Downregulation of CD30, Resistance to MMAE, and Upregulation of MDR1 Are All Associated with Resistance to Brentuximab Vedotin

Abstract

Background: Both Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) express surface CD30. Brentuximab vedotin (BV) is an antibody-drug conjugate that delivers a potent cytotoxic agent, monomethyl auristatin E (MMAE), specifically to cells expressing surface CD30. Although BV elicits a high response rate (75% in HL and 86% in ALCL), the majority of patients who do not attain complete response (CR) will eventually develop resistance to BV. It is not known whether resistance to BV is through a) CD 30 alterations b) resistance to cytotoxic agent MMAE or c) overexpression of drug exporters. We developed 2 BV-resistant cell models and obtained primary lymphoma samples from patients with relapsed/progressive disease post BV therapy. We examined CD30 expression, MMAE resistance, drug exporter expression, and gene expression profiles in vitro and in vivo to determine mechanisms of resistance to BV. Methods: HL cell line(L428) and ALCL cell line (KARPAS 299) were used for in vitro experiments. The selection of BV resistant cell model (L428R and KARPAS 299R) used two different approaches (pulsatile or constant exposure). Both BV resistance and MMAE resistance were confirmed by MTS assays. CD30 expression was measured by flow cytometry,qRT-PCR, and Western blotting. Drug exporter expression was measured using qRT-PCR to MDR1, MRP1, and MRP3. In vivo experiments utilized primary tumor samples from 15 HL and 4 ALCL patients who had developed relapsed/progressive disease post BV treatment. CD30 expression was assessed by immunohistocytochemistry (IHC). Gene expression profiling was performed in both parental and resistant HL and ALCL cells, and in 4 ALCL primary tumor samples using Affymetrix whole genome GeneChip® Human Genome U133 2.0 Plus. Results: MTS assay showed the IC50 of KARPAS 299R to BV shifted from 24 +/- 10 ng/ml to 28 +/- 9 ug/ml, an 1183-fold increase. MTS assay also showed the IC50 of KARPAS 299R to MMAE only increased 2-fold when compared to KARPAS 299. Flow cytometry showed downregulation of surface CD30 expression in KARPAS 299R as compared to KARPAS 299 parental (59% vs. 96%, median intensity 78 +/- 17 vs. 591 +/- 51). This downregulation was confirmed by qRT-PCR and Western blotting for CD30. As KARPAS 299R is a mixed cell population, we sorted them into CD30+ and CD30- subpopulations. We then analyzed for BV sensitivity based on CD 30 expression status in KARPAS 299R. MTS assay showed that KARPAS 299R CD30+ cells were equally as resistant to BV as KARPAS 299R CD30- cells (figure 1A). IHC performed in 4 ALCL primary tumor samples showed persistent CD 30 expression in relapsed/progressive tumor specimens post BV treatment. Gene expression profiling on KARPAS 299R showed downregulation of CD30 as compared to KARPAS 299. Gene expression profiling on pre- and post-treatment ALCL samples (8) did not show significant differences in CD30 expression. The top four upregulated genes in relapsed/progressive samples as compared to pretreatment samples were LCE3D, WNT3, TNNT, CITED2. The top four downregulated genes in relapsed/progressive samples as compared to pretreatment samples were CXCL13, C4orf7, MS4A1, and IGJ. MTS assay showed that the IC50 of L428R to BV has shifted from 32 +/- 11 ug/ml to 391 +/- 92 ug/ml, a 12-fold increase. MTS assays showed the IC50 of L428R to MMAE has increased 99-folds when compared to L428 (figure 1B). No difference was seen in CD 30 expression by flow cytometry, qRT-PCR, or western blotting between L428R vs. L428. IHC performed in 15 HL primary tumors show persistent CD30 expression in relapsed/progressive tumor specimen post-BV treatment. qRT-PCR showed upregulation of MDR1mRNA in L428R as compared to L428. Gene expression profiling on L428R showed upregulation of MDR1 as compared to L428. Conclusion: Downregulation of CD30 is seen in BV-resistant ALCL cell model. However, sensitivity to BV did not depend solely on the level of CD30 expression as CD30+ cell subpopulations still exhibited resistance to BV in vitro. Upregulation of MDR-1 and resistance to MMAE were seen in BV-resistant HL cells, rather than downregluation of CD30. Downregulation of CD30 was not seen in HL or ALCL primary tumors. Further work is ongoing to explore/validate potential targets derived from gene expression profiling in ALCL primary tumors. Figure 1A Sensitivity to BV is not related to CD30 expression Figure 1A. Sensitivity to BV is not related to CD30 expression Figure 1B. Figure 1B. Viability of L-428 parental versus BV-resistant cells Disclosures Chen: Seattle Genetics, Inc.: Consultancy, Research Funding, Speakers Bureau, Travel expenses Other.

ChAcNLS, a Novel Modification to Antibody-Conjugates Permitting Target Cell-Specific Endosomal Escape, Localization to the Nucleus, and Enhanced Total Intracellular Accumulation

“…However, this delivery system is plagued by cellular mechanisms that reduce intracellular drug levels and ultimately results in resistance. 7,8 For example, lymphoma cells resistant to brentuximab vedotin were able to down regulate CD30 expression and increase the expression of MDR 1. Breast cancer cells resistant to trastuzumab emtansine also down regulated Her2 expression and increased expression of MDR pumps.…”

“…This recirculation of bile acids is also a likely contributing aspect for the improved tumor targeting by 64 Cu-A14-ChAcNLS. For applications toward targeted chemotherapy, because these drugs transported by current ACs are readily exported outside of the cell, 7,8 this new advancement in AC technology will most likely have an impact in the retention of chemotherapeutics due to the fact that the drug remains attached to the AC and are sheltered away from lysosome proteases and subsequent exportation. Further testing will have to be conducted to see if this strategy has a therapeutic advantage over the current system of intracellular drug accumulation by ACs.…”

“…Despite this method of targeted intracellular drug accumulation, most patients eventually progress. 3−6 Important factors that cause cancer cell resistance have been shown to be the overexpression of multidrug resistant (MDRs) transporters and the down regulation of target receptor cell surface levels for chemo- therapeutics 7,8 and intracellular dehalogenation for radioiodinated ACs. 9 Receptor recycling pathways and their increased use by cancer cells has also been implicated to reduce the intracellular accumulation of the internalized AC.…”

The Role of Specific ATP-Binding Cassette Transporters in the Acquired Resistance to Pyrrolobenzodiazepine Dimer–Containing Antibody–Drug Conjugates

“…One of the most common ways of cells to become resistant to an antibody-based therapy is the loss of antigen expression, or epitope modification, to prevent antibody binding and internalisation or signaling [6]. This mechanism has not only been seen with naked antibody therapy, but also with ADCs in vitro and clinically [7][8][9]. Our study showed that the binding and subsequent internalisation of the CD25 and HER2 antibodies was not affected in the acquired resistant cell lines and the apparent affinity of the antibody to all the cell lines was unchanged.…”

“…Both pre-clinical and clinical studies have been carried out on currently approved ADCs and the mechanism of acquired resistance generally falls into two major categories; related to the antibody portion of the ADC, or related to the drug/drug-linker portion of the ADC [5]. Tumor cells that have become refractory to monoclonal antibody (mAb) therapy often show downregulation of the target antigen or impaired mAb-antigen complex internalisation [6], a resistance mechanism that has also been reported with trastuzumab emtansine (T-DM1) [7,8], brentuximab vedotin (BV) [9] and gemtuzumab ozogamicin (GO) [10].…”