TARGETED MODIFIED IL-1 FAMILY MEMBERS

Abstract

The present disclosure relates to a modified Interleukin-1 (IL-1) family member cytokine, with reduced activity via its cytokine receptor, wherein said interleukin-1 family member cytokine is specifically delivered to target cells. Preferably, the IL-1 family member cytokine is a mutant, more preferably it is a mutant IL-1 with low affinity to the IL-1 receptor, wherein said mutant IL-1 is specifically delivered to target cells. The targeting is preferably realized by fusion of the modified IL-1 family member cytokine to a targeting moiety, preferably an antibody or antibody-like molecule. The disclosure relates further to the use of such targeted modified IL-1 family member cytokine to treat diseases.

Claims

1. A targeting construct, comprising a modified IL-1 family cytokine characterized by a reduced affinity for its receptor, and a targeting moiety.

2. The targeting construct, according to claim 1, wherein said modified IL-1 family cytokine is a mutant IL-1 family cytokine.

3. The targeting construct according to claim 1 or 2, wherein said targeting moiety is targeting to a marker expressed on a IL-1 family cytokine receptor expressing cell.

4. The targeting construct, according to claim 2, wherein said mutant IL-1 family cytokine is a mutant IL-1β.

5. The targeting construct, according to claim 4, wherein said targeting moiety is targeting to a marker expressed on an IL-1R1 and/or IL-1RacP expressing cell.

6. The targeting construct according to any of the preceding claims, wherein said targeting moiety is directed to a tissue specific marker.

7. The targeting construct according to any of the claims 1-5, wherein said targeting moiety is directed to Her2.

8. The targeting construct according to any of the previous claims, wherein said targeting moiety is an antibody.

9. The targeting construct according to claim 8, wherein said antibody is a nanobody.

10. The targeting construct according to any of the preceding claims, wherein the modified IL-1 family cytokine is an IL-1β mutant, selected from the group consisting of A117G/P118G, R120GR120X, L122A, T125G/L126G, R127G, Q130X, Q131G, K132A, S137G/Q138Y, L145G, H146X, L145A/L147A, Q148X, Q148G/Q150G, Q150G/D151A, M152G, F162A, F162A/Q164E, F166A, Q164E/E167K, N169G/D170G, I172A, V174A, K208E, K209A K209X, K209A/K210A, K219X, and E221KX, E221S/N224A, N224S/K225S, E244K and N245Q

11. A targeting construct according to any of the claims 1-10 for use as a medicament.

12. The targeting construct according to any of the claims 1-10 for use in stimulation of the immune response.

13. The targeting construct according to any of the claims 1-10 for use in treatment of cancer.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0018] FIG. 1: Schematic representation of the IL-1β-nanobody fusion proteins

[0019] FIG. 2: Concentration dependency of the induction of the NFκB activity by wild type and mutant Q148G IL-1 Her2 nanobody fusions (A) and other selected mutants (B), in mock transfected cells, or cells transfected with signaling deficient Her2.

[0020] FIG. 3: Effect of wild type and mutant (Q148G, L145A/L147A, F162A/Q164E) IL-1 Her2 nanobody fusions on nuclear translocation of endogenous NF-κB p65 in mock transfected cells, or cells transfected with signaling deficient Her2.

[0021] FIG. 4: Induction of the NFκB activity by wild type and 5 different IL-1 mutants, fused to an anti-murine leptin receptor nanobody, on cells expressing the murine leptin receptor (mLR) or not (no mLR).

[0022] FIG. 5: Concentration dependency of the induction of the NFκB activity by IL1 double mutants fused to the Her2 nanobody in mock transfected cells, or cells transfected with signaling deficient Her2.

EXAMPLES

Materials and Methods to the Examples

Cloning of IL-1-Nanobody Fusion Proteins.

[0023] The 4-10 nanobody directed against the murine leptin receptor is described in Zabeau et al. (2012) and in the patent WO 2006/053883. The anti-Her2 nanobody 1R59B is described in Vaneycken et al. (2011). Both nanobodies were cloned with a C-terminal His tag in the pMET7 eukaryotic expression vector. A codon-optimized sequence encoding the mature IL-1β protein, preceded by the SigK leader peptide, and equipped with an N-terminal HA tag, was generated via gene synthesis (Invitrogen Gene Art). To generate the IL-1β-nanobody fusion proteins, the IL-1β sequence was cloned 5′ to the nanobody sequence in pMet7, with a 13×GGS linker separating the cytokine and nanobody moieties. (FIG. 1)

IL-1β Mutants.

[0024] IL-16 mutants expected to have reduced binding affinity for the IL-1R were selected based on literature and analysis of published crystal structures of human IL-1β complexed with its receptor. Mutations in the hIL-1β moiety were created via site-directed mutagenesis (QuickChange, Stratagene) using the mutagenesis primers as indicated in table I:

TABLE-US-00001 TABLE I mutants and primers used Fw primer Rev primer  1 A117G/ CCGACTACGCTGGCGGCAGTGACGGTGTCA GCAGTTCAGGCTTCTGACACCGTCACTG P118G GAAGCCTGAACTGC CCGCCAGCGTAGTCGG  2 R120A CTGGCGGCAGCGCCCCTGTCGCTAGCCTGA CGCAGGGTGCAGTTCAGGCTAGCGACAG ACTGCACCCTGCG GGGCGCTGCCGCCAG  3 R120G GCGGCAGCGCCCCTGTCGGAAGCTTGAACT GCAGGGTGCAGTTCAAGCTTCCGACAGG GCACCCTGC GGCGCTGCCGC  4 L122A CGCTGGCGGCAGTGCCCCTGTCAGAAGCGC GCTGTCCCGCAGGGTGCAGTTCGCGCTT GAACTGCACCCTGCGGGACAGC CTGACAGGGGCACTGCCGCCAGCG  5 T125G/ CGCCCCTGTCAGAAGCCTGAACTGCGGCGG GCTTTTCTGCTGGCTGTCCCGGCCGCCG L126G CCGGGACAGCCAGCAGAAAAGC CAGTTCAGGCTTCTGACAGGGGCG  6 R127G AGAAGCCTGAACTGCACACTGGGGGACAGC GACCAGGCTTTTCTGCTGGCTGTCCCCC CAGCAGAAAAGCCTGGTC AGTGTGCAGTTCAGGCTTCT  7 Q130A CCCTGCGGGACAGCGCGCAGAAAAGCCTGG CCAGGCTTTTCTGCGCGCTGTCCCGCAG GG  8 Q130W CTGCACCCTGCGGGACAGCTGGCAGAAAAG GCTCATGACCAGGCTTTTCTGCCAGCTG CCTGGTCATGAGC TCCCGCAGGGTGCAG  9 Q131G CTGCGGGACAGCCAGGGGAAGAGCCTGGTC CGCTCATGACCAGGCTCTTCCCCTGGCT ATGAGCG GTCCCGCAG 10 K132A GCACCCTGCGGGACAGCCAGCAGGCTAGCC GGCCGCTCATGACCAGGCTAGCCTGCTG TGGTCATGAGCGGCC GCTGTCCCGCAGGGTGC 11 S137G/ CAGCAGAAAAGCCTGGTCATGGGGTACCCC GCAGTGCCTTCAGCTCGTAGGGGTACCC Q138Y TACGAGCTGAAGGCACTGC CATGACCAGGCTTTTCTGCTG 12 L145G GCCCCTACGAGCTGAAGGCAGGTCATCTGC CCATGTCCTGGCCCTGCAGATGACCTGC AGGGCCAGGACATGG CTTCAGCTCGTAGGGGC 13 H146A CGAGCTGAAGGCACTGGCTCTTCAGGGCCA CCATGTCCTGGCCCTGAAGAGCCAGTGC GGACATGG CTTCAGCTCG 14 H146G CCTACGAGCTGAAGGCACTGGGTCTGCAGG CCATGTCCTGGCCCTGCAGACCCAGTGC GCCAGGACATGG CTTCAGCTCGTAGG 15 H146E GCTGAAGGCACTGGAGCTGCAGGGCCAGG CCTGGCCCTGCAGCTCCAGTGCCTTCAG C 16 H146N AGCTGAAGGCACTGAATCTGCAGGGCCAG CTGGCCCTGCAGATTCAGTGCCTTCAGC T 17 H146R CTGAAGGCACTGCGTCTGCAGGGCCAG CTGGCCCTGCAGACGCAGTGCCTTCAG 18 L145A/ GCGGCCCCTACGAGCTGAAGGCAGCGCATG CCATGTCCTGGCCCTGCGCATGCGCTGC L147A CGCAGGGCCAGGACATGG CTTCAGCTCGTAGGGGCCGC 19 Q148E GGCACTGCATCTGGAGGGCCAGGACAT ATGTCCTGGCCCTCCAGATGCAGTGCC 20 Q148G GAAGGCACTGCATCTGGGTGGCCAGGACAT GCTGTTCCATGTCCTGGCCACCCAGATG GGAACAGC CAGTGCCTTC 21 Q148L GCACTGCATCTGCTGGGCCAGGACATG CATGTCCTGGCCCAGCAGATGCAGTGC 22 Q148G/ CGAGCTGAAGGCACTGCATCTGGGGGGCGG CCTGCTGTTCCATGTCCCCGCCCCCCAG Q150G GGACATGGAACAGCAGG ATGCAGTGCCTTCAGCTCG 23 Q150G/ GCACTGCATCTGCAGGGCGGGGCCATGGAA GCTGAACACGACCTGCTGTTCCATGGCC D151A CAGCAGGTCGTGTTCAGC CCGCCCTGCAGATGCAGTGC 24 M152G GCACTGCATCTGCAGGGCCAGGACGGGGAA GCTCATGCTGAACACCACCTGCTGTTCC CAGCAGGTGGTGTTCAGCATGAGC CCGTCCTGGCCCTGCAGATGCAGTGC 25 F162A CATGGAACAGCAGGTGGTGTTCAGCATGAG GTCGTTGCTTTCCTCGCCCTGCACGGCG CGCCGTGCAGGGCGAGGAAAGCAACGAC CTCATGCTGAACACCACCTGCTGTTCCA TG 26 F162A/ GCAGGTCGTGTTCAGCATGAGCGCCGTGGA GGATCTTGTCATTGCTTTCCTCGCCCTC Q164E GGGCGAGGAAAGCAATGACAAGATCC CACGGCGCTCATGCTGAACACGACCTGC 27 F166A CCGACTTCACCATGCAGGCCGTCTCCAGCG CCAGATCTGCTGCCGCCGCTGGAGACGG GCGGCAGCAGATCTGG CCTGCATGGTGAAGTCGG 28 Q164E/ GCATGAGCTTCGTGGGGGGCAAGGAAAGCA GGCCACGGGGATCTTGTCATTGCTTTCC E167K ATGACAAGATCCCCGTGGCC TTGCCCCCCACGAAGCTCATGC 29 N169G/ GCAGGGCGAGGAAAGCGGCGGCAAGATCCC CTTCTCTTTCAGGCCTAGGGCCACGGGG D170G CGTGGCCCTAGGCCTGAAAGAGAAG ATCTTGCCGCCGCTTTCCTCGCCCTGC 30 I172A GAAAGCAACGACAAGGCCCCCGTGGCCCTG CCCAGGGCCACGGGGGCCTTGTCGTTGC GG TTTC 31 V174A GCAACGACAAGATCCCCGCGGCCCTGGGCC CTTTCAGGCCCAGGGCCGCGGGGATCTT TGAAAG GTCGTTGC 32 K208E GCAGCTGGAAAGCGTGGATCCCAAGAACTA GCGTTTTTCCACTCTTTTTCTCGGGGTA CCCCGAGAAAAAGATGGAAAAACGC GTTCTTGGGATCCACGCTTTCCAGCTGC 33 K209A CCCCAAGAACTACCCCAAGGCAAAGATGGA GTTGAACACGAAGCGCTTTTCCATCTTT AAAGCGCTTCGTGTTCAAC GCCTTGGGGTAGTTCTTGGGG 34 K209D GCAGCTGGAAAGCGTGGATCCCAAGAACTA GCGTTTTTCCATCTTGTCCTTGGGGTAG CCCCAAGGACAAGATGGAAAAACGC TTCTTGGGATCCACGCTTTCCAGCTGC 35 K209A/ CCCCAAGAACTACCCCAAGGCAGCGATGGA GAACACGAAGCGTTTTTCCATCGCTGCC K210A AAAACGCTTCGTGTTC TTGGGGTAGTTCTTGGGG 36 K219S AAAAACGCTTCGTGTTCAACAGCATCGAGA GAGCTTGTTGTTGATCTCGATGCTGTTG TCAACAACAAGCTC AACACGAAGCGTTTTT 37 K219Q AAAAACGCTTCGTGTTCAACCAGATCGAGA CTTGTTGTTGATCTCGATCTGGTTGAAC TCAACAACAAG ACGAAGCGTTTTT 38 E221S GCTTCGTGTTCAACAAGATCTCGATCAACA ACTCGAGCTTGTTGTTGATCGAGATCTT ACAAGCTCGAGT GTTGAACACGAAGC 39 E221K CTTCGTGTTCAACAAGATCAAGATCAACAA TCGAGCTTGTTGTTGATCTTGATCTTGT CAAGCTCGA TGAACACGAAG 40 K219S/ GGAAAAACGCTTCGTCTTCAACAGCATCTC CGAACTCGAGCTTGTTGTTGATCGAGAT E221S GATCAACAACAAGCTCGAGTTCG GCTGTTGAAGACGAAGCGTTTTTCC 41 E221S/ CGCTTCGTGTTCAACAAGATCTCGATCAAC CTCGAACTCGAGCTTGGCGTTGATCGAG N224A GCCAAGCTCGAGTTCGAG ATCTTGTTGAACACGAAGCG 42 N224S/ CAACAAGATCGAGATCAACAGCAGCCTCGA CTGGGCGCTCTCGAATTCGAGGCTGCTG K225S ATTCGAGAGCGCCCAG TTGATCTCGATCTTGTTG 43 E244K CCCCAACTGGTACATCAGTACTAGTCAGGC GGAACACGGGCATATTCTTGGCCTGACT CAAGAATATGCCCGTGTTCC AGTACTGATGTACCAGTTGGGG 44 N245Q CAGCACTAGTCAGGCCGAGCAGATGCCCGT GGTGCCGCCCAGGAAGACGGGCATCTGC CTTCCTGGGCGGCACC TCGGCCTGACTAGTGCTG 45 E244K/ CATCAGCACTAGTCAGGCCAAGCAGATGCC GGTGCCGCCCAGGAAGACGGGCATCTGC N245Q CGTCTTCCTGGGCGGCACC TTGGCCTGACTAGTGCTGATG  46* R120G/ GCGGCAGCGCCCCTGTCGGAAGCTTGAACT GCAGGGTGCAGTTCAAGCTTCCGACAGG Q131G GCACCCTGC GGCGCTGCCGC  47* R120G/ CGAGCTGAAGGCACTGGCTCTTCAGGGCCA CCATGTCCTGGCCCTGAAGAGCCAGTGC H146A GGACATGG CTTCAGCTCG  49* R120G/ GCGGCCCCTACGAGCTGAAGGCAGCGCATG CCATGTCCTGGCCCTGCGCATGCGCTGC L145A/ CGCAGGGCCAGGACATGG CTTCAGCTCGTAGGGGCCGC L147A   48** R120G/ GCGGCAGCGCCCCTGTCGGAAGCTTGAACT GCAGGGTGCAGTTCAAGCTTCCGACAGG Q148G GCACCCTGC GGCGCTGCCGC  50* R120G/ GCAGGTCGTGTTCAGCATGAGCGCCGTGGA GGATCTTGTCATTGCTTTCCTCGCCCTC F162A/ GGGCGAGGAAAGCAATGACAAGATCC CACGGCGCTCATGCTGAACACGACCTGC Q164E  51* R120G/ GCAGCTGGAAAGCGTGGATCCCAAGAACTA GCGTTTTTCCACTCTTTTTCTCGGGGTA K208E CCCCGAGAAAAAGATGGAAAAACGC GTTCTTGGGATCCACGCTTTCCAGCTGC   52** Q131G/ CTGCGGGACAGCCAGGGGAAGAGCCTGGTC CGCTCATGACCAGGCTCTTCCCCTGGCT Q148G ATGAGCG GTCCCGCAG   53** Q148G/ GCAGGTCGTGTTCAGCATGAGCGCCGTGGA GGATCTTGTCATTGCTTTCCTCGCCCTC F162A/ GGGCGAGGAAAGCAATGACAAGATCC CACGGCGCTCATGCTGAACACGACCTGC Q164E   54** Q148G/ GCAGCTGGAAAGCGTGGATCCCAAGAACTA GCGTTTTTCCATCTTTTTCTCGGGGTAG K208E CCCCGAGAAAAAGATGGAAAAACGC TTCTTGGGATCCACGCTTTCCAGCTGC *double/triple-mutants were created using R120G as template. **double/triple-mutants were created using Q148G as template.

Production of IL-1β Fusion Proteins.

[0025] IL-1β fusion proteins were produced in HEK293T cells. For small-scale production, HEK293T cells were seeded in 6-well plates at 400000 cells/well in DMEM supplemented with 10% FCS. After 24 hours, culture medium was replaced by medium with reduced serum (DMEM/5% FCS) and cells were transfected using linear PEI. Briefly, PEI transfection mix was prepared by combining 1 μg expression vector with 5 μg PEI in 160 μl DMEM, incubated for 10 minutes at RT and added to the wells dropwise. After 24 hours, transfected cells were washed with DMEM and layered with 1.5 ml OptiMem/well for protein production. Conditioned media were recuperated after 48 hours, filtered through 0.45 μfilters and stored at −20° C. IL-1β content in the conditioned media was determined by Elisa according to the manufacturer's instructions (R&D Systems).

NF-κB Reporter Gene Assay.

[0026] To assess IL-1R activation, we used HEK-Blue™ IL-1β cells that stably express the IL-1R (Invivogen) and transfected them transiently with an NF-κB luciferase reportergene. Briefly, HEK-Blue™ IL-1β cells were seeded in culture medium (DMEM/10% FCS) in 96-well plates (10000 cells/well) and transfected the next day using the calciumphosphate precipitation method with the indicated amounts of expression plasmids and 5 ng/well of the 3 KB-Luc reportergene plasmid (Vanden Berghe et al., 1998). 24 hours post-transfection, culture medium was replaced by starvation medium (DMEM) and 48 hours post-transfection, cells were induced for 6 hours with fusion proteins. After induction, cells were lysed and luciferase activity in lysates was determined using the Promega Firefly Luciferase Assay System on a Berthold centro LB960 luminometer.

Analysis of NF-κB Nuclear Translocation Via Confocal Microscopy.

[0027] For confocal imaging, 10.sup.5 HEK293-T cells/well (in 6-well plate) were seeded on glass coverslips (Zeiss), coated with poly-L-lysine (Sigma). The next day, cells were transfected with 200 ng/well of empty vector or HER2Δcyt expression plasmid using the calcium phosphate precipitation method. After 48 hours, cells were treated for 30 minutes with vehicle (medium) or IL1-Her2 nanobody fusion protein (10 ng/ml). Next, cells were rinsed with 1×PBS and fixed for 15 minutes at room temperature in 4% paraformaldehyde. After three washes with 1×PBS, cells were permeabilized with 0.1% Triton X-100 in 1×PBS for 10 minutes and blocked in 1% BSA in 1×PBS for another 10 minutes at room temperature. Samples were then incubated for 1 hour at 37° C. with rabbit anti-p65 antibody (Santa Cruz C20, diluted 1:800) and mouse anti-Flag Antibody (Sigma M2, 1:2000). After four washes in 1×PBS, cells were incubated for 1 hour at room temperature with anti-rabbit Alexa 488 and anti-mouse Alexa 594 fluorochrome-conjugated secondary antibodies (both diluted 1:800). After secondary antibody incubation, cells were washed four times in 1×PBS and nuclei were stained with DAPI (2 μg/ml). After a final wash step in 1×PBS, coverslips were mounted using propyl gallate. Images were acquired using a 60×1.35 NA objective on an Olympus IX-81 laser scanning confocal microscope and analyzed using Fluoview 1000 software.

Example 1: IL-1β-Ligand and IL-1β-Nanobody Fusion Proteins

[0028] FIG. 1 shows a scheme of the IL-1β-nanobody fusion proteins constructed with either WT hIL-1β or the hIL1β mutants described in table I.

Example 2: IL-1β Activity of Selected Mutant IL-1β-Nanobody Fusions is Restored on Cells Expressing the Nb Targets

[0029] Wild type IL-1β and 45 IL-1β mutants (Table I) were fused to a well-characterized nanobody recognizing Her2 (1 R59B). The IL-1β-nanobody fusion proteins were tested on HEK-Blue™ IL-13 cells, transiently transfected with an NF-κB reportergene plasmid (5 ng/well) and a Her2Δcyt (signalling-deficient) expression plasmid (2 ng/well). Cells were treated for 6 hours with IL-1β-Her2 nanobody fusions (dose response ranging from 0.4 to 250 ng/ml). As demonstrated in FIG. 2A, the IL-1β-Q148G-Her2 nanobody fusion displayed a reduced ability to activate NF-κB as compared to the WT IL1-β-Her2 nanobody fusion. Importantly, targeting of the Q148G mutant to Her2Δcyt-expressing cells restored its activity and produced a dose-response curve for NF-κB activation that perfectly parallels that of the WT IL-1β on mock-transfected cells. Also evident from this figure is a strong targeting effect for the WT IL-1β Her2 nanobody fusion. Similar “activation by targeting” effects were observed for six other IL-1β mutants (R120G, Q131G, H146A, H145A/L147A, F162A/Q164E and K208E) fused to the Her2 nanobody (FIG. 2B).

[0030] To obtain further proof for the “activation by targeting” concept, we next explored whether we could visualize the selective activation of NF-κB in Her2-expressing cells by the IL-1β-Her2 nanobody fusions via confocal microscopy. We measured activation of endogenous NF-κB by assaying its nuclear translocation. As evident from FIG. 3, only the WT IL-1β-Her2 nanobody fusion promoted translocation of endogenous NF-κB in cells that do not express Her2. Whereas they did not promote detectable NF-κB translocation in mock-transfected cells, the three tested mutant IL1-β-Her2 nanobody fusions triggered NF-κB nuclear translocation in cells that also stained positive for Her2, indicating they only act on targeted cells.

[0031] To evaluate whether the “activation by targeting” concept also works using a nanobody to an unrelated membrane protein, we fused WT IL-1β and five of the disabled IL-1β mutants (R120G, Q131G, H146A, Q148G, K209A) to a previously characterized nanobody recognizing the mLR (4-10). An experiment similar to that reported for the IL-1β-Her2 nanobody fusion (FIG. 2) was performed using HEK-Blue™ IL-1β cells, transiently transfected with a mLR expression plasmid (10 ng/well). Similar to the results obtained with the Her2 nanobody fusion proteins, all investigated mutant IL-1β nanobody fusions (tested at 12.5 ng/ml) showed a reduced ability, as compared to the WT fusion, to activate NF-κB on cells that do not express mLRs. However, targeting by the mLR nanobody moiety partially restored the activity of the selected mutants (FIG. 4).

[0032] Because the IL-1β mutants described above retained significant residual biological activity, we combined different mutations to obtain double/triple mutants with reduced basal activity. Nine double/triple mutants were tested (cf. table I mutants 46 to 54) and from these, six mutant proteins (Q131G/Q148G, Q148G/K208E, R120G/Q131G, R120G/Q131G, R120G/H146A, R120G/K208E, R120G/F162A/Q164E) displayed no residual activity (using the same assay for measuring NF-κB as in FIG. 2) on Her2-negative cells, whilst partially restored activity was apparent on cells overexpressing Her2Δcyt (FIG. 5).

[0033] These data altogether indicate that targeting partially inactive mutant IL-1β, by fusing it to a nanobody recognizing a cell surface receptor, can restore its activity on nanobody target cells, probably by forced receptor interaction through a membrane concentration effect. The fact that activation by targeting can be accomplished using nanobodies recognizing different classes of membrane proteins indicates broad applicability of the “activation by targeting” concept.

[0034] Because these data provide proof of concept for the ability of targeting mutant IL-1 family members to selected cell types, restoring their activity on these target cells only, nanobodies are produced that allow targeting IL-1 family members to physiologically relevant IL-1β target cells. In view of the important role of IL-1 family members as T- and NK-cell activators, the nanobodies are designed to specifically target IL-1 to T- and NK-cell subsets. More specifically nanobodies targeting CCR6, which are predominantly expressed on Th17 cells as well as nanobodies targeting CD8 on cytotoxic T cells are developed and fused to the members of the IL1-family, preferably IL-1β.

Example 3: Effect of IL-1β-Nanobody Fusions on IL-17 Production by Primary Human T Cells

[0035] Primary human T cells were isolated from buffy coats. First, PBMC's were isolated by lymphoprep density gradient centrifugation and incubated O/N with 0.5 ng/ml rhIL-2 for recovery. Next, T-cells were isolated using the pan-T cell isolation kit (Miltenyi Biotec) according to the manufacturer's instructions. Briefly, T cells were resuspended (1×10.sup.6/ml) in RPMI-1640 supplemented with 10% FCS and CD3/CD28 activating microbeads (Miltenyi Biotec). Next, cells (100 μl/well) were plated in U-bottom 96-well plates and stimulated for 96 hours with the indicated concentrations of IL-1β variants. After an additional 6 hours stimulation with PMA/ionomycin (both at 100 nM), supernatants were recovered and IL-17 levels were determined by Elisa (R&D Systems). Additional cytokines are evaluated via Luminex technology.

[0036] For selected mutant IL-1β-nanobody fusions (e.g. with a nanobody targeting CCR6) target cell-specific IL-17 and IFNγ production are evaluated by intracellular staining using a flow cytometric approach.

[0037] Also, to corroborate selectivity for the Th17 population, binding to PBMC subpopulations is measured via double staining using the Flag tag and selected CD markers, followed by flow cytometric analysis.

[0038] Finally, in a clinically relevant in vitro model of human Th17 cell function, the adjuvant activity of the IL-1β-nanobody fusions is assessed. In view of the need for more efficacious vaccines against Bordetella pertussis (or adjuvants for the existing vaccines), we determined whether the selected fusion proteins enhance the human Th17 response in a coculture model of naïve T cells with B. pertussis-treated monocyte-derived dendritic cells (MDDCs). Human MDDCs are isolated from buffy coats (using the monocyte isolation kit II, Miltenyi Biotec), treated with different ratios of B. pertussis for 48 hours and then cocultured with naïve allogeneic T cells for 12 days. After restimulation with anti-CD3/anti-CD28, the cytokine profiles in supernatants are determined using Elisa/Luminex technology (cfr. supra).

Example 4: Effect of IL-1β-Nanobody Fusions on CTLs

[0039] To assess whether IL-1β-CD8 nanobody fusions can specifically enhance the function of CD8+ T cells, human PBMC's are isolated by lymphoprep density gradient centrifugation from buffy coats and stimulated for 24 hours with CD3/CD28 activating microbeads (Miltenyi Biotec) in combination with wt or mutant IL1β-CD8 Nb fusions. The effect of these fusion proteins on CD8+ T cell activation is evaluated by performing intracellular staining for active (phosphorylated) NF-κB and IFNγ. In addition, to investigate whether the IL-1β-nanobody fusions affect CTL degranulation, PBMC's (2×10.sup.6 cells/ml) are differentiated for 48 hours in the presence of phytohaemagglutinin (PHA, 1 μg/ml) and IL-2 (100 IU/ml) in combination with increasing doses of the IL-1β fusion proteins. Next, to induce degranulation, cells are stimulated for 3 hours with CD3/CD28 dynabeads and analysed by flow cytometry. Degranulation is measured via detection of cell surface CD107a, a well-established marker for natural killer activity. In all flow cytometric analyses on leukocyte pools, anti-CD8 staining is included to allow monitoring of the cell-type specificity of the IL-1β-CD8 Nb effects.

[0040] Finally to assess whether the IL-1β-CD8 nanobody fusions promote anti-tumor activity in vivo, C57BL/6 mice are injected subcutaneously with TC1 tumor cells, which produce the E6 and E7 antigenic oncoproteins from HPV16. This model was previously used to demonstrate that IL-1β promotes CD8+ T cell-mediated, antigen-specific, anti-tumor responses (Ben-Sasson, 2013). Briefly, mice are immunized four days after tumor injection with a vaccine containing the HPV16E7.sub.49-57 peptide, combined with DOTAP and LPS, and with our without WT or mutant IL-1β-CD8 Nb fusions or IL-1β-GFP Nb fusions. Tumor size is monitored for 18 days post-immunization.

Example 5: In Vivo Experiments—Vaccine Adjuvans Effect

[0041] In a first series of experiments C57BL/6 mice are treated iv/ip with different doses of WT and mutant IL-1β-nanobody fusions and unfused IL-1β, to monitor acute toxicity. Venous blood is collected at different times post treatment by tail venopuncture and the cytokine profile in serum is determined by Luminex assay. In addition, via flow cytometric analysis intracellular cytokine levels (IL-17, IFNγ) and activation of IL-1R (as assessed by measuring phospho-NF-κB levels) are determined in selected leukocyte subsets.

[0042] When optimal doses have been established, their adjuvant activity is assessed in a murine vaccination protocol. Briefly, C57BL/6 mice are immunized ip with acellular pertussis vaccine (Pa). The Pa vaccine is composed of 5 μg/mouse of purified recombinant detoxified pertussis toxin (PT9K/129G)+filamentous hemagglutinin (FHA) (composition according to Brereton et al., 2011). 24 hours after immunization, selected mutant IL1β-Nb or PBS are administered ip or iv. Animals are boosted after 28 days. One set of animals is sacrificed 14 days after the second immunization and splenocytes are isolated and restimulated in vitro with medium or FHA for 3 days. Cytokine levels in culture supernatants (IL-17, IFNγ, IL-2, IL-10, IL-5, IL-4, etc.) are determined via Luminex technology. A second set of mice is challenged with B. pertussis on day 14 post-boost and sacrificed 2 h and 5 and 10 days post-challenge. Lungs are isolated and CFU in lung homogenates will be quantified on Bordet-Gengou agar plates. Cytokine levels in lung homogenates are determined as in splenocyte supernatants.

[0043] In addition, blood is sampled (from the tail vene) before immunization and then every 14 days for determination of B. pertussis-specific IgG levels in serum.

Example 6: Direct Antitumor Effect of IL-1β-Nanobody Fusions

[0044] To investigate the direct anti-tumour activity of selected IL1-nanobody fusions, we use human A375 melanoma cells, which were shown to be highly susceptible to IL-1-induced cytostatic effects (Morinaga et al., 1990). To allow targeting of mutant IL-1 family members to the A375 cells, a stable A375 clone expressing a cell surface marker to which high-affinity nanobodies are already available (i.e. CD20) is generated. The sensitivity of this cell line, as compared to the parental A375 cells, to the antiproliferative effect of the mutant IL1-nanobody fusion, is investigated in vitro using the XTT proliferation assay. In vivo anti-tumour activity of the mutant IL-1-nanobody fusions is investigated using an A375 xenotransplant model. Briefly, athymic nude mice are inoculated subcutaneously with A375 cells (parental or expressing a surface marker for targeting) and tumor growth is monitored for four weeks in animals treated with PBS or mutant IL1-nanobody fusions.

Example 7: Extension of Principle to IL18: Application in Tumor Models

[0045] To assess the indirect anti-tumour activity of IL1 family members, experiments are conducted to address the efficacy of selected mutant IL-18-nanobody fusions using the Meth A syngeneic mouse sarcoma model according to the protocol that was used previously to demonstrate anti-tumour activity of IL-18 (Micallef et al., 1997). IL18 variants used in these experiments consist of mutant IL-18s fused to nanobodies targeting immune cells with tumoricidal properties (i.e. CTLs, NK-cells). The mice are treated with the construct, and a significant reduction of the tumor is noted when compared to the mock treated control.

REFERENCES

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