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WO2024213635A1 - Protéines de liaison à l'antigène associées à l'interféron destinées à être utilisées dans le traitement ou la prévention d'une infection par le virus de l'hépatite delta - Google Patents

Protéines de liaison à l'antigène associées à l'interféron destinées à être utilisées dans le traitement ou la prévention d'une infection par le virus de l'hépatite delta Download PDF

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Publication number
WO2024213635A1
WO2024213635A1 PCT/EP2024/059835 EP2024059835W WO2024213635A1 WO 2024213635 A1 WO2024213635 A1 WO 2024213635A1 EP 2024059835 W EP2024059835 W EP 2024059835W WO 2024213635 A1 WO2024213635 A1 WO 2024213635A1
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Prior art keywords
seq
interferon
antigen binding
sequence
agonistic
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PCT/EP2024/059835
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English (en)
Inventor
Antoine Alam
Xavier MARNIQUET
Pascal Bernard JALAGUIER
Kara Lynn CARTER
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Evotec International Gmbh
Sanofi
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Publication of WO2024213635A1 publication Critical patent/WO2024213635A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Interferon-Associated Antigen Binding Proteins for Use for the Treatment or Prevention of Hepatitis delta virus Infection
  • the present invention relates to methods for treating or preventing Hepatitis delta virus infection in a subject.
  • the present invention also relates to 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 treating or preventing Hepatitis delta virus infection.
  • This includes interferon-fused antibodies or interferon-fused antigen binding fragments thereof, which are also referred to herein as “IF As”.
  • the present invention also relates to pharmaceutical compositions comprising such interferon-associated antigen binding proteins or nucleic acids or expression vectors for use in therapy, more particularly for use in treating Hepatitis delta virus infection.
  • the present invention further provides methods of treatment using such interferon-associated antigen binding proteins or nucleic acids or expression vectors or pharmaceutical compositions.
  • Said interferon-associated antigen binding proteins afford beneficial improvements over the current state of the art, for example in that they may effectively reduce viral burden in Hepatitis delta virus-infected cells and/or inhibit Hepatitis delta virus infection and/or rescue cells from Hepatitis delta virus-induced cell death and/or rescue cells from Hepatitis delta virus-induced cytopathic effect.
  • Hepatitis D is the most severe viral hepatitis known (Romeo et al., Gastroenterology 136: 1629-1638 (2009)). Unlike Hepatitis B virus (HBV) which is part of the Hepadnavidae family (partial double strand DNA), the Hepatitis delta virus (HDV) genome is composed of a negative single strand RNA (He et al., J. Med. Virol. 27: 31-33 (1989)) and belongs to the Kolmioviridae family (Kuhn et al., Arch. Virol. 165: 3023-3072 (2020)).
  • HBV Hepatitis B virus
  • HDV Hepatitis delta virus
  • HDV as a viroid needs HBV surface proteins for secretion and entry into target liver cells (Branch et al., Science 223: 450-455 (1984)). Thus, during their life cycle, the two viruses share the same cellular receptor (NTCP) for their entry step.
  • NTCP cellular receptor
  • HDV due to its small size and limited protein coding capacity, HDV also relies on the host machinery for its protein production with a specific RNA editing step to produce 2 proteins L-HDVAg and S-HDVAg from one RNA sequence.
  • a prenylation step involving the host enzyme Famesyl- transferase, is also required for HDV and is essential for the HDV ribonucleoprotein to be able to interact with the HBs protein from HBV and to finalize the assembly and release from the cell (Goodrum et al, 2021).
  • These specificities of each virus are not exhaustive and many host-factors, which are involved in the respective viral replication, differ and are not detailed here.
  • the host response to the infection is also different, in particular regarding the innate immune response.
  • HBV virus employs active strategies to evade innate immune responses and induce immunosuppression in chronic HBV infection
  • an HDV infection induces the type-1 and type-3 IFN pathways (Zhang et al, 2018).
  • HDV also shows some capabilities to escape immune responses and a certain degree of resistance to interferon activity.
  • the IFN therapy which demonstrates benefits in HBV-infected patients, remains one of the therapeutic approaches also used in HBV/HDV patients.
  • CHD chronic hepatitis delta
  • HCC hepatocellular carcinoma
  • recent meta-analyses estimate that 13-14.6 % of HBsAg carriers are infected with HDV worldwide (15-50 million of people) (Stockdale et al., J. Hepatol. 73: 523-532 (2020 and Miao et al., J. Infec. Dis. 221 : 1677-1687 (2020)).
  • HBV/HDV infections are defined in two different categories:
  • Pegylated interferon-IFNa pegylated interferon-IFNa
  • Pegasys pegylated interferon-IFNa
  • BLV Bulevirtide
  • Myrcludex B previously known as Myrcludex B and acting as an entry inhibitor by preventing L- HBsAg interaction with his cellular receptor (NTCP)
  • NTCP his cellular receptor
  • Lonafarnib which was initially developed to inhibit famesylation of RAS for anti-cancer applications, is also currently in phase 3 clinical trials (Bordier et al., J. Virol.
  • Novel methods for treating and preventing Hepatitis delta virus infection are needed.
  • methods for reducing viral burden in Hepatitis delta virus- infected cells, inhibiting Hepatitis delta virus infection, rescuing cells from Hepatitis delta virus-induced cell death and/or rescuing cells from Hepatitis delta virus- induced cytopathic effect are needed.
  • the invention relates to a cluster of differentiation factor 40 (CD40) agonist or a functional fragment thereof for use in the treatment or prevention of a Hepatitis delta virus infection, wherein the CD40 agonist or a functional fragment thereof is administered in combination with an interferon (IFN) or a functional fragment thereof.
  • CD40 cluster of differentiation factor 40
  • IFN interferon
  • the invention further relates to an interferon (IFN) or a functional fragment thereof for use in the treatment or prevention of a Hepatitis delta virus infection, wherein the IFN or a functional fragment thereof is administered in combination with a cluster of differentiation factor 40 (CD40) agonist or a functional fragment thereof.
  • IFN interferon
  • CD40 cluster of differentiation factor 40
  • the invention also relates to a combination of a cluster of differentiation factor 40 (CD40) agonist or a functional fragment thereof and an interferon (IFN) or a functional fragment thereof, for use in the treatment or prevention of a Hepatitis delta virus infection.
  • CD40 cluster of differentiation factor 40
  • IFN interferon
  • the invention in another aspect relates to an interferon-associated antigen binding protein comprising (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 or prevention of a Hepatitis delta virus infection.
  • an interferon-associated antigen binding protein comprising (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 or prevention of a Hepatitis delta virus infection.
  • the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof may comprise (a) a heavy chain or a fragment thereof comprising a complementarity determining region (CDR) CDRH1 that is at least 90% identical to SEQ ID NO 56, a CDRH2 that is at least 90% identical to SEQ ID NO 57, and a CDRH3 that is at least 90% identical to SEQ ID NO 58; and (b) a light chain or a fragment thereof comprising a CDRL1 that is at least 90% identical to SEQ ID NO 52, a CDRL2 that is at least 90% identical to SEQ ID NO 53, and a CDRL3 that is at least 90% identical to SEQ ID NO 54.
  • CDR complementarity determining region
  • the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof may comprise (a) a heavy chain or a fragment thereof comprising a complementarity determining region (CDR) CDRH1 that is identical to SEQ ID NO 56, a CDRH2 that is identical to SEQ ID NO 57, and a CDRH3 that is identical to SEQ ID NO 58; and (b) a light chain or a fragment thereof comprising a CDRL1 that is identical to SEQ ID NO 52, a CDRL2 that is identical to SEQ ID NO 53, and a CDRL3 that is identical to SEQ ID NO 54.
  • CDR complementarity determining region
  • the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof, comprises a light chain variable region VL comprising the sequence as set forth in SEQ ID NO 51, or a sequence at least 90% identical thereto; and/or a heavy chain variable region VH comprising the sequence as set forth in SEQ ID NO 55, or a sequence at least 90% identical thereto.
  • the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof comprises a light chain (LC) that comprises a sequence as set forth in SEQ ID NO 3, or a sequence at least 90% identical thereto; and/or a heavy chain (HC) that comprises a sequence selected from the group consisting of SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO 49 and SEQ ID NO 48, or a sequence at least 90% identical thereto.
  • LC light chain
  • HC heavy chain
  • the IFN or the functional fragment thereof may be selected from the group consisting of a Type I IFN, a Type II IFN and a Type III IFN, or a functional fragment thereof.
  • the type I IFN or the functional fragment thereof is IFNa or IFNP, or a functional fragment thereof.
  • the IFN or the functional fragment thereof is IFNa2a, or a functional fragment thereof.
  • the IFNa2a comprises the sequence as set forth in SEQ ID NO 17, or a sequence at least 90% identical thereto.
  • the IFN or the functional fragment thereof is IFN0, or a functional fragment thereof.
  • the IFN0 comprises the sequence as set forth in SEQ ID NO 14, or a sequence at least 90% identical thereto.
  • the IFN or the functional fragment thereof is fused to a light chain of the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof, preferably to the C-terminus.
  • the IFN or the functional fragment thereof is fused to a heavy chain of the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof, preferably to the C-terminus.
  • the agonistic anti-CD40 antibody or an agonistic antigen binding fragment thereof, and the IFN or the functional fragment thereof are fused to each other via a linker.
  • the linker comprises a sequence as set forth in SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 24, SEQ ID NO 25 or SEQ ID NO 26.
  • the interferon-associated antigen binding protein is an interferon-fused agonistic anti-CD40 antibody or an interferon-fused agonistic antigen binding fragment thereof comprising one of the sequence combinations disclosed in Table 9, in particular Table 9A or Table 9B, more particularly Table 9A.
  • the use comprises administering the interferon-associated antigen binding protein to a subject in need thereof by means of genetic delivery with RNA or DNA sequences encoding the interferon-associated antigen binding protein, or a vector or vector system encoding the interferon- associated antigen binding protein.
  • the interferon-associated antigen binding protein is comprised in a pharmaceutical composition.
  • Fig. 1 This schematic drawing depicts exemplary interferon-associated antigen binding protein formats.
  • the interferon-associated antigen binding protein is an interferon-fused agonistic anti-CD40 antibody or an interferon-fused agonistic antigen binding fragment thereof.
  • IFNs are associated via linkers to different positions on the antibody or the antigen binding fragment thereof N-terminal or C- terminal part of the light chain (LC) or the heavy chain (HC).
  • IFNs are chosen from Type I, Type II and Type III interferon families.
  • FIG. 2A depicts an exemplary map of a pcDNA3.1 plasmid encoding SEQ ID NO 32 under the control of the pCMV promoter.
  • Fig. 2B shows examples of SDS PAGE in reduced conditions of some IF As, with IFNa or IFN0 fused either at the heavy chain or the light chain. Migration of the parental CP870,893 is also shown on the left.
  • FIG. 3A-3B graphically depict a dose dependent effect of a number of IF A molecules with IFN0 fusions on activating the CD40-mediated NFKB pathway reporter assay in HEK-BlueTM CD40L cells.
  • Fig. 3A shows examples of anti-CD40 activities for IFAs with IFN0 fused to the C-terminal part of the heavy chain (HC).
  • Fig. 3B shows examples of anti-CD40 activities for IFAs with IFN0 fused to the N- terminal part of the LC (IF A34) or the HC (IF A36) and the corresponding fusions on the C-terminal part (IFA35 and IFA37). Purification yield of the latter group of IFAs was very low, thus to test their activity, the supernatants from HEK transfected cells were used and serially diluted to evaluate the anti-CD40 activity on HEK-BlueTM CD40L cells.
  • Figs. 3C-3D graphically depict a dose dependent effect of a number of IF A molecules with IFN0 fusions on activating the Type I IFN- pathway in reporter HEK- Blue-IFN-a/0 cells.
  • Fig. 3C shows examples of IFN activity for IFAs with IFN0 fused to the C-terminal part of the HC.
  • Fig. 3D shows examples of IFN activity for IFAs with IFN0 fused to the N-terminal part of the LC (IFA34) or the HC (IFA36) and the corresponding fusions on the C-terminal part (IFA35 and IFA37).
  • IFA34 the N-terminal part of the LC
  • IFA36 the HC
  • the same supernatants from HEK transfected cells as in Fig. 3B were used and serially diluted to evaluate the IFN activity.
  • Parental antibody CP870,893 was used as negative control and recombinant human IFN0 was used as
  • Fig. 4A graphically depicts a dose effect of a number of IFA molecules with IFNa fusions on activating the CD40-mediated NFKB pathway reporter assay in HEK-BlueTM CD40L cells.
  • Fig. 4B graphically depicts a dose effect of a number of IFA molecules with IFNa fusions on activating the Type I IFN-mediated pathway in reporter HEK-Blue- IFN-a/
  • the activity of Pegasys is indicated in the insert in the lower right comer.
  • Fig. 4C graphically depicts the effect of IFA molecules with IFNa fusions and HL linker on HC (IFA38) or LC (IFA39) on activating the CD40-mediated NFKB pathway reporter assay in HEK-BlueTM CD40L cells.
  • Fig. 4D graphically depicts the effect of IFA38 and IFA39 on activation of the Type I IFN-pathway in reporter HEK-Blue-IFNa/0 cells.
  • Figs. 5A-5C graphically depict a dose dependent effect of Pegasys on freshly isolated primary human hepatocytes (PHH) after HBV/HDV co-infection. Briefly, at the day of co-infection, cells were infected both with HBV and HDV for 4 days and incubated at 37°C, 5% CO2. Then, all media was replaced with fresh media containing Pegasys in a 10-fold serial dilution at days 4 and 7, respectively. Finally, at day 10 post infection (p.i.) secreted HBsAg was measured with CLIA assay (Fig. 5A). Relative intracellular HBV RNA (Fig. 5B) and HDV RNA (Fig. 5C) expression was quantified by specific qRT-PCR reactions.
  • Fig. 5D graphically depicts a propagation assessment in Huh7.5-hNTCP with Pegasys conditioned PHH medium. Briefly, supernatants collected at the end of the kinetic of the co-infection (day 10) as described in Fig. 5A-5C were directly transferred to non-infected Huh7.5-hNTCP growing cell line for 6 days. At the end of the kinetic total RNA was extracted and the relative intracellular HDV RNA expression was quantified by a specific qRT-PCR reaction.
  • Figs. 5E-5G graphically depict a dose dependent effect of IFA44 on freshly isolated primary human hepatocytes (PHH) after HBV/HDV co-infection.
  • Fig. 5H graphically depicts a propagation assessment in Huh7.5-hNTCP with IFA44 conditioned PHH medium. Briefly, supernatants collected at the end of the kinetic of the co-infection (day 10) as described in Figs. 5E-5G were directly transferred to non-infected Huh7.5-hNTCP growing cell line for 6 days. At the end of the kinetic total RNA was extracted and the relative intracellular HDV RNA expression was quantified by a specific qRT-PCR reaction.
  • Figs. 5I-5K graphically depict a dose dependent effect of IFA25 on freshly isolated primary human hepatocytes (PHH) after HBV/HDV co-infection. Briefly, at the day of co-infection, cells were infected both with HBV and HDV for 4 days and incubated at 37°C, 5% CO2. Then, all media was replaced with fresh media containing IFA25 in a 10-fold serial dilution at days 4 and 7, respectively. Finally, at day 10 p.i. secreted HBsAg was measured with CLIA assay (Fig. 51). Relative Intracellular HBV RNA (Fig. 5J) and HDV RNA (Fig. 5K) expression was quantified by specific qRT-PCR reactions.
  • Fig. 5L graphically depicts a propagation assessment in Huh7.5-hNTCP with IFA25 conditioned PHH medium. Briefly, supernatants collected at the end of the kinetic of the co-infection (day 10) as described in Figs. 5I-5K were directly transferred to non-infected Huh7.5-hNTCP growing cell line for 6 days. At the end of the kinetic total RNA was extracted and the relative intracellular HDV RNA expression was quantified by a specific qRT-PCR reaction.
  • Figs. 5M-5O graphically depict a dose dependent effect of IFA25 on freshly isolated primary human hepatocyte after HBV/HDV co-infection with a different kinetic. Briefly, at the day of co-infection, cells were infected both with HBV and HDV for 3 days and incubated at 37°C, 5% CO2. Then, all media was replaced with fresh media containing IFA25 in a 10-fold serial dilution at days 3 and 6 respectively. Finally, at day 9 p.i. secreted HBsAg was measured with CLIA assay (Fig. 5M). Relative Intracellular HB V RNA (Fig. 5N) and HDV RNA (Fig. 50) expression was quantified by specific qRT-PCR reactions.
  • Fig. 5P graphically depicts a propagation assessment in Huh7.5-hNTCP with IFA25 conditioned PHH medium. Briefly, supernatants collected at the end of the kinetic of the co-infection (day 9) as described in Figs. 5M-5O were directly transferred to non-infected Huh7.5-hNTCP growing cell line for 6 days. At the end of the kinetic total RNA was extracted and the relative intracellular HDV RNA expression was quantified by a specific qRT-PCR reaction.
  • Figs. 5Q-5S graphically depict antiviral effects of IFA25 compared to reference controls on freshly isolated primary human hepatocytes (PHH) after HBV/HDV co-infection. Briefly, at the day of co-infection, cells were infected for 3 days and incubated at 37°C, 5% CO2. Then, all media was replaced with fresh media containing EVI5 (1 pg/ml), IFA25 (0.1 and 1 pg/ml), IFA202 (Ipg/ml), Roferon-A (lOOOUI/ml), Myrcludex-B (MyrB; lOOnM) and FTI-277 (lOpM) (in that order from left to right as shown in Figs.
  • EVI5 1 pg/ml
  • IFA25 0.1 and 1 pg/ml
  • IFA202 Ipg/ml
  • Roferon-A lOOOUI/ml
  • Myrcludex-B MyrB; l
  • Fig. 5T graphically depicts a propagation assessment in Huh7.5-hNTCP with IFA25 conditioned PHH medium or reference controls conditioned PHH medium. Briefly, supernatants collected at the end of the kinetic of the co-infection (day 9) as described in Figs. 5Q-5S were directly transferred to non-infected Huh7.5-hNTCP growing cell line for 6 days. At the end of the kinetic total RNA was extracted and the relative intracellular HDV RNA expression was quantified by a specific qRT- PCR reaction.
  • Figs. 6A-6C graphically depict a dose dependent effect of IFA25 on freshly isolated primary human hepatocytes (PHH) after HBV/HDV super-infection. Briefly, at the day of infection, cells were infected for 5 days with HBV only and incubated at 37°C, 5% CO2. Then, cells were super-infected by HDV for 3 extra days and incubated at 37°C, 5% CO2. All media was replaced with fresh media containing IFA25 in a 10-fold serial dilution at days 8 and 11 respectively. Finally, at day 14 p.i. secreted HBsAg was measured with CLIA assay (Fig. 6A). Relative Intracellular HBV RNA (Fig. 6B) and HDV RNA (Fig. 6C) expression was quantified by specific qRT-PCR reactions.
  • Fig. 6D graphically depicts a propagation assessment in Huh7.5-hNTCP with IFA25 conditioned PHH medium. Briefly, supernatants collected at the end of the kinetic of the super-infection (day 14) as described in Figs. 6A-6C were directly transferred to non-infected Huh7.5-hNTCP growing cell line for 6 days. At the end of the kinetic total RNA was extracted and the relative intracellular HDV RNA expression was quantified by a specific qRT-PCR reaction.
  • Figs. 6E-6G graphically depict a dose dependent effect of IFA25 compared to reference controls on freshly isolated primary human hepatocytes (PHH) after HBV/HDV super-infection. Briefly, at the day of infection, cells were infected for 5 days with HBV only and incubated at 37°C, 5% CO2. Then, cells were super-infected by HDV for 3 extra days and incubated at 37°C, 5% CO2.
  • Fig. 6H graphically depicts propagation assessment in Huh7.5-hNTCP with IFA25 conditioned PHH medium or reference controls conditioned PHH medium. Briefly, supernatants collected at the end of the kinetic of the infection (day 14) were directly transferred to non-infected Huh7.5-hNTCP growing cell line for 6 days. At the end of the kinetic of the infection total RNA was extracted and the relative intracellular HDV RNA expression was quantified by a specific qRT-PCR reaction.
  • Fig. 7 depicts results from an in vitro Cytokines Release Assay of Human Whole Blood Cells (WBCs): Example of data obtained after stimulation of WBCs from 4 healthy volunteer donors. WBC were left Non-Stimulated (NS), treated with LPS (10 ng/mL) or with IFA1 (1 pg/mL) for 24 h. Supernatants were collected and submitted to cytokines release quantification using the MSD u-Plex kit for human cytokines. Results represent the mean of two independent stimulations from each donor. The profile of CXCL10 (IP10), IL6, IL1J3 and TNFa are shown.
  • IP10 CXCL10
  • Tables lla-b These tables summarize data obtained after in vitro stimulation of whole blood cells (WBCs) obtained from healthy volunteers. Each IFA was tested on WBCs from four different donors. WBCs were left Non-Treated (NT), treated with LPS (10 ng/mL) or with IFAs (1 pg/mL) for 24 h. Supernatants were collected and submitted to cytokines release quantification using the MSD u-Plex kit for human cytokines. Results represent the mean 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 0.5 mg/kg (IFAs) or 0.3 mg/kg (Pegasys) intravenous bolus injection to mice. Data expressed as mean +/- SD on semi -logarithmic scale. Samples were collected up to 10 days after administration. ELISA assay using anti-IFNa as secondary antibody for quantification method was used for IFA27, IFA29 and IFA30 (Fig. 8A) and for IFA25, IFA26 and IFA28 (Fig. 8B). ELISA assay using anti-IgG2 as secondary antibody for quantification method was used for IFA25 and IFA27 (Fig. 8C). Fig. 8D: Pegasys quantification was done using human IFNa matched antibody pairs. The marked line (LLOQ) denotes the limit of detection for the Pegasys assay.
  • Table 12A PK Report Summary: PK parameters for CP870,893, IFA27, IFA29 and IFA30 following single intravenous administration of 0.5 mg/kg to male CD1 Swiss mice. PK parameters for CP870,893 were explored in a 7-day experiment and those for IFA27, IFA29 and IFA30 in 10-day experiments (quantification for IFA27 was performed using 2 different ELISA approaches).
  • Table 12B PK Report Summary: PK parameters for CP870,893, Pegasys and for three different IFAs (IFA25, IFA26 and IFA28) following single intravenous bolus administration of 0.5 mg/kg to male CD1 Swiss mice. PK parameters for CP870,893 and IFA25, IFA26, IFA28 and Pegasys were explored in 21-day experiments (quantification for IFA25 was performed using 2 different ELISA approaches).
  • Fig. 9A depicts CD40 agonistic activity in a dose dependent manner of IFA50 and IFA51 with no Fc region in comparison to the parental anti-CD40 antibody in reporter HEK-BlueTM CD40L cells.
  • Fig. 9B depicts the IFNa activity in a dose dependent manner of IFA50 and IFA51 in reporter HEK-BlueTM hIFN-a/0 cells.
  • Fig. 10A depicts CD40 agonistic activity in a dose dependent manner of IFNE based IFA49, in comparison to parental anti-CD40 antibody, in HEK-BlueTM CD40L reporter cells.
  • IFA49 corresponds to fusion of IFNE to the HC via a peptide linker.
  • Fig. 10B depicts the IFN activity in a dose dependent manner of IFA49 on reporter HEK-BlueTM hIFN-a/0 reporter cells which are activated by Type I interferons.
  • Fig. HA depicts CD40 agonistic activity in a dose dependent manner of IFNco based IFA46, in comparison to parental anti-CD40 antibody, in HEK-BlueTM CD40L reporter cells.
  • IFA46 correspond to fusion of IFNro to the LC via a peptide linker.
  • Fig. 11B depicts the IFN activity in a dose dependent manner of IFA46 on reporter HEK-BlueTM hIFN-a/0 reporter cells which are activated by Type I interferons.
  • Fig. 12A depicts CD40 agonistic activity in a dose dependent manner of IFNy based IFAs (IFA42 and IFA43), in comparison to parental anti-CD40 antibody, in HEK-BlueTM CD40L reporter cells.
  • IFA42 corresponds to fusion of IFNy to the LC via a peptide linker
  • IFA43 corresponds to fusion of IFNy to the HC via a peptide linker.
  • Fig. 12B depicts the IFN activity in a dose dependent manner of IFA42 and IFA43 in reporter HEK-Blue-hlFNy cells.
  • Fig. 13A depicts CD40 agonistic activity in a dose dependent manner of IFNX based IFAs (IFA44 and IFA45), in comparison to parental anti-CD40 antibody, in HEK-BlueTM CD40L reporter cells.
  • IFA44 corresponds to fusion of IF NX to the LC via a peptide linker
  • IFA45 correspond to fusion of IFNA. to the HC via a peptide linker.
  • Fig. 13B depicts the IFN activity in a dose dependent manner of IFA44 and IFA45 in reporter HEK-Blue-hlFN cells.
  • Fig. 14 shows examples of SDS PAGE in reduced conditions of some IF As, with IFNa or IFN0 fused on the heavy chain of 3G5-antiCD40 antibody. Migration of the parental 3G5 antiCD40 antibody is also shown on the left.
  • Figs. 15A-B graphically show a dose dependent effect of a number of 3G5- based IFA molecules with IFNP fusions on activating the CD40-mediated NFKB pathway reporter assay in HEK-BlueTM CD40L cells. Comparison to the parental antibody 3G5 (designated in this figure as CDX-3G5) is likewise shown.
  • Fig. 15A shows examples of anti-CD40 activities for IFAs with fusion of IFNP to the C- terminal part of the heavy chain (HC).
  • Figs. 15C-D graphically show a dose dependent effect of a number of IFA molecules with IFNP fusions on activating the Type I IFN-pathway in reporter HEK- Blue-IFN-a/p cells.
  • Fig. 15C shows examples of IFN activity for IFAs with fusion of IFNp to the C-terminal part of the HC.
  • Fig. 15D shows IFN activity of IFAs with IFNP fused on the light chain; the production level of these proteins was very low and thus an example of activity for two IFAs is shown in Fig. 15D using the same supernatant as in Fig. 15B.
  • Fig. 16A graphically shows a dose effect of four IFAs molecules with IFNa fusions on activating the CD40-mediated NFKB pathway reporter assay in HEK- BlueTM CD40L cells. Comparison to the parental antibody 3G5 (designated in this figure as CDX-3G5) is likewise shown.
  • Fig. 16B graphically shows a dose effect of a number of IFAs molecules with IFNa fusions on activating the Type I IFN-mediated pathway in reporter HEK-Blue- IFN-a/p cells.
  • Fig. 17 In vitro Cytokines Release Assay of Human Whole Blood Cells (WBCs): Example of data obtained after stimulation of WBCs from 4 healthy volunteer donors. WBCs were left non-treated (NT), treated with LPS (10 ng/mL) or with IFA109 (1 pg/mL) for 24 h. Supernatants were collected and submitted to cytokines release quantification using the MSD u-Plex kit for human cytokines. Results represent the mean of two independent stimulations from each donor. The profile of CXCL10 (IP10), IL6, IL10 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 WBCs from four different donors. WBCs were left Non-Treated (NT), treated with LPS (lO ng/mL) or with IFA109 (1 pg/mL) for 24 h. Supernatants were collected and submitted to cytokines release quantification using the MSD u-Plex kit for human cytokines. Results represent the mean of two independent stimulations from each donor and are expressed in pg/mL (nd: not detected).
  • Figs. 18A-C graphically depict viral parameters monitored at day 7 following HBV/HDV Coinfection in Cryo-PHH after compounds treatment in primary infection.
  • Fig. 18A depicts HDVRNA viremia quantification by qRT-PCR.
  • Fig. 18B depicts HBsAg secretion quantification by CLIA assay.
  • Fig. 18C depicts Tubulin (upper band) and HD-Ag (lower bands) protein expression in western blot from intracellular cryo-PHH.
  • FIG. 19 depicts relative intracellular HDV RNA expression measurement by qRT-PCR at day 7 in cryo-PHH after the secondary infection from conditioned- supernatants at fixed-volume (Fig. 19A) and fixed-Vge/cell (Fig. 19B).
  • Figs. 20A-D graphically depict viral parameters monitored at day 7 following HBV/HDV Coinfection with two different treatment modalities (Concomitant or 1- day post-Coinfection).
  • Fig. 20A depicts HDV RNA viremia quantification by qRT- PCR after treatments given at the time of infection.
  • Fig. 20B depicts HDV RNA viremia quantification by qRT-PCR after treatments given at 1-day post-Coinfection.
  • Fig. 20C depicts effect of treatments given at the time of Coinfection on HBsAg secretion quantification by CLIA.
  • Fig. 20D depicts effect of treatments given at 1- day post-Coinfection on HBsAg secretion quantification by CLIA assay.
  • Figs. 21 A-E graphically depict viral parameters monitored at day 7 following HBV/HDV genotype-3 Coinfection in Cryo-PHH after IFA25 and Pegasys treatment.
  • Fig. 21A depicts HDV RNA viremia quantification by qRT-PCR.
  • Fig. 21B depicts HD-Ag expression in western blot from intracellular cryo-PHH.
  • Fig. 21C depicts HBsAg secretion quantification by CLIA assay.
  • Fig. 21D depicts HBeAg secretion quantification by CLIA assay.
  • Fig. 21E HBV DNA viremia quantification by ddPCR.
  • Figs. 22A-G graphically depict the assessment of Myrcludex-B compatibility with IFA25 and Pegasys (control) on viral parameters monitored at day 7 following HBV/HDV Coinfection.
  • Fig. 22A depicts inhibitory concentration estimation from a dose range effect of Myrcludex-B given at the time of infection on HDV RNA viremia quantification by qRT-PCR.
  • Fig. 22B depicts effect of IFA25 dose range given in addition to Myrcludex-B on HDV RNA viremia quantification by qRT -PCR.
  • Fig. 22C depicts effect of Pegasys dose range given in addition to Myrcludex-B on HDV RNA viremia quantification by qRT-PCR.
  • Fig. 22A-G graphically depict the assessment of Myrcludex-B compatibility with IFA25 and Pegasys (control) on viral parameters monitored at day 7 following HBV/HDV Coinfection.
  • Fig. 22A depicts inhibitory concentration estimation from a
  • FIG. 22D depicts effect of IFA25 dose range given in addition to Myrcludex-B on HBsAg secretion quantified by CLIA assay.
  • Fig. 22E depicts effect of Pegasys dose range given in addition to Myrcludex-B on HBsAg secretion quantification by CLIA assay.
  • Fig. 22F depicts effect of IFA25 dose range given in addition to Myrcludex-B on cell viability assessed by Cell TiterGlo assay.
  • Fig. 22G depicts effect of Pegasys dose range given in addition to Myrcludex-B on cell viability assessed by Cell TiterGlo assay.
  • Figs. 23A-I graphically depict the assessment of Tenofovir compatibility with IFA25 and Pegasys (control) on viral parameters monitored at day 7 following HBV/HDV Coinfection.
  • Fig. 23A depicts inhibitory concentration estimation from a dose range effect of Tenofovir given at the time of infection on HBV DNA viremia quantification by qRT-PCR.
  • Fig. 23B depicts effect of IFA25 dose range given in addition to tenofovir on HBV DNA viremia quantification by qRT-PCR.
  • Fig. 23C depicts effect of Pegasys dose range given in addition to Tenofovir on HBV DNA viremia quantification by qRT-PCR.
  • Fig. 23A-I graphically depict the assessment of Tenofovir compatibility with IFA25 and Pegasys (control) on viral parameters monitored at day 7 following HBV/HDV Coinfection.
  • Fig. 23A depicts inhibitory concentration estimation from a dose range effect of Tenofovir given at the time of
  • FIG. 23D depicts effect of IFA25 dose range given in addition to Tenofovir on HBsAg secretion quantified by CLIA assay.
  • Fig. 23E depicts effect of Pegasys dose range given in addition to Tenofovir on HBsAg secretion quantification by CLIA assay.
  • Fig. 23F depicts effect of IFA25 dose range given in addition to tenofovir on HDV RNA viremia quantification by qRT-PCR.
  • Fig. 23G depicts effect of Pegasys dose range given in addition to tenofovir on HDV RNA viremia quantification by qRT-PCR.
  • FIG. 23H depicts effect of IFA25 dose range given in addition to Tenofovir on cell viability assessed by Cell TiterGlo assay.
  • Fig. 231 depicts effect of Pegasys dose range given in addition to Tenofovir on cell viability assessed by Cell TiterGlo assay.
  • any of the definitions and embodiments described and/or claimed herein are intended to be definitions and embodiments applicable to all aspects, embodiments, items and matters of the invention.
  • teaching and explanations provided herein in respect of suitable ways or embodiments of preparing, formulating and administering the interferon- associated antigen binding proteins of the invention, or nucleic acids encoding or expressing same, and routes of their administration, suitable dosages and administration regimens therefor apply mutatis mutandis to the cluster of differentiation factor 40 (CD40) agonists or functional fragments thereof, the interferons (IFNs) or functional fragments thereof, or nucleic acids encoding or expressing same, or the combinations thereof as described or claimed herein.
  • CD40 cluster of differentiation factor 40
  • IFNs interferons
  • the present invention is based in part on the discovery of a therapy that is based on the use of “interferon-associated antigen-binding proteins”, variants or derivatives thereof comprising (I) an agonistic anti-CD40 antibody or an agonistic antigen binding fragment thereof, and (II) an interferon (IFN) or a functional fragment thereof in Hepatitis delta virus therapy.
  • “interferon-associated antigen-binding proteins” variants or derivatives thereof comprising (I) an agonistic anti-CD40 antibody or an agonistic antigen binding fragment thereof, and (II) an interferon (IFN) or a functional fragment thereof in Hepatitis delta virus therapy.
  • Said interferon-associated antigenbinding proteins reduce viral burden in Hepatitis delta virus-infected cells, inhibit Hepatitis delta virus infection, rescue cells from Hepatitis delta virus-induced cell death and from Hepatitis delta virus-induced cytopathic effect and enhance the IFN pathway in uninfected and infected cells, and may even act in a synergistic fashion.
  • Hepatitis delta virus therapy comprising administering an interferon-associated antigen-binding protein to a Hepatitis delta virus-infected cell, or a subject infected with Hepatitis delta virus, is provided. [0075]
  • the invention may be more readily understood in the light of the selected terms defined below.
  • CD40 refers to “Cluster of differentiation 40”, a member of the tumor necrosis factor receptor (TNFR) superfamily.
  • Table B is derived from the HUGO Gene Nomenclature Committee (HGNC) (see, Gray et al. Nucleic Acids Res. 43: DI 079- 1085 (2015); HGNC Database, HUGO Gene Nomenclature Committee (HGNC), EMBL Outstation - Hinxton, European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, CB10 1SD, UK www.genenames.org) .
  • the Approved Symbol denotes the HGNC symbol applied to a particular gene and the Approved Name corresponds to the full spelling of the gene.
  • Previous Symbols denotes any previous symbol used by HGNC or refer to a particular gene. Synonyms refer to alternative, synonymous names for a particular gene.
  • CD40 is a costimulatory protein found on antigen presenting cells (e.g., B cells, dendritic cells, monocytes), hematopoietic precursors, endothelial cells, smooth muscle cells, epithelial cells, as well as the majority of human tumors (Grewal & Flavell, Ann. Rev. Immunol., 1996, 16: 111-35; Toes & Schoenberger, Seminars in Immunology, 1998, 10(6): 443-8).
  • antigen presenting cells e.g., B cells, dendritic cells, monocytes
  • hematopoietic precursors hematopoietic precursors
  • CD40L natural ligand CD 154
  • TRAF1, TRAF2, TRAF6 and TRAF5 interact with CD40 and serve as mediators of the signal transduction.
  • CD40 signaling activates both the canonical and the noncanonical NF- KB pathways.
  • a “CD40 agonist” refers to a compound (e.g., protein, a fusion protein, a polypeptide, an antibody, an antigen-binding fragment of an antibody or the like) that activates CD40.
  • a CD40 agonist may be an agonistic antibody directed against CD40 or a functional fragment thereof, or a soluble CD40 agonist including but not limited to its natural ligand or a functional fragment thereof.
  • a CD40 agonist is an agonistic antibody directed against CD40.
  • a CD40 agonist is CD40L.
  • a functional fragment refers to a fragment of a substance that retains one or more functional activities of the original substance, preferably all of the functional activities.
  • a functional fragment of a CD40 agonist refers to a fragment of a CD40 agonist that retains a function of the CD40 agonist as described and/or claimed herein, e.g., it activates a target CD40.
  • ligand refers to any substance capable of binding, or of being bound, to another substance.
  • a ligand may be a peptide, a polypeptide, a protein, an aptamer, a polysaccharide, a sugar molecule, a carbohydrate, a lipid, an oligonucleotide, a polynucleotide, a synthetic molecule, an inorganic molecule, an organic molecule, and any combination thereof.
  • the ligand is a polypeptide.
  • the term “antibody” refers to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM).
  • Each heavy chain comprises a heavy chain variable region (abbreviated VH or VH) and a heavy chain constant region (CH or CH).
  • the heavy chain constant region comprises three domains, CHI, CH2 and CH3.
  • Each light chain comprises a light chain variable region (abbreviated VL or VL) and a light chain constant region (CL or CL).
  • the light chain constant region comprises one domain (CL1).
  • VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions (CDRs)”, interspersed with regions that are more conserved, termed “framework regions” (FR).
  • CDRs complementarity determining regions
  • FR frame regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • Framework regions can aid in maintaining the proper conformation of the CDRs to promote binding between the antigen binding region and an antigen.
  • immunoglobulin G a tetrameric glycoprotein.
  • each tetramer is composed of two identical pairs of polypeptide chains, each pair having one light (about 25 kDa) and one heavy chain (about 50-70 kDa).
  • the amino-terminal portion of each chain includes a variable region of about 100 to 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 assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chains.
  • Heavy chains are classified as mu (p), delta (5), gamma (y), alpha (a), and epsilon (e), and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • Several of these may be further divided into subclasses or isotypes, e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2.
  • Different isotypes have different effector functions; for example, IgGl and IgG3 isotypes have antibody-dependent cellular cytotoxicity (ADCC) activity.
  • ADCC antibody-dependent cellular cytotoxicity
  • the agonistic antiCD40 antibodies or agonistic antigen binding fragments thereof comprised in the interferon-associated antigen binding proteins according to the invention are of the IgG class.
  • the agonistic antiCD40 antibodies or agonistic antigen binding fragments thereof comprised in the interferon-associated antigen binding proteins according to the invention are of the IgGl or IgG3 subclasses.
  • the agonistic antiCD40 antibodies or agonistic antigen binding fragments thereof comprised in the interferon-associated antigen binding proteins according to the invention are of the IgGl subclass.
  • the agonistic antiCD40 antibodies or agonistic antigen binding fragments thereof comprised in the interferon-associated antigen binding proteins according to the invention are of the IgG2 or IgG4 subclasses. In specifically preferred embodiments, the agonistic antiCD40 antibodies or agonistic antigen binding fragments thereof comprised in the interferon-associated antigen binding proteins according to the invention are of the IgG2 subclass.
  • the agonistic antiCD40 antibodies or agonistic antigen binding fragments thereof comprised in the interferon-associated antigen binding proteins according to the invention comprise a light chain of the K class.
  • the agonistic antiCD40 antibodies or agonistic antigen binding fragments thereof comprised in the interferon-associated antigen binding proteins according to the invention comprise a light chain of the 1 class.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, wherein the heavy chain additionally includes 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)).
  • antibody further includes, but is not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to as “antibody mimetics”), chimeric antibodies, humanized antibodies, human antibodies, and fragments thereof, respectively.
  • antibody mimetics sometimes referred to as “antibody mimetics”
  • antibody includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, derivatives, variants, antigen binding fragments, and muteins thereof, examples of which are described below.
  • the term “agonistic CD40 antibody” or “agonistic anti- CD40 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 may be determined using surface plasmon resonance, preferably using the BIAcore® system.
  • the agonistic anti-CD40 antibody may 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 the agonistic anti-CD40 antibody may be measured using a whole blood surface molecule upregulation assay or using an in vitro reporter cell assay, e.g., using HEK-BlueTM CD40L cells (InvivoGen Cat. #: hkb-cd40), as described in greater detail in Example I.
  • HEK-BlueTM CD40L cells InvivoGen Cat. #: hkb-cd40
  • SEAP NFKB-inducible secreted embryonic alkaline phosphatase
  • the interferon-associated antigen binding proteins activate both the CD40 and an IFN pathway.
  • the interferon-associated antigen binding protein activates the CD40 pathway with an ECso of less than 400, 300, 200, 150, 100, 70, 60, 50, 40, 30, 25, 20, or 15 ng/mL, wherein CD40 activity is preferably determined using an in vitro reporter cell assay, optionally using HEK-BlueTM CD40L cells, as described for instance in Example I.
  • the interferon-associated antigen binding protein activates the CD40 pathway with an EC so ranging from 10 to 200 ng/mL. In even more specific embodiments, the interferon-associated antigen binding protein activates the CD40 pathway with an ECso ranging from 10 to 50 ng/mL, preferably 10 to 30 ng/mL.
  • Suitable agonistic anti-CD40 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).
  • Exemplary light and heavy chain sequences of the agonistic anti-CD40 antibody CP870,893 are shown in Table 7.
  • Exemplary light and heavy chain sequences of the agonistic anti-CD40 antibody 3G5 are shown in Table 8.
  • agonistic antigen binding fragment of an agonistic anti-CD40 antibody refers to a fragment of an agonistic anti-CD40 antibody that retains one or more functional activities of the original antibody, such as the ability to bind to and act as an agonist of CD40 signaling in a cell, e.g., it mediates CD40 pathway signaling. Such 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 cleavage of an anti-CD40 antibody.
  • Agonistic antigen binding fragments include, but are not limited to, a Fab fragment, a diabody (heavy chain variable domain on the same polypeptide as a light chain variable domain, connected via a short peptide linker that is too short to permit pairing between the two domains on the same chain), a Fab’ fragment, a F(ab’)2 fragment, a Fv fragment, domain antibodies and singlechain antibodies, and can be derived from any mammalian source, including but not limited to human, mouse, rat, camelid or rabbit.
  • variable region refers to a portion of the light and/or heavy chains of an antibody, typically including approximately the amino-terminal 120 to 130 amino acids in the heavy chain and about 100 to 110 amino terminal amino acids in the light chain. Variable regions of different antibodies differ extensively in amino acid sequence even among antibodies derived from the same species or of the same class.
  • Exemplary VL and VH domain sequences of the agonistic anti-CD40 antibody CP870,893 are shown in Table 1.
  • the variable region of an antibody typically determines specificity of a particular antibody for its target as it contains the CDRs.
  • Table 1 also shows exemplary CDR sequences of the agonistic anti-CD40 antibody CP870,893.
  • Bold italicized sequences correspond to CDR regions according to the Kabat definition.
  • Delineation of a CDR and identification of residues comprising the binding site of an antibody may be accomplished by solving the structure of the antibody and/or solving 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 methods of analysis can be employed to identify or approximate the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition and the contact definition. [0095] The Kabat definition is a standard for numbering the residues in an antibody and is typically used to identify CDR regions. See, e.g., Johnson & Wu, Nucleic Acids Res., 28: 214-8 (2000).
  • the Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account positions of certain structural loop regions. See, e.g., Chothia etal., J. Mol. Biol., 196: 901-17 (1986); Chothia et al., Nature, 342: 877-83 (1989).
  • the AbM definition uses an integrated suite of computer programs produced by Oxford Molecular Group that model antibody structure. See, e.g., Martin et al., Proc Natl Acad Sci (USA), 86:9268-9272 (1989); “AbMTM, A Computer Program for Modeling Variable Regions of Antibodies,” Oxford, UK; Oxford Molecular, Ltd.
  • the AbM definition models the tertiary structure of an antibody from primary sequence using a combination of knowledge 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 contact definition is based on an analysis of the available complex crystal structures. See, e.g., MacCallum et al., J. Mol. Biol., 5:732-45 (1996).
  • 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 can be grafted to framework regions (FRs) from the same, or another, species.
  • the CDRs of the light and heavy chain variable regions of an agonistic anti-CD40 antibody, or an agonistic antigen binding fragment thereof can be grafted to consensus human FRs.
  • consensus human FRs in certain embodiments, FRs from several human heavy chain or light chain amino acid sequences are aligned to identify a consensus amino acid sequence.
  • the FRs of the heavy chain or light chain of an agonistic anti-CD40 antibody, or an agonistic antigen binding fragment thereof are replaced with the FRs from a different heavy chain or light chain.
  • rare amino acids in the FRs of the heavy and light chains of an agonistic anti-CD40 antibody, or an agonistic antigen binding fragment thereof are not replaced, while the rest of the FR amino acids are replaced. Rare amino acids are specific amino acids that are in positions in which they are not usually found in FRs.
  • the grafted variable regions from an agonistic anti-CD40 antibody, or an agonistic antigen binding fragment thereof can 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.
  • the grafted variable regions are part of a single chain Fv antibody. CDR grafting is described, e.g., in U.S. Patent Nos.
  • An “Fc” region typically comprises two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains.
  • a “Fab fragment” comprises one full-length light chain as well as the CHI and variable regions of one heavy chain (the combination of the VH and CHI regions is referred to herein as “fab region heavy chain”).
  • a “Fab’ fragment” comprises one light chain and a portion of one heavy chain that contains the VH domain and the CHI domain and also the region between the CHI and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab’ fragments to form an F(ab’)2 molecule.
  • a “F(ab’)i fragment” contains two light chains and two heavy chains containing a portion of the constant region between the CHI and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains.
  • a F(ab’)2 fragment thus is composed of two Fab’ fragments that are held together by a disulfide bond between the two heavy chains.
  • the “Fv region” comprises the variable regions from both the heavy and light chains, but lacks the constant regions.
  • Single-chain antibodies are Fv molecules in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen binding region.
  • Single chain antibodies are discussed in detail in International Patent Application Publication No. WO 88/01649 and United States Patent Nos. 4,946,778 andNo. 5,260,203, the disclosures of which are incorporated by reference.
  • a “domain antibody” is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain.
  • two or more VH regions are covalently joined with a peptide linker to create a bivalent domain antibody.
  • the two VH regions of a bivalent domain antibody can target the same or different antigens.
  • An antibody or antigen binding protein such as an interferon-associated antigen binding protein according to the invention, preferably binds to its target antigen with a dissociation constant (Ka) of ⁇ 10' 7 M.
  • the antibody or antigen binding protein binds its antigen with “high affinity” when the Ka is ⁇ 5 x 10' 9 M, and with “very high affinity” when the K is ⁇ 5 x 10' 10 M. More preferably, the antibody or antigen binding protein has a Ka of ⁇ 10' 9 M. In some embodiment, the off-rate is ⁇ 1 x 10' 5 . In other embodiments, the antibody or antigen binding protein will bind to human CD40 with a Ka of between about 10' 9 M and 10' 13 M, and in yet another embodiment the antibody or antigen binding protein will bind with a Ka ⁇ 5 x 10' 10 . As will be appreciated by one of skill in the art, in some embodiments, any or all of the antigen binding fragments can bind to CD40. Preferably, said constants are determined using surface plasmon resonance, more preferably using the BIAcore® system.
  • surface plasmon resonance means an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore® system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, N.J.). For further descriptions, see Jonsson et al. (1993) Ann. Biol. Clin. 51 : 19-26.
  • K on means the on rate constant for association of a binding protein (e.g., an antibody or antigen binding protein) to the antigen to form the, e.g., antigen binding protein/antigen complex.
  • K on also means “association rate constant”, or “ka”, as is used interchangeably herein.
  • association rate constant or “ka”, as is used interchangeably herein. This value indicating the binding rate of a binding protein to its target antigen or the rate of complex formation between a binding protein, e.g., an antibody or an antigen binding protein, and antigen also is shown by the equation below:
  • K O ff means the off rate constant for dissociation, or “dissociation rate constant”, of a binding protein (e.g., an antibody or antigen binding protein) from the, e.g., antigen binding protein/antigen complex as is known in the art.
  • This value indicates the dissociation rate of a binding protein, e.g., an antibody or an antigen binding protein, from its target antigen or separation of Ab- Ag complex over time into free antibody and antigen as shown by the equation below:
  • KT and “equilibrium dissociation constant” means the value obtained in a titration measurement at equilibrium, or by dividing the dissociation rate constant (K o ff) by the association rate constant (K on ).
  • the association rate constant, the dissociation rate constant and the equilibrium dissociation constant are used to represent the binding affinity of a binding protein (e.g., an antibody or an antigen binding protein) to an antigen.
  • Methods for determining association and dissociation rate constants are well known in the art. Using fluorescence-based techniques offers high sensitivity and the ability to examine samples in physiological buffers at equilibrium.
  • BIAcore® biological interaction analysis
  • KinExA® Kineetic Exclusion Assay
  • An antigen binding protein according to the invention may bind to one target with an affinity at least one order of magnitude, preferably at least two orders of magnitude higher than for a second target.
  • target refers to a molecule or a portion of a molecule capable of being bound by an antigen binding protein.
  • a target can have one or more epitopes. It will therefore be understood that the target may serve as “antigen” for the “antigen binding protein” of the present invention.
  • epitope includes any determinant capable of being bound by an antigen binding protein, such as an antibody.
  • An epitope is a region of an antigen that is bound by an antigen binding protein that targets that antigen, and when the antigen is a protein, includes specific amino acids that directly contact the antigen binding protein. Most often, epitopes reside on proteins, but in some instances can reside on other kinds of molecules, such as nucleic acids.
  • Epitope determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural characteristics, and/or specific charge characteristics.
  • antibodies specific for a particular target antigen will preferentially/specifically recognize an epitope on the target antigen in a complex mixture of proteins and/or macromolecules.
  • the agonistic anti-CD40 antibody, or the 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 CDRL1, CDRL2 and CDRL3 sequences within SEQ ID NO 3; and three heavy chain CDRs that are at least 90% identical to the CDRH1, CDRH2 and CDRH3 sequences within SEQ ID NO 6.
  • CDRs light chain complementarity determining regions
  • the agonistic anti-CD40 antibody, or the agonistic antigen binding fragment thereof may also comprise three light chain complementarity determining regions (CDRs) that are identical to the CDRL1, CDRL2 and CDRL3 sequences within SEQ ID NO 3; and three heavy chain CDRs that are identical to the CDRH1, CDRH2 and CDRH3 sequences within SEQ ID NO 6.
  • each CDR is defined in accordance with the Kabat definition, the Chothia definition, the AbM definition, or the contact definition of CDR; preferably wherein each CDR is defined in accordance with the CDR definition of Kabat or the CDR definition of Chothia.
  • each CDR is defined in accordance with the Kabat definition.
  • each CDR is defined in accordance with the Chothia definition.
  • the agonistic anti-CD40 antibody, or the 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 a fragment thereof comprising a complementarity determining region (CDR) CDRH1 that is at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO 56, a CDRH2 that is at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO 57, and a CDRH3 that is at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO 58; and (b) a light chain or a fragment thereof comprising a CDRL1 that is at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO 52, a CDRL2 that is at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ
  • the agonistic anti-CD40 antibody, or the agonistic antigen binding fragment thereof comprises (a) a heavy chain or a fragment thereof comprising a complementarity determining region (CDR) CDRH1 that is identical to SEQ ID NO 56, a CDRH2 that is identical to SEQ ID NO 57, and a CDRH3 that is identical to SEQ ID NO 58; and (b) a light chain or a fragment thereof comprising a CDRL1 that is identical to SEQ ID NO 52, a CDRL2 that is identical to SEQ ID NO 53, and a CDRL3 that is identical to SEQ ID NO 54.
  • CDR complementarity determining region
  • the agonistic anti-CD40 antibody, or the agonistic antigen binding fragment thereof comprises a light chain variable region VL comprising the sequence as set forth in SEQ ID NO 51, or a sequence at least 90%, at least 95%, at least 98% or at least 99% identical thereto; and/or a heavy chain variable region VH comprising the sequence as set forth in 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 agonistic anti-CD40 antibody or an agonistic antigen binding fragment thereof, comprising a Fab region heavy chain comprising an amino acid sequence as set forth in SEQ ID NO 12, or a sequence at least 90%, at least 95%, at least 98% or at least 99% identical thereto.
  • the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof comprises a light chain (LC) that comprises a sequence as set forth in SEQ ID NO 3, or a sequence at least 90%, at least 95%, at least 98% or at least 99% identical thereto; and/or a heavy chain (HC) that comprises a sequence selected from the group consisting of SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO 49, SEQ ID NO 12 and SEQ ID NO 50, or a sequence at least 90%, at least 95%, at least 98% or at least 99% identical thereto.
  • LC light chain
  • HC heavy chain
  • the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof comprises a light chain (LC) that comprises a sequence as set forth in SEQ ID NO 3, or a sequence at least 90%, at least 95%, at least 98% or at least 99% identical thereto; and/or a heavy chain (HC) that comprises a sequence as set forth in SEQ ID NO 6, or a sequence at least 90%, at least 95%, at least 98% or at least 99% identical thereto.
  • LC light chain
  • HC heavy chain
  • the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof comprises a light chain (LC) that comprises a sequence as set forth in SEQ ID NO 3, or a sequence at least 90%, at least 95%, at least 98% or at least 99% identical thereto; and/or a heavy chain (HC) that comprises a sequence as set forth in SEQ ID NO 9, or a sequence at least 90%, at least 95%, at least 98% or at least 99% identical thereto.
  • LC light chain
  • HC heavy chain
  • the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof comprises a light chain (LC) that comprises a sequence as set forth in SEQ ID NO 3, or a sequence at least 90%, at least 95%, at least 98% or at least 99% identical thereto; and/or a heavy chain (HC) that comprises a sequence as set forth in SEQ ID NO 49, or a sequence at least 90%, at least 95%, at least 98% or at least 99% identical thereto.
  • LC light chain
  • HC heavy chain
  • the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof comprises a light chain (LC) that comprises a sequence as set forth in SEQ ID NO 3, or a sequence at least 90%, at least 95%, at least 98% or at least 99% identical thereto; and/or a heavy chain (HC) that comprises a sequence as set forth in SEQ ID NO 12, or a sequence at least 90%, at least 95%, at least 98% or at least 99% identical thereto.
  • LC light chain
  • HC heavy chain
  • the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof comprises a light chain (LC) that comprises a sequence as set forth in SEQ ID NO 3, or a sequence at least 90%, at least 95%, at least 98% or at least 99% identical thereto; and/or a heavy chain (HC) that comprises a sequence as set forth in SEQ ID NO 50, or a sequence at least 90%, at least 95%, at least 98% or at least 99% identical thereto.
  • LC light chain
  • HC heavy chain
  • the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof comprises a light chain (LC) that comprises a sequence as set forth in SEQ ID NO 59, or a sequence at least 90%, at least 95%, at least 98% or at least 99% identical thereto; and/or a heavy chain (HC) that comprises a sequence selected from the group consisting of SEQ ID NO 61, SEQ ID NO 63 and SEQ ID NO 65, or a sequence at least 90%, at least 95%, at least 98% or at least 99% identical thereto.
  • LC light chain
  • HC heavy chain
  • the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof comprises a light chain (LC) that comprises a sequence as set forth in SEQ ID NO 59, or a sequence at least 90%, at least 95%, at least 98% or at least 99% identical thereto; and/or a heavy chain (HC) that comprises a sequence as set forth in SEQ ID NO 61, or a sequence at least 90%, at least 95%, at least 98% or at least 99% identical thereto.
  • LC light chain
  • HC heavy chain
  • the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof comprises a light chain (LC) that comprises a sequence as set forth in SEQ ID NO 59, or a sequence at least 90%, at least 95%, at least 98% or at least 99% identical thereto; and/or a heavy chain (HC) that comprises a sequence as set forth in SEQ ID NO 63, or a sequence at least 90%, at least 95%, at least 98% or at least 99% identical thereto.
  • LC light chain
  • HC heavy chain
  • the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof comprises a light chain (LC) that comprises a sequence as set forth in SEQ ID NO 59, or a sequence at least 90%, at least 95%, at least 98% or at least 99% identical thereto; and/or a heavy chain (HC) that comprises a sequence as set forth in SEQ ID NO 65, or a sequence at least 90%, at least 95%, at least 98% or at least 99% identical thereto.
  • LC light chain
  • HC heavy chain
  • a “variant” of a polypeptide comprises an amino acid sequence wherein one, two, three, four, five or more amino acid residues are inserted into, deleted from and/or substituted into the amino acid sequence relative to another polypeptide sequence.
  • the variant comprises up to ten insertions, deletions and/or substitutions, more preferably up to eight insertions, deletions and/or substitutions. More specifically, the variant may comprise up to ten, more preferably up to eight insertions. The variant may also comprise up to ten, more preferably up to eight deletions. In even more preferred embodiments, the variant comprises up to ten substitutions, most preferably up to eight substitutions. In some embodiments, these substitutions are conservative amino acid substitution as described below.
  • a "variant" of a polynucleotide sequence comprises one or more mutations within the polynucleotide sequence relative to another polynucleotide sequence, wherein one, two, three, four, five or more nucleic acid residues are inserted into, deleted from and/or substituted into the nucleic acid sequence.
  • the variant comprises up to ten insertions, deletions and/or substitutions, more preferably up to eight insertions, deletions and/or substitutions. More specifically, the variant may comprise up to ten, more preferably up to eight insertions. The variant may also comprise up to ten, more preferably up to eight deletions.
  • the variant comprises up to ten substitutions, most preferably up to eight substitutions.
  • Said one, two, three, four, five or more mutations can cause one, two, three, four, five or more amino acid exchanges within the amino acid sequence the variant encodes for as compared to another amino acid sequence (i.e. a “non-silent mutation”).
  • Variants also include nucleic acid sequences wherein one, two, three, four, five or more codons have been replaced by their synonyms which does not cause an amino acid exchange and is thus called a “ silent mutation” .
  • identity refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent identity” means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. Preferably, identity is determined over the full length of a sequence.
  • the expression “at least 90% identical” includes embodiments wherein the described or claimed sequence is at least 90%, preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98% or still more preferably at least 99% identical to the reference sequence.
  • gaps in alignments are preferably addressed by a particular mathematical model or computer program (z.e., an “algorithm”).
  • Methods that can be used to calculate the identity of the 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.
  • the sequences being compared are typically aligned in a way that gives the largest match between the sequences.
  • One example of a computer program that can be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI).
  • GAP is used to align the two polypeptides or polynucleotides for which the percent sequence identity is to be determined.
  • the sequences are aligned for optimal matching of their respective amino acid or nucleotide (the “matched span”, as determined by the algorithm).
  • a gap opening penalty (which is calculated as 3x the average diagonal, wherein the “average diagonal” is the average of the diagonal of the comparison matrix being used; the “diagonal” is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1/10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSum 62 are used in conjunction with the algorithm.
  • a standard comparison matrix (see, Dayhoff et al. , 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919 for the BLOSum 62 comparison matrix) is also used by the algorithm.
  • Certain alignment schemes for aligning two amino acid sequences may result in matching of only a short region of the two sequences, 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 so desired to result in 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 can encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties.
  • Naturally occurring residues can be divided into classes based on common side chain properties:
  • non-conservative substitutions can involve the exchange of a member of one of these classes for a member from another class.
  • Such substituted residues can be introduced, for example, into regions of a human antibody that are homologous with non-human antibodies, or into the non-homologous regions of the molecule.
  • the hydropathic index of amino acids can be considered.
  • Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cy stine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (- 0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (- 3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • the substitution of like amino acids can be made effectively on the basis of hydrophilicity.
  • the greatest local average hydrophilicity of a protein as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.
  • hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4).
  • the substitution of amino acids whose hydrophilicity values are within ⁇ 2 is included, in certain embodiments, those which are within ⁇ 1 are included, and in certain embodiments, those within ⁇ 0.5 are included.
  • Exemplary amino acid substitutions are set forth in Table 2.
  • a skilled artisan will be able to determine suitable variants of the interferon-associated antigen binding proteins as set forth herein using well-known techniques.
  • one skilled in the art can identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity.
  • even areas that can be important for biological activity or for structure can be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.
  • One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar proteins or protein domains. In view of such information, one skilled in the art can predict the alignment of amino acid residues of interferon-associated antigen binding protein, an antibody or an antigen binding fragment thereof or an interferon or a functional fragment thereof as described herein with respect to its three dimensional structure. In certain embodiments, one skilled in the art can choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues can be involved in important interactions with other molecules. Moreover, one skilled in the art can generate test variants containing a single amino acid substitution at each desired amino acid residue.
  • variants can then be screened using activity assays known to those skilled in the art. Such variants can be used to gather information about suitable variants. For example, if one discovered that a change to a particular amino acid residue resulted in destroyed, undesirably reduced, or unsuitable activity, variants with such a change can be avoided. In other words, based on information gathered from such experiments, one skilled in the art can readily determine the amino acids where further substitutions should be avoided either alone or in combination with other mutations.
  • amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and/or (5) confer or modify other physicochemical or functional properties on such polypeptides.
  • single or multiple amino acid substitutions in certain embodiments, conservative amino acid substitutions can be made in the naturally-occurring sequence (in certain embodiments, in the portion of the polypeptide outside the domain(s) forming intermolecular contacts).
  • a conservative amino acid substitution typically may not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence).
  • a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence.
  • Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden & J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al., Nature, 354: 105 (1991), which are each incorporated herein by reference.
  • derivative refers to a molecule that includes a chemical modification other than an insertion, deletion, or substitution of amino acids (or nucleic acids).
  • derivatives comprise covalent modifications, including, but not limited to, chemical bonding with polymers, lipids, or other organic or inorganic moieties.
  • a chemically modified interferon-associated antigen binding protein can have a greater circulating half-life than an interferon-associated antigen binding protein that is not chemically modified.
  • a chemically modified interferon-associated antigen binding protein can have improved targeting capacity for desired cells, tissues, and/or organs.
  • a derivative interferon-associated antigen binding protein is covalently modified to include one or more water-soluble polymer attachments, including, but not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. See, e.g., U.S. Patent Nos: 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 and 4,179,337.
  • a derivative interferon- associated antigen binding protein comprises one or more polymer, including, but not limited to, monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures of such polymers.
  • polymer including, but not limited to, monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and poly
  • a derivative of an interferon-associated antigen binding protein as described herein is covalently modified with polyethylene glycol (PEG) subunits.
  • PEG polyethylene glycol
  • one or more water-soluble polymer is bonded at one or more specific position, for example at the amino terminus, of a derivative.
  • one or more water-soluble polymer is randomly attached to one or more side chains of a derivative.
  • PEG is used to improve the therapeutic capacity of the interferon-associated antigen binding protein. Certain such methods are discussed, for example, in U.S. Patent No. 6,133,426, which is hereby incorporated by reference for any purpose.
  • interferon-associated antigen binding protein variants include glycosylation variants wherein the number and/or type of glycosylation site has been altered compared to the amino acid sequences of a parent polypeptide.
  • protein variants comprise a greater number of N-linked glycosylation sites than the native protein.
  • protein variants comprise a lesser number of N-linked glycosylation sites than the native protein.
  • An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X can be any amino acid residue except proline.
  • substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain.
  • substitutions which eliminate this sequence will remove an existing N- linked carbohydrate chain.
  • rearrangement of N-linked carbohydrate chains wherein one, two, three, four, five or more N-linked glycosylation sites (typically those that are naturally occurring) are eliminated and one or more new N-linked sites are created.
  • Additional preferred variants include cysteine variants wherein one or more cysteine residues are deleted from or substituted for another amino acid (e.g., serine) as compared to the parent amino acid sequence.
  • Cysteine variants can be useful when antibodies must be refolded into a biologically active conformation such as after the isolation of insoluble inclusion bodies. Cysteine variants generally have fewer cysteine residues than the native protein, and typically have an even number to minimize interactions resulting from unpaired cysteines.
  • Hepatitis delta virus belongs to the Kolmioviridae family.
  • the Hepatitis delta virus genome is composed of a negative single strand RNA.
  • treat Hepatitis delta virus infection and “treatment of Hepatitis delta virus infection” refers to one or more of: (i) reducing viral burden in Hepatitis delta virus-infected cells; (ii) inhibiting Hepatitis delta virus infection; (iii) rescuing cells from Hepatitis delta virus-induced cell death; (iv) rescuing cells from Hepatitis delta virus-induced cytopathic effect; (v) decreasing one or more Hepatitis delta virus-related disorders; and (vi) decreasing one or more Hepatitis delta virus-related symptoms in a subject.
  • viral load refers to the number of viral particles in a cell, an organ or a 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.
  • viral load is usually measured using international units per milliliter (lU/mL).
  • Viral load or viral titer may alternatively be determined as so-called viral genome equivalent.
  • a higher viral burden, titer, or viral load often correlates with the severity of an active viral infection. Accordingly, reducing the viral load or viral titer correlates with a reduced number of infectious viral particles, e.g., in the serum.
  • Viral load is usually determined using nucleic acid amplification based tests (NATs or NAATss).
  • NAT/NAAT tests utilize, for example, PCR, (quantitative) reverse transcription polymerase chain reaction (RT-PCR or qRT-PCR), nucleic acid sequence based amplification (NASBA) or probe-based assays. Due to the ease of detection of viral nucleic acids using nucleic acid amplification based tests, the viral load is useful in clinical settings to monitor success during treatment.
  • patient and “subject” are used interchangeably and include human and non-human animal subjects, preferably human subjects, as well as those with formally diagnosed disorders, those without formally recognized disorders, those receiving medical attention, those at risk of developing the disorders, etc.
  • Hepatitis delta virus-related disorder refers to a disorder that results from infection of a subject by Hepatitis delta virus.
  • a “Hepatitis delta virus-related symptom,” a “symptom of Hepatitis delta virus infection” or a “Hepatitis delta virus-related complication” includes one or more physical dysfunctions related to Hepatitis delta virus infection.
  • an “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 a tumor cell.
  • IFNs include type I IFNs (e.g., IFNa, IFN , IFNs, IFNK, IFNT, IFN ⁇ and IFNco), type II IFNs (e g., IFNy) and type III IFNs (e g., IFNX1, IFNX2 and IFNX3).
  • IFN includes without limitation full-length IFN, a variant or a 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 a full-length IFN.
  • a functional fragment refers to a fragment of a substance that retains one or more functional activities of the original substance.
  • a functional fragment of an interferon refers to a fragment of an interferon that retains an IFN function as described herein, e.g., it mediates IFN pathway signaling.
  • the IFN may increase one or more IFN receptor activities by at least about 20% when added to a cell, tissue or organism expressing a cognate IFN receptor (IFNAR for IFNa, IFNBR for IFN0, etc).
  • the interferon activates IFN receptor activity by at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 85%.
  • the activity of the IFN i.e., the “IFN activity” may be measured, e.g., using an in vitro reporter cell assay, e.g., using HEK-BlueTM IFN-a/0 cells (InvivoGen, Cat.
  • HEK-BlueTM IFN-X InvivoGen, Cat. #: hkb-ifnl
  • HEK-BlueTM Dual IFN-y cells InvivoGen, Cat. #: hkb-ifng
  • SEAP embryonic alkaline phosphatase
  • the interferon-associated antigen binding proteins activate both the CD40 and an IFN pathway.
  • the interferon-associated antigen binding protein activates the IFN pathway with an ECso of less than 100, 60, 50, 40, 30, 20, 10, or 1 ng/mL, preferably with an ECso of less than 11 ng/mL, more preferably with an ECso of less than 6 ng/mL, wherein IFN activity is preferably determined using an in vitro reporter cell assay, optionally using HEK-BlueTM IFN-cells, as described for instance in Example I.
  • the IFN pathway is the IFNa (interferon alpha), IFN0 (interferon beta), IFNE (interferon epsilon), IFNco (interferon omega), IFNy (interferon gamma), or IFNA, (interferon lambda) pathway.
  • an interferon-associated antigen binding protein as described herein comprises full-length IFN, a variant or a 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 a full-length IFN.
  • the IFN is a human IFN.
  • an interferon-associated antigen binding protein as described herein comprises an IFN or a functional fragment thereof selected from the group consisting of a Type I IFN, a Type II IFN and a Type III IFN, or a functional fragment thereof.
  • the IFN or the functional fragment thereof is a Type I IFN, or a functional fragment thereof.
  • the type I IFN or the functional fragment thereof is IFNa, IFN0, IFNco or IFNs, or a functional fragment thereof.
  • the type I IFN or the functional fragment thereof is IFNa or IFN0, or a functional fragment thereof.
  • the type I IFN or the functional fragment thereof is IFN a, or a functional fragment thereof.
  • the type I IFN or the functional fragment thereof is IFN 0, or a functional fragment thereof.
  • the type I IFN or the functional fragment thereof is IFNco, or a functional fragment thereof.
  • the type I IFN or the functional fragment thereof is IFNE, or a functional fragment thereof.
  • the IFN or the functional fragment thereof is IFNa, IFN0, lENy, IFNA,, IFNE or IFNco, or a functional fragment thereof.
  • the IFN or a functional fragment thereof is IFNa or IFN0, or a functional fragment thereof.
  • the IFN or the functional fragment thereof is IFNa, or a functional fragment thereof.
  • the IFN or functional fragment thereof is IFNa2a, or a functional fragment thereof.
  • the IFNa2a may comprise the sequence as set forth in SEQ ID NO 17, or a sequence at least 90% identical thereto.
  • the IFN or the functional fragment thereof is IFN0, or a functional fragment thereof.
  • the IFN0 may comprise the sequence as set forth in SEQ ID NO 14, or a sequence at least 90% identical thereto.
  • the IFN0 or the functional fragment thereof may comprise one or two amino acid substitution(s) relative to SEQ ID NO 14, selected from C17S and N80Q.
  • the IFN0 or the functional fragment thereof comprises the amino acid substitution C17S relative to SEQ ID NO 14.
  • the IFN0 comprises the amino acid sequence as set forth in SEQ ID NO 15.
  • the IFN0 comprises the amino acid substitutions C17S and N80Q relative to SEQ ID NO 14.
  • the IFN0 comprises the amino acid sequence as set forth in SEQ ID NO 16.
  • the IFN or the functional fragment thereof is IFNy or IFNA, or a functional fragment thereof.
  • the IFN or functional fragment thereof is IFNy, or a functional fragment thereof.
  • the IFNy comprises the sequence as set forth in SEQ ID NO 19, or a sequence at least 90% identical thereto.
  • the IFN or functional fragment thereof is IFNA,, or a functional fragment thereof.
  • the IFNA or the functional fragment thereof is IFNA2, or a functional fragment thereof.
  • the IFNA2 may comprise the sequence as set forth in SEQ ID NO 18, or a sequence at least 90% identical thereto.
  • the IFN or the functional fragment thereof is IFNe, or a functional fragment thereof.
  • the IFNE may comprise the sequence as set forth in SEQ ID NO 80, or a sequence at least 90% identical thereto.
  • the IFN or the functional fragment thereof is IFN®, or a functional fragment thereof.
  • the IFN® may comprise the sequence as set forth in SEQ ID NO 79, or a sequence at least 90% identical thereto.
  • the expression level of one or more IFN signaling pathway biomarkers is altered, i.e., upregulated or downregulated, in a Hepatitis delta virus-infected cell treated with an interferon-associated antigen binding protein described herein.
  • the expression level of one or more IFN pathway biomarkers is upregulated in a Hepatitis delta virus- infected cell treated with an interferon-associated antigen binding protein described herein.
  • a “biomarker” is to be understood as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.
  • a suitable IFN pathway biomarker featured herein is a chemokine, e.g., a C-X-C chemokine, selected from the group consisting of CXCL9, CXCL10 and CXCL11.
  • a suitable biomarker induced by the IFN pathway is CXCL9, CXCL10 and/or CXCL11, and also the interferon stimulated gene ISG20.
  • Cytokine induction or release may be quantified using techniques known in the art, such as ELISA. Alternatively, induction may also be determined using RNA-based assays such as RNAseq or qRT-PCR.
  • upregulation may refer to an at least at 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5- fold or at least 10-fold increased expression or secretion of these cytokines.
  • the expression level of pro- inflammatory cytokines is not significantly upregulated in human Whole Blood cells upon treatment with an interferon-associated antigen binding protein of the invention.
  • the expression level of IL10 is not significantly upregulated in human Whole Blood cells upon treatment with an interferon-associated antigen binding protein of the invention.
  • the expression level of IL 1 is not significantly upregulated in human Whole Blood cells upon treatment with an interferon-associated antigen binding protein of the invention.
  • the expression level of IL2 is not significantly upregulated in a Hepatitis delta virus-infected cell upon treatment with an interferon- associated antigen binding protein of the invention. In some embodiments, the expression levels of IL10 and IL10 are not significantly upregulated in a Hepatitis delta virus-infected cell upon treatment with an interferon-associated antigen binding protein of the invention. In some embodiments, the expression levels of IL10 and IL2 are not significantly upregulated in a Hepatitis delta virus-infected cell upon treatment with an interferon-associated antigen binding protein of the invention.
  • the expression levels of IL 10 and IL2 are not significantly upregulated in a Hepatitis delta virus-infected cell upon treatment with an interferon- associated antigen binding protein of the invention. In some embodiments, the expression levels of IL10, IL10 and IL2 are not significantly upregulated in a Hepatitis delta virus-infected cell upon treatment with an interferon-associated antigen binding protein of the invention.
  • association generally refers to a covalent or non- covalent linkage of two (or more) molecules. Associated proteins are created by joining two or more distinct 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, the interferon-associated antigen binding proteins activate both the CD40 and an IFN pathway.
  • An associated protein encompasses monomeric and multimeric, e.g., dimeric, trimeric, tetrameric or the like, complexes of distinct associated or fused proteins.
  • non-covalent linkage results from strong interactions between two protein surface regions, usually via ionic, Van-der-Waals, and/or hydrogen bond interactions.
  • an 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.
  • the IFN or the functional fragment thereof is non- covalently associated with the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof. In more specific embodiments, the IFN or the functional fragment thereof is non-covalently associated with the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof via ionic, Van-der-Waals, and/or hydrogen bond interactions.
  • the IFN or the functional fragment thereof is covalently associated with the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof.
  • the IFN or the functional fragment thereof is fused to the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof.
  • the IFN or the functional fragment thereof may be fused to a light chain of the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof.
  • the IFN or the functional fragment thereof is fused to the N-terminus of a light chain of the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof.
  • the IFN or the functional fragment thereof is fused to the C-terminus of a light chain of the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof.
  • the IFN or the functional fragment thereof may be also be fused to a heavy chain of the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof.
  • the IFN or the functional fragment thereof is fused to the N-terminus of a heavy chain of the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof.
  • the IFN or the functional fragment thereof is fused to the C-terminus of a heavy chain of the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof.
  • the agonistic anti-CD40 antibody or an agonistic antigen binding fragment thereof, and the IFN or the functional fragment thereof may be fused to each other via a linker.
  • linker refers to any moiety that covalently joins one or more agonistic anti-CD40 antibody or an agonistic antigen binding fragment thereof to one or more interferon, or a functional fragment thereof.
  • a linker is a peptide linker.
  • peptide linker refers to a peptide adapted to link two or more moieties.
  • a peptide linker referred to herein may have one or more of the properties outlined in the following. The sequences of peptide linker according to certain exemplary embodiments are set forth in Table 7.
  • a peptide linker may have any length, i.e., comprise any number of amino acid residues.
  • the linker comprises at least 1, at least 2, at least 3, at least 4, at least 5 amino acids.
  • the linker may comprise at least 4 amino acids.
  • the linker may comprise at least 11 amino acids.
  • the linker may comprise at least 12 amino acids.
  • the linker may comprise at least 13 amino acids.
  • the linker may comprise at least 15 amino acids.
  • the linker may comprise at least 20 amino acids.
  • the linker may comprise at least 21 amino acids.
  • the linker may comprise at least 24 amino acids.
  • a linker is typically long enough to provide an adequate degree of flexibility to prevent the linked moieties from interfering with each other’s activity, e.g., the ability of a moiety to bind to a receptor.
  • the linker comprises up to 10, up to 20, up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, or up to 100 amino acids.
  • the linker may comprise up to 80 amino acids.
  • the linker may comprise up to 40 amino acids.
  • the linker may comprise up to 24 amino acids.
  • the linker may comprise up to 21 amino acids.
  • the linker may comprise up to 20 amino acids.
  • the linker may comprise up to 15 amino acids.
  • the linker may comprise up to 13 amino acids.
  • the linker may comprise up to 12 amino acids.
  • the linker may comprise up to 11 amino acids.
  • the linker may comprise up to 4 amino acids.
  • the linker is selected from the group comprising rigid, flexible and/or helix-forming linkers. It is understood that helix-forming linkers can also be rigid linkers, since an a-helix has less degrees of freedom than a peptide assuming a more random-coil conformation.
  • the linker is a rigid linker.
  • An exemplary rigid linker comprises a sequence as set forth in SEQ ID NO 20. Further exemplary rigid linkers comprise a sequence as set forth in SEQ ID NO 22 or SEQ ID NO 23.
  • the linker is a helix-forming linker. Exemplary helix-forming linkers comprise a sequence as set forth in SEQ ID NO 22 or SEQ ID NO 23.
  • the linker is a flexible linker. Exemplary flexible linkers comprise a sequence as set forth in SEQ ID NO 21, SEQ ID NO 24, SEQ ID NO 25 or SEQ ID NO 26.
  • the linker can also have different chemical properties.
  • a linker can be selected from acidic, basic or neutral linkers.
  • acidic linkers contain one or more acidic amino acid, 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.
  • the linker is an acidic linker.
  • Exemplary acidic linkers comprise a sequence as set forth in SEQ ID NO 22 or SEQ ID NO 23.
  • the linker is a basic linker.
  • the linker is a neutral linker.
  • Exemplary neutral linkers comprise a sequence as set forth in SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 24, SEQ ID NO 25 or SEQ ID NO 26.
  • the linker is Gly-Ser or a Gly-Ser-Thr linker composed of multiple glycine, serine and, where applicable, threonine residues.
  • the linker comprises the amino acids glycine and serine.
  • the linker comprises the sequence as set forth in SEQ ID NO 21, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26.
  • the linker further comprises the amino acid threonine.
  • the linker comprises the sequence as set forth in SEQ ID NO 21.
  • the interferon-associated antigen binding protein comprises a linker comprising a sequence selected from the sequences as set forth in SEQ ID NOs 20 to 26, preferably from the sequences as set forth in SEQ ID NO 24, SEQ ID NO 25 or SEQ ID NO 26.
  • the linker comprises a sequence as set forth in SEQ ID NO 24.
  • the linker comprises a sequence as set forth in SEQ ID NO 25.
  • the linker comprises a sequence as set forth in SEQ ID NO 26.
  • the interferon-associated antigen binding protein comprises no amino acids other than those forming (I) said agonistic anti-CD40 antibody, or agonistic antigen binding fragment thereof and (II) said IFN or functional fragment thereof.
  • the interferon-associated antigen binding protein comprises no amino acids other than those forming (I) said agonistic anti-CD40 antibody, or agonistic antigen binding fragment thereof, (II) said IFN or functional fragment thereof and (III) said linker.
  • the IFN or a functional fragment thereof is fused to the C-terminus of a heavy chain of the agonistic anti-CD40 antibody, or the agonistic antigen binding fragment thereof, via the linker, as set forth in Table 3A or Table 3B.
  • the heavy chain of the agonistic anti-CD40 antibody, or the agonistic antigen binding fragment thereof may comprise a sequence as set forth in 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.
  • the IFNa2a may comprise the sequence as set forth in SEQ ID NO 17.
  • the IFNJ3 may comprise the sequence as set forth in SEQ ID NO 14, SEQ ID NO 15 or SEQ ID NO 16.
  • the IFN0 may comprise the sequence as set forth in SEQ ID NO 14.
  • the IFN C17S may comprise the sequence as set forth in SEQ ID NO 15.
  • the IFNp_C17S,N80Q may comprise the sequence as set forth in SEQ ID NO 16.
  • the IFNy may comprise the sequence as set forth in SEQ ID NO 19.
  • the IFNX2 may comprise the sequence as set forth in SEQ ID NO 18.
  • the IFNE may comprise the sequence as set forth in SEQ ID NO 80.
  • the IFNco may comprise the sequence as set forth in SEQ ID NO 79.
  • the linkers referred to are those listed in Table 7.
  • the interferon-associated antigen binding protein further comprises a light chain of an agonistic anti-CD40 antibody, or an agonistic antigen binding fragment thereof.
  • a heavy chain comprises a sequence as set forth in SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO 12, SEQ ID NO 48, or SEQ ID NO 49 and a light chain comprises a sequence as set forth in SEQ ID NO 3.
  • a heavy chain comprises a sequence as set forth in SEQ ID NO 61 or SEQ ID NO 63 and a light chain comprises a sequence as set forth in SEQ ID NO 59.
  • Interferon or a functional fragment thereof fused to the C-terminus of a heavy chain of the anti-CD40 antibody or an agonistic antigen binding fragment thereof [00187]
  • the IFN or a functional fragment thereof is fused to the N-terminus of a heavy chain of the agonistic anti-CD40 antibody, or the agonistic antigen binding fragment thereof, via the linker, as set forth in Table 4A or Table 4B.
  • the heavy chain of the agonistic anti-CD40 antibody, or the agonistic antigen binding fragment thereof may comprise a sequence as set forth in 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.
  • the IFNa2a may comprise the sequence as set forth in SEQ ID NO 17.
  • 3 may comprise the sequence as set forth in SEQ ID NO 14, SEQ ID NO 15 or SEQ ID NO 16.
  • the IFNp may comprise the sequence as set forth in SEQ ID NO 14.
  • 3_C17S may comprise the sequence as set forth in SEQ ID NO 15.
  • 3_C17S,N80Q may comprise the sequence as set forth in SEQ ID NO 16.
  • the IFNy may comprise the sequence as set forth in SEQ ID NO 19.
  • the IFNZ2 may comprise the sequence as set forth in SEQ ID NO 18.
  • the IFNE may comprise the sequence as set forth in SEQ ID NO 80.
  • the IFNco may comprise the sequence as set forth in SEQ ID NO 79.
  • the linkers referred to are those listed in Table 7.
  • the interferon-associated antigen binding protein further comprises a light chain of an agonistic anti-CD40 antibody, or an agonistic antigen binding fragment thereof.
  • a heavy chain comprises a sequence as set forth in 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 a light chain comprises a sequence as set forth in SEQ ID NO 3.
  • a heavy chain comprises a sequence as set forth in SEQ ID 61, SEQ ID 63 or SEQ ID 65 and a light chain comprises a sequence as set forth in SEQ ID NO 59.
  • the IFN is fused to the C-terminus of a light chain of the agonistic anti-CD40 antibody, or the agonistic antigen binding fragment thereof, via the linker, as set forth in Table 5A or Table 5B.
  • the light chain of the agonistic anti-CD40 antibody, or the agonistic antigen binding fragment thereof may comprise a sequence as set forth in SEQ ID NO 3.
  • the light chain may comprise a sequence as set forth in SEQ ID NO 59.
  • the IFNa2a may comprise the sequence as set forth in SEQ ID NO 17.
  • the IFNP may comprise the sequence as set forth in SEQ ID NO 14, SEQ ID NO 15 or SEQ ID NO 16.
  • the IFN0 may comprise the sequence as set forth in SEQ ID
  • the IFN0 C17S may comprise the sequence as set forth in SEQ ID NO 15.
  • the IFNP_C17S,N80Q may comprise the sequence as set forth in SEQ ID NO 16.
  • the IFNy may comprise the sequence as set forth in SEQ ID NO 19.
  • the IFNX.2 may comprise the sequence as set forth in SEQ ID NO 18.
  • the IFNE may comprise the sequence as set forth in SEQ ID NO 80.
  • the IFNco may comprise the sequence as set forth in SEQ ID NO 79.
  • the linkers referred to are those listed in Table 7.
  • the interferon-associated antigen binding protein further comprises a heavy chain of an agonistic anti-CD40 antibody, or an agonistic antigen binding fragment thereof.
  • a light chain comprises a sequence as set forth in SEQ ID NO 3 and a heavy chain comprises a sequence as set forth in 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.
  • a light chain comprises a sequence as set forth in SEQ ID NO 59 and a heavy chain comprises a sequence as set forth in SEQ ID NO 61, SEQ ID NO 63 or SEQ ID NO 65.
  • the IFN is fused to the N-terminus of a light chain of the agonistic anti-CD40 antibody, or the agonistic antigen binding fragment thereof, via the linker, as set forth in Table 6A or Table 6B.
  • the light chain of the agonistic anti-CD40 antibody, or the agonistic antigen binding fragment thereof may comprise a sequence as set forth in SEQ ID NO 3 or SEQ ID NO 59.
  • the IFNa2a may comprise the sequence as set forth in SEQ ID NO 17.
  • the IFN0 may comprise the sequence as set forth in SEQ ID NO 14, SEQ ID NO 15 or SEQ ID NO 16.
  • the IFN0 may comprise the sequence as set forth in SEQ ID NO 14.
  • the IFN0 C17S may comprise the sequence as set forth in SEQ ID NO 15.
  • the IFN0_C17S,N8OQ may comprise the sequence as set forth in SEQ ID NO 16.
  • the IFNy may comprise the sequence as set forth in SEQ ID NO 19.
  • the IFN 2 may comprise the sequence as set forth in SEQ ID NO 18.
  • the IFNE may comprise the sequence as set forth in SEQ ID NO 80.
  • the IFN® may comprise the sequence as set forth in SEQ ID NO 79.
  • the linkers referred to are those listed in Table 7
  • the interferon-associated antigen binding protein further comprises a heavy chain of an agonistic anti-CD40 antibody, or an agonistic antigen binding fragment thereof.
  • a light chain comprises a sequence as set forth in SEQ ID NO 3 and a heavy chain comprises a sequence as set forth in 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.
  • a light chain comprises a sequence as set forth in SEQ ID NO 59 and a heavy chain comprises a sequence as set forth in 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 a light chain of the anti-CD40 antibody or an agonistic antigen binding fragment thereof
  • the interferon-associated antigen binding protein comprises an interferon-fused agonistic anti-CD40 antibody or an interferon-fused agonistic antigen binding fragment thereof comprising a sequence selected from SEQ ID NOs 28-47 or SEQ ID NOs 66-75.
  • the interferon-associated antigen binding protein comprises an interferon-fused agonistic anti-CD40 antibody or an interferon-fused agonistic antigen binding fragment thereof comprising a sequence selected from SEQ ID NOs 81-88.
  • the interferon-associated antigen binding protein comprises an interferon-fused agonistic anti-CD40 antibody or an interferon-fused agonistic antigen binding fragment thereof comprising a sequence selected from SEQ ID NOs 89-90.
  • the interferon-associated antigen binding protein is an interferon-fused agonistic anti- CD40 antibody or an interferon-fused agonistic antigen binding fragment thereof comprising a sequence selected from SEQ ID NOs 28-47 or SEQ ID NOs 66-75.
  • the interferon-associated antigen binding protein is an interferon-fused agonistic anti-CD40 antibody or an interferon-fused agonistic antigen binding fragment thereof comprising a sequence selected from SEQ ID NOs 81-88. In other exemplary embodiments, the interferon-associated antigen binding protein is an interferon-fused agonistic anti-CD40 antibody or an interferon-fused agonistic antigen binding fragment thereof comprising a sequence selected from SEQ ID NOs 89-90.
  • the interferon-associated antigen binding protein comprises an interferon-fused agonistic anti-CD40 antibody or an interferon- fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 89.
  • the interferon-associated antigen binding protein is an interferon-fused agonistic anti-CD40 antibody or an interferon- fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 89.
  • the interferon-associated antigen binding protein comprises an interferon-fused agonistic anti-CD40 antibody or an interferon- fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 90.
  • the interferon-associated antigen binding protein is an interferon-fused agonistic anti-CD40 antibody or an interferon- fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 90.
  • the interferon-associated antigen binding protein comprises an interferon-fused agonistic anti-CD40 antibody or an interferon- fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 81.
  • the interferon-associated antigen binding protein is an interferon-fused agonistic anti-CD40 antibody or an interferon- fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 81.
  • the interferon-associated antigen binding protein comprises an interferon-fused agonistic anti-CD40 antibody or an interferon- fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 82.
  • the interferon-associated antigen binding protein is an interferon-fused agonistic anti-CD40 antibody or an interferon- fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 82.
  • the interferon-associated antigen binding protein comprises an interferon-fused agonistic anti-CD40 antibody or an interferon- fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 83.
  • the interferon-associated antigen binding protein is an interferon-fused agonistic anti-CD40 antibody or an interferon- fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 83.
  • the interferon-associated antigen binding protein comprises an interferon-fused agonistic anti-CD40 antibody or an interferon- fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 84.
  • the interferon-associated antigen binding protein is an interferon-fused agonistic anti-CD40 antibody or an interferon- fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 84.
  • the interferon-associated antigen binding protein comprises an interferon-fused agonistic anti-CD40 antibody or an interferon- fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 85.
  • the interferon-associated antigen binding protein is an interferon-fused agonistic anti-CD40 antibody or an interferon- fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 85.
  • the interferon-associated antigen binding protein comprises an interferon-fused agonistic anti-CD40 antibody or an interferon- fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 86.
  • the interferon-associated antigen binding protein is an interferon-fused agonistic anti-CD40 antibody or an interferon- fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 86.
  • the interferon-associated antigen binding protein comprises an interferon-fused agonistic anti-CD40 antibody or an interferon- fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 87.
  • the interferon-associated antigen binding protein is an interferon-fused agonistic anti-CD40 antibody or an interferon- fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 87.
  • the interferon-associated antigen binding protein comprises an interferon-fused agonistic anti-CD40 antibody or an interferon- fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 88.
  • the interferon-associated antigen binding protein is an interferon-fused agonistic anti-CD40 antibody or an interferon- fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 88.
  • the interferon-associated antigen binding protein comprises an interferon-fused agonistic anti-CD40 antibody or an interferon- fused agonistic antigen binding fragment thereof comprising a sequence selected from SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42 or SEQ ID NO 43.
  • the interferon-associated antigen binding protein is an interferon-fused agonistic anti-CD40 antibody or an interferon-fused agonistic antigen binding fragment thereof comprising a sequence selected from SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42 or SEQ ID NO 43.
  • the interferon- associated antigen binding protein comprises an interferon-fused agonistic anti- CD40 antibody or an interferon-fused agonistic 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.
  • the interferon- associated antigen binding protein is an interferon-fused agonistic anti-CD40 antibody or an interferon-fused agonistic 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.
  • the interferon-associated antigen binding protein comprises an interferon-fused agonistic anti-CD40 antibody or an interferon-fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 38.
  • the interferon-associated antigen binding protein is an interferon-fused agonistic anti- CD40 antibody or an interferon-fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 38.
  • the interferon-associated antigen binding protein comprises an interferon-fused agonistic anti-CD40 antibody or an interferon-fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 39.
  • the interferon-associated antigen binding protein is an interferon-fused agonistic anti- CD40 antibody or an interferon-fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 39.
  • the interferon-associated antigen binding protein comprises an interferon-fused agonistic anti-CD40 antibody or an interferon-fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 40.
  • the interferon-associated antigen binding protein is an interferon-fused agonistic anti- CD40 antibody or an interferon-fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 40.
  • the interferon-associated antigen binding protein comprises an interferon-fused agonistic anti-CD40 antibody or an interferon-fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 41.
  • the interferon-associated antigen binding protein is an interferon-fused agonistic anti- CD40 antibody or an interferon-fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 41.
  • the interferon-associated antigen binding protein comprises an interferon-fused agonistic anti-CD40 antibody or an interferon-fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 42.
  • the interferon-associated antigen binding protein is an interferon-fused agonistic anti- CD40 antibody or an interferon-fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 42.
  • the interferon-associated antigen binding protein comprises an interferon-fused agonistic anti-CD40 antibody or an interferon-fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 43.
  • the interferon-associated antigen binding protein is an interferon-fused agonistic anti- CD40 antibody or an interferon-fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 43.
  • the interferon-associated antigen binding protein comprises an interferon-fused agonistic anti-CD40 antibody or an interferon-fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 72.
  • the interferon-associated antigen binding protein is an interferon-fused agonistic anti- CD40 antibody or an interferon-fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 72.
  • the interferon-associated antigen binding protein comprises an interferon-fused agonistic anti-CD40 antibody or an interferon-fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 73.
  • the interferon-associated antigen binding protein is an interferon-fused agonistic anti- CD40 antibody or an interferon-fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 73.
  • the interferon-associated antigen binding protein comprises an interferon-fused agonistic anti-CD40 antibody or an interferon-fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 74.
  • the interferon-associated antigen binding protein is an interferon-fused agonistic anti- CD40 antibody or an interferon-fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 74.
  • the interferon-associated antigen binding protein comprises an interferon-fused agonistic anti-CD40 antibody or an interferon-fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 75.
  • the interferon-associated antigen binding protein is an interferon-fused agonistic anti- CD40 antibody or an interferon-fused agonistic binding fragment thereof comprising a sequence as set forth in SEQ ID NO 75.
  • Table 7 Sequences of exemplary interferon-associated antigen binding protein and components thereof based on the antiCD40 antibody CP870,893. Italic sequences correspond to signal peptides. Bold italic sequences in SEQ ID NOs 3 and 6 correspond to CDR regions. Bold non-italic sequences correspond to linkers. Mutated amino acids are underlined.
  • Table 8 Sequences of exemplary interferon-associated antigen binding protein and components thereof based on the antiCD40 antibody 3G5. Italic sequences correspond to signal peptides. Bold non-italic sequences correspond to linkers. Mutated amino acids are underlined.
  • the interferon-associated antigen binding proteins described herein are interferon-fused antigen binding proteins comprising polypeptides derived from those specified in Table 9, in particular Table 9A or Table 9B, more particularly 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.
  • the interferon-associated antigen binding proteins described herein are interferon-fused antigen binding proteins consisting of polypeptides derived from those specified in Table 9, in particular Table 9A or Table 9B, more particularly 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.
  • the interferon-fused antibody comprises the sequences as set forth in SEQ ID NO 38 and SEQ ID NO 3.
  • the interferon-fused antibody comprises the sequences as set forth in SEQ ID NO 39 and SEQ ID NO 3.
  • the interferon-fused antibody comprises the sequences as set forth in SEQ ID NO 40 and SEQ ID NO 3. In other more preferred embodiments, the interferon-fused antibody comprises the sequences as set forth in SEQ ID NO 41 and SEQ ID NO 9. In other more preferred embodiments, the interferon-fused antibody comprises the sequences as set forth in SEQ ID NO 42 and SEQ ID NO 9. In other more preferred embodiments, the interferon-fused antibody comprises the sequences as set forth in SEQ ID NO 43 and SEQ ID NO 9.
  • the interferon-associated antigen binding proteins described herein are interferon-fused antigen binding proteins comprising polypeptides derived from those specified in Table 10 below. In preferred embodiments, the interferon-associated antigen binding proteins described herein are interferon-fused antigen binding proteins consisting of polypeptides derived from those specified in Table 10 below.
  • Table 10 Polypeptide combinations found in preferred interferon-fused antigen binding proteins of the invention based on the antiCD40 antibody 3G5, their mean EC so values with regard to the activation of CD40 and IFN-pathways. Each sequence combination as indicated is comprised twice in the respective IF A. SN: supernatant.
  • a combination of polynucleotides encoding an interferon- associated antigen binding protein is provided. Methods of making an interferon- associated antigen binding protein comprising expressing these polynucleotides are also provided.
  • a nucleic acid encoding an IFN or a functional fragment thereof being fused to an agonistic anti-CD40 antibody or an agonistic antigen binding fragment thereof, as disclosed herein is provided.
  • the nucleic acid is encoding 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 the sequences set forth in SEQ ID NOs 89 to 90, or a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to a nucleic acid encoding any of these sequences.
  • said nucleic acid is at least 95%, at least 98% or at least 99% identical to a nucleic acid encoding any of SEQ ID NOs 89 to 90.
  • the nucleic acid is encoding 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 the sequences set forth in SEQ ID NOs 81 to 88, or a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to a nucleic acid encoding any of these sequences.
  • said nucleic acid is at least 95%, at least 98% or at least 99% identical to a nucleic acid encoding any of SEQ ID NOs 81 to 88.
  • the nucleic acid is encoding 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 the sequences set forth in SEQ ID NOs 28 to 47, or a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to a nucleic acid encoding any of these sequences.
  • said nucleic acid is at least 95%, at least 98% or at least 99% identical to a nucleic acid encoding any of SEQ ID NOs 28 to 47.
  • the nucleic acid is encoding 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 the sequences set forth in SEQ ID NOs 66 to 75, or a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to a nucleic acid encoding any of these sequences.
  • said nucleic acid is at least 95%, at least 98% or at least 99% identical to a nucleic acid encoding any of SEQ ID NOs 66 to 75.
  • nucleic acid encodes an IFN or a functional fragment thereof being fused to a light chain of the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof
  • the nucleic acid may further encode a heavy chain of the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof.
  • the heavy chain of the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof comprises a sequence as set forth in SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 48, SEQ ID NO 49, or SEQ ID NO 50, or a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to a nucleic acid encoding any of these sequences.
  • said nucleic acid is at least 95%, at least 98% or at least 99% identical to a nucleic acid encoding SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 48, SEQ ID NO 49, or SEQ ID NO 50.
  • the heavy chain of the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof comprises a sequence as set forth in SEQ ID NO 61, SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64 or SEQ ID NO 65, or a nucleic acid sequence at least at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to a nucleic acid encoding any of these sequences.
  • said nucleic acid is at least 95%, at least 98% or at least 99% identical to a nucleic acid encoding SEQ ID NO 61, SEQ ID NO 62, SEQ ID NO 63, SEQ ID NO 64 or SEQ ID NO 65.
  • nucleic acid encodes an IFN or a functional fragment thereof being fused to the heavy chain of the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof
  • the nucleic acid may further encode a light chain of the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof.
  • the light chain of the agonistic anti- CD40 antibody or the agonistic antigen binding fragment thereof comprises a sequence as set forth in SEQ ID NO 3, SEQ ID NO 4 or SEQ ID NO 5, or a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to a nucleic acid encoding any of these sequences.
  • said nucleic acid is at least 95%, at least 98% or at least 99% identical to a nucleic acid encoding SEQ ID NO 3, SEQ ID NO 4 or SEQ ID NO 5.
  • the light chain of the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof comprises a sequence as set forth in SEQ ID NO 59 or SEQ ID NO 60, or a nucleic acid sequence at least at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to a nucleic acid encoding any of these sequences.
  • said nucleic acid is at least 95%, at least 98% or at least 99% identical to a nucleic acid encoding SEQ ID NO 59 or SEQ ID NO 60.
  • the nucleic acids described herein may comprise a sequence encoding a sequence to increase the yield (e.g. a solubility tag) or facilitate purification of the expressed proteins (i.e., a purification tag).
  • Purification tags are known to a person skilled in the art and may be selected from glutathione S- transferase (GST) tags, maltose binding protein (MBP) tags, calmodulin binding peptide (CBP) tags, intein-chitin binding domain (intein-CBD) tags, Streptavidin/Biotin-based tags (such as biotinylation signal peptide (BCCP) tags, Streptavidin-binding peptide (SBP) tags, His-patch ThioFusion tags, tandem affinity purification (TAP) tags, Small ubiquitin-like modifier (SUMO) tags, HaloTag® (Promega), Profinity eXactTM system (Bio-Rad).
  • GST glutathione S- transferase
  • MBP maltose binding protein
  • CBP calmodulin binding peptide
  • intein-chitin binding domain intein-CBD
  • Streptavidin/Biotin-based tags such as biotinylation
  • the purification tag may be a polyhistidine tag (e.g., a Hise-, His?-, Hiss-, His9- or Hisio- tag).
  • the purification tag may be a Strep-tag (e.g., a Strep- tag® or a Strep-tag II®; IB A Life Sciences).
  • the purification tag may be a maltose binding protein (MBP) tag.
  • MBP maltose binding protein
  • the nucleic acid sequence may further comprise a sequence encoding a cleavage site for removal of the purification tag.
  • cleavage sequences are known to a person skilled in the art and may be selected from a sequence recognized and cleaved by an endoprotease or an exoprotease.
  • an endoprotease for the removal of a purification tag may be selected from: Enteropeptidase, Thrombin, Factor Xa, TEV protease or Rhinovirus 3C protease.
  • an exoprotease for the removal of a purification tag may be selected from: Carboxypeptidase A, Carboxypeptidase B or DAPase.
  • the protease for the removal of a purification tag is TEV protease.
  • the nucleic acid comprises a sequence encoding a Hise-tag and a TEV cleavage site. In an even more specific preferred embodiment, said nucleic acid comprises a sequence encoding a sequence as set forth in SEQ ID NO 27.
  • the nucleic acid molecules of the invention may also comprise a sequence encoding a signal peptide.
  • the skilled person is aware of the various signal peptides available to direct the expressed protein to the desired site of folding, assembly and/or maturation as well as to effect secretion of the final protein into the medium to facilitate downstream processing.
  • the signal peptide is a secretory signal peptide.
  • the encoded signal peptide may comprise a sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2.
  • the signal peptide comprises the sequence as set forth in SEQ ID NO: 1.
  • the signal peptide comprises the sequence as set forth in SEQ ID NO: 2.
  • Signal peptide 1 was used for synthesis of the polypeptide sequences as set forth in 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
  • Such signal peptide that is initially present at the N-terminus of the respective sequence of the polypeptide is cleaved during synthesis.
  • Signal peptide 2 (SEQ ID NO 2) was used for synthesis of the polypeptide sequences as set forth in 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. Such signal peptide that is initially present at the N-terminus of the respective sequence of the polypeptide is cleaved during synthesis.
  • polynucleotides encoding an IFN or a functional fragment thereof being fused to the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof as disclosed herein are typically inserted in an expression vector for introduction into host cells that may be used to produce the desired quantity of the described or claimed interferon-associated antigen binding proteins. Accordingly, in certain aspects, the invention provides expression vectors comprising polynucleotides disclosed herein and host cells comprising these vectors and polynucleotides.
  • vectors used in accordance with the present invention as a vehicle for introducing into and expressing a desired gene in a cell.
  • vectors may easily be selected from the group consisting of plasmids, phages, viruses and retroviruses.
  • vectors compatible with the present invention will comprise 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.
  • one class of vector utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV), or SV40 virus.
  • animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV), or SV40 virus.
  • Others involve the use of polycistronic systems with internal ribosome binding sites.
  • cells which have integrated the DNA into their chromosomes may be selected by introducing one or more markers which allow selection of transfected host cells.
  • the marker may provide for prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics) or resistance to heavy metals such as copper.
  • the selectable marker gene can either be directly linked to the DNA sequences to be expressed, or introduced into the same cell by co-transformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals. In some embodiments the cloned variable region genes, one of them fused with a gene encoding an IFN or a functional fragment thereof, are inserted into an expression vector along with the heavy and light chain constant region genes (such as human genes) synthesized as discussed above.
  • a vector system of the invention may comprise more than one vector.
  • a vector system may comprise a first vector for the expression of an IFN or a functional fragment thereof fused to a light chain of the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof and a second vector for expression of a heavy chain of the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof.
  • such a vector system may comprise a first vector for the expression of an IFN or a functional fragment thereof fused to a heavy chain of the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof and a second vector for expression of a light chain of the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof.
  • an interferon-associated antigen binding protein as described herein may be expressed using polycistronic constructs.
  • multiple gene products of interest such as those encoding an IFN or a functional fragment thereof being fused to a heavy chain of an agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof and encoding a light chain of said antibody, or those encoding an IFN or a functional fragment thereof being fused to a light chain of an agonistic anti-CD40 antibody or an agonistic antigen binding fragment thereof and encoding a heavy chain of said antibody or an agonistic antigen binding fragment thereof may be produced from a single polycistronic construct.
  • These systems advantageously use an internal ribosome entry site (IRES) to provide relatively high levels of polypeptides in eukaryotic host cells.
  • IRES sequences are disclosed in U.S. Pat. No. 6,193,980, which is incorporated by reference herein. Those skilled in the art will appreciate that such expression systems may be used to effectively produce the full range of polypeptides disclosed in the instant application.
  • the expression vector may be introduced into an appropriate host cell. That is, the host cell may be transformed.
  • Introduction of a plasmid into the host cell can be accomplished by various techniques well known to those of skill in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus. See, e.g., Ridgway, A. A. G. “Mammalian Expression Vectors” Chapter 24.2, pp.
  • the term “transformation” shall be used in a broad sense to refer to the introduction of DNA into a recipient host cell that changes the genotype and consequently results in a change in the recipient cell.
  • host cells refer to cells that have been transformed with vectors constructed using recombinant DNA techniques and encoding at least one heterologous gene.
  • the terms “cell” and “cell culture” are used interchangeably to denote the source of the interferon-associated antigen binding protein unless it is clearly specified otherwise.
  • recovery of polypeptide from the “cells” may mean either from spun down whole cells, or from the cell culture containing both the medium and the suspended cells.
  • the host cell line used for expression of an interferon-associated antigen binding protein is of eukaryotic or prokaryotic origin.
  • expression may include the transcription and translation of more than one polypeptide chain (such as a heavy and a light chain of the antibody moiety of an interferon-associated antigen binding protein), which associate to form the final interferon-associated antigen binding protein.
  • the host cell line used for expression of an interferon-associated antigen binding protein is of bacterial origin.
  • the host cell line used for expression of an interferon- associated antigen binding protein is of mammalian origin; those skilled in the art can determine particular host cell lines which are best suited for the desired gene product to be expressed therein.
  • Exemplary host cell lines include, but are not limited to, CHO KI GS knockout from Horizon, DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (a derivative of CVI with SV40 T antigen), R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line), SP2/O (mouse myeloma), BFA-lclBPT (bovine endothelial cells), RAJI (human lymphocyte), HEK 293 (human kidney).
  • HEK FS SI 1/ 254 cells may be used.
  • CHO KI GS from Horizon may be used.
  • the cell line provides for altered glycosylation, e.g., afucosylation, of the antibody expressed therefrom (e.g., PER.C6® (Crucell) or FUT8-knock-out CHO cell lines (POTELLIGENTTM cells) (Biowa, Princeton, NJ)).
  • PER.C6® Crucell
  • FUT8-knock-out CHO cell lines POTELLIGENTTM cells
  • NSO cells may be used.
  • Host cell lines are typically available from commercial services, the American Tissue Culture Collection or from published literature.
  • the host used for expression of an interferon-associated antigen binding protein is a non-human transgenic animal or transgenic plant.
  • Interferon-associated antigen binding proteins of the invention can also be produced transgenically through the generation of a non-human animal (e.g., mammal) or plant that is transgenic for the sequences of interest and production of the interferon-associated antigen binding protein in a recoverable form therefrom.
  • interferon-associated antigen binding proteins can be produced in, and recovered from, the milk of goats, cows, or other mammals. See, e.g., US. Patent Nos 5,827,690, 5,756,687, 5,750,172, and 5,741,957.
  • Exemplary plant hosts are Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc.
  • non-human transgenic animals or 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 by standard transgenic techniques. See Hogan and United States Patent 6,417,429.
  • the transgenic cells used for making the transgenic animal can be embryonic stem cells or somatic cells.
  • the transgenic non-human organisms can be chimeric, nonchimeric heterozygotes, and nonchimeric homozygotes.
  • the transgenic non-human animals have a targeted disruption and replacement by a targeting construct that encodes the sequence(s) of interest.
  • the interferon-associated antigen binding proteins may be made in any transgenic animal.
  • 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 bodily fluids.
  • a solution of an interferon-associated antigen binding protein can be purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose and/or (immuno-) affinity chromatography.
  • One or more genes encoding an interferon-associated antigen binding protein can also be expressed in non-mammalian cells such as bacteria or yeast or plant cells.
  • non-mammalian cells such as bacteria or yeast or plant cells.
  • various unicellular non-mammalian microorganisms such as bacteria can also be transformed; i.e. those capable of being grown in cultures or fermentation.
  • Bacteria which are susceptible to transformation, include members of the enterob acteriaceae, such as strains of Escherichia coli or Salmonella, Bacillaceae, such as Bacillus suhlilis: Pneumococcus,' Streptococcus, and Haemophilus influenzae.
  • interferon-associated antigen binding proteins when expressed in bacteria, interferon-associated antigen binding proteins according to the invention or components thereof (i.e., agonistic anti-CD40 antibodies or agonistic antigen binding fragments thereof, and IFNs or functional fragments of IFNs) can become part of inclusion bodies.
  • the desired interferon-associated antigen binding proteins may then need to be isolated, optionally also refolded, and purified.
  • eukaryotic microbes may also be used. Saccharomyces cerevisiae, or common baker’s yeast, is the most commonly used among eukaryotic microorganisms although a number of other strains are commonly available.
  • Saccharomyces cerevisiae or common baker’s yeast
  • yeast is the most commonly used among eukaryotic microorganisms although a number of other strains are commonly available.
  • the plasmid YRp7 for example, (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7: 141 (1979); Tschemper et al., Gene, 10:157 (1980)) is commonly used.
  • This plasmid already contains the TRP1 gene, which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85: 12 (1977)).
  • the presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
  • a nucleic acid sequence encoding an interferon-associated antigen binding protein can be inserted into a vector and used as a therapeutic vector, e.g., a vector that expresses an interferon-associated antigen binding protein of the invention.
  • a therapeutic vector e.g., a vector that expresses an interferon-associated antigen binding protein of the invention.
  • suitable, functional expression constructs and therapeutic expression vectors is known to one of ordinary skill in the art.
  • the interferon-associated antigen binding protein may be administered to a subject by means of genetic delivery with RNA or DNA sequences, a vector or vector system encoding the interferon-associated antigen binding protein.
  • Therapeutic vectors can be delivered to a subj ect by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see, e.g., Chen et al., PNAS 91 :3054-3057 (1994)).
  • the pharmaceutical preparation of a therapeutic vector can include the vector in an acceptable diluent.
  • An interferon-associated antigen binding protein encoding nucleic acid, or nucleic acids can be incorporated into a gene construct to be used as a part of a therapy protocol to deliver nucleic acids encoding an interferon-associated antigen binding protein.
  • Expression vectors for in vivo transfection and expression of an interferon-associated antigen binding protein are provided.
  • Expression constructs of such components may be administered in any biologically effective carrier, e.g., any formulation or composition capable of effectively delivering the component nucleic acid sequence to cells in vivo, as are known to one of ordinary skill in the art.
  • Approaches include, but are not limited to, insertion of the subject nucleic acid sequence(s) in viral vectors including, but not limited to, recombinant retroviruses, adenovirus, adeno-associated virus and herpes simplex virus- 1, recombinant bacterial or eukaryotic plasmids and the like.
  • Retrovirus vectors and adeno-associated viral vectors can be used as a recombinant delivery system for the transfer of exogenous nucleic acid sequences in vivo, particularly into humans.
  • Such vectors provide efficient delivery of genes into cells, and the transferred nucleic acids can be stably integrated into the chromosomal DNA of the host.
  • a replication-defective retrovirus can be packaged into virions, which can be used to infect a target cell through the use of a helper virus by standard techniques.
  • retroviruses include pLJ, pZIP, pWE and pEM, which are known to those of ordinary skill in the art.
  • suitable packaging virus 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.
  • adenovirus-derived delivery vectors are provided.
  • the genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral 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 the adenovirus strain Ad type 5 dl324 or other strains of adenovirus are known to those of ordinary skill in the art.
  • Recombinant adenoviruses can be advantageous in certain circumstances in that they are not capable of infecting non-dividing cells and can be used to infect a wide variety of cell types, including epithelial cells (Rosenfeld, et al. (1992), supra).
  • the virus particle is relatively stable and amenable to purification and concentration and, as above, can be modified so as to affect the spectrum of infectivity.
  • introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell, but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situ where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA).
  • the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other delivery vectors (Berkner, et al. (1998), supra; Haj-Ahmand and Graham, J. Virol. 57:267 (1986)).
  • AAV adeno-associated virus
  • AAV is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle.
  • AAV is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flotte, et al., Am. J. Respir. Cell. Mol. Biol.
  • Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb.
  • An AAV vector such as that described in Tratschin, et al., Mol. Cell. Biol. 5:3251-3260 (1985) can be used to introduce DNA into cells.
  • a variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat, et al., Proc. Natl. Acad. Sci.
  • non-viral methods can also be employed to cause expression of a nucleic acid sequence encoding an interferon-associated antigen binding protein in the tissue of a subject.
  • Most non-viral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules.
  • non-viral delivery systems rely on endocytic pathways for the uptake of the subject gene by the targeted cell.
  • Exemplary delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes.
  • Other embodiments include plasmid injection systems such as are described in 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).
  • the delivery systems can be introduced into a subject by any of a number of methods, each of which is familiar in the art.
  • a pharmaceutical preparation of the delivery system can be introduced systemically, e.g., by intravenous injection.
  • Specific transduction of the protein in the target cells occurs predominantly from specificity of transfection provided by the delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof.
  • initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized.
  • the delivery vehicle can be introduced by catheter (see, U.S. Pat. No. 5,328,470) or by stereotactic injection (e.g., Chen, et al., PNAS 91 : 3054-3057 (1994)).
  • the pharmaceutical preparation of the therapeutic construct can consist essentially of the delivery system in an acceptable diluent, or can comprise a slow release matrix in which the delivery vehicle is imbedded.
  • the complete delivery system can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can comprise one or more cells, which produce the delivery system.
  • the invention provides methods of treating a patient in need thereof (e.g., a patient infected with Hepatitis delta virus) 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.
  • a patient in need thereof e.g., a patient infected with Hepatitis delta virus
  • the invention also provides for a use of an interferon- associated antigen binding protein, or a nucleic acid sequence (e.g., mRNA) that encodes an interferon-associated antigen binding protein, as disclosed herein, in the preparation of a medicament for the treatment of Hepatitis delta virus infection.
  • kits and methods for the treatment of disorders and/or symptoms e.g., Hepatitis delta virus-related disorders and/or Hepatitis delta virus-related symptoms, in a mammalian subject in need of such treatment.
  • the subject is a human.
  • interferon-associated antigen binding proteins, or nucleic acid sequences that encode them, of the present invention are useful in a number of different applications.
  • the subject interferon-associated antigen binding proteins, or nucleic acid sequences that encode them are useful for reducing viral burden in Hepatitis delta virus-infected cells, inhibiting Hepatitis delta virus infection, rescuing Hepatitis delta virus-infected cells from cell death and/or from Hepatitis delta virus-induced cytopathic effect.
  • the subject interferon-associated antigen binding proteins, or nucleic acid sequences that encode them are useful for reducing one or more symptoms and/or complications associated with Hepatitis delta virus infection, as described herein (infra).
  • this invention also relates to a method of treating one or more disorders, symptoms and/or complications associated with Hepatitis delta virus infection in a human or other animal by administering to such human or animal an effective, non-toxic amount of an interferon-associated antigen binding protein, or a nucleic acid sequence that encodes it.
  • an effective, non-toxic amount of an interferon-associated antigen binding protein, or a nucleic acid sequence that encodes it would be for the purpose of treating Hepatitis delta virus infection.
  • a “therapeutically active amount” of an interferon-associated antigen binding protein of the present invention may vary according to factors such as the disease stage (e.g., acute vs. chronic), age, sex, medical complications (e.g., HIV co-infection, immunosuppressed conditions or diseases) and weight of the subject, and the ability of the interferon-associated antigen binding protein to elicit a desired response in the subject.
  • the dosage regimen may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • compositions provided in the current invention may be used to prophylactically treat non-infected cells or therapeutically treat any Hepatitis delta virus-infected cells comprising an antigenic marker that allows for the targeting of the Hepatitis delta virus-infected cells by an interferon-associated antigen binding protein.
  • the treatment or prevention of a Hepatitis delta virus infection may entail administering a CD40 agonist or a functional fragment thereof e.g., “in combination” with an IFN or a functional fragment thereof, and vice versa. If the CD40 agonist or the functional fragment thereof and the IFN or the functional fragment thereof are present in distinct pharmaceutical compositions, such administration in combination may be performed by simultaneous administration. Alternatively, the combined administration may be achieved in that case via sequential administration. For example, the CD40 agonist or the functional fragment thereof may be administered prior to the IFN or the functional fragment thereof. Alternatively, the IFN or the functional fragment thereof may be administered prior to the CD40 agonist or the functional fragment thereof.
  • the administration will be performed in such a manner that the combined administration will lead to an enhancement of the beneficial effects of the treatment on, e.g., reducing viral burden in Hepatitis delta virus-infected cells, inhibiting Hepatitis delta virus infection, rescuing cells from Hepatitis delta virus-induced cell death and/or from Hepatitis delta virus-induced cytopathic effect preferably compared to the administration of the IFN or the functional fragment thereof alone.
  • a skilled artisan will be readily able to determine suitable administration regimens, associated dosages, administration intervals, and, where sequential administration of distinct pharmaceutical compositions is chosen, intervals between the administration of said distinct pharmaceutical compositions, where such enhancement is achieved.
  • the interferon-associated antigen binding proteins of the invention or nucleic acid sequences (including vectors or vector systems) that encode them are comprised in a pharmaceutical composition.
  • Methods of preparing and administering interferon-associated antigen binding proteins, or nucleic acid sequences that encode them, of the current invention to a subject are well known to or can be readily determined by those skilled in the art using this specification and the knowledge in the art as a guide.
  • the route of administration of the interferon- associated antigen binding proteins, or nucleic acid sequences that encode them, of the current invention may be oral, parenteral, by inhalation or topical.
  • a form for administration would be a solution for injection, in particular for intravenous or intraarterial injection or drip.
  • a suitable pharmaceutical composition for injection may comprise a buffering agent (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), optionally a stabilizing agent (e.g. human albumin), etc.
  • the buffering agent is acetate.
  • the buffering agent is formate.
  • the buffering agent is citrate.
  • the surfactant may be selected from the list comprising pluronics, PEG, sorbitan esters, polysorbates, triton, tromethamine, lecithin, cholesterol and tyloxapal.
  • the surfactant is polysorbate.
  • the surfactant is polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80 or polysorbate 100, preferably polysorbate 20 or polysorbate 80.
  • the interferon-associated antigen binding proteins, or nucleic acid sequences that encode them can be delivered directly to the site of the adverse cellular population (e.g., the liver) thereby increasing the exposure of the diseased tissue to the therapeutic agent.
  • Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1 M, e.g., 0.05 M phosphate buffer, or 0.8% saline.
  • compositions suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and will typically be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of 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 and the like.
  • isotonic agents will be included, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • sterile injectable solutions can be prepared by incorporating an active compound such as an interferon-associated antigen binding protein, or a nucleic acid sequence encoding said interferon-associated antigen binding protein, of the present invention by itself or in combination with other active agents in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization.
  • an active compound such as an interferon-associated antigen binding protein, or a nucleic acid sequence encoding said interferon-associated antigen binding protein, of the present invention by itself or in combination with other active agents in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • exemplary methods of preparation include vacuum drying and freeze-drying, which yields a powder of an active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the preparations for injections are processed, filled into containers such as ampules, bags, bottles, syringes or vials, and sealed under aseptic conditions according to methods known in the art. Further, the preparations may be packaged and sold in the form of a kit. Such articles of manufacture will typically have labels or package inserts indicating that the associated compositions are useful for treating a subject suffering from Hepatitis delta virus infection.
  • compositions of the present invention for the treatment of the above described Hepatitis delta virus infection-related conditions vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic.
  • the patient is a human, but non-human mammals including transgenic mammals, in particular non-human primates, can also be treated.
  • Treatment dosages may be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.
  • the dosage can range, e.g., from about 0.0001 to about 100 mg/kg, and more usually about 0.01 to about 5 mg/kg (e.g., about 0.02 mg/kg, about 0.25 mg/kg, about 0.5 mg/kg, about 0.75 mg/kg, about 1 mg/kg, about 2 mg/kg, etc.), of the host body weight.
  • dosages can be about 1 mg/kg body weight or about 10 mg/kg body weight or within the range of about 1 to about 10 mg/kg, e.g., at least about 1 mg/kg. Doses intermediate in the above ranges are also intended to be within the scope of the current invention.
  • Subjects can be administered such doses daily, on alternative days, weekly or according to any other schedule determined by empirical analysis.
  • An exemplary treatment entails administration in multiple dosages over a prolonged period, for example, of at least six months. Additional exemplary treatment regimens entail administration about once per every two weeks or about once a month or about once every 3 to 6 months.
  • Exemplary dosage schedules include about 1 to about 10 mg/kg or about 15 mg/kg on consecutive days, about 30 mg/kg on alternate days or about 60 mg/kg weekly.
  • Interferon-associated antigen binding proteins, or nucleic acid sequences expressing any of these can be administered on multiple occasions. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of interferon-associated antigen binding proteins of components thereof in the patient. Alternatively, interferon- associated antigen binding proteins, or nucleic acid sequences expressing any of these 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 the interferon-associated antigen binding proteins in the patient.
  • the term “half-life” or “ti/2”, as referred to herein, relates to the stability and/or the rate of excretion of a compound, such as the interferon-associated antigen binding proteins of the invention.
  • the half-life of a compound is usually measured in the serum and denotes the time after administration that 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 serum half-life in mice.
  • the half-life of the interferon- associated antigen binding protein is at least 50 h, at least 60 h, at least 70 h, at least 80 h, at least 90 h or at least 100 h.
  • the half-life of the interferon-associated antigen binding protein is at least 100 h.
  • the half-life of the interferon-associated antigen binding protein in mice ranges from 116 to 158 h.
  • the half-life of a protein is related to its clearance.
  • volume of distribution refers to the theoretical volume that would be necessary to contain the total amount of an administered compound such as the interferon- associated antigen binding protein of the invention at the same concentration that it is observed in the blood plasma and relates to the distribution of said compound between plasma and the rest of the body after oral or parenteral dosing.
  • the volume of distribution Vss of the interferon-associated antigen binding protein is below 500 mL/kg, below 400 mL/kg, below 300 mL/kg, below 200 mL/kg, or below 100 mL/kg.
  • the volume of distribution Vss of the interferon-associated antigen binding protein is below 100 mL/kg. In some embodiments, the volume of distribution Vss of the interferon-associated antigen binding protein in mice ranges from 50 to 98 mL/kg.
  • systemic exposure Another related pharmacokinetic parameter is the systemic exposure.
  • systemic exposure might be represented by plasma (serum or blood) concentrations or the AUCs of parent compound and/or metabolite(s).
  • the interferon-associated antigen binding proteins of the invention circulate in the blood with higher systemic exposure (AUC (0-inf)) than their parental antibody.
  • the parental antibody is CP870,893. In other embodiments, the parental antibody is 3G5.
  • the systemic exposure of the interferon-associated antigen binding protein is at least 600 pg*h/mL, at least 700 pg*h/mL, at least 800 pg*h/mL, at least 900 pg*h/mL or at least 1000 pg*h/mL, preferably at least 1000 pg*h/mL.
  • the systemic exposure of the interferon-associated antigen binding protein in mice ranges from 1033 pg*h/mL to 1793 pg*h/mL.
  • an interferon-associated antigen binding protein of the present invention may be administered in a pharmaceutically effective amount for the in vivo treatment of mammalian disorders.
  • an interferon-associated antigen binding protein will be formulated to facilitate administration and promote stability of the active agent.
  • a pharmaceutical composition in accordance with the present invention can comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, nontoxic buffers, preservatives and the like.
  • a pharmaceutically effective amount of an interferon-associated antigen binding protein typically is an amount sufficient to mediate one or more of: a reduction of Hepatitis delta virus- induced cell death and a stimulation of the IFN signaling pathway in an infected cell.
  • the pharmaceutical compositions of the present invention may be administered in single or multiple doses to provide for a pharmaceutically effective amount of the interferon-associated antigen binding protein.
  • interferon-associated antigen binding proteins may be administered to a human or other animal in accordance with the aforementioned methods of treatment in an amount sufficient to produce a therapeutic effect.
  • the interferon-associated antigen binding proteins, or nucleic acid sequences expressing any of them can be administered to such human or other animal in a conventional dosage form prepared by combining the interferon-associated antigen binding proteins, or nucleic acid sequences expressing any of them, with a conventional pharmaceutically acceptable carrier or diluent according to known techniques.
  • the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well- known variables.
  • a cocktail comprising one or more species of interferon-associated antigen binding proteins, or nucleic acid sequences expressing any of them, described in the current invention may prove to be effective.
  • Interferon-Fused Antibodies based on agonistic anti-CD40 antibody CP870.893 and characterization on reporter cells
  • IFAs The sequence combinations of exemplary IFAs, designed with CP870,893 agonistic anti-CD40 antibody as backbone antibody, with the location of IFNs and the nature of the linkers are listed in Table 7 and Table 9. IFN was fused via a linker at the N- or the C-terminal part of the Light Chain (LC) or the Heavy Chain (HC), as indicated in Table 7. Nucleic acids encoding the HC, the LC or the fusions were synthesized with optimized mammalian expression codons and cloned into a eukaryotic expression vector such as pcDNA3.1 (Invitrogen).
  • Fig. 2A depicts an exemplary map of a pcDNA3.1 plasmid encoding Seq ID NO 32 under the control of the pCMV promoter.
  • the 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 pm filters. Purification process was performed in two purification steps, on AktaExpress chromatography system (GE Healthcare) using Protein A MabSelect Sure 5mL 1.6/2.5 cm column (GE Healthcare) at a Flow rate of 5 mL/min. Sample binding was done in D-PBS1X pH 7.5 buffer, and elution with Glycine/HCl 0.1 M pH 3.0 buffer.
  • Elution peak was stored in a loop then injected on HiTrap desalting 26/10 column (GE Healthcare) with a flow rate of 10 mL/min in D-PBSIXpH 7.5 buffer. Elution peak was collected on a 96-well microplate (2 mL fractions). Pool was performed according to the UV peak profile. After filtration on 0.22 pm filters (Sartorius MiniSart), quality control was performed including Bacterial Endotoxins using Endosafe® nexgen-PTSTM (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 NuPAGE gel System (Invitrogen) in MES SDS running buffer. The production yield is indicated in Table 9. For some IF As, the production yield was very low. In that case, the agonistic CD40 activity and the IFN activity were assessed directly using the supernatant containing IF As without any further purification.
  • HEK-BlueTM CD40L cells InvivoGen Cat. #: hkb-cd40
  • 3 cells InvivoGen, Cat. #: hkb-ifnaP
  • HEK-BlueTM CD40L cells were generated by stable transfection of HEK293 cells with the human CD40 gene and aNFKB-inducible Secreted Embryonic Alkaline Phosphatase (SEAP) construct (Invivogen) to measure the bioactivity of CD40 agonists. Stimulation of CD40 leads to NFKB induction and then production of SEAP, which is detected in the supernatant using QUANTI-BlueTM (Invivogen, Cat. # rep-qbs2).
  • SEAP Secreted Embryonic Alkaline Phosphatase
  • HEK-BlueTM IFN-cells are designed to monitor the activation of the JAK/STAT/ISGF3 pathways induced by type I-IFNs. Activation of this pathway induces the production and release of SEAP. Levels of SEAP are readily assessable in the supernatant using QUANTI-BlueTM.
  • HEK-BlueTM IFN-a/p are used to monitor the activity of human IFNa or IFNp.
  • HEK-BlueTM Dual IFN-y cells InvivoGen, Cat. #: hkb-ifng
  • HEK-BlueTM IFN-X InvivoGen, Cat. # hkb-ifnl
  • HEK-BlueTM IFN-A cells are designed to monitor the activity of IFNX.
  • HEK-BlueTM Dual IFN-y cells allow the detection of bioactive human IFNy.
  • Fig. 3 shows examples of dose responses of IF As, where IFNP or a mutated version thereof as specified in Tables 7 was fused to the HC as indicated in Table 7, on HEK-BlueTM CD40L (Figs. 3A-3B) and HEK-BlueTM IFN-a/p cells (Figs. 3C- 3D).
  • Agonistic anti-CD40 activities of IFAs are summarized in Table 9 and examples are shown in Fig. 3A and Fig. 3B. Results indicate that all tested IFAs are functional to activate both the CD40 pathway and the IFN-a/p pathway in a dose dependent manner.
  • the ECso values for agonistic CD40 are ranging from 11.1 ng/mL to 192 ng/mL (Table 9).
  • the mean ECso value for the parental antibody is 48ng/mL and 57ng/mL in the experiment shown in Fig. 3.
  • IFAs with the IFN fused to the N-terminus of the HC or the LC were also able to activate the CD40 pathway, but the precise EC50 values could not be determined for these IFAs since the activity was directly determined from the supernatant and not using purified proteins (Fig. 3B).
  • IFN activity of various IFAs is summarized in Table 9 and examples are shown in Figs. 3C to 3D.
  • the IFN activity is variable depending on the linker sequence with EC50 values ranging from 0.14 ng/mL to 4.5 ng/mL (Fig. 3C and Table 9).
  • Fig. 3D shows that IFAs with IFNP fused to the N-terminal part exhibit high IFN activity.
  • the parental antibody used as negative control did not show any activity, whereas recombinant IFNP did show a strong dose-dependent response.
  • Fig. 4 shows examples of dose responses of IF As, where IFNa was fused to the HC or the LC as indicated in Table 7, on HEK-BlueTM CD40L (Fig. 4A and Fig.4 C) and HEK-BlueTM IFN-a/p cells (Fig. 4B and Fig. 4D). Results indicate that all tested IF As are functional to activate both the CD40 pathway and the IFNa/p pathway in a dose-dependent manner. Surprisingly, for all the IFNa-based IFAs, the potency on CD40 pathway was reproducibly higher than that of the parental antibody.
  • the ECso values for IFNa-based IFAs ranged from 11.1 ng/mL to 22.7 ng/mL and the EC50 for CP870,893 ranged from 30 ng/mL to 80 ng/mL (mean EC50 value: 48 ng/mL).
  • IFN activity of IFAs is variable depending on the linker sequence with EC50 values ranging from 1.6 ng/mL to 5.1 ng/mL.
  • PEGylated IFNa2a (Pegasys®) was also active in a dose-dependent manner with an EC50 value of around 1 ng/mL.
  • Suitable constructs according to the invention can also be interferon- associated antigen binding proteins without an Fc region.
  • a construct encoding the heavy chain of the fab fragment of CP870,893 fused to a TEV-His tag was designed (SEQ ID NO 50) and cloned into the expression plasmid pcDNA3.1. This construct is cotransfected in HEK cells as described earlier, with LCs fused via different linkers to different IFNs such as SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 41, SEQ ID NO 42, or SEQ ID NO 43. Proteins and/or supernatants are evaluated in reporter cells.
  • HBV genotype D inoculum was prepared from the supernatant of HepAD38 cells, which were initially described in Ladner and colleagues (Antimi crob Agents Chemother, 41(8): 1715-20, 1997; doi: 10.1128/AAC.41.8.1715). The 0.22- micron filtrated supernatant of HepAD38 was precipitated with PEG8000 (8% final) to generate the viral stock.
  • An HDV genotype 1 inoculum was prepared from the supernatant of Huh7 cells co-transfected with pSVLD3 et pHB2.7 cells as described by C. Sureau (Methods Mol Biol, 640:463-73, 2010; doi: 10.1007/978-l-60761-688-7_25). This 0.22-micron filtrated supernatant was PEG8000 (8% final) precipitated to generate the HDV stock. lib - Virus quantification
  • Huh7.5-hNTCP cells were generated starting from Huh7.5 cells (described in Blight et al., J Virol., 77(5):3181-90, 2003; doi: 10.1128/j vi.77.5.3181-3190.2003) that were stably transduced with a lentivirus encoding hNTCP gene (Gene ID: 6554).
  • Huh7.5-hNTCP cells were cultivated in the same medium as used for PHH (see Il.d below), but without DMSO. Cells were seeded in 24-well plates at 0.1 million cells/well 24 hours before use.
  • Il.d Primary human hepatocytes (PHH) freshly isolated from liver resection
  • PHH were isolated from liver resection.
  • the procedure of isolation of PHH and infection with HBV was essentially adapted after the work of Gripon and colleagues (J. Virol., 62(l l):4136-43, 1988; doi: 10.1128/JVI.62.11.4136- 4143.1988, and Virology, 192(2): 534-40, 1993; doi: 10.1006/viro.1993.1069).
  • Myrcludex-B consists of a myristoylated peptidic sequence (Myristoyl-GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNP NKDHWPEANKVG; SEQ ID NO 94) and was synthesized (with purity grade: >95%) on request by GenScript (Order No. 778876; Ref: SC1208). The peptide was diluted in water, stored at -20°C, and was used at a final concentration of 100 nM.
  • IFN alpha 2A (Roferon-A; 6 million UI Injectable in 0.5 mL pre-prepared syringe) was used at a final concentration of 1000 lU/mL.
  • FTI-277 HC1 was purchased from Selleckchem (catalog No. S7465, >99% purity). Stock solution was at lOmM in DMSO and the final working concentration was at 10 pM.
  • a multiplicity of infection of 250 HBV vge/cell and 25 HDV vge/cell were used to infect PHH.
  • the day of coinfection cells were exposed overnight with an inoculum containing HBV and HDV virus in PEG8000 at 4% final concentration.
  • cells were washed to remove viral inoculum and were kept for 3-4 days post-infection (p.i.) before receiving the treatments.
  • p.i. 3-4 days post-infection
  • As and controls were extra- temporally diluted in complete medium from mother stocks.
  • Treatments were repeated twice for each treatment kinetic, one treatment after 3 or 4 days p.i., respectively, and the second treatment after 6 or 7 days p.i., respectively.
  • Analyses were performed on the supernatant and on cell extracts recovered at the end of the kinetic of the coinfection at 9 or 10 days, respectively.
  • a multiplicity of infection of 250 HBV vge/cell and 25 HDV vge/cell were used to infect PHH.
  • the day of infection cells were first infected with HBV overnight and kept for 5 days post-infection to establish a long-lasting “chronic” HBV infection. Then, cells were super-infected by HDV for 3 extra days.
  • As and controls were extra-temporally diluted in complete medium from mother stocks. Treatments were done at 8 days and 11 days p.i.. Analyses were performed on the supernatant and on cell extracts recovered at the end of the kinetic of superinfection at 14 days.
  • Huh7.5-hNTCP cells were used for a secondary infection as a surrogate test to assess viral propagation.
  • Cells were infected with supernatants, which were recovered from PHH cultures at the end of the kinetic of co-infection or superinfection and adjusted to 4% final with PEG8000.
  • the secondary infection was stopped at 6-day p.i. and intracellular HDV RNA quantification analyses were performed as described below.
  • HBsAg in the supernatants of infected non-treated (NT) and treated PHH cells were quantified with CLIA kits according to manufacturer’s instructions (Autobio; ref: CL0310-2).
  • the luminescence signals of calibration standards and samples were acquired with Luminoskan (Thermo Fischer Scientific).
  • a calibration curve was generated using signals of the standards and a four-parameter logistic (4-PL) fitting curve. Sample concentrations were determined by interpolating the sample signal with the standard curve. The concentrations obtained were exported to GraphPad-Prism and percentages were calculated as compared to the non-treated (NT) infected condition of each cell culture plate, NT being defined at 100%.
  • IC50 Concentration of compound that gives a response halfway between Top and Bottom
  • IC50 concentration of compound that gives a response halfway between Top and Bottom
  • the adjustment was obtained by non-linear regression using GraphPad PRISM V9.1.0 software.
  • HBsAg the top was constrained to 100 and the bottom was constrained to be positive if needed.
  • the geometric mean of ICsos of several donors and its 95% confidence interval were calculated for each experiment, each parameter and each treatment.
  • Complementary DNA was quantified by qRT-PCR with either HBV or HDV specific primers and the “Luna® Universal qPCR Master Mix” reagent (BioLabs; ref: M3003E).
  • HBV primers HBV-F (5’- AGCTACTGTGGAGTTACTCTCGT-3’; SEQ ID NO 95) and HBV-R (5’- CAAAGAATTGCTTGCCTGAGTG-3’; SEQ ID NO 96).
  • HDV primers HDV-F (5’- CGGGCCGGCTACTCTTCT-3’; SEQ ID NO 97) and HDV-R (5’- AAGGAAGGCCCTCGAGAACA-3’; SEQ ID NO 98). More details can be found in Michelet et al. (J Hep Report, 4(3): 100415, 2021; doi:
  • IC50 concentration of compound that gives a response halfway between Top and Bottom
  • HBV RNA, HDV RNA and HDV RNA re-infection (viral propagation) parameters in triplicate The adjustment was obtained by non-linear regression using GraphPad PRISM V9.1.0 software.
  • HBV RNA, HDV RNA and HDV RNA re-infection parameters the top was constrained to 0 (corresponding to 1 on the antilog scale).
  • the geometric mean of IC50S of several donors and its 95% confidence interval were calculated for each experiment, each parameter and each treatment.
  • HBsAg secretion was reduced by IFA44 treatment with IC50 values of 505.54 pM and 51.94 pM for donor 1 and 2, respectively (Fig. 5E).
  • IFA44 treatment resulted in a reduction of intracellular HBV RNA expression with estimated IC50 values of 134.07 pM and 59.82 pM for donor 1 and 2, respectively, (Fig. 5F) and in a reduction of intracellular HDV RNA expression with estimated IC50 values of 1844.49 pM and 386.22 pM for donor 1 and 2, respectively (Fig. 5G).
  • IC50 values of 56.33 pM and 210.25 pM were estimated for donor 1 and 2, respectively (Fig. 5H).
  • HBsAg secretion was reduced by IFA25 treatment with IC50 values of 20.63 pM and 11.50 pM for donor 1 and 2, respectively (Fig. 51).
  • IFA25 treatment resulted in a reduction of intracellular HBV RNA expression with estimated IC50 values of 57.76 pM and 61.89 pM for donor 1 and 2, respectively (Fig. 5J), and in a reduction of intracellular HDV RNA expression with estimated IC50 values of 219.34 pM and 344.70 pM for donor 1 and 2, respectively (Fig. 5K).
  • HBsAg secretion was reduced by IFA25 treatment with IC50 values of 7.76 pM, 14.51 pM and 15.05 pM for donor 1 to 3, respectively (Fig. 5M).
  • IFA25 treatment resulted in a reduction of intracellular HBV RNA expression with estimated IC50 values of 221.39 pM, 88.54 pM and 82.96 pM for donor 1-3, respectively (Fig. 5N), and in a reduction of intracellular HDV RNA expression with estimated IC50 values of 143.56 pM, 523.55 pM and 854.04 pM for donor 1-3, respectively (Fig. 50).
  • Results are expressed in percentage of secreted HBsAg of the co-infected non-treated control for the CLIA assay or as fold change of RNA expression compared to the co-infected nontreated control for RNA quantification (Figs. 5Q- 5T). Treatment with the isotype control antibody EVI5 showed a similar profile as in the coinfected non-treated condition for all tested viral parameters.
  • IFA202 the isotype antibody with the same IFN alpha2A fusion as IFA25 (but without the anti- CD40 agonistic activity) demonstrated a limited antiviral effect with an average reduction of 25.13% of HBsAg secretion, 18.35% of intracellular HBV expression, no effect on intracellular HDV expression and a good reduction of 61.64% on HDV propagation.
  • Roferon also demonstrated limited antiviral effects with an average of 59.83 % inhibition of HBsAg secretion, 29.01% and 3.64% inhibition of both intracellular HBV and HDV RNA expression and a strong inhibition of HDV reinfection with 91.55% compared to the coinfected nontreated condition.
  • IFA25 is the most potent antiviral molecule evaluated in this experiment with a significant average reduction of HBsAg secretion of 88.79% at 0. Ipg/ml and 97.31% at Ipg/ml compared to the co-infected non-treated control (Fig. 5Q), a significant average reduction of intracellular HBV RNA expression of 68.75% at O.lpg/ml and 79.93% at Ipg/ml compared to the coinfected non-treated control (Fig.
  • IFA25 treatment resulted in a reduction of HBsAg secretion with estimated IC50 values of 23.43 pM, 35.63 pM and 31.40 pM for donor 1 to 3, respectively (Fig. 6A).
  • IFA25 treatment also resulted in a reduction of intracellular HBV RNA expression with estimated IC50 values of 157.13 pM, 92.64 pM and 317.65 pM for donor 1-3, respectively (Fig. 6B) and in a reduction of intracellular HDV RNA expression with estimated IC50 values of 472.67 pM for donor 3 respectively (Fig. 6C, light grey line).
  • No reliable fitting model for intracellular HDV RNA IC50 determination could be applied for donors 1 and 2.
  • Results are expressed in percentage of secreted HBsAg of the superinfected non-treated control for the CLIA assay or as fold change of RNA expression compared to the super-infected non-treated control for RNA quantification (Fig. 6E- 6H) Treatment with the isotype control antibody EVI5 showed a similar profile as in the coinfected non-treated condition for all tested viral parameters.
  • IFA202 the isotype antibody with the same IFN alpha2A fusion as IFA25 (but without the anti- CD40 agonistic activity) demonstrated a limited antiviral effect with an average reduction of 21.39% of HBsAg secretion, 8.58% of intracellular HBV expression, no effect on intracellular HDV expression and an average reduction of 21.68% on HDV propagation.
  • Roferon also demonstrated limited antiviral effects with an average of 40.75 % inhibition of HBsAg secretion, 24.68% inhibition of intracellular HBV and no effect on intracellular HDV RNA expression.
  • a good inhibition of HDV reinfection with 53.78% compared to the coinfected nontreated condition was shown.
  • IFA25 is the most potent antiviral molecule evaluated in this assay with a significant average reduction of HBsAg secretion of 74.21% at O. lpg/ml and 85.71% at Ipg/ml compared to the super-infected nontreated control (Fig. 6E), a significant average reduction of intracellular HBV RNA expression of 63.64% at Ipg/ml and 73.85% at Ipg/ml compared to the super-infected nontreated control (Fig.
  • WBC ex vivo stimulation assay was used to investigate release of cytokines following IFA stimulation.
  • WBC were collected from four healthy donors, diluted 1/3 in RPMH640 (72400-021, Gibco) and distributed in sterile reaction tubes (300 pl).
  • Cells were left unstimulated, stimulated with LPS (LipoPolySaccharide) K12 (tlrl-eklps, Invivogen) at 10 ng/mL as a positive control or with IFAs at 1 pg/mL and incubated for 24 h at 37 C.
  • Supernatants were then collected and frozen at -20 C until the day of analysis.
  • cytokines Human pro-inflammatory cytokines were analyzed using multiplexing MSD assay (K15067L-4) which measures Tumor Necrosis Factor (TNF)-a, Interleukin (IL)-lp, IL-2, IL-6, IL-8, IL-10, IL-12/IL-23p40 and IFNy. MSD plates were analyzed on the 1300 MESO QuickPlex SQ120 apparatus (MSD).
  • Fig. 7 depicts exemplary results from an in vitro Cytokine Release Assessment of Human WBC either non-stimulated, treated with LPS or with IF Al.
  • TMB Tetramethylbenzidin, Tebu Bio
  • TMBW-1000-01 Tetramethylbenzidin, Tebu Bio
  • the reaction was stopped by adding IM HC1. Plates were read at 450-650 nm with an Ensight plate reader (Perkin Elmer). Quantification of Pegasys was assessed using similar protocol steps but using human IFNa matched antibody pairs from eBioscience/Invitrogen. Capture was performed using 100 pL of human anti-IFNa antibody (eBioscience/Invitrogen; BMS216MST), at 1 pg/mL in sodium carbonate (0.05 M,pH 9.6, C-3041, Sigma). For the detection, a secondary anti-IFNa conjugate HRP antibody (1/1000, Affymetrix eBioscience/BMS216MST; 15501707) in PBS - 0.05% Tween20 - 1% Milk was applied.
  • CP870,893, IFA25, IFA26, IFA27, IFA28, IFA29 and IFA30 were administrated at 0.5 mg/kg and Pegasys at 0.3 mg/kg i.v. bolus to male CD 1 -Swiss mice and blood samples were collected at different time points.
  • Examples of quantification of circulating molecules using the ELISA approach described above and revealed with anti-IFNa-conjugated HRP are shown in Fig. 8A and 8B, while examples of quantification revealed with anti-IgG2- conjugated HRP are shown in Fig. 8C; Pegasys quantification is shown in Fig. 8D.
  • PK parameters for CP870,893 were explored in a 7-day experiment and those for IFA27, IFA29 and IFA30 in 10- day experiments (quantification for IFA27 was performed using 2 different ELISA approaches).
  • Table 12B the PK parameters for CP870,893 and IFA25, IFA26, IFA28 and Pegasys were explored in 21 -day experiments (quantification for IFA25 was performed using 2 different ELISA approaches).
  • the pharmacokinetic parameters summarized in Table 12A/B indicate that these IFAs surprisingly circulate in the blood with higher systemic exposure (AUC (0-inf)) ranging from 1033 pg.h/mL to 2552 pg.h/mL for IFAs in comparison to 590 or 797 pg.h/mL, respectively, for the parental antibody CP870,893 (up to 3.2 fold), also reflecting lower clearance values for IF As.
  • the volume of distribution Vss was low and ranked from 50 to 105 mL/kg, slightly higher than the plasma vascular volume (50 mL/kg) in this species. For all IF As, the clearance was ranked as low (0.28 to 0.49 mL/h/kg).
  • IFNa was linked to the LC part with a (G4S)2 (IFA50) or (G4S)3 (IFA51) linker.
  • IFN epsilon IFNs
  • IFNs third type I interferon
  • IFA49 IFA49
  • Fig. 10A Evaluation on HEK-BlueTM hlFN- a/p cells (which are in fact activated by any type I interferon) showed that IFA49 is also able to activate the IFN-I-pathways (Fig. 10B).
  • EC50 values are reported in
  • IFN lambda type III Interferon
  • IFNX type III Interferon
  • Interferon-Fused Antibodies based on anti-CD40 antibody 3G5 and characterization on reporter cells
  • IFAs designed with 3G5 anti- CD40 antibody (Celldex) as backbone antibody, with the location of IFNs and the nature of the linkers are listed in Table 8 and Table 10.
  • IFN was fused via a linker at the C-terminal part of the Light Chain (LC) or the Heavy Chain (HC), as indicated in Table 8.
  • Nucleic acids encoding the HC, the LC or the fusions were synthesized with optimized mammalian expression codons and cloned into a eukaryotic expression vector such as pcDNA3.1 (Invitrogen).
  • IFA production was performed as described earlier and the production yield is indicated in Table 10.
  • the production yield was very low, mainly for the fusion of IFNP to the C-terminal part of the LC.
  • the agonistic CD40 and the IFN activities were assessed directly using the supernatant containing IFAs without any further purification.
  • Reduced SDS-PAGE analysis of purified IFAs indicated the presence of two major bands corresponding to the HC and LC. When IFN was fused to the HC, a shift of its molecular weight was observed. (Fig. 14).
  • Fig. 15- shows examples of dose responses of IFAs, where IFNp was fused to the HC or the LC of 3G5, on HEK-BlueTM CD40L and HEK-BlueTM IFN-a/p cells (Fig. 15). Results summarized in Table 10 indicate that all tested IFN -based IFAs are functional and able to activate both the CD40 pathway and the IFNa/p pathway in a dose-dependent manner.
  • FIG. 15A and Fig. 15B Examples of CD40 activity are shown in Fig. 15A and Fig. 15B. Fusion of IFNP to the C-terminal part of the HC demonstrates high variable anti-CD40 activity and in all cases lower than the parental antibody with EC so values ranging from 30 ng/mL to 190.5 ng/mL (Fig. 15A and Table 10). The mean ECso value for the parental 3G5 antibody is 9.3 ng/mL.
  • IFN activity of IFAs were tested on HEK-BlueTM IFN-a/p cells and results are summarized in Table 10. Examples are shown in Fig. 15C-D.
  • the IFN activity is variable depending on the linker sequence with ECso values ranging from 0.45 ng/mL to 10,3 ng/mL (Fig. 15C).
  • IFN activity is still detected even after a 10000-fold dilution of the supernatant (Fig. 15D).
  • Figs. 16A-B show examples of dose responses of IF As, where IFNa was fused to the HC of 3G5, on HEK-BlueTM CD40L (Fig. 16A) and HEK-BlueTM IFNa/p cells (Fig. 16B).
  • Results indicate that all IF As display a functional activation of both the CD40 pathway and the IFNa/p pathway in a dose-dependent manner (mean ECso values are reported in Table 10).
  • the potency on CD40 pathway was similar to the parental antibody with the mean EC so values ranging from 11.74 ng/mL to 14.2 ng/mL (Fig. 16A and Table 10).
  • the mean ECso value for the parental 3G5 antibody is 9.3ng/mL.
  • IFNa-based IF The IFN activities of IFNa-based IF As were tested on HEK-BlueTM IFN-a/p cells and demonstrate very high activity.
  • the mean ECso values for the IFN activity of these IFAs ranged from 0.04 ng/mL to 0.12 ng/mL (Fig. 16B and Table 10).
  • Suitable constructs according to the invention can also be interferon- associated antigen binding proteins without an Fc region.
  • a construct encoding the heavy chain of the Fab fragment of 3G5 fused to a TEV-His tag was designed (SEQ ID NO 65) and cloned into the expression plasmid pcDNA3.1. This construct is cotransfected in HEK cells as described earlier, with LCs fused via different linkers to IFNs such as SEQ ID NO 70, or SEQ ID NO 71. Proteins and/or supernatants are evaluated in reporter cells and/or their effect on Hepatitis delta virus-infected cells.
  • constructs for use in therapy will no longer contain the TEV-His tag. These constructs are likewise embodiments of the invention. Interferon-associated antigen binding proteins without the Fc part will be active against Hepatitis delta virus infection.
  • a WBC ex vivo stimulation assay was used to investigate release of cytokines following IFA stimulation as described previously (see IILa).
  • IFA109 An example with IFA109 is shown in Fig. 17 and Table 13. The results indicate that all IF As induce CXCL10 release. They did not induce IL-10, IL- 1 [3 and IL-2, and they induced only very low to moderate level of IFNy, IL-6 and TNF-a, thus suggesting a favorable safety profile with regard to the induction of inflammatory cytokines.
  • HBV genotype D inoculum was prepared from the supernatant of HepAD38 cells, which were initially described in Ladner and colleagues (Antimi crob Agents Chemother, 41(8): 1715-20, 1997; doi: 10.1128/AAC.41.8.1715). The 0.22- micron filtrated supernatant of HepAD38 was centrifuged with centricon for virus concentration to generate the viral stock.
  • An HDV genotype 1 inoculum was prepared from the supernatant of Huh7 cells co-transfected with pSVLD3 and pHB2.7 cells as described by C. Sureau (Methods Mol Biol, 640:463-73, 2010; doi: 10.1007/978-1 -60761-688-7_25). This 0.22-micron filtrated supernatant was used to generate the HDV stock. VIII. b - Virus stocks quantification
  • the titers of these stocks were determined by ddPCR for HBV genotype D and by qRT-PCR for HDV genotype-1 and are expressed in viral genome equivalent (Vge in copy/mL).
  • Cryopreserved-PHH from Bioreclamation IVT (batch DSX; product number F00995-P) were used as in vitro cellular model for HBV/HDV Coinfection experiments.
  • Cells were plated at least 24h before Coinfection in PHH plating medium [William’s E glutaMAX (Gibco; 32551-020); 10% FBS (GE healthcare; SH30066.03); 5mg/ml Insulin (Sigma; I9278-5ML); 25mg/ml Hydrocortison (Sigma; H2270-100MG) and 1% Penicillin-streptomycin (Gibco; 15140122)].
  • PHH plating medium was changed for fresh one.
  • PHH were maintained in PHH infection medium [invitrogro HI medium (BioIVT; Z99009); 5% FBS (GE healthcare; SH30066.03); 2% DMSO (Sigma; D2650-100ml) and 1% Penicillin-streptomycin (Gibco; 15140122)].
  • PHH infection medium Invitrogro HI medium (BioIVT; Z99009); 5% FBS (GE healthcare; SH30066.03); 2% DMSO (Sigma; D2650-100ml) and 1% Penicillin-streptomycin (Gibco; 15140122)].
  • a multiplicity of infection 500 HBV genotype D Vge/cell and 5 pl per well of 96-well plate or 20pl per well of 24-well plate for HDV genotype- 1 were used.
  • the day of Coinfection cells were exposed overnight with an inoculum containing HBV and HDV virus in PEG8000 at 4% final in PHH infection medium. At day one, cells were washed to
  • naive cryopreserved-PHH were used for a second round of infection as a surrogate test to assess viral propagation.
  • This second round of infection was performed either at fixed-volume (i.e. same volume of conditioned-supernatant from primary infection was used to infect the naive PHH) or at fixed-Vge/cell (i.e. adjusted-volume of conditioned-supernatant from primary infection to have same HDV load to infect the naive PHH).
  • fixed-volume i.e. same volume of conditioned-supernatant from primary infection was used to infect the naive PHH
  • fixed-Vge/cell i.e. adjusted-volume of conditioned-supernatant from primary infection to have same HDV load to infect the naive PHH.
  • relative HDV intracellular RNA expression was measured by qRT-PCR and normalised to Coinfected nontreated condition.
  • HBs antigen (HBsAg) quantification in the supernatant was carried out according to commercial AutoBio CLIA kit protocol (#CL0310-2). First, the samples were diluted 10 times in 96-well plate format or 20 times in 24-well plate format in infection cell culture medium solution. In the assay plate, 50 pl of standards, controls, and diluted samples were placed in the wells. Then, 50 pl of "Enzyme conjugate” solution was added to each well and the plate was incubated for one hour at 37° C. Subsequently, the plate was washed 6 times with 300 pl of washing solution from the kit using an automated plate washer.
  • HBV DNA quantification in the supernatant was carried out by qPCR quantification with QuantStudio 7 Pro real-time PCR instrument, 96-Well 0.1 ml from ThermoFisher Scientific. Briefly viral DNA was first extracted from cell monolayer with NucleoSpin 96 Virus kit (Macherey-Nagel; 740691). Then, 5 pl of Eluted viral DNA was used as template for HBV DNA viremia quantification with TaqMan fast universal PCR master Mix (Thermofisher scientific; 4366073) and Taqman-FAM-MGB assay with commercial set of HBV primers and Probe (Thermofisher scientific; Vi03453405 si).
  • HBV viremia quantification a standard curve was built with HBV standard reference. For each sample, Ct quantification fluorescence was interpolated using the standard curve to obtain concentration in HBV DNA copy/ml. The final concentration was obtained by multiplying the concentration by the dilution factor.
  • HD V RNA quantification in the supernatant was carried out by qRT-PCR quantification with QuantStudio 7 Pro real-time PCR instrument, 96-Well 0.1 ml from ThermoFisher Scientific. Briefly viral RNA was first extracted from cell monolayer with NucleoSpin 96 Virus kit (Macherey-Nagel; 740691).
  • HDV RNA viremia quantification with TaqMan master mix fast virus 1-Step (Thermofisher scientific; 4444434) and Taqman-FAM-MGB assay with custom set of HDV primers and Probe (Thermofisher scientific; fwd 5’-TGGACGTGCGTCCTCCT-3’, SEQ ID NO 99; rev 5’-TCTTCGGGTCGGCATGG-3’, SEQ ID NO IOO; and probe 5’- ATGCCCAGGTCGGAC-3’; SEQ ID NO 101).
  • HDV viremia quantification a standard curve was built with HDV standard reference. For each sample, Ct quantification fluorescence was interpolated using the standard curve to obtain concentration in HDV RNA copy/ml. The final concentration was obtained by multiplying the concentration by the dilution factor.
  • Myrcludex-B was able to strongly decrease HDV RNA level, by 60% at InM and more than 99% at lOnM. Similar profil was observed of HBsAg level. Finally, Lonafamib induced a dose dependent decrease in HDV RNA level, from 20% at 1 pM to 80% decrease at lOpM. On HBsAg level, the effect of Lonafarnib was detected, only at the highest dose of lOpM.
  • HDV antigens were evaluated by western blot analysis (Fig. 18C).
  • HDV L and S antigens were detected, and Tubulin protein was used as loading control.
  • No HDV Antigen was detected in non-infected condition whereas in the Coinfection condition, without treatment, the level of HDV antigens were high.
  • Treatments with EVI5 and CP870.893 did not affect HDV antigen bands.
  • IFA202 and Myrcludex-B demonstrated a dose dependent decrease of the HDV antigen bands intensity, with a higher potency for Myrcludex-B at highest dose which nearly abolished the HDV antigen expression.
  • IFA25 was able to abolish the HDV antigen bands intensity, at its 2 dose levels thus being the most potent treatment to decrease HDV antigen parameter in these experimental conditions.
  • Lonafamib showed a different pattern with an increase of the HDV Antigen bands intensity thus reflecting an intracellular accumulation of HDV antigen compared to the coinfected non-treated condition.
  • Pegasys treatment was able to decrease the viral parameters in these different treatment designs, however its potency was always lower compared to IFA25.
  • Myrcludex-B treatment induced a decrease in HDV RNA and HBsAg level only in co-treatment design.
  • Myrcludex-B was not able to limit the HDV infection.
  • Tenofovir treatment did not impact HDV RNA level whatever the treatment design, it was only able to slightly decrease the HBsAg level in the co-treatment setting.
  • HBV genotype D inoculum was prepared from the supernatant of HepAD38 cells, which were initially described in Ladner and colleagues (Antimi crob Agents Chemother, 41(8): 1715-20, 1997; doi: 10.1128/AAC.41.8.1715). The 0.22- micron filtrated supernatant of HepAD38 was centrifuged with centricon for virus concentration to generate the viral stock.
  • An HDV genotype 3 inoculum was prepared from the supernatant of Huh-7 cells co-transfected with PL2032 (a 2-fold antigenome HDV-3) and pHB2.7. The 0.22-micron filtrated supernatant of Huh-7 was clarified by centrifugation to generate the HDV genotype 3 stock.
  • IFA25 and Pegasys were added at day 1 postCoinfection at two different concentrations for IFA25 (0.1 and 1 nM) and at a single concentration for Pegasys (1 nM).
  • IX. e Viral parameters analysis on HDV genotype-3 in HBV/HDV Coinfection experiments in cryopreserved PHH
  • HBV DNA was quantified in the supernatant by qPCR analysis as described in section Vlll.h.
  • HDV antigen in cell lysate was analyzed by Western Blot as described in section Vlll.j.
  • Secreted HBsAg and HBeAg were quantified in the supernatant by CLIA Assay, respectively CLIA kit CL0310-2 and CL0312-2, as described in section VIII. g.
  • RNA viremia was quantified by ddPCR.
  • RNA extraction with Nucleospin 96 virus kit as described in section VIII.
  • Eluted RNA was converted in cDNA in the presence of superscript VILO cDNA synthesis kit (Invitrogen; 11754-250) in PCR thermocycler (Bio-rad; T100) for 60 min at 42° C and 5 minutes at 85° C.
  • 5 pl of cDNA was subjected to droplet generation in a reaction mix of ddPCR Supermix for probes (Bio-rad, 1863010) and taqman custom primers and probe in autoDG instrument (Bio-rad; 773BR1339).
  • ddPCR plate move to PCR Thermocycler (Bio-rad; T100) for DNA amplification; 10 min. at 95° C; (30 sec. at 94°C and 1 min. at 58°C) 40 cycles and a final stage 10 min. at 98° C. Final quantification with Droplet Digital System (Biorad; QX-200). HDV viremia is expressed in copy/ml.
  • HBV genotype D and HDV genotype-1 viruses used in example X were obtained and tittered as described in example VIII in section VIII. a and Vlll.b respectively.
  • Cryopreserved PHH were coinfected with the viruses following the protocol described in section VIII.c.
  • HBV Viremia was quantified in the supernatant by PCR analysis as described in section Vlll.h.
  • HDV Viremia was quantified in the supernatant by PCR analysis as described in section Vlll.i.
  • Secreted HBsAg was quantified in the supernatant by CLIA Assay as described in section VIII. g.
  • Cell viability was assessed at 7-day post-Coinfection by CellTiter-Glo assay (Promega; G8462). Briefly, supernatant was harvested and a volume-volume solution of CellTiter-Glo solution and PBS was added to the cell monolayer. The mixture was mixed in contact of the cells for at least 2 minutes on an orbital shaker to allow cell lysis. Then luminescence was recorded on Envision (Perkin). Results were expressed as percentage of cell viability related to Coinfected nontreated control.
  • HBsAg parameter On HBsAg parameter, the effect of Myrcludex-B alone was of 70% and treatment with IFA25 in addition to Myrcludex-B further decrease HBsAg secretion down to 95% at highest dose (Fig. 22D) whereas Pegasys decrease HBsAg to a lesser extent of 80% (Fig. 22E). Finally, the cell viability was assessed using a Cell TiterGlo assay which was normalized to the coinfected non-treated condition. As shown on Fig. 22F and Fig. 22G, the level in Uninfected conditions were either similar or slightly lower compared to the coinfected conditions thus showing that the Coinfection did not induce any cell toxicity.
  • the CTG assay resulted in a slightly higher variability in the Pegasys experiment as shown by the greater error bars in Fig. 22G compared to the experiment with IFA25 in Fig. 22F.
  • the conditions where no cytotoxicity is observed can range from about 85% to about 107%.
  • the treatment with Myrcludex-B alone also did not impact the cell viability.
  • IFA25 or Pegasys in dose range, respectively on Fig. 22F and Fig- 22G did not induce any or only minor cell cytotoxicity as shown by the cell viability above 80% even at the highest dose of lOnM.
  • Fig. 22F, 22G and Fig. 23H and 231 demonstrated that, among the doses tested of IFA25 (from 0,001 nM to lOnM), 50% cytotoxicity concentrations (CC50), i.e. the concentration required to reduce cell viability by 50%, were reached in no experiment. Furthermore, in this dose range, the anti-HDV effects of IFA25, observed on HDV viremia and HBsAg already at low concentrations, were very potent (see Figs. 22B, 22D and 23B, 23D). Taken together, the demonstrated data confirm a favorable effective and safe profile of IF A treatment. Equivalents
  • An interferon-associated antigen binding protein comprising
  • IFN Interferon
  • the interferon-associated antigen binding protein for the use of item 1 or 2, wherein the agonistic anti-CD40 antibody, or the agonistic antigen binding fragment thereof, comprises three light chain complementarity determining regions (CDRs) that are identical to the CDRL1, CDRL2 and CDRL3 sequences within SEQ ID NO 3; and three heavy chain CDRs that are identical to the CDRH1, CDRH2 and CDRH3 sequences within SEQ ID NO 6.
  • CDRs light chain complementarity determining regions
  • each CDR is defined in accordance with the Kabat definition, the Chothia definition, the AbM definition, or the contact definition of CDR; preferably wherein each CDR is defined in accordance with the CDR definition of Kabat or the CDR definition of Chothia.
  • CDR complementarity determining region
  • a light chain or a fragment thereof comprising a CDRL1 that is at least 90% identical to SEQ ID NO 52, a CDRL2 that is at least 90% identical to SEQ ID NO 53, and a CDRL3 that is at least 90% identical to SEQ ID NO 54; preferably (II)
  • CDR complementarity determining region
  • a light chain or a fragment thereof comprising a CDRL1 that is at least 95% identical to SEQ ID NO 52, a CDRL2 that is at least 95% identical to SEQ ID NO 53, and a CDRL3 that is at least 95% identical to SEQ ID NO 54; more preferably (III)
  • CDR complementarity determining region
  • CDR complementarity determining region
  • a light chain or a fragment thereof comprising a CDRL1 that is at least 99% identical to SEQ ID NO 52, a CDRL2 that is at least 99% identical to SEQ ID NO 53, and a CDRL3 that is at least 99% identical to SEQ ID NO 54.
  • CDR complementarity determining region
  • a light chain or a fragment thereof comprising a CDRL1 that is identical to SEQ ID NO 52, a CDRL2 that is identical to SEQ ID NO 53, and a CDRL3 that is identical to SEQ ID NO 54.
  • the interferon-associated antigen binding protein for the use of any one of the preceding items, wherein the agonistic anti-CD40 antibody, or the agonistic antigen binding fragment thereof, comprises a light chain variable region VL comprising the sequence as set forth in SEQ ID NO 51, or a sequence at least 90% identical thereto; and/or a heavy chain variable region VH comprising the sequence as set forth in SEQ ID NO 55, or a sequence at least 90% identical thereto; preferably wherein the agonistic anti-CD40 antibody, or the agonistic antigen binding fragment thereof, comprises a light chain variable region VL comprising a sequence that is at least 95% identical to the sequence as set forth in SEQ ID NO 51; and/or a heavy chain variable region VH comprising a sequence that is at least 9
  • a heavy chain of the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof comprises a Fab region heavy chain comprising an amino acid sequence as set forth in SEQ ID NO 12, or a sequence at least 90%, preferably 95%, more preferably 98%, or still more preferably 99% identical thereto.
  • interferon-associated antigen binding protein for the use of any one of the preceding items, wherein the IFN or the functional fragment thereof is a human interferon.
  • interferon-associated antigen binding protein for the use of any one of the preceding items, wherein the IFN or the functional fragment thereof is selected from the group consisting of a Type I IFN, a Type II IFN and a Type III IFN, or a functional fragment thereof.
  • interferon-associated antigen binding protein for the use of any one of the preceding items, wherein the IFN or the functional fragment thereof is a Type I IFN, or a functional fragment thereof.
  • interferon-associated antigen binding protein for the use of item 16, wherein the type I IFN or the functional fragment thereof is IFNa, IFN0, IFN ⁇ , or IFNE, or a functional fragment thereof.
  • interferon-associated antigen binding protein for the use of item 16, wherein the type I IFN or the functional fragment thereof is IFNa or IFN0, or a functional fragment thereof.
  • interferon-associated antigen binding protein for the use of item 22, wherein the IFN or the functional fragment thereof is IFNa, or a functional fragment thereof.
  • interferon-associated antigen binding protein for the use of item 23, wherein the IFN or functional fragment thereof is IFNa2a, or a functional fragment thereof.
  • interferon-associated antigen binding protein for the use of item 24, wherein the IFNa2a comprises the sequence as set forth in SEQ ID NO 17, or a sequence at least 90%, preferably 95%, more preferably 98%, or still more preferably 99% identical thereto.
  • interferon-associated antigen binding protein for the use of item 22, wherein the IFN or the functional fragment thereof is IFNP, or a functional fragment thereof.
  • interferon-associated antigen binding protein for the use of item 26, wherein the IFNP comprises the sequence as set forth in SEQ ID NO 14, or a sequence at least 90%, preferably 95%, more preferably 98%, or still more preferably 99% identical thereto.
  • interferon-associated antigen binding protein for the use of item 26, wherein the IFNP or the functional fragment thereof comprises one or two amino acid substitution(s) relative to SEQ ID NO 14, selected from C17S and N80Q.
  • interferon-associated antigen binding protein for the use of item 28, wherein the IFNP or the functional fragment thereof comprises the amino acid substitution C17S relative to SEQ ID NO 14.
  • interferon-associated antigen binding protein for the use of item 31, wherein the IFN0 comprises the amino acid sequence as set forth in SEQ ID NO 16.
  • interferon-associated antigen binding protein for the use of item 21, wherein the IFN or a functional fragment thereof is IFNy or IFNX, or a functional fragment thereof.
  • interferon-associated antigen binding protein for the use of item 33, wherein the IFN or a functional fragment thereof is IFNy, or a functional fragment thereof.
  • interferon-associated antigen binding protein for the use of item 34, wherein the IFNy comprises the sequence as set forth in SEQ ID NO 19, or a sequence at least 90%, preferably 95%, more preferably 98%, or still more preferably 99% identical thereto.
  • interferon-associated antigen binding protein for the use of item 33, wherein the IFN or a functional fragment thereof is IF NX, or a functional fragment thereof.
  • interferon-associated antigen binding protein for the use of item 36, wherein the IFN or the functional fragment thereof is IFNX2, or a functional fragment thereof.
  • interferon-associated antigen binding protein for the use of item 37, wherein the IFN 2 comprises the sequence as set forth in SEQ ID NO 18, or a sequence at least 90%, preferably 95%, more preferably 98%, or still more preferably 99% identical thereto.
  • interferon-associated antigen binding protein for the use of any one of the preceding items, wherein the IFN or the functional fragment thereof is non- covalently associated with the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof.
  • 40 The interferon-associated antigen binding protein for the use of item 39, wherein the IFN or the functional fragment thereof is non-covalently associated with the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof via ionic, Van-der-Waals, and/or hydrogen bond interactions.
  • interferon-associated antigen binding protein for the use of any one of items 1 to 38, wherein the IFN or the functional fragment thereof is covalently associated with the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof.
  • interferon-associated antigen binding protein for the use of item 41, wherein the IFN or the functional fragment thereof is fused to the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof.
  • interferon-associated antigen binding protein for the use of item 42, wherein the IFN or the functional fragment thereof is fused to a light chain of the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof.
  • interferon-associated antigen binding protein for the use of item 43, wherein the IFN or the functional fragment thereof is fused to the N-terminus of the light chain of the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof.
  • interferon-associated antigen binding protein for the use of item 43, wherein the IFN or the functional fragment thereof is fused to the C-terminus of the light chain of the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof.
  • interferon-associated antigen binding protein for the use of item 42, wherein the IFN or the functional fragment thereof is fused to a heavy chain of the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof.
  • interferon-associated antigen binding protein for the use of item 46 wherein the IFN or the functional fragment thereof is fused to the N-terminus of the heavy chain of the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof.
  • interferon-associated antigen binding protein for the use of any one of items 42 to 48, wherein the agonistic anti-CD40 antibody or an agonistic antigen binding fragment thereof, and the IFN or the functional fragment thereof are fused to each other via a linker.
  • interferon-associated antigen binding protein for the use of item 49, wherein the interferon-associated antigen binding protein comprises no amino acids other than those forming (I) said agonistic anti-CD40 antibody, or agonistic antigen binding fragment thereof, (II) said IFN or functional fragment thereof and (III) said linker.
  • interferon-associated antigen binding protein for the use of any one of items 1 to 49, wherein the interferon-associated antigen binding protein comprises no amino acids other than those forming (I) said agonistic anti- CD40 antibody, or agonistic antigen binding fragment thereof and (II) said IFN or functional fragment thereof.
  • interferon-associated antigen binding protein for the use of any one of items 49 to 50, wherein the linker is a peptide linker.
  • interferon-associated antigen binding protein for the use of item 52, wherein the linker comprises at least 1, at least 2, at least 3, at least 4, or at least 5 amino acids.
  • interferon-associated antigen binding protein for the use of item 53, wherein the linker comprises at least 4 amino acids.
  • interferon-associated antigen binding protein for the use of item 53, wherein the linker comprises at least 11 amino acids.
  • interferon-associated antigen binding protein for the use of item 53, wherein the linker comprises at least 12 amino acids.
  • interferon-associated antigen binding protein for the use of item 53, wherein the linker comprises at least 20 amino acids.
  • interferon-associated antigen binding protein for the use of item 53, wherein the linker comprises at least 21 amino acids.
  • interferon-associated antigen binding protein for the use of item 53, wherein the linker comprises at least 24 amino acids.
  • interferon-associated antigen binding protein for the use of item 52, wherein the linker comprises up to 10, up to 20, up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, or up to 100 amino acids.
  • interferon-associated antigen binding protein for the use of item 62, wherein the linker comprises up to 80 amino acids.
  • interferon-associated antigen binding protein for the use of item 62, wherein the linker comprises up to 40 amino acids.
  • interferon-associated antigen binding protein for the use of item 62, wherein the linker comprises up to 24 amino acids.
  • interferon-associated antigen binding protein for the use of item 62, wherein the linker comprises up to 21 amino acids.
  • interferon-associated antigen binding protein for the use of item 62, wherein the linker comprises up to 20 amino acids.
  • interferon-associated antigen binding protein for the use of item 62, wherein the linker comprises up to 15 amino acids.
  • interferon-associated antigen binding protein for the use of item 62, wherein the linker comprises up to 13 amino acids.
  • interferon-associated antigen binding protein for the use of item 62, wherein the linker comprises up to 4 amino acids.
  • interferon-associated antigen binding protein for the use of any one of items 52 to 72, wherein the linker is selected from the group comprising acidic, basic and neutral linkers.
  • interferon-associated antigen binding protein for the use of item 73, wherein the linker is an acidic linker.
  • interferon-associated antigen binding protein for the use of item 73 or 74, wherein the linker comprises a sequence as set forth in SEQ ID NO 22 or SEQ ID NO 23.
  • interferon-associated antigen binding protein for the use of item 73, wherein the linker is a neutral linker.
  • interferon-associated antigen binding protein for the use of item 73 or 77, wherein the linker comprises a sequence as set forth in SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 24, SEQ ID NO 25 or SEQ ID NO 26.
  • interferon-associated antigen binding protein for the use of any one of items 52 to 78, wherein the linker is selected from the group comprising rigid, flexible and helix-forming linkers.
  • interferon-associated antigen binding protein for the use of item 79, wherein the linker is a rigid linker.
  • interferon-associated antigen binding protein for the use of item 79 or 80, wherein the linker comprises a sequence as set forth in SEQ ID NO 20, SEQ ID NO 22 or SEQ ID NO 23.
  • the linker comprises a sequence as set forth in SEQ ID NO 21, SEQ ID NO 24, SEQ ID NO 25 or SEQ ID NO 26.
  • interferon-associated antigen binding protein for the use of item 79, wherein the linker is a helix-forming linker.
  • interferon-associated antigen binding protein for the use of item 79 or 84, wherein the linker comprises a sequence as set forth in SEQ ID NO 22 or SEQ ID NO 23.
  • interferon-associated antigen binding protein for the use of any one of items 52 to 74, 76, 77, 79, 80, 82 or 84, wherein the linker comprises the amino acids glycine and serine.
  • interferon-associated antigen binding protein for the use of item 86, wherein the linker comprises the sequence as set forth in SEQ ID NO 21, SEQ ID NO 24, SEQ ID NO 25, or SEQ ID NO 26.
  • interferon-associated antigen binding protein for the use of item 86, wherein the linker further comprises the amino acid threonine.
  • interferon-associated antigen binding protein for the use of item 88, wherein the linker comprises the sequence as set forth in SEQ ID NO 21.
  • interferon-associated antigen binding protein for the use of item 52, wherein the linker comprises a sequence selected from the sequences as set forth in SEQ ID NOs 20 to 26.
  • interferon-associated antigen binding protein for the use of item 90, wherein the linker comprises a sequence selected from the sequences as set forth in SEQ ID NO 24, SEQ ID NO 25 or SEQ ID NO 26.
  • interferon-associated antigen binding protein for the use of item 91, wherein the linker comprises a sequence as set forth in SEQ ID NO 24.
  • interferon-associated antigen binding protein for the use of item 91 wherein the linker comprises a sequence as set forth in SEQ ID NO 25.
  • linker comprises a sequence as set forth in SEQ ID NO 26.
  • interferon-associated antigen binding protein for the use of any one of items 49, 50 or 52 to 94, wherein the IFN or a functional fragment thereof is fused to the C-terminus of a heavy chain of the agonistic anti-CD40 antibody, or the agonistic antigen binding fragment thereof, via the linker as set forth in Table 3, in particular Table 3A or Table 3B, more particularly Table 3A.
  • the interferon-associated antigen binding protein for the use of item 95, wherein the heavy chain of the agonistic anti-CD40 antibody, or the agonistic antigen binding fragment thereof, comprises a sequence as set forth in SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO 12, SEQ ID NO 48 or SEQ ID NO 49.
  • interferon-associated antigen binding protein for the use of items 95 or 96, wherein the IFNa2a comprises the sequence as set forth in SEQ ID NO 17.
  • interferon-associated antigen binding protein for the use of items 95 or 96, wherein the IFN0 comprises the sequence as set forth in SEQ ID NO 14, SEQ ID NO 15 or SEQ ID NO 16.
  • interferon-associated antigen binding protein for the use of item 98, wherein the IFN0 comprises the sequence as set forth in SEQ ID NO 14.
  • interferon-associated antigen binding protein for the use of item 98, wherein the IFN0 comprises the sequence as set forth in SEQ ID NO 15.
  • interferon-associated antigen binding protein for the use of item 98, wherein the IFN0 comprises the sequence as set forth in SEQ ID NO 16.
  • interferon-associated antigen binding protein for the use of item 95 or 96, wherein the IFNy comprises the sequence as set forth in SEQ ID NO 19.
  • interferon-associated antigen binding protein for the use of item 95 or 96, wherein the IFN 2 comprises the sequence as set forth in SEQ ID NO 18.
  • interferon-associated antigen binding protein for the use of any one of items 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.
  • interferon-associated antigen binding protein for the use of item 104, wherein the light chain comprises a sequence as set forth in SEQ ID NO 3.
  • interferon-associated antigen binding protein for the use of any one of items 49, 50 or 52 to 94, wherein the IFN or a functional fragment thereof is fused to the N-terminus of a heavy chain of the agonistic anti-CD40 antibody, or the agonistic antigen binding fragment thereof, via the linker as set forth in Table 4, in particular Table 4A or Table 4B, more particularly Table 4A.
  • the interferon-associated antigen binding protein for the use of item 106, wherein the heavy chain of the agonistic anti-CD40 antibody, or the agonistic antigen binding fragment thereof, comprises a sequence as set forth in SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO 49, SEQ ID NO 48, or SEQ ID NO 12.
  • interferon-associated antigen binding protein for the use of items 106 or 107, wherein the IFNa2a comprises the sequence as set forth in SEQ ID NO
  • interferon-associated antigen binding protein for the use of items 106 or 107, wherein the IFNP comprises the sequence as set forth in SEQ ID NO 14, SEQ ID NO 15 or SEQ ID NO 16.
  • interferon-associated antigen binding protein for the use of item 109, wherein the IFNP comprises the sequence as set forth in SEQ ID NO 14.
  • interferon-associated antigen binding protein for the use of item 109, wherein the IFNP comprises the sequence as set forth in SEQ ID NO 15.
  • interferon-associated antigen binding protein for the use of item 109, wherein the IFNP comprises the sequence as set forth in SEQ ID NO 16.
  • interferon-associated antigen binding protein for the use of items 106 or 107, wherein the IFNy comprises the sequence as set forth in SEQ ID NO 19.
  • interferon-associated antigen binding protein for the use of items 106 or 107, wherein the IFNX2 comprises the sequence as set forth in SEQ ID NO
  • interferon-associated antigen binding protein for the use of any one of items 106 to 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.
  • interferon-associated antigen binding protein for the use of item 115, wherein the light chain comprises a sequence as set forth in SEQ ID NO 3.
  • interferon-associated antigen binding protein for the use of any one of items 49, 50 or 52 to 94, wherein the IFN or a functional fragment thereof is fused to the C-terminus of a light chain of the agonistic anti-CD40 antibody, or the agonistic antigen binding fragment thereof, via the linker as set forth in Table 5, in particular Table 5A or Table 5B, more particularly Table 5A.
  • the interferon-associated antigen binding protein for the use of item 117, wherein the light chain of the agonistic anti-CD40 antibody, or the agonistic antigen binding fragment thereof, comprises a sequence as set forth in SEQ ID NO 3.
  • interferon-associated antigen binding protein for the use of items 117 or 118, wherein the IFNa2a comprises the sequence as set forth in SEQ ID NO 17.
  • interferon-associated antigen binding protein for the use of items 117 or 118, wherein the IFNP comprises the sequence as set forth in SEQ ID NO 14, SEQ ID NO 15 or SEQ ID NO 16.
  • interferon-associated antigen binding protein for the use of item 120, wherein the IFNP comprises the sequence as set forth in SEQ ID NO 14.
  • interferon-associated antigen binding protein for the use of item 120, wherein the IFN comprises the sequence as set forth in SEQ ID NO 15.
  • interferon-associated antigen binding protein for the use of item 120, wherein the IFNP comprises the sequence as set forth in SEQ ID NO 16.
  • interferon-associated antigen binding protein for the use of item 131 wherein the IFNP comprises the sequence as set forth in SEQ ID NO 15. 134.
  • interferon-associated antigen binding protein for the use of items 128 or 129, wherein the IFNy comprises the sequence as set forth in SEQ ID NO 19.
  • interferon-associated antigen binding protein for the use of items 128 or 129, wherein the IFNA.2 comprises the sequence as set forth in SEQ ID NO 18.
  • interferon-associated antigen binding protein for the use of any one of items 128 to 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.
  • interferon-associated antigen binding protein for the use of item 137, wherein the heavy chain of the agonistic anti-CD40 antibody comprises a sequence as set forth in SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO 49, SEQ ID NO 48, or SEQ ID NO 12.
  • interferon-associated antigen binding protein for the use of any one of items 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 NO 46, SEQ ID NO 47, 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, SEQ ID NO 88 and SEQ ID NO 90.
  • interferon-associated antigen binding protein for the use of item 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.
  • interferon-associated antigen binding protein for the use of items 139 or 140, wherein the interferon-associated antigen binding protein is an interferon-fused agonistic anti-CD40 antibody or an interferon-fused agonistic antigen binding fragment thereof comprising one of the sequence combinations disclosed in Table 9, in particular Table 9A or Table 9B, more particularly Table 9A.
  • the interferon-associated antigen binding protein for the use of item 141, wherein the interferon-associated antigen binding protein is an interferon- fused agonistic anti-CD40 antibody or an interferon-fused agonistic antigen binding fragment thereof comprising the sequences as set forth in SEQ ID NO 38 and SEQ ID NO 3.
  • interferon-associated antigen binding protein for the use of item 141, wherein the interferon-associated antigen binding protein is an interferon- fused agonistic anti-CD40 antibody or an interferon-fused agonistic antigen binding fragment thereof comprising the sequences as set forth in SEQ ID NO 39 and SEQ ID NO 3.
  • the interferon-associated antigen binding protein for the use of item 141, wherein the interferon-associated antigen binding protein is an interferon- fused agonistic anti-CD40 antibody or an interferon-fused agonistic antigen binding fragment thereof comprising the sequences as set forth in SEQ ID NO 40 and SEQ ID NO 3.
  • interferon-associated antigen binding protein for the use of item 141, wherein the interferon-associated antigen binding protein is an interferon- fused agonistic anti-CD40 antibody or an interferon-fused agonistic antigen binding fragment thereof comprising the sequences as set forth in SEQ ID NO 41 and SEQ ID NO 9.
  • interferon-associated antigen binding protein for the use of item 141, wherein the interferon-associated antigen binding protein is an interferon- fused agonistic anti-CD40 antibody or an interferon-fused agonistic antigen binding fragment thereof comprising the sequences as set forth in SEQ ID NO 43 and SEQ ID NO 9. 148.
  • the interferon-associated antigen binding protein for the use of any one of items 1 to 147, wherein the interferon-associated antigen binding protein activates both the CD40 and an IFN pathway.
  • interferon-associated antigen binding protein for the use of item 148, wherein CD40 activity is determined using a whole blood surface molecule upregulation assay or an in vitro reporter cell assay.
  • interferon-associated antigen binding protein for the use of item 149, wherein CD40 activity is determined using an in vitro reporter cell assay, optionally using HEK-BlueTM CD40L cells.
  • interferon-associated antigen binding protein for the use of any one of items 148 to 150, wherein the interferon-associated antigen binding protein activates the CD40 pathway with an ECso of less than 400, 300, 200, 150, 100, 70, 60, 50, 40, 30, 25, 20, or 15 ng/mL.
  • interferon-associated antigen binding protein for the use of item 151, wherein the interferon-associated antigen binding protein activates the CD40 pathway with an ECso ranging from 10 to 200 ng/mL.
  • interferon-associated antigen binding protein for the use of item 152, wherein the interferon-associated antigen binding protein activates the CD40 pathway with an EC so ranging from 10 to 50 ng/mL, preferably 10 to 30 ng/mL.
  • interferon-associated antigen binding protein for the use of any one of items 148 to 153, wherein the interferon-associated antigen binding protein activates the IFN pathway with an ECso of less than 100, 60, 50, 40, 30, 20, 10, or 1 ng/mL.
  • interferon-associated antigen binding protein for the use of any one of items 148 to 154, wherein the interferon-associated antigen binding protein activates the IFN pathway with an ECso of less than 11 ng/mL, preferably less than 6 ng/mL.
  • interferon-associated antigen binding protein for the use of any one of items 148 to 155, wherein the IFN pathway is the IFNa, IFN0, IFNE, IFNy, IFN® or IFNA, pathway.
  • IFN0 activity is determined using an in vitro reporter cell assay, optionally using HEK-BlueTM IFN-a/0 cells.
  • interferon-associated antigen binding protein for the use of item 156, wherein IFNa activity is determined using an in vitro reporter cell assay, optionally using HEK-BlueTM IFN-a/0 cells.
  • interferon-associated antigen binding protein for the use of item 156, wherein IFNy activity is determined using an in vitro reporter cell assay, optionally using HEK-BlueTM Dual IFN-y cells.
  • interferon-associated antigen binding protein for the use of item 156, wherein IFNA. activity is determined using an in vitro reporter cell assay, optionally using HEK-BlueTM IFN-X cells.
  • interferon-associated antigen binding protein for the use of any one of the preceding items, in particular items 148 to 160, wherein the expression level of one or more IFN pathway biomarkers is upregulated in a Hepatitis delta virus-infected cell upon treatment with the interferon-associated antigen binding protein, preferably at least 1.5-fold, more preferably at least 2-fold, most preferably at least 3 -fold, as compared to the expression level of said biomarkers in said Hepatitis delta virus-infected cell that has not been treated with the interferon-associated antigen binding protein.
  • interferon-associated antigen binding protein for the use of item 161, wherein the IFN pathway biomarker is a chemokine.
  • interferon-associated antigen binding protein for the use of item 162, wherein the IFN pathway biomarker is the interferon stimulated gene ISG20.
  • interferon-associated antigen binding protein for the use of item 162, wherein the IFN pathway biomarker is a C-X-C chemokine, selected from the group consisting of CXCL9, CXCL10 and CXCL11.
  • interferon-associated antigen binding protein for the use of item 164, wherein the IFN pathway biomarker is CXCL10.
  • interferon-associated antigen binding protein for the use of any one of the preceding items, in particular items 148 to 165, wherein the expression level of one or more of IL 10, IL 10 and IL2 is not significantly upregulated in a Hepatitis delta virus-infected cell upon treatment with the interferon- associated antigen binding protein, as compared to the expression level of said interleukins in said Hepatitis delta virus-infected cell that has not been treated with the interferon-associated antigen binding protein.
  • interferon-associated antigen binding protein for the use of any one of the preceding items, wherein the systemic exposure of the interferon-associated antigen binding protein is increased compared to antibody CP870,893, preferably by at least 10%, more preferably by at least 15%, most preferably by at least 25%.
  • interferon-associated antigen binding protein for the use of any one of the preceding items, wherein the systemic exposure of the interferon-associated antigen binding protein is at least 1000 pg*h/mL.
  • interferon-associated antigen binding protein for the use of item 168, wherein the systemic exposure of the interferon-associated antigen binding protein ranges from 1033 pg*h/mL to 1793 pg*h/mL.
  • interferon-associated antigen binding protein for the use of any one of the preceding items, wherein the half-life of the interferon-associated antigen binding protein is at least 100 h.
  • interferon-associated antigen binding protein for the use of item 170, wherein the half-life of the interferon-associated antigen binding protein ranges from 116 to 158 h.
  • interferon-associated antigen binding protein for the use of any one of the preceding items, wherein the clearance rate of the interferon-associated antigen binding protein is below 0.5 mL/h/kg.
  • interferon-associated antigen binding protein for the use of item 172, wherein the clearance of the interferon-associated antigen binding protein ranges from 0.28 to 0.49 mL/h/kg.
  • interferon-associated antigen binding protein for the use of any one of items 1 to 173, wherein the volume of distribution Vss of the interferon- associated antigen binding protein is below 100 mL/kg. 175.
  • interferon-associated antigen binding protein for the use of any one of the preceding items, wherein the use comprises administering the interferon- associated antigen binding protein to a subject in need thereof by means of genetic delivery with RNA or DNA sequences encoding the interferon- associated antigen binding protein, or a vector or vector system encoding the interferon-associated antigen binding protein.
  • interferon-associated antigen binding protein for the use of any one of items 1 to 176, wherein the interferon-associated antigen binding protein is comprised in a pharmaceutical composition.
  • interferon-associated antigen binding protein for the use of item 177, wherein the pharmaceutical composition is suitable for oral, parenteral, or topical administration or for administration by inhalation.
  • interferon-associated antigen binding protein for the use of item 178, wherein the pharmaceutical composition is suitable for oral administration.
  • interferon-associated antigen binding protein for the use of item 178, wherein the pharmaceutical composition is suitable for topical administration.
  • interferon-associated antigen binding protein for the use of item 178, wherein the pharmaceutical composition is suitable for administration by inhalation.
  • interferon-associated antigen binding protein for the use of item 178, wherein the pharmaceutical composition is suitable for parenteral administration.
  • the interferon-associated antigen binding protein for the use of any one of items 177 to 184, wherein the pharmaceutical composition comprises at least one buffering agent.
  • the interferon-associated antigen binding protein for the use of item 190 wherein the surfactant is selected from the list comprising pluronics, PEG, sorbitan esters, polysorbates, triton, tromethamine, lecithin, cholesterol and tyloxapal.
  • interferon-associated antigen binding protein for the use of item 193, wherein the surfactant is polysorbate 80.
  • the interferon-associated antigen binding protein for the use according to any one of items 1 to 196, wherein the agonistic anti-CD40 antibody or agonistic antigen binding fragment thereof comprises three light chain complementarity determining regions (CDRs) CDRL1, CDRL2 and CDRL3 that are at least 90% identical to the respective CDRL1, CDRL2 and CDRL3 sequences within SEQ ID NO 3; and three heavy chain CDRs, CDRH1, CDRH2 and CDRH3, that are at least 90% identical to the respective CDRH1, CDRH2 and CDRH3 sequences within SEQ ID NO 6.
  • CDRs light chain complementarity determining regions
  • the interferon-associated antigen binding protein for the use according to any one of items 1 to 197, wherein the agonistic anti-CD40 antibody or agonistic antigen binding fragment thereof comprises three light chain complementarity determining regions (CDRs) CDRL1, CDRL2 and CDRL3 that are identical to the respective CDRL1, CDRL2 and CDRL3 sequences within SEQ ID NO 3; and three heavy chain CDRs, CDRH1, CDRH2 and CDRH3, that are identical to the respective CDRH1, CDRH2 and CDRH3 sequences within SEQ ID NO 6.
  • CDRs light chain complementarity determining regions
  • each CDR is defined in accordance with the Kabat definition, the Chothia definition, the AbM definition, or the contact definition of CDR; preferably wherein each CDR is defined in accordance with the CDR definition of Kabat or the CDR definition of Chothia.
  • the interferon-associated antigen binding protein for its use according to any one of items 2 to 199, wherein no amino acid substitutions, insertions or deletions are present within the complementarity determining regions of the heavy chain and light chain variable regions of the agonistic anti-CD40 antibody or agonistic antigen binding fragment thereof.
  • the interferon-associated antigen binding protein for its use according to any one of items 2 to 199, wherein no, one or two amino acid substitutions, insertions or deletions are independently present within each of the complementarity determining regions of the heavy chain and light chain variable regions of the agonistic anti-CD40 antibody or agonistic antigen binding fragment thereof.
  • interferon-associated antigen binding protein or the polynucleotide or polynucleotides for their use according to any one of items 2 to 204, wherein the interferon-associated antigen binding protein, or the polynucleotide or polynucleotides are to be administered in addition to a host targeting agent.
  • a cluster of differentiation factor 40 (CD40) agonist or a functional fragment thereof for use in the treatment or prevention of a Hepatitis delta virus infection, wherein the CD40 agonist or a functional fragment thereof is administered in combination with an interferon (IFN) or a functional fragment thereof.
  • IFN interferon
  • IFN interferon
  • CD40 cluster of differentiation factor 40
  • CD40 cluster of differentiation factor 40
  • IFN interferon
  • CD40 agonist or functional fragment thereof the IFN or functional fragment thereof, or the combination for their use according to any one of the preceding matters, wherein the CD40 agonist or functional fragment thereof is a polypeptide or functional fragment thereof, or an antibody or functional fragment thereof.
  • the agonistic anti-CD40 antibody or agonistic antigen binding fragment thereof comprises three light chain complementarity determining regions (CDRs) CDRL1, CDRL2 and CDRL3 that are identical to the respective CDRL1, CDRL2 and CDRL3 sequences within SEQ ID NO 3; and three heavy chain CDRs, CDRH1, CDRH2 and CDRH3, that are identical to the respective CDRH1, CDRH2 and CDRH3 sequences within SEQ ID NO
  • CDR complementarity determining region
  • a light chain or a fragment thereof comprising a CDRL1 that is at least 90% identical to SEQ ID NO 52, a CDRL2 that is at least 90% identical to SEQ ID NO 53, and a CDRL3 that is at least 90% identical to SEQ ID NO 54; preferably (II)
  • CDR complementarity determining region
  • a light chain or a fragment thereof comprising a CDRL1 that is at least 95% identical to SEQ ID NO 52, a CDRL2 that is at least 95% identical to SEQ ID NO 53, and a CDRL3 that is at least 95% identical to SEQ ID NO 54; more preferably (III)
  • CDR complementarity determining region
  • CDR complementarity determining region
  • a light chain or a fragment thereof comprising a CDRL1 that is at least 99% identical to SEQ ID NO 52, a CDRL2 that is at least 99% identical to SEQ ID NO 53, and a CDRL3 that is at least 99% identical to SEQ ID NO 54.
  • a heavy chain or a fragment thereof comprising a complementarity determining region (CDR) CDRH1 that is identical to SEQ ID NO 56, a CDRH2 that is identical to SEQ ID NO 57, and a CDRH3 that is identical to SEQ ID NO 58; and b. a light chain or a fragment thereof comprising a CDRL1 that is identical to SEQ ID NO 52, a CDRL2 that is identical to SEQ ID NO 53, and a CDRL3 that is identical to SEQ ID NO 54.
  • CDR complementarity determining region
  • the agonistic anti-CD40 antibody or agonistic antigen binding fragment thereof comprises a light chain (LC) that comprises a sequence as set forth in SEQ ID NO 3, or a sequence at least 90% identical thereto; and/or a heavy chain (HC) that comprises a sequence selected from the group consisting of SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO 49 and SEQ ID NO 48, or a sequence at least 90% identical thereto preferably wherein the agonistic anti-CD40 antibody or agonistic antigen binding fragment thereof comprises a light chain (LC) that comprises a sequence that is at least 95% identical to the sequence as set forth in SEQ ID NO 3; and/or a heavy chain (HC) that comprises a sequence that is at least 95% identical to a sequence as set forth within the group of sequences consisting of SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO 49 and SEQ ID NO 48
  • the CD40 agonist or functional fragment thereof, the IFN or functional fragment thereof, or the combination for their use according to matter 31, wherein the IFNa2a comprises the sequence as set forth in SEQ ID NO 17, or a sequence at least 90%, preferably 95%, more preferably 98%, or still more preferably 99% identical thereto.
  • a heavy chain or a fragment thereof comprising a complementarity determining region (CDR) CDRH1 that is identical to SEQ ID NO 56, a CDRH2 that is identical to SEQ ID NO 57, and a CDRH3 that is identical to SEQ ID NO 58; and b. a light chain or a fragment thereof comprising a CDRL1 that is identical to SEQ ID NO 52, a CDRL2 that is identical to SEQ ID NO 53, and a CDRL3 that is identical to SEQ ID NO 54; and wherein the IFN or a functional fragment thereof is fused to said antibody or antigen binding fragment thereof.
  • CDR complementarity determining region
  • the CD40 agonist or functional fragment thereof, the IFN or functional fragment thereof, or the combination for their use according to matter 83, wherein the heavy chain of the agonistic anti-CD40 antibody, or the agonistic antigen binding fragment thereof, comprises a sequence as set forth in SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO 12, SEQ ID NO 48 or SEQ ID NO 49.
  • the CD40 agonist or functional fragment thereof, the IFN or functional fragment thereof, or the combination for their use according to matter 103, wherein the light chain of the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof, comprises a sequence as set forth in SEQ ID NO 3.
  • the CD40 agonist or functional fragment thereof, the IFN or functional fragment thereof, or the combination for their use according to matter 113, wherein the light chain of the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof, comprises a sequence as set forth in SEQ ID NO 3.
  • the interferon-associated antigen binding protein comprises a sequence selected from SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, 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, SEQ ID NO 88 and SEQ ID NO 90.
  • An interferon-associated antigen binding protein comprising
  • Interferon an Interferon (IFN) or a functional fragment thereof for use in the treatment or prevention of a Hepatitis delta virus infection.
  • CDR complementarity determining region
  • a light chain or a fragment thereof comprising a CDRL1 that is at least 90% identical to SEQ ID NO 52, a CDRL2 that is at least 90% identical to SEQ ID NO 53, and a CDRL3 that is at least 90% identical to SEQ ID NO 54.
  • CDR complementarity determining region
  • the interferon-associated antigen binding protein for the use of any one of matters 132 to 134, wherein the agonistic anti-CD40 antibody, or the agonistic antigen binding fragment thereof, comprises a light chain variable region VL comprising the sequence as set forth in SEQ ID NO 51, or a sequence at least 90% identical thereto; and/or a heavy chain variable region VH comprising the sequence as set forth in SEQ ID NO 55, or a sequence at least 90% identical thereto. 136.
  • the interferon-associated antigen binding protein for the use of any one of matters 132 to 135, wherein the agonistic anti-CD40 antibody or the agonistic antigen binding fragment thereof comprises a light chain (LC) that comprises a sequence as set forth in SEQ ID NO 3, or a sequence at least 90% identical thereto; and/or a heavy chain (HC) that comprises a sequence selected from the group consisting of SEQ ID NO 6, SEQ ID NO 9, SEQ ID NO 12, SEQ ID NO 49 and SEQ ID NO 48, or a sequence at least 90% identical thereto.
  • LC light chain
  • HC heavy chain
  • interferon-associated antigen binding protein for the use of any one of matters 132 to 136, wherein the IFN or the functional fragment thereof is selected from the group consisting of a Type I IFN, a Type II IFN and a Type III IFN, or a functional fragment thereof.
  • interferon-associated antigen binding protein for the use of matter 137, wherein the type I IFN or the functional fragment thereof is IFNa or IFNp, or a functional fragment thereof.
  • interferon-associated antigen binding protein for the use of any one of matters 132 to 138, wherein the IFN or the functional fragment thereof is IFNa2a, or a functional fragment thereof, and wherein preferably the IFNa2a comprises the sequence as set forth in SEQ ID NO 17, or a sequence at least 90% identical thereto.
  • interferon-associated antigen binding protein for the use of any one of matters 132 to 138, wherein the IFN or the functional fragment thereof is IFNP or a functional fragment thereof, and wherein preferably the IFNP comprises the sequence as set forth in SEQ ID NO 14, or a sequence at least 90% identical thereto.
  • interferon-associated antigen binding protein for the use of any one of matters 132 to 142, wherein the agonistic anti-CD40 antibody or an agonistic antigen binding fragment thereof, and the IFN or the functional fragment thereof, are fused to each other via a linker, and wherein preferably the linker comprises a sequence as set forth in SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 24, SEQ ID NO 25 or SEQ ID NO 26.
  • interferon-associated antigen binding protein for the use of any one of matters 132 to 143, wherein the interferon-associated antigen binding protein is an interferon-fused agonistic anti-CD40 antibody or an interferon-fused agonistic antigen binding fragment thereof comprising one of the sequence combinations disclosed in Table 9, in particular Table 9A or Table 9B, more particularly Table 9A.
  • interferon-associated antigen binding protein for the use of any one of matters 132 to 144, wherein the use comprises administering the interferon- associated antigen binding protein to a subject in need thereof by means of genetic delivery with RNA or DNA sequences encoding the interferon- associated antigen binding protein, or a vector or vector system encoding the interferon-associated antigen binding protein.
  • RNA or DNA sequences encoding said interferon-associated antigen binding protein, or the vector or vector system encoding said interferon-associated antigen binding protein are comprised in a pharmaceutical composition.
  • An interferon-associated antigen binding protein comprising an interferon- fused agonistic anti-CD40 antibody or interferon-fused agonistic antigen binding fragment thereof comprising the amino acid sequences as set forth in SEQ ID NO 38 and SEQ ID NO 3, for use in the treatment or prevention of a Hepatitis delta virus infection.
  • An interferon-associated antigen binding protein comprising an interferon- fused agonistic anti-CD40 antibody or interferon-fused agonistic antigen binding fragment thereof comprising the amino acid sequences as set forth in SEQ ID NO 39 and SEQ ID NO 3, for use in the treatment or prevention of a Hepatitis delta virus infection.
  • An interferon-associated antigen binding protein comprising an interferon- fused agonistic anti-CD40 antibody or interferon-fused agonistic antigen binding fragment thereof comprising the amino acid sequences as set forth in SEQ ID NO 40 and SEQ ID NO 3, for use in the treatment or prevention of a Hepatitis delta virus infection.
  • An interferon-associated antigen binding protein comprising an interferon- fused agonistic anti-CD40 antibody or interferon-fused agonistic antigen binding fragment thereof comprising the amino acid sequences as set forth in SEQ ID NO 41 and SEQ ID NO 9, for use in the treatment or prevention of a Hepatitis delta virus infection.
  • An interferon-associated antigen binding protein comprising an interferon- fused agonistic anti-CD40 antibody or interferon-fused agonistic antigen binding fragment thereof comprising the amino acid sequences as set forth in SEQ ID NO 42 and SEQ ID NO 9, for use in the treatment or prevention of a Hepatitis delta virus infection.
  • An interferon-associated antigen binding protein comprising an interferon- fused agonistic anti-CD40 antibody or interferon-fused agonistic antigen binding fragment thereof comprising the amino acid sequences as set forth in SEQ ID NO 43 and SEQ ID NO 9, for use in the treatment or prevention of a Hepatitis delta virus infection.
  • the CD40 agonist or functional fragment thereof, the IFN or functional fragment thereof, the combination, or the interferon-associated antigen binding protein for their use according to any one of matters 153 to 155, wherein the interferon-associated antigen binding protein activates the CD40 pathway with an ECso of less than 400, 300, 200, 150, 100, 70, 60, 50, 40, 30, 25, 20, or 15 ng/mL.
  • the CD40 agonist or functional fragment thereof, the IFN or functional fragment thereof, the combination, or the interferon-associated antigen binding protein for their use according to any one of matters 1 to 165, wherein the expression level of one or more IFN pathway biomarkers is upregulated in a Hepatitis delta virus-infected cell upon treatment with the interferon-associated antigen binding protein, preferably at least 1.5-fold, more preferably at least 2-fold, most preferably at least 3 -fold, as compared to the expression level of said biomarkers in said Hepatitis delta virus-infected cell that has not been treated with the interferon-associated antigen binding protein.
  • RNA or DNA sequences encoding said CD40 agonist or functional fragment thereof, said IFN or functional fragment thereof, said combination or said interferon-associated antigen binding protein; or b. a vector or vector system encoding said CD40 agonist or functional fragment thereof, said IFN or functional fragment thereof, said combination or said interferon-associated antigen binding protein.
  • the CD40 agonist or functional fragment thereof, the IFN or functional fragment thereof, the combination, or the interferon-associated antigen binding protein for their use according to matter 181, wherein said interferon- associated antigen binding protein is expressed in said subject from a polynucleotide or polynucleotides administered to said subject.
  • the CD40 agonist or functional fragment thereof, the IFN or functional fragment thereof, the combination, or the interferon-associated antigen binding protein for their use according to any one of matters 1 to 182, wherein the interferon-associated antigen binding protein is comprised in a pharmaceutical composition.
  • CD40 agonist or functional fragment thereof, the IFN or functional fragment thereof, the combination, or the interferon-associated antigen binding protein for their use according to matter 183 or matter 184, wherein the pharmaceutical composition is suitable for oral, parenteral, or topical administration or for administration by inhalation.
  • CD40 agonist or functional fragment thereof, the IFN or functional fragment thereof, the combination, or the interferon-associated antigen binding protein for their use according to matter 185, wherein the pharmaceutical composition is suitable for oral administration.
  • CD40 agonist or functional fragment thereof, the IFN or functional fragment thereof, the combination, or the interferon-associated antigen binding protein for their use according to matter 185, wherein the pharmaceutical composition is suitable for administration by inhalation.
  • CD40 agonist or functional fragment thereof, the IFN or functional fragment thereof, the combination, or the interferon-associated antigen binding protein for their use according to matter 183 or matter 184, wherein the pharmaceutical composition is suitable for injection, preferably for intravenous or intraarterial injection or drip.
  • CD40 agonist or functional fragment thereof, the IFN or functional fragment thereof, the combination, or the interferon-associated antigen binding protein for their use according to any one of matters 183 to 196, wherein the pharmaceutical composition comprises a surfactant.
  • CD40 agonist or functional fragment thereof, the IFN or functional fragment thereof, the combination, or the interferon-associated antigen binding protein for their use according to matter 199, wherein the surfactant is polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80 or polysorbate 100.
  • the interferon-associated antigen binding protein for its use according to any one of matters 132 to 203, wherein the agonistic anti-CD40 antibody or agonistic antigen binding fragment thereof comprises three light chain complementarity determining regions (CDRs) CDRL1, CDRL2 and CDRL3 that are at least 90% identical to the respective CDRL1, CDRL2 and CDRL3 sequences within SEQ ID NO 3; and three heavy chain CDRs, CDRH1, CDRH2 and CDRH3, that are at least 90% identical to the respective CDRH1, CDRH2 and CDRH3 sequences within SEQ ID NO 6.
  • CDRs light chain complementarity determining regions
  • the interferon-associated antigen binding protein for its use according to any one of matters 132 to 204, wherein the agonistic anti-CD40 antibody or agonistic antigen binding fragment thereof comprises three light chain complementarity determining regions (CDRs) CDRL1, CDRL2 and CDRL3 that are identical to the respective CDRL1, CDRL2 and CDRL3 sequences within SEQ ID NO 3; and three heavy chain CDRs, CDRH1, CDRH2 and CDRH3, that are identical to the respective CDRH1, CDRH2 and CDRH3 sequences within SEQ ID NO 6.
  • CDRs light chain complementarity determining regions
  • each CDR is defined in accordance with the Kabat definition, the Chothia definition, the AbM definition, or the contact definition of CDR; preferably wherein each CDR is defined in accordance with the CDR definition of Kabat or the CDR definition of Chothia.
  • interferon-associated antigen binding protein for its use according to any one of matters 132 to 206, wherein no, one or two amino acid substitutions, insertions or deletions are independently present within each of the complementarity determining regions of the heavy chain and light chain variable regions of the agonistic anti-CD40 antibody or agonistic antigen binding fragment thereof.
  • the CD40 agonist or functional fragment thereof, the IFN or functional fragment thereof, the combination, the interferon-associated antigen binding protein, or the polynucleotide or polynucleotides for their use according to any one of matters 1 to 211, wherein the CD40 agonist or functional fragment thereof, the IFN or functional fragment thereof, the combination, the interferon-associated antigen binding protein, or the polynucleotide or polynucleotides are to be administered in addition to a host targeting agent.
  • the CD40 agonist or functional fragment thereof, the IFN or functional fragment thereof, the combination, the interferon-associated antigen binding protein, or the polynucleotide or polynucleotides for their use according to any one of matters 1 to 211, wherein the CD40 agonist or functional fragment thereof, the IFN or functional fragment thereof, the combination, the interferon-associated antigen binding protein, or the polynucleotide or polynucleotides are to be administered in addition to a direct acting antiviral agent.
  • the present invention is also directed to a CD40 agonist or functional fragment thereof, an IFN or functional fragment thereof, a combination of the two, an interferon-associated antigen binding protein or a polynucleotide or polynucleotides for their use according to any one of the matters or items identified herein, wherein the CD40 agonist comprises SEQ ID NO 59, SEQ ID NO 61 or SEQ ID NO 63 as disclosed in Table 8 and/or wherein the interferon-associated binding protein is an interferon-fused agonistic anti-CD40 antibody comprising one of the sequence combinations disclosed in Table 10, or an interferon-fused agonistic antigen binding fragment thereof.
  • the present invention is furthermore also directed to methods for the treatment or prevention of a Hepatitis delta virus infection in a subject, in particular a Hepatitis delta virus infection as identified in matters 210 and 211, wherein a therapeutically effective amount of the CD40 agonist or functional fragment thereof, the IFN or functional fragment thereof, the combination of the two, the interferon-associated antigen binding protein or the polynucleotide or polynucleotides as referred to in any one of the aspects, embodiments, matters or items identified herein, or in the preceding paragraph, is administered to said subject.
  • the subject to be treated is preferably a mammal, and most preferably a human subject.

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Abstract

La présente invention concerne des méthodes de traitement ou de prévention d'une infection par le virus de l'hépatite delta chez un patient par administration d'une association d'un agoniste du cluster de différenciation 40 (CD40) et un d'un interféron (IFN), par exemple une protéine de liaison à l'antigène associée à l'IFN, telle qu'un anticorps fusionné à l'IFN ou des acides nucléiques et des vecteurs d'expression codant pour ceux-ci. La présente invention concerne également l'utilisation de compositions pharmaceutiques correspondantes pour une utilisation dans le traitement d'une infection par le virus de l'hépatite delta.
PCT/EP2024/059835 2023-04-12 2024-04-11 Protéines de liaison à l'antigène associées à l'interféron destinées à être utilisées dans le traitement ou la prévention d'une infection par le virus de l'hépatite delta WO2024213635A1 (fr)

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Citations (34)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4179337A (en) 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
US4301144A (en) 1979-07-11 1981-11-17 Ajinomoto Company, Incorporated Blood substitute containing modified hemoglobin
US4496689A (en) 1983-12-27 1985-01-29 Miles Laboratories, Inc. Covalently attached complex of alpha-1-proteinase inhibitor with a water soluble polymer
US4640835A (en) 1981-10-30 1987-02-03 Nippon Chemiphar Company, Ltd. Plasminogen activator derivatives
US4670417A (en) 1985-06-19 1987-06-02 Ajinomoto Co., Inc. Hemoglobin combined with a poly(alkylene oxide)
WO1988001649A1 (fr) 1986-09-02 1988-03-10 Genex Corporation Molecules de liaison de chaines de polypeptide simples
US4791192A (en) 1986-06-26 1988-12-13 Takeda Chemical Industries, Ltd. Chemically modified protein with polyethyleneglycol
WO1989002468A1 (fr) 1987-09-11 1989-03-23 Whitehead Institute For Biomedical Research Fibroblastes transduits et leurs applications
WO1989005345A1 (fr) 1987-12-11 1989-06-15 Whitehead Institute For Biomedical Research Modification genetique de cellules endotheliales
WO1989007136A2 (fr) 1988-02-05 1989-08-10 Whitehead Institute For Biomedical Research Hepatocytes modifies et leurs utilisations
US4868116A (en) 1985-07-05 1989-09-19 Whitehead Institute For Biomedical Research Introduction and expression of foreign genetic material in epithelial cells
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
US4980286A (en) 1985-07-05 1990-12-25 Whitehead Institute For Biomedical Research In vivo introduction and expression of foreign genetic material in epithelial cells
WO1992007573A1 (fr) 1990-10-31 1992-05-14 Somatix Therapy Corporation Modification genetique de cellules endotheliales
US5260203A (en) 1986-09-02 1993-11-09 Enzon, Inc. Single polypeptide chain binding molecules
US5328470A (en) 1989-03-31 1994-07-12 The Regents Of The University Of Michigan Treatment of diseases by site-specific instillation of cells or site-specific transformation of cells and kits therefor
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US5565332A (en) 1991-09-23 1996-10-15 Medical Research Council Production of chimeric antibodies - a combinatorial approach
US5741957A (en) 1989-12-01 1998-04-21 Pharming B.V. Transgenic bovine
US5750172A (en) 1987-06-23 1998-05-12 Pharming B.V. Transgenic non human mammal milk
US5756687A (en) 1993-03-09 1998-05-26 Genzyme Transgenics Corporation Isolation of components of interest from milk
US5827690A (en) 1993-12-20 1998-10-27 Genzyme Transgenics Corporatiion Transgenic production of antibodies in milk
US5859205A (en) 1989-12-21 1999-01-12 Celltech Limited Humanised antibodies
US6054297A (en) 1991-06-14 2000-04-25 Genentech, Inc. Humanized antibodies and methods for making them
US6133426A (en) 1997-02-21 2000-10-17 Genentech, Inc. Humanized anti-IL-8 monoclonal antibodies
US6193980B1 (en) 1995-12-06 2001-02-27 Cambridge University Technical Services, Limited Replication defective herpes simplex virus comprising heterologous inserts
US6417429B1 (en) 1989-10-27 2002-07-09 The Scripps Research Institute Transgenic plants expressing assembled secretory antibodies
US6517529B1 (en) 1999-11-24 2003-02-11 Radius International Limited Partnership Hemodialysis catheter
WO2003040170A2 (fr) * 2001-11-09 2003-05-15 Pfizer Products Inc. Anticorps anti-cd40
WO2007130493A2 (fr) * 2006-05-03 2007-11-15 Regents Of The University Of Colorado Combinaison adjuvante synergique d'anticorps agoniste cd40/interféron de type 1, conjugués contenant une telle combinaison et utilisation en tant qu'agent thérapeutique pour améliorer l'immunité cellulaire
WO2018036852A1 (fr) * 2016-08-25 2018-03-01 F. Hoffmann-La Roche Ag Dosage intermittent d'un anticorps anti-csf-1r en combinaison avec un agent d'activation de macrophages
WO2021110562A1 (fr) * 2019-12-03 2021-06-10 Evotec International Gmbh Protéines de liaison à l'antigène associées à l'interféron et leurs utilisations
WO2021110561A1 (fr) * 2019-12-03 2021-06-10 Evotec International Gmbh Protéines de liaison à l'antigène associées à l'interféron, destinées à être utilisées dans le traitement d'infection par le virus de l'hépatite b
WO2022258720A1 (fr) * 2021-06-09 2022-12-15 Evotec International Gmbh Protéines de liaison à l'antigène associées à l'interféron destinées à être utilisées pour le traitement ou la prévention d'une infection à coronavirus

Patent Citations (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4179337A (en) 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
US4301144A (en) 1979-07-11 1981-11-17 Ajinomoto Company, Incorporated Blood substitute containing modified hemoglobin
US4640835A (en) 1981-10-30 1987-02-03 Nippon Chemiphar Company, Ltd. Plasminogen activator derivatives
US4496689A (en) 1983-12-27 1985-01-29 Miles Laboratories, Inc. Covalently attached complex of alpha-1-proteinase inhibitor with a water soluble polymer
US4670417A (en) 1985-06-19 1987-06-02 Ajinomoto Co., Inc. Hemoglobin combined with a poly(alkylene oxide)
US4868116A (en) 1985-07-05 1989-09-19 Whitehead Institute For Biomedical Research Introduction and expression of foreign genetic material in epithelial cells
US4980286A (en) 1985-07-05 1990-12-25 Whitehead Institute For Biomedical Research In vivo introduction and expression of foreign genetic material in epithelial cells
US4791192A (en) 1986-06-26 1988-12-13 Takeda Chemical Industries, Ltd. Chemically modified protein with polyethyleneglycol
WO1988001649A1 (fr) 1986-09-02 1988-03-10 Genex Corporation Molecules de liaison de chaines de polypeptide simples
US5260203A (en) 1986-09-02 1993-11-09 Enzon, Inc. Single polypeptide chain binding molecules
US5750172A (en) 1987-06-23 1998-05-12 Pharming B.V. Transgenic non human mammal milk
WO1989002468A1 (fr) 1987-09-11 1989-03-23 Whitehead Institute For Biomedical Research Fibroblastes transduits et leurs applications
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
WO1989005345A1 (fr) 1987-12-11 1989-06-15 Whitehead Institute For Biomedical Research Modification genetique de cellules endotheliales
WO1989007136A2 (fr) 1988-02-05 1989-08-10 Whitehead Institute For Biomedical Research Hepatocytes modifies et leurs utilisations
US5585089A (en) 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
US5693762A (en) 1988-12-28 1997-12-02 Protein Design Labs, Inc. Humanized immunoglobulins
US5693761A (en) 1988-12-28 1997-12-02 Protein Design Labs, Inc. Polynucleotides encoding improved humanized immunoglobulins
US6180370B1 (en) 1988-12-28 2001-01-30 Protein Design Labs, Inc. Humanized immunoglobulins and methods of making the same
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US5328470A (en) 1989-03-31 1994-07-12 The Regents Of The University Of Michigan Treatment of diseases by site-specific instillation of cells or site-specific transformation of cells and kits therefor
US6417429B1 (en) 1989-10-27 2002-07-09 The Scripps Research Institute Transgenic plants expressing assembled secretory antibodies
US5741957A (en) 1989-12-01 1998-04-21 Pharming B.V. Transgenic bovine
US5859205A (en) 1989-12-21 1999-01-12 Celltech Limited Humanised antibodies
WO1992007573A1 (fr) 1990-10-31 1992-05-14 Somatix Therapy Corporation Modification genetique de cellules endotheliales
US6054297A (en) 1991-06-14 2000-04-25 Genentech, Inc. Humanized antibodies and methods for making them
US5565332A (en) 1991-09-23 1996-10-15 Medical Research Council Production of chimeric antibodies - a combinatorial approach
US5756687A (en) 1993-03-09 1998-05-26 Genzyme Transgenics Corporation Isolation of components of interest from milk
US5827690A (en) 1993-12-20 1998-10-27 Genzyme Transgenics Corporatiion Transgenic production of antibodies in milk
US6193980B1 (en) 1995-12-06 2001-02-27 Cambridge University Technical Services, Limited Replication defective herpes simplex virus comprising heterologous inserts
US6133426A (en) 1997-02-21 2000-10-17 Genentech, Inc. Humanized anti-IL-8 monoclonal antibodies
US6517529B1 (en) 1999-11-24 2003-02-11 Radius International Limited Partnership Hemodialysis catheter
WO2003040170A2 (fr) * 2001-11-09 2003-05-15 Pfizer Products Inc. Anticorps anti-cd40
WO2007130493A2 (fr) * 2006-05-03 2007-11-15 Regents Of The University Of Colorado Combinaison adjuvante synergique d'anticorps agoniste cd40/interféron de type 1, conjugués contenant une telle combinaison et utilisation en tant qu'agent thérapeutique pour améliorer l'immunité cellulaire
WO2018036852A1 (fr) * 2016-08-25 2018-03-01 F. Hoffmann-La Roche Ag Dosage intermittent d'un anticorps anti-csf-1r en combinaison avec un agent d'activation de macrophages
WO2021110562A1 (fr) * 2019-12-03 2021-06-10 Evotec International Gmbh Protéines de liaison à l'antigène associées à l'interféron et leurs utilisations
WO2021110561A1 (fr) * 2019-12-03 2021-06-10 Evotec International Gmbh Protéines de liaison à l'antigène associées à l'interféron, destinées à être utilisées dans le traitement d'infection par le virus de l'hépatite b
WO2022258720A1 (fr) * 2021-06-09 2022-12-15 Evotec International Gmbh Protéines de liaison à l'antigène associées à l'interféron destinées à être utilisées pour le traitement ou la prévention d'une infection à coronavirus

Non-Patent Citations (75)

* Cited by examiner, † Cited by third party
Title
ARMENTANO ET AL., PROC. NATL. ACAD. SCI. USA, vol. 87, 1990, pages 6141 - 6145
BERKNER ET AL., BIOTECHNIQUES, vol. 6, 1988, pages 616
BLANK ET AL., J. HEPATOL., vol. 65, 2016, pages 483 - 489
BLIGHT ET AL., J VIROL., vol. 77, no. 5, 2003, pages 3181 - 90
BORDIER ET AL., J. VIROL., vol. 76, 2002, pages 10465 - 10472
BRANCH ET AL., SCIENCE, vol. 223, 1984, pages 450 - 455
C. SUREAU, METHODS MOL BIOL, vol. 640, 2010, pages 463 - 73
CARILLO ET AL., SIAMJ. APPLIED MATH., vol. 48, 1988, pages 1073
CHANG ET AL., J. VIROL., vol. 82, 2008, pages 1118 - 1127
CHEN ET AL., PNAS, vol. 91, 1994, pages 3054 - 3057
CHOTHIA ET AL., J. MOL. BIOL., vol. 196, 1986, pages 901 - 17
CHOTHIA ET AL., NATURE, vol. 342, 1989, pages 877 - 83
COHEN ET AL., GENE THER, vol. 7, no. 22, 2000, pages 1896 - 905
DAYHOFF ET AL., ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, vol. 5, 1978, pages 345 - 352
DEVEREUX ET AL., NUCL. ACID RES., vol. 12, 1984, pages 387
EGLITIS ET AL., SCIENCE, vol. 230, 1985, pages 1395 - 1398
FERRY ET AL., PROC. NATL. ACAD. SCI. USA, vol. 88, 1991, pages 8377 - 8381
FLOTTE ET AL., AM. J. RESPIR. CELL. MOL. BIOL., vol. 7, 1992, pages 349 - 356
FLOTTE ET AL., J. BIOL. CHEM., vol. 268, 1993, pages 3781 - 3790
GOVINDARAJAN ET AL., HEPATOLOGY, vol. 6, 1986, pages 640 - 44
GRAY ET AL., NUCLEIC ACIDS RES., vol. 43, 2015, pages D1079 - 1085
GREWALFLAVELL, ANN. REV. LMMUNOL., vol. 16, 1996, pages 111 - 35
GRIPON, J. VIROL., vol. 62, no. 11, 1988, pages 4136 - 43
HAJ-AHMANDGRAHAM, J. VIROL., vol. 57, 1986, pages 267
HE ET AL., J. MED. VIROL., vol. 27, 1989, pages 31 - 33
HEINJE, G.: "Current Protocols in Molecular Biology", 1987, JOHN WILEY & SONS, INC.
HENIKOFF ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 89, 1992, pages 10915 - 10919
HERMONAT ET AL., PROC. NATL. ACAD. SCI. USA, vol. 81, 1984, pages 6466 - 6470
HOGAN ET AL.: "Transgenic Animal Technology: A Laboratory Handbook", 1999, COLD SPRING HARBOR PRESS
HUANG CHI-RUEI ET AL: "Hepatitis D virus infection, replication and cross-talk with the hepatitis B virus", WORLD JOURNAL OF GASTROENTEROLOGY, vol. 20, no. 40, 28 October 2014 (2014-10-28), CN, pages 14589, XP093082489, ISSN: 1007-9327, DOI: 10.3748/wjg.v20.i40.14589 *
HWU ET AL., J. IMMUNOL., vol. 150, 1993, pages 4104 - 4115
JOHNSONWU, NUCLEIC ACIDS RES., vol. 28, 2000, pages 214 - 8
JONES ET AL., NATURE, vol. 321, 1986, pages 522 - 525
JONES, GENETICS, vol. 85, 1977, pages 12
JONSSON ET AL., ANN. BIOL. CLIN., vol. 51, 1993, pages 19 - 26
KAY ET AL., HUMAN GENE THERAPY, vol. 3, 1992, pages 641 - 647
KINGSMAN ET AL., GENE, vol. 7, 1979, pages 141
KUHN ET AL., ARCH. VIROL., vol. 165, 2020, pages 3023 - 3072
KYTE ET AL., J. MOL. BIOL., vol. 157, 1982, pages 105 - 131
LADNER, ANTIMICROB AGENTS CHEMOTHER, vol. 41, no. 8, 1997, pages 1715 - 20
MACCALLUM ET AL., J. MOL. BIOL., vol. 5, 1996, pages 732 - 45
MARTIN ET AL., PROC NATL ACAD SCI (USA, vol. 86, 1989, pages 9268 - 9272
MCLAUGHLIN ET AL., J. VIROL., vol. 62, 1989, pages 1963 - 1973
MEULI ET AL., J. INVEST. DERMATOL., vol. 116, no. 1, 2001, pages 131 - 135
MIAO ET AL., J. INFEC. DIS., vol. 221, 2020, pages 1677 - 1687
MICHELET ET AL., J HEP REPORT, vol. 4, no. 3, 2021, pages 100415
MILLER, BLOOD, vol. 76, 1990, pages 271 - 78
MR GREENJ. SAMBROOK: "Molecular Cloning: A Laboratory Manual", 2013, COLD SPRING HARBOR LABORATORY
MUZYCZKA ET AL., CURR. TOPICS IN MICRO. AND IMMUNOL., vol. 158, 1992, pages 97 - 129
NEEDLEMAN ET AL., J. MOL. BIOL., vol. 48, 1970, pages 443 - 453
NEGRO ET AL., COLD SPRING HARB. PERSPECT. MED., vol. 4, 2014, pages a021550
RIDGWAY, A. A. G.: "Vectors", 1988, BUTTERWORTHS, article "Mammalian Expression Vectors", pages: 470 - 472
RIECHMANN ET AL., NATURE, vol. 332, 1988, pages 323 - 327
ROMEO ET AL., GASTROENTEROLOGY, vol. 136, 2009, pages 1629 - 1638
ROSENFELD ET AL., CELL, vol. 68, 1992, pages 143 - 155
ROSENFELD ET AL., SCIENCE, vol. 252, 1991, pages 1802 - 1805
SAMUDRALA ET AL.: "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach", PROTEINS, STRUCTURE, FUNCTION AND GENETICS SUPPL., vol. 3, 1999, pages 194 - 198, XP001146416
SARACCO ET AL., J. HEPATOL., vol. 5, 1987, pages 274 - 281
SMEDILE ET AL., LANCET, vol. 2, 1982, pages 945 - 947
SMEDILE ET AL., PROG. CLIN. BIOL RES., vol. 143, 1983, pages 237 - 243
STINCHCOMB ET AL., NATURE, vol. 282, 1979, pages 39
STOCKDALE ET AL., J. HEPATOL., vol. 73, 2020, pages 523 - 532
TAM ET AL., GENE THER., vol. 7, no. 21, 2000, pages 1867 - 74
THORNTON ET AL., NATURE, vol. 354, 1991, pages 105
TOESSCHOENBERGER, SEMINARS IN IMMUNOLOGY, vol. 10, no. 6, 1998, pages 443 - 8
TRATSCHIN ET AL., J. VIROL., vol. 51, 1984, pages 611 - 619
TRATSCHIN ET AL., MOL. CELL. BIOL., vol. 4, 1985, pages 2072 - 2081
TSCHEMPER ET AL., GENE, vol. 10, 1980, pages 157
VAN BEUSECHEM ET AL., PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 10892 - 10895
VERHOEYEN ET AL., SCIENCE, vol. 239, 1988, pages 1534 - 1536
VIROLOGY, vol. 192, no. 2, 1993, pages 534 - 40
WILSON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 3014 - 3018
WINTER, FEBS LETTS., vol. 430, 1998, pages 92 - 94
WONDISFORD ET AL., MOL. ENDOCRINOL., vol. 2, 1988, pages 32 - 39
YURDAYDIN ET AL., J. VIRAL. HEPAT., vol. 17, 2010, pages 749 - 756

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