Targeting of human interferon antagonists

Abstract

The present invention relates to a fusion protein, comprising a cytokine antagonist and a targeting moiety, preferably an antibody or anti-body like molecule. In a preferred embodiment, the cytokine antagonist is a modified cytokine which binds to the receptor, but doesn't induce the receptor signalling. The invention relates further to a fusion protein according to the invention for use in treatment of cancer and immune- or inflammation-related disorders.

Claims

1. A composition comprising a fusion protein comprising an interferon antagonist and a targeting moiety, wherein: the interferon antagonist is a human IFNα2 comprising an R120E mutation which provides antagonism; and the targeting moiety comprises a variable domain of camelid heavy chain antibody (VHH) or a variable domain of new antigen receptor (VNAR) directed to CD20 which provides B cell-specific targeting of antagonistic activity.

2. The composition according to claim 1, wherein the human IFNα2 comprises a second mutation that decreases binding activity of the interferon antagonist.

3. The composition according to claim 2, wherein the second mutation is R149A.

4. A pharmaceutical composition comprising the composition according of claim 3; and a suitable excipient.

5. The composition of claim 3, further comprising a linker, connecting the interferon antagonist and a targeting moiety.

6. A pharmaceutical composition comprising the composition according of claim 5; and a suitable excipient.

7. The composition according to claim 2, wherein the second mutation is L153A.

8. A pharmaceutical composition comprising the composition according of claim 7; and a suitable excipient.

9. The composition of claim 7, further comprising a linker, connecting the interferon antagonist and a targeting moiety.

10. A pharmaceutical composition comprising the composition according of claim 9; and a suitable excipient.

11. A pharmaceutical composition comprising the composition according of claim 1; and a suitable excipient.

12. The composition of claim 1, further comprising a linker, connecting the interferon antagonist and a targeting moiety.

13. A pharmaceutical composition comprising the composition according of claim 12; and a suitable excipient.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Representation of the structural elements of the nanobody-hIFNα2-R120E fusion protein.

(2) FIG. 2: Quantification of the luciferase activity induced by 10 pM hIFNα2 in the presence or absence (untreated) of the 4-11-hIFNα2-R120E fusion protein on HL116 (A) and HL116-mLR10 (B) cells.

(3) FIG. 3: Quantification of the luciferase activity induced by 1 pM IFNβ in the presence or absence (untreated) of the 4-11-hIFNα2-R120E fusion protein on HL116 (A) and HL116-mLR10 (B) cells.

(4) FIG. 4: FACS analysis of pY701-STAT1 in CD19 positive and negative human PBMCs left untreated (left panel), treated with 50 pM of hIFNα2 (center) or with 50 pM of hIFNα2 in the presence of the CD20-targeted IFN antagonist.

(5) FIG. 5: Density of the Daudi cell cultures treated by the following components:

(6) A: Untreated

(7) B: hIFNα2. 2 pM

(8) C: hIFNα2. 2 pM+2HCD25-20xGGS-hIFNα2-R120E. 1 μg/ml

(9) D: hIFNα2. 2 pM+2HCD25-20xGGS-hIFNα2-R120E. 0.1 μg/ml

(10) E: hIFNα2. 2 pM+2HCD25-20xGGS-hIFNα2-R120E-R149A. 3 μg/ml

(11) F: hIFNα2. 2 pM+2HCD25-20xGGS-hIFNα2-R120E-R149A. 1 μg/ml

(12) G: hIFNα2. 2 pM+2HCD25-20xGGS-hIFNα2-R120E-L153A. 3 μg/ml

(13) H: hIFNα2. 2 pM+2HCD25-20xGGS-hIFNα2-R120E-L153A. 1 μg/ml

EXAMPLES

(14) Materials & Methods to the Examples

(15) Nanobody-IFN Antagonist Fusion Construction.

(16) Using the QuikChange II-E Site-Directed Mutagenesis Kit (Agilent), the mutation R120E which abrogates IFN-IFNAR1 binding and confers the antagonistic behaviour of human IFNα2 (Pan et al., 2008), (PCT/US2009/056366), was introduced into the pMET7 SIgK-HA-4.11-His-PAS-ybbr-IFNα2 construct (PCT/EP2013/050787), which is a fusion between a nanobody against the murine leptin receptor and the human IFNα2.

(17) Production of the Nanobody-IFN Antagonist Fusion Protein

(18) Hek 293T cells were transfected with the protein fusion constructs using the standard lipofectamin method (Invitrogen). 48 hours after the transfection culture mediums were harvested and stored at −20° C.

(19) Cell Lines

(20) Hek 293T cells were grown in DMEM supplemented with 10% FCS. The HL116 clone (Uze et al., 1994) is derived from the human HT1080 cell line. It contains the firefly luciferase gene controlled by the IFN-inducible 6-16 promoter. The derived HL116-mLR10 clone which expresses the murine leptin receptor was described (PCT/EP2013/050787).

(21) Measurement of the Luciferase Activities

(22) Antagonistic IFN activities were measured by quantifying the inhibition of the luciferase activity induced in HL116 cells and on the HL116-mLR10 expressing the mLR by IFNα2 or IFNβ. The IC50 values were calculated using nonlinear data regression with Prism software (GraphPad). Luciferase activities were determined on a Berthold Centro LB960 luminometer using a luciferase substrate buffer (20 mM Tricine, 1.07 mM (MgCO3)4Mg(OH)2•5H2O, 2.67 mM MgSO4•7H2O, 0.1 mM EDTA, 33.3 mM dithiothreitol, 270 μM coenzyme A, 470 μM luciferin, 530 μM ATP, final pH 7.8) after 6 hr IFN stimulation.

Example 1: The Nanobody-IFNα2-R120E Fusion Protein

(23) The nanobody 4-11, directed against the murine leptin receptor was fused to the IFNα2 mutant R120E as described in the materials and methods

(24) FIG. 1 shows a schematic representation of the nanobody-IFN antagonist fusion protein constructed with the nanobody 4-11 against the murine leptin receptor and the human IFNα2-R120E (numbering as in Piehler et al., 2000).

Example 2: Targeted Inhibition of IFNα Activity on mLR-Expressing Cells

(25) Parental HL116 cells and the derived HL116-mLR10 cells which express the mouse leptin receptor were treated for 6 hours with 10 pM IFNα2 in the presence of several dilutions of culture medium conditioned by Hek 293T cells expressing the 4-11-IFNα2-R120E fusion protein. The 10 pM IFNα2 dose was chosen because it corresponds to the IFNα2 EC50 on both cell lines. Cells were then lysed and the IFN-induced luciferase activity was quantified. At the higher concentration tested, the 4-11-IFNα2-R120E fusion protein was unable to inhibit IFNα2 action on untargeted HL116 cells (FIG. 2A). In contrast, its dose-dependent inhibition effect is clear on HL116-mLR10 cells which express the target of the 4-11 nanobody (FIG. 2B).

Example 3: Targeted Inhibition of IFNβ Activity on mLR-Expressing Cells

(26) Among the subtypes which constitute the human type I IFN, the IFNβ shows the highest affinity for the IFNα/β receptor. We thus tested whether the 4-11-IFNα2-R120E fusion protein exerts also an antagonistic activity against IFNβ action.

(27) Parental HL116 cells and the derived HL116-mLR10 cells which express the mouse leptin receptor were treated for 6 hours with 1 pM IFNβ in the presence of several dilutions of culture medium conditioned by Hek 293T cells expressing the 4-11-IFNα2-R120E fusion protein. The 1 pM IFNβ dose was chosen because it corresponds to the IFNβ EC50 on both cell lines. Cells were then lysed and the IFN-induced luciferase activity was quantified. At the higher concentration tested, the 4-11-IFNα2-R120E fusion protein was unable to inhibit IFNα2 action on untargeted HL116 cells (FIG. 3A). In contrast, its dose-dependent inhibition effect is clear on HL116-mLR10 cells which express the target of the 4-11 nanobody (FIG. 3B).

Example 4: Specific Inhibition of IFNα2-Induced STAT1 Phosphorylation in B-Cells within Human Whole PBMCs

(28) The type I IFN antagonist IFNα2-R120E was fused to the anti-human CD20 nanobody 2HCD25 through a linker sequence made with 20 repeats of GGS motif. The fusion protein was produced in E. coli and purified by Immobilized Metal Affinity chromatography (IMAC). Human peripheral blood mononuclear cells (PBMCs) are expected to contain ≈4% of B-cells which can be characterized by the cell surface expression of CD19. The large majority of circulating B-cells are also positive for the expression of CD20.

(29) PBMCs were isolated over ficoll gradient (histopaque-1077, Sigma-Aldrich) from blood samples of healthy donors. Cells were left untreated or were incubated for 15 minutes with 50 pM of human IFNα2 in the absence or presence of 10 μg/ml of the 2HCD25 nanobody—IFNα2-R120E fusion protein.

(30) Cells were then fixed (BD Fix Buffer I), permeabilized (BD Perm Buffer III) and labelled with PE-labelled anti pSTAT1 (BD#612564) and APC-labelled anti human CD19 (BD #555415). FACS data were acquired using a BD FACS Canto and analyzed using Diva (BD Biosciences) software for the fluorescence associated with pSTAT1 in CD19 positive and negative cell populations.

(31) FIG. 4 shows that the IFN antagonist linked to the nanobody specific for CD20 inhibits the IFN action specifically in the major part of the B cell population, leaving intact the IFN response in the CD19 negative cell population.

Example 5: The CD20-Targeted Type I IFN Antagonist Inhibits the Antiproliferative Activity of Type I IFN

(32) Having established that the fusion protein of the 2HCD25 nanobody and IFNα2-R120E inhibits IFN-induced STAT1 phosphorylation specifically in B-cells, we tested if it can inhibit the antiproliferative activity of type I IFN. In addition, we evaluated the effect of the IFN mutations L153A and R149A that decrease the affinity of IFNα2 for IFNAR2 by a factor of 10 and 100, respectively, in combination with the inhibiting mutation R120E.

(33) Daudi cells are a human lymphoblastoid B-cell line expressing CD20. Daudi cells were seeded at 2.0×105 cells/ml and were left untreated or cultured for 72 h in the presence of 2 pM IFNα2 alone or in combination with various CD20-targeted IFN antagonists. They were then counted to estimate the efficacy of the inhibition of proliferation induced by IFNα2. FIG. 5 shows that the CD20-targeted IFN antagonist fully inhibits the antiproliferative activity of IFNα2. It also shows that decreasing the IFN-IFNAR2 affinity decreases the antagonistic activity, proving that the inhibitory effect is indeed due to the binding of the targeted antagonist.

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