Multispecific antibody containing LRRC15 antigen-binding domain

Abstract

The present invention relates to a multispecific antibody containing an LRRC15 antigen-binding domain and the use thereof. Particularly, provided in the present invention is a multispecific antibody, which contains a first targeting domain that targets to a leucine-rich repeat containing 15 (LRRC15) molecule highly expressed on the surface of a tumor cell, also contains a second targeting domain that binds to a CD40 molecule on the surface of an antigen presenting cell (APC) or a CD3 molecule on the surface of a T cell, and optionally, contains a third targeting domain that targets to PD-1, CLDN18.2 or FAP, and / or a fourth targeting domain that targets to TGF-β. The LRRC15-related multispecific antibody of the present invention can effectively activate T cells and reduce tumor stroma impact, thereby providing effective treatment for tumors and fibrotic diseases.

Description

Multispecific antibodies comprising LRRC15 antigen-binding domains Technical Field

[0001] The present invention relates to the field of cancer treatment, and more particularly, to a multispecific antibody comprising an anti-LRRC15 antibody or an immunoreactive fragment thereof for treating cancer. Background Art

[0002] Cancer is generally defined as a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. Conventional cancer treatments aim to remove cancerous tissue and prevent its spread. These treatment options include surgery, chemotherapy, radiation therapy, hormone therapy, targeted therapies, and palliative care. Treatment is typically tailored to the type, location, and grade of the cancer, as well as the patient's health and preferences. However, these therapies have limitations, as they may be ineffective, particularly when the cancer has metastasized. Furthermore, chemotherapy and radiation therapy have a range of side effects related to cytotoxicity.

[0003] Current promising areas of cancer treatment include targeted therapies using antibodies. Another promising area is developing treatments that harness the immune system to attack and kill tumor cells.

[0004] Leucine-rich repeat containing 15 (LRRC15) is a member of the LRR (leucine-rich repeat) superfamily. LRRC15 is selectively overexpressed in tumor cells, including breast cancer, brain tumors, pancreatic cancer, lung cancer, melanoma, highly aggressive sarcomas, and breast cancer bone metastases, while expression levels are relatively low in normal tissues. Furthermore, it is highly expressed in stromal fibroblasts of many solid tumors (e.g., breast, head and neck, lung, and pancreatic). Therefore, LRRC15 is a candidate for targeted therapies such as antibody-drug conjugates, T cell engagers, and CAR-T cells.

[0005] CD40 and CD40L are co-stimulatory molecules that connect innate and adaptive immunity. CD40 on antigen-presenting cells (APCs) binds to CD40 ligand (CD40L), primarily expressed by activated T cells, inducing APC activation. In turn, APCs trigger cytotoxic T lymphocytes (CTLs). CD40 and CD40L are co-stimulatory molecules that connect innate and adaptive immunity. Antibodies to CD40 can bind with higher affinity and activate the CD40 signaling pathway, thereby activating the immune system.

[0006] The tight junction protein Claudin is a tetraspanin integrin found on epithelial and endothelial cells. Claudin 18.2, a subtype of Claudin 18, is rarely expressed in normal tissues but is abnormally expressed during the development and progression of various primary malignancies, including gastric / gastroesophageal junction (GC / GEJ) cancer, breast cancer, colon cancer, head and neck cancer, bronchial cancer, and non-small cell lung cancer. Astellas' Claduin 18.2 monoclonal antibody has completed Phase III clinical trials and achieved promising results, and is expected to become the first marketed drug targeting this target.

[0007] PD-1 / PD-L1 are important targets in immuno-oncology (IO) therapy. PD-L1 is highly expressed in most tumors, and binding to PD-1 on the surface of T cells transmits inhibitory signals to T cells. Therefore, blocking PD-1 / PD-L1 can effectively activate T cells to kill tumor cells. Since the launch of nivolumab, the first PD-1 antibody, in 2014, several PD-1 / PD-L1 monoclonal antibodies have achieved promising clinical therapeutic effects.

[0008] Fibroblast activation protein α (FAP) is a type II transmembrane serine protease. Its expression is low in most tissues, but it is highly expressed in tumors such as pancreatic and breast cancer. Furthermore, similar to LRRC15, FAP is primarily expressed on cancer-associated fibroblasts (CAFs) within the stroma of most epithelial cancers. FAP itself can influence tumor growth through multiple mechanisms, including promoting tumor proliferation, migration, and angiogenesis. Therefore, drugs targeting FAP could effectively target both tumors and their fibrotic cells for therapeutic purposes.

[0009] Transforming growth factor β (TGF-β) is a multifunctional cytokine belonging to the transforming growth factor superfamily. Its key function is to regulate inflammatory processes and it also plays an important role in stem cell differentiation and T cell regulation and differentiation. Dysregulation of TGF-β signaling can disrupt immune tolerance and promote inflammation, fibrosis, cancer, and autoimmune diseases. TGF-β receptor II can bind and capture TGF-β molecules, blocking TGF-β expression and preventing its inhibitory effects on immune cells in the tumor microenvironment. Multispecific antibodies combining TGF-β receptor II with antibodies such as LRRC15 or FAP have shown promise in treating tumors and diseases such as tumor fibrosis.

[0010] Although a growing 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 involved in cancer growth and progression to metastasis. Although the immune system is a major mechanism for preventing cancer, cancer cells resist immune surveillance. Therefore, there is a pressing need in the art for improved therapeutic combinations of antagonists or antibodies and methods for treating cancer with these agents. Summary of the Invention

[0011] The present invention provides bispecific / trispecific antibodies comprising an LRRC15 antigen-binding domain, and also provides methods for treating diseases such as cancer using the antibodies and antibody conjugates of the present invention, and pharmaceutical compositions and preparations thereof.

[0012] In a first aspect of the present invention, a multispecific antibody is provided, comprising:

[0013] a first targeting domain comprising one or more LRRC15 antigen binding domains;

[0014] a second targeting domain comprising one or more CD40 antigen binding domains, or one or more CD3 antigen binding domains;

[0015] Optionally, it comprises a third targeting domain that binds to a target protein selected from the group consisting of PD-1, Claudin18.2 (CLDN18.2), and FAP; and / or a fourth targeting domain that binds to TGF-β.

[0016] In another preferred embodiment, the targeting domain is in the form of a single-chain Fv (scFv), a Fab fragment, a single-domain antibody (sdAb), a fragment variable (Fv) heterodimer, a TriFab or a combination thereof.

[0017] 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 following group:

[0018] In another preferred embodiment, the LRRC15 antigen binding domain comprises the following three heavy chain variable region CDRs:

[0019] HCDR1 having the amino acid sequence shown in SEQ ID NO: 33;

[0020] HCDR2 having the amino acid sequence shown in SEQ ID NO: 34; and

[0021] HCDR3 having the amino acid sequence shown in SEQ ID NO: 35;

[0022] and, the following three light chain variable region CDRs:

[0023] LCDR1, which has the amino acid sequence shown in SEQ ID NO:45;

[0024] LCDR2 having the amino acid sequence shown in SEQ ID NO: 46; and

[0025] LCDR3 having the amino acid sequence shown in SEQ ID NO:47.

[0026] In another preferred embodiment, the LRRC15 antigen binding domain comprises a heavy chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, 2, 3, 4 or 5.

[0027] In another preferred embodiment, the LRRC15 antigen binding domain comprises a light chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 6, 7, 8, 9 or 10.

[0028] In another preferred example, the LRRC15 antigen binding domain 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: 6.

[0029] In another preferred example, the LRRC15 antigen binding domain 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: 7.

[0030] In another preferred example, the LRRC15 antigen binding domain comprises a heavy chain variable region as shown in SEQ ID NO: 3, and a light chain variable region as shown in SEQ ID NO: 8.

[0031] In another preferred example, the LRRC15 antigen binding domain comprises a heavy chain variable region as shown in SEQ ID NO: 4, and a light chain variable region as shown in SEQ ID NO: 9.

[0032] In another preferred example, the LRRC15 antigen binding domain comprises a heavy chain variable region as shown in SEQ ID NO: 5, and a light chain variable region as shown in SEQ ID NO: 10.

[0033] In another preferred embodiment, the LRRC15 antigen binding domain is selected from the group consisting of scFv, Fab, or a combination thereof.

[0034] In another preferred embodiment, the LRRC15 antigen binding domain is scFv.

[0035] In another preferred embodiment, the LRRC15 antigen binding domain is a Fab comprising a heavy chain variable region as shown in SEQ ID NO: 1, 2, 3, 4 or 5, and a light chain variable region as shown in SEQ ID NO: 6, 7, 8, 9 or 10; and

[0036] The heavy chain constant region CH1 is shown in SEQ ID NO: 55, 56 or 64, and the light chain constant region CL is shown in SEQ ID NO: 66, 67 or 68.

[0037] In another preferred embodiment, the multispecific antibody further comprises an Fc fragment.

[0038] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4.

[0039] In another preferred embodiment, the Fc fragment is an Fc fragment derived from IgG1, which has an amino acid sequence as shown in SEQ ID NO: 57 or 58.

[0040] In another preferred embodiment, the Fc fragment derived from IgG1 has a mutation selected from the following group:

[0041] N297A,

[0042] L234F / L235E / P331S,

[0043] L234A / L235A / P329G

[0044] Y349C / K370E / K409D / K439E,

[0045] S354C / D356K / E357K / D399K,

[0046] S354C / T366W,

[0047] Y349C / T366S / L368A / Y407V,

[0048] L234F / L235E / P331S / Y349C / K370E / K409D / K439E,

[0049] L234F / L235E / P331S / S354C / D356K / E357K / D399K,

[0050] L234F / L235E / P331S / S354C / T366W,

[0051] L234F / L235E / P331S / Y349C / T366S / L368A / Y407V,

[0052] L234A / L235A / P329G / Y349C / K370E / K409D / K439E,

[0053] L234A / L235A / P329G / S354C / D356K / E357K / D399K,

[0054] L234A / L235A / P329G / S354C / T366W,

[0055] L234A / L235A / P329G / Y349C / T366S / L368A / Y407V,

[0056] L234F / L235E / P331S / S354C / T366W,

[0057] L234F / L235E / P331S / Y349C / T366S / L368A / Y407V,

[0058] N297A / Y349C / K370E / K409D / K439E,

[0059] N297A / S354C / D356K / E357K / D399K,

[0060] N297A / S354C / T366W,

[0061] N297A / Y349C / T366S / L368A / Y407V;

[0062] C220S / L234A / L235A / P329G / S354C / D356K / E357K / D399K,

[0063] C220S / L234A / L235A / P329G / Y349C / K370E / K409D / K439E; and / or

[0064] S267E,

[0065] S267E / G236D,

[0066] S267E / S239D,

[0067] S267E / L328F.

[0068] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in any one of SEQ ID NOs: 59-62, or an amino acid sequence as shown in any one of SEQ ID NOs: 96-98.

[0069] In another preferred embodiment, the Fc fragment is an Fc fragment derived from IgG4, which has the amino acid sequence shown in SEQ ID NO: 65.

[0070] In another preferred embodiment, the Fc fragment derived from IgG4 has a mutation selected from the following group:

[0071] Y349C / K370E / R409D / K439E,

[0072] S354C / E356K / E357K / D399K,

[0073] S354C / T366W,

[0074] Y349C / T366S / L368A / Y407V; and / or

[0075] S228P / S267E,

[0076] S228P / S267E / G236D,

[0077] S228P / S267E / S239D,

[0078] S228P / S267E / L328F.

[0079] In another preferred embodiment, the multispecific antibody is a bispecific, trispecific or tetraspecific antibody.

[0080] In another preferred embodiment, the multispecific antibody is a bispecific antibody.

[0081] In another preferred embodiment, the bispecific antibody comprises:

[0082] and a first targeting domain comprising one or more LRRC15 antigen binding domains; and a second targeting domain comprising one or more CD40 antigen binding domains.

[0083] In another preferred embodiment, the bispecific antibody comprises:

[0084] and a first targeting domain comprising one or more LRRC15 antigen binding domains; and a second targeting domain comprising one or more CD3 antigen binding domains.

[0085] In another preferred embodiment, the multispecific antibody is a trispecific antibody.

[0086] In another preferred embodiment, the trispecific antibody comprises:

[0087] a first targeting domain comprising one or more LRRC15 antigen binding domains;

[0088] a second targeting domain comprising one or more CD40 antigen binding domains; and

[0089] The third targeting domain is a PD-1 antigen binding domain.

[0090] In another preferred embodiment, the trispecific antibody comprises:

[0091] a first targeting domain comprising one or more LRRC15 antigen binding domains;

[0092] a second targeting domain comprising one or more CD40 antigen binding domains; and

[0093] The third targeting domain is a Claudin18.2 antigen binding domain.

[0094] In another preferred embodiment, the trispecific antibody comprises:

[0095] a first targeting domain comprising one or more LRRC15 antigen binding domains;

[0096] a second targeting domain comprising one or more CD3 antigen binding domains; and

[0097] The third targeting domain is a FAP antigen binding domain.

[0098] In another preferred embodiment, the antibody is a tetraspecific antibody.

[0099] In another preferred embodiment, the tetraspecific antibody comprises:

[0100] a first targeting domain comprising one or more LRRC15 antigen binding domains;

[0101] a second targeting domain comprising one or more CD3 antigen binding domains;

[0102] a third targeting domain, said third targeting domain being a FAP antigen binding domain; and

[0103] The fourth targeting domain is a TGF-β antigen binding domain.

[0104] In another preferred example, the CD40 antigen-binding domain comprises a heavy chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 11, and a light chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 12.

[0105] In another preferred example, the CD40 antigen-binding domain comprises a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 13-23, 114-116, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 12.

[0106] In another preferred example, the CD40 antigen-binding domain comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 11, and a light chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 24-26.

[0107] In another preferred example, the CD40 antigen-binding domain comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 15, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 12.

[0108] In another preferred example, the CD40 antigen-binding domain comprises a heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 27, and a light chain variable region with at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 28.

[0109] In another preferred embodiment, the CD40 antigen binding domain is selected from the group consisting of scFv, Fab, or a combination thereof.

[0110] In another preferred example, the CD3 antigen-binding domain comprises a heavy chain variable region having at least 80% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 82-84, 117, and a light chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 85.

[0111] In another preferred embodiment, the CD3 antigen binding domain is selected from the group consisting of scFv, Fab, or a combination thereof.

[0112] In another preferred example, the PD-1 antigen-binding domain comprises a heavy chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 29, and a light chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 30.

[0113] In another preferred embodiment, the PD-1 antigen binding domain is selected from the group consisting of scFv, Fab, or a combination thereof.

[0114] In another preferred example, the Claudin18.2 antigen binding domain comprises a heavy chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:31, and a light chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:32.

[0115] In another preferred embodiment, the Claudin18.2 antigen binding domain is selected from the following group: scFv, Fab, or a combination thereof.

[0116] In another preferred example, the FAP antigen-binding domain comprises a heavy chain variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 86, and a light chain variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 87; or a heavy chain variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 88, and a light chain variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 89; or a heavy chain variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 90, and a light chain variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 91; or a heavy chain variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 92, and a light chain variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 93.

[0117] In another preferred embodiment, the FAP antigen binding domain is selected from the group consisting of scFv, Fab, or a combination thereof.

[0118] In another preferred embodiment, the TGF-β antigen binding domain is the TGF-β receptor: TGF-βRII.

[0119] In another preferred embodiment, the TGF-β antigen binding domain comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 94 or 95.

[0120] In another preferred embodiment, the multispecific antibody has a structure shown in the following formula I (a in Figure 1):

[0121] In the formula, “-” is each independently a peptide bond or a connecting peptide; “║” is a connecting bond between peptide chains;

[0122] Fab1 is the first targeting domain, and the Fab1 is an anti-LRRC15 Fab;

[0123] scFv2 is a second targeting domain, and the scFv2 is an anti-CD40 scFv;

[0124] Fc1 is the Fc fragment.

[0125] In another preferred embodiment, the “║” is a disulfide bond.

[0126] In another preferred example, the anti-LRRC15 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 1, 2, 3, 4 or 5, and a light chain variable region as shown in SEQ ID NO: 6, 7, 8, 9 or 10.

[0127] In another preferred example, the LRRC15 Fab 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.

[0128] In another preferred embodiment, the anti-CD40 scFv comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 11, 13-23, 114-116, and a light chain variable region as shown in any one of SEQ ID NOs: 12, 24-26; or

[0129] The heavy chain variable region is shown in SEQ ID NO: 27, and the light chain variable region is shown in SEQ ID NO: 28.

[0130] In another preferred embodiment, the anti-CD40 scFv comprises the heavy chain variable region shown in SEQ ID NO: 15 and the light chain variable region shown in SEQ ID NO: 12.

[0131] In another preferred embodiment, the anti-CD40 scFv comprises the heavy chain variable region shown in SEQ ID NO: 11 and the light chain variable region shown in SEQ ID NO: 12.

[0132] In another preferred embodiment, the anti-CD40 scFv comprises the heavy chain variable region shown in SEQ ID NO: 19 and the light chain variable region shown in SEQ ID NO: 12.

[0133] In another preferred embodiment, the anti-CD40 scFv comprises the heavy chain variable region shown in SEQ ID NO: 115 and the light chain variable region shown in SEQ ID NO: 12.

[0134] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0135] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0136] In another preferred embodiment, the Fc fragment has the amino acid sequence shown in SEQ ID NO: 59.

[0137] In another preferred example, the amino acid sequence of "HC1 (heavy chain)-Fc1-scFv2" in the "Fab1-Fc1-scFv2" is shown in SEQ ID NO: 69, and the amino acid sequence of "LC1 (light chain)" is shown in SEQ ID NO: 70.

[0138] In another preferred example, the amino acid sequence of "HC1 (heavy chain)-Fc1-scFv1" in the "Fab1-Fc1-scFv1" is shown in SEQ ID NO:71, and the amino acid sequence of "LC1 (light chain)" is shown in SEQ ID NO:70.

[0139] In another preferred example, the amino acid sequence of "HC1 (heavy chain)-Fc1-scFv1" in the "Fab1-Fc1-scFv1" is shown in SEQ ID NO: 118, and the amino acid sequence of "LC1 (light chain)" is shown in SEQ ID NO: 70.

[0140] In another preferred example, the amino acid sequence of "HC1 (heavy chain)-Fc1-scFv1" in the "Fab1-Fc1-scFv1" is shown in SEQ ID NO: 119, and the amino acid sequence of "LC1 (light chain)" is shown in SEQ ID NO: 70.

[0141] In another preferred embodiment, the multispecific antibody has a structure as shown in the following formula II (b in FIG1 ):

[0142] In the formula, “-” is each independently a peptide bond or a connecting peptide; “║” is a connecting bond between peptide chains;

[0143] scFv1 is the first targeting domain, and the scFv1 is an anti-LRRC15 scFv;

[0144] Fab2 is the second targeting domain, and the Fab2 is an anti-CD40 Fab;

[0145] Fc1 is the Fc fragment.

[0146] In another preferred embodiment, the “║” is a disulfide bond.

[0147] In another preferred embodiment, the anti-LRRC15 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 1, 2, 3, 4 or 5, and a light chain variable region as shown in SEQ ID NO: 6, 7, 8, 9 or 10.

[0148] In another preferred embodiment, the LRRC15 scFv comprises the heavy chain variable region shown in SEQ ID NO: 5, and the light chain variable region shown in SEQ ID NO: 10.

[0149] In another preferred embodiment, the anti-CD40 Fab comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 11, 13-23, 114-116, and a light chain variable region as shown in any one of SEQ ID NOs: 12, 24-26; or

[0150] The heavy chain variable region is shown in SEQ ID NO: 27, and the light chain variable region is shown in SEQ ID NO: 28.

[0151] In another preferred embodiment, the anti-CD40 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 27, and a light chain variable region as shown in SEQ ID NO: 28.

[0152] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0153] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0154] In another preferred example, the Fc fragment has the amino acid sequence shown in SEQ ID NO: 59.

[0155] In another preferred example, the amino acid sequence of "HC2 (heavy chain)-Fc1-scFv1" in the "Fab2-Fc1-scFv1" is shown in SEQ ID NO:72, and the amino acid sequence of "LC2 (light chain)" is shown in SEQ ID NO:73.

[0156] In another preferred embodiment, the multispecific antibody has a structure as shown in the following formula III (c in Figure 1):

[0157] In the formula, “-” is each independently a peptide bond or a connecting peptide; “║” is a connecting bond between peptide chains;

[0158] Fab1 is the first targeting domain, and the Fab1 is an anti-LRRC15 Fab;

[0159] scFv2 is a second targeting domain, and the scFv2 is an anti-CD40 scFv;

[0160] Fab3 is the third targeting domain, and the Fab3 is anti-PD-1 Fab or anti-CLDN18.2 Fab;

[0161] Fc2 and Fc3 are Fc fragments.

[0162] In another preferred embodiment, the anti-LRRC15 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 1, 2, 3, 4 or 5, and a light chain variable region as shown in SEQ ID NO: 6, 7, 8, 9 or 10.

[0163] In another preferred example, the LRRC15 Fab 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.

[0164] In another preferred embodiment, the anti-CD40 scFv comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 11, 13-23, 114-116, and a light chain variable region as shown in any one of SEQ ID NOs: 12, 24-26; or

[0165] The heavy chain variable region is shown in SEQ ID NO: 27, and the light chain variable region is shown in SEQ ID NO: 28.

[0166] In another preferred embodiment, the anti-CD40 scFv comprises the heavy chain variable region shown in SEQ ID NO: 11 and the light chain variable region shown in SEQ ID NO: 12.

[0167] In another preferred example, the anti-PD-1 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 29, and a light chain variable region as shown in SEQ ID NO: 30.

[0168] In another preferred example, the anti-CLDN18.2 Fab comprises the heavy chain variable region shown in SEQ ID NO: 31, and the light chain variable region shown in SEQ ID NO: 32.

[0169] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0170] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0171] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in SEQ ID NO: 60 or 62.

[0172] In another preferred embodiment, the amino acid sequence of "HC1 (heavy chain)-Fc2-scFv2" in the "Fab1-Fc2-scFv2" is shown in SEQ ID NO: 74, and the amino acid sequence of "LC1 (light chain)" is shown in SEQ ID NO: 70;

[0173] The amino acid sequence of "HC3 (heavy chain)-Fc3-scFv2" in the "Fab3-Fc3-scFv2" is shown in SEQ ID NO:75, and the amino acid sequence of "LC3 (light chain)" is shown in SEQ ID NO:76.

[0174] In another preferred embodiment, the amino acid sequence of "HC1 (heavy chain)-Fc2-scFv2" in the "Fab1-Fc2-scFv2" is shown in SEQ ID NO: 74, and the amino acid sequence of "LC1 (light chain)" is shown in SEQ ID NO: 70;

[0175] The amino acid sequence of "HC3 (heavy chain)-Fc3-scFv2" in the "Fab3-Fc3-scFv2" is shown in SEQ ID NO:77, and the amino acid sequence of "LC3 (light chain)" is shown in SEQ ID NO:78.

[0176] In another preferred embodiment, the multispecific antibody has a structure as shown in the following formula IV (d in FIG1 ):

[0177] In the formula, “-” is each independently a peptide bond or a connecting peptide; “║” is a connecting bond between peptide chains;

[0178] scFv1 is the first targeting domain, and the scFv1 is an anti-LRRC15 scFv;

[0179] Fab2 is the second targeting domain, and the Fab2 is an anti-CD40 Fab;

[0180] scFv3 is the third targeting domain, and the scFv3 is an anti-PD-1 scFv or an anti-CLDN18.2 scFv;

[0181] Fc2 and Fc3 are Fc fragments.

[0182] In another preferred embodiment, the anti-LRRC15 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 1, 2, 3, 4 or 5, and a light chain variable region as shown in SEQ ID NO: 6, 7, 8, 9 or 10.

[0183] In another preferred embodiment, the LRRC15 scFv comprises the heavy chain variable region shown in SEQ ID NO: 5, and the light chain variable region shown in SEQ ID NO: 10.

[0184] In another preferred embodiment, the anti-CD40 Fab comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 11, 13-23, 114-116, and a light chain variable region as shown in any one of SEQ ID NOs: 12, 24-26; or

[0185] The heavy chain variable region is shown in SEQ ID NO: 27, and the light chain variable region is shown in SEQ ID NO: 28.

[0186] In another preferred embodiment, the anti-CD40 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 27, and a light chain variable region as shown in SEQ ID NO: 28.

[0187] In another preferred example, the anti-PD-1 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 29, and a light chain variable region as shown in SEQ ID NO: 30.

[0188] In another preferred embodiment, the anti-CLDN18.2 scFv comprises the heavy chain variable region shown in SEQ ID NO: 31 and the light chain variable region shown in SEQ ID NO: 32.

[0189] In another preferred embodiment, the anti-PD-1 scFv and anti-CLDN18.2 scFv contain cysteine ​​(C) mutations of VH44 and VL100 to improve the stability of the multispecific antibody.

[0190] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0191] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0192] In another preferred example, the Fc fragment has an amino acid sequence as shown in SEQ ID NO: 60 or 61.

[0193] In another preferred embodiment, the amino acid sequence of "HC2 (heavy chain)-Fc2-scFv1" in the "Fab2-Fc2-scFv1" is shown in SEQ ID NO: 79, and the amino acid sequence of "LC2 (light chain)" is shown in SEQ ID NO: 73;

[0194] The amino acid sequence of "HC2 (heavy chain)-Fc3-scFv3" in the "Fab2-Fc3-scFv3" is shown in SEQ ID NO:80, and the amino acid sequence of "LC2 (light chain)" is shown in SEQ ID NO:73.

[0195] In another preferred embodiment, the amino acid sequence of "HC2 (heavy chain)-Fc2-scFv1" in the "Fab2-Fc2-scFv1" is shown in SEQ ID NO: 79, and the amino acid sequence of "LC2 (light chain)" is shown in SEQ ID NO: 73;

[0196] The amino acid sequence of "HC2 (heavy chain)-Fc3-scFv3" in the "Fab2-Fc3-scFv3" is shown in SEQ ID NO:81, and the amino acid sequence of "LC2 (light chain)" is shown in SEQ ID NO:73.

[0197] In another preferred embodiment, the multispecific antibody has a structure as shown in the following formula V (a in FIG2 ):

[0198] In the formula, “-” is each independently a peptide bond or a connecting peptide; “║” is a connecting bond between peptide chains;

[0199] scFv1 is the first targeting domain, and the scFv1 is an anti-LRRC15 scFv;

[0200] scFv2 is the second targeting domain, and the scFv2 is an anti-CD3 scFv;

[0201] Fc2 and Fc3 are Fc fragments.

[0202] In another preferred embodiment, the anti-LRRC15 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 1, 2, 3, 4 or 5, and a light chain variable region as shown in SEQ ID NO: 6, 7, 8, 9 or 10.

[0203] In another preferred embodiment, the LRRC15 scFv comprises the heavy chain variable region shown in SEQ ID NO: 5, and the light chain variable region shown in SEQ ID NO: 10.

[0204] In another preferred embodiment, the anti-CD3 scFv comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 82-84, 117, and a light chain variable region as shown in SEQ ID NO: 85.

[0205] In another preferred example, the anti-CD3 scFv comprises the heavy chain variable region shown in SEQ ID NO: 83 and the light chain variable region shown in SEQ ID NO: 85.

[0206] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0207] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0208] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in SEQ ID NO: 97 or 98.

[0209] In another preferred embodiment, the amino acid sequence of the "scFv1-scFv2-Fc2" is shown in SEQ ID NO: 99; the amino acid sequence of the Fc3 is shown in SEQ ID NO: 98.

[0210] In another preferred embodiment, the multispecific antibody has a structure as shown in the following formula VI (b in FIG2 ):

[0211] In the formula, “-” is each independently a peptide bond or a connecting peptide; “║” is a connecting bond between peptide chains;

[0212] scFv1 is the first targeting domain, and the scFv1 is an anti-LRRC15 scFv;

[0213] scFv2 is the second targeting domain, and the scFv2 is an anti-CD3 scFv;

[0214] Fab3 is the third targeting domain, and the Fab3 is an anti-FAP Fab;

[0215] Fc2 and Fc3 are Fc fragments.

[0216] In another preferred embodiment, the anti-LRRC15 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 1, 2, 3, 4 or 5, and a light chain variable region as shown in SEQ ID NO: 6, 7, 8, 9 or 10.

[0217] In another preferred embodiment, the LRRC15 scFv comprises the heavy chain variable region shown in SEQ ID NO: 5, and the light chain variable region shown in SEQ ID NO: 10.

[0218] In another preferred embodiment, the anti-CD3 scFv comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 82-84, 117, and a light chain variable region as shown in SEQ ID NO: 85.

[0219] In another preferred example, the anti-CD3 scFv comprises the heavy chain variable region shown in SEQ ID NO: 83 and the light chain variable region shown in SEQ ID NO: 85.

[0220] In another preferred example, the anti-FAP Fab comprises a heavy chain variable region as shown in SEQ ID NO: 86, and a light chain variable region as shown in SEQ ID NO: 87; or a heavy chain variable region as shown in SEQ ID NO: 88, and a light chain variable region as shown in SEQ ID NO: 89; or a heavy chain variable region as shown in SEQ ID NO: 90, and a light chain variable region as shown in SEQ ID NO: 91; or a heavy chain variable region as shown in SEQ ID NO: 92, and a light chain variable region as shown in SEQ ID NO: 93.

[0221] In another preferred example, the anti-FAP Fab comprises a heavy chain variable region as shown in SEQ ID NO: 86, and a light chain variable region as shown in SEQ ID NO: 87.

[0222] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0223] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0224] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in SEQ ID NO: 96 or 97.

[0225] In another preferred example, the amino acid sequence of the "scFv1-scFv2-Fc2" is shown in SEQ ID NO: 99; the amino acid sequence of "HC3 (heavy chain)-Fc3" in the "Fab3-Fc3" is shown in SEQ ID NO: 100, and the amino acid sequence of "LC3 (light chain)" is shown in SEQ ID NO: 101.

[0226] In another preferred embodiment, the multispecific antibody has a structure as shown in the following formula VI (c in FIG2 ):

[0227] In the formula, “-” is each independently a peptide bond or a connecting peptide; “║” is a connecting bond between peptide chains;

[0228] Fab1 is the first targeting domain, and the Fab1 is an anti-LRRC15 Fab;

[0229] scFv2 is the second targeting domain, and the scFv2 is an anti-CD3 scFv;

[0230] scFv3 is the third targeting domain, and the scFv3 is an anti-FAP scFv;

[0231] Fc2 and Fc3 are Fc fragments.

[0232] In another preferred example, the anti-LRRC15 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 1, 2, 3, 4 or 5, and a light chain variable region as shown in SEQ ID NO: 6, 7, 8, 9 or 10.

[0233] In another preferred embodiment, the LRRC15 Fab 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: 6.

[0234] In another preferred embodiment, the anti-CD3 scFv comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 82-84, 117, and a light chain variable region as shown in SEQ ID NO: 85.

[0235] In another preferred embodiment, the anti-CD3 scFv comprises the heavy chain variable region shown in SEQ ID NO: 83 and the light chain variable region shown in SEQ ID NO: 85.

[0236] In another preferred example, the anti-FAP scFv comprises the heavy chain variable region shown in SEQ ID NO: 86, and the light chain variable region shown in SEQ ID NO: 87; or the heavy chain variable region shown in SEQ ID NO: 88, and the light chain variable region shown in SEQ ID NO: 89; or the heavy chain variable region shown in SEQ ID NO: 90, and the light chain variable region shown in SEQ ID NO: 91; or the heavy chain variable region shown in SEQ ID NO: 92, and the light chain variable region shown in SEQ ID NO: 93.

[0237] In another preferred example, the anti-FAP scFv comprises the heavy chain variable region shown in SEQ ID NO: 86, and the light chain variable region shown in SEQ ID NO: 87.

[0238] In another preferred embodiment, the anti-FAP scFv comprises cysteine ​​(C) mutations of VH44 and VL100 to improve the stability of the multispecific antibody.

[0239] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0240] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0241] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in SEQ ID NO: 96 or 97.

[0242] In another preferred example, the amino acid sequence of the "scFv3-scFv2-Fc2" is shown in SEQ ID NO: 102; the amino acid sequence of "HC1 (heavy chain)-Fc3" in the "Fab1-Fc3" is shown in SEQ ID NO: 103, and the amino acid sequence of "LC1 (light chain)" is shown in SEQ ID NO: 70.

[0243] In another preferred embodiment, the multispecific antibody has a structure shown in the following formula VII (d in FIG2 ):

[0244] In the formula, “-” is each independently a peptide bond or a connecting peptide; “║” is a connecting bond between peptide chains;

[0245] scFv1 is the first targeting domain, and the scFv1 is an anti-LRRC15 scFv;

[0246] scFv2 is the second targeting domain, and the scFv2 is an anti-CD3 scFv;

[0247] Fab3 is the third targeting domain, and the Fab3 is an anti-FAP Fab;

[0248] R4 is the fourth targeting domain, and the R4 is TGF-βRII.

[0249] Fc2 and Fc3 are Fc fragments.

[0250] In another preferred embodiment, the anti-LRRC15 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 1, 2, 3, 4 or 5, and a light chain variable region as shown in SEQ ID NO: 6, 7, 8, 9 or 10.

[0251] In another preferred embodiment, the LRRC15 scFv comprises the heavy chain variable region shown in SEQ ID NO: 5, and the light chain variable region shown in SEQ ID NO: 10.

[0252] In another preferred embodiment, the anti-CD3 scFv comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 82-84, 117, and a light chain variable region as shown in SEQ ID NO: 85.

[0253] In another preferred embodiment, the anti-CD3 scFv comprises the heavy chain variable region shown in SEQ ID NO: 83 and the light chain variable region shown in SEQ ID NO: 85.

[0254] In another preferred example, the anti-FAP Fab comprises a heavy chain variable region as shown in SEQ ID NO: 86, and a light chain variable region as shown in SEQ ID NO: 87; or a heavy chain variable region as shown in SEQ ID NO: 88, and a light chain variable region as shown in SEQ ID NO: 89; or a heavy chain variable region as shown in SEQ ID NO: 90, and a light chain variable region as shown in SEQ ID NO: 91; or a heavy chain variable region as shown in SEQ ID NO: 92, and a light chain variable region as shown in SEQ ID NO: 93.

[0255] In another preferred example, the anti-FAP Fab comprises a heavy chain variable region as shown in SEQ ID NO: 86, and a light chain variable region as shown in SEQ ID NO: 87.

[0256] In another preferred embodiment, the amino acid sequence of the TGF-βRII is shown in SEQ ID NO: 94 or 95.

[0257] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0258] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0259] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in SEQ ID NO: 96 or 97.

[0260] In another preferred example, the amino acid sequence of the "scFv1-scFv2-Fc2-R4" is shown in SEQ ID NO: 104; the amino acid sequence of "HC3 (heavy chain)-Fc3-R4" in the "Fab3-Fc3-R4" is shown in SEQ ID NO: 105, and the amino acid sequence of "LC3 (light chain)" is shown in SEQ ID NO: 101.

[0261] In another preferred embodiment, the multispecific antibody has a structure as shown in the following formula VIII (e in FIG2 ):

[0262] In the formula, “-” is each independently a peptide bond or a connecting peptide; “║” is a connecting bond between peptide chains;

[0263] Fab1 is the first targeting domain, and the Fab1 is an anti-LRRC15 Fab;

[0264] scFv2 is the second targeting domain, and the scFv2 is an anti-CD3 scFv;

[0265] scFv3 is the third targeting domain, and the scFv3 is an anti-FAP scFv;

[0266] Fc2 and Fc3 are Fc fragments.

[0267] In another preferred example, the anti-LRRC15 Fab comprises a heavy chain variable region as shown in SEQ ID NO: 1, 2, 3, 4 or 5, and a light chain variable region as shown in SEQ ID NO: 6, 7, 8, 9 or 10.

[0268] In another preferred embodiment, the LRRC15 Fab 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: 6.

[0269] In another preferred embodiment, the anti-CD3 scFv comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 82-84, 117, and a light chain variable region as shown in SEQ ID NO: 85.

[0270] In another preferred embodiment, the anti-CD3 scFv comprises the heavy chain variable region shown in SEQ ID NO: 83 and the light chain variable region shown in SEQ ID NO: 85.

[0271] In another preferred example, the anti-FAP scFv comprises the heavy chain variable region shown in SEQ ID NO: 86, and the light chain variable region shown in SEQ ID NO: 87; or the heavy chain variable region shown in SEQ ID NO: 88, and the light chain variable region shown in SEQ ID NO: 89; or the heavy chain variable region shown in SEQ ID NO: 90, and the light chain variable region shown in SEQ ID NO: 91; or the heavy chain variable region shown in SEQ ID NO: 92, and the light chain variable region shown in SEQ ID NO: 93.

[0272] In another preferred example, the anti-FAP scFv comprises the heavy chain variable region shown in SEQ ID NO: 86, and the light chain variable region shown in SEQ ID NO: 87.

[0273] In another preferred embodiment, the anti-FAP scFv comprises cysteine ​​(C) mutations of VH44 and VL100 to improve the stability of the multispecific antibody.

[0274] In another preferred embodiment, the amino acid sequence of the TGF-βRII is shown in SEQ ID NO: 94 or 95.

[0275] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0276] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0277] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in SEQ ID NO: 96 or 97.

[0278] In another preferred example, the amino acid sequence of the "scFv3-scFv2-Fc2-R4" is shown in SEQ ID NO: 106; the amino acid sequence of "HC1 (heavy chain)-Fc3-R4" in the "Fab1-Fc3-R4" is shown in SEQ ID NO: 107, and the amino acid sequence of "LC1 (light chain)" is shown in SEQ ID NO: 70.

[0279] In another preferred embodiment, the multispecific antibody has a structure as shown in the following formula IX (f in FIG2 ):

[0280] In the formula, “-” is each independently a peptide bond or a connecting peptide; “║” is a connecting bond between peptide chains;

[0281] scFv1 is the first targeting domain, and the scFv1 is an anti-LRRC15 scFv;

[0282] scFv2 is the second targeting domain, and the scFv2 is an anti-CD3 scFv;

[0283] scFv3 is the third targeting domain, and the scFv3 is an anti-FAP scFv;

[0284] Fc2 and Fc3 are Fc fragments.

[0285] In another preferred embodiment, the anti-LRRC15 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 1, 2, 3, 4 or 5, and a light chain variable region as shown in SEQ ID NO: 6, 7, 8, 9 or 10.

[0286] In another preferred embodiment, the LRRC15 scFv comprises the heavy chain variable region shown in SEQ ID NO: 5, and the light chain variable region shown in SEQ ID NO: 10.

[0287] In another preferred embodiment, the anti-CD3 scFv comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 82-84, 117, and a light chain variable region as shown in SEQ ID NO: 85.

[0288] In another preferred example, the anti-CD3 scFv comprises the heavy chain variable region shown in SEQ ID NO: 83 and the light chain variable region shown in SEQ ID NO: 85.

[0289] In another preferred example, the anti-FAP scFv comprises the heavy chain variable region shown in SEQ ID NO: 86, and the light chain variable region shown in SEQ ID NO: 87; or the heavy chain variable region shown in SEQ ID NO: 88, and the light chain variable region shown in SEQ ID NO: 89; or the heavy chain variable region shown in SEQ ID NO: 90, and the light chain variable region shown in SEQ ID NO: 91; or the heavy chain variable region shown in SEQ ID NO: 92, and the light chain variable region shown in SEQ ID NO: 93.

[0290] In another preferred example, the anti-FAP scFv comprises the heavy chain variable region shown in SEQ ID NO: 86, and the light chain variable region shown in SEQ ID NO: 87.

[0291] In another preferred embodiment, the anti-FAP scFv comprises cysteine ​​(C) mutations of VH44 and VL100 to improve the stability of the multispecific antibody.

[0292] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0293] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0294] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in SEQ ID NO: 97 or 98.

[0295] In another preferred embodiment, the amino acid sequence of the "scFv3-scFv1-scFv2-Fc2" is shown in SEQ ID NO: 108; the amino acid sequence of the Fc3 is shown in SEQ ID NO: 98.

[0296] In another preferred embodiment, the multispecific antibody has a structure as shown in the following formula X (g in FIG2 ):

[0297] In the formula, “-” is each independently a peptide bond or a connecting peptide; “║” is a connecting bond between peptide chains;

[0298] scFv1 is the first targeting domain, and the scFv1 is an anti-LRRC15 scFv;

[0299] scFv2 is the second targeting domain, and the scFv2 is an anti-CD3 scFv;

[0300] scFv3 is the third targeting domain, and the scFv3 is an anti-FAP scFv;

[0301] Fc2 and Fc3 are Fc fragments.

[0302] In another preferred embodiment, the anti-LRRC15 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 1, 2, 3, 4 or 5, and a light chain variable region as shown in SEQ ID NO: 6, 7, 8, 9 or 10.

[0303] In another preferred embodiment, the LRRC15 scFv comprises the heavy chain variable region shown in SEQ ID NO: 5, and the light chain variable region shown in SEQ ID NO: 10.

[0304] In another preferred embodiment, the anti-CD3 scFv comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 82-84, 117, and a light chain variable region as shown in SEQ ID NO: 85.

[0305] In another preferred embodiment, the anti-CD3 scFv comprises the heavy chain variable region shown in SEQ ID NO: 83 and the light chain variable region shown in SEQ ID NO: 85.

[0306] In another preferred example, the anti-FAP scFv comprises the heavy chain variable region shown in SEQ ID NO: 86, and the light chain variable region shown in SEQ ID NO: 87; or the heavy chain variable region shown in SEQ ID NO: 88, and the light chain variable region shown in SEQ ID NO: 89; or the heavy chain variable region shown in SEQ ID NO: 90, and the light chain variable region shown in SEQ ID NO: 91; or the heavy chain variable region shown in SEQ ID NO: 92, and the light chain variable region shown in SEQ ID NO: 93.

[0307] In another preferred example, the anti-FAP scFv comprises the heavy chain variable region shown in SEQ ID NO: 86, and the light chain variable region shown in SEQ ID NO: 87.

[0308] In another preferred embodiment, the anti-FAP scFv comprises cysteine ​​(C) mutations of VH44 and VL100 to improve the stability of the multispecific antibody.

[0309] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0310] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0311] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in SEQ ID NO: 97 or 98.

[0312] In another preferred embodiment, the amino acid sequence of the "scFv1-scFv3-scFv2-Fc2" is shown in SEQ ID NO: 109; the amino acid sequence of the Fc3 is shown in SEQ ID NO: 98.

[0313] In another preferred embodiment, the multispecific antibody has a structure as shown in the following formula XI (h in FIG. 2 ):

[0314] In the formula, “-” is each independently a peptide bond or a connecting peptide; “║” is a connecting bond between peptide chains;

[0315] scFv1 is the first targeting domain, and the scFv1 is an anti-LRRC15 scFv;

[0316] scFv2 is the second targeting domain, and the scFv2 is an anti-CD3 scFv;

[0317] scFv3 is the third targeting domain, and the scFv3 is an anti-FAP scFv;

[0318] Fc2 and Fc3 are Fc fragments.

[0319] In another preferred embodiment, the anti-LRRC15 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 1, 2, 3, 4 or 5, and a light chain variable region as shown in SEQ ID NO: 6, 7, 8, 9 or 10.

[0320] In another preferred embodiment, the LRRC15 scFv comprises the heavy chain variable region shown in SEQ ID NO: 5, and the light chain variable region shown in SEQ ID NO: 10.

[0321] In another preferred embodiment, the anti-CD3 scFv comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 82-84, 117, and a light chain variable region as shown in SEQ ID NO: 85.

[0322] In another preferred embodiment, the anti-CD3 scFv comprises the heavy chain variable region shown in SEQ ID NO: 83 and the light chain variable region shown in SEQ ID NO: 85.

[0323] In another preferred example, the anti-FAP scFv comprises the heavy chain variable region shown in SEQ ID NO: 86, and the light chain variable region shown in SEQ ID NO: 87; or the heavy chain variable region shown in SEQ ID NO: 88, and the light chain variable region shown in SEQ ID NO: 89; or the heavy chain variable region shown in SEQ ID NO: 90, and the light chain variable region shown in SEQ ID NO: 91; or the heavy chain variable region shown in SEQ ID NO: 92, and the light chain variable region shown in SEQ ID NO: 93.

[0324] In another preferred example, the anti-FAP scFv comprises the heavy chain variable region shown in SEQ ID NO: 86, and the light chain variable region shown in SEQ ID NO: 87.

[0325] In another preferred example, the anti-FAP scFv comprises the heavy chain variable region shown in SEQ ID NO: 92, and the light chain variable region shown in SEQ ID NO: 93.

[0326] In another preferred embodiment, the anti-FAP scFv comprises cysteine ​​(C) mutations of VH44 and VL100 to improve the stability of the multispecific antibody.

[0327] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0328] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0329] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in SEQ ID NO: 97 or 98.

[0330] In another preferred example, the amino acid sequence of the "scFv1-scFv2-Fc2" is shown in SEQ ID NO:99; the amino acid sequence of the "Fc3-scFv3-scFv2" is shown in SEQ ID NO:110.

[0331] In another preferred embodiment, the multispecific antibody has a structure as shown in the following formula XII (i in Figure 2):

[0332] In the formula, “-” is each independently a peptide bond or a connecting peptide; “║” is a connecting bond between peptide chains;

[0333] scFv1 is the first targeting domain, and the scFv1 is an anti-LRRC15 scFv;

[0334] scFv2 is the second targeting domain, and the scFv2 is an anti-CD3 scFv;

[0335] scFv3 is the third targeting domain, and the scFv3 is an anti-FAP scFv;

[0336] Fc2 and Fc3 are Fc fragments.

[0337] In another preferred embodiment, the anti-LRRC15 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 1, 2, 3, 4 or 5, and a light chain variable region as shown in SEQ ID NO: 6, 7, 8, 9 or 10.

[0338] In another preferred embodiment, the LRRC15 scFv comprises the heavy chain variable region shown in SEQ ID NO: 5, and the light chain variable region shown in SEQ ID NO: 10.

[0339] In another preferred embodiment, the anti-CD3 scFv comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 82-84, 117, and a light chain variable region as shown in SEQ ID NO: 85.

[0340] In another preferred embodiment, the anti-CD3 scFv comprises the heavy chain variable region shown in SEQ ID NO: 83 and the light chain variable region shown in SEQ ID NO: 85.

[0341] In another preferred example, the anti-FAP scFv comprises the heavy chain variable region shown in SEQ ID NO: 86, and the light chain variable region shown in SEQ ID NO: 87; or the heavy chain variable region shown in SEQ ID NO: 88, and the light chain variable region shown in SEQ ID NO: 89; or the heavy chain variable region shown in SEQ ID NO: 90, and the light chain variable region shown in SEQ ID NO: 91; or the heavy chain variable region shown in SEQ ID NO: 92, and the light chain variable region shown in SEQ ID NO: 93.

[0342] In another preferred example, the anti-FAP scFv comprises the heavy chain variable region shown in SEQ ID NO: 86, and the light chain variable region shown in SEQ ID NO: 87.

[0343] In another preferred embodiment, the anti-FAP scFv comprises cysteine ​​(C) mutations of VH44 and VL100 to improve the stability of the multispecific antibody.

[0344] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0345] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0346] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in SEQ ID NO: 97 or 98.

[0347] In another preferred example, the amino acid sequence of the "scFv1-scFv2-Fc2" is shown in SEQ ID NO:99; the amino acid sequence of the "Fc3-scFv2-scFv3" is shown in SEQ ID NO:111.

[0348] In another preferred embodiment, the multispecific antibody has a structure as shown in Formula XIII or Formula XIV below (j and k in Figure 2):

[0349] In the formula, “-” is each independently a peptide bond or a connecting peptide; “║” is a connecting bond between peptide chains;

[0350] scFv1 is the first targeting domain, and the scFv1 is an anti-LRRC15 scFv;

[0351] scFv2 is the second targeting domain, and the scFv2 is an anti-CD3 scFv;

[0352] scFv3 is the third targeting domain, and the scFv3 is an anti-FAP scFv;

[0353] Fc2 and Fc3 are Fc fragments.

[0354] In another preferred embodiment, the anti-LRRC15 scFv comprises a heavy chain variable region as shown in SEQ ID NO: 1, 2, 3, 4 or 5, and a light chain variable region as shown in SEQ ID NO: 6, 7, 8, 9 or 10.

[0355] In another preferred embodiment, the LRRC15 scFv comprises the heavy chain variable region shown in SEQ ID NO: 5, and the light chain variable region shown in SEQ ID NO: 10.

[0356] In another preferred embodiment, the anti-CD3 scFv comprises a heavy chain variable region as shown in any one of SEQ ID NOs: 82-84, 117, and a light chain variable region as shown in SEQ ID NO: 85.

[0357] In another preferred example, the anti-CD3 scFv comprises the heavy chain variable region shown in SEQ ID NO: 83 and the light chain variable region shown in SEQ ID NO: 85.

[0358] In another preferred example, the anti-FAP scFv comprises the heavy chain variable region shown in SEQ ID NO: 86, and the light chain variable region shown in SEQ ID NO: 87; or the heavy chain variable region shown in SEQ ID NO: 88, and the light chain variable region shown in SEQ ID NO: 89; or the heavy chain variable region shown in SEQ ID NO: 90, and the light chain variable region shown in SEQ ID NO: 91; or the heavy chain variable region shown in SEQ ID NO: 92, and the light chain variable region shown in SEQ ID NO: 93.

[0359] In another preferred example, the anti-FAP scFv comprises the heavy chain variable region shown in SEQ ID NO: 86, and the light chain variable region shown in SEQ ID NO: 87.

[0360] In another preferred embodiment, the anti-FAP scFv comprises cysteine ​​(C) mutations of VH44 and VL100 to improve the stability of the multispecific antibody.

[0361] In another preferred embodiment, the Fc fragment is derived from IgG1 or IgG4

[0362] In another preferred embodiment, the Fc fragment is derived from IgG1.

[0363] In another preferred embodiment, the Fc fragment has an amino acid sequence as shown in SEQ ID NO: 97 or 98.

[0364] In another preferred embodiment, the amino acid sequence of the "scFv3-scFv2-Fc2" is shown in SEQ ID NO: 102; the amino acid sequence of the "Fc3-scFv1-scFv2" is shown in SEQ ID NO: 112.

[0365] In another preferred example, the amino acid sequence of the "scFv3-scFv2-Fc2" is shown in SEQ ID NO: 102; the amino acid sequence of the "Fc3-scFv2-scFv1" is shown in SEQ ID NO: 113.

[0366] The second aspect of the present invention provides a polynucleotide encoding the multispecific antibody according to the first aspect of the present invention.

[0367] The third aspect of the present invention provides an expression vector, which comprises the polynucleotide as described in the second aspect of the present invention.

[0368] In another preferred embodiment, the expression vector includes a prokaryotic expression vector and a eukaryotic expression vector.

[0369] The fourth aspect of the present invention provides a host cell, which comprises the expression vector as described in the third aspect of the present invention, or the polynucleotide as described in the second aspect of the present invention is integrated into its genome.

[0370] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0371] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, HEK 293T cells, and CHO cells.

[0372] The fifth aspect of the present invention provides a use of the multispecific antibody according to the first aspect of the present invention for preparing a medicament for treating cancer / tumor and / or fibrotic disease.

[0373] In another preferred embodiment, the cancer / tumor is a cancer / tumor with high expression of LRRC15.

[0374] In another preferred embodiment, the cancer / tumor is a cancer / tumor with high expression of LRRC15 and high expression of PD-1.

[0375] In another preferred embodiment, the cancer / tumor is a cancer / tumor with high expression of LRRC15 and high expression of CLDN18.2.

[0376] In another preferred embodiment, the cancer / tumor is a cancer / tumor with high expression of LRRC15 and high expression of FAP.

[0377] In another preferred embodiment, the cancer / tumor includes solid tumors and blood tumors.

[0378] In another preferred embodiment, the cancer / tumor is a solid tumor.

[0379] In another preferred embodiment, the cancer / tumor is an epithelial cancer / tumor.

[0380] In another preferred embodiment, the cancer / tumor is selected from the group consisting of breast cancer, head and neck cancer, lung cancer, pancreatic cancer, ovarian cancer, melanoma, malignant glioma, sarcoma, bone cancer, colon cancer, bronchial cancer, or a combination thereof.

[0381] In another preferred embodiment, the fibrotic disease includes fibrosis of multiple organs such as lung, liver, kidney, heart, brain, spleen, muscle and skin, as well as tumor-associated fibroblastic lesions.

[0382] The sixth aspect of the present invention provides an immunoconjugate, wherein the conjugate comprises:

[0383] (i) the multispecific antibody according to the first aspect of the present invention; and

[0384] (ii) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0385] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (for example, cisplatin) or any form of nanoparticles, etc.

[0386] The seventh aspect of the present invention provides a pharmaceutical composition, comprising: (a) the multispecific antibody as described in the first aspect of the present invention, or the immunoconjugate as described in the sixth aspect of the present invention; and (b) a pharmaceutically acceptable carrier.

[0387] In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

[0388] The eighth aspect of the present invention provides the use of the immunoconjugate according to the sixth aspect of the present invention for preparing a medicament for treating cancer / tumor and / or fibrotic disease.

[0389] In another preferred embodiment, the cancer / tumor is a cancer / tumor with high expression of LRRC15.

[0390] In another preferred embodiment, the cancer / tumor is a cancer / tumor with high expression of LRRC15 and high expression of PD-1.

[0391] In another preferred embodiment, the cancer / tumor is a cancer / tumor with high expression of LRRC15 and high expression of CLDN18.2.

[0392] In another preferred embodiment, the cancer / tumor is a cancer / tumor with high expression of LRRC15 and high expression of FAP.

[0393] In another preferred embodiment, the cancer / tumor includes solid tumors and blood tumors.

[0394] In another preferred embodiment, the cancer / tumor is a solid tumor.

[0395] In another preferred embodiment, the cancer / tumor is an epithelial cancer / tumor.

[0396] In another preferred embodiment, the cancer / tumor is selected from the group consisting of breast cancer, head and neck cancer, lung cancer, pancreatic cancer, ovarian cancer, melanoma, malignant glioma, sarcoma, bone cancer, colon cancer, bronchial cancer, or a combination thereof.

[0397] In another preferred embodiment, the fibrotic disease includes fibrosis of multiple organs such as lung, liver, kidney, heart, brain, spleen, muscle and skin, as well as tumor-associated fibroblastic lesions.

[0398] The ninth aspect of the present invention provides a method for treating cancer / tumor and / or fibrotic disease, comprising administering the multispecific antibody described in the first aspect of the present invention, or the immunoconjugate described in the sixth aspect of the present invention to a subject in need.

[0399] In another preferred embodiment, the subject in need thereof is a human or non-human mammal.

[0400] In another preferred embodiment, the cancer / tumor is a cancer / tumor with high expression of LRRC15.

[0401] In another preferred embodiment, the cancer / tumor is a cancer / tumor with high expression of LRRC15 and high expression of PD-1.

[0402] In another preferred embodiment, the cancer / tumor is a cancer / tumor with high expression of LRRC15 and high expression of CLDN18.2.

[0403] In another preferred embodiment, the cancer / tumor is a cancer / tumor with high expression of LRRC15 and high expression of FAP.

[0404] In another preferred embodiment, the cancer / tumor includes solid tumors and blood tumors.

[0405] In another preferred embodiment, the cancer / tumor is a solid tumor.

[0406] In another preferred embodiment, the cancer / tumor is an epithelial cancer / tumor.

[0407] In another preferred embodiment, the cancer / tumor is selected from the group consisting of breast cancer, head and neck cancer, lung cancer, pancreatic cancer, ovarian cancer, melanoma, malignant glioma, sarcoma, bone cancer, colon cancer, bronchial cancer, or a combination thereof.

[0408] In another preferred embodiment, the fibrotic disease includes fibrosis of multiple organs such as lung, liver, kidney, heart, brain, spleen, muscle and skin, as well as tumor-associated fibroblastic lesions.

[0409] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0410] Figure 1 shows two bispecific antibody structures (a and b) consisting of the LRRC15 antigen-binding domain and the CD40 antigen-binding domain; and two trispecific antibody structures (c and d) consisting of the LRRC15 antigen-binding domain and the CD40 antigen-binding domain, CLND18.2 or PD-1 antigen-binding domain.

[0411] FIG1A shows an exemplary bispecific antibody structure of FIG1a , designated 1540s.

[0412] FIG1B shows another exemplary bispecific antibody structure of FIG1a, designated 1540s-3.

[0413] FIG1C shows an exemplary bispecific antibody structure of FIG1b , designated 40d15.

[0414] FIG1D shows an exemplary trispecific antibody of FIG1c, designated 151840-L06.

[0415] FIG1E shows another exemplary trispecific antibody of FIG1C , designated 15P140-L06.

[0416] FIG1F shows an exemplary trispecific antibody of FIG1d, designated 40d1815.

[0417] FIG1G shows another exemplary trispecific antibody of FIG1d , designated 40dP115.

[0418] Figure 2 shows the bispecific antibody structure consisting of the LRRC15 antigen-binding domain and the CD3 antigen-binding domain (a), the trispecific antibody structure consisting of the aforementioned two domains plus the FAP antigen-binding domain (b, c, f, g, h, I, j, k), and the tetraspecific antibody structure consisting of the aforementioned three domains plus the TGF-β binding domain (d, e).

[0419] FIG2A shows an exemplary bispecific antibody structure of FIG2a, designated 153.

[0420] FIG2B shows an exemplary trispecific antibody structure of FIG2b, designated 315F.

[0421] FIG2C shows an exemplary trispecific antibody structure of FIG2C , designated F315.

[0422] FIG2D shows an exemplary tetraspecific antibody structure of FIG2d , designated 315Fb.

[0423] FIG2E shows an exemplary tetraspecific antibody structure of FIG2e , designated F315b.

[0424] FIG2F shows an exemplary trispecific antibody structure of FIG2f , designated F153.

[0425] Figure 2G shows an exemplary trispecific antibody structure of Figure 2g, designated 15F3.

[0426] Figure 2H shows an exemplary trispecific antibody structure of Figure 2h, designated 153F.

[0427] Figure 2I shows an exemplary trispecific antibody structure of Figure 2i, designated 1533F.

[0428] FIG2J shows an exemplary trispecific antibody structure of FIG2j , designated F3153.

[0429] Figure 2K shows an exemplary trispecific antibody structure of Figure 2k, designated F3315.

[0430] [Corrected 24.04.2025 according to Rule 91] Figures 3A and 3B show the results of the detection of DC cell activation by the constructed bispecific antibody targeting LRRC15 and CD40.

[0431] FIG4A shows the detection results of the constructed bispecific antibody targeting LRRC15 and CD40 activating the CD40 reporter gene signal.

[0432] FIG4B shows the detection results of the constructed bispecific antibody targeting LRRC15 and CD40 activating the CD40 reporter gene signal.

[0433] Figure 4C shows the detection results of the CD3 reporter gene signal activated by the constructed trispecific antibody targeting LRRC15, FAP and CD3.

[0434] FIG5 shows the detection results of cytokine release stimulated by PBMCs using the constructed bispecific antibody targeting LRRC15 and CD40.

[0435] FIG6A shows the results of tumor volume changes in a mouse KPC model after administration of a bispecific antibody targeting LRRC15 and CD40.

[0436] FIG6B shows the results of body weight changes in the mouse KPC model after administration of a bispecific antibody targeting LRRC15 and CD40.

[0437] Figure 7A shows the results of tumor volume changes in the mouse MC38 model after administration of a control monoclonal antibody and a bispecific antibody targeting LRRC15 and CD40, respectively.

[0438] Figure 7B shows the results of body weight changes in the MC38 mouse model after administration of a control monoclonal antibody and a bispecific antibody targeting LRRC15 and CD40, respectively.

[0439] Figure 7C shows the expression of ALT and AST in MC38 model mice induced by the control monoclonal antibody and the bispecific antibody targeting LRRC15 and CD40 24 hours after administration.

[0440] FIG8 shows the effects of bispecific antibodies targeting LRRC15 and CD40 on body weight in cynomolgus monkeys. DETAILED DESCRIPTION

[0441] After extensive and in-depth research, the inventors unexpectedly developed a class of multispecific antibodies containing LRRC15 antigen-binding domains for the first time. The multispecific T cell binder contains a first targeting domain that targets the leucine-rich repeat sequence 15 (LRRC15) molecule highly expressed on the surface of tumor cells, which comprises an LRRC15 antibody or an antigen-binding fragment thereof, and also comprises a second targeting domain that binds to the CD40 molecule on the surface of antigen-presenting cells (APCs) or the CD3 molecule on the surface of T cells, and optionally comprises a third targeting domain that targets PD-1, CLDN18.2 or FAP, and / or a fourth targeting domain that targets TGF-β. The LRRC15-related multispecific antibodies of the present invention can effectively activate T cells and reduce the matrix effects of tumors, and can provide effective treatment for tumors and tumor-related fibrosis. On this basis, the present invention was completed.

[0442] As used herein, the term "leucine-rich repeat 15" or "LRRC15" refers to the protein encoded by the LRRC15 gene in humans. The leucine-rich repeat (LRR) domain is evolutionarily conserved among proteins associated with innate immunity. LRRC15 is expressed on stromal fibroblasts of many solid tumors (e.g., breast, head and neck, lung, pancreas) and directly on a subset of cancer cells of mesenchymal origin (e.g., sarcomas, melanomas, glioblastomas), while most normal tissues show low or no expression.

[0443] As used herein, the term "CD," "cluster of differentiation," or "cluster of differentiation molecules" refers to cell surface markers, such as CD40, that can be used to identify and characterize leukocytes.

[0444] 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 to activate APCs, leading to the upregulation of CD80, CD86, and other co-stimulatory molecules to stimulate CD8+ antigen-specific T cell responses.

[0445] The term "Fc fragment" or "Fc" refers to a portion of an antibody that does not have antigen binding activity but was initially observed to crystallize readily, and is therefore named an Fc fragment (for fragment crystallizability). This fragment corresponds to a paired CH2 and CH3 domain and is the portion 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 specific uses, a specific IgG subclass may be preferred. For example, IgG1 is more effective than IgG2 and IgG4 in mediating ADCC and CDC. Therefore, when effector function is undesirable, IgG2 Fc may be preferred. However, molecules containing IgG2 Fc are generally more difficult to prepare and may not be as stable as molecules containing IgG1 Fc. On the other hand, cross-binding to FcγRIIB is crucial for enhancing the co-stimulatory signal of CD40 or CD137, and the binding affinity of IgG4 is approximately 2 times higher than that of IgG1 and 10 times higher than that of IgG2. Mutations such as S267E and L328F can further enhance the cross-binding of IgG Fc to FcγRIIB. In addition, the effector function of an antibody can be increased or decreased by introducing one or more mutations into Fc (see, e.g., Strohl, Curr. Opin. Biotech., 20: 685-691, 2009).

[0446] As used herein, the terms "antibody" or "immunoglobulin" are heterotetrameric glycoproteins of approximately 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. 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; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0447] As used herein, the term "variable" refers to certain parts of the variable region in an antibody that are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are generally in a β-pleated configuration and are connected by three CDRs that form a connecting loop, and in some cases can form a partial β-pleated structure. The CDRs in each chain are closely together through the FR region and form the antigen-binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume 1, pages 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cytotoxicity of the antibody.

[0448] The antibodies of the present application may include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific, bispecific, human, humanized, primatized, chimeric, and single-chain antibodies. The antibodies disclosed herein may be from any animal origin, including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies.

[0449] The term "antibody fragment" or "antigen-binding fragment" is used to refer to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies. Regardless of the structure, antibody fragments bind to the same antigen recognized by the intact antibody. The term "antibody fragment" includes DARTs and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein comprising an immunoglobulin variable region that acts like an antibody by binding to a specific antigen to form a complex. "Single-chain fragment variable region" or "scFv" refers to a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin. In some aspects, the domains are connected to a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility and serine or threonine for solubility, and can connect 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, this protein still retains the specificity of the original immunoglobulin. Regarding IgG, the 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, wherein the light chain is connected to the heavy chain from the mouth of the "Y" and extends through the variable region.

[0450] As mentioned above, the variable region allows the antibody to selectively recognize and specifically bind to the epitope on the antigen. That is, the VL domain and VH domain of the antibody or the complementary determining region (CDR) subset of the antibody combine to form a variable region that limits the three-dimensional antigen binding site. This four-membered antibody structure forms the 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) on each of the VH and VL chains. In some cases, for example, some immunoglobulin molecules are derived from camelid species or are engineered based on camelid immunoglobulins. Alternatively, the immunoglobulin molecule can be composed of a heavy chain without a light chain or a light chain without a heavy chain.

[0451] In naturally occurring antibodies, the six CDRs present in each antigen-binding domain are short, non-continuous amino acid sequences that are specifically positioned to form an "antigen-binding domain" because the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining amino acids in the antigen-binding domain, referred to as the "framework" region, exhibit less inter-molecular variability. The framework region primarily adopts a β-sheet conformation, and the CDRs form loops that connect and, in some cases, form part of the β-sheet structure. Therefore, the framework region plays the role of forming a scaffold that positions the CDRs in the correct direction through interchain non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface that is complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. Since it has been precisely defined, a person of ordinary skill in the art can easily identify the amino acids comprising the CDRs and framework regions, respectively, for any given heavy or light chain variable region.

[0452] As used herein, the term "light chain constant region (CL)" includes the amino acid sequence CL derived from an antibody light chain (eg, SEQ ID NO: 66, 67, 68). Preferably, the light chain constant region includes at least one of a constant kappa domain or a constant lambda domain.

[0453] As used herein, the term "heavy chain constant region (CH)" includes an amino acid sequence derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region comprises at least one of the following: a CH1 domain, 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 should be understood that the heavy chain constant region can be modified such that its amino acid sequence differs from that of a naturally occurring immunoglobulin molecule.

[0454] In one embodiment of the present invention, the prepared multispecific antibody comprises a crossmab structure, i.e., the heavy chain CH1 and the light chain CL exchange part of the amino acid sequence to prevent mispairing. Such an exemplary structure comprises CH1 as shown in SEQ ID NO: 56 and CL as shown in SEQ ID NO: 68.

[0455] 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 to the original antibody, antibody fragment, or antibody domain, and (2) specifically binds to the same target to which the original antibody, antibody fragment, or antibody domain specifically binds. It will be understood that where sequence identity is expressed in the form of "at least x% identical" or "at least x% identical," such embodiments include any and all numerical percentages equal to or above the lower limit. Furthermore, it will be understood that where an amino acid sequence is presented in this application, it should be construed as further disclosing or encompassing an amino acid sequence that is 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% identical to that amino acid sequence.

[0456] Included within the scope of the multispecific molecules of the present invention are various compositions and methods, including: asymmetric IgG-like antibodies (e.g., triomabs / quadromas); knobs-into-holes antibodies; cross monoclonal antibodies (Cross MAbs); electrostatically matched antibodies; LUZ-Y; chain exchange engineered domain (SEED) bodies; Fab exchange antibodies, symmetric IgG-like antibodies; two-in-one antibodies; cross-linked monoclonal antibodies, mAb2; Cov X-body; dual variable domain (DVD)-Ig fusion protein; IgG-like bispecific antibody; Ts2Ab; BsAb; scFv / Fc fusion; double (scFv)2-Fabs; F(ab)2 fusion protein; dual-action or Bis-Fab; Dock-and-Lock (DNL); Fab-Fv; scFv-based antibodies and diabody-based antibodies (e.g., bispecific T cell engagers (BiTEs); tandem diabodies (Tandab); DARTs; single-chain diabodies; TCR-like antibodies; human serum albumin scFv fusion proteins, combodies, and IgG / non-IgG fusion proteins.

[0457] As used herein, the phrase "multispecific antibody" refers to a molecule comprising at least two targeting domains with different binding specificities. In some embodiments, a 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 antibody is a bispecific antibody. In other embodiments, the multispecific antibody is a trispecific antibody. In other embodiments, the multispecific antibody is a tetraspecific antibody.

[0458] As used herein, the phrase "bispecific" refers to a molecule comprising at least two targeting domains with different binding specificities. Each targeting domain is capable of specifically binding to a target molecule and, when bound to the target molecule, inhibits the biological function of the target molecule. In some embodiments, a bispecific antibody is a polymer 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 fragment thereof that specifically binds to a target protein. In some embodiments, a bispecific inhibitor is a bispecific antibody.

[0459] The terms "bispecific antibody" and "bispecific molecule" are used interchangeably herein and refer to antibodies 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) on one of its two binding arms (a pair of HC / LC) and binds to a different antigen (or epitope) on its second arm (another pair of HC / LC). In these embodiments, the bispecific antibody has two different antigen-binding arms (both in specificity and CDR sequences) and is monovalent for each antigen it binds.

[0460] In other embodiments, bispecific antibodies are full-length antibodies that can bind to two different antigens (or epitopes) in each of their two binding arms (two pairs of HC / LC). In these embodiments, the bispecific antibodies have two identical antigen-binding arms with the same specificity and the same CDR sequences, and are bivalent for each antigen to which they bind.

[0461] The terms "trispecific antibody" and "trispecific molecule" are used interchangeably herein and refer to molecules comprising three targeting domains with three different binding specificities. Each targeting domain is capable of specifically binding to a target molecule and, upon binding to the target molecule, inhibiting the biological function of the target molecule. In some embodiments, a trispecific antagonist is a polymer 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 fragment thereof that specifically binds to a target protein.

[0462] In a preferred embodiment of the present invention, a bispecific antibody targeting LRRC15 and CD40 was constructed, having the structures shown in Figures ab and 1 . An exemplary molecule of structure a is shown in Figures 2A or 2B , wherein the anti-LRRC15 Fab can be constructed using the VH and VL of any anti-LRRC15 antibody described herein; and the anti-CD40 scFv can be constructed using the VH and VL of any anti-CD40 antibody described herein. An exemplary molecule of structure b is shown in Figure 2C , wherein the anti-CD40 Fab can be constructed using the VH and VL of any anti-CD40 antibody described herein; and the anti-LRRC15 scFv can be constructed using the VH and VL of any anti-LRRC15 antibody described herein.

[0463] In another preferred embodiment of the present invention, a trispecific antibody targeting LRRC15, CD40, and CLDN18.2 is constructed, having the structures shown in Figure 1 (cd). An exemplary molecule of structure c is shown in Figure 2D, wherein the anti-LRRC15 Fab can be a Fab constructed using the VH and VL of any anti-LRRC15 antibody described herein, the anti-CD40 scFv can be a scFv constructed using the VH and VL of any anti-CD40 antibody described herein, and the anti-CLDN18.2 Fab can be a Fab constructed using the VH and VL of any anti-CLDN18.2 antibody described herein. d Structure An exemplary molecule is shown in Figure 2F, wherein the anti-LRRC15 scFv can be a scFv constructed using the VH and VL of any anti-LRRC15 antibody described in the present invention, 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-CLDN18.2 scFv can be a scFv constructed using the VH and VL of any anti-CLDN18.2 antibody described in the present invention.

[0464] In another preferred embodiment of the present invention, a trispecific antibody targeting LRRC15, CD40, and PD-1 was constructed, having the structures shown in Figure 1 (cd). An exemplary molecule of structure c is shown in Figure 2E , wherein the anti-LRRC15 Fab can be constructed using the VH and VL of any anti-LRRC15 antibody described herein, the anti-CD40 scFv can be constructed using the VH and VL of any anti-CD40 antibody described herein, and the anti-PD-1 Fab can be constructed using the VH and VL of any anti-PD-1 antibody described herein. An exemplary molecule of structure d is shown in Figure 2G , wherein the anti-LRRC15 scFv can be constructed using the VH and VL of any anti-LRRC15 antibody described herein, the anti-CD40 Fab can be constructed using the VH and VL of any anti-CD40 antibody described herein, and the anti-PD-1 scFv can be constructed using the VH and VL of any anti-PD-1 antibody described herein.

[0465] In another preferred embodiment of the present invention, a bispecific antibody targeting LRRC15 and CD3 was constructed, having the structure shown in FIG2 a.

[0466] In another preferred embodiment of the present invention, a trispecific antibody targeting LRRC15, CD3 and FAP was constructed, having the structures shown in b, c, f, g, h, i, j, k in FIG. 2 .

[0467] In another preferred embodiment of the present invention, a tetraspecific antibody targeting LRRC15, CD3, FAP and TGF-β was constructed, having the structures shown in Figure 2 d and e.

[0468] When constructing a multispecific antibody using an scFv composed of the antibody VH and VL of the present invention, a cysteine ​​(C) mutation can be introduced into the scFv to increase the stability of the multispecific antibody, for example, a cysteine ​​mutation can be introduced at position 44 of VH and at position 100 of VL. As used herein, the term "cysteine ​​(C) mutation of VH44 and VL100" refers to replacing the amino acid at position 44 of the scFv VH with cysteine, and replacing the amino acid at position 100 of the scFv VL with cysteine.

[0469] The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments thereof. The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or artificially synthesized DNA. DNA may be single-stranded or double-stranded. 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 a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence encoding an amino acid sequence identical to that of the polypeptide of the present invention, but having a different coding region sequence.

[0470] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0471] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and / or non-coding sequences.

[0472] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficol 1, 42°C; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptides shown in SEQ ID NO.4 and SEQ ID NO.9.

[0473] The full-length nucleotide sequence of the antibody of the present invention or its fragments can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis methods. One feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then ligating them, very long fragments of sequence can be obtained. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0474] Once the relevant sequence is obtained, recombinant methods can be used to obtain it in large quantities. This is typically accomplished by cloning it into a vector, transferring it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) referred to in the present invention include biomolecules in isolated form.

[0475] Currently, DNA sequences encoding proteins of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into protein sequences of the present invention by chemical synthesis.

[0476] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0477] Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 or Sf9; and animal cells such as CHO, COS7, and 293 cells.

[0478] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated using the CaCl2 method, using procedures well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0479] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0480] The recombinant polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods utilizing its 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, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0481] The antibodies of the present invention may be used alone or in combination with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or any combination of these.

[0482] Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product.

[0483] Therapeutic agents that can be conjugated include, but are not limited to, insulin, IL-2, interferon, calcitonin, GHRH peptide, intestinal peptide analogs, albumin, antibody fragments, cytokines, and hormones.

[0484] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising the above-mentioned antibody or active fragment thereof or fusion protein thereof, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the properties of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): oral, respiratory, intratumoral, intraperitoneal, intravenous, or topical administration.

[0485] The pharmaceutical composition of the present invention can be used to treat cancer / tumor, especially solid tumor, especially solid tumor with high expression of LCRR15.

[0486] The pharmaceutical composition of the present invention contains 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 its conjugate) and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured 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 per day. In addition, the pharmaceutical composition of the present invention can also be used in conjunction with other therapeutic agents.

[0487] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is generally at least about 10 μg / kg body weight, and in most cases does not exceed about 8 mg / kg body weight. Preferably, the dose is about 10 μg / kg body weight to about 1 mg / kg body weight. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician.

[0488] The main advantages of the present invention include:

[0489] (1) Cancer-associated fibroblasts (CAFs) are an important component of the microenvironment of most solid tumors. These cells promote cancer progression by supporting tumor cell growth, participating in the remodeling of the tumor extracellular matrix, promoting angiogenesis, and regulating tumor-induced inflammation. CAFs coexist with other immune cells such as endothelial cells and cells expressing proteins such as CD40 in the cancer stroma. Therefore, because LRRC15 is specifically expressed on CAFs, bi / tri / quadruplex antibodies can be designed against this target to bring immune activation signals to the tumor stroma, thereby facilitating the transition of cold tumors to hot tumors in the treatment of most solid tumors.

[0490] (2) Compared with common tumor-associated antigen (TAA) targets, the expression of LRRC15 on various solid tumors and CAFs makes the application of the bi / tri / tetraspecific antibodies containing the LRRC15 antigen binding domain of the present invention more extensive.

[0491] (3) Compared with monospecific antibodies, the bi / tri / tetraspecific antibodies of the present invention have better anti-tumor effects in vivo and in vitro, and are safer.

[0492] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0493] Sequences used to prepare the multispecific T cell engagers of the present invention

[0494] LRRC15 antibody sequence

[0495] CD40 antibody sequence

[0496] PD-1 antibody sequence

[0497] Claudin 18.2 antibody sequence

[0498] CD3 antibody sequence

[0499] FAP antibody sequence

[0500] TGF-βRII sequence

[0501] IgG1 CH sequence

[0502] Among them, the bold part is the Fc region.

[0503] CH1 sequence

[0504] Fc region sequence

[0505] The Fc region may comprise the following mutations:

[0506] In a preferred embodiment of the present invention, the Fc region has the following sequence:

[0507] IgG4 CH sequence

[0508] Among them, the bold part is the Fc region.

[0509] The Fc region may comprise the following mutations:

[0510] In another preferred embodiment of the present invention, the C-terminal lysine (K) of the Fc region is removed to reduce the formation of charge variants of the antibody.

[0511] CL sequence

[0512] Optional linker sequences

[0513] Example 1 Preparation of the multispecific antibody of the present invention

[0514] Based on the four LRRC15 antibody sequences (L15-06, L15-08, L15-03 and L15-07) obtained by phage screening, the mutant sequence of L15-06 (L15-06C), the CD40 antibody sequence, the PD-1 antibody sequence, the Claudin18.2 antibody sequence, the CD3 antibody sequence, the FAP antibody sequence and the TGF-βRII sequence, a variety of multispecific antibody molecules were designed.

[0515] The molecular sequence fragment gene of the multispecific antibody is obtained by the method of total gene synthesis, and then the target sequence is inserted into the expression vector by conventional gene cloning means (see, for example, Lo. BKC methods in Molecular Biology. Volume 248, 2004. Antibody Engineering).

[0516] HEK293E cells were transfected with a vector carrying a multispecific molecule. Cultured for 7 days at 37°C, 5% CO2, in F17 medium (1 L F17 + 10 mL 10% PF68 + 30 mL 200 mM L-glutamine) to produce the desired molecule. After expression, the supernatant was harvested and purified to yield the multispecific T cell binder molecule for subsequent analysis.

[0517] Using the methods described above, the following exemplary multispecific molecules were prepared:

[0518] A bispecific antibody consisting of a LRRC15 antigen-binding domain and a CD40 antigen-binding domain with the structure shown in FIG1 ab; a trispecific antibody consisting of a LRRC15 antigen-binding domain, a CD40 antigen-binding domain, and a PD-1 or Claudin18.2 antigen-binding domain with the structure shown in cd.

[0519] One bispecific antibody with structure a in Figure 1 was named 1540s, and its structure is shown in Figure 1A. Another bispecific antibody with structure a was named 1540s-3, and its structure is shown in Figure 1B. In addition, two other bispecific antibodies with structure a were named 1540s-7 and 1540s-16.

[0520] A bispecific antibody with the structure b in FIG1 is named 40d15, and its structure is shown in FIG1C .

[0521] A trispecific antibody of structure c in Figure 1 was named 151840-L06, and its structure is shown in Figure 1D ; another bispecific antibody of structure c was named 15P140-L06, and its structure is shown in Figure 1E .

[0522] A trispecific antibody with structure d in FIG1 was named 40d1815, and its structure is shown in FIG1F ; another trispecific antibody with structure d was named 40dP115, and its structure is shown in FIG1G .

[0523] A bispecific antibody consisting of a LRRC15 antigen-binding domain and a CD3 antigen-binding domain as shown in the structure a in Figure 2; a trispecific antibody consisting of a LRRC15 antigen-binding domain, a CD3 antigen-binding domain, and a FAP antigen-binding domain as shown in the structures b, c, f, g, h, i, j, k; and a tetraspecific antibody consisting of a LRRC15 antigen-binding domain, a CD3 antigen-binding domain, a FAP antigen-binding domain, and a TGF-β binding domain as shown in the structures d and e.

[0524] A bispecific antibody with the structure a in FIG2 is named 153, and its structure is shown in FIG2A .

[0525] A trispecific antibody with the structure b in FIG2 is named 315F, and its structure is shown in FIG2B .

[0526] A trispecific antibody with the structure c in Figure 2 was named F315, and its structure is shown in Figure 2C.

[0527] A tetraspecific antibody with the structure in Figure 2d was named 315Fb, and its structure is shown in Figure 2D.

[0528] A tetraspecific antibody with the structure in Figure 2e was named F315b, and its structure is shown in Figure 2E.

[0529] A trispecific antibody with the structure f in Figure 2 was named F153, and its structure is shown in Figure 2F.

[0530] A trispecific antibody with the structure g in FIG2 is named 15F3, and its structure is shown in FIG2G .

[0531] A trispecific antibody with the structure h in Figure 2 was named 153F, and its structure is shown in Figure 2H.

[0532] A trispecific antibody of structure i in FIG2 is named 1533F, and its structure is shown in FIG2I .

[0533] A trispecific antibody with the structure j in Figure 2 was named F3153, and its structure is shown in Figure 2J.

[0534] A trispecific antibody with the structure k in Figure 2 was named F3315, and its structure is shown in Figure 2K.

[0535] Exemplary molecules prepared in this example are as follows:

[0536] Example 2 Detection of Binding Affinity of Bispecific Antibody Molecules Targeting LRRC15 and CD40 to CD40

[0537] The binding affinity of the bispecific antibody 1540s and its mutants 1540s-1 to 1540s-17 to CD40 was tested. Mutants 1540s-1 to 1540s-17 have the same structure as 1540s, except that the variable region sequences in the CD40 antigen-binding domain are replaced with VH1-7, VL8-10, and VH11-17, respectively.

[0538] Dilute the test antibody in Octet Buffer (1X PBS + 0.02% Tween) and add 200 μl / well to a 96-well black plate. Add serially diluted hCD40-His protein in Octet Buffer to the same 96-well plate at 200 μl / well. Add 200 μl / well of Octet Buffer to the same 96-well plate. Add 200 μl / well of regeneration solution (glycine solution) to the same 96-well plate. Prepare the probe cartridge and add 200 μl / well of Octet Buffer to the probe wells to be used. Then, place the probes (AHC2.0 assay probes) in the probe wells. Place the 96-well plate and probe cartridge in the ForteBio assay. Analyze using OctetBLI Analysis software to calculate KD values. The results are shown in Tables 1-1 to 1-3.

[0539] The results showed that the 1540s mutants all exhibited reduced affinity for CD40 compared to the original molecule. Some mutations made binding of the 1540s molecule to CD40 virtually impossible to detect, and were labeled as NA.

[0540] Table 1-1

[0541] Table 1-2

[0542] Table 1-3

[0543] Table 1-4

[0544] Example 3 Performance testing of bispecific antibody molecules targeting LRRC15 and CD40

[0545] 3.1 Activation of DCs by Bispecific Antibody Molecules

[0546] Monocytes were isolated from PBMC using a monocyte isolation kit (cat#130-096-537, Miltenyi). 20 ng / ml of GM-CSF and IL-4 were added to induce monocyte differentiation into DC cells, and DC cells were harvested after 7 days. CHO cells expressing LRRC15 (wild-type CHO cells that do not express LRRC15 were used as a control) were washed with PBMC buffer (RPMI-1640+10% FBS+1% Penstrep) and resuspended at 2E6 cells / ml with gradient dilutions of the test antibody. Prepared DC cells were added to the resuspension to make the E / T cell ratio 3:1. Incubate at 37°C for 24 hours. The supernatant was taken and the IL-12p40 content was tested using an IL-12p40 detection kit.

[0547] The results are shown in Figures 3A and 3B. The results in Figure 3A show that the four bispecific antibodies all stimulated the activation of DC cells better than the monoclonal antibody control, among which 1540s and 1540s-3 had better effects and were better than 1540-L06 (Fc region with L234F / L235E / P331S / S267E mutations, other amino acid sequences were the same as 1540s, and the complete sequence can be found in WO2023104214A1) and 40d15. At the same time, in the absence of target cells, the four bispecific antibodies had no effective activation of DC cells, showing better safety. The results in Figure 3B show the effects of 1540s-3, 1540s-7, 1540s-16 and the control monoclonal antibody Sel icrelumab on DC cells induced by another PBMC donor. The results also showed that the three bispecific antibodies had signals that were better than the monoclonal antibodies. At the same time, in the absence of target cells, the three bispecific antibodies had no effective activation and had better safety than the control monoclonal antibody.

[0548] 3.2.1 Activation of CD40 reporter gene by bispecific antibody molecules

[0549] CD40 effector cells (Promega) in the logarithmic growth phase were collected and centrifuged at 300g for 5 min. The cells were resuspended in 1 ml of assay buffer (1640 medium + 1% FBS), counted, and the cell density was adjusted to 1.5E5 / ml using assay buffer.

[0550] After mixing evenly, the CD40 effector cell suspension was seeded into a 96-well white plate at 100 μl / well and incubated overnight (18–22 hours) at 37°C in 5% CO2.

[0551] The next day, harvest CHO-hLRRC15 cells in the logarithmic growth phase and centrifuge at 300g for 5 minutes. Resuspend the cells in 1ml of assay buffer (1640 medium + 1% FBS), count them, and adjust the cell density to 8E5 / ml with assay buffer. Remove the 96-well plate that was lined with CD40 effector cells the day before, gently discard the medium, and add the CHO-hLRRC15 cell suspension to the 96-well plate at 50μl / well. Add the serially diluted antibodies to the 96-well plate at 25μl / well in duplicate. Incubate in a CO2 incubator for 5-6 hours. Remove the 96-well plate and add BRITELITE PLUS reagent at 75μl / well. Incubate at room temperature in the dark for 5 minutes. Measure the Lumi readings using a microplate reader. Graph the graph using Prism software and calculate the EC50 value.

[0552] The results are shown in Figures 4A and 4B. The results in Figure 4A show that 1540s and 1540s-3 can strongly activate the CD40 reporter gene signal in the presence of target cells, but the activation of this signal is greatly weakened in the absence of target cells (wild-type CHOK1). 40d15 has the weakest activation in the absence of target cells, has a higher safety profile, and its efficacy is also higher than the control monoclonal antibodies selicrelumab and dacetuzuamb (the control antibody is a sequence-identical antibody biosimilar). Figure 4B shows another independent experiment comparing the effects of another group of bispecific antibodies and the control monoclonal antibody. The results show that 1540s, 1540s-3, 1540s-7, and 1540s-16 all have better activation effects than the control monoclonal antibody, and they only have a small amount of activation signals compared to the control monoclonal antibody in the absence of target cells (wild-type CHOK1), showing good efficacy and safety.

[0553] 3.2.2 Activation of CD3 reporter gene by trispecific antibody molecules

[0554] The experiment was conducted using CD3 effector cells (Promega) in logarithmic growth phase. Other experimental procedures were similar to those for the CD40 reporter gene system described above. The results are shown in Figure 4C. All four triple antibodies effectively activated the CD3 reporter gene system, with minimal background signal in the absence of target cells (wild-type CHOK1). However, in the presence of target cells expressing LRRC15 or FAP, 153F exhibited superior activation compared to the other antibodies.

[0555] 3.3 Ability of bispecific antibodies to stimulate PBMC to release cytokines

[0556] The ability of antibody molecules to stimulate PBMC to release cytokines was measured, using monoclonal antibodies L15-06 (abbreviated as L06), selicrelumab, and dacetuzuamb as control samples, and CD3 antibody (OKT3) as a positive control. The specific method is as follows:

[0557] Thaw and dilute PBMC cells from three different donors (D1, D2, D3) to 10E7 cells / ml in 96-well plates with PBMC buffer (RPMI-1640 + 10% FBS + 1% Penstrep), then culture in a 37°C incubator for 48 hours. Dilute the test antibody to 10nM in PBMC buffer and add it to the 96-well plate containing PBMC cells, incubate at 37°C for 24 hours. TM The Cytometric Bead Array (CBA) kit captures and detects the cytokine content in the culture supernatant, which can show the cytokine release of PBMC from different donors stimulated by each antibody.

[0558] The results are shown in Figure 5. The results show that none of the three bispecific antibodies significantly promoted the release of the six cytokines, while the CD3 antibody (OKT3) used as a positive control showed varying degrees of cytokine release. The untreated group, which did not contain the antibody, served as a negative control and showed no significant cytokine release.

[0559] Example 4 In vivo efficacy testing of bispecific antibody molecules targeting LRRC15 and CD40

[0560] 4.1 Drug efficacy in the KPC model

[0561] CD40 humanized C57BL / 6 mice were inoculated with KPC cells (5×10 5 cells / mouse), and the average tumor volume grew to about 170 mm 3 The mice were then divided into groups and dosed (the control group was given PBS, and the experimental groups were given 1540s-3, 1540s-7, and 1540s-16, respectively). Intraperitoneal injection was administered once every 5 days for a total of 5 doses, with a dose of 10 mg / kg. 1540s-3, 1540s-7, and 1540s-16 all showed significant anti-tumor effects, with TGIs of 82%, 88%, and 54%, respectively (Figure 6A). At the same time, none of the three bispecific antibodies caused changes in mouse body weight (Figure 6B), indicating that these bispecific antibodies are effective and safe.

[0562] 4.2 Drug efficacy in the MC38 model

[0563] CD40 humanized C57BL / 6 mice were inoculated with MC38-huLRRC15 cells (1×10 6 cells / mouse), and the average tumor volume grew to about 220 mm 3The mice were then divided into groups and dosed (the control group was given PBS, and the experimental groups were given 1540s, 1540s-3, and 40d15, respectively; the dosing schedule is shown in Figure 7A). All three bispecific antibodies in the experimental group demonstrated excellent anti-tumor effects, similar to the anti-tumor effects of the control Selicrelumab (Seli). On day 18 after administration, the TGIs were 106% (1540s), 105% (1540s-3), 107% (40d15), and 106% (Seli), respectively (Figure 7A). Continuous observation showed that the bispecific antibodies continued to inhibit tumor growth, but tumor recurrence occurred in the control Selicrelumab group. In terms of body weight, the control monoclonal antibody Selicrelumab group experienced a larger decrease in body weight, however, 1540s, 1540s-3, and 40d15 did not cause a significant decrease in body weight in mice, indicating that 1540s, 1540s-3, and 40d15 are safer than Selicrelumab (Figure 7B). 24 hours after administration, the ALT and AST levels in the serum of mice in the control Seli monoclonal antibody group increased sharply, indicating its potential hepatotoxicity risk, while the ALT / AST release levels of the three bispecific antibodies were similar to those of the Vehicle (PBS group), and no obvious increase in ALT and AST was found (Figure 7C).

[0564] Example 5 Pre-toxicity experiment in cynomolgus monkeys

[0565] A preliminary toxicological evaluation of 1540s-3 and 1540s-7 was conducted in cynomolgus monkeys, with intravenous injection once a week for 4 consecutive weeks (a total of 5 times) at a dose of 30 mg / kg.

[0566] The results showed that there was no significant change in body weight (as shown in Figure 8), no obvious abnormalities in clinical observation, and no gross abnormal changes were found in the gross anatomy. No significant cytokine changes were detected 6 hours, 24 hours, 72 hours, 168 hours after the first dose, and 24 hours after the second to fifth doses. The IL-12 level was at the lower limit of quantification (9.77 pg / mL) at each detection time point (Table 2). In contrast, there are literature reports that Selicrelumab can significantly activate IL-12 production and show significant spleen enlargement when administered subcutaneously at 5 mg / kg. This shows that 1540s-3 and 1540s-7 have significantly improved safety compared to Selicrelumab.

[0567] Table 2 Note: BLQ in the table means below the lower limit of quantification.

[0568] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that 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 that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A multispecific antibody, characterized in that The multispecific antibody comprises: a first targeting domain comprising one or more LRRC15 antigen binding domains; a second targeting domain comprising one or more CD40 antigen binding domains, or one or more CD3 antigen binding domains; Optionally, it comprises a third targeting domain that binds to a target protein selected from the group consisting of PD-1, Claudin18.2 (CLDN18.2), and FAP; and / or a fourth targeting domain that binds to TGF-β.

2. The multispecific antibody according to claim 1, wherein The multispecific antibody is a bispecific, trispecific or tetraspecific antibody.

3. The multispecific antibody according to claim 2, wherein The bispecific antibody comprises: A first targeting domain, wherein the first targeting domain comprises one or more LRRC15 antigen binding domains; and a second targeting domain, wherein the second targeting domain comprises one or more CD40 antigen binding domains, or one or more CD3 antigen binding domains.

4. The multispecific antibody according to claim 2, wherein The trispecific antibody comprises: a first targeting domain comprising one or more LRRC15 antigen binding domains; a second targeting domain comprising one or more CD40 antigen binding domains; and The third targeting domain is a Claudin18.2 or PD-1 antigen binding domain.

5. The multispecific antibody according to claim 2, wherein The trispecific antibody comprises: a first targeting domain comprising one or more LRRC15 antigen binding domains; a second targeting domain comprising one or more CD3 antigen binding domains; and The third targeting domain is a FAP antigen binding domain.

6. The multispecific antibody according to claim 2, wherein The tetraspecific antibody comprises: a first targeting domain comprising one or more LRRC15 antigen binding domains; a second targeting domain comprising one or more CD3 antigen binding domains; a third targeting domain, said third targeting domain being a FAP antigen binding domain; and The fourth targeting domain is a TGF-β antigen binding domain.

7. The multispecific antibody according to claim 1, wherein 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:

8. The multispecific antibody according to claim 1, wherein The CD40 antigen-binding domain comprises a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 11, 13-23, 114-116, and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 12; or A heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 11, and a light chain variable region comprising the amino acid sequence shown in any one of SEQ ID NOs: 12, 24-26; or The heavy chain variable region is shown in SEQ ID NO: 27, and the light chain variable region is shown in SEQ ID NO:

28.

9. The multispecific antibody according to claim 1, wherein The CD3 antigen-binding domain comprises a heavy chain variable region having at least 80% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 82-84, 117, and a light chain variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO:

85.

10. The multispecific antibody according to claim 1, wherein The PD-1 antigen binding domain comprises a heavy chain variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO: 29, and a light chain variable region having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO:

30.

11. The multispecific antibody according to claim 1, wherein The Claudin18.2 antigen binding domain comprises a heavy chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:31, and a light chain variable region having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

32.

12. The multispecific antibody according to claim 1, wherein The FAP antigen-binding domain comprises a heavy chain variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 86, and a light chain variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 87; or a heavy chain variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 88, and a light chain variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 89; or a heavy chain variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 90, and a light chain variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 91; or a heavy chain variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 92, and a light chain variable region having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:

93.

13. A polynucleotide encoding the multispecific antibody of any one of claims 1-12.

14. Use of the multispecific antibody according to claim 1 for preparing a medicament for treating cancer / tumor and / or fibrotic disease.

15. A pharmaceutical composition comprising: (a) the multispecific antibody of claim 1; and (b) a pharmaceutically acceptable carrier.

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