Patent Application: US-98347498-A

Abstract:
a recombinant heteromultimeric protein including at least a polypeptide fusion molecule a consisting of a c4bp α - chain c - terminal fragment and a polypeptide fragment heterologous to said α - chain , and a polypeptide fusion molecule b consisting of a c4bp β - chain c - terminal fragment and a polypeptide fragment heterologous to said β - chain , wherein and are linked in the c - terminal portion to form said multimeric protein .

Description:
the advantage of using cr1 in a multimeric construct according to the invention results from the studies carried out by the inventors on the physiological fate of cr1 in the normal subject . thus , the inventors have been able to determine the parameters of a physiological catabolism of erythrocytic cr1 and its relationships with the genetic polymorphism of erythrocytic cr1 density . they have also been able to clarify the catabolism of erythrocytic cr1 in lupus patients , that is patients who are suffering from disseminated lupus erythematosus . the distribution among lupus patients and normal subjects of the different genotypes of length polymorphism and cr1 c3b / c4b - binding site number polymorphism has also been studied . recombinant cr1 molecules which make it possible to change the erythrocytic cr1 density in order to restore the physiological state of the erythrocytes or to produce “ armed ” erythrocytes having “ supraphysiological ” densities of cr1 have then been prepared . the potential of soluble cr1 was demonstrated in different models , in particular models of experimental myocardial ischemia and of arthus phenomenon . a molecule of multimeric soluble cr1 is produced and its anti - inflammatory power , its plasma life span and its distribution space are studied in the animal . reduced monomers are coupled chemically to erythrocytes by means of their free sh group . in this way , erythrocytes are armed with supraphysiological densities of cr1 , with the cr1 being presented in a functional manner , and the ability of the erythrocytes to bind artificial c3b - opsonized hbs antigen / anti - hbs antibody immune complexes is then studied . the results which were obtained with an anti - rh ( d ) antibody are shown in example i below . the constructs which were used for carrying out the c4bp - cr1 transduction are depicted in fig3 , 4 and 8 , in plasmids pmamneo , pcdm8 and pkc3b . the complementary dna encoding cr1 had been inserted into the xho i and not i sites in plasmid pcdm8 ( due to the kindness of t . j . bartow , d . t . fearon and w . wong , john hopkins hospital , baltimore , u . s . a .). the sequence encoding the extramembrane moiety of cr1 is extracted by digesting this plasmid with the restriction enzymes xho i and bal i in a moiety of c4bp is amplified using the primers ( seq id nos 1 and 2 respectively ) 5 ′- gagacccccgaaggctgtga - 3 ′, and 5 ′- ctcgagttatagttctttatcccaagtgg - 3 ′, with this second primer containing a stop codon and a xho i restriction site . the sequences encoding the c - terminal moiety of c4bp and the extramembrane moiety of cr1 are inserted into pmamneo ( clontech , palo alto , usa ) at the xho i site ( fig3 ) cr1 - c4bp by digestion with xho i , and inserted into plasmid pcdm8 ( invitrogen , san diego , usa ). the sequence encoding the cr1 - c4bp fusion protein was extracted from pmamneo cr1 - c4bp by digestion with xho i and inserted into plasmid pcdm8 ( invitrogen , san diego , usa ) ( fig4 ). the sequence encoding the cr1 - c4bp fusion protein was extracted from pmamneo cr1 - c4bp by digestion with xho i , and inserted into plasmid pkc3b ( fig8 ). a c - terminal fragment of the α chain of c4bp was recopied from genomic dna by means of pcr . it is found in one single exon . the minimum size is beyond the second cysteine proceeding from the c - terminal end , with the optimum size being a few amino acids beyond that , creating a spacer of from 5 to 10 amino acids , that is 58 aa in all . the maximum size selected is of 6 scrs , in order to avoid the c3b - c4b - binding site . this maximum fragment is synthesized from a cdna prepared from c4bp mrna , since the fragment is made up of several exons . the optimum fragment of the c - terminal moiety of c4bp is recopied once again by means of pcr using primers which are provided at their ends with arms containing enzyme restriction sites which are adequate for inserting the fragment into a given vector which already contains the gene for the protein which it is desired to multimerize . an enzyme site close to the c - terminal moiety of this protein , or which is located in its extramembrane moiety , is selected which enables the multimerizing fragment to be inserted in the 3 ′ position . the 3 ′ end of the multimerizing fragment is linked either to a site in the vector or to a site beyond in the gene for the protein of interest . that part of the gene for the protein of interest which is located 3 ′ of the multimerizing fragment is in any case no longer translated since the multimerizing fragment contains a stop codon . it is therefore in this way possible to modify an expression vector containing the gene for a given protein very readily by simply inserting the fragment without any other alteration . the vectors pcdm8 , st4 and pmamneo have been used for the different examples of applying the multimeric system according to the invention . the skilled person will always know how to find vectors which currently exist or which could be developed and which are / could be able to exhibit the optimum efficacy for transducing the fusion protein into cells and expressing it . heteromultimeric molecules combining erythrocytic functions and cr1 are produced within the context of preventing anti - rh ( d ) alloimmunization . they will make it possible to bind cr1 readily to erythrocytes , thereby ensuring the achievement of cr1 densities which can be fully controlled . the antibody molecule used to generate the anti - rh ( d ) scfv was produced and sequenced in philippe rouger &# 39 ; s laboratory at the institut national de transfusion sanguine [ national blood transfusion institute ] ( ints ) ( 9 ). construction of vectors which comprise the sequence encoding the anti - rh ( d ) scfv and the terminal moiety of the α chain of c4bp . an epitope site of an anti - rhesus antibody was first of all reduced down to a structure of the scfv ( denoting single - chain fv ) type for expression in e . coli by means of transfecting with a phage vector . constructs of the scfv type are antibody fragments which represent the variable moiety of the antibody and only contain one single chain . this technique has been described by g . winter ( 10 ). the sequence encoding this scfv was then transferred into an expression vector after adding the multimerizing system . we have described above the construction of expression vectors which carry the sequence encoding the scfv of the anti - rh ( d ) antibody and which are depicted in fig6 and 7 . the c - terminal moiety of c4bp was amplified using the primers ( seq id nos 3 and 4 , respectively ) 5 ′- gcggccgcagagacccccgaaggctgtg - 31 , which contains a not i restriction site , and 5 ′- ccactttggataaagaactataa - 3 ′, which contains a xho i restriction site . this fragment was inserted into the not i site 3 ′ of the anti - rh ( d ) scfv gene . this sequence was then inserted into plasmid pcdm8 and inserted between the hind iii and xho i sites of pkc3b . this construct is depicted in fig5 . fig5 depicts another construct of anti - rh ( d ) scfv - c4bp in plasmid pkc 3b . the plasmids are then used to transduce animal cells , in particular cho dhfr − cells . the cho - dhfr − cells ( american type culture collection , rockville , usa ) were transfected using the calcium phosphate technique ( calcium phosphate transfection kit , 5 prime 3 prime inc ., boulder , u . s . a .). these cells are cultured in ham medium lacking in hypoxanthine and thymidine ( biochrom , vindelle , france ) containing 10 % dialysed calf serum ( fcs , gibco brl , paisley , scotland ) and 1 % glutamine ( sigma , st . louis , usa ). the functionality of the reconstituted multimeric proteins , which were either c4bp - scfv or c4bp - rh ( d )/ cr1 , was studied . success was achieved in producing functional multimeric scfv , as was demonstrated ( i ) by biosynthetic labeling and immunoprecipitation followed by sds page analysis , ( ii ) by detecting erythrocyte - bound multi anti - rh ( d ) scfvs by flow cytometry , ( iii ) by the ability of multi anti - rh ( d ) scfv chimeras to agglutinate papainated red blood corpuscles at weak ionic strength , ( iiii ) by analyzing molecular interactions using an instrument for detecting evanescent waves ( iasys fisons ), with the instrument verifying the binding of the multimeric anti - rh ( d ) anti scfv chimera to erythrocytes . subsequently , the feasibility of creating heteromultimers which combine different functions was established by preparing an anti - rhesus d antibody / cr1 chimera . its ability to function was demonstrated by flow cytometry . the results which were obtained by flow cytometry are depicted in fig1 . it is clear from this figure that , if tracks 3 and 5 , in which the red blood corpuscles are rh + and rh −, respectively , are compared , it is observed that only the red blood corpuscles which possess the surface antigen are agglutinated . this figure also shows that this agglutination is not due to the effect of the papain since the papainated rh + red blood corpuscles are not agglutinated by cr1 . it was therefore possible to bind a supraphysiological density of cr1 molecules on erythrocytes which had been previously papainated , and therefore depleted of cr1 , by binding a mixed multimeric anti - rh ( d ) antibody / cr1 chimera to the rhesus d molecules . even though the humoral immune response is unable to eradicate the hiv virus , it is nevertheless effective against a large number of infectious agents against which neutralizing antibodies are regularly produced by vaccinated subjects . natural antibodies can confer protection against a large number of bacterial or viral infectious agents which infect other species . certain antigenic motifs have been characterized as targets for this type of antibody in connection with their role in the peracute vascular rejection of xenogeneic transplants . the potentially unfavorable role of the humoral immune response and of complement activation with regard to infection with hiv virus has been demonstrated . under certain circumstances , opsonization of the virions can facilitate ingestion by macrophages or binding of the virions to lymphocytes by way of cell receptors for complement or the igg fc fragment . on the other hand , the hiv virus , as an enveloped virus , is extremely sensitive to the lytic action of complement when the latter has been sufficiently activated to initiate its final lytic path and binding of its membrane attack complex . xenogenic retroviruses are destroyed extremely efficiently by normal human serum , due to the existence of natural antibodies , by way of activating complement . in the 1970s , this phenomenon had even led to the conclusion that the human species was naturally immune to retroviruses . the sought - after aim is to reroute a lytic humoral immune response toward hiv virions , with attachment to the virions being effected by the cd4 component of a recombinant heteromultimeric protein . for this , the inventors have taken into account the fact that while the hiv virus exhibits a very high degree of variability , this variability is nevertheless limited by the necessity of having constantly to preserve an ability to bind to cd4 , which ability is required for the virus to penetrate the cell and is essential for the virus . in a reciprocal manner , the cd4 molecule is able to bind to all hiv virions , in contrast to a large number of neutralizing antibodies , which have a spectrum of efficacy which is restricted to a small number of subtypes . the soluble cd4 molecule inhibits cell infection by hiv . however , the concentrations which are required for it to have this effect , particularly for neutralizing wild - type isolates , render its clinical use impracticable . various attempts have been made to improve its half - life and its avidity , and / or supply an fc gamma function which effects cell binding or complement activation using constructs of the bivalent or tetravalent cd4 / igg type . the inventors have developed a heptameric multivalent cd4 molecule using a multimerizing c4bp system whose biological efficacy in vitro was demonstrated by inhibiting infection of susceptible cells by hiv at inhibitory concentrations which are common for monomeric soluble cd4 . in the present example , rather than inhibiting cell penetration by the virus , the inventors have sought to destroy the virus extracellularly using soluble molecules which are able , on the one hand , to bind to the virion and , on the other , to supply an antigenic effector function which elicits destruction of the virus by means of an antibody response which depends on complement which pre - exists in the individual patient . this response is directed against an antigen which has nothing to do with the hiv but in relation to which the immune system has nevertheless been demonstrated to exhibit neutralizing and lytic efficacy . in other words , since the hiv virus has the ability to “ disguise ” itself , to “ hide ” itself or to “ raise decoys ”, the inventors have sought to embellish the virus with targets which the immune system of the individual patient knows how to identify and deal with efficiently . in order to do this , two plasmid constructs were made which respectively carry cd4 or the fragment of cd4 which carries the “ ligand ” fraction and an antigen . the last 183 nucleotides of the sequence encoding c4bp were amplified by means of pcr , on genomic dna , using the following primers ( seq id nos 1 and 5 , respectively ): 5 ′- gagacccccgaaggctgtgtga - 3 , and 5 ′- atttctagagagttatagttctttatccaaagtgga - 3 ′, with this latter primer containing a stop codon and a restriction site for xba i . this pcr fragment was linked at its 5 ′ end to the following double - stranded synthetic oligonucleotide sequence ( seq id no : 6 ); 5 ′ ccgggacaggtcctgctggaatccaacatcaaggttctgcccacag - 3 ′. this fragment encoding the c - terminal end of the extramembrane moiety of cd4 and having an ava i site at its 5 ′ end . this sequence was inserted into the ava i and xba i sites in plasmid st4 cd4 , containing the construct encoding soluble cd4 , and this construct is depicted in fig1 . it is firstly a matter of trying to determine which parameters direct the anti - gp120 antibody response of infected subjects to complement activation up to the c3 amplification loop , resulting in opsonization which is relatively advantageous for the individual patient , without , for all that , being accompanied by activation of the final common pathway which would result in lysis of the virion . the role of surface molecules which inhibit activation of complement and which the virion has taken from the cell surface has been demonstrated . the shedding of envelope particles when antibody is being bound also militates against terminal activation of complement . activation of the final common complement pathway requires the c3 to have a density which is critical for activation in order to initiate c5 conversion . this conversion is not brought about by igg being bound to gp120 epitopes which are too distant from each other . the use of the constructs of the invention to supply a “ cluster ” of antigens for each binding site on the virion thus makes it possible to trigger adequate local activation of complement . various categories of antigen have been considered : vaccinating antigens , bacterial antigens against which humans are universally immunized , and xenogenic antigens which are the targets of natural antibodies . vaccinating antigens which exist in the form of cloned genes and which encode a protein which can be expressed in eukaryotic cells ( hbs antigen , tetanus toxoid , etc . ), bacterial antigens to which there exists strong immunity in humans ( escherichia coli , klebsiella , shigella flagellin or salmonella antigen ), molecules which possess protein sequences which accept xenogenic glycosylations may also be envisaged after they have been produced by animal cells which possess strong glycosyl transferase activities which will attach to the proteins carbohydrates which are the targets of natural antibodies and which are known to react strongly in xenotransplants ( for example : the alpha - galactosyl group , which is an impediment to xenogeneic pig / man transplants ). these miniantibodies will be used as agents for binding heterochimeras to erythrocytes , of which chimeras they only represent one valency of the c4bp β type , which valency is linked to a multimeric antigenic molecule of the heptameric c4bp α type . the most effective antigenic system will therefore be selected in a readily quantifiable screening test . it will then be transferred into a recombinant cd4 / antigen target chimera whose different cd4 / antigen ratios ( 1cd4 / 7 antigens or ncd4 / m antigens ) will be tested in an in - vitro model of the inhibition of viral infection . these miniantibodies will be used as agents for binding heterochimeras to erythrocytes , of which chimeras they only represent one valency of the c4bp beta type , which valency is linked to a multimeric antigenic molecule of the heptameric c4bp alpha type . the most effective antigenic system will therefore be selected in a readily quantifiable screening test . it will then be transferred into a recombinant cd4 / antigen target chimera whose different cd4 / antigen ratios ( 1cd4 / 7 antigens or ncd4 / m antigens ) will be tested in an in - vitro model of the inhibition of viral infection . the most interesting target antigens were inserted into heteromultimeric constructs containing cd4 and tested for their ability to enable hiv virions to be destroyed in the presence of human serum and complement , with residual infectivity being evaluated in an in - vitro test of the inhibition of cell infection . in addition to the abovementioned constructs , the techniques employed for transfecting and culturing cells were as follows : dhfr − cho cells ( american type culture collection , rockville , usa ) were transfected using the calcium phosphate technique ( calcium phosphate transfection kit , 5 prime 3 prime inc ., boulder , u . s . a .). the cells are cultured in ham medium lacking hypoxanthine and thymidine ( biochrom , vindelle , france ) but which contains 10 % dialyzed calf serum ( fcs gibco brl , paisley , scotland ) and 1 % glutamine ( sigma , st . louis , usa ). the cells which have been transfected with pmamneo are selected on the basis of their ability to resist neomycin ( g418 , 0 . 7 microg / ml ) ( sigma ). dexamethasone ( 0 . 8 microg / ml ) is used to induce the production of mcr1 in the cells which are transfected with the pmamneo cr1 - c4bp . in their experiments , the inventors used an appliance for culturing cells continuously in hollow fibers in order to produce recombinant proteins on the scale of a few milligrams , or a few tens of milligrams , of recombinant proteins . most of the experiments can be carried out using crude or concentrated culture supernatants . small - scale purified preparations have also been prepared . the oligonucleotide syntheses which were employed for constructing the vectors were carried out in order to fit the c - terminal c4bp fragment to each construct . the nucleotide sequences were also determined on an automated fluorescence sequencer in order to check the constructs . the multimeric proteins of the invention , and their use in producing a medicament for prophylactic or therapeutic purposes , or their use as a diagnostic or research tool , are very powerful . using them can be an efficient tool for analyzing physiological mechanisms in the immune response as well as for understanding the physiopathology of certain disorders of the immune system . these molecules should make it possible to intervene in the immune system in a more sophisticated manner , thereby opening up the possibility of being better able to study large numbers of physiopathological mechanisms in vitro . in certain cases , this immunointervention will open up the route to manipulating the immune system in vivo for therapeutic purposes . the molecules are , therefore , at one and the same time tools for carrying out clinical physiopathological research and in vitro experimental research and also therapeutic tools for use in vivo . 1 . matsuguchi t ., okamura s ., aso t ., niho y ., molecular cloning of the cdna coding for prp : identity of prp as c4bp . biochem biophys res commun 1989 ; 1 : 139 – 44 . 2 . scharfstein j ., ferreira a ., gigli i ., nussenzweig v ., human c4 - 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348 - p . 552 – 554 . m . cafferty j ., griffiths a ., winter g ., chiswell . “ phage antibodies filamentous phase displaying antibody variable domains ”.