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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

The cytotoxicity of YP218 Fv-PE38 in primary cell lines from mesothelioma patients and in an NCI-H226 orthotopic mesothelioma xenograft model. (a) Cytotoxicity of YP218 Fv-PE38 and SS1P in primary cell lines from patients. (b) Anti-tumor activity of YP218 Fv-PE38 and SS1P in a mouse xenograft model with NCI-H226. Error bars indicate standard errors. The p values were calculated by comparing with controls in nonparametric Mann-Whitney test. Tumor cells were injected on Day 0. IT, immunotoxin.
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f7: The cytotoxicity of YP218 Fv-PE38 in primary cell lines from mesothelioma patients and in an NCI-H226 orthotopic mesothelioma xenograft model. (a) Cytotoxicity of YP218 Fv-PE38 and SS1P in primary cell lines from patients. (b) Anti-tumor activity of YP218 Fv-PE38 and SS1P in a mouse xenograft model with NCI-H226. Error bars indicate standard errors. The p values were calculated by comparing with controls in nonparametric Mann-Whitney test. Tumor cells were injected on Day 0. IT, immunotoxin.

Mentions: We also found that the immunotoxin with higher affinity generally had a stronger cytotoxicity (Supplemental Table 3, p < 0.0001 in Spearman’s rank correlation test). As shown in Fig. 5e, at its IC50 concentration, the amount of surface-bound immunotoxin molecules were similar among various immunotoixns for each cell line although they bind different epitopes. Therefore, the different anti-tumor activities (IC50s) of different immunotoxins may be attributed to their different binding affinities for mesothelin instead of their different epitopes. If we assume that each fluorophore corresponds to one antigen, then 104–105 immunotoxin molecules per cell are necessary and sufficient to kill 50% of target tumor cells. Interestingly, while ovarian cancer cells (OVCAR8 and NCI-ADR-RES) and mesothelioma (NCI-H226) require 105 immunotoxin molecules to achieve its IC50, lung adenocarcinoma cells (L55 and NCI-H322M) need fewer immunotoxin molecules (104). The combination of SS1P and YP218 immunotoxins showed an additive effect and no obvious synergistic effect in EKVX (a cell line with low mesothelin expression levels) (Fig. 6). In addition to exhibiting slightly more cytotoxicity than SS1P in the NCI-H226 human mesothelioma line (Fig. 4b), YP218 Fv-PE38 also showed higher affinity than other rabbit anti-mesothelin immunotoxins on most of the cell lines (Supplemental Table 3). In light of this, we decided to further compare the cytotoxicity of YP218 Fv-PE38 and SS1P in vitro with four primary cell lines isolated from malignant mesothelioma patients (Fig. 7a, Supplemental Table 3). We found that YP218 Fv-PE38 showed greater cytotoxicity than SS1P in NCI-M-19 and comparable cytotoxicity in NCI-M-16 and NCI-M-21, whereas NCI-M-18 was resistant to both immunotoxins.


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)

The cytotoxicity of YP218 Fv-PE38 in primary cell lines from mesothelioma patients and in an NCI-H226 orthotopic mesothelioma xenograft model. (a) Cytotoxicity of YP218 Fv-PE38 and SS1P in primary cell lines from patients. (b) Anti-tumor activity of YP218 Fv-PE38 and SS1P in a mouse xenograft model with NCI-H226. Error bars indicate standard errors. The p values were calculated by comparing with controls in nonparametric Mann-Whitney test. Tumor cells were injected on Day 0. IT, immunotoxin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: The cytotoxicity of YP218 Fv-PE38 in primary cell lines from mesothelioma patients and in an NCI-H226 orthotopic mesothelioma xenograft model. (a) Cytotoxicity of YP218 Fv-PE38 and SS1P in primary cell lines from patients. (b) Anti-tumor activity of YP218 Fv-PE38 and SS1P in a mouse xenograft model with NCI-H226. Error bars indicate standard errors. The p values were calculated by comparing with controls in nonparametric Mann-Whitney test. Tumor cells were injected on Day 0. IT, immunotoxin.
Mentions: We also found that the immunotoxin with higher affinity generally had a stronger cytotoxicity (Supplemental Table 3, p < 0.0001 in Spearman’s rank correlation test). As shown in Fig. 5e, at its IC50 concentration, the amount of surface-bound immunotoxin molecules were similar among various immunotoixns for each cell line although they bind different epitopes. Therefore, the different anti-tumor activities (IC50s) of different immunotoxins may be attributed to their different binding affinities for mesothelin instead of their different epitopes. If we assume that each fluorophore corresponds to one antigen, then 104–105 immunotoxin molecules per cell are necessary and sufficient to kill 50% of target tumor cells. Interestingly, while ovarian cancer cells (OVCAR8 and NCI-ADR-RES) and mesothelioma (NCI-H226) require 105 immunotoxin molecules to achieve its IC50, lung adenocarcinoma cells (L55 and NCI-H322M) need fewer immunotoxin molecules (104). The combination of SS1P and YP218 immunotoxins showed an additive effect and no obvious synergistic effect in EKVX (a cell line with low mesothelin expression levels) (Fig. 6). In addition to exhibiting slightly more cytotoxicity than SS1P in the NCI-H226 human mesothelioma line (Fig. 4b), YP218 Fv-PE38 also showed higher affinity than other rabbit anti-mesothelin immunotoxins on most of the cell lines (Supplemental Table 3). In light of this, we decided to further compare the cytotoxicity of YP218 Fv-PE38 and SS1P in vitro with four primary cell lines isolated from malignant mesothelioma patients (Fig. 7a, Supplemental Table 3). We found that YP218 Fv-PE38 showed greater cytotoxicity than SS1P in NCI-M-19 and comparable cytotoxicity in NCI-M-16 and NCI-M-21, whereas NCI-M-18 was resistant to both immunotoxins.

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