Bispecific antibody against hepatitis b surface antigen (HBSAG) and CD28, and use thereof

Abstract

Provided are a bispecific antibody targeting hepatitis B surface antigen (HBsAg) and CD28, and a use thereof. The bispecific antibody can simultaneously act as a conditional CD28 agonist and an HBsAg neutralizing antibody, and can only conditionally activate CD28 in the presence of HBsAg. The bispecific antibody can not only enhance antiviral responses, but also limit systemic toxicity caused by CD28 activation. In addition, the bispecific antibody can also act in combination with another specific / multispecific antibody, thereby enhancing the antiviral effect.

Description

Bispecific antibodies against hepatitis B surface antigen (HBsAg) and CD28 and their applications

[0001] This application claims priority to Chinese application No. 2025103844202, filed on March 28, 2025, the contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of antibody technology, and more specifically, this invention relates to a bispecific antibody targeting hepatitis B surface antigen (HBsAg) and CD28 and its uses. Background Technology

[0003] Globally, approximately 400 million people are chronically infected with hepatitis B virus (HBV), representing 3.5% of the total population. According to the World Health Organization, the Western Pacific and Africa region is a high-incidence area for chronic hepatitis B (prevalence rate of 6%). Acute HBV infection can develop into chronic hepatitis B (with a morbidity rate of 95% in newborns and 5% in adults), with a poor prognosis, leading to cirrhosis, liver failure, and hepatocellular carcinoma. Currently used antiviral drugs for treating HBV infection (such as tenofovir and entecavir) can inhibit viral replication but cannot clear the HBV covalently closed circular DNA (HBV cccDNA) carried by infected hepatocytes. Once patients stop taking medication, reinfection with HBV may occur. Therefore, complete cure of chronic hepatitis B depends on clearing hepatocytes carrying HBV cccDNA. Studies have found that HBV clearance is associated with sustained control of the virus by effector T cells, while the progression of chronic hepatitis B infection is thought to be limited by virus-specific T cell responses. Therefore, there is an urgent need to develop a novel immunotherapy for chronic hepatitis B to restore HBV-specific T cell-mediated responses to virus-infected hepatocytes.

[0004] T-cell redirection bispecific antibodies are molecules containing more than two binding domains. The first domain specifically binds to cell surface antigens on target cells / tissues (e.g., tumor-associated / pathogenic specific antigens); the second domain specifically binds to T-cell surface antigens (e.g., CD3 or CD28). This dual-target binding molecule can redirect T cells to the target cells / tissues, thereby eliminating the target cells.

[0005] T cell activation is a highly regulated process that typically requires two signaling events to function effectively: the first signal is initiated after the MHC antigen complex binds to the T cell receptor (TCR), helping to distinguish between "self" and "non-self" antigens; the second signal is initiated by activating co-stimulatory molecules. While the first recognition signal activates T cells and triggers T cell-mediated cytotoxicity, the absence of the second co-stimulatory signal leads to cell tolerance, where T cells continue to recognize tumor antigens but do not mount an immune response against tumor cells. The second co-stimulatory signal prevents T cell tolerance and further activates T cells, enhancing their cytotoxicity against target cells.

[0006] CD28 is a homodimeric transmembrane glycoprotein belonging to the immunoglobulin (Ig) superfamily, primarily expressed by T cells. CD28 provides a costimulatory signal that determines the magnitude of T cell responses to antigens and participates in T cell activation and survival. It is well known that CD28 costimulatory signals, in conjunction with BiTEs, induce T cell cytotoxicity. Therefore, CD28 is an ideal therapeutic target for T cell connector drugs. CD28 agonist antibodies can be divided into two categories: (i) CD28 hyperagonist antibodies and (ii) CD28 conventional agonist antibodies. Under normal circumstances, activation... T cells require the participation of the T cell antigen receptor (TCR, signal 1) and the stimulation signal of CD28 (signal 2). CD28 superagonists are CD28-specific monoclonal antibodies that can autonomously activate T cells without the involvement of the T cell receptor. Theralizumab (TGN1412) is a humanized IgG4-type CD28 superagonist that directly stimulates T cells; relevant information was described in a 2006 clinical trial. Conventional CD28 agonist antibodies only enhance T cell activation in the presence of the T cell receptor signal (signal 1). FR104 (WO2011 / 101791) is a conventional CD28 agonist antibody described in the prior art; it is a humanized CD28 agonist derived from a murine antibody called CD28.3. Another conventional CD28 agonist antibody is a 9.3 mAb described in the prior art that can activate T cells in the presence of TCR-CD3 signaling pathway activation.

[0007] Currently, bispecific antibodies targeting T-cell adaptors developed around molecules such as SP34 have significant adverse reactions and need to be combined with CD28-targeting agonists to regulate T-cell activation responses.

[0008] HBsAg refers to the envelope antigen of the hepatitis B virus. HBV surface proteins include small, medium, and large surface proteins, which are transcribed and translated from an open reading frame (ORF) and differ from each other at the N-terminus. The large surface antigen contains portions not present in either the medium or small surface antigens, while the medium surface antigen contains portions present in the large surface antigen but not in the small surface antigen. The sequence contained in the small surface antigen is present in the C-terminal portions of the medium and large surface antigens. It is hypothesized that although viral particles are released into intracellular vesicles, many HBV surface proteins still integrate into the endoplasmic reticulum membrane. During vesicle transport, this membrane can fuse with the cell membrane, resulting in HBV surface proteins being displayed on the surface of infected cells. Therefore, HBsAg provides an ideal therapeutic target for treating chronic hepatitis B and related diseases.

[0009] T-cell immunity plays a crucial role in controlling viral infection and eliminating infected and malignant cells. In chronic HBV infection, the virus persists in the body through various mechanisms, including immunosuppression, T-cell exhaustion, and the establishment of a latency period. However, viral infection typically induces virus antigen-specific immunity, including antigen-specific CD8 T cells. These cells can easily recognize and control or kill infected cells through the release of cytokines or cytotoxic T-cell (CTL)-mediated killing processes. Therefore, activation and / or expansion of virus antigen-specific T cells in vivo can provide a therapeutic strategy for chronic viral infection. Summary of the Invention

[0010] The purpose of this invention is to provide a bispecific antibody targeting hepatitis B surface antigen (HBsAg) and CD28 and its uses.

[0011] The present invention aims to provide a bispecific antibody that specifically binds to HBsAg and human CD28; to provide a nucleotide molecule encoding the bispecific antibody; to provide an expression vector comprising the nucleotide molecule; to provide a host cell comprising the expression vector; to provide a method for preparing the bispecific antibody; to provide a pharmaceutical composition comprising the bispecific antibody; and to provide the use of the bispecific antibody in the preparation of a drug.

[0012] In a first aspect of the invention, a multispecific antibody / antigen binding molecule is provided, the multispecific antibody comprising:

[0013] (1) Humanized CD28 antibody / antigen binding domain that specifically binds to CD28

[0014] The humanized CD28 antibody comprises a VH region of the amino acid sequence as shown in SEQ ID NO: 9, 10, 11, 12, 13, or 14, and a VL region of the amino acid sequence as shown in SEQ ID NO: 15, 16, 17, 18, 19, or 20; and

[0015] (2) Antibody / antigen binding domains that specifically bind to one or more viral / tumor antigens.

[0016] In another preferred embodiment, the humanized CD28 antibody comprises a VH region of the amino acid sequence as shown in SEQ ID NO:9, 10, 11, 12, 13, or 14, or the humanized CD28 antibody comprises a VH region of the amino acid sequence as shown in SEQ ID NO:9, 10, 11, 12, 13, or 14 with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, and a VL region of the amino acid sequence as shown in SEQ ID NO:15, 16, 17, 18, 19, or 20, or the humanized CD28 antibody comprises a VL region of the amino acid sequence as shown in SEQ ID NO:9, 10, 11, 12, 13, or 14, with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, or the humanized CD28 antibody comprises a VH region of the amino acid sequence as shown in SEQ ID NO:15, 16, 17, 18, 19, or 20, or the humanized CD28 antibody comprises a VL ... The sequences shown in NO:15, 16, 17, 18, 19, or 20 have a VL region with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity amino acid sequence.

[0017] In another preferred embodiment, the structure of the humanized CD28 antibody is selected from the group consisting of: (i) Fab fragment; (ii) CrossFab fragment; (iii) Fv fragment; or (iv) single-chain Fv (scFv).

[0018] In another preferred embodiment, the structure of the antibody against the virus / tumor antigen is selected from the group consisting of (i) Fab fragments; (ii) CrossFab fragments; (iii) Fv fragments; or (iv) single-chain Fv (scFv).

[0019] In another preferred embodiment, the antibodies against the virus / tumor antigen include: single-chain antibodies, double-chain antibodies, nanobodies, monoclonal antibodies, chimeric antibodies, murine antibodies, humanized antibodies, and bispecific antibodies.

[0020] In another preferred embodiment, the virus / tumor antigen is selected from the following group: DNA viruses, RNA viruses, and protein viruses (such as prions) classified by genetic material; true viruses (Euviruses, or simply viruses) and subviruses (including viroids, virusoids, and prions) classified by viral structure; bacteriophages (bacterial viruses), plant viruses (such as tobacco mosaic virus), and animal viruses (such as avian influenza virus, smallpox virus, and HIV) classified by morphology; and spherical viruses, rod-shaped viruses, brick-shaped viruses, coronaviruses (such as SARS-CoV-2), filamentous viruses, rotaviruses, enveloped spherical viruses, viruses with spherical heads, and insect viruses encapsulated within inclusion bodies. It can be an antigen presented by the virus itself or an antigen formed after the virus infects a cell.

[0021] In another preferred embodiment, the multispecific antibody is a bispecific antibody, the bispecific antibody comprising:

[0022] a) Humanized CD28 antibody / antigen-binding domain D1 that specifically binds to CD28; and

[0023] b) The second antigen-binding domain D2, which specifically binds to hepatitis B virus surface antigen (HBsAg).

[0024] HBsAg includes hepatitis B virus small surface antigen, hepatitis B virus medium surface antigen, or hepatitis B virus large surface antigen.

[0025] In another preferred embodiment, the bispecific antibody is an IgG1 isotype.

[0026] In another preferred embodiment, the bispecific antibody further comprises alanine at position 234, alanine at position 235, and glycine at position 329 of the heavy chain, wherein the residue numbers are based on the EU index.

[0027] In another preferred embodiment, D1 and / or D2 are IgG antibodies.

[0028] In another preferred embodiment, the IgG antibody is an IgG1, IgG2, IgG3, or IgG4 antibody.

[0029] In another preferred embodiment, D1 and / or D2 are scFv.

[0030] In another preferred embodiment, D2 is an anti-HBsAg scFv.

[0031] In another preferred embodiment, D2 is an IgG antibody against HBsAg.

[0032] In another preferred embodiment, D2 is a CrossFab antibody against HBsAg.

[0033] In another preferred embodiment, D1 is a CrossMab antibody against CD28.

[0034] In another preferred embodiment, D1 is an anti-CD28 IgG antibody.

[0035] In another preferred embodiment, the D2 comprises one, two, three or more scFvs that are anti-HBsAg.

[0036] In another preferred embodiment, the D2 comprises one, two, three or more CrossFab antibodies against HBsAg.

[0037] In another preferred embodiment, the D1 comprises one, two, three or more CrossFab antibodies against CD28.

[0038] In another preferred embodiment, the bispecific antibody has the structure shown in Formula 1 or Formula 3:

[0039] Among them, VH CD28 Heavy chain variable region representing anti-human CD28 antibody;

[0040] VL CD28 The light chain variable region representing the anti-human CD28 antibody;

[0041] CL λ(或Lamda) Represents the light chain constant region of the anti-human IgG type;

[0042] CH1 represents the heavy chain constant region CH1 of the lgG type;

[0043] VH HBS Heavy chain variable region representing anti-HBsAg antibody;

[0044] VL HBS The light chain variable region representing the anti-HBsAg antibody;

[0045] CL K(或Kappa) Represents the constant region of the light chain in the lgG type;

[0046] CH1, CH2, and CH3 represent the heavy chain constant regions CH1, CH2, and CH3 of the lgG type, respectively;

[0047] L1 is the connector;

[0048] "~" represents a disulfide bond or a covalent bond;

[0049] "-" represents a peptide bond.

[0050] In another preferred embodiment, the bispecific antibody has the structure shown in Formula 1:

[0051] in,

[0052] VH CD28 Heavy chain variable region representing anti-human CD28 antibody;

[0053] VL CD28 The light chain variable region representing the anti-human CD28 antibody;

[0054] CL λ(或Lamda) Represents the light chain constant region of the anti-human IgG type;

[0055] CH1 represents the heavy chain constant region CH1 of the lgG type;

[0056] VH HBS Heavy chain variable region representing anti-HBsAg antibody;

[0057] VL HBS The light chain variable region representing the anti-HBsAg antibody;

[0058] CL K(或Kappa) Represents the constant region of the light chain in the lgG type;

[0059] CH1, CH2, and CH3 represent the heavy chain constant regions CH1, CH2, and CH3 of the lgG type, respectively;

[0060] L1 is the connector;

[0061] "~" represents a disulfide bond or a covalent bond;

[0062] "-" represents a peptide bond.

[0063] In another preferred embodiment, the heavy chain variable region of the anti-HBsAg antibody includes the following three complementarity-determining regions (HCDRs):

[0064] HCDR1 as shown in SEQ ID NO.31;

[0065] HCDR2 as shown in SEQ ID NO.32; and

[0066] HCDR3 as shown in SEQ ID NO.33; and

[0067] The light chain variable region of the anti-HBsAg antibody includes the following three complementarity-determining regions (LCDRs):

[0068] LCDR1 as shown in SEQ ID NO.34;

[0069] LCDR2 as shown in SEQ ID NO.35; and

[0070] LCDR3 as shown in SEQ ID NO.36.

[0071] In another preferred embodiment, the heavy chain variable region of the anti-human CD28 antibody includes the following three complementarity-determining regions (HCDRs):

[0072] HCDR1 as shown in SEQ ID NO.1;

[0073] HCDR2 as shown in SEQ ID NO.2; and

[0074] HCDR3 as shown in SEQ ID NO.3; and

[0075] The light chain variable region of the anti-human CD28 antibody includes the following three complementarity-determining regions (LCDRs):

[0076] LCDR1 as shown in SEQ ID NO.4;

[0077] LCDR2 as shown in SEQ ID NO.5; and

[0078] LCDR3 as shown in SEQ ID NO.6.

[0079] In another preferred embodiment, the bispecific antibody comprises:

[0080] Anti-HBsAg antibody comprising the VH region of the amino acid sequence shown in SEQ ID NO:37 and the VL region of the amino acid sequence shown in SEQ ID NO:38; and

[0081] Anti-human CD28 antibody comprising a VH region containing an amino acid sequence as shown in SEQ ID NO:9, 10, 11, 12, 13, or 14, and a VL region containing an amino acid sequence as shown in SEQ ID NO:15, 16, 17, 18, 19, or 20.

[0082] In another preferred embodiment, the anti-human CD28 antibody is a CrossFab fragment; the CrossFab fragment contains VH-CL. Lamda Fragments and VL-CH1 fragments; the VH-CL Lamda The fragment is fused to the N-terminus of the anti-HBsAg antibody heavy chain via a glycine-serine linker; VL-CH1 is the fusion of VL with the CH1 domain of the heavy chain constant region; VL of the anti-HBsAg domain is fused with the light chain constant region, which is Kappa and contains the amino acid sequence shown in SEQ ID NO:49.

[0083] In another preferred embodiment, the anti-HBsAg antibody is a CrossFab fragment; the CrossFab fragment contains VH-CL. Lamda Fragments and VL-CH1 fragments; the VH-CLLamda The fragment is fused to the N-terminus of the anti-CD28 antibody heavy chain via a glycine-serine linker; VL-CH1 is the fusion of VL with the CH1 domain of the heavy chain constant region; VL of the anti-CD28 antibody domain is fused with the light chain constant region, which is Kappa and contains the amino acid sequence shown in SEQ ID NO:49.

[0084] In another preferred embodiment, the CL Lamda It contains the amino acid sequence shown in SEQ ID NO:50.

[0085] In another preferred embodiment, the CH1 sequence comprises an amino acid sequence as shown in SEQ ID NO:51.

[0086] In another preferred embodiment, the connective sequence is selected from (GGGGS)n shown in SEQ ID NO:39, (GGGSG)n shown in SEQ ID NO:40, (GSGGG)n shown in SEQ ID NO:41, (GSGGGP)n shown in SEQ ID NO:42, (GGSEPS)n shown in SEQ ID NO:43, (GGEGGGP)n shown in SEQ ID NO:44, (GGEGGGSEGGGS)n shown in SEQ ID NO:45, (GGGSGGGGG)n shown in SEQ ID NO:46, or a combination thereof, wherein n = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0087] In another preferred embodiment, for the structure of Formula 1, the CD28 antibody is a humanized 9.3 mAb, and the HBsAg antibody is G12, VH CD28 -CL Lamda -L1-VH HBS The amino acid sequence of -CH1-CH2-CH3 is shown in SEQ ID NO:64, VL CD28 The amino acid sequence of -CH1 is shown in SEQ ID NO:75, VL HBS -CL Kappa The amino acid sequence is shown in SEQ ID NO:76, and the last three peptide chains form a polymer through disulfide bonds.

[0088] In another preferred embodiment, for the structure of Formula 3, the CD28 antibody is a humanized 9.3 mAb, and the HBsAg antibody is G12, VH HBS -CL Lamda -L1-VH CD28 The amino acid sequence of -CH1-CH2-CH3 is shown in SEQ ID NO:77, VL HBSThe amino acid sequence of -CH1 is shown in SEQ ID NO:83, VL CD28 -CL Kappa The amino acid sequence is shown in SEQ ID NO:89, and the last three peptide chains form a polymer through disulfide bonds.

[0089] In another preferred embodiment, the bispecific antibody further includes the structure shown in Formula 2:

[0090] in,

[0091] VH CD28 Heavy chain variable region representing anti-human CD28 antibody;

[0092] VL CD28 The light chain variable region representing the anti-human CD28 antibody;

[0093] CL represents the constant region of the light chain in the lgG type;

[0094] CH1, CH2, and CH3 represent the heavy chain constant regions CH1, CH2, and CH3 of the lgG type, respectively;

[0095] VH HBS Heavy chain variable region representing the single-stranded fragment (scFv) that binds HBsAg;

[0096] VL HBS The light chain variable region represents the single-stranded fragment (scFv) that binds HBsAg;

[0097] L1 and L2 are each independent connectors;

[0098] "~" represents a disulfide bond or a covalent bond;

[0099] "-" represents a peptide bond.

[0100] In another preferred embodiment, the heavy chain variable region of the HBsAg-binding single-stranded fragment (scFv) includes the following three complementary determinant regions (HCDRs):

[0101] HCDR1 as shown in SEQ ID NO.21;

[0102] HCDR2 as shown in SEQ ID NO.22; and

[0103] HCDR3 as shown in SEQ ID NO.23; and

[0104] The light chain variable region of the HBsAg-binding single-chain fragment (scFv) includes the following three complementary determinant regions (LCDRs):

[0105] LCDR1 as shown in SEQ ID NO.24;

[0106] LCDR2 as shown in SEQ ID NO.25; and

[0107] LCDR3 as shown in SEQ ID NO.26.

[0108] In another preferred embodiment, the heavy chain variable region of the anti-human CD28 antibody includes the following three complementarity-determining regions (HCDRs):

[0109] HCDR1 as shown in SEQ ID NO.1;

[0110] HCDR2 as shown in SEQ ID NO.2; and

[0111] HCDR3 as shown in SEQ ID NO.3; and

[0112] The light chain variable region of the anti-human CD28 antibody includes the following three complementarity-determining regions (LCDRs):

[0113] LCDR1 as shown in SEQ ID NO.4;

[0114] LCDR2 as shown in SEQ ID NO.5; and

[0115] LCDR3 as shown in SEQ ID NO.6.

[0116] In another preferred embodiment, the bispecific antibody comprises:

[0117] A single-stranded fragment (scFv) containing the VH region of the amino acid sequence shown in SEQ ID NO:27 and the VL region of the amino acid sequence shown in SEQ ID NO:28, which binds HBsAg; and

[0118] Anti-human CD28 antibody comprising a VH region containing an amino acid sequence as shown in SEQ ID NO:9, 10, 11, 12, 13, or 14, and a VL region containing an amino acid sequence as shown in SEQ ID NO:15, 16, 17, 18, 19, or 20.

[0119] In another preferred embodiment, the L1 connector sequence is selected from (GGGGS)n shown in SEQ ID NO:39, (GGGSG)n shown in SEQ ID NO:40, (GSGGG)n shown in SEQ ID NO:41, (GSGGGP)n shown in SEQ ID NO:42, (GGSEPS)n shown in SEQ ID NO:43, (GGEGGGP)n shown in SEQ ID NO:44, (GGEGGGSEGGGS)n shown in SEQ ID NO:45, (GGGSGGGGG)n shown in SEQ ID NO:46, or a combination thereof, wherein n = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0120] In another preferred embodiment, the L2 linker is a “YOL” linker; preferably, the “YOL” linker comprises an amino acid sequence such as SEQ ID NO:29.

[0121] In another preferred embodiment, for the structure of Formula 2, the CD28 antibody is a humanized 9.3 mAb, and the scFv of the HBsAg is C8, VH HBS -L2-VL HBS -L1-VH CD28 The amino acid sequence of -CH1-CH2-CH3 is shown in SEQ ID NO:52, VL CD28 The amino acid sequence of -CL is shown in SEQ ID NO:63, and the last two identical heavy and light chains form a polymer through disulfide bonds.

[0122] In another preferred embodiment, VL in Equation 2 CD28 -CL is VL CD28 -CL κ .

[0123] In a second aspect of the invention, a polynucleotide molecule is provided, the polynucleotide molecule encoding a multispecific antibody as described in the first aspect of the invention.

[0124] In a third aspect of the invention, an expression vector is provided, the expression vector containing the polynucleotide molecule described in the second aspect of the invention.

[0125] In another preferred embodiment, the expression vector is a virus or plasmid, preferably a bacteriophage or bacteriophage particle.

[0126] In a fourth aspect of the invention, a host cell is provided, the host cell containing an expression vector as described in the third aspect of the invention or its genome having integrated the polynucleotide molecule as described in the second aspect of the invention.

[0127] In another preferred embodiment, the host cell is selected from the group consisting of COS, CHO, 293F, NSO, sf9, sf21, DH5α, BL21(DE3) or TG1, more preferably E. coli TG1, BL21(DE3) cells (expressing single-chain antibodies or Fab antibodies) or 293F, CHO-K1 cells (expressing full-length IgG antibodies).

[0128] In another preferred embodiment, the host cell is a eukaryotic cell, preferably a CHO cell or a 293F cell.

[0129] In a fifth aspect of the present invention, a method for preparing a multispecific antibody as described in the first aspect of the present invention is provided, the method comprising the following steps:

[0130] a) Under expression conditions, host cells as described in the fourth aspect of the present invention are cultured to express multispecific antibodies;

[0131] b) Isolate and purify the multispecific antibody described in step a).

[0132] In a sixth aspect of the invention, an immunoconjugate is provided, the immunoconjugate comprising:

[0133] (a) a specific antibody as described in the first aspect of the invention; and

[0134] (b) The coupling part selected from the following group: detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes.

[0135] In a seventh aspect of the invention, a pharmaceutical composition is provided comprising an effective amount of a multispecific antibody as described in the first aspect of the invention and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0136] In an eighth aspect of the invention, the use of a multispecific antibody as described in the first aspect of the invention, or a pharmaceutical composition as described in the seventh aspect of the invention, in the preparation of a medicament for treating diseases caused by HBV infection is provided.

[0137] In another preferred embodiment, the disease caused by the anti-HBV infection is selected from: hepatitis, liver fibrosis, cirrhosis, liver cancer, or a combination thereof.

[0138] In a ninth aspect of the invention, a method for treating a disease caused by HBV infection is provided, the method comprising administering to a subject in need a multispecific antibody as described in the first aspect of the invention, an immunoconjugate as described in the sixth aspect of the invention, or a pharmaceutical composition as described in the seventh aspect of the invention.

[0139] In another preferred embodiment, the method further includes co-administration with other bispecific antibodies.

[0140] In another preferred embodiment, the other bispecific antibody includes an HBsAg / CD3 bispecific antibody.

[0141] In another preferred embodiment, the disease caused by HBV infection is selected from the group consisting of: acute hepatitis B, chronic hepatitis B, cirrhosis, hepatocellular carcinoma, liver failure, glomerulonephritis, polyarteritis nodosa, polyarthritis, or peripheral neuropathy.

[0142] In a tenth aspect of the invention, a CD28 antibody or antigen-binding fragment thereof is provided, the antibody or antigen-binding fragment comprising a VH region of an amino acid sequence as shown in SEQ ID NO:9, 10, 11, 12, 13, or 14, or the antibody or antigen-binding fragment comprising a VH region of an amino acid sequence as shown in SEQ ID NO:9, 10, 11, 12, 13, or 14 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, and a VL region of an amino acid sequence as shown in SEQ ID NO:15, 16, 17, 18, 19, or 20, or the antibody or antigen-binding fragment comprising an amino acid sequence as shown in SEQ ID NO:9, 10, 11, 12, 13, or 14. The sequences shown in NO:15, 16, 17, 18, 19, or 20 have a VL region with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity amino acid sequence.

[0143] In another preferred embodiment, the sequence of the six CDRs of VH and VL of the CD28 antibody or its antigen-binding fragment is as shown in SEQ ID No:1-6, or differs from the six CDRs shown in SEQ ID No:1-6 by only 1-3 amino acids.

[0144] This invention also provides a treatment method using the aforementioned HBsAg / CD28 bispecific antigen-binding molecule to target / kill HBsAg-positive hepatocytes, wherein the HBsAg / CD28 bispecific antigen-binding molecule is combined with other HBsAg bispecific antigen-binding molecules that bind CD3 (e.g., the HBsAg / CD3 bispecific antibody BsAb2-6-011 (see WO2024005713A1, the heavy chain of BsAb2-6-011 is arranged from the N-terminus to the C-terminus as follows: heavy chain VH3-CH1-Fc-C8scFv, the amino acid sequence is shown in SEQ ID NO:49, light chain VL2-CL, the amino acid sequence is shown in SEQ ID NO:53) and / or other anti-HBV drug compositions. BsAb2-6-011 is abbreviated as BsAb2-6 and is used interchangeably in this invention.

[0145] One of the positive and progressive effects of the present invention is that the bispecific antibody can simultaneously act as a conditional agonist of CD28 and a neutralizing antibody against HBsAg. Furthermore, it conditionally activates CD28 only in the presence of HBsAg, thereby enhancing the antiviral response and limiting the systemic toxicity of CD28 activation.

[0146] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0147] Figure 1 is a schematic diagram of the structure of the bispecific antibody of the present invention:

[0148] (1A) Two Fab tandem HBsAg / CD28 bispecific antibodies, comprising (a) the first peptide chain fused with the N or C terminus of the heavy chain of the 9.3 antibody VH-CL fragment and the heavy chain of the hepatitis B surface antigen binding antibody (Anti-HBsAg) G12, the second peptide chain of the 9.3 antibody light chain VL-CH1, and the third peptide chain of the antibody G12 light chain VL-CL; or (b) the first peptide chain fused with the N or C terminus of the heavy chain of the hepatitis B surface antigen binding antibody G12 and the heavy chain of the 9.3 antibody, the second peptide chain of the G12 antibody light chain VL-CH1, and the third peptide chain of the 9.3 antibody light chain VL-CL;

[0149] (1B) HBsAg / CD28 bispecific antibody in the form of scFv-IgG, wherein a single-chain fragment of anti-hepatitis B surface antigen (HBsAg) (Anti-HBsAg scFv) is fused to the N or C terminus of the heavy chain of anti-CD28 9.3 IgG antibody (Anti-CD28 IgG).

[0150] Figure 2 shows the ELISA binding activity of humanized 9.3 monoclonal antibody to human CD28.

[0151] Figure 3 shows the purity of the bispecific antibody of the present invention as characterized by SEC-HPLC.

[0152] Figure 4 shows the SPR characterization of the binding affinity of the bispecific antibodies of the present invention, HBsAg (4A) and human CD28 (4B).

[0153] Figure 5 shows the ELISA binding activity of the BsAb2-1 series of bispecific antibodies against HBsAg antigens (HBsAg P1, HBsAg P2, HBsAg P3) and human CD28 antigens (hCD28 P1, hCD28 P2, hCD28 P3), respectively.

[0154] Figure 6 shows the ELISA and flow cytometry binding activity characterization of the bispecific antibody of the present invention. (6A) The ELISA binding activity of the bispecific antibody of the present invention with human CD28 and HBsAg recombinant protein was dose-dependent. (6B) The binding activity of the bispecific antibody of the present invention with CD28-positive human leukemia T cell line and HBsAg-positive cells was dose-dependent. The readings are the average fluorescence intensity of BsAb2-1-006 and BsAb2-37-006 bound to CD28+Jurkat cells and HBs(+)HepG2-LMS-660 cells.

[0155] Figure 7 shows that the NFAT reporter gene signal of the BsAb2-1 series bispecific antibody combined with the HBsAg / CD3 bispecific antibody of the present invention is dose-dependent.

[0156] Figure 8 shows the dose-dependent NFAT reporter gene signaling of the bispecific antibody combined with the HBsAg / CD3 bispecific antibody of the present invention. T cell activation induced by the combination of BsAb2-6-011 and BsAb2-1-006 or BsAb2-37-006 was measured by the NFAT reporter gene assay under conditions of S antigen pre-coating (8A) and uncoated groups as negative controls (8B), respectively.

[0157] Figure 9 shows the dose-dependent effect of the bispecific antibody of the present invention combined with the HBsAg / CD3 bispecific antibody in inducing T cell proliferation. The expression levels of CD25 on the surface of CD4+ and CD8+ T cells were determined by flow cytometry (9A and 9B). T cells were labeled with Violet and the proliferation levels of CD4+ and CD8+ subset T cells were determined by flow cytometry (9C and 9D).

[0158] Figure 10 illustrates the cytotoxicity of T cells induced by the bispecific antibody combined with the HBsAg / CD3 bispecific antibody of the present invention against HBs(+) cell lines. HBs(+) HepG2-LMS-660 cells (10A) and HBs(+) HepAD38 cells (10B) were co-incubated with human PBMCs supplemented with BsAb2-6-011, BsAb2-1-006, BsAb2-37-006, BsAb2-6-011+BsAb2-1-006, or BsAb2-37-006 (E:T = 10:1).

[0159] Figure 11 shows the in vitro neutralizing efficacy of the bispecific antibody of the present invention in an HBV-infected Huh7-NTCP cell model. The levels of HBsAg (11A) and HBeAg (11B) in the cell supernatant were detected using an HBeAg / HBsAg enzyme-linked immunosorbent assay kit.

[0160] Figure 12 illustrates the in vitro antiviral activity of human T cells mediated by the combination of the bispecific antibody of the present invention and the HBsAg / CD3 bispecific antibody in a primary human hepatocyte model infected with HBV.

[0161] (12A) BsAb2-6-011 with or without BsAb2-1-006 / BsAb2-37-006 induces cytotoxicity of human T cells against HBV-infected primary human hepatocytes, as determined using the CellTiter-Glo cell viability assay kit.

[0162] (12B) HBsAg / CD3 combined with HBsAg / CD28 bispecific antibody induced T cell activation in the presence of HBV-infected primary human hepatocytes, and the expression level of CD69 on the surface of T cells was detected by flow cytometry.

[0163] (12C) is the induction of cytokine release from T cells by HBsAg / CD3 combined with HBsAg / CD28 bispecific antibody in the presence of HBV-infected primary human hepatocytes.

[0164] (12D) and (12E) BsAb2-6-011 with or without BsAb2-1-006 / BsAb2-37-006 induced in vitro antiviral activity in human T cells. The levels of HBeAg and HBsAg in the cell supernatant were detected using an HBeAg / HBsAg enzyme-linked immunosorbent assay kit.

[0165] Figure 13 shows the in vivo antiviral activity of human T cells mediated by HBsAg / CD3 and HBsAg / CD28 bispecific antibodies in a chimeric mouse model of HBV-infected liver.

[0166] (A) uPa mice that had undergone human liver transplantation were infected with HBV (genotype D). Treatment began six weeks later, with each mouse receiving 1x10^7 PBMCs, grouped, and injected with BsAb2-6-011+ / BsAb2-37-006, BsAb2-37-006, or PBS;

[0167] (B) The HBV viral DNA copy number (HBV DNA) in mouse serum was quantified by qPCR, and (C) HBsAg, (D) HBeAg, and (E) HSA in mouse serum were detected using an ELISA kit. Mice were sacrificed 11 days after treatment, and HBV pgRNA in mouse liver was quantified by RT-qPCR (F), and T cell characterization was analyzed by flow cytometry (G).

[0168] Figure 14 shows the in vivo antitumor activity of human T cells mediated by HBsAg / CD3 and HBsAg / CD28 bispecific antibodies in a HepAD38 tumor-bearing mouse model; (A) Experimental flowchart; (B) and (C) Tumor growth curves; (C) The right side shows the curves of a single animal, with each curve representing the tumor volume change of a mouse; (D) Mouse body weight change curve; (EG) Cell counts of different T cell subsets in peripheral blood were detected by flow cytometry. Detailed Implementation

[0169] Through extensive and in-depth research, the inventors have developed a bispecific antibody targeting HBsAg and CD28. This bispecific antibody can simultaneously act as a conditional agonist of CD28 and a neutralizing antibody against HBsAg. Furthermore, it conditionally activates CD28 only in the presence of HBsAg, thereby enhancing the antiviral response and limiting the systemic toxicity of CD28 agonists.

[0170] the term

[0171] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing this invention, it should be understood that it is not limited to the specific methods and experimental conditions described, as such methods and conditions can be varied.

[0172] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0173] In this invention, the terms "antibody (Ab)" and "immunoglobulin G (IgG)" refer to heterotetraglycoproteins with the same structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by a constant region, which consists of three domains: CH1, CH2, and CH3. Each light chain has a variable region (VL) at one end and a constant region at the other end, with the light chain constant region including a domain CL; the light chain constant region pairs with the CH1 domain of the heavy chain constant region, and the light chain variable region pairs with the heavy chain variable region. Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC). Heavy chain constant regions include IgG1, IgG2, IgG3, and IgG4 isotypes; light chain constant regions include κ (Kappa) or λ (Lamda). The heavy and light chains of an antibody are covalently linked by disulfide bonds between the CH1 domain of the heavy chain and the CL domain of the light chain. The two heavy chains of an antibody are covalently linked by interpeptide disulfide bonds formed between their hinge regions.

[0174] The "immunoglobulin antibody IgG" described in this invention is a molecule of approximately 150 kDa, composed of four peptide chains. It contains two identical γ heavy chains of approximately 50 kDa and two identical light chains of approximately 25 kDa, thus exhibiting a tetrameric quaternary structure. The two heavy chains are interconnected by disulfide bonds and each is linked to a light chain. The resulting tetramer has two identical halves, forming a fork-like or Y-like shape, with each end of the fork containing an identical antigen-binding site. IgG antibodies can be classified into several subclasses (e.g., IgG1, 2, 3, 4) based on minor differences in the amino acid sequence of the constant region of the heavy chain.

[0175] In this invention, the term "bispecific antibody (or bispecific antibody)" refers to an antibody molecule that can simultaneously and specifically bind to two antigens (targets) or two epitopes. Based on symmetry, bispecific antibodies can be classified into structurally symmetrical and asymmetrical molecules. Based on the number of binding sites, bispecific antibodies can be classified into bivalent, trivalent, tetravalent, and multivalent molecules.

[0176] In this invention, the term "monoclonal antibody (MABS)" refers to an antibody obtained from a substantially homogeneous population, meaning that the individual antibodies in this population are identical, except for a few possible naturally occurring mutations. Monoclonal antibodies target a single antigenic site with high specificity. Moreover, unlike conventional polyclonal antibody formulations (which are typically mixtures of different antibodies targeting different antigenic determinants), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized through hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" indicates the antibody's characteristic of being obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring any special method to produce the antibody.

[0177] As used herein, the term "antigen-binding fragment" refers to a Fab fragment, Fab' fragment, F(ab')2 fragment, or single Fv fragment that has antigen-binding activity. Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fv fragments; or (iv) single-chain Fv (scFv). As used herein, the term "antigen-binding fragment" also encompasses other engineered molecules such as domain-specific antibodies, single-domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-transplanted antibodies, bisomal antibodies, trisomal antibodies, tetrasomal antibodies, microantibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains.

[0178] In this invention, the terms "Fab" and "Fc" refer to the ability of papain to cleave an antibody into two identical Fab fragments and one Fc fragment. The Fab fragment consists of the VH and CH1 domains of the antibody's heavy chain and the VL and CL domains of its light chain. The Fc fragment, or crystallizable fragment (Fc), consists of the CH2 and CH3 domains of the antibody. The Fc fragment has no antigen-binding activity and is the site of interaction between the antibody and effector molecules or cells. The term "F(ab')2 fragment" or "F(ab')2 fragment antibody" refers to the antibody obtained by digesting an entire IgG antibody with pepsin, removing most of the Fc region while retaining some hinge regions intact. It has two antigen-binding F(ab) portions linked together by disulfide bonds.

[0179] In this invention, the term "scFv" or "single-chain variable region fragment scFv" refers to a single-chain antibody fragment (scFv), which is a fusion protein containing the variable regions of the immunoglobulin heavy chain VH and light chain VL, with VH and VL linked by a linker of 15 to 25 amino acids, wherein the fusion protein retains the same antigen specificity as the complete immunoglobulin.

[0180] In this invention, the term "Fv fragment" or "Fv antibody" refers to the smallest antibody fragment containing variable regions of the antibody heavy chain and light chain, but no constant regions, and having all antigen-binding sites. Generally, an Fv fragment also includes a polypeptide linker between the VH and VL domains and is capable of forming the structure required for antigen binding.

[0181] In this invention, the term "variable" refers to the fact that certain portions of the variable region in an antibody differ in sequence, resulting in the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of the antibody. It is concentrated in three segments within the variable regions of the heavy and light chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable regions are called frame regions (FRs). The variable regions of the natural heavy and light chains each contain four FR regions, which are generally β-sheet configurations, linked by three CDRs forming a linking loop, and in some cases may form a partial β-sheet structure. The CDRs in each chain are closely packed together through the FR regions and together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)).

[0182] As used herein, the term "linker" or "connector" refers to an insertion into an immunoglobulin domain that provides sufficient mobility for both the light and heavy chain domains to fold into an exchangeable dual variable region immunoglobulin of one or more amino acid residues. In this invention, preferred linkers are linkers L1 and / or L2.

[0183] Suitable examples of linkers include monoglycine (Gly) or serine (Ser) residues, and the identification and sequence of amino acid residues in the linker can vary depending on the type of secondary structural element that needs to be achieved in the linker.

[0184] In this invention, the terms "antibody," "binding," and "specific binding" refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and its targeted antigen. Typically, antibodies bind at a rate of less than approximately 10... -7 M, for example, less than approximately 10 -8 M, 10 -9 M, 10 -10 M, 10 -11The antibody binds to the antigen with an equilibrium dissociation constant (KD) of M or smaller. In this invention, the term "KD" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. For example, the binding affinity between the antibody and the antigen can be determined using surface plasmon resonance (SPR) in a BIACORE instrument or using ELISA to determine the relative affinity of antibody-antigen binding.

[0185] The term "fusion" in the context of polypeptides or polynucleotides refers to a form of polypeptide or polynucleotide that does not exist in its natural state. One non-limiting example is that it can be achieved by combining polynucleotides or polypeptides that do not normally appear together.

[0186] The term "cross-reactivity" refers to the ability of the antibody described herein to bind to tumor-associated antigens from different species. For example, the antibody described herein that binds to human TAAs can also bind to TAAs from other species (e.g., cynomolgus monkey TAAs). Cross-reactivity can be measured by detecting specific reactivity with purified antigens in binding assays (e.g., SPR, ELISA), or binding to cells physiologically expressing TAAs, or otherwise interacting functionally with cells physiologically expressing TAAs. Examples of analyses known in the art for measuring binding affinity include surface plasmon resonance (e.g., Biacore) or similar techniques (e.g., Kinexa or Octet).

[0187] Bispecific antigen-binding molecules (bispecific antibodies)

[0188] The bispecific antibody described in this article refers to a bispecific antibody composed of a CD28-binding domain (Anti-CD28) and an HBsAg-binding domain (Anti-HBsAg), which can simultaneously act as a conditional agonist of CD28 and a neutralizing antibody against HBsAg. This multispecific antibody conditionally activates CD28 only in the presence of HBsAg, which is highly expressed in HBV-infected hepatocytes. Furthermore, anti-HBsAg antibodies can neutralize HBsAg in serum, inhibiting further HBV infection. This combination provides a mechanism of action that both enhances the antiviral response and limits the systemic toxicity of CD28 agonists.

[0189] The CD28 antibody / binding domain (Anti-CD28) or fragment thereof of this application can be in various forms with CD28 binding activity, such as IgG structure, Fab form, scFab form, F(ab'), scFv or si-scFv form, and can be linked with various forms of second antigen-binding molecules to form bispecific antigen-binding molecules.

[0190] The HBsAg antibody / binding domain (Anti-HBsAg) or fragment thereof of this application can be in various forms with HBsAg binding activity, such as IgG structural form, Fab form, scFab form, F(ab'), scFv or si-scFv form, and can be linked with various forms of second antigen-binding molecules to form bispecific antigen-binding molecules.

[0191] In this invention, the bispecific antibody also includes its conserved variants, which are polypeptides formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the bispecific antibody of this invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0192] Table A

[0193] Virus-associated antigens

[0194] The term "virus-associated antigen" in this application refers to antigens associated with various known viruses. Viruses include, based on genetic material classification (DNA viruses, RNA viruses, protein viruses such as prions); based on viral structure classification (euviruses and subviruses, including viroids, virusoids, and prions); based on host type classification (bacteriophages (bacterial viruses), plant viruses (such as tobacco mosaic virus), animal viruses (such as avian influenza virus, smallpox virus, HIV, etc.); and based on morphology (spherical viruses, baculoviruses, brick-shaped viruses, coronaviruses (such as SARS-CoV-2), filamentous viruses, rotaviruses, enveloped spherical viruses, viruses with spherical heads, and insect viruses encapsulated within inclusion bodies). These antigens can be antigens presented by the virus itself or antigens formed after the virus infects cells.

[0195] The term "hepatitis B virus" as used herein is interchangeable with "HBV" and refers to the well-known hepatotropic DNA virus belonging to the category of hepatic DNA viruses. The HBV genome is a partially double-stranded circular DNA with overlapping reading frames. Based on size, there are four transcripts encoded by the HBV genome (which may be referred to herein as "genes" or "open reading frames"). These contain open reading frames called C, X, P, and S. The core protein is encoded by gene C (HBcAg). Hepatitis B e antigen (HBeAg) is produced through the proteolytic processing of the pre-core (pre-C) protein. DNA polymerase is encoded by gene P. Gene S is the gene encoding the surface antigen (HBsAg). The HBsAg gene is a long open reading frame containing three in-frame "start" (ATG) codons, resulting in three polypeptides of different sizes called large, medium, and small S antigens, namely large surface antigen, medium surface antigen, and small surface antigen, pre-S1+pre-S2+S, pre-S2+S, or S. In addition to decorating the HBV envelope, the surface antigen is also part of the subviral particle. It is produced in much larger quantities than the viral particle and plays a role in immune tolerance and isolation of anti-HBsAg antibodies, thereby allowing infectious particles to evade immune detection.

[0196] Tumor antigens

[0197] In this application, "tumor antigen" refers to antigenic substances that newly emerge or are overexpressed during the occurrence and development of tumors. Based on their specificity, tumor antigens are classified into tumor-specific antigens and tumor-associated antigens.

[0198] Tumor-specific antigens (TSA) are novel antigens that are unique to tumor cells or exist only in certain tumor cells and not in normal cells. These antigens are confirmed through tumor transplantation between syngeneic animals, hence they are also called tumor-specific transplantation antigens (TSTA) or tumor rejection antigens (TRA). Chemically or physically induced tumor antigens, spontaneous tumor antigens, and virus-induced tumor antigens are among the majority of these.

[0199] Tumor-associated antigens (TAAs) are antigens that are not specific to tumor cells but are also present on normal cells and other tissues, although their levels increase significantly during cell carcinogenesis. These antigens only exhibit quantitative changes and do not possess strict tumor specificity. Embryonic antigens are a typical example.

[0200] Tumor antigens are classified according to the induction and occurrence of tumors: tumor antigens induced by chemical or physical factors, tumor antigens induced by viruses, antigens of spontaneous tumors, embryonic antigens, differentiation antigens, and overexpressed antigens.

[0201] Pharmaceutical Composition

[0202] Another aspect of the present invention provides a composition comprising the above-described bispecific antibody capable of specifically binding to HBsAg and CD28 and one or more pharmaceutically acceptable carriers, diluents, or excipients. Preferably, the composition is a pharmaceutical composition.

[0203] The bispecific antibodies provided by this invention can be combined with pharmaceutically acceptable carriers to form drug formulations that exert their therapeutic effects more stably. These formulations ensure the conformational integrity of the amino acid core sequence of the bispecific antibodies of this invention, while also protecting the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamination, or oxidation). Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, with a pH typically around 5-8, preferably around 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated drug composition can be administered via conventional routes, including (but not limited to): intravenous injection, intravenous infusion, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (e.g., intraperitoneal), intracranial injection, or intracavitary injection. Generally, liquid formulations are stable for at least one year at 2°C-8°C, and lyophilized formulations are stable for at least six months at 30°C. The bispecific antibody formulations can be commonly used in the pharmaceutical industry, such as suspensions, injections, and lyophilized formulations.

[0204] In this invention, the term "pharmaceutical composition" refers to the bispecific antibody of this invention that can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical formulation composition to exert its therapeutic effect more stably. These formulations can ensure the conformational integrity of the amino acid core sequence of the bispecific antibody disclosed in this invention, while also protecting the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamination, or oxidation).

[0205] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described bispecific antibody (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. Furthermore, the bispecific antibody of the present invention can also be used with other therapeutic agents.

[0206] When using a pharmaceutical composition, a safe and effective amount of the bispecific antibody or its immunoconjugate is administered to a mammal. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is between about 10 micrograms per kilogram of body weight and about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0207] application

[0208] In another aspect, the present invention provides the use of the above-mentioned bispecific antibody that can specifically bind to HBsAg and CD28, or the above-mentioned pharmaceutical composition, in the preparation of a medicament for treating diseases caused by HBV infection.

[0209] The diseases caused by HBV infection described in this invention preferably include, but are not limited to: hepatitis, liver fibrosis, cirrhosis, liver cancer, or combinations thereof.

[0210] The bispecific antibodies and their compositions of the present invention can also be administered in combination with other drugs for treating diseases caused by HBV infection to achieve a more effective antiviral effect, including but not limited to: HBsAg-CD3 bispecific antibodies.

[0211] When the bispecific antibody and its composition of the present invention are administered to animals, including humans, the dosage varies depending on the patient's age and weight, disease characteristics and severity, and route of administration. The dosage can be determined with reference to the results of animal experiments and various other factors, but the total dosage should not exceed a certain range.

[0212] Compared with the prior art, the main advantages of the present invention include:

[0213] 1. The present invention provides a humanized CD28 antibody, which is specific and has low immunogenicity.

[0214] 2. This invention provides for the first time a bispecific antibody that can simultaneously target HBsAg and CD28.

[0215] 3. The bispecific antibody of the present invention can conditionally activate CD28 through HBsAg crosslinking on the basis of TCR-CD3 signaling pathway activation, further activating T cells, which can both enhance antiviral response and limit the systemic toxicity of CD28 activation.

[0216] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0217] Example 1. Production of HBsAg / CD28 bispecific antibody

[0218] 1. Humanization of anti-CD28 antibody (9.3mAb)

[0219] The CDR sequence of the mouse 9.3 mAb was analyzed using the Kabat system. The results showed that the amino acid sequences of HCDR1-3 of the mouse 9.3 mAb are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; the VH sequence of the mouse 9.3 mAb is shown in SEQ ID NO:7; the amino acid sequences of LCDR1-3 of the mouse 9.3 mAb are shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; and the VL sequence of the mouse 9.3 mAb is shown in SEQ ID NO:8.

[0220] By chimeric integration of the skeletal regions of the heavy and light chain variable regions of human immunoglobulins, the humanization of mouse-derived 9.3 mAb is improved.

[0221] By comparing the variable region of the mouse 9.3mAb heavy chain with the protein sequences of known human immunoglobulin heavy chains, it was confirmed that the variable region of the mouse 9.3mAb heavy chain has 62.9% homology with human IGHV4-4*08, and can be used as a chimeric receptor (acceptor) for the mouse 9.3mAb heavy chain CDR.

[0222] By comparing the variable region of the mouse 9.3mAb light chain with the protein sequences of known human germline immunoglobulin light chains, it was confirmed that the variable region of the mouse 9.3mAb light chain has 70.7% homology with human germline IGKV7-3*01, and can be used as a chimeric receptor (acceptor) for the mouse 9.3mAb light chain CDR.

[0223] To enhance the affinity of mouse 9.3mAb antibodies, reverse mutations can be performed on some key amino acids in the chimeric heavy chain and light chain backbone regions, respectively.

[0224] SEQ ID NO:9 (humanized 9.3mAb VH1), SEQ ID NO:10 (humanized 9.3mAb VH2), SEQ ID NO:11 (humanized 9.3mAb VH3), SEQ ID NO:12 (humanized 9.3mAb VH4), SEQ ID NO:13 (humanized 9.3mAb VH5), and SEQ ID NO:14 (humanized 9.3mAb VH6) are the amino acid sequences of the variable region of the heavy chain of humanized 9.3mAb.

[0225] SEQ ID NO:15 (humanized 9.3mAb VL1), SEQ ID NO:16 (humanized 9.3mAb VL2), SEQ ID NO:17 (humanized 9.3mAb VL3), SEQ ID NO:18 (humanized 9.3mAb VL4), SEQ ID NO:19 (humanized 9.3mAb VL5), and SEQ ID NO:20 (humanized 9.3mAb VL6) are the amino acid sequences of the variable region of the light chain of the humanized 9.3mAb. Table 1 lists the amino acid sequences of the 9.3mAb before and after humanization.

[0226] Table 1. Amino acid sequence listing of 9.3 mAb

[0227] Table 2 shows the binding affinity constants of chimeric 9.3mAb and humanized 9.3mAb with human CD28, with KD values ​​of 6.146^6. -9 M and 1.09~1.63^ -9 M. Compared to the chimeric 9.3 mAb, the affinity level of the humanized 9.3 mAb was increased by 2 times, a result consistent with the results of the ELISA binding assay (see Figure 2).

[0228] Table 2. Affinity characterization of humanized 9.3 mAb

[0229] 2. Construction and production of expression vectors for HBsAg / CD28 bispecific antibodies

[0230] Previously, we commissioned Zhixiang Biotechnology to prepare more than twenty structures of HBsAg / CD28 bispecific antibodies, and obtained BsAb2-1, BsAb2-36, and BsAb2-37 lead molecules through physicochemical characterization and high-throughput reporter gene screening. We also commissioned Genewiz to synthesize the DNA sequences encoding humanized 9.3 mAbs of BsAb2-1, BsAb2-36, and BsAb2-37 bispecific antibodies, and subcloned them into the pcDNA3.1 vector using the restriction enzyme sites XbaI and NheI, respectively.

[0231] The BsAb2-1 series of bispecific antibodies are fused to the N-terminus of the heavy chain of HBsAg / C8 scFv (SEQ ID NO:30) and either mouse 9.3 mAb (bispecific antibody number BsAb2-1) or humanized 9.3 mAb (bispecific antibody numbers BsAb2-1-001 to BsAb2-1-036). Their structures are shown in Figure 1B, and schematic diagrams are shown in Equation 2. Specifically, the corresponding light and heavy chain sequences of each bispecific antibody, as well as the combinations of humanized VH and VL of the 9.3 antibody, are shown in Table 3. Taking BsAb2-1-001 as an example, the CD28 antibody is a humanized 9.3 mAb, the scFv of HBsAg is C8, and the heavy chain (abbreviated as HC, specific amino acid sequences are detailed in the HC sequence on the left side of Table 3) is arranged from the N-terminus to the C-terminus as VH. HBS -L2-VL HBS -L1-VH CD28 -CH1-CH2-CH3, the light chain (abbreviated as LC, see the LC sequence on the left side of Table 3 for details) is VL. CD28 -CL, where the last two identical heavy and light chains form a polymer via disulfide bonds; where VH HBS -L2-VL HBS That is, C8 scFv (SEQ ID NO:10) is a single-chain antibody against HBsAg.

[0232] The BsAb2-37 series of bispecific antibodies are synthesized by fusing 9.3CrossFab with HBsAg antibody G12, i.e., mouse 9.3VH-CL. Lamda (Bispecific antibody BsAb2-37) or humanized 9.3VH-CL LamdaThe fragments of (bispecific antibodies BsAb2-37-001 to BsAb2-27-036) are fused to the N-terminus of the heavy chain of G12, and their structures are shown in Figure 1A(a) or as shown in Formula 1. Specifically, the corresponding light and heavy chain sequences of each bispecific antibody and the combination of humanized VH and VL of the 9.3 mAb are shown in Table 3. For example, in BsAb2-37-001, the CD28 antibody is a humanized 9.3 mAb, the HBsAg antibody is G12, and the heavy chain (abbreviated as HC, the specific amino acid sequence is detailed in Table 3) is arranged from the N-terminus to the C-terminus as VH. CD28 -CL Lamda -L1-VH HBS -CH1-CH2-CH3, the first light chain (abbreviated as VL-CH1) is VL. CD28 -CH1, the second light chain (LC) is G12 VL HBS -CL Kappa The last three peptide chains form a polymer through disulfide bonds.

[0233] Similarly, the BsAb2-36 series of bispecific antibodies are synthesized by fusing G12 CrossFab with anti-CD28 antibody 9.3, i.e., G12 VH-CL. Lamda The fragment is fused to the N-terminus of the heavy chain of a murine or humanized 9.3 mAb antibody (bispecific antibody BsAb2-36 and its derivatives BsAb2-36-001 to BsAb2-36-036), and its structure is shown in Figure 1A(b) or a schematic diagram as shown in Equation 3. Specifically, the corresponding light and heavy chain sequences of each bispecific antibody and the combination of humanized VH and VL of the 9.3 mAb are shown in Table 3. For example, in BsAb2-36-001, the CD28 antibody is a humanized 9.3 mAb, the HBsAg antibody is G12, and the heavy chain (HC) is arranged from the N-terminus to the C-terminus as VH. HBS -CL Lamda -L1-VH CD28 -CH1-CH2-CH3, the first light chain (abbreviated as VL-CH1) is VL. HBS -CH1, the second light chain (LC for short) is VL CD28 -CL Kappa The last three peptide chains form a polymer through disulfide bonds.

[0234] Table 3. Light and heavy chain sequences of the corresponding bispecific antibodies, and combinations of humanized VH and VL forms of anti-CD28 antibodies.

[0235] 3. Expression in Expi293 cells

[0236] Bispecific antibodies were expressed using a transient co-transfection method employing the Expi293 cell expression system (Invitrogen) to transfect plasmids encoding the antibody light and heavy chains (heavy chain and heavy chain variants were transfected with light chain and light chain variants in a 1:1 ratio). In short, a mixture of plasmids encoding the antibody light and heavy chains and Expi293 fectin (Invitrogen) was transfected into serum-free Expi293 cells and cultured in suspension in shaking Erlenmeyer flasks. 100 mL of 1x10 6 Expi293 cells / mL were seeded in 1L shaking Erlenmeyer flasks and cultured at 125 rpm and 8% CO2. The next day, the Expi293 cell density reached 2.5 x 10⁻⁶ cells / mL. 6 When the cell count / mL and viability >95%, prepare 10 mL of Opti-MEM (Invitrogen) and add 100 μg of total plasmid DNA (1 μg / mL, 50 μg heavy chain plasmid and 50 μg light chain plasmid), which is solution A; prepare another 10 mL of Opti-MEM and add 260 μL of Expi293 fectin (2.6 μL / mL), which is solution B; after incubating the mixture of solution A and solution B at room temperature for 20 minutes, slowly add Expi293 cell culture medium. Add enhancers 1 and 2 according to the instructions on the second day after transfection. After 5 days of suspension culture, collect the supernatant containing secreted antibodies, or purify the antibody directly from the supernatant, or freeze the supernatant.

[0237] 4. Purification

[0238] Proteins were purified from filtered cell culture supernatant using standard affinity chromatography procedures. In short, BsAb2-1, BsAb2-36, and BsAb2-37 were purified using 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). After obtaining the target proteins at pH 3.0, the sample solution pH was immediately adjusted to 7.0. Protein aggregates were removed using size exclusion chromatography (Superdex200, GE Healthcare) with PBS (pH 7.2) or 20 mM histidine, 50 mM NaCl (pH 5.5) as the mobile phase to improve antibody purity. If necessary, samples could be pooled and concentrated using a MillIPORE Amicon Ultra (30 MWCO) ultrafiltration tube to obtain antibody standards, which were then aliquoted and stored at -20°C or -80°C. We can also provide some samples for protein analysis and characterization using SDS-PAGE or SEC-HPLC.

[0239] 5. Protein assay

[0240] The concentration of antibody standards was determined at a wavelength of 280 nm. In addition, the molar extinction coefficient could be calculated based on the antibody amino acid sequence (Pace et al., Protein Science, 1995, 4, 2411-1423).

[0241] Example 2. Purity characterization of HBsAg / CD28 bispecific antibody

[0242] Using an HPLC system (Agilent Technologies 1260 Infinity II) and X Bridge A SEC column (3.5 μm, 7.8 × 300 mm, Waters Corporation) was used to determine the purity of the bispecific antibody generated in Example 1 by size exclusion chromatography, and the molecular weight was determined by SEC-HPLC. Mobile phase A consisted of 67 mM Na₂HPO₄, 33 mM NaH₂PO₄, and 100 mM Na₂SO₄, with a pH of 7.0. The flow rate was set to 0.5 mL / min, with 100% mobile phase A, and the column temperature was 30 °C for 30 minutes. 100 μg of protein was injected into the column and detected at 280 nm and 10 Hz. Data processing was performed using CDS2 (Agilent).

[0243] Figure 3 shows the high purity of the BsAb2-1-006, BsAb2-36-006, and BsAb2-37-006 antibodies, indicating that these molecules have good pharmaceutical development potential.

[0244] Example 3. Surface plasmon resonance derivatization to characterize the binding affinity of HBsAg / CD28 bispecific antibody.

[0245] The binding affinity of the HBsAg / CD28 construct to HBsAg and human CD28 was characterized using a Biacore T200. Specifically, the CM5 Biacore sensor, via amine conjugation to Peirce™ protein A, captured the HBsAg / CD28 bispecific antibody. The experiment was performed in an HBS-EP buffer consisting of 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, and 0.05% v / v surfactant P20. Different concentrations of HBsAg dilutions (from 50 nM to 800 nM, 3-fold dilution) and different concentrations of human CD28 dilutions (from 6.25 nM to 100 nM, 3-fold dilution) were prepared using HBS-EP buffer and injected at a flow rate of 30 μL / min. Binding of HBsAg or human CD28 to the captured bispecific antibody was monitored over 120 seconds, and dissociation of HBsAg or human CD28 was monitored over 300 seconds. Regeneration conditions were 30 μL / min x 30 sec, pH 1.5 Gly. All binding kinetic experiments were conducted at 25 °C. A 1:1 binding model was fitted using Biacore T200 Evaluation software 3.2 to determine the kinetic binding (Ko) and dissociation (Kd) rate constants, which are summarized in Table 4.

[0246] Table 4. Binding affinity of HBsAg / CD28 bispecific antibody to human CD28 and HBsAg

[0247] Figure 4A shows the binding kinetics of BsAb2-1-006 and BsAb2-37-006 constructs with HBsAg, with KD values ​​of 2.06 and 2.06, respectively. ^-9 M and 2.49 ^-9 M. Figure 4B shows the binding kinetics of BsAb2-1-006 and BsAb2-37-006 constructs to human CD28, with KD values ​​of 7.12 and 7.12, respectively. ^-9 M and 1.09 ^-8 M. Furthermore, the binding kinetics of the BsAb2-36-006 construct to HBsAg are similar to those of the BsAb2-37-006 construct.

[0248] Example 4. Cell-binding activity of HBsAg / CD28 bispecific antibody

[0249] The binding characteristics of the HBsAg / CD28 bispecific antibody generated in Example 1 to CD28 on the surface of human T cell line and HBsAg on the surface of human hepatocellular carcinoma cell line HepG2-LMS-660 were analyzed using flow cytometry. Target cells were harvested and the cell density was adjusted to 2 x 10⁻⁶ cells in FACS buffer (PBS containing 2% BSA). 6 Cells / mL. 50 μL of cell suspension was co-incubated with 50 μL of BsAb2-1-006, BsAb2-36-006, and BsAb2-37-006 diluted in FACS buffer (final concentrations from 300 nM to 0.017 nM). Parental monoclonal antibodies C8 mAb, G12 mAb, and 9.3 mAb served as positive controls, and human IgG isotype antibody as a negative control. The mixture was then aliquoted into 50 μL wells of a 96-well U-type plate. After incubation at room temperature for 60 minutes, the cells were centrifuged (3 min, 300 x g) and washed twice with 150 μL / well FACS buffer. The cells were resuspended in 100 μL of 5000-fold diluted APC-labeled anti-human Fc secondary antibody (Jackson, #615-605-214), incubated at room temperature for 30 minutes, and then washed three times with 150 μL / well FACS. Finally, cells were collected by centrifugation, resuspended in 100 μL of FACS buffer, analyzed by flow cytometry (Beckman), and the data were processed using FlowJo and GraphPad Prism.

[0250] The binding affinity of the BsAb2-1 series of bispecific antibodies to HBsAg antigens (HBsAg P1, HBsAg P2, HBsAg P3) and human CD28 antigens (hCD28 P1, hCD28 P2, hCD28 P3) was validated by ELISA assays. The results showed that the binding affinity of different humanized 9.3 antibodies to recombinant human CD28 protein was comparable to that of mouse 9.3. Furthermore, the binding affinity of the BsAb2-1 series of bispecific antibodies to recombinant HBsAg protein remained unchanged (Figure 5).

[0251] Figure 6A shows that the EC50 value of BsAb2-1-006 binding to the HBsAg recombinant protein is 0.061 nM, which is comparable to the binding activity of its parent C8mAb (EC50 value = 0.08 nM). Similarly, the EC50 of BsAb2-37-006 binding to recombinant HBsAg protein was 0.094 nM, slightly better than its parent G12 mAb (EC50 = 0.118 nM). For the CD28 arm, the EC50 values ​​of BsAb2-1-006 and BsAb2-37-006 binding to recombinant human CD28 protein were 0.103 nM and 0.155 nM, respectively, while the EC50 of its parent mAb 9.3 was 0.061 nM. The EC50 values ​​of BsAb2-36-006 binding to recombinant HBsAg protein and recombinant human CD28 protein were 1.10 nM and 0.27 nM, respectively. These results are highly consistent with the binding affinity characterization results of the HBsAg / CD28 bispecific antibody.

[0252] Figure 6B shows that the EC50 value of BsAb2-1-006 binding to HBsAg(+) HepG2-LMS-660 cells was 0.2829 nM, slightly better than the EC50 value of its parent C8 mAb (0.5017 nM). Similarly, the EC50 value of BsAb2-37-006 binding to HepG2-LMS-660 cells was 0.1129 nM, while the EC50 value of its parent G12 mAb was 0.1388 nM. Regarding the CD28 arm, the EC50 values ​​of BsAb2-1-006 and BsAb2-37-006 binding to CD28(+) human T cells were 0.2834 nM and 0.1043 nM, respectively, while the EC50 of its parent mAb 9.3 was 0.016 nM. Furthermore, the binding affinity of BsAb2-36-006 to HBsAg(+)HepG2-LMS-660 cells / human CD3+ T cells was similar to that of BsAb2-37-006. This result is highly consistent with the binding affinity characterization results of the HBsAg / CD28 bispecific antibody.

[0253] Example 5. HBsAg / CD28 bispecific antibody promotes HBsAg-specific T cell activation

[0254] Jurkat engineered cells, which incorporate the firefly luciferase gene stably expressed under the control of NFAT response elements, are generated by integrating this gene into the genome of Jurkat cells. This technology can be used to evaluate HBsAg / CD28 bispecific antibody-supported TCR-CD3 stimulation-mediated T cell activation and nonspecific activation of the NFAT signaling pathway. Specifically, NFAT-Luc Jurkat cells were resuscitated and grown in RPMI-1640 medium containing 10% fetal bovine serum and 1 mg / mL G418. For high-throughput screening, cells were collected and adjusted to a density of 2 x 10⁶ cells using 384 plates pre-coated with 2 μg / mL HBsAg on a white, transparent bottom (incubated at 37°C for 2 hours). 6 Jurkat cells were seeded at a rate of 20 μL per well into 384-well plates and co-cultured with 0.1 nM HBsAg / CD3 bispecific antibody (BsAb2-6-011) in combination with different dilutions of HBsAg / CD28 bispecific antibody (concentrations from 27 nM to 0.001 nM). To ensure that the HBsAg / CD28 bispecific antibody strictly depends on the TCR-CD3 signaling pathway and HBsAg bridging to induce the NFAT reporter gene, HBsAg pre-coated wells containing 27 nM HBsAg / CD28 bispecific antibody but not HBsAg / CD3 bispecific antibody, or HBsAg uncoated wells containing 27 nM HBsAg / CD28 and HBsAg / CD3, were used as negative controls. After incubation at 37°C and 5% CO2 for 6 hours, 15 μL of LOneGlo luciferase substrate (Promega#E6120) was added to each well. Fluorescence was measured on a TECAN INFINITE M PLEX plate reader after 5 minutes of incubation, and the readout data was processed using GraphPad Prism.

[0255] As shown in Figure 7, BsAb2-1 series bispecific antibodies and HBsAg / CD3 bispecific antibodies can jointly stimulate NFAT expression levels in T cells, and the enhancement effects of different humanized BsAb2-1 molecules show similar dose-dependent effects.

[0256] The results, shown in Figures 8A and 8B, reveal that the expression levels of NFAT in T cells stimulated by both HBsAg / CD28 and HBsAg / CD3 bispecific antibodies enhance HBsAg / CD3 bispecific antibody-induced T cell activation in a dose-dependent manner. The EC50 values ​​of BsAb2-1-006 and BsAb2-37-006 were 0.12 and 0.45 nM, respectively. No T cell activation was observed in the absence of either HBsAg or HBsAg / CD3 bispecific antibodies. Furthermore, BsAb2-36-006 exhibited a similar effect to BsAb2-37-006 in promoting HBsAg-specific T cell activation.

[0257] Example 6. HBsAg / CD28 bispecific antibody can promote HBsAg-specific T cell response.

[0258] The ability of the HBsAg / CD28 bispecific antibody generated in Example 1 to enhance the proliferation of HBsAg / CD3 bispecific antibody-mediated human primary T cells under HBsAg cross-linking was detected by flow cytometry. U-bottom 96-well plates were pre-coated with 2 μg / mL recombinant HBsAg protein (incubated at 37°C for 2 hours); simultaneously, human peripheral blood mononuclear cells (PBMCs) were rapidly thawed in a 37°C water bath and cultured at 1 x 10⁻⁶ cells / well. 6 PBMCs were transferred to preheated RPMI-1640 medium at a density of cells / mL and labeled with 1 μM CellTrace Violet. The labeled PBMCs were resuspended in RPMI-1640 complete medium and the cell density was adjusted to 2 x 10⁻⁶ cells / mL. 6Cells / mL. Simultaneously, remove the HBsAg coating solution from the 96-well plate and wash three times with PBS. Add 50 μL of PBMC cell suspension to each well, along with 0.1 nM HBsAg / CD3 bispecific antibody (BsAb2-6-011) and different concentrations of diluted HBsAg / CD28 bispecific antibody (from 9 nM to 0.004 nM). To ensure that the HBsAg / CD28 bispecific antibody strictly depends on HBsAg bridging and co-stimulates T cell proliferation with the CD3 signaling pathway, HBsAg pre-coated wells containing 9 nM HBsAg / CD28 but not HBsAg / CD3, or HBsAg uncoated wells containing 9 nM HBsAg / CD28 and 0.1 nM HBsAg / CD3 bispecific antibody, served as negative controls. After incubation at 37°C and 5% CO2 for 3 days, cells were collected by centrifugation (5 minutes, 300x g), stained with APC-labeled anti-human CD4, FITC-labeled anti-human CD8, and PE-labeled anti-human CD25 antibodies, and washed twice with FACS buffer. The expression levels of CD25 and Violet in (Beckman) CD4+ T cells, CD8+ T cells, and total T cells were detected by flow cytometry, and the data were processed using FlowJo and GraphPad Prism.

[0259] The results are shown in Figure 9. The percentages of CD25 and Violet-positive T cells stimulated by both HBsAg / CD28 and HBsAg / CD3 bispecific antibodies indicate that both antibodies induced T cell proliferation in a dose-dependent manner. Figure 9A shows the dose-dependent increase in the percentage of CD25+CD4+ T cells mediated by BsAb2-1-006, BsAb2-37-006 combined with BsAb2-6-011. Figure 9B shows the dose-dependent increase in the percentage of CD25+CD8+ T cells mediated by BsAb2-1-006, BsAb2-37-006 combined with BsAb2-6-011. Figure 9C shows the dose-dependent proliferation of CD4+ T cells mediated by BsAb2-1-006, BsAb2-37-006 combined with BsAb2-6-011. Figure 9D shows that the CD8+ T cell proliferation mediated by BsAb2-1-006, BsAb2-37-006 in combination with BsAb2-6-011 was dose-dependent. Furthermore, BsAb2-36-006 exhibited a similar T cell proliferation-inducing effect to BsAb2-37-006.

[0260] Example 7. HBsAg / CD28 bispecific antibody promotes the lysis of HBsAg(+)HepG2-LMS-660 target cells by human T cells.

[0261] The cytotoxic effect of HBsAg / CD28 combined with HBsAg / CD3 bispecific antibody-induced T cell cytotoxicity against HBs(+) target cells was monitored in real time using the xCELLigence Real-Time Cell Analyzer (RTCA, Aligent). Specifically, E-Plate 96 culture plates were coated with Collagen dilution (PBS 1:30 dilution) and incubated at 37°C for 30 min. The RTCA was started, the assay program was set, and 50 μL of M10 complete cell culture medium was added to each well of the pre-coated E-Plate 96 culture plate. Baseline measurements were taken. HepG2-LMS-660 or HepAD38 cells in logarithmic growth phase were collected by trypsin digestion with 0.25% trypsin, resuspended in M10 complete culture medium, and the cell concentration was adjusted to 2 x 10⁻⁶ cells / well. 5 Cells / mL, at 100 μL / well, 2 x 10 4 Target cells were added per well to 96-well E-Plate microplates (E-plates), incubated for 5 min, and then subjected to RTCA for 16 hours to continuously monitor cell growth. The next day, resuspended human PBMCs were collected and the density adjusted to 4 x 102 6 Cells / mL; simultaneously prepare 3-fold diluted M10 complete medium containing HBsAg / CD3 bispecific antibody (concentration range 27 nM to 0.012 nM) alone or with serially diluted HBsAg / CD28 bispecific antibody (0.003 nM / 0.01 nM BsAb2-37-006 or 0.1 nM / 0.3 nM BsAb2-1-006, respectively); co-culture 50 μL of effector T cells (effector cells: target cells 10:1) and 50 μL of serially diluted HBsAg / CD3+HBsAg / CD28 bispecific antibody with HBs(+) target cells. HBsAg(-) HepG2 cells mixed with HBsAg / CD3+HBsAg / CD28 antibody serve as a negative control. All treatment groups were continuously monitored for cell growth at a final volume of 200 μL using RTCA for 72 hours. The RTCA assay was then stopped, the files were saved, and the continuous cell growth curve (Plot) was exported.

[0262] Figure 10A shows the growth of HBsAg(+) HepG2-LMS-660 cells monitored by XCelligence. The results indicate that BsAb2-6-011 monotherapy successfully induced T cell activation and specifically lysed HepG2-LMS-660 cells. Regarding combination therapy, treatment with the suboptimal dose of BsAb2-6-011 in combination with BsAb2-1-006 and BsAb2-37-006 produced a strong synergistic effect, with a target cell killing rate of 100%. For HepAD38 cells (Figure 10B), BsAb2-6-011 monotherapy failed to adequately induce T cell activation and lead to effective specific target cell lysis, possibly due to low S antigen expression levels on the cell surface. However, when the suboptimal dose of BsAb2-6-011 was used in combination with BsAb2-1-006 and BsAb2-37-006, respectively, a strong synergistic effect was observed, with a 100% killing rate of HepAD38. In the absence of BsAb2-6-011, neither BsAb2-1-006 nor BsAb2-37-006 showed T cell activation capacity, demonstrating that the synergistic stimulatory effect of the HBsAg / CD28 bispecific antibody is strictly dependent on the presence of the TCR-CD3 signaling pathway (provided by the suboptimal concentration of BsAb2-6-011). BsAb2-6-011 monotherapy, BsAb2-6-011 in combination with BsAb2-1-006, or BsAb2-37-006 failed to induce non-specific killing of HBs(-) negative HepG2 cells by T cells. In addition, BsAb2-36-006 has a similar effect to BsAb2-37-006 in inducing T cells to kill target cells.

[0263] Example 8. Neutralizing activity of HBsAg / CD28 bispecific antibody in an HBV-infected Huh7-NTCP cell model

[0264] The in vitro neutralizing potency of the HBsAg / CD28 bispecific antibody generated in Example 1 was evaluated using an HBV-infected Huh7-NTCP cell model. Huh7-NTCP cells were collected in preheated DMEM + 10% FBS + 2.5% DMSO medium, and the cell density was adjusted to 3 x 10⁻⁶ cells / mL. 6 cells / mL. Add 100 μL / well (1 x 10⁻⁶ cells / mL). 4Huh7-NTCP cells (per well) were seeded into collagen-pre-coated 96-well plates and cultured overnight. The next day, HBV virus dilutions were prepared and incubated with 100, 10, or 0 nM BsAb2-1-006, BsAb2-37-006, C8 mAb, or G12 mAb (HBV virus only as a system control), and then added sequentially to the Huh7-NTCP cell wells. The next day, HBV virus was removed, and Huh7-NTCP cells were cultured in fresh DMEM complete medium for 5-10 days. The concentrations of HBeAg and HBsAg in the supernatant were determined according to the instructions of the HBeAg / HBsAg ELISA kit (readings represent neutralization capacity).

[0265] Figures 11A and 11B show that both BsAb2-1-006 and BsAb2-37-006 exhibited strong neutralizing activity at a dose of 100 nM, successfully inhibiting HBsAg secretion. The HBsAg level in the BsAb2-1-006 treatment group was as low as 0.06 IU / mL, and the HBsAg level in the BsAb2-37-006 treatment group decreased to 0.013 IU / mL, consistent with the neutralizing activity of the parental C8 mAb and G12 mAb. Similarly, both BsAb2-1-006 and BsAb2-37-006 successfully inhibited HBeAg secretion, reducing HBeAg levels to 0.016 and 0.055 PEIU / mL, respectively. In contrast, the HBeAg level in the HBV virus group (0 nM) was approximately 4 PEIU / mL, indicating successful infection with this detection system. In addition, BsAb2-36-006 has similar antibody neutralizing activity to BsAb2-37-006.

[0266] Example 9. Antiviral activity of HBsAg / CD28 bispecific antibody in HBV-infected primary human hepatocytes

[0267] To evaluate the in vitro antiviral activity of HBsAg / CD28 combined with HBsAg / CD3 bispecific antibody-mediated human T cell immunity, HBV-infected primary human hepatocytes were co-cultured with the antibody, and their killing ability was monitored. Specifically, pre-warmed medium (InvitroGRO CP medium + 10% FBS + 1% PS) was added to the thawed primary human hepatocytes, and the cell density was adjusted to 8 x 10⁶ cells / mL. 5 cells / mL. 100 μL / well (8 x 10⁻⁶ cells / mL). 4PHH cells were seeded at a density of 10 cells / well into collagen-pre-coated 96-well plates and cultured overnight. On the day of infection, HBV virus was diluted with PHH complete medium containing 4% PEG8000 and added to the PHH wells. On the second day, HBV virus was removed, and PHH cells were cultured in fresh complete medium for 7–10 days. The HBeAg concentration in the supernatant was measured at regular intervals (read as infection rate) to monitor the viral infection stage. On day 11, serially diluted BsAb2-6-011 (100, 33, 11, 3.7, 1.2, 0 nM) was mixed with BsAb2-1-006 (10 and 50 nM) or BsAb2-37-006 (2 and 10 nM) and added to the infected PHH cell wells, along with 2.4 x 10⁻⁶ cells / well. 5 PBMCs / wells (effective cells:target cells:3:1), with uninfected PHH cells serving as a systemic control. After 7 days of co-culture, PHH cell lysis was assessed using a CellTiter-Glo luminescent cell viability assay kit (Promega, C7570); the expression level of CD69 on the surface of T cells was detected by flow cytometry; and the secretion of cytokines (R&D system, DY285B and DY202) and viral markers (AntuBio Diagnostics) in the co-culture supernatant was quantified using an enzyme-linked immunosorbent assay kit according to the manufacturer's instructions.

[0268] Figure 12A shows that BsAb2-6-011 only partially induced T cell activation and specific PHH lysis. The addition of BsAb2-1-006 or BsAb2-37-006 as co-stimulatory signals showed a strong synergistic effect on PHH cell lysis, with a killing rate as high as 60%–80%. This is consistent with the increase in CD69 levels on the T cell surface and the secretion of cytokines in the supernatant (Figures 12B and 12C). Regarding antiviral activity, the combined treatment group showed a significant decrease in HBeAg, accompanied by a decrease in HBsAg (Figures 12D and 12E). This indicates that the T-cell cytotoxicity mediated by the combined HBsAg / CD28 and HBsAg / CD3 bispecific antibodies has a significant clearance effect on HBV-infected PHH, meaning that the combined T-cell immune response mediated by the combined HBsAg / CD28 and HBsAg / CD3 bispecific antibodies can effectively control HBV infection. Furthermore, in HBV-infected primary human hepatocytes, BsAb2-36-006 exhibits similar antiviral activity to BsAb2-37-006.

[0269] Example 10. Antiviral activity of HBsAg / CD28 bispecific antibody in a human liver / immune double-humanized mouse model infected with HBV.

[0270] To evaluate whether combination therapy with HBsAg / CD3 and HBsAg / CD28 bispecific antibodies is effective in treating HBV infection, its antiviral activity was evaluated in a human liver / immune dual-humanized mouse model of HBV infection.

[0271] The references report that 1x10 6 Human hepatocytes were transplanted into homologous USG mice, ultimately resulting in human liver chimeric urokinase-type plasminogen activator deficiency (uPA). - / - Severe immunodeficiency (SCI) / interleukin-2 receptor γ chain defective (IL2Rr- / -) mice (USG mice). Transplantation success rate was estimated by measuring human serum albumin (HSA) in the mouse serum. Animals with high levels of human chimerism (HSA > 2 mg / mL in serum) were selected for HBV peritoneal injection (1 x 10⁻⁶ mg / mL). 7 HBV gene copy number (in mice) was used to construct a human liver chimeric mouse model infected with HBV. Six weeks after virus inoculation, 2 x 10⁻⁶ HBV gene copies / mouse were injected via tail vein. 6 Human PBMCs underwent immune reconstitution under three different administration regimens: (1) solvent only; (2) 0.5 mpk HBsAg / CD28 (BsAb2-37-006); (3) HBsAg / CD3 and HBsAg / CD28 (0.5 mpk BsAb2-6 and 0.5 mpk BsAb2-37-006). Due to GvHD-induced rejection, this experiment was terminated on day 11 (Figure 13A). The HBsAg / HBeAg levels in mouse serum were measured at different time points using an ELISA kit according to the manufacturer's instructions. DNA and hepatocyte RNA were extracted from mouse serum using a DNA / RNA purification kit (QIAGEN), and HBV DNA copy number and hepatic RNA content were measured by q(RT)-PCR. Phenotypic analysis of peripheral blood T cells was performed using CD3 / CD4 / CD8 staining and flow cytometry.

[0272] Eleven days after treatment, serum HBV DNA decreased by 1.6 log in mice treated with the combination of BsAb2-6 and BsAb2-37-006, while no decrease was observed in the solvent group or the BsAb2-6-011 monotherapy group (Fig. 13B). The combination therapy also resulted in a 2 log decrease in HBsAg levels and a 0.5 log decrease in HBeAg levels (Figs. 13C and 13D). Simultaneously, human serum albumin (HSA) in mice treated with the combination of BsAb2-6-011 and BsAb2-37-006 decreased to 1.86 log, indicating that the combined HBsAg / CD3 and HBsAg / CD28 bispecific antibodies induced T-cell cytotoxicity and effectively cleared virus-infected human hepatocytes (Fig. 13E). HBV pgRNA levels were significantly lower in the combination therapy group compared to the solvent group or BsAb2-6-011 monotherapy (Fig. 13F). The lack of TCR-CD3 primary signaling provided by the HBsAg / CD3 bispecific antibody meant that the BsAb2-37-0006 monotherapy group failed to adequately induce T cell activation and redirection to HBs+ target cells. T cell phenotypic analysis confirmed this hypothesis. As shown in Figure 13G, the percentages of human CD45+PBMCs and CD45+CD3+ T cells demonstrated that human PBMCs were successfully colonized in the livers of all chimeric mice; however, T cell expansion was only observed in the HBs / CD3 and HBs / CD28 co-administration groups.

[0273] Example 11. Antitumor activity of HBsAg / CD28 bispecific antibody in the HepAD38 CDX model

[0274] The antitumor activity of the HBsAg-specific T-cell connective was evaluated using a HepAD38 xenograft CDX model. Previous studies found that different doses of the HBsAg / CD3 bispecific antibody BsAb2-6 (BsAb2-6-011) inhibited tumor cell growth; among them, the 1 mpk BsAb2-6 group had a TGI of 43%, showing significantly better tumor inhibition than the 0.3 mpk BsAb2-6 group. However, this treatment failed to achieve sustained tumor suppression, and all animals eventually died due to tumor burden at different dose levels. These results indicate that the HBsAg / CD3 connective activates T cells through HBsAg cross-linking, thereby producing a limited tumor-suppressive effect, and further enhancement of co-stimulatory signals is needed to further enhance T-cell effector function.

[0275] This embodiment continues to evaluate the ability of BsAb2-1-006 / BsAb2-37-006 to enhance the antitumor activity of HBsAg / CD3 in vivo on the HepAD38 CDX model. The dosing regimen (Figure 14A) was as follows: After tumor establishment, mice were intravenously injected twice weekly with the following treatments: (i) PBS group; (ii) BsAb2-1-006 2 mg / kg; (iii) BsAb2-37-006 2 mg / kg; (iv) BsAb2-6 1 mg / kg and BsAb2-1-006 0.2 mg / kg; (v) BsAb2-6 1 mg / kg and BsAb2-1-006 2 mg / kg; (vi) BsAb2-6 1 mg / kg and BsAb2-37-006 0.2 mg / kg; (vii) BsAb2-6 1 mg / kg and BsAb2-37-006 2 mg / kg. Each group had n = 5. The differences between each drug administration group and the PBS group were calculated using repeated two-way ANOVA (*p<0.05, **p<0.01, ***p<0.0001, ****p<0.0001).

[0276] The results, as shown in Figures 14B and 14C, indicate that both HBsAg / CD28, administered alone and in combination with HBsAg / CD28, effectively delayed tumor progression. At the endpoint, the antitumor activity of BsAb2-6 (1 mpk) and BsAb2-1-006 (2 mpk) was statistically superior to the PBS group (****p<0.0001), but not significantly different from BsAb2-1 monotherapy. The combination of BsAb2-6 (1 mpk) and BsAb2-37-006 effectively controlled tumor progression in all animals, with **p<0.1 and ****p<0.0001 compared to BsAb2-37-006 monotherapy and the PBS group, respectively. Contrary to in vitro results, monotherapy with BsAb2-1-006 and BsAb2-37-006 (1 mpk) showed some antitumor activity. This is attributed to the activation of TCR-CD3 signaling to some extent during CIK cell preparation. On day 10 after treatment initiation, animals in the control group experienced weight loss due to tumor progression (Figure 14D). Mice treated with BsAb2-1-006 / BsAb2-37-006 alone or in combination with BsAb2-6 initially experienced weight loss but subsequently recovered, with no further weight loss due to tumor progression. Furthermore, no drug-induced toxicity was observed in the treated mice in this experiment.

[0277] Furthermore, the number of T cells in the peripheral blood of mice was correlated with the tumor growth inhibition observed after each drug treatment. Compared with the BsAb2-37-006 monotherapy group, the combination therapy of BsAb2-6 and BsAb2-37-006 significantly increased the number of CD4, CD8 and total T cells, *p<0.5 (Figure 14E-G).

[0278] It is evident that the combined use of BsAb2-6 with BsAb2-1-006 / BsAb2-37-006 can effectively control tumor regression.

[0279] In conclusion, HBs / CD3 and HBs / CD28 have been shown to be a novel immunization approach for the prevention and treatment of chronic HBV infection, and warrant further preclinical and clinical research.

[0280] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0281] The sequence described in this article:

Claims

1. A multispecific antibody / antigen binding molecule, characterized in that, The multispecific antibodies include: (1) Humanized CD28 antibody / antigen binding domain that specifically binds to CD28 The humanized CD28 antibody comprises a VH region containing an amino acid sequence as shown in SEQ ID NO:9, 10, 11, 12, 13, or 14, or the humanized CD28 antibody comprises a VH region containing an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identical to the sequence shown in SEQ ID NO:9, 10, 11, 12, 13, or 14. and The humanized CD28 antibody may contain a VL region of an amino acid sequence as shown in SEQ ID NO:15, 16, 17, 18, 19, or 20, or a VL region of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:15, 16, 17, 18, 19, or 20; and (2) Antibody / antigen binding domains that specifically bind to one or more viral / tumor antigens.

2. The multispecific antibody / antigen binding molecule as described in claim 1, characterized in that, The multispecific antibody is a bispecific antibody, and the bispecific antibody includes: a) Humanized CD28 antibody / antigen-binding domain that specifically binds to CD28; and b) A second antigen-binding domain that specifically binds to hepatitis B virus surface antigen (HBsAg). HBsAg includes hepatitis B virus small surface antigen, hepatitis B virus medium surface antigen, or hepatitis B virus large surface antigen.

3. The multispecific antibody / antigen binding molecule as described in claim 2, characterized in that, The bispecific antibody has the structure shown in Formula 1 or Formula 3: in, VH CD28 Heavy chain variable region representing anti-human CD28 antibody; VL CD28 The light chain variable region representing the anti-human CD28 antibody; CL λ(或Lamda) Represents the light chain constant region of the anti-human IgG type; CH1 represents the heavy chain constant region CH1 of the lgG type; VH HBS Heavy chain variable region representing anti-HBsAg antibody; VL HBS The light chain variable region representing the anti-HBsAg antibody; CL K(或Kappa) Represents the constant region of the light chain in the lgG type; CH1, CH2, and CH3 represent the heavy chain constant regions CH1, CH2, and CH3 of the lgG type, respectively; L1 is the connector; "~" represents a disulfide bond or a covalent bond; "-" represents a peptide bond.

4. The multispecific antibody / antigen binding molecule as described in claim 3, characterized in that, The heavy chain variable region of the anti-HBsAg antibody contains the following three complementarity-determining regions (HCDRs): HCDR1 as shown in SEQ ID NO.31; HCDR2 as shown in SEQ ID NO.32; and HCDR3 as shown in SEQ ID NO.33; and The light chain variable region of the anti-HBsAg antibody includes the following three complementarity-determining regions (LCDRs): LCDR1 as shown in SEQ ID NO.34; LCDR2 as shown in SEQ ID NO.35; and LCDR3 as shown in SEQ ID NO.

36.

5. The multispecific antibody / antigen binding molecule as described in claim 3, characterized in that, The heavy chain variable region of the anti-human CD28 antibody contains the following three complementarity-determining regions (HCDRs): HCDR1 as shown in SEQ ID NO.1; HCDR2 as shown in SEQ ID NO.2; and HCDR3 as shown in SEQ ID NO.3; and The light chain variable region of the anti-human CD28 antibody includes the following three complementarity-determining regions (LCDRs): LCDR1 as shown in SEQ ID NO.4; LCDR2 as shown in SEQ ID NO.5; and LCDR3 as shown in SEQ ID NO.

6.

6. The multispecific antibody / antigen binding molecule as described in claim 3, characterized in that, The bispecific antibody includes: An HBsAg antibody comprising the VH region of the amino acid sequence shown in SEQ ID NO:37 and the VL region of the amino acid sequence shown in SEQ ID NO:38; and Anti-human CD28 antibody comprising a VH region containing an amino acid sequence as shown in SEQ ID NO:9, 10, 11, 12, 13, or 14, and a VL region containing an amino acid sequence as shown in SEQ ID NO:15, 16, 17, 18, 19, or 20.

7. The multispecific antibody / antigen binding molecule as described in claim 3, characterized in that, The L1 connector sequence is selected from (GGGGS)n shown in SEQ ID NO:39, (GGGSG)n shown in SEQ ID NO:40, (GSGGG)n shown in SEQ ID NO:41, (GSGGGP)n shown in SEQ ID NO:42, (GGSEPS)n shown in SEQ ID NO:43, (GGEGGGP)n shown in SEQ ID NO:44, (GGEGGGSEGGGS)n shown in SEQ ID NO:45, (GGGSGGGGG)n shown in SEQ ID NO:46, or a combination thereof, wherein each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

8. The multispecific antibody / antigen binding molecule as described in claim 2, characterized in that, The bispecific antibody also includes the structure shown in Formula 2: in, VH CD28 Heavy chain variable region representing anti-human CD28 antibody; VL CD28 The light chain variable region representing the anti-human CD28 antibody; CL represents the light chain constant region CH1 of the lgG type; CH1, CH2, and CH3 represent the heavy chain constant regions CH1, CH2, and CH3 of the lgG type, respectively; VH HBS Heavy chain variable region representing the single-stranded fragment (scFv) that binds HBsAg; VL HBS The light chain variable region represents the single-stranded fragment (scFv) that binds HBsAg; L1 and L2 are each independent connectors; "~" represents a disulfide bond or a covalent bond; "-" represents a peptide bond.

9. The multispecific antibody / antigen binding molecule as described in claim 2 or 8, characterized in that, The heavy chain variable region of the HBsAg-binding single-stranded fragment (scFv) contains the following three complementarity-determining regions (HCDRs): HCDR1 as shown in SEQ ID NO.21; HCDR2 as shown in SEQ ID NO.22; and HCDR3 as shown in SEQ ID NO.23; and The light chain variable region of the HBsAg-binding single-chain fragment (scFv) includes the following three complementary determinant regions (LCDRs): LCDR1 as shown in SEQ ID NO.24; LCDR2 as shown in SEQ ID NO.25; and LCDR3 as shown in SEQ ID NO.

26.

10. The multispecific antibody / antigen binding molecule as described in claim 2 or 8, characterized in that, The heavy chain variable region of the anti-human CD28 antibody contains the following three complementarity-determining regions (HCDRs): HCDR1 as shown in SEQ ID NO.1; HCDR2 as shown in SEQ ID NO.2; and HCDR3 as shown in SEQ ID NO.3; and The light chain variable region of the anti-human CD28 antibody includes the following three complementarity-determining regions (LCDRs): LCDR1 as shown in SEQ ID NO.4; LCDR2 as shown in SEQ ID NO.5; and LCDR3 as shown in SEQ ID NO.

6.

11. The multispecific antibody / antigen binding molecule as described in claim 8, characterized in that, The bispecific antibody includes: A single-stranded fragment (scFv) containing the VH region of the amino acid sequence shown in SEQ ID NO:27 and the VL region of the amino acid sequence shown in SEQ ID NO:28, which binds HBsAg; and Anti-human CD28 antibody comprising a VH region containing an amino acid sequence as shown in SEQ ID NO:9, 10, 11, 12, 13, or 14, and a VL region containing an amino acid sequence as shown in SEQ ID NO:15, 16, 17, 18, 19, or 20.

12. The multispecific antibody / antigen binding molecule as described in claim 8, characterized in that, The L1 linker sequence is selected from the group consisting of (GGGGS)n shown in SEQ ID NO:39, (GGGSG)n shown in SEQ ID NO:40, (GSGGG)n shown in SEQ ID NO:41, (GSGGGP)n shown in SEQ ID NO:42, (GGSEPS)n shown in SEQ ID NO:43, (GGEGGGP)n shown in SEQ ID NO:44, (GGEGGGSEGGGS)n shown in SEQ ID NO:45, (GGGSGGGGG)n shown in SEQ ID NO:46, or combinations thereof, wherein each n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and / or The L2 linker is a "YOL" linker; preferably, the "YOL" linker includes an amino acid sequence such as SEQ ID NO:

29.

13. A polynucleotide molecule, characterized in that, The polynucleotide molecule encodes a multispecific antibody / antigen binding molecule as described in any one of claims 1-12.

14. An expression carrier, characterized in that, The expression vector contains the polynucleotide molecule as described in claim 13.

15. A host cell, characterized in that, The host cell contains the expression vector as described in claim 14 or its genome is integrated with the polynucleotide molecule as described in claim 13.

16. A method for preparing a multispecific antibody / antigen binding molecule as described in any one of claims 1-12, characterized in that, The preparation method includes the following steps: a) Under expression conditions, host cells as described in claim 15 are cultured to express multispecific antibody / antigen binding molecules; b) Isolate and purify the multispecific antibody described in step a).

17. An immunoconjugate, characterized in that, The immunoconjugate includes: (a) the multispecific antibody / antigen binding molecule as described in any one of claims 1-12; and (b) Coupled portions selected from the following group: detectable markers, drugs, toxins, cytokines, radionuclides, and / or enzymes.

18. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises an effective amount of the multispecific antibody / antigen binding molecule as described in any one of claims 1-12 and one or more pharmaceutically acceptable carriers, diluents or excipients.

19. Use of the multispecific antibody / antigen binding molecule as described in any one of claims 1-12, or the pharmaceutical composition as described in claim 18, in the preparation of a medicament for treating diseases caused by HBV infection; Preferably, the disease caused by the anti-HBV infection is selected from: hepatitis, liver fibrosis, cirrhosis, liver cancer, or a combination thereof.

20. A CD28 antibody or its antigen-binding fragment, characterized in that, The antibody or its antigen-binding fragment comprises a VH region of the amino acid sequence shown in SEQ ID NO:9, 10, 11, 12, 13, or 14, or the antibody or its antigen-binding fragment comprises a VH region of the amino acid sequence shown in SEQ ID NO:9, 10, 11, 12, 13, or 14 with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology, and a VL region of the amino acid sequence shown in SEQ ID NO:15, 16, 17, 18, 19, or 20, or the antibody or its antigen-binding fragment comprises a VL region of the amino acid sequence shown in SEQ ID NO:15, 16, 17, 18, 19, or 20 with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology.

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