Abstract
Antibody-drug conjugates (ADC) are effective antibody-based therapeutics for hematopoietic and lymphoid tumors. However, there is need to identify new targets for ADCs, particularly for solid tumors and cancers with unmet needs. From a hybridoma library developed against cancer cells, we selected the mouse monoclonal antibody 33B7, which was able to bind to, and internalize, cancer cell lines. This antibody was used for identification of the target by immunoprecipitation and mass spectrometric analysis, followed by target validation. After target validation, 33B7 binding and target positivity were tested by flow cytometry and western blot analysis in several cancer cell lines. The ability of 33B7 conjugated to saporin to inhibit in vitro proliferation of PTFRN positive cell lines was investigated, as well as the 33B7 ADC in vivo effect on tumor growth in athymic mice. All flow cytometry and in vitro internalization assays were analyzed for statistical significance using a Welsh’s T-test. Animal studies were analyzed using Two-Way Analysis of Variance (ANOVA) utilizing post-hoc Bonferroni analysis, and/or Mixed Effects analysis. The 33B7 cell surface target was identified as Prostaglandin F2 Receptor Negative Regulator (PTGFRN), a transmembrane protein in the Tetraspanin family. This target was confirmed by showing that PTGFRN-expressing cells bound and internalized 33B7, compared to PTGFRN negative cells. Cells able to bind 33B7 were PTGFRN-positive by Western blot analysis. In vitro treatment PTGFRN-positive cancer cell lines with the 33B7-saporin ADC inhibited their proliferation in a dose-dependent fashion. 33B7 conjugated to saporin was also able to block tumor growth in vivo in mouse xenografts when compared to a control ADC. These findings show that screening antibody libraries for internalizing antibodies in cancer cell lines is a good approach to identify new cancer targets for ADC development. These results suggest PTGFRN is a possible therapeutic target via antibody-based approach for certain cancers.
Highlights
Antibody-Drug Conjugates (ADCs) are a combination of biological and small-molecule drugs that have recently received increased interest as therapeutic choices in oncology
The present paper describes the properties of this antibody 33B7, identifies its target as Prostaglandin F2 Receptor Negative Regulator (PTGFRN), and shows its efficacy as an ADC in several cancer cell lines
PTGFRN is a cell-surface protein that is upregulated in certain cancer types, including head and neck (A431) and, notably, pediatric medulloblastoma (DAOY), an aggressive cancer with limited therapeutic options
Summary
Antibody-Drug Conjugates (ADCs) are a combination of biological and small-molecule drugs that have recently received increased interest as therapeutic choices in oncology. T-DM1 is an ADC used to treat HER-2 overexpressing breast cancer It is derived from the existing anti-HER2 monoclonal antibody Trastuzumab to which emtansine has been conjugated via a non-reducible thioether linker. Patients whose HER-2 expression was immunohistologically scored as lower than 3+ and considered “HER-2 negative” and ineligible for Trastuzumab therapy, were able to benefit from Kadcyla therapeutic effects These improvements are likely because the monoclonal antibody is capable of binding to HER-2 and blocking its dimerization, interfering with its signaling for increased proliferation, while conjugated emtansine further propagates the anticancer effect by triggering apoptosis via mitotic arrest [8]. There is still interest in identifying more cancer-related targets that can be the basis for new ADC therapy, for solid tumors with unmet needs for targeted therapies
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