<p><b>A)</b> Immunohistochemistry with the LEM2/15 anti MT1-MMP antibody in a T... more <p><b>A)</b> Immunohistochemistry with the LEM2/15 anti MT1-MMP antibody in a TMA (n = 40) of Low-grade GBMs (LGG) and High-grade GBMs (HGG) patient tissues (representative images). Right panel: MT1-MMP staining intensity score (0–3). <b>B)</b> Immunoblotting and <b>C)</b> FACS analyses of the indicated cell lines using the LEM2/15 anti MT1-MMP antibody.</p
<p><b>A)</b> Representative fused PET/CT images of coronal and sagittal planes ... more <p><b>A)</b> Representative fused PET/CT images of coronal and sagittal planes at 2 and 4 days p.i. containing TS543 brain tumors with <sup>89</sup>Zr-DFO-LEM2/15 (left panels) and <sup>89</sup>Zr-DFO-IgG1 as isotype control (right panels) <b>B)</b> Tumor-to-blood ratios for <sup>89</sup>Zr-DFO-LEM2/15 (black bars) and <sup>89</sup>Zr-DFO-IgG1 (grey bars) in nude mice bearing orthotopic TS453 xenografts at 2 and 4 days p.i. derived from PET imaging. Values are presented as mean±SD (n = 3–5). <b>C)</b> Representative brain images of double immunostaining for MT1-MMP (pink) and CD31 vascular marker (brown) (left panel) and for MT1-MMP (pink) and Ki67 proliferation marker (brown) (right panel) from orthotopic TS453 xenografts used for PET imaging. Higher-magnification are shown in the bottom panels. Scale bars: 20 μm. Significant differences: p<0.05 (*) and p<0.0001 (***).</p
GCET1 (germinal center B cell–expressed transcript-1) gene codes for a serpin expressed in germin... more GCET1 (germinal center B cell–expressed transcript-1) gene codes for a serpin expressed in germinal center (GC) B cells. Following the observation that follicular lymphoma cases exhibit an increased level of Gcet1 expression, compared with follicular hyperplasia, we have characterized Gcet1 protein expression in human tissues, cell lines, and a large series of lymphomas. To this end, we have performed immunohistochemical and Western blot analyses using a newly generated monoclonal antibody that is reactive in paraffin-embedded tissues. Our results demonstrate that Gcet1 is expressed exclusively by neoplasms hypothetically to be arrested at the GC stage of differentiation, including follicular lymphoma, nodular lymphocyte predominant Hodgkin lymphoma, and a subset of diffuse large B-cell lymphoma, T-cell/histiocyte rich B-cell lymphoma, and Burkitt lymphoma. Within these tumors, Gcet-1 protein expression is restricted to a subset of GC B cells, establishing the existence of a distinc...
Human single-chain Fv directed against fibroblast growth factor receptor 3 (FGFR3) have been show... more Human single-chain Fv directed against fibroblast growth factor receptor 3 (FGFR3) have been shown to block proliferation of RT112 bladder carcinoma cells in vitro. Here, we examined the ability of the recombinant gelonin toxin (rGel) to enhance this inhibitory effect in vitro and in vivo on the bladder cancer cell line RT112 and the corresponding xenografts. Immunotoxins were genetically engineered by fusing FGFR3-specific Fv fragments (3C) to the NH2 terminus of rGel and expressed as a soluble protein in Escherichia coli. The 3C/rGel fusion construct showed an IC50 of 200 nmol/L against log-phase RT112 cells compared with 1,500 nmol/L for free rGel. Immunofluorescence studies showed that the 3C/rGel construct internalized rapidly into the cytoplasm of RT112 cells within 1 h of exposure. The mechanism of immunotoxin-induced cell death was found to be mediated by apoptosis. RT112 tumor xenografts in severe combined immunodeficient mice treated with 50 mg/kg 3C/rGel exhibited conside...
In the last years we have been working in the production of plant-derivedoral vaccines based onch... more In the last years we have been working in the production of plant-derivedoral vaccines based onchimeric,plant virus particles (CVPs). The CVP technology involves the modification of cowpea mosaic virus (CPMV), which can be easily manipulated to display epitopes from viral pathogens on its surface. These CVPs can be easily obtained in large quantities from infected plants of Vigna unguiculataat
Rabbit haemorrhagic disease virus (RHDV) causes an important disease in rabbits. The virus capsid... more Rabbit haemorrhagic disease virus (RHDV) causes an important disease in rabbits. The virus capsid is composed of a single 60 kDa protein. The capsid protein gene was cloned in Escherichia coli using the pET3 system, and the antigenic structure of RHDV VP60 was dissected using 11 monoclonal antibodies (MAbs) and 12 overlapping fragments of the protein expressed in E. coli. Two antigenic regions were found. Ten out of the 11 MAbs recognized different discontinuous epitopes in the most immunodominant region of the viral capsid. This domain was located between residues 31 and 250 of the VP60 N terminus. The other MAb revealed the presence of an antigenic site within 102 aa of the C terminus. This MAb did not recognize the major cleavage product of the full-length 60 kDa protein. These results indicate that, in contrast to other caliciviruses such as Norwalk virus (NV), the 36 kDa cleavage product probably forms the N-terminal region of VP60. However, as in NV, the cleavage region appear...
Fifteen horses were experimentally infected with African horse sickness virus (AHSV) serotype 4. ... more Fifteen horses were experimentally infected with African horse sickness virus (AHSV) serotype 4. To learn more about the time course of production and specificity of AHSV-specific antibodies, sera were analyzed by immunoblot analysis. Only animals that survived for more than 9 days were able to develop a humoral immune response detectable by immunoblotting. The earliest serological markers corresponded mainly to VP5, VP6, and NS2 and to a lesser extent to VP3, NS1, and NS3. Neutralizing antibodies to VP2 were not detected by immunoblotting, suggesting that they are mostly conformation dependent. VP7-specific antibodies were detected later in infection. These results make NS2 and VP6 the most attractive candidates for the rapid diagnosis of the infection.
The peptidoglycan layer surrounding the photosynthetic organelles (cyanelles) of the protist Cyan... more The peptidoglycan layer surrounding the photosynthetic organelles (cyanelles) of the protist Cyanophora paradoxa is thought to be a relic of their cyanobacterial ancestors. The separation of muropeptides by gel filtration and reverse-phase high-performance liquid chromatography revealed four different muropeptide monomers. A number of muropeptides were identical in retention behavior to muropeptides of Escherichia coli, while others had remarkably long retention times with respect to their sizes, as indicated by gel filtration. Molecular mass determination by plasma desorption and matrix-assisted laser desorption ionization mass spectrometry showed that these unusual muropeptides had molecular masses greater by 112 Da or a multiple thereof than those of ones common to both species. Fast atom bombardment-tandem mass spectrometry of these reduced muropeptide monomers allowed the localization of the modification to D-glutamic acid. High-resolution fast atom bombardment-mass spectrometr...
NIH mice were vaccinated subcutaneously or intranasally with chimaeric cow pea mosaic virus (CPMV... more NIH mice were vaccinated subcutaneously or intranasally with chimaeric cow pea mosaic virus (CPMV) constructs expressing a 17-mer peptide sequence from canine parvovirus (CPV) as monomers or dimers on the small or large protein surface subunits. Responses to the chimaeric virus particles (CVPs) were compared with those of mice immunized with the native virus or with parvovirus peptide conjugated to keyhole limpet haemocyanin (KLH). The characteristics of the immune response to vaccination were examined by measuring serum and mucosal antibody responses in ELISA, in vitro antigen-induced spleen cell proliferation and cytokine responses. Mice made strong antibody responses to the native plant virus and peptide-specific responses to two of the four CVP constructs tested which were approximately 10-fold lower than responses to native plant virus. The immune response generated by the CVP constructs showed a marked TH1 bias, as determined by a predominantly IgG(2a) isotype peptide-specific antibody response and the release of IFN-gamma but not IL-4 or IL-5 from lymphocytes exposed to antigen in vitro. In comparison, parvovirus peptide conjugated to KLH generated an IgG(1)-biased (TH2) response. These data indicate that the presentation of peptides on viral particles could be used to bias the immune response in favor of a TH1 response.Anti-viral and anti-peptide IgA were detected in intestinal and bronchial lavage fluid of immunized mice, demonstrating that a mucosal immune response to CPV can be generated by systemic and mucosal immunization with CVP vaccines. Serum antibody from both subcutaneously-vaccinated and intranasally-vaccinated mice showed neutralizing activity against CPV in vitro.
An antigen delivery system based on hybrid recombinant parvovirus-like particles (VLPs) formed by... more An antigen delivery system based on hybrid recombinant parvovirus-like particles (VLPs) formed by the self-assembly of the capsid VP2 protein of porcine (PPV) or canine parvovirus (CPV) expressed in insect cells with the baculovirus system has been developed. PPV:VLPs containing a CD8(+) epitope from the LCMV nucleoprotein evoked a potent CTL response and were able to protect mice against a lethal infection with the virus. Also, PPV:VLPs containing the C3:T epitope from poliovirus elicited a CD4(+)3 log(10) units) against poliovirus. The possibility of combining different types of epitopes in different positions of a single particle to stimulate different branches of the immune system paves the way to the production of more potent vaccines in a simple and cheap way.
A vaccine based upon a recombinant plant virus (CPMV-PARVO1), displaying a peptide derived from t... more A vaccine based upon a recombinant plant virus (CPMV-PARVO1), displaying a peptide derived from the VP2 capsid protein of canine parvovirus (CPV), has previously been described. To date, studies with the vaccine have utilized viable plant chimaeric particles (CVPs). In this study, CPMV-PARVO1 was inactivated by UV treatment to remove the possibility of replication of the recombinant plant virus in a plant host after manufacture of the vaccine. We show that the inactivated CVP is able to protect dogs from a lethal challenge with CPV following parenteral immunization with the vaccine. Dogs immunized with the inactivated CPMV-PARVO1 in adjuvant displayed no clinical signs of disease and shedding of CPV in faeces was limited following CPV challenge. All immunized dogs elicited high titres of peptide-specific antibody, which neutralized CPV in vitro. Levels of protection, virus shedding and VP2-specific antibody were comparable to those seen in dogs immunized with the same VP2- peptide coupled to keyhole limpet hemocyanin (KLH). Since plant virus-derived vaccines have the potential for cost-effective manufacture and are not known to replicate in mammalian cells, they represent a viable alternative to current replicating vaccine vectors for development of both human and veterinary vaccines.
Vav proteins are phosphorylation-dependent guanine nucleotide exchange factors (GEFs) that cataly... more Vav proteins are phosphorylation-dependent guanine nucleotide exchange factors (GEFs) that catalyze the activation of members of the Rho family of guanosine triphosphatases (GTPases). The current regulatory model holds that the nonphosphorylated, catalytically inactive state of these GEFs is maintained by intramolecular interactions among the amino-terminal domains and the central catalytic core, which block the binding of Vav proteins to GTPases. We showed that this autoinhibition is mechanistically more complex, also involving the bivalent association of the carboxyl-terminal Src homology 3 (SH3) region of Vav with its catalytic and pleckstrin homology (PH) domains. Such interactions occurred through proline-rich region-independent mechanisms. Full release from this double-locked state required synergistic weakening effects from multiple phosphorylated tyrosine residues, thus providing an optimized system to generate gradients of Vav GEF activity depending on upstream signaling inputs. This mechanism is shared by mammalian and Drosophila melanogaster Vav proteins, suggesting that it may be a common regulatory feature for this protein family.
Proceedings of the National Academy of Sciences, 2013
Antibody cancer therapies rely on systemically accessible targets and suitable antibodies that ex... more Antibody cancer therapies rely on systemically accessible targets and suitable antibodies that exert a functional activity or deliver a payload to the tumor site. Here, we present proof-of-principle of in vivo selection of human antibodies in tumor-bearing mice that identified a tumor-specific antibody able to deliver a payload and unveils the target antigen. By using an ex vivo enrichment process against freshly disaggregated tumors to purge the repertoire, in combination with in vivo biopanning at optimized phage circulation time, we have identified a human domain antibody capable of mediating selective localization of phage to human prostate cancer xenografts. Affinity chromatography followed by mass spectrometry analysis showed that the antibody recognizes the proteasome activator complex PA28. The specificity of soluble antibody was confirmed by demonstrating its binding to the active human PA28αβ complex. Whereas systemically administered control phage was confined in the lumen of blood vessels of both normal tissues and tumors, the selected phage spread from tumor vessels into the perivascular tumor parenchyma. In these areas, the selected phage partially colocalized with PA28 complex. Furthermore, we found that the expression of the α subunit of PA28 [proteasome activator complex subunit 1 (PSME1)] is elevated in primary and metastatic human prostate cancer and used anti-PSME1 antibodies to show that PSME1 is an accessible marker in mouse xenograft tumors. These results support the use of PA28 as a tumor marker and a potential target for therapeutic intervention in prostate cancer.
<p><b>A)</b> Immunohistochemistry with the LEM2/15 anti MT1-MMP antibody in a T... more <p><b>A)</b> Immunohistochemistry with the LEM2/15 anti MT1-MMP antibody in a TMA (n = 40) of Low-grade GBMs (LGG) and High-grade GBMs (HGG) patient tissues (representative images). Right panel: MT1-MMP staining intensity score (0–3). <b>B)</b> Immunoblotting and <b>C)</b> FACS analyses of the indicated cell lines using the LEM2/15 anti MT1-MMP antibody.</p
<p><b>A)</b> Representative fused PET/CT images of coronal and sagittal planes ... more <p><b>A)</b> Representative fused PET/CT images of coronal and sagittal planes at 2 and 4 days p.i. containing TS543 brain tumors with <sup>89</sup>Zr-DFO-LEM2/15 (left panels) and <sup>89</sup>Zr-DFO-IgG1 as isotype control (right panels) <b>B)</b> Tumor-to-blood ratios for <sup>89</sup>Zr-DFO-LEM2/15 (black bars) and <sup>89</sup>Zr-DFO-IgG1 (grey bars) in nude mice bearing orthotopic TS453 xenografts at 2 and 4 days p.i. derived from PET imaging. Values are presented as mean±SD (n = 3–5). <b>C)</b> Representative brain images of double immunostaining for MT1-MMP (pink) and CD31 vascular marker (brown) (left panel) and for MT1-MMP (pink) and Ki67 proliferation marker (brown) (right panel) from orthotopic TS453 xenografts used for PET imaging. Higher-magnification are shown in the bottom panels. Scale bars: 20 μm. Significant differences: p<0.05 (*) and p<0.0001 (***).</p
GCET1 (germinal center B cell–expressed transcript-1) gene codes for a serpin expressed in germin... more GCET1 (germinal center B cell–expressed transcript-1) gene codes for a serpin expressed in germinal center (GC) B cells. Following the observation that follicular lymphoma cases exhibit an increased level of Gcet1 expression, compared with follicular hyperplasia, we have characterized Gcet1 protein expression in human tissues, cell lines, and a large series of lymphomas. To this end, we have performed immunohistochemical and Western blot analyses using a newly generated monoclonal antibody that is reactive in paraffin-embedded tissues. Our results demonstrate that Gcet1 is expressed exclusively by neoplasms hypothetically to be arrested at the GC stage of differentiation, including follicular lymphoma, nodular lymphocyte predominant Hodgkin lymphoma, and a subset of diffuse large B-cell lymphoma, T-cell/histiocyte rich B-cell lymphoma, and Burkitt lymphoma. Within these tumors, Gcet-1 protein expression is restricted to a subset of GC B cells, establishing the existence of a distinc...
Human single-chain Fv directed against fibroblast growth factor receptor 3 (FGFR3) have been show... more Human single-chain Fv directed against fibroblast growth factor receptor 3 (FGFR3) have been shown to block proliferation of RT112 bladder carcinoma cells in vitro. Here, we examined the ability of the recombinant gelonin toxin (rGel) to enhance this inhibitory effect in vitro and in vivo on the bladder cancer cell line RT112 and the corresponding xenografts. Immunotoxins were genetically engineered by fusing FGFR3-specific Fv fragments (3C) to the NH2 terminus of rGel and expressed as a soluble protein in Escherichia coli. The 3C/rGel fusion construct showed an IC50 of 200 nmol/L against log-phase RT112 cells compared with 1,500 nmol/L for free rGel. Immunofluorescence studies showed that the 3C/rGel construct internalized rapidly into the cytoplasm of RT112 cells within 1 h of exposure. The mechanism of immunotoxin-induced cell death was found to be mediated by apoptosis. RT112 tumor xenografts in severe combined immunodeficient mice treated with 50 mg/kg 3C/rGel exhibited conside...
In the last years we have been working in the production of plant-derivedoral vaccines based onch... more In the last years we have been working in the production of plant-derivedoral vaccines based onchimeric,plant virus particles (CVPs). The CVP technology involves the modification of cowpea mosaic virus (CPMV), which can be easily manipulated to display epitopes from viral pathogens on its surface. These CVPs can be easily obtained in large quantities from infected plants of Vigna unguiculataat
Rabbit haemorrhagic disease virus (RHDV) causes an important disease in rabbits. The virus capsid... more Rabbit haemorrhagic disease virus (RHDV) causes an important disease in rabbits. The virus capsid is composed of a single 60 kDa protein. The capsid protein gene was cloned in Escherichia coli using the pET3 system, and the antigenic structure of RHDV VP60 was dissected using 11 monoclonal antibodies (MAbs) and 12 overlapping fragments of the protein expressed in E. coli. Two antigenic regions were found. Ten out of the 11 MAbs recognized different discontinuous epitopes in the most immunodominant region of the viral capsid. This domain was located between residues 31 and 250 of the VP60 N terminus. The other MAb revealed the presence of an antigenic site within 102 aa of the C terminus. This MAb did not recognize the major cleavage product of the full-length 60 kDa protein. These results indicate that, in contrast to other caliciviruses such as Norwalk virus (NV), the 36 kDa cleavage product probably forms the N-terminal region of VP60. However, as in NV, the cleavage region appear...
Fifteen horses were experimentally infected with African horse sickness virus (AHSV) serotype 4. ... more Fifteen horses were experimentally infected with African horse sickness virus (AHSV) serotype 4. To learn more about the time course of production and specificity of AHSV-specific antibodies, sera were analyzed by immunoblot analysis. Only animals that survived for more than 9 days were able to develop a humoral immune response detectable by immunoblotting. The earliest serological markers corresponded mainly to VP5, VP6, and NS2 and to a lesser extent to VP3, NS1, and NS3. Neutralizing antibodies to VP2 were not detected by immunoblotting, suggesting that they are mostly conformation dependent. VP7-specific antibodies were detected later in infection. These results make NS2 and VP6 the most attractive candidates for the rapid diagnosis of the infection.
The peptidoglycan layer surrounding the photosynthetic organelles (cyanelles) of the protist Cyan... more The peptidoglycan layer surrounding the photosynthetic organelles (cyanelles) of the protist Cyanophora paradoxa is thought to be a relic of their cyanobacterial ancestors. The separation of muropeptides by gel filtration and reverse-phase high-performance liquid chromatography revealed four different muropeptide monomers. A number of muropeptides were identical in retention behavior to muropeptides of Escherichia coli, while others had remarkably long retention times with respect to their sizes, as indicated by gel filtration. Molecular mass determination by plasma desorption and matrix-assisted laser desorption ionization mass spectrometry showed that these unusual muropeptides had molecular masses greater by 112 Da or a multiple thereof than those of ones common to both species. Fast atom bombardment-tandem mass spectrometry of these reduced muropeptide monomers allowed the localization of the modification to D-glutamic acid. High-resolution fast atom bombardment-mass spectrometr...
NIH mice were vaccinated subcutaneously or intranasally with chimaeric cow pea mosaic virus (CPMV... more NIH mice were vaccinated subcutaneously or intranasally with chimaeric cow pea mosaic virus (CPMV) constructs expressing a 17-mer peptide sequence from canine parvovirus (CPV) as monomers or dimers on the small or large protein surface subunits. Responses to the chimaeric virus particles (CVPs) were compared with those of mice immunized with the native virus or with parvovirus peptide conjugated to keyhole limpet haemocyanin (KLH). The characteristics of the immune response to vaccination were examined by measuring serum and mucosal antibody responses in ELISA, in vitro antigen-induced spleen cell proliferation and cytokine responses. Mice made strong antibody responses to the native plant virus and peptide-specific responses to two of the four CVP constructs tested which were approximately 10-fold lower than responses to native plant virus. The immune response generated by the CVP constructs showed a marked TH1 bias, as determined by a predominantly IgG(2a) isotype peptide-specific antibody response and the release of IFN-gamma but not IL-4 or IL-5 from lymphocytes exposed to antigen in vitro. In comparison, parvovirus peptide conjugated to KLH generated an IgG(1)-biased (TH2) response. These data indicate that the presentation of peptides on viral particles could be used to bias the immune response in favor of a TH1 response.Anti-viral and anti-peptide IgA were detected in intestinal and bronchial lavage fluid of immunized mice, demonstrating that a mucosal immune response to CPV can be generated by systemic and mucosal immunization with CVP vaccines. Serum antibody from both subcutaneously-vaccinated and intranasally-vaccinated mice showed neutralizing activity against CPV in vitro.
An antigen delivery system based on hybrid recombinant parvovirus-like particles (VLPs) formed by... more An antigen delivery system based on hybrid recombinant parvovirus-like particles (VLPs) formed by the self-assembly of the capsid VP2 protein of porcine (PPV) or canine parvovirus (CPV) expressed in insect cells with the baculovirus system has been developed. PPV:VLPs containing a CD8(+) epitope from the LCMV nucleoprotein evoked a potent CTL response and were able to protect mice against a lethal infection with the virus. Also, PPV:VLPs containing the C3:T epitope from poliovirus elicited a CD4(+)3 log(10) units) against poliovirus. The possibility of combining different types of epitopes in different positions of a single particle to stimulate different branches of the immune system paves the way to the production of more potent vaccines in a simple and cheap way.
A vaccine based upon a recombinant plant virus (CPMV-PARVO1), displaying a peptide derived from t... more A vaccine based upon a recombinant plant virus (CPMV-PARVO1), displaying a peptide derived from the VP2 capsid protein of canine parvovirus (CPV), has previously been described. To date, studies with the vaccine have utilized viable plant chimaeric particles (CVPs). In this study, CPMV-PARVO1 was inactivated by UV treatment to remove the possibility of replication of the recombinant plant virus in a plant host after manufacture of the vaccine. We show that the inactivated CVP is able to protect dogs from a lethal challenge with CPV following parenteral immunization with the vaccine. Dogs immunized with the inactivated CPMV-PARVO1 in adjuvant displayed no clinical signs of disease and shedding of CPV in faeces was limited following CPV challenge. All immunized dogs elicited high titres of peptide-specific antibody, which neutralized CPV in vitro. Levels of protection, virus shedding and VP2-specific antibody were comparable to those seen in dogs immunized with the same VP2- peptide coupled to keyhole limpet hemocyanin (KLH). Since plant virus-derived vaccines have the potential for cost-effective manufacture and are not known to replicate in mammalian cells, they represent a viable alternative to current replicating vaccine vectors for development of both human and veterinary vaccines.
Vav proteins are phosphorylation-dependent guanine nucleotide exchange factors (GEFs) that cataly... more Vav proteins are phosphorylation-dependent guanine nucleotide exchange factors (GEFs) that catalyze the activation of members of the Rho family of guanosine triphosphatases (GTPases). The current regulatory model holds that the nonphosphorylated, catalytically inactive state of these GEFs is maintained by intramolecular interactions among the amino-terminal domains and the central catalytic core, which block the binding of Vav proteins to GTPases. We showed that this autoinhibition is mechanistically more complex, also involving the bivalent association of the carboxyl-terminal Src homology 3 (SH3) region of Vav with its catalytic and pleckstrin homology (PH) domains. Such interactions occurred through proline-rich region-independent mechanisms. Full release from this double-locked state required synergistic weakening effects from multiple phosphorylated tyrosine residues, thus providing an optimized system to generate gradients of Vav GEF activity depending on upstream signaling inputs. This mechanism is shared by mammalian and Drosophila melanogaster Vav proteins, suggesting that it may be a common regulatory feature for this protein family.
Proceedings of the National Academy of Sciences, 2013
Antibody cancer therapies rely on systemically accessible targets and suitable antibodies that ex... more Antibody cancer therapies rely on systemically accessible targets and suitable antibodies that exert a functional activity or deliver a payload to the tumor site. Here, we present proof-of-principle of in vivo selection of human antibodies in tumor-bearing mice that identified a tumor-specific antibody able to deliver a payload and unveils the target antigen. By using an ex vivo enrichment process against freshly disaggregated tumors to purge the repertoire, in combination with in vivo biopanning at optimized phage circulation time, we have identified a human domain antibody capable of mediating selective localization of phage to human prostate cancer xenografts. Affinity chromatography followed by mass spectrometry analysis showed that the antibody recognizes the proteasome activator complex PA28. The specificity of soluble antibody was confirmed by demonstrating its binding to the active human PA28αβ complex. Whereas systemically administered control phage was confined in the lumen of blood vessels of both normal tissues and tumors, the selected phage spread from tumor vessels into the perivascular tumor parenchyma. In these areas, the selected phage partially colocalized with PA28 complex. Furthermore, we found that the expression of the α subunit of PA28 [proteasome activator complex subunit 1 (PSME1)] is elevated in primary and metastatic human prostate cancer and used anti-PSME1 antibodies to show that PSME1 is an accessible marker in mouse xenograft tumors. These results support the use of PA28 as a tumor marker and a potential target for therapeutic intervention in prostate cancer.
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Papers by J. Martinez-Torrecuadrada