BACKGROUND
autoimmunity is an important cause of disease in humans, it is estimated to affect at least 3% to 5% of the human population and depends on a failure of the mechanisms normally responsible for maintaining self-tolerance .
although many factors causing these diseases, including the genes that may predispose to autoimmunity, have been identified, the aetiology of most autoimmune diseases remains obscure.
much interest has focused on the analysis of the immune factors leading to the tissue lesions.
in some cases the cellular immune response stimulated by lymphokines seems to play a major role, whereas in others the humoral antibody response is deemed prevalent.
functional genomics may offer a solution to these problems by using biological systems which allow the massive interaction between an autoimmune patient's cloned antibody repertoire and individual antigens.
one of these systems is phage display, a technique which involves the coupling of phenotype to genotype in a selectable format.
it has been extensively used in molecular biology to study protein-protein interactions and one of the most successful applications of phage display has been the isolation of monoclonal antibodies to purified antigens  <cit> .
in addition to libraries from naive or immunized sources, phage antibody libraries have also been made from patients suffering from autoimmune diseases.
this work has been most extensively carried out with thyroid disease  <cit> , systemic lupus erythematosus  <cit> , paraneoplastic encephalomyelitis  <cit> , myasthenia gravis  <cit>  and type  <dig> diabetes mellitus  <cit> .
in a recent work we described the antibody response in celiac disease   <cit> .
this is a genetic illness strongly linked to hla dq <dig>  characterized by flattening of the intestinal mucosa and malabsorption.
the pathogenesis is precipitated by dietary exposure to wheat gluten and similar proteins in rye, barley and possibly oats  <cit> .
the disease is characterized by the presence of specific antibodies recognizing gliadins, food proteins and an endomysial autoantigen, identified as tissue transglutaminase   <cit> .
we recently made and selected phage antibody libraries from the dna isolated from cd patient lymphocytes and were able to isolate single-chain antibody fragments  to ttg showing their specific production by intestinal lymphocytes, indicating that the site of synthesis of these antibodies is the intestinal mucosa  <cit> .
scfvs isolated from different patients recognized the same ttg epitopes and by elisa competition experiments we demonstrated that the number of epitopes recognized was restricted to two, distinguished by the ability of the antibodies to recognize mouse ttg  <cit> .
the activities of these in vitro selected antibodies mimics those found in the serum of cd patients, and preliminary work suggests similar biological activity indicating that in vivo studies of these antibodies may provide useful information on the pathogenic roles of these antibodies in cd.
the isolation of disease-specific antibodies, as well as the related gene, is the first step in the generation of animal models through in vivo antibody gene expression.
in vivo gene expression has been implemented using a number of different techniques, including injection of naked dna into the muscle, with or without electroporation  <cit> , coating gold particles with dna and injecting them with a gene gun  <cit> .
however, expression of recombinant protein is usually transient and only low levels are reached, using these methods.
recently gene transfer systems mediated by vectors based on the adeno-associated virus, have been shown to mediate sustained and prolonged titres of engineered antibody  <cit> .
in the present work we describe the production of a series of miniantibody constructs composed of a human autoimmune anti-ttg scfv combined with antibody constant fc regions from human, rat and mouse.
this was sought as a simple approach to rapidly obtain sustained in vivo production of antibodies with specific specificities.
in addition to providing appropriate effectors domains, fusion to constant regions also prolongs half life.
this represents an innovative tool for the in vivo studies of the pathogenic properties of cloned autoimmune antibody fragments.
RESULTS
in a previous paper  <cit>  we described the isolation of human iga scfvs to ttg from phage antibody libraries obtained from the intestinal lymphocytes of cd patients.
two of these scfvs, indicated as  <dig>  ttg, cross-reactive to rodent ttg, and  <dig>  ttg, specific for human enzyme, were the reference antibody fragments used to make the series of constructs reported in fig.
<dig>  the construction of the series pmb-sv <dig> is reported in materials and methods section.
it is characterized by a bsshii-nhei cassette for the subcloning of scfvs from the pdan <dig> phagemid vector, a nhei-spei cassette for the cloning of ch <dig> and ch <dig> domains of fc from different species, and an sv <dig> tag at the 3' end for uniform detection.
expression of the miniantibodies was driven by a cmv promoter and a leader sequence at the n-terminus was included to allow mammalian secretion.
the two selected scfvs  were cloned into the  <dig> different vectors, generating a total of  <dig> different constructs.
all vectors were checked by dna sequencing and the purified plasmidic dnas were transfected into hek 293t cells.
the secretion of the miniantibodies in the culture medium was analyzed after  <dig> h by elisa on plates coated with either human or mouse ttg.
the results, determined by recognition of the sv <dig> tag at the c-terminus of the constructs , or with species specific antibodies , for the  <dig>  miniantibodies show that they were all able to recognize both antigens with o.d.
values ranging from  <dig>   to  <dig>  .
similar results were obtained with the  <dig>  miniantibodies, except that recognition was specific for the human enzyme, with no binding to mouse ttg .
stable cell lines for all the constructs were established by growing transfected cells in the selective agent for hygromicin resistance.
supernatant from individual clones were screened for the best elisa reactivity and then expanded for further experiments.
the average yield of miniantibody production was in the 5– <dig> mg/liter range using standard culture flask.
the miniantibodies were analyzed by western blotting under reducing and non reducing conditions and after treatment of the purified miniantibodies with glycosidase pngasef to assay the level of glycosylation.
the results for the miniantibody mb-mog- <dig>  are reported, as an example, in fig.
3a.
the predicted molecular weight of the miniantibodies, result of the fusion of the scfv with constant domains and sv <dig> tag, is about  <dig> kda.
we found bands of the predicted molecular weight in the samples treated with reducing agent and a slight increase in the electrophoretic mobility in the deglycosylated samples, indicating that the miniantibodies are glycosylated in hek 239t cells.
under non reducing, non denaturing conditions, a high molecular weight band, explained by the interchain disulfide bond in the hinge region, was obtained.
the ability of the miniantibodies to activate the classic complement pathway was tested with an in vitro assay, as reported in fig.
<dig>  in which c1q deposition was measured using an elisa assay.
the results showed that human and rat miniantibodies, of igg <dig> and igg2b origin respectively, were able to bind c1q, the first component of the classic complement cascade, as described in the literature  <cit> , whereas the mouse miniantibody of igg <dig> origin could not bind c1q  <cit> .
the miniantibodies were analyzed for their ability to recognize ttg on histological sections.
these experiments were undertaken in view of the possible use of these miniantibodies in in vivo studies.
the immunolabeling of histological sections of mouse muscle is shown in fig.
3b, using purified mb-mog- <dig>  and  <dig> .
staining was only detected for mb-mog- <dig>  with specific recognition of the extracellular ttg present at the muscular endomysium and perimysium.
the same pattern was found when miniantibodies with the human or rat fc were used .
for the same reasons, an assay on the inhibition of ttg activity by miniantibodies was tested.
we have already shown that scfvs to ttg isolated from the intestinal lymphocytes of cd patients inhibit the in vitro transamidation activity of ttg  <cit> .
fig.
<dig> shows that the coupling of 5-pentylamine to gliadin  is catalyzed by mouse purified recombinant ttg, which is inhibited by miniantibody mb-mog- <dig> , but not miniantibody mb-mog- <dig> , confirming the specificity observed by elisa.
the inhibition closely mirrors the values previously described for the scfv  <cit>  in a similar assay.
in comparing the two miniantibodies with the commercial ttg-specific monoclonal antibody cub <dig>  the results show that the percentage of enzyme activity inhibition is higher with the miniantibodies compared to cub <dig>  using the same enzyme to antibody ratio.
we believe that this is due to both a difference in affinity and the different epitope recognized by the cd derived antibodies.
the in vivo expression of selected miniantibodies was studied by using dna vaccination protocols.
according to this method of gene transfer, dna is delivered directly to the muscle of the animal where it is internalized by the muscular fibers and expressed if an appropriate eukaryotic promoter is present.
in our case, in order to evaluate the possibility of using miniantibodies for in vivo studies of the biological activities of autoimmune antibodies, we used an anti-ttg scfv that recognizes mouse ttg, and a second construct which did not cross-react with rodent ttg.
in the latter case, the purpose was to monitor the serum level of an antibody not sequestered by ttg at the tissue level, which may occur, if an antibody recognizing mouse ttg is used.
<dig> μg of purified pmb-mog- <dig>  and pmb-mog- <dig>  dna were injected twice into the quadriceps of eight balb/c mice at an interval of  <dig> days.
the mice were periodically examined for the presence of reactive miniantibodies to ttg at serum levels by elisa.
as outlined in fig.
<dig>  for both constructs low but detectable levels of miniantibodies to ttg were measured in the blood samples taken up to  <dig> days after the injection.
mouse sera were also investigated for the possible presence of antibodies raised against the miniantibody molecule.
this was done by elisa, adsorbing purified mb-mog- <dig>  and mb-mog- <dig>  on plastic wells.
no evidence of an induced immune response was found, with sera from the  <dig>  or  <dig>  treated mice all negative at  <dig> days .
at the end of the experiments the mice were sacrificed and the hindquarter muscle examined by in situ pcr for the presence of miniantibodies dna.
the result, depicted in fig.
<dig>  showed a positive labeling for scattered muscular fibers, indicating the continued presence of the plasmid dna.
discussion
engineered antibodies are increasingly being used as therapeutic agents in numerous cases including oncology, autoimmunity, inflammation and infectious diseases  <cit> .
combinatorial approaches have been applied to scfvs isolated from phage display libraries, modifying the reactive v regions by fusion with a range of molecules to improve the antibody stability and avidity  <cit> , to alter the effector functions  <cit> , to balance the pharmacokinetics  <cit> , to facilitate the purification  <cit> , or to combine different antibodies giving rise to bifunctional antibodies  <cit> .
in the case of the present work, the fusion of human autoimmune scfvs to the fc domains of different species had the goal of using such constructs for expression in vivo and possibly generate an autoimmune animal model.
such direct scfv-fc fusions have been widely used and expressed in yeast  <cit>  and mammalian cells  <cit> , with activity similar to full length iggs in most assays, with the advantages of dimerization and effector functions, provided by the fused fc domain.
the most critical passage of this approach was the preservation of the antibody reactivity after fusion with fc domains from other species.
this was implemented by the creation of a novel mammalian expression vector in which either scfv or fc domain could be easily switched using compatible restriction sites.
as demonstrated by elisa, all the chimeric constructs recognized the ttg antigen, and western blotting showed the expected higher molecular weight bands corresponding to the dimeric form.
the binding activity of the constructs with the scfv  <dig> , crossreactive to rodent ttg, was also preserved to mouse ttg tested in elisa as well as in an immunochemical assay on histological section of mouse muscle, as was the ability to inhibit the crosslinking activity of ttg.
complement activation measured by c1q deposition was confirmed for the human and rat miniantibody constructs reflecting the presence of the ch <dig> domain and correct glycosylation produced by the transfected 293t cell.
injection of plasmid dna was used to induce in vivo expression of miniantibodies.
this approach derives from extensive studies on dna vaccination in which naked plasmid dna, coding for an antigenic protein, is transfected into muscle cells in vivo either by injection  <cit>  or using a "gene gun"  <cit> .
this results in expression of the vector-encoded antigen, which induces cellular and humoral responses  <cit> .
in a similar vein, injection of dna coding for an anti-tumoral scfv has also been carried out  <cit> .
although other studies have demonstrated both cellular and humoral responses against human scfvs in mice  <cit>  with a reduction of the therapeutic potential, this feature can also be exploited to generate anti-idiotypic responses against scfvs derived from mouse lymphomas  <cit> .
in these experiments, the effectiveness of the anti-idiotypic response was enhanced by fusing the cloned antibody expressed by the tumoral cells to an additional ch <dig> antigenic region, as originally suggested by syrengelas et al.
<cit> .
in the present study, since a human scfv xenogenic for mice was used, we sought to minimize the host immune response by using the scfv fused to a ch2-ch <dig> syngenic mouse fc region.
the outcomes of the experiments have confirmed the validity of this approach, with a detectable production of miniantibodies in the serum for at least  <dig> days and a peak of production after  <dig> days.
CONCLUSIONS
the reactivity of the serum miniantibodies in elisa together with the apparent lack of humoral response against the miniantibody molecule, led us to conclude that the human scfv-mouse fc fusion miniantibodies are poorly immunogenic in the mouse under the experimental conditions used here.
in conclusion, our results indicate that chimeric proteins generated by fusion of human scfvs to human, murine and rat fc regions are effectively produced and secreted by cultured cells; the polypeptides dimerize, forming disulfide bridges, so increasing the valency of the miniantibody; the miniantibodies retain the antigen recognition both in elisa and immunohistology and the ability to activate complement.
the inhibitory properties of the scfv are preserved and, upon intramuscular injection of the plasmid, the elisa antibody titre is still detectable after  <dig> days, suggesting the absence of an immune response by the host when a syngenic fc fragment is present in the construct.
while this indicates that the approach is functional and will be useful in the in vivo study of the role of autoimmune antibodies, in the absence of complicating immunological factors, the period of expression obtained here is relatively short when compared to the long time scale involved in most autoimmune diseases, which occurs over a period of years.
for this reason it may be appropriate to explore alternative in vivo expression methods, such as adeno-associated virus vector, which has been shown to induce expression over a prolonged period.

 autoimmunity is an important cause of disease in humans, it is estimated to affect at least 3% to 5% of the human population and depends on a failure of the mechanisms normally responsible for maintaining self-tolerance. although many factors causing these diseases, including the genes that may predispose to autoimmunity, have been identified, the aetiology of most autoimmune diseases remains obscure. much interest has focused on the analysis of the immune factors leading to the tissue lesions. in some cases the cellular immune response stimulated by lymphokines seems to play a major role, whereas in others the humoral antibody response is deemed prevalent. functional genomics may offer a solution to these problems by using biological systems which allow the massive interaction between an autoimmune patient's cloned antibody repertoire and individual antigens. one of these systems is phage display, a technique which involves the coupling of phenotype to genotype in a selectable format. it has been extensively used in molecular biology to study protein-protein interactions and one of the most successful applications of phage display has been the isolation of monoclonal antibodies to purified antigens. in addition to libraries from naive or immunized sources, phage antibody libraries have also been made from patients suffering from autoimmune diseases. this work has been most extensively carried out with thyroid disease, systemic lupus erythematosus, paraneoplastic encephalomyelitis, myasthenia gravis and type diabetes mellitus. in a recent work we described the antibody response in celiac disease. this is a genetic illness strongly linked to hla dq characterized by flattening of the intestinal mucosa and malabsorption. the pathogenesis is precipitated by dietary exposure to wheat gluten and similar proteins in rye, barley and possibly oats. the disease is characterized by the presence of specific antibodies recognizing gliadins, food proteins and an endomysial autoantigen, identified as tissue transglutaminase. we recently made and selected phage antibody libraries from the dna isolated from cd patient lymphocytes and were able to isolate single-chain antibody fragments to ttg showing their specific production by intestinal lymphocytes, indicating that the site of synthesis of these antibodies is the intestinal mucosa. scfvs isolated from different patients recognized the same ttg epitopes and by elisa competition experiments we demonstrated that the number of epitopes recognized was restricted to two, distinguished by the ability of the antibodies to recognize mouse ttg. the activities of these in vitro selected antibodies mimics those found in the serum of cd patients, and preliminary work suggests similar biological activity indicating that in vivo studies of these antibodies may provide useful information on the pathogenic roles of these antibodies in cd. the isolation of disease-specific antibodies, as well as the related gene, is the first step in the generation of animal models through in vivo antibody gene expression. in vivo gene expression has been implemented using a number of different techniques, including injection of naked dna into the muscle, with or without electroporation, coating gold particles with dna and injecting them with a gene gun. however, expression of recombinant protein is usually transient and only low levels are reached, using these methods. recently gene transfer systems mediated by vectors based on the adeno-associated virus, have been shown to mediate sustained and prolonged titres of engineered antibody. in the present work we describe the production of a series of miniantibody constructs composed of a human autoimmune anti-ttg scfv combined with antibody constant fc regions from human, rat and mouse. this was sought as a simple approach to rapidly obtain sustained in vivo production of antibodies with specific specificities. in addition to providing appropriate effectors domains, fusion to constant regions also prolongs half life. this represents an innovative tool for the in vivo studies of the pathogenic properties of cloned autoimmune antibody fragments.  in a previous paper we described the isolation of human iga scfvs to ttg from phage antibody libraries obtained from the intestinal lymphocytes of cd patients. two of these scfvs, indicated as ttg, cross-reactive to rodent ttg, and ttg, specific for human enzyme, were the reference antibody fragments used to make the series of constructs reported in fig. the construction of the series pmb-sv is reported in materials and methods section. it is characterized by a bsshii-nhei cassette for the subcloning of scfvs from the pdan phagemid vector, a nhei-spei cassette for the cloning of ch and ch domains of fc from different species, and an sv tag at the 3' end for uniform detection. expression of the miniantibodies was driven by a cmv promoter and a leader sequence at the n-terminus was included to allow mammalian secretion. the two selected scfvs were cloned into the different vectors, generating a total of different constructs. all vectors were checked by dna sequencing and the purified plasmidic dnas were transfected into hek 293t cells. the secretion of the miniantibodies in the culture medium was analyzed after h by elisa on plates coated with either human or mouse ttg. the results, determined by recognition of the sv tag at the c-terminus of the constructs, or with species specific antibodies, for the miniantibodies show that they were all able to recognize both antigens with o.d. values ranging from to. similar results were obtained with the miniantibodies, except that recognition was specific for the human enzyme, with no binding to mouse ttg. stable cell lines for all the constructs were established by growing transfected cells in the selective agent for hygromicin resistance. supernatant from individual clones were screened for the best elisa reactivity and then expanded for further experiments. the average yield of miniantibody production was in the 5– mg/liter range using standard culture flask. the miniantibodies were analyzed by western blotting under reducing and non reducing conditions and after treatment of the purified miniantibodies with glycosidase pngasef to assay the level of glycosylation. the results for the miniantibody mb-mog- are reported, as an example, in fig. 3a. the predicted molecular weight of the miniantibodies, result of the fusion of the scfv with constant domains and sv tag, is about kda. we found bands of the predicted molecular weight in the samples treated with reducing agent and a slight increase in the electrophoretic mobility in the deglycosylated samples, indicating that the miniantibodies are glycosylated in hek 239t cells. under non reducing, non denaturing conditions, a high molecular weight band, explained by the interchain disulfide bond in the hinge region, was obtained. the ability of the miniantibodies to activate the classic complement pathway was tested with an in vitro assay, as reported in fig. in which c1q deposition was measured using an elisa assay. the results showed that human and rat miniantibodies, of igg and igg2b origin respectively, were able to bind c1q, the first component of the classic complement cascade, as described in the literature, whereas the mouse miniantibody of igg origin could not bind c1q. the miniantibodies were analyzed for their ability to recognize ttg on histological sections. these experiments were undertaken in view of the possible use of these miniantibodies in in vivo studies. the immunolabeling of histological sections of mouse muscle is shown in fig. 3b, using purified mb-mog- and. staining was only detected for mb-mog- with specific recognition of the extracellular ttg present at the muscular endomysium and perimysium. the same pattern was found when miniantibodies with the human or rat fc were used. for the same reasons, an assay on the inhibition of ttg activity by miniantibodies was tested. we have already shown that scfvs to ttg isolated from the intestinal lymphocytes of cd patients inhibit the in vitro transamidation activity of ttg. fig. shows that the coupling of 5-pentylamine to gliadin is catalyzed by mouse purified recombinant ttg, which is inhibited by miniantibody mb-mog-, but not miniantibody mb-mog-, confirming the specificity observed by elisa. the inhibition closely mirrors the values previously described for the scfv in a similar assay. in comparing the two miniantibodies with the commercial ttg-specific monoclonal antibody cub the results show that the percentage of enzyme activity inhibition is higher with the miniantibodies compared to cub using the same enzyme to antibody ratio. we believe that this is due to both a difference in affinity and the different epitope recognized by the cd derived antibodies. the in vivo expression of selected miniantibodies was studied by using dna vaccination protocols. according to this method of gene transfer, dna is delivered directly to the muscle of the animal where it is internalized by the muscular fibers and expressed if an appropriate eukaryotic promoter is present. in our case, in order to evaluate the possibility of using miniantibodies for in vivo studies of the biological activities of autoimmune antibodies, we used an anti-ttg scfv that recognizes mouse ttg, and a second construct which did not cross-react with rodent ttg. in the latter case, the purpose was to monitor the serum level of an antibody not sequestered by ttg at the tissue level, which may occur, if an antibody recognizing mouse ttg is used. μg of purified pmb-mog- and pmb-mog- dna were injected twice into the quadriceps of eight balb/c mice at an interval of days. the mice were periodically examined for the presence of reactive miniantibodies to ttg at serum levels by elisa. as outlined in fig. for both constructs low but detectable levels of miniantibodies to ttg were measured in the blood samples taken up to days after the injection. mouse sera were also investigated for the possible presence of antibodies raised against the miniantibody molecule. this was done by elisa, adsorbing purified mb-mog- and mb-mog- on plastic wells. no evidence of an induced immune response was found, with sera from the or treated mice all negative at days. at the end of the experiments the mice were sacrificed and the hindquarter muscle examined by in situ pcr for the presence of miniantibodies dna. the result, depicted in fig. showed a positive labeling for scattered muscular fibers, indicating the continued presence of the plasmid dna. discussion engineered antibodies are increasingly being used as therapeutic agents in numerous cases including oncology, autoimmunity, inflammation and infectious diseases. combinatorial approaches have been applied to scfvs isolated from phage display libraries, modifying the reactive v regions by fusion with a range of molecules to improve the antibody stability and avidity, to alter the effector functions, to balance the pharmacokinetics, to facilitate the purification, or to combine different antibodies giving rise to bifunctional antibodies. in the case of the present work, the fusion of human autoimmune scfvs to the fc domains of different species had the goal of using such constructs for expression in vivo and possibly generate an autoimmune animal model. such direct scfv-fc fusions have been widely used and expressed in yeast and mammalian cells, with activity similar to full length iggs in most assays, with the advantages of dimerization and effector functions, provided by the fused fc domain. the most critical passage of this approach was the preservation of the antibody reactivity after fusion with fc domains from other species. this was implemented by the creation of a novel mammalian expression vector in which either scfv or fc domain could be easily switched using compatible restriction sites. as demonstrated by elisa, all the chimeric constructs recognized the ttg antigen, and western blotting showed the expected higher molecular weight bands corresponding to the dimeric form. the binding activity of the constructs with the scfv, crossreactive to rodent ttg, was also preserved to mouse ttg tested in elisa as well as in an immunochemical assay on histological section of mouse muscle, as was the ability to inhibit the crosslinking activity of ttg. complement activation measured by c1q deposition was confirmed for the human and rat miniantibody constructs reflecting the presence of the ch domain and correct glycosylation produced by the transfected 293t cell. injection of plasmid dna was used to induce in vivo expression of miniantibodies. this approach derives from extensive studies on dna vaccination in which naked plasmid dna, coding for an antigenic protein, is transfected into muscle cells in vivo either by injection or using a "gene gun". this results in expression of the vector-encoded antigen, which induces cellular and humoral responses. in a similar vein, injection of dna coding for an anti-tumoral scfv has also been carried out. although other studies have demonstrated both cellular and humoral responses against human scfvs in mice with a reduction of the therapeutic potential, this feature can also be exploited to generate anti-idiotypic responses against scfvs derived from mouse lymphomas. in these experiments, the effectiveness of the anti-idiotypic response was enhanced by fusing the cloned antibody expressed by the tumoral cells to an additional ch antigenic region, as originally suggested by syrengelas et al.. in the present study, since a human scfv xenogenic for mice was used, we sought to minimize the host immune response by using the scfv fused to a ch2-ch syngenic mouse fc region. the outcomes of the experiments have confirmed the validity of this approach, with a detectable production of miniantibodies in the serum for at least days and a peak of production after days.  the reactivity of the serum miniantibodies in elisa together with the apparent lack of humoral response against the miniantibody molecule, led us to conclude that the human scfv-mouse fc fusion miniantibodies are poorly immunogenic in the mouse under the experimental conditions used here. in conclusion, our results indicate that chimeric proteins generated by fusion of human scfvs to human, murine and rat fc regions are effectively produced and secreted by cultured cells; the polypeptides dimerize, forming disulfide bridges, so increasing the valency of the miniantibody; the miniantibodies retain the antigen recognition both in elisa and immunohistology and the ability to activate complement. the inhibitory properties of the scfv are preserved and, upon intramuscular injection of the plasmid, the elisa antibody titre is still detectable after days, suggesting the absence of an immune response by the host when a syngenic fc fragment is present in the construct. while this indicates that the approach is functional and will be useful in the in vivo study of the role of autoimmune antibodies, in the absence of complicating immunological factors, the period of expression obtained here is relatively short when compared to the long time scale involved in most autoimmune diseases, which occurs over a period of years. for this reason it may be appropriate to explore alternative in vivo expression methods, such as adeno-associated virus vector, which has been shown to induce expression over a prolonged period. 
phage display antibody libraries have been made from the lymphocytes of patients suffering from autoimmune diseases in which the antibodies are known to play a role in the pathogenesis or are important for the diagnosis of the disease. this can be proposed as a general method to study the pathogenic role of autoimmune antibodies in autoimmune diseases. these are produced by genetic fusion of anti-ttg scfv to human, mouse or rat fc domains, making them suitable for in vivo expression. finally, the in vivo expression in mice leads to detectable serum levels with no apparent gross immune response by the host. in this work we demonstrated the usefulness of a method for the in vivo expression of miniantibodies specific to transglutaminase, corresponding to the autoimmune specificity of celiac disease. the results obtained here indicate that the miniantibody molecule is efficiently secreted, and that the reactivity to the antigen is retained even after fusion to heterologous fc domains. in the case of celiac disease, the immune response is directed against the autoantigen tissue transglutaminase.
 phage display antibody libraries have been made from the lymphocytes of patients suffering from autoimmune diseases in which the antibodies are known to play a role in the pathogenesis or are important for the diagnosis of the disease. in the case of celiac disease, the immune response is directed against the autoantigen tissue transglutaminase. however, despite numerous studies, the role of these antibodies in the pathogenesis of this disease has not been elucidated.  we were able to engineer specific anti-transglutaminase antibody fragments in the form called "miniantibody". these are produced by genetic fusion of anti-ttg scfv to human, mouse or rat fc domains, making them suitable for in vivo expression. the results obtained here indicate that the miniantibody molecule is efficiently secreted, and that the reactivity to the antigen is retained even after fusion to heterologous fc domains. further analysis demonstrate that the molecule is secreted as homodimeric, mimicking original antibody structure. finally, the in vivo expression in mice leads to detectable serum levels with no apparent gross immune response by the host.  in this work we demonstrated the usefulness of a method for the in vivo expression of miniantibodies specific to transglutaminase, corresponding to the autoimmune specificity of celiac disease. this can be proposed as a general method to study the pathogenic role of autoimmune antibodies in autoimmune diseases. 