Patent Application: US-201615278854-A

Abstract:
this disclosure relates to a modified α - helical bundle cytokine , with reduced activity via an α - helical bundle cytokine receptor , wherein the α - helical bundle cytokine is specifically delivered to target cells . preferably , the α - 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 α - helical bundle cytokine to a targeting moiety , preferably an antibody . this disclosure relates further to the use of such targeted modified α - 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 .

Description:
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 . the nanobody 4 - 10 is also described in zabeau et al . ( 2012 ). 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 . 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 ). the anti her2 scfv was obtained from andrea plückthun ( wörn 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 . control nanobody against gfp was obtained from katrien van impe ( university ghent ). the ifnα2 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 . 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 .) the coding sequence of the ifnα2 , 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 : 5 ′ ggggggtccggaccatcaccatcaccatcaccatcaccatcaccctgcttctcccgc ctccccagcatcacctgccagcccagcaagtgatagcctggaatttattgc3 ′ ( seq id no : 1 ), reverse : 5 ′ cgtctagatcattccttacttcttaaac3 ′ ( 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 . in a similar way , the human mutant q124r was fused to the 1r59b nanobody and to the anti - pd - l2 nanobody . 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 - hi s - pas - ybbr - ifna2 - l i 53a 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 . alternatively , sequences encoding the different nanobody - ifn fusions were subcloned into the baculovirus transfer plasmid pbac - 3 ( novagen ). proteins were produced by insect cells using the bacvector kit ( novagen ) and purified to homogeneity using the hispur ni - nta purification kit ( pierce , thermo scientific ) and gel filtration . protein concentrations were measured by absorbance at 280 nm . the hl116 clone ( uzé 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 & amp ; d and streptavidin - apc ( bd bioscience ). 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 . 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 . the mouse btg9a cells were described in uzé et al . ( 1990 ). 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 . 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 . the expression of the interferon inducible gene 6 - 16 was quantified by rt - pcr relative to gapdh or β - 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 ). 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 β - 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 ); β - actin forward : aga - ggg - aaa - tcg - tgc - gtg - ac ( seq id no : 5 ); β - 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 β - 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 ). the antiviral assay was performed using the emc virus and scoring the virus replication - dependent cytopathic effect as described in stewart ( 1979 ). btg9a cells expressing human her2 were treated with 200 pm to 2 nm of 1r59b - ifna2 - q124r for 10 to 30 minutes . cells were lysed in ripa , and analyzed by western blot on an odyssey fc ( licor bioscience ) after 7 % sds - page ( 40 μg / lane ). phospho - 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 ). stat1 phosphorylated on y701 were detected by facs using the stat1 - py701 ( pe ) ( becton dickinson # 612564 ) and the manufacturer instruction for the phosflow ™ technology . the sequence of the targeted leptin constructs is given in fig1 . the l86 that is indicated is the amino acid that is mutated either to s or n . fig1 shows a schematic representation of the nanobody - ifn fusion proteins constructed with either ifnα2 wild - type or the l153a and r149a mutants . ifn activity of the nanobody - ifn fusion proteins is targeted toward murine leptin receptor expressing cells the three nanobody fusion proteins with ifnα2 wt , ifnα2 l153a or r149a were assayed on both hl116 and hl116 - mlr - clone 10 cells , which express the murine leptin receptor . the ifnα2 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 ( fig2 a 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 fig2 a with fig2 c and fig2 b with fig2 d ). 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 . 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 - ifnα2r149a 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 - ifnα2r149a 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 fig2 b and 2d , black curves ). fig3 shows clearly that the renilla luciferase activity is not induced upon stimulation of the co - culture with the nanobody - ifnα2r149a , indicating that the targeted ifn activity is delivered only on cells expressing the antigen recognized by the nanobody . 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 ( fig4 a and 4b ). in order to prove that the targeting was nanobody specific , hl116 cells expressing the mlr were incubated for 6 hours with either the ifn - α2 ( 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 fig5 , 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 . 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 ( fig6 ). the ifn activity of the nanobody - ifn fusion proteins on cells expressing the leptin receptor is mediated by both ifn receptor chains 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 . fig7 shows that both anti - ifnar1 and anti - ifnar2 neutralizing antibodies inhibit the ifn activity of the nanobody - ifna2 - r149a . target - specific induction of antiviral activity by 4 - 11 - ifna2 - r149a in cells expressing the murine leptin receptor 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 fig8 . 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 . 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 - α2 ( 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 . 9 × 10 3 and 478 × 10 3 , respectively as reported by gaborit et al . ( 2011 )). fig9 a and 9b show the ec 50 determination of ifna2 activity ( fig9 a ) and 2r5a - ifna2 - r149a activity ( fig9 b ) for the induction of the ifn - inducible gene 6 - 16 on bxpc3 and bt474 cell lines . fig9 a shows that bxpc3 and bt474 cell lines exhibit the same sensitivity to ifn - α2 . fig9 b 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 . 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 . 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 fig1 . there is clearly a targeting - specific induction in the her2 - expressing cells , whereas there is no significant expression detected in untransfected btg9a cells . 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 fig1 . 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 . to check whether targeting of her2 is resulting in her2 activation , her2 phosphorylation was controlled in targeted cells . the results are shown in fig1 , clearly demonstrating that no phosphorylated her2 could be detected in 1r59b - ifna2 - q124r targeted cells , irrespective of the concentration or time of treatment . the anti pd - l2 nb122 - ifna2 - q124r construct activity is targeted on mouse primary cells expressing pd - l2 cells from a mouse peritoneal cavity were isolated and treated in vitro with nb122 - ifna2 - q124r or natural mifnα / β 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 . the pd - l2 - positive cell population represents 20 % of the total cell population present in the mouse peritoneal cavity . the results are shown in fig1 . 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 . 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 . in vivo injection of 122 - ifna2 - q124r construct induces an ifn response only in pd - l2 - expressing cells mice were injected ( ip or iv ) with either pbs , nb122 - ifna2 - q124r or a control nb ( against gfp ) fused to ifna2 - q124r . thirty minutes post - injection , mice were killed , cells from the peritoneal cavity were recovered by washing the peritoneal cavity with pbs , fixed ( phoslow fix buffer i bd # 557870 ), permeabilized ( phosflow penn buffer iii , bd # 558050 ), labelled with abs against pd - l2 ( apc ) ( bd # 560086 ) and stat1 - py701 ( pe ) ( bd # 612564 ) and analyzed by facs . the results are shown in fig1 . 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 pd - l2 - positive cell population ) can be seen when the mice are injected with the targeted mutant ifn . 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 . fig1 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 . targeting of mutant leptin to the leptin receptor , using a truncated tnfα receptor 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 . additional transfection of ba / f3 - mlr cells with the human tnfα receptor 1 ( htnfr1 ) lacking its intracellular domain introduces a non - functional receptor , which can function as a membrane - bound extracellular marker . 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 . chimeric proteins were produced by transient transfection of hek293t cells with expression plasmids . supernatant was 0 . 45 μm 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 fig1 , for two experiments using a different leptin construct ( see fig1 ). 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 - 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