Impact of Drug Conjugation on Pharmacokinetics and Tissue

ARTICLE pubs.acs.org/bc

Impact of Drug Conjugation on Pharmacokinetics and Tissue Distribution of Anti-STEAP1 Antibody
Drug Conjugates in Rats C. Andrew Boswell,† Eduardo E. Mundo,† Crystal Zhang,† Daniela Bumbaca,† Nicole R. Valle,‡ Katherine R. Kozak,§ Aimee Fourie,§ Josefa Chuh,§ Neelima Koppada,|| Ola Saad,|| Herman Gill,^ Ben-Quan Shen,† Bonnee Rubinfeld,# Jay Tibbitts,† Surinder Kaur,|| Frank-Peter Theil,† Paul J. Fielder,† Leslie A. Khawli,*,† and Kedan Lin*,† Department of Pharmacokinetic & Pharmacodynamic Sciences ‡Department of Investigative Safety Assessment §Department of Assay & Automation Technology Department of BioAnalytical Research & Development ^Department of Biomedical Imaging and # Department of Cancer Targets, Genentech Research & Early Development, South San Francisco, California 94080, United States )

†

bS Supporting Information ABSTRACT: Antibody
drug conjugates (ADCs) are designed to combine the exquisite specificity of antibodies to target tumor antigens with the cytotoxic potency of chemotherapeutic drugs. In addition to the general chemical stability of the linker, a thorough understanding of the relationship between ADC composition and biological disposition is necessary to ensure that the therapeutic window is not compromised by altered pharmacokinetics (PK), tissue distribution, and/or potential organ toxicity. The six-transmembrane epithelial antigen of prostate 1 (STEAP1) is being pursued as a tumor antigen target. To assess the role of ADC composition in PK, we evaluated plasma and tissue PK profiles in rats, following a single dose, of a humanized anti-STEAP1 IgG1 antibody, a thio-antiSTEAP1 (ThioMab) variant, and two corresponding thioether-linked monomethylauristatin E (MMAE) drug conjugates modified through interchain disulfide cysteine residues (ADC) and engineered cysteines (TDC), respectively. Plasma PK of total antibody measured by enzyme-linked immunosorbent assay (ELISA) revealed ∼45% faster clearance for the ADC relative to the parent antibody, but no apparent difference in clearance between the TDC and unconjugated parent ThioMab. Total antibody clearances of the two unconjugated antibodies were similar, suggesting minimal effects on PK from cysteine mutation. An ELISA specific for MMAE-conjugated antibody indicated that the ADC cleared more rapidly than the TDC, but total antibody ELISA showed comparable clearance for the two drug conjugates. Furthermore, consistent with relative drug load, the ADC had a greater magnitude of drug deconjugation than the TDC in terms of free plasma MMAE levels. Antibody conjugation had a noticeable, albeit minor, impact on tissue distribution with a general trend toward increased hepatic uptake and reduced levels in other highly vascularized organs. Liver uptakes of ADC and TDC at 5 days postinjection were 2-fold and 1.3-fold higher, respectively, relative to the unmodified antibodies. Taken together, these results indicate that the degree of overall structural modification in anti-STEAP1MMAE conjugates has a corresponding level of impact on both PK and tissue distribution.

’ INTRODUCTION Prostate cancer represents one-quarter of newly diagnosed cancer cases in men and is surpassed only by lung cancer in the number of male deaths.1 Despite significant efforts toward improving the diagnosis2,3 and treatment4
7 of prostate tumors, there are few therapeutic options for metastatic prostate cancer, and developing effective treatments remains a critical priority. Antibody-based therapies are currently being developed against specific antigens that are expressed in prostate tumors. One such molecular target is the six-transmembrane epithelial antigen of the prostate 1 (STEAP1),8 a cell-surface antigen that is overexpressed in the majority of human epithelial prostate cancers, but with restricted expression in normal tissues.8
10 Although the normal functional role for STEAP1 is not fully understood, its r 2011 American Chemical Society

restricted expression makes it an ideal antigen for antibodymediated therapy. The development of antibody
drug conjugates (ADCs) is a particularly promising therapeutic approach that combines the antigen targeting specificity of monoclonal antibodies with the cytotoxic potency of chemotherapeutic drugs.11
20 Conjugation of cytotoxic drugs to antibodies can be achieved either through lysine side-chain amines or through cysteine sulfhydryl groups that are typically activated by reduction of interchain disulfide bonds. As such, a humanized IgG1 anti-STEAP1 conventional Received: April 27, 2011 Revised: August 25, 2011 Published: September 13, 2011 1994

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Figure 1. (a) Conceptual depiction of sites of anti-STEAP1 modification. The auristatin linker-drug, MC-vc-PAB-MMAE (green), was conjugated randomly through thioether bonds to cysteine thiols at the hinge region (left) or site-specifically to exactly two engineered thiols (center), while the radiometal chelate, DOTA (yellow), was randomly conjugated to lysine residues through amide bonds (right). (b) A space-filling three-dimensional model of an IgG1 (PDB code: 1igy) conjugated to a single molecule each of DOTA (left) and MC-vc-PAB-MMAE (right) is shown to depict the relative molecular sizes of the antibody, linker-drug, and chelate. Both MMAE and DOTA (without In3+) were energy-minimized separately using the MM2 force field within CambridgeSoft Chem3D Pro version 7.0.0, exported as a PDB file, and merged with the IgG in the PyMOL Molecular Graphics System.49 DOTA and MMAE are conjugated to LYS115 and CYS114 (mutated from ALA114 in 1igy), respectively. Carbons, oxygens, and nitrogens in DOTA and MMAE are colored white, blue, and red, respectively. Heavy chains bearing DOTA and MMAE are colored in yellow and blue, respectively.

antibody can be conjugated through cysteine residues to a potent antimitotic auristatin drug, monomethyl auristatin E (MMAE) via a protease-labile linker, maleimidocaproyl-valine-citrulline-p-

aminobenzyloxycarbonyl (MC-vc-PAB, abbreviated as -vchenceforth).21
23 The peptidic vc-MMAE linker is designed to be stable in plasma, and has much improved stability in systemic 1995

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Bioconjugate Chemistry circulation compared to earlier chemically labile linkers such as hydrazone.22 Drug conjugation in the presence of four sets of interchain disulfide bonds gives rise to a heterogeneous ADC mixture that can be described in terms of a drug to antibody ratio (DAR) distribution and an average DAR. For instance, the antiSTEAP1 ADC with an average DAR of 4 (illustrated in Figure 1) is just one possible molecular species of a mixture that may be composed of zero to eight drugs per antibody covalently attached via the -vc- linker. This heterogeneity may ultimately lead to different PK, efficacy, and toxicity properties of each fraction; for example, fractions with higher DAR have, in some cases, been reported to clear more rapidly and contributed to more severe toxicity.24 Other reports have demonstrated, however, similar efficacy, tolerability, and PK between preparations having heterogeneous (0
8) and homogeneous (4) DAR.25 Recently, to control the heterogeneity of ADCs and to explore a novel strategy for potentially increasing the therapeutic window, a novel thio-anti-STEAP1 drug conjugate (TDC) was developed with site-specific conjugation through two engineered reactive thiols using ThioMab antibody technology.15 The molecular structures in Figure 1a illustrate two ADC variants that differ in how the drug is covalently attached to anti-STEAP1. The linker-drug (vc-MMAE) is conjugated to the antibody through a thioether bond between the linker maleimide moiety and (i) a cysteine thiol that normally forms the interchain disulfide bond at the hinge region in the anti-STEAP1 ADC (Figure 1a left) or (ii) a site-specific, engineered thiol in the antiSTEAP1 TDC (Figure 1a center). The relative sizes of the antibody and MMAE are depicted in Figure 1b. Note that the theoretical drug load for the anti-STEAP1 ADC is a heterogeneous distribution of 0, 2, 4, 6, and 8 DAR, while the TDC is homogeneous with a DAR of 2. Anti-STEAP1 ADCs, including TDCs, have exhibited antitumor activity in explant and xenograft models and are being investigated for the treatment of prostate cancer.26 The present study investigates the potential impact of MMAE drug conjugation on antibody pharmacokinetics (PK) and tissue distribution of ADCs prepared through interchain thiol residues in a monoclonal antibody (mAb) or through site-specific, engineered cysteines in a ThioMab.15 The anti-STEAP1 antibody and corresponding ADCs do not cross react with rat STEAP1; therefore, this species is suitable for evaluation of antigen-independent PK, biodistribution, and toxicity. In this context, the PK of four different molecular entities and biodistributions of their radiolabeled (via indium-111-DOTA, Figure 1) counterparts were compared following a single intravenous dose in rats. The rationale for selection of test molecules was to determine the potential effects of varying the site of drug conjugation and/or the DAR characteristics relative to unconjugated antibodies. Furthermore, we compared the PK profiles measured by both gamma counting and total antibody ELISA in order to bridge radiometric data from both terminal tissue distribution harvests and nonradioactive PK studies.

’ EXPERIMENTAL PROCEDURES Antibody/ThioMab Production and MMAE Conjugation. The anti-STEAP1 antibody and anti-STEAP1-vc-MMAE conjugate (ADC) were prepared as previously described.22 Briefly, the maleimido drug derivative was incubated with reduced mAbs for 1 h at 4 C, followed by quenching with excess cysteine. Methods for construction and production of the thio-anti-STEAP1 (ThioMab) variant were reported previously.15 Briefly, a cysteine residue was

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engineered at Ala114 position of the anti-STEAP1 heavy chain to produce its ThioMab variant from which the thio-anti-STEAP1-vcMMAE conjugate (TDC) was produced. The DAR for each immunoconjugate was determined by hydrophobic interaction chromatography analysis as described earlier.15 DOTA Conjugation and Characterization. Proteins were conjugated to 1,4,7,10-tetraazacyclododecane-N,N0 ,N00 ,N000 -tetraacetic acid (DOTA) for indium-111 (111In) complexation by random modification of lysine residues (Figure 1a right). Aliquots containing 2
6 mg of the fully human antibodies antiSTEAP1, thio-anti-STEAP1, and the corresponding vc-MMAE conjugates were exchanged from formulation buffer into aqueous 50 mM sodium borate, pH 8.5 using illustra NAP5 columns (GE Healthcare Life Sciences, Piscataway, NJ). A quantity of 5 mol equiv of the N-hydroxysuccinimidyl ester of DOTA in 0.68
1.66 μL of dimethylformamide was added to the 600 μL boratebuffered protein solutions. Reaction mixtures were gently agitated (300 rpm) for 1 h at 37 C on a Thermomixer (Eppendorf North America, Hauppauge, NY). Reaction was terminated by promptly applying the mixtures to NAP5 columns pre-equilibrated in aqueous 0.3 M ammonium acetate buffer, pH 7.0. The resulting purified DOTA-mAb conjugates were stored at 4 C. The binding specificity and affinity to STEAP1 for all four antibody variants were characterized by total antibody ELISA (see below). Determination of the average number of covalently attached chelates for each DOTA conjugate by radiometric assay was performed by modification of previously reported procedures.27,28 An aliquot of 10 μL of each mAb-DOTA conjugate (2
4 mg/mL in 0.3 M ammonium acetate pH 7.0) was added to 10 μL of a standardized InCl3 solution (323 μM, 3.23 nmol, containing >150 000 cpm/μL of 111InCl). The reaction was incubated at 37 C for 3 h, after which 5 μL of 50 mM EDTA and 75 μL of 0.3 M ammonium acetate pH 7 were added, followed by further incubation for 5 min at 25 C. The entire mixture was loaded onto a NAP5 column pre-equilibrated in PBS and allowed to settle, followed by an additional 400 μL of PBS. The radiolabeled antibody fraction was eluted separately with an additional 500 μL of PBS. The radioactivity in the eluent and remaining on the NAP5 column was measured using a 1480 WIZARD Gamma Counter (Wallac, Turku, Finland) in the energy window for the 245 keV photon peak of 111In and with automatic background and decay correction. The number of chelates per antibody molecule was calculated from the ratio of counts in the eluted product to the total number of counts using the method of Meares and co-workers.27 The number of chelates per antibody was also estimated by LC-MS. Prior to analysis, all protein samples (100 μL, 0.25
0.5 mg/mL) were deglycosylated overnight at 37 C using 2 μL peptide N-glycosidase (PNGase F, Prozyme). Anti-STEAP1 ADC samples were also further subjected to a reduction step using 10 μL of 200 mM dithiothreitol (DTT). Samples were injected onto a Pepswift Monolithic PS-DVB column (0.5  5 mm, ID 500 μm, Dionex) using an HTS PAL autosampler (LEAP Technologies) with a cooling stack set at 4 C. The column temperature was maintained at 70 C using a column heater (Keystone Scientific). The LC separation was conducted using an Express LC-100 liquid chromatography system (Eksigent Technologies, Dublin, CA) at a flow rate of 15 μL/min. Mobile phase A was water with 0.1% formic acid, and mobile phase B was acetonitrile with 0.1% formic acid. The gradient condition was maintained at 2% B for 4 min, ramped to 40% B in 8 min, kept at 40% B for 3 min, increased to 100% B in 1.5 min, 1996

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Bioconjugate Chemistry retained at 100% B for 1 min, returned back to 2% B in 0.8 min, and finally equilibrated for 0.7 min before the next injection (for a total run time of 15 min). For the first 6 min, the LC flow was diverted to waste. The eluate was then directed to a Q-STAR XL quadrupole time-of-flight (TOF) mass spectrometer (AB Sciex, Foster City, CA) operated with a turbo ionspray source maintained at 200 C in the positive ion mode. The declustering potential (DP) and focusing potential (FP) were optimized at 100 and 300 V, respectively. Data analysis was performed using the Analyst QS 1.1 software and a Bayesian Protein Reconstruct algorithm for mass spectral deconvolution (AB Sciex, Foster City, CA). Radiochemistry. A 2 μL (820 μCi; 30.3 MBq) aliquot of 111 InCl (MDS Nordion, Ottawa, ON) was added to a 20 μL aliquot of each ammonium acetate-buffered DOTA
protein conjugate. Reaction mixtures were gently agitated (300 rpm) for 1 h at 37 C on a Thermomixer. A 5 μL aliquot of 50 mM aqueous EDTA challenge solution was added, followed by an additional 73 μL aliquot of aqueous 0.3 M ammonium acetate buffer, pH 7.0. Each radiolabeled protein was purified using NAP5 columns pre-equilibrated in PBS. Purity of each radioimmunoconjugate was assessed by size-exclusion radiometric high-performance liquid chromatography (HPLC) (isocratic, PBS, 0.5 mL/min) on an Agilent 1100 series HPLC system operated through ChemStation software and equipped with a Biosep-SEC-S 3000 column (Phenomenex) and a raytest Ramona 90 radioactive flow monitor. Pharmacokinetic Studies. All experimental animal studies were conducted according to protocols that were reviewed and approved by the Institutional Animal Care and Use Committees (IACUC) of Genentech Laboratory Animal Research (LAR). Male Sprague
Dawley rats ranging from 8 to 10 weeks old and weighing approximately 250
300 g at the initiation of the study were randomly assigned to 4 groups (n = 4 per group), and administered an intravenous bolus (5 mg/kg) of test article. Blood samples were collected from each animal via the femoral vein for up to 28 days and used to derive plasma for total and conjugated antibody concentration determination using an ELISA and the concentration of free MMAE released in vivo from anti-STEAP1 conjugates in plasma using LC-MS/MS detection (see below). Plasma concentration
time data were used to estimate relevant PK parameters using WinNonlin software (v 5.2.1 Pharsight Corporation, Mountain View, CA). To better compare the profile between radiometry and ELISA data, radiometric (In-111) blood PK data (see below) were normalized to the same dose level. Tissue Distribution. Juvenile male Sprague
Dawley rats (Harlan) with weight range of 75
100 g received a single bolus intravenous injection of 111In-labeled conjugates. Dosing solutions were prepared by mixing the 111In-DOTA-labeled test articles with the corresponding non-DOTA-conjugated molecules to achieve a total protein dose of 10 mg/kg and radioactive dose of approximately 400 μCi/kg (14.8 MBq/kg) in no more than 200 μL of PBS. A 500 μL aliquot of whole blood was terminally collected at 1 h, 1 day, 2 days, 5 days, and 7 days in lithium heparinized tubes via cardiac puncture under inhaled isoflurane anesthesia. The following tissues were subsequently harvested: lungs, liver, kidneys, heart, spleen, right femur, gastrocnemius muscle, stomach, small intestine, and large intestine. All tissues were rinsed with PBS, blot-dried, weighed, frozen on dry ice, and stored at
70 C. Blood, tissues, and 5 μL aliquots of dosing solution standards were counted for radioactivity using a 1480 WIZARD Gamma Counter in the energy window for the

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245-keV photon peak of 111In and with automatic background and decay correction. Average counts per minute (cpm) were converted to percentage of injected dose per gram of tissue (%ID/g) and plotted with standard deviations. Statistical significance was determined by one-way ANOVA followed by Tukey’s post-test in GraphPad Prism v 5.04. Total Antibody ELISA. Nunc MaxiSorp 384-well plates (Nalge Nunc International, Rochester, NY) were coated with either antiidiotypic antibody 5093 (Genentech, South San Francisco, CA) or donkey antihuman Fc (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and incubated overnight at 4 C. The plates were washed 3 times with 0.05% Tween-20 in PBS buffer (pH 7.4). Diluted standards plasma samples were added to the wells and incubated on a shaker for 2 h at room temperature. The plates were washed 6 times and a detection antibody, either goat antihuman IgG antibody conjugated to horseradish peroxidase (Bethyl Laboratories, Montgomery, TX) or goat antihuman Fc conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), was added to the wells and incubated on a shaker for 1 h at room temperature. The plates were washed 6 times and developed using TMB peroxidase substrate (Moss Inc., Pasadena, Maryland). Both assay ranges were 0.164
40 ng/mL with a minimum dilution of 1:100 (limit of detection = 16.4 ng/mL). TDC assays were characterized as having acceptable (80-120%) DAR analyte recovery (data not shown). For ADC assays, DAR reagents were not available to characterize analyte recovery at the time of the original study sample analysis. However, data reported from the assay herein were confirmed using a more recent assay where DAR recovery was characterized and found to be acceptable) 80
120%; data not shown). Conjugated Antibody ELISA. Nunc MaxiSorp 384-well plates were coated with anti-MMAE antibody (Seattle Genetics Inc., Bothell, WA) and incubated overnight at 4 C. The plates were washed 3 times with 0.05% Tween-20 in PBS buffer (pH 7.4). Diluted standards and the ADC or TDC plasma samples were added to the wells and incubated for 2 h at room temperature. The plates were washed 6 times and a detection antibody, either goat antihuman IgG antibody conjugated to horseradish peroxidase (Bethyl Laboratories, Montgomery, TX) or goat antihuman Fc conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), was added on a shaker for 1 h at room temperature. All plates were developed using TMB peroxidase substrate. The ADC assay range was 0.0655-16 ng/mL with a minimum dilution of 1:100 (limit of detection = 6.6 ng/mL). The TDC range was 0.164
40 ng/mL with a minimum dilution of 1:100 (limit of detection = 16.4 ng/mL). TDC and ADC assays were characterized for DAR analyte recovery as described for the total antibody ELISA. Free MMAE Assay. The concentration of free MMAE in plasma was determined by liquid chromatography tandem mass spectrometry (LC-MS/MS). Briefly, plasma samples were protein precipitated with 100 μL of 80/20 acetonitrile/water containing 2 nM monomethylauristatin F (MMAF) as an internal standard, and analyzed for MMAE by TurboIon Spray using an API 3000 mass spectrometer (Applied Biosystems, Foster City, CA). The HPLC system used for analysis was a Shimadzu HPLC LC-10Avp system (Shimadzu Scientific Instruments, Columbia, MD), equipped with a Short Hot Pocket column heater (Keystone Scientific, Inc., Bellefonte, PA) and analytical column (Phenomenex Synergi MAX-RP 80A, C12, 4 μm, 2.0  50 mm) 1997

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Figure 2. Representative hydrophobic interaction chromatography (HIC) of anti-STEAP1 ADC with average drug-to-antibody ratio (DAR) of approximately 3.5. The peak assignments are consistent with the conjugation of the linker
drug being primarily limited to the interchain cysteines of the antibody and leading to a DAR distribution of mainly 0, 2, 4, 6, or 8.

Table 1. Summary of Analytical Data Obtained during Characterization, DOTA Conjugation, and 111In Radiolabeling of AntiSTEAP1 (mAb), Thio-anti-STEAP1 (ThioMab), and the Two Corresponding MMAE Conjugates (ADC and TDC) test material

# MMAE (HIC)a

# DOTA (radiometric)b

# DOTA (MS)c

ELISA recovery (%)d

111

In labeling yield (%)e

In labeling purity (%)f

111

mAb

N/A

2.20 ( 0.148

2.6

73

82

98

ADC ThioMab

3.1 N/A

1.50 ( 0.0787* 2.47 ( 0.273

1.7 3.2

73 80

61 84

97 98

1.67 ( 0.139*

2.3

82

78

99

TDC

1.7

a

HIC: hydrophobic interaction chromatography. b Performed in triplicate. c Estimated from differences between mass spectrometry (MS) peaks before and after DOTA conjugation. d Measured by ELISA after DOTA conjugation to verify immunoreactivity. e Radiolabeling performed using ∼30 MBq 111 InCl and ∼40 mg DOTA-mAb in 0.3 M ammonium acetate pH 7 at 37 C for 1 h. f Derived by integration of size exclusion radiochromatogram. * P < 0.05 vs mAb by unpaired t test.

at 50 C. Multiple reaction monitoring scan mode was used for quantitation. For MMAE quantitation, transition 732.7/170.3 was monitored for MMAF (internal standard) and 718.7/152.2 for MMAE. The LC-MS/MS assay had a lower limit of quantitation of 0.018 ng/mL (0.025 nM) with linearity demonstrable up to 18 ng/mL (25 nM) using a sample volume of 0.025 mL.

’ RESULTS Antibody/ThioMab Production and MMAE Conjugation. The presence of a heterogeneous mixture of DAR species was demonstrated by hydrophobic interaction chromatography (HIC) as shown in a representative chromatogram for the antiSTEAP1 ADC (Figure 2). The calculated average DARs for the ADC and TDC used in PK and tissue distribution studies were 3.1 and 1.7 per mAb, respectively (Table 1). DOTA Conjugation and Characterization. All four DOTA conjugates retained antigen binding, measured as percent recovery in a STEAP1-specific ELISA (Table 1). Radiometric measurement of the average number of DOTA chelates attached per antibody molecule gave values ranging from 1.5 to 1.7 for the drug conjugates and from 2.2 to 2.5 for the non-drug-conjugated molecules (Table 1). These values were in rough agreement with estimates based on shifts of mass peaks before and after DOTA conjugation, which showed a range of 1.7
2.3 for the drug conjugates and 2.6
3.2 for the non-drug-conjugated molecules (Table 1; see also Supporting Information).

Radiochemistry. Slightly lower radiochemical yields (Table 1) were obtained for the ADC (61%) and TDC (78%) than for the corresponding non-drug-conjugated antibody and ThioMab (82% and 84%, respectively). Size-exclusion HPLC demonstrated radiochemically pure (g97%) products (Table 1) with no evidence of unconjugated 111In (see Supporting Information). Pharmacokinetic Studies. The anti-STEAP1 total antibody ELISA quantifies the antibody moiety irrespective of drug conjugation status, as the assay format is designed to capture both unconjugated antibody and ADC variants. The anti-STEAP1 conventional mAb and ThioMab showed similar PK profiles (Figure 3a), indicating that cysteine mutations had little impact on antibody clearance. Indeed, the clearances of the conventional mAb and ThioMab at rates of 7.27 ( 1.71 and 9.65 ( 2.33 mL/ day/kg, respectively, were comparable (Table 2). The two drug conjugates had similar clearances, with 10.5 ( 1.42 and 9.56 ( 2.53 mL/day/kg for the ADC and TDC, respectively. The ADC total antibody cleared approximately 45% more rapidly than the corresponding unconjugated mAb at rates of 10.5 ( 1.42 and 7.27 ( 1.71 mL/day/kg (P < 0.05), respectively. In contrast, the TDC had a similar clearance compared with the ThioMab at rates of 9.56 ( 2.53 and 9.65 ( 2.33 mL/day/kg, respectively. This reflects the differential impact on the overall clearance of the antibody depending on site and degree of conjugation. The anti-STEAP1 conjugate ELISA format is designed to measure any anti-STEAP1 antibody conjugated with one or more MMAE drugs. This assay is not sensitive to incremental 1998

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changes in drug load, but can estimate the overall complete drug loss from the ADC or TDC as unconjugated mAb is not measured. The anti-STEAP1 ADC and TDC showed markedly different conjugate PK profiles (Figure 4a). A summary of the PK parameters for both anti-STEAP1 total antibody and conjugate in plasma is presented in Table 2. The anti-STEAP1 ADC cleared 3 times as fast in terms of conjugated antibody relative to total antibody (33.6 ( 4.27 and 10.5 ( 1.42 mL/day/kg, respectively). However, it is difficult to distinguish whether biotransformation of

ADC is dominated by MMAE release (deconjugation) versus proteolytic degradation of the antibody itself (total antibody clearance). In contrast, the TDC showed only 1.3 times faster clearance in terms of conjugated antibody compared to total antibody clearance (12.5 ( 2.41 and 9.56 ( 2.53 mL/day/kg, respectively). The clearance of the anti-STEAP1 ADC in terms of conjugated antibody was almost three times faster than that of the TDC (33.6 ( 4.27 and 12.5 ( 2.41 mL/day/kg, respectively). Overall, the volume of distribution (V1) for all groups approximated to plasma volume in rats (31 mL/kg for a 250 g rat29), ranging from 39.4 ( 0.704 to 47.3 ( 8.82 mL/kg. Plasma concentration
time profiles of free MMAE, measured by LC-MS/MS, following administration of the two MMAE-conjugated

Figure 3. Mean total antibody concentrations in plasma of antiSTEAP1, thio anti-STEAP1, and the two corresponding MMAE drug conjugates in male rats following intravenous administration at 5 mg/kg. Error bars are standard deviations. Data from (a) ELISA-derived PK (solid lines) only and (b) both PK and radiometric tissue distribution (dotted lines) are included. Data for 111In-labeled molecules in (b) was dose normalized to 5 mg/kg for comparison with ELISA-derived data.

Figure 4. (a) Mean total and conjugated antibody concentrations (by ELISA) in plasma of anti-STEAP1 ADC and TDC in male rats following intravenous administration at 5 mg/kg. (b) Mean free MMAE concentrations (by LC-MS/MS) in plasma following administration of antiSTEAP1 ADC and TDC in male rats. Error bars in both (a) and (b) are standard deviations.

Table 2. Summary of Total Anti-STEAP1 Antibody Pharmacokinetics in Plasma of Anti-STEAP1 (mAb), Thio-anti-STEAP1 (ThioMab), and the Two Corresponding MMAE Drug Conjugates (ADC and TDC) in Male Rats (n =4) test material

dose (mg/kg)

analyte

CLa (mL/day/kg)

V1 (mL/kg)

mAb

5

total antibody

7.27 ( 1.71

41.4 ( 2.31

ADC

5

total antibody

10.5 ( 1.42*

44.8 ( 6.00

conjugated antibody

33.6 ( 4.27*

47.3 ( 8.82

ThioMab

5

total antibody

9.65 ( 2.33

39.4 ( 0.704

TDC

5

total antibody

9.56 ( 2.53

42.9 ( 2.04*

conjugated antibody

12.5 ( 2.41

a

42.2 ( 1.79*

*

*

Abbreviations: CL, clearance; V1, volume of distribution of the central compartment (i.e., volume of initial dilution compartment). P < 0.05 vs mAb by unpaired t test. 1999

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Figure 5. Tissue distribution of 111In-DOTA-labeled anti-Steap1 (black), thio-anti-Steap1 (gray), and the corresponding MMAE conjugates (white and striped, respectively) in rats at (a) 24 h and (b) 120 h after intravenous injection. Data are presented as percentage of injected dose per gram of tissue (mean %ID/g ( SD for 3 rats per group). Statistically significant differences are indicated by brackets (*P < 0.05).

anti-STEAP1 antibodies are presented in Figure 4b. Although observed free MMAE concentrations were generally very low (∼0.2 ng/mL or less) and detected only up to day 4 postdose for both conjugated antibodies, the anti-STEAP1 ADC showed a higher concentration of plasma MMAE at time points ranging from 6 h through 4 days. The observed free MMAE exposure measured by both Cmax and AUC was approximately four times higher for the ADC than for the TDC (Cmax: 0.19 ( 0.02 vs 0.04 ( 0.02 ng/mL; AUC: 0.45 ( 0.05 vs 0.14 ( 0.03 ng/mL days, respectively). These differences seem to exceed the roughly 2-fold expected difference based on DAR; however, it is difficult to distinguish between deconjugation versus total antibody catabolism in explaining greater drug loss from the ADC relative to the TDC. Tissue Distribution. Radiometrically derived (dose-normalized) and ELISA-derived total antibody PK data were largely comparable (Figure 3b), indicating similar blood exposures of all four test articles. At 24 h postinjection, significantly (P < 0.05) higher hepatic uptake of 111In was observed for anti-STEAP1 ADC

(1.76 ( 0.0878) than for unconjugated anti-STEAP1 (1.10 ( 0.183%ID/g) (Figure 5a). Conversely, anti-STEAP1 TDC demonstrated lower cardiac uptake of 111In at 24 h (1.04 ( 0.0497%ID/g) than unconjugated anti-STEAP1 (1.34 ( 0.108%ID/g). At 120 h postinjection, elevated hepatic uptake of 111In for anti-STEAP1 ADC relative to unconjugated anti-STEAP1 was maintained (0.791 ( 0.0648 vs 0.386 ( 0.00617%ID/g) with a similar but less pronounced trend for anti-STEAP1 TDC and its corresponding unconjugated ThioMab (0.610 ( 0.0155 vs 0.454 ( 0.0157%ID/g) (Figure 5b). Significantly (P < 0.05) higher uptake for anti-STEAP1 ADC (1.24 ( 0.173) relative to anti-STEAP1 (0.925 ( 0.0888) was also observed at 5 d in spleen. Meanwhile, thio-anti-STEAP1 uptake in both heart (0.768 ( 0.0252%ID/g) and lungs (1.50 ( 0.188%ID/g) was higher than for all other variants, which ranged from 0.628 ( 0.0476 to 0.691 ( 0.0323%ID/g in heart and from 0.923 ( 0.131%ID/g to 0.962 ( 0.0975%ID/g in lungs. Similar trends 2000

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Bioconjugate Chemistry were observed in additional tissue distribution data at 1, 48, and 168 h postinjection (see Supporting Information).

’ DISCUSSION Considerable effort has been applied to understanding the PK,30,31 tissue distribution,20 metabolism,32
34 and pharmacologic effects19,35,36 of ADCs. Still, the inherent heterogeneity of these complex macromolecular entities remains a prominent challenge in understanding their properties in vivo during nonclinical development. For example, conjugation through interchain disulfides leads to ADCs with DAR ranging from 0 to 8, with each fraction potentially exhibiting a unique efficacy, PK, and toxicity profile. In addition, the existing analytical methods available for antibodies and small molecule drugs have inherent technical limitations for complex ADCs. For example, the performance of ELISA is sensitive to the heterogeneity of the analytes, and susceptible to variation in recovery of each DAR fraction.31 Assays used in this study demonstrated 80-120% DAR analyte recovery (data not shown); however, it is not known if these analytes are structurally identical to DAR species formed in vivo. The recent introduction of ThioMabs presents an opportunity to control the drug load and site of attachment of drug molecules, thereby limiting heterogeneity by conjugation through genetically engineered cysteine sites15 and potentially improving the overall PK characteristics. In this context, the current single-dose study encompasses the evaluation of an anti-STEAP1 antibody, the corresponding ThioMab variant possessing engineered cysteines, and two corresponding thioether-linked MMAE conjugates (ADC and TDC, respectively) to assess the impact of conjugation site and drug load on PK and tissue distribution. Plasma PK of anti-STEAP1 total antibody was measured by both ELISA and radiometric detection. Anti-STEAP1 total antibody clearances of the two unconjugated antibodies were similar, suggesting minimal impact on PK due to cysteine mutation (Figure 3). The PK profile comparison between unconjugated and conjugated variants reflects the impact of drug conjugation on PK and disposition of the antibodies. An increase in total antibody clearance of approximately 45% was observed for the anti-STEAP1 ADC, while no change in clearance was observed for the TDC, relative to their non-drug-conjugated counterparts. The exact magnitude of the increased clearance of the ADC may be somewhat affected by the inherent technical limitations of ELISA for mixture analysis; however, the trend is clearly apparent. Indeed, this trend toward faster plasma clearance of immunoconjugates relative to unconjugated antibodies (Figure 3a) is consistent with previous studies.24 This suggests that controlled conjugation with lower DAR and without structural disruption of disulfide bonds (i.e., TDC) leads to minimal alteration in antibody PK behaviors. However, generalizations to other ADC platforms37 should not be assumed, and the relationship between conjugation method and biological disposition must be independently established. In addition, a very good overall agreement existed between ELISA- and radiometrically derived PK, indicating that DOTA conjugation had no dramatic effect on disposition kinetics (Figure 3b). The enzyme labile, peptidic vc-MMAE linker is designed to be stable in plasma, and has greatly improved stability in systemic circulation and a superior safety profile compared to chemically labile linkers such as hydrazone.22 However, ADCs in blood may release some MMAE (i.e., deconjugate), resulting in changes in

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the DAR distribution of the drug-loaded species and the unconjugated antibody. To gain further mechanistic insights into ADC disposition and deconjugation, the plasma PK of conjugated antibody was measured by ELISA. The conjugate antibody assay format is designed to capture any antibody with at least one conjugated drug, such that the clearance encompasses both complete deconjugation and antibody clearance. Its comparison with total antibody clearance sheds light on the relative contribution of each process. The anti-STEAP1 ADC (average DAR = 3.1) showed a marked (approximately 3-fold) difference between conjugated antibody clearance and total antibody clearance. A similar trend between clearance rates of total antibody and conjugate for other immunoconjugates has been previously reported.38 In contrast, deconjugation had much less impact (roughly 1.3-fold) on the overall clearance in TDC (Table 2 and Figure 4a). Deconjugation from anti-STEAP1 ADC and TDC involves release of small molecules that may ultimately result in cytotoxic free MMAE in circulation. Very low levels of free MMAE (below 1 ng/mL) were detected for either ADC or TDC (Figure 4b). This, in part, reflects the stability of the linker and relatively limited deconjugation of MMAE from conjugated antibody; it is also likely that the result of the rapid distribution and clearance rates of MMAE in relation to its production. Alternatively, there may be additional drug catabolites other than free MMAE (e.g., Cys-MC-vc-PAB-MMAE) that are not detected by the MMAEspecific free drug assay. Although the absolute MMAE concentrations in plasma are very low, a correlation may exist between stability and MMAE levels in plasma. In this case, greater than 3-fold (based on AUC) higher free MMAE plasma levels were observed compared to the TDC. However, the higher initial drug load (roughly 2-fold) in the ADC likely contributes to the higher level of MMAE observed in plasma. Nevertheless, one plausible explanation for these observations is that conjugation through a precise number (two) of engineered cysteines induced fewer disturbances in the overall antibody structure relative to the unmodified antibody and a correspondingly more stable molecule. Since most ADC targets are selected for their low and restricted expression in normal tissues, exploitation of the rat as a nonbinding species provides an initial assessment of antigenindependent disposition of ADCs. Overall, tissue distribution trends were similar for all four radiolabeled antibody platforms at all time points and were consistent with the expected behavior for a typical nonbinding humanized antibody in rats (Figure 5, see also Supporting Information). Concentrations in blood and in all tissues decreased over time, with the exception of kidneys, where nearly constant uptake values were sustained. This is likely due to the continual clearance of low molecular weight 111In-labeled metabolites (e.g., 111In-DOTA-lysine) being generated in liver and in other sites of IgG catabolism.39 Elevated hepatic uptake for anti-STEAP1 ADC and TDC relative to the non-drugconjugated proteins may be rationalized by higher hydrophobicities of the drug conjugates, resulting in greater reticuloendothelial system clearance. Blood-corrected uptake for liver and other tissues may be calculated from the %ID/g values reported herein based on reported fractional vascular volumes in rodents; however, this correction would affect antibodies and drug conjugates to the same extent due to similar blood exposures.40,41 Robust characterization of all four derivatives was necessary in order to ensure that observed differences, if any, in biological disposition were indeed due to drug conjugation, as opposed to inconsistencies in radiolabeling (Table 1). The lower observed 2001

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Bioconjugate Chemistry radiochemical yields for the ADC and TDC than for the corresponding non-drug-conjugated molecules may reflect the influence of MMAE in sterically blocking lysine residues for DOTA conjugation. Although approximately 80
90 lysine residues are present in a typical IgG1,42 it is likely that many of these may not be solvent-accessible, especially following conjugation of MMAE. Even though slightly higher incorporation of DOTA was obtained for the non-MMAE-conjugated molecules, the effects should be minor given that much larger changes in isoelectric point (i.e., pI) would be necessary to invoke a difference in tissue distribution or PK.42 Furthermore, the ADC and TDC herein were, in fact, modified on average by approximately one fewer molecule of DOTA than the respective non-MMAE-conjugated counterparts as measured by both radiometric assay and mass spectrometry (Table 1). If the differences in hepatic uptake (Figure 5), for instance, were due to DOTA conjugation, then the trend observed herein would be inconsistent with a previous report that overconjugation with DOTA causes increased hepatic accumulation.43 It is therefore more likely that the conjugation of hydrophobic drug moieties, not the slightly lower DOTA conjugation yield, is responsible for the increased hepatic uptake of anti-STEAP1-MMAE conjugates relative to the mAb and ThioMab. Additionally, a STEAP1-specific ELISA confirmed that the immunoreactivities of the four variants were largely retained following DOTA conjugation (Table 1). The choice of 111In-DOTA as a probe was influenced by a desire to increase the likelihood of detecting any differences in tissuespecific uptake among the four tested platforms. Antibodies labeled with metal radionuclides via DOTA or other polyaminopolycarboxylate chelators tend to accumulate in antigen-expressing tissues following receptor-mediated endocytosis due to the residualizing properties of this charged, highly polar probe.44 The exploitation of DOTA as a carrier for 111In, a medium-energy gamma emitting radionuclide with a 2.7 day decay half-life,45 and other metallic radionuclides is well-documented.46
48 Conjugation with a small molecule drug through specific sites on mAbs can potentially alter mAb hydrophobicity, charge, polarity, and PK.42 ADCs with higher drug loads possess higher intrinsic potency; however, they may also exhibit faster blood clearance in terms of total antibody.24 However, it should be emphasized that each antibody and drug platform should be considered on a case-by-case basis; for instance, a lysine-modified trastuzumab-maytansinoid ADC (DAR 3.3) showed similar PK compared with the analogous TDC (DAR 1.8) modified through engineered cysteines.37 In addition, the increased release of free drug or toxic intermediates into systemic circulation or nontarget organs could potentially shrink the therapeutic window. The ability to define the DAR combined with the knowledge of PK and distribution will afford us new opportunities in optimizing ADCs. In conclusion, an anti-STEAP1 antibody, a ThioMab variant possessing engineered cysteines, and two corresponding thioether-linked MMAE conjugates were studied in order to assess the impact of drug conjugation methodology on PK and tissue distribution. MMAE conjugation through native cysteine thiols comprising interchain disulfide bonds (average DAR 3.1) resulted in an accelerated clearance of total antibody, while conjugation through engineered cysteine thiols (average DAR 1.7) led to only a marginal difference in clearance. In addition, an ELISA assay specific for MMAE-conjugated antibody indicated that the ADC had faster clearance than the TDC, which is due in part to a greater magnitude of drug deconjugation from the ADC. Although very low in both cases, free MMAE measured in plasma

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was higher for the ADC relative to the TDC. Overall, modification by either method had a noticeable, albeit minor, impact on tissue distribution with a general trend toward increased hepatic uptake and reduced levels in other highly vascular organs. Taken together, these results indicate that the degree of overall structural modification of the antibody in an ADC has a corresponding level of impact on the PK behavior and stability in vivo. Accordingly, in addition to understanding the general chemical stability of the linker, it is important to consider the degree of structural modification of ADCs, the DAR distributions, and the sites of modifications to gain insight into the potential impacts on PK behavior and distribution.

’ ASSOCIATED CONTENT

bS

Supporting Information. Size exclusion HPLC radiochromatograms of the radioimmunoconjugates used for tissue distribution. Additional tissue distribution data at 1 h, 2 days, and 7 days post intravenous injection. Mass spectrometry data before and after DOTA conjugation. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Authors

*Kedan Lin, Ph.D., MS 463A, Genentech, Inc., South San Francisco, CA 94080. Tel. 650-225-8090; E-mail: lin.kedan@ gene.com. *Leslie A. Khawli, Ph.D., MS 463A, Genentech, Inc., South San Francisco, CA 94080. Tel. 650-225-6509; E-mail: [email protected].

’ DISCLOSURE All authors are employees of Genentech, a member of the Roche Group, and hold financial interest in Roche. ’ ACKNOWLEDGMENT The authors would like to thank Michelle Schweiger, Kirsten Messick, Noore Kadri, Misia Bruski, Michael Reich, Jose Imperio, Sheila Ulufatu, Shannon Stainton, Cynthia Young, Nina Ljumanovic, Bernadette Johnstone, and Jason Ho for excellent animal studies support, Tracy Lou for assistance with study coordination, Jakub Baudys for assay support, and Paul Polakis for helpful scientific discussions. ’ REFERENCES (1) Jemal, A., Siegel, R., Xu, J., and Ward, E. (2010) Cancer statistics, 2010. CA Cancer J. Clin. 60, 277–300. (2) Hricak, H., Choyke, P. L., Eberhardt, S. C., Leibel, S. A., and Scardino, P. T. (2007) Imaging prostate cancer: a multidisciplinary perspective. Radiology 243, 28–53. (3) Ravizzini, G., Turkbey, B., Kurdziel, K., and Choyke, P. L. (2009) New horizons in prostate cancer imaging. Eur. J. Radiol. 70, 212–26. (4) Denmeade, S. R., and Isaacs, J. T. (2002) A history of prostate cancer treatment. Nat. Rev. Cancer 2, 389–96. (5) Lin, G. A., Aaronson, D. S., Knight, S. J., Carroll, P. R., and Dudley, R. A. (2009) Patient decision aids for prostate cancer treatment: a systematic review of the literature. CA Cancer J. Clin. 59, 379–90. (6) Rosenthal, S. A., and Sandler, H. M. (2010) Treatment strategies for high-risk locally advanced prostate cancer. Nat. Rev. Urol. 7, 31–8. (7) Shepard, D. R., and Raghavan, D. (2009) Innovations in the systemic therapy of prostate cancer. Nat. Rev. Clin. Oncol. 7, 13–21. 2002

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