Patent Application: US-66163305-A

Abstract:
chemokine binding activity of viral tnf receptors and related proteins . the invention relates to a c - terminal domain of viral tumour necrosis factor receptors crmb or crmd or ctd homologues from poxvirus and their functional homologues , including derivatives , and fragments , for use in binding chemokines and their analogues and / or to enhance the immunomodulatory properties of tnfrs or in bloking binding of chemokines to their corresponding cell surface receptors and / or to modulate chemokine biological activity .

Description:
the invention , and materials and methods applicable to carrying out embodiments thereof , are further illustrated in the following examples , but without intent to limit its scope . cpv , ev and vv were propagated in vitro by infecting confluent monolayers of bsc - i cells . orf 264 of strain cms of camelpox virus , corresponding to the crmb gene was amplified by pcr using oligonucleotides cmlv 264 eco ( 5 ′- gcggaattcatgaagtccgtattatactcg ) and cmlv 264 xho ( 5 ′- gcgctcgagtaaaaagtgggtgggtttgg ) and purified cmlv dna as a template . the pcr product was cloned into ecori / xhoi digested pbaci ( novagen ) to generate plasmid pra1 . the absence of mutations in the amplified gene was confirmed by dna sequencing . the dna corresponding to the vav ( strain bangladesh 1975 ; orf 188 ) was obtained by multiple - site directed mutagenesis of plasmid pra1 using the “ quikchange multi site - directed mutagenesis kit ( stratagene )” following the manufacturer &# 39 ; s instructions . the mutations introduced are shown in fig6 . after several consecutive rounds of site - directed mutagenesis , plasmid pra105 was obtained . this plasmid contains the sequence coding for vav ( bsh 1975 strain ) crmb fused to a c - terminal his tag provided by the original pbac1 plasmid . the presence of all the mutations and absence of unwanted additional mutations was confirmed by direct sequencing . 1 . 3 .— construction of recombinant baculoviruses and vaccinia viruses expressing crmd from ev or cpv for expression in the baculovirus system , dna encoding full length or truncated versions of crmd ( table 3 ) was pcr - amplified with the specific oligonucleotides ( table 4 ), and cloned into pbac - 1 . recombinant baculoviruses were generated by homologous recombination in sf21 insect cells transfected with recombinant pbac - 1 plasmids and the linearized baculovirus dna as described ( alcami et al ., 1998 ). vtnfrs were also expressed from a w expression system . the genes of interest were cloned into pmj601 for expression of the gene from a strong vv promoter ( davison & amp ; moss , 1990 ). the gene of interest is inserted into the thymidine kinase locus of the vv genome by homologous recombination , and the recombinant w selected in the presence of bromodeoxiuridine and identified by colour selection ( expression of β - galactosidase and staining with x - gal ). vtnfrs were expressed from vv western reserve , a virus strain that does not encode tnf binding activity ( alcami et al ., 1999 ). 1 . 4 .— generation of recombinant baculoviruses expressing vav crmb , vav crmb crd ( 1 - 4 ), vav crmb ctd , ev e12 , ev e184 , cpv v218 and vav crmb crd ( 1 - 4 ) fused to cpv 35 kda protein the full length vav crmb gene fused to a c - terminal his tag was subcloned into ecori / sphi digested pfastbac1 ( invitrogen ) to generate pra107 . the n - terminal domain of vav crmb including the four crds and corresponding to residues 1 ( m ) to 192 ( c ) was amplified by pcr using oligonucleotides vav 188 eco ( 5 ′- gcggaattcatgaagtccgtattatacttg ) and vav 188 crds1 - 4 xho ( 5 ′- gcgctcgagacacgatgtgtcgttaactgg ) using pra107 as a template . the amplified fragment was cloned in - frame with a c - terminal his tag provided by the ecori / xhoi digested pbac1 ( novagen ) to generate pra99 . pra99 was digested with ecori / sphi and the fragment carrying the vav crmb crds 1 to 4 fused to the his tag cloned into pfastbac1 as before to generate pra106 . the ctd of vav crmb ( residues t194 to l348 ) was pvr amplified with oligonucleotides vav 188 cter - pfimi ( 5 ′- cgcccacccaatggaactaggacgaccac - taccgg ) and h347r 3 using pra107 as a template . this fragment was digested with pfmii and xhoi and cloned into pra107 digested with the same enzymes . this generates pra108 , a plasmid that encodes a fusion protein composed of the 29 n - terminal residues of vav crmb ( which includes the predicted signal peptide ) followed by the ctd of crmb and an additional his tag . the absence of unwanted mutations in pra106 , pra107 and pra108 was confirmed by sequencing the complete inserts in all cases . the ev gene e12 was amplified by pcr using oligonucleotides e1 ( 5 ′- gcggga - tccatgataaacataaacataaacacaatac ) and e2 ( 5 ′- gcggcggccgcat - taatagttctagtagcgcaag ) and purified ev ( strain naval . cam ) dna as a template . the pcr product was cloned into bam hi / not i digested pbac1 ( novagen ) generating plasmid pms51 . the e12 gene was subcloned into bam hi / xho 1 - digested pra106 to give plasmid pah18 , which contains the e12 gene fused to a c - terminal sequence coding for a his - tag in a pfastbac ( invitrogen ) backbone . the cpv brighton red strain orf v218 was pcr - amplified using oligonucleotides 5 ′ v218 ecori ( 5 ′- cgcgaattcatgatgatatacggattaatagc ) and 3 ′ v218 sali ( 5 ′- gcggtcgacaccatcgacaccactcatc ) and purified viral dna as a template . the pcr product was cloned into eco ri / xho i digested pra106 to generate plasmid pah17 , which contains the cpv v218 gene fused to a c - terminal sequence coding for a his - tag in a pfastbac ( invitrogen ) backbone . the fragment corresponding to residues s23 to v246 of the cpv ( strain brighton red ) 35 kda protein was pcr - amplified using oligonucleotides 5 ′ 35 kbr - s23 ( 5 ′- cgcctcgagtcattctcatcctcatcctc ) and 3 ′ 35 kbr (- stop )( 5 ′- cgcctcgaggacacacgctataagttttgc ) and purified viral dna as a template . the pcr product was cloned into xho i digested pra106 to generate plasmid pra109 . this plasmid carries the sequence encoding vav crmb crd ( 1 - 4 ) fused in frame to the sequence encoding cpv 35 kda vckbp without its signal peptide and with a c - terminal his tag in a pfastbac backbone ( invitrogen ). recombinant baculoviruses were obtained using the bac - to - bac expression system ( invitrogen ) as described by the manufacturer . briefly the plasmids pra106 , pra107 , pra108 , pra109 , pah17 and pah18 were transformed into competent dh10bac bacteria , were a transposition event generates the corresponding recombinant bacmids . these were purified and transfected into high5 insect cells and three days after transfection , the recombinant baculoviruses vbac106 , vbac107 , vbac108 , vbac109 , vbacah17 and vbacah18 harvested from the cell culture supernatants . these viruses were further amplified in one single step to generate a high titer recombinant virus stock for protein production . the e184 gene from ev was pcr - amplified with the oligonucleotides 5 ′ e184 ( 5 ′- gcggaattcatgtataaaaaactaataacgttt ) and 3 ′ e184 xhoi ( 5 ′- cgcctcgagaaaatcatattttgaataatatgta ) and purified ev dna as template . the pcr product was cloned into the pra106 plasmid digested with ecori and xhoi , generating pah11 . the correct dna sequence was confirmed . plasmid pah11 was used to generate the recombinant baculovirus vbacah11 , as described above , expressing the ev e184 protein fused to a histidine tag to facilitate the purification of the protein . to express the recombinant proteins , high5 cells were infected at high multiplicity ( 10 pfu / cell ) and the supernantants from infected cultures harvested at three days post infection ( d . p . i .). the presence of proteins in these supernatants was confirmed by western blot and / or , in the case of the full length and n - terminal vav crmb constructs , a tnf binding assay in solution ( alcami et al ., 1999 ). the samples were concentrated to approximately 2 . 5 ml on a stirred ultrafiltration cell ( amicon ) using ym - 3 membranes ( millipore ) and buffer exchanged into binding buffer ( 50 mm phosphate , 300 mm nacl , 10 mm imidazole , ph 7 . 4 ) on pd - 10 desalting columns ( amersham - pharamcia biosciences ). the his - tagged recombinant proteins were affinity - purified using vivapure metal chelate columns ( vivascience ) following the manufacturer &# 39 ; s recommendations . the purity and amount of purified proteins was checked on coomassie - blue stained sds - polyacrylamide gels . for tnf protection and chemotaxis inhibition assays , recombinant proteins were dialyzed against pbs . cytokine binding specificity and affinity constants were determined using a biacore x biosensor ( biacore , uppsala , sweden ). for ligand screening experiments , purified recombinant proteins were amine - coupled to cm5 chips to a level of approx . 5000 ru ( 5000 pg / mm 2 ) in each case . commercially available cytokines ( peprotech , r & amp ; d systems ) were then injected at 100 nm in hbs - ep buffer ( 10 mm hepes , 150 mm nacl , 3 mm edta , 0 . 005 % ( v / v ) surfactant p20 ; ph 7 . 4 ) at a flow rate of 5 μl / min and association and dissociation monitored . the surface was regenerated after each injection using 10 mm glycine - hcl ph2 . 0 . for kinetic analysis , the recombinant proteins were immobilised at low densities ( rmax & lt ; 200 ru ). different concentrations of the corresponding analyte were then injected at a flow rate of 30 μl / min over a 2 min period and allowed to dissociate for an additional 5 min . all biacore sensorgrams were analysed using biaevaluation 3 . 2 software . bulk refractive index changes were removed by subtracting the reference flow cell responses and the average response of a blank injection was subtracted from all analyte sensorgrams to remove systematic artifacts . kinetic data were globally fitted to a 1 : 1 langmuir model . tnf - induced cytotoxicity assays were performed on mouse l929 cells as described previously ( loparev et al ., 1998 , saraiva & amp ; alcami , 2001 , schreiber et al ., 1997 , smith et al ., 1996 ). tnf ( 20 ng / ml ) ( r & amp ; d systems ) was preincubated for 2 h at 37 c with purified recombinant proteins of complete dmem supplemented with actinomycin d ( 4 μg / ml ) ( sigma ). the mixture was then added to 2 × 10 4 cells seeded the day before in 96 - well plates and cell death assessed 16 to 18 h later using the “ celltiter 96 ® aqueous one solution cell proliferation assay ” ( promega ) following the manufacturer &# 39 ; s recommendations . the migration of molt - 4 cells was assessed using 24 well transwell ® plates with 3 μm pore size filters ( costar ) as described previously ( zaballos et al ., 1999 ). briefly , human ccl25 ( 100 nm ) ( r & amp ; d systems ) alone or in the presence of increasing amounts of purified recombinant protein was incubated at 37 c for 30 minutes and placed in the lower compartment . after this period , 5 × 10 5 molt - 4 cells were added in 100 μl complete rpmi containing 0 . 1 % fcs to the top well and the plate incubated in a 37 ° c ., 5 % co 2 , 95 % humidified incubator . migration of molt - 4 cells towards the bottom well was determined after 4 hours by flow cytometry . searching for novel viral secreted proteins that bind chemokines , we performed cross - linking assays with 125i - cxcl8 and identified a novel secreted vckbp encoded by the poxvirus ev ( fig3 a ). this activity was absent in vv samples and of higher molecular size than the 35 kda vckbp encoded by vv and other poxviruses , a protein that can cross - link cxcl8 but does not block its biological activity due to its low affinity for cxcl8 ( alcami et al ., 1998 , graham et al ., 1997 , lalani et al ., 1998 ). unexpectedly , we found that the vtnfr crmd encoded by ev , known to bind tnf , has the additional property of binding cxcl8 ( fig3 b ). using truncated versions of the crmd protein expressed in the vv expression system , we have shown that the 3 n - terminal crds of crmd are necessary to block tnf activity ( fig4 a , b , c ) and the ctd is not necessary for tnf binding but confers crmd the ability to bind chemokines ( fig3 b ). different domains of crmd appear to be involved in tnf and chemokine binding since cross - linking of cxcl8 to crmd cannot be blocked in the presence of tnf ( fig3 c ) and binding of tnf to crmd is not inhibited in the presence of cxcl8 ( not shown ). using the surface plasmon resonance ( spr , biacore x ) technology we have tested the potential interaction of purified ev crmd to all commercially available chemokines of human and mouse origin , and have identified that crmd binds with high affinity several chemokines ( fig5 ). to test whether the other vtnfr encoding an extended ctd binds chemokines , we expressed crmb from cpv in the baculovirus system and showed that it binds cxcl8 in cross - linking assays ( fig3 d ). crmb is also encoded by the human pathogen vav , the causative agent of smallpox . we have generated the crmb gene of vav by extensive site - directed mutagenesis of the crmb gene from the related camelpox virus ( fig6 ) and expressed the protein in the baculovirus system . we first tested that purified vav crmb protein binds tnf ( fig7 ) and inhibits tnf biological activity ( fig8 a ). crmb was also tested for binding to all available human chemokines by spr and we demonstrated that crmb binds with high affinity a number of chemokines ( fig9 ). a truncated version of vav crmb lacking the ctd did not bind chemokines ( fig7 ). moreover , expression and purification of the ctd of vav crmb has demonstrated that the ctd encodes the chemokine binding activity of crmb ( fig7 ). this is corroborated by spa analysis showing that tnf and chemokines bind to different sites in crmb ( not shown ). 2 . 3 .— the vtnfr crmb encoded by vav blocks migration of molt4 cells induced by ccl25 in vitro the high affinity of the vtnfrs crmb and crmd for some chemokines suggests that these viral proteins may act as decoy receptors sequestering chemokines and preventing chemokines from binding to their specific receptors on leukocytes and inducing signals that trigger cell migration . we show that vav crmb inhibits the migration of molt4 cell , expressing relevant receptors , in response to the chemokine ccl25 ( fig8 b ). 2 . 4 .— proteins related to the ctd of vtnfrs ( ctd homologues ) bind chemokines as indicated above , three proteins encoded by poxviruses have amino acid sequence similarity to the ctd of the vtnfrs ( crmb and crmd ) ( fig1 and 2 ). these proteins , named ctd1 , ctd2 and ctd3 have an n - terminal signal peptide suggesting that they are secreted . expression of two of these proteins , ev e12 ( ctd1 ), ev184 ( ctd2 ) and cpv b21r ( ctd3 ), in the baculovirus system has shown that both proteins are secreted into the medium . purified ev e12 protein was tested for binding to all mouse chemokines and found to bind several chemokines with high affinity ( table 2 ). in addition , we have determined by spr that the purified proteins cpv b21r and v e184 bind ccl21 , ccl24 , ccl25 , ccl27 , ccl28 , cxcl10 , cxcl11 , cxcl12β , cxcl13 and cxcl14 from mouse , and human ccl26 the vv wr b7r ( ctd2 ) has been shown to be translocated to the lumen of the endoplasmic reticulum and to be retained inside the cell rather than being secreted ( price et al ., 2000 ). some of the ctd homologues may function to block the activity of chemokines such as ccl27 that have been known to be expressed inside the cell as well ( gortz et al ., 2002 ). a vv mutant lacking the b7r gene is attenuated in a murine intradermal model ( price et al ., 2000 ). 2 . 5 .— the vtnfrs crmd from ev and crmb from vav bind other tnf ligand superfamily members tnf is a member of a large family of immune mediators with structural similarities , known as the tnf ligand ( tnfl ) superfamily ( locksley et al ., 2001 , wallach , 2001 ). ev crmd and vav crmb were tested by spr for binding to all commercially available tnf ligand superfamily members and found to bind april ( tnfl13 ) ( crmd kd 110 μm ; crmb kd 2 nm ) and light ( tnfl14 ) ( crmd kd 140 nm ; crmb kd 2 nm ). this suggests that crmd and crmb ( and maybe other vtnfrs ) may inhibit the biological activity of several tnf ligand superfamily members . 2 . 6 .— the ctd of vtnfrs may influence the ability of the crds to bind tnf with high affinity as shown above , a truncated vtnfr crmd comprising the n - terminal crd ( 1 , 2 ) looses affinity for tnf and does not block tnf biological activity , while crd ( 1 - 3 ) binds tnf and inhibits its activity ( fig4 a , b , c ). surprisingly , when crd ( 1 , 2 ) is expressed fused to ctd it recovers tnf inhibitory activity , showing that ctd may enhance the tnf binding activity of the n - terminal crds of the vtnfr ( fig4 d ). 2 . 7 .— virus - encoded proteins are formed of domains that bind immune ligands independently the data shown here indicate that the vtnfrs crmb and crmd are composed of two independent domains ( fig1 and 2 ). the n - terminal crds have the ability to bind tnf while the ctd binds chemokines in an independent fashion . this is demonstrated by the finding that crd ( 1 , 4 ) from crmb retains tnf binding activity and ctd from crmb has chemokine binding activity when expressed independently . the concept that these regions of viral proteins represent structural modules or domains that bind immune mediators is emphasized by the finding that : ( 1 ) purified ctd of vav crmb binds chemokines ( fig7 ); ( 2 ) the ctd of crmb and crmd encoded by cpv can be exchanged and still confer chemokine binding activity ( not shown ); ( 3 ) three different proteins related to vtnfr ctd ( ev e12 , ev e184 and cpv b21r ) encode chemokine binding activity ( table 2 ); and ( 4 ) fusion of the cpv 35 kda vckbp to the crd ( 1 , 4 ) of vav crmb confers this protein the ability of binding chemokines without affecting the tnf - inhibitory activity ( fig1 ). therefore , these viral domains may be exchanged or combined to generate immune modulatory proteins that block the activity of several cytokines . we propose that the ctd of vtnfrs defines a novel protein structure / domain that binds immune proteins such as chemokines or other proteins involved in the immune system and confers vtnfrs the ability to bind other immunomodulatory proteins in addition to tnf . alcami , a . 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