EA047752B1 - INTERFERON-ASSOCIATED ANTIGEN-BINDING PROTEINS AND THEIR APPLICATION - Google Patents

Abstract

The invention relates to novel interferon-associated antigen-binding proteins, as well as nucleic acids, vectors, and vector systems encoding such interferon-associated antigen-binding proteins. The invention also relates to compositions containing such interferon-associated antigen-binding proteins, nucleic acids, vectors, and vector systems. The novel interferon-associated antigen-binding proteins provide useful improvements over the current state of the art, for example, in that they effectively disrupt viral replication and thereby reduce the HBV viral load. Thus, the invention also relates to the medical use of such interferon-associated antigen-binding proteins, nucleic acids, vectors, vector systems, and compositions, for example, in the treatment of hepatitis B virus (HBV) infection and/or to reduce one or more symptoms of HBV infection in a patient. The invention further relates to host cells containing such nucleic acids, vectors and vector systems, as well as to methods for producing interferon-associated antigen-binding proteins according to the invention using said host cells.

Description

Field of technology

The present invention relates to novel interferon-associated antigen-binding proteins based on the agonist CD40 antibody CP870,893, as well as to nucleic acids, vectors and vector systems encoding such interferon-associated antigen-binding proteins. The present invention also relates to compositions comprising such interferon-associated antigen-binding proteins, nucleic acids, vectors and vector systems. The novel interferon-associated antigen-binding proteins provide useful improvements over the state of the art, for example, in that they effectively disrupt viral replication and thereby reduce the HBV viral load. Thus, the present invention also relates to medical uses of such interferon-associated antigen-binding proteins, nucleic acids, vectors, vector systems and compositions, for example in the treatment of hepatitis B virus (HBV) infection and/or to reduce one or more symptoms of HBV infection in a patient. The present invention further relates to host cells containing such nucleic acids, vectors and vector systems, as well as to methods for producing interferon-associated antigen-binding proteins according to the invention using said host cells.

State of the art

HBV infects more than 300 million people worldwide and is a common cause of liver disease and liver cancer (Liang (2009) Hepatology 49:S13). HBV is a small DNA virus with unusual retrovirus-like properties that replicates via an intermediate RNA (pregenomic RNA, pgRNA) and can integrate into the host genome. Unique features of the HBV replication cycle confer the virus with the ability to persist in infected cells. HBV infection results in a wide spectrum of liver disease, ranging from acute liver disease (including fulminant liver failure) to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Acute HBV infection may be asymptomatic or present with symptomatic acute hepatitis. Ninety to ninety-five percent of children and 5 to 10 percent of adults infected with HBV fail to clear the virus and become chronically infected. Many chronically infected people have mild liver disease with little or no long-term morbidity or mortality. Other people with chronic HBV infection develop active disease that can progress to cirrhosis and liver cancer. These patients require close monitoring and therapeutic intervention.

New therapies for the treatment of HBV infection by modulating HBV infection in the cell are needed. Specifically, methods are needed to effectively disrupt viral replication, reduce the HBV viral load in HBV-infected cells, reduce the transcription of HBV covalently closed circular DNA in HBV-infected cells, and/or reduce the amount of HBV pregenomic RNA in HBV-infected cells.

The essence of the invention

The invention relates to an interferon-associated antigen-binding protein comprising (I) an agonist CD40 antibody or an agonist antigen-binding fragment thereof, and (II) an interferon (IFN) or a functional fragment thereof, wherein the agonist CD40 antibody or agonist antigen-binding fragment thereof comprises (a) three light chain complementarity determining regions (CDRs) that are at least 90% identical to the sequences CDRL1, CDRL2 and CDRL3 of SEQ ID NO: 3; and three heavy chain CDRs that are at least 90% identical to the sequences CDRH1, CDRH2 and CDRH3 of SEQ ID NO: 6; wherein each CDR is defined according to the Kabat definition, the Chothia definition, the AbM definition or the CDR contact definition; preferably wherein each CDR is defined according to the Kabat CDR definition or the Chothia CDR definition;

(b) three light chain complementarity determining regions (CDRs) that are identical to the sequences CDRL1, CDRL2 and CDRL3 of SEQ ID NO: 3; and three heavy chain CDRs that are identical to the sequences CDRH1, CDRH2 and CDRH3 of SEQ ID NO: 6; wherein each CDR is defined according to the Kabat definition, the Chothia definition, the AbM definition or the CDR contact definition; preferably wherein each CDR is defined according to the Kabat CDR definition or the Chothia CDR definition;

(c) a heavy chain or fragment thereof comprising a complementarity determining region (CDR) CDRH1 that is at least 90% identical to SEQ ID NO: 56, CDRH2 that is at least 90% identical to SEQ ID NO: 57, and CDRH3 that is at least 90% identical to SEQ ID NO: 58; and a light chain or fragment thereof comprising CDRL1 that is at least 90% identical to SEQ ID NO: 52, CDRL2 that is at least 90% identical to SEQ ID NO: 53, and CDRL3 that is at least 90% identical to SEQ ID NO: 54;

(d) a heavy chain or fragment thereof comprising a complementarity determining region (CDR) CDRH1 that is identical to SEQ ID NO: 56, CDRH2 that is identical to SEQ ID NO: 57, and CDRH3 that is

- 1 047752 light chain identical to SEQ ID NO: 58; and a light chain or fragment thereof comprising CDRL1, which is identical to SEQ ID NO: 52, CDRL2, which is identical to SEQ ID NO: 53, and CDRL3, which is identical to SEQ ID NO: 54;

(e) a light chain variable region VL comprising the sequence of SEQ ID NO: 51, or a sequence at least 90% identical thereto; and/or a heavy chain variable region VH comprising the sequence of SEQ ID NO: 55, or a sequence at least 90% identical thereto;

(f) a heavy chain of the Fab region comprising the amino acid sequence of SEQ ID NO: 12, or a sequence at least 90% identical thereto; or (g) a light chain (LC) that comprises the sequence of SEQ ID NO: 3, or a sequence at least 90% identical thereto; and/or a heavy chain (HC) that comprises a sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 49 and SEQ ID NO: 48, or a sequence at least 90% identical thereto.

According to this aspect of the invention, the IFN or functional fragment thereof is selected from the group consisting of type I IFN, type II IFN and type III IFN or a functional fragment thereof. In a preferred embodiment, the type I IFN or functional fragment thereof is IFNa or IFNe or a functional fragment thereof.

According to one embodiment, the IFN or functional fragment thereof is IFNa2a or a functional fragment thereof. Preferably, IFNa2a comprises the sequence of SEQ ID NO: 17, or a sequence at least 90% identical thereto.

According to another embodiment, the IFN or functional fragment thereof is IFNe or a functional fragment thereof. In a preferred embodiment, IFNe comprises the sequence of SEQ ID NO: 14, or a sequence at least 90% identical thereto.

According to a further embodiment, the IFN or a functional fragment thereof is fused to the light chain of the CD40 agonist antibody or its agonist antigen-binding fragment. Preferably, the IFN or a functional fragment thereof is fused to the C-terminus of the light chain of the CD40 agonist antibody or its agonist antigen-binding fragment.

According to another embodiment, the IFN or a functional fragment thereof is fused to the heavy chain of the CD40 agonist antibody or an agonist antigen-binding fragment thereof. In a preferred embodiment, the IFN or a functional fragment thereof is fused to the C-terminus of the heavy chain of the CD40 agonist antibody or an agonist antigen-binding fragment thereof.

According to another embodiment, the agonist CD40 antibody or agonist antigen-binding fragment thereof and the IFN or functional fragment thereof are fused to each other via a linker. In a preferred embodiment, the linker comprises the sequence of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26.

According to another embodiment, the interferon-associated antigen-binding protein comprises a sequence selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47.

According to a further embodiment, the interferon-associated antigen-binding protein is an interferon-fused agonist CD40 antibody or an interferon-fused agonist antigen-binding fragment thereof comprising one of the sequence combinations disclosed in Table 8.

According to another aspect, the invention relates to a nucleic acid encoding an interferon-associated antigen-binding protein according to the invention. In a preferred embodiment, the nucleic acid further encodes a secretory signal peptide.

According to a further aspect, the invention relates to a vector comprising said nucleic acid.

According to another aspect, the invention relates to a vector system comprising (I) a first vector comprising a nucleic acid encoding an IFN or a functional fragment thereof fused to the light chain of an agonist CD40 antibody or an agonist antigen-binding fragment thereof, an interferon-associated antigen-binding protein of the present invention; and a second vector comprising a nucleic acid encoding the heavy chain of an agonist CD40 antibody or an agonist antigen-binding fragment thereof, an interferon-associated antigen-binding protein of the present invention; or (II) a first vector comprising a nucleic acid encoding an IFN or a functional fragment thereof fused to the heavy chain of an agonist CD40 antibody or an agonist antagonist

- 2 047 752 antigen-binding fragment of the interferon-associated antigen-binding protein of the present invention; and a second vector comprising a nucleic acid encoding the light chain of an agonistic CD40 antibody or its agonistic antigen-binding fragment of the interferon-associated antigen-binding protein of the present invention.

According to another aspect, the invention relates to a composition, preferably a pharmaceutical composition, comprising an interferon-associated antigen-binding protein, nucleic acid, vector or vector system according to the invention.

According to a further aspect, the invention relates to a host cell comprising a nucleic acid, a vector or a vector system according to the invention. According to another aspect, the invention relates to a method for producing an interferon-associated antigen-binding protein according to the invention, comprising culturing said host cell and isolating said interferon-associated antigen-binding protein.

According to another aspect, the invention relates to an interferon-associated antigen-binding protein, nucleic acid, vector, vector system or composition according to the invention for use as a medicine.

According to another aspect, the invention relates to an interferon-associated antigen-binding protein, nucleic acid, vector, vector system or composition according to the invention for use in the treatment of hepatitis B virus (HBV) infection and/or for reducing one or more symptoms of HBV infection in a patient.

Brief description of the drawings

Fig. 1: This schematic drawing shows exemplary formats of an interferon-associated antigen-binding protein. The interferon-associated antigen-binding protein is an interferon-fused agonist antibody against CD40 or an interferon-fused agonist antigen-binding fragment thereof. IFNs are linked via linkers to different positions on the antibody or its antigen-binding fragment: to the N-terminal or C-terminal portion of the light chain (LC) or heavy chain (HC). In particular, IFNs are selected from the type I, type II and type III interferon families.

Fig. 2A shows an exemplary map of plasmid pcDNA3.1 encoding SEQ ID NO: 32 under the control of the pCMV promoter. The nucleic acid sequence encoding SEQ ID NO: 32 (SEQ ID NO: 59) is also shown on the right. Italics: signal peptide sequence; black: CP870, coding sequence of the 893 heavy chain; underlined: coding sequence of the HL linker; bold: coding sequence of IFNe.

Figure 2B shows examples of SDS-PAGE under reduced conditions of some IFAs, with IFNa or IFNe fused to either the heavy chain or the light chain. Migration of parental CP870,893 is also shown on the left.

Figures 3A-3B depict the dose-response effect of a series of IFNe-fused IFA molecules on CD40-mediated NFkB pathway activation in HEK-Blue™ CD40L reporter cells. Figure 3A shows examples of anti-CD40 activities for IFA with IFNe fused to the C-terminal portion of the heavy chain (HC). Figure 3B shows examples of anti-CD40 activities for IFA with IFNe fused to the N-terminal portion of LC (IFA34) or HC (IFA36) and the corresponding fusion variants at the C-terminal portion (IFA35 and IFA37). The purification yield of the latter group of IFAs was very low, so supernatants of transfected HEK cells were used to test their activity and serially diluted to assess anti-CD40 activity on HEK-Blue™ CD40L cells.

Figures 3C-3D graphically depict the dose-dependent effect of a series of IFA molecules with IFNe fusion variants on the activation of the type I IFN pathway in HEK-Blue-IFN-α/β reporter cells. Figure 3C shows examples of IFN activity for IFA with IFNe fused to the C-terminal portion of HC. Figure 3D shows examples of IFN activity for IFA with IFNe fused to the N-terminal portion of LC (IFA34) or HC (IFA36) and with the corresponding fusion variants at the C-terminal portion (IFA35 and IFA37). The same supernatants from transfected HEK cells as in Figure 3B were used for the assessment of IFN activity and were serially diluted. The parent antibody CP870,893 was used as a negative control and recombinant human IFNe was used as a positive control. NS: Not stimulated.

Fig. 4A graphically depicts the dose response of a series of IFA molecules with IFNa fusion variants on CD40-mediated NFkB pathway activation in HEK-Blue™ CD40L reporter cells.

Fig. 4B graphically depicts the dose response of a series of IFA molecules with IFNa fusion variants on the activation of the type I IFN pathway in HEK-Blue-IFN-a/e reporter cells. The activity of Pegasys is indicated in the insert at the lower right.

Fig. 4C graphically depicts the effect of IFA molecules with fusion variants of IFNa and an HL linker on HC (IFA38) or LC (IFA39) on the activation of the CD40-mediated NFkB pathway in HEK-Blue™ CD40L reporter cells.

Fig. 4D graphically depicts the effect of IFA38 and IFA39 on activation of the type I IFN pathway in

- 3,047,752 HEK-Blue-IFNa/β reporter cells.

Figure 5 shows the dose-dependent effect of INEβ-based IFA on HBeAg release from HBV-infected primary hepatocytes. IFA1, IFA12: IFNe fusion to the C-terminus of LC via HL or RL linkers, respectively. IFA2 and IFA13: IFNe_C17S fusion to the C-terminus of LC via HL or RL linkers, respectively.

Fig. 6A shows the dose-dependent effect of IFA25, IFA26 and IFA27 on HBeAg release from HBV-infected primary human hepatocytes.

Fig. 6B shows the dose-dependent effect of IFA28, IFA29 and IFA30 on HBeAg release from HBV-infected primary human hepatocytes.

Fig. 6C shows the dose-dependent antiviral activity (HBeAg release) of HL-linked IFAs (IFA38 and IFA39) against HBV-infected PHH.

Figures 6D-6H depict the dose-dependent antiviral activity of 4 IFA molecules containing fusion IFNa via a peptide linker against HBV-infected human primary hepatocytes. Figure 6D: Graphical representation of the study design. Figure 6E: Effect of IFA on HBeAg release compared to Pegasys. Figure 6F: Effect of IFA on HBsAg release compared to Pegasys. Figure 6G: Effect of IFA on pgRNA level compared to Pegasys. Figure 6H: Effect of IFA on CXCL10 release compared to Pegasys.

Figure 7 shows the results of an in vitro human whole blood cell (WBC) cytokine release assay: example data obtained after stimulation of leukocytes from 4 healthy volunteer donors. Leukocytes were left unstimulated (NS), treated with LPS (10 ng/mL) or IFA1 (1 μg/mL) for 24 h. Supernatants were collected and quantified for cytokine release using the MSD u-Plex Human Cytokine Kit. Results represent the average of two independent stimulations from each donor. Profiles of CXCL10 (IP10), IL6, IL1p, and TNFa are shown.

Tables 9a-b: These tables summarize the data obtained following in vitro stimulation of whole blood cells (WBCs) obtained from healthy volunteers. Each IFA was tested on WBCs from four different donors. WBCs were left untreated (NT), treated with LPS (10 ng/mL), or IFA (1 μg/mL) for 24 h. Supernatants were collected and quantified for cytokine release using the MSD u-Plex Human Cytokine Kit. Results represent the mean of two independent stimulations from each donor and are expressed as pg/mL (nd: not detected).

Fig. 8: Pharmacokinetic profile of IFA25, IFA26, IFA27, IFA28, IFA29, and IFA30 following intravenous bolus injection of 0.5 mg/kg (IFA) or 0.3 mg/kg (Pegasys) in mice. Data are expressed as mean +/- standard deviation on a semi-log scale. Samples were collected for 10 days after administration. An ELISA assay using anti-IFNa as a secondary antibody for quantification was used for IFA27, IFA29, and IFA30 (Fig. 8A) and for IFA25, IFA26, and IFA28 (Fig. 8B). An ELISA assay using anti-IgG2 as a secondary antibody for quantification was used for IFA25 and IFA27 (Fig. 8C). Fig. 8D. Pegasys was quantified using human IFNa-matched antibody pairs. The marked line (LLOQ) represents the limit of detection for the Pegasys assay.

Table 10A: Summary of pharmacokinetic data: pharmacokinetic parameters for CP870, 893, IFA27, IFA29, and IFA30 following a single 0.5 mg/kg intravenous administration to male CD1 Swiss mice. PK parameters for CP870,893 were investigated in a 7-day experiment and those for IFA27, IFA29, and IFA30 in a 10-day experiment (IFA27 was quantified using 2 different ELISA assays).

Table 10B: PK parameters for CP870,893, Pegasys and three different IFAs (IFA25, IFA26 and IFA28) after a single 0.5 mg/kg intravenous bolus administration in male CD1 Swiss mice. PK parameters for CP870,893 and IFA25, IFA26, IFA28 and Pegasys were studied in 21-day experiments (IFA25 quantification was performed using 2 different ELISA approaches).

Figure 9A shows the dose-dependent CD40 agonist activity of IFA50 and IFA51 without the Fc region compared to the parental anti-CD40 antibody in HEK-Blue™ CD40L reporter cells. Figure 9B shows the dose-dependent IFNa activity of IFA50 and IFA51 in HEK-Blue™ hIFN-α/β reporter cells. Figure 9C: Effect of IFA50 and IFA51 on HBeAg release from HBV-infected PHH.

Figure 10A shows the dose-dependent IFNα-based CD40 agonist activity of IFA49 compared to the parental anti-CD40 antibody in HEK-Blue™ CD40L reporter cells. IFA49 corresponds to the fusion of IFNα to HC via a peptide linker. Figure 10B shows the dose-dependent IFNα activity of IFA49 on HEK-Blue™ hIFN-α/β reporter cells that are activated by type I interferons. Figure 10C: Effect of IFA49 on HbeAg release from HBV-infected PHH.

Fig. 11A shows the dose-dependent agonist activity of CD40 for IFA46 based on

- 4 047 752 compared to the parental anti-CD40 antibody in HEK-Blue™ CD40L reporter cells. IFA46 corresponds to the fusion of ΙΗΝω to LC via a peptide linker. Figure 11B shows the dose-dependent IFN activity of IFA46 on HEK-Blue™ hIFN-α/β reporter cells that are activated by type I interferons. Figure 11C: Effect of IFA46 on HbeAg release from HBV-infected PHH.

Figure 12A shows the dose-dependent CD40 agonist activity of IFNy-based IFAs (IFA42 and IFA43) compared to the parental CD40 antibody in HEK-Blue™ CD40L reporter cells. IFA42 corresponds to the fusion of IFNy to LC via a peptide linker, and IFA43 corresponds to the fusion of IFNy to HC via a peptide linker. Figure 12B shows the dose-dependent IFN activity of IFA42 and IFA43 in HEK-Blue-hIFN/.. reporter cells. Figure 12C: Effect of IFA42 and IFA43 on HBeAg release from HBV-infected PHH.

Figure 13A shows the dose-dependent CD40 agonist activity of IFN/β-based IFAs (IFA44 and IFA45) compared to the parental CD40 antibody in HEKBlue™ CD40L reporter cells. IFA44 corresponds to the fusion of IFN/β to LC via a peptide linker, and IFA45 corresponds to the fusion of IFN/β to HC via a peptide linker. Figure 13B shows the dose-dependent IFN activity of IFA44 and IFA45 in HEK-Blue-hlFN/β reporter cells. Figure 13C: Effect of IFN/β-based IFAs (IFA44 and IFA45) on HbeAg release from HBV-infected PHH.

The above and other features and advantages of the present invention will become more apparent from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings.

Detailed description

The present invention is based in part on the discovery of a therapy based on the use of interferon-associated antigen-binding proteins, variants or derivatives thereof comprising (i) an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof and (ii) interferon (IFN) or a functional fragment thereof in the therapy of hepatitis B virus (HBV). Said interferon-associated antigen-binding proteins inhibit the transcription of hepatitis B virus covalently closed circular DNA (cccDNA) into HBV pregenomic RNA (pgRNA) in HBV-infected cells, inhibit the release of hepatitis B e antigen (HBeAg) from HBV-infected cells, and enhance the IFN pathway in uninfected and HBV-infected hepatocytes, in particular in uninfected and HBV-infected primary human hepatocytes, and in a synergistic manner. A proposed therapy for HBV involves the administration of interferon-associated antigen-binding protein into an HBV-infected cell or patient infected with HBV.

The invention can be more easily understood in light of the selected terms defined below.

As used herein, the term CD40 refers to cluster of differentiation 40, a member of the tumor necrosis factor receptor (TNFR) superfamily. CD40 is a costimulatory protein found on antigen-presenting cells (e.g., B cells, dendritic cells, monocytes), hematopoietic progenitors, endothelial cells, smooth muscle cells, epithelial cells, and most human tumors (Grewal & Flavell, Ann. Rev. lmmunol., 1996, 16: 111-35; Toes & Schoenberger, Seminars in Immunology, 1998, 10(6):443-8). Binding of the natural ligand CD154 (CD40L) on TH cells to CD40 activates antigen-presenting cells and induces a variety of downstream effects. The TNF receptor-associated factor adaptor proteins TRAF1, TRAF2, TRAF6, and TRAF5 interact with CD40 and serve as mediators of signal transduction. Ultimately, CD40 signaling activates both the canonical and noncanonical NF-kB pathways.

Agonistic antibodies against CD40 and their antigen-binding fragments

As used herein, the term "antibody" refers to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains linked together by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated VH or VH) and a heavy chain constant region (CH or CH). The heavy chain constant region contains three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated VL or VL) and a light chain constant region (CL or CL). The light chain constant region contains one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Framework regions may help maintain the proper conformation of the CDRs to ensure binding between the antigen-binding region and the antigen.

The most commonly used immunoglobulin for therapeutic purposes is immunoglobulin G (or IgG), a tetrameric glycoprotein. In naturally occurring immunoglobulin, each tetramer consists of two identical pairs of polypeptide chains, each pair having one light

- 5,047,752 (approximately 25 kDa) and one heavy chain (approximately 50-70 kDa). The amino-terminal portion of each chain includes a variable region of approximately 100-110 or more amino acids, primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region, primarily responsible for effector function. Immunoglobulins can be classified into different classes depending on the amino acid sequence of the constant domain of their heavy chains.

Heavy chains are classified as mu (μ), delta (δ), gamma (γ), alpha (α), and epsilon (ε) and define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Some of them can be further divided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Different isotypes have different effector functions; for example, the IgG1 and IgG3 isotypes have antibody-dependent cellular cytotoxicity (ADCC). In preferred embodiments, the agonist CD40 antibodies or agonist antigen-binding fragments thereof contained in the interferon-associated antigen-binding proteins of the invention belong to the IgG class. In more preferred embodiments, the agonist CD40 antibodies or agonist antigen-binding fragments thereof contained in the interferon-associated antigen-binding proteins of the invention belong to the IgG1 or IgG3 subclasses. In particularly preferred embodiments, the agonist CD40 antibodies or agonist antigen-binding fragments thereof contained in the interferon-associated antigen-binding proteins of the invention belong to the IgG1 subclass. In other more preferred embodiments, the agonist CD40 antibodies or agonist antigen-binding fragments thereof contained in the interferon-associated antigen-binding proteins of the invention belong to the IgG2 or IgG4 subclasses. In particularly preferred embodiments, the agonist CD40 antibodies or agonist antigen-binding fragments thereof contained in the interferon-associated antigen-binding proteins of the invention belong to the IgG2 subclass.

Human light chains are classified as kappa (κ) and lambda (λ) light chains. Accordingly, in some embodiments, the agonist CD40 antibodies or agonist antigen-binding fragments thereof contained in the interferon-associated antigen-binding proteins of the invention comprise a class κ light chain. In other embodiments, the agonist CD40 antibodies or agonist antigen-binding fragments thereof contained in the interferon-associated antigen-binding proteins of the invention comprise a class λ light chain. In the light and heavy chains, the variable and constant regions are joined by a J region of about 12 or more amino acids, with the heavy chain further comprising a D region of about 10 additional amino acids. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N. Y. (1989)).

The term antibody further includes, but is not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to as antibody mimetics), chimeric antibodies, humanized antibodies, human antibodies, and fragments thereof, respectively. Unless otherwise specified, the term antibody includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, derivatives, variants, antigen-binding fragments, and muteins thereof, examples of which are described below.

As used herein, the term "CD40 agonist antibody" or "anti-CD40 agonist antibody" refers to an antibody that binds to CD40 and mediates CD40 signaling. In a preferred embodiment, it binds to human CD40. As described below, binding to CD40 can be determined by surface plasmon resonance, preferably using the BIAcore® system. The CD40 agonist antibody can increase one or more CD40 activities by at least about 20% when added to a cell, tissue, or organism expressing CD40. In some embodiments, the antibody activates CD40 activity by at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 85%. The CD40 activity of the anti-CD40 agonist antibody can be measured using a whole blood cell surface molecule upregulation assay or using an in vitro reporter cell assay, such as using HEK-Blue™ CD40L cells (InvivoGen Cat. #: hkb-cd40), as described in more detail in Example I. These reporter cells were generated by stably transfecting HEK293 cells with the human CD40 gene and an NFkB-inducible secreted embryonic alkaline phosphatase (SEAP) construct to measure CD40 agonist activity. Stimulation of CD40 results in NFkB activation and thus SEAP production, which can be detected in the supernatant using chromogenic substrates such as QUANTI-Blue™.

In the context of the present invention, interferon-associated antigen-binding proteins activate both CD40 and the IFN pathway. In some embodiments, the interferon-associated antigen-binding protein activates the CD40 pathway with an EC50 of less than 400, 300, 200, 150, 100, 70, 60, 50, 40, 30, 25, 20, or 15 ng/mL. In more specific embodiments, the interferon

- 6 047752 the associated antigen-binding protein activates the CD40 pathway with an EC50 in the range of 10 to 200 ng/ml. In even more specific embodiments, the interferon-associated antigen-binding protein activates the CD40 pathway with an EC50 in the range of 10 to 50 ng/ml, preferably 10 to 30 ng/ml.

Examples of light and heavy chain sequences of the anti-CD40 agonist antibody CP870,893 are shown in Table 7.

As used herein, the term "agonist antigen-binding fragment of an agonist CD40 antibody" refers to a fragment of an agonist CD40 antibody that retains one or more functional activities of the parent antibody, such as the ability to bind to and act as an agonist of CD40 signaling in a cell, such as mediating the CD40 signaling pathway. Such a fragment can compete with the intact antibody for binding to CD40.

Agonist antigen-binding fragments of an agonist CD40 antibody may be produced by recombinant DNA techniques or may be produced by enzymatic or chemical cleavage of an anti-CD40 antibody. Agonist antigen-binding fragments include, but are not limited to, a Fab fragment, a diabody (a heavy chain variable domain on the same polypeptide as the light chain variable domain joined by a short peptide linker too short to pair the two domains on the same chain), a Fab' fragment, an F(ab')2 fragment, an Fv fragment, domain antibodies, and single chain antibodies, and which may be obtained from any mammalian source, including, but not limited to, human, mouse, rat, camel, or rabbit.

The term variable region or variable domain refers to a portion of the light and/or heavy chain of an antibody, typically comprising about 120 to 130 N-terminal amino acids in the heavy chain and about 100 to 110 N-terminal amino acids in the light chain. The variable regions of different antibodies vary widely in amino acid sequence, even among antibodies derived from the same species or class. Exemplary VL and VH domain sequences of the CD40 agonist antibody CP870,893 are shown in Table 1. The variable region of an antibody typically determines the specificity of a particular antibody for its target because it contains the CDRs. Also shown in Table 1 are exemplary CDR sequences of the CD40 agonist antibody CP870,893.

Table 1

Heavy/light chain variable regions and CDRs of the CD40 agonist antibody CP870,893. Sequences in bold italics correspond to the CDRs as defined by Kabat. 1

Anti-CD40 antibody regions Subsequence anti-CD40 antibody, V _L domain (SEQ ID NO: 51) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPG KAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQANIFPLTFGGGTKVEIK anti-CD40 antibody, CDRL1 (SEQ ID NO: 52) RASQGIYSWLA anti-CD40 antibody, CDRL2 (SEQ ID NO: 53) TASTLQS anti-CD40 antibody, CDRL3 (SEQ ID NO: 54) QQANIFPLT anti-CD40 antibody, V _H domain (SEQ ID NO: 55) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQ APGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTA YMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQ GTLVTVSS anti-CD40 antibody, CDRH1 (SEQ ID NO: 56) TGYYMH anti-CD40 antibody, CDRH2 (SEQ ID NO: 57) WINPDSGGTNYAQKFQG anti-CD40 antibody, CDRH3 (SEQ ID NO: 58) DQPLGYCTNGVCSYFDY

Delineation of the CDRs and identification of residues containing the antibody binding site can be accomplished by determining the structure of the antibody and/or determining the structure of the antibody-ligand complex. This can be accomplished by any of a variety of methods known to those skilled in the art, such as X-ray crystallography. Various analytical methods can be used to identify or approximate regions of the CDRs. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, and the contact definition.

The Kabat definition is the standard for numbering residues in an antibody and is commonly used

- 7 047752 to identify the CDR regions. See, e.g., Johnson & Wu, Nucleic Acids Res., 28: 214–8 (2000). The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account the positions of certain structural loop regions. See, e.g., Chothia et al., J. Mol. Biol., 196: 901–17 (1986); Chothia et al., Nature, 342: 877–83 (1989). The AbM definition uses an integrated suite of computer programs produced by the Oxford Molecular Group that model the structure of antibodies. See, e.g., Martin et al., Proc Natl Acad Sci (USA), 86:9268–9272 (1989); AbMTM, A Computer Program for Modeling Variable Regions of Antibodies, Oxford, UK; Oxford Molecular, Ltd. The AbM definition models the tertiary structure of an antibody from the primary sequence using a combination of database information and ab initio methods such as those described by Samudrala et al., Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach, in PROTEINS, Structure, Function and Genetics Suppl. 3:194–198 (1999). Contact definition is based on analysis of available complex crystal structures. See, e.g., MacCallum et al., J. Mol. Biol., 5:732–45 (1996).

In some embodiments, the complementarity determining regions (CDRs) of the variable regions of the light and heavy chains of the CD40 agonist antibody or agonist antigen-binding fragment thereof may be grafted to framework regions (FRs) of the same or a different species. In some embodiments, the CDRs of the variable regions of the light and heavy chains of the CD40 agonist antibody or agonist antigen-binding fragment thereof may be grafted to human consensus FRs. To generate human consensus FRs, in some embodiments, FRs from multiple amino acid sequences of a human heavy or light chain are aligned to identify a consensus amino acid sequence. In some embodiments, the FRs of a heavy chain or light chain of the CD40 agonist antibody or agonist antigen-binding fragment thereof are replaced with FRs from another heavy chain or light chain. In some embodiments, rare amino acids in the FRs of the heavy and light chains of the CD40 agonist antibody or agonist antigen-binding fragment thereof are not replaced, while the remaining amino acids of the FR are replaced. Rare amino acids are specific amino acids that are in positions in which they are not typically found in FRs. In some embodiments, the grafted variable regions of the CD40 agonist antibody or agonist antigen-binding fragment thereof can be used with a constant region that is different from the constant region of the CD40 agonist antibody or agonist antigen-binding fragment thereof. In some embodiments, the grafted variable regions are part of a single-chain Fv antibody. CDR grafting is described, for example, in U.S. Pat. 6180370, 6054297, 56937625, 859205, 5693761, 5565332, 5585089, and 5530101, and in Jones et al., Nature, 321: 522–525 (1986); Riechmann et al., Nature, 332: 323–327 (1988); Verhoeyen et al., Science, 239:1534–1536 (1988), Winter, FEBS Letts., 430:92–94 (1998), which are hereby incorporated by reference for any purpose.

The Fc region typically contains two heavy chain fragments containing the CH2 and CH3 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domains.

The Fab fragment contains one full-length light chain, as well as the CH1 and variable regions of one heavy chain (the combination of the VH and CH1 regions is referred to herein as the heavy chain fab region).

The Fab' fragment comprises one light chain and a portion of one heavy chain that contains the VH domain and the CH1 domain, as well as a region between the CH1 and CH2 domains such that a disulfide bond can form between the two heavy chains between the chains of the two Fab' fragments to form an F(ab')2 molecule.

The F(ab')2 fragment contains two light chains and two heavy chains containing part of the constant region between the CH1 and CH2 domains, so that an interchain disulfide bond is formed between the two heavy chains. Thus, the F(ab')2 fragment consists of two Fab' fragments that are held together by a disulfide bond between the two heavy chains.

The Fv region includes the variable regions of both the heavy and light chains, but does not contain the constant regions.

Single chain antibodies are Fv molecules in which the variable regions of the heavy and light chains are joined by a flexible linker to form a single polypeptide chain that forms the antigen-binding region. Single chain antibodies are discussed in detail in International Patent Application Publication No. WO 88/01649 and U.S. Patent Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated by reference.

A domain antibody is an immunologically functional fragment of an immunoglobulin that contains only the variable region of the heavy chain or the variable region of the light chain. In some cases, two or more VH regions are covalently joined with a peptide linker to create a bivalent domain antibody. The two VH regions of a bivalent domain antibody may target the same or different antigens.

- 8 047752

An antibody or antigen-binding protein, such as an interferon-associated antigen-binding protein of the invention, preferably binds its target antigen with a dissociation constant (Kd) of <10 ^-7 M. The antibody or antigen-binding protein binds its antigen with high affinity when the Kd is <5*10 ^-9 M, and with very high affinity when the Kd is <5x10 ^-10 M. More preferably, the antibody or antigen-binding protein has a Kd <10 ^-9 M. In some embodiments, the dissociation rate is <1*10 ^-5 . In other embodiments, the antibody or antigen-binding protein will bind to human CD40 with a Kd of from about 10 ^-9 M to 10 ^-13 M, and in yet another embodiment, the antibody or antigen-binding protein will bind with a Kd <5*10 ^-10 As will be appreciated by one of skill in the art, in some embodiments, any or all of the antigen-binding fragments can bind to CD40. Preferably, said constants are determined using surface plasmon resonance, more preferably using the BIAcore® system.

The term surface plasmon resonance refers to an optical phenomenon that allows the analysis of biospecific interactions in real time by detecting changes in the concentration of proteins in a biosensor matrix, for example, using the BIAcore® system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, NJ). For further descriptions, see Jonsson et al. (1993) Ann. Biol., Clin. 51:19-26. The term Kon refers to the rate constant of association of a binding protein (e.g., an antibody or antigen-binding protein) with an antigen to form, for example, an antigen-binding protein/antigen complex. The term Kon or onrate also refers to the association rate constant or ka, as used interchangeably herein. This value, which represents the rate of binding of a binding protein to its target antigen or the rate of formation of a complex between a binding protein, e.g., an antibody or antigen-binding protein, and an antigen, is also represented by the equation below:

Antibody (Ab) + antigen (Ag) ^ Ab-Ag

The term Koff or dissociation rate means the dissociation rate constant for dissociation or the dissociation rate constant of a binding protein (e.g., an antibody or antigen-binding protein) from, for example, an antigen-binding protein/antigen complex, as known in the art. This value represents the rate of dissociation of a binding protein, e.g., an antibody or antigen-binding protein, from a target antigen or the partition of an Ab-Ag complex over time into free antibody and antigen, as shown in the equation below:

Ab+Ag ^ Ab-Ag

The terms Kd and equilibrium dissociation constant refer to the value obtained by measuring a titration at equilibrium or by dividing the dissociation rate constant (Koff) by the association rate constant (Kon). The association rate constant, dissociation rate constant, and equilibrium dissociation constant are used to represent the binding affinity of a binding protein (e.g., an antibody or antigen-binding protein) for an antigen. Methods for determining association and dissociation rate constants are well known in the art. The use of fluorescence-based methods provides high sensitivity and the ability to study samples in physiological buffers at equilibrium. Other experimental approaches and instruments such as the BIAcore® (Biomolecular Interaction Assay) assay (e.g., an instrument available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden) can be used. In addition, the KinExA® (Kinetic Exclusion Assay) assay, available from Sapidyne Instruments (Boise, Id.), can also be used.

The antigen-binding protein of the invention may bind to one target with an affinity of at least one order of magnitude higher, preferably at least two orders of magnitude higher, than for a second target.

The term target refers to a molecule or part of a molecule capable of binding to an antigen-binding protein. In some embodiments, the target may have one or more epitopes. It will therefore be understood that the target may serve as an antigen for the antigen-binding protein of the present invention.

The term epitope includes any determinant capable of binding to an antigen-binding protein such as an antibody. An epitope is a region of an antigen that is bound by an antigen-binding protein that targets that antigen and, when the antigen is a protein, includes specific amino acids that are in direct contact with the antigen-binding protein. Epitopes are most commonly present on proteins, but in some cases may be present on other types of molecules such as nucleic acids. Epitopic determinants may include chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural characteristics and/or specific charge characteristics. In general, antibodies specific for a particular target antigen preferentially/specifically recognize an epitope on the target antigen in a complex mixture of proteins and/or macromolecules.

In exemplary embodiments, the CD40 agonist antibody or agonist thereof

- 9 047 752 the agonist antigen-binding fragment forming part (I) of the interferon-associated antigen-binding proteins of the invention comprises three light chain complementarity determining regions (CDRs) that are at least 90% identical to the sequences CDRL1, CDRL2 and CDRL3 of SEQ ID NO: 3; and three heavy chain CDRs that are at least 90% identical to the sequences CDRH1, CDRH2 and CDRH3 of SEQ ID NO: 6. The CD40 agonist antibody or agonist antigen-binding fragment thereof may also comprise three light chain complementarity determining regions (CDRs) that are identical to the sequences CDRL1, CDRL2 and CDRL3 of SEQ ID NO: 3; and three heavy chain CDRs that are identical to the sequences of CDRH1, CDRH2, and CDRH3 in SEQ ID NO: 6. In such embodiments, each CDR is defined according to the Kabat definition, the Chothia definition, the AbM definition, or a contact definition of CDRs; preferably, wherein each CDR is defined according to the Kabat definition of CDRs or the Chothia definition of CDRs. In particular embodiments, each CDR is defined according to the Kabat definition. In other particular embodiments, each CDR is defined according to the Chothia definition.

Alternatively, an agonist CD40 antibody or agonist antigen-binding fragment thereof forming part (I) of the interferon-associated antigen-binding proteins of the invention comprises (a) a heavy chain or fragment thereof comprising a complementarity determining region (CDR) CDRH1 that is at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 56, a CDRH2 that is at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 57, and a CDRH3 that is at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 58; and (b) a light chain or fragment thereof comprising a CDRL1 that is at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 52, a CDRL2 that is at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 53, and a CDRL3 that is at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID 54.

In some embodiments, an agonist CD40 antibody or agonist antigen-binding fragment thereof comprises (a) a heavy chain or fragment thereof comprising a complementarity determining region (CDR) CDRH1 that is identical to SEQ ID NO: 56, CDRH2 that is identical to SEQ ID NO: 57, and CDRH3 that is identical to SEQ ID NO: 58; and (b) a light chain or fragment thereof comprising CDRL1 that is identical to SEQ ID NO: 52, CDRL2 that is identical to SEQ ID NO: 53, and CDRL3 that is identical to SEQ ID NO: 54.

More particularly, the CD40 agonist antibody or agonist antigen-binding fragment thereof comprises a light chain variable region VL comprising the sequence of SEQ ID NO: 51 or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto; and/or a heavy chain variable region VH comprising the sequence of SEQ ID NO: 55 or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

The interferon-associated antigen-binding proteins of the invention may also comprise an agonist CD40 antibody or agonist antigen-binding fragment thereof comprising a heavy chain Fab region comprising the amino acid sequence of SEQ ID NO: 12 or a sequence at least 90%, at least 95%, at least 98% or at least 99% identical thereto.

In some embodiments, the agonist CD40 antibody or agonist antigen-binding fragment thereof comprises a light chain (LC) that comprises the sequence of SEQ ID NO: 3 or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto; and/or a heavy chain (HC) that comprises a sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 49, SEQ ID NO: 12, and SEQ ID NO: 50, or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

In more specific embodiments, the CD40 agonist antibody or agonist antigen-binding fragment thereof comprises a light chain (LC) that comprises the sequence of SEQ ID NO: 3 or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto; and/or a heavy chain (HC) that comprises the sequence of SEQ ID NO: 6 or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

In more specific embodiments, the CD40 agonist antibody or agonist antigen-binding fragment thereof comprises a light chain (LC) that comprises the sequence of SEQ ID NO: 3 or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto; and/or a heavy chain (HC) that comprises the sequence of SEQ ID NO: 9 or a sequence at least 90% identical thereto.

- 10 047752 at least 95%, at least 98% or at least 99% identical to it.

In other more specific embodiments, the CD40 agonist antibody or agonist antigen-binding fragment thereof comprises a light chain (LC) that comprises the sequence of SEQ ID NO: 3 or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto; and/or a heavy chain (HC) that comprises the sequence of SEQ ID NO: 49 or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

In other more specific embodiments, the CD40 agonist antibody or agonist antigen-binding fragment thereof comprises a light chain (LC) that comprises the sequence of SEQ ID NO: 3, or a sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto; and/or a heavy chain (HC) that comprises the sequence of SEQ ID NO: 12, or a sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

In other more specific embodiments, the CD40 agonist antibody or agonist antigen-binding fragment thereof comprises a light chain (LC) that comprises the sequence of SEQ ID NO: 3 or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto; and/or a heavy chain (HC) that comprises the sequence of SEQ ID NO: 50 or a sequence at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

Variants and derivatives of interferon-associated antigen-binding protein or its components

A variant polypeptide (e.g., an interferon-associated antigen-binding protein, an interferon-fused agonist anti-CD40 antibody, or an interferon-fused agonist antigen-binding fragment thereof, an antibody, an antigen-binding protein, or an IFN, or components thereof) comprises an amino acid sequence wherein one, two, three, four, five or more amino acid residues are inserted, deleted, and/or substituted in the amino acid sequence compared to another polypeptide sequence. Preferably, the variant comprises up to ten insertions, deletions, and/or substitutions, more preferably up to eight insertions, deletions, and/or substitutions. More particularly, the variant may comprise up to ten, more preferably up to eight insertions. The variant may also comprise up to ten, more preferably up to eight deletions. In even more preferred embodiments, the variant comprises up to ten substitutions, most preferably up to eight substitutions. In some embodiments, these substitutions are conservative amino acid substitutions as described below.

A variant of a polynucleotide sequence (e.g., RNA or DNA) comprises one or more mutations in the polynucleotide sequence compared to another polynucleotide sequence, wherein one, two, three, four, five or more nucleic acid residues are inserted, deleted and/or substituted in the nucleic acid sequence. Preferably, the variant comprises up to ten insertions, deletions and/or substitutions, more preferably up to eight insertions, deletions and/or substitutions. More specifically, the variant may comprise up to ten, more preferably up to eight insertions. The variant may also comprise up to ten, more preferably up to eight deletions. In even more preferred embodiments, the variant comprises up to ten substitutions, most preferably up to eight substitutions. Said one, two, three, four, five or more mutations may cause one, two, three, four, five or more amino acid substitutions in the amino acid sequence that the variant encodes compared to the other amino acid sequence (i.e., a non-silent mutation). Variants also include nucleic acid sequences in which one, two, three, four, five or more codons are replaced by their synonyms, which does not cause an amino acid change and is therefore called a silent mutation.

The term identity or homology in the context of polypeptide or nucleotide sequence variants refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, determined by alignment and comparison of the sequences. Percent identity means the percentage of identical residues between amino acids or nucleotides in the molecules being compared and is calculated based on the size of the smallest of the molecules being compared. Preferably, the identity is determined over the entire length of the sequence. It is understood that the expression at least 80% identical includes embodiments in which the claimed sequence is at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the reference sequence. The expression at least 90% identical includes embodiments in which the claimed sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the reference sequence.

For calculation of percent identity, gaps in alignments (if any) are preferred.

- 11 047752 are carefully eliminated using a specific mathematical model or computer program (i.e., algorithm). Methods that can be used to determine the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology (Lesk, A. M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

In calculating percent identity, the sequences being compared are usually aligned in a way that provides the greatest match between the sequences. One example of a computer program that can be used to determine percent identity is the GCG software package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI). The GAP computer algorithm is used to align two polypeptides or polynucleotides for which percent sequence identity is to be determined. The sequences are aligned for optimal alignment to their corresponding amino acid or nucleotide (the consensus interval, as determined by the algorithm). A gap opening penalty (which is calculated as 3x the mean diagonal, where the mean diagonal is the mean of the diagonal of the comparison matrix used; the diagonal is the score or number assigned to each perfect amino acid match using a particular comparison matrix) and a gap widening penalty (which is typically 1/10 of the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSum 62, are used in conjunction with the algorithm. In some embodiments, a standard comparison matrix (see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. U. S. A. 89:10915-10919 for the BLOSum 62 comparison matrix) is also used by the algorithm.

The following are examples of parameters that can be used to determine the percent identity of polypeptides or nucleotide sequences using the GAP program:

Algorithm: Needleman et al., 1970, J. Mol. Biol., 48:443-453

Comparison matrix: BLOSum 62 from Henikoff et al., 1992, above

Penalty for space: 12 (but no penalty for trailing spaces)

Gap Length Penalty: 4

Similarity threshold: 0

Some alignment schemes for aligning two amino acid sequences may result in a match of only a short region of the two sequences, and this small aligned region may have a very high sequence identity, even if there is no significant relationship between the two full-length sequences. Accordingly, the selected alignment method (GAP program) can be adjusted, if desired, to obtain an alignment that covers at least 50 or at least 100, preferably the full length, contiguous amino acids of the target polypeptide.

Conservative amino acid substitutions may involve unnatural amino acid residues that are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid fragments.

Naturally occurring residues can be divided into classes based on common side chain properties:

1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

3) acidic: Asp, Glu;

4) basic: His, Lys, Arg;

5) residues that affect chain orientation: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe.

For example, non-conservative substitutions may involve the replacement of a member of one of these classes with a member of another class. Such substituted residues may be introduced, for example, into regions of a human antibody that are homologous to non-human antibodies, or into non-homologous regions of the molecule.

When making changes to the interferon-associated antigen-binding protein according to some embodiments, the hydropathic index of amino acids can be taken into account. Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics. These are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The Importance of the Hydropathic Index of an Amino Acid in Imparting Interactive Biological Functionality to a Protein

- 12 047752 sical function is known to those skilled in the art. Kyte et al., J. Mol. Biol., 157:105-131 (1982). It is known that certain amino acids can be replaced with other amino acids having a similar hydropathic index or value and still retain similar biological activity. When making changes based on the hydropathic index, in some embodiments, amino acids whose hydropathic indices are within ±2 are included. In some embodiments, those that are within ±1 are included, and in some embodiments, those that are within ±0.5 are included.

It is also understood in the art that the substitution of such amino acids can be effectively carried out based on hydrophilicity. In some embodiments, the highest local average hydrophilicity of a protein, determined by the hydrophilicity of its neighboring amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values were assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (3.4). When making changes based on similar hydrophilicity values, some embodiments include substitution of amino acids whose hydrophilicity values are within ±2, some embodiments include amino acids whose values are within ±1, and some embodiments include amino acids whose values are within ±0.5.

Examples of amino acid substitutions are given in Table 2.

Table 2. Amino acid substitutions.

Initial balances Illustrative Substitutions Preferred Substitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Sir, Ala Ser Gln Asn Asn Glu Asp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe, Norleucine Leu Leu Norleucine, Ile, Val, Met, Ala, Phe Ile Lys Arg, 1,4 diaminobutyric acid, Gln, Asn Arg Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Tyr Leu Pro Ala, Gly Ala Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Ala, Norleucine Leu

In light of the present invention, one skilled in the art will be able to determine suitable variants of the interferon-associated antigen-binding proteins as described herein using well-known techniques. In some embodiments, one skilled in the art can identify suitable regions of the molecule that can be altered without disrupting activity by targeting regions that are not believed to be important for activity. In some embodiments, residues and portions of the molecule that are conserved among similar polypeptides can be identified. In some embodiments, even regions that may be important for biological activity or structure can be subject to conservative amino acid substitutions without disrupting biological activity or without adversely affecting the structure of the polypeptide.

In addition, one skilled in the art may review structure-function studies that identify residues in similar polypeptides that are important for activity or structure.

- 13 047752 ry. In view of such a comparison, it is possible to predict the importance of amino acid residues in a protein that correspond to amino acid residues important for activity or structure in similar proteins. A person skilled in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues.

The skilled artisan can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar proteins or protein domains. Taking into account such information, the skilled artisan can predict the alignment of amino acid residues of the interferon-associated antigen-binding protein, the antibody or antigen-binding fragment thereof, or the interferon or functional fragment thereof, as described herein, in relation to its three-dimensional structure. In some embodiments, the skilled artisan can decide not to make radical changes to amino acid residues that are expected to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. In addition, the skilled artisan can create test variants containing one amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays known to those skilled in the art. Such variants can be used to gather information about suitable variants. For example, if it is found that a change in a particular amino acid residue results in a disruption, an undesirable decrease, or an unacceptable activity, variants with such a change can be avoided. In other words, based on the information obtained from such experiments, a person skilled in the art can easily determine amino acids in which further substitutions should be avoided, either alone or in combination with other mutations.

According to some embodiments, the amino acid substitutions are substitutions that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity, and/or (5) impart or modify other physicochemical or functional properties of such polypeptides. According to some embodiments, one or more amino acid substitutions (in some embodiments, conservative amino acid substitutions) may be made in the native sequence (in some embodiments, in the portion of the polypeptide outside the domain(s) that form intermolecular contacts). In some embodiments, a conservative amino acid substitution typically cannot significantly alter the structural characteristics of the original sequence (e.g., the substituting amino acid must not break a helix found in the parent sequence or disrupt other types of secondary structure characteristic of the original sequence). Examples of secondary and tertiary structures of polypeptides known in the art are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden & J. Tooze, eds., Garland Publishing, New York, N. Y. (1991)); and Thornton et al., Nature, 354:105 (1991), each of which is incorporated herein by reference.

The term "derivative" refers to a molecule that includes a chemical modification other than the insertion, deletion, or substitution of amino acids (or nucleic acids). In some embodiments, derivatives comprise covalent modifications, including, but not limited to, chemical bonding to polymers, lipids, or other organic or inorganic moieties. In some embodiments, a chemically modified interferon-associated antigen-binding protein may have a longer half-life in the bloodstream than an interferon-associated antigen-binding protein that is not chemically modified. In some embodiments, a chemically modified interferon-associated antigen-binding protein may have improved targeting ability to desired cells, tissues, and/or organs. In some embodiments, the derivative of an interferon-associated antigen-binding protein is covalently modified to include one or more water-soluble polymer attachments, including, but not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. See, for example, U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, and 4,179,337. In some embodiments, the interferon-associated antigen-binding protein derivative comprises one or more polymers, including, but not limited to, monomethoxypolyethylene glycol, dextran, cellulose or other carbohydrate-based polymers, poly(N-vinylpyrrolidone)-polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), and polyvinyl alcohol, and mixtures of such polymers.

In some embodiments, a derivative of an interferon-associated antigen-binding protein as described herein is covalently modified with polyethylene glycol (PEG) subunits. In some embodiments, one or more water-soluble polymers are linked at one or more specific positions, such as at the N-terminus of the derivative. In some

- 14 047 752 In some embodiments, one or more water-soluble polymers are randomly attached to one or more side chains of the derivative. In some embodiments, PEG is used to improve the therapeutic ability of the interferon-associated antigen-binding protein. Some such methods are discussed, for example, in U.S. Patent No. 6,133,426, which is incorporated herein by reference for all purposes.

In some embodiments, interferon-associated antigen-binding protein variants include glycosylation variants in which the number and/or type of glycosylation site is altered compared to the amino acid sequences of the parent polypeptide. In some embodiments, protein variants comprise a greater number of N-linked glycosylation sites than the native protein. In other embodiments, protein variants comprise a lesser number of N-linked glycosylation sites than the native protein. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated X can be any amino acid residue except proline. Substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions that eliminate this sequence remove an existing N-linked carbohydrate chain. Also proposed are rearrangements of N-linked carbohydrate chains in which one, two, three, four, five or more N-linked glycosylation sites (typically found in nature) are deleted and one or more new N-linked sites are created. Additional preferred variants include cysteine variants in which one or more cysteine residues are deleted or replaced with a different amino acid (e.g., serine) compared to the parent amino acid sequence. Cysteine variants may be useful when antibodies need to be converted to a biologically active conformation, such as after isolation of insoluble inclusion bodies. Cysteine variants typically have fewer cysteine residues than the native protein and typically have an even number of them to minimize interactions arising from unpaired cysteines.

HBV and HBV marker

As used herein, the term hepatitis B virus or HBV refers to the double-stranded DNA virus that causes hepatitis B, which belongs to a family of closely related DNA viruses called hepadnaviruses. Hepadnaviruses preferentially infect liver cells, but small amounts of hepadnavirus DNA can be found in the kidneys, pancreas, and mononuclear cells. However, infection in these sites is not associated with extrahepatic disease.

The HBV virion, or Dane particle, consists of an outer lipid envelope and an icosahedral nucleocapsid core of protein. The nucleocapsid encloses the viral DNA and DNA polymerase, which has reverse transcriptase activity similar to retroviruses. The outer envelope contains embedded proteins that aid in viral binding to and entry into susceptible cells. The virus is one of the smallest enveloped animal viruses, with a virion diameter of 42 nm, but pleomorphic forms exist, including filamentous and spherical bodies without a core. These particles are not infectious and consist of a lipid and protein that forms part of the virion surface, called the surface antigen (HBsAg), which is produced in excess during the viral life cycle. HBV includes HBsAg, HBcAg (and its splice variant HBeAg), DNA polymerase, and Hbx. HBV is one of the few known non-retroviral viruses that uses reverse transcription in its replication process.

The HBV nucleocapsid contains a relatively small and partially duplex 3.2 kb circular DNA, the viral polymerase, and the core protein. The genome contains only four long open reading frames. The pre-SS (pre-surface-surface) region of the genome encodes three viral surface antigens by differentially initiating translation at each of the three in-frame initiation codons.

The most abundant HBV protein is the 24 kDa S protein (known as HBsAg). The pre-C-C (pre-core-core) region encodes HBcAg (HBV core antigen) and HBeAg (HBV e antigen). HBeAg is not required for viral replication and plays no role in viral assembly, but is nevertheless a useful indicator of active viral replication. Since HBeAg is secreted by HBV-infected hepatocytes, it can be detected in blood using standard diagnostic tests (such as ELISA) and thus used as a laboratory marker of viremic HBV infection (Testoni et al., Serum hepatitis B core-related antigen (HBcrAg) correlates with covalently closed circular DNA. J Hepatol. 2019, 70, 615625.htip://dx.doi.org/10.1016/j.jhep.2018.11.030).

The P-coding region is specific for the viral polymerase, a multifunctional enzyme involved in DNA synthesis and RNA encapsidation. The X open reading frame encodes the viral X protein (HBx), which modulates host cell signaling and can directly or indirectly influence host and viral gene expression.

The HBV life cycle is thought to begin when the virus attaches to the host cell membrane via envelope proteins. It has been suggested that HBV binds to a receptor on

- 15 047752 plasma membrane, which is predominantly expressed on human hepatocytes via the pre-S1 domain of the large envelope protein as an initial step in HBV infection. However, the nature of the receptor remains controversial. The viral membrane then fuses with the cellular membrane and the viral genome is released into the cells.

HBV replication can be regulated by a variety of factors, including hormones, growth factors, and cytokines. Once the viral genome reaches the nucleus, the cellular DNA repair machinery converts the partial double-stranded DNA (dsDNA; also called HBV relaxed circular DNA (cDNA)) genome into covalently closed circular DNA (cccDNA). The resulting cccDNA serves as a template for host RNA Pol-II for further transcription of pregenomic RNA and subgenomic RNA (Allweiss L and Dandri M, The Role of cccDNA in HBV Maintenance. Viruses 2017, 9(6):156; doi:10. 3390/v9060156; Nur K. Mohd-Ismail , Zijie Lim, Jayantha Gunaratne and Yee-Joo Tan, Mapping the Interactions of HBV cccDNA with Host Factors. Int. J. Mol. Sci. 2019, 20(17):4276; doi:10. 3390/ijms20174276).

Pregenomic RNA is bifunctional, serving both as a template for viral DNA synthesis and as a messenger for translation of pre-C, C, and P. Subgenomic RNAs function exclusively to translate the envelope and X protein. All viral RNA is transported into the cytoplasm, where it is translated to form the viral envelope, core, and polymerase proteins, as well as HBx and HBcAg.

HBV core particles assemble in the cytosol, during which time one molecule of pregenomic RNA is incorporated into the assembling viral core. Following encapsidation of the viral RNA, reverse transcription begins. Synthesis of two strands of viral DNA occurs sequentially. The first strand of DNA consists of an encapsidated RNA template; during or after synthesis of this strand, the RNA template is degraded and synthesis of the second strand of DNA continues, using the newly produced first strand of DNA as a template. Some nuclei bearing the mature genome are transported back to the nucleus, where their newly minted DNA genomes can be converted to cccDNA to maintain a stable intranuclear pool of transcriptional templates.

HBV surface antigen (HBsAg) proteins are initially synthesized and polymerized in the rough endoplasmic reticulum. These proteins are transported to the post-ER and preGolgi compartments where nucleocapsid budding occurs. The assembled HBV virion and subviral particles are transported to the Golgi apparatus for further modification of surface protein glycans and are then secreted from the host cell to complete the life cycle.

In specific embodiments, the interferon-associated antigen-binding proteins, nucleic acids, vectors, vector systems, methods, and compositions described herein can be used to treat HBV infection. As used herein, the terms treat HBV infection and treat HBV infection refer to one or more of: (i) reducing HBV viral load/titer; (ii) reducing cccDNA transcription; (iii) reducing pregenomic RNA levels in cells; (iv) alleviating one or more HBV-associated diseases; and (v) alleviating one or more HBV-associated symptoms in a patient.

The terms viral load and viral titer refer to the amount of viral particles in a cell, organ, or bodily fluid such as blood or serum. Viral load or viral titer is often expressed as viral particles or infectious particles per ml, depending on the type of assay. Today, viral load is usually measured in international units per milliliter (IU/ml). Viral load or viral titer can also be defined as the equivalent of the so-called viral genome. A higher viral load, titer, or viral load often correlates with the severity of an active viral infection. Accordingly, a decrease in viral load or viral titer correlates with a decrease in the number of infectious viral particles, for example, in serum. Viral load is usually determined using nucleic acid amplification tests (NAT or NAAT). NAT/NAAT tests use, for example, PCR, (quantitative) reverse transcription polymerase chain reaction (RT-PCR or qRT-PCR), nucleic acid sequence-based amplification (NASBA) or probe-based assays. Real-time PCR assays for the quantitative detection of hepatitis B virus DNA have been described, for example, in Liu et al., Virol J 14, 94 (2017) doi:10. 1186/s12985-017-0759-8. Due to the ease of detecting viral DNA by PCR, viral load is useful in the clinical setting to monitor the success of treatment. A viral load >10,000 copies/mL (2000 IU/mL) is a strong predictor of the risk of hepatocellular carcinoma, independent of HBeAg status.

The terms patient and subject are used interchangeably and include humans and non-human animals, preferably humans, as well as individuals with formally diagnosed disorders, individuals without formally recognized disorders, individuals receiving medical care, individuals at risk of developing disorders, etc.

In particular embodiments, the interferon-associated antigen binding protein, nucleic acids, vectors, vector systems, methods, and compositions described herein can be used to reduce the HBV viral load/titer in an HBV-infected cell (e.g., in a cell culture, in an HBV-infected organ, or in an HBV-infected patient). The HBV viral load/titer can be reduced by about 20%,

- 16 047752

25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to an untreated HBV-infected cell culture or the same patient before treatment. In some embodiments, the HBV viral load/titer is reduced by at least 20%, at least 25%, at least 30%, at least 35%, at least

40%, at least 45%, at least 50%, at least 55%, at least

60%, at least 65%, at least 70%, at least 75%, at least

80%, at least 85%, at least 90%, or at least 95%. Preferably, the HBV viral load/titer is reduced by at least 35%, more preferably by at least 50%. In some embodiments, the viral load/titer is determined by PCR or quantitative RT-PCR.

In particular embodiments, the interferon-associated antigen binding protein, nucleic acids, vectors, vector systems, methods, and compositions described herein can be used to reduce the transcription of HBV cccDNA in an HBV-infected cell (e.g., in a cell culture, in an HBV-infected organ, or in an HBV-infected patient). The transcription of cccDNA can be reduced by about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to an untreated HBV-infected cell culture or the same patient prior to treatment. In some embodiments, HBV cccDNA transcription is reduced by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. Preferably, HBV cccDNA transcription is reduced by at least 35%, more preferably at least 50%. In some embodiments, HBV cccDNA transcription is detected by PCR or qPCR.

In particular embodiments, the interferon-associated antigen binding protein, nucleic acids, vectors, vector systems, methods and compositions described herein can be used to reduce the level of HBV pregenomic RNA in an HBV infected cell (e.g., in a cell culture, in an HBV infected organ, or in an HBV infected patient). HBV pregenomic RNA levels can be reduced by about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to an untreated HBV infected cell culture or the same patient before treatment. In some embodiments, the level of HBV pregenomic RNA is reduced by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. Preferably, the level of HBV pregenomic RNA is reduced by at least 35%, more preferably by at least 50%. In some embodiments, the level of HBV pregenomic RNA is determined by quantitative RT-PCR.

As used herein, the term HBV-associated disorder refers to a disorder that results from infection of a patient with HBV. HBV-associated disorders include, but are not limited to, acute hepatitis, chronic hepatitis, icteric hepatitis, fulminant hepatitis, subfulminant hepatitis, and symptoms and/or complications resulting from any of these disorders.

As used herein, the term HBV-associated symptom, HBV infection symptom, or HBV-associated complication includes one or more physical dysfunctions associated with HBV infection. Symptoms and complications of HBV include, but are not limited to, cirrhosis, hepatocellular carcinoma (HCC), membranous glomerulonephritis (MGN), death, acute necrotizing vasculitis (polyarteritis nodosa), membranous glomerulonephritis, papular acrodermatitis of childhood (Gianotti-Crosti syndrome), HBV-associated nephropathy (eg, membranous glomerulonephritis), immune-mediated hematologic disorders (eg, essential mixed cryoglobulinemia, aplastic anemia), portal hypertension, ascites, encephalopathy, jaundice, pruritus, pale stools, steatorrhea, polyarteritis nodosa, glomerular disease, abnormal ALT levels, abnormal AST levels, abnormal alkaline phosphatase levels, increased bilirubin, anorexia, malaise, fever, nausea, vomiting, etc.

Interferons

As used herein, the term interferon or IFN refers to a cytokine or derivative thereof that is typically produced and released by cells in response to the presence of a pathogen or tumor cell. IFNs include type I IFNs (e.g., IFNa, IFNe, IFN::. IFNk, IFNt, IFNZ, and ZEXω), type II IFNs (e.g., IFNy), and type III IFNs (e.g., IFN/1, UPNX2, and IFN/3). The term interferon or IFN includes, but is not limited to, full-length IFN, a variant, or a derivative thereof.

- 17 047752 (e.g., a chemically (e.g., pegylated) modified derivative or mutein), or a functionally active fragment thereof, that retains one or more signaling activities of a full-length IFN.

As used herein, the term "functional fragment" refers to a fragment of a substance that retains one or more functional activities of the parent substance. For example, a functional fragment of interferon refers to a fragment of interferon that retains the function of IFN as described herein, such as mediating the IFN signaling pathway.

IFN can increase the activity of one or more IFN receptors by at least about 20% when added to a cell, tissue, or organism expressing a related IFN receptor (IFNAR for IFNa, IFNBR for IFNe, etc.). In some embodiments, the interferon activates IFN receptor activity by at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 85%. IFN activity (i.e., IFN activity) can be measured, for example, using an in vitro reporter cell assay, for example, using HEK-Blue™ IFN-α/β cells (InvivoGen, Cat. #: hkb-ifnae), HEK-Blue™ IFNλ cells (InvivoGen, Cat. #: hkb-ifnl), or HEK-Blue™ Dual IFN-γ cells (InvivoGen, Cat. #: hkb-ifng), as described in more detail in Example I. These reporter cells were generated by stably transfecting HEK293 cells with human IFN receptor genes and a secreted embryonic alkaline phosphatase (SEAP) construct controlled by an IFN-stimulated response element to measure interferon activity. HEK-Blue™ IFN cells are designed to monitor activation of the JAK/STAT/ISGF3 pathways induced by type I, type II, or type III interferons. Activation of these pathways induces the production and release of SEAP.

In the context of the present invention, interferon-associated antigen-binding proteins activate both CD40 and the IFN pathway. In some embodiments, the interferon-associated antigen-binding protein activates the IFN pathway with an EC50 of less than 100, 60, 50, 40, 30, 20, 10 or 1 ng/ml, preferably with an EC50 of less than 11 ng/ml, more preferably with an EC50 of less than 6 ng/ml. In some of these embodiments, the IFN pathway is the IFNa (interferonalpha), IFNe (interferon-beta), IFN: (interferon-epsilon), IFNio (interferon omega), IFNy (interferon-gamma) or IFNX (interferon-lambda) pathway.

According to some exemplary embodiments, the interferon-associated antigen-binding protein described herein comprises a full-length IFN, a variant or derivative (e.g., a chemically (e.g., pegylated) modified derivative or mutein) thereof, or a functionally active fragment thereof that retains one or more signaling activities of a full-length IFN. In some embodiments, the IFN is a human IFN.

In some embodiments, an interferon-associated antigen-binding protein as described herein comprises an IFN or functional fragment thereof selected from the group consisting of type I IFN, type II IFN, and type III IFN, or functional fragments thereof.

In specific embodiments, the IFN or functional fragment thereof is a type I IFN or a functional fragment thereof. In specific embodiments, the type I IFN or functional fragment thereof is IFNa, IFNe, IFNio, or IFN:, or a functional fragment thereof. In more specific embodiments, the type I IFN or functional fragment thereof is IFNa or IFNe, or a functional fragment thereof. In other more specific embodiments, the type I IFN or functional fragment thereof is IFNa or a functional fragment thereof. In other more specific embodiments, the type I IFN or functional fragment thereof is IFNe or a functional fragment thereof. In other more specific embodiments, the type I IFN or functional fragment thereof is IFN ω or a functional fragment thereof. In other more specific embodiments, the type I IFN or functional fragment thereof is IFN: or a functional fragment thereof.

In particular embodiments, the IFN or functional fragment thereof is IFNa, IFNe, IFNy, IFNZ, IFN:, or IFNio, or a functional fragment thereof. In particular embodiments, the IFN or functional fragment thereof is IFNa or IFNe, or a functional fragment thereof.

In some embodiments, the IFN or functional fragment thereof is IFNa or a functional fragment thereof. In more specific embodiments, the IFN or a functional fragment thereof is IFNa2a or a functional fragment thereof. IFNa2a may comprise the sequence of SEQ ID NO: 17 or a sequence at least 90% identical thereto.

In some embodiments, the IFN or functional fragment thereof is IFNe or a functional fragment thereof. IFNe may comprise the sequence of SEQ ID NO: 14 or

- 18 047752 a sequence identical thereto by at least 90%. IFNe or a functional fragment thereof may comprise one or two amino acid substitutions relative to SEQ ID NO: 14 selected from C17S and N80Q. In some embodiments, IFNe or a functional fragment thereof comprises an amino acid substitution C17S relative to SEQ ID NO: 14. In some embodiments, IFNe comprises the amino acid sequence of SEQ ID NO: 15. In other embodiments, IFNe comprises the amino acid substitutions C17S and N80Q relative to SEQ ID NO: 14. In other embodiments, IFNe comprises the amino acid sequence of SEQ ID NO: 16.

In some embodiments, the IFN or functional fragment thereof is IFNY or IFNL or a functional fragment thereof. In particular embodiments, the IFN or functional fragment thereof is IFNy or a functional fragment thereof. In more particular embodiments, IFNy comprises the sequence of SEQ ID NO: 19 or a sequence at least 90% identical thereto. In other particular embodiments, the IFN or functional fragment thereof is IFNL or a functional fragment thereof. In more particular embodiments, IFNL or a functional fragment thereof is IFNL2 or a functional fragment thereof. IFNL2 may comprise the sequence of SEQ ID NO: 18 or a sequence at least 90% identical thereto.

In some embodiments, the IFN or functional fragment thereof is IFNi: or a functional fragment thereof. IFNi: may comprise the sequence of SEQ ID NO: 61 or a sequence at least 90% identical thereto.

In some embodiments, the IFN or functional fragment thereof is IFNe! or a functional fragment thereof. IFNio may comprise the sequence of SEQ ID NO: 60 or a sequence at least 90% identical thereto.

In some embodiments, the expression level of one or more IFN signaling pathway biomarkers is altered, i.e., up-regulated or down-regulated, in HBV-infected cells treated with the interferon-associated antigen-binding protein described herein. According to some exemplary embodiments, the expression level of one or more IFN pathway biomarkers is increased in HBV-infected cells treated with the interferon-associated antigen-binding protein described herein. In this context, a biomarker should be understood as a characteristic that is objectively measured and assessed as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention.

According to some embodiments, a suitable IFN pathway biomarker provided herein is a chemokine, such as a C-X-C chemokine selected from the group consisting of CXCL9, CXCL10, and CXCL11. In some exemplary embodiments, a suitable IFN pathway-inducible biomarker is CXCL9, CXCL10, and/or CXCL11, as well as the interferon-stimulated gene ISG20. Cytokine induction or release can be quantified using methods known in the art, such as ELISA. Alternatively, induction can also be determined using RNA-based assays, such as RNAseq or qRT-PCR. In some embodiments, upregulation may refer to at least a 1.5-fold, at least a 2-fold, at least a 2.5-fold, at least a 3-fold, at least a 4-fold, at least a 5-fold, or at least a 10-fold increase in the expression or secretion of these cytokines.

In these or other exemplary embodiments, the expression level of proinflammatory cytokines, such as IL10, IL1e and/or IL2, is not significantly upregulated in human whole blood cells upon treatment with the interferon-associated antigen-binding protein of the invention. In some embodiments, the expression level of IL-10 is not significantly upregulated in human whole blood cells upon treatment with the interferon-associated antigen-binding protein of the invention. In some embodiments, the expression level of IL1e is not significantly upregulated in human whole blood cells upon treatment with the interferon-associated antigen-binding protein of the invention. In some embodiments, the expression level of IL2 is not significantly upregulated in HBV-infected cells upon treatment with the interferon-associated antigen-binding protein of the invention. In some embodiments, the expression levels of IL10 and IL1e are not significantly upregulated in HBV-infected cells upon treatment with the interferon-associated antigen-binding protein of the invention. In some embodiments, the expression levels of IL10 and IL2 are not significantly increased in HBV-infected cells upon treatment with the interferon-associated antigen-binding protein of the invention. In some embodiments, the expression levels of IL1e and IL2 are not significantly increased in HBV-infected cells upon treatment with the interferon-associated antigen-binding protein of the invention. In some embodiments, the expression levels of IL10, IL1e, and IL2 are not significantly increased in HBV-infected cells upon treatment with the interferon-associated antigen-binding protein of the invention.

- 19 047752 calling protein according to the invention.

Interferon-associated antigen-binding proteins

The term associated, as used herein, generally refers to a covalent or non-covalent association of two (or more) molecules. Associated proteins are created by joining two or more different peptides or proteins, resulting in a protein with one or more functional properties derived from each of the parent proteins. In the context of the present invention, interferon-associated antigen-binding proteins activate both the CD40 pathway and the IFN pathway. An associated protein encompasses monomeric and multimeric, such as dimeric, trimeric, tetrameric or the like, complexes of different associated or fusion proteins. In this context, a non-covalent association results from strong interactions between two protein surface regions, typically through ionic, van der Waals and/or hydrogen bonds. A covalent bond, on the other hand, requires the presence of actual chemical bonds such as peptide bonds, disulfide bridges, etc. The term fusion as used herein generally refers to the covalent connection of two or more different peptides or proteins via a peptide bond. Thus, a fusion protein refers to a single protein created by connecting two or more different peptides or proteins via a peptide bond with one or more functional properties derived from each of the original proteins. In some embodiments, two or more different peptides or proteins may be fused to each other via one or more peptide linkers (L).

In all aspects of the invention, the interferon-associated antigen-binding protein is a protein comprising an agonist anti-CD40 antibody or an agonist antigen-binding fragment thereof and an IFN or a functional fragment thereof.

In some embodiments, the IFN or functional fragment thereof is non-covalently linked to the agonist CD40 antibody or agonist antigen-binding fragment thereof. In more specific embodiments, the IFN or functional fragment thereof is non-covalently linked to the agonist CD40 antibody or agonist antigen-binding fragment thereof through ionic interactions, van der Waals and/or hydrogen bonds.

In other embodiments, the IFN or functional fragment thereof is covalently linked to the agonist CD40 antibody or agonist antigen-binding fragment thereof. In preferred embodiments, the IFN or functional fragment thereof is fused to the agonist CD40 antibody or agonist antigen-binding fragment thereof. The IFN or functional fragment thereof can be fused to the light chain of the agonist CD40 antibody or agonist antigen-binding fragment thereof. In some embodiments, the IFN or functional fragment thereof is fused to the N-terminus of the light chain of the agonist CD40 antibody or agonist antigen-binding fragment thereof. In other embodiments, the IFN or functional fragment thereof is fused to the C-terminus of the light chain of the agonist CD40 antibody or agonist antigen-binding fragment thereof. An IFN or a functional fragment thereof may also be fused to the heavy chain of an agonist CD40 antibody or an agonist antigen-binding fragment thereof. In some embodiments, the IFN or a functional fragment thereof is fused to the N-terminus of the heavy chain of an agonist CD40 antibody or an agonist antigen-binding fragment thereof. In other embodiments, the IFN or a functional fragment thereof is fused to the C-terminus of the heavy chain of an agonist CD40 antibody or an agonist antigen-binding fragment thereof. In any of these embodiments, the agonist CD40 antibody or an agonist antigen-binding fragment thereof and the IFN or a functional fragment thereof may be fused to each other via a linker.

The term linker or L, as used herein, refers to any moiety that covalently connects one or more agonist CD40 antibodies or agonist antigen-binding fragments thereof to one or more interferons or functional fragments thereof. In exemplary embodiments, the linker is a peptide linker. The term peptide linker, as used herein, refers to a peptide adapted to link two or more moieties. A peptide linker referred to herein may have one or more of the properties described below. Sequences of a peptide linker according to some exemplary embodiments are presented in Table 7.

The peptide linker may be of any length, i.e., contain any number of amino acid residues. In exemplary embodiments, the linker contains at least 1, at least 2, at least 3, at least 4, at least 5 amino acids. The linker may contain at least 4 amino acids. The linker may contain at least 11 amino acids. The linker may contain at least 12 amino acids. The linker may contain at least 13 amino acids. The linker may contain at least 15 amino acids. The linker may contain at least 20 amino acids. The linker may contain at least 21 amino acids. The linker may contain at least 24 amino acids.

The linker is usually of sufficient length to provide sufficient flexibility for

- 20 047752 preventing interference of linked portions with each other's activity, such as the ability of a fragment to bind to a receptor. In exemplary embodiments, the linker comprises up to 10, up to 20, up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, or up to 100 amino acids. The linker may comprise up to 80 amino acids. The linker may comprise up to 40 amino acids. The linker may comprise up to 24 amino acids. The linker may comprise up to 21 amino acids. The linker may comprise up to 20 amino acids. The linker may comprise up to 15 amino acids. The linker may comprise up to 13 amino acids. The linker may comprise up to 12 amino acids. The linker may comprise up to 11 amino acids. The linker may comprise up to 4 amino acids.

In some embodiments, the linker is selected from the group consisting of rigid, flexible, and/or helix-forming linkers. It is understood that helix-forming linkers can also be rigid linkers, since the α-helix has fewer degrees of freedom than a peptide adopting a more random helical conformation. In some embodiments, the linker is a rigid linker. An exemplary rigid linker comprises the sequence of SEQ ID NO: 20. Additional exemplary rigid linkers comprise the sequence of SEQ ID NO: 22 or SEQ ID NO: 23. In related embodiments, the linker is a helix-forming linker. Examples of helix-forming linkers comprise the sequence of SEQ ID NO: 22 or SEQ ID NO: 23. In other embodiments, the linker is a flexible linker. Examples of flexible linkers comprise the sequence SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26.

The linker may also have different chemical properties. The linker may be selected from acidic, basic or neutral linkers. Typically, acidic linkers comprise one or more acidic amino acids, such as Asp or Glu. Basic linkers typically comprise one or more basic amino acids, such as Arg, His and Lys. Both types of amino acids are very hydrophilic. In some embodiments, the linker is an acidic linker. Examples of acidic linkers comprise the sequence of SEQ ID NO: 22 or SEQ ID NO: 23. In other embodiments, the linker is a basic linker. In other embodiments, the linker is a neutral linker. Examples of neutral linkers comprise the sequence of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26.

In preferred embodiments, the linker is a Gly-Ser or a GlySer-Thr linker consisting of several glycine, serine and, where applicable, threonine residues. In some of these embodiments, the linker comprises the amino acids glycine and serine. In more specific embodiments, the linker comprises the sequence of SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26. In some embodiments, the linker further comprises the amino acid threonine. In a more specific embodiment, the linker comprises the sequence of SEQ ID NO: 21.

In typical embodiments of the present invention, the interferon-associated antigen-binding protein comprises a linker comprising a sequence selected from the sequences SEQ ID NO: 20-26, preferably from the sequences SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26. In a preferred embodiment, the linker comprises the sequence SEQ ID NO: 24. In another preferred embodiment, the linker comprises the sequence SEQ ID NO: 25. In another preferred embodiment, the linker comprises the sequence SEQ ID NO: 26.

In various embodiments of any of the aspects of the invention, the interferon-associated antigen-binding protein does not comprise amino acids other than the amino acids forming (i) said agonist CD40 antibody or agonist antigen-binding fragment thereof and (ii) said IFN or functional fragment thereof. In related embodiments, the interferon-associated antigen-binding protein does not comprise amino acids other than the amino acids forming (i) said agonist CD40 antibody or agonist antigen-binding fragment thereof, (ii) said IFN or functional fragment thereof and (iii) said linker.

Illustrative embodiments representing various configurations of (i) an agonist CD40 antibody or agonist antigen-binding fragment thereof, (ii) an interferon (IFN) or functional fragment thereof, and (iii) a linker are described below.

In some preferred embodiments, the IFN or functional fragment thereof is fused to the C-terminus of the heavy chain of the CD40 agonist antibody or agonist antigen-binding fragment thereof via a linker as set forth in Table 3A or Table 3B. In these embodiments, the heavy chain of the CD40 agonist antibody or agonist antigen-binding fragment thereof may comprise the sequence of SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 48, or SEQ ID NO: 49. IFNa2a may comprise the sequence of SEQ ID NO: 17. IFNe may comprise the sequence of SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. IFNe may comprise the sequence of SEQ ID NO: 14. IFNe_C17S may comprise the sequence of SEQ ID

- 21 047752

NO: 15. IFNe_C17S, N80Q may comprise the sequence of SEQ ID NO: 16. IFNy may comprise the sequence of SEQ ID NO: 19. IFNX2 may comprise the sequence of SEQ ID NO: 18. IFNo: may comprise the sequence of SEQ ID NO: 61. IFNco may comprise the sequence of SEQ ID NO: 60. The mentioned linkers are listed in Table 7.

In embodiments wherein IFN is fused to the C-terminus of the heavy chain of the CD40 agonist antibody or agonist antigen-binding fragment thereof, the interferon-associated antigen-binding protein further comprises a light chain of the CD40 agonist antibody or agonist antigen-binding fragment thereof. In more specific embodiments, the heavy chain comprises the sequence of SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 48, or SEQ ID NO: 49, and the light chain comprises the sequence of SEQ ID NO: 48 or SEQ ID NO: 49. in SEQ ID NO: 3.

Table 3 Interferon or its functional fragment fused to the C-terminus of the heavy chain of an anti-CD40 antibody or its agonist antigen-binding fragment.

A IFNa2a IFNe IFNe_C17S IFNe_C17S, N80Q IFNy IRSH RL linker antiCD40_H C--RL-IFNa2a anraCD40_HC-RL--IFNe antiCD 40_HC--RL-IFNe_C17S antiCD40_HC --RL-IFNe_C17S, N80Q anrnCD40_HC --RL-IFNy aniiiiCD40 H C--RL-IRSH GST linker antiCD40_H C--GST-IFNa2a anraCD40_HC-GST--IFNe antiCD 40_HC-GST-IFNe C17S antiCD40_HC --GST-IFNe_C17S, N80Q anrnCD40_HC --GST--IFNy aniiiiCD40 H C--GST-IRSH HL linker antiCD40_H C--HL-IFNa2a anraCD40_HC-HL--IFNe antiCD 40_HC--HL-IFNe_C17S antiCD40_HC --HL-IFNe_C17S, N80Q anrnCD40_HC --HL-IFNy aniiiiCD40 H C--HL-IRSH HL2 linker antiCD40_H C--HL2-IFNa2a anraCD40_HC-HL2--IFNe antiCD 40_HC--HL2-IFNe_C17S antiCD40_HC --HL2-- IFNe_C17S, N80Q anrnCD40_HC --HL2--IFNy aniiiiCD40 H C--HL2-IRSH (G4S)2 linker antiCD40_H C--(G4S)2IFNa2a anraCD40_HC-(G4S)2-IFNP antiCD 40_HC-(G4S)2-IFNP C17S antiCD40_HC --(G4S)2-IFNe_C17S, N80Q anrnCD40_HC --(G4S)2-IFNy aniiiCD40 H C--(G4S)2-IRS (G4S)3 linker antiCD40_H C--(G4S)3IFNa2a anraCD40_HC-(G4S)3-IFNP antiCD 40_HC-(G4S)3-IFNP C17S antiCD40_HC --(G4S)3-IFNe_C17S, N80Q anrnCD40_HC --(G4S)3-IFNy aniiiCD40 H C--(G4S)3-ШШ2 (G4S)4 linker antiCD40_H C--(G4S)4IFNa2a anraCD40_HC-(G4S)4-IFNe antiCD 40_HC-(G4S)4-IFNe_C17S antiCD40_HC --(G4S)4-IFNe_C17S, N80Q anrnCD40_HC --(G4S)4-IFNy anrnCD40_H C--(G4S)4-ШШ2

B IF№ IFNtt, RL linker antiCD40_HC-RL-IF№ antiCD40_HC--RLIFNtt, GST linker antiCD40_HC--GST-IF№ antiCD40_HC--GST-IFNtt, HL linker antiCD40_HC-HL-IF№ antiCD40_HC--HL-IFNtt, HL2 linker antiCD40_HC-HL2-IF№ antiCD40_HC--HL2-IFNtt, (G4S)2 linker antiCD40_HC-(G4S)2--IFNo. antiCD40_HC-(G4S)2--IFNtt> (G4S)3 linker antiCD40_HC-(G4S)3--IFNo. antiCD40_HC-(G4S)3--IFNtt> (G4S)4 linker antiCD40_HC-(G4S)4--IFNo. antiCD40_HC-(G4S)4--IFNtt>

In some preferred embodiments, the IFN or functional fragment thereof is fused to the N-terminus of the heavy chain of the CD40 agonist antibody or agonist antigen binding fragment thereof via a linker as set forth in Table 4A or Table 4B. In these embodiments, the heavy chain of the CD40 agonist antibody or agonist antigen-binding fragment thereof may comprise the sequence of SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 48, SEQ ID NO: 49, or SEQ ID NO: 50. IFNa2a may comprise the sequence of SEQ ID NO: 17. IFNe may comprise the sequence of SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. IFNe may comprise the sequence of SEQ ID NO: 14. IFNe_C17S may comprise the sequence of SEQ ID NO: 15. IFNe_C17S, N80Q may comprise the sequence of SEQ ID NO: 16. IFNy may comprise the sequence of SEQ ID NO: 19. IF^2 may comprise the sequence of SEQ ID NO: 18. IF#: may contain the sequence SEQ ID NO: 61. IF^ may contain the sequence SEQ ID NO: 60. The mentioned linkers are listed in Table 7.

In embodiments wherein IFN is fused to the N-terminus of the heavy chain of the CD40 agonist antibody or agonist antigen-binding fragment thereof, the interferon-associated antigen-binding protein further comprises a light chain of the CD40 agonist antibody or agonist antigen-binding fragment thereof. In more specific embodiments, the heavy chain comprises the sequence of SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 48, SEQ ID NO: 49, or SEQ ID NO: 50, and the light chain comprises the sequence of SEQ ID NO: 3.

Table 4 Interferon or its functional fragment fused to the N-terminus of the heavy chain of an anti-CD40 antibody or its agonist antigen-binding fragment.

A IFNa2a IFNe IFNe_C17S IFNe_C17S, N80Q IFNy I№ RL linker IFNa2a-RL-antiCD40 _ HC IFNe--RL-aniiCD40 11C IFNe_C17S-RL-antiCD40_HC IFNe_C17S, N80Q--RL-antiCD40_HC IFNY--RL-aniiiCD40 11 C IFNMRL-aniiCD40 11 C GST linker IFNa2a-GST-antiCD40 _ HC IFNe--GST-aniiCD40 11C IFNe_C17S-GST-antiCD40_HC IFNe_C17S, N80Q--GST-antiCD40_HC IFNy-GST-aniiCD40 11 C IFNM- GST-aniiCD40 11 C HL linker IFNa2a-HL-antiCD40 _ HC IFNe--HL-aniiCD40 11C IFNe_C17S-HL-antiCD40_HC IFNe_C17S, N80Q--HL-antiCD40_HC IFNy--HL-aniiCD40 11 C IFNMHL-aniiCD40 11 C HL2 linker IFNa2a-HL2-antiCD40 _ HC IFNe--HL2-aniiCD40 11C IFNe_C17S-- HL2-antiCD40_HC IFNe_C17S, N80Q--HL2-antiCD40_HC IFNy-HL2-aniiCD40 11 C IFNM- HL2-aniiCD40 11 C (G4S) 2 linker IFNa2a-- (G4S)2-- antiCD40 _ HC IFNe-(G4S)2-aniiCD40 11C IFNe_C17S-(G4S)2-antiCD40_HC IFNe_C17S, N80Q--(G4S)2- antiCD40_HC IFNy-(G4S)2-aniiCD40 11 C IFNM(G4S)2aniiiCD40 11 C (G4S) 3 linker IFNa2a-- (G4S)3-- antiCD40 _ HC IFNe-(G4S)3-aniiCD40 11C IFNe_C17S-(G4S)3-antiCD40_HC IFNe_C17S, N80Q--(G4S)3- - antiCD40_HC IFNy-(G4S)3-aniiCD40 11 C IFNM(G4S)3aniiiCD40 11 C (G4S) 4 linker IFNa2a-(G4S)4-antiCD40 _ HC IFNe-(G4S)4-aniiCD40 11C IFNe_C17S-(G4S)4-antiCD40_HC IFNe_C17S, N80Q--(G4S)4- - antiCD40_HC IFNy-(G4S)4-aniiCD40 11 C IFNM(G4S)4aniiiCD40 11 C

B IFN IFNtt, RL linker IFNs-RL-antiCD40 HC IFNo--RL-antiCD40 HC GST linker IFNs--GST— antiCD40_HC IFN<o--GST-antiCD40_HC

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HL linker JFNs-HL-antiCD40 HC IFNo--HL-antiCD40 HC HL2 linker JFNs--HL2-antiCD40 HC IFN<o--HL2-antiCD40 HC (G4S)2 linker JFNs--(G4S)2—antiCD40 HC IFNo--(G4S)2-antiCD40 HC (G4S)3 linker JFNs--(G4S)3—antiCD40 HC IFNo--(G4S)3-antiCD40 HC (G4S)4 linker JFNs--(G4S)4—antiCD40_HC IFNo--(G4S)4-antiCD40_HC

In some preferred embodiments, the IFN is fused to the C-terminus of the light chain of the CD40 agonist antibody or agonist antigen-binding fragment thereof via a linker as set forth in Table 5A or Table 5B. In these embodiments, the light chain of the CD40 agonist antibody or agonist antigen-binding fragment thereof may comprise the sequence of SEQ ID NO: 3. IFNa2a may comprise the sequence of SEQ ID NO: 17. IFNe may comprise the sequence of SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. IFNe may comprise the sequence of SEQ ID NO: 14. IFNe_C17S may comprise the sequence of SEQ ID NO: 15. IFNe_C17S, N80Q may comprise the sequence of SEQ ID NO: 16. IFNy may comprise the sequence of SEQ ID NO: 19. IFNX2 may comprise the sequence of SEQ ID NO: 18. IFNo: may comprise the sequence of SEQ ID NO: 61. IFNio may comprise the sequence of SEQ ID NO: 60. The mentioned linkers are listed in Table 7.

In embodiments wherein IFN is fused to the C-terminus of the light chain of the CD40 agonist antibody or agonist antigen-binding fragment thereof, the interferon-associated antigen-binding protein further comprises the heavy chain of the CD40 agonist antibody or agonist antigen-binding fragment thereof. In more specific embodiments, the light chain comprises the sequence of SEQ ID NO3, and the heavy chain comprises the sequence of SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 49, SEQ ID NO: 48, SEQ ID NO: 50, or SEQ ID NO: 12.

Table 5. Interferon or its functional fragment fused to the C-terminus of the light chain of an anti-CD40 antibody or its agonist antigen-binding fragment.

A IFNa2a IFNe IFNe_C17S IFNe_C17S, N80Q IFNy I№ RL linker antiCD40_L C-RL-- IFNa2a an- iiiCD40 L C--RL-- IFNe antiCD40_L C--RL-IFNe_C17S antiCD40_LC --RL-IFNe_C17S, N80Q aniiiiCD40 L C--RL-IFNy aniiiiCD40 LC --RL--IFW2 GST linker antiCD40_L C--GST- IFNa2a aniiiiCD40 L C--GST-IFNe antiCD40_L C--GST-IFNe_C17S antiCD40_LC --GST— IFNe_C17S, N80Q aniiiiCD40 L C--GST-IFNy an- iiiCD40 LC --GST-I№ HL linker antiCD40_L C--HL-IFNa2a an- iiiCD40 L C--HL-- IFNe antiCD40_L C--HL-IFNe_C17S antiCD40_LC --HL-IFNe_C17S, N80Q aniiiiCD40 L C--HL-IFNy an- iiiCD40 LC --HL-I№ HL2 linker antiCD40_L C--HL2- IFNa2a aniiiiCD40 L C--HL2-IFNe antiCD40_L C--HL2-- IFNe_C17S antiCD40_LC --HL2-- IFNe_C17S, N80Q aniiiiCD40 L C--HL2-IFNy an- iiiCD40 LC --HL2-I№ (G4S)2 linker antiCD40_L C--(G4S)2-IFNa2a aniiiiCD40 L C--(G4S)2-IFNP antiCD40_L C--(G4S)2-IFNe_C17S antiCD40_LC --(G4S)2IFNe_C17S, N80Q aniiiiCD40 L C--(G4S)2-IFNy an- iiiCD40 LC --(G4S)2-I№ (G4S)3 linker antiCD40_L C--(G4S)3-IFNa2a aniiiiCD40 L C--(G4S)3-IFNP antiCD40_L C--(G4S)3-IFNe_C17S antiCD40_LC --(G4S)3IFNe_C17S, N80Q aniiiiCD40 L C--(G4S)3-IFNy anrnCD40_LC --(G4S)3-ШШ2 (G4S)4 linker antiCD40_L C--(G4S)4-IFNa2a aniiiiCD40 L C--(G4S)4-IFNP antiCD40_L C--(G4S)4-IFNe_C17S antiCD40_LC --(G4S)4IFNe_C17S, N80Q aniiiiCD40 L C--(G4S)4-IFNy anrnCD40_LC --(G4S)4-ШШ2

B | IFN | IFNo

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RL linker angiCD40_LC-RL-IFN angiCD40_LC--RL-IFNtt, GST linker angiCD40_LC-GST-IFN antiCD40_LC--GST-IFNtt, HL linker angiCD40_LC-HL-IFN angiCD40_LC--HL-IFNtt, HL2 linker antiCD40_LC-HL2-IFN antiCD40_LC--HL2-IFNtt, (G4S)2 linker antiCD40_LC-(G4S)2--IFN antiCD40_LC-(G4S)2--IFN<o (G4S)3 linker antiCD40_LC-(G4S)3--IFN antiCD40_LC-(G4S)3--IFN<o (G4S)4 linker antiCD40_LC-(G4S)4--IFN antiCD40_LC-(G4S)4--IFN<o

In some preferred embodiments, the IFN is fused to the N-terminus of the light chain of the CD40 agonist antibody or agonist antigen-binding fragment thereof via a linker as set forth in Table 6A or Table 6B. In these embodiments, the light chain of the CD40 agonist antibody or agonist antigen-binding fragment thereof may comprise the sequence of SEQ ID NO: 3. IFNa2a may comprise the sequence of SEQ ID NO: 17. IFNe may comprise the sequence of SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. IFNe may comprise the sequence of SEQ ID NO: 14. IFNe_C17S may comprise the sequence of SEQ ID NO: 15. IFNe_C17S, N80Q may comprise the sequence of SEQ ID NO: 16. IFNy may comprise the sequence of SEQ ID NO: 19. IFNX2 may comprise the sequence of SEQ ID NO: 18. IFNo: may comprise the sequence of SEQ ID NO: 61. IFNio may comprise the sequence of SEQ ID NO: 60. The mentioned linkers are listed in Table 7.

In embodiments wherein IFN is fused to the N-terminus of the light chain of the CD40 agonist antibody or agonist antigen-binding fragment thereof, the interferon-associated antigen-binding protein further comprises the heavy chain of the CD40 agonist antibody or agonist antigen-binding fragment thereof. In more specific embodiments, the light chain comprises the sequence of SEQ ID NO: 3, and the heavy chain comprises the sequence of SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 49, SEQ ID NO: 48, SEQ ID NO: 12, or SEQ ID NO: 50.

Table 6. Interferon or its functional fragment fused to the N-terminus of the light chain of an anti-CD40 antibody or its agonist antigen-binding fragment.

A IFNa2a IFNP IFNP C17S IFNP C17S, N80Q IFNy IRSH RL linker IFNa2a--RL-angiCD40_L C IFNe--RLанiiiCD40 L C IFNe_C17S-RL-antiCD40_L C IFNe_C17S, N80Q--RLangiCD40_LC ZFNy--RL-aniiiCD40 L C IF^2--RLанiiiCD40 LC GST linker IFNa2a-GST-angiCD40_L C IFNe-GST-aniiCD40 L C IFNe_C17S-GST-antiCD40_L C IFNe_C17S, N80Q--GSTangCD40_LC IFNy-GST-aniiCD40 L C IRSH- GST-aniiCD40 LC HL linker IFNa2a--HL-angiCD40_L C IFNe--HLанiiiCD40 L C IFNe_C17S-HL-antiCD40_L C IFNe_C17S, N80Q--HLangiCD40_LC IFNy--HL-aniiCD40 L C IF^2--HLанiiiCD40 LC HL2 linker IFNa2a-HL2-angiCD40_L C IFNe-HL2-aniiCD40 L C IFNe_C17S-HL2-antiCD40_L C IFNe_C17S, N80Q--HL2angiCD40_LC IFNy-HL2-aniiCD40 L C IRSH- HL2-aniiCD40 LC (G4S)2 linker IFNa2a-- (G4S)2— angiCD40_L C IFNe-(G4S)2-aniiCD40 L C IFNe_C17S-(G4S)2-antiCD40_L C IFNe_C17S, N80Q--(G4S)2angiCD40_LC IFNy-(G4S)2-aniiCD40 L C IRSh- (G4S)2-an-iiiCD40 LC

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(G4S)3 linker IFNa2a-- (G4S)3— antiCD40_L C IFNe-(G4S)3-aniiCD40 L C IFNe_C17S-(G4S)3-antiCD40_L C IFNe_C17S, N80Q--(G4S)3antiCD40_LC IFNy-(G4S)3-aniiCD40 L C ShSh- (G4S)3-an- iiiCD40 LC (G4S)4 linker IFNa2a-- (G4S)4— antiCD40_L C IFNe-(G4S)4-aniiCD40 L C IFNe_C17S-(G4S)4-antiCD40_L C IFNe_C17S, N80Q--(G4S)4antiCD40_LC IFNy-(G4S)4-aniiCD40 L C ShSh(G4S)4-aniiiCD40 LC B IFN IFNo RL linker IFNε--RL--antiCD40 LC IFNω--RL--antiCD40 LC GST linker IFNε--GST--antiCD40 LC IFNω--GST--antiCD40 LC HL linker IFNε--HL--antiCD40 LC IFNω--HL--antiCD40 LC HL2 linker IFNε--HL2--antiCD40 LC IFNω--HL2--antiCD40 LC (G4S)2 linker JFNs--(G4S)2antiCD40 LC IFNω--(G4S)2--antiCD40_LC (G4S)3 linker JFNs--(G4S)3antiCD40 LC IFNω--(G4S)3--antiCD40_LC (G4S)4 linker JFNs--(G4S)4antiCD40 _LC IFNω--(G4S)4--antiCD40_LC

Examples of sequences contained in the interferon-associated antigen-binding proteins of the invention or their precursors are listed in Table 7.

In exemplary preferred embodiments, the interferon-associated antigen-binding protein comprises an interferon-fused agonist anti-CD40 antibody or an interferon-fused agonist antigen-binding fragment thereof comprising a sequence selected from SEQ ID NOs: 28-47. In other exemplary embodiments, the interferon-associated antigen-binding protein comprises an interferon-fused agonist anti-CD40 antibody or an interferon-fused agonist antigen-binding fragment thereof comprising a sequence selected from SEQ ID NOs: 62-69. In exemplary preferred embodiments, the interferon-associated antigen-binding protein comprises an interferon-fused agonist anti-CD40 antibody or an interferon-fused agonist antigen-binding fragment thereof comprising a sequence selected from SEQ ID NOs: 28-47. In other exemplary embodiments, the interferon-associated antigen-binding protein is an interferon-fused agonist anti-CD40 antibody or an interferon-fused agonist antigen-binding fragment thereof comprising a sequence selected from SEQ ID NO: 62-69.

In some exemplary embodiments, the interferon-associated antigen-binding protein comprises an interferon-fused anti-CD40 agonist antibody or an interferon-fused agonist binding fragment thereof comprising the sequence of SEQ ID NO: 62. In another exemplary embodiment, the interferon-associated antigen-binding protein is an interferon-fused anti-CD40 agonist antibody or an interferon-fused agonist binding fragment thereof comprising the sequence of SEQ ID NO: 62.

In some exemplary embodiments, the interferon-associated antigen-binding protein comprises an interferon-fused anti-CD40 agonist antibody or an interferon-fused agonist binding fragment thereof comprising the sequence of SEQ ID NO: 63. In another exemplary embodiment, the interferon-associated antigen-binding protein is an interferon-fused anti-CD40 agonist antibody or an interferon-fused agonist binding fragment thereof comprising the sequence of SEQ ID NO: 63.

In some exemplary embodiments, the interferon-associated antigen-binding protein comprises an interferon-fused anti-CD40 agonist antibody or an interferon-fused agonist binding fragment thereof comprising the sequence of SEQ ID NO: 64. In another exemplary embodiment, the interferon-associated antigen-binding protein is an interferon-fused anti-CD40 agonist antibody or an interferon-fused agonist binding fragment thereof comprising the sequence of SEQ ID NO: 64.

In some exemplary embodiments, the interferon-associated antigen-binding protein comprises an interferon-fused agonist CD40 antibody or an interferon-fused

- 26 047752 interferon agonist binding fragment thereof comprising the sequence of SEQ ID NO: 65. In another exemplary embodiment, the interferon-associated antigen-binding protein is an interferon-fused agonist CD40 antibody or an interferon-fused agonist binding fragment thereof comprising the sequence of SEQ ID NO: 65.

In some exemplary embodiments, the interferon-associated antigen-binding protein comprises an interferon-fused anti-CD40 agonist antibody or an interferon-fused agonist binding fragment thereof comprising the sequence of SEQ ID NO: 66. In another exemplary embodiment, the interferon-associated antigen-binding protein is an interferon-fused anti-CD40 agonist antibody or an interferon-fused agonist binding fragment thereof comprising the sequence of SEQ ID NO: 66.

In some exemplary embodiments, the interferon-associated antigen-binding protein comprises an interferon-fused anti-CD40 agonist antibody or an interferon-fused agonist binding fragment thereof comprising the sequence of SEQ ID NO: 67. In another exemplary embodiment, the interferon-associated antigen-binding protein is an interferon-fused anti-CD40 agonist antibody or an interferon-fused agonist binding fragment thereof comprising the sequence of SEQ ID NO: 67.

In some exemplary embodiments, the interferon-associated antigen-binding protein comprises an interferon-fused anti-CD40 agonist antibody or an interferon-fused agonist binding fragment thereof comprising the sequence of SEQ ID NO: 68. In another exemplary embodiment, the interferon-associated antigen-binding protein is an interferon-fused anti-CD40 agonist antibody or an interferon-fused agonist binding fragment thereof comprising the sequence of SEQ ID NO: 68.

In some exemplary embodiments, the interferon-associated antigen-binding protein comprises an interferon-fused agonist anti-CD40 antibody or an interferon-fused agonist binding fragment thereof comprising the sequence of SEQ ID NO: 69. In another exemplary embodiment, the interferon-associated antigen-binding protein is an interferon-fused agonist anti-CD40 antibody or an interferon-fused agonist binding fragment thereof comprising the sequence of SEQ ID NO: 69.

In more preferred embodiments, the interferon-associated antigen-binding protein comprises an interferon-fused agonist CD40 antibody or an interferon-fused agonist antigen-binding fragment thereof comprising a sequence selected from SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43. In more preferred embodiments, the interferon-associated antigen-binding protein is an interferon-fused agonist CD40 antibody or an interferon-fused agonist antigen-binding fragment thereof comprising a sequence selected from SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43.

In an even more preferred embodiment, the interferon-associated antigen-binding protein comprises an interferon-fused agonist CD40 antibody or an interferon-fused agonist binding fragment thereof comprising the sequence of SEQ ID NO: 38. In yet another even more preferred embodiment, the interferon-associated antigen-binding protein is an interferon-fused agonist CD40 antibody or an interferon-fused agonist binding fragment thereof comprising the sequence of SEQ ID NO: 38.

In another even more preferred embodiment, the interferon-associated antigen-binding protein comprises an interferon-fused agonist CD40 antibody or an interferon-fused agonist binding fragment thereof comprising the sequence of SEQ ID NO: 39. In another even more preferred embodiment, the interferon-associated antigen-binding protein is an interferon-fused agonist CD40 antibody or an interferon-fused agonist binding fragment thereof comprising the sequence of SEQ ID NO: 39.

In another even more preferred embodiment, the interferon-associated antigen-binding protein comprises an interferon-fused agonist CD40 antibody or an interferon-fused agonist binding fragment thereof comprising the sequence of SEQ ID NO: 40. In another even more preferred embodiment, the interferon-associated antigen-binding protein is an interferon-fused agonist CD40 antibody or an interferon-fused agonist binding fragment thereof,

- 27 047752 containing the sequence SEQ ID NO: 40.

In another even more preferred embodiment, the interferon-associated antigen-binding protein comprises an interferon-fused agonist CD40 antibody or an interferon-fused agonist binding fragment thereof comprising the sequence of SEQ ID NO: 41. In another even more preferred embodiment, the interferon-associated antigen-binding protein is an interferon-fused agonist CD40 antibody or an interferon-fused agonist binding fragment thereof comprising the sequence of SEQ ID NO: 41.

In another even more preferred embodiment, the interferon-associated antigen-binding protein comprises an interferon-fused agonist CD40 antibody or an interferon-fused agonist binding fragment thereof comprising the sequence of SEQ ID NO: 42. In another even more preferred embodiment, the interferon-associated antigen-binding protein is an interferon-fused agonist CD40 antibody or an interferon-fused agonist binding fragment thereof comprising the sequence of SEQ ID NO: 42.

In another even more preferred embodiment, the interferon-associated antigen-binding protein comprises an interferon-fused agonist CD40 antibody or an interferon-fused agonist binding fragment thereof comprising the sequence of SEQ ID NO: 43. In another even more preferred embodiment, the interferon-associated antigen-binding protein is an interferon-fused agonist CD40 antibody or an interferon-fused agonist binding fragment thereof comprising the sequence of SEQ ID NO: 43.

Table 7. Sequences of an exemplary interferon-associated antigen-binding protein and its components based on the anti-CD40 antibody CP870,893. Sequences in italics correspond to signal peptides. Sequences in bold italics in SEQ ID NOs: 3 and 6 correspond to CDR regions. Sequences in bold, not italics, correspond to linkers. Mutated amino acids are underlined.

Name/SEQ ID number Subsequence Signal peptide 1 (SEQ ID NO: 1) MGWSCIILFLVATATGVHS Signal peptide 2 (SEQ ID NO: 2) MDMRVPAQLLGLLLLWLRGARC antibody against CD40 light chain (SEQ ID NO: 3) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTA STLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVT KSFNRGEC anti-CD40 light chain with signal peptide 1 (SEQ ID NO: 4) MGWSCIILFLVATATGVHSDIQMTQSPSSVSASVGDRVTITCRASQGIYSWL AWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC

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anti-CD40 light chain with signal peptide 2 (SEQ ID NO: 5) MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSVSASVGDRVTITCRASQGI YSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPE DFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVV CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC anti-CD40 antibody heavy chain hIgG2 dK (SEQ ID NO: 6) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWM GWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCAR DQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SS NFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG anti-CD40 antibody heavy chain hIgG2 dK with signal peptide 1 (SEQ ID NO: 7) MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTG YYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTA YMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSAS TKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVE CPPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKG LPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPG anti-CD40 antibody heavy chain hIgG2 dK with signal peptide 2 (SEQ ID NO: 8) MDMRVPAQLLGLLLLWLRGARCQVQLVQSGAEVKKPGASVKVSCKASG YTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDT SISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVT VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG FPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVER KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV QFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKC KVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPG anti-CD40 antibody heavy chain hIgG2 (SEQ ID NO: 9) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWM GWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCAR DQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SS NFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK anti-CD40 antibody heavy chain hIgG2 with signal peptide 1 (SEQ ID NO: 10) MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTG YYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTA YMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSAS TKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVE CPPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKG LPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK

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anti-CD40 antibody heavy chain hIgG2 with signal peptide 2 (SEQ ID NO: 11) MDMRVPAQLLGLLLLWLRGARCQVQLVQSGAEVKKPGASVKVSCKASG YTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDT SISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVT VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG FPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVER KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV QFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKC KVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK antibody against CD40 hIgG1 heavy chain -NNAS (SEQ ID NO: 48) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWM GWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCAR DQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SS SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNNASRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK antibody against CD40 hIgG1 heavy chain -NNASdK (SEQ ID NO: 49) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWM GWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCAR DQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SS SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNNASRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G anti-CD40 antibody hIgG2 Fab region, heavy chain (SEQ ID NO: 12) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWM GWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCAR DQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SS NFGTQTYTCNVDHKPSNTKVDKTVERKCCVE anti-CD40 antibody hIgG2 Fab region, heavy chain with signal peptide 1 (SEQ ID NO: 13) MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTG YYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTA YMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSAS TKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVE antiCD40antibo dy hIgG2, Fab region, heavy chain --TEV--6His fragment (SEQ ID NO: 50) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWM GWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCAR DQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SS NFGTQTYTCNVDHKPSNTKVDKTVERKCCVEENLYFQSHHHHHH IFNe (SEQ ID NO: 14) MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQF QKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVL EEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRN FYFINRLTGYLRN IFNeC17S (SEQ ID NO: MSYNLLGFLQRSSNFQSQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQ KEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLE

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15) EKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNF YFINRLTGYLRN IFNeC17S, N80Q (SEQ ID NO: 16) MSYNLLGFLQRSSNFQSQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQ KEDAALTIYEMLQNIFAIFRQDSSSTGWQETIVENLLANVYHQINHLKTVLE EKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNF YFINRLTGYLRN IFNa2a (SEQ ID NO: 17) CDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAE TIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGV GVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLST NLQESLRSKE IFNX2 (SEQ ID NO: 18) VPVARLHGALPDARGCHIAQFKSLSPQELQAFKRAKDALEESLLLKDCRCH SRLFPRTWDLRQLQVRERPMALEAELALTLKVLEATADTDPALVDVLDQP LHTLHHILSQFRACIQPQPTAGPRTRGRLHHWLYRLQEAPKKESPGCLEAS VTFNLFRLLTRDLNCVASGDLCV ifny (SEQ ID NO: 19) QDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIV SFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVT DLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQ IFNo (SEQ ID NO: 60) KSLFLSTNMQERLRSKDRDLGSS IFN (SEQ ID NO: 61) LDLKLIIFQQRQVNQESLKLLNKLQTLSIQQCLPHRKNFLLPQKSLSPQQYQ KGHTLAILHEMLQQIFSLFRANISLDGWEENHTEKFLIQLHQQLEYLEALM GLEAEKLSGTLGSDNLRLQVKMYFRRIHDYLENQDYSTCAWAIVQVEISRC LFFVFSLTEKLSKQGRPLNDMKQELTTEF RSPR RL linker (SEQ ID NO: 20) PAPA GST linker (SEQ ID NO: 21) SGGTSGSTSGTGS HL linker (SEQ ID NO: 22) AAAAAKEAAAKA HL2 linker (SEQ ID NO: 23) AEAAAKEAAAKAAEAAAKEAAAKA (G4S)2 linker (SEQ ID NO: 24) GGGGSGGGGS (G4S)3 linker (SEQ ID NO: 25) GGGGSGGGGSGGGGS (G4S)4 linker (SEQ ID NO: 26) GGGGSGGGGSGGGGSGGGGS TEV-6His fragment (SEQ ID NO: 27) ENLYFQSHHHHHH antiCD40_LC --HL--IFNe (SEQ ID NO: 28) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTA STLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVT KSFNRGECAEAAAKEAAAKAMSYNLLGFLQRSSNFQCQKLLWQLNGRLE YCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNE TIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHY LKAKEYSHCAWTIVVRVEILRNFYFINRLTGYLRN antiCD40_LC --HL-- IFNP C17S DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTA STLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ

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(SEQ ID NO: 29) SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGECAEAAAKEAAAKAMSYNLLGFLQRSSNFQSQKLLWQLNGRLE YCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNE TIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMS SLHLKRYYGRILHY LKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN anTuCD40_hIgG 2_dK_HC-RL--IFNe (SEQ ID NO: 30) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWM GWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCAR DQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SS NFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPA PAMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQ QFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLAN VYHQINHLKT VLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEIL RNFYFINRLTGYLRN aHTuCD40_hIg G2_dK_HC-RL-IFNe_C17S (SEQ ID NO: 31) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWM GWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCAR DQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SS NFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGPA PAMSYNLLGFLQRSSNFQSQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQ FQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLAN VYHQINHLKTV LEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILR NFYFINRLTGYLRN anTuCD40_hIgG 2_dK_HC-HL--IFNe (SEQ ID NO: 32) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWM GWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCAR DQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SS NFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAE AAAKEAAAKAMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFD IPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTG WNETIVENLLANVY HQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCA WTIVRVEILRNFYFINRLTGYLRN aHTuCD40_hIg G2_dK_HC-HL-IFNe_C17S (SEQ ID NO: 33) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWM GWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCAR DQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SS NFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAE AAAKEAAAKAMSYNLLGFLQRSSNFQSQKLLWQLNGRLEYCLKDRMNFD IPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTG WNETIVENLLANVY HQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCA WTIVRVEILRNFYFINRLTGYLRN antiCD40_LC --RL--IFNe (SEQ ID NO: 34) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTA STLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVT

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KSFNRGECPAPAMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNF DIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANV YHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHC AWTIVRVEILRNFYFINRLTGYLRN aHTuCD40_LC --RL-IFNe_C17S (SEQ ID NO: 35) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTA STLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVT KSFNRGECPAPAMSYNLLGFLQRSSNFQSQKLLWQLNGRLEYCLKDRMNF DIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANV YHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHC AWTIVRVEILRNFYFINRLTGYLRN aHTuCD40_LC --GST-IFNe_C17S (SEQ ID NO: 36) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTA STLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVT KSFNRGECSGGTSGSTSGTGSMSYNLLGFLQRSSNFQSQKLLWQLNGRLEY CLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNET IVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYL KAKEYSHCAWTIVVRVEILRNFYFINRLTGYLRN aHTuCD40_LC --HL2-- IFNe_C17S (SEQ ID NO: 37) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTA STLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVT KSFNRGECAEAAAKEAAAKAAEAAAKEAAAKAMSYNLLGFLQRSSNFQS QKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFA IFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSL HLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN aHTuCD40_hIg G2_dK_HC-(G4S)2-IFNa2a (SEQ ID NO: 38) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWM GWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCAR DQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SS NFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGG GGSGGGGSCDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEF GNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTE LYQQLND LEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRA EIMRSFSLSTNLQESLRSKE aHTuCD40_hIg G2_dK_HC-(G4S)3-IFNa2a (SEQ ID NO: 39) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWM GWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCAR DQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SS NFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGG GGSGGGGSGGGGSCDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGF PQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDK FYTELYQ QLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWE VVRAEIMRSFSLSTNLQESLRSKE aHTuCD40_hIg G2_dK_HC-(G4S)4-IFNa2a (SEQ ID NO: 40) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWM GWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCAR DQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SS NFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ

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VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGG GGSGGGGSGGGGSGGGGSCDLPQTHSLGSRRTLMLLAQMRKISLFSCLKD RHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDE TLLDKF YTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKY SPCAWEVVRAEIMRSFSLSTNLQESLRSKE aHTuCD40_LC --(G4S)2— IFNa2a (SEQ ID NO: 41) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTA STLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVT KSFNRGECGGGGSGGGGSCDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDR HDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFY TELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYS PCAWEVVRAEIMRSSFSLSTNLQESLRSKE aHTuCD40_LC --(G4S)3— IFNa2a (SEQ ID NO: 42) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTA STLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVT KSFNRGECGGGGSGGGGSGGGGSCDLPQTHSLGSRRTLMLLAQMRKISLFS CLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETL LDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLK EKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE aHTuCD40_LC --(G4S)4— IFNa2a (SEQ ID NO: 43) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTA STLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVT KSFNRGECGGGGSGGGGSGGGGSGGGGSCDLPQTHSLGSRRTLMLLAQM RKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSA AWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQ RITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE IFNe--(G4S)3- aHTuCD40_LC ) (SEQ ID NO: 44) MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQF QKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVL EEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRN FYFINRLTGYLRNGGGGSGGGGSGGGGSDIQMTQSPSSVSA SVGDRVTITC RASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC aHTuCD40_LC --(G4S)4—IFNe (SEQ ID NO: 45) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTA STLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVT KSFNRGECGGGGSGGGGSGGGGSGGGGSMSYNLLGFLQRSSNFQCQKLL WQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQ DSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLK RYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN IFNe--(G4S)3- aHTuCD40_HC _IgG1 NNASdK (SEQ ID NO: 46) MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQF QKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVL EEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRN FYFINRLTGYLRNGGGGSGGGGSGGGGSQVQLVQSGAEV KKPGASVKVSC KASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVT MTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNNASRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

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aHTuCD40_HC_IgG1_NNAS_dK--(G4S)4-IFNe (SEQ ID NO: 47) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWM GWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCAR DQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SS SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE EQYNNASRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GGGGGSGGGGSGGGGSGGGGSMSYNLLGFLQRSSNFQCQKLLWQLNGRL EYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQ NIFAIFRQDSSSTGWN ETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILH YLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN aHTuCD40_hIg G2 dK_HC-HL--IFNa2A (SEQ ID NO: 62) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWM GWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCAR DQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SS NFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAE AAAKEAAAKACDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQ EEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFY TELYQQL NDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVV RAEIMRSFSLSTNLQESLRSKE aHTuCD40_LC -derivative-HL--IFNa2A (SEQ ID NO: 63) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTA STLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVT KSFNRGEKSLSLSPGAEAAAKEAAAKACDLPQTHSLGSRRTLMLLAQMRK ISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAW DETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRIT LYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE antiCD40_LC --(G4S)4IFNy (SEQ ID NO: 64) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTA STLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVT KSFNRGECGGGGSGGGGSGGGGSGGGGSQDPYVKEAENLKKYFNAGHSD VADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETI KEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPA AKTGKRKRSQMLFRGRRASQ antiCD40_hIg G2 dK_HC-(G4S)4--IFNy (SEQ ID NO: 65) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWM GWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCAR DQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SS NFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGG GGSGGGGSGGGGSGGGGSQDPYVKEAENLKKYFNAGHSDVADNGTLFLG ILKNWKEESDRKIMQSQIVSFYFKLFKSVKDDQSIQK ETIKEDMNVKFFN SNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQ MLFRGRRASQ

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antiCD40_LC --(G4S)4— (SEQ ID NO: 66) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTA STLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVT KSFNRGECGGGGSGGGGSGGGGSGGGGSVPVARLHGALPDARGCHIAQFK SLSPQELQAFKRAKDALEESLLLKDCRCHSRLFPRTWDLRQLQVRERPMAL EAELALTLKVLEATADTDPALVDVLDQPLHTLHHILSQFRACIQPQPTAGPR TRGRLHHWLYRLQEAPKKESPGCLEASVTFNLFRLLTRDLNCVASGDLCV antiCD40_hIg G2 dK_HC-(G4S)4-I№ (SEQ ID NO: 67) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWM GWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCAR DQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SS NFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGG GGSGGGGSGGGGSGGGGSVPVARLHGALPDARGCHIAQFKSLSPQELQAF KRAKDALEESLLLKDCRCHSRLFPRTWDLRQLQVRERPMALEAELALTLK V LEATADTDPALVDVLDQPLHTLHHILSQFRACIQPQPTAGPRTRGRLHHW LYRLQEAPKKESPGCLEASVTFNLFRLLTRDLNCVASGDLCV antiCD40_LC --(G4S)4IFNo (SEQ ID NO: 68) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTA STLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVT KSFNRGECGGGGSGGGGSGGGGSGGGGSLGCDLPQNHGLLSRNTLVLLHQ MRRISPFLCLKDRRDFRFPQEMVKGSQLQKAHVMSVLHEMLQQIFSLFHTE RSSAAWNMTLLDQLHTGLHQQLQHLETCLLQVVGEGESAGAISSPALTLR RYFQGIRVYLKEKKYSDCAWEVVRMEIMKSLFLSTNMQERLRSKDRDLGS S antiCD40_hIg G2 dK_HC-(G4S)4--IFN (SEQ ID NO: 69) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWM GWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCAR DQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SS NFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGG GGSGGGGSGGGGSGGGGSLDLKLIIFQQRQVNQESLKLLNKLQTLSIQQCL PHRKNFLLPQKSLSPQQYQKGHTLAILHEMLQQIFSLFRAN ISLDGWEENHT EKFLIQLHQQLEYLEALMGLEAEKLSGTLGSDNLRLQVKMYFRRIHDYLEN QDYSTCAWAIVQVEISRCLFFVFSLTEKLSKQGRPLNDMKQELTTEFRSPR

In preferred embodiments, the interferon-associated antigen-binding proteins described herein are interferon fusion proteins comprising polypeptides derived from the polypeptides set forth in Table 8, particularly Table 8A or Table 8B, more particularly Table 8A below, and especially the polypeptides of SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 or SEQ ID NO: 43 above. In preferred embodiments, the interferon-associated antigen-binding proteins described herein are interferon fusion proteins consisting of polypeptides derived from the polypeptides set forth in Table 8, particularly Table 8A or Table 8B, more particularly Table 8A below, and especially from the polypeptides of SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43 above. In more preferred embodiments, the interferon fusion antibody comprises the sequences of SEQ ID NO: 38 and SEQ ID NO: 3. In other more preferred embodiments, the interferon fusion antibody comprises the sequences of SEQ ID NO: 39 and SEQ ID NO: 3. In other more preferred embodiments, the interferon fusion antibody comprises the sequences of SEQ ID NO: 40 and SEQ ID NO: 3. In other more preferred embodiments, the interferon fusion antibody comprises the sequences of SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43 above.

- 36 047752 interferon fused antibody comprises the sequences of SEQ ID NO: 41 and SEQ ID NO: 9. In other more preferred embodiments, the interferon fused antibody comprises the sequences of SEQ ID NO: 42 and SEQ ID NO: 9. In other more preferred embodiments, the interferon fused antibody comprises the sequences of SEQ ID NO: 43 and SEQ ID NO: 9.

Table 8. Combinations of polypeptides found in preferred interferon fusion antigen-binding proteins of the invention, their mean EC50 values for activation of CD40 and IFN pathways, and their productivity (i.e. yield per liter of culture). Each sequence combination, as indicated, is contained in duplicate in the corresponding IFA. SN: supernatant.

A

Interferon-fused antibody (IFA) Combination of sequences CD40 EC50 (ng/ml) IFNe EC ₅₀ (ng/ml) IFNa EC ₅₀ (ng/ml) productivity (mg/l) IFA1 (SEQ ID NO: 28) + (SEQ ID NO: 9) 74.1 1.64 16.7 IFA2 (SEQ ID NO: 29) + (SEQ ID NO: 9) 111 0.14 17.8 IFA8 (SEQ ID NO: 30) + (SEQ ID NO: 3) 39.7 2.9 6.45 IFA9 (SEQ ID NO: 31) + (SEQ ID NO: 3) 42.6 0,7 3,4 IFA10 (SEQ ID NO: 32) + (SEQ ID NO: 3) 26.5 4,5 6.9 IFA11 (SEQ ID NO: 33) + (SEQ ID NO: 3) 42.8 1.78 5.1 IFA12 (SEQ ID NO: 34) + (SEQ ID NO: 9) 105 3.64 21.2 IFA13 (SEQ ID NO: 35) + (SEQ ID NO: 9) 192 0,7 11,5 IFA19 (SEQ ID NO: 36) + (SEQ ID NO: 9) 110 1,3 5,6 IFA20 (SEQ ID NO: 37) + (SEQ ID NO: 9) 182 2.34 4.2 IFA25 (SEQ ID NO: 38) + (SEQ ID NO: 3) 13.3 5.1 21 IFA26 (SEQ ID NO: 39) + (SEQ ID NO: 3) 15.35 4 8.6 IFA27 (SEQ ID NO: 40) + (SEQ ID NO: 3) 17 2,4 9.3 IFA28 (SEQ ID NO: 41) + (SEQ ID NO: 9) 12.8 4,5 75 IFA29 (SEQ ID NO: 42) + (SEQ ID NO: 9) 11,1 2 56.6 IFA30 (SEQ ID NO: 43) + (SEQ ID NO: 9) 11.3 1,6 46.6

- 37 047752

IFA34 (SEQ ID NO: 44) + (SEQ ID NO: 49) Active (SN) Active (SN) No significant productivity IFA35 (SEQ ID NO: 45) + (SEQ ID NO: 49) Active (SN) Active (SN) No significant productivity IFA36 (SEQ ID NO: 46) + (SEQ ID NO: 3) Active (SN) Active (SN) No significant productivity IFA37 (SEQ ID NO: 47) + (SEQ ID NO: 3) Active (SN) Active (SN) No significant productivity

B

Interferon Fusion Antibody (IFA) Combination of sequences CD40 EC50 (ng/ml) IFNa EC50 (ng/ml) EC50 (ng/ml) IFNy EC50 (ng/ml) IFN EC50 (ng/ml) IFNo EC50 (ng/ml) productivity (mg/l) IFA38 (SEQ ID NO: 62) + (SEQ ID NO: 3) 22.7 3.77 1.32 IFA39 (SEQ ID NO: 63) + (SEQ ID NO: 9) 17,5 2.95 1.25 IFA42 (SEQ ID NO: 64) + (SEQ ID NO: 9) 65.6 15.4 0.72 IFA43 (SEQ ID NO: 65) + (SEQ ID NO: 3) 50.8 <0.001 0.55 IFA44 (SEQ ID NO: 66) + (SEQ ID NO: 9) 41.4 0.153 0.91 IFA45 (SEQ ID NO: 67) + (SEQ ID NO: 3) 25.8 <0.00 1 1.09 IFA46 (SEQ ID NO: 68) + (SEQ ID NO: 9) 86.3 0.493 0.89 IFA49 (SEQ ID NO: 69) + (SEQ ID NO: 3) 65.8 78.2 0.61 IFA50 (SEQ ID NO: 41) + (SEQ ID NO: 50) 128 1.36 0.57 IFA51 (SEQ ID NO: 42) + (SEQ ID NO: 50) 123 1.43 0.48

Nucleic acids and expression vectors

In one aspect, a combination of polynucleotides encoding an interferon-associated antigen-binding protein is provided. Methods for producing an interferon-associated antigen-binding protein are also provided, comprising expressing these polynucleotides.

In some embodiments, there is provided a nucleic acid encoding an IFN or a functional fragment thereof fused to an agonist CD40 antibody or an agonist antigen-binding fragment thereof as disclosed herein. In some exemplary embodiments, the nucleic acid encodes an IFN or a functional fragment thereof fused to

- 38 047 752 with an agonistic CD40 antibody or agonistic antigen-binding fragment thereof according to any of SEQ ID NOs: 62-69, or a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding any of these sequences. In some exemplary embodiments, said nucleic acid is at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding any of SEQ ID NOs: 62-69. In preferred embodiments, the nucleic acid encodes an IFN or a functional fragment thereof fused to an agonist CD40 antibody or agonist antigen-binding fragment thereof according to any of SEQ ID NOs: 28-47, or a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding any of these sequences. In even more specific embodiments, said nucleic acid is at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding any of SEQ ID NOs: 28-47.

In those embodiments where the nucleic acid encodes an IFN or a functional fragment thereof fused to the light chain of an agonist CD40 antibody or an agonist antigen-binding fragment thereof, the nucleic acid may further encode the heavy chain of an agonist CD40 antibody or an agonist antigen-binding fragment thereof. In more specific embodiments, the heavy chain of the CD40 agonist antibody or agonist antigen-binding fragment thereof comprises the sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 48, SEQ ID NO: 49, or SEQ ID NO: 50, or a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding any of these sequences. In even more specific embodiments, said nucleic acid is at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 48, SEQ ID NO: 49, or SEQ ID NO: 50.

In those embodiments where the nucleic acid encodes an IFN or a functional fragment thereof fused to a heavy chain of an agonist CD40 antibody or an agonist antigen-binding fragment thereof, the nucleic acid may further encode a light chain of the agonist CD40 antibody or an agonist antigen-binding fragment thereof. In more particular embodiments, the light chain of the agonist CD40 antibody or agonist antigen-binding fragment thereof comprises the sequence of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, or a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding any of these sequences. In even more specific embodiments, said nucleic acid is at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

In some embodiments, the nucleic acids described herein may comprise a sequence encoding a sequence to enhance yield (e.g., a solubility moiety) or to facilitate purification of expressed proteins (i.e., a purification moiety). Purification moieties are known to those skilled in the art and may be selected from glutathione-S-transferase (GST) moieties, maltose binding protein (MBP) moieties, calmodulin binding peptide (CBP) moieties, intein-chitin binding domain (intein-CBD) moieties, streptavidin/biotin-based moieties (such as biotinylation signal peptide (BCCP) moieties, streptavidin-binding peptide (SBP) moieties, His-patch ThioFusion moieties, tandem affinity purification (TAP) moieties, small ubiquitin-like modifier (SUMO) moieties, HaloTag® (Promega) moieties, the Profinity eXact™ System (Bio-Rad). In some embodiments, the purification moiety may be a polyhistidine moiety (e.g., His6, His7, His8, His9- or His10-). In other embodiments, the purification moiety may be a fragment of Strep (e.g., Strep-tag® or a Strep-tag II®; IBA Life Sciences). In other embodiments, the purification moiety may be a fragment of maltose binding protein (MBP).

In some embodiments, the nucleic acid sequence may further comprise a sequence encoding a cleavage site for removing the purification moiety. Such cleavage sequences are known to those skilled in the art and may be selected from sequences recognized and cleaved by an endoprotease or exoprotease. In some embodiments, the endoprotease for removing the purification moiety may be selected from: enteropeptidase, thrombin, factor Xa, TEV protease, or rhinovirus 3C protease. In some variations, the endoprotease may be selected from: enteropeptidase, thrombin, factor Xa, TEV protease, or rhinovirus 3C protease.

- 39 047752 embodiments, the exoprotease for removing the purification moiety may be selected from: carboxypeptidase A, carboxypeptidase B or DAPase. In preferred embodiments, the protease for removing the purification moiety is TEV protease. In a more specific preferred embodiment, the nucleic acid comprises a sequence encoding a His6 moiety and a TEV cleavage site. In an even more specific preferred embodiment, said nucleic acid comprises a sequence encoding SEQ ID NO: 27.

The nucleic acid molecules of the invention may also comprise a sequence encoding a signal peptide. The skilled artisan knows the various signal peptides available to direct the expressed protein to a desired site of folding, assembly and/or maturation, as well as to ensure secretion of the final protein into the medium to facilitate subsequent processing. Thus, in some embodiments, the signal peptide is a secretory signal peptide. The encoded signal peptide may comprise the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the signal peptide comprises the sequence of SEQ ID NO: 1. In other embodiments, the signal peptide comprises the sequence of SEQ ID NO: 2.

Signal peptide 1 (SEQ ID NO: 1) was used to synthesize the polypeptide sequences SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID 36, SEQ ID NO: 37, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 or SEQ ID NO: 50. This signal peptide, which is initially present at the N-terminus of the corresponding polypeptide sequence, is cleaved during synthesis.

Signal peptide 2 (SEQ ID NO: 2) was used to synthesize the polypeptide sequences SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, and SEQ ID NO: 43. This signal peptide, which is initially present at the N-terminus of the corresponding polypeptide sequence, is cleaved during synthesis.

For the synthesis of polypeptide sequences SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69, the signal peptide MGWSCIILFLVATATGVHS (SEQ ID NO: 1) was used. Such a signal peptide, which is initially present at the N-terminus of the corresponding polypeptide sequence, is cleaved during synthesis.

Polynucleotides encoding an IFN or a functional fragment thereof fused to an agonist CD40 antibody or an agonist antigen-binding fragment thereof as disclosed herein are typically inserted into an expression vector for introduction into host cells that can be used to produce a desired amount of the claimed interferon-associated antigen-binding proteins. Accordingly, in some aspects, the invention relates to expression vectors comprising the polynucleotides disclosed herein and to host cells comprising these vectors and the polynucleotides.

The term vector or expression vector is used herein for the purposes of the description and claims to designate vectors used in accordance with the present invention as a vehicle for introducing and expressing a desired gene in a cell. As is known to those skilled in the art, such vectors can be readily selected from the group consisting of plasmids, phages, viruses and retroviruses. In general, vectors compatible with the present invention will contain a selection marker, appropriate restriction sites to facilitate cloning of the desired gene and the ability to be introduced and/or replicated in eukaryotic or prokaryotic cells.

Numerous expression vector systems can be used for the purposes of the present invention. For example, one class of vectors uses DNA elements derived from animal viruses such as bovine papillomavirus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV) or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites. In addition, cells that have integrated the DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. The marker can provide prototrophy to an auxotrophic host, resistance to biocides (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can either be directly linked to the DNA sequence to be expressed or can be introduced into the same cell by cotransformation. Additional elements may also be necessary for optimal mRNA synthesis. These elements may include signal sequences, splicing signals, as well as transcriptional promoters, enhancers and termination signals. In some embodiments, cloned variable region genes, one of which is fused to a gene encoding IFN or a functional fragment thereof, are inserted into an expression vector along with heavy and light chain constant region genes (such as human genes) synthesized as described above.

In other embodiments, the vector system of the invention may comprise more than one vector. In some embodiments, the vector system may comprise a first vector for expressing an IFN or a functional fragment thereof fused to the light chain of an agonist antigen.

- 40 047752 anti-CD40 antibodies or an agonistic antigen-binding fragment thereof, and a second vector for expressing the heavy chain of the anti-CD40 agonistic antibody or an agonistic antigen-binding fragment thereof. Alternatively, such a vector system may comprise a first vector for expressing an IFN or a functional fragment thereof fused to the heavy chain of the anti-CD40 agonistic antibody or an agonistic antigen-binding fragment thereof, and a second vector for expressing the light chain of the anti-CD40 agonistic antibody or an agonistic antigen-binding fragment thereof.

In other embodiments, an interferon-associated antigen-binding protein as described herein can be expressed using polycistronic constructs. In such expression systems, multiple gene products of interest, such as products encoding an IFN or a functional fragment thereof that is fused to the heavy chain of an agonist CD40 antibody or an agonist antigen-binding fragment thereof and encoding the light chain of said antibody, or encoding an IFN or a functional fragment thereof fused to the light chain of an agonist CD40 antibody or an agonist antigen-binding fragment thereof and encoding the heavy chain of said antibody or agonist antigen-binding fragment thereof, can be produced from a single polycistronic construct. These systems advantageously utilize an internal ribosome entry site (IRES) to provide relatively high levels of polypeptides in eukaryotic host cells. Compatible IRES sequences are disclosed in U.S. Pat. 6, 193, 980, which is incorporated herein by reference. Those skilled in the art will appreciate that such expression systems can be used to efficiently produce the full range of polypeptides disclosed in the present application.

More generally, once the vector or DNA sequence encoding the interferon-associated antigen-binding protein of the present invention has been obtained, the expression vector can be introduced into an appropriate host cell. That is, the host cell can be transformed. Introduction of the plasmid into the host cell can be accomplished by a variety of methods well known to those skilled in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus. See, for example, Ridgway, A. A. G. Mammalian Expression Vectors Chapter 24. 2, pp. 470-472 Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, MA 1988). The transformed cells are grown under conditions suitable for the production of light chains and heavy chains and assayed for heavy and/or light chain protein synthesis. Examples of assay methods include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence-activated cell sorter (FACS) analysis, immunohistochemistry, etc.

As used herein, the term transformation should be used in a broad sense to refer to the introduction of DNA into a recipient host cell that alters the genotype and therefore results in a change in the recipient cell.

In the same vein, host cells refer to cells that have been transformed with vectors constructed using recombinant DNA techniques and encoding at least one heterologous gene. In descriptions of methods for isolating polypeptides from recombinant hosts, the terms cell and cell culture are used interchangeably to refer to the source of the interferon-associated antigen-binding protein unless otherwise specified. In other words, isolating a polypeptide from cells may mean either centrifugation of whole cells or a cell culture containing both medium and suspended cells.

In one embodiment, the host cell line used to express the interferon-associated antigen-binding protein is of eukaryotic or prokaryotic origin. As used herein, the term expression may include transcription and translation of more than one polypeptide chain (such as the heavy and light chains of an antibody fragment of the interferon-associated antigen-binding protein) that are associated to form the final interferon-associated antigen-binding protein. In one embodiment, the host cell line used to express the interferon-associated antigen-binding protein is of bacterial origin. In one embodiment, the host cell line is used to express a mammalian interferon-associated antigen-binding protein; those skilled in the art can determine particular host cell lines that are best suited to express the desired gene product. Exemplary host cell lines include, but are not limited to, Horizon's CHO K1 GS knockout, DG44 and DUXB11 (DHFR minus Chinese hamster ovary lines), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (SV40 T-antigen derivative of CVI), R1610 (Chinese hamster fibroblasts), BALBC/3T3 (mouse fibroblasts), HAK (hamster kidney line), SP2/O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), HEK 293 (human kidney cells). In a preferred embodiment, HEK FS S11/254 cells can be used. In another preferred embodiment, Horizon's CHO K1 GS can be used. In one embodiment, the cells

- 41 047752 the host cell line provides altered glycosylation, such as afucosylation, of the antibody expressed therefrom (e.g., PER. C6® (Crucell) or FUT8 knockout CHO cell lines (POTELLIGENTTM cells) (Biowa, Princeton, NJ)). In one embodiment, NS0 cells can be used. Host cell lines are generally available from commercial services, the American Tissue Culture Collection, or the published literature.

In one embodiment, the host used to express the interferon-associated antigen-binding protein is a transgenic non-human animal or a transgenic plant.

The interferon-associated antigen-binding proteins of the present invention can also be produced transgenically by creating a non-human animal (e.g., a mammal) or plant that is transgenic for the sequences of interest and by obtaining therefrom an interferon-associated antigen-binding protein in a reducible form. In connection with transgenic production in mammals, the interferon-associated antigen-binding proteins can be obtained and isolated from the milk of goats, cows, or other mammals. See, for example, U.S. Patent Nos. 5,827,690, 5,756,687, 5,750,172, and 5,741,957. Examples of host plants include Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc. Methods for expressing antibodies in plants, including the description of promoters and vectors, as well as plant transformation, are known in the art. See, e.g., U.S. Patent 6,517,529, incorporated herein by reference. In some embodiments, transgenic non-human animals or transgenic plants are produced by introducing one or more nucleic acid molecules encoding an interferon-associated antigen-binding protein of the invention into an animal or plant using standard transgenic techniques. See Hogan and U.S. Patent 6,417,429. The transgenic cells used to produce the transgenic animal can be embryonic stem cells or somatic cells. Transgenic non-human organisms can be chimeric, non-chimeric heterozygotes, and non-chimeric homozygotes. See, e.g., Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual 2nd ed., Cold Spring Harbor Press (1999); Jackson et al., Mouse Genetics and Transgenics: A Practical Approach, Oxford University Press (2000); and Pinkert, Transgenic Animal Technology: A Laboratory Handbook, Academic Press (1999). In some embodiments, the transgenic non-human animals have a target disruption and a replacement with a targeting construct that encodes the sequence(s) of interest. The interferon-associated antigen-binding proteins can be produced in any transgenic animal. In a preferred embodiment, the non-human animals are mice, rats, sheep, pigs, goats, cattle, or horses. The transgenic non-human animal expresses the interferon-associated antigen-binding proteins in blood, milk, urine, saliva, tears, mucus, and other body fluids.

In vitro production allows for scale-up of production to obtain large quantities of the desired interferon-associated antigen-binding proteins. Methods for culturing mammalian cells under tissue culture conditions are known in the art and include homogeneous suspension culture, such as in an airlift reactor or a continuous stirred tank reactor, or immobilized or mechanically entrapped cell culture, such as in hollow fibers, microcapsules, on agarose microspheres or in ceramic cartridges. If necessary and/or desired, the interferon-associated antigen-binding protein solution can be purified by conventional chromatographic techniques, such as gel filtration, ion exchange chromatography, DEAE cellulose chromatography and/or immunoaffinity chromatography.

One or more genes encoding an interferon-associated antigen-binding protein may also be expressed in non-mammalian cells such as bacteria, yeast, or plant cells. In this regard, it should be understood that various single-celled non-mammalian microorganisms such as bacteria may also be transformed; i.e., those that can be grown in culture or fermented. Bacteria that are susceptible to transformation include members of the Enterobacteriaceae family such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; Streptococcus pneumoniae; and Haemophilus influenzae. It is further understood that when expressed in bacteria, the interferon-associated antigen-binding proteins of the invention or components thereof (i.e., agonistic anti-CD40 antibodies or agonistic antigen-binding fragments thereof and IFN or functional fragments of IFN) may become part of inclusion bodies. The desired interferon-associated antigen-binding proteins may then need to be isolated, optionally also refolded and purified.

In addition to prokaryotes, eukaryotic microbes can also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used eukaryotic microorganism, although a number of other strains are commonly available. For example, the YRp7 plasmid is commonly used for expression in Saccharomyces (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)). This plasmid already contains the TRP1 gene, which is a selection marker for a mutant strain of yeast lacking the ability to

- 42 047752 grow in tryptophan, such as ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). The presence of trp 1 damage as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

Therapeutic vectors

A nucleic acid sequence encoding an interferon-associated antigen-binding protein can be inserted into a vector and used as a therapeutic vector, for example, a vector that expresses the interferon-associated antigen-binding protein of the invention. The construction of suitable functional expression constructs and therapeutic expression vectors is known to those skilled in the art. Thus, in some embodiments, the interferon-associated antigen-binding protein can be administered to a patient by genetic delivery with RNA or DNA sequences, a vector, or a vector system encoding the interferon-associated antigen-binding protein.

Therapeutic vectors can be delivered to a patient, for example, by intravenous injection, topical administration (see U.S. Patent No. 5,328,470), or stereotactic injection (see, for example, Chen et al., PNAS 91:3054-3057 (1994)). A pharmaceutical preparation of a therapeutic vector can include the vector in a suitable diluent.

The nucleic acid encoding the interferon-associated antigen-binding protein or nucleic acids can be included in a gene construct for use as part of a therapeutic protocol for the delivery of nucleic acids encoding the interferon-associated antigen-binding protein. Expression vectors for in vivo transfection and expression of the interferon-associated antigen-binding protein are provided.

Expression constructs of such components may be administered in any biologically effective carrier, such as any formulation or composition capable of effectively delivering the component nucleic acid sequence to cells in vivo, as known to those skilled in the art. Approaches include, but are not limited to, inserting the nucleic acid sequence(s) in question into viral vectors, including, but not limited to, recombinant retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex virus-1, recombinant bacterial or eukaryotic plasmids, and the like.

Retroviral vectors and adeno-associated viral vectors can be used as a recombinant delivery system for transferring exogenous nucleic acid sequences in vivo, particularly to humans. Such vectors provide efficient gene delivery to cells, and the transferred nucleic acids can be stably integrated into the host chromosomal DNA.

The development of specialized cell lines (called packaging cells) that produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses have been characterized for use in gene transfer for gene therapy (for review, see, e.g., Miller, Blood 76:271–78 (1990)). Replication-defective retrovirus can be packaged into virions that can be used to infect a target cell with a helper virus by standard methods. Protocols for producing recombinant retroviruses and infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, et al., (eds.) Greene Publishing Associates, (1989), Sections 9. 10–9. 14 and other standard laboratory manuals. Non-limiting examples of suitable retroviruses include pLJ, pZIP, pWE, and pEM, which are known to those skilled in the art. Examples of suitable packaging virus lineages include *Crip, *Cre, *2, and *Am. (See, for example, Eglitis, et al., Science 230:1395-1398 (1985); Danos and Mulligan, Proc. Natl. Acad. Sci. USA 85:6460-6464 (1988); Wilson, et al., Proc. Natl. Acad. Sci. USA 85:3014-3018 (1988); Armentano, et al., Proc. Nat. l. Acad. Sci. USA 87:6141-6145 (1991); al., Science 254:1802-1805 (1991); van Beusechem, et al., Proc. Natl. Acad. Sci. USA 89:7640-7644 (1992); Kay, et al., Human Gene Therapy 3:641-647 (1992); Dai, et al., Proc. Natl. Acad. Sci. USA 89:10892-10895 (1992); Hwu, et al., J. Immunol. 150:4104-4115 (1993); US Patent No. 4868116; US Patent No. 4980286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT WO application 89/05345; and PCT application WO 92/07573).

In another embodiment, delivery vectors derived from an adenovirus are provided. The adenovirus genome can be manipulated such that it encodes and expresses a gene product of interest, but is inactivated in terms of its ability to replicate in the normal lytic virus life cycle. See, e.g., Berkner, et al., BioTechniques 6:616 (1988); Rosenfeld, et al., Science 252:431-434 (1991); and Rosenfeld, et al., Cell 68:143-155 (1992). Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 d1324 or other adenovirus strains (e.g., Ad2, Ad3, Ad7, etc.) are known to those skilled in the art. Recombinant adenoviruses may be advantageous in certain circumstances because they are unable to infect non-dividing cells and can be used to infect a wide range of cell types, including epithelial cells (Rosenfeld, et al. (1992), supra). In addition, the virus particle is relatively stable and amenable to purification and concentration and, as noted above, can

- 43 047752 be modified to affect the spectrum of infectivity. In addition, the introduced adenoviral DNA (and the foreign DNA it contains) is not integrated into the host cell genome but remains episomal, thereby avoiding potential problems that may arise from in situ insertional mutagenesis when the introduced DNA is integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kbp) compared with other delivery vectors (Berkner et al. (1998), supra; Haj-Ahmand and Graham, J. Virol. 57:267 (1986)).

Another viral vector system suitable for delivery of a nucleic acid sequence encoding an interferon-associated antigen-binding protein is the adeno-associated virus (AAV). AAV is a naturally occurring defective virus that requires another virus, such as adenovirus or herpes virus, as a helper virus for efficient replication and a productive life cycle. (For review, see Muzyczka, et al., Curr. Topics in Micro. and Immunol. 158:97-129 (1992)). It is also one of the few viruses that can integrate its DNA into non-dividing cells and exhibits a high frequency of stable integration (see, e.g., Flotte et al., Am. J. Respir. Cell. Mol. Biol., 7:349-356 (1992); Samulski, et al., J. Virol. 63:3822-3828 (1989); and McLaughlin, et al., J. Virol. 62:1963-1973 (1989)). Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. The space for exogenous DNA is limited to approximately 4.5 kb. An AAV vector such as that described by Tratschin, et al., Mol. Cell. Biol., 5:3251-3260 (1985) can be used to introduce DNA into cells. Various nucleic acids have been introduced into various cell types using AAV vectors (see, e.g., Hermonat, et al., Proc. Natl. Acad. Sci. USA 81:6466–6470 (1984); Tratschin, et al., Mol. Cell. Biol., 4:2072–2081 (1985); Wondisford, et al., Mol. Endocrinol. 2:32–39 (1988); Tratschin, et al., J. Virol. 51:611–619 (1984); and Flotte, et al., J. Biol. Chem. 268:3781–3790 (1993)).

In addition to viral transfer methods, non-viral methods can also be used to induce expression of a nucleic acid sequence encoding an interferon-associated antigen-binding protein in a patient's tissue. Most non-viral gene transfer methods rely on the normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. In some embodiments, non-viral delivery systems rely on endocytic pathways for the uptake of the gene in question by the target cell. Examples of delivery systems of this type include liposome systems, polylysine conjugates, and artificial viral envelopes. Other embodiments include plasmid injection systems such as those described by Meuli, et al., J. Invest. Dermatol. 116 (1): 131-135 (2001); Cohen, et al., Gene Ther 7 (22): 1896-905 (2000); or Tam, et al., Gene Ther. 7 (21): 1867-74 (2000).

In a clinical setting, delivery systems can be administered to a patient by any of a number of methods, each of which is known in the art. For example, a pharmaceutical preparation of the delivery system can be administered systemically, such as by intravenous injection. Specific transduction of the protein into target cells occurs primarily due to the transfection specificity provided by the delivery vehicle, the cell-type or tissue-type expression due to transcriptional regulatory sequences controlling receptor gene expression, or a combination of both. In other embodiments, the initial delivery of the recombinant gene is more limited, with administration to the animal being fairly localized. For example, the delivery vehicle can be administered by catheter (see U.S. Patent 5,328,470) or by stereotactic injection (e.g., Chen, et al., PNAS 91: 3054-3057 (1994)).

The pharmaceutical preparation of the therapeutic construct may consist essentially of the delivery system in a suitable diluent or may comprise a slow-release matrix in which the delivery vehicle is incorporated. Alternatively, when the entire delivery system can be obtained intact from recombinant cells, for example using retroviral vectors, the pharmaceutical preparation may comprise one or more cells that produce the delivery system.

Treatment methods

In one aspect, the invention relates to methods of treating a patient (e.g., a patient infected with HBV), comprising administering an effective amount of an interferon-associated antigen-binding protein or a nucleic acid sequence (e.g., mRNA) that encodes an interferon-associated antigen-binding protein as disclosed herein. The invention also relates to the use of an interferon-associated antigen-binding protein or a nucleic acid sequence (e.g., mRNA) that encodes an interferon-associated antigen-binding protein as disclosed herein in the preparation of a medicament for the treatment of HBV. More particularly, the invention relates to the use of an interferon-associated antigen-binding protein or a nucleic acid sequence (e.g., mRNA) that encodes an interferon-associated antigen-binding protein as disclosed herein in the preparation of a medicament for the treatment of disorders and/or symptoms, such as HBV-associated disorders and/or HBV-associated symptoms. In some embodiments, the present invention relates to kits and methods for treating

- 44 047 752 disorders and/or symptoms, such as HBV-related disorders and/or symptoms associated with HBV, in a mammal in need of such treatment. In some exemplary embodiments, the patient is a human.

The interferon-associated antigen-binding proteins or nucleic acid sequences encoding them of the present invention can be used in a number of different applications. For example, in one embodiment, the subject interferon-associated antigen-binding proteins or nucleic acid sequences encoding them are useful for reducing the release of HBeAg from an HBV-infected cell. In some embodiments, the interferon-associated antigen-binding proteins of the invention reduce the release of HBeAg by primary hepatocytes in vitro by at least 10% at 1 ng/ml, at least 20% at 1 ng/ml, at least 30% at 1 ng/ml, at least 40% at 1 ng/ml, at least 50% at 1 ng/ml, at least 60% at 1 ng/ml, at least 70% at 1 ng/ml, at least 80% at 1 ng/ml, or at least 85% at 1 ng/ml. In some embodiments, the interferon-associated antigen-binding proteins of the invention reduce the release of HBeAg by primary hepatocytes in vitro by at least 12% at 1 ng/ml. In some embodiments, the interferon-associated antigen-binding proteins of the invention reduce the release of HBeAg by primary hepatocytes in vitro by up to 90% at 1 ng/mL. In related embodiments, the interferon-associated antigen-binding protein reduces the release of HBeAg at an EC50 of less than 30 ng/mL, preferably at an EC50 of less than 10 ng/mL, more preferably at an EC50 of less than 1 ng/mL.

In another embodiment, the subject interferon-associated antigen-binding proteins or nucleic acid sequences that encode them are useful for reducing transcription of pgRNA cccDNA in an HBV-infected cell.

In another embodiment, the subject interferon-associated antigen-binding proteins or nucleic acid sequences that encode them are useful for reducing one or more symptoms and/or complications associated with HBV infection, as described herein (below).

In some embodiments, the subject interferon-associated antigen-binding proteins or nucleic acid sequences that encode them are useful for reducing one or more disorders, symptoms and/or complications associated with chronic HBV infection, such as chronic liver inflammation (chronic hepatitis) leading to cirrhosis over several years; hepatocellular carcinoma (HCC); development of membranous glomerulonephritis (MGN); risk of death; acute necrotizing vasculitis (polyarteritis nodosa), membranous glomerulonephritis and papular acrodermatitis of childhood (Gianotti-Crosti syndrome); HBV-associated nephropathy (e.g., membranous glomerulonephritis), immune-mediated hematological disorders (e.g., essential mixed cryoglobulinemia, aplastic anemia); and the like.

In some embodiments, the subject interferon-associated antigen-binding proteins or nucleic acid sequences that encode them are useful for reducing one or more symptoms and/or complications associated with acute HBV infection, such as acute viral hepatitis (which begins with general malaise, loss of appetite, nausea, vomiting, body aches, mild fever, and dark urine, and then progresses to the development of jaundice, fulminant liver failure, and/or serum sickness-like syndrome); loss of appetite; joint and muscle pain; low-grade fever; abdominal pain; nausea; vomiting; jaundice; bloating; and the like.

Accordingly, the present invention also relates to a method of treating one or more disorders, symptoms and/or complications associated with HBV infection in a human or other animal by administering to such human or animal an effective, non-toxic amount of an interferon-associated antigen-binding protein or a nucleic acid sequence that encodes it. A person skilled in the art will be able to determine, using routine experimentation, what effective, non-toxic amount of an interferon-associated antigen-binding protein or a nucleic acid sequence that encodes it will be used to treat HBV infection.

For example, a therapeutically active amount of the interferon-associated antigen-binding protein of the present invention may vary depending on factors such as the stage of the disease (e.g., acute versus chronic), age, sex, medical complications (e.g., HIV co-infection, immunosuppressed conditions or diseases) and weight of the patient, as well as the ability of the interferon-associated antigen-binding protein to elicit a desired response in the patient. The dosage regimen may be adjusted to provide an optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced depending on the severity of the therapeutic situation.

In general, the compositions provided in the present invention can be used for

- 45 047752 prophylactic treatment of uninfected cells or therapeutic treatment of any HBV-infected cells containing an antigen marker that provides targeting of HBV-infected cells by interferon-associated antigen-binding protein.

Pharmaceutical compositions and their administration

In some embodiments, the interferon-associated antigen-binding proteins of the invention or the nucleic acid sequences (including vectors or vector systems) that encode them are included in a pharmaceutical composition. Methods for producing and administering to a patient the interferon-associated antigen-binding proteins or the nucleic acid sequences that encode them of the present invention are well known or can be readily determined by those skilled in the art using this disclosure and knowledge in the art as a guide. The route of administration of the interferon-associated antigen-binding proteins or the nucleic acid sequences that encode them of the present invention may be oral, parenteral, inhalation or topical. The term parenteral as used herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. Although all these forms of administration are clearly considered to be within the scope of the present invention, the form for administration will be a solution for injection, in particular for intravenous or intraarterial injection or drip administration. Typically, a suitable pharmaceutical composition for injection may contain a buffering agent (e.g., acetate, phosphate or citrate buffer), a surfactant (e.g., polysorbate), optionally a stabilizing agent (e.g., human albumin), etc. In some embodiments, the buffering agent is acetate. In another embodiment, the buffering agent is formate. In yet another embodiment, the buffering agent is citrate. In related embodiments, the surfactant may be selected from the list consisting of pluronics, PEG, sorbitan esters, polysorbates, triton, tromethamine, lecithin, cholesterol and tyloxapal. In preferred embodiments, the surfactant is polysorbate. In more preferred embodiments, the surfactant is polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80 or polysorbate 100, preferably polysorbate 20 or polysorbate 80.

In some embodiments, interferon-associated antigen-binding proteins or nucleic acid sequences that encode them can be delivered directly to the site of an adverse cell population (e.g., the liver), thereby increasing the exposure of the therapeutic agent to the affected tissue.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. In the compositions and methods of the present invention, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1 M, such as 0.05 M phosphate buffer or 0.8% saline. Other common parenteral solutions include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate or fixed oils. Intravenous carriers include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, etc. Preservatives and other additives such as antimicrobial agents, antioxidants, chelating agents, inert gases, etc. may also be present. In particular, pharmaceutical compositions suitable for injection include sterile aqueous solutions (where soluble in water) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and must be fluid to the extent that it can be easily drawn into a syringe. It must be stable under the conditions of manufacture and storage and is generally preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants.

Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents will be included in the composition, for example, sugars, polyhydric alcohols such as mannitol, sorbitol or sodium chloride. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

In any case, sterile injectable solutions can be prepared by including

- 46 047 752 of an active compound, such as an interferon-associated antigen-binding protein, or a nucleic acid sequence encoding said interferon-associated antigen-binding protein of the present invention, alone or in combination with other active agents in the required amount in an appropriate solvent with one or a combination of ingredients listed herein, as required, followed by filtered sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, exemplary methods of preparation include vacuum drying and lyophilization to obtain a powder of the active ingredient together with any additional desired ingredient from a previously sterile-filtered solution thereof. The injectable preparations are processed, filled into containers such as ampoules, bags, jars, syringes or vials, and sealed under aseptic conditions according to methods known in the art. In addition, the preparations may be packaged and sold as a kit. Such manufactured articles typically have labels or package inserts indicating that the corresponding compositions are suitable for the treatment of a patient suffering from HBV infection.

Effective doses of the compositions of the present invention for treating the above-described conditions associated with HBV infection vary depending on many different factors, including the route of administration, the target site, the physiological state of the patient, whether the patient is a human or an animal, other medications being administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals, including transgenic mammals, in particular non-human primates, can also be treated. Treatment doses can be titrated using conventional methods known to those skilled in the art to optimize safety and efficacy.

For treatment with an interferon-associated antigen-binding protein, the dosage can range, for example, from about 0.0001 to about 100 mg/kg, more often from about 0.01 to about 5 mg/kg (e.g., about 0.02 mg/kg, about 0.25 mg/kg, about 0.5 mg/kg, about 0.75 mg/kg, about 1 mg/kg, about 2 mg/kg, etc.) of the host body weight. For example, dosages can be about 1 mg/kg body weight or about 10 mg/kg body weight, or in the range of about 1 to about 10 mg/kg, such as at least about 1 mg/kg. Doses intermediate in the above ranges are also within the scope of the present invention. Patients can be administered such doses daily, on alternate days, weekly, or according to any other schedule determined by empirical analysis. Typical treatment involves administration of multiple doses over an extended period of time, such as for at least six months. Additional exemplary treatment regimens include administration about once every two weeks, or about once a month, or about once every 3 to 6 months. Exemplary dosing regimens include about 1 to about 10 mg/kg, or about 15 mg/kg on consecutive days, about 30 mg/kg every other day, or about 60 mg/kg weekly.

The interferon-associated antigen-binding proteins or nucleic acid sequences expressing any of them may be administered repeatedly. The intervals between single doses may be weekly, monthly or yearly. The intervals may also be irregular, as indicated by measuring the levels of interferon-associated antigen-binding proteins or their components in the patient's blood. Alternatively, the interferon-associated antigen-binding proteins or nucleic acid sequences expressing any of them may be administered as a sustained-release formulation, in which case less frequent administration is required. The dosage and frequency vary depending on the half-life of the interferon-associated antigen-binding proteins in the patient.

The term half-life or t1/2 as referred to herein refers to the stability and/or clearance rate of a compound, such as the interferon-associated antigen-binding proteins of the invention. In practice, the half-life of a compound is typically measured in serum and refers to the time after administration when the serum concentration is 50% of the serum concentration at the time of administration. The interferon-associated antigen-binding proteins of the invention are characterized by a long half-life in the serum of mice. In some embodiments, the half-life of the interferon-associated antigen-binding protein is at least 50 hours, at least 60 hours, at least 70 hours, at least 80 hours, at least 90 hours, or at least 100 hours. In some embodiments, the half-life of the interferon-associated antigen-binding protein is at least 100 hours. In preferred embodiments, the half-life of the interferon-associated antigen-binding protein in mice is from 116 to 158 hours.

The half-life of a protein is related to its clearance. The term clearance or clearance rate as used herein refers to the volume of plasma cleared of protein per unit time. The clearance of the interferon-associated antigen-binding proteins of the invention is low. In some

- 47 047752 In some embodiments, the clearance of the interferon-associated antigen-binding protein is less than 10 ml/hr/kg, less than 5 ml/hr/kg, less than 2.5 ml/hr/kg, less than 1 ml/hr/kg, or less than 0.5 ml/hr/kg. In some embodiments, the clearance of the interferon-associated antigen-binding protein is less than 5 ml/hr/kg. In some embodiments, the clearance of the interferon-associated antigen-binding protein is less than 1 ml/hr/kg. In some embodiments, the clearance of the interferon-associated antigen-binding protein in mice is in the range of 0.28-0.49 ml/hr/kg.

The terms volume of distribution, VD, VSS or apparent volume of distribution as used herein refer to the theoretical volume that would be required to contain the total amount of an administered compound, such as an interferon-associated antigen-binding protein of the invention, at the same concentration as in blood plasma and refers to the distribution of said compound between the plasma and the rest of the body following oral or parenteral dosing. In some embodiments, the volume of distribution Vss of the interferon-associated antigen-binding protein is less than 500 ml/kg, less than 400 ml/kg, less than 300 ml/kg, less than 200 ml/kg or less than 100 ml/kg. In some embodiments, the volume of distribution Vss of the interferon-associated antigen-binding protein is less than 100 ml/kg. In some embodiments, the volume of distribution Vss of the interferon-associated antigen-binding protein in mice is from 50 to 98 ml/kg.

Another related pharmacokinetic parameter is systemic exposure. As used herein, the terms systemic exposure, AUC, or area under the curve refer to the integral of the concentration-time curve. Systemic exposure may be represented by plasma (serum or blood) concentrations or AUC of the parent compound and/or metabolite(s). The interferon-associated antigen-binding proteins of the invention circulate in the blood with a higher systemic exposure (AUC(0-inf)) than their parent antibody (CP870,893). In some embodiments, the systemic exposure to the interferon-associated antigen-binding protein is at least 600 μg*hr/mL, at least 700 μg*hr/mL, at least 800 μg*hr/mL, at least 900 μg*hr/mL, or at least 1000 μg*hr/mL, preferably at least 1000 μg*hr/mL. In some embodiments, the systemic exposure to the interferon-associated antigen-binding protein in mice is in the range of 1033 μg*hr/mL to 1793 μg*hr/mL.

As discussed previously, the interferon-associated antigen-binding protein of the present invention can be administered in a pharmaceutically effective amount for the treatment of mammalian diseases in vivo. In this regard, it should be understood that, as disclosed, the interferon-associated antigen-binding protein will be included in the formulation to facilitate administration and ensure stability of the active agent.

The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as saline, non-toxic buffers, preservatives, and the like. A pharmaceutically effective amount of an interferon-associated antigen-binding protein is typically an amount sufficient to mediate one or more of the following: a decrease in the release of HBeAg from an HBV-infected cell; a decrease in the transcription of pgRNA in an HBV-infected cell; and stimulation of the IFN signaling pathway in an infected cell. Of course, the pharmaceutical compositions of the present invention can be administered in one or more doses to provide a pharmaceutically effective amount of an interferon-associated antigen-binding protein.

In accordance with the scope of the present invention, interferon-associated antigen-binding proteins or nucleic acid sequences expressing any of them can be administered to a human or other animal in accordance with the above-mentioned methods of treatment in an amount sufficient to produce a therapeutic effect. Interferon-associated antigen-binding proteins or nucleic acid sequences expressing any of them can be administered to such a human or other animal in a conventional dosage form obtained by combining the interferon-associated antigen-binding proteins or nucleic acid sequences expressing any of them together with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. One skilled in the art will understand that the form and properties of the pharmaceutically acceptable carrier or diluent are determined by the amount of active ingredient with which it is to be combined, the route of administration and other well-known parameters. Those skilled in the art will also appreciate that a mixture comprising one or more of the interferon-associated antigen-binding proteins or nucleic acid sequences expressing any of them described in the present invention may be effective.

It should be understood that the methods described in the present invention are not limited to the specific methods and experimental conditions disclosed herein, as such methods and conditions may vary. It is also understood that the term used in the present application

- 48 047752 minology is intended solely for the purpose of describing specific embodiments and is not intended to be limiting.

In addition, the experiments described herein, unless otherwise indicated, employ conventional molecular and cell biological and immunological techniques known to those skilled in the art. Such techniques are well known to those skilled in the art and are fully described in the literature. See, e.g., Ausubel, et al. ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N. Y. (1987-2008), including all appendices, Molecular Cloning: A Laboratory Manual (Fourth Edition) by M. R. Green and J. Sambrook and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition).

Unless otherwise defined, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In case of any hidden ambiguity, the definitions provided herein take precedence over any dictionary or external definition. Unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular. The use of or means and/or, unless otherwise specified. The use of the term including, as well as other forms such as includes and included, is not limiting. The use of the term containing includes the term consisting of, unless otherwise specified.

In general, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, as well as the hybridization described herein, are well known and widely used in the art. The methods and techniques described herein are generally performed in accordance with conventional methods well known in the art and as described in various general and more specific references that are cited and discussed in the present description unless otherwise indicated. Enzymatic reactions and purification methods are performed in accordance with the manufacturer's specifications, as is customary in the art or as described herein. The nomenclatures used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry, as well as the laboratory procedures and methods described herein, are well known and widely used in the art. Standard methods are used for chemical synthesis, chemical analysis, pharmaceuticals, formulations, and for delivery and treatment of patients.

The section headings used in this specification are for organizational purposes only and are not to be construed as limiting the subject matter described. The contents of articles, patents, and patent applications, and all other documents and electronically accessible information mentioned or cited in this specification are hereby incorporated by reference in their entirety to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any material and information from any such articles, patents, patent applications, or other physical and electronic documents.

Although the present invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention, using the present description as a guide. Having now described certain embodiments in detail, they will be better understood with reference to the following examples, which are included for illustrative purposes only and are not intended to be limiting.

Examples

Example I

Generation of interferon fusion antibodies (IFA) and characterization of reporter cells a - IFA design

Sequence combinations of illustrative IFAs constructed using the CD40 agonist antibody CP870,893 as a scaffold antibody, with the location of the IFN and the nature of the linkers are listed in Table 7 and Table 8. The IFN was fused via a linker at the N- or C-terminal portion of the light chain (LC) or heavy chain (HC) as listed in Table 7. Nucleic acids encoding the HC, LC, or fusions were synthesized with codons optimized for mammalian expression and cloned into a eukaryotic expression vector such as pcDNA3.1 (Invitrogen). Fig. 2A shows an illustrative map of the pcDNA3.1 plasmid encoding SEQ ID NO: 32 under the control of the pCMV promoter.

b - obtaining IFA

Freestyle 293-F cells (Invitrogen) were transiently cotransfected with plasmids encoding both HC and LC at a HC/LC ratio of 4/6. Six days after transfection, the supernatant was collected, centrifuged, and filtered through 0.22 μm filters. Purification was performed in two steps on an AktaExpress chromatography system (GE Healthcare) using a Protein A MabSelect Sure 5 ml 1.6/2.5 cm column (GE Healthcare) at a flow rate of 5 ml/min. Sample binding was performed in

- 49 047752 in D-PBS1X buffer pH 7.5 and eluted with Glycine/HCl 0.1 M pH 3 buffer. The elution peak was saved in a loop and then injected onto a HiTrap 26/10 desalting column (GE Healthcare) at a flow rate of 10 ml/min in D-PBS1X buffer pH 7.5. The elution peak was collected on a 96-well microplate (2 ml fractions). The pool was collected according to the UV peak profile. After filtration on 0.22 µm filters (Sartorius MiniSart), quality control including bacterial endotoxins was performed using Endosafe® nexgen-PTS™ (Charles River), size exclusion chromatography: using a SEC 200 Increase 10/300 column (GE Healthcare) for purity and oligomers, and SDS-PAGE under reducing and non-reducing conditions on the NuPAGE gel system (Invitrogen) in MES SDS running buffer. The yields are listed in Table 8. For some IFAs, the yields were very low. In these cases, CD40 agonist activity and IFN activity were assessed directly using the supernatant containing the IFAs without any further purification.

Analysis of purified IFA on SDS-PAGE under reducing conditions revealed the presence of two major bands corresponding to HC and LC. When IFN (any member of the IFN family) was fused to HC, a shift in its molecular weight was observed, and the same phenomenon was observed for LC fused to any IFN (Fig. 2B).

c - IFA characterization on reporter cells

HEK-Blue™ CD40L cells (InvivoGen cat.#: hkb-cd40) or HEK-Blue™ IFN-α/β cells (InvivoGen, cat.#: hkb-ifnae) were used to monitor, respectively, the activation of the NFkB pathway by CD40 agonists or the IFN pathway induced by type I IFN.

HEK-Blue™ CD40L cells were generated by stably transfecting HEK293 cells with the human CD40 gene and an NFkB-inducible secreted embryonic alkaline phosphatase (SEAP) construct (Invivogen) to measure the biological activity of CD40 agonists. CD40 stimulation results in the induction of NFkB and subsequently the production of SEAP, which is detected in the supernatant using QUANTI-Blue™ (Invivogen, cat.# rep-qbs2).

HEK-Blue™ IFN cells are designed to monitor the activation of the JAK/STAT/ISGF3 pathway induced by type I IFN. Activation of this pathway induces the production and release of SEAP. SEAP levels are readily detectable in the supernatant using QUANTI-Blue™.

HEK-Blue™ IFN-α/β is used to monitor human IFNa or IFNe activity.

Cells were seeded in 96-well plates (50,000 cells/well) and stimulated with the indicated concentration for each IFA or control and incubated at 37°C for 24 hours. Supernatants were then collected and SEAP levels were quantified after supernatant incubation for approximately 30 minutes using QuantiBlue™ and optical density (O.D.) at 620 nm on an Ensight or PheraStar plate reader (Lab Biotech).

HEK-Blue™ Dual IFN-γ cells (InvivoGen, Cat. #: hkb-ifng) or HEK-Blue™ IFN-λ (InvivoGen, Cat. #: hkb-ifnl) can be used to monitor type II and type III interferon activity, respectively. HEK-Blue™ IFN-λ cells are intended to monitor ZEXλ activity. HEK-Blue™ Dual IFN-γ cells detect biologically active human IENy.

d - Functional activity of IFNa/β-based IFA on reporter cells.

Figure 3 shows examples of the dose-dependent effects of IFA where IFNe or its mutant version as indicated in Table 7 was fused to HC as indicated in Table 7 on HEK-Blue™ CD40L cells (Figures 3A-3B) and HEK-Blue™ IFN-α/β cells (Figures 3C-3D). The agonist anti-CD40 activity of the IFA is summarized in Table 8 and examples are shown in Figures 3A and 3B. The results indicate that all IFA tested are able to activate both the CD40 and IFN-α/β pathways in a dose-dependent manner. For fusions to the C-terminus of HC or LC, the EC50 values for agonist CD40 ranged from 11.1 ng/ml to 192 ng/ml (Table 8). The mean EC50 value for the parent antibody was 48 ng/ml and 57 ng/ml in the experiment shown in Fig. 3. IFA with IFN fused to the N-terminus of HC or LC were also able to activate the CD40 pathway, but exact EC50 values could not be determined for these IFAs because activity was determined directly from the supernatant and not using purified proteins (Fig. 3B).

The IFN activity of the different IFAs is summarized in Table 8, and examples are shown in Figs. 3C through 3D. For fusions of IEN[i or mutated IEN[i (as indicated in Table 7) to the C-terminus of HC or LC, the IFN activity varied depending on the linker sequence, with EC50 values ranging from 0.14 ng/ml to 4.5 ng/ml (Fig. 3C and Table 8). Fig. 3D shows that IFAs with IENII fused to the N-terminal portion exhibited high IFN activity. The parental antibody, used as a negative control, showed no activity, whereas recombinant IEN[i did exhibit a strong dose-dependent response. Overall, these results demonstrate that fusions of IEN[i or its mutant version, as indicated in Table 7, with the antibody, regardless of localization, retains both biological functions, although with differences in terms of effectiveness.

Figure 4 shows examples of the dose-dependent response of IEA, where IEN β was fused to HC or LC, as indicated in Table 7, on HEK-Blue™ CD40L cells (Figure 4A and Figure 4C) and HEK-Blue™ IFN-α/β cells (Figure

- 50 047752

4B and Fig. 4D). The results show that all tested IFAs are able to activate both the CD40 and IFNa/β pathways in a dose-dependent manner. Surprisingly, for all IFNa-based IFAs, the potency against the CD40 pathway was reproducibly higher than that of the parent antibody. The EC50 values for the IFNa-based IFAs ranged from 11.1 ng/mL to 22.7 ng/mL, and the EC50 for CP870,893 ranged from 30 ng/mL to 80 ng/mL (mean EC50: 48 ng/mL).

The IFN activity of IFA varied depending on the linker sequence with EC50 values ranging from 1.6 ng/mL to 5.1 ng/mL. In the same assay, pegylated IFNa2a (Pegasys®) also showed dose-dependent activity with an EC50 value of approximately 1 ng/mL.

e- Generation and characterization of Fc-less IFA.

Suitable constructs of the invention may also be interferon-associated antigen-binding proteins lacking the Fc region. A construct encoding the heavy chain of the fab fragment of CP870893 fused to a TEV-His fragment (SEQ ID NO: 50) was constructed and cloned into the expression plasmid pcDNA3. 1. This construct was co-transfected into HEK cells as described previously with LC fused via different linkers to different IFNs such as SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 42 or SEQ ID NO: 43. The proteins and/or supernatants were assessed in reporter cells and/or their effect on HBV infection in PHH. It will be clear to those skilled in the art that the constructs for use in therapy will no longer contain the TEV-His fragment. These constructs are also embodiments of the invention. Interferon-associated antigen binding proteins without the Fc portion will be active against HBV infection. Then, two IFAs were obtained and their functional characterization is described in Example V: IFA50: (SEQ ID NO: 41) + (SEQ ID NO: 50) and IFA51: (SEQ ID NO: 42) + (SEQ ID NO: 50).

EXAMPLE II

Effect of ELISA on HBV-infected primary hepatocytes

II. a - Effect of IFA on HBV infection in primary human hepatocytes

The effect of IFA on HBV infection in primary human hepatocytes (PHH) was investigated. PHH cells were seeded in 96-well plates (70,000 cells/well) in William's E GlutaMAX medium (32551020, Gibco) supplemented with 10% fetal calf serum (FCS) (SH30066.02, Hyclone), insulin (19278-5ML, Sigma), hydrocortisone (H2270-100MG, Sigma), and penicillin/streptomycin (15140, Gibco). After four hours, the cells were washed and the medium was replaced. The following day, the medium was replaced with medium containing Matrigel (0.25 mg/ml; 356231, Corning). Cells were infected 48 hours after seeding at an MOI of 500 to 1000 vge/cell (viral genome equivalent) in InVitroGRO HI medium (Z99009, Bioreclamation IVT) supplemented with 5% FCS, 4% PEG 8000 (81268, Sigma), 2% DMSO (DMSO-100ML, Sigma), and 1% penicillin/streptomycin. Sixteen hours after infection, cells were washed three times with PBS. Four days after infection, cells were left untreated or treated with serially diluted IFA as shown in the figures. Three days after treatment, culture supernatants were collected and stored at -80°C for further protein detection.

II. b. - Evaluation of HBV e-antigen (HBeAg) release

HBV e antigen (HBeAg) levels in cell culture supernatant were measured by ELISA as described by the manufacturer and results were expressed as PEI units (HBeAg CLIA 96T/K: CL0312-2; Autobio) or luminescence.

II. c. - Evaluation of HBV s-antigen (HBsAg) release

Quantification of HBsAg in the supernatant was performed according to the AutoBio HBsAg CLIA kit protocol (#CL0310-2), the main steps of which were as follows: First, the samples were diluted 1/5 in 1XPBS. Then, 50 μl of standards, controls, and diluted samples were added to the wells. 50 μl of Enzyme Conjugate Solution was added to each well, followed by incubation for one hour at 37°C. The plates were then washed six times with 300 μl of the kit wash solution using a plate washer. Then, 50 μl of Substrate Solution (volumetric mixture in reagents A and B) was added to each well and incubation was carried out for 10 minutes in the dark. The plates were then read on a PHERASTAR microplate reader (BMG Labtech) in luminescence mode.

II. d. - pgRNA quantification

Quantitative PCR technique was used to compare the expression level of pgRNA from infected cells treated with the test compounds. pgRNA from infected cells was quantified in 96-well plates using QuantStudio 12K Flex. cDNA was generated by RT reverse transcription followed by qPCR with the TaqMan Fast Virus One-Step Assay (ThermoFisher, Cat# 4444434). The results were AACt processed and normalized to the housekeeping gene GUSB in duplicate. pgRNA was amplified using the following primers and probe: (forward: CCTCACCATACTGCACTCA, reverse: GAGGGAGTTCTTCTTCTAGG, AGTGTGGATTCGCACTCCTCCAGC as probe). The GUSB gene was amplified

- 51 047752 was purified using the Thermo Fisher TaqMan assay (Hs99999908-m1).

II. e - release of CXCL10

CXCL10 release was assessed using an ELISA kit according to the manufacturer's instructions (BioLegend 439904). Samples were diluted 1/50 and assessed for luminescence on an EnSight microplate reader at 450 nm.

II. f - Effect of IFNa/β-based IFA on HBV infection

Several IFAs were tested for their ability to reduce HBeAg secretion following PHH HBV infection. In Fig. 5, an IFA with IFNe or its mutant version fused at the C-terminus of LC was used. The results show that all IFAs tested strongly reduce HBeAg release. Indeed, even at the lowest concentration tested (1 ng/ml), depending on the IFA, inhibition of HBeAg release by 70% to 90% was observed, indicating that they have potent antiviral activity. Notably, 100% inhibition could not be achieved in this experiment, since treatment was started four days after infection, at which time an existing pool of HBeAg (mRNA and protein) is already present in the cell and continues to be produced further.

The effect of IFA fused to IFNa was also tested in HBV-infected PHH. Fig. 6 shows that these IFAs are highly potent against HBV infection with EC50 values ranging from 0.06 ng/mL to 0.2 ng/mL for IFAs with IFNa2a fused at the C-terminus of HC (IFA25: 0.16 ng/mL, IFA26: 0.1 ng/mL; IFA27: 0.06 ng/mL; and IFA38: ~0.2 ng/mL (~2.2 pM); Fig. 6A and Fig. 6C) and from 0.15 ng/mL to 0.36 ng/mL for IFAs with IFNa2a fused at the C-terminus of LC (IFA28: 0.36 ng/mL; IFA29: 0.15 ng/mL; IFA30: 0.31 ng/mL; and IFA39: ~0.3 ng/mL (~3 pM); Fig. 6B and Fig. 6C). To compare the antiviral effect of Pegasys with IFA38 and IFA39, the results are expressed in pM and show that the EC50 for Pegasys is ~250 pM compared to ~2.2 pM for IFA38 and ~3 pM for IFA39, indicating that IFA is much more potent than Pegasys.

II. g - A short course of treatment is sufficient to induce potent antiviral activity

To evaluate the effect of short-term IFA treatment of HBV-infected primary hepatocytes, cells were infected and incubated for 4 days, treated with IFA25, IFA27, IFA28, IFA30 or Pegasys dose-dependently for 24 h, washed and then incubated with fresh medium without any treatment. After 3 days, supernatants were collected to assess the release of HBeAG (Fig. 6E), HBsAG (Fig. 6F) and CXCL10 (Fig. 6H), cells were lysed and RNA was extracted for pgRNA quantification (Fig. 6G). The results show that all IFA tested were able to inhibit HbeAG and HBsAG release as well as pgRNA expression in a dose-dependent manner. Pegasys alone was only able to inhibit HBeAG release and reduce pgRNA levels. In this regard, IFA is at least 2 log more potent than Pegasys in viral parameters. Surprisingly, although all IFA tested showed dose-dependent inhibition of HBsAg release, no reduction was observed with Pegasys even at the highest concentration. Analysis of CXCL10, a biomarker of the IFN pathway, showed that IFA is also much more potent than Pegasys.

Example III

Release of cytokines

III.a - Cytokine release assay (CRA) from human whole blood cells

An ex vivo whole blood cell (WBC) stimulation assay was used to investigate cytokine release following IFA stimulation. Leukocytes were collected from four healthy donors, diluted 1/3 in RPMI1640 (72400-021, Gibco), and distributed into sterile reaction tubes (300 μl). Cells were left unstimulated, stimulated with LPS (LipoPolySaccharide) K12 (tlrl-eklps, Invivogen) at 10 ng/ml as a positive control, or IFA at 1 μg/ml, and incubated for 24 h at 37°C. Supernatants were then collected and frozen at -20°C until the day of analysis.

Human proinflammatory cytokines were analyzed using the MSD multiplex assay (K15067L-4), which measures tumor necrosis factor (TNF)-a, interleukin (IL)-1e, IL-2, IL-6, IL-8, IL-10, IL-12/IL-23p40, and IFNy. MSD plates were analyzed on a 1300 MESO QuickPlex SQ120 (MSD).

Figure 7 shows typical results of in vitro cytokine release assays from human leukocytes, either unstimulated or treated with LPS or IFA1.

Further results of the IFNe-/mutated IFNe- and IFNa-based IFA testing are shown in Tables 9a and 9b. The results show that in all donors, LPS induced very high levels of inflammatory cytokines (IL-1e, TNF-a, IL-6, IL-12p40, and IFNy). It also induced IP10 (CXCL10), which is a biomarker of the IFN pathway, and moderate levels of IL-10. Two IFNe- (Table 9a) and six IFNa- (Table 9b) based IFAs were tested. All of them induced the IP10 biomarker. However, they did not induce IL-10, IL-1 β, and IL-2, and induced only very low to moderate levels of IFNy, IL-6, and TNF-a.

- 52 047752 moderate, indicating a favorable safety profile with respect to the induction of inflammatory cytokines.

Example IV

Pharmacokinetic studies

IV. a - Development of an ELISA assay for the quantitative determination of IFA

For quantitative ELISA, 96-well plates (Maxisorp 96-well plates, THERMO Scientifique; 442404) were coated overnight at 4°C with 100 μl of recombinant human CD40/TNFRSF5 Fc Chimera protein consisting of the extracellular domain of human CD40 fused to the Fc portion of human IgG1 (CD40-Fc; R&D Systems; 1493-CDB-050) at 0.5 μg/ml in sodium carbonate (0.05 M, pH 9.6, C-3041, Sigma). After emptying by inversion, the plates were then incubated for 1 h at 37°C with PBS-0.05% Tween20-1% milk (SIGMA; 70166-500 g) followed by a wash with PBS-0.05% Tween20. Samples and controls (100 μl of 1/2 serial dilutions) were then incubated for 90 min at 37°C, followed by three washes (PBS - 0.05% Tween20) and incubation with HRP-conjugated anti-IgG2 secondary antibody (1/5000, ab99779, Abcam) or HRP-conjugated anti-IFNa antibody (1/1000, eBIOSCIENCE/Invitrogen; BMS216MST) in PBS-0.05% Tween20-1% milk. After three washes with PBS 0.05% Tween2, TMB (tetramethylbenzidine, Tebu Bio; TMBW-1000-01) was added and the plates were incubated for 20 min in the dark. The reaction was stopped by adding 1 M HCl. Plates were read at 450–650 nm using an Ensight plate reader (Perkin Elmer). Pegasys quantification was assessed using similar protocol steps but using human IFNa antibody pairs from eBioscience/Invitrogen. Capture was performed using 100 μl human anti-IFNa antibody (eBioscience/Invitrogen; BMS216MST) at 1 μg/ml in sodium carbonate (0.05 M, pH 9.6, C-3041, Sigma). Detection was achieved using anti-IFNa secondary antibody conjugated to HRP (1/1000, Affymetrix eBioscience/BMS216MST; 15501707) in PBS-0.05% Tween20-1% milk.

IV. b - In vivo bioavailability in mice

To determine PK parameters, CP870,893, IFA25, IFA26, IFA27, IFA28, IFA29, and IFA30 at 0.5 mg/kg and Pegasys at 0.3 mg/kg were administered as an i.v. bolus to male CD1-Swiss mice and blood samples were collected at different time points. Examples of quantification of circulating molecules using the ELISA approach described above and detected with HRP-conjugated anti-IFNa antibody are shown in Figs. 8A and 8B, while examples of quantification detected with HRP-conjugated anti-IgG2 antibody are shown in Fig. 8C; Pegasys quantification is shown in Fig. 8D. In one set of experiments, summarized in Table In Table 10A, the PK parameters for CP870,893 were tested in a 7-day experiment and those for IFA27, IFA29, and IFA30 were tested in a 10-day experiment (IFA27 was quantified using 2 different ELISA approaches). In another set of experiments, summarized in Table 10B, the PK parameters for CP870, 893 and IFA25, IFA26, IFA28, and Pegasys were tested in 21-day experiments (IFA25 was quantified using 2 different ELISA approaches).

Following a short distribution phase, the pharmacokinetic profiles of IFA are characterized by a long serum half-life of 116 to 218 hours (Table 10A and Table 10B). Very similar pharmacokinetic profiles were obtained for the 6 IFA tested with high circulating levels even ten days after single-dose administration. The pharmacokinetic parameters summarized in Table 10A/B show that these IFA unexpectedly circulate in the blood with higher systemic exposure (AUC(0-inf)) ranging from 1033 μg.hr/mL to 2552 μg.hr/mL for IFA compared to 590 or 797 μg.hr/mL, respectively, for the parent antibody CP870,893 (up to 3.2-fold), also reflecting the lower clearance values for IFA. The volume of distribution Vss was low, ranging from 50 to 105 ml/kg, slightly higher than the plasma vascular volume (50 ml/kg) in this species. For all IFA, clearance was estimated to be low (0.28-0.49 ml/h/kg). Interestingly, the clearance of Pegasys (1.4 ml/h/kg) was up to 7-fold higher than that of IFA (e.g. 0.2 ml/h/kg for IFA27), demonstrating higher systemic exposure to IFA.

Example V

V. a - Functional activity of IFA without Fc fragment towards reporter cells and HBV infection

To determine whether the Fc portion of IFA is required for activity, fusions of IFNa with the C-terminal LC portion linked to the HC Fab fragment were designed and produced. IFNa was linked to the LC portion via a (G4S)2 (IFA50) or (G4S)3 (IFA51) linker.

Assessment on HEK-Blue™ CD40L cells showed that these IFAs still exhibited CD40 agonist activity (Fig. 9A) and activated the CD40 pathway with EC50 values of approximately 128 ng/mL (IFA 50) and 123 ng/mL (IFA51), respectively.

Assessment of IFN activity on HEK-Blue™ IFN-a/β cells showed that both IFA tested exhibited IFN activity (Fig. 9B). The EC50 values are shown in Table 8B and were approximately 1.36

- 53,047,752 ng/ml for IFA50 and 1.43 ng/ml for IFA51.

Both IFAs were tested for HBV infection as described previously, and both IFAs exhibited potent antiviral activity with EC50 values of approximately 4.1 pM (IFA50) and 2.7 pM (IFA51), respectively.

V. b - Functional activity of IFN-based IFAs: towards reporter cells and HBV infection.

Fusions of CP870,893 to a third type I interferon (IFNepsilon; IFN:) were also designed and produced. These IFAs were tested in HEK-Blue™ CD40L cells and could be demonstrated to retain CD40 agonist activity. The results for one such IFA (IFA49) are shown in Fig. 10A. Evaluation of HEK-Blue™ hIFN-α/β cells (which are essentially activated by any type I interferon) showed that IFA49 was also able to activate the IFN-I pathway (Fig. 10B). EC50 values are presented in Table 8B. In addition, IFA49 was also tested for HBV infection in primary hepatocytes and showed activity similar to Pegasys (Fig. 10C).

These results demonstrate that IFN-IFA retain both IFN and CD40 agonist activity (i.e., are bifunctional) and have antiviral activity.

V. c - Functional activity of ΓΕΝω-based IFA towards reporter cells and HBV infection

Fusions of CP870,893 to a fourth type I interferon (IFN omega; IFNio) were also designed and produced. These IFAs were tested in HEK-Blue™ CD40L cells and the results showed that they retained CD40 agonist activity. The results for one such IFA (IFA46) are shown in Fig. 11A. Assessment of HEK-Blue™ hIFN-α/β cells (which are essentially activated by any type I interferon) showed that IFA46 was also able to activate the IFN-I pathway (Fig. 11B). EC50 values are presented in Table 8B. In addition, IFA46 was also tested for HBV infection in primary hepatocytes and showed activity similar to Pegasys (Fig. 11C).

These results demonstrate that IFN-containing IFAs retain both IFN and CD40 agonist activity (i.e., are bifunctional) and have antiviral activity.

V. d - Functional activity of IFNy-based IFA on reporter cells in HBV infection

Fusions of CP870,893 to type II interferon (IFN gamma; IFNy) were also designed and produced. Evaluation of these IFAs on HEK-Blue™ CD40L cells demonstrated that they retained CD40 agonist activity regardless of whether IFNy was bound to the C-terminal portion of LC (IFA42) or HC (IFA43) (Fig. 12A). Evaluation of these IFAs on HEK-Blue™-IFNY cells (Fig. 12B) demonstrated that they were also capable of activating the IFNy pathway. IFNy activity differed slightly between IFA42 (EC50: 15 ng/mL) and IFA43 (EC50: < 0.01 ng/mL). EC50 values are presented in Table 8B. In addition, IFA42 and IFA43 were tested in a dose-dependent manner against HBV infection in primary hepatocytes as described previously. The results show that both IFAs reduce HbeAg release in a dose-dependent manner (Fig. 12C), indicating that IFAs with type II IFN are active against HBV infection.

Taken together, these results demonstrate that IFNy IFAs retain both IFN activity and CD40 agonist activity (i.e., are bifunctional) and have antiviral activity.

V. e - Functional activity of IFNA-based IFA on reporter cells and against HBV infection

Fusions of CP870,893 to type III interferon (IFN lambda; IFNA) were also designed and produced. These IFAs were tested in HEK-Blue™ CD40L cells and the results demonstrated that they also retain CD40 agonist activity regardless of whether IFNA was bound to the C-terminal portion of LC (IFA44) or HC (IFA45) (Fig. 13A). Evaluation of these IFAs in HEK-Blue™-IFNA cells showed that they were also able to activate the IFNA pathway (Fig. 13B). EC50 values are presented in Table 8B. These results also demonstrate that IFNA IFAs retain both IFN and CD40 agonist activity (i.e., are bifunctional).

IFA44 and IFA45 were tested at a single dose versus Pegasys against HBV infection in primary hepatocytes as described previously. The results show that both IFA types reduced HbeAg release by 65% and 78%, respectively. Under these conditions, Pegasys inhibited HbeAg release by 81%. These results indicate that type III IFN IFAs are active against HBV infection with EC50 values for both IFAs tested <10 nM (Fig. 13C).

Equivalents

The invention may be embodied in other specific forms without departing from its scope or essential characteristics. The above embodiments are therefore to be considered in all respects as illustrative and not restrictive of the invention. The scope of the invention is therefore indicated in the appended claims rather than in the foregoing description, and all changes coming within the meaning and range of equivalence of the claims are therefore intended to be included in the present description.

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Objects

In view of the above, it should be understood that the present invention also relates to the following objects:

1. An interferon-associated antigen-binding protein comprising (I) an agonist CD40 antibody or agonist antigen-binding fragment thereof, and (II) an interferon (IFN) or a functional fragment thereof, wherein the agonist CD40 antibody or agonist antigen-binding fragment thereof comprises (a) three light chain complementarity determining regions (CDRs) that are at least 90% identical to the sequences CDRL1, CDRL2, and CDRL3 of SEQ ID NO: 3; and three heavy chain CDRs that are at least 90% identical to the sequences CDRH1, CDRH2, and CDRH3 of SEQ ID NO: 6; wherein each CDR is defined according to the Kabat definition, the Chothia definition, the AbM definition, or the Contact CDR definition; preferably wherein each CDR is defined according to the Kabat CDR definition or the Chothia CDR definition;

(b) three light chain complementarity determining regions (CDRs) that are identical to the sequences CDRL1, CDRL2 and CDRL3 of SEQ ID NO: 3; and three heavy chain CDRs that are identical to the sequences CDRH1, CDRH2 and CDRH3 of SEQ ID NO: 6; wherein each CDR is defined according to the Kabat definition, the Chothia definition, the AbM definition or the contact definition of CDRs; preferably wherein each CDR is defined according to the Kabat definition of CDRs or the Chothia definition of CDRs;

(c) a heavy chain or fragment thereof comprising a complementarity determining region (CDR) CDRH1 that is at least 90% identical to SEQ ID NO: 56, CDRH2 that is at least 90% identical to SEQ ID NO: 57, and CDRH3 that is at least 90% identical to SEQ ID NO: 58; and a light chain or fragment thereof comprising CDRL1 that is at least 90% identical to SEQ ID NO: 52, CDRL2 that is at least 90% identical to SEQ ID NO: 53, and CDRL3 that is at least 90% identical to SEQ ID NO: 54;

(d) a heavy chain or fragment thereof comprising a complementarity determining region (CDR) CDRH1 that is identical to SEQ ID NO: 56, CDRH2 that is identical to SEQ ID NO: 57, and CDRH3 that is identical to SEQ ID NO: 58; and a light chain or fragment thereof comprising CDRL1 that is identical to SEQ ID NO: 52, CDRL2 that is identical to SEQ ID NO: 53, and CDRL3 that is identical to SEQ ID NO: 54;

(e) a light chain variable region VL comprising the sequence of SEQ ID NO: 51, or a sequence at least 90% identical thereto; and/or a heavy chain variable region VH comprising the sequence of SEQ ID NO: 55, or a sequence at least 90% identical thereto;

(f) a heavy chain of the Fab region comprising the amino acid sequence of SEQ ID NO: 12, or a sequence at least 90% identical thereto; or (g) a light chain (LC) that comprises the sequence of SEQ ID NO: 3, or a sequence at least 90% identical thereto; and/or a heavy chain (HC) that comprises a sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 49 and SEQ ID NO: 48, or a sequence at least 90% identical thereto.

2. The interferon-associated antigen-binding protein of claim 1, wherein HC comprises the sequence of SEQ ID NO: 6, or a sequence at least 90% identical thereto.

3. The interferon-associated antigen-binding protein of claim 1, wherein HC comprises the sequence of SEQ ID NO: 9, or a sequence at least 90% identical thereto.

4. The interferon-associated antigen-binding protein of claim 1, wherein HC comprises the sequence of SEQ ID NO: 49, or a sequence at least 90% identical thereto.

5. The interferon-associated antigen-binding protein of claim 1, wherein HC comprises the sequence of SEQ ID NO: 48, or a sequence at least 90% identical thereto.

6. An interferon-associated antigen-binding protein according to any of the preceding claims, wherein the IFN or a functional fragment thereof is human interferon.

7. An interferon-associated antigen-binding protein according to any one of the preceding claims, wherein the IFN or a functional fragment thereof is selected from the group consisting of type I IFN, type II IFN and type III IFN, or a functional fragment thereof.

8. An interferon-associated antigen-binding protein according to any one of the preceding claims, wherein the IFN or functional fragment thereof is type I IFN or a functional fragment thereof.

9. The interferon-associated antigen-binding protein of claim 8, wherein the type I IFN or a functional fragment thereof is IFNa, IFNe, IFNω or IFNα or a functional fragment thereof.

10. The interferon-associated antigen-binding protein of claim 8, wherein the type I IFN or its functional

- 55 047752 the functional fragment is IFNa or IFNe or a functional fragment thereof.

11. The interferon-associated antigen-binding protein of claim 8, wherein the type I IFN or a functional fragment thereof is IFNio or a functional fragment thereof.

12. The interferon-associated antigen-binding protein of claim 8, wherein the type I IFN or a functional fragment thereof is IFNα or a functional fragment thereof.

13. An interferon-associated antigen-binding protein according to any one of claims 1 to 6, wherein the IFN or a functional fragment thereof is IFNa, IFNe, IFNy, ΚΗλ, IFNe·) or IFN or a functional fragment thereof.

14. The interferon-associated antigen-binding protein of claim 13, wherein the IFN or functional fragment thereof is IFNa or IFNe or a functional fragment thereof.

15. The interferon-associated antigen-binding protein of claim 14, wherein the IFN or functional fragment thereof is IFNa or a functional fragment thereof.

16. The interferon-associated antigen-binding protein of claim 15, wherein the IFN or a functional fragment thereof is IFNa2a or a functional fragment thereof.

17. The interferon-associated antigen-binding protein of claim 16, wherein IFNa2a comprises the sequence of SEQ ID NO: 17, or a sequence at least 90% identical thereto.

18. The interferon-associated antigen-binding protein of claim 14, wherein the IFN or functional fragment thereof is IFNe or a functional fragment thereof.

19. The interferon-associated antigen-binding protein of claim 18, wherein IFNe comprises the sequence of SEQ ID NO: 14, or a sequence at least 90% identical thereto.

20. The interferon-associated antigen-binding protein of claim 18, wherein IFNe or a functional fragment thereof comprises one or two amino acid substitutions relative to SEQ ID NO: 14, selected from C17S and N80Q.

21. The interferon-associated antigen-binding protein of claim 20, wherein IFNe or a functional fragment thereof comprises an amino acid substitution C17S relative to SEQ ID NO: 14.

22. The interferon-associated antigen-binding protein of claim 21, wherein IFNe comprises the amino acid sequence of SEQ ID NO: 15.

23. The interferon-associated antigen-binding protein of claim 20, wherein IFNe or a functional fragment thereof comprises amino acid substitutions C17S and N80Q relative to SEQ ID NO: 14.

24. The interferon-associated antigen-binding protein of claim 23, wherein IFNe comprises the amino acid sequence of SEQ ID NO: 16.

25. The interferon-associated antigen-binding protein of claim 13, wherein the IFN or a functional fragment thereof is IFNy or a functional fragment thereof.

26. The interferon-associated antigen-binding protein of claim 25, wherein the IFN or functional fragment thereof is IFNy or a functional fragment thereof.

27. The interferon-associated antigen-binding protein of claim 26, wherein IFNy comprises the sequence of SEQ ID NO: 19, or a sequence at least 90% identical thereto.

28. The interferon-associated antigen-binding protein of claim 25, wherein the IFN or a functional fragment thereof is or a functional fragment thereof.

29. The interferon-associated antigen-binding protein of claim 28, wherein or a functional fragment thereof is IFN-2 or a functional fragment thereof.

30. The interferon-associated antigen-binding protein of claim 29, wherein IF^2 comprises the sequence of SEQ ID NO: 18, or a sequence at least 90% identical thereto.

31. The interferon-associated antigen-binding protein of any one of the preceding claims, wherein the IFN or a functional fragment thereof is non-covalently linked to an agonist CD40 antibody or an agonist antigen-binding fragment thereof.

32. The interferon-associated antigen-binding protein of claim 31, wherein the IFN or a functional fragment thereof is non-covalently linked to the agonist CD40 antibody or agonist antigen-binding fragment thereof via ionic, van der Waals and/or hydrogen bond interactions.

33. The interferon-associated antigen-binding protein of any one of claims 1 to 30, wherein the IFN or a functional fragment thereof is covalently linked to an agonist CD40 antibody or an agonist antigen-binding fragment thereof.

34. The interferon-associated antigen-binding protein of claim 33, wherein the IFN or a functional fragment thereof is fused to an agonistic CD40 antibody or an agonistic antigen-binding fragment thereof.

35. The interferon-associated antigen-binding protein of claim 34, wherein the IFN or a functional fragment thereof is fused to the light chain of an agonist antibody against CD40 or an agonist antigen-binding fragment thereof.

36. The interferon-associated antigen-binding protein of claim 35, wherein the IFN or its functional

- 56 047752 the N-terminus of the light chain of an agonist CD40 antibody or an agonist antigen-binding fragment thereof.

37. The interferon-associated antigen-binding protein of claim 35, wherein the IFN or a functional fragment thereof is fused to the C-terminus of the light chain of an agonist antibody against CD40 or an agonist antigen-binding fragment thereof.

38. The interferon-associated antigen-binding protein of claim 34, wherein the IFN or a functional fragment thereof is fused to the heavy chain of an agonist antibody against CD40 or an agonist antigen-binding fragment thereof.

39. The interferon-associated antigen-binding protein of claim 38, wherein the IFN or a functional fragment thereof is fused to the N-terminus of the heavy chain of an agonist antibody against CD40 or an agonist antigen-binding fragment thereof.

40. The interferon-associated antigen-binding protein of claim 38, wherein the IFN or a functional fragment thereof is fused to the C-terminus of the heavy chain of an agonist antibody against CD40 or an agonist antigen-binding fragment thereof.

41. The interferon-associated antigen-binding protein of any one of claims 34-40, wherein the agonist CD40 antibody or agonist antigen-binding fragment thereof and the IFN or functional fragment thereof are fused to each other via a linker.

42. The interferon-associated antigen-binding protein of claim 41, wherein the interferon-associated antigen-binding protein does not comprise amino acids other than the amino acids forming (I) said agonist CD40 antibody or agonist antigen-binding fragment thereof, (II) said IFN or functional fragment thereof, and (III) said linker.

43. The interferon-associated antigen-binding protein of any one of claims 1 to 41, wherein the interferon-associated antigen-binding protein does not comprise amino acids other than the amino acids forming (i) said agonist CD40 antibody or agonist antigen-binding fragment thereof, and (ii) said IFN or functional fragment thereof.

44. The interferon-associated antigen-binding protein of any one of claims 41-42, wherein the linker is a peptide linker.

45. The interferon-associated antigen-binding protein of claim 44, wherein the linker comprises at least 1, at least 2, at least 3, at least 4, or at least 5 amino acids.

46. Interferon-associated antigen-binding protein of at least 4 amino acids.

47. Interferon-associated antigen-binding protein of at least 11 amino acids.

48. Interferon-associated antigen-binding protein of at least 12 amino acids.

49. Interferon-associated antigen-binding protein of at least 13 amino acids.

50. Interferon-associated antigen-binding protein of at least 15 amino acids.

51. Interferon-associated antigen-binding protein of at least 20 amino acids.

52. Interferon-associated antigen-binding protein of at least 21 amino acids.

53. Interferon-associated antigen-binding protein

p.45, where the linker contains

p.45, where the linker contains

p.45, where the linker contains

p.45, where the linker contains

p.45, where the linker contains

p.45, where the linker contains

p.45, where the linker contains

p.45, wherein the linker comprises at least 24 amino acids

54. The interferon-associated antigen-binding protein of claim 44, wherein the linker comprises up to 10, up to 20, up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, or up to 100 amino acids.

55. The interferon-associated antigen-binding protein of claim 54, wherein the linker comprises up to 80 amino acids.

56. The interferon-associated antigen-binding protein of claim 54, wherein the linker comprises up to 40 amino acids.

57. The interferon-associated antigen-binding protein of claim 54, wherein the linker comprises up to 24 amino acids.

58. The interferon-associated antigen-binding protein of claim 54, wherein the linker comprises up to 21 amino acids.

59. The interferon-associated antigen-binding protein of claim 54, wherein the linker comprises up to 20 amino acids.

60. The interferon-associated antigen-binding protein of claim 54, wherein the linker comprises up to 15 amino acids.

61. The interferon-associated antigen-binding protein of claim 54, wherein the linker comprises up to 13 amino acids.

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62. The interferon-associated antigen-binding protein of claim 54, wherein the linker comprises up to 12 amino acids.

63. The interferon-associated antigen-binding protein of claim 54, wherein the linker comprises up to 11 amino acids.

64. The interferon-associated antigen-binding protein of claim 54, wherein the linker comprises up to 4 amino acids.

65. An interferon-associated antigen-binding protein according to any one of claims 44-64, wherein the linker is selected from the group consisting of acidic, basic and neutral linkers.

66. The interferon-associated antigen-binding protein of claim 65, wherein the linker is an acidic linker.

67. The interferon-associated antigen-binding protein of claim 65 or 66, wherein the linker comprises the sequence of SEQ ID NO: 22 or SEQ ID NO: 23.

68. The interferon-associated antigen-binding protein of claim 65, wherein the linker is a basic linker.

69. The interferon-associated antigen-binding protein of claim 65, wherein the linker is a neutral linker.

70. The interferon-associated antigen-binding protein of claim 65 or 69, wherein the linker comprises the sequence of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26.

71. The interferon-associated antigen-binding protein of any one of claims 44-70, wherein the linker is selected from the group consisting of rigid, flexible and helix-forming linkers.

72. The interferon-associated antigen-binding protein of claim 71, wherein the linker is a rigid linker.

73. The interferon-associated antigen-binding protein of claim 71 or 72, wherein the linker comprises the sequence of SEQ ID NO: 20, SEQ ID NO: 22 or SEQ ID NO: 23.

74. The interferon-associated antigen-binding protein of claim 71, wherein the linker is a flexible linker.

75. The interferon-associated antigen-binding protein of claim 71 or 74, wherein the linker comprises the sequence of SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26.

76. The interferon-associated antigen-binding protein of claim 71, wherein the linker is a helix-forming linker.

77. The interferon-associated antigen-binding protein of claim 71 or 76, wherein the linker comprises the sequence of SEQ ID NO: 22 or SEQ ID NO: 23.

78. The interferon-associated antigen-binding protein according to any one of claims 44-66, 68, 69, 71, 72, 74 or 76, wherein the linker comprises the amino acids glycine and serine.

79. The interferon-associated antigen-binding protein of claim 78, wherein the linker comprises the sequence of SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26.

80. The interferon-associated antigen-binding protein according to claim 78, wherein the linker additionally comprises the amino acid threonine.

81. The interferon-associated antigen-binding protein of claim 80, wherein the linker comprises the sequence of SEQ ID NO: 21.

82. The interferon-associated antigen-binding protein of claim 44, wherein the linker comprises a sequence selected from SEQ ID NO: 20-26.

83. The interferon-associated antigen-binding protein of claim 82, wherein the linker comprises a sequence selected from SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26.

84. The interferon-associated antigen-binding protein of claim 83, wherein the linker comprises the sequence of SEQ ID NO: 24.

85. The interferon-associated antigen-binding protein of claim 83, wherein the linker comprises the sequence of SEQ ID NO: 25.

86. The interferon-associated antigen-binding protein of claim 83, wherein the linker comprises the sequence of SEQ ID NO: 26.

87. The interferon-associated antigen-binding protein of any one of claims 41, 42 or 44-86, wherein the IFN or a functional fragment thereof is fused to the C-terminus of the heavy chain of the CD40 agonist antibody or an agonist antigen-binding fragment thereof via a linker from Table 3, in particular from Table 3A or Table 3B, more in particular from Table 3A.

88. The interferon-associated antigen-binding protein of claim 87, wherein the heavy chain of the agonist antibody against CD40 or its agonist antigen-binding fragment comprises the sequence of SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 49, SEQ ID NO: 48 or SEQ ID NO: 12.

89. The interferon-associated antigen-binding protein according to claim 87 or 88, wherein IFNa2a comprises the sequence of SEQ ID NO: 17.

90. The interferon-associated antigen-binding protein of claim 87 or 88, wherein IFNe comprises the sequence of SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

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91. The interferon-associated antigen-binding protein of claim 90, wherein IFNe comprises the sequence of SEQ ID NO: 14.

92. The interferon-associated antigen-binding protein of claim 90, wherein IFNe comprises the sequence of SEQ ID NO: 15.

93. The interferon-associated antigen-binding protein of claim 90, wherein IFNe comprises the sequence of SEQ ID NO: 16.

94. The interferon-associated antigen-binding protein of claim 87 or 88, wherein IFNy comprises the sequence of SEQ ID NO: 19.

95. The interferon-associated antigen-binding protein according to claim 87 or 88, wherein III^2 comprises the sequence SEQ ID NO: 18.

96. The interferon-associated antigen-binding protein of any one of claims 87-95, wherein the interferon-associated antigen-binding protein further comprises a light chain of an agonist antibody against CD40 or an agonist antigen-binding fragment thereof.

97. The interferon-associated antigen-binding protein of claim 96, wherein the light chain comprises the sequence of SEQ ID NO: 3.

98. The interferon-associated antigen-binding protein of any one of claims 41, 42 or 44-86, wherein the IFN or a functional fragment thereof is fused to the N-terminus of the heavy chain of the CD40 agonist antibody or an agonist antigen-binding fragment thereof via a linker from Table 4, in particular from Table 4A or Table 4B, more in particular from Table 4A.

99. The interferon-associated antigen-binding protein of claim 98, wherein the heavy chain of the agonist CD40 antibody or agonist antigen-binding fragment thereof comprises the sequence of SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 48, SEQ ID NO: 49 or SEQ ID NO: 50, preferably the sequence of SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 48 or SEQ ID NO: 49.

100. The interferon-associated antigen-binding protein of claim 98 or 99, wherein IFNa2a comprises the sequence of SEQ ID NO: 17.

101. The interferon-associated antigen-binding protein of claim 98 or 99, wherein IFNe comprises the sequence of SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

102. The interferon-associated antigen-binding protein of claim 101, wherein IFNe comprises the sequence of SEQ ID NO: 14.

103. The interferon-associated antigen-binding protein of claim 101, wherein IFNe comprises the sequence of SEQ ID NO: 15.

104. The interferon-associated antigen-binding protein of claim 101, wherein IFNe comprises the sequence of SEQ ID NO: 16.

105. The interferon-associated antigen-binding protein of claim 98 or 99, wherein IFNy comprises the sequence of SEQ ID NO: 19.

106. The interferon-associated antigen-binding protein according to claim 98 or 99, wherein IF^2 comprises the sequence of SEQ ID NO: 18.

107. The interferon-associated antigen-binding protein of any one of claims 98-106, wherein the interferon-associated antigen-binding protein further comprises a light chain of an agonist antibody against CD40 or an agonist antigen-binding fragment thereof.

108. The interferon-associated antigen-binding protein of claim 107, wherein the light chain comprises the sequence of SEQ ID NO: 3.

109. The interferon-associated antigen-binding protein of any one of claims 41, 42 or 44-86, wherein the IFN or a functional fragment thereof is fused to the C-terminus of the light chain of the CD40 agonist antibody or an agonist antigen-binding fragment thereof via a linker from Table 5, in particular from Table 5A or Table 5B, more in particular from Table 5A.

110. The interferon-associated antigen-binding protein of claim 109, wherein the light chain of the agonist antibody against CD40 or its agonist antigen-binding fragment comprises the sequence of SEQ ID NO: 3.

111. The interferon-associated antigen-binding protein according to claim 109 or 110, wherein IFNa2a comprises the sequence of SEQ ID NO: 17.

112. The interferon-associated antigen-binding protein of claim 109 or 110, wherein IFNe comprises the sequence of SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

113. The interferon-associated antigen-binding protein of claim 112, wherein IFNe comprises the sequence of SEQ ID NO: 14.

114. The interferon-associated antigen-binding protein of claim 112, wherein IFNe comprises the sequence of SEQ ID NO: 15.

115. The interferon-associated antigen-binding protein of claim 112, wherein IFNe comprises the sequence of SEQ ID NO: 16.

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116. The interferon-associated antigen-binding protein according to claim 109 or 110, wherein ZFNy comprises the sequence of SEQ ID NO: 19.

117. The interferon-associated antigen-binding protein of claim 109 or 110, wherein IFNL2 comprises the sequence of SEQ ID NO: 18.

118. The interferon-associated antigen-binding protein of any one of claims 109-117, wherein the interferon-associated antigen-binding protein further comprises the heavy chain of an agonistic antibody against CD40 or an agonistic antigen-binding fragment thereof.

119. The interferon-associated antigen-binding protein of claim 118, wherein the heavy chain of the CD40 agonist antibody comprises the sequence of SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 48, SEQ ID NO: 49 or SEQ ID NO: 50, preferably the sequence of SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 48 or SEQ ID NO: 49.

120. The interferon-associated antigen-binding protein of any one of claims 41, 42 or 44-86, wherein the IFN is fused to the N-terminus of the light chain of the CD40 agonist antibody or agonist antigen-binding fragment thereof via a linker from Table 6, in particular from Table 6A or Table 6B, more in particular from Table 6A.

121. The interferon-associated antigen-binding protein of claim 120, wherein the light chain of the agonist antibody against CD40 or its agonist antigen-binding fragment comprises the sequence of SEQ ID NO: 3.

122. The interferon-associated antigen-binding protein according to claim 120 or 121, wherein IFNa2a comprises the sequence of SEQ ID NO: 17.

123. The interferon-associated antigen-binding protein of claim 120 or 121, wherein IFNe comprises the sequence of SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

124. The interferon-associated antigen-binding protein of claim 123, wherein IFNe comprises the sequence of SEQ ID NO: 14.

125. The interferon-associated antigen-binding protein of claim 123, wherein IFNe comprises the sequence of SEQ ID NO: 15.

126. The interferon-associated antigen-binding protein of claim 123, wherein IFNe comprises the sequence of SEQ ID NO: 16.

127. The interferon-associated antigen-binding protein of claim 120 or 121, wherein IFNy comprises the sequence of SEQ ID NO: 19.

128. The interferon-associated antigen-binding protein according to claim 120 or 121, wherein IFNL2 comprises the sequence of SEQ ID NO: 18.

129. The interferon-associated antigen-binding protein of any one of claims 120-128, wherein the interferon-associated antigen-binding protein further comprises a heavy chain of an antibody against CD40 or an agonist antigen-binding fragment thereof.

130. The interferon-associated antigen-binding protein of claim 129, wherein the heavy chain of the CD40 agonist antibody comprises the sequence of SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 48, SEQ ID NO: 49 or SEQ ID NO: 50, preferably the sequence of SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 48 or SEQ ID NO: 49.

131. The interferon-associated antigen-binding protein of any one of claims 1 to 130, wherein the interferon-associated antigen-binding protein comprises a sequence selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47.

132. The interferon-associated antigen-binding protein of claim 131, wherein the interferon-associated antigen-binding protein comprises a sequence selected from SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43.

133. The interferon-associated antigen-binding protein of claim 131 or 132, wherein the interferon-associated antigen-binding protein is an interferon-fused agonist CD40 antibody or an interferon-fused agonist antigen-binding fragment thereof, comprising one of the combinations of sequences disclosed in Table 8, particularly Table 8A, or Table 8B, more particularly Table 8A.

134. The interferon-associated antigen-binding protein of claim 133, which is an interferon-fused agonist antibody against CD40 or an interferon-fused agonist antigen-binding fragment thereof, comprising the sequences of SEQ ID NO: 38 and SEQ ID NO: 3.

135. The interferon-associated antigen-binding protein of claim 133, which is an interferon-fused agonist antibody against CD40 or an interferon-fused agonist antigen-binding fragment thereof, comprising the sequences of SEQ ID NO: 39 and SEQ ID NO: 3.

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136. The interferon-associated antigen-binding protein of claim 133, which is an interferon-fused agonist antibody against CD40 or an interferon-fused agonist antigen-binding fragment thereof, comprising the sequences of SEQ ID NO: 40 and SEQ ID NO: 3.

137. The interferon-associated antigen-binding protein of claim 133, which is an interferon-fused agonist antibody against CD40 or an interferon-fused agonist antigen-binding fragment thereof, comprising the sequences of SEQ ID NO: 41 and SEQ ID NO: 9.

138. The interferon-associated antigen-binding protein of claim 133, which is an interferon-fused agonist antibody against CD40 or an interferon-fused agonist antigen-binding fragment thereof, comprising the sequences of SEQ ID NO: 42 and SEQ ID NO: 9.

139. The interferon-associated antigen-binding protein of claim 133, which is an interferon-fused agonist antibody against CD40 or an interferon-fused agonist antigen-binding fragment thereof, comprising the sequences of SEQ ID NO: 43 and SEQ ID NO: 9.

140. The interferon-associated antigen-binding protein of any one of claims 1 to 139, wherein the interferon-associated antigen-binding protein activates both the CD40 pathway and the IFN pathway.

141. The interferon-associated antigen-binding protein of claim 140, wherein CD40 activity is determined using a whole blood surface molecule upregulation assay or an in vitro reporter cell assay.

142. The interferon-associated antigen-binding protein of claim 141, wherein CD40 activity is determined using an in vitro reporter cell assay, optionally using HEK-Blue ^™ CD40L cells.

143. The interferon-associated antigen-binding protein of any one of claims 140-142, wherein the interferon-associated antigen-binding protein activates the CD40 pathway with an EC50 of less than 400, 300, 200, 150, 100, 70, 60, 50, 40, 30, 25, 20 or 15 ng/ml.

144. The interferon-associated antigen-binding protein of claim 143, wherein the interferon-associated antigen-binding protein activates the CD40 pathway with an EC50 in the range of 10 to 200 ng/ml.

145. The interferon-associated antigen-binding protein of claim 144, wherein the interferon-associated antigen-binding protein activates the CD40 pathway with an EC50 in the range of 10 to 50 ng/ml, preferably 10 to 30 ng/ml.

146. The interferon-associated antigen-binding protein of any one of claims 140-145, wherein the interferon-associated antigen-binding protein activates the IFN pathway with an EC50 of less than 100, 60, 50, 40, 30, 20, 10, or 1 ng/ml.

147. The interferon-associated antigen-binding protein of any one of claims 140-146, wherein the interferon-associated antigen-binding protein activates the IFN pathway with an EC50 of less than 11 ng/ml, preferably less than 6 ng/ml.

148. The interferon-associated antigen-binding protein of any one of claims 140-147, wherein the IFN pathway is the IFNa, IFNe, IFNi, IFNy, IFNe·) or IFN pathway.

149. The interferon-associated antigen-binding protein of claim 148, wherein the IFNe activity is determined using an in vitro reporter cell assay, optionally using HEK-Blue™ IFN-α/β cells.

150. The interferon-associated antigen-binding protein of claim 148, wherein the IFNa activity is determined using an in vitro reporter cell assay, optionally using HEK-Blue™ IFN-α/β cells.

151. The interferon-associated antigen-binding protein of claim 148, wherein the IFNy activity is determined using an in vitro reporter cell assay, optionally using HEK-Blue™ Dual IFN-γ cells.

152. The interferon-associated antigen-binding protein of claim 148, wherein the IFN-λ activity is determined using an in vitro reporter cell assay, optionally using HEK-Blue™ IFN-λ cells.

153. The interferon-associated antigen-binding protein of any one of the preceding claims, wherein the interferon-associated antigen-binding protein reduces the release of HBeAg by primary hepatocytes in vitro by at least 12% at 1 ng/ml.

154. An interferon-associated antigen-binding protein for use according to claim 153, wherein the interferon-associated antigen-binding protein reduces the release of HBeAg by primary hepatocytes in vitro by up to 90% at 1 ng/ml.

155. The interferon-associated antigen-binding protein of claim 153, wherein the interferon-associated antigen-binding protein reduces the release of HBeAg with an EC50 of less than 30 ng/ml.

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156. The interferon-associated antigen-binding protein of claim 155, wherein the interferon-associated antigen-binding protein reduces the release of HBeAg with an EC50 of less than 10 ng/ml.

157. The interferon-associated antigen-binding protein of claim 156, wherein the interferon-associated antigen-binding protein reduces the release of HBeAg with an EC50 of less than 1 ng/ml.

158. The interferon-associated antigen-binding protein of claim 156, wherein the interferon-associated antigen-binding protein reduces the release of HBeAg with an EC50 of less than 0.1 ng/ml.

159. The interferon-associated antigen-binding protein of any one of the preceding claims, wherein the interferon-associated antigen-binding protein is capable of increasing the expression level of one or more IFN pathway biomarkers in an HBV-infected cell, preferably at least 1.5-fold, more preferably at least 2-fold, most preferably at least 3-fold.

160. The interferon-associated antigen-binding protein of claim 159, wherein the IFN pathway biomarker is a chemokine.

161. The interferon-associated antigen-binding protein of claim 160, wherein the IFN pathway biomarker is the interferon-stimulated gene ISG20.

162. The interferon-associated antigen-binding protein of claim 160, wherein the IFN pathway biomarker is a C-X-C chemokine selected from the group consisting of CXCL9, CXCL10, and CXCL11.

163. The interferon-associated antigen-binding protein of claim 162, wherein the IFN pathway biomarker is CXCL10.

164. The interferon-associated antigen-binding protein of any one of the preceding claims, wherein the interferon-associated antigen-binding protein slightly increases the expression level of one or more of IL10, ΓΒ1β and IL2 in an HBV-infected cell.

165. The interferon-associated antigen-binding protein of any one of the preceding claims, wherein the systemic exposure of the interferon-associated antigen-binding protein is increased compared to the antibody CP870,893, preferably by at least 10%, more preferably by at least 15%, most preferably by at least 25%.

166. The interferon-associated antigen-binding protein of any one of the preceding claims, wherein the systemic exposure of the interferon-associated antigen-binding protein is at least 1000 μg*h/ml.

167. The interferon-associated antigen-binding protein of claim 166, wherein the systemic exposure of the interferon-associated antigen-binding protein is in the range of 1033 μg*hr/ml to 1793 μg*hr/ml.

168. The interferon-associated antigen-binding protein of any one of the preceding claims, wherein the half-life of the interferon-associated antigen-binding protein is at least 100 hours.

169. The interferon-associated antigen-binding protein of claim 168, wherein the half-life of the interferon-associated antigen-binding protein is from 116 to 158 hours.

170. The interferon-associated antigen-binding protein of any one of the preceding claims, wherein the clearance rate of the interferon-associated antigen-binding protein is below 0.5 ml/h/kg.

171. The interferon-associated antigen-binding protein of claim 170, wherein the clearance of the interferon-associated antigen-binding protein is in the range of 0.28 to 0.49 ml/h/kg.

172. The interferon-associated antigen-binding protein according to any one of the preceding claims, wherein the volume of distribution Vss of the interferon-associated antigen-binding protein is less than 100 ml/kg.

173. The interferon-associated antigen-binding protein according to claim 172, wherein the volume of distribution Vss of the interferon-associated antigen-binding protein is in the range of 50 to 98 ml/kg.

174. The interferon-associated antigen-binding protein of any one of the preceding claims, wherein the interferon-associated antigen-binding protein is suitable for administration to a patient via genetic delivery using nucleic acid sequences encoding the interferon-associated antigen-binding protein, or a vector or vector system encoding the interferon-associated antigen-binding protein.

175. A nucleic acid encoding an interferon-associated antigen-binding protein according to any one of the preceding claims.

176. A nucleic acid encoding IFN or a functional fragment thereof fused to an agonistic CD40 antibody or an agonistic antigen-binding fragment thereof according to any one of claims 34-174.

177. The nucleic acid of claim 176, encoding an IFN or a functional fragment thereof fused to the light chain of an agonist antibody against CD40 or an agonist antigen-binding fragment thereof, and further encoding the heavy chain of an agonist antibody against CD40 or an agonist antigen-binding fragment thereof.

178. The nucleic acid of claim 177, wherein the heavy chain of the agonist antibody against CD40 or its agonist antigen-binding fragment comprises the sequence of SEQ ID NO: 6, SEQ

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ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 48, SEQ ID NO: 49 or SEQ ID NO: 50, or a nucleic acid sequence that is at least 95% identical to a nucleic acid encoding any of these sequences.

179. The nucleic acid of claim 176, encoding IFN or a functional fragment thereof fused to the heavy chain of an agonistic CD40 antibody or an agonistic antigen-binding fragment thereof, and additionally encoding the light chain of an agonistic CD40 antibody or an agonistic antigen-binding fragment thereof.

180. The nucleic acid of claim 179, wherein the light chain of the agonist CD40 antibody or agonist antigen-binding fragment thereof comprises the sequence of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, or a nucleic acid sequence that is at least 95% identical to a nucleic acid encoding any of these sequences.

181. A nucleic acid encoding an IFN or a functional fragment thereof fused to an agonist CD40 antibody or an agonist antigen-binding fragment thereof of any of the sequences of SEQ ID NO: 28-47, or a nucleic acid sequence that is at least 95% identical to a nucleic acid encoding any of these sequences.

182. The nucleic acid of claims 175-181, wherein the nucleic acid additionally encodes a fragment for purification.

183. The nucleic acid of claim 182, wherein the fragment for purification is a His fragment.

184. The nucleic acid of claim 182 or 183, wherein the nucleic acid further encodes a cleavage site for cleavage of the fragment for purification.

185. The nucleic acid of claim 184, wherein the nucleic acid comprises a sequence encoding the amino acid sequence of SEQ ID NO: 27.

186. The nucleic acid of claims 175-185, wherein the nucleic acid additionally encodes a signal peptide.

187. The nucleic acid of claim 186, wherein the signal peptide is a secretory signal peptide.

188. The nucleic acid of claim 186 or 187, wherein the signal peptide comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

189. The nucleic acid of claim 188, wherein the signal peptide comprises the sequence SEQ ID NO: 1.

190. The nucleic acid according to claim 188, wherein the signal peptide comprises the sequence SEQ ID NO: 2.

191. A vector or vector system containing a nucleic acid according to any one of claims 175-190.

192. A vector system comprising (I) a first vector comprising a nucleic acid encoding an IFN or a functional fragment thereof fused to the light chain of an agonist CD40 antibody or an agonist antigen-binding fragment thereof of an interferon-associated antigen-binding protein according to any one of claims 1 to 174, and a second vector comprising a nucleic acid encoding the heavy chain of an agonist CD40 antibody or an agonist antigen-binding fragment thereof of an interferon-associated antigen-binding protein according to any one of claims 1 to 174; or (II) a first vector comprising a nucleic acid encoding an IFN or a functional fragment thereof fused to the heavy chain of an agonist CD40 antibody or an agonist antigen-binding fragment thereof of an interferon-associated antigen-binding protein according to any one of claims 1 to 174, and a second vector comprising a nucleic acid encoding the light chain of an agonist CD40 antibody or an agonist antigen-binding fragment thereof of an interferon-associated antigen-binding protein according to any one of claims 1 to 174.

193. A vector system comprising (I) a first vector according to claim 191 for expressing an IFN or a functional fragment thereof fused to a light chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof, and a second vector for expressing a heavy chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof; or (II) a first vector according to claim 191 for expressing an IFN or a functional fragment thereof fused to a heavy chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof, and a second vector for expressing a light chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof.

194. A composition comprising an interferon-associated antigen-binding protein according to any one of claims 1-174, a nucleic acid according to any one of claims 175-190, a vector according to claim 191 or a vector system according to any one of claims 191-193.

195. The composition according to claim 194, wherein the composition is a pharmaceutical composition.

196. The composition according to claim 195, wherein the pharmaceutical composition is suitable for oral, parenteral or topical administration or for administration by inhalation.

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197. The composition according to claim 196, wherein the pharmaceutical composition is suitable for oral administration.

198. The composition according to claim 196, wherein the pharmaceutical composition is suitable for topical application.

199. The composition according to claim 196, wherein the pharmaceutical composition is suitable for administration by inhalation.

200. The composition according to claim 196, wherein the pharmaceutical composition is suitable for parenteral administration.

201. The composition according to claim 200, wherein the pharmaceutical composition is suitable for intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration.

202. The composition according to claim 201, wherein the pharmaceutical composition is suitable for injection, preferably for intravenous or intraarterial injection or drip administration.

203. A composition according to any one of claims 195-202, comprising at least one buffering agent.

204. The composition according to claim 203, wherein the buffering agent is acetate, formate or citrate.

205. The composition of claim 204, wherein the buffering agent is acetate.

206. The composition according to claim 204, wherein the buffering agent is formate.

207. The composition according to claim 204, wherein the buffering agent is citrate.

208. The composition according to any one of claims 195-207, wherein the pharmaceutical composition contains a surfactant.

209. The composition according to claim 208, wherein the surfactant is selected from the list containing pluronics, PEG, sorbitan esters, polysorbates, triton, tromethamine, lecithin, cholesterol and tyloxapal.

210. The composition according to claim 209, wherein the surfactant is a polysorbate.

211. The composition of claim 210, wherein the surfactant is polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, or polysorbate 100.

212. The composition according to claim 211, wherein the surfactant is polysorbate 20.

213. The composition according to claim 211, wherein the surfactant is polysorbate 80.

214. A composition according to any one of claims 195-213, comprising a stabilizing agent, wherein the stabilizing agent is optionally albumin.

215. A host cell comprising a nucleic acid according to any one of claims 175-190, a vector according to claim 191, or a vector system according to any one of claims 191-193.

216. A method for producing an interferon-associated antigen-binding protein according to any one of claims 1-174, comprising culturing a host cell according to claim 215 and isolating said interferon-associated antigen-binding protein.

217. A transgenic non-human animal or transgenic plant comprising a nucleic acid according to any one of claims 175-190, a vector according to claim 191 or a vector system according to any one of claims 191-193, wherein the transgenic non-human animal or transgenic plant expresses said nucleic acid.

218. A method for producing an interferon-associated antigen-binding protein according to any one of claims 1-174, comprising the step of isolating the interferon-associated antigen-binding protein from a transgenic animal other than a human or a transgenic plant according to claim 217.

219. An interferon-associated antigen-binding protein according to any one of claims 1-174, a nucleic acid according to any one of claims 175-190, a vector according to claim 191, a vector system according to any one of claims 191-193 or a composition according to any one of claims 195-214 for use as a medicinal product.

220. An interferon-associated antigen-binding protein according to any one of claims 1-174, a nucleic acid according to any one of claims 175-190, a vector according to claim 191, a vector system according to any one of claims 191-193 or a composition according to any one of claims 195-214 for use in the treatment of an infection caused by the hepatitis B virus (HBV) and/or for reducing one or more symptoms of an HBV infection in a patient.

221. An interferon-associated antigen-binding protein, nucleic acid, vector, vector system or composition for use according to claim 220, wherein treating an infection caused by the hepatitis B virus (HBV) comprises reducing one or more symptoms of the HBV infection in a patient.

222. An interferon-associated antigen-binding protein according to any one of claims 1-174, a nucleic acid according to any one of claims 175-190, a vector according to claim 191, a vector system according to any one of claims 191-193 or a composition according to any one of claims 195-214 for use in reducing the HBV viral load in an HBV-infected cell culture or in an HBV-infected patient compared to untreated HBV-infected or in the same patient before treatment.

223. An interferon-associated antigen-binding protein, nucleic acid, vector, vector system, or composition for use according to claim 222, wherein the HBV viral load is reduced by about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

224. An interferon-associated antigen-binding protein, nucleic acid, vector, vector system or composition for use according to claim 222 or 223, wherein the HBV viral load is determined using PCR or quantitative RT-PCR.

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225. An interferon-associated antigen-binding protein according to any one of claims 1-174, a nucleic acid according to any one of claims 175-190, a vector according to claim 191, a vector system according to any one of claims 191-193 or a composition according to any one of claims 195-214 for use in reducing the titre of HBV virus in an HBV-infected cell culture or in an HBV-infected patient compared to an untreated HBV-infected cell culture or in the same patient before treatment.

226. An interferon-associated antigen-binding protein, nucleic acid, vector, vector system or composition for use according to claim 225, wherein the HBV virus titer is reduced by about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

227. An interferon-associated antigen-binding protein, nucleic acid, vector, vector system or composition for use according to claim 225 or 226, wherein the HBV virus titer is determined using PCR or quantitative RT-PCR.

228. An interferon-associated antigen-binding protein according to any one of claims 1-174, a nucleic acid according to any one of claims 175-190, a vector according to claim 191, a vector system according to any one of claims 191-193 or a composition according to any one of claims 195-214 for use in reducing the transcription of HBV cccDNA in an HBV-infected cell culture or in an HBV-infected patient compared to an untreated HBV-infected cell culture or in the same patient before treatment.

229. An interferon-associated antigen-binding protein, nucleic acid, vector, vector system, or composition for use according to claim 228, wherein cccDNA transcription is reduced by about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

230. An interferon-associated antigen-binding protein, nucleic acid, vector, vector system or composition for use according to claim 228 or 229, wherein the transcription of cccDNA is determined using quantitative PCR.

231. An interferon-associated antigen-binding protein according to any one of claims 1-174, a nucleic acid according to any one of claims 175-190, a vector according to claim 191, a vector system according to any one of claims 191-193 or a composition according to any one of claims 195-214 for use in reducing the level of HBV pregenomic RNA in an HBV-infected cell culture or in an HBV-infected patient compared to an untreated HBV-infected cell culture or in the same patient before treatment.

232. An interferon-associated antigen-binding protein, nucleic acid, vector, vector system, or composition for use according to claim 231, wherein the level of HBV pregenomic RNA is reduced by about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

233. An interferon-associated antigen-binding protein, nucleic acid, vector, vector system or composition for use according to claim 231 or 232, wherein the level of HBV pregenomic RNA is determined using quantitative RT-PCR.

Table 9A IFN-β-based IFA IFN-γ IL-10 IL12p4 0 IL1 β IL2 IL6 IP10 TNFa NT Donor 1 Nd nd nd nd nd nd 457 nd Donor 2 Nd nd 101.4 nd nd nd 672.7 4.6 Donor 3 Nd nd nd nd nd nd 302.3 nd Donor 4 Nd nd 104 nd nd nd 648.2 nd LPS Donor 1 2023 148 7757,1 5116 nd 20709,6 6646,7 1706,3 Donor 2 4675,6 57.2 6265,6 6263,7 20.7 11070,1 39539,4 2987,1 Donor 3 1537,3 192.9 1750 3137,6 nd 16837,7 6141 944.9 Donor 4 2360,7 299.7 1676,5 6423 18.6 20654 22848,2 1107.2 IFA1 Donor 1 98.1 nd nd nd nd 16.3 46033,6 43.7 Donor 2 Nd nd 118.8 nd nd 11.8 43545,5 36.6 Donor 3 nd nd nd nd nd nd 23562,1 34 Donor 4 nd nd nd nd nd nd 31922,5 57.1 IFA2 Donor 1 nd nd nd nd nd 18.6 43382,3 41 Donor 2 nd nd 114.2 nd nd 17.4 43283,4 33.8 Donor 3 nd nd nd nd nd nd 25961,4 32.2 Donor 4 109.4 nd nd Nd nd nd 38445 66 Table 9B IFN-α-based IFA IFN-γ IL-10 IL12p4 0 IL1 β IL2 IL6 IP10 TNFa NT Donor 5 12.6 0,6 91.6 0.9 0.9 3.9 270.3 2,1 Donor 6 5 1,1 129.9 19.9 #div/0! 423.2 1052.7 16 Donor 7 16,5 2 143.7 22.1 2,1 426.9 1025 12.6 Donor 8 9.7 0,1 58.3 1,8 #div/0! 2.6 594.2 2,2 LPS Donor 5 10848,1 46.6 8463,3 8712,3 10,5 30713,2 20538,9 1738,3

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Donor 6 2467,1 175.6 5364.9 6557,9 3,3 31735,5 17262,6 2583,3 Donor 7 3310,1 248.6 6814,8 9123,9 16.6 39139,8 59939,2 6270,1 Donor 8 2555,6 138.5 2942.9 6767,5 9.6 31756,7 20062.7 1265,5 IFA25 Donor 5 495,5 1,5 99.5 1.9 5.5 30,5 39637,5 30.4 Donor 6 312.2 2 129.8 16,5 4 51.8 61963,8 71.4 Donor 7 271.2 2.9 130.3 9.1 4,4 75 133442, 5 30.3 Donor 8 441.6 1.9 74.8 6.8 3.2 44.3 95647,9 87.4 IFA26 Donor 5 330.4 2 98.1 2,1 6.4 29.3 37880,2 32.1 Donor 6 303.7 3,3 150.8 17.1 3.1 53 72944,8 45.7 Donor 7 180.3 2 135.6 9.2 4.9 75.2 154696, 3 29.7 Donor 8 421.4 2.8 95.7 6.8 4.1 42.1 79768,5 89.1 IFA27 Donor 5 430.7 2,2 127.8 3.1 7.1 32.9 40214,1 61.3 Donor 6 286.5 2 148.5 16.8 2,1 66 83445 70.1 Donor 7 350.3 4.7 117.6 9.3 4,4 73.5 195844, 6 105.6 Donor 8 440,1 2.6 68.6 8.9 0,6 46.9 102676, 8 43.4 IFA28 Donor 5 620,1 2.7 127.3 3,4 8.7 35 40958,5 24.6 Donor 6 264.7 2 170.3 13.6 2,4 45.7 62333,3 33 Donor 7 289.6 2.7 144.8 13.7 3.9 77.1 176521, 8 59.6 Donor 8 436.2 2,5 74.4 4.9 2,3 36.8 79217,6 37.6 IFA29 Donor 5 692.7 1,3 108.7 2,3 3.7 33.9 55062,8 30.3 Donor 6 183.1 2,2 158.8 11.6 0.4 44.4 58665,4 44.3 Donor 7 235.5 2.6 127.6 9.6 2 65.6 136893, 2 90.5 Donor 8 301,1 3 77.7 5.8 0,6 33.8 69226,3 48 IFA30 Donor 5 709.7 1,2 110.6 2.9 5.5 38 63040,7 36.5 Donor 6 122.9 2 153 14.9 1,7 46.1 67861,2 37.4 Donor 7 64.6 1 114 10 2.9 75.5 149093 32.7 Donor 8 206 1.9 71.1 6.8 1,8 37.9 85986,9 40,5

Table 10A Matrix Connection Dose lifetime period Method С0 (μg/m l) AUC (0-last) (mcg.h/ml) THyavG (h) AUC (0inf) (mcg.h/m l) % extrapolation T1/21 (h) CI (ml/h/kg) Vd ml/k g Whey SR87 0.893 0.5 mg/kg 168h ELISA -IgG2 7.15 241 168 590 59 264 (long) 0.35 (low) 296 (low) Whey IFA2 7 0.5 mg/kg 240h ELISA -IgG2 14.7 1501 240 2552 41 218 (long) 0.2 (low) 55 (low) Whey IFA2 7 0.5 mg/kg 240h IFA -IFN 16.9 1318 240 1793 26 125 (long) 0.28 (low) 50 (low) Whey IFA2 9 0.5 mg/kg 240h IFA -IFN 11.6 804 240 1033 22 116 (long) 0.49 (low) 78 (low) Whey IFA3 0 0.5 mg/kg 240h IFA -IFN 8,12 741 240 1089 31 158 (long) 0.46 (low) 98 (low)

Table 10B

Matrix Connection Dose lifetime period Method С0 (μg/m l) AU C (0last) (µg .h/ml) Tlas t (h) AU C (0inf) (µg .h/ml) % extrapolation and ^T 1^ (h) CI (ml/h/kg) Vd ml/kg

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Claims (22)

Serum IFA2 5 0.5 mg/kg 504h IFA- IFN 7.4 5 1328 50 4 1500 11 154 (long) 0.34 (low) 73 (low) Serum IFA2 6 0.5 mg/kg 504h IFA- IFN 8.2 988 33 6 1027 3.8 59 (long) 0.49 (low) 57 (low) Serum IFA2 8 0.5 mg/kg 504h IFA- IFN 9.3 8 1048 50 4 1264 17 213 (long) 0.4 (low) 105 (low) Serum Pegas ys 0.3 mg/kg 504h ELISAIFNa-specific 8.3 210 16 8 215 2 30 (medium) 1.4 (low) 62 (low) Serum CP87 0.893 0.5 mg/kg 504h ELISA IgG2 11.9 527 16 8 797 34 116 (long) 0.63 (low) 96 (low) Serum IFA2 5 0.5 mg/kg 504h IFA IgG2 11.8 1292 24 0 1971 34 155 (long) 0.26 (low) 56 (low) CLAUSE OF THE INVENTION 1. An interferon-associated antigen-binding protein comprising (I) an agonist anti-CD40 antibody or an agonist antigen-binding fragment thereof, and (II) an interferon (IFN) or a functional fragment thereof, wherein the IFN or functional fragment thereof is fused to the light chain of an agonist anti-CD40 antibody or an agonist antigen-binding fragment thereof; and wherein the agonist anti-CD40 antibody or agonist antigen-binding fragment thereof comprises: a heavy chain or fragment thereof comprising a complementarity determining region (CDR) CDRH1 which is identical to SEQ ID NO: 56, CDRH2 which is identical to SEQ ID NO: 57, and CDRH3 which is identical to SEQ ID NO: 58; and a light chain or fragment thereof comprising CDRL1 which is identical to SEQ ID NO: 52, CDRL2 which is identical to SEQ ID NO: 53, and CDRL3 which is identical to SEQ ID NO: 54. 2. The interferon-associated antigen-binding protein of claim 1, wherein the IFN or a functional fragment thereof is fused to an agonistic CD40 antibody or an agonistic antigen-binding fragment thereof via a linker. 3. The interferon-associated antigen-binding protein of claim 1 or 2, wherein the agonist antibody against CD40 or agonist antigen-binding fragment thereof comprises: (a) a light chain variable region VL comprising the sequence of SEQ ID NO: 51 or a sequence at least 90% identical thereto; and/or a heavy chain variable region VH comprising the sequence of SEQ ID NO: 55 or a sequence at least 90% identical thereto; (b) a heavy chain of the Fab region comprising the amino acid sequence of SEQ ID NO: 12 or a sequence at least 90% identical thereto; or (c) a light chain (LC) that comprises the sequence of SEQ ID NO: 3 or a sequence at least 90% identical thereto; and/or a heavy chain (HC) that comprises a sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 49 and SEQ ID NO: 48, or a sequence at least 90% identical thereto. 4. An interferon-associated antigen-binding protein according to any one of claims 1 to 3, wherein the IFN or a functional fragment thereof is selected from the group consisting of type I IFN, type II IFN and type III IFN, or a functional fragment thereof. 5. The interferon-associated antigen-binding protein of claim 4, wherein the type I IFN or a functional fragment thereof is IFNa or IFNe or a functional fragment thereof. 6. An interferon-associated antigen-binding protein according to any of the preceding claims, wherein the IFN or a functional fragment thereof is IFNa2a or a functional fragment thereof; wherein preferably IFNa2a comprises the sequence of SEQ ID NO: 17 or a sequence at least 90% identical thereto. 7. The interferon-associated antigen-binding protein of any one of claims 1 to 5, wherein the IFN or a functional fragment thereof is IFNe or a functional fragment thereof; wherein preferably IFNe comprises the sequence of SEQ ID NO: 14 or a sequence identical thereto in - 67 047752 at least 90%. 8. An interferon-associated antigen-binding protein according to any of the preceding claims, wherein the IFN or a functional fragment thereof is fused to the light chain of an agonist CD40 antibody or an agonist antigen-binding fragment thereof; preferably, wherein the IFN or a functional fragment thereof is fused to the C-terminus of the light chain of an agonist CD40 antibody or an agonist antigen-binding fragment thereof. 9. The interferon-associated antigen-binding protein of any one of claims 1 to 7, wherein the IFN or a functional fragment thereof is fused to the heavy chain of an agonist CD40 antibody or an agonist antigen-binding fragment thereof; preferably, wherein the IFN or a functional fragment thereof is fused to the C-terminus of the heavy chain of an agonist CD40 antibody or an agonist antigen-binding fragment thereof. 10. The interferon-associated antigen-binding protein of any one of the preceding claims, wherein the agonist CD40 antibody or agonist antigen-binding fragment thereof and the IFN or functional fragment thereof are fused to each other via a linker, and wherein the linker comprises the sequence of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26. 11. The interferon-associated antigen-binding protein of any one of the preceding claims, comprising a sequence selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47. 12. The interferon-associated antigen-binding protein of any one of the preceding claims, wherein the interferon-associated antigen-binding protein is an interferon-fused agonist anti-CD40 antibody or an interferon-fused agonist antigen-binding fragment thereof comprising one of the combinations of sequences disclosed in Table 8, particularly Table 8A, or Table 8B, more particularly Table 8A. 13. The interferon-associated antigen-binding protein of any one of claims 1-6 or 9-12, wherein the interferon-associated antigen-binding protein is an interferon-fused agonist CD40 antibody or an interferon-fused agonist antigen-binding fragment thereof comprising the sequence of SEQ ID NO: 38 and SEQ ID NO: 3. 14. The interferon-associated antigen-binding protein of any one of claims 1-6 or 9-12, wherein the interferon-associated antigen-binding protein is an interferon-fused agonist CD40 antibody or an interferon-fused agonist antigen-binding fragment thereof comprising the sequence of SEQ ID NO: 40 and SEQ ID NO: 3. 15. A nucleic acid encoding an interferon-associated antigen-binding protein according to any one of claims 1 to 14. 16. The nucleic acid of claim 15, wherein the nucleic acid additionally encodes a secretory signal peptide. 17. An expression vector containing a nucleic acid according to claim 15 or 16. 18. An expression vector system comprising (I) a first vector comprising a nucleic acid encoding an IFN or a functional fragment thereof fused to the light chain of an agonist CD40 antibody or an agonist antigen-binding fragment thereof of an interferon-associated antigen-binding protein according to any one of claims 1 to 12; and a second vector comprising a nucleic acid encoding the heavy chain of an agonist CD40 antibody or an agonist antigen-binding fragment thereof of an interferon-associated antigen-binding protein according to any one of claims 1 to 12; or (II) a first vector comprising a nucleic acid encoding an IFN or a functional fragment thereof fused to the heavy chain of an agonist CD40 antibody or an agonist antigen-binding fragment thereof of an interferon-associated antigen-binding protein according to any one of claims 1 to 14; and a second vector comprising a nucleic acid encoding the light chain of an agonistic antibody against CD40 or an agonistic antigen-binding fragment thereof of the interferon-associated antigen-binding protein of any one of claims 1 to 14. 19. A pharmaceutical composition comprising an interferon-associated antigen-binding protein according to any one of claims 1 to 14, a nucleic acid according to claim 15 or 16, a vector according to claim 17, or a vector system according to claim 18. 20. A host cell containing a nucleic acid according to claim 15 or 16, a vector according to claim 17, or a vector system according to claim 18. 21. A method for producing an interferon-associated antigen-binding protein according to any one of claims 1 to 14, comprising culturing a host cell according to claim 20 and isolating said interferon-associated antigen-binding protein. 22. The use of an interferon-associated antigen-binding protein according to any one of claims 1 to 14, a nucleic acid according to claim 15 or 16, a vector according to claim 17, a vector system according to claim 18 or a composition according to claim 19 as a medicinal product for the treatment of an infection caused by the hepatitis B virus (HBV), -