Antitumor activity of SS(dsFv)PE38 and SS1(dsFv)PE38, recombinant antimesothelin immunotoxins against human gynecologic cancers grown in organotypic culture in vitro.
OBJECTIVE
Mesothelin, a cell surface glycoprotein overexpressed in ovarian cancer, mesotheliomas, and some squamous cell carcinomas, is an attractive candidate for targeted therapy because it is not shed in significant amounts into the bloodstream and is not present in significant amounts on normal human tissues except for mesothelial cells. The objective of this study was to determine the antitumor activity of SS1(dsFv)PE38, a recombinant antimesothelin immunotoxin, against human gynecologic tumors grown in short-term culture in vitro.


METHODS
Tumor cells obtained from primary cultures of five ovarian and one cervical tumor were mixed with an equal proportion of NIH-3T3 fibroblasts and plated inside collagen gels in tissue culture plates. After 4-7 days of growth, these organotypic cultures were treated with media alone, SS1(dsFv)PE38, and a control immunotoxin RFB4(dsFv)PE38, which targets the CD22 antigen not present on gynecologic tumors, every other day x 3. The organotypic culture gels were then formalin fixed, paraffin embedded, and evaluated for immunotoxin sensitivity using light microscopic examination of H&E-stained slides and also evaluated for apoptosis using the terminal deoxynucleotidyl transferase-mediated nick end labeling assay.


RESULTS
Tumors expressing mesothelin showed a significant dose-dependent sensitivity to SS1(dsFv)PE38 even at concentrations as low as 1 ng/ml, whereas no antitumor activity was seen at 100 ng/ml in tumors that did not express mesothelin. This activity was specifically attributable to mesothelin targeting because RFB4 (dsFv)-PE38 had no activity against mesothelin-expressing tumors.


CONCLUSIONS
These results demonstrate that ovarian and cervical tumor cells obtained from patients can be grown in short-term culture using an organotypic culture model. Our results also show low concentrations of an immunotoxin targeting mesothelin is cytotoxic to mesothelin-expressing human tumors by inducing apoptosis.
Hassan
Raffit
R
Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
Lerner
Megan R
MR
Benbrook
Doris
D
Lightfoot
Stan A
SA
Brackett
Daniel J
DJ
Wang
Qing-Cheng
QC
Pastan
Ira
I
D016475:3T3 Cells; D000818:Animals; D000911:Antibodies, Monoclonal; D017209:Apoptosis; D002478:Cells, Cultured; D018920:Coculture Techniques; D003094:Collagen / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004768:Enterotoxins; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D058851:GPI-Linked Proteins; D005833:Genital Neoplasms, Female / Q000188:drug therapy*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000494:pharmacology; D007150:Immunohistochemistry; D000922:Immunotoxins / Q000494:pharmacology*; D020287:In Situ Nick-End Labeling; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors*; D000090204:Mesothelin; D051379:Mice; D011506:Proteins; D011994:Recombinant Proteins; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D004768:Enterotoxins; D058851:GPI-Linked Proteins; D007128:Immunoglobulin Fragments; D000922:Immunotoxins; D008562:Membrane Glycoproteins; C000715528:Msln protein, mouse; D011506:Proteins; D011994:Recombinant Proteins; C474514:SS(dsFv)PE38; C474515:SS1(dsFv)PE38; C055966:immunoglobulin Fv; D003094:Collagen; D000090204:Mesothelin