Multi-specific antibody specifically binding to HBV pre-s1 antigen, PD-l1 antigen and CD3 antigen and use thereof

Abstract

The present invention relates to a multi-specific antibody or an antigen-binding fragment that specifically binds to an HBV preS1 antigen, PD-L1 antigen and CD3 antigen, and a preparation method therefor and the use thereof.

Description

Multispecific antibodies specifically binding to HBV pre-S1 antigen, PD-L1 antigen and CD3 antigen and applications thereof

[0001] The contents of the sequence listing submitted below are incorporated into this application by reference in its entirety: Text name: Multispecific antibodies specifically binding to HBV preS1 antigen, PD-L1 antigen and CD3 antigen and their applications.xml, Record date: 2025.01.22, Size: 100KB). Technical Field

[0002] The present application relates to multispecific antibodies that specifically bind to HBV preS1 antigen, PD-L1 antigen and CD3 antigen, as well as pharmaceutical compositions comprising multispecific antibodies that specifically bind to HBV preS1 antigen, PD-L1 antigen and CD3 antigen, as well as preparation methods and uses thereof, including methods for preventing and treating HBV infection or diseases related to HBV infection using the same. Background Art

[0003] Approximately 2 billion people worldwide are infected with the hepatitis B virus (HBV). Currently, over 350 million people are chronically infected with HBV and are at high risk of developing cirrhosis, liver failure, or liver cancer. Over 50% of liver cancers worldwide are caused by HBV infection. HBV-related liver disease remains a major public health problem, resulting in approximately 1 million deaths annually (Lavanchy, D. Journal of viral hepatitis vol. 11, 2 (2004): 97-107).

[0004] The HBV genome is a partially double-stranded, relaxed circular DNA (rcDNA) approximately 3.2 kb in length, consisting of a complete coding negative strand (-) and an incomplete non-coding positive strand (+) (Lamontagne, R Jason et al. Hepatoma research vol. 2 (2016): 163-186.; Glebe, Dieter, and Corinna M Bremer. Seminars in liver disease vol. 33, 2 (2013): 103-12.). The coding negative strand contains four overlapping open reading frames (ORFs) (preC / C, P, preS / S, and X), of which the preS / S open reading frame encodes the large (L), medium (M), and small (S) envelope proteins. The L protein contains pre-S1, pre-S2, and S domains, the M protein contains pre-S2 and S domains, and the S protein contains only the S domain (Sheu, SY, and SJ Lo. Virology vol. 188, 1 (1992): 353-7.). All three surface antigens are known to stimulate the production of HBV neutralizing antibodies. The pre-S1 domain of the L protein is considered to be a key determinant of HBV entry and mediates the interaction between the virus and receptors on hepatocytes (Gripon, P et al. Virology vol. 213, 2 (1995): 292-9.; Le Seyec, J et al. Journal of virology vol. 73, 3 (1999): 2052-7.; Chouteau, P et al. Journal of virology vol. 75, 23 (2001): 11565-72.; Le Duff, Yann et al. Journal of virology vol. 83, 23 (2009): 12443-51.). Existing research indicates that sodium taurocholate cotransporting polypeptide (NTCP), primarily expressed in the liver, is a high-affinity functional receptor for HBV pre-S1 (Yan, Huan et al. eLife vol. 3 10.7554 / eLife.00049.13 Nov. 2012). Therefore, antibodies that inhibit the binding of pre-S1 to its receptor could effectively inhibit HBV infection. However, currently developed neutralizing antibodies targeting HBV have limited efficacy due to their short serum half-life, low specific activity, and high immunogenicity.

[0005] In addition, the currently available antiviral drugs for treating HBV infection achieve a certain therapeutic effect by suppressing viral replication, but the covalently closed circular DNA (cccDNA) of HBV virus is retained in the nucleus of infected hepatocytes, and once the patient stops taking the drug, it is possible to cause the reactivation of HBV infection. Therefore, if the infection is to be completely cured, it is essential to eliminate the HBV infected cells (Protzer et al., Nat Immunol Rev 12: 2013-213, 2012) carrying the cccDNA. The HBV infected cells carrying cccDNA exhibit viral surface proteins on their surface. Even if the virus is released into the intracellular vesicle, it is also the same, because many HBV surface proteins remain integrated in the intracellular membrane of the endoplasmic reticulum, and during the vesicle transfer process, the intracellular membrane can fuse with the cell membrane, thereby causing HBV surface proteins to be displayed on the surface of infected cells.

[0006] HBV-specific immune activation is of great significance for achieving functional cure of HBV and effectively clearing infected cells carrying HBV nucleic acid. T cells play an important role in functional cure of HBV, thus promoting the development of related immunotherapy, aiming to enhance or restore specific CD8 T cells in chronic hepatitis B patients. + T cell activity. Currently reported therapeutic vaccines can reactivate inactivated CD8 T cells in patients with chronic hepatitis B. + T cells. At the same time, the activity of T cells can be regulated by engineering HBV-specific T cells or antigen-chimeric T cell receptors to control or even eliminate HBV infection in preclinical models. However, the production of such T cells requires operation in specialized facilities. In comparison, multispecific antibodies are simple to produce and easy to administer, and can reactivate T cells in vivo. Activated T cells can be enriched in the infected liver and effectively kill infected liver cells. Therefore, multispecific antibodies have broad application prospects compared to other therapies.

[0007] Multispecific antibodies targeting activated T cells have demonstrated promising clinical efficacy in treating tumors such as non-Hodgkin's lymphoma and acute lymphoblastic leukemia. For example, Roche's CD3-CD20 bispecific antibody, glofitamab, dually targets T cells, bringing them closer to B cells. This activates them to release cancer-killing proteins and causes them to proliferate, leading to the rapid breakdown of cancerous B cells and thus treating diffuse large B-cell lymphoma (see WO201620309A1). CD3 (cluster of differentiation 3) is a protein complex composed of four subunits: the CD3γ chain, the CD3δ chain, and two CD3ε chains. CD3 associates with the T cell receptor and the ζ chain to generate activation signals in T lymphocytes. The CD3 molecule is a complex consisting of a CD3γ chain, a CD3δ chain, and two CD3ε chains that associate into three dimers (εγ, εδ, ζζ) (Guy, CS et al. (2009) "Organization of Proximal Signal Initiation at the TCR:CD3 Complex," Immunol Rev. 232(1):7-21;). The CD3 complex binds to the TCR and plays a role in the T cell activation process.

[0008] PD-L1 is constitutively expressed on immune cells such as T cells, B cells, macrophages, and dendritic cells (DCs), and is also expressed on a variety of non-hematopoietic cells, including vascular endothelium, pancreatic islets, and placental syncytiotrophoblasts (Keir et al., 2008). Furthermore, PD-L1 is expressed by tumor cells as an “adaptive immune mechanism” to evade antitumor responses (Ohaegbulam KC et al., 2015) and is upregulated by any inflammatory stimulus (Yamazaki et al., 2002). When proinflammatory cytokines secreted by infiltrating tumor-reactive cytotoxic T lymphocytes (CTLs) induce PD-L1 expression in the tumor microenvironment, PD-L1 binding to PD-1 on CTLs can downregulate antitumor immunity. Therefore, blocking the PD-L1 / PD-1 pathway is an ideal therapeutic approach to restore and enhance antitumor immune responses. In preclinical models, anti-PD-1Ab and anti-PD-L1Ab have shown anti-tumor effects in various tumor models (especially those using immunogenic tumors) by enhancing tumor antigen-specific T cell responses (including cytokine production, survival, cell motility, and glycolysis) (Chang et al. 2015; Zinselmeyer et al., 2013; Okazaki et al., 2013). In addition to its role in tumors, PD-L1 antigen is also highly expressed in patients with HBV infection, causing T cell exhaustion through the interaction of PD-1 / PD-L1, thereby leading to persistent HBV infection (Mol Immunol. 2008; 45(4): 963-70; Cell Death Dis. 2015 Mar 19; 6: e1694). Currently developed PD-L1 antibodies such as Envafolimab (ASC-22, Alphamab Oncology) have shown some effectiveness in treating HBV infection, but a safe and effective functional cure for HBV infection is still under exploration.

[0009] Therefore, in order to achieve a more effective functional cure for HBV-infected patients, the present invention aims to form a multispecific antibody (e.g., a trispecific antibody) targeting multiple antigens including HBV preS1 antigen, PDL1 antigen, and T cell CD3 antigen. On the one hand, the PD-L1 molecule, which serves as an immune checkpoint, is upregulated in HBV-infected liver cells. The designed CD3-preS1-PDL1 multifunctional molecule is believed to be able to target the preS1 antigen and PD-L1 antigen on HBV-infected liver cells, while at the same time binding to CD3 on the surface of T cells, bringing T cells closer to HBV-infected liver cells. By targeting the preS1 antigen and PDL1 antigen on HBV-infected cells, as well as the CD3 antigen on T cells, a multispecific antibody is formed to bridge the infected liver cells with the T cells, thereby effectively activating specific T cells by activating the CD3 signaling pathway, theoretically achieving the killing of HBV-infected liver cells. On the other hand, the PD-L1 binding portion in the multispecific antibody can inhibit PD1 / PD-L1 from transmitting immunosuppressive signals by binding to PD-L1 on the surface of T cells, thereby reversing the immune exhaustion of T cells. Furthermore, this multispecific antibody can also bind to the PD-L1 target on DC cells, block the PD-L1 / PD-1 signaling pathway, promote DC cell activation and inhibit T cell exhaustion, thereby playing a killing role on HBV-infected cells.

[0010] The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are incorporated by reference in their entirety.

[0011] Application Overview

[0012] In some embodiments, the present application provides multispecific antibodies (e.g., trispecific antibodies) that specifically bind to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen, as well as pharmaceutical compositions comprising multispecific antibodies (e.g., trispecific antibodies) that specifically bind to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen. In other embodiments, the present application also provides methods for preventing and / or treating HBV infection-related diseases using the above-mentioned multispecific antibodies (e.g., trispecific antibodies) or pharmaceutical compositions thereof. In other embodiments, the present application also provides uses of the above-mentioned multispecific antibodies (e.g., trispecific antibodies) or pharmaceutical compositions thereof in the preparation of medicaments for preventing and / or treating HBV infection.

[0013] In some embodiments, the present application provides a multispecific antibody comprising a first antigen-binding domain that specifically binds to the PD-L1 antigen, a second antigen-binding domain that specifically binds to the HBV preS1 antigen, and a third antigen-binding domain that specifically binds to the CD3 antigen, wherein the first antigen-binding domain that specifically binds to the PD-L1 antigen comprises: a heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 37; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 38; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 39; and a light chain variable region (V L ), the V L comprising: a light chain complementary determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 40; a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41; and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42; the second antigen binding domain that specifically binds to the HBV preS1 antigen comprises: a heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 1; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable region (V L ), the V L comprising: a light chain complementary determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 4; a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and the third antigen binding domain that specifically binds to the CD3 antigen comprises: (a) a heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 11; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 14; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and a light chain variable region (V L ), the V Lcomprising: a light chain complementary determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 20; a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 23; and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 26; (b) a heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 12; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 15; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18; and a light chain variable region (V L ), the V L comprising: a light chain complementary determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 21; a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 24; and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 27; or (c) a heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 13; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and a light chain variable region (V L ), the V L It comprises: a light chain complementary determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 22; a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 25; and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 28.

[0014] In some embodiments, according to any multispecific antibody described herein, the first antigen-binding domain that specifically binds to the PD-L1 antigen comprises: (a) V H , comprising the amino acid sequence shown in any one of SEQ ID NOs: 43 and 73 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 43 and 73; and V L , comprising the amino acid sequence shown in any one of SEQ ID NOs: 46 and 75 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 46 and 75; (b) V H, comprising the amino acid sequence of SEQ ID NO: 43 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 43; and V L , comprising the amino acid sequence of SEQ ID NO: 45 or a variant thereof having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 45; or (c) V H , comprising the amino acid sequence of SEQ ID NO: 43 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 43; and V L , comprising the amino acid sequence of SEQ ID NO: 47 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 47; the second antigen-binding domain that specifically binds to the HBV preS1 antigen comprises: V H , comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 7; and V L , comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 8; and the third antigen-binding domain that specifically binds to the CD3 antigen comprises: (a) V H , comprising the amino acid sequence of SEQ ID NO: 29 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 29; and V L , comprising the amino acid sequence of SEQ ID NO: 33 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 33; (b) V H , comprising the amino acid sequence shown in any one of SEQ ID NOs: 30 and 69 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 30 and 69; and V L , comprising the amino acid sequence shown in any one of SEQ ID NOs: 34 and 71 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 34 and 71; or (c) V H , comprising the amino acid sequence of SEQ ID NO: 31 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 31; and V L, which comprises the amino acid sequence of SEQ ID NO: 35 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 35.

[0015] In some embodiments, the multispecific antibodies described herein comprise an Fc. In other embodiments, the Fc region described herein is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD. In some embodiments, the Fc region comprises an Fc variant. In some embodiments, the Fc region is glycosylated. In some embodiments, the Fc region is aglycosylated. In some embodiments, the Fc region is reduced in fucosylation or afucosylated. In some embodiments, the Fc variant comprises a substitution at position 297. In some embodiments, the substitution at position 297 is 297Q. In some embodiments, the variable Fc region comprises a substitution at one or more of positions 234, 235, 239, 282, 289, 297, 312, 324, 330, 335, 337, 339, 356, 359, 361, 383, 384, 398, 400, 440, 422, and 442, numbered according to the EU index.

[0016] In some embodiments, the structure of the multispecific antibody (eg, trispecific antibody) is selected from CrossMab2+1-scFv, tri-IgG-(scFv)2, or tri-IgG-scFv.

[0017] In some embodiments, the multispecific antibody (e.g., trispecific antibody) has a CrossMab2+1-scFv structure. In some embodiments, the multispecific antibody comprises five polypeptide chains: wherein,

[0018] A polypeptide chain from N-terminus to C-terminus contains V H 2-C H 1-C H 2-C H 3-LV H 1-L3-V L 1 structure, where V H 2 is the heavy chain variable region that specifically binds to HBV preS1 antigen; V H 1 is the heavy chain variable region that specifically binds to the PD-L1 antigen; V L 1 is the light chain variable region that specifically binds to the PD-L1 antigen; L and L3 are connecting peptides; C H 1 is the heavy chain constant region C H 1 domain; C H 2 is the heavy chain constant region C H 2 domains; C H3 is the heavy chain constant region C H 3 domains;

[0019] Both polypeptide chains contain V from N-terminus to C-terminus L 2-C L , where V L 2 is the light chain variable region that specifically binds to HBV preS1 antigen, C L is the light chain constant region;

[0020] A polypeptide chain from N-terminus to C-terminus contains V H 2-C H 1-V H 3-C L , where V H 2 is the heavy chain variable region that specifically binds to HBV preS1 antigen; V H 3 is the heavy chain variable region that specifically binds to the CD3 antigen; C H 1 is the heavy chain constant region C H 1 domain; C L is a light chain constant region; wherein the polypeptide chain further comprises an Fc, the Fc comprising a C H 2 and C H 3 domains; and

[0021] A polypeptide chain from N-terminus to C-terminus contains V L 3-C H 1, where V L 3 is the light chain variable region that specifically binds to the CD3 antigen, C H 1 is the heavy chain constant region C H 1 domain.

[0022] Where V H 2-C H 1 and V L 2-C L Composition of antigen binding domain (Fab) that specifically binds to HBV preS1 antigen, V H 1-L3-V L 1 constitutes an antigen binding domain (scFv) that can specifically bind to PD-L1 antigen, V H 3-C L and V L 3-C H 1 constitutes the antigen binding domain (Fab) that specifically binds to the CD3 antigen.

[0023] In some embodiments, the multispecific antibody (e.g., a trispecific antibody) comprises: the amino acid sequence of SEQ ID NO: 55 or a variant thereof, which has about 80% sequence identity with the amino acid sequence of SEQ ID NO: 55; and / or the amino acid sequence of SEQ ID NO: 56 or a variant thereof, which has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 56; and / or the amino acid sequence of SEQ ID NO: 57 or a variant thereof, which has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 57; and / or the amino acid sequence of SEQ ID NO: 58 or a variant thereof, which has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 58.

[0024] In some embodiments, the multispecific antibody (e.g., a trispecific antibody) comprises: the amino acid sequence of SEQ ID NO: 55 or a variant thereof, which has about 80% sequence identity with the amino acid sequence of SEQ ID NO: 55; and / or the amino acid sequence of SEQ ID NO: 56 or a variant thereof, which has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 56; and / or the amino acid sequence of SEQ ID NO: 62 or a variant thereof, which has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 62; and / or the amino acid sequence of SEQ ID NO: 58 or a variant thereof, which has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 58.

[0025] In other embodiments, the multispecific antibody (e.g., trispecific antibody) has a tri-IgG-(scFv)2 structure. In some embodiments, the multispecific antibody comprises four polypeptide chains: wherein,

[0026] A polypeptide chain from N-terminus to C-terminus contains V H 2-C H 1-C H 2-C H 3-LV H 1-L3-V L 1 structure, where V H 2 is the heavy chain variable region that specifically binds to HBV preS1 antigen; V H 1 is the heavy chain variable region that specifically binds to the PD-L1 antigen; V L 1 is the light chain variable region that specifically binds to the PD-L1 antigen; L and L3 are connecting peptides; C H 1 is the heavy chain constant region C H 1 domain; C H 2 is the heavy chain constant region C H2 domains; C H 3 is the heavy chain constant region C H 3 domains;

[0027] Both polypeptide chains contain V from N-terminus to C-terminus L 2-C L structure, where V L 2 is the light chain variable region that specifically binds to HBV preS1 antigen, C L is the light chain constant region; and

[0028] A polypeptide chain from N-terminus to C-terminus contains V H 2-C H 1-C H 2-C H 3-LV H 3-L3-V L 3 structure, in which V H 2 is the heavy chain variable region that specifically binds to HBV preS1 antigen; V H 3 is the heavy chain variable region that specifically binds to the CD3 antigen; V L 3 is the light chain variable region that specifically binds to the CD3 antigen; L and L3 are connecting peptides; C H 1 is the heavy chain constant region C H 1 domain; C H 2 is the heavy chain constant region C H 2 domains; C H 3 is the heavy chain constant region C H 3 domains.

[0029] Where V H 2-C H 1 and V L 2-C L Composition of antigen binding domain (Fab) that specifically binds to HBV preS1 antigen, V H 1-L3-V L 1 constitutes an antigen binding domain (scFv) that can specifically bind to PD-L1 antigen, V H 3-L3-V L 3 constitutes an antigen binding domain (scFv) that specifically binds to the CD3 antigen.

[0030] In some embodiments, the multispecific antibody (e.g., a trispecific antibody) comprises: the amino acid sequence of SEQ ID NO: 64 or a variant thereof, which has about 80% sequence identity with the amino acid sequence of SEQ ID NO: 64; and / or the amino acid sequence of SEQ ID NO: 65 or a variant thereof, which has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 65; and / or the amino acid sequence of SEQ ID NO: 66 or a variant thereof, which has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 66.

[0031] In other embodiments, the multispecific antibody (e.g., trispecific antibody) has a tri-IgG-scFv structure. In some embodiments, the multispecific antibody comprises four polypeptide chains: wherein,

[0032] A polypeptide chain from N-terminus to C-terminus contains V H 2-C H 1-C H 2-C H 3-LV H 3-L3-V L 3 structure, in which V H 2 is the heavy chain variable region that specifically binds to HBV preS1 antigen; V H 3 is the heavy chain variable region that specifically binds to the CD3 antigen; V L 3 is the light chain variable region that specifically binds to the CD3 antigen; L and L3 are connecting peptides; C H 1 is the heavy chain constant region C H 1 domain; C H 2 is the heavy chain constant region C H 2 domains; C H 3 is the heavy chain constant region C H 3 domains;

[0033] A polypeptide chain from N-terminus to C-terminus contains V L 2-C L structure, where V L 2 is the light chain variable region that specifically binds to HBV preS1 antigen, C L is the light chain constant region;

[0034] A polypeptide chain from N-terminus to C-terminus contains V H 1-C L structure, where V H 1 is the light chain variable region that specifically binds to the PD-L1 antigen, C L is a light chain constant region; wherein the polypeptide chain further comprises an Fc, the Fc comprising a C H 2 and C H3 domains; and

[0035] A polypeptide chain from N-terminus to C-terminus contains V L 1-C H 1 structure, where V L 1 is the light chain variable region that specifically binds to the PD-L1 antigen, C L is the light chain constant region.

[0036] Where V H 2-C H 1 and V L 2-C L Composition of antigen binding domain (Fab) that specifically binds to HBV preS1 antigen, V H 3-L3-V L 3 constitutes an antigen binding domain (scFv) that can specifically bind to CD3 antigen, V H 1-C L and V L 1-C H 1Antigen binding domain (Fab) that specifically binds to PD-L1 antigen.

[0037] In some embodiments, the multispecific antibody (e.g., a trispecific antibody) comprises: the amino acid sequence of SEQ ID NO: 77 or a variant thereof, which has about 80% sequence identity with the amino acid sequence of SEQ ID NO: 77; and / or the amino acid sequence of SEQ ID NO: 66 or a variant thereof, which has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 66; and / or the amino acid sequence of SEQ ID NO: 80 or a variant thereof, which has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 80; and / or the amino acid sequence of SEQ ID NO: 79 or a variant thereof, which has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 79.

[0038] In some embodiments, the multispecific antibody (e.g., a trispecific antibody) comprises: the amino acid sequence of SEQ ID NO: 77 or a variant thereof, which has about 80% sequence identity with the amino acid sequence of SEQ ID NO: 77; and / or the amino acid sequence of SEQ ID NO: 66 or a variant thereof, which has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 66; and / or the amino acid sequence of SEQ ID NO: 78 or a variant thereof, which has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 78; and / or the amino acid sequence of SEQ ID NO: 79 or a variant thereof, which has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 79.

[0039] In certain embodiments, a method for treating a desired individual disease or condition is provided, comprising administering an effective amount of any multispecific antibody (e.g., trispecific antibody) as described above or a pharmaceutical composition comprising the same to the individual. In certain embodiments, there is provided the use of any multispecific antibody (e.g., trispecific antibody) as described above in the preparation of a pharmaceutical composition for treating a desired individual disease or condition. In certain embodiments, there is provided the use of any multispecific antibody (e.g., trispecific antibody) as described above or a pharmaceutical composition comprising the same in the preparation of a medicine for treating a disease or condition. In certain embodiments, the disease or condition includes HBV infection or a disease or condition associated with HBV infection. In certain embodiments, the disease or condition includes hepatitis B, liver failure, cirrhosis or liver cancer.

[0040] In one aspect, the present application provides a method for treating and / or preventing a disease or condition in an individual in need thereof, comprising administering to the individual an effective amount of a multispecific antibody (e.g., a trispecific antibody) and / or any pharmaceutical composition described herein.

[0041] In some embodiments, according to any of the methods described herein, the disease or condition comprises HBV infection or a disease associated with HBV infection. In some embodiments, the disease or condition comprises hepatitis B, liver failure, cirrhosis, or liver cancer.

[0042] In some embodiments, the present application provides an isolated nucleic acid molecule encoding any multispecific antibody (e.g., a trispecific antibody) as described above. In some embodiments, a vector is provided comprising any nucleic acid molecule as described above. In some embodiments, a host cell is provided comprising any multispecific antibody (e.g., a trispecific antibody), any nucleic acid molecule, or any vector as described above. In some embodiments, a method for preparing a multispecific antibody (e.g., a trispecific antibody) that specifically binds to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen is provided, comprising: a) culturing any host cell as described above under conditions effective to express a multispecific antibody (e.g., a trispecific antibody) that specifically binds to HBV preS1 antigen, PDL1 antigen, and CD3 antigen; and b) obtaining the expressed multispecific antibody (e.g., a trispecific antibody) in the host cell.

[0043] Also provided are pharmaceutical compositions, kits, and articles of manufacture comprising any of the multispecific antibodies (eg, trispecific antibodies), nucleic acid molecules, vectors, or host cells described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1A shows the results of the Jurkat-Raji reporter cell assay in which the exemplary humanized anti-human PD-L1 antibody Hum-7 promoted increased IL2 secretion. Figure 1B shows the results of the Jurkat-Raji reporter cell assay in which the exemplary humanized anti-human PD-L1 antibodies Hum-5 and SBT451 promoted increased IL2 secretion.

[0045] Figure 2A shows the results of enhancing T cell activity, as represented by IL2 secretion, with the humanized anti-human PD-L1 antibody SBT451 in SEB-stimulated PBMCs. Figure 2B shows the results of enhancing T cell activity, as represented by IL2 secretion, with the humanized anti-human PD-L1 antibodies Hum-5 and Hum-7 in SEB-stimulated PBMCs.

[0046] Figure 3A shows a schematic diagram of the CrossMab 2+1-scFv multispecific antibody structure; Figure 3B shows a schematic diagram of the tri-IgG-(scFv)2 multispecific antibody structure; Figure 3C shows a schematic diagram of the tri-IgG-scFv multispecific antibody structure; and Figure 3D shows a schematic diagram of the tri-DVD-Ig multispecific antibody structure.

[0047] Figure 4A shows the binding affinity of K127-9-Mo-mutCrossMab2+1-Ig1-SBT451scFv of CrossMab2+1-scFv structure, K127-9-Ig1-SBT451scFv-MoscFv of tri-IgG-(scFv)2 structure, and K127-9-SBT451-Ig1-MoscFv of tri-IgG-scFv structure to human CD3E-CD3D antigen analyzed by ELISA. Figure 4B shows the binding affinity of POC trispecific antibodies including K127-9-2c11-mutCrossMab2+1-scFv structure of CrossMab2+1-scFv and K127-9-Ig1-S70scFv-2c11scFv of tri-IgG-(scFv)2 structure to mouse CD3E-CD3D antigen analyzed by ELISA.

[0048] Figure 5A shows the binding affinity of K127-9-Mo-mutCrossMab2+1-Ig1-SBT451scFv in the CrossMab2+1-scFv structure, K127-9-Ig1-SBT451scFv-MoscFv in the tri-IgG-(scFv)2 structure, and K127-9-SBT451-Ig1-MoscFv in the tri-IgG-scFv structure to the HBV preS1 antigen as analyzed by ELISA. Figure 5B shows the binding affinity of POC trispecific antibodies, including K127-9-2c11-mutCrossMab2+1-Ig1-S70scFv in the CrossMab2+1-scFv structure and K127-9-Ig1-S70scFv-2c11scFv in the tri-IgG-(scFv)2 structure, to the HBV preS1 antigen as analyzed by ELISA.

[0049] Figure 6A shows the binding affinity of K127-9-Mo-mutCrossMab2+1-Ig1-SBT451scFv in the CrossMab2+1-scFv structure, K127-9-Ig1-SBT451scFv-MoscFv in the tri-IgG-(scFv)2 structure, and K127-9-SBT451-Ig1-MoscFv in the tri-IgG-scFv structure to the human PD-L1 antigen as analyzed by ELISA. Figure 6B shows the binding affinity of POC trispecific antibodies, including K127-9-2c11-mutCrossMab2+1-Ig1-S70scFv in the CrossMab2+1-scFv structure and K127-9-Ig1-S70scFv-2c11scFv in the tri-IgG-(scFv)2 structure, to the mouse PD-L1 antigen as analyzed by ELISA.

[0050] Figure 7 shows the binding affinity of K127-9-Mo-mutCrossMab2+1-Ig1-SBT451scFv of the CrossMab2+1-scFv structure and K127-9-Ig1-SBT451scFv-MoscFv of the tri-IgG-(scFv)2 structure to cynomolgus monkey CD3E-CD3D antigen analyzed by ELISA.

[0051] Figure 8A shows the cytotoxicity of K127-9-Mo-mutCrossMab2+1-Ig1-SBT451 scFv with a CrossMab2+1-scFv structure against 293T-preS1 target cells. Figure 8B shows the cytotoxicity of POC trispecific antibodies, including K127-9-2c11-mutCrossMab2+1-Ig1-S70 scFv with a CrossMab2+1-scFv structure and K127-9-Ig1-S70 scFv-2c11 scFv with a tri-IgG-(scFv)2 structure, against CT26-preS1 target cells.

[0052] Detailed description of this application

[0053] In one aspect, the present application provides a multispecific antibody (e.g., a trispecific antibody) that specifically binds to HBV preS1, PD-L1, and CD3 antigens. In another aspect, the present application also provides a pharmaceutical composition comprising a multispecific antibody (e.g., a trispecific antibody) that specifically binds to HBV preS1, PD-L1, and CD3 antigens. In another aspect, the present application also provides a method for treating a disease or condition in an individual in need thereof, comprising administering to the individual an effective amount of a multispecific antibody (e.g., a trispecific antibody) that specifically binds to HBV preS1, PD-L1, and CD3 antigens, or a pharmaceutical composition comprising the same. In another aspect, the present application provides a use of a multispecific antibody (e.g., a trispecific antibody) that specifically binds to HBV preS1, PD-L1, and CD3 antigens, or a pharmaceutical composition comprising the same, in the preparation of a medicament for preventing or treating HBV infection.

[0054] Antibodies or antigen-binding fragments that specifically bind to HBV preS1 were identified through a combination of scFv yeast library screening, affinity maturation, and appropriately designed biochemical and biological experiments. Antibodies or antigen-binding fragments that specifically bind to human PD-L1 were identified through a combination of hybridoma screening, humanization, and appropriately designed biochemical and biological experiments. Multispecific antibodies (e.g., trispecific antibodies) that specifically bind to HBV preS1, PD-L1, and CD3 antigens were also prepared. When used as multispecific antibodies (e.g., trispecific antibodies) to prevent and / or treat related diseases, synergistic effects can be achieved.

[0055] The present application also provides nucleic acids encoding multispecific antibodies (e.g., trispecific antibodies) that specifically bind to HBV preS1, PD-L1, and CD3 antigens, as well as methods for preparing and using multispecific (e.g., trispecific antibodies) antibodies that specifically bind to HBV preS1, PD-L1, and CD3 antigens, and pharmaceutical compositions comprising any of the above antibodies or antigen-binding fragments.

[0056] definition

[0057] As used herein, "HBV preS1 antigen" refers to one of the components of the large (L) envelope protein of the hepatitis B virus (HBV) surface antigen. The pre-S1, pre-S2, and S domains collectively constitute the L protein. The M protein contains the pre-S2 and S domains, while the S protein consists solely of the S domain (Sheu, SY, and SJ Lo. Virology vol. 188, 1 (1992): 353-7).

[0058] As used herein, "CD3" refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed CD3, as well as any form of CD3 produced by processing in cells. The term also encompasses naturally occurring variants of CD3, such as splice variants or allelic variants. In one aspect, the “CD3 antigen” is a complex comprising a CD3γ chain, a CD3δ chain, and two CD3ε chains associated as three dimers (εγ, εδ, ζζ) (Guy, CS et al. (2009) “Organization of Proximal Signal Initiation at the TCR: CD3 Complex,” Immunol Rev. 232(1):7-21; Call, ME et al. (2007) “Common Themes In The Assembly And Architecture Of Activating Immune Receptors,” Nat. Rev. Immunol. 7:841-850; Weiss, A. (1993) “T Cell Antigen Receptor Signal Transduction: A Tale Of Tails And Cytoplasmic Protein-Tyrosine Kinases,” Cell 73:209-212). In one aspect, the "CD3 antigen" described in the present invention is the epsilon subunit of CD3 (CD3ε), also known as CD3E, T3E, or T-cell surface glycoprotein CD3 epsilon chain.

[0059] The "PD-L1 antigen" described herein is programmed cell death ligand 1 (PD-L1), also known as B7-H1 and CD274 (Dong et al., 1999; Freeman et al., 2000). It is a 290-amino acid type I transmembrane protein belonging to the immunoglobulin (Ig) superfamily and a B7 homolog. PD-L1 comprises an Ig-V and Ig-C-like extracellular domain, a transmembrane domain, and a short cytoplasmic tail domain that does not contain a canonical signaling motif (Dong et al., 1999; Keir et al., 2008; Lin et al., 2008).

[0060] As described in the present application, "treatment" or "treating" is a method for obtaining beneficial or desired results, including clinical results. In view of the purpose of the present application, the beneficial or desired clinical results, including but not limited to one or more of the following: alleviating one or more symptoms caused by the disease, alleviating the degree of the disease, stabilizing the disease (for example, preventing or delaying disease worsening), preventing or delaying the spread of the disease (for example, systemic spread), preventing or delaying disease recurrence, delaying or slowing down disease progression, improving the disease state, alleviating the disease (partial or complete), reducing the dosage of one or more other drugs required for the treatment of the disease, delaying disease progression, improving or improving quality of life, gaining weight, and / or prolonging life. At the same time, "treatment" also includes the reduction of infection pathology results (for example, for HBV virus infection, viral load, degree of liver damage). The method of the present application takes into account any one or more aspects of these treatments.

[0061] The term "prevent" and similar words, such as "prevented", "preventing", "prevention" or "prophylactic", etc., refer to a method for preventing, inhibiting or reducing the likelihood of the occurrence or recurrence of a disease or condition (such as HBV infection). It also refers to delaying the occurrence or recurrence of a disease or condition, or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, "prevention" and similar words also include reducing the intensity, impact, symptoms and / or burden of a disease or condition before it occurs or recurs. As used herein, "prevention" and similar words also include reducing the risk and susceptibility of a disease or condition to occur or recur, such as HBV infection.

[0062] Antibodies or antigen-binding fragments As described in this application, the term "antibody" is broad and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (such as trispecific antibodies), full-length antibodies and antigen-binding fragments thereof, as long as they exhibit the desired antigen-binding activity. A full-length antibody includes two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in the two chains typically include three highly variable loops, known as complementary determining regions (CDRs), light chain (LC) CDRs include LC-CDR1, LC-CDR2 and LC-CDR3, and heavy chain (HC) CDRs include HC-CDR1, HC-CDR2 and HC-CDR3. The CDR boundaries of the antibodies or antigen-binding fragments disclosed herein may be defined or identified by the Kabat, Chothia, or Al-Lazikani conventions (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDR regions of the heavy or light chain are interposed between flanking segments called framework regions (FRs), which are more highly conserved than the CDR regions and form a scaffold that supports the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are classified based on the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by having heavy chains of type α, δ, ε, γ, and μ, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0063] As used herein, the term "antigen-binding fragment" includes an antibody fragment, including, for example, a diabody, Fab, Fab', F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, a multispecific dsFv (dsFv-dsFv'), a disulfide-stabilized diabody (dsdiabody), a single-chain antibody (scFv), an scFv dimer (divalent diabody), a multispecific antibody composed of an antibody fragment comprising one or more CDRs, a single-domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that can bind to an antigen but does not comprise a complete antibody structure. Fab (fragment antigen-binding) as used herein is a fragment comprising the V domain of an antibody. L domain, V H domain, C Ldomain, and C H Antigen-binding fragments also include fusion proteins comprising the above-mentioned antibody fragments. Antigen-binding fragments are capable of binding to the same antigen as the parent antibody or parent antibody fragment (such as a parent scFv). In some embodiments, the antigen-binding fragment may include one or more CDRs from a specific human antibody, which are transplanted into the framework regions of one or more different human antibodies.

[0064] As described in the present application, the term "multispecific antibody" refers to an antibody molecule (e.g., bispecific antibody, trispecific antibody) that has binding specificity for at least two different antigens or epitopes in one molecule. Preferably, the multispecific antibody is a trispecific antibody. As described in the present application, the term "trispecific antibody" refers to an antibody molecule that has binding specificity for three different antigens or epitopes in one molecule. In some preferred embodiments of the present application, the structure of the trispecific antibody can be transformed and obtained on the basis of the structure of the bispecific antibody. The production process of multispecific antibodies (e.g., trispecific antibodies) includes the design of complete molecules, synthesis and cloning of the nucleotide sequence of each domain, expression in mammalian cells, and purification of the final product. Exemplary multispecific antibodies include bispecific antibodies, whose structures are known in the art, including, for example, CrossMab2+1 structure, IgG-(scFv)2 structure, IgG-scFv structure, DVD-Ig structure, etc. (e.g., see review document Labrijn AF, et al. Nat Rev Drug Discov. 2019 Aug; 18(8): 585-608).

[0065] As described in this application, the term "antigen binding domain" refers to the part of an antigen binding molecule that specifically binds to an antigen. More specifically, the term "antigen binding domain" refers to a part of an antibody that includes a region that specifically binds to and is complementary to part or all of an antigen. If it is a large antigen, the antigen binding molecule may only bind to a specific part of the antigen, which is called an antigen epitope. For example, the antigen binding domain can be provided by one or more variable regions (also referred to as variable domains). Preferably, the antigen binding domain includes the variable region of the antibody light chain (V L ) and antibody heavy chain variable region (V H In one aspect, the antigen-binding domain is capable of binding to its antigen and blocking or partially blocking the function of the antigen. Antigen-binding domains that specifically bind to HBV preS1 antigen, PDL1 antigen, or CD3 antigen include antibodies and antigen-binding fragments as further defined herein.

[0066] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody or antibody portion binds. If two antibodies or antibody portions exhibit competitive binding to an antigen, they likely bind to the same epitope on the antigen.

[0067] As described herein, a first antibody "competes" for binding to a second antibody for an HBV preS1 antigen target when the first antibody, at equimolar concentrations, inhibits binding of the second antibody to the HBV preS1 antigen target by at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%), and vice versa. PCT Publication WO 03 / 48731 describes a high-throughput antibody "epitope binning" method based on cross-competition.

[0068] As used herein, the terms "specifically bind," "specifically recognize," or "specific for" refer to a measurable and reproducible interaction, such as the binding of a target to an antibody that can confirm the presence of the target in a heterogeneous population of molecules, including biomolecules. For example, an antibody's ability to specifically recognize a target (which may be an epitope) means that the antibody binds to the target with greater affinity, avidity, greater ease, and / or greater persistence than it binds to other targets. In some embodiments, an antibody that specifically recognizes an antigen reacts with one or more antigenic determinants of the antigen with an affinity that is at least 10 times greater than its binding affinity to other targets.

[0069] As used herein, an "isolated" antibody is an antibody that (1) is not related to naturally occurring proteins, (2) does not contain other proteins from the same source, (3) is expressed by cells of a different species, or (4) does not exist in nature.

[0070] As used herein, the term "isolated nucleic acid" refers to a nucleic acid of genomic, cDNA, or synthetic origin, or a combination thereof. Depending on its origin, the "isolated nucleic acid" (1) is unrelated to all or part of a polynucleotide found in nature, (2) is operably linked to a polynucleotide to which it is not naturally associated, or (3) does not occur in nature as part of a longer sequence.

[0071] As used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen binding sites found within the variable regions of heavy and light chain polypeptides. In the literature Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Kabat et al., USDept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196: 901-917 (1987); Al-Lazikani B. et al. al., J. Mol. Biol., 273: 927-948 (1997); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc MPet These specific regions have been described in, for example, Desmond et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001), wherein these definitions include overlap or subsets of amino acid residues when compared to one another. However, any definition used to designate a CDR of an antibody or grafted antibody or variant thereof is included within the scope of the term as defined and used herein. Table 1 lists the positions of the amino acid residues included in the CDRs defined by the various references cited above for comparison. Algorithms and binding interfaces for CDR prediction are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38: D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43: D432-D438 (2015). The contents of the references cited in this paragraph are incorporated herein by reference in their entirety for use in this application and in one or more claims that may be included in this application.

[0072] Table 1: CDR Definition

[0073] 1The amino acid residue numbers refer to the nomenclature of Kabat et al.

[0074] 2 The amino acid residue numbers refer to the nomenclature of Chothia et al.

[0075] 3 The amino acid residue numbers refer to the nomenclature of MacCallum et al.

[0076] 4 The amino acid residue numbers refer to the nomenclature of Lefranc et al.

[0077] 5 The amino acid residue numbers refer to the nomenclature of Honegger and Plückthun.

[0078] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to the corresponding sequence in an antibody from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they have the biological activity described in the present application (see US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)).

[0079] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This fragment is a dimer formed by a heavy chain variable region and a light chain variable region tightly non-covalently linked. Six hypervariable loops (3 loops each in the light chain and heavy chain) are derived from the folding of these two domains. The hypervariable loops provide the antibody with amino acid residues for binding to the antigen and give the antibody specificity for binding to the antigen. However, even a single variable region (or half of an Fv fragment, which contains only 3 CDRs specific for the antigen) has the ability to recognize and bind to the antigen, although its affinity is lower than that of the complete binding site.

[0080] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is a Fv that contains V molecules linked into a single polypeptide chain. H and V L In some embodiments, the scFv polypeptide further comprises a V H and V LThe linker polypeptide between the domains allows the scFv to form an ideal structure for antigen binding. For an overview of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0081] The term "diabodies" refers to double-chain antibodies H and V L A small antibody fragment prepared by constructing an scFv fragment (see above) with a short linker (e.g., 5-10 residues) between the chains, so that the variable regions pair between the chains rather than within the chains, resulting in a bivalent fragment, that is, a fragment with two antigen-binding sites. A multispecific diabody is a heterodimer of two "crossover" scFv fragments, in which the V H and V L The domains are located on different polypeptide chains. Diabodies are fully described in EP 404,097; WO 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0082] The "humanized" form of a non-human (such as a rodent) antibody is a chimeric antibody that includes minimal sequences from a non-human antibody. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which the hypervariable region (HVR) residues of the recipient antibody are replaced by hypervariable region residues from a non-human species such as a mouse, rat, rabbit or non-human primate with ideal antibody specificity, affinity and performance (donor antibody). In some cases, residues in the human immunoglobulin framework region (FR) are replaced by corresponding non-human residues. In addition, a humanized antibody can include residues that are not present in either the recipient antibody or the donor antibody. These modifications can further improve the performance of the antibody. Typically, a humanized antibody will comprise essentially at least one, usually two, variable regions, in which all or substantially all of the hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin, and all or substantially all of the framework regions are human immunoglobulin sequences. The human antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin. For details, see Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992).

[0083] "Percent amino acid sequence homology (%)" or "homology" or "identity" of the polypeptide and antibody sequences identified herein is defined as the percentage of identical amino acid residues between a candidate sequence and the polypeptide sequence being compared, when conservative substitutions are considered part of the sequence homology. Percent amino acid sequence homology can be determined by a variety of alignment methods within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm needed to maximize alignment over the full length of the compared sequences. However, for the purposes of this application, percent amino acid sequence homology values ​​are generated using the sequence alignment computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004).

[0084] The term "Fc (fragment crystallizable)" or "Fc region" refers to a polypeptide comprising the constant region of a complete antibody, excluding the CH1 domain and, in some cases, part of the hinge, in either monomeric or multimeric form. The original immunoglobulin source of the natural Fc is preferably human, and can be any immunoglobulin, for example, IgG1, IgG2, IgG3, or IgG4. The natural Fc is composed of monomeric polypeptides that can be linked into dimer or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent associations. The Fc region of an immunoglobulin generally comprises the C1 domain of the heavy chain constant region. H 2 domains and C H 3 domains, and optionally including C H 4 domains.

[0085] In some embodiments, each of the two Fc monomers in the Fc dimer comprises an amino acid substitution that promotes heterodimerization of the two monomers. In some embodiments, heterodimerization of the Fc monomers can be promoted by introducing different but compatible substitutions such as "knob-into-hole" residue pairs into the two Fc monomers. The "knob-into-hole" technology is also disclosed in U.S. Patent Publication No. 8,216,805. In some embodiments, one Fc monomer comprises a knob mutation T366W, and the other Fc monomer comprises a hole mutation T366S, L358A, and Y407V. In some embodiments, two Cys residues (S354C on the "knob" side and Y349C on the "hole" side) that form a stabilized disulfide bridge are introduced.

[0086] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR described herein is an FcR that binds to an IgG antibody (a gamma receptor), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (activating receptor) and FcγRIIB (inhibiting receptor), which have similar amino acid sequences and differ primarily in the cytoplasmic domain. The cytoplasmic domain of the activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM). The cytoplasmic domain of the inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) (see M.in Annu. Rev. Immunol. 15:203-234 (1997). The term also includes allotypes, such as the FcγRIIIA allotypes: FcγRIIIA-Phe158, FcγRIIIA-Val158, FcγRIIA-R131, and / or FcγRIIA-H131. FcRs are described in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991), Capel et al., Immunomethods 4:25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). The term FcR in this application encompasses other types of FcRs, including those identified in the future. The term FcR also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgGs to the newborn (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

[0087] The term "FcRn" refers to the neonatal Fc receptor (FcRn). FcRn is structurally similar to the major histocompatibility complex (MHC) and consists of an α chain non-covalently bound to β2 microglobulin. The various functions of the neonatal Fc receptor FcRn are described in Ghetie and Ward (2000) Annu. Rev. Immunol. 18, 739-766. FcRn plays an important role in the passive transport of immunoglobulins (IgGs) from mother to newborn and in regulating serum IgG levels. As a salvage receptor, FcRn can bind and transport endocytosed IgGs in their intact form within and between cells, protecting them from the default degradation pathway.

[0088] The "C" of the human IgG heavy chain constant region H The "I domain" typically extends from amino acid position 118 to amino acid position 215 (EU numbering system).

[0089] The "hinge region" is generally defined as extending from Glu 216 to Pro 230 of human IgG1 (Burton, Molec. Immunol. 22: 161-206 (1985)). By placing the first and last cysteine ​​residues that form inter-heavy chain disulfide bonds in the same positions as in IgG1, the hinge regions of other IgG subtypes can be aligned with the IgG1 sequence.

[0090] Human IgG Fc region "C HThe "2 domain" usually extends from amino acid position 231 to amino acid position 340. H The 2 domain is unique in that it does not pair closely with another region. Instead, it is located between the two C H Two N-terminally linked branched sugar chains are inserted between the two domains. It is speculated that sugars may serve as a substitute for domain-to-domain pairing to help maintain C H 2 domains are stabilized. Burton, Molec Immunol. 22: 161-206 (1985).

[0091] “C H The 3" domain includes the C-terminal residue extending from the C H The 2 domains (from amino acid 341 to the C-terminus of the antibody sequence, usually amino acid residue 446 or 447 of IgG).

[0092] A "functional Fc fragment" possesses the "effector functions" of a native Fc region sequence. Exemplary "effector functions" include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptor; BCR). Such effector functions generally require the Fc region to be bound to a binding domain (e.g., an antibody variable region) and can be assessed using a variety of experimental methods known in the art.

[0093] Antibodies with IgG Fc variants having "altered" FcR binding affinity or ADCC activity have enhanced or diminished FcR binding activity and / or ADCC activity compared to the parent polypeptide or a polypeptide comprising a native Fc sequence. Fc variants that exhibit "enhanced binding" to an FcR have a higher binding affinity (e.g., a lower apparent Kd or IC50 value) for at least one FcR compared to the parent polypeptide or a polypeptide comprising a native IgG Fc sequence. In some embodiments, the binding ability is enhanced by 3-fold, e.g., 5, 10, 25, 50, 60, 100, 150, 200, or even up to 500-fold, or the binding ability is increased by 25% to 1000%, compared to the parent polypeptide. Fc variants that exhibit "decreased binding" to an FcR have a lower affinity (e.g., a higher apparent Kd or IC50 value) for at least one FcR compared to the parent polypeptide. The binding ability is decreased by 40% or more compared to the parent polypeptide.

[0094] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a form of cytotoxicity that refers to the binding of secreted Ig to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to target cells bearing antigen and subsequently kill the target cells using cytotoxins. Antibodies "arm" the cytotoxic cells and are required for this killing. Of the major cell types that mediate ADCC, NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of a target molecule, an in vitro ADCC assay can be performed, as described in U.S. Patent Nos. 5,500,362 or 5,821,337. Effector cells suitable for such experiments include peripheral blood mononuclear cells (PBMC) and natural killer cells (NK). Alternatively, or in addition, the ADCC activity of the target molecule can also be assessed in vivo, for example, as described in the animal model disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998).

[0095] Polypeptides comprising Fc variants exhibit "enhanced ADCC activity" or are capable of more effectively mediating ADCC in the presence of human effector cells, compared to polypeptides comprising wild-type IgG Fc polypeptides or parent polypeptides. When tested in substantially the same quantity as polypeptides comprising wild-type IgG Fc polypeptides (or parent polypeptides), such polypeptides comprising Fc variants are capable of more effectively mediating ADCC, both in vitro and in vivo. Such variants are generally identified using any in vitro ADCC assay known in the art, such as assays or methods for identifying ADCC activity, such as in animal models. In some embodiments, such variants mediate ADCC 5- to 100-fold more efficiently, such as 25- to 50-fold more efficiently, than wild-type Fc (or parent polypeptide).

[0096] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate structural subclass) that binds to the cognate antigen. To assess complement activation, a CDC assay, such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), can be performed. Polypeptide variants with altered Fc region amino acid sequences and increased or decreased C1q binding capacity are described in U.S. Patent No. 6,194,551 B1 and WO 99 / 51642. The contents of these patent publications are expressly incorporated herein by reference. See also Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0097] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. A nucleotide sequence encoding a protein or RNA may also include introns, for example, a nucleotide sequence encoding a protein may contain introns in some forms.

[0098] The term "operably linked" refers to a functional connection between a regulatory sequence and a heterologous nucleotide sequence, thereby enabling expression of the latter. For example, a first nucleotide sequence is operably linked to a second nucleotide sequence when the first nucleotide sequence is in a functional relationship with the second nucleotide sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, if necessary, can link two protein coding regions in the same reading frame.

[0099] "Homologous" refers to the sequence similarity or sequence homology between two polypeptides or between two nucleic acid molecules. If the same base or amino acid monomer subunit is present at the same position in two compared sequences, for example, adenine is present at the same position in two DNA molecules, then the two DNA molecules are homologous at that position. The percentage homology between two sequences refers to the ratio of the number of matching or homologous positions shared by the two sequences to the total number of positions multiplied by 100. For example, if 6 out of 10 positions in two sequences are matched or homologous, then the homology between the two sequences is 60%. For example, the DNA sequences ATTGCC and TATGGC have 50% homology. Generally speaking, when aligning two sequences, the comparison is performed with the goal of obtaining maximum homology.

[0100] An "effective amount" of an antibody (including a multispecific antibody) or composition disclosed herein is an amount sufficient to achieve a specific purpose. An "effective amount" can be determined empirically and by known methods related to the purpose.

[0101] The term "therapeutically effective amount" refers to the amount of the antibody (including multispecific antibodies) or composition described herein that can effectively treat an individual's disease or symptoms. That is, an amount sufficient to reduce or improve the severity and / or duration of the disease or one or more of its symptoms; to prevent the development of the disease, cause the symptoms to subside, prevent the recurrence, development, onset or progression of one or more symptoms associated with the disease, detect the disease, or enhance / improve the preventive or therapeutic effect of another therapy (such as a prophylactic or therapeutic agent). In the case of HBV infection, the therapeutically effective amount of the antibody or composition disclosed herein is the amount of the binding molecule that can effectively prevent or treat the condition caused by HBV infection. The antibody or composition disclosed herein can, to some extent, prevent and / or kill existing HBV-infected cells, and it can be cytostatic or cytotoxic. In some embodiments, a therapeutically effective amount refers to an amount that can prolong the patient's survival. In some embodiments, a therapeutically effective amount refers to an amount that can improve the patient's progression-free survival.

[0102] As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" refers to a material that is free of biological activity or other undesirable properties, e.g., a material that can be added to a pharmaceutical composition administered to a patient without causing a significant adverse biological reaction, or that does not interact in a deleterious manner with any other component contained in the composition. Pharmaceutically acceptable carriers or excipients preferably meet the required standards for toxicology or manufacturing testing and / or are included in the inactive ingredient guide compiled by the U.S. Food and Drug Administration.

[0103] The embodiments of the present application described herein should be understood to include "consisting of" and / or "consisting essentially of" embodiments.

[0104] In this application, "about" is mentioned as a numerical value or parameter, including (and describing) variations of the value or parameter itself. For example, the description of "about X" includes the description of "X".

[0105] As used herein, reference to a value or parameter "not" generally refers to and describes "other than" a value or parameter. For example, "the method cannot be used to treat type X infection" means that the method is generally used to treat other types of infection except type X.

[0106] As used in this application and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0107] Antibodies or antigen-binding domains that specifically bind to PD-L1, CD3, or HBV preS1 antigen

[0108] In one aspect, the present application provides antibodies or antigen-binding domains that specifically bind to PD-L1, CD3, or HBV preS1 antigens, including, but not limited to, humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antibody molecules comprising heavy and / or light chain CDRs as described herein. In one aspect, the antibody or antigen-binding domain is an isolated antibody that binds to PD-L1, CD3, or HBV preS1 antigens. Contemplated antibodies or antigen-binding domains that specifically bind to PD-L1, CD3, or HBV preS1 antigens include all or fragments of full-length antibodies (e.g., full-length IgG1, IgG2, or IgG4) that specifically bind to PD-L1, CD3, or HBV preS1 antigens, single-chain antibodies that specifically bind to PD-L1, CD3, or HBV preS1 antigens, multispecific (e.g., trispecific) antibodies that bind to PD-L1, CD3, or HBV preS1 antigens, immunoconjugates that specifically bind to PD-L1, CD3, or HBV preS1 antigens, and the like. In some embodiments, the antibody or antigen-binding domain that specifically binds to PD-L1, CD3, or HBV preS1 antigen is a Fab, Fab', F(ab)'2, Fab'-SH, single-chain antibody (scFv), Fv fragment, dAb, Fd, nanobody, or diabody. In some embodiments, the antibody or antigen-binding domain that specifically binds to PD-L1, CD3, or HBV preS1 antigen refers to an antibody or antigen-binding domain that binds to PD-L1, CD3, or HBV preS1 antigen with an affinity that is at least 10 times greater (including, for example, 10 ... 2 , 10 3 , 10 4 , 10 5 , 10 6 , or 10 7 In some embodiments, a non-target is an antigen that is not PD-L1, CD3, or HBV preS1 antigen.

[0109] Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation analysis (RIA). Kd values ​​can be determined by methods known in the art, such as surface plasmon resonance (SPR) technology or biolayer interferometry (BLI) technology.

[0110] In certain aspects, the antibody or antigen-binding domain that specifically binds to PD-L1, CD3 or HBV preS1 antigen is capable of (a) promoting, mediating or enhancing ADCC activity against HBV-infected cells, and / or (b) promoting, mediating or enhancing ADCP activity against HBV-infected cells, and / or (c) promoting, mediating or enhancing neutralization activity against HBV-infected cells.

[0111] Although this application generally discusses antibodies or antigen-binding domains that specifically bind to PD-L1, CD3, or HBV preS1 antigens comprising human sequences (e.g., human heavy and light chain variable regions comprising human CDR sequences), non-human antibodies are also contemplated. In some embodiments, the non-human antibodies comprise human CDR sequences and non-human framework region sequences for antibodies or antigen-binding domains that specifically bind to PD-L1, CD3, or HBV preS1 antigens as described herein. In some embodiments, the non-human framework region sequences include any sequence used to generate heavy and / or light chain variable regions using one or more human CDR sequences as described herein, including, for example, mammals such as mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffaloes), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmosets, macaques), and the like. In some embodiments, the non-human antibody or antigen-binding domain that specifically binds to PD-L1, CD3, or HBV preS1 antigen comprises an antibody or antigen-binding domain that specifically binds to PD-L1, CD3, or HBV preS1 antigen generated by grafting one or more human CDR sequences described herein into a non-human framework region (e.g., a murine or chicken framework region sequence).

[0112] Antibodies or antigen-binding domains that specifically bind to PD-L1 antigen

[0113] In one aspect, the present application provides an antibody or antigen-binding domain that specifically binds to PD-L1.

[0114] In some embodiments, the antibody or antigen-binding domain that specifically binds to the PD-L1 antigen binds to PD-L1. In some embodiments, the antibody or antigen-binding domain that specifically binds to the PD-L1 antigen is specific for the PD-L1 antigen and does not cross-react with species or other non-PD-L1 antigens. In some embodiments, the antibody or antigen-binding domain that specifically binds to the PD-L1 antigen cross-reacts with other non-PD-L1 antigens.

[0115] In some embodiments, the antibody or antigen binding domain that specifically binds to the PD-L1 antigen comprises: a heavy chain variable region (V H ), the V Hcomprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 37; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 38; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 39, or the V H A variant comprising up to about 5 amino acid substitutions in the HC-CDRs; and a light chain variable region (V L ), the V L comprising: a light chain complementary determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 40; a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41; and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0116] In some embodiments, the antibody or antigen-binding domain that specifically binds to the PD-L1 antigen comprises: (a) V H , comprising the amino acid sequence of any one of SEQ ID NOs: 43 and 73, or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 43 and 73; and V L , comprising the amino acid sequence of any one of SEQ ID NOs: 46 and 75, or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 46 and 75; (b) V H , comprising the amino acid sequence of SEQ ID NO:43 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:43; and V L , comprising the amino acid sequence of SEQ ID NO: 45 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 45; (c) V H, comprising the amino acid sequence of SEQ ID NO:43 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:43; and V L , which comprises the amino acid sequence of SEQ ID NO:47 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:47.

[0117] The sequences of exemplary antibodies or antigen-binding domains that specifically bind to the PD-L1 antigen are shown in Tables 6 and 7, wherein the CDR numbering is performed according to the Kabat (S70) or IMGT (Hum-5, SBT451, Hum-7) definitions. Those skilled in the art will recognize that there are a variety of known algorithms for predicting the positions of CDRs and defining the light and heavy chain variable regions of antibodies or antigen-binding domains. H and / or V L Sequences, but based on prediction algorithms other than those exemplified in the table below, are also within the scope of this application.

[0118] Antibodies or antigen-binding domains that specifically bind to HBV preS1 antigen

[0119] In one aspect, the present application provides antibodies or antigen-binding domains that specifically bind to HBV preS1 antigen.

[0120] In some embodiments, the antibody or antigen-binding domain that specifically binds to the HBV preS1 antigen binds to HBV preS1. In some embodiments, the antibody or antigen-binding domain that specifically binds to the HBV preS1 antigen is specific for the HBV preS1 antigen and does not cross-react with species or other non-HBV preS1 antigens. In some embodiments, the antibody or antigen-binding domain that specifically binds to the HBV preS1 antigen cross-reacts with other non-HBV preS1 antigens.

[0121] In some embodiments, the antibody or antigen binding domain that specifically binds to the HBV preS1 antigen comprises: a heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 1; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, or the VH A variant comprising up to about 5 amino acid substitutions in the HC-CDRs; and a light chain variable region (V L ), the V L comprising: a light chain complementary determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 4; a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0122] In some embodiments, the antibody or antigen binding domain that specifically binds to the HBV preS1 antigen comprises: H , comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 7; and V L , which comprises the amino acid sequence of SEQ ID NO:8 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:8.

[0123] Sequences of exemplary antibodies or antigen-binding domains that specifically bind to HBV preS1 antigen are shown in Tables 2 and 3, wherein CDR numbering is performed according to the Kabat definition. Those skilled in the art will recognize that there are various known algorithms for predicting the positions of CDRs and defining the light and heavy chain variable regions of antibodies or antigen-binding domains. CDRs, VDRs, and VDRs comprising antibodies or antigen-binding domains as described herein are provided. H and / or V L Sequences, but based on prediction algorithms other than those exemplified in the table below, of antibodies or antigen binding domains are also within the scope of this application.

[0124] Antibodies or antigen-binding domains that specifically bind to CD3 antigen

[0125] In some embodiments, the present application provides antibodies or antigen-binding domains that specifically bind to the CD3 antigen.

[0126] In some embodiments, the antibodies or antigen-binding domains described herein that specifically bind to the CD3 antigen specifically bind to an epitope in the CD3 antigen. In some embodiments, the antibodies or antigen-binding domains that specifically bind to the CD3 antigen are specific for the CD3 antigen and do not cross-react with species or other non-CD3 antigens. In some embodiments, the antibodies or antigen-binding domains that specifically bind to the CD3 antigen cross-react with other non-CD3 antigens.

[0127] In some embodiments, the antibody or antigen binding domain that specifically binds to the CD3 antigen comprises:

[0128] (i) Heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 11; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 14; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17, or the V H A variant comprising up to about 5 amino acid substitutions in the HC-CDRs; and a light chain variable region (V L ), the V L comprising: a light chain complementary determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 20; a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 23; and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 26, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0129] (ii)V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 27, or the V L a variant comprising up to about 5 amino acid substitutions in its LC-CDRs; or

[0130] (iii)V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 13, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 22, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 28, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0131] In some embodiments, the antibody or antigen binding domain that specifically binds to the CD3 antigen comprises:

[0132] (i)V H , comprising the amino acid sequence of SEQ ID NO: 29 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 29; and V L , comprising the amino acid sequence of SEQ ID NO: 33 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 33; (ii) V H , comprising the amino acid sequence of any one of SEQ ID NOs: 30 and 69 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 30 and 69; and V L , which comprises the amino acid sequence shown in any one of SEQ ID NOs: 34 and 71 or a variant thereof, wherein the variant has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 34 and 71; or

[0133] (iii)V H, comprising the amino acid sequence of SEQ ID NO: 31 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 31; and V L , which comprises the amino acid sequence of SEQ ID NO:35 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:35.

[0134] In some embodiments, the antibody or antigen binding domain that specifically binds to the CD3 antigen can be selected from the antibodies or antigen binding domains that specifically bind to the CD3 antigen described in patent applications US20030180799A1, CN106029696B, and CN107074956B. The sequences of exemplary antibodies or antigen binding domains that specifically bind to the CD3 antigen are shown in Tables 4 and 5, wherein the CDRs are numbered according to the Kabat definition. Those skilled in the art will recognize that there are a variety of known algorithms for predicting the positions of CDRs and defining the light and heavy chain variable regions of antibodies or antigen binding domains. The CDRs, V H and / or V L Sequences, but based on prediction algorithms other than the antibody or antigen binding domains exemplified in the table below, are also within the scope of this application.

[0135] In some embodiments, the above amino acid substitutions are limited to the "exemplary substitutions" shown in Table 12 of the present application. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" shown in Table 12 of the present application.

[0136] In some embodiments, the present application provides antibodies that can competitively bind to PD-L1, CD3, or HBV preS1 with any of the above-mentioned antibodies that specifically bind to PD-L1, CD3, or HBV preS1 antigens. In some embodiments, the present application provides antibodies that competitively bind to the same epitope as any of the above-mentioned antibodies that specifically bind to PD-L1, CD3, or HBV preS1 antigens.

[0137] In some embodiments, competition assays can be used to identify monoclonal antibodies that compete for binding to PD-L1, CD3, or HBV preS1 with the antibodies described herein that specifically bind to PD-L1, CD3, or HBV preS1. Competition assays can determine whether two antibodies bind to the same epitope by recognizing the same or spatially overlapping epitopes or by competitively inhibiting binding of one antibody to the antigen. In certain embodiments, such competing antibodies bind to the same epitope as the antibodies described herein. Some exemplary competition assays include, but are not limited to, the general assays described in Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Detailed exemplary methods for resolving epitopes bound by antibodies are described in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ). In some embodiments, each antibody is said to bind to the same epitope if it blocks 50% or more of the binding of the other antibody. In some embodiments, the antibody that competes with the antibody described herein that specifically binds to PD-L1, CD3, or HBV preS1 antigen is a chimeric antibody, a humanized antibody, or a fully human antibody.

[0138] A multispecific antibody that specifically binds to HBV preS1 antigen, PD-L1, and CD3 antigens

[0139] On the one hand, the present application provides a multispecific antibody (preferably, a trispecific antibody), which includes a first antigen-binding domain that specifically binds to the PD-L1 antigen, a second antigen-binding domain that specifically binds to the HBV preS1 antigen, and a third antigen-binding domain that specifically binds to the CD3 antigen.

[0140] In some embodiments, the first antigen binding domain that specifically binds to the PD-L1 antigen comprises: a heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 37; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 38; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 39; and a light chain variable region (V L ), the V LIt comprises: a light chain complementary determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO:40; a LC-CDR2 comprising the amino acid sequence of SEQ ID NO:41; and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO:42.

[0141] In some embodiments, the second antigen binding domain that specifically binds to the HBV preS1 antigen comprises: a heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 1; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable region (V L ), the V L It comprises: a light chain complementarity determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 4; a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0142] In some embodiments, the third antigen binding domain that specifically binds to the CD3 antigen comprises: (a) a heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 11; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 14; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and a light chain variable region (V L ), the V L comprising: a light chain complementary determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 20; a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 23; and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 26; (b) a heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 12; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 15; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18; and a light chain variable region (V L ), the V Lcomprising: a light chain complementary determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 21; a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 24; and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 27; or (c) a heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 13; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and a light chain variable region (V L ), the V L It comprises: a light chain complementary determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 22; a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 25; and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 28.

[0143] In some embodiments, the first antigen binding domain that specifically binds to the PD-L1 antigen comprises: (a) V H , comprising the amino acid sequence of any one of SEQ ID NOs: 43 and 73, or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 43 and 73; and V L , comprising the amino acid sequence of any one of SEQ ID NOs: 46 and 75, or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 46 and 75; (b) V H , comprising the amino acid sequence of SEQ ID NO:43 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:43; and V L , comprising the amino acid sequence of SEQ ID NO: 45 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 45; (c) V H, comprising the amino acid sequence of SEQ ID NO:43 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:43; and V L , which comprises the amino acid sequence of SEQ ID NO:47 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:47.

[0144] In some embodiments, the second antigen binding domain that specifically binds to the HBV preS1 antigen comprises: H , comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 7; and V L , which comprises the amino acid sequence of SEQ ID NO:8 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:8.

[0145] In some embodiments, the third antigen binding domain that specifically binds to the CD3 antigen comprises: (a) V H , comprising the amino acid sequence of SEQ ID NO: 29 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 29; and V L , comprising the amino acid sequence of SEQ ID NO: 33 or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 33; (b) V H , V H , comprising the amino acid sequence of any one of SEQ ID NOs: 30 and 69, or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 30 and 69; and V L, comprising the amino acid sequence of any one of SEQ ID NOs: 34 and 71 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 34 and 71; or (c) V H , comprising the amino acid sequence of SEQ ID NO: 31 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 31; and V L , which comprises the amino acid sequence of SEQ ID NO:35 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO:35.

[0146] In some embodiments, the present application provides a multispecific antibody (preferably, a trispecific antibody) comprising a first antigen-binding domain that specifically binds to the PD-L1 antigen, a second antigen-binding domain that specifically binds to the HBV preS1 antigen, and a third antigen-binding domain that specifically binds to the CD3 antigen, wherein the first antigen-binding domain comprises: V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 37, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 39; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 40, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42; and wherein the second antigen binding domain comprises: V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and V L , the V Lcomprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and wherein the third antigen binding domain comprises: V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18; and V L , the V L It comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:21, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:24, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:27.

[0147] In some embodiments, the present application provides a multispecific antibody (preferably, a trispecific antibody) comprising a first antigen-binding domain that specifically binds to the PD-L1 antigen, a second antigen-binding domain that specifically binds to the HBV preS1 antigen, and a third antigen-binding domain that specifically binds to the CD3 antigen, wherein the first antigen-binding domain comprises: V H , comprising the amino acid sequence of any one of SEQ ID NOs: 43 and 73, or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 43 and 73; and V L , comprising the amino acid sequence of any one of SEQ ID NOs: 46 and 75, or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 46 and 75; and wherein said second antigen binding domain comprises: V H , comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 7; and V L, comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 8; and wherein the third antigen binding domain comprises: V H , comprising the amino acid sequence of any one of SEQ ID NOs: 30 and 69, or a variant thereof, said variant having at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 30 and 69; and V L , which comprises the amino acid sequence shown in any one of SEQ ID NOs: 34 and 71 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 34 and 71.

[0148] CrossMab2+1-scFv structured multispecific antibody

[0149] In some embodiments, a multispecific antibody (referred to as CrossMab2+1-scFv structure in this application, preferably, a trispecific antibody) obtained by modification based on the bispecific CrossMab2+1 structure is based on the CrossMab 2+1 bispecific antibody structure (see, for example, CN103748114B, CN106661120B, Klein C, et al. Engineering therapeutic bispecific antibodies using CrossMab technology. Methods. 2019 Feb 1; 154: 21-31.), in which a scFv fragment that binds to another antigen is connected to the C-terminus of one of the heavy chains. Such a multispecific antigen-binding molecule comprises two monomers, one of which is composed of two polypeptide chains and contains two antigen-binding domains, one of which is an scFv (in a preferred embodiment, referred to herein as the first antigen-binding domain) and the other is a Fab (in a preferred embodiment, referred to herein as the second antigen-binding domain); the other monomer is composed of three polypeptide chains and contains two antigen-binding domains, both of which are Fab (in a preferred embodiment, referred to herein as the third and fourth antigen-binding domains). These antigen-binding domains can bind to the same and / or different antigens.

[0150] In a preferred embodiment of the present application: (i) the first antigen binding domain is a scFv molecule that specifically binds to a first antigen, the second antigen binding domain and the fourth antigen binding domain are Fab molecules that specifically bind to a second antigen, and the third antigen binding domain is a Fab molecule that specifically binds to a third antigen; (ii) preferably, the third antigen binding domain can be a crossover Fab molecule, wherein C H 1-C L Between, V H -V L Between, or V H -C H 1 and V L -C L exchanged between them to ensure the correct pairing between the light and heavy chains of the antibody; (iii) preferably, the C H 1. C L The structural domain contains amino acid mutations that change the charge properties to ensure correct pairing between the antibody light and heavy chains; and (iv) the second antigen-binding domain and the third antigen-binding domain are each fused at the C-terminus of the heavy chain of their Fab to the N-terminus of one of the subunits of the Fc domain, and the fourth antigen-binding domain is fused at the C-terminus of the heavy chain of its Fab to the N-terminus of the heavy chain of the Fab of the third antigen-binding domain; and, (v) the first antigen-binding domain is fused to the C-terminus of one of the subunits of the Fc domain. In a preferred embodiment of the present application, a schematic diagram of the typical structure of the trispecific antibody is shown in Figure 3A.

[0151] In other preferred embodiments, the multispecific antibody further comprises two Fcs, which include C H 2 and C H 3 domains. In some embodiments, one of the Fc C H The amino acid residues in the 3 domain are replaced by amino acid residues with larger side chain volume to form a "knob". At the same time, the C H The amino acid residues in the 3 domain are replaced with amino acid residues with smaller side chain volume to form a "hole". The combination of the "knob" and the "hole" can promote the mutual binding of heterodimers. In other embodiments, in Fab, the light chain constant region (C L ) and heavy chain constant region C H 1 domain (C H 1) positions are replaced with each other; or, the heavy chain variable region (V H ) and light chain variable region (V L ) positions are replaced with each other; or the light chain constant region (C L ) and heavy chain constant region C H 1 domain (CH 1) and the heavy chain variable region (V H ) and light chain variable region (V L ) are replaced with each other at the same time to avoid mispairing between the light and heavy chains.

[0152] In some embodiments, the Fc is derived from wild-type human IgG1 Fc. In other embodiments, the C H The 3 domain includes, but is not limited to, the following amino acid substitutions: S354C, T366W, Y349C, T366S, L368A, and Y407V, wherein the numbering is according to the EU index as in Kabat.

[0153] In some embodiments, the multispecific antibody (preferably, a trispecific antibody) as described above is a heterodimer composed of a first monomer and a second monomer, wherein the first monomer is composed of a first heavy chain and a first light chain, and comprises two antigen binding domains (a first antigen binding domain and a second antigen binding domain); the second monomer is composed of a second heavy chain, a second light chain, and a third light chain, and comprises two antigen binding domains (a third antigen binding domain and a fourth antigen binding domain). In a preferred embodiment of the present application, the first antigen binding domain specifically binds to a first antigen (e.g., PD-L1), the second antigen binding domain and the fourth antigen binding domain have the same sequence, specifically bind to a second antigen (e.g., HBV preS1), and the third antigen binding domain specifically binds to a third antigen (e.g., CD3). In some preferred embodiments, the sequences of the first light chain and the second light chain are the same.

[0154] In some embodiments, the first heavy chain of the multispecific antibody (preferably, a trispecific antibody) comprises from N-terminus to C-terminus: V H 2-C H 1-C H 2-C H 3-LV H 1-L3-V L 1 structure. In other embodiments, the first heavy chain of the multispecific antibody (preferably, a trispecific antibody) comprises from N-terminus to C-terminus: V H 2-C H 1-C H 2-C H 3-LV L 1-L3-V H 1 structure. In some embodiments, the first light chain of a multispecific antibody (preferably, a trispecific antibody) comprises, from N-terminus to C-terminus: V L 2-C L structure.

[0155] Among them, V H 1 and VL 1 is the heavy chain variable region and light chain variable region of the first antigen binding domain; V H 2 and V L 2 are the heavy chain variable region and light chain variable region of the second antigen binding domain respectively; L and L3 are connecting peptides; C H 1 is the heavy chain constant region C H 1 domain; C H 2 is the heavy chain constant region C H 2 domains; C H 3 is the heavy chain constant region C H 3 domains; C L V is the light chain constant region. H 1-L3-V L 1 or V L 1-L3-V H 1 constitutes the first antigen binding domain (scFv) that binds to the first antigen; V H 2-C H 1 and V L 2-C L It forms a second antigen-binding domain (Fab) that binds a second antigen.

[0156] In some embodiments, the second heavy chain of the multispecific antibody (preferably, a trispecific antibody) comprises from N-terminus to C-terminus: V H 2-C H 1-V H 3-C L In some embodiments, the second light chain of the multispecific antibody (preferably, a trispecific antibody) comprises from N-terminus to C-terminus: V L 2-C L In some embodiments, the third light chain of the multispecific antibody (preferably, a trispecific antibody) comprises from N-terminus to C-terminus: V L 3-C H 1.

[0157] In some embodiments, the second heavy chain further comprises an Fc comprising a C H 2 and C H 3 domains.

[0158] In some embodiments, the second heavy chain of the multispecific antibody (preferably, a trispecific antibody) comprises from N-terminus to C-terminus: V H 2-C H 1-V H 3-C L -C H 2-C H 3 structure. In some embodiments, the second light chain of the multispecific antibody (preferably, a trispecific antibody) comprises from N-terminus to C-terminus: VL 2-C L In some embodiments, the third light chain of the multispecific antibody (preferably, a trispecific antibody) comprises from N-terminus to C-terminus: V L 3-C H 1 structure.

[0159] Among them, V H 2 and V L 2 are the heavy chain variable region and light chain variable region of the fourth antigen binding domain; V H 3 and V L 3 are the heavy chain variable region and light chain variable region of the third antigen binding domain; C H 1 is the heavy chain constant region C H 1 domain, C L Is the light chain constant region. H 2-C H 1 and V L 2-C L Composition of the fourth antigen binding domain (Fab) that binds to the second antigen; V H 3-C L and V L 3-C H 1 constitutes the third antigen-binding domain (Fab) that binds to a third antigen.

[0160] In some preferred embodiments, the sequences of the first light chain and the second light chain are identical.

[0161] In some embodiments, C in the first monomer or the second monomer of the multispecific antibody (preferably, a trispecific antibody) H 1 and C L The positions of V H 2 and V L 2 can be interchangeable. In other embodiments, V H 3 and V L The positions of 3 can be interchanged.

[0162] In some embodiments, the C of the first heavy chain H The amino acid residues in the 1 domain are replaced by negatively charged amino acid residues, and the first light chain C L The amino acid residues in the domain are replaced with positively charged amino acid residues; and the C H The amino acid residues in the 1 domain are replaced by negatively charged amino acid residues, and the second light chain C L The amino acid residues in the domain are replaced with positively charged amino acid residues.

[0163] In other embodiments, the C of the first heavy chain HThe amino acid residues in the 1 domain are replaced by positively charged amino acid residues, and the first light chain C L The amino acid residues in the domain are replaced with negatively charged amino acid residues; and the C H The amino acid residues in the 1 domain are replaced by positively charged amino acid residues, and the second light chain C L In some embodiments, the amino acid residues in the C H 1 domain includes, but is not limited to, the following amino acid substitutions: K148E, K214E, wherein the numbering is according to the EU index as in Kabat; the C L The domain includes, but is not limited to, the following amino acid substitutions: E128R, Q129K, wherein the numbering is according to the EU index as in Kabat.

[0164] In some embodiments, the C of the first heavy chain H The amino acid residues in the 3 domain are replaced by amino acid residues with larger side chain volume to form a "knob", and the C of the second heavy chain H In some other embodiments, the amino acid residues in the C domain of the second heavy chain are replaced with amino acid residues with smaller side chain volume to form a "hole". H The amino acid residues in the 3 domain are replaced by amino acid residues with larger side chain volume to form a "knob", and the C of the first heavy chain H The amino acid residues in the C3 domain are replaced with amino acid residues with smaller side chain volume to form a "hole". In some embodiments, the C H The 3 domain includes, but is not limited to, the following amino acid substitutions: S354C, T366W, Y349C, T366S, L368A, and Y407V, wherein the numbering is according to the EU index as in Kabat.

[0165] In some preferred embodiments of the present application, V H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to the PD-L1 antigen binding domain, V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to the antigen binding domain of HBV preS1, V H 3 and V L 3 are the heavy chain variable region and light chain variable region of the antigen binding domain that specifically binds to CD3. H 1-L3-V L 1 constitutes an antigen binding domain (scFv) that specifically binds to PD-L1 antigen, V H 2-C H 1 and V L2-C L Composition of antigen binding domain (Fab) that specifically binds to HBV preS1 antigen, V H 3-C L and V L 3-C H 1 constitutes the antigen binding domain (exchange Fab) that binds to the CD3 antigen.

[0166] In some preferred embodiments of the present application, the multispecific antibody (preferably, a trispecific antibody) described in the present application has a CrossMab 2+1-scFv structure, wherein the first antigen binding domain in the structure specifically binds to the PD-L1 antigen, the second antigen binding domain and the fourth antigen binding domain specifically bind to the HBV pre-S1 antigen, and the third antigen binding domain specifically binds to the CD3 antigen. In some embodiments, the multispecific antibody can simultaneously bind to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen. In some embodiments, the first antigen binding domain scFv that specifically binds to the PD-L1 antigen includes a genetically engineered cysteine ​​mutation, which is achieved by H and V L Two cysteine ​​mutations were introduced into the interface to obtain a disulfide-bond-stabilized multispecific antibody.

[0167] In some embodiments, the multispecific antibody (preferably, a trispecific antibody) as described herein comprises: the amino acid sequence of SEQ ID NO: 55 or a variant thereof, wherein the variant has about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO: 55.

[0168] In some embodiments, the multispecific antibody (preferably, a trispecific antibody) as described herein comprises: the amino acid sequence of SEQ ID NO: 56 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO: 56.

[0169] In some embodiments, the multispecific antibody (preferably, a trispecific antibody) as described herein comprises: the amino acid sequence of SEQ ID NO: 57 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO: 57.

[0170] In some embodiments, the multispecific antibody (preferably, a trispecific antibody) as described herein comprises: the amino acid sequence of SEQ ID NO: 58 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO: 58.

[0171] In some embodiments, the multispecific antibody (preferably, a trispecific antibody) as described herein comprises: the amino acid sequence of SEQ ID NO: 62 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO: 62.

[0172] In some embodiments, the multispecific antibody (preferably, a trispecific antibody) as described herein comprises: the amino acid sequence of SEQ ID NO: 55 or a variant thereof, wherein the variant has about 80% sequence identity with the amino acid sequence of SEQ ID NO: 55; and / or the amino acid sequence of SEQ ID NO: 56 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 56; and / or the amino acid sequence of SEQ ID NO: 57 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 57; and / or the amino acid sequence of SEQ ID NO: 58 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 58.

[0173] In some embodiments, the multispecific antibody (preferably, a trispecific antibody) as described herein comprises: the amino acid sequence of SEQ ID NO: 55 or a variant thereof, wherein the variant has about 80% sequence identity with the amino acid sequence of SEQ ID NO: 55; and / or the amino acid sequence of SEQ ID NO: 56 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 56; and / or the amino acid sequence of SEQ ID NO: 62 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 62; and / or the amino acid sequence of SEQ ID NO: 58 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 58.

[0174] Multispecific antibody with Tri-IgG-(scFv)2 structure

[0175] In some embodiments, a multispecific antibody structure (referred to herein as tri-IgG-(scFv) 2 , preferably, a trispecific antibody) is obtained by modifying a bispecific IgG-(scFv) 2 structure (see, for example, Coloma MJ, Morrison SL. Design and production of novel tetravalent bispecific antibodies. Nat Biotechnol. 1997 Feb; 15(2): 159-63) as a basis, wherein two scFv fragments that bind to different antigens are respectively connected to the C-termini of the two heavy chains of an IgG antibody to achieve multispecificity (preferably, trispecificity). In a preferred embodiment of the present application, a schematic diagram of the typical structure of the trispecific antibody is shown in FIG3B .

[0176] In some embodiments, the multispecific antibody (preferably, a trispecific antibody) as described above has a tri-IgG-(scFv)2 structure, which is composed of two monomers, each of which contains two antigen-binding domains, one of which is a Fab and the other is a scFv. In some embodiments, the multispecific antibody contains an Fc, which contains a C H 2 and C H In some embodiments, the scFv is linked to the carboxyl terminus of the Fc via a linker peptide (L).

[0177] In some embodiments, the multispecific antibody (preferably, a trispecific antibody) as described above is a heterodimer composed of two different monomers, including a total of 4 polypeptide chains, forming 4 antigen binding domains. Among them, the first monomer is composed of a first heavy chain and a first light chain, comprising two antigen binding domains (scFv and Fab, in a preferred embodiment of the present application, referred to as the first antigen binding domain and the second antigen binding domain, respectively); the second monomer is composed of a second heavy chain and a second light chain, comprising two antigen binding domains (scFv and Fab, in a preferred embodiment of the present application, referred to as the third antigen binding domain and the fourth antigen binding domain). In a more preferred embodiment of the present application, the first antigen binding domain specifically binds to a first antigen (e.g., PD-L1), the second antigen binding domain and the fourth antigen binding domain have the same sequence, specifically bind to a second antigen (e.g., HBV preS1), and the third antigen binding domain specifically binds to a third antigen (e.g., CD3). In some preferred embodiments, the sequences of the first light chain and the second light chain are the same.

[0178] In some embodiments, the first heavy chain of the multispecific antibody (preferably, a trispecific antibody) comprises from N-terminus to C-terminus: VH 2-C H 1-C H 2-C H 3-LV H 1-L3-V L 1 structure. In other embodiments, the first heavy chain of the multispecific antibody (preferably, a trispecific antibody) comprises from N-terminus to C-terminus: V H 2-C H 1-C H 2-C H 3-LV L 1-L3-V H 1 structure. In some embodiments, the first light chain of the multispecific antibody (preferably, a trispecific antibody) comprises from N-terminus to C-terminus: V L 2-C L structure.

[0179] Among them, V H 1 and V L 1 are the heavy chain variable region and light chain variable region of the first antigen binding domain, V H 2 and V L 2 are the heavy chain variable region and light chain variable region of the second antigen binding domain, C H 1 is the heavy chain constant region C H 1 domain, C L V is the light chain constant region, L and L3 are connecting peptides. H 1-L3-V L 1 or V L 1-L3-V H 1 constitutes the first antigen-binding domain (scFv) of a multispecific antibody (preferably, a trispecific antibody) that binds to a first antigen, V H 2-C H 1 and V L 2-C L The second antigen-binding domain (Fab) that binds to a second antigen constitutes a multispecific antibody (preferably, a trispecific antibody).

[0180] In some embodiments, the second heavy chain of the multispecific antibody (preferably, a trispecific antibody) comprises from N-terminus to C-terminus: V H 2-C H 1-C H 2-C H 3-LV H 3-L3-V L 3 structure. In other embodiments, the second heavy chain of the multispecific antibody (preferably, a trispecific antibody) comprises from N-terminus to C-terminus: V H 2-C H 1-C H2-C H 3-LV L 3-L3-V H 3 Structure. In some embodiments, the second light chain of the multispecific antibody (preferably, a trispecific antibody) comprises, from N-terminus to C-terminus: V L 2-C L structure.

[0181] Among them, V H 2 and V L 2 are the heavy chain variable region and light chain variable region of the fourth antigen binding domain, V H 3 and V L 3 are the heavy chain variable region and light chain variable region of the third antigen binding domain, C H 1 is the heavy chain constant region C H 1 domain, C L V is the light chain constant region, L and L3 are connecting peptides. H 2-C H 1 and V L 2-C L The fourth antigen-binding domain (Fab) that binds to the second antigen constitutes a multispecific antibody (preferably a trispecific antibody), V H 3-L3-V L 3 or V L 3-L3-V H 3. A third antigen-binding domain (scFv) that binds to a third antigen and constitutes a multispecific antibody (preferably, a trispecific antibody).

[0182] In some preferred embodiments, the sequences of the first light chain and the second light chain are identical.

[0183] In some embodiments, the C of the first heavy chain H The amino acid residues in the 3 domain are replaced by amino acid residues with larger side chain volume to form a "knob", and the C of the second heavy chain H In some other embodiments, the amino acid residues in the C domain of the second heavy chain are replaced with amino acid residues with smaller side chain volume to form a "hole". H The amino acid residues in the 3 domain are replaced by amino acid residues with larger side chain volume to form a "knob", and the C of the first heavy chain H The amino acid residues in the C3 domain are replaced with amino acid residues with smaller side chain volume to form a "hole". In some embodiments, the C H The 3 domain includes, but is not limited to, the following amino acid substitutions: S354C, T366W, Y349C, T366S, L368A, and Y407V, wherein the numbering is according to the EU index as in Kabat.

[0184] In some preferred embodiments of the present application, V H 1 and V L 1 are the heavy chain variable region and light chain variable region of the antigen binding domain that specifically bind to PD-L1, V H 1-L3-V L 1 or V L 1-L3-V H 1 constitutes an antigen binding domain (scFv) that can specifically bind to the PD-L1 antigen; V H 2 and V L 2 are the heavy chain variable region and light chain variable region of the antigen binding domain that specifically bind to HBV preS1, V H 2-C H 1 and V L 2-C L Composition of antigen binding domain (Fab) that can specifically bind to HBV preS1 antigen; V H 3 and V L 3 are the heavy chain variable region and light chain variable region of the antigen binding domain that specifically binds to CD3, V H 3-L3-V L 3 or V L 3-L3-V H 3 constitutes an antigen-binding domain (scFv) capable of specifically binding to CD3 antigen.

[0185] In some preferred embodiments of the present application, the multispecific antibody (preferably, a trispecific antibody) has a tri-IgG-(scFv)2 structure, wherein the first antigen-binding domain in the structure specifically binds to the PD-L1 antigen, the second antigen-binding domain and the fourth antigen-binding domain specifically bind to the HBV preS1 antigen, and the third antigen-binding domain specifically binds to the CD3 antigen. In some embodiments, the multispecific antibody (preferably, a trispecific antibody) can simultaneously bind to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen.

[0186] In some embodiments, the first antigen binding domain scFv that specifically binds to the PD-L1 antigen and the third antigen binding domain scFv that specifically binds to the CD3 antigen include genetically engineered cysteine ​​mutations. H and V L Two cysteine ​​mutations were introduced into the interface to obtain a disulfide-bond-stabilized multispecific antibody.

[0187] In some embodiments, the multispecific antibody (preferably, a trispecific antibody) as described herein comprises: the amino acid sequence of SEQ ID NO: 64 or a variant thereof, wherein the variant has about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO: 64.

[0188] In some embodiments, the multispecific antibody (preferably, a trispecific antibody) as described herein comprises: the amino acid sequence of SEQ ID NO: 65 or a variant thereof, wherein the variant has about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO: 65.

[0189] In some embodiments, the multispecific antibody (preferably, a trispecific antibody) as described herein comprises: the amino acid sequence of SEQ ID NO: 66 or a variant thereof, wherein the variant has about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO: 66.

[0190] In some embodiments, the multispecific antibody (preferably, a trispecific antibody) as described herein comprises: the amino acid sequence of SEQ ID NO: 64 or a variant thereof, wherein the variant has about 80% sequence identity with the amino acid sequence of SEQ ID NO: 64; and / or the amino acid sequence of SEQ ID NO: 65 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 65; and / or the amino acid sequence of SEQ ID NO: 66 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 66.

[0191] Multispecific antibody with Tri-IgG-scFv structure

[0192] In some embodiments, a multispecific antibody structure (referred to in this application as Tri-IgG-scFv, preferably, a trispecific antibody) obtained by modification based on the bispecific Hetero H, CrossMab structure (see, for example, Klein C, et al. The use of CrossMAb technology for the generation of bi-and multispecific antibodies. MAbs. 2016 Aug-Sep; 8(6): 1010-20) is a structure based on the Hetero H, CrossMab bispecific antibody, in which a scFv fragment that binds to another antigen is connected to the Fc terminus of one of the heavy chains to achieve multispecificity (preferably, trispecificity). Hetero H, CrossMab technology is based on the exchange of antibody domains within one Fab arm of a bispecific IgG antibody, which can be the exchange of complete Fab domains (CrossMab Fab ), or exchange of only the variable regions in Fab (CrossMab VH-VL ) or exchange of only the constant regions (CrossMab CH1-CL In some embodiments of the present application, preferably, the multispecific antibody (preferably, a trispecific antibody) has an exchange of constant regions (CrossMab CH1-CL In a preferred embodiment of the present application, a schematic diagram of the typical structure of the trispecific antibody is shown in FIG3C .

[0193] In some embodiments, the multispecific antibodies (preferably, trispecific antibodies) described herein have a tri-IgG-scFv structure, which is a trivalent multispecific antibody composed of two monomers, one of which is composed of two polypeptide chains and contains two antigen-binding domains, one of which is a Fab and the other is a scFv; the other monomer is composed of two polypeptide chains and contains one antigen-binding domain, which is a Fab. In some embodiments, the multispecific antibody (preferably, a trispecific antibody) contains two Fcs, the Fc containing C H 2 and C H The scFv is linked to the carboxyl terminus of one of the Fc domains via a linker peptide (L).

[0194] In some embodiments, the tri-IgG-scFv structure as described above is a heterodimer composed of two different monomers, including a total of 4 polypeptide chains, forming 3 antigen binding domains. Among them, the first monomer is composed of a first heavy chain and a first light chain, comprising two antigen binding domains (Fab and scFv, in a preferred embodiment of the present application, referred to as the second antigen binding domain and the third antigen binding domain, respectively); the second monomer is composed of a second heavy chain and a second light chain, comprising an antigen binding domain (Fab, in a preferred embodiment of the present application, referred to as the first antigen binding domain). In a preferred embodiment of the present application, the multispecific antibody (preferably, a trispecific antibody) has a tri-IgG-scFv structure, wherein the first antigen binding domain specifically binds to a first antigen (e.g., PD-L1), the second antigen binding domain specifically binds to a second antigen (e.g., HBV preS1), and the third antigen binding domain specifically binds to a third antigen (e.g., CD3).

[0195] In some embodiments, the first heavy chain of the multispecific antibody (preferably, a trispecific antibody) comprises from N-terminus to C-terminus: V H 2-C H 1-C H 2-C H 3-LV H 3-L3-V L 3 structure. In other embodiments, the first heavy chain of the multispecific antibody (preferably, a trispecific antibody) comprises from N-terminus to C-terminus: V H 2-C H 1-C H 2-C H 3-LV L 3-L3-V H 3 Structure. In some embodiments, the first light chain of a multispecific antibody (preferably, a trispecific antibody) comprises, from N-terminus to C-terminus: V L 2-C L structure.

[0196] Among them, V H 2 and V L 2 are the heavy chain variable region and light chain variable region of the second antigen binding domain, V H 3 and V L 3 are the heavy chain variable region and light chain variable region of the third antigen binding domain, C H 1 is the heavy chain constant region C H 1 domain, C L V is the light chain constant region, L and L3 are connecting peptides. H 2-C H 1 and V L 2-C LThe second antigen-binding domain (Fab) that binds to the second antigen constitutes a multispecific antibody (preferably, a trispecific antibody), V H 3-L3-V L 3 or V L 3-L3-V H 3. A third antigen-binding domain (scFv) that binds to a third antigen and constitutes a multispecific antibody (preferably, a trispecific antibody).

[0197] In some embodiments, the second heavy chain of the multispecific antibody (preferably, a trispecific antibody) comprises, from N-terminus to C-terminus: V H 1-C L In some embodiments, the second light chain of the multispecific antibody (preferably, a trispecific antibody) comprises from N-terminus to C-terminus: V L 1-C H 1 structure.

[0198] In some embodiments, the second heavy chain further comprises an Fc comprising a C H 2 and C H 3 domains.

[0199] In some embodiments, the second heavy chain of the multispecific antibody (preferably, a trispecific antibody) comprises from N-terminus to C-terminus: V H 1-C L -C H 2-C H 3 structure; In some embodiments, the second light chain of the multispecific antibody (preferably, a trispecific antibody) comprises from N-terminus to C-terminus: V L 1-C H 1 structure.

[0200] Among them, V H 1 and V L 1 are the heavy chain variable region and light chain variable region of the first antigen binding domain, C H 1 is the heavy chain constant region C H 1 domain, C L Is the light chain constant region. H 1-C L and V L 1-C H 1 constitutes the first antigen binding domain (Fab) that binds the first antigen.

[0201] In some embodiments, C in the first monomer or the second monomer of the multispecific antibody (preferably, a trispecific antibody) L and C H 1 can be interchanged. In some embodiments, V H 1 and V L1 can be replaced with each other. In other embodiments, V H 2 and V L The positions of 2 can be interchanged.

[0202] In some embodiments, the C of the first heavy chain H The amino acid residues in the 3 domain are replaced by amino acid residues with larger side chain volume to form a "knob", and the C of the second heavy chain H In some other embodiments, the amino acid residues in the C domain of the second heavy chain are replaced with amino acid residues with smaller side chain volume to form a "hole". H The amino acid residues in the 3 domain are replaced by amino acid residues with larger side chain volume to form a "knob", and the C of the first heavy chain H The amino acid residues in the C3 domain are replaced with amino acid residues with smaller side chain volume to form a "hole". In some embodiments, the C H The 3 domain includes, but is not limited to, the following amino acid substitutions: S354C, T366W, Y349C, T366S, L368A, and Y407V, wherein the numbering is according to the EU index as in Kabat.

[0203] In some preferred embodiments of the present application, V H 1 and V L 1 are the heavy chain variable region and light chain variable region of the antigen binding domain that specifically bind to PD-L1, V H 2 and V L 2 are the heavy chain variable region and light chain variable region of the antigen binding domain that specifically bind to HBV preS1, V H 3 and V L 3 are the heavy chain variable region and light chain variable region of the antigen binding domain that specifically binds to CD3. H 1-C L and V L 1-C H 1 constitutes an antigen-binding domain (Fab) that can specifically bind to the PD-L1 antigen; V H 2-C H 1 and V L 2-C L Composition of antigen binding domain (Fab) that can specifically bind to HBV preS1 antigen; V H 3-L3-V L 3 or V L 3-L3-V H 3 constitutes an antigen-binding domain (scFv) capable of specifically binding to CD3 antigen.

[0204] In some preferred embodiments of the present application, the multispecific antibodies (preferably, trispecific antibodies) described herein have a tri-IgG-scFv structure, wherein the first antigen-binding domain in the structure specifically binds to the PD-L1 antigen, the second antigen-binding domain specifically binds to the HBV preS1 antigen, and the third antigen-binding domain specifically binds to the CD3 antigen. In some embodiments, the multispecific antibodies can simultaneously bind to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen.

[0205] In some embodiments, the third antigen binding domain scFv that specifically binds to the CD3 antigen includes genetically engineered cysteine ​​mutations by H and V L Two cysteine ​​mutations were introduced into the interface to obtain a disulfide-bond-stabilized multispecific antibody.

[0206] In some embodiments, the multispecific antibody (preferably, a trispecific antibody) as described herein comprises: the amino acid sequence of SEQ ID NO: 77 or a variant thereof, wherein the variant has about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO: 77.

[0207] In some embodiments, the multispecific antibody (preferably, a trispecific antibody) as described herein comprises: the amino acid sequence of SEQ ID NO: 66 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO: 66.

[0208] In some embodiments, the multispecific antibody (preferably, a trispecific antibody) as described herein comprises: the amino acid sequence of SEQ ID NO: 80 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO: 80.

[0209] In some embodiments, the multispecific antibody (preferably, a trispecific antibody) as described herein comprises: the amino acid sequence of SEQ ID NO: 79 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO: 79.

[0210] In some embodiments, the multispecific antibody (preferably, a trispecific antibody) as described herein comprises: the amino acid sequence of SEQ ID NO: 78 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence of SEQ ID NO: 78.

[0211] In some embodiments, the multispecific antibody (preferably, a trispecific antibody) as described herein comprises: an amino acid sequence of SEQ ID NO: 77 or a variant thereof, wherein the variant has about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 77; and / or an amino acid sequence of SEQ ID NO: 66 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 66; and / or an amino acid sequence of SEQ ID NO: 80 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 80; and / or an amino acid sequence of SEQ ID NO: 79 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 79. ID NO: 79 has at least about 80% (eg, at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0212] In some embodiments, the multispecific antibody (preferably, a trispecific antibody) as described herein comprises: an amino acid sequence of SEQ ID NO: 77 or a variant thereof, wherein the variant has about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 77; and / or an amino acid sequence of SEQ ID NO: 66 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 66; and / or an amino acid sequence of SEQ ID NO: 78 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 78; and / or an amino acid sequence of SEQ ID NO: 79 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 79; ID NO: 79 has at least about 80% (eg, at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0213] Multispecific antibodies with Tri-DVD-Ig structure

[0214] In some embodiments, a multispecific antibody structure (referred to herein as tri-VD-Ig, preferably a trispecific antibody) obtained by modifying a bispecific dual variable region immunoglobulin molecule (DVD-Ig) structure (see, for example, patent document US 7,612,181, B2) is a normal bispecific anti-IgG antibody V L and V H The N-termini of the V L and V H In a preferred embodiment of the present application, the schematic diagram of the typical structure of the trispecific antibody is shown in Figure 3D.

[0215] In some embodiments, according to any multispecific antibody described herein (preferably, a trispecific antibody), it has a tri-DVD-IgG structure, which is a tetravalent multispecific antibody composed of two monomers, wherein each monomer is composed of two polypeptide chains, including two antigen-binding domains, one of which is an Fv and the other is a Fab, and the two regions are connected in series by a connecting peptide (L). In some embodiments, wherein the multispecific antibody (preferably, a trispecific antibody) further comprises two Fc, which comprises C H 2 and C H3 domains.

[0216] In some embodiments, the tri-DVD-Ig structure as described above is a heterodimer composed of two different monomers, including a total of 4 polypeptide chains, forming 4 antigen binding domains. Among them, the first monomer consists of a first heavy chain and a first light chain, comprising two antigen binding domains (Fab and Fv, in a preferred embodiment of the present application, referred to as the first antigen binding domain and the second antigen binding domain, respectively); the second monomer consists of a second heavy chain and a second light chain, comprising two antigen binding domains (Fab and Fv, in a preferred embodiment of the present application, referred to as the third antigen binding domain and the fourth antigen binding domain, respectively).

[0217] In a preferred embodiment of the present application, the multispecific antibody (preferably, a trispecific antibody) has a tri-DVD-Ig structure, wherein the first antigen binding domain specifically binds to a first antigen (e.g., PD-L1), the second antigen binding domain and the fourth antigen binding domain have the same sequence and specifically bind to a second antigen (e.g., HBV preS1), and the third antigen binding domain specifically binds to a third antigen (e.g., CD3).

[0218] In some embodiments, the first heavy chain of the multispecific antibody (preferably, a trispecific antibody) comprises from N-terminus to C-terminus: V H 2-LV H 1-C H 1 structure. In some embodiments, the first light chain of the multispecific antibody comprises, from N-terminus to C-terminus: V L 2-LV L 1-C L structure.

[0219] In some embodiments, wherein the first heavy chain further comprises an Fc comprising a C H 2 and C H 3 domains.

[0220] In some embodiments, the first heavy chain of the multispecific antibody comprises, from N-terminus to C-terminus: V H 2-LV H 1-C H 1-C H 2-C H 3 structure. In some embodiments, the first light chain of the multispecific antibody comprises, from N-terminus to C-terminus: V L 2-LV L 1-C L structure.

[0221] Where V H 1 and V L1 is the heavy chain variable region and light chain variable region of the first antigen binding domain; V H 2 and V L 2 are the heavy chain variable region and light chain variable region of the second antigen binding domain respectively; L is a connecting peptide; C H 1 is the heavy chain constant region C H 1 domain; C L Is the light chain constant region. H 2 and V L 2. The second antigen-binding domain (Fv) that binds to the second antigen that constitutes the multispecific antibody; V H 1-C H 1 and V L 1-C L The multispecific antibody comprises a first antigen-binding domain (Fab) that binds to a first antigen.

[0222] In some embodiments, the second heavy chain of the multispecific antibody (preferably, a trispecific antibody) comprises from N-terminus to C-terminus: V H 2-LV H 3-C H 1 structure. In some embodiments, the second light chain of the multispecific antibody (preferably, a trispecific antibody) comprises from N-terminus to C-terminus: V L 2-LV L 3-C L structure.

[0223] In some embodiments, wherein the second heavy chain further comprises an Fc comprising a C H 2 and C H 3 domains.

[0224] In some embodiments, the second heavy chain of the multispecific antibody (preferably, a trispecific antibody) comprises from N-terminus to C-terminus: V H 2-LV H 3-C H 1-C H 2-C H 3 structure. In some embodiments, the second light chain of the multispecific antibody (preferably, a trispecific antibody) comprises from N-terminus to C-terminus: V L 2-LV L 3-C L structure.

[0225] Where V H 2 and V L 2 are the heavy chain variable region and light chain variable region of the fourth antigen binding domain; V H 3 and V L 3 are the heavy chain variable region and light chain variable region of the third antigen binding domain respectively; L is the connecting peptide; CH 1 is the heavy chain constant region C H 1 domain; C L Is the light chain constant region. H 2 and V L 2. A fourth antigen-binding domain (Fv) that binds to a second antigen and constitutes a multispecific antibody (preferably, a trispecific antibody); V H 3-C H 1 and V L 3-C L The third antigen-binding domain (Fab) that constitutes a multispecific antibody (preferably, a trispecific antibody) binds to a third antigen.

[0226] In some embodiments, the C of the first heavy chain H The amino acid residues in the 3 domain are replaced by amino acid residues with larger side chain volume to form a "knob", and the C of the second heavy chain H In some other embodiments, the amino acid residues in the C domain of the second heavy chain are replaced with amino acid residues with smaller side chain volume to form a "hole". H The amino acid residues in the 3 domain are replaced by amino acid residues with larger side chain volume to form a "knob", and the C of the first heavy chain H The amino acid residues in the C3 domain are replaced with amino acid residues with smaller side chain volume to form a "hole". In some embodiments, the C H The 3 domain includes, but is not limited to, the following amino acid substitutions: S354C, T366W, Y349C, T366S, L368A, and Y407V, wherein the numbering is according to the EU index as in Kabat.

[0227] In some preferred embodiments of the present application, the V H 1 and V L 1 are the heavy chain variable region and light chain variable region of the antigen binding domain that specifically bind to PD-L1, V H 2 and V L 2 are the heavy chain variable region and light chain variable region of the antigen binding domain that specifically bind to HBV preS1, V H 3 and V L 3 are the heavy chain variable region and light chain variable region of the antigen binding domain that specifically binds to CD3. H 1-C H 1 and V L 1-C L Composition of the antigen binding domain (Fab) that specifically binds to the PD-L1 antigen; V H 2 and V L 2 constitutes an antigen binding domain (Fv) that specifically binds to HBV preS1 antigen; V H 3-CH 1 and V L 3-C L It consists of an antigen-binding domain (Fab) that specifically binds to the CD3 antigen.

[0228] In some preferred embodiments of the present application, the multispecific antibody (preferably, a trispecific antibody) described herein has a tri-DVD-Ig structure, wherein the first antigen-binding domain in the structure specifically binds to the PD-L1 antigen, the second antigen-binding domain and the fourth antigen-binding domain specifically bind to the HBV preS1 antigen, and the third antigen-binding domain specifically binds to the CD3 antigen. In some embodiments, the multispecific antibody (preferably, a trispecific antibody) can simultaneously bind to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen.

[0229] Connector peptide

[0230] Connecting peptides (or, may be referred to as "linkers") can be used to connect the domains and / or regions of the chimeric heavy chains of a multispecific antibody (preferably, a trispecific antibody) into a continuous molecule. In some embodiments, the multispecific antibody (preferably, a trispecific antibody) may include additional linkers, such as a flexible linker connecting the variable heavy chain and light chain of an scFv. In some embodiments, the multispecific antibody (preferably, a trispecific antibody) may include additional linkers, such as a flexible linker connecting the variable heavy chain and light chain of an scFv and other linkers for connecting other binding units to the core structure of the multispecific antibody (preferably, a trispecific antibody).

[0231] In one embodiment, the present invention relates to a polypeptide chain comprising at least 4 residues. The position of a typical, non-limiting example of a joint is a polypeptide chain comprising at least 4 residues. The position of this type of joint may be flexible, hydrophilic, and they themselves seldom or will not form a secondary structure (joint site or flexible joint site). After the molecular assembly is complete, at least 4 amino acid whose joints can be used to connect domains and / or districts close to each other. Longer joints can also be used. In certain embodiments, the joint can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 100, 125, 150, 175 or 200 residues. When using multiple joints to connect the various parts of a molecule, the joint can be identical or different (for example, identical or different length and / or amino acid sequence).

[0232] In some aspects, the connecting peptide (or linker) comprises or is composed of a glycine-serine linker. As described in the present application, the term "glycine-serine linker" refers to a peptide consisting of glycine and serine residues. Exemplary glycine-serine linkers include amino acid sequences of the general formula (Gly4Ser)n, where n is a positive integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10). A preferred glycine-serine linker is (Gly4Ser)2, i.e., GGGGSGGGGS (SEQ ID NO: 91) and (Gly4Ser)4, i.e., GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 92). Another preferred glycine-serine linker is (Gly4Ser)3, i.e., GGGGSGGGGSGGGGS (SEQ ID NO: 93). In other aspects, two or more glycine-serine linkers are connected in series in a connecting peptide. In certain aspects, the connecting peptide comprises at least a portion of the hinge region (e.g., from an IgG1, IgG2, IgG3, or IgG4 molecule) and a series of glycine-serine residues (e.g., a glycine-serine linker, such as (Gly4Ser) n ).

[0233] In some embodiments, the connecting peptide comprises a glycine-serine or all-glycine linker and a portion or a modified portion of the hinge region. In some embodiments, the connecting peptide comprises a glycine-serine or all-glycine linker and a portion or a modified portion of the hinge region. H The connecting peptide of 3 (eg, L) comprises the amino acid sequence GGGGSGGGGTGGGGS (SEQ ID NO: 103).

[0234] In some embodiments, the multispecific antibody (preferably, trispecific antibody) may optionally include additional connecting peptides in addition to the connecting peptide (e.g., L) connecting the antigen binding domain to the Fc. The length and sequence of these additional connecting peptides are independently selected. For example, the multispecific antibody (preferably, trispecific antibody) may further include a flexible connecting peptide (L3) connecting the variable heavy chain and light chain (V HSCFV and V LSCFV ). This flexible connecting peptide can include a glycine-serine linker. Typically, this linker does not include a hinge portion. In some embodiments, the flexible connecting peptide (L3) connecting the variable heavy chain and light chain of the scFv comprises the sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 92).

[0235] The connecting peptide (or linker) sequence can be a single amino acid or polypeptide sequence. In some embodiments, the connecting peptide comprises the amino acid sequence ASTKGP (SEQ ID NO: 94) or the amino acid sequence TVAAP (SEQ ID NO: 95).

[0236] Exemplary antibody sequences are shown in Tables 2 to 10, where CDR numbering is based on the EU index in Kabat or IMGT. Those skilled in the art will recognize that there are a variety of known algorithms for predicting the positions of CDRs and defining the variable regions of antibody light and heavy chains. The CDRs, V domains, and CDRs of antibodies or antigen-binding domains that specifically bind to HBV preS1 antigen, antibodies or antigen-binding domains that specifically bind to PD-L1 antigen, antibodies or antigen-binding domains that specifically bind to CD3 antigen, and multispecific antibodies described herein are described. H and / or V L Sequences, but antibodies or antigen-binding domains based on prediction algorithms other than those exemplified in the table below are also within the scope of this application. The antibody or antigen-binding domain that specifically binds to the CD3 antigen can be selected from the anti-CD3 antigen antibodies or antigen-binding domains described in US20030180799A1, CN106029696B, and CN107074956B, which are incorporated into this application by reference.

[0237] Table 2: CDR sequences of exemplary antibodies or antigen-binding domains that specifically bind to HBV preS1 antigen

[0238] Table 3: V variants of exemplary antibodies or antigen-binding domains that specifically bind to HBV preS1 antigen H &V L sequence

[0239] Table 4: CDR sequences of exemplary antibodies or antigen binding domains that specifically bind to the CD3 antigen

[0240] Table 5-1: V values ​​of exemplary antibodies or antigen-binding domains that specifically bind to the CD3 antigen H &V L sequence

[0241] Table 5-2: V values ​​of exemplary antibodies or antigen-binding domains that specifically bind to the CD3 antigen H &V L Cysteine ​​variant sequences

[0242] Table 6: CDR sequences of exemplary antibodies or antigen-binding domains that specifically bind to the PDL1 antigen

[0243] Table 7-1: V values ​​of exemplary antibodies or antigen-binding domains that specifically bind to the PD-L1 antigen H &V L sequence

[0244] Table 7-2: V values ​​of exemplary antibodies or antigen-binding domains that specifically bind to the PD-L1 antigen H &V L Cysteine ​​variant sequences

[0245] Table 8: Exemplary antibody constant region sequences

[0246] Table 9-1: Partial heavy chain and light chain sequences of exemplary multispecific antibodies that specifically bind to HBV preS1 antigen, CD3 antigen, and PDL1 antigen.

[0247] Table 9-2: Partial heavy chain and light chain sequences of exemplary multispecific antibodies with tri-IgG-scFv structure that specifically bind to HBV preS1 antigen, CD3 antigen, and PDL1 antigen

[0248] Table 10-1: Full-length heavy and light chain sequences of exemplary multispecific antibodies that specifically bind to HBV preS1 antigen, CD3 antigen, and PDL1 antigen.

[0249] Table 10-2: Full-length heavy and light chain sequences of exemplary multispecific antibodies with tri-IgG-(scFv)2 structures that specifically bind to HBV preS1 antigen, CD3 antigen, and PDL1 antigen

[0250] Table 10-3: Full-length heavy and light chain sequences of exemplary multispecific antibodies with tri-IgG-scFv structures that specifically bind to HBV preS1 antigen, CD3 antigen, and PDL1 antigen

[0251] Table 10-4: Full-length heavy and light chain sequences of exemplary multispecific antibodies that specifically bind to HBV preS1 and CD3 antigens (CrossMab2+1)

[0252] Table 11: Exemplary connecting peptide (or linker) sequences

[0253] Binding affinity

[0254] Binding affinity can be expressed as Kd, Koff, Kon or Ka. As used in this application, the term "Koff" refers to the rate constant at which the antigen binding domain dissociates from the antigen binding domain / antigen complex, as determined by a kinetic selection device. The term "Kon" refers to the association rate constant at which the antibody binds to the antigen to form the antigen binding domain / antigen complex. The dissociation constant "Kd" used in this application refers to the dissociation constant during a specific antibody-antigen interaction, and refers to the antigen concentration required when the antigen occupies half of all antibody binding domains and reaches equilibrium in a solution of antibody molecules, which is equal to Koff / Kon. The determination of Kd assumes that all binding molecules are in solution. In cases where the antigen binding domain is attached to the cell wall, for example in a yeast expression system, the corresponding dissociation rate constant is expressed as EC50, which is a good approximation of Kd. The affinity binding constant Ka is the reciprocal of the dissociation constant Kd.

[0255] The equilibrium dissociation constant (Kd) can be used as an indicator of the affinity of the antigen-binding domain for the antigen. For example, simple analysis can be performed using antibodies labeled with various markers using the Scatchard method and a Biacore instrument (manufactured by Amersham Biosciences) to analyze the interaction between biomolecules by surface plasmon resonance according to the user manual or the accompanying kit. The Kd values ​​obtained using these methods are expressed in units of M. An antibody that specifically binds to a target may have, for example, a Kd of ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, ≤10 -12 M or ≤10 -13 Kd value of M.

[0256] The binding specificity of an antibody can be determined experimentally by methods known in the art, including, but not limited to, Western blots, ELISA, RIA, ECL, IRMA, EIA, BIAcore assays, and peptide scanning.

[0257] In some embodiments, the antibody or antigen binding domain that specifically binds to HBV preS1, PDL1 or CD3 antigen specifically binds to HBV preS1, PDL1 or CD3 antigen target with a Kd value of 10 -7 M to 10-13 M (e.g. 10 - 7 M to 10 -13 M, 10 -8 M to 10 -13 M, 10 -9 M to 10 -13 M or 10 -10 M to 10 -12 M). Therefore, in some embodiments, the Kd value of the binding between the antibody or antigen binding domain that specifically binds to HBV preS1, PDL1 or CD3 antigen and HBV preS1, PDL1 or CD3 antigen is 10 -7 M to 10 -13 M, 1×10 -7 M to 5×10 -13 M, 10 -7 M to 10 -12 M, 10 -7 M to 10 -11 M, 10 -7 M to 10 -10 M, 10 -7 M to 10 -9 M, 10 -8 M to 10 -13 M, 1×10 -8 M to 5×10 -13 M, 10 -8 M to 10 -12 M, 10 -8 M to 10 -11 M, 10 -8 M to 10 -10 M, 10 -8 M to 10 -9 M, 5×10 -9 M to 1×10 -13 M, 5×10 -9 M to 1×10 -12 M, 5×10 -9 M to 1×10 -11 M, 5×10 -9 M to 1×10 -10 M, 10 -9 M to 10 -13 M, 10 -9 M to 10 -12 M, 10 -9 M to 10 -11 M, 10 -9 M to 10 -10 M, 5×10 -10 M to 1×10 -13 M, 5×10 -10M to 1×10 -12 M, 5×10 -10 M to 1×10 -11 M, 10 -10 M to 10 -13 M, 1×10 -10 M to 5×10 -13 M, 1×10 -10 M to 1×10 -12 M, 1×10 -10 M to 5×10 -12 M, 1×10 -10 M to 1×10 -11 M, 10 -11 M to 10 -13 M, 1×10 -11 M to 5×10 -13 M, 10 -11 M to 10 -12 M, 10 -12 M to 10 -13 In some embodiments, the Kd value of the binding between the antibody or antigen binding domain that specifically binds to HBV preS1, PDL1 or CD3 antigen and the HBV preS1, PDL1 or CD3 antigen is 10 -7 M to 10 -13 M.

[0258] In some embodiments, the Kd value of binding between an antibody or antigen-binding domain that specifically binds to HBV preS1, PDL1, or CD3 antigen and a non-target is higher than the Kd value of binding between the antibody or antigen-binding domain that specifically binds to HBV preS1, PDL1, or CD3 antigen and the target, and in some embodiments cited herein, the binding affinity of an antibody or antigen-binding domain that specifically binds to HBV preS1, PDL1, or CD3 antigen and the target (e.g., HBV preS1, PDL1, or CD3 antigen) is higher than the binding affinity of an antibody or antigen-binding domain that specifically binds to HBV preS1, PDL1, or CD3 antigen and the non-target. In some embodiments, the non-target refers to an antigen other than HBV preS1, PDL1, or CD3 antigen. In some embodiments, the Kd value of the antibody or antigen binding domain that specifically binds to the HBV preS1, PDL1 or CD3 antigen and the non-HBV preS1, PDL1 or CD3 antigen target is at least 10 times the Kd of the antibody or antigen binding domain that specifically binds to the HBV preS1, PDL1 or CD3 antigen and the target HBV preS1, PDL1 or CD3 antigen, for example, 10-10 2 times, 10 2 -10 3 times, 10 3 -104 times, 10 4 -10 5 times, 10 5 -10 6 times, 10 6 -10 7 times, 10 7 -10 8 times, 10 8 -10 9 times, 10 9 -10 10 times, 10 10 -10 11 times, 10 11 -10 12 times.

[0259] Preparation of nucleic acids, vectors, and antibodies

[0260] Nucleic acids and vectors

[0261] Nucleic acid molecules encoding antibodies or antigen-binding domains that specifically bind to HBV preS1 antigen, antibodies or antigen-binding domains that specifically bind to CD3 antigen, antibodies or antigen-binding domains that specifically bind to PD-L1 antigen, and multispecific antibodies are also contemplated.

[0262] In some embodiments, the present application provides a nucleic acid (or a set of nucleic acids) encoding an antibody or antigen-binding domain that specifically binds to the HBV preS1 antigen, or a nucleic acid encoding an antibody or antigen-binding domain that specifically binds to the CD3 antigen, or an antibody or antigen-binding domain that specifically binds to the PD-L1 antigen, or a nucleic acid encoding a multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen, including any of the antibodies or antigen-binding domains that specifically bind to the HBV preS1 antigen, or antibodies or antigen-binding domains that specifically bind to the CD3 antigen, or antibodies or antigen-binding domains that specifically bind to the PD-L1 antigen, or a multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen described herein. In some embodiments, the nucleic acid (or set of nucleic acids) encoding the antibody, antigen-binding domain, or multispecific antibody described herein may further include a nucleic acid sequence encoding a polypeptide tag (e.g., a protein purification tag, a His-tag, an HA tag).

[0263] At the same time, the present application also provides an antibody or antigen-binding domain that specifically binds to HBV preS1 antigen, an antibody or antigen-binding domain that specifically binds to CD3 antigen, an antibody or antigen-binding domain that specifically binds to PD-L1 antigen, or a multispecific antibody that specifically binds to HBV preS1 antigen, PD-L1 antigen and CD3 antigen; or a nucleic acid molecule encoding the antibody or antigen-binding domain; or an isolated host cell containing a vector carrying the nucleic acid molecule.

[0264] The present application also includes variants of these nucleic acid sequences. For example, variants include nucleotide sequences that hybridize to a nucleic acid sequence encoding an antibody, antigen-binding fragment, or multispecific antibody of the present application under at least moderately stringent hybridization conditions.

[0265] The present application also provides a vector into which the nucleic acid sequence of the present application can be inserted.

[0266] In brief, a natural or synthetic nucleic acid encoding an antibody, antigen-binding fragment, or multispecific antibody is inserted into a suitable expression vector such that the nucleic acid is operably linked to 5' and 3' regulatory elements, such as a promoter (e.g., a lymphocyte-specific promoter) and a 3' untranslated region (UTR), to express the antibody, antigen-binding fragment, or multispecific antibody. The vector is suitable for replication and integration in eukaryotic host cells. Typical cloning and expression vectors contain transcriptional and translational terminators, initiation sequences, and promoters that regulate the expression of the target nucleic acid sequence.

[0267] The nucleic acids described herein can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Nucleic acid delivery methods are known in the art. For example, see US Pat. Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. In some embodiments, the present application also provides gene therapy vectors.

[0268] Nucleic acids can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0269] In addition, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described in, for example, Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology or molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and slow viruses. Typically, suitable vectors include a replication origin that works in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (see, for example, WO 01 / 96584; WO 01 / 29058; and US Pat. No. 6,326,193).

[0270] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Techniques known in the art can be applied to insert the selected gene into a vector and package it in retroviral particles. The recombinant virus is then isolated and delivered to the cells of the subject in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for long-term stable integration of transgenes and their propagation in daughter cells. Lentiviral vectors have additional advantages over tumor-derived retroviruses, such as mouse leukemia viruses, because they can transduce non-dividing cells, such as hepatocytes. At the same time, they also have the additional advantage of low immunogenicity.

[0271] Other promoter elements, such as enhancers, regulate the frequency of transcription initiation. These are typically located 30-110 bp upstream of the start site, although many promoters have recently been found to contain functional elements downstream of the start site. The spacing between promoter elements is often flexible, so that promoter function is maintained even when elements are swapped or moved relative to one another. In the thymidine kinase (Tk) promoter, activity begins to decline only when the spacing between promoter elements increases to 50 bp.

[0272] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a very strong constitutive promoter sequence that can drive high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is the elongation factor 1α (EF-1α) promoter. However, other constitutive promoters may also be used, including but not limited to, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus long terminal repeat (HIV-LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, including but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In addition, this application should not be limited to the use of only constitutive promoters. Inducible promoters are also considered in this application. The use of an inducible promoter provides a molecular switch that can activate expression of an operably linked polynucleotide sequence when such expression is desired, and deactivate expression when such expression is not desired. Inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0273] In some embodiments, expression of the antibody, antigen-binding fragment, or multispecific antibody is inducible. In some embodiments, the nucleic acid sequence encoding the antibody, antigen-binding fragment, or multispecific antibody is operably linked to an inducible promoter, including any of the inducible promoters described herein.

[0274] Inducible promoter

[0275] The use of an inducible promoter provides a molecular switch that turns on the expression of a polynucleotide sequence operably linked thereto when expression is desired, and turns off expression when expression is not desired. Exemplary inducible promoters suitable for use in eukaryotic cells include, but are not limited to, hormone-regulated elements (e.g., see Mader, S. and White, JH (1993) Proc. Natl. Acad. Sci. USA 90:5603-5607), synthetic ligand-regulated elements (see Spencer, DM et al (1993) Science 262:1019-1024), and ionizing radiation-regulated elements (see Manome, Y. et al (1993) Biochemistry 32:10607-10613; Datta, R. et al (1992) Proc. Natl. Acad. Sci. USA 89:1014-10153). Other exemplary inducible promoters suitable for use in in vivo or in vitro mammalian systems are described in Gingrich et al. (1998) Annual Rev. Neurosci 21:377-405. In some embodiments, the inducible promoter system used to express antibodies, antigen-binding fragments, or multispecific antibodies is the Tet system. In some embodiments, the inducible promoter system used to express antibodies, antigen-binding fragments, or multispecific antibodies is the E. coli lac repression system.

[0276] An exemplary inducible promoter system used in this application is the Tet system. This system is based on the Tet system described by Gossen et al. (1993). In an exemplary embodiment, the target polynucleotide is controlled by a promoter comprising one or more Tet operator (TetO) sites. In the inactive state, the Tet repressor (TetR) binds to the TetO site and inhibits transcription of the promoter. In the active state, for example, in the presence of an inducer such as tetracycline (Tc), anhydrotetracycline, doxycycline (Dox) or its active analogs, the inducer releases TetR from TetO, thereby causing transcription to occur. Doxycycline is a member of the tetracycline antibiotic family, and its chemical name is 1-dimethylamino-2,4a,5,7-pentahydroxy-11-methyl-4,6-dioxy-1,4a,11,11a,12,12a-hexahydrotetraene-3-carboxamide.

[0277] In one embodiment, TetR is codon-optimized for expression in mammalian cells, such as mouse or human cells. Due to the degeneracy of the genetic code, most amino acids are encoded by more than one codon, so that the sequence of a given nucleic acid has a large number of variants without any change in the amino acid sequence it encodes. However, many organisms differ in codon usage, also known as "codon preference" (i.e., the preference for using a specific codon for a given amino acid). Codon preference is generally associated with the presence of a dominant tRNA species for a particular codon, which in turn improves the efficiency of mRNA translation. Therefore, coding sequences derived from specific species (e.g., prokaryotes) can be customized by codon optimization to improve their expression in different species (e.g., eukaryotes).

[0278] Other specific variants of the Tet system include the following "Tet-Off" and "Tet-On" systems. In the Tet-off system, transcription is inactivated in the presence of Tc or Dox. In this system, a tetracycline-regulated transcription activator protein (tTA), composed of a fusion of TetR and the strong transcriptional activation domain of herpes simplex virus VP16, regulates the expression of the target nucleic acid under the transcriptional control of a tetracycline-responsive promoter element (TRE). The TRE element consists of a TetO sequence fused in series with a promoter (usually a minimal promoter sequence derived from the immediate early promoter of human cytomegalovirus). In the absence of Tc or Dox, tTA binds to TRE and activates transcription of the target gene. In the presence of Tc or Dox, tTA cannot bind to TRE and the target gene cannot be expressed.

[0279] In contrast, in the Tet-On system, transcription is activated in the presence of either Tc or Dox. The Tet-On system is based on the reverse tetracycline-regulated transcriptional activator rtTA. Like tTA, rtTA is a fusion protein consisting of the TetR repressor and the VP16 transactivation domain. However, a four-amino acid change in the DNA-binding region of TetR alters rtTA's binding properties, allowing it to only recognize the tetO sequence within the target transgene's TRE in the presence of Dox. Therefore, in the Tet-On system, rtTA can activate transcription of its TRE-regulated target gene only in the presence of Dox.

[0280] Another inducible promoter system is the lac repressor system of Escherichia coli (see Brown et al., Cell 49: 603-612 (1987)). The lac repressor system functions by regulating the transcription of a target polynucleotide operably linked to a promoter comprising the lac operator (lacO). The lac repressor (lacR) binds to LacO, thereby preventing transcription of the target polynucleotide. Expression of the target polynucleotide is induced by a suitable inducing agent, for example, isopropyl-β-D-thiogalactopyranoside (IPTG).

[0281] In order to evaluate the expression of a polypeptide or portion thereof, the expression vector to be introduced into the cell may also contain a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from a cell population transfected or infected by a viral vector. In other aspects, the selectable marker can be carried on a separate DNA fragment and used in co-transfection experiments. Either the selectable marker gene or the reporter gene can be flanked by suitable regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo and similar genes.

[0282] Reporter genes can be used to identify potential transfected cells and evaluate the function of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by a recipient organism or tissue, and encodes a polypeptide whose expression is expressed as some easily detectable properties, such as enzymatic activity. After DNA is introduced into the recipient cell, the expression of the reporter gene is detected at the appropriate time. Suitable reporter genes can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein (eg, Ui-Tel et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and can be prepared by known techniques or obtained commercially. Generally, a construct with a minimum 5' flanking region that can show the highest expression level of a reporter gene is identified as a promoter. This type of promoter region can be connected to a reporter gene and used to assess the ability of certain substances to regulate promoter-driven transcription.

[0283] In some embodiments, nucleic acids encoding any one of the antibodies or antigen-binding fragments or multispecific antibodies described herein are provided. In some embodiments, the nucleic acid includes one or more nucleic acid sequences encoding the heavy and light chains of the antibodies or antigen-binding fragments or multispecific antibodies. In some embodiments, each of the one or more nucleic acid sequences is contained in a separate vector. In some embodiments, at least some nucleic acid sequences are contained in the same vector. In some embodiments, all nucleic acid sequences are contained in the same vector. The vector can be selected from, for example, mammalian expression vectors and viral vectors (such as vectors derived from retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses).

[0284] Methods for introducing genes into cells and expressing them are known in the art. In the context of expression vectors, the vectors can be readily introduced into host cells, such as mammalian cells, bacteria, yeast, or insect cells, by any method known in the art. For example, expression vectors can be introduced into host cells by physical, chemical, or biological methods.

[0285] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, biolistic methods, microinjection, electroporation, and the like. Methods for preparing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). In some embodiments, polynucleotides are introduced into host cells by calcium phosphate transfection.

[0286] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus type 1, adenoviruses, and adeno-associated viruses, among others. See, for example, US Pat. Nos. 5,350,674 and 5,585,362.

[0287] Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as polymer complexes, nanocapsules, microspheres, magnetic beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in vivo and in vitro is a liposome (e.g., an artificial membrane vesicle).

[0288] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. It is contemplated that a lipid formulation is used to import nucleic acid into a host cell (in vitro, in vitro or in vivo). On the other hand, the nucleic acid can be combined with lipids. The nucleic acid combined with lipids can be wrapped into the aqueous interior of the liposome, dispersed in the lipid bilayer of the liposome, connected to the liposome by a linker molecule combined with the liposome and the oligonucleotide, embedded in the liposome, form a complex with the liposome, be dispersed in a solution containing lipids, mix with lipids, combine with lipids, be suspended in lipids, be contained in micelles or mix with micelles, or otherwise combine with lipids. The compositions related to lipids, lipid / DNA or lipid / expression vectors are not limited to any particular structure in solution. For example, they may exist with a bilayer structure, with micelles or with a "collapsed" structure. They can also simply be dispersed in solution and may form aggregates of uneven size or shape. Lipids are fatty substances and can be naturally occurring or synthetic lipids. For example, lipids include fat droplets naturally present in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0289] Regardless of the method used to introduce the exogenous nucleic acid into the host cell or otherwise expose the cell to the inhibitor of the present application, a variety of experiments can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such experiments include, for example, "molecular biology" experiments well known to those skilled in the art. For example, Southern and Northern blotting, RT-PCR and PCR; "biochemical" experiments, such as detecting the presence or absence of a particular polypeptide, such as by immunological methods (ELISAs and Western blots) or by the experiments described in this application are all within the scope of this application. Preparation of antibodies or antigen-binding fragments and multispecific antibodies

[0290] In some embodiments, the antibody or antigen-binding fragment (e.g., an antibody or antigen-binding fragment that specifically binds to HBV preS1 antigen, CD3 antigen, or PD-L1 antigen) is a monoclonal antibody. In some embodiments, the antibody or antigen-binding fragment or multispecific antibody is derived from a monoclonal antibody. In some embodiments, the antibody, antigen-binding fragment, or multispecific antibody comprises a V from a monoclonal antibody. H and V L In some embodiments, the antibody, antigen-binding fragment or multispecific antibody further comprises a C from a monoclonal antibody. H 1 and C Lregion, or variants thereof. Monoclonal antibodies can be prepared using methods known in the art, such as hybridoma methods, yeast display, phage display methods, or recombinant DNA methods. In addition, exemplary yeast display and phage display methods are described in this application and in the following examples. Multispecific antibodies can be prepared using methods known in the art, such as chemical coupling, hybridoma methods, and genetic engineering methods.

[0291] In the hybridoma method, hamsters, mice or other suitable host animals are usually immunized with an immunizing agent to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro. The immunizing agent may include a polypeptide or fusion protein of the target protein. Typically, if human cells are needed, peripheral blood lymphocytes (PBLs) are used, and if non-human mammalian derived cells are needed, spleen cells or lymph node cells are used. Lymphocytes are fused with immortalized cell lines using an appropriate fusion agent, such as polyethylene glycol, to form hybridoma cells. Immortalized cell lines are typically transformed mammalian cells, especially myeloma cells of rodent, bovine and human origin. Rat or mouse myeloma cell lines are typically used. Hybridoma cells can be cultured in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), hybridoma cell culture medium typically includes hypoxanthine, aminopterin, and thymidine (HAT medium), which prevents the growth of HGPRT-deficient cells.

[0292] In some embodiments, the immortalized cell line is effectively fused, ensures high-level stable expression of the antibody by the selected antibody-producing cells, and is sensitive to certain culture media, such as HAT medium. In some embodiments, the immortalized cell line is a mouse myeloma cell line, which can be obtained from, for example, the Salk Cell Collection in San Diego, California and the American Type Culture Collection in Manassas, Virginia. Human myeloma and mouse-human hybrid myeloma cell lines are also described for use in preparing human monoclonal antibodies.

[0293] The presence of monoclonal antibodies against the polypeptide in the culture medium of the hybridoma cells can then be determined. The binding specificity of the monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or in vitro binding experiments, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques or analytical methods are known in the art. The binding affinity of the monoclonal antibodies can be determined by Scatchard analysis as described in, for example, Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0294] After identifying the desired hybridoma cells, the target clones can be subcloned by limiting dilution and cultured by standard methods. Suitable culture media for this purpose include, for example, modified Eagle medium (DMEM) and RPMI-1640 culture medium. Alternatively, hybridoma cells can be grown in mammalian ascites.

[0295] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0296] In some embodiments, according to any of the antibodies or antigen-binding fragments or multispecific antibodies described herein, the antibodies or antigen-binding fragments or multispecific antibodies comprise sequences selected from clones of an antibody library (e.g., a phage library displaying scFv or Fab fragments). The clones can be identified by methods for screening combinatorial libraries of antibody fragments having the desired activity. For example, various methods are known in the art for generating phage display libraries and screening these libraries to obtain antibodies with the desired binding properties. These methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and in, for example, McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al. It is further described in Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0297] In some phage display methods, V H and V L All components of the gene are randomly recombined in a phage library, and then phage that can bind to the antigen is screened, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage usually display antibody fragments in the form of scFv fragments or Fab fragments. Library phage of immune origin provides high affinity antibodies to immunogens without the need to construct hybridoma cells. Alternatively, natural libraries (e.g., from humans) can be cloned to provide a single source of antibodies to a variety of non-self antigens and self antigens without any immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, natural libraries can also be prepared by cloning non-rearranged V-gene fragments from stem cells and using PCR primers containing random sequences to encode CDR3 hypervariable regions and complete rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, US Pat. No. 5,750,373 and US Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936 and 2009 / 0002360.

[0298] The antibody or antigen-binding fragment or multispecific antibody is prepared by screening the library for an antigen-binding portion that can specifically bind to a target (e.g., HBV preS1 antigen, CD3 antigen, or PD-L1 antigen) through phage display. The library can be a human scFv phage display library with at least 1×10 9 (For example, at least 1×10 9 , 2.5×10 9 , 5×10 9 , 7.5×10 9 , 1×10 10 , 2.5×10 10 , 5×10 10 , 7.5×10 10 or 1×10 11) kinds of diverse unique human antibody fragments. In some embodiments, the library is a human natural library, constructed by DNA extracted from PMBCs and spleens of healthy subjects, comprising all human heavy and light chain subfamilies. In some embodiments, the library is a human natural library, constructed by DNA extracted from PMBCs isolated from patients with various diseases, such as patients with autoimmune diseases, cancer patients, and patients with infectious diseases. In some embodiments, the library is a semisynthetic human library, wherein the heavy chain CDR3 is completely random, and all amino acids (except cysteine) are present at any given position with the same probability. (See, for example, Hoet, RM et al., Nat. Biotechnol. 23 (3): 344-348, 2005). In some embodiments, the heavy chain CDR3 length of the semisynthetic human library is between 5 and 24 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24) amino acids. In some embodiments, the library is a fully synthetic phage display library. In some embodiments, the library is a non-human phage display library.

[0299] Phage clones with high affinity for the target antigen (e.g., HBV preS1 antigen or CD3 antigen or PD-L1 antigen) can be screened by iterative binding of phage to the target antigen, wherein the target antigen is bound to a solid support (e.g., beads for solution panning or mammalian cells for cell panning), followed by removal of unbound phage and elution of specifically bound phage. Subsequently, the bound phage clones are eluted and used to infect suitable host cells, such as E. coli XL1-Blue, for expression and purification. Phage clones that specifically bind to the target antigen can be enriched by multiple rounds of panning (e.g., 2, 3, 4, 5, 6 or more rounds), such as solution panning, cell panning, or a combination of both. Specific binding of the enriched phage clones to the target antigen can be detected by any method known in the art, including, for example, ELISA and FACS.

[0300] Monoclonal antibodies can also be prepared by recombinant DNA methods, such as those described in US Patent No. 4,816,567. The DNA encoding the monoclonal antibodies described in this application can be easily isolated and sequenced by conventional methods (e.g., by oligonucleotide probes that specifically bind to the genes encoding the light and heavy chains of the mouse antibody). Hybridoma cells as described above or antigen-specific phage clones of this application can be used as a source of such DNA. After isolation, the DNA can be placed in an expression vector, which is then transfected into a host cell, such as a simian COS cell, a Chinese hamster ovary carcinoma (CHO) cell, or a myeloma cell that does not produce immunoglobulins, to obtain monoclonal antibodies synthesized in the recombinant host cell. The DNA can also be modified, for example, by replacing the homologous non-human sequence with the coding sequence for the human heavy and light chain constant structure and / or framework region (U.S. Patent No. 4,816,567; Morrison et al., supra), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such non-immunoglobulin polypeptides can replace the constant region of the antibody in the present application, or can replace one antigen binding site in the variable region of the antibody in the present application to form a chimeric bivalent antibody. In some embodiments, additional variable regions directed against different epitopes or antigens can be included to generate chimeric multispecific antibodies.

[0301] The antibody can be a monovalent antibody. Methods for preparing monovalent antibodies are known in the art. For example, a method involves recombinant expression of immunoglobulin light chains and modified heavy chains. The heavy chain is typically truncated at any position in the Fc region to prevent cross-linking of the heavy chains. Alternatively, the relevant cysteine ​​residues are replaced with other amino acid residues or deleted to prevent cross-linking.

[0302] In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce antibody fragments, particularly Fab fragments, can be accomplished using any method known in the art.

[0303] Chemical coupling is the earliest technology used to prepare multispecific antibodies. In 1985, Brennan first used the chemical conjugation of two monoclonal antibody G1 fragments to prepare multispecific antibodies (Brennan M, et al. Preparation of bispecific antibodies by chemical recombination of monoclonal immunoglobulin G1 fragments [J]. Science, 1985, 229(4708):81-83). There are two main methods for chemical conjugation: one is to directly conjugate two monoclonal antibodies or their derivatives to form a multispecific antibody; the other is to first dissociate the two monoclonal antibodies into free light and heavy chains through various physical and chemical methods, and then recombine these light and heavy chains. The advantages of chemical conjugation are rapidity, ease of operation, and high recovery rate, but it can also easily damage the antigen-binding domain of the antibody, affecting antibody activity, and easily form multimers.

[0304] The use of hybridoma cell lines to prepare multispecific antibodies involves fusing two different hybridoma cell lines through cell fusion technology, and then identifying and isolating cells that can produce specific therapeutic antibodies (Kohler, G, et al. Continuous cultures of fused cells secreting antibodies of predefined specificity [J]. J Immunol., 2005, 174(5): 2453-2455). Because two hybridoma cells can produce two different light-heavy chains, and these light-heavy chains can be randomly combined, the multispecific antibodies prepared using this method have a high degree of randomness and low production efficiency.

[0305] Genetic engineering technology is currently also used to prepare a variety of multispecific antibodies (Roland E K. Antibody-cytokine fusion proteins [J]. Arch Biochem Biophys., 2012, 526 (2): 194-205). Using genetic engineering to edit recombinant antibodies can solve the problem of random combination by limiting the selectivity of the two pairs of light-heavy chain binding in various ways. KiH (Knob into hole) and CrossMab are two commonly used technologies currently used to improve the light-heavy chain pairing problem. KiH technology, that is, in C H 3 domains introduced asymmetric mutation structures (“knob” mutation refers to the mutation in C HIn the 3 domain, a large amino acid residue is substituted for a smaller residue, while a "hole" mutation refers to the use of a small amino acid residue to replace a larger residue). The Fc region of the engineered multispecific antibody is more inclined to heterodimerization rather than homodimerization due to steric hindrance (Ridgway JB, et al. "Knobs-into-holes" engineering of antibody C H 3 domains for heavy chain heterodimerization[J].Protein Eng.1996,9(7):617-621). In the glycosylated C H Introducing a Y349C mutation into the 3 domain can form disulfide bonds between glycosylated heavy chains and enhance the stability of KiH (Kuglstatter A, et al. Structural differences between glycosylated, disulfide-linked heterodimeric knob-into-hole Fc fragment and its homodimeric knob-knob and hole-hole side products[J]. Protein Eng Des Sel., 2017, 30(9): 649-656).

[0306] In addition to steric effects, the charge effects of amino acid residues have also been exploited to enhance heterodimerization between the two heavy chains of multispecific antibodies. Through structural modeling and molecular design, one chain is positively charged, while the paired chain is negatively charged. This promotes heavy chain heterodimer formation through the pattern of like charges repelling and unlike charges attracting. Mutations such as K409D and D399K, K409D / K392D and D399K / E356K, or E356K / E357K / D399K and K370E / K409D / K439E in both chains have all been shown to enhance heterodimerization to some extent (IGAWA T, et al. Methods for producing polypeptides by regulating polypeptide; association: US, 20100015133A1[P]. 2006). Combining the steric effects of KiH with charge effects is also a strategy for enhancing heterodimerization.

[0307] CrossMab technology is a new antibody pairing technology developed by Roche based on KiH technology. It is a technology that exchanges the domains of the light chain and heavy chain of one Fab in a multispecific antibody, while the other does not. The exchanged light chain will contain a fragment of the homologous heavy chain, making it unable to pair with the unexchanged heavy chain, thereby ensuring the correct combination between the light chain and the heavy chain (Schaefer W, et al. Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies [J]. Proc Natl Acad Sci USA, 2011, 108 (27): 11187-11192). The structure includes "CrossMab Fab", "CrossMab V H -V L ” or “CrossMab C H 1-C L " and other forms.

[0308] Antibody variable regions with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant regions. Preferably, the fusion is to an immunoglobulin heavy chain constant region, which includes at least part of the hinge, C H 2 and C H 3 domains. In some embodiments, the heavy chain constant region C contains the necessary sites for light chain binding H The 1 domain is present in at least one fusion. DNA encoding the immunoglobulin heavy chain fusion, and if desired, DNA encoding the immunoglobulin light chain, is inserted into separate expression vectors and co-transfected into a suitable host organism. In some embodiments, antibody variable regions directed against different epitopes or different antigens can be fused to immunoglobulin constant region sequences to produce chimeric multispecific antibodies.

[0309] Fully human and humanized antibodies

[0310] The antibody or antigen-binding fragment or multispecific antibody can be a humanized antibody or a fully human antibody. The humanized form of a non-human (such as mouse) antibody portion is a chimeric immunoglobulin, immunoglobulin chain or its fragment (such as Fv, Fab, Fab', F(ab')2, scFv or other antigen-binding subsequences of an antibody), which generally includes a minimum sequence derived from a non-human immunoglobulin. Humanized antibodies include human immunoglobulins, immunoglobulin chains or their fragments (receptor antibodies), wherein the residues of the receptor CDR are replaced by non-human (donor antibody) CDR residues with desired specificity, affinity and performance, such as mouse, rat or rabbit CDRs. In certain embodiments, human immunoglobulin Fv framework region residues are replaced by corresponding non-human residues. Humanized antibodies can also include amino acid residues that are neither part of the receptor antibody nor in the CDR or framework region sequences introduced. Typically, a humanized antibody comprises at least one, typically two variable regions, wherein all or substantially all of the CDR regions correspond to the CDR regions of non-human immunoglobulins, and all or substantially all of the framework regions are human immunoglobulin consensus sequences.

[0311] Typically, a humanized antibody contains one or more amino acid residues introduced from a non-human source. Those non-human amino acid residues are generally referred to as "imported" residues, typically from the "imported" variable region. According to some embodiments, humanization can be performed essentially according to the following method of Winter and colleagues (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by replacing the corresponding sequences of a human antibody with rodent CDRs or CDR sequences. Thus, this "humanized" antibody portion (US Patent No. 4,816,567), which is substantially less than a complete human antibody, has its variable regions replaced by corresponding sequences from a non-human source. In practice, the humanized antibody portion is a typical human antibody portion in which some CDR residues and possibly some framework region residues are substituted by residues from analogous sites in rodent antibodies.

[0312] Generating partially human antibodies is an alternative to humanization. For example, it is now possible to prepare transgenic animals (e.g., mice) that can produce a complete library of fully human antibodies upon immunization without producing endogenous immunoglobulins. For example, it has been reported that homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice completely inhibits endogenous antibody production. Transferring the human germline immunoglobulin gene array into such germline mutant mice can produce fully human antibodies upon antigen stimulation, see, for example, akobovits et al., PNAS USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immunol., 7:33 (1993); US Patent Nos. 5,545,806, 5,569,825, 5,591,669; 5,545,807; and WO 97 / 17852. Alternatively, fully human antibodies can be prepared by introducing human immunoglobulin loci into transgenic animals (e.g., mice in which endogenous immunoglobulin genes have been partially or completely silenced). Upon antigen stimulation, the production of fully human antibodies is found to be very similar to that produced in humans in all aspects, including gene rearrangement, assembly, and antibody libraries. This method is described in, for example, US Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016, and Marks et al., Bio / Technology, 10:779-783 (1992); Lonberg et al., Nature, 368:856-859 (1994); Morrison, Nature, 368:812-813 (1994); Fishwild et al., Nature Biotechnology, 14:845-851 (1996); Neuberger, Nature Biotechnology, 14:826 (1996); Lonberg and Described in Huszar, Intern. Rev. Immunol., 13:65-93 (1995).

[0313] Human antibodies or human antibody portions can also be produced by in vitro activated B cells (see US Patents 5,567,610 and 5,229,275) or by using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). The techniques of Cole et al. and Boerner et al. can also be used to prepare fully human monoclonal antibodies. See Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985) and Boerner et al., J. Immunol., 147 (1):86-95 (1991).

[0314] Variants of antibodies or antigen-binding fragments

[0315] In some embodiments, amino acid sequence variants of the antibodies or antigen-binding fragments provided herein (e.g., antibodies that specifically bind to HBV preS1 antigen, CD3 antigen, or PD-L1 antigen, or multispecific antibodies that specifically bind to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen) are also contemplated. For example, it may be desirable to improve the binding affinity and / or other biological activities of the antibody or antigen-binding fragment. The amino acid sequence variants of the antigen-binding entity can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antigen-binding entity or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequence of the antigen-binding entity. The final construct can be achieved by any combination of deletions, insertions, and substitutions of amino acid residues to impart the desired characteristics, such as antigen binding.

[0316] In some embodiments, variants of antibodies or antigen-binding fragments having one or more amino acid substitutions are provided. The target sites of the substitution mutations include hypervariable regions (HVRs) and framework regions (FRs). Amino acid substitutions can be introduced into the target antibody to screen for products of desired activity, for example, improved biological activity, maintaining / improving antigen binding ability, reduced immunogenicity, or improved ADCC, ADCP or CDC. In some embodiments, the amino acid substitutions described herein are limited to the "exemplary substitutions" in Table 12 of the present application. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" in Table 12 of the present application.

[0317] Conservative substitutions are shown in Table 12 below.

[0318] Amino acids are divided into different categories based on the properties of their side chains:

[0319] a. Hydrophobic amino acids: Norleucine, Met, Ala, Val, Leu, Isoleucine;

[0320] b. Neutral hydrophilic amino acids: cysteine ​​(Cys), serine (Ser), threonine (Thr), asparagine (Asn), and glutamine (Gln);

[0321] c. Acidic amino acids: Aspartic acid Asp, glutamic acid Glu;

[0322] d. Basic amino acids: Histidine (His), Lysine (Lys), Arginine (Arg);

[0323] e. Amino acids that affect chain direction: glycine Gly, proline Pro;

[0324] f. Aromatic amino acids: tryptophan Trp, tyrosine Tyr, phenylalanine Phe.

[0325] Non-conservative amino acid substitutions include substituting one class for another.

[0326] An exemplary substitution variant is an affinity-matured antibody, which can be conveniently produced using, for example, affinity maturation techniques based on phage display. In short, one or more CDR residues are mutated, the variant antibody portion is displayed on a phage, and variants with specific biological activity (e.g., based on PBMC killing assays or binding affinity to target cells) are screened. Changes (e.g., substitutions) can be made in the HVRs region to obtain improved PBMC-based killing assays or antibody affinity to target cells. Changes can be made in the "hotspots" of the HVR, i.e., residues encoded by codons that undergo high-frequency mutations during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and / or at specific deterministic residues (SDRs), and the resulting variant V H and V L Methods for constructing and reselecting affinity maturation from secondary libraries have been described in some literature, for example, Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)).

[0327] In some affinity maturation embodiments, diversity is introduced into the variable genes selected for affinity maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide directed mutagenesis). A secondary library is then created. The library is screened to identify antibody variants with the desired affinity. Another method for introducing diversity includes HVR-mediated methods, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding are specifically identified, for example, using alanine scanning mutagenesis or modeling. Typically, CDR-H3 and CDR-L3 regions are particularly key targets.

[0328] In some embodiments, substitutions, insertions or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes that do not substantially reduce binding affinity (e.g., conservative substitutions provided herein) may be produced in HVRs. These changes may occur outside of HVR "hot spots" or SDRs regions. In some embodiments, the variant V provided above may be modified to include a nucleotide sequence that is not substantially modified. H and V L Sequences, each HVR is either unchanged or contains no more than 1, 2 or 3 amino acid substitutions.

[0329] A method for identifying an antibody that can bind to a target amino acid or region by a mutation method is called "alanine scanning mutagenesis," as described in Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, one or a group of target residues (e.g., charged residues such as arginine, aspartic acid, histidine, lysine, and glutamic acid) are replaced by neutral or negatively charged amino acids (e.g., alanine or glutamic acid) to determine whether the antibody-antigen interaction is affected. Substitutions can be further introduced at the position of the amino acid to demonstrate that the position has functional sensitivity to the initial substitution. Alternatively or in addition, the contact sites between the antibody and the antigen are identified by the crystal structure of the antigen-antibody complex. These contact site residues and adjacent residues can be targeted or eliminated as replacement candidates. Variants are screened to determine whether they have the desired properties.

[0330] Insertions of amino acid sequences include fusions at the amino and / or carboxyl termini ranging in length from one residue to polypeptides containing 100 or more residues, and also include insertions of one or more amino acid residues within a sequence. Examples of terminal insertions include antigen-binding portions having a methionyl residue at the N-terminus. Other insertional variants of the antigen-binding portion include fusions to the N-terminus or C-terminus of the antigen-binding portion to an enzyme (e.g., ADEPT) or a polypeptide that increases the serum half-life of the antigen-binding portion.

[0331] Fc variants

[0332] In some embodiments, one or more amino acid modifications are introduced into the Fc region of an antibody or antigen-binding fragment described herein (e.g., a full-length antibody that specifically binds to the HBV preS1 antigen, a full-length antibody that specifically binds to the CD3 antigen, a full-length antibody that specifically binds to the PD-L1 antigen, a multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen, or a fusion protein comprising the antibody, antigen-binding fragment, or multispecific antibody), thereby generating an Fc variant. In some embodiments, the Fc variant has enhanced ADCC potency, which is generally associated with Fc-binding receptors (FcRs). In some embodiments, the Fc variant has reduced ADCC potency. There are many examples of how changes or mutations in the Fc sequence affect its potency. For example, WO 00 / 42072 and Shields et al. J Biol. Chem. 9(2):6591-6604 (2001) describe antibody variants with enhanced or reduced binding to FcRs. The contents of these publications are incorporated herein by reference.

[0333] Antibody-dependent cell-mediated cytotoxicity (ADCC) is the mechanism of action of therapeutic antibodies against tumor cells. ADCC is a cell-mediated immune defense in which effector cells of the immune system actively lyse target cells (e.g., infected cells) when antigens on the target cell membrane are bound by a specific antigen-binding moiety (e.g., an antibody that specifically binds to HBV preS1, an antibody that specifically binds to CD3, an antibody that specifically binds to PD-L1, or a multispecific antibody that specifically binds to HBV preS1, PD-L1, and CD3). The ADCC effect typically involves antibody-activated natural killer (NK) cells. NK cells express the Fc receptor CD16. This receptor recognizes and binds to the Fc portion of antibody molecules bound to the target cell surface. The most common Fc receptors on the surface of NK cells are CD16 or FcγRIII. Binding of Fc receptors to the Fc region of an antibody leads to NK cell activation, release of lytic granules, and subsequent apoptosis of the target cell.

[0334] In some embodiments, the present application also provides an antibody or antigen-binding domain that specifically binds to the HBV preS1 antigen, an antibody or antigen-binding domain that specifically binds to the CD3 antigen, an antibody or antigen-binding domain that specifically binds to the PD-L1 antigen, or a multispecific antibody variant that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen (e.g., a full-length antibody that specifically binds to the HBV preS1 antigen, a full-length antibody that specifically binds to the CD3 antigen, a full-length antibody that specifically binds to the PD-L1 antigen, or a variant of a multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen), which comprises an Fc region having one or more effector functions, making it an ideal candidate antibody for the application, in which the in vivo half-life of the antibody or antigen-binding domain that specifically binds to the HBV preS1 antigen, the antibody or antigen-binding domain that specifically binds to the CD3 antigen, the antibody or antigen-binding domain that specifically binds to the PD-L1 antigen, or the multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen is important, but certain effector functions (such as CDC and ADCC) are unnecessary or deleterious. The reduction / elimination of CDC and / or ADCC activity can be confirmed by performing in vitro and / or in vivo cytotoxicity assays. For example, an Fc receptor (FcR) binding assay can be performed to confirm that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. Among the main cells mediating ADCC, NK cells only express FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet Annu. Rev. Immunol. 9: 457-492 (1991). Non-limiting examples of in vitro assessment of ADCC activity of a molecule of interest are described in US Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); US Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive detection methods can be used (see, e.g., ACTI TM Flow cytometry nonradioactive cytotoxicity assay (Cell Technology, Inc. Mountain View, Calif.) and CYTOTOX 96TM Non-radioactive cytotoxicity assays (Promega, Madison, Wis.) can be used. Effector cells used in such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, ADCC activity of the target molecule can be tested in vivo, for example, in animal models as described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody does not bind to C1q and thus lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life can be determined using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0335] Antibodies with reduced effector function comprising one or more substitutions at residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region ( US Pat. No. 6,737,056 ). These Fc variants include Fc variants with substitutions at two or more residues at positions 265, 269, 270, 297, and 327, including an Fc variant known as "DANA" in which residues 265 and 297 are substituted with alanine ( US Pat. No. 7,332,581 ).

[0336] Such antibody variants with increased or decreased binding to FcRs have been described (see, eg, US Pat. No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0337] In some embodiments, alterations in the Fc region result in altered (ie, increased or decreased) opsonization, as described in Moore et al., MAbs. 2(2): 181-189 (2010).

[0338] In some embodiments, a variant of an antibody or antigen-binding domain that specifically binds to the HBV preS1 antigen, an antibody or antigen-binding domain that specifically binds to the CD3 antigen, an antibody or antigen-binding domain that specifically binds to the PD-L1 antigen, or a multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen (e.g., a full-length antibody that specifically binds to the HBV preS1 antigen, a full-length antibody that specifically binds to the CD3 antigen, a full-length antibody that specifically binds to the PD-L1 antigen, or a multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen) is provided, comprising an Fc variant having one or more amino acid substitutions that can extend half-life and / or enhance binding to an Fc receptor (FcRn). Antibodies with extended half-life and improved FcRn binding are described in US 2005 / 0014934A1 (Hinton et al.). These antibodies comprise one or more amino acid substitutions in the Fc region that enhance binding of the Fc region to FcRn. These Fc variants comprise one or more substitutions at residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 in the Fc region, such as a substitution at residue 434 in the Fc region ( U.S. Pat. No. 7,371,826 ).

[0339] See also Duncan & Winter, Nature 322:738-40 (1988); US Pat. No. 5,648,260; US Pat. No. 5,624,821 and WO 94 / 29351 for additional examples of Fc variants.

[0340] Also contemplated are antibodies or antigen-binding domains that specifically bind to HBV preS1 antigen, antibodies or antigen-binding domains that specifically bind to CD3 antigen, antibodies or antigen-binding domains that specifically bind to PD-L1 antigen, or multispecific antibodies that specifically bind to HBV preS1 antigen and CD3 antigen (e.g., full-length antibodies that specifically bind to HBV preS1 antigen, full-length antibodies that specifically bind to CD3 antigen, full-length antibodies that specifically bind to PD-L1 antigen, or multispecific antibodies that specifically bind to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen) comprising any one of the Fc variants described herein or a combination thereof.

[0341] Glycosylation variants

[0342] In some embodiments, the antibody or antigen-binding domain that specifically binds to the HBV preS1 antigen, the antibody or antigen-binding domain that specifically binds to the CD3 antigen, the antibody or antigen-binding domain that specifically binds to the PD-L1 antigen, or the multispecific antibody that specifically binds to the HBV preS1 antigen, the PDL1 antigen, and the CD3 antigen (e.g., a full-length antibody that specifically binds to the HBV preS1 antigen, a full-length antibody that specifically binds to the CD3 antigen, a full-length antibody that specifically binds to the PD-L1 antigen, or a multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen) provided herein is altered to increase or decrease the degree of glycosylation of the antibody or antigen-binding domain that specifically binds to the HBV preS1 antigen, the antibody or antigen-binding domain that specifically binds to the CD3 antigen, the antibody or antigen-binding domain that specifically binds to the PD-L1 antigen, or the multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen. By changing the amino acid sequence of an antibody or antigen-binding domain that specifically binds to the HBV preS1 antigen, an antibody or antigen-binding domain that specifically binds to the CD3 antigen, an antibody or antigen-binding domain that specifically binds to the PD-L1 antigen, or a multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen, or a polypeptide portion thereof to add or remove one or more glycosylation sites, it is possible to conveniently add or delete glycosylation sites on the antibody or antigen-binding domain that specifically binds to the HBV preS1 antigen, the antibody or antigen-binding domain that specifically binds to the CD3 antigen, the antibody or antigen-binding domain that specifically binds to the PD-L1 antigen, or a multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen.

[0343] Wherein the antibody or antigen binding domain that specifically binds to HBV preS1 antigen, the antibody or antigen binding domain that specifically binds to CD3 antigen, the antibody or antigen binding domain that specifically binds to PD-L1 antigen, or the multispecific antibody that specifically binds to HBV preS1 antigen, PD-L1 antigen and CD3 antigen comprises an Fc region, and the sugars attached thereto can be changed. Natural antibodies produced by mammalian cells generally comprise branched biantennary oligosaccharides, which are generally attached to the Fc region C through an N-link. H2 domains are linked to Asn297 of the HBV preS1 domain, see, for example, Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharide may comprise a variety of sugars, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as trehalose linked to the GlcNAc in the "stem" portion of the biantennary oligosaccharide structure. In some embodiments, the antibody or antigen-binding domain that specifically binds to the HBV preS1 antigen, the antibody or antigen-binding domain that specifically binds to the CD3 antigen, the antibody or antigen-binding domain that specifically binds to the PD-L1 antigen, or the multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen of the present application may be oligosaccharide-modified to produce an antibody or antigen-binding domain that specifically binds to the HBV preS1 antigen, the antibody or antigen-binding domain that specifically binds to the CD3 antigen, the antibody or antigen-binding domain that specifically binds to the PD-L1 antigen, or a multispecific antibody variant that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen with certain improved properties.

[0344] C with Fc region H The N-glycans linked to the two domains are heterogeneous. Antibodies or Fc fusion proteins produced in CHO cells are fucosylated by fucosyltransferase activity (see Shoji-Hosaka et al., J. Biochem. 2006, 140:777-83). Typically, a small fraction of naturally occurring non-fucosylated IgGs can be detected in human serum. N-glycosylation of the Fc region is important for its binding to FcγRs; however, non-fucosylated N-glycans enhance the binding ability of Fc to FcγRIIIa. Enhanced binding to FcγRIIIa results in an enhanced ADCC effect, which is advantageous in certain antibody therapeutic applications requiring cytotoxicity.

[0345] In some embodiments, when Fc-mediated cytotoxicity is not desired, enhanced effector function may be detrimental. In some embodiments, the Fc fragment or C H 2 domains are non-glycosylated. In some embodiments, by H The N-glycosylation site in domain 2 was mutated to prevent its glycosylation.

[0346] In some embodiments, provided are variants of antibodies or antigen-binding domains that specifically bind to HBV preS1 antigen, antibodies or antigen-binding domains that specifically bind to CD3 antigen, antibodies or antigen-binding domains that specifically bind to PD-L1 antigen, or multispecific antibodies that specifically bind to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen (e.g., full-length antibodies that specifically bind to HBV preS1 antigen, full-length antibodies that specifically bind to CD3 antigen, full-length antibodies that specifically bind to PD-L1 antigen, or multispecific antibodies that specifically bind to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen), which comprise an Fc region, wherein the carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose, which may enhance ADCC function. Specifically, the present application provides antibodies or antigen-binding domains that specifically bind to the HBV preS1 antigen, antibodies or antigen-binding domains that specifically bind to the CD3 antigen, antibodies or antigen-binding domains that specifically bind to the PD-L1 antigen, or multispecific antibody variants that specifically bind to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen, which have reduced fucose relative to the same antibodies or antigen-binding domains that specifically bind to the HBV preS1 antigen, the CD3 antigen, the PD-L1 antigen, or multispecific antibodies that specifically bind to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen produced in wild-type CHO cells. That is, they are characterized by having a lower amount of fucose than antibodies produced in native CHO cells (e.g., CHO cells that produce native glycosylation forms, CHO cells that contain the native FUT8 gene). In some embodiments, the N-linked glycans of the antibody or antigen-binding domain that specifically binds to the HBV preS1 antigen, the antibody or antigen-binding domain that specifically binds to the CD3 antigen, the antibody or antigen-binding domain that specifically binds to the PD-L1 antigen, or the multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen have less than 50%, 40%, 30%, 20%, 10%, or 5% fucose. For example, the fucose content of the antibody or antigen-binding domain that specifically binds to the HBV preS1 antigen, the antibody or antigen-binding domain that specifically binds to the CD3 antigen, the antibody or antigen-binding domain that specifically binds to the PD-L1 antigen, or the multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen may be 1%-80%, 1%-65%, 5%-65%, or 20%-40%.In some embodiments, the N-linked glycans of the antibody or antigen-binding domain that specifically binds to the HBV preS1 antigen, the antibody or antigen-binding domain that specifically binds to the CD3 antigen, the antibody or antigen-binding domain that specifically binds to the PD-L1 antigen, or the multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen do not contain fucose, i.e., the antibody or antigen-binding domain that specifically binds to the HBV preS1 antigen, the antibody or antigen-binding domain that specifically binds to the CD3 antigen, the antibody or antigen-binding domain that specifically binds to the PD-L1 antigen, or the multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen do not contain fucose at all, or are devoid of fucose or are defucosylated. The fucose content is determined by calculating the average fucose content within the sugar chains attached to Asn297 relative to the total amount of all sugar structures (such as complex, hybrid, or mannose structures) attached to Asn297 as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue at position 297 of the Fc region (EU Fc region residue numbering system). However, due to minor sequence variations in antibodies, Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. These fucosylation variants may have enhanced ADCC function. See, for example, US Patent Publication Nos. US 2003 / 0157108 (Presta, L.) and US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249(2004);Cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation function (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986); US Pat Appl No US2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11), and gene knockout cell lines, such as CHO cells in which the α-1,6-fucosyltransferase gene, FUT8, is knocked out (see Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4): 680-688 (2006); and WO2003 / 085107).

[0347] Variants of the antibody or antigen-binding domain that specifically binds to the HBV preS1 antigen, the antibody or antigen-binding domain that specifically binds to the CD3 antigen, the antibody or antigen-binding domain that specifically binds to the PD-L1 antigen, or the multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen (e.g., a full-length antibody that specifically binds to the HBV preS1 antigen, a full-length antibody that specifically binds to the CD3 antigen, a full-length antibody that specifically binds to the PD-L1 antigen, or a multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen) further provide a bisected oligosaccharide, for example, wherein a biantennary oligosaccharide attached to the Fc region of the antibody or antigen-binding domain that specifically binds to the HBV preS1 antigen, the antibody or antigen-binding domain that specifically binds to the CD3 antigen, the antibody or antigen-binding domain that specifically binds to the PD-L1 antigen, or the multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen is bisected by GlcNAc. Such an antibody or antigen-binding domain that specifically binds to the HBV preS1 antigen, an antibody or antigen-binding domain that specifically binds to the CD3 antigen, an antibody or antigen-binding domain that specifically binds to the PD-L1 antigen, or a multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen (e.g., a full-length antibody that specifically binds to the HBV preS1 antigen, a full-length antibody that specifically binds to the CD3 antigen, a full-length antibody that specifically binds to the PD-L1 antigen, or a multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen) variant may have reduced fucosylation and / or enhanced ADCC function. Examples of such antibody variants are described in WO 2003 / 011878 (Jean-Mairet et al.); US Pat. No. 6,602,684 (Umana et al.); US 2005 / 0123546 (Umana et al.), and Ferrara et al., Biotechnology and Bioengineering, 93(5):851-861 (2006). Also provided are antibodies or antigen-binding domains that specifically bind to the HBV preS1 antigen, antibodies or antigen-binding domains that specifically bind to the CD3 antigen, antibodies or antigen-binding domains that specifically bind to the PD-L1 antigen, or multispecific antibodies that specifically bind to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen (e.g., full-length antibodies that specifically bind to the HBV preS1 antigen, full-length antibodies that specifically bind to the CD3 antigen, full-length antibodies that specifically bind to the PD-L1 antigen, or multispecific antibodies that specifically bind to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen) variants that have at least one galactose residue in the oligosaccharide attached to the Fc region.Such antibodies or antigen-binding domains that specifically bind to the HBV preS1 antigen, antibodies or antigen-binding domains that specifically bind to the CD3 antigen, antibodies or antigen-binding domains that specifically bind to the PD-L1 antigen, or multispecific antibody variants that specifically bind to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen may have enhanced CDC function. Such variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0348] In some embodiments, the antibody or antigen-binding domain that specifically binds to the HBV preS1 antigen, the antibody or antigen-binding domain that specifically binds to the CD3 antigen, the antibody or antigen-binding domain that specifically binds to the PD-L1 antigen, or the multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen (e.g., a full-length antibody that specifically binds to the HBV preS1 antigen, a full-length antibody that specifically binds to the CD3 antigen, a full-length antibody that specifically binds to the PD-L1 antigen, or a multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen) variant comprises an Fc region that can bind to FcγRIII. In some embodiments, the antibody or antigen-binding domain that specifically binds to HBV preS1 antigen, the antibody or antigen-binding domain that specifically binds to CD3 antigen, the antibody or antigen-binding domain that specifically binds to PD-L1 antigen, or the multispecific antibody that specifically binds to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen (e.g., a full-length antibody that specifically binds to HBV preS1 antigen, a full-length antibody that specifically binds to CD3 antigen, a full-length antibody that specifically binds to PD-L1 antigen, or a multispecific antibody that specifically binds to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen) variant comprising an Fc region has ADCC activity in the presence of human effector cells (e.g., T cells), or has ADCC activity compared to other same antibodies or antigen-binding domains that specifically bind to HBV preS1 antigen, antibodies or antigen-binding domains that specifically bind to CD3 antigen, antibodies or antigen-binding domains that specifically bind to PD-L1 antigen, or multispecific antibodies that specifically bind to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen (e.g., a full-length antibody that specifically binds to HBV preS1 antigen, a full-length antibody that specifically binds to CD3 antigen, a full-length antibody that specifically binds to PD-L1 antigen, or a multispecific antibody that specifically binds to HBV In the presence of human effector cells, the present invention has enhanced ADCC activity compared with a full-length antibody that specifically binds to the HBV preS1 antigen, a full-length antibody that specifically binds to the CD3 antigen, a full-length antibody that specifically binds to the PD-L1 antigen, or a multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen.

[0349] Cysteine ​​engineered variants

[0350] In some embodiments, it is desirable to prepare a cysteine-engineered antibody or antigen-binding domain that specifically binds to the HBV preS1 antigen, an antibody or antigen-binding domain that specifically binds to the CD3 antigen, an antibody or antigen-binding domain that specifically binds to the PD-L1 antigen, or a multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen (e.g., a full-length antibody that specifically binds to the HBV preS1 antigen, a full-length antibody that specifically binds to the CD3 antigen, a full-length antibody that specifically binds to the PD-L1 antigen, or a multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen), in which one or more amino acid residues are substituted with a cysteine ​​residue. In some embodiments, the substituted residue occurs at an accessible site of the antibody or antigen-binding domain that specifically binds to the HBV preS1 antigen, the antibody or antigen-binding domain that specifically binds to the CD3 antigen, the antibody or antigen-binding domain that specifically binds to the PD-L1 antigen, or the multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen. By replacing those residues with cysteine, reactive sulfhydryl groups are located at accessible sites of the antibody or antigen-binding domain that specifically binds to the HBV preS1 antigen, the antibody or antigen-binding domain that specifically binds to the CD3 antigen, the antibody or antigen-binding domain that specifically binds to the PD-L1 antigen, or the multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen, which can be used to conjugate the antibody or antigen-binding domain that specifically binds to the HBV preS1 antigen, the antibody or antigen-binding domain that specifically binds to the CD3 antigen, the antibody or antigen-binding domain that specifically binds to the PD-L1 antigen, or the multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen to other moieties, such as drug moieties or linker-drug moieties, to prepare antibody or antigen-binding domain that specifically binds to the HBV preS1 antigen, the antibody or antigen-binding domain that specifically binds to the CD3 antigen, the antibody or antigen-binding domain that specifically binds to the PD-L1 antigen, or the multispecific antibody that specifically binds to the HBV preS1 antigen, the PD-L1 antigen, and the CD3 antigen immunoconjugates as further described herein.Cysteine-engineered antibodies or antigen-binding domains that specifically bind to HBV preS1 antigen, antibodies or antigen-binding domains that specifically bind to CD3 antigen, antibodies or antigen-binding domains that specifically bind to PD-L1 antigen, or multispecific antibodies that specifically bind to HBV preS1 antigen, PD-L1 antigen, and CD3 antigens (e.g., full-length antibodies that specifically bind to HBV preS1 antigen, full-length antibodies that specifically bind to CD3 antigen, full-length antibodies that specifically bind to PD-L1 antigen, or multispecific antibodies that specifically bind to HBV preS1 antigen, PD-L1 antigen, and CD3 antigens) can be prepared according to, for example, U.S. Pat. No.

[0351] Prepared as described in 7,521,541.

[0352] derivative

[0353] In some embodiments, the multispecific antibodies provided herein that specifically bind to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen (e.g., multispecific antibodies that specifically bind to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen) may be further modified to include other non-protein moieties known in the art and readily available. Suitable moieties for derivatizing the multispecific antibodies that specifically bind to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde has advantages in manufacturing due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the multispecific antibody that specifically binds to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on the following considerations, including but not limited to, whether the characteristics or function of the multispecific antibody that specifically binds to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen needs to be improved, whether the multispecific antibody derivative that specifically binds to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen is used to treat a specific condition, etc.

[0354] Pharmaceutical composition

[0355] The present application also provides compositions (e.g., pharmaceutical compositions, also referred to herein as formulations) comprising any multispecific antibody that specifically binds to HBV preS1, PD-L1, and CD3 antigens, nucleic acids encoding the antibodies or antigen-binding fragments, vectors comprising nucleic acids encoding the antibodies or antigen-binding fragments, or host cells comprising the nucleic acids or vectors described herein. In some embodiments, a pharmaceutical composition is provided comprising any multispecific antibody that specifically binds to HBV preS1 and CD3 antigens described herein and a pharmaceutically acceptable carrier.

[0356] Suitable multispecific antibody formulations that specifically bind to HBV preS1, PD-L1, and CD3 antigens can be prepared by mixing a multispecific antibody having the desired purity that specifically binds to HBV preS1, PD-L1, and CD3 antigens with an optional pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) and preparing the formulation in the form of a lyophilized formulation or a liquid formulation. Acceptable carriers, excipients, or stabilizers are nontoxic to the recipient at the dosages and concentrations employed and include buffers, preservatives; low molecular weight (less than 10 residues) polypeptides; proteins; hydrophilic polymers; amino acids; monosaccharides, disaccharides, and other carbohydrates; chelating agents; sugars; salt-forming counterions; metal complexes; and / or nonionic surfactants. Exemplary formulations are described in WO98 / 56418, which is expressly incorporated herein by reference. Lyophilized formulations suitable for subcutaneous administration are described in WO97 / 04801. Such lyophilized formulations can be reconstituted into high protein concentration formulations with suitable diluents, and the reconstituted formulations can be administered subcutaneously to the individuals to be treated in the present application. Cationic liposomes or liposomes can be used to deliver the multispecific antibodies specifically binding to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen in the present application to cells.

[0357] In addition to the multispecific antibodies that specifically bind to HBV preS1, PD-L1, and CD3, the formulations described herein may also contain one or more other active substances necessary for treating a specific condition, preferably substances with complementary activities and no adverse reactions with each other. For example, in addition to the multispecific antibodies that specifically bind to HBV preS1, PD-L1, and CD3, it may be desirable to further include other therapeutically active substances, such as antibiotics. These molecules are present in combination in amounts effective for the intended purpose. The effective amount of the other active substances depends on the amount of the multispecific antibodies that specifically bind to HBV preS1, PD-L1, and CD3 in the formulation, the disease or condition, the treatment modality, and other factors as described above. These drugs are generally used at the same dosages and routes of administration as described herein, or at 1% to 99% of currently used dosages.

[0358] The multispecific antibodies that specifically bind to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen can also be encapsulated in microcapsules prepared, for example, by coacervation techniques and interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions. Sustained-release formulations can be prepared.

[0359] Sustained-release formulations of multispecific antibodies that specifically bind to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen can be prepared. Suitable examples of sustained-release formulations include solid hydrophobic polymer semipermeable matrices containing antibodies (or fragments thereof), which are in the form of shaped articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactic acid (US Pat. No.

[0360] 3,773,919), L-glutamic acid and L-glutamic acid ethyl ester copolymers, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT TM(injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D(-)-3-hydroxybutyric acid. Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for more than 100 days, certain hydrogels can release proteins for shorter periods of time. When encapsulated antibodies remain in the body for a long time, they can denature or aggregate due to exposure to a humid environment at 37°C, potentially leading to loss of bioactivity or altered immunogenicity. Rational strategies can be designed to stabilize multispecific antibodies that specifically bind to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen based on the corresponding mechanism. For example, if the aggregation mechanism is found to be through the formation of intermolecular SS bonds through thiodisulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing in acidic solutions, controlling the water content, using appropriate additives, and developing specific polymer matrix compositions.

[0361] In some embodiments, the multispecific antibody that specifically binds to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen is formulated in a buffer containing citrate, sodium chloride, acetate, succinate, glycine, polysorbate 80 (Tween 80), or any combination thereof. In some embodiments, the multispecific antibody that specifically binds to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen is formulated in a buffer with a pH between 4 and 9.

[0362] Preparations for in vivo administration must be sterile. This can be readily achieved, for example, by filtration through sterile filtration membranes.

[0363] Methods for preventing or treating HBV infection

[0364] In some embodiments, the present application provides a method for preventing or treating HBV infection in an individual, comprising administering to the individual an effective amount of any multispecific antibody specifically binding to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen as described herein, or a composition comprising the same.

[0365] In some embodiments, the present application also provides the use of any of the multispecific antibodies that specifically bind to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen, or a composition comprising the same, in the preparation of a medicament for preventing or treating HBV infection.

[0366] In some embodiments, the method for treating HBV infection further provides a therapeutic or prophylactic effect for diseases and / or conditions associated with HBV infection. In certain aspects, a method for preventing HBV infection in an individual is provided, comprising administering to the individual an effective amount of a composition comprising any of the multispecific antibodies described herein or a pharmaceutical composition comprising the multispecific antibody.

[0367] Diseases and / or conditions associated with HBV infection include, but are not limited to, hepatitis B, liver failure, cirrhosis, or liver cancer. In some embodiments, the method of preventing or treating HBV infection reduces mortality caused by HBV infection.

[0368] In some embodiments, a method for preventing or treating HBV infection in an individual is provided, comprising administering to the individual an effective amount of the multispecific antibody described herein that specifically binds to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen, or a composition comprising the multispecific antibody, wherein the multispecific antibody comprises a first antigen-binding domain that specifically binds to PD-L1 antigen, a second antigen-binding domain that specifically binds to HBV preS1 antigen, and a third antigen-binding domain that specifically binds to CD3 antigen.

[0369] In some embodiments, provided is the use of a multispecific antibody that specifically binds to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen as described herein, or a composition comprising the multispecific antibody in the preparation of a medicament for preventing or treating hepatitis B virus infection, wherein the multispecific antibody comprises a first antigen-binding domain that specifically binds to PD-L1 antigen, a second antigen-binding domain that specifically binds to HBV preS1 antigen, and a third antigen-binding domain that specifically binds to CD3 antigen.

[0370] In some embodiments, a method for preventing and / or treating HBV infection in an individual is provided, comprising administering to the individual an effective amount of a multispecific antibody comprising specific binding to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen as described herein, or a composition comprising the multispecific antibody, wherein the method is more effective than administering an equivalent dose of an antibody that specifically binds to HBV preS1 antigen, an equivalent dose of an antibody that specifically binds to PD-L1 antigen, or an equivalent dose of an antibody that specifically binds to CD3 antigen.

[0371] In some embodiments, the method comprises administering an effective amount of a multispecific antibody described herein that specifically binds to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen, which enhances ADCC activity against HBV-infected cells by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold compared to administering an equivalent dose of an antibody that specifically binds to HBV preS1 antigen, an equivalent dose of an antibody that specifically binds to PD-L1 antigen, or an equivalent dose of an antibody that specifically binds to CD3 antigen.

[0372] In some embodiments, the method comprises administering an effective amount of a multispecific antibody described herein that specifically binds to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen, which enhances the neutralization activity against HBV-infected cells by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold compared to administering an equivalent dose of an antibody that specifically binds to HBV preS1 antigen, an equivalent dose of an antibody that specifically binds to PD-L1 antigen, or an equivalent dose of an antibody that specifically binds to CD3 antigen.

[0373] In some embodiments, the method comprises administering an effective amount of a multispecific antibody described herein that specifically binds to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen, which enhances ADCP activity against HBV-infected cells by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold compared to administering an equivalent dose of an antibody that specifically binds to HBV preS1 antigen, an equivalent dose of an antibody that specifically binds to PD-L1 antigen, or an equivalent dose of an antibody that specifically binds to CD3 antigen.

[0374] In some embodiments, the method comprises administering an effective amount of a multispecific antibody described herein that specifically binds to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen, and the method reduces the HBsAg content in the patient's serum by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold compared to administering an equivalent dose of an antibody that specifically binds to HBV preS1 antigen, an equivalent dose of an antibody that specifically binds to PD-L1 antigen, or an equivalent amount of an antibody that specifically binds to CD3 antigen.

[0375] Products and kits

[0376] In some embodiments of the present application, a product is provided, comprising a substance that can be used to prevent or treat HBV infection in an individual, or for delivering a multispecific antibody (e.g., a multispecific antibody that specifically binds to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen) to cells attached to pathogens expressing HBV preS1 antigen or PD-L1 antigen. The product may include a container and a label or package insert on or accompanying the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be made of a variety of materials, such as glass or plastic. Typically, the container contains a composition that is effective for treating the disease or condition described herein and has a sterile port (e.g., the container can be an intravenous infusion bag or a vial with a cap pierceable by a hypodermic needle). At least one active substance in the composition is an antibody, antigen-binding fragment, or multispecific antibody described herein. The label or package insert indicates the specific condition that the composition can be used to treat. The label or package insert further includes instructions for administering the multispecific antibody or pharmaceutical composition to a patient. Articles of manufacture and kits comprising the combination therapies described herein are contemplated.

[0377] A package insert is an instruction sheet typically included in the commercial packaging of a therapeutic product that contains information regarding the indications, usage, dosage, administration, contraindications, and / or warnings associated with the use of such therapeutic product. In some embodiments, the package insert indicates that the composition can be used to treat bacterial infections. In some embodiments, the package insert indicates that the composition can be used to treat HBV infection.

[0378] In addition, the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, or dextrose solution. Other materials desirable from a commercial and user perspective may also be included, including other buffers, diluents, filters, needles, and syringes.

[0379] Also provided are kits that can be used for various purposes, such as for preventing or treating HBV infection in an individual, or for delivering a multispecific antibody that specifically binds to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen to cells attached to pathogens expressing HBV preS1 antigen or PD-L1 antigen, optionally in combination with a product. The kits of the present application include one or more containers containing a multispecific antibody or antigen-binding fragment that specifically binds to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen, and in some embodiments, further contain another agent (e.g., an agent described herein) and / or instructions for use consistent with any of the methods described herein. The kit may further include instructions for selecting an appropriate individual for treatment. Instructions for use included with the kits herein are typically written instructions on a label or package insert (e.g., a paper sheet included with the kit), although machine-readable instructions (e.g., instructions on a magnetic or optical storage disc) are also acceptable.

[0380] For example, in some embodiments, the kit comprises a composition of a multispecific antibody that specifically binds to HBV preS1, PD-L1, and CD3. In some embodiments, the kit comprises: a) a composition comprising any of the multispecific antibodies described herein that specifically bind to HBV preS1, PD-L1, and CD3, and b) at least one other agent in an effective amount that can enhance the effect (e.g., therapeutic effect, detection effect) of the multispecific antibody that specifically binds to HBV preS1, PD-L1, and CD3. In some embodiments, the kit comprises: a) a composition comprising any of the multispecific antibodies described herein that specifically bind to HBV preS1, PD-L1, and CD3, and b) instructions for administering the multispecific antibody composition that specifically binds to HBV preS1, PD-L1, and CD3 to an individual for treating HBV infection in the individual. In some embodiments, a kit includes: a) a composition comprising any of the multispecific antibodies described herein that specifically bind to HBV preS1, PD-L1, and CD3 antigens, and b) at least one other agent in an effective amount that can enhance the efficacy (e.g., therapeutic efficacy, detection efficacy) of the multispecific antibody that specifically binds to HBV preS1, PD-L1, and CD3 antigens, and c) instructions for administering the multispecific antibody composition that specifically binds to HBV preS1, PD-L1, and CD3 antigens and the other agent to an individual for treating HBV infection in the individual. The multispecific antibody that specifically binds to HBV preS1, PD-L1, and CD3 antigens and the other agent can be present in separate containers or in the same container. For example, the kit can include one specific composition or two or more compositions, wherein one composition includes the multispecific antibody that specifically binds to HBV preS1, PD-L1, and CD3 antigens and the other composition includes the other agent.

[0381] In some embodiments, the kit comprises a nucleic acid (or a set of nucleic acids) encoding a multispecific antibody that specifically binds to HBV preS1, PD-L1, and CD3. In some embodiments, the kit comprises: a) a nucleic acid (or a set of nucleic acids) encoding a multispecific antibody that specifically binds to HBV preS1, PD-L1, and CD3, and b) a host cell that expresses the nucleic acid (or nucleic acids). In some embodiments, the kit comprises: a) a nucleic acid (or a set of nucleic acids) encoding a multispecific antibody that specifically binds to HBV preS1, PD-L1, and CD3, and b) instructions for: i) expressing the multispecific antibody that specifically binds to HBV preS1, PD-L1, and CD3 in a host cell, ii) preparing a composition comprising the multispecific antibody that specifically binds to HBV preS1, PD-L1, and CD3, and iii) administering the composition comprising the multispecific antibody that specifically binds to HBV preS1, PD-L1, and CD3 to an individual to prevent or treat HBV infection in the individual. In some embodiments, the kit comprises: a) a nucleic acid (or a set of nucleic acids) encoding a multispecific antibody that specifically binds to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen, b) a host cell that expresses the nucleic acid (or a set of nucleic acids), and c) instructions for use, suitable for: i) expressing the multispecific antibody that specifically binds to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen in the host cell, ii) preparing a composition comprising the multispecific antibody that specifically binds to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen, and iii) administering the composition comprising the multispecific antibody that specifically binds to HBV preS1 antigen, PD-L1 antigen, and CD3 antigen to an individual to prevent or treat HBV infection in the individual.

[0382] The test kits described herein are packaged in a suitable form. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed polyester film or plastic bags), etc. The test kits may optionally provide other components, such as buffers and instructional information. Therefore, the application also provides articles, which include vials (e.g., sealed vials), bottles, jars, flexible packaging, etc.

[0383] Instructions for use of a multispecific antibody composition comprising a specific binding agent for HBV preS1, PD-L1, and CD3 typically include information such as dosage, dosing cycle, and route of administration. The container may be a unit dose, a bulk package (e.g., a multi-dose package), or a subunit dose. For example, a kit is provided that contains a sufficient dose of a multispecific antibody as described herein that specifically binds to HBV preS1, PD-L1, and CD3 to provide long-term, effective treatment for an individual, e.g., for one week, eight days, nine days, ten days, eleven days, twelve days, thirteen days, two weeks, three weeks, four weeks, six weeks, eight weeks, three months, four months, five months, seven months, eight months, nine months, or longer. The kit may also contain multiple unit doses of a multispecific antibody that specifically binds to HBV preS1, PD-L1, and CD3, the pharmaceutical composition, and instructions for use, and is packaged in an amount sufficient for storage and use in a pharmacy, e.g., a hospital pharmacy or compounding pharmacy.

[0384] Those skilled in the art will recognize that several embodiments are possible within the scope and purpose of this application. The application will now be described in more detail with reference to the following non-limiting examples. The following examples further illustrate the application, but should not be construed as limiting its scope in any way. DETAILED DESCRIPTION

[0385] Example 1: Screening, preparation and characterization of full-length anti-PD-L1 antibodies

[0386] Screening and preparation of full-length PDL1 antibodies

[0387] Mice were immunized with purified human PD-L1 antigen, B cells were collected, and fused with myeloma cells to obtain hybridoma cells. A series of hybridoma antibodies were obtained through ELISA screening and PD1 / PD-L1 binding inhibition experiments, and they were sequenced. The sequenced VH&VL sequences were constructed into the human IgG1 framework region to obtain anti-human PD-L1 chimeric antibodies. Subsequently, the chimeric antibody Mab3-16 was used as a parent for humanization and back mutation to obtain a series of humanized antibodies Hum5, Hum-6 (also known as SBT451), and Hum-7, and their biological activity was assayed. The specific amino acid sequences of the above antibodies are shown in Tables 6 and 7 (numbered according to the definition of Kabat (S70) or IMGT (Hum-5, Hum-6, Hum-7)).

[0388] Binding affinity constant determination assay

[0389] The binding affinity constants of humanized anti-PD-L1 antibodies were determined using BLI technology. Briefly, the humanized anti-human PD-L1 antibodies Hum5, SBT451, and Hum-7 were captured using an anti-human Fc sensor (AHC, Fortebio) at a loading concentration of 2 μg / mL with a capture time of 300 seconds. The antibody capture sensor was used to detect different concentrations of human PD-L1 antigen (Sino biological, 10084-H08H) or cynomolgus monkey PD-L1 antigen from 50 nM to less than 1 nM with an association time of 300 seconds and a dissociation time of 1200 seconds. Kinetic data were analyzed using Data Analysis HT 10.0 software.

[0390] The results are shown in Table 13. The humanized anti-PD-L1 antibodies Hum-5, SBT451, and Hum-7 can effectively bind to the human PD-L1 antigen, among which the binding activity of SBT451 and Hum-7 antibodies is comparable to that of their parent antibody Mab3-16.

[0391] In addition, the above-mentioned humanized antibody has cross-binding activity with crab-eating macaque PD-L1, and its affinity is comparable to that for human PD-L1.

[0392] Table 13: Binding affinity constants of anti-PD-L1 antibodies to PD-L1 antigen

[0393] SEE reporter cell assay

[0394] The ability of humanized anti-PD-L1 antibodies to increase IL-2 production was tested using a Staphylococcal enterotoxin E (SEE) reporter cell assay. The Jurkat 6E-1 cell line (ATCC) is derived from human acute T-lymphoblastic leukemia. This cell line is characterized by the production of large amounts of IL-2 upon stimulation. Jurkat 6E-1 cells were transfected with the human PD-1 gene. Raji cells (purchased from Invivogen), derived from human B lymphocytes expressing human PD-L1, were used as antigen-presenting cells (APCs). When superantigens such as Staphylococcal enterotoxin E (SEE) were presented to T cells by the APCs, the anti-PD-L1 antibody's ability to enhance IL-2 production was observed.

[0395] In short, 1×10 6 / ml of Raji B cells expressing human PD-L1 were pre-incubated with 2-3ng / ml of SEE superantigen (Toxin Technology #ET404), and then an equal volume of 2×10 6Human PD-1-expressing Jurkat cells (prepared) were gently shaken to mix. 100 μl of this cell mixture and 100 μl of the humanized anti-PD-L1 antibody to be tested (1:3 serial dilution) were transferred to a 96-well U-shaped plate and incubated at 37°C with 5% CO2. The culture supernatant was collected after 48 hours. Human IL-2 levels were quantified using an IL-2 ELISA kit (Biolegend MAX Human IL-2 ELISA Kit).

[0396] The results are shown in Figures 1A-1B . Humanized anti-PD-L1 antibodies Hum-5, SBT451 ( Figure 1B ), and Hum-7 ( Figure 1A ) can effectively promote the secretion level of IL-2, and their activity is comparable to that of the parent antibody Mab 3-16 and the positive control antibody Ref (Atezolizumab, Roche).

[0397] Detection of the activity of anti-human PD-L1 antibody in promoting IL-2 secretion by PBMCs stimulated with Staphylococcal enterotoxin B

[0398] The PD-1 / PD-L1 signaling pathway inhibits TCR / CD28 co-stimulatory signals, which can be manifested as reduced production of cytokines such as IL-2 and IFN-γ. Therefore, inhibiting the PD-1 pathway by blocking the interaction between PD-1 and PD-L1 with PD-L1 antibodies can enhance T cell activation. In this experiment, after antigen-presenting cells (APCs) presented superantigens such as Staphylococcal enterotoxin B (SEB) to T cells, the ability of anti-human PD-L1 antibodies to promote IL-2 production was observed. This mechanism involves cross-linking of the T cell receptor (TCR) with MHC class II molecules, activating CD4+ T cells through the TCR / CD28 pathway.

[0399] Briefly, PBMC cells from healthy human donors were plated in 96-well U-bottom plates at 1×10 5 Cells were then added with serial dilutions of the humanized anti-PD-L1 antibody to be tested, the positive control antibody Ref, or the negative control antibody IgG1 (Biolegend, catalog #403501), along with Staphylococcal enterotoxin B (purchased from Toxin Technology) at a final concentration of 100 ng / ml. The cells were cultured at 37°C with 5% CO2 for 3 days, and the cell culture supernatant was collected. IL-2 levels in the cell culture supernatant were measured using an ELISA (Biolegend MAX Human IL-2 ELISA Kit).

[0400] The results are shown in Figure 2A. The exemplary humanized anti-PD-L1 antibody SBT451 can effectively and dose-dependently promote the secretion of cytokine IL-2 by PBMC cells, indicating that the anti-human PD-L1 antibody can effectively enhance the activity of T cells, and the activity is comparable to that of the parent antibody Mab3-16 and the positive control antibody Ref.

[0401] Other humanized antibodies Hum-5 and Hum-7 could also effectively promote PBMC cells to secrete IL-2 ( Figure 2B ).

[0402] Example 2: Screening and preparation of full-length anti-HBV pre-S1 antibodies

[0403] The sequence of the known HBV pre-S1 antibody K127-9 (see Patent Publication No. WO2023 / 066171A) was used to construct and prepare the subsequent anti-CD3-preS1-PDL1 trispecific antibody. The amino acid sequence of the K127-9 antibody is shown in Tables 2 and 3 (numbered according to the Kabat definition).

[0404] Example 3: Preparation of anti-CD3 antibodies

[0405] With reference to the disclosed contents in the prior art, the heavy chain variable regions (V ) of the anti-CD3 antibodies Bl (patent application US20030180799A1), Mo (patent CN106029696B, clone hu40G5c), Te (patent CN107074956B, clone CD3B146), and 2C11 (anti-mouse CD3e antibody (Leo et al., (1987) Proc. Natl. Acad. Sci. USA 84:1374)) were obtained by gene synthesis. H ) and light chain variable region (V L ) coding sequence for the construction and preparation of trispecific antibodies. Among them, the V of the anti-CD3 antibody used in the examples of the present application is H and V L The amino acid sequences are shown in Tables 4 and 5 (numbering according to Kabat definition). At the same time, a full-length anti-CD3 monoclonal antibody (IgG1 format) was constructed and prepared as a control.

[0406] Example 4: Preparation of trispecific antibodies with different structures that specifically bind to HBV pre-S1, PD-L1, and CD3 antigens

[0407] In the following sequence design: V H and V L Represent the heavy chain variable region and light chain variable region of the antibody, for example, K127-9V H Represents the heavy chain variable region of K127-9, MoVL Represents the light chain variable region of Mo, etc.; C H Represents the constant region of the antibody heavy chain, including C H 1. C H 2 and C H 3 domains; mC H 1 represents the constant region of the antibody heavy chain with amino acid mutations; scFv is composed of the V H and V L A single-chain antibody connected by a linker peptide; IgG1 Fc represents the Fc region of the IgG1 subclass antibody, which contains C H 2 and C H 3 domains; C L represents the light chain constant region; mC L Represents the antibody light chain constant region with amino acid mutations.

[0408] 4.1 Construction of a trispecific antibody with a CrossMab 2+1-scFv structure:

[0409] Sequence design: The CrossMab 2+1-scFv trispecific antibody structure is based on the CrossMab 2+1 bispecific antibody structure, with a scFv fragment connected to the C-terminus of one of the Fcs to form an antigen-binding module that can specifically bind to another different antigen. CrossMab 2+1-scFv is a tetravalent multispecific antibody structure composed of two monomers, one of which contains two antigen-binding domains, one of which is Fab and the other is scFv; the other monomer contains two antigen-binding domains, both of which are Fab. The CrossMab2+1-scFv trispecific antibody structure used in this example includes a total of 5 polypeptide chains, namely two heavy chains and three light chains, and the heavy chain contains an Fc domain. In the design of this structure, Hetero H, CrossMab technology was applied to ensure the correct pairing between the light and heavy chains of the antibody.

[0410] In the trispecific antibody of this structure used in the examples of this application, a knobs-in-holes (KIH) structure was designed in the Fc region, that is, a monomer C H The threonine (T) at position 366 in region 3 is replaced by tryptophan (W) to form a "knobs" structure, and the C of the other paired monomer is replaced by HIn region 3, threonine (T) at position 366 is replaced by serine (S), leucine (L) at position 368 is replaced by alanine (A), and tyrosine (Y) at position 407 is replaced by valine (V) to form a "hole" structure, which promotes heterologous heavy chain dimerization by relying on the decrease in steric hindrance after mutation and the covalent disulfide bond formed in the hinge region, wherein the numbering is according to the EU index as in Kabat; and two Cys residue mutations are introduced to form a stabilizing disulfide bridge (S354C on the "knob" side and Y349C on the "hole" side).

[0411] In addition, by H 1 and C L The introduction of amino acids with opposite charges at specific amino acid positions in the domains can increase the ratio of desired multispecific antibodies to unwanted byproducts, for example, in the constant domain C of one of the Fab molecules. L The amino acid at position 128 is replaced with arginine (R), and the amino acid at position 129 is replaced with lysine (K), respectively (numbering is according to Kabat); and wherein in the constant domain C of the corresponding Fab molecule H 1, the amino acid at position 148 is replaced by glutamic acid (E), and the amino acid at position 214 is replaced by glutamic acid (E) (numbering is in accordance with Kabat) (see patent document CN106661120B). Specifically, K127-9 V H 1-mC H C in 1 H K148E and K214E mutations were introduced into region 1, as well as K127-9 V L 1-mC L C in L In addition, by introducing E128R and Q129K mutations in V H and V L Two cysteine ​​mutations were introduced into the interface to obtain a disulfide-stabilized multispecific antibody, i.e., in the V H The G44C mutation was introduced into the V region of SBT451scFv. L In addition, the leucine (L) at position 234 and the leucine (L) at position 235 of the antibody hinge region were replaced with alanine (A), and the C HThe proline (P) at position 331 in region 2 is replaced with serine (S) to form the combined mutation LALAPS. This mutation combination can weaken the binding of antibody Fc to FcR receptors CD64, CD32A, CD16 and human complement component C1q, thereby weakening antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The numbering is in accordance with the EU index as in Kabat. A schematic diagram of the CrossMab 2+1-scFv trispecific antibody structure used in the examples of the present application is shown in Figure 3A. Table 14-1 is the composition of the anti-preS1-PDL1-CD3 trispecific antibody with the CrossMab2+1-scFv structure used in the examples, and the specific amino acid sequences of its partial heavy chain, full-length heavy chain and light chain are shown in Table 9-1 and Table 10-1, respectively.

[0412] The construction process of multispecific antibodies: According to the operating instructions, two heavy chain expression vectors (two) expressing two heavy chains and three light chain expression vectors (two of the three light chains are K127-9V L -mC L The amino acid sequences of the light chains are the same, so there are 2 light chain expression vectors in total), for a total of 4 expression vectors.

[0413] Expression and purification of trispecific antibodies: According to the operating instructions, the four expression plasmids of the above trispecific antibodies were co-transfected into HEK293F cells, and the transfected 293F cells were cultured at 37°C, 5% CO2, and 120 rpm for 6 days, and the cell culture fluid was collected separately.

[0414] The expressed antibodies were purified using protein A resin (MabCap At 4FF 5ml pre-packed column, catalog number SA023C15, Changzhou Tiandi Renhe Biotechnology Co., Ltd.). The specific operation is as follows: 6 times the column volume of PBS buffer (containing 50mM PBS and 0.15M NaCl, pH7.2) was used to balance the protein A column at a flow rate of 5ml / min. The pH of the culture supernatant was adjusted to 7.2, and the sample was loaded at room temperature at a flow rate of 5ml / min. Subsequently, the column was balanced again with 6-10 times the column volume of PBS buffer at a flow rate of 5ml / min. After complete equilibrium, elution was performed with 6 times the column volume of eluent (containing 0.1M Gly, 150mM NaCl, pH3.2), and the eluted antibodies were collected. The solution was exchanged into PBS using an ultrafiltration tube and concentrated to a certain concentration.

[0415] Molecular sieve chromatography (superdex 200pg, column volume 100ml) was then used to remove aggregate components in the antibody. Briefly, a molecular sieve chromatography column was balanced with 1 column volume of PBS buffer (containing 50mM PBS and 0.15M NaCl, pH 7.2) at a flow rate of 1ml / min. A volume of no more than 5ml of antibody sample was loaded, and the molecular sieve chromatography column was then rinsed with PBS buffer, and UV280 was detected to collect the components corresponding to the monomer peak. The solution was then ultrafiltered into PBS using an ultrafiltration tube and concentrated to a certain concentration.

[0416] Table 14-1: Composition of heavy and light chains of the trispecific antibody CrossMab2+1-scFv structure that specifically binds to HBV pre-S1, PD-L1, and CD3 antigens

[0417] 4.2 Construction of trispecific antibodies with tri-IgG-(scFv)2 structure:

[0418] Sequence design: The tri-specific antibody of tri-IgG-(scFv)2 structure is modified on the basis of IgG-(scFv)2 bispecific antibody, that is, two scFv fragments binding to different antigens are respectively connected to the Fc termini of the two heavy chains of an IgG antibody, so as to realize the trispecificity of the antibody molecule. For the tri-specific antibody of tri-IgG-(scFv)2 structure used in the examples of the present application, a knob-in-hole structure (KIH) is also designed in the Fc region, and two Cys residue mutations (S354C on the "knob" side and Y349C on the "hole" side) that form a stabilizing disulfide bridge are introduced. In addition, the combined mutation LALAPS is introduced. For details, see Section 4.1. In addition, by H and V L Two cysteine ​​mutations were introduced into the interface to obtain a disulfide-stabilized multispecific antibody, namely, in the V H The G44C mutation was introduced into the V region of SBT451scFv. L Q100C mutation was introduced into the V region of MoscFv; H The G44C mutation was introduced into the V region of MoscFv. L A Q105C mutation was introduced into the IgG2 region. A schematic diagram of the tri-specific antibody structure of tri-IgG-(scFv)2 used in the Examples of this application is shown in Figure 3B. Table 14-2 shows the composition of the tri-specific antibody structure of tri-IgG-(scFv)2 used in the Examples, and the specific amino acid sequences of its full-length heavy and light chains are shown in Table 10-2.

[0419] Construction process of trispecific antibodies: According to the operating instructions, two heavy chain expression vectors (2) expressing two heavy chains and one light chain expression vector (1) expressing the light chain were constructed by seamless cloning.

[0420] Expression and purification of trispecific antibodies: The specific operation steps are the same as described in 4.1.

[0421] Table 14-2: Composition of heavy and light chains of trispecific antibodies that specifically bind to HBV pre-S1, PD-L1, and CD3 antigens.

[0422] 4.3 Construction of trispecific antibodies with tri-IgG-scFv structure:

[0423] Sequence design: The tri-IgG-scFv trispecific antibody structure is based on the Hetero H, CrossMab bispecific antibody structure, and the Fc terminus of one heavy chain of the IgG antibody is connected to a scFv fragment that binds to another antigen to achieve trispecificity. For the tri-IgG-scFv trispecific antibody used in the examples of this application, a knob-in-hole structure (KIH) was designed in the Fc region, and two Cys residue mutations (S354C on the "knob" side and Y349C on the "hole" side) that form a stabilizing disulfide bridge were introduced. In addition, the combined mutation LALAPS was introduced. For details, see Section 4.1. In addition, by H and V L Two cysteine ​​mutations were introduced into the interface to obtain a disulfide-stabilized multispecific antibody, namely, in the V H The G44C mutation was introduced into the V region of MoscFv. L A Q105C mutation was introduced into the scFv region. A schematic diagram of this structure is shown in Figure 3C. Table 14-3 shows the composition of the tri-specific antibody with a tri-IgG-scFv structure used in the Examples. Tables 9-2 and 10-3 show the specific amino acid sequences of the partial heavy and light chains and the full-length heavy and light chains, respectively.

[0424] Construction process of trispecific antibodies: According to the operating instructions, two heavy chain expression vectors (2) expressing two heavy chains and two light chain expression vectors (2) expressing two light chains were constructed by seamless cloning.

[0425] Expression and purification of trispecific antibodies: The specific operation steps are the same as described in 4.1.

[0426] Table 14-3: Composition of heavy and light chains of trispecific antibodies that specifically bind to HBV pre-S1, PD-L1, and CD3 antigens in the tri-IgG-scFv structure

[0427] 4.4 Construction of trispecific antibodies for proof-of-concept:

[0428] Sequence design: Since the above-mentioned anti-PD-L1 antibody binding domain has no cross-binding activity with the mouse PD-L1 antigen, and the above-mentioned anti-CD3 binding domain has no cross-binding activity with the mouse CD3 antigen, in order to verify the effect of the constructed anti-CD3-preS1-PDL1 trispecific antibody in the mouse model, the present application constructed an anti-CD3-preS1-PDL1 trispecific antibody for proof of concept (POC) for in vivo activity verification of the anti-CD3-preS1-PDL1 trispecific antibody in mice. A three-antibody POC molecule with a CrossMab2+1-scFv structure was constructed according to the steps described in 4.1; a three-antibody POC molecule with an IgG-(scFv)2 structure was constructed according to the steps described in 4.2. The anti-CD3 antigen binding domain in the designed POC molecule adopts the sequence of the anti-mouse CD3e antibody 2c11 (Leo et al., (1987) Proc. Natl. Acad. Sci. USA 84: 1374), and the V H The G44C mutation was introduced into the V region of 2c11scFv. L The P100C mutation was introduced into the anti-PD-L1 antigen binding domain, which was based on the sequence of the mouse antibody S70 (refer to Clin Cancer Res (2020) 26 (15): 4154–4167), and the V H The G44C mutation was introduced into the V region of S70scFv. L Table 14-4 shows the composition of the POC trispecific antibody used in the Examples, and Tables 9-1, 10-1, and 10-2 show the specific amino acids of the heavy and light chains.

[0429] Expression and purification of POC trispecific antibodies: The specific operation steps are the same as described in 4.1.

[0430] Table 14-4: Composition of heavy and light chains of POC trispecific antibodies that specifically bind to HBV pre-S1 antigen, PD-L1 antigen, and CD3 antigen

[0431] Example 5: ELISA detection of the binding activity of trispecific antibodies to CD3E & CD3D antigens

[0432] 5.1 ELISA binding assay of trispecific antibodies

[0433] Binding of the trispecific antibodies to the human CD3E-CD3D (i.e., CD3ε-CD3δ) antigen was determined using an ELISA binding assay. Briefly, 100 μl / well of 1 μg / ml human CD3E-CD3D antigen (ACRO, #CDD-H52W1) was added to an ELISA plate (Corning #9018) and incubated overnight at 4°C. The next day, the plate was washed three times with 0.5% PBST. Next, 100 μl / well of 4% milk blocking buffer was added and incubated at room temperature for 1 hour. The plate was then washed three times with 0.5% PBST. Subsequently, 100 μl / well of a serial dilution (initial concentration 10 μg / ml, 3-fold serial dilution, a total of 11 concentrations) of the trispecific antibody to be tested or a control antibody was added and incubated at 37°C for 1 hour. The control antibody was the full-length anti-CD3 monoclonal antibody Mo-IgG1. After washing the plate six times with 0.5% PBST, 100 μl / well of a goat anti-human kappa chain AP tag secondary antibody (1:2500 dilution, SouthernBiotech, #2060-04) was added and incubated at 37°C for 1 hour. The plate was then washed six times with 0.5% PBST. Finally, PNPP (Sigma, Cat. No. 0410-01L) was added for color development at 37°C for 15 minutes. The OD405 was measured, and a binding curve was generated using PRISM software to analyze the binding affinity of the antibody to the human CD3E-CD3D antigen and calculate the EC. 50 value.

[0434] The results are shown in Figure 4A . The anti-CD3-preS1-PDL1 trispecific antibodies, including K127-9-Mo-mutCrossMab2+1-Ig1-SBT451scFv with CrossMab2+1-scFv structure, K127-9-Ig1-SBT451scFv-MoscFv with IgG-(scFv)2 structure, and K127-9-SBT451-Ig1-MoscFv with tri-IgG-scFv structure, can all effectively bind to human CD3E-CD3D antigen, but their binding activity is weaker than that of the anti-CD3 monoclonal antibody Mo-IgG1.

[0435] 5.2 ELISA Binding Assay for POC Trispecific Antibodies

[0436] The binding activity of the POC trispecific antibody to the mouse CD3E-CD3D antigen (ACRO, G162-227DF1-145) was detected by ELISA binding assay. The specific operation steps were as described in 5.1, wherein the test antibodies were diluted to an initial concentration of 20 μg / ml and then diluted 4-fold in a series of 8 concentrations.

[0437] The results are shown in Figure 4B. The POC trispecific antibodies, including the CrossMab2+1-scFv structure K127-9-2c11-mutCrossMab2+1-Ig1-S70scFv and the IgG-(scFv)2 structure K127-9-Ig1-S70scFv-2c11scFv, were able to effectively bind to mouse CD3E and CD3D antigens. Among them, the CrossMab2+1-scFv structure trispecific antibody K127-9-2c11-mutCrossMab2+1-Ig1-S70scFv had a binding activity approximately 4-fold lower than that of the anti-CD3 monoclonal antibody 2c11.

[0438] Example 6: ELISA detection of the binding activity of trispecific antibodies to HBV pre-S1 antigen

[0439] 6.1 ELISA binding assay of trispecific antibodies

[0440] The binding of the trispecific antibodies to the HBV pre-S1 antigen was detected by ELISA binding assay. The specific operation steps were as described in 5.1, wherein the antigen coated on the ELISA plate was purified HBV pre-S1-His antigen (see Patent Publication No. WO2023 / 066171A), and the control antibody was the anti-HBV pre-S1 monoclonal antibody K127-9.

[0441] The results are shown in Figure 5A. The exemplary anti-CD3-preS1-PDL1 molecules, including the CrossMab2+1-scFv structure molecule K127-9-Mo-mutCrossMab2+1-Ig1-SBT451scFv and the IgG-(scFv)2 structure molecule K127-9-Ig1-SBT451scFv-MoscFv, can effectively bind to the HBV preS1 antigen, and their binding activity is comparable to that of the anti-HBV preS1 monoclonal antibody K127-9.

[0442] The K127-9-SBT451-Ig1-MoscFv molecule with a tri-IgG-scFv structure can also effectively bind to HBV preS1 antigen.

[0443] 6.2 ELISA Binding Assay for POC Trispecific Antibodies

[0444] The binding activity of POC trispecific antibodies to HBV preS1 antigen was detected by ELISA binding assay. The specific operation steps are as described in 6.1.

[0445] The results are shown in Figure 5B . The POC trispecific antibodies, including K127-9-2c11-mutCrossMab2+1-Ig1-S70scFv with a CrossMab2+1-scFv structure and K127-9-Ig1-S70scFv-2c11scFv with an IgG-(scFv)2 structure, were able to effectively bind to the HBV preS1 antigen, and their binding activity was basically equivalent to that of the anti-HBV pre-S1 monoclonal antibody K127-9.

[0446] Example 7: ELISA detection of the binding activity of trispecific antibodies to PD-L1 antigen

[0447] 7.1 ELISA binding assay of trispecific antibodies

[0448] The binding of the trispecific antibodies to the human PD-L1 antigen was detected by ELISA binding assay. The specific steps were as described in 5.1, where the antigen coated on the ELISA plate was purified human PDL1-Fc fusion protein, and the control antibody was the anti-PD-L1 monoclonal antibody SBT451.

[0449] The results are shown in Figure 6A. The anti-CD3-preS1-PDL1 trispecific antibodies, including K127-9-Mo-mutCrossMab2+1-Ig1-SBT451scFv with CrossMab2+1-scFv structure, K127-9-Ig1-SBT451scFv-MoscFv with IgG-(scFv)2 structure, and K127-9-SBT451-Ig1-MoscFv with tri-IgG-scFv structure, can all effectively bind to human PD-L1 antigen, and their binding activity is comparable to that of the anti-PD-L1 monoclonal antibody SBT451.

[0450] 7.2 ELISA Binding Assay for POC Trispecific Antibodies

[0451] The binding activity of the POC trispecific antibody to the mouse PD-L1 antigen was detected by ELISA binding assay. The specific operation steps are as described in 7.1, wherein the antigen coated on the ELISA plate is the mPDL1-Fc antigen (PD1-M5251, ACRO), and the control antibody is the anti-PD-L1 monoclonal antibody S70-Ig1.

[0452] The results are shown in Figure 6B. The POC trispecific antibodies, including K127-9-2c11-mutCrossMab2+1-scFv structure of CrossMab2+1-scFv and K127-9-Ig1-S70scFv-2c11scFv of IgG-(scFv)2 structure, can effectively bind to mouse PD-L1 antigen.

[0453] Example 8: Biacore detection of affinity constants of trispecific antibodies for CD3 antigen

[0454] The affinity constants of the trispecific antibodies for the human CD3 antigen were determined using a Biacore T200 (GE Healthcare). Briefly, anti-human Fc was coupled to a CM5 chip using a human anti-capture kit and an amino coupling kit. The anti-CD3-preS1-PDL1 trispecific antibodies to be tested were diluted to 2 μg / ml in 1× HBS-EP buffer (pH 7.4). The capture time was 30 s and the flow rate was 30 μL / min. Fc1 was used as a reference channel without capturing the antibody, while Fc2, Fc3, and Fc4 were used for capturing the antibody. The analyte, human CD3E-CD3D, was added and allowed to bind to the ligand for 120 s and dissociate for 600 s at a flow rate of 30 μL / min. The Kon, Koff, and Kd values ​​of the trispecific antibodies are shown in Table 15.

[0455] Table 15: Affinity of trispecific antibodies specifically binding to CD3, HBV preS1 and PD-L1 antigens

[0456] Example 9: Cross-species binding activity of trispecific antibodies against cynomolgus monkey CD3 antigen

[0457] The binding activity of the anti-CD3-preS1-PDL1 trispecific antibody to be tested and the cynomolgus monkey CD3E-CD3D antigen (ACRO, CDD-C52W4) was detected by ELISA binding assay. The specific operation steps are as described in 5.1.

[0458] The results are shown in Figure 7 , and exemplary anti-CD3-preS1-PDL1 trispecific antibodies, including K127-9-Mo-mutCrossMab2+1-Ig1-SBT451scFv with a CrossMab2+1-scFv structure and K127-9-Ig1-SBT451scFv-MoscFv with an IgG-(scFv)2 structure, have species cross-reactivity with cynomolgus macaque CD3 antigen.

[0459] The tri-IgG-scFv structure of K127-9-SBT451-Ig1-MoscFv also showed species cross-reactivity with cynomolgus monkey CD3 antigen (data not shown).

[0460] Example 10: Detection of the killing activity of trispecific antibodies on PBMCs to target cells 293T-PreS1 in vitro 10.1 Detection of the killing activity of trispecific antibodies on 293T-preS1 target cells

[0461] First, 293T-preS1 target cells were constructed. Briefly, the nucleic acid sequence encoding the HBV preS1 antigen protein (GenBank: ACJ48741.1, 1-119AA) was subcloned into the pcDNA3 expression vector. This expression vector plasmid was then prepared according to established standard molecular biology methods. 293T cells were then transfected with this expression vector plasmid. Successful transfected clones were then selected and expanded to obtain 293T-preS1 target cells. These cells were then cultured in DMEM supplemented with 10% FBS, 1% PS, and 4 μg / ml blasticidin.

[0462] Then, the cell density was 1×10 5 The above 293T-preS1 target cells were plated in a 96-well plate at a density of 1×10 5 100 μl / well of effector PBMC (Shanghai Miaoshun, Cat. No. PB050C) with a concentration of 50 μl / well was added at a 2:1 effector cell:target cell ratio. After vortexing and mixing, 50 μl / well of the trispecific antibodies to be tested or the control antibody at different concentrations (initial concentration: 40 μg / ml, followed by 5-fold serial dilutions for a total of 8 concentrations) were added. The test antibodies were K127-9-Mo-mutCrossMab2+1-scFv (CrossMab2+1-Ig1-SBT451scFv), K127-9-Ig1-SBT451scFv-MoscFv (IgG-(scFv)2), and K127-9-SBT451-Ig1-MoscFv (tri-IgG-scFv). The control antibodies were the full-length anti-CD3 monoclonal antibody Mo-IgG1 and the anti-HBV preS1 monoclonal antibody K127-9. After mixing, place the cell culture plate in a CO2 incubator and incubate for 72 hours. TM The Cytotoxicity Assay Kit (Promega, #G9291) was used as described in the kit's operating instructions. Staining solution and lysis solution were added to the cell culture plate, and fluorescence was then detected. Graphpad Prism 8.0 was used to generate curves for analyzing the biological activity of the candidate trispecific antibodies.

[0463] Figure 8A shows that the trispecific antibody K127-9-Mo-mutCrossMab2+1-Ig1-SBT451scFv with a CrossMab2+1-scFv structure can effectively kill 293T-preS1 target cells, while the control antibody showed no killing activity against target cells. Other trispecific antibodies, including K127-9-Ig1-SBT451scFv-MoscFv with an IgG-(scFv)2 structure and K127-9-SBT451-Ig1-MoscFv with a tri-IgG-scFv structure, can also effectively kill 293T-preS1 target cells (data not shown). 10.2 Detection of the killing activity of POC trispecific antibodies against CT26-preS1 target cells

[0464] CT26-PreS1 target cells were obtained by transfecting CT26 cells with a PreS1 expression vector plasmid. PBMCs were prepared by lysing erythrocytes from mouse peripheral blood and cultured in 1640 medium (containing 10% FBS + 1% penicillin / streptomycin). 50 μl / well of the serially diluted POC trispecific antibody to be tested (initial concentration: 200 μg / ml, 5-fold serial dilution) was added to a 96-well plate at a density of 2×10 5 / ml mouse PBMC cells 100 μl / well, and the density was 2×10 5 50 μl / well of CT26-PreS1 target cells (100 μg / ml) were added, and the control antibodies were anti-CD3 monoclonal antibody 2c11 and anti-HBV preS1 monoclonal antibody K127-9. After thorough mixing, the cell culture plate was placed in a CO2 incubator and incubated for 72 hours. TM The Cytotoxicity Assay Kit (Promega, #G9291) was used as described in the kit's operating instructions. Staining solution and lysis solution were added to the cell culture plate, and fluorescence was then detected. Graphpad Prism 8.0 was used to generate curves for analyzing the biological activity of the POC trispecific antibody.

[0465] The results are shown in Figure 8B. The exemplary POC trispecific antibodies, including K127-9-2c11-mutCrossMab2+1-scFv structure of CrossMab2+1-Ig1-S70scFv and K127-9-Ig1-S70scFv-2c11scFv of IgG-(scFv)2 structure, were able to effectively kill CT26-preS1 target cells, while the control antibody did not show killing activity.

[0466] Example 11: Effects of trispecific antibodies in HBV transgenic mouse models

[0467] The HBV transgenic mouse (HBV-Tg) model was developed by Vital River Pharmaceuticals to evaluate the in vivo pharmacodynamic activity of the anti-CD3-preS1-PDL1 trispecific antibody in inhibiting HBV replication. The HBV-Tg preparation process is as follows: a linearized DNA fragment 1.28 times the length of the HBV (type A, GenBank: AF305422.1) genome was injected into the pronuclei of C57BL / 6NCrl mouse embryos to obtain transgenic positive mice. Peripheral blood HBV DNA copy number analysis showed that the copy number of HBV DNA was retained at 10 7 ~10 8 IU / ml transgenic founder mice. The HBV-Tg mouse strain was established by mating hemizygous wild-type C57BL / 6NCrl mice. These mice can produce complete, infectious viral particles, and the level of HBV replication is comparable to that of patients with chronic hepatitis B. In addition to high titers of HBV DNA, high levels of HBsAg and HBeAg can also be detected in the peripheral blood. Because HBV antigens are continuously expressed during the embryonic stage, inducing immune tolerance in mice, these mice do not exhibit immunopathological changes similar to those seen in human hepatitis B.

[0468] The model mice were administered intraperitoneally with the POC molecule K127-9-2c11-mutCrossMab2+1-Ig1-S70scFv, corresponding to the anti-CD3-preS1-PDL1 trispecific antibody to be tested. A control group of mice was injected with a combination of the anti-CD3-preS1 bispecific antibody K127-9-2c11-mutCrossMab2+1-Ig1 (this antibody differs from the trispecific antibody K127-9-2c11-mutCrossMab2+1-Ig1 only in that it lacks the PD-L1-binding domain, namely the S70 scFv; the remaining components are identical in structure and sequence to the trispecific antibody) and the anti-PD-L1 monoclonal antibody S70. Dosing was performed twice weekly for four consecutive weeks. Blood samples were collected on the second day after administration, and HBsAg levels in the supernatant were measured using an HBsAg ELISA kit (Mike Biotech).

[0469] The results showed that the anti-CD3-preS1-PDL1 trispecific antibody K127-9-2c11-mutCrossMab2+1-Ig1-S70scFv was able to reduce the HBsAg content in mouse serum, and its activity was better than the combination of the anti-CD3-preS1 bispecific antibody 2c11-K127-9 and the anti-PD-L1 monoclonal antibody S70 (data not shown).

Claims

1. A multispecific antibody comprising a first antigen-binding domain that specifically binds to the PD-L1 antigen, a second antigen-binding domain that specifically binds to the HBV preS1 antigen, and a third antigen-binding domain that specifically binds to the CD3 antigen, wherein: The first antigen binding domain that specifically binds to the PD-L1 antigen comprises: Heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 37, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 39; and a light chain variable region (V L ), the V L comprising: a light chain complementarity determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 40, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 41, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 42; The second antigen binding domain that specifically binds to the HBV preS1 antigen comprises: V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; And, the third antigen binding domain that specifically binds to the CD3 antigen comprises: (a)V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 11, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and V L , the V L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 20, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 23, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 26; (b)V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 18; and V L , the V L comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 27; or (c)V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 13, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and V L , the V L It comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 22, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:

28.

2. The multispecific antibody according to claim 1, wherein: The first antigen binding domain that specifically binds to the PD-L1 antigen comprises: (a)V H , comprising the amino acid sequence shown in any one of SEQ ID NOs: 43 and 73 or a variant thereof, wherein the variant has at least 80% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 43 and 73; and V L , comprising the amino acid sequence shown in any one of SEQ ID NOs: 46 and 75 or a variant thereof, wherein the variant has at least 80% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 46 and 75; (b)V H , comprising the amino acid sequence of SEQ ID NO: 43 or a variant thereof, said variant having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 43; and V L , comprising the amino acid sequence of SEQ ID NO: 45 or a variant thereof, said variant having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 45; or (c)V H , comprising the amino acid sequence of SEQ ID NO: 43 or a variant thereof, said variant having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 43; and V L , comprising the amino acid sequence of SEQ ID NO: 47 or a variant thereof, said variant having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 47; The second antigen binding domain that specifically binds to the HBV preS1 antigen comprises: V H , comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 7; and V L , comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 8; And, the third antigen binding domain that specifically binds to the CD3 antigen comprises: (a)V H , comprising the amino acid sequence of SEQ ID NO: 29 or a variant thereof, said variant having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 29; and V L , comprising the amino acid sequence of SEQ ID NO: 33 or a variant thereof, said variant having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 33; (b)V H , comprising the amino acid sequence shown in any one of SEQ ID NOs: 30 and 69 or a variant thereof, wherein the variant has at least 80% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 30 and 69; and V L , which comprises the amino acid sequence shown in any one of SEQ ID NOs: 34 and 71 or a variant thereof, wherein the variant has at least 80% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 34 and 71; or (c)V H , comprising the amino acid sequence of SEQ ID NO: 31 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 31; and V L , comprising the amino acid sequence of SEQ ID NO: 35 or a variant thereof, wherein the variant has at least 80% sequence identity with the amino acid sequence of SEQ ID NO:

35.

3. The multispecific antibody according to any one of claims 1-2, wherein the structure thereof is selected from CrossMab2+1-scFv, tri-IgG-(scFv)2 or tri-IgG-scFv.

4. The multispecific antibody according to any one of claims 1 to 3, which has a CrossMab2+1-scFv structure and comprises five polypeptide chains: in, A polypeptide chain from N-terminus to C-terminus contains V H 2-C H 1-C H 2-C H 3-LV H 1-L3-V L 1 structure, where V H 2 is the heavy chain variable region that specifically binds to HBV preS1 antigen; V H 1 is the heavy chain variable region that specifically binds to the PD-L1 antigen; V L 1 is the light chain variable region that specifically binds to the PD-L1 antigen; L and L3 are connecting peptides; C H 1 is the heavy chain constant region C H 1 domain; C H 2 is the heavy chain constant region C H 2 domains; C H 3 is the heavy chain constant region C H 3 domains; Both polypeptide chains contain V from N-terminus to C-terminus L 2-C L , where V L 2 is the light chain variable region that specifically binds to HBV preS1 antigen, C L is the light chain constant region; A polypeptide chain from N-terminus to C-terminus contains V H 2-C H 1-V H 3-C L , where V H 2 is the heavy chain variable region that specifically binds to the HBV preS1 antigen; V H 3 is the heavy chain variable region that specifically binds to the CD3 antigen; C H 1 is the heavy chain constant region C H 1 domain; C L is a light chain constant region; preferably, the polypeptide chain further comprises an Fc comprising a C H 2 and C H 3 domains; and A polypeptide chain from N-terminus to C-terminus contains V L 3-C H 1, where V L 3 is the light chain variable region that specifically binds to the CD3 antigen, C H 1 is the heavy chain constant region C H 1 domain.

5. The multispecific antibody according to claim 4, comprising: The amino acid sequence of SEQ ID NO: 55 or a variant thereof, wherein the variant has 80% sequence identity with the amino acid sequence of SEQ ID NO: 55; and / or The amino acid sequence of SEQ ID NO: 56 or a variant thereof, said variant having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 56; and / or The amino acid sequence of SEQ ID NO: 57 or a variant thereof, said variant having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 57; and / or The amino acid sequence of SEQ ID NO: 58 or a variant thereof, wherein the variant has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:

58.

6. The multispecific antibody according to claim 4, comprising: The amino acid sequence of SEQ ID NO: 55 or a variant thereof, wherein the variant has 80% sequence identity with the amino acid sequence of SEQ ID NO: 55; and / or The amino acid sequence of SEQ ID NO: 56 or a variant thereof, said variant having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 56; and / or The amino acid sequence of SEQ ID NO: 62 or a variant thereof, said variant having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 62; and / or The amino acid sequence of SEQ ID NO: 58 or a variant thereof, wherein the variant has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:

58.

7. The multispecific antibody according to any one of claims 1 to 3, which has tri-IgG-(scFv)2, comprising four polypeptide chains: in, A polypeptide chain from N-terminus to C-terminus contains V H 2-C H 1-C H 2-C H 3-LV H 1-L3-V L 1 structure, where V H 2 is the heavy chain variable region that specifically binds to HBV preS1 antigen; V H 1 is the heavy chain variable region that specifically binds to the PD-L1 antigen; V L 1 is the light chain variable region that specifically binds to the PD-L1 antigen; L and L3 are connecting peptides; C H 1 is the heavy chain constant region C H 1 domain; C H 2 is the heavy chain constant region C H 2 domains; C H 3 is the heavy chain constant region C H 3 domains; Both polypeptide chains contain V from N-terminus to C-terminus L 2-C L structure, where V L 2 is the light chain variable region that specifically binds to HBV preS1 antigen, C L is the light chain constant region; and A polypeptide chain from N-terminus to C-terminus contains V H 2-C H 1-C H 2-C H 3-LV H 3-L3-V L 3 structure, in which V H 2 is the heavy chain variable region that specifically binds to HBV preS1 antigen; V H 3 is the heavy chain variable region that specifically binds to the CD3 antigen; V L 3 is the light chain variable region that specifically binds to the CD3 antigen; L and L3 are connecting peptides; C H 1 is the heavy chain constant region C H 1 domain; C H 2 is the heavy chain constant region C H 2 domains; C H 3 is the heavy chain constant region C H 3 domains.

8. The multispecific antibody according to claim 7, comprising: The amino acid sequence of SEQ ID NO: 64 or a variant thereof, wherein the variant has 80% sequence identity with the amino acid sequence of SEQ ID NO: 64; and / or The amino acid sequence of SEQ ID NO: 65 or a variant thereof, said variant having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 65; and / or The amino acid sequence of SEQ ID NO: 66 or a variant thereof, wherein the variant has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:

66.

9. The multispecific antibody according to any one of claims 1 to 3, which has a tri-IgG-scFv structure and comprises four polypeptide chains: in, A polypeptide chain from N-terminus to C-terminus contains V H 2-C H 1-C H 2-C H 3-LV H 3-L3-V L 3 structure, in which V H 2 is the heavy chain variable region that specifically binds to HBV preS1 antigen; V H 3 is the heavy chain variable region that specifically binds to the CD3 antigen; V L 3 is the light chain variable region that specifically binds to the CD3 antigen; L and L3 are connecting peptides; C H 1 is the heavy chain constant region C H 1 domain; C H 2 is the heavy chain constant region C H 2 domains; C H 3 is the heavy chain constant region C H 3 domains; A polypeptide chain from N-terminus to C-terminus contains V L 2-C L structure, where V L 2 is the light chain variable region that specifically binds to HBV preS1 antigen, C L is the light chain constant region; A polypeptide chain from N-terminus to C-terminus contains V H 1-C L structure, where V H 1 is the light chain variable region that specifically binds to the PD-L1 antigen, C L is a light chain constant region; preferably, the polypeptide chain further comprises an Fc comprising a C H 2 and C H 3 domains; and A polypeptide chain from N-terminus to C-terminus contains V L 1-C H 1 structure, where V L 1 is the light chain variable region that specifically binds to the PD-L1 antigen, C L is the light chain constant region.

10. The multispecific antibody according to claim 9, comprising: The amino acid sequence of SEQ ID NO: 77 or a variant thereof, said variant having 80% sequence identity with the amino acid sequence of SEQ ID NO: 77; and / or The amino acid sequence of SEQ ID NO: 66 or a variant thereof, said variant having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 66; and / or The amino acid sequence of SEQ ID NO: 80 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 80; and / or The amino acid sequence of SEQ ID NO: 79 or a variant thereof, wherein the variant has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:

79.

11. The multispecific antibody according to claim 9, comprising: The amino acid sequence of SEQ ID NO: 77 or a variant thereof, said variant having 80% sequence identity with the amino acid sequence of SEQ ID NO: 77; and / or The amino acid sequence of SEQ ID NO: 66 or a variant thereof, said variant having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 66; and / or The amino acid sequence of SEQ ID NO: 78 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 78; and / or The amino acid sequence of SEQ ID NO: 79 or a variant thereof, wherein the variant has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:

79.

12. An isolated nucleic acid molecule encoding the multispecific antibody of any one of claims 1-11.

13. A vector comprising the nucleic acid molecule of claim 12.

14. An isolated host cell comprising the multispecific antibody of any one of claims 1-11, the nucleic acid molecule of claim 12, or the vector of claim 13.

15. A method for preparing the multispecific antibody according to any one of claims 1 to 11, comprising: a) culturing the host cell of claim 14 under conditions effective to express the antibody; and b) obtaining the expressed antibody from the host cell.

16. A pharmaceutical composition comprising the multispecific antibody of any one of claims 1 to 11, the nucleic acid molecule of claim 12, the vector of claim 13, the isolated host cell of claim 14, or the multispecific antibody produced by the method of claim 15, and a pharmaceutically acceptable carrier or excipient.

17. Use of the multispecific antibody that specifically binds to PD-L1 antigen, HBV preS1 antigen, and CD3 antigen according to any one of claims 1 to 11, the nucleic acid molecule according to claim 12, the vector according to claim 13, the host cell according to claim 14, the multispecific antibody produced by the method according to claim 15, or the pharmaceutical composition according to claim 16 in the preparation of a medicament for treating a disease or condition in an individual in need thereof.

18. The use according to claim 17, wherein the disease or condition comprises HBV infection or a disease associated with HBV infection.

19. The use according to claim 18, wherein the disease or condition comprises hepatitis B, liver failure, cirrhosis or liver cancer.

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