Treatment of chronic hepatitis B based on the synergistic effect of interferon and immune checkpoint inhibitor antibodies

Abstract

The present invention provides a treatment for chronic hepatitis B based on the synergistic effect of interferon and an immune checkpoint inhibitor antibody. Specifically, the present invention provides a fusion protein comprising an operably linked PD-L1-binding polypeptide and an interferon (IFN), which is a heterodimeric or homodimeric protein. The fusion protein of the present invention can be administered intravenously to locally exert the antiviral and immunomodulatory effects of IFN in the liver, thereby improving efficacy and reducing side effects.

Description

[Technical Field]

[0001] The present invention relates to the technical fields of genetic engineering and biomedicine, and specifically to the application of interferon (IFN) and anti-PD-L1 fusion proteins in the treatment of chronic hepatitis B, and their application in the prevention of viral infections, and the treatment of chronic viral infections and in vivo latent viral recurrent infections. [Background technology]

[0002] Hepatitis B virus (HBV) is a DNA virus belonging to the Hepadnaviridae family. The complete HBV is a particulate virus with a diameter of 42 nanometers, consisting of two parts: an envelope and a nucleocapsid. The envelope contains small (S), middle (M), and large (L) envelope proteins (HBsAg), glycoproteins, and cellular lipids. The core particle contains the core protein (HBcAg), a 3.2-kb partial circular double-stranded DNA, and polymerase. HBV attaches to the surface of hepatocytes and mediates endocytosis of the virus through binding of the preS1 region of the large envelope protein (L-HBsAg) to the viral receptor sodium ion-taurocholate transport polypeptide (NTCP), allowing HBV to enter the cell and establish infection. Only humans, chimpanzees, and tree shrews are susceptible to HBV. HBV infection can cause acute or chronic hepatitis, liver fibrosis, cirrhosis, and liver cancer. Currently, there are approximately 240 million chronic hepatitis B patients worldwide, and approximately 1 million people die each year from HBV-related cirrhosis or liver cancer. Whether HBV infection develops into chronic hepatitis B (CHB) depends on the host's age and immune system status. Approximately 80-90% of infants infected with HBV develop chronic hepatitis B. 30-50% of infants infected with HBV before the age of 6 develop chronic hepatitis B. Less than 5% of adult HBV infections develop chronic hepatitis B.

[0003] HBV infection is a global health problem. Eliminating viral infection and reducing complications associated with CHB, including liver fibrosis, cirrhosis, and liver cancer, are the primary goals of current CHB treatment. Currently, the drugs approved for CHB treatment are type I interferons (IFNs) and five nucleoside(t)ide analogs (NAs). Type I interferons promote dendritic cell (DC) maturation, activate T cells by processing and presenting antigens, and act as a bridge between innate and adaptive immunity. After a limited period of treatment (usually 48 weeks), IFNs can modulate immune activity in a small number of CHB patients and / or suppress HBV replication through one or more antiviral interferon-stimulated gene (ISG) products. Type I interferon receptors (IFN-αRs) are widely expressed in tissues, and as the dose and duration of administration increase, they can cause serious side effects in patients. These include flu-like symptoms (e.g., fever, headache), nausea, fatigue, leukopenia, anemia, and thrombocytopenia. These symptoms severely impact patients' quality of life, leading many patients to discontinue the medication due to intolerance to these side effects. Furthermore, type I interferons suppress antiviral immune responses by upregulating the expression of the immunosuppressive molecule programmed death ligand 1 (PD-L1), thereby reducing therapeutic efficacy. How to overcome the side effects and immunosuppression associated with systemic administration of type I interferons remains an important issue. Nucleoside (acid) analogs inhibit HBV replication by inhibiting viral reverse transcriptase. Compared to type I interferon, the advantages of nucleoside (acid) analogs are that they can be administered orally, are highly safe, and can significantly reduce serum HBV-DNA levels. However, they rarely achieve HBsAg serological conversion (a functional cure indicator) and may require lifelong administration.Because covalently closed circular DNA (cccDNA) molecules remain in the liver during HBV infection, there is currently no effective method to silence or eliminate cccDNA, making it impossible to completely cure CHB. The current consensus for CHB treatment is functional cure (elimination of HBsAg in the serum accompanied by production of anti-HBsAg, and concomitant reduction of HBV-DNA levels in the serum to below the detection limit), which is the ideal endpoint that can be achieved with current CHB treatments.

[0004] Programmed cell death protein 1 (PD1) is an important immune checkpoint molecule that can suppress immune responses by inhibiting T cell TCR signaling. PD1 ligand PD-L1 is commonly elevated in the liver of CHB patients. At the same time, in vivo T cells of CHB patients overexpress the inhibitory receptor PD1. PD1-PD-L1 binding reduces T cell function, resulting in an ineffective immune response in the liver and the inability to suppress or eliminate HBV. Currently, PD1 / PD-L1 blockade has been applied to the treatment of various tumors. PD1 / PD-L1 blockade has also been reported in HBV treatment studies. Treatment of HBeAg-negative CHB patients with nivolumab restored HBV-specific immune responses to some extent, but the rate of functional cure was low (only 1 in 10 patients achieved serum HBsAg elimination). Further research is needed to determine how PD1 / PD-L1 blockade can be used to increase the rate of functional cure in CHB.

[0005] Since the successful development of a preventive hepatitis B vaccine in 1982, it has been widely used worldwide, demonstrating good protective effects and significantly reducing the HBV infection rate. However, it is unable to produce neutralizing antibodies in chronic hepatitis B patients and carriers. Inducing a specific immune response in chronic hepatitis B patients, whose immune systems are already resistant to HBV, is relatively difficult. Therefore, research into therapeutic chronic hepatitis B vaccines remains a long and arduous undertaking. Currently, research into therapeutic hepatitis B vaccines mainly involves DNA vaccines, DC vaccines, and protein vaccines. DNA vaccines are nucleic acid sequences that encode antigens using plasmids or modified viruses as vectors. Due to their sustained in vivo antigen synthesis and inherent immunomodulatory capabilities, DNA vaccines can induce relatively strong and sustained humoral and cellular immunity. However, HBV transgenic mice, non-human primate animal models, and clinical results have shown that DNA vaccines lack effective therapeutic effects. Antigen-bearing DC cells act as vaccines to promote specific immune responses, and DC vaccines can induce specific cytotoxic T lymphocyte (CTL) responses in HBV transgenic mouse models. However, HBV-related RNA or DNA levels in the liver are not significantly reduced, and the number of CTLs induced in the transgenic mice is insufficient, indicating dysfunction. Researchers have used HBV preventive vaccines (HBsAg as the antigen and Alum as the adjuvant) to increase the vaccine dose and frequency, add preS1 / preS2 antigens, or modify the structural form of the recombinant protein. Clinical trials have shown a reduction in HBV DNA, a return to normal ALT levels, and serological conversion of HBeAg in a small number of patients, but none of these methods were able to eliminate HBV DNA or induce serological conversion of HBsAg. Summary of the Invention [Problem to be solved by the invention]

[0006] Currently, the drugs approved for the treatment of CHB patients, whether type I interferons (IFNs) or nucleoside (acid) analogs (NAs), have not achieved a functional cure. Systemic administration of IFNs has a low response rate and significant side effects. To overcome the limitations and shortcomings of existing IFNs in the treatment of CHB patients, and considering the high expression of PD-L1 in the liver of CHB patients, the present invention provides a fusion protein of an anti-PD-L1 antibody and IFN (anti-PD-L1-IFN). This fusion protein uses anti-PD-L1 to target IFN to the liver, allowing IFN's antiviral and immunomodulatory effects to be exerted locally, improving efficacy and reducing side effects. The anti-PD-L1-IFN fusion protein targets IFN to the liver to exert its HBV-suppressing effect while also targeting IFN to DC cells in the liver, improving DC cell function and further promoting HBV-specific T cell function, exerting immune checkpoint inhibition, overcoming PD-L1-mediated immunosuppression, and achieving immunomodulatory effects. IFN-induced elevation of PD-L1 expression in the liver favors more effective liver targeting of anti-PD-L1-IFN fusion proteins. In an HBV-carrier mouse model, significant liver accumulation of anti-PD-L1-IFN fusion proteins was observed, and the therapeutic efficacy of anti-PD-L1-IFN fusion proteins was significantly greater than that of untargeted IFN or anti-PD-L1, or a physical combination of untargeted IFN and anti-PD-L1. The antiviral effect of anti-PD-L1-IFN fusion proteins reduces HBsAg and HBV-DNA levels in the liver and peripheral blood and their immunomodulatory effects, breaking immune tolerance and creating a "blank period." Combined with active vaccination, this can induce HBV-specific T cell and B cell responses in the host, achieving a functional cure for chronic hepatitis B.

[0007] The present invention further relates to mixtures of anti-PD-L1-IFN and HBV antigens (or other viral and tumor antigens), or homo- or heterodimeric or multimeric fusion proteins consisting of anti-PD-L1-IFN and HBV antigens (or other viral and tumor antigens), for prophylactic or therapeutic vaccine applications.

[0008] The object of the present invention is achieved by the following technical solutions: [Means for solving the problem]

[0009] The present invention provides a fusion protein comprising an operably linked PD-L1 binding polypeptide and an interferon (IFN).

[0010] In a specific embodiment of the invention, said fusion protein is a heterodimeric or homodimeric protein.

[0011] Heterodimers of fusion proteins described in the present invention (which may be referred to as anti-PD-L1-IFN heterodimeric fusion proteins) include, for example, two forms: 1) In the first type, the PD-L1-binding polypeptide is in the form of Fab (also called hetero Fab), and the fusion protein comprises a first polypeptide, a second polypeptide, and a third polypeptide, and the first polypeptide, second polypeptide, and third polypeptide are different from each other, wherein the first polypeptide comprises an IFN and an immunoglobulin Fc region, and the IFN is located N- or C-terminal to the Fc region; the second polypeptide comprises a PD-L1-binding polypeptide heavy chain Fab region and an immunoglobulin Fc region, and the PD-L1-binding polypeptide heavy chain Fab region is located N- or C-terminal to the Fc region; and the third polypeptide is a PD-L1-binding polypeptide light chain.

[0012] Preferably, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NO: 23 or SEQ ID NO: 25, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 21, and the third polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 3. 2) In a second type of form, the PD-L1-binding polypeptide is in the form of an scFv (also referred to as a hetero scFv) and comprises a first polypeptide and a second polypeptide, which are distinct from each other, wherein the first polypeptide comprises an IFN and an immunoglobulin Fc region, and the IFN is located at the N-terminus or C-terminus of the Fc region; and the second polypeptide comprises a PD-L1-binding polypeptide scFv and an immunoglobulin Fc region, and the PD-L1-binding polypeptide scFv is located at the N-terminus or C-terminus of the Fc region; Preferably, the first polypeptide comprises the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:23, and the second polypeptide comprises the amino acid sequence shown in SEQ ID NO:9.

[0013] The Fc region contained in the first polypeptide and the Fc region contained in the second polypeptide are derived from the same or different subtypes of immunoglobulins.

[0014] Homodimers of fusion proteins (which may be referred to as anti-PD-L1-IFN homodimeric proteins) according to the present invention include, for example, two forms: 1) In the first type, the PD-L1-binding polypeptide is in the form of Fab (also called homo Fab), and the fusion protein comprises four polypeptides, wherein the first polypeptide and the second polypeptide are the same, and the third polypeptide and the fourth polypeptide are the same, and the first polypeptide and the second polypeptide comprise, from N- to C-terminus, IFN, a PD-L1-binding polypeptide heavy chain Fab region, and an immunoglobulin Fc region (optionally, the IFN, PD-L1-binding polypeptide heavy chain Fab region, and immunoglobulin Fc region can be combined and arranged in any order), and preferably comprise the amino acid sequence set forth in SEQ ID NO:7, and the third polypeptide and the fourth polypeptide are the PD-L1-binding polypeptide light chain, and preferably comprise the amino acid sequence set forth in SEQ ID NO:3.

[0015] 2) In a second type of form, the PD-L1-binding polypeptide is in the form of an scFv (also called homo scFv), and the fusion polypeptide comprises a first polypeptide and a second polypeptide, where the first polypeptide and the second polypeptide are the same. The first polypeptide and the second polypeptide comprise, from N- to C-terminus, IFN, a PD-L1-binding polypeptide scFv, and an immunoglobulin Fc domain (optionally, the IFN, PD-L1-binding polypeptide scFv, and immunoglobulin Fc domain can be combined and arranged in any order), and preferably comprise the amino acid sequence set forth in SEQ ID NO: 11.

[0016] In a specific embodiment of the present invention, the interferon (IFN) may be selected from type I interferon, type I interferon mutants, preferably mouse IFNα4 (e.g., amino acids 1 to 162 of SEQ ID NO: 1 or SEQ ID NO: 19) and human IFNα2 (e.g., amino acids 1 to 165 of SEQ ID NO: 23 or SEQ ID NO: 25), and the IFN may be of human or mouse origin.

[0017] In a specific embodiment of the present invention, the immunoglobulin Fc region may be selected from the constant region amino acid sequences of IgG1, IgG2, IgG3, or IgG4, preferably IgG1. IgG1 is the most common antibody subtype for antibody drugs, possessing a relatively long serum half-life and the ability to strongly induce antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Alternatively, mutations in the Fc region may be used to form a non-ADCC form. Here, the no-ADCC form of the Fc region refers to a human IgG1 Fc segment mutated into three amino acids: L234A, L235A, and P329G (abbreviated as the LALA-PG mutant). This mutant completely loses the ability to bind to Fcγ receptors (FcγR), thereby reducing cytotoxicity (ADCC). Mutation of an Fc region to reduce cytotoxicity is a technique known in the art; see, for example, Tilman Schlothauer et al., "Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions," Protein Engineering, Design & Selection, 2016, vol. 29, no. 10, pp. 457-466. In an embodiment of the present invention, the Fc region in the four types of fusion proteins listed in Figure 1a can be replaced with a mutant Fc region (i.e., a no-ADCC form) that has an effect similar to that of the wild-type.

[0018] In specific embodiments of the invention, the PD-L1-binding polypeptide may be selected from an anti-PD-L1 antibody (whole antibody), a single-chain antibody (scFv), a Fab fragment, or a F(ab')2 fragment, and the anti-PD-L1 antibody is preferably selected from Tecentriq, Bavencio, Imfinzi, KN035, CS1001, KL-A167, SHR-1316, or YW243.55.S70, and more preferably a Fab fragment that binds to PD-L1.

[0019] Specifically, the present invention provides the following technical solutions: 1. A fusion protein comprising an operably linked PD-L1-binding polypeptide and an interferon (IFN).

[0020] 2. The fusion protein according to item 1, which is a heterodimeric protein or a homodimeric protein.

[0021] 3. It is a heterodimeric protein, and the PD-L1-binding polypeptide is in the form of a Fab; optionally comprising a first polypeptide, a second polypeptide, and a third polypeptide, wherein the first polypeptide comprises an IFN and an immunoglobulin Fc region, and the IFN is located N-terminal or C-terminal to the Fc region; the second polypeptide comprises a PD-L1-binding polypeptide heavy chain Fab region and an immunoglobulin Fc region, and the PD-L1-binding polypeptide heavy chain Fab region is located N-terminal or C-terminal to the Fc region; and the third polypeptide is a PD-L1-binding polypeptide light chain; Preferably, the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 19 or SEQ ID NO: 23 or SEQ ID NO: 25, the second polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 21, and the third polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3; Or, a heterodimeric protein, and the PD-L1-binding polypeptide is in the form of an scFv; Optionally, the fusion protein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises an IFN and an immunoglobulin Fc region, and the IFN is located N-terminal or C-terminal to the Fc region; and the second polypeptide comprises a PD-L1-binding polypeptide scFv and an immunoglobulin Fc region, and the PD-L1-binding polypeptide scFv is located N-terminal or C-terminal to the Fc region; Preferably, the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 23, and the second polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9. The fusion protein of item 1.

[0022] 4. The fusion protein is a homodimeric protein and the PD-L1-binding polypeptide is in the form of a Fab; optionally comprising the same first and second polypeptides, and the same third and fourth polypeptides, wherein the first and second polypeptides comprise an IFN, a PD-L1-binding polypeptide heavy chain Fab region, and an immunoglobulin Fc region, and preferably comprise or consist of the amino acid sequence set forth in SEQ ID NO:7; and the third and fourth polypeptides are the light chain of a PD-L1-binding polypeptide, and preferably comprise or consist of the amino acid sequence set forth in SEQ ID NO:3; Or, a homodimeric protein, and the PD-L1-binding polypeptide is in the form of an scFv; Item 1. The fusion protein of Item 1, optionally comprising two identical first and second polypeptides, wherein the first and second polypeptides comprise IFN, a PD-L1-binding polypeptide scFv, and an immunoglobulin Fc region, and preferably comprise or consist of the amino acid sequence set forth in SEQ ID NO: 11.

[0023] 5. The fusion protein according to item 1, wherein the interferon (IFN) may be selected from type I interferon, type I interferon mutants, preferably mouse IFNα4 and human IFNα2, and the IFN may be of human or mouse origin.

[0024] 6. The fusion protein of item 3 or 4, wherein the immunoglobulin Fc region may be selected from the constant region amino acid sequence of IgG1, IgG2, IgG3, or IgG4 (preferably IgG1) or a variant thereof.

[0025] 7. The fusion protein of clause 1, wherein the PD-L1-binding polypeptide may be selected from an anti-PD-L1 antibody or an antigen-binding fragment thereof, such as a single-chain antibody (scFv), Fab fragment, or F(ab')2 fragment, and the anti-PD-L1 antibody is preferably selected from Tecentriq, Bavencio, Imfinzi, KN035, CS1001, KL-A167, SHR-1316, or YW243.55.S70, and more preferably the PD-L1-binding polypeptide is a Fab fragment of a PD-L1 antibody.

[0026] 8. Use of the fusion protein according to any one of paragraphs 1 to 7 in the preparation of a medicament for treating chronic hepatitis B.

[0027] 9. A pharmaceutical preparation or composition comprising the fusion protein according to any one of items 1 to 7 as an active ingredient.

[0028] 10. A polynucleotide encoding the fusion protein according to any one of items 1 to 7.

[0029] 11. A vector comprising the polynucleotide of paragraph 10, which is optionally a plasmid vector or a viral vector.

[0030] 12. A cell comprising the polynucleotide of Item 10 or the vector of Item 11, which expresses the fusion protein of any one of Items 1 to 7, and which is preferably a non-human mammalian cell, preferably selected from CHO and HEK293 cells.

[0031] 13. A pharmaceutical composition or kit comprising the fusion protein and a vaccine according to any one of items 1 to 7, wherein the fusion protein and the vaccine are preferably administered sequentially or simultaneously, and the administration methods include intravenous injection, subcutaneous immunization, or intramuscular immunization, or simultaneous subcutaneous immunization and intramuscular immunization, etc.; Optionally, the antigen in the vaccine may be an HBV envelope protein (S-HBsAg, M-HBsAg, L-HBsAg) or a polypeptide thereof, a nucleocapsid protein (HBcAg) or a polypeptide thereof, a polymerase protein or a polypeptide thereof, or an HBxAg protein or a polypeptide thereof, preferably an HBV envelope protein or a polypeptide thereof; or the antigen in the vaccine may be a novel coronavirus receptor binding region antigen or an influenza virus HA1 antigen; Optionally, the vaccine further comprises an adjuvant, the adjuvant comprising an aluminum adjuvant, a liposome, or CpG, etc.

[0032] 14. Use of the fusion protein of any one of clauses 1 to 7 or a combination of the fusion protein of any one of clauses 1 to 7 with a vaccine in the preparation of a medicament or kit for the prevention of a viral infection (e.g., an acute viral infection) or the treatment of a chronic or latent viral infection, optionally wherein the virus includes, but is not limited to, HBV, coronavirus, or influenza virus.

[0033] The fusion protein (anti-PD-L1-IFN) of the present invention uses anti-PD-L1 to target IFN to the liver, locally exerting the antiviral and immunomodulatory effects of IFN, improving efficacy and reducing side effects. The anti-PD-L1-IFN fusion protein targets IFN to the liver to exert its HBV-suppressing effect, while also targeting IFN to DC cells in the liver, improving DC cell function and further promoting HBV-specific T cell function, exerting immune checkpoint inhibitory effects, overcoming PD-L1-mediated immunosuppression, and achieving immunomodulatory effects. IFN-induced elevation of PD-L1 in the liver is favorable for more effective targeting of the anti-PD-L1-IFN fusion protein. The antiviral effects of the anti-PD-L1-IFN fusion protein reduce HBsAg and HBV-DNA levels in the liver and peripheral blood, as well as their immunoregulatory effects, thereby breaking immune tolerance and creating a "blank period." In combination with active vaccination, this can induce HBV-specific T cell and B cell immunity in the host, or the immune induction following the combination of the anti-PD-L1-IFN fusion protein and vaccine can produce antiviral neutralizing antibodies and T cell responses, achieving a functional cure for chronic hepatitis B.

[0034] Furthermore, the present invention can be used as an immunotherapy platform for preventing acute infections with coronaviruses and influenza viruses, and for preventing and treating chronic infections with viruses such as HBV, HPV, and EBV. The fusion proteins of the present invention can be used as vaccine adjuvants by subcutaneous or intramuscular injection after mixing with vaccines to exert an effect on lymph node drainage; because dendritic cells (DCs) in lymph nodes highly express PD-L1, fusion proteins comprising anti-PD-L antibodies and IFN can more effectively target DCs, stimulating their activation, antigen capture, and antigen presentation functions, thereby enhancing immune responses to antigens in vaccines, significantly improving vaccine efficacy, and enhancing neutralizing antibody and T cell responses to the antigens, thereby eliminating viruses and achieving the goals of preventing or treating the corresponding viruses (not limited to the specific viruses listed in the examples).

[0035] In summary, the fusion proteins of the present invention can be administered intravenously to locally exert the antiviral and immunomodulatory effects of IFN in the liver, improving efficacy and reducing side effects. The antiviral effects of the fusion proteins of the present invention reduce HBV virus and its antigen levels in the liver and peripheral blood, and the immunomodulatory effects of the PD-L1-binding polypeptides reduce HBV immune tolerance. In this case, when combined with active vaccination, HBV-specific T cell and B cell immune responses can be induced in the host. Furthermore, intramuscular immunization after directly mixing the fusion proteins of the present invention with vaccines can induce and produce antiviral neutralizing antibodies and T cell responses, thereby achieving functional cure of chronic viral infections or prevention of viral infections, and treatment of chronic viral infections and in vivo latent viral recurrent infections.

[0036] Terms and Definitions Unless otherwise specified, all terms and definitions used in this application have the meanings commonly used and known to those skilled in the art.

[0037] The term "administration" as used herein refers to systemic and / or local administration. The term "systemic administration" refers to non-local administration, where the administered substance may affect several organs or tissues throughout the body; or the administered substance may pass through several organs or tissues throughout the body to reach the target site. Those skilled in the art will understand that systemic administration encompasses various forms of administration, including, but not limited to, parenteral administration, intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, oral administration, etc. The term "local administration" refers to administration at or near a specific site. Those skilled in the art will understand that local administration encompasses various forms of administration, such as direct injection at or near a specific site.

[0038] As used herein, the term "therapeutically effective amount" refers to the dosage of an anti-PD-L1-IFN fusion protein of the invention required to achieve a therapeutic objective, such as significantly reducing or negating HBsAg in the serum of a hepatitis B patient, or improving immune cell function. The effective amount can be determined for a particular objective through practice and according to conventional methods. In particular, the therapeutically effective amount may be the amount required to achieve an objective, such as reducing HBsAg or negating it with or without HBsAb production, reducing or negating HBeAg, reducing or making HBV-DNA undetectable, reducing liver necroinflammation or restoring liver function, or activating immune cells, in the serum of a hepatitis B patient.

[0039] As used herein, the term "antibody" includes, for example, monoclonal antibodies, polyclonal antibodies, single-chain antibodies, and antibody fragments that exhibit the desired biological or immunological activity. As used herein, the term "immunoglobulin" (Ig) is used interchangeably with antibodies that can specifically target liver tissue.

[0040] The terms "use", "application" or "use" as used herein may refer to applications aimed at treating a disease, or may refer to non-therapeutic applications such as, for example, scientific research.

[0041] In the present invention, the PD-L1-binding polypeptide may be selected from an anti-PD-L1 intact antibody, a single-chain antibody (scFv), a Fab fragment, or a F(ab')2 fragment, where the anti-PD-L1 intact antibody is a commercially available conventional anti-PD-L1 antibody, such as Tecentriq, Bavencio, Imfinzi, KN035, CS1001, KL-A167, SHR-1316, or YW243.55.S70. [Effects of the Invention]

[0042] The beneficial effects of the present invention are as follows: 1. The anti-PD-L1-IFN fusion protein of the present invention has two structures, a heterodimer and a homodimer, both of which have effective antiviral activity in vitro. In vivo, the heterodimer has superior liver targeting, serum stability, and more effective HBV suppression.

[0043] 2. The anti-PD-L1-IFN fusion protein of the present invention demonstrates that anti-PD-L1 antibodies (PD-L1-binding polypeptides) can be used to specifically deliver IFN to liver tissue with minimal side effects. The present invention lays the foundation for the development of novel drugs targeting IFN for the treatment of CHB.

[0044] 3. Combining the anti-PD-L1-IFN fusion protein of the present invention with active vaccine immunization, whether with an HBsAg / CpG vaccine or a commercial hepatitis B prophylactic vaccine, can achieve functional cure in a portion of HBV carrier mice.

[0045] 4. The anti-PD-L1-IFN fusion protein according to the present invention is an immune adjuvant platform that can be used to prevent multiple acute viral infections, treat chronic or latent viral infections, and treat virus-associated tumors. [Brief explanation of the drawings]

[0046] [Figure 1]Anti-PD-L1-IFN fusion proteins and control proteins were constructed. (a) Schematic diagram of the four anti-PD-L1-IFN fusion proteins in heterodimeric or homodimeric forms. Fab, antigen-binding fragment; scFv, single-chain variable region fragment. (b) The four anti-PD-L1-IFN fusion proteins were expressed in 293F cells and purified prior to SDS-PAGE analysis under non-reducing and reducing conditions. M: molecular weight indicator; 1. Fab (PD-L1) heterodimer (hetero Fab); 2. Fab (PD-L1) homodimer (homo Fab); 3. scFv (PD-L1) heterodimer (hetero scFv); 4. scFv (PD-L1) homodimer (homo scFv). (c) Schematic diagram of three control fusion proteins: IFNα-Fc, anti-PD-L1 (Fab), and anti-PD-L1 (scFv). (d) These three control fusion proteins were expressed in 293F cells and purified before analysis by non-reducing and reducing SDS-PAGE. M: molecular weight marker; 5. IFNα-Fc; 6. anti-PD-L1 (Fab); 7. anti-PD-L1 (scFv). (e) Schematic diagram of anti-PD-L1-IFN fusion proteins with no ADCC Fc segment. The fusion proteins were expressed in 293F cells and purified before analysis by non-reducing and reducing SDS-PAGE. M: molecular weight marker.

[0047] [Figure 2]All four anti-PD-L1-IFN fusion proteins maintained the antiviral infection and viral copy suppression activities of IFN. (a) An antiviral infection bioassay was used to detect the biological activity of IFN in the four anti-PD-L1-IFN fusion proteins. L929 cells (6 × 104 cells / well) and various fusion proteins (6 dilutions) were cultured in 24-well plates for 12 hours, then infected with vesicular stomatitis virus-green fluorescent protein (VSV-GFP) (4 × 105 pfu / well). After a further 30 hours of culture, the percentage of virus-infected cells (%GFP+) was detected by flow cytometry. (b) The MFI of virus-infected cells (GFP+) was detected by flow cytometry. (c) The inhibition rate of VSV-GFP virus-infected L929 cells by the four anti-PD-L1-IFN fusion proteins.

[0048] [Figure 3] All four anti-PD-L1-IFN fusion proteins maintained their affinity for PD-L1. (a-e) Biolayer interferometry (BLI) was used to measure the binding and dissociation curves of the homo-scFv fusion protein (a), hetero-scFv fusion protein (b), homo-Fab fusion protein (c), hetero-Fab fusion protein (d), and anti-PD-L1 (Fab) (e) to PD-L1. (f) The affinity constants of the four anti-PD-L1-IFN fusion proteins and anti-PD-L1 (Fab) to PD-L1 were calculated.

[0049] [Figure 4]Heter Fab and hetero scFv fusion proteins had higher accumulation in the liver and blood stability. (a–c) HBV carrier mice were intravenously injected with the indicated fusion proteins (0.1687 nmol / mouse). The concentrations of each fusion protein were measured by ELISA in tissues at 3 hours (a), tissues at 72 hours (b), or serum at different time points (c). Data represent the mean ± SEM and are representative of at least two independent experiments. *P<0.05; **P<0.01; ns indicates not significant.

[0050] [Figure 5] Heter Fab fusion proteins inhibited HBV in vivo in a dose-dependent manner. (a) Male C57BL / 6J mice (4-6 weeks old) were intravenously injected with 1 × 10 GC AAV-HBV1.3 / mouse. After 5 weeks, mice with a stable serum HBsAg content of >1000 IU / mL were designated as HBV carrier mice and used in subsequent experiments. On days 1 and 4, HBV carrier mice (6 groups, 3 mice per group) were intravenously treated with PBS or 5 μg, 10 μg, 20 μg, 40 μg, or 80 μg / mouse of hetero Fab fusion proteins. Blood was collected at designated time points, and serum was collected. (b-d) Serum HBsAg (b) and HBeAg (c) levels were detected by ELISA at each time point. Mice were weighed, and weight changes (d) were recorded. (e) HBV-DNA levels were detected by real-time PCR in serum on days 0 and 7. Data represent the mean ± SEM and are representative of at least two independent experiments.

[0051] [Figure 6]The in vivo HBV suppression effect of hetero Fab or hetero scFv fusion proteins was higher than that of homo Fab or homo scFv fusion proteins. (a) Male C57BL / 6J mice (4-6 weeks old) were intravenously injected with 1 × 10 GC AAV-HBV1.3 / mouse. After 5 weeks, mice with a stable serum HBsAg content of >1000 IU / mL were used as HBV carrier mice (day 0) for subsequent experiments. On days 1 and 4, HBV carrier mice (5 groups, 3 mice per group) were intravenously treated with PBS or four fusion proteins (equimolar IFNα and anti-PD-L1): homo-scFv fusion protein (0.0844 nmol / mouse), hetero-scFv fusion protein (0.1687 nmol / mouse), homo-Fab fusion protein (0.0844 nmol / mouse), or hetero-Fab fusion protein (0.1687 nmol / mouse). Blood was collected at designated time points, and serum was collected. (b, c) HBsAg (b) and HBeAg (c) levels in serum at each time point were detected by ELISA. (d) HBV-DNA levels in serum on days 0, 7, 14, and 21 were detected by real-time PCR. Data represent the mean ± SEM and are representative of at least two independent experiments. *P<0.05; **P<0.01; ns indicates not significant.

[0052] [Figure 7]The in vivo suppression of HBV by hetero-Fab fusion proteins had a synergistic and positive feedback effect. (a) C57BL / 6J male mice (4-6 weeks old) were intravenously injected with 1 × 10 GC AAV-HBV1.3 / mouse. After 5 weeks, mice with a stable serum HBsAg content of >1000 IU / mL were used as HBV carrier mice for subsequent experiments. (b–d) HBV carrier mice (5 groups, 3 mice per group) were intravenously treated with PBS, IFNα-Fc (0.0844 nmol / mouse), anti-PD-L1 (Fab) (0.0844 nmol / mouse), a mixture of IFNα-Fc and anti-PD-L1 (Fab) (0.0844 nmol + 0.0844 nmol / mouse), or hetero Fab fusion protein (0.1687 nmol / mouse) on days 1 and 4. Blood was collected at designated time points, and serum was collected. HBsAg (b) and HBeAg (c) levels in serum were detected by ELISA. (d) HBV-DNA levels in serum were detected by real-time PCR on days 0, 7, 14, and 19. (e, f) HBV carrier mice (3 groups, 6 mice per group) were intravenously treated with PBS, IFNα-Fc (0.0844 nmol / mouse), or hetero Fab fusion protein (0.1687 nmol / mouse) on day 1. Three days later, liver tissues were collected and PD-L1 levels on CD45- (e) and CD45+ (f) cells were analyzed by flow cytometry. The left panel shows a representative flow diagram, and the right panel shows statistical data for MFI (PD-L1). Data are mean ± SEM and represent the results of at least two independent experiments. *P<0.05; **P<0.01; ns indicates not significant.

[0053] [Figure 8]Heter Fab fusion proteins can enhance DC cell function and further promote HBsAg-specific T cell function, breaking immune tolerance. (a) C57BL / 6J male mice (4-6 weeks old) were intravenously injected with 1 × 10 GC AAV-HBV1.3 / mouse. After 5 weeks, mice with a stable serum HBsAg concentration >1000 IU / mL were designated HBV carrier mice and used for subsequent experiments. On day 1, HBV carrier mice were intravenously treated with PBS or equimolar fusion proteins (0.1687 nmol / mouse, equimolar IFNα and anti-PD-L1). Three days later, flow cytometry was used to detect the expression of CD80, CD86, and MHC1 on DC cells and IFN-γ+ CD4+ and CD8+ T cells in the liver and spleen. (b, c) Liver mononuclear cells (b) and splenocytes (c) were collected, and CD86 expression on their DCs was analyzed by flow cytometry. (d) Liver mononuclear cells were collected, stimulated with HBsAg (ayw, 5 μg / mL) for 12 hours, and then cultured for 6 hours in the presence of brefeldin A (5 μg / mL). IFN-γ+ CD4+ and CD8+ T cells were analyzed by flow cytometry. (e) Liver mononuclear cells were collected, and HBsAg-specific T cells and ENV190 (CD8 epitope)-specific T cells were detected by T cell ELISPOT. (f) T cells were depleted by intraperitoneal injection of 200 μg of anti-CD4 and 200 μg of anti-CD8 antibodies twice a week (injected the day before hetero Fab fusion protein treatment), and HBV carrier mice were intravenously injected with hetero Fab fusion protein (0.1687 nmol / mouse) twice (3 days apart). In the control group, T cell depletion was not performed. HBV carrier mice were treated twice (3 days apart) intravenously with PBS or hetero Fab fusion protein (0.1687 nmol / mouse). Blood was collected at the corresponding time points, and serum HBsAg levels were detected by ELISA. (g) HBV carrier mice or Rag1- / - HBV carrier mice were treated twice (3 days apart) intravenously with PBS or hetero Fab fusion protein (0.1687 nmol / mouse). Blood was collected at the corresponding time points, and serum HBsAg levels were detected by ELISA.(h) DC2.4 cells were incubated with HBsAg-FITC (1 μg / mL) for 4 hours at 37°C, followed by the addition of 0.391 μg / mL of IFN α-Fc or 1 μg / mL of hetero Fab (equimolar IFN α). The phagocytic activity of DC2.4 toward HBsAg-FITC was analyzed by flow cytometry. Data represent the mean ± SEM and are representative of at least two independent experiments. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

[0054] [Figure 9] Heter Fab fusion proteins increased the expression of CD80 and MHCI on DC cells. (a) The gating strategy for CD80 and CD86 on DC cells was used to detect them by flow cytometry. (b-d) The treatment methods for HBV carrier mice were as described in Figure 8a. On day 4, liver mononuclear cells and splenocytes were collected, and the expression of CD80 (b) and MHCI (d) on DC cells in the liver and CD80 (c) on splenic DC cells were analyzed by flow cytometry. Data represent the mean ± SEM and are representative of at least two independent experiments. *P<0.01; ****P<0.0001; ns indicates not significant.

[0055] [Figure 10]Heter Fab fusion proteins enhanced the function of HBsAg-specific T cells. (a) IFN-γ+ CD4+ and CD8+ T cells were detected by flow cytometry using a gating strategy. (b, c) Treatment of HBV carrier mice was as described in Figure 8a. On day 4, liver mononuclear cells and splenocytes were collected and stimulated with HBsAg (5 μg / mL) for 12 hours, followed by 6 hours of culture in the presence of brefeldin A (5 μg / mL). IFN-γ+ CD4+ and CD8+ T cells were analyzed by flow cytometry. A representative flow scattergram (b) and a statistical plot of the proportions of IFN-γ+ CD4+ and CD8+ T cells in the spleen (c) are shown. Data are the mean ± SEM (five mice per group from two independent experiments). ****P<0.0001.

[0056] [Figure 11]Combining hetero-scFv fusion protein with HBsAg / aluminum adjuvant vaccination achieved functional cure in some HBV carrier mice. (a) Male C57BL / 6J mice (4-6 weeks old) were intravenously injected with 1 × 10 GC AAV-HBV1.3 / mouse. Five weeks later, mice with a stable serum HBsAg concentration >1000 IU / mL were designated as HBV carrier mice and used for subsequent experiments. On days 1 and 4, HBV carrier mice (5 groups, 3 mice per group) were intravenously treated with PBS, IFNα-Fc (0.0844 nmol / mouse), anti-PD-L1 (scFv) (0.0844 nmol / mouse), or hetero-scFv fusion protein (0.1687 nmol / mouse). Simultaneously, HBsAg (ayw, 2 μg) / aluminum adjuvant fusion protein immunizations were conducted on days 4, 11, 18, and 25, and a single HBsAg (ayw, 2 μg) / aluminum adjuvant immunization group was also established. Blood samples were collected at designated time points. (b, c) Serum HBsAg (b) and anti-HBsAg (c) levels were measured by ELISA. (d, e) Serum HBsAg (d) and anti-HBsAg (e) levels were measured by ELISA at the end of the experiment (day 118). (f) Serum HBV-DNA levels were measured by real-time PCR on days 0 and 118. One mouse in the hetero scFv + HBsAg / aluminum adjuvant group (df) achieved a functional cure (elimination of HBsAg and HBV-DNA in serum accompanied by production of anti-HBsAg). Data represent the mean±SEM and are representative of at least two independent experiments.

[0057] [Figure 12]Combining hetero-scFv fusion protein with HBsAg / CpG vaccination achieved functional cure in most HBV carrier mice. (a) Male C57BL / 6J mice (4-6 weeks old) were intravenously injected with 1 × 10 GC AAV-HBV1.3 / mouse. Five weeks later, mice with a serum HBsAg content stably higher than 1000 IU / mL were designated as HBV carrier mice and used in subsequent experiments. These mice were designated as day 0. On days 1 and 4, HBV carrier mice (four groups total) were intravenously treated with PBS or hetero-scFv fusion protein (0.1687 nmol / mouse). Two groups (heter scFv+HBsAg / CpG and HBsAg / CpG groups) were immunized four times with HBsAg (2 μg) and CpG adjuvant on days 4, 11, 18, and 25. Blood samples were collected at designated time points. (b, c) HBsAg (b) and anti-HBsAg (c) levels in serum were detected by ELISA at each time point. (d, e) HBsAg (d) and anti-HBsAg (e) levels in serum were detected by ELISA at the end of the experiment (day 104). (f) HBV-DNA levels in serum on day 0 and at the end of the experiment (day 104) were detected by real-time PCR. (g) HBsAg levels in liver were detected by ELISA at the end of the experiment (day 104). Three mice in the hetero scFv + HBsAg / CpG group (d–f) achieved functional cure (elimination of serum HBsAg and HBV-DNA accompanied by production of anti-HBsAg). (h, i) At the end of the experiment (day 104), splenic lymphocytes were collected from mice in the PBS group and the combination treatment group (heter scFv + HBsAg / CpG) to detect HBsAg-specific T and B lymphocytes. 1 × 106 cells per well were stimulated with 5 μg / mL HBsAg (ayw) for 48 hours, and specific T cell responses (h) were detected by T cell ELISPOT assay, and specific B cell responses (i) were detected by B cell ELISPOT assay. Data represent the mean ± SEM and are representative of at least two independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

[0058] [Figure 13] Combining hetero-scFv fusion protein with a commercial prophylactic hepatitis B vaccine resulted in functional cure of some HBV carrier mice. (a) Male C57BL / 6J mice (4-6 weeks old) were intravenously injected with 1 × 10 GC AAV-HBV1.3 / mouse. Five weeks later, mice with a serum HBsAg content consistently higher than 1000 IU / mL were designated as HBV carrier mice and used in subsequent experiments. These mice were designated as day 0. On days 1 and 4, HBV carrier mice (four groups total) were treated intravenously with PBS or hetero-scFv fusion protein (0.1687 nmol / mouse). Two groups (heter scFv+HBsAg / CpG and HBsAg / CpG groups) were immunized four times with the commercial prophylactic hepatitis B vaccine rHBsAg (adw, 2 μg) on ​​days 4, 11, 18, and 25. Blood samples were collected at designated time points. (b, c) HBsAg (b) and anti-HBsAg (c) levels in serum at each time point were detected by ELISA. (d, e) HBsAg (d) and anti-HBsAg (e) levels in serum at the end of the experiment (day 174) were detected by ELISA. (f) HBV-DNA levels in serum on days 0 and 174 were detected by real-time PCR. Two mice in the hetero scFv + rHBsAg group in (d–f) achieved functional cure (elimination of HBsAg and HBV-DNA in serum accompanied by production of anti-HBsAg). Data represent the mean ± SEM and are representative of at least two independent experiments.

[0059] [Figure 14]Combining hetero-Fab fusion protein with HBsAg / CpG vaccination achieved functional cure in most HBV carrier mice. (a) Male C57BL / 6J mice (4-6 weeks old) were intravenously injected with 1 × 10 GC AAV-HBV1.3 / mouse. Five weeks later, mice with a serum HBsAg concentration stably higher than 1000 IU / mL were designated as HBV carrier mice and used in subsequent experiments. These mice were designated as day 0. On days 1 and 4, HBV carrier mice (four groups total) were treated intravenously with PBS or hetero-Fab fusion protein (0.1687 nmol / mouse). Two groups (hetero Fab + HBsAg / CpG and HBsAg / CpG groups) were immunized four times with HBsAg (2 μg each) and CpG adjuvant on days 4, 11, 18, and 25. Blood samples were collected at designated time points. (b, c) HBsAg (b) and anti-HBsAg (c) levels in serum were measured by ELISA. (d, e) HBsAg (d) and anti-HBsAg (e) levels in serum on day 157 were measured by ELISA. (f) HBV-DNA levels in serum on days 0 and 157 were measured by real-time PCR. Three mice in the hetero Fab + HBsAg / CpG group (d–f) achieved functional cure (elimination of HBsAg and HBV-DNA in serum accompanied by production of anti-HBsAg). (g, h) Serum from three functionally cured mice in the combination treatment group (hetero Fab + HBsAg / CpG) and from other groups was intravenously injected in 100 μL volumes into untreated HBV carrier mice. HBsAg levels (g) and HBV-DNA levels (h) were detected in the serum of the HBV carrier mice before and 24 hours after injection by ELISA and real-time PCR, respectively. (i, j) HepG2-NTCP cells and 1 × 107 vg HBV were incubated in serum from HBV carrier mice treated with PBS, HBsAg / CpG, hetero Fab, or hetero Fab + HBsAg / CpG (n = 5 / group).After 24 hours of culture, HepG2-NTCP cells were washed three times with culture medium and then cultured in maintenance medium. The supernatant was collected and replaced with fresh maintenance medium every two days. HBsAg levels (i) and HBeAg levels (j) in the supernatant were detected by ELISA. Data are mean ± SEM and represent the results of at least two independent experiments. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

[0060] [Figure 15] Subcutaneous immunization of chronically HBV-infected mice with hetero Fab fusion protein mixed with a commercial prophylactic hepatitis B vaccine demonstrated therapeutic vaccine activity. (A) HBV carrier mice were subcutaneously immunized three times, one week apart, with hetero Fab fusion protein (three gradient concentrations: 0.02 μg, 0.2 μg, and 2 μg) mixed with a commercial prophylactic hepatitis B vaccine (2 μg). Anti-HBsAg and HBsAg levels were measured in mouse serum. (B) Anti-HBsAg antibody levels in mouse serum. (C) HBsAg antigen levels in mouse serum. Data represent the mean ± SEM and are representative of at least two independent experiments. *P<0.05; **P<0.01.

[0061] [Figure 16]Anti-PD-L1-IFNα was used as a targeted adjuvant to help overcome immune tolerance to rHBsAg vaccine in HBV carrier mice. (a) Equimolar amounts (0.01687 nmol, calculated as a single subunit) of the fusion protein were administered subcutaneously to HBV carrier mice either alone or mixed with 1 μg of rHBsAg vaccine. (b, d) ELISA was used to detect serum HBsAg (b) and anti-HBsAg (d) levels after subcutaneous administration of each fusion protein alone. (c, e) ELISA was used to detect serum HBsAg (c) and anti-HBsAg (e) levels after subcutaneous administration of each fusion protein mixed with rHBsAg vaccine. (f) Serum ALT and AST levels were measured using a kit. Data represent the mean + SEM.

[0062] [Figure 17] Anti-PD-L1-IFNα as a targeted adjuvant effectively promoted rHBsAg vaccine immunity and induced HBsAg seroconversion. (a) HBV carrier mice were treated as in Figure 16(a), and serum HBsAg and anti-HBsAg levels were measured by ELISA at each time point. (b-d) At the end of the experiment, draining lymph nodes and spleens were collected, and single-cell suspensions were prepared. HBsAg-specific B cells were detected in the draining lymph nodes (b) and spleen (c) by B cell ELISPOT, and HBsAg-specific T cells in the spleen (d) by T cell ELISPOT. Data are shown as mean + SEM (a) or mean ± SEM (b-d).

[0063] [Figure 18]Anti-PD-L1-IFNα as an immunoadjuvant effectively enhanced immunity against novel coronavirus receptor-binding domain (RBD) antigen, influenza virus HA1 antigen, and OVA antigen. Wild-type mice were subcutaneously immunized with a mixture of anti-PD-L1-IFNα (2μg / mouse) and RBD antigen (3.4μg / mouse), or RBD antigen alone (3.4μg / mouse). Ten days after the primary immunization with anti-PD-L1-IFN fusion protein, sera were collected from the immunized mice, and anti-RBD antibody levels in the serum were detected by ELISA (Figure 18a). Wild-type mice were subcutaneously immunized with a mixture of anti-PD-L1-IFNα (2μg / mouse) and HA1 antigen (2μg / mouse), or HA1 antigen alone (2μg / mouse). Ten days after the primary immunization, sera were collected from the immunized mice, and anti-HA1 antibody levels in the serum were detected by ELISA (Figure 18b). Wild-type mice were subcutaneously immunized with a mixture of anti-PD-L1-IFNα (2 μg / mouse) and OVA antigen (2 μg / mouse), or with OVA antigen alone (2 μg / mouse). Ten days after the first immunization, serum was collected from the immunized mice, and anti-OVA antibody levels in the serum were detected by ELISA (Figure 18c). Data represent the mean ± SEM.

[0064] [Figure 19]Combining anti-PD-L1-IFNα (human) fusion protein with rHBsAg vaccination reduced serum HBsAg levels in HBV carrier mice. Mouse IFNα4 in the anti-PD-L1-IFNα fusion protein was replaced with human-derived IFNα2 (Q124R), which can bind to mouse IFNAR. This human interferon fusion protein was designated anti-PD-L1-IFNα (human). HBV carrier mice (two groups, four groups total) were intravenously treated with PBS or anti-PD-L1-IFNα (human) fusion protein (20μg / mouse) on days 1 and 4. Two groups of mice were selected from those injected with PBS or anti-PD-L1-IFNα (human) fusion protein, and were subcutaneously immunized four times with rHBsAg vaccine (1 μg / mouse) and aluminum adjuvant on days 4, 11, 18, and 25 (i.e., the anti-PD-L1-IFNα (human) + rHBsAg and rHBsAg vaccine alone + PBS groups). Blood samples were taken at designated time points, and serum was collected. HBsAg levels in the serum at each time point were detected by ELISA (Figure 19). Data represent the mean + SEM.

[0065] [Figure 20] Subcutaneous administration of human anti-PD-L1-IFNα as a targeted immune adjuvant effectively promoted rHBsAg vaccine immunity and reduced serum HBsAg levels. HBV carrier mice were subcutaneously immunized with a mixture of human anti-PD-L1-IFNα (2 μg / mouse) and rHBsAg (1 μg / mouse). The specific treatment regimen was the same as in Figure 16(a). Serum HBsAg levels were measured at each time point by ELISA (Figure 20). Data represent the mean + SEM. DETAILED DESCRIPTION OF THE INVENTION

[0066] The present invention will be described in more detail by the following examples, but it should be understood that the present invention is not limited to the following.

[0067] Experimental materials and methods Construction of mice and HBV carrier mouse model Male (4-6 weeks old) C57BL / 6J mice were purchased from SPF (Beijing) Biotechnology Co., Ltd. (Beijing, China). All experimental mice were housed in an SPF (specific pathogen-free) level ABSL-2 (secondary biosafety laboratory) infection animal facility. All experiments were performed in accordance with the regulations of the Institute of Biophysics, Chinese Academy of Sciences, Animal Experiment Lot Number: ABSL-2-201902. Animal care and experiments were performed in accordance with the guidelines of the Institute of Biophysics, Chinese Academy of Sciences, and in accordance with protocols approved by the Institutional Laboratory Animal Care and Use Committee (IACUC).

[0068] The AAV-HBV1.3 virus used to construct the HBV carrier experimental animal model was purchased from Guangzhou Paijin Biotechnology Co., Ltd. (Guangzhou, China). The virus was the ayw serotype and the D genotype. Mice were infected via tail vein injection at a dose of 1 × 10 11 The dose was 200 μL / mouse (diluted in saline). After 5 weeks, stably infected mice (serum HBsAg ≥ 1000 IU / mL, i.e., HBV carrier mice) were used for the experiment. Blood was collected from the ophthalmic vein during the experiment, and serum HBsAg, HBeAg, anti-HBsAg, and HBV-DNA levels were measured.

[0069] Cell lines and key reagents The L929 cell line, a mouse fibroblast cell line (purchased from ATCC, USA), was used to detect IFN bioactivity in vitro. HepG2-NTCP (purchased from Qingqi (Shanghai) Biotechnology Development Co., Ltd.) was used for in vitro HBV infection and inhibition experiments. HBsAg and HBeAg in mouse serum were measured using an ELISA detection kit (Shanghai Kehua Bio-Engineering Co., Ltd.; Shanghai, China). HBsAg protein 111-140 amino acid polypeptide (ayw serotype, amino acid sequence PGSSTTSTGPCRTCMTTAQGTSMYPSCCCT) was synthesized by Shanghai GL Biochemical Co., Ltd. (Shanghai, China). This polypeptide was used to coat 96-well plates and measure anti-HBsAg levels in mouse serum. HBV-DNA in mouse serum was quantitatively measured using real-time PCR, using a reagent kit from Shengxiang Bioscience and Technology Co., Ltd. (Changsha, China). Mouse liver DNA was extracted using a gDNA kit (Tiangen Biotech, China). HBV-DNA, 3.5 kb RNA, and total RNA contents in mouse livers were measured by real-time PCR using a SYBR Premix Ex Taq kit purchased from TaKaRa (Japan). Prophylactic hepatitis B vaccine was purchased from Dalian Hanxin Biopharmaceutical Co., Ltd. (Dalian, China). HBsAg(ayw) protein (purchased from Meridian Life Science, USA) was used in mouse vaccination and enzyme-linked immunospot (ELISPOT) experiments; the ELISPOT kit was purchased from BD, USA. Aluminum adjuvant (Alu-Gel-S) was purchased from Serva Electrophoresis GmbH (Germany), and CpG-1826 adjuvant was synthesized by Shanghai Jierui Bioengineering Co., Ltd. (Shanghai, China). ELISA quantitatively detected human IgG Fc and used goat anti-human IgG and goat anti-human IgG-HRP (Beijing Kangwei Century Biotechnology Co., Ltd.; Beijing, China).

[0070] Construction and activity analysis of anti-PD-L1-IFN and control fusion proteins a. Fusion protein construction Hetero-Fab heterodimer: The mouse IFNα4 cDNA sequence was cloned and inserted into the N-terminus of human IgG1 Fc via a (G4S)4 linker to obtain the mIFNα4-Fc nucleotide sequence (SEQ ID NO: 2). The mIFNα4-Fc nucleotide sequence (SEQ ID NO: 2) was cloned into the pEE6.4 vector (Lonza) and expressed as the first polypeptide of the heterodimer, mIFNα4-Fc (SEQ ID NO: 1). The anti-PD-L1 heavy chain Fab segment nucleotide sequence was cloned and inserted into the N-terminus of human IgG1 Fc to obtain the anti-PD-L1 heavy chain Fab-Fc nucleotide sequence (SEQ ID NO: 6). The anti-PD-L1 heavy chain Fab-Fc nucleotide sequence (SEQ ID NO: 6) was cloned into the pEE12.4 vector (Lonza) and expressed as the second polypeptide of the heterodimer, anti-PD-L1 heavy chain Fab-Fc (SEQ ID NO: 5). The nucleotide sequence encoded by the anti-PD-L1 light chain (SEQ ID NO: 4) was cloned into the pEE12.4 vector (Lonza) and expressed as the third polypeptide of the heterodimer, the anti-PD-L1 light chain (SEQ ID NO: 3). The heterodimerization of IFNα and the PD-L1-binding polypeptide was generated using a previously reported knobs-to-holes technique. The plasmids were transiently transfected into 293F cells at a ratio of 2:1:2. The supernatant was collected 7 days after transfection. The fusion protein was purified using a protein A-agarose gel column according to the manufacturer's instructions (Repligen).

[0071] Hetero-scFv heterodimer: The cDNA sequence of mouse IFNα4 was cloned and inserted into the N-terminus of human IgG1 Fc using a (G4S)4 linker to obtain the mIFNα4-Fc base sequence (SEQ ID NO: 2). The mIFNα4-Fc base sequence (SEQ ID NO: 2) was then cloned into the pEE6.4 vector (Lonza) and expressed to obtain the first polypeptide of the heterodimer, mIFNα4-Fc (SEQ ID NO: 1). Based on the patent (Patent No.: US8217149B2), the light and heavy chain variable regions of the PD-L1 binding polypeptide (scFv) (YW243.55.S70) sequence were synthesized. The light and heavy chain variable region sequences were linked using a GGGGSGGGGSGGGGS linker, and human IgG1 Fc was inserted at the C-terminus of the heavy chain to obtain the scFv(PD-L1)-Fc sequence (SEQ ID NO: 10). The scFv(PD-L1)-Fc sequence (SEQ ID NO: 10) was then cloned into the pEE12.4 vector (Lonza) and expressed as the second polypeptide of the heterodimer, scFv(PD-L1)-Fc (SEQ ID NO: 9). The heterodimer of IFNα and the PD-L1 binding polypeptide was generated using the previously reported knobs-to-holes technique. The plasmid was transiently transfected into 293F cells at a 2:1 ratio. The supernatant was collected 7 days after transfection, and the fusion protein was purified using a protein A-agarose gel column according to the manufacturer's instructions (Repligen).

[0072] Homo Fab homodimer: The C-terminus of the mouse IFNα4 cDNA sequence was ligated to the N-terminus of the anti-PD-L1 heavy chain Fab segment and inserted into the N-terminus of human IgG1 Fc (WT) to obtain the homo Fab homodimer sequence (SEQ ID NO: 8). This sequence (SEQ ID NO: 8) was cloned into the pEE12.4 vector (Lonza) to express the first and second polypeptides (SEQ ID NO: 7) of the homodimeric fusion protein. The sequence encoded by the anti-PD-L1 light chain (SEQ ID NO: 4) was cloned into the pEE12.4 vector (Lonza) to express the third and fourth polypeptides (SEQ ID NO: 3) of the homodimeric fusion protein. After transfection of the plasmids at a 1:1 ratio, spontaneous homodimeric protein formation occurred due to Fc (WT) dimerization. Supernatant was collected on day 7 post-transfection. The fusion protein was purified using a protein A-agarose gel column according to the operating manual (Repligen).

[0073] Homo scFv homodimer: The C-terminus of the mouse IFNα4 cDNA sequence was linked to the N-terminus of scFv(PD-L1)-Fc(WT) to obtain the homo scFv homodimer sequence (SEQ ID NO: 12). This sequence (SEQ ID NO: 12) was cloned into the pEE12.4 vector (Lonza) to express the first and second polypeptides (SEQ ID NO: 11) of the homodimeric fusion protein. After transfection, the homodimeric protein spontaneously formed through dimerization of Fc(WT). The supernatant was collected 7 days after transfection. The fusion protein was purified using a protein A-agarose gel column according to the manufacturer's instructions (Repligen).

[0074] In the present invention, the Fc region in the above dimers may be replaced with an Fc variant with no ADCC form of the Fc region, or mouse IFN α4 may be replaced with human IFN α2, and one skilled in the art would expect that any of these variations would not substantially affect the effects of the present invention. For example, the inventors replaced the Fc region in the Heter Fab heterodimer with an Fc variant with no ADCC form of the Fc region, and / or replaced mouse IFN α4 with human IFN α2, and named the resulting fusion proteins Anti-PD-L1-IFNα(no ADCC) and anti-PD-L1-IFNα(human), respectively.

[0075] IFNα-Fc: The cDNA sequence of mouse IFNα4 was cloned and inserted into the N-terminus of human IgG1 Fc (WT) via a (G4S)4 linker to obtain the IFNα-Fc nucleotide sequence (SEQ ID NO: 14). The nucleotide sequence encoding IFNα-Fc (SEQ ID NO: 14) was cloned into the pEE6.4 vector (Lonza), and the first and second polypeptides, IFNα-Fc (SEQ ID NO: 13), were expressed. After transfection, dimerization of Fc (WT) spontaneously formed a dimeric protein. The supernatant was collected 7 days after transfection. The fusion protein was purified using a protein A-agarose gel column according to the manufacturer's instructions (Repligen).

[0076] Anti-PD-L1 (Fab) whole antibody: The nucleotide sequence of the anti-PD-L1 heavy chain Fab segment was cloned and inserted into the N-terminus of human IgG1 Fc (WT) to obtain the anti-PD-L1 heavy chain Fab-Fc nucleotide sequence (SEQ ID NO: 16). The anti-PD-L1 heavy chain Fab-Fc nucleotide sequence (SEQ ID NO: 16) was cloned into the pEE12.4 vector (Lonza) to express the anti-PD-L1 heavy chain Fab-Fc polypeptide (SEQ ID NO: 15). The nucleotide sequence encoded by the anti-PD-L1 light chain (SEQ ID NO: 4) was cloned into the pEE12.4 vector (Lonza) to express the anti-PD-L1 light chain polypeptide (SEQ ID NO: 3). After transfection of the plasmids at a 2:3 ratio, spontaneous dimeric protein formation occurred due to Fc (WT) dimerization. Supernatants were collected on day 7 post-transfection. The fusion protein was purified using a protein A-agarose gel column according to the operating manual (Repligen).

[0077] Anti-PD-L1 (scFv) antibody: Based on the patent (patent number: US8217149B2), the light and heavy chain variable regions of the PD-L1 binding polypeptide (scFv) (YW243.55.S70) sequence were synthesized. The light and heavy chain variable region sequences were linked with a GGGGSGGGGSGGGGS linker, and human IgG1 Fc (WT) was inserted at the C-terminus of the heavy chain to obtain the anti-PD-L1 (scFv) sequence (SEQ ID NO: 18). The anti-PD-L1 (scFv) sequence (SEQ ID NO: 18) was then cloned into the pEE12.4 vector (Lonza) to express the first and second polypeptides, anti-PD-L1 (scFv) (SEQ ID NO: 17). After transfection, the dimeric protein spontaneously formed due to Fc (WT) dimerization. The supernatant was collected on day 7 post-transfection. The fusion protein was purified using a protein A-agarose gel column according to the operating manual (Repligen).

[0078] b. In vitro functional experiments: SDS-PAGE was used to detect the purity of the fusion protein; L929 cells (mouse fibroblasts) (purchased from ATCC, USA) were incubated with the fusion protein and infected with vesicular stomatitis virus-green fluorescent protein (VSV-GFP), a virus commonly used in the art (purchased from Yunzhou Biosciences). The antiviral biological activity of IFN was detected; Biolayer Interferometry (BLI) was used to analyze the affinity of IFN / IFN receptor, anti-PD-L1 / PD-L1; and flow cytometry (FACS) was used to analyze the affinity of anti-PD-L1-IFN and IFN-R. + , PD-L1 + The ability to bind to cells was analyzed.

[0079] IFN bioactivity was measured using L929 cells, which are susceptible to VSV-GFP infection. Cells were cultured overnight at 37°C with serially diluted IFNα-Fc or four anti-PD-L1-IFN fusion proteins. The next day, cells were infected with VSV-GFP at an MOI of 5 and cultured for an additional 16 hours. Cells were then harvested and fixed with 4% PFA. Data were acquired using a FACS LSRFortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (TreeStar). GFP + Cells were defined as virus-infected cells, and MFI characterized the virus copy intensity.

[0080] The binding affinity of the anti-PD-L1-IFN fusion protein to PD-L1 protein was measured using Biolayer Interferometry (BLI) on an Octet Red 96 system (Pall ForteBio). Binding experiments were performed in binding buffer (PBS, 0.05% Tween 20, pH 7.4) at 37°C. SA sensors were first loaded with biotinylated PD-L1 protein (10 μg / mL) in a 96-well plate. The SA sensors were then transferred to the binding wells to generate a baseline, then to the sample protein wells (concentrations ranging from 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, and 6.25 nM) for 120 seconds, and finally to the binding buffer well for 180 seconds for dissociation. The affinity constant, K, was calculated using a kinetic analysis model. D =k d / k a (d: dissociation; a: binding).

[0081] c. In vivo pharmacokinetic analysis: In vivo tissue distribution and blood drug concentration measurement of Anti-PD-L1-IFN fusion protein.

[0082] Tissue distribution assay: HBV carrier mice were intravenously injected with IFNα-Fc or four types of anti-PD-L1-IFN fusion proteins (0.1687 nmol / mouse). After 3 or 72 hours, each mouse tissue was perfused and collected, and the supernatants were homogenized by centrifugation. The concentrations of the fusion proteins in the homogenized supernatants of each tissue were measured by ELISA.

[0083] Measurement of blood drug concentrations: HBV carrier mice were intravenously injected with IFNα-Fc or four types of anti-PD-L1-IFN fusion proteins (0.1687 nmol / mouse). Blood samples were taken at 0.5, 6, 24, 72, 96, and 120 hours. Serum was collected by centrifugation and the concentration of fusion proteins in the serum at each time point was measured by ELISA.

[0084] d. In vivo toxicity analysis The experimental mice were administered within a gradient weighing range, and their weights were recorded and the changes in their weights were calculated.

[0085] Effects of Anti-PD-L1-IFN fusion protein on DC cells A mouse model of chronic HBV infection (HBV carrier mice) was established and treated intravenously with anti-PD-L1-IFN. Flow cytometry (FACS) was used to detect the levels of costimulatory molecules (CD80, CD86) and MHC1 molecules on the surface of mouse spleen and liver DC cells, and to assess the phagocytic ability of DC cells against HBsAg antigen.

[0086] The effects of Anti-PD-L1-IFN on adaptive immune cells The levels of IFN-γ secreted by T cells in the spleen and liver of HBV carrier mice after anti-PD-L1-IFN treatment were detected by cytokine intracellular staining.

[0087] Virological indicator analysis after treatment of HBV carrier mice a. Detecting serum HBsAg, anti-HBsAg, HBeAg and HBV-DNA levels; b. HBsAg levels in the liver were detected.

[0088] Combination therapy Anti-PD-L1-IFN fusion protein was combined with HBsAg vaccine to treat HBV carrier mice, and the combination therapy was explored according to the following two treatment programs. Serum HBsAg antigen, anti-HBsAg antibody, and HBV-DNA levels were analyzed.

[0089] a. First, intravenous administration of anti-PD-L1-IFN fusion protein and then subcutaneous immunization in combination with a commercial HBsAg vaccine; b. Anti-PD-L1-IFN fusion protein was mixed with a commercial HBsAg vaccine and administered subcutaneously.

[0090] In vivo neutralization and in vitro inhibition experiments of HBV antisera In vivo HBV neutralization experiments: Serum was collected from HBV carrier mice treated with PBS, HBsAg / CpG, hetero Fab, or hetero Fab + HBsAg / CpG, and then intravenously injected into untreated HBV carrier mice (100 μL per mouse). Serum was collected from these mice 24 hours later, and HBsAg and HBV-DNA levels in the serum before and after treatment were detected by ELISA and real-time PCR, respectively.

[0091] In vitro HBV infection inhibition experiment: 1 x 10 7 Inoculate vg HBV into 48-well plate HepG2-NTCP cells (8 × 10 per well) 4 Cells were inoculated into HBV-positive cells (cells) and simultaneously cultured for 24 hours with serum from HBV-carrier mice treated with PBS, HBsAg / CpG, hetero Fab, or hetero Fab + HBsAg / CpG. The cells were washed three times with medium and then cultured in maintenance medium. The maintenance medium was Williams's E medium (Gibco) supplemented with 5 μg / mL transferrin, 5 ng / mL sodium selenate, 3 μg / mL insulin (insulin-transferrin-sodium selenate; Corning), 2 mM L-glutamine, 10 ng / mL epidermal growth factor (Sigma-Aldrich), 2% dimethyl sulfoxide, 100 U / mL penicillin, and 100 μg / mL streptomycin. Supernatants were collected every two days, replaced with fresh maintenance medium, and HBsAg and HBeAg levels in the supernatants collected at different time points were detected by ELISA.

[0092] HBsAg antigen-specific T cells and B cells were detected by ELISPOT. After the animal experiments, mouse lymph nodes, spleens, and livers were collected and single-cell suspensions were prepared. IFN-γ production was measured using an IFN-γ ELISPOT kit for T cell ELISPOT assays according to the manufacturer's protocol (BD Biosciences). B cell ELISPOT protocol: Culture plates were coated with PBS-diluted HBsAg (ayw type, final concentration 5 μg / mL), 100 μL per well, and left overnight at 4°C. The liquid was discarded, and 200 μL of medium was added per well for washing. The plate was then sealed at 37°C for 2 hours. The single-cell suspension was then placed in a 96-well culture plate and incubated for 5–6 hours in a 37°C incubator. After washing five times with wash buffer, 100 μL of detection antibody (biotin-anti-mouse IgG) was added per well and incubated at room temperature for 2 hours. After washing five times with wash buffer, 100 μL of HRP-conjugated streptavidin was added per well and incubated at room temperature for 1 hour. After washing five times with wash buffer, 100 μL of substrate was added per well and color development was allowed to continue for 5–20 minutes. The plate was then rinsed with clean water to terminate color development. The formed spots were counted using a fluorescent enzyme-linked spot analyzer (Cellular Technology Ltd., USA).

[0093] statistical analysis Data are presented as mean ± SEM. Statistical analysis was performed using an unpaired Student's two-tailed t-test. Analysis was performed using GraphPad Prism version 6.0 (GraphPad Software). * , ** , *** and **** The statistical significance of P<0.05, P<0.01, P<0.001 and P<0.0001 was indicated, respectively.

[0094] Example 1: Anti-PD-L1-IFN fusion proteins were constructed, all of which retained IFN bioactivity and PD-L1 affinity Systemic administration of type I IFNs results in low response rates and serious side effects. Targeting cytokine-bearing antibodies has proven to be an effective strategy for local delivery of immunomodulatory molecules. Given the high expression of PD-L1 in the livers of CHB patients, anti-PD-L1 was used to target IFNs to the liver, allowing them to exert their antiviral and immunomodulatory effects locally, improving the response rate and reducing side effects.

[0095] To achieve this goal, the present invention proposes constructing a fusion protein, anti-PD-L1-IFN, using anti-PD-L1 and IFNα. Fusion proteins comprising a PD-L1-binding polypeptide Fab fragment [Fab(PD-L1)] and IFNα were constructed in the form of heterodimers (heterodimers) or homodimers (homodimers), designated heteroFab or homoFab. Fusion proteins comprising a PD-L1-binding polypeptide single-chain variable region fragment [scFv(PD-L1)] and IFNα were also constructed in the form of heterodimers or homodimers, designated heteroscFv or homoscFv (Figure 1a) and their SDS-PAGE gel patterns (Figure 1b). IFNα-Fc, anti-PD-L1(Fab) and anti-PD-L1(scFv) control fusion proteins (Figure 1c) and their SDS-PAGE gel patterns (Figure 1d) were also constructed. At the same time, the inventors used a heterodimer of hetero-Fab as an example, replacing the Fc region with a non-ADCC form of the Fc region to construct anti-PD-L1-IFN α (no ADCC), and presented an SDS-PAGE gel image (Figure 1e). The in vitro antiviral activity of the four anti-PD-L1-IFN fusion proteins was evaluated. Experiments showed that all four anti-PD-L1-IFN fusion proteins effectively inhibited vesicular stomatitis virus-green fluorescent protein (VSV-GFP) infection in L929 cells (Figures 2a and 2c), while also effectively inhibiting VSV-GFP replication (Figure 2b). The IFNs in the four anti-PD-L1-IFN fusion proteins demonstrated good biological activity.Next, biolayer interferometry (BLI) was used to assess the affinity of the four anti-PD-L1-IFN fusion proteins and anti-PD-L1 (Fab) to PD-L1. Experiments showed that all four anti-PD-L1-IFN fusion proteins could bind to PD-L1, and the affinity of the hetero Fab or homo Fab fusion proteins to PD-L1 was close to that of the anti-PD-L1 (Fab) to PD-L1, but higher than that of the hetero scFv or homo scFv fusion proteins to PD-L1 (Figures 3a-f).

[0096] Taken together, these data demonstrate that both anti-PD-L1-IFN heterodimeric and homodimeric fusion proteins can effectively inhibit VSV-GFP infection and replication, and that both fusion proteins maintain the biological activity of IFN. At the same time, both fusion proteins maintain PD-L1 binding, with the affinity of the hetero Fab or homo Fab for PD-L1 being higher than that of the hetero scFv or homo scFv for PD-L1.

[0097] Example 2: Heter Fab or hetero scFv fusion proteins were able to deliver IFN to liver tissue and had better stability in blood In addition to their antiviral and immunomodulatory effects, IFNs also induce the expression of the immunosuppressive molecule PD-L1, which binds to PD-L1 to transmit inhibitory signals and reduce immune cell activity, thereby suppressing immune cell responses to HBV. To overcome this counteracting effect and achieve mutual promotion of immune (re)activation in the liver, the fusion protein anti-PD-L1-IFN constructed in this invention can target the liver by taking advantage of the high expression of PD-L1 in liver tissue, and by increasing PD-L1 expression in liver tissue, the anti-PD-L1-IFN fusion protein can further accumulate in the liver.

[0098] To test this hypothesis, we first investigated the in vivo kinetics of the four anti-PD-L1-IFN fusion proteins in HBV-carrier mice. Equimolar amounts (0.1687 nmol / mouse) of the four anti-PD-L1-IFN or IFNα-Fc fusion proteins were intravenously injected into HBV-carrier mice (5 groups, 3 mice each). After 3 hours, samples were collected and the distribution of the fusion proteins in each tissue was examined. Experiments showed that the accumulated amount of hetero Fab or hetero scFv fusion proteins in the liver was significantly higher than that of IFNα-Fc, as well as that of homo Fab or homo scFv (Figure 4a). 72-hour tissue distribution results also showed that the accumulated amount of hetero Fab or hetero scFv fusion proteins in the liver was significantly higher than that of IFNα-Fc, as well as that of homo Fab or homo scFv (Figure 4b). Blood concentration measurements showed that within the first few hours after intravenous injection of equimolar doses (0.1687 nmol / mouse) of the four anti-PD-L1-IFN or IFN α-Fc fusion proteins, the blood concentrations of the hetero Fab or hetero scFv fusion proteins were higher than those of the other forms of fusion proteins (Figure 4c).

[0099] In summary, the cumulative liver levels of hetero Fab or hetero scFv fusion proteins were significantly higher than those of homo Fab, homo scFv, or IFNα-Fc, and this significant difference could be maintained for at least 72 hours, indicating that hetero Fab or hetero scFv fusion proteins have a better liver targeting effect. At the same time, the blood concentrations of hetero Fab or hetero scFv fusion proteins were higher than those of homo Fab, homo scFv, or IFNα-Fc within the first few hours after injection, indicating that hetero Fab or hetero scFv fusion proteins have better stability in the blood.

[0100] Example 3: The in vivo HBV inhibitory effect of hetero Fab or hetero scFv fusion proteins was higher than that of homo Fab or homo scFv Given that the four anti-PD-L1-IFN fusion proteins effectively suppressed VSV-GFP activity in vitro, we further investigated their antiviral activity in HBV carrier mice. We first examined whether the dose-dependent suppression of HBV activity by hetero Fab fusion proteins in vivo was achieved. Five concentrations of hetero Fab fusion proteins were tested: 5 μg, 10 μg, 20 μg, 40 μg, and 80 μg / mouse / injection. The proteins were administered intravenously twice, three days apart (Figure 5a). The experiment demonstrated a dose-dependent suppression of HBV activity in HBV carrier mice (Figures 5b and c). Furthermore, increasing the dose significantly reduced the mice's body weight, indicating greater toxicity with higher doses (Figure 5d). All five doses tested reduced serum HBV-DNA below the detection limit by day 7, demonstrating its relative sensitivity to IFN (Figure 5e). Considering the antiviral activity and in vivo toxicity, the dose of 20 μg / mouse / dose was relatively less toxic (mouse body weight decreased by less than 4%, and recovered after a slight decrease) (Fig. 5d) and relatively highly suppressed HBV activity (serum HBsAg level increased by an average of 2 log on day 7). 10The optimal in vivo dose of the hetero Fab fusion protein was 20 μg / mouse / injection (0.1687 nmol / mouse / injection), which was used in subsequent experiments. The four anti-PD-L1-IFN fusion proteins were then compared for their in vivo HBV activity suppression. The hetero Fab dose was 20 μg / mouse / injection (0.1687 nmol / mouse / injection), and the doses of the other three fusion proteins were calculated based on equimolar amounts of IFNα and anti-PD-L1. Each fusion protein was intravenously injected twice, 3 days apart (Figure 6a). Experimental results showed that the HBV suppression effects of hetero Fab or hetero scFv fusion proteins were better than those of homo Fab or homo scFv fusion proteins (Figures 6b, c). Furthermore, serum HBV-DNA levels after hetero Fab treatment fell below the detection limit on day 7 and recovered slowly (Figure 6d).

[0101] In summary, the hetero Fab or hetero scFv fusion proteins suppressed HBV activity in vivo more effectively than the homo Fab or homo scFv, which was consistent with the significantly higher liver accumulation of the hetero Fab or hetero scFv fusion proteins compared with the homo Fab or homo scFv.

[0102] Example 4: Heter Fab fusion proteins inhibit HBV in vivo with synergistic and positive feedback effects HBV carrier mice were intravenously injected with the fusion protein on days 1 and 4 (Figure 7a). Experimental results showed that the in vivo HBV-suppressing effect of the hetero Fab fusion protein was superior to that of IFN α-Fc or anti-PD-L1 (Fab) alone, or the physical mixture of IFN α-Fc and anti-PD-L1 (Fab), and that the hetero Fab fusion protein significantly reduced HBsAg and HBeAg levels in the serum of HBV carrier mice on day 7 (Figure 7b, c). Furthermore, serum HBV-DNA levels in the IFN α-Fc group, the physical mixture of IFN α-Fc and anti-PD-L1 (Fab) group, and the hetero Fab group were significantly reduced on day 7, but the recovery of serum HBV-DNA levels in the hetero Fab group was slower, indicating the long-lasting efficacy of the hetero Fab fusion protein (Figure 7d).

[0103] Since type I interferon can induce PD-L1 expression, we detected the expression of PD-L1 in the liver after hetero Fab treatment. As a result, hetero Fab inhibited the expression of CD45 in the liver. - and CD45 + It was shown that PD-L1 expression on the cells could be significantly increased, and increased PD-L1 expression could further promote hetero Fab accumulation in the liver (Fig. 7e, f).

[0104] In summary, the mixture of IFN α-Fc and anti-PD-L1 did not produce the synergistic effect of the hetero Fab fusion protein to inhibit HBV in vivo, and liver targeting was essential for the HBV-inhibitory effect of the fusion protein. At the same time, the hetero Fab had a positive feedback effect, further enhancing the accumulation of the hetero Fab in the liver and resulting in better HBV inhibition.

[0105] Example 5: Heter Fab fusion protein could improve DC cell function and further promote HBsAg-specific T cell function to break immune tolerance Type I interferon plays an important role in promoting DC cross-presentation and activating T cells. Therefore, we investigated whether hetero Fab treatment could improve DC cell function. HBV carrier mice were treated with a single dose of hetero Fab (0.1687 nmol / mouse), and three days later, DC cells in the liver and spleen were analyzed by flow cytometry (Fig. 8a). Experimental results showed that hetero Fab treatment significantly increased the expression of CD86, CD80, and MHCI on DC cells in the liver and spleen compared with other fusion protein treatments (Fig. 8b, c and Fig. 9a-d). The increase in these molecules favored DC cells for T cell activation. Indeed, hetero Fab could activate T cell function in HBV carrier mice. Flow cytometry and ELISPOT experiments demonstrated the expression of HBsAg-specific IFN-γ in the liver and spleen. + CD4 + and CD8 + The results showed that the number of T cells significantly increased after treatment with hetero Fab fusion protein (Fig. 8d, e and Fig. 10a-c). To verify whether the specific T cells activated after treatment with hetero Fab fusion protein could exert an HBV-suppressing effect, T cells were deleted in HBV carrier mice before and during hetero Fab administration. As a result, the antiviral effect of the hetero Fab fusion protein was significantly reduced (Fig. 8f), and Rag1 - / - Similar results were obtained using HBV carrier mice (Fig. 8g). These data indicated that T cells activated by hetero Fab fusion proteins can suppress HBV. We also investigated whether hetero Fab fusion proteins enhance the antigen uptake capacity of DC cells. We found that hetero Fab fusion proteins enhanced DC2.4 cell uptake of HBsAg-FITC antigen compared with hIgG or IFN α-Fc treatment (Fig. 8h). In summary, these data demonstrated that hetero Fab fusion proteins promote DC maturation and antigen presentation, thereby enhancing HBV-specific T cell function.

[0106] Example 6: Combining hetero scFv fusion proteins with HBsAg / aluminum adjuvant immunization resulted in functional cure of some HBV carrier mice Heter Fab or hetero scFv fusion proteins suppressed HBV in vivo, significantly reducing serum HBsAg, HBeAg, and HBV-DNA levels, but this was only maintained for a certain period (approximately 14 days) before returning to baseline levels. Treatment with hetero Fab or hetero scFv fusion proteins alone did not result in functional treatment of HBV carrier mice. High virus and antigen loading confer immune tolerance to HBV. We used hetero Fab or hetero scFv fusion proteins to rapidly remove HBsAg and HBV-DNA from serum, enhance DC and specific T cell function, and disrupt HBV immune tolerance, creating a "blank period." This, combined with active vaccination, stimulates host cellular and humoral immunity, potentially achieving a functional cure for CHB. We compared HBsAg(ayw) antigen immunization alone, the combination of an anti-PD-L1 antibody and HBsAg(ayw) antigen, the combination of an IFNα-Fc fusion protein and HBsAg(ayw) antigen, and the combination of a hetero scFv fusion protein and HBsAg(ayw) antigen, all of which were adjuvanted with aluminum. The fusion protein was administered intravenously twice (at 3-day intervals) and subcutaneously with HBsAg four times (at 7-day intervals) (Figure 11a). Experimental results showed that the combination of the hetero scFv fusion protein and HBsAg(ayw) antigen produced the best response (Figures 11b and 11c), with one of three HBV carrier mice achieving functional cure, i.e., serum HBsAg was eliminated and anti-HBsAg was produced (Figures 11d and 11e), and serum HBV-DNA was near the limit of detection (Figure 11f). Other treatment methods failed to achieve functional cure in HBV carrier mice (Fig. 11d-f).

[0107] In summary, treatment combining hetero scFv fusion protein with HBsAg / aluminum adjuvant immunization achieved functional cure in some HBV carrier mice.

[0108] Example 7: Combining hetero scFv fusion proteins with HBsAg / CpG immunization achieved functional cure of most HBV carrier mice The results of Example 6 demonstrated that combining hetero scFv with active vaccine immunization (using aluminum adjuvant) achieved functional cure in some HBV carrier mice. We attempted to improve the therapeutic effect by modifying the treatment method. First, we replaced the aluminum adjuvant used in Example 6 with a CpG adjuvant to promote humoral and cellular immunity in the body and better eliminate HBV. The treatment strategy employed, as shown in Figure 12a, consisted of two intravenous injections (3 days apart) of the fusion protein and four subcutaneous immunizations (7 days apart) of HBsAg(ayw) / CpG. The experimental results showed that combining hetero scFv fusion protein with active immunization of HBsAg(ayw) / CpG achieved functional cure in the majority of HBV carrier mice, with 3 out of 5 HBV carrier mice achieving functional cure, improving the cure rate to 60% (Figures 12b-f). At the end of the experiment (day 104), HBsAg antigen levels in the livers of mice were measured, and it was found that the HBsAg antigen levels in the livers of the combination treatment group (heter scFv + HBsAg / CpG) were significantly lower than those of the other three groups (Fig. 12g). At the same time, HBV-specific T cells and B cells in the spleens of mice were measured at the end of the experiment (day 104). The results showed that the combination treatment group showed a significant increase in both HBsAg-specific T cells and B cells (Fig. 12h, i), indicating that the combination treatment group functionally cured most HBV carrier mice by simultaneously inducing both cellular and humoral immunity.

[0109] According to a review, the therapeutic effect of CpG adjuvant was superior to that of aluminum adjuvant, improving the rate of functional cure. The combination of hetero scFv fusion protein with HBsAg(ayw) / CpG immunization induced specific cellular and humoral immunity, achieving functional cure in the majority of HBV carrier mice and significantly reducing HBsAg levels in the liver. The combined treatment group not only reduced HBV viral load in the serum but also in the liver.

[0110] Example 8: Combining hetero scFv fusion proteins with a commercial hepatitis B prophylactic vaccine allows functional cure of some HBV carrier mice In Example 7, the combination treatment group achieved functional cure in the majority of HBV carrier mice. Whether treatment was performed directly in combination with a commercial hepatitis B vaccine (e.g., EngerixB, serotype adw), or by combining the hetero scFv fusion protein with EngerixB immunization, HBV carrier mice (Figure 13a) were treated. Experiments showed that serum HBsAg levels in mice in the combination treatment group tended to decrease (Figure 13b). Concurrently, anti-HBsAg antibodies were produced and maintained for a relatively long period of time, with serum anti-HBsAg antibodies detectable on day 174 (Figure 13c). The combination treatment group achieved functional cure in two of five HBV carrier mice (40%) (Figures 13d-f).

[0111] In summary, combining hetero scFv fusion proteins with a commercial hepatitis B prophylactic vaccine also enabled functional cure of some HBV carrier mice, making the clinical translation of the fusion protein more convenient.

[0112] Example 9: Combining hetero Fab fusion proteins with HBsAg / CpG immunization achieved functional cure in most HBV carrier mice The treatment strategy employed was the same as in Example 7, in which the fusion protein was intravenously injected twice (at 3-day intervals) and HBsAg(ayw) / CpG was administered four times (at 7-day intervals) (Figure 14a). The experimental results showed that the serum HBsAg levels of HBV carrier mice in the combination treatment group tended to decrease (Figure 14b), and the corresponding serum anti-HBsAg antibody levels in the combination treatment group were the highest and maintained for the longest period (Figure 14c). At the end of the experiment, three mice (a total of five mice) in the combination treatment group had HBsAg negative status (Figure 14d), and accordingly, three mice maintained relatively high serum anti-HBsAg antibody levels (Figure 14e), and serum HBV-DNA levels were close to or below the detection limit (Figure 14f). This means that the combination treatment group achieved functional cure in three HBV carrier mice. Sera from these three mice with relatively high antibody levels were intravenously injected (100 μL per mouse) into untreated HBV carrier mice. Compared with sera from mice in the other groups, sera from the three mice with relatively high antibody levels in the combination treatment group were able to neutralize HBV in vivo (Fig. 14g, h). Simultaneously, experiments to suppress HBV infection in vitro demonstrated that anti-HBsAg antibody-containing sera produced from functionally cured mice in the combination treatment group were also able to suppress HBV-infected cells in vitro (Fig. 14i, j).

[0113] In summary, treatment combining hetero Fab fusion protein with HBsAg(ayw) / CpG immunization was able to functionally cure 60% of HBV carrier mice, and the serum anti-HBsAg antibody levels in the combined treatment group peaked around day 60 and then gradually decreased, but were maintained for a relatively long period of time. The produced anti-HBsAg antibodies were able to neutralize HBV in vivo and protect the body from HBV reinfection.

[0114] Example 10: Heter Fab fusion proteins were mixed with a commercial prophylactic hepatitis B vaccine to form a therapeutic vaccine, which could produce anti-HBsAg antibodies and reduce HBsAg levels in serum. The treatment strategy employed was similar to that described in Example 8. HBV carrier mice were subcutaneously immunized with hetero Fab fusion protein mixed with a commercial prophylactic hepatitis B vaccine, with a total of three immunizations at weekly intervals (Figure 15A). Serum was collected from the mice, and anti-HBsAg and HBsAg levels in the serum were detected. Mixing hetero Fab fusion protein with a commercial prophylactic hepatitis B vaccine induced and produced stronger antibody responses than immunization with a commercial prophylactic hepatitis B vaccine alone (Figure 15B). The HBsAg levels in the mixed immunization group were significantly lower than those in the single vaccine immunization group (Figure 15C), suggesting that mixing hetero Fab fusion protein with a commercial prophylactic hepatitis B vaccine is useful for breaking HBsAg immune tolerance.

[0115] In summary, combined immunization of hetero Fab fusion proteins with a commercial prophylactic hepatitis B vaccine had a good therapeutic effect in HBV carrier mice, inducing and producing anti-HBsAg antibodies and reducing serum HBsAg levels.

[0116] Example 11: Anti-PD-L1-IFNα targeted interferon as an immune adjuvant helped HBV carrier mice overcome immune tolerance to rHBsAg vaccine HBV carrier mice were subcutaneously immunized with equimolar amounts (0.01687 nmol, calculated as a single subunit) of anti-PD-L1, IFNα-Fc, anti-PD-L1+IFNα-Fc (anti-PD-L1 and IFNα-Fc mixture), and anti-PD-L1-IFNα alone or mixed with 1 μg rHBsAg vaccine (Figure 16a). ELISA was used to detect changes in serum HBsAg and anti-HBsAg levels. The experimental results showed that only the combination of anti-PD-L1-IFNα and rHBsAg vaccine produced a relatively high anti-HBsAg antibody response and reduced serum HBsAg levels, while none of the other groups produced an antibody response or reduced serum HBsAg levels (Figures 16b-e). Furthermore, neither ALT nor AST levels increased after subcutaneous immunization with the combination (Figure 16f).

[0117] In summary, anti-PD-L1-IFNα targeted interferon can effectively promote rHBsAg vaccine immunity as an immune adjuvant, and has a favorable safety profile.

[0118] Example 12: Combined immunization with anti-PD-L1-IFNα and rHBsAg vaccine induced relatively high antibody responses HBV carrier mice were subcutaneously immunized with 2 μg or 10 μg of anti-PD-L1-IFNα mixed with 1 μg of rHBsAg vaccine. Seven days after the primary immunization, they received the same dose of co-immunization, followed by booster immunizations with rHBsAg vaccine alone 3 and 5 weeks after the primary immunization. Changes in serum HBsAg and anti-HBsAg levels were measured by ELISA. Experimental results showed that the anti-HBsAg antibody response was significantly enhanced in the anti-PD-L1-IFNα and rHBsAg vaccine co-immunization group, while HBsAg levels were significantly reduced (Figure 17a). HBsAg-specific B and T cell responses were also significantly enhanced in the co-immunization group (Figures 17b-d).

[0119] In summary, anti-PD-L1-IFNα, as an immune adjuvant, could significantly enhance anti-HBsAg antibody response levels and HBsAg-specific T cell responses, and could also eliminate HBsAg antigen in HBV carrier mice.

[0120] Example 13: Anti-PD-L1-IFNα as an immune adjuvant effectively enhances immunity against novel coronavirus receptor binding domain (RBD) antigen, influenza virus HA1 antigen, and OVA antigen Mice were immunized subcutaneously with a mixture of anti-PD-L1-IFNα (2μg / mouse) and RBD antigen (3.4μg / mouse), for a total of two immunizations, 14 days apart. 28 days after the first immunization, sera were collected from the immunized mice, and anti-RBD antibody levels in the serum were detected by ELISA (Figure 18a). Mice were immunized subcutaneously with a mixture of anti-PD-L1-IFNα (2μg / mouse) and HA1 antigen (2μg / mouse). 10 days after the first immunization, sera were collected from the immunized mice, and anti-HA1 antibody levels in the serum were detected by ELISA (Figure 18b). Mice were immunized subcutaneously with a mixture of anti-PD-L1-IFNα (2μg / mouse) and OVA antigen (2μg / mouse). 10 days after the first immunization, sera were collected from the immunized mice, and anti-OVA antibody levels in the serum were detected by ELISA (Figure 18c). The experimental results showed that compared with single antigen immunization, combined subcutaneous immunization significantly increased the corresponding antibody levels in serum.

[0121] In summary, anti-PD-L1-IFNα can be used as an immune adjuvant to promote immunity to non-hepatitis B antigens, such as the novel coronavirus receptor-binding domain (RBD) antigen, influenza virus HA1 antigen, and OVA antigen, and significantly enhance the immune function of the vaccine by significantly improving antibody levels.

[0122] Example 14: Combining anti-PD-L1-IFNα (human) fusion protein with rHBsAg vaccination reduced serum HBsAg levels in HBV carrier mice On days 1 and 4, HBV carrier mice (two groups each, a total of four groups) were treated intravenously with either PBS or anti-PD-L1-IFNα (human) fusion protein (20 μg / mouse). One group of mice (two groups in total) was collected from the PBS- or anti-PD-L1-IFNα (human) fusion protein-injected mice, and these mice were subcutaneously immunized four times with rHBsAg (1 μg / mouse) on days 4, 11, 18, and 25 (i.e., anti-PD-L1-IFNα (human) + rHBsAg and rHBsAg alone + PBS groups). Serum HBsAg levels at each time point were measured by ELISA. The experimental results demonstrated that combining intravenous anti-PD-L1-IFNα (human) injection with subcutaneous rHBsAg immunization produced the best therapeutic effect, achieving a significant reduction in serum HBsAg levels (Figure 19).

[0123] In summary, replacing the interferon in the fusion protein from mouse IFNα4 with human IFNα2(Q124R) also had the effect of promoting rHBsAg vaccine immunity.

[0124] Example 15: Anti-PD-L1-IFNα (human) as a targeted immune adjuvant effectively promoted rHBsAg vaccine immunity HBV carrier mice were subcutaneously immunized with a mixture of anti-PD-L1-IFNα (human) (2μg / mouse) and rHBsAg (1μg / mouse), and serum HBsAg levels were measured by ELISA at various time points. The experimental results showed that subcutaneous immunotherapy with a mixture of anti-PD-L1-IFNα (human) and rHBsAg vaccine reduced serum HBsAg levels (Figure 20).

[0125] In summary, anti-PD-L1-IFNα (human) also acts as an immune adjuvant, and when mixed with rHBsAg and administered subcutaneously, it was able to overcome immune tolerance and promote the effects of vaccine immunity.

[0126] The above description is merely a better embodiment of the present invention, and does not limit the present invention. Any modifications, equivalent replacements, improvements, etc. that may be made within the spirit and principle of the present invention shall fall within the scope of protection of the present invention.

[0127] Sequence Listing SEQ ID NO. 1: Amino acid sequence of mIFNα4-Fc(hole) in heterodimers of heteroFab or heteroscFv (where the underlined bold sequence is the linker sequence) [ka]

[0128] SEQ ID NO.2: Nucleotide sequence of mIFNα4-Fc(hole) in heterodimers of heteroFab or heteroscFv (where the underlined bold sequence is the linker sequence) [ka]

[0129] SEQ ID NO.3: Amino acid sequence of the anti-PD-L1 light chain in the Heter Fab heterodimer, homo Fab homodimer, or anti-PD-L1 (Fab) whole antibody [ka]

[0130] SEQ ID NO.4: Nucleotide sequence of the anti-PD-L1 light chain in the Heter Fab heterodimer, homo Fab homodimer, or anti-PD-L1 (Fab) whole antibody [ka]

[0131] SEQ ID NO.5: Amino acid sequence of the anti-PD-L1 heavy chain Fab knob in the heterodimer [ka]

[0132] SEQ ID NO.6: Nucleotide sequence of the anti-PD-L1 heavy chain Fab knob in the heterodimer [ka]

[0133] SEQ ID NO.7: Amino acid sequence of mIFNα4-PD-L1 heavy chain Fab-Fc (hIgG1-Fc,WT) in Homo Fab homodimer (where the underlined bold sequence is the linker sequence) [ka]

[0134] SEQ ID NO.8: mIFNα4-PD-L1 heavy chain Fab fragment-Fc (hIgG1-Fc,WT) nucleotide sequence in Homo Fab homodimer (where the underlined bold sequence is the linker sequence) [ka]

[0135] SEQ ID NO.9: Amino acid sequence of scFv(PD-L1)-Fc(knob) in Heter scFv heterodimer (where the underlined bold sequence is the linker sequence) [ka]

[0136] SEQ ID NO.10: Nucleotide sequence of scFv(PD-L1)-Fc(knob) in Heter scFv heterodimer (where the underlined bold sequence is the linker sequence) [ka]

[0137] SEQ ID NO.11: Amino acid sequence of mIFNα4-scFv(PD-L1)-Fc(hIgG1-Fc,WT) in Homo scFv homodimer (where the underlined bold sequence is the linker sequence) [ka]

[0138] SEQ ID NO.12: Nucleotide sequence of mIFNα4-scFv(PD-L1)-Fc (hIgG1-Fc, WT) in Homo scFv homodimer (where the underlined bold sequence is the linker sequence) [ka]

[0139] SEQ ID NO. 13: Amino acid sequence of IFNα-Fc (hIgG1-Fc, WT) (where the underlined bold sequence is the linker sequence) [ka]

[0140] SEQ ID NO. 14: Nucleotide sequence of IFNα-Fc (hIgG1-Fc,WT) (where the underlined bold sequence is the linker sequence) [ka]

[0141] SEQ ID NO.15: Amino acid sequence of Anti-PD-L1 (Fab) complete antibody heavy chain (Fc is hIgG1-Fc, WT) [ka]

[0142] SEQ ID NO.16: Nucleotide sequence of Anti-PD-L1 (Fab) complete antibody heavy chain (Fc is hIgG1-Fc, WT) [ka]

[0143] SEQ ID NO.17: Amino acid sequence of Anti-PD-L1(scFv) (Fc is hIgG1-Fc,WT) (wherein the underlined bold sequence is the linker sequence) [ka]

[0144] SEQ ID NO.18: Nucleotide sequence of Anti-PD-L1(scFv) (Fc is hIgG1-Fc,WT) (wherein the underlined bold sequence is the linker sequence) [ka]

[0145] SEQ ID NO.19: Amino acid sequence of mIFNα4-Fc (no ADCC) in the Anti-PD-L1-IFNα heterodimer (wherein the underlined bold sequence is the linker sequence) [ka]

[0146] SEQ ID NO.20: Nucleotide sequence of mIFNα4-Fc (no ADCC) in the Anti-PD-L1-IFNα heterodimer (where the underlined bold sequence is the linker sequence) [ka]

[0147] SEQ ID NO.21: Amino acid sequence of the anti-PD-L1 heavy chain Fab-Fc (no ADCC) in the anti-PD-L1-IFNα heterodimer [ka]

[0148] SEQ ID NO.22: Nucleotide sequence of the anti-PD-L1 heavy chain Fab-Fc (no ADCC) in the anti-PD-L1-IFNα heterodimer [ka]

[0149] SEQ ID NO.23: Amino acid sequence of human IFNα2-Fc(hole) in the Anti-PD-L1-IFNα(human) heterodimer (wherein the underlined bold sequence is the linker sequence) [ka]

[0150] SEQ ID NO.24: Nucleotide sequence of human IFNα2-Fc(hole) in the Anti-PD-L1-IFNα(human) heterodimer (where the underlined bold sequence is the linker sequence) [ka]

[0151] SEQ ID NO.25: Amino acid sequence of human IFNα2-Fc (no ADCC) in the Anti-PD-L1-IFNα (human) heterodimer (wherein the underlined bold sequence is the linker sequence) [ka]

[0152] SEQ ID NO.26: Nucleotide sequence of human IFNα2-Fc (no ADCC) in the Anti-PD-L1-IFNα (human) heterodimer (wherein the underlined bold sequence is the linker sequence) [ka]

Claims

1. A fusion protein comprising an operably linked PD-L1-binding polypeptide and an interferon (IFN).

2. The fusion protein of claim 1 , which is a heterodimeric protein or a homodimeric protein.

3. a heterodimeric protein, and the PD-L1-binding polypeptide is in a Fab form; optionally, comprising a first polypeptide, a second polypeptide, and a third polypeptide, wherein the first polypeptide comprises IFN and an immunoglobulin Fc region, and the IFN is located at the N-terminus or C-terminus of the Fc region; the second polypeptide comprises a PD-L1-binding polypeptide heavy chain Fab region and an immunoglobulin Fc region, and the PD-L1-binding polypeptide heavy chain Fab region is located at the N-terminus or C-terminus of the Fc region; and the third polypeptide is a PD-L1-binding polypeptide light chain; Preferably, the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NO: 23 or SEQ ID NO: 25, the second polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 21, and the third polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3; Or, a heterodimeric protein, and the PD-L1-binding polypeptide is in the form of an scFv; optionally, comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises an IFN and an immunoglobulin Fc region, and the IFN is located at the N-terminus or C-terminus of the Fc region; and the second polypeptide comprises a PD-L1-binding polypeptide scFv and an immunoglobulin Fc region, and the PD-L1-binding polypeptide scFv is located at the N-terminus or C-terminus of the Fc region; Preferably, the first polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 23, and the second polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:

9. The fusion protein of claim 1.

4. a homodimeric protein, and the PD-L1-binding polypeptide is in a Fab form; optionally comprising the same first and second polypeptides, and the same third and fourth polypeptides, wherein the first and second polypeptides comprise IFN, a PD-L1-binding polypeptide heavy chain Fab region, and an immunoglobulin Fc region, and preferably comprise or consist of the amino acid sequence set forth in SEQ ID NO:7; and the third and fourth polypeptides are the light chains of a PD-L1-binding polypeptide, and preferably comprise or consist of the amino acid sequence set forth in SEQ ID NO:3; Or, a homodimeric protein, and the PD-L1-binding polypeptide is in the form of an scFv; Optionally, the antibody comprises two identical first and second polypeptides, wherein the first and second polypeptides comprise an IFN, a PD-L1-binding polypeptide scFv, and an immunoglobulin Fc region, and preferably comprise or consist of the amino acid sequence set forth in SEQ ID NO:

11. The fusion protein of claim 1.

5. The fusion protein of claim 1, wherein the interferon IFN may be selected from type I interferon, type I interferon variants, preferably mouse IFNα4 and human IFNα2, and the IFN may be of human or mouse origin.

6. 5. The fusion protein of claim 3 or 4, wherein the immunoglobulin Fc region may be selected from the constant region amino acid sequence of IgG1, IgG2, IgG3 or IgG4 (preferably IgG1) or a variant thereof.

7. The PD-L1-binding polypeptide may be an anti-PD-L1 antibody or an antigen-binding fragment thereof, such as a single chain antibody (scFv), a Fab fragment, or a F(ab') ２ The fusion protein of claim 1, wherein the anti-PD-L1 antibody is preferably selected from Tecentriq, Bavencio, Imfinzi, KN035, CS1001, KL-A167, SHR-1316, or YW243.55.S70, and more preferably the PD-L1-binding polypeptide is a Fab fragment of a PD-L1 antibody.

8. Use of the fusion protein of any one of claims 1 to 7 in the preparation of a medicament for treating chronic hepatitis B.

9. A pharmaceutical preparation or composition comprising the fusion protein according to any one of claims 1 to 7 as an active ingredient.

10. A polynucleotide encoding the fusion protein of any one of claims 1 to 7.

11. A vector comprising the polynucleotide of claim 10, optionally a plasmid vector or a viral vector.

12. 12. A cell comprising the polynucleotide of claim 10 or the vector of claim 11 and expressing the fusion protein of any one of claims 1 to 7, preferably a non-human mammalian cell, preferably selected from CHO and HEK293 cells.

13. A pharmaceutical composition or kit comprising the fusion protein and vaccine according to any one of claims 1 to 7, wherein the fusion protein and the vaccine are preferably administered sequentially or simultaneously, and the administration methods include intravenous injection, subcutaneous immunization, or intramuscular immunization, respectively, or simultaneous subcutaneous immunization and intramuscular immunization, etc. Optionally, the antigen in the vaccine may be an HBV envelope protein (S-HBsAg, M-HBsAg, L-HBsAg) or a polypeptide thereof, a nucleocapsid protein (HBcAg) or a polypeptide thereof, a polymerase protein or a polypeptide thereof, or an HBxAg protein or a polypeptide thereof, preferably an HBV envelope protein or a polypeptide thereof, or the antigen in the vaccine may be a novel coronavirus receptor binding region antigen or an influenza virus HA1 antigen; Optionally, the vaccine further comprises an adjuvant, the adjuvant comprising an aluminum adjuvant, a liposome, or CpG, or the like.

14. 10. Use of a fusion protein according to any one of claims 1 to 7 or a combination of a fusion protein according to any one of claims 1 to 7 with a vaccine in the preparation of a medicament or kit for the prevention of viral infections (e.g. acute viral infections) or the treatment of chronic or latent viral infections, optionally wherein the viruses include but are not limited to HBV, coronavirus, influenza virus.

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