Bispecific antibody, isolated nucleic acid molecule, recombinant vector, host cell, pharmaceutical composition, use of the bispecific antibody, and method for treating or preventing a tumor.

Description

1 / 101 “BIESPECIFIC ANTIBODY, ISOLATED NUCLEIC ACID MOLECULE, RECOMBINANT VECTOR, HOST CELL, PHARMACEUTICAL COMPOSITION AND USE OF THE BIESPECIFIC ANTIBODY” Cross-reference to related applications

[001] This application is based on and claims priority to Chinese Patent Application No. CN202310327266.6, filed on March 29, 2023, which is incorporated herein by reference in its entirety. Field of invention

[002] This disclosure relates to the field of biomedicine and refers to a bispecific antibody, a pharmaceutical composition comprising the bispecific antibody and its use. Background of the Invention

[003] Tumors, especially malignant tumors, are serious and potentially fatal diseases in the world today, being the second leading cause of death among various diseases. In recent years, the incidence of the disease has increased significantly. Malignant tumors are characterized by poor therapeutic effect, a high rate of late metastasis, and a poor prognosis. Although conventional treatment methods (such as radiotherapy, chemotherapy, and surgical treatment) currently adopted clinically significantly alleviate pain and prolong survival, these methods have major limitations, and it is difficult to further improve their effectiveness.

[004] Lymphocyte-activating gene 3 (LAG3), or CD223, is a type I transmembrane protein composed of 498 amino acids and a member of the immunoglobulin superfamily (IgSF). LAG3 is primarily expressed on activated CD4+ and CD8+ T cells. In addition, LAG3 is also expressed on cells such as natural killer (NK) cells, B cells, regulatory T cells (Tregs), and plasmacytoid dendritic cells (pDCs). (Ruffo Elisa, Wu Richard C, Bruno Tullia C et al. Lymphocyte-activation gene 3 (LAG3): The next immune Petition 870250099246, dated 10 / 30 / 2025, pp. 142 / 270 2 / 101 receiving checkpoint. [J]. Semin Immunol, 2019, 42: 101305).

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

[006] 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. The LAG3 molecule, through its D1 domain (which contains a proline-rich loop structure), forms a dimeric molecule to specifically bind to the MHC-II molecule in the first signaling axis of CD4+ T cell activation, CD3-TCRMHC-II, so that, in one aspect, a signal transduction pathway for T cell activation is blocked and, in another aspect, an intracellular segment of the LAG3 molecule (KIEELE motif) generates an immunosuppressive signal to negatively regulate CD4+ T cell activity. The LAG3 molecule can promote Treg cell differentiation, participate in downstream signaling of the signal transducer and transcription activator 5 and thus increase the inhibitory effect of Treg cells.This is one of the mechanisms by which tumors escape death by the immune system (Andrews Lawrence P,. Petition 870250099246, dated 10 / 30 / 2025, pp. 143 / 270 3 / 101 Marciscano Ariel E, Drake Charles G, et al., LAG3 (CD223) as a target for immunotherapy against cancer. [J]. Immunol Rev, 2017, 276: 80-96).

[007] Several studies have demonstrated that LAG3 is overexpressed in tumor-infiltrating CD8+ T cells of various malignant tumors. For example, in ovarian cancer, tumor-infiltrating CD8+ T cells specific for New York esophageal squamous cell carcinoma antigen 1 (NY-ESO-1) express high levels of PD-1 and LAG3 and have a reduced capacity to produce IFN-γ and TNF-α, leading to lymphocyte inactivation. Galectin-3 and LSECtin interact primarily with LAG3 to regulate CD8+ T cell activation and function. Furthermore, melanoma antigen-specific T cells isolated from patients with metastatic melanoma show a significant upregulation in the expression of LAG3 and other immune checkpoint molecules CTLA-4 and TIM-3. (Liu Hao, Li Xinying, Luo Longlong et al.)Advances in research on the biological function of the lymphocyte-activating gene-3 (LAG-3) molecule and clinical application of drugs with antibodies targeting LAG-3 [J]. Chinese Journal of Pharmacology and Toxicology, 2019, 33(01): 70-78).

[008] Currently, several drugs with LAG3 antibodies have entered the clinical research phase, among which Relatlimab, from Bristol Myers Squibb, is the one that has progressed the most, with 10 clinical studies underway. The vast majority of these studies involve combination therapy of Relatlimab with nivolumab for the treatment of tumors such as hematological malignancies, melanoma, glioblastoma, renal cell carcinoma, non-small cell lung cancer and similar cancers.

[009] Ecto-5'-nucleotidase, or CD73 protein, is a multifunctional glycoprotein encoded by an NT5E gene and with a protein molecular weight of 70 kDa. It is anchored to the cell membrane by glycosylphosphatidylinositol (GPI) (Zimmermann H. Biochem J. 1992; 285:345-365). Petition 870250099246, dated 10 / 30 / 2025, pp. 144 / 270 4 / 101

[010] CD73 is widely distributed on the surface of human tissue cells, and research has shown that its high expression occurs in various solid tumors, specifically in cancer cells, dendritic cells, regulatory T cells (Tregs), natural killer cells (NK cells), myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), and similar cells, in a tumor microenvironment. An important characteristic of the tumor microenvironment is hypoxia. Hypoxia induces the upregulation of molecules such as hypoxia-inducible factor-1 (HIF-1), leading to widespread expression of CD73 in the tumor microenvironment (Synnestvedt K et al. J Clin Invest. 2002; 110:993-1002).Analysis of clinical tumor samples has shown that high CD73 expression is a potential biomarker and is closely related to poor prognosis in several types of tumors, including breast cancer, lung cancer, ovarian cancer, kidney cancer, gastric cancer, head and neck cancer, and similar cancers.

[011] CD73 possesses both hydrolase and non-hydrolase activity. The enzymatic and non-enzymatic functions of CD73 act simultaneously in the tumor-related process and mutually promote and maintain tumor progression. Increasingly recent studies have demonstrated that CD73 is an essential regulatory molecule for the proliferation, metastasis, and invasion of tumor cells in vitro, as well as for tumor angiogenesis and the tumor immune escape mechanism in vivo, being an important immunosuppressive mechanism mediated by the CD73-adenosine metabolic signaling pathway. CD39, upstream of CD73, can catalyze ATP to generate adenosine monophosphate (AMP). The generated AMP is converted into adenosine by CD73, and adenosine binds to an adenosine receptor (A2AR) downstream.A2AR inhibits a series of signaling pathways related to immune activation, such as LCK, MAPK, and PKC, and inhibits the immunosuppressive effect of T cells by activating protein kinase A (PKA) and Csk kinase, playing a role in... Petition 870250099246, dated 10 / 30 / 2025, pp. 145 / 270 5 / 101 Thus, an immunosuppressive role that allows the tumor to achieve immune escape (Antonioli L, et al. Nat Rev Cancer. 2013; 13:842-857). Preclinical studies in animal models have demonstrated that CD73 expressed on immune and non-immune cells can promote immune escape, tumor progression and metastasis, with the inhibition of cytotoxic T cell (CTL) and NK cell functions by CD73-adenosine signals related to Treg cells being the most significant.

[012] For the treatment of solid tumors, an important aspect to overcome drug resistance and improve the therapeutic effect is to alleviate the inhibitory effect of the tumor microenvironment (TME) on immune effector cells. The TME is a very complex system composed of various cells, intercellular matrix, enzymes, cytokines, metabolites, etc. It has characteristics of significantly low hydrogen, low pH and high pressure, and is therefore very different from normal tissues. Hypoxia or ATP enrichment caused by chemoradiotherapy to kill tumor cells promotes the CD39-CD73 adenosine signaling cascade reaction, which is beneficial for the proliferation and function of various cancer-promoting cells, but not for cancer-inhibiting cells (Regateiro, FS, Cobbold, SP & Waldmann, H. Clin. Exp. Immunol. 2013; 171:1-7).

[013] The use of antibodies targeting CD73 or the elimination of the CD73 gene in animal models can effectively block tumor growth and metastasis. Recently, the use of CD73 monoclonal antibodies, small RNA interference technology, specific APCP inhibitors, and similar technologies has achieved remarkable therapeutic effects in antitumor treatment in animal experiments, providing a new form of antitumor treatment. Evidence from in vivo studies has demonstrated that targeted CD73 blockade will be an effective means of treatment for patients with tumors. Petition 870250099246, dated 10 / 30 / 2025, pp. 146 / 270 6 / 101

[014] The relationship between CD73 overexpression and tumor subtype, prognosis, and response in patients has demonstrated that CD73 may be an important marker for future tumor treatment and individual detection. Therefore, the study of the CD73 target is indispensable.

[015] Currently, there is a need to develop a new anti-LAG3 antibody and a bispecific antibody that targets both CD73 and LAG3. Brief Description of the Invention

[016] Through intensive studies and creative efforts, the inventors obtained a bispecific anti-LAG3-anti-CD73 antibody. The inventors surprisingly found that the bispecific anti-LAG3-anti-CD73 antibody of the present disclosure (also referred to, in short, as the antibody or antibody of the present disclosure) exhibits good antitumor activity. The present disclosure is detailed below.

[017] One aspect of the present disclosure relates to a bispecific antibody comprising a first protein functional region and a second protein functional region, wherein: The first functional protein region targets LAG3, and the second functional protein region targets a different LAG3 target (e.g., CD73 or PD-1), wherein the first functional protein region is an anti-LAG3 antibody or an antigen-binding fragment thereof, and the anti-LAG3 antibody comprises a variable heavy chain region and a variable light chain region; the variable heavy chain region comprises HCDR1, HCDR2, and HCDR3, and the variable light chain region comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 has the amino acid sequence shown in SEQ ID NO: 9, Petition 870250099246, dated 10 / 30 / 2025, pp. 147 / 270 7 / 101 HCDR2 has the amino acid sequence shown in SEQ ID NO: 10, and HCDR3 has the amino acid sequence shown in SEQ ID NO: 11; LCDR1 has the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 17. LCDR2 has the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 15, and LCDR3 has the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 16.

[018] In some embodiments of this disclosure, the bispecific antibody is provided, in which LCDR1 has the amino acid sequence shown in SEQ ID NO: 12, LCDR2 has the amino acid sequence shown in SEQ ID NO: 13, and LCDR3 has the amino acid sequence shown in SEQ ID NO: 14; or LCDR1 has the amino acid sequence shown in SEQ ID NO: 12, LCDR2 has the amino acid sequence shown in SEQ ID NO: 15, and LCDR3 has the amino acid sequence shown in SEQ ID NO: 16; or LCDR1 has the amino acid sequence shown in SEQ ID NO: 17, Petition 870250099246, dated 10 / 30 / 2025, pp. 148 / 270 8 / 101 LCDR2 has the amino acid sequence shown in SEQ ID NO: 15 and LCDR3 has the amino acid sequence shown in SEQ ID NO: 14; or LCDR1 has the amino acid sequence shown in SEQ ID NO: 12, LCDR2 has the amino acid sequence shown in SEQ ID NO: 13 and LCDR3 has the amino acid sequence shown in SEQ ID NO: 16; or LCDR1 has the amino acid sequence shown in SEQ ID NO: 12, LCDR2 has the amino acid sequence shown in SEQ ID NO: 15, and LCDR3 has the amino acid sequence shown in SEQ ID NO: 14; or LCDR1 has the amino acid sequence shown in SEQ ID NO: 17, LCDR2 has the amino acid sequence shown in SEQ ID NO: 13, and LCDR3 has the amino acid sequence shown in SEQ ID NO: 14; or LCDR1 has the amino acid sequence shown in SEQ ID NO: 17, Petition 870250099246, dated 10 / 30 / 2025, pp. 149 / 270 9 / 101 LCDR2 has the amino acid sequence shown in SEQ ID NO: 13, and LCDR3 has the amino acid sequence shown in SEQ ID NO: 16; or LCDR1 has the amino acid sequence shown in SEQ ID NO: 17, LCDR2 has the amino acid sequence shown in SEQ ID NO: 15, and LCDR3 has the amino acid sequence shown in SEQ ID NO: 16.

[019] In some embodiments of this disclosure, the bispecific antibody is provided, wherein HCDR1 has the amino acid sequence shown in SEQ ID NO: 9, HCDR2 has the amino acid sequence shown in SEQ ID NO: 10, and HCDR3 has the amino acid sequence shown in SEQ ID NO: 11; LCDR1 has the amino acid sequence shown in SEQ ID NO: 12, LCDR2 has the amino acid sequence shown in SEQ ID NO: 13, and LCDR3 has the amino acid sequence shown in SEQ ID NO: 14. ID NO: 14.

[020] In some embodiments of this disclosure, the bispecific antibody is provided, in which: HCDR1 has the amino acid sequence shown in SEQ. Petition 870250099246, dated 10 / 30 / 2025, pages 150 / 270 10 / 101 ID NO: 9, HCDR2 has the amino acid sequence shown in SEQ ID NO: 10, HCDR3 has the amino acid sequence shown in SEQ ID NO: 11; LCDR1 has the amino acid sequence shown in SEQ ID NO: 12, LCDR2 has the amino acid sequence shown in SEQ ID NO: 15 and LCDR3 has the amino acid sequence shown in SEQ ID NO: 16.

[021] In some embodiments of this disclosure, it is The bispecific antibody is provided, in which: HCDR1 has the amino acid sequence shown in SEQ ID NO: 9, HCDR2 has the amino acid sequence shown in SEQ ID NO: 10, HCDR3 has the amino acid sequence shown in SEQ ID NO: 11; LCDR1 has the amino acid sequence shown in SEQ ID NO: 17, LCDR2 has the amino acid sequence shown in SEQ ID NO: 15 and LCDR3 has the amino acid sequence shown in SEQ ID NO: 14.

[022] In some embodiments of this disclosure, it is The bispecific antibody is provided, in which: HCDR1 has the amino acid sequence shown in SEQ. Petition 870250099246, dated 10 / 30 / 2025, pp. 151 / 270 11 / 101 ID NO: 9, HCDR2 has the amino acid sequence shown in SEQ ID NO: 10, HCDR3 has the amino acid sequence shown in SEQ ID NO: 11; LCDR1 has the amino acid sequence shown in SEQ ID NO: 12, LCDR2 has the amino acid sequence shown in SEQ ID NO: 13 and LCDR3 has the amino acid sequence shown in SEQ ID NO: 16.

[023] In some embodiments of this disclosure, it is The bispecific antibody is provided, in which: HCDR1 has the amino acid sequence shown in SEQ ID NO: 9, HCDR2 has the amino acid sequence shown in SEQ ID NO: 10, HCDR3 has the amino acid sequence shown in SEQ ID NO: 11; LCDR1 has the amino acid sequence shown in SEQ ID NO: 12, LCDR2 has the amino acid sequence shown in SEQ ID NO: 15 and LCDR3 has the amino acid sequence shown in SEQ ID NO: 14.

[024] In some embodiments of this disclosure, it is The bispecific antibody is provided, in which: HCDR1 has the amino acid sequence shown in SEQ. Petition 870250099246, dated 10 / 30 / 2025, pp. 152 / 270 12 / 101 ID NO: 9, HCDR2 has the amino acid sequence shown in SEQ ID NO: 10, HCDR3 has the amino acid sequence shown in SEQ ID NO: 11; LCDR1 has the amino acid sequence shown in SEQ ID NO: 17, LCDR2 has the amino acid sequence shown in SEQ ID NO: 13 and LCDR3 has the amino acid sequence shown in SEQ ID NO: 14.

[025] In some embodiments of this disclosure, it is The bispecific antibody is provided, in which: HCDR1 has the amino acid sequence shown in SEQ ID NO: 9, HCDR2 has the amino acid sequence shown in SEQ ID NO: 10, HCDR3 has the amino acid sequence shown in SEQ ID NO: 11; LCDR1 has the amino acid sequence shown in SEQ ID NO: 17, LCDR2 has the amino acid sequence shown in SEQ ID NO: 13 and LCDR3 has the amino acid sequence shown in SEQ ID NO: 16.

[026] In some embodiments of this disclosure, it is The bispecific antibody is provided, in which: HCDR1 has the amino acid sequence shown in SEQ. Petition 870250099246, dated 10 / 30 / 2025, pp. 153 / 270 13 / 101 ID NO: 9, HCDR2 has the amino acid sequence shown in SEQ ID NO: 10, HCDR3 has the amino acid sequence shown in SEQ ID NO: 11; LCDR1 has the amino acid sequence shown in SEQ ID NO: 17, LCDR2 has the amino acid sequence shown in SEQ ID NO: 15 and LCDR3 has the amino acid sequence shown in SEQ ID NO: 16.

[027] In some embodiments of this disclosure, the bispecific antibody is provided, in which: The variable region of the heavy chain of the anti-LAG3 antibody is selected from the amino acid sequences presented in SEQ ID NO: 2 and SEQ ID NO: 44, and the variable region of the light chain of the anti-LAG3 antibody is selected from the amino acid sequences presented in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 45; Preferably, the variable region of the anti-LAG3 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the anti-LAG3 antibody light chain has the amino acid sequence shown in SEQ ID NO: 4; The variable region of the anti-LAG3 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the anti-LAG3 antibody light chain has the amino acid sequence shown in SEQ ID NO: 6; the variable region of the heavy chain of the anti-LAG3 antibody has Petition 870250099246, dated 10 / 30 / 2025, pp. 154 / 270 14 / 101 the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the anti-LAG3 antibody light chain has the amino acid sequence shown in SEQ ID NO: 8; The variable region of the anti-LAG3 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the anti-LAG3 antibody light chain has the amino acid sequence shown in SEQ ID NO: 45; The variable region of the anti-LAG3 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 44, and the variable region of the anti-LAG3 antibody light chain has the amino acid sequence shown in SEQ ID NO: 4; The variable region of the anti-LAG3 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 44, and the variable region of the anti-LAG3 antibody light chain has the amino acid sequence shown in SEQ ID NO: 6; The variable region of the anti-LAG3 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 44, and the variable region of the anti-LAG3 antibody light chain has the amino acid sequence shown in SEQ ID NO: 8; or the variable region of the anti-LAG3 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 44, and the variable region of the anti-LAG3 antibody light chain has the amino acid sequence shown in SEQ ID NO: 45.

[028] In some embodiments of the present disclosure, a bispecific antibody is provided, in which the anti-LAG3 antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, a complementarity-determining region fragment, a single-chain antibody, a humanized antibody, and an antibody Petition 870250099246, dated 10 / 30 / 2025, pages 155 / 270 15 / 101 chimerical.

[029] In some embodiments of the present disclosure, the bispecific antibody is provided, wherein the anti-LAG3 antibody comprises a non-CDR region derived from a human antibody.

[030] In some embodiments of the present disclosure, the bispecific antibody is provided, which is a bispecific anti-LAG3anti-CD73 antibody.

[031] In some embodiments of this disclosure, the bispecific antibody is provided, in which: The second functional protein region is an anti-CD73 antibody or its antigen-binding fragment, and the anti-CD73 antibody comprises a variable heavy chain region and a variable light chain region; the variable heavy chain region comprises HCDR1 with the amino acid sequence presented in SEQ ID NO: 25, HCDR2 with the amino acid sequence presented in SEQ ID NO: 26, and HCDR3 with the amino acid sequence presented in SEQ ID NO: 27; and the variable light chain region comprises LCDR1 with the amino acid sequence presented in SEQ ID NO: 28, LCDR2 with the amino acid sequence presented in SEQ ID NO: 29, and LCDR3 with the amino acid sequence presented in SEQ ID NO: 30.

[032] In some embodiments of the present disclosure, the bispecific antibody is provided, wherein the variable region of the anti-CD73 antibody heavy chain is selected from the amino acid sequences presented in SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 46; the variable region of the anti-CD73 antibody light chain is selected from the amino acid sequences presented in SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 47 and SEQ ID NO: 62; preferably, Petition 870250099246, dated 10 / 30 / 2025, pp. 156 / 270 16 / 101 the variable region of the heavy chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 31, and the variable region of the light chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 32; The variable region of the heavy chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 31, and the variable region of the light chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 34; The variable region of the heavy chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 31, and the variable region of the light chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 36; The variable region of the heavy chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 31, and the variable region of the light chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 37; The variable region of the heavy chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 31, and the variable region of the light chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 47; The variable region of the heavy chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 31, and the variable region of the light chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 62; The variable region of the anti-CD73 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 33, and the variable region of the anti-CD73 antibody light chain has the amino acid sequence shown in SEQ ID NO: 32; Petition 870250099246, dated 10 / 30 / 2025, pp. 157 / 270 17 / 101 the variable region of the heavy chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 33, and the variable region of the light chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 34; The variable region of the heavy chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 33, and the variable region of the light chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 36; The variable region of the heavy chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 33, and the variable region of the light chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 37; The variable region of the heavy chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 33, and the variable region of the light chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 47; The variable region of the heavy chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 33, and the variable region of the light chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 62; The variable region of the heavy chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the light chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 32; The variable region of the heavy chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the light chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 34; Petition 870250099246, dated 10 / 30 / 2025, pp. 158 / 270 18 / 101 the variable region of the heavy chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the light chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 36; The variable region of the heavy chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the light chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 37; The variable region of the anti-CD73 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the anti-CD73 antibody light chain has the amino acid sequence shown in SEQ ID NO: 47; The variable region of the heavy chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the light chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 62; The variable region of the heavy chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 46, and the variable region of the light chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 32; The variable region of the heavy chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 46, and the variable region of the light chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 34; The variable region of the heavy chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 46, and the variable region of the light chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 36; Petition 870250099246, dated 10 / 30 / 2025, pp. 159 / 270 19 / 101 the variable region of the heavy chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 46, and the variable region of the light chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 37; The variable region of the heavy chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 46, and the variable region of the light chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 47; or the variable region of the heavy chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 46, and the variable region of the light chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 62.

[033] In some embodiments of this disclosure, the bispecific antibody is provided, in which: The anti-LAG3 antibody or the anti-CD73 antibody further comprises a constant region derived from a human antibody; Preferably, the constant region of the anti-LAG3 antibody or the anti-CD73 antibody is selected from a constant region of human IgG1, IgG2, IgG3, or IgG4; Preferably, the anti-LAG3 antibody comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is the C region of the Ig gamma-1 chain (e.g., as set forth in SEQ ID NO: 18) or the C region of the Ig gamma-4 chain (e.g., as set forth in SEQ ID NO: 20), and the light chain constant region is the C region of the Ig kappa chain (e.g., as set forth in SEQ ID NO: 19); Preferably, the anti-CD73 antibody comprises a constant region of the heavy chain and a constant region of the light chain, wherein the Petition 870250099246, dated 10 / 30 / 2025, pages 160 / 270 20 / 101 The constant region of the heavy chain is the C region of the gamma-1 chain of Ig (e.g., as established in SEQ ID NO: 18) or the C region of the gamma-4 chain of Ig (e.g., as established in SEQ ID NO: 20), and the constant region of the light chain is the C region of the kappa chain of Ig (e.g., as established in SEQ ID NO: 19).

[034] Amino acid sequence of the constant region of the IgG1 heavy chain: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 18).

[035] In some embodiments of the present disclosure, the bispecific antibody is provided, which is characterized by any of the following (1) to (4): (1) The anti-LAG3 antibody is of the human IgG1 subtype, where, according to the EU numbering system, the constant region of the antibody heavy chain has the following mutations: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A and G237A; (2) the anti-LAG3 antibody is of the human IgG4 subtype, in which, according to the EU numbering system, the constant region of the antibody heavy chain has the following mutations: F234A and L235A; Petition 870250099246, dated 10 / 30 / 2025, pp. 161 / 270 21 / 101 F234A and G237A; L235A and G237A; or F234A, L235A and G237A; (3) the anti-CD73 antibody is of the human IgG1 subtype, in which, according to the EU numbering system, the constant region of the antibody heavy chain has the following mutations: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A and G237A; or (4) the anti-CD73 antibody is of the human IgG4 subtype, where, according to the EU numbering system, the constant region of the antibody heavy chain has the following mutations: F234A and L235A; F234A and G237A; L235A and G237A; or F234A, L235A and G237A.

[036] In some embodiments of this disclosure, the bispecific antibody is provided, in which: The bispecific antibody is in the IgG-scFv form; Preferably, the first functional protein region is an immunoglobulin and the second functional protein region is a single-chain antibody; or preferably, the first functional protein region is a single-strand antibody and the second functional protein region is an immunoglobulin directed to a target other than LAG3 (e.g., CD73 or PD-1). Petition 870250099246, dated 10 / 30 / 2025, pp. 162 / 270 22 / 101

[037] In some embodiments of this disclosure, the bispecific antibody is provided, comprising: a first functional protein region targeting LAG3 and a second functional protein region targeting CD73, wherein the first functional protein region is an anti-LAG3 antibody, the anti-LAG3 antibody is an immunoglobulin, and the second functional protein region is a single-chain anti-CD73 antibody; or the first functional protein region is a single-chain anti-LAG3 antibody, the second functional protein region is an anti-CD73 antibody, and the anti-CD73 antibody is an immunoglobulin.

[038] In some embodiments of this disclosure, the bispecific antibody is provided, in which: A variable region of the immunoglobulin heavy chain comprises HCDR1-HCDR3 with amino acid sequences established in SEQ ID NOs: 9-11, respectively, and a variable region of the immunoglobulin light chain comprises LCDR1-LCDR3 with amino acid sequences established in SEQ ID NOs: 12-14, respectively; a variable region of the single-chain antibody heavy chain comprises HCDR1-HCDR3 with amino acid sequences established in SEQ ID NOs: 25-27, respectively, and a variable region of the single-chain antibody light chain comprises LCDR1-LCDR3 with amino acid sequences established in SEQ ID NOs: 28-30, respectively;or a variable region of the heavy chain of the single-chain antibody comprises HCDR1-HCDR3 with amino acid sequences established in SEQ ID NOs: 9-11, respectively, and a variable region of the light chain of the single-chain antibody comprises LCDR1-LCDR3 with amino acid sequences established in SEQ ID NOs: 12-14; Petition 870250099246, dated 10 / 30 / 2025, pp. 163 / 270 23 / 101 respectively; a variable region of the immunoglobulin heavy chain comprises HCDR1-HCDR3 with amino acid sequences established in SEQ ID NOs: 25-27, respectively, and a variable region of the immunoglobulin light chain comprises LCDR1-LCDR3 with amino acid sequences established in SEQ ID NOs: 28-30, respectively.

[039] In some embodiments of this disclosure, the bispecific antibody is provided, in which: The variable region of the immunoglobulin heavy chain is selected from the amino acid sequences presented in SEQ ID NO: 2 and SEQ ID NO: 44, and the variable region of the immunoglobulin light chain is selected from the amino acid sequences presented in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 45; the variable region of the single-chain antibody heavy chain is selected from the amino acid sequences presented in SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 46, and the variable region of the single-chain antibody light chain is selected from the amino acid sequences presented in SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 47 and SEQ ID NO: 62;or the variable region of the heavy chain of the single-chain antibody is selected from the amino acid sequences presented in SEQ ID NO: 2 and SEQ ID NO: 44, and the variable region of the light chain of the single-chain antibody is selected from the amino acid sequences presented in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 45; the variable region of the heavy chain of the immunoglobulin is selected from the amino acid sequences presented in SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 46, and the variable region of the light chain of the immunoglobulin is selected from the amino acid sequences presented in SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 47 and SEQ; Petition 870250099246, dated 10 / 30 / 2025, pp. 164 / 270 24 / 101 ID NO: 62.

[040] In some embodiments of the present disclosure, the bispecific antibody is provided, which is selected from any of the following (1) to (24): (1) the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 4; the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 31, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 32; (2) the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 4; the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 33, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 34; (3) the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 4; the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 36; (4) the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the immunoglobulin light chain has the amino acid sequence shown Petition 870250099246, dated 10 / 30 / 2025, pages 165 / 270 25 / 101 in SEQ ID NO: 4; the variable region of the heavy chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the light chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 37; (5) the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 4; the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 46, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 47; (6) the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 4; the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 62; (7) the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 44, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 45; the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 31, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 32; (8) the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 44, and the variable region of the immunoglobulin light chain has the amino acid sequence Petition 870250099246, dated 10 / 30 / 2025, pp. 166 / 270 26 / 101 presented in SEQ ID NO: 45; the variable region of the heavy chain of the single-chain antibody has the amino acid sequence presented in SEQ ID NO: 33, and the variable region of the light chain of the single-chain antibody has the amino acid sequence presented in SEQ ID NO: 34; (9) the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 44, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 45; the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 36; (10) the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 44, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 45; the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 37; (11) the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 44, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 45; the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 46, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 47; (12) the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 44, and the variable region of the immunoglobulin light chain has the amino acid sequence Petition 870250099246, dated 10 / 30 / 2025, pp. 167 / 270 27 / 101 presented in SEQ ID NO: 45; the variable region of the heavy chain of the single-chain antibody has the amino acid sequence presented in SEQ ID NO: 35, and the variable region of the light chain of the single-chain antibody has the amino acid sequence presented in SEQ ID NO: 62; (13) the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 4; the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 31, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 32; (14) the variable region of the heavy chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the light chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 4; the variable region of the heavy chain of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 33, and the variable region of the light chain of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 34; (15) the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 4; the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 36; (16) the variable region of the heavy chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the light chain of the single-chain antibody has the sequence Petition 870250099246, dated 10 / 30 / 2025, pp. 168 / 270 28 / 101 of amino acids presented in SEQ ID NO: 4; the variable region of the immunoglobulin heavy chain has the amino acid sequence presented in SEQ ID NO: 35, and the variable region of the immunoglobulin light chain has the amino acid sequence presented in SEQ ID NO: 37; (17) the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 4; the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 46, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 47; (18) the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 4; the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 62; (19) the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 44, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 45; the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 31, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 32; (20) the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 44, and Petition 870250099246, dated 10 / 30 / 2025, pp. 169 / 270 29 / 101 the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 45; the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 33, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 34; (21) the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 44, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 45; the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 36; (22) the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 44, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 45; the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 37; (23) the variable region of the heavy chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 44, and the variable region of the light chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 45; the variable region of the heavy chain of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 46, and the variable region of the light chain of Petition 870250099246, dated 10 / 30 / 2025, pages 170 / 270 30 / 101 immunoglobulin has the amino acid sequence shown in SEQ ID NO: 47; and (24) the variable region of the heavy chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 44, and the variable region of the light chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 45; The variable region of the heavy chain of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the light chain of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 62.

[041] In some embodiments of the present disclosure, the bispecific antibody is provided, in which the immunoglobulin is of the human IgG1 subtype and, according to the EU numbering system, the constant region of the immunoglobulin heavy chain has the following mutations: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A and G237A.

[042] In this disclosure, the letters before the position number represent amino acids before the mutation, and the letters after the position number represent amino acids after the mutation, unless otherwise specified.

[043] In some embodiments of the present disclosure, according to the EU numbering system, the constant region of the immunoglobulin heavy chain also has one or more mutations selected from: N297A, D265A, D270A, P238D, L328E, E233D, H268D, P271G, Petition 870250099246, dated 10 / 30 / 2025, pp. 171 / 270 31 / 101 A330R, C226S, C229S, E233P, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, G236R, G236A, L328R, A330S, P331S, H268A, E318A and K320A.

[044] In some embodiments of this disclosure, a bispecific antibody is provided, in which the immunoglobulin is of the human IgG4 subtype and, according to the EU numbering system, the constant region of the immunoglobulin heavy chain has the following mutations: F234A and L235A; F234A and G237A; L235A and G237A; or F234A, L235A and G237A.

[045] In some embodiments of the present disclosure, a bispecific antibody is provided, in which the first protein functional region and the second protein functional region are each independently 1, 2 or more in number; Preferably, the first protein functional region and the second protein functional region are linked directly or through a linker; Preferably, the variable region of the heavy chain and the variable region of the light chain of the single-chain anti-LAG3 antibody are linked directly or via a ligand; Preferably, the variable region of the heavy chain and the variable region of the light chain of the single-chain anti-CD73 antibody are linked directly or via a ligand; Preferably, the linker is, independently, the polypeptide established in SEQ ID NO: 48 (GGGGS) or a polypeptide formed by the concatenation of a plurality of (e.g., 2, 3, 4, 5, or 6) polypeptides established in SEQ ID NO: 48; Preferably, the linker is, regardless, a Petition 870250099246, dated 10 / 30 / 2025, pp. 172 / 270 32 / 101 polypeptide with one or more glycines attached to the C-terminus of the polypeptide shown in SEQ ID NO: 48 or a polypeptide formed by the concatenation of a plurality of (e.g., 2, 3, 4, 5 or 6) polypeptides shown in SEQ ID NO: 48.

[046] In some embodiments of the present disclosure, a bispecific antibody is provided, in which each single-chain antibody is attached to the C-terminus or the N-terminus of one of the two heavy chains of the immunoglobulin.

[047] In some embodiments of this disclosure, the bispecific antibody is provided, comprising: There is a first functional protein region targeting LAG3 and a second functional protein region targeting CD73; the first functional protein region is 1 in number and the second functional protein region is 2 in number. where the first functional protein region is an immunoglobulin and the second functional protein region is a single-chain antibody; An immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 38, and an immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 39; a variable region of the heavy chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 35, and a variable region of the light chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 62; or a variable region of the heavy chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 46, and a variable region of the light chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 47; Petition 870250099246, dated 10 / 30 / 2025, pp. 173 / 270 33 / 101 each single-chain antibody is attached to the C-terminus of one of the two heavy chains of the immunoglobulin; the first protein functional region and the second protein functional region are linked by a first linker; the variable region of the heavy chain of the single-chain antibody and the variable region of the light chain of the single-chain antibody are linked by a second linker; the first linker and the second linker are identical or different; Preferably, the first ligand and the second ligand each have an amino acid sequence selected independently from SEQ ID NO: 42 and SEQ ID NO: 43; Preferably, the first ligand and the second ligand have the amino acid sequence shown in SEQ ID NO: 43; Preferably, according to the EU numbering system, the constant region of the heavy chain (SEQ ID NO: 38) of the immunoglobulin comprises the following mutations: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A and G237A.

[048] In some embodiments of this disclosure, the bispecific antibody is provided, comprising: a first functional protein region targeting LAG3, and a second functional protein region targeting CD73, the first functional protein region is 2 in number, and the second functional protein region is 1 in number; where the first functional protein region is a single-chain antibody and the second functional protein region is an immunoglobulin; an immunoglobulin heavy chain has the sequence of Petition 870250099246, dated 10 / 30 / 2025, pp. 174 / 270 34 / 101 amino acids presented in SEQ ID NO: 40 and an immunoglobulin light chain has the amino acid sequence presented in SEQ ID NO: 41; a variable region of the heavy chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 2 and a variable region of the light chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 4; or a variable region of the heavy chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 44 and a variable region of the light chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 45; Each single-chain antibody is attached to the C-terminus of one of the two heavy chains of the immunoglobulin; the first protein functional region and the second protein functional region are linked by a first linker; the variable region of the heavy chain of the single-chain antibody and the variable region of the light chain of the single-chain antibody are linked by a second linker; the first linker and the second linker are identical or different; Preferably, the first ligand and the second ligand each have an amino acid sequence selected independently from SEQ ID NO: 42 and SEQ ID NO: 43; Preferably, the first ligand and the second ligand both have the amino acid sequence shown in SEQ ID NO: 43; Preferably, according to the EU numbering system, the constant region of the heavy chain (SEQ ID NO: 40) of the immunoglobulin comprises the following mutations: L234A and L235A; L234A and G237A; Petition 870250099246, dated 10 / 30 / 2025, pages 175 / 270 35 / 101 L235A and G237A; or L234A, L235A and G237A.

[049] The bispecific antibody is provided in accordance with any embodiment of this disclosure, which is for use in the treatment or prevention of a tumor, wherein: 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, gastric cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and renal cancer; Preferably, lung cancer is non-small cell lung cancer; Preferred cause of hematological malignancy is leukemia; Preferably, esophageal cancer is squamous cell carcinoma of the esophagus.

[050] Another aspect of the present disclosure relates to an isolated nucleic acid molecule encoding the bispecific antibody according to any of the embodiments of the present disclosure.

[051] Yet another aspect of the present disclosure relates to a recombinant vector comprising the nucleic acid molecule isolated from the present disclosure.

[052] Yet another aspect of the present disclosure relates to a host cell comprising the isolated nucleic acid molecule of the present disclosure or the recombinant vector of the present disclosure.

[053] Another aspect of the present disclosure relates to a method for preparing the bispecific antibody according to any of the embodiments of the present disclosure, which comprises culturing the cell. Petition 870250099246, dated 10 / 30 / 2025, pp. 176 / 270 36 / 101 host of the present disclosure under suitable conditions and isolate the bispecific antibody from cell cultures.

[054] Another aspect of the present disclosure relates to a pharmaceutical composition comprising the bispecific antibody according to any of the embodiments of the present disclosure; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable auxiliary material.

[055] Another aspect of this disclosure relates to the use of the bispecific antibody according to any of the embodiments of this disclosure in the preparation of a medicament to treat or prevent a tumor; 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, gastric cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and renal cancer; Preferably, lung cancer is non-small cell lung cancer; Preferred cause of hematological malignancy is leukemia; Preferably, esophageal cancer is squamous cell carcinoma of the esophagus.

[056] Another aspect of the present disclosure relates to a method for treating or preventing a tumor, which comprises a step of administering to a needy individual an effective amount of the bispecific antibody according to any of the embodiments of the present disclosure; Preferably, the tumor is selected from one or more of Petition 870250099246, dated 10 / 30 / 2025, pp. 177 / 270 37 / 101 ovarian cancer, esophageal cancer, melanoma, hematologic malignancy, glioblastoma, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, gastric cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer; Preferably, lung cancer is non-small cell lung cancer; Preferred cause of hematological malignancy is leukemia; Preferably, esophageal cancer is squamous cell carcinoma of the esophagus.

[057] In one or more embodiments of the present disclosure, a method is provided for treating or preventing a tumor, wherein the bispecific anti-LAG3-anti-CD73 antibody is administered in a single dose of 0.1-100 mg per kg of body weight, preferably 1-15 mg, 1-12 mg, 1-10 mg (for example, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg or 10 mg) or 6-10 mg per kg of body weight; or the bispecific anti-LAG3-anti-CD73 antibody is administered to each individual in a single dose of 10-1000 mg (for example, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg or about 1000 mg), preferably 50-500 mg, 100-400 mg, 150-300 mg, 150-250 mg or 200 mg; Ideally, administration is performed once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks, or 3 weeks; Preferably, one route of administration is intravenous infusion by drip or intravenous injection.

[058] In some embodiments, the administration of the bispecific anti-LAG3-anti-CD73 antibody is carried out in 2-week cycles. Petition 870250099246, dated 10 / 30 / 2025, pp. 178 / 270 38 / 101 (14 days) or 3 weeks (21 days) and, preferably, the bispecific anti-LAG3-anti-CD73 antibody is administered intravenously on the first day (D1) of each cycle. For example, the bispecific anti-LAG3-anti-CD73 antibody is administered once every two weeks (q2w) or three weeks (q3w).

[059] However, it should be noted that the total daily dose of the drug (e.g., the pharmaceutical composition) or the pharmaceutically active ingredient (e.g., the bispecific anti-LAG3-antiCD73 antibody) of this disclosure should be determined by an attending physician within the scope of reliable medical judgment. For each specific patient, the specific therapeutically effective dose is determined based on a variety of factors, including the specific type and severity of the tumor to be treated, the specific formulation of the drug employed, the patient's age, body weight, general health status, sex and diet, the time of administration, the route of administration, the excretion rate, the duration of treatment, other drugs used concomitantly, and similar factors well known in the medical field.For example, practice in this area involves starting administration at doses below those needed to achieve the desired therapeutic effect and gradually increasing the dose until the desired effect is achieved.

[060] In this publication, unless otherwise defined, the scientific and technical terms used herein have the meanings generally understood by specialists in the field. Furthermore, the laboratory operations of cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are routine procedures widely used in the corresponding fields. For a better understanding of this publication, definitions and explanations of related terms are provided below.

[061] As used herein, the term EC50 refers to the concentration for 50% of the maximum effect, that is, the concentration that can Petition 870250099246, dated 10 / 30 / 2025, pp. 179 / 270 39 / 101 cause 50% of the maximum effect.

[062] As used herein, the term antibody refers to an immunoglobulin molecule that generally consists of two pairs of polypeptide chains (each pair with a light chain (L) and a heavy chain (H)). Antibody light chains are classified into κ and λ light chains. Heavy chains are classified into μ, δ, γ, α, or ε. Antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE. In the light and heavy chains, the variable region and the constant region are linked by a J region of about 12 or more amino acids, and the heavy chain further comprises a D region of about 3 or more amino acids. Each heavy chain consists of a variable heavy chain region (VH) and a constant heavy chain region (CH). The constant heavy chain region consists of 3 domains (CH1, CH2, and CH3). Each light chain consists of a variable light chain region (VL) and a constant light chain region (CL).The constant region of the light chain consists of a CL domain. The constant region of the antibody can mediate the binding of immunoglobulins to tissues or host factors, including the binding of various immune system cells (e.g., effector cells) to the first component (C1q) of the classical complement system. The VH and VL regions can be subdivided into hypervariable regions (called complementarity-determining regions (CDRs)), among which conservative regions called structure regions (FRs) are distributed. Each VH and VL consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form an antibody binding site. The amino acid assignment to the regions or domains is based on Bethesda MD., Kabat's 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 Petition 870250099246, of 10 / 30 / 2025, p. 180 / 270. 40 / 101 883, or the definition of the IMGT numbering system, see the definition in Ehrenmann F, Kaas Q, Lefranc MP, IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T-cell receptors, MHC, IgSF and MhcSF[J]., Nucleic Acid Research, 2009; 38(suppl_1): D301-D307.

[063] The term antibody is not limited to any specific method for antibody production. For example, antibody includes recombinant antibody, monoclonal antibody and polyclonal antibody. The antibody may be an antibody of different isotypes, such as IgG (e.g., subtype IgG1, IgG2, IgG3 or IgG4), IgA1, IgA2, IgD, IgE or IgM.

[064] As used herein, the terms “mAb” and “monoclonal antibody” refer to an antibody or antibody fragment derived from a group of highly homologous antibodies, that is, from a group of antibody molecules identical except for natural mutations that may occur spontaneously. The monoclonal antibody is highly specific for a single epitope on an antigen. The polyclonal antibody, in relation to the monoclonal antibody, generally comprises at least two or more different antibodies that generally recognize different epitopes on an antigen. Monoclonal antibodies can generally be obtained using hybridoma technology, first reported by Kohler et al. (Kohler 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 technology (see, for example, U.S. Patent 4,816,567).

[065] As used herein, the term “humanized antibody” refers to an antibody or antibody fragment obtained when all or part of the CDR regions of a human immunoglobulin (recipient antibody) are replaced by the CDR regions of a non-human antibody (donor antibody). Petition 870250099246, dated 10 / 30 / 2025, pp. 181 / 270 41 / 101 where the donor antibody may be a non-human antibody (e.g., mouse, rat, or rabbit) with expected specificity, affinity, or reactivity. Furthermore, some amino acid residues in the structural regions (FRs) of the recipient antibody may also be replaced by amino acid residues from the corresponding non-human antibodies or by amino acid residues from other antibodies to further improve or optimize antibody performance. For more details on humanized antibodies, see, 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.

[066] As used in this document, the term “isolate” refers to obtaining it by artificial means from a natural state. If a particular “isolated” substance or component is present in nature, it may have been altered from its natural environment, or it may have been isolated from the natural environment, or both. For example, a particular non-isolated polynucleotide or polypeptide occurs naturally in a particular living animal, and the same polynucleotide or polypeptide with high purity isolated from such a natural state is termed an isolated polynucleotide or polypeptide. The term “isolate” does not exclude the existence of artificial or synthetic substances or other impurities that do not affect the activity of the substance.

[067] As used in this document, the term “vector” refers to a nucleic acid carrier into which a polynucleotide can be inserted. When a vector allows the expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction, or transfection, so that the elements of the genetic substance carried by the vector can be expressed in the host cell. Vectors are well known to specialists in the field, including, among others: plasmids; Petition 870250099246, dated 10 / 30 / 2025, pp. 182 / 270 42 / 101 Phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC); phages, such as λ phages or M13 phages; and animal viruses. Animal viruses that can be used as vectors include, among others, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40). A vector may comprise a variety of elements that control expression, including, among others, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also comprise a replication initiation site.

[068] As used in this document, the term “host cell” refers to cells into which vectors can be introduced, including, but not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast or Aspergillus cells, insect cells such as Drosophila S2 or Sf9 cells, or animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[069] As used in this document, the term “specific binding” refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen to which it is directed. In some embodiments, an antibody that binds specifically to an antigen (or an antibody that is specific for an antigen) means that the antibody binds to the antigen with an affinity (KD) less than about 10-5 M, for example, less than about 10-6 M, 10-7 M, 10-8 M, 10-9 M or 10-10 M or less.

[070] As used in this document, the term “KD” refers to a dissociation equilibrium constant for a specific interaction. Petition 870250099246, dated 10 / 30 / 2025, pp. 183 / 270 The antibody-antigen ratio (KD) is used to describe the binding affinity between the antibody and the antigen. A smaller dissociation equilibrium constant indicates a stronger antibody-antigen bond and a higher affinity between the antibody and the antigen. Generally, antibodies bind to antigens (e.g., PD-1 protein) with a dissociation equilibrium constant (KD) less than about 10⁻⁵ M, such as less than about 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, or 10⁻¹⁰ M or less. KD can be determined using methods known to experts in the field, for example, using a Fortebio molecular interaction instrument.

[071] As used herein, the terms monoclonal antibody and mAb have the same meaning and are used interchangeably; the terms polyclonal antibody and pAb have the same meaning and are used interchangeably. In addition, in the present disclosure, amino acids are generally represented by one- or three-letter abbreviations known in the field. For example, alanine may be represented by A or Ala.

[072] As used in this document, the term “pharmaceutically acceptable auxiliary material” refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, that 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, among others, pH regulators, surfactants, adjuvants, and ionic strength enhancers. For example, pH regulators include, among others, phosphate buffer; surfactants include, among others, cationic, anionic, or non-ionic surfactants such as Tween-80; ionic strength enhancers include, among others, sodium chloride.

[073] As used in this document, the term “effective quantity” refers to a quantity sufficient to obtain, or at least obtain Petition 870250099246, dated 10 / 30 / 2025, pp. 184 / 270 44 / 101 partially, a desired effect. For example, a prophylactically effective amount against a disease (e.g., a tumor) refers to an amount sufficient to prevent, stop, or delay the development of the disease (e.g., a tumor); a therapeutically effective amount refers to an amount sufficient to cure or at least partially stop the disease and its complications in patients suffering from the disease. It is undoubtedly within the competence of specialists in the field to determine such an effective amount. For example, the effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the general state of the patient's immune system, the patient's general condition such as age, body weight and sex, the route of administration and other treatments administered concomitantly, etc.

[074] As used in this document, when referring to the amino acid sequence of lymphocyte activation gene 3 (LAG3), this includes the full length of the LAG3 protein, or the LAG3 ECD extracellular fragment of LAG3, or a fragment comprising LAG3 ECD, and also includes a full-length fusion protein of the LAG3 protein or a fusion protein of LAG3 ECD, such as a fragment fused to a mouse or human IgG Fc protein fragment (mFc or hFc). However, those skilled in the art will recognize that mutations or variations (including, but not limited to, substitutions, deletions, and / or additions) in the amino acid sequence of the LAG3 protein may occur naturally or be artificially introduced without affecting its biological functions. Therefore, in this disclosure, the term “LAG3” or “LAG3 protein” will 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 fragments in their natural or artificial variants.

[075] As used here, when referring to the sequence of Petition 870250099246, dated 10 / 30 / 2025, pp. 185 / 270 45 / 101 amino acids of CD73, includes the full length of the CD73 protein, or the CD73 ECD extracellular fragment of CD73, or a fragment comprising CD73 ECD, and also includes a full-length CD73 protein fusion protein or a CD73 ECD fusion protein, as a fragment fused to a murine or human IgG Fc protein fragment (mFc or hFc). However, those skilled in the art will understand that mutations or variations (including, but not limited to, substitutions, deletions and / or additions) may occur naturally or artificially introduced in the amino acid sequence of the CD73 protein without affecting its biological functions. Therefore, in the present disclosure, the term “CD73” or “CD73 protein” shall include all such sequences, including their natural or artificial variants. Furthermore, when describing a sequence fragment of the CD73 protein, it shall also include the corresponding sequence fragments in their natural or artificial variants.

[076] In this disclosure, the terms “first” (e.g., first protein functional region or first ligand) and “second” (e.g., second protein functional region or second ligand) are used for purposes of distinction or clarity of expression and have no typical sequential meanings unless otherwise specified. Beneficial Effects of This Disclosure

[077] This disclosure achieves one or more of the following technical effects: (1) The bispecific antibody of the present disclosure can specifically bind to LAG3 and effectively block LAG3 binding to MHCII, thereby specifically relieving LAG3 immunosuppression in an organism; (2) the bispecific antibody of the present disclosure can effectively inhibit the enzymatic activity reaction of CD73; Petition 870250099246, dated 10 / 30 / 2025, pages 186 / 270 46 / 101 (3) the bispecific antibody of the present disclosure can promote the secretion of IFN-γ by T cells; (4) the first protein functional region and the second protein functional region in the bispecific antibody of this disclosure have a synergistic effect; (5) the bispecific antibody of the present disclosure can effectively inhibit tumor growth in a xenograft tumor model in mice; (6) The antibody in this disclosure can effectively treat or prevent tumors. Brief Description of the Figures

[078] Figure 1: Results of assays for binding activity of H9L8(hG4WT), H9L9(hG4WT) and H9L10(hG4WT) to the human antigen LAG3mG1Fc by ELISA.

[079] Figure 2: Results of assays for the binding activity of H9L8(hG4WT), H9L9(hG4WT) and H9L10(hG4WT) to human LAG3 antigen on the surface of the 293T-LAG3 cell by FACS.

[080] Figure 3: Results of assays for H9L8(hG4WT), H9L9(hG4WT) and H9L10(hG4WT) competing with MHC II antigen on the Raji cell membrane surface for binding to human LAG3-mG1Fc by competitive flow cytometry.

[081] Figure 4: Results of assays for the biological activity of anti-LAG3 antibodies in promoting IFN-γ secretion by mixed lymphocyte reaction (MLR).

[082] Figure 5: Results of assays for the biological activity of anti-LAG3 antibodies in promoting IL-2 secretion by mixed lymphocyte reaction (MLR).

[083] Figure 6: Results of the assays for biological activity Petition 870250099246, dated 10 / 30 / 2025, pages 187 / 270 47 / 101 of anti-LAG3 antibodies in blocking the interaction between LAG3 and MHC-II.

[084] Figure 7: Results of assays for the binding of LA5EV1 and LA5EV2 to the NT5E-His antigen by ELISA.

[085] Figure 8: Results of assays for the binding of NTLAV2 and NTLAV7 to the NT5E-His antigen by ELISA.

[086] Figure 9: Results of the assay for the binding of NTLAV8 to the NT5E-His antigen by ELISA.

[087] Figure 10: Test results for the connection of LA5EV1 and LA5EV2 to the huLAG3-mG1Fc antigen by ELISA.

[088] Figure 11: Test results for the bonding of NTLAV2 and NTLAV7 to huLAG3-mG1 Fc antigen by ELISA.

[089] Figure 12: Results of the NTLAV8 binding assay to the huLAG3-mG1Fc antigen by ELISA.

[090] Figure 13: Affinity constant of the LA5EV1 antibody for hNT5E(1-552)-His.

[091] Figure 14: Affinity constant of the LA5EV2 antibody for hNT5E(1-552)-His.

[092] Figure 15: Affinity constant of the NTLAV2 antibody for hNT5E(1-552)-His.

[093] Figure 16: Affinity constant of the NTLAV7 antibody for hNT5E(1-552)-His.

[094] Figure 17: Affinity constant of the NTLAV8 antibody for hNT5E(1-552)-His.

[095] Figure 18: Affinity constant of the 19F3H2L3(hG1DM) antibody for hNT5E(1-552)-His.

[096] Figure 19: Affinity constant of the LA5EV1 antibody for huLAG3-mG1Fc.

[097] Figure 20: Affinity constant of the LA5EV2 antibody for Petition 870250099246, dated 10 / 30 / 2025, pages 188 / 270 48 / 101 huLAG3-mG1Fc. stop stop stop

[098] Figure huLAG3-mG1Fc.

[099] Figure huLAG3-mG1Fc.

[0100] Figure huLAG3-mG1Fc.

[0101] Figure 21: 22: 23: Constant Constant Constant of of of 24: Constant affinity affinity affinity of the antibody antibody antibody affinity NTLAV2 NTLAV7 NTLAV8 antibody H9L8(hG4WT) to huLAG3-mG1Fc.

[0102] Figure 25: Affinity constant of the Relatlimab antibody for huLAG3-mG1Fc.

[0103] Figure 26: Results of assays for binding activity of NTLAV8, LA5EV2, H9L8(hG4WT) and Relatlimab for LAG3 on the surface of the 293T-LAG3 cell by FACS.

[0104] Figure 27: Results of assays for binding activity of NTLAV8, LA5EV2, 19F3H2L3(hG1DM) and CPI-006 for CD73 on the surface of the U87-MG cell by FACS.

[0105] Figure 28: Results of assays for NTLAV8, LA5EV2, Relatlimab and H9L8 competing with MHC II on the surface of the Raji cell for binding to the human antigen LAG3-mG1Fc by FACS.

[0106] Figure 29: Results of blocking the interaction between LAG3 and MHCII by the antibodies NTLAV8 and H9L8(hG4WT).

[0107] Figure 30: Results of assays for the inhibition of CD73 enzymatic activity on the cell membrane surface by bispecific anti-CD73-anti-LAG3 antibodies.

[0108] Figure 31: Results of assays for the biological activity of bispecific anti-CD73-anti-LAG3 antibodies in promoting IFN-γ secretion by mixed lymphocyte reaction. Petition 870250099246, dated 10 / 30 / 2025, pp. 189 / 270 49 / 101

[0109] Figure 32: Results of assays for the potential cellular phagocytic activity mediated by bispecific anti-CD73-anti-LAG3 antibodies in CHO-K1-LAG3-CD73 target cells.

[0110] Figure 33: Results of the pharmacodynamic evaluation of the bispecific anti-CD73-anti-LAG3 antibody in a murine model subcutaneously grafted with tumor cells.

[0111] Figure 34: Results of the effects of the bispecific anti-CD73-anti-LAG3 antibody on body weight in a murine model subcutaneously grafted with tumor cells. Detailed Description of the Invention

[0112] The methods of carrying out this disclosure will be described in detail below with reference to examples. Experts in the field will understand that the following examples are for illustrative purposes only and should not be construed as limitations to the scope of this disclosure. Examples where specific technologies or conditions are not specified are carried out in accordance with the technologies or conditions described in publications in the field (e.g., see Molecular Cloning: A Laboratory Manual, by J. Sambrook et al. and translated by Huang Peitang et al., third edition, Science Press) or in accordance with the product information leaflet. The reagents or instruments used are commercially available conventional products unless their manufacturers are specified.

[0113] The positive control antibody, Relatlimab, has sequences referenced to U.S. Patent Publication No. US20160326248A1, wherein, for the heavy chain amino acid sequence, see SEQ ID NO: 1 of this patent publication, and for the light chain amino acid sequence, see SEQ ID NO: 2 of this patent publication. Relatlimab is an anti-LAG3 antibody.

[0114] Heavy chain amino acid sequence of Petition 870250099246, dated 10 / 30 / 2025, pp. 190 / 270 50 / 101 Relatlimabe: QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPG KGLEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYC AFGYSDYEYNWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTK TYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTL MISRTP EVTCVVVDVS QEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQ EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 23).

[0115] Amino acid sequence of the light chain of Relatlimab: EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAP RLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTF GQGTNLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC (SEQ ID NO: 24).

[0116] The 14C12H1L1(hG1TM) antibody is an anti-PD-1 antibody prepared by Akeso Biopharma Inc.

[0117] Amino acid sequence of the heavy chain of 14C12H1L1(hG1TM): EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPG KGLDWVATISGGGRYTYYPDSVKRFTISRDNSKNNLYLQMNSLRAEDTALYY CANRYGEAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS Petition 870250099246, dated 10 / 30 / 2025, pp. 191 / 270 51 / 101 KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 21).

[0118] Amino acid sequence of the 14C12H1L1(hG1TM) light chain: DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKS PKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPL TFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC (SEQ ID NO: 22).

[0119] Amino acid sequence of human LAG3-mG1 Fc: LQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQH QPDSGPPAAAPGHPLAPPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPL QPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLG QASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPH HHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVY AGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLED VSQAGTYTCHIHLQEQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSG QERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYF TELSSPGAQRSGRAPGALPAGHLKLENLYFQGPRPPCPCPCPCPCPPCPPLL GGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQT QTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGS VRAPQVYVLPPPEEEMTKKQVTLTCCMVTDFMPEDIYVEWTEKN EPVLDSDGSYFMYSKLRVEKNWVERNSYSCSVVHEGLHNHHTTKSFSRTP GK (SEQ ID NO: 60).

[0120] Amino acid sequence of hNT5E(1-552)-His: WELTILHTNDVHSRLEQTSEDSSSKCVNASRCMGGVARLFTKV QQIRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVAHFMNALRYDAMALGNHEF DNGVEGLIEPLLKEAKFPILSANIKAKGPLASQISGLYLPYKVLPVGDEVVGIVG YTSKETPFLSNPGTNLVFEDEITALQPEWDKLKTLNVNKIIALGHSGFMDKLIAQ Petition 870250099246, de 30 / 10 / 2025, pág. 192 / 270 52 / 101 KVRGVDVVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPVVQAYAF GKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQEL GKTIVYLDGSSQSCRFRECNMGNLICDAMINNNLRHTDEMFWNHVSMCILNG GGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQ STGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVIL PNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKFSTG SHHHHHH (SEQ ID NO: 61).

[0121] The 293T-LAG3 cell line was constructed by Akeso Biopharma Inc. The 293T-LAG3 cell line was produced by viral infection of HEK293T cells using 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), where the lentivirus expression vector used was plenti6.3 / V5huLAG3FL-BSD (LAG3, Genebank ID: NP_002277.4; plenti6.3 / V5-BSD vector, acquired from Invitrogen, Cat. No. K5315-20).

[0122] The Raji-PDL1 cell line was constructed by Akeso Biopharma Inc. The Raji-PDL1 cell line was produced by viral infection of Raji cells using 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), where the lentivirus expression vector used was plenti6.3 / V5-PDL1 (PDL1, Genebank ID: NP_054862.1; plenti6.3 / V5 vector, acquired from Invitrogen, Cat. No. K5315-20).

[0123] The Jurkat-NFAT-PD1-LAG3 cell line was constructed by Akeso Biopharma Inc. The Jurkat-NFAT-PD1-LAG3 cell line was produced by viral infection of PD-1 effector cells (CPM, manufacturer: Promega, Cat. No. J112A) using third-generation lentiviral systems (see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Petition 870250099246, dated 10 / 30 / 2025, pp. 193 / 270 53 / 101 Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D and Naldini L., J Virol., 1998. 72(11): 8463-8471), in which the lentivirus expression vector used was pCDHhuLAG3FL-RFP-NEO (LAG3, Genebank ID: NP_002277.4; pCDH-CMVMCS-EF1-RFP+Neo vector, acquired from Youbio, Cat. No. VT9005).

[0124] The CHO-K1-LAG3-CD73 cell line was constructed by Akeso Biopharma Inc. The CHO-K1-LAG3-CD73 cell line was produced by viral infection of CHO-K1 cells (manufacturer: Institute of Basic Medical Sciences of the Chinese Academy of Medical Sciences, Cat. No. 3111C0001CCC000004) using 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), wherein the lentivirus expression vectors used were plenti6.3 / V5-huLAG3FL-BSD (LAG3, Genebank) ID: NP_002277.4; plenti6.3 / V5-BSD vector, acquired from Invitrogen, Cat. No. K5315-20) and pure pCDH-GFP-NT5EFL (NT5E, Genebank ID: NP_002517.1; pure pCDH vector, acquired from Youbio).

[0125] The sequence information for the positive control antibody MEDI9447 is as follows. MEDI9447 is an anti-CD73 antibody.

[0126] Amino acid sequence of the heavy chain of MEDI9447: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAYSWVRQAPGK GLEWVSAISGSGGRTYYADSVKRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARLGYGRVDEWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKRVEPKSCDKTHTCPPPCPAPEFEGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 56). Petition 870250099246, dated 10 / 30 / 2025, pp. 194 / 270 54 / 101

[0127] Amino acid sequence of the MEDI9447 light chain: QSVLTQPPSASGTPGQRVTISCSGSLSNIGRNPVNWYQQLPGT APKLLIYLDNLRLSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCATWDDSH PGWTFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVT VAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVT HEGSTVEKTVAPTECS (SEQ ID NO: 57).

[0128] The sequence information for the positive control antibody CPI-006 is as follows. CPI-006 is an anti-CD73 antibody.

[0129] Amino acid sequence of the heavy chain of CPI-006: QVQLVQSGAEVEKPGASVKVSCKASGYTFTSYWITWVRQAPGQG LEWMGDIYPGSGNTNYNEKFKTRVTITADKSTSTAYMELSSLRSEDTAVYYCAKE GGLTTEDYALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 58).

[0130] Amino acid sequence of the CPI-006 light chain: EIVLTQSPATLSLSPGERATLSCRASKNVSTSGYSYMHWYQQK PGQAPRLLIYLASNLESGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQHSRE LPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC (SEQ ID NO: 59). Example of Preparation 1: Design and Preparation of AntiLAG3 Antibodies 1. Antibody Design

[0131] The inventors creatively designed a series of Petition 870250099246, dated 10 / 30 / 2025, pp. 195 / 270 55 / 101 antibody sequences based on the known sequence of the LAG3 protein (NCBI Reference Sequence: NP_002277.4), its three-dimensional crystal structure, etc. Through extensive screening and testing, finally, humanized monoclonal antibodies that bind specifically to LAG3 were obtained, named H9L8, H9L9, and H9L10, respectively. The amino acid sequences of the variable regions of the heavy and light chains of the monoclonal antibodies and their coding sequences are as follows.

[0132] Nucleotide sequence of the variable region of the H9v heavy chain of H9L8 (360 bp): CAGGTGCAGCTGCAGCAGTGGGGAGCTGGACTGCTGAAACC TAGCGAGACACTGAGCCTGACCTGTGCTGTGTACGGCGGATCTATCAGCG ATTACTACTGGAACTGGATCAGGCAGCCCCCTGGAAAGGGACTGGAATGG ATCGGAGAGATCAACTACAGGGCACCACCAACTCCAATCCCTCTCTGAA GAGCAGGGTGACACTGAGCCTCGACACAAGCAAGAATCAGTTCAGCCTGA AGCTGAGGTCCGTGACCGCTGCTGATACAGCTGTGTACTACTGTGCCTTC GGCTACAGCGATTACGAGTACGATTGGTTCGACCCTTGGGGCCAGGGAAC ACTGGTTACAGTGAGCTCC (SEQ ID NO: 1).

[0133] Amino acid sequence of the variable region of the H9v heavy chain of H9L8 (120 aa): QVQLQQWGAGLLKPSETLSLTCAVYGGSISDYYWNWIRQPPGK GLEWIGEINYRGTTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAF GYSDYEYDWFDPWGQGTLVTVSS (SEQ ID NO: 2).

[0134] Nucleotide sequence of the variable region of the L8v light chain of H9L8 (321 bp): GAGATCGTTCTGACCCAGAGCCCAGCTACACTGAGCCTGTCT CCTGGAGAGAGGGCTACACTGTCCTGCAGAGCTAGCCAGACCATCAGCA GCTACCTGGCTTGGTACCAGCAGAAGCCTGGCCAAGCTCCAAGGCTGCTG ATCTACGACGCCTCTAATAGGGCCACCGGCATCCCTGCTAGATTCTCTGG Petition 870250099246, dated 10 / 30 / 2025, pp. 196 / 270 56 / 101 AAGCGGCAGCGGAACCGACTTTACACTGACAATCAGCTCCCTGGAGCCCG AGGATTTCGCTGTTTACTACTGTCAGCAGCGCAGCAACTGGCCCATCACAT TCGGACAGGGCACAAATCTGGAGATCAAG (SEQ ID NO: 3).

[0135] Amino acid sequence of the variable region of the L8v light chain of H9L8 (107 aa): EIVLTQSPATLSLSPGERATLSCRASQTISSYLAWYQQKPGQAP RLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITF GQGTNLEIK (SEQID NO: 4).

[0136] The nucleotide sequence of the variable region of the H9v heavy chain of H9L9 is identical to the nucleotide sequence of the variable region of the H9v heavy chain of H9L8, as established in SEQ ID NO: 1.

[0137] The amino acid sequence of the variable region of the H9v heavy chain of H9L9 is identical to the amino acid sequence of the variable region of the H9v heavy chain of H9L8, as established in SEQ ID NO: 2.

[0138] Nucleotide sequence of the variable region of the L9v light chain of H9L9 (321 bp): GAGATCGTTCTGACCCAGAGCCCAGCTACACTGAGCCTGTCT CCTGGAGAGAGGGCTACACTGTCCTGCAGAGCTAGCCAGACCATCAGCA GCTACCTGGCTTGGTACCAGCAGAAGCCTGGCCAAGCTCCAAGGCTGCTG ATCTACGACGGCTCTAATAGGGCCACCGGCATCCCTGCTAGATTCTCTGG AAGCGGCAGCGGAACCGACTTTACACTGACAATCAGCTCCCTGGAGCCCG AGGATTTCGCTGTTTACTACTGTCAGCAGCGCAGCAACTGGCCCCTCACAT TCGGACAGGGCACAAATCTGGAGATCAAG (SEQ ID NO: 5).

[0139] Amino acid sequence of the variable region of the L9v light chain of H9L9 (107 bp): EIVLTQSPATLSLSPGERATLSCRASQTISSYLAWYQQKPGQAP RLLIYDGSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTF GQGTNLEIK (SEQ ID NO: 6). Petition 870250099246, dated 10 / 30 / 2025, pp. 197 / 270 57 / 101

[0140] The nucleotide sequence of the variable region of the H9v heavy chain of H9L10 is identical to the nucleotide sequence of the variable region of the H9v heavy chain of H9L8, as established in SEQ ID NO: 1.

[0141] The amino acid sequence of the variable region of the H9v heavy chain of H9L10 is identical to the amino acid sequence of the variable region of the H9v heavy chain of H9L8, as established in SEQ ID NO: 2.

[0142] Nucleotide sequence of the variable region of the L10v light chain of H9L10 (321 bp): GAGATCGTTCTGACCCAGAGCCCAGCTACACTGAGCCTGTCT CCTGGAGAGAGGGCTACACTGTCCTGCAGAGCTAGCCAGTCCATCAGCAG CTACCTGGCTTGGTACCAGCAGAAGCCTGGCCAAGCTCCAAGGCTGCTGA TCTACGACGGCTCTAATAGGGCCACCGGCATCCCTGCTAGATTCTCTGGA AGCGGCAGCGGAACCGACTTTACACTGACAATCAGCTCCCTGGAGCCCGA GGATTTCGCTGTTTACTACTGTCAGCAGCGCAGCAACTGGCCCATCACATT CGGACAGGGCACAAATCTGGAGATCAAG (SEQ ID NO: 7).

[0143] Amino acid sequence of the variable region of the L10v light chain of H9L10 (107 bp): EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAP RLLIYDGSNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITF GQGTNLEIK (SEQ ID NO: 8).

[0144] The amino acid sequences of the H9L8 antibody CDRs are as follows (according to the IMGT numbering system): HCDR1: GGSISDYY (SEQ ID NO: 9); HCDR2: INYRGTT (SEQ ID NO: 10); HCDR3: AFGYSDYEYDWFDP (SEQ ID NO: 11); LCDR1: QTISSY (SEQ ID NO: 12); LCDR2: DAS (SEQ ID NO: 13); LCDR3: QQRSNWPIT (SEQ ID NO: 14). Petition 870250099246, dated 10 / 30 / 2025, pp. 198 / 270 58 / 101

[0145] The amino acid sequences of the H9L9 antibody CDRs are as follows (according to the IMGT numbering system): HCDR1: GGSISDYY (SEQ ID NO: 9); HCDR2: INYRGTT (SEQ ID NO: 10); HCDR3: AFGYSDYEYDWFDP (SEQ ID NO: 11); LCDR1: QTISSY (SEQ ID NO: 12); LCDR2: DGS (SEQ ID NO: 15); LCDR3: QQRSNWPLT (SEQ ID NO: 16).

[0146] The amino acid sequences of the antibody CDRs H9L10 are as follows (according to the IMGT numbering system): HCDR1: GGSISDYY (SEQ ID NO: 9); HCDR2: INYRGTT (SEQ ID NO: 10); HCDR3: AFGYSDYEYDWFDP (SEQ ID NO: 11); LCDR1: QSISSY (SEQ ID NO: 17); LCDR2: DGS (SEQ ID NO: 15); LCDR3: QQRSNWPIT (SEQ ID NO: 14). 2. Expression and purification of humanized antibodies H9L8(hG4WT), H9L9(hG4WT) and H9L10(hG4WT)

[0147] The heavy chain cDNA sequences (the coding sequences of the variable regions are shown in SEQ ID NO: 1; the constant regions were the C regions of the gamma-4 chain of Ig) of H9L8(hG4WT), H9L9(hG4WT) and H9L10(hG4WT), the light chain cDNA sequence (the coding sequence of the variable region is shown in SEQ ID NO: 3; the constant region was the C region of the kappa chain of human Ig) of H9L8(hG4WT), the light chain cDNA sequence (the coding sequence of the variable region is shown in SEQ ID NO: 5; the constant region was the C region of the kappa chain of human Ig) of H9L9(hG4WT) and the light chain cDNA sequence (the sequence Petition 870250099246, dated 10 / 30 / 2025, pp. 199 / 270 The variable region encoding 59 / 101 is presented in SEQ ID NO: 7; the constant region was the C region of the human Ig kappa chain) of H9L10(hG4WT) were cloned separately into pUC57simple vectors (provided by GenScript), and the pUC57simple-H9, pUC57simple-L8, pUC57simple-L9, and pUC57simple-L10 plasmids were obtained, respectively. The pUC57simple-H9, pUC57simple-L8, pUC57simple-L9, and pUC57simple-L10 plasmids were digested (HindIII and EcoRI). The heavy and light chains isolated by electrophoresis were subcloned separately into pcDNA3.1 vectors, and the recombinant plasmids were extracted for cotransfection of 293F cells. After 7 days of cell culture, the culture solution was separated by high-speed centrifugation, and the supernatant was concentrated and loaded onto a HiTrap MabSelect SuRe column. The protein was eluted in a single step with an elution buffer. The target sample was isolated, and the buffer was changed to PBS.

[0148] Amino acid sequence of the constant region of the heavy chain of H9L8(hG4WT), H9L9(hG4WT) or H9L10(hG4WT): ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDK RVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSC SVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 20).

[0149] Amino acid sequence of the constant region of the light chain of H9L8(hG4WT), H9L9(hG4WT) or H9L10(hG4WT): RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC (SEQ ID NO: 19). Petition 870250099246, dated 10 / 30 / 2025, pages 200 / 270 60 / 101 3. Expression and purification of the humanized antibody H9L8(hG1WT)

[0150] Based on the variable region of the H9L8(hG4WT) antibody described above, the H9L8(hG1WT) antibody was obtained using the C region of the gamma-1 Ig chain as the constant region of the heavy chain.

[0151] Amino acid sequence of the heavy chain of H9L8(hG1WT): QVQLQQWGAGLLKPSETLSLTCAVYGGSISDYYWNWIRQPPG KGLEWIGEINYRGTTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYC AFGYSDYEYDWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 38).

[0152] Amino acid sequence of the H9L8(hG1WT) light chain: EIVLTQSPATLSLSPGERATLSCRASQTISSYLAWYQQKPGQAP RLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITF GQGTNLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC (SEQ ID NO: 39). Example of Preparation 2: Preparation of Human Anti-Lysozyme Antibody from Chicken Eggs

[0153] The human anti-chicken egg lysozyme IgG antibody sequence (anti-HEL, or human IgG, abbreviated as hIgG) was derived from the variable region sequence of the Fab F10.6.6 sequence. In the study reported by Petition 870250099246, dated 10 / 30 / 2025, pages 201 / 270 61 / 101 Acierno et al., entitled “Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies” (Acierno et al., J Mol Biol., 2007; 374(1): 130-46), the preparation method was as follows: Nanjing GenScript Biotech was tasked with performing amino acid codon optimization and gene synthesis on heavy and light chain genes (complete sequence or variable region) of the human IgG antibody. Consulting the standard technologies presented in the Handbook of Molecular Cloning: A Laboratory Manual (Third Edition) and using standard molecular cloning techniques such as PCR, enzymatic digestion, DNA gel extraction, ligation transformation, colony PCR, or identification by enzymatic digestion, the heavy and light chain genes were subcloned into the antibody heavy chain expression vector and the antibody light chain expression vector of the mammalian expression system, respectively. The heavy and light chain genes of the recombinant expression vectors were subsequently sequenced and analyzed.After verifying sequence correctness, a medium to large quantity of endotoxin-free expression plasmids was prepared, and the heavy and light chain expression plasmids were transiently co-transfected into HEK293 cells for recombinant antibody expression. After 7 days of culture, cell culture solutions were collected and subjected to affinity purification on an rProtein A (GE) column, and the quality of the resulting antibody sample was determined using standard SDSPAGE and SEC-HPLC analytical techniques. Unless otherwise indicated, hIgG1, hIgG1DM, and hIgG4WT used in this disclosure were anti-HEL isotype control antibodies with constant region sequences of hIgG1, hG1DM, and hG4WT, respectively, prepared in the laboratory of Akeso Biopharma Inc. Petition 870250099246, dated 10 / 30 / 2025, pages 202 / 270 62 / 101 Example of Preparation 3: Design and Preparation of AntiCD73 Antibodies

[0154] 1. For sequences and preparation methods of anti-CD73 antibodies 19F3, 19F3H1L1, 19F3H2L2 and 19F3H2L3, reference was made to Chinese Patent Application CN113527489A, and part of the sequences is cited below: 19F3, 19F3H1L1, 19F3H2L2, and 19F3H2L3 have 3 identical heavy chain CDRs and 3 identical light chain CDRs (according to the IMGT numbering system): HCDR1: GYSFTGYT (SEQ ID NO: 25) HCDR2: INPYNAGT (SEQ ID NO: 26) HCDR3: ARSEYRYGGDYFDY (SEQ ID NO: 27) LCDR1: QSLLNSSNQKNY (SEQ ID NO: 28) LCDR2: FAS (SEQ ID NO: 29) LCDR3: QQHYDTPYT (SEQ ID NO: 30).

[0155] Amino acid sequence of the variable region of the 19F3 heavy chain, with CDR sequences underlined: EVQLQQSGPELVKPGASMRMSCKASGYSFTGYTMNWVKQSH GKNLEWIGLINPYNAGTSYNQKFKGKATLTVDKSSSTAYMELLSLTSEDSAVY YCARSEYRYGGDYFDYWGQGTTLTVSS (SEQ ID NO: 31).

[0156] Amino Sequence of amino acids from the variable region of the 19F3 light chain, with CDR sequences underlined: DIVMTQSPSSLAMSVGQKVTMSCKSSQSLLNSSNQKNYLAWYQ QKPGQSPKLLVYFASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQ HYDTPYTFGGGTKLEIK (SEQ ID NO: 32).

[0157] Amino acid sequence of the variable region of the heavy chain of 19F3H1L1, with CDR sequences underlined: QVQLQQSGAEVVKPGASMKMSCKASGYSFTGYTMNWVKQAH GQNLEWIGLINPYNAGTSYNQKFQGKATLTVDKSTSTAYMELSSLRSEDTAVY Petition 870250099246, dated 10 / 30 / 2025, pp. 203 / 270 63 / 101 YCARSEYRYGGDYFDYWGQGTTLTVSS (SEQ ID NO: 33).

[0158] Amino acid sequence of the variable region of the 19F3H1L1 light chain, with CDR sequences underlined: DIVMTQSPSSLAMSVGERVTMSCKSSQSLLNSSNQKNYLAWYQ QKPGQAPKLLVYFASTRESGVPDRFSGSGSGTDFTLTISSVQAEDLADYFCQ QHYDTPYTFGGGTKLEIK (SEQ ID NO: 34).

[0159] Amino acid sequence of the variable region of the heavy chain of 19F3H2L2, with CDR sequences underlined: QVQLVQSGAEVVKPGASVKVSCKASGYSFTGYTMNWVRQAPG QNLEWIGLINPYNAGTSYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYY CARSEYRYGGDYFDYWGQGTTLTVSS (SEQ ID NO: 35).

[0160] Amino acid sequence of the variable region of the 19F3H2L2 light chain, with CDR. Underlined sequences: DIVMTQSPSSLAVSVGERVTISCKSSQSLLNSSNQKNYLAWYQQ KPGQAPKLLIYFASTRESGVPDRFSGSGSGTDFTLTISSVQAEDVADYYCQQH YDTPYTFGGGTKLEIK (SEQ ID NO: 36).

[0161] Amino acid sequence of the variable region of the heavy chain of 19F3H2L3, with CDR sequences underlined: QVQLVQSGAEVVKPGASVKVSCKASGYSFTGYTMNWVRQAPG QNLEWIGLINPYNAGTSYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYY CARSEYRYGGDYFDYWGQGTTLTVSS (SEQ ID NO: 35).

[0162] Amino acid sequence of the variable region of the 19F3H2L3 light chain, with CDR sequences Underlined: DIVMTQSPSSLAVSVGERVTISCKSSQSLLNSSNQKNYLAWYQQ KPGQAPKLLIYFASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQH YDTPYTFGGGTKLEIK (SEQ ID NO: 37). 2. Preparation of the humanized antibody 19F3H2L3

[0163] The constant regions of the heavy chain were all the Petition 870250099246, dated 10 / 30 / 2025, pp. 204 / 270 64 / 101 region C of the gamma-1 chain of Ig, and the constant regions of the light chain were all region C of the kappa chain of Ig.

[0164] The heavy chain cDNA and light chain cDNA of 19F3H2L3 were cloned separately into pUC57simple vectors (provided by GenScript) to obtain pUC57simple-19F3H2 and pUC57simple-19F3L3, respectively. Based on standard techniques described in Molecular Cloning: A Laboratory Manual (Third Edition), the full-length heavy and light chain genes synthesized by digestion with EcoRI and HindIII were subcloned separately into pcDNA3.1 expression vectors via digestion with a restriction enzyme (EcoRI and HindIII) to obtain the pcDNA3.1-19F3H2 and pcDNA3.1-19F3L3 expression plasmids, and the heavy / light chain genes from the recombinant expression plasmids were subjected to sequencing analysis. Next, the designed gene combination, comprising the corresponding recombinant light and heavy chain plasmids (pcDNA3.1-19F3H2 / pcDNA3.1-19F3L3), was co-transfected into 293F cells, and the culture solution was collected and purified.After verifying the correctness of the sequences by sequencing, endotoxin-free expression plasmids were prepared and transiently transfected into HEK293 cells for antibody expression. After 7 days, the cell culture solution was collected and subjected to affinity purification on a Protein A column to obtain the humanized antibody.

[0165] Heavy chain amino acid sequence of 19F3H2L3: QVQLVQSGAEVVKPGASVKVSCKASGYSFTGYTMNWVRQAPG QNLEWIGLINPYNAGTSYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYY CARSEYRYGGDYFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST Petition 870250099246, dated 10 / 30 / 2025, pages 205 / 270 65 / 101 YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 40).

[0166] Amino acid sequence of the 19F3H2L3 light chain: DIVMTQSPSSLAVSVGERVTISCKSSQSLLNSSNQKNYLAWYQQ KPGQAPKLLIYFASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQH YDTPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC (SEQ ID NO: 41). 3. Preparation of the humanized antibody 19F3H2L3(hG1DM)

[0167] The constant region of the light chain of the antibody 19F3H2L3(hG1DM) was the C region of the kappa chain of Ig, as established in SEQ ID NO: 19.

[0168] Based on the constant region of the heavy chain of the C region of the Ig gamma-1 chain (SEQ ID NO: 18), a humanized antibody was obtained by introducing a leucine-to-alanine point mutation at position 234 (L234A) and a leucine-to-alanine point mutation at position 235 (L235A), and was designated as 19F3H2L3(hG1DM).

[0169] The heavy chain cDNA and light chain cDNA of 19F3H2L3(hG1DM) were cloned separately into pUC57simple vectors (provided by GenScript) to obtain pUC57simple-19F3H2(hG1DM) and pUC57simple-19F3L3, respectively. Based on standard techniques described in Molecular Cloning: A Laboratory Manual (Third Edition), the complete heavy and light chain genes synthesized by digestion with EcoRI and HindIII were subcloned separately into pcDNA3.1 expression vectors via digestion with a restriction enzyme (EcoRI and HindIII) to obtain the pcDNA3.1-19F3H2(hG1DM) and expression plasmids. Petition 870250099246, dated 10 / 30 / 2025, pp. 206 / 270 66 / 101 pcDNA3.1-19F3L3, and the heavy / light chain genes of the recombinant expression plasmids were subjected to sequencing analysis. Subsequently, the designed gene combination, comprising the corresponding recombinant light and heavy chain plasmids (pcDNA3.119F3H2(hG1DM) / pcDNA3.1-19F3L3), was co-transfected into 293F cells, and the culture solution was collected and purified. After verifying sequence correctness by sequencing, endotoxin-free expression plasmids were prepared and transiently transfected into HEK293 cells for antibody expression. After 7 days, the cell culture solution was collected and subjected to affinity purification on a Protein A column to obtain the humanized antibody.

[0170] Complete heavy chain of 19F3H2L3(hG1DM): QVQLVQSGAEVVKPGASVKVSCKASGYSFTGYTMNWVRQAPG QNLEWIGLINPYNAGTSYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYY CARSEYRYGGDYFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 49).

[0171] Complete light chain of 19F3H2L3(hG1 DM): DIVMTQSPSSLAVSVGERVTISCKSSQSLLNSSNQKNYLAWYQQ KPGQAPKLLIYFASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQH YDTPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC (SEQ ID NO: 50). Petition 870250099246, dated 10 / 30 / 2025, pp. 207 / 270 67 / 101 Example of Preparation 4: Design and Preparation of Bispecific Anti-CD73-Anti-LAG3 Antibodies 1. Sequence Design

[0172] The structural formats of the bispecific antibodies NTLAV2, NTLAV7, NTLAV8, LA5EV1 and LA5EV2 of this disclosure are in Morrison Format (IgG-scFv), i.e., the C-terminus of two heavy chains of an IgG antibody are linked, each µm, to the scFv fragment of another antibody, and the main design of the heavy and light chain composition is shown in Table 1 below. Table 1. Diagram of the composition of heavy and light chains of bispecific antibodies. Anticorpo biespecífico n°. Porção of imunoglobulina Ligante porção scFv Corrente pesada Cadeia le Região Variável de Cadeia Pesada Ligante Região Variável da Cadeia leve NTLAV2 19FH2 (SEQ ID N°: 40) 19L3 (SEQ ID N°: 41) (GGGGS)4 (SEQ ID N°: 43) H9v(M) (SEQ ID N°: 44) (GGGGS)4 (SEQ ID N°: 43) L8v(M) (SEQ ID N°: 45) NTLAV7 19FH2 (SEQ ID N°: 40) 19L3 (SEQ ID N°: 41) (GGGGS)3 (SEQ ID N°: 42) H9v (SEQ ID N°: 2) (GGGGS)4 (SEQ ID N°: 43) L8v (SEQ ID N°: 4) NTLAV8 19FH2 (SEQ ID N°: 40) 19L3 (SEQ ID N°: 41) (GGGGS)3 (SEQ ID N°: 42) H9v(M) (SEQ ID N°: 44) (GGGGS)4 (SEQ ID N°: 43) L8v(M) (SEQ ID N°: 45) LA5EV1 H9 (SEQ ID N°: 38) L8 (SEQ ID N°: 39) (GGGGS)4 (SEQ ID N°: 43) 19FH2v (SEQ ID N°: 35) (GGGGS)4 (SEQ ID N°: 43) 19L3v(M2) (SEQ ID N°: 62) LA5EV2 H9 (SEQ ID N°: 38) L8 (SEQ ID N°: 39) (GGGGS)4 (SEQ ID N°: 43) 19FH2v(M) (SEQ ID N°: 46) (GGGGS)4 (SEQ ID N°: 43) 19L3v(M1) (SEQ ID N°: 47)

[0173] Na Tabela 1 acima: (1) Sequence of amino acids do ligante (GGGGS)3: GGGGSGGGGSGGGGS (SEQ ID NO: 42) (2) Amino acid sequence of the ligand (GGGGS)4: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 43).

[0174] Those marked with a v in the lower right corner refer to the corresponding variable region of the heavy chain or the corresponding variable region of the light chain. For those without the v marking, the Petition 870250099246, dated 10 / 30 / 2025, pp. 208 / 270 68 / 101 The corresponding heavy or light chain is the total length comprising the constant region. Unless otherwise indicated, for all amino acid sequences of these variable regions or the complete lengths and nucleotide sequences encoding them, reference has been made to the corresponding sequences described in the preparation examples above.

[0175] In Table 1 described above, H9V(M), L8V(M), 19FH2V(M), 19L3V(M1), and 19L3V(M2) in the scFv fragments of the antibodies were generated by introducing specific amino acid mutations in the corresponding structural regions of H9V, L8V, 19FH2V, and 19L3V, thus effectively optimizing the antibody structures and increasing antibody efficacy. Amino acid sequence of H9V(M): QVQLQQWGAGLLKPSETLSLTCAVYGGSISDYYWNWIRQPPGK CLEWIGEINYRGTTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAF GYSDYEYDWFDPWGQGTLVTVSS (SEQ ID NO: 44).

[0176] Amino acid sequence of L8V(M): EIVLTQSPATLSLSPGERATLSCRASQTISSYLAWYQQKPGQAP RLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITF GCGTNLEIK (SEQ ID NO: 45).

[0177] Amino acid sequence of 19FH2V(M): QVQLVQSGAEVVKPGASVKVSCKASGYSFTGYTMNWVRQAPG QCLEWIGLINPYNAGTSYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYY CARSEYRYGGDYFDYWGQGTTLTVSS (SEQ ID NO: 46).

[0178] Sequence of amino acids of 19L3V(M1): DIVMTQSPSSLAVSVGERVTISCKSSQSLLNSSNQKNYLAWYQQ KPGQAPKLLIYFASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQH YDTPYTFGCGTKLEIKR (SEQ ID NO: 47).

[0179] Sequence of amino acids of 19L3V(M2): DIVMTQSPSSLAVSVGERVTISCKSSQSLLNSSNQKNYLAWYQQ Petition 870250099246, on 10 / 30 / 2025, page. 209 / 270 69 / 101 KPGQAPKLLIYFASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQH YDTPYTFGGGTKLEIKR (SEQ ID NO: 62).

[0180] Based on the bispecific antibodies designed in Table 1 described above, by introducing a leucine-to-alanine point mutation at position 234 (L234A), a leucine-to-alanine point mutation at position 235 (L235A), and a glycine-to-alanine point mutation at position 237 (G237A) in the heavy chains, bispecific antibodies comprising the above mutation sites were obtained, designated as NTLAV2(hG1TM), NTLAV7(hG1TM), NTLAV8(hG1TM), LA5EV1(hG1TM), and LA5EV2(hG1TM).

[0181] Amino acid sequence of the NTLAV2(hG1TM) heavy chain: QVQLVQSGAEVVKPGASVKVSCKASGYSFTGYTMNWVRQAPG QNLEWIGLINPYNAGTSYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYY CARSEYRYGGDYFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGKGGGGS GGGGSGGGGSGGGSQVQLQQWGAGLLKPSETLSLTCAVYGGSISDYYWN WIRQPPGKCLEWIGEINYRGTTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAA DTAVYCAFGYSDYEYDWFDPWGQGTLVTVSSGGGGSGGGGSGGGGSGG GGSEIVLTQSPATLSLSPGERATLSCRASQTISSYLAWYQQKPGQAPRLLIYDA SNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGCGTNL EIK (SEQ ID NO: 51).

[0182] Sequência de aminoados da caiade pesada de Petition 870250099246, de 30 / 10 / 2025, pág. 210 / 270 70 / 101 NTLAV7(hG1TM): QVQLVQSGAEVVKPGASVKVSCKASGYSFTGYTMNWVRQAPG QNLEWIGLINPYNAGTSYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYY CARSEYRYGGDYFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGG GGSGGGGSQVQLQQWGAGLLKPSETLSLTCAVYGGSISDYYWNWIRQPPGK GLEWIGEINYRGTTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYCAF GYSDYEYDWFDPWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTQ SPATLSLSPGERATLSCRASQTISSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTNLEIK (SEQ ID NO: 52).

[0183] Amino acid sequence of the heavy chain of NTLAV8(hG1TM): QVQLVQSGAEVVKPGASVKVSCKASGYSFTGYTMNWVRQAPG QNLEWIGLINPYNAGTSYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYY CARSEYRYGGDYFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGG GGSGGGGSQVQLQQWGAGLLKPSETLSLTCAVYGGSISDYYWNWIRQPPGK Petition 870250099246, dated 10 / 30 / 2025, pages 211 / 270 71 / 101 CLEWIGEINYRGTTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAF GYSDYEYDWFDPWGQGTLVTVSSGGGGSGGGSGGGGSGGGGSEIVLTQ SPATLSLSPGERATLSCRASQTISSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGCGTNLEIK (SEQ ID NO: 53).

[0184] Amino acid sequence of the heavy chain of LA5EV1(hG1TM): QVQLQQWGAGLLKPSETLSLTCAVYGGSISDYYWNWIRQPPGK GLEWIGEINYRGTTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYCAF GYSDYEYDWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICVNNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASVFLFPPKPKDT LMISRTPEVTVCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSG GGGSGGGGSQVQLVQSGAEVVKPGASVKVSCKASGYSFTGYTMNWVRQAP GQNLEWIGLINPYNAGTSYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVY YCARSEYRYGGDYFDYWGQGTTLTVSSGGGGSGGGGSGGGGSDI VMTQSPSSLAVSVGERVTISCKSSQSLLNSSNQKNYLAWYQQKPGQAPKLLIY FASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYDTPYTFGGG TKLEIKR (SEQ ID NO: 54).

[0185] Amino acid sequence of the heavy chain of LA5EV2(hG1TM): QVQLQQWGAGLLKPSETLSLTCAVYGGSISDYYWNWIRQPPGK GLEWIGEINYRGTTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYCAF GYSDYEYDWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT Petition 870250099246, de 30 / 10 / 2025, pág. 212 / 270 72 / 101 YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAGAPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSG GGGSGGGSQVQLVQSGAEVVKPGASVKVSCKASGYSFTGYTMNWVRQAP GQCLEWIGLINPYNAGTSYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVY YCARSEYRYGGDYFDYWGQGTTLTVSSGGGGSGGGSGGGSGGGSGGGGSDI VMTQSPSSLAVSVGERVTISCKSSQSLLNSSNQKNYLAWYQQKPGQAPKLLIY FASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYDTPYTFGCG TLKLEIKR (SEQ ID NO: 55).

[0186] Unless otherwise indicated, in subsequent experiments and examples, the bispecific antibodies NTLAV2(hG1TM), NTLAV7(hG1TM), NTLAV8(hG1TM), LA5EV1(hG1TM) and LA5EV2(hG1TM) carrying the mutation sites described above are named NTLAV2, NTLAV7, NTLAV8, LA5EV1 and LA5EV2, respectively. 2. Antibody expression and purification

[0187] The heavy chain and light chain cDNA sequences of NTLAV2, NTLAV7, NTLAV8, LA5EV1, and LA5EV2 were cloned separately into pUC57simple vectors (provided by GenScript) to obtain the plasmids pUC57simple-NTLAV2H / pUC57simple-NTLAV2L, pUC57simpleNTLAV7H / pUC57simple-NTLAV7L, pUC57simple-NTLAV8H / pUC57simpleNTLAV8L, pUC57simple-LA5EV1H / pUC57simple-LA5EV1L, and pUC57simpleLA5EV2H / pUC57simple-LA5EV2H, respectively.

[0188] Plasmids pUC57simple-NTLAV2H / pUC57simpleNTLAV2L, pUC57simple-NTLAV7H / pUC57simple-NTLAV7L, pUC57simpleNTLAV8H / pUC57simple-NTLAV8L, pUC57simple-LA5EV1H / pUC57simpleLA5EV1L and pUC57simple-LA5EV2H / pUC57simple-LA5EV2H were digested Petition 870250099246, dated 10 / 30 / 2025, pp. 213 / 270 73 / 101 (HindlII and EcoRI). The heavy and light chains isolated by electrophoresis were subcloned separately into pcDNA3 vectors, and recombinant plasmids were extracted for cotransfection into 293F cells. After 7 days of cell culture, the culture solution was separated by high-speed centrifugation, and the supernatant was concentrated and loaded onto a HiTrap MabSelect SuRe column. The protein was eluted in a single step with an elution buffer. The target sample was isolated, and the buffer was exchanged in PBS. Example 1: Anti-LAG3 Antibody Binding Activity Assays for Antigen by ELISA

[0189] An ELISA plate was coated with 2 μg / mL of human LAG3mG1Fc and incubated overnight at 4 °C. The antigen-coated ELISA plate was then washed once with PBST and blocked with a PBS solution containing 1% BSA (blocking solution) at 37 °C for 2 h. After blocking, the ELISA plate was washed 3 times with PBST. Serially diluted antibodies with PBST solution (dilution gradients for the antibodies are shown in Table 2) were added. The ELISA plate containing the test antibodies was incubated at 37 °C for 30 min and then washed 3 times with PBST. After washing, a working solution of HRP-labeled goat anti-human IgG (H+L) secondary antibody (Jackson, Cat. No. 109-035-088), diluted 1:5000, was added, and the plate was then incubated at 37 °C for 30 min. After incubation, the plate was washed 4 times with PBST.Color development was then performed with TMB (Neogen, 308177) in the dark for 5 min, and a stopping solution was added to interrupt the color development reaction. The ELISA plate was immediately placed in a microplate reader, and the OD at a wavelength of 450 nm in each well of the ELISA plate was measured. The data were analyzed and processed using SoftMax Pro 6.2.1 software. Petition 870250099246, dated 10 / 30 / 2025, pp. 214 / 270 74 / 101

[0190] The test results are presented in Table 2, Figure 1 and Table 3 (referring to Figure 1). Table 2. Results of the H9L8(hG4WT) bonding tests. H9L9(hG4WT) and H9L10(hG4WT) to human LAG3-mG1 Fc antigen by elisa LAG3-mG1Fc human, 2 μg / mL, 50 μL / well Antibody dilution (pg / mL) H9L8 (hG4WT) H9L9 (hG4WT) H9L10 (hG4WT) Relatlimab 1 2.403 2.463 2.485 2.555 2.438 2.471 2.568 2.589 0.3 2.414 2.399 2.420 2.412 2.345 2.419 2.468 2.557 0.1 2.201 2.141 2.200 2.135 2.121 2.107 2.260 2.327 0.03 1.734 1.647 1.783 1.691 1.637 1.657 1.868 1.975 0.01 1.104 1.065 1.089 1.054 1.019 1.018 1.220 1.288 0.003 0.592 0.558 0.567 0.560 0.525 0.540 0.649 0.725 0.001 0.282 0.277 0.298 0.287 0.275 0.282 0.324 0.344 0 0.122 0.123 0.125 0.118 0.121 0.126 0.119 0.121 Secondary IgG antibody against goat anti-human (H+L), HRP (1:5000) EC50(nM) 0.123 0.129 0.140 0.097 Table 3. Results relating to the assays for binding activity of H9L8(hG4WT), H9L9(hG4WT) and H9L10(hG4WT) to the human antigen LAG3-mG1 Fc by ELISA (Figure 1) Parameter Estimated Value Standard Error Confidence Range H9L8 R2 = 1.000 EC50 = 0.123 A 0.121 0.022 [0.060, 0.182] B 0.990 0.038 [0.885, 1.096] C 0.123 0.005 [0.108, 0.138] D 2.505 0.025 [2.436, 2.574] H9L9 R2 = 1.000 EC50 = 0.129 A 0.120 0.014 [0.082, 0.158] B 0.974 0.023 [0.910, 1.038] C 0.129 0.004 [0.119, 0.138] D 2.562 0.016 [2.517, 2.606] H9L10 R2 = 1.000 EC50 = 0.140 A 0.123 0.009 [0.097, 0.148] B 0.973 0.016 [0.928, 1.018] C 0.140 0.003 [0.132, 0.147] D 2.518 0.011 [2.487, 2.549] Petition 870250099246, dated 10 / 30 / 2025, pp. 215 / 270 75 / 101 Parameter Estimated value Standard error Confidence interval Relatlimab R2 = 1.000 EC50 = 0.097 A 0.117 0.016 [0.072, 0.162] B 0.982 0.025 [0.912, 1.052] C 0.097 0.003 [0.089, 0.105] D 2.614 0.017 [2.568, 2.661] Curve fit: 4y = D + -4t A

[0191] The results show that: H9L8(hG4WT), H9L9(hG4WT) and H9L10(hG4WT) were able to bind effectively to the human antigen LAG3-mG1Fc in a dose-dependent manner and exhibited binding activity comparable to that of the positive control antibody Relatlimab. Example 2: Assays for Anti-LAG3 Antibody Binding Activity to Human LAG3 Antigen on the Cell Surface by Flow Cytometry

[0192] Antibody labeling and flow cytometry detection: 293T-LAG3 cells expressing the human LAG3 antigen were digested with conventional pancreatin, and the number of cells in each collection tube was 3 χ 105. LAG3 antibody dilutions prepared using 1% PBSA (PBS containing 1% BSA) at final concentrations of 0.0123 nM, 0.123 nM, 1.23 nM, 3.7 nM, 11.1 nM, 33.3 nM, 100 nM, and 300 nM, respectively, were incubated with 293T-LAG3 cells expressing LAG3 on ice for 1 h. After centrifugation and repeated washing with 1% PBSA, 100 μL of FITC goat anti-human IgG antibody (acquired from Jackson, Cat. No. 109-095-098) (diluted 1:300) were added to each tube, and the mixture was incubated on ice in the dark for 40 minutes. After washing with 1% PBSA, 200 μL of 1% PBSA were added to resuspend the cells. Fluorescence signals were detected using the FITC channel in a flow cytometer.

[0193] The results of the binding of humanized anti-LAG3 antibodies to 293T-LAG3 cells are shown in Figure 2. The EC50 values ​​for the binding of anti-LAG3 antibodies to the antigen on the surface of the Petition 870250099246, dated 10 / 30 / 2025, pages 216 / 270 76 / 101 cells 293T-LAG3 are shown in Table 4. Table 4. Results of assays for the binding activity of anti-LAG3 antibodies to the antigen on the surface of 293T-LAG3 cells by flow cytometry. EC50 antibody (nM) Relatlimab 4.289 H9L8(hG4WT) 4.862 H9L9(hG4WT) 4.525 H9L10(hG4WT) 3.925

[0194] As can be seen in Figure 2, the anti-LAG3 antibodies were able to bind effectively to the target LAG3 protein on the surface of the 293T-LAG3 cell, and the binding activity of the anti-LAG3 antibodies H9L8(hG4WT), H9L9(hG4WT) and H9L10(hG4WT) to the antigen on the surface of the 293T-LAG3 cell was comparable to that of the positive control antibody Relatlimab. Example 3: Assays for Anti-LAG3 Antibodies Competing with MHC II on the Raji Cell Membrane Surface for Binding to Human Fc LAG3mG1 Antigen by Competitive Flow Cytometry

[0195] Raji cells (medium: 1640 + 10% FBS) (Cell Resource Center, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, Cat. No. TCHu 44) were added to EP tubes at 300,000 cells per sample. 1000 μL of 1% PBSA (PBS containing 1% BSA) was added. The mixture was centrifuged at 600 χ g for 5 min, and the supernatant was discarded. 100 μL of hIgG1 (prepared by Akeso Biopharma Inc., Lot No. 20190410) at a final concentration of 300 nM was added to each tube, and the mixture was incubated on ice for 1 h; 200 μL of 1% PBSA were added to Raji cells after incubation, and the mixture was centrifuged at 600 χ²g for 5 min, followed by removal of the supernatant. According to the experimental design, antibodies diluted to the corresponding concentrations were added to additional clean EP tubes at 60 μL / tube. 60 μL of human LAG3-mG1Fc (prepared by Akeso Biopharma Inc., Lot No. 20190508) were... Petition 870250099246, dated 10 / 30 / 2025, pp. 217 / 270 77 / 101 added to each corresponding antibody tube, and the mixture was thoroughly mixed and pre-incubated on ice for 30 min, so that the final antibody concentrations were 300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.23 nM, 0.123 nM, and 0.0123 nM. The final concentration of human LAG3-mG1Fc was 3 nM. 100 μL of the pre-incubated antibody and protein mixture were added to the cells. The resulting mixture was thoroughly mixed and incubated on ice in the dark for 1 h; 200 μL of 1% PBSA were added, and the mixture was centrifuged at 600 x g for 5 min, followed by removal of the supernatant, and then the pellet was washed twice; 100 μL of an anti-mouse APC antibody (acquired from Biolegend, Cat.No. 405308) (diluted in a 1:400 ratio) were added, and the mixture was thoroughly mixed and incubated on ice in the dark for 40 min; 200 μL of 1% PBSA were added, and the mixture was centrifuged at 600 xg for 5 min, followed by removal of the supernatant; 200 μL of 1% PBSA were added to each tube to resuspend the cells, and then the suspension was transferred to a sample loading tube for analysis in a flow cytometer.

[0196] The results are presented in Figure 3 and Table 5. Through fluorescence analysis and curve fitting, the EC50 values ​​for the competitive binding of the antibodies Relatlimab, H9L8(hG4WT), H9L9(hG4WT) and H9L10(hG4WT) were calculated to be 1.153 nM, 1.459 nM, 1.482 nM and 1.435 nM, respectively. Table 5. Results of the fluorescence intensity analysis of Relatlimab, H9L8(hG4WT), H9L9(hG4WT), and H9L10(hG4WT) in competition with MHC II on the surface of Raji cells for binding to human LAG3mG1 Fc antigen by FACS. EC50 antibody (nM) Relatlimab 1.153 H9L8(hG4WT) 1.459 H9L9(hG4WT) 1.482 H9L10(hG4WT) 1.435 Petition 870250099246, dated 10 / 30 / 2025, pp. 218 / 270 78 / 101

[0197] The results show that the antibodies H9L8(hG4WT), H9L9(hG4WT) and H9L10(hG4WT) were able to competitively bind to LAG3 and effectively block LAG3 binding to MHC II on the surface of Raji cells in a dose-dependent manner, exhibiting activity comparable to that of the positive control antibody Relatlimab. Example 4: Assays for biological activity of anti-LAG3 antibodies in promoting IFN-Γ and IL-2 secretion by mixed lymphocyte reaction (MLR) 1. Assays for the biological activity of anti-LAG3 antibodies in promoting IFN-γ secretion in the Raji-PDLI mixed lymphocyte reaction system.

[0198] Raji-PDL1 cells were conventionally subcultured. PBMCs (from healthy donors) were thawed, cultured in 10 mL of complete medium 1640, and stimulated with SEB (staphylococcal enterotoxin B) (Dianotech, Cat. No. S010201) at 0.5 μg / mL for two days. Raji-PDL1 cells were treated with MMC (Stressmarq, Cat. No. SIH-246-10MG) at a working concentration of 2 μg / mL and incubated at 37 °C in a 5% CO2 incubator for 1 h; PBMCs stimulated with SEB for 2 days and Raji-PDL1 cells treated with MMC for 1 h were collected, washed twice with PBS, and then resuspended in complete medium (i.e., RPMI 1640 + 10% FBS) and counted. PBMCs and Raji-PDL1 cells were added separately to a 96-well U-shaped plate (Corning, Model No. 3799) at 10 χ 104 cells / well and co-cultured.According to the experimental design, the antibodies (the final concentrations of each antibody were 300 nM, 30 nM, and 3 nM when used alone or in combination) were added and co-cultured with the cells in an incubator for 3 days; after 3 days, the cells were centrifuged at 250 χ² g for 5 min, and the cell culture supernatant was collected and analyzed for IFN-γ by ELISA.

[0199] As shown in Figure 4, the mixed culture of PBMCs Petition 870250099246, dated 10 / 30 / 2025, pp. 219 / 270 79 / 101 human and Raji-PDL1 cells promoted IFN-γ secretion in PBMCs, and the addition of antibodies to the mixed culture system can significantly induce additional IFN-γ secretion in PBMCs. In terms of the level of activity in promoting IFN-γ secretion, the anti-LAG3 antibodies H9L8(hG4WT), H9L9(hG4WT), and H9L10(hG4WT), each in combination with 14C12H1L1(hG1TM), and the positive control antibody Relatlimab in combination with 14C12H1L1(hG1TM), were all able to promote IFN-γ secretion, with comparable activities. 2. Assays for the biological activity of anti-LAG3 antibodies in promoting IL-2 secretion in the RAJI-PDL1 mixed lymphocyte reaction system.

[0200] Raji-PDL1 cells were conventionally subcultured. PBMCs were thawed, cultured in 10 mL of complete medium 1640, and stimulated with SEB (staphylococcal enterotoxin B, acquired from Dianotech, Cat. No. S010201) at 0.5 μg / mL for two days. Raji-PDL1 cells were treated with MMC (Stressmarq, Cat. No. SIH-246-10MG) at a working concentration of 2 μg / mL and incubated at 37 °C in a 5% CO2 incubator for 1 h. PBMCs stimulated with SEB for 2 days and Raji-PDL1 cells treated with MMC for 1 h were collected, washed twice with PBS, and then resuspended in complete medium (i.e., RPMI 1640 + 10% FBS) and counted. PBMCs and Raji-PDL1 cells were added separately to a 96-well U-shaped plate (Corning, Model No. 3799) at 10 x 10⁴ cells / well and co-cultured.According to the experimental design, the antibodies (the final concentrations of each antibody were 300 nM, 30 nM, and 3 nM when used alone or in combination) were added and co-cultured with the cells for 3 days; after 3 days, the cells were centrifuged at 250 xg for 5 min, and the cell culture supernatant was collected and analyzed for IL-2 by ELISA.

[0201] As shown in Figure 5, the mixed culture of PBMCs Petition 870250099246, dated 10 / 30 / 2025, pages 220 / 270 Human 80 / 101 cells (from healthy donors) and Raji-PDL1 cells promoted IL-2 secretion in PBMCs to some extent, and the addition of antibodies to the mixed culture system could significantly induce additional IL-2 secretion in PBMCs, exhibiting a significant dose-dependent relationship. In terms of the level of activity in promoting IL-2 secretion, the anti-LAG3 antibodies H9L8(hG4WT), H9L9(hG4WT), and H9L10(hG4WT), each in combination with 14C12H1L1(hG1TM), and the positive control antibody Relatlimab in combination with 14C12H1L1(hG1TM), all could promote IL-2 secretion, with comparable activities. Example 5: Assays for Evaluating the Biological Activity of Anti-LAG3 Antibodies in Blocking the Interaction between LAG3 and MHC-II (Method of Gene (Reporter)

[0202] Jurkat-NFAT-PD1-LAG3 cells and Raji cells were used as a reporter gene system. After the addition of a SEE superantigen, a TCR-NFAT signaling pathway was activated to induce luciferase expression. LAG3 in Jurkat cells bound to MHC-II in Raji cells, so that the NFAT signaling pathway was inhibited and luciferase expression was downregulated. The antibody, by specifically binding to LAG3, relieved the inhibition and increased luciferase expression.

[0203] Jurkat-NFAT-PD1-LAG3 cells and Raji cells (acquired from the Cell Resource Center, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, Cat. No. TCHu 44) were collected and centrifuged at 110 xg for 5 min, followed by removal of the supernatant. The cells were then resuspended in a 1640 + 10% FBS medium and counted. Jurkat-NFAT-PD1-LAG3 cells were seeded in a 96-well black-bottom plate (Corning, Model No. 3916) at 10⁵ cells / well (30 μL / well); according to the experimental design, antibodies (at final concentrations of 0.3 nM, 3 nM, and 300 nM) were added at 10 μL / well, and the mixture was pre-incubated at 37 Petition 870250099246, dated 10 / 30 / 2025, pp. 221 / 270 81 / 101 °C in a 5% CO2 incubator for 30 min. Meanwhile, SEE (Staphylococcal Enterotoxins E, acquired from Toxin Technology, Cat. No. ET404) (at a final concentration of 0.05 ng / mL) was added to the Raji cells, and the mixture was incubated at 37 °C in a 5% CO2 incubator for 30 min. After 30 min, the SEE-treated Raji cells were added to the 96-well plate containing Jurkat-NFAT-PD1-LAG3 cells described above at 2 χ¹⁰⁴ cells / well (40 μL / well), with the final volume of each well being 80 μL. The mixture was thoroughly mixed and incubated at 37 °C in a 5% CO2 incubator for 6 h. After incubation, the culture plate was removed and allowed to reach room temperature. The Bright-Glo™ Luciferase Assay System (purchased from Promega, Cat. No. E2650) was added at 80 μL / well, and the mixture was incubated in the dark for 2 min. Then, the URL values ​​were read. The hG4WT(hIgG4) isotypic control was manufactured by Akeso Biopharma Inc., Lot No. 20190910.

[0204] As shown in Figure 6, the anti-LAG3 antibodies H9L8(hG4WT), H9L9(hG4WT), H9L10(hG4WT) and the positive control antibody Relatlimab were able to block the interaction between LAG3 and MHC-II and thus increase luciferase expression. The activities of the anti-LAG3 antibodies H9L8(hG4WT), H9L9(hG4WT) and H9L10(hG4WT) were superior to those of the control antibody Relatlimab. Example 6: Assays for Binding Activity of Bispecific Anti-CD73-Anti-LAG3 Antibodies to Antigen by ELISA 1. Tests for binding activity of LA5EV1, LA5EV2, NTLAV2, NTLAV7 and NTLAV8 for NT5E-His antigen by indirect ELISA.

[0205] The specific method was as follows: An ELISA plate was coated with 1 μg / mL of NT5E-His and incubated overnight at 4 °C. The antigen-coated ELISA plate was then washed once with PBST and blocked with a PBS solution. Petition 870250099246, dated 10 / 30 / 2025, pp. 222 / 270 82 / 101 containing 1% BSA (blocking solution) at 37 °C for 2 h. After blocking, the ELISA plate was washed 3 times with PBST. Serially diluted antibodies with PBST solution (dilution gradients for the antibodies are shown in Tables 6 to 8) were added. The ELISA plate containing the test antibodies was incubated at 37 °C for 30 min and then washed 3 times with PBST. After washing, a working solution of HRP-labeled FC (H+L) anti-human goat IgG secondary antibody (Jackson, Cat. No. 109-035-098), diluted 1:5000, was added, and the plate was then incubated at 37 °C for 30 min. After incubation, the plate was washed 4 times with PBST. The color development was then carried out with TMB (Neogen, 308177) in the dark for 5 min, and a stopping solution was added to interrupt the color development reaction.The ELISA plate was immediately placed in a microplate reader, and the OD at a wavelength of 450 nm in each well of the ELISA plate was measured. The data were analyzed and processed using SoftMax Pro 6.2.1 software.

[0206] The results are shown in Tables 6 to 8 and Figures 7 to 9 and Tables 9 to 11 (referring to Figures 7 to 9, respectively).

[0207] The results indicate that, under the same experimental conditions, LA5EV1, LA5EV2, NTLAV2, NTLAV7, NTLAV8 and 19F3H2L3(hG1DM) could effectively bind to the NT5E-His antigen in a dose-dependent manner. Table 6. Results of the binding tests of LA5EV1 and LA5EV2 to NT5EHis by ELISA. Antibody dilution concentration (nM) Coating: NT5E-His (1 μg / mL) EC50 (nM) 2.5 0.833 3333 0.277 7778 0.092 5926 0.030 8642 0.010 2881 0.0034 294 0.0011 431 0.0003 810 0.0001 270 0.000 0423 0 LA5EV1 3.022 3.004 2.882 2.436 1.415 0.617 0.273 0.137 0.084 0.069 0.060 0.059 0.036 3.057 3.010 2.886 2.379 1.325 0.598 0.279 0.132 0.078 0.067 0.058 0.062 LA5EV2 3.103 3.073 2.971 2.613 1.607 0.739 0.327 0.162 0.090 0.065 0.059 0.058 0.029 Petition 870250099246, dated 10 / 30 / 2025, pp. 223 / 270 83 / 101 Antibody dilution concentration (nM) Coating: NT5E-His (1 μg / mL) EC50 (nM) 2.5 0.833 3333 0.277 7778 0.092 5926 0.030 8642 0.010 2881 0.0034 294 0.0011 431 0.0003 810 0.0001 270 0.000 0423 0 3.125 3.120 3.032 2.575 1.631 0.772 0.331 0.154 0.088 0.066 0.059 0.058 19F3H2L3(hG1DM) 3.121 3,132 3,045 2,939 2,520 1,435 0,601 0,272 0,124 0,079 0,063 0,061 0,011 3,072 3,055 3,020 2,916 2,534 1,531 0,645 0,279 0,132 0,083 0,069 0,065 Secondary antibody IgG Fc anti-human goat, HRP (1:5000) Table 7. Results of NTLAV2 and NTLAV7 binding assays to NT5EHis by ELISA. Antibody dilution concentration (nM) Coating: NT5E-His (1 pg / mL) EC50 (nM) 2.5 0.833 3333 0.277 7778 0.092 5926 0.030 8642 0.010 2881 0.003 4294 0.001 1431 0.000 3810 0.000 1270 0.000 0423 0 NTLAV2 3.062 3.065 2.941 2.481 1.430 0.614 0.259 0.130 0.084 0.068 0.066 0.074 0.037 3.100 3.080 2.947 2.424 1.310 0.577 0.241 0.121 0.085 0.062 0.061 0.061 NTLAV7 3.173 3.151 3.068 2.529 1.352 0.587 0.249 0.123 0.075 0.065 0.058 0.058 3.036 3.131 3.092 3.041 2.559 1.422 0.656 0.268 0.142 0.080 0.062 0.061 0.061 19F3H2L3(hG1DM) 3.164 3.151 3,097 2,962 2,489 1,441 0,627 0,267 0,132 0,079 0,065 0,059 0,011 3,109 3,108 3,051 2,921 2,544 1,486 0,669 0,297 0,139 0,086 0,070 0,060 Goat anti-human IgG Fc secondary antibody, HRP (1:5000) Table 8. Results of the NTLAV8 to NT5E-His binding assay by ELISA Antibody dilution concentration (nM) Coating: NT5E-His (1 pg / mL) EC50 (nM) 2.5 0.833 3333 0.277 7778 0.092 5926 0.030 8642 0.010 2881 0.003 4294 0.001 1431 0.000 3810 0.000 1270 0.000 0423 0 NTLAV8 3.036 3.006 2.830 2.166 1.091 0.466 0.219 0.113 0.077 0.067 0.066 0.065 0.05 3.067 3.051 2,870 2,178 1,094 0,467 0,197 0,102 0,075 0,061 0,053 0,063 19F3H2L3(hG1DM) 3,162 3,156 3,073 2,913 2,308 1,202 0,528 0,221 0,111 0,073 0,062 0,057 0,015 3,119 3,109 3,051 2,849 2,246 1,179 0,513 0,216 0,113 0,097 0,070 0,065 Secondary antibody IgG Fc anti-human goat, HRP (1:5000) Table 9. Results of the assays for binding of LA5EV1 and LA5EV2 to the NT5E-His antigen by ELISA (Figure 7) Parameter Estimated value Standard error Confidence range LASEV1 R2 = 0.999 EC50 = 0.036 A 0.080 0.016 [0.044, 0.117] B 1.265 0.049 [1.152, 1.379] C 0.036 0.001 [0.033, 0.039] Petition 870250099246, dated 10 / 30 / 2025, pp. 224 / 270 84 / 101 Parameter Estimated Value Standard Error Confidence Interval D 3.074 0.027 [3.012, 3.135] LASEV2 R2 = 1.000 EC50 = 0.029 A 0.078 0.016 [0.041, 0.114] B 1.229 0.045 [1.125, 1.333] C 0.029 9.90e-4 [0.027, 0.032] D 3.155 0.025 [3.097, 3.214] 19F3H2L3 (hG1DM) R2 = 1.000 EC50 = 0.011 A 0.083 0.015 [0.048, 0.119] B 1.323 0.043 [1.223, 1.423] C 0.011 3.09e-4 [0.010, 0.012] D 3.098 0.018 [3.056, 3.140] Curve Fit: 4 y = D + -4-2^ 1+Φ Table 10. Results of assays for NTLAV2 and NTLAV7 binding to NT5E-HIS antigen by ELISA (Figure 8) Parameter Estimated Value Standard Error Confidence Range NTLAV2 R2 = 1.000 EC50 = 0.037 A 0.082 0.015 [0.047, 0.117] B 1.311 0.049 [1.199, 1.424] C 0.037 0.001 [0.034, 0.040] D 3.123 0.025 [3.065, 3.180] NTLAV7 R2 = 0.999 EC50 = 0.036 A 0.084 0.022 [0.033, 0.135] B 1.343 0.073 [1.175, 1.511] C 0.036 0.002 [0.033, 0.040] D 3.194 0.037 [3.109, 3.279] 19F3H2L3 (hG1DM) R2 = 1.000 EC50 = 0.011 A 0.082 0.016 [0.045, 0.120] B 1.270 0.043 [1.171, 1.369] C 0.011 3.43e-4 [0.011,0.012] D 3.142 0.019 [3.097, 3.187] Curve Fit: 4 y = D + -4-2^ 1+Φ Table 11. Results of the assay for NTLAV8 binding to the antigen. NT5E-HIS by ELISA (Figure 9) Parameter Estimated Value Standard Error Confidence Range NTLAV8 R2 = 1.000 EC50 = 0.050 A 0.079 0.014 [0.046, 0.112] B 1.304 0.048 [1.193, 1.415] C 0.050 0.002 [0.046, 0.054] Petition 870250099246, dated 10 / 30 / 2025, pp. 225 / 270 85 / 101 Parameter Estimated value Standard error Confidence interval D 3.101 0.027 [3.040, 3.162] 19F3H2L3 (hG1DM) R2 = 1.000 EC50 = 0.015 A 0.081 0.013 [0.050, 0.112] B 1.266 0.036 [1.183, 1.349] C 0.015 3.93e-4 [0.014, 0.016] D 3.151 0.017 [3.111, 3.191] Curve fit: 4 y = D + -41+Φ 2. Assays for binding activity of LA5EV1, LA5EV2, NTLAV2, NTLAV7 and NTLAV8 to the huLAG3-mG1 Fc antigen by indirect ELISA.

[0208] The specific method was as follows: An ELISA plate was coated with 2 μg / mL of huLAG3-mG1 Fc and incubated overnight at 4 °C. The antigen-coated ELISA plate was then washed once with PBST and blocked with a PBS solution containing 1% BSA (blocking solution) at 37 °C for 2 h. After blocking, the ELISA plate was washed 3 times with PBST. Serially diluted antibodies with PBST solution (dilution gradients for the antibodies are shown in Tables 12 to 14) were added. The ELISA plate containing the test antibodies was incubated at 37 °C for 30 min and then washed 3 times with PBST. After washing, a working solution of FC (H+L) secondary antibody anti-human goat IgG labeled with HRP (Jackson, Cat. No. 109-035-098), diluted in a 1:5000 ratio, was added, and the plate was then incubated at 37 °C for 30 min. After incubation, the plate was washed 4 times with PBST.Color development was then performed with TMB (Neogen, 308177) in the dark for 5 min, and a stopping solution was added to interrupt the color development reaction. The ELISA plate was immediately placed in a microplate reader, and the OD at a wavelength of 450 nm in each well of the ELISA plate was measured. The data were analyzed and processed using SoftMax Pro 6.2.1 software.

[0209] The results of the test are presented in Tables 12 to 14 and in Figures 10 to 12 and Tables 15 to 17 (referring to Figures 10 to 12, respectively). Petition 870250099246, dated 10 / 30 / 2025, pages 226 / 270 86 / 101

[0210] The results indicate that, under the same experimental conditions, LA5EV1, LA5EV2, NTLAV2, NTLAV7, NTLAV8 and Relatlimab were able to bind effectively to the huLAG3-mG1Fc antigen in a dose-dependent manner. Table 12. Results of the bonding tests of LA5EV1 and LA5EV2 to HULAG3-MG1 FC by ELISA Antibody dilution concentration (nM) Coating: huLAG3-mG1Fc ^g / mL) EC50 (nM) 2.5 0.833 3333 0.277 7778 0.092 5926 0.030 8642 0.010 2881 0.003 4294 0.001 1431 0.000 3810 0.000 1270 0.000 0423 0 LA5EV1 3.150 3.079 2.920 2.396 1.337 0.576 0.295 0.160 0.117 0.104 0.093 0.100 0.043 3.180 3.075 2.942 2.361 1.217 0.533 0.247 0.145 0.113 0.101 0.097 0.097 LA5EV2 3.220 3.147 3.032 2.565 1.467 0.615 0.292 0.156 0.124 0.103 0.095 0.098 0.036 3.253 3.153 3.042 2.606 1.436 0.610 0.281 0.156 0.118 0.103 0.097 0.100 Relatlimab 3.154 3.059 2.855 2.043 1.028 0.463 0.230 0.140 0.114 0.107 0.099 0.110 0.056 3.107 3.034 2.843 2.155 1.118 0.511 0.255 0.165 0.137 0.126 0.125 0.139 Secondary antibody IgG Fc anti-human goat, HRP (1:5000) Table 13. Results of the NTLAV2 and NTLAV7 bonding tests to HULAG3-MG1 FC by ELISA Antibody dilution concentration (nM) Coating: huLAG3-mG1Fc ^g / mL) EC50 (nM) 2.5 0.833 3333 0.277 7778 0.092 5926 0.030 8642 0.010 2881 0.003 4294 0.001 1431 0.000 3810 0.000 1270 0.000 0423 0 NTLAV2 2.890 2.680 1.997 1.181 0.540 0.268 0.152 0.116 0.107 0.105 0.105 0.111 0.173 2.934 2.648 1.916 1.009 0.489 0.225 0.138 0.110 0.101 0.098 0.101 0.102 NTLAV7 3.023 2.770 2.023 1.069 0.463 0.230 0.134 0.107 0.098 0.093 0.109 0.102 0.158 3.007 2.833 2.232 1.176 0.538 0.242 0.147 0.114 0.102 0.102 0.102 0.103 Relatlimab 3.187 3,084 2,862 2.024 0.986 0.454 0.223 0.147 0.119 0.120 0.108 0.109 0.059 3.143 3.056 2.823 2.146 1.096 0.474 0.241 0.163 0.147 0.123 0.120 0.116 Secondary antibody IgG Fc anti-human goat, HRP (1:5000) Table 14. Results of the NTLAV8 to HuLAG3-mG1 Fc binding assay by ELISA. Antibody dilution concentration (nM) Coating: huLAG3-mG1Fc ^g / mL) EC50 (nM) 2.5 0.833 3333 0.277 7778 0.092 5926 0.030 8642 0.010 2881 0.003 4294 0.001 1431 0.000 3810 0.000 1270 0.000 0423 0 NTLAV8 2.849 2.578 1.764 0.894 0.395 0.219 0.133 0.109 0.101 0.102 0.107 0.118 0.231 2.896 2,543 1,688 0,798 0,367 0,196 0,125 0,113 0,097 0,091 0,094 0,103 Relatlimab 3,174 3,076 2,836 1,954 0,943 0,418 0,217 0,139 0,116 0,106 0,104 0,108 0,066 3,087 3,021 2,765 1,945 0,948 0,438 0,240 0,155 0,130 0,118 0,127 0,131 Secondary antibody IgG Fc anti-human goat, HRP (1:5000) Petition 870250099246, dated 10 / 30 / 2025, pp. 227 / 270 87 / 101 Table 15. Results of assays for binding of LA5EV1 and LA5EV2 to the HULAG3-MG1 Fc antigen by ELISA (Figure 10) Parameter Estimated Value Standard Error Confidence Range LASEV1 R2 = 1.000 EC50 = 0.043 A 0.114 0.014 [0.083, 0.146] B 1.294 0.044 [1.193, 1.396] C 0.043 0.001 [0.040, 0.046] D 3.171 0.024 [3.115, 3.227] LASEV2 R2 = 1.000 EC50 = 0.036 A 0.116 0.015 [0.081, 0.151] B 1.349 0.050 [1.234, 1.464] C 0.036 0.001 [0.034, 0.039] D 3.229 0.025 [3.172, 3.287] Relatlimab R2 = 1.000 EC50 = 0.056 A 0.128 0.012 [0.101, 0.155] B 1.256 0.038 [1.168, 1.343] C 0.056 0.002 [0.053, 0.060] D 3.165 0.023 [3.112, 3.219] Curve Fit: 4 y = D + -4-2^ i+Φ Table 16. Test Results for Connecting Ntlav2 and Ntlav7 to Hulag3-Mg1 fc Antigen By Elisa (Figure 11) Parameter Estimated Value Standard Error Confidence Range NTLAV2 R2 = 1.000 EC50 = 0.173 A 0.105 0.007 [0.089, 0.121] B 1.090 0.024 [1.034, 1.146] C 0.173 0.005 [0.163, 0.184] D 3.086 0.026 [3.027, 3.145] NTLAV7 R2 = 1.000 EC50 = 0.158 A 0.107 0.008 [0.089, 0.125] B 1.211 0.029 [1.144, 1.277] C 0.158 0.004 [0.149, 0.166] D 3.136 0.024 [3.079, 3.192] Relatlimab R2 = 1.000 EC50 = 0.059 A 0.128 0.010 [0.106, 0.150] B 1.263 0.031 [1.191, 1.334] C 0.059 0.001 [0.056, 0.062] D 3.194 0.019 [3.150, 3.237] Curve fit: 4 y = D + -41+Φ Table 17. Results of the Ntlav8 Antigen Binding Assay Hulag3-Mg1fc By Elisa (Figure 12) Parameter Estimated value Standard error Confidence interval NATLAV8 R2 = 1.000 EC50 - 0.231 A 0.109 0.008 [0.089, 0.128] B 1.179 0.034 [1.101, 1.258] C 0.231 0.007 [0.214, 0.248] Petition 870250099246, dated 10 / 30 / 2025, pp. 228 / 270 88 / 101 Parameter Estimated Value Standard Error Confidence Interval D 3.060 0.034 [2.982, 3.138] Relatlimab R2 = 1.000 EC50 = 0.066 A 0.129 0.012 [0.100, 0.157] B 1.272 0.042 [1.176, 1.368] C 0.066 0.002 [0.062, 0.071] D 3.177 0.026 [3.118, 3.236] Curve Fit: 4 y = D + -4-2^ 1+Φ Example 7: Determination of the Kinetic Parameters of Humanized Antibodies LA5EV1, LA5EV2, NTLAV2, NTLAV7, and NTLAV8 1. Determination of the kinetic parameters of binding of humanized antibodies LA5EV1, LA5EV2, NTLAV2, NTLAV7 and NTLAV8 to hNT5E(1552)-His

[0211] The sample dilution buffer was PBS containing 0.02% Tween-20 and 0.1% BSA (pH 7.4). 5 μg / mL of hNT5E(1-552)-His were immobilized on the HIS1K sensor to an immobilization height of 0.15 nm. The sensor was equilibrated in buffer for 60 s, then the binding of the hNT5E(1-552)-His immobilized on the sensor to antibodies at concentrations of 2.469-200 nM (triple dilution) was determined for 120 s. The antibodies were then dissociated in buffer for 300 s. The sensor was regenerated 4 times in 10 mM glycine at pH 1.7, each time for 5 s. The sample plate agitation speed was 1000 rpm, the determination temperature was 37 °C, and the frequency was 5.0 Hz. Data were analyzed by 1:1 model fitting to obtain affinity constants. The data acquisition software was Fortebio Data Acquisition 12.0, and the data analysis software was Fortebio Data Analysis 12.0. Table 18. Kinetic parameters of humanized antibody binding. LA5EV1, LA5EV2, NTLAV2, NTLAV7 AND NTLAV8 A hNT5E(1-552)-His Antibody Maximum analyte signal height (nm) KD (M) Kon (1 / Ms) SE (kon) Kdis (1 / s) SE (kdis) Rmax (nm) LA5EV1 0.2736 7.93E-10 5.79E+05 1.75E+03 4.60E04 2.64E06 0.25 0.36 Petition 870250099246, dated 10 / 30 / 2025, pp. 229 / 270 89 / 101 Antibody Maximum analyte signal height (nm) KD (M) Kon (1 / Ms) SE (kon) Kdis (1 / s) SE (kdis) Rmax (nm) LA5EV2 0.2278 1.21E09 4.95E+05 1.36E+03 5.99E04 2.50E06 0.210.36 NTLAV2 0.2014 4.08E- 10 3.35E+05 1.23E+03 1.37E04 3.22E06 0.180.30 NTLAV7 0.2267 3.22E- 10 3.88E+05 1.39E+03 1.25E- 04 3.15E- 06 0,200,28 NTLAV8 0.2032 3.40E- 10 3.75E+05 1.71E+03 1.28E- 04 4.01E- 06 0.180.25 19F3H2L3(hG1DM) 0.1677 5.05E- 10 5.93E+05 2.46E+03 3.00E04 3.54E06 0.140.17

[0212] KD is the affinity constant; kon is the antigen-antibody binding rate; kdis is the antigen-antibody dissociation rate; KD = kdis / kon.

[0213] The results are presented in Table 18 and Figures 13 to 18.

[0214] The results indicate that the humanized antibodies LA5EV1, LA5EV2, NTLAV2, NTLAV7 and NTLAV8 showed good affinity for the hNT5E(1-552)-His antigen. 2. Determination of the kinetic parameters of binding of humanized antibodies LA5EV1, LA5EV2, NTLAV2, NTLAV7 and NTLAV8 to HULAG3mGIFc

[0215] The sample dilution buffer was PBS containing 0.02% Tween-20 and 0.1% BSA (pH 7.4). 10 μg / mL of huLAG3-mG1Fc were immobilized on the AMC sensor to an immobilization height of 0.8 nm. The sensor was equilibrated in buffer for 60 s, then the binding of the huLAG3mG1Fc immobilized on the sensor to antibodies at concentrations of 6.17-500 nM (triple dilution) was determined for 100 s. The antibodies were then dissociated in buffer for 400 s. The sensor was regenerated 4 times in 10 mM glycine at pH 1.7, each time for 5 s. The sample plate stirring speed was 1000 rpm, the determination temperature was 30 °C, and the frequency was 5.0 Hz. The data were analyzed by 1:1 model fitting to obtain constants of Petition 870250099246, dated 10 / 30 / 2025, pages 230 / 270 90 / 101 affinity. The data acquisition software was Fortebio Data Acquisition 12.0, and the data analysis software was Fortebio Data Analysis 12.0.

[0216] The results are shown in Table 19 and Figures 19 to 25.

[0217] The results indicate that the humanized antibodies LA5EV1, LA5EV2, NTLAV2, NTLAV7 and NTLAV8 showed good affinity for the huLAG3-mG1 Fc antigen. Table 19. Kinetic parameters of humanized antibody binding. LA5EV1, LA5EV2, NTLAV2, NTLAV7 AND NTLAV8 TO HULAG3-MG1FC Antibody Maximum analyte signal height (nm) KD (M) Kon (1 / Ms) SE (kon) Kdis (1 / s) SE (kdis) Rmax (nm) LA5EV1 0.9935 1.25E10 5.98E+05 3.30E+03 7.46E05 2.68E06 0.82- 1.46 LA5EV2 0.9481 1.52E10 5.00E+05 2.84E+03 7.58E05 2.80E06 0.801.51 NTLAV2 0.8142 1.23E09 4.36E+05 2.47E+03 5.35E04 3.08E06 0.63- 1.07 NTLAV7 0.8167 1.04E09 2.98E+05 1.68E+03 3.09E04 3.11E06 0.67- 1.30 NTLAV8 0.8058 1.47E09 3.44E+05 1.88E+03 5.05E04 3.05E06 0.63- 1.13 H9L8(hG4WT) 0.8777 3.00E- 10 4.05E+05 2.33E+03 1.21E04 2.94E06 0.73- 1.29 Relatlimab 0.6674 4.47E- 11 6.32E+05 2.87E+03 2.82E05 2.22E06 0.630.88 Example 8: Assays for Binding Activity of Anti-CD73-Anti-LAG3 Bispecific Antibodies to CD73 / LAG3 Antigen by FACS 1. Assays for binding activity of bispecific anti-CD73anti-LAG3 antibodies to LAG3 on the surface of the 293T-LAG3 membrane by FACS.

[0218] 293T-LAG3 cells in logarithmic growth phase were collected and transferred to a 96-well flow cytometry plate at 3 χ¹⁰⁵ cells / well. 100 μL of 1% PBSA was added and the mixture was centrifuged at 350 χ¹⁰ g for 5 min, followed by removal of the supernatant. 100 μL of each antibody diluted with 1% PBSA was added (to final concentrations of 300 nM, Petition 870250099246, dated 10 / 30 / 2025, pp. 231 / 270 91 / 101 (100 nM, 33.33 nM, 11.11 nM, 3.7 nM, 1.23 nM, 0.41 nM, 0.041 nM, 0.0041 nM, and 0.00041 nM). The mixture was gently mixed and then incubated on ice for 1 h. 200 μL of 1% PBSA was added, and the mixture was centrifuged at 350 χ²g for 5 min, followed by removal of the supernatant. The plate was washed twice. A 300-fold diluted Alexa Fluor® 647-labeled anti-human mouse IgG secondary antibody (Southern Biotech, Cat. No. 904031) was added for resuspension. The mixture was thoroughly mixed and incubated on ice in the dark for 0.5 h. 200 μL of 1% PBSA were added and the mixture was centrifuged at 350 χ²g for 5 min, followed by removal of the supernatant. The plate was washed twice. 200 μL of 1% PBSA were added to resuspend the cell pellets and the suspension was analyzed in a flow cytometer. The experimental results are presented in Table 20 and Figure 26.

[0219] The results demonstrate that NTLAV8, LA5EV2, H9L8(hG4WT) and Relatlimab specifically bound to the LAG3 receptor on the cell membrane surface of 293T-LAG3. Table 20. Results of tests for the binding activity of NTLAV8, LA5EV2, H9L8(hG4WT) and Relatlimab for LAG3 on the cell surface 293T-LAG3 by FACS Relatlimab H9L8(hG4WT) LA5EV2 NTLAV8 EC50 antibody (nM) 0.6026 0.5011 0.3989 0.7587 2. ASSAYS for BINDING ACTIVITY OF BIESPECIFIC ANTI-CD73ANTI-LAG3 ANTIBODY FOR CD73 ON THE CELL MEMBRANE SURFACE U87-MG by FACS

[0220] U87-MG cells (ATCC, Cat. No. HTB-14) in logarithmic growth phase were collected and transferred to a 96-well plate at 3 χ¹⁰⁵ cells / well. 200 μL of 1% PBSA were added, and the mixture was centrifuged at 750 χ¹⁰ g for 5 min, followed by removal of the supernatant. 100 μL of each antibody diluted with 1% PBSA (at the concentrations) were added. Petition 870250099246, dated 10 / 30 / 2025, pp. 232 / 270 (92 / 101 final values ​​of 300 nM, 100 nM, 33.33 nM, 11.11 nM, 3.7 nM, 1.23 nM, 0.41 nM, 0.041 nM, 0.0041 nM, and 0.00041 nM). The mixture was thoroughly mixed gently and then incubated on ice for 1 h. 200 μL of 1% PBSA was added, and the mixture was centrifuged at 750 χ²g for 5 min, followed by removal of the supernatant. The plate was washed twice. A 300-fold diluted Alexa Fluor® 647-labeled anti-human mouse IgG secondary antibody (Southern Biotech, Cat. No. 904031) was added for resuspension. The mixture was thoroughly mixed and incubated on ice in the dark for 0.5 h. 100 μL of 1% PBSA was added and the mixture was centrifuged at 750 χ² g for 5 min, followed by removal of the supernatant. The plate was washed twice with 200 μL of 1% PBSA. 200 μL of 1% PBSA was added to resuspend the cell pellets and the suspension was analyzed in a flow cytometer.

[0221] The experimental results are presented in Table 21 and Figure 27.

[0222] The results show that NTLAV8, LA5EV2, 19F3H2L3(hG1DM) and CPI-006 specifically bound to CD73 on the surface membrane of the U87-MG cell, and the ability of NTLAV8 and LA5EV2 to bind to CD73 on the surface membrane of the U87-MG cell was greater than that of the positive control CPI-006. Table 21. Results of assays for the binding activity of NTLAV8, LA5EV2, 19F3H2L3(HG1 DM) and CPI-006 to CD73 on the cell surface. U87-MG by FACS Antibody CPI-006 19F3H2L3(hG1DM) LA5EV2 NTLAV8 EC50 (nM) 8.472 6.059 2.162 5.456 Example 9: Assays for Bispecific Anti-CD73-Anti-LAG3 Antibodies Competing with MHC II on the cell membrane surface Raji for Binding to Human LAG3-MG1 Fc Antigen by FACS

[0223] According to the experimental design, the antibodies Petition 870250099246, dated 10 / 30 / 2025, pp. 233 / 270 93 / 101 and LAG3-mG1Fc were diluted and thoroughly mixed in a 1:1 ratio, so that the final concentration of LAG3-mG1Fc was 3 nM, and the final concentrations of the antibodies were 300 nM, 100 nM, 33.33 nM, 11.11 nM, 3.7 nM, 1.23 nM, 0.41 nM, 0.041 nM, 0.0041 nM, or 0.00041 nM. The mixture was incubated on ice for 30 minutes. Raji cells were conventionally collected and seeded in a 96-well V-bottom plate at 3 χ¹⁰⁵ cells / well. 1% PBSA was added, and the mixture was centrifuged at 500χ¹ g for 5 min, followed by removal of the supernatant. 150 μg / mL of mouse isotype IgG control (Thermofisher, Cat. No. 10400C) was added, and the mixture was incubated on ice for 20 min and then centrifuged at 500χ g for 5 min, followed by removal of the supernatant. 100 μL of an antibody-protein pre-incubation solution was added to resuspend the cells, and a blank control, a negative control, and an isotype control were designed.The plate was incubated on ice in the dark for 1 h. 100 μL of 1% PBSA was added and the mixture was centrifuged at 500 χ²g for 5 min, followed by removal of the supernatant. The plate was washed twice with 200 μL of 1% PBSA. 100 μL of APC-labeled anti-mouse goat IgG secondary antibody diluted 300-fold (Biolegend, Cat. No. 405308) was added to resuspend the cells, and 100 μL of 1% PBSA was added to resuspend the blank control. The mixture was incubated on ice in the dark for 30 min. 100 μL of 1% PBSA was added and the mixture was centrifuged at 500 χ²g for 5 min, followed by removal of the supernatant. The plate was washed twice with 200 μL of 1% PBSA. 200 μL of 1% PBSA was added to resuspend the cells, and the mixture was analyzed using a flow cytometer.

[0224] The EC50 values ​​of the samples are presented in Table 22. The results are presented in Figure 28. The results indicate that the antibodies NTLAV8, LA5EV2, Relatlimab (positive control) and Petition 870250099246, dated 10 / 30 / 2025, pp. 234 / 270 94 / 101 H9L8(hG4WT) were able to competitively bind to human LAG3-mG1 Fc and effectively block LAG3-mG1Fc binding to MHC II on the Raji cell surface in a dose-dependent manner. Table 22. Results of the analysis of the fluorescence intensities of NTLAV8, LA5EV2, Relatlimab, and H9L8 in competition with MHC II on the surface of the Raji cell for binding to the human LAG3-MG1 Fc antigen by FACS. Antibody LA5EV2 NTLAV8 Relatlimab H9L8(hG4WT) EC50 (nM) 0.9989 0.9740 0.9448 1.440 Example 10: Blocking Assays for Bispecific Anti-CD73 Anti-LAG3 Antibodies

[0225] Jurkat-NFAT-PD1-LAG3 cells (constructed by Akeso Biopharma Inc.) and Raji cells (Cell Resource Center, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, Cat. No. TCHu 44) were collected and centrifuged at 110 x g for 5 min, followed by removal of the supernatant. The cells were resuspended in 1640 medium (containing 10% FBS) and counted. Jurkat-NFAT-PD1-LAG3 cells were seeded in a 96-well black-bottom plate (Corning, Model No. 3916) at 10 x 10⁴ cells / well. According to the experimental design, antibodies (at final concentrations of 900 nM, 300 nM, 100 nM, 33.3 nM, 3.3 nM, 0.33 nM, 0.03 nM, and 0.003 nM) were added, and the mixture was pre-incubated at 37 °C in a 5% CO2 incubator for 30 min. SEE (staphylococcal enterotoxin E) (at a final concentration of 0.05 ng / mL, Toxin Technology, Cat. No. ET404) and Raji cells were incubated at 37 °C in a 5% CO2 incubator for 30 min.After incubation for 30 min, Raji cells were added to the 96-well plate at 2 χ 104 cells / well (the final system volume was 80 μL). The mixture was thoroughly mixed and incubated at 37 °C in an incubator with 5% CO2 for 16 h. The culture plate was removed and allowed to reach room temperature. Petition 870250099246, dated 10 / 30 / 2025, pages 235 / 270 95 / 101 ambient. The Firefly Glo Luciferase Reporter Gene Assay kit (Yeasen, Cat. No. 11404ES80) was added at 80 μL / well, and the mixture was incubated in the dark for 2 min. Then, URL values ​​were read.

[0226] The results are shown in Figure 29.

[0227] The results show that both NTLAV8 and H9L8(hG4WT) were able to effectively block the inhibition of the signaling pathway mediated by the interaction between LAG3 and MHCII. Example 11: Assays for Inhibition of CD73 Enzyme Activity on the Cell Membrane Surface by Bispecific Anti-CD73 / Anti-LAG3 Antibodies

[0228] The groups in the experiment were defined as follows: ATP control group, AMP + ATP control group, negative control group (cells + AMP + ATP), APCP control group (cells + APCP + AMP + ATP), isotypic control group (cells + isotypic control antibody + AMP + ATP), and antibody group (cells + corresponding antibody + AMP + ATP).

[0229] ATP refers to adenosine triphosphate, AMP refers to adenosine monophosphate and APCP refers to α,β-methylene adenosine-5'-bisphosphate, which is a specific inhibitor of CD73.

[0230] Logarithmic-phase, good-condition U87-MG cells (ATCC, Cat. No. HTB-14) were collected, resuspended in analytical medium (i.e., a serum-free RPMI-1640 culture solution), and counted. U87-MG cells were seeded in a 96-well plate at 2.5 χ¹⁰⁴ cells / 60 μE / well. 120 μM of APCP and diluted antibodies (initial concentration of 300 nM, diluted in a 3-fold gradient (10-fold dilution of the last two low concentrations)) were prepared in analytical medium. The APCP or diluted antibodies described above were added to the 96-well plate at 60 μE / well, and the plate was incubated at 37 °C for 1 h. Then, [the text abruptly ends here, so the translation stops here as well.] Petition 870250099246, dated 10 / 30 / 2025, pages 236 / 270 96 / 101 AMP (AMP was acquired from TCI, Cat. No. A0158) was diluted with an analytical medium to a concentration of 600 μM at 60 μL / well, and the mixture was thoroughly mixed and incubated for 3 h. After 3 h, 100 μL of cell culture supernatant were removed from each well and transferred to a new 96-well plate, to which CTG (CellTiter-Glo® One Solution Assay) (Promega, Cat. No. G8461) was added at 40 μL / well. The plate was gently tapped to mix well and left to stand in the dark at room temperature for 5 min. After 5 min of rest, ATP (5'-ATP 2Na Hydrate, acquired from TCI, Cat. No. A0157) was added at a concentration of 300 μM, prepared with TM buffer (prepared by Akeso Biopharma Inc.), at 10 μL / well. The plate was gently tapped to mix well and left to stand in the dark, at room temperature, for 5 min. Finally, the data were read on a multi-label microplate assay instrument (PerkinElmer, Model No. 2140-0020).

[0231] The experimental results are presented in Table 23 and Figure 30. The results demonstrate that the anti-CD73-antiLAG3 antibodies LA5EV2, NTLAV2, and NTLAV8 and the positive control drug MEDI9447 for the CD73 target were able to inhibit, in a dose-dependent manner, the enzymatic activity of CD73 endogenously expressed by U87-MG to catalyze the conversion of AMP to adenosine, thus reducing the URL (URL) value of the mean fluorescence intensity in a dose-dependent manner; LA5EV2 and NTLAV8 exhibited stronger activity in inhibiting the enzymatic activity of CD73 than MEDI9447. Table 23. Results of the inhibition of CD73 enzymatic activity. Endogenously expressed in cells by bispecific antibodies antiCD73-anti-LAG3 Antibody MEDI9447 LA5EV2 NTLAV2 NTLAV8 EC50 (nM) 0.1520 0.6097 0.9399 0.8160 Maximum inhibition rate (%) 58.26 74.69 98.24 98.80 Petition 870250099246, dated 10 / 30 / 2025, pp. 237 / 270 97 / 101 Example 12: Assays for Biological Activity of Anti-CD73-Anti-LAG3 Bispecific Antibodies in Promoting IFN-r Secretion by Lymphocyte Mixed Reaction (MLR)

[0232] Raji-PDL1 cells (constructed by Akeso Biopharma Inc.) were conventionally subcultured; meanwhile, PBMCs (from healthy donors) were thawed, cultured in 10 mL of complete 1640 medium, and stimulated with SEB (staphylococcal enterotoxin B, toxin technology, Cat. No. BT202) to a final concentration of 0.5 μg / mL for two days. After two days, Raji-PDL1 cells were conventionally collected and resuspended in an analytical medium (i.e., RPMI-1640 + 10% FBS), and then MMC (Mito-mycin C, Stressmarq, Cat. No. SIH-24610MG) was added to a final concentration of 2 μg / mL. The mixture was incubated at 37 °C in an incubator with 5% CO2 for 1 h. PBMCs stimulated with SEB for two days and Raji-PDL1 cells treated with MMC for 1 h were conventionally collected and washed twice with an analytical medium.The two cell types were resuspended in an analytical medium, counted, and added to a 96-well U-shaped plate (Corning, Model No. 3799) at 1 χ¹⁰⁵ cells / well for co-culture. According to the experimental design, AMP (5'-adenylic acid) (at a final concentration of 200 μM, 200TCI, Cat. No. A0158) and antibodies (at final concentrations of 300 nM, 30 nM, and 3 nM) were added, and a negative control group (PBMC + Raji-PDL1 + AMP), an isotypic control group, and similar groups were established. The cells were co-cultured in an incubator for three days. After three days, the cells were centrifuged at 250 χ²g for 5 min, and the cell culture supernatant was collected and analyzed for IFN-γ by ELISA.

[0233] As shown in Figure 31, in the mixed culture system of human PBMCs and Raji-PDL1 cells, the addition of antibodies can significantly induce IFN-γ secretion in the system, and the anti-antibodies Petition 870250099246, dated 10 / 30 / 2025, pp. 238 / 270 98 / 101 CD73-anti-LAG3 LA5EV2 and NTLAV8 exhibited superior activity to antibodies 19F3H2L3(hG1DM) and MEDI9447 targeting only CD73 and to the control antibodies H9L8(hG4WT) and Relatlimab targeting only LAG3. Example 13: Potential Cellular Phagocytic Activity Mediated by Bispecific Anti-CD73-Anti-LAG3 Antibodies in CHO-K1LAG3-CD73 Target Cells

[0234] Cryopreserved MBMM (induced from C57 mice, acquired from Guangdong GemPharmatech Co., Ltd.) was thawed and cultured overnight in DMEM medium + 10% FBS + 100 ng / mL M-CSF (murine M-CSF, peprotech, Cat. No. 315-02).

[0235] CHO-K1-CD73-LAG3 target cells (constructed by Cells from Akeso Biopharma Inc. were collected, centrifuged at 170xg for 5 min, washed once with PBS, and counted. CFSE (Biolegend, Cat. No. 423801) was diluted with PBS to a final concentration of 2.5 μM. An adequate amount of diluted CFSE was collected to resuspend the cells (staining density: 1 χ¹⁰⁷ cells / mL), and the cells were incubated in an incubator for 20 min. 6 mL of complete DMEM medium (containing 10% FBS) was added to stop staining. The cells were centrifuged at 170 χ¹⁰g for 5 min, followed by removal of the supernatant. 1 mL of complete DMEM medium was added, and the cells were incubated in an incubator for 10 min. The antibodies were diluted with complete DMEM medium to final concentrations of 0.01 nM, 0.1 nM, 1 nM, 10 nM, and 100 nM, and negative and isotype controls were defined. Target cells were added to a 96-well V-bottom plate at 1.5 χ¹⁰⁵ cells / well, and the antibodies were added.The mixture was thoroughly mixed and incubated on ice for 40 min. The mixture was centrifuged at 170 χ²g for 5 min and washed twice with complete DMEM medium. Macrophages (MBMMs) were collected and... Petition 870250099246, dated 10 / 30 / 2025, pp. 239 / 270 99 / 101 cells were centrifuged at 750 χ² g for 5 min, followed by removal of the supernatant. The cells were counted, resuspended in complete DMEM medium, adjusted to a concentration of 5 χ²¹⁰⁴ cells / 100 μL, and added to a 96-well V-bottom plate containing target cells. The cells were resuspended, thoroughly mixed, and incubated in an incubator at 37 °C for 2 h. 100 μL of 1% PBSA at room temperature was added to each well, and the mixture was centrifuged at 750 χ² g for 5 min, followed by removal of the supernatant. The plate was washed once with 200 μL of 1% PBSA. A mouse / human anti-CD11b APC antibody (Biolegend, Cat. No. 101212) diluted 500-fold with 1% PBSA was added to the corresponding samples at 100 μL / well, and the mixture was thoroughly mixed and incubated on ice for 40 min. 100 μL of 1% PBSA was added to each well, and the mixture was centrifuged at 750 χ²g for 5 min, followed by removal of the supernatant. The plate was washed once with 200 μL of 1% PBSA.The cells were resuspended in 200 μL of 1% PBSA and analyzed using a flow cytometer.

[0236] The results are shown in Figure 32.

[0237] The results show that the antibody 19F3H2L2(G1WT), targeting only CD73, was able to promote phagocytosis of CHO-K1-CD73-LAG3 cells by macrophages (MBMMs) and had an ADCP effect, while the anti-CD73-anti-LAG3 antibodies LA5EV2 and NTLAV8 did not have an ADCP effect. Example 14: Pharmacodynamic Evaluation of the Bispecific Anti-CD73-Anti-LAG3 Antibody in a Mouse Model Subcutaneously Grafted with Tumor Cells

[0238] To determine the in vivo antitumor activity of the bispecific anti-CD73-anti-LAG3 antibody, MIA-PaCa-2 cells (acquired from the Cell Bank of the China Types Culture Collection Center) were first subcutaneously inoculated into 6-7 week old female NCG mice (acquired from Guangdong GemPharmatech Co., Ltd.). The day of Petition 870250099246, dated 10 / 30 / 2025, pages 240 / 270 The 100 / 101 grouping was defined as D0. The route of administration was intraperitoneal (IP) injection, once a week, totaling 4 doses. The specific dosing regimen and modeling are shown in Table 24. After administration, the length and width of the tumors in each group were measured, and the tumor volume was calculated. The tumor volume was calculated according to the following formula: long tumor diameter χ² / short tumor diameter². The data were processed using GraphPad Prism 5 statistical processing software. Table 24. Dosage regimen of the bispecific anti-CD73-antiLAG3 antibody for the treatment of the xenograft cell tumor model. NCG mouse MIA-PaCa-2 Group Number of animals Inoculation Administration Normal group 6 MIA-PaCa-2 cells (5 million), inoculated subcutaneously Normal saline solution, IP, weekly χ² 4 Model group 6 NCG mice were subcutaneously inoculated with 5 million MIA-PaCa-2 cells, and each mouse was then intraperitoneally injected with 5 million hPBMCs (non-activated) on day 5 after inoculation. When the mean tumor volume reached approximately 140 mm³, the mice were pooled for administration (D0). hIgG1.7.5 mg / kg, IP, weeklyx4 19F3H2L3 (hG1DM) 6 7.5mg / kg, IP, weeklyx4 Relatlimab 6 7.5mg / kg, IP, weeklyx4 NTLAV8 6 10 mg / kg, IP, weeklyx4 19F3H2L3 (hG1DM)+ Relatlimab 6 7.5mg / kg+7.5mg / kg, IP, weeklyx4

[0239] Note: Doses of 19F3H2L3(hG1DM) at 7.5 mg / kg, Relatlimab at 7.5 mg / kg and NTLAV8 at 10 mg / kg were based on equal molar concentrations.

[0240] The results are shown in Figure 33. The results demonstrate that, compared to the isotypic control antibody, the bispecific anti-CD73-anti-LAG3 NTLAV8 antibody and the control antibodies Relatlimab and 19F3H2L3(hG1DM) were able to effectively inhibit tumor growth in mice. At the same dose, the antitumor effect Petition 870250099246, dated 10 / 30 / 2025, pp. 241 / 270 The 101 / 101 ratio of the bispecific anti-CD73-anti-LAG3 NTLAV8 antibody was superior to that of monotherapy with the control antibody Relatlimab or 19F3H2L3(hG1 DM) and to that of the combination therapy of Relatlimab and 19F3H2L3(hG1DM).

[0241] Furthermore, as shown in Figure 34, tumor-bearing mice exhibited good tolerance to all drugs tested, and the drugs tested in each group had no significant effect on the body weight of tumor-bearing mice.

[0242] Although the specific embodiments of this disclosure have been described in detail, those skilled in the art will understand that various modifications and substitutions may be made to these details in accordance with all the teachings disclosed, and all such changes fall within the scope of protection of this disclosure. The full scope of this disclosure is provided by the appended claims and any equivalents thereof. Petition 870250099246, dated 10 / 30 / 2025, pages 242 / 270

Claims

1 / 26 Claims 1. A BIESPECIFIC ANTIBODY, characterized by comprising 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 other than LAG3 (e.g., CD73 or PD-1), wherein the first protein functional region is an anti-LAG3 antibody or an antigen-binding fragment thereof, and the anti-LAG3 antibody comprises a variable heavy chain region and a variable light chain region; the variable heavy chain region comprises HCDR1, HCDR2, and HCDR3, and the variable light chain region comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 has the amino acid sequence presented in SEQ ID NO: 9, HCDR2 has the amino acid sequence presented in SEQ ID NO: 10, and HCDR3 has the amino acid sequence presented in SEQ ID NO: 11; LCDR1 has the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 17.LCDR2 has the amino acid sequence presented in SEQ ID NO: 13 or SEQ ID NO: 15, and LCDR3 has the amino acid sequence presented in SEQ ID NO: 14 or SEQ ID NO:

16.

2. ANTIBODY, according to claim 1, characterized by: LCDR1 having the amino acid sequence presented in SEQ ID NO: 12, LCDR2 having the amino acid sequence presented in SEQ ID NO: 13, and LCDR3 having the amino acid sequence presented in SEQ ID NO: 14; or LCDR1 having the amino acid sequence presented in SEQ ID NO: 12, LCDR2 having the amino acid sequence presented in SEQ ID NO: 15, and LCDR3 having the amino acid sequence presented in SEQ ID NO: 16; or LCDR1 possesses the amino acid sequence presented in SEQ ID NO: 17,LCDR2 has the amino acid sequence presented in SEQ ID NO: 15 and LCDR3 has the amino acid sequence presented in SEQ ID NO: 14; or LCDR1 has the amino acid sequence presented in SEQ ID NO: 12, LCDR2 has the amino acid sequence presented in SEQ ID NO: 13 and LCDR3 has the amino acid sequence presented in SEQ ID NO: 16; or LCDR1 has the amino acid sequence presented in SEQ ID NO: 12, LCDR2 has the amino acid sequence presented in SEQ ID NO: 15 and LCDR3 has the amino acid sequence presented in SEQ ID NO: 14; or LCDR1 has the amino acid sequence shown in SEQ ID NO: 17, LCDR2 has the amino acid sequence shown in SEQ ID NO: 13 and LCDR3 has the amino acid sequence shown in SEQ ID NO: 14; or LCDR1 has the amino acid sequence shown in SEQ ID NO: 17,LCDR2 having the amino acid sequence shown in SEQ ID NO: 13 and LCDR3 having the amino acid sequence shown in SEQ ID NO: 16; or LCDR1 having the amino acid sequence shown in SEQ ID NO: 17, LCDR2 having the amino acid sequence shown in SEQ ID NO: 15 and LCDR3 having the amino acid sequence shown in SEQ ID NO:

16.

3. ANTIBODY, according to any one of claims 1 to 2, characterized by: Petition 870250099246, dated 10 / 30 / 2025, p. 245 / 270 4 / 26 The variable region of the heavy chain of the anti-LAG3 antibody should be selected from the amino acid sequences presented in SEQ ID NO: 2 and SEQ ID NO: 44, and the variable region of the light chain of the anti-LAG3 antibody should be selected from the amino acid sequences presented in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 45; preferably, the variable region of the heavy chain of the anti-LAG3 antibody has the amino acid sequence presented in SEQ ID NO: 2,The variable region of the anti-LAG3 antibody light chain has the amino acid sequence shown in SEQ ID NO: 4; the variable region of the anti-LAG3 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the anti-LAG3 antibody light chain has the amino acid sequence shown in SEQ ID NO: 6; the variable region of the anti-LAG3 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the anti-LAG3 antibody light chain has the amino acid sequence shown in SEQ ID NO: 8; the variable region of the anti-LAG3 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the anti-LAG3 antibody light chain has the amino acid sequence shown in SEQ ID NO: 45; the variable region of the anti-LAG3 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 44.and the variable region of the anti-LAG3 antibody light chain has the amino acid sequence shown in SEQ ID NO: 4; the variable region of the anti-LAG3 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 44, and the variable region of the anti-LAG3 antibody light chain has the amino acid sequence shown in SEQ ID NO: 6; the variable region of the anti-LAG3 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 44, and the variable region of the anti-LAG3 antibody light chain has the amino acid sequence shown in SEQ ID NO: 8; or the variable region of the heavy chain of the anti-LAG3 antibody has the amino acid sequence shown in SEQ ID NO: 44, and the variable region of the light chain of the anti-LAG3 antibody has the amino acid sequence shown in SEQ ID NO:

45.

4. ANTIBODY, according to any one of claims 1 to 3,characterized by the anti-LAG3 antibody or its antigen-binding fragment being selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, a complementarity-determining region fragment, a single-chain antibody, a humanized antibody, and a chimeric antibody.

5. ANTIBODY, according to any one of claims 1 to 4, characterized by the anti-LAG3 antibody comprising a non-CDR region derived from a human antibody.

6. ANTIBODY, according to any one of claims 1 to 5, characterized by the second protein functional region being an anti-CD73 antibody or its antigen-binding fragment, and the anti-CD73 antibody comprising a variable heavy chain region and a variable light chain region; the variable heavy chain region comprises HCDR1 with the amino acid sequence presented in SEQ ID NO: 25,HCDR2 with the amino acid sequence shown in SEQ ID NO: 26 and HCDR3 with the amino acid sequence shown in SEQ ID NO: 27; and the variable region of the light chain comprising LCDR1 with the amino acid sequence shown in SEQ ID NO: 28, LCDR2 with the amino acid sequence shown in SEQ ID NO: 29 and LCDR3 with the amino acid sequence shown in SEQ ID NO:

30.

7. ANTIBODY, according to claim 6, characterized in that the variable region of the heavy chain of the anti-CD73 antibody is selected from the amino acid sequences shown in SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 46; The variable region of the anti-CD73 antibody light chain should be selected from the amino acid sequences presented in SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 47, and SEQ ID NO: 62; preferably,The variable region of the anti-CD73 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 31, and the variable region of the anti-CD73 antibody light chain has the amino acid sequence shown in SEQ ID NO: 32; the variable region of the anti-CD73 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 31, and the variable region of the anti-CD73 antibody light chain has the amino acid sequence shown in SEQ ID NO: 34; the variable region of the anti-CD73 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 31, and the variable region of the anti-CD73 antibody light chain has the amino acid sequence shown in SEQ ID NO: 36; the variable region of the anti-CD73 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 31.and the variable region of the anti-CD73 antibody light chain has the amino acid sequence shown in SEQ ID NO: 37; the variable region of the anti-CD73 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 31, and the variable region of the anti-CD73 antibody light chain has the amino acid sequence shown in SEQ ID NO: 47; the variable region of the anti-CD73 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 31, and the variable region of the anti-CD73 antibody light chain has the amino acid sequence shown in SEQ ID NO: 62; the variable region of the anti-CD73 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 33,The variable region of the anti-CD73 antibody light chain has the amino acid sequence shown in SEQ ID NO: 32; the variable region of the anti-CD73 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 33, and the variable region of the anti-CD73 antibody light chain has the amino acid sequence shown in SEQ ID NO: 34; the variable region of the anti-CD73 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 33, and the variable region of the anti-CD73 antibody light chain has the amino acid sequence shown in SEQ ID NO: 36; the variable region of the anti-CD73 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 33, and the variable region of the anti-CD73 antibody light chain has the amino acid sequence shown in SEQ ID NO: 37; the variable region of the anti-CD73 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 33.and the variable region of the anti-CD73 antibody light chain has the amino acid sequence shown in SEQ ID NO: 47; the variable region of the anti-CD73 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 33, and the variable region of the anti-CD73 antibody light chain has the amino acid sequence shown in SEQ ID NO: 62; the variable region of the anti-CD73 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the anti-CD73 antibody light chain has the amino acid sequence shown in SEQ ID NO: 32; the variable region of the anti-CD73 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 35,The variable region of the anti-CD73 antibody light chain has the amino acid sequence shown in SEQ ID NO: 34; the variable region of the anti-CD73 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the anti-CD73 antibody light chain has the amino acid sequence shown in SEQ ID NO: 36; the variable region of the anti-CD73 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the anti-CD73 antibody light chain has the amino acid sequence shown in SEQ ID NO: 37; the variable region of the anti-CD73 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the anti-CD73 antibody light chain has the amino acid sequence shown in SEQ ID NO: 47; the variable region of the anti-CD73 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 35.and the variable region of the anti-CD73 antibody light chain has the amino acid sequence shown in SEQ ID NO: 62; the variable region of the anti-CD73 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 46, and the variable region of the anti-CD73 antibody light chain has the amino acid sequence shown in SEQ ID NO: 32; the variable region of the anti-CD73 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 46, and the variable region of the anti-CD73 antibody light chain has the amino acid sequence shown in SEQ ID NO: 34; the variable region of the anti-CD73 antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 46,and the variable region of the light chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 36; the variable region of the heavy chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 46, and the variable region of the light chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 37; the variable region of the heavy chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 46, and the variable region of the light chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 47; or the variable region of the heavy chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO: 46, and the variable region of the light chain of the anti-CD73 antibody has the amino acid sequence shown in SEQ ID NO:

62.

8. ANTIBODY, according to any one of claims 1 to 7,characterized by the anti-LAG3 antibody or the anti-CD73 antibody further comprising a constant region derived from a human antibody; preferably, the constant region of the anti-LAG3 antibody or Petition 870250099246, of 10 / 30 / 2025, p. 251 / 270 10 / 26 of the anti-CD73 antibody is selected from a constant region of human IgG1, IgG2, IgG3 or IgG4; preferably, the anti-LAG3 antibody comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is the C region of the Ig gamma-1 chain (for example, as set forth in SEQ ID NO: 18) or the C region of the Ig gamma-4 chain (for example, as set forth in SEQ ID NO: 20), and the light chain constant region is the C region of the Ig kappa chain (for example, as set forth in SEQ ID NO: 19); Preferably, the anti-CD73 antibody comprises a constant heavy chain region and a constant light chain region.where the constant region of the heavy chain is the C region of the Ig gamma-1 chain (for example, as set forth in SEQ ID NO: 18) or the C region of the Ig gamma-4 chain (for example, as set forth in SEQ ID NO: 20), and the constant region of the light chain is the C region of the Ig kappa chain (for example, as set forth in SEQ ID NO: 19).

9. ANTIBODY, according to any one of claims 1 to 8, characterized in being defined by any one of the following items (1) to (4): (1) the anti-LAG3 antibody is of the human IgG1 subtype, in which, according to the EU numbering system, the constant region of the antibody heavy chain has the following mutations: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A and G237A;, (2) Petition 870250099246, of 10 / 30 / 2025, p. 252 / 270 11 / 26 the anti-LAG3 antibody is of the human IgG4 subtype, in which, according to the EU numbering system, the constant region of the antibody heavy chain presents the following mutations: F234A and L235A; F234A and G237A; L235A and G237A; or F234A, L235A and G237A; (3) the anti-CD73 antibody is of the human IgG1 subtype, in which, according to the EU numbering system, the constant region of the antibody heavy chain has the following mutations: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A and G237A; or (4) the anti-CD73 antibody is of the human IgG4 subtype, in which, according to the EU numbering system, the constant region of the antibody heavy chain has the following mutations: F234A and L235A; F234A and G237A; L235A and G237A; or F234A, L235A and G237A.

10. ANTIBODY, according to any one of claims 1 to 9, characterized in that the bispecific antibody is in the IgG-scFv form; Preferably, the first functional protein region is an immunoglobulin and the second functional protein region is a single-stranded antibody;or preferably, the first functional protein region is a single-chain antibody and the second functional protein region is an immunoglobulin directed to a target other than LAG3 (e.g., CD73 or PD-1).

11. ANTIBODY, according to any one of claims 1 to 10, characterized by comprising: a first functional protein region directed to LAG3 and a second functional protein region directed to CD73, wherein the first functional protein region is an anti-LAG3 antibody, the anti-LAG3 antibody is an immunoglobulin and the second functional protein region is a single-chain anti-CD73 antibody;or the first functional protein region is a single-chain anti-LAG3 antibody, the second functional protein region is an anti-CD73 antibody, and the anti-CD73 antibody is an immunoglobulin.

12. ANTIBODY, according to any one of claims 10 to 11, characterized by: a variable region of the immunoglobulin heavy chain comprising HCDR1-HCDR3 with amino acid sequences presented in SEQ ID NOs: 9 to 11, respectively, and a variable region of the immunoglobulin light chain comprising LCDR1-LCDR3 with amino acid sequences presented in SEQ ID NOs: 12 to 14, respectively;a variable region of the heavy chain of the single-chain antibody comprising HCDR1-HCDR3 with amino acid sequences presented in SEQ ID NOs: 25 to 27, respectively, and a variable region of the light chain of the single-chain antibody comprising LCDR1-LCDR3 with amino acid sequences shown in SEQ ID NOs: 28 to 30, respectively; or a variable region of the heavy chain of the single-chain antibody comprising HCDR1-HCDR3 with amino acid sequences presented in SEQ ID NOs: 9 to 11, respectively, and a variable region of the light chain of the single-chain antibody comprising LCDR1-LCDR3 with amino acid sequences presented in SEQ ID NOs: 12 to 14, respectively;a variable region of the immunoglobulin heavy chain comprising HCDR1-HCDR3 with amino acid sequences presented in SEQ ID NOs: 25 to 27, respectively, and a variable region of the immunoglobulin light chain comprising LCDR1-LCDR3 with amino acid sequences presented in SEQ ID NOs: 28 to 30, respectively.

13. ANTIBODY, according to any one of claims 10 to 12, characterized: by the variable region of the immunoglobulin heavy chain being selected from the amino acid sequences presented in SEQ ID NO: 2 and SEQ ID NO: 44, and the variable region of the immunoglobulin light chain being selected from the amino acid sequences presented in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 45;the variable region of the heavy chain of the single-chain antibody is selected from the amino acid sequences presented in SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 46, and the variable region of the light chain of the single-chain antibody is selected from the amino acid sequences presented in SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 47 and SEQ ID NO: 62; or by the variable region of the heavy chain of the single-chain antibody being selected from the amino acid sequences presented in SEQ ID NO: 2 and SEQ ID NO: 44, and the variable region of the light chain of the single-chain antibody being selected from the amino acid sequences Petition 870250099246, of 10 / 30 / 2025, p. 255 / 270 14 / 26 presented in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 45;the variable region of the immunoglobulin heavy chain being selected from the amino acid sequences presented in SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 46, and the variable region of the immunoglobulin light chain being selected from the amino acid sequences presented in SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 47 and SEQ ID NO:

62.

14. ANTIBODY, according to any one of claims 10 to 13, characterized in that the bispecific antibody is selected from any one of the following (1) to (24): (1) the variable region of the immunoglobulin heavy chain has the amino acid sequence presented in SEQ ID NO: 2, and the variable region of the immunoglobulin light chain has the amino acid sequence presented in SEQ ID NO: 4;(1) the variable region of the heavy chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 31, and the variable region of the light chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 32; (2) the variable region of the heavy chain of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the light chain of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 4; the variable region of the heavy chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 33, and the variable region of the light chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 34;(3) the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 4; the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 36; (4) the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 4; the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 37; (5) the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 4; the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 46, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 47; (6) the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 4; the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 35, and Petition 870250099246, of 10 / 30 / 2025, page 257 / 270 16 / 26 the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 62; (7) the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 44, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 45; the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 31, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 32; (8) the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 44, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 45; the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 33, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 34; (9) the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 44, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 45; the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 36; (10) the variable region of the immunoglobulin heavy chain has the amino acid sequence presented in SEQ ID NO: 44, and the variable region of the immunoglobulin light chain has the amino acid sequence presented in SEQ ID NO: 45; the variable region of the single-chain antibody heavy chain has the amino acid sequence presented in SEQ ID NO: 35, and the variable region of the single-chain antibody light chain has the amino acid sequence presented in SEQ ID NO: 37; (11) the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 44, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 45; the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 46, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 47; (12) the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 44, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 45; the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 62; (13) the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 4; the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID Petition 870250099246, of 10 / 30 / 2025, page 259 / 270 18 / 26 NO: 31, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 32; (14) the variable region of the heavy chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the light chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 4; the variable region of the heavy chain of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 33, and the variable region of the light chain of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 34; (15) the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 4; the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 36; (16) the variable region of the heavy chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the light chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 4; the variable region of the heavy chain of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the light chain of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 37; (17) the variable region of the single-chain antibody heavy chain Petition 870250099246, 10 / 30 / 2025, p. 260 / 270 19 / 26 has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 4; the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 46, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 47; (18) the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 2, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 4; the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 62; (19) the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 44, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 45; the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 31, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 32; (20) the variable region of the heavy chain of the single-chain antibody has the amino acid sequence presented in SEQ ID NO: 44, and the variable region of the light chain of the single-chain antibody has the amino acid sequence presented in SEQ ID NO: 45; the variable region of the heavy chain of the immunoglobulin has the amino acid sequence presented in SEQ ID Petition 870250099246, of 10 / 30 / 2025, page 261 / 270 20 / 26 NO: 33, and the variable region of the light chain of the immunoglobulin has the amino acid sequence presented in SEQ ID NO: 34; (21) the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 44, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 45; the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 36; (22) the variable region of the single-chain antibody heavy chain has the amino acid sequence shown in SEQ ID NO: 44, and the variable region of the single-chain antibody light chain has the amino acid sequence shown in SEQ ID NO: 45; the variable region of the immunoglobulin heavy chain has the amino acid sequence shown in SEQ ID NO: 35, and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO: 37; (23) the variable region of the heavy chain of the single-chain antibody has the amino acid sequence presented in SEQ ID NO: 44, and the variable region of the light chain of the single-chain antibody has the amino acid sequence presented in SEQ ID NO: 45; the variable region of the heavy chain of the immunoglobulin has the amino acid sequence presented in SEQ ID NO: 46, and the variable region of the light chain of the immunoglobulin has the amino acid sequence presented in SEQ ID NO: 47; and (24) the variable region of the heavy chain of the single-chain antibody Petition 870250099246, of 10 / 30 / 2025, p. 262 / 270 21 / 26 has the amino acid sequence presented in SEQ ID NO: 44, and the variable region of the light chain of the single-chain antibody has the amino acid sequence presented in SEQ ID NO: 45; the variable region of the heavy chain of the immunoglobulin has the amino acid sequence presented in SEQ ID NO: 35,and the variable region of the immunoglobulin light chain has the amino acid sequence shown in SEQ ID NO:

62.

15. ANTIBODY, according to any one of claims 1 to 14, characterized in that the first protein functional region and the second protein functional region are each independently 1, 2 or more in number; preferably, the first protein functional region and the second protein functional region are linked directly or via a linker; preferably, the variable region of the heavy chain and the variable region of the light chain of the single-chain anti-LAG3 antibody are linked directly or via a linker; preferably, the variable region of the heavy chain and the variable region of the light chain of the single-chain anti-CD73 antibody are linked directly or via a linker; preferably, the linker is independently,the polypeptide shown in SEQ ID NO: 48 or a polypeptide formed by the concatenation of a plurality of (e.g., 2, 3, 4, 5 or 6) polypeptides shown in SEQ ID NO: 48; preferably, the linker is independently a polypeptide with one or more glycines attached to the C-terminus of the polypeptide shown in SEQ ID NO: 48 or a polypeptide formed by the concatenation of a plurality of (e.g., 2, 3, 4, 5 or 6) polypeptides shown in SEQ ID NO:

48.

16. ANTIBODY, according to any of the Petition 870250099246, dated 10 / 30 / 2025, pp. 263 / 270 22 / 26 claims 10 to 15, characterized in that each single-chain antibody is attached to the C-terminus or the N-terminus of one of two immunoglobulin heavy chains.

17. ANTIBODY, according to any one of claims 1 to 16, characterized by comprising: a first functional protein region targeting LAG3 and a second functional protein region targeting CD73,The first protein functional region is 1 in number and the second protein functional region is 2 in number; wherein the first protein functional region is an immunoglobulin and the second protein functional region is a single-chain antibody; a heavy chain of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 38 and a light chain of the immunoglobulin has the amino acid sequence shown in SEQ ID NO: 39; a variable region of the heavy chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 35, and a variable region of the light chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 62; or a variable region of the heavy chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 46.and a variable region of the light chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 47; each single-chain antibody is linked to the C-terminus of one of the two heavy chains of the immunoglobulin; the first protein functional region and the second protein functional region are linked by a first linker; the variable region of the heavy chain of the single-chain antibody and the variable region of the light chain of the single-chain antibody are linked by a second linker; the first linker and the second linker are identical or different; preferably, the first linker and the second linker each have an amino acid sequence independently selected from SEQ ID NO: 42 and SEQ ID NO: 43; preferably, the first linker and the second linker have the amino acid sequence shown in SEQ ID NO: 43; preferably,According to the EU numbering system, the constant region of the immunoglobulin heavy chain comprises the following mutations: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A and G237A.

18. ANTIBODY, according to any one of claims 1 to 16, characterized by comprising: a first functional protein region directed to LAG3, and a second functional protein region directed to CD73, the first functional protein region being 2 in number, and the second functional protein region being 1 in number; wherein the first functional protein region is a single-chain antibody, and the second functional protein region is an immunoglobulin; an immunoglobulin heavy chain having the amino acid sequence presented in SEQ ID NO: 40, and an immunoglobulin light chain having the amino acid sequence presented in SEQ ID NO: 41; a variable region of the heavy chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 2,and Petition 870250099246, dated 10 / 30 / 2025, pp. 265 / 270 24 / 26 a variable region of the light chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 4; or a variable region of the heavy chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 44, and a variable region of the light chain of the single-chain antibody has the amino acid sequence shown in SEQ ID NO: 45; each single-chain antibody is linked to the C-terminus of one of the two heavy chains of the immunoglobulin; the first protein functional region and the second protein functional region are linked by a first linker; the variable region of the heavy chain of the single-chain antibody and the variable region of the light chain of the single-chain antibody are linked by a second linker; the first linker and the second linker are identical or different; preferably, the first linker and the second linker have,each, an amino acid sequence independently selected from SEQ ID NO: 42 and SEQ ID NO: 43; preferably, the first and second ligands both have the amino acid sequence shown in SEQ ID NO: 43; preferably, according to the EU numbering system, the constant region of the immunoglobulin heavy chain comprises the following mutations: L234A and L235A; L234A and G237A; L235A and G237A; or L234A, L235A and G237A.

19. ANTIBODY, according to any one of claims 1 to 18, characterized in that it is for treating or preventing a tumor, wherein: Petition 870250099246, dated 10 / 30 / 2025, p. 266 / 270 25 / 26 Preferably, the tumor is selected from one or more of the following: ovarian cancer, esophageal cancer, melanoma, hematological malignancy, glioblastoma, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, gastric cancer, breast cancer, brain cancer,Pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer; preferably, lung cancer is non-small cell lung cancer; preferably, hematological malignancy is leukemia; preferably, esophageal cancer is squamous cell carcinoma of the esophagus.

20. ISOLATED NUCLEIC ACID MOLECULE, characterized in that the isolated nucleic acid molecule encodes the bispecific antibody, as defined in any one of claims 1 to 18.

21. RECOMBINANT VECTOR, characterized in that it comprises the isolated nucleic acid molecule, as defined in claim 20.

22. HOST CELL, characterized in that it comprises the isolated nucleic acid molecule, as defined in claim 20, or the recombinant vector, as defined in claim 21.

23. PHARMACEUTICAL COMPOSITION, characterized in that it comprises the bispecific antibody, as defined in any one of claims 1 to 18, wherein, optionally,The pharmaceutical composition further comprises a pharmaceutically acceptable auxiliary material.

24. USE OF THE BIESPECIFIC ANTIBODY, as defined in any of claims 1 to 18, or of the pharmaceutical composition, as defined in claim 23, characterized in that it is in the preparation of a medicament to treat or prevent a tumor, wherein: preferably, the tumor is selected from one or more of the following: ovarian cancer, esophageal cancer, melanoma, hematologic neoplasm, glioblastoma, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, gastric cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer and renal cancer; preferably, the lung cancer is non-small cell lung cancer; Preferably, the hematological neoplasm is leukemia; preferably,Esophageal cancer is squamous cell carcinoma of the esophagus. Petition 870250099246, dated 10 / 30 / 2025, pp. 268 / 270.

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