Patent Application: US-31237403-A

Abstract:
the invention provides a protein framework which allows active polypeptides e . g . ligands or antigens to be displayed at increased concentration . the inventors show that the lectin binding domains of collectins can be replaced by a polypeptide of interest and that polypeptide can be multimerised by the framework of the collectin and as a result displayed in greater number on a single structure . the inventors show that the activity of polypeptides such as those of the tnf superfamily are significantly enhanced when displayed in this way .

Description:
specifically , and by way of example , the present inventors have generated two forms of soluble cd154 ( fig1 a ); the first is a novel dodecameric fusion protein between lung surfactant protein - d ( sp - d ) and cd154 ( sp - d - cd154 ), and the second is a trimeric form of cd154 ( tcd154 ). these two forms of cd154 allowed the direct investigation of the effect of cd40 oligomerisation on the downstream signalling events without the use of cross - linking antibodies . moreover , to gain insights into the mechanism by which multimerisation enhances the biological activity of cd154 , the affinity and kinetics of the interaction between soluble trimeric and dodecameric forms of cd154 and cd40 were determined . to express a dodecameric form of cd154 , the lectin domains of sp - d were replaced by the c - terminal extracellular domain of murine cd154 . sp - d is a c - type lectin produced by epithelial cells , mainly in the lung , that preferentially forms dodecamers , consisting of four trimeric subunits ( fig1 a ) [ 16 ]. sp - d binds to pathogenic micro - organisms in the lung and enhances their uptake and killing by alveolar macrophages and neutrophils [ 17 ]. the lectin domains of sp - d perform a dual function ; the binding to carbohydrate structures on invading micro - organisms as well as the interaction with receptors on cells of the innate immune system [ 17 - 19 ]. the sp - d polypeptide chain consists of an n - terminal region , which forms inter - chain disulphide bonds that stabilises the overall structure , a collagenous region , an a helical coiled - coil and a c - terminal lectin domain [ 20 , 21 ]. the trimerisation of the lectin domains is mediated by the a helical coiled - coil , referred to as the neck region [ 21 ]. the sp - d - cd154 fusion protein preserves the orientation of cd154 with respect to cd40 binding and thus mimics the orientation ( type ii ) of membrane - bound cd154 . a construct expressing soluble trimeric cd154 ( tcd154 ) was also prepared which consisted of the extracellular domain of cd154 fused at its n - terminus to the neck region of sp - d . analysis of the purified sp - d - cd154 and tcd154 by sds - page under reducing conditions revealed bands corresponding to proteins with a molecular mass of ˜ 58 and ˜ 30 kda , respectively ( fig1 b ). these values are consistent with the predicted molecular mass of the polypeptide chains ( tcd154 , 28 kda ; sp - d - cd154 , 48 kda ), plus 1 ( tcd154 ) or 2 ( sp - d - cd154 ) typical n - linked carbohydrates . under non - reducing conditions , sp - d - cd154 gave four other bands in addition to the ˜ 58 kda band , corresponding to higher molecular mass proteins (& gt ; 58 kda ), consistent with the presence of inter - chain disulphide bonds ( fig1 b ). a previous study has shown that the substitution of the two conserved cysteine residues within the n - terminal region of sp - d with serine resulted in the production of exclusively trimeric form of sp - d , suggesting that these residues are required for the assembly of the four trimeric subunits into a dodecamer [ 20 ]. the apparent molecular mass of sp - d - cd154 determined by size - exclusion chromatography under non - denaturing conditions was ˜ 600 kda , consistent with assembly of sp - d - cd154 into a dodecamer ( fig2 a ). under the same chromatography conditions tcd154 had an apparent molecular mass of ˜ 100 kda , suggesting that it forms a non - covalent homotrimer ( fig2 b ). oligomeric requirement of cd154 for the induction of b cell proliferation and expression of icam - 1 , cd86 and mhc class ii both tcd154 and sp - d - cd154 induced the proliferation of murine splenic b cells in a concentration dependent manner ( fig3 ). this effect was observed with either whole splenic cultures , or purified b cells ( data not shown ). multimeric sp - d - cd154 was ˜ 8 - fold more potent than tcd154 in inducing b cell proliferation ( fig3 ). furthermore , the proliferative response elicited by sp - d - cd154 or tcd154 was completely abolished by the addition of anti - cd154 mab ( mr1 ), confirming that this response is entirely dependent on cd154 and not any other part of the fusion protein ( data not shown ). the inventors then examined it other cd40 - mediated functions are also influenced by the oligomeric nature of cd154 . cd40 signaling upregulates the expression of costimulatory molecules on b cells and other antigen presenting cells , a process required for the priming and activation of both cd4 and cd8 t cells [ 4 - 6 , 8 ]. the inventors analysed the expression of icam - 1 , cd86 and mhc class ii on b cells 24 hours after incubation with either tcd154 or sp - d - cd154 ( 5 nm ). both tcd154 and sp - d - cd154 triggered upregulation of icam - 1 , cd86 and mhc class ii , however when compared to tcd154 , sp - d - cd154 consistently induced higher levels of icam - 1 and cd86 expression ( fig4 a ). when compared to untreated cells , a 3 . 8 - and 3 . 6 - fold increase in the level of icam - 1 and cd86 , respectively , were obtained using sp - d - cd154 , whereas stimulation with tcd154 produced a 2 - and 1 . 4 - fold increase in the level of icam - 1 and cd86 , respectively . in contrast , tcd154 and sp - d - cd154 induced similar levels of mhc class ii expression ( fig4 a ). analysis of the forward scatter of b cells ( fig4 b ), a measure of their relative size and activation status , revealed that activation with tcd154 triggered only a small increase in their size ( mean forward scatter = 368 ), when compared to cells activated with sp - d - cd154 ( mean forward scatter = 409 ). the inventors addressed whether the differences in the activities of sp - d - cd154 and tcd154 can be attributed to the activation of nf - κb . oligomerization of cd40 results in the recruitment of several members of the traf family leading to the activation of nf - κb [ 1 ]. nf - κb is normally sequestered in the cytoplasm through interaction with iκb proteins [ 22 ]. phosporylation of iκb proteins leads to their degradation via a proteosome - mediated pathway , resulting in the release and translocation of nf - κb into the nucleus , where it can activate the transcription of target genes [ 22 ]. the inventors &# 39 ; results demonstrate that both tcd154 and sp - d - cd154 were equally effective in inducing rapid phosphorylation of iκbα ( followed by its degradation ( fig5 ). these results suggest that the differences in the downstream activities of sp - d - cd154 and tcd154 are unlikely to be due to differential iκbα phosphorylation , and imply the involvement of other cd40 signalling pathways , such as the activation of jak3 / stat [ 23 ], c - jun n - terminal kinase [ 24 ], or p38 mitogen activated protein kinase ( mapk ) [ 25 ]. the observation that tcd154 and sp - d - cd154 were equally effective in upregulating the expression of mhc class ii in b cells ( fig4 a ) indicates that the signalling pathways that mediate this process are likely to be distinct from those required for triggering the proliferation , or the expression of icam - 1 and cd86 . in agreement with this , an inhibitor of p38 mapk was shown to inhibit cd40 - induced b cell proliferation and upregulation of icam - 1 expression , but not cd40 , cd95 , dr3 , traf1 / 4 or ciap2 expression , suggesting that cd40 - induced functions are mediated by different signaling pathways [ 25 ]. the lower activity of tcd154 when compared with sp - d - cd154 could be the result of its relatively low affinity for cd40 . to address this question the inventors analysed the affinity and kinetics of the interaction between tcd154 or sp - d - cd154 and cd40 using the biacore ™ biosensor , which measures protein - protein interaction in real time . a murine anti - human fc mab was covalently coupled to the dextran matrix , and either tcd154 or spd - cd154 was then injected over this mab in order to determine the level of non - specific binding . specific binding was determined by first injecting murine cd40 - human fc fusion protein which bound to the immobilised anti - human fc mab , and then injecting ( 31 . 3 - 250 nm ) tcd154 or spd - cd154 ( fig6 ). co - injection of the anti - cd154 mab mr1 and tcd154 or sp - d - cd154 completely abolished the binding of tcd154 or sp - d - cd154 to cd40 ( data not shown ). the association ( k a ) and dissociation ( k d ) rate constants were determined using the biaevaluation 2 . 1 software ( biacore ). tcd154 bound to cd40 with high apparent affinity ( k d = 2 nm ) and dissociated very slowly ( k d = 1 . 8 × 10 − 4 s − 1 ; t 1 / 2 ˜ 64 min ). using the same technique , the inventors have previously shown [ 26 ] that soluble trimeric ox40 ligand , a member of the tnf superfamily , binds to its receptor with a similar apparent affinity ( k d = 3 . 8 nm ). sp - d - cd154 also bound to cd40 with a high apparent affinity ( k d = 1 . 3 nm ), and dissociated with similar kinetics ( k d = 1 . 4 × 10 − 4 s − 1 ; t 1 / 2 ˜ 83 min ) to tcd154 . these results suggest that the higher activity of sp - d - cd154 when compared to tcd154 is unlikely to be due to differences in their apparent affinity ( avidity ) for cd40 , since both soluble forms of cd154 bound to cd40 with high avidity and dissociated very slowly following binding . sp - d - cd154 can potentially bind to twelve cd40 molecules , compared to three molecules with tcd154 [ 9 ], implying that the extent of receptor oligomerisation may influence the signals generated by cd40 . within the x - shaped sp - d molecule , the adjacent arms are separated by a distance of either ˜ 20 nm or ˜ 90 nm ( fig1 a ) [ 16 ]. thus if all four arms of sp - d - cd154 engage cell surface - expressed cd40 , two clusters are likely to form each with six closely associated cd40 molecules . the cytoplasmic adapter proteins , traf2 and traf3 have been shown to bind to a trimerised form of the cytoplasmic tail of cd40 with low affinity ( k d = 3 - 13 μm ), and the affinity for the interaction with traf6 was estimated to be even lower , although the k d of this interaction was not determined [ 27 ]. therefore , the close association of six cd40 receptors may provide a high avidity platform , which facilitates a more stable interaction with downstream adapter proteins , such as the trafs . alternatively , the association of six or more cd40 receptors into clusters may trigger signaling more effectively by a proximity induced mechanism as described for the activation of caspase 8 [ 28 ]. taken together , the data presented here demonstrates conclusively that tcd154 , which binds to cd40 with high apparent affinity , is sufficient to trigger signalling in b cells , however , higher order oligomers provide a more potent stimulus . recent studies have shown that during cell - cell interaction , receptors and ligands segregate within contact zones resulting in the formation of supramolecular clusters [ 29 ]. if similar clusters exist between cd154 and cd40 during cell - cell interaction , then this would generate high order oligomeric complexes that are more effective in signalling than single trimeric units . finally , the strategy described here could be adapted for other proteins and particularly for other members of the tnf superfamily , where trimeric forms are known to be ineffective [ 12 - 14 ]. the use of the sp - d multimerisation platform for the construction of soluble and highly active members of the tnf superfamily may prove to be particularly useful for the generation of immunotherapeutic agents . one potential candidate is the cd154 molecule itself , which is essential for the priming of cytotoxic t cell responses such as those required for the generation of a protective anti - tumour response [ 7 ]. the region encoding amino acid ( aa ) residues 1 - 257 of sp - d was amplified from a plasmid containing full - length human sp - d . the 5 ′ oligonucleotide introduced a xbai site , and the 3 ′ oligonucleotide incorporated a linker ( gggns ), an ecori site and a downstream bamhi site . the digested pcr fragment was ligated into pee14 ( lonza biologics ) at the xbai and bcli sites to produce pee14 / sp - d . the extracellular domain of cd154 , ( aa residues 50 - 260 ), was amplified using cdna from 48 h concanavalin a activated mouse splenocytes , introducing 5 ′ and 3 ′ ecori sites . the pcr product was cloned into the ecori site of pee14 / sp - d . the predicted amino acid sequence at the junction between sp - d and the n - terminus of cd154 is ( sp - d ) lfpng / gggns / ldkve ( cd154 ). a tcd154 construct encoding the rat cd4 leader , the α helical coiled - coil domain of sp - d ( aa 223 - 257 ) and the extracellular domain of cd154 was prepared by a three - step overlaping pcr strategy . the pcr fragment was digested with hindiii and xbai and ligated into pee14 . the expression constructs were transfected into cho - k1 as described previously [ 30 ]. recombinant proteins were purified from tissue culture supernatant by affinity chromatography using mr - 1 mab column [ 31 ]. tcd154 was further purified by size - exclusion chromatography using a superdex ™ 200 hr10 / 30 column ( amersham pharmacia biotech ab ). the extinction coefficients at 280 nm , e ( 0 . 1 %, 1 cm ) for sp - d - cd154 ( 0 . 453 ) and tcd154 ( 0 . 678 ) were estimated from the amino acid sequence using the protparam tool ( www . expasy . ch [ 32 ]). mouse splenocytes ( 5 × 10 5 / ml ) were cultured in rpmi 1640 , 10 % ( v / v ) foetal calf serum and 25 μm 2 - me using u - shaped 96 well plates . after 72 h of culture , wells were pulsed with 0 . 5 μci of [ 3 h ] thymidine for the final 16 h of culture . mouse splenocytes ( 1 . 25 × 10 6 / ml ) in 2 ml cultures were treated with either sp - d - cd154 or tcd154 ( 5 nm ) or left untreated for 24 hours . cells were incubated with pe - labelled anti - cd19 mab ( serotec ) and fitc - labelled mabs ( 10 μg / ml ) to icam - 1 ( yn1 . 4 . 7 ), cd86 ( gl - 1 ), and mhc class ii ( n22 ) in pbs , 0 . 2 % ( w / v ) bsa , 1 % ( v / v ) mouse serum . mouse splenocytes ( 5 × 10 5 / ml ) were cultured in serum free media with sp - d - cd154 , tcd154 ( 5 nm ) or media alone . cells were lysed , and the equivalent of 5 × 10 4 cells were analysed by sds - page . the levels of total and phophorylated iκb - α were detected by western blotting ( phosphoplus ® iκb - α ( ser - 32 ) antibody kit , new england biolabs ). all experiments were performed at the indicated flow rates in hepes buffered saline ( 150 mm nacl , 0 . 005 % ( v / v ) surfactant p20 ( biacore ), 10 mm hepes , ph 7 . 4 ). mabs were covalently bound to the carboxylated dextran matrix using the amine coupling kit ( biacore ) as described previously [ 26 ]. for analysis of the expression of sp - d - cd154 and tcd154 by cho - k1 clones , culture supernatant was injected at 4 μl / min ( 7 . 5 min ) over mr - 1 mab . to assess binding of sp - d - cd154 or tcd154 to murine cd40 , cd40 - human fc fusion protein was first injected over covalently bound anti - human fc mab ( sb2h2 ) at a flow rate of 5 μl / min ( 6 min ) followed by injection ( 6 min ) of sp - d - cd154 or tcd154 ( 31 . 3 nm - 250 nm ). communication between cells of the immune system through cell - cell interactions is critical for the initiation and maintenance of an appropriate immune response . cell - cell interactions are mediated by glycoproteins ( also known as receptors and ligands ) that are anchored to the cell surface of immune cells normally through a stretch of hydrophobic residues known as the transmembrane domain . a signal within an immune cell is initiated when the extracellular domain of a specific receptor is bound to a specific glycoprotein known as the ligand . it is the extracellular domain of the ligand alone that is responsible for binding to the receptor , and as a result of this , a signalling cascade is initiated which may activate for example a lymphocyte to react against an invading organism . such signals may be artificially induced , for example in order to enhance an immune response during vaccination , or to stimulate an immune response against certain diseases such as cancer , by providing an exogenous form of the stimulatory ligands . this can be achieved by preparing a soluble recombinant form of the ligand containing the extracellular receptor - binding domain , or a protein , such as an antibody fragment , that is capable of binding to the receptor and inducing signalling . many receptors , such as members of the tumour necrosis ( tnf ) receptor superfamily , require clustering to mediate their signals . this is normally attained through presentation of the natural membrane - bound ligand in a highly multimeric fashion . multimerisation is acquired at two different levels . first , certain ligands adopt a native oligomeric fold , for example trimers . second , these ligands when presented on the cell surface appear as an array of highly multimeric proteins . this invention describes methods - to generate soluble proteins ( ligands ) that artificially mimic the highly multimeric natural membrane - bound forms , with the aim of using these proteins therapeutically to modulate immune responses . the following is an example of how this technology may be applied to the cd40 ligand ( also known as cd154 ) molecule . the same strategy could be applied to other members of this family of molecules including , cd27 ligand , cd30 ligand , cd95 ligand ( fas ), cd134 ligand , cd137 ligand and trail , all of which have been shown to have important roles in immune regulation as well as the control of survival and death of normal and malignant cells . cd40 is a member of the tnf receptor superfamily and is expressed on a number of cells including b cells , various antigen presenting cells ( apcs ) fibroblasts , epithelial cells and endothelial cells . cd40 binds to cd154 , a member of the tnf family that is expressed mainly on activated cd4 + t helper cells . cd40 - cd154 interaction plays an important role in the generation of humoral and cellular immune responses . mice that have been rendered deficient for cd40 or cd154 are immuno - compromised with respect to antibody production and ig - class switching , and are unable to mount an effective response to infectious pathogens such as leishmania . humans that have mutations in dc154 develop a severe form of immunodeficiency , known as hyper igm syndrome , that is characterised by high levels of igm and low levels of iga , ige and igg , the absence of germinal centres and the inability to mount a thymus - dependent humoral response . it is now clear that cd40 plays a critical role in activating apcs and is important for generating cytotoxic t lymphocytes ( ctls ). recent date show that during an immune response an activation signal is delivered to the apc via cd40 through interaction with cd154 on the t helper cell . this activation signal “ conditions ” the apcs and empowers them to stimulate ctls . although the nature of the “ conditioning ” induced by cd40 - triggering on apcs is not fully understood , it probably involves a combination of improved antigen processing , increased expression of co - stimulatory and adhesion molecules and up - regulation of cytokine production . there is now convincing evidence to show that professional apcs , such as dendritic cells are capable of presenting tumour antigens to ctls by a process known as cross - priming . an antibody that cross - links cd40 on the surface of apcs can therefore replace the requirement of the help provided by cd4 + t helper cells expressing cd154 . these observations suggest that in cases where t helper responses are compromised or absent , as the case may be in cancer , stimulation of cd40 on professional apcs could be used to provoke an immune response . recently , nakajima et al . have shown that transfection of the otherwise non - immunogenic p815 mastocytoma with the cdna for membrane - bound full length cd154 triggers a specific immune response that results in their prompt rejection . futhermore these cd154 expressing p815 tumour cells were able to elicit protective immunity against subsequent challenge with parental p815 cells , thus leading the authors to suggest that this approach could be useful as a new strategy for immuno - gene therapy for tumours . it has recently been demonstrated that treatment of b - cell lymphoma - bearing mice with cd40 monoclonal antibody ( mab ) generates a rapid cd4 + t cell independent ctl response capable of eradicating the syngeneic tumour cells . the therapeutic activity of the cd40 mab is dependent on the presence of an intact fc region , which is required for the cross - linking of several cd40 molecules on the apcs . these results are consistent with the current understanding of the requirements for signalling by members of the tnf receptor superfamily . however , the immunogenicity of either murine or chimerized mabs together with any potential immunotoxicity resulting from cross - linking of fc receptors on effector cells will very likely preclude their use in man . a method for the preparation of soluble recombinant highly multimeric cd154 the extracellular domain of cd154 has been shown to form a homotrimer and to adopt a similar fold to that of tnf - α and lymphotoxin - α . a number of recent studies utilising soluble tnf - α , fas ligand and cd30 ligand ( hargreaves and al - shamkhani , unpublished ) have suggested that further cross - linking of the timers may be necessary to produce the full biological activity of the natural membrane - bound form . therefore , a novel highly multimeric soluble fusion protein consisting of the extracellular domain of cd154 and specific domains of lung surfactant protein - d was produced in chinese hamster ovary cells ( cho ). this chimeric protein is likely to be non - immunogenic as all of its components will be of human origin . the spd - cd154 chimeric protein was purified by affinity chromatography . characterisation by gel filtration chromatography and sds - page shows that the purified protein is a homogenous product consisting of 12 polypeptide chains that associate together to form 4 trimeric cd40 ligand subunits . an in vitro b cell proliferation assay was used to assess the biological activity of spd - cd154 . the inventors have shown that spd - 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