Targeted and mutated human-interferon fusion proteins

Abstract

This disclosure relates to a modified -helical bundle cytokine, with reduced activity via an -helical bundle cytokine receptor, wherein the -helical bundle cytokine is specifically delivered to target cells. Preferably, the -helical bundle cytokine is a mutant, more preferably it is a mutant interferon, with low affinity to the interferon receptor, wherein the mutant interferon is specifically delivered to target cells. The targeting is realized by fusion of the modified -helical bundle cytokine to a targeting moiety, preferably an antibody. This disclosure relates further to the use of such targeted modified -helical bundle cytokine to treat diseases. A preferred embodiment is the use of a targeted mutant interferon, to treat diseases, preferably viral diseases and tumors.

Claims

1. A composition comprising a targeting construct, wherein the targeting construct comprises: a mutated human interferon alpha 2, the mutated human interferon alpha 2 having a mutation selected from R149A, L153A, and M148A and a reduced affinity for IFNAR2 as compared to the wild-type human interferon alpha 2; and a targeting moiety, the targeting moiety comprising an antibody directed to PD-L2, CD20, Her2, or DC-STAMP, wherein the targeting moiety restores the reduced affinity of the mutated human interferon alpha 2 for IFNAR2 on targeted cells.

2. The composition of claim 1, wherein the antibody is a single-chain antibody.

3. The composition of claim 2, further comprising a linker, wherein the linker connects the mutated human interferon alpha 2 and the single-chain antibody.

4. A pharmaceutical composition comprising the targeting construct of claim 3 and a suitable excipient.

5. A pharmaceutical composition comprising the targeting construct of claim 2 and a suitable excipient.

6. The composition of claim 1, wherein the antibody is a variable domain of a camelid heavy chain antibody (VHH).

7. The composition of claim 6, further comprising a linker, wherein the linker connects the mutated human interferon alpha 2 and the variable domain of the camelid heavy chain antibody (VHH).

8. A pharmaceutical composition comprising the targeting construct of claim 7 and a suitable excipient.

9. A pharmaceutical composition comprising the targeting construct of claim 6 and a suitable excipient.

10. The composition of claim 1, further comprising a linker, wherein the linker connects the mutated human interferon alpha 2 and the targeting moiety.

11. A pharmaceutical composition comprising the targeting construct of claim 10 and a suitable excipient.

12. A pharmaceutical composition comprising the targeting construct of claim 1 and a suitable excipient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

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

(2) FIG. 2: Firefly luciferase activity induced by the indicated IFN preparation on HL116 cells (panels A and B) or HL116 cells expressing the murine leptin receptor (mLR) (panels C and D). Panels A and C on the one hand, and panels B and D on the other hand, were generated in two separate experiments. Consequently, only vertical comparison (panel A versus panel C or panel B versus panel D) is possible.

(3) FIG. 3: Renilla (light grey) and Firefly (dark grey) luciferase activity induced by the nanobody-IFN2R149A or by the IFN2 (7 pM) in a 1:1 coculture of cells expressing the leptin receptor and an IFN-inducible firefly luciferase or in cells expressing an IFN-inducible renilla luciferase but devoid of leptin receptor. Luciferase activities are expressed as a percentage of the luciferase activities induced by 3 nM IFN 2.

(4) FIGS. 4A and 4B: Activity of the purified constructs targeting the mLR: FIG. 4A, Quantification of their specific activities on cells expressing the target (HL116-mLR) or on cells lacking the target (HL116). FIG. 4B, Calculation of the targeting efficiencies of the different constructs.

(5) FIG. 5: Activity of the construct 4-11-IFNA2-R149A in presence and absence of the unconjugated leptin receptor binding nanobody. HL116 cells expressing the mLR were incubated for 6 hours with either the IFN-2 (IFNA2) or the IFNA2-R149A fused to the nanobody 4-11 (Nanobody-IFNA2-R149A) at their respective EC50 concentration in the presence or absence (control) of a 100-fold molar excess of free 4-11 nanobody.

(6) FIG. 6: Targeting the mutant IFN using the leptin binding nanobody 4-10.

(7) FIG. 7: Firefly luciferase activity induced in HL116 cells expressing the mLR by the nanobody-IFN2R149A in the presence of anti IFNAR1 monoclonal antibody 64G12 (Benoit et al., J. Immunol. 150:707-716, 1993) or anti IFNAR2 monoclonal antibody MMHAR2 (PBL Interferon Source).

(8) FIGS. 8A and 8B: Specificity of the targeting of 4-11-IFNA2-R149A to cells expressing the mLR. FIG. 8A, Cytopathic effect of the EMCV on HL116 cells (dark gray symbols) or on HL116-mLR (light grey symbols) of parental IFNA2 (upper left panel) or of the 4-11-IFNA2-R149A (lower left panel). FIG. 8B, Upper panel: calculated EC50 for antiviral activity; lower panel: calculated targeting efficiencies.

(9) FIGS. 9A and 9B: Specific activities (EC50) of IFN2 (FIG. 9A) and the nanobody-IFN2R149A (2R5A; FIG. 9B) on BXPC3 and BT474 cell lines, which express different number of Her2 molecule at their surface (10.910.sup.3 and 47810.sup.3, respectively). The ordinate scale of FIG. 9A cannot be compared to the ordinate scale of FIG. 9B.

(10) FIG. 10: Targeting of the 1R59B-IFNA2-Q124R to human Her2 expressing mouse cells. Quantification of the OASL2 mRNA expression in BTG9A cells with and without Her2 expression.

(11) FIG. 11: Targeting of mutant IFNA2 to human Her2 expressing mouse cells, using a single chain antibody. Quantification of the ISG15 mRNA expression in BTG9A cells with and without Her2 expression.

(12) FIG. 12: Control of the activation of Her2 phosphorylation: Lanes 13 to 76: no phosphorylated Her2 in extract of BTG9A cells expressing human Her2 treated with different concentration (200 pM for lanes 3 to 5, 2 nM for lane 6) and time (lane 3: 5 minutes, lanes 4 and 6: 10 minutes, lane 5: 30 minutes) with the construct 1R59B-IFNA2-Q124R. Lanes 7 and 8: control for the detection of phosphorylated Her2 in the human BT474 cell line. Lane 1: extract of BTG9A cells. Lane 2: extract of BTG9A cells expressing human Her2.

(13) FIG. 13: Targeting the anti-PD-L2 122-IFNA2-Q124R to mouse primary cells endogenously expressing PD-L2. The activation is measured as STAT phosphorylation. The light gray area represents the PD-L2-negative cell population; the dark gray area represents the PD-L2-positive population.

(14) FIG. 14: In vivo targeting of 122-IFNA2-Q124R to PD-L2 expressing cells. Mice were injected intraperitoneally (IP) or intravenous (IV) with either PBS, a control construct (nanobody against GFP fused to mutant IFNA2-Q124R, indicated as control) or a targeted mutant IFN (targeted to PD-L2, Nb122-IFN2-Q124R, indicated as 122-Q124R. The light gray area represents the PD-L2-negative cell population; the dark gray area represents the PD-L2-positive population.

(15) FIG. 15: Dose response curve after IV injection of 122-IFN-Q124R in mice. The light gray area represents the PD-L2-negative cell population; the dark gray area represents the PD-L2-positive population.

(16) FIG. 16: Leptin-dependent growth induced by targeted mutant leptin: the loss in activity of a mutant leptin can be rescued in Ba/F3 cells expressing the human TNFR1. First two panels, experiment using the H6-leptin construct; second two panels, experiment using the mleptin construct. H6 indicated the his tag (6his).

(17) FIG. 17: construction of the targeted leptin constructs (SEQ ID NOS:9 and 10).

DETAILED DESCRIPTION

Examples

(18) Materials and Methods to the Examples

(19) Nanobodies and ScFv

(20) The nanobody 4-11 directed against the murine leptin receptor was described in Zabeau et al. (2012), and in the patent application WO 2006/053883. Its coding sequence is cloned into the mammalian expression vector pMET7 (Takebe et al., 1988) in fusion with the SIgk leader peptide, the HA tag and albumin. Plasmid name: pMET7 SIgK-HA-4.11-Albumin.

(21) The nanobody 4-10 is also described in Zabeau et al. (2012).

(22) The anti Her2 nanobodies 1R59B and 2R5A are described in Vaneycken et al. (2011). They were fused to the human IFNA2-Q124R and to the human IFNA2-R149A in the pMET7 vector. Fusion protein was produced by transfection of 293T cells.

(23) The anti PD-L2 nanobody 122 was from Johan Grooten (VIB, Gent, Belgium). It was fused to the human IFNA2-Q124R in the pMET7 vector. The fusion protein was produced by transfection of 293T cells and purified using the HisPur Ni-NTA purification kit (Pierce, Thermo Scientific).

(24) The anti TNF nanobody was obtained from Claude Libert (VIB).

(25) The anti Her2 ScFv was obtained from Andrea Plckthun (Wrn et al., 1998). It was fused to the human IFNA2-Q124R in the pMET7 vector. The fusion protein was produced by transfection of 293T cells.

(26) Control nanobody against GFP was obtained from Katrien Van Impe (University Ghent).

(27) Interferons

(28) The IFN2 and the mutants L153A and R149A, which show an IFNAR2 affinity reduced by a factor 10 and 100, respectively, have been described in Roisman et al. (2001). IFN coding sequences are cloned in the pT3T7 vector (Stratagene) in fusion with the ybbR tag. Plasmid names: pT7T3ybbR-IFNa2, pT7T3ybbR-IFNa2-L153A, pT7T3ybbR-IFNa2-R149A.

(29) The human IFNA2 Q124R has a high affinity for the murine IFNAR1 chain and a low affinity for the murine IFNAR2 chain. (Weber et al., 1987.)

(30) Nanobody-IFN Fusion Construction

(31) The coding sequence of the IFN2, wild-type, L153A and R149A were synthesized by PCR from the corresponding pT3T7ybbR IFNa2 plasmids using the Expand High Fidelity PCR system from Roche Diagnostics and the following primers: Forward: 5GGGGGGTCCGGACCATCACCATCACCATCACCATCACCATCACCCTGCTTCTCCCGC CTCCCCAGCATCACCTGCCAGCCCAGCAAGTGATAGCCTGGAATTTATTGC3 (SEQ ID NO:1), Reverse: 5CGTCTAGATCATTCCTTACTTCTTAAAC3 (SEQ ID NO:2). This PCR introduces a His tag and a series of five Proline-Alanine-Serine (PAS) repeats at the amino terminal extremity of the IFNs. The PCR products were digested with BspEI and XbaI and cloned into BspEI-XbaI digested pMET7 SIgK-HA-4.11-Albumin vector to obtain pMET7 SIgK-HA-4.11-His-PAS-ybbr-IFNA2, pMET7 SIgK-HA-4.11-His-PAS-ybbr-IFNA2-L153A and pMET7 SIgK-HA-4.11-His-PAS-ybbr-IFNA2-R149A.

(32) In a similar way, the human mutant Q124R was fused to the 1R59B nanobody and to the anti-PD-L2 nanobody.

(33) Production of the Nanobody-IFN Fusion Protein

(34) HEK293T cells were grown in DMEM supplemented with 10% FCS. They were transfected with pMET7 SIgK-HA-4.11-His-PAS-ybbr-IFNA2, pMET7 SIgK-HA-4.11-His-PAS-ybbr-IFNA2-L153A pMET7 SIgK-HA-4.11-His-PAS-ybbr-IFNA2-R149A, pMET7 SIgK-HA-2R5A-His-PAS-ybbr-IFNA2-R149A, pMET7 SIgK-HA-1R59B-His-PAS-ybbr-IFNA2-Q124R, pMET7 SIgK-HA-4D5-His-PAS-ybbr-IFNA2-Q124R or pMET7 SIgK-HA-122-His-PAS-ybbr-IFNA2-Q124R using lipofectamin (Invitrogen). 48 hours after the transfection, culture mediums were harvested and stored at 20 C.

(35) Alternatively, sequences encoding the different nanobody-IFN fusions were subcloned into the baculovirus transfer plasmid pBAC-2 (Novagen). Proteins were produced by insect cells using the BACVECTOR kit (reagents and kits designed for efficient and reliable construction of recombinant baculovirus and expression of target proteins in insect cells, Novagen) and purified to homogeneity using the HisPur Ni-NTA purification kit (Pierce, Thermo Scientific) and gel filtration. Protein concentration were measured by absorbance at 280 nm.

(36) IFN Reporter Cell Lines

(37) The HL116 clone (Uz 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 HL116 cells were co-transfected with an expression vector encoding the short isoform of the murine leptin receptor (pMET7 mLRsh-FLAG, Eyckerman et al., 1999) and pSV2neo (Southern and Berg 1982). Stable transfected clones were isolated in G418-containing medium. The clone 10 was selected after analysis of the surface expression level of the murine leptin receptor by FACS, using the biotinylated anti-mouse leptin receptor antibody BAF497 from R&D and streptavidin-APC (BD Bioscience).

(38) HT1080 cells were cotransfected with p6-16-RL, a plasmid encoding the Renilla luciferase (from pRL-null, Promega) controlled by the IFN-inducible 6-16 promoter (from p1.8gpt-5, Pellegrini et al., 1989), pBB3 (Bourachot et al., 1982) and salmon sperm DNA (Sigma). Stable transfected clones were isolated in HAT-containing medium. The clone 4 was selected for a high level of renilla luciferase activity induction upon IFN induction.

(39) The human pancreatic carcinoma BXPC3 (Tan et al., 1986; ATCC: CRL 1687) and breast cancer BT474 (Lasfargues et al., 1979; ATCC: HTB-20) cell lines were obtained from ATCC.

(40) The mouse BTG9A cells were described in Uz et al. (1990).

(41) Measurement of the Luciferase Activities

(42) IFN-specific activities were measured by quantifying the luciferase activity induced in HL116 cells and on the HL116 clone 10 expressing the mLR. The EC50 were calculated using non-linear data regression with GraphPad Prism software.

(43) Luciferase activities were determined on a Berthold centro LB960 luminometer using either the Firefly Luciferase Assay System or the Dual-Luciferase Reporter Assay System from Promega after six hours IFN stimulation.

(44) Quantitative RT-PCR

(45) The expression of the interferon inducible gene 6-16 was quantified by RT-PCR relative to GAPDH or -actin. Cells were treated with targeted or control IFN for 4 hours. Total RNA was purified with RNEASY columns (Qiagen). Reverse transcriptions were primed with random primers and performed using Moloney murine leukemia virus reverse transcriptase (Invitrogen). Quantitative real-time PCR (qRT-PCR) was performed using a LIGHTCYCLER as described (Coccia et al., 2004).

(46) For Her2, the transfection culture medium was assayed on murine BTG9A and BTG9A cells expressing human Her2 for expression of the OASL2 gene relative to the expression of the -actin gene by quantitative RT-PCR using a LIGHTCYCLER (Roche) and the following primers: OASL2 forward: CAC-GAC-TGT-AGG-CCC-CAG-CGA (SEQ ID NO:3); OASL2 reverse: AGC-AGC-TGT-CTC-TCC-CCT-CCG (SEQ ID NO:4); -actin forward: AGA-GGG-AAA-TCG-TGC-GTG-AC (SEQ ID NO:5); -actin reverse: CAA-TAG-TGA-TGA-CCT-GGC-CGT (SEQ ID NO:6). In a similar way, the ISG expression in Her2 targeted cells was measured using the same -actin primers and the following primer ISG15 primers: ISG15 forward: GAG-CTA-GAG-CCT-GCA-GCA-AT (SEQ ID NO:7); ISG15 reverse: TTC-TGG-GCA-ATC-TGC-TTC-TT (SEQ ID NO:8).

(47) Antiviral Assay

(48) The antiviral assay was performed using the EMC virus and scoring the virus replication-dependent cytopathic effect as described in Stewart (1979).

(49) Measurement of Her2 Phosphorylation

(50) BTG9A cells expressing human Her2 were treated with 200 pM to 2 nM of 1 R59B-IFNA2-Q124R for 10 to 30 min. Cells were lysed in RIPA, and analysed by western blot on an ODYSSEY FC (gels imaging system using visible, near-infrared or chemiluminesce signals, Licor Bioscience) after 7% SDS-PAGE (40 g lane). Phopho-Her2 was detected with the anti Her2 Y-P 1248 (Upstate #06-229) and the Goat anti rabbit secondary antibody IRDye 680 (Licor Bioscience #926-32221).

(51) Measurement of STAT1 Phosphorylation

(52) STAT1 phosporylated on Y701 were detected by FACS using the STAT1-PY701 (PE) (Beckton Dickinston #612564) and the manufacturer instruction for the PHOSFLOW (flow cytometry-based protein phosphorylation detection) technology.

(53) Targeted Leptin Constructs

(54) The sequence of the targeted leptin constructs is given in FIG. 17. The L86 that is indicated is the amino acid that is mutated either to S or N.

Example 1: The Nanobody-Interferon Fusion Proteins

(55) FIG. 1 shows a schematic representation of the nanobody-IFN fusion proteins constructed with either IFN2 wild-type or the L153A and R149A mutants.

Example 2: IFN Activity of the Nanobody-IFN Fusion Proteins is Targeted Toward Murine Leptin Receptor Expressing Cells

(56) The three nanobody fusion proteins with IFN2 WT, IFN2 L153A or R149A were assayed on both HL116 and HL116-mLR-clone 10 cells, which express the murine leptin receptor. The IFN2 alone was also assayed in this assay system in order to check that the two cell clones do not differ in their IFN responsiveness. Indeed, both HL116 and HL116-mLR-clone 10 cells are equally sensitive to this IFN (FIGS. 2A and 2C, black symbols). The IFN activity of the three nanobody-IFN fusion proteins is, however, dramatically increased in cells expressing the leptin receptor compared to parental HL116 cells (compare FIG. 2A with FIG. 2C and FIG. 2B with FIG. 2D).

(57) It was estimated that cells expressing the leptin receptor are 10-, 100- and 1000-fold more sensitive than parental HL116 cells to the nanobody-IFN WT, L153A and R149A, respectively. Since the affinities for IFNAR2 of the IFN mutant L153A and R149A are 0.1 and 0.01 relative to the WT, there is a correlation between the loss of activity caused by mutations in the IFNAR2 binding site and the targeting efficiency by the nanobody.

(58) In order to determine whether the IFN activity of the nanobody-IFN fusion proteins is delivered only on cells expressing the nanobody target or also on neighboring cells, the nanobody-IFN2R149A was assayed on a coculture of HL116-mLR-clone10 and HT1080-6-16 renilla luciferase clone4. Both cell types will express luciferase activity in response to IFN stimulation, but cells expressing the target of the nanobody will display a firefly luciferase activity, whereas cells devoid of leptin receptor will display a renilla luciferase activity. The dilution of the nanobody-IFN2R149A protein was chosen at 1/30, a dilution that induces a maximal response in cells carrying the leptin receptor and a minimal response on cells devoid of the nanobody target (see FIGS. 2B and 2D, black curves). FIG. 3 shows clearly that the renilla luciferase activity is not induced upon stimulation of the co-culture with the nanobody-IFN2R149A, indicating that the targeted IFN activity is delivered only on cells expressing the antigen recognized by the nanobody.

(59) The efficacy of the targeting is further illustrated by comparing the activity of wild-type and two types of mutant IFN (L153A and R149A) when added to HL116 expressing or not expressing the murine leptin receptor that is used for the targeting. The results clearly show that the activity of the mutants is higher when the construct is targeted, and that the effect of targeting for the mutant is bigger than for wild-type (FIGS. 4A and 4B).

(60) In order to prove that the targeting was nanobody specific, HL116 cells expressing the mLR were incubated for 6 hours with either the IFN-2 (indicated as IFNA2) or the IFNA2-R149A fused to the nanobody 4-11 (Nanobody-IFNA2-R149A) at their respective EC50 concentration in the presence or absence (control) of a 100-fold molar excess of free 4-11 nanobody. Cells were lysed and the IFN-induced luciferase activities were measured. As shown in FIG. 5, the non-targeted IFN is not inhibited by the free nanobody, while the targeted construct is strongly inhibited, showing the specific effect of the targeting.

(61) The targeting to the leptin receptor is independent of the epitope on the receptor: using the anti-leptin receptor nanobody 4-10 (Zabeau et al., 2012), which recognizes a different domain on the receptor than the nanobody 4-11, a similar activation can be obtained using a targeted mutant IFN (FIG. 6).

Example 3: The IFN Activity of the Nanobody-IFN Fusion Proteins on Cells Expressing the Leptin Receptor is Mediated by Both IFN Receptor Chains

(62) In order to determine whether the IFN activity of the nanobody-IFN fusion proteins needs the activation of the IFN receptor, HL116 cells expressing the murine leptin receptor were pretreated with neutralizing antibodies against IFNAR1 or IFNAR2, and then stimulated with the nanobody-IFNA2-R149A fusion protein. The activity of the IFN-induced luciferase was measured. FIG. 7 shows that both anti-IFNAR1 and anti-IFNAR2 neutralizing antibodies inhibit the IFN activity of the nanobody-IFNA2-R149A.

Example 4: Target-Specific Induction of Antiviral Activity by 4-11-IFNA2-R149A in Cells Expressing the Murine Leptin Receptor

(63) Antiviral activity is an integrated part of the IFN response, implying the expression of several genes. Therefore, the antiviral activity on mLR-expressing cells was controlled, after targeting the mutant R149A IFN using the anti-leptin receptor antibody 4-11. The results are summarized in FIG. 8. The activity was measured as the cytopathic effect on HL116 cells, with or without leptin receptor expression. The specific antiviral activity of the 4-11-IFNA2-R149A nanobody-IFN fusion protein is 716-fold higher when assayed on leptin receptor-expressing cells compared to HL116 cells.

Example 5: Targeting of IFN Activity on Her2 Expressing Cells

(64) In order to demonstrate that the concept is not restricted to cytokine receptor targeting, we generated similar fusion protein using the nanobody 2R5A against Her2 (Vaneycken et al., 2011) and the mutant IFN alpha2 R149A (2R5A-IFNA2-R149A). This molecule was assayed on BXPC3 (Pancreatic cancer, from ATCC) and BT474 (Breast cancer, from ATCC) cell lines and compared with the activity of IFN-2 (IFNA2) for the induction of the 6-16 IFN-inducible gene as determined relative to GAPDH by quantitative RT-PCR. The BXPC3 and BT474 cells lines differ by their number of Her2 molecules expressed at their surface (10.910.sup.3 and 47810.sup.3, respectively as reported by Gaborit et al. (2011)).

(65) FIGS. 9A and 9B show the EC.sub.50 determination of IFNA2 activity (FIG. 9A) and 2R5A-IFNA2-R149A activity (FIG. 9B) for the induction of the IFN-inducible gene 6-16 on BXPC3 and BT474 cell lines. FIG. 9A shows that BXPC3 and BT474 cell lines exhibit the same sensitivity to IFN-2. FIG. 9B shows that the 2R5A-IFNA2-R149A nanobody-IFN fusion protein is much more potent on the BT474 cell line, which expresses 40-fold more Her2 molecule than BXPC3.

(66) In conclusion, the concept that consists of targeting type I IFN activity on cells expressing a specific cell surface antigen, as shown on human cells expressing the mouse leptin receptor, can be extended to untransfected human cells expressing another cell surface molecule from a different structural family, at a level naturally found in several types of breast carcinoma.

Example 6: Targeting of Mutant IFNA2-Q149R to Mouse Cells Expressing Human Her2

(67) Mutant human IFNA2 Q149R was targeted to murine cells, expressing the human Her2, using the nanobody 1R59B in the 1R59B-IFNA2-Q124R. The IFNA2 Q124R has a high affinity for the murine IFNAR1 chain and a low activity for the murine IFNAR2 chain (Weber et al., 1987). The induction by IFN was measured as expression of the OASL2 messenger RNA, by RT-QPCR. The results are shown in FIG. 10. There is clearly a targeting-specific induction in the Her2-expressing cells, whereas there is no significant expression detected in untransfected BTG9A cells.

(68) Similar results were obtained when the Her2-specific ScFv against Her2 was used to target the mutant IFN Q124R. In this case, the IFN induction was measured using the ISG15 messenger RNA expression. The results are shown in FIG. 11. Again, a specific induction of ISG15 is seen in the cells expressing Her2, while there is little effect of the mutant IFN on the cells that do not express Her2.

Example 7: The Construct 1R59B-IFNA2-Q124R does not Activate the Phosphorylation of Her2

(69) To check whether targeting of Her2 is resulting in Her2 activation, Her2 phosphorylation was controlled in targeted cells. The results are shown in FIG. 12, clearly demonstrating that no phosphorylated Her2 could be detected in 1R59B-IFNA2-Q124R targeted cells, irrespective of the concentration or time of treatment.

Example 8: The Anti PD-L2 Nb122-IFNA2-Q124R Construct Activity is Targeted on Mouse Primary Cells Expressing PD-L2

(70) Cells from a mouse peritoneal cavity were isolated and treated in vitro with Nb122-IFNA2-Q124R or natural mIFN/ for 30 minutes. Cells were, fixed, permeabilized, labelled with antibodies against PD-L2 (APC) (BD #560086) and STAT1-PY701 (PE) (BD #612564) and analyzed by FACS.

(71) The PD-L2-positive cell population represents 20% of the total cell population present in the mouse peritoneal cavity.

(72) The results are shown in FIG. 13. It is clear from this figure that in untreated cells, or in non-targeted, murine IFN-treated cells, the peaks of STAT1-P for PD-L2 expressing and non-expressing cells coincide. Moreover, a clear induction in STAT1-P can be seen by murine IFN treatment. Treatment with the targeted mutant IFN, however, results in a specific shift in the STAT1-P only for the PD-L2 expressing cells.

(73) The same result is obtained if the IFN response of splenocytes is analyzed in a similar experiment. The PD-L2-positive cell population represents 1% of the total cell population present in mouse spleen, indicating that also a minor cell population can be targeted in an efficient way.

Example 9: In Vivo Injection of 122-IFNA2-Q124R Construct Induces an IFN Response Only in PD-L2-Expressing Cells

(74) Mice were injected (IP or IV) with either PBS, Nb122-IFNA2-Q124R or a control Nb (against GFP) fused to IFNA2-Q124R. 30 min post injection, mice were killed, cells from the peritoneal cavity were recovered by washing the peritoneal cavity with PBS, fixed (PHOSFLOW (flow cytometry-based protein phosphorylation detection) Fix buffer I BD #557870), permeabilized (PHOSFLOW (flow cytometry-based protein phosphorylation detection) Perm buffer III, BD #558050), labelled with Abs against PD-L2 (APC) (BD #560086) and STAT1-PY701 (PE) (BD #612564) and analysed by FACS. The results are shown in FIG. 14. STAT1-P coincides in PD-L2 positive and negative cells treated with either PBS or control nanobody. However, a clear induction in STAT1-P (only in the PDL2 positive cell population) can be seen when the mice are injected with the targeted mutant IFN.

(75) As a control, STAT1-P was checked in mice, iv injected with different doses of natural mouse IFN (10,000, 100,000 or 1,000,000 units), and no difference in STAT1-P could be detected between the PD-L2-positive and PD-L2-negative cells.

(76) FIG. 15 shows a similar dose response curve after iv injection of the Nb122-IFNA2-Q124R construct. A shift in STAT1-P in the PD-L2 expressing cells can be noticed even at the lowest dose of 64 ng.

Example 10: Targeting of Mutant Leptin to the Leptin Receptor, Using a Truncated TNF Receptor

(77) Ba/F3 cells are growth-dependent on IL-3. After transfection with the mLR, Ba/F3 cells also proliferate with leptin. Leptin mutants with reduced affinity for their receptor are less potent in inducing and sustaining proliferation of Ba/F3-mLR cells. Leptin mutant L86S has a moderate, and mutant L86N has a strong, reduction in affinity and, hence, a moderate and strong reduced capacity to induce proliferation, respectively.

(78) Additional transfection of Ba/F3-mLR cells with the human TNF Receptor 1 (hTNFR1) lacking its intracellular domain introduces a non-functional receptor, which can function as a membrane-bound extracellular marker.

(79) Chimeric proteins consisting of leptin and a nanobody against human TNFR1 (here nb96) will bind to cells carrying the mLR and to cells carrying the hTNFR1. Chimeric proteins with leptin mutants L86S and L86N have reduced affinity for the LR but retain their affinity for the hTNFR1.

(80) Chimeric proteins were produced by transient transfection of Hek293T cells with expression plasmids. Supernatant was 0.45 m filtered and serially diluted in 96-well plates for the assay. A serial dilution of purified recombinant leptin was used as a reference. 3000 to 10000 cells were plated per well and proliferation was measured by staining with XTT four or five days later. OD was measured at 450 nm. The results are shown in FIG. 16, for two experiments using a different leptin construct (see FIG. 17). For both constructs, an hTNFR depending growth stimulation can be seen for the mutant constructs, whereas the hTNFR expression does not affect the growth of the cells treated with wt (non-targeted) leptin. It is clear from these results that the targeting can compensate for the negative effect of the mutation.

REFERENCES

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