New Insights in Tissue Distribution, Metabolism...

Title Page

New Insights in Tissue Distribution, Metabolism and

Excretion of [3H]-labeled Antibody Maytansinoid

Conjugates in Female Tumor Bearing Nude Rats

Markus Walles, Bettina Rudolph, Thierry Wolf, Julien Bourgailh, Martina Suetterlin,

Thomas Moenius, Gisela Peraus, Olivier Heudi, Walid Elbast, Christian Lanshoeft,

Sanela Bilic

Novartis Institutes for Biomedical Research, Drug Metabolism and Pharmacokinetics,

Basel, Switzerland (M.W, B.R., T.W., J.B., M.S., T.M., G.P., O.H., W.E., C.L.),

Translational Clinical Oncology, East Hannover, NJ, USA (S.B.)

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on April 27, 2016 as DOI: 10.1124/dmd.115.069021

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Running Title Page

Running title: DME-properties of antibody maytansinoid conjugates (41, max. 60

characters)

Address correspondence to: Dr. Markus Walles, Novartis Pharma AG, Novartis

Institutes for Biomedical Research, Drug Metabolism and Pharmacokinetics,

Fabrikstrasse 14, 1.12, CH-4002 Basel, Switzerland. E-mail:

[email protected]; phone +41798346242; Fax: +41616968582

Number of:

Text pages 36

Tables 7

Figures 9

References 32

Number of words in abstract 250

Number of words in introduction 750

Number of words in discussion 1493

Abbreviations: ADC, antibody drug conjugate; AUC, Area under the curve; AUClast ,

area under the curve from 0 h to last measured time point; anti-Dig-Fab-POD, Anti-

Digoxigenin-Fab-Peroxidase; Cmax, maximum concentration; CV, coefficient of

variation; cps, counts per second; DAR, payload (“drug”) to antibody ratio; DME,

distribution, metabolism, and excretion; GSH, glutathione; ELISA, enzyme linked

immuno sorbent assay; HPLC, high performance liquid chromatography; LESA-µLC-

MS/MS, liquid extraction surface analysis coupled to micro LC-MS/MS; LC-MS,

Liquid chromatography coupled to mass spectrometry; μLC, micro LC; LSC, liquid


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scintillation counting; LYS, lysine; μLC, micro-liquid chromatography; MCC,

4(maleimidylmethyl) cyclohexane-1-carboxylate; MSe, MS/MS experiments with

collisional energy switching; NEM, N-ethyl-maleimide; QWBA, quantitative whole-

body autoradiography; RA, radioactivity; SD, standard deviation; SEC, size exclusion

chromatography; MCC, (Succinimidyl)trans-4(maleimidylmethyl)cyclohexane-1-

carboxylate;TCEP,Tris(2-carboxyethyl) phosphine; T-DM1 trastuzumab-MCC-DM1

maytansinoid conjugate


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Abstract

For antibody drug conjugates (ADCs), the fate of the cytotoxic payload in vivo needs

to be well understood in order to mitigate toxicity risks and properly design the first in

patient studies. Therefore, a distribution, metabolism and excretion (DME) study with

a radiolabeled rat cross reactive ADC ([3H]DM1-LNL897) targeting the p-cadherin

receptor was conducted in female tumor bearing nude rats. Although multiple

components (total radioactivity, conjugated ADC, total ADC, DM1 payload and

catabolites) needed to be monitored with different technologies (LSC, LC-MS, ELISA,

SEC), the pharmacokinetic data were nearly superimposable with the various

techniques. [3H]DM1-LNL897 was cleared with half-lives of 51-62 h and LNL897

related radioactivity showed a minor extent of tissue distribution. The highest tissue

concentrations of [3H]DM1-LNL897 related radioactivity were measured in tumor.

Complimentary LESA-µLC-MS/MS data proved that the LYS-MCC-DM1 catabolite

was the only detectable component distributed evenly in the tumor and liver tissue.

The mass balance was complete with up to 13.8 + 0.482% of the administered

radioactivity remaining in carcass 168 h post-dose. LNL897 derived radioactivity was

mainly excreted via feces (84.5 + 3.12%) and through urine only to a minor extent

(4.15 + 0.462%). In serum, the major part of radioactivity could be attributed to ADC

while small molecule disposition products were the predominant species in excreta.

We showed that there is a difference in metabolite profiles depending if derivatization

methods for DM1 were applied. Maysine and a cysteine conjugate of DM1 could be

identified in serum and excreta besides previously published LYS-MCC-DM1 and

MCC-DM1.


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Introduction

Antibody–drug conjugates (ADCs) constitute a therapeutic modality in which a

cytotoxic agent (payload) is chemically linked via cleavable or non-cleavable linker to

a monoclonal antibody (mAb) that recognizes a tumor-associated antigen. To date,

three ADCs have been approved for therapeutic use: Gemtuzumab ozogamicin for

acute myelogenous leukemia which has been withdrawn from the market,

Brentuximab vedotin for Hodgkin’s lymphoma and the most recently approved

trastuzumab ado-emtansine for second-line treatment of HER2-positive metastatic

breast cancer). While the goal of using ADCs as therapeutics is to minimize or

localize toxicity with the antibody intended to target specific tissue(s); toxic changes

with these agents can been seen in multiple tissues (Van der Heiden et al., 2006; De

Claro et al., 2012, Poon et al., 2013, Yan et al., 2016). Toxicities observed with ADCs

may be associated with either the components of the ADC (antibody, linker and

payload), or the metabolites. Therefore, the fate of the ADCs in vivo needs to be well

understood in order to mitigate toxicity risks and properly design the first in patient

studies. Up to now, little has been published about the distribution and uptake of

ADCs (Saad et al., 2015). Therefore, tissue distribution as well as metabolism and

excretion data can be very useful to support pharmacokinetic and

pharmacodynamics (PK/PD) as well as safety assessment in that they provide

insights of drug levels at the site of action e.g. in tumors (Wada et al., 2014). In

addition, they may be used for mechanistic investigations, e.g. to explore sites of

unexpected drug clearance or for characterization of target mediated drug disposition

(TMDD). Therefore these data can be considered as a powerful tool to learn about

disposition, PK/PD and toxicity of ADCs.


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The bioanalytical and DME properties of antibody maytansinoid based ADCs have

been recently reviewed (Erickson and Lambert, 2012; Han and Zhao, 2014, Kraynov

E et al., 2016, Thudium et al., 2014; Marcoux et al., 2015, Kaur et al., 2013, Beck et

al., 2015, Deslandes, 2014) The fate of antibody maytansinoid conjugates was

investigated in naive or tumor bearing mice and naive rats with iodine labels on the

antibody or a tritium label on DM1 (Xie and Blättler, 2006; Shen et al, 2012a; Saad et

al., 2015). The measured tissue exposures were low compared to blood and the

main components in serum were attributed to ADC. The main catabolites in serum

and excreta differed depending on whether the ADC utilized a cleavable linker or a

non-cleavable linker. With a cleavable linker, the main catabolite in mouse liver was

DM1 (Sun et al., 2011), while with the non cleavable MCC linker the main catabolites

detected in rat were MCC-DM1 and LYS-MCC-DM1 (Sun et al., 2011, Saad et al.,

2015). As released DM1 was reported to bind to proteins (Davis et al, 2012), usually

derivatization methods using NEM and TCEP are applied (Shen et al., 2012a).

This manuscript describes a DME study for an ADC with tritium labeled DM1 and

MCC linker conducted in female tumor bearing nude rats against p-cadherin which is

overexpressed in mammary carcinomas (Albergaria et al., 2011). A p-cadherin

targeted ADC approach is currently explored in the clinic being a novel promising

therapy for patients suffering from breast cancer (Menezes et al., 2015).

The key questions we wanted to address in this study were if the tritium labeled ADC

with higher specific activity would be sufficiently stable to perform quantitative whole

body autoradiography (QWBA) to study the tissue distribution in depth. We were

particular interested to investigate the extent of distribution between tumor and other

tissues as this can differ and to see if QWBA can give additional insights compared to


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the before published tissue harvesting methods (Shen et al., 2012a). In order to

investigate if the radioactivity in tumor can be attributed mainly to intact ADC or small

molecule catabolites, tumors were analyzed at different time points to establish

kinetics. In addition, complimentary investigations using the newly developed Liquid

Extraction Surface Analysis micro-Liquid Chromatography tandem Mass

Spectrometry (LESA-µLC-MS/MS) technique (Lanshoeft et al., 2016) were performed

on tumor tissue for the first time.

For the bioanalytical methods applied, we wanted to investigate if size exclusion

chromatography coupled to radioactivity detection (SEC-RA) provides similar results

for pharmacokinetic assessments of ADC like the ELISA assay and/or provide

additional benefits.

Lastly, as the catabolism of antibody maytansinoid conjugates has been previously

described (Widdison et al., 2015, Shen et al., 2012a), these current data compare the

metabolic profiles with and without applying derivatization methods to gain new

insights into catabolism.

Material and Methods

Test compound and reference standards. For the purpose of this study, an ADC

with an MCC linker was labeled with tritium in the chemically stable aromatic

methoxygroup of the DM1-moiety. The reference standards LYS-MCC-DM1, MCC-

DM1, as well as DM1 (structures described in Sun et al., 2011 and Figure 8) were

kindly provided by ImmunoGen, Inc. (Waltham, MA, US).

In vivo experiments for radiolabeled ADC. The animal studies were approved by

Animal Care and Use Committees of the Kanton Basel, Switzerland. Nude rats were


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provided by TaconicArtemis (Cologne, Germany). Tumor transplantation was

performed in accordance to animal license. For the tumor transplantation the human

breast carcinoma cell line HCC70 was cultured under standard conditions. The cells

were harvested, re-suspended and injected subcutaneously into the hind left flank of

the female nude rats. The tumors were allowed to grow until they reached a mean

volume of about ~250– 400 mm3 before dosing with [3H]DM1-LNL897 was initiated.

For dosing, [3H]DM1-LNL897 was dissolved in water containing 10 mM citric acid and

135 mM sodium chloride (pH 5.5). The rats received an intravenous bolus injection in

the vena saphena of nominal 10 mg radiolabeled ADC/kg under a light inhalation

anesthesia using an oxygen/isoflurane mixture (97/3, v/v; isoflurane: Forene®, Abbott

AG, Zug, Switzerland). The total amount of radioactivity administered was nominal 50

MBq/kg.

After dosing, blood was collected from the sublingual vein of three tumor bearing

nude rats at 1, 24, 96 and 168 h post-dose. From each time point the blood was

processed to serum by allowing it to clot for at least 30 min at room temperature

followed by centrifugation at 2500 × g for 15 min at 4ºC. A small aliquot was taken for

total radioactivity detection and the remainder material was snap frozen and stored at

-80ºC until further analysis. From a second group of tumor bearing nude rats, tumors

were collected at the same time points as the ones where blood has been collected

(N=1 rat/time point). In addition, selected animals (N=3 tumor bearing nude rats)

were housed in metabolic cages, for urine and feces collection allowing

determination of the route of excretion and the mass balance. Quantitative whole-

body autoradiography (QWBA) was performed in a last group of animals at 1, 24, 72,

96, 168 and 264 h post-dose using one animal per time point.


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Analytical Methods

Radiometric analysis. Radioactive signals in the different matrices were quantified

by liquid scintillation counting, using Liquid Scintillation Systems 3500 TR from

Packard Instr. Co. (Meriden, CT, USA). For quench correction, an external standard

ratio method was used. Quench correction curves were established by means of

sealed standards (Packard). Background values were prepared for each batch of

samples according to the respective matrix. The limit of detection was defined as 1.8

times the background value. All determinations of radioactivity were conducted in

weighed samples. In order to determine possible formation of tritiated water, samples

were analyzed twice: direct measurements of radioactivity and after drying of the

sample (at 37ºC for at least 12 h). Dried or non-dried serum and urine were mixed

with scintillation cocktail directly, whereas whole blood and feces samples were

solubilized before radioactivity analysis. Measured radioactivity for blood, plasma and

tissues was converted into concentrations (mol equivalents per volume or gram)

considering the specific radioactivity and assuming a matrix density of 1 g/mL.

Quantitative whole-body autoradiography (QWBA). Rats were sacrificed by deep

isoflurane inhalation. After deep anesthesia, blood for radioactivity determination by

LSC was collected from the sublingual vein. The animals were then submerged in n-

hexane/dry ice at -70°C for approximately 30 minutes. The embedded carcasses

were stored at -18°C and all subsequent procedures were performed at temperatures

below -20°C to minimize diffusion of radiolabeled materials into thawed tissues.

Sections were prepared as follows: in brief, 40 µm thick lengthwise dehydrated

whole-body sections were prepared and exposed to Fuji BAS III imaging plates (Fuji

Photo Film Co., Ltd., Tokyo, Japan) in a lead-shielded box at room temperature for


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two weeks. Afterwards, the sections were scanned in a Fuji BAS 5000 phosphor

imager (Fuji Photo Film) at a 50 µm scanning step. The concentrations of total

radiolabeled components in the tissues were determined by comparative

densitometry and digital analysis of the autoradiogram as described in Schweitzer et

al., 1987; blood samples of known concentrations of total radiolabeled components

processed under the same conditions were used as calibrators.

LESA-µLC-MS/MS. For mass spectrometric detection, whole-body sections were

lyophilized at -40°C overnight and mounted on metal glass slides plates, after which

they were placed on a Perfection V370 flatbed scanner (Epson, Kloten, Switzerland)

to acquire an optical image. A PAL auto-sampler from CTC Analytics AG (Zwingen,

Switzerland) was utilized for the extraction of the analytes from the tissue surface at

pre-defined sampling spots using a standard 100 µL PAL syringe from Hamilton

Company (Reno, NV, USA). Droplets on the sections were deposited and extracted

using a modified CTC autosampler (Schlieren, Switzerland). The extracted analytes

were injected into an Ekspert™ microLC 200 system from Eksigent (Dublin, CA,

USA) coupled to an API4000 triple quadrupole mass analyzer system (AB SCIEX,

Toronto, Canada). The extraction with 2 µL of extraction solvent resulted in with a

spatial resolution of 2 mm, observing specific transitions for MCC-DM1 and LYS-

MCC-DM1 in mass range from m/z 100 to 1300. A detailed overview of principle

experimental set-up has been described recently (Lanshoeft et al., 2016).

Quantification of whole and conjugated ADC by ELISA. In serum samples total

antibody concentrations (reflecting conjugated and unconjugated forms of the ADC)

were quantified using an ELISA. With this ELISA all ADCs species available in the

systemic circulation independent of the status of DM1 conjugation (DAR 0-8) was


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measured. In addition, conjugated antibody concentrations (ADC) of all ADCs

bearing at least one conjugated DM1 were also measured using an ELISA. The

ELISA assay for the measurement of conjugated ADC used an anti-maytansine

antibody (provided by ImmunoGen Inc, Waltham, MA, USA) as capturing reagent,

which was immobilized on microtiter plates. Digoxigenin labeled anti-human specific

antibody followed by anti-Dig-Fab-POD was used for detection by colorimetric read-

out. The same concept was used for the total Ab assay. However, in this case anti-

human specific antibodies were used for capturing and detection.

For measurement of ADC, biotinylated anti-maytansinoid monoclonal antibody

immobilized on a SA coated microtiter plate was used as capturing antibody. A

second antibody, digoxygenin labeled anti-human Fc (Fragment crystallizable)

monoclonal antibody (R10) was used, which binds to the Fc part of the ADC and was

detected by anti-Digoxigen in Fragment antigen binding region (Fab) labeled with

POD and 3,3΄-5,5΄-tetramethylbenzidine (TMB) as substrate. The optical density was

measured at 450 nm. The same concept was used for the total antibody assay (Ab).

However, in this case monoclonal R10 antibody (anti-human Fc specific monoclonal

antibody) was used to capture the ADC and a polyclonal goat anti-human Fc

antibody was used for detection.

The lower limit of quantification (LLOQ) for both assays (ADC and total Ab) was

0.0991 μg/mL DM1-LNL897 in 100% matrix. The upper limit of quantification (ULOQ)

for both assays (ADC and total Ab) was 2.13 μg/mL DM1-LNL897 in 100% matrix.

Determination of payload (“drug”) to antibody ratio (DAR) and “drug” load

distribution in dosing solution. The DAR as well as the distribution of the different

ADC species (D: number of DM1 molecules covalently attached to antibody: D0, D1,


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D2, D3, D4, D5, D6, D7, D8) in the dosing solution was determined using HPLC with

MS detection.

To calculate the ADC distribution by mass spectrometry (MS), the dosing solution (20

µL) was diluted into (80 µL) of mobile phase A (0.1% formic acid in water). The

diluted dosing solution was injected (50 µL) and separated on a MassPrep™ on-line

desalting cartridge 10 × 2.1 mm (Waters, Milford, MA, USA) with a flow rate of 0.4

mL/min. The mobile phase was composed of 0.1% formic acid in water (Eluent A)

and acetonitrile containing 0.1% formic acid (Eluent B). The gradient condition was

maintained at 10%B for 1 min, ramped to 80%B in 5 min, kept at 80%B for the next 4

min, and returned back to 10%B in 1 min. After the chromatography, the effluent was

split into a ratio of 1:3 with the smaller portion directed into the MS interface and the

bigger portion used for radiodetection. Mass measurements were made online using

a Synapt G2-Si HDMS mass spectrometer from Waters (Milford, MA, USA) operating

under MassLynx (version 4.1) for instrument control, data acquisition and data

processing. From 0 to 12 min, the mass spectrometer was operating in positive

electrospray (ESI) ionization mode and scanning from m/z 400-4000 with resolution

of 9000.

The total ion chromatogram (TIC) was selected and the corresponding TOF-MS

mass spectrum was extracted at a representative time window (containing signals

from ADCs of interest) to determine the drug load distribution. The characteristic

mass spectrum was deconvoluted using the MaxEnt1 algorithm in MassLynx. The

resulting processed mass spectrum (see Figure 1) showed different groups of peaks

corresponding to the different number of conjugated payloads. Various glycan forms

were also observed for each group of a specific payload, since no deglycosylation


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procedure was applied. The area under each peak was measured using the

Radiostar software version 5.12.0.3 (Berthold Technologies, Bad Wildbad, Germany)

and the sum of the peak areas for each group was determined. The relative

percentage ratio of individual ADC species to total peak area corresponds to the

relative abundance of each ADC species (distribution).

�� 100 ��

��

with:

RAAi: Relative abundance of each ADC species in % (see distribution calculation

above; for the assessment it was assumed that all ADC species have the same

ionization efficiency).

Determination of ADC in serum and excreta by SEC and radiometry. A HPLC

system model 1100 (Agilent Technologies, Santa Clara, CA, USA) equipped with a

binary capillary pump, a column oven, an auto-sampler, and a DAD detector was

used. 15 µL of each serum sample was injected on a Yarra SEC 3000; 300 x 7.8 mm

3 µm SEC column (Yarra SEC 3000; 300 × 7.8 mm 3 µm from Phenomenex Inc.

(Torrance, CA, USA)) equipped with a GFC 3000m 4x3 mm ID guard column (GFC

3000m 4x3 mm ID from Phenomenex Inc.). The column was maintained at 60°C. The

ADC was eluted isocratically with 200 mM ammonium acetate (pH 7) containing 15%

isopropanol and an isocratic at a flow rate of 0.40 mL/min which was maintained for

60 min. The column effluent was collected in 6 second fractions on yttrium silicate

scintillator-coated 96-weel well plates (LumaPlates; Packard BioScience, Groningen,

the Netherlands) from 0 to 40 min using a fraction collector GX271 (Gilson, Villiers-le-

Bel, France). The plates were dried and counted for 5 min in triplicate on a Packard


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TopCount instrument (PerkinElmer, Waltham, MA, USA). To determine the presence

of intact monomeric ADC [3H]-DM1-LNL897 in excreta, aliquots of urine and feces

pools (Group 2 T0-168 h) were centrifuged at 20000 × g for 15 minutes at 12°C, and

15 µL of the respective supernatants were directly injected onto the SEC column as

previously described.

Determination of DM1 and catabolites in serum, urine and feces. Urine and feces

samples from three animals collected between 0-168 h were pooled by combining

equal percentages (by weight) of individual fractions, while for serum equal volumes,

taken from three rats at the same time post-dose, were mixed. Since DM1 has a thiol

function, it can dimerize in solution under mild oxidative conditions and/or form

disulfide bonds with thiol-containing molecules like cysteine in serum (Shen et al.,

2012a). Therefore, before extraction, serum pools as well as urine pools and feces

homogenized pools were split into two equal parts, after which one was incubated

with an equal amount (v/v) of 0.5 M Tris(2-carboxyethyl) phosphine (TCEP) (Sigma

Aldrich GmbH, Steinheim, Germany) for 2 h at room temperature in the dark. The

TCEP is used as a reducing agent to reduce any disulfides (including S-bound DM1)

in the sample. The resulting free thiol groups were then alkylated by addition of a

4-fold amount (v/v) of 0.2 M N-ethyl-maleimide (NEM, Sigma Aldrich GmbH,

Steinheim, Germany) to the incubation solutions, in order to prevent further

reaction. The samples were stored for two additional hours after addition of NEM

(at room temperature in the dark) before further extraction.

The treated and untreated serum and feces samples were then extracted by protein

precipitation as follows:


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Aliquots of sample pools with and without TCEP treatment were stirred for one hour

with four volumes of acetonitrile followed by centrifugation at 10,000 × g for 30 min.

After decanting the supernatants, the radioactivity in the pellets was determined and

the recoveries of extraction were calculated. The supernatants were evaporated to

dryness under a stream of nitrogen at room temperature and the obtained residues

were reconstituted in two volumes (equivalent to original feces/serum volume) of

water/acetonitrile (95/5 v/v).

All extracts as well as TCEP treated/untreated urine samples were directly injected

(injection volume 20-50 microliter) into a UPLC system (Waters) with off-line

radioactivity and MS detection. The UPLC system was equipped with two pumps, a

column oven, a diode array detector and an autosampler (CTC PAL 2777) from CTC

Analytics, Zwingen, Switzerland. The chromatographic system was in line with a

fraction collector GX271 (Gilson, Villiers-le-Bel, France) and a Quadrupole-time-of-

flight tandem mass spectrometer, Synapt HDMS (Waters) operating under Masslynx,

version 4.1 for instrument control, data acquisition and data processing. Samples

were injected on a reverse phase C18 column Acquity HSS T3; 150 × 2.1 mm; 1.8

µm particles (Waters, Baden-Dättwil, Switzerland) equipped with a guard column

Acquity; 5 × 2.1 mm; 1.5 µm particles. The column was maintained at 40°C , and the

flow rate on the column was 0.45 mL/min. Eluent A was made up of 20 mM

ammonium formate including 0.1% of tri-fluoroacetic acid (pH 3.5), and eluent B was

made up of acetonitrile. The gradient was maintained at 5% B for 2 min, ramped to

35% B in 3 min, increased slowly to 55% B in 15 min, then to 90% B in 5 min, kept at

90% B for 5 min, returned back to 5% B in 5 min and finally equilibrated for 5 min

before next injection (for a total run time of 40 min). After elution from the column, the


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effluent was split into a ratio of 1:6 with the smaller portion directed to the MS source,

while the remaining flow was collected in 3 second fractions on 96 LumaPlates

(Packard BioScience) from 0-30 min. The plates were dried and counted (up to 30

min per well, depending on the level of radioactivity) on a Packard TopCount

instrument (PerkinElmer, Waltham, MA, USA).

From the relative peak areas, the concentrations of individual radiolabeled

components in serum and the amounts of individual radiolabeled components in the

excreta were calculated as follows:

%100%100,,iSP

serumRAserumi

RPARCC ⋅⋅=

and

%100%100,,iSP

excretaRAexcretai

RPARAA ⋅⋅=

with:

Ci,serum: Concentration of radiolabeled component i in serum (on molar basis);

CRA,serum: Concentration of total radiolabeled components (radioactivity) in serum (on

molar basis); RSP: Recovery of radioactivity after sample preparation (%); RPAi:

Relative peak area of radiolabeled component i in radiochromatogram (% of total

area under the radiochromatogram); Ai,excreta: Amount of radiolabeled component i in

excreta (urine, feces; % of dose); ARA,excreta: Amount of total radiolabeled components

(radioactivity) in excreta (urine, feces; % of dose).

Determination of ADC and its catabolites in tumor tissues. For metabolic

profiling, collected tumor tissues were homogenized separately using a Covaris

CryoPrep (Woburn, MA, USA). After extraction, samples were analyzed by LC-MS.


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Each tumor was weighed and placed in the center of the flexible pouch of a TT05

Tissue Tube using tweezers. The sample was frozen by immersing the flexible pouch

into liquid nitrogen, and the tissue was pulverized using a Covaris CryoPrep. The

tissue particles were then transferred into a 2 mL pre-weighed Protein LoBind tube

from Eppendorf (Hamburg, Germany), weighed and homogenized with 10 parts of

water (w/w) using a PT1200 Polytron (Kinematica, Eschbach, Germany). Afterwards,

an aliquot of each tumor homogenate was treated with TCEP (according to the

derivatization method described before) and a second one was processed without

treatment. Both samples were extracted as follows: a 4-fold amount (v/w) of

acetonitrile was added to the treated and un-treated tumor homogenate and the

mixture was stirred (750 rpm) for up to 3 h, followed by centrifugation at 20000 × g for

30 min at 12°C. The remaining supernatant was evaporated to dryness under a

stream of nitrogen at room temperature. The residues of un-treated tissues were then

reconstituted in one volume (equivalent to original tumor homogenate volume) of

water/acetonitrile (95/5, v/v) while residues of derivatized tissue samples were

reconstituted in two volumes of water/acetonitrile (95/5, v/v). Tumor extracts (80 µL)

were injected onto an UPLC system with off-line radioactivity and MS detection for

determination of free drugs and catabolites (see instrumentation and conditions for

analysis of free DM1 and catabolites in serum, urine and feces). To determine the

presence of intact monomeric ADC [3H]DM1-LNL897 in tumors, homogenized tissues

were centrifuged at 18000 × g for 20 min at 12°C and the recovered supernatants

were directly injected (25 µL) on the SEC system described previously.

The radioactivity amount in tumor homogenates was measured by LSC as described

before. All determinations of radioactivity were conducted in weighed samples. The


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radioactivity was transformed into concentrations of total radiolabeled components

considering the specific radioactivity of [3H]-DM1-LNL897, which was derived by the

determination of the radioactivity in the dose solution

C��,��

��

with:

CRA,tumor: Concentration of radiolabeled component in tumor (in pmol.g-1); RAtumor:

Total radioactivity measured in tumor (dpm); Mmass: Molecular mass of ADC (g.mol-1);

RAspec.: Specific radioactivity in dosing solution (MBq.mg-1); Wtumor: Tumor weight (g);

60: Conversion factor (Bq to dpm).

Peaks in the radiochromatograms were integrated manually using the Radiostar

software (Berthold Technologies, Bad Wildbad, Germany). From the relative peak

areas, the concentrations of individual radiolabeled components in tumor were

estimated as follows:

�,�� ,�� 100% � ��100%

with:

Ci,tumor: Concentration of radiolabeled component i in tumor (pmol.g-1); CRA,tumor:

Concentration of radiolabeled component in tumor (in pmol.g-1); RSP: Recovery of

radioactivity after sample preparation (%); RPAi: Relative peak area of radiolabeled

component i in radiochromatogram (% of total area under the radiochromatogram);

DAR: Drug Antibody Ratio.

Pharmacokinetic analysis. Pharmacokinetic parameters in serum and tissues were

calculated using Phoenix™ WinNonlin® software (Pharsight Corp., Mountain View,

CA, USA). The pharmacokinetic estimations were based on a non-compartmental

analysis model. The tissue to blood Cmax and AUClast ratios were calculated where


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possible. The AUClast was calculated using the linear trapezoidal rule. The half-life

was calculated for those tissues and matrices where at least 3 measurable data

points were available in the terminal phase of the tissue concentration time course.


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Results

Purity, composition and stability of dosing solutions.

The radiopurity of [3H]DM1-LNL897 ADC in the dosing solution (DS) was determined

by SEC to be 94.5% pure (ADC monomer) with 2.94% accounted for by soluble

aggregates and 2.56% by small molecule components (data not shown). In addition

to radiochemical purity measurements of [3H]DM1-LNL897 ADC in the dosing

solution, the relative distribution of different ADC species (0 to 8 DM1 molecules

attached to antibody) as well as the DAR were determined by MS (Figure 1). The

deconvoluted mass spectrum revealed a relative area ratio of 3.3%, 11.7%, 19.7%,

22.7%, 18.2%, 12.5%, 6.3%, 3.9% and 1.8% for antibody drug conjugate D0, D1, D2,

D3, D4, D5, D6, D7 and D8, respectively (where Dn corresponds to the number of

covalently bound payload DM1). After five days at 4 °C, the sample was re-measured

and a comparable distribution was observed (1.2%, 10.4%, 20.5%, 25.2%, 19.9%,

13.6%, 5.8%, 2.6% and 0.8% for ADC D0 to D8, respectively). The calculated DAR

at day 1 and after five days was found to be 3.3. These results demonstrate that

[3H]DM1-LNL897 ADC was stable (over a period of 5 days at 4°C) in the dosing

solution without significant loss of DM1 from the ADC.

Pharmacokinetics of total radiolabeled components, total and conjugated

antibody after intravenous dosing of nominal 10 mg/kg [3H]DM1-LNL897 ADC.

The concentrations of total radiolabeled components, as well as total and conjugated

antibody in serum were measurable throughout the observation period up to 168 h

post-dose (Table 1, Figure 2). The ADC was eliminated with mean half-lives ranging

from 51.3 h (based on radioactivity measurements by LSC) to 62.2 h (based on


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conjugated antibody measured by ELISA). The AUClast of total antibody and

conjugated antibody amounted to 62.3 and 73.4 h μM, respectively (Table 1), which

represented ~99.8% and 117% of the AUClast of total radiolabeled components,

respectively. The concentration data acquired by SEC and radioactivity

measurements (SEC-RA) were in good agreement with ELISA derived data (Table 1

and Figure 2).

Tissue distribution of total radiolabeled components as determined by QWBA

after intravenous dosing of nominal 10 mg/kg of [3H]DM1-LNL897 to female

tumor bearing nude rats.

The tissue distribution data are summarized in Table 2. Representative QWBA

images are shown in Figure 3.

Total radiolabeled components were extravascularly distributed throughout the body

to a minor extent. In selected tissues shown in Table 2 and Figure 3, total

radiolabeled components were quantifiable up to 264 h post-dose. In 20 out of 36

tissues, Tmax was rapidly reached at 1 h post-dose, the first time point analyzed. In

10 out of 35 tissues, Tmax was reached at 24 h post-dose, the second time point

analyzed. In the remaining tissues, Tmax was reached at 72 h post-dose. Only hair

(tactile) showed a Tmax of 168 h post-dose. The highest Cmax was determined for

lung (0.679 nmol/g), tumor (0.575 nmol/g), liver (0.595 nmol/g), adrenal gland

(medulla) (0.573 nmol/g), ovarian tissue (0.556 nmol/g), spleen (red pulp) (0.506

nmol/g) and kidney (medulla)(0.494 nmol/g). In all tissues analyzed, exposure based

on Cmax was lower in comparison to blood (QWBA) (1.15 nmol/g).


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The highest AUClast was determined for tumor (85.6 h·nmol/g), adrenal gland

(medulla) (52.7 h nmol/g), spleen (red pulp) (49.2 h nmol/g), liver (48.5 h·nmol/g),

ovarian tissue (46.1 h·nmol/g) and uterus mucosa (43.7 h·nmol/g). In almost all

tissues analyzed, exposure based on AUClast was lower in comparison with blood

(QWBA) (55.5 h·nmol/g). The only exception was tumor tissue, where the exposure

based on AUClast was 1.54 fold higher than in blood indicating clear distribution into

tumor as target tissue of the ADC.

At 264 h post-dose, total radiolabeled components were still detected in the analyzed

tissues, which is in line with the high fraction of radioactivity determined in the

carcass at the end of the excretion experiment (13.8 ± 0.482% of dose; Table 7). In

many tissues a half–life (T1/2) was calculated. The tissues showed varying half-lives

ranging from 62.4 h for stomach (glandular) to 94.2 h for eye (choroid).

Metabolic stability of [3H]-label for DM1 and ADC. As we have synthesized

radiolabeled ADC with a high specific activity, the formation of tritiated water was

retested and calculated according to Tse and Jaffe, 1991. Total radiolabeled

components measured in urine condensates of individual rats after lyophilization

indicated that a very low fraction of the administered radioactivity was transformed

into tritiated water (0.826 -1.01% of dose), indicating that the [3H]label showed an

acceptable metabolic stability (data not shown).

Characterization of [3H]DM1-LNL897 and catabolites.

In Figure 1, the deconvoluted mass spectrum of [3H]DM1-LNL897 in the dosing

solution is depicted. The ADC peaks were distributed in a m/z range of 1800-3800

amu, corresponding to a charge state distribution of 40 to 80 charges on the intact


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[3H]DM1-LNL897 (data not shown). The resolving power of the Q-TOF (approx.

9000) allowed baseline separation of the various glycosylation forms of the antibody

with molecular weights differences of 160 Da (nominal mass) representing galactose

units (Beck et al, 2013). By applying a collision energy of 45 eV in the trapping cell

(MSe), two characteristic fragment ions at m/z 547 and m/z 485 were observed

corresponding to intact maysine and maysine after loss of water and carbon dioxide,

respectively as described in Liu et al., 2005. The presence of these two signature

ions confirmed that the detected proteins correspond to the [3H]DM1-LNL897 as they

are characteristic for the fragmentation of the payload DM1.

In general, the metabolite identification was challenging due to the low abundance of

catabolites.

The structural characterization of metabolites in different matrices (serum, urine,

feces, tumor) was carried out by LC-MS/MS and peaks were assigned as shown in

Figure 4 to Figure 7. However the poor ionization by electrospray of this class of DM1

containing species, combined with their very low concentration in biological samples,

created significant technical challenges for their identification using LC-MS/MS. As a

result no mass spectral data could be acquired for components P1, P1.8 (front

peaks, Figure 4), and as well as for peaks summarized as un-identified residual

radioactivity in Figure 6. Nevertheless, structural proposals and identification of

metabolites like MCC-DM1 and LYS-MCC-DM1, which have been characterized

before for ADCs using the same payload/linker combination (Erickson et al, 2010,

Sun et al, 2011, Shen et al, 2012a) were supported by comparison with synthetic

reference standards whenever possible. Other metabolites like M6 (maysine), which

has been previously published as disposition product of maytansine (Suchocki and


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Sneden, 1987) and M7 (cysteine conjugate) were characterized in serum samples

following protein precipitation by LC-MS/MS (Figure 4).

In excreta samples (urine, Figure 6 and feces, Figure 7) metabolites M1 (MCC-DM1)

and M2 (LYS-MCC-DM1) were identified. Mass spectral data of all characterized

components are summarized in Table 3. Additional information about the chemical

structures of metabolites was acquired by reduction of the disulfide bonds using

TCEP and by alkylation of the resulting free thiol groups with NEM. This

derivatization process was previously tested using a spiked rat serum with DM1-

dimer (positive control) by LC-MS. The presence or absence of NEM conjugated

DM1 in derivatized biological samples indicated that DM1 was conjugated to

endogenous thiol-containing molecules via disulfide bonds. The structures of all

identified disposition products of LNL897 are shown in Figure 8.

Metabolite profiles in serum. Size exclusion off-line radio-profiles of [3H]DM1-

LNL897 in serum ([3H]-DM1-MCC-Ab, Figure 2) were recorded up to 168 h after

dosing. Intact antibody drug conjugate (ADC) was the most abundant radiolabeled

compound in serum (81.0% of [3H]-AUC0-168h of total radiolabeled components).

Additional peaks in front of the main compound, suggesting the presence of soluble

ADC aggregates and/or other ADC/protein complexes, accounted together for 17.3%

of the [3H]-AUC0-168h. The sum of [3H]-AUC0-168h for circulating intact ADC

monomer and aggregates accounted for 62.6 µM·h (Table 4A), which is slightly lower

than the 73.4 µM·h measured by the ELISA method (Table 1). The total radiolabeled

components corresponding to free small molecule components accounted together

for only 1.99% of [3H]-AUC0-168h.


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The concentration of total radiolabeled components measured at 1 h, 24 h, 96 h, and

168 h in serum supernatant samples following protein precipitation were 13.1, 4.74,

1.66 and 0.475 nM which resulted in a [3H]-AUC0-168h of 519 nM·h or 0.824% of

detected small molecule components (Table 4B). These results suggested that free

small molecule components in serum represent only a minor proportion compared to

the intact ADC or other protein bound DM1 containing components in precipitated

pellets. The metabolite profiles of the serum supernatant fractions (Figure 4) showed

two early eluting components: P1 and P1.8. For both of these, no mass spectral data

could be acquired. As the front peaks were still present after drying and solvent

evaporation, prior to off-line radioactivity measurement, presence of tritiated water in

the front peaks could be excluded. The [3H]-AUC0-168h for P1 and P1.8 accounted

for 0.395% and 0.0640% of total radiolabeled components (Table 4B). MCC-DM1

(M1), LYS-MCC-DM1 (M2), maysine (M6) and a DM1-cysteine conjugate (M7) were

identified by MS and accounted for 0.0575%, 0.0611%, 0.0664% and 0.0907% of

[3H]-AUC0-168h, respectively. Additional unidentified peaks were observed in serum

extracts and accounted together for 0.0888% of [3H]-AUC0-168h (Table 4B).

After derivatization a new peak corresponding to derivatized DM1 (M5, DM1-NEM)

appeared (Figure 4) and accounted for 0.635% of AUC0-168h (Table 4B). The

radiochromatographic background was higher after derivatization between 5 and 15

minutes post injection, which potentially indicates that the antibody degraded during

the procedure, forming artefacts.

The recoveries of radioactivity after extraction of un-treated serum samples were

found to be 0.9%, 0.8%, 0.8%, and 0.7%, for time points 1 h, 24 h, 96 h, and 168 h,


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respectively. For TCEP derivatized samples recoveries were 4.1%, 3.9%, 4.8%, and

5.2% at the equivalent time points.

Metabolite profiles in tumor. In tumor, the concentration of total radiolabeled

components was between 110 and 137 pmol·g-1 for the four rats. The derived

concentrations for [3H]DM1-LNL897 and LYS-MCC-DM1 are listed in Table 5.

Analysis of rat tumors collected after 1 h, 24 h, 72 h and 168 h also demonstrated

that the intact [3H]DM1-LNL897 was the major component at 1 h post-dose,

increased slightly at 24 h post-dose and then decreased with time. The fraction

representing the small molecules increased from 1 to 72 h post-dose. After 168 h

post dose, the fraction representing the intact ADC was small compared to fraction

representing the small molecule catabolites (Figure 5). The metabolite profiles

following protein precipitation (Figure 5) showed LYS-MCC-DM1 (M2) to be the major

catabolite, which had a concentration in the tissue between 159-307 pmol·g-1 (Table

5). An additional minor radiolabeled component, eluting between 7 and 8 minutes,

was detected but could not be identified by MS due to the low concentration and the

poor ionization by electrospray.

Radio-chromatograms of tumor homogenates treated with TCEP (reducing agent),

resulted in a new small peak eluting at 15 min, which corresponds to the retention

time of DM1 derivatized with N-ethyl maleimide (data not shown).

Concentrations of catabolites in tumor and liver tissues determined by LESA-

µLC-MS/MS. Complimentary tumor data analyzed by LESA-µLC-MS/MS from tumors

of different animals were acquired and compared to radioactivity measurements

(Figure 9). Three different sampling locations on two separate slices could be probed

and the results suggested a homogenous distribution of LYS-MCC-DM1 catabolite


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(Table 6). Although these tumors were collected from different animals, and

measured by a different technique, the measured concentration data are in alignment

with the radioprofiling data (Figure 9). The sensitivity of LESA-µLC-MS/MS allowed

specific detection of main catabolite LYS-MCC-DM1 (M2) in tumor sections.

Metabolite profiles in urine. Analysis of rat urine (0-168 h) by size exclusion

chromatography (data not shown) showed no peak corresponding to the retention

time of the intact [3H]DM1-LNL897 (based on retention time of standard). The

remaining part of excreted radioactivity represented small molecule components

which were analyzed by UPLC/MS with off-line radioactivity detection in presence

and in absence of reducing agent TCEP. Results showed that only a small fraction of

the dosed radioactivity, equivalent to 4.15% between 0-168 h (Table 7) was

recovered in urine. The metabolite profile in urine pool showed M2 (LYS-MCC-DM1)

as the major identified radiolabeled component (1.69% of dose) and M7 (DM1-

cysteine conjugate) representing 0.621% of dose (Table 7). Other minor radiolabeled

components like M1 (MCC-DM1) and M6 (maysine) accounted for 0.0946% and

0.0265% of the dose, respectively. In addition to the above components the radio-

profile showed un-resolved baseline radioactivity and a front peak (P1) which

combined accounted for 1.41% of dose (Table 7 and Figure 6).

Treatment of the urine pool with TCEP resulted in a new small peak in the radio-

chromatograms (0.127% of dose). This eluted after M1 and corresponded to DM1

derivatized with NEM (M5).

Metabolite profiles in feces. 84.5% of the dose was excreted in feces between 0-

168 h (Table 7). The most abundant metabolites in feces were M1 (MCC-DM1),

which accounted for 42.5% of the dose and M2 (LYS-MCC-DM1) which represented


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12.7% of the dose (see Figure 7 and Table 7). Metabolite M7 (DM1-cysteine

conjugate) was identified based on retention time and accounted for 1.96% of dose.

Additional unknown radiolabeled components, such as front peak P1 (0.320% of

dose) and P24 representing 1.79% of dose, were detected in rat feces between 0-

168 h. The sum of residual un-resolved radioactivity accounted for 16.8% of the

dose.

Elimination of [3H]DM1-LNL897

Analysis of rat excreta (urine and feces extracts) by SEC (data not shown), showed

negligible peaks corresponding to the retention time of the intact [3H]DM1-LNL897

(based on retention time of standard). The remaining radioactivity excreted

represented metabolites. Hence, metabolism/catabolism of [3H]DM1-LNL897 is one

major elimination pathway.

The primary metabolic pathways of [3H]DM1-LNL897 were cleavage before the

terminal lysine leading to metabolite M2 (LYS-MCC-DM1) and cleavage after the

terminal lysine leading to M1 (MCC-DM1). Additional minor metabolites were

characterized in urine and feces like the cysteine conjugate (M7) and M6 (detected in

serum and urine only) corresponding to maysine (Figure 8).

Several additional minor components could be detected (P1, P24, and un-resolved

baseline radioactivity see Table 5, and Figure 6), but could not be characterized with

the applied analytical methods. No free DM1 or DM1 dimer could be detected in

untreated excreta samples.


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Excretion of radioactivity and mass balance.

Within 168 h after administration, 4.15 ± 0.462% and 84.5 ± 3.12% (Table 7) of the

administered radioactivity was excreted in urine and feces, respectively.

At the end of the experiment, i.e. at 168 h post-dose, 13.8 ± 0.482% of the

administered radioactivity was determined in the carcass. Together with the

radioactivity recovered in the cage wash (2.55 ± 0.818%), the recovery of

radioactivity was complete (105 ± 3.27%).


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Discussion

This study presents the DME-properties of the rat cross reactive [3H] DM1-LNL897

ADC after intravenous dosing to female tumor bearing nude rats.

Pharmacokinetics and serum profiles

The pharmacokinetic parameters of the radiolabeled ADC were determined by

different analytical methods (ELISA, SEC, MS) as described by Shen et al., 2012a.

By comparison of AUClast of total radiolabeled components with ELISA data (Table

1) it is obvious that total radiolabeled components mainly reflect the conjugated

antibody, suggesting that the MCC linker of the ADC was stable in circulation as

previously reported, which is different from previous observed results with T-DM1

(Shen et al., 2012a). In our opinion possible changes in the manufacturing process,

could contribute to these findings but need further investigations. The high stability of

LNL897 in circulation is further supported by catabolite profiling and recovery

measurements which were below 1% for all time points. No DM1 could be directly

detected at 1 h post-dose and about 16.3 nM of the DM1 derivate (DM1-NEM, M5;

data not shown) could be detected at the same time point after derivatization, which

indicates that DM1 was likely bound to endogenous substrates (GSH, cysteine) or

proteins via S-S bonds. The treatment of ADC dosing solution with increasing

concentrations of TCEP have shown in increased release of DM1. At the same time

control analysis have shown that stability of thioether bond is not affected by

presence of TCEP and that only species with disulfide bonds are effected. Based on

this experiment we concluded that minor amounts of DM1 are directly bound to

cysteines. These cysteine bound DM1 species could be released as cysteine–DM1


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conjugates (like M7) after lysosomal processing. However, glutathione conjugates of

DM1 after intravenous dosing of DM1 to rats have been reported recently (Shen et

al., 2015). Further degradation of glutathione conjugates to cysteine could also lead

to formation of M7.

Compared to ELISA data, the SEC–RA profiles provided more insights about the

circulating components. As shown in Figure 2, SEC-RA allows the differentiation

between ADC and higher molecular weight aggregates/complexes while for the

ELISA assay it is unclear how these additional aggregates/complexes impact the

detection. When the main ADC peak and earlier eluting small peak were summed,

serum concentrations were approximately the same as those measured by the

ELISA method (Table 1, Table 4A and Figure 2).

With SEC-RA profiles, it is only possible to distinguish between fractions of large

molecules and small molecules. For the late eluting peaks, which are attributed to

small molecules, it is possible that these peaks contain multiple co-eluting

components. Therefore, the supernatants after protein precipitation were injected and

re-profiled on an analytical LC-MS system.

The metabolite profiles in serum and urine showed qualitative and quantitative

differences depending on if the previously described derivatization method was

applied or not (Table 4b, Figure 4 and Figure 6). A front peak, as well as metabolites

M7, M6 and M1, was detected with both sample preparation methods, whereas M5

was only detected after applying the derivatization method. It is worth mentioning that

only limited control experiments were conducted for the ADME studies. As possible

S-S bond formation was identified as the main liability, the sample methods without

TCEP were tested before with DM1 dimer to make sure to not cause any reduction.


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A front peak was also detected during the ADME study in rats for T-DM1 and was

attributed to tritiated water (Shen et al., 2012a). As our samples were dried during

processing, the front peak cannot be fully attributed to tritiated water. Hence the

presence of further polar catabolites eluting in the front peak cannot be excluded.

Under conditions of the derivatization, M7 could form DM1-NEM (M5). M5 was only

detected after derivatization, and the recoveries of radioactivity from serum were also

slightly higher (up to 5%), which could indicate that protein bound-DM1 was released

by the derivatization method. On the other hand, it cannot be excluded that the

derivatization method destroys antibody to a minor extent, which could also lead to a

slightly higher amount of radioactivity in the solution.

Distribution

In previous studies, either tritium labels on the payload with lower specific activity

(Shen at al., 2012a) or with 125I labels on the antibody were synthesized, of which

only the iodinated material was used to perform QWBA studies (Saad et al., 2015). In

our study, the confirmed stability of the ADC in the dosing solution allowed for the

QWBA experiment to be performed with a tritium label on the DM1 payload.

In rats, p-cadherin is mainly expressed in tumor and tissues like tongue, skin,

esophagus and bladder (internal data, not shown). After intravenous dosing of the

ADC, the tissue distribution as determined by QWBA was comparably low in all

tissues analyzed with the exception of tumor tissue where the highest AUClast was

determined being 1.54 fold higher than in blood (Table 2). In the remaining tissues,

the exposure based on AUClast was 0.184 to 0.950 fold the AUClast of blood. This

demonstrates a clear distribution of ADC related radiolabeled components into tumor


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as target tissue. The exposure in other tissues also indicates that non target specific

distribution occurs, which is also nicely illustrated in Figure 3. Besides tumor, there

was no accumulation or tissue retention of radioactivity over time in any of the tissues

analysed (data not shown). Like small molecules, biotherapeutics are also cleared

through kidney and liver (Hamuro and Kishnani, 2012). Although these tissues are

highlighted in the depicted QWBA pictures, it needs to be recognized that the organs

are also highly perfused by blood which contains large amounts of ADC (Figure 3).

However, as QWBA data only show distribution of total radiolabeled components and

only the DM1 part of ADC was radiolabeled, it is unclear whether the observed

radioactivity in tissues could be attributed to intact ADC, released DM1 or another

catabolite so further investigations were conducted.

Figure 5 shows the tumor profiles acquired by SEC-RA and it is obvious that after 1 h

tumor tissue mainly contained ADC while after 24 h the catabolite LYS-MCC-DM1

(M2) was the predominant component (Table 5).

To see if the catabolite M2 was evenly distributed in tumor tissue, the newly

developed LESA-µLC-MS/MS (Lanshoeft et al., 2016) was applied. The acquired

quantitative LESA data showed no significant difference over tumor tissue (Table 6)

and the measured concentration data were well in line with results from radioactivity

profiling in tissues (Figure 9), considering that analyzed tumor samples were

collected from different rats with different tumor sizes.

In line with previous results, the LYS-MCC-DM1 catabolite was confirmed as main

component in the liver (data not shown) by applying the LESA-µLC-MS/MS method


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on QWBA slices which is in line with early observations (Lambert and Erickson,

2012).

The main advantage of LESA-µLC-MS/MS method is that no radiolabel is ultimately

needed and that no sample processing is required so that it can provide

complimentary data. For quantitative assessments without radiolabel, more thorough

validation using internal standards to correct for different tissue responses would

need to be applied (Lanshoeft et al., 2016).

Elimination

Like serum, differences in the catabolite profiles in urine were observed with and

without derivatization. In untreated urine LYS-MCC-DM1 (M2) and the cysteine

conjugated DM1 (M7) were the main components followed by maysine (M6) and

MCC-DM1 (M1). After applying the derivatization method, the peak corresponding to

M7 was reduced and M5 appeared in the sample (Figure 6). This could indicate that

either the derivatization was not complete or that further uncharacterized

components co-elute under the M7 peak.

These findings are in line with previous observations (Shen et al., 2012a) except that

maysine (M6) as well as the cysteine conjugate of DM1 (M7) have not been reported

before. In a recent rat in vivo study with DM1, maytansinol has been reported as a

catabolite (Shen et al., 2015) but it remains unclear if the detected maysine is a

downstream product of maytansinol.

The presence of MCC-DM1 in plasma and excreta suggests that this is probably

cleaved off in plasma or other tissues from the ADC. Different mechanisms for

hydrolysis of the maleimide ring which could lead to loss of DM1 have been recently


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described for cysteine linked ADCs (Shen et al. 2012b), but not for lysine linked

ADCs. It also cannot be excluded during synthesis the reactive NHS esters can react

and couple with other amino acids like serine, and threonine (Hermanson et al.,

2013) which could lead to formation and release of MCC-DM1. Further investigations

are required to investigate how MCC-DM1 is released.

The elimination of LNL897 was moderately fast and results were aligned with

previously reported results (Shen et al. 2012a). After 1 week a complete mass

balance was obtained (105 + 3%). 88.7% of dose was detected in the collected

excreta and 13.8% in the carcass (data not shown). To obtain a complete excretion

profile (to reduce the amount of dose retained in the carcass), a longer sampling time

up to two weeks is recommended for future DME studies of ADCs.

Conclusion

We have demonstrated that the use of a tritium label on the payload of an ADC is

suitable even for longer term DME and QWBA studies. We have also shown that a

diverse set of analytical methods is required to investigate all DME properties of an

ADC and that the combination of QWBA and LESA-µLC-MS/MS is a powerful tool

which can give new insights into the distribution of these constructs. Label free

distribution studies may also be possible in future if the quantitative abilities of the

LESA method can be further improved. For metabolism investigations of antibody

maytansoid conjugates, it is important to pay attention to the sample preparation

method, especially to the use of reductive agents, as this has an impact on the

observed catabolite profile. In this study we identified a DM1-cysteine conjugate and

maysine as additional catabolites. MCC-DM1 was also identified as a major

component in excreta which suggests that there must be additional mechanisms


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present that cause its release which need to further investigated. The methods

presented provide useful perspectives for the further development of in vivo

applications of ADCs as therapeutics.


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Acknowledgements

We sincerely thank Wayne Widdison, XiuXia Sun and Kate Lai of ImmunoGen, Inc

for discussions on study design of radiolabeled synthesis and support for release

analysis.

We acknowledge also Prakash Mistry from Oncology, Basel for implementing the

tumors in female rats and Alexander David James for thorough review of the

manuscript.

We also acknowledge Wolfgang Marterer for giving insight into the synthetic

strategies.


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Authorship Contributions

Participated in research design: Rudolph, Walles, Wolf, Peraus, Heudi, Bilic, Moenius

Conducted experiments: Wolf, Lanshoeft, Bourgailh, Suetterlin

Contributed with new reagents or analytical tools: Elbast, Lanshoeft, Moenius

Performed data analysis: Rudolph, Walles, Wolf

Wrote or contributed to the writing of the manuscript: Walles, Wolf, Rudolph, Heudi,

Lanshoeft, Elbast


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Figure Legends Figure 1: Measurement of [3H]DM1-ADC species distribution in dosing solution

The dosing solution was analyzed by LC-MS using a desalting cartridge.

Representative deconvoluted mass spectra at day 1 (day of dosing) and at day 5 of

the dosing solution being stored at 4°C were compared and are shown here. The

different groups of peaks were annotated by Dn where n corresponds to the number

of conjugated DM1 molecules per antibody. Each group is composed of 4 main

peaks and several minor components which correspond to the different glycosylation

forms. As no deglycosylation procedure was used for this analysis, the [3H]DM1-

LNL897 distribution in percentage refers to the relative area ratios of each

group.Profiles were acquired by size exclusion chromatography coupled to

radioactivity detection.

Figure 2: Off-line SEC radiochromatograms of serum samples

Upper graph contains time concentration serum profiles (logarithmic scale) acquired

by LSC, ELISA and SEC RA

Figure 3: Selected whole-body autoradiograms after a single nominal 10 mg/kg i.v.

dose of [3H]DM1-LNL897 in female tumor-bearing nude rats.

Selected lengthwise whole-body sections (A-H) through tumor bearing nude rats are

displayed: A and B: 1 h post-dose; C and D: 24 h post-dose; E and F: 72 h post-

dose; G and H: 168 h post-dose.


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Selected major organs are labeled in the following order: a) tumor, b) blood, c)

kidney, d) stomach, e) liver, f) heart, g) thymus, h) brain, j) intestinal tract, k)

tongue/mouth, l) spinal cord, m) salivary gland.

Figure 4: Radiochromatograms of serum samples 1 h post-dose acquired by HPLC

with off-line radioactivity detection.

Figure 5: Size exclusion radiochromatograms and HPLC radioprofiles (embedded) of

tumors homogenates at T1h and at T168h post-dose after a single intravenous

administration of nominal10 mg/kg [3H]DM1-LNL897.

Figure 6: Metabolite profiles in urine

Off-line radiochromatograms in the presence and absence of reducing agent TCEP

of urine pools (0-168 h) following a single intravenous administration of nominal 10

mg/kg [3H]DM1-LNL897 to female tumor-bearing nude rats (n=3). Proteins contained

in the urine pools were previously precipitated using acetonitrile and only the soluble

metabolites were profiled by LC/MS with off-line radioactivity detection.

Figure 7: Metabolite profiles in feces

Off-line radiochromatograms in the presence and absence of reducing agent TCEP

of feces pools (0-168 h) following a single intravenous administration of nominal 10

mg/kg [3H]DM1-LNL897 to female tumor-bearing nude rats (n=3). Proteins contained

in the feces pools were previously precipitated using acetonitrile and only the soluble

metabolites were profiled by LC/MS with off-line radioactivity detection.


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Figure 8: Identified disposition products of [3H]DM1-LNL897

Figure 9: Comparison of tumor concentration data acquired by LC-with radioactivity

detection and LESA-μLC-MS/MS


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Tables

Table 1 Measured concentrations and derived pharmacokinetic data of [3H]DM1-LNL897

Concentrations of total radiolabeled components measured by LSC, total and conjugated antibody measured by ELISA, conjugated antibody measured by SEC-RA (expressed in µM) and corresponding pharmacokinetic parameters in serum of female tumor-bearing nude rats after a single intravenous administration of [3H]DM1-LNL897 (nominal dose: 10 mg/kg).

Total Radioactivity (LSC)

Total Antibody (ELISA) Conjugated Antibody (ELISA)

Conjugated Antibody (SEC) c

Time (h) Mean SD CV(%) Mean SD CV(%) Mean SD CV(%)

1 1.39 0.0764 5.48 1.25 0.0557 4.46 1.42 0.0279 1.97 1.31

24 0.585 0.0286 4.89 0.577 0.0128 2.22 0.664 0.00873 1.31 0.483

96 0.197 0.0159 8.05 0.214 0.0034 1.61 0.268 0.012 4.47 0.147

168 0.0835 0.00683 8.18 0.107 0.00979 9.17 0.133 0.0133 9.98 0.0457

Actual Dose (mg/kg) 10 0.0808 0.807 10 0.0808 0.807 10 0.0808 0.807 10

Tmax (h) 1.0 a [1.0-1.0] b 1.0 a [1.0-1.0] b 1.0 a [1.0-1.0] b

Cmax (µM) 1.39 0.0764 5.48 1.25 0.0557 4.46 1.42 0.0279 1.97

Cmax/Dose (µM)/(mg/kg) 0.139 0.00717 5.16 0.125 0.0055 4.43 0.142 0.0021 1.49

Tlast (h) 168 a [168-168] b 168 a [168-168] b 168 a [168-168] b

AUClast (h•µM) 62.4 2.08 3.34 62.3 0.749 1.2 73.4 1.33 1.81

AUCinf (h•µM) 68.6 1.72 2.5 71.5 0.721 1.01 85.4 2.77 3.25

T1/2 [h] 51.3 2.45 4.77 59.2 4.02 6.79 62.2 4.09 6.58

T1/2 range [h] [24-168] [24-168] [24-168]

Vss (L/kg) nc nc. 0.617 0.00295 4.77

a: Median

b: [range]

c: To calculate concentrations aggregate peak and ADC peak were summed up.

nc: not calculated

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

DM

D Fast Forw

ard. Published on April 27, 2016 as D

OI: 10.1124/dm

d.115.069021 at ASPET Journals on October 5, 2020 dmd.aspetjournals.org Downloaded from


Table 2 Pharmacokinetic parameters of total radiolabeled components in selected tissues of female tumor-bearing nude rats following a single nominal 10 mg/kg i.v. dose of [3H]DM1-LNL897.

Tissues Tmax (h)

Cmax (nmol/g)

Cmax T/B ratio

AUClast (h*nmol/g)

AUClast T/B ratio

Tlast (h)

T1/2 (h)

Range for T1/2 (h)

Blood (LSC) 1.0 0.902 0.784 44.4 0.800 264 76.6 96-264

Blood (QWBA) 1.0 1.15 1.0 55.5 1.0 264 75.5 96-264

Adrenal gland (cortex) 1.0 0.333 0.290 23.3 0.420 264 91.4 72-264

Adrenal gland (medulla) 1.0 0.573 0.498 52.7 0.950 264 nc nc

Bone marrow 1.0 0.390 0.339 20.6 0.371 264 85.7 72-264

Choroid plexus 24.0 0.190 0.165 15.9 0.287 264 81.3 72-264

Esophagus 72.0 0.142 0.123 12.8 0.231 264 80.9 96-264

Eye (choroid) 24.0 0.174 0.151 17.0 0.306 264 94.2 72-264

Eye (ciliary body) 72.0 0.0719 0.0625 12.7 0.229 264 67.5 96-264

Fat (brown) 1.0 0.106 0.0922 12.8 0.231 264 nc nc

Hair (follicle) 24.0 0.129 0.112 28.2 0.508 264 nc nc

Hair (tactile) 168 0.139 0.121 24.7 0.445 264 nc nc

Heart 1.0 0.391 0.340 19.7 0.355 264 81.3 72-264

Intestinal wall (colon) 24.0 0.109 0.0948 11.7 0.211 264 nc nc

Intestinal wall (small int.) 24.0 0.281 0.244 24.8 0.447 264 65.3 96-264

Kidney (CM junction) 1.0 0.377 0.328 29.4 0.530 264 nc nc

Kidney (cortex) 1.0 0.358 0.311 33.8 0.609 264 nc nc

Kidney (medulla) 1.0 0.494 0.430 38.7 0.697 264 73.1 96-264

Liver 1.0 0.595 0.517 48.5 0.874 264 85.8 72-264

Lung 1.0 0.679 0.590 39.3 0.708 264 85.4 96-264

Lymph nodes (sub.) 1.0 0.443 0.385 42.2 0.761 264 88.4 96-264

Ovarian tissue 1.0 0.556 0.483 46.1 0.831 264 68.7 96-264

Pancreas 1.0 0.125 0.109 10.2 0.184 264 74.2 72-264

Pineal body 1.0 0.265 0.230 17.1 0.308 264 85.9 72-264

Pituitary gland 24.0 0.279 0.243 28.4 0.512 264 80.6 72-264

Preputial gland 24.0 0.211 0.183 21.3 0.384 264 nc nc

Skin 72.0 0.0676 0.0588 10.7 0.193 264 76.3 96-264

Spleen 1.0 0.475 0.413 43.4 0.782 264 78.3 96-264

Spleen (red pulp) 1.0 0.506 0.440 49.2 0.887 264 84.7 96-264

Spleen (white pulp) 1.0 0.203 0.177 19.9 0.359 264 86.7 24-264

Stomach (glandular) 24.0 0.121 0.105 10.7 0.193 264 62.4 96-264

Thyroid gland 1.0 0.144 0.125 15.9 0.287 264 nc nc

Tongue 24.0 0.145 0.126 12.4 0.223 264 81.8 96-264

Tooth (pulp) 1.0 0.478 0.416 41.6 0.750 264 nc nc

Tumor 24.0 0.575 0.500 85.6 1.54 264 nc nc

Uterus 72.0 0.177 0.154 27.5 0.496 264 83.7 96-264

Uterus mucosa 72.0 0.265 0.230 43.7 0.788 264 88.0 96-264

T/B ratio: tissue to blood ratio of Cmax and AUClast, normalized to the respective blood value estimated by QWBA. nc: not calculated due to limited data set available or the adjusted r2<0.75.


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Table 3 Elemental compositions and mass spectral data of [3H]-DM1-LNL897metabolites and derivatized DM1.

Name LC Rt Full-MS a

Type of ion Elemental composition

Fragments observed in MS/MS

(min) (m/z) (m/z)

M7 (Cysteine conjugate) 7.0 (857) [M+H]+ C38H54N4O12S2Cl 795, 547, 485, 467, 435, 299,

140

M2b (LYS-MCC-DM1)

9.0 9.5

1125 (1103)

[M+Na]+ [M+H]+

C53H76N6O15SCl 1085, 1041, 1023, 1009, 557, 547, 539, 529, 521, 511, 485, 467, 453, 435

M6 (Maysine) 13.0

569 (547)

[M+Na]+ [M+H]+

C28H36N2O7Cl 529, 453, 425

M1b (MCC-DM1)

14.0 14.5

997 (975) 957 913

[M+Na]+ [M+H]+ [M+H]+-H2O [M+H]+-H2O-CO2

C47H64N4O14SCl 957, 913, 881, 547, 529, 485, 467, 453, 435, 411, 383, 326, 298, 280, 140

M5 (derivatized DM1) 15.0

885 880 (863) 845 801

[M+Na]+

[M+NH4]+

[M+H]+ [M+H]+-H2O [M+H]+-H2O-CO2

C41H56N4O12SCl 845, 801, 547, 485, 453, 435, 299, 271, 214

M4 (DM1) 15.8

760 (738) 720 676

[M+Na]+ [M+H]+ [M+H]+-H2O [M+H]+-H2O-CO2

C35H49N3O10SCl 720, 676, 658, 644, 626, 547, 529, 485, 467, 453, 435, 174, 146

a: Mass to charge (m/z) used for MS/MS fragmentation is shown in brackets. b: Metabolites M1 and M2 consist of two diastereoisomers which results in the formation of double peaks.


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Table 4A Concentrations (µM) and AUC values (µM.h) of [3H]DM1-LNL897 in serum.

Concentrations (µM) and pharmacokinetic parameters of intact antibody drug conjugates (ADC), ADC complexes and/or aggregates as well as free small molecule components in serum of tumor-bearing nude rats following a single intravenous administration of 10 mg/kg [3H]DM1-LNL897. Data were derived from SEC-LC with off-line radiodetection obtained after direct injection of serum.

Sample collection time (h)

1 24 96 168

Concentration AUC0-168h AUCinf

Metabolite / Component µM µM·h % µM·h

Aggregates / complexes a 0.119 0.102 0.0558 0.0200 11.0 17.3 12.9

LNL897 monomer 1.31 0.483 0.147 0.0457 51.6 81.0 54.4

Total ADC b 1.42 0.664 0.268 0.133 73.4 nc 85.4

Free small molecule components c 0.0294 0.0106 0.00472 0.00199 1.27 1.99 1.44

Sum of additional metabolites nd nd nd nd

Total components detected 1.46 0.593 0.207 0.0679 63.7 100 68.2

Total components in original sample 1.46 0.593 0.207 0.0679 63.7 100 68.2

nd: not detected nc: not calculated a: Total radioactivity corresponding to ADC aggregates and/or complexes with endogenous proteins in the serum. b: Values italic – conjugated antibody (LNL897 monomer + potential aggregates / complexes) concentrations measured by ELISA method. c: Radioactivity corresponding to small molecule containing components of [3H]DM1-LNL897


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Table 4B Concentration and AUC data of catabolites in serum

Concentrations (nM) and pharmacokinetic parameters of metabolites in serum of tumor-bearing nude rats following a single intravenous administration of nominal 10 mg/kg [3H]DM1-LNL897. Data were derived from SEC-LC with off-line radiodetection obtained after injection of extracted serum.

Sample collection time (h)

1 24 96 168

Concentration AUC0-168h AUCinf

Metabolite / Component nM nM.h % nM.h

P1 (front peak) 5.81 2.47 0.754 0.215 249 0.395 262

P1.8 (front peak) 0.700 0.408 0.158 0.0321 40.3 0.0640 42.1

M7 (Cysteine conjugate) 1.58 0.515 0.166 0.0463 57.1 0.0907 59.9

M2 a (LYS-MCC-DM1) nd 0.408 0.232 0.0676 38.5 0.0611 42.5

M6 (Maysine) 1.06 0.419 0.116 0.0248 41.9 0.0664 43.1

M1a (MCC-DM1) 1.72 0.327 nd nd 36.2 0.0575

M5 (derivatized DM1) d 16.3 2.93 0.821 0.189 400 0.635 410

Sum of additional metabolites b 2.23 0.199 0.230 0.0895 56.0 0.0888 60.6

Total components detected 13.1 4.74 1.66 0.475 519 0.824 549

Lost during sample processing c 1443 588 205 67.4 62469 99.2 nc

Total components in original sample 1456 593 207 67.9 62988 100 67500

nd: not detected nc: not calculated a: Peaks were identified by retention time only b: Corresponds to the sum of the residual radioactivity measured in each radio-chromatograms c: Including ADC’s and/or aggregates/complexes d: DM1-NEM measured after TCEP treatment (corresponds to possible di-sulfide bonded DM1 with endogenous components in serum


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Table 5 Concentrations of total radiolabeled components and metabolite M2 (LYS-MCC-DM1) in rat tumors

Amount (% of dose) and concentrations (nmol/g) of total radiolabeled components as well as concentrations (nmol/g) of LNL897 and of the principal metabolite M2 (Lys-MCC-DM1) in tumors at different time points after a single intravenous administration of nominal 10 mg/kg [3H]DM1-LNL897. Data were derived from metabolic patterns obtained by SEC analysis with off-line radioactivity detection and UPLC/MS/off-line radio-detection for small molecules. Time Total radiolabeled components LNL897 concentration M2 (LYS-MCC-DM1 )

(h) (% of dose) (nmol/g)a (nmol/g)a (nmol/g)b (nmol/g)b

Rat 1 1 0.262 0.0858 0.0377 0.125 0.00159

Rat 2 24 0.590 0.282 0.0516 0.170 0.263

Rat 3 72 0.351 0.244 0.0275 0.0907 0.307

Rat 4 168 0.242 0.142 0.00935 0.0309 0.188

a: concentration expressed as protein b: concentration expressed as DM1 conjugated (DAR of 3.3)


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Table 6 Peak area of LYS-MCC-DM1 (M2) in tumor sections obtained with LESA-µLC-MS/MS (cps)

1 h 24 h 72 h 168 h

1 93 2550 3410 1550

2 0 2490 2800 1610

3 29 1740 2750 2430

4 0 2540 3010 1780

5 0 3620 1400 2220

6 0 3030 2280 1310

Mean 20 2662 2608 1817

SD 37 626 697 427

CV 184.1 23.5 26.7 23.5

Time post-dose

1

2

Plate Sample


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Table 7 Amounts (% of dose) of LNL897 catabolites excreted in urine, and feces of rats after a single intravenous administration of nominal 10 mg/kg [3H]DM1-LNL897

Data were derived from metabolic patterns obtained by LC analysis with off-line radioactivity detection (pools of n=3 rats).

Excretion (% of dose)

Urine Feces Total excretion

Excretion period (h) 0-168h 0-168 h 0-168 h

P1 (front peak) 0.0943 0.320 0.414

M7 (Cysteine conjugate) 0.621 1.96 2.58

M2 (Lys-MCC-DM1) 1.69 12.7 14.4

M6 (Maysine) 0.0265 nd 0.0265

M1 (MCC-DM1) 0.0946 42.5 42.6

P24 nd 1.79 1.79

Sum of additional componentsa) 1.32 16.8 18.2

Total components detected 3.85 76.1 80.0

Lost during sample processing 0.303 8.37 8.67

Total components in original sample 4.15b 84.5c 88.7

Carcass 13.8d

Cage wash 2.55e

nd: not detected a: Corresponds to the sum of the residual radioactivity measured in each radio-chromatograms b: SD was 0.462;CV was 11.1% c: SD was 3.12; CV was 3.69% d: measured after 168 h; SD was 0.482, CV was 3.49% e: measured after 168 h; SD was 0.818, CV was 32%


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Figure 1In

tens

ity

D0 3.3 %

D1 11.7 %

D2 19.7 %

D3 22.7 %

D4 18.2 %

D5 12.5 %

D6 6.3 % D7

3.9 % D8 1.8 %

150 151 152 153 154 155 156 157 158 159

Inte

nsity

Mass (kDa)

D0 1.2 %

D1 10.4 %

D2 20.5 %

D3 25.2 %

D4 19.9 %

D5 13.6 % D6

5.8 % D7 2.6 %

D8 0.8 %

Day 1

Day 5


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Figure 2

0 10 20 30 40 50 60 70

Rad

ioac

tivity

Retention time (min.)

T1h

T24h

T96h

T168h

Agg

rega

tes

/ Com

plex

es

0.05

0.5

5

0 48 96 144

Mean total radiolabeled components(LSC)Mean total ADC (ELISA)

Mean total Ab (ELISA)

Mean total ADC (SEC-RA)

Time (h)

[mM

]

[3H

]DM

1-M

CC

-Ab


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Figure 4R

adio

activ

ity

AP1

M7M6

M1

0 5 10 15 20 25 30

Rad

ioac

tivity


B

P1M6

M5

M1

P1.8


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Figure 5

Rad

ioac

tivity

A

[3H]DM1-MCC-Ab

0 10 20 30 40 50

Rad

ioac

tivity


B M2

0 5 10 15

Rad

ioac

tivity


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Figure 6R

adio

activ

ity

A

P1

M7

M2

Residual radioactivity

M1

M6

0 5 10 15 20 25

Rad

ioac

tivity


B

P1

M2

M5

M1

M6

Residual radioactivity


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Figure 7R

adio

activ

ity

A

P1 M7

M2

M1

P24

0 5 10 15 20 25 30

Rad

ioac

tivity


B

P1

M2M5

M1

P24


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��

� ��

�

��

��

��

�

��

�

��

�

�

��

�

��

�

��

�

�

M1 (MCC-DM1) s, u, f

M2 (LYS-MCC-DM1) s, u, f, t, l

DM:

DAR: 3.3n

��

� ��

�

��

�

��

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�

��

��

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M7: s, u, f

M4 (DM1) s, u, f, t

�

��

�

�

��

�

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�

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M6 (Maysine) s

�� identified after derivatization as maleimide conjugate

DS: dosing solution

s: serum

u: urine

f: feces

t: tumor

l: liver

[ H]DM1-LNL897: DS, s, t

Figure 8

��

� ��

��

� ��

+[Cysteine]


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0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0

0 . 0 0 00 . 0 5 00 . 1 0 00 . 1 5 00 . 2 0 00 . 2 5 00 . 3 0 00 . 3 5 0

L C - R A L E S A - µ L C - M S / M S

T i m e p o s t - d o s e ( h )

Conc

entra

tion L

C-RA

(nmo

l/g)

F i g u r e 9

05 0 01 0 0 01 5 0 02 0 0 02 5 0 03 0 0 0

LESA

-µLC-

MS/M

S pea

k area

(cps

)


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