PK assays for antibodydrug conjugates: case study ...

R esearch A rticle S pecial Focus I ssue: A ntibody – drug

conjugates

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PK assays for antibody–drug conjugates: case study with ado-trastuzumab emtansine Background: Antibody–drug conjugates (ADCs) combine the characteristics of large-molecule biologics and small-molecule drugs and are heterogeneous mixtures that can biotransform in vivo, resulting in additional complexity. ADC bioanalytical strategies require novel analytical methods, as well as existing large- and small-molecule methods. Because ADCs in late-stage clinical development are relatively new, regulatory guidelines and standard industry best practices for developing strategies for bioanalytical PK assays are still being established. Results: A PK assay strategy was developed that included comprehensive novel reagent and assay characterization approaches for the ADC ado-trastuzumab emtansine (T-DM1). Conclusion: The bioanalytical strategy was successfully applied to the drug development of T-DM1 and ensured that key analytes were accurately measured in support of nonclinical and clinical development. Antibody–drug conjugates (ADCs) are complex molecules with monoclonal antibodies covalently bound to cytotoxic drugs via a chemical linker [1,2] . ADCs specifically bind to targeted cell surface antigens overexpressed on tumor cells. Upon binding, the ADCs are internalized and trafficked to lysosomes, from which the cytotoxic drug is released within the cell. The use and targeted delivery of highly potent cytotoxic drugs is designed to enhance the antitumor effects of the molecule while reducing systemic toxicity [3–5] . There are currently several novel ADCs in preclinical, early clinical or late-stage clinical development for the treatment of solid and hematologic tumors [3–7] . Kadcyla™ (ado-trastuzumab emtansine [T-DM1]) is an ADC that recently received US FDA approval for the treatment of advanced breast cancers strongly positive for HER2. The HER2 receptor tyrosine kinase plays an important role in cell proliferation, differentiation and survival during morphogenesis [8] . Over expression of HER2 occurs in approximately 25–30% of tumors in patients with breast cancer. HER2 overexpression is associated with aggressive tumor growth and poor clinical outcomes [9,10] . Trastuzumab, a humanized antibody directed against the extracellular domain (ECD) of HER2, is approved for the treatment of HER2-overexpressing breast cancer. However, a significant proportion of patients do not respond to trastuzumab or tend to relapse following treatment [11,12] . T-DM1 is a promising novel therapeutic for this population of patients.

T-DM1 is composed of trastuzumab and DM1, an antimicrotubule agent derived from maytansine. Trastuzumab is linked to DM1 primarily through lysine residues using a stable, noncleavable, thioether linker – succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC). This method of conjugation results in a heterogeneous mixture because the antibody can have varying numbers of cytotoxic drugs attached to it. The T-DM1 drug-to-antibody ratio (DAR) ranges from 0 to 8 with an average DAR of approximately 3.5. The combination of a monoclonal antibody with a cytotoxic drug forms a complex molecular entity that combines the characteristics of a large-molecule biologic with those of a small-molecule drug. Thus, it is necessary to devise a bioanalytical strategy for PK analyses to suit both of these classes of molecules. In addition, due to the presence of mixtures with different DARs and the potential for biotransformations, novel methods are required to understand the changing DAR mixtures present in plasma/serum [13,14] . Our bioanalytical strategies for T-DM1 included the structural characterization of T-DM1 in plasma/serum in vitro and in vivo using affinity capture capillary LC–MS to understand the DAR analyte properties present in circulation [15] . After qualitative characterization in serum/plasma, three validated assays capable of quantifying the analytes in circulation were used to characterize the PK and stability of T-DM1 in nonclinical and clinical studies:

10.4155/BIO.13.72 © 2013 Future Science Ltd

Bioanalysis (2013) 5(9), 1025–1040

Randall Dere*1, Joo-Hee Yi1, Corinna Lei1, Ola M Saad1, Catherine Huang1, Yanhong Li1, Jakub Baudys2 & Surinder Kaur1 Department of Bioanalytical Sciences, Genentech, 1 DNA Way, South San Francisco, CA 94080-4990, USA 2 Centers for Disease Control & Prevention, Atlanta, GA, USA *Author for correspondence: Tel.: +1 650 225 3519 Fax: +1 650 742 4933 E-mail: [email protected] 1

ISSN 1757-6180

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R esearch A rticle | Key Terms Kadcyla™: Genentech’s first

US FDA-approved antibody–drug conjugate; targets HER2-positive tumors.

Drug-to-antibody ratio:

Molar ratio (moles of drug per mole of antibody); adotrastuzumab emtansine has an average drug-to-antibody ratio of 3.5, which represents the average drug load.

Biotransformations:

Structural changes occurring to molecules in a biological matrix such as serum/plasma in vitro or in vivo.

Dere, Yi, Lei et al.

An ELISA designed to measure the amount of total trastuzumab (all DARs including DAR 0) (Figure 1A) ;

linker [18] , antibody-conjugated drug could not be measured directly using an affinity capture/ drug release approach that relies on the ability of the linker to be enzymatically cleaved [15,19] . As an alternative to measuring antibody-conjugated drug, conjugated antibody was measured using ELISA. To increase the efficiency of sample ana lysis, the total- and conjugated-trastuzumab ELISAs were developed with the same assay ranges. This allowed for the same set of test samples to be analyzed in both assays. This paper describes the validated quantitative assays mentioned above, used to characterize nonclinical and clinical PK with selected examples of study data.

n

An ELISA to measure conjugated trastuzumab (all DARs except DAR 0) (Figure 1B) ;

n

A small-molecule LC–MS/MS assay to measure the amount of released DM1 catabolite (Figure 1C) .

n

Other small-molecule catabolites, such as lysine-N-maleimidomethyl cyclohexane-1-carboxylate (MCC)-DM1 and MCC-DM1 were also measured in a limited exploratory manner [16,17] . Because T-DM1 utilizes a noncleavable A

B Streptavidin-HRP

F(ab´)2 anti-human IgG-HRP

Biotin-HER2 ECD

HER2 ECD coat Total-trastuzumab ELISA

Anti-drug Ab coat Conjugated-trastuzumab ELISA

C Plasma sample containing DM1 Reduction of organic disulfides by TCEP DM1-S-S-R

TCEP

T-DM1

DM1-SH + R-SH

*

Protein precipitation with 100% ACN

DM1 dimer DM1-SH Derivitization of DM1 with NEM to form DM1–NEM complex pH > 5 DM1-S-NEM DM1-SH + NEM Inject sample for SPE LC–MS/MS detection of DM1–NEM

*X

Other DM1-S-S-X DM1

**

DM1 disulfide bound in T-DM1

*

DM1-albumin

‘Releasable’ DM1 bound in T-DM1 via: • Disulfides (∼0.4% of DM1) DM1 not conjugated to T-DM1: • Free DM1 in plasma • Bound via disulfide linkages to plasma components (DM1 dimer, albumin, cysteine and glutathione) Bioanalysis © Future Science Group (2013)

Figure 1. T-DM1 assays. (A) Total-trastuzumab ELISA; (B) conjugated-trastuzumab ELISA; (C) DM1 catabolite LC–MS/MS assay. Ab: Antibody; ACN: Acetonitrile; DM1: Antimicrotubule agent derived from maytansine; ECD: Extracellular domain; HRP: Horseradish peroxidase; NEM: N-ethylmaleimide; TCEP: Tris(2-carboxyethyl)phosphine; T-DM1: Ado-trastuzumab emtansine.

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PK assays for ADCs: case study with ado-trastuzumab emtansine Experimental Total-trastuzumab ELISA Total-trastuzumab ELISAs are validated assays designed to quantify all T-DM1 DARs, including conjugated T-DM1, as well as partially unconjugated and fully unconjugated T-DM1 that are immunoreactive to HER2 ECD. As a part of validation, data from minimum and maximum incubation times were assessed to confirm acceptable assay performance (±20% of nominal). For nonclinical, nonhuman primate samples, microtiter plates (96 wells; Nunc, NY, USA) were coated with recombinant HER2 ECD, p105HER-2, (Genentech, Inc., CA, USA) at 0.5 µg/ml in coating buffer (0.05 M carbonate/bicarbonate buffer; pH 9.6). After 16–72 h incubation at 2–8°C, the coat solution was removed and nonspecific binding sites were blocked by adding assay diluent (phosphate-buffered saline [PBS]; 0.5% bovine serum albumin [BSA]; and 0.05% polysorbate 20; 0.05% Proclin 300) for 1–2 h at ambient temperature with agitation. Plates were washed with wash buffer (PBS; 0.05% polysorbate 20; pH 7.4); eight standards ranging from 0.16 to 20 ng/ml, controls (94, 327 and 1200 ng/ml) and samples were subsequently added. T-DM1 (Genentech, Inc.) with an average DAR of 3.5 was used as the standard. Samples were diluted a minimum of 1/100 in sample diluent (PBS; 0.5% BSA; 0.05% polysorbate 20; 0.05% Proclin 300; 0.25% 3-[3-cholamidopropyldimethylammonio]-1-propanesulfonate; 0.2% bovine g-globulin; 5mM EDTA; and 0.35 M sodium chloride at pH 7.4 ± 0.1). Subsequent dilutions were performed using sample diluent containing 1% normal cynomolgus monkey serum. Plates were incubated for 2 h at ambient temperature with agitation. After washing, F(ab´)2 goat anti-human IgG Fc conjugated to horseradish peroxidase (HRP; Jackson Immuno Research, PA, USA) was diluted in assay diluent and added for detection. Plates were incubated for 1 h at ambient temperature with agitation and then washed. Bound HRP conjugate was detected using tetramethyl benzidine peroxidase substrate (Moss, Inc., MD, USA). Color was allowed to develop for 10–20 min at ambient temperature without agitation; the enzymatic reaction was stopped by adding 1 M phosphoric acid. Absorbance was measured at 450 nm against a reference wavelength of either 620 or 630 nm, using a microplate reader. Totaltrastuzumab concentrations were calculated by interpolation from a four-parameter fit of the future science group

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standard curve. Figure 2A shows a representative standard curve and curve fit for the total trastuzumab ELISA. For clinical samples the assay format was similar to the nonclinical format except that the diluted assay standards, controls and test samples contained human serum. Human serum samples were initially diluted 1/100 in assay diluent with subsequent dilutions in sample diluent containing 1% normal human serum (NHS). The standard curve was also diluted in sample diluent. Conjugated-trastuzumab

ELISA The conjugated-trastuzumab ELISAs are validated assays designed to quantify all T-DM1 DARs except DAR 0 (present in the reference standard or resulting from complete deconjugation). As a part of validation, data from minimum and maximum incubation times were assessed to confirm acceptable assay performance (±20% of nominal). For nonclinical, nonhuman primate samples, microtiter plates (96 wells; Nunc) were coated with a murine monoclonal anti-DM1 antibody (Genentech, Inc.) at 1 µg/ml in coating buffer (0.05 M carbonate/bicarbonate buffer; pH 9.6). After 16–72 h incubation at 2–8°C, the coat solution was removed and nonspecific binding sites were blocked by adding assay diluent (PBS; 0.5% BSA; 0.05% polysorbate 20; 0.05% Proclin 300) for 1 to 2 h at ambient temperature with agitation. Plates were washed; eight standards ranging from 0.16 to 20 ng/ml, controls (94.3, 194 and 1300 ng/ml) and samples were subsequently added. T-DM1 with an average DAR of 3.5 was used as the standard. Samples were diluted a minimum of 1/100 in sample diluent (PBS; 0.5% BSA; 0.05% polysorbate 20; 0.05% Proclin 300; 0.25% 3-[3-cholamidopropyl-dimethylammonio]-1-propanesulfonate; 0.2% bovine g-globulin; 5 mM EDTA; and 0.35 M sodium chloride at pH 7.4 ± 0.1). Subsequent dilutions were performed using sample diluent containing 1% normal cynomolgus monkey serum. Plates were incubated for 2 h at ambient temperature with agitation. Recombinant HER2 ECD conjugated to biotin was diluted in assay diluent and added after the plates were washed. Plates were incubated for 1 h at ambient temperature with agitation and then washed. Amdex™ Streptavidin-HRP (GE Healthcare, WI, USA) diluted in assay diluent was added for detection. Plates were incubated for 1 h at ambient temperature with agitation and then washed. Bound HRP conjugate was detected using tetramethyl benzidine peroxidase www.future-science.com

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A

Absorbance (450–620 nm)

2

1.5

1

0.5

0 0.1

1 10 Total-trastuzumab concentration (ng/ml) A

B

C

D

R2

0.0668

1.11

15.5

2.42

0.998

4-P fit: y = (A - D)/(1 + (x/C)^B) + D: Standard (total trastuzumab: concentration vs OD)

Absorbance (450–620 nm)

B

100

2

1.5

1

0.5

0 0.1

1 10 Conjugated-trastuzumab concentration (ng/ml) 4-P fit: y = (A - D)/(1 + (x/C)^B) + D:

Standard (T-DM1: concentration vs OD)

100

A

B

C

D

R2

0.00635

0.989

7.41

2.35

0.999

Figure 2. Representative standard curves and curve fits. (A) Total-trastuzumab ELISA; (B) conjugated-trastuzumab ELISA. 4-P fit: Four-parameter fit; R2: Coefficient of determination; OD: Optical density; T-DM1: Ado-trastuzumab emtansine.

substrate (Moss, Inc., MD, USA). Color was allowed to develop for 10–20 min at ambient temperature without agitation; the enzymatic reaction was stopped by adding 1 M phosphoric acid. Absorbance was measured at 450 nm against a reference wavelength of either 620 or 1028


630 nm using a microplate reader. Conjugatedtrastuzumab concentrations were calculated by interpolation from a four-parameter fit of the standard curve. Figure 2B shows a representative standard curve and curve fit for the conjugated-trastuzumab ELISA. future science group

PK assays for ADCs: case study with ado-trastuzumab emtansine For clinical samples, the assay format was similar to the nonclinical format except that the diluted assay standards, controls and test samples contained human serum. Human serum samples were initially diluted 1/100 in assay diluent with subsequent dilutions in sample diluent containing 1% NHS. The standard curve was also diluted in sample diluent. Linearity

of sample dilution in ELISA During validation each ELISA was evaluated for linearity of dilution. Known concentrations of T-DM1 were added at mid and high levels (with respect to the standard curve range) to cynomolgus monkey (n = 6), and human breast cancer patient serum samples (n = 10). Additionally, trastuzumab was added to individual breast cancer patient serum samples (n = 5) and breast cancer patient serum pools (n = 2). Cynomolgus monkey and human serum samples were diluted to the minimum dilution in sample diluent, followed by three serial 1/2 dilutions in either 1% cynomolgus monkey serum/sample diluent or 1% NHS/sample diluent, respectively. These samples were then analyzed to determine total- and conjugated-trastuzumab concentrations. Assessment

of the impact of varying DAR on ELISA accuracy T-DM1 with average DARs of 2.58, 3.07, 3.40, 3.94 and 4.10 were diluted to approximately 20 ng/ml in a sample diluent containing 1% NHS. The samples were then diluted serially twofold to fall within the reporting range of the assays. Affinity

capture LC–MS

Preparation of HER2 ECD & anti-DM1 antibody-modified paramagnetic beads

Approximately 1 mg of streptavidin-coated paramagnetic beads (Dynabeads M-280, Invitrogen, CA, USA) was washed twice with 400 µl HBS-EP buffer (GE Healthcare) using a KingFisher 96 magnetic particle processor (Thermo Electron Corporation, WA, USA). The beads were added to 2 ml 96 square-well plates (Analytical Sales and Service Inc., Pompton Plains, NJ, USA) containing either approximately 13 µg biotinylated HER2 ECD or biotinylated anti-DM1 antibody in 400 µl HBS-EP per well. The plates were covered with an aluminum foil plate sealer and incubated at room temperature for 2 h with agitation. The beads were washed twice with 400 µl HBS-EP buffer. future science group


Affinity capture & elution

Approximately 50 µl of plasma samples containing T-DM1 were added to 400 µl HBS-EP in 96-well plates, followed by the addition of either HER2 ECD- or anti-DM1 antibody-modified paramagnetic beads. The plates were covered with an aluminum foil plate sealer and incubated at room temperature for 2 h with gentle agitation. The beads were washed twice with 400 µl HBS-EP followed by a deglycosylation step. The beads were transferred to a plate containing 300 µl HBS-EP, 32 µl of 80 mM sodium phosphate buffer and 4 µl PNGase F (ProZyme) and incubated overnight at 37°C with agitation. Following deglycosylation the beads were washed twice with 500 µl HBS-EP, twice with 500 µl water and finally with 500 µl 10% acetonitrile in water. Analyte was eluted by incubating in 50 µl 30% acetonitrile in water with 1% formic acid at room temperature for 15 min with agitation. After removing the beads, 40 µl of the eluate was transferred to a 96-well injection plate and centrifuged at 3000 rpm for 5 min at 2–8°C before injecting onto LC–MS. DM1

catabolite LC–MS/MS assay DM1 concentrations in nonclinical and clinical plasma samples were determined using validated LC–MS/MS methods. The DM1 catabolite LC–MS/MS assay was designed with a reduction step to measure free DM1 present in circulation or any disulfide bound forms of released DM1 (e.g., dimers, glutathione, cysteine, and albumin adducts); that is, all forms of DM1 in plasma except DM1 covalently bound through the MCC linker to lysines in T-DM1. Additional forms of DM1 still conjugated to the antibody that would be measured in this assay may exist in very minimal quantities. These include DM1 that may be disulfide linked to the T-DM1 antibody through available cysteines, or DM1 where the thioether succinimide linker may have been oxidized to the corresponding sulfoxide [20] . An unoxidized thioether, as in the MCC-DM1 linker, is stable and does not show any thio-succinimide cleavage product even when incubated with reducing agent (dithiothreitol or tris[2-carboxyethyl] phosphine) [20] . However, as Fishkin et al. have shown, this is in contrast to sulfoxides [20] . If the sulfoxide was to be formed, thermal instability could lead to cleavage and additional maytansinoid byproducts forming, and furthermore, when incubated in the presence of reducing agent, these oxidized maytansinoid species including the sulfoxide www.future-science.com

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R esearch A rticle | Key Term Drug-to-antibody ratio distribution: Proportion of

individual drug-to-antibody ratio analytes.


result solely in conversion to the released form of DM1. Thus, the assay approach used herein was conservative and measured all disulfide-bound forms of DM1, or releasable DM1. It is hypothesized that the contribution of minor amounts of DM1, released from cysteine-linked or thioether oxidized T-DM1 during sample analysis, likely resulted in some overquantification of DM1 (see section titled ‘Discussion’). DM1 standards ranging from 1 to 500 nM, controls (3.0, 250 and 400 nM) and samples (30 µl) were added to 1.5 ml microtubes; 3 µl of 26 mM tris(2-carboxyethyl) phosphine in 5 mM ammonium acetate was added to each tube. The tubes were vortexed and then incubated at 37°C for 10 min. Precipitation solution (125 µl acetonitrile) was added to each tube and vortexed. Tubes were subsequently centrifuged for 5 min at 14,000 rpm at room temperature using a microcentrifuge (5417-C, Eppendorf AG Hamburg, Germany). After centrifugation, 100 µl of supernatant was transferred to fresh microtubes; 3.0 µl derivatizing agent (12.5 mM N-ethylmaleimide [NEM] in dimethyl sulfoxide) was then added to each tube. The tubes were mixed and incubated at 37°C for 20 min. To each tube, 5 µl of a working solution of internal standard – 200 nM maytansine in acetonitrile – was added and mixed. Supernatant was transferred to 96-well polypropylene v-bottom plates (Porvair Sciences, Leatherhead, UK) prior to analysis by LC–MS/MS. Online SPE was conducted using either a Symbiosis or Prospekt2 system and HySphere C18 HD 7 µm cartridges (Spark Holland BV, The Netherlands). The NEM-derivatized samples were eluted into an API3000 LC–MS/MS system (AB Sciex, MA, USA). The column used was a Synergi MAX-RP 80A (50 × 1.0 mm; 4 µm) column and mobile phase (MP) A was 5 mM ammonium acetate with 0.1% formic acid in H2O. LC separation was conducted at a flow rate of 500 µl/min, using a linear gradient from 0 to 98% MP B (5 mM ammonium acetate with 0.1% formic acid in 95:5 acetonitrile:H2O) over 1.4 min, followed by 98% MP B for 1 min, back to 0% MP B over 0.3 min, and then equilibrated for 1 min. The API3000 system was operated using SRM in positive ionization mode at a temperature of 450°C. Data were acquired using Analyst software (AB Sciex). Sample stability of T-DM1 in human plasma

T-DM1 stability in human lithium heparin plasma was assessed. DM1 was analyzed as the 1030


indicator for instability of T-DM1. The T-DM1 stability samples were prepared by adding 500 µg/ml T-DM1 to human lithium heparin plasma. The stability samples were stored at -20 and -70°C. Stability of T-DM1 in plasma stored at -20°C was assessed in triplicate for 1, 3, 5 and 10 weeks. Stability of T-DM1 in plasma stored at -70°C was assessed in triplicate for 10, 11, 14, 18 and 22 weeks. Nonclinical

TK study in cynomolgus monkeys PK studies were conducted at Covance Laboratories (WI, USA) in compliance with NIH guidelines for the care and use of laboratory animals and approved by the Institutional Animal Care and Use Committee. T-DM1 was administered to cynomolgus monkeys (n = 14/dose group) by intravenous infusion once every 3 weeks for a total of four doses, at doses of 0.3, 3.0 or 30 mg/kg. Serum and plasma samples were collected for up to 105 days following dosing for the total-trastuzumab and conjugated-trastuzumab ELISA, as well as the DM1 catabolite LC–MS/MS assay analyses. In addition, serum was collected for antitherapeutic antibody analysis. Phase I

clinical study Clinical studies were carried out in accordance with approval by the relevant institutional review boards. All patients provided written informed consent before participating. In Phase I, multicenter, open-labeled dose escalation studies, T-DM1 was administered to 15 metastatic breast cancer patients by intravenous infusion once every 3 weeks at a dose of 3.6 mg/ kg [21] . If patients met retreatment criteria, additional treatment cycles were administered. Serum and plasma samples were collected for the total-trastuzumab and conjugated-trastuzumab ELISAs, as well as the DM1 catabolite LC–MS/MS assay analyses. In addition, serum was collected for antitherapeutic antibody analysis. Results Reagent characterization using affinity capture LC–MS T-DM1 is a complex mixture with DARs ranging from 0 to 8. After administration, the proportion of the individual DARs, or DAR distribution, may change over time due to the faster clearance of the higher DAR species compared with that of the lower DAR species and/or drug future science group

PK assays for ADCs: case study with ado-trastuzumab emtansine


30 HER2 ECD Anti-DM1

Composition (%)

25 20 15 10 5 0

DAR 0

DAR 1

DAR 2

DAR 3

DAR 4

DAR 5

DAR 6

DAR 7

T-DM1 analyte

Figure 3. Comparison of T-DM1 drug-to-antibody ratio distribution from affinity capture LC–MS using an anti-DM1 antibody capture and a HER2 extracellular domain capture. For simplicity, only DAR 0 to DAR 7 are shown (the level of DAR 8 was too low to allow a comparison). DAR: Drug-to-antibody ratio; DM1: Antimicrotubule agent derived from maytansine; ECD: Extracellular domain; T-DM1: Ado-trastuzumab emtansine.

deconjugation [22] . We have previously shown that cynomolgus monkeys dosed with T-DM1 have a change in DAR distribution over time in vivo, where there is a gradual shift to lower DAR species [15] . Based on this information, the standard calibration curve of the assay does not represent the analytes measured at later PK time

points. In order to ensure the assay is appropriate, even when the analyte and standard curve compositions are different, the ELISA capture reagents used in our assays must be able to bind to each of the individual T-DM1 DAR molecules without any selective loss or bias. The HER2 ECD and anti-DM1 antibody used as Buffer Cynomolgus monkey plasma Human plasma

100 90

Relative intensity (%)

80 70 60 50 40 30 20 10 0

DAR 1

DAR 2

DAR 3

DAR 4 T-DM1 analytes

DAR 5

DAR 6

DAR 7

Figure 4. Comparison of T-DM1 drug-to-antibody ratio distribution from affinity capture LC–MS of T-DM1 in buffer, cynomolgus monkey and human plasma using an anti-DM1 antibody capture. Peaks normalized to the DAR 3 component of T-DM1 in each species. For simplicity, only DAR 1 to DAR 7 are shown (the level of DAR 8 was too low to allow a comparison). DAR: Drug-to-antibody ratio; DM1: Antimicrotubule agent derived from maytansine; T-DM1: Ado-trastuzumab emtansine.


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Box 1. Assay validation parameters for a large- and small-molecule assay. Large-molecule assay LOD LLOQ ULOQ Minimum dilution Accuracy Precision Linearity of dilution:

Ado-trastuzumab emtansine

Trastuzumab Cross-reactivity:

Unconjugated humanized anti-HER2 extracellular domain monoclonal antibody Interference:

HER2 extracellular domain

Hemoglobin

Lipid Reagent stability:

Analyte stability in serum

Standard stock

Coat stock

Conjugate stock

Standard curve in sample diluent

Standard curve in sample diluent containing 1% species serum (normal human or cynomolgus monkey)

Small-molecule assay Selectivity/specificity Response function Calibration curves Precision Accuracy LLOQ Higher LOQ Stability:

Autosampler stability

Freezing and thawing stability

Bench-top stability

Assay

validation In the absence of regulatory guidelines or established industry best practices for ADC assay validations, we have used validation approaches typically used for large and small molecules and added additional experiments unique to ADCs. Our approach to assay validation has included using qualified assays for discovery research studies and validated assays for investigational new drug-enabling preclinical as well as clinical studies. Large-molecule

Long-term stability Dilution of samples Carryover

capture reagents for the total-trastuzumab and conjugated-trastuzumab ELISAs, respectively, were therefore further characterized by affinity capture LC–MS [15] . A comparison of the T-DM1 DAR distribution isolated from nonclinical plasma, using either recombinant HER2 ECD or anti-DM1 antibody as the capture probe, showed a comparable DAR distribution for DAR 1–DAR 7 (Figure 3) . As expected, 1032

DAR 0 binding was not observed for the antiDM1 reagent. These data are consistent with the data previously reported for human plasma [15] . In addition, the T-DM1 DAR distribution isolated from either cynomolgus monkey or human plasma using an anti-DM1 antibody as the capture probe showed a comparable DAR distribution to T-DM1 reference standard in buffer (Figure 4) . For simplicity, only DAR 0 to DAR 7 are shown (the level of DAR 8 was too low to allow a comparison). Both the lesser (e.g., DAR 1) and higher (e.g., DAR 6 and DAR 7) conjugated DAR components, which are present in lower abundance within the mixture were captured appropriately. As expected, only the HER2 ECD capture method was capable of measuring unconjugated antibody; unconjugated antibody was not observed in the mass spectrum after affinity capture with anti-DM1 antibody. Although these data indicate that the capture reagents demonstrated no apparent selective loss or bias to the individual DARs, it is possible that reagents immobilized onto affinity beads could demonstrate different immunoreactivity as compared with immobilization onto ELISA plates.


ligand-binding assay validation During validation, typical performance charac teristics for large molecules, such as accuracy, precision, dilutional linearity and specificity, were evaluated (Box 1) . Assay performance characteristics for the nonclinical and clinical totaltrastuzumab and conjugated-trastuzumab ELISAs are shown in Tables 1 & 2 , respectively. The standard curve range for both ELISAs in nonclinical and clinical matrices was 0.16–20 ng/ml with a minimum dilution of 1/100. Each assay was checked for accuracy by spiking known concentrations of T-DM1 reference standard into individual serum samples at three levels. Overall, total trastuzumab and conjugated trastuzumab future science group



Table 1. Assay performance of validated PK assays used in nonclinical studies. Nonclinical PK assay

Standard curve range

MQC or LLOQ

Accuracy (%)

Intra-assay precision (%CV)

Inter-assay precision (%CV)

Total trastuzumab

0.16–20 ng/ml

MQC: 40 ng/ml

3–6

9–13

Conjugated trastuzumab DM1 catabolite†

0.16–20 ng/ml

MQC: 30 ng/ml

3–6

6–8

1.00–500 nM (0.737–369 ng/ml)

LLOQ: 1.00 nM (0.737 ng/ml)

Recovery: 93–102 Recovery: 91–111 Bias: -8.9–4.4

6.4–10.2

2.9–19.1

DM1 concentrations were determined in plasma samples. MQC: Minimum quantifiable concentration in neat serum samples. †

nonclinical PK ELISAs had a recovery range from 91 to 111%, whereas the corresponding clinical ELISAs had a slightly wider recovery range from 90 to 118% and 91 to 127%, respectively. It is not clear why there is a slightly wider recovery range for the clinical assays, however, it is most likely due to the use of serum collected from metastatic breast cancer patients. Although the assays were evaluated for soluble target interference, differences in endogenous levels of shed HER2 ECD in cancer patient sera may contribute to the slightly wider recovery range observed. Precision for all PK ELISAs was acceptable (±20% of nominal). Overall, intraand inter-assay precision across all four nonclinical and clinical assays ranged from 2 to 10%CV (nonclinical) and 4 to 13%CV (clinical). To assess linearity of sample dilution for the trastuzumab assay, both T-DM1 and trastuzumab were evaluated in cynomolgus monkey and human breast cancer patient serum samples. For T-DM1, the difference between concentration values from consecutive dilutions within a sample dilution series did not exceed 8% in cynomolgus monkey serum and 16% for human breast cancer patient serum (data not shown). For trastuzumab, the difference between concentration values from consecutive dilutions within a sample dilution series did not exceed 19% in breast cancer patient serum (data not shown).

Small-molecule

assay validation The DM1 catabolite LC–MS/MS assay measured DM1 (also referred to as ‘free DM1’) concentrations in lithium-heparin plasma (Figure 1C) . The assay was designed to measure DM1 and any disulfide-bound forms of released DM1 (e.g., dimers, glutathione, cysteine and albumin adducts), while excluding DM1 linked to lysine residues of trastuzumab via the MCC linker. Because DM1 contains a free sulfhydryl, DM1 released from T-DM1 is likely to dimerize or react with other thiol-containing molecules in plasma. Therefore, to avoid underquantification of released DM1, plasma samples were treated with a reducing agent to release disulfide-bound DM1. The free thiol was then blocked with NEM to prevent any further reactions. Hence, the LC–MS/MS assay-quantified DM1-NEM. We used typical small-molecule assay validation parameters for investigational new drugenabling nonclinical and clinical studies (Box 1) . Nonclinical and clinical assay performance for the DM1 catabolite LC–MS/MS assay is shown in Tables 1 & 2 , respectively. The standard curve range for the DM1 assay was 1.00–500 nM with no requirement of a minimum dilution necessary. The LLOQ was 1.00 nM (0.737 ng/ml). Accuracy was evaluated at four levels, which included the LLOQ. Bias across both nonclinical and clinical assays ranged from -8.9 to 14.9%.

Table 2. Assay performance of validated PK assays used in clinical studies. Clinical PK assay

Standard curve range

MQC or LLOQ

Accuracy

Intra-assay precision (%CV)

Inter-assay precision (%CV)

Total trastuzumab

0.16–20 ng/ml

MQC: 40 ng/ml

3–7

4–8

Conjugated trastuzumab

0.16–20 ng/ml

MQC: 40 ng/ml

2–10

5–6

DM1†

1.00–500 nM (0.737–369 ng/ml)

LLOQ: 1.00 nM (0.737 ng/ml)

% recovery: 90–118 % recovery: 91–127 % bias: 0.8–14.9

6.2–9.1

8.8–17.7

DM1 concentrations were determined in plasma samples. MQC: Minimum quantifiable concentration in neat serum samples. †



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Table 3. Recovery for samples containing both trastuzumab and T-DM1 in the total-trastuzumab ELISA. Trastuzumab concentration (ng/ml)

T-DM1 concentration (ng/ml)

Targeted total-trastuzumab concentration (ng/ml)

Obtained concentration (ng/ml)

Recovery (%)

0 100 200 400 400

400 400 200 100 0

400 500 400 500 400

368 486 413 554 478

92 97 103 111 120

T-DM1: Ado-trastuzumab emtansine.

Intra-and inter-assay precision across both nonclinical and clinical assays ranged from 6.2 to 10.2%CV and 2.9 to 19.1%CV, respectively. Assay

characterization unique to ADCs As mentioned previously, the total-trastuzumab ELISA was designed to measure antibody with DAR equal to or greater than 0, including conjugated, partially unconjugated and fully unconjugated T-DM1. Therefore, additional experiments were performed during validation to demonstrate the ability of the assay to quantify both conjugated and completely unconjugated trastuzumab with acceptable accuracy. Known concentrations of T-DM1 and trastuzumab reference standards were added to cynomolgus monkey serum at three levels and

Conjugated-trastuzumab ELISA (ng/ml)

10,000

1000

100

10 10

100

1000

10,000

Total-trastuzumab ELISA (ng/ml) Linear regression value calculated as y = 1.04x + 1.12; R2 = 0.995

Figure 5. Scatter plot comparing serum T-DM1 concentrations obtained by using the total-trastuzumab and conjugated-trastuzumab ELISAs. R2: Coefficient of determination; T-DM1: Ado-trastuzumab emtansine.

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analyzed. The recovery for samples containing both trastuzumab and T-DM1 ranged from 97 to 111%. When only trastuzumab was added to cynomolgus monkey serum, recovery was 120% (Table 3) . Known concentrations of T-DM1 reference standard were added to 12 serum samples at three levels and analyzed in both assays. A scatter plot of values obtained from both assays is presented in Figure 5. Linear regression yielded a coefficient of determination (R 2) of 0.995 and a slope of 1.04. As expected, there appears to be acceptable equivalency between the totaltrastuzumab and conjugated-trastuzumab ELISAs. After administration the ADC can biotransform, dynamically changing the in vivo DAR distribution of the analyte mixture over time. Therefore, a standard calibration curve consisting of the reference standard may not be appropriate for quantifying in vivo analytes at all PK time points. To assess the impact of varying DARs on the assay accuracy, the total-trastuzumab and conjugated-trastuzumab ELISAs were characterized using T-DM1, with average DARs of 2.58, 3.07, 3.40, 3.94 and 4.10. Each T-DM1 DAR sample appeared to dilute linearly in both ELISAs (data not shown). Recovery in the conjugated-trastuzumab ELISA was calculated relative to the concentration obtained by the total-trastuzumab ELISA (Table 4) . Recoveries in the conjugate T-DM1 ELISA ranged from 102 to 106%. Additional experiments unique to ADCs also included the assessment of free-DM1 analyte stability in the presence of T-DM1. As T-DM1 concentrations may decrease during storage due to instability, free DM1 concentrations may increase due to the liberation of additional DM1 from the ADC during storage. For a relatively stable linker such as SMCC, the molar concentration of the free DM1 in study samples future science group


Nonclinical

& clinical PK Our assays have performed robustly and provided consistent data across multiple nonclinical and clinical T-DM1 studies [21,23–25] . Figure 7 shows representative PK curves for a 30 mg/kg multiple-dose intravenous administration of T-DM1 in cynomolgus monkeys. In general, nonclinical data indicated that the PK of conjugated trastuzumab is bi-exponential, with a mean serum clearance (CL) ranging from 42 ml/kg/day (0.3 mg/kg dose) to 10 ml/kg/day (30 mg/kg dose) and terminal half-life (t½,b ) of approximately 0.9 days (0.3 mg/kg dose) to 5 days (30 mg/kg dose). Total-trastuzumab PK was also bi-exponential, with a volume of distribution similar to that of conjugated trastuzumab. Conjugated trastuzumab has a CL approximately two- to 2.5-times faster and a t½,b approximately two-times shorter than that of total trastuzumab. DM1 concentrations in plasma were highest immediately after dosing, but were approximately 10,000-times lower than the concentrations of T-DM1 (~49-times lower by molar concentrations). Figure 8 shows representative PK curves for the first cycle of T-DM1 administered to metastatic breast cancer patients. Across multiple Phase I to Phase III clinical studies, conjugated trastuzumab demonstrated a predictable PK future science group

Table 4. Recovery in the conjugated-trastuzumab ELISA calculated relative to the concentration obtained by the total-trastuzumab ELISA. Drug-toantibody ratio

Average Average total trastuzumab conjugated trastuzumab (ng/ml) (ng/ml)

T-DM1 recovery† (%)

2.58 3.07 3.40 3.94 4.10

26.3 24.6 23.5 20.0 22.3

106 103 102 102 102

27.7 25.3 23.9 20.5 22.7

T-DM1 recovery = 100 × (average conjugated trastuzumab/average total trastuzumab). T-DM1: Ado-trastuzumab emtansine. †

profile, characterized by CL ranging from 7 to 13 ml/day/kg and t½,b of approximately 4 days. In addition, total trastuzumab had a slower CL (~3–6 ml/day/kg) and longer t½,b (~9 to 11 days) compared with conjugated trastuzumab. The linker stability was demonstrated by consistently low maximum systemic plasma DM1 concentrations, averaging to approximately 6 ng/ml with an administration of T-DM1 3.6 mg/kg as measured using our conservative DM1 catabolite LC–MS/MS assay described above. There was no evidence of DM1 accumulation in plasma following repeated dosing of T-DM1. Maximum DM1 levels did not exceed 60 ng/ml in individual patients after repeated T-DM1 administration. Discussion The T-DM1 molecule contains a large- molecule antibody as well as a small-molecule 120 DM1 concentration difference from T = 0 (%)

is low compared with the antibody-conjugated drug. In our experience, free DM1 measured in nonclinical/clinical studies represented less than 1% of the molar concentration of the antibodyconjugated drug. Therefore, even a minor release of DM1 from T-DM1 during storage or oxidation of the thioether can have a large impact on the measurement of free drug. For example, deconjugation of 1% of DM1 from T-DM1 during storage would result in an approximately 100% increase in the measurement of free DM1. Figure 6 shows the percentage difference in DM1 concentration over time for a known concentration of T-DM1 added to human plasma and stored at either -20 or -70°C, compared with a freshly prepared sample (T = 0). The dashed line at 20% DM1 concentration difference represents the upper limit of acceptable sample stability. Samples stored at -20°C for 3 weeks or longer yielded differences greater than 20%. For samples stored at -70°C, differences greater than 20% were observed with the 22-week time point. We have therefore conservatively assigned clinical sample stability times of 1 and 18 weeks for storage at -20 and -70°C, respectively.


-20°C storage -70°C storage Upper limit of sample stability acceptability

100 80 60 40 20 0 0 -20

5

10

15

20

25

Time (week)

Figure 6. DM1 analyte stability in the presence of T-DM1. Time 0 had 0.4% free DM1. DM1: Antimicrotubule agent derived from maytansine; T = 0: Freshly prepared sample; T-DM1: Ado-trastuzumab emtansine.


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Mean concentration (µg/ml)

1000 100 10 1 0.1 0.01 0.001 0.0001 0

20

40

60 Time (day)

80

100

Total-trastuzumab ELISA

DM1 catabolite assay

Conjugated-trastuzumab ELISA

LLOQ value of 0.737 ng/ml for the DM1 assay

120

Figure 7. Representative mean concentration–time curves after every 3 week dosing of T-DM1 in cynomolgus monkeys. Plasma DM1 concentrations below LLOQ were replaced by half of the LLOQ. DM1: Antimicrotubule agent derived from maytansine.

drug. Therefore, a bioanalytical assay strategy that encompasses both attributes is required. Antibodies have well defined tertiary structures and are therefore well suited to analysis by ligand-binding assays (LBAs), for example, ELISA [26,27] . In contrast, small molecules lack

a tertiary structure and pose a potential steric hindrance for capture and detection reagents. Therefore, developing LBAs for small molecules can be challenging. Small molecules are thus typically analyzed using LC–MS/MS techniques.

Mean concentration (µg/ml)

1000 100 10 1 0.1 0.01 0.001 0.0001 0

5

10

Time (day)

15

20

Total-trastuzumab ELISA

DM1 catabolite assay

Conjugated-trastuzumab ELISA

LLOQ value of 0.737 ng/ml for the DM1 assay

25

Figure 8. Representative mean concentration–time curves for the first cycle of T-DM1 administered every 3 weeks to metastatic breast cancer patients. Plasma DM1 concentrations below LLOQ were replaced by half of the LLOQ. DM1: Antimicrotubule agent derived from maytansine.

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PK assays for ADCs: case study with ado-trastuzumab emtansine Regulatory guidelines and standard industry best practices for developing bioanalytical PK assay strategies for ADCs are still being established. We developed an assay strategy consisting of three validated assays capable of quantifying DAR analytes in circulation to characterize the PK and stability of T-DM1 in nonclinical and clinical studies. These validated assays included: n An ELISA designed to measure total trastuzumab; An ELISA to measure conjugated trastuzumab;

n

A small-molecule LC–MS/MS assay to measure the amount of free DM1 catabolite.

n

The total-trastuzumab ELISA can be used to determine PK parameters for total trastuzumab, for example, CL or t½,b, and confirm that these parameters are in a range typical for trastuzumab and not compromised significantly by conjugation of the antibody [17,28] . Comparison of data from the total-antibody assay and conjugated-antibody assay may provide some general qualitative insights. For example, the conjugated-trastuzumab ELISA was designed to measure all trastuzumab DARs except DAR 0. After administration, circulating levels of both total trastuzumab and conjugated trastuzumab are expected to decrease over time. However, if the level of conjugated trastuzumab in vivo decreases significantly faster than the level of total trastuzumab, it would suggest a high level of fully unconjugated antibody (DAR 0) in circulation. Conversely, similar PK results for total trastuzumab and conjugated trastuzumab, along with low levels of DM1 catabolite, would suggest that T-DM1 is relatively stable in vivo. Overall, the PK data from the three key assays indicated that T-DM1 was relatively stable. It is noteworthy that the conjugated-trastuzumab ELISA does not provide direct information on the drug load. The assay capture reagent cannot differentiate between conjugated trastuzumab containing only DAR 1 and one containing higher DARs. An assay format using the anti-DM1 antibody as the detection reagent, instead of the capture reagent, could theoretically be more sensitive to drug load. However, this format has been reported to demonstrate a nonlinear response when the standard curve and analyte mixture are not identical [29] . The antibody-conjugated drug assay, which measures the concentration of all drug molecules that are covalently bound to the antibody, provides accurate data for the drug load [15] . However, this future science group


assay format is not compatible with ADCs using noncleavable linkers, such as T-DM1. ADCs present unique challenges for LBAs. T-DM1 biotransformation in vivo can lead to changes in DAR distribution, resulting in dynamically changing mixtures due to drug loss (deconjugation) and/or the faster clearance of the higher DAR species compared with that of the lower DAR species [13,14] . The ELISA calibration curves are prepared from the product reference standard that has a fixed DAR distribution similar to only the earliest PK time points. Thus, it is important to ensure that the calibration curve is appropriate for quantifying the dynamically changing mixture of ADC for PK evaluation. The impact of a changing in vivo DAR distribution on the accuracy of the assay may be assessed using ADC molecules with a variety of DARs [29] . Another technical challenge is that the binding of the assay reagents to the analytes may vary with the amount of drug conjugated to the antibody (drug load). Binding of assay reagents directed against the antibody portion of the ADC may be sterically hindered if the ADC has a high drug load. In contrast, if drug load is low, assay reagents directed against the drug portion of the ADC may not bind sufficiently due to low avidity [15] . It would be preferable to assess the assay recovery using individually isolated DARs for each component in the mixture; however, it is not feasible to obtain individual DARs for a complex mixture such as T-DM1 with the currently existing technology. Therefore, to assess the impact of varying DARs, the totaltrastuzumab and conjugated-trastuzumab ELISAs were characterized using T-DM1 with a range of average DARs of 2.58, 3.07, 3.40, 3.94 and 4.10. The targeted average DARs were achieved by adjusting the amount of SMCC linker added to the trastuzumab during production. Recovery of T-DM1 DAR samples in the conjugated-trastuzumab ELISA was based on measured total-trastuzumab ELISA concentrations (Table 4) . The recovery ranged from 102 to 106%, which confirmed that the assays were able to accurately measure T-DM1 across the range of DARs tested. For both nonclinical and clinical studies reported herein, total-trastuzumab concentrations followed a pattern similar to that of the conjugated trastuzumab. As expected, the total-trastuzumab and conjugated-trastuzumab concentrations at time = 0 and Cmax, were similar. The differences in the concentrations of the www.future-science.com

Key Term Drug load: Amount of drug conjugated to the antibody.

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conjugated trastuzumab and the total trastuzumab following T-DM1 administration, in addition to the presence of DM1 in plasma, suggest that in vivo T-DM1 is deconjugating over time. DM1 plasma concentrations decreased in a manner similar to that of conjugated trastuzumab. It is noteworthy that DM1 plasma concentrations measured by LC–MS/MS were highest immediately following each dose of T-DM1. This was unexpected for a catabolite concentration–time profile. The assay sample preparation involved a sulfhydryl reduction step to allow measurement of released DM1 that may subsequently dimerize or form disulfide bonds with plasma proteins or peptides. However, the DM1 concentration–time profile suggested the presence of a minor amount of cysteine-conjugated DM1 in T-DM1 or oxidation of the thioether. While the majority of DM1 is linked to trastuzumab via the nonreducible MCC linker, a minor amount of approximately 0.4% of DM1 is releasable, either being disulfide bound to the antibody via cysteine residues or having undergone thioether oxidation; this amount of DM1 appears to be liberated from T-DM1 during the reduction step in control experiments. From additional T-DM1 characterization during conjugation, there is also a low percentage of free thiols per mole of antibody measured (data not shown), and given the conjugation process for preparation of T-DM1, it is possible that antibody–DM1 disulfide bond formation could occur. As noted by Fishken et al., it is also possible that by oxidation of the thioether linkage this may also lead to DM1 release when subjected to reducing conditions [20] . Thus, in study samples, DM1 that may be disulfide bound to T-DM1 via cysteine residues or through an oxidized thioether would also have been measured in the assay, resulting in some conservative over-quantification of DM1. Conclusion The bioanalysis of ADCs in vivo has been challenging due to their complex and heterogeneous structures. Designing a ‘one size fits all’ assay strategy for ADCs is not possible due to differences in linker conjugation chemistry, the type of linker (e.g., cleavable or noncleavable) and the drug. A comprehensive bioanalytical strategy using multiple immunoassay and MS methods may be required to understand the fate of the ADC in vivo. ADC reference standards are heterogeneous mixtures that become even more complex in vivo 1038


due to drug deconjugation and degradation of the antibody. Therefore, assay reagents require additional characterization to ensure that they can detect all DAR species expected to form in vivo without selective loss or bias. If individually isolated DARs cannot be obtained, ADC mixtures with average DARs spanning over a wide range may be utilized. Comprehensive reagent and assay characterization may require the development of novel analytical techniques in order to ensure that the assays accurately measure each in vivo analyte. For T-DM1, we have developed a comprehensive bioanalytical strategy using multiple analytical techniques. This bioanalytical strategy, incorporating small-and large-molecule techniques, has been successfully applied to the drug development of T-DM1. Our strategy was based on some considerations specific for T-DM1; for example, the activity of naked trastuzumab, the noncleavable nature of the linker and the relative stability of linker in vivo. One unique aspect of T-DM1 included the presence of a sulfhydryl in DM1. Thus, the DM1 catabolite measurement involved a conservative strategy incorporating a sulfydryl reduction step to allow measurement of all released forms of the potent drug. This assay also measured additional forms of DM1 that might still be conjugated to the antibody and might exist in very minimal quantities, resulting in a very conservative assessment of levels of circulating DM1 that could be used to help understand potential safety liabilities of T-DM1. The total-trastuzumab ELISA provides data that are likely to be clinically relevant as the trastuzumab component itself is active. These data, combined with the conjugated-trastuzumab ELISA data, provided insights about T-DM1 clearance mechanisms. In summary, our bioanalytical strategy, using three key assays, provided a valuable approach to help understand safety and efficacy. Future perspective With the number of ADC therapeutics in clinical development increasing, an understanding of how ADCs biotransform in vivo will continue to be important to ensure assays accurately measure the analytes present in circulation. As more information is acquired, we will gain a greater understanding of which analytes correlate best with observed safety and efficacy end points. With this knowledge it may be possible to reduce the number of assays used to describe and monitor the ADC’s clinical pharmacology. future science group

PK assays for ADCs: case study with ado-trastuzumab emtansine Acknowledgements The authors would like to thank the following for their assistance in acquiring PK and assay data: S Girish, D Lu, J Tibbitts, D Leipold, M Khadkhodayan, E Mann, K Xu and L Liu.


with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research Financial & competing interests disclosure RC Dere, J-H Yi, C Lei, OM Saad, C Huang, Y Li and S Kaur are employees of Genentech, a member of the Roche group, and hold stock in Roche. J Baudys is a former Genentech employee. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Executive summary

The bioanalysis of antibody–drug conjugates (ADCs) is challenging, especially with regard to the development of PK assays to measure ADCs in plasma and serum.

Novel techniques may be required to identify drug-to-antibody ratio (DAR) analytes present in circulation after ADC administration. After these analytes are identified, appropriate assays can then be developed to generate a comprehensive PK profile in vivo.

ADCs may deconjugate in vivo changing the DAR distribution. Therefore, comprehensive reagent and assay characterization may be required to demonstrate that all DAR species expected to form in vivo can be detected without selectivity or bias.

It may be necessary to develop PK assay strategies on a case-by-case basis due to: The activity of the naked antibody; The molecular properties of the linker and drug; Possible catabolite formation; The dynamic nature of the ADC mixture in vivo.

References n nn

of interest of considerable interest

1

n

2

3

4

5

6

Carter PJ, Senter PD. Antibody–drug conjugates for cancer therapy. Cancer J. 14(3), 154–169 (2008).

prostate tumors to PSMA ADC, an auristatin-conjugated antibody to prostatespecific membrane antigen. Mol. Cancer Ther. 10(9), 1728–1739 (2011). 7

Antibody–drug conjugates (ADCs) are reviewed from a design perspective where the critical parameters for ADC development are discussed. Schrama D, Reisfeld RA, Becker JC. Antibody targeted drug as cancer therapeutics. Nat. Rev. Drug Discov. 5(2), 147–159 (2006). Polakis P. Arming antibodies for cancer therapy. Curr. Opin. Pharmacol. 5(4), 382–387 (2005). Alley SC, Okeley NM, Senter PD. Antibody–drug conjugates: targeted drug delivery for cancer. Curr. Opin. Chem. Biol. 14(4), 529–537 (2010). Ryan MC, Kostner H, Gordon KA et al. Targeting pancreatic and ovarian carcinomas using the auristatin-based anti-CD70 antibody–drug conjugate SGN-75. Br. J. Cancer 103(5), 676–684 (2010). Wang X, Ma D, Olson W, Heston WDW. In vitro and in vivo responses of advanced


Younes A, Kim S, Romaguera J et al. Phase I multidose-escalation study of the anti-CD19 maytansinoid immunoconjugate SAR3419 administered by intravenous infusion every 3 weeks to patients with relapsed/refractory B-cell lymphoma. J. Clin. Oncol. 30(22), 2776–2782 (2012).

8

Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network. Nat. Rev. Mol. Cell. Biol. 2(2), 127–137 (2001).

9

Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235(4785), 177–182 (1987).

10 Slamon DJ, Godolphin W, Jones LA et al.

Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244(4905), 707–712 (1989). 11 Nahta R, Yu D, Hung MC, Hortobagyi GN,

Esteva FJ. Mechanisms of disease: understanding resistance to HER2-targeted therapy in human breast cancer. Nat. Clin. Pract. Oncol. 3(5), 269–280 (2006).


12 Slamon DJ, Leyland-Jones B, Shak S et al.

Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N. Engl. J. Med. 344(11), 783–792 (2001). 13 Xu K, Liu L, Saad OM, Baudys J et al.

Characterization of intact antibody–drug conjugates from plasma/serum in vivo by affinity capture capillary liquid chromatography–mass spectrometry. Anal. Biochem. 412(1), 56–66 (2011). nn

Describes a novel method to determine the drug-to-antibody ratio distribution in biological fluids.

14 Shen BQ, Xu K, Liu L et al. Conjugation site

modulates the in vivo stability and therapeutic activity of antibody–drug conjugates. Nat. Biotechnol. 30(2), 184–189 (2012). nn

Assesses the impact of the conjugation site on the in vivo stability and activity of an ADC. The article also demonstrates that hydrolysis of the succinimide ring in a linker containing a maleimide group effectively stabilizes the ADCs resulting in increased stability.

15 Kaur S, Xu K, Saad O et al. A perspective on

bioanalytical assay strategies for the

1039



development of antibody–drug conjugate biotherapeutics. Bioanalysis 5(2), 201–226 (2013). nn

Describes the rationale behind designing effective assay strategies for ADCs. The article provides highlights of data from a variety of nonclinical and clinical case studies.

16 Shen BQ, Bumbaca D, Saad O et al.

Catabolic fate and pharmacokinetic characterization of trastuzumab emtansine: an emphasis on preclinical and clinical catabolism. Curr. Drug Metab. 13(7), 901–910 (2012). 17 Girish S, Gupta M, Wang B et al. Clinical

pharmacology of T-DM1: an anti-body-drug conjugate in development for the treatment of HER2-positive cancer. Cancer Chemother. Pharmacol. 69(5), 1229–1240 (2012). 18 Phillips GDL, Li G, Dugger DL et al.

Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody–cytotoxic drug conjugate. Cancer Res. 68(22), 9280–9290 (2008). 19 Sanderson RJ, Hering MA, James SF et al.

In vivo drug–linker stability of anti-CD30 dipeptide-linked auristatin immunoconjugate. Clin. Cancer Res. 11(2 Pt 1), 843–852 (2005). 20 Fishkin N, Maloney EK, Chari RV, Singh R.

from thioether linked antibody–drug conjugates (ADCs) under oxidative conditions. Chem. Commun. 47, 10752–10754 (2011).

25 Beeram M, Krop IE, Buris HS et al. A

Phase 1 study of weekly dosing of trastuzumab emtansine (T-DM1) in patients with advanced human epidermal growth factor 2-positive breast cancer. Cancer 118(23), 5733–5740 (2012).

21 Krop IE, Beeram M, Modi S et al. Phase I

study of trastuzumab–DM1, an HER2 antibody–drug conjugate, given every 3 weeks to patients with HER2-positive metastatic breast cancer. J. Clin. Oncol. 28(16), 2698–2704 (2010). 22 Hamblett KJ, Senter PD, Chace DF et al.

Effects of drug loading on the anti-tumor activity of a monoclonal antibody drug conjugate. Clin. Cancer Res. 10(20), 7063–7070 (2004).

26 Engvall E, Perlmann P. Enzyme-linked

immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 8(9), 871–874 (1971). 27 Lequin RM. Enzyme immunoassay (EIA)/

enzyme-linked immunosorbent assay (ELISA). Clin. Chem. 51(12), 2415–2418 (2005). 28 Bruno R, Washington CB, Lu J-F et al.

23 Burris HA 3rd, Rugo HS, Vukelja SJ et al.

Phase II study of the antibody drug conjugate trastuzumab–DM1 for the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer after prior HER2-directed therapy. J. Clin. Oncol. 29(4), 398–405 (2011).

Population pharmacokinetics of trastuzumab in patients with HER2+ metastatic breast cancer. Cancer Chemother. Pharmacol. 56, 361–369 (2005). 29 Stephan JP, Chan P, Lee C et al. Anti-CD22–

MCC-DM1 and MC–MMAF conjugates: impact of assay format on pharmacokinetic parameters determination. Bioconjug. Chem. 19(8), 1673–1683 (2008).

24 Krop IE, LoRusso P, Miller KD et al. A

Phase II study of trastuzumab emtansine in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer who were previously treated with trastuzumab, lapatinib, an anthracycline, a taxane, and capecitabine. J. Clin. Oncol. 30(26), 3234–3241 (2012).

nn

Compares various total antibody and conjugated antibody assay formats, and the impact of the assay format on the detection of enriched mixtures with different drug-to-antibody ratios.

A novel pathway for maytansinoid release

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