Bispecific antibody, immunoconjugate, and use thereof

Abstract

Provided is a bispecific antibody targeting HBsAg and DEC-205; the invention further relates to an antibody-drug conjugate containing the antibody, a preparation method therefor, and the use thereof.

Description

Bispecific antibodies and immunoconjugates and their uses

[0001] Cross-referencing

[0002] This application is based on and claims priority to CN application number 202410935164.7, filed on July 12, 2024, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] This application relates to the field of biopharmaceuticals, specifically to a bispecific antibody targeting HBsAg and DEC-205, as well as antibody-drug conjugates containing the antibody, their preparation methods, and applications. Background Technology

[0004] Chronic hepatitis B virus (HBV) infection is a major cause of liver fibrosis, cirrhosis, and liver cancer, posing a significant threat to global public health. Currently, clinical treatments for chronic HBV infection mainly consist of two classes of drugs: interferons and nucleoside / nucleotide analogs (NAs). While these drugs and their combinations can control viral replication and alleviate disease progression to some extent, they are unlikely to achieve a clinical cure for chronic hepatitis B (CHB). High levels of HBsAg in patients with chronic hepatitis B are a significant factor contributing to immune tolerance; inhibiting HBsAg levels helps reverse host immune tolerance to HBV. Developing innovative treatments and methods that can effectively clear the virus, especially those that can effectively clear HBsAg or significantly reduce HBsAg levels, is urgent and necessary. Therapeutic monoclonal antibodies are a rapidly developing new targeted therapy approach in recent years, widely used in the treatment of tumors, autoimmune diseases, and infectious diseases due to their high specificity and good safety profile. Currently, various monoclonal antibodies targeting HBsAg have been developed, and their half-life has been extended through antibody engineering to rapidly and persistently clear viral antigens from peripheral blood. However, reported therapeutic HBV antibodies vary considerably in their ability to clear HBsAg from the circulating system, and suffer from problems such as high therapeutic doses, frequent administration, poor clearance of high initial HBsAg titers, and rapid HBsAg rebound. Furthermore, long-term use can easily alter HBsAg immune characteristics, potentially leading to drug resistance and immune escape. Therefore, simply clearing the virus or antigens from the bloodstream is insufficient to effectively control viral infection and cannot meet the needs of HBV treatment.

[0005] Toll-like receptors (TLRs) are pattern recognition receptors that play a crucial role in innate and adaptive immune responses. TLR7, TLR8, and TLR9 are located in the endosome compartment of various antigen-presenting cells. By recognizing exogenous nucleic acids, they activate downstream signaling pathways, upregulate the transcription of inflammatory cytokines, and promote cell activation. Based on the immune activation effect of the TLR pathway, immunomodulators or agonists targeting TLRs can exert good immunomodulatory effects. Gilead's GS-9620 (vesatolimod), a TLR7 agonist, has shown good therapeutic effects in HBV animal models, reducing viremia and cccDNA expression, and promoting functional cure in the animal models. However, free small molecule agonists, due to their predominantly lipid-soluble nature and lack of tissue or specific cell targeting, can lead to systemic activation, thus facing significant obstacles in clinical research.

[0006] In conclusion, new drugs still need to be developed for the treatment of chronic hepatitis B.

[0007] Antibody-drug conjugates (ADCs) are novel cancer therapeutics that conjugate small toxin molecules to monoclonal antibodies via linkers. Due to the potent cytotoxic effects of the conjugated toxin molecules, combined with the targeted specificity of antibodies, they show great promise in the field of cancer treatment. With the development and maturation of conjugation technology, an immune agonist antibody-drug conjugate (ISAC) has emerged in recent years, primarily for cancer immunotherapy. This drug, based on ADCs, replaces the small toxin molecules with immune agonists, which are then targeted to the tumor microenvironment via antibody molecules. Through immune regulation of myeloid cells in tumor tissue, it reshapes the immune microenvironment and promotes tumor regression. Bolt Therapeutics conjugated the TLR7 / 8 agonist T785 with trastuzumab, which targets HER2, to construct ISAC (BDC-1001), which has shown good therapeutic efficacy in various HER2+ tumor-bearing mouse models. On September 28, 2023, the U.S. Food and Drug Administration (FDA) granted orphan drug designation to the cutting-edge drug BDC-1001 for the treatment of gastric cancer patients, including cancers at the gastroesophageal junction. In addition, several other companies have pipelines of isotropic immunoglobulins (ISACs), with primary targets including HER2, Nectin4, CD73, and TROP2, and conjugated immune agonists including TLR7, TLR8, TLR9, and STING.

[0008] Currently, this type of ISAC drug is still in the early stages of research and efficacy verification. Although conjugating small molecule agonists to monoclonal antibodies has limited the delivery range of agonists to some extent, monoclonal antibodies still pose a risk of activating a large number of peripheral immune cells when administered systemically, because they mediate endocytosis through the interaction between Fc and Fc receptors. Therefore, the problem of low tolerable drug dose still exists in clinical practice, and a fine balance between efficacy and safety is still needed. Summary of the Invention

[0009] DEC-205 is a type C lectin receptor, mainly expressed on the surface of dendritic cells (DCs). It mediates ligand uptake and delivers it to acidic protein degradation vesicles for processing and related antigen presentation. The DEC-205 receptor can also recycle to the cell surface. In this application, the inventors fused a DEC-205-targeting antibody to an HBsAg monoclonal antibody and introduced or omitted a loss-of-function mutation in the Fc domain of the antibody, obtaining a high-affinity HBsAg / DEC-205 bispecific antibody with excellent properties. This bispecific antibody was conjugated with a TLR7 / 8 agonist to construct an Immune-Stimulating Bis-antibody Conjugate (ISBC) targeting DCs. This ISBC mediates efficient DC uptake and phagocytosis, achieving sustained and effective viral clearance, while limiting the targeted delivery range of the agonist, which helps to promote the reversal of chronic hepatitis B immune tolerance at higher tolerable doses.

[0010] Based on the above, the technical solution of this application involves the following aspects.

[0011] 1. Bispecific antibodies

[0012] In one aspect, this application provides a bispecific antibody comprising a first antigen-binding domain that specifically binds to HBsAg and a second antigen-binding domain that specifically binds to receptors on the surface of dendritic cells (such as DEC-205, Clec9A, Dectin 1, XCR1, CD204, CD206, CD209, etc.).

[0013] In some embodiments, the bispecific antibody comprises a first antigen-binding domain that specifically binds to HBsAg and a second antigen-binding domain that specifically binds to DEC-205.

[0014] In some embodiments, the first antigen-binding domain and the second antigen-binding domain can be any antibody form, and can each be independently selected from full-length antibodies (e.g., IgG antibodies) or their antigen-binding fragments (e.g., scFv, Fab, scFab), or nanobodies, etc.

[0015] In some embodiments, the first antigen-binding domain comprises a nanobody that forms a domain capable of specifically binding HBsAg.

[0016] In some embodiments, the first antigen-binding domain includes a first heavy chain variable region (VH) and a first light chain variable region (VL), which together form a domain capable of specifically binding HBsAg.

[0017] The second antigen-binding domain includes a nanobody that forms a domain capable of specifically binding to DEC-205.

[0018] The second antigen-binding domain includes a second heavy chain variable region (VH) and a second light chain variable region (VL), which together form a domain capable of specifically binding to DEC-205.

[0019] In some embodiments, the first antigen-binding domain includes:

[0020] Nanobodies, or

[0021] A first heavy chain variable region (VH) and a first light chain variable region (VL), the first heavy chain variable region (VH) and the first light chain variable region (VL) together form a domain capable of specifically binding HBsAg; and / or

[0022] The second antigen-binding domain includes:

[0023] Nanobodies, or

[0024] The second heavy chain variable region (VH) and the second light chain variable region (VL) together form a domain that can specifically bind to DEC-205.

[0025] In some embodiments, the bispecific antibody further comprises an Fc domain, the Fc domain comprising a second constant region (CH2) and a third constant region (CH3) of the heavy chain.

[0026] In some embodiments, the second constant region (CH2) of the heavy chain contains modifications of one or more amino acids that can eliminate the binding of bispecific antibodies and Fcγ receptors, thereby preventing nonspecific activation of the immune response.

[0027] In some embodiments, the Fc domain is derived from the Fc domain of a human immunoglobulin. In some embodiments, the human immunoglobulin is IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc domain is derived from the Fc domain of mouse IgG1.

[0028] In some embodiments, the second constant region (CH2) of the heavy chain contains an amino acid sequence as shown in SEQ ID NO:44 or 46.

[0029] In some embodiments, the heavy chain third constant region (CH3) contains an amino acid sequence as shown in SEQ ID NO:45.

[0030] In some embodiments, the first heavy chain (HC) variable region (VH) comprises:

[0031] (i) HCDR1 containing the amino acid sequence shown in SEQ ID NO:5, HCDR2 containing the amino acid sequence shown in SEQ ID NO:6, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:7; wherein the CDRs are defined by the IMGT numbering system;

[0032] (ii) HCDR1 comprising the amino acid sequence shown in SEQ ID NO:11, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:12, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO:13; wherein the CDRs are defined by the Chothia numbering system; or,

[0033] (iii) HCDR1 containing the amino acid sequence shown in SEQ ID NO:16, HCDR2 containing the amino acid sequence shown in SEQ ID NO:17, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:13; wherein the CDRs are defined by the AbM or Kabat numbering system.

[0034] And / or,

[0035] The first light chain (LC) variable region (VL) includes:

[0036] (i) LCDR1 comprising the amino acid sequence shown in SEQ ID NO:8, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:9, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:10; wherein the CDRs are defined by the IMGT numbering system; or,

[0037] (ii) LCDR1 comprising the amino acid sequence shown in SEQ ID NO:14, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:15, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:10; wherein the CDRs are defined by the Chothia, Kabat, or AbM numbering system.

[0038] In some embodiments, the first antigen-binding domain includes a first heavy chain variable region (VH) and a first light chain variable region (VL), wherein the first heavy chain variable region (VH) comprises:

[0039] (i) The amino acid sequence as shown in SEQ ID NO:1, or

[0040] (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the amino acid sequence shown in SEQ ID NO:1, or

[0041] (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:1; and / or,

[0042] The first light chain variable region (VL) includes:

[0043] (i) The amino acid sequence as shown in SEQ ID NO:2, or

[0044] (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the amino acid sequence shown in SEQ ID NO:2; or

[0045] (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:2.

[0046] In some implementations, the permutation is a conservative permutation.

[0047] In some embodiments, the amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity does not involve changes to the CDR sequence.

[0048] In some embodiments, the first heavy chain variable region (VH) contains an amino acid sequence as shown in SEQ ID NO:1, and / or the first light chain variable region (VL) contains an amino acid sequence as shown in SEQ ID NO:2.

[0049] In some embodiments, the second heavy chain (HC) variable region (VH) comprises:

[0050] (i) HCDR1 containing the amino acid sequence shown in SEQ ID NO:18, HCDR2 containing the amino acid sequence shown in SEQ ID NO:19, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:20; wherein the CDRs are defined by the IMGT numbering system.

[0051] (ii) HCDR1 comprising the amino acid sequence shown in SEQ ID NO:24, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:25, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO:26; wherein the CDRs are defined by the Chothia numbering system.

[0052] (iii) HCDR1 comprising the amino acid sequence shown in SEQ ID NO:29, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:30, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO:26; wherein the CDRs are defined by the Kabat numbering system; or,

[0053] (iv) HCDR1 comprising the amino acid sequence shown in SEQ ID NO:31, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:32, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO:26; wherein the CDRs are defined by the AbM numbering system.

[0054] And / or,

[0055] The second light chain (LC) variable region (VL) comprises:

[0056] (i) LCDR1 comprising the amino acid sequence shown in SEQ ID NO:21, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:22, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:23; wherein the CDRs are defined by the IMGT numbering system; or,

[0057] (ii) LCDR1 comprising the amino acid sequence shown in SEQ ID NO:27, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:28, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:23; wherein the CDRs are defined by the Chothia, Abm, or IMGT numbering system.

[0058] In some embodiments, the second antigen-binding domain includes a second heavy chain variable region (VH) and a second light chain variable region (VL), wherein the second heavy chain variable region (VH) comprises:

[0059] (i) The amino acid sequence as shown in SEQ ID NO:3, or

[0060] (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the amino acid sequence shown in SEQ ID NO:3, or

[0061] (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:3; and / or,

[0062] The second light chain variable region (VL) comprises:

[0063] (i) The amino acid sequence as shown in SEQ ID NO:4, or

[0064] (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the amino acid sequence shown in SEQ ID NO:4; or

[0065] (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:4.

[0066] In some implementations, the permutation is a conservative permutation.

[0067] In some embodiments, the amino acid sequence of sequence b having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% does not involve changes to the CDR sequence.

[0068] In some embodiments, the second heavy chain variable region (VH) comprises an amino acid sequence as shown in SEQ ID NO:3, and / or, the second light chain variable region (VL) comprises an amino acid sequence as shown in SEQ ID NO:4.

[0069] In some implementations, the first antigen-binding domain is Fab, and the second antigen-binding domain is scFv.

[0070] In some embodiments, the second antigen domain comprises an amino acid sequence as shown in SEQ ID NO:36.

[0071] In some embodiments, the bispecific antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a first heavy chain variable region, a heavy chain CH1 region, a heavy chain CH2 region, a heavy chain CH3 region, a second heavy chain variable region, and a second light chain variable region, and the light chain comprises the first light chain variable region and a light chain constant region.

[0072] In some embodiments, the heavy chain from the N end to the C end includes: a first heavy chain variable region, a heavy chain CH1 region, a heavy chain CH2 region, a heavy chain CH3 region, a second heavy chain variable region, and a second light chain variable region, and the light chain from the N end to the C end includes: a first light chain variable region and a light chain constant region.

[0073] In some embodiments, adjacent structural domains of the heavy chain are optionally connected by or without joints, and adjacent structural domains of the light chain are optionally connected by or without joints.

[0074] In some embodiments, the peptide linkers are each independently the same or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers). In some embodiments, the peptide linkers are each independently selected from peptide linkers containing one or more glycine (G) and / or serine (S), such as those possessing (GGGGS). n The structure shown is provided, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, each peptide linker independently comprises an amino acid sequence as shown in SEQ ID NO:36.

[0075] In some embodiments, the heavy chain from the N end to the C end includes: a first heavy chain variable region, a heavy chain CH1 region, a hinge region, a heavy chain CH2 region, a heavy chain CH3 region, a joint, a second heavy chain variable region, a joint, and a second light chain variable region.

[0076] In some embodiments, the heavy chain CH1 region contains an amino acid sequence as shown in SEQ ID NO:42, the heavy chain CH2 region contains an amino acid sequence as shown in SEQ ID NO:44 or 46, the heavy chain CH3 region contains an amino acid sequence as shown in SEQ ID NO:45, and / or, the light chain constant region contains an amino acid sequence as shown in SEQ ID NO:35.

[0077] In some embodiments, the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:39 or 40, and / or the light chain comprises an amino acid sequence as shown in SEQ ID NO:41.

[0078] 2. Nucleic acids, vectors, and host cells

[0079] On the other hand, this application provides isolated nucleic acid molecules or groups of nucleic acid molecules containing nucleotide sequences encoding bispecific antibodies as described above.

[0080] The nucleic acid can be obtained using methods known in the art, such as isolation from a phage display library, a yeast display library, immunization of animals, immortalized cells (e.g., mouse B cell hybridoma cells, EBV-mediated immortalized B cells), or chemical synthesis. Based on codon degeneracy in the art, in some embodiments, the nucleotide sequence can be substituted according to codon degeneracy. In some embodiments, the nucleotide sequence is codon-optimized.

[0081] On the other hand, this application provides a vector containing isolated nucleic acid molecules or groups of nucleic acid molecules as described above.

[0082] In some embodiments, the vector of this application is, for example, a plasmid, a granulosome, a bacteriophage, a lentivirus, etc. In some embodiments, the vector is capable of expressing the bispecific antibody of this application in a subject (e.g., a mammal, such as a human).

[0083] In some embodiments, the nucleotide sequences encoding the heavy and light chains of the bispecific antibody are located in different carrier molecules.

[0084] In some implementations, the vector is a cloning vector or an expression vector.

[0085] On the other hand, this application provides a host cell comprising the isolated nucleic acid molecules or groups of nucleic acid molecules as described above, or the vector as described above. The host cell can be a eukaryotic cell (e.g., mammalian cells, insect cells, yeast cells) or a prokaryotic cell (e.g., *E. coli*). Suitable eukaryotic cells include, but are not limited to, NSO cells, Vero cells, HeLa cells, COS cells, CHO cells, ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In some embodiments, the host cell of this application is a mammalian cell, such as Expi-293F cells.

[0086] 3. Preparation of bispecific antibodies

[0087] The bispecific antibody of this application can be prepared by various methods known in the art, such as through genetic engineering recombination technology. For example, DNA molecules encoding the heavy and light chains of the bispecific antibody of this application can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into an expression vector and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the bispecific antibody of this application.

[0088] On the other hand, this application provides a method for preparing the bispecific antibody as described above, comprising culturing host cells as described above under conditions that allow the expression of the bispecific antibody, and recovering the bispecific antibody from the cultured host cell culture.

[0089] 4. Application of bispecific antibodies

[0090] On the other hand, this application provides the use of the bispecific antibody as described above in the preparation of antibody-drug conjugates.

[0091] On the other hand, this application provides a composition comprising the bispecific antibody as described above, or the isolated nucleic acid molecule or nucleic acid molecule group as described above, or the carrier as described above, or the host cell as described above. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier and / or excipient. In some embodiments, the composition further comprises additional pharmaceutically active agents.

[0092] In some embodiments, the additional pharmaceutically active agent is a drug for treating hepatitis B, particularly chronic hepatitis B.

[0093] 5. Couplings

[0094] The bispecific antibody of this application can be derivatized, for example, by being linked to another molecule. Generally, antibody derivatization does not adversely affect its binding to HBsAg and DEC-205. Therefore, the bispecific antibody of this application is also intended to include such derivatized forms. For example, the bispecific antibody of this application can be functionally linked (through chemical coupling, gene fusion, non-covalent linkage, or other means) to one or more other molecular groups, such as another antibody, a detection reagent, a pharmaceutical reagent, and / or a protein or polypeptide (e.g., avidin or a multihistidine tag) capable of mediating the binding of the bispecific antibody to another molecule.

[0095] Therefore, in another aspect, this application provides a conjugate comprising the bispecific antibody as described above and a conjugate portion thereto.

[0096] In some embodiments, the conjugation portion is selected from protein tags. Such protein tags are well known in the art, and examples include, but are not limited to, His, Flag, GST, MBP, HA, Myc, GFP, or biotin, and those skilled in the art know how to select appropriate protein tags (e.g., purification tags, detection tags, or tracer tags) according to the desired purpose. In some exemplary embodiments, the C-terminus of the bispecific antibody of this application is linked to a purification tag.

[0097] In some embodiments, the coupling portion is selected from detectable markers, such as enzymes (e.g., horseradish peroxidase), radionuclides, fluorescent dyes, luminescent substances (e.g., chemiluminescent substances), or biotin. The detectable markers described in this application can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent substances such as acridinium esters), magnetic beads (e.g., The labeling includes thermometric markers such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.), and biotin for binding avidin (e.g., streptavidin) modified with the aforementioned markers. In some embodiments, such markers are suitable for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable markers described above can be linked to the bispecific antibody of this application via linkers of varying lengths to reduce potential steric hindrance.

[0098] In some embodiments, the coupling portion comprises a therapeutic agent. In some embodiments, the therapeutic agent is selected from TLR agonists, STING, etc. In some embodiments, the therapeutic agent is selected from TLR agonists. In some embodiments, the therapeutic agent is selected from TLR7 agonists, TLR8 agonists, and any combination thereof. In some embodiments, the therapeutic agent is selected from TLR7 agonists. In some embodiments, the TLR agonist is selected from imidazoquinoline derivatives, pyrimidine derivatives, purine derivatives, benzothiophene derivatives, synthetic oligonucleotides, and any combination thereof. In some embodiments, the TLR agonist is selected from GS-9620, GS-9688, JNJ-64794964, IMDQ, SZU-101, R848, and any combination thereof. In some embodiments, the TLR agonist is IMDQ.

[0099] In some embodiments, the conjugate is an antibody-drug conjugate, comprising:

[0100] The target molecule is selected from the bispecific antibodies mentioned above;

[0101] The therapeutic agent; and

[0102] Linker for connecting the target molecule and the therapeutic agent.

[0103] In some embodiments, the therapeutic agent is coupled to a naked amino or thiol group in the bispecific antibody via the linker. Those skilled in the art can decide whether to modify the antibody to form a naked thiol or amino group to facilitate the coupling of the therapeutic agent.

[0104] In some embodiments, the connector is selected from the structural segment shown in Formula A.

[0105] -L1-L2-L3-

[0106] A

[0107] L1 is a structural fragment linked to the bispecific antibody shown.

[0108] L2 is the structural segment connecting L1 and L3;

[0109] L3 is the structural segment that connects L2 and the therapeutic agent.

[0110] In some implementations, L1 is selected from:

[0111] Where h is 0, 1, 2, 3, 4, 5 or 6.

[0112] In some implementations, L1 is

[0113] In some implementations, L2 is selected from:

[0114] -(CH2) m -,-(CH2) t O-, -(CH2CH2O) r -,

[0115] Where m, t, and r are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12;

[0116] R is selected from: hydrogen, halogen, methyl, ethyl, nitro, methoxy, and ethoxy;

[0117] Each p is independently 0, 1, 2, 3 or 4.

[0118] In some implementations, m, t, and r are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0119] In some implementations, L2 is -(CH2CH2O). r - where r is defined as described above.

[0120] In some implementations, L3 is selected from: -S(O)-, -S(O)2-, -C(O)-, -(CH2) g -C(O)-,-(CH2) g -S(O)- and -(CH2) g -S(O)2-, where each g is independently selected from 1, 2, 3, 4.

[0121] In some implementations, each g is independently selected from 1, 2, and 3. In some implementations, each g is independently 1 or 2. In some implementations, each g is independently 2.

[0122] In some implementations, L3 is -C(O)- or -(CH2). g -C(O)-, where g is defined as described above.

[0123] In some embodiments, the bispecific antibody in the conjugate is a modified or unmodified antibody. In some embodiments, the bispecific antibody is a deacetylated SAT(PEG)4 modified antibody.

[0124] In some embodiments, the coupling agent is selected from the coupling agents shown in Formula I.

[0125] Wherein, Ab represents the bispecific antibody.

[0126] The definitions of L1, L2, and L3 are as described above.

[0127] D represents the therapeutic agent.

[0128] n represents DAR, which is a number between 1 and 10, including integers and decimals.

[0129] In some embodiments, n is a number between 1 and 8. In some embodiments, n is a number between 1 and 6. In some embodiments, n is a number between 1 and 5. In some embodiments, n is a number between 1 and 4. In some embodiments, n is a number between 1 and 3. In some embodiments, n is a number between 1.5 and 2.5. In some embodiments, n is a number between 1.8 and 2.2. In some embodiments, n is approximately 1. In some embodiments, n is approximately 2. In some embodiments, n is approximately 3. In some embodiments, n is approximately 4. In some embodiments, n is approximately 5. In some embodiments, n is approximately 6. In some embodiments, n is approximately 7. In some embodiments, n is approximately 8. In some embodiments, n is approximately 9. In some embodiments, n is approximately 10.

[0130] In some embodiments, the coupling agent is the coupling agent shown in Formula II.

[0131] The definitions of Ab and n are as described above.

[0132] 6. Pharmaceutical Composition

[0133] On the other hand, this application also provides pharmaceutical compositions comprising the conjugates as described above, and pharmaceutically acceptable carriers and / or excipients.

[0134] In some embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent. In some embodiments, the additional pharmaceutically active agent is a drug for treating hepatitis B, particularly chronic hepatitis B, including therapeutic antibodies against hepatitis B, recombinant protein vaccines, agonist antibodies that target and activate receptors, etc.

[0135] In some embodiments, the conjugate is present in the pharmaceutical composition in an effective amount.

[0136] 7. Therapeutic applications

[0137] On the other hand, this application provides the use of the bispecific antibody or conjugate or pharmaceutical composition as described above in the preparation of a medicament for treating hepatitis B, particularly chronic hepatitis B, in a subject.

[0138] On the other hand, this application provides a method for treating and / or adjunctive treating a disease in a subject, the method comprising administering to a subject in need an effective amount of the bispecific antibody as described above, or the conjugate as described above, or the pharmaceutical composition as described above, wherein the disease is hepatitis B, particularly chronic hepatitis B.

[0139] The conjugates and pharmaceutical compositions of this application can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use. The pharmaceutical compositions of this application should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the required dose of the antibody of this application into a suitable solvent, and optionally, simultaneously incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by sterile filtration. Alternatively, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier, such as sterile pyrogen-free water, before use.

[0140] Furthermore, the conjugates of this application can be present in the pharmaceutical composition in unit dose form for ease of administration.

[0141] The conjugates and pharmaceutical compositions of this application may be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, bladder, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / method of administration is parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose. In some preferred embodiments, the conjugates and pharmaceutical compositions of this application are administered by intravenous infusion or injection.

[0142] The pharmaceutical composition of this application may include conjugates of this application in a "therapeutic effective amount" or a "preventive effective amount." A "preventive effective amount" refers to an amount sufficient to prevent, stop, or delay the onset of a disease. A "therapeutic effective amount" refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. The therapeutic effective amount of the bispecific antibody of this application may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the method of drug administration, and other concurrent treatments, etc.

[0143] In this application, the dosing regimen can be adjusted to obtain the optimal target response (e.g., treatment or prevention). For example, it can be administered as a single dose, multiple times over a period of time, or the dose can be reduced or increased proportionally according to the urgency of the treatment situation.

[0144] In this application, the subject can be a mammal, such as a human.

[0145] Abbreviations: CDR (Complementarity-Determining Region) of immunoglobulin variable region; FR (Antibody Framework Region: Amino acid residues other than CDR residues in the antibody variable region); VH (Antibody Heavy Chain Variable Region); VL (Antibody Light Chain Variable Region); IgG (Immunoglobulin G); IMGT (Based on the international ImMunoGeneTics information system initiated by Lefranc et al.). For the (IMGT) numbering system, see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003. Kabat's immunoglobulin alignment and numbering system, proposed by Elvin A. Kabat (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). Chothia's immunoglobulin numbering system, proposed by Chothia et al., is a classic rule for identifying CDR boundaries based on the location of structural loop regions (see, for example, Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883). Abm. Immunoglobulin numbering system proposed by Martin et al., see, for example, Martin AC, et al. Modeling antibody hypervariable loops: a combined algorithm. Proc Natl Acad Sci US A. 1989 Dec; 86(23):9268-72. mAb Monoclonal antibody EC50 Concentration producing 50% efficacy or binding IC50 Concentration producing 50% inhibition PCR Polymerase chain reaction HRP Horseradish peroxidase ADCC Antibody-dependent cytotoxicity FACS Flow cytometry HCDR1 Complementarity-determining region 1 in the variable region of immunoglobulin heavy chain HCDR2 Complementarity-determining region 2 in the variable region of immunoglobulin heavy chain HCDR3 Complementarity-determining region 3 in the variable region of immunoglobulin heavy chain LCDR1 Complementarity-determining region 1 in the variable region of immunoglobulin light chain LCDR2 Complementarity-determining region 2 in the variable region of immunoglobulin light chain LCDR3 Complementarity-determining region 3 in the variable region of immunoglobulin light chain

[0146] Terminology Definition

[0147] In this application, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, biochemistry, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this application, definitions and explanations of relevant terms are provided below.

[0148] As used herein, the term "TLR receptor" refers to Toll-like receptors, an important class of innate immune pattern recognition receptors in the immune system of organisms. These receptors specifically recognize relatively conserved antigen molecules (or pathogen-associated molecular patterns) during the evolution of pathogenic microorganisms, enabling effective detection of pathogen invasion and induction of innate immune responses. Ten TLR receptors have been identified in humans, namely TLR1-TLR10. TLR3, TLR7, TLR8, and TLR9 are located on the endosomes and lysosomal membranes of cells, while the rest are located on the cytoplasmic membrane. In the embodiments of this application, TLR7 / 8 is preferably used as one of the drug molecule targets. The natural ligands of TLR7 / 8 molecules are single-stranded linear RNA.

[0149] As used herein, the term "TLR receptor agonist" refers to a macromolecular (protein or nucleic acid) or small molecule agonist that can specifically bind to and activate the TLR receptor, promoting the transduction of downstream signals of the TLR receptor and activating innate immune cells. In the embodiments of this application, TLR7 / 8 agonists are preferably used to construct nanobody-drug conjugates. In addition to IMDQ used in the embodiments of this application, other usable TLR7 / 8 agonists include SZU-101 and R848.

[0150] As used in this article, the term "DEC-205" refers to a type C lectin receptor primarily expressed on the surface of dendritic cells (DCs). It mediates ligand uptake and delivery to acidic protein-degrading vesicles for processing and related antigen presentation. The DEC-205 receptor can also recycle to the cell surface. Several studies have explored targeted antigen delivery using antigen-conjugated DEC-205 monoclonal antibodies. Furthermore, the combined use of DEC-205 monoclonal antibody fusion antigen and adjuvant can promote DC maturation and antigen presentation, thereby activating adaptive immune cells and potentially for antiviral or antitumor therapy. Without adjuvant, however, DC tolerance develops, further promoting Treg cell differentiation, which could be used to treat autoimmune diseases.

[0151] As used herein, the term "antibody" is used in the broadest sense to encompass a wide variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired antigen-binding activity. For example, an immunoglobulin molecule can consist of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both the light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a variable region (VL) and a constant region (CL). The constant region consists of a single CL domain. While not directly involved in antibody-antigen binding, the constant domain exhibits various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding sites. The distribution of amino acids in different regions or domains can follow the definitions in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883.

[0152] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0153] As used herein, the term “complementarity-determining region” or “CDR” refers to the amino acid residues in the variable region of an antibody responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, such as the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272) or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, those skilled in the art will readily identify the CDR as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0154] In this application, the CDR contained in the antibody or its antigen-binding fragment can be determined according to various numbering systems known in the art. In some embodiments, the CDR contained in the antibody or its antigen-binding fragment is determined using the IMGT, Kabat, Chothia, or AbM numbering systems.

[0155] As used herein, the term “framework region” or “FR” residues refer to the amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0156] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0157] As used herein, the term "bispecific antibody" refers to an antibody that has binding specificity to two different antigens (or epitopes), comprising two antigen-binding domains specific for binding to different antigens (or epitopes), thereby enabling it to bind to two different binding sites and / or target molecules. The individual antigen-binding domains of a bispecific antibody can be independently selected from full-length antibodies (e.g., IgG antibodies) or their antigen-binding fragments (e.g., Fv, Fab, scFab, scFv, F(ab')2, or SdAb), nanobodies, etc. In some cases, the individual antigen-binding domains are linked by peptide linkers.

[0158] As used herein, the term "Fv fragment" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as the Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to antigens, although its affinity may be lower than that of a complete binding site.

[0159] As used herein, the terms "Fc fragment" or "Fc domain" refer to an antibody fragment formed by the second and third constant regions of the first heavy chain of an antibody, or an antibody fragment formed by the second and third constant regions of the second heavy chain of an antibody, or an antibody fragment formed by the combination of the second and third constant regions of the first heavy chain and the second and third constant regions of the second heavy chain via disulfide bonds. Antibody Fc fragments have various functions but do not participate in antigen binding.

[0160] As used herein, the term "scFv" refers to a single polypeptide chain containing VL and VH domains linked by a linker. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 (SEQ ID NO:36) may be used, but variants thereof may also be used. In some cases, a disulfide bond may also exist between the VH and VL domains of the scFv.

[0161] As used herein, the term "Fab fragment" refers to an antibody fragment consisting of VL, VH, CL, and CH1 domains, typically comprising one peptide chain containing VL and CL and another peptide chain containing VH and CH1. However, those skilled in the art will understand that the Fab domains may be arranged according to the aforementioned natural orientation, but may also include domain substitutions or exchanges that facilitate proper VH and VL pairing (e.g., domain exchanges in the form of Crossmab). The term "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments connected by a disulfide bridge on the hinge region. The term “scFab” refers to a single polypeptide chain containing VL, VH, CL and CH1 domains, wherein adjacent domains are optionally linked by a linker. In a typical scFab structure, the single polypeptide chain contained in scFab contains from the N-terminus to the C-terminus: (1) VL, CL, VH and CH1, wherein CL and VH are typically linked by a peptide linker (e.g., a flexible peptide linker), or (1) VH, CH1, VL and CL, wherein CH1 and VL are typically linked by a peptide linker (e.g., a flexible peptide linker).

[0162] As used herein, the term "nanobody" refers to an antibody that, like a conventional antibody, can selectively bind to a specific antigen. A naturally occurring antibody lacking the light chain exists in alpaca peripheral blood; this antibody contains only a heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, and is called a heavy chain antibody (HcAb). The antibody fragment located in the variable region of the heavy chain antibody is called a VHH single-domain antibody, also known as a nanobody, which contains the sequences CDR1 (complementarity-determining region 1), CDR2 (complementarity-determining region 2), and CDR3 (complementarity-determining region 3).

[0163] As used herein, the terms "monoclonal antibody," "monoclonal antibody," and "mAb" have the same meaning and are used interchangeably. They refer to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules; that is, a group of identical antibody molecules, except for the possibility of spontaneous natural mutations. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Furthermore, the modifier "monoclonal" only indicates that the antibody is derived from a highly homologous group of antibodies and should not be construed as requiring preparation by any specific method.

[0164] As used herein, the terms “specific binding,” “specificity,” or “specific to…” refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (K0) of that interaction. D or half-maximal effect concentration (EC50) 50 The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. In some embodiments, an antibody that specifically binds to a certain antigen (or an antibody that is specific to a certain antigen) means that the antibody binds to an antigen at a concentration of less than about 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or lower affinity (K) D () binds to the antigen.

[0165] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of antigen binding site / antigen complex formation and dissociation. The "binding rate constant" (k...) a or k on ) and "dissociation rate constant" (k dis or k off Both can be calculated from concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). dis / k on The ratio is equal to the dissociation constant K. D (See Davies et al., Annual Rev Biochem, 1990; 59:439-473). K can be measured by any effective method. D k on and k dis The dissociation constant can be measured using bioluminescence interferometry (e.g., the ForteBio Octet method) in some implementations. Alternatively, surface plasmon resonance techniques (e.g., Biacore) or Kinexa can be used.

[0166] As used herein, the term “about” is understood to mean within + / -20%, + / -18%, + / -15%, + / -12%, + / -10%, + / -9%, + / -8%, + / -7%, + / -6%, + / -5%, + / -4%, + / -3%, + / -2%, + / -1%, + / -0.5%, + / -0.4%, + / -0.3%, + / -0.2%, and + / -0.1% of the stated value. Unless otherwise apparent from the context, all numerical values ​​provided herein are modified by the term “about”.

[0167] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0168] Expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Typically, in cloning vectors, this sequence is the one that enables the vector to replicate independently of the host chromosomal DNA, and it includes an origin of replication or an autonomous replication sequence. As used herein, the term "expression vector" refers to a vector containing recombinant polynucleotides that include expression regulatory sequences effectively linked to the nucleotide sequence to be expressed. Expression vectors contain sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or an in vitro expression system. Expression vectors include all those known in the art, such as entrapments, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0169] As used herein, the term "host cell" refers to cells that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, NSO cells, Vero cells, HeLa cells, COS cells, CHO cells (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44 cells), ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells.

[0170] The twenty common amino acids referred to herein are written in accordance with conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this application, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. And in this application, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0171] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit occupies the same location (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be performed using, for example, a computer program such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0172] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the essential properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, conservative substitution generally refers to replacing a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0173] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining agents, absorption delaying agents, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintaining agents include, but are not limited to, sugars, NaCl, and their analogues. Absorption delaying agents include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Stabilizers have the meaning commonly understood by those skilled in the art for stabilizing the desired activity of the active ingredient in a pharmaceutical product, including but not limited to monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate).

[0174] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that retains the bioavailability and properties of a compound or conjugate, which is biologically or otherwise desirable for use in pharmaceuticals. In many cases, the conjugates disclosed herein can form acid and / or base salts in the presence of amino and / or carboxyl or similar groups. Pharmaceutically acceptable acid addition salts can consist of inorganic and organic acids. Inorganic acids that can be derived to form salts include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Organic acids that can be derived to form salts include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc. Pharmaceutically acceptable base addition salts can consist of inorganic and organic bases. Inorganic bases that can be derived to form salts include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum; particularly preferred are ammonium, potassium, sodium, calcium, and magnesium salts. Organic bases that can be derived to form salts include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, etc., specifically, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. Many such salts are known in the art, such as those described in WO87 / 05297 by Johnston et al., published on September 11, 1987 (which is incorporated herein by reference in its entirety).

[0175] In cases where the compound name used in this article differs from the chemical structural formula, the chemical structural formula shall prevail.

[0176] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom (e.g., a tumor) in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this application, a beneficial or desired clinical outcome includes, but is not limited to, alleviating symptoms, reducing the extent of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to expected survival (if no treatment was received).

[0177] As used herein, the term "subject" refers to a mammal, such as a primate mammal, like a human. In some embodiments, the subject (e.g., a human) suffers from hepatitis B, particularly chronic hepatitis B.

[0178] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for treating a disease means an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0179] It should also be noted that the dosage and method of administration of the conjugate in this application depend on many factors, including the patient's age, weight, sex, natural health condition, nutritional status, the activity intensity of the compound, the time of administration, metabolic rate, the severity of the condition, and the subjective judgment of the treating physician. The preferred dosage is between 0.01 and 1000 mg / kg body weight / day.

[0180] As used herein, the term “and / or” should be considered as a specific disclosure of each of two or more specified features or elements, and any combination of two or more features or elements. Therefore, the term “and / or” as used in phrases, such as “A and / or B” herein, is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Attached Figure Description

[0181] Figure 1 shows a schematic diagram of the structures of the bispecific antibodies BIS-WT and BIS-DA;

[0182] Figure 2 shows the band sizes of antibodies 73-WT and 73-DA, as well as bispecific antibodies BIS-WT and BIS-DA, identified by SDS-PAGE.

[0183] Figure 3 shows the binding activity of 73-WT, 73-DA, BIS-WT, and BIS-DA to HBsAg and DEC-205 proteins detected by ELISA.

[0184] Figure 4 shows the binding activity of 73-WT, 73-DA, BIS-WT, and BIS-DA to the DEC-205 protein on the surface of CHO-LY75 cells as verified by flow cytometry.

[0185] Figure 5 shows the phagocytic effect of 73-WT, 73-DA, BIS-WT, BIS-DA-mediated BMDC and cDC1 on antigens, as verified by flow cytometry.

[0186] Figure 6 shows a schematic diagram of the structure of ISBC-WT and ISBC-DA;

[0187] Figure 7 shows the band sizes before and after identification of bispecific antibody-conjugated agonists by SDS-PAGE, where lanes 1-4 are BIS-WT, ISBC-WT, BIS-DA, and ISBC-DA, respectively.

[0188] Figure 8 shows the conjugation of the immunostimulant bispecific antibody-drug conjugate ISBC-DA using hydrophobic interaction chromatography (HIC).

[0189] Figure 9 shows the deconvolution results of the mean DAR value of the immune agonist bispecific antibody-drug conjugate ISBC-DA determined by LC-MS.

[0190] Figure 10 shows the results of flow cytometry analysis of HBsAg phagocytosis by BMDCs mediated by BIS-WT, ISBC-WT, BIS-DA, and ISBC-DA.

[0191] Figure 11 shows the expression of BMDC surface maturation markers mediated by BIS-WT, ISBC-WT, BIS-DA, and ISBC-DA, as detected by flow cytometry.

[0192] Figure 12 shows the monitoring results of serum HBsAg in mice with chronic AAV-HBV infection after treatment with 73-WT, 73-DA, BIS-WT, BIS-DA, ISBC-WT, ISBC-DA, and PBS.

[0193] Figure 13 shows the changes in viral markers in the liver of mice after ISBC-DA treatment, where (a) shows the HBsAg level in the liver of mice; bd shows the levels of HBV-DNA (b), total HBV RNA (c), and 3.5Kb RNA (d) in the liver;

[0194] Figure 14 shows the HBcAg level in the liver of mice after ISBC-DA treatment;

[0195] Figure 15 shows the results of liver injury identification in mice, where a and b show the changes in ALT and AST in mouse serum during treatment; c and d show the ALT and AST levels in mouse serum at the treatment endpoint; and e shows the H&E staining results of mouse liver at the treatment endpoint.

[0196] Sequence information

[0197] Information on some of the sequences involved in this application is shown in Table 1 below.

[0198] Table 1. Information on partial sequences Detailed Implementation

[0199] The embodiments of this application will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of this application. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0200] Example 1: Construction and expression of bispecific antibodies

[0201] This embodiment constructs bispecific antibodies BIS-WT and BIS-DA targeting HBsAg and DEC-205 (structural schematic diagrams are shown in Figure 1), as well as antibodies 73-WT and 73-DA targeting HBsAg.

[0202] 1.1 An M1D antibody targeting HBsAg was screened from a monkey anti-liquid library, and a high-affinity antibody 73 was obtained through pH-dependent modification and humanization. The sequence information of antibody 73 is as follows:

[0203] The amino acid sequences of CDR1-3 in the heavy chain variable region are shown in SEQ ID NO:5-7; the amino acid sequences of CDR1-3 in the light chain variable region are shown in SEQ ID NO:8-10; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1; and the amino acid sequence of the hydrogen chain variable region is shown in SEQ ID NO:2. The CDRs are defined by the IMGT numbering system.

[0204] The NLDC-145 sequence was obtained by retrieving the antibody gene sequence from hybridoma cells HB290. The NLDC-145 sequence information is as follows:

[0205] The amino acid sequences of CDR1-3 in the heavy chain variable region are shown in SEQ ID NO:18-20; the amino acid sequences of CDR1-3 in the light chain variable region are shown in SEQ ID NO:21-23; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:3; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:4. The CDRs are defined by the IMGT numbering system.

[0206] The heavy chain variable region shown in SEQ ID NO:3 is connected in series to the light chain variable region shown in SEQ ID NO:4 through the (GGGGS)3 connector shown in SEQ ID NO:36 to obtain NLDC-145scFv(NLDC-145VH-(GGGGS)3-NLDC-145VL) shown in SEQ ID NO:47.

[0207] The variable region sequence of the heavy chain 73 (73VH shown in SEQ ID NO:1) was ligated to the mouse IgG1 heavy chain constant region shown in SEQ ID NO:33 or the mutant mouse IgG1 heavy chain constant region (D265A) shown in SEQ ID NO:34 to obtain the 73-WT heavy chain sequence shown in SEQ ID NO:37 or the 73-DA heavy chain sequence shown in SEQ ID NO:38. The 73-WT heavy chain sequence shown in SEQ ID NO:37 or the 73-DA heavy chain sequence shown in SEQ ID NO:38 was then tandemly connected to the NLDC-145scFv shown in SEQ ID NO:47 via the (GGGGS)3 adapter shown in SEQ ID NO:36 to obtain the BIS-WT heavy chain sequence shown in SEQ ID NO:39 or the BIS-DA heavy chain sequence shown in SEQ ID NO:40. These heavy chain sequences were then constructed into the PTT5 vector, and heavy chain plasmids were synthesized by General Biosystems (Anhui) Co., Ltd.

[0208] The light chain sequences of BIS-WT, BIS-DA, 73-WT, and 73-DA are identical, all being the light chain sequence shown in SEQ ID NO:41. The variable region sequence of the light chain of 73 (73-VL shown in SEQ ID NO:2) was ligated to the mouse light chain constant region shown in SEQ ID NO:35 to obtain the light chain sequence shown in SEQ ID NO:41. This light chain sequence was also constructed into the PTT5 vector, and the light chain plasmid was synthesized by General Biosystems (Anhui) Co., Ltd.

[0209] 1.2 The obtained heavy chain plasmid and light chain plasmid were rapidly transfected into DH5α Escherichia coli competent cells (Shenzhen Kangti), heat-shocked for 90 s, incubated on ice for 2 min, and cultured overnight at 37°C on ampicillin-resistant LB agar plates. Single colonies were picked and cultured at 37°C for 16 h on ampicillin-resistant LB liquid medium at 180 rpm. The bacterial cells were collected, and plasmids were extracted using an endotoxin-free plasmid extraction kit (Tiangen).

[0210] 1.3 Expi-293F cells were transiently transfected with two plasmids to express 73-WT, 73-DA, BIS-WT, and BIS-DA. The cell density was approximately 4 × 10⁻⁶ cells / year. 6200 mL of Expi-293F cells (95% viability) were cultured. 0.25 mg each of the light and heavy chain plasmids were filtered through a 0.22 μm filter and added to 5 mL of CD05 medium. Simultaneously, 2 mg of PEI was added to 5 mL of CD05 medium, vortexed for 8 seconds, and incubated for 2 minutes. Then, 7 mL of the PEI mixture was added to the plasmid mixture, vortexed for 8 seconds, and incubated for 8 minutes. Finally, the mixture was pipetted dropwise into 200 mL of Expi-293F cell solution, gently mixing as it was added. The cells were cultured in a 5% CO2, 37°C shaker for 4 hours. After 4 hours, 200 mL of freestyle medium was added, and the cells were cultured again in a shaker for 7-8 days for expression.

[0211] 1.4 Collect the cell expression supernatant of 73-WT, 73-DA, BIS-WT, and BIS-DA cells, and centrifuge at 10,000 rpm for 25 min. Filter the cell supernatant through a 0.22 μm filter membrane for later use. AKTA purification instrument operation: Set the software flow rate to 8 mL / min and the maximum pressure to 0.3 MPa. First, thoroughly flush the instrument tubing with solution B (100 mM citric acid monohydrate), reduce the flow rate to 2 mL / min, load the protein A medium-pressure chromatography column, and equilibrate the protein A medium with 95% solution A (200 mM disodium hydrogen phosphate dodecahydrate) at a flow rate of 8 mL / min until the baseline level stabilizes, which takes about 15 min. Load the sample at a flow rate of 8 mL / min; the UV value will rise and remain at a certain level, this peak is the breakthrough peak. After loading the sample, equilibrate again with 95% solution A, and the peak value will drop to the baseline level and stabilize. Elute with 70% solution B; during this process, the peak value will first rise and then drop to the baseline, this elution peak formation process is the elution of the target protein, collect the eluent from this process. Rinse the tubing for contaminating proteins with 100% solution B, then fill the tubing and protein A column with 20% ethanol, remove the column, and store at 4°C. The eluted proteins were dialyzed into 20 mM PBS via a dialysis bag at 4°C for 24 h. Antibody concentration was measured using a microplate reader or BCA. If the antibody concentration was <0.5 mg / mL, it was concentrated using a 10 kDa Millipore concentrator at 3000 rpm for 10 min. The antibodies were aliquoted and stored at -20°C for later use.

[0212] Example 2: Verification of the properties of bispecific antibodies

[0213] 2.1 Antibody SDS-PAGE Validation

[0214] Take 5 μg of each antibody obtained in Example 1, mix them separately with reducing and non-reducing loading buffers at working concentrations, heat, and then add SurePAGE. TMElectrophoresis was started in the precast gel at 100V for 120 minutes. After rapid staining, the gel was imaged using a high-resolution gel imaging system (Bio-Red). The results are shown in Figure 2, where bands of the correct size can be seen for antibodies 73-WT, 73-DA, BIS-WT, and BIS-DA.

[0215] 2.2 Verification of protein-level binding activity

[0216] The binding activity of the antibody to HBsAg was detected using an ELISA plate (Wantai Biopharmaceutical) coated with HBsAg. DEC-205 protein was diluted to 1 μg / mL with PBS and coated into ELISA plates (100 μL per well). After incubation overnight at 4°C, the plates were washed five times with PBST, blocked overnight with blocking buffer I, and then dried for use in detecting the binding activity of the antibody to DEC-205 protein.

[0217] Antibodies 73-WT, 73-DA, BIS-WT, and BIS-DA were serially diluted 3-fold at an initial concentration of 100 nM, resulting in 12 serial dilutions. 100 μL of each diluted antibody was added to plates containing HBsAg and DEC-205, and incubated at 37°C for 1 h. The plates were then washed 5 times with PBST. Next, Goat Anti-mouse IgG-HRP secondary antibody (diluted 1:5000 with ED11 buffer) was added, and the plates were incubated at 37°C for 30 min. The plates were washed 5 times with PBST, and 100 μL of a 1:1 mixture of substrate A and B solutions was added to each well. The plates were incubated at 37°C for 10-15 min, and a blue gradient was observed. The reaction was terminated by adding 50 μL of stop solution to each well, and the absorbance was measured at 450 / 630 nm.

[0218] As shown in Figure 3, antibodies 73-WT, 73-DA, BIS-WT, and BIS-DA all exhibited concentration-dependent binding to HBsAg. Bispecific antibodies BIS-WT and BIS-DA also showed concentration-dependent binding to DEC-205, while antibodies 73-WT and 73-DA did not show a specific binding trend to DEC-205.

[0219] 2.3 Validation of antibody binding activity to DEC-205 at the cellular level

[0220] CHO cells expressing DEC-205 (CHO-LY75), previously constructed in our laboratory, were used to detect the cellular-level binding activity of anti-DEC-205. Antibodies were diluted to equimolar concentrations with PBS; antibodies 73-WT and 73-DA were diluted to 7.5 μg / mL, and bispecific antibodies BIS-WT and BIS-DA were diluted to 10 μg / mL. Each antibody was resuspended in 100 μL of water at a concentration of approximately 5 × 10⁶ cells / mL. 5After incubating CHO-LY75 cells at 4°C for 1 hour, unbound antibodies were washed away with PBS. The secondary antibody Goat-anti-mIgG-Alex flour 488 was diluted 1:1000 with PBS, and each sample was resuspended in 100 μL of the working concentration of secondary antibody. The cells were incubated at 4°C for 30 minutes, and unbound antibodies were washed away with PBS. After filtering through a 70 μm sieve, the samples were analyzed by flow cytometry (LSRFortessaX-20; BD). The results are shown in Figure 4. The bispecific antibodies BIS-WT and BIS-DA specifically bound to CHO cells expressing DEC-205.

[0221] 2.4 Validation of antibody-mediated antigen phagocytosis

[0222] DEC-205 receptor is a phagocytic receptor. To verify the ability of bispecific antibodies BIS-WT and BIS-DA to mediate the phagocytosis of antigens by dendritic cells (DCs), bone marrow-derived dendritic cells (BMDCs) were induced in vitro (see Sci Immunol. 2023; 8(79):eabn6612.). HBsAg was labeled with the pHrodo™ Deep Red antibody labeling kit. The labeled HBsAg showed bright fluorescence after entering late endosomes and lysosomes. 1 μg / mL of fluorescently labeled HBsAg was mixed with 10 μg / mL of bispecific antibodies BIS-WT and BIS-DA or 7.5 μg / mL of antibodies 73-WT and 73-DA, and approximately 5 × 10⁻⁶ HBsAg was added to each of the following solutions: 5 BMDCs were co-incubated at 37°C for 2 hours. Unbound antigens and antibodies were washed away, and the cells were filtered through a 70 μm sieve and analyzed by flow cytometry. Mouse spleen cDC1 cells were also isolated and validated using the same experimental method. The results, shown in Figure 5, demonstrate that the bispecific antibodies BIS-WT and BIS-DA specifically mediate the phagocytosis of HBsAg by BMDCs and spleen-derived cDC1 cells, delivering it to late endosomes and lysosomes.

[0223] Example 3: Construction of the immune agonist bispecific antibody-drug conjugate ISBC

[0224] 3.1 Linking of small molecule agonists to linkers

[0225] The TLR7 / 8 agonist IMDQ was linked to Mal-PEG6-NHS ester (maleimide-hexaethylene glycol-succinimide acrylate, CAS No.:1599472-25-9) via an amino reaction to obtain Mal-PEG6-IMDQ.

[0226] 3.2 Antibody Modification and Thiol Exposure

[0227] SAT(PEG)4 was added to the antibody at a molar ratio of 1:10 to the linker, and the reaction was carried out at room temperature for 2 hours by rotation. Unreacted SAT(PEG)4 was removed through an ultrafiltration tube (10 kDa, Millipore) at 3500 rpm and 4°C. The ultrafiltration buffer was PBS containing 10 mM EDTA. The thiol groups were exposed by deacetylation. A deacetylation reagent containing hydroxylamine chloride was mixed with the SAT(PEG)4-modified antibody at a volume ratio of 1:10 and reacted at room temperature for 2 hours. Unreacted deacetylation reagent was then removed through an ultrafiltration tube to obtain deacetylated SAT(PEG)4-modified bispecific antibodies BIS-WT and BIS-DA, whose schematic structures are shown in the following formula, where Ab represents the bispecific antibody BIS-WT or BIS-DA.

[0228] 3.3 Conjugation of Mal-PEG6-IMDQ with deacetylated SAT(PEG)4-modified bispecific antibodies BIS-WT or BIS-DA

[0229] Mal-PEG6-IMDQ was mixed with the antibody at a molar ratio of 4:1. The reaction buffer contained 1 mM EDTA and 25% DMSO (dimethyl sulfoxide). After mixing, the mixture was allowed to stand at room temperature for 3 h, and then incubated overnight at 4 °C. Unreacted small molecules and excess DMSO were removed using an ultrafiltration tube (3500 rpm, 25 °C). The ultrafiltration buffer was PBS containing 1 mM EDTA. The resulting immunostimulator bispecific antibody conjugates ISBC, represented by Formula II, including ISBC-WT and ISBC-DA (see Figure 6 for structural formulas), were sterilized and stored at -20 °C for later use. In Formula II, Ab represents the bispecific antibody BIS-WT or BIS-DA, and n represents DAR (Drug-to-Antibody Ratio).

[0230] Example 4: Verification of the basic properties of the immunostimulant bispecific antibody-drug conjugate ISBC

[0231] 4.1 SDS-PAGE Validation

[0232] To confirm that the size and structure remained unchanged after antibody-coupled agonist, reduction and non-reduction electrophoresis were performed. 5 μg of bispecific antibodies BIS-WT and BIS-DA, as well as ISBC-WT and ISBC-DA obtained in Example 3, were mixed with working concentrations of reduction and non-reduction loading buffers, heated, and then added to SurePAGE. TMElectrophoresis was initiated in the precast gel at 100V for 120 min. After rapid Cochlear staining, the images were obtained using a high-resolution gel imaging system (Bio-Red). The results are shown in Figure 7. The light and heavy chain bands of the antibody were of the correct size after conjugation, indicating that the size and structure of antibodies BIS-WT and BIS-DA remained unchanged after conjugation with the agonist.

[0233] 4.2 Validation by Hydrophobic Interaction Chromatography (HIC)

[0234] The test samples were BIS-DA (represented as BIS in Figure 8), the bispecific antibody BIS-DA modified with deacetylated SAT(PEG)4 (represented as BIS-SAT(PEG)4 in Figure 8), and ISBC-DA (represented as ISBC in Figure 8).

[0235] The test sample (concentration 3-4 mg / mL) was mixed with mobile phase A at a volume ratio of 1:1 (final antibody concentration 2 mg / mL). After mixing, the mixture was centrifuged at 13000 rpm for 10 minutes, and the supernatant was collected for HIC-HPLC detection. The results are shown in Figure 8. Compared with the unconjugated BIS-DA, the main peak of ISBC-DA after conjugation with the agonist showed a significant shift.

[0236] 4.3 LC-MS Validation: ISBC-DA after agonist conjugation was desugared and then analyzed by LC-MS to obtain the primary raw spectrum. The deconvolution result is shown in Figure 9. The calculated average DAR value was 2.03. The DAR value of ISBC-WT was approximately 2.

[0237] Example 5: Functional validation of the immune agonist bispecific antibody-drug conjugate ISBC

[0238] 5.1 BMDC Verification of ISBC-Mediated Phagocytosis

[0239] 10 μg / mL of bispecific antibodies (BIS-WT and BIS-DA) and ISBC (ISBC-WT and ISBC-DA) were respectively mixed with 1 μg / mL of fluorescently labeled HBsAg and approximately 5 × 10⁻⁶ mmol / L of HBsAg. 5 BMDCs were co-incubated, and after 24 hours, unbound antigens and antibodies or ISBCs were washed away with PBS. After filtration through a 70 μm sieve, the samples were detected by flow cytometry. The results are shown in Figure 10. Compared with the unconjugated bispecific antibody, the agonist-conjugated ISBC-WT and ISBC-DA significantly enhanced the antigen-phagocytic ability of DCs.

[0240] 5.2 BMDC Validation of ISBC's Effect on Promoting DC Activation

[0241] 10 μg / mL of bispecific antibodies (BIS-WT and BIS-DA), ISBC (ISBC-WT and ISBC-DA), and IMDQ were respectively mixed with 1 μg / mL of fluorescently labeled HBsAg and approximately 5 × 10⁻⁶ mmol / L of HBsAg. 5 BMDCs were co-incubated for 24 hours. Unbound antigens and antibodies, or ISBCs, were washed away with PBS. The cells were resuspended in 50 μL of a flow cytometry antibody mixture containing working concentrations of anti-CD80-PerCP5.5, anti-CD86-PE, anti-CD40-APC, and Zombie Aqua. After staining at 4°C for 30 min, excess flow cytometry antibody was washed away with PBS. The cells were filtered through a 70 μm sieve and analyzed by flow cytometry. The results are shown in Figure 11. Compared with bispecific antibodies without conjugated agonists, ISBC-WT and ISBC-DA significantly promoted the upregulation of DC maturation markers CD80, CD86, and CD40 expression levels.

[0242] Example 6: Therapeutic effect of the immune agonist bispecific antibody-drug conjugate ISBC in an HBV mouse model

[0243] 6.1 Establishment of the AAV-HBV infection model

[0244] Female C57 / B6 mice (Shanghai Silex Laboratory Animal Co., Ltd.) aged 4-6 weeks were ordered. AAV-HBV (HBV-B type, adw serotype, Guangzhou Paizhen Biotechnology Co., Ltd.) was diluted with PBS and injected via tail vein, 200 μL / mouse. Four weeks post-infection, blood was collected from the orbital sinus, and serum was analyzed using an HBsAg detection kit (chemiluminescence method, Wantai Biopharmaceutical). Mice were grouped according to HBsAg titer as follows: PBS, 73-WT, BIS-WT, ISBC-WT, 73-DA, BIS-DA, and ISBC-DA.

[0245] 6.2 Antiviral effect of ISBC in AAV-HBV mouse model

[0246] Equimolar doses were administered: 73-WT and 73-DA at 7.5 mg / kg, and BIS-WT, BIS-DA, ISBC-WT, and ISBC-DA at 10 mg / kg via intraperitoneal injection, once weekly for 4 weeks. Blood was collected from the orbital rim every 3-4 days, and again every 1-2 weeks after treatment to collect serum and measure HBsAg titers. Results are shown in Figure 12. Wild-type antibody 73-WT and bispecific antibody BIS-WT, due to their Fc-mediated ADCP function, exhibited longer viral inhibition times compared to mutant antibody 73-DA and bispecific antibody BIS-DA. Weak antigen clearance was only detected on the day of administration for 73-DA and BIS-DA; subsequent antigen titers in mouse serum remained at baseline. In the 73-WT group, serum antigen titers decreased rapidly after administration and rebounded slowly within 2 months after treatment. In the BIS-WT group, serum HBsAg titers rebounded rapidly after treatment. In contrast, mice in the ISBC-WT and ISBC-DA groups achieved long-term viral control, maintaining low levels of viral antigens in their bodies long after treatment ended, indicating that ISBC has excellent therapeutic efficacy. Comparing ISBC-DA and ISBC-WT, it was found that all 10 mice in the ISBC-DA treatment group had good control of viral infection, while in the ISBC-WT treatment group, 3 out of 10 mice showed a rebound in viral titer to baseline after treatment, indicating that ISBC-DA had a better therapeutic efficacy than ISBC-WT.

[0247] The levels of HBsAg and viral nucleic acid in mouse liver tissue were measured at the treatment endpoint, and the results are shown in Figure 13. The results showed that the HBsAg level in the liver of mice treated with ISBC-DA was significantly reduced, while no similar effect was observed in other control groups (Figure 13a). Viral DNA was further extracted from liver tissue lysate, and the HBV-DNA titer was quantitatively detected using a probe method. The results showed that the HBV-DNA load in the liver of mice treated with ISBC-DA was significantly reduced (Figure 13b). Simultaneously, RNA was isolated from mouse liver using an animal tissue RNA extraction kit, and the HBV-RNA level was detected using SYBR Green real-time quantitative PCR. It was found that ISBC-DA treatment also significantly reduced the HBV-RNA content in the liver (Figures 13c and d). The presence of HBcAg is an important marker of HBV infection. Immunohistochemical detection was performed on the liver tissue of mice after treatment, and the results are shown in Figure 14. The results showed that HBcAg staining mainly exhibited nuclear distribution. Compared with the PBS control group, 73-DA, and BIS-DA treatment group, the HBcAg level in the liver tissue of mice in the ISBC-DA treatment group was significantly decreased (Figure 14), indicating that HBV-infected hepatocytes were significantly reduced after ISBC-DA treatment. These results collectively demonstrate that ISBC-DA treatment effectively inhibits the active replication of HBV in the liver and successfully achieves long-term control of HBV infection.

[0248] To comprehensively evaluate the safety of ISBC-DA treatment, serum ALT and AST levels in mice were systematically monitored during and at the treatment endpoint, and histopathological analysis was performed using H&E staining of liver tissue. Images were acquired using a fully automated digital slide scanner (Zeiss AxioScan7). Image processing was performed using SlideViewer. As shown in Figure 15, the results indicated that serum ALT and AST levels in the treatment group remained within the normal range, and histopathological examination of liver tissue revealed no significant inflammatory infiltration or structural damage (Figure 15). These data suggest that ISBC-DA treatment exerts a significant antiviral effect without causing significant liver damage, preliminarily confirming its favorable safety profile.

[0249] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and not to limit them; although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of this application or equivalent substitutions can be made to some technical features without departing from the spirit of the technical solutions of this application, and all such modifications and substitutions should be covered within the scope of the technical solutions claimed in this application.

Claims

1. A bispecific antibody comprising a first antigen-binding domain that specifically binds to HBsAg and a second antigen-binding domain that specifically binds to receptors on the surface of dendritic cells (such as DEC-205, Clec9A, Dectin 1, XCR1, CD204, CD206, CD209, etc.). Preferably, the bispecific antibody comprises a first antigen-binding domain that specifically binds to HBsAg and a second antigen-binding domain that specifically binds to DEC-205.

2. The bispecific antibody of claim 1, wherein, The first antigen-binding domain and the second antigen-binding domain can be any antibody form, and can be independently a full-length antibody (e.g., IgG antibody) or its antigen-binding fragment (e.g., scFv, Fab, scFab) or nanobody, etc.

3. The bispecific antibody of claim 1 or 2, wherein, The first antigen-binding domain includes: Nanobodies, or A first heavy chain variable region (VH) and a first light chain variable region (VL), the first heavy chain variable region (VH) and the first light chain variable region (VL) together form a domain capable of specifically binding HBsAg; and / or The second antigen-binding domain includes: Nanobodies, or The second heavy chain variable region (VH) and the second light chain variable region (VL) together form a domain that can specifically bind to DEC-205.

4. The bispecific antibody of any one of claims 1-3, further comprising an Fc domain, said Fc domain comprising a second constant region (CH2) and a third constant region (CH3) of the heavy chain. Preferably, the second constant region (CH2) of the heavy chain contains modifications of one or more amino acids that can eliminate the binding of bispecific antibodies and Fcγ receptors, thereby avoiding nonspecific activation of the immune response.

5. The bispecific antibody of claim 4, wherein the second constant region (CH2) of the heavy chain comprises an amino acid sequence as shown in SEQ ID NO:44 or 46. Preferably, the third constant region (CH3) of the heavy chain contains an amino acid sequence as shown in SEQ ID NO:

45.

6. The bispecific antibody according to any one of claims 3-5, wherein: The first heavy chain (HC) variable region (VH) includes: (i) HCDR1 containing the amino acid sequence shown in SEQ ID NO:5, HCDR2 containing the amino acid sequence shown in SEQ ID NO:6, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:

7. (ii) HCDR1 comprising the amino acid sequence shown in SEQ ID NO:11, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:12, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO:13; or, (iii) HCDR1 containing the amino acid sequence shown in SEQ ID NO:16, HCDR2 containing the amino acid sequence shown in SEQ ID NO:17, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:

13. And / or, The first light chain (LC) variable region (VL) includes: (i) LCDR1 comprising the amino acid sequence shown in SEQ ID NO:8, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:9, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:10; or, (ii) LCDR1 containing the amino acid sequence shown in SEQ ID NO:14, LCDR2 containing the amino acid sequence shown in SEQ ID NO:15, and LCDR3 containing the amino acid sequence shown in SEQ ID NO:

10.

7. The bispecific antibody according to any one of claims 3-6, wherein, The first heavy chain variable region (VH) includes: (i) The amino acid sequence as shown in SEQ ID NO:1, or (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the amino acid sequence shown in SEQ ID NO:1, or (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:1; and / or, The first light chain variable region (VL) includes: (i) The amino acid sequence as shown in SEQ ID NO:2, or (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the amino acid sequence shown in SEQ ID NO:2; or (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:2; Preferably, the substitution is a conservative substitution; Preferably, the amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity does not involve changes to the CDR sequence.

8. The bispecific antibody according to any one of claims 3-7, wherein: The second heavy chain (HC) variable region (VH) includes: (i) HCDR1 containing the amino acid sequence shown in SEQ ID NO:18, HCDR2 containing the amino acid sequence shown in SEQ ID NO:19, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:

20. (ii) HCDR1 containing the amino acid sequence shown in SEQ ID NO:24, HCDR2 containing the amino acid sequence shown in SEQ ID NO:25, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:

26. (iii) HCDR1 comprising the amino acid sequence shown in SEQ ID NO:29, HCDR2 comprising the amino acid sequence shown in SEQ ID NO:30, and HCDR3 comprising the amino acid sequence shown in SEQ ID NO:26; or, (iv) HCDR1 containing the amino acid sequence shown in SEQ ID NO:31, HCDR2 containing the amino acid sequence shown in SEQ ID NO:32, and HCDR3 containing the amino acid sequence shown in SEQ ID NO:

26. And / or, The second light chain (LC) variable region (VL) comprises: (i) LCDR1 comprising the amino acid sequence shown in SEQ ID NO:21, LCDR2 comprising the amino acid sequence shown in SEQ ID NO:22, and LCDR3 comprising the amino acid sequence shown in SEQ ID NO:23; or, (ii) LCDR1 containing the amino acid sequence shown in SEQ ID NO:27, LCDR2 containing the amino acid sequence shown in SEQ ID NO:28, and LCDR3 containing the amino acid sequence shown in SEQ ID NO:

23.

9. The bispecific antibody according to any one of claims 3-8, wherein, The second heavy chain variable region (VH) includes: (i) The amino acid sequence as shown in SEQ ID NO:3, or (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the amino acid sequence shown in SEQ ID NO:3, or (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:3; and / or, The second light chain variable region (VL) comprises: (i) The amino acid sequence as shown in SEQ ID NO:4, or (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the amino acid sequence shown in SEQ ID NO:4; or (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:4; Preferably, the substitution is a conservative substitution; Preferably, the amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity does not involve changes to the CDR sequence.

10. The bispecific antibody according to any one of claims 3-9, wherein, The first antigen-binding domain is Fab, and the second antigen-binding domain is scFv. Preferably, the second antigen domain comprises an amino acid sequence as shown in SEQ ID NO:

36.

11. The bispecific antibody of claim 10, comprising a heavy chain and a light chain, wherein, The heavy chain includes a first heavy chain variable region, a heavy chain CH1 region, a heavy chain CH2 region, a heavy chain CH3 region, a second heavy chain variable region, and a second light chain variable region. The light chain includes a first light chain variable region and a light chain constant region.

12. The bispecific antibody of claim 11, wherein, The heavy chain from N end to C end includes: the first heavy chain variable region, the heavy chain CH1 region, the heavy chain CH2 region, the heavy chain CH3 region, the second heavy chain variable region, and the second light chain variable region. The light chain from N end to C end includes: the first light chain variable region and the light chain constant region.

13. The bispecific antibody of claim 12, wherein, The adjacent structural domains of the heavy chain are optionally connected by or without joints, and the adjacent structural domains of the light chain are optionally connected by or without joints. Preferably, each of the peptide linkers is independently the same or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers); preferably, each of the peptide linkers is independently selected from peptide linkers containing one or more glycine (G) and / or serine (S), for example, possessing (GGGGS). n The structure shown, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; preferably, each peptide linker independently comprises an amino acid sequence as shown in SEQ ID NO:

36.

14. The bispecific antibody of any one of claims 11-13, wherein the heavy chain comprises, from the N-terminus to the C-terminus: a first heavy chain variable region, a heavy chain CH1 region, a hinge region, a heavy chain CH2 region, a heavy chain CH3 region, a linker, a second heavy chain variable region, a linker, and a second light chain variable region. Preferably, the heavy chain CH1 region contains an amino acid sequence as shown in SEQ ID NO:42, the heavy chain CH2 region contains an amino acid sequence as shown in SEQ ID NO:44 or 46, the heavy chain CH3 region contains an amino acid sequence as shown in SEQ ID NO:45, and / or, the light chain constant region contains an amino acid sequence as shown in SEQ ID NO:

35.

15. The bispecific antibody according to any one of claims 11-14, wherein, The heavy chain comprises an amino acid sequence as shown in SEQ ID NO:39 or 40, and / or the light chain comprises an amino acid sequence as shown in SEQ ID NO:

41.

16. An isolated nucleic acid molecule or group of nucleic acid molecules, comprising a nucleotide sequence encoding a bispecific antibody of any one of claims 1-15.

17. A vector comprising the isolated nucleic acid molecule or nucleic acid molecule group of claim 16.

18. The carrier of claim 17, wherein, The nucleotide sequences encoding the heavy and light chains of the bispecific antibody are located in different carrier molecules.

19. The carrier of claim 17 or 18, wherein, The vector is a cloning vector or an expression vector.

20. A host cell comprising the isolated nucleic acid molecule or group of nucleic acid molecules of claim 16, or the vector of any one of claims 17-19.

21. A method for preparing the bispecific antibody of any one of claims 1-15, comprising culturing the host cell of claim 20 under conditions allowing expression of the bispecific antibody, and recovering the bispecific antibody from the cultured host cell culture.

22. Use of the bispecific antibody of any one of claims 1-15 in the preparation of antibody-drug conjugates.

23. A composition comprising a bispecific antibody of any one of claims 1-15, or an isolated nucleic acid molecule or group of nucleic acid molecules of claim 16, or a vector of any one of claims 17-19, or a host cell of claim 20.

24. A conjugate comprising the bispecific antibody of any one of claims 1-15 and a conjugate thereof; Preferably, the coupling portion comprises a therapeutic agent, such as a TLR agonist, STING, etc. Preferably, the therapeutic agent is selected from TLR agonists; Preferably, the therapeutic agent is selected from TLR7 agonists, TLR8 agonists, and any combination thereof; Preferably, the therapeutic agent is selected from TLR7 agonists; Preferably, the TLR agonist is selected from imidazoquinoline derivatives, pyrimidine derivatives, purine derivatives, benzothiophene derivatives, synthetic oligonucleotides, and any combination thereof; Preferably, the TLR agonist is selected from GS-9620, GS-9688, JNJ-64794964, IMDQ, SZU-101, R848, and any combination thereof. Preferably, the TLR agonist is IMDQ.

25. The conjugate of claim 24, which is an antibody-drug conjugate, comprising: The targeting molecule is selected from the bispecific antibodies of any one of claims 1-15; The therapeutic agent; and Linker for connecting the target molecule and the therapeutic agent; Preferably, the therapeutic agent is coupled to the naked amino or thiol group of the bispecific antibody via the linker.

26. The coupling of claim 25, wherein the connector is selected from the structural segment shown in formula A, -L1-L2-L3- A L1 is a structural fragment linked to the bispecific antibody shown. L2 is the structural segment connecting L1 and L3; L3 is the structural segment that connects L2 and the therapeutic agent; Preferably, L1 is selected from: Where h is 0, 1, 2, 3, 4, 5 or 6; Preferably, L1 is Preferably, L2 is selected from: -(CH2) m -,-(CH2) t O-,-(CH2CH2O) r -, Where m, t, and r are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12; R is selected from: hydrogen, halogen, methyl, ethyl, nitro, methoxy, and ethoxy; Each p is independently 0, 1, 2, 3 or 4; Preferably, L2 is -(CH2CH2O) r - where r is defined as described above; Preferably, L3 is selected from: -S(O)-, -S(O)2-, -C(O)-, -(CH2) g -C(O)-,-(CH2) g -S(O)- and -(CH2) g -S(O)2-, where each g is independently selected from 1, 2, 3, 4; Preferably, L3 is -C(O)- or -(CH2). g -C(O)-, where g is defined as described above.

27. The conjugate of any one of claims 24-26, wherein the bispecific antibody is a modified or unmodified antibody; Preferably, the bispecific antibody is a deacetylated SAT(PEG)4 modified antibody.

28. The coupling compound of any one of claims 24-27, wherein the coupling compound is selected from the coupling compound shown in Formula I. in, Ab represents the aforementioned bispecific antibody. The definitions of L1, L2, and L3 are as described in claim 26. D represents the therapeutic agent. n represents DAR, which is a number between 1 and 10 (including integers and decimals), such as numbers between 1 and 8, numbers between 1 and 6, numbers between 1 and 5, numbers between 1 and 4, numbers between 1 and 3, numbers between 1.5 and 2.5, and numbers between 1.8 and 2.

2. Preferably, n is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10; Preferably, the coupling agent is the coupling agent shown in Formula II. The definitions of Ab and n are as described above.

29. A pharmaceutical composition comprising the conjugate of any one of claims 24-28, and a pharmaceutically acceptable carrier and / or excipient. Preferably, the conjugate is present in the pharmaceutical composition in an effective amount.

30. The pharmaceutical composition of claim 29 further comprises an additional pharmaceutically active agent. Preferably, the additional pharmaceutically active agent is a drug for treating hepatitis B, especially chronic hepatitis B, including hepatitis B therapeutic antibodies, recombinant protein vaccines, agonist antibodies that target and activate receptors, etc.

31. Use of the bispecific antibody of any one of claims 1-15, or the conjugate of any one of claims 24-28, or the pharmaceutical composition of any one of claims 29-30 in the preparation of a medicament for treating hepatitis B, particularly chronic hepatitis B, in a subject.

32. A method for treating and / or adjunctive treating a disease in a subject, the method comprising administering to a subject in need an effective amount of a bispecific antibody of any one of claims 1-15, or a conjugate of any one of claims 24-28, or a pharmaceutical composition of any one of claims 29-30, wherein the disease is hepatitis B, particularly chronic hepatitis B.

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