Patent Application: US-95923001-A

Abstract:
the present invention relates to the use of antibodies against glucose - 6 - phosphate isomerase and like protein for diagnosis of arthritis and the use of said protein for treatment of arthritis . it is also aimed at a process for isolating monoclonal antibodies capable of transferring arthritis and antibodies thereof , as well as a method for determining the anti - arthritic potential of a composition .

Description:
we have previously shown (( kouskoff et al ., 1996 ), u . s . pat . no . 5 , 675 , 060 ) that mice carrying all the genetic elements needed for the appearance of krn arthritis , but deficient in b lymphocytes , are free of disease . to explore the possibility that arthritis development in the krn model depends critically on a particular b cell product , we tried to provoke disease in non - arthritic mice by transfer of serum from k / b × n mice . severe joint swelling appeared in the animals injected with serum from arthritic k / b × n donors , but not in those receiving serum from non - arthritic b × n controls ( fig1 a ). disease could be induced with as little as 100 μl of k / b × n serum , and showed up reproducibly . it was provoked very rapidly , measured by either clinical score or ankle thickness , evident already within two days after serum injection . arthritis could be obtained upon injecting sera from arthritic donors into normal mice , lymphocyte - deficient rag o / o hosts , or b cell deficient k / b × n mice ( k / b × n mice - μmt o / o ) ( fig1 b ). these results show that serum components from k / b × n mice are sufficient to confer arthritis , although the more aggressive disease seen with the latter animals indicates that k / b × n t cells probably play an accessory enhancing role in the effector phase . the arthritis provoked by serum transfer presents all of the histological features of the disease in regular k / b × n mice , including invasion of inflammatory cells , hyperplasia of synoviocytes , pannus formation , and cartilage destruction ( fig2 a and b ). the arthritis induced by serum injection is transient . in mice that had received a single pair of injections , joint inflammation began to subside after about 15 days 110 ( fig1 c ); after 30 days , some of the joints appeared quite normal , even in the animals that had initially been fully arthritic . disease transience could be overcome by repeated injections of serum from arthritic mice ( fig1 c ). that this was true even for rag o / o recipients suggests that instability of the serum compound is the explanation for the transient nature of the disease . the arthritogenic serum factor is a b cell - produced immunoglobulin ( ig ): after fractionation of serum from k / b × n mice into igg and non - igg components , only the igg fraction is capable of provoking arthritis in rag o / o hosts ; its potency is similar to that of whole serum ( relative to the starting volume ) ( fig1 d ), as are the histological features of the disease it induced ( data not shown ). thus , an ig produced by b lymphocytes is key to arthritis development in k / b × n mice . we then attempted to define their molecular targets . at the onset , one could have imagined that these could be antibodies directed against specific components of the joint , somehow generated by the interaction of transgenic t cells reactive against the ag7 molecule on the surface of b cells : this could prevent the normal tolerance of b cells towards self - components , or induce polyclonal b cell stimulation and the synthesis of ig reactive against self ( perhaps polymeric ) components . we addressed this question : 1 ) by immunohistochemical analysis of rago / o mice after transfer of ig from k / b × n mice . these analyses showed deposition of transferred ig not only in the synovial tissue of the joint , but also in lining membranes of many other organs ( spleen , kidney , liver , muscle ; data not shown ). 2 ) by western blot analysis : whole protein extracts were prepared from the ankle joint and from several other organs of rag o / o mice ( to avoid the presence of ig in the extract ), electrophoresed on denaturing poyacrylamide gels ( sds - page ), blotted , and probed with serum from k / b × n mice . ig binding was revealed by probing with hrp - conjugated anti - mouse igg . as can be seen on fig3 , a single dominant protein band could be detected on these blots , at approximately 60 kd mw . this band was seen repeatedly when sera from a number of k / b × n arthritic mice were used , but not with sera from control non arthritic littermates . other proteins were detected with some of the sera , but inconsistently and they were always weaker than the 60 kd band . we then attempted to identify this 60 kd protein . to this end . igg from a large pool of k / b × n sera was purified by affinity chromatography on proteing columns . this purified ig ( 1 , 5 mg ) was covalently bound to cnbr - activated sepharose . this matrix was then used as an immunoadsorbant on which were loaded 50 mg of np - 40 extract from kidney of rag o / o mice . the column was washed extensively , and bound proteins eluted at acid ph . they were analyzed by sds - page and coomassie staining . as expected from the results described above , a dominant band of 60 kd was seen . this band was excised , the protein digested in the gel slice by trypsin , as described in ( rosenfeld et al ., 1992 ), and the resulting peptides resolved by reverse - phase hplc . several peptides were identified . three of them were then sequenced by automated edman degradation on an applied biosystems 470a instrument . the sequences were compared to public databases , using the blast program on the swissprot database . all three were found to belong to the protein glucose - 6 - phosphate isomerase ( aka phosphohexose - isomerase ; ec 5 . 3 . 1 . 9 ; swissprot database number p 06745 ; sequence accession # 1230741 ; hereafter abbreviated as gpi ). their positions are shown in fig4 . another of the peptides obtained was also found to belong to gpi , on the basis of mass spectrometry analysis which fit exactly with the molecular weight predicted from the sequence ( fig4 ). the molecular weight of gpi ( 62 636 kd ) is very concordant with the mw of the target of k / b × n serum predicted from the western blots , within the precision of mw estimation by page . gpi is an essential enzyme of the glycolytic pathway . it is an enzyme expressed in essentially all tissues , whether normal or tumoral , with some quantative variations , from the earliest stages of embryogenesis to late in life of the animals ( west et al ., 1990 ; hallbook et al ., 1989 ; warner et al ., 1985 ). it is normally a cytoplasmic enzyme , but soluble gpi can be found in serum ; it is found elevated in tumor patients ( various organs ), but not in a manner that make it a reliable marker ( see for example ( neri et al ., 1983 ; schwemmer et al ., 1985 ; paulick et al ., 1987 ; gomm et al ., 1988 ; gurney et al ., 1986 )). gpi has also been purified independently as “ neuroleukin ”. “ maturation factor ”, or “ autocrine motility factor ”, secreted factors of often limited potency as a neurotrophic agent , or as agents promoting cell migration or tumor cell differentiation ( gurney et al ., 1986 ; faik et al ., 1988 ; niinaka et al ., 1998 ; xu et al ., 1996 ). genetic deficiencies in gpi result in hemolytic anemia syndromes ( see for example ( kanno et al ., 1996 ; baronciani et al ., 1996 )). to confirm that gpi is the protein recognized by the igs present in k / b × n serum , we produced recombinant gpi in e . coli . the coding sequence of mouse gpi was amplified by polymerase chain reaction , and the product cloned in the plasmid pgex - 4t - 3 ( pharmacia ). the recombinant protein , a fusion product with glutathione - s - transferase , was purified by affinity chromatography on glutathione - sepharose 4b column . the product was characterized on sds - page , and showed the expected size ( data not shown ) the gel was blotted , and strips were probed with sera from k / b × n or non - transgenic littermate controls . as can be seen in fig5 , all k / b × n sera reacted strongly , while control sera were negative , confirming that the gpi protein is the molecular target of anti - 60 kd antibodies in k / b × n serum . the recombinant protein was also used in enzyme linked immunosorbent assays ( elisa ) to detect reactivity to gpi in sera of k / b × n mice of different ages . as previously described ( kouskoff et al ., 1996 ), krn arthritis appears at 28 – 32 days of age . as shown in fig6 , the assay detected significant reactivity above background , up to dilutions of 1 / 20 000 , in sera from k / b × n mice . no such reactivity was seen in control littermates . the assay can thus serve as a good diagnostic test of arthritis in these mice . do anti - gpi antibodies constitute all the pathogenic specificities of k / b × n serum , and can their removal eliminate the pathogenic potential of the serum . to address this question , we coupled 5 mg of recombinant gpi or of control gst protein , prepared as above , to cnbr - conjugated sepharose . pooled sera from k / b × n mice were applied sequentially to these columns . the bound proteins were eluted at acid ph , and tested by transfer into naive mice , along with an aliquot of the starting material and of flow - through fractions . as shown in fig7 , all arthritogenic activity was found in the fraction bound to the gpi - conjugated column , and none in the flow - through , even though the latter contained the majority of the immunoglobulin . these results demonstrate that anti - gpi antibodies are indeed the pathogenic ig in serum from arthritic k / b × n mice , and that it can be adsorbed with recombinant gpi protein . from these data , we conclude that anti - gpi antibodies produced in the transgenic mice provoke arthritis . we have found ( unpublished data ) that t cells expressing the krn receptor are specifically stimulated by antigen - presenting - cells exposed to gpi antigen . these t cells in turn stimulate b cells producing anti - gpi immunoglobulin , which internalize gpi effectively and thus receive help from t cells more readily than non - specific b cells ( lanzavecchia , 1985 ). it is thus proposed that self - reactive t cells against gpi or related circulating proteins , present in limited amounts in the circulation and thus unable to induce a paralyzing tolerance of the immune system , could induce similar arthritogenic antibodies in human conditions such as ra . the transfer of serum from arthritic k / b × n offers a model wherein potential anti - arthritic formulations can be tested by administration concomitant with the arthritogenic serum or immunoglobulins . as a proof of principle we tested the ability of anti - c5 monoclonal antibody to interfere with arthritogenesis on the k / b × n model . we chose this reagent because of prior evidence implicating complement components , and in particular c5 , in the generation of joint lesions in other mouse models of arthritis and in human ra patients ( watson et al , 1987 ; wang et al , 1995 ). mice were injected with arthritogenic serum from k / b × n mice , and at the same time with the anti - c5 monoclonal antibody bb5 . 1 , known to block c5 activity ( frei et al , 1987 ). as can be seen from fig8 , mice injected with the anti - c5 monoclonal were protected from disease . if immunoglobulins present in the serum of k / b × n mice are able to transfer arthritis , it should be possible to isolate monoclonal antibodies ( mabs ) also able to transfer disease to naïve recipients . splenocytes from 30 and 50 days old k / b × n mice were fused according to standard protocols for hybridoma derivation ( de st groth and scheidegger , 1980 ), and hybridomas selected in hat medium in limiting dilution conditions in 96 - well plates . hybrids were screened for production of immunoglobulins reactive to gpi by testing culture supernatants in an elisa assay with recombinant gpi as a bound antigen and anti - mouse igg as a developing reagent . several positive wells were selected for expansion , cloning by limiting dilution and testing for stability of the hybridoma lines . eleven stable hybridoma lines producing anti - gpi igg were thus obtained ( see table 1 below ). the ability of these monoclonals to transfer disease was tested by protein - g purification of mg amounts of the igg produced by these hybridomas , and intraveneous injection into na ve balb / c recipients . as can be seen on fig9 , the coupled injection of anti - gpi immunoglobulin from the 6 . 121 and 2 . 99 hybridoma cells provoked arthritis in the recipients , but none of the antibodies were able to induce disease when injected alone . among these antibodies , some works , such as the 2 . 99 and 6 . 121 antibodies , while others don &# 39 ; t . however , step e ) of the process for isolating antibodies capable of transferring arthritis according to the invention allows to select the working ones . baronciani , l ., zanella , a ., bianchi , p ., et al . 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