Pharmaceutical composition and application

Abstract

The invention belongs to the field of oncotherapy and molecular immunology, and particularly relates to a pharmaceutical composition and application thereof. Specifically, the pharmaceutical composition comprises an anti-LAG3 (Lagranoside 3) and anti-PD-1 (programmed death-1) bispecific antibody and an anti-CTLA4-anti-PD-1 bispecific antibody. The pharmaceutical composition disclosed by the invention can be used for effectively treating or preventing tumors and has a good application prospect.

Description

[0001] Cross-references to related applications

[0002] This application is based on and claims priority to CN application No. 202410580093.3, filed on May 10, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the fields of tumor treatment and molecular immunology, specifically relating to drug combinations and their uses. Specifically, the drug combination comprises an anti-LAG3-anti-PD-1 bispecific antibody and an anti-CTLA4-anti-PD-1 bispecific antibody. Background Technology

[0004] Cancer, especially malignant cancer, is a serious threat to human health worldwide, ranking second among all causes of death. Moreover, its incidence has shown a significant upward trend in recent years. Malignant cancers have poor treatment outcomes, high rates of late-stage metastasis, and generally poor prognoses. While current clinical treatments such as radiotherapy, chemotherapy, and surgery have significantly alleviated pain and prolonged survival, these methods have considerable limitations, making further improvements in efficacy difficult.

[0005] Lymphocyte-activation gene 3 (LAG3), also known as CD223, is a type I transmembrane protein composed of 498 amino acids and belongs to the immunoglobulin superfamily (IgSF). LAG3 is mainly expressed on activated CD4+. + T cells and CD8 + T cells, as well as natural killer (NK) cells, B cells, regulatory T cells (Treg), and plasmacytoid dendritic cells (pDC) also express LAG3. (Ruffo Elisa, Wu Richard C, Bruno Tullia C et al. Lymphocyte-activation gene 3 (LAG3): The next immune checkpoint receptor.[J].SeminImmunol,2019,42:101305.)

[0006] The LAG3 gene is located on human chromosome 12 (20p13.3), adjacent to the CD4 gene, and both share the same exons and introns. Although their amino acid sequence homology is only about 20%, LAG3 and CD4 molecules show high structural similarity. Major histocompatibility complex class II (MHC II), hepatic sinusoidal endothelial cell lectin (LSECtin), and galectin-3 are related ligands of LAG3. MHC II molecules are the major ligands of LAG3, and their affinity for MHC II molecules (Kd: 60 nmol·L⁻¹) is 100 times that of CD4, indicating that LAG3 can effectively compete with CD4 for binding to MHC II molecules and inhibit T cell activation.

[0007] In the tumor microenvironment, the expression of the immunosuppressive molecule LAG3 can be detected 24 hours after T cell activation, leading to T cell dysfunction or apoptosis. LAG3 forms a dimer with CD4 through its D1 domain (containing a proline-rich ring structure). + The specific binding of MHCII molecules in the first signaling axis of T cell activation, "CD3-TCR-MHCII," blocks the signal transduction pathway of T cell activation on the one hand, and on the other hand, the intracellular segment of LAG3 (Keele motif) generates an immunosuppressive signal that downregulates CD4. + T cell activity. LAG3 molecules can promote Treg cell differentiation, participate in signal transduction and downstream signaling of transcription activator factor 5, thereby enhancing the inhibitory effect of Treg cells. This is one of the mechanisms by which tumors escape the killing effect of the immune system (Andrews Lawrence P, Marciscano Ariel E, Drake Charles G et al. LAG3 (CD223) as a cancer immunotherapy target. [J]. Immunol Rev, 2017, 276: 80-96.).

[0008] Multiple studies have shown that LAG3 plays a role in the tumor-invasive CD8 complex of various malignant tumors. + Overexpression in T cells. For example, in ovarian cancer, tumor-infiltrating New York esophageal squamous cell carcinoma 1 antigen (NY-ESO-1)-specific CD8+. + T cells express high levels of PD-1 and LAG3, resulting in reduced ability to produce IFN-γ and TNF-α, which in turn leads to lymphocyte inactivation. Galectin-3 and LSECtin primarily interact with LAG3 to regulate CD8. +T cell activation and function. Furthermore, melanoma antigen-specific T cells isolated from melanoma metastasis patients showed significant upregulation of LAG3 and other immune checkpoint molecules CTLA-4 and TIM-3 (Liu Hao, Li Xinying, Luo Longlong, et al. Research progress on molecular biological function of lymphocyte activation gene 3 and its clinical application of antibody drugs [J]. Chinese Journal of Pharmacology and Toxicology, 2019, 33(01):70-78.). Currently, several LAG3 antibody drugs have entered the clinical research stage, among which Bristol-Myers Squibb's Relatlimab has made the fastest progress, with 10 clinical studies underway.

[0009] The transmembrane receptor PD-1 (programmed cell death-1) is a member of the CD28 family and is expressed on activated T cells, B cells, and myeloid cells. The receptors for PD-1, PDL1 and PDL2, both belong to the B7 superfamily. PDL1 is expressed on a variety of cells, including T cells, B cells, endothelial cells, and epithelial cells, while PDL2 is expressed only on antigen-presenting cells such as dendritic cells and macrophages.

[0010] PD-1 plays a crucial role in negatively regulating T cell activation. PD-1-mediated negative regulation of T cells is a key mechanism for tumor immune evasion. PD-L1 expressed on the tumor surface can bind to PD-1 on the surface of immune cells, thereby inhibiting the killing of tumor tissue by immune cells through the PD-1 / PD-L1 signaling pathway. Tumors with high PD-L1 expression are often difficult to detect (Hamanishi et al., Proc. Natl. Acad. Sci. USA 2007; 104:3360-5). An effective method to antagonize PD-1 and thus inhibit the PD-1 / PD-L1 signaling pathway is in vivo injection of anti-PD-1 antibodies.

[0011] PD-1 antibodies have broad-spectrum anti-tumor prospects and amazing efficacy. Antibodies targeting the PD-1 pathway will bring breakthrough progress in the treatment of various tumors: for the treatment of non-small cell lung cancer, renal cell carcinoma, ovarian cancer, melanoma (Homet MB, Parisi G., et al., Anti-PD-1 Therapy in Melanoma. Semin Oncol. 2015 Jun; 42(3):466-473), hematological malignancies and anemia (Held SA, Heine A, et al., Advances in immunotherapy of chronic myeloid leukemia CML. Curr Cancer Drug Targets. 2013 Sep; 13(7):768-74).

[0012] Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and CD28 molecules share very similarities in gene structure, chromosomal location, sequence homology, and gene expression. Both are receptors for the co-stimulatory molecule B7 and are primarily expressed on the surface of activated T cells. CTLA4 binding to B7 inhibits the activation of mouse and human T cells, playing a negative regulatory role in T cell activation.

[0013] CTLA4 antibodies (or anti-CTLA4 monoclonal antibodies) or CTLA4 ligands can prevent CTLA4 from binding to its natural ligand, thereby blocking the transmission of negative regulatory signals by CTLA4 to T cells and enhancing the responsiveness of T cells to various antigens. In vivo and in vitro studies have yielded largely consistent results in this regard. Currently, CTLA4 monoclonal antibodies are in clinical trials or have been approved for the treatment of prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, and malignant melanoma (Grosso JF., Jure-Kunkel MN., 2013, Cancer Immun. 13:5.).

[0014] CTLA4 antibodies can produce specific therapeutic effects on diseases and exert high efficacy, supplementing the shortcomings of traditional drugs, thus opening up new avenues for gene therapy.

[0015] Bispecific antibodies, also known as bifunctional antibodies, are specific antibody drugs that simultaneously target and bind to two different antigens. They can be produced through immunosorting purification or obtained through genetic engineering. Genetic engineering offers advantages in terms of flexibility in optimizing binding sites, considering synthetic forms, and yield. Currently, more than 45 forms of bispecific antibodies have been proven to exist (Müller D, Kontermann RE. Bispecific antibodies for cancer immunotherapy: Current perspectives. BioDrugs 2010; 24:89-98). The IgG-ScFv form, also known as the Morrison pattern (Coloma MJ, Morrison SL. Design and production of novel tetravalent bispecific antibodies. Nat Biotechnol. Nature Biotechnology, 1997; 15:159-163), has advantages in antibody engineering, expression, and purification due to its similarity to naturally occurring IgG, and has been proven to be an ideal form of bispecific antibody (Miller BR, Demarest SJ, et al., Stability engineering of scFvs for the development of bispecific and multivalent antibodies. Protein Eng Des Sel 2010; 23:549-57; Fitzgerald J, Lugovskoy A. Rational engineering of antibody therapeutics targeting multiple oncogene pathways. MAbs 2011; 3:299-309).

[0016] In conclusion, the development of monoclonal or bispecific antibodies targeting LAG3, PD-1, and CTLA4 with lower toxicity and greater efficacy, along with corresponding combination therapy regimens, has significant clinical implications. Summary of the Invention

[0017] Through in-depth research and creative work, the inventors have obtained a drug combination comprising anti-LAG3-anti-PD-1 bispecific antibody and anti-CTLA4-anti-PD-1 bispecific antibody. The inventors have surprisingly discovered that the combined use of anti-LAG3-anti-PD-1 bispecific antibody and anti-CTLA4-anti-PD-1 bispecific antibody exhibits effective pharmacological effects in inhibiting tumors, and these effects are superior to either anti-LAG3-anti-PD-1 bispecific antibody monotherapy or anti-CTLA4-anti-PD-1 bispecific antibody monotherapy.

[0018] This leads to the following invention:

[0019] One aspect of the present invention relates to a pharmaceutical combination comprising an effective amount of a first antibody and an effective amount of a second antibody, wherein the first antibody is an anti-LAG3-anti-PD-1 bispecific antibody and the second antibody is an anti-CTLA4-anti-PD-1 bispecific antibody.

[0020] In some embodiments of the present invention, the drug combination, wherein:

[0021] (1) The anti-LAG3-anti-PD-1 bispecific antibody includes:

[0022] Targeting the first protein functional region of LAG3, and

[0023] Targeting the second protein functional region of PD-1;

[0024] in,

[0025] The first protein functional region includes a heavy chain variable region VH1 and a light chain variable region VL1. VH1 includes HCDR1 shown in SEQ ID NO:5, HCDR2 shown in SEQ ID NO:6, and HCDR3 shown in SEQ ID NO:7; and VL1 includes LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:9, and LCDR3 shown in SEQ ID NO:10, or VL1 includes LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:46, and LCDR3 shown in SEQ ID NO:47, or VL1 includes LCDR1 shown in SEQ ID NO:48, LCDR2 shown in SEQ ID NO:46, and LCDR3 shown in SEQ ID NO:10.

[0026] The second protein functional region includes a heavy chain variable region VH2 and a light chain variable region VL2. The VH2 includes HCDR1 shown in SEQ ID NO:26, HCDR2 shown in SEQ ID NO:27, and HCDR3 shown in SEQ ID NO:28; and the VL2 includes LCDR1 shown in SEQ ID NO:29, LCDR2 shown in SEQ ID NO:30, and LCDR3 shown in SEQ ID NO:31.

[0027] (2) The anti-CTLA4-anti-PD-1 bispecific antibody includes:

[0028] Targeting the third protein functional region of PD-1, and

[0029] Targeting the fourth protein functional region of CTLA4;

[0030] in,

[0031] The third protein functional region comprises a heavy chain variable region VH3 and a light chain variable region VL3. The VH3 comprises HCDR1 shown in SEQ ID NO:26, HCDR2 shown in SEQ ID NO:27, and HCDR3 shown in SEQ ID NO:28; and the VL3 comprises LCDR1 shown in SEQ ID NO:29, LCDR2 shown in SEQ ID NO:30, and LCDR3 shown in SEQ ID NO:31.

[0032] The fourth protein functional region includes a heavy chain variable region VH4 and a light chain variable region VL4. VH4 includes HCDR1 shown in SEQ ID NO:67, HCDR2 shown in SEQ ID NO:68, and HCDR3 shown in SEQ ID NO:69; and VL4 includes LCDR1 shown in SEQ ID NO:70, LCDR2 shown in SEQ ID NO:71, and LCDR3 shown in SEQ ID NO:72.

[0033] In some embodiments of the present invention, the drug combination, wherein...

[0034] The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is selected from SEQ ID NO:4, SEQ ID NO:42 and SEQ ID NO:44.

[0035] In some embodiments of the present invention, the drug combination, wherein...

[0036] The amino acid sequence of VH2 is selected from SEQ ID NO:15 and SEQ ID NO:19, and the amino acid sequence of VL2 is selected from SEQ ID NO:17, SEQ ID NO:21 and SEQ ID NO:38;

[0037] Preferably, the amino acid sequence of VH2 is as shown in SEQ ID NO:19, and the amino acid sequence of VL2 is selected from SEQ ID NO:17, SEQ ID NO:21 and SEQ ID NO:38.

[0038] In some embodiments of the present invention, the drug combination, wherein...

[0039] The amino acid sequence of VH3 is selected from SEQ ID NO:15 and SEQ ID NO:19, and the amino acid sequence of VL3 is selected from SEQ ID NO:17, SEQ ID NO:21 and SEQ ID NO:38;

[0040] Preferably, the amino acid sequence of VH3 is as shown in SEQ ID NO:19, and the amino acid sequence of VL3 is selected from SEQ ID NO:17, SEQ ID NO:21 and SEQ ID NO:38.

[0041] In some embodiments of the present invention, the drug combination, wherein...

[0042] The amino acid sequence of VH4 is selected from SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:61 and SEQ ID NO:63, and the amino acid sequence of VL4 is selected from SEQ ID NO:52, SEQ ID NO:56, SEQ ID NO:60, SEQ ID NO:62 and SEQ ID NO:64;

[0043] Preferably,

[0044] The amino acid sequence of VH4 is shown in SEQ ID NO:50, and the amino acid sequence of VL4 is shown in SEQ ID NO:52;

[0045] The amino acid sequence of VH4 is shown in SEQ ID NO:54, and the amino acid sequence of VL4 is shown in SEQ ID NO:56;

[0046] The amino acid sequence of VH4 is shown in SEQ ID NO:58, and the amino acid sequence of VL4 is shown in SEQ ID NO:60.

[0047] The amino acid sequence of VH4 is shown in SEQ ID NO:61, and the amino acid sequence of VL4 is shown in SEQ ID NO:62; or

[0048] The amino acid sequence of VH4 is shown in SEQ ID NO:63, and the amino acid sequence of VL4 is shown in SEQ ID NO:64.

[0049] In some embodiments of the present invention, the drug combination, wherein...

[0050] (1) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:21.

[0051] (2) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:38.

[0052] (3) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:42; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:21.

[0053] (4) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:42; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:38.

[0054] (5) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:44; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:21; or

[0055] (6) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:44; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:38.

[0056] In some embodiments of the present invention, the drug combination, wherein...

[0057] (1) The amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:21; the amino acid sequence of VH4 is shown in SEQ ID NO:54, and the amino acid sequence of VL4 is shown in SEQ ID NO:56.

[0058] (2) The amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:21; the amino acid sequence of VH4 is shown in SEQ ID NO:58, and the amino acid sequence of VL4 is shown in SEQ ID NO:60.

[0059] (3) The amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:21; the amino acid sequence of VH4 is shown in SEQ ID NO:61, and the amino acid sequence of VL4 is shown in SEQ ID NO:62.

[0060] (4) The amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:21; the amino acid sequence of VH4 is shown in SEQ ID NO:63, and the amino acid sequence of VL4 is shown in SEQ ID NO:64.

[0061] (5) The amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:38; the amino acid sequence of VH4 is shown in SEQ ID NO:54, and the amino acid sequence of VL4 is shown in SEQ ID NO:56.

[0062] (6) The amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:38; the amino acid sequence of VH4 is shown in SEQ ID NO:58, and the amino acid sequence of VL4 is shown in SEQ ID NO:60.

[0063] (7) The amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:38; the amino acid sequence of VH4 is shown in SEQ ID NO:61, and the amino acid sequence of VL4 is shown in SEQ ID NO:62; or

[0064] (8) The amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:38; the amino acid sequence of VH4 is shown in SEQ ID NO:63, and the amino acid sequence of VL4 is shown in SEQ ID NO:64.

[0065] In some embodiments of the present invention, the drug combination, wherein...

[0066] (1) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:21; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:21; the amino acid sequence of VH4 is shown in SEQ ID NO:54, and the amino acid sequence of VL4 is shown in SEQ ID NO:56;

[0067] (2) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:21; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:21; the amino acid sequence of VH4 is shown in SEQ ID NO:58, and the amino acid sequence of VL4 is shown in SEQ ID NO:60.

[0068] (3) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:21; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:21; the amino acid sequence of VH4 is shown in SEQ ID NO:61, and the amino acid sequence of VL4 is shown in SEQ ID NO:62.

[0069] (4) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:21; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:21; the amino acid sequence of VH4 is shown in SEQ ID NO:63, and the amino acid sequence of VL4 is shown in SEQ ID NO:64.

[0070] (5) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:21; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:38; the amino acid sequence of VH4 is shown in SEQ ID NO:54, and the amino acid sequence of VL4 is shown in SEQ ID NO:56.

[0071] (6) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:21; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:38; the amino acid sequence of VH4 is shown in SEQ ID NO:58, and the amino acid sequence of VL4 is shown in SEQ ID NO:60.

[0072] (7) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:21; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:38; the amino acid sequence of VH4 is shown in SEQ ID NO:61, and the amino acid sequence of VL4 is shown in SEQ ID NO:62.

[0073] (8) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:21; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:38; the amino acid sequence of VH4 is shown in SEQ ID NO:63, and the amino acid sequence of VL4 is shown in SEQ ID NO:64.

[0074] (9) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:38; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:21; the amino acid sequence of VH4 is shown in SEQ ID NO:54, and the amino acid sequence of VL4 is shown in SEQ ID NO:56.

[0075] (10) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:38; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:21; the amino acid sequence of VH4 is shown in SEQ ID NO:58, and the amino acid sequence of VL4 is shown in SEQ ID NO:60.

[0076] (11) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:38; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:21; the amino acid sequence of VH4 is shown in SEQ ID NO:61, and the amino acid sequence of VL4 is shown in SEQ ID NO:62.

[0077] (12) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:38; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:21; the amino acid sequence of VH4 is shown in SEQ ID NO:63, and the amino acid sequence of VL4 is shown in SEQ ID NO:64.

[0078] (13) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:38; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:38; the amino acid sequence of VH4 is shown in SEQ ID NO:54, and the amino acid sequence of VL4 is shown in SEQ ID NO:56;

[0079] (14) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:38; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:38; the amino acid sequence of VH4 is shown in SEQ ID NO:58, and the amino acid sequence of VL4 is shown in SEQ ID NO:60.

[0080] (15) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:38; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:38; the amino acid sequence of VH4 is shown in SEQ ID NO:61, and the amino acid sequence of VL4 is shown in SEQ ID NO:62; or

[0081] (16) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:38; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:38; the amino acid sequence of VH4 is shown in SEQ ID NO:63, and the amino acid sequence of VL4 is shown in SEQ ID NO:64.

[0082] In some embodiments of the present invention, the drug combination, wherein:

[0083] The first, second, third, and fourth protein functional regions are independently selected from immunoglobulins and single-chain antibodies;

[0084] Preferably, the first protein functional region is an immunoglobulin and the second protein functional region is a single-chain antibody; or the first protein functional region is a single-chain antibody and the second protein functional region is an immunoglobulin.

[0085] Preferably, the third protein functional region is an immunoglobulin and the fourth protein functional region is a single-chain antibody; or the third protein functional region is a single-chain antibody and the fourth protein functional region is an immunoglobulin.

[0086] Preferably, the anti-LAG3-anti-PD-1 bispecific antibody is a bispecific antibody in the form of IgG-scFv; and / or the anti-CTLA4-anti-PD-1 bispecific antibody is a bispecific antibody in the form of IgG-scFv;

[0087] Preferably, the anti-LAG3-anti-PD-1 bispecific antibody is a tetravalent bispecific antibody in the form of IgG-scFv; and / or the anti-CTLA4-anti-PD-1 bispecific antibody is a tetravalent bispecific antibody in the form of IgG-scFv.

[0088] In some embodiments of the present invention, the drug combination is wherein a single-chain antibody is attached to the C-terminus of each of the two heavy chains of the immunoglobulin.

[0089] In some embodiments of the present invention, the drug combination, wherein...

[0090] The immunoglobulin contains a constant region derived from human antibodies;

[0091] Preferably, the constant region is independently selected from the constant regions of human IgG1, IgG2, IgG3 or IgG4.

[0092] In some embodiments of the present invention, the drug combination, wherein...

[0093] The immunoglobulin contains a heavy chain constant region, which is independently an Ig gamma-1 chain C region (e.g., as shown in SEQ ID NO:39) or an Ig gamma-4 chain C region (e.g., as shown in SEQ ID NO:45); and

[0094] The immunoglobulin contains a light chain constant region, which is the Ig kappa chain C region (e.g., as shown in SEQ ID NO:40).

[0095] In some embodiments of the present invention, the drug combination, wherein...

[0096] The immunoglobulin is independently human IgG1 subtype.

[0097] The immunoglobulin contains a heavy chain constant region.

[0098] According to the EU numbering system, the heavy chain constant region of the immunoglobulin contains the following mutations:

[0099] L234A and L235A,

[0100] L234A and G237A,

[0101] L235A and G237A,

[0102] or

[0103] L234A, L235A, and G237A;

[0104] Preferably, the first protein functional region is an immunoglobulin, the amino acid sequence of its heavy chain is shown in SEQ ID NO:11, and the amino acid sequence of its light chain is shown in SEQ ID NO:12;

[0105] Preferably, the functional region of the second protein is an immunoglobulin, the amino acid sequence of its heavy chain is shown in SEQ ID NO:34, and the amino acid sequence of its light chain is shown in SEQ ID NO:25;

[0106] or

[0107] Preferably, the third protein functional region is an immunoglobulin, the amino acid sequence of its heavy chain is shown in SEQ ID NO:66, and the amino acid sequence of its light chain is shown in SEQ ID NO:25.

[0108] In some embodiments of the present invention, the drug combination, wherein...

[0109] The immunoglobulin is independently human IgG4 subtype.

[0110] The immunoglobulin comprises a heavy chain constant region, wherein, according to the EU numbering system, the heavy chain constant region of the immunoglobulin contains the following mutations:

[0111] F234A and L235A,

[0112] F234A and G237A,

[0113] L235A and G237A,

[0114] or

[0115] F234A, L235A, and G237A;

[0116] Preferably, the first protein functional region is an immunoglobulin, the amino acid sequence of its heavy chain is shown in SEQ ID NO:13, and the amino acid sequence of its light chain is shown in SEQ ID NO:12;

[0117] Preferably, the functional region of the second protein is an immunoglobulin, the amino acid sequence of its heavy chain is shown in SEQ ID NO:32, and the amino acid sequence of its light chain is shown in SEQ ID NO:25;

[0118] or

[0119] Preferably, the third protein functional region is an immunoglobulin, the amino acid sequence of its heavy chain is shown in SEQ ID NO:32, and the amino acid sequence of its light chain is shown in SEQ ID NO:25.

[0120] In some embodiments of the present invention, the drug combination wherein the first and second protein functional regions are directly connected or connected via connectors; and the third and fourth protein functional regions are directly connected or connected via connectors.

[0121] Preferably, the linker is independently a polypeptide shown in SEQ ID NO:73, or a polypeptide consisting of multiple (e.g., 2, 3, 4, 5 or 6) polypeptides shown in SEQ ID NO:73 linked together;

[0122] Preferably, the linker is a polypeptide to which one or more glycine residues are linked at the C-terminus of the polypeptide shown in SEQ ID NO:73 or a polypeptide consisting of multiple (e.g., 2, 3, 4, 5 or 6) polypeptides shown in SEQ ID NO:73 in tandem.

[0123] In some embodiments of the present invention, the drug combination wherein the first protein functional region, the second protein functional region, the third protein functional region and the fourth protein functional region are independently one, two or more.

[0124] Preferably, the first protein functional region is one, and the second protein functional region is two.

[0125] Preferably, the first protein functional region has two regions, and the second protein functional region has one region.

[0126] Preferably, there is one third protein functional region and two fourth protein functional regions.

[0127] In some embodiments of the present invention, the drug combination, wherein:

[0128] The first protein functional region is one immunoglobulin, and the second protein functional region is two single-chain antibodies; or the first protein functional region is two single-chain antibodies, and the second protein functional region is one immunoglobulin.

[0129] And / or, the third protein functional region is one immunoglobulin, and the fourth protein functional region is two single-chain antibodies.

[0130] In some embodiments of the present invention, the drug combination, wherein...

[0131] The anti-LAG3-anti-PD-1 bispecific antibody includes:

[0132] Targeting the first protein functional region of LAG3, and

[0133] Targeting the second protein functional region of PD-1;

[0134] The first protein has one functional region, and the second protein has two functional regions;

[0135] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0136] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:2, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:4.

[0137] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:19, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:21 or SEQ ID NO:38; (e.g.:

[0138] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:2, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:4; the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:19, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:21; or

[0139] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:2, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:4; the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:19, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:38.

[0140] The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin;

[0141] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0142] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NOs:35-37;

[0143] Preferably, the amino acid sequences of the first linker and the second linker are both as shown in SEQ ID NO:36.

[0144] In some embodiments of the present invention, the drug combination, wherein...

[0145] The anti-LAG3-anti-PD-1 bispecific antibody includes:

[0146] Targeting the first protein functional region of LAG3, and

[0147] Targeting the second protein functional region of PD-1;

[0148] The first protein has one functional region, and the second protein has two functional regions;

[0149] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0150] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:11 or SEQ ID NO:13, and the amino acid sequence of its light chain is shown in SEQ ID NO:12.

[0151] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:19, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:21 or SEQ ID NO:38; (e.g.:

[0152] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:11, and the amino acid sequence of its light chain is shown in SEQ ID NO:12; the amino acid sequence of the variable region of the heavy chain of the single-chain antibody is shown in SEQ ID NO:19, and the amino acid sequence of the variable region of the light chain of the single-chain antibody is shown in SEQ ID NO:21.

[0153] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:11, and the amino acid sequence of its light chain is shown in SEQ ID NO:12; the amino acid sequence of the variable region of the heavy chain of the single-chain antibody is shown in SEQ ID NO:19, and the amino acid sequence of the variable region of the light chain of the single-chain antibody is shown in SEQ ID NO:38.

[0154] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:13, and the amino acid sequence of its light chain is shown in SEQ ID NO:12; the amino acid sequence of the variable region of the heavy chain of the single-chain antibody is shown in SEQ ID NO:19, and the amino acid sequence of the variable region of the light chain of the single-chain antibody is shown in SEQ ID NO:21; or

[0155] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:13, and the amino acid sequence of its light chain is shown in SEQ ID NO:12; the amino acid sequence of the variable region of the heavy chain of the single-chain antibody is shown in SEQ ID NO:19, and the amino acid sequence of the variable region of the light chain of the single-chain antibody is shown in SEQ ID NO:38.

[0156] The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin;

[0157] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0158] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NOs:35-37;

[0159] Preferably, the amino acid sequences of the first linker and the second linker are both as shown in SEQ ID NO:36.

[0160] In some embodiments of the present invention, the drug combination, wherein...

[0161] The anti-LAG3-anti-PD-1 bispecific antibody includes:

[0162] Targeting the first protein functional region of LAG3, and

[0163] Targeting the second protein functional region of PD-1;

[0164] The first protein has two functional regions, and the second protein has one functional region;

[0165] Wherein, the first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin;

[0166] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:4.

[0167] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:19, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:21.

[0168] The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin;

[0169] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0170] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NOs:35-37;

[0171] Preferably, the amino acid sequences of the first linker and the second linker are both as shown in SEQ ID NOs:36.

[0172] In some embodiments of the present invention, the drug combination, wherein...

[0173] The anti-LAG3-anti-PD-1 bispecific antibody includes:

[0174] Targeting the first protein functional region of LAG3, and

[0175] Targeting the second protein functional region of PD-1;

[0176] The first protein has two functional regions, and the second protein has one functional region;

[0177] Wherein, the first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin;

[0178] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:4.

[0179] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:34 or SEQ ID NO:32, and the amino acid sequence of its light chain is shown in SEQ ID NO:25; (e.g.:

[0180] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:4; the amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:34, and the amino acid sequence of its light chain is shown in SEQ ID NO:25; or

[0181] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:4; the amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:32, and the amino acid sequence of its light chain is shown in SEQ ID NO:25.

[0182] The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin;

[0183] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0184] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NOs:35-37;

[0185] Preferably, the amino acid sequences of the first linker and the second linker are both as shown in SEQ ID NOs:36.

[0186] In some embodiments of the present invention, the drug combination, wherein...

[0187] The anti-CTLA4-anti-PD-1 bispecific antibody includes:

[0188] Targeting the third protein functional region of PD-1, and

[0189] Targeting the fourth protein functional region of CTLA4;

[0190] in,

[0191] The third protein functional region consists of one unit, and the fourth protein functional region consists of two units.

[0192] The third protein functional region is an immunoglobulin, and the fourth protein functional region is a single-chain antibody.

[0193] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:19, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:21.

[0194] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:61 or SEQ ID NO:63, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:62 or SEQ ID NO:64 (e.g., the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:61, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:62; the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:61, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:64; the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:63, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:62; or, the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:63, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:64).

[0195] The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin;

[0196] The third protein functional region is connected to the fourth protein functional region via a third linker; and the heavy chain variable region of the single-chain antibody is connected to the light chain variable region of the single-chain antibody via a fourth linker; the third linker and the fourth linker may be the same or different;

[0197] Preferably, the amino acid sequences of the third linker and the fourth linker are independently selected from SEQ ID NOs:35-37;

[0198] Preferably, the amino acid sequences of the third linker and the fourth linker are both as shown in SEQ ID NO:36.

[0199] In some embodiments of the present invention, the drug combination, wherein...

[0200] The anti-CTLA4-anti-PD-1 bispecific antibody includes:

[0201] Targeting the third protein functional region of PD-1, and

[0202] Targeting the fourth protein functional region of CTLA4;

[0203] in,

[0204] The third protein functional region consists of one unit, and the fourth protein functional region consists of two units.

[0205] The third protein functional region is an immunoglobulin, and the fourth protein functional region is a single-chain antibody.

[0206] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:66, and the amino acid sequence of its light chain is shown in SEQ ID NO:25.

[0207] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:61 or SEQ ID NO:63, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:62 or SEQ ID NO:64 (e.g., the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:61, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:62; the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:61, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:64; the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:63, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:62; or, the amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:63, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:64).

[0208] The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin;

[0209] The third protein functional region is connected to the fourth protein functional region via a third linker; and the heavy chain variable region of the single-chain antibody is connected to the light chain variable region of the single-chain antibody via a fourth linker; the third linker and the fourth linker may be the same or different;

[0210] Preferably, the amino acid sequences of the third linker and the fourth linker are independently selected from SEQ ID NOs:35-37;

[0211] Preferably, the amino acid sequences of the third linker and the fourth linker are both as shown in SEQ ID NO:36.

[0212] In some embodiments of the present invention, the drug combination, wherein...

[0213] The anti-LAG3-anti-PD-1 bispecific antibody is the anti-LAG3-anti-PD-1 bispecific antibody BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02 or Bi-PGV02 of the present invention;

[0214] The anti-CTLA4-anti-PD-1 bispecific antibody is one of the antibodies BiAb001(hG1TM), BiAb002(hG1TM), BiAb003(hG1TM), or BiAb004(hG1TM) of the present invention.

[0215] In some embodiments of the present invention, the drug combination, wherein...

[0216] The anti-LAG3-anti-PD-1 bispecific antibody is the anti-LAG3-anti-PD-1 bispecific antibody BS-PL021A of the present invention;

[0217] The anti-CTLA4-anti-PD-1 bispecific antibody is one of the antibodies BiAb001(hG1TM), BiAb002(hG1TM), BiAb003(hG1TM), or BiAb004(hG1TM) of the present invention.

[0218] In some embodiments of the present invention, the drug combination, wherein...

[0219] The anti-LAG3-anti-PD-1 bispecific antibody is the anti-LAG3-anti-PD-1 bispecific antibody BS-PL022B of the present invention.

[0220] The anti-CTLA4-anti-PD-1 bispecific antibody is one of the antibodies BiAb001(hG1TM), BiAb002(hG1TM), BiAb003(hG1TM), or BiAb004(hG1TM) of the present invention.

[0221] In some embodiments of the present invention, the drug combination, wherein...

[0222] The anti-LAG3-anti-PD-1 bispecific antibody is the anti-LAG3-anti-PD-1 bispecific antibody BS-PL023C of the present invention;

[0223] The anti-CTLA4-anti-PD-1 bispecific antibody is one of the antibodies BiAb001(hG1TM), BiAb002(hG1TM), BiAb003(hG1TM), or BiAb004(hG1TM) of the present invention.

[0224] In some embodiments of the present invention, the drug combination, wherein...

[0225] The anti-LAG3-anti-PD-1 bispecific antibody is the anti-LAG3-anti-PD-1 bispecific antibody Bs-PLV02 of the present invention;

[0226] The anti-CTLA4-anti-PD-1 bispecific antibody is one of the antibodies BiAb001(hG1TM), BiAb002(hG1TM), BiAb003(hG1TM), or BiAb004(hG1TM) of the present invention.

[0227] In some embodiments of the present invention, the drug combination, wherein...

[0228] The anti-LAG3-anti-PD-1 bispecific antibody is the anti-LAG3-anti-PD-1 bispecific antibody Bi-PGV02 of the present invention;

[0229] The anti-CTLA4-anti-PD-1 bispecific antibody is one of the antibodies BiAb001(hG1TM), BiAb002(hG1TM), BiAb003(hG1TM), or BiAb004(hG1TM) of the present invention.

[0230] In some embodiments of the present invention, the drug combination, wherein the first antibody and the second antibody are mixed together, is a pharmaceutical composition.

[0231] In some embodiments of the present invention, the drug combination, wherein the first antibody and the second antibody are in separate packages, is a combination product.

[0232] In some embodiments of the present invention, the drug combination wherein the mass ratio of the first antibody to the second antibody is (10:1) to (1:5), preferably (5:1) to (1:3) or (3:1) to (1:2), more preferably 2:1.

[0233] In some embodiments of the present invention, the drug combination wherein the molar ratio of the first antibody to the second antibody is (10:1) to (1:10), preferably (5:1) to (1:5), (3:1) to (1:3) or (2:1) to (1:2), and more preferably 1:1.

[0234] In some embodiments of the present invention, the drug combination comprises an effective amount of a first antibody and an effective amount of a second antibody, wherein the first antibody is an anti-LAG3-anti-PD-1 bispecific antibody, and the second antibody is an anti-CTLA4-anti-PD-1 bispecific antibody, wherein:

[0235] The anti-LAG3-anti-PD-1 bispecific antibody includes:

[0236] Targeting the first protein functional region of LAG3, and

[0237] Targeting the second protein functional region of PD-1;

[0238] The first protein has one functional region, and the second protein has two functional regions;

[0239] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0240] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:2, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:4.

[0241] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:19, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:21 or SEQ ID NO:38.

[0242] The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin;

[0243] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0244] and

[0245] The anti-CTLA4-anti-PD-1 bispecific antibody includes:

[0246] Targeting the third protein functional region of PD-1, and

[0247] Targeting the fourth protein functional region of CTLA4;

[0248] in,

[0249] The third protein functional region consists of one unit, and the fourth protein functional region consists of two units.

[0250] The third protein functional region is an immunoglobulin, and the fourth protein functional region is a single-chain antibody.

[0251] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:19, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:21.

[0252] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:61 or SEQ ID NO:63, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:62 or SEQ ID NO:64.

[0253] The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin;

[0254] The third protein functional region is connected to the fourth protein functional region via a third linker; and the heavy chain variable region of the single-chain antibody is connected to the light chain variable region of the single-chain antibody via a fourth linker; the third linker and the fourth linker may be the same or different;

[0255] Preferably, the molar ratio or mass ratio of the anti-LAG3-anti-PD-1 bispecific antibody to the anti-CTLA4-anti-PD-1 bispecific antibody is (5:1) to (1:2), (4:1) to (1:2), (3:1) to (1:2), (2:1) to (1:2), (5:1) to (1:1), (4:1) to (1:1), (3:1) to (1:1), (2:1) to (1:1), 5:1, 4:1, 3:1, 2.5:1, 2:1, 1.5:1 or 1:1;

[0256] Preferably, the drug combination further comprises an effective amount of a chemotherapy drug, wherein the chemotherapy drug is a chemotherapy drug of the CapeOX regimen (also known as the Xelox regimen) (e.g., oxaliplatin and capecitabine), a chemotherapy drug of the mFOLFOX6 regimen, or a chemotherapy drug of the FOLFIRI regimen.

[0257] In some embodiments of the present invention, the drug combination comprises an effective amount of a first antibody and an effective amount of a second antibody, wherein the first antibody is an anti-LAG3-anti-PD-1 bispecific antibody, and the second antibody is an anti-CTLA4-anti-PD-1 bispecific antibody, wherein:

[0258] The anti-LAG3-anti-PD-1 bispecific antibody includes:

[0259] Targeting the first protein functional region of LAG3, and

[0260] Targeting the second protein functional region of PD-1;

[0261] The first protein has one functional region, and the second protein has two functional regions;

[0262] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0263] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:2, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:4.

[0264] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:19, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:21 or SEQ ID NO:38.

[0265] The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin;

[0266] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0267] The amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NOs:35-37;

[0268] and

[0269] The anti-CTLA4-anti-PD-1 bispecific antibody includes:

[0270] Targeting the third protein functional region of PD-1, and

[0271] Targeting the fourth protein functional region of CTLA4;

[0272] in,

[0273] The third protein functional region consists of one unit, and the fourth protein functional region consists of two units.

[0274] The third protein functional region is an immunoglobulin, and the fourth protein functional region is a single-chain antibody.

[0275] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:19, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:21.

[0276] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:61 or SEQ ID NO:63, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:62 or SEQ ID NO:64.

[0277] The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin;

[0278] The third protein functional region is connected to the fourth protein functional region via a third linker; and the heavy chain variable region of the single-chain antibody is connected to the light chain variable region of the single-chain antibody via a fourth linker; the third linker and the fourth linker may be the same or different;

[0279] The amino acid sequences of the third and fourth linkers are independently selected from SEQ ID NOs:35-37;

[0280] Preferably, the molar ratio or mass ratio of the anti-LAG3-anti-PD-1 bispecific antibody to the anti-CTLA4-anti-PD-1 bispecific antibody is (5:1) to (1:2), (4:1) to (1:2), (3:1) to (1:2), (2:1) to (1:2), (5:1) to (1:1), (4:1) to (1:1), (3:1) to (1:1), (2:1) to (1:1), 5:1, 4:1, 3:1, 2.5:1, 2:1, 1.5:1 or 1:1;

[0281] Preferably, the drug combination further comprises an effective amount of a chemotherapy drug, wherein the chemotherapy drug is a chemotherapy drug of the CapeOX regimen (also known as the Xelox regimen) (e.g., oxaliplatin and capecitabine), a chemotherapy drug of the mFOLFOX6 regimen, or a chemotherapy drug of the FOLFIRI regimen.

[0282] In some embodiments of the present invention, the drug combination comprises an effective amount of a first antibody and an effective amount of a second antibody, wherein the first antibody is an anti-LAG3-anti-PD-1 bispecific antibody, and the second antibody is an anti-CTLA4-anti-PD-1 bispecific antibody, wherein:

[0283] The anti-LAG3-anti-PD-1 bispecific antibody includes:

[0284] Targeting the first protein functional region of LAG3, and

[0285] Targeting the second protein functional region of PD-1;

[0286] The first protein has one functional region, and the second protein has two functional regions;

[0287] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0288] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:2, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:4.

[0289] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:19, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:21 or SEQ ID NO:38.

[0290] The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin;

[0291] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0292] The amino acid sequences of both the first linker and the second linker are shown in SEQ ID NO:36;

[0293] and

[0294] The anti-CTLA4-anti-PD-1 bispecific antibody includes:

[0295] Targeting the third protein functional region of PD-1, and

[0296] Targeting the fourth protein functional region of CTLA4;

[0297] in,

[0298] The third protein functional region consists of one unit, and the fourth protein functional region consists of two units.

[0299] The third protein functional region is an immunoglobulin, and the fourth protein functional region is a single-chain antibody.

[0300] The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:19, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:21.

[0301] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:61 or SEQ ID NO:63, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:62 or SEQ ID NO:64.

[0302] The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin;

[0303] The third protein functional region is connected to the fourth protein functional region via a third linker; and the heavy chain variable region of the single-chain antibody is connected to the light chain variable region of the single-chain antibody via a fourth linker; the third linker and the fourth linker may be the same or different;

[0304] The amino acid sequences of the third linker and the fourth linker are both shown in SEQ ID NO:36;

[0305] Preferably, the molar ratio or mass ratio of the anti-LAG3-anti-PD-1 bispecific antibody to the anti-CTLA4-anti-PD-1 bispecific antibody is (5:1) to (1:2), (4:1) to (1:2), (3:1) to (1:2), (2:1) to (1:2), (5:1) to (1:1), (4:1) to (1:1), (3:1) to (1:1), (2:1) to (1:1), 5:1, 4:1, 3:1, 2.5:1, 2:1, 1.5:1 or 1:1;

[0306] Preferably, the drug combination further comprises an effective amount of a chemotherapy drug, wherein the chemotherapy drug is a chemotherapy drug of the CapeOX regimen (also known as the Xelox regimen) (e.g., oxaliplatin and capecitabine), a chemotherapy drug of the mFOLFOX6 regimen, or a chemotherapy drug of the FOLFIRI regimen.

[0307] In some embodiments of the present invention, the drug combination comprises an effective amount of a first antibody and an effective amount of a second antibody, wherein the first antibody is an anti-LAG3-anti-PD-1 bispecific antibody, and the second antibody is an anti-CTLA4-anti-PD-1 bispecific antibody, wherein:

[0308] The anti-LAG3-anti-PD-1 bispecific antibody includes:

[0309] Targeting the first protein functional region of LAG3, and

[0310] Targeting the second protein functional region of PD-1;

[0311] The first protein has one functional region, and the second protein has two functional regions;

[0312] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0313] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:11 or SEQ ID NO:13, and the amino acid sequence of its light chain is shown in SEQ ID NO:12.

[0314] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:19, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:21 or SEQ ID NO:38.

[0315] The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin;

[0316] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0317] and;

[0318] The anti-CTLA4-anti-PD-1 bispecific antibody includes:

[0319] Targeting the third protein functional region of PD-1, and

[0320] Targeting the fourth protein functional region of CTLA4;

[0321] in,

[0322] The third protein functional region consists of one unit, and the fourth protein functional region consists of two units.

[0323] The third protein functional region is an immunoglobulin, and the fourth protein functional region is a single-chain antibody.

[0324] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:66, and the amino acid sequence of its light chain is shown in SEQ ID NO:25.

[0325] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:61 or SEQ ID NO:63, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:62 or SEQ ID NO:64.

[0326] The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin;

[0327] The third protein functional region is connected to the fourth protein functional region via a third linker; and the heavy chain variable region of the single-chain antibody is connected to the light chain variable region of the single-chain antibody via a fourth linker; the third linker and the fourth linker may be the same or different;

[0328] Preferably, the molar ratio or mass ratio of the anti-LAG3-anti-PD-1 bispecific antibody to the anti-CTLA4-anti-PD-1 bispecific antibody is (5:1) to (1:2), (4:1) to (1:2), (3:1) to (1:2), (2:1) to (1:2), (5:1) to (1:1), (4:1) to (1:1), (3:1) to (1:1), (2:1) to (1:1), 5:1, 4:1, 3:1, 2.5:1, 2:1, 1.5:1 or 1:1;

[0329] Preferably, the drug combination further comprises an effective amount of a chemotherapy drug, wherein the chemotherapy drug is a chemotherapy drug of the CapeOX regimen (also known as the Xelox regimen) (e.g., oxaliplatin and capecitabine), a chemotherapy drug of the mFOLFOX6 regimen, or a chemotherapy drug of the FOLFIRI regimen.

[0330] In some embodiments of the present invention, the drug combination comprises an effective amount of a first antibody and an effective amount of a second antibody, wherein the first antibody is an anti-LAG3-anti-PD-1 bispecific antibody, and the second antibody is an anti-CTLA4-anti-PD-1 bispecific antibody, wherein:

[0331] The anti-LAG3-anti-PD-1 bispecific antibody includes:

[0332] Targeting the first protein functional region of LAG3, and

[0333] Targeting the second protein functional region of PD-1;

[0334] The first protein has one functional region, and the second protein has two functional regions;

[0335] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0336] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:11 or SEQ ID NO:13, and the amino acid sequence of its light chain is shown in SEQ ID NO:12.

[0337] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:19, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:21 or SEQ ID NO:38.

[0338] The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin;

[0339] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0340] The amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NOs:35-37;

[0341] and

[0342] The anti-CTLA4-anti-PD-1 bispecific antibody includes:

[0343] Targeting the third protein functional region of PD-1, and

[0344] Targeting the fourth protein functional region of CTLA4;

[0345] in,

[0346] The third protein functional region consists of one unit, and the fourth protein functional region consists of two units.

[0347] The third protein functional region is an immunoglobulin, and the fourth protein functional region is a single-chain antibody.

[0348] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:66, and the amino acid sequence of its light chain is shown in SEQ ID NO:25.

[0349] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:61 or SEQ ID NO:63, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:62 or SEQ ID NO:64.

[0350] The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin;

[0351] The third protein functional region is connected to the fourth protein functional region via a third linker; and the heavy chain variable region of the single-chain antibody is connected to the light chain variable region of the single-chain antibody via a fourth linker; the third linker and the fourth linker may be the same or different;

[0352] The amino acid sequences of the third and fourth linkers are independently selected from SEQ ID NOs:35-37;

[0353] Preferably, the molar ratio or mass ratio of the anti-LAG3-anti-PD-1 bispecific antibody to the anti-CTLA4-anti-PD-1 bispecific antibody is (5:1) to (1:2), (4:1) to (1:2), (3:1) to (1:2), (2:1) to (1:2), (5:1) to (1:1), (4:1) to (1:1), (3:1) to (1:1), (2:1) to (1:1), 5:1, 4:1, 3:1, 2.5:1, 2:1, 1.5:1 or 1:1;

[0354] Preferably, the drug combination further comprises an effective amount of a chemotherapy drug, wherein the chemotherapy drug is a chemotherapy drug of the CapeOX regimen (also known as the Xelox regimen) (e.g., oxaliplatin and capecitabine), a chemotherapy drug of the mFOLFOX6 regimen, or a chemotherapy drug of the FOLFIRI regimen.

[0355] In some embodiments of the present invention, the drug combination comprises an effective amount of a first antibody and an effective amount of a second antibody, wherein the first antibody is an anti-LAG3-anti-PD-1 bispecific antibody, and the second antibody is an anti-CTLA4-anti-PD-1 bispecific antibody, wherein:

[0356] The anti-LAG3-anti-PD-1 bispecific antibody includes:

[0357] Targeting the first protein functional region of LAG3, and

[0358] Targeting the second protein functional region of PD-1;

[0359] The first protein has one functional region, and the second protein has two functional regions;

[0360] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0361] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:11 or SEQ ID NO:13, and the amino acid sequence of its light chain is shown in SEQ ID NO:12.

[0362] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:19, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:21 or SEQ ID NO:38.

[0363] The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin;

[0364] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0365] The amino acid sequences of both the first linker and the second linker are shown in SEQ ID NO:36;

[0366] and

[0367] The anti-CTLA4-anti-PD-1 bispecific antibody includes:

[0368] Targeting the third protein functional region of PD-1, and

[0369] Targeting the fourth protein functional region of CTLA4;

[0370] in,

[0371] The third protein functional region consists of one unit, and the fourth protein functional region consists of two units.

[0372] The third protein functional region is an immunoglobulin, and the fourth protein functional region is a single-chain antibody.

[0373] The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:66, and the amino acid sequence of its light chain is shown in SEQ ID NO:25.

[0374] The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:61 or SEQ ID NO:63, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:62 or SEQ ID NO:64.

[0375] The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin;

[0376] The third protein functional region is connected to the fourth protein functional region via a third linker; and the heavy chain variable region of the single-chain antibody is connected to the light chain variable region of the single-chain antibody via a fourth linker; the third linker and the fourth linker may be the same or different;

[0377] The amino acid sequences of the third linker and the fourth linker are both shown in SEQ ID NO:36;

[0378] Preferably, the molar ratio or mass ratio of the anti-LAG3-anti-PD-1 bispecific antibody to the anti-CTLA4-anti-PD-1 bispecific antibody is (5:1) to (1:2), (4:1) to (1:2), (3:1) to (1:2), (2:1) to (1:2), (5:1) to (1:1), (4:1) to (1:1), (3:1) to (1:1), (2:1) to (1:1), 5:1, 4:1, 3:1, 2.5:1, 2:1, 1.5:1 or 1:1;

[0379] Preferably, the drug combination further comprises an effective amount of a chemotherapy drug, wherein the chemotherapy drug is a chemotherapy drug of the CapeOX regimen (also known as the Xelox regimen) (e.g., oxaliplatin and capecitabine), a chemotherapy drug of the mFOLFOX6 regimen, or a chemotherapy drug of the FOLFIRI regimen.

[0380] In some embodiments of the present invention, the drug combination wherein, calculated based on the mass of the first antibody or the second antibody, the unit dose of the first antibody and the second antibody is independently 100mg-2500mg, 100mg-2000mg, 100mg-1500mg, 100mg-1200mg, 100mg-1000mg, 200mg-800mg, 200mg-500mg, 300mg-600mg, 400mg-500mg, or 450mg.

[0381] In some embodiments of the present invention, the pharmaceutical combination further comprises one or more pharmaceutically acceptable excipients; preferably, it further comprises a product instruction manual.

[0382] In some embodiments of the present invention, the drug combination wherein the first antibody and the second antibody are pharmacodynamic ingredients (APIs) or active ingredients.

[0383] In some embodiments of the present invention, the drug combination comprises a first antibody and a second antibody as its active ingredient (API).

[0384] In some embodiments of the present invention, the drug combination comprises a first antibody, a second antibody, and pharmaceutically acceptable excipients.

[0385] In some embodiments of the present invention, the pharmaceutical combination, wherein the first antibody and the second antibody are in separate packages, the pharmaceutical combination is a combination product, and each of the separate packages contains the first antibody and a pharmaceutically acceptable excipient, and the second antibody and a pharmaceutically acceptable excipient.

[0386] In some embodiments of the present invention, the drug combination further comprises an effective amount of an oncology chemotherapy drug, such as an alkylating agent, antimetabolite, antitumor antibiotic, herbal anticancer drug, hormone or immunomodulator, preferably oxaliplatin and / or capecitabine.

[0387] In some embodiments of the present invention, the drug combination further comprises an effective amount of a chemotherapy drug, wherein the chemotherapy drug is a chemotherapy drug of the CapeOX regimen (also known as the Xelox regimen), a chemotherapy drug of the mFOLFOX6 regimen, or a chemotherapy drug of the FOLFIRI regimen.

[0388] In some embodiments of the present invention, the drug combination further comprises an effective amount of a chemotherapy drug, wherein the chemotherapy drug is oxaliplatin and capecitabine.

[0389] In some embodiments of the present invention, the drug combination further comprises an effective amount of a chemotherapy drug, wherein the chemotherapy drug is oxaliplatin, leucovorin (LV), and 5-fluorouracil (5-FU).

[0390] In some embodiments of the present invention, the drug combination further comprises an effective amount of a chemotherapy drug, wherein the chemotherapy drug is irinotecan, leucovorin calcium, and 5-fluorouracil.

[0391] In some embodiments of the present invention, the drug combination comprises the active pharmaceutical ingredient (API) or active ingredient consisting of the anti-LAG3-anti-PD-1 bispecific antibody, the anti-CTLA4-anti-PD-1 bispecific antibody, and a chemotherapy drug, wherein the chemotherapy drug is a chemotherapy drug of the CapeOX regimen (also known as the Xelox regimen), a chemotherapy drug of the mFOLFOX6 regimen, or a chemotherapy drug of the FOLFIRI regimen.

[0392] In some embodiments of the present invention, the drug combination comprises the pharmacodynamic ingredient (API) or active ingredient consisting of the anti-LAG3-anti-PD-1 bispecific antibody, the anti-CTLA4-anti-PD-1 bispecific antibody, and a chemotherapy drug, wherein the chemotherapy drug is oxaliplatin and capecitabine.

[0393] In some embodiments of the present invention, the drug combination comprises the pharmacodynamic ingredient (API) or active ingredient consisting of the anti-LAG3-anti-PD-1 bispecific antibody, the anti-CTLA4-anti-PD-1 bispecific antibody, and a chemotherapy drug, wherein the chemotherapy drug is oxaliplatin, leucovorin calcium, and 5-fluorouracil.

[0394] In some embodiments of the present invention, the drug combination comprises the pharmacologically active ingredient (API) or active ingredient consisting of the anti-LAG3-anti-PD-1 bispecific antibody, the anti-CTLA4-anti-PD-1 bispecific antibody, and a chemotherapy drug, wherein the chemotherapy drug is irinotecan, leucovorin calcium, and 5-fluorouracil.

[0395] In some embodiments of the present invention, the drug combination wherein the first antibody, the second antibody, and the tumor chemotherapy drug are pharmacodynamic ingredients (APIs) or active ingredients.

[0396] In some embodiments of the present invention, the drug combination comprises a first antibody, a second antibody, a tumor chemotherapy drug, and pharmaceutically acceptable excipients.

[0397] In some embodiments of the present invention, the drug combination, wherein the unit formulation comprises 1-10000 mg (preferably 10-1000 mg, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg or 200 mg) of the anti-LAG3-anti-PD-1 bispecific antibody according to any aspect of the present invention and 1-10000 mg (preferably 1-1000 mg, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, 200 mg or 100 mg) of the anti-CTLA4-anti-PD-1 bispecific antibody according to any aspect of the present invention, and optionally one or more chemotherapeutic agents according to the present invention (such as platinum-based drugs and / or fluorouracil-based antitumor drugs); wherein the anti-LAG3-anti-PD-1 bispecific antibody, the anti-CTLA4-anti-PD-1 bispecific antibody and the chemotherapeutic agent are each packaged separately.

[0398] The pharmaceutical combination according to any one of the present invention is used for the treatment or prevention of tumors;

[0399] Preferably, the tumor is selected from one or more of the following: pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, head and neck cancer, brain cancer, pharyngeal cancer, nasopharyngeal cancer, thyroid cancer, mesothelioma, lung cancer, liver cancer, stomach cancer, biliary tract cancer, kidney cancer, fallopian tube cancer, endometrial cancer, cervical cancer, bladder cancer, urothelial carcinoma, prostate cancer, testicular cancer, skin cancer, melanoma, myeloma, plasma cell carcinoma, vulvar cancer, leukemia, lymphoma, bone cancer, and osteosarcoma.

[0400] Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell carcinoma of the lung;

[0401] Preferably, the gastric cancer is gastric adenocarcinoma or esophageal junction adenocarcinoma;

[0402] Preferably, the lymphoma is Hodgkin lymphoma or non-Hodgkin lymphoma;

[0403] Preferably, the esophageal cancer is esophageal squamous cell carcinoma.

[0404] In some embodiments of the invention, the drug combination is wherein the first antibody and the antibody are administered independently before or after surgery, and / or before or after radiotherapy.

[0405] In some embodiments of the present invention, the drug combination, wherein...

[0406] The single dose of the first antibody and the second antibody is independently 0.1-100 mg per kilogram of body weight, preferably 5-50 mg or 5-15 mg per kilogram of body weight;

[0407] The first antibody and the second antibody are administered independently once every half day, every 1 day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 8 days, every 10 days, every 1 week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 1 month, every 2 months, every 3 months, every 4 months, every 5 months, or every 6 months.

[0408] and / or

[0409] The administration methods include intravenous infusion, intravenous injection, subcutaneous injection, intraperitoneal injection, or intratumoral injection.

[0410] In some embodiments of the present invention, the drug combination, wherein one or more doses of the unit formulation of the present invention are administered to a subject in need, preferably, the anti-LAG3-anti-PD-1 bispecific antibody, the anti-CTLA4-anti-PD-1 bispecific antibody and the chemotherapy drug in the unit formulation are each administered separately.

[0411] In some embodiments of the present invention, the drug combination, wherein the single-dose dose of the anti-LAG3-anti-PD-1 bispecific antibody and / or the anti-CTLA4-anti-PD-1 bispecific antibody of any one of the present invention is 0.1-100 mg per kilogram of body weight, preferably 1-10 mg; or, the single-dose dose of the anti-LAG3-anti-PD-1 bispecific antibody and / or the anti-CTLA4-anti-PD-1 bispecific antibody of any one of the present invention is 10-1000 mg per subject, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, or 200 mg.

[0412] Preferably, the medication is administered twice daily to approximately once every other day, or once every 3 days, 4 days, 5 days, 6 days, 8 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months.

[0413] Preferably, the administration method is intravenous infusion, intravenous injection, subcutaneous injection, intraperitoneal injection, or intratumoral injection.

[0414] In some embodiments of the present invention, the drug combination wherein the first antibody is administered at a single dose of 15 mg per kilogram of body weight independently, the second antibody is administered at a single dose of 10 mg per kilogram of body weight independently, and the first antibody and / or the second antibody are administered in a 3-week cycle.

[0415] In some embodiments of the present invention, the drug combination wherein the first antibody is administered at a single dose of 25 mg per kilogram of body weight, the second antibody is administered at a single dose of 10 mg per kilogram of body weight, and the first antibody and / or the second antibody are administered in a 3-week cycle.

[0416] In some embodiments of the present invention, the drug combination wherein the first antibody is administered at a single dose of 40 mg per kilogram of body weight, the second antibody is administered at a single dose of 10 mg per kilogram of body weight, and the first antibody and / or the second antibody are administered in a 3-week cycle.

[0417] In some embodiments of the present invention, the drug combination wherein the first antibody is administered at a single dose of 15 mg / kg body weight independently, the second antibody is administered at a single dose of 10 mg / kg body weight independently, and the oxaliplatin is administered at a single dose of 130 mg / kg body weight independently. 2 The single-dose capecitabine dose is independently 1000 mg / m². 2 The first antibody, the second antibody, oxaliplatin and / or capecitabine are administered in a 3-week cycle.

[0418] In some embodiments of the present invention, the drug combination wherein the first antibody is administered at a single dose of 25 mg / kg body weight independently, the second antibody is administered at a single dose of 10 mg / kg body weight independently, and the oxaliplatin is administered at a single dose of 130 mg / kg body weight independently. 2 The single-dose capecitabine dose is independently 1000 mg / m². 2 The first antibody, the second antibody, oxaliplatin and / or capecitabine are administered in a 3-week cycle.

[0419] In some embodiments of the present invention, the drug combination wherein the first antibody is administered at a single dose of 40 mg / kg body weight independently, the second antibody is administered at a single dose of 10 mg / kg body weight independently, and the oxaliplatin is administered at a single dose of 130 mg / kg body weight independently. 2 The single-dose capecitabine dose is independently 1000 mg / m². 2 The first antibody, the second antibody, oxaliplatin and / or capecitabine are administered in a 3-week cycle.

[0420] Another aspect of the present invention relates to the use of the pharmaceutical combination described in any one of the present invention in the preparation of a medicament for treating or preventing tumors;

[0421] Preferably, the tumor is selected from one or more of the following: pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, head and neck cancer, brain cancer, pharyngeal cancer, nasopharyngeal cancer, thyroid cancer, mesothelioma, lung cancer, liver cancer, stomach cancer, biliary tract cancer, kidney cancer, fallopian tube cancer, endometrial cancer, cervical cancer, bladder cancer, urothelial carcinoma, prostate cancer, testicular cancer, skin cancer, melanoma, myeloma, plasma cell carcinoma, vulvar cancer, leukemia, lymphoma, bone cancer, and osteosarcoma.

[0422] Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell carcinoma of the lung;

[0423] Preferably, the gastric cancer is gastric adenocarcinoma or esophageal junction adenocarcinoma;

[0424] Preferably, the lymphoma is Hodgkin lymphoma or non-Hodgkin lymphoma;

[0425] Preferably, the esophageal cancer is esophageal squamous cell carcinoma.

[0426] In some embodiments of the invention, the described use is wherein the first antibody and the antibody are administered independently before or after surgery, and / or before or after radiotherapy.

[0427] In some embodiments of the present invention, the described uses, wherein...

[0428] The single dose of the first antibody and the second antibody is independently 0.1-100 mg per kilogram of body weight, preferably 5-50 mg or 5-15 mg per kilogram of body weight;

[0429] The first antibody and the second antibody are administered independently once every half day, every day, every two days, every three days, every four days, every five days, every six days, every eight days, every ten days, every week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every month, every two months, every three months, every four months, every five months, or every six months.

[0430] and / or

[0431] The administration methods include intravenous infusion, intravenous injection, subcutaneous injection, intraperitoneal injection, or intratumoral injection.

[0432] In some embodiments of the present invention, the described use includes administering one or more doses of the unit formulation of the present invention to a subject in need, preferably, wherein the anti-LAG3-anti-PD-1 bispecific antibody, the anti-CTLA4-anti-PD-1 bispecific antibody, and the chemotherapy drug in the unit formulation are administered separately.

[0433] In some embodiments of the present invention, the described uses include a single dose of 0.1-100 mg per kilogram of body weight, preferably 1-10 mg, of the anti-LAG3-anti-PD-1 bispecific antibody and / or the anti-CTLA4-anti-PD-1 bispecific antibody of the present invention; or a single dose of 10-1000 mg per subject, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, or 200 mg of the anti-LAG3-anti-PD-1 bispecific antibody and / or the anti-CTLA4-anti-PD-1 bispecific antibody of the present invention.

[0434] Preferably, the medication is administered twice daily to approximately once every other day, or once every 3 days, 4 days, 5 days, 6 days, 8 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months.

[0435] Preferably, the administration method is intravenous infusion, intravenous injection, subcutaneous injection, intraperitoneal injection, or intratumoral injection.

[0436] In some embodiments of the present invention, the use is described in that the single dose of the first antibody is independently 15 mg per kilogram of body weight, the single dose of the second antibody is independently 10 mg per kilogram of body weight, and the first antibody and / or the second antibody are administered in a 3-week cycle.

[0437] In some embodiments of the present invention, the use is described in that the single dose of the first antibody is independently 25 mg per kilogram of body weight, the single dose of the second antibody is independently 10 mg per kilogram of body weight, and the first antibody and / or the second antibody are administered in a 3-week cycle.

[0438] In some embodiments of the present invention, the use is described in that the single dose of the first antibody is independently 40 mg per kilogram of body weight, the single dose of the second antibody is independently 10 mg per kilogram of body weight, and the first antibody and / or the second antibody are administered in a 3-week cycle.

[0439] In some embodiments of the present invention, the described uses include, wherein the single dose of the first antibody is independently 15 mg per kilogram of body weight, the single dose of the second antibody is independently 10 mg per kilogram of body weight, and the single dose of oxaliplatin is independently 130 mg / m². 2 The single-dose capecitabine dose is independently 1000 mg / m². 2 The first antibody, the second antibody, oxaliplatin and / or capecitabine are administered in a 3-week cycle.

[0440] In some embodiments of the present invention, the described uses include, wherein the single dose of the first antibody is independently 25 mg per kilogram of body weight, the single dose of the second antibody is independently 10 mg per kilogram of body weight, and the single dose of oxaliplatin is independently 130 mg / m². 2 The single-dose capecitabine dose is independently 1000 mg / m². 2 The first antibody, the second antibody, oxaliplatin and / or capecitabine are administered in a 3-week cycle.

[0441] In some embodiments of the present invention, the described uses include, wherein the single dose of the first antibody is independently 40 mg per kilogram of body weight, the single dose of the second antibody is independently 10 mg per kilogram of body weight, and the single dose of oxaliplatin is independently 130 mg / m². 2 The single-dose capecitabine dose is independently 1000 mg / m². 2 The first antibody, the second antibody, oxaliplatin and / or capecitabine are administered in a 3-week cycle.

[0442] Another aspect of the invention relates to a method of treating or preventing tumors, comprising the step of administering to a subject in need an effective amount of any of the drug combinations of the present invention;

[0443] Preferably, the tumor is selected from one or more of the following: pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, head and neck cancer, brain cancer, pharyngeal cancer, nasopharyngeal cancer, thyroid cancer, mesothelioma, lung cancer, liver cancer, stomach cancer, biliary tract cancer, kidney cancer, fallopian tube cancer, endometrial cancer, cervical cancer, bladder cancer, urothelial carcinoma, prostate cancer, testicular cancer, skin cancer, melanoma, myeloma, plasma cell carcinoma, vulvar cancer, leukemia, lymphoma, bone cancer, and osteosarcoma.

[0444] Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell carcinoma of the lung;

[0445] Preferably, the gastric cancer is gastric adenocarcinoma or esophageal junction adenocarcinoma;

[0446] Preferably, the lymphoma is Hodgkin lymphoma or non-Hodgkin lymphoma;

[0447] Preferably, the esophageal cancer is esophageal squamous cell carcinoma.

[0448] In some embodiments of the present invention, the method of treating or preventing tumors is wherein the first antibody and the antibody are administered independently before or after surgery, and / or before or after radiotherapy.

[0449] In some embodiments of the present invention, the method for treating or preventing tumors, wherein,

[0450] The single dose of the first antibody and the second antibody is independently 0.1-100 mg per kilogram of body weight, preferably 5-50 mg or 5-15 mg per kilogram of body weight;

[0451] The first antibody and the second antibody are administered independently once every half day, every 1 day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 8 days, every 10 days, every 1 week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 1 month, every 2 months, every 3 months, every 4 months, every 5 months, or every 6 months.

[0452] and / or

[0453] The administration methods include intravenous infusion, intravenous injection, subcutaneous injection, intraperitoneal injection, or intratumoral injection.

[0454] In some embodiments of the present invention, the method of treating or preventing tumors includes administering one or more doses of the unit formulation of the present invention to a subject in need, preferably, the anti-LAG3-anti-PD-1 bispecific antibody, the anti-CTLA4-anti-PD-1 bispecific antibody and the chemotherapy drug in the unit formulation are administered separately.

[0455] In some embodiments of the present invention, the method for treating or preventing tumors, wherein the single-dose dose of the anti-LAG3-anti-PD-1 bispecific antibody and / or the anti-CTLA4-anti-PD-1 bispecific antibody of any one of the present invention is 0.1-100 mg per kilogram of body weight, preferably 1-10 mg; or, the single-dose dose of the anti-LAG3-anti-PD-1 bispecific antibody and / or the anti-CTLA4-anti-PD-1 bispecific antibody of any one of the present invention is 10-1000 mg per subject, preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg, or 200 mg.

[0456] Preferably, the medication is administered twice daily to approximately once every other day, or once every 3 days, 4 days, 5 days, 6 days, 8 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months.

[0457] Preferably, the administration method is intravenous infusion, intravenous injection, subcutaneous injection, intraperitoneal injection, or intratumoral injection.

[0458] In some embodiments of the present invention, the method for treating or preventing tumors is wherein the single dose of the first antibody is independently 15 mg per kilogram of body weight, the single dose of the second antibody is independently 10 mg per kilogram of body weight, and the first antibody and / or the second antibody are administered in a 3-week cycle.

[0459] In some embodiments of the present invention, the method for treating or preventing tumors is wherein the single dose of the first antibody is independently 25 mg per kilogram of body weight, the single dose of the second antibody is independently 10 mg per kilogram of body weight, and the first antibody and / or the second antibody are administered in a 3-week cycle.

[0460] In some embodiments of the present invention, the method for treating or preventing tumors is wherein the single dose of the first antibody is independently 40 mg per kilogram of body weight, the single dose of the second antibody is independently 10 mg per kilogram of body weight, and the first antibody and / or the second antibody are administered in a 3-week cycle.

[0461] In some embodiments of the present invention, the method for treating or preventing tumors includes a single dose of the first antibody independently of 15 mg per kilogram of body weight, a single dose of the second antibody independently of 10 mg per kilogram of body weight, and a single dose of oxaliplatin independently of 130 mg / m². 2 The single-dose capecitabine dose is independently 1000 mg / m².2 The first antibody, the second antibody, oxaliplatin and / or capecitabine are administered in a 3-week cycle.

[0462] In some embodiments of the present invention, the method for treating or preventing tumors includes a single dose of the first antibody independently of 25 mg / kg body weight, a single dose of the second antibody independently of 10 mg / kg body weight, and a single dose of oxaliplatin independently of 130 mg / m². 2 The single-dose capecitabine dose is independently 1000 mg / m². 2 The first antibody, the second antibody, oxaliplatin and / or capecitabine are administered in a 3-week cycle.

[0463] In some embodiments of the present invention, the method for treating or preventing tumors includes a single dose of the first antibody independently of 40 mg / kg body weight, a single dose of the second antibody independently of 10 mg / kg body weight, and a single dose of oxaliplatin independently of 130 mg / m². 2 The single-dose capecitabine dose is independently 1000 mg / m². 2 The first antibody, the second antibody, oxaliplatin and / or capecitabine are administered in a 3-week cycle.

[0464] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0465] As used in this article, the term EC 50 The half-maximal effect concentration refers to the concentration that produces 50% of the maximum effect.

[0466] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one "light" (L) chain and one "heavy" (H) chain). Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by "J" regions of approximately 12 or more amino acids, and the heavy chain also contains "D" regions of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antibody binding sites. The allocation of amino acids to various regions or domains follows Bethesda Md, Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 1987; 196: 901-917; Chothia et al. Nature 1989; 342: 878-883, or the definition of the IMGT numbering system, see Ehrenmann F, Kaas Q, Lefranc M P. IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF[J]. Nucleic acids research, 2009; 38(suppl_1): D301-D307.

[0467] In particular, the heavy chain may also contain more than three CDRs, such as six, nine, or twelve. For example, in the bispecific antibody of the present invention, the heavy chain may be an IgG antibody with a ScFv attached to its C-terminus, in which case the heavy chain contains nine CDRs.

[0468] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0469] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules—that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies exhibit high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies can usually be obtained using the hybridoma technique first reported by Kohler et al. G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity[J]. Nature, 1975; 256(5517):495), but it can also be obtained using recombinant DNA technology (see USPatent 4,816,567).

[0470] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR region of a human immunoglobulin (receptor antibody) with the CDR region of a non-human antibody (donor antibody), wherein the donor antibody can be a non-human antibody (e.g., mouse, rat, or rabbit) with the expected specificity, affinity, or reactivity. Furthermore, some amino acid residues in the framework region (FR) of the receptor antibody may also be replaced by amino acid residues of the corresponding non-human antibody, or by amino acid residues of other antibodies, to further improve or optimize the antibody's performance. For more detailed information on humanized antibodies, please refer to, for example, Jones et al., Nature 1986; 321:522-525; Reichmann et al., Nature, 1988; 332:323-329; Presta, Curr. Op. Struct. Biol. 1992; 2:593-596; and Clark, Immunol. Today 2000; 21:397-402. In some cases, the antigen-binding fragment of the antibody is a diabetic, where V... H and V L The domain is expressed on a single polypeptide chain, but the linker is too short to allow pairing between two domains on the same chain, thus forcing the domain to pair with a complementary domain on another chain and creating two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 1993; 90:6444-6448 and Poljak RJ et al., Structure 1994; 2:1121-1123).

[0471] As used herein, the term "single-chain fragment variable (ScFv)" refers to an antibody heavy chain variable region (V) linked by linkers. H ) and antibody light chain variable region (V L The molecules of V. L and V H The domain enables the formation of monovalent molecules by linker pairing with single polypeptide chains (see, for example, Birdet et al., Science 1988; 242:423-426 and Huston et al., Proc. Natl. Acad. Sci. USA 1988; 85:5879-5883). Such scFv molecules can have a general structure: NH2-V L -Connector-V H -COOH or NH2-V H-Connector-V L -COOH. Suitable prior art adapters consist of a repeating GGGGS (SEQ ID NO:73) amino acid sequence or a variant thereof. For example, an adapter having the amino acid sequence (GGGGS)4 (SEQ ID NO:36) can be used, but variants thereof can also be used (Holliger et al, Proc. Natl. Acad. Sci. USA 1993; 90:6444-6448). Other connectors that can be used in this invention are described by Alfthan et al, Protein Eng. 1995; 8:725-731, Choi et al, Eur. J. Immunol. 2001; 31:94-106, Hu et al, Cancer Res. 1996; 56:3055-3061, Kipriyanov et al, J. Mol. Biol. 1999; 293:41-56 and Roovers et al, Cancer Immunology, Immunotherapy, 2001, 50(1):51-59.

[0472] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds to an antigen at a concentration of less than about 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or lower affinity (K) D The antigen is bound to the antigen. In some embodiments of the invention, the term "targeted" refers to specific binding.

[0473] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide containing a fragment of the full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the “antigen-binding moiety.” See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of the intact antibody. In some cases, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabody antibodies, and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0474] As used herein, the term “Fd fragment” refers to an antibody fragment consisting of the VH and CH1 domains; the term “Fv fragment” refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody; the term “dAb fragment” refers to an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544-546 (1989)); the term “Fab fragment” refers to an antibody fragment consisting of the VL, VH, CL, and CH1 domains; and the term “F(ab')2 fragment” refers to an antibody fragment containing two Fab fragments connected by a disulfide bridge on the hinge region.

[0475] As used in this article, the term "K" D "" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, antibodies have an equilibrium dissociation constant of less than approximately 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 - 9 M or 10 -10 M or a smaller dissociation equilibrium constant (K) D K binds to an antigen (e.g., PD-1 protein). K can be measured using methods known to those skilled in the art. D For example, measurements can be taken using a Fortebio molecular interaction analyzer.

[0476] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0477] As used herein, the term "pharmaceuticalally acceptable excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0478] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing disease (e.g., cancer) means an amount sufficient to prevent, stop, or delay the onset of disease (e.g., cancer); an effective amount for treating disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0479] As used herein, when referring to PD-1 protein, it includes the full-length PD-1 protein, or the extracellular fragment PD-1ECD of PD-1, or a fragment containing PD-1ECD; it also includes fusion proteins of the full-length PD-1 protein or fusion proteins of PD-1ECD, such as fragments fused with a fragment of the Fc protein (mFc or hFc) of mouse or human IgG. However, those skilled in the art will understand that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of PD-1 protein without affecting its biological function. Therefore, in this invention, the term "PD-1 protein" should include all such sequences, including its natural or artificial variants. Furthermore, when describing a sequence fragment of PD-1 protein, it also includes the corresponding sequence fragment from its natural or artificial variants.

[0480] As used herein, when referring to the amino acid sequence of lymphocyte-activation gene 3 (LAG3), it includes the full-length LAG3 protein, or the extracellular fragment LAG3 ECD, or a fragment containing LAG3 ECD; it also includes fusion proteins of the full-length LAG3 protein or fusion proteins of LAG3 ECD, such as fragments fused with a fragment of the Fc protein (mFc or hFc) of mouse or human IgG. However, those skilled in the art will understand that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of the LAG3 protein without affecting its biological function. Therefore, in this invention, the term "LAG3 protein" should include all such sequences, including its natural or artificial variants. Furthermore, when describing a sequence fragment of the LAG3 protein, it also includes the corresponding sequence fragment from its natural or artificial variants.

[0481] As used herein, when referring to the amino acid sequence of the CTLA4 protein (Cytotoxic T-Lymphocyte-associated Antigen 4), it includes the full-length CTLA4 protein, or the extracellular fragment CTLA4ECD of CTLA4, or a fragment containing CTLA4ECD; it also includes fusion proteins of CTLA4ECD, such as fragments fused with a fragment of the Fc protein (mFc or hFc) of mouse or human IgG. However, those skilled in the art will understand that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of the CTLA4 protein without affecting its biological function. Therefore, in this invention, the term "CTLA4 protein" should include all such sequences, including its natural or artificial variants. Furthermore, when describing a sequence fragment of the CTLA4 protein, it also includes the corresponding sequence fragment from its natural or artificial variants.

[0482] In this invention, unless otherwise specified, the terms "first" (e.g., first protein functional region, first antibody, first linker, etc.), "second" (e.g., second protein functional region, second antibody, second linker, etc.), "third" (e.g., third protein functional region, third linker, etc.) and "fourth" (e.g., fourth protein functional region, fourth linker, etc.) are used for distinguishing references or clarifying expressions, and do not have a typical sequential meaning.

[0483] In this invention, unless otherwise specified, the numbers 1, 2, 3 or 4 in VH1, VH2, VH3, VH4, VL1, VL2, VL3 and VL4 are for reference or clarity of expression and do not have a typical meaning of order.

[0484] Beneficial effects of the invention

[0485] This invention achieves one or more of the following effects:

[0486] (1) The drug combination of the present invention can effectively prevent or treat tumors.

[0487] (2) The combination of anti-LAG3-anti-PD-1 bispecific antibody and anti-CTLA4-anti-PD-1 bispecific antibody has a pharmacological effect of effectively inhibiting tumors, and the effect is better than that of anti-LAG3-anti-PD-1 bispecific antibody monotherapy or anti-CTLA4-anti-PD-1 bispecific antibody monotherapy, achieving a synergistic effect. Attached Figure Description

[0488] Figure 1 : Mixed lymphocyte reaction detection of anti-LAG3-anti-PD-1 bispecific antibody combined with anti-CTLA4-anti-PD-1 bispecific antibody to promote IL-2 secretion.

[0489] Figure 2 : Mixed lymphocyte reaction detection of anti-LAG3-anti-PD-1 bispecific antibody combined with anti-CTLA4-anti-PD-1 bispecific antibody to promote IFN-γ secretion. Detailed Implementation

[0490] The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the examples, they are performed according to the techniques or conditions described in the literature in the art (e.g., refer to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press, translated by Huang Peitang et al.) or according to the product instructions. Reagents or instruments whose manufacturers are not specified are conventional products that can be purchased commercially.

[0491] In the embodiments of the present invention, the isotype control antibody used, namely hIgG4, is an antibody targeting human anti-egg lysosome (HEL). Its variable region sequence comes from Acierno et al.'s "Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies" (Acierno et al. J Mol Biol. 2007; 374(1):130-46.). The hIgG4 heavy chain constant region uses Ig gamma-4chain C region, ACCESSION: P01861.1 as the heavy chain constant region and introduces S228P mutation to improve stability. The Igkappa chain C region, ACCESSION: P01834, is the light chain constant region. hIgG4 was prepared in the laboratory of Zhongshan Kangfang Biopharmaceutical Co., Ltd.

[0492] Isotype control B12hG1 heavy chain amino acid sequence

[0493] QVQLVQSGAEVKKPGASVKVSCQASGYRFSNFVIHWVRQAPGQRFEWMGWINPYNGNKEFSAKFQDRVTFTADTSANTAYMELRSLRSADTAVYYCARVGPYSWDDSPQDNYYMD VWGKGTTVIVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:74)

[0494] Isotype control B12hG1 light chain amino acid sequence

[0495] EIVLTQSPGTLSLSPGERATFSCRSSHSIRSRRVAWYQHKPGQAPRLVIHGVSNRASGISDRFSGSGSGTDFTLTITRVEPEDFALYYCQVYGASSYTFGQGTKLERKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQID NO:75)

[0496] The Raji-PDL1 cell line was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. The Raji-PDL1 cell line was obtained by viral infection of Raji cells. The virus preparation used 3rd Generation Lentiviral Systems, see, for example, AThird Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini LJ Virol. 1998. 72(11): 8463-8471. The lentiviral expression vector used was plenti6.3 / V5-PDL1 (where PDL1, Genebank ID: NP_054862.1; vector plenti6.3 / V5, purchased from Invitrogen, catalog number: K5315-20).

[0497] Anti-PD-1 monoclonal antibody Opdivo (purchased from BMS, lot number ABS3203).

[0498] Anti-CTLA4 monoclonal antibody Yervoy (purchased from BMS, lot number AAT3892).

[0499] The anti-LAG3 monoclonal antibody Relatlimab was manufactured in-house by Zhongshan Kangfang Biotechnology.

[0500] Preparation Example 1: Design and Preparation of Anti-LAG3 Antibody

[0501] 1. Antibody design

[0502] Based on the existing LAG3 protein sequence (NCBI Reference Sequence: NP_002277.4) and its three-dimensional crystal structure, the inventors creatively designed a series of antibody sequences. Through extensive screening and testing, humanized monoclonal antibodies that specifically bind to LAG3 were finally obtained, named H7L8, H7L9, and H7L10, respectively. The amino acid sequences and their coding sequences of the heavy and light chain variable regions of these monoclonal antibodies are as follows.

[0503] The nucleic acid sequence (360bp) of the H7L8 heavy chain variable region H7v:

[0504] CAGGTGCAGCTGCAGCAGTGGGGAGCTGGACTGCTGAAACCTAGCGAGACACTGAGCCTGACCTGTGCTGTGTACGGCGGATCTATCAGCGATTACTACTGGAACTGGATCAGGCAGCCCCTGGAAAGGGACTGGAATGGATCGGAGAGATCAACCACAGGGGCACCACCAACTCCAATCC CTCTCTGAAGAGCAGGGTGACACTGAGCCTCGACACAAGCAAGAATCAGTTCAGCCTGAAGCTGAGGTCCGTGACCGCTGCTGATACAGCTGTGTACTACTGTGCCTTCGGCTACAGCGATTACGAGTACGATTGGTTCGACCCTTGGGGCCAGGGAACACTGGTTACAGTGAGCTCC(SEQ ID NO:1)

[0505] The amino acid sequence (120 aa) of the heavy chain variable region H7v of H7L8:

[0506] QVQLQQWGAGLLKPSETLSLTCAVYGGSISDYYWNWIRQP PGKGLEWIGEINHRGTTNSNPSLKSRVTLSLDTSKNQFSLKLRS VTAADTAVYYCAFGYSDYEYDWFDPWGQGTLVTVSS(SEQ ID NO:2)

[0507] The nucleic acid sequence (321 bp) of the L8v variable region of the H7L8 light chain:

[0508] GAGATCGTTCTGACCCAGAGCCCAGCTACACTGAGCCTGTCTCCTGGAGAGAGGGCTACACTGTCCTGCAGAGCTAGCCAGACATCAGCAGCTACCTGGCTTGGTACCAGCAGAAGCCTGGCCAAGCTCCAAGGCTGCTGATCTACGACGCCTCTAATAGG GCCACCGGCATCCCTGCTAGATTCTCTGGAAGCGGCAGCGGAACCGACTTTACACTGACAATCAGCTCCCTGGAGCCCGAGGATTTCGCTGTTTACTACTGTCAGCAGCGCAGCAACTGGCCCATCACATTCGGACAGGGCACAAATCTGGAGATCAAG(SEQ ID NO:3)

[0509] The amino acid sequence (107aa) of the L8v variable region of the H7L8 light chain:

[0510] EIVLTQSPATLSLSPGERATLSCRASQTISSYLAWYQQKPGQ APRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPITFGQGTNLEIK(SEQ ID NO:4)

[0511] The nucleic acid sequence of the heavy chain variable region H7v of H7L9 is the same as that of the heavy chain variable region H7v of H7L8, i.e., SEQ ID NO:1.

[0512] The amino acid sequence of the heavy chain variable region H7v of H7L9 is the same as that of the heavy chain variable region H7v of H7L8, i.e., SEQ ID NO:2.

[0513] The nucleic acid sequence (321 bp) of the L9v variable region of the H7L9 light chain:

[0514] GAGATCGTTCTGACCCAGAGCCCAGCTACACTGAGCCTGTCTCCTGGAGAGAGGGCTACACTGTCCTGCAGAGCTAGCCAGACATCAGCAGCTACCTGGCTTGGTACCAGCAGAAGCCTGGCCAAGCTCCAAGGCTGCTGATCTACGACGGCTCTAATAGG GCCACCGGCATCCCTGCTAGATTCTCTGGAAGCGGCAGCGGAACCGACTTTACACTGACAATCAGCTCCCTGGAGCCCGAGGATTTCGCTGTTTACTACTGTCAGCAGCGCAGCAACTGGCCCCTCACATTCGGACAGGGCACAAATCTGGAGATCAAG(SEQ ID NO:41)

[0515] The amino acid sequence (107 bp) of the L9v variable region of the light chain of H7L9:

[0516] EIVLTQSPATLSLSPGERATLSCRASQTISSYLAWYQQKPGQ APRLLIYDGSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPLTFGQGTNLEIK(SEQ ID NO:42)

[0517] The nucleic acid sequence of the heavy chain variable region H7v of H7L10 is the same as that of the heavy chain variable region H7v of H7L8, i.e., SEQ ID NO:1.

[0518] The amino acid sequence of the heavy chain variable region H7v of H7L10 is the same as that of the heavy chain variable region H7v of H7L8, i.e., SEQ ID NO:2.

[0519] The nucleic acid sequence (321 bp) of the L10v variable region of the H7L10 light chain:

[0520] GAGATCGTTCTGACCCAGAGCCCAGCTACACTGAGCCTGTCTCCTGGAGAGAGGGCTACACTGTCCTGCAGAGCTAGCCAGTCCATCAGCAGCTACCTGGCTTGGTACCAGCAGAAGCCTGGCCAAGCTCCAAGGCTGCTGATCTACGACGGCTCTAATAGG GCCACCGGCATCCCTGCTAGATTCTCTGGAAGCGGCAGCGGAACCGACTTTACACTGACAATCAGCTCCCTGGAGCCCGAGGATTTCGCTGTTTACTACTGTCAGCAGCGCAGCAACTGGCCCATCACATTCGGACAGGGCACAAATCTGGAGATCAAG(SEQ ID NO:43)

[0521] The amino acid sequence (107 bp) of the L10v variable region of the light chain of H7L10:

[0522] EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQ APRLLIYDGSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPITFGQGTNLEIK(SEQ ID NO:44)

[0523] The amino acid sequence of the CDR of antibody H7L8 is as follows (according to the IMGT numbering system):

[0524] HCDR1:GGSISDYY(SEQ ID NO:5);

[0525] HCDR2: INHRGTT (SEQ ID NO: 6);

[0526] HCDR3:AFGYSDYEYDWFDP(SEQ ID NO:7);

[0527] LCDR1: QTISSY(SEQ ID NO:8);

[0528] LCDR2: DAS (SEQ ID NO: 9);

[0529] LCDR3: QQRSNWPIT (SEQ ID NO: 10).

[0530] The amino acid sequence of the CDR of antibody H7L9 is as follows (according to the IMGT numbering system):

[0531] HCDR1:GGSISDYY(SEQ ID NO:5);

[0532] HCDR2: INHRGTT (SEQ ID NO: 6);

[0533] HCDR3:AFGYSDYEYDWFDP(SEQ ID NO:7);

[0534] LCDR1: QTISSY(SEQ ID NO:8);

[0535] LCDR2: DGS (SEQ ID NO:46);

[0536] LCDR3: QQRSNWPLT (SEQ ID NO: 47).

[0537] The amino acid sequence of the CDR of antibody H7L10 is as follows (according to the IMGT numbering system):

[0538] HCDR1:GGSISDYY(SEQ ID NO:5);

[0539] HCDR2: INHRGTT (SEQ ID NO: 6);

[0540] HCDR3:AFGYSDYEYDWFDP(SEQ ID NO:7);

[0541] LCDR1: QSISSY(SEQ ID NO:48);

[0542] LCDR2: DGS (SEQ ID NO:46);

[0543] LCDR3: QQRSNWPIT (SEQ ID NO: 10).

[0544] 2. Expression and purification of humanized antibody H7L8 (hG1WT)

[0545] The heavy chain cDNA sequence of H7L8 (hG1WT) (the variable region coding sequence is shown in SEQ ID NO:1; the constant region is the Ig gamma-1 chain C region, SEQ ID NO:39) and the light chain cDNA sequence (the variable region coding sequence is shown in SEQ ID NO:3; the constant region is P01834.1 (human Ig kappa chain C region, SEQ ID NO:40) were cloned into the pUC57simple vector (provided by GenScript) to obtain pUC57simple-H7 and pUC57simple-L8 plasmids, respectively. The pUC57simple-H7 and pUC57simple-L8 plasmids were digested with HindIII and EcoRI, respectively, and the heavy and light chains were recovered by electrophoresis and subcloned into the pcDNA3.1 vector. The recombinant plasmids were extracted and co-transfected into 293F cells. After 7 days of cell culture, the culture medium was centrifuged at high speed, the supernatant was concentrated, and the cells were loaded into HiTrap MabSelect. SuRe column, use ElutionBuffer to elute protein in one step and recover target sample and change to PBS.

[0546] H7L8(hG1WT) heavy chain constant region amino acid sequence

[0547] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:39)

[0548] H7L8(hG1WT) light chain constant region amino acid sequence

[0549] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:40)

[0550] 3. Design of humanized antibody H7L8 (hG1TM)

[0551] Based on H7L8(hG1WT), the inventors obtained the humanized antibody H7L8(hG1TM) with constant region mutations by introducing point mutations at position 234 (L234A) of leucine to alanine (L235A) at position 235 (according to the EU numbering system, the same below) and at position 237 (G237A) of glycine to alanine (G237A) in its heavy chain. The amino acid sequence of the heavy chain H7(hG1TM) of H7L8(hG1TM) is shown in SEQ ID NO:11; the amino acid sequence of the light chain L8 is shown in SEQ ID NO:12.

[0552] The preparation of humanized antibody H7L8 (hG1TM) can be carried out by referring to the method in step 2 above.

[0553] The amino acid sequence of the heavy chain H7(hG1TM) of H7L8(hG1TM).

[0554] QVQLQQWGAGLLKPSETLSLTCAVYGGSISDYYWNWIRQPPGKGLEWIGEINHRGTTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYDWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:11)

[0555] Amino acid sequence of the light chain L8 of H7L8 (hG1TM)

[0556] EIVLTQSPATLSLSPGERATLSCRASQTISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTNLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:12)

[0557] 4. Design of the humanized antibody H7L8 (hG4DM)

[0558] Based on H7L8(hG1WT), the inventors kept the variable region of the antibody unchanged and used the Ig gamma-4chain Cregion as the heavy chain constant region. They obtained the humanized antibody H7L8(hG4DM) with constant region mutations by introducing a point mutation from phenylalanine to alanine (F234A) at position 234 and a point mutation from leucine to alanine (L235A) at position 235 in the heavy chain constant region. The heavy chain amino acid sequence of H7L8(hG4DM) is shown in SEQ ID NO:13; the light chain amino acid sequence is shown in SEQ ID NO:12.

[0559] The amino acid sequence of the heavy chain H7(hG4DM) of H7L8(hG4DM):

[0560] QVQLQQWGAGLLKPSETLSLTCAVYGGSISDYYWNWIRQPPGKGLEWIGEINHRGTTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYDWFDPWGQ GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:13)

[0561] The amino acid sequence of the light chain L8 of H7L8(hG4DM) is the same as that of the light chain of H7L8(hG1TM), which is SEQ ID NO:12.

[0562] 5. Expression and purification of humanized antibodies H7L8(hG4WT), H7L9(hG4WT), and H7L10(hG4WT)

[0563] The heavy chain cDNA sequences of H7L8(hG4WT), H7L9(hG4WT), and H7L10(hG4WT) (variable region coding sequence as shown in SEQ ID NO:1; constant region is Ig gamma-4chain C region, as shown in SEQ ID NO:45) and the light chain cDNA sequences of H7L8(hG4WT) (variable region coding sequence as shown in SEQ ID NO:3; constant region is human Ig kappa chain C region, as shown in SEQ ID NO:40), H7L9(hG4WT) light chain (variable region coding sequence as shown in SEQ ID NO:42; constant region is human Ig kappa chain C region, as shown in SEQ ID NO:40), and H7L10(hG4WT) light chain (variable region coding sequence as shown in SEQ ID NO:44; constant region is human Ig kappa chain C region, as shown in SEQ ID NO:45) are included. (As shown in NO:40) plasmids were cloned into the pUC57simple (provided by GenScript) vector to obtain pUC57simple-H7, pUC57simple-L8, pUC57simple-L9, and pUC57simple-L10 plasmids, respectively. The pUC57simple-H7, pUC57simple-L8, pUC57simple-L9, and pUC57simple-L10 plasmids were digested with HindIII and EcoRI, and the heavy and light chains obtained by electrophoresis were subcloned into the pcDNA3.1 vector, respectively. The recombinant plasmids were then extracted and co-transfected into 293F cells. After 7 days of cell culture, the culture medium was centrifuged at high speed, the supernatant was concentrated, and the sample was loaded onto a HiTrap MabSelect SuRe column. Proteins were eluted with Elution Buffer in one step, the target sample was recovered, and the medium was changed to PBS.

[0564] H7L8(hG4WT), H7L9(hG4WT), or H7L10(hG4WT) heavy chain constant region amino acid sequence:

[0565] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:45)

[0566] The amino acid sequence of the light chain constant region of H7L8(hG4WT), H7L9(hG4WT), or H7L10(hG4WT):

[0567] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:40)

[0568] Preparation Example 2: Design and preparation of anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1

[0569] The amino acid sequences of the heavy and light chains of the anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1, as well as the sequences encoding nucleic acids, are completely identical to those of 14C12 and 14C12H1L1 in Chinese Patent Publication CN 106967172A (or CN 106977602A).

[0570] (1) Heavy chain variable region sequence and light chain variable region sequence of 14C12

[0571] Nucleic acid sequence of the heavy chain variable region of 14C12: (354 bp)

[0572] GAGGTCAAACTGGTGGAGAGCGGCGGCGGGCTGGTGAAGCCCGGCGGGTCACTGAAACTGAGCTGCGCCGCTTCCGGCTTCGCCTTTAGCTCCTACGACATGTCATGGGTGAGGCAGACCCCTGAGAAGCGCCTGGAATGGGTCGCTACTATCAGCGGAGGCGGGCGATACACCTACTATCCTGACTCTGTCAAAGGGAGATTCACAATTAGTCGGGATAACGCCAGAAATACTCTGTATCTGCAGATGTCTAGTCTGCGGTCCGAGGATACAGCTCTGTACTATTGTGCAAACCGGTACGGCGAAGCATGGTTTGCCTATTGGGGACAGGGCACCCTGGTGACAGTCTCTGCC(SEQ ID NO:14)

[0573] Amino acid sequence of the heavy chain variable region of 14C12: (118aa)

[0574] EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQT PEKRLEWVATISGGGRYTYYPDSVKGRFTISRDNARNTLYLQM SSLRSEDTALYYCANRYGEAWFAYWGQGTLVTVSA(SEQ ID NO:15)

[0575] Nucleic acid sequence of the light chain variable region of 14C12: (321bp)

[0576] GACATTAAGATGACACAGTCCCCTTCCTCAATGTACGCTAGCCTGGGCGAGCGAGTGACCTTCACATGCAAAGCATCCCAGGACATCAACACATACCTGTCTTGGTTTCAGCAGAAGCCAGGCAAAAGCCCCAAGACCCTGATCTACCGGGCCAATAGACTGGTGGACGGGGTCCCCAGCAGATTCTCCGGATCTGGCAGTGGGCAGGATTACTCCCTGACCATCAGCTCCCTGGAGTATGAAGACATGGGCATCTACTATTGCCTGCAGTATGATGAGTTCCCTCTGACCTTTGGAGCAGGCACAAAACTGGAACTGAAG(SEQ ID NO:16)

[0577] The amino acid sequence of the variable region of the 14C12 light chain is: (107aa)

[0578] DIKMTQSPSSMYASLGERVTFTCKASQDINTYLSWFQQKPG KSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIY YCLQYDEFPLTFGAGTKLELK(SEQ ID NO:17)

[0579] (2) Heavy chain variable region sequence and light chain variable region sequence, heavy chain sequence and light chain sequence of humanized monoclonal antibody 14C12H1L1

[0580] The nucleic acid sequence of the heavy chain variable region 14C12H1v of 14C12H1L1: (354bp)

[0581] GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGGAAAGGGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGGAGATACACCTACTA TCCTGACAGCGTCAAGGCCCGGTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTTTGCCTATTGGGGGGCAGGGAACCCTGGTGACAGTCTCTAGT(SEQ ID NO:18)

[0582] The amino acid sequence of the heavy chain variable region 14C12H1v of 14C12H1L1 is: (118aa)

[0583] EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQ APGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYL QMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSS(SEQ ID NO:19)

[0584] The nucleic acid sequence of the light chain variable region 14C12L1v of 14C12H1L1: (321bp)

[0585] GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGACAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGGGCTAATAGACTGGTGTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCCGGGACAAAACTGGAGCTGAAG(SEQ ID NO:20)

[0586] Amino acid sequence of the light chain variable region 14C12L1v of 14C12H1L1: (107aa)

[0587] DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKP GKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMA TYYCLQYDEFPLTFGAGTKLELK(SEQ ID NO:21)

[0588] Nucleic acid sequence of the heavy chain 14C12H1 of 14C12H1L1: (1344bp)

[0589]

[0590] Amino acid sequence of the heavy chain 14C12H1 of 14C12H1L1: (448 aa)

[0591] EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:23)

[0592] Nucleic acid sequence of the light chain 14C12L1 of 14C12H1L1: (642 bp)

[0593] GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGACAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGGGCTAATAGACTGGTGTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCCGGGACAAAACTGGAGCTGAAGCGAACTGTGGCCGCTCCCTCCGTCTTCATTTTTCCCCCTTCTGACGAACAGCTGAAATCAGGCACAGCCAGCGTGGTCTGTCTGCTGAACAATTTCTACCCTAGAGAGGCAAAAGTGCAGTGGAAGGTCGATAACGCCCTGCAGTCCGGCAACAGCCAGGAGAGTGTGACTGAACAGGACTCAAAAGATAGCACCTATTCCCTGTCTAGTACACTGACTCTGTCCAAGGCTGATTACGAGAAGCACAAAGTGTATGCATGCGAAGTGACACATCAGGGACTGTCAAGCCCCGTGACTAAGTCTTTTAACCGGGGCGAATGT(SEQ ID NO:24)

[0594] Amino acid sequence of the light chain 14C12L1 of 14C12H1L1: (214aa)

[0595] DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:25)

[0596] Antibodies 14C12 and 14C12H1L1 have the same CDR, as follows (according to the IMGT numbering system):

[0597] HCDR1:GFAFSSYD (SEQ ID NO:26)

[0598] HCDR2:ISGGGRYT(SEQ ID NO:27)

[0599] HCDR3:ANRYGEAWFAY(SEQ ID NO:28)

[0600] LCDR1:QDINTY(SEQ ID NO:29)

[0601] LCDR2:RAN(SEQ ID NO:30)

[0602] LCDR3:LQYDEFPLT(SEQ ID NO:31)

[0603] Heavy chain variable region sequence and light chain variable region sequence of 14C12H1L1(M)

[0604] Based on 14C12H1L1, individual amino acids in its backbone region (light chain) were mutated to obtain 14C12H1L1(M).

[0605] The heavy chain variable region 14C12H1(M) of 14C12H1L1(M):

[0606] It is identical to the heavy chain variable region 14C12H1 of 14C12H1L1, i.e., the amino acid sequence is as shown in SEQ ID NO:19.

[0607] The light chain variable region 14C12L1(M) of 14C12H1L1(M):

[0608] DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPG KSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATY YCLQYDEFPLTFGAGTKLELKR(SEQ ID NO:38)

[0609] Preparation Example 3: Design of Humanized Antibody 14C12H1L1 (hG4DM)

[0610] Based on 14C12H1L1, the inventors retained the variable region of the antibody and used the Ig gamma-4chain C region as the constant region of the heavy chain. They obtained the humanized antibody 14C12H1L1(hG4DM) with constant region mutations by introducing a point mutation from phenylalanine to alanine (F234A) at position 234 and a point mutation from leucine to alanine (L235A) at position 235 within the constant region of the heavy chain. The amino acid sequence of the heavy chain 14C12H1(hG4DM) is shown in SEQ ID NO:32; the amino acid sequence of the light chain is shown in SEQ ID NO:25.

[0611] 14C12H1L1(hG4DM) heavy chain amino acid sequence

[0612] EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGT LVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQID NO:32)

[0613] The amino acid sequence of the light chain of 14C12H1L1(hG4DM) is the same as that of the light chain of 14C12L1, which is SEQ ID NO:25.

[0614] Preparation Example 4: Sequence Design of Humanized Antibody 14C12H1L1 (hG1TM)Based on the humanized antibody 14C12H1L1, the inventors, following the EU numbering system, introduced a point mutation from leucine to alanine (L234A) at position 234, a point mutation from leucine to alanine (L235A) at position 235, and a point mutation from glycine to alanine (G237A) at position 237 in its heavy chain hinge region, thus obtaining a mutated humanized 14C12H1L1 (hG1TM).

[0615] The nucleic acid sequence of the heavy chain 14C12H1(hG1TM) of 14C12H1(hG1TM): (1344bp)

[0616]

[0617] The amino acid sequence of the heavy chain 14C12H1(hG1TM) of 14C12H1(hG1TM) is: (448aa)

[0618] EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:34)

[0619] The nucleic acid sequence of the light chain of 14C12H1L1(hG1TM) is the same as that of SEQ ID NO:24.

[0620] The amino acid sequence of the light chain of 14C12H1L1(hG1TM) is the same as that of the light chain of 14C12H1L1, i.e., SEQ ID NO:25.

[0621] Preparation Example 5: Design and preparation of anti-LAG3-anti-PD-1 bispecific antibody

[0622] 1. Sequence Design

[0623] The bispecific antibodies BS-PL021A, Bs-PL022B, BS-PL023C, Bs-PLV02 and Bi-PGV02 of the present invention have a Morrison pattern (IgG-scFv), that is, the scFv fragment of another antibody is attached to the C-terminus of both heavy chains of an IgG antibody. The main components of their heavy and light chains are designed as shown in Table 1 below.

[0624]

[0625] In Table 1 above:

[0626] (1) Linker (GGGGS) 3 amino acid sequence:

[0627] GGGGSGGGGSGGGGS(SEQ ID NO:35)

[0628] Linker (GGGGS) 4 amino acid sequence:

[0629] GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:36)

[0630] Linker (GGGGS) 4G amino acid sequence:

[0631] GGGGSGGGGSGGGGSGGGGSG (SEQ ID NO:37)

[0632] (2) Those marked with "v" in the lower right corner refer to the variable region of the corresponding heavy chain or the variable region of the corresponding light chain. Those without "v" are the full-length heavy or light chains containing the constant region. The amino acid sequences of these variable regions or full-length chains and their encoding nucleic acid sequences are all based on the corresponding sequences described in the preparation examples above.

[0633] 2. Antibody expression and purification

[0634] The heavy chain cDNA sequences and light chain cDNA sequences of Bs-PL021A, Bs-PL022B, Bs-PL023C, and Bs-PLV02 were cloned into the pUC57simple vector (provided by GenScript) to obtain the plasmids pUC57simple-Bs-PL021AH / pUC57simple-Bs-PL021AL, pUC57simple-Bs-PL022BH / pUC57simple-Bs-PL022BL, pUC57simple-Bs-PL023CH / pUC57simple-Bs-PL023CL, pUC57simple-Bs-PLV02H / pUC57simple-Bs-PLV02L, and pUC57simple-Bi-PGV02 / pUC57simple-Bi-PGV02.

[0635] The plasmids pUC57simple-Bs-PL021AH / pUC57simple-Bs-PL021AL, pUC57simple-Bs-PL022BH / pUC57simple-Bs-PL022BL, pUC57simple-Bs-PL023CH / pUC57simple-Bs-PL023CL, pUC57simple-Bs-PLV02H / pUC57simple-Bs-PLV02L, and pUC57simple-Bi-PGV02 / pUC57simple-Bi-PGV02 were digested with HindIII and EcoRI, respectively. The heavy and light chains obtained by electrophoresis were subcloned into the pcDNA3.1 vector, and the recombinant plasmids were extracted and co-transfected into 293F cells. After 7 days of cell culture, the culture medium was centrifuged at high speed, the supernatant was concentrated and then loaded onto a HiTrap MabSelect SuRe column. Proteins were eluted in one step with Elution Buffer and the target sample was recovered and the medium was changed to PBS.

[0636] Preparation Example 6: Sequence Design of Anti-CTLA4 Antibody

[0637] The heavy and light chain amino acid sequences and the encoding nucleic acid sequences of the anti-CTLA4 antibody 4G10 and its humanized antibodies 4G10H1L1 and 4G10H3L3 are identical to those of 4G10, 4G10H1L1, and 4G10H3L3 in Chinese Patent Publication CN 106967172A.

[0638] (1) Heavy chain variable region sequence and light chain variable region sequence of 4G10

[0639] Nucleic acid sequence of the variable region of the 4G10 heavy strand: (372bp)

[0640] CAGGTCAAGCTGCAGGAGTCTGGACCTGAGCTGGTGAAGCCTGGAGCTTCAATGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACACCATGAACTGGGTGAAGCAGAGCCATGGAAAGAACCTTGAATGGATTGGACTTATTAATCCTTACAATAATATTACTAACTACAACCAGAAGTTCATGGGCAAGGCCACATTTACTGTAGACAAGTCATCCAGCACAGCCTACATGGAACTCCTCAGACTGACATCTGAAGACTCTGGAGTCTATTTCTGTGCAAGACTCGACTATAGGTCTTATTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTAT(SEQ ID NO:49)

[0641] Amino acid sequence of the 4G10 heavy chain variable region: (124 aa)

[0642] QVKLQESGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGKNLEWIGLINPYNNITNYNQKFMGKATFTVDKSSSTAYMELLRLTSEDSGVYFCARLDYRSYWGQGTLVTVSAAKTTPPSVY(SEQ ID NO:50)

[0643] Nucleic acid sequence of the 4G10 light chain variable region: (378 bp)

[0644] CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTTTGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTAGTCTAATAGGTGGTACCAACAACCGAGCTCCAGGTGTTCCTGCCAGATT CTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCATTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAGGCCAGCCCAAGTCTTCGCCATCAGTCACCCTGTTTCAAGGGCAATTCTGC(SEQ ID NO:51)

[0645] The amino acid sequence of the variable region of the 4G10 light chain is: (126 aa)

[0646] QAVVTQESALTTSPGETVTTLTCRSSTGAVTTSNFANWVQEKPDHLFTSLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGQPKSSPSVTLFQGQFC(SEQ ID NO:52)

[0647] The amino acid sequences of the three CDR regions in the variable region of the 4G10 heavy chain are as follows (according to the IMGT numbering system):

[0648] HCDR1:GYSFTGYT(SEQ ID NO:67)

[0649] HCDR2:INPYNNIT (SEQ ID NO:68)

[0650] HCDR3:ARLDYRSY (SEQ ID NO:69)

[0651] The amino acid sequences of the three CDR regions in the variable region of the 4G10 light chain are as follows (according to the IMGT numbering system):

[0652] LCDR1:TGAVTTSNF(SEQ ID NO:70)

[0653] LCDR2:GTN(SEQ ID NO:71)

[0654] LCDR3:ALWYSNHWV (SEQ ID NO:72)

[0655] (2) Heavy chain variable region sequence and light chain variable region sequence of humanized monoclonal antibody 4G10H1L1

[0656] Nucleic acid sequence of the 4G10H1L1 heavy chain variable region (4G10H1V): (345bp)

[0657] CAGGGTGCAGCTGGTGGAGTCTGGGGCCGAGCTGGTGAAGCCCGGCGCCTCCATGAAGATCTCTTGCAAGGCCAGCGGATACAGTTTCACTGGCTATACCATGAACTGGGTCAAACAGGCTTCCAGGACAGGGACTGGAGTGGATCGGGCTGATTAATCCTTACAACAACATCACC AACTACAACCAGAAGTTCATGGGAAAAGCAACCTTTACAGTGGACAAGAGCATTTCCACAGCCTACATGGAACTGAGCCGGCTGACTTCAGACGATAGCGGGGTCTATTTTTGTGCAAGGCTGGATTATCGCTCTTACTGGGGGCAGGGAACTCTGGTCACTGTCTCCGCT(SEQ ID NO:53)

[0658] The amino acid sequence of the variable region of the 4G10H1L1 heavy chain is: (115 aa)

[0659] QVQLVESGAELVKPGASMKISCKASGYSFTGYTMNWVKQ APGQGLEWIGLINPYNNITNYNQKFMGKATFTVDKSISTAYMEL SRLTSDDSGVYFCARLDYRSYWGQGTLVTVSA(SEQ ID NO:54)

[0660] Nucleic acid sequence of the 4G10H1L1 light chain variable region (4G10L1V): (327bp)

[0661] CAGGCTGTCGTCACTCAGGAACCTTCACTGACTGTGAGCCCAGGAGGAACTGTCACCCTGACATGCGGAAGCTCCACCGGAGCAGTGACCACATCCAACTTCGCCAATTGGGTCCAGGAAAAGCCAGGCCAGGCATTTCGATCCCTGATCGGAGGCACAAACAAT CGGGCTTCTTGGGTGCCCGCAAGATTCTCAGGAAGCCTGCTGGGGGGAAAAGCCGCTCTGACCATTAGTGGCGCTCAGCCTGAGGACGAAGCCGAGTACTTCTGCGCTCTGTGGTATAGCAACCACTGGGTTGTTTGGCGGGGGAACAAAGCTGACTGTGCTG(SEQ ID NO:55)

[0662] The amino acid sequence of the variable region of the 4G10H1L1 light chain is: (109 aa)

[0663] QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFANWVQE KPGQAFRSLIGGTNNRASWVPARFSGSLLGGKAALTISGAQPED EAEYFCALWYSNHWVFGGGTKLTVL(SEQ ID NO:56)

[0664] (3) Heavy chain variable region sequence and light chain variable region sequence of humanized monoclonal antibody 4G10H3L3

[0665] Nucleic acid sequence of the 4G10H3L3 heavy chain variable region (4G10H3V): (345bp)

[0666] CAGGTGCAGCTGGTCGAGTCTGGGGCCGAAGTGAAGAAACCCGGCGCCTCAGTGAAGGTCAGCTGCAAGGCCAGCGGGTACAGTTTCACTGGATATACCATGAACTGGGTCCGACAGGCCCCTGGCCAGGGGCTGGAGTGGATCGGCCTGATTAACCCTTACAACAACATCACTAACTACGCACAGAAGTTCCAGGGGAGAGTGACCTTTACAGTGGACACCAGCATTTCCACAGCCTACATGGAACTGTCCCGGCTGAGATCTGACGATACAGGCGTGTACTTCTGCGCTAGGCTGGATTACCGCAGCTATTGGGGACAGGGCACACTGGTGACTGTCAGCGCA(SEQ ID NO:57)

[0667] Amino acid sequence of the heavy chain variable region of 4G10H3L3 (4G10H3V): (115 aa)

[0668] QVQLVESGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQ APGQGLEWIGLINPYNNITNYAQKFQGRVTFTVDTSISTAYMEL SRLRSDDTGVYFCARLDYRSYWGQGTLVTVSA(SEQ ID NO:58)

[0669] Nucleic acid sequence of the light chain variable region of 4G10H3L3 (4G10L3V): (327 bp)

[0670] CAGGCTGTCGTCACTCAGGAACCTTCACTGACCGTGTCTCCTGGCGGGACTGTCACCCTGACATGCGGCAGCTCCACAGGGGCCGTGACCACAAGTAACTTCCCAAATTGGGTCCAGCAGAAGCCAGGACAGGCTCCCCGGAGTCTGATCGGAGGCACCAACAAC AAGGCCAGCTGGACACCCGCACGGTTCAGCGGCAGCCTGCTGGGCGGCAAGGCCGCTCTGACAATTAGCGGAGCCCAGCCTGAGGACGAAGCCGAGTACTATTGCGCTCTGTGGTACTCCAACCACTGGGTGTTCGGCGGCGGCACCAAGCTGACTGTGCTG(SEQ ID NO:59)

[0671] The amino acid sequence of the variable region of the 4G10H3L3 light chain (4G10L3V): (109 aa)

[0672] QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFPNWVQQ KPGQAPRSLIGTNNKASWTPARFSGSLLGGKAALTISGAQPED EAEYYCALWYSNHWVFGGGTKLTVL(SEQ ID NO:60)

[0673] Preparation Example 7: Sequence Design of Bispecific Antibodies BiAb001(M), BiAb002(M), BiAb003(M) and BiAb004(M)

[0674] The bispecific antibodies BiAb001(M), BiAb002(M), BiAb003(M) and BiAb004(M) have a Morrison pattern (IgG-scFv), in which the C-terminus of both heavy chains of an IgG antibody is linked to the scFv fragment of another antibody via a linker. The design composition of their heavy and light chains is shown in Table 2 below.

[0675] Table 2: Sequence design of BiAb001(M), BiAb002(M), BiAb003(M) and BiAb004(M)

[0676]

[0677] In Table 2 above:

[0678] The amino acid sequence of Linker1 is GGGGSGGGGSGGGGS (SEQ ID NO:35)

[0679] The amino acid sequence of Linker2 is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:36)

[0680] In addition, in Table 2 above, the scFv fragments of the BiAb001(M), BiAb002(M), BiAb003(M), and BiAb004(M) antibodies, specifically the 4G10H1V(M), 4G10L1V(M), 4G10H3V(M), and 4G10L3V(M), are derived from 4G10H1V, 4G10L1V, 4G10H3V, and 4G10L3V respectively, with mutations made to individual amino acids in their backbone regions, as follows:

[0681] (1) 4G10H1V(M): (115aa, amino acid sequence based on 4G10H1V, mutation sites are marked with underscores)

[0682] QVQLVESGAELVKPGASMKISCKASGYSFTGYTMNWVKQ APGQ C LEWIGLINPYNNITNYNQKFMGKATFTVDKSISTAYMEL SRLTSDDSGVYFCARLDYRSYWGQGTLVTVSA(SEQ ID NO:61)

[0683] (2) 4G10L1V(M): (110aa, amino acid sequence based on 4G10L1V, mutation sites are marked with underscores)

[0684] QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFANWVQE KPGQAFRSLIGGTNNRASWVPARFSGSLLGGKAALTISGAQPED EAEYFCALWYSNHWVFG C GTKLTVL R (SEQ ID NO:62)

[0685] (3) 4G10H3V(M): (115aa, amino acid sequence based on 4G10H3V, mutation sites are marked with underscores)

[0686] QVQLVESGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQ APGQ CLEWIGLINPYNNITNYAQKFQGRVTFTVDTSISTAYMEL SRLRSDDTGVYFCARLDYRSYWGQGTLVTVSA(SEQ ID NO:63)

[0687] (4) 4G10L3V(M): (110aa, amino acid sequence mutation sites based on 4G10L3V are underlined)

[0688] QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNFPNWVQQ KPGQAPRSLIGGTNNKASWTPARFSGSLLGGKAALTISGAQPED EAEYYCALWYSNHWVFG C GTKLTVL R (SEQ ID NO:64)

[0689] To distinguish it from the mutated antibody described below, BiAb004(M) is referred to as BiAb004(hG1WT) in the embodiments or preparation examples of this invention. The BiAb004(M) described above is the "wild type", which uses Ig gamma-1 chain C region, ACCESSION: P01857 as the heavy chain constant region and Ig kappa chain C region, ACCESSION: P01834 as the light chain constant region.

[0690] Preparation Example 8: Design of amino acid mutations in the non-variable region of the humanized bispecific antibody BiAb004

[0691] Based on the BiAb004(hG1WT) obtained in Preparation Example 7, the inventors introduced a point mutation from leucine to alanine (L234A) at position 234, a point mutation from leucine to alanine (L235A) at position 235, and a point mutation from glycine to alanine (G237A) at position 237 in its heavy chain, thus obtaining BiAb004(hG1TM).

[0692] DNA sequence of the heavy chain of the immunoglobulin portion of BiAb004(hG1TM): (1344bp)

[0693]

[0694] The amino acid sequence of the heavy chain of the immunoglobulin moiety in BiAb004(hG1TM) is: (448aa)

[0695] EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:66)

[0696] BiAb004(hG1TM) and BiAb004(hG1WT) light chains have the same DNA sequence and encode the same amino acid sequence.

[0697] Example 1: Detection of anti-LAG3-anti-PD-1 bispecific antibodies using mixed lymphocyte reaction The combination of anti-CTLA4 and anti-PD-1 bispecific antibodies promotes the secretion of cytokines IL-2 and IFN-γ.

[0698] According to the separation liquid Ficoll-Paque TM Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated using the Plus (Cytiva, catalog number: 17544203) instruction manual. Two days prior to the experiment, PBMCs were resuscitated and cultured in RPMI 1640 + 10% FBS medium at 37°C in a 5% CO2 incubator. SEB (Toxin Technology, catalog number: BT202, final concentration: 0.5 μg / mL) was added, and the cells were cultured for another two days.

[0699] On the day of the experiment, Raji-PDL1 cells were collected, centrifuged at 110xg for 5 min, resuspended in RPMI 1640 + 10% FBS medium for counting, and MMC (Mito-mycin C, Stressmarq, catalog number: SIH-246-10MG, final concentration 2μg / mL) was added and incubated at 37℃ in a 5% CO2 incubator for 1 h.

[0700] Collect PBMCs and Raji-PDL1 cells treated with SEB for two days, wash twice with analytical medium, resuspend and count; seed PBMCs at 100,000 / well and Raji-PDL1 cells at 100,000 / well into 96-well U-shaped plates. Add drugs to the PBMC+Raji-PDL1 mixture according to the following experimental design:

[0701] Except for the Yervoy+Relatlimab+Opdivo group, where the final concentrations of Opdivo antibody were 0.6 nM, 6 nM, 60 nM, and 600 nM, and the BS-PL022B+BiAb004(hG1TM) group, where the final concentrations of the two antibodies were 0.3 nM, 3 nM, 15 nM, 30 nM, 150 nM, and 300 nM, the final concentrations of all other antibodies in the single-use and combination groups were 0.3 nM, 3 nM, 30 nM, and 300 nM. Negative controls (PBMC+Raji-PDL1) and isotype controls (B12hG1 group) were also included.

[0702] After adding the drug, continue culturing for 3 days. After 3 days, centrifuge at 250xg for 5 min (Beckman centrifuge), collect the cell supernatant, and use the Dakota kit to detect the levels of IFN-γ and IL-2.

[0703] The results are as follows Figure 1 and Figure 2 As shown.

[0704] Figure 1 The results showed that the anti-LAG3-anti-PD-1 bispecific antibody BS-PL022B alone, the anti-CTLA4-anti-PD-1 bispecific antibody BiAb004 (hG1TM) alone, or the combination of BS-PL022B and BiAb004 (hG1TM) could all effectively promote IL-2 secretion by PBMCs. Moreover, the combination of anti-LAG3-anti-PD-1 bispecific antibody BS-PL022B and anti-CTLA4-anti-PD-1 bispecific antibody BiAb004 (hG1TM) was significantly more effective in promoting IL-2 secretion by PBMCs than any of the single-drug groups.

[0705] Figure 2The results showed that the anti-LAG3-anti-PD-1 bispecific antibody BS-PL022B alone, the anti-CTLA4-anti-PD-1 bispecific antibody BiAb004 (hG1TM) alone, or the combination of BS-PL022B and BiAb004 (hG1TM) could all effectively promote IFN-γ secretion by PBMCs. Moreover, the combination of anti-LAG3-anti-PD-1 bispecific antibody BS-PL022B and anti-CTLA4-anti-PD-1 bispecific antibody BiAb004 (hG1TM) was significantly better than the single-drug groups and the positive control group Yervoy+Relatlimab+Opdivo in promoting IFN-γ secretion by PBMCs.

[0706] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of this invention. The full scope of this invention is given by the appended claims and any equivalents thereof.

Claims

1. A drug combination comprising an effective amount of a primary antibody and an effective amount of a secondary antibody, wherein, The first antibody is an anti-LAG3-anti-PD-1 bispecific antibody, and the second antibody is an anti-CTLA4-anti-PD-1 bispecific antibody.

2. The pharmaceutical combination according to claim 1, wherein: (1) The anti-LAG3-anti-PD-1 bispecific antibody includes: Targeting the first protein functional region of LAG3, and Targeting the second protein functional region of PD-1; in, The first protein functional region includes a heavy chain variable region VH1 and a light chain variable region VL1. VH1 includes HCDR1 shown in SEQ ID NO:5, HCDR2 shown in SEQ ID NO:6, and HCDR3 shown in SEQ ID NO:7; and VL1 includes LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:9, and LCDR3 shown in SEQ ID NO:10, or VL1 includes LCDR1 shown in SEQ ID NO:8, LCDR2 shown in SEQ ID NO:46, and LCDR3 shown in SEQ ID NO:47, or VL1 includes LCDR1 shown in SEQ ID NO:48, LCDR2 shown in SEQ ID NO:46, and LCDR3 shown in SEQ ID NO:

10. The second protein functional region includes a heavy chain variable region VH2 and a light chain variable region VL2. The VH2 includes HCDR1 shown in SEQ ID NO:26, HCDR2 shown in SEQ ID NO:27, and HCDR3 shown in SEQ ID NO:28; and the VL2 includes LCDR1 shown in SEQ ID NO:29, LCDR2 shown in SEQ ID NO:30, and LCDR3 shown in SEQ ID NO:

31. (2) The anti-CTLA4-anti-PD-1 bispecific antibody includes: Targeting the third protein functional region of PD-1, and Targeting the fourth protein functional region of CTLA4; in, The third protein functional region comprises a heavy chain variable region VH3 and a light chain variable region VL3. The VH3 comprises HCDR1 shown in SEQ ID NO:26, HCDR2 shown in SEQ ID NO:27, and HCDR3 shown in SEQ ID NO:28; and the VL3 comprises LCDR1 shown in SEQ ID NO:29, LCDR2 shown in SEQ ID NO:30, and LCDR3 shown in SEQ ID NO:

31. The fourth protein functional region includes a heavy chain variable region VH4 and a light chain variable region VL4. VH4 includes HCDR1 shown in SEQ ID NO:67, HCDR2 shown in SEQ ID NO:68, and HCDR3 shown in SEQ ID NO:69; and VL4 includes LCDR1 shown in SEQ ID NO:70, LCDR2 shown in SEQ ID NO:71, and LCDR3 shown in SEQ ID NO:

72.

3. The drug combination according to claim 2, characterized in that... Any one or more of the following items (1) to (7): (1) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is selected from SEQ ID NO:4, SEQ ID NO:42 and SEQ ID NO:44; (2) The amino acid sequence of VH2 is selected from SEQ ID NO:15 and SEQ ID NO:19, and the amino acid sequence of VL2 is selected from SEQ ID NO:17, SEQ ID NO:21 and SEQ ID NO:38; (3) The amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is selected from SEQ ID NO:17, SEQ ID NO:21 and SEQ ID NO:38; (4) The amino acid sequence of VH3 is selected from SEQ ID NO:15 and SEQ ID NO:19, and the amino acid sequence of VL3 is selected from SEQ ID NO:17, SEQ ID NO:21 and SEQ ID NO:38; (5) The amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is selected from SEQ ID NO:17, SEQ ID NO:21 and SEQ ID NO:38; (6) The amino acid sequence of VH4 is selected from SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:61 and SEQ ID NO:63, and the amino acid sequence of VL4 is selected from SEQ ID NO:52, SEQ ID NO:56, SEQ ID NO:60, SEQ ID NO:62 and SEQ ID NO:64; (7) The amino acid sequence of VH4 is shown in SEQ ID NO:50, and the amino acid sequence of VL4 is shown in SEQ ID NO:52; The amino acid sequence of VH4 is shown in SEQ ID NO:54, and the amino acid sequence of VL4 is shown in SEQ ID NO:56; The amino acid sequence of VH4 is shown in SEQ ID NO:58, and the amino acid sequence of VL4 is shown in SEQ ID NO:

60. The amino acid sequence of VH4 is shown in SEQ ID NO:61, and the amino acid sequence of VL4 is shown in SEQ ID NO:62; or The amino acid sequence of VH4 is shown in SEQ ID NO:63, and the amino acid sequence of VL4 is shown in SEQ ID NO:

64.

4. The pharmaceutical combination according to any one of claims 2 to 3, wherein, (1) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:

21. (2) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:

38. (3) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:42; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:

21. (4) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:42; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:

38. (5) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:44; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:21; or (6) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:44; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:

38.

5. The pharmaceutical combination according to any one of claims 2 to 4, wherein, (1) The amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:21; the amino acid sequence of VH4 is shown in SEQ ID NO:54, and the amino acid sequence of VL4 is shown in SEQ ID NO:

56. (2) The amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:21; the amino acid sequence of VH4 is shown in SEQ ID NO:58, and the amino acid sequence of VL4 is shown in SEQ ID NO:

60. (3) The amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:21; the amino acid sequence of VH4 is shown in SEQ ID NO:61, and the amino acid sequence of VL4 is shown in SEQ ID NO:

62. (4) The amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:21; the amino acid sequence of VH4 is shown in SEQ ID NO:63, and the amino acid sequence of VL4 is shown in SEQ ID NO:

64. (5) The amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:38; the amino acid sequence of VH4 is shown in SEQ ID NO:54, and the amino acid sequence of VL4 is shown in SEQ ID NO:

56. (6) The amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:38; the amino acid sequence of VH4 is shown in SEQ ID NO:58, and the amino acid sequence of VL4 is shown in SEQ ID NO:

60. (7) The amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:38; the amino acid sequence of VH4 is shown in SEQ ID NO:61, and the amino acid sequence of VL4 is shown in SEQ ID NO:62; or (8) The amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:38; the amino acid sequence of VH4 is shown in SEQ ID NO:63, and the amino acid sequence of VL4 is shown in SEQ ID NO:

64.

6. The pharmaceutical combination according to any one of claims 2 to 5, wherein, (1) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:21; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:21; the amino acid sequence of VH4 is shown in SEQ ID NO:54, and the amino acid sequence of VL4 is shown in SEQ ID NO:56; (2) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:21; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:21; the amino acid sequence of VH4 is shown in SEQ ID NO:58, and the amino acid sequence of VL4 is shown in SEQ ID NO:

60. (3) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:21; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:21; the amino acid sequence of VH4 is shown in SEQ ID NO:61, and the amino acid sequence of VL4 is shown in SEQ ID NO:

62. (4) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:21; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:21; the amino acid sequence of VH4 is shown in SEQ ID NO:63, and the amino acid sequence of VL4 is shown in SEQ ID NO:

64. (5) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:21; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:38; the amino acid sequence of VH4 is shown in SEQ ID NO:54, and the amino acid sequence of VL4 is shown in SEQ ID NO:

56. (6) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:21; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:38; the amino acid sequence of VH4 is shown in SEQ ID NO:58, and the amino acid sequence of VL4 is shown in SEQ ID NO:

60. (7) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:21; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:38; the amino acid sequence of VH4 is shown in SEQ ID NO:61, and the amino acid sequence of VL4 is shown in SEQ ID NO:

62. (8) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:21; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:38; the amino acid sequence of VH4 is shown in SEQ ID NO:63, and the amino acid sequence of VL4 is shown in SEQ ID NO:

64. (9) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:38; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:21; the amino acid sequence of VH4 is shown in SEQ ID NO:54, and the amino acid sequence of VL4 is shown in SEQ ID NO:

56. (10) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:38; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:21; the amino acid sequence of VH4 is shown in SEQ ID NO:58, and the amino acid sequence of VL4 is shown in SEQ ID NO:

60. (11) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:38; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:21; the amino acid sequence of VH4 is shown in SEQ ID NO:61, and the amino acid sequence of VL4 is shown in SEQ ID NO:

62. (12) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:38; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:21; the amino acid sequence of VH4 is shown in SEQ ID NO:63, and the amino acid sequence of VL4 is shown in SEQ ID NO:

64. (13) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:38; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:38; the amino acid sequence of VH4 is shown in SEQ ID NO:54, and the amino acid sequence of VL4 is shown in SEQ ID NO:56; (14) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:38; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:38; the amino acid sequence of VH4 is shown in SEQ ID NO:58, and the amino acid sequence of VL4 is shown in SEQ ID NO:

60. (15) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:38; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:38; the amino acid sequence of VH4 is shown in SEQ ID NO:61, and the amino acid sequence of VL4 is shown in SEQ ID NO:62; or (16) The amino acid sequence of VH1 is shown in SEQ ID NO:2, and the amino acid sequence of VL1 is shown in SEQ ID NO:4; the amino acid sequence of VH2 is shown in SEQ ID NO:19, and the amino acid sequence of VL2 is shown in SEQ ID NO:38; the amino acid sequence of VH3 is shown in SEQ ID NO:19, and the amino acid sequence of VL3 is shown in SEQ ID NO:38; the amino acid sequence of VH4 is shown in SEQ ID NO:63, and the amino acid sequence of VL4 is shown in SEQ ID NO:

64.

7. The pharmaceutical combination according to any one of claims 1 to 6, characterized in that... Any one or more of the following items (1) to (23): (1) The first protein functional region, the second protein functional region, the third protein functional region and the fourth protein functional region are independently selected from immunoglobulins and single-chain antibodies; (2) The first protein functional region is an immunoglobulin and the second protein functional region is a single-chain antibody; or the first protein functional region is a single-chain antibody and the second protein functional region is an immunoglobulin. (3) The third protein functional region is an immunoglobulin and the fourth protein functional region is a single-chain antibody; or the third protein functional region is a single-chain antibody and the fourth protein functional region is an immunoglobulin. (4) The anti-LAG3-anti-PD-1 bispecific antibody is a bispecific antibody in the form of IgG-scFv; and / or the anti-CTLA4-anti-PD-1 bispecific antibody is a bispecific antibody in the form of IgG-scFv; (5) The anti-LAG3-anti-PD-1 bispecific antibody is a tetravalent bispecific antibody in the form of IgG-scFv; and / or the anti-CTLA4-anti-PD-1 bispecific antibody is a tetravalent bispecific antibody in the form of IgG-scFv; (6) The immunoglobulin contains a constant region derived from human antibodies; (7) The constant region is independently selected from the constant regions of human IgG1, IgG2, IgG3 or IgG4; (8) The immunoglobulin contains a heavy chain constant region, which is independently an Ig gamma-1 chain C region or an Ig gamma-4 chain C region; and The immunoglobulin contains a light chain constant region, which is the Ig kappa chain C region. (9) The immunoglobulin contains a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region contains the amino acid sequence shown in SEQ ID NO:39 or SEQ ID NO:45, and the light chain constant region contains the amino acid sequence shown in SEQ ID NO:40; (10) The immunoglobulin is independently human IgG1 subtype. According to the EU numbering system, the heavy chain constant region of the immunoglobulin contains the following mutations: L234A and L235A, L234A and G237A, L235A and G237A, or L234A, L235A, and G237A; (11) The first protein functional region is an immunoglobulin, the amino acid sequence of its heavy chain is shown in SEQ ID NO:11, and the amino acid sequence of its light chain is shown in SEQ ID NO:

12. (12) The second protein functional region is an immunoglobulin, the amino acid sequence of its heavy chain is shown in SEQ ID NO:34, and the amino acid sequence of its light chain is shown in SEQ ID NO:

25. (13) The third protein functional region is an immunoglobulin, the amino acid sequence of its heavy chain is shown in SEQ ID NO:66, and the amino acid sequence of its light chain is shown in SEQ ID NO:

25. (14) The immunoglobulin is independently human IgG4 subtype. According to the EU numbering system, the heavy chain constant region of the immunoglobulin contains the following mutations: F234A and L235A, F234A and G237A, L235A and G237A, or F234A, L235A, and G237A; (15) The first protein functional region is an immunoglobulin, the amino acid sequence of its heavy chain is shown in SEQ ID NO:13, and the amino acid sequence of its light chain is shown in SEQ ID NO:

12. (16) The functional region of the second protein is an immunoglobulin, the amino acid sequence of its heavy chain is shown in SEQ ID NO:32, and the amino acid sequence of its light chain is shown in SEQ ID NO:

25. (17) The third protein functional region is an immunoglobulin, the amino acid sequence of its heavy chain is shown in SEQ ID NO:32, and the amino acid sequence of its light chain is shown in SEQ ID NO:

25. (18) The first and second protein functional regions are directly connected or connected through a connector; the third and fourth protein functional regions are directly connected or connected through a connector. (19) The first and second protein functional regions are connected by a linker; the third and fourth protein functional regions are connected by a linker; the linker is independently a polypeptide shown in SEQ ID NO:73, a polypeptide consisting of multiple polypeptides shown in SEQ ID NO:73 in series, or a polypeptide having one or more glycine residues linked to the C-terminus of the polypeptide shown in SEQ ID NO:73 or a polypeptide consisting of multiple polypeptides shown in SEQ ID NO:73 in series; (20) The first protein functional region, the second protein functional region, the third protein functional region and the fourth protein functional region are independently one, two or more; (21) The first protein functional region is 1, and the second protein functional region is 2; (22) The first protein has two functional regions and the second protein has one functional region; (23) The third protein functional region is 1, and the fourth protein functional region is 2.

8. The pharmaceutical combination according to any one of claims 1 to 7, wherein, The anti-LAG3-anti-PD-1 bispecific antibody includes: Targeting the first protein functional region of LAG3, and Targeting the second protein functional region of PD-1; The first protein has one functional region, and the second protein has two functional regions; Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:2, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:

4. The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:19, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:21 or SEQ ID NO:

38. The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin; The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different; Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NOs:35-37; Preferably, the amino acid sequences of the first linker and the second linker are both as shown in SEQ ID NO:

36.

9. The pharmaceutical combination according to any one of claims 1 to 8, wherein, The anti-LAG3-anti-PD-1 bispecific antibody includes: Targeting the first protein functional region of LAG3, and Targeting the second protein functional region of PD-1; The first protein has one functional region, and the second protein has two functional regions; Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:11 or SEQ ID NO:13, and the amino acid sequence of its light chain is shown in SEQ ID NO:

12. The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:19, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:21 or SEQ ID NO:

38. The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin; The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different; Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NOs:35-37; Preferably, the amino acid sequences of the first linker and the second linker are both as shown in SEQ ID NO:

36.

10. The pharmaceutical combination according to any one of claims 1 to 7, wherein, The anti-LAG3-anti-PD-1 bispecific antibody includes: Targeting the first protein functional region of LAG3, and Targeting the second protein functional region of PD-1; The first protein has two functional regions, and the second protein has one functional region; Wherein, the first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin; The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:

4. The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:19, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:

21. The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin; The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different; Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NOs:35-37; Preferably, the amino acid sequences of the first linker and the second linker are both as shown in SEQ ID NOs:

36.

11. The pharmaceutical combination according to any one of claims 1 to 7, wherein, The anti-LAG3-anti-PD-1 bispecific antibody includes: Targeting the first protein functional region of LAG3, and Targeting the second protein functional region of PD-1; The first protein has two functional regions, and the second protein has one functional region; Wherein, the first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin; The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:

4. The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:34 or SEQ ID NO:32, and the amino acid sequence of its light chain is shown in SEQ ID NO:

25. The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin; The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different; Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NOs:35-37; Preferably, the amino acid sequences of the first linker and the second linker are both as shown in SEQ ID NOs:

36.

12. The pharmaceutical combination according to any one of claims 1 to 11, wherein, The anti-CTLA4-anti-PD-1 bispecific antibody includes: Targeting the third protein functional region of PD-1, and Targeting the fourth protein functional region of CTLA4; in, The third protein functional region consists of one unit, and the fourth protein functional region consists of two units. The third protein functional region is an immunoglobulin, and the fourth protein functional region is a single-chain antibody. The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:19, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:

21. The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:61 or SEQ ID NO:63, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:62 or SEQ ID NO:

64. The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin; The third protein functional region is connected to the fourth protein functional region via a third linker; and the heavy chain variable region of the single-chain antibody is connected to the light chain variable region of the single-chain antibody via a fourth linker; the third linker and the fourth linker may be the same or different; Preferably, the amino acid sequences of the third linker and the fourth linker are independently selected from SEQ ID NOs:35-37; Preferably, the amino acid sequences of the third linker and the fourth linker are both as shown in SEQ ID NO:

36.

13. The pharmaceutical combination according to any one of claims 1 to 12, wherein, The anti-CTLA4-anti-PD-1 bispecific antibody includes: Targeting the third protein functional region of PD-1, and Targeting the fourth protein functional region of CTLA4; in, The third protein functional region consists of one unit, and the fourth protein functional region consists of two units. The third protein functional region is an immunoglobulin, and the fourth protein functional region is a single-chain antibody. The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:66, and the amino acid sequence of its light chain is shown in SEQ ID NO:

25. The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:61 or SEQ ID NO:63, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:62 or SEQ ID NO:

64. The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin; The third protein functional region is connected to the fourth protein functional region via a third linker; and the heavy chain variable region of the single-chain antibody is connected to the light chain variable region of the single-chain antibody via a fourth linker; the third linker and the fourth linker may be the same or different; Preferably, the amino acid sequences of the third linker and the fourth linker are independently selected from SEQ ID NOs:35-37; Preferably, the amino acid sequences of the third linker and the fourth linker are both as shown in SEQ ID NO:

36.

14. The pharmaceutical combination according to any one of claims 1 to 13, characterized in that... Any one or more of the following items (1) to (9): (1) The first antibody and the second antibody are mixed together, and the drug combination is a drug composition; (2) The first antibody and the second antibody are in separate packages, and the drug combination is a combination product; (3) The mass ratio of the first antibody to the second antibody is (10:1) to (1:5), (5:1) to (1:3), (3:1) to (1:2) or 2:1; (4) The molar ratio of the first antibody to the second antibody is (10:1) to (1:10), (5:1) to (1:5), (3:1) to (1:3), (2:1) to (1:2) or 1:1; (5) The unit dose of the first antibody and the second antibody, calculated according to the mass of the first antibody or the second antibody, is independently 100mg-2500mg, 100mg-2000mg, 100mg-1500mg, 100mg-1200mg, 100mg-1000mg, 200mg-800mg, 200mg-500mg, 300mg-600mg, 400mg-500mg or 450mg; (6) The drug combination also includes effective amounts of tumor chemotherapy drugs, alkylating agents, antimetabolites, antitumor antibiotics, herbal anticancer drugs, hormones or immunomodulators, oxaliplatin or capecitabine; (7) The drug combination further comprises an effective amount of chemotherapy drugs, wherein the chemotherapy drugs are chemotherapy drugs of the CapeOX regimen (e.g., oxaliplatin and capecitabine), chemotherapy drugs of the mFOLFOX6 regimen, or chemotherapy drugs of the FOLFIRI regimen. (8) The active ingredient of the drug combination consists of the anti-LAG3-anti-PD-1 bispecific antibody, the anti-CTLA4-anti-PD-1 bispecific antibody and chemotherapy drugs, wherein the chemotherapy drugs are oxaliplatin and capecitabine; (9) The drug combination further comprises one or more pharmaceutically acceptable excipients; preferably, it also comprises instructions for use.

15. The pharmaceutical combination according to any one of claims 1 to 14, comprising any one of the following (1) to (6): (1) The anti-LAG3-anti-PD-1 bispecific antibody includes: Targeting the first protein functional region of LAG3, and Targeting the second protein functional region of PD-1; The first protein has one functional region, and the second protein has two functional regions; Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:2, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:

4. The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:19, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:21 or SEQ ID NO:

38. The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin; The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different; and The anti-CTLA4-anti-PD-1 bispecific antibody includes: Targeting the third protein functional region of PD-1, and Targeting the fourth protein functional region of CTLA4; in, The third protein functional region consists of one unit, and the fourth protein functional region consists of two units. The third protein functional region is an immunoglobulin, and the fourth protein functional region is a single-chain antibody. The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:19, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:

21. The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:61 or SEQ ID NO:63, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:62 or SEQ ID NO:

64. The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin; The third protein functional region is connected to the fourth protein functional region via a third linker; and the heavy chain variable region of the single-chain antibody is connected to the light chain variable region of the single-chain antibody via a fourth linker; the third linker and the fourth linker may be the same or different; (2) The anti-LAG3-anti-PD-1 bispecific antibody includes: Targeting the first protein functional region of LAG3, and Targeting the second protein functional region of PD-1; The first protein has one functional region, and the second protein has two functional regions; Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:2, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:

4. The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:19, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:21 or SEQ ID NO:

38. The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin; The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different; The amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NOs:35-37; and The anti-CTLA4-anti-PD-1 bispecific antibody includes: Targeting the third protein functional region of PD-1, and Targeting the fourth protein functional region of CTLA4; in, The third protein functional region consists of one unit, and the fourth protein functional region consists of two units. The third protein functional region is an immunoglobulin, and the fourth protein functional region is a single-chain antibody. The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:19, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:

21. The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:61 or SEQ ID NO:63, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:62 or SEQ ID NO:

64. The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin; The third protein functional region is connected to the fourth protein functional region via a third linker; and the heavy chain variable region of the single-chain antibody is connected to the light chain variable region of the single-chain antibody via a fourth linker; the third linker and the fourth linker may be the same or different; The amino acid sequences of the third and fourth linkers are independently selected from SEQ ID NOs:35-37; (3) The anti-LAG3-anti-PD-1 bispecific antibody includes: Targeting the first protein functional region of LAG3, and Targeting the second protein functional region of PD-1; The first protein has one functional region, and the second protein has two functional regions; Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:2, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:

4. The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:19, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:21 or SEQ ID NO:

38. The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin; The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different; The amino acid sequences of both the first linker and the second linker are shown in SEQ ID NO:36; and The anti-CTLA4-anti-PD-1 bispecific antibody includes: Targeting the third protein functional region of PD-1, and Targeting the fourth protein functional region of CTLA4; in, The third protein functional region consists of one unit, and the fourth protein functional region consists of two units. The third protein functional region is an immunoglobulin, and the fourth protein functional region is a single-chain antibody. The amino acid sequence of the heavy chain variable region of the immunoglobulin is shown in SEQ ID NO:19, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:

21. The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:61 or SEQ ID NO:63, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:62 or SEQ ID NO:

64. The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin; The third protein functional region is connected to the fourth protein functional region via a third linker; and the heavy chain variable region of the single-chain antibody is connected to the light chain variable region of the single-chain antibody via a fourth linker; the third linker and the fourth linker may be the same or different; The amino acid sequences of the third linker and the fourth linker are both shown in SEQ ID NO:36; (4) The anti-LAG3-anti-PD-1 bispecific antibody includes: Targeting the first protein functional region of LAG3, and Targeting the second protein functional region of PD-1; The first protein has one functional region, and the second protein has two functional regions; Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:11 or SEQ ID NO:13, and the amino acid sequence of its light chain is shown in SEQ ID NO:

12. The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:19, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:21 or SEQ ID NO:

38. The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin; The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different; and; The anti-CTLA4-anti-PD-1 bispecific antibody includes: Targeting the third protein functional region of PD-1, and Targeting the fourth protein functional region of CTLA4; in, The third protein functional region consists of one unit, and the fourth protein functional region consists of two units. The third protein functional region is an immunoglobulin, and the fourth protein functional region is a single-chain antibody. The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:66, and the amino acid sequence of its light chain is shown in SEQ ID NO:

25. The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:61 or SEQ ID NO:63, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:62 or SEQ ID NO:

64. The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin; The third protein functional region is connected to the fourth protein functional region via a third linker; and the heavy chain variable region of the single-chain antibody is connected to the light chain variable region of the single-chain antibody via a fourth linker; the third linker and the fourth linker may be the same or different; (5) The anti-LAG3-anti-PD-1 bispecific antibody includes: Targeting the first protein functional region of LAG3, and Targeting the second protein functional region of PD-1; The first protein has one functional region, and the second protein has two functional regions; Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:11 or SEQ ID NO:13, and the amino acid sequence of its light chain is shown in SEQ ID NO:

12. The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:19, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:21 or SEQ ID NO:

38. The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin; The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different; The amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NOs:35-37; and The anti-CTLA4-anti-PD-1 bispecific antibody includes: Targeting the third protein functional region of PD-1, and Targeting the fourth protein functional region of CTLA4; in, The third protein functional region consists of one unit, and the fourth protein functional region consists of two units. The third protein functional region is an immunoglobulin, and the fourth protein functional region is a single-chain antibody. The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:66, and the amino acid sequence of its light chain is shown in SEQ ID NO:

25. The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:61 or SEQ ID NO:63, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:62 or SEQ ID NO:

64. The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin; The third protein functional region is connected to the fourth protein functional region via a third linker; and the heavy chain variable region of the single-chain antibody is connected to the light chain variable region of the single-chain antibody via a fourth linker; the third linker and the fourth linker may be the same or different; The amino acid sequences of the third and fourth linkers are independently selected from SEQ ID NOs:35-37; (6) The anti-LAG3-anti-PD-1 bispecific antibody includes: Targeting the first protein functional region of LAG3, and Targeting the second protein functional region of PD-1; The first protein has one functional region, and the second protein has two functional regions; Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody; The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:11 or SEQ ID NO:13, and the amino acid sequence of its light chain is shown in SEQ ID NO:

12. The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:19, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:21 or SEQ ID NO:

38. The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin; The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the single-chain antibody and the light chain variable region of the single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different; The amino acid sequences of both the first linker and the second linker are shown in SEQ ID NO:36; and The anti-CTLA4-anti-PD-1 bispecific antibody includes: Targeting the third protein functional region of PD-1, and Targeting the fourth protein functional region of CTLA4; in, The third protein functional region consists of one unit, and the fourth protein functional region consists of two units. The third protein functional region is an immunoglobulin, and the fourth protein functional region is a single-chain antibody. The amino acid sequence of the heavy chain of the immunoglobulin is shown in SEQ ID NO:66, and the amino acid sequence of its light chain is shown in SEQ ID NO:

25. The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO:61 or SEQ ID NO:63, and the amino acid sequence of the light chain variable region of the single-chain antibody is shown in SEQ ID NO:62 or SEQ ID NO:

64. The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin; The third protein functional region is connected to the fourth protein functional region via a third linker; and the heavy chain variable region of the single-chain antibody is connected to the light chain variable region of the single-chain antibody via a fourth linker; the third linker and the fourth linker may be the same or different; The amino acid sequences of the third linker and the fourth linker are both shown in SEQ ID NO:36; Preferably, in any one of items (1) to (6) above, the molar ratio or mass ratio of the anti-LAG3-anti-PD-1 bispecific antibody to the anti-CTLA4-anti-PD-1 bispecific antibody is (5:1) to (1:2), (4:1) to (1:2), (3:1) to (1:2), (2:1) to (1:2), (5:1) to (1:1), (4:1) to (1:1), (3:1) to (1:1), (2:1) to (1:1), 5:1, 4:1, 3:1, 2.5:1, 2:1, 1.5:1 or 1:1; Preferably, the drug combination comprises any one of (1) to (6) and an effective amount of a chemotherapy drug, wherein the chemotherapy drug is oxaliplatin and capecitabine.

16. The pharmaceutical combination according to any one of claims 1 to 15, for the treatment or prevention of tumors; Preferably, the tumor is selected from one or more of the following: pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, head and neck cancer, brain cancer, pharyngeal cancer, nasopharyngeal cancer, thyroid cancer, mesothelioma, lung cancer, liver cancer, stomach cancer, biliary tract cancer, kidney cancer, fallopian tube cancer, endometrial cancer, cervical cancer, bladder cancer, urothelial carcinoma, prostate cancer, testicular cancer, skin cancer, melanoma, myeloma, plasma cell carcinoma, vulvar cancer, leukemia, lymphoma, bone cancer, and osteosarcoma. Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell carcinoma of the lung; Preferably, the gastric cancer is gastric adenocarcinoma or esophageal junction adenocarcinoma; Preferably, the lymphoma is Hodgkin lymphoma or non-Hodgkin lymphoma; Preferably, the esophageal cancer is esophageal squamous cell carcinoma.

17. The pharmaceutical combination according to any one of claims 1 to 16, characterized in that... Any one or more of the following items (1) to (10): (1) The first antibody and the antibody are administered independently before or after surgery, and / or before or after radiotherapy; (2) The single dose of the first antibody and the second antibody is independently 0.1-100 mg, 5-50 mg or 5-15 mg per kilogram of body weight; (3) The first antibody and the second antibody are administered independently once every half day, every 1 day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 8 days, every 10 days, every 1 week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 1 month, every 2 months, every 3 months, every 4 months, every 5 months or every 6 months; (4) The administration method is intravenous drip, intravenous injection, subcutaneous injection, intraperitoneal injection or intratumoral injection; (5) The single dose of the first antibody is independently 15 mg per kilogram of body weight, and the single dose of the second antibody is independently 10 mg per kilogram of body weight. The first antibody and / or the second antibody are administered in a cycle of 3 weeks. (6) The single dose of the first antibody is independently 25 mg per kilogram of body weight, and the single dose of the second antibody is independently 10 mg per kilogram of body weight. The first antibody and / or the second antibody are administered in a cycle of 3 weeks. (7) The single dose of the first antibody is independently 40 mg per kilogram of body weight, and the single dose of the second antibody is independently 10 mg per kilogram of body weight. The first antibody and / or the second antibody are administered in a cycle of 3 weeks. (8) The single dose of the first antibody is independently 15 mg per kilogram of body weight, the single dose of the second antibody is independently 10 mg per kilogram of body weight, and the single dose of oxaliplatin is independently 130 mg / m². 2 The single-dose capecitabine dose is independently 1000 mg / m². 2 The first antibody, the second antibody, oxaliplatin and / or capecitabine are administered in a 3-week cycle. (9) The single dose of the first antibody is independently 25 mg per kilogram of body weight, the single dose of the second antibody is independently 10 mg per kilogram of body weight, and the single dose of oxaliplatin is independently 130 mg / m². 2 The single-dose capecitabine dose is independently 1000 mg / m². 2 The first antibody, the second antibody, oxaliplatin and / or capecitabine are administered in a 3-week cycle. (10) The single dose of the first antibody is independently 40 mg per kilogram of body weight, the single dose of the second antibody is independently 10 mg per kilogram of body weight, and the single dose of oxaliplatin is independently 130 mg / m². 2 The single-dose capecitabine dose is independently 1000 mg / m². 2 The first antibody, the second antibody, oxaliplatin and / or capecitabine are administered in a 3-week cycle.

18. Use of the pharmaceutical combination according to any one of claims 1 to 17 in the preparation of a medicament for treating or preventing tumors; Preferably, the tumor is selected from one or more of the following: pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, head and neck cancer, brain cancer, pharyngeal cancer, nasopharyngeal cancer, thyroid cancer, mesothelioma, lung cancer, liver cancer, stomach cancer, biliary tract cancer, kidney cancer, fallopian tube cancer, endometrial cancer, cervical cancer, bladder cancer, urothelial carcinoma, prostate cancer, testicular cancer, skin cancer, melanoma, myeloma, plasma cell carcinoma, vulvar cancer, leukemia, lymphoma, bone cancer, and osteosarcoma. Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer, and squamous cell carcinoma of the lung; Preferably, the gastric cancer is gastric adenocarcinoma or esophageal junction adenocarcinoma; Preferably, the lymphoma is Hodgkin lymphoma or non-Hodgkin lymphoma; Preferably, the esophageal cancer is esophageal squamous cell carcinoma.

19. The use according to claim 18, characterized in that... Any one or more of the following items (1) to (10): (1) The first antibody and the antibody are administered independently before or after surgery, and / or before or after radiotherapy; (2) The single dose of the first antibody and the second antibody is independently 0.1-100 mg, 5-50 mg or 5-15 mg per kilogram of body weight; (3) The first antibody and the second antibody are administered independently once every half day, every 1 day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 8 days, every 10 days, every 1 week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 1 month, every 2 months, every 3 months, every 4 months, every 5 months or every 6 months; (4) The administration method is intravenous drip, intravenous injection, subcutaneous injection, intraperitoneal injection or intratumoral injection; (5) The single dose of the first antibody is independently 15 mg per kilogram of body weight, and the single dose of the second antibody is independently 10 mg per kilogram of body weight. The first antibody and / or the second antibody are administered in a cycle of 3 weeks. (6) The single dose of the first antibody is independently 25 mg per kilogram of body weight, and the single dose of the second antibody is independently 10 mg per kilogram of body weight. The first antibody and / or the second antibody are administered in a cycle of 3 weeks. (7) The single dose of the first antibody is independently 40 mg per kilogram of body weight, and the single dose of the second antibody is independently 10 mg per kilogram of body weight. The first antibody and / or the second antibody are administered in a cycle of 3 weeks. (8) The single dose of the first antibody is independently 15 mg per kilogram of body weight, the single dose of the second antibody is independently 10 mg per kilogram of body weight, and the single dose of oxaliplatin is independently 130 mg / m². 2 The single-dose capecitabine dose is independently 1000 mg / m². 2 The first antibody, the second antibody, oxaliplatin and / or capecitabine are administered in a 3-week cycle. (9) The single dose of the first antibody is independently 25 mg per kilogram of body weight, the single dose of the second antibody is independently 10 mg per kilogram of body weight, and the single dose of oxaliplatin is independently 130 mg / m². 2 The single-dose capecitabine dose is independently 1000 mg / m². 2 The first antibody, the second antibody, oxaliplatin and / or capecitabine are administered in a 3-week cycle. (10) The single dose of the first antibody is independently 40 mg per kilogram of body weight, the single dose of the second antibody is independently 10 mg per kilogram of body weight, and the single dose of oxaliplatin is independently 130 mg / m². 2 The single-dose capecitabine dose is independently 1000 mg / m². 2 The first antibody, the second antibody, oxaliplatin and / or capecitabine are administered in a 3-week cycle.

Images