, mCD4, mCD25, mFOXP3, mKI-67, AH1 (gp70) tetramer, mCD226, mCD96) and qRT-PCR (CXCL10, CXCL11, TNF-α, IFN-γ) on Day 7.

The results are shown in figures 19 to 20(b). As shown in figure 19, a significant decrease in both tumor growth and weight were observed on day 7 in anti-TIGIT/anti-4-1BB bispecific antibody-treated mice, and as shown in figure 20(a), a significant increase in tumor-infiltrating leukocytes was observed in anti-TIGIT/anti-4-1BB bispecific antibody-treated mice. In addition, anti-TIGIT/anti-4-1BB bispecific antibody induced the depletion of immunosuppressive Treg cells specifically in tumors, unlike TIGIT sdAb. And anti-TIGIT/anti-4-1BB bispecific antibody treatment induced significantly higher activation of cytotoxic T lymphocytes compared to TIGIT sdAb. As shown in figure 20(b), anti-TIGIT/anti-4-1BB bispecific antibody treatment induced significantly higher activation of myeloid cells compared to TIGIT sdAb.

Interestingly, Treg depletion was only observed in anti-TIGIT/anti-4-1BB bispecific antibody-treated mice, which showed an increased CD8+ T cell/Treg ratio. It was reported that 4-1BB is predominantly expressed in intra-tumoral Treg cells compared to peripheral Treg cells and other immune cells and TIGIT and 4-1BB expression are increased on tumor infiltrating lymphocytes (TILs). In this study approximately 15-fold higher 4-1BB expression was observed in intra-tumoral Tregs compared to other intra-tumoral immune cells, such as CD8+ T cells and NK cells. TIGIT expression in Tregs was approximately two-fold higher than that in CD8+ T cells and NK cells. More importantly, there was no decrease in intra-tumoral CD8+ T cell or CD4+ T cell numbers. Mouse CD226 as well as CD96 expression were induced after treatment of anti-TIGIT/anti-4-1BB bispecific antibody, suggesting that CD226-mediated stimulatory signaling may promote T cell activation under TIGIT-mediated suppression. This result suggests that the anti-TIGIT/anti-4-1BB bispecific antibody-mediated crosslinking of TIGIT or 4-1BB to FcyR may proactively trigger FcγR activation. Based on the macrophage activation signatures, anti-TIGIT/anti-4-1BB bispecific antibody significantly induced expression of CXCL10, CXCL11, IFN-γ, and TNF-α, indicating FcγR-mediated myeloid cell activation.

Example 6. Combination study ( In vivo ) of anti-TIGIT/anti-4-1BB bispecific antibody

6-1. Efficacy Evaluation of anti-TIGIT/anti-4-1BB bispecific antibody and combination therapy of anti-TIGIT/anti-4-1BB bispecific antibody with anti-PD-1 antibody in the treatment of the CT26-hPD-L1 mouse colon cancer Model in BALB/c-hPD-1/hPD-L1/h4-1BB mice

PD-1/PD-L1 blockades are the most extensively studied immune checkpoint inhibitors (ICIs) and co-inhibition of TIGIT and PD-1/PD-L1 represents a promising therapeutic approach for cancer treatment. Although co-inhibition of TIGIT and PD-1/PD-L1 enhanced anti-tumor immunity and treatment outcomes in preclinical and clinical studies, the response rates were insufficient potentially owing to the activities of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs).

To confirm the superior anti-cancer performance of anti-TIGIT/anti-4-1BB bispecific antibody over parent anti-TIGIT antibody, the combined efficacies of anti-TIGIT/anti-4-1BB bispecific antibody and anti-PD-1/PD-L1 antibody were compared to anti-TIGIT antibody and anti-PD-1/PD-L1 antibody treatment. 30 Female BALB/c-hPD-1/hPD-L1/h4-1BB knock-in mice (6-8 weeks old, supplied by GemPharmatech) were subcutaneously injected with CT26-hPD-L1 tumor cells (1Х10⁶/0.1 mL/mouse) (Gempharmatech (Nanjing, China)) in the lower right flank for tumor development. 13 days post inoculation, 30 tumor-bearing animals were randomly enrolled into six study groups when the mean tumor size reached 197.99 mm³. Each group consisted of 5 mice. The six groups were G1: Human IgG1 (3 mg/kg), G2: parent anti-TIGIT sdAb (1.6 mg/kg), G3: anti-TIGIT/anti-4-1BB bispecific antibody VH28/1A10M12 (2.6 mg/kg), G4: Keytruda (Pembrolizumab (MSD, Cat No.: 7009224200) (0.3 mg/kg), G5: parent anti-TIGIT sdAb + Keytruda combination (1.6 mg/kg + 0.3 mg/kg) and G6: VH28/1A10M12 + Keytruda combination (2.6 mg/kg + 0.3 mg/kg). Treatment began on the day of grouping (Day 0). All test antibodies were intraperitoneally administered to tumor-bearing mice twice weekly for a total of six times. The tumor volume and body weight were measured and recorded twice weekly.

The obtained results are shown in the figures 21(a) to 21(h). As shown in the figures 21(a) to 21(h), combination of anti-TIGIT and Keytruda as well as monotherapy of each antibody showed moderate antitumor efficacy. However, anti-TIGIT/anti-4-1BB bispecific antibody showed better tumor growth inhibition, with complete tumor regression in 3 out of 5 mice. The combination of anti-TIGIT/anti-4-1BB bispecific antibody and Keytruda initially prevented tumor growth in all mice, though tumor recurrence was observed in two mice.

Additionally, mice in anti-TIGIT/anti-4-1BB bispecific antibody treatment had significantly longer survival times after receiving treatment compared to anti-TIGIT monospecific antibodies or the combination of anti-TIGIT monospecific antibody and anti-PD-1 antibody. Also, with anti-PD-1 antibody demonstrated excellent survival times (figure 21(b)). In other words, combination of anti-TIGIT/anti-4-1BB bispecific antibody with anti-PD-1 antibody showed potent tumor growth inhibition.

6-2. Efficacy evaluation of anti-TIGIT/anti-4-1BB bispecific antibody and combination therapy of anti-TIGIT/anti-4-1BB bispecific antibody with anti-PD-L1 antibody in the treatment of the MC38 mouse colon cancer Model in C57BL/6-h4-1BB/hTIGIT mice

To confirm the combination synergy, we tested another in vivo study with MC38 in hTIGIT/h4-1BB transgenic mice. 35 Female C57BL/6-h4-1BB/hTIGIT knock-in mice (6-8 weeks old, supplied by Biocytogen) were subcutaneously injected with MC38 tumor cells (5Х10⁵/0.1 mL/mouse) in the upper right flank for tumor development. 7 days post inoculation, 35 tumor-bearing animals were randomly enrolled into sevenstudy groups when the mean tumor size reached about 101 mm³. Each group consisted of 5 mice. The three groups were G1: Human IgG1 (3 mg/kg), G2: anti-TIGIT/anti-4-1BB bispecific antibody VH28/1A10M12 (0.3 mg/kg) and G3: anti-TIGIT/anti-4-1BB bispecific antibody VH28/1A10M12 (3 mg/kg), G4: Atezolizumab (anti-PD-L1 antibody, inhouse production, CoA-R22090 Lot No.: PPS0283) (3 mg/kg), G5: Tiragolumab (inhouse production, CoA-R23093 Lot No.: PPS0781) (10 mg/kg), G6: anti-TIGIT/anti-4-1BB bispecific antibody VH28/1A10M12 + Atezolizumab (0.3 mg/kg + 3 mg/kg) and G7: Tiragolumab + Atezolizumab (10 mg/kg + 3 mg/kg). Treatment began on the day of grouping (Day 0). All test antibodies were intraperitoneally administered to tumor-bearing mice once every three days for a total of six times. The tumor volume and body weight were measured and recorded twice weekly.

The results are shown in figures 22(a) to 22(i). As shown in the figures, the anti-TIGIT/anti-4-1BB bispecific antibody exhibited dose-dependent tumor growth inhibition, with complete tumor regression in the 3 mg/kg group, while 10 mg/kg of tiragolumab failed to inhibit tumor progression (figure 22(c) to 22(i)). Atezolizumab (3 mg/kg), which binds to mouse PD-L1, did not inhibit tumor progression. However, a combination of low dose of the anti-TIGIT/anti-4-1BB bispecific antibody (0.3 mg/kg) and atezolizumab (3 mg/kg) showed synergistic anti-tumor efficacy, resulting in complete regression of tumors in all the mice tested. In contrast, the combination of tiragolumab and atezolizumab did not result in complete regression. In summary, these data suggested that anti-TIGIT/anti-4-1BB bispecific antibody exhibited more potent in anti-tumor activity through TIGIT inhibition and 4-1BB activation and better therapeutic option with the combination of anti-PD-L1 therapies for cancer patients.

Claims

A bispecific antibody or antigen-binding fragment thereof comprising:

(i) a first antigen binding site that binds to TIGIT (T-cell immunoreceptor with immunoglobulin and ITIM domain) and

(ii) a second antigen binding site that binds to 4-1BB.
The bispecific antibody or antigen-binding fragment thereof according to claim 1,

wherein the first antigen binding site comprises any one of the following:

(a) a heavy chain CDR1 of SEQ ID NO: 2; a heavy chain CDR2 of SEQ ID NO: 4; and a heavy chain CDR3 of SEQ ID NO: 6;

(b) a heavy chain CDR1 of SEQ ID NO: 37; a heavy chain CDR2 of SEQ ID NO: 38; a heavy chain CDR3 of SEQ ID NO: 39; a light chain CDR1 of SEQ ID NO: 31; a light chain CDR2 of SEQ ID NO: 32; a light chain CDR3 of SEQ ID NO: 33;

(c) a heavy chain CDR1 of SEQ ID NO: 47; a heavy chain CDR2 of SEQ ID NO: 48; a heavy chain CDR3 of SEQ ID NO: 49; a light chain CDR1 of SEQ ID NO: 42; a light chain CDR2 of SEQ ID NO: 43; a light chain CDR3 of SEQ ID NO: 44;

(d) a heavy chain CDR1 of SEQ ID NO: 57; a heavy chain CDR2 of SEQ ID NO: 58; a heavy chain CDR3 of SEQ ID NO: 59; a light chain CDR1 of SEQ ID NO: 52; a light chain CDR2 of SEQ ID NO: 53; a light chain CDR3 of SEQ ID NO: 54; and

(e) a heavy chain CDR1 of SEQ ID NO: 67; a heavy chain CDR2 of SEQ ID NO: 68; a heavy chain CDR3 of SEQ ID NO: 69; a light chain CDR1 of SEQ ID NO: 62; a light chain CDR2 of SEQ ID NO: 63; a light chain CDR3 of SEQ ID NO: 64.
The bispecific antibody or antigen-binding fragment thereof according to claim 2,

wherein the first antigen binding site comprises any one of the following:

(a) a single-domain antibody having a heavy chain CDR1 of SEQ ID NO: 2; a heavy chain CDR2 of SEQ ID NO: 4; and a heavy chain CDR3 of SEQ ID NO: 6;

(b) a Fv fragment or scFv having a heavy chain CDR1 of SEQ ID NO: 37; a heavy chain CDR2 of SEQ ID NO: 38; a heavy chain CDR3 of SEQ ID NO: 39; a light chain CDR1 of SEQ ID NO: 31; a light chain CDR2 of SEQ ID NO: 32; a light chain CDR3 of SEQ ID NO: 33;

(c) a Fv fragment or scFv having a heavy chain CDR1 of SEQ ID NO: 47; a heavy chain CDR2 of SEQ ID NO: 48; a heavy chain CDR3 of SEQ ID NO: 49; a light chain CDR1 of SEQ ID NO: 42; a light chain CDR2 of SEQ ID NO: 43; a light chain CDR3 of SEQ ID NO: 44;

(d) a Fv fragment or scFv having a heavy chain CDR1 of SEQ ID NO: 57; a heavy chain CDR2 of SEQ ID NO: 58; a heavy chain CDR3 of SEQ ID NO: 59; a light chain CDR1 of SEQ ID NO: 52; a light chain CDR2 of SEQ ID NO: 53; a light chain CDR3 of SEQ ID NO: 54; and

(e) a Fv fragment or scFv having a heavy chain CDR1 of SEQ ID NO: 67; a heavy chain CDR2 of SEQ ID NO: 68; a heavy chain CDR3 of SEQ ID NO: 69; a light chain CDR1 of SEQ ID NO: 62; a light chain CDR2 of SEQ ID NO: 63; a light chain CDR3 of SEQ ID NO: 64.
The bispecific antibody or antigen-binding fragment thereof according to claim 2,

wherein the first antigen binding site thereof comprise:

a heavy chain framework 1 (H-FR1) comprising an amino acid sequence of SEQ ID NO: 1 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 1;

a heavy chain framework 2 (H-FR2) comprising an amino acid sequence of SEQ ID NO: 3 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 3;

a heavy chain framework 3 (H-FR3) comprising an amino acid sequence of SEQ ID NO: 5 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 5; and

a heavy chain framework 4 (H-FR4) comprising an amino acid sequence of SEQ ID NOs: 7 or a peptide having at least 95%, at least 90%, at least 85% or at least 80% sequence identity to an amino acid sequence of SEQ ID NO:7.
The bispecific antibody or antigen-binding fragment thereof according to claim 3,

wherein the first antigen binding site comprises any one of the following:

(a) a heavy chain variable region of SEQ ID NO: 8;

(b) a heavy chain variable region of SEQ ID NO: 36 and a light chain variable region of SEQ ID NO: 30;

(c) a heavy chain variable region of SEQ ID NO: 46 and a light chain variable region of SEQ ID NO: 41;

(d) a heavy chain variable region of SEQ ID NO: 56 and a light chain variable region of SEQ ID NO: 51; and

(e) a heavy chain variable region of SEQ ID NO: 66 and a light chain variable region of SEQ ID NO: 61.
The bispecific antibody or antigen-binding fragment thereof according to claim 1,

wherein the second antigen binding site comprises:

(a) a light chain CDR1 of SEQ ID NO: 10;

(b) a light chain CDR2 of SEQ ID NO: 12;

(c) a light chain CDR3 of SEQ ID NO: 14;

(d) a heavy chain CDR1 of SEQ ID NO: 18;

(e) a heavy chain CDR2 of SEQ ID NO: 20; and

(f) a heavy chain CDR3 of SEQ ID NO: 22.
The bispecific antibody or antigen-binding fragment thereof according to claim 6,

wherein the second antigen binding site comprises a light chain variable region of SEQ ID NO: 16 and a heavy chain variable region of SEQ ID NO: 24.
The bispecific antibody or antigen-binding fragment thereof according to claim 1 to claim 7,

wherein the bispecific antibody is a mouse antibody, a chimeric antibody, a humanized antibody or a fully human antibody.
The bispecific antibody or antigen-binding fragment thereof according to claim 1 to claim 7,

wherein the bispecific antibody is independently selected from the group consisting of a whole IgG, sdAb (single-domain antibody), Fab, Fab', F(ab')2, xFab, scFab, dsFv, Fv, scFv, IgG-scFv, sdAb-Fc, sdAb-Fc-scFv, scFv-Fc, scFab-Fc, diabody, minibody, scAb, dAb, half-IgG and combinations thereof.
The bispecific antibody or antigen-binding fragment thereof according to claim 1 to claim 7,

wherein the bispecific antibody comprises an Fc region of an IgG1, IgG2, IgG3, or IgG4 antibody, or a hybrid Fc region or a constant region.
The bispecific antibody or antigen-binding fragment thereof according to claim 1 to claim 7,

wherein the first antigen binding site is covalently linked with the second antigen binding site.
The bispecific antibody or antigen-binding fragment thereof according to claim 1 to claim 7,

wherein the first antigen binding site or the second antigen binding site are linked with a peptide linker of SEQ ID NOs: 25 or 26.
The bispecific antibody or antigen-binding fragment thereof according to claim 1 to claim 7,

wherein the bispecific antibody or antigen-binding fragment comprise hinge region of SEQ ID NO: 27.
A pharmaceutical composition comprising the bispecific antibody or antigen-binding fragment thereof according to claim 1 to claim 13 and a pharmaceutically acceptable carrier.
The pharmaceutical composition of claim 14,

wherein the bispecific antibody or antigen-binding fragment thereof according to claim 1 to claim 13 with at least one of the inhibitors of an immune checkpoint molecule.
The pharmaceutical composition of claim 15,

wherein the immune checkpoint molecule is at least one selected from the group consisting of PD-1 and PD-L1.
A pharmaceutical composition for use in a method of preventing or treating cancer in a patient, comprising the bispecific antibody or antigen-binding fragment thereof according to claim 1 to claim 13.
The pharmaceutical composition of claim 17,

wherein the patient is also being administered with at least one of the inhibitors of an immune checkpoint molecule continuously or discontinuously.
The pharmaceutical composition of claim 17,

wherein the cancer is immune checkpoint inhibitor-resistant cancer.
The pharmaceutical composition of claim 17,

wherein the cancer is TIGIT-positive cancer.
The pharmaceutical composition of claim 17,

wherein the cancer is selected from the group consisting of leukemia, rectal cancer, endometrial cancer, nephroblastoma, basal cell carcinoma, nasopharyngeal cancer, bone tumor, esophageal cancer, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, follicular thyroid cancer, hepatocellular carcinoma, oral cancer, renal cell carcinoma, multiple myeloma, mesothelioma, osteosarcoma, myelodysplastic syndrome, mesenchymal tumor, soft tissue sarcoma, liposarcoma, gastrointestinal stromal tumor, malignant peripheral nerve sheath tumor (MPNST), Ewing sarcoma, leiomyosarcoma, mesenchymal chondrosarcoma, lymphosarcoma, fibrosarcoma, rhabdomyosarcoma, teratoma, neuroblastoma, medulloblastoma, glioma, benign skin tumor, Burkitt's lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, marginal zone lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy-cell leukemia, myelodysplastic syndrome (MDS), chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML), neuroectodermal tumor, epithelial tumor, cutaneous T-cell lymphoma (CTCL), peripheral T cell lymphoma (PTCL), pancreatic cancer, hematological malignancies, kidney cancer, tumor vasculature, breast cancer, renal cancer, ovarian cancer, epithelial ovarian cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, pancreatic cancer, skin cancer, bladder cancer, testicular cancer, uterine cancer, prostate cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), Head and neck squamous cell carcinoma (HNSCC), glioblastoma multiforme (GBM), brain cancer, colon cancer, squamous cell carcinoma, melanoma, myeloma, cervical cancer, thyroid cancer, head and neck cancer and adrenal cancer.
A method for preventing or treating a cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the bispecific antibody or antigen-binding fragment thereof according to claim 1 to claim 13.
The method of claim 22,

wherein the patient is also being administered at least one of the inhibitors of an immune checkpoint molecule continuously or discontinuously.
The method of claim 23,

wherein the immune checkpoint molecule is at least one selected from the group consisting of PD-1 and PD-L1.
A use of the bispecific antibody or antigen-binding fragment thereof according to claim 1 to claim 13 in the manufacture of medicament for treating or preventing a cancer.
A method for restoring T cells in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the bispecific antibody or antigen-binding fragment thereof according to claim 1 to claim 13.
An isolated nucleic acid encoding the bispecific antibody or antigen-binding fragment thereof according to claim 1 to claim 13.
A vector comprising the isolated nucleic acid according to claim 27.
A host cell comprising the vector according to claim 28.