EA046818B1 - INTERFERON-ASSOCIATED ANTIGEN-BINDING PROTEINS FOR THE TREATMENT OF INFECTION CAUSED BY HEPATITIS B VIRUS - Google Patents

Description

Field of technology

The present invention relates to novel interferon-associated antigen-binding proteins, as well as nucleic acids and expression vectors encoding such interferon-associated antigen-binding proteins, for use in therapy, more particularly for use in the treatment of hepatitis B virus (HBV) infection. The present invention also relates to pharmaceutical compositions containing such interferon-associated antigen-binding proteins or nucleic acids or expression vectors for use in therapy, more particularly for use in the treatment of hepatitis B virus (HBV) infection. The present invention further relates to methods of treatment using such interferon-associated antigen-binding proteins or nucleic acids, or expression vectors, or pharmaceutical compositions. These new 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.

State of the art

HBV affects 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 properties similar to retroviruses that replicates through an RNA intermediate (pregenomic RNA, pgRNA) and can integrate into the host genome. The unique features of the HBV replication cycle give the virus a special ability to persist in infected cells. HBV infection leads to a wide range of liver diseases, ranging from acute (including fulminant liver failure) to chronic hepatitis, cirrhosis and hepatocellular carcinoma. Acute HBV infection may be asymptomatic or present with symptomatic acute hepatitis. 90-95% of children and 5-10% of adults infected with HBV cannot clear the virus and become chronically infected. In many chronically infected people, liver disease is mild, with little or no long-term morbidity or mortality. Other people with chronic HBV infection develop active disease, which can progress to cirrhosis and liver cancer. These patients require close monitoring and require therapeutic intervention.

New methods of treating HBV infection by modulating HBV infection in the cell are needed. In particular, methods are needed to effectively disrupt viral replication, reduce HBV viral load in HBV-infected cells, reduce 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 containing (I) an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof, and (II) interferon (IFN) or a functional fragment thereof for use in the treatment of hepatitis B virus (HBV) infection.

In accordance with this aspect of the invention, an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof may comprise: (a) a heavy chain or fragment thereof containing a complementarity determining region (CDR) of CDRH1 that is at least 90% identical to SEQ ID NO: 56, CDRH2, which is at least 90% identical to SEQ ID NO::: 57, and CDRH3, which is at least 90% identical to SEQ ID NO: 58; and (b) 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. Alternatively, an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof may comprise (a) a heavy chain or fragment thereof containing a complementarity determining region (CDR) of CDRH1 that 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 (b) 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.

In accordance with one embodiment, an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof comprises a VL light chain variable region comprising the sequence of SEQ ID NO: 51, or a sequence having at least 90% identity thereto; and/or a VH heavy chain variable region comprising the sequence SEQ ID NO: 55, or a sequence at least 90% identical thereto.

In another embodiment, an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof comprises a light chain (LC) that contains the sequence of SEQ ID NO: 3, or a sequence that is at least 90% identical thereto; and/or a heavy chain (HC) that contains 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 identical thereto

- 1 046818 at least 90%.

In accordance with a further embodiment, the IFN or a functional fragment thereof may be selected from the group consisting of type I IFN, type II IFN and type III IFN or a functional fragment thereof. Preferably, the type I IFN or a functional fragment thereof is IFNa or IFNe or a functional fragment thereof.

In another embodiment, the IFN or a functional fragment thereof is IFNa2a or a functional fragment thereof. In accordance with a preferred embodiment, IFNa2a contains the sequence SEQ ID NO: 17, or a sequence identical to it at least 90%.

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

In another embodiment, the IFN or a functional fragment thereof is fused to the light chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof, preferably the C-terminus.

In a further embodiment, the IFN or a functional fragment thereof is fused to the heavy chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof, preferably to the C-terminus.

In another embodiment, an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof and an IFN or a functional fragment thereof are fused to each other through a linker. In a preferred embodiment, the linker contains the sequence SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26.

In another embodiment, the interferon-associated antigen-binding protein is an interferon-fusion agonist anti-CD40 antibody or an interferon-fusion agonist antigen-binding fragment thereof containing one of the combinations of sequences disclosed in Table 1. 9.

In another embodiment, the use comprises administering the interferon-associated antigen-binding protein to a patient by genetically delivering RNA or DNA sequences encoding the interferon-associated antigen-binding protein or a vector or vector system encoding the interferon-associated antigen-binding protein.

According to yet another embodiment, the interferon-associated antigen binding protein is contained in a pharmaceutical composition.

Brief description of drawings

Fig. 1: This schematic drawing shows exemplary interferon-associated antigen binding protein formats. Interferon-associated antigen-binding protein is an agonistic anti-CD40 antibody fused to interferon or an agonistic antigen-binding fragment thereof fused to interferon. IFNs are associated through linkers with different positions on the antibody or its antigen-binding fragment: with the N-terminal or C-terminal part 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.

In 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: 78) is also shown on the right. Italics: signal peptide sequence; black: heavy chain coding sequence CP870.893; underlined: HL linker coding sequence; bold: IFNe coding sequence.

In fig. 2B shows examples of SDS-PAGE under reduced conditions for certain IFAs, with IFNa or IFNe fused to either the heavy chain or the light chain. The migration of the parent CP870,893 is also shown on the left.

In fig. 3A-3B graphically depict the dose-dependent effect of a number of IFA molecules with IFNe fusions on activation of the CD40-mediated NFkB reporter pathway in HEK-Blue™ CD40L cells. In fig. 3A shows examples of anti-CD40 activities for IFA with IFNe fused to the C-terminal portion of the heavy chain (HC). In fig. 3B shows examples of anti-CD40 activities for IFAs 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 final batch of IFAs was very low, so supernatants from transfected HEK cells were used to test their activity and serially diluted to evaluate anti-CD40 activity on HEK-Blue™ CD40L cells.

In fig. 3C-3D graphically depict the dose-dependent effect of a number of IFA molecules with IFNe fusions on activation of the type I IFN pathway in HEK-Blue-IFN-a/e reporter cells. In fig. Figure 3C shows examples of IFN activity for IFA with IFNe fused to the C-terminal portion of HC. In fig. 3D by

- 2 046818 shows examples of IFN activity for IFA with IFNe fused to the N-terminal part of the LC (IFA34) or HC (IFA36), and the corresponding fusion variants at the C-terminal part (IFA35 and IFA37). To assess IFN activity, the same supernatants from transfected EK cells were used as in Fig. 3B and serially diluted. The parental antibody CP870,893 was used as a negative control, and recombinant human IFNe was used as a positive control. NS: Not stimulated.

In fig. 4A graphically depicts the dose effect of a number of IFA molecules with IFNa fusions on CD40-mediated activation of the NFkB reporter pathway in HEK-Blue™ CD40L cells.

In fig. 4B graphically depicts the dose effect of a number of IFA molecules with IFNa fusions on activation of the type I IFN-mediated pathway in HEK-Blue-IFN-α/β reporter cells. Pegasys activity is listed on the insert in the lower right corner.

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

In fig. 4D graphically depicts the effect of IFA38 and IFA39 on type I IFN pathway activation in HEK-Blue-IFNa/β reporter cells.

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

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

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

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

In fig. 6D-6H shows the dose-dependent antiviral activity of 4 IFA molecules fused to IFNa via a peptide linker against primary human hepatocytes infected with HBV. Fig. 6D: Graphical representation illustrating the study design. Fig. 6E: Effect of IFA on HBeAg release compared to Pegasys. Fig. 6F: Effect of IFA on HBsAg release compared to Pegasys. Fig. 6G: effect of IFA on pgRNA levels compared to Pegasys. Fig. 6H: Effect of IFA on CXCL10 release compared to Pegasys.

In fig. Figure 7 shows the results of an in vitro cytokine release assay from human whole blood cells (WBCs): an example of 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 subjected to quantitation of cytokine release using the MSD u-Plex Human Cytokine Kit. Results are the average of two independent stimulations from each donor. Profiles of CXCL10 (IP10), IL6, IL1p and TNFa are shown.

Table 11a-b: These tables summarize data obtained after in vitro stimulation of whole blood cells (leukocytes) obtained from healthy volunteers. Each IFA was tested on leukocytes from four different donors. Leukocytes were left untreated (NT), treated with LPS (10 ng/ml) or IFA (1 μg/ml) for 24 h. Supernatants were collected and subjected to quantitation of cytokine release using the MSD u-Plex Human Cytokine Kit. Results are the average of two independent stimulations from each donor and are expressed in pg/ml (nd: not detected).

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

Table 12A: Summary of Pharmacokinetic Data: Pharmacokinetic parameters for CP870,893, IFA27, IFA29 and IFA30 following a single 0.5 mg/kg intravenous dose in male CD1 Swiss mice. PK parameters for CP870,893 were studied in a 7-day experiment, and parameters for IFA27, IFA29 and IFA30 in a 10-day experiment (IFA27 quantification was carried out using 2 different ELISA approaches).

Table 12B: PK data summary: PK parameters for CP870,893, Pegasys and for three different IFAs

- 3 046818 (IFA25, IFA26 and IFA28) after a single intravenous bolus of 0.5 mg/kg in male CD1 Swiss mice. PK parameters for CP870,893 and IFA25, IFA26, IFA28 and Pegasys were examined in 21-day experiments (IFA25 quantification was performed using 2 different ELISA approaches).

In fig. 9A shows the dose-dependent CD40 agonist activity of IFA50 and IFA51 lacking the Fc region compared to the parental anti-CD40 antibody in HEK-Blue™ CD40L reporter cells. In fig. 9B shows dose-dependent IFNa activity for IFA50 and IFA51 in HEK-Blue™ hIFN-α/β reporter cells. Fig. 9C: Effect of IFA50 and IFA51 on HBeAg release from HBV-infected PHH.

In fig. 10A shows the dose-dependent CD40 agonist activity of IFA49 based on IF#: compared to the parental anti-CD40 antibody in HEK-Blue™ CD40L reporter cells. IFA49 corresponds to the fusion of IFNo: with HC via a peptide linker. In fig. 10B shows the dose-dependent IFN activity of IFA49 on HEK-Blue™ hIFN-α/β reporter cells, which are activated by type I interferons. Fig. 10C: effect of IFA49 on HbeAg release from HBV-infected PHH.

In fig. 11A shows the dose-dependent CD40 agonist activity of IFNm-based IFA46 compared to the parental anti-CD40 antibody in HEK-Blue™ CD40L reporter cells. IFA46 corresponds to the fusion of IFNm to LC via a peptide linker. In fig. 11B shows the dose-dependent IFN activity of IFA46 on HEK-Blue™ hIFN-α/β reporter cells, which are activated by type I interferons. Fig. 11C: Effect of IFA46 on HbeAg release from HBV-infected PHH.

In fig. 12A shows the dose-dependent CD40 agonist activity of IFNy-based IFAs (IFA42 and IFA43) compared to the parent 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. In fig. 12B shows dose-dependent IFN activity for IFA42 and IFA43 in HEK-Blue-hIFNY reporter cells; fig. 12C: Effect of IFA42 and IFA43 on HbeAg release from HBV-infected PHH.

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

In fig. 14 shows examples of SDS-PAGE under reduced conditions for certain IFAs with IFNa or IFNe fused to the heavy chain of the anti-CD40 antibody 3G5. The migration of the parental anti-CD40 antibody 3G5 is also shown on the left.

In fig. 15A-B graphically depict the dose-dependent effect of a series of 3G5-based IFA molecules with IFNe fusions on activation of the CD40-mediated NFkB reporter pathway in the HEK-Blue™ CD40L cell assay. Comparison with the parent antibody 3G5 (denoted CDX-3G5 in this figure) is similarly shown. In fig. 15A shows examples of anti-CD40 activities for IFAs with an IFNe fusion to the C-terminal heavy chain (HC). The purification yield of IFAs with IFNe fusions on the light chain was very low, so supernatants from transfected HEK cells were used to test their activity and serially diluted to evaluate anti-CD40 activity on HEK-Blue™ CD40L cells; an example of activity is shown in Fig. 15, and the supernatant containing band 3G5 was used as a control.

In fig. 15C-D graphically shows the dose-dependent effect of a number of IFA molecules with IFNe fusions on activation of the type I IFN pathway in HEK-Blue-IFN-α/β reporter cells. In fig. 15C shows examples of IFN activity for IFA with IFNe fused to the C-terminal portion of HC. In fig. 15D shows IFN activity of IFA with IFNe fused to a light chain; The level of production of these proteins was very low and therefore an example of the activity of the two IFAs is shown in FIG. 15D using the same supernatant as in FIG. 15V.

In fig. 16A graphically shows the dose effect of four IFNa fusion IFA molecules on NFkB pathway activation mediated by CD40b reporter assay in HEKBlue™ CD40L cells. Comparison with the parent antibody 3G5 (denoted CDX-3G5 in this figure) is similarly shown.

In fig. 16B graphically shows the dose effect of a number of IFA molecules with IFNa fusions on activation of the type I IFN-mediated pathway in HEK-Blue-IFN-a/e reporter cells.

In fig. 17 shows the dose-dependent effect of type I IFN-based IFA on the release of HBeAg from HBV-infected PHH. In fig. 17A shows the results obtained with IFA based

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IFNe (IFA106, IFA107, IFA108 and IFA109) with IFNe fused to the C-terminal part of the HC. In fig. 17B shows the results obtained with four IFNa-based IFAs (IFA121, IFA122, IFA123 and IFA124) with IFNa fused to the C-terminal portion of the HC. NI-NT: uninfected, untreated. MOI: multiplicity of infection.

Fig. 18: In vitro cytokine release assay from human whole blood cells (WBC): example of data obtained after WBC stimulation from 4 healthy volunteer donors. Leukocytes were left untreated (NT), treated with LPS (10 ng/ml) or IFA109 (1 μg/ml) for 24 h. Supernatants were collected and subjected to quantitation of cytokine release using the MSD u-Plex Human Cytokine Kit. Results are the average of two independent stimulations from each donor. Profiles of CXCL10 (IP10), IL6, IL1β and TNFa are shown.

Table 13. This table summarizes data obtained after in vitro stimulation of whole blood cells obtained from healthy volunteers. IFA109 was tested on leukocytes from four different donors. Leukocytes were left untreated (NT), treated with LPS (10 ng/ml) or IFA109 (1 μg/ml) for 24 h. Supernatants were collected and subjected to quantitation of cytokine release using the MSD u-Plex Human Cytokine Kit. Results are the average of two independent stimulations from each donor and are expressed in pg/ml (nd: not detected).

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

Detailed description

The present invention is based in part on the discovery of a therapy using interferon-associated antigen-binding proteins, variants or derivatives thereof, containing (i) an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof and (ii) interferon (IFN) or a functional fragment thereof in the treatment of an infection, caused by the hepatitis B virus (HBV). These 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. HBV therapy is proposed that involves administering interferon-associated antigen-binding protein to an HBV-infected cell or patient infected with HBV.

The invention may 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 (eg, B cells, dendritic cells, monocytes), hematopoietic progenitors, endothelial cells, smooth muscle cells, epithelial cells, and in 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 various downstream effects. The TNF receptor-associated factor adapter proteins TRAF1, TRAF2, TRAF6 and TRAF5 interact with CD40 and serve as mediators of signal transduction. Ultimately, CD40 signaling activates both canonical and non-canonical NF-κB 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 by disulfide bonds, as well as multimers thereof (eg, IgM). Each heavy chain contains 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 contains 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, located from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The framework regions can help maintain the proper conformation of the CDR to promote 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

- 5 046818 This tetramer consists of two identical pairs of polypeptide chains, each pair having one light chain (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 agonistic anti-CD40 antibodies or agonistic antigen-binding fragments thereof contained in the interferon-associated antigen-binding proteins of the invention are of the IgG class. In more preferred embodiments, the agonistic anti-CD40 antibodies or agonistic antigen-binding fragments thereof contained in the interferon-associated antigen-binding proteins of the invention are of the IgG1 or IgG3 subclasses. In particularly preferred embodiments, the agonistic anti-CD40 antibodies or agonistic antigen-binding fragments thereof contained in the interferon-associated antigen-binding proteins of the invention are of the IgG1 subclass. In other more preferred embodiments, the agonistic anti-CD40 antibodies or agonistic antigen-binding fragments thereof contained in the interferon-associated antigen-binding proteins of the invention are of the IgG2 or IgG4 subclasses. In particularly preferred embodiments, the agonistic anti-CD40 antibodies or agonistic antigen-binding fragments thereof contained in the interferon-associated antigen-binding proteins of the invention are of the IgG2 subclass.

Human light chains are classified as kappa (k) and lambda (λ) light chains. Accordingly, in some embodiments, the agonistic anti-CD40 antibodies or agonistic antigen-binding fragments thereof contained in the interferon-associated antigen binding proteins of the invention contain a class k light chain. In other embodiments, the agonistic anti-CD40 antibodies or agonistic antigen-binding fragments thereof contained in the interferon-associated proteins of the invention contain a class k light chain. associated antigen-binding proteins of the invention contain a light chain of class λ. In the light and heavy chains, the variable and constant regions are connected by a J region of about 12 or more amino acids, with the heavy chain further including a D region of about 10 more 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, miniantibodies, domain antibodies, synthetic antibodies (sometimes called antibody mimetics), chimeric antibodies, humanized antibodies, human antibodies, and fragments thereof, respectively. Unless otherwise specified, the term antibody includes, in addition to antibodies containing two full-length heavy chains and two full-length light chains, their derivatives, variants, antigen-binding fragments and muteins, 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 using surface plasmon resonance, preferably using the BIAcore® system. An agonistic anti-CD40 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%. CD40 activity of an anti-CD40 agonist antibody can be measured using a whole blood cell surface molecule up-regulation 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 leads to activation of NFkB and thus the production of SEAP, 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 the OE40 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,

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100, 70, 60, 50, 40, 30, 25, 20 or 15 ng/ml. In more specific embodiments, the interferon-associated antigen binding protein activates the CD40 pathway with an EC50 ranging from 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 suitable CD40 agonistic antibodies include, but are not limited to, CP870,893 (Pfizer/Roche), SGN-40 (Seattle Genetics), ADC-1013 (Janssen/Alligator BioSciences), Chi Lob 7/4 (University of Southampton), dacetumumab (Seattle Genetics), APX005M (Apexigen, Inc.), 3G5 (Celldex), and CDX-1140 (Celldex). Examples of the light and heavy chain sequences of the agonistic anti-CD40 antibody CP870,893 are shown in Table 1. 7. Example sequences of the light and heavy chains of the agonistic anti-CD40 antibody 3G5 are shown in table. 8.

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

Agonistic antigen binding fragments of an agonistic anti-CD40 antibody can be produced by recombinant DNA techniques or can be produced by enzymatic or chemical digestion of an anti-CD40 antibody. Agonistic antigen-binding fragments include, but are not limited to, a Fab fragment, a diantibody (a heavy chain variable domain on the same polypeptide as the light chain variable domain, linked by a short peptide linker too short to pair between two domains on the same chain ), Fab' fragment, F(ab')2 fragment, Fv fragment, domain antibodies and single chain antibodies, and which can be obtained from any mammalian source, including, but not limited to, human, mouse, rat, camel or a 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 greatly in amino acid sequence, even among antibodies derived from the same species or class. Example sequences of the VL and VH domains of the CD40 agonistic antibody CP870,893 are shown in Table 1. 1. The variable region of an antibody typically determines the specificity of a particular antibody for its target because it contains the CDR. In table 1 also shows exemplary CDR sequences of the CD40 agonist antibody CP870,893.

Table 1 Heavy/light chain variable regions and CDRs of the agonistic anti-CD40 antibody CP870,893. Sequences in bold italics correspond to CDR regions as defined by Kabat. 1 ____________________________________________

Anti-CD40 antibody regions Subsequence anti-CD40 antibody, V _L DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWY

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domain (SEQIDNO51) QQKPGKAPNLLTYTASTLQSGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQANIFPLTFGGGTKVEIK antibody against CD40, CDRL1 (SEQ ID NO 52) RASQGIYSWLA antibody against CD40, CDRL2 (SEQ ID NO 53) TASTLQS antibody against CD40, CDRL3 (SEQ ID NO 54) QQANIFPLT anti-CD40 antibody, Vh domain (SEQ ID NO 55) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYM HWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGR VTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPL GYCTNGVCSYFDYWGQGTLVTVSS antibody against CD40, CDRH1 (SEQ ID NO 56) TGYYMH antibody against CD40, CDRH2 (SEQ ID NO 57) WINPDS GGTNYAQKFQG antibody against CD40, CDRH3 (SEQ ID NO 58) DQPLGYCTNGVCSYFDY

Delineation of 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 techniques known to those skilled in the art, such as x-ray crystallography. Various analysis techniques can be used to identify or approximate CDR regions. Examples of such methods include, but are not limited to, Kabat detection, Chothia detection, AbM detection, and contact detection.

The Kabat definition is a standard for numbering residues in an antibody and is commonly used to identify CDR regions. See, for example, 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, for example, Chothia et al., J. Mol. Biol., 196: 901-17 (1986); Chothia et al., Nature, 342: 877-83 (1989). AbM determination uses an integrated set of computer programs produced by the Oxford Molecular Group that model the structure of antibodies. See, for example, 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. AbM determination models the tertiary structure of an antibody from a primary sequence using a combination of information databases 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). The determination of contact is based on the analysis of existing complex crystal structures. See, for example, MacCallum et al., J. Mol. Biol., 5:732-45 (1996).

In some embodiments, the complementarity determining regions (CDRs) of the light and heavy chain variable regions of an agonistic anti-CD40 antibody or an agonistic 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 light and heavy chain variable regions of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof may be grafted to human consensus FRs. To generate human consensus FRs, in some embodiments, FRs from multiple human heavy or light chain amino acid sequences are aligned to identify a consensus amino acid sequence. In some embodiments, the heavy chain or light chain FRs of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof are replaced with FRs from another heavy chain or light chain. In some embodiments, rare amino acids in the FR of the heavy and light chains of an agonistic anti-CD40 antibody or an agonistic 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 found in positions in

- 8 046818 which they are not usually found in FR. In some embodiments, the transplanted variable regions of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof may be used with a constant region that is different from the constant region of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof. In some embodiments, the transplanted variable regions are part of a single chain Fv antibody. CDR transplantation is described, for example, in US patent No. 6180370, 6054297, 5693762, 5859205, 5693761, 5565332, 5585089 and 5530101, as well as 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 all purposes.

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

The Fab' fragment includes one light chain and part of one heavy chain, which contains the VH domain and the CH1 domain, as well as the region between the CH1 and CH2 domains, so that a disulfide bond can be formed between the two heavy chains between the chains of the two Fab' fragments with the formation of the 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 variable regions of both the heavy and light chains, but does not contain constant regions.

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

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

An antibody or antigen binding protein, such as the interferon-associated antigen binding protein of the invention, preferably binds its target antigen with a dissociation constant (Kd) of <10 ^-7 M. An antibody or antigen binding protein binds its antigen with high affinity when the Kd is <5x10 ^-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 <1x10 ^-5 . In other embodiments, the antibody or antigen binding protein will bind to human CD40 with a Kd of about 10 ^-9 M to 10 ^-13 M, and in yet another embodiment, the antibody or antigen binding protein will bind with a Kd <5x10 ^-10 . As will be appreciated by one of ordinary skill in the art, in some embodiments, any or all of the antigen binding moieties may 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, New Jersey ). 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 (eg, antibody or antigen binding protein) with an antigen to form, for example, an antigen binding protein/antigen complex. The term Kon or onrate also means the association rate constant or ka, as used interchangeably herein. This value, indicating the rate of binding of a binding protein to its target antigen or the rate of formation of a complex between a binding protein, such as an antibody or an 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 refers to the dissociation rate constant for disso

- 9 046818 cyation or dissociation rate constant of a binding protein (eg, antibody or antigen binding protein) from, for example, an antigen binding protein/antigen complex, as is known in the art. This value indicates the rate of dissociation of a binding protein, such as an antibody or antigen-binding protein, from a target antigen or separation 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 mean 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 (eg, antibody or antigen-binding protein) to 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 examine samples in physiological buffers at equilibrium. Other experimental approaches and tools can be used, such as the BIAcore® (Biomolecular Interaction Assay) assay (eg, instrument available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden). In addition, the KinExA® assay (kinetic exclusion assay), available from Sapidyne Instruments (Boise, Id.), can also be used.

The antigen binding protein of the invention can bind to one target with an affinity that is at least one order of magnitude higher, preferably at least two orders of magnitude higher, than that of 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. Therefore, it will be understood that the target can 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 associated with an antigen-binding protein that targets that antigen, and when the antigen is a protein, it includes specific amino acids that directly contact the antigen-binding protein. Most often, epitopes are present on proteins, but in some cases they can be present on other types of molecules, such as nucleic acids. Epitope determinants may include reactive 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. Typically, 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 illustrative embodiments, an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof 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 in SEQ ID NO: 3; and three heavy chain CDRs that are at least 90% identical to the sequences CDRH1, CDRH2 and CDRH3 in SEQ ID NO: 6. An agonistic anti-CD40 antibody or an agonistic antigen binding fragment thereof may also contain three light chain complementarity determining regions (CDRs) that identical to the sequences CDRL1, CDRL2 and CDRL3 in SEQ ID NO: 3; and three heavy chain CDRs that are identical to the sequences CDRH1, CDRH2, and CDRH3 in SEQ ID NO: 6. In such embodiments, each CDR is defined in accordance with the Kabat definition, the Chothia definition, the AbM definition, or the contact CDR definition; preferably, where each CDR is defined in accordance with the Kabat CDR definition or the Chothia CDR definition. In specific embodiments, each CDR is defined in accordance with the Kabat definition. In other specific embodiments, each CDR is defined in accordance with the Chothia definition.

Alternatively, an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof forming part (I) of the interferon-associated antigen-binding proteins of the invention may comprise (a) a heavy chain or fragment thereof containing a complementarity determining region (CDR) of CDRH1 that is at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 56, CDRH2, which is at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 57, and 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 containing CDRL1 that is at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 52, CDRL2, which is at least 90%

- 10 046818 is at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 53, and CDRL3, which is at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID 54.

In some embodiments, an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof comprises: (a) a heavy chain or fragment thereof containing a complementarity determining region (CDR) of CDRH1 that is identical to SEQ ID NO: 56, CDRH2 that is identical to SEQ ID NO: 57, and CDRH3, which is identical to SEQ ID NO: 58; and (b) 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.

More specifically, the agonistic anti-CD40 antibody or agonistic antigen-binding fragment thereof comprises a VL light chain variable region comprising the sequence of SEQ ID NO: 51, or a sequence of at least 90%, at least 95%, at least 98% or at least 99% identical to it; 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 anti-CD40 agonistic antibody or an agonistic antigen-binding fragment thereof comprising a Fab region heavy chain containing the amino acid sequence of SEQ ID NO: 12, or at least 90% of the sequence %, at least 98% or at least 99% identical to it.

In some embodiments, an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof comprises a light chain (LC) that contains the sequence of SEQ ID NO: 3, or a sequence of at least 90%, at least 95%, at least 98% or at least 99% identical to it; and/or a heavy chain (HC) that contains 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 that is at least 90%, at least 95%, at least 98%, or at least 99% identical.

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

In more specific embodiments, an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof comprises a light chain (LC) that contains the sequence of SEQ ID NO: 3, or the sequence of at least 90%, at least 95%, 98% or at least 99% identical to it; and/or a heavy chain (HC) that contains the sequence of SEQ ID NO: 9 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 agonistic antibody or an agonistic antigen-binding fragment thereof comprises a light chain (LC) that contains the sequence of SEQ ID NO: 3, or at least 90% of the sequence, or at least 95% of the sequence. , at least 98% or at least 99% identical to it; and/or a heavy chain (HC) that contains the sequence of SEQ ID NO: 49 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, an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof comprises a light chain (LC) that contains the sequence of SEQ ID NO: 3 or a sequence of at least 90%, at least 95%, of at least 98% or at least 99% identical to it; and/or a heavy chain (HC) that contains 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, an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof comprises a light chain (LC) that contains the sequence of SEQ ID NO: 3 or at least 90% of the sequence, at least 95% of the sequence, or at least 95% of the sequence. 98% or at least 99% identical to it; and/or a heavy chain (HC) that contains the sequence of SEQ ID NO: 50, or a sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto .

In some embodiments, an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof comprises a light chain (LC) that contains the sequence of SEQ ID NO: 59 or at least 90% of the sequence, at least 95% of the sequence, or at least 98% of the sequence. or at least 99% identical to it; and/or a heavy chain (HC) that contains a sequence selected from the group consisting of SEQ ID NO: 61, SEQ ID NO: 63 and SEQ ID NO:

- 11 046818

65, or a sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

In more specific embodiments, an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof comprises a light chain (LC) that contains the sequence of SEQ ID NO: 59 or at least 90% of the sequence, at least 95% of the sequence, or at least 98 of the sequence. % or at least 99% identical to it; and/or a heavy chain (HC) that contains the sequence of SEQ ID NO: 61 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 agonistic antibody or an agonistic antigen-binding fragment thereof comprises a light chain (LC) that contains the sequence of SEQ ID NO: 59 or at least 90% of the sequence, at least 95% of the sequence, or at least 95% of the sequence. 98% or at least 99% identical to it; and/or a heavy chain (HC) that contains the sequence of SEQ ID NO: 63 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 agonistic antibody or an agonistic antigen-binding fragment thereof comprises a light chain (LC) that contains the sequence of SEQ ID NO: 59 or at least 90% of the sequence, at least 95% of the sequence, or at least 95% of the sequence. 98% or at least 99% identical to it; and/or a heavy chain (HC) that contains the sequence of SEQ ID NO: 65 or a sequence that is 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 polypeptide variant (e.g., an interferon-associated antigen-binding protein, an interferon-fusion agonist anti-CD40 antibody, or an interferon-fusion agonist antigen-binding fragment thereof, an antibody, an antigen-binding protein, or an IFN, or components thereof) contains an amino acid sequence wherein one, two, three, four, five or more amino acid residues are inserted, deleted and/or replaced in an amino acid sequence compared to another polypeptide sequence. Preferably, the embodiment contains up to ten insertions, deletions and/or substitutions, more preferably up to eight insertions, deletions and/or substitutions. More specifically, the embodiment may contain up to ten, more preferably up to eight, inserts. The variant may also contain up to ten, more preferably up to eight, deletions. In even more preferred embodiments, the variant contains up to ten substitutions, most preferably up to eight substitutions. In some embodiments, these substitutions are conservative amino acid substitutions, as described below.

A polynucleotide sequence variant (e.g., RNA or DNA) contains one or more mutations in the polynucleotide sequence relative to another polynucleotide sequence, wherein one, two, three, four, five or more nucleic acid residues are inserted, deleted, and/or substituted in the sequence nucleic acid. Preferably, the embodiment contains up to ten insertions, deletions and/or substitutions, more preferably up to eight insertions, deletions and/or substitutions. More specifically, the embodiment may contain up to ten, more preferably up to eight, inserts. The variant may also contain up to ten, more preferably up to eight, deletions. In even more preferred embodiments, the variant contains up to ten substitutions, most preferably up to eight substitutions. The 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 another 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 amino acid substitution 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 sequence alignment and comparison. Percent identity refers to 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 molecule being compared. Preferably, identity is determined along the entire length of the sequence. It is understood that the expression is 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 is 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% , is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the reference sequence.

- 12 046818

To calculate percent identity, gaps in alignments (if any) are preferably eliminated using a specific mathematical model or computer program (ie, 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.

When calculating percent identity, the compared sequences are usually aligned to provide the greatest match between 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 the percentage of sequence identity must be determined. Sequences are aligned to optimally match their corresponding amino acid or nucleotide (consensus interval as determined by the algorithm). The gap opening penalty (which is calculated as 3x the average diagonal, where the average diagonal is the average 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 the gap expansion penalty (which is usually 1/10 penalty for opening a space), 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 comparison matrix BLOSum 62) is also used using the algorithm.

The following are examples of parameters that can be used to determine the percentage 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

Space penalty: 12 (but no penalty for trailing spaces)

Space length penalty: 4

Similarity threshold: 0

Some alignment schemes for aligning two amino acid sequences may result in only a short region of the two sequences matching, and this small aligned region may have very high sequence identity even though 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 spans at least 50 or at least 100, preferably the entire length, of contiguous amino acids of the target polypeptide.

Conservative amino acid substitutions may involve unnatural amino acid residues that are typically inserted 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 affecting chain orientation: Gly, Pro; And

6) aromatic: Trp, Tyr, Phe.

For example, non-conservative substitutions may involve replacing 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 in accordance with some embodiments, the hydropathic index of amino acids can be taken into account. Each amino acid was 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).

- 13 046818

The importance of the hydropathic index of an amino acid in imparting interactive biological function to a protein is known to those skilled in the art. Kyte et al., J. Mol. Biol., 157:105-131 (1982). It is known that some amino acids can be replaced by other amino acids that have a similar hydropathic index or indicator, and at the same time maintain similar biological activity. When making changes based on the hydropathic index, in some embodiments, the implementation includes substitution of amino acids whose hydropathic indices are within ±2. 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 replacement of such amino acids can be effectively carried out on the basis of hydrophilicity. In some embodiments, the highest local average hydrophilicity of a protein, as determined by the hydrophilicity of its neighboring amino acids, correlates with its immunogenicity and antigenicity, i.e. with the biological properties of protein.

These amino acid residues were assigned the following hydrophilicity values: 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, in some embodiments include amino acids whose hydrophilicity values are within ±1, and in some embodiments include amino acids whose hydrophilicity values are which are within ±0.5.

Examples of amino acid substitutions are given in table. 2.

Table 2. Amino acid substitutions.2

Initial balances Illustrative Substitutions Preferred Substitutions Ala Vai, Leu, He Vai Arg Lys, Gin, Asn Lys Asn Gin Gin Asp Glu Glu Cys Ser, Ala Ser Gin Asn Asn Glu Asp Asp Gly Pro,Ala Ala His Asn, Gin, Lys, Arg Arg He Leu, Vai, Met, Ala, Phe, Norleucine Leu Leu Norleucine, He, Vai, Met, Ala, Phe lie Lys Arg, 1,4 diaminobutyric acid, Gin, Asn Arg Met Leu, Phe, He Leu Phe Leu, Vai, Tie, 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 Vai He, Met, Leu, Phe, Ala, Norleucine Leu

In light of the present invention, one skilled in the art will be able to identify suitable variants of interferon-associated antigen binding proteins as set forth herein using well known methods. In some embodiments, one of ordinary skill in the art can identify suitable regions of the molecule that can be altered without interfering with activity by targeting regions not believed to be important for activity. In some embodiments, residues and portions of molecules 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 subjected to conservative amino acid substitutions without disrupting biological activity or adversely affecting the structure of the polypeptide.

In addition, one of ordinary skill in the art can review structure-function studies that identify residues in similar polypeptides that are important for activity or structure. In view of this 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. One skilled in the art may select chemically similar amino acid substitutions for such

- 14,046,818 predicted important amino acid residues.

One skilled in the art can also analyze the three-dimensional structure and the amino acid sequence in relation to this structure in similar proteins or protein domains. Given such information, one of ordinary skill in the art can predict the alignment of amino acid residues of an interferon-associated antigen binding protein, an antibody, or an antigen binding fragment thereof, or an interferon or functional fragment thereof, as described herein, with respect to its three-dimensional structure. In some embodiments, one skilled in the art may decide not to make radical changes to amino acid residues that are expected to be on the surface of the protein because such residues may be involved in important interactions with other molecules. In addition, one skilled in the art 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 options can be used to collect information about suitable options. For example, if a change to a particular amino acid residue is found to result in disruption, undesired reduction, or unacceptable activity, variants with that change can be avoided. In other words, based on the information obtained from such experiments, one skilled in the art can easily determine amino acids in which further substitutions should be avoided, either alone or in combination with other mutations.

In some embodiments, amino acid substitutions are substitutions that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for protein complex formation, (4) alter binding affinity and/or (5) impart or modify other physicochemical or functional properties of such polypeptides. In some embodiments, one or more amino acid substitutions (in some embodiments, conservative amino acid substitutions) may be made to a portion of the polypeptide outside the crosslinking domain(s). a conservative amino acid substitution usually cannot significantly change the structural characteristics of the original sequence (eg, the replacement amino acid must not break a helix found in the original sequence or disrupt other types of secondary structure characterizing the original sequence. Examples of secondary and tertiary structures of polypeptides known in the art). 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 involves a chemical modification other than the insertion, deletion, or substitution of amino acids (or nucleic acids). In some embodiments, the derivatives contain 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, the chemically modified interferon-associated antigen-binding protein may have an improved ability to target desired cells, tissues and/or organs. In some embodiments, the interferon-associated antigen binding protein derivative 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, US Pat. 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, monomethoxy polyethylene glycol, dextran, cellulose, or other carbohydrate-based polymers, poly(N-vinylpyrrolidone)-polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyols (eg glycerol) and polyvinyl alcohol, as well as mixtures of such polymers.

In some embodiments, the interferon-associated antigen binding protein derivative 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, for example, at the N-terminus of the derivative. 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 interferon-associated antigen binding

- 15 046818 protein. Some such methods are discussed, for example, in US Pat. No. 6,133,426, which is incorporated herein by reference for all purposes.

In some embodiments, variants of the interferon-associated antigen binding protein include glycosylation variants in which the number and/or type of glycosylation site is changed compared to the amino acid sequences of the parent polypeptide. In some embodiments, the protein variants contain a greater number of N-linked glycosylation sites than the native protein. In other embodiments, protein variants contain fewer N-glycosylation sites than the native protein. The N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, where the amino acid residue designated as X can be any amino acid residue except proline. The 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. Rearrangement of N-linked carbohydrate chains has also been proposed, in which one, two, three, four, five or more N-linked glycosylation sites (typically found in nature) are removed 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 by a different amino acid (eg, serine) from the original amino acid sequence. Cysteine variants may be useful when antibodies need to be converted to a biologically active conformation, for example after isolation of insoluble inclusion bodies. Cysteine variants typically have fewer cysteine residues than the native protein and usually 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 prefer to infect liver cells, but small amounts of hepadnavirus DNA can be found in the kidneys, pancreas, and mononuclear cells. However, infection at these sites is not associated with extrahepatic disease.

HBV virion, i.e. Dane particle, consists of an outer lipid shell and an icosahedral nucleocapsid core consisting of protein. The nucleocapsid contains viral DNA and DNA polymerase, which has reverse transcriptase activity similar to retroviruses. The outer shell contains embedded proteins that are involved in the binding of the virus to sensitive cells and penetration into them. 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 nucleus. These particles are non-infectious and consist of a lipid and protein that form part of the surface of the virion, called surface antigen (HBsAg), which is produced in excess during the life cycle of the virus. 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 during its replication process.

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

The most common 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 does not play any role in virus assembly, but is nonetheless a useful indicator of active viral replication. Because HBeAg is secreted by HBV-infected hepatocytes, it can be detected in the 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, 70,615-625.

The P-coding region is specific for viral polymerase, a multifunctional enzyme involved in DNA synthesis and RNA encapsulation. The X open reading frame encodes the viral protein X (HBx), which modulates host cell signal transduction 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 through envelope proteins. It has been proposed that HBV binds to a plasma membrane receptor that is predominantly expressed on human hepatocytes through the pre-S1 domain of the major envelope protein as an initial step in HBV infection. However, the nature of the receptor remains controversial. The viral membrane then fuses with the cell membrane and the viral genome is released into the cells.

- 16 046818

HBV replication can be regulated by various factors, including hormones, growth factors, and cytokines. After the viral genome reaches the nucleus, the cellular DNA repair machinery converts the partial double-stranded DNA (dsDNA; also called relaxed circular HBV DNA (cDNA)) genome into covalently closed circular DNA (cccDNA). The resulting cccDNA is 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 Int. 2019, 20(17): doi:10. .3390/ijms20174276).

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

HBV core particles assemble in the cytosol, and during this process one molecule of pregenomic RNA is incorporated into the assembling viral core. After encapsidation of the viral RNA, reverse transcription begins. The synthesis of two strands of viral DNA occurs sequentially. The first strand of DNA consists of an encapsulated RNA template; During or after the synthesis of this strand, the RNA template is degraded and the synthesis of the second DNA strand continues, using the newly obtained first DNA strand as a template. Some nuclei carrying the mature genome are transported back to the nucleus, where their newly minted DNA genomes can be converted into cccDNA to maintain a stable intranuclear pool of transcription 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 follows. The assembled HBV virion and subviral particles are transported to the Golgi apparatus for further modification of surface protein glycans and then secreted from the host cell to complete the life cycle.

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 the viral load/titer of the HBV virus; (ii) reduction of cccDNA transcription; (iii) reducing the level of pregenomic RNA in cells; (iv) ameliorating one or more diseases associated with HBV; and (v) reducing one or more HBV-related symptoms in the patient.

The terms viral load and viral titer refer to the number 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). The viral load or viral titer can also be defined as the equivalent of the so-called viral genome. Higher viral load, titer, or viral load often correlates with the severity of 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 quantifying hepatitis B virus DNA have been described, for example, in Liu et al., Virol J14, 94 (2017) doi:10. 1186/s12985-017-0759-8. Because of the ease of detection of viral DNA by PCR, viral load is useful in clinical settings to monitor success during treatment. Viral load >10,000 copies/mL (2000 IU/mL) is a strong predictor of hepatocellular carcinoma risk, regardless of HBeAg status.

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

In specific embodiments, the interferon-associated antigen-binding protein, nucleic acids, vectors, vector systems, methods, and compositions described herein can be used to reduce the viral load/titer of the HBV virus in an HBV-infected cell (e.g., a culture cell, an organ, infected with HBV, or in a patient infected with HBV). HBV viral load/titer can be reduced by approximately 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared with 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%, by at least 25%, by at least 30%, by at least 35%, by at least

- 17 046818

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, viral load/virus titer is determined using PCR or quantitative RT-PCR.

In specific embodiments, interferon-associated antigen binding protein, nucleic acids, vectors, vector systems, methods and compositions described herein can be used to reduce 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). cccDNA transcription can be reduced by approximately 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90 %, 95% or 100% compared with an untreated HBV-infected cell culture or the same patient before 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%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by by at least 85%, by at least 90%, or by at least 95%. Preferably, HBV cccDNA transcription is reduced by at least 35%, more preferably by at least 50%. In some embodiments, HBV cccDNA transcription is determined using PCR or qPCR.

In specific 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., a cell culture, an organ, infected with HBV, or in a patient infected with HBV). HBV pregenomic RNA levels can be reduced by approximately 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%, by at least 85%, by at least 90%, or by 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 using quantitative RT-PCR.

As used herein, the term HBV-related disorder refers to a disorder that results from a patient being infected with HBV. HBV-related 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-related symptom, HBV infection symptom, or HBV-related 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, acrodermatitis papularis in children (Gianotti-Crosti syndrome), HBV-associated nephropathy (eg, membranous glomerulonephritis), immune-mediated hematological 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 levels, anorexia, malaise, fever, nausea, vomiting and the like.

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 (eg, IFNa, IFNe, IFNo: IFNk, IFNt, IFNZ and ΙΕΝω), type II IFNs (eg, ZFNy) and type III IFNs (eg, IFNXl, IFNX2 and IFNX3). The term interferon or IFN includes, but is not limited to, full-length IFN, a variant or derivative thereof (eg, a chemically (eg, pegylated) modified derivative or mutein), or a functionally active fragment thereof that retains one or more of the signaling activities of full-length IFN.

As used herein, the term functional fragment refers to a fragment

- 18 046818 substances that retain one or more functional activities of the original substance. For example, a functional interferon fragment refers to a fragment of interferon that retains the function of IFN, as described herein, for example, it mediates IFN pathway signaling.

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 cognate IFN receptor (IFNAR for IFNa, IFNBR for IFNe, etc.). In some embodiments, 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 (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 JAK/STAT/ISGF3 pathway activation 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 the CD40, tuk and IFN pathways. 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 an IFNa (interferon alpha), IFNe (interferon beta), IFNi (interferon epsilon), IFNio (interferon omega), IFNy (interferon gamma) or IFA (interferon lambda) pathway ).

In accordance with some illustrative embodiments, an interferon-associated antigen binding protein described herein comprises a full-length IFN, a variant or derivative thereof (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 full-length IFN. In some embodiments, the IFN is human IFN.

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

In certain embodiments, the IFN or a functional fragment thereof is a type I IFN or a functional fragment thereof. In specific embodiments, the type I IFN or a functional fragment thereof is IFNa, IFNe, IFNio, or IFNo: or a functional fragment thereof. In more specific embodiments, the type I IFN or a functional fragment thereof is IFNa or IFNe or a functional fragment thereof. In other more specific embodiments, the type I IFN or a functional fragment thereof is IFN a or a functional fragment thereof. In other more specific embodiments, the type I IFN or functional fragment thereof is IFN e or a functional fragment thereof. In other more specific embodiments, the type I IFN or functional fragment thereof is IFNio 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 specific embodiments, the IFN or a functional fragment thereof is IFNa, IFNe, IFNy, IFA, IFNo: or IFNio or a functional fragment thereof. In certain embodiments, the IFN or a functional fragment thereof is IFNa or IFNe or a functional fragment thereof.

In some embodiments, the IFN or a 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 contain the sequence SEQ ID NO: 17, or a sequence identical to it at least 90%.

In some embodiments, the IFN or a functional fragment thereof is IFNe or a functional fragment thereof. IFNe may contain the sequence SEQ ID NO: 14 or a sequence that is at least 90% identical thereto. IFNe or a functional fragment thereof may contain 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

- 19 046818 amino acid substitution C17S relative to SEQ ID NO: 14. In some embodiments, ΙΕΝβ comprises the amino acid sequence of SEQ ID NO: 15. In other embodiments, IFNe contains amino acid substitutions C17S and N80Q relative to SEQ ID NO: 14. In other embodiments, IFNe contains the amino acid sequence SEQ ID NO: 16.

In some embodiments, the IFN or a functional fragment thereof is IFNY or IFN/ or a functional fragment thereof. In certain embodiments, the IFN or a functional fragment thereof is IFNy or a functional fragment thereof. In more specific embodiments, IFNy comprises the sequence of SEQ ID NO: 19 or a sequence that is at least 90% identical thereto. In other specific embodiments, the IFN or a functional fragment thereof is IFN. or a functional fragment thereof. In more specific embodiments, IFN. or the functional fragment thereof is IFN/.2 or a functional fragment thereof. IFN/.2 may contain the sequence of SEQ ID NO: 18, or a sequence identical to it at least 90%.

In some embodiments, the IFN or a functional fragment thereof is an IFN or a functional fragment thereof. The IFN may contain the sequence of SEQ ID NO: 80, or a sequence that is at least 90% identical thereto.

In some embodiments, the IFN or a functional fragment thereof is IFNe! or a functional fragment thereof. IFNio may contain the sequence SEQ ID NO: 79, or a sequence identical to it at least 90%.

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

In accordance with 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 illustrative embodiments, a suitable IFN pathway-inducible biomarker is CXCL9, CXCL10, and/or CXCL11, as well as the interferon-stimulated gene ISG20. The induction or release of cytokines 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, the upregulation may be at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold -fold, at least 5-fold or at least 10-fold increase in the expression or secretion of these cytokines.

In these or other illustrative embodiments, the expression level of pro-inflammatory 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 slightly increased in human whole blood cells upon treatment with an interferon-associated antigen binding protein of the invention. In some embodiments, the expression level of IL1 β is slightly increased in human whole blood cells upon treatment with an interferon-associated antigen binding protein of the invention. In some embodiments, the expression level of IL2 is slightly increased in HBV-infected cells upon treatment with an interferon-associated antigen binding protein of the invention. In some embodiments, the expression levels of IL10 and IL1e 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 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 IL1[/ 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, IL1β, and IL2 are not significantly increased in HBV-infected cells upon treatment with the interferon-associated antigen binding protein of the invention.

Interferon-associated antigen-binding proteins

The term associated, as used herein, generally refers to the covalent or non-covalent bond of two (or more) molecules. Associated proteins are created by

- 20 046818 combining two or more different peptides or proteins, resulting in a protein with one or more functional properties derived from each of the original proteins. In the context of the present invention, interferon-associated antigen binding proteins activate both the CD40 and IFN pathways. Associated protein encompasses monomeric and multimeric, eg dimeric, trimeric, tetrameric or the like, complexes of various associated or fusion proteins. In this context, non-covalent bonding results from strong interactions between two regions of the protein surface, typically through ionic, van der Waals and/or hydrogen bonds. Covalent bonding, on the other hand, requires the presence of actual chemical bonds such as peptide bonds, disulfide bridges, etc. As used herein, the term fusion generally refers to the joining of two or more different peptides or proteins in a covalent manner through a peptide bond. Thus, a fusion protein refers to a single protein created by joining 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 can be fused to each other through one or more peptide linkers (L).

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

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

In other embodiments, the IFN or a functional fragment thereof is covalently linked to an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof. In preferred embodiments, the IFN or a functional fragment thereof is fused to an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof. IFN or a functional fragment thereof can be fused to the light chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof. In some embodiments, the IFN or a functional fragment thereof is fused to the N-terminus of the light chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof. In other embodiments, the IFN or a functional fragment thereof is fused to the C-terminus of the light chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof. IFN or a functional fragment thereof can also be fused to the heavy chain of an agonistic anti-CD40 antibody or an agonistic 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 agonistic anti-CD40 antibody or an agonistic 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 agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof. In any of these embodiments, an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof and an 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 links one or more agonistic anti-CD40 antibodies or agonistic antigen-binding fragments thereof to one or more interferons or functional fragments thereof. In typical 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 as used herein may have one or more of the properties described below. The peptide linker sequences in accordance with some illustrative embodiments are presented in table. 7.

The peptide linker can be of any length, i.e. contain any number of amino acid residues. In illustrative 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 typically of sufficient length to provide a sufficient degree of flexibility to prevent the linked portions from interfering with each other's activity, such as the ability of the moiety to bind to a receptor. In illustrative embodiments, the linker contains 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 can contain up to 80 amino acids. The linker can contain up to 40 amino acids. The linker can contain up to 24 amino acids

- 21 046818 lot. The linker can contain up to 21 amino acids. The linker can contain up to 20 amino acids. The linker can contain up to 15 amino acids. The linker can contain up to 13 amino acids. The linker can contain up to 12 amino acids. The linker can contain up to 11 amino acids. The linker can contain up to 4 amino acids.

In some embodiments, the linker is selected from the group consisting of rigid, flexible, and/or helical linkers. It is clear that linkers that form a helix can also be rigid linkers, since an α-helix has fewer degrees of freedom than a peptide adopting a more random helix conformation. In some embodiments, the linker is a rigid linker. An exemplary rigid linker contains the sequence SEQ ID NO: 20. Additional exemplary rigid linkers contain the sequence SEQ ID NO: 22 or SEQ ID NO: 23. In related embodiments, the linker is a helix-forming linker. Examples of helix linkers include the sequence SEQ ID NO: 22 or SEQ ID NO: 23. In other embodiments, the linker is a flexible linker. Examples of flexible linkers contain 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 contain one or more acidic amino acids, such as Asp or Glu. Basic linkers typically contain 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 contain the sequence 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 include the sequence 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 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 contains the sequence SEQ ID NO: 21.

In exemplary embodiments of the present invention, the interferon-associated antigen binding protein comprises a linker comprising a sequence selected from SEQ ID NO: 20-26, preferably from SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26. B in a preferred embodiment, the linker contains the sequence SEQ ID NO: 24. In another preferred embodiment, the linker contains the sequence SEQ ID NO: 25. In another preferred embodiment, the linker contains 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 contain amino acids other than the amino acids forming (I) the specified agonistic anti-CD40 antibody or agonistic antigen-binding fragment thereof and (II) the specified IFN or a functional fragment thereof. In related embodiments, the interferon-associated antigen-binding protein does not contain amino acids other than the amino acids forming (I) said agonistic anti-CD40 antibody or agonistic antigen-binding fragment thereof, (II) said IFN or a functional fragment thereof, and (III) said linker.

Exemplary embodiments representing various configurations of (I) an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof, (II) an interferon (IFN) or a functional fragment thereof, and (III) a linker are described below.

In some preferred embodiments, the IFN or a functional fragment thereof is fused to the C-terminus of the heavy chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof via a linker as set forth in Table. 3A or table 3B. In these embodiments, the heavy chain of an agonistic anti-CD40 antibody or an agonistic 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, SEQ ID NO: 61 or SEQ ID NO: 63. IFNa2a may contain the sequence SEQ ID NO: 17. IFNe may contain the sequence SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. IFNe may contain the sequence SEQ ID NO: 14. IFNe_C17S may contain the sequence SEQ ID NO: 15. IFNe_C17S, N80Q may contain the sequence SEQ ID NO: 16. IFNy may contain the sequence SEQ ID NO: 19. IFNX2 may contain the sequence SEQ ID NO: 18. IF№: may contain the sequence SEQ ID NO: 80. IFNio may contain the sequence SEQ ID NO: 79. Mentioned linkers are listed in table. 7.

In embodiments wherein the IFN is fused to the C-terminus of the pro-agonist antibody heavy chain

- 22 046818 anti-CD40 antibody or an agonistic antigen-binding fragment thereof, the interferon-associated antigen-binding protein further comprises a light chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof. In more specific embodiments, the heavy chain comprises the sequence 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 SEQ ID NO: 48 or SEQ ID NO: 49 to SEQ ID NO: 3. In other more specific embodiments, the heavy chain contains the sequence of SEQ ID NO: 61 or SEQ ID NO: 63 and the light chain contains the sequence of SEQ ID NO: 59.

Table 3 Interferon or a functional fragment thereof fused to the C-terminus of the heavy chain of an anti-CD40 antibody or an agonistic antigen-binding fragment thereof

A IFNa2a IFNp IFNp_CI7 S IFNp_C17S, N80Q IFNy IFN7.2 RL link P antiСО40_Н C—RL— IFNa2a antiCO4 0-HC- RL-IFNp antiCO4 0-HCRLIFNp_C17 S antiСО40_HC-RL— ΙΡΝβ C17S, N80Q antiCO4 0-HC- RL-IFNy antiСО40_Н C—RL—IFNX2 GST link P antiСО40_Н C-GSTIFNa2a antiCO4 0-HCGSTIFNp antiCO4 0-HCGSTIFNp_C17 S antiC D40_HC-GSTIFN3_C17S, N80Q antiCO4 0-HCGSTIFNy antiСО40_Н C-GST- IFNX2 HL link P antiСО40_Н C—HL— IFNa2a antiCO4 0-HC- HL-IFNp antiCO4 0-HCHLIFNp_C17 antiC D40_HC-HLIFNp_C17S, N80Q antiCO4 0-HC- HL-IFNy antiСО40_Н C—HL— IFNX2 HL2 link P (G4S) 2 link P (G4S) 3 link P (G4S) 4 link P antiСО40_Н C-HL2IFNa2a antiСО40_Н C-(G4S)2— IFNa2a antiC D40H C-(G4S)3— IFNa2a antiСО40_Н C-(G4S)4— IFNa2a antiCB4 0-HCHL2—IFNp antiCO4 0-HC(G4S)2IFNp antiCB4 0_HC-(G4S)3IFNp antiCO4 0-HC(G4S)4IFNp S antiСО4 0-HCHL2IFNp_C17 S antiСО4 0-HC(G4S)2— IFNp_C17 S antiСО4 0_HC(G4S)3— IFNp_C17 S antiСО4 0-HC(G4S)4IFNp_C17 S antiСО40_HC-HL2IFNp_C17S, N80Q antiСО40_ HC(G4S)2IFNp_C17S , N80Q antiСО40_ HC(G4S)3IFNp_C17S , N80Q antiСО40_ HC(G4S)4IFNp_C17S , N80Q antiСО4 0-HCHL2— IFNy antiСО4 0-HC(G4S)2IFNy antiСО4 0_HC-(G4S)3ifn ₇ antiСО4 0-HC(G4S)4IFNy antiCH40_H C-HL2- ΙΡΝλ2 antiСО40_Н C—(G4S)2— IFNX2 antiСО40_Н C—(G4S)3—IFNX2 antiСО40_Н C—(G4S)4— IFNX2

IN IFNe IFNco RL linker antiCO40_HCRL—IFNe aHTHCD40_HC-RLIFNoj GST linker antiCO40_HCGST-IFNe antiСО40_НС-GSTIFNco HL linker antiCO40_HCHL—IFNe antiCO40_NS-НН- IFNco HL2 linker antiCO40_NS- HL2-IFNe antiCO40_NS-Hb2- IFNoj (G4S)2 linker antiCO40_NS— (G4S)2-IFNe antiCO40_NS- (G4S)2-IFNro (G4S)3 linker antiCO40_HC(G4S)3-IFNe antiCO40_NS- (G4S)3—IFNffl (G4S)4 linker antiСО40_НС— (G4S)4-IFNe antiCO40_HC(G4S)4-IFN(D

In some preferred embodiments, the IFN or a functional fragment thereof is fused to the N-terminus of the heavy chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof via a linker as set forth in Table. 4A or table 4B. In these embodiments, the heavy chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof may comprise the sequence SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 61, SEQ ID NO: 63 or SEQ ID NO: 65. IFNa2a

- 23 046818 may contain the sequence SEQ ID NO: 17. IFNe may contain the sequence SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16. IFNe may contain the sequence SEQ ID NO: 14. IFNe_C17S may contain the sequence SEQ ID NO: 15. IFNe_C17S, N80Q may contain the sequence SEQ ID NO: 16. IFNy may contain the sequence SEQ ID NO: 19. IFNX2 may contain the sequence SEQ ID NO: 18. Π ^; \ε may contain the sequence SEQ ID NO: 80. IFNω may contain the sequence SEQ ID NO: 79. Mentioned linkers are listed in table. 7.

In embodiments where the IFN is fused to the N-terminus of the heavy chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof, the interferon-associated antigen-binding protein further comprises a light chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof. In more specific embodiments, the heavy chain comprises the sequence 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 SEQ ID NO: 3. In other more specific embodiments, the heavy chain contains the sequence of SEQ ID NO: 61, SEQ ID NO: 63, or SEQ ID NO: 65 and the light chain contains the sequence of SEQ ID NO: 59.

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

A IFNa2a ΙΡΝβ IFNP_C1 7S IFNP_C17S, N80Q IFNy IFNX2 RL link P IFNa2a—RL- antiСО40_Н С IFNp-RLantiСО40_NS IFNp_C17 S-RLantiCO4 0_HC IFNp_C17S, N80Q-RLantiСО40_Н C IFNy—RLantiСО4 0_HC IFNX2— RLantiCO4 0_HC GST link P IFNa2a— GSTantiСО40_Н С IFNp-GSTantiCO40 NS IFNp_C17 S-GSTantiCO4 0_HC IFNp_C17S, N80Q-GSTantiСО40_Н C IFNy— GST— antiСО4 0_HC IFNX2— GSTantiCO4 0_HC HL link R IFNa2a—HL- antiСО40_Н С IFNp-HLantiСО40_ NS IFNp_C17 S-HLantiCO4 0_HC IFNp_C17S, N80Q-HLantiСО40_Н C IFNy—HLantiSV4 0_HC Π?Νλ2HLantiСО4 0_HC HL2 link R IFNa2a— HL2—antiСО40_Н С IFNP-HL2antiCO40_NS IFN3_C17 S-HL2antiСО4 0_HC IFN3_C17S, N80Q-HL2antiСО40_Н C IFNy— HL2antiСО4 0_HC Π?Νλ2HL2— antiСО4 0_HC (G4S) 2 link R IFNa2a— (G4S)2antiСО40_Н С IFNp(G4S)2—antiCO40_NS IFNp_C17 S(G4S)2—antiСО4 0_HC IFNp_C17S, N80Q- (G4S)2—antiСО40_Н C IFNy— (G4S)2antiСО4 0_HC Π?Νλ2(G4S)2antiСО4 0_HC (G4S) 3 link R IFNa2a— (G4S)3antiСО40_Н С IFNp(G4S)3—antiCO40_NS IFNp_C17 S(G4S)3antiCO4 0_HC IFNp_C17S, N80Q- (G4S)3antiCO40_H C IFNy— (G4S)3antiСО4 0_HC Π?Νλ2(G4S)3antiСО4 0_HC (G4S) 4 IFNa2a— (G4S)4— IFNp(G4S)4— IFNp_C17 S- IFNp_C17S, N80Q- IFNy—(G4S)4- Π?Νλ2- (G4S)4-

- 24 046818

link P antiСО40_Н C antiSLE40_ NS (G4S)4—antiCO4 0 NS (G4S)4—antiСО40_Н С antiCO4 0_NS antiC D4 (LHC

IN IFNe IFNm RL link P IFNs-RLantiСО40_Н С IFNo-RLantiСО40_ NS GST link P IFNe-GSTantiСО40_Н С IFNo-GST- antiCO40_ NS HL link P IFNe-HLantiСО40_Н С IFNo-HLantiCO40_ NS HL2 link P IFNe—HL2—antiСО40_Н С IFNo—HL2—antiCO40_NS (G4S) 2 link P IFNe— (G4S)2—antiСО40_Н С IFNco(G4S)2—antiCO40_NS (G4S) 3 link R IFNe— (G4S)3antiСО40_Н С IFNio— (G4S)3antiCO40_NS (G4S) 4 link P IFNe— (G4S)4—antiСО40_Н С IFNco— (G4S)4—antiCO40_NS

In some preferred embodiments, the IFN is fused to the C-terminus of the light chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof via a linker as set forth in Table. 5A or table 5B. In these embodiments, the light chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof may comprise the sequence of SEQ ID NO: 3. In other embodiments, the light chain may comprise the sequence of SEQ ID NO: 59. IFNa2a may comprise the sequence of SEQ ID NO: 17. IFNe may contain the sequence SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16. IFNe may contain the sequence SEQ ID NO: 14. IFNe_C17S may contain the sequence SEQ ID NO: 15. IFNe_C17S, N80Q may contain the sequence SEQ ID NO : 16. IFNy may contain the sequence SEQ ID NO: 19. IFNX2 may contain the sequence SEQ ID NO: 18. Π ^; \ε may contain the sequence SEQ ID NO: 80. IFNω may contain the sequence SEQ ID NO: 79. Mentioned linkers are listed in table. 7.

In embodiments where the IFN is fused to the C-terminus of the light chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof, the interferon-associated antigen-binding protein further comprises a heavy chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof. In more specific embodiments, the light chain comprises the sequence SEQ ID NO: 3 and the heavy chain comprises the sequence 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. In other more specific embodiments, the light chain comprises the sequence SEQ ID NO: 59 and the heavy chain comprises the sequence SEQ ID NO: 61, SEQ ID NO: 63, or SEQ ID NO: 65.

- 25 046818

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

A IFNa2a ΙΡΝβ IFNPC17 S IFNP_C17S, N80Q IFNy IFNX2 RL link P antiC D40LC -RL—IFNa2a antiSP40 -LC-RL- IFNp antiCB40 _LC—RL—IFNp_C17 S antiCO40_ LC-RL-- IFNp_C17S, N80Q antiCO40 _LC—RL—IFNy antiCO40 -LC-RLIFNX2 GST link P antiSV40_T.S -GST-IFNa2a antiC4)40_LC—GSTIFNp antiSG)40 _LC-GSTIFNp_C17 S antiCB40_LC-GSTIFNp_C17S, N80Q antiCH40 _LC-GSTIFNy antiCH40_LC—GSTIFNF2 HL link P antiСО40_EC -HL-IFNa2a antiCO40 -LC-HL- TFNp antiCO40 _LC-HL IFNp_C17 S antiCB40_LC-HL -IFNp_C17S, N80Q antiCO40 _LC -HL-IFNy antiCO40 -LC-HL- IFNX2 HL2 link P antiSV40_EC -HL2—IFNa2a antiSP40 _LC— HL2— IFNp antiСО40_LC-HL2IFNp_C17 S antiСО40_LC-HL2IFNp_C17S, N80Q antiCO40 _LC-HL2IFNy antiCO40 _LCHL2IFNX2 (G4S)2 link R aHTiiCD40_LC -(G4S)2IFNa2a antiCB40 _LC— (G4S)2IFNp antiCO40_LC-(G4S)2IFNp_C17 S antiCO40_ LC-(G4S)2- IFNp_C17S, N80Q antiCO40_LC-(G4S)2IFNy antiCO40 _LC(G4S)2IFNX2 (G4S)3 link R antiCO40_bs -(G4S)3- IFNa2a aiiTiiCD40 _LC— (G4S)3IFNp aimiCD40_LC-(G4S)3IFNp_C17 S antiCO40_ LC-(G4S)3- IFNp_C17S, N80Q airrnCD40_LC-(G4S)3IFNy antiCO40 _LC- (G4S)3- IFNX2 (G4S)4 link R aHTiiCD40_LC -(G4S)4IFNa2a antiCO40 _LC— (G4S)4IFNp antiСО40_LC (G4S)4IFNp_C17 S antiCO40_ LC-(G4S)4- IFNp_C17S, N80Q antiCO40 _LC (G4S)4IFNy antiCO40 _LC(G4S)4IFNX2

IN IFNe IINo) RL linker aiTinCD40_L C-RL-IFNa aHTiiCD40_LC -RL-IFNro GST linker aHTnCD40_L C-GSTIFNa antiCO40_bs -GST-IFNro HL linker antiCO40 L C-HL-IFNs antiSP40 LC -HL-IFNro HL2 linker antiCO40_b C-HL2-IFN8 aHTiiCD40_LC -HL2-IFNro (G4S)2 linker antiCO40_b C-(G4S)2- IFNe aHTiiCD40_LC -(G4S)2FFNco (G4S)3 linker antiCB40_b C-(G4S)3- IFNs aHTiiCD40_LC -(G4S)3IFNw (G4S)4 linker aHTnCD40_L C-(G4S)4- IFNs aHTiiCD40_LC -(G4S)4FFNco

In some preferred embodiments, the IFN is fused to the N-terminus of the light chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof via a linker, as set forth in Table. 6A or table 6B. In these embodiments, the light chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof may comprise the sequence of SEQ ID NO: 3 or SEQ ID NO: 59. IFNa2a may comprise the sequence of SEQ ID NO: 17. IFNe may contain the sequence of SEQ ID NO: 14 , SEQ ID NO: 15 or SEQ ID NO: 16. IFNe may contain the sequence SEQ ID NO: 14. IFNe_C17S may contain the sequence SEQ ID NO: 15. IFNe_C17S, N80Q may contain the sequence SEQ ID NO: 16. IFNy may contain the sequence SEQ ID NO: 19. IFN/.2 may contain the sequence of SEQ ID NO: 18. IFN may contain the sequence of SEQ ID NO: 80. IFNo may contain the sequence of SEQ ID NO: 79. Said linkers are listed in table. 7.

In embodiments wherein the IFN is fused to the N-terminus of the pro-agonist antibody light chain

- 26 046818 anti-CD40 or an agonistic antigen-binding fragment thereof, the interferon-associated antigen-binding protein further comprises a heavy chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof. In more specific embodiments, the light chain comprises the sequence SEQ ID NO: 3 and the heavy chain comprises the sequence 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. In other more specific embodiments, the light chain comprises the sequence SEQ ID NO: 59 and the heavy chain comprises the sequence SEQ ID NO: 61, SEQ ID NO: 63, or SEQ ID NO: 65.

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

A IFNa2a IFNp IFNp_C17S IFNp_C17S, N80Q IFNy 1IX/.2 RL linker IFNa2a- RLantiCO40 _LC IFNp-RL- antiCO40 _LC IFNp_C17S -RLantiCO40_LC IFNp_C17S, N80Q-RLa _H TiiCD40_LC IFNy—RLantiСО40 _LC IFNX2— RLantiCO40 _LC GST linker IFNa2a— GSTantiCO40_LC IFNp- GSTantiCO40_LC IFNp_C17S -GST—antiСО40_LC IFNp_C17S, N80Q-GST- aHTiiCD40_LC IFNyGSTantiСО40 _LC IFNX2GSTantiCO40 _LC HL linker IFNa2a- HLantiCO40 _LC IFNp-HL- antiCO40 _LC IFNp_C17S -HLantiСО40_LC IFNp_C17S, N80Q-HLa _H THCD40_LC IFNy-HLantiСО40 _LC IFNX2— HLantiCO40 _LC HL2 linker IFNa2a— HL2antiSV40 _LC ΙΡΝβ- HL2—antiS1E40 _LC IFNp_C17S -HL2antiСО40_LC IFN3_C17S, N80Q-HL2- aHTiiCD40_LC IFNyHL2antiСО40 _LC IFNX2HL2—antiCB40 _LC (G4S) 2 linker IFNa2a(G4S)2—antiСО40 _LC IFNp(G4S)2antiСО40 _LC IFNp_C17S —(G4S)2— antiCB40_ LC IFNp_C17S, N80Q—(G4S)2- aHTiiCD40_LC IFNy(G4S)2antiСО40 _LC IFNX2— (G4S)2— atmiCD40_LC (G4S) 3 linker IFNa2a— (G4S)3antiCO40 _LC IFNp(G4S)3antiСО40 _LC IFNp_C17S -(G4S)3aptiCO40_ LC IFNp_C17S, N80Q-(G4S)3- antiCO40 LC IFNy(G4S)3antiСО40 L.C. IFNX2(G4S)3antiCO40 L.C. (G4S) 4 linker IFNa2a(G4S)4—antiСО40 _LC IFNp(G4S)4antiCO40 _LC IFNp_C17S —(G4S)4— antiСО40_ LC IFNp_C17S, N80Q—(G4S)4- aHTiiCD40_LC IFNy(G4S)4antiСО40 _LC IFNX2— (G4S)4—antiCO40 _LC

IN IFNs IFNo RL linker IFNe-RL- antiCO40 LC IFNro-RLantiCO40 LC GST linker IFNs-GST- antiCO40 LC IFNro-GSTantiCO40 LC HL linker IFNa-HL- antiCO40 LC IFNro-HL- antiCO40 LC HL2 linker IFN8-HL2- antiCO40 LC IFN<d-HL2antiСО40 LC (G4S)2 linker IFNs-(G4S)2- antiCO40 LC IFNro-(G4S)2antiСО40 LC (G4S)3 linker IFN8-(G4S)3- antiCO40 LC IFN(D-(G4S)3antiCO40 LC (G4S)4 linker IFN8-(G4S)4- antiCO40 LC IFN(D-(G4S)4antiCO40 LC

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

In typical preferred embodiments, the interferon-associated antigen-binding protein comprises an interferon-fusion agonist anti-CD40 antibody or an interferon-fusion agonist antigen-binding fragment thereof comprising a sequence selected from SEQ ID NOs: 28-47 or SEQ ID NOs: 66-75. In other illustrative embodiments, the interferon-associated antigen-binding protein comprises an interferon-fusion agonist anti-CD40 antibody or an interferon-fusion agonist antigen-binding fragment thereof comprising a sequence selected from SEQ ID NOs: 81-88. In illustrative preferred embodiments, the interferon-associated antigen-binding protein is an interferon-fusion agonist anti-CD40 antibody or an interferon-fusion agonist antigen-binding fragment thereof comprising a sequence selected from SEQ ID NOs: 28-47 or SEQ ID NOs: 66-75. In other illustrative embodiments

- 27 046818 interferon-associated antigen-binding protein is an interferon-fusion agonist anti-CD40 antibody or an interferon-fusion agonist antigen-binding fragment thereof containing a sequence selected from SEQ ID NOs: 81-88.

In some illustrative embodiments, the interferon-associated antigen-binding protein comprises an interferon-fusion agonist anti-CD40 antibody or an interferon-fusion agonistic binding fragment thereof comprising the sequence SEQ ID NO: 81. In another illustrative embodiment, the interferon-associated antigen-binding protein is an interferon-fusion an anti-CD40 agonist antibody or an interferon fusion agonist binding fragment thereof containing the sequence SEQ ID NO: 81.

In some illustrative embodiments, the interferon-associated antigen-binding protein comprises an interferon-fusion agonist anti-CD40 antibody or an interferon-fusion agonistic binding fragment thereof comprising the sequence SEQ ID NO: 82. In another illustrative embodiment, the interferon-associated antigen-binding protein is an interferon-fusion an agonistic anti-CD40 antibody or an interferon fusion agonist binding fragment thereof containing the sequence SEQ ID NO: 82.

In some illustrative embodiments, the interferon-associated antigen-binding protein comprises an interferon-fusion agonist anti-CD40 antibody or an interferon-fusion agonistic binding fragment thereof comprising the sequence SEQ ID NO: 83. In another illustrative embodiment, the interferon-associated antigen-binding protein is an interferon-fusion an agonistic anti-CD40 antibody or an interferon fusion agonist binding fragment thereof containing the sequence SEQ ID NO: 83.

In some illustrative embodiments, the interferon-associated antigen-binding protein comprises an interferon-fusion agonist anti-CD40 antibody or an interferon-fusion agonistic binding fragment thereof comprising the sequence SEQ ID NO: 84. In another illustrative embodiment, the interferon-associated antigen-binding protein is an interferon-fusion an anti-CD40 agonist antibody or an interferon fusion agonist binding fragment thereof containing the sequence SEQ ID NO: 84.

In some illustrative embodiments, the interferon-associated antigen-binding protein comprises an interferon-fusion agonist anti-CD40 antibody or an interferon-fusion agonistic binding fragment thereof comprising the sequence SEQ ID NO: 85. In another illustrative embodiment, the interferon-associated antigen-binding protein is an interferon-fusion an agonistic anti-CD40 antibody or an interferon fusion agonist binding fragment thereof containing the sequence SEQ ID NO: 85.

In some illustrative embodiments, the interferon-associated antigen-binding protein comprises an interferon-fusion agonist anti-CD40 antibody or an interferon-fusion agonistic binding fragment thereof comprising the sequence SEQ ID NO: 86. In another illustrative embodiment, the interferon-associated antigen-binding protein is an interferon-fusion an agonistic anti-CD40 antibody or an interferon fusion agonist binding fragment thereof containing the sequence SEQ ID NO: 86.

In some illustrative embodiments, the interferon-associated antigen-binding protein comprises an interferon-fusion agonist anti-CD40 antibody or an interferon-fusion agonistic binding fragment thereof comprising the sequence SEQ ID NO: 87. In another illustrative embodiment, the interferon-associated antigen-binding protein is an interferon-fusion an agonistic anti-CD40 antibody or an interferon fusion agonist binding fragment thereof containing the sequence SEQ ID NO: 87.

In some illustrative embodiments, the interferon-associated antigen-binding protein comprises an interferon-fusion agonist anti-CD40 antibody or an interferon-fusion agonistic binding fragment thereof comprising the sequence SEQ ID NO: 88. In another illustrative embodiment, the interferon-associated antigen-binding protein is an interferon-fusion an agonistic anti-CD40 antibody or an interferon fusion agonist binding fragment thereof containing the sequence SEQ ID NO: 88.

In more preferred embodiments, the interferon-associated antigen-binding protein comprises an interferon-fusion agonist anti-CD40 antibody or an interferon-fusion agonist antigen-binding fragment thereof comprising a sequence

- 28 046818 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, an interferon-associated antigen-binding the protein is an interferon-fusion agonist anti-CD40 antibody or an interferon-fusion agonist antigen-binding fragment thereof containing 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 other more preferred embodiments, the interferon-associated antigen-binding protein comprises an interferon-fusion agonist anti-CD40 antibody or an interferon-fusion agonist antigen-binding fragment thereof comprising a sequence selected from SEQ ID NO: 72, SEQ ID NO : 73, SEQ ID NO: 74 and SEQ ID NO: 75. In other more preferred embodiments, the interferon-associated antigen binding protein is an interferon fusion agonist anti-CD40 antibody or an interferon fusion agonist antigen binding fragment thereof comprising a sequence selected from SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 and SEQ ID NO:75.

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

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

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

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

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

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

In another even more preferred embodiment, the interferon-associated antigen-binding protein comprises an interferon-fused anti-CD40 agonist antibody or an interferon-fused agonistic binding fragment thereof comprising the sequence SEQ ID NO: 72. In another even more preferred embodiment, the interferon-associated antigen-binding protein is a fusion an interferon agonist anti-CD40 antibody or an interferon fusion agonist binding fragment thereof containing the sequence SEQ ID NO: 72.

- 29 046818

In another even more preferred embodiment, the interferon-associated antigen-binding protein comprises an interferon-fusion agonist anti-CD40 antibody or an interferon-fusion agonist binding fragment thereof comprising the sequence SEQ ID NO: 73. In another even more preferred embodiment, the interferon-associated antigen-binding protein is a fusion an interferon agonist anti-CD40 antibody or an interferon fusion agonist binding fragment thereof containing the sequence SEQ ID NO: 73.

In another even more preferred embodiment, the interferon-associated antigen-binding protein comprises an interferon-fused anti-CD40 agonist antibody or an interferon-fused agonistic binding fragment thereof comprising the sequence SEQ ID NO: 74. In another even more preferred embodiment, the interferon-associated antigen-binding protein is a fusion an interferon agonist anti-CD40 antibody or an interferon fusion agonist binding fragment thereof containing the sequence SEQ ID NO: 74.

In another even more preferred embodiment, the interferon-associated antigen-binding protein comprises an interferon-fused anti-CD40 agonist antibody or an interferon-fused agonistic binding fragment thereof comprising the sequence SEQ ID NO: 75. In another even more preferred embodiment, the interferon-associated antigen-binding protein is a fusion an interferon agonist anti-CD40 antibody or an interferon fusion agonist binding fragment thereof containing the sequence SEQ ID NO: 75.

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

Name/SEQ GO number Subsequence Signal peptide 1 (SEQ ID NO 1) MGWSCIILFLVATATGVHS Signal peptide 2 (SEQ ID NO 2) MDMRVPAQLLGLLLLWLRGARC

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anti-CD40 antibody light chain (SEQ ID NO 3) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAP NLLIYTASTLQSGVPSRFSGSGSGTDFTISSLQPEDFATYYCQQA NIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHK VYACEVTHQGLSSPVTKSFNRGEC anti-CD40 antibody light chain with signal peptide 1 (SEQ ID NO 4) MGWSCIILFLVATATGVHSDIQMTQSPSSVSASVGDRVTrrCRASQ G1YSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTL TISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDS KDST YS LS STLTLS KAD YEKHKVYACEVTHQGLS SPVTKSFN RGEC anti-CD40 antibody light chain with signal peptide 2 (SEQ ID NO 5) MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSVSASVGDRVTITC RASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSG TDFTLTIS S LQPEDFATYYCQQANIFPLTFGGGTKVEIKRT VAAPS V FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC anti-CD40 antibody heavy chain hIgG2 dK (SEQ ID NO 6) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPG QGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNR LRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSAS TKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVT TLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTT PPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG anti-CD40 antibody heavy chain hIgG2 dK with signal peptide 1 (SEQ ID NO 7) MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKAS GYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGR VTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCS YFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSNF GTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPA PIEKTISKTKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPG anti-CD40 antibody heavy chain hIgG2 dK with signal MDMRVPAQLLGLLLLWLRGARCQVQLVQSGAEVKKPGASVKVS CKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQK FQGRVTMTRDTSISTAYMELNRLRLSDTAVYYCARDQPLGYCTN GVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC

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peptide 2 (SEQ ID NO 8) LVKD YFPEPVT VS WNS GALTS GVHTFP A VLQS S GLYSLS S VVT VPS SNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVS NKGL PAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPG anti-CD40 antibody heavy chain hIgG2 (SEQ ID NO 9) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQ GLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLR SDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKG PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVT VPSSNFGTQTYTCNVDHKPSNTKVDKT VERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTV VHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK anti-CD40hIgG2 antibody heavy chain with signal peptide 1 (SEQ ID NO 10) MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKAS GYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGR VTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCS YFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSNF GTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPA PIEKTISKTKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLS LSPGK anti-CD40 antibody heavy chain hIgG2 with signal peptide 2 (SEQTDNO 11) MDMRVPAQLLGLLLLWLRGARCQVQLVQSGAEVKKPGASVKVS CKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQK FQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTN GVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPS SNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPS VFLFPPKPKDTLM1SRTPEVTCVVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGL PAPIEKTISKTKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK heavy chain antibody against CD40hIgGl NNAS (SEQ ID NO 48) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQ GLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLR SDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNNASRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTP

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PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK anti-CD40hIgGl antibody heavy chain NNAS-dK (SEQ ID NO 49) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQ GLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLR SDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNNASRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG heavy chain Fab region of anti-CD40 antibody hIgG2 (SEQ ID NO 12) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQ GLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLR SDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKG PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVT VPSSNFGTQTYTCNVDHKPSNTKVDKT VERKCCVE heavy chain Fab region of anti-CD40 antibody hIgG2 with signal peptide 1 (SEQ ID NO 13) MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKAS GYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGR VTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCS YFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSNF GTQTYTCNVDHKPSNTKVDKTVERKCCVE heavy chain Fab region antibodies against CD40 hIgG2 -TEV-6His fragment (SEQ ID NO 50) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQ GLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLR SDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKG PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVT VPSSNFGTQTYTCNVDHKPSNTKVDKT VERKCCVEENLYFQSHHHHHH ΙΡΝβ dM (SEQ ID NO 76) SYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQ LQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVY HQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKE YSHCAWTIVRVEILRNFYFINRLTGYLRN IFNpdMC17S (SEQ ID NO 77) SYNLLGFLQRSSNFQSQKLLWQLNGRLEYCLKDRMNFDIPEEIKQ LQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVY HQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKE YSHCAWTIVRVEILRNFYFINRLTGYLRN ΙΓΝβ (SEQ ID NO 14) MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIK QLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANV YHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAK

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EYSHCAWTIVRVETLRNFYFTNRLTGYLRN IFNpCHS (SEQ ID NO 15) MSYNLLGFLQRSSNFQSQKLLWQLNGRLEYCLKDRMNFDIPEEIK QLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANV YHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAK EYSHCAWTIVRVEILRNFYFINRLTGYLRN IFNpC17S, N80Q (SEQ ID NO 16) MSYNLLGFLQRSSNFQSQKLLWQLNGRLEYCLKDRMNFDIPEEIK QLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWQETIVENLLANV YHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAK EYSHCAWTIVRVEILRNFYFINRLTGYLRN IFNa2a (SEQ ID NO 17) CDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGN QFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQ LNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSP CAWEVVRAEIMRSFSLSTNLQESLRSKE IFNA2 (SEQ ID NO 18) VPVARLHGALPDARGCHIAQFKSLSPQELQAFKRAKDALEESLLL KDCRCHSRLFPRTWDLRQLQVRERPMALEAELALTLKVLEATAD TDPALVDVLDQPLHTLHHILSQFRACIQPQPTAGPRTRGRLHHWL YRLQEAPKKESPGCLEASVTFNLFRLLTRDLNCVASGDLCV IFNy (SEQ ID NO 19) QDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKI MQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKR DDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQ MLFRGRRASQ ΙΕΝω (SEQ ID NO 79) LGCDLPQNHGLLSRNTLVLLHQMRRISPFLCLKDRRDFRFPQEMV KGSQLQKAHVMSVLHEMLQQIFSLFHTERSSAAWNMTLLDQLHT GLHQQLQHLETCLLQVVGEGESAGAISSPALTLRRYFQGIRVYLKE KKYSDCAWEVVRMETMKSLFLSTNMQERLRSKDRDLGSS ΙΕΝε (SEQ ID NO 80) LDLKLIIFQQRQVNQESLKLLNKLQTLSIQQCLPHRKNFLLPQKSLS PQQYQKGHTLAILHEMLQQIFSLFRANISLDGWEENHTEKFLIQLH QQLEYLEALMGLEAEKLSGTLGSDNLRLQVKMYFRRIHDYLENQ DYSTCAWAIVQVEISRCLFFVFSLTEKLSKQGRPLNDMKQELTTEF RSPR RL linker (SEQ ID NO 20) PAPA GST linker (SEQ ID NO 21) SGGTSGSTSGTGS HL linker (SEQ ID NO 22) AEAAAAKEAAAKA HL2 linker (SEQ ID NO 23) AEAAAKEAAAKAEAEAAKEAAAKA (G4S)2 linker GGGGSGGGGS

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(SEQ ID NO 24) (G4S)3 linker (SEQ ID NO 25) GGGGSGGGGSGGGGS (G4S)4 linker (SEQ ID NO 26) GGGGSGGGGSGGGGSGGGGS TEV-6His fragment (SEQ ID NO 27) ENLYFQSHHHHHH antiСО40_EC—HL-ΙΕΝβ (SEQ ID NO 28) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPN LLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANI FPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKV YACEVTHQGLSSPVTKSFNRGECAEAAAKEAAAKAMS YNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQL QQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYH QINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEY SHCAWTIVRVEILRNFYFINRLTGY LRN antiCO40_EC— HL-IENP_C17S (SEQ ID NO 29) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPN LLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANI FPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKV YACEVTHQGLSSPVTKSFNRGECAEAAAKEAAAKAMS YNLLGFLQRSSNFQSQKLLWQLNGRLEYCLKDRMNFDIPEEIKQL QQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYH QINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEY SHCAWTIVRVEILRNFYFINRLTGY LRN aHTHCD40_hIgG 2_dK_HC—RLΙΡΝβ (SEQ ID NO 30) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQ GLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLR SDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKG PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVT VPSSNFGTQTYTCNVDHKPSNTKVDKT VERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTV VHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGPAPAMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRM NFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETI VENLLANVYHQINHLKTVLEEKLEKEDFT RGKLMSSLHLKRYYG RILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN aHTHCD40_hIgG 2_dK_HC—RL—IFN3_C17S (SEQ ID NO 31) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQ GLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLR SDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKG PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVT VPSSNFGTQTYTCNVDHKPSNTKVDKT

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DSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGPAPAMSYNLLGFLQRSSNFQSQKLLWQLNGRLEYCLKDRM NFDIPEEIKQLQQFQKED AALTIYEMLQNIFAIFRQDS S STGWNETI VENLLANVYHQTNHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYG AKEYSHCAWTIVVRVEILRNFYFINRLTGYLRN aHT[iCD40_h!gG 2_dK_HC—HLΤΡΝβ (SEQ ID NO 32) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQ GLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLR SDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKG PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVT VPSSNFGTQTYTCNVDHKPSNTKVDKT VERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTV VHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGAEAAAKEAAAKAMSYNLLGFLQRSSNFQCQKLLWQLNGR LEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQD SSSTGWNETIVENLLANVYHQINHLKTVLEEKLE KEDFTRGKLMS SLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYL RN aHTiiCD40_hIgG 2_dK_HC--HLIFNp_C17S (SEQ ID NO 33) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQ GLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLR SDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKG PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVT VPSSNFGTQTYTCNVDHKPSNTKVDKT VERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTV VHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGAEAAAKEAAAKAMSYNLLGFLQRSSNFQSQKLLWQLNGRL EYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSS STGWNETIVENLLANVYHQINHLKTVLEEKLE KEDFTRGKLMSSL HLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN antiCO40_EC—RL-IFNp (SEQ ID NO 34) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPN LLIYT ASTLQSGVPSRFS GS GS GTDFTLTIS SLQPEDFAT YYCQQANI FPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEK HKVYACEVTHQGLSSPVTKSFNRGECPAPAMSYNLLGFLQRS SNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAAL TIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLE EKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRV EILRNFYFINRLTG YLRN antiCO40_EC— DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPN LLIYT ASTLQSGVPSRFS GS GS GTDFTLTIS SLQPEDFAT YYCQQANI

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RL-TFNp_C17S (SEQ ID NO 35) FPLTFGGGTKVETKRTVAAPSVFTFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGECPAPAMSYNLLGFLQRS SNFQSQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAAL TIYEM LQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLE EKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRV EILRNNFYFINRLTGYLRN aHTHCD40_LC- GSTIFNp_C17S (SEQ ID NO 36) YEK HKVYACEVTHQGLSSPVTKSFNRGECSGGTSGSTSGTGSMS YNLLGFLQRSSNFQSQKLLWQLNGRLEYCLKDRMNFDIPEEIKQL QQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYH QINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEY SHCAWTIVRVEILRNFY FINRLTGYLRN antiCO40_EC- HL2IFNp_C17S (SEQ ID NO 37) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPN LLIYT ASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANI FPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKV YACEVTHQGLSSPVTKSFNRGECAEAAAKEAAAKAAE AAAKEAAAKAMSYNLLGFLQRSSNFQSQKLLWQLNGRLEYCLK DRMNFDIPEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGW NETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKR YYGRILHYLKAKEYSHCAWTIVR VEILRNNFYFINRLTGYLRN aHTiiCD40_hIgG 2_dK_HC— (G4S)2-IENa2a (SEQ ID NO 38) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQ GLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLR SDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKG PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVT VPSSNFGTQTYTCNVDHKPSNTKVDKT VERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTV VHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGGGGGSGGGGSCDLPQTHSLGSRRTLMLLAQMRKISLFSCLK DRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWD ETLLDKFYTELYQQLNDLEACVIQGVGVTETPLM KEDSILAVRKY FQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE aHTiiCD40_hIgG 2_dK_HC— (G4S)3-IFNa2a (SEQ ID NO 39) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQ GLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLR SDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKG PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVT VPSSNFGTQTYTCNVDHKPSNTKVDKT VERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTV VHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS

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LSPGGGGGSGGGGSGGGGSCDLPQTHSLGSRRTLMLLAQMRKTS LFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDS SAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSIL AVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRS KE aHTnCD40_h!gG 2 dK HC(G4S)4-IFNa2a (SEQ ID NO 40) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQ GLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLR SDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKG PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVT VPSSNFGTQTYTCNVDHKPSNTKVDKT VERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTV VHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGGGGGSGGGGSGGGGSGGGGSCDLPQTHSLGSRRTLMLLA QMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNL FSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVT ETPLM KEDSTLAVRKYFQRTTLYLKEKKYSPCAWEVVRAETMRSFSLSTNL QESLRSKE antiCO40_EC(G4S)2-IFNa2a (SEQ ID NO 41) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPN LLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANI FPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKV YACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSCDLPQ THSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKA ETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLE ACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWE VVRAEIMRSFSLSTNLQES LRSKE antiCO40_EC(G4S)3-IFNa2a (SEQ ID NO 42) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPN LLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANI FPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKV YACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGG SCDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGN QFQKAET1PVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQ LNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSP CAWEVVRAEIMRSFSLSTNLQ ESLRSKE antiCO40_EC— (G4S)4-IFNa2a (SEQ ID NO 43) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPN LLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANI FPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKV YACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGG SGGGGSCDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFP QEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFY TELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLK EKKYSPCAWEVVRAEIMRSF SLSTNLQESLRSKE

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ΙΡΝβ—(G4S)3—antiCO40_bC) (SEQ ID NO 44) MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIK QLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANV YHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAK EYSHCAWTIVRVEILRNFYFINRLTGYLRNGGGGSGGGGSGGGG SDIQMTQSPSSV SASVGDRVTITCRASQGIYSWLAWYQQKPGKAP NLLIYT ASTLQSGVPSRFS GS GS GTDFTLTIS S LQPEDFAT YYCQQA NIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK GLSSPVTKSFNRGEC antiSV40_EC— (G4S)4-IFNp (SEQ ID NO 45) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPN LLIYT ASTLQSGVPSRFS GS GS GTDFTLTIS SLQPEDFATYYCQQANI FPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD HKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGG SGGGGSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMN FDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIV ENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRI LHYLKAKEYSHCAWTI VRVEILRNNFYFINRLTGYLRN ΙΡΝβ—(G4S)3— antiСО40_НС_1 gGl_NNAS_dK (SEQ ID NO 46) MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIK QLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANV YHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAK EYSHCAWTIVRVEILRNFYFINRLTGYLRNGGGGSGGGGSGGGG SQVQLVQSGA EVKKPGASVKVSCKASGYTFTGYYMHWVRQAPG QGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRL RSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFP A VLQS S GLYS LS S V VT VPS S YTLPPSRDELTKNQVSLTCLV KGFYPSDTAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG antiСО40_НС_1 gGl_NNAS_dK-(G4S)4—ΙΕΝβ (SEQ ID NO 47) GLYS LS S V VT VPS S S LGTQT YICNVNHKPS NTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNNASRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGGGGGSGGGGSGGGGSGGGGSMSYNLLGFLQRSSNFQ CQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYE MLQNIFAIFRQDSSS TGWNETIVENLLANVYHQINHLKTVLEEKLE KEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILR NFYFINRLTGYLRN

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aHTiiCD40_hIgG 2 dK_HC-HLIFNa2A (SEQ ID NO 81) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQ GLEWMGWINPDS GGTNYAQKFQGRVTMTRDTS IS TA YMELNRLR SDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKG PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYS SREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGAEAAAKEAAAKACDLPQTHSLGSRRTLMLLAQMRKISLFSC LKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAA WDETLLDKFYTELYQQLNDLEACVIQGVGVTET PLMKEDSILAVR KYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE antiCO40_ECderivative—HL—IFNa2A (SEQ ID NO 82) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPN LLIYT ASTLQS GVPSRFS GS GS GTDFTLTIS SLQPEDFAT YYCQQANI FPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YE KHKVYACEVTHQGLSSPVTKSFNRGEKSLSLSPGAEAAAKEA AAKACDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQE EFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTE LYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKE KKYSPCAWEVV RAEIMRSSFSLSTNLQESLRSKE aHTHCD40_LC(G4S)4—IFNy (SEQ ID NO 83) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPN LLIYT ASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANI FPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKV YACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGG SGGGGSQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWK EESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFN SNKKKRDDFEKLTNYSVTDLNVQRKATHELTQVMAELSPAAKTGK RKRSQMLFRGRRASQ aHTiiCD40_hIgG 2 dK_HC(G4S)4—IFNy (SEQ ID NO 84) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQ GLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTA YMELNRLR SDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKG PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSV VTVPSSNFGTQTYTCNVDHKPSNTKVDKT VERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTV VHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGGGGGSGGGGSGGGGSGGGGSQDPYVKEAENLKKYFNAG HSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDD QSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNY SVTDLNVQRK AIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQ

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antiCB40_bs- (G4S)4-IFNX2 (SEQ ID NO 85) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPN LLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANI FPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKV YACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGG SGGGGSVPVARLHGALPDARGCHIAQFKSLSPQELQAFKRAKDA LEESLLLKDCRCHSRLFPRTWDLRQLQVRERPMALEAELALTLKV LEATADTDPALVDVLDQPLHTLHHILSQFRACIQPQPTAGPRTRGR LHHWLYRLQEAPKKESPGCLEASVTFNLFRLLTRDLNC VASGDLC V aHTHCD40_hI _g G 2 dKHC(G4S)4-IFNX2 (SEQ ID NO 86) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQ GLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLR SDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKG PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVT VPSSNFGTQTYTCNVDHKPSNTKVDKT VERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTV VHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGGGGGSGGGGSGGGGSGGGGSVPVARLHGALPDARGCHIA QFKSLSPQELQAFKRAKDALEESLLLKDCRCHSRLFPRTWDLRQL QVRERPMALEAELALTLKVLEATADTDPALVDVLDQPLHTLHHIL SQFR ACIQPQPTAGPRTRGRLHHWLYRLQEAPKKESPGCLEASVTF NLFRLLTRDLNCVASGDLCV antiCO40 LC(G4S)4-IFNm (SEQ ID NO 87) DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPN LLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANI FPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKV YACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGG SGGGGSLGCDLPQNHGLLSRNTLVLLHQMRRISPFLCLKDRRDFR FPQEMVKGSQLQKAHVMSVLHEMLQQIFSLFHTERSSAAWNMTL LDQLHTGLHQQLQHLETCLLQVVGEGESAGAISSPALTLRRYFQGI RVYLKEKKYSDCAWEVVRMEIMKSLFL STNMQERLRSKDRDLGS S aHTiiCD40_hIgG 2 dK.HC(G4S)4—IFNe (SEQ ID NO 88) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQ GLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLR SDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKG PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVT VPSSNFGTQTYTCNVDHKPSNTKVDKT VERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTV VHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGGGGGSGGGGSGGGGSGGGGSLDLKLIIFQQRQVNQESLKL LNKLQTLSIQQCLPHRKNFLLPQKSLSPQQYQKGHTLAILHEMLQQ IFSLFRANISLDGWEENHTEKFLIQLHQQLE YLEALMGLEAEKLSG TLGSDNLRLQVKMYFRRIHDYLENQDYSTCAWAIVQVEISRCLFF VFSLTEKLSKQGRPLNDMKQELTTEFRSPR

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Table 8. Sequences of exemplary interferon-associated antigen binding protein and its components based on the anti-CD40 antibody 3G5. Sequences in italics correspond to signal peptides. Sequences in bold and not in italics correspond to linkers. Mutated amino acids are underlined.

Name/SEQ ID number Subsequence antiCO40_light chain (SEQ ID NO 59) EIVMTQSPATLSVSPGERATLSCRASQSVRSNLAWYQQKPGQAP RLLIYGASTRATGIPARFSGSGSGTEFTLTINSLQSEDFAVYYCQQ HNKWHFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC antiCO40_light chain with signal peptide 1 (SEQ ID NO 60) MGWSCIILFLVATATGVHSEIVMTQSPATLSVSPGERATLLSCRAS QS VRSNLAWYQQKPGQAPRLLIYGASTRATGIPARFS GS GS GTE FTLTINSLQSEDFAVYYCQQHNKWITFGQGTRLEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC antiCO40_heavy chain hIgG2 dK (SEQ ID NO 61) QVQLVES GGGVVQPGKSLRLSC AAS GFTFS SNGIHWVRQ APGK GLEWVAVIWSDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSL RAEDTAVYYCARASGSGSYYNFFDYWGQGTLVTVSSASTKGPS VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVP SSNFGTQTYTCNVDHKPSNTKVD KTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNY KTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG antiCO40_heavy chain hIgG2 dK with signal peptide 1 (SEQ ID NO 62) MGWSCIILFLVATATGVHSQVQLVESGGGVVQPGKSLRLSCAAS GFTFSSNGIHWVRQAPGKGLEWVAVIWSDGSNKFYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCARASGSGSYYNFFDY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSNFGT QTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLF PPKPKDTLMTSRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLP

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APIEKTTSKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG Anti-CD40 heavy chain Fab region hIgG2 (SEQ ID NO 63) QVQLVES GGGVVQPGKSLRLSC AAS GFTFS SNGIHWVRQ APGK GLEWVAVIWSDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSL RAEDTAVYYCARASGSGSYYNFFDYWGQGTLVTVSSASTKGPS VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFP A VLQS S GLYSLS S V VT VPS S NFGTQT YTCN VDHKPS NTKVD KTVERKCCVE Anti-CD40 heavy chain Fab region hIgG2 with signal peptide 1 (SEQ ID NO 64) MGWSCIILFLVATATGVHSQVQLVESGGGVVQPGKSLRLSCAAS GFTFSSNGIHWVRQAPGKGLEWVAVIWSDGSNKFYADSVKGRF TISRDNSKNTLYLQMNSLRAEDTAVYYCARASGSGSYYNFFDY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSNFGT QTYTCNVDHKPSNTKVDKTVERKCCVE Anti-CD40 antibody heavy chain Fab region hIgG2 -TEV-6His fragment (SEQ ID NO 65) QVQLVES GGGVVQPGKSLRLSC AAS GFTFS SNGIHWVRQ APGK GLEWVAVIWSDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSL RAEDTAVYYCARASGSGSYYNFFDYWGQGTLVTVSSASTKGPS VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVP SSNFGTQTYTCNVDHKPSNTKVD KTVERKCCVEENLYFQSHHHHHH aHTHCD40_hIgG2 dK_HC-RLIFNpdM (SEQ ID NO 66) QVQLVESGGGVVQPGKSLRLSCAASGFTFSSNGIHWVRQAPGK GLEWVAVIWSDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSL RAEDTAVYYCARASGSGSYYNFFDYWGQGTLVTVSSASTKGPS VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSNF GTQTYTCNVDHKPSNTKVD KTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNY KTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGPAPASYNLLGFLQRSSNFQCQKLLWQLNGRLE YCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDS SSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMS SLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGY LRN

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aHTiiCD40_hIgG2 dK_HC-RLIFNpdM_C17S (SEQ ID NO 67) QVQLVES GGGVVQPGKSLRLSC AAS GFTFS SNGIHWVRQ APGK GLEWVAVIWSDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSL RAEDTAVYYCARASGSGSYYNFFDYWGQGTLVTVSSASTKGPS VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVP SSNFGTQTYTCNVDHKPSNTKVD KTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNY KTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGPAPASYNLLGFLQRSSNFQSQKLLWQLNGRLE YCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDS SSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKL MS SLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGY LRN aHTiiCD40_hIgG2 dK_HC-HLIFNpdM (SEQ ID NO 68) QVQLVESGGGVVQPGKSLRLSCAASGFTFSSNGIHWVRQAPGK GLEWVAVIWSDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSL RAEDTAVYYCARASGSGSYYNFFDYWGQGTLVTVSSASTKGPS VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFP A VLQS S GLYSLS S V VT VPS S NFGTQT YTCN VDHKPS NTKVD KTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ VYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGAEAAAKEAAAKASYNLLGFLQRSSNFQCQKL LWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQ NIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTV LEEKLEKE DFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRN FYFINRLTGYLRN aHTHCD40_hIgG2 dK_HC-HLIFN3dM_C17S (SEQ ID NO 69) QVQLVES GGGVVQPGKSLRLSC A AS GFTFS SNGIHWVRQ APGK GLEWVAVIWSDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSL RAEDTAVYYCARASGSGSYYNFFDYWGQGTLVTVSSASTKGPS VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFP A VLQS S GLYSLS S V VT VPS S NFGTQT YTCN VDHKPS NTKVD KTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ VYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGAEAAAKEAAAKASYNLLGFLQRSSNFQSQKL LWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQ NIFAIFRQDSSSTGWNETIVENLLANVYHQINHL KTVLEEKLEKE DFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRN FYFINRLTGYLRN

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antiSV40_EC- HL2-IFNp_C17S (SEQ ID NO 70) EIVMTQSPATLSVSPGERATLSCRASQSVRSNLAWYQQKPGQAP RLLIYGASTRATGIPARFSGSGSGTEFTLTINSLQSEDFAVYYCQQ HNKWITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECAEAAAKEA AAKAAEAAAKEAAAKAMSYNLLGFLQRSSNFQSQKLLWQLNG RLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFR QDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGK LMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNF YFINRL TGYLRN antiCO40_EC(G4S)3IFNp_C17S (SEQ ID NO 71) EIVMTQSPATLSVSPGERATLSCRASQSVRSNLAWYQQKPGQAP RLLIYGASTRATGIPARFSGSGSGTEFTLTINSLQSEDFAVYYCQQ HNKWITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECGGGGSGG GGSGGGGSMSYNLLGFLQRSSNFQSQKLLWQLNGRLEYCLKD RMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGW NETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLK RYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLT GYLRN aHTiiCD40_hIgG2 dK_HC—(G4S)2—IFNa2a (SEQ ID NO 72) QVQLVESGGGVVQPGKSLRLSCAASGFTFSSNGIHWVRQAPGK GLEWVAVIWSDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSL RAEDTAVYYCARASGSGSYYNFFDYWGQGTLVTVSSASTKGPS VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSNF GTQTYTCNVDHKPSNTKVD KTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNY KTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGGGGGSGGGGSCDLPQTHSLGSRRTLMLLAQM RKTSLFSCLKDRHDFGFPQEEFGNQFQKAETTPVLHEMTQQTFNLF STKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPL MKEDSILAVR KYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLS TNLQESLRSKE aHTHCD40_h!gG2 dK_HC-(G4S)3IFNa2a (SEQ ID NO 73) QVQLVES GGGVVQPGKSLRLSC A AS GFTFS SNGTHWVRQ APGK GLEWVAVIWSDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSL RAEDTAVYYCARASGSGSYYNFFDYWGQGTLVTVSSASTKGPS VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSS NFGTQTYTCNVDHKPSNTKVD KTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNY KTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGGGGGSGGGGSGGGGSCDLPQTHSLGSRRTL MLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMI QQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVG EDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIM RSFSLSTNLQESLRSKE

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aHTiiCD40_hIgG2 dK_HC—(G4S)4IFNa2a (SEQ ID NO 74) QVQLVESGGGVVQPGKSLRLSCAASGFTFSSNGIHWVRQAPGK GLEWVAVIWSDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSL RAEDTAVYYCARASGSGSYYNFFDYWGQGTLVTVSSASTKGPS VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSNF GTQTYTCNVDHKPSNTKVD KTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNY KTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSCDLPQTHSLG SRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPV LHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACV IQGVGVTETPL MKEDSILAVRKYFQRITLYLKEKKYSPCAWEVV RAEIMRSFSLSTNLQESLRSKE aHTiiCD40_hIgG2 dK_HC-HLIFNa2a (SEQ ID NO 75) QVQLVES GGGVVQPGKSLRLSC AAS GFTFS SNGIHWVRQ APGK GLEWVAVIWSDGSNKFYADSVKGRFTISRDNSKNTLYLQMNSL RAEDTAVYYCARASGSGSYYNFFDYWGQGTLVTVSSASTKGPS VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFP A VLQS S GLYSLS S V VT VPS S NFGTQT YTCN VDHKPS NTKVD KTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQ VYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGAEAAAKEAAAKACDLPQTHSLGSRRTLMLLA QMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIF NLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQG VGVTET PLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFS LSTNLQESLRSKE

In preferred embodiments, the interferon-associated antigen-binding proteins described herein are interferon-fusion antigen-binding proteins containing polypeptides derived from the polypeptides listed in Table 1. 9, specifically, in table. 9A or table 9B, more specifically in table. 9A 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 preferred embodiments, the interferon-associated antigen-binding proteins described herein are interferon-fusion antigen-binding proteins consisting of polypeptides derived from the polypeptides listed in Table. 9, specifically, in table. 9A or table 9B, more specifically in table. 9A 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 contains the sequences SEQ ID NO: 38 and SEQ ID NO: 3. In other more preferred embodiments, the interferon fusion antibody contains the sequences SEQ ID NO: 39 and SEQ ID NO: 3. In other more preferred embodiments, the interferon fusion antibody contains the sequences SEQ ID NO: 39 and SEQ ID NO: 3. in preferred embodiments, the interferon fusion antibody comprises the sequences SEQ ID NO: 40 and SEQ ID NO: 3. In other more preferred embodiments, the interferon fusion antibody comprises the sequences SEQ ID NO: 41 and SEQ ID NO: 9. In other more preferred embodiments In another more preferred embodiment, the interferon fusion antibody contains the sequences SEQ ID NO: 42 and SEQ ID NO: 9. In other more preferred embodiments, the interferon fusion antibody contains the sequences SEQ ID NO: 43 and SEQ ID NO: 9.

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Table 9. Combinations of polypeptides found in the preferred interferon-fusion antigen-binding proteins of the invention based on the anti-CD40 antibody CP870,893, their average EC50 values for activation of the CD40 and IFN pathways and their productivity (i.e., yield per liter of culture) . Each sequence combination, as indicated, is contained twice in the corresponding IFA. SN: supernatant.

A

Interferon fusion antibody (IFA) Combination of sequences CD40 EC ₅₀ (ng/ml) IFNp EC ₅₀ (ng/ml) IFNaECso (ng/ml) Product content (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

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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 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

Interferon fusion antibody (IFA) Combination of sequences CD40 EQo (ng/m l) IFNa ec ₅₀ (ng/m l) IFNZ ec ₅₀ (ng/m l) IFNy ec ₅₀ (ng/m l) IFNe ec ₅₀ (ng/m l) IFNo) ec ₅₀ (ng/m l) Product intensity (mg/l) IFA38 (SEQ ID NO 81) +(SEQ ID NO 3) 22.7 3.77 1.32

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IF AZ 9 (SEQ ID NO 82) + (SEQ ID NO 9) 17.5 2.95 1.25 IFA42 (SEQ ID NO 83) + (SEQ ID NO 9) 65.6 15.4 0.72 IFA43 (SEQ ID NO 84) + (SEQ ID NO 3) 50.8 <0.00 1 0.55 IFA44 (SEQ ID NO 85) + (SEQ ID NO 9) 41.4 0.153 0.91 IFA45 (SEQ ID NO 86) + (SEQ ID NO 3) 25.8 <0.00 1 1.09 IFA46 (SEQ ID NO 87) + (SEQ ID NO 9) 86.3 0.493 0.89 IFA49 (SEQ ID NO 88) + (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

In other preferred embodiments, the interferon-associated antigen-binding proteins described herein are interferon-fusion antigen-binding proteins comprising polypeptides derived from the polypeptides listed in Table. 10 below. In preferred embodiments, the interferon-associated antigen-binding proteins described herein are interferon-fusion antigen-binding proteins consisting of polypeptides derived from the polypeptides listed in Table. 10 below.

Table 10. Combinations of polypeptides found in the preferred interferon-fusion antigen-binding proteins of the invention based on the anti-CD40 antibody 3G5, their average EC50 values for activation of the CD40 and IFN pathways. Each sequence combination, as indicated, is contained twice in the corresponding IFA. SN: supernatant._________________

Interfero HOM fusion antibody (IFA) Combination of sequences CD40EC50 (ng/ml) IFNp EC50 (ng/ml) IFNa EC50 (ng/ml) productivity mg/l IFA 106 IFA 107 IFA 108 IFA 109 IFA114 IFA115 IFA121 (seq ID NO 66)+ (seq ID NO 59) (seq ID NO 67)+ (seq ID NO 59) (seq ID NO 68)+ (seq ID NO 59) (seq ID NO: 69)+ (seq ID NO: 59) (seq ID NO 70) + (seq ID NO 61) (seq ID NO 71) + (seq ID NO 61) (seq ID NO 72) + (seq ID NO 59) 190.5 141.5 37.3 thirty active (SN) active (SN) 14.2 10.30 2.03 1.27 0.45 active (SN) active (SN) 0.12 0.36 0.28 0.59 0.4 No significantly productivity No significantly productivity 22.6 IFA 122 (seq ID NO 73) + (seq ID NO 59) 11.74 0.07 16.8 IFA 123 (seq ID NO 74) + (seq ID NO 59) 12.85 0.05 17.2 IFA 124 (seq ID NO 75) + (seq ID NO 59) 12.14 0.04 21.6

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Nucleic acids and expression vectors

In one aspect, a combination of polynucleotides encoding an interferon-associated antigen-binding protein is provided. Also proposed are methods for producing interferon-associated antigen-binding protein, including the expression of these polynucleotides.

In some embodiments, a nucleic acid encoding an IFN or a functional fragment thereof fused to an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof, as disclosed herein, is provided. In some illustrative embodiments, the nucleic acid encodes an IFN or a functional fragment thereof fused to an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof according to any of SEQ ID NOs: 81-88, or at least 80% of the nucleic acid sequence is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the nucleic acid encoding any of these sequences. In some illustrative embodiments, the nucleic acid is at least 95%, at least 98%, or at least 99% identical to the nucleic acid encoding any of SEQ ID NOs: 81-88. In preferred embodiments, the nucleic acid encodes IFN or a functional fragment thereof fused to an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof according to any of SEQ ID NOs: 28-47, or at least 80% of the nucleic acid sequence is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the 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 the nucleic acid encoding any of SEQ ID NOs: 28-47. In other preferred embodiments, the nucleic acid encodes IFN or a functional fragment thereof fused to an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof according to any of SEQ ID NOs: 66-75, or at least 80% of the nucleic acid sequence is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the 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 the nucleic acid encoding any of SEQ ID NOs: 66-75.

In those embodiments where the nucleic acid encodes IFN or a functional fragment thereof fused to the light chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof, the nucleic acid may further encode the heavy chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof. In more specific embodiments, the heavy chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof comprises the sequence 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 at least 80% nucleic acid sequence, at least 85% % with at least 90%, at least 95%, at least 98%, or at least 99% identical to the 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 the 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 other more specific embodiments, the heavy chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof comprises the sequence SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, or SEQ ID NO: 65 or the sequence nucleic acid that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the nucleic acid encoding any from these sequences. In such other, even more specific embodiments, said nucleic acid is at least 95%, at least 98%, or at least 99% identical to the nucleic acid encoding SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64 or SEQ ID NO: 65.

In those embodiments where the nucleic acid encodes IFN or a functional fragment thereof fused to a heavy chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof, the nucleic acid may further encode a light chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof. In more specific embodiments, the light chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof comprises at least 80% of the sequence of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, or a nucleic acid sequence by at least 85%, by at least 90%, by at least 95%, by at least 98% or according

- 50 046818 at least 99% identical to the 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 the nucleic acid encoding SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In other more specific embodiments, the light chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof comprises at least 80% of SEQ ID NO: 59 or SEQ ID NO: 60 or a nucleic acid sequence. , at least 90%, at least 95%, at least 98%, or at least 99% identical to the 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 the nucleic acid encoding SEQ ID NO: 59 or SEQ ID NO: 60.

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

In some embodiments, the nucleic acid sequence may further comprise a sequence encoding a cleavage site to remove a purification fragment. Such cleavage sequences are known to one skilled in the art and can be selected from sequences recognized and cleaved by an endoprotease or exoprotease. In some embodiments, the endoprotease to remove the purification moiety may be selected from: enteropeptidase, thrombin, factor Xa, TEV protease, or rhinovirus 3C protease. In some embodiments, the exoprotease to remove the purification fragment may be selected from: carboxypeptidase A, carboxypeptidase B, or DAPase. In preferred embodiments, the purification moiety removal protease is a TEV protease. In a more specific preferred embodiment, the nucleic acid comprises a sequence encoding a His6 fragment 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 contain a sequence encoding a signal peptide. One skilled in the art will recognize the various signal peptides available to direct the expressed protein to the 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 SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the signal peptide comprises the sequence SEQ ID NO: 1. In other embodiments, the signal peptide comprises the sequence SEQ ID NO: 2.

Signal peptide 1 (SEQ ID NO: 1) was used to synthesize 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 NO: 36, SEQ ID NO: 37, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75. Such a 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: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87 and SEQ ID NO: 88, used signal

- 51 046818 ny peptide MGWSCIILFLVATATGVHS (SEQ ID NO: 1). Such a signal peptide, which is initially present at the N-terminus of the corresponding polypeptide sequence, is cleaved during synthesis.

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

The term vector or expression vector is used herein for purposes of description and claims to refer to 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. Typically, 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 enter and/or replicate 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 papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV) or SV40 virus. Others suggest the use of polycistronic systems with internal ribosome binding sites. In addition, cells that have integrated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. The marker may provide prototrophy to an auxotrophic host, resistance to biocides (eg antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can either be directly linked to the DNA sequence being expressed or can be introduced into the same cell by co-transformation. Additional elements may also be required 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 contain 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 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. Alternatively, such a vector system may comprise a first vector for expressing 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.

In other embodiments, 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 those encoding an IFN or a functional fragment thereof that is fused to the heavy chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof and encoding a light chain of said antibody, or encoding an IFN or a functional fragment thereof, fused to the light chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof, and encoding the heavy chain of said antibody or an agonistic antigen-binding fragment thereof, can be derived from a single polycistronic construct. These systems predominantly utilize the internal ribosome entry site (IRES) to provide relatively high levels of polypeptides to eukaryotic host cells. Compatible IRES sequences are disclosed in US Pat. No. 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 herein.

More generally, once a vector or DNA sequence encoding an interferon-associated antigen-binding protein of the present invention has been obtained, the expression vector can be introduced into a suitable host cell. That is, the host cell can be transformed. Introduction of a plasmid into a host cell can be accomplished by various methods well known to those skilled in the art. These include, but are not limited to, trans

- 52 046818 infection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, fusion of cells 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). Transformed cells are grown under conditions suitable for light chain and heavy chain production 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) assay, immunohistochemistry, and the like.

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 changes the genotype and therefore results in a change in the recipient cell.

In the same spirit, 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 interferon-associated antigen-binding protein unless otherwise indicated. In other words, isolating the polypeptide from cells can 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 eukaryotic or prokaryotic in 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 interferon-associated antigen-binding protein antibody fragment) that are coupled 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, a host cell line is used to express a mammalian interferon-associated antigen binding protein; those skilled in the art can determine the specific host cell lines that are best suited to express the desired gene product. Typical host cell lines include, but are not limited to, CHO K1 GS knockout from Horizon, DG44 and DUXB11 (Chinese hamster ovary lines, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS ( derivative of CVI with T-antigen SV40), 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 may be used. In another preferred embodiment, CHO K1 GS from Horizon can be used. In one embodiment, the cell line provides altered glycosylation, such as afucosylation of an antibody expressed from it (eg, 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 usually 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 non-human transgenic animal or transgenic plant.

The interferon-associated antigen-binding proteins of the present invention can also be produced transgenically by generating a non-human animal (eg, mammal) or plant that is transgenic for the sequences of interest and obtaining the interferon-associated antigen-binding protein therefrom in a reducible form. In connection with transgenic production in mammals, interferon-associated antigen-binding proteins can be prepared and isolated from the milk of goats, cows, or other mammals. See, for example, US Patent Nos. 5827690, 5756687, 5750172 and 5741957. 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 to those skilled in the art. See, for example, US Pat. No. 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 the animal or plant using standard transgenic techniques. See Hogan and US Pat. No. 6,417,429. The transgenic cells used to produce a transgenic animal may be embryonic stem cells or somatic cells. Non-human transgenic organisms can be chimeric, non-chimeric heterozygotes, and non-chimeric homozygotes. See, for example, Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual 2nd ed., Cold Spring Harbor Press (1999); Jackson et

- 53 046818 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 non-human transgenic animals have the target disruption and are replaced with a targeting construct that encodes the sequence(s) of interest. Interferon-associated antigen-binding proteins can be obtained from any transgenic animal. In a preferred embodiment, the non-human animals are mice, rats, sheep, pigs, goats, cattle or horses. The non-human transgenic animal expresses said interferon-associated antigen-binding proteins in blood, milk, urine, saliva, tears, mucus and other body fluids.

In vitro production allows production to be scaled up to produce 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, for example, in an airlift or continuous stirred tank reactor, or in immobilized or mechanically entrapped cell culture, for example, in hollow fibers, microcapsules, agarose microspheres or ceramic cartridges. If necessary and/or desired, the interferon-associated antigen binding protein solution can be purified by conventional chromatography methods, for example, gel filtration, ion exchange chromatography, DEAE cellulose chromatography and/or immunoaffinity chromatography.

One or more genes encoding 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, can also be transformed; those. those that can be grown in cultures 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; Pneumococcus; Streptococci and Haemophilus influenzae. In addition, it should be understood that when expressed in bacteria, the interferon-associated antigen-binding proteins of the invention or components thereof (ie, agonistic anti-CD40 antibodies or agonistic antigen-binding fragments thereof and IFN or functional IFN fragments) may become part of inclusion bodies. The desired interferon-associated antigen-binding proteins may then need to be isolated, and 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 yeast strain lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). The presence of trp1 damage as a feature of the host yeast 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 via 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, local administration (see US Pat. No. 5328470) or stereotactic injection (see, for example, Chen et al., PNAS 91:3054-3057 (1994)). The therapeutic vector pharmaceutical preparation may include the vector in a suitable diluent.

The interferon-associated antigen-binding protein-encoding nucleic acid or nucleic acids may be included in a gene construct for use as part of a therapeutic protocol for delivering interferon-associated antigen-binding protein-encoding nucleic acids. Expression vectors have been proposed for in vivo transfection and expression of interferon-associated antigen-binding protein.

Expression constructs of such components can be administered in any biologically effective vehicle, for example, any formulation or composition capable of effectively delivering the component nucleic acid sequence to cells in vivo, as known to one of ordinary skill in the art. Approaches include, but are not limited to, insertion of the nucleic acid sequence(s) of interest into viral vectors, including, but not limited to, recombinant

- 54 046818 new 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 to transfer exogenous nucleic acid sequences in vivo, particularly to humans. Such vectors ensure efficient delivery of genes into 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 a review, see e.g. Miller, Blood 76: 271-78 (1990)). The replication-defective retrovirus can be packaged into virions, which can be used to infect a target cell with a helper virus using 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 viral lines 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. Sci. USA 85:3014-3018 (1988); Proc. Natl. Sci. USA 87:6141-6145; Natl. Acad. Sci. USA 88:8039-8043 (1991); Proc. Natl. Sci. USA 88:8377-8381 (1991); -1805 (1991); van Beusechem, et al., Proc. Natl. Sci. USA 89:7640-7644 (1992); , et al., Proc. Natl. Sci. USA 89:10892-10895 (1992); 4980286; PCT application WO 89/07136; PCT application WO 89/02468; PCT application WO 89/05345 and PCT application WO 92/07573).

In another embodiment, adenovirus-derived delivery vectors are provided. The adenovirus genome can be manipulated so that it encodes and expresses the gene product of interest, but is inactivated from its ability to replicate during the normal lytic virus life cycle. See, for example, 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 Ad type 5 adenovirus strain d1324 or other adenovirus strains (eg 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 nondividing 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 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 the potential problems that can 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 kb) compared to other delivery vectors (Berkner et al. (1998), supra; Haj-Ahmand and Graham, J. Virol. 57:267 ( 1986)).

Another viral vector system suitable for delivering a nucleic acid sequence encoding an interferon-associated antigen-binding protein is adeno-associated virus (AAV). AAV is a naturally occurring defective virus that requires another virus, such as an 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 nondividing 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. 63:38223828 (1989); Vectors containing as little as 300 base pairs of AAV can be packaged and integrated. 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, for example, Hermonat, et al., Proc. Natl. Acad. Sci. USA 81:6466-6470 (1984); Tratschin, et al., Mol Cell. Biol., 4:2072-2081 (1985); Mol. Endocrinol. 2:32-39 (1988); 1984); and Flotte, et al., J. Biol. 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 interferon-associated antigen-binding protein in patient tissue. Most non-viral methods do not

- 55 046818 gene transfer is based on the usual 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 uptake of the gene of interest into 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, all of which are known in the art. For example, the pharmaceutical delivery system may be administered systemically, such as by intravenous injection. Specific protein transduction in target cells occurs primarily due to transfection specificity provided by the delivery vehicle, cell- or tissue-type expression due to transcriptional regulatory sequences controlling receptor gene expression, or a combination thereof. In other embodiments, the initial delivery of the recombinant gene is more limited, with the administration to the animal being fairly localized. For example, the delivery vehicle can be administered by catheter (see US Pat. No. 5,328,470) or by stereotactic injection (eg, Chen, et al., PNAS 91: 3054-3057 (1994)).

A pharmaceutical preparation of a therapeutic construct may consist essentially of a delivery system in a suitable diluent or may comprise a slow release matrix into which the delivery vehicle is embedded. Alternatively, when the complete delivery system can be produced intact from recombinant cells, for example, in the form of retroviral vectors, the pharmaceutical preparation may contain one or more cells that produce the delivery system.

Treatment options

In one aspect, the invention provides methods for 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 (eg, mRNA) that encodes an interferon-associated antigen-binding protein, as disclosed herein, in the preparation of a medicament for the treatment of HBV. In some embodiments, the present invention provides kits and methods for treating disorders and/or symptoms, such as HBV-related disorders and/or HBV-related symptoms, in a mammal in need of such treatment. In some illustrative embodiments, the patient is a human.

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

In another embodiment, the subject interferon-associated antigen-binding proteins or the 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 the nucleic acid sequences that encode them, are useful for reducing one or more disorders, symptoms and/or complications associated with chronic HBV infection, for example, chronic liver inflammation (chronic hepatitis) leading to

- 56 046818 to cirrhosis for 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 (eg, membranous glomerulonephritis); immune-mediated hematological disorders (eg, essential mixed cryoglobulinemia, aplastic anemia); etc.

In some embodiments, the subject interferon-associated antigen-binding proteins, or the nucleic acid sequences that encode them, are useful for reducing one or more symptoms and/or complications associated with acute HBV infection, for example, acute viral hepatitis (which begins with general ill health, 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-like syndrome); loss of appetite; joint and muscle pain; low-grade fever; stomach ache; nausea; vomiting; jaundice; bloating; etc.

Accordingly, the present invention also provides 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 nucleic acid sequence, which encodes it. One of ordinary skill in the art will be able to determine, through routine experimentation, what effective, non-toxic amount of interferon-associated antigen binding protein or the nucleic acid sequence that encodes it will be used to treat HBV infection.

For example, the 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 (eg, acute versus chronic), age, sex, medical complications (eg, coinfection with HIV, conditions or diseases with immunosuppressed) and the patient's weight, as well as the ability of interferon-associated antigen-binding protein to induce the desired response in the patient. The dosage regimen may be adjusted to ensure 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 of the present invention can be used for the prophylactic treatment of uninfected cells or the therapeutic treatment of any HBV-infected cells containing an antigenic marker that allows interferon-associated antigen binding protein to target the HBV-infected cells.

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 preparing and administering to a patient the interferon-associated antigen-binding proteins, or 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 of the art as a guide. The route of administration of the interferon-associated antigen-binding proteins or nucleic acid sequences that encode them according to the present invention may be oral, parenteral, inhalation or topical. The term parenteral as used herein includes intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. Although all of 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 intra-arterial injection or drip administration. Typically, a suitable pharmaceutical composition for injection may contain a buffering agent (eg, acetate, phosphate or citrate buffer), a surfactant (eg, polysorbate), optionally a stabilizing agent (eg, 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 including Pluronics, PEGs, sorbitan esters, polysorbates, Triton, tromethamine, lecithin, cholesterol, and tyloxapal. In preferred embodiments, the surfactant is a 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

- 57 046818 intravenously into a site of unfavorable cell population (for example, the liver), thereby increasing the exposure of the therapeutic agent to the affected tissue.

Formulations 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 vehicles include water, alcohol/aqueous solutions, emulsions or suspensions, including saline and buffer media. In the compositions and methods of the present invention, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1 M, for example, 0.05 M phosphate buffer or 0.8% saline. Other common parenteral solutions include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents, inert gases, and the like. In particular, pharmaceutical compositions suitable for injection include sterile aqueous solutions (where dissolved in water) or dispersions and sterile powders for the immediate 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 syringed. It must be stable under the conditions of production and storage and is usually protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (eg, glycerin, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of 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 such as sugars, polyhydric alcohols such as mannitol, sorbitol or sodium chloride will be included in the composition. 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 an active compound, such as 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 the ingredients listed here, as necessary, followed by sterilization by filtration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains the basic dispersion medium and the necessary other ingredients 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. Injectable preparations are processed, packaged into containers such as ampoules, bags, jars, syringes or vials, and sealed under aseptic conditions in accordance with methods known in the art. In addition, the drugs may be packaged and sold as a kit. Such manufactured products typically have labels or package inserts indicating that the respective compositions are suitable for treating a patient suffering from HBV infection.

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

For treatment with interferon-associated antigen binding protein, the dosage may vary, for example, from about 0.0001 to about 100 mg/kg, more often from about 0.01 to about 5 mg/kg (for example, about 0.02 mg/kg, about 0 .25 mg/kg, approximately 0.5 mg/kg, approximately 0.75 mg/kg, approximately 1 mg/kg, approximately 2 mg/kg, etc.) based on the host’s body weight. For example, dosages may 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 to the above ranges are also within the scope of the present invention. Patients can be given these doses

- 58 046818 daily, on alternative days, weekly or in accordance with any other scheme determined by empirical analysis. Typical treatment entails administering multiple doses over a long period of time, such as at least six months. Additional exemplary treatment regimens include administration about once every two weeks, or about once per month, or about once every 3-6 months. Exemplary dosage regimens include from 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.

Interferon-associated antigen-binding proteins or nucleic acid sequences expressing any of them can be administered multiple times. The intervals between single doses may be a week, a month or a year. 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, interferon-associated antigen-binding proteins or nucleic acid sequences expressing any of them can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of interferon-associated antigen-binding proteins in the patient.

The term half-life or t1/2, as used herein, refers to the stability and/or rate of clearance of a compound, such as the interferon-associated antigen-binding proteins of the invention. In practice, the half-life of a compound is usually 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 mouse serum. 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 In 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 interferon-associated antigen-binding proteins according to the invention is low. In some embodiments, the clearance of interferon-associated antigen binding protein is less than 10 ml/hour/kg, less than 5 ml/hour/kg, less than 2.5 ml/hour/kg, less than 1 ml/hour/kg, or less than 0.5 ml /h/kg. In some embodiments, the clearance of interferon-associated antigen binding protein is less than 5 ml/hour/kg. In some embodiments, the clearance of interferon-associated antigen binding protein is less than 1 ml/hour/kg. In some embodiments, the clearance of interferon-associated antigen binding protein in mice is in the range of 0.28-0.49 ml/hour/kg.

The terms volume of distribution, VD, VSS or apparent volume of distribution as used herein refer to the theoretical volume that would be necessary to contain the total amount of administered compound, such as the interferon-associated antigen-binding protein of the invention, at the same concentration as in blood plasma, and refers to the distribution of the specified compound between the plasma and the rest of the body after oral or parenteral dosing. In some embodiments, the volume of distribution of the Vss 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 of Vss interferon-associated antigen binding protein is less than 100 ml/kg. In some embodiments, the volume of distribution of Vss interferon-associated antigen binding protein in mice is between 50 and 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 the parent antibody. In some embodiments, the parent antibody is CP870893. In other embodiments, the parent antibody is 3G5. In some embodiments, the systemic exposure to interferon-associated antigen binding protein is at least 600 μg*h/ml, at least 700 μg*h/ml, at least 800 μg*h/ml, at least 900 μg*h/ ml or at least 1000 μg*h/ml, preferably at least 1000 μg*h/ml. In some embodiments, systemic exposure to interferon-associated antigen binding protein in mice ranges from 1033 μg*h/ml to 1793 μg*h/ml.

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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 diseases of mammals in vivo. In this regard, it should be understood that, as disclosed, interferon-associated antigen binding protein will be included in the formulation to facilitate administration and ensure stability of the active agent.

The pharmaceutical composition in accordance with the present invention may contain a pharmaceutically acceptable, non-toxic, sterile carrier such as saline, non-toxic buffers, preservatives and the like. A pharmaceutically effective amount of interferon-associated antigen binding protein is generally an amount sufficient to mediate one or more of the following: reducing the release of HBeAg from an HBV-infected cell; decreased transcription of pgRNA in an HBV-infected cell; and stimulation of the IFN signaling pathway in the 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 interferon-associated antigen binding protein.

It is within the scope of the present invention that 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 prepared by combining 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 methods. One skilled in the art will appreciate 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 containing one or more types of 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, since such methods and conditions may vary. It is also understood that the terminology used herein 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, use conventional molecular and cellular 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, for example, 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 specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In the event of any implied ambiguity, the definitions provided herein shall take precedence over any dictionary or external definition. Unless the context otherwise requires, terms in the singular shall include the plural and terms in the plural shall include the singular. The use of or means and/or unless otherwise noted. The use of the term including, as well as other forms such as includes and included, is not intended to be 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, is well known and widely used in the art. The methods and techniques disclosed herein are generally performed in accordance with conventional methods well known in the art and as described in various general and more specific references cited and discussed herein unless otherwise indicated. Enzymatic reactions and purification methods are carried out in accordance with the manufacturer's specifications, as is routinely done 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 for both delivery and treatment of patients.

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Section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. The contents of articles, patents and patent applications, and all other documents and electronically available information mentioned or cited herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication had been specifically and separately indicated for inclusion by reference. Applicants reserve the right to physically include in this application any materials 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 purposes of illustration only and are not intended to be limiting.

Examples

Example I: Generation of Interferon Fusion Antibodies (IFA) Based on the Anti-CD40 Agonist Antibody CP870,893 and Characterization of Reporter Cells a - IFA Design

Sequence combinations of exemplary IFAs constructed using the CD40 agonist antibody CP870,893 as the scaffold antibody, the location of the IFNs, and the nature of the linkers are listed in Table 1. 7 and 9. IFN was fused through a linker at the N- or C-terminal part of the light chain (LC) or heavy chain (HC), as indicated in table. 7. Nucleic acids encoding HC, LC or fusions were synthesized with codons optimized for mammalian expression and cloned into a eukaryotic expression vector such as pcDNA3.1 (Invitrogen). In fig. 2A shows an exemplary map of plasmid pcDNA3.1 encoding SEQ ID NO: 32 under the control of the pCMV promoter.

b - receiving 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. The purification process was carried out in two stages 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 D-PBS1X pH 7.5 buffer and elution with Glycine/HCl 0.1 M pH 3 buffer. The elution peak was retained in the loop, then injected into 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 in 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 was carried out including bacterial endotoxins using Endosafe® nexgen-PTS™ (Charles River), size exclusion chromatography: using SEC 200 Increase 10/300 column (GE Healthcare) to determine purity and oligomers and SDS-PAGE under reducing and non-reducing conditions on a NuPAGE gel system (Invitrogen) in MES SDS running buffer. The product yield is indicated in table. 9. For some IFAs the yield was very low. In this case, CD40 agonist activity and IFN activity were assessed directly using the IFA-containing supernatant without any further purification.

SDS-PAGE analysis of the purified IFAs under reducing conditions showed 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, activation of the NFkB pathway by CD40 agonists or the type I IFN-induced IFN pathway.

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. Stimulation of CD40 leads to the induction of NFkB and subsequently to 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 JAK/STAT/ISGF3 pathway activation induced by type I IFN. Activation of this pathway induces the production and release of SEAP. SEAP levels are easily determined in the supernatant using QUANTI-Blue™.

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

Cells were seeded in 96-well plates (50,000 cells per well) and stimulated with the indicated

- 61 046818 concentration for each IFA or control and incubated at 37°C for 24 hours. Supernatants were then collected and SEAP levels were quantified after incubating the supernatant for approximately 30 minutes using QuantiBlue™ and assessing 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) cells can be used to monitor the activity of type II and type III interferons , respectively. HEK-Blue™ IFN-λ cells are designed to monitor IFNX activity. HEK-Blue™ Dual IFN-γ cells enable the detection of biologically active human IFN-γ.

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

In fig. 3 shows examples of dose-dependent effects of IFA, where IFNe or its mutant version, as indicated in table. 7 were fused with HC as indicated in Table. 7, on HEK-Blue™ CD40L cells (Figs. 3A-3B) and HEK-Blue™ IFN-α/β cells (Figs. 3C-3D). The agonistic anti-CD40 activity of IFA is summarized in Table. 9, and examples are shown in Fig. 3A and 3B. The results indicate that all IFAs tested are able to activate both the CD40 and IFN-α/β pathways in a dose-dependent manner. For HC or LC CTerminal fusions, EC50 values for agonistic CD40 range from 11.1 ng/ml to 192 ng/ml (Table 9). The average EC50 for the parent antibody was 48 ng/ml and 57 ng/ml in the experiment shown in FIG. 3. IFAs with IFN fused to the N-terminus of HC or LC were also able to activate the CD40 pathway, but precise 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 various IFAs is summarized in Table. 9, and examples are shown in Fig. 3C-3D. For fusions of IFNe or mutated IFNe (as listed in Table 7) to the C terminus of HC or LC, IFN activity varies depending on the linker sequence with EC50 values ranging from 0.14 ng/ml to 4.5 ng/ml (Fig. .3C and table 9). In fig. 3D shows that IFAs with IFNe fused to the N-terminal portion exhibit high IFN activity. The parental antibody used as a negative control showed no activity, whereas recombinant IFNe did exhibit a strong dose-dependent response. Overall, these results demonstrate that the IFNe fusion or its mutant version, as indicated in Table. 7, with the antibody, regardless of location, retains both biological functions, although with differences in terms of effectiveness.

In fig. Figure 4 shows examples of dose-dependent IFA responses where IFNa was fused to HC or LC, as indicated in Table. 7, on HEK-Blue™ CD40L cells (Figs. 4A and 4C) and HEK-Blue™ IFN-a/β cells (Figs. 4B and 4D). The results show that all IFAs tested are able to activate both the CD40 and IFNa/β pathways in a dose-dependent manner. Surprisingly, for all IFNa-based IFAs, potency against the CD40 pathway was reproducibly higher than that of the parent antibody. EC50 values for IFNa-based IFA ranged from 11.1 ng/mL to 22.7 ng/mL, and EC50 for CP870,893 ranged from 30 to 80 ng/mL (mean EC50: 48 ng/mL).

The IFN activity of IFA varies 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 exhibited dose-dependent activity with an EC50 value of approximately 1 ng/mL.

e - Generation and characterization of IFA without Fc region

Suitable constructs of the invention may also be interferon-associated antigen-binding proteins without the Fc region. A construct encoding the fab fragment heavy chain CP870893 fused to a TEV-His fragment (SEQ ID NO: 50) was constructed and cloned into the pcDNA3.1 expression plasmid. This construct was cotransfected into HEK cells as described previously, with LC fused through different linkers with 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. Proteins and/or supernatants are assessed in reporter cells and/or their effect on HBV infection in PHH. One skilled in the art will appreciate that constructs for use in therapy will no longer contain the TEV-His moiety. These designs are also embodiments of the invention. Interferon-associated antigen-binding proteins without the Fc portion will be active against HBV infection. Two IFAs were then prepared 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 next day the medium was replaced with medium

- 62 046818 containing Matrigel (0.25 mg/ml; 356231, Corning). Cells were infected 48 h after seeding at an MOI of 500 to 1000 vge/cell 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 postinfection, cells were left untreated or treated with serially diluted IFAs as shown in the figures. Three days after treatment, culture supernatants were collected and stored at −80°C for further protein detection.

II. b. - Assessment 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 in PEI units (HBeAg CLIA 96T/K: CL0312-2; Autobio) or luminescence.

II. With. - Assessment 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, samples were diluted 1/5 in 1X PBS. 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 1 hour at 37°C. The plates were then washed 6 times with 300 µl of the kit wash solution using a plate washer. Then, 50 μl of Substrate Solution (volume mixture in reagents A and B) was added to each well and incubated for 10 min in the dark. The plates were then read on a PHERASTAR microplate reader (BMG Labtech) in luminescence mode.

II. d. - quantitative assessment of pgRNA

The qPCR technique was used to compare the expression levels of pgRNAs from infected cells treated with test compounds. Quantification of pgRNA from infected cells was performed in 96-well plates using QuantStudio 12K Flex. cDNA was obtained by RT reverse transcription followed by one-step qPCR with TaqMan Fast Virus assay (ThermoFisher, cat. no. 4444434). The results were processed by the AACt method and normalized to the housekeeping gene GUSB, duplicating the results. pgRNA was amplified using the following primers and probe: (forward: CCTCACCATACTGCACTCA, reverse: GAGGGAGTTCTTCTTCTAGG, AGTGTGGATTCGCACTCCTCCAGC as probe). The GUSB gene was amplified using a TaqMan assay from Thermo Fisher (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 luminescence was assessed on an EnSight microplate reader at 450 nm.

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

Several IFAs have been tested for their ability to reduce HBeAg secretion following PHH HBV infection. In fig. 5 used IFA with IFNe or its mutant version fused at the C terminus of the LC. The results show that all IFAs tested strongly reduced HBeAg release. Indeed, even at the lowest concentration tested (1 ng/ml), 70 to 90% inhibition of HBeAg release was observed depending on the IFA, indicating that they have potent antiviral effects. It is noteworthy that in this experiment it was not possible to achieve 100% inhibition, since treatment began four days after infection and at this time the existing pool of HBeAg (mRNA and protein) is already present in the cell and continues to be produced thereafter.

The effect of IFAs fused to IFNa was also tested in HBV-infected PHH. In fig. 6 shows that these IFAs are very effective 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-terminal 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 6C) and from 0.15 to 0. .36 ng/ml for IFA 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 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 more effective 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 primary HBV-infected hepatocytes, cells were infected and incubated for 4 days, treated with IFA25, IFA27, IFA28, IFA30 or Pegasys depending on the dose for 24 h, washed and then incubated with fresh medium without any or processing. After 3 days, supernatants were collected to assess the level of release of HBeAG (Fig. 6E), HBsAG (Fig. 6F), and CXCL10 (Fig. 6H), cells were lysed, and RNA was extracted to quantify

- 63 046818 vein determination of pgRNA (Fig. 6G). The results show that all IFAs tested were able to inhibit the release of HbeAG and HBsAG, as well as pgRNA expression, in a dose-dependent manner. Pegasys by itself was only able to inhibit HBeAG release and reduce pgRNA levels. In this regard, IFA is at least 2 log more active than Pegasys in terms of viral parameters. Surprisingly, although all IFAs tested demonstrated 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 IFAs are also much more effective than Pegasys.

Example III. Release of cytokines

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

An ex vivo whole blood cell (WBC) stimulation assay was used to examine 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 unstimulated, stimulated with LPS (LipoPolySaccharide) K12 (tlrl-eklps, Invivogen) at a concentration of 10 ng/ml as a positive control or IFA at a concentration of 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 pro-inflammatory 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).

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

Further results of IFA testing based on IFNe-/mutated IFNe- and IFNa are shown in table. 11a and 11b. The results show that in all donors, LPS induces 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 IFAs based on IFNe- (Table 11a) and six IFNa- (Table 11b) were tested. All of them induced the IP10 biomarker. However, they did not induce IL-10, IL-1e and IL-2 and only induced very low to moderate levels of IFNy, IL-6 and TNF-a, suggesting a favorable safety profile with regard to the induction of inflammatory cytokines.

Example IV. Pharmacokinetic studies

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

For ELISA quantitation, 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 being emptied 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 serial 1/2 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 with anti-IFNa antibody conjugated to HRP (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. The plates were read at 450–650 nm using an Ensight plate reader (Perkin Elmer). Quantification of Pegasys was assessed using similar protocol steps but using antibody pairs corresponding to human IFNa from eBioscience/Invitrogen. Capture was performed using 100 μl of human anti-IFNa antibody (eBioscience/Invitrogen; BMS216MST) at a concentration of 1 μg/ml in sodium carbonate (0.05 M, pH 9.6, C-3041, Sigma). For detection, a secondary antibody against IFNa conjugated to HRP (1/1000, Affymetrix eBioscience/BMS216MST; 15501707) in PBS - 0.05% Tween20-1% milk was used.

IV. b - Bioavailability in vivo in mice

To determine PK parameters, CP870,893, IFA25, IFA26, IFA27, IFA28, IFA29 and IFA30 at 0.5 mg/kg and Pegasys at a dose of 0.3 mg/kg were administered i.v. bolus into male CD1-Swiss mice, and blood samples were collected at different time points. Examples of quantitation of circulating molecules using the ELISA approach described above and detected with an anti-IFNa antibody conjugated to HRP are shown in FIG. 8A and 8B, while examples of quantitation 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. 12A,

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PK parameters for CP870,893 were studied in a 7-day experiment, and parameters for IFA27, IFA29 and IFA30 in a 10-day experiment (IFA27 quantification was carried out using 2 different ELISA approaches). In another set of experiments, summarized in Table. 12B, PK parameters for CP870,893 and IFA25, IFA26, IFA28 and Pegasys were examined in 21-day experiments (IFA25 quantitation was performed using 2 different ELISA approaches).

After a short distribution phase, the pharmacokinetic profiles of IFA are characterized by a long serum half-life of 116 to 218 hours (Tables 12A and 12B). Very similar pharmacokinetic profiles were obtained for the 6 IFAs tested, with high circulating levels even ten days after a single dose. Pharmacokinetic parameters summarized in table. 12A/B show that these IFAs surprisingly circulate in the blood with higher systemic exposure (AUC(0-inf)) ranging from 1033 μg. h/ml up to 2552 mcg. h/ml for IFA compared to 590 or 797 μg. h/ml, respectively, for the parent antibody CP870,893 (up to 3.2 times), which also reflects the lower clearance values for IFA. The volume of distribution of Vss was low, ranging from 50 to 105 ml/kg, slightly larger than the plasma vascular volume (50 ml/kg) in this species. For all IFAs, clearance was estimated to be low (0.28-0.49 ml/h/kg). Interestingly, the clearance of Pegasys (1.4 ml/h/kg) is up to 7 times higher than the clearance of IFA (eg, 0.2 ml/h/kg for IFA27), demonstrating higher systemic exposure of IFA.

Example V

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

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

Evaluation on HEK-Blue™ CD40L cells showed that these IFAs still exhibit CD40 agonist activity (Figure 9A) and activate 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-α/β cells showed that both IFAs tested exhibited IFN activity (Figure 9B). EC50 values are given in table. 9B and are approximately 1.36 ng/ml for IFA50 and 1.43 ng/ml for IFA51.

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

V. b - Functional activity of IFA based on IFNo: against reporter cells and HBV infection.

Fusions of CP870,893 with the third type I interferon (IFNepsilon; IF#:) 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 IFN-I pathways (Figure 10B). EC50 values are presented in table. 9B. In addition, IFA49 was also tested for HBV infection in primary hepatocytes and showed activity similar to Pegasys (Fig. 10C).

These results demonstrate that IFAs with IF#: retain both IFN and CD40 agonist activity (i.e., are bifunctional) and have antiviral activity.

V.c - Functional activity of IFNco-based IFA against reporter cells and HBV infection

Fusions of CP870,893 to a fourth type I interferon (IFN omega; IFNio) have also been designed and produced. These IFAs were tested on HEK-Blue™ CD40L cells and the results showed that they retain CD40 agonist activity. The results for one such IFA (IFA46) are shown in FIG. 11A. Evaluation of HEK-Blue™ hIFN-α/β cells (which are essentially activated by any type I interferon) showed that IFA46 was also able to activate IFN-I pathways (Figure 11B). EC50 values are presented in table. 9B. In addition, IFA46 was also tested for HBV infection in primary hepatocytes and showed activity similar to Pegasys (Fig. 11C).

These results demonstrate that IFAs with IFNco retain both IFN and CD40 agonist activity (ie, are bifunctional) and have antiviral activity.

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

Fusions of CP870,893 with type II interferon (IFN gamma; IFNy) have also been designed and produced. Evaluation of these IFAs in HEK-Blue™ CD40L cells demonstrates that they retain CD40 agonist activity regardless of whether IFNy is bound to the C-terminal portion of LC (IFA42) or HC (IFA43) (Figure 12A). Evaluation of these IFAs on HEK-Blue™-IFNY cells (Figure 12B) showed that they are also capable of activating the IFNy pathway. IFNy activity was slightly different between IFA42 (EC50: 15 ng/ml) and IFA43 (EC50: < 0.01 ng/ml). EC50 values are presented in table. 9B. In addition, IFA42 and IFA43

- 65 046818 was tested in a dose-dependent manner for HBV infection in primary hepatocytes as previously described. The results show that both IFAs reduce HbeAg release in a dose-dependent manner (Fig. 12C), indicating that type II IFAs are active in HBV infection.

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

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

Fusions of CP870,893 with type III interferon (IFN lambda; IFN/) have also been designed and produced. These IFAs were tested in HEK-Blue™ CD40L cells and the results showed that they also retain CD40 agonist activity regardless of whether IFN/ is bound to the C-terminal portion of LC (IFA44) or HC (IFA45) (Figure 13A ). Evaluation of these IFAs on HEK-Blue™-IFN/ cells. showed that they are also able to activate the IFN/ pathway (Fig. 13B). EC50 values are presented in table. 9B. These results also demonstrate that IFAs with IFN/ retain both IFN and CD40 agonist activity (ie, are bifunctional).

IFA44 and IFA45 were tested at a single dose versus Pegasys for HBV infection in primary hepatocytes as previously described. The results show that both types of IFA 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 (Figure 13C).

Example VI. Generation of interferon fusion antibodies (IFA) based on anti-CD40 antibody 3G5 and characterization of reporter cells

VI. a - IFA design

Sequence combinations of exemplary IFAs constructed with anti-CD40 antibody 3G5 (Celldex) as the scaffold antibody, the location of the IFNs, and the nature of the linkers are listed in Table 1. 7 and 10. IFN was fused through a linker at the C-terminal part of the light chain (LC) or heavy chain (HC), as indicated in Table. 7. Nucleic acids encoding HC, LC or fusions were synthesized with codons optimized for mammalian expression and cloned into a eukaryotic expression vector such as pcDNA3.1 (Invitrogen).

VI. b - receiving IFA

IFA preparation was carried out as described previously, and the product yield is shown in table. 10. For some IFAs the product yield was very low, mainly for the fusion of IFNe to the C-terminal part of LC. For these IFAs, CD40 and IFN agonistic activities were assessed directly using the IFA-containing supernatant without any further purification. SDSPAGE analysis of the purified IFAs under reducing conditions showed the presence of two major bands corresponding to HC and LC. When IFN fused with HC, a change in its molecular weight was observed (Fig. 14).

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

Characterization of 3G5 IFA on reporter cells was performed on HEK-Blue™ CD40L cells (Figures 15A-B and 16A) and HEK-Blue™ IFN-α/β cells (Figures 15C-D and 16B) as previously described (see I. c).

VI. With. 1. IFA based on IFNe

In fig. 15 shows examples of dose-dependent IFA responses where IFNe was fused to HC or LC 3G5 on HEK-Blue™ CD40L and HEK-Blue™ IFN-α/β cells (FIG. 15). The results are summarized in table. 10 show that all IFNe-based IFAs tested are functional and capable of activating both the CD40 and IFNa/β pathways in a dose-dependent manner.

Examples of CD40 activity are shown in FIG. 15A and 15B. The IFNe fusion to the C-terminal portion of HC exhibits highly variable activity against CD40 and is in all cases lower than that of the parent antibody, with EC50 values ranging from 30 ng/ml to 190.5 ng/ml (Fig. 15A and Table 10 ). The mean EC50 for the parent antibody 3G5 is 9.3 ng/mL.

For fusions at the C-terminal portion of LC, product yield was very low and activity was assessed using supernatants containing IFA after overexpression in HEK cells. Evaluation of these supernatants on HEK-Blue™ CD40L (Figure 15B) demonstrates that these IFAs are active in the CD40 pathway. For 3G5, CD40 agonist activity was still detected when the supernatant was diluted 300-fold. Conversely, a 1/10 dilution was required to observe the activity of supernatants containing IFA (Fig. 15B).

IFN activity of IFA was tested on HEK-Blue™ IFN-α/β cells, and the results are summarized in table. 10. Examples are shown in FIG. 15C-D. For IFNe fusions in the C-terminal portion of the HC, IFN activity varies depending on the linker sequence with EC50 values ranging from 0, 45 to 10.3 ng/ml (Fig. 15C). For IFA with an IFNe fusion in the C-terminal portion of the LC-containing supernatant, IFN activity was still detectable even after a 10,000-fold dilution of the supernatant (Figure 15D).

VI. With. 2. IFA based on IFNa

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In fig. 16 shows examples of the dose-dependent response of IFA, where IFNa was fused to HC 3G5, on HEK-Blue™ CD40L cells (Fig. 16A) and HEK-Blue™ IFNa/β (Fig. 16B).

The results show that all IFAs demonstrate functional activation of both the CD40 and IFNa/β pathways in a dose-dependent manner (mean EC50 values are presented in Table 10).

For all IFNa-based IFAs, activity against the CD40 pathway was similar to that of the parent antibody, with mean EC50 values ranging from 11.74 to 14.2 ng/ml (Figure 16A and Table 10). The average EC50 value for the parent antibody 3G5 is 9.3 ng/ml.

IFN activity IFNa-based IFAs were tested on HEK-Blue™ IFN-a/β cells and demonstrated very high activity. The average EC50 values for IFN activity of these IFAs ranged from 0.04 to 0.12 ng/ml (Fig. 16B and Table 10).

VI. d - Generation and characterization of IFA without Fc region

Suitable constructs of the invention may also be interferon-associated antigen-binding proteins without the Fc region. A construct encoding the heavy chain of the 3G5 Fab fragment fused to a TEV-His fragment (SEQ ID NO: 65) was constructed and cloned into the pcDNA3.1 expression plasmid. This construct was cotransfected into HEK cells as described previously, with LC fused through different linkers to IFN, such as SEQ ID NO:: 70 or SEQ ID NO:: 71. Proteins and/or supernatants are assessed in reporter cells and/or their impact on HBV infection in PHH. One skilled in the art will appreciate that constructs for use in therapy will no longer contain the TEV-His moiety. These designs are also embodiments of the invention. Interferon-associated antigen-binding proteins without the Fc portion will be active against HBV infection.

Example VII.

VII. a - Effect of IFNa/β-based IFA on HBV infection

VII. A. 1. IFA based on IFNe

FAs fused to IFNe were tested for their ability to reduce HBeAg release following HBV PHH infection as previously described, and examples are shown in FIG. 17A. The results indicate that these IFAs are HBV. For all IFAs tested, dose-dependent inhibition was observed, with a reduction of 12% to 52% obtained at 1 ng/ml and a maximum reduction of approximately 85% observed with IFA109 at 100 ng/ml. 100% inhibition cannot be achieved because treatment active in the infection began four days after infection, at which time the existing pool of HBeAg (mRNA and protein) is already present in the cell and continues to be produced thereafter.

VII. A. 2. IFA based on IFNa

IFAs fused to IFNa were also tested for their ability to reduce HBeAg release following HBV PHH infection as previously described, and examples are shown in FIG. 17B. The results show that these IFNa-based IFAs are very effective against HBV infection. For all IFAs tested, dose-dependent inhibition was also observed, with a reduction of 61 to 80% obtained at 1 ng/ml, and the maximum reduction (85 to 92%) was almost achieved at 100 ng/ml for all IFAs. These results demonstrate that IFNa-based IFAs are very potent antiviral molecules against HBV.

Example VIII. Cytokine release assay (CRA) from human whole blood cells

An ex vivo leukocyte stimulation assay was used to examine cytokine release following IFA stimulation as described previously (see a). An example with IFA109 is shown in FIG. 18 and in table. 13. The results show that all IFAs induce the release of CXCL10. They did not induce IL-10, IL1β and IL-2 and only induced very low to moderate levels of IFNy, IL-6 and TNF-a, suggesting a favorable safety profile with regard to the induction of inflammatory cytokines.

Equivalents

The invention may be embodied in other specific forms without departing from its scope or essential characteristics. Thus, the above embodiments should be considered in all respects as illustrative and not limiting of the invention. The scope of the invention is therefore set forth in the appended claims rather than in the preceding description, and it is therefore intended that all modifications falling within the meaning and range of the claims are included in the present specification.

Objects

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

1. Interferon-associated antigen-binding protein, containing:

(I) an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof; and (II) an interferon (IFN) or a functional fragment thereof for use in the treatment of hepatitis B virus (HBV) infection.

2. An interferon-associated antigen-binding protein for use according to claim 1, wherein the agonistic anti-CD40 antibody or agonistic antigen-binding fragment thereof comprises three op.

- 67 046818 light chain complementarity determining regions (CDRs) that are at least 90% identical to the sequences CDRL1, CDRL2 and CDRL3 in SEQ ID NO: 3; and three heavy chain CDRs that are at least 90% identical to the sequences CDRH1, CDRH2 and CDRH3 in SEQ ID NO: 6.

3. An interferon-associated antigen-binding protein for use according to claim 1 or 2, wherein the agonistic anti-CD40 antibody or agonistic antigen-binding fragment thereof contains three light chain complementarity determining regions (CDRs) that are identical to the sequences CDRL1, CDRL2 and CDRL3 in SEQ ID NO: 3; and three heavy chain CDRs that are identical to the sequences CDRH1, CDRH2 and CDRH3 in SEQ ID NO: 6.

4. An interferon-associated antigen-binding protein for use according to claim 2 or 3, wherein each CDR is defined in accordance with the Kabat definition, the Chothia definition, the AbM definition or the contact CDR definition; preferably, where each CDR is defined in accordance with the Kabat CDR definition or the Chothia CDR definition.

5. Interferon-associated antigen-binding protein for use according to claim 1, wherein the agonistic anti-CD40 antibody or agonistic antigen-binding fragment thereof comprises:

(a) a heavy chain or fragment thereof containing a complementarity determining region (CDR) of 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 which is at least 90% identical to SEQ ID NO: 58; and (b) 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.

6. Interferon-associated antigen-binding protein for use according to claim 1, wherein the agonistic anti-CD40 antibody or agonistic antigen-binding fragment thereof comprises:

(a) a heavy chain or fragment thereof containing a complementarity determining region (CDR) of 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 (b) 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.

7. An interferon-associated antigen-binding protein for use as claimed in any of the preceding claims, wherein the CD40 agonist antibody or an agonistic antigen-binding fragment thereof comprises a VL light chain variable region comprising the sequence SEQ ID NO: 51 or a sequence at least 90% identical thereto ; and/or a VH heavy chain variable region comprising the sequence SEQ ID NO: 55 or a sequence at least 90% identical thereto.

8. An interferon-associated antigen-binding protein for use according to any of the preceding claims, wherein the heavy chain of the agonistic anti-CD40 antibody or agonistic antigen-binding fragment thereof comprises a heavy chain of a Fab region comprising the amino acid sequence of SEQ ID NO: 12, or a sequence of at least 90% identical to it.

9. An interferon-associated antigen-binding protein for use as claimed in any of the preceding claims, wherein the anti-CD40 agonist antibody or an agonistic antigen-binding fragment thereof comprises a light chain (LC) that contains the sequence of SEQ ID NO: 3, or at least 90% of the sequence identical to it; and/or a heavy chain (HC) that contains 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 identical thereto at least 90%.

10. An interferon-associated antigen-binding protein for use according to claim 9, wherein the HC contains the sequence of SEQ ID NO: 6, or a sequence that is at least 90% identical thereto.

11. An interferon-associated antigen-binding protein for use according to claim 9, wherein the HC contains the sequence of SEQ ID NO: 9, or a sequence identical to it by at least 90%.

12. An interferon-associated antigen-binding protein for use according to claim 9, wherein the HC contains the sequence of SEQ ID NO: 49, or a sequence that is at least 90% identical thereto.

13. An interferon-associated antigen-binding protein for use according to claim 9, wherein the HC contains the sequence of SEQ ID NO: 48, or a sequence that is at least 90% identical thereto.

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

15. An interferon-associated antigen-binding protein for use according to any of the preceding claims, wherein the IFN or a functional fragment thereof is selected from the group consisting of IFN type

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I, type II IFN and type III IFN, or a functional fragment thereof.

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

17. An interferon-associated antigen-binding protein for use according to claim 16, wherein the type I IFN or a functional fragment thereof is IFNa, IFNe, IFNm or IFN: or a functional fragment thereof.

18. An interferon-associated antigen-binding protein for use according to claim 16, wherein the type I IFN or a functional fragment thereof is IFNa or IFNe or a functional fragment thereof.

19. An interferon-associated antigen-binding protein for use according to claim 16, wherein the type I IFN or a functional fragment thereof is a ΣΕΚω or a functional fragment thereof.

20. An interferon-associated antigen-binding protein for use according to claim 16, wherein the type I IFN or a functional fragment thereof is an IFN: or a functional fragment thereof.

21. An interferon-associated antigen-binding protein for use according to any one of claims 114, wherein the IFN or a functional fragment thereof is IFNa, IFNe, IFNy, ΓΡΗλ, IFNe·) or IFN:, or a functional fragment thereof.

22. An interferon-associated antigen-binding protein for use according to claim 21, wherein the IFN or a functional fragment thereof is IFNa or IFNe or a functional fragment thereof.

23. An interferon-associated antigen-binding protein for use according to claim 22, wherein the IFN or a functional fragment thereof is IFNa or a functional fragment thereof.

24. An interferon-associated antigen-binding protein for use according to claim 23, wherein the IFN or a functional fragment thereof is IFNa2a or a functional fragment thereof.

25. An interferon-associated antigen-binding protein for use according to claim 24, wherein IFNa2a contains the sequence of SEQ ID NO: 17, or a sequence identical to it by at least 90%.

26. An interferon-associated antigen-binding protein for use according to claim 22, wherein the IFN or a functional fragment thereof is IFNe or a functional fragment thereof.

27. An interferon-associated antigen-binding protein for use according to claim 26, wherein IFNe contains the sequence of SEQ ID NO: 14 or a sequence that is at least 90% identical thereto.

28. An interferon-associated antigen-binding protein for use according to claim 26, wherein IFNe or a functional fragment thereof contains one or two amino acid substitutions relative to SEQ ID NO: 14 selected from C17S and N80Q.

29. Interferon-associated antigen-binding protein for use according to claim 28, where IFNe or a functional fragment thereof contains the amino acid substitution C17S relative to SEQ ID NO: 14.

30. An interferon-associated antigen-binding protein for use according to claim 29, wherein IFNe comprises the amino acid sequence of SEQ ID NO: 15.

31. Interferon-associated antigen-binding protein for use according to claim 28, where IFNe or a functional fragment thereof contains amino acid substitutions C17S and N80Q relative to SEQ ID NO: 14.

32. An interferon-associated antigen-binding protein for use according to claim 31, wherein IFNe comprises the amino acid sequence of SEQ ID NO: 16.

33. An interferon-associated antigen-binding protein for use according to claim 21, wherein the IFN or a functional fragment thereof is IFNγ or IFNA or a functional fragment thereof.

34. An interferon-associated antigen-binding protein for use according to claim 33, wherein the IFN or a functional fragment thereof is IFNy or a functional fragment thereof.

35. An interferon-associated antigen-binding protein for use according to claim 34, wherein IFNy comprises the sequence of SEQ ID NO: 19 or a sequence that is at least 90% identical thereto.

36. An interferon-associated antigen-binding protein for use according to claim 33, wherein the IFN or a functional fragment thereof is an IFNA or a functional fragment thereof.

37. An interferon-associated antigen-binding protein for use according to claim 36, wherein the IFNA or a functional fragment thereof is IFNL2 or a functional fragment thereof.

38. An interferon-associated antigen-binding protein for use according to claim 37, wherein IFNL2 comprises the sequence of SEQ ID NO: 18 or a sequence that is at least 90% identical thereto.

39. Interferon-associated antigen-binding protein for use according to any of the previous claims, wherein IFN or a functional fragment thereof is non-covalently linked to an agonist

- 69 046818 anti-CD40 antibody or an agonistic antigen-binding fragment thereof.

40. An interferon-associated antigen-binding protein for use according to claim 39, wherein the IFN or a functional fragment thereof is non-covalently linked to an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof via ionic, van der Waals and/or hydrogen bonding interactions.

41. An interferon-associated antigen-binding protein for use according to any one of claims 138, wherein the IFN or a functional fragment thereof is covalently linked to an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof.

42. An interferon-associated antigen-binding protein for use according to claim 41, wherein the IFN or a functional fragment thereof is fused to an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof.

43. An interferon-associated antigen-binding protein for use according to claim 42, wherein the IFN or a functional fragment thereof is fused to the light chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof.

44. An interferon-associated antigen-binding protein for use according to claim 43, wherein the IFN or a functional fragment thereof is fused to the N-terminus of the light chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof.

45. An interferon-associated antigen-binding protein for use according to claim 43, wherein the IFN or a functional fragment thereof is fused to the C-terminus of the light chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof.

46. An interferon-associated antigen-binding protein for use according to claim 42, wherein the IFN or a functional fragment thereof is fused to the heavy chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof.

47. An interferon-associated antigen-binding protein for use according to claim 46, wherein the IFN or a functional fragment thereof is fused to the N-terminus of the heavy chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof.

48. An interferon-associated antigen-binding protein for use according to claim 46, wherein the IFN or a functional fragment thereof is fused to the C-terminus of the heavy chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof.

49. An interferon-associated antigen-binding protein for use according to any one of claims 42 to 48, wherein the agonistic anti-CD40 antibody or agonistic antigen-binding fragment thereof and IFN or a functional fragment thereof are fused to each other through a linker.

50. An interferon-associated antigen-binding protein for use according to claim 49, wherein the interferon-associated antigen-binding protein does not contain amino acids other than the amino acids forming (I) the specified agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof, (II) the specified IFN or its a functional fragment, and (III) said linker.

51. An interferon-associated antigen-binding protein for use according to any one of claims 149, wherein the interferon-associated antigen-binding protein does not contain amino acids other than the amino acids forming: (I) the specified agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof, and (II) the specified IFN or a functional fragment thereof.

52. An interferon-associated antigen-binding protein for use according to any one of claims 49 to 50, wherein the linker is a peptide linker.

53. An interferon-associated antigen-binding protein for use according to claim 52, wherein the linker contains at least 1, at least 2, at least 3, at least 4 or at least 5 amino acids.

54. An interferon-associated antigen-binding protein for use according to claim 53, wherein the linker contains at least 4 amino acids.

55. An interferon-associated antigen-binding protein for use according to claim 53, wherein the linker contains at least 11 amino acids.

56. An interferon-associated antigen-binding protein for use according to claim 53, wherein the linker contains at least 12 amino acids.

57. An interferon-associated antigen-binding protein for use according to claim 53, wherein the linker contains at least 13 amino acids.

58. An interferon-associated antigen-binding protein for use according to claim 53, wherein the linker contains at least 15 amino acids.

59. An interferon-associated antigen-binding protein for use according to claim 53, wherein the linker contains at least 20 amino acids.

60. An interferon-associated antigen-binding protein for use according to claim 53, wherein the linker contains at least 21 amino acids.

61. An interferon-associated antigen-binding protein for use according to claim 53, wherein the linker contains at least 24 amino acids.

- 70 046818

62. Interferon-associated antigen-binding protein for use according to claim 52, where the linker contains 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.

63. Interferon-associated antigen-binding protein for use according to claim 62, where the linker contains up to 80 amino acids.

64. Interferon-associated antigen-binding protein for use according to claim 62, where the linker contains up to 40 amino acids.

65. Interferon-associated antigen-binding protein for use according to claim 62, where the linker contains up to 24 amino acids.

66. Interferon-associated antigen-binding protein for use according to claim 62, where the linker contains up to 21 amino acids.

67. Interferon-associated antigen-binding protein for use according to claim 62, where the linker contains up to 20 amino acids.

68. Interferon-associated antigen-binding protein for use according to claim 62, where the linker contains up to 15 amino acids.

69. Interferon-associated antigen-binding protein for use according to claim 62, where the linker contains up to 13 amino acids.

70. Interferon-associated antigen-binding protein for use according to claim 62, where the linker contains up to 12 amino acids.

71. Interferon-associated antigen-binding protein for use according to claim 62, where the linker contains up to 11 amino acids.

72. Interferon-associated antigen-binding protein for use according to claim 62, where the linker contains up to 4 amino acids.

73. An interferon-associated antigen-binding protein for use according to any one of claims 52 to 72, wherein the linker is selected from the group consisting of acidic, basic and neutral linkers.

74. An interferon-associated antigen-binding protein for use according to claim 73, wherein the linker is an acidic linker.

75. An interferon-associated antigen-binding protein for use according to claim 73 or 74, wherein the linker contains the sequence SEQ ID NO: 22 or SEQ ID NO: 23.

76. An interferon-associated antigen-binding protein for use according to claim 73, wherein the linker is a primary linker.

77. An interferon-associated antigen-binding protein for use according to claim 73, wherein the linker is a neutral linker.

78. An interferon-associated antigen-binding protein for use according to claims 73 or 77, wherein the linker comprises the sequence SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26.

79. An interferon-associated antigen-binding protein for use according to any one of claims 52 to 78, wherein the linker is selected from the group consisting of rigid, flexible and helix linkers.

80. An interferon-associated antigen-binding protein for use according to claim 79, wherein the linker is a rigid linker.

81. An interferon-associated antigen-binding protein for use according to claim 79 or 80, wherein the linker comprises the sequence SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 23.

82. An interferon-associated antigen-binding protein for use according to claim 79, wherein the linker is a flexible linker.

83. An interferon-associated antigen-binding protein for use according to claim 79 or 82, wherein the linker comprises the sequence SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26.

84. An interferon-associated antigen-binding protein for use according to claim 79, wherein the linker is a helix-forming linker.

85. An interferon-associated antigen-binding protein for use according to claim 79 or 84, wherein the linker contains the sequence SEQ ID NO: 22 or SEQ ID NO: 23.

86. Interferon-associated antigen-binding protein for use according to any one of claims 52-74, 76, 77, 79, 80, 82 or 84, where the linker contains the amino acids glycine and serine.

87. An interferon-associated antigen-binding protein for use according to claim 86, wherein the linker comprises the sequence of SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26.

88. An interferon-associated antigen-binding protein for use according to claim 86, wherein the linker further comprises the amino acid threonine.

89. An interferon-associated antigen-binding protein for use according to claim 88, wherein the linker contains the sequence SEQ ID NO:: 21.

90. An interferon-associated antigen-binding protein for use according to claim 52, wherein the linker contains a sequence selected from the sequences of SEQ ID NO: 20-26.

91. An interferon-associated antigen-binding protein for use according to claim 90, wherein the linker comprises a sequence selected from SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26.

- 71 046818

92. An interferon-associated antigen-binding protein for use according to claim 91, wherein the linker contains the sequence SEQ ID NO: 24.

93. An interferon-associated antigen-binding protein for use according to claim 91, wherein the linker contains the sequence SEQ ID NO: 25.

94. An interferon-associated antigen-binding protein for use according to claim 91, wherein the linker contains the sequence SEQ ID NO: 26.

95. An interferon-associated antigen-binding protein for use according to any one of claims 49, 50 or 52-94, wherein IFN or a functional fragment thereof is fused to the C-terminus of the heavy chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof via the linker of Table. 3, specifically from table. 3A or table 3B, more specifically from table. 3A.

96. An interferon-associated antigen-binding protein for use according to claim 95, wherein the heavy chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof 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.

97. An interferon-associated antigen-binding protein for use according to claim 95 or 96, wherein IFNa2a contains the sequence SEQ ID NO: 17.

98. An interferon-associated antigen-binding protein for use according to claim 95 or 96, wherein IFNe comprises the sequence SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

99. An interferon-associated antigen-binding protein for use according to claim 98, wherein IFNe contains the sequence SEQ ID NO: 14.

100. An interferon-associated antigen-binding protein for use according to claim 98, wherein IFNe contains the sequence SEQ ID NO: 15.

101. An interferon-associated antigen-binding protein for use according to claim 98, wherein IFNe contains the sequence SEQ ID NO: 16.

102. An interferon-associated antigen-binding protein for use according to claim 95 or 96, wherein IFNy contains the sequence SEQ ID NO: 19.

103. An interferon-associated antigen-binding protein for use according to claim 95 or 96, wherein IF^2 contains the sequence SEQ ID NO: 18.

104. An interferon-associated antigen-binding protein for use according to any one of claims 95 to 103, wherein the interferon-associated antigen-binding protein further comprises a light chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof.

105. An interferon-associated antigen-binding protein for use according to claim 104, wherein the light chain comprises the sequence SEQ ID NO: 3.

106. An interferon-associated antigen-binding protein for use according to any one of claims 49, 50 or 52-94, wherein IFN or a functional fragment thereof is fused to the N-terminus of the heavy chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof via the linker of Table. 4, specifically from table. 4A or 4B, more specifically from table. 4A.

107. An interferon-associated antigen-binding protein for use according to claim 106, wherein the heavy chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof 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.

108. An interferon-associated antigen-binding protein for use according to claims 106 or 107, wherein IFNa2a contains the sequence SEQ ID NO: 17.

109. An interferon-associated antigen-binding protein for use according to claim 106 or 107, wherein IFNe comprises the sequence of SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

110. An interferon-associated antigen-binding protein for use according to claim 109, wherein IFNe contains the sequence SEQ ID NO: 14.

111. An interferon-associated antigen-binding protein for use according to claim 109, wherein IFNe contains the sequence SEQ ID NO: 15.

112. An interferon-associated antigen-binding protein for use according to claim 109, wherein IFNe contains the sequence SEQ ID NO: 16.

113. An interferon-associated antigen-binding protein for use according to claim 106 or 107, wherein IFNy contains the sequence SEQ ID NO: 19.

114. An interferon-associated antigen-binding protein for use according to claim 106 or 107, wherein IFNX2 contains the sequence SEQ ID NO: 18.

115. An interferon-associated antigen-binding protein for use according to any one of claims 106-114, wherein the interferon-associated antigen-binding protein further comprises a light chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof.

116. Interferon-associated antigen-binding protein for use according to claim 115, where

- 72 046818 This chain contains the sequence SEQ ID NO: 3.

117. An interferon-associated antigen-binding protein for use according to any one of claims 49, 50 or 52-94, wherein IFN or a functional fragment thereof is fused to the C-terminus of the light chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof via the linker of Table. 5, specifically, from table. 5A or table 5B, more specifically, from table. 5A.

118. An interferon-associated antigen-binding protein for use according to claim 117, wherein the light chain of the agonistic anti-CD40 antibody or agonistic antigen-binding fragment thereof comprises the sequence of SEQ ID NO: 3.

119. An interferon-associated antigen-binding protein for use according to claim 117 or 118, wherein IFNa2a contains the sequence SEQ ID NO: 17.

120. An interferon-associated antigen-binding protein for use according to claims 117 or 118, wherein IFNe comprises the sequence of SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

121. An interferon-associated antigen-binding protein for use according to claim 120, wherein IFNe contains the sequence SEQ ID NO: 14.

122. An interferon-associated antigen-binding protein for use according to claim 120, wherein IFNe contains the sequence SEQ ID NO: 15.

123. An interferon-associated antigen-binding protein for use according to claim 120, wherein IFNe contains the sequence SEQ ID NO: 16.

124. An interferon-associated antigen-binding protein for use according to claims 117 or 118, wherein IFNy contains the sequence SEQ ID NO:: 19.

125. An interferon-associated antigen-binding protein for use according to claims 117 or 118, wherein IF^2 contains the sequence SEQ ID NO: 18.

126. An interferon-associated antigen-binding protein for use according to any one of claims 117-125, wherein the interferon-associated antigen-binding protein further comprises a heavy chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof.

127. An interferon-associated antigen-binding protein for use according to claim 126, wherein the anti-CD40 agonist antibody heavy chain 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.

128. An interferon-associated antigen-binding protein for use according to any one of claims 49, 50 or 52-94, wherein the IFN is fused to the N-terminus of the light chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment through the linker of Table. 6, specifically from table. 6A or table 6B, more specifically from table. 6A.

129. An interferon-associated antigen-binding protein for use according to claim 128, wherein the light chain of the agonistic anti-CD40 antibody or agonistic antigen-binding fragment thereof comprises the sequence of SEQ ID NO: 3.

130. An interferon-associated antigen-binding protein for use according to claims 128 or 129, wherein IFNa2a contains the sequence SEQ ID NO: 17.

131. An interferon-associated antigen-binding protein for use according to claims 128 or 129, wherein IFNe comprises the sequence of SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

132. An interferon-associated antigen-binding protein for use according to claim 131, wherein IFNe contains the sequence SEQ ID NO: 14.

133. An interferon-associated antigen-binding protein for use according to claim 131, wherein IFNe contains the sequence SEQ ID NO: 15.

134. An interferon-associated antigen-binding protein for use according to claim 131, wherein IFNe contains the sequence SEQ ID NO: 16.

135. An interferon-associated antigen-binding protein for use according to claim 128 or 129, wherein IFNy contains the sequence SEQ ID NO: 19.

136. An interferon-associated antigen-binding protein for use according to claim 128 or 129, wherein IFNX2 contains the sequence SEQ ID NO: 18.

137. An interferon-associated antigen-binding protein for use according to any one of claims 128-136, wherein the interferon-associated antigen-binding protein further comprises a heavy chain of an anti-CD40 antibody or an agonistic antigen-binding fragment thereof.

138. An interferon-associated antigen-binding protein for use according to claim 137, wherein the anti-CD40 agonist antibody heavy chain 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.

139. An interferon-associated antigen-binding protein for use according to any one of claims 1 to 138, 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

- 73 046818

NO: 46 and SEQ ID NO: 47.

140. An interferon-associated antigen-binding protein for use according to claim 139, 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.

141. An interferon-associated antigen-binding protein for use according to claim 139 or 140, where the interferon-associated antigen-binding protein is an interferon-fused agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof fused to interferon, containing one of the combinations of sequences disclosed in table. 9, specifically in table. 9A or table 9B, more specifically in table. 9A.

142. An interferon-associated antigen-binding protein for use according to claim 141, wherein the interferon-associated antigen-binding protein is an interferon-fusion agonist anti-CD40 antibody or an interferon-fusion agonist antigen-binding fragment thereof comprising the sequences of SEQ ID NO: 38 and SEQ ID NO: 3.

143. An interferon-associated antigen-binding protein for use according to claim 141, wherein the interferon-associated antigen-binding protein is an interferon-fusion agonist anti-CD40 antibody or an interferon-fusion agonist antigen-binding fragment thereof comprising the sequences of SEQ ID NO: 39 and SEQ ID NO: 3.

144. An interferon-associated antigen-binding protein for use according to claim 141, wherein the interferon-associated antigen-binding protein is an interferon-fusion agonist anti-CD40 antibody or an interferon-fusion agonist antigen-binding fragment thereof comprising the sequences of SEQ ID NO: 40 and SEQ ID NO: 3.

145. An interferon-associated antigen-binding protein for use according to claim 141, wherein the interferon-associated antigen-binding protein is an interferon-fusion agonist anti-CD40 antibody or an interferon-fusion agonist antigen-binding fragment thereof comprising the sequences of SEQ ID NO: 41 and SEQ ID NO: 9.

146. An interferon-associated antigen-binding protein for use according to claim 141, wherein the interferon-associated antigen-binding protein is an interferon-fusion agonist anti-CD40 antibody or an interferon-fusion agonist antigen-binding fragment thereof comprising the sequences of SEQ ID NO: 42 and SEQ ID NO: 9.

147. An interferon-associated antigen-binding protein for use according to claim 141, wherein the interferon-associated antigen-binding protein is an interferon-fusion agonist anti-CD40 antibody or an interferon-fusion agonist antigen-binding fragment thereof comprising the sequences of SEQ ID NO: 43 and SEQ ID NO: 9.

148. An interferon-associated antigen-binding protein for use according to any one of claims 1 to 147, wherein the interferon-associated antigen-binding protein activates both the CD40 pathway and the IFN pathway.

149. An interferon-associated antigen-binding protein for use according to claim 148, wherein CD40 activity is determined using a whole blood surface molecule up-regulation assay or an in vitro reporter cell assay.

150. An interferon-associated antigen-binding protein for use according to claim 149, wherein CD40 activity is determined using an in vitro reporter cell assay, optionally using HEK-Blue™ CD40L cells.

151. An interferon-associated antigen-binding protein for use according to any one of claims 148-150, 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.

152. An interferon-associated antigen-binding protein for use according to claim 151, wherein the interferon-associated antigen-binding protein activates the CD40 pathway with an EC50 in the range of 10 to 200 ng/ml.

153. An interferon-associated antigen-binding protein for use according to claim 152, wherein the interferon-associated antigen-binding protein activates the CD40 pathway with an EC50 ranging from 10 to 50 ng/ml, preferably from 10 to 30 ng/ml.

154. An interferon-associated antigen-binding protein for use according to any one of claims 148-153, 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.

155. Interferon-associated antigen-binding protein for use according to any one of claims 148-154, 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.

156. An interferon-associated antigen-binding protein for use according to any one of claims 148 to 155, wherein the IFN pathway is an IFNa, IFNe, IFNo: pathway. ZFNy, IFNio or ΖΕΧλ.

157. An interferon-associated antigen-binding protein for use according to claim 156, wherein IFNe activity is determined using an in vitro reporter cell assay, optionally using

- 74 046818 induction of HEK-Blue™ IFN-α/β cells.

158. An interferon-associated antigen-binding protein for use according to claim 156, wherein IFNa activity is determined using an in vitro reporter cell assay, optionally using HEK-Blue™ IFN-α/β cells.

159. An interferon-associated antigen-binding protein for use according to claim 156, wherein IFNy activity is determined using an in vitro reporter cell assay, optionally using HEK-Blue™ Dual IFN-y cells.

160. Interferon-associated antigen-binding protein for use according to claim 156, wherein the activity is IFN. determined using an in vitro reporter cell assay, optionally using HEK-Blue™ IFN-λ cells.

161. An interferon-associated antigen-binding protein for use as claimed in any 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.

162. An interferon-associated antigen-binding protein for use according to claim 161, 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.

163. An interferon-associated antigen-binding protein for use according to claim 161, wherein the interferon-associated antigen-binding protein reduces the release of HBeAg with an EC50 of less than 30 ng/ml.

164. An interferon-associated antigen-binding protein for use according to claim 163, wherein the interferon-associated antigen-binding protein reduces the release of HBeAg with an EC50 of less than 10 ng/ml.

165. An interferon-associated antigen-binding protein for use according to claim 164, wherein the interferon-associated antigen-binding protein reduces the release of HBeAg with an EC50 of less than 1 ng/ml.

166. An interferon-associated antigen-binding protein for use according to claim 164, wherein the interferon-associated antigen-binding protein reduces the release of HBeAg with an EC50 of less than 0.1 ng/ml.

167. An interferon-associated antigen-binding protein for use according to any of the preceding claims, in particular according to claims 148-165, wherein the level of expression of one or more biomarkers of the IFN pathway is up-regulated in an HBV-infected cell upon treatment with the interferon-associated antigen-binding protein, preferably at least 1.5 times, more preferably at least 2 times, most preferably at least 3 times.

168. An interferon-associated antigen-binding protein for use according to claim 167, wherein the IFN pathway biomarker is a chemokine.

169. An interferon-associated antigen-binding protein for use according to claim 168, wherein the IFN pathway biomarker is an interferon-stimulated gene ISG20.

170. An interferon-associated antigen-binding protein for use according to claim 168, wherein the IFN pathway biomarker is a C-X-C chemokine selected from the group consisting of CXCL9, CXCL10 and CXCL11.

171. An interferon-associated antigen-binding protein for use according to claim 170, wherein the IFN pathway biomarker is CXCL10.

172. An interferon-associated antigen-binding protein for use according to any of the previous claims, in particular according to claims 148-171, wherein the expression level of one or more of IL10, ΓΕ1β and IL2 is not significantly up-regulated in the HBV-infected cell upon treatment with interferon- associated antigen binding protein.

173. Interferon-associated antigen-binding protein for use according to any of the previous claims, wherein the systemic exposure of interferon-associated antigen-binding protein is increased compared to the CP870, 893 antibody, preferably by at least 10%, more preferably by at least 15%, most preferably by at least 25%.

174. Interferon-associated antigen-binding protein for use according to any of the previous claims, wherein the systemic exposure of interferon-associated antigen-binding protein is at least 1000 μg*h/ml.

175. Interferon-associated antigen-binding protein for use according to claim 174, wherein the systemic exposure of interferon-associated antigen-binding protein is in the range of 1033 to 1793 μg*h/ml.

176. An interferon-associated antigen-binding protein for use according to any of the preceding claims, wherein the half-life of the interferon-associated antigen-binding protein is at least 100 hours.

177. An interferon-associated antigen-binding protein for use according to claim 176, wherein the half-life of the interferon-associated antigen-binding protein is from 116 to 158 hours.

- 75 046818

178. Interferon-associated antigen-binding protein for use according to any of the previous claims, where the clearance rate of interferon-associated antigen-binding protein is below 0.5 ml/h/kg.

179. An interferon-associated antigen-binding protein for use according to claim 178, wherein the clearance of the interferon-associated antigen-binding protein is in the range of 0.28 to 0.49 ml/h/kg.

180. An interferon-associated antigen-binding protein for use according to any one of claims 1 to 179, wherein the volume of distribution Vss of the interferon-associated antigen-binding protein is below 100 ml/kg.

181. Interferon-associated antigen-binding protein for use according to claim 180, wherein the volume of distribution of Vss interferon-associated antigen-binding protein is from 50 to 98 ml/kg.

182. An interferon-associated antigen-binding protein for use as defined in any of the preceding claims, wherein the use includes administering the interferon-associated antigen-binding protein to a patient by genetic delivery using RNA or DNA sequences encoding interferon-associated antigen-binding protein, or a vector or vector system encoding interferon -associated antigen-binding protein.

183. Interferon-associated antigen-binding protein for use according to any one of claims 1 to 182, wherein the interferon-associated antigen-binding protein is contained in the pharmaceutical composition.

184. An interferon-associated antigen-binding protein for use according to claim 183, wherein the pharmaceutical composition is suitable for oral, parenteral or topical administration or for administration by inhalation.

185. An interferon-associated antigen-binding protein for use according to claim 184, wherein the pharmaceutical composition is suitable for oral administration.

186. An interferon-associated antigen-binding protein for use according to claim 184, wherein the pharmaceutical composition is suitable for topical administration.

187. An interferon-associated antigen-binding protein for use according to claim 184, wherein the pharmaceutical composition is suitable for administration by inhalation.

188. Interferon-associated antigen-binding protein for use according to claim 184, wherein the pharmaceutical composition is suitable for parenteral administration.

189. An interferon-associated antigen-binding protein for use according to claim 188, wherein the pharmaceutical composition is suitable for intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration.

190. An interferon-associated antigen-binding protein for use according to claim 189, wherein the pharmaceutical composition is suitable for injection, preferably for intravenous or intra-arterial injection or drip administration.

191. Interferon-associated antigen-binding protein for use according to any one of claims 183-190, wherein the pharmaceutical composition contains at least one buffering agent.

192. An interferon-associated antigen-binding protein for use according to claim 191, wherein the buffering agent is acetate, formate or citrate.

193. An interferon-associated antigen-binding protein for use according to claim 192, wherein the buffering agent is acetate.

194. An interferon-associated antigen-binding protein for use according to claim 192, wherein the buffering agent is formate.

195. An interferon-associated antigen-binding protein for use according to claim 192, wherein the buffering agent is citrate.

196. Interferon-associated antigen-binding protein for use according to any one of claims 183 to 195, wherein the pharmaceutical composition contains a surfactant.

197. An interferon-associated antigen-binding protein for use according to claim 196, wherein the surfactant is selected from the list containing Pluronics, PEGs, sorbitan esters, polysorbates, Triton, tromethamine, lecithin, cholesterol and tyloxapal.

198. An interferon-associated antigen-binding protein for use according to claim 197, wherein the surfactant is a polysorbate.

199. An interferon-associated antigen-binding protein for use according to claim 198, wherein the surfactant is polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80 or polysorbate 100.

200. An interferon-associated antigen-binding protein for use according to claim 199, wherein the surfactant is polysorbate 20.

201. An interferon-associated antigen-binding protein for use according to claim 199, wherein the surfactant is polysorbate 80.

202. Interferon-associated antigen-binding protein for use in any of

- 76 046818 claims 183-201, where the pharmaceutical composition contains a stabilizing agent, optionally where the stabilizing agent is albumin.

Table 11A. IFN-β-based IFA

IFN-γ IL-10 IL12p4 0 IL1 β IL2 IL6 IP10 TNF a 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, s 57.1 TFA2 Donor 1 nd nd nd nd nd 18.6 43382, 41 Donor 2 nd nd 114.2 nd nd 17.4 J 43283, l 33.8 Donor 3 nd nd nd nd nd nd 25961, l 32.2 Donor 4 109.4 nd nd Nd nd nd 38445 66

- 77 046818

Table 11B. IFN-α-based IFA

IFN-γ IL10 IL12p 40 IL1 β IL2 IL6 IP10 TNF a 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 1 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 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 59.6 ,8 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

- 78 046818

Table 12A

Mat rik s Connection Dose lifetime period Method CO (µg/ml) AU C (0last) (µ g.h/ml) Tlast (h) AUC (0-inf) (mcg.h/ml) % extrapolation Tl/2t (h) CI (ml/h/kg) v _D ml/k g Sy thief otka SR87 0.893 0.5 mg/kg 168h ELISAIgG2 7.15 241 168 590 59 264 (long) 0.35 (low) 296 (low kaya) Sy thief otka IFA27 0.5 mg/kg 240h ELISAIgG2 14.7 150 1 240 2552 41 218 (long) 0.2 (low) 55 (low kaya) Sy thief otka IFA27 0.5 mg/kg 240h ELISAIFN 16.9 131 8 240 1793 26 125 (long) 0.28 (low) 50 (low kaya) Sy thief otka IFA29 0.5 mg/kg 240h ELISAIFN 11.6 804 240 1033 22 116 (long) 0.49 (low) 78 (low kaya) Sy thief OTK IFA30 0.5 mg/kg 240h ELISAIFN 8.12 741 240 1089 31 158 (long) 0.46 (low) 98 (low kaya)

Table 12B

Matri KS Connection Acute dose Method CO (µ g/m l) AU C (0last) T|as (h) AU C (0inf) % extra rapolation T]/2t (h) CI (ml/h/kg) V _D ml/k g period d (µg .h/m l) (µg .h/m l) And Otka cheese IFA2 5 0.5 mg/kg 504h ELISA1FN 7.4 5 132 8 504 150 0 eleven 154 (long) 0.34 (low) 73 (low) Otka cheese IFA2 6 0.5 mg/kg 504h ELISAIFN 8.2 988 336 102 7 3.8 59 (long) 0.49 (low) 57 (low) Otka cheese IFA2 8 0.5 mg/kg 504h ELISAIFN 9.3 8 104 8 504 126 4 17 213 (long) 0.4 (low) 105 (low) Otka cheese Pcgas ys 0.3 mg/kg 504h IFAIFNaspecial features KIY 8.3 210 168 215 2 30 (average II) 1.4 (low) 62 (low) Otka cheese SR87 0.893 0.5 mg/kg 504h ELISAIgG2 And, 9 527 168 797 34 116 (long) 0.63 (low) 96 (low) Otka cheese IFA2 5 0.5 mg/kg 504h ELISAIgG2 I, 8 129 2 240 197 1 34 155 (long) 0.26 (low) 56 (low)

Table 13

Condition donor IFN-γ IL-10 IL12p40 IL1 β IL2 IL6 IP10 TNFa NT D1 nd nd Nd nd nd nd 457 Nd D2 Nd Nd 101.4 Nd Nd Nd 672.7 4.63 D3 Nd Nd Nd Nd Nd Nd 302.3 Nd D4 Nd Nd 104 Nd Nd Nd 648.2 Nd LPS D1 2023 148 7757 5116 Nd 20710 6647 1706 D2 4676 57 6266 6264 21 11070 39539 2987 D3 1537 193 1750 3138 Nd 16838 6141 945 D4 2361 300 1677 6423 19 20654 22848 1107 IFA109 D1 nd nd Nd nd nd 13.48 44495.49 43.63 D2 Nd Nd 116.29 Nd Nd 10.6 44030.74 37.52 D3 Nd Nd Nd Nd Nd Nd 31506.88 62.17 D4 Nd Nd 103.45 Nd Nd nd 45005.31 133.02

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Claims (14)

CLAIM 1. The use of interferon-associated antigen-binding protein containing: (I) an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof; and (II) an interferon (IFN) or a functional fragment thereof for treating hepatitis B virus (HBV) infection, wherein the IFN or a functional fragment thereof is fused to an agonistic anti-CD40 antibody or a functional fragment thereof. agonistic antigen-binding fragment. 2. Use according to claim 1, wherein the IFN or a functional fragment thereof is fused to an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof via a linker. 3. Use according to claim 1 or 2, wherein the agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof contains: (a) a heavy chain or fragment thereof containing a complementarity determining region (CDR) of 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 which is at least 90% identical to SEQ ID NO: 58; and (b) 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. 4. Use according to claim 1 or 2, wherein the agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof contains: (a) a heavy chain or fragment thereof containing a complementarity determining region (CDR) of 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 (b) 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. 5. Use according to any of the preceding claims, wherein the agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof comprises a VL light chain variable region comprising the sequence of SEQ ID NO: 51 or a sequence at least 90% identical thereto; and/or a VH heavy chain variable region comprising the sequence SEQ ID NO: 55 or a sequence at least 90% identical thereto. 6. Use according to any of the preceding claims, wherein the agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof comprises a light chain (LC) that contains the sequence of SEQ ID NO: 3 or a sequence at least 90% identical thereto; and/or a heavy chain (HC) that contains 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 identical thereto at least 90%. 7. Use according to any 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. Use according to claim 7, wherein the type I IFN or functional fragment thereof is IFNa or IFNe or a functional fragment thereof. 9. Use according to any of the preceding claims, wherein the IFN or a functional fragment thereof is IFNa2a or a functional fragment thereof, and wherein preferably IFNa2a comprises the sequence SEQ ID NO: 17 or a sequence identical thereto at least 90%. 10. Use according to any one of claims 1 to 8, wherein the IFN or a functional fragment thereof is IFNe or a functional fragment thereof and wherein preferably the IFNe contains the sequence SEQ ID NO: 14 or a sequence identical thereto at least 90%. 11. Use according to any of the preceding claims, wherein the IFN or a functional fragment thereof is fused to the light chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof, preferably to the C-terminus. 12. Use according to any one of claims 1 to 10, wherein the IFN or a functional fragment thereof is fused to the heavy chain of an agonistic anti-CD40 antibody or an agonistic antigen-binding fragment thereof, preferably to the C-terminus. 13. Use as claimed in any of the preceding claims, wherein the CD40 agonistic antibody or an agonistic antigen-binding fragment thereof and the IFN or a functional fragment thereof are fused to each other through a linker, and wherein the linker comprises the sequence SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26. 14. Use according to any of the previous claims, wherein the interferon-associated antigen-binding protein is an interferon-fusion agonistic anti-CD40 antibody or an interferon-fusion agonistic antigen-binding fragment thereof containing one of the combinations of sequences disclosed in table. 9, specifically in table. 9A or table 9B, more specifically in table. 9A. -