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New high affinity monoclonal antibodies recognize non-overlapping epitopes on mesothelin for monitoring and treating mesothelioma.

Zhang YF, Phung Y, Gao W, Kawa S, Hassan R, Pastan I, Ho M - Sci Rep (2015)

Bottom Line: These antibodies do not compete for mesothelin binding with the immunotoxin SS1P that binds Region I of mesothelin.Furthermore, we have engineered a humanized YP218 Fv that retains full binding affinity for mesothelin-expressing cancer cells.In conclusion, with their unique binding properties, these antibodies may be promising candidates for monitoring and treating mesothelioma and other mesothelin-expressing cancers.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, Bethesda, MD 20892, United States.

ABSTRACT
Mesothelin is an emerging cell surface target in mesothelioma and other solid tumors. Most antibody drug candidates recognize highly immunogenic Region I (296-390) on mesothelin. Here, we report a group of high-affinity non-Region I rabbit monoclonal antibodies. These antibodies do not compete for mesothelin binding with the immunotoxin SS1P that binds Region I of mesothelin. One pair of antibodies (YP218 and YP223) is suitable to detect soluble mesothelin in a sandwich ELISA with high sensitivity. The new assay can also be used to measure serum mesothelin concentration in mesothelioma patients, indicating its potential use for monitoring patients treated with current antibody therapies targeting Region I. The antibodies are highly specific and sensitive in immunostaining of mesothelioma. To explore their use in tumor therapy, we have generated the immunotoxins based on the Fv of these antibodies. One immunotoxin (YP218 Fv-PE38) exhibits potent anti-tumor cytotoxicity towards primary mesothelioma cell lines in vitro and an NCI-H226 xenograft tumor in mice. Furthermore, we have engineered a humanized YP218 Fv that retains full binding affinity for mesothelin-expressing cancer cells. In conclusion, with their unique binding properties, these antibodies may be promising candidates for monitoring and treating mesothelioma and other mesothelin-expressing cancers.

No MeSH data available.


Related in: MedlinePlus

Construction and characterization of anti-mesothelin immunotoxins. (a) Live cell binding analysis of anti-mesothelin Fv-PE38 (immunotoxins) via flow cytometry. The functional binding affinity was summarized in Supplemental Table 3. The order of binding affinity varies between different cell lines. (b) Cytotoxicity of anti-mesothelin Fv-PE38 (immunotoxins) in vitro. The order of cytotoxicity varies between different cell lines. The WST signal measures dehydrogenase activities, which is directly proportional to the number of living cells. Error bars indicate standard errors. IT, immunotoxin.
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f4: Construction and characterization of anti-mesothelin immunotoxins. (a) Live cell binding analysis of anti-mesothelin Fv-PE38 (immunotoxins) via flow cytometry. The functional binding affinity was summarized in Supplemental Table 3. The order of binding affinity varies between different cell lines. (b) Cytotoxicity of anti-mesothelin Fv-PE38 (immunotoxins) in vitro. The order of cytotoxicity varies between different cell lines. The WST signal measures dehydrogenase activities, which is directly proportional to the number of living cells. Error bars indicate standard errors. IT, immunotoxin.

Mentions: To determine whether the new antibodies can be used as therapeutic candidates, we constructed immunotoxins. We produced similar single chain Fv-PE38 fusion proteins for the rabbit Abs and compared them with the SS1P immunotoxin. We first compared the cytotoxicity and affinity of YP3 Fv-PE38, YP218 Fv-PE38, YP223 Fv-PE38 and SS1P in vitro with several solid tumor cell lines (Figs. 4,5a, Supplemental Table 3). The immunotoxins have potent anti-tumor activity on ovarian cancer (OVCAR8 and NCI-ADR-RES), lung adenocarcinomas (L55, EKVX and NCI-H322M) and mesothelioma (NCI-H226 and M30) cells. The efficacy of these immunotoxins may be possibly influenced by the following factors: 1) surface mesothelin expression level, 2) immunotoxin affinity, 3) cell context and 4) immunotoxin binding epitope. To analyze the immunotoxins, we quantitatively measured the surface mesothelin expression level for these cell lines by flow cytometry (Fig. 5b). We found that the IC50s of SS1P and YP218 Fv-PE38 were inversely correlated with the mesothelin surface expression level, if the outlier, Panc 3.014, was excluded from the analysis (Fig. 5c,d). Pancreatic cancer cells (e.g., Panc 3.014) were very resistant to anti-mesothelin immunotoxins as previously described2425.


New high affinity monoclonal antibodies recognize non-overlapping epitopes on mesothelin for monitoring and treating mesothelioma.

Zhang YF, Phung Y, Gao W, Kawa S, Hassan R, Pastan I, Ho M - Sci Rep (2015)

Construction and characterization of anti-mesothelin immunotoxins. (a) Live cell binding analysis of anti-mesothelin Fv-PE38 (immunotoxins) via flow cytometry. The functional binding affinity was summarized in Supplemental Table 3. The order of binding affinity varies between different cell lines. (b) Cytotoxicity of anti-mesothelin Fv-PE38 (immunotoxins) in vitro. The order of cytotoxicity varies between different cell lines. The WST signal measures dehydrogenase activities, which is directly proportional to the number of living cells. Error bars indicate standard errors. IT, immunotoxin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440525&req=5

f4: Construction and characterization of anti-mesothelin immunotoxins. (a) Live cell binding analysis of anti-mesothelin Fv-PE38 (immunotoxins) via flow cytometry. The functional binding affinity was summarized in Supplemental Table 3. The order of binding affinity varies between different cell lines. (b) Cytotoxicity of anti-mesothelin Fv-PE38 (immunotoxins) in vitro. The order of cytotoxicity varies between different cell lines. The WST signal measures dehydrogenase activities, which is directly proportional to the number of living cells. Error bars indicate standard errors. IT, immunotoxin.
Mentions: To determine whether the new antibodies can be used as therapeutic candidates, we constructed immunotoxins. We produced similar single chain Fv-PE38 fusion proteins for the rabbit Abs and compared them with the SS1P immunotoxin. We first compared the cytotoxicity and affinity of YP3 Fv-PE38, YP218 Fv-PE38, YP223 Fv-PE38 and SS1P in vitro with several solid tumor cell lines (Figs. 4,5a, Supplemental Table 3). The immunotoxins have potent anti-tumor activity on ovarian cancer (OVCAR8 and NCI-ADR-RES), lung adenocarcinomas (L55, EKVX and NCI-H322M) and mesothelioma (NCI-H226 and M30) cells. The efficacy of these immunotoxins may be possibly influenced by the following factors: 1) surface mesothelin expression level, 2) immunotoxin affinity, 3) cell context and 4) immunotoxin binding epitope. To analyze the immunotoxins, we quantitatively measured the surface mesothelin expression level for these cell lines by flow cytometry (Fig. 5b). We found that the IC50s of SS1P and YP218 Fv-PE38 were inversely correlated with the mesothelin surface expression level, if the outlier, Panc 3.014, was excluded from the analysis (Fig. 5c,d). Pancreatic cancer cells (e.g., Panc 3.014) were very resistant to anti-mesothelin immunotoxins as previously described2425.

Bottom Line: These antibodies do not compete for mesothelin binding with the immunotoxin SS1P that binds Region I of mesothelin.Furthermore, we have engineered a humanized YP218 Fv that retains full binding affinity for mesothelin-expressing cancer cells.In conclusion, with their unique binding properties, these antibodies may be promising candidates for monitoring and treating mesothelioma and other mesothelin-expressing cancers.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, Bethesda, MD 20892, United States.

ABSTRACT
Mesothelin is an emerging cell surface target in mesothelioma and other solid tumors. Most antibody drug candidates recognize highly immunogenic Region I (296-390) on mesothelin. Here, we report a group of high-affinity non-Region I rabbit monoclonal antibodies. These antibodies do not compete for mesothelin binding with the immunotoxin SS1P that binds Region I of mesothelin. One pair of antibodies (YP218 and YP223) is suitable to detect soluble mesothelin in a sandwich ELISA with high sensitivity. The new assay can also be used to measure serum mesothelin concentration in mesothelioma patients, indicating its potential use for monitoring patients treated with current antibody therapies targeting Region I. The antibodies are highly specific and sensitive in immunostaining of mesothelioma. To explore their use in tumor therapy, we have generated the immunotoxins based on the Fv of these antibodies. One immunotoxin (YP218 Fv-PE38) exhibits potent anti-tumor cytotoxicity towards primary mesothelioma cell lines in vitro and an NCI-H226 xenograft tumor in mice. Furthermore, we have engineered a humanized YP218 Fv that retains full binding affinity for mesothelin-expressing cancer cells. In conclusion, with their unique binding properties, these antibodies may be promising candidates for monitoring and treating mesothelioma and other mesothelin-expressing cancers.

No MeSH data available.


Related in: MedlinePlus