Sensitive ELISA Method for the Measurement of Catabolites of

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Article pubs.acs.org/molecularpharmaceutics

Sensitive ELISA Method for the Measurement of Catabolites of Antibody−Drug Conjugates (ADCs) in Target Cancer Cells Paulin L. Salomon and Rajeeva Singh* ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States

ABSTRACT: A new, sensitive ELISA method has been developed which measures catabolites in cells and media upon processing of antibody−drug conjugates (ADCs) by target cancer cells. This ELISA method, exemplified for maytansinoid ADCs, uses competitive inhibition by a maytansinoid analyte of the binding of biotinylated antimaytansine antibody to an immobilized BSA−maytansinoid conjugate. Synthetic standards of several maytansinoid catabolites derived from ADCs with different linkers were tested and showed similar inhibition curves, with an EC50 of about 0.1 nM (0.03 pmol in an assay volume of 0.25 mL). This high sensitivity allowed quantification of catabolites from a methanolic cell extract and from the medium, generated from an ADC in 1 day using only about 1 million cells. The processing of anti-EpCAM and anti-CanAg ADCs with noncleavable linker (SMCC-DM1), disulfide linker (SPDB-DM4), and charged sulfonate-bearing disulfide linker (sulfo-SPDB-DM4), each containing an average of about four maytansinoid molecules per antibody, were compared in colon cancer cell lines (COLO 205 and HT-29). An 8−10-fold higher total level of catabolite was observed for anti-CanAg ADCs than for anti-EpCAM ADCs upon processing by COLO 205 cells, consistent with a higher cell-surface expression of CanAg. In a multidrug resistant HCT-15 colon cancer cell line, the anti-EpCAM-SPDB-DM4 linker conjugate was not cytotoxic and showed a significantly lower level of catabolite within cells compared to that in medium, presumably due to Pgp-mediated efflux of the nonpolar DM4 catabolite. In contrast, sulfo-SPDB-DM4 and SMCC-DM1 linker conjugates were cytotoxic, which correlated with higher amounts of catabolites found within the HCT-15 cells relative to amounts in medium. In a nonmultidrug resistant HT-29 cell line, the antiEpCAM-SPDB-DM4 linker conjugate was cytotoxic, with most of the catabolite found in cells and little in the medium. In conclusion, this highly sensitive ELISA method for measurement of ADC catabolite is convenient for screening multiple ADC parameters such as linkers and antibodies in a number of cell lines, does not require concentration of sample or extraction of media, and is complementary to other reported methods such as radiolabeling of ADCs or mass spectrometry. KEYWORDS: antibody−drug conjugate, ADC, catabolite, assay, degradation, processing, ELISA, maytansinoid, linker, disulfide, noncleavable, multidrug resistant, MDR

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INTRODUCTION Antibody−drug conjugates (ADCs) constitute a new class of therapeutic agents being developed against cancer.1,2 Two ADCs are currently approved by the FDA for cancer treatment: (i) brentuximab vedotin (Adcetris) for the treatment of patients with CD30-positive relapsed or refractory Hodgkin lymphoma and systemic anaplastic large cell lymphoma, and (ii) ado-trastuzumab emtansine (Kadcyla) for the treatment of patients with HER2-positive late-stage (metastatic) breast cancer.3,4 A large number of ADCs targeting different antigens and employing various cytotoxic drugs are currently being tested in the clinic against several types of cancer.2,5 An ADC has three components: (a) a monoclonal antibody developed to bind preferentially to a cell-surface antigen target, (b) a cytotoxic small molecule of sufficient potency that can kill the target cancer cell upon release from the antibody, and (c) a linker that joins this molecule to the antibody and is designed © 2015 American Chemical Society

to be stable in the circulation while allowing the release of the cytotoxic agent upon internalization and degradation of the ADC in target cancer cell.5 Most of the ADCs currently undergoing clinical evaluation contain an average of 3−4 molecules of the cytotoxic agent per antibody molecule, although a few ADCs have a higher number of about 6−8 molecules linked per antibody molecule.3,4,6,7 The cytotoxic agent is linked at lysine or cysteine residues. ADCs in clinical development target a diverse number of cell-surface antigens expressed on cancer cells using a large array of different, tumor-specific monoclonal antibodies.2 These Special Issue: Antibody-Drug Conjugates Received: Revised: Accepted: Published: 1752

January 12, 2015 February 19, 2015 March 4, 2015 March 4, 2015 DOI: 10.1021/acs.molpharmaceut.5b00028 Mol. Pharmaceutics 2015, 12, 1752−1761

Article

Molecular Pharmaceutics

Figure 1. Structures of maytansinoid ADCs used in this study and their catabolites.

para-aminobenzylcarbamate (PABC) spacer and a valinecitrulline (val-cit) peptide linker.3,16,17 The calicheamicin ADCs use a hybrid linker containing both an acid-cleavable hydrazone and a stable disulfide.7,18 A carbonate linker is used for SN38 ADCs.19 The preclinical optimization of an ADC typically involves preparation of multiple conjugates, which are tested for their in vitro activity and in vivo therapeutic index.4,5 A quantitative measurement of the catabolites of the ADC in target cancer cells is important both for correlation with cytotoxic activity and for preclinical optimization to select the appropriate antibody, linker, and cytotoxic agent. Two quantitative methods have been reported for ADC catabolism, which either use mass spectrometry for quantification of catabolites or use HPLC and liquid scintillation counting to analyze radioactive catabolites generated from radiolabeled ADCs.17,20−22 Using ADCs prepared with 3H-labeled maytansinoids, it was shown that noncleavable linker SMCC-DM1 conjugates of anti-HER2 and anti-CanAg C242 antibodies were catabolized by lysosomal proteolysis in antigen-expressing

ADCs employ various cytotoxic small molecules to inhibit essential intracellular functions, such as maytansinoids (DM1, DM4) and auristatins (MMAE, MMAF), which inhibit microtubule dynamics, calicheamicin and pyrrolobenzodiazepine dimer, which inhibit DNA, and doxorubicin and SN38, which inhibit DNA topoisomerases.6−13 Several types of linkers are used in ADCs, ranging from noncleavable thioether linkers to cleavable linkers such as disulfide, peptide, hydrazone, and carbonate.5 The noncleavable thioether linkers include 4-(Nmaleimidomethyl)cyclohexane carboxylic acid N-hydroxysuccinimide ester (SMCC) and maleimidocaproyl (MC), used for DM1 and MMAF ADCs, respectively.4,9 Two disulfide linker designs are employed in DM4 ADCs, N-succinimidyl 4-(2pyridyldithio)butanoate linker (SPDB) and a charged sulfonate-bearing linker (sulfo-SPDB), both of which result in a DM4-linker disulfide having enhanced stability due to steric hindrance through incorporation of methyl substituents at the carbon atom adjacent to the disulfide bond.10,14,15 Brentuximab vedotin and other MMAE ADCs employ a self-immolative 1753

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cancer cells to yield a lysine-linked maytansinoid catabolite, lysine-SMCC-DM1.20,21 The catabolism of disulfide-linker SPDB-DM4 conjugate yielded lysine-SPDB-DM4 catabolite generated by proteolytic processing, a nonpolar DM4 catabolite generated from further disulfide bond cleavage, and S-methyl DM4 generated by S-methylation.21,22 The maytansinoid catabolites were found not only intracellularly, as expected, but also in the medium for the high CanAg antigen-expressing COLO 205 cancer cells, presumably due to the generation of high intracellular catabolite levels resulting in the export of catabolites.22 The SPDB-DM4 and sulfo-SPDB-DM4 disulfide linker conjugates are often more active in preclinical in vivo models than the noncleavable linker SMCC-DM1 conjugate, potentially due to the additional bystander cytotoxic activity of exported nonpolar, DM4 and S-methyl DM4 catabolites, which can enter and kill neighboring bystander cells that cannot be killed directly either due to low accessibility or low antigen levels.15,23 Figure 1 shows structures of catabolites generated from maytansinoid ADCs with noncleavable (SMCC-DM1) and disulfide (SPDB-DM4 and sulfo-SPDB-DM4) linkers.14,22 Using an anti-CD30-val-cit-PABC-MMAE conjugate prepared with 14C-labeled MMAE, and also by quantitative mass spectrometry for a nonradioactive conjugate, it was shown that CD30-expressing cancer cells generated MMAE catabolite that was exported into the medium and exerted additional cytotoxic activity toward bystander cells.17 We report here a new, quantitative ELISA method for analyzing as low as 0.01 picomoles of catabolites (intracellular and media) from a cancer cell line treated with an ADC. This highly sensitive ELISA method is convenient, does not require concentration of cell extract or media, can be carried out simultaneously for many samples in multiwell cell culture plates (for treatment of cells with conjugates) and multiwell ELISA plates (for catabolite quantification), and does not require specialized facilities for handling radioactivity or for mass spectrometry. This ELISA method is based on the competitive inhibition by a maytansinoid catabolite of the binding of biotinylated antimaytansine antibody to immobilized BSA− maytansinoid conjugate in microtiter plates, such that the signal of binding of biotinylated antimaytansine antibody to plate (detected using streptavidin−peroxidase conjugate and a chromophoric peroxidase substrate) decreases with increasing concentration of the maytansinoid catabolite. An ELISA method has been described previously for assaying free MMAE drug upon release from val-cit-PABC-MMAE conjugate using cathepsin B, but it was not used for analyzing ADC catabolism by cells.24 This previously reported MMAE competition ELISA method used horseradish peroxidase (HRP)−MMAE conjugate which was mixed with free MMAE drug and then added to wells coated with anti-MMAE antibody, where the binding of HRP−MMAE was in competition with free MMAE in a dose-dependent manner with an EC50 of 2.4 nM drug.24 In contrast, the new ELISA method reported here, with an EC50 of about 0.1 nM drug, shows a 20-fold improvement in sensitivity compared to the previously reported MMAE competition ELISA. We have used this new, competition ELISA method to analyze catabolite levels in cells and media from several cancer cell lines, including multidrug resistant cancer cells, generated upon treatment with anti-CanAg and anti-EpCAM antibody−maytansinoid conjugates with different linkers.

Article

EXPERIMENTAL SECTION

Materials. Human colon cancer cell lines, COLO 205, HT29, and HCT-15 were from American Type Culture Collection (ATCC). Cell culture media were from ATCC or Life Sciences. CellTiter-Glo reagent was from Promega. Streptavidin−Horseradish Peroxidase (HRP) conjugate was from Jackson ImmunoResearch Laboratories. A monoclonal antimaytansine antibody (mouse IgG 1 ; K d ∼0.1 nM), developed at ImmunoGen, was biotinylated with succinimidyl-6(biotinamido)hexanoate reagent (NHS-LC-biotin; Pierce) to an average modification of about 8 biotin molecules per antibody molecule. Bovine serum albumin (BSA; Sigma) was modified using DM4 maytansinoid and noncleavable SMCC linker to an average of 1.8 DM4 molecules per BSA molecule using a procedure similar to that previously described.10,14 Conjugates of humanized anti-CanAg antibody (huC242) and chimeric anti-EpCAM antibody with SMCC-DM1, SPDBDM4, and sulfo-SPDB-DM4 were prepared as described previously.10,14 The anti-CanAg antibody (huC242) SMCCDM1 and SPDB-DM4 conjugates contained an average of 4.2 and 4.9 maytansinoid molecules per antibody, and the antiEpCAM SMCC-DM1, SPDB-DM4, and sulfo-SPDB-DM4 conjugates contained an average of 4.3, 3.5, and 3.5 maytansinoid molecules per antibody, respectively. EpCAM expression is sensitive to the density of cells and the day of plating;25 typically, cells were plated 1 day before conjugate treatment and were not confluent. The cells were maintained at 37 °C in a humidified atmosphere that contained 5% CO2. Lysine-SMCC-DM1, DM4-Me (S-methyl DM4), and DM4NEM (N-ethylmaleimide-capped DM4) standards for ELISA were chemically synthesized.10,22 Chemical reagents were from Sigma-Aldrich. The compositions of some of the frequently used buffers were the following: carbonate buffer (15 mM sodium carbonate, 35 mM sodium bicarbonate); TBS-T (20 mM Tris buffer, pH 7.5, containing 150 mM NaCl and 0.1% Tween 20); and blocking buffer (10 mg/mL BSA in TBS-T). The antigen numbers per cell for the cell lines, based on reported values, are CanAg, COLO 205 (∼4 × 106); EpCAM, COLO 205 (∼9 × 105); EpCAM, HT-29 (∼5−7 × 105); EpCAM, HCT-15 (∼7 × 105 per cell).14,26 Treatment of Cells with ADCs for Catabolism Experiments. Cells were plated in a 6-well plate (typically about 0.5 million cells per well) and incubated overnight at 37 °C in incubator to achieve ∼70% confluency the next day. The medium was removed, and ADC was added at a saturating concentration of 2 μg/mL (antibody-based concentration), or a control containing a mixture of 2 μg/mL ADC and 200-fold molar excess of unconjugated antibody (400 μg/mL), for a short exposure of ∼2 h in an incubator at 37 °C. After the short exposure, the conjugate-containing medium was removed, and cells were washed three times with 3 mL of medium before addition of 2 mL of fresh medium. The cells were incubated for an additional period of about 1 day at 37 °C, to allow processing of ADC before harvest. The harvested medium was clarified by centrifugation to separate any detached cells. Cells were lysed using 150 μL ice-cold methanol and scraped off the plate with a cell scraper. Each well was rinsed with an additional 150 μL of methanol, which was combined with the cell pellet of detached cells derived from clarification of the medium. The pooled methanolic extract and cell suspension mixtures were stored at −20 °C and clarified by centrifugation (16000g, 5 min) before analysis by ELISA. One well of untreated cells was 1754

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Figure 2. (A) ELISA inhibition curves of synthetic catabolite standards, lysine-SMCC-DM1, DM4-Me (S-methyl DM4), and DM4-NEM (Nethylmaleimide capped DM4). (B) ELISA measurement of catabolites generated upon processing of anti-EpCAM-SMCC-DM1 ADC (E-SMCCDM1) in colon cancer COLO 205 cells. Cells were incubated for about 2 h with a saturating concentration (2 μg/mL) of ADC, without or with an excess of unconjugated anti-EpCAM antibody (400 μg/mL), washed, and further incubated for 1 day of processing. Catabolite amounts in methanolic cell extracts and media were analyzed using ELISA. The antigen number per cell for EpCAM in COLO 205 cells is ∼9 × 105 per cell.

incubated with streptavidin−HRP (1 μg/mL; 100 μL each well) for about 30 min at ambient temperature, followed by washes and incubation with ABTS/H2O2 substrate (0.5 mg/mL ABTS, 0.03% H2O2, in 0.1 M citrate buffer, pH 4.2). The absorbance was measured using a multiwell plate reader at 405 nm, typically within 5−10 min. Nonlinear 4-PL fit (Graph Pad Prism) was used to estimate the maytansinoid concentrations in samples (tested at several dilutions) using the curve fit derived from maytansinoid standards. The catabolite estimates showed coefficients of variation (CV) ranging from 7 to 28%. In separate experiments, control media samples (generated from cells after 1 day ADC-processing) were treated with a 3fold volume of ice-cold methanol and incubated typically for a minimum of 6 h at −20 °C to precipitate any dissociated conjugate, and supernatants were assayed by competition ELISA to demonstrate that inhibition was caused by catabolites in media and not by dissociated conjugate. Alternate HRP development methods such as TMB, acid-stoppage of development, and a chemiluminescent substrate (Luminol) were tested and found to be compatible, but typically, they were not used. The nonspecific catabolite amount detected for control treatment (mixture of conjugate with excess unconjugated antibody) was much lower than the amount with conjugate alone and was typically subtracted from the amount with conjugate alone to calculate the specifically processed amount of catabolite in cells and in the medium. In Vitro Cytotoxicity of ADCs. HT-29 and HCT-15 cells (3000 cells per well) were incubated in a 96-well white cellculture plate (clear-bottom; Greiner Bio-One) with anti-CanAg and anti-EpCAM SMCC-DM1, SPDB-DM4, and sulfo-SPDBDM4 conjugates at various concentrations (10−11−10−8 M range) for 3−4 days in an incubator at 37 °C. Control wells contained a mixture of conjugate and 200-fold molar excess of unconjugated antibody. After 3−4 days of continuous incubation, at which significant morphological cytotoxicity was observed, CellTiter-Glo reagent was added, and luminescence measured. COLO 205 cells were treated with 2 μg/mL ADC (and controls containing mixtures of conjugates with 200-fold molar excess of unconjugated antibody) for a short-term of about 2 h at 37 °C, washed to remove unbound

trypsinized, trypan blue was added, and cells were counted using a hemocytometer to calculate the total number of cells per well. Cell viability typically was 90% or higher, showing little cell death after 1 day, as expected. For suspension cells (COLO 205), about 1−2 million cells in 2 mL of medium were treated for about 2 h with conjugate at 37 °C as described above, before centrifugation (400 rpm, ∼65g, 5 min) and washing (two times with 5 mL medium). The cells were resuspended in 2 mL of fresh medium and transferred to a 6well plate for incubation for about 1 day at 37 °C to allow ADC processing, after which the methanolic cell extract (in a total volume of 150 μL of methanol) and medium samples were prepared. Quantitation of Maytansinoid Catabolites by ELISA. Maytansinoid catabolites were analyzed by their competition toward the binding of biotinylated antimaytansine antibody to immobilized BSA−DM4 conjugate, where increasing concentrations of maytansinoid catabolite resulted in decreasing signals of bound biotinylated antimaytansine antibody, which was detected using streptavidin−HRP conjugate. BSA−DM4 conjugate (2 μg/mL in carbonate buffer; 100 μL per well) was added to 96-well Immulon-2HB plate and coated overnight at 4 °C. The wells were then blocked with 300 μL of blocking buffer for 1 h at ambient temperature, followed by a wash with TBS-T buffer. Separately, mixtures of biotinylated antimaytansine antibody (16 ng/mL) and maytansinoid catabolites or maytansinoid standards (typically lysine-SMCC-DM1, ranging in concentrations from 0.031 to 2 nM in a total volume of ∼1 mL blocking buffer were preincubated for >2 h at ambient temperature (or overnight at 4 °C), and then the samples were added in triplicate (0.25 mL each) to the wells containing the coated BSA−DM4 conjugate. A relatively large volume (0.25 mL per well) was typically used for this step to maximize the sample amount and to decrease the methanol concentration (6% or lower) in the diluted cell lysate sample, so as to eliminate further processing steps such as concentration of cell lysates or extraction of media. For diluted samples containing 6% methanol, a similar methanol amount of 6% was used for the maytansinoid standards. After an incubation for ∼2 h at ambient temperature, the wells were washed and then 1755

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Figure 3. Cytotoxicity of anti-CanAg and anti-EpCAM ADCs in colon cancer COLO 205 cells. COLO 205 cells were treated for 2 h with saturating concentrations (2 μg/mL) of anti-CanAg or anti-EpCAM ADCs, washed, and then incubated with fresh media for 3 days. Controls for specificity contained 200-fold molar excess of unconjugated antibody (400 μg/mL). (A) Cytotoxicity of anti-CanAg (C) conjugates with SMCC-DM1 and SPDB-DM4 linkers (C-SMCC-DM1 and C-SPDB-DM4, respectively). (B) Cytotoxicity of anti-EpCAM (E) conjugates with SMCC-DM1, SPDBDM4, and sulfo-SPDB-DM4 linkers (E-SMCC-DM1, E-SPDB-DM4, and E-sulfo-SPDB-DM4, respectively). The antigen numbers per cell for CanAg and EpCAM antigens in COLO 205 cells are ∼4 × 106 and 9 × 105 per cell, respectively.

ADC. After 1 day, the medium and the methanolic extract of the cells were assayed by competition ELISA, as described above. It has been reported that treatment of EpCAMexpressing COLO 205 cells with [3H]-DM1-labeled antiEpCAM-SMCC-DM1 conjugate generates lysine-SMCC-[3H]DM1 as the sole catabolite in a 1 day processing measurement.26 The generation of the maytansinoid catabolite in target cell causes suppression of microtubule dynamic instability, G2/ M phase mitotic arrest, and ultimately cytotoxicity, which is typically measured after about 4 days.21,26,27 The amount of maytansinoid catabolite in COLO 205 cell extracts generated in 1 day upon treatment with E-SMCC-DM1 conjugate was measured by ELISA as 1.17 pmol catabolite per million cells. This catabolite generation was through antigenmediated processing as a control treatment with a mixture of conjugate and 200-fold molar excess of unconjugated antiEpCAM antibody resulted in only 0.11 pmol catabolite per million cells (10% of specific level; Figure 2B). Similarly, 2.16 pmol catabolite per million cells was detected in the medium upon conjugate treatment, compared to a control treatment with conjugate and excess unconjugated antibody, which produced only 0.12 pmol catabolite per million cells in the medium (5% of specific level; Figure 2B). The catabolite amounts measured both in cells and in the medium, therefore, were generated by antigen-mediated processing. A control experiment was carried out to demonstrate that the catabolite detected in medium was a small-molecule catabolite and not protein-bound drug. The medium was treated with methanol (3:1, v/v) to precipitate intact conjugate and was found to contain about 75% of the catabolite level compared to untreated medium (data not shown). In an independent experiment, it was verified that a spiked-in conjugate in medium was removed using precipitation with methanol, based on ELISA (data not shown). In another experiment, the time course of catabolite formation was measured after a 1 h treatment with E-SMCC-DM1, followed by washing and incubation for 11 or 24 h. The amount of total catabolites increased by about 50% from 11 to 24 h (1.75 to 2.63 pmol per million cells), with the increase mostly in the medium (0.83 to 1.60 pmol/106 cells) and little increase in the cells (0.92 to 1.03

conjugate, followed by further incubation with fresh medium for 3 days at 37 °C, after which their viability was measured using CellTiter-Glo reagent.

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RESULTS Measurement of Maytansinoid Catabolites by ELISA. Here we explore an ELISA method designed for quantitative measurements of maytansinoid catabolites generated by cellular metabolism of maytansinoid ADCs. The assay involves mixing biotinylated antimaytansine antibody with maytansinoid catabolites (or maytansinoid standards) to allow binding, followed by the addition of the mixture to immobilized BSA−maytansinoid conjugate, and detection of bound biotinylated antimaytansine antibody using streptavidin−horseradish peroxidase (HRP) conjugate. Increasing concentrations of maytansinoid catabolites (or standards) result in decreasing amounts of bound biotinylated antimaytansine antibody (Figure 2A). The ELISA method performed in a consistent manner for different maytansinoid catabolites, lysine-SMCC-DM1, Smethyl DM4, and N-ethylmaleimide-capped DM4 (DM4NEM), as shown in Figure 2A. Several other maytansinoid derivatives also have been tested and show similar inhibition in ELISA (data not shown). The ELISA method is highly sensitive for measuring catabolites, showing an EC50 of about 0.03 pmol of catabolite in a volume of 0.25 mL (about 0.1 nM), which is 20-fold more sensitive than a previously reported ELISA method.24 This high sensitivity allows measurement of catabolites generated by cancer cell lines upon treatment with ADCs toward multiple targets. ADC Catabolite Measurement in Target Cancer Cells. To explore the specificity of ADC catabolism by target cells, we treated a colon cancer cell line, COLO 205, for a shortexposure of about 2 h with a saturating concentration of antiEpCAM antibody (E)-maytansinoid conjugate bearing noncleavable SMCC linker (E-SMCC-DM1; 2 μg/mL), or with a mixture of E-SMCC-DM1 conjugate and a 200-fold molar excess of unconjugated anti-EpCAM antibody (400 μg/mL). The cells were washed to remove excess conjugate and further incubated for about 1 day at 37◦C to allow catabolism of the 1756

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Table 1. Catabolite Amounts Generated by Colon Cancer COLO 205 Cells in 1 Day after a Pulsed Incubation with Anti-CanAg (C) and Anti-EpCAM (E) ADCs conjugate

total catabolite (pmol/106 cells)a

catabolite in cells (pmol/106 cells)

catabolite in medium (pmol/106 cells)

C-SPDB-DM4 C-SMCC-DM1 E-SPDB-DM4 E-sulfo-SPDB-DM4 E-SMCC-DM1

19.2 20.1 2.04 2.59 2.67

2.67 2.41 1.26 1.96 0.98

16.7 17.7 0.78 0.63 1.69

a COLO 205 cells were treated for 2 h with saturating concentrations (2 μg/mL) of anti-CanAg or anti-EpCAM ADCs, washed, and then incubated with fresh media for 1 day. Total catabolite amounts were calculated by adding the amounts in cells and in media. The antigen numbers per cell for CanAg and EpCAM antigens in COLO 205 cells are ∼4 × 106 and 9 × 105 per cell, respectively.

Figure 4. Cytotoxicity and processing of anti-EpCAM ADCs in colon cancer HT-29 cells. (A) Cytotoxicity of anti-EpCAM ADCs in HT-29 cells. HT-29 cells were treated with several concentrations of anti-EpCAM (E) conjugates bearing SPDB-DM4 and SMCC-DM1 linkers (E-SPDB-DM4 and E-SMCC-DM1, respectively) continuously for 4 days and analyzed for cell viability using CellTiter-Glo reagent. (B) Processing of anti-EpCAM (E) conjugates in HT-29 cells. Cells were treated for a 2 h pulse with saturating concentrations of anti-EpCAM ADCs (2 μg/mL), washed, and further incubated with fresh media for 1 day for processing. Controls for specificity contained 200-fold molar excess of unconjugated anti-EpCAM antibody (400 μg/mL). The antigen number per cell for EpCAM in HT-29 cells is ∼5−7 × 105 per cell.

pmol/106 cells), presumably due to a significant export of maytansinoid catabolites above an intracellular level of about 1 pmol catabolite per million cells. Comparison of Catabolism of Anti-EpCAM and AntiCanAg Conjugates with Different Linkers in Cancer Cell Lines. We used the ELISA method to measure the processing of anti-EpCAM (E) and anti-CanAg (C) conjugates with different linkers in colon cancer cell lines, COLO 205 and HT29. COLO 205 cells have been reported to express high levels of CanAg, or CA242 antigen (∼4 × 106 per cell) and a 73% processing of bound ADC in 1 day, consistent with high levels of catabolite generation and export upon treatment with [3H]maytansinoid-labeled, noncleavable or disulfide linker conjugates (C-SMCC-[3H]-DM1, C-SPDB-[3H]-DM4), and specific cytotoxicity demonstrated at low concentrations of conjugates.14,21,26,28 The cytotoxicity in COLO 205 cells was measured using a pulsed, 2 h exposure of the conjugate (2 μg/ mL; ∼12 nM), after which unbound conjugate was washed off, and cells were incubated with fresh medium at 37◦C for 3 days before their viability was measured. Both C-SMCC-DM1 and C-SPDB-DM4 conjugates showed near complete cell-killing, with approximately 4% cell viability compared to the untreated cells (Figure 3A). The cell-killing of each conjugate was abrogated in the control sample treated with a mixture of conjugate and 200-fold excess of unconjugated antibody, demonstrating the specificity of cell-killing by conjugate when added alone (Figure 3A). Using a similar short-term conjugate treatment, followed by wash and 1 day incubation, catabolite levels were analyzed by ELISA in cells (methanolic extract) and in the medium. The total levels of catabolites were calculated by

adding the amounts in cells and in the medium. As shown in Table 1, very high levels of total catabolites (about 20 pmol per million cells) were observed for C-SMCC-DM1 and C-SPDBDM4 conjugates, of which the levels in the medium (∼17−18 pmol per million cells) were much higher than those in the cells (∼2.5−2.7 pmol per million cells), presumably due to export resulting from high catabolite levels generated in cells. The catabolism of C-SMCC-DM1 and C-SPDB-DM4 conjugates by COLO 205 cells was antigen-specific, based on much lower catabolite generation for control treatments with mixtures containing excess unconjugated antibody (data not shown). Upon short-term treatment of COLO 205 cells with antiEpCAM conjugates bearing disulfide linkers (E-SPDB-DM4, Esulfo-SPDB-DM4) or noncleavable linker (E-SMCC-DM1), followed by washes to remove excess conjugate, and incubation at 37 ◦C for 3 days, the cell viabilities (35%, 18% and 4.3%, respectively) were lower compared to untreated cells. These cell-killing effects were specific based on their abrogation in mixtures of conjugate with excess unconjugated antibody (Figure 3B). The catabolite levels in cells and in the medium were determined by ELISA after a short-term conjugate treatment and a 1 day processing of cell-bound anti-EpCAM conjugates (Table 1). The total amounts of catabolites for antiEpCAM (E) conjugates (∼2−2.7 pmol per million cells) were much lower than those for anti-CanAg (C) conjugates (∼20 pmol per million cells), as expected based on the much higher cell-surface expression of CanAg than of EpCAM in COLO 205 cells (∼4 × 106 and 9 × 105 per cell, respectively; Table 1),14,26 and they are consistent with the overall lower extent of cellkilling effects by E conjugates compared to C conjugates 1757

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Molecular Pharmaceutics

Figure 5. Cytotoxicity and processing of anti-EpCAM ADCs in multidrug-resistant colon cancer HCT-15 cells. (A) Cytotoxicity of anti-EpCAM ADCs in HCT-15 cells. HCT-15 cells were treated with several concentrations of anti-EpCAM (E) conjugates bearing SPDB-DM4, sulfo-SPDBDM4, and SMCC-DM1 linkers (E-SPDB-DM4, E-sulfo-SPDB-DM4, and E-SMCC-DM1, respectively) continuously for 4 days and analyzed for cell viability using CellTiter-Glo reagent. (B) Processing of anti-EpCAM (E) conjugates. Cells were treated for a 2 h pulse with saturating concentrations of anti-EpCAM ADCs (2 μg/mL), washed, and further incubated with fresh media for 1 day for processing. Controls for specificity contained 200fold molar excess of unconjugated anti-EpCAM antibody (400 μg/mL). The antigen number per cell for EpCAM in HCT-15 cells is ∼7 × 105 per cell.

(MDR) Cell Line, HCT-15. Anti-EpCAM (E) conjugates with different linkers were previously tested for cytotoxic activity in a native MDR1 (P-glycoprotein, Pgp)-expressing multidrug resistant colon cancer cell line, HCT-15, and in Pgp-transfected COLO 205 cells (COLO 205-MDR). It was observed that a noncleavable linker conjugate, E-SMCC-DM1, and a disulfide linker conjugate bearing a charged, sulfonate group, E-sulfoSPDB-DM4, were significantly more cytotoxic than another disulfide linker conjugate, E-SPDB-DM4.14 We prepared new batches of these conjugates and tested their activities toward the multidrug resistant HCT-15 cells (∼7 × 105 EpCAM per cell) in a 4 day continuous incubation cytotoxicity assay, and we observed, in concordance with the published data, that ESMCC-DM1 and E-sulfo-SPDB-DM4 were significantly more cytotoxic (IC50 ∼ 0.5 nM) than E-SPDB-DM4, which was was not cytotoxic even at 4 nM (Figure 5A). The cytotoxic activities of E-SMCC-DM1 and E-sulfo-SPDB-DM4 conjugates were EpCAM-specific because their activities were abrogated upon incubations with conjugate mixtures containing 200-fold excess unconjugated antibody. We employed the ELISA method to analyze catabolite levels in HCT-15 cells and in the medium upon 1 day processing of these ADCs. The total amount of catabolite generated from E-sulfo-SPDB-DM4 was higher (0.70 pmol per million cells) compared to E-SPDB-DM4 and ESMCC-DM1 (0.40 pmol per million cells each). However, a very high proportion of exported catabolite was detected in the medium for E-SPDB-DM4 even though the total catabolite level (0.40 pmol per million cells) is relatively low. Unlike ESPDB-DM4, the intracellular catabolite amounts in HCT-15 cells were higher than in medium for both E-sulfo-SPDB-DM4 and E-SMCC-DM1 (Figure 5B). It is hypothesized that due to active Pgp export of DM4 catabolite, the intracellular catabolite accumulation for E-SPDB-DM4 (0.08 pmol per million cells) was below the threshold needed for cytotoxic activity. In contrast, the relatively higher intracellular levels for E-sulfoSPDB-DM4 and E-SMCC-DM1 (0.50 and 0.30 pmol per million cells) presumably were sufficient to be cytotoxic toward the multidrug resistant HCT-15 cells.

(Figure 3A,B). Among the E conjugates, the total catabolite levels were slightly greater for E-SMCC-DM1 and E-sulfoSPDB-DM4 (2.7 and 2.6 pmol per million cells) compared to that for E-SPDB-DM4 (2 pmol per million cells) and correlate with the greater cell killing by E-SMCC-DM1 and E-sulfoSPDB-DM4 compared to E-SPDB-DM4 (Figure 3B). The cytotoxicity for E-SMCC-DM1 was greater than for E-sulfoSPDB-DM4, presumably due to a greater efflux of the lysineSMCC-DM1 catabolite out of the lysosome into the cytosol, where the catabolite bound to microtubules and was partly exported into the medium. In contrast, the E-sulfo-SPDB-DM4 catabolites could remain trapped in the lysosomes of these cells, consistent with a higher catabolite level detected in the cell extract but a lower level in the medium for E-sulfo-SPDB-DM4 compared to E-SMCC-DM1 (Table 1). The catabolism and cytotoxicity of anti-EpCAM and antiCanAg ADCs were measured in another colon cancer cell line, HT-29. The anti-CanAg conjugates were not cytotoxic in HT29 cells and did not show significant amounts of catabolites (data not shown), consistent with the report of low and heterogeneous CanAg expression in HT-29 cells.28 The antiEpCAM conjugates were active in HT-29 cells (∼5−7 × 105 EpCAM per cell), where E-SPDB-DM4 showed slightly greater cytotoxic activity than E-SMCC-DM1 upon continuous incubation for 4 days (IC50 ∼ 0.8 and 2 nM, respectively; Figure 4A). The total amount of catabolites after 1-day processing of bound conjugates was assayed using ELISA, and found to be about 50% greater for E-SPDB-DM4 than for ESMCC-DM1, which correlates with the greater cytotoxicity of E-SPDB-DM4 in HT-29 cells (Figure 4B). For E-SPDB-DM4, most of the catabolite was found in the cells and little in the medium, suggesting a relatively low efflux at this total level of catabolite (0.65 pmol per million cells) in a nonmultidrug resistant cell line. The relative cytotoxicities of E-SPDB-DM4 and E-SMCC-DM1 conjugates were different in the two cell lines tested, COLO 205 and HT-29, and correlated with the extent of their processing, which could be readily tested using ELISA. Comparison of Catabolism of Anti-EpCAM (E) Conjugates with Different Linkers in a Multidrug Resistant 1758

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DISCUSSION A highly sensitive ELISA method has been developed to analyze maytansinoids at levels as low as 0.01 pmol. The maytansinoid analyte is premixed with a small amount of biotinylated antimaytansine antibody such that it competes with the binding of biotinylated antimaytansine antibody to an immobilized BSA−maytansinoid conjugate. Increasing levels of maytansinoid analyte result in decreasing signals of captured biotinylated antimaytansine antibody (detected using streptavidin−HRP conjugate and a chromophoric HRP substrate). Several maytansinoid catabolites expected from degradation of ADCs with different noncleavable and disulfide linkers were tested and showed similar inhibition curves with EC50 values of about 0.03 pmol. This EC50 of 0.03 pmol is similar to the amount of antibody used (0.025 pmol; or, 0.05 pmol total binding capacity, assuming two binding sites per antibody), indicating similar, high-affinity binding of these catabolites to biotinylated antimaytansine antibody. Similar EC50 values for different catabolites allowed the ease of use of one standard to test multiple catabolites. A relatively large assay volume (0.25 mL per well) avoids need for sample concentration and allows dilution of organic solvents in samples (such as methanolic extracts of cells) to low, noninterfering levels in final ELISA mixtures. This ELISA method for maytansinoid is about 20-fold more sensitive than a reported ELISA method for MMAE auristatin.24 The sensitivity of the ELISA was further improved by 2-fold using lower amounts of biotinylated antimaytansine antibody and a chemiluminescent HRP substrate; however, this sensitivity enhancement was not needed for routine use. The sensitivity of the maytansinoid ELISA method reported here (EC50 = 0.03 pmol) is also significantly higher than previously reported for radiolabeled drugs, which at 0.03 pmol would have low, near background scintillation counts of about 10−20 cpm (reported specific radioactivities for 3H-maytansinoids, ∼300− 850 mCi/mmol, and for 14C-MMAE conjugate, 7.9 μCi/ mg).17,29 This ELISA method can be implemented to screen the efficiency of catabolism of multiple ADC variants prepared with different linkers, drugs, and antibodies, and these data can be correlated with in vitro cytotoxicity. The method involves exposing target cell lines to ADCs for a short-term to allow binding, removing excess ADC by wash, and incubating cells for 1 day to allow processing, after which catabolites are assayed in cells and in the medium using ELISA. Due to the high sensitivity of the ELISA method, cells from 1 well of a 6-well plate (about 1 million cells) are sufficient for catabolite analysis, which can be done simultaneously for many samples in a 96well ELISA plate. The competition ELISA method was employed to assay catabolites generated in target cancer cells upon degradation of ADCs derived from antibodies directed toward EpCAM and CanAg (CA242) antigens, bearing noncleavable linker, SMCCDM1, and disulfide linkers, SPDB-DM4 and sulfo-SPDB-DM4. Colon cancer cell lines, COLO 205 and HT-29, showed processing of anti-EpCAM ADCs, with a greater amount of catabolite generated in COLO 205 cells and exported into the medium. COLO 205 cells express very high levels of CanAg antigen (∼4 × 106 per cell), which correlated with very high levels of catabolites generated and exported into the medium for anti-CanAg ADCs. In nonmultidrug resistant cell lines expresing high levels of antigen, it appears that a significant amount of catabolite is detected in the medium when the levels

of maytansinoid catabolites in cells are above 1 pmol per million cells, suggesting significant maytansinoid efflux above levels about 10−18 mol per cell, or about 700 000 maytansinoid molecules per cell. However, significant cell killing is observed even at levels of maytansinoid catabolites below 1 pmol per million cells, as was observed for anti-EpCAM ADCs in HT-29 cells. Assuming the volume of a single cell as 5 picoliter,30 the intracellular concentration of maytansinoid at this high, typically observed upper limit of about 1 pmol per million cells (10−18 mol per cell) is calculated as about 0.2 μM, which is comparable to the 0.1−0.4 μM concentration range of 3HDM1-S-Me uptake that was previously reported.27 For the CD30-targeting ADC, brentuximab vedotin, which uses the microtubule-directed MMAE drug, a similar intracellular concentration of about 0.3 μM catabolized MMAE was reported for nonmultidrug resistant L540cy cells under conditions where significant MMAE export into medium was observed, suggesting comparable high, intracellular accumulation levels for maytansinoids and auristatins, both which bind and inhibit microtubules.17,31 In a Pgp-expressing multidrug resistant (MDR) cell line, HCT-15, however, we observed that the anti-EpCAM-SPDBDM4 conjugate, which was not cytotoxic, showed significant levels of catabolite export into medium and little retention in cells, even though the total catabolite level of 0.40 pmol per million cells was relatively low. In contrast, sulfo-SPDB-DM4 and SMCC-DM1 conjugates, which were cytotoxic, showed greater intracellular catabolite levels and lower levels in media. The total catabolite generation was higher for sulfo-SPDBDM4 conjugate than for SPDB-DM4, presumably aided by the hydrophilic sulfo-SPDB linker. It is hypothesized that the much lower potency of anti-EpCAM-SPDB-DM4 conjugate compared to sulfo-SPDB-DM4 and SMCC-DM1 linker conjugates in HCT-15 cells is due to Pgp-export of nonpolar DM4 catabolite for SPDB-DM4, resulting in low intracellular catabolite level. The major catabolites of SMCC-DM1 and sulfo-SPDB-DM4 conjugates are lysine-SMCC-DM1 and lysine-sulfo-SPDB-DM4, respectively, which presumably are poor substrates for Pgp and better retained by cells leading to their cytotoxic activity. In conclusion, a highly sensitive ELISA method has been developed for measuring catabolites of maytansinoid ADCs, which does not require any sample concentration or media extraction steps and has been applied to analyze catabolite levels in cells and media for several cancer cell lines using ADCs derived from different target antibodies and multiple linkers. This convenient ELISA method is complementary to other methods of catabolite quantitation such as chromatographic analysis of radioactive catabolites from radiolabeled ADCs and mass spectrometry of catabolites from nonradioactive ADCs, which also yield information about the structures of the catabolites. We believe that the maytansinoid ELISA method can be extended to other drug catabolites using antibodies generated against the drug molecules.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: 781-895-0600. Fax: 781-895-0611. Notes

The authors declare the following competing financial interest(s): The authors are employees of ImmunoGen, Inc. 1759

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in a human multiple myeloma xenograft and in monkeys. Clin. Cancer Res. 2005, 11, 5257−5264. (13) Jeffrey, S. C.; Burke, P. J.; Lyon, R. P.; Meyer, D. W.; Sussman, D.; Anderson, M.; et al. A potent anti-CD70 antibody-drug conjugate combining a dimeric pyrrolobenzodiazepine drug with site-specific conjugation technology. Bioconjug Chem. 2013, 24, 1256−1263. (14) Zhao, R. Y.; Wilhelm, S. D.; Audette, C.; Jones, G.; Leece, B. A.; Lazar, A. C.; et al. Synthesis and evaluation of hydrophilic linkers for antibody-maytansinoid conjugates. J. Med. Chem. 2011, 54, 3606− 3623. (15) Kellogg, B. A.; Garrett, L.; Kovtun, Y.; Lai, K. C.; Leece, B.; Miller, M.; et al. Disulfide-linked antibody-maytansinoid conjugates: optimization of in vivo activity by varying the steric hindrance at carbon atoms adjacent to the disulfide linkage. Bioconjug Chem. 2011, 22, 717−727. (16) Doronina, S. O.; Toki, B. E.; Torgov, M. Y.; Mendelsohn, B. A.; Cerveny, C. G.; Chace, D. F.; et al. Development of potent monoclonal antibody auristatin conjugates for cancer therapy. Nat. Biotechnol. 2003, 21, 778−784. (17) Okeley, N. M.; Miyamoto, J. B.; Zhang, X.; Sanderson, R. J.; Benjamin, D. R.; Sievers, E. L.; et al. Intracellular activation of SGN-35, a potent anti-CD30 antibody-drug conjugate. Clin. Cancer Res. 2010, 16, 888−897. (18) Hamann, P. R.; Hinman, L. M.; Hollander, I.; Beyer, C. F.; Lindh, D.; Holcomb, R.; et al. Gemtuzumab ozogamicin, a potent and selective anti-CD33 antibody-calicheamicin conjugate for treatment of acute myeloid leukemia. Bioconjug Chem. 2002, 13, 47−58. (19) Govindan, S. V.; Cardillo, T. M.; Moon, S. J.; Hansen, H. J.; Goldenberg, D. M. CEACAM5-targeted therapy of human colonic and pancreatic cancer xenografts with potent labetuzumab-SN-38 immunoconjugates. Clin. Cancer Res. 2009, 15, 6052−6061. (20) Erickson, H. K.; Lewis Phillips, G. D.; Leipold, D. D.; Provenzano, C. A.; Mai, E.; Johnson, H. A.; et al. The effect of different linkers on target cell catabolism and pharmacokinetics/ pharmacodynamics of trastuzumab maytansinoid conjugates. Mol. Cancer Ther. 2012, 11, 1133−1142. (21) Erickson, H. K.; Park, P. U.; Widdison, W. C.; Kovtun, Y. V.; Garrett, L. M.; Hoffman, K.; et al. Antibody-maytansinoid conjugates are activated in targeted cancer cells by lysosomal degradation and linker-dependent intracellular processing. Cancer Res. 2006, 66, 4426− 4433. (22) Erickson, H. K.; Widdison, W. C.; Mayo, M. F.; Whiteman, K.; Audette, C.; Wilhelm, S. D.; et al. Tumor delivery and in vivo processing of disulfide-linked and thioether-linked antibody-maytansinoid conjugates. Bioconjug Chem. 2010, 21, 84−92. (23) Kovtun, Y. V.; Audette, C. A.; Ye, Y.; Xie, H.; Ruberti, M. F.; Phinney, S. J.; et al. Antibody-drug conjugates designed to eradicate tumors with homogeneous and heterogeneous expression of the target antigen. Cancer Res. 2006, 66, 3214−3221. (24) Sanderson, R. J.; Hering, M. A.; James, S. F.; Sun, M. M.; Doronina, S. O.; Siadak, A. W.; et al. In vivo drug-linker stability of an anti-CD30 dipeptide-linked auristatin immunoconjugate. Clin. Cancer Res. 2005, 11, 843−852. (25) Denzel, S.; Maetzel, D.; Mack, B.; Eggert, C.; Barr, G.; Gires, O. Initial activation of EpCAM cleavage via cell-to-cell contact. BMC Cancer 2009, 9, 402. (26) Kovtun, Y. V.; Audette, C. A.; Mayo, M. F.; Jones, G. E.; Doherty, H.; Maloney, E. K.; et al. Antibody-maytansinoid conjugates designed to bypass multidrug resistance. Cancer Res. 2010, 70, 2528− 2537. (27) Oroudjev, E.; Lopus, M.; Wilson, L.; Audette, C.; Provenzano, C.; Erickson, H.; et al. Maytansinoid-antibody conjugates induce mitotic arrest by suppressing microtubule dynamic instability. Mol. Cancer Ther. 2010, 9, 2700−2713. (28) Calvete, J. A.; Newell, D. R.; Wright, A. F.; Rose, M. S. In vitro and in vivo antitumor activity of ZENECA ZD0490, a recombinant ricin A-chain immunotoxin for the treatment of colorectal cancer. Cancer Res. 1994, 54, 4684−4690.

ACKNOWLEDGMENTS We thank Dr. Nathan Fishkin, Dr. Xiuxia Sun, and Dr. Wayne Widdison for helpful discussions. We thank Timothy Boit for help in ELISA optimization experiments. We thank Dr. Yelena Kovtun and Gregory Jones for EpCAM antigen expression data in HT-29 cells. We are thankful to Dr. Ravi Chari, Dr. Thomas Keating, and Dr. John M. Lambert for their careful review of the manuscript.

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ABBREVIATIONS: ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; HRP, horseradish peroxidase; DM1, N2′deacetyl-N-2′(3-mercapto-1-oxopropyl)-maytansine; DM4, N2′deacetyl-N-2′(4-mercapto-4-methyl-1-oxopentyl)-maytansine; MMAE, monomethylauristatin E; SMCC, N-succinimidyl 4-(Nmaleimidomethyl) trans-cyclohexane 1-carboxylate; SPDB, Nsuccinimidyl 4-(2-pyridyldithio)butanoate; sulfo-SPDB, Nsuccinimidyl 4-(2-pyridyldithio)-2-sulfo-butanoate

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DOI: 10.1021/acs.molpharmaceut.5b00028 Mol. Pharmaceutics 2015, 12, 1752−1761