Multispecific T cell engagers containing the LRRC15 antigen-binding domain

Abstract

The present invention relates to multispecific T cell engagers and their applications. Specifically, the present invention provides a multispecific T cell engager that comprises a first targeting domain of a leucine-rich repeat-containing protein 15 (LRRC15) molecule highly expressed on the surface of a target tumor cell, and further comprises a second targeting domain and / or a third targeting domain of a binding T cell surface CD molecule. The LRRC15-associated multispecific T cell engager of the present invention can effectively activate T cells and reduce the matrix effect of tumors, and can be used as an effective therapeutic agent for tumor therapy, particularly for tumors with high LRRC15 expression.

Description

[Technical field]

[0001] The present invention relates to the field of cancer therapy, and more specifically to multispecific T Cells Engagers comprising anti-LRRC15 antibodies or immune response fragments thereof, for use in the treatment of cancer. [Background technology]

[0002] Cancer is usually defined as a disease related to abnormal cell growth, which can invade or spread to other parts of the body. Conventional cancer treatment aims to remove the cancerous tissue and prevent its spread. Such treatment options include surgery, chemotherapy, radiation therapy, hormonal therapy, targeted therapy, and palliative care. Usually, it is treated according to the type, location, and level of cancer, as well as the health and preferences of the patient. However, these therapies are ineffective and limited, especially in cases where the cancer has metastasized. Moreover, chemotherapy and radiation therapy have a series of side effects related to cytotoxicity.

[0003] There are currently prospects for cancer treatment that involve targeted therapy using antibodies. Another promising area is developing therapeutic methods that utilize the immune system to attack and kill tumor cells.

[0004] Leucine-rich repeat containing protein 15 (LRRC15) is a member of the LRR (leucine-rich repeat containing protein) superfamily. LRRC15 is relatively highly expressed in tumor cells, including breast cancer, brain tumor, pancreatic cancer, lung cancer, melanoma, highly invasive sarcoma, breast cancer bone metastasis, etc., but is relatively low expressed in normal cells. In addition, it is also expressed at high levels in stromal fibroblasts of many solid tumors (e.g., breast, head and neck, lung, pancreas). Therefore, LRRC15 is a candidate for targeted therapies, such as antibody-drug conjugates, T cell engagers, and CAR-T cells.

[0005] CD3, CD28 and CD137 are receptors present on T cells. T cells are activated by antigen presenting cells via CD3, CD28 and CD137, which utilize MHC class I and class II activation signals, CD80 and CD86, and 4-1BBL, respectively. CD3 is part of the T cell receptor (TCR) and is the signaling component of the receptor. CD3 has three subunits, epsilon, delta and gamma. Epsilon is associated with both delta and zeta, which are responsible for signal transduction together. CD3 signal is considered to be signal 1, which is necessary for T cell activation. The coreceptors CD28 and CD137 are considered to be signal 2. Both signal 1 and signal 2 are necessary for full activation, proliferation and survival of T cells.

[0006] CD40 ligand (CD40L) is expressed primarily by activated T cells. CD40L binds to CD40 on antigen-presenting cells (APCs), thereby inducing APC activation. In turn, APCs trigger cytotoxic T lymphocytes (CTLs). CD40 and CD40L are costimulatory molecules that link innate and adaptive immunity.

[0007] Although an increasing number of therapeutic monoclonal antibodies have been approved for the treatment of various cancers, resistance to these antibodies is often observed, given the many different molecular pathways by which cancers grow and progress toward metastasis. The immune system is the main mechanism for preventing cancer, but cancer cells resist immune surveillance. Thus, there is an urgent need in the art for improved therapeutic binding antagonists or antibodies, as well as methods for treating cancer with these agents. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention provides bi / trispecific T cell engagers comprising an LRRC15 antigen binding domain. The bi / trispecific T cell engager molecules are capable of binding to LRRC15 and activating CD (cluster of differentiation) molecules (e.g., CD3, CD28, CD137 and CD40). Methods of treating diseases such as cancer using the antibodies and antibody conjugates of the invention, pharmaceutical compositions and products thereof are further provided. [Means for solving the problem]

[0009] A first aspect of the present invention provides multispecific T Cell Engagers, the multispecific T Cell Engagers comprising: a first targeting domain comprising one or more LRRC15 antigen binding domains; and / or a second targeting domain that binds to a target protein selected from the group consisting of CD3, CD28, CD40, or CD137. and an optional third targeting domain that binds to a target protein selected from the group consisting of CD3, CD28, CD40, or CD137.

[0010] In another preferred embodiment, the targeting domain is in the form of a single domain antibody (sdAb), a fragment variable (Fv) heterodimer, a single chain Fv (scFv), a Fab fragment, a TriFab, or a combination thereof.

[0011] In another preferred embodiment, the LRRC15 antigen-binding domain comprises: HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 32, 35, 38, or 41; HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 33, 36, 39, or 42, and Three heavy chain variable region CDRs, such as an HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 34, 37, 40, or 43; and, LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 44 or 49; LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 45, 47, or 50, and and three light chain variable region CDRs, such as an LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 46, 48, 51, or 52.

[0012] In another preferred embodiment, the LRRC15 antigen binding domain comprises three heavy chain variable region CDRs (HCDRs) and three light chain variable region CDRs (LCDRs) selected from the group consisting of: [Table 1]

[0013] In another preferred embodiment, the LRRC15 antigen-binding domain comprises: HCDR1 having the amino acid sequence as set forth in SEQ ID NO: 32, HCDR2 having the amino acid sequence as set forth in SEQ ID NO: 33, and Three heavy chain variable region CDRs, such as an HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 34; and, LCDR1 having the amino acid sequence as set forth in SEQ ID NO: 44, LCDR2 having the amino acid sequence as set forth in SEQ ID NO: 45, and and three light chain variable region CDRs, such as LCDR3 having the amino acid sequence as set forth in SEQ ID NO:46.

[0014] In another preferred embodiment, the LRRC15 antigen-binding domain comprises: HCDR1 having the amino acid sequence as set forth in SEQ ID NO: 35, HCDR2 having the amino acid sequence as set forth in SEQ ID NO: 36, and Three heavy chain variable region CDRs, such as an HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 37; and, LCDR1 having the amino acid sequence as set forth in SEQ ID NO: 44, LCDR2 having the amino acid sequence as set forth in SEQ ID NO: 47, and and three light chain variable region CDRs, such as LCDR3 having the amino acid sequence as set forth in SEQ ID NO:48.

[0015] In another preferred embodiment, the LRRC15 antigen-binding domain comprises a heavy chain variable region having at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO:1, 2, 3, or 4, and a light chain variable region having at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO:5, 6, 7, or 8.

[0016] In another preferred embodiment, the LRRC15 antigen binding domain comprises a heavy chain variable region as set forth in SEQ ID NO:1, and a light chain variable region as set forth in SEQ ID NO:5.

[0017] In another preferred embodiment, the LRRC15 antigen binding domain comprises a heavy chain variable region as set forth in SEQ ID NO:2, and a light chain variable region as set forth in SEQ ID NO:6.

[0018] In another preferred embodiment, the LRRC15 antigen binding domain comprises a heavy chain variable region as set forth in SEQ ID NO:3, and a light chain variable region as set forth in SEQ ID NO:7.

[0019] In another preferred embodiment, the LRRC15 antigen binding domain comprises a heavy chain variable region as set forth in SEQ ID NO:4 and a light chain variable region as set forth in SEQ ID NO:8.

[0020] In another preferred embodiment, the LRRC15 antigen binding domain comprises a heavy chain variable region as set forth in SEQ ID NO:53 and a light chain variable region as set forth in SEQ ID NO:54.

[0021] In another preferred embodiment, the LRRC15 antigen-binding domain is selected from the group consisting of an scFv, a Fab, or a combination thereof.

[0022] In another preferred embodiment, the LRRC15 antigen-binding domain is an scFv.

[0023] In another preferred embodiment, the LRRC15 antigen binding domain is a Fab, which comprises a heavy chain variable region as set forth in SEQ ID NO: 1, 2, 3, 4 or 53, and a light chain variable region as set forth in SEQ ID NO: 5, 6, 7, 8 or 54, and It comprises a heavy chain constant region CH1 as shown in SEQ ID NO: 26 or 28, and a light chain constant region CL as shown in SEQ ID NO: 30 or 31.

[0024] In another preferred embodiment, the second targeting domain is selected from the group consisting of a CD3 antigen-binding domain, a CD28 antigen-binding domain, a CD40 antigen-binding domain, a CD137 antigen-binding domain, a CD40L sequence, or a CD137L sequence.

[0025] In another preferred embodiment, the third targeting domain is selected from the group consisting of a CD3 antigen-binding domain, a CD28 antigen-binding domain, a CD40 antigen-binding domain, a CD137 antigen-binding domain, a CD40L sequence, or a CD137L sequence.

[0026] In another preferred embodiment, the multispecific T cell engager further comprises an Fc fragment.

[0027] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4.

[0028] In another preferred embodiment, the Fc fragment is an IgG1-derived Fc fragment having the amino acid sequence as shown in SEQ ID NO:27.

[0029] In another preferred embodiment, the Fc fragment derived from IgG1 is N297A, L234F / L235E / P331S, Y349C / K370E / K409D / K439E, S354C / D356K / E357K / D399K, S354C / T366W, Y349C / T366S / L368A / Y407V, L234F / L235E / P331S / Y349C / K370E / K409D / K439E, L234F / L235E / P331S / S354C / D356K / E357K / D399K, L234F / L235E / P331S / S354C / T366W, L234F / L235E / P331S / Y349C / T366S / L368A / Y407V, N297A / Y349C / K370E / K409D / K439E, N297A / S354C / D356K / E357K / D399K, N297A / S354C / T366W, N297A / Y349C / T366S / L368A / Y407V, and / or Has a mutation selected from the group consisting of S267E, S267E / G236D, S267E / S239D, S267E / L328F.

[0030] In another preferred example, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NOs:57 to 60.

[0031] In another preferred embodiment, the Fc fragment is an Fc fragment derived from IgG4 having the amino acid sequence as shown in SEQ ID NO:29.

[0032] In another preferred embodiment, the Fc fragment derived from IgG4 is Y349C / K370E / R409D / K439E, S354C / E356K / E357K / D399K, S354C / T366W, Y349C / T366S / L368A / Y407V, and / or S228P / S267E, S228P / S267E / G236D, S228P / S267E / S239D, Has a mutation selected from the group consisting of S228P / S267E / L328F.

[0033] In another preferred embodiment, the multispecific T cell engager is a bi / trispecific T cell engager.

[0034] In another preferred embodiment, the multispecific T cell engager is a bispecific T cell engager.

[0035] In another preferred embodiment, the bispecific T cell engager comprises: The first targeting domain comprises one or more LRRC15 antigen binding domains, and a second targeting domain that is a CD3 antigen binding domain.

[0036] In another preferred embodiment, the bispecific T cell engager comprises: The first targeting domain comprises one or more LRRC15 antigen binding domains, and a second targeting domain that is a CD40 antigen binding domain.

[0037] In another preferred embodiment, the bispecific T cell engager comprises: The antibody comprises a first targeting domain that comprises one or more LRRC15 antigen-binding domains, and a second targeting domain that is a CD137 antigen-binding domain.

[0038] In another preferred embodiment, the bispecific T cell engager comprises: The antibody comprises a first targeting domain that comprises one or more LRRC15 antigen binding domains, and a second targeting domain that is one or more CD40L sequences.

[0039] In another preferred embodiment, the bispecific T cell engager comprises: The antibody comprises a first targeting domain that comprises one or more LRRC15 antigen binding domains, and a second targeting domain that is one or more CD137L sequences.

[0040] In another preferred embodiment, the multispecific T cell engager is a trispecific T cell engager.

[0041] In another preferred embodiment, the trispecific T cell engager comprises: a first targeting domain comprising one or more LRRC15 antigen binding domains; a second targeting domain that is a CD3 antigen binding domain; and and a third targeting domain which is a CD28 antigen binding domain.

[0042] In another preferred embodiment, the multispecific T cell engager is a trispecific T cell engager.

[0043] In another preferred embodiment, the trispecific T cell engager comprises: a first targeting domain comprising one or more LRRC15 antigen binding domains; a second targeting domain that is a CD3 antigen binding domain; and and a third targeting domain which is a CD40 antigen binding domain.

[0044] In another preferred embodiment, the multispecific T cell engager is a trispecific T cell engager.

[0045] In another preferred embodiment, the trispecific T cell engager comprises: a first targeting domain comprising one or more LRRC15 antigen binding domains; a second targeting domain that is a CD3 antigen binding domain; and and a third targeting domain which is a CD137 antigen binding domain.

[0046] In another preferred embodiment, the multispecific T cell engager is a trispecific T cell engager.

[0047] In another preferred embodiment, the trispecific T cell engager comprises: a first targeting domain comprising one or more LRRC15 antigen binding domains; a second targeting domain that is a CD3 antigen binding domain; and and a third targeting domain which is the CD40L sequence.

[0048] In another preferred embodiment, the trispecific T cell engager comprises: a first targeting domain comprising one or more LRRC15 antigen binding domains; a second targeting domain that is a CD3 antigen binding domain; and and a third targeting domain which is the CD137L sequence.

[0049] In another preferred embodiment, the CD3 antigen-binding domain comprises a heavy chain variable region having at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 11, 9, 10, or 12, and a light chain variable region having at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 13.

[0050] In another preferred embodiment, the CD3 antigen-binding domain is selected from the group consisting of scFv, Fab, or a combination thereof.

[0051] In another preferred embodiment, the CD28 antigen binding domain comprises a heavy chain variable region having at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO:14, and a light chain variable region having at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO:15.

[0052] In another preferred embodiment, the CD28 antigen-binding domain is selected from the group consisting of scFv, Fab, or a combination thereof.

[0053] In another preferred embodiment, the CD40 antigen-binding domain comprises a heavy chain variable region having at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO:16, and a light chain variable region having at least 80% sequence identity to the amino acid sequence as set forth in SEQ ID NO:17.

[0054] In another preferred embodiment, the CD40 antigen-binding domain comprises a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO:55, and a light chain variable region having at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO:56.

[0055] In another preferred embodiment, the CD40 antigen-binding domain is selected from the group consisting of scFv, Fab, or a combination thereof.

[0056] In another preferred embodiment, the CD137 antigen-binding domain comprises a heavy chain variable region having at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 18 or 20, and a light chain variable region having at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 19 or 21.

[0057] In another preferred embodiment, the CD137 antigen-binding domain is selected from the group consisting of scFv, Fab, or a combination thereof.

[0058] In another preferred embodiment, the CD40L sequence is the RBD receptor binding domain sequence of CD40L having the amino acid sequence as set forth in SEQ ID NO:23.

[0059] In another preferred embodiment, the CD137L sequence is the RBD receptor binding domain sequence of CD40L having the amino acid sequence as shown in SEQ ID NO:25.

[0060] In another preferred embodiment, the multispecific T cell engager has the structure as shown in formula I: [ka] In the formula, each "-" is independently a peptide bond or a linking peptide; [ka] is the connecting bond between the peptide chains.

[0061] scFv1 is a first targeting domain, said scFv1 being an anti-LRRC15 scFv; scFv2 is a second targeting domain, said scFv2 being an anti-CD3 scFv; Fc1 and Fc2 are each independently an Fc fragment.

[0062] In another preferred embodiment, the anti-LRRC15 scFv comprises a heavy chain variable region as set forth in SEQ ID NO: 1, 2, 3, 4 or 53, and a light chain variable region as set forth in SEQ ID NO: 5, 6, 7, 8 or 54.

[0063] In another preferred embodiment, the LRRC15 scFv comprises a heavy chain variable region as set forth in SEQ ID NO:53, and a light chain variable region as set forth in SEQ ID NO:54.

[0064] In another preferred embodiment, the anti-CD3 scFv comprises a heavy chain variable region as set forth in SEQ ID NO:11, 9, 10, or 12, and a light chain variable region as set forth in SEQ ID NO:13.

[0065] In another preferred embodiment, the anti-CD3 scFv comprises a heavy chain variable region as shown in SEQ ID NO:11 and a light chain variable region as shown in SEQ ID NO:13.

[0066] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4.

[0067] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0068] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in SEQ ID NO:57 or 58.

[0069] In another preferred example, the Fc1 has an amino acid sequence as shown in SEQ ID NO:57, and the Fc2 has an amino acid sequence as shown in SEQ ID NO:58.

[0070] In another preferred example, the amino acid sequence of the "scFv1-scFv2-Fc1" is as shown in SEQ ID NO:64, and the amino acid sequence of the "Fc2" is as shown in SEQ ID NO:58.

[0071] In another preferred embodiment, the multispecific T cell engager has a structure as shown in formula II below (FIG. 1A-c): [ka] In the formula, each "-" is independently a peptide bond or a linking peptide; [ka] is the connecting bond between the peptide chains.

[0072] scFv1 and Fab1 are first targeting domains, said scFv1 being an anti-LRRC15 scFv and said Fab1 being an anti-LRRC15 Fab; scFv2 is a second targeting domain, said scFv2 being an anti-CD3 scFv; Fc1 and Fc2 are each independently an Fc fragment.

[0073] In another preferred embodiment, the anti-LRRC15 scFv comprises a heavy chain variable region as set forth in SEQ ID NO: 1, 2, 3, 4 or 53, and a light chain variable region as set forth in SEQ ID NO: 5, 6, 7, 8 or 54.

[0074] In another preferred embodiment, the anti-LRRC15 Fab comprises a heavy chain variable region as set forth in SEQ ID NO: 1, 2, 3, 4, or 53, and a light chain variable region as set forth in SEQ ID NO: 5, 6, 7, 8, or 54.

[0075] In another preferred embodiment, the LRRC15 scFv comprises a heavy chain variable region as set forth in SEQ ID NO:53, and a light chain variable region as set forth in SEQ ID NO:54.

[0076] In another preferred embodiment, the anti-LRRC15 Fab comprises a heavy chain variable region as shown in SEQ ID NO:2 and a light chain variable region as shown in SEQ ID NO:6.

[0077] In another preferred embodiment, the anti-CD3 scFv comprises a heavy chain variable region as set forth in SEQ ID NO:11, 9, 10, or 12, and a light chain variable region as set forth in SEQ ID NO:13.

[0078] In another preferred embodiment, the anti-CD3 scFv comprises a heavy chain variable region as shown in SEQ ID NO:11 and a light chain variable region as shown in SEQ ID NO:13.

[0079] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4.

[0080] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0081] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in SEQ ID NO:57 or 58.

[0082] In another preferred example, the Fc1 has an amino acid sequence as shown in SEQ ID NO:57, and the Fc2 has an amino acid sequence as shown in SEQ ID NO:58.

[0083] In another preferred embodiment, the amino acid sequence of the "scFv1-scFv2-Fc1" is as shown in SEQ ID NO:64.

[0084] In another preferred example, the amino acid sequence of "HC1 (heavy chain)-Fc2" in the "Fab1-Fc2" is as shown in SEQ ID NO: 81, and the amino acid sequence of "LC1 (light chain)" is as shown in SEQ ID NO: 84.

[0085] In another preferred embodiment, the multispecific T cell engager has the structure as shown in formula III below (FIG. 2A-c): [ka] In the formula, each "-" is independently a peptide bond or a linking peptide; [ka] is the connecting bond between the peptide chains.

[0086] Fab1 is a first targeting domain, said Fab1 being an anti-LRRC15 Fab; scFv3 is a second targeting domain, said scFv3 being an anti-CD40 scFv; Fc3 is an Fc fragment.

[0087] In another preferred embodiment, [ka] is a disulfide bond.

[0088] In another preferred embodiment, the anti-LRRC15 Fab comprises a heavy chain variable region as set forth in SEQ ID NO: 1, 2, 3, 4, or 53, and a light chain variable region as set forth in SEQ ID NO: 5, 6, 7, 8, or 54.

[0089] In another preferred embodiment, the LRRC15 Fab comprises a heavy chain variable region as set forth in SEQ ID NO:1, and a light chain variable region as set forth in SEQ ID NO:5.

[0090] In another preferred embodiment, the anti-CD40 scFv comprises a heavy chain variable region as set forth in SEQ ID NO:16 and a light chain variable region as set forth in SEQ ID NO:17.

[0091] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4.

[0092] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0093] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in SEQ ID NO:59 or 60.

[0094] In another preferred example, the amino acid sequence of "HC1 (heavy chain)-Fc3-scFv3" in the "Fab1-Fc3-scFv3" is as shown in SEQ ID NO: 70, and the amino acid sequence of "LC1 (light chain)" is as shown in SEQ ID NO: 83.

[0095] In another preferred embodiment, the multispecific T cell engager has the structure as shown in formula IV (FIG. 3A-c): [ka] In the formula, each "-" is independently a peptide bond or a linking peptide; [ka] is the connecting bond between the peptide chains.

[0096] Fab1 is a first targeting domain, said Fab1 being an anti-LRRC15 Fab; scFv4 is a second targeting domain, said scFv4 being an anti-CD137 scFv; Fc3 is an Fc fragment.

[0097] In another preferred embodiment, [ka] is a disulfide bond.

[0098] In another preferred embodiment, the anti-LRRC15 Fab comprises a heavy chain variable region as set forth in SEQ ID NO: 1, 2, 3, 4, or 53, and a light chain variable region as set forth in SEQ ID NO: 5, 6, 7, 8, or 54.

[0099] In another preferred embodiment, the LRRC15 Fab comprises a heavy chain variable region as set forth in SEQ ID NO:1, and a light chain variable region as set forth in SEQ ID NO:5.

[0100] In another preferred embodiment, the anti-CD137 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 18 or 20, and a light chain variable region as shown in SEQ ID NO: 19 or 21.

[0101] In another preferred embodiment, the anti-CD137 scFv comprises a heavy chain variable region as set forth in SEQ ID NO:20, and a light chain variable region as set forth in SEQ ID NO:21.

[0102] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4.

[0103] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0104] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in SEQ ID NO:59 or 60.

[0105] In another preferred example, the amino acid sequence of "HC1 (heavy chain)-Fc3-scFv4" in the "Fab1-Fc3-scFv4" is as shown in SEQ ID NO: 77, and the amino acid sequence of "LC1 (light chain)" is as shown in SEQ ID NO: 83.

[0106] A second aspect of the invention provides an anti-LRRC15 antibody, or antigen-binding fragment thereof, comprising: HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 32, 35, 38, or 41; HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 33, 36, 39, or 42, and Three heavy chain variable region CDRs, such as an HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 34, 37, 40, or 43; and, LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 44 or 49; LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 45, 47, or 50, and and three light chain variable region CDRs, such as an LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 46, 48, 51, or 52.

[0107] In another preferred embodiment, the anti-LRRC15 antibody or antigen-binding fragment thereof comprises three heavy chain variable region CDRs (HCDRs) and three light chain variable region CDRs (LCDRs) selected from the group consisting of: [Table 2]

[0108] In another preferred embodiment, the anti-LRRC15 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO:1, 2, 3, or 4, and a light chain variable region having at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO:5, 6, 7, or 8.

[0109] In another preferred embodiment, the anti-LRRC15 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID NO:1, and a light chain variable region as set forth in SEQ ID NO:5.

[0110] In another preferred embodiment, the anti-LRRC15 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID NO:2, and a light chain variable region as set forth in SEQ ID NO:6.

[0111] In another preferred embodiment, the anti-LRRC15 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID NO:3, and a light chain variable region as set forth in SEQ ID NO:7.

[0112] In another preferred embodiment, the anti-LRRC15 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID NO:4, and a light chain variable region as set forth in SEQ ID NO:8.

[0113] In another preferred embodiment, the anti-LRRC15 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID NO:53, and a light chain variable region as set forth in SEQ ID NO:54.

[0114] In another preferred embodiment, the antibody or antigen-binding fragment thereof is a human antibody, a murine antibody, a humanized antibody or a chimeric antibody.

[0115] In another preferred embodiment, the antibody or antigen-binding fragment thereof is a human antibody.

[0116] A third aspect of the invention provides a polynucleotide, said polynucleotide encoding a multispecific T cell engager according to the first aspect of the invention or an antibody according to the second aspect of the invention.

[0117] A fourth aspect of the invention provides an expression vector, said expression vector comprising a polynucleotide according to the third aspect of the invention.

[0118] In another preferred embodiment, the expression vector includes a prokaryotic expression vector and a eukaryotic expression vector.

[0119] A fifth aspect of the invention provides a host cell, said host cell comprising an expression vector according to the fourth aspect of the invention or having integrated in its genome a polynucleotide according to the third aspect of the invention.

[0120] In another preferred embodiment, the host cell comprises a prokaryotic or eukaryotic cell.

[0121] In another preferred embodiment, the host cell is selected from the group consisting of E. coli, yeast cells, HEK 293T cells, and CHO cells.

[0122] A sixth aspect of the present invention provides the use of a multispecific T cell engager according to the first aspect of the present invention for use in the preparation of a medicament for treating cancer / tumours.

[0123] In another preferred embodiment, the cancer / tumor is a LRRC15 highly expressed cancer / tumor.

[0124] In another preferred embodiment, the cancer / tumor comprises a solid tumor and a hematological tumor.

[0125] In another preferred embodiment, the cancer / tumor is a solid tumor.

[0126] In another preferred embodiment, the cancer / tumor is selected from the group consisting of breast cancer, head and neck tumor, lung cancer, pancreatic cancer, ovarian cancer, melanoma, glioblastoma, sarcoma, bone cancer, or a combination thereof.

[0127] A seventh aspect of the present invention provides an immunoconjugate, said conjugate comprising: (i) a multispecific T cell engager according to the first aspect of the invention, or an antibody or antigen-binding fragment thereof according to the second aspect of the invention, and (ii) a coupling moiety selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0128] In another preferred embodiment, the conjugate is selected from a fluorescent or luminescent marker, a radioactive marker, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product, a radionuclide, a biotoxin, a cytokine (e.g., IL-2, etc.), an antibody, an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle / nanorod, a viral particle, a liposome, a nanomagnetic particle, a prodrug activating enzyme (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), a chemotherapeutic agent (e.g., cisplatin) or a nanoparticle of any form, etc.

[0129] An eighth aspect of the present invention provides a pharmaceutical composition comprising (a) a multispecific T cell engager according to the first aspect of the invention, or an antibody or antigen-binding fragment thereof according to the second aspect of the invention, and (b) a pharma- ceutical acceptable carrier.

[0130] In another preferred embodiment, the pharmaceutical composition is in an injectable form.

[0131] A ninth aspect of the invention provides a method of treating cancer / tumours comprising administering to a subject in need thereof a multispecific T cell engager according to the first aspect of the invention.

[0132] In another preferred embodiment, the subject in need thereof is a human or non-human mammal.

[0133] In another preferred embodiment, the cancer / tumor is a LRRC15 highly expressed cancer / tumor.

[0134] In another preferred embodiment, the cancer / tumor comprises a solid tumor and a hematological tumor.

[0135] In another preferred embodiment, the cancer / tumor is a solid tumor.

[0136] In another preferred embodiment, the cancer / tumor is selected from the group consisting of breast cancer, head and neck tumor, lung cancer, pancreatic cancer, ovarian cancer, melanoma, glioblastoma, sarcoma, bone cancer, or a combination thereof. Effect of the Invention

[0137] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (e.g., in the Examples) can be combined with each other to form new or preferred technical solutions, which will not be repeated here due to space limitations. [Brief description of the drawings]

[0138] [Figure 1A-1D]FIG. 1A shows three types of dual antibody structure configurations (ac) composed of LRRC15 antigen-binding domain and CD3 antigen-binding domain, and two types of triple antibody structure configurations (de) composed of LRRC15 antigen-binding domain, CD3 antigen-binding domain, and CD28 antigen-binding domain, where a shows an antibody structure representing a combination of anti-CD3 scFv and anti-LRRC15 Fab (CH1-VH+CK-VL), where IgG1 Fc forms a heterodimer through charge pairing or knob and hole mutation, b shows an antibody structure (scFv-scFv-Fc×Fc) formed by anti-LRRC15 scFv and anti-CD3 scFv, where IgG1 Fc forms a heterodimer through charge pairing or knob and hole mutation, c shows an antibody structure (scFv-scFv-Fc×Fc) formed by anti-LRRC15 scFv, anti-CD3 scFv, and anti-LRRC15 1A shows the molecular structure formed by Fab (scFv-scFv-Fc×Fab-Fc), where IgG1 Fc forms a heterodimer through charge pairing or knob and hole mutation, d shows the molecular structure formed by anti-LRRC15 scFv, anti-CD3 scFv and anti-CD28 Fab (scFv-scFv-Fc×Fab-Fc), where IgG1 Fc forms a heterodimer through charge pairing or knob and hole mutation, and e shows the molecular structure formed by anti-CD28 scFv, anti-CD3 scFv and anti-LRRC15 Fab (scFv-scFv-Fc×Fab-Fc), where IgG1 Fc forms a heterodimer through charge pairing or knob and hole mutation. FIG. 1B shows an exemplary bispecific antibody structure of b in FIG. 1A, designated 153-L06C. Figure 1C shows an exemplary bispecific antibody structure of c in Figure 1A, designated 315-L06C08. Figure 1D shows an exemplary bispecific antibody structure of a in Figure 1A, designated 315-L06. [Figure 1E] FIG. 1E shows an exemplary trispecific antibody of FIG. 1A e, designated 28315-L06. [Figure 2A-2B] Figure 2A shows four types of bispecific antibody structure configurations composed of LRRC15 antigen binding domain and CD40 antigen binding domain, where a shows the molecular structure formed by anti-LRRC15 antibody and CD40L (IgG-RBD x IgG-RBD), b shows the molecular structure formed by anti-LRRC15 antibody and CD40L (IgG-trimeric RBD x Fab-Fc), c shows the molecular structure formed by anti-LRRC15 antibody and anti-CD40 scFv (IgG-scFv x IgG-scFv), and d shows the molecular structure formed by anti-CD40 antibody and anti-LRRC15 scFv (IgG-scFv x IgG-scFv). Figure 2B shows an exemplary bispecific antibody structure of c in Figure 2A, named 1540-L06. [Fig. 2C-2E] Figure 2C shows an exemplary bispecific antibody structure of a in Figure 2A, designated 1540L-L06. Figure 2D shows an exemplary bispecific antibody structure of b in Figure 2A, designated 1540TL-L06. Figure 2E shows an exemplary bispecific antibody structure of d in Figure 2A, designated 4015-L06. [Figure 3A-1] The figures show four types of dual antibody structural forms composed of an LRRC15 antigen-binding domain and a CD137 antigen-binding domain, where a shows the molecular structure formed by an anti-LRRC15 antibody and CD137L (IgG-RBD x IgG-RBD), b shows the molecular structure formed by an anti-LRRC15 antibody and CD137L (IgG-trimeric RBD x Fab-Fc), c shows the molecular structure formed by an anti-LRRC15 antibody and anti-CD137 scFv (IgG-scFv x IgG-scFv), and d shows the molecular structure formed by an anti-CD137 antibody and anti-LRRC15 scFv (IgG-scFv x IgG-scFv). [Figure 3A-2]The figures show four types of dual antibody structural forms composed of an LRRC15 antigen-binding domain and a CD137 antigen-binding domain, where a shows the molecular structure formed by an anti-LRRC15 antibody and CD137L (IgG-RBD x IgG-RBD), b shows the molecular structure formed by an anti-LRRC15 antibody and CD137L (IgG-trimeric RBD x Fab-Fc), c shows the molecular structure formed by an anti-LRRC15 antibody and anti-CD137 scFv (IgG-scFv x IgG-scFv), and d shows the molecular structure formed by an anti-CD137 antibody and anti-LRRC15 scFv (IgG-scFv x IgG-scFv). [Figure 3B-3D] Figure 3B shows an exemplary bispecific antibody structure of c in Figure 3A, designated 15BB-L06. Figure 3C shows an exemplary bispecific antibody structure of a in Figure 3A, designated 15BBL-L06. Figure 3D shows an exemplary bispecific antibody structure of b in Figure 3A, designated 15BBTL-L06. [Figure 3E] FIG. 3E shows an exemplary bispecific antibody structure of d in FIG. 3A, designated BB15-L06. [Figure 4A-4B] Figure 4A shows FACS detection of anti-LRRC15 antibody binding to a cell line expressing human LRRC15, and Figure 4B shows FACS detection of anti-LRRC15 antibody binding to a cell line expressing mouse LRRC15. [Fig. 4C-4D] Figure 4C shows FACS detection of anti-LRRC15 antibodies binding to cell lines expressing cynomolgus monkey LRRC15. Figure 4D shows FACS detection between LRRC15 antibodies competitively binding to cell lines expressing human LRRC15. [Figure 5A] 1 shows FACS detection of binding of bispecific antibodies targeting LRRC15 and CD3 (153-L06C and 315-L06C08) to cell lines expressing human LRRC15. [Figure 5B] 1 shows FACS detection of binding of bispecific antibodies targeting LRRC15 and CD3 (153-L06C and 315-L06C08) to a cell line expressing cynomolgus monkey LRRC15. [Figure 6] 1 shows the change curves of the percentage of T cells expressing CD25 according to different antibody concentrations after stimulation of PBMCs with bispecific antibodies (153-L06C, 315-L06C08). [Figure 7] FIG. 1 shows the change curves of IFN-γ release from PBMCs as a function of different antibody concentrations after stimulation of the PBMCs with bispecific antibodies (153-L06C, 315-L06C08). [Figure 8] 1 shows the change curves of the ratio of proliferating T cells according to different antibody concentrations after stimulation of T cells with bispecific antibodies (153-L06C, 315-L06C08). [Figure 9] 1 shows the change curves of the target cell killing ratio according to different antibody concentrations after stimulation of PBMCs with bispecific antibodies (153-L06C, 315-L06C08). [Figure 10-1] Shown are the results of bispecific antibodies (153-L06C, 315-L06C08) that stimulate PBMCs to release cytokines. [Figure 10-2] Shown are the results of bispecific antibodies (153-L06C, 315-L06C08) that stimulate PBMCs to release cytokines. [Figure 11] 1 shows the binding curves of a bispecific antibody targeting LRRC15 and CD137 (15BB-L06) with 4-1BB protein, and the ELISA detection of the binding curves of a bispecific antibody targeting LRRC15 and CD40 (1540-L06) with CD40 protein. [Figure 12] The results of stimulating IL-2 release from PBMCs by a bispecific antibody (15BB-L06) are shown. [Figure 13] FIG. 1 shows the results of stimulating IL-12p40 release from DCs by a bispecific antibody (1540-L06). [Figure 14] FIG. 1 shows the results of stimulation of cytokine (TNF-α and IL-6) release from PBMCs by bispecific antibodies (15BB-L06; 1540-L06). [Figure 15A] Changes in tumor volume in NCI-H1650 PBMC mice during the treatment period are shown. [Figure 15B] The figure shows a comparison of tumor weights between groups in NCI-H1650 PBMC mice after the end of administration. [Fig. 15C-15D] Figure 15C shows the antitumor effect in MC38 model mice after administration of 153-L06C or 15BB-L06. Figure 15D shows the antitumor effect in MC38 model mice after administration of 1540-L06. [Figure 16] A comparison of the killing effects of 153-L06 and 315-L06 is shown. [Figure 17] 1 shows a comparison of the killing effects between 153-L06C, 315-L06C08, 315-L08C06 and 28315-L06. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0139] As a result of extensive and detailed research, the present inventors have unexpectedly developed for the first time a multispecific T cell engager comprising an LRRC15 antigen-binding domain. The multispecific T cell engager comprises a first targeting domain that targets highly expressed leucine-rich repeat-containing protein 15 (LRRC15) molecule on the surface of tumor cells, the first targeting domain being an LRRC15 antibody or an antigen-binding fragment thereof, and further comprises a second targeting domain and / or a third targeting domain that binds to a CD4 molecule on the surface of T cells. The multispecific T cell engager of the present invention can simultaneously bind to tumor cells and T cells, and can be used as an effective therapeutic agent for tumor therapy, particularly for tumors with high LRRC15 expression. Based on this, the present invention was completed.

[0140] As used herein, the term "leucine-rich repeat-containing protein 15" or "LRRC15" refers to the protein encoded by the LRRC15 gene in humans. The leucine-rich repeat-containing protein (LRR) domain is evolutionarily conserved in proteins related to innate immunity. LRRC15 is expressed directly on matrix stromal fibroblasts in many solid tumors (e.g., breast, head and neck, lung, pancreas) and on a subset of cancer cells of mesenchymal origin (e.g., sarcoma, melanoma, glioblastoma), but is expressed poorly or not at all in most normal tissues.

[0141] As used herein, the terms "CD", "cluster of differentiation" or "cluster of differentiation molecules" refer to cell surface markers that can be used to identify and characterize white blood cells, such as CD3, CD28 and CD40. CD3 is the signaling component of the T cell receptor (TCR) complex. Because CD3 is required for T cell activation, drugs that target it, usually monoclonal antibodies, are being investigated as immune stimulants for cancer treatment.

[0142] CD28 is a major costimulatory molecule required to generate T cell-mediated immune responses. After interacting with its ligands CD80 and CD86, CD28 transmits activation signals, triggering the expression of anti-apoptotic proteins and enhancing the synthesis of various cytokines, including IL-2. CD28 costimulatory receptors are present on all T cells. Compared with antibodies against other CD28 family members CTLA-4, PD-1 or their ligands, agonistic antibodies against CD28 are associated with clinically severe adverse events, the latter acting as checkpoint inhibitors to overcome tumor immune tolerance and can be used in cancer immunotherapy.

[0143] CD40 is a cell surface receptor belonging to the tumor necrosis factor-R (TNF-R) family and is constitutively expressed on APCs such as B cells and dendritic cells. CD40L (CD154) is a type II transmembrane glycoprotein that is inducible and restricted to hematopoietic cells such as platelets, granulocytes, and activated T / B / NK cells. CD40L binds to CD40 and activates APCs, resulting in upregulation of CD80, CD86, and other costimulatory molecules, stimulating CD8+ antigen-specific T cell responses.

[0144] The term "Fc fragment" or "Fc" refers to the portion of an antibody that was originally observed to have no antigen-binding activity but to be readily crystallized, hence the name Fc fragment (for fragment crystallizable). Such fragments correspond to the paired CH2 and CH3 domains and are the part of the antibody molecule that interacts with effector molecules and cells. The Fc fragments described herein can be derived from IgG1, IgG2 and IgG4 antibodies. For certain applications, certain IgG subclasses may be preferred. For example, IgG1 is more effective than IgG2 and IgG4 in mediating ADCC and CDC. Thus, IgG2 Fc may be preferred when effector functions are undesirable. However, IgG2 Fc-containing molecules are usually more difficult to prepare and may be less stable than IgG1 Fc-containing molecules. On the other hand, cross-binding to FcγRIIB is important for enhancing the costimulatory signal of CD40 or CD137, but the binding affinity of IgG4 is about two-fold higher than that of IgG1 and ten-fold higher than that of IgG2. Mutations such as S267E and L328F may further enhance cross-binding of IgG Fc with FcγRIIB. Furthermore, by introducing one or more mutations into the Fc, the effector functions of the antibody can be increased or decreased (see, e.g., Strohl, Curr. Opin. Biotech., 20:685-691, 2009).

[0145] As used herein, the term "antibody" or "immunoglobulin" refers to a heterotetrameric glycoprotein of about 150,000 daltons, composed of two identical light chains (L) and two identical heavy chains (H) with the same structural characteristics. Each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end, where the constant region of the light chain faces the first constant region of the heavy chain, and the variable region of the light chain faces the variable region of the heavy chain. Certain amino acid residues form an interface between the variable regions of the light and heavy chains.

[0146] As used herein, the term "variable" refers to the difference in sequence of certain parts of the variable region in an antibody, which forms the binding and specificity of various specific antibodies to a particular antigen. However, variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments called the complementarity determining regions (CDRs) or hypervariable regions of the light and heavy chain variable regions. The more conserved parts of the variable regions are called framework regions (FRs). Natural heavy and light chain variable regions each contain four FR regions, which are mostly in a β-folded configuration connected by three CDRs that form a connecting ring, and can sometimes form a partial β-folded structure. The CDRs of each chain are closely juxtaposed by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant regions are involved directly in binding the antibody to an antigen, but they also exhibit various effector functions, such as those involved in antibody-dependent cellular toxicity of the antibody.

[0147] The antibodies of the present application include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific, bispecific, human, humanized, primatized, chimeric and single chain antibodies. The antibodies disclosed herein can be from any animal source, including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken antibodies.

[0148] The term "antibody fragment" or "antigen-binding fragment" is used to refer to portions of antibodies such as F(ab')2, F(ab)2, Fab', Fab, Fv, single chain Fvs (scFv), single chain antibodies, disulfide-linked Fvs (sdFv), fragments containing VL or VH domains, fragments generated from Fab expression libraries, and anti-idiotypic (anti-Id) antibodies. Regardless of structure, antibody fragments all bind to the same antigen recognized by the intact antibody. The term "antibody fragment" includes DART and diabodies. The term "antibody fragment" further includes synthetic or engineered proteins containing immunoglobulin variable regions, which function like antibodies by binding to specific antigens to form complexes. "Single chain fragment variable region" or "scFv" refers to a fusion protein of the variable regions of the immunoglobulin heavy (VH) and light (VL) chains. In some embodiments, the domains are connected by a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine to increase flexibility and serine or threonine to increase solubility, and can be attached to the N-terminus of VH or the C-terminus of VL, or vice versa. Despite the removal of the constant region and the introduction of the linker, such proteins still retain the specificity of the original immunoglobulin. For IgG, a standard immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of about 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000. The four chains are usually connected by disulfide bonds in a "Y" configuration, where the light chains bracket the heavy chains from the mouth of the "Y" and extend through the variable regions.

[0149] As mentioned above, the variable region allows the antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the VL domain and the VH domain of an antibody or a subset of the antibody's complementarity determining regions (CDRs) combine to form the variable region that defines a three-dimensional antigen-binding site. Such a quaternary antibody structure forms an antigen-binding site present at the end of each arm of each Y configuration. More specifically, the antigen-binding site is defined by three CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) in the VH and VL chains, respectively. In some cases, for example, a particular immunoglobulin molecule is derived from a camelid species or engineered based on camelid immunoglobulins. Alternatively, the immunoglobulin molecule can be composed of only heavy chains without light chains, or only light chains without heavy chains.

[0150] In naturally occurring antibodies, the six CDRs present in each antigen-binding domain are short non-contiguous amino acids specifically positioned to form the "antigen-binding domain" when the antibody adopts a three-dimensional structure in an aqueous environment. Other amino acids in the antigen-binding domain, called the "framework" domain, have less intermolecular variability. The framework regions primarily adopt a β-folded structure, and the CDRs form loops, the connections of which may form part of the β-folded structure. Thus, the framework regions act to form a scaffold that orients the CDRs through non-covalent interactions between the chains. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. The complementary surface promotes non-covalent binding of the antibody to its cognate epitope. Because of their precise definition, one skilled in the art can easily identify the amino acids that comprise the CDRs and framework regions, respectively, for any given heavy or light chain variable region.

[0151] As used herein, the term "light chain constant region (CL)" comprises the amino acid sequence CL (SEQ ID NO: 30 or 31) derived from an antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain.

[0152] As used herein, the term "heavy chain constant region (CH)" includes an amino acid sequence derived from an immunoglobulin heavy chain. A polypeptide containing a heavy chain constant region includes at least one of a CH1 domain (SEQ ID NO: 26 or 28), a hinge region (e.g., an upper, middle and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. It is understood that the heavy chain constant regions are modified such that their amino acid sequences differ from naturally occurring immunoglobulin molecules.

[0153] As used herein, a "variant" of an antibody, antibody fragment, or antibody domain refers to an antibody, antibody fragment, or antibody domain that (1) has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the original antibody, antibody fragment, or antibody domain, and (2) specifically binds to the same target as the original antibody, antibody fragment, or antibody domain. When expressing sequence identity in the form of "at least x% identical" or "at least x% identity," it is understood that such embodiments include any numerical percentage above the lower limit. Furthermore, it is understood that when an amino acid sequence is present in this application, it should be interpreted as disclosing or including having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence.

[0154] Included within the scope of the multispecific molecules of the invention are a variety of compositions and methods, including asymmetric IgG-like antibodies (e.g., triomab / quadroma), knobs-into-holes antibodies, cross MAb, electrostatic match antibodies, LUZ-Y, strand-exchange engineered domain (SEED) bodies, Fab-exchange antibodies, symmetric IgG class antibodies, two-in-one antibodies, cross-linked monoclonal antibodies, mAb2, Cov These include X-bodies, dual variable domain (DVD)-Ig fusion proteins, IgG-like bispecific antibodies, Ts2Ab, BsAb, scFv / Fc fusions, dual (scFv)2-Fabs, F(ab)2 fusion proteins, duplex or Bis-Fab, Dock-and-Lock (DNL), Fab-Fv, scFv-based antibodies and bispecific antibodies (e.g., bispecific T cell engagers (BiTEs), tandem biantibodies (Tandabs), DARTs, single chain biantibodies, TCR-like antibodies, human serum albumin scFv fusion proteins, COMBODIES and IgG / non-IgG fusion proteins.

[0155] As used herein, the phrase "multispecific T cell engager" refers to a molecule comprising at least two targeting domains with different binding specificities, where at least one targeting domain specifically binds to a T cell surface antigen. In some embodiments, the multispecific inhibitor is a polypeptide comprising a scaffold and two or more immunoglobulin antigen binding domains that target different antigens or epitopes. In certain embodiments, the multispecific T cell engager is a bispecific antibody. In some other embodiments, the multispecific T cell engager is a trispecific antibody.

[0156] As used herein, the phrase "bispecific" refers to a molecule that comprises at least two targeting domains with different binding specificities. Each targeting domain is capable of specifically binding to a target molecule and inhibiting a biological function of the target molecule upon binding to the target molecule. In some embodiments, the bispecific T cell engager is a polymeric molecule having two or more peptides. In some embodiments, the targeting domain comprises an antigen binding domain or CDR of an antibody. In some embodiments, the targeting domain comprises a ligand or a fragment thereof that specifically binds to a target protein. In some embodiments, the bispecific inhibitor is a bispecific antibody.

[0157] The terms "bispecific antibody" and "bispecific T cell engager" are used interchangeably herein and refer to an antibody that can specifically bind to two different antigens (or epitopes). In some embodiments, a bispecific antibody is a full-length antibody that binds to one antigen (or epitope) with one of its two binding arms (a pair of HC / LC) and to a different antigen (or epitope) with its second arm (another pair of HC / LC). In these embodiments, a bispecific antibody has two different antigen binding arms (both in specificity and CDR sequences) and each antigen it binds is monovalent.

[0158] In some other embodiments, bispecific antibodies are full-length antibodies capable of binding to two different antigens (or epitopes) with each of their two binding arms (two HC / LC pairs). In these embodiments, bispecific antibodies have two identical antigen-binding arms, with the same specificity and the same CDR sequences, and each antigen to which they bind is all bivalent.

[0159] The terms "trispecific antibody" and "trispecific T cell engager" are used interchangeably herein and refer to a molecule comprising three targeting domains with three different binding specificities. Each targeting domain is capable of specifically binding to a target molecule and inhibiting a biological function of the target molecule upon binding to the target molecule. In some embodiments, the trispecific antagonist is a polymeric molecule comprising two or more peptides. In some embodiments, the targeting domain comprises an antigen binding domain or CDR of an antibody. In some embodiments, the targeting domain comprises a ligand or a fragment thereof that specifically binds to a target protein.

[0160] In a preferred embodiment of the present invention, a bispecific antibody targeting LRRC15 and CD3 was constructed having a structure as shown in ac in Figure 1A. An exemplary molecule of the a structure is as shown in Figure 1D, in which the anti-CD3 scFv can be an scFv constructed using the VH and VL of any anti-CD3 antibody described in the present invention, and the anti-LRRC15 Fab can be an Fab constructed using the VH and VL of any anti-LRRC15 antibody described in the present invention. An exemplary molecule of the b structure is as shown in Figure 1B, in which the anti-LRRC15 scFv can be an scFv constructed using the VH and VL of any anti-LRRC15 antibody described in the present invention, and the anti-CD3 scFv can be an scFv constructed using the VH and VL of any anti-CD3 antibody described in the present invention. Exemplary molecules of the c structure are as shown in FIG. 1C, in which the anti-LRRC15 scFv can be an scFv constructed using the VH and VL of any anti-LRRC15 antibody described in the present invention, the anti-CD3 scFv can be an scFv constructed using the VH and VL of any anti-CD3 antibody described in the present invention, and the anti-LRRC15 Fab can be an Fab constructed using the VH and VL of any anti-LRRC15 antibody described in the present invention.

[0161] In another preferred embodiment of the present invention, a bispecific antibody targeting LRRC15, CD3 and CD28 was constructed having a structure as shown in de in Figure 1A. An exemplary molecule of the e structure is as shown in Figure 1E, in which the anti-CD28 scFv can be an scFv constructed using the VH and VL of any anti-CD28 antibody described in the present invention, the anti-CD3 scFv can be an scFv constructed using the VH and VL of any anti-CD3 antibody described in the present invention, and the anti-LRRC15 Fab can be an Fab constructed using the VH and VL of any anti-LRRC15 antibody described in the present invention.

[0162] In another preferred embodiment of the present invention, a bispecific antibody targeting LRRC15 and CD40 was constructed with the structure as shown in ad in Figure 2A. An exemplary molecule of the a structure is as shown in Figure 2C, in which the anti-LRRC15 Fab can be a Fab constructed using the VH and VL of any anti-LRRC15 antibody described in the present invention, and the CD40L can be any CD40L described in the present invention. An exemplary molecule of the b structure is as shown in Figure 2D, in which the anti-LRRC15 Fab can be a Fab constructed using the VH and VL of any anti-LRRC15 antibody described in the present invention, and the CD40L can be any CD40L described in the present invention. An exemplary molecule of the c structure is as shown in Figure 2B, in which the anti-LRRC15 Fab can be a Fab constructed using the VH and VL of any anti-LRRC15 antibody described in the present invention, and the anti-CD40 scFv can be a scFv constructed using the VH and VL of any anti-CD40 antibody described in the present invention. An exemplary molecule of the d structure is as shown in FIG. 2E, in which the anti-CD40 Fab can be a Fab constructed using the VH and VL of any anti-CD40 antibody described in the present invention, and the anti-LRRC15 scFv can be a scFv constructed using the VH and VL of any anti-LRRC15 antibody described in the present invention.

[0163] In another preferred embodiment of the present invention, a bispecific antibody targeting LRRC15 and CD137 was constructed with the structure as shown in ad in Figure 3A. An exemplary molecule of the a structure is as shown in Figure 3C, in which the anti-LRRC15 Fab can be a Fab constructed using the VH and VL of any anti-LRRC15 antibody described in the present invention, and the CD137L can be any CD137L described in the present invention. An exemplary molecule of the b structure is as shown in Figure 3D, in which the anti-LRRC15 Fab can be a Fab constructed using the VH and VL of any anti-LRRC15 antibody described in the present invention, and the CD137L can be any CD137L described in the present invention. An exemplary molecule of the c structure is as shown in Figure 3B, in which the anti-LRRC15 Fab can be a Fab constructed using the VH and VL of any anti-LRRC15 antibody described in the present invention, and the anti-CD137 scFv can be a scFv constructed using the VH and VL of any anti-CD137 antibody described in the present invention. An exemplary molecule of the d structure is as shown in FIG. 3E, in which the anti-CD137 Fab can be a Fab constructed using the VH and VL of any anti-CD137 antibody described in the present invention, and the anti-LRRC15 scFv can be a scFv constructed using the VH and VL of any anti-LRRC15 antibody described in the present invention.

[0164] The present invention further provides a polynucleotide molecule encoding the antibody or a fragment thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical to the coding region sequence of the antibody of the present invention or may be a degenerate variant. As used herein, the term "degenerate variant" in the present invention refers to a nucleic acid sequence that encodes a nucleic acid sequence having the same amino acid sequence as the polypeptide of the present invention but a different sequence in the coding region.

[0165] A polynucleotide encoding a mature polypeptide of the present invention includes a coding sequence encoding only the mature polypeptide, a coding sequence for the mature polypeptide and various additional coding sequences, a coding sequence for the mature polypeptide (and any additional coding sequences) and non-coding sequences.

[0166] The term "polynucleotide encoding a polypeptide" includes a polynucleotide that encodes the polypeptide, or further includes a polynucleotide that contains additional coding and / or non-coding sequences.

[0167] The present invention further relates to polynucleotides that hybridize with the above sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the above polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refers to (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60oC, or (2) addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42oC, or (3) hybridization that occurs only when the identity between the two sequences is at least 90% or more, more preferably 95% or more. Furthermore, the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides shown in SEQ ID NO.4 and SEQ ID NO.9.

[0168] The full-length nucleotide sequence of the antibody of the present invention or its fragments can usually be obtained by PCR amplification, recombinant methods or artificial synthesis.Especially when the length of the fragment is short, a feasible method is to use artificial synthesis to synthesize the relevant sequence.Generally, multiple small fragments are first synthesized, and then connected to obtain a very long fragment of sequence.In addition, the coding sequence of the heavy chain and an expression tag (e.g., 6His) can be fused to form a fusion protein.

[0169] Once the relevant sequence is obtained, recombinant methods can be used to obtain the relevant sequence in large quantities. Typically, this is achieved by cloning it into a vector, then transforming it into a cell, and then isolating the relevant sequence from the host cell that has been propagated by conventional methods. The biological molecules involved in the present invention (nucleic acids, proteins, etc.) include biological molecules that exist in isolated form.

[0170] Currently, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) can be obtained entirely by chemical synthesis. The DNA sequences can then be introduced into a variety of existing DNA molecules (or vectors, etc.) and cells known in the art. Furthermore, mutations can be introduced into the protein sequences of the present invention by chemical synthesis. The present invention further relates to vectors containing said appropriate DNA sequences and appropriate promoter or control sequences, which can be used to transform appropriate host cells so as to express the protein.

[0171] The host cell may be a prokaryotic cell, such as, for example, a bacterial cell, or a lower eukaryotic cell, such as, for example, a yeast cell, or a higher eukaryotic cell, such as, for example, a mammalian cell. Representative examples include bacterial cells of E. coli, Streptomyces, Chinese hamster bacteria, fungal cells such as yeast, insect cells of Drosophila S2 or Sf9, animal cells such as CHO, COS7 and 293 cells, and the like.

[0172] Transformation of host cells with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of absorbing DNA can be obtained by treating with CaCl2 method after exponential growth phase, the steps used being well known in the art. Another method is to use MgCl2. Transformation can also be carried out by electroporation method if necessary. When the host is a eukaryote, DNA transfection methods such as calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection and electroporation, liposome packaging, etc. can be selected.

[0173] The obtained transformant can be cultured in a conventional manner to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the medium used for the culture can be selected from a variety of conventional media. The culture is performed under conditions suitable for the growth of the host cell. After the host cell has grown to an appropriate cell density, the selected promoter is induced using an appropriate method (e.g., temperature change or chemical induction), and the cell is further cultured for a certain period of time.

[0174] The recombinant polypeptide in the method can be expressed intracellularly or in the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various isolation methods using physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation, treatment with protein precipitants (salting out), centrifugation, osmotic sterilization, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid phase chromatography (HPLC) and various other liquid chromatography techniques, as well as combinations of these methods.

[0175] The antibodies of the present invention can be used alone, can be conjugated or coupled to a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modulating moiety, or any combination of these substances.

[0176] Detectable markers for diagnostic purposes include, but are not limited to, fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product.

[0177] Therapeutic agents that can be coupled include, but are not limited to, insulin, IL-2, interferons, calcitonin, GHRH peptides, gut peptide analogs, albumin, antibody fragments, cytokines, and hormones.

[0178] The present invention further provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising the above-mentioned antibody or an active fragment thereof or a fusion protein thereof and a pharma- ceutically acceptable carrier. Usually, these substances can be formulated in a non-toxic, inert, pharma- ceutically acceptable aqueous vector medium, where the pH is usually about 5-8, preferably about 6-8, and the pH value varies depending on the nature of the substance to be formulated and the disease to be treated. The formulated pharmaceutical composition can be administered by a conventional route, where it includes (but is not limited to) oral, respiratory, intratumoral, intravenous, or topical administration.

[0179] The pharmaceutical composition of the present invention can be used to treat cancer / tumors, particularly solid tumors, especially solid tumors with high expression of LCRR15.

[0180] The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned monoclonal antibody of the present invention (or a complex thereof) and a pharma- ceutically acceptable carrier or excipient. Such vectors include, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The drug formulation must be consistent with the method of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection by conventional methods, for example, using saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions must be prepared under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 μg / kg body weight to about 10 mg / kg body weight daily. In addition, the pharmaceutical composition of the present invention can also be used with other therapeutic agents.

[0181] When using the pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, where the safe and effective amount is usually at least about 10 μg / kg body weight, and in most cases will not exceed about 8 mg / kg body weight, and preferably the dosage is from about 10 μg / kg body weight to about 1 mg / kg body weight. Of course, the specific dosage must take into account factors such as the route of administration and the health condition of the patient, all of which are within the skill of a skilled physician.

[0182] The main advantages of the present invention are: (1) Cancer-associated stromal fibroblasts (CAFs) are important components of the microenvironment of most solid tumors, and these cells promote cancer progression by supporting tumor cell growth, participating in tumor extracellular matrix remodeling, promoting angiogenesis, and regulating tumor-induced inflammation. CAFs coexist with other cells, such as immune cells and endothelial cells, that express CD40 within the cancer matrix. Thus, since LRRC15 is specifically expressed in CAFs, i.e., dual / triplex antibodies can be designed against this target to deliver immune activation signals to the tumor matrix, thereby helping to convert cold tumors into hot tumors in the treatment of most solid tumors.

[0183] (2) Compared with general tumor-associated antigen (TAA) targets, the expression of LRRC15 on various solid tumors and CAFs makes the application of the bi / trispecific antibody containing the LRRC15 antigen-binding domain of the present invention broader.

[0184] (3) Due to the blocking effect of the tumor matrix, it is difficult for conventional TAA-targeting biantibody to directly deliver immunomodulatory agents to the location of tumor cells. However, the LRRC15 biantibody, one end of which is targeted and expressed on CAFs, can easily induce the activation of immune cells to play a role in the tumor matrix by using the other end to bind to activation-regulating targets (e.g., targeting TNFRSF).

[0185] (4) The LRRC15-related double / triple antibodies of the present invention can effectively activate T cells and reduce the matrix effect of tumors.

[0186] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and do not limit the scope of the present invention. In the following examples, the experimental methods that do not show specific conditions are usually in accordance with conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions suggested by manufacturers. Unless otherwise specified, percentages and parts are calculated by weight percentages and parts by weight.

[0187] Sequences used to prepare the multispecific T cell engagers of the invention LRRC15 antibody sequence [Table 3]

[0188] [Table 4]

[0189] CD3 antibody sequence [Table 5]

[0190] CD28 antibody sequence [Table 6]

[0191] CD40 antibody sequence [Table 7]

[0192] CD137 antibody sequence [Table 8]

[0193] CD40 ligand (CD40L) sequence The CD40LRBD mutant can use one of the 112-261 segment, the 113-261 segment, the 116-261 segment, the 117-261 segment, the 119-261 segment, and the 121-261 segment in the CD40 ligand sequence. [Table 9]

[0194] CD137 ligand (CD137L) sequence The CD137LRBD mutant can use one of the 52-248 segment, the 58-248 segment, the 71-248 segment, the 80-248 segment, the 85-248 segment, the 52-254 segment, the 58-254 segment, the 71-254 segment, the 80-254 segment, and the 85-254 segment in the CD137 ligand sequence. [Table 10]

[0195] IgG1 CH sequence [Table 11]

[0196] The Fc region may comprise the following mutations: [Table 12]

[0197] In a preferred embodiment of the invention, the Fc region has the following sequence: [Table 13]

[0198] IgG4 CH sequence [Table 14]

[0199] The Fc region may comprise the following mutations: [Table 15]

[0200] CL sequence [Table 16]

[0201] Selectable linker sequences [Table 17]

[0202] Example 1. Preparation and validation of anti-LRRC15 antibodies The LRRC15 antibodies of the present invention are screened from a phage library, and phage screening techniques are described in (e.g., Kay, B. Winter, J. & McCafferty, J. (eds.) (1996) Phage Display of Polypeptides and Proteins: A Laboratory Manual (Academic Press)). During the screening process, specific binding molecules are screened against the extracellular domain of LRRC15 by ELISA and FACS methods, and then the sequence is confirmed by sequencing. Finally, four candidate antibodies are obtained: L15-06, L15-08, L15-03 and L15-07.

[0203] Furthermore, the binding ability of the LRRC15 antibody of the present invention to CHO cells expressing LRRC15 is tested. Here, the CHO cells expressing LRRC15 are obtained by full gene synthesis of the full-length sequence gene fragments of human, mouse and monkey LRRC15, and then these fragments are inserted into the backbone of a stable expression vector to obtain an expression plasmid vector. These three plasmids are transfected into CHO cells, and the monoclonal cell lines are screened under the pressure of puromycin, that is, the flow cytometry detection cell lines used in this embodiment.

[0204] LRRC15-expressing CHO cells are washed with FACS buffer (0.5% BSA in PBS) and resuspended with 2E6 cells / ml and serially diluted LRRC15 antibodies. The resuspension is incubated on ice for 30 min, then washed twice with FACS buffer and incubated with secondary antibodies on ice for 30 min. The resuspended cells are washed again with FACS buffer before detection by flow cytometry. Based on the measured mean fluorescence intensity, half the maximal effective concentration (EC 50 ) is measured.

[0205] The results of the analysis are shown in Figure 4A-C. The binding ability of the four antibodies to cells expressing human LRRC15 is similar, but the binding ability to cells expressing mouse LRRC15 is significantly different, with L15-06 binding the strongest, followed by L15-08, L15-03 weakly, and L15-07 being the weakest. Furthermore, all four antibodies have strong monkey cross-binding ability.

[0206] In addition, we also carry out a competitive experiment on antibody binding. After binding the four monoclonal antibody molecules to LRRC15-expressing CHO cells, we use biotin-labeled monoclonal antibody molecules to competitively bind, and then use SA-FITC to detect the signal intensity, and the more the signal decreases, the stronger the competition. The results show that L15-08 has little competitive effect on L15-L06, which indicates that L15-06 and L15-08 bind to different epitopes of LRRC15 (Figure 4D).

[0207] Example 2. Preparation of multispecific T cell engager molecules of the invention Various multispecific antibody molecules are designed based on the four different LRRC15 antibody sequences obtained in Example 1 and the antibody sequence derived from L15-06 (L15-06C). The molecular sequence fragment genes of the multispecific antibodies are obtained by the method of total gene synthesis, and then the target sequences are inserted into an expression vector by conventional gene cloning methods (see, for example, Lo. BKC methods in Molecular Biology. Volume 248, 2004. Antibody Engineering).

[0208] HEK293E cells are transfected with vectors carrying multispecific molecules. They are cultured at 37°C, 5% CO2 for 7 days and the corresponding molecules can be produced in F17 medium (1L F17 + 10mL 10% PF68 + 30mL 200mM L-glutamine). After expression is complete, the supernatant is collected and purified to obtain the multispecific T cell engager molecules, which can then be used for various experimental assays.

[0209] Using the above method, the following multispecific molecules were obtained: (1) A bispecific antibody composed of an LRRC15 antigen-binding domain and a CD3 antigen-binding domain having the structure shown in FIG. 1A ac, and a trispecific antibody composed of an LRRC15 antigen-binding domain, a CD3 antigen-binding domain, and a CD28 antigen-binding domain having the structure shown in de.

[0210] The bispecific antibody of the a-structure in FIG. 1A is named 315-L06, the structure of which is shown in FIG. 1D. The bispecific antibody with b structure is named 153-L06C, the structure of which is shown in FIG. 1B; another bispecific antibody with b structure is named 153-L07, the difference from 153-L06C is that the anti-LRRC15 scFv therein uses the VH and VL of L15-07; another bispecific antibody with b structure is named 153-L06, the difference from 153-L06C is that the anti-LRRC15 scFv therein uses the VH and VL of L15-06; The bispecific antibody with the c structure is named 315-L06C08, the structure of which is as shown in FIG. 1C; another bispecific antibody with the c structure is named 315-L0608, the difference from 315-L06C08 is that the anti-LRRC15 scFv thereof uses the VH and VL of L15-06; another bispecific antibody with the c structure is named 315-L0806, the difference from 315-L06C08 is that the anti-LRRC15 scFv thereof uses the VH and VL of L15-08, and the anti-LRRC15 Fab uses the VH and VL of L15-06; another bispecific antibody with the c structure is named 315-L08C06, the difference from 315-L06C08 is that the anti-LRRC15 The scFv uses the VH and VL of L15-08C, and the anti-LRRC15 Fab uses the VH and VL of L15-06; The trispecific antibody of the e structure was named 28315-L06, and its structure is as shown in FIG. 1E.

[0211] (2) A bispecific antibody composed of an LRRC15 antigen-binding domain and a CD40 antigen-binding domain as shown in FIG. 2A ad.

[0212] The bispecific antibody of the a-structure in FIG. 2A is named 1540L-L06, the structure of which is shown in FIG. 2C. The bispecific antibody of the b-structure was named 1540TL-L06, and the structure is as shown in FIG. 2D. The bispecific antibody of the c structure was named 1540-L06, the structure of which is shown in FIG. 2B. The bispecific antibody with the d structure is named 4015-L06, the structure of which is as shown in FIG. 2E; another bispecific antibody with the d structure is named 40D15-L06C, the difference from 4015-L06 is that the anti-CD40 IgG therein uses the VH and VL of CD40d, and the anti-LRRC15 scFv therein uses the VH and VL of L15-06C; another bispecific antibody with the d structure is named 40D15-L06CS, the difference from 4015-L06 is that the anti-CD40 IgG therein uses the VH and VL of CD40d, the Fc fragment is replaced with the fragment shown in SEQ ID NO: 60, and the anti-LRRC15 scFv therein uses the VH and VL of L15-06C.

[0213] (3) A bispecific antibody composed of an LRRC15 antigen-binding domain and a CD137 antigen-binding domain as shown in FIG. 3A ad. The bispecific antibody of the a-structure in FIG. 3A is named 15BBL-L06, the structure of which is shown in FIG. 3C. The bispecific antibody of the b-structure was named 15BBTL-L06, the structure of which is shown in FIG. 3D. The bispecific antibody of the c structure is named 15BB-L06, the structure of which is as shown in FIG. 3B ; another bispecific antibody of the c structure is named 15BBe-L06, the difference from 15BB-L06 is that the anti-CD137 scFv therein uses the VH and VL of CD137e; The bispecific antibody with the d structure was named BB15-L06, the structure of which is as shown in FIG. 3E, and another bispecific antibody with the d structure was named BBe15-L06, the difference from BB15-L06 is that the anti-CD137 IgG therein uses the VH and VL of CD137e.

[0214] Exemplary molecules prepared in this example are as follows: [Table 18]

[0215] Example 3. Verification of binding of bispecific antibody molecules targeting LRRC15 and CD3 to target molecules LRRC15 and CD3 targeting bispecific antibody molecules (153-L06C, structure as shown in FIG. 1B; 315-L06C08, structure as shown in FIG. 1C) and control sample BM bispecific antibody molecules (sequence see WO2021022304A2) were tested for binding to CHO cells expressing human LRRC15 and CHO cells expressing cynomolgus monkey LRRC15, methods as described in Example 1.

[0216] As a result, in the case of CHO cells expressing human LRRC15, 315-L06C08 exhibited binding EC 50 The lowest value indicates a stronger binding capacity, and 153-L06C has the highest binding plateau, indicating that it can bind more antibody molecules at a saturating dose (Figure 5A).

[0217] In the case of CHO cells expressing cynomolgus monkey LRRC15, the binding propensity of each molecule is similar to that of CHO cells expressing human LRRC15 (Figure 5B).

[0218] Example 4. Performance detection of bispecific antibody molecules targeting LRRC15 and CD3 4.1. T cell activation experiments of bispecific antibody molecules targeting LRRC15 and CD3 The ability of bispecific antibody molecules (153-L06C and 315-L06C08) targeting LRRC15 and CD3 to activate T cells was measured, and the above BM molecules were used as control samples. The specific method is as follows.

[0219] LRRC15-expressing CHO cells (wild-type CHO not expressing LRRC15 is used as a control) are washed with PBMC buffer (RPMI-1640 + 10% FBS + 1% pen-strep) and resuspended at 2E6 cells / ml and serially diluted antibodies to be tested. Prepared PBMC cells are added to the resuspension so that the E / T cell ratio is 10:1. Incubate at 37°C for 48 hours, then wash twice with FACS buffer and co-incubate with anti-CD3 and anti-CD25 secondary antibodies for 30 minutes on ice. Resuspended cells are washed again with FACS buffer and then detected by flow cytometry. Based on the measured mean fluorescence intensity, half the maximum effective concentration (EC 50 ) to calculate

[0220] CD25 is an important marker for T cell activation. We show that all three bibody molecules have an activating effect on T cells in the presence of target cells, but none of them affect T cell activation in the presence of cells that do not express the target protein. Here, we show that activated ECs from the BM 50 has the smallest activation dose but the smallest maximum percentage of activated cells. 153-L06C has the largest activation dose but also the largest maximum percentage of activated cells, with 315-L06C08 in between (Figure 6).

[0221] 4.2. Experiments on stimulation of IFN-γ release from PBMCs by bispecific antibody molecules targeting LRRC15 and CD3 The ability of bispecific antibody molecules targeting LRRC15 and CD3 (153-L06C and 315-L06C08) to stimulate IFN-γ release from PBMCs was measured, and the above BM molecules were used as control samples.

[0222] Wash LRRC15 expressing CHO cells (wild type CHO not expressing LRRC15 is used as a control) with PBMC buffer (RPMI-1640 + 10% FBS + 1% pen-strep) and resuspend at 2E6 cells / ml and serially diluted antibodies to be tested. Add prepared PBMC cells to the resuspension to give an E / T cell ratio of 10:1. Incubate at 37°C for 48 hours. Collect the supernatant and test for IFN-γ content using an IFN-γ detection kit.

[0223] The results show that the three bibody molecules can stimulate PBMCs to release IFN-γ in the presence of target cells, but not in the absence of target cells, where BM has the weakest stimulatory effect. 153-L06C has the highest maximum IFN-γ release, but the EC value of 315-L06C08 for IFN-γ release is lower than that of 315-L06C08. 50 There is a large difference when compared with the values ​​(Figure 7).

[0224] 4.3. Experiments on stimulation of T cell proliferation by bispecific antibody molecules targeting LRRC15 and CD3 The ability of bispecific antibody molecules (153-L06C and 315-L06C08) targeting LRRC15 and CD3 to stimulate T cell proliferation was measured, and the above BM molecules were used as control samples. The specific method is as follows.

[0225] T cells in PBMCs are isolated and labeled with CFSE using a Pan T cell isolation kit (cat# 130-096-535, Miltenyi). In each well of a 96-well plate, 50 μl of labeled T cells (containing 5E4 cells) are mixed with 100 μl of 1E4 LRRC15-expressing CHO cells (wild-type CHO cells that do not express LRRC15 are used as control) and resuspended with 50 μl of serially diluted antibodies to be tested. Incubate at 37°C for 5 days. Then detect by flow cytometry after washing twice with FACS buffer. Based on the proliferation of T cells under different conditions, the half-maximal effective concentration (EC) of various antibodies is determined according to the corresponding CFSE staining signal value. 50 ) is measured.

[0226] The results show that both 153-L06C and 315-L06C08 can promote cell proliferation in the presence of target cells, but in the presence of cells that do not express LRRC15, none of the three dual antibody molecules show a significant effect on promoting proliferation. Here, BM has little effect on promoting T cell proliferation. 153-L06C has the highest percentage of maximally proliferating T cells, but the EC of 315-L06C08 that promotes proliferation is 50 There is a large difference when compared with the values ​​(Figure 8).

[0227] 4.4. Experiments on stimulation of PBMC killing of target cells by bispecific antibody molecules targeting LRRC15 and CD3 The ability of bispecific antibody molecules (153-L06C and 315-L06C08) targeting LRRC15 and CD3 to stimulate PBMCs to kill target cells was measured, and the above BM molecules were used as control samples. The specific method is as follows.

[0228] Wash LRRC15-expressing CHO cells using PBMC buffer (RPMI-1640 + 10% FBS + 1% pen-strep) and resuspend at 2E6 cells / ml with serial dilutions of the antibody to be tested. Add prepared PBMC cells to the resuspension to give an E / T cell ratio of 10:1. Incubate at 37°C for 48 hours. Add CytoTox-Glo™ Cytotoxicity Detection Reagent to the incubated 96-well plate. Measure the killing of target cells by various antibodies based on the measured chemiluminescence intensity.

[0229] The results show that the three double antibody molecules can stimulate PBMCs to kill target cells in the presence of target cells, but show no obvious killing effect in the absence of cells expressing LRRC15. Here, BM has the weakest killing effect and has obvious hook-like effectors. 153-L06C has the highest maximum killing ratio, but 315-L06C08 kills ECs. 50 There is a large difference when compared with the values ​​(Figure 9).

[0230] 4.5. Experiments of stimulating cytokine release from PBMCs by bispecific antibody molecules targeting LRRC15 and CD3 The ability of bispecific antibody molecules targeting LRRC15 and CD3 (153-L06C and 315-L06C08, the structures of which are shown in Figures 1B and 1C) to stimulate PBMCs to release cytokines was examined, and the above BM molecules were used as control samples, and CD3 antibody (OKT3) was used as a positive control. The specific method is as follows:

[0231] PBMC cells from four different donors (D1, D2, D3, D4) are thawed and diluted to 10E7 cells / ml in 96-well plates using PBMC buffer (RPMI-1640 + 10% FBS + 1% pen-strep) and then cultured for 48 hours in a 37°C incubator. Antibodies to be tested are diluted to 10 nM in PBMC buffer and added to the 96-well plate containing the PBMC cells and incubated at 37°C for 24 hours. BD TM Cytometric Bead Array (CBA) kits can be used to capture and detect cytokine content in culture supernatants to demonstrate how different antibodies stimulate cytokine release from target donor PBMCs.

[0232] As a result, the three double antibody molecules did not show a significant promoting effect on the release of six cytokines, but the CD3 antibody (OKT3) used as a positive control showed that all of them released cytokines to various degrees. Untreated, which is the group without antibody, was used as a negative control, and all of them showed no obvious cytokine release (Figure 10).

[0233] Example 5. Verification of binding between bispecific antibody molecules targeting LRRC15 and CD137 / CD40 and target molecules A bispecific antibody molecule targeting LRRC15 and CD137 (15BB-L06, the structure of which is shown in FIG. 3B) binds to 4-1BB protein, and the binding of a bispecific antibody molecule targeting LRRC15 and CD40 (1540-L06, the structure of which is shown in FIG. 2B) to CD40 protein is detected by ELISA. The specific method is as follows.

[0234] Coat the antigen protein of 4-1BB or CD40 overnight at 4℃, wash three times with 0.05% PBST solution, add 200μl of 1% BSA to each well of the 96-well plate and block for 1 hour at room temperature. Wash the plate three times with 0.05% PBST. Resuspend serially diluted antibody molecules (15BB-L06 or 1540-L06) with 1% BSA and then add 100μl per well to the 96-well plate in duplicate. Wash the plate three times with 0.05% PBST. Add anti-hIgG-HRP secondary antibody and incubate for 30 minutes at room temperature. Wash the plate three times with 0.05% PBST. Then add TMB reagent to develop color and read at 450nm on a microplate reader. Based on the measured average OD450 value, half the maximum effective concentration (EC 50 ) is measured.

[0235] The results show that 15BB-L06 can bind to 4-1BB, and 1540-L06 can bind to CD40 (FIG. 11). In addition, the 4-1BB antibody portion or the CD40 antibody portion in the bispecific antibody molecule is in the form of an scFv, which does not cause excessive activation after the antibody binds to an antigen, compared with its IgG form, thereby reducing the risk of the bispecific antibody molecule during administration.

[0236] Example 6. Performance detection of bispecific antibody molecules targeting LRRC15 and CD137 / CD40 The ability of a bispecific antibody targeting LRRC15 and CD137 (15BB-L06) to stimulate PBMCs to release IL-2 and the ability of a bispecific antibody targeting LRRC15 and CD40 (1540-L06) to stimulate DCs to release IL-12p40 will be measured.

[0237] Pre-add 100 μl of 10 μg / ml CD3 antibody (OKT3, eBioscience) per well to a 96-well plate and incubate for 2 hours at 37°C. Wash LRRC15-expressing CHO cells (wild-type CHO cells not expressing LRRC15 are used as controls) with PBMC buffer (RPMI-1640 + 10% FBS + 1% pen-strep) and resuspend at 2E6 cells / ml and serially diluted antibodies to be tested. Add the prepared PBMC cells to the resuspension so that the E / T cell ratio is 10:1. Incubate for 48 hours at 37°C. Collect the supernatant and test the IL-2 content with an IL-2 detection kit.

[0238] The results show that 15BB-L06 releases more IL-2 than Urelumab (4-1BB monospecific antibody) in the presence of target cells, but in the presence of cells that do not express the target protein, the release of IL-2 is similar to that of the control IgG, reflecting the effectiveness of the target specificity of the antibody molecule. The control monoclonal antibody Urelumab does not have this specificity (Figure 12).

[0239] Isolate mononuclear cells from PBMC using a mononuclear cell isolation kit (cat#130-096-537, Miltenyi). Add 20ng / ml GM-CSF and IL-4 to induce differentiation of mononuclear cells into DC cells, and harvest DC cells after 7 days. Wash LRRC15-expressing CHO cells (wild-type CHO not expressing LRRC15 is used as a control) with PBMC buffer (RPMI-1640+10%FBS+1% pen-strep) and resuspend at 2E6 cells / ml and serially diluted antibodies to be tested. Add DC cells prepared in the resuspension to achieve an E / T cell ratio of 3:1. Incubate at 37°C for 24 hours. Collect the supernatant and test the content of IL-12p40 with an IL-12p40 detection kit.

[0240] The results show that 1540-L06 releases slightly less IL-12p40 than Selicrelumab (a CD40 monospecific antibody) in the presence of target cells, but in the presence of cells that do not express the target protein, the release of IL-12p40 is significantly reduced, reflecting the effectiveness of the antibody's molecular targeting specificity. The control monoclonal antibody Selicrelumab does not have this specificity (Figure 13). In addition, the ability of the above-mentioned double antibody molecule to stimulate PBMCs to release cytokines (TNF-α and IL-6) was measured, and CD3 antibody (OKT3) was used as a positive control. The specific method is as follows:

[0241] Thaw and dilute PBMC cells from five different donors to 10E7 cells / ml using PBMC buffer (RPMI-1640+10%FBS+1% pen-strep) in a 96-well plate, then culture in a 37°C incubator for 48 hours. Dilute the antibody to be tested to 10nM in PBMC buffer and add it to the 96-well plate containing PBMC cells, and incubate at 37°C for 24 hours. Use TNF-α and IL-6 detection kits to capture and detect the content of cytokines in the culture supernatant, respectively, to show how different antibodies stimulate the cytokine release status from PBMCs of different donors.

[0242] The results show that the two double antibody molecules 1540-L06 and 15BB-L06 have no obvious promoting effect on the two cytokines TNF-α and IL-6 (FIG. 14).

[0243] Example 7. In vivo efficacy studies of bispecific antibody molecules targeting LRRC15 7.1.NCI-H1650 model NOG mice were cultured with NCI-H1655 cells (1 × 10 7 After 15 days, PBMCs were inoculated, and the average tumor volume was approximately 200 mm 3 When the mice reached 100 mm in size, they were divided into groups and started to be treated (the control group was treated with PBS, and the experimental groups were treated with 153-L06C and 315-L06C08, respectively). The mice were injected intravenously twice a week for 7 consecutive doses. During this period, the tumor volume was measured (Figure 15A) and the mice were observed for any abnormalities.

[0244] The results showed that under equimolar doses, 153-L06C (0.2 mg / kg) and 315-L06C08 (0.288 mg / kg) showed similar antitumor effects, with TGIs of 37% and 39%, respectively. The animals tolerated well during the administration period. After the experiment was completed, the tumors were harvested and weighed, and the results of tumor weight more clearly reflected the antitumor effects of 153-L06C and 315-L06C08, with tumor weights being 55% and 57% of those of the control group, respectively (Figure 15B).

[0245] 7.2.MC38 model Humanized hCD3e / h4-1BB double gene knock-in C57BL / 6 mice were transfected with MC38-huLRRC15 cells (2 × 10 6 The average tumor volume was approximately 120 mm 3 When the mice reach adulthood, they are divided into groups and begin to receive treatment (the control group receives saline, and the experimental groups receive 153-L06C and 15BB-L06, respectively) by intravenous injection, twice a week for four consecutive doses.

[0246] The results showed that both 153-L06C (1 mg / kg) and 15BB-L06 (1 mg / kg) exhibited similar antitumor effects, with TGIs of 24% and 43%, respectively (Figure 15C).

[0247] Humanized hCD40 gene knock-in C57BL / 6 mice were cultured with MC38-huLRRC15 cells (2 × 10 6 The average tumor volume was approximately 120 mm 3 When the tumors reached 100 mm, they were divided into groups and started to be administered (the control group was administered saline, and the experimental group was administered 1540-L06). After intravenous injection twice a week (the first two doses were 1 mg / kg, and the third dose was 6 mg / kg) for three consecutive doses, and the dose was adjusted to 6 mg / kg, 1540-L06 showed obvious antitumor effect, with a TGI of 59% (Figure 15D).

[0248] Comparative Example 1. Performance comparison of multispecific T cell engager molecules with different structures The ability of the bispecific antibody molecules (153-L06 and 315-L06) targeting LRRC15 and CD3 prepared in Example 2 to stimulate PBMCs to kill target cells is measured. For specific methods, see Example 4.4.

[0249] The results showed that the killing effect of 153-L06 was significantly superior to that of 315-L06, indicating that molecules having the 153-L06 structure have a greater ability to stimulate target cell killing (Figure 16).

[0250] The bispecific antibody molecules (153-L06C, 315-L06C08, 315-L08C06) targeting LRRC15 and CD3 prepared in Example 2 and the trispecific antibody molecule 28315-L06 targeting LRRC15, CD3 and CD28 are measured for their ability to stimulate PBMCs to kill target cells. For specific methods, see Example 4.4.

[0251] The results showed that the killing effect of 315-L06C08 was significantly superior to those of the other molecules, indicating that molecules having the 315-L06C08 structure have superior ability to stimulate killing of target cells (Figure 17).

[0252] All documents mentioned in the present invention are incorporated by reference in this application as if each document was incorporated by reference individually. Furthermore, after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalents are also included in the scope defined by the appended claims of this application.

Claims

1. Multispecific T Cell Engagers, comprising: The multispecific T cell engager comprises: a first targeting domain comprising one or more LRRC15 antigen binding domains; and / or a second targeting domain that binds to a target protein selected from the group consisting of CD3, CD28, CD40, or CD137; and / or and an optional third targeting domain that binds a target protein selected from the group consisting of CD3, CD28, CD40, or CD137.

2. The targeting domain is characterized in that it is in the form of a single domain antibody (sdAb), a fragment variable (Fv) heterodimer, a single chain Fv (scFv), a Fab fragment, a TriFab, or a combination thereof. The multispecific T cell engager of claim 1 .

3. The LRRC15 antigen-binding domain is characterized in that it comprises a heavy chain variable region having at least 80% sequence identity with an amino acid sequence as set forth in SEQ ID NO: 1, 2, 3, 4 or 53, and a light chain variable region having at least 80% sequence identity with an amino acid sequence as set forth in SEQ ID NO: 5, 6, 7, 8 or 54. The multispecific T cell engager of claim 1 .

4. The LRRC15 antigen-binding domain is characterized in that it comprises a heavy chain variable region as shown in SEQ ID NO: 1 and a light chain variable region as shown in SEQ ID NO:

5. The multispecific T cell engager of claim 3.

5. The LRRC15 antigen-binding domain is characterized in that it comprises a heavy chain variable region as shown in SEQ ID NO: 53 and a light chain variable region as shown in SEQ ID NO:

54. The multispecific T cell engager of claim 3.

6. The second targeting domain is selected from the group consisting of a CD3 antigen-binding domain, a CD28 antigen-binding domain, a CD40 antigen-binding domain, a CD137 antigen-binding domain, a CD40L sequence, or a CD137L sequence. The multispecific T cell engager of claim 1 .

7. The third targeting domain is selected from the group consisting of a CD3 antigen-binding domain, a CD28 antigen-binding domain, a CD40 antigen-binding domain, a CD137 antigen-binding domain, a CD40L sequence, or a CD137L sequence. The multispecific T cell engager of claim 1 .

8. An anti-LRRC15 antibody or an antigen-binding fragment thereof, The antibody or antigen-binding fragment thereof comprises: HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 32, 35, 38, or 41; HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 33, 36, 39, or 42, and three heavy chain variable region CDRs, such as HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 34, 37, 40, or 43; and, LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 44 or 49; LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 45, 47, or 50; and The anti-LRRC15 antibody or antigen-binding fragment thereof, characterized in that it comprises three light chain variable region CDRs, such as an LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 46, 48, 51, or 52.

9. 2. Use of the multispecific T cell engager of claim 1, comprising: The above use, characterized in that it is used in the preparation of a medicament for treating cancer / tumor.

10. The cancer / tumor is characterized in that it is a cancer / tumor with high expression of LRRC15.

10. The use according to claim 9.

11. 1. An immune complex comprising: The complex is (i) a multispecific T cell engager according to claim 1, or an antibody or antigen-binding fragment thereof according to claim 8, and (ii) a coupling moiety selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

12. 1. A pharmaceutical composition comprising: The pharmaceutical composition comprises: (a) the multispecific T cell engager of claim 1, or the antibody or antigen-binding fragment thereof of claim 8; and (b) a pharma- ceutical acceptable carrier.

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