Abstract
Acute myeloid leukemia (AML) is a high-risk malignancy with overall poor outcomes across the age spectrum. Attempts to intensify cytotoxic chemotherapeutic regimens in recent decades have been limited by toxic acute and late effects, establishing the necessity of targeted therapy development. Immunotherapy is a highly promising treatment strategy in AML; however, its development has been limited as agents with potent anti-leukemic activity often confer significant hematopoietic toxicities. We previously reported the identification of an ideal potential immunotherapeutic target, mesothelin (MSLN), a cell surface protein expressed in 37% of childhood and young adult AML as well as older adult AML that, importantly, is absent in normal hematopoietic cells. Mesothelin is overexpressed in a number of solid tumors and has been studied as a target in solid tumors with various immunotherapeutic agents, including the antibody-drug conjugate (ADC) anetumab ravtansine (AR), comprised of an anti-MSLN antibody conjugated to the maytansinoid tubulin inhibitor DM4. We evaluated AR as well as a novel MSLN-targeted ADC for their ability to induce MSLN-directed cytotoxicity in several cell lines engineered to express MSLN.
As we have previously reported, the leukemia cell line K562 transduced to overexpress MSLN (K562+MSLN) was highly sensitive to AR in vitro with an IC50 of 1.4 nM (Fig. 1a). Furthermore, AR was highly active in vivo in K562+MSLN xenograft-bearing mice. We sought to extend the study of AR's cytotoxicity in AML, to illustrate its potential as a therapeutic agent, in 3 additional AML cell lines, MV4;11, Kasumi-1, and Me-1, lentivirally-transduced to express MSLN, with cell-surface MSLN levels confirmed by flow cytometry. In vitro cytotoxicity assays demonstrated potent MSLN-dependent cytotoxicity of AR in MV4;11+MSLN cells with IC50 0.009 nM, compared to 1.9-3.8 nM in control experiments with parental cells or isotype control (IC) treatment (Fig. 1b). AR had limited MSLN-dependent activity in Kasumi-1+MSLN, with an IC50 of 28 nM, compared to 76-87 nM in controls (Fig. 1c) and no MSLN-dependent cytotoxicity in Me-1+MSLN, compared to controls (Fig. 1d). Previous reports suggest that DM4 is a substrate of the drug resistance protein P-glycoprotein (ABCB1). We evaluated drug resistance protein expression, including ABCB1, MRP1-6, LRP1, and BRCP, using RNASeq in representative AR-sensitive and AR-resistant cell lines, MV4;11 and Kasumi-1, respectively, and examined published RNASeq data for K562 and Me-1, and found that P-glycoprotein expression correlated with resistance to AR, measured by the IC50 (R2 = 0.995, p = 0.002).
To optimize ADC targeting of MSLN in AML, we created a novel MSLN-targeted ADC using an alternative MSLN antibody with an alternative chemotherapeutic payload, pyrrolobenzodiazepine (PBD) dimer, and molecular linker (anti-MSLN-PBD) as well as 3 control compounds: an anti-MSLN ADC with a DM4 payload (anti-MSLN-DM4) and isotype controls comprised of anti-Tetanus toxin antibody conjugated to PBD (IC-PBD) or DM4 (IC-DM4). In vitro cytotoxicity assays in the 4 aforementioned MSLN+ leukemia cell lines revealed potent MSLN-dependent cell kill with the novel anti-MSLN-PBD compared to controls in all but Me-1+MSLN, with IC50 of 1.6 pM-8.3 nM (Fig. 1e-h). Notably, unlike AR, anti-MSLN-PBD was able to confer MSLN-dependent cytotoxicity in Kasumi-1+MSLN. Sensitivity to anti-MSLN-PBD was independent of P-glycoprotein expression.
Mesothelin is a cell-surface protein previously recognized as an attractive immunotherapeutic target in solid tumors that is aberrantly expressed in a sizeable subset of AML patients of all ages, and herein we report successful targeting of MSLN in leukemia cells in vitro with antibody-drug conjugates. AR, an ADC well-tolerated in phase I testing for solid tumors, showed dramatic MSLN-dependent cytotoxicity in 2 cell lines at clinically-achievable doses. To optimize MSLN targeting in AML, we created a novel MSLN-targeted ADC with a PBD dimer payload, which conferred a high degree of MSLN-dependent cytotoxicity in vitro in 3 of 4 MSLN+ leukemia cell lines tested. Mesothelin-directed ADCs hold great promise for the treatment of MSLN+ AML across the age spectrum and warrant additional pre-clinical and clinical evaluation as potential novel immunotherapeutics for AML.
Disclosures
No relevant conflicts of interest to declare.
P-glycoprotein Expression in Solid Tumors – An Analysis
