Anti-tigit / Anti-PD-l1 bispecific antibody and use thereof

Abstract

The present invention relates to an anti-TIGIT / anti-PD-L1 bispecific antibody including a first domain that specifically binds to PD-L1 and a second domain that specifically binds to TIGIT. The anti-TIGIT / anti-PD-L1 bispecific antibody shows significantly excellent cancer treatment efficacy compared to the single and combination treatment of existing anti-PD-L1 and anti-TIGIT monoclonal antibodies. Therefore, the pharmaceutical composition including the anti-TIGIT / anti-PD-L1 bispecific antibody as an active ingredient can be used as an anti-cancer immunotherapy through activation of immune cells.

Description

ANTI-TIGIT / ANTI-PD-L1 BISPECIFIC ANTIBODY AND USE THEREOF

[0001] The present invention relates to an anti-TIGIT / anti-PD-L1 bispecific antibody and use thereof.

[0002] The expression of PD-L1(Programmed cell death-ligand 1) is increased in various tumor cells and is known as an immune checkpoint that suppresses the anticancer action of T cells and NK cells through its binding to PD-1 expressed on T cells and NK cells. Currently, various drugs targeting PD-L1, such as PD-L1-targeting antibodies and PD-1-targeting antibodies, are being developed. These drugs prevent the binding between PD-L1 and PD-1, thereby activating T cells and exhibiting an anticancer effect.

[0003] Meanwhile, likewise even in a T cell immunoglobulin and ITIM domain (TIGIT), which includes an immunoreceptor tyrosine-based inhibitory motif (ITIM), is an inhibitory receptor expressed on activated T cells, exhausted T cells, NK cells, and Treg cells. TIGIT transmits inhibitory signals into immune cells through its binding to a poliovirus receptor (PVR), and it has been reported that PVR expression is mainly observed in tumors, and is also expressed in the placenta. In particular, it has been reported that TIGIT expression is increased in CD8+ T cells and Treg cells among tumor-infiltrating lymphocytes (TILs), and it has been confirmed that antitumor efficacy can be achieved in several animal models by blocking the inhibition of immune cell activation by TIGIT. The prevention of the binding between PD-1 and PD-L1 and the prevention of the binding between TIGIT and PVR play important roles in anti-tumor immunity, and there is a need for molecules that can prevent each of these bindings for therapeutic purposes.

[0004] The present invention aims to develop a dual-targeting substance that simultaneously binds to PD-L1, which is overexpressed in various cancers, and TIGIT, which is involved in suppressing immune cell activation, thereby providing a best-in-class drug with excellent anticancer efficacy and improved side effects compared to competitive drugs.

[0005] In the case of PVR expressed in tumors, it can bind not only to TIGIT on immune cells but also to CD226. CD226, which is an activating receptor, interacts with CD155 and CD112 ligands, and activates the functions of T cells and NK cells through an immunoreceptor tyrosine-based activation motif (ITAM). Compared to single / co-treatment of a PD-L1 targeting monoclonal antibody and a TIGIT targeting monoclonal antibody, the bispecific antibody according to the present invention can have the effect of preventing the inhibition of immune cell activation by way of binding to TIGIT thereby preventing its binding to PVR, while simultaneously preventing the inhibition of immune cell activation caused by the PD-1 / PD-L1 binding by way of binding to PD-L1 of tumors; additionally, through its role as a T cell engager that recruits immune cells to tumor sites, CD226, which is expressed on tumor-recruited immune cells, may increase immune cell activation through its binding to PVR on nearby tumors. Mutations that increase antibody-dependent cell-mediated cytotoxicity (ADCC) efficacy through enhancement of an additional Fc-effector function by improving the anticancer efficacy of therapeutic antibodies have been reported. Enhancement of Fc-effector functions and improvement of antibody efficacy may be achieved by improving the binding affinity of antibodies to activating FcγRs. The development of antibodies through mutations that induce enhancement of various typical Fc-effector functions has been reported.

[0006] In an aspect of the present invention, the Fc-effector functions of antibodies were improved by using AAA mutations (S298A / E333A / K334A), which is one of the above-mentioned mutations; additionally, the FcRn binding affinity was significantly increased by introducing the LS mutations (M428L / N434S), which causes an increase in the FcRn binding affinity, and as a result, the blood half-life can be improved.

[0007] Compared to the existing single and combination treatment of anti-PD-L1 and anti-TIGIT monoclonal antibodies, the efficacy of a bispecific antibody as an excellent anti-tumor therapeutic agent is achieved by enhancing T cell engager efficacy and Fc-effector function and improvement of blood half-life.

[0008] [Prior Art Document]

[0009] [Patent Document]

[0010] (Patent Document 1) U.S. Patent No. 9,713,641 (July 25, 2017)

[0011] An object of the present invention is to provide an anti-TIGIT / anti-PD-L1 bispecific antibody, which includes a first domain that specifically binds to PD-L1; and a second domain that specifically binds to TIGIT.

[0012] Another object of the present invention is to provide a pharmaceutical composition for treating cancer, which includes the anti-TIGIT / anti-PD-L1 bispecific antibody as an active ingredient.

[0013] Still another object of the present invention is to provide a nucleic acid encoding the amino acid sequence of a first domain and the amino acid sequence of a second domain of the anti-TIGIT / anti-PD-L1 bispecific antibody.

[0014] Still another object of the present invention is to provide a recombinant expression vector which includes the nucleic acid.

[0015] Still another object of the present invention is to provide a host cell transformed with the recombinant expression vector.

[0016] Still another object of the present invention is to provide a method for preparing an anti-TIGIT / anti-PD-L1 bispecific antibody, which includes culturing the host cell.

[0017] [1] In an aspect of the present invention, the present invention relates to an anti-TIGIT / anti-PD-L1 bispecific antibody, which includes a first domain that specifically binds to PD-L1, and a second domain that specifically binds to TIGIT.

[0018] [2] In the anti-TIGIT / anti-PD-L1 bispecific antibody of [1] above,

[0019] the first domain may include:

[0020] a heavy chain variable region (VH), which includes:

[0021] a heavy chain CDR1 represented by an amino acid sequence of SEQ ID NO: 1;

[0022] a heavy chain CDR2 represented by an amino acid sequence of SEQ ID NO: 2;

[0023] a heavy chain CDR3 represented by an amino acid sequence of SEQ ID NO: 3, and

[0024] a light chain variable region (VL), which includes:

[0025] a light chain CDR1 represented by an amino acid sequence of SEQ ID NO: 4;

[0026] a light chain CDR2 represented by an amino acid sequence of SEQ ID NO: 5; and

[0027] a light chain CDR3 represented by an amino acid sequence of SEQ ID NO: 6.

[0028] [3] In the anti-TIGIT / anti-PD-L1 bispecific antibody of [1] or [2] above,

[0029] the second domain may include:

[0030] a heavy chain variable region (VH), which includes:

[0031] a heavy chain CDR1 represented by an amino acid sequence of SEQ ID NO: 7 or 8;

[0032] a heavy chain CDR2 represented by an amino acid sequence of SEQ ID NO: 9 or 10;

[0033] a heavy chain CDR3 represented by an amino acid sequence of SEQ ID NO: 11 or 12, and

[0034] a light chain variable region (VL), which includes:

[0035] a light chain CDR1 represented by an amino acid sequence of SEQ ID NO: 13 or 14;

[0036] a light chain CDR2 represented by an amino acid sequence of SEQ ID NO: 15 or 16; and

[0037] a light chain CDR3 represented by an amino acid sequence of SEQ ID NO: 17 or 18.

[0038] [4] In the anti-TIGIT / anti-PD-L1 bispecific antibody of any one of [1] to [3] above, the first domain may include an Fc region of an immunoglobulin, which is the Fc region consisting of any one of the Fc region of IgG1, IgG2, IgG3, IgG4 and IgD, or a hybrid Fc containing a combination thereof.

[0039] [5] In the anti-TIGIT / anti-PD-L1 bispecific antibody of any one of [1] to [4] above, the first domain may include an IgG1 Fc region.

[0040] [6] In the anti-TIGIT / anti-PD-L1 bispecific antibody of any one of [1] to [4] above, wherein in the IgG1 Fc region represented by an amino acid sequence of SEQ ID NO: 29, the first domain may include any one or more mutations selected from the group consisting of S239D, F243L, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, P396L, M428L, and N434S.

[0041] [7] In the anti-TIGIT / anti-PD-L1 bispecific antibody of any one of [1] to [4] above, wherein in the IgG1 Fc region represented by an amino acid sequence of SEQ ID NO: 29, the first domain may include any one or more mutations among the following (i) to (iv):

[0042] (i) S239D, A330L, and I332E;

[0043] (ii) F243L, R292P, Y300L, V305I, and P396L;

[0044] (iii) S298A, E333A, and K334A; and

[0045] (iv) M428L and N434S.

[0046] [8] In the anti-TIGIT / anti-PD-L1 bispecific antibody of any one of [1] to [4] above, wherein the first domain may include an IgG1 Fc region that includes one or more mutations in the amino acid sequence of SEQ ID NO: 29, and wherein the mutations may be selected from the group consisting of S239D, A330L, and I332E.

[0047] [9] In the anti-TIGIT / anti-PD-L1 bispecific antibody of any one of [1] to [4] above, wherein the first domain may include an IgG1 Fc region that includes one or more mutations in the amino acid sequence of SEQ ID NO: 29, and wherein the mutations may be selected from the group consisting of F243L, R292P, Y300L, V305I, P396L, M428L, and N434S.

[0048]

[0010] In the anti-TIGIT / anti-PD-L1 bispecific antibody of any one of [1] to [4] above, wherein the first domain may include an IgG1 Fc region that includes one or more mutations in the amino acid sequence of SEQ ID NO: 29, and wherein the mutations may be selected from the group consisting of S298A, E333A, K334A, M428L, and N434S.

[0049]

[0011] In the anti-TIGIT / anti-PD-L1 bispecific antibody of any one of [1] to [4] above, the first domain may include an IgG1 Fc region represented by an amino acid sequence of SEQ ID NO: 30.

[0050]

[0012] In the anti-TIGIT / anti-PD-L1 bispecific antibody of any one of [1] to [4] above, the first domain may include an IgG1 Fc region represented by an amino acid sequence of SEQ ID NO: 31.

[0051]

[0013] In the anti-TIGIT / anti-PD-L1 bispecific antibody of any one of [1] to [4] above, the first domain may include an IgG1 Fc region represented by an amino acid sequence of SEQ ID NO: 32.

[0052]

[0014] In the anti-TIGIT / anti-PD-L1 bispecific antibody of any one of [1] to [4] above, the first domain may include an anti-PD-L1 heavy chain variable region represented by an amino acid sequence of SEQ ID NO: 19.

[0053]

[0015] In the anti-TIGIT / anti-PD-L1 bispecific antibody of any one of [1] to

[0014] above, the first domain may include an anti-PD-L1 light chain variable region represented by an amino acid sequence of SEQ ID NO: 20.

[0054]

[0016] In the anti-TIGIT / anti-PD-L1 bispecific antibody of any one of [1] to

[0015] above, the second domain may include an anti-TIGIT heavy chain variable region represented by any one amino acid sequence selected from the group consisting of SEQ ID NOS: 21 to 24.

[0055]

[0017] In the anti-TIGIT / anti-PD-L1 bispecific antibody of any one of [1] to

[0016] above, the second domain may include an anti-TIGIT light chain variable region represented by any one amino acid sequence selected from the group consisting of SEQ ID NOS: 25 to 28.

[0056]

[0018] In the anti-TIGIT / anti-PD-L1 bispecific antibody of any one of [1] to [4] above, the first domain may include a heavy chain and a light chain; and the second domain may be an scFv including a heavy chain variable region and a light chain variable region, wherein a C-terminus of the heavy chain of the first domain and an N-terminus of the heavy chain variable region of the second domain may be linked through a linker.

[0057]

[0019] In the anti-TIGIT / anti-PD-L1 bispecific antibody of any one of [1] to [4] above, the first domain may include a heavy chain and a light chain; and the second domain may be an scFv including a heavy chain variable region and a light chain variable region, wherein a C-terminus of the heavy chain of the first domain and an N-terminus of the light chain variable region of the second domain may be linked through a linker.

[0058]

[0020] In the anti-TIGIT / anti-PD-L1 bispecific antibody of any one of [1] to [3] above, the first domain may be an scFv including a heavy chain variable region and a light chain variable region; and the second domain may include a heavy chain and a light chain, wherein a C-terminus of the heavy chain of the second domain and an N-terminus of the heavy chain variable region of the first domain may be linked through a linker.

[0059]

[0021] Still another aspect of the present invention relates to an anti-TIGIT / anti-PD-L1 bispecific antibody, which is any one selected from the following:

[0060] 1) an anti-TIGIT / anti-PD-L1 bispecific antibody including the amino acid sequences of SEQ ID NOS: 33 to 36;

[0061] 2) an anti-TIGIT / anti-PD-L1 bispecific antibody including the amino acid sequences of SEQ ID NOS: 37 to 40;

[0062] 3) an anti-TIGIT / anti-PD-L1 bispecific antibody including the amino acid sequences of SEQ ID NOS: 41 to 44;

[0063] 4) an anti-TIGIT / anti-PD-L1 bispecific antibody including the amino acid sequences of SEQ ID NOS: 45 to 48;

[0064] 5) an anti-TIGIT / anti-PD-L1 bispecific antibody including the amino acid sequences of SEQ ID NOS: 49 to 52;

[0065] 6) an anti-TIGIT / anti-PD-L1 bispecific antibody including the amino acid sequences of SEQ ID NOS: 53 to 56; and

[0066] 7) an anti-TIGIT / anti-PD-L1 bispecific antibody including the amino acid sequences of SEQ ID NOS: 57 to 60.

[0067]

[0022] Still another aspect of the present invention relates to a pharmaceutical composition for treating cancer, which includes an anti-TIGIT / anti-PD-L1 bispecific antibody according to any one of [1] to

[0021] above as an active ingredient.

[0068]

[0023] In

[0022] above, the cancer may be one selected from the group consisting of skin cancer, liver cancer, hepatocellular carcinoma, stomach cancer, breast cancer, lung cancer, ovarian cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, colorectal cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, thyroid cancer, parathyroid cancer, kidney cancer, esophageal cancer, bile duct cancer, testicular cancer, rectal cancer, head and neck cancer, cervical spine cancer, ureteral cancer, osteosarcoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, glioma, myeloma, neuroblast-derived CNS tumor, monocytic leukemia, B-cell derived leukemia, T-cell derived leukemia, B-cell derived lymphoma, T-cell derived lymphoma, and mast cell derived tumor.

[0069]

[0024] Still another aspect of the present invention relates to a nucleic acid, which encodes an amino acid sequence of the first domain and an amino acid sequence of the second domain of the anti-TIGIT / anti-PD-L1 bispecific antibody according to any one of [1] to

[0021] above.

[0070]

[0025] Still another aspect of the present invention relates to a recombinant expression vector, which includes the nucleic acid of

[0024] above.

[0071]

[0026] Still another aspect of the present invention relates to a host cell, which is transformed with the recombinant expression vector of

[0025] above.

[0072]

[0027] Still another aspect of the present invention relates to a method for preparing an anti-TIGIT / anti-PD-L1 bispecific antibody, which includes culturing the host cell of

[0026] above.

[0073]

[0028] Still another aspect of the present invention relates to an anti-TIGIT / anti-PD-L1 bispecific binding protein, which includes a first domain that specifically binds to PD-L1; and a second domain that specifically binds to TIGIT.

[0074]

[0029] Still another aspect of the present invention relates to an anti-TIGIT / anti-PD-L1 bispecific binding molecule, which includes a first domain that specifically binds to PD-L1; and a second domain that specifically binds to TIGIT.

[0075] The anti-TIGIT / anti-PD-L1 bispecific antibody according to the present invention can bind to TIGIT and PD-L1 in a very specific and potent manner. As a result, the anti-TIGIT / anti-PD-L1 bispecific antibody according to the present invention shows excellent cancer treatment efficacy compared to the single and combination treatment of existing anti-PD-L1 and anti-TIGIT monoclonal antibodies. Therefore, a pharmaceutical composition including the anti-TIGIT / anti-PD-L1 bispecific antibody, as an active ingredient, according to the present invention may be used as an anti-cancer immunotherapy through activation of immune cells.

[0076] Fig. 1 shows a schematic diagram of an anti-TIGIT / anti-PD-L1 bispecific antibody.

[0077] Fig. 2 shows the results of SDS-PAGE analysis of the anti-TIGIT / anti-PD-L1 bispecific antibodies of the present invention (B101041, B101042, B101643, B101644, B101645, and B101646).

[0078] Fig. 3 shows the measurement results of the binding affinity of the anti-TIGIT / anti-PD-L1 bispecific antibodies of the present invention (B204011, B101042, B101643, B101644, B101645, and B101646) for each antigen, in which Fig. 3a shows the measurement results of binding affinity for human PD-L1, and Fig. 3b shows the measurement results of binding affinity for human TIGIT.

[0079] Fig. 4 shows the measurement results of dual binding affinity of the anti-TIGIT / anti-PD-L1 bispecific antibodies of the present invention (B101041, B101042, B101643, B101644, B101645, and B101646) for human TIGIT and human PD-L1.

[0080] Fig. 5 shows the results of confirming the binding potency of the anti-TIGIT / anti-PD-L1 bispecific antibodies of the present invention (B101041 and B204011) for TIGIT-overexpressing CHO-S cells.

[0081] Fig. 6 shows the results of confirming the binding potency of the anti-TIGIT / anti-PD-L1 bispecific antibodies of the present invention (B101042 and B101643) for TIGIT-overexpressing CHO-S cells.

[0082] Fig. 7 shows the results of confirming the binding potency of the anti-TIGIT / anti-PD-L1 bispecific antibodies of the present invention (B101644, B101645, and B101646) for TIGIT-overexpressing CHO-S cells.

[0083] Fig. 8 shows the results of confirming the binding potency of the anti-TIGIT / anti-PD-L1 bispecific antibodies of the present invention (B101041, B101042, and B204011) for human tumor cells.

[0084] Fig. 9 shows the results of confirming the binding potency of the anti-TIGIT / anti-PD-L1 bispecific antibodies of the present invention (B101643 and B101644) for human tumor cells.

[0085] Fig. 10 shows the results of confirming the binding potency of the anti-TIGIT / anti-PD-L1 bispecific antibodies of the present invention (B101645 and B101646) for human tumor cells.

[0086] Fig. 11 shows the results of confirming the binding affinity of the anti-TIGIT / anti-PD-L1 bispecific antibodies of the present invention (B101643 and B101646) for human FcRn.

[0087] Fig. 12 shows the ADCC activity results of the anti-TIGIT / anti-PD-L1 bispecific antibodies of the present invention (B101042 and B101643) for human tumor cells, H1975.

[0088] Fig. 13 shows the ADCC activity results of the anti-TIGIT / anti-PD-L1 bispecific antibodies of the present invention (B101645 and B101646) for human tumor cells, H1975.

[0089] Fig. 14 shows the ADCC activity results of the anti-TIGIT / anti-PD-L1 bispecific antibodies of the present invention (B101644) for human tumor cells, MDA-MB-231.

[0090] Fig. 15 shows the ADCC activity results of the anti-TIGIT / anti-PD-L1 bispecific antibodies of the present invention (B101645 and B101646) for human tumor cells, MDA-MB-231.

[0091] Fig. 16 shows the ADCC activity results of the anti-TIGIT / anti-PD-L1 bispecific antibodies of the present invention (B101644, B101645, and B101646) for human TIGIT-overexpressing CHO-S cells.

[0092] Fig. 17 shows the ADCC activity results of the anti-TIGIT / anti-PD-L1 bispecific antibody of the present invention (B204011) for primary Treg cells.

[0093] Fig. 18 shows the ADCC activity results of the anti-TIGIT / anti-PD-L1 bispecific antibody of the present invention (B101644) for primary Treg cells.

[0094] Fig. 19 shows the ADCC activity results of the anti-TIGIT / anti-PD-L1 bispecific antibodies of the present invention (B101645 and B101646) for primary Treg cells.

[0095] Fig. 20 shows the CD8 T cell-mediated cancer cell killing efficacy of the anti-TIGIT / anti-PD-L1 bispecific antibodies of the present invention (B101643 and B101644) against human tumor cells, H1975.

[0096] Fig. 21 shows the CD8 T cell-mediated cancer cell killing efficacy of the anti-TIGIT / anti-PD-L1 bispecific antibodies of the present invention (B101645 and B101646) against human tumor cells, H1975.

[0097] Fig. 22 shows the NK cell-mediated cancer cell killing efficacy of the anti-TIGIT / anti-PD-L1 bispecific antibody of the present invention (B101041) against human tumor cells, MDA-MB-231.

[0098] Fig. 23 shows the NK cell-mediated cancer cell killing efficacy of the anti-TIGIT / anti-PD-L1 bispecific antibodies of the present invention (B101042 and B101644) against human tumor cells, MDA-MB-231.

[0099] Fig. 24 shows the NK cell-mediated cancer cell killing efficacy of the anti-TIGIT / anti-PD-L1 bispecific antibodies of the present invention (B101645 and B101646) against human tumor cells, MDA-MB-231.

[0100] Fig. 25 shows the NK cell-mediated killing efficacy of the anti-TIGIT / anti-PD-L1 bispecific antibody of the present invention (B101644) against human Treg cells.

[0101] Fig. 26 shows the NK cell-mediated killing efficacy of the anti-TIGIT / anti-PD-L1 bispecific antibodies of the present invention (B101645 and B101646) against human Treg cells.

[0102] Fig. 27 shows the NK cell-mediated cancer cell killing efficacy of the anti-TIGIT / anti-PD-L1 bispecific antibody of the present invention (B101646) against human tumor cells, BxPC3.

[0103] Fig. 28 shows the NK cell-mediated cancer cell killing efficacy of the anti-TIGIT / anti-PD-L1 bispecific antibody of the present invention (B101646) against human tumor cells, Cal-27.

[0104] Fig. 29 shows the NK cell-mediated cancer cell killing efficacy of the anti-TIGIT / anti-PD-L1 bispecific antibody of the present invention (B101646) against human tumor cells, SK-OV3.

[0105] Fig. 30 shows the PK profile of the anti-TIGIT / anti-PD-L1 bispecific antibodies of the present invention (B101645 and B101646) in BALB / c Jh- / -mice.

[0106] Fig. 31 shows the results of animal efficacy evaluation of the anti-TIGIT / anti-PD-L1 bispecific antibody of the present invention (B101646) for each group.

[0107] Fig. 32 shows the results of animal efficacy evaluation of the anti-TIGIT / anti-PD-L1 bispecific antibody of the present invention (B101646) for each individual in each group.

[0108] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

[0109] In one aspect, the present invention relates to an anti-TIGIT / anti-PD-L1 bispecific antibody, which includes a first domain that specifically binds to PD-L1 and a second domain that specifically binds to TIGIT.

[0110] As used herein, "antibody" refers to an immunoglobulin molecule that has immunological reactivity for a specific antigen, and a protein molecule that acts as a receptor that specifically recognizes the antigen. The antibody may be used as a concept encompassing whole antibody and an antigen-binding fragment.

[0111] As used herein, "bispecific antibody" refers to an antibody that can bind to two different antigens simultaneously. In particular, when the type of antigen to which a bispecific antibody binds is appropriately selected, immune cells such as T cells may exhibit toxicity only against specific target cells, such as cancer cells, but not against other normal cells. Therefore, bispecific antibodies may be useful for treatments requiring T cell-mediated killing because they can minimize side effects while maximize therapeutic efficacy. The bispecific antibody that specifically binds to PD-L1 and TIGIT according to the present invention may be referred to as "anti-TIGIT / anti-PD-L1 bispecific antibody."

[0112] The anti-TIGIT / anti-PD-L1 bispecific antibody according to the present invention may include a first domain, which specifically binds to PD-L1 as one of the variable regions of the antibody, and a second domain, which specifically binds to TIGIT as another variable region.

[0113] As used herein, "a first domain" refers to a region that specifically binds to PD-L1 in the anti-TIGIT / anti-PD-L1 bispecific antibody, and "a second domain" refers to a region that specifically binds to TIGIT in the anti-TIGIT / anti-PD-L1 bispecific antibody.

[0114] In the anti-TIGIT / anti-PD-L1 bispecific antibody according to the present invention, the first domain that specifically binds to PD-L1 may include:

[0115] a heavy chain variable region (VH), which includes:

[0116] a heavy chain CDR1 represented by an amino acid sequence of SEQ ID NO: 1;

[0117] a heavy chain CDR2 represented by an amino acid sequence of SEQ ID NO: 2;

[0118] a heavy chain CDR3 represented by an amino acid sequence of SEQ ID NO: 3, and

[0119] a light chain variable region (VL), which includes:

[0120] a light chain CDR1 represented by an amino acid sequence of SEQ ID NO: 4;

[0121] a light chain CDR2 represented by an amino acid sequence of SEQ ID NO: 5; and

[0122] a light chain CDR3 represented by an amino acid sequence of SEQ ID NO: 6.

[0123] As used herein, "PD-L1" is a protein mainly expressed on the surface of cancer (tumor) cells, and is also referred to as CD274 and B7-H1. PD-L1 inhibits the anticancer action of T cells and NK cells through a binding to PD-1 expressed on T cells and NK cells, inhibits the activity of T cells thereby reducing autoimmune responses against self-cells, and inhibits immune attacks against tumor cells or infected cells.

[0124] As used herein, PD-L1 is a term that collectively refers to any variant, isoform, and species homolog of PD-L1 naturally expressed by cells, and preferably refers to human PD-L1, but is not limited thereto, and may be a term that includes PD-L1 of other animals, such as monkeys, mice, rats,etc.

[0125] As used herein, "anti-PD-L1 antibody" refers to an antibody that binds to PD-L1, thereby causing inhibition of the biological activity of PD-L1.

[0126] In the anti-TIGIT / anti-PD-L1 bispecific antibody according to the present invention, the first domain that specifically binds to PD-L1 may include a heavy chain CDR and a light chain CDR represented by the following amino acid sequences.

[0127] Amino acids of heavy chain CDRs of the first domain that specifically binds to PD-L1H-CDR1H-CDR2H-CDR3SYAYS(SEQ ID NO: 1)GIIPSFGTANYAQKFQG(SEQ ID NO: 2)GPIVATITPLDY(SEQ ID NO: 3)

[0128] Amino acids of light chain CDRs of the first domain that specifically binds to PD-L1L-CDR1L-CDR2L-CDR3GGENIGRKTVH(SEQ ID NO: 4)YDSDRPS(SEQ ID NO: 5)LVWDSSSDHRI(SEQ ID NO: 6)

[0129] Additionally, the anti-TIGIT / anti-PD-L1 bispecific antibody according to the present invention may include a heavy chain variable region (VH), which includes a sequence having at least 80% sequence homology, preferably at least 90% sequence homology, and more preferably at least 99% sequence homology to each of a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1; a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2; and a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3; and a light chain variable region (VL), which includes a sequence having at least 80% sequence homology, preferably at least 90% sequence homology, and more preferably at least 99% sequence homology to each of a light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4; a light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5; and a light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6.

[0130] The first domain may include an anti-PD-L1 heavy chain variable region of represented by the amino acid sequence of the following SEQ ID NO: 19. The first domain may include an anti-PD-L1 light chain variable region represented by the amino acid sequence of the following SEQ ID NO: 20.

[0131] SequenceSEQ ID NO:An anti-PD-L1 heavy chain variable regionQMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAYSWVRQAPGQGLEWMGGIIPSFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGPIVATITPLDYWGQGTLVTVSS19An anti-PD-L1 light chain variable regionSYVLTQPPSVSVAPGKTATIACGGENIGRKTVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCLVWDSSSDHRIFGGGTKLTVL20

[0132] Meanwhile, in the anti-TIGIT / anti-PD-L1 bispecific antibody of the present invention, the second domain that specifically binds to TIGIT may include:

[0133] a heavy chain variable region (VH), which includes:

[0134] a heavy chain CDR1 represented by an amino acid sequence of SEQ ID NO: 7 or 8;

[0135] a heavy chain CDR2 represented by an amino acid sequence of SEQ ID NO: 9 or 10; and

[0136] a heavy chain CDR3 represented by an amino acid sequence of SEQ ID NO: 11 or 12, and

[0137] a light chain variable region (VL), which includes:

[0138] a light chain CDR1 represented by an amino acid sequence of SEQ ID NO: 13 or 14;

[0139] a light chain CDR2 represented by an amino acid sequence of SEQ ID NO: 15 or 16; and

[0140] a light chain CDR3 represented by an amino acid sequence of SEQ ID NO: 17 or 18.

[0141] As used herein, "TIGIT" is a protein expressed on the surface of immune cells, such as T cells, NK cells, and dendritic cells, and it binds to poliovirus receptor (PVR, CD155) on the surface of cancer cells to thereby inhibit the activity of immune cells. PVR on the surface of cancer cells can bind not only to TIGIT but also to CD226 on immune cells. CD226, also known as DNAX Accessory Molecule-1 (DNAM-1), is an activating receptor involved in the activation of T cells and natural killer (NK) cells, and CD226 interacts with CD155 and CD112 ligands and activates the functions of T cells and NK cells through an immunoreceptor tyrosine-based activation motif (ITAM).

[0142] As used herein, TIGIT is a term that collectively refers to any mutant, isoform, and species homolog of TIGIT naturally expressed by cells, and preferably refers to human TIGIT, but is not limited thereto, and may be a term that includes TIGIT of other animals, such as monkeys, mice, rats,etc.

[0143] As used herein, "anti-TIGIT antibody" refers to an antibody that binds to TIGIT and thereby inhibit the biological activity of TIGIT.

[0144] In the anti-TIGIT / anti-PD-L1 bispecific antibody according to the present invention, the second domain that specifically binds to TIGIT may include a heavy chain CDR and a light chain CDR represented by any one of the following amino acid sequences.

[0145] Amino acids of heavy chain CDRs of the second domain that specifically binds to TIGITH-CDR1H-CDR2H-CDR3SYYMS(SEQ ID NO: 7)SIGSGSPSSTYYADSVKG(SEQ ID NO: 9)SSYSGGNGYYYYAYAFDY(SEQ ID NO: 11)NYAMS(SEQ ID NO: 8)GISPSGSSIYYADSVQG(SEQ ID NO: 10)AIRTCSLSHCYYYYGMDV(SEQ ID NO: 12)

[0146] Amino acids of light chain CDRs of the second domain that specifically binds to TIGITL-CDR1L-CDR2L-CDR3RASQSVSSSYLA(SEQ ID NO: 13)GASSRAT(SEQ ID NO: 15)QQGYHRYAT(SEQ ID NO: 17)SSSSSNIGSNAVN(SEQ ID NO: 14)YDNQRPS(SEQ ID NO: 16)ATWDYSLSGYV(SEQ ID NO: 18)

[0147] Additionally, the anti-TIGIT / anti-PD-L1 bispecific antibody according to the present invention may include a heavy chain variable region (VH), which includes a sequence having at least 80% sequence homology, preferably at least 90% sequence homology, and more preferably at least 99% sequence homology to each of a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 7 or 8; a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 9 or 10; and a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 11 or 12; and a light chain variable region (VL), which includes a sequence having at least 80% sequence homology, preferably at least 90% sequence homology, and more preferably at least 99% sequence homology to each of a light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 13 or 14; a light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 15 or 16; and a light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 17 or 18.

[0148] The second domain may include an anti-TIGIT heavy chain variable region represented by any one of the amino acid sequences selected from the group consisting of SEQ ID NOS: 21 to 24. The second domain may include an anti-TIGIT light chain variable region represented by any one of the amino acid sequences selected from the group consisting of SEQ ID NOS: 25 to 28.

[0149] SequenceSEQ ID NO:An anti-TIGIT heavy chain variable region (1)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKGLEWVSSIGSGSPSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYSGGNGYYYYAYAFDYWGQGTLVTVSS21An anti-TIGIT heavy chain variable region (2)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKCLEWVSSIGSGSPSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYSGGNGYYYYAYAFDYWGQGTLVTVSS22An anti-TIGIT heavy chain variable region (3)EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSGISPSGSSIYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAIRTCSLSHCYYYYGMDVWGQGTLVTVSS23An anti-TIGIT heavy chain variable region (4)EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKCLEWVSGISPSGSSIYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAIRTCSLSHCYYYYGMDVWGQGTLVTVSS24An anti-TIGIT light chain variable region (1)EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGYHRYATFGQGTKVEIK25An anti-TIGIT light chain variable region (2)EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGYHRYATFGQCTKVEIK26An anti-TIGIT light chain variable region (3)QSVLTQPPSASGTPGQRVTISCSSSSSNIGSNAVNWYQQLPGTAPKLLIYYDNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGGGTKLTVL27An anti-TIGIT light chain variable region (4)QSVLTQPPSASGTPGQRVTISCSSSSSNIGSNAVNWYQQLPGTAPKLLIYYDNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVL28

[0150]

[0151] In an embodiment, in order to introduce a disulfide bond, interface engineering, in which the amino acid glycine (G) in the heavy chain variable region and the light chain variable region of the second domain are substituted with cysteine (C), may be applied. For example, the sequences to which the interface engineering is applied may be amino acid the sequences represented by SEQ ID NOS: 22, 24, 26, and 28. Structural stability may be improved by the introduction of a disulfide bond.

[0152] Additionally, in the anti-TIGIT / anti-PD-L1 bispecific antibody according to the present invention, the first domain may include an IgG1 Fc region.

[0153] In an embodiment, the IgG1 Fc region may be represented by the following amino acid sequences.

[0154] SequenceSEQ ID NO:Wild-type IgG1 FcASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK29IgG1 FcMutation (1)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK30IgG1 FcMutation (2)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTLRVVSILTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK31IgG1 FcMutation (3)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK32

[0155] In an embodiment, the IgG1 Fc region may be a wild-type represented by the amino acid sequence of SEQ ID NO: 29.

[0156] Additionally, the anti-TIGIT / anti-PD-L1 bispecific antibody according to the present invention may include mutations that can induce the enhancement of an Fc-effector function. More specifically, the Fc-effector function can be enhanced by improving the binding affinity for activating FcγRs or FcRn binding affinity, and the anticancer efficacy of the antibody can be improved by increasing the antibody-dependent cellular cytotoxicity (ADCC) efficacy by enhancing the Fc-effector function. More specifically, the Fc-effector function may be enhanced by improving the binding affinity for activating FcγRs or FcRn binding affinity, and the anticancer efficacy of an antibody may be improved by increasing the antibody-dependent cellular cytotoxicity (ADCC) efficacy through the enhancement of the Fc-effector function. The mutation that enhances the Fc-effector function may be any one of the following mutations (i) to (iii):

[0157] (i) S239D, A330L, and I332E;

[0158] (ii) F243L, R292P, Y300L, V305I, and P396L; and

[0159] (iii) S298A, E333A, and K334A.

[0160] As used herein, the position of the amino acid in indicating the position of a mutation is designated according to the nomenclature by EU numbering.

[0161] Additionally, the anti-TIGIT / anti-PD-L1 bispecific antibody according to the present invention may include a mutation that improves blood half-life. The mutation that improves blood half-life may be an LS mutation (M428L / N434S).

[0162] In an embodiment, the anti-TIGIT / anti-PD-L1 bispecific antibody according to the present invention is a mutation that induces the enhancement of the Fc-effector function and / or a mutation that improves blood half-life, and may include any one or more mutations selected from the group consisting of S239D, F243L, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, P396L, M428L, and N434S.

[0163] In an embodiment, the anti-TIGIT / anti-PD-L1 bispecific antibody according to the present invention may include one or more of the following mutations (i) to (iv) in the IgG1 Fc region represented by the amino acid sequence of SEQ ID NO: 29:

[0164] (i) S239D, A330L, and I332E;

[0165] (ii) F243L, R292P, Y300L, V305I, and P396L;

[0166] (iii) S298A, E333A, and K334A; and

[0167] (iv) M428L and N434S.

[0168] In an embodiment, the first domain of the anti-TIGIT / anti-PD-L1 bispecific antibody according to the present invention may an IgG1 Fc region that includes one or more mutations in the amino acid sequence of SEQ ID NO: 29, in which the mutations may be selected from the group consisting of S239D, A330L, and I332E.

[0169] In an embodiment, the first domain of the anti-TIGIT / anti-PD-L1 bispecific antibody according to the present invention may include an IgG1 Fc region that includes one or more mutations in the amino acid sequence of SEQ ID NO: 29, in which the mutations may be selected from the group consisting of F243L, R292P, Y300L, V305I, P396L, M428L, and N434S.

[0170] In an embodiment, the first domain of the anti-TIGIT / anti-PD-L1 bispecific antibody according to the present invention may include an IgG1 Fc region that includes one or more mutations in the amino acid sequence of SEQ ID NO: 29, in which the mutations may be selected from the group consisting of S298A, E333A, K334A, M428L, and N434S.

[0171] In an embodiment, the first domain of the anti-TIGIT / anti-PD-L1 bispecific antibody according to the present invention may include an IgG1 Fc region represented by any one of the amino acid sequences of SEQ ID NOS: 30 to 32.

[0172] The anti-TIGIT / anti-PD-L1 bispecific antibody including the above-mentioned mutations may exhibit excellent antitumor therapeutic efficacy through enhanced T cell engager efficacy as a bispecific antibody and enhanced Fc-effector function and improved blood half-life, compared to the single and combination treatment of anti-PD-L1 and anti-TIGIT monoclonal antibodies.

[0173] Additionally, the anti-TIGIT / anti-PD-L1 bispecific antibody according to the present invention also includes antibodies in which part of the amino acid sequence is substituted through conservative substitution.

[0174] As used herein, "conservative substitution" refers to a modification of a polypeptide that includes substituting one or more amino acids with amino acids having similar biochemical properties that do not result in loss of biological or biochemical function of the polypeptide. A "conservative substitution of amino acids" refers to a substitution that substitutes an amino acid residue with an amino acid residue having a similar side chain. The groups of amino acid residues having similar side chains are defined and well known in the art. These groups include amino acids having basic side chains (e.g., lysine, arginine, and histidine), amino acids having acidic side chains (e.g., aspartic acid and glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), amino acids having non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), amino acids having beta-branched side chains (e.g., threonine, valine, and isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). The antibodies of the present invention may still have activity even with conservative substitution of amino acids.

[0175] Meanwhile, as used herein, "linker" refers a linking moiety capable of linking two different fusion partners (e.g., biological polymers,etc.) using a hydrogen bond, electrostatic interaction, van der Waals force, disulfide bond, salt bridge, hydrophobic interaction, covalent bond,etc.

[0176] Specifically, the "linker", in addition to simply linking each of the fusion partners, may also serve to provide a certain size of gap between the fusion partners or serve as a hinge that provides flexibility or rigidity to the fused entity. The linker may be a non-peptide linker or peptide linker, and may include those directly linked by a peptide bond, a disulfide bond,etc., and the peptide linker may include a plurality of amino acid sequences or an amino acid sequence with a form where a motif is repeated.

[0177] The linker may be a peptide, which consists of 10 to 50 amino acid residues and includes one or more residues selected from the group consisting of glycine (G), serine (S), alanine (A), lysine (K), and glutamic acid (E).

[0178] The linker may be a linker selected from the group consisting of (GGGGS)n, GS(GGGGS)n, GS(EEEA)n, (EEEA)n, GS(EAAAK)n, and (EAAAK)n, wherein n may be an integer of 1 to 10.

[0179] The part linked directly or indirectly through the linker is not particularly limited.

[0180] As used herein, the terms "Fc region", "Fc fragment", or "Fc" refers to a protein, which includes a heavy chain constant region 1 (CH1), a heavy chain constant region 2 (CH2), and a heavy chain constant region 3 (CH3) of an immunoglobulin, but does not include variable regions of heavy and light chains and a light chain constant region 1 (CL1) of an immunoglobulin.

[0181] Additionally, in the phrase "immunoglobulin Fc or a variant thereof" used herein, the term "variant" refers to one in which some amino acids in the Fc region are substituted or one prepared by combining different types of Fc regions. The IgG Fc region or a variant thereof may be an Fc of IgG1, IgG2, IgG3, or IgG4, or a variant thereof. Additionally, the Fc region variant may be a variant that does not include the hinge region of the heavy chain constant region, or may include the hinge region.

[0182] In another aspect of the present invention, the Fc of the immunoglobulin or a variant thereof may be a hybrid Fc consisting of any one of Fc of IgG1, IgG2, IgG3, IgG4, and IgD, or a combination thereof.

[0183] In another embodiment of the present invention, the Fc of the immunoglobulin or a variant thereof may be an IgG1 Fc or a variant thereof, and the human IgG1 Fc may consist of the amino acid sequence represented by SEQ ID NO: 29. The human IgG Fc or variant thereof may have an amino acid sequence, which is at least 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 29.

[0184] In an embodiment, in the anti-TIGIT / anti-PD-L1 bispecific antibody, the first domain may include a heavy chain and a light chain, and the second domain may be an scFv including a heavy chain variable region and a light chain variable region, in which the C-terminus of the heavy chain of the first domain and the N-terminus of the heavy chain variable region of the second domain may be linked through a linker.

[0185] In an embodiment, in the anti-TIGIT / anti-PD-L1 bispecific antibody, the first domain may include a heavy chain and a light chain, and the second domain may be an scFv including a heavy chain variable region and a light chain variable region, in which the C-terminus of the heavy chain of the first domain and the N-terminus of the light chain variable region of the second domain may be linked through a linker.

[0186] In an embodiment, in the anti-TIGIT / anti-PD-L1 bispecific antibody, the first domain may include a heavy chain and a light chain, and the second domain may be an scFv including a heavy chain variable region and a light chain variable region, in which the C-terminus of the heavy chain of the second domain and the N-terminus of the heavy chain variable region of the first domain may be linked through a linker.

[0187] The anti-TIGIT / anti-PD-L1 bispecific antibody of the present invention may be:

[0188] 1) an anti-TIGIT / anti-PD-L1 bispecific antibody comprising the amino acid sequences of SEQ ID NOS: 33 to 36;

[0189] 2) an anti-TIGIT / anti-PD-L1 bispecific antibody comprising the amino acid sequences of SEQ ID NOS: 37 to 40;

[0190] 3) an anti-TIGIT / anti-PD-L1 bispecific antibody comprising the amino acid sequences of SEQ ID NOS: 41 to 44;

[0191] 4) an anti-TIGIT / anti-PD-L1 bispecific antibody comprising the amino acid sequences of SEQ ID NOS: 45 to 48;

[0192] 5) an anti-TIGIT / anti-PD-L1 bispecific antibody comprising the amino acid sequences of SEQ ID NOS: 49 to 52;

[0193] 6) an anti-TIGIT / anti-PD-L1 bispecific antibody comprising the amino acid sequences of SEQ ID NOS: 53 to 56; and

[0194] 7) an anti-TIGIT / anti-PD-L1 bispecific antibody comprising the amino acid sequences of SEQ ID NOS: 57 to 60.

[0195] In still another aspect, the present invention relates to a pharmaceutical composition for treating cancer, which includes the anti-TIGIT / anti-PD-L1 bispecific antibody as an active ingredient.

[0196] The pharmaceutical composition of the present invention may include the anti-TIGIT / anti-PD-L1 bispecific antibody as an active ingredient.

[0197] In the present invention, the anti-TIGIT / anti-PD-L1 bispecific antibody, by binding to PD-L1 of a tumor, can prevent the binding between PD-1 and PD-L1 thereby preventing the inhibition of immune cell activity due to the PD-1 / PD-L1 binding, while, by simultaneously binding to TIGIT, can prevent the inhibition of immune cell activity due to the TIGIT / PVR binding. Additionally, in the present invention, the pharmaceutical composition of the present invention has excellent anticancer efficacy since the anti-TIGIT / anti-PD-L1 bispecific antibody acts as a T cell engager, that is, the anti-TIGIT / anti-PD-L1 bispecific antibody recognizes tumor cells, and increases immune cell activation by binding of PVR of a tumor adjacent to CD226, which is expressed on immune cells recruited to a tumor.

[0198] As used herein, "treatment" refers to any act of improving or beneficially changing the symptoms of a disease by administering the composition according to the present invention; "prevention" refers to any act of inhibiting or delaying a disease by administering the composition according to the present invention; and "improvement" refers to any act of improving a bad condition of a disease by administering or ingesting the pharmaceutical composition of the present invention to a subject.

[0199] The target disease for "treatment," "prevention", or "improvement" of the present invention is cancer (tumor).

[0200] As used herein, "cancer" or "tumor" refers to or means a physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation.

[0201] The cancer or cancer type that can be treated with the pharmaceutical composition of the present invention is not particularly limited and includes both solid cancer and blood cancer. Examples of such cancers may be selected from the group consisting of skin cancer such as melanoma, liver cancer, hepatocellular carcinoma, stomach cancer, breast cancer, lung cancer, ovarian cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, colorectal cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, thyroid cancer, parathyroid cancer, kidney cancer, esophageal cancer, bile duct cancer, testicular cancer, rectal cancer, head and neck cancer, cervical spine cancer, ureteral cancer, osteosarcoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, glioma, myeloma, neuroblast-derived CNS tumor, monocytic leukemia, B-cell derived leukemia, T-cell derived leukemia, B-cell derived lymphoma, T-cell derived lymphoma, and mast cell derived tumor, but is not limited thereto.

[0202] The present invention provides a pharmaceutical composition, which includes a therapeutically effective amount of an anti-TIGIT / anti-PD-L1 bispecific antibody and a pharmaceutically acceptable carrier.

[0203] A "pharmaceutically acceptable carrier" is a substance that may be added to an active ingredient to aid in formulating or stabilizing a formulation and that does not cause significant detrimental toxic effects to patients.

[0204] The carrier refers to an additive or diluent that does not irritate patients and does not inhibit the biological activity and properties of the compound to be administered. In compositions formulated as liquid solutions, acceptable pharmaceutical carriers are those that are sterile and biocompatible, and saline, sterile water, a Ringer's solution, buffered saline, an albumin injection solution, a dextrose solution, a maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these ingredients may be used, and other common additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Additionally, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations (e.g., aqueous solutions, suspensions, emulsions,etc.), pills, capsules, granules, or tablets.

[0205] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the preparation of extemporaneous sterile injectable solutions or dispersions. The use of these media and agents for pharmaceutically active substances is known in the art.

[0206] The composition is preferably formulated into a parenteral injection. The composition may be formulated as a solution agent, a microemulsion agent, a liposome agent, or other ordered structures suitable for high drug concentrations. The carrier may be a solvent or dispersion medium including, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol,etc.), and a suitable mixture thereof. In some cases, the composition may include isotonic agents, for example, sugars, polyalcohols (e.g., mannitol, sorbitol) or sodium chloride. Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients described above, as needed, followed by sterile microfiltration. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle which includes a basic dispersion medium and other required ingredients from those described above. For sterile powders for the preparation of sterile injectable solutions, some preparation methods are vacuum drying and freeze-drying (lyophilization), which produce powder of the active ingredient and any additional desired ingredient from a previously sterile-filtered solution thereof.

[0207] The administration dose of the pharmaceutical composition according to the present invention is not particularly limited, but may vary depending on various factors including the patient's health conditions and weight, severity of disease, type of drug, route of administration, and time of administration. The pharmaceutical composition according to the present invention may be administered to mammals including rats, mice, livestock, humans,etc., in a single or multiple doses per day by any typically accepted route, such as, but not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or rectal administration.

[0208] As used herein, "therapeutically effective amount" refers to the amount of a combination of anti-TIGIT / anti-PD-L1 bispecific antibodies required to produce a measurable benefitin vivoin a patient in need of treatment. The exact amount may vary depending on numerous factors including, but not limited to, the ingredients and physical characteristics of the therapeutic composition, the population of intended patients, considerations of individual patients,etc., and may be readily determined by those of ordinary skill in the art. Taking these factors into full consideration, it is important to administer the minimum dose sufficient to achieve the maximum effect without adverse effects, and this dose may be readily determined by experts in this field.

[0209] In still another aspect, the present invention relates to a method for treating cancer and inhibiting the growth of cancer by administering, to a subject in need of treatment, the anti-TIGIT / anti-PD-L1 bispecific antibody according to the present invention, or a pharmaceutical composition including the same.

[0210] The pharmaceutical composition according to the present invention may be administered in a pharmaceutically effective amount to treat cancer cells or metastasis of cancer or to inhibit the growth of cancer.

[0211] The pharmaceutically effective amount may vary depending on various factors such as the type of cancer, the patient's age, weight, characteristics and severity of symptoms, type of current treatment, number of treatments, form and route of administration,etc., and may be readily determined by experts in the corresponding field.

[0212] The pharmaceutical composition of the present invention may be administered in combination with other therapeutic agents and may be administered sequentially or simultaneously with the other therapeutic agents. Such administration may be a single or multiple administration. Taking all of these factors into consideration, it is important to administer the amount that can achieve the maximum effect with the minimum amount without side effects, and this may easily be determined by those of ordinary skill in the art.

[0213] As used herein, "subject" may refer to a mammal, and preferably a human, suffering from or at risk of a condition or disease that can be alleviated, inhibited, or treated by administering the anti-TIGIT / anti-PD-L1 bispecific antibody according to the present invention, or a pharmaceutical composition including the same.

[0214] In still another aspect, the present invention relates to a nucleic acid, which encodes the amino acid sequence of the first domain and the amino acid sequence of the second domain of the anti-TIGIT / anti-PD-L1 bispecific antibody according to the present invention.

[0215] As used herein, a "nucleic acid" may be present in a cell, a cell lysate, or in a partially purified or substantially pure form. The nucleic acid is "isolated" or "rendered substantially pure" when purified away from other cellular components or other contaminants, such as nucleic acids or proteins from other cells, by standard techniques including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. The nucleic acid of the present invention may be, for example, DNA or RNA, and may or may not include intron sequences.

[0216] In still another aspect, the present invention relates to a recombinant expression vector including the nucleic acid. For the expression of the anti-TIGIT / anti-PD-L1 bispecific antibodies according to the present invention, DNA encoding partial or full-length light and heavy chains may be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using hybridomas expressing target antibodies) and inserted into an expression vector "operably linked" to transcriptional and translational control sequences.

[0217] As used herein, "operatively linked" may mean that a gene encoding an antibody is ligated into a vector such that transcriptional and translational control sequences within the vector perform their intended function of controlling transcription and translation of the antibody gene. Expression vectors and expression control sequences are selected to be compatible with the host cell for expression to be used. The antibody light chain gene and the antibody heavy chain gene may be inserted into separate vectors, or both genes may be inserted into the same expression vector. The antibody may be inserted into the expression vector by standard methods (e.g., ligation into a complementary restriction enzyme site on the gene fragment of the antibody and the vector, or blunt-end ligation when no restriction enzyme site is present at all). In some cases, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene may be cloned into a vector such that the signal peptide is linked to be suitable for the frame to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a protein other than immunoglobulin). Additionally, the recombinant expression vector carries control sequences that control the expression of the antibody chain gene in a host cell. The "control sequence" may include promoters, enhancers, and other expression control elements (e.g., polyadenylation signal) that control the transcription or translation of the antibody chain gene. Those of ordinary skill in the art would be able to recognize that the design of the expression vector may vary depending on factors such as the choice of a host cell to be transformed, the level of protein expression,etc., by selecting different control sequences.

[0218] In still another aspect, the present invention relates to a host cell transformed with the recombinant expression vector.

[0219] The host cell according to the present invention is preferably selected from the group consisting of animal cells, plant cells, yeast,E. coli, and insect cells, but is not limited thereto.

[0220] Specifically, the host cell according to the present invention may be a prokaryotic cell, such asEscherichia coli,Bacillus subtilis,Streptomycessp.,Pseudomonassp.,Proteus mirabilis, orStaphylococcussp. Additionally, the cell may be a eukaryotic cell, such as fungi (e.g.,Aspergillussp.), yeasts (e.g.,Pichia pastoris,Saccharomyces cerevisiae,Schizosaccharomycessp. andNeurospora crassa), other lower eukaryotic cells, and eukaryotic cells (e.g., cells from insects).

[0221] Additionally, the host cell may be derived from plants or mammals. Preferably, monkey kidney cells 7 (COS7) cells, NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, and HEK293 cells may be used, but are not limited thereto. Particularly preferably, CHO cells may be used.

[0222] The nucleic acid or the vector is transfected into a host cell. For "transfection" purposes, a variety of different techniques, such as electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, and lipofection, may be used to introduce an exogenous nucleic acid (DNA or RNA) into prokaryotic or eukaryotic host cells. A variety of expression host / vector combinations may be used to express the anti-TIGIT / anti-PD-L1 bispecific antibody according to the present invention. Expression vectors suitable for eukaryotic hosts may include, for example, expression control sequences derived from SV40, bovine papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus, and retrovirus, but are not limited thereto. Expression vectors to be used in bacterial hosts may include, for example, bacterial plasmids obtained fromEscherichia coli(e.g., pET, pRSET, pBluescript, pGEX2T, pUC vectors, col E1, pCR1, pBR322, pMB9, and derivatives thereof), plasmids having a broader host range (e.g., RP4), phage DNA that can be illustrated in a variety of phage lambda derivatives (e.g., λgt10, λgt11, and NM989), and other DNA phages (e.g., M13 and filamentous single-stranded DNA phages), but are not limited thereto.

[0223] In still another aspect, the present invention relates to a method for preparing an anti-TIGIT / anti-PD-L1 bispecific antibody, which includes the step of culturing a host cell.

[0224] When the recombinant expression vector capable of expressing the anti-TIGIT / anti-PD-L1 bispecific antibody according to the present invention is introduced into a mammalian host cell, the antibody may be prepared by culturing the host cell for a period of time sufficient to induce the expression of the antibody in the host cell, more preferably for a period of time sufficient to induce the secretion of the antibody into the culture medium in which the host cell is cultured.

[0225] In some cases, the expressed antibody may be separated from the host cell and purified to homogeneity. The separation or purification of the antibody may be performed by separation and purification methods commonly used for proteins, such as chromatography. The chromatography may include, for example, affinity chromatography including a protein A column and a protein G column, ion exchange chromatography, and hydrophobic chromatography. In addition to the various kinds of chromatography, antibodies may be separated and purified by combining filtration, ultrafiltration, salting out, dialysis,etc.

[0226] In still another aspect, the present invention relates to an anti-TIGIT / anti-PD-L1 bispecific binding protein, which includes a first domain that specifically binds to PD-L1 and a second domain that specifically binds to TIGIT.

[0227] The bispecific binding protein refers to a polypeptide or protein, which includes a first domain that specifically binds to PD-L1 and a second domain that specifically binds to TIGIT, thereby being capable of specifically binding to PD-L1 and TIGIT.

[0228] The descriptions overlapping with the above-described anti-TIGIT / anti-PD-L1 bispecific antibody are omitted.

[0229] In still another aspect, the present invention relates to an anti-TIGIT / anti-PD-L1 bispecific binding molecule, which includes a first domain that specifically binds to PD-L1 and a second domain that specifically binds to TIGIT.

[0230] The bispecific binding molecule refers to any biochemical molecule, which includes a first domain that specifically binds to PD-L1 and a second domain that specifically binds to TIGIT, thereby being capable of specifically binding to PD-L1 and TIGIT.

[0231] The descriptions overlapping with the above-described anti-TIGIT / anti-PD-L1 bispecific antibody are omitted.

[0232] Hereinafter, the present invention will be described in more detail through examples. It will be apparent to those skilled in the art that these examples are intended only to illustrate the present invention and that the scope of the present invention is not to be construed as being limited by these examples.

[0233] Preparation Examples

[0234] 1. Anti-TIGIT / anti-PD-L1 bispecific antibody: design of materials

[0235] An anti-TIGIT / anti-PD-L1 bispecific antibody was designed by combining two types of anti-TIGIT antibodies and one type of anti-PD-L1 antibody.

[0236] For example, the scFv of an anti-TIGIT monoclonal antibody may be introduced into the backbone of the an anti-PD-L1 IgG1 with a (G4S)2linker.

[0237] B101041 and B204011 were prepared by a combination of a first domain, which specifically binds to PD-L1 that includes heavy chain / light chain variable regions represented by the amino acid sequences of SEQ ID NOS: 1 to 6; and a second domain, which specifically binds to TIGI that includes heavy chain / light chain variable regions represented by the amino acid sequences of SEQ ID NOS: 7, 9, 11, 13, 15, and 17.

[0238] B101042, B101643, B101644, B101645, and B101646 were prepared by a combination of a first domain, which specifically binds to PD-L1 that includes heavy chain / light chain variable regions represented by the amino acid sequences of SEQ ID NOS: 1 to 6; and a second domain, which specifically binds to TIGI that includes heavy chain / light chain variable regions represented by the amino acid sequences of SEQ ID NOS: 8, 10, 12, 14, 16, and 18.

[0239] The Fc effector function was added to B101644, while B101645 and B101646 have been engineered to improve Fc effector function and blood half-life. The mutations applied to B101644 to improve Fc effector function were S239D / A330L / I332E, the mutations applied to B101645 were F243L / R292P / Y300L / V305I / P396L, and the mutations applied to B161046 were S298A / E333A / K334A. B101645 and B101646 were introduced with the M428L / N434S mutations to improve blood half-life.

[0240] 2. Anti-TIGIT / anti-PD-L1 bispecific antibody: sequence

[0241] The amino acid sequences of the anti-TIGIT / anti-PD-L1 bispecific antibodies (i.e., B101041, B204011, B101042, B101643, B101644, B101645, and B101646) according to one embodiment are as follows.

[0242] Amino acid sequence of B101041B101041 (bispecific antibody)Amino Acid Sequence (N→C)SEQ ID NO:Heavy chain of anti-PD-L1 antibodyQMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAYSWVRQAPGQGLEWMGGIIPSFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGPIVATITPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK33LinkerGGGGSGGGGSGGGGS34scFv of anti-TIGIT antibodyVHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKCLEWVSSIGSGSPSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYSGGNGYYYYAYAFDYWGQGTLVTVSS35LinkerGGGGSGGGGSGGGGSVLEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGYHRYATFGQCTKVEIKLight chain of anti-PD-L1 antibodySYVLTQPPSVSVAPGKTATIACGGENIGRKTVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCLVWDSSSDHRIFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS36

[0243] Amino acid sequence of B204011B204011 (bispecific antibody)Amino Acid Sequence (N→C)SEQ ID NO:Heavy chain of anti-TIGIT antibodyEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKGLEWVSSIGSGSPSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYSGGNGYYYYAYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK37LinkerGGGGSGGGGS38scFv of anti-PD-L1 antibodyVHQMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAYSWVRQAPGQGLEWMGGIIPSFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGPIVATITPLDYWGQGTLVTVSS39LinkerGGGGSGGGGSGGGGSGGGGSVLSYVLTQPPSVSVAPGKTATIACGGENIGRKTVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCLVWDSSSDHRIFGGGTKLTVLLight chain of anti-TIGIT antibodyEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGYHRYATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC40

[0244] Amino acid sequence of B101042B101042 (bispecific antibody)Amino Acid Sequence (N→C)SEQ ID NO:Heavy chain of anti-PD-L1 antibodyQMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAYSWVRQAPGQGLEWMGGIIPSFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGPIVATITPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK41LinkerGGGGSGGGGS42scFv of anti-TIGIT antibodyVLEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQGYHRYATFGQGTKVEIK43LinkerGGGGSGGGGSGGGGSGGGGSVHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYYMSWVRQAPGKGLEWVSSIGSGSPSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYSGGNGYYYYAYAFDYWGQGTLVTVSSLight chain of anti-PD-L1 antibodySYVLTQPPSVSVAPGKTATIACGGENIGRKTVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCLVWDSSSDHRIFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS44

[0245] Amino acid sequence of B101643B101643 (bispecific antibody)Amino Acid Sequence (N→C)SEQ ID NO:Heavy chain of anti-PD-L1 antibodyQMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAYSWVRQAPGQGLEWMGGIIPSFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGPIVATITPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK45LinkerGGGGSGGGGS46scFv of anti-TIGIT antibodyVLQSVLTQPPSASGTPGQRVTISCSSSSSNIGSNAVNWYQQLPGTAPKLLIYYDNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVL47LinkerGGGGSGGGGSGGGGSGGGGSVHEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKCLEWVSGISPSGSSIYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAIRTCSLSHCYYYYGMDVWGQGTLVTVSSLight chain of anti-PD-L1 antibodySYVLTQPPSVSVAPGKTATIACGGENIGRKTVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCLVWDSSSDHRIFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS48

[0246] Amino acid sequence of B101644B101644 bispecific antibodyAmino Acid Sequence (N→C)SEQ ID NO:Heavy chain of anti-PD-L1 antibodyQMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAYSWVRQAPGQGLEWMGGIIPSFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGPIVATITPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK49LinkerGGGGSGGGGS50scFv of anti-TIGIT antibodyVLQSVLTQPPSASGTPGQRVTISCSSSSSNIGSNAVNWYQQLPGTAPKLLIYYDNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVL51LinkerGGGGSGGGGSGGGGSGGGGSVHEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKCLEWVSGISPSGSSIYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAIRTCSLSHCYYYYGMDVWGQGTLVTVSSLight chain of anti-PD-L1 antibodySYVLTQPPSVSVAPGKTATIACGGENIGRKTVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCLVWDSSSDHRIFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS52

[0247] Amino acid sequence of B101645B101645 (bispecific antibody)Amino Acid Sequence (N→C)SEQ ID NO:Heavy chain of anti-PD-L1 antibodyQMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAYSWVRQAPGQGLEWMGGIIPSFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGPIVATITPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTLRVVSILTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK53LinkerGGGGSGGGGS54scFv of anti-TIGIT antibodyVLQSVLTQPPSASGTPGQRVTISCSSSSSNIGSNAVNWYQQLPGTAPKLLIYYDNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVL55LinkerGGGGSGGGGSGGGGSGGGGSVHEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKCLEWVSGISPSGSSIYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAIRTCSLSHCYYYYGMDVWGQGTLVTVSSLight chain of anti-PD-L1 antibodySYVLTQPPSVSVAPGKTATIACGGENIGRKTVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCLVWDSSSDHRIFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS56

[0248] Amino acid sequence of B101646B101646 bispecific antibodyAmino Acid Sequence (N→C)SEQ ID NO:Heavy chain of anti-PD-L1 antibodyQMQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAYSWVRQAPGQGLEWMGGIIPSFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGPIVATITPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK57LinkerGGGGSGGGGS58scFv of anti-TIGIT antibodyVLQSVLTQPPSASGTPGQRVTISCSSSSSNIGSNAVNWYQQLPGTAPKLLIYYDNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDYSLSGYVFGCGTKLTVL59LinkerGGGGSGGGGSGGGGSGGGGSVHEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKCLEWVSGISPSGSSIYYADSVQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAIRTCSLSHCYYYYGMDVWGQGTLVTVSSLight chain of anti-PD-L1 antibodySYVLTQPPSVSVAPGKTATIACGGENIGRKTVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCLVWDSSSDHRIFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS60

[0249] 3. Preparation of anti-TIGIT / anti-PD-L1 bispecific antibody

[0250] 3.1. Gene cloning for expression of anti-TIGIT / anti-PD-L1 bispecific antibody

[0251] In order to express the anti-TIGIT / anti-PD-L1 bispecific antibody, the nucleotide sequences encoding the amino acid sequences shown in Preparation Example 2 above were synthesized by requesting to Macrogen Co., Ltd., and the corresponding nucleotide sequences were inserted into the pcDNA 3.4 (ThermoFisher) expression vector using restriction enzymes to prepare a vector expressing the heavy chain, scFv, and the light chain.

[0252] 3.2. Expression and purification of anti-TIGIT / anti-PD-L1 bispecific antibodies in ExpiCHO cells

[0253] The pcDNA3.4 expression vector cloned in Preparation Example 3.1 above was transiently transfected into ExpiCHO cell line (ThermoFisher), harvested on day 8, and purified. In order to purify the anti-TIGIT / anti-PD-L1 bispecific antibody in the culture medium, affinity purification was performed using MabSelect Sure Protein A resin (Cytiva).

[0254] Specifically, the culture medium was loaded onto and bound to MabSelect SuRe Protein A resin (Cytiva) equilibrated with 1X PBS (pH 7.4). After completion of the binding, the MabSelect SuRe Protein A resin was washed with 1X PBS (pH 7.4), and eluted using a 0.1 M glycine (pH 3.0) solution to obtain the final material.

[0255]

[0256] Examples

[0257] 1. SDS-PAGE analysis of anti-TIGIT / anti-PD-L1 bispecific antibody

[0258] In order to confirm the purity of the anti-TIGIT / anti-PD-L1 bispecific antibody purified in Preparation Example 3.2 above, SDS-PAGE was performed under reducing and non-reducing conditions. 5 μg of the purified anti-TIGIT / anti-PD-L1 bispecific antibody was mixed with non-reducing sample buffer (GenDEPOT, Cat# L1200-001) and reducing sample buffer (GenDEPOT, Cat# L1100-001), and the mixture was heated at 80℃ for 5 minutes. The denatured proteins were loaded onto SDS-PAGE gels (NuPAGE Bis-Tris gels, 4-12%) and electrophoresed at 90 V for 140 minutes. The proteins on the gel were then stained with a Coomassie blue solution to visualize them. The SDS PAGE results for each material are shown in Fig. 2.

[0259] 2. SEC-HPLC analysis of anti-TIGIT / anti-PD-L1 bispecific antibody

[0260] After connecting the developing solvent to pump A of the HPLC system (Thermo, Ultimate 3000), the system was operated. After exchanging with the developing solvent, the SEC column (Tosoh, TSK gel G3000SWXL, 7.8 × 300 mm, 5 μm, 300 Å) as connected thereto. In order to stabilize the column, the system was operated at a speed of 1 mL / min for 1 hour and stabilized. The developing solvent was flowed while confirming whether the baseline was stabilized in the chromatogram. After filtering the proteins to be analyzed through a 0.22 μm filter, 100 μg of each sample was injected to analyze the purity of each protein. As shown in Table 15, the SEC-HPLC purity of each of the anti-TIGIT / anti-PD-L1 bispecific antibodies was confirmed.

[0261] Results of SEC-HPLC analysis for anti-TIGIT / anti-PD-L1 bispecific antibodiesPurity (%)B10104176.0B20401199.6B10104290.2B10164393.4B10164493.4B10164594.4B10164692.8

[0262] 3. Evaluation of binding affinity of anti-TIGIT / anti-PD-L1 bispecific antibodies for human TIGIT and PD-L1

[0263] 3.1. Surface Plasmon Resonance (SPR) Analysis

[0264] Quantitative analysis of the binding of anti-TIGIT / anti-PD-L1 bispecific antibodies to human TIGIT and PD-L1 proteins was performed using BIACORE T200. Experimental conditions using surface plasmon resonance (SPR) were as follows.

[0265] The anti-TIGIT / anti-PD-L1 bispecific antibodies were immobilized on Series S Sensor Chip CM5 (Cytiva, BR100530) by amine coupling. A recombinant human TIGIT protein (Acro Biosystems, TIT-H5254) was flowed across the chip at 30 μL / min for 120 seconds at concentrations of 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.63 nM, and 7.81 nM, followed by a dissociation step for 600 seconds. Regeneration was performed using 50 mM NaOH.

[0266] A recombinant human PD-L1 protein (Sino Biological, 10084-H08H) was flowed across the chip at 30 μL / min for 200 seconds at concentrations of 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, 1.56 nM, and 0.78 nM, followed by a dissociation step for 300 seconds. Regeneration was performed using 50 mM NaOH.

[0267] In order to measure binding affinity of the human PD-L1, a CM5 chip (GE Healthcare) on which each anti-TIGIT / anti-PD-L1 bispecific antibody was immobilized, was used. 50 mM NaOH was used as regeneration buffer, and HBS-EP (pH 7.4, GE Healthcare) was used for a running buffer and buffers for antibody dilution and antigen dilution.

[0268] Human TIGIT-Fc protein (Acro Biosystems, TIT-H5254) was subjected to a 2-fold serial dilution starting from 500 nM, and analyzed at a total of seven concentrations. In the association phase of the human TIGIT protein, the association time was set at 120 seconds and the flow rate was set at 30 μL / min, whereas in the dissociation phase, the dissociation time was set at 600 seconds and the flow rate was set at 30 μL / min. In the regeneration phase, the flow rate was set at 30 μL / min and the contact time was set at 30 seconds. The analysis program used was BIACORE Evaluation software, and fitting was performed using a 1:1 binding model. The results are shown in Table 16 below.

[0269] KD values of anti-TIGIT / anti-PD-L1 bispecific antibodies (B101041, B101042, B101643, B101644, B101645, and B101646) against human TIGITKD(M)ka(1 / Ms)kd(1 / s)Anti-TIGIT mAb-11.393 × 10-98.168 × 1041.137 × 10-4Anti-TIGIT mAb-22.745 × 10-92.040 × 1045.602 × 10-5B1010411.868 × 10-98.104 × 1041.514 × 10-4B1010421.853 × 10-97.570 × 1041.403 × 10-4B1016433.953 × 10-96.548 × 1042.589 × 10-4B1016446.504 × 10-93.893 × 1042.532 × 10-5B1016456.237 × 10-93.611 × 1042.252 × 10-4B1016465.943 × 10-93.679 × 1042.186 × 10-4

[0270] Human PD-L1-his protein (Sino Biological, 10084-H08H) was subjected to a 2-fold serial dilution starting from 50 nM, and analyzed at a total of seven concentrations. In the association phase of the human PD-L1 protein, the association time was set at 200 seconds and the flow rate was set at 30 μL / min, whereas in the dissociation phase, the dissociation time was set at 300 seconds and the flow rate was set at 30 μL / min. In the regeneration phase, the flow rate was set at 30 μL / min and the contact time was set at 30 seconds. The analysis program used was BIACORE Evaluation software, and fitting was performed using a 1:1 binding model. The results are shown in Table 17 below.

[0271] KD values of anti-TIGIT / anti-PD-L1 bispecific antibodies (B204011, B101643, B101644, B101645, and B101646) against human PD-L1KD(M)ka(1 / Ms)kd(1 / s)Anti-PD-L1 mAb1.170 × 10-92.400 × 1062.809 × 10-3B2040117.359 × 10-94.002 × 1052.945 × 10-3B1016432.757 × 10-97.713 × 1052.126 × 10-3B1016443.046 × 10-96.983 × 1052.127 × 10-3B1016455.128 × 10-94.084 × 1052.095 × 10-3B1016464.978 × 10-94.458 × 1052.219 × 10-3

[0272] 3.2. Single ELISA and dual binding ELISA assays of TIGIT / PD-L1

[0273] In order to confirm the binding affinity of anti-TIGIT / anti-PD-L1 bispecific antibodies for human TIGIT and PD-L1 proteins, single ELISA assay for each of TIGIT and PD-L1 and TIGIT / PD-L1 dual binding ELISA assay were performed.

[0274] For single ELISA assay of PD-L1, His-conjugated human PD-L1 protein (Sino biological, Cat# 10084-H08H) was dispensed at 0.05 μg / 100 μL / well into a 96-well plate (Nunc, Cat# 469949) and coated at 4℃ for 16 hours. For TIGIT single ELISA assay, His-conjugated human TIGIT protein (Novoprotein, Cat# DY998) was dispensed at 0.05 μg / 100 μL / well into a 96-well plate (Nunc, Cat# 469949) and coated at 4℃ for 16 hours. After washing the resultant 3 times with washing buffer (PBST), 200 μL of blocking buffer (PBST including 3% BSA) was added to each well and reacted at room temperature for 2 hours. After washing 3 times with washing buffer, 100 μL / well of a test material serially diluted 2-fold starting from 1 μg / mL was added thereto and reacted at room temperature for 2 hours. Then, after washing the resultant 3 times with washing buffer, 100 μL / well of HRP-tagged anti-Human IgG antibody (Jackson ImmunoResearch Inc., Cat# 109-035-098) was diluted 5000x and reacted at room temperature for 1 hour. Finally, after washing the resultant 3 times with washing buffer, 100 μL of TMB solution (Bio-rad, Cat# 172-1066) was added and the color was developed for 10 minutes. The reaction was stopped by adding 100 μL of 2 N H2SO4thereto, and the absorbance was measured at 450 nm / 595 nm. The binding affinity of each of anti-TIGIT / anti-PD-L1 bispecific antibody substances for each antigen is as shown in Figs. 3a and 3b and Tables 18 and 19.

[0275] Measurement of binding affinity of anti-TIGIT / anti-PD-L1 bispecific antibodies (B204011, B101042, B101643, B101644, B101645, and B101646) for human PD-L1PD-L1B204011B101042B101643B101644B101645B101646Top3.4412.7883.4113.4223.2753.509Bottom0.054970.061100.056570.067520.039720.09731EC50(ng / mL)50.1320.877.8846.8798.4485.878HillSlope0.82900.77501.2381.3361.151.37R squared0.99960.99850.99980.99930.99960.9989

[0276] Measurement of binding affinity of anti-TIGIT / anti-PD-L1 bispecific antibodies (B204011, B101042, B101643, B101644, B101645, and B101646) for human TIGITTIGITB204011B101042B101643B101644B101645B101646Top3.7373.3763.4283.4203.3853.419Bottom0.091820.0037350.090250.062840.077400.07077EC50(ng / mL)18.4325.5723.9324.4221.7115.61HillSlope1.0431.2351.2461.2741.4301.347R squared0.99940.99950.99870.99890.99860.9992

[0277] For TIGIT / PD-L1 dual binding ELISA, His-conjugated human PD-L1 protein (Sino Biological, Cat# 10084-H08H) was dispensed at 0.05 μg / 100 μL / well into a 96-well plate (Nunc, Cat# 469949) and coated at 4℃ for 16 hours. After washing the resultant 3 times with washing buffer (PBST), 200 μL of blocking buffer (PBST including 3% BSA) was added to each well and reacted at room temperature for 2 hours.

[0278] For B101042 and B101643, after washing the resultant 3 times with washing buffer, 100 μL / well of test materials serially diluted 3 times starting from 10 μg / mL or 1 μg / mL was added thereto and reacted at room temperature for 2 hours. For B101644, B101645, and B101646, 100 μL / well of test materials serially diluted 3 times starting from 1 μg / mL was added thereto and reacted at room temperature for 2 hours. For the bispecific antibody developed later, 100 μL / well of a test material, which was serially diluted 3-fold from 1 μg / mL to 0.037 μg / mL and then serially diluted 1.5-fold to 0.0048 μg / mL, was added thereto and reacted at room temperature for 2 hours. Biotin-tagged human TIGIT protein (Novoprotein, Cat# CJ58) was added to a 96-well plate at 0.05 μg / 100 μL / well and reacted at room temperature for 2 hours. Then, after washing the resultant 3 times with washing buffer, HRP-conjugated Streptavidin (R&D Systems, Cat# DY998) was diluted 200x and treated, and reacted at room temperature for 1 hour. Finally, after washing the resultant 3 times with washing buffer, 100 μL of TMB solution (Bio-rad, Cat# 172-1066) was added and the color was developed for 10 minutes. The reaction was stopped by adding 100 μL of 2 N H2SO4, and the absorbance was measured at 450 nm / 595 nm. As a result, the binding affinity of the anti-TIGIT / anti-PD-L1 bispecific antibody for the dual binding antigen is as shown in Fig. 4 and Table 20.

[0279] Measurement of dual binding affinity of anti-TIGIT / anti-PD-L1 bispecific antibodies (B101041, B101042, B101643, B101644, B101645, and B101646) for human TIGIT and human PD-L1B101041B101042B101643B101644B101645B101646Top3.6153.4443.2853.4743.4023.523Bottom0.15240.11860.091800.087360.095830.09715EC50(ng / mL)5.79211.1615.806.50810.749.378HillSlope0.89150.96980.87931.3361.3911.400R squared0.99900.99940.99950.99830.99870.9992

[0280] 3.3. Confirmation of cell binding potency of anti-TIGIT / anti-PD-L1 bispecific antibody to human TIGIT expressing cells

[0281] In order to evaluate the ability of anti-TIGIT / anti-PD-L1 bispecific antibodies to bind to human TIGIT-overexpressing CHO-S cells, a corresponding test was performed. CHO-S cells overexpressing human TIGIT protein were dispensed at 105cells per well, washed once with binding buffer (3% BSA in PBS), and the antibodies to be compared were diluted 5-fold to a total of 12 sections starting from 1,000 nM, and reacted at 4℃ for 1 hour. After washing twice with binding buffer, the anti-TIGIT / anti-PD-L1 bispecific antibodies bound to cells were detected by reacting with fluorescently-labeled (Alexa Fluor 488) anti-human IgG1 antibodies (Invitrogen, Cat# A-11013) at 4℃ for 30 minutes. The intensity of fluorescent labels bound to anti-TIGIT / anti-PD-L1 bispecific antibodies was measured using a BD FAC Symphony™ A3 flow cytometry analyzer, and the binding ability of anti-TIGIT / anti-PD-L1 bispecific antibodies to CHO-S cells overexpressing human TIGIT protein was evaluated based on the mean fluorescence intensity (MFI) using FlowJo software (Figs. 5 to 7 and Tables 21 to 23).

[0282] Confirmation of binding potency of anti-TIGIT / anti-PD-L1 bispecific antibodies (B101041 and B204011) to human TIGIT-expressing CHO-S cellsEC50(nM)EMAX(MFI)B1010415.1426679B2040110.1645976

[0283] Confirmation of binding potency of anti-TIGIT / anti-PD-L1 bispecific antibodies (B101042 and B101643) to human TIGIT-expressing CHO-S cellsEC50(nM)EMAX(MFI)B1010427.1576474B1016435.2495836

[0284] Confirmation of binding potency of anti-TIGIT / anti-PD-L1 bispecific antibodies (B101644, B101645, and B101646) to human TIGIT-expressing CHO-S cellsEC50(nM)EMAX(MFI)B1016440.08986667B1016450.07096718B1016460.09966684

[0285] 3.4. Confirmation of cell binding potency to human PD-L1 expressing tumor cells

[0286] In order to evaluate the ability of anti-TIGIT / anti-PD-L1 bispecific antibodies to bind to MDA-MB-231 cell line expressing human PD-L1, a corresponding test was performed. MDA-MB-231 cells, a human breast cancer cell line expressing PD-L1, were dispensed at 105cells per well, and anti-TIGIT / anti-PD-L1 bispecific antibodies were diluted 5-fold starting from the highest concentration and reacted at 4℃ for 60 minutes. Cell-bound anti-TIGIT / anti-PD-L1 bispecific antibodies were detected by reacting with fluorescently-labeled anti-human IgG1 antibodies (Invitrogen, Cat# A-11013) at 4℃ for 30 minutes. The intensity of fluorescent labeling bound to anti-TIGIT / anti-PD-L1 bispecific antibodies was measured using a BD FACSCanto™ and an A3 BD FACSymphony™ A3 flow cytometry analyzer, and the binding ability was evaluated based on the mean fluorescence intensity (MFI) of the anti-TIGIT / anti-PD-L1 bispecific antibodies bound to tumor cells using FlowJo software (Figs. 8 to 10 and Tables 24 to 26).

[0287] Confirmation of cell binding potency of anti-TIGIT / anti-PD-L1 bispecific antibodies (B101041, B101042, and B204011) to human tumor cellsEC50(nM)EMAX(MFI)B1010410.14513071B1010420.15393236B2040110.46313641

[0288] Confirmation of cell binding potency of anti-TIGIT / anti-PD-L1 bispecific antibodies (B101643 and B101644) to human tumor cellsEC50(nM)EMAX(MFI)B1016430.203611315B1016440.214012499

[0289] Confirmation of cell binding potency of anti-TIGIT / anti-PD-L1 bispecific antibodies (B101645 and B101646) to human tumor cellsEC50(nM)EMAX(MFI)B1016450.19435232B1016460.17545234

[0290] 3.5. Comparison / analysis of binding affinity of anti-TIGIT / anti-PD-L1 bispecific antibodies for human FcRn

[0291] 3.5.1. Comparison / analysis of hFcRn binding affinity through FcRn binding immunoassay

[0292] In the anti-TIGIT / anti-PD-L1 bispecific antibodies developed in the present invention, the M428L / N434S mutations known to enhance FcRn binding affinity was introduced to improve the blood half-life (B101646). In order to confirm the improvement in human FcRn binding affinity by the introduced mutations, the LummitTMFcRn Binding Immunoassay system (W1151) from Promega was utilized, and the assay was performed based on the method recommended by the manufacturer. Each substance including anti-TIGIT / anti-PD-L1 bispecific antibody was serially diluted and incubated with LargeBiT-tagged Tracer and SmallBiT-tagged hFcRn protein at room temperature for 1 hour. Then, Lumit FcRn Detection reagent was added thereto, and 3 minutes later, the reaction value (RLU, relative light unit) was measured using a microplate reader (Victor5) to compare the binding affinity for hFcRn. As a result, as shown in Fig. 11 and Table 27, the anti-TIGIT / anti-PD-L1 bispecific antibody B101646 was confirmed to have significantly superior hFcRn binding affinity (EC50) compared to anti-PD-L1 mAb used as a control material and anti-TIGIT / anti-PD-L1 bispecific antibody B101643 which does not introduce mutations that improve FcRn binding affinity (Fig. 11 and Table 27).

[0293] Comparison of binding affinity of anti-TIGIT / anti-PD-L1 bispecific antibodies (B101643 and B101646) for human FcRnB101646B101643Anti-PD-L1 mAbBottom46092147153.1Top145882148507149790HillSlope-1.018-0.8593-1.115EC50(nM)2.43575.75327.6R squared0.99970.99870.9995

[0294] 3.5.2. Analysis of hFcRn binding affinity of anti-TIGIT / anti-PD-L1 bispecific antibodies using SPR

[0295] For the affinity analysis of anti-TIGIT / anti-PD-L1 bispecific antibodies for human FcRn, recombinant human FcRn protein (ACROBiosystems, FCM-H8286) was immobilized on Series S Sensor Chip CM5 (Cytiva, BR100530) by amine coupling. For the affinity analysis, each antibody was performed under pH 6.0 and pH 7.4 buffer conditions. An anti-TIGIT / anti-PD-L1 bispecific antibody and an anti-PD-L1 antibody (Anti-PD-L1 mAb) as a control were flowed across the chip at 30 μL / min for 200 seconds at concentrations of 1000 nM, 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.63 nM, 7.81 nM, 3.91 nM, and 1.95 nM, followed by a 150-second dissociation step. The affinity analysis for human FcRn was performed by diluting anti-TIGIT / anti-PD-L1 bispecific antibodies in buffers at pH 6.0 and pH 7.4, respectively (Table 28).

[0296] Affinity level of anti-TIGIT / anti-PD-L1 bispecific antibodies (SPR, B101643 and B101646) for human FcRnKD(M)Anti-PD-L1 mAb4.738 × 10-7B1016434.738 × 10-7B1016465.812 × 10-8

[0297]

[0298] Experimental Examples

[0299] 1. Evaluation ofin vitroefficacy of anti-TIGIT / anti-PD-L1 bispecific antibodies

[0300] 1.1. Confirmation of ADCC activity of anti-TIGIT / anti-PD-L1 bispecific antibodies on human PD-L1-expressing tumor cells

[0301] The experiment was performed to evaluate the antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cells by FcγRIIIa signaling of anti-TIGIT / anti-PD-L1 bispecific antibodies. This test was performed using the FcγRIIIa ADCC Bioassay kit (Promega, Cat# G7018), and was performed according to the method recommended by the manufacturer. Non-small cell lung cancer cells H1975, colon cancer cells RKO, and breast cancer cells MDA-MB-231 of human origin, which were used as target cells, were seeded at 30,000 cells per well in a 96-well plate and cultured in a CO2incubator at 37℃ for 16 hours. After replacing the culture medium of the target cells with RPMI1640 medium including 25 μL of 4% low IgG serum, and the target cells were treated with 25 μL of diluted anti-TIGIT / anti-PD-L1 bispecific antibody and reacted for 10 minutes. FcγRIIa-H effector cells, which are effector cells, were dispensed in an amount of 25 μL each, and finally, the anti-TIGIT / anti-PD-L1 bispecific antibody variants were serially diluted 5-fold starting from the highest concentration of 6.67 nM and treated. The Bio-GloTM reagent was dispensed in an amount of 75 μL into each well and reacted at room temperature for 10 minutes. The 96-well plate was taken out to room temperature, treated with the Bio-Glo reagent, left to stand for 10 minutes thereat, and the ADCC efficacy was evaluated by measuring the reaction value (RLU, relative light unit) using a microplate reader (Victor5) capable of measuring luminescence. As a result, in H1975 and MDA-MB-231 cancer cells, the anti-TIGIT / anti-PD-L1 bispecific antibodies showed significantly superior ADCC activity (EC50and EMAX) compared to the control group treated with anti-PD-L1 mAb alone or the group treated with a combination of anti-PD-L1 mAb and anti-TIGIT mAb-2 (Figs. 12 to 15 and Tables 29 to 32).

[0302] ADCC activity of anti-TIGIT / anti-PD-L1 bispecific antibodies (B101042 and B101643) on human tumor cells H1975EC50(nM)EMAX(RLU)Anti-PD-L1 mAb0.0402655520B1010420.0405778807B1016430.05490109735

[0303] ADCC activity of anti-TIGIT / anti-PD-L1 bispecific antibodies (B101645 and B101646) on human tumor cells H1975EC50(nM)EMAX(RLU)Anti-PD-L1 mAb0.0136153440Combo(Anti-TIGIT mAb-2+ Anti-PD-L1 mAb)0.0205167247B1016450.00791078485B1016460.0129933622

[0304] ADCC activity of anti-TIGIT / anti-PD-L1 bispecific antibody (B101644) on human tumor cells MDA-MB-231EC50(nM)EMAX(RLU)Anti-PD-L1 mAb0.04728234629Combo(Anti-TIGIT mAb-2+Anti-PD-L1 mAb)0.04310218125B1016440.0089921428175

[0305] ADCC activity of anti-TIGIT / anti-PD-L1 bispecific antibodies (B101645 and B101646) on human tumor cells MDA-MB-231EC50(nM)EMAX(RLU)Anti-PD-L1 mAb0.0310209066Combo(Anti-TIGIT mAb-2+ Anti-PD-L1 mAb)0.0278216242B1016450.00891164242B1016460.0128952467

[0306] 1.2. Confirmation of ADCC activity of anti-TIGIT / anti-PD-L1 bispecific antibodies on human TIGIT-overexpressing cells

[0307] The experiment was performed to evaluate the antibody-dependent cell-mediated cytotoxicity (ADCC) of TIGIT-expressing cells by FcγRIIIa signaling of anti-TIGIT / anti-PD-L1 bispecific antibodies. This test was performed using the FcγRIIIa ADCC Bioassay kit (Promega, Cat# G7018), and was performed according to the method recommended by the manufacturer. Human TIGIT-overexpressing CHO-S cells, which were used as target cells, were maintained by subculturing in a CO2incubator at 37℃. The required number of target cells were replaced with RPMI1640 medium including 4% low IgG serum, dispensed into a 96-well plate in an amount of 25 μL each, and the target cells were treated with 25 μL each of diluted anti-TIGIT / anti-PD-L1 bispecific antibodies, and reacted for 10 minutes. FcγRIIa-H effector cells, which are effector cells, were dispensed in an amount of 25 μL each, and finally, the anti-TIGIT / anti-PD-L1 bispecific antibody variants were serially diluted 5-fold starting from the highest concentration of 6.67 nM and treated. The Bio-GloTMreagent was dispensed in an amount of 75 μL into each well and reacted at room temperature for 10 minutes. The 96-well plate was taken out to room temperature, treated with the Bio-Glo reagent, left to stand for 10 minutes thereat, and the ADCC efficacy was evaluated by measuring the reaction value (RLU, relative light unit) using a microplate reader (Victor5) capable of measuring luminescence. As a result, B101644, B101645, and B101646 showed significantly superior ADCC activity (EC50and EMAX) compared to the group treated with a combination of anti-PD-L1 mAb and anti-TIGIT mAb-2, which were used as the control materials (Fig. 16 and Table 33).

[0308] Confirmation of ADCC activity of anti-TIGIT / anti-PD-L1 bispecific antibodies (B101644, B101645, and B101646) on human TIGIT-overexpressing CHO-S cellsEC50(nM)EMAX(Fold induction of RLU)Combo(Anti-PD-L1 mAb + Anti-TIGIT mAb-2)0.44802.402B1016440.055314.54B1016450.032412.12B1016460.04998.429

[0309] 1.3. Confirmation of ADCC activity on human Treg cells

[0310] The experiment was performed to evaluate the antibody-dependent cell-mediated cytotoxicity (ADCC) of Treg cells by FcγRIIIa signaling of anti-TIGIT / anti-PD-L1 bispecific antibodies. This test was performed using the FcγRIIIa ADCC Bioassay kit (Promega, Cat# G7018), and was performed according to the method recommended by the manufacturer. Primary Treg cells with increased human TIGIT and PD-L1 expression, which were used as target cells, were maintained by subculturing in a CO2incubator at 37℃. The required number of target cells were replaced with RPMI1640 medium including 4% low IgG serum, dispensed into a 96-well plate in an amount of 25 μL each, and the target cells were treated with 25 μL each of diluted anti-TIGIT / anti-PD-L1 bispecific antibodies, and reacted for 10 minutes. FcγRIIa-H effector cells, which are effector cells, were dispensed in an amount of 25 μL each, and finally, the anti-TIGIT / anti-PD-L1 bispecific antibody variants were serially diluted 5-fold starting from the highest concentration of 4.8 nM and treated. The Bio-GloTM reagent was dispensed in an amount of 75 μL into each well and reacted at room temperature for 10 minutes. The 96-well plate was taken out to room temperature, treated with the Bio-Glo reagent, left to stand for 10 minutes thereat, and the ADCC efficacy was evaluated by measuring the reaction value (RLU, relative light unit) using a microplate reader (Victor5) capable of measuring luminescence. As a result, the anti-TIGIT / anti-PD-L1 bispecific antibodies showed significantly superior ADCC activity (EC50and EMAX) compared to the group treated with a combination of anti-PD-L1 mAb and anti-TIGIT mAb-1 (or anti-TIGIT mAb-2), which were used as control materials (Figs. 17 to 19 and Tables 34 to 36).

[0311] Confirmation of ADCC activity of anti-TIGIT / anti-PD-L1 bispecific antibody (B204011) on primary Treg cellsEC50(nM)EMAX(Fold induction of RLU)Anti-TIGIT mAb-10.09711.840Anti-PD-L1 mAbInterruptedInterruptedB2040110.00447.466Combo (1 / 2 Anti-PD-L1 mAb + 1 / 2 Anti-TIGIT mAb-1)0.33311.685

[0312] Confirmation of ADCC activity of anti-TIGIT / anti-PD-L1 bispecific antibody (B101644) on primary Treg cellsEC50(nM)EMAX(Fold induction of RLU)Anti-TIGIT mAb-20.56635.180Anti-PD-L1 mAb0.00931.804B101644 (6.67 nM)0.011215.84Combo(1 / 2 Anti-PD-L1 mAb + 1 / 2 Anti-TIGIT mAb-2)1.06104.744

[0313] Confirmation of ADCC activity of anti-TIGIT / anti-PD-L1 bispecific antibody (B101645 and B101646) on primary Treg cellsEC50(nM)EMAX(Fold induction of RLU)Anti-TIGIT mAb-20.16581.694Anti-PD-L1 mAbAmbiguousAmbiguousB1016450.024915.07B1016460.03359.401Combo(1 / 2 Anti-PD-L1 mAb + 1 / 2 Anti-TIGIT mAb-2)0.40991.779

[0314] 1.4. Confirmation of CD8 T cell-mediated cancer cell killing activity of anti-TIGIT / anti-PD-L1 bispecific antibodies on tumor cells H1975 expressing human PD-L1 and PVR

[0315] Human non-small cell lung cancer cells H1975, as target cells, were resuspended in PBS, stained with 5 μM CellTrace CFSE Dye (Invitrogen, Cat# C34554), washed with 10% FBS RPMI medium, and dispensed into each well of a 96-well plate at a concentration of 10,000 cells / 100 μL. The next day, human CD8 T cells (Stem Cell Technology, Cat# 70027) were thawed and resuspended in complete medium (RPMI medium including 10% FBS and 1× Pen / Strep). For the evaluation of the activity of anti-TIGIT / anti-PD-L1 bispecific antibodies, serial dilutions of anti-TIGIT / anti-PD-L1 bispecific antibodies were prepared using RPMI1640 medium including 10% FBS. During the culture period, culture medium including anti-CD3 (OKT3, Biolegend, Cat#317326) and rhIL-2 (Sigma, Cat# 11147528001) was prepared for activation. The medium including the target cells was removed from the 96-well plate and add 100 uL of fresh 10% FBS RPMI medium was added thereto. For activation purposes, 50 μL of the culture medium prepared and 50 μL of the pre-diluted anti-TIGIT / anti-PD-L1 bispecific antibodies were dispensed into each 96-well plate including the target cells, and the reaction was performed in a CO2incubator at 37℃ for 48 hours. After completion of the reaction, Fixable Viability Dye eFluor® 780 (Invitrogen, Cat# 65-0865-14) was diluted 1:500 to prepare a staining buffer, and target cells and NK cells were stained at 4℃ for 20 minutes. The target cells and CD8 T cells were washed and fixed with 1% PFA and subjected to FACS analysis. As a result, it was confirmed that the anti-TIGIT / anti-PD-L1 bispecific antibodies showed superior CD8 T cell-mediated cancer cell killing efficacy in H1975 carcinoma compared to the group treated with a combination of anti-PD-L1 mAb and anti-TIGIT mAb-2, which were used as control materials (Figs. 20 and 21 and Tables 37 and 38).

[0316] Confirmation of CD8 T cell-mediated cancer cell killing efficacy of anti-TIGIT / anti-PD-L1 bispecific antibodies (B101643 and B101644) on human tumor cells H1975% of Dead Tumor Cell (Average)hIgG25.9Combo(Anti-PD-L1 mAb + Anti-TIGIT mAb-2)28.23B10164337.3B10164449.53

[0317] Confirmation of CD8 T cell-mediated cancer cell killing efficacy of anti-TIGIT / anti-PD-L1 bispecific antibodies (B101645 and B101646) on human tumor cells H1975% of Dead Tumor Cells (Average)hIgG18.9Combo(Anti-PD-L1 mAb + Anti-TIGIT mAb-2)23.6B10164539.3B10164641.2

[0318] 1.5. Confirmation of NK cell-mediated cancer cell killing activity of anti-TIGIT / anti-PD-L1 bispecific antibodies on tumor cells MDA-MB-231 expressing human PD-L1 and PVR

[0319] The day before the test, human CD56+NK cells (Stem Cell Technology, Cat# 70037) were activated for 16 hours in complete medium (MEM-alpha medium including 12.5% FBS, 12.5% horse serum, 1× Pen / Strep) including 10 ng / mL of rhIL-2 (Sigma, Cat# 11147528001), 30 ng / mL of rhIL-15 (R&D systems, Cat# 247-ILB-025 / CF), and 55 nM 2-mercaptoethanol (Gibco, Cat# 21985023). On the same day, human breast cancer cells MDA-MB-231, as target cells, were resuspended in PBS, stained with 5 μM CellTrace CFSE Dye (Invitrogen, Cat# C34554), washed with 10% FBS RPMI medium, and dispensed into each well of a 96-well plate at a density of 25,000 cells / 100 μL. On the day of the test, in order to evaluate the activity of the anti-TIGIT / anti-PD-L1 bispecific antibodies, serial dilutions of anti-TIGIT / anti-PD-L1 bispecific antibodies were prepared using 10% FBS RPMI1640 medium, and NK cells separated from the plate using accutase were resuspended in the MEM-alpha medium prepared above. The culture medium of the stained breast cancer cells MDA-MB-231 was removed, and 50 μL of the anti-TIGIT / anti-PD-L1 bispecific antibodies, which had been serially diluted 5-fold to a final concentration of 24 nM, was dispensed into each 96-well plate including the target cells. Then, the NK cells prepared in MEM-alpha medium were dispensed into each 96-well plate at a density of 50,000 cells / 50 μL, and the reaction was performed in a CO2incubator at 37℃for 48 hours. After completion of the reaction, Fixable Viability Dye eFluor® 780 (Invitrogen, Cat# 65-0865-14) was diluted 1:500 to prepare a staining buffer, and the target cells and NK cells were stained at 4℃ for 20 minutes. The target cells and NK cells were washed and fixed with 1% PFA and subjected to FACS analysis. As a result, the anti-TIGIT / anti-PD-L1 bispecific antibodies were confirmed to have an excellent level of NK cell-mediated cancer cell killing efficacy, compared to the groups treated with each of anti-PD-L1 mAb and anti-TIGIT mAb-2, which were used as control materials, alone or in combination (Figs. 22 to 24 and Tables 39 to 41).

[0320] Confirmation of NK cell-mediated cancer cell killing efficacy of anti-TIGIT / anti-PD-L1 bispecific antibody (B101041) on human tumor cells MDA-MB-231EC50(nM)% of Dead Tumor CellsB1010410.063479.27Combo(Anti-PD-L1 mAb + Anti-TIGIT mAb-2)0.081868.9

[0321] Confirmation of NK cell-mediated cancer cell killing efficacy of anti-TIGIT / anti-PD-L1 bispecific antibodies (B101042 and B101644) on human tumor cells MDA-MB-231EC50(nM)% of Dead Tumor CellsCombo(Anti-PD-L1 mAb + Anti-TIGIT mAb-2)0.244841.14B1010425.56056.26B1016440.119564.41

[0322] Confirmation of NK cell-mediated cancer cell killing efficacy of anti-TIGIT / anti-PD-L1 bispecific antibodies (B101645 and B101646) on human tumor cells MDA-MB-231EC50(nM)% of Dead Tumor CellsCombo(Anti-PD-L1 mAb + Anti-TIGIT mAb-2)0.036057.87B1016450.048168.99B1016460.051766.07

[0323] 1.6. Confirmation of NK cell-mediated cell killing activity of anti-TIGIT / anti-PD-L1 bispecific antibodies on Treg cells expressing human PD-L1 and TIGIT

[0324] Human Treg cells, as target cells, were cultured and maintained for 4 to 5 days, and on the day before the test, human CD56+NK cells (Stem Cell Technology, Cat# 70037) were activated for 16 hours by adding them in complete medium (MEM-alpha medium including 12.5% FBS, 12.5% horse serum, 1× Pen / Strep) including 10 ng / mL rhIL-2 (Sigma, Cat# 11147528001), 30 ng / mL rhIL-15 (R&D systems, Cat# 247-ILB-025 / CF), and 55 nM 2mercaptoethanol (Gibco, Cat# 21985023). On the day of the test, the Treg cells maintained were resuspended in PBS, stained with 5 μM CellTrace CFSE Dye (Invitrogen, Cat# C34554), washed with 10% FBS RPMI medium, and dispensed into each well of a 96-well plate at a density of 25,000 cells / 100 μL. To evaluate the activity of anti-TIGIT / anti-PD-L1 bispecific antibodies, an anti-TIGIT / anti-PD-L1 bispecific antibody was prepared by serial dilution using 10% FBS RPMI1640 medium, and NK cells separated from the plate using accutase were resuspended in the MEM-alpha medium prepared above. The anti-TIGIT / anti-PD-L1 bispecific antibodies were serially diluted 5-fold to a final concentration of 24 nM, and 50 μL each of the same was dispensed into a 96-well plate including the target cells. The NK cells prepared in MEM-ahpha medium were dispensed into a 96-well plate at a density of 50,000 cells / 50 μL, and reacted in a CO2incubator at 37℃ for 48 hours. After completion of the reaction, Fixable Viability Dye eFluor® 780 (Invitrogen, Cat# 65-0865-14) was diluted 1:500 to prepare a staining buffer, and the target cells and the NK cells were stained at 4℃ for 20 minutes. The target cells and the NK cells were washed, fixed with 1% PFA, and then subjected to FACS analysis. As a result, the anti-TIGIT / anti-PD-L1 bispecific antibodies were confirmed to have an excellent level of NK cell-mediated Treg cell killing efficacy compared to the anti-PD-L1 mAb and anti-TIGIT mAb-2, which were used as control materials, alone or in combination (Figs. 25 and 26 and Tables 42 and 43).

[0325] Confirmation of NK cell-mediated killing efficacy of anti-TIGIT / anti-PD-L1 bispecific antibody (B101644) on human Treg cellsEC50(nM)% of Dead Tumor CellsB1016440.495890.23Combo(Anti-PD-L1 mAb + Anti-TIGIT mAb-2)0.248978.56

[0326] Confirmation of NK cell-mediated killing efficacy of anti-TIGIT / anti-PD-L1 bispecific antibodies (B101645 and B101646) on human Treg cellsEC50(nM)% of Dead Tumor CellsB1016450.093077.1B1016460.850085.3Combo(Anti-PD-L1 mAb + Anti-TIGIT mAb-2)0.129563.65

[0327] 1.7. Confirmation of NK cell-mediated cancer cell killing activity of anti-TIGIT / anti-PD-L1 bispecific antibodies on tumor cells BxPC-3, CAL-27, and SK-OV3 which express human PD-L1 and PVR

[0328] On the day before the test, human CD56+NK cells (Stem Cell Technology, Cat# 70037) were activated for 16 hours by adding them in complete medium (MEM-alpha medium including 12.5% FBS, 12.5% horse serum, 1× Pen / Strep) including 10 ng / mL rhIL-2 (Sigma, Cat# 11147528001), 30 ng / mL rhIL-15 (R&D systems, Cat# 247-ILB-025 / CF), and 55 nM 2mercaptoethanol (Gibco, Cat# 21985023). On the same day, human pancreatic cancer cells BxPC-3, head and neck cancer cells CAL-27, and ovarian cancer cells SK-OV3, which were target cells and were separated from the plates, were each resuspended in PBS, stained with 5 μM CellTrace CFSE Dye (Invitrogen, Cat# C34554), washed with 10% FBS RPMI medium, and dispensed into each well of a 96-well plate at a density of 25,000 cells / 100 μL. On the day of the test, in order to evaluate the activity of anti-TIGIT / anti-PD-L1 bispecific antibodies, serial dilutions of the anti-TIGIT / anti-PD-L1 bispecific antibodies were prepared using 10% FBS RPMI1640 medium, 10% FBS DMEM medium, and 10% FBS McCoy’s 5a medium, respectively. The NK cells separated from the plate using accutase were resuspended in the MEM-alpha medium prepared above. The culture media for each of the stained pancreatic cancer cells BxPC-3, head and neck cancer cell CAL-27, and ovarian cancer cell SK-OV3 were removed, and the anti-TIGIT / anti-PD-L1 bispecific antibodies, which were serially diluted 5-fold to a final concentration of 24 nM, were dispensed into a 96-well plate including the target cells (50 μL each). The NK cells prepared in MEM-ahpha medium above were dispensed into each well of a 96-well plate (at a density of 50,000 cells / 50 μL) and reacted in a CO2incubator at 37℃ for 24 to 48 hours (BxPC3, Cal-27: 24 hours, and SK-OV3: 48 hours). After completion of the reaction, Fixable Viability Dye eFluor® 780 (Invitrogen, Cat# 65-0865-14) was diluted 1:500 to prepare a staining buffer, and the target cells and the NK cells were stained at 4℃ for 20 minutes. Target cells (pancreatic cancer cells BxPC-3, head and neck cancer cells CAL-27, and ovarian cancer cells SK-OV3) and NK cells were washed, fixed with 1% PFA, and subjected to FACS analysis. As a result, the anti-TIGIT / anti-PD-L1 bispecific antibodies were confirmed to have an excellent level of NK cell-mediated cancer cell killing efficacy, compared to the co-treatment of anti-PD-L1 mAb and anti-TIGIT mAb-2 (Combo), or co-treatment of anti-PD-L1 mAb2 and anti-TIGIT mAb-3 (Combo 2), which were used as control materials (Figs. 27 to 29 and Tables 44 to 46).

[0329] Confirmation of NK cell-mediated cancer cell killing efficacy of anti-TIGIT / anti-PD-L1 bispecific antibody (B101646) on human tumor cell BxPC3EC50(nM)% of Dead Tumor CellsB1016460.066773.50Combo(Anti-PD-L1 mAb + Anti-TIGIT mAb-2)ND61.66Combo2(Anti-PD-L1 mAb-2 + Anti-TIGIT mAb-3)0.028466.55

[0330] Confirmation of NK cell-mediated cancer cell killing efficacy of anti-TIGIT / anti-PD-L1 bispecific antibody (B101646) on human tumor cell Cal-27EC50(nM)% of Dead Tumor CellsB1016460.174683.26Combo(Anti-PD-L1 mAb + Anti-TIGIT mAb-2)30.140063.29Combo2(Anti-PD-L1 mAb-2 + Anti-TIGIT mAb-3)0.005422.51

[0331] Confirmation of NK cell-mediated cancer cell killing efficacy of anti-TIGIT / anti-PD-L1 bispecific antibody (B101646) on human tumor cell SK-OV3EC50(nM)% of Dead Tumor CellsB1016460.093721.74Combo2(Anti-PD-L1 mAb-2 + Anti-TIGIT mAb-3)Ambiguous11.20

[0332] 2. Pharmacokinetic measurements of anti-TIGIT / anti-PD-L1 bispecific antibodies in Balb / C Jh- / -mice

[0333] 2.1. Experimental method for measuring pharmacodynamics

[0334] After completion of quarantine, 6-week-old Balb / c mice were acclimated for about 1 week. On the test day, test animals were divided into groups according to body weight, and then the test material was administered intravenously once at a dose of 3 mg / kg. In order to collect blood directly from the jugular vein outside the body, the head and right arm of each mouse were held with one hand so that the left clavicle was well exposed, and about 0.1 mL or less of blood was collected at 0.083, 1, 4, 8, 24 (D1), 48 (D2), 96 (D4), 168 (D7), and 336 (D14) hours after the administration (9 points, 9 mice / group × 6 groups (G1 to G6), divided into 3 groups of 3 mice per group, and blood was collected alternately at each time).

[0335] After blood collection, the injection site was pressed for about 10 seconds to stop the bleeding and held in place for no longer than 1 minute. The collected blood was transferred to a new EP tube and left at room temperature for 30 to 40 minutes for serum separation. Then, the blood was centrifuged at 2,500 rpm for 15 minutes and the serum was separated into one for analysis (50 μL) and one for storage. The serum was frozen until analysis, and the concentration of each test material in the serum was measured using ELISA. Pharmacodynamic evaluation of bispecific antibody materials was performed by analyzing blood concentration using single ELISA and dual binding ELISA for a single target.

[0336] The analysis method for the PD-L1 binding portion through a single target ELISA is as follows. Human PD-L1-his protein (Sino Biologics, 10084-H02H) was dispensed at a density of 0.25 μg / 100 μL / well into a 96-well plate (Nunc, Cat# 469949) and then coated at 4℃ for 16 hours. After washing the resultant 3 times with washing buffer (PBST), 200 μL of blocking buffer (PBST including 3% BSA) was added to each well and reacted at room temperature for 2 hours. After washing the resultant 3 times with washing buffer, 100 μL / well of the diluted test material was added thereto and reacted at room temperature for 2 hours. After washing the resultant 3 times with washing buffer, HRP-conjugated anti-human IgG-Fc antibody (Jackson ImmunoResearch, 109-035-098) was diluted 1:5000 and treated, and reacted at room temperature for 1 hour. Finally, after washing the resultant 3 times with washing buffer, 100 μL of TMB solution (Bio-rad, Cat# 172-1066) was added thereto and the color was developed for 10 minutes. The reaction was stopped by adding 100 μL of 2 N H2SO4thereto, and the absorbance was measured at 450 nm / 595 nm.

[0337] Additionally, the method for analyzing the TIGIT binding portion through single target ELISA is as follows. Human TIGIT-his protein (Novoprotein, CJ58) was dispensed at 0.5 μg / 100 μL / well into a 96-well plate (Nunc, Cat# 469949) and coated at 4℃ for 16 hours. After washing the resultant 3 times with washing buffer (PBST), 200 μL of blocking buffer (PBST including 3% BSA) was added to each well and reacted at room temperature for 2 hours. After washing the resultant 3 times with washing buffer, 100 μL / well of the diluted test material was added thereto and reacted at room temperature for 2 hours. Then, after washing the resultant 3 times with washing buffer, HRP-conjugated anti-human IgG-Fc antibody (Jackson ImmunoResearch, 109-035-098) was diluted 1:5000 and treated, and reacted at room temperature for 1 hour. Finally, after washing the resultant 3 times with washing buffer, 100 μL of TMB solution (Bio-rad, Cat# 172-1066) was added thereto and the color was developed for 10 minutes. The reaction was stopped by adding 100 μL of 2 N H2SO4thereto, and the absorbance was measured at 450 nm / 595 nm.

[0338] The analysis method through dual binding ELISA that simultaneously targets the binding portions for PD-L1 and TIGIT is as follows. Human PD-L1-his protein (Sino Biologics, 10084-H02H) was dispensed at a density of 0.25 μg / 100 μL / well into a 96-well plate (Nunc, Cat# 469949) and then coated at 4℃ for 16 hours. After washing the resultant 3 times with washing buffer (PBST), 200 μL of blocking buffer (PBST including 3% BSA) was added to each well and reacted at room temperature for 2 hours. After washing the resultant 3 times with washing buffer, 100 μL / well of the diluted test material was added thereto and reacted at room temperature for 2 hours. Then, after washing the resultant 3 times with washing buffer, biotinylation human TIGIT (prepared in-house) was treated thereon and reacted for 2 hours. After washing the resultant 3 times with washing buffer, the resultant was treated with Streptavidin-HRP (R&D systems, 890903) diluted 1:200 and reacted for 1 hour. Thereafter, after washing the resultant 3 times with washing buffer, 100 μL of TMB solution (Bio-rad, Cat# 172-1066) was added thereto and the color was developed for 10 minutes. The reaction was stopped by adding 100 μL of 2 N H2SO4thereto, and the absorbance was measured at 450 nm / 595 nm.

[0339] After obtaining the blood concentration profile after intravenous administration through each of single / dual ELISA analyses, non-compartment analysis (NCA) was performed using Phoenix 8.3 to calculate pharmacokinetic parameters, such as area under the blood curve (AUClast, AUCinf), half-life, clearance (CL), volume of distribution at steady state (Vdss), and elimination rate constant (Ke), and used them as evaluation items. If necessary, residual serum was used to determine whether ADA occurred. The PK parameters for each test material were calculated from the concentration of the test material in the blood at each time point, and thein vivokinetic characteristics were compared and evaluated.

[0340] 2.2. Results of measurements of pharmacodynamic activity

[0341] PK parameter analysis was performed through a non-compartmental analysis using Phoenix® WinNonlin®, version 8.2. For each animal, the following PK parameters were calculated based on the serum concentrations of each test material. The mean blood concentration-time trends when anti-TIGIT / anti-PD-L1 bispecific antibodies were intravenously administered to mice and PK parameters derived therefrom are as follows (Fig. 30 and Table 47).

[0342] PK profile of anti-TIGIT / anti-PD-L1 bispecific antibodies (B101645 and B101646) in BALB / c Jh- / - miceTest articleB101645B101646RouteIVIVTargetPD-L1TIGITDualPD-L1TIGITDualDose (mg / kg)333333AUC0-96h(μg·hr / mL)1,982,9122,003,8471,631,3652,078,2272,083,0872,043,830AUC0-336h(μg·hr / mL)2,328,8932,219,2701,881,1853,366,7702,965,4633,127,834T1 / 2(hr)43.933.137.6107.693.587.6CL (mL / hr / kg)0.02140.02250.02650.01340.01590.0151Vdss(mL / kg)63.652.572.2106.793.995.5

[0343] 3. Evaluation of animal efficacy of anti-TIGIT / anti-PD-L1 bispecific antibodies

[0344] In a hPD-L1 KI CT26 / B cell deficient syngeneic mouse model, the efficacy of an anti-TIGIT / anti-PD-L1 bispecific antibody alone at different doses and the efficacies of a bispecific antibody compared to a co-treatment of monoclonal antibodies were evaluated. Balb / c-HK-dKO mice, which were prepared not to express B cells, were subcutaneously transplanted with one million hPD-L1 KI CT26 tumor cells, which were prepared to express human PD-L1 but not mouse PD-L1. After 8 days, the mice were randomly divided into groups so that the mean tumor volume of each test group could be about 100 mm3, and then antibody treatment was initiated (n = 8 per group). Thereafter, according to the determined dose, negative control antibody (isotype Ab), co-treatment of anti-TIGIT mAb-2 and anti-PD-L1 mAb, and B101646 were administered intraperitoneally twice a week for 4 weeks, and the resulting tumor volumes were measured.

[0345] The antitumor effect of the test material compared to the negative control antibody was evaluated using a method for calculating the tumor growth inhibition rate. As shown in Figs, 31 and 32 and Table 48, B101646 showed a significantly higher tumor growth inhibition rate compared to the negative control antibody. An excellent tumor growth inhibition rate of B101646 was confirmed compared to co-treatment of anti-TIGIT mAb-2 and anti-PD-L1 mAb at the same dose, and in particular, starting from 10 mpk, the inhibition rate gradually increased compared to the co-treatment, thereby showing a significant antitumor effect (Figs. 31 and 32 and Table 48).

[0346] Evaluation results of animal efficacy of anti-TIGIT / anti-PD-L1 bispecific antibody (B101646)TGITVGroupD0D2D5D7D9D12D14D16D19D21D23D26G20.00%11.58%23.61%36.40%45.54%51.63%56.26%61.54%68.20%70.51%72.51%75.77%G30.00%11.33%28.07%34.49%44.67%54.53%62.02%68.79%76.75%81.55%83.53%86.98%G40.00%10.76%27.09%36.83%48.07%61.08%66.39%72.89%79.16%82.66%82.83%83.92%G50.00%11.90%27.42%37.39%41.49%46.94%50.50%57.69%67.47%72.64%75.58%77.32%G60.00%13.03%37.18%52.48%64.55%75.10%81.85%86.70%91.16%93.62%94.65%96.17%G70.00%12.98%30.76%49.71%62.24%75.60%82.75%88.14%94.15%96.28%97.27%98.53%

[0347] Note: The calculation of TGITVwas based on G1

Claims

1.An anti-TIGIT / anti-PD-L1 bispecific antibody, comprising:a first domain that specifically binds to PD-L1; anda second domain that specifically binds to TIGIT.2.The anti-TIGIT / anti-PD-L1 bispecific antibody of claim 1, wherein the first domain comprises:a heavy chain variable region (VH) comprising:a heavy chain CDR1 represented by an amino acid sequence of SEQ ID NO: 1;a heavy chain CDR2 represented by an amino acid sequence of SEQ ID NO: 2; anda heavy chain CDR3 represented by an amino acid sequence of SEQ ID NO: 3, anda light chain variable region (VL) comprising:a light chain CDR1 represented by an amino acid sequence of SEQ ID NO: 4;a light chain CDR2 represented by an amino acid sequence of SEQ ID NO: 5; anda light chain CDR3 represented by an amino acid sequence of SEQ ID NO: 6.3.The anti-TIGIT / anti-PD-L1 bispecific antibody of claim 1 or 2, wherein the second domain comprises:a heavy chain variable region (VH) comprising:a heavy chain CDR1 represented by an amino acid sequence of SEQ ID NO: 7 or 8;a heavy chain CDR2 represented by an amino acid sequence of SEQ ID NO: 9 or 10; anda heavy chain CDR3 represented by an amino acid sequence of SEQ ID NO: 11 or 12, anda light chain variable region (VL) comprising:a light chain CDR1 represented by an amino acid sequence of SEQ ID NO: 13 or 14;a light chain CDR2 represented by an amino acid sequence of SEQ ID NO: 15 or 16; anda light chain CDR3 represented by an amino acid sequence of SEQ ID NO: 17 or 18.4.The anti-TIGIT / anti-PD-L1 bispecific antibody of any one of claims 1 to 3, wherein the first domain comprises an Fc region of an immunoglobulin, which is the Fc region consisting of any one of the Fc region of IgG1, IgG2, IgG3, IgG4 and IgD, or a hybrid Fc containing a combination thereof.5.The anti-TIGIT / anti-PD-L1 bispecific antibody of any one of claims 1 to 4, wherein the first domain comprises an IgG1 Fc region.6.The anti-TIGIT / anti-PD-L1 bispecific antibody of any one of claims 1 to 4, wherein in the IgG1 Fc region represented by an amino acid sequence of SEQ ID NO: 29, the first domain comprises any one or more mutations selected from the group consisting of S239D, F243L, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, P396L, M428L, and N434S.7.The anti-TIGIT / anti-PD-L1 bispecific antibody of any one of claims 1 to 4, wherein in the IgG1 Fc region represented by an amino acid sequence of SEQ ID NO: 29, the first domain comprises any one or more mutations among the following (i) to (iv):(i) S239D, A330L, and I332E;(ii) F243L, R292P, Y300L, V305I, and P396L;(iii) S298A, E333A, and K334A; and(iv) M428L and N434S.8.The anti-TIGIT / anti-PD-L1 bispecific antibody of any one of claims 1 to 4, wherein the first domain comprises an IgG1 Fc region that comprises one or more mutations in the amino acid sequence of SEQ ID NO: 29, andthe mutations are selected from the group consisting of S239D, A330L, and I332E.9.The anti-TIGIT / anti-PD-L1 bispecific antibody of any one of claims 1 to 4, wherein the first domain comprises an IgG1 Fc region that comprises one or more mutations in the amino acid sequence of SEQ ID NO: 29, andthe mutations are selected from the group consisting of F243L, R292P, Y300L, V305I, P396L, M428L, and N434S.10.The anti-TIGIT / anti-PD-L1 bispecific antibody of any one of claims 1 to 4, wherein the first domain comprises an IgG1 Fc region that comprises one or more mutations in the amino acid sequence of SEQ ID NO: 29, andthe mutations are selected from the group consisting of S298A, E333A, K334A, M428L, and N434S.11.The anti-TIGIT / anti-PD-L1 bispecific antibody of any one of claims 1 to 4, wherein the first domain comprises an IgG1 Fc region represented by an amino acid sequence of SEQ ID NO: 30.12.The anti-TIGIT / anti-PD-L1 bispecific antibody of any one of claims 1 to 4, wherein the first domain comprises an IgG1 Fc region represented by an amino acid sequence of SEQ ID NO: 31.13.The anti-TIGIT / anti-PD-L1 bispecific antibody of any one of claims 1 to 4, wherein the first domain comprises an IgG1 Fc region represented by an amino acid sequence of SEQ ID NO: 32.14.The anti-TIGIT / anti-PD-L1 bispecific antibody of any one of claims 1 to 13, wherein the first domain comprises an anti-PD-L1 heavy chain variable region represented by an amino acid sequence of SEQ ID NO: 19.15.The anti-TIGIT / anti-PD-L1 bispecific antibody of any one of claims 1 to 14, wherein the first domain comprises an anti-PD-L1 light chain variable region represented by an amino acid sequence of SEQ ID NO: 20.16.The anti-TIGIT / anti-PD-L1 bispecific antibody of any one of claims 1 to 15, wherein the second domain comprises an anti-TIGIT heavy chain variable region represented by any one amino acid sequence selected from the group consisting of SEQ ID NOS: 21 to 24.17.The anti-TIGIT / anti-PD-L1 bispecific antibody of any one of claims 1 to 16, wherein the second domain comprises an anti-TIGIT light chain variable region represented by any one amino acid sequence selected from the group consisting of SEQ ID NOS: 25 to 28.18.The anti-TIGIT / anti-PD-L1 bispecific antibody of any one of claims 1 to 4,wherein the first domain comprises a heavy chain and a light chain,wherein the second domain is an scFv comprising a heavy chain variable region and a light chain variable region, andwherein a C-terminus of the heavy chain of the first domain and an N-terminus of the heavy chain variable region of the second domain are linked through a linker.19.The anti-TIGIT / anti-PD-L1 bispecific antibody of any one of claims 1 to 4,wherein the first domain comprises a heavy chain and a light chain,wherein the second domain is an scFv comprising a heavy chain variable region and a light chain variable region, andwherein a C-terminus of the heavy chain of the first domain and an N-terminus of the light chain variable region of the second domain are linked through a linker.20.The anti-TIGIT / anti-PD-L1 bispecific antibody of any one of claims 1 to 3,wherein the first domain is an scFv comprising a heavy chain variable region and a light chain variable region,wherein the second domain comprises a heavy chain and a light chain, andwherein a C-terminus of the heavy chain of the second domain and an N-terminus of the heavy chain variable region of the first domain are linked through a linker.21.An anti-TIGIT / anti-PD-L1 bispecific antibody, which is any one selected from the following:1) an anti-TIGIT / anti-PD-L1 bispecific antibody comprising the amino acid sequences of SEQ ID NOS: 33 to 36;2) an anti-TIGIT / anti-PD-L1 bispecific antibody comprising the amino acid sequences of SEQ ID NOS: 37 to 40;3) an anti-TIGIT / anti-PD-L1 bispecific antibody comprising the amino acid sequences of SEQ ID NOS: 41 to 44;4) an anti-TIGIT / anti-PD-L1 bispecific antibody comprising the amino acid sequences of SEQ ID NOS: 45 to 48;5) an anti-TIGIT / anti-PD-L1 bispecific antibody comprising the amino acid sequences of SEQ ID NOS: 49 to 52;6) an anti-TIGIT / anti-PD-L1 bispecific antibody comprising the amino acid sequences of SEQ ID NOS: 53 to 56; and7) an anti-TIGIT / anti-PD-L1 bispecific antibody comprising the amino acid sequences of SEQ ID NOS: 57 to 60.22.A pharmaceutical composition for treating cancer, comprising the anti-TIGIT / anti-PD-L1 bispecific antibody according to any one of claims 1 to 21 as an active ingredient.23.The pharmaceutical composition of claim 22, wherein the cancer is selected from the group consisting of skin cancer, liver cancer, hepatocellular carcinoma, stomach cancer, breast cancer, lung cancer, ovarian cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, colorectal cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, thyroid cancer, parathyroid cancer, kidney cancer, esophageal cancer, bile duct cancer, testicular cancer, rectal cancer, head and neck cancer, cervical spine cancer, ureteral cancer, osteosarcoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, glioma, myeloma, neuroblast-derived CNS tumor, monocytic leukemia, B-cell derived leukemia, T-cell derived leukemia, B-cell derived lymphoma, T-cell derived lymphoma, and mast cell derived tumor.24.A nucleic acid encoding an amino acid sequence of the first domain and an amino acid sequence of the second domain of the anti-TIGIT / anti-PD-L1 bispecific antibody according to any one of claims 1 to 21.25.A recombinant expression vector comprising the nucleic acid of claim 24.26.A host cell transformed with the recombinant expression vector of claim 25.27.A method for preparing an anti-TIGIT / anti-PD-L1 bispecific antibody, comprising culturing the host cell of claim 26.28.An anti-TIGIT / anti-PD-L1 bispecific binding protein, comprising:a first domain that specifically binds to PD-L1; anda second domain that specifically binds to TIGIT.29.An anti-TIGIT / anti-PD-L1 bispecific binding molecule, comprising:a first domain that specifically binds to PD-L1; anda second domain that specifically binds to TIGIT.

Images