Bispecific antibodies against HBV surface antigen (HBsAg) and CD3 and uses thereof

Abstract

Provided are bispecific antibodies capable of specifically targeting both HBV surface antigen (HBsAg) and CD3 protein, and pharmaceutical compositions comprising them. In particular, provided are nucleic acid molecules encoding the bispecific antibodies or fragments thereof, and vectors comprising the nucleic acid molecules. Also provided are uses of the bispecific antibodies or fragments thereof, the nucleic acid molecules, and the vectors in the preparation of medicaments for preventing or treating HBV infection and other related diseases.

Description

[Technical Field]

[0001] The present invention relates to bispecific homodimeric antibodies and their use in the treatment of hepatitis B virus infection and associated hepatocellular carcinoma. [Background technology]

[0002] According to WHO data, over 400 million people worldwide are infected with hepatitis B virus, representing 3.5% of the total population, with the highest prevalence rates of approximately 6% in the Western Pacific and African regions. Acute HBV infection can develop into chronic hepatitis B virus (CHB), which has a poor prognosis (90% of newborns and 5% of adult patients, respectively), resulting in cirrhosis, liver failure, and hepatocellular carcinoma (HCC), causing 6 million deaths each year.

[0003] Current clinical management of CHB patients includes antiviral drugs such as tenofovir and entecavir, which suppress viral replication but do not eliminate HBV covalently closed circular DNA (cccDNA)-harboring hepatocytes, potentially leading to relapse of HBV infection when medication is discontinued. While clearance of HBV infection has been reported to be associated with sustained viral control by effector T cells, the progression of chronic infection is thought to result from limited virus-specific T cell responses. Therefore, there is an urgent need to develop novel immunotherapeutic approaches for CHB that restore HBV-specific T cell-mediated responses to virally infected hepatocytes.

[0004] A T cell redirecting bispecific antibody refers to a molecule that contains more than two binding domains, where one domain specifically binds to a cell surface antigen (e.g., tumor-associated / pathogenic-specific) on a target cell / tissue, and the second domain of the molecule specifically binds to a T cell antigen (e.g., CD3). This dual-target binding molecule can redirect T cells to the target cell / tissue, leading to elimination of the target cell.

[0005] CD3 refers to a human antigen expressed on T cells as part of the TCR / CD3 complex, which contains either TCR alpha / beta or TCR gamma / delta heterodimers coexpressed on the cell surface with the invariant subunits of CD3 labeled gamma, delta, epsilon, and zeta. Human CD3ε is described in UniProt P07766 (CD3E_HUMAN). An anti-CD3ε antibody described in the state of the art is SP34 (Yang SJ, The Journal of Immunology (1986) 137:1097-1100). SP34 is available from Pharmigen. Another anti-CD3 antibody described in the state of the art is UCHT-1 (see WO 2000041474). A further anti-CD3 antibody described in the state of the art is BC-3 (Fred Hutchison Cancer Research Institute; used in a phase I / II clinical trial for GvHD, Anasetti et al., Transplantation 54:844 (1992)). SP34 differs from UCHT-1 and BC3 in that SP34 recognizes an epitope present only on the ε chain of CD3 (see Salmeron et al., 1991), whereas UCHT-1 and BC-3 recognize epitopes contributed by both the ε and δ chains. The sequence of an antibody having the same sequence as that of antibody SP34 is mentioned in International Publication No. 2008119565, International Publication No. 2008119566, International Publication No. 2008119567, International Publication No. 2010037836, International Publication No. 2010037837, and International Publication No. 2010037838. A sequence that is 96% identical to the VH of antibody SP34 is mentioned in U.S. Patent No. 8,236,308 (International Publication No. 2007042261). However, for many existing CD3 antibodies, including SP34, there are problems of significant adverse reactions and poor druggability.

[0006] HBsAg refers to the envelope antigen of hepatitis B virus (HBV) displayed on the surface of virus-infected cells. HBV S / L / M surface proteins are small, medium, and large surface proteins, which are transcribed and translated from a single open frame and differ from each other in their N-terminal portions. Thus, large surface antigens contain portions not present in either medium or small surface antigens, while medium surface antigens contain portions present in large antigens but not small antigens. Small antigens consist of sequences contained in the C-terminal portions of both medium and large antigens. It is presumed that this occurs despite the virus being released into intracellular vesicles because a large number of HBV surface proteins (HBsAg) remain integrated into the intracellular membrane of the endoplasmic reticulum. During the vesicular transport process, these intracellular membranes may fuse with the cellular membrane, resulting in the presentation of HBV surface proteins on the surface of infected cells. Therefore, HBsAg provides an ideal target for virus-directed immunotherapeutic intervention to treat CHB. Summary of the Invention

[0007] The present application provides a structural form suitable for an anti-HBsAg / CD3 bispecific antibody, in which one antibody is in the form of an IgG and the other antibody is in the form of an scFv or VHH nanobody. Preferably, the CD3 antibody is in the form of an IgG1 structural form, and the HBsAg antibody is in the form of an scFv and is fused to the N-terminus or C-terminus of the CD3 antibody heavy chain. On the other hand, the present application also provides a CD3 antibody or antigen-binding fragment thereof with significantly reduced affinity for CD3, and a bispecific or multispecific antigen-binding molecule comprising the CD3 antibody or antigen-binding fragment thereof.

[0008] A first aspect of the present invention is a T cell redirecting bispecific homomeric molecule anti-HBsAg x anti-CD3, comprising: (a) a first polypeptide chain comprising a heavy chain of an anti-CD3 antibody (anti-CD3 VH-CH1-Fc) fused to a single-chain fragment (scFv) that binds to a hepatitis B virus surface antigen (HBsAg), wherein the HBsAg comprises a small surface antigen, a hepatitis B virus middle surface antigen, or a hepatitis B virus large surface antigen; (b) a second polypeptide chain comprising the light chain of an anti-human CD3 antibody (anti-CD3 VL-CL); The present invention provides a bispecific antibody comprising:

[0009] In some embodiments, the HBsAg scFv is fused to the N-terminus or C-terminus of the heavy chain of the anti-CD3 antibody, preferably to the C-terminus of the heavy chain of the anti-CD3 antibody. In some embodiments, the HBsAg scFv is fused to the N-terminus or C-terminus of the heavy chain of the anti-CD3 antibody via a glycine-serine linker. In some embodiments, the glycine-serine linker is (GGGGS) of SEQ ID NO: 33. n , SEQ ID NO: 34 (GGGSG) n , SEQ ID NO: 35 (GSGGG) n , SEQ ID NO: 36 (GSGGGP) n , SEQ ID NO: 37 (GSEPS) n , SEQ ID NO: 38 (GGEGGGP) n , SEQ ID NO: 39 (GGEGGGSEGGGS) n , SEQ ID NO: 40 (GGGSGGGG) n or combinations thereof (wherein n=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0010] In some embodiments, the HBsAg scFv comprises HCDRs 1 to 3 and LCDRs 1 to 3 as set forth in SEQ ID NOs: 1 to 6, respectively.

[0011] In some embodiments, the CD3 antibody comprises HCDRs 1 to 3 and LCDRs 1 to 3 as set forth in SEQ ID NOs: 11 to 16, respectively.

[0012] In some embodiments, the HBsAg scFv comprises a VH binding domain and a VL binding domain, wherein the VH comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:7 and the VL comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:8.

[0013] In some embodiments, the VH and VL domains are linked by a "YOL" linker, preferably the "YOL" linker comprises the amino acid sequence of SEQ ID NO:9.

[0014] In some embodiments, the HBsAg scFv comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:10.

[0015] In some embodiments, the CD3 antibody fraction of the HBsAg / CD3 bispecific antibody is a humanized antibody with significantly reduced affinity for CD3. In some embodiments, the humanized anti-human CD3 antibody comprises a VH domain and a VL domain, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. In some embodiments, the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 29.

[0016] In some preferred embodiments, the amino acid sequence of the VL domain of the CD3 antibody is as set forth in SEQ ID NO: 25. In some other preferred embodiments, the amino acid sequence of the VH domain of the CD3 antibody comprises SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, and the amino acid sequence of the VL domain of the CD3 antibody is as set forth in SEQ ID NO: 25 or has at least 90% sequence identity thereto.

[0017] In some embodiments, the bispecific antibody comprises a heavy chain constant region and a conventional variant thereof selected from the IgG1, IgG2, IgG3, or IgG4 subtype, and a light chain constant region and a conventional variant thereof selected from the kappa or lambda subtype. Preferably, the constant region belongs to the IgG1-kappa isotype. In some embodiments, the bispecific antibody further comprises an alanine at positions 234 and 235 of the heavy chain, where residue numbering is according to the EU Index.

[0018] In some embodiments, the bispecific antibody comprises a first polypeptide chain and a second polypeptide chain, wherein (a) the first polypeptide chain comprises, from N-terminal to C-terminal direction: i. domain I comprising three subdomains, wherein subdomain IA comprising a variable heavy region (VH) binds to an epitope of a hepatitis B virus small surface antigen, a hepatitis B virus middle surface antigen, or a hepatitis B virus large surface antigen as set forth in SEQ ID NO:7; subdomain IB comprises a "YOL" linker sequence as set forth in SEQ ID NO:9; and subdomain IC comprising a variable light region (VL) binds to an epitope of a hepatitis B virus small surface antigen, a hepatitis B virus middle surface antigen, or a hepatitis B virus large surface antigen as set forth in SEQ ID NO:8. (b) The second polypeptide chain comprises, from the N-terminus to the C-terminus, the following: (i) Domain I, which binds to an epitope of a hepatitis B virus small surface antigen, a hepatitis B virus middle surface antigen, or a hepatitis B virus large surface antigen; (ii) Domain II, which comprises a glycine-serine linker sequence as set forth in any one of SEQ ID NOs: 33 to 40, and has a copy number n of 1 to 10; (iii) Domain III, whose variable heavy region (VH) binds to an epitope of a human CD3 sequence as set forth in any one of SEQ ID NOs: 19 to 24; and (iv) Domain IV, which is a constant region of a heavy chain sequence as set forth in SEQ ID NO: 30 or SEQ ID NO: 31. (b) The second polypeptide chain comprises, from the N-terminus to the C-terminus: (i) Domain I, whose variable light region (VL) binds to an epitope of a human CD3 sequence as set forth in any one of SEQ ID NOs: 25 to 29; and (ii) Domain II, which is a constant region of a light chain sequence as set forth in SEQ ID NO: 32.

[0019] In some embodiments, the bispecific antibody comprises a first polypeptide chain and a second polypeptide chain, wherein (a) the first polypeptide chain comprises, from N-terminus to C-terminus: i. Domain I, a variable heavy region (VH) that binds to an epitope of a human CD3 sequence such as that set forth in any one of SEQ ID NOs: 19 to 24; ii. Domain II, a constant region of the heavy chain sequence such as that set forth in SEQ ID NO: 30 or SEQ ID NO: 31; iii. Domain III, comprising a glycine-serine linker sequence such as that set forth in any one of SEQ ID NOs: 33 to 40, wherein the copy number of Domain III is n=1 to 10; (iv) Domain IV comprising three subdomains, wherein subdomain IVA comprising a variable heavy region (VH) binds to an epitope of the hepatitis B virus small surface antigen, the hepatitis B virus middle surface antigen, or the hepatitis B virus large surface antigen as set forth in SEQ ID NO: 7, subdomain IVB comprises a "YOL" linker sequence as set forth in SEQ ID NO: 9, and subdomain IVC comprising a variable light region (VL) binds to an epitope of the hepatitis B virus small surface antigen, the hepatitis B virus middle surface antigen, or the hepatitis B virus large surface antigen as set forth in SEQ ID NO: 8. (b) The second polypeptide chain comprises, from N-terminal to C-terminal, i. domain I, wherein the variable light region (VL) binds to an epitope of the human CD3 sequence as set forth in any one of SEQ ID NOs: 25 to 29; ii. domain II, a constant region of the light chain sequence as set forth in SEQ ID NO: 32.

[0020] In some embodiments, the bispecific antibody comprises a first polypeptide chain and a second polypeptide chain, wherein (a) the first polypeptide chain comprises, from N-terminus to C-terminus: i. Domain I comprising a single-chain Fv region (scFv) as set forth in SEQ ID NO: 10, wherein the scFv fragment binds to an epitope of a hepatitis B virus small surface antigen, a hepatitis B virus middle surface antigen, or a hepatitis B virus large surface antigen; ii. Domain II comprised of a glycine-serine linker sequence as set forth in any one of SEQ ID NOs: 33 to 40, wherein the copy number of Domain II is n = 1 to 10; iii. Domain III, a variable heavy region (VH), which binds to an epitope of a human CD3 sequence as set forth in any one of SEQ ID NOs: 19 to 24; and iv. Domain IV of the constant region of the heavy chain sequence as set forth in SEQ ID NO: 30 or SEQ ID NO: 31. (b) The second polypeptide chain comprises, from the N-terminus to the C-terminus, the following: i. a variable light region (VL) comprising Domain I, which binds to an epitope of a human CD3ε sequence as set forth in any one of SEQ ID NOs: 25 to 29; and ii. a constant region domain II of a light chain sequence as set forth in SEQ ID NO: 32.

[0021] In some embodiments, the bispecific antibody comprises a first polypeptide chain and a second polypeptide chain, wherein (a) the first polypeptide chain comprises, from N-terminus to C-terminus: i. Domain I, a variable heavy region (VH) that binds to an epitope of a human CD3 sequence such as that set forth in any one of SEQ ID NOs: 19 to 24; ii. Domain II, a constant region of the heavy chain sequence such as that set forth in SEQ ID NO: 30 or SEQ ID NO: 31; iii. Domain III, comprising a glycine-serine linker sequence such as that set forth in any one of SEQ ID NOs: 33 to 40, wherein the copy number of Domain III is n = 1 to 10; and iv. Domain IV, comprising a single-chain Fv region (scFv) as set forth in SEQ ID NO: 10, wherein the scFv fragment binds to an epitope of a hepatitis B virus small surface antigen, a hepatitis B virus middle surface antigen, or a hepatitis B virus large surface antigen. and (b) a second polypeptide chain comprising, from N-terminal to C-terminal, the following: i. a variable light region (VL) comprising Domain I that binds to an epitope of a human CD3 sequence as set forth in any one of SEQ ID NOs: 25-29; ii. a constant region of a light chain sequence as set forth in SEQ ID NO: 32, comprising Domain II.

[0022] In some embodiments, the glycine-serine linker sequence is as set forth in SEQ ID NO: 31, with copy number n='4'.

[0023] In some embodiments, the first polypeptide chain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 41 to 52, and the second polypeptide chain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 53 to 57.

[0024] In some embodiments, the first polypeptide chain comprises the amino acid sequence as set forth in any one of SEQ ID NOs: 41 to 46, and the second polypeptide chain comprises the amino acid sequence as set forth in SEQ ID NO: 53. In an exemplary embodiment, the bispecific antibody is BsAb2-5-011.

[0025] In some embodiments, the first polypeptide chain comprises the amino acid sequence as set forth in any one of SEQ ID NOs: 41 to 46, and the second polypeptide chain comprises the amino acid sequence as set forth in SEQ ID NO: 54. In an exemplary embodiment, the bispecific antibody is BsAb2-5-030.

[0026] In some embodiments, the first polypeptide chain comprises the amino acid sequence as set forth in any one of SEQ ID NOs: 47 to 52, and the second polypeptide chain comprises the amino acid sequence as set forth in SEQ ID NO: 54. In an exemplary embodiment, the bispecific antibody is BsAb2-6-017.

[0027] In some embodiments, the first polypeptide chain comprises the amino acid sequence as set forth in any one of SEQ ID NOs: 47 to 52, and the second polypeptide chain comprises the amino acid sequence as set forth in SEQ ID NO: 53. In exemplary embodiments, the bispecific antibodies are BsAb2-6-001, BsAb2-6-006, BsAb2-6-011, and BsAb2-6-016.

[0028] The present application further provides nucleic acid molecules encoding the above-described bispecific antibodies.

[0029] The present application further provides an expression vector comprising a nucleic acid molecule as described above.

[0030] The present application further provides a pharmaceutical composition comprising the bispecific antibody, the nucleic acid molecule, or the vector and a pharmaceutically acceptable excipient, diluent, or carrier.

[0031] The present application further provides use of the bispecific antibody, the nucleic acid molecule, the vector, the host cell, or the pharmaceutical composition in the preparation of a medicament for preventing or treating HBV infection and other related diseases. In some embodiments, the disease caused by HBV infection is hepatitis, liver fibrosis, cirrhosis, or liver cancer.

[0032] The present application further provides a CD3 antibody or an antigen-binding fragment thereof. Surprisingly, the CD3 antibody of the present application has significantly reduced binding affinity to CD3, and it has been found that preparation of a bispecific antigen-binding molecule using the CD3 antibody of the present application can overcome the drawbacks of the prior art, such as cytokine storm, T cell exhaustion, significant side effects, and a narrow dosing window induced by excessive T cell activation due to the high affinity of the CD3 antibody.

[0033] In some embodiments, a CD3 antibody or antigen-binding fragment thereof comprising a VH-binding domain and a VL-binding domain, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24; and / or A CD3 antibody or antigen-binding fragment thereof, wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29.

[0034] In some embodiments, a CD3 antibody or antigen-binding fragment thereof (the amino acid sequence of the VH domain is as set forth in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, and the amino acid sequence of the VL domain is as set forth in SEQ ID NO: 25).

[0035] The present application further provides bispecific or multispecific antigen-binding molecules comprising a CD3 antibody or antigen-binding fragment as described above.

[0036] In some embodiments, the bispecific antigen-binding molecule comprises a second antigen-binding domain in addition to the CD3 antibody or antigen-binding fragment thereof.

[0037] In some embodiments, the second antigen-binding domain comprises a virus-associated antigen-binding domain or a tumor antigen-binding domain.

[0038] In some embodiments, the second antigen-binding domain comprises an antibody or fragment thereof capable of binding to the second antigen, a TCR or soluble fragment thereof, a receptor or receptor extracellular domain corresponding to any antigen, a ligand or ligand extracellular domain, and "derivatives" and "analogs" of the above domains.

[0039] In some embodiments, the second antigen-binding domain is a hepatitis B surface antigen-binding domain, and the hepatitis B surface antigen comprises a hepatitis B virus small surface antigen, a hepatitis B virus middle surface antigen, or a hepatitis B virus large surface antigen.

[0040] In some embodiments, the hepatitis B surface antigen binding domain is an anti-hepatitis B surface antigen single chain antibody (HBsAg scFv).

[0041] The present application further provides nucleic acid molecules encoding the CD3 antibody or antigen-binding fragment. Nucleic acid molecules encoding bispecific or multispecific antigen-binding molecules comprising the CD3 antibody or antigen-binding fragment as described above.

[0042] Beneficial effects of the present application over the prior art: The present application constructs a bispecific antibody format that specifically binds to CD3 and HBsAg, with an HBsAg scFv fused to the C-terminus of the heavy chain of the CD3 antibody (in the form of IgG), which has the properties of simultaneously targeting HBV-infected target cells and T cells, cross-linking T cells to the target cells, enhancing target cell killing, and enhancing neutralization of HBV virus in the blood. The bispecific antibody of the present application can eradicate cells carrying HBV cccDNA. The bispecific antibody of the present application can effectively treat HBV-induced infection and liver cancer by killing multiple target cells and the HBV virus in the blood. Meanwhile, the bispecific antibody of the present application is structurally stable, easy to express and purify, and has good pharmacological applicability.

[0043] On the other hand, the present application provides a CD3 antibody or antigen-binding fragment thereof with significantly reduced affinity for CD3. The preparation of bispecific and multispecific antigen-binding molecules comprising a CD3 antibody or antigen-binding fragment thereof can overcome the drawbacks of the prior art, such as cytokine storm, T cell exhaustion, significant side effects, and a narrow dosing window induced by excessive transitional T cell activation due to the high activity of CD3 antibodies.

[0044] It will be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention.

[0045] The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are incorporated herein by reference in their entirety. [Brief explanation of the drawings]

[0046] [Figure 1A] Figure 1 shows useful schematic diagrams of four alternative representations of the bispecific anti-HBsAg / anti-CD3 antibodies of the invention. Note that the IgG and scFv domains can be swapped. (A) and (B) each comprise a single-chain fragment (scFv) that binds to the hepatitis B virus surface antigen (HBsAg) fused to the N / C terminus of the heavy chain of an anti-CD3 IgG antibody, and (C) and (D) each comprise a single-chain fragment (scFv) that binds to the human CD3 antigen fused to the N / C terminus of the heavy chain of an anti-HBsAg IgG antibody. [Figure 1B] Figure 1 shows useful schematic diagrams of four alternative representations of the bispecific anti-HBsAg / anti-CD3 antibodies of the invention. Note that the IgG and scFv domains can be swapped. (A) and (B) each comprise a single-chain fragment (scFv) that binds to the hepatitis B virus surface antigen (HBsAg) fused to the N / C terminus of the heavy chain of an anti-CD3 IgG antibody, and (C) and (D) each comprise a single-chain fragment (scFv) that binds to the human CD3 antigen fused to the N / C terminus of the heavy chain of an anti-HBsAg IgG antibody. [Figure 1C]Figure 1 shows useful schematic diagrams of four alternative representations of the bispecific anti-HBsAg / anti-CD3 antibodies of the invention. Note that the IgG and scFv domains can be swapped. (A) and (B) each comprise a single-chain fragment (scFv) that binds to the hepatitis B virus surface antigen (HBsAg) fused to the N / C terminus of the heavy chain of an anti-CD3 IgG antibody, and (C) and (D) each comprise a single-chain fragment (scFv) that binds to the human CD3 antigen fused to the N / C terminus of the heavy chain of an anti-HBsAg IgG antibody. [Figure 1D] Figure 1 shows useful schematic diagrams of four alternative representations of the bispecific anti-HBsAg / anti-CD3 antibodies of the invention. Note that the IgG and scFv domains can be swapped. (A) and (B) each comprise a single-chain fragment (scFv) that binds to the hepatitis B virus surface antigen (HBsAg) fused to the N / C terminus of the heavy chain of an anti-CD3 IgG antibody, and (C) and (D) each comprise a single-chain fragment (scFv) that binds to the human CD3 antigen fused to the N / C terminus of the heavy chain of an anti-HBsAg IgG antibody. [Figure 2A] Figure 1 shows HbsAg-binding ELISA of anti-HbsAg / anti-CD3 bispecific antibodies: BsAb2-5 (A), (B) and bsAb2-6 (C), (D), respectively, bind to HbsAg. [Figure 2B] Figure 1 shows HbsAg-binding ELISA of anti-HbsAg / anti-CD3 bispecific antibodies: BsAb2-5 (A), (B) and bsAb2-6 (C), (D), respectively, bind to HbsAg. [Figure 2C] Figure 1 shows HbsAg-binding ELISA of anti-HbsAg / anti-CD3 bispecific antibodies: BsAb2-5 (A), (B) and bsAb2-6 (C), (D), respectively, bind to HbsAg. [Figure 2D] Figure 1 shows HbsAg-binding ELISA of anti-HbsAg / anti-CD3 bispecific antibodies: BsAb2-5 (A), (B) and bsAb2-6 (C), (D), respectively, bind to HbsAg. [Figure 3A]Figure 1 shows human CD3ε binding ELISA of anti-HbsAg / anti-CD3 bispecific antibodies: BsAb2-5 (A), (B) and bsAb2-6 (C), (D), respectively, bind to human CD3ε. [Figure 3B] Figure 1 shows human CD3ε binding ELISA of anti-HbsAg / anti-CD3 bispecific antibodies: BsAb2-5 (A), (B) and bsAb2-6 (C), (D), respectively, bind to human CD3ε. [Figure 3C] Figure 1 shows human CD3ε binding ELISA of anti-HbsAg / anti-CD3 bispecific antibodies: BsAb2-5 (A), (B) and bsAb2-6 (C), (D), respectively, bind to human CD3ε. [Figure 3D] Figure 1 shows human CD3ε binding ELISA of anti-HbsAg / anti-CD3 bispecific antibodies: BsAb2-5 (A), (B) and bsAb2-6 (C), (D), respectively, bind to human CD3ε. [Figure 4A] Figure 1 shows rhesus CD3ε binding ELISA of anti-HbsAg / anti-CD3 bispecific antibodies: BsAb2-5 (A), (B) and bsAb2-6 (C), (D), respectively, bind to rhesus CD3ε. [Figure 4B] Figure 1 shows rhesus CD3ε binding ELISA of anti-HbsAg / anti-CD3 bispecific antibodies: BsAb2-5 (A), (B) and bsAb2-6 (C), (D), respectively, bind to rhesus CD3ε. [Figure 4C] Figure 1 shows rhesus CD3ε binding ELISA of anti-HbsAg / anti-CD3 bispecific antibodies: BsAb2-5 (A), (B) and bsAb2-6 (C), (D), respectively, bind to rhesus CD3ε. [Figure 4D] Figure 1 shows rhesus CD3ε binding ELISA of anti-HbsAg / anti-CD3 bispecific antibodies: BsAb2-5 (A), (B) and bsAb2-6 (C), (D), respectively, bind to rhesus CD3ε. [Figure 5A]Figure 1 shows the dose-dependent binding of anti-HbsAg / anti-CD3 bispecific antibodies to CD3- and HBsAg-positive cells, respectively. Mean fluorescence intensity for bsAb2-5 (A) and bsAb2-6 (B) binding to CD3+ T cells, and for bsAb2-6 (C) binding to HepG2-LMS-660 cells. [Figure 5B] Figure 1 shows the dose-dependent binding of anti-HbsAg / anti-CD3 bispecific antibodies to CD3- and HBsAg-positive cells, respectively. Mean fluorescence intensity for bsAb2-5 (A) and bsAb2-6 (B) binding to CD3+ T cells, and for bsAb2-6 (C) binding to HepG2-LMS-660 cells. [Figure 5C] Figure 1 shows the dose-dependent binding of anti-HbsAg / anti-CD3 bispecific antibodies to CD3- and HBsAg-positive cells, respectively. Mean fluorescence intensity for bsAb2-5 (A) and bsAb2-6 (B) binding to CD3+ T cells, and for bsAb2-6 (C) binding to HepG2-LMS-660 cells. [Figure 6A] Figure 1 shows dose-dependent NFAT reporter gene induction by anti-HBsAg / anti-CD3 bispecific antibodies. BsAb2-5 (A) and bsAb2-6 (B)-induced T cell activation was determined by NFAT reporter gene, with the non-HBsAg-coated group serving as a negative control. [Figure 6B] Figure 1 shows dose-dependent NFAT reporter gene induction by anti-HBsAg / anti-CD3 bispecific antibodies. BsAb2-5 (A) and bsAb2-6 (B)-induced T cell activation was determined by NFAT reporter gene, with the non-HBsAg-coated group serving as a negative control. [Figure 7A] Figure 1 shows dose-dependent T cell proliferation induced by anti-HBsAg / anti-CD3 bispecific antibodies. BsAb2-5 (A) and bsAb2-6 (B)-induced T cell proliferation was determined by CSFE-labeled flow cytometry analysis. [Figure 7B]Figure 1 shows dose-dependent T cell proliferation induced by anti-HBsAg / anti-CD3 bispecific antibodies. BsAb2-5 (A) and bsAb2-6 (B)-induced T cell proliferation was determined by CSFE-labeled flow cytometry analysis. [Figure 8] Figure 1 shows the cytokine profile of human PBMCs stimulated with anti-HBsAg / anti-CD3 bispecific antibodies. Cytokine levels in the supernatant of bsAb2-6-stimulated human PBMCs were measured using the huTh1 / Th2 CBA Kit II (BD, 551809). (A) IFN-γ, (B) TNF-α, (C) IL-10, (D) IL-6, (E) IL-4, and (F) IL-2. [Figure 9] Anti-HBsAg / anti-CD3 bispecific antibodies redirected T cell cytotoxicity against HBs cell lines. HBs HepAD38 cells were incubated with bsAb2-6-001, bsAb2-6-006, bsAb 2-6-011, bsAb2-6-016, bsAb2-6-017, or bsAb2-5-011, and activated T cells were used as effector cells (E:T = 3:1). [Figure 10] FIG. 1 shows the binding affinity of anti-HBsAg / anti-CD3 bispecific antibodies and recombinant human CD3ε as measured by surface plasmon resonance (SPR). [Figure 11] FIG. 1 shows the binding affinity of anti-HBsAg / anti-CD3 bispecific antibodies and cynomolgus CD3ε as measured by surface plasmon resonance (SPR). [Figure 12] FIG. 1 shows the binding affinity of anti-HBsAg / anti-CD3 bispecific antibodies and HBsAg as measured by surface plasmon resonance (SPR). [Figure 13] FIG. 1 shows SEC-HPLC characterization of anti-HBsAg / anti-CD3 bispecific antibodies. [Figure 14]Figure 1 shows the in vitro HBV neutralization efficacy of anti-HBsAg / anti-CD3 bispecific antibodies in Huh7-NTCP cells. Newly produced HBeAg in the supernatant is measured by HBeAg ELISA kit as a marker of successful HBV infection. [Figure 15A] Figure 1 shows the anti-tumor activity of anti-HBsAg / anti-CD3 bispecific antibodies in the HBs+HepAD38 CDX model. (A) Mean tumor volume in mice treated with vehicle, control antibody, or bsAb2-6-011 at the indicated doses. Error bars represent SEM. (B) Characterization of human leukocytes in HepAD38 xenograft-bearing mice after treatment with bsAb2-6-011 or control antibody, stained for human CD45 and determined by flow cytometry at endpoint. (C) Mean body weight of mice treated with vehicle, control antibody, or bsAb2-6-011 at the indicated doses. Error bars represent SEM. [Figure 15B] Figure 1 shows the anti-tumor activity of anti-HBsAg / anti-CD3 bispecific antibodies in the HBs+HepAD38 CDX model. (A) Mean tumor volume in mice treated with vehicle, control antibody, or bsAb2-6-011 at the indicated doses. Error bars represent SEM. (B) Characterization of human leukocytes in HepAD38 xenograft-bearing mice after treatment with bsAb2-6-011 or control antibody, stained for human CD45 and determined by flow cytometry at endpoint. (C) Mean body weight of mice treated with vehicle, control antibody, or bsAb2-6-011 at the indicated doses. Error bars represent SEM. [Figure 15C]Figure 1 shows the anti-tumor activity of anti-HBsAg / anti-CD3 bispecific antibodies in the HBs+HepAD38 CDX model. (A) Mean tumor volume in mice treated with vehicle, control antibody, or bsAb2-6-011 at the indicated doses. Error bars represent SEM. (B) Characterization of human leukocytes in HepAD38 xenograft-bearing mice after treatment with bsAb2-6-011 or control antibody, stained for human CD45 and determined by flow cytometry at endpoint. (C) Mean body weight of mice treated with vehicle, control antibody, or bsAb2-6-011 at the indicated doses. Error bars represent SEM. [Figure 16A] Figure 1 shows the pharmacokinetic profile of anti-HBsAg / anti-CD3 bispecific antibody in cynomolgus monkeys. (A) Pharmacokinetic profile of bsAb2-6-011 in blood samples before and at various time points after administration. (B) Body weight change in 5 mpk bsAb2-6-011 treated cynomolgus monkeys. [Figure 16B] Figure 1 shows the pharmacokinetic profile of anti-HBsAg / anti-CD3 bispecific antibody in cynomolgus monkeys. (A) Pharmacokinetic profile of bsAb2-6-011 in blood samples before and at various time points after administration. (B) Body weight change in 5 mpk bsAb2-6-011 treated cynomolgus monkeys. DETAILED DESCRIPTION OF THE INVENTION

[0047] definition In order that the present invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0048] The term "bispecific antigen-binding molecule" in this application refers to a molecule capable of binding two different antigens or epitopes, comprising two different antigen-binding domains that are functionally linked or coupled by chemical coupling, genetic fusion, non-covalent bonding, or other means. The two different antigen-binding domains are selected from an antibody or a fragment thereof, a TCR or a soluble fragment thereof, a receptor or receptor extracellular domain corresponding to any antigen, a ligand or ligand extracellular domain, and "derivatives" and "analogs" of the above domains.

[0049] The term "antibody", as used in the present invention, encompasses not only complete antibodies, but also fragments, polypeptide sequences, and derivatives and analogs thereof that have antigen-binding activity.

[0050] The term antigen-binding fragment refers to one or more portions of a full-length antibody, which portions retain the ability to bind an antigen (e.g., HER2) in competition with the intact antibody for specific binding to the antigen. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some embodiments, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), and chimeric antibodies containing at least a portion of an antibody sufficient to confer peptide-specific antigen-binding ability. Antigen-binding fragments of antibodies (e.g., the antibody fragments described above) may be obtained from a given antibody (e.g., monoclonal antibody 2E12) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and antigen-binding fragments of antibodies may be screened for specificity in the same manner as intact antibodies. The term "Fd fragment" refers to an antibody fragment comprising the VH and CH1 structural domains; the term "Fv fragment" refers to an antibody fragment comprising the VL and VH structural domains of a single arm of an antibody; the term "dAb fragment" refers to an antibody fragment comprising the VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" refers to an antibody fragment consisting of the VL, VH, CL, and CH1 structural domains of an antibody; and "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region.

[0051] The terms "derivative" and "analog" refer to polypeptides that maintain substantially the same biological function or activity as an antibody, a TCR or soluble fragment thereof, an antigen receptor or receptor extracellular domain, or an antigen ligand or ligand extracellular domain. A derivative or analog of the present invention can be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) have been substituted, where such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substitution at one or more amino acid residues, or (iii) a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence, a signal peptide, a sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). Based on the teachings herein, these derivatives and analogs are within the scope of those well known to those skilled in the art.

[0052] In this application, "virus-associated antigens" refer to antigens associated with various known viruses, including DNA viruses, RNA viruses, and protein viruses (e.g., prions) classified by genetic material; euviruses and subviruses (viroids, viroids, prions) classified by viral structure; phages (bacterial viruses), plant viruses (e.g., tobacco mosaic virus), and animal viruses (e.g., avian influenza virus, smallpox virus, HIV, etc.) classified by host type; and morphologically classified spherical viruses, baculoviruses, brick-shaped viruses, coronaviruses (e.g., SARS-CoV-2), filoviridae, rotaviruses, enveloped spherical viruses, and viruses with spherical heads. In this application, "virus-associated antigens" may be antigens presented by the virus itself or antigens formed by the virus after infection of cells.

[0053] As used herein, "hepatitis B virus" is used interchangeably with the term "HBV" and refers to a well-known non-cytopathic hepatotropic DNA virus belonging to the Hepadnaviridae family.

[0054] The HBV genome is a partially double-stranded circular DNA with overlapping reading frames. There are four size-based transcripts (referred to herein as "genes" or "open reading frames") encoded by the HBV genome. These contain open reading frames designated C, X, P, and S. The core protein is encoded by gene C (HBcAg). Hepatitis B e antigen (HBeAg) is produced by proteolytic processing of the pre-core (pre-C) protein. DNA polymerase is encoded by gene P. Gene S encodes the surface antigen (HBsAg). The HBsAg gene is one long open reading frame containing three in-frame "start" (ATG) codons that give rise to three differently sized polypeptides designated large, middle, and small S antigens: pre-S1 + pre-S2 + S, pre-S2 + S, or S. Surface antigens not only decorate the HBV envelope but are also part of subviral particles that are produced in great excess compared to virion particles and play a role in immune tolerance and sequestration of anti-HBsAg antibodies, thereby allowing infectious particles to escape immune detection.

[0055] In this application, "tumor antigen" refers to an antigenic substance that appears or is overexpressed during the process of tumor formation and development. Tumor antigens are classified into tumor-specific antigens and tumor-associated antigens according to their specificity.

[0056] Tumor-specific antigens (TSAs) are new antigens that are unique to tumor cells or that exist only in certain tumor cells and do not exist in normal cells. These antigens are also known as tumor-specific transplantation antigens (TSTAs) or tumor rejection antigens (TRAs) because they are identified by tumor transplantation between animals of the same strain. Chemically or physically induced tumor antigens, spontaneous tumor antigens, and virus-induced tumor antigens are the main categories.

[0057] Tumor-associated antigens (TAAs) are antigens that are not specific to tumor cells and are present in normal cells and other tissues, but their levels increase significantly when the cells become cancerous. These antigens only show quantitative changes without strict tumor specificity. For example, embryonic antigens are typical examples.

[0058] CD3ε has the same meaning as CD3 epsilon in this application.

[0059] The term "fusion," when used in reference to a polypeptide or polynucleotide, refers to a form of the polypeptide or polynucleotide that does not exist in its natural state, a non-limiting example of which may be achieved by combining polynucleotides or polypeptides that do not normally occur together.

[0060] The term "cross-reactivity" refers to the ability of the antibodies described herein to bind to antigens from various species. For example, an antibody described herein that binds to human CD3 can also bind to CD3 from other species (e.g., cynomolgus monkey CD3). Cross-reactivity can be measured by detecting specific reactivity with purified antigens in binding assays (e.g., SPR, ELISA), or by detecting binding to cells that physiologically express the antigen or interaction with the function of cells that physiologically express the antigen. Examples of assays known in the art for determining binding affinity include surface plasmon resonance (e.g., Biacore) or similar techniques (e.g., Kinexa or Octet).

[0061] The invention is illustrated by the following non-limiting examples.

[0062] In the following examples, experimental methods for which specific conditions are not listed are usually in accordance with conventional conditions, such as those in Molecular Cloning: Laboratory Manual by Sambrook et al. (Molecular Cloning-A Laboratory Manual), or methods recommended by manufacturers. Percentages and parts are by weight unless otherwise specified. Unless otherwise specified, reagents and materials contained herein can be commercially obtained or prepared by those skilled in the art according to general knowledge. Any methods and materials similar or equivalent to those described can be used in this application. The preferred methods and materials described herein are merely illustrative and do not limit the content of this application. [Example]

[0063] Preparation of anti-HBsAg / anti-CD3 bispecific antibody Humanization of anti-CD3 mAb The sequences of the CDR regions of the mouse anti-CD3 mAb were analyzed using the Kabat system. The results showed that the amino acid sequences of HCDR1 to 3 of the mouse anti-CD3 mAb were as shown in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, respectively, the VH sequence of the mouse anti-CD3 mAb was as shown in SEQ ID NO:17, the amino acid sequences of LCDR1 to 3 of the mouse anti-CD3 mAb were as shown in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively, and the VL sequence of the mouse anti-CD3 mAb was as shown in SEQ ID NO:18.

[0064] To improve the humanization of murine anti-CD3 mAb by chimerizing framework regions of human immunoglobulin heavy and light chain variable regions.

[0065] Comparison of the protein sequences of the mouse anti-CD3 mAb heavy chain variable regions with known human germline immunoglobulin heavy chain protein sequences revealed that the mouse anti-CD3 mAb heavy chain variable regions were identical to those of the human germline IGHV3-73. * 01 and IGHV3-23 * 05. They were confirmed to have 79% and 74% homology with 05. They can be used as chimeric receptors (acceptors) for the mouse anti-CD3 mAb heavy chain CDR.

[0066] Comparison of the protein sequences of the mouse-derived anti-CD3 mAb light chain variable regions with known human germline immunoglobulin light chain protein sequences confirmed that the mouse anti-CD3 mAb light chain variable regions are 60.0% and 57% homologous to the human germline IGLV7-43*01 and IGLV8-61*01, respectively, and can be used as chimeric receptors (acceptors) for the mouse anti-CD3 mAb light chain CDRs.

[0067] To improve the affinity of the murine anti-CD3 mAb antibody, back mutations can be made to several key amino acids in the framework regions of the chimeric heavy and light chains, respectively. SEQ ID NO: 19 (humanized CD3 mAb VH1), SEQ ID NO: 20 (humanized CD3 mAb VH2), SEQ ID NO: 21 (humanized CD3 mAb VH3), SEQ ID NO: 22 (humanized CD3 mAb VH4), SEQ ID NO: 23 (humanized CD3 mAb VH5), and SEQ ID NO: 24 (humanized CD3 mAb VH6) are the amino acid sequences of the heavy chain variable regions of humanized anti-CD3 mAbs, and SEQ ID NO: 25 (humanized CD3 mAb VL1), SEQ ID NO: 26 (humanized CD3 mAb VL2), SEQ ID NO: 27 (humanized CD3 mAb VL3), SEQ ID NO: 28 (humanized CD3 mAb VL4), and SEQ ID NO: 29 (humanized CD3 mAb VL5) are the amino acid sequences of the light chain variable regions of humanized anti-CD3 mAbs.

[0068] Cloning and production of anti-HBsAg / anti-CD3 bispecific antibodies The DNA sequence encoding the bispecific anti-HBsAg / anti-CD3 antibody was synthesized by Genewiz and subcloned into the pcDNA3.1 vector via the restriction enzyme sites XbaI and NheI, respectively. The amino acid sequence encoded by the DNA is shown in Table 1.

[0069] The bispecific antibodies of the BsAb2-5 series are anti-HBsAg antibody C8 scFv (SEQ ID NO: 10) fused to the N-terminus of the heavy chain of a mouse anti-CD3 mAb (bispecific antibody No. BsAb2-5) or a humanized anti-CD3 mAb (bispecific antibody Nos. BsAb2-5-001 to BsAb2-5-030). The bispecific antibodies of the BsAb2-6 series are anti-HBsAg antibody C8 scFv (SEQ ID NO: 10) fused via a linker to the C-terminus of the heavy chain of a mouse anti-CD3 Ab (bispecific antibody No. BsAb2-6) or a humanized anti-CD3 mAb (bispecific antibody Nos. BsAb2-6-001 to BsAb2-6-030). Specifically, the corresponding light and heavy chain sequences of each bispecific antibody, as well as the combination of humanized VH and VL of the anti-CD3 antibody, are shown in Table 1. For example, in BsAb2-5-001, the heavy chain is arranged from N-terminus to C-terminus as C8 scFv, VH1-CH1-Fc, and the light chain is VL1-CL; finally, two identical heavy and light chains form a homodimer through disulfide bonds; C8 scFv (SEQ ID NO: 10) is a single-chain antibody against HBsAg, and VH1 and VL1 are heavy chain variable region variant 1 and light chain variable region variant 1 of humanized mouse-derived anti-CD3 mAb, respectively. In BsAb2-6-012, the heavy chain is arranged from N-terminus to C-terminus in the following order: VH3-CH1-Fc, and C8 scFv; the light chain is VL2-CL; C8 scFv (SEQ ID NO: 10) is a single-chain antibody against HBsAg; VH3 and VL2 are heavy chain variable region variant 3 and light chain variable region variant 2, respectively, of a humanized mouse-derived antibody anti-CD3 mAb.

[0070] [Table 1]

[0071] Expression in Expi293 cells Bispecific antibodies are expressed by transient cotransfection of the respective expression plasmids (e.g., encoding the heavy and modified heavy chains, and the corresponding light and modified light chains, in a 1:1 ratio) using the Expi293 system (Invitrogen, #A14635CN) according to the manufacturer's instructions. Briefly, Expi293 cells growing in suspension in serum-free Expi293 expression medium in shake flasks or stirred fermenters are transfected with a mixture of the two expression plasmids and Expi293fectin. For 250 mL shake flasks, Expi293 cells are seeded at a density of 1E6 cells / mL in 100 mL and incubated at 125 rpm and 8% CO2. The next day, cells are transfected at a cell density of 2.5E6 cells / mL with a mixture of: A) 5 mL of Opti-MEM with 100 µg of total plasmid DNA (1 µg / mL, 50 µg of heavy chain plasmid and the corresponding light chain in an equimolar ratio) and B) 5 mL of Opti-MEM with 260 µL of Expi293fectin (2.6 µL / mL). Enhancers 1 and 2 are added the day after transfection according to the manufacturer's instructions. The supernatant containing the secreted antibody is harvested after 5 days, and the antibody is either purified directly from the supernatant or frozen and stored.

[0072] purification Proteins were purified from filtered cell culture supernatants according to standard protocols. Briefly, supernatants of BsAb2-5 and BsAb2-6 constructs were subjected to one-step Protein A affinity chromatography (equilibration buffer: 20 mM sodium citrate, 20 mM sodium phosphate, pH 7.5; elution buffer: 20 mM sodium citrate, pH 3.0). Elution was achieved at pH 3.0, followed by immediate pH neutralization of the sample. Aggregated proteins were separated from monomeric antibody by size-exclusion chromatography (Superdex200, GE Healthcare) in PBS or 20 mM histidine, 50 mM NaCl, pH 5.5. Monomeric molecular fractions were pooled, concentrated (if necessary) using a MILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrator, and frozen and stored at -20°C or -80°C. A portion of the sample may be submitted for subsequent protein analysis characterization, for example by SDS-PAGE or size exclusion chromatography (SEC) or mass spectrometry.

[0073] Protein determination The concentration of the purified antibody is determined by optical density (OD) at 280 nm using the molar extinction coefficient calculated based on the amino acid sequence according to Pace et al., Protein Science, 1995, 4, 2411-1423. [Example]

[0074] Binding characteristics of anti-HBsAg / anti-CD3 bispecific antibodies The anti-HBsAg / anti-CD3 bispecific antibodies produced in Example 1 were analyzed by standard ELISA techniques for their binding properties to HBsAg, human CD3ε, and rhesus CD3ε recombinant proteins, respectively. Figures 2A-2D and Table 2 show that the humanized anti-CD3 mAb-derived bsAb2-5 and bsAb2-6 constructs had strong binding to recombinant HBsAg.

[0075] [Table 2]

[0076] The binding characteristics of the BsAb2-5 and BsAb2-6 constructs to human and rhesus CD3ε are shown in Figures 3, 4 and Table 2. Compared to other molecules, the resulting bispecific antibodies combined with the VL1 of bsAb2-5-001, bsAb2-5-006, bsAb2-5-011, bsAb2-5-016, bsAb2-5-021, bsAb2-5-026, bsAb2-6-001, bsAb2-6-006, bsAb2-6-011, bsAb2-6-016, bsAb2-6-021, and bsAb2-6-026 molecules showed a significantly reduced ability to bind to human and rhesus CD3ε recombinant proteins. On the other hand, the resulting bispecific antibodies in combination with VL4 of bsAb2-5-004, bsAb2-5-009, bsAb2-5-014, bsAb2-5-019, bsAb2-5-024, bsAb2-5-029, bsAb2-6-004, bsAb2-6-009, bsAb2-6-014, bsAb2-6-019, BsAb2-6-024, and bsAb2-6-029 molecules did not show cross-binding to human and rhesus CD3ε.

[0077] The anti-HBsAg / anti-CD3 T cell bispecific antibodies produced in Example 1 were analyzed by flow cytometry for their binding properties to CD3 expressed on the human leukemia T cell line Jurkat and HBsAg on the surface of the human hepatocellular carcinoma cell line HepG2-LMS-660. Specifically, the two target cells were harvested, counted, and adjusted to 2E6 cells per mL in FACS buffer (PBS with 2% BSA). 50 μL of the cell suspension was further aliquoted per well into a U-bottom 96-well plate, along with 50 μL of anti-HBsAg / anti-CD3 bispecific antibody or a negative control human IgG isotype diluted in FACS buffer, resulting in final concentrations ranging from 300 nM to 0.017 nM. After 60 minutes of incubation at room temperature, the cells were centrifuged (3 minutes, 300 × g) and washed twice with 150 μL of FACS buffer per well. The cells were then resuspended in 100 μL of 1:5000 diluted APC-labeled anti-human Fc secondary antibody (Jackson, #615-605-214) and incubated at room temperature for 30 minutes, followed by three washes with 150 μL / well of FACS buffer. The cells were finally collected by centrifugation, resuspended in 100 μL of FACS buffer, and subjected to flow cytometry (Beckman), and data were processed using FlowJo and GraphPad Prism.

[0078] Flow cytometry analysis of the binding profiles of BsAb2-5 and BsAb2-6 constructs to CD3ε expressed on the surface of Jurkat cells is shown in Figures 5A and 5B. All tested bsAb2-6 constructs were able to bind to CD3ε on the surface of Jurkat cells. Clones bsAb2-6, bsAb2-6-017, bsAb2-6-018, and bsAb2-6-020 exhibited the best binding profiles of all tested variants, while bispecific antibodies obtained by combining the VL1 of BsAb2-6-001, 006, 011, 016, 021, and 026 exhibited dramatically reduced binding profiles compared to bsAb2-6-017. Similarly, bsAb2-5-006, bsAb2-5-011, and bsAb2-5-021 showed significantly less binding to hCD3ε than the parent bsAb2-5. This result was consistent with the binding characteristics with the hCD3ε recombinant protein in Table 2.

[0079] Figure 5C shows the binding characteristics of BsAb2-5 and BsAb2-6 constructs to HBsAg on the surface of HepG2-LMS-660 cells. All tested BsAb2-5 and BsAb2-6 constructs were able to bind to HBsAg on the surface of HepG2-LMS-660 cells with similar binding affinities. The results showed that the binding affinity to HBsAg was unchanged between BsAb2-5 and BsAb2-6 constructs. [Example]

[0080] T cell activation induced by anti-HBsAg / anti-CD3 bispecific antibodies. NFAT reporter (luc) Jurkat cells purchased from BPS Bioscience stably express the firefly luciferase gene under the control of an NFAT response element integrated into the Jurkat cells. These cells can be used to evaluate the function of anti-HBsAg / anti-CD3 bispecific antibodies, along with nonspecific activation of the NFAT signaling pathway. Specifically, NFAT reporter-luc cells were thawed and grown in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1 mg / mL geneticin. For high-throughput screening, 25,000 Jurkat cells per well were seeded in 20 μL of complete RPMI-1640 medium into a clear-bottom white 384-well plate pre-coated with 2 μg / mL HBsAg. A 3-fold dilution of anti-HBsAg / anti-CD3 bispecific antibodies in 10 μL of RPMI-1640 medium was added, resulting in final concentrations ranging from 27 nM to 0.032 nM. To ensure that the anti-HBsAg / anti-CD3 bispecific antibody induces the NFAT reporter gene strictly cross-linked with HBsAg, wells containing 27 nM anti-HBsAg / anti-CD3 but not pre-coated with HBsAg serve as negative controls. After 6 hours of incubation at 37°C and 5% CO2, 15 μL of OneGlo luciferase substrate (Promega #E6120) is added to each well. Luminescence is measured after 5 minutes of incubation on a TECAN INFINITE M PLEX plate reader, and the readout data is processed using GraphPad Prism.

[0081] Figures 6A and 6B show that the anti-HBsAg / anti-CD3 bispecific antibodies induced T cell activation only in the presence of HBsAg antigen. BsAb2-6-017 had the most potent ability to activate T cells, while the VL1 combination-derived BsAb2-6-001, BsAb2-6-006, BsAb2-6-011, BsAb2-6-016, BsAb2-6-021, and BsAb2-6-026 T cells had much weaker activation abilities and their EC 50Values varied from 5-fold to 10-fold. Similarly, the parental bsAb 2-5 had a potency on T cell activation comparable to that of BsAb 2-6-017, whereas the structurally identical BsAbs 2-5-001, 2-5-006, 2-5-011, 2-5-016, 2-5-021, and 2-5-026 failed to induce T cell activation. [Example]

[0082] T cell proliferation induced by anti-HBsAg / anti-CD3 bispecific antibody The anti-HBsAg / anti-CD3 bispecific antibodies produced in Example 1 were analyzed by flow cytometry for their ability to induce proliferation of human primary T cells in the presence of HBsAg. Specifically, U-bottom 96-well plates were pre-coated overnight at 4°C with 2 μg / mL of HBsAg recombinant protein before cell seeding. Frozen human PBMCs were quickly thawed in a 37°C water bath, then transferred to pre-warmed RPMI-1640 medium at a density of 1E6 cells / mL and stained with 1 μM CFSE for 5 minutes at room temperature, protected from light. The staining reaction was then stopped by adding an equal volume of RPMI-1640 medium supplemented with 10% FBS, and the cells were further washed once with pre-warmed complete RPMI-1640 medium to remove free CFSE. The CFSE-labeled total PBMCs were then suspended in complete RPMI-1640 medium at a density of 2E6 cells / mL. Meanwhile, the 2 μg / mL HBsAg coating solution was removed from the 96-well plate, followed by three PBS washes. 50 μL of cell suspension was added to each well along with a 5-fold dilution of anti-HBsAg / anti-CD3 bispecific antibody in 50 μL of RPMI-1640 medium, resulting in a final concentration of 50 nM to 0.016 nM. To exclude nonspecific T cell proliferation induced by the anti-HBsAg / anti-CD3 bispecific antibody without HBsAg, a negative control well was prepared containing 50 nM anti-HBsAg / anti-CD3 and CFSE-labeled PBMCs without HBsAg coating. After 3 days of incubation at 37°C and 5% CO2, the cells were collected by centrifugation (5 min, 300 × g) and washed twice with 150 μL of FACS wash buffer per well. Total cells were analyzed by flow cytometry (Beckman) and FlowJo software. The percent level of total T cell proliferation is determined by all CFSE dilution peaks.

[0083] Figures 7A-7C show T cell proliferation induced by anti-HBsAg / anti-CD3 bispecific antibodies in the presence of HBsAg antigen. BsAb2-6-017 induced the most potent T cell proliferation, while the VL1 combination-derived BsAb2-6-001, BsAb2-6-006, BsAb2-6-011, BsAb2-6-016, BsAb2-6-021, and BsAb2-6-026 showed weaker induction of T cell proliferation. Similarly, the parental bsAb2-5 showed comparable potency to that of BsAb2-6-017 in T cell proliferation, while the structurally identical BsAb2-5-001, BsAb2-5-006, BsAb2-5-011, and BsAb2-5-021 failed to induce T cell proliferation. [Example]

[0084] Cytokine profile of T cells induced by anti-HBsAg / anti-CD3 bispecific antibody The anti-HBsAg / anti-CD3 T cell bispecific antibodies produced in Example 1 were analyzed for their efficacy in inducing cytokine secretion in human primary T cells with or without HBsAg. Specifically, U-bottom 96-well plates were pre-coated with 2 μg / mL of HBsAg recombinant protein overnight at 4°C before cell seeding. Frozen human PBMCs were quickly thawed in a 37°C water bath and then transferred to pre-warmed RPMI-1640 complete medium at a density of 1 million cells per mL. After three PBS washes, 100 μL of PBMCs were seeded onto 2 μg / mL HBsAg-coated 96-well plates with the addition of 5-fold diluted anti-HBsAg / anti-CD3 bispecific antibodies in 10 μL of RPMI-1640 medium, resulting in final concentrations of 50 nM to 0.016 nM. To exclude nonspecific cytokine secretion induced by the anti-HBsAg / anti-CD3 bispecific antibody without HBsAg, wells containing 50 nM anti-HBsAg / anti-CD3 and primary PBMCs but without HBsAg coating served as negative controls. After 3 days of incubation at 37°C and 5% CO2, the supernatants were collected by centrifugation (20 min, 3000 × g) and transferred to a new 96-well plate for CBA analysis according to the manufacturer's instructions (BD, #551809). IFN-γ, TNF-α, IL-2, IL-4, IL-6, and IL-10 produced by human Th1 / Th2 cells were determined, and the readout data were processed using GraphPad Prism.

[0085] Figures 8A-8F show the successful induction of T cell activation by anti-HBsAg / anti-CD3 bispecific antibodies in the presence of HBsAg antigen, and therefore the secretion of Th1 / Th2 class cytokines (IFN-γ, IL-2, IL-4, IL-6, IL-10, TNF-α). BsAb2-6-017 induced stronger T cell activation accompanied by greater cytokine secretion, while the VL1 combinations BsAb2-6-001, BsAb2-6-011, and BsAb2-6-016 induced less T cell activation accompanied by less cytokine secretion. Under the same conditions, BsAb2-5-001 failed to induce T cells to secrete the relevant cytokines. [Example]

[0086] T cell cytotoxicity induced by anti-HBsAg / anti-CD3 bispecific antibodies The anti-HBsAg / anti-CD3 bispecific antibodies produced in Example 1 were analyzed for their potency in inducing cytotoxic T cell-mediated lysis of the HBsAg-positive HepAD38 cell line in response to HBsAg cross-linking. Specifically, E-Plate 96 plates were coated with collagen (1:30 dilution) at 37°C for 30 minutes, followed by addition of M10 complete medium and then subjected to xCelligence Real Time Cell Analyzer (RTCA) to set the baseline of the assay program. HepAD38 cells were harvested and cultured at 1 x 10 5 Resuspend target cells (1 x 10) in M10 complete medium at a density of 1 x 10 cells / mL. 4 100 μL / well of 100 cells / well was added to an E-Plate 96 and incubated for 5 minutes. The cell growth curve was continuously monitored overnight by RTCA. The next day, human peripheral blood total T cells induced with 400 IU / mL of human IL-2 for 7 days were collected as effector cells at a cell density of 3 × 10 5The concentration was adjusted to 100 cells / mL. Meanwhile, a 3-fold dilution of anti-HBsAg / anti-CD3 bispecific antibody (concentration range: 300 nM to 1.23 nM) in M10 complete medium was prepared. 50 μL of effector T cells (effector cell:target cell ratio = 3:1) were mixed with 50 μL of gradient-diluted anti-HBsAg / anti-CD3 bispecific antibody and added to HepAD38 culture wells. To detect nonspecific killing of HepAD38 cells by effector T cells, an additional group containing effector T cells and HepAD38 cells but without antibody was used as a negative control. After 72 hours of continuous monitoring, cell growth curves (plots) were exported.

[0087] Figure 9 shows that exemplary anti-HBsAg / anti-CD3 bispecific antibodies induced T cells to kill HBsAg-positive hepatocellular carcinoma cells, HepAD38. The results showed that HBsAg / CD3 bispecific antibodies exerted differential cytolytic effects on HepAD38 cells in a dose-dependent manner. bsAb2-6-017 induced the most potent T cell lytic effect on HepAD38 cells at a dose of 33 nM. bsAb2-6-011 and bsAb2-6-016 stimulated T cell killing at a dose of 300 nM, whereas bsAb2-6-001 and bsAb2-6-006 also showed incomplete killing under the same conditions. However, bsAb2-5-001 failed to induce T cell killing of HepAD38 cells under the same conditions. [Example]

[0088] Surface plasmon resonance (SPR) characterization of an anti-HBsAg / anti-CD3 bispecific antibody The binding affinities of the anti-HBsAg / anti-CD3 constructs to HBsAg and human / cynomolgus CD3ε were characterized by SPR using a Biacore T800 (GE Healthcare). Specifically, the anti-HBsAg / anti-CD3 bispecific antibody was immobilized on a Protein A chip at a flow rate of 10 μL / min, and a gradient dilution of huCD3ε, cynoCD3ε, or HBsAg antigen was used as the mobile phase. The binding conditions were 10 μL / min × 180 s. The dissociation conditions were 10 μL / min × 400 s, and the regeneration conditions were 30 μL / min × 30 s at pH 1.5 Gly. Figures 10 and 11 show the kinetic curves of the bsAb-2-5 and bsAb-2-6 constructs binding to human and cynomolgus CD3 recombinant proteins, respectively. Figure 12 shows the kinetic curves of bsAb2-5 and bsAb2-6 constructs binding to HBsAg.

[0089] As shown in Table 3, Figure 10 and Figure 12, the exemplary HBsAg / CD3 bispecific antibodies BsAb2-6-001, BsAb2-6-006, BsAb2-6-011, BsAb2-6-016, BsAb2-6-017 and BsAb2-5-001 were measured at 10 -11 It had comparable binding affinity to HBsAg, with a KD of 10 M, but 10 to human CD3ε. -8 ~10 -10 The constructs had differential binding affinities ranging from M to M. All of these constructs showed good cross-reactivity to cynomolgus monkey CD3ε (seen in Figure 11), indicating that they can be used as a non-human primate model for preclinical safety and toxicity studies of anti-HBsAg / anti-CD3 bispecific antibodies.

[0090] [Table 3] [Example]

[0091] Purity determination of anti-HBsAg / anti-CD3 bispecific antibodies by size-exclusion chromatography. The bispecific antibody produced in Example 1 was analyzed for purity by size exclusion chromatography. SEC-HPLC was performed using an HPLC system (1260 Infinity II model, Agilent Technologies) and an XBridge BEH200Å SEC Column (3.5 μm, 7.8 × 300 mm; Waters). Mobile phase A consisted of 67 mM NaHPO, 33 mM NaHPO, and 100 mM NaSO at pH 7.0. The flow rate was set at 0.5 mL / min of 100% mobile phase A for 30 min at a column temperature of 30°C. 100 μg of protein was injected onto the column, and detection was performed at 280 nm and 10 Hz. Data processing was performed using CDS2 (Agilent).

[0092] FIG. 13 shows the high purity of the anti-HBsAg / anti-CD3 bispecific antibody, suggesting that such a molecule has good pharmacological potential. [Example]

[0093] In vitro HBV neutralization efficacy of anti-HBsAg / anti-CD3 bispecific antibodies The anti-HBsAg / anti-CD3 bispecific antibodies produced in Example 1 are analyzed for their potency in neutralizing HBV in vitro using Huh7-NTCP cells. Specifically, Huh7-NTCP cells were harvested at a density of 3.5E6 cells / mL in pre-warmed DMEM medium supplemented with 10% FBS and 2.5% DMSO. Huh7-NTCP was then injected into a collagen-coated 96-well plate at 100 μL / well (1 × 10 4Huh7-NTCP cells were seeded at 100, 10, and 0 nM (HBV virus alone as a systematic control) of bsAb2-6-011 and C8 mAb, respectively, and then added to the Huh7-NTCP cells. On day 2, the HBV virus was removed, and Huh7-NTCP cells were incubated with fresh DMEM complete medium for another 5–10 days. The supernatant HBeAg concentration (readout as neutralization capacity) was determined according to the HBeAg ELISA kit protocol.

[0094] Figure 14 shows that both bsAb2-6-001 and bsAb2-6-011 exert potent neutralizing activity at doses of 10 nM and 100 nM, while HBeAg levels are low, in the single digits of PEIU / mL. This is consistent with the potency of the parent C8 mAb. In comparison, the HBeAg levels for the HBV virus group were approximately 60 PEIU / mL, indicating successful infection of the assay system. [Example]

[0095] HBs + In vivo antitumor activity of anti-HBsAg / anti-CD3 bispecific antibodies against HepAD38 CDX xenografts The in vivo anti-tumor activity of the anti-HBsAg / anti-CD3 bispecific antibody produced in Example 1 is evaluated in immunodeficient mice inoculated with the HBs+HepAD38 cell line. In this model, HepAD38 cells (1E7 cells per mouse) were inoculated into VitalStar NPG mice (NOD.Prkdc scid The tumors were subcutaneously implanted into the right flank of Il2rg- / - mice. One week later, the mice were reconstituted with 1.5E7 human T cells purified from healthy donors. Two weeks later, tumors grew to 100–200 mm. 3Mice were sorted when tumor size reached 1.0 and hCD45 ratio reached >5%. Treatment was initiated by intravenous injection of bsAb2-6-011 at a dose of 0.3 mpk on day 0, followed by two injections of 1 mpk weekly. The PBS-treated group was set as the vehicle, and the anti-RSV IgG-treated group was set as the isotype control. Tumor size and body weight were measured every 2-3 days. Tumor volume (mm 3 ) is measured from caliper 0.5 x length x width 2 It was calculated as:

[0096] The results are shown in Figure 15. bsAb2-6-011 effectively reduced HepAD38 tumor size at doses of 0.3 mpk and 1 mpk, whereas the vehicle and isotype control groups had no effect on tumor growth (Figure 15A). After 26 days of treatment, the average tumor size in mice treated with bsAb2-6-011 was significantly smaller than that in mice treated with PBS and the isotype control.

[0097] Figure 15B shows the percentage of human leukocytes in the blood of mice as determined at endpoint. + The percentage of T cells (HBsAg / CD3 T cells) was comparable in all groups, whereas a slight enhancement was observed in mice treated with 1mkp bsAb2-6-011, indicating T cell activation after treatment with the anti-HBsAg / anti-CD3 bispecific antibody. No significant changes in mouse weight were observed in any treatment group (seen in Figure 15C). [Example]

[0098] Pharmacokinetic properties of anti-HBsAg / anti-CD3 bispecific antibodies The pharmacokinetics of one of the anti-HBsAg / anti-CD3 bispecific antibodies in cynomolgus monkeys was examined according to the study described below. This study was conducted at Pharmalegacy. Three male and three female cynomolgus monkeys were assigned to three groups and received a single intravenous injection of bsAb2-6-011 at doses of 0.1 mpk, 1 mpk, and 5 mpk. The pharmacokinetic profile of bsAb2-6-011 was assessed by determining plasma samples obtained before dosing and at various time points up to 7 days post-dosing. The total concentration of bsAb2-6-011 was determined by sandwich ELISA.

[0099] As shown in Figure 16A, the pharmacokinetic profile of bsAb2-6-011 showed a fast initial distribution and clearance in cynomolgus monkeys, with T half is approximately 63 hours. Furthermore, no significant weight change was observed in cynomolgus monkeys treated with 5 mpk bsAb2-6-011 (seen in Figure 16B).

Claims

1. (a) A first polypeptide chain comprising a heavy chain of an anti-CD3 antibody fused with a single-chain fragment (scFv) that binds to hepatitis B virus surface antigen (HBsAg), wherein the HBsAg comprises a small surface antigen, a medium-sized surface antigen of hepatitis B virus, or a large surface antigen of hepatitis B virus. (b) A second polypeptide chain containing the light chain of the anti-human CD3 antibody. A bispecific antibody containing this antibody.

2. The bispecific antibody according to claim 1, wherein the HBsAg scFv is fused to the N-terminus or C-terminus of the heavy chain of the anti-CD3 antibody.

3. The bispecific antibody according to claim 1, wherein the HBsAg scFv is fused to the heavy chain of the anti-CD3 antibody via a glycine-serine linker.

4. The bispecific antibody according to claim 1, wherein the glycine-serine linker is selected from (GGGGGS)n as shown in SEQ ID NO: 33, (GGGSG)n as shown in SEQ ID NO: 34, (GSGGGG)n as shown in SEQ ID NO: 35, (GSGGGP)n as shown in SEQ ID NO: 36, (GSEPS)n as shown in SEQ ID NO: 37, (GGEGGGP)n as shown in SEQ ID NO: 38, (GGEGGGSEGGGGS)n as shown in SEQ ID NO: 39, (GGGSGGGG)n as shown in SEQ ID NO: 40, or a combination thereof (wherein n = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

5. The HBsAg scFv includes, respectively, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 1 to 6, and / or The bispecific antibody according to claim 1, wherein the CD3 antibody comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, as shown in SEQ ID NOs. 11 to 16.

6. The bispecific antibody according to claim 5, wherein the scFv comprises a VH binding domain and a VL binding domain, the VH comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 7, and the VL comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

8.

7. The bispecific antibody according to claim 6, wherein the VH domain and the VL domain are linked by a "YOL" linker, and preferably the "YOL" linker includes the amino acid sequence of SEQ ID NO:

9.

8. The bispecific antibody according to claim 7, wherein the scFv comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

10.

9. The bispecific antibody according to claim 1, wherein the anti-CD3 antibody comprises a VH binding domain and a VL binding domain, the VH comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, and the VL comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO:

29.

10. The bispecific antibody according to claim 1, which is an IgG1-kappa isotype.

11. The bispecific antibody according to claim 1, further comprising alanine at position 234 and alanine at position 235 of the heavy chain, wherein the residue numbering follows the EU Index.

12. The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, or SEQ ID NO: 52, and / or The bispecific antibody according to claim 1, wherein the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO:

57.

13. A nucleic acid molecule encoding the bispecific antibody according to any one of claims 1 to 12.

14. An expression vector comprising the nucleic acid molecule described in claim 13.

15. A host cell comprising the nucleic acid molecule described in claim 13.

16. A pharmaceutical composition comprising a bispecific antibody according to any one of claims 1 to 12 and a pharmaceutically acceptable excipient, diluent, or carrier.

17. A pharmaceutical composition comprising a nucleic acid molecule according to claim 13 and a pharmaceutically acceptable excipient, diluent, or carrier.

18. The pharmaceutical composition according to claim 16 for use in the treatment of diseases caused by HBV infection.

19. The pharmaceutical composition according to claim 17 for use in the treatment of diseases caused by HBV infection.

20. The pharmaceutical composition according to claim 18, wherein the disease caused by the HBV infection includes hepatitis, hepatic fibrosis, cirrhosis, and liver cancer.

21. The pharmaceutical composition according to claim 19, wherein the disease caused by the HBV infection includes hepatitis, hepatic fibrosis, cirrhosis, and liver cancer.

22. A CD3 antibody or its antigen-binding fragment comprising a VH-binding domain and a VL-binding domain, wherein the VH comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, and / or A CD3 antibody or its antigen-binding fragment, wherein the VL contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO:

29.

23. The CD3 antibody or antigen-binding fragment according to claim 22, wherein the amino acid sequence of the VH domain is as shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, and the amino acid sequence of the VL domain is as shown in SEQ ID NO:

25.

24. A bispecific antigen-binding molecule comprising the CD3 antibody or its antigen-binding fragment according to claim 22 or 23.

25. A nucleic acid molecule encoding the CD3 antibody or its antigen-binding fragment according to claim 22 or 23.

26. A nucleic acid molecule encoding a bispecific antigen-binding molecule as described in claim 24.

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