Anti-lag3 bispecific antibodies, pharmaceutical compositions, and uses

Abstract

The application belongs to the field of biological medicine, and relates to a bispecific antibody of an anti-LAG3 antibody, a pharmaceutical composition thereof and purposes. Specifically, the bispecific antibody comprises a first protein functional region and a second protein functional region, wherein the first protein functional region targets LAG3, and the second protein functional region targets a target point different from LAG3, wherein the first protein functional region is an anti-LAG3 antibody or an antigen binding fragment thereof, and comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-HCDR3 with the amino acid sequences shown in SEQ ID NOs:5-7 respectively, and the light chain variable region comprises LCDR1-LCDR3 with the amino acid sequences shown in SEQ ID NOs:8-10 respectively. The bispecific antibody of the application has superior affinity and specificity, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a bispecific antibody against LAG3, its pharmaceutical composition, and its uses. Specifically, the bispecific antibody is an anti-LAG3-anti-PD-1 bispecific antibody. Background Technology

[0002] 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.

[0003] 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 primarily expressed on activated CD4+ cells. + 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].Semin Immunol, 2019, 42: 101305.).

[0004] 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.

[0005] 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 "CD3-TCR-MHCII" signaling axis, the first signaling axis of T cell activation, blocks the signal transduction pathway of T cell activation on the one hand, and on the other hand, the intracellular domain of LAG3 (Keele motif) generates immunosuppressive signals that downregulate 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, which is one of the mechanisms by which tumors escape the killing 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.).

[0006] 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 in antibody drugs [J]. Chinese Journal of Pharmacology and Toxicology, 2019, 33(01):70-78.).

[0007] Currently, several LAG3 antibody drugs have entered the clinical research stage. Among them, Bristol-Myers Squibb's Relatlimab is progressing the fastest, with 10 clinical studies underway. The vast majority of these studies involve the combination of Relatlimab and nivolumab for the treatment of hematologic malignancies, melanoma, glioblastoma, renal cell carcinoma, and non-small cell lung cancer.

[0008] 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.

[0009] 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.

[0010] 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).

[0011] 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 bifunctional antibody (Miller BR, Demarest SJ, et al., Stability engineering of scFvs for the development of bispecific and multivalent antibodies. Protein Eng Des Sel2010; 23:549-57; Fitzgerald J, Lugovskoy A. Rational engineering of antibodytherapeutics targeting multiple oncogene pathways. MAbs 2011; 3:299-309).

[0012] Currently, there is a need to develop new anti-LAG3 antibodies and bifunctional antibody drugs that simultaneously target PD-1 and LAG3. Summary of the Invention

[0013] Through in-depth research and creative labor, the inventors have obtained an anti-LAG3 antibody and, based on this, prepared an anti-LAG3-anti-PD-1 bispecific antibody. The inventors have surprisingly discovered that the anti-LAG3 antibody (also simply referred to as the antibody or the antibody of this invention) and the anti-LAG3-anti-PD-1 bispecific antibody (also simply referred to as the bispecific antibody or the bispecific antibody of this invention) of this invention possess superior affinity and / or specificity, and are even superior to positive control antibodies (e.g., Nivolumab, Pembrolizumab, Relatlimab, etc.) in one or more aspects. Therefore, the following invention is provided:

[0014] One aspect of the present invention relates to an anti-LAG3 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein,

[0015] The heavy chain variable region comprises: amino acid sequences as shown in SEQ ID NOs: 5-7, namely HCDR1-HCDR3; and the light chain variable region comprises: amino acid sequences as shown in SEQ ID NOs: 8-10, namely LCDR1-LCDR3.

[0016] The heavy chain variable region comprises: amino acid sequences as shown in SEQ ID NOs: 5-7, namely HCDR1-HCDR3; and the light chain variable region comprises: amino acid sequences as shown in SEQ ID NO: 8, SEQ ID NO: 46 and SEQ ID NO: 47, namely LCDR1-LCDR3;

[0017] or

[0018] The heavy chain variable region comprises: amino acid sequences HCDR1-HCDR3 as shown in SEQ ID NOs: 5-7 respectively; and the light chain variable region comprises: amino acid sequences LCDR1-LCDR3 as shown in SEQ ID NO: 48, SEQ ID NO: 46 and SEQ ID NO: 10 respectively.

[0019] In some embodiments of the present invention, the antibody or its antigen-binding fragment, wherein...

[0020] The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 4;

[0021] The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 42.

[0022] or

[0023] The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 44.

[0024] In some embodiments of the present invention, the antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody, humanized antibody, chimeric antibody, or biantibody.

[0025] In some embodiments of the present invention, the antibody or its antigen-binding fragment is wherein the antibody is in an EC50 concentration of less than 0.2 nM, for example less than 0.15 nM, less than 0.1 nM, less than 0.08 nM, 0.06 nM, or less than 0.05 nM or smaller. 50 Combined with human LAG3-mFc; preferably, the EC 50 Measured by indirect ELISA method.

[0026] In some embodiments of the present invention, the antibody or its antigen-binding fragment, wherein...

[0027] The antibody includes a non-CDR region, and the non-CDR region is derived from a species other than rodents, such as from human antibodies.

[0028] In some embodiments of the present invention, the antibody or its antigen-binding fragment, wherein...

[0029] The antibody described herein has a constant region derived from human antibodies;

[0030] Preferably, the constant region of the antibody is selected from the constant regions of human IgG1, IgG2, IgG3 or IgG4.

[0031] In some embodiments of the present invention, the antibody or its antigen-binding fragment, wherein...

[0032] The heavy chain constant region of the anti-LAG3 antibody is the Ig gamma-1 chain C region (e.g., as shown in SEQ ID NO: 39) or the Ig gamma-4 chain C region (e.g., as shown in SEQ ID NO: 45); the light chain constant region is the Ig kappa chain C region (e.g., as shown in SEQ ID NO: 40).

[0033] In some embodiments of the present invention, the antibody or its antigen-binding fragment, wherein...

[0034] The antibody is a human IgG1 subtype.

[0035] According to the EU numbering system, the heavy chain constant region of the antibody has the following mutation:

[0036] L234A and L235A,

[0037] L234A and G237A,

[0038] L235A and G237A,

[0039] or

[0040] L234A, L235A, and G237A;

[0041] Preferably, the amino acid sequence of the heavy chain of the antibody is as shown in SEQ ID NO: 11, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 12.

[0042] In some embodiments of the present invention, the antibody or its antigen-binding fragment, wherein...

[0043] The antibody is human IgG4 subtype.

[0044] According to the EU numbering system, the heavy chain constant region of the antibody has the following mutation:

[0045] F234A and L235A,

[0046] F234A and G237A,

[0047] L235A and G237A,

[0048] or

[0049] F234A, L235A, and G237A;

[0050] Preferably, the amino acid sequence of the heavy chain of the antibody is as shown in SEQ ID NO: 13, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 12.

[0051] In some embodiments of the present invention, the anti-LAG3 antibody is a monoclonal antibody.

[0052] In some embodiments of the present invention, the anti-LAG3 antibody is in the form of an immunoglobulin.

[0053] In some embodiments of the present invention, the anti-LAG3 antibody is a single-chain antibody.

[0054] Another aspect of the present invention relates to antibody-drug conjugates (ADCs), which comprise an antibody or an antigen-binding fragment thereof and a small molecule drug, wherein the antibody or antigen-binding fragment thereof is any one of the anti-LAG3 antibodies or antigen-binding fragments thereof described in the present invention; preferably, the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is an anti-tumor chemotherapy drug.

[0055] The chemotherapy drugs can be conventional tumor chemotherapy drugs, such as alkylating agents, antimetabolites, antitumor antibiotics, herbal anticancer drugs, hormones, immunomodulators, etc.

[0056] In one or more embodiments of the present invention, the antibody-drug conjugate wherein the antibody or its antigen-binding fragment is linked to a small molecule drug via a linker; the linker may be a linker known to those skilled in the art, for example, the linker may be a hydrazone bond, a disulfide bond or a peptide bond.

[0057] In one or more embodiments of the present invention, the antibody-drug conjugate wherein the molar ratio of the antibody or its antigen-binding fragment to the small molecule drug is 1:(2-4), for example 1:2, 1:3 or 1:4.

[0058] Another aspect of the present invention relates to a bispecific antibody comprising a first protein functional region and a second protein functional region, wherein:

[0059] The first protein's functional region targets LAG3.

[0060] The second protein functional region targets a different target than LAG3 (e.g., PD-1).

[0061] Wherein, the first protein functional region is the antibody or antigen-binding fragment described in any one of the present invention;

[0062] Preferably, the bispecific antibody is in IgG-scFv mode;

[0063] Preferably, the first protein functional region is an antibody as described in any one of the present invention, and the second protein functional region is a single-chain antibody; or

[0064] Preferably, the first protein functional region is a single-chain antibody, and the second protein functional region is an antibody as described in any one of the present invention.

[0065] The bispecific antibody of the present invention is an anti-LAG3-anti-PD-1 bispecific antibody.

[0066] In some embodiments of the present invention, the bispecific antibody is wherein the first protein functional region and the second protein functional region are directly connected or connected through a linker fragment.

[0067] Preferably, the connecting segment is (GGGGS)m, where m is a positive integer, such as 1, 2, 3, 4, 5 or 6;

[0068] Preferably, the connection segment is (GGGGS)nG, where n is a positive integer, such as 1, 2, 3, 4, 5 or 6.

[0069] In some embodiments of the present invention, the bispecific antibody wherein the first protein functional region and the second protein functional region are independently one, two, or more.

[0070] In some embodiments of the present invention, the bispecific antibody is wherein the single-chain antibody is attached to the C-terminus of the heavy chain of the antibody.

[0071] In some embodiments of the present invention, the bispecific antibody includes:

[0072] Targeting the first protein functional region of LAG3, and

[0073] Targeting the second protein functional region of PD-1;

[0074] in,

[0075] The first protein functional region is the anti-LAG3 antibody described in any one of the present invention, and the anti-LAG3 antibody is in the form of an immunoglobulin.

[0076] The second protein functional region is an anti-PD-1 single-chain antibody.

[0077] In some embodiments of the present invention, the bispecific antibody, wherein the anti-PD-1 single-chain antibody comprises a heavy chain variable region and a light chain variable region, wherein,

[0078] The heavy chain variable region comprises: amino acid sequences as shown in SEQ ID NOs: 26-28, HCDR1-HCDR3; and

[0079] The light chain variable region comprises: amino acid sequences as shown in SEQ ID NOs: 29-31, namely LCDR1-LCDR3.

[0080] In some embodiments of the present invention, the bispecific antibody, wherein the anti-PD-1 single-chain antibody,

[0081] The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 15, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 17; or

[0082] The amino acid sequence of its heavy chain variable region 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 or SEQ ID NO: 38.

[0083] In some embodiments of the present invention, the bispecific antibody, wherein the heavy chain variable region and the light chain variable region of the anti-PD-1 single-chain antibody are directly linked or linked through a linker fragment;

[0084] Preferably, the connecting segment is (GGGGS)m, where m is a positive integer, such as 1, 2, 3, 4, 5 or 6;

[0085] Preferably, the connection segment is (GGGGS)nG, where n is a positive integer, such as 1, 2, 3, 4, 5 or 6.

[0086] In some embodiments of the present invention, the bispecific antibody, wherein,

[0087] The bispecific antibody includes:

[0088] Targeting the first protein functional region of LAG3, and

[0089] Targeting the second protein functional region of PD-1;

[0090] The first protein has one functional region, and the second protein has two functional regions;

[0091] Wherein, the first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody;

[0092] 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;

[0093] 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.

[0094] The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin;

[0095] The first protein functional region and the second protein functional region are connected by a first linker fragment; 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 fragment; the first linker fragment and the second linker fragment may be the same or different;

[0096] Preferably, the amino acid sequences of the first linker fragment and the second linker fragment are independently selected from SEQ ID NOs: 35-37;

[0097] Preferably, the amino acid sequences of the first linker fragment and the second linker fragment are both as shown in SEQ ID NO:36.

[0098] In some embodiments of the present invention, the bispecific antibody includes:

[0099] Targeting the first protein functional region of LAG3, and

[0100] Targeting the second protein functional region of PD-1;

[0101] Wherein, the first protein functional region is an anti-LAG3 single-chain antibody, the second protein functional region is an anti-PD-1 antibody, and the anti-PD-1 antibody is in the form of an immunoglobulin;

[0102] The anti-LAG3 single-chain antibody comprises a heavy chain variable region and a light chain variable region, wherein,

[0103] The heavy chain variable region comprises: amino acid sequences HCDR1-HCDR3 as shown in SEQ ID NOs: 5-7; and

[0104] The light chain variable region comprises: amino acid sequences as shown in SEQ ID NOs: 8-10, namely LCDR1-LCDR3.

[0105] In some embodiments of the present invention, the bispecific antibody, wherein the anti-LAG3 single-chain antibody,

[0106] The amino acid sequence of its heavy chain variable region 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;

[0107] The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 2, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 42; or

[0108] The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 2, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 44.

[0109] In some embodiments of the present invention, the bispecific antibody, wherein the heavy chain variable region and the light chain variable region of the anti-LAG3 single-chain antibody are directly linked or linked through a linker fragment;

[0110] Preferably, the connecting segment is (GGGGS)m, where m is a positive integer, such as 1, 2, 3, 4, 5 or 6;

[0111] Preferably, the connection segment is (GGGGS)nG, where n is a positive integer, such as 1, 2, 3, 4, 5 or 6.

[0112] In some embodiments of the present invention, the bispecific antibody, wherein the anti-PD-1 antibody comprises a heavy chain variable region and a light chain variable region, wherein,

[0113] The heavy chain variable region comprises: amino acid sequences as shown in SEQ ID NOs: 26-28, HCDR1-HCDR3; and

[0114] The light chain variable region comprises: amino acid sequences as shown in SEQ ID NOs: 29-31, namely LCDR1-LCDR3.

[0115] In some embodiments of the present invention, the bispecific antibody, wherein the anti-PD-1 antibody,

[0116] The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 15, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 17; or

[0117] The amino acid sequence of its heavy chain variable region 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 or SEQ ID NO: 38.

[0118] In some embodiments of the present invention, the bispecific antibody, wherein the anti-PD-1 antibody has a heavy chain constant region of Ig gamma-1 chain C region (e.g., as shown in SEQ ID NO: 39) or Ig gamma-4 chain C region (e.g., as shown in SEQ ID NO: 45); and a light chain constant region of Ig kappa chain C region (e.g., as shown in SEQ ID NO: 40).

[0119] In some embodiments of the present invention, the bispecific antibody, wherein,

[0120] The anti-PD-1 antibody is the human IgG1 subtype.

[0121] According to the EU numbering system, the anti-PD-1 antibody has the following mutations:

[0122] L234A and L235A,

[0123] L234A and G237A,

[0124] L235A and G237A,

[0125] or

[0126] L234A, L235A, and G237A;

[0127] Preferably, the amino acid sequence of the heavy chain of the anti-PD-1 antibody is as shown in SEQ ID NO: 34, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 25.

[0128] In some embodiments of the present invention, the bispecific antibody, wherein,

[0129] The anti-PD-1 antibody is the human IgG4 subtype.

[0130] According to the EU numbering system, the anti-PD-1 antibody has the following mutations:

[0131] F234A and L235A,

[0132] F234A and G237A,

[0133] L235A and G237A,

[0134] or

[0135] F234A, L235A, and G237A;

[0136] Preferably, the amino acid sequence of the heavy chain of the anti-PD-1 antibody is as shown in SEQ ID NO: 32, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 25.

[0137] In some embodiments of the present invention, the bispecific antibody, wherein,

[0138] The bispecific antibody includes:

[0139] Targeting the first protein functional region of LAG3, and

[0140] Targeting the second protein functional region of PD-1;

[0141] The first protein has two functional regions, and the second protein has one functional region;

[0142] Wherein, the first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin;

[0143] 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.

[0144] 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.

[0145] The single-chain antibody is attached to the C-terminus of the two heavy chains of the immunoglobulin;

[0146] The first protein functional region and the second protein functional region are connected by a first linker fragment; 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 fragment; the first linker fragment and the second linker fragment may be the same or different;

[0147] Preferably, the amino acid sequences of the first linker fragment and the second linker fragment are independently selected from SEQ ID NOs: 35-37;

[0148] Preferably, the amino acid sequences of the first linker fragment and the second linker fragment are both as shown in SEQ ID NO:36.

[0149] In some embodiments of the present invention, the bispecific antibody comprises an immunoglobulin molecule linked to two single-chain antibody molecules; preferably, the two single-chain antibody molecules are identical.

[0150] Another aspect of the invention relates to an isolated nucleic acid molecule that encodes the anti-LAG3 antibody described in any one of the inventions, or that encodes the bispecific antibody described in any one of the inventions.

[0151] Another aspect of the invention relates to a recombinant vector comprising the isolated nucleic acid molecules of the invention.

[0152] Another aspect of the invention relates to a host cell comprising the isolated nucleic acid molecules of the invention, or comprising the recombinant vector of the invention.

[0153] Another aspect of the present invention relates to a method for preparing the antibody or antigen-binding fragment thereof as described in any one of the present invention, or the bispecific antibody as described in any one of the present invention, comprising culturing the host cells of the present invention under suitable conditions, and recovering the antibody or antigen-binding fragment thereof or bispecific antibody from the cell culture.

[0154] Another aspect of the present invention relates to a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described in any one of the present invention, the antibody-drug conjugate as described in any one of the present invention, or the bispecific antibody as described in any one of the present invention; optionally, it further comprises pharmaceutically acceptable excipients.

[0155] Another aspect of the present invention relates to the use of any antibody or antigen-binding fragment thereof described in any one of the present invention, any antibody-drug conjugate described in any one of the present invention, or any bispecific antibody described in any one of the present invention in the preparation of medicaments for treating and / or preventing tumors or anemia;

[0156] Preferably, the tumor is selected from one or more of the following: ovarian cancer, esophageal cancer, melanoma, hematologic malignancy, glioblastoma, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, gastrointestinal cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer.

[0157] Preferably, the lung cancer is non-small cell lung cancer;

[0158] Preferably, the hematologic malignancy is leukemia;

[0159] Preferably, the esophageal cancer is esophageal squamous cell carcinoma.

[0160] The antibody or its antigen-binding fragment according to any one of the present invention, the antibody-drug conjugate according to any one of the present invention, or the bispecific antibody according to any one of the present invention, is used for the treatment and / or prevention of tumors or anemia.

[0161] Preferably, the tumor is selected from one or more of the following: ovarian cancer, esophageal cancer, melanoma, hematologic malignancy, glioblastoma, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, gastrointestinal cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer.

[0162] Preferably, the lung cancer is non-small cell lung cancer;

[0163] Preferably, the hematologic malignancy is leukemia;

[0164] Preferably, the esophageal cancer is esophageal squamous cell carcinoma.

[0165] Another aspect of the present invention relates to a method for treating and / or preventing tumors or anemia, comprising the step of administering to a subject in need an effective amount of any of the antibodies or antigen-binding fragments thereof described in any one of the present invention, any of the antibody-drug conjugates described in any one of the present invention, or any of the bispecific antibodies described in any one of the present invention;

[0166] Preferably, the tumor is selected from one or more of the following: ovarian cancer, esophageal cancer, melanoma, hematologic malignancy, glioblastoma, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, gastrointestinal cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer.

[0167] Preferably, the lung cancer is non-small cell lung cancer;

[0168] Preferably, the hematologic malignancy is leukemia;

[0169] Preferably, the esophageal cancer is esophageal squamous cell carcinoma.

[0170] 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.

[0171] As used in this article, the term EC 50 The half-maximal effect concentration (50% of maximal effect) refers to the concentration that produces a 50% maximal effect.

[0172] 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 the 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.

[0173] 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.

[0174] 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.

[0175] 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 are usually obtained using the hybridoma technique first reported by Kohler et al. (Köhler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity[J]. nature, 1975; 256(5517): 495), but can also be obtained using recombinant DNA techniques (see USPatent 4,816,567).

[0176] 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).

[0177] 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 for single polypeptide chains (see, for example, Bird 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 -Connection fragment-VH -COOH or NH2-V H -Connection fragment-V L -COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 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.

[0178] As used herein, the terms "separated" or "isolated" refer to substances obtained artificially from their natural state. If a substance or component is found in nature as a "separated" entity, it may be due to an alteration of its natural environment, the separation of the substance from its natural environment, or both. For example, a certain unseparated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide isolated from this natural state is called a separated one. The terms "separated" or "isolated" do not exclude the presence of artificial or synthetic substances, nor do they exclude the presence of other impurities that do not affect the substance's activity.

[0179] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0180] As used herein, the term “host cell” refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0181] 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 () binds to the antigen.

[0182] 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 -6M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or a smaller dissociation equilibrium constant (K) D K binds to antigens (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.

[0183] 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.

[0184] As used herein, the term "pharmaceutically acceptable carrier and / or 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.

[0185] 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.

[0186] As used herein, when referring to the amino acid sequence of the PD-1 protein (NCBI GenBank: NM_005018), it includes the full-length PD-1 protein, or the extracellular fragment of PD-1, PD-1 ECD, or a fragment containing PD-1 ECD; it also includes fusion proteins of the full-length PD-1 protein or fusion proteins of PD-1 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 PD-1 protein without affecting its biological function. Therefore, in this invention, the term "added protein" should include all such sequences, including their natural or artificial variants. Furthermore, when describing a sequence fragment of the PD-1 protein, it also includes the corresponding sequence fragment from its natural or artificial variants.

[0187] 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 "added protein" should include all such sequences, including their 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.

[0188] In this invention, unless otherwise specified, the terms "first" (e.g., first protein functional region) and "second" (e.g., second protein functional region) are used for reference or clarity of expression and do not have a typical sequential meaning.

[0189] Beneficial effects of the invention

[0190] This invention achieves one or more of the following effects:

[0191] (1) The anti-LAG3 antibody of the present invention has superior affinity and specificity;

[0192] (2) The bispecific antibodies of the present invention, such as BS-PL021A, BS-PL022B, and BS-PL023C, can bind to LAG3 specifically and effectively block the binding of LAG3 to MHC II, thereby specifically relieving the immunosuppression of LAG3 on the body.

[0193] (3) The bispecific antibodies of the present invention, such as BS-PL021A, BS-PL022B and BS-PL023C, can bind to PD-1 specifically and can effectively block the binding of PD-1 to PDL1, specifically relieve the immunosuppression of the body by PD-1 and activate the immune response.

[0194] (4) The first and second protein functional regions in the bispecific antibody of the present invention have a synergistic effect.

[0195] (5) The bispecific antibody of the present invention, especially BS-PL022B, completely eliminates its binding activity with Fc receptors FcγRI, FcγRIIb, FcγRIIa_H131, FcγRIIIa_V158 and / or FcγRIIIa_F158, thereby eliminating its ADCC or ADCP activity.

[0196] (6) The bispecific antibody of the present invention, especially BS-PL022B, completely eliminates its binding activity with complement C1q, thereby eliminating its CDC activity. Attached Figure Description

[0197] Figure 1 The binding activity of BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, 14C12H1L1 (hG1TM) to antigen PD-1-mFc was detected by indirect ELISA method.

[0198] Figure 2 The binding activity of BS-PL021A, BS-PL022B, BS-PL023C, BS-PLV02, Relatlimab, H7L8 (hG1WT) to antigen LAG3-mFc was determined by indirect ELISA.

[0199] Figure 3 The competitive ELISA method was used to detect the activity of anti-LAG3-anti-PD-1 bispecific antibody in competitively binding to human PD-1-mFc-Biotin with human PDL1-mFc.

[0200] Figure 4 FACS detection results of the binding activity of anti-LAG3-anti-PD-1 bispecific antibody to PD-1 on the surface of 293T-PD1 membrane.

[0201] Figure 5 FACS detection results of the binding activity of anti-LAG3-anti-PD-1 bispecific antibody to LAG3 on the 293T-LAG3 membrane surface.

[0202] Figure 6 Competitive flow cytometry was used to determine the results of anti-LAG3-anti-PD-1 bispecific antibody competing with PDL1 to bind to cell membrane surface antigen PD-1.

[0203] Figure 7 Competitive flow cytometry was used to determine the results of MHC II assay, which showed that anti-LAG3-anti-PD-1 bispecific antibody competitively binds to cell membrane surface antigen LAG3.

[0204] Figure 8A : Detection results of LAG3-MHCII binding blocked by anti-LAG3-anti-PD-1 bispecific antibody.

[0205] Figure 8B : Detection results of LAG3-MHCII binding blocked by anti-LAG3-anti-PD-1 bispecific antibody.

[0206] Figure 9A Detection results of the blocking of PD-1 and PD-L1 binding by anti-LAG3-anti-PD-1 bispecific antibody.

[0207] Figure 9B Detection results of the blocking of PD-1 and PD-L1 binding by anti-LAG3-anti-PD-1 bispecific antibody.

[0208] Figure 10A The detection results show that the anti-LAG3 and anti-PD-1 bispecific antibodies simultaneously block the binding of LAG3 to MHCII and PD-1 to PD-L1.

[0209] Figure 10B The detection results show that the anti-LAG3 and anti-PD-1 bispecific antibodies simultaneously block the binding of LAG3 to MHCII and PD-1 to PD-L1.

[0210] Figure 11 Results of anti-LAG3-anti-PD-1 bispecific antibody bridging assay.

[0211] Figure 12A Results of the bioactivity assay of anti-LAG3-anti-PD-1 bispecific antibody in promoting IFN-γ secretion by mixed lymphocyte reaction (MLR).

[0212] Figure 12BResults of the bioactivity assay of anti-LAG3-anti-PD-1 bispecific antibody in promoting IL-2 secretion by mixed lymphocyte reaction (MLR).

[0213] Figure 13 Results of affinity constant detection between BS-PL022B and FcγRI.

[0214] Figure 14 Results of the affinity constant detection between H7L8(hG1WT) and FcγRI.

[0215] Figure 15 Results of affinity constant detection between BS-PL022B and FcγRIIIa_V158.

[0216] Figure 16 Figure: Detection results of affinity constants between H7L8(hG1WT) and FcγRIIIa_V158.

[0217] Figure 17 Results of affinity constant detection for BS-PL022B and FcγRIIIa_F158.

[0218] Figure 18 Results of affinity constant detection between H7L8(hG1WT) and FcγRIIIa_F158.

[0219] Figure 19 Results of affinity constant detection between BS-PL022B and FcγRIIa_H131.

[0220] Figure 20 Results of affinity constant detection for H7L8(hG1WT) and FcγRIIa_H131.

[0221] Figure 21 Results of affinity constant detection between BS-PL022B and FcγRIIb.

[0222] Figure 22 Results of the affinity constant detection between H7L8(hG1WT) and FcγRIIb.

[0223] Figure 23 Results of affinity constant test between BS-PL022B and C1q.

[0224] Figure 24 Results of the affinity constant test between H7L8(hG1WT) and C1q.

[0225] Figure 25 Results of ADCP effect of BS-PL022B.

[0226] Figure 26Efficacy of anti-LAG3-anti-PD-1 bispecific antibody in BALB / c-hPD1 / hLAG3 mouse CT26 xenograft model. * P < 0.05, ** P < 0.01, *** P < 0.001, VS isotype control group (two-way ANOVA)

[0227] Figure 27 Effect of anti-LAG3-anti-PD-1 bispecific antibody on body weight in BALB / c-hPD1 / hLAG3 mouse CT26 xenograft model. Detailed Implementation

[0228] 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 should be 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. For example, MDA-MB-231 cells and U87-MG cells can be purchased from ATCC.

[0229] BALB / c mice were purchased from the Guangdong Provincial Medical Laboratory Animal Center.

[0230] Nivolumab was purchased from BMS, lot number: ABA0330. Nivolumab is an anti-PD-1 antibody.

[0231] Pembrolizumab was purchased from MSD Ireland (Carlow), catalog number: S023942. Pembrolizumab is an anti-PD-1 antibody.

[0232] The positive control antibody, Relatlimab, has its sequence referenced in US Patent Publication US20160326248A1. The heavy chain amino acid sequence refers to SEQ ID NO: 1 in that patent publication, and the light chain amino acid sequence refers to SEQ ID NO: 2 in that patent publication. Relatlimab is an anti-LAG-3 antibody.

[0233] The 293T-PD1 cell line was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. The 293T-PD1 cell line was obtained by viral infection of HEK293T cells. The virus preparation used 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L. JVirol. 1998. 72(11):8463-8471. The lentiviral expression vector used was plenti6.3 / V5-PD1FL-BSD (PD1, Genebank ID: NM_005018; vector plenti6.3 / V5-BSD, purchased from Invitrogen, product number: K5315-20).

[0234] The 293T-LAG3 cell line was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. The 293T-LAG3 cell line was obtained by viral infection of HEK293T cells. The virus preparation used 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L. J Virol. 1998. 72(11):8463-8471. The lentiviral expression vector used was plenti6.3 / V5-huLAG3FL-BSD (LAG3, Genebank ID: NM_002277.4; vector plenti6.3 / V5-BSD, purchased from Invitrogen, product number: K5315-20).

[0235] 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. DullT, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L. J Virol.1998. 72(11):8463-8471. The lentiviral expression vector used was plenti6.3 / V5-PDL1 (PDL1, Genebank ID: NP_054862.1; vector plenti6.3 / V5, purchased from Invitrogen, catalog number: K5315-20).

[0236] The Jurkat-NFAT-PD1-LAG3 cell line was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. The Jurkat-NFAT-PD1-LAG3 cell line was prepared by viral infection of PD-1 effector cells (CPM, Promega, catalog number: J112A). The virus preparation used 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L. J Virol. 1998. 72(11):8463-8471. The lentiviral expression vector used was pCDH-huLAG3FL-RFP-NEO (where LAG3, Genebank ID: NM_002277.4; vector pCDH-CMV-MCS-EF1-RFP+Neo, purchased from UBO Biotechnology, product number: VT9005).

[0237] The CHO-K1-PD1 cell line was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. The CHO-K1-PD1 cell line was obtained by viral infection of CHO-K1 cells. The virus preparation used 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L. JVirol. 1998. 72(11):8463-8471. The lentiviral expression vector used was pCDH-CMV-PD-1FL-Puro (PD1, Genebank ID: NM_005018; vector pCDH-CMV-Puro, purchased from Youbao Biotechnology, product number: VT1480).

[0238] The CHO-K1-LAG3 cell line was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. The CHO-K1-LAG3 cell line was obtained by viral infection of CHO-K1 cells. The virus preparation used 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L. J Virol. 1998. 72(11):8463-8471. The lentiviral expression vector used was plenti6.3 / V5-huLAG3FL-BSD (LAG3, Genebank ID: NM_002277.4; vector plenti6.3 / V5-BSD, purchased from Invitrogen, product number: K5315-20).

[0239] The Jurkat-NFAT-CD64-CD32R cell line was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. The Jurkat-NFAT-CD64-CD32R cell line was obtained from Jurkat cells through viral infection. The virus preparation used 3rd Generation Lentivirus Systems; see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L. J Virol. 1998. 72(11):8463-8471. The lentiviral expression vectors used were pCDH-NFAT-Hygro (the pCDH-Hygro vector was modified by our laboratory based on pCDH-CMV-MCS-EF1-Puro (purchased from UBO Biotechnology, catalog number: VT1480), pcDH-hFCGR1AFL-Neo (the pCDH-Neo vector was modified by our laboratory based on pCDH-CMV-MCS-EF1-Puro (purchased from UBO Biotechnology, catalog number: VT1480)), and pCDH-hFCGR2A(H167)-puro (where hFCGR2A(H167), Genebank ID: P12318; and the pCDH-CMV-MCS-EF1-Puro vector was purchased from UBO Biotechnology, catalog number: VT1480).

[0240] The CHO-K1-PD1-LAG3 cell line was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. The CHO-K1-PD1-LAG3 cell line was obtained from CHO-K1 cells via viral infection. The virus preparation used 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L. J Virol. 1998. 72(11):8463-8471. The lentiviral expression vectors used were pCDH-hPD1-FL-puro (PD-1, Genebank ID: NM_005018; its vector pCDH-CMV-MCS-EF1-Puro was purchased from Youbao Biotechnology, catalog number: VT1480) and plenti6.3 / V5-huLAG3FL-BSD (LAG3, Genebank ID: NM_005018). ID: NM_002277.4; Carrier: plenti6.3 / V5-BSD, purchased from Invitrogen, part number: K5315-20)

[0241] Preparation Example 1: Design and Preparation of Anti-LAG3 Antibody

[0242] 1. Antibody design

[0243] 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.

[0244] The nucleic acid sequence (360 bp) of the H7L8 heavy chain variable region H7v:

[0245] CAGGTGCAGCTGCAGCAGTGGGGAGCTGGACTGCTGAAACCTAGCGAGACACTGAGCCTGACCTGTGCTGTGTACGGCGGATCTATCAGCGATTACTACTGGAACTGGATCAGGCAGCCCCCTGGAAAGGGACTGGAATGGATCGGAGAGATCAACCACAGGGGCACCACCAACTCCAATCCCTCTCTGAAGAGCAGGGTGACACTGAGCCTCGACACAAGCAAGAATCAGTTCAGCCTGAAGCTGAGGTCCGTGACCGCTGCTGATACAGCTGTGTACTACTGTGCCTTCGGCTACAGCGATTACGAGTACGATTGGTTCGACCCTTGGGGCCAGGGAACACTGGTTACAGTGAGCTCC (SEQ ID NO:1)

[0246] Amino acid sequence (120 aa) of the heavy chain variable region H7v of H7L8:

[0247] QVQLQQWGAGLLKPSETLSLTCAVYGGSISDYYWNWIRQPPGKGLEWIGEINHRGTTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYDWFDPWGQGTLVTVSS (SEQ ID NO: 2)

[0248] Nucleic acid sequence (321 bp) of the light chain variable region L8v of H7L8:

[0249] GAGATCGTTCTGACCCAGAGCCCAGCTACACTGAGCCTGTCTCCTGGAGAGAGGGCTACACTGTCCTGCAGAGCTAGCCAGACATCAGCAGCTACCTGGCTTGGTACCAGCAGAAGCCTGGCCAAGCTCCAAGGCTGCTGATCTACGACGCCTCTAATA GGGCCACCGGCATCCCTGCTAGATTCTCTGGAAGCGGCAGCGGAACCGACTTTACACTGACAATCAGCTCCCTGGAGCCCGAGGATTTCGCTGTTACTACTGTCAGCAGCGCAGCAACTGGCCCATCACATTCGGACAGGGCACAAATCTGGAGATCAAG (SEQ ID NO: 3)

[0250] The amino acid sequence (107aa) of the L8v variable region of the H7L8 light chain:

[0251] EIVLTQSPATLSLSPGERATLSCRASQTISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTNLEIK (SEQID NO: 4)

[0252] 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.

[0253] 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.

[0254] The nucleic acid sequence (321 bp) of the L9v variable region of the H7L9 light chain:

[0255] GAGATCGTTCTGACCCAGAGCCCAGCTACACTGAGCCTGTCTCCTGGAGAGAGGGCTACACTGTCCTGCAGAGCTAGCCAGACATCAGCAGCTACCTGGCTTGGTACCAGCAGAAGCCTGGCCAAGCTCCAAGGCTGCTGATCTACGACGGCTCTAATAGG GCCACCGGCATCCCTGCTAGATTCTCTGGAAGCGGCAGCGGAACCGACTTTACACTGACAATCAGCTCCCTGGAGCCCGAGGATTTCGCTGTTTACTACTGTCAGCAGCGCAGCAACTGGCCCCTCACATTCGGACAGGGCACAAATCTGGAGATCAAG (SEQ ID NO: 41)

[0256] The amino acid sequence (107 bp) of the L9v variable region of the H7L9 light chain:

[0257] EIVLTQSPATLSLSPGERATLSCRASQTISSYLAWYQQKPGQAPRLLIYDGSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIK (SEQ ID NO: 42)

[0258] 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.

[0259] 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.

[0260] The nucleic acid sequence (321 bp) of the L10v variable region of the H7L10 light chain:

[0261] GAGATCGTTCTGACCCAGAGCCCAGCTACACTGAGCCTGTCTCCTGGAGAGAGGGCTACACTGTCCTGCAGAGCTAGCCAGTCCATCAGCAGCTACCTGGCTTGGTACCAGCAGAAGCCTGGCCAAGCTCCAAGGCTGCTGATCTACGACGGCTCTAATAGG GCCACCGGCATCCCTGCTAGATTCTCTGGAAGCGGCAGCGGAACCGACTTTACACTGACAATCAGCTCCCTGGAGCCCGAGGATTTCGCTGTTTACTACTGTCAGCAGCGCAGCAACTGGCCCATCACATTCGGACAGGGCACAAATCTGGAGATCAAG (SEQ ID NO: 43)

[0262] The amino acid sequence (107 bp) of the L10v variable region of the light chain of H7L10:

[0263] EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDGSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTNLEIK (SEQ ID NO: 44)

[0264] The amino acid sequence of the CDR of antibody H7L8 is as follows (according to the IMGT numbering system):

[0265] HCDR1:GGSISDYY (SEQ ID NO: 5);

[0266] HCDR2: INHRGTT (SEQ ID NO: 6);

[0267] HCDR3:AFGYSDYEYDWFDP (SEQ ID NO: 7);

[0268] LCDR1: QTISSY (SEQ ID NO: 8);

[0269] LCDR2: DAS (SEQ ID NO: 9);

[0270] LCDR3: QQRSNWPIT (SEQ ID NO: 10).

[0271] The amino acid sequence of the CDR of antibody H7L9 is as follows (according to the IMGT numbering system):

[0272] HCDR1:GGSISDYY (SEQ ID NO: 5);

[0273] HCDR2: INHRGTT (SEQ ID NO: 6);

[0274] HCDR3:AFGYSDYEYDWFDP (SEQ ID NO: 7);

[0275] LCDR1: QTISSY (SEQ ID NO: 8);

[0276] LCDR2: DGS (SEQ ID NO: 46);

[0277] LCDR3: QQRSNWPLT (SEQ ID NO: 47).

[0278] The amino acid sequence of the CDR of antibody H7L10 is as follows (according to the IMGT numbering system):

[0279] HCDR1:GGSISDYY (SEQ ID NO: 5);

[0280] HCDR2: INHRGTT (SEQ ID NO: 6);

[0281] HCDR3:AFGYSDYEYDWFDP (SEQ ID NO: 7);

[0282] LCDR1: QSISSY (SEQ ID NO: 48);

[0283] LCDR2: DGS (SEQ ID NO: 46);

[0284] LCDR3: QQRSNWPIT (SEQ ID NO: 10).

[0285] 2. Expression and purification of humanized antibody H7L8 (hG1WT)

[0286] 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, and the heavy and light chains were recovered by electrophoresis and subcloned into the pcDNA3.1 vector, respectively. The recombinant plasmids were extracted and co-transfected into 293F cells. Cells were cultured for 7 days. After 1 day, the culture medium was centrifuged at high speed, the supernatant was concentrated and then loaded onto a HiTrapMabSelect SuRe column. Proteins were eluted in one step with Elution Buffer and the target sample was recovered and the medium was changed to PBS.

[0287] H7L8(hG1WT) heavy chain constant region amino acid sequence

[0288] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 39)

[0289] H7L8(hG1WT) light chain constant region amino acid sequence

[0290] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 40)

[0291] 3. Design of humanized antibody H7L8 (hG1TM)

[0292] 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 (L235A) and at position 237 (G237A) of glycine to alanine (G237A) on 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.

[0293] The preparation of humanized antibody H7L8 (hG1TM) can be carried out by referring to the method in step 2 above.

[0294] The amino acid sequence of the heavy chain H7(hG1TM) of H7L8(hG1TM).

[0295] QVQLQQWGAGLLKPSETLSLTCAVYGGSISDYYWNWIRQPPGKGLEWIGEINHRGTTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYDWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11)

[0296] Amino acid sequence of the light chain L8 of H7L8 (hG1TM)

[0297] EIVLTQSPATLSLSPGERATLSCRASQTISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTNLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQID NO: 12)

[0298] 4. Design of the humanized antibody H7L8 (hG4DM)

[0299] Based on H7L8(hG1WT), the inventors retained the variable region of the antibody and used the Ig gamma-4 chain C region 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 within 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.

[0300] The amino acid sequence of the heavy chain H7(hG4DM) of H7L8(hG4DM):

[0301] QVQLQQWGAGLLKPSETLSLTCAVYGGSISDYYWNWIRQPPGKGLEWIGEINHRGTTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYDWFDPWGQ GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 13)

[0302] 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.

[0303] 5. Expression and purification of humanized antibodies H7L8(hG4WT), H7L9(hG4WT), and H7L10(hG4WT)

[0304] The cDNA sequences of the heavy chains of H7L8(hG4WT), H7L9(hG4WT), and H7L10(hG4WT) (variable region coding sequence as shown in SEQ ID NO: 1; constant region is Ig gamma-4 chain C region, as shown in SEQ ID NO: 45) and the cDNA sequences of the light chains 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) (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) (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 Figure 40) plasmids were cloned into the pUC57simple vector (provided by GenScript) 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 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 replaced with PBS.

[0305] H7L8(hG4WT), H7L9(hG4WT), or H7L10(hG4WT) heavy chain constant region amino acid sequence:

[0306] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 45)

[0307] The amino acid sequence of the light chain constant region of H7L8(hG4WT), H7L9(hG4WT), or H7L10(hG4WT):

[0308] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 40)

[0309] Preparation Example 2: Design and preparation of anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1

[0310] The heavy and light chain amino acid sequences of the anti-PD-1 antibody 14C12 and its humanized antibody 14C12H1L1, as well as the encoding nucleic acid sequences, are completely identical to 14C12 and 14C12H1L1 in Chinese Patent Publication CN 106967172A (or CN 106977602A).

[0311] (1) 14C12 heavy chain variable region sequence and light chain variable region sequence

[0312] Nucleic acid sequence of the variable region of the 14C12 heavy chain: (354 bp)

[0313] GAGGTCAAACTGGTGGAGAGCGGCGGCGGGCTGGTGAAGCCCGGCGGGTCACTGAAACTGAGCTGCGCCGCTTCCGGCTTCGCCTTTAGCTCCTACGACATGTCATGGGTGAGGCAGACCCCTGAGAAGCGCCTGGAATGGGTCGCTACTATCAGCGGAGGCGGGCGATACACCTACTATCCTGACTCTGTCAAAGGGAGATTCACAATTAGTCGGGATAACGCCAGAAATACTCTGTATCTGCAGATGTCTAGTCTGCGGTCCGAGGATACAGCTCTGTACTATTGTGCAAACCGGTACGGCGAAGCATGGTTTGCCTATTGGGGACAGGGCACCCTGGTGACAGTCTCTGCC (SEQ ID NO: 14)

[0314] Amino acid sequence of the heavy chain variable region of 14C12: (118 aa)

[0315] EVKLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQTPEKRLEWVATISGGGRYTYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYCANRYGEAWFAYWGQGTLVTVSA (SEQ ID NO: 15)

[0316] Nucleic acid sequence of the light chain variable region of 14C12: (321 bp)

[0317] GACATTAAGATGACACAGTCCCCTTCCTCAATGTACGCTAGCCTGGGCGAGCGAGTGACCTTCACATGCAAAGCATCCCAGGACATCAACACATACCTGTCTTGGTTTCAGCAGAAGCCAGGCAAAAGCCCCAAGACCCTGATCTACCGGGCCAATAGACTGGTGGACGGGGTCCCCAGCAGATTCTCCGGATCTGGCAGTGGGCAGGATTACTCCCTGACCATCAGCTCCCTGGAGTATGAAGACATGGGCATCTACTATTGCCTGCAGTATGATGAGTTCCCTCTGACCTTTGGAGCAGGCACAAAACTGGAACTGAAG (SEQ ID NO: 16)

[0318] The amino acid sequence of the variable region of the light chain at 14C12 is: (107 aa)

[0319] DIKMTQSPSSMYASLGERVTFTCKASQDINTYLSWFQQKPGKSPKTLIYRANRLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPLTFGAGTKLELK (SEQ ID NO: 17)

[0320] (2) Heavy chain variable region sequence and light chain variable region sequence, heavy chain sequence and light chain sequence of humanized monoclonal antibody 14C12H1L1

[0321] The nucleic acid sequence of the heavy chain variable region 14C12H1v of 14C12H1L1: (354 bp)

[0322] GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTCACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACATGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACTATCTCAGGAGGCGGGAGATACACCTAC TATCCTGACAGCGTCAAGGCCCGGTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGAACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTACGGGGAAGCATGGTTTTGCCTATTGGGGGGCAGGGAACCCTGGTGACAGTCTCTAGT (SEQ ID NO: 18)

[0323] The amino acid sequence of the heavy chain variable region 14C12H1v of 14C12H1L1: (118 aa)

[0324] EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSS (SEQ ID NO: 19)

[0325] The nucleic acid sequence of the light chain variable region 14C12L1v of 14C12H1L1: (321 bp)

[0326] GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGACAGGGTCACCTTCACATGCCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGGGCTAATAGACTG GTGTTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCCGGGACAAAACTGGAGCTGAAG (SEQ ID NO: 20)

[0327] The amino acid sequence of the light chain variable region 14C12L1v of 14C12H1L1: (107 aa)

[0328] DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELK (SEQ ID NO: 21)

[0329] The nucleic acid sequence of the heavy chain 14C12H1L1: (1344 bp)

[0330]

[0331] Amino acid sequence of the heavy chain 14C12H1 of 14C12H1L1: (448 aa)

[0332] EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 23)

[0333] Nucleic acid sequence of the light chain 14C12L1 of 14C12H1L1: (642 bp)

[0334] GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGACAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGAGCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGGGCTAATAGACTGGTGTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAGCGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGGCAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCCGGGACAAAACTGGAGCTGAAGCGAACTGTGGCCGCTCCCTCCGTCTTCATTTTTCCCCCTTCTGACGAACAGCTGAAATCAGGCACAGCCAGCGTGGTCTGTCTGCTGAACAATTTCTACCCTAGAGAGGCAAAAGTGCAGTGGAAGGTCGATAACGCCCTGCAGTCCGGCAACAGCCAGGAGAGTGTGACTGAACAGGACTCAAAAGATAGCACCTATTCCCTGTCTAGTACACTGACTCTGTCCAAGGCTGATTACGAGAAGCACAAAGTGTATGCATGCGAAGTGACACATCAGGGACTGTCAAGCCCCGTGACTAAGTCTTTTAACCGGGGCGAATGT (SEQ ID NO: 24)

[0335] Amino acid sequence of the light chain 14C12L1 of 14C12H1L1: (214 aa)

[0336] DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 25)

[0337] Antibodies 14C12 and 14C12H1L1 have the same CDR, as follows (according to the IMGT numbering system):

[0338] HCDR1: GFAFSSYD (SEQ ID NO: 26)

[0339] HCDR2: ISGGGRYT (SEQ ID NO: 27)

[0340] HCDR3: ANRYGEAWFAY (SEQ ID NO: 28)

[0341] LCDR1: QDINTY (SEQ ID NO: 29)

[0342] LCDR2: RAN (SEQ ID NO: 30)

[0343] LCDR3: LQYDEFPLT (SEQ ID NO: 31)

[0344] Heavy chain variable region sequence and light chain variable region sequence of 14C12H1L1(M)

[0345] Based on 14C12H1L1, individual amino acids in its backbone region (light chain) were mutated to obtain 14C12H1L1(M).

[0346] The heavy chain variable region 14C12H1(M) of 14C12H1L1(M):

[0347] 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.

[0348] The light chain variable region 14C12L1(M) of 14C12H1L1(M):

[0349] DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKR (SEQ ID NO: 38)

[0350] Preparation Example 3: Design of Humanized Antibody 14C12H1L1 (hG4DM)

[0351] Based on 14C12H1L1, the inventors retained the variable region of the antibody and used the Ig gamma-4 chain 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.

[0352] 14C12H1L1 (hG4DM) heavy chain amino acid sequence

[0353] EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGT LVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQID NO: 32)

[0354] 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.

[0355] Preparation Example 4: Sequence Design of Humanized Antibody 14C12H1L1 (hG1TM)

[0356] 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).

[0357] The nucleic acid sequence of the heavy chain 14C12H1(hG1TM) of 14C12H1(hG1TM): (1344 bp)

[0358]

[0359] The amino acid sequence of the heavy chain 14C12H1(hG1TM) of 14C12H1(hG1TM) is: (448 aa)

[0360] EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 34)

[0361] The nucleic acid sequence of the light chain of 14C12H1L1(hG1TM) is the same as that of SEQ ID NO: 24.

[0362] 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.

[0363] Preparation Example 5: Design and Preparation of Anti-LAG3 / PD-1 Bifunctional Antibody

[0364] 1. Sequence Design

[0365] The bifunctional antibodies BS-PL021A, Bs-PL022B, BS-PL023C, and Bs-PLV02 in this invention have a Morrison pattern (IgG-scFv), meaning that the scFv fragment of another antibody is attached to the C-terminus of both heavy chains of an IgG antibody.

[0366] The main components of its heavy and light chains are designed as shown in Table 1 below.

[0367] Table 1: Compositional Design of Heavy and Light Chains in Bifunctional Antibodies

[0368]

[0369] In Table 1 above:

[0370] (1) Linker fragment (GGGGS) 3 amino acid sequence:

[0371] GGGGSGGGGSGGGGS (SEQ ID NO: 35)

[0372] The 4-amino acid sequence of the linker fragment (GGGGS):

[0373] GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 36)

[0374] Linkage fragment (GGGGS) 4G amino acid sequence:

[0375] GGGGSGGGGSGGGGSGGGGSG (SEQ ID NO: 37)

[0376] (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 not marked with "v" are the full length of the corresponding heavy chain or light chain, including the constant region. The amino acid sequences of these variable regions or full lengths and their encoding nucleic acid sequences are all based on the corresponding sequences described in the preparation examples above.

[0377] 2. Antibody expression and purification

[0378] 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), yielding pUC57simple-Bs-PL021AH / pUC57simple-Bs-PL021AL, pUC57simple-Bs-PL022BH / pUC57simple-Bs-PL022BL, pUC57simple-Bs-PL023CH / pUC57simple-Bs-PL023CL, and pUC57simple-Bs-PLV02H / pUC57simple-Bs-PLV02L, respectively.

[0379] pUC57simple-Bi-PGV02 / pUC57simple-Bi-PGV02 plasmid.

[0380] Plasmids pUC57simple-Bs-PL021AH / pUC57simple-Bs-PL021AL, pUC57simple-Bs-PL022BH / pUC57simple-Bs-PL022BL, pUC57simple-Bs-PL023CH / pUC57simple-Bs-PL023CL, and pUC57simple-Bs-PLV02H / pUC57simple-Bs-PLV02L were respectively added to the plasmids.

[0381] The pUC57simple-Bi-PGV02 / pUC57simple-Bi-PGV02 cells were digested with HindIII and EcoRI. 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 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.

[0382] Preparation Example 6: Preparation of fusion proteins PD-1-mFc, PD-1-hFc and PDL1-hFc

[0383] The preparation of fusion proteins PD-1-mFc, PD-1-hFc and PDL1-hFc and their SDS-PAGE electrophoresis detection were carried out entirely according to Preparation Example 1 of Chinese Patent Publication CN106632674A.

[0384] In this preparation example, the amino acid sequences and their encoding nucleic acid sequences of the fusion proteins PD-1-mFc, PD-1-hFc and PDL1-hFc are the same as those of PD-1-mFc, PD-1-hFc and PDL1-hFc in Preparation Example 1 of Chinese Patent Publication CN106632674A.

[0385] The fusion proteins PD-1-mFc, PD-1-hFc and PDL1-hFc were prepared.

[0386] Preparation Example 7: Preparation of Human Anti-Egg Lysosomal Antibody

[0387] Human anti-Hen Egg Lysozyme IgG (anti-HEL, i.e., human IgG, abbreviated as hIgG) has a sequence derived from the variable region of the FabF10.6.6 sequence in Acierno et al.'s study on Affinity maturation increases thestability and plasticity of the Fv domain of anti-protein antibodies (Acierno et al. J Mol Biol. 2007; 374(1): 130-46.). The preparation method is as follows:

[0388] For human IgG, Nanjing GenScript Biotech was commissioned to perform codon optimization and gene synthesis of the heavy and light chain (full sequence or variable region) genes of the antibody. Following standard techniques described in *Molecular Cloning: A Laboratory Manual (3rd Edition)*, standard molecular cloning techniques including PCR, enzyme digestion, DNA gel recovery, ligation transformation, and colony PCR or enzyme digestion identification were used to subclone the heavy and light chain genes into antibody heavy chain expression vectors and antibody light chain expression vectors in mammalian expression systems, respectively. Further sequencing analysis of the heavy and light chain genes in the recombinant expression vectors was performed. After sequencing verification, endotoxin-free expression plasmids were prepared in large quantities and transiently co-transfected into HEK293 cells for recombinant antibody expression. After 7 days of culture, cell culture medium was collected and purified using rProtein A column (GE). The harvested antibody samples were quality-assessed using standard analytical techniques such as SDS-PAGE and SEC-HPLC.

[0389] Experimental Example 1: ELISA method for determining the binding activity of anti-LAG3-anti-PD-1 bispecific antibody to antigen.

[0390] 1. The binding activity of BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, 14C12H1L1 (hG1TM) to antigen PD-1-mFc was determined by indirect ELISA. The specific method is as follows:

[0391] Human PD-1-mFc, 0.5 μg / mL, was coated onto an ELISA plate and incubated overnight at 4°C. The plate was then washed once with PBST, followed by blocking with 1% BSA in PBS at 37°C for 2 hours. After blocking, the plate was washed three times with PBST. Serially diluted antibodies (see Table 2 for antibody dilution gradients) were added, and the plate was incubated at 37°C for 30 minutes. After incubation, the plate was washed three times with PBST. Following washing, a 1:5000 dilution of HRP-labeled goat anti-human IgG FC (H+L) (Jackson, catalog number: 109-035-098) secondary antibody was added, and the plate was incubated at 37°C for 30 minutes. After incubation, the plate was washed four times with PBST, followed by TMB (Neogen, 308177) for 5 minutes in the dark. The reaction was then terminated with stop solution. Immediately place the microplate into the microplate reader and select a 450nm wavelength to read the OD values ​​of each well. Analyze the data using SoftMax Pro 6.2.1 software.

[0392] The test results are shown in Table 2 and Figure 1 As shown.

[0393] Table 2: ELISA detection of the binding of BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, 14C12H1L1 (hG1TM) to human PD-1-mFc

[0394]

[0395] Depend on Figure 1 It was found that BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, and 14C12H1L1 (hG1TM) could effectively bind to the antigen human PD-1-mFc, and the binding efficiency was dose-dependent. Quantitative absorbance analysis of the bound antibodies was performed, and curve simulation calculations were used to obtain the EC50 binding efficiency of antibodies BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, and 14C12H1L1 (hG1TM) (as a control). 50 The values ​​are 0.066 nM, 0.074 nM, 0.046 nM, 0.103 nM, and 0.02 nM, respectively.

[0396] The results showed that, under the same experimental conditions, the PD-1-mFc binding activities of BS-PL021A, BS-PL022B, and BS-PL023C were basically equivalent to those of the positive control 14C12H1L1 (hG1TM) at the same target site, suggesting that BS-PL021A, BS-PL022B, BS-PL023C, and Bs-PLV02 have effective PD-1-mFc binding activities.

[0397] 2. The binding activity of BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, Relatlimab, H7L8 (hG1WT) to antigen LAG3-mFc was determined by indirect ELISA.

[0398] The specific method is as follows:

[0399] Human LAG3-mFc (Kangfang Biotechnology, batch number: 20200417), 2 μg / mL, was coated onto an ELISA plate and incubated overnight at 4°C. The plate was then washed once with PBST, followed by blocking with 1% BSA in PBS solution at 37°C for 2 hours. After blocking, the plate was washed three times with PBST. Serially diluted antibodies (see Table 3 for antibody dilution gradient) were added, and the plate was incubated at 37°C for 30 minutes. After incubation, the plate was washed three times with PBST. Finally, HRP-labeled goat anti-human IgG FC (H+L) (Jackson, catalog number: 109-035-098) secondary antibody working solution diluted 1:5000 was added, and the plate was incubated at 37°C for 30 minutes. After incubation, wash the plate four times with PBST, then add TMB (Neogen, 308177) and incubate in the dark for 5 minutes. Stop the reaction by adding stop solution. Immediately place the plate in a microplate reader and read the OD values ​​of each well at 450 nm. Analyze the data using SoftMax Pro 6.2.1 software.

[0400] The test results are shown in Table 3 and Figure 2 As shown.

[0401] Table 3: ELISA detection of the binding of BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, Relatlimab, H7L8(hG1WT) to LAG3-mFc

[0402]

[0403] Depend on Figure 2It was found that BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, Relatlimab, and H7L8(hG1WT) could effectively bind to the human LAG3-mFc antigen, and the binding efficiency was dose-dependent. The absorbance intensities at each dose are shown in Table 3. Quantitative analysis of the bound antibodies was performed, and curve simulation calculations were used to obtain the EC50 binding efficiencies of antibodies BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, Relatlimab (as a positive control), and H7L8(hG1WT) (as a control). 50 The values ​​are 0.073 nM, 0.081 nM, 0.377 nM, 0.685 nM, 0.106 nM, and 0.045 nM, respectively.

[0404] The above experimental results indicate that, under the same experimental conditions, BS-PL021A, BS-PL022B, and H7L8(hG1WT) possess effective binding activity against LAG3-mFc, and the binding activity of BS-PL021A, BS-PL022B, and H7L8(hG1WT) against human LAG3-mFc is stronger than that of the target-positive drug Relatlimab. In particular, H7L8(hG1WT) exhibits significantly stronger binding activity against human LAG3-mFc than the target-positive drug Relatlimab.

[0405] Experimental Example 2: Competitive ELISA method was used to determine the activity of anti-LAG3-anti-PD-1 bispecific antibody in competitively binding to human PD-1-mFc-Biotin with human PDL1-mFc.

[0406] Human PD-L1-mFc (PD-L1 Genbank ID: NP_054862.1, mFc SEQ ID NO:) was coated onto an ELISA plate at a concentration of 2 μg / mL and incubated overnight at 4°C. After incubation, the plate was blocked with 1% BSA in PBS at 37°C for 2 hours. After blocking, the plate was washed three times and patted dry. The antibody was serially diluted to seven concentrations using a 3-fold dilution starting at 80 nM (final concentration 40 nM) on a dilution plate, with a blank control included. An equal volume of 1.2 μg / mL (final concentration 0.6 μg / mL) of human PD-1-mFc-Biotin solution was added, and the mixture was incubated at room temperature for 10 minutes. The resulting mixture was then added to the coated ELISA plate and incubated at 37°C for 30 minutes. After incubation, wash the plate three times with PBST and blot dry. Add SA-HRP (KPL, 14-30-00) working solution and incubate at 37°C for 30 minutes. After incubation, wash the plate four times and blot dry. Then add TMB (Neogen, 308177) and develop the color in the dark for 5 minutes. Add stop solution to terminate the color development reaction. Immediately place the microplate in a microplate reader and read the OD values ​​of each well at 450 nm. Analyze the data using SoftMax Pro 6.2.1 software.

[0407] Test results as follows Figure 3 As shown in Table 4, the OD values ​​for each dose are listed below. By quantitatively analyzing the absorbance of the bound antibody, the competitive binding EC20 was obtained by simulating the antibody's ability to block the binding of human PD-1-mFc-Biotin to its ligand human PDL1-mFc. 50 (Table 4).

[0408] Table 4: Activity assay results of BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, 14C12H1L1 (hG1TM) competing with human PD-1-mFc for binding to human PD-1-mFc-Biotin.

[0409]

[0410] The results showed that BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, and 14C12H1L1 (hG1TM) (as a control) effectively blocked the binding of human PD-1-mFc-Biotin to its ligand human PDL1-mFc, and the blocking efficiency showed a dose-dependent relationship. BS-PL021A, BS-PL022B, BS-PL023C, Bs-PLV02, and 14C12H1L1 (hG1TM) blocked the EC50 of human PD-1-mFc-Biotin binding to its ligand human PDL1-mFc. 50 The concentrations were 3.031 nM, 3.462 nM, 2.982 nM, 5.045 nM, and 2.606 nM, respectively. The efficiency of BS-PL021A, BS-PL022B, and BS-PL023C in blocking the binding of human PD-1-mFc-Biotin to its ligand human PDL1-mFc was basically equivalent to that of 14C12H1L1 (hG1TM).

[0411] Experiment Example 3: FACS detection of binding activity of anti-LAG3-anti-PD-1 bispecific antibody

[0412] 1. FACS detection of the binding activity of anti-LAG3-anti-PD-1 bispecific antibody to PD-1 on the surface of 293T-PD1 membrane.

[0413] Collect 293T-PD1 cells in logarithmic growth phase, at a ratio of 3 × 10⁻⁶. 5 Transfer cells per well to a 96-well plate with a conical bottom. Add 100 μL of 1% PBSA, centrifuge at 350×g for 5 min, and discard the supernatant. Add 100 μL of antibody diluted with PBSA (final concentrations of 300, 100, 33.3, 11.1, 3.7, 1.23, 0.123, and 0.0123 nM), mix gently, and incubate on ice for 1 h. Add 100 μL of 1% PBSA, centrifuge at 350×g for 5 min, discard the supernatant, and wash twice with 200 μL of 1% PBSA. Resuspend the cells in 400-fold diluted FITC-labeled goat anti-human IgG secondary antibody (Jackson, catalog number: 109-095-098), mix well, and incubate on ice in the dark for 0.5 h. Add 100 μL of 1% PBSA, centrifuge at 350 x g for 5 min, discard the supernatant, and wash twice with 200 μL of 1% PBSA. Resuspend the cell pellet in 400 μL of 1% PBSA and transfer to a flow cytometry tube for FACSCalibur analysis.

[0414] The experimental results are shown in Table 5 and Figure 4As shown, 14C12H1L1 (hG1TM), BS-PL021A, BS-PL022B, Bs-PLV02, Bi-PGV02, Nivolumab, and Pembrolizumab can all specifically bind to the PD-1 receptor on the surface of the 293T-PD1 cell membrane.

[0415] Table 5: FACS detection of PD-1 binding activity on the surface of 14C12H1L1 (hG1TM), BS-PL021A, BS-PL022B, Bs-PLV02, Bi-PGV02, Nivolumab, and Pembrolizumab 293T-PD1 cells.

[0416]

[0417] Under the same experimental conditions, EC2 binding of 14C12H1L1 (hG1TM), BS-PL021A, BS-PL022B, Bs-PLV02, Bi-PGV02, Nivolumab, Pembrolizumab and 293T-PD1 cells was observed. 50 The concentrations were 5.351 nM, 6.851 nM, 6.066 nM, 6.866 nM, 7.206 nM, 3.073 nM, and 3.970 nM, respectively. These experimental results indicate that, under the same experimental conditions, the binding activity of 14C12H1L1 (hG1TM), BS-PL021A, BS-PL022B, Bs-PLV02, and Bi-PGV02 to 293T-PD1 cells was comparable to that of the control antibodies Nivolumab and Pembrolizumab, suggesting that 14C12H1L1 (hG1TM), BS-PL021A, BS-PL022B, Bs-PLV02, and Bi-PGV02 effectively bind to PD-1 on the cell membrane surface of 293T-PD1 cells.

[0418] 2. FACS detection of the binding activity of anti-LAG3-anti-PD-1 bispecific antibody to LAG3 on the surface of 293T-LAG3 cell membrane.

[0419] Log-phase 293T-LAG3 cells were routinely digested with trypsin at a concentration of 3 × 10⁻⁶. 5Transfer cells per well to a 96-well plate with a conical bottom. Add 100 μL of 1% PBSA, centrifuge at 350×g for 5 min, and discard the supernatant. Add 100 μL of antibody diluted with 1% PBSA (final concentrations of 300, 100, 33.3, 11.1, 3.7, 1.23, 0.123, 0.0123, and 0.00123 nM), mix well, and incubate on ice for 1 h. Add 100 μL of 1% PBSA, centrifuge at 350×g for 5 min, discard the supernatant, and wash twice with 200 μL of 1% PBSA. Resuspend the cells in 300-fold diluted FITC-labeled goat anti-human IgG secondary antibody (Jackson, catalog number: 109-095-098), mix well, and incubate on ice in the dark for 0.5 h. Add 500 μL PBSA, centrifuge at 350 × g for 5 min, discard the supernatant, and wash twice with 200 μL 1% PBSA. Resuspend the cell pellet in 300 μL 1% PBSA and transfer to a flow cytometer for FACSCalibur analysis.

[0420] The experimental results are shown in Table 6 and Figure 5 As shown.

[0421] Table 6: FACS detection of the binding activity of BS-PL022B, Relatlimab to LAG33 on the surface of 293T-LAG3 cells.

[0422]

[0423] The results showed that, under the same experimental conditions, BS-PL022B, Relatlimab, and EC2 cells bound to LAG3 on the surface of 293T-LAG3 cells... 50 The concentrations were 3.213 nM and 4.113 nM, respectively, indicating that BS-PL022B had a higher binding activity to LAG3 on the cell membrane surface of 293T-LAG3 than Relatlimab.

[0424] The above experimental results indicate that both BS-PL022B and the same target positive drug Relatlimab can bind specifically to LAG-3 on the surface of 293T-LAG3 cells in a dose-dependent manner. This suggests that BS-PL022B has effective binding activity to LAG3 on the 293T-LAG3 cell membrane, and its binding ability is stronger than that of Relatlimab.

[0425] Experimental Example 4: Competitive binding of anti-LAG3-anti-PD-1 bispecific antibodies to cell membrane surface antigens

[0426] 1. Competitive flow cytometry was used to determine the binding of anti-LAG3-anti-PD-1 bispecific antibody to cell membrane surface antigen PD-1 in competition with PDL1.

[0427] 293T-PD1 cells were routinely digested and processed at a ratio of 3×10⁻⁶. 5 Transfer cells per well to a conical 96-well plate and wash with 100 μL of 1% PBSA. Add serially diluted antibodies (final concentrations of 300, 100, 33.3, 11.1, 3.7, 1.23, 0.123, and 0.0123 nM) at 100 μL per sample and incubate on ice for 30 min. Add 100 μL of PDL-1-mFc to each tube, mix well to a final concentration of 20 nM, and incubate on ice for 1 h. Centrifuge at 350 × g for 5 min, discard the supernatant, and wash twice with 200 μL of 1% PBSA. Add 100 μL of 400-fold diluted FITC goat anti-mouse IgG / IgM antibody (BD, catalog number: 555988), or 100 μL of 1% PBSA for blank samples, mix well, and incubate on ice in the dark for 30 min. After washing and centrifugation, the sample is resuspended, transferred to a flow cytometer tube, and tested.

[0428] The results are as follows Figure 6 As shown, the EC values ​​of each sample 50 The values ​​are shown in Table 7. The competitive binding ECGs of antibodies Nivolumab, Pembrolizumab, 14C12H1L1 (hG1TM), and BS-PL022B were calculated using quantitative fluorescence analysis and curve fitting. 50 The values ​​are 3.608 nM, 2.769 nM, 2.511 nM, and 5.123 nM, respectively.

[0429] Table 7: Fluorescence intensity analysis of Nivolumab, Pembrolizumab, 14C12H1L1 (hG1TM), and BS-PL022B competitively binding to 293T-PD-1 surface antigen using FACS

[0430]

[0431] The results showed that the BS-PL022B antibody effectively blocked the binding of PD-L1 to PD-1 on the surface of 293T-PD1 host cells in a dose-dependent manner.

[0432] 2. Competitive flow cytometry was used to determine the binding of anti-LAG3-anti-PD-1 bispecific antibody to cell membrane surface antigen MHC II by LAG3-mG1Fc.

[0433] Dilute the antibody and LAG3-mG1Fc according to the experimental design, mix them 1:1 to achieve a final concentration of 3 nM for LAG3-mG1Fc (manufactured by Kangfang, batch number 20190508), and final antibody concentrations of 300, 100, 33.3, 11.1, 3.7, 1.23, 0.123, 0.0123, and 0.00123 nM. Incubate on ice for 30 min. Collect Raji cells (Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, catalog number: THu 44), seed 300,000 cells per sample into 96-well conical plates, add 1% PBSA, centrifuge at 500xg for 5 min and discard the supernatant. Resuspend the cells in 100 μL of antibody and protein pre-incubation buffer. Design blank control (cells + PBSA + PBSA), negative control (cells + PBSA + secondary antibody), and isotype control. Incubate on ice in the dark for 1 h. Add 100 μL of... Centrifuge at 500xg for 5 min with 1% PBSA and discard the supernatant; resuspend cells in 200 μL of 1% PBSA, centrifuge at 500xg for 5 min, discard the supernatant, and wash once more; resuspend cells in 100 μL of APC goat antimouse IgG secondary antibody (BioLegend, catalog number: 405308) (1:300 dilution), or resuspend the blank control in 100 μL of 1% PBSA and incubate on ice in the dark for 30 min; add 100 μL of 1% PBSA, centrifuge at 500xg for 5 min, and discard the supernatant; resuspend cells in 200 μL of 1% PBSA, centrifuge at 500xg for 5 min, discard the supernatant, and wash once more; resuspend cells in 200 μL of 1% PBSA, transfer to flow cytometry tubes, and perform the assay.

[0434] The results are as follows Figure 7 Table 8 shows the EC values ​​of each sample. 50 The values ​​are shown in the table. The competitive binding ECGs of antibodies Relatlimab and BS-PL022B were calculated using quantitative fluorescence analysis and curve fitting. 50 The values ​​are 0.9689 nM and 1.306 nM, respectively.

[0435] Table 8: Fluorescence intensity analysis of Relalimab and BS-PL022B competing for binding to Raji cell surface antigen by FACS

[0436]

[0437] The results showed that the BS-PL022B antibody effectively blocked the binding of LAG-3 to MHC II on the surface of Raji host cells in a dose-dependent manner.

[0438] Experimental Example 5: Anti-LAG3-Anti-PD-1 Bispecific Antibody Blockade Experiment

[0439] 1. Anti-LAG3-anti-PD-1 bispecific antibody blocks the binding of LAG3 to MHCII.

[0440] Jurkat-NFAT-PD1-LAG3 cells (constructed by Kangfang Biotechnology, P9, viability: 97.75%) and Raji cells (Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, catalog number: THu 44) were collected, centrifuged at 110×g for 5 min, the supernatant was discarded, and the cells were resuspended in 1640 medium (containing 10% FBS) and counted. Jurkat-NFAT-PD1-LAG3 cells were then divided into 10×10⁻⁶ cells per cell line. 4 25 μL / well was seeded into a 96-well black-background plate (Corning, model: 3916). Antibodies were added according to the experimental design, 30 μL / well (final concentrations of 900 nM, 300 nM, 100 nM, 33.3 nM, 3.3 nM, 0.3 nM, 0.03 nM, and 0.003 nM), and pre-incubated at 37°C in a 5% CO2 incubator for 30 min. SEE (Staphylococcal enterotoxin E) (final concentration 0.05 ng / mL, Toxin Technology, catalog number: ET404) and Raji cells were incubated at 37°C in a 5% CO2 incubator for 30 min. After 30 min of incubation, Raji cells were seeded at 2 × 10⁶ cells / well. 4 Add 25 μL / well to each well of a 96-well plate, bringing the final volume to 80 μL. Mix well and incubate at 37°C in a 5% CO2 incubator for 16 hours. Remove the plate, allow it to equilibrate to room temperature, and add Bright-Glo... TM Luciferase Assay System (Promega, catalog number: E2650) 80 μL / well, incubate in the dark for 2 min and then read the RLU value.

[0441] The results are as follows Figure 8A , Figure 8B As shown in Table 9.

[0442] Table 9: Anti-LAG3-anti-PD-1 bispecific antibodies block the binding of LAG3 to MHCII

[0443]

[0444] The results showed that Bs-PLV02, Bi-PGV02, BS-PL022B, and Relatlimab blocked the binding of LAG3 to MHCII in ECG. 50The nM values ​​were 1.21 nM, 1.483 nM, 0.9762 nM, and 8.563 nM, respectively. Bs-PLV02, Bi-PGV02, and BS-PL022B showed stronger ability to block the binding of LAG3 to MHCII than the positive control antibody Relatlimab.

[0445] 2. Anti-LAG3-anti-PD-1 bispecific antibody blocks the binding of PD-1 and PD-L1.

[0446] PDL1 aAPC / CHO-K1 cells (Promega, catalog number: J1081A) 4×10 4 100 μL of cells / well was seeded overnight in a 96-well flat-bottom black plate (Corning, model: 3916) and cultured. The culture medium was Ham F-12 + 10% FBS. The next day, the culture medium was removed from the plate, and 5 × 10⁶ PD1 effector cells (Promega, catalog number: J1121A) were added. 4 Cells / well, 40 μL / well (medium: 1640 + 10% FBS); Add antibody, 40 μL / well (final concentrations: 1000 nM, 300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.23 nM, 0.123 nM and 0.0123 nM), and simultaneously set up isotype control and negative control groups, with a final volume of 80 μL / well; Incubate in an incubator for 6 h. Remove the culture plate, equilibrate to room temperature, add 80 μL / well of Bright-Glo™ Luciferase Assay System (Promega, catalog number: E2650), incubate in the dark for 2 min, and then read the RLU value.

[0447] The results are as follows Figure 9A , Figure 9B As shown in Table 10.

[0448] Table 10: Anti-LAG3-anti-PD-1 bispecific antibodies block the binding of PD-1 and PD-L1

[0449]

[0450] The results showed that nivolumab, pembrolizumab, 14C12H1L1 (hG1TM), and BS-PL022B blocked ECGs that bind to PD-1 and PD-L1. 50The nM values ​​were 4.089 nM, 1.281 nM, 5.219 nM, and 20.01 nM, respectively. The results indicate that nivolumab, pembrolizumab, 14C12H1L1 (hG1TM), and BS-PL022B can all block the binding of PD-1 and PD-L1.

[0451] 3. The anti-LAG3-anti-PD-1 bispecific antibody simultaneously blocks the binding of LAG-3 to MHCII and PD-1 to PD-L1.

[0452] Jurkat-NFAT-PD1-LAG3 and Raji-PDL1 cells were collected, centrifuged at 110×g for 5 min, the supernatant was removed, and the cells were resuspended in 1640 medium (containing 10% FBS) and counted.

[0453] Jurkat-NFAT-PD1-LAG3 cells 10×10 4 Raji-PDL1 cells were seeded into 96-well black-background plates (Corning, model: 3916) at 25 μL / well. Antibody was added according to the experimental design at 30 μL / well (final concentrations of 3000, 1000, 300, 30, 3, 0.3, 0.03, and 0.003 nM). The plates were pre-incubated at 37°C in a 5% CO2 incubator for 30 min. SEE (Staphylococcal enterotoxin E) (final concentration 0.1 ng / mL) was added to the Raji-PDL1 cells, and the plates were incubated at 37°C in a 5% CO2 incubator for 30 min. After 30 min of incubation, the Raji-PDL1 cells were seeded at a density of 3 × 10⁻⁶ cells / well. 4 Add 25 μL / well to each well of a 96-well plate, bringing the final volume to 80 μL. Mix well and incubate at 37°C in a 5% CO2 incubator for 15 hours. Remove the plate, allow it to equilibrate to room temperature, and add Bright-Glo... TM Luciferase Assay System (Promega, catalog number: E2650) 80 μL / well, incubate in the dark for 5 min and then read the RLU value.

[0454] The results are as follows Figure 10A , Figure 10B As shown in Table 11.

[0455] Table 11: Anti-LAG3-anti-PD-1 bispecific antibodies simultaneously block the binding of LAG-3 to MHCII and PD-1 to PD-L1.

[0456]

[0457] The results showed that Pembrolizumab, Relatlimab, 14C12H1L1 (hG1TM), 14C12H1L1 (hG1TM) + Relatlimab, and BS-PL022B simultaneously blocked ECGs that inhibited PD1 and PD-L1 binding as well as LAG3 binding to MHCII. 50 The nM values ​​were 24.01 nM, 5.525 nM, 44.86 nM, 29.75 nM, and 16.21 nM, respectively. Pembrolizumab, Relatlimab, 14C12H1L1 (hG1TM), 14C12H1L1 (hG1TM) + Relatlimab, and Bs-PL022B could all simultaneously block the binding of PD-1 and PD-L1 as well as the binding of LAG3 to MHCII, and the blocking ability of Bs-PL022B was stronger than that of other antibodies.

[0458] Experiment Example 6: Anti-LAG3-anti-PD-1 Bispecific Antibody Bridging Assay

[0459] CHO-K1 (Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, catalog number: 3111C0001CCC000004), CHO-K1-PD1 (constructed by Kangfang Biotechnology), and CHO-K1-LAG3 (constructed by Kangfang Biotechnology) cells were routinely digested, centrifuged at 170×g for 5 min, and the supernatant was discarded. Cells were resuspended in complete culture medium, and cell counts and viability were determined. CHO-K1-PD1 cells were stained with CFSE (CFSE Cell Division Tracker Kit, Biolegend, catalog number: 423801) at a concentration of 1 μM, 1 mL / 10×10⁻⁶. 6 CHO-K1-LAG3 cells stained with Far Red (Thermofisher, catalog number: C34564) (treatment concentration 0.3 μM, 1 mL / 10 × 10⁻⁶ cells). 6 CHO-K1 cells were stained with Far Red or CFSE and incubated for 20 min. Staining was terminated with complete culture medium, centrifuged at 170×g for 5 min, the supernatant was discarded, and the cells were incubated again with complete culture medium for 10 min. The cells were then centrifuged at 170×g for 5 min, the supernatant was discarded, and the washing was repeated once. The cells were resuspended in complete culture medium and counted. The stained CHO-K1-PD1, CHO-K1-LAG3, and CHO-K1 cells were then divided into groups of 1.5×10⁻⁶ cells. 5Transfer cells / well to a conical 96-well plate, add buffer (PBS + 1% human serum) (human serum, Zhongke Chenyu, catalog number: 168014-100mL), centrifuge and discard the supernatant; add antibody to CHO-K1-PD1 according to the experimental design (final concentrations of 30, 3, 1, 0.3, 0.1 nM), add buffer or antibody to CHO-K1-LAG3 (final concentrations of 30 nM, 3 nM, 1 nM, 0.3 nM, 0.1 nM), add buffer to CHO-K1, 100 μL / well, incubate on ice for 60 min.

[0460] Add 100 μL buffer, centrifuge at 350 × g for 5 min, discard the supernatant, and wash twice with 200 μL buffer. Resuspend CHO-K1-LAG3 and CHO-K1 cells in 100 μL buffer respectively, and transfer to the corresponding CHO-K1-PD1 sample wells, mix well, and centrifuge at 1.5 × 10⁻⁶. 5 Incubate cells / well on ice in the dark for 40 min. Resuspend cells in 200 μL buffer, transfer to flow cytometry tubes, and perform analysis.

[0461] The results are as follows Figure 11 As shown. Compared with the isotype control, the anti-LAG3-anti-PD-1 bispecific antibody can bind to both CHO-K1-PD1 and CHO-K1-LAG3 cells simultaneously, bridging the two cells together, while 14C12H1L1 (hG1TM) and Relatlimab, even when used in combination, do not have this effect.

[0462] Experiment Example 7: MLR Detection of the Biological Activity of Anti-LAG3-Anti-PD-1 Bispecific Antibody in Promoting IFN-γ and IL-2 Secretion

[0463] 1. Detection of the bioactivity of anti-LAG3-anti-PD-1 bispecific antibody in promoting IFN-γ secretion in the Raji-PDL1 mixed lymphocyte reaction system.

[0464] Raji-PDL1 cells were routinely passaged and cultured; PBMCs were revived and cultured in 10 mL of 1640 complete medium, and stimulated for two days with 0.5 μg / mL SEB (Staphylococcal enterotoxin B) (Denotec, catalog number: S010201). Raji-PDL1 cells were treated with MMC (Stressmarq, catalog number: SIH-246-10MG) to a final concentration of 2 μg / mL and incubated at 37°C in a 5% CO2 incubator for 1 hour; PBMCs stimulated with SEB for 2 days and Raji-PDL1 cells treated with MMC for 1 hour were collected, washed twice with PBS; after resuspending in complete medium, cells were counted and cultured at 10 × 10⁻⁶ cells each. 4Cells / well were added to a U-shaped 96-well plate (Corning, model: 3799) for co-culture. Antibodies were added according to the experimental design (final concentrations of 300 nM, 30 nM, and 3 nM), and the cells were co-cultured in an incubator for 3 days. After 3 days, the cells were centrifuged at 1200 rpm for 5 min, and the cell culture supernatant was collected for IFN-γ detection by ELISA.

[0465] like Figure 12A As shown, co-culture of human PBMCs and Raji-PDL1 cells significantly promoted IFN-γ secretion from PBMCs. Simultaneous addition of antibody to the co-culture system significantly induced further IFN-γ secretion from PBMCs. Regarding the level of IFN-γ secretion-promoting activity, the BS-PL022B antibody was superior to the PD-1 single-target antibody 14C12H1L1 (hG1TM) and the LAG-3 single-target control antibody Relatlimab. Even compared to the combination of 14C12H1L1 (hG1TM) and Relatlimab, BS-PL022B exhibited better IFN-γ secretion-promoting potential at two different antibody concentration levels (30 nM and 300 nM).

[0466] 2. Detection of the bioactivity of anti-LAG-3 and anti-PD-1 bispecific antibodies in promoting IL-2 secretion in the Raji-PDL1 mixed lymphocyte reaction system.

[0467] Raji-PDL1 cells were routinely passaged and cultured; PBMCs were revived and cultured in 10 mL of 1640 complete medium, then stimulated for two days with 0.5 μg / mL SEB (Staphylococcal enterotoxin B) (Denotec, catalog number: S010201). Raji-PDL1 cells were then treated with MMC (Stressmarq, catalog number: SIH-246-10MG) at a final concentration of 2 μg / mL and incubated at 37°C in a 5% CO2 incubator for 1 hour. PBMCs stimulated with SEB for 2 days and Raji-PDL1 cells treated with MMC for 1 hour were collected, washed twice with PBS, resuspended in complete medium, counted, and then cultured at 10 × 10⁻⁶ cells each. 4 Cells / well were added to a U-shaped 96-well plate (Corning, model: 3799) for co-culture. Antibodies were added according to the experimental design (final concentrations of 300 nM, 30 nM, and 3 nM), and the cells were co-cultured for 3 days. After 3 days, the cells were centrifuged at 1200 rpm for 5 min, and the cell culture supernatant was collected for IL-2 detection by ELISA.

[0468] like Figure 12BAs shown, co-culture of human PBMCs and Raji-PDL1 cells promoted IL-2 secretion in PBMCs. Simultaneous addition of the antibody to the co-culture system significantly induced further IL-2 secretion from PBMCs in a dose-dependent manner. Regarding IL-2 secretion-promoting activity, BS-PL022B exhibited superior IL-2 secretion-promoting potential at all three antibody concentration levels compared to the PD-1 single-target antibody 14C12H1L1 (hG1TM) and the LAG-3 single-target control antibody Relatlimab. Even compared to the combination of 14C12H1L1 (hG1TM) and Relatlimab, BS-PL022B showed superior IL-2 secretion-promoting potential at all three antibody concentration levels.

[0469] Experimental Example 8: Affinity Detection of BS-PL022B with Fc Receptor FcγRI

[0470] The Fc receptor FcγRI (also known as CD64) can bind to the Fc terminus of IgG antibodies, participating in antibody-dependent cell-mediated cytotoxicity (ADCC). The ability of therapeutic antibodies to bind to the Fc receptor affects their safety and efficacy. In this study, the affinity constant between BS-PL022B and FcγRI was measured using a Fortebio Octet molecular interaction analyzer to evaluate the antibody's ADCC activity.

[0471] The experimental method for detecting the affinity constants of corresponding antibodies with FcγRI using the Fortebio Octet molecular interaction analyzer is briefly described below: The sample dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. A 1 μg / mL solution of FcγRI (purchased from Sinobio) was added to the HIS1K sensor and immobilized for 50 seconds to allow FcγRI to adhere to the sensor surface. The binding and dissociation parameters of the antibody with FcγRI were measured in buffer solutions at antibody concentrations ranging from 3.12 to 50 nM (two-fold serial dilution). The sample plate was vibrated at 1000 rpm, the detection temperature was 30 °C, and the frequency was 5.0 Hz. Data were analyzed using a 1:1 model to obtain the affinity constants.

[0472] The results of the affinity constant determination between FcγRI and BS-PL022B are shown in Table 12 and Figure 13 - Figure 14 As shown.

[0473] Table 12: Kinetic parameters of BS-PL022B combined with FcγRI

[0474]

[0475] N / A indicates that the antibody did not bind to the antigen or the binding signal was extremely low. The results were not analyzed, so no corresponding data was obtained.

[0476] The results showed that H7L8(hG1WT) could bind to FcγRI with an affinity constant of 6.59E-09 M. BS-PL022B did not bind to FcγRI or had an extremely low binding signal, so the results were not analyzed and no corresponding data were obtained.

[0477] The results showed that the binding activity of BS-PL022B to FcγRI was effectively eliminated.

[0478] Experimental Example 9: Affinity determination of BS-PL022B with Fc receptor FcγRIIIa and its subtypes

[0479] (1) Determination of the affinity constant between FcγRIIIa_V158 and BS-PL022B

[0480] The Fc receptor FcγRIIIa_V158 (also known as CD16a_V158) can bind to the Fc terminus of IgG antibodies, mediating ADCC effects. In this experiment, the affinity constant between BS-PL022B and FcγRIIIa_V158 was measured using a Fortebio Octet molecular interaction analyzer to evaluate the ADCC activity of the antibody.

[0481] The experimental method for detecting the affinity constants of corresponding antibodies using the Fortebio Octet molecular interaction analyzer is briefly described below: Sample dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. 5 μg / mL FcγRIIIa_V158 was immobilized on the HIS1K sensor for 60 s. The sensor equilibrated in the buffer for 60 s. The immobilized FcγRIIIa_V158 bound to each antibody (31.25-500 nM, 2-fold dilution) for 60 s. The antibody dissociated in the buffer for 60 s. The sample plate vibrated at 1000 rpm, the detection temperature was 30℃, and the frequency was 5.0 Hz. Data were analyzed using a 1:1 model to obtain the affinity constants.

[0482] The results of the affinity constant determination between FcγRIIIa_V158 and BS-PL022B are shown in Table 13 and Figure 15 - Figure 16 As shown.

[0483] Table 13: Kinetic parameters of BS-PL022B combined with FcγRIIIa_V158

[0484]

[0485] N / A indicates that the antibody did not bind to the antigen or the binding signal was extremely low. The results were not analyzed, so no corresponding data was obtained.

[0486] The results showed that H7L8(hG1WT) could bind to FcγRIIIa_V158 with an affinity constant of 8.77E-08M. BS-PL022B was not analyzed because it did not bind to FcγRIIIa_V158 or had an extremely low binding signal.

[0487] The results showed that the binding activity of BS-PL022B to FcγR IIIa_V158 was effectively eliminated.

[0488] (2) Determination of the affinity constant between FcγRIIIa_F158 and BS-PL022B

[0489] The Fc receptor FcγRIIIa_F158 (also known as CD16a_F158) can bind to the Fc terminus of IgG antibodies, mediating ADCC. In this experiment, the affinity constant between BS-PL022B and FcγRIIIa_F158 was detected using a Fortebio Octet molecular interaction analyzer to evaluate the ADCC activity of each antibody.

[0490] The experimental method for detecting the affinity constant of TF01 and FcγRIIIa_F158 using the Fortebio Octet molecular interaction analyzer is briefly described below: The sample dilution buffer was PBS (0.02% Tween-20, 0.1% BSA, pH 7.4). 5 μg / mL FcγRIIIa_F158 was immobilized on the HIS1K sensor for 120 s. The sensor equilibrated in the buffer for 60 s. The immobilized FcγRIIIa_F158 bound to various antibodies (31.25-500 nM, 2-fold dilution) for 60 s. The antibodies dissociated in the buffer for 60 s. The sample plate was vibrated at 1000 rpm, the detection temperature was 30℃, and the frequency was 5.0 Hz. The data were analyzed using a 1:1 model to obtain the affinity constant.

[0491] The results of the affinity constant determination between FcγRIIIa_F158 and BS-PL022B are shown in Table 14 and Figure 17 - Figure 18 As shown.

[0492] Table 14: Kinetic parameters of TF01FcγRIIIa_F158 bonding

[0493]

[0494] N / A indicates that the antibody did not bind to the antigen or the binding signal was extremely low. The results were not analyzed, so no corresponding data was obtained.

[0495] The results showed that H7L8(hG1WT) could bind to FcγRIIIa_F158 with an affinity constant of 3.64E-07M. BS-PL022B did not bind to FcγRIIIa_F158 or had a very low binding signal. The results were not analyzed, so no corresponding data were obtained.

[0496] The results showed that the binding activity of BS-PL022B to FcγRIIIa_F158 was effectively eliminated.

[0497] Experimental Example 10: Affinity determination of BS-PL022B with Fc receptor FcγRIIa and its subtypes

[0498] (1) Determination of the affinity constant between FcγRIIa_H131 and BS-PL022B

[0499] The Fc receptor FcγRIIa_H131 (also known as CD32a_H131) can bind to the Fc terminus of IgG antibodies, participating in antibody-dependent cell-mediated phagocytosis (ADCP) or antibody-dependent cell-mediated cytotoxicity (ADCC). The ability of therapeutic antibodies to bind to the Fc receptor affects their safety and efficacy. In this study, the affinity constant of BS-PL022B to FcγRIIa_H131 was detected using a Fortebio Octet molecular interaction analyzer to evaluate the binding ability of each test antibody to the Fc receptor.

[0500] The experimental method for detecting the affinity constant of BS-PL022B and FcγRIIa_H131 using the Fortebio Octet molecular interaction analyzer is briefly described below: The sample dilution buffer was PBS (0.02% Tween-20, 0.1% BSA, pH 7.4). 5 μg / mL of FcγRIIa_H131 was immobilized on the NTA sensor at a height of approximately 1.0 nm. The sensor was equilibrated in the buffer for 60 s. The immobilized FcγRIIa_H131 then bound to various antibodies at concentrations ranging from 12.5 to 200 nM (two-fold serial dilution) for 60 s. The antibodies then dissociated in the buffer for 60 s. The sample plate was vibrated at 1000 rpm, the detection temperature was 30 °C, and the frequency was 5.0 Hz. The data were analyzed using a 1:1 model to obtain the affinity constant.

[0501] The results of the affinity constant determination between FcγRIIa_H131 and BS-PL022B are shown in Table 15 and Figure 19 - Figure 20 As shown.

[0502] Table 15: Kinetic parameters of BS-PL022B combined with FcγRIIa_H131

[0503]

[0504] N / A indicates that the antibody did not bind to the antigen or the binding signal was extremely low. The results were not analyzed, so no corresponding data was obtained.

[0505] The results showed that H7L8(hG1WT) could bind to FcγRIIa_H131 with an affinity constant of 1.78E-07M. BS-PL022B did not bind to FcγRIIa_H131 or had a very low binding signal. The results were not analyzed, so no corresponding data were obtained.

[0506] The results showed that the binding activity of BS-PL022B to FcγRIIa_H131 was effectively eliminated.

[0507] Experimental Example 11: Determination of the affinity constant between FcγRIIb and BS-PL022B

[0508] The Fc receptor FcγRIIb (also known as CD32b) can bind to the Fc terminus of IgG antibodies. In this experiment, the affinity constants of each test antibody with FcγRIIb were detected using a FortebioOctet molecular interaction analyzer to evaluate the binding ability of BS-PL022B to the Fc receptor.

[0509] The experimental method for detecting the affinity constants of BS-PL022B and FcγRIIb using the Fortebio Octet molecular interaction analyzer is briefly described below: The sample dilution buffer was PBS (0.02% Tween-20, 0.1% BSA, pH 7.4). 5 μg / mL FcγRIIb was immobilized on the NTA sensor at a height of approximately 1.0 nm. The sensor was equilibrated in the buffer for 60 s. hFCGR2B-his immobilized on the sensor then bound to various antibodies at concentrations ranging from 12.5 to 200 nM (two-fold serial dilution) for 60 s. The antibodies then dissociated in the buffer for 60 s. The sample plate was vibrated at 1000 rpm, the detection temperature was 30 °C, and the frequency was 5.0 Hz. The data were analyzed using a 1:1 model to obtain the affinity constants.

[0510] The results of the affinity constant determination between FcγRIIb and BS-PL022B are shown in Table 16 and Figure 21 - Figure 22 As shown.

[0511] Table 16: Kinetic parameters of BS-PL022B combined with FcγRIIb

[0512]

[0513] N / A indicates that the antibody did not bind to the antigen or the binding signal was extremely low. The results were not analyzed, so no corresponding data was obtained.

[0514] The results showed that H7L8(hG1WT) could bind to FcγRIIb with an affinity constant of 1.21E-07M. BS-PL022B did not bind to FcγRIIb or had an extremely low binding signal, so the results were not analyzed and no corresponding data were obtained.

[0515] The results showed that the binding activity of BS-PL022B to FcγRIIb was effectively eliminated.

[0516] Experimental Example 12: Determination of the Affinity between BS-PL022B and C1q

[0517] Serum complement C1q can bind to the Fc terminus of IgG antibodies, mediating the CDC effect. The ability of therapeutic antibodies to bind to C1q affects the safety and efficacy of the antibody. In this experiment, the affinity constant between BS-PL022B and C1q was detected using a Fortebio Octet molecular interaction analyzer to evaluate the CDC activity of the antibody.

[0518] The experimental method for detecting the affinity constant between the corresponding antibody and C1q using the Fortebio Octet molecular interaction analyzer is briefly described below: The sample dilution buffer was PBS (0.02% Tween-20, 0.1% BSA, pH 7.4). 50 μg / mL antibody was immobilized on the FAB2G sensor at a height of approximately 2.0 nm. The sensor was equilibrated in the buffer for 60 s. The antibody immobilized on the sensor then bound to the antigen C1q at a concentration of 0.625–10 nM (two-fold serial dilution) for 60 s. The antigen and antibody then dissociated in the buffer for 60 s. The sample plate was vibrated at 1000 rpm, the detection temperature was 30°C, and the detection frequency was 5.0 Hz. Data were analyzed using a 1:1 model to obtain the affinity constant. Fortebio Data Acquisition 7.0 and Fortebio Data Analysis 7.0 were used for data acquisition and analysis.

[0519] The results of the affinity constant determination between BS-PL022B and C1q are shown in Table 17 and Figure 23 - Figure 24 As shown.

[0520] Table 17: Kinetic parameters of BS-PL022B combined with C1q

[0521]

[0522] N / A indicates that the antibody did not bind to the antigen or the binding signal was extremely low. The results were not analyzed, so no corresponding data was obtained.

[0523] The results showed that H7L8(hG1WT) could bind to C1q with an affinity constant of 1.75E-09M. BS-PL022B did not bind to C1q or had an extremely low binding signal, so the results were not analyzed and no corresponding data were obtained.

[0524] The results showed that the binding activity of BS-PL022B to C1q was effectively eliminated.

[0525] Experimental Example 13: Antibody-mediated phagocytic activity of BS-PL022B against CHO-K1-PD1-LAG3

[0526] Jurkat-NFAT-CD64-CD32R (constructed by Kangfang Biotechnology) and CHO-K1-PD1-LAG3 (constructed by Kangfang Biotechnology) cells were routinely collected, centrifuged at 110xg for 5 min, and the supernatant was discarded. Cells were resuspended in 1640 + 4% FBS, counted, and cell viability was measured to adjust cell concentration. According to the experimental design, antibodies were diluted to 50 nM, 5 nM, and 0.5 nM with 1640 + 4% FBS (working concentrations were 10 nM, 1 nM, 0.1 nM or 5 nM, 0.5 nM, 0.05 nM), and control antibodies were diluted to 50 nM. (Working concentration: 10 nM); Add 40 μL / sample of Jurkat-NFAT-CD64-CD32R cell suspension to a 96-well black plate (40,000 cells / well); Add 40 μL / sample of target cells CHO-K1-PD1-LAG3 cells (40,000 cells / well) to the sample containing Jurkat-NFAT-CD64-CD32R; Add 20 μL / well of antibody to the corresponding sample and mix well; Set up blank control and isotype control, and incubate in an incubator for 5 h; Add 50 μL / well of Bright-Glo™ Luciferase Assay System (Promega, catalog number: E2650) to the sample, mix well, and read the plate.

[0527] The results are as follows Figure 25 As shown.

[0528] The results showed that 14C12H1L1(G1WT)+H7L8(hG1WT) and Nivolumab+Relatlimab had ADCP effects at the same concentration, while BS-PL022B did not have ADCP effects.

[0529] Experimental Example 14: Pharmacodynamic Evaluation of Anti-LAG3-Anti-PD-1 Bispecific Antibody in a Mouse Tumor Cell Subcutaneous Transplantation Model

[0530] To detect the in vivo tumor-suppressive activity of the anti-LAG3-anti-PD-1 bispecific antibody, CT26 colon cancer cells (purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) were first inoculated subcutaneously on the upper right thigh of 7.1-7.3-week-old female BALB / c-hPD1 / hLAG3 mice. The day of grouping was defined as day D0. Administration was via intraperitoneal injection (ip), twice weekly (BIW), for a total of 6 administrations. Model establishment and specific administration methods are shown in Table 18. After administration, the length and width of the tumors in each group were measured, and the tumor volume was calculated.

[0531] Table 18: Dosing regimens for anti-LAG3-anti-PD-1 bispecific antibody therapy in BALB / c-hPD1 / hLAG3 mouse CT26 colon cancer xenograft model

[0532]

[0533] Note: The doses of isotype control 15 mg / kg, Relatlimab 15 mg / kg and BS-PL022B 20 mg / kg are equimolar concentrations; the doses of Relatlimab 3 mg / kg and BS-PL022B 4 mg / kg are equimolar concentrations.

[0534] The results are as follows Figure 26 As shown in the figure. The results showed that, compared with the isotype control antibody, both the anti-LAG3-anti-PD-1 bispecific antibody BS-PL022B and the positive control antibody Relatlimab effectively inhibited the growth of tumors in mice. The results indicate that the anti-LAG3-anti-PD-1 bispecific antibody BS-PL022B has a significantly better tumor-suppressing effect than the positive control antibody Relatlimab.

[0535] In addition, such as Figure 27 As shown, tumor-bearing mice tolerated the test drug BS-PL022B well, and there was no effect on the body weight of tumor-bearing mice in any group.

[0536] 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 bispecific antibody comprising a first protein functional region and a second protein functional region, wherein: The first protein's functional region targets LAG3. The second protein's functional region targets a different target than LAG3. The first protein functional region is an anti-LAG3 antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises: amino acid sequences as shown in SEQ ID NOs: 5-7, namely HCDR1-HCDR3; and the light chain variable region comprises: amino acid sequences as shown in SEQ ID NOs: 8-10, namely LCDR1-LCDR3.

2. The bispecific antibody according to claim 1, wherein, The second protein's functional region targets PD-1.

3. The bispecific antibody according to claim 1, wherein, The amino acid sequence of the heavy chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the anti-LAG3 antibody is shown in SEQ ID NO:

4.

4. The bispecific antibody according to claim 1, wherein, The anti-LAG3 antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fv, humanized antibody or chimeric antibody.

5. The bispecific antibody according to claim 1, wherein, The anti-LAG3 antibody or its antigen-binding fragment is a single-chain antibody.

6. The bispecific antibody according to claim 1, wherein, The anti-LAG3 antibody was expressed at an EC50 concentration of less than 0.2 nM. 50 Combined with human LAG3-mFc; wherein, the EC 50 Measured by indirect ELISA method.

7. The bispecific antibody according to claim 6, wherein, The anti-LAG3 antibody was expressed at an EC50 concentration of less than 0.15 nM. 50 Combined with human LAG3-mFc.

8. The bispecific antibody according to claim 6, wherein, The anti-LAG3 antibody was prepared at an EC50 concentration of less than 0.1 nM. 50 Combined with human LAG3-mFc.

9. The bispecific antibody according to claim 6, wherein, The anti-LAG3 antibody was expressed at an EC50 concentration of less than 0.08 nM. 50 Combined with human LAG3-mFc.

10. The bispecific antibody according to claim 6, wherein, The anti-LAG3 antibody was expressed at an EC50 concentration of less than 0.06 nM. 50 Combined with human LAG3-mFc.

11. The bispecific antibody according to claim 6, wherein, The anti-LAG3 antibody was expressed at an EC50 concentration of less than 0.05 nM. 50 Combined with human LAG3-mFc.

12. The bispecific antibody according to claim 1, wherein, The anti-LAG3 antibody includes a non-CDR region, and the non-CDR region is derived from a human antibody.

13. The bispecific antibody according to claim 1, wherein, The anti-LAG3 antibody has a constant region derived from human antibodies.

14. The bispecific antibody according to claim 1, wherein, The anti-LAG3 antibody has a constant region, and the constant region is selected from the constant regions of human IgG1, IgG2, IgG3 or IgG4.

15. The bispecific antibody according to claim 1, wherein, The anti-LAG3 antibody comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is the heavy chain constant region of IgG1 or the heavy chain constant region of IgG4; and the light chain constant region is the constant region of the immunoglobulin κ light chain.

16. The bispecific antibody according to claim 15, wherein, The amino acid sequence of the heavy chain constant region of IgG1 is shown in SEQ ID NO:

39.

17. The bispecific antibody according to claim 15, wherein, The amino acid sequence of the heavy chain constant region of IgG4 is shown in SEQ ID NO:

45.

18. The bispecific antibody according to claim 15, wherein, The amino acid sequence of the constant region of the immunoglobulin κ light chain is shown in SEQ ID NO:

40.

19. The bispecific antibody according to claim 1, wherein, The anti-LAG3 antibody is the human IgG1 subtype. According to the EU numbering system, the heavy chain constant region of the antibody has the following mutation: L234A and L235A, L234A and G237A, L235A and G237A, or L234A, L235A, and G237A.

20. The bispecific antibody according to claim 19, wherein, The amino acid sequence of the heavy chain of the anti-LAG3 antibody is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain is shown in SEQ ID NO:

12.

21. The bispecific antibody according to claim 1, wherein, The anti-LAG3 antibody is the human IgG4 subtype. According to the EU numbering system, the heavy chain constant region of the antibody has the following mutation: F234A and L235A, F234A and G237A, L235A and G237A, or F234A, L235A, and G237A.

22. The bispecific antibody according to claim 21, wherein, The amino acid sequence of the heavy chain of the anti-LAG3 antibody is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain is shown in SEQ ID NO:

12.

23. The bispecific antibody according to claim 1, wherein, The bispecific antibody is in IgG-scFv mode.

24. The bispecific antibody according to claim 1, wherein, The first protein functional region is an antibody against LAG3, 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 antibody that targets a different target than LAG3.

25. The bispecific antibody according to claim 1, wherein, The first and second protein functional regions are directly connected or connected through a linker fragment.

26. The bispecific antibody according to claim 25, wherein, The connection segment is (GGGGS)m, where m is a positive integer.

27. The bispecific antibody according to claim 26, wherein, m can be 1, 2, 3, 4, 5, or 6.

28. The bispecific antibody according to claim 25, wherein, The connection segment is (GGGGS)nG, where n is a positive integer.

29. The bispecific antibody according to claim 28, wherein, n can be 1, 2, 3, 4, 5, or 6.

30. The bispecific antibody according to claim 24, wherein, The first protein functional region is an antibody against LAG3, the second protein functional region is a single-chain antibody, and there is one first protein functional region and two second protein functional regions; or The first protein functional region is a single-chain antibody, and the second protein functional region is an antibody that targets a different target than LAG3. There are two first protein functional regions and one second protein functional region.

31. The bispecific antibody according to claim 24, wherein, The single-chain antibody is attached to the C-terminus of the antibody's heavy chain.

32. The bispecific antibody according to any one of claims 1 to 31, comprising: Targeting the first protein functional region of LAG3, and Targeting the second protein functional region of PD-1; in, The first protein functional region is an anti-LAG3 antibody, and the anti-LAG3 antibody is in the form of an immunoglobulin. The second protein functional region is an anti-PD-1 single-chain antibody.

33. The bispecific antibody according to claim 32, wherein, The anti-PD-1 single-chain antibody comprises a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region comprises: amino acid sequences as shown in SEQ ID NOs: 26-28, HCDR1-HCDR3; and The light chain variable region comprises: amino acid sequences as shown in SEQ ID NOs: 29-31, namely LCDR1-LCDR3.

34. The bispecific antibody according to claim 32, wherein, The anti-PD-1 single-chain antibody, The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 15, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 17; or The amino acid sequence of its heavy chain variable region 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 or SEQ ID NO:

38.

35. The bispecific antibody according to claim 32, wherein, The heavy chain variable region and light chain variable region of the anti-PD-1 single-chain antibody are directly linked or linked through a linker fragment.

36. The bispecific antibody according to claim 35, wherein, The connection segment is (GGGGS)m, where m is a positive integer.

37. The bispecific antibody according to claim 36, wherein, m can be 1, 2, 3, 4, 5, or 6.

38. The bispecific antibody according to claim 35, wherein, The connection segment is (GGGGS)nG, where n is a positive integer.

39. The bispecific antibody according to claim 38, wherein, n can be 1, 2, 3, 4, 5, or 6.

40. The bispecific antibody according to claim 32, wherein, The 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 fragment; 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 fragment; the first linker fragment and the second linker fragment may be the same or different.

41. The bispecific antibody according to claim 40, wherein, The amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NOs: 35-37.

42. The bispecific antibody according to claim 40, wherein, The amino acid sequences of both the first linker fragment and the second linker fragment are shown in SEQ ID NO:

36.

43. The bispecific antibody according to any one of claims 1 to 31, comprising: Targeting the first protein functional region of LAG3, and Targeting the second protein functional region of PD-1; Wherein, the first protein functional region is an anti-LAG3 single-chain antibody, the second protein functional region is an anti-PD-1 antibody, and the anti-PD-1 antibody is in the form of an immunoglobulin; The anti-LAG3 single-chain antibody comprises a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region comprises: amino acid sequences HCDR1-HCDR3 as shown in SEQ ID NOs: 5-7; and The light chain variable region comprises: amino acid sequences as shown in SEQ ID NOs: 8-10, namely LCDR1-LCDR3.

44. The bispecific antibody according to claim 43, wherein, The anti-LAG3 single-chain antibody, The amino acid sequence of its heavy chain variable region 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.

45. The bispecific antibody according to claim 43, wherein, The heavy chain variable region and light chain variable region in the anti-LAG3 single-chain antibody are directly linked or linked through a linker fragment.

46. ​​The bispecific antibody according to claim 45, wherein, The connection segment is (GGGGS)m, where m is a positive integer.

47. The bispecific antibody according to claim 46, wherein, m can be 1, 2, 3, 4, 5, or 6.

48. The bispecific antibody according to claim 45, wherein, The connection segment is (GGGGS)nG, where n is a positive integer.

49. The bispecific antibody according to claim 48, wherein, m can be 1, 2, 3, 4, 5, or 6.

50. The bispecific antibody according to claim 43, wherein, The anti-PD-1 antibody comprises a heavy chain variable region and a light chain variable region, wherein, The heavy chain variable region comprises: amino acid sequences as shown in SEQ ID NOs: 26-28, HCDR1-HCDR3; and The light chain variable region comprises: amino acid sequences as shown in SEQ ID NOs: 29-31, namely LCDR1-LCDR3.

51. The bispecific antibody according to claim 43, wherein, The anti-PD-1 antibody, The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 15, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 17; or The amino acid sequence of its heavy chain variable region 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 or SEQ ID NO:

38.

52. The bispecific antibody according to claim 43, wherein, The anti-PD-1 antibody comprises a heavy chain variable region and a light chain variable region. Its heavy chain constant region is the heavy chain constant region of IgG1 or the heavy chain constant region of IgG4; and its light chain constant region is the constant region of the immunoglobulin κ light chain.

53. The bispecific antibody according to claim 52, wherein, The amino acid sequence of the heavy chain constant region of IgG1 is shown in SEQ ID NO:

39.

54. The bispecific antibody according to claim 52, wherein, The amino acid sequence of the heavy chain constant region of IgG4 is shown in SEQ ID NO:

45.

55. The bispecific antibody according to claim 52, wherein, The amino acid sequence of the constant region of the immunoglobulin κ light chain is shown in SEQ ID NO:

40.

56. The bispecific antibody according to claim 43, wherein, The anti-PD-1 antibody is the human IgG1 subtype. According to the EU numbering system, the anti-PD-1 antibody has the following mutations: L234A and L235A, L234A and G237A, L235A and G237A, or L234A, L235A, and G237A.

57. The bispecific antibody according to claim 56, wherein, The amino acid sequence of the heavy chain of the anti-PD-1 antibody is shown in SEQ ID NO: 34, and the amino acid sequence of the light chain is shown in SEQ ID NO:

25.

58. The bispecific antibody according to claim 43, wherein, The anti-PD-1 antibody is the human IgG4 subtype. According to the EU numbering system, the anti-PD-1 antibody has the following mutations: F234A and L235A, F234A and G237A, L235A and G237A, or F234A, L235A, and G237A.

59. The bispecific antibody according to claim 58, wherein, The amino acid sequence of the heavy chain of the anti-PD-1 antibody is shown in SEQ ID NO: 32, and the amino acid sequence of the light chain is shown in SEQ ID NO:

25.

60. The bispecific antibody according to claim 43, wherein, The 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 fragment; 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 fragment; the first linker fragment and the second linker fragment may be the same or different.

61. The bispecific antibody according to claim 60, wherein, The amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NOs: 35-37.

62. The bispecific antibody according to claim 60, wherein, The amino acid sequences of both the first linker fragment and the second linker fragment are shown in SEQ ID NO:

36.

63. An isolated nucleic acid molecule encoding the bispecific antibody as described in any one of claims 1 to 62.

64. A recombinant vector comprising the isolated nucleic acid molecule of claim 63.

65. A host cell comprising the isolated nucleic acid molecule of claim 63, or comprising the recombinant vector of claim 64.

66. A method for preparing the bispecific antibody according to any one of claims 1 to 62, comprising culturing the host cell of claim 65 under suitable conditions, and recovering the bispecific antibody from the cell culture.

67. A pharmaceutical composition comprising the bispecific antibody as described in any one of claims 1 to 62.

68. The pharmaceutical composition of claim 67, further comprising pharmaceutically acceptable excipients.

69. Use of the bispecific antibody according to any one of claims 1 and 3 to 31 in the preparation of a medicament for treating or preventing tumors, wherein, The tumor is selected from one or more of the following: ovarian cancer, melanoma, leukemia, colon cancer, rectal cancer, and glioblastoma.

70. Use of the bispecific antibody according to any one of claims 2 and 32 to 62 in the preparation of a medicament for treating or preventing tumors, wherein, The tumor is selected from one or more of the following: ovarian cancer, melanoma, leukemia, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, liver cancer, gastrointestinal cancer, breast cancer, brain cancer, pancreatic cancer, head and neck cancer, and kidney cancer.

71. The use according to claim 70, wherein, The lung cancer in question is non-small cell lung cancer.

72. Use of the bispecific antibody according to any one of claims 2 and 32 to 62 in the preparation of a medicament for treating or preventing tumors, wherein, The tumor is selected from one or more of glioblastoma, thyroid cancer, esophageal cancer, colon cancer, and rectal cancer.

73. The use according to claim 72, wherein, The esophageal cancer mentioned is esophageal squamous cell carcinoma.

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