MMAE ADC EIA Kit

Tumor glycans constitute attractive targets for therapeutic antibodies. The sialylated glycocalyx plays a prominent role in cancer progression and immune evasion. Here, we describe the characterization of the mAb, FG129, which targets tumor-associated sialylated glycan, and demonstrate its potential for multimodal cancer therapy. FG129, obtained through BALB/c mouse immunizations with liposomes containing membrane glycan extracts from the colorectal cancer cell line LS180, is an mIgGlic that targets sialyl-di-Lewis(a)-containing glycoproteins. FG129, as well as its chimeric human IgG1 variant, CH129, binds with nanomolar functional affinity to a range of colorectal, pancreatic, and gastric cancer cell lines. FG129 targets 74% (135/182) of pancreatic, 50% (46/92) of gastric, 36% (100/281) of colorectal, 27% (89/327) of ovarian, and 21% (42/201) of non-small cell lung cancers, by IHC. In our pancreatic cancer cohort, high FG129 glyco-epitope expression was significantly associated with poor prognosis (P = 0.004). Crucially, the glyco-epitope displays limited normal tissue distribution, with FG129 binding weakly to a small percentage of cells within gallbladder, ileum, liver, esophagus, pancreas, and thyroid tissues. Owing to glyco-epitope internalization, we validated payload delivery by CH129 through monomethyl auristatin E (MMAE) or maytansinoid (DM1 and DM4) conjugation. All three CH129 drug conjugates killed high-binding colorectal and pancreatic cancer cell lines with (sub)nanomolar potency, coinciding with significant in vivo xenograft tumor control by CH129-vcMMAE. CH129, with its restricted normal tissue distribution, avid tumor binding, and efficient payload delivery, is a promising candidate for the treatment of sialyl-di-Lewis a expressing solid tumors, as an antibody-drug conjugate or as an alternative cancer immunotherapy modality.

Brentuximab vedotin (BV) is an antibody-drug conjugate that specifically delivers the potent cytotoxic drugmonomethyl auristatin E (MMAE) to CD30-positive cells. BV is FDA approved for treatment of relapsed/refractory Hodgkin lymphoma and anaplastic large cell lymphoma (ALCL); however, many patients do not achieve complete remission and develop BV-resistant disease. We selected for BV-resistant Hodgkin lymphoma (L428) and ALCL (Karpas-299) cell lines using either constant (ALCL) or pulsatile (Hodgkin lymphoma) exposure to BV. We confirmed drug resistance by MTS assay and analyzed CD30 expression in resistant cells by flow cytometry, qRT-PCR, and Western blotting. We also measured drug exporter expression, MMAE resistance, and intracellular MMAE concentrations in BV-resistant cells. In addition, tissue biopsy samples from 10 Hodgkin lymphoma and 5 ALCL patients who had relapsed or progressed after BV treatment were analyzed by immunohistocytochemistry for CD30 expression. The resistant ALCL cell line, but not the Hodgkin lymphoma cell line, demonstrated downregulated CD30 expression compared with the parental cell line. In contrast, the Hodgkin lymphoma cell line, but not the ALCL cell line, exhibited MMAE resistance and increased expression of the MDR1 drug exporter compared with the parental line. For both Hodgkin lymphoma and ALCL, samples from patients relapsed/resistant on BV persistently expressed CD30 by immunohistocytochemistry. One Hodgkin lymphoma patient sample expressed MDR1 by immunohistocytochemistry. Although loss of CD30 expression is a possible mode of BV resistance in ALCL in vitro models, this has not been confirmed in patients. MMAE resistance and MDR1 expression are possiblemodes of BV resistance for Hodgkin lymphoma both in vitro and in patients. (C)2015 AACR.