Antibody-Maytansinoid Conjugates Are Activated in
`Targeted Cancer Cells by Lysosomal Degradation and
`Linker-Dependent Intracellular Processing
`(cid:160)
`Hans K. Erickson, Peter U. Park, Wayne C. Widdison, et al.
`Cancer Res(cid:160)(cid:160)
`
`2006;66:4426-4433.
`
`(cid:160)
`(cid:160)
`(cid:160)
`(cid:160)
`
`(cid:160)
`(cid:160)
`
`Updated version
`(cid:160)
`Supplementary
`Material
`(cid:160)
`
`Access the most recent version of this article at:
`http://cancerres.aacrjournals.org/content/66/8/4426
`(cid:160)
`Access the most recent supplemental material at:
`http://cancerres.aacrjournals.org/content/suppl/2006/04/14/66.8.4426.DC1.html
`(cid:160)
`
`
`
`
`
`Cited Articles
`(cid:160)
`Citing articles
`(cid:160)
`
`This article cites by 22 articles, 11 of which you can access for free at:
`
`http://cancerres.aacrjournals.org/content/66/8/4426.full.html#ref-list-1
`(cid:160)
`This article has been cited by 33 HighWire-hosted articles. Access the articles at:
`
` http://cancerres.aacrjournals.org/content/66/8/4426.full.html#related-urls
`(cid:160)
`
`E-mail alerts
`(cid:160)
`Reprints and
`Subscriptions
`(cid:160)
`Permissions
`(cid:160)
`
`Sign up to receive free email-alerts
`
` related to this article or journal.
`
`To order reprints of this article or to subscribe to the journal, contact the AACR Publications
`.
`pubs@aacr.org
`Department at
`(cid:160)
`To request permission to re-use all or part of this article, contact the AACR Publications
`.
`permissions@aacr.org
`Department at
`(cid:160)
`
`
`
`
`
`Downloaded from Downloaded from cancerres.aacrjournals.org on June 10, 2014. © 2006 American Association for Cancercancerres.aacrjournals.org on June 10, 2014. © 2006 American Association for Cancer
`
`
`
`
`
`Research. Research.
`
`IMMUNOGEN 2175, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`Research Article
`
`Antibody-Maytansinoid Conjugates Are Activated in Targeted Cancer
`Cells by Lysosomal Degradation and Linker-Dependent
`Intracellular Processing
`
`Hans K. Erickson, Peter U. Park, Wayne C. Widdison, Yelena V. Kovtun, Lisa M. Garrett,
`Karen Hoffman, Robert J. Lutz, Victor S. Goldmacher, and Walter A. Bla¨ttler
`
`ImmunoGen, Inc., Cambridge, Massachusetts
`
`Abstract
`Antibody-drug conjugates are targeted anticancer agents
`consisting of a cytotoxic drug covalently linked to a mono-
`clonal antibody for tumor antigen–specific activity. Once
`bound to the target cell-surface antigen, the conjugate must
`be processed to release an active form of the drug, which can
`reach its intracellular target. Here, we used both biological
`and biochemical methods to better define this process for
`antibody-maytansinoid conjugates. In particular, we exam-
`ined the metabolic fate in cells of huC242-maytansinoid
`conjugates containing either a disulfide linker (huC242-
`SPDB-DM4) or a thioether linker (huC242-SMCC-DM1). Using
`cell cycle analysis combined with lysosomal inhibitors, we
`showed that lysosomal processing is required for the activity
`of antibody-maytansinoid conjugates,
`irrespective of the
`linker. We also identified and characterized the released
`maytansinoid molecules from these conjugates, and measured
`their rate of release compared with the kinetics of cell cycle
`arrest. Both conjugates are efficiently degraded in lysosomes
`to yield metabolites consisting of the intact maytansinoid drug
`and linker attached to lysine. The lysine adduct is the sole
`metabolite from the thioether-linked conjugate. However, the
`lysine metabolite generated from the disulfide-linked conju-
`gate is reduced and S-methylated to yield the lipophilic and
`potently cytotoxic metabolite, S-methyl-DM4. These findings
`provide insight into the mechanism of action of antibody-
`maytansinoid conjugates in general, and more specifically,
`identify a biochemical mechanism that may account for the
`significantly enhanced antitumor efficacy observed with
`disulfide-linked conjugates. (Cancer Res 2006; 66(8): 4426-33)
`
`Introduction
`
`Antibody-drug conjugates are designed to selectively eliminate
`cancer cells that express the target cell surface antigen ( for
`recent reviews, see refs. 1–3). Several conjugates are currently in
`clinical evaluation (3) and one conjugate, gemtuzumab ozoga-
`micin, is licensed for the treatment of refractory acute myeloid
`leukemia (4).
`Antibody-drug conjugates are structurally composed of a
`monoclonal antibody that binds specifically to a target antigen, a
`
`Note: Supplementary data for this article are available at Cancer Research Online
`(http://cancerres.aacrjournals.org/).
`H.K. Erickson and P.U. Park contributed equally to this work.
`Requests for reprints: Hans K. Erickson, ImmunoGen, Inc., 128 Sidney Street,
`Cambridge, MA 02139. Phone: 617-995-2500; Fax: 617-995-2510; E-mail: hans.erickson@
`immunogen.com.
`I2006 American Association for Cancer Research.
`doi:10.1158/0008-5472.CAN-05-4489
`
`small chemical cytotoxic drug, and a linker that stably connects the
`two. We have developed conjugates that contain maytansinoid
`drugs, which are derivatives of the natural cytotoxic polyketide,
`maytansine (5). In particular, we have developed maytansinoid
`conjugates of the humanized monoclonal antibody, huC242, which
`binds to the CanAg antigen expressed on colorectal, pancreatic,
`and certain non–small cell lung cancers (6). During the preclinical
`development, we compared several conjugates of huC242 that
`differed in the chemical nature of the linker between huC242 and
`the maytansinoid and established that one conjugate, huC242-
`SPDB-DM4, had the highest activity in several human xenograft
`tumor models in mice and the widest therapeutic window as
`defined by the largest difference between the minimal effective
`dose and the maximal tolerated dose. Based on these results,
`huC242-SPDB-DM4 was selected for clinical development and is
`currently being tested in a phase I trial.
`Here, we report
`that
`the huC242-maytansinoid conjugate,
`huC242-SMCC-DM1, with a ‘‘noncleavable’’
`linker containing a
`thioether bond was at least as potent in vitro as the selected
`conjugate, huC242-SPDB-DM4, which has a ‘‘cleavable’’
`linker
`containing a disulfide bond. This was surprising because
`huC242-SMCC-DM1 displayed significantly lower in vivo activity
`in multiple xenograft tumor models. To investigate this conun-
`drum, we undertook a series of experiments to elucidate the
`mechanism of cell killing by the conjugates. The results delineate
`an activation process, for both conjugates, that requires lysosomal
`degradation of the antibody component of the conjugate. However,
`distinct maytansinoid metabolites produced by intracellular
`processing of huC242-SPDB-DM4 were identified and character-
`ized, providing a likely mechanism for its superior antitumor
`efficacy.
`
`Materials and Methods
`
`Materials. RPMI 1640 and glutamine were from Cambrex Bioscience
`(Walkersville, MD). Ultima Flo M scintillation fluid was from Perkin-Elmer
`Life and Analytical Sciences (Wellesley, MA) Gentamicin sulfate was from
`Life Technologies (Gaithersburg, MD). N-Ethylmaleimide and all other
`chemicals were obtained from Sigma (St. Louis, MO). All antibodies used,
`huC242, Tras, and huB4, are humanized IgG1 antibodies. The maytansi-
`q
`noids [3H]DM4, D-[3H]DM4, S-methyl-DM4,
`lysine-N
`-SMCC-DM1, and
`q
`-SPDB-DM4, and the conjugates, huC242-SPDB-[3H]DM4,
`lysine-N
`huC242-SMCC-[3H]DM4, Tras-SMCC-[3H]DM4, and huB4-SPDB-[3H]DM4,
`were prepared at ImmunoGen following published procedures (7, 8).
`Treatment of COLO 205 cells with antibody-drug conjugates. COLO
`
`205 cells (6  106) suspended in 3 mL culture medium containing an
`antibody-drug conjugate at a concentration of 10-7 mol/L of conjugated
`antibody were incubated at 37jC for 3 to 30 hours. The cells and the
`medium were then separated by centrifugation (2,000  g, 5 minutes). The
`supernatant (3 mL) was chilled on ice, mixed with 4 mL ice-cold acetone,
`and kept at 80jC for at least l hour or until further processing. The cells
`
`Cancer Res 2006; 66: (8). April 15, 2006
`
`4426
`
`www.aacrjournals.org
`
`Downloaded from
`
`on June 10, 2014. © 2006 American Association for Cancercancerres.aacrjournals.org
`
`
`Research.
`
`IMMUNOGEN 2175, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`were suspended in 3 mL HBBS buffer and sedimented by centrifugation,
`then resuspended in 0.3 mL TBS containing 0.5% bovine serum albumin.
`At
`this point,
`if alkylation was desired for capping of
`free thiols,
`N-ethylmaleimide was added to 7.5 mmol/L, and the solution was mixed
`and kept at room temperature for 30 minutes. The cell suspension was then
`mixed with 0.6 mL ice-cold acetone. The samples were placed at 80jC for
`at least 1 hour or until further processing. Precipitated protein was removed
`by centrifugation at 2,500  g and the supernatants were acidified with
`
`5% acetic acid and evaporated to dryness. The samples were dissolved in
`0.12 mL of 20% aqueous CH3CN containing 0.025% trifluoroacetic acid
`(TFA), aliquots of 0.1 mL were submitted to high-performance liquid
`chromatography (HPLC).
`In vivo studies. The antitumor activity of the conjugates was assessed in
`female CB-17 severe combined immunodeficient mice (SCID mice; Taconic
`Labs, Germantown, NY) bearing HT-29 tumors as described previously (9).
`Conjugates were administered i.v. to groups of six mice.
`Cell cycle studies. Exponentially growing COLO 205 cells were
`
`resuspended at 1  105/mL to 2  105/mL before drug or conjugate
`
`treatment. The nuclei of the cells were stained with propidium iodide as
`previously described (10). DNA content analysis was done using
`FACSCalibur (Becton Dickinson, San Jose, CA). For each sample, 10,000
`events were collected and FL2-A histograms were generated. The cell cycle
`analysis software, ModFit LT 3.1 (Verity Software House, Topsham, ME)
`was used to determine the percentage of cells in different phases of the cell
`cycle.
`Analytic methods. All maytansinoids were separated on an analytic
`
`C-18 column (0.46  25 cm) equilibrated with 20% aqueous CH3CN
`containing 0.025% TFA and developed with a linear gradient of 2% CH3CN/
`min and a flow rate of 1 mL/min. The effluent was directed to a diode array
`detector followed either by a Radiomatic 150 h-counter (Perkin-Elmer Life
`and Analytic Sciences) where the effluent was mixed continuously with
`3 mL scintillation cocktail before it was directed to a 0.5 mL flow cell or
`a Bruker Daltonics Esquire 3000 electrospray mass spectrometer depending
`on the application. The data sampling interval was 6 seconds. The peak
`areas of the chromatograms were converted to picomoles as follows:
`A standard curve to relate the peak of radioactivity (mV2) on the
`chromatograms to cpm of tritium was prepared by injecting 1,000 to
`50,000 cpm of a stock solution of [3H]DM4 (250 mCi/mmol) diluted in 20%
`aqueous CH3CN containing 0.025% TFA. Duplicates for each sample were
`submitted to HPLC as described above.
`
`Results
`In vitro potency and in vivo activity of huC242-maytansinoid
`conjugates. The disulfide-linked antibody-maytansinoid conju-
`gate, huC242-SPDB-DM4, and the thioether-linked conjugate,
`huC242-SMCC-DM1, were first assayed for their cytotoxic potency
`against antigen-positive COLO 205 cells and antigen-negative
`Namalwa cells (both sensitive to maytansine with IC50 values of
`f30 to 60 pmol/L for both cell
`lines) using an 3-(4,5-
`dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)–
`based assay. The conjugates displayed similar potencies with
`IC50 values of 40 pmol/L against COLO 205 cells and 20 to 80
`nmol/L against Namalwa cells upon a 4-day exposure of the cells
`to the conjugates (Fig. 1A). The antitumor activity of the two
`conjugates was assessed in SCID mice bearing s.c. HT-29 or COLO
`205 tumors (both CanAg-positive human colon adenocarcinoma).
`The activity of huC242-SPDB-DM4 at a single dose of 50 Ag/kg
`(concentration based on conjugated DM4) in eradicating HT-29
`tumors was greater
`than that of huC242-SMCC-DM1 when
`administered as five daily injections at a dose of 150 Ag/kg/d
`(Fig. 1B). HuC242-SPDB-DM4 also displayed greater activity than
`huC242-SMCC-DM1 when both were administered as five daily
`injections at a dose of 150 Ag/kg/d in SCID mice bearing COLO
`205 tumors ( five of five tumor free mice at 122 days compared
`
`Intracellular Activation of Antibody-Maytansinoid
`
`Figure 1. Cytotoxicity of huC242-SPDB-DM4 and huC242-SMCC-DM1 and
`their effects on the cell cycle progression in the presence of the lysosomal
`inhibitor BafA1. A, surviving fractions of antigen-positive COLO 205 (closed
`symbols ) and antigen-negative Nalmawa cells (open symbols ) were measured
`using an MTT assay after exposure to different concentrations of huC242-SPDB-
`DM4 (diamonds ) or huC242-SMCC-DM1 (squares ) for 4 days, and plotted
`versus conjugate concentrations. B, antitumor activities of huC242-SPDB-DM4
`and huC242-SMCC-DM1 in SCID mice bearing HT-29 human colon tumor
`xenografts. Doses are based on the amount of conjugated DM4 or DM1.
`Tumor-bearing mice were treated with PBS (closed circles, left and right ), a
`single dose of 50 Ag/kg huC242-SPDB-DM4 (open diamonds), 150 Ag/kg
`huC242-SPDB-DM4 (closed diamonds ), or five daily injections of huC242-
`SMCC-DM1 at a dose of 150 Ag/kg (open squares ). C, FACS histograms
`(FL2-A) display DNA contents of asynchronous, exponentially growing COLO
`205 cells treated for 20 hours with 0.66 Amol/L nocodazole, 10-8 mol/L
`DM1-SMe, 3  10-9 mol/L huC242-SMCC-DM1, or 3  10
`9 mol/L huC242-
`SPDB-DM4 in the presence of 30 nmol/L BafA1 or no treatment.
`
`with two of five tumor free mice at 122 days; data not shown).
`These results show that although both conjugates display similar
`cytotoxicity in vitro, their efficacy against tumors in vivo differs
`significantly.
`Effect of lysosomal inhibitors on cell cycle arrest induced by
`huC242-maytansinoid conjugates. Maytansinoids inhibit tubulin
`polymerization, which leads to cell cycle arrest in the G2-M phase
`(11); therefore, we checked if this activity was also present in both
`conjugates. Samples of asynchronously growing cells were exposed
`to conjugate (3 nmol/L), DM1-SMe (10 nmol/L; see Fig. 5 for
`structure), or nocodazole (0.66 Amol/L), another inhibitor of
`microtubule polymerization, at 37jC for 20 hours. The DNA
`content of the cells was then analyzed by flow cytometry. In
`cultures treated with nocodazole or DM1-SMe, >50% of the cells
`were arrested in the G2-M phase (Fig. 1C, first row), compared with
`only 10% of untreated COLO 205 cells (typically f60% in G1 and
`30% in S). In samples treated with either of the two conjugates,
`f70% to 80% of the cells were arrested at the G2-M phase,
`indicating that the cell cycle effects of maytansinoid conjugates are
`similar to those of the free maytansinoids (Fig. 1C). No cell cycle
`arrest was induced by the conjugates in cells lacking the target
`antigen (data not shown).
`To test whether uptake and processing of conjugates through
`lysosomes was necessary for their activity against cancer cells, we
`examined G2-M arrest induced by conjugates in the presence of
`bafilomycin A1 (BafA1), a lysosomal inhibitor. BafA1 selectively
`
`www.aacrjournals.org
`
`4427
`
`Cancer Res 2006; 66: (8). April 15, 2006
`
`Downloaded from
`
`on June 10, 2014. © 2006 American Association for Cancercancerres.aacrjournals.org
`
`
`Research.
`
`IMMUNOGEN 2175, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`Cancer Research
`
`inhibits V-ATPase, a proton pump present in endosomes and
`lysosomes, which leads to neutralization of the pH in these vesicles
`(12, 13). The pH neutralization blocks trafficking from late
`endosomes to lysosomes and lysosomal processing, yet modestly
`affects the rate of internalization and recycling and does not
`inhibit trafficking between endosomes and trans-Golgi (14–16). We
`also found that the rate of
`internalization of a fluorescently
`modified huC242 was not affected by BafA1 (data not shown). The
`treatment of COLO 205 cells with BafA1 alone did not significantly
`alter the distribution of cells between the phases of the cell cycle
`(Fig. 1C). However, in the presence of BafA1, G2-M arrest induced
`by either huC242-SMCC-DM1 or huC242-SPDB-DM4 was almost
`completely abolished (6-9% in G2-M; Fig. 1C). In contrast, BafA1
`treatment had a modest effect on the extent of G2-M arrest caused
`by the free maytansinoid, DM1-SMe or by nocodazole (37-39% in
`G2-M; Fig. 1C). Similar results were also found when chloroquine,
`another lysosomal
`inhibitor that neutralizes pH by a different
`mechanism (16, 17), was used (Supplementary Fig. S1). These
`findings were the first indication of the importance of lysosomal
`processing in the activation of both huC242-SMCC-DM1 and
`huC242-SPDB-DM4.
`Isolation of maytansinoid metabolites from huC242-may-
`tansinoid conjugates. To examine the fate of the maytansinoid
`drug upon incubation of target cells with an antibody-maytansi-
`noid conjugate, we prepared conjugates with maytansinoids that
`were 3H-labeled at the C-20 methoxy group (see Fig. 5). Radio-
`labeled maytansinoid conjugates, huC242-SPDB-[3H]DM4 (250
`mCi/mmol) and huC242-SMCC-[3H]DM1 (214 mCi/mmol), exhibit
`in vitro cytotoxicities similar to nonradiolabeled conjugate samples
`(data not shown). Cultures of 2  106 COLO 205 cells were exposed
`7 mol/L 3H-labeled conjugates for periods of 5, 9, and 26
`to 10
`hours, separated into cell and conditioned medium fractions, and
`each sample was extracted with acetone. We determined that
`cells treated under these conditions remained viable with intact
`
`plasma membranes for at least the first 26 hours of exposure as
`determined by trypan blue staining (data not shown). The acetone
`extracts were analyzed for 3H-labeled metabolites by reversed-
`phase HPLC. The chromatograms in Fig. 2A display signals for
`the amount of radioactivity in the acetone-extracted samples from
`the thioether-linked huC242-SMCC-[3H]DM1-treated cells. A con-
`trol was prepared from an acetone extract of the conjugate used
`in the experiment to identify free maytansinoid species present in
`the huC242-SMCC-[3H]DM1 sample before the exposure to cells
`(Fig. 2A, g ). Two new, partially separated peaks of radioactivity
`with retention times of f18.7 and 19.2 minutes, respectively,
`were identified in extracts from cell pellets. These metabolites are
`readily detectable after a 5-hour exposure, and increase by 9 and
`26 hours (Fig. 2A, a, c, e). These same metabolites were also detect-
`able in the culture medium but not until the 9-hour time point, and
`then were readily detectable in the 26-hour sample. In separate
`experiments, the two metabolites were isolated and found by mass
`q
`spectrometry (MS) to be the two isomers of
`lysine-N
`-SMCC-
`[3H]DM1 (R or S configuration at the carbon of the thioether bond
`formed in the conjugation reaction; both peaks M+ = 1,103.5; M +
`Na = 1,125.5).
`The results from an analogous experiment with the disulfide-
`linked huC242-SPDB-[3H]DM4 conjugate are shown in Fig. 2B. Three
`distinct peaks of radioactivity with retention times of 20.5, 26.5, and
`27 minutes were observed in the chromatograms derived from the
`cell pellets treated with huC242-SPDB-[3H]DM4 for 5 and 9 hours
`(Fig. 2B, a and c). Cells treated for 26 hours yielded largely one
`peak with a retention time of 27 minutes (Fig. 2B, e), indicating that
`the metabolites eluting at 20.5 and 26.5 minutes had either been
`converted to the metabolite eluting at 27 minutes or were no longer
`present in the cells. The peak at 26.5 minutes has the same retention
`time as DM4. To further study this metabolite and to assess whether
`any of the other metabolites contained a free sulfhydryl group, an
`acetone extract from a 9-hour exposure was treated with
`
`Figure 2. Maytansinoid metabolites formed upon treatment of COLO 205 cells with huC242-SMCC-[3H]DM1 (A) or huC242-SPDB-[3H]DM4 (B ). Metabolites
`were extracted with acetone from the cell pellet and the spent medium, respectively, and then analyzed by HPLC. The effluent from the column was monitored for tritium
`using an in-line flow scintillation analyzer with an output in mV2; thus, the chromatograms show retention time on the abscissa and mV2 as a measure of [3H]
`on the ordinate. a to f, chromatograms associated with the medium and cell pellets from COLO 205 cells that were treated for 5, 9, and 26 hours with huC242-SMCC-
`[3H]DM1 (A) or huC242-SPDB-[3H]DM4 (B). g (A ) and h (B ), chromatograms obtained from the acetone extract of the huC242-SMCC-[3H]DM1 and huC242-
`SPDB-[3H]DM4 conjugate sample, respectively, used in the experiment. g (B), chromatogram derived from cells that were treated equally to the cell pellet from the
`9-hour incubation (as in c ), except that the sample was additionally exposed to N-ethylmaleimide (NEM ) before chromatography to alkylate any sulfhydryl groups.
`
`Cancer Res 2006; 66: (8). April 15, 2006
`
`4428
`
`www.aacrjournals.org
`
`Downloaded from
`
`on June 10, 2014. © 2006 American Association for Cancercancerres.aacrjournals.org
`
`
`Research.
`
`IMMUNOGEN 2175, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`Intracellular Activation of Antibody-Maytansinoid
`
`Figure 3. Accumulation of maytansinoid
`metabolites inside and outside of COLO 205 cells
`treated with 3H-maytansinoid conjugates and
`their correlation with mitotic arrest. A, areas
`associated with the peaks of radioactivity for the
`metabolites in Fig. 2 were quantified and converted
`to picomoles as described in Materials and
`Methods. a to c, changes in the amount of various
`metabolites in the samples over a 26-hour
`q
`incubation period. a, accumulation of lysine-N
`-
`SMCC-DM1 in the medium (open circles ) and cells
`(closed circles ) following treatment of COLO 205
`cells with huC242-SMCC-[3H]DM1. b, changes
`in the amounts of maytansinoid metabolites in the
`cell pellet (closed symbols ) and in the medium
`(open symbols ) following treatment of COLO 205
`with huC242-SPDB-[3H]DM4: S -methyl-DM4 +
`q
`DM4 (squares ), lysine-N
`-SPDB-DM4 (circles ),
`S-cysteinyl-DM4 (diamonds ). c, change of the
`sum of all metabolites present for both conjugates,
`huC242-SMCC-[3H]DM1 (circles) and huC242-
`SPDB-[3H]DM4 (squares ), either in the cell pellet
`(solid symbols ) or in the cell pellet and medium
`together (open symbols ). B, COLO 205 cells were
`synchronized in S phase with a 24 hours of
`treatment of 2 Ag/mL aphidicolin. The cells were
`released from S phase by the removal of
`8 mol/L
`aphidicolin and incubated with 10
`DM1-SMe, 3  10
`9 mol/L huC242-SMCC-DM1,
`3  10
`9 mol/L huC242-SPDB-DM4, or left
`untreated. FACS analysis was done at 5, 10, and
`18 hours after aphidicolin release.
`
`N-ethylmaleimide and then subjected to HPLC (Fig. 2B, g ). Following
`alkylation, the peak at 26.5 minutes, observed in the 5- and 9-hour
`samples, was replaced with a new peak at 25 minutes. This new peak
`coelutes with a standard of the purified DM4-N-ethylmaleimide
`reaction product (data not shown), suggesting that the metabolite at
`26.5 minutes is indeed DM4.
`The corresponding chromatograms associated with the
`conditioned medium from treated cells are displayed in
`Fig. 2B (b, d , and f ). After 5 hours of exposure, no metabolites
`can be detected; the peaks present are also visible in the control
`chromatogram of the acetone extract of the conjugate sample
`(Fig. 2B, h). After 9 hours of exposure, however, the unknown
`metabolite with a retention time of 20.5 minutes that was
`observed in cells after 5 hours can be detected in the medium,
`and after 26 hours, two additional metabolites with retention
`times of 18.5 and 27 minutes were present. The unique
`metabolite in the medium that elutes at 18.5 minutes was
`found to have the same retention time as the mixed disulfide
`between DM4 and cysteine (S-cysteinyl-DM4; data not shown).
`RPMI 1640 contains 0.21 mmol/L cystine, and separate experi-
`ments have shown that DM4 under these conditions reacts
`rapidly (t 1/2 < 1 hour) to form S-cysteinyl-DM4 through a thiol-
`disulfide interchange reaction (data not
`shown). Thus,
`the
`S-cysteinyl-DM4 disulfide compound would be expected to form
`in the medium if the DM4 observed in the cell pellet leaves the
`cells. To further test whether any of the metabolites in the
`medium were disulfide compounds, a cell supernatant sample
`from a 26-hour exposure was divided into two equal portions of
`which one was subjected directly to chromatography, and the
`other portion was
`treated with DTT and selenol before
`chromatography to reduce all disulfide bonds (18). Following
`reduction, all of the peaks of radioactivity in the chromatograms
`disappeared except the peak eluting at 27 minutes and a single
`
`new peak appeared with a retention time of 26.5 minutes, which
`is that of DM4 (Supplementary Fig. S2). Thus, the metabolites
`eluting at 18.5 and 20.5 minutes, but not the metabolite at 27
`minutes, are DM4 species containing a disulfide-linked substit-
`uent. In separate experiments, the unknown metabolites eluting
`at 20.5 and 27 minutes were found to have a mass consistent
`q
`with lysine-N
`-SPDB-DM4 (M + Na = 1048.4) and S-methyl-DM4
`(M + Na = 816.4/M + K = 832.5), respectively.
`In the above experiments, 2  106 COLO 205 cells were incubated
`7 mol/L conjugate. From this large
`in 3 mL medium containing 10
`amount of conjugate, <20% of the conjugate-bound maytansinoid
`was recovered in the identified maytansinoid metabolites after
`incubation with the cells for 26 hours (Fig. 3A, c; dotted line
`represents 20% level). In a separate experiment following the fate
`of huC242-SPDB-[3H]DM4 that was bound to cells at 4jC, we
`determined that within a 22-hour period at 37jC, 73% of the
`conjugated DM4 bound to COLO 205 cells was converted into
`the four maytansinoid metabolites—DM4, S-cysteinyl-DM4, lysine-
`q
`N
`-SPDB-DM4, and S-methyl-DM4 (Supplementary Fig. S3). We
`conclude that these metabolites represent the majority (if not all)
`of the metabolites formed in the cellular activation process.
`Moreover, production of these maytansinoid metabolites was
`not observed when COLO 205 cells were incubated with disulfide-
`linked or SMCC-linked maytansinoid conjugates, which do not bind
`target antigens on these cells (Supplementary Fig. S4).
`The kinetics for the accumulation of metabolites and cell
`cycle arrest. The peak areas of radioactivity associated with the
`metabolites generated from huC242-SMCC-[3H]DM1 and from
`huC242-SPDB-[3H]DM4 (Fig. 2) were determined and converted
`to picomoles of maytansinoid as described in Materials and
`Methods. The results are shown in Fig. 3A as accumulation of
`the maytansinoid metabolites over time from the addition of
`the conjugates to the cells (T = 0). Figure 3A (a) shows the
`
`www.aacrjournals.org
`
`4429
`
`Cancer Res 2006; 66: (8). April 15, 2006
`
`Downloaded from
`
`on June 10, 2014. © 2006 American Association for Cancercancerres.aacrjournals.org
`
`
`Research.
`
`IMMUNOGEN 2175, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`Cancer Research
`
`q
`-SMCC-DM1, the single metabolite from
`accumulation of lysine-N
`huC242-SMCC-[3H]DM1, in the cell pellet and in the medium. The
`concentration of the metabolite in the cells reaches a steady state
`after 9 hours of incubation, which may be due to the saturation of
`an intracellular binding site. This maytansinoid metabolite begins
`to appear in the medium at 9 hours, suggesting efficient efflux of
`q
`the charged lysine-N
`-SMCC-DM1 species across the plasma
`membrane. Figure 3A (b) shows the accumulation of the three
`stable metabolites generated from huC242-SPDB-[3H]DM4, in the
`cell pellet and in the medium. The intracellular amount of
`q
`the disulfide compound, lysine-N
`-SPDB-DM4, declines over the
`observed period of 26 hours, presumably through disulfide cleavage
`to DM4 in the intracellular reducing environment, followed by
`subsequent conversion of the resulting free thiol, DM4, to the
`stable S-methyl-DM4 derivative. Very little, if any, DM4 was observed
`inside the cells after 9 hours of
`incubation,
`indicating rapid
`methylation probably catalyzed by a methyl transferase. The total
`intracellular accumulation of metabolites reaches a steady state
`after 9 hours of exposure to either conjugate and the levels of
`metabolites at the steady state are about the same for both
`conjugates (Fig. 3A, c), suggesting that the conjugates share the
`same rate-limiting step with respect to production of
`the
`metabolites and that these metabolites likely bind to the same
`intracellular target.
`Because only f9 hours is required for intracellular accumu-
`lation of metabolites to reach the steady-state level, we
`investigated whether G2-M arrest induced by either conjugate
`can occur within that time frame. COLO 205 cells were first
`synchronized with a 24-hour treatment of aphidicolin, a reversible
`DNA replication inhibitor that blocks cells in S phase (19).
`Because the majority of the asynchronous population of COLO
`205 cells were in G1 phase, most of the aphidicolin-treated cells
`arrested in the beginning of S phase, which cannot be
`distinguished from G1 phase in the fluorescence-activated cell
`sorting (FACS) analysis (Fig. 3B, top picture). The synchronized
`cells were then released from the arrest by the removal of
`aphidicolin, and either left untreated, or treated with DM1-SMe
`or with either conjugate. Untreated cells immediately continue
`with DNA synthesis, and the majority is in G2-M phase (59%)
`after 5 hours (Fig. 3B), and has undergone mitosis at 10 hours
`and are in G1 phase (76%). In contrast, the majority of cells
`treated with DM1-SMe or either conjugate are in G2-M phase
`(75% to 85%) after 10 and 18 hours,
`indicating that sufficient
`amounts of active drug species have accumulated intracellularly
`
`to induce mitotic arrest when the aphidicolin-released cells
`reached mitosis. Therefore, COLO 205 cells require as little as 5
`hours, but not more than 10 hours to produce enough of active
`drug metabolites to cause cell cycle arrest. Thus, the kinetics of
`G2-M phase arrest induced by these conjugates correlate with the
`time required to reach the steady-state level of
`intracellular
`accumulation of the drug metabolites.
`Activity of maytansinoid metabolites. The two major
`q
`metabolites, S-methyl-DM4 and lysine-N
`-SMCC-DM1, were syn-
`thesized and tested for their in vitro cytotoxicity against COLO 205
`cells and Namalwa cells. S-methyl-DM4 was highly cytotoxic with
`q
`an IC50 value of 2 pmol/L against both cell lines, whereas lysine-N
`-
`SMCC-DM1 was f105-fold less potent against both cell lines with
`an IC50 value of 0.1 Amol/L (data not shown). This difference may
`be explained by the different charge status of the compounds. The
`charged lysine derivatives are expected to penetrate cell mem-
`branes very inefficiently such that high external concentrations are
`needed to reach the required toxic intracellular concentrations,
`whereas penetration of cell membranes is efficient for the neutral
`lipophilic S-methyl-DM4 compound.
`q
`Accumulation of the lysine-N
`-SMCC-DM1 metabolite from the
`noncleavable conjugate is coincident with the observed forma-
`q
`-SPDB-DM4
`tion of the potent S-methyl-DM4, DM4, and lysine-N
`q
`from the cleavable conjugate. This suggests that the lysine-N
`-
`SMCC-DM1 metabolite is as potent as the metabolites from the
`cleavable conjugate when delivered intracellularly and that all of
`the maytansinoid metabolites are active when produced in the
`cell. The cell cycle arrest of both the cleavable and noncleavable
`conjugates was abrogated in the presence of
`the lysosomal
`q
`inhibitor BafA1 (Fig. 1C). If the lysine-N
`-SMCC-DM1 metabolite
`and the other metabolites observed from the cleavable conjugate
`arrest cells, BafA1 should prevent their formation. To investi-
`gate this possibility, COLO 205 cells were incubated at 37jC for
`7 mol/L huC242-SMCC-[3H]DM1 or huC242-
`22 hours with 10
`SPDB-[3H]DM4 conjugate in the presence or in the absence of
`300 nmol/L BafA1. We found that BafA1 abolished the formation
`q
`of both the lysine-N
`-SMCC-DM1 from the thioether-linked
`conjugate, and the S-methyl-DM4 from the disulfide linked
`conjugate (Fig. 4A). In addition, no metabolites were detected in
`the medium of cells treated with either conjugate in the
`presence of BafA1 (data not shown). These results suggest that
`the lysine metabolites are toxic when delivered intracellularly
`and that lysosomal processing is necessary for the production of
`all the observed metabolites.
`
`Figure 4. The role of the major
`metabolites in cellular toxicity. A, influence
`of BafA1 on the cellular metabolism of
`huC242-SMCC-[3H]DM1 and huC242-
`SPDB-[3H]DM4 in the presence or absence
`of BafA1. The chromatograms are from
`acetone extracts of COLO 205 cells treated
`with huC242-SMCC-[3H]DM1 or huC242-
`SPDB-[3H]DM4 for 22 hours in the
`presence of BafA1 or no treatment.
`B, accumulation of maytansinoid
`metabolites formed upon treatment of
`COLO 205 cells with huC242-SPDB-D-
`[3H]DM4 versus huC242-SPDB-[3H]DM4.
`Total amounts of maytansinoid metabolites
`generated from huC242-SPDB-D-[3H]DM4
`(closed squares ) and from huC242-SPDB-
`[3H]DM4 (closed circles ). Th