90Y Labeling of monoclonal antibody MOv18 and preclinical validation for radioimmunotherapy of human ovarian carcinomas

Abstract

The monoclonal antibody (mAb) MOv18 binds the membrane alpha isoform of the folate receptor (FR) which is overexpressed in human ovarian carcinoma cells. Exploiting the targeting capacity of this mAb, we developed and preclinically validated a protocol for the stable labeling of the mAb with 90Y, an isotope which has shown promise in cancer radioimmunotherapy. MOv18 was derivatized with the stable macrocyclic ligand p-isothiocyanatobenzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10- tetraacetic acid (Bz-DOTA). MOv18-Bz-DOTA conjugates were labeled with 90Y or 111In under metal-free and good laboratory practice conditions. At the optimal Bz-DOTA/mAb derivatization ratio of 4-5, conjugates maintained binding activity up to 6 months, were efficiently labeled with 90Y or 111In (mean labeling yield 85 and 64%, associated to a final mean specific activity of 74 and 37 MBq/mg) and displayed a mean immunoreactivity of 60 and 58%, respectively. The radiolabeled preparations were stable in human serum, with >97% radioactivity associated to mAb at 48 h after labeling. The ability of 90Y- and 111In-MOv18 to localize FR on tumors in vivo was analyzed in nude mice bearing tumors induced by isogenic cell lines differing only in the presence or absence of the relevant antigen [A431FR (FR-positive) and A431tMock (FR-negative)]. In vivo biodistribution in organs other than tumor was comparable in non-tumor-, A431tMock- and A431FR-bearing mice, whereas the median tumor uptake of the radiolabeled reagents, expressed as area under the curve (AUC) and maximum uptake (Umax), was significantly higher (sixfold to sevenfold) in A431FR than in A431tMock tumors (P=0.0465 and P=0.0332, respectively). Mean maximum uptake (% ID/g) for 90Y-MOv18 was 53.7 and 7.4 in A431FR and A431tMock respectively; corresponding values for 111In-Mov18 were 45.0 and 11.3. These data demonstrate the feasibility of 90Y-labeling of MOv18 without compromising antibody binding ability and the immunoreagent-specific localization in vivo on FR-expressing tumors, suggesting the suitability of 90Y-MOv18 for clinical studies.

Antitumor Effects of a Human Dimeric Antibody Fragment 131I-AFRA-DFM5.3 in a Mouse Model for Ovarian Cancer

“…This aspect was examined using a well-defined subcutaneous tumor model system (21). In this in vivo system, despite a rapid elimination, only 131 I-AFRA-DFM5.3-that is, the radiolabeled dimer specifically recognizing FR on tumor cells-localized to FR-positive tumor masses in an amount sufficient to significantly slow tumor growth.…”

“…The following human tumor cell lines were used: ovarian carcinoma IGROV-1 (a gift from Dr. Jean Bénard, Institute Gustave Roussy), OVCAR3 (American Type Culture Collection), and A431-FR and A431-MK (epidermoid carcinoma cells transfected with FR or empty vector, respectively), isolated as previously described (21). All cell lines were grown in RPMI 1640 medium supplemented with 10% fetal calf serum and 2 mM glutamine; stably transfected cells were grown with the addition of G418 at 800 mg/mL (Gibco).…”

“…Radiolabeling with 131 I was done using IODO-GEN-precoated iodination tubes (Pierce) by the Chizzonite method, as previously described (4,21). Radiopharmaceuticals were characterized for radiochemical purity by instant thin layer chromatography, integrity by sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectric point (pI) by isoelectrofocusing using the Phast System (GE Healthcare), and immunoreactivity as previously described (4), using A431FR, IGROV-1, or OVCAR3 as positive target cells and A431MK as negative target cells.…”

Strategy to Prevent Drug-Related Hypersensitivity in Folate-Targeted Hapten Immunotherapy of Cancer

“…Interestingly, carboplatin desensitization is one example where short desensitization protocols (e.g., less than 6 h) have an unacceptable failure rate in patients with carboplatin allergy, but longer infusion times (over days) is well tolerated without recurrence of the allergic reaction and with good antitumor responses (25). Immmune-based therapies of FR-positive cancers can be pursued with a variety of strategies including administration of anti-FR monoclonal antibodies (MOv18, MOv19, and LK26) (26)(27)(28), radioimmunotherapy (29,30), treatment with bispecific ligand/antibodies (31,32), administration of genetically modified T cells (33), and vaccination with DNA (34) and mRNA-transfected dendritic cells (35,36). Here, we offer a unique approach whereby metastatic FR-positive tumors can be non-invasively marked with a folate-hapten conjugate.…”

Development of radioimmunotherapeutic and diagnostic antibodies: an inside-out view

“…154 Immuno-specific localization of 90 Y-labeled c-MOv18 on FR-expressing tumors has been demonstrated, suggesting that further studies are warranted. 155 The same Ab labeled with the α-emitting halogen, 211 At, has also been investigated. 44…”