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Enhanced Delivery of SN-38 to Human Tumor Xenografts with an Anti-Trop-2-SN-38 Antibody Conjugate (Sacituzumab Govitecan) Robert M. Sharkey, William J. McBride, Thomas M. Cardillo, Serengulam V. Govindan, Yang Wang, Edmund A. Rossi, Chien-Hsing Chang, David M. Goldenberg Immunomedics, Inc., Morris Plains, NJ 07950
Correspondence should be addressed to: David M. Goldenberg, Immunomedics, Inc., 300 The American Road, Morris Plains, NJ, 07950. E-mail: [email protected]; Telephone: 973-605-8200; Fax: 973-605-8282
Conflict of Interest Disclosure: All authors are employees of Immunomedics, Inc. Keywords: Antibody-drug conjugate, Trop-2, SN-38 Running Title: Enhanced tumor targeting of SN-38 with sacituzumab govitecan Word Count Translational Relevance = 135 Abstract = 248 Introduction through Discussion = 4989 Tables = 3 Figures = 3 (no color) Supplementary Information provided separately
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Translational Relevance Sacituzumab govitecan (IMMU-132) is an antibody-drug conjugate composed of a humanized anti-Trop2 IgG coupled with a high ratio of SN-38 molecules. This conjugate was shown previously to improve therapeutic responses in human cancer xenografts compared to irinotecan, its prodrug. Recently, clinical trials with IMMU-132 have shown objective responses in several types of Trop-2-expressing epithelial cancers. In this report, we examine the advantage of delivering SN-38 with IMMU-132 compared to irinotecan in nude mice bearing 2 human tumor xenografts, as well as assess the concentrations of SN-38 and SN-38G in the serum, liver and intestine. A significant targeting advantage for IMMU-132 was observed, in terms of the delivery of SN-38 in a fully active form to the tumors, improved tumor/blood ratios, and reduced intestinal uptake that support the reduced rate and severity of diarrhea in patients.
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Abstract Purpose: This study examined the delivery of SN-38 to Trop-2-expressing tumors and assessed the constitutive products in the serum, liver, and small intestine in nude mice bearing human tumor xenografts (Capan-1 or NCI-N87) given a single injection of irinotecan (40 mg/kg; ~0.8 mg/mouse, containing ~460 µg SN-38 equivalents) or sacituzumab govitecan (IMMU-132), an antibody-drug conjugate composed of a humanized anti-Trop-2 IgG coupled site-specifically with an average of 7.6 molecules of SN-38. Experimental Design: At select times, tissues were extracted and concentrations of the products measured by reversed-phase HPLC. Results: In serum, >98% irinotecan cleared within 5 min; peak levels of SN-38 and SN-38G (glucuronidated SN-38) were detected in equal amounts at this time, and no longer detected after 6-8 h. IMMU-132 was detected in the serum over 3 days, and at each interval, ≥95% of total SN-38 was bound to the antibody. Intact IMMU-132 cleared with a half-life of 14 h, which closely reflected the in vitro rate of SN-38 release from the conjugate in mouse serum (i.e., 17.5 h), while the IgG portion of the conjugate cleared with a half-life of 67.1 h. In vitro and in vivo studies disclosed IgG-bound SN-38 was protected from glucuronidation. Area-under-the-curve (AUC) analysis indicated IMMU-132 delivers 20-fold to as much as 136-fold more SN-38 to tumors than irinotecan, with tumor/blood ratios favoring IMMU-132 by 20- to 40-fold. Intestinal concentrations of SN-38/SN-38G also were 9-fold lower with IMMU-132. Conclusion: These studies confirm a superior SN-38 tumor delivery by IMMU-132 compared to irinotecan.
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Introduction Antibody-drug conjugates (ADC) represent a new therapy class based on the proposition of their being able to deliver cytotoxic agents more specifically to their intended target with less collateral damage. However, this technology has faced many challenges, but with 3 conjugates gaining FDA approval, each using drugs that are active at picomolar concentrations (so called ultra-toxic drugs), it appeared that the major hurdles facing this technology had been overcome, and that a core platform on which similar agents could be developed was available (1-5). Our group departed from the current popular approach, and instead developed an ADC with a more moderately-toxic agent, SN-38. SN-38, a topoisomerase I inhibitor, is a highly potent drug with activity in the low nanomolar range, administered in a prodrug form, irinotecan. Irinotecan’s pharmacology is well known, but complex, limiting its bioavailability. First, only a small portion of irinotecan is converted to SN-38, a process that occurs primarily in the liver, but also in the intestine and plasma, as well as in some tumors (6). Compounding a poor conversion rate is the fact that a sizable portion of SN-38 is readily converted to less active forms, most notably SN-38G, a glucuronidated product whose concentration is often 4- to 30-times higher than SN-38 (7). Enterohepatic recirculation exposes SN-38G to bacterial enzymes in the intestine that reconvert SN-38G to SN-38, which is the most likely cause of late diarrhea, a common and serious toxicity associated with irinotecan therapy (8). SN-38’s lactone ring is another vulnerable conversion site, with irinotecan and SN-38 generally found in equilibrium between the less active carboxylate and the fully potent lactone ring forms (6). Our antibody-SN-38 ADC platform emerged empirically by examining a number of different linkers. Selecting the best conjugate relied on efficacy studies in human tumor xenograft mouse models, with the linker designation CL2 providing the best responses (9, 10). Interestingly, in vitro serum stability studies of the initial three candidate conjugates revealed SN-38 was released at different rates, with one having a half-life of ~10 h, while another was highly stable; yet, the selected CL2-linked 4
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conjugate had an intermediate stability in serum of 36 h. This derivative was later modified to simplify scale-up production for clinical use, with the new derivative designated CL2A (11). Numerous animal studies have shown that IMMU-132 improves therapeutic responses compared to irinotecan, suggesting IMMU-132 delivers more SN-38 to the tumors (11-13) . The principal goal of this investigation was to quantify this difference, evaluating concentrations of the constitutive products in tumors and critical normal tissues (serum, liver, intestine), in order to gain a better perspective of the efficacy and toxicity properties of this conjugate. Materials and Methods Reagents. IMMU-132 was prepared from the hRS7 anti-Trop-2 IgG and the CL2A-SN-38 drug carrier, as described previously (11), with hydrophobic interactive chromatography (HIC) and liquid chromatography mass spectroscopy (LC-MS) showing a drug-antibody ratio of 7.6 (13). Conjugates prepared under these conditions have ~16 µg SN-38/mg IgG. The final lyophilized product was reconstituted in sterile saline immediately before use. Irinotecan was purchased from AREVA pharmaceuticals, Inc. (Elizabethtown, KY), and diluted in sterile saline to the desired concentration. Standards for SN-38, SN-38G, and irinotecan were purchased from Toronto Research Chemicals (Toronto, Ontario, Canada). Study Design. Table 1 lists the 3 sets of studies performed for this analysis, 2 in nude mice bearing xenografts of the human pancreatic cancer cell line, Capan-1, and one with the human gastric carcinoma cell line, NCI-N87. Capan-1 and NCI-N87 were purchased from the American Type Culture Collection (ATCC) (Manassas, VA), and were authenticated by short tandem repeat (STR) assay by the ATCC and routinely tested for mycoplasma. Surface Trop-2 expression on Capan-1 and NCI-N87 cells in culture are 157,000 and 247,000 molecules, respectively (12).
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All animal studies were approved by Rutgers School of Biomedical and Health Sciences Institutional Animal Care and Use Committee. Initiation of subcutaneous tumors in NCr female athymic nude (nu/nu) mice (4-8 weeks old; Taconic Farms; Germantown, NY) is described in the Supplementary Information. Mice were administered intravenously with single doses containing a fixed amount of IMMU-132 (1.0 mg), whereas irinotecan was given at a dose of 40 mg/kg, using the average pre-study weight of the animals to determine a fixed dose for that group. By mass, 1.0 mg of irinotecan contains approximately 0.58 mg SN-38. Animals in the first and third study were examined at 5 different intervals, while animals in the 2nd study were examined only at 2-3 intervals. At the time of necropsy, mice were anesthetized and bled by cardiac puncture, with subsequent removal of tissues of interest that were weighed and frozen. Serum samples were analyzed by ELISA for detection of the IMMU-132 and hRS7 IgG (see Supplementary Information) or by HPLC analysis (below). HPLC analysis of SN-38, SN-38G, and irinotecan required extraction of the harvested tissues. A precipitating reagent reported by Hirose et al. that contained equal parts of methanol, ethylene glycol, and 1.0 M zinc sulfate was used for extraction/precipitation of proteins (14). Prior to extraction, serum volume was extended by diluting with an equal part of water; water was also added to the tissues (10 parts water to 1 part tissue), which were then homogenized on ice using a Powergen-35 homogenizer equipped with a 7 x 110 mm tip for 2 min at 30,000 rpm (Fisher Scientific, Pittsburgh, PA). An equal volume of the precipitating reagent was added to diluted serum or to a portion of the tissue homogenates prior to separation of solids. Each sample was spiked first with an internal standard, 10hydroxycaptothecin (10-HCPT; 9 µL 3.33 mg/mL stock solution in DMSO; Toronto Research Chemicals). The samples were vortexed for 15 sec and then centrifuged to collect the clarified supernatant, which was then then applied on a Phenomenex Chromolith high-resolution C-18 analytical column (EMD Millipore Corp., Billerica, MA), using a linear gradient composed of 85:15 of buffer A/B to 100% buffer B (buffer A: 50 mM KH2PO4 and 4 mM sodium 1-decanesulfonate in water, pH 3.5; buffer B: 600 mL buffer 6
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A plus 400 mL acetonitrile), with a flow rate of 1 mL/min and fluorescence detection with excitation wavelength at 373 nm, and emission wavelength of 540 nm. Portions of the homogenates and diluted serum taken from animals given IMMU-132 were subjected first to an acid hydrolysis step that ensured all SN-38 was released from the IMMU-132 conjugate. For this process, 0.15 mL of 6.0 M HCL was added to 0.15 mL of the homogenate or diluted serum spiked with 10-HCPT, then heated to 50oC overnight before neutralizing with 6.0 M NaOH. An equal volume of the precipitating reagent was added, vortexed, and centrifuged. Thus, the previous processing method measures the amount of free SN-38/SN-38G in the IMMU-132 and irinotecan samples, while the acid-hydrolysis processing isolates the total amount of SN-38/SN-38G specifically in the IMMU-132 sample (designated SN-38 [TOTAL]). The HPLC analysis method is described in Supplementary Information. AUCs were calculated using GraphPad Prism version 6.0 for Windows (GraphPad Software, La Jolla, CA), using only the trapezoidal area where detectable levels of product were measured in ≥3 consecutive intervals. Procedures that monitored SN-38 release rates in mouse serum and in vitro glucuronidation are provided in Supplementary Information. RESULTS Quantitative HPLC Methodological Assessments Prior to initiating these studies, various aspects of the procedures used for the analysis of tissue samples were assessed. Details are reported in Supplementary Information (Fig. S1, Tables S1 and S2). Overall, these studies provided sufficient evidence that the procedures developed for these assessments yielded reliable and reproducible results. The minimum sensitivity of detection in serum was 20 ng/mL and 110 ng/mL in tissue homogenates.
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Of particular importance was the finding that the addition of the precipitating reagent to serum freshly spiked with IMMU-132 recovered only ~7% of the expected amount of SN-38 associated with IMMU-132, indicating that the SN-38 bound to the IgG was not released effectively during the extraction process. Therefore, the acid-hydrolyzing step was introduced, which subsequently showed the expected amount of SN-38 associated with IMMU-132. Furthermore, triplicate samples of serum spiked with SN38G that were first processed by acid hydrolysis and then extracted found only 3.0 ± 0.2% of the SN-38G was converted to SN-38, indicating there was a negligible impact on the determination of SN-38G in the acid-hydrolyzed samples. Concentrations of Products in Serum Although animals given irinotecan in Study 1 examined serum taken over 24 h, none of the products was detected at this time, and therefore subsequent studies focused on intervals over 6-8 h. Measurements of IMMU-132 and its SN-38 products were assayed over 72 h, with detection by both ELISAs and SN-38 [TOTAL] over this entire period of time, but unbound SN-38 and SN-38G were within detectable levels only through 48 and 6 h, respectively (Fig. 1A-D). In Study 1, irinotecan concentrations at 5 min averaged 1.7 ± 0.2% injected dose per mL (% ID/mL), indicating that >98% of the irinotecan already had cleared from the blood. Both SN-38 and SN38G were detected at 5 min, illustrating rapid conversion and glucuronidation (Fig. 1A). Concentrations of SN-38 and SN-38G were similar over 6 h (AUC5 min-6 h of SN-38 and SN-38G were 2.52 and 2.84 µg/mL·h, respectively) (Supplementary Table S3). The combined AUCs of SN-38 and SN-38G divided by the AUC of irinotecan estimated the conversion rate to be ~25%, confirming the more efficient conversion in mice compared to humans (6, 15, 16). In the IMMU-132-treated animals, at 1 h SN-38 [TOTAL] concentrations accounted for 58.7 ± 3.3% ID/mL of the estimated amount of SN-38 equivalents. At each interval examined, free SN-38 levels 8
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were 95% of the total SN-38 in the serum is bound to the IgG, (c) the amount of free SN-38G in the serum is only a fraction of the free SN-38, and (d) there is no evidence of glucuronidation of SN-38 bound to IgG. Analysis of serum samples taken from patients given 8 to 10 mg/kg of IMMU-132 on days 1 and 8 of 21-day cycles has found free SN-38 concentrations at levels of ~100 ng/mL 30 minutes after 17
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the end of a 2-3 h infusion, while the total SN-38 levels average ~4000 ng/mL (34). These levels decrease steadily, being undetectable within 2-3 days; SN-38G levels in sera never exceeded SN-38. Thus, we suspect the levels of free SN-38 in the serum of patients receiving IMMU-132 therapy, which are similar to the peak levels of SN-38 seen in patients given irinotecan therapy (18), contribute to the dose-limiting neutropenia in patients given IMMU-132, whereas the significantly lower levels of SN-38G with IMMU-132 reduces the incidence of severe diarrhea. In conclusion, this ADC platform departs from the current practice using stably-linked ultra-toxic agents (subnanomolar), employing instead a moderately-toxic agent (low nanomolar) that is released in serum. IMMU-132 was found to deliver much higher levels of SN-38 to tumors than irinotecan and, importantly, all of the SN-38 delivered to the tumor by IMMU-132 is released in its most potent form. In relationship to gastrointestinal toxicity, the much lower amounts of SN-38G in the serum and significantly lower amounts of SN-38/SN38G in the intestine with IMMU-132 are expected to reduce the risk for severe diarrhea in patients, which is confirmed in clinical studies (34-38). Thus, IMMU-132 is an ADC that appears to have unique properties that combine to make an effective therapeutic agent in the solid cancers tested.
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Acknowledgements We thank Jennifer Inghrim, Nalini Sathyanarayan, Jayson Jebson, Roberto Arrojo, and Preeti Trisal, Maria Zalath for their technical assistance.
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Table 1. Examination of SN-38 concentrations in tissues of nude mice bearing 2 human cancer xenografts. IMMU-132 Irinotecan (SN-38 (SN-38 Intervals equivalents)a equivalents)a Tissues examined Study Tumor (N = 3 animals/interval)
1
b
2
3
Capan-1
Irinotecan 5 min, 1, 2, 6, and 24 h IMMU-132 1, 6, 24, 48, 72 h
Tumor, serum, liver, small intestine contents
1.0 mg (16 µg)
773 µg (448 µg)
Capan-1
Irinotecan 1 and 6 h IMMU-132 1, 6, and 24 h
Tumor, serum, liver, small and large intestine contents
1.0 mg (16 µg)
808 µg (468 µg)
NCI-N87
Irinotecan 5 min, 1, 2, 6, and 8 h IMMU-132 1, 6, 24, 48, 72 h
Tumor, serum
1.0 mg (16 µg)
840 µg (486 µg)
a
SN-38 equivalents for IMMU-132 based on spectrophotometric determinations of protein and SN-38 concentrations. SN-38 equivalents for irinotecan based on mass, with SN-38 representing approximately 58% of irinotecan’s mass. .
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Table 2. Concentrations of products over time (AUC) in mice bearing Capan-1 or NCI-N87 tumors administered irinotecan or IMMU-132a. Tissue Capan-1 Study 1 only [Study 1+2] NCI-N87
Irinotecan-treated SN-38 SN-38G Irinotecan 0.40 [1.09] 2.11
1.08 [1.04] 0.31
48.37 [46.73] 45.69
IMMU-132-treated SN-38 [TOTAL]
SN-38 deliver ratio IMMU-132/Irinotecan
54.25 [49.02] 43.88
135.6 [45.0] 20.8
a
AUC are expressed as (µg/g·h). In Study 1, SN-38 AUC derived from 5 min – 2 h; SN-38G and irinotecan from 5 min to 6 h. AUC from 5 min to 6 h for irinotecan-treated mice combining Studies 1 and 2. For IMMU-132-treated animals SN-38 [TOTAL] AUC are derived from 1 h to 72 h.
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Table 3. Determination of tumor/serum AUC ratiosa. Capan-1 [Study 1+2] Serum Irinotecan-treated SN-38 3.27 1.09 SN-38G 4.02 Irinotecan 27.48 IMMU-132-treated Free SN-38 Free SN-38G SN-38 [TOTAL]
3.87 1.00 157.21
49.02
NCI-N87
Tumor/serum AUC ratio Capan-1 NCI-N87
2.11
0.33
0.65
43.88
12.7
11.3
a
AUCs, expressed as µg/g·h were averaged from all 3 studies, with values derived from 5 min to 8 h for irinotecan-treated animals or from 1 h to 72 h for IMMU-132-treated animals.
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Figure Legends
Figure 1. Clearance of constitutive products in tumor-bearing nude mice given irinotecan or IMMU132. (A) Study 1, Capan-1-beaing mice; irinotecan. (B) Study 1; IMMU-132. (C) Study 3, NCI-N87-bearing mice; irinotecan. (D) Study 3; IMMU-132. Area-under-the-curve (AUC) estimates are provided in the table for products with 3 or more samples. Figure 2. Concentrations of products extracted from Capan-1 and NCI-N87 tumors isolated from animals given irinotecan or IMMU-132. (A) Concentrations of SN-38, SN-38G and irinotecan in Capan-1 tumors from mice given irinotecan in Study 1 and Study 2. In Study 1, SN-38 was below the threshold of detection in the 6-h sample, whereas SN-38G and irinotecan was detected in all samples. Study 2 only examined samples taken at 1 and 6 h. (B) Concentrations of products in NCI-N87 tumors from animals given irinotecan (Study 3). (C) Uptake and clearance of SN-38 [TOTAL] in mice bearing Capan-1 (Studies 1 and 2 combined) and NCI-N87 (Study 3). No other products were detected in the tumors. (D) Comparison of SN-38 [TOTAL] concentrations in Capan-1 xenografts from mice in Study 1 and Study 2. Figure 3. Contents of the upper small intestine analyzed for the various products in mice treated with irinotecan or IMMU-132 (Studies 1 and 2 combined, N = 3 to 6). Irinotecan animals were examined from 5 min to 24 h, while IMMU-132-treated animals were examined 1 to 72 h, but no products were found at detectable levels after 24 h.
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Enhanced Delivery of SN-38 to Human Tumor Xenografts with an Anti-Trop-2-SN-38 Antibody Conjugate (Sacituzumab Govitecan) Robert M Sharkey, William J McBride, Thomas M. Cardillo, et al. Clin Cancer Res Published OnlineFirst June 23, 2015.
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Enhanced Delivery of SN-38 to Human Tumor Xenografts with an Anti-Trop-2-SN-38 Antibody Conjugate (Sacituzumab Govitecan) Robert M. Sharkey, William J. McBride, Thomas M. Cardillo, Serengulam V. Govindan, Yang Wang, Edmund A. Rossi, Chien-Hsing Chang, David M. Goldenberg Immunomedics, Inc., Morris Plains, NJ 07950
Correspondence should be addressed to: David M. Goldenberg, Immunomedics, Inc., 300 The American Road, Morris Plains, NJ, 07950. E-mail: [email protected]; Telephone: 973-605-8200; Fax: 973-605-8282
Conflict of Interest Disclosure: All authors are employees of Immunomedics, Inc. Keywords: Antibody-drug conjugate, Trop-2, SN-38 Running Title: Enhanced tumor targeting of SN-38 with sacituzumab govitecan Word Count Translational Relevance = 135 Abstract = 248 Introduction through Discussion = 4989 Tables = 3 Figures = 3 (no color) Supplementary Information provided separately
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Translational Relevance Sacituzumab govitecan (IMMU-132) is an antibody-drug conjugate composed of a humanized anti-Trop2 IgG coupled with a high ratio of SN-38 molecules. This conjugate was shown previously to improve therapeutic responses in human cancer xenografts compared to irinotecan, its prodrug. Recently, clinical trials with IMMU-132 have shown objective responses in several types of Trop-2-expressing epithelial cancers. In this report, we examine the advantage of delivering SN-38 with IMMU-132 compared to irinotecan in nude mice bearing 2 human tumor xenografts, as well as assess the concentrations of SN-38 and SN-38G in the serum, liver and intestine. A significant targeting advantage for IMMU-132 was observed, in terms of the delivery of SN-38 in a fully active form to the tumors, improved tumor/blood ratios, and reduced intestinal uptake that support the reduced rate and severity of diarrhea in patients.
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Abstract Purpose: This study examined the delivery of SN-38 to Trop-2-expressing tumors and assessed the constitutive products in the serum, liver, and small intestine in nude mice bearing human tumor xenografts (Capan-1 or NCI-N87) given a single injection of irinotecan (40 mg/kg; ~0.8 mg/mouse, containing ~460 µg SN-38 equivalents) or sacituzumab govitecan (IMMU-132), an antibody-drug conjugate composed of a humanized anti-Trop-2 IgG coupled site-specifically with an average of 7.6 molecules of SN-38. Experimental Design: At select times, tissues were extracted and concentrations of the products measured by reversed-phase HPLC. Results: In serum, >98% irinotecan cleared within 5 min; peak levels of SN-38 and SN-38G (glucuronidated SN-38) were detected in equal amounts at this time, and no longer detected after 6-8 h. IMMU-132 was detected in the serum over 3 days, and at each interval, ≥95% of total SN-38 was bound to the antibody. Intact IMMU-132 cleared with a half-life of 14 h, which closely reflected the in vitro rate of SN-38 release from the conjugate in mouse serum (i.e., 17.5 h), while the IgG portion of the conjugate cleared with a half-life of 67.1 h. In vitro and in vivo studies disclosed IgG-bound SN-38 was protected from glucuronidation. Area-under-the-curve (AUC) analysis indicated IMMU-132 delivers 20-fold to as much as 136-fold more SN-38 to tumors than irinotecan, with tumor/blood ratios favoring IMMU-132 by 20- to 40-fold. Intestinal concentrations of SN-38/SN-38G also were 9-fold lower with IMMU-132. Conclusion: These studies confirm a superior SN-38 tumor delivery by IMMU-132 compared to irinotecan.
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Introduction Antibody-drug conjugates (ADC) represent a new therapy class based on the proposition of their being able to deliver cytotoxic agents more specifically to their intended target with less collateral damage. However, this technology has faced many challenges, but with 3 conjugates gaining FDA approval, each using drugs that are active at picomolar concentrations (so called ultra-toxic drugs), it appeared that the major hurdles facing this technology had been overcome, and that a core platform on which similar agents could be developed was available (1-5). Our group departed from the current popular approach, and instead developed an ADC with a more moderately-toxic agent, SN-38. SN-38, a topoisomerase I inhibitor, is a highly potent drug with activity in the low nanomolar range, administered in a prodrug form, irinotecan. Irinotecan’s pharmacology is well known, but complex, limiting its bioavailability. First, only a small portion of irinotecan is converted to SN-38, a process that occurs primarily in the liver, but also in the intestine and plasma, as well as in some tumors (6). Compounding a poor conversion rate is the fact that a sizable portion of SN-38 is readily converted to less active forms, most notably SN-38G, a glucuronidated product whose concentration is often 4- to 30-times higher than SN-38 (7). Enterohepatic recirculation exposes SN-38G to bacterial enzymes in the intestine that reconvert SN-38G to SN-38, which is the most likely cause of late diarrhea, a common and serious toxicity associated with irinotecan therapy (8). SN-38’s lactone ring is another vulnerable conversion site, with irinotecan and SN-38 generally found in equilibrium between the less active carboxylate and the fully potent lactone ring forms (6). Our antibody-SN-38 ADC platform emerged empirically by examining a number of different linkers. Selecting the best conjugate relied on efficacy studies in human tumor xenograft mouse models, with the linker designation CL2 providing the best responses (9, 10). Interestingly, in vitro serum stability studies of the initial three candidate conjugates revealed SN-38 was released at different rates, with one having a half-life of ~10 h, while another was highly stable; yet, the selected CL2-linked 4
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conjugate had an intermediate stability in serum of 36 h. This derivative was later modified to simplify scale-up production for clinical use, with the new derivative designated CL2A (11). Numerous animal studies have shown that IMMU-132 improves therapeutic responses compared to irinotecan, suggesting IMMU-132 delivers more SN-38 to the tumors (11-13) . The principal goal of this investigation was to quantify this difference, evaluating concentrations of the constitutive products in tumors and critical normal tissues (serum, liver, intestine), in order to gain a better perspective of the efficacy and toxicity properties of this conjugate. Materials and Methods Reagents. IMMU-132 was prepared from the hRS7 anti-Trop-2 IgG and the CL2A-SN-38 drug carrier, as described previously (11), with hydrophobic interactive chromatography (HIC) and liquid chromatography mass spectroscopy (LC-MS) showing a drug-antibody ratio of 7.6 (13). Conjugates prepared under these conditions have ~16 µg SN-38/mg IgG. The final lyophilized product was reconstituted in sterile saline immediately before use. Irinotecan was purchased from AREVA pharmaceuticals, Inc. (Elizabethtown, KY), and diluted in sterile saline to the desired concentration. Standards for SN-38, SN-38G, and irinotecan were purchased from Toronto Research Chemicals (Toronto, Ontario, Canada). Study Design. Table 1 lists the 3 sets of studies performed for this analysis, 2 in nude mice bearing xenografts of the human pancreatic cancer cell line, Capan-1, and one with the human gastric carcinoma cell line, NCI-N87. Capan-1 and NCI-N87 were purchased from the American Type Culture Collection (ATCC) (Manassas, VA), and were authenticated by short tandem repeat (STR) assay by the ATCC and routinely tested for mycoplasma. Surface Trop-2 expression on Capan-1 and NCI-N87 cells in culture are 157,000 and 247,000 molecules, respectively (12).
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All animal studies were approved by Rutgers School of Biomedical and Health Sciences Institutional Animal Care and Use Committee. Initiation of subcutaneous tumors in NCr female athymic nude (nu/nu) mice (4-8 weeks old; Taconic Farms; Germantown, NY) is described in the Supplementary Information. Mice were administered intravenously with single doses containing a fixed amount of IMMU-132 (1.0 mg), whereas irinotecan was given at a dose of 40 mg/kg, using the average pre-study weight of the animals to determine a fixed dose for that group. By mass, 1.0 mg of irinotecan contains approximately 0.58 mg SN-38. Animals in the first and third study were examined at 5 different intervals, while animals in the 2nd study were examined only at 2-3 intervals. At the time of necropsy, mice were anesthetized and bled by cardiac puncture, with subsequent removal of tissues of interest that were weighed and frozen. Serum samples were analyzed by ELISA for detection of the IMMU-132 and hRS7 IgG (see Supplementary Information) or by HPLC analysis (below). HPLC analysis of SN-38, SN-38G, and irinotecan required extraction of the harvested tissues. A precipitating reagent reported by Hirose et al. that contained equal parts of methanol, ethylene glycol, and 1.0 M zinc sulfate was used for extraction/precipitation of proteins (14). Prior to extraction, serum volume was extended by diluting with an equal part of water; water was also added to the tissues (10 parts water to 1 part tissue), which were then homogenized on ice using a Powergen-35 homogenizer equipped with a 7 x 110 mm tip for 2 min at 30,000 rpm (Fisher Scientific, Pittsburgh, PA). An equal volume of the precipitating reagent was added to diluted serum or to a portion of the tissue homogenates prior to separation of solids. Each sample was spiked first with an internal standard, 10hydroxycaptothecin (10-HCPT; 9 µL 3.33 mg/mL stock solution in DMSO; Toronto Research Chemicals). The samples were vortexed for 15 sec and then centrifuged to collect the clarified supernatant, which was then then applied on a Phenomenex Chromolith high-resolution C-18 analytical column (EMD Millipore Corp., Billerica, MA), using a linear gradient composed of 85:15 of buffer A/B to 100% buffer B (buffer A: 50 mM KH2PO4 and 4 mM sodium 1-decanesulfonate in water, pH 3.5; buffer B: 600 mL buffer 6
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A plus 400 mL acetonitrile), with a flow rate of 1 mL/min and fluorescence detection with excitation wavelength at 373 nm, and emission wavelength of 540 nm. Portions of the homogenates and diluted serum taken from animals given IMMU-132 were subjected first to an acid hydrolysis step that ensured all SN-38 was released from the IMMU-132 conjugate. For this process, 0.15 mL of 6.0 M HCL was added to 0.15 mL of the homogenate or diluted serum spiked with 10-HCPT, then heated to 50oC overnight before neutralizing with 6.0 M NaOH. An equal volume of the precipitating reagent was added, vortexed, and centrifuged. Thus, the previous processing method measures the amount of free SN-38/SN-38G in the IMMU-132 and irinotecan samples, while the acid-hydrolysis processing isolates the total amount of SN-38/SN-38G specifically in the IMMU-132 sample (designated SN-38 [TOTAL]). The HPLC analysis method is described in Supplementary Information. AUCs were calculated using GraphPad Prism version 6.0 for Windows (GraphPad Software, La Jolla, CA), using only the trapezoidal area where detectable levels of product were measured in ≥3 consecutive intervals. Procedures that monitored SN-38 release rates in mouse serum and in vitro glucuronidation are provided in Supplementary Information. RESULTS Quantitative HPLC Methodological Assessments Prior to initiating these studies, various aspects of the procedures used for the analysis of tissue samples were assessed. Details are reported in Supplementary Information (Fig. S1, Tables S1 and S2). Overall, these studies provided sufficient evidence that the procedures developed for these assessments yielded reliable and reproducible results. The minimum sensitivity of detection in serum was 20 ng/mL and 110 ng/mL in tissue homogenates.
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Of particular importance was the finding that the addition of the precipitating reagent to serum freshly spiked with IMMU-132 recovered only ~7% of the expected amount of SN-38 associated with IMMU-132, indicating that the SN-38 bound to the IgG was not released effectively during the extraction process. Therefore, the acid-hydrolyzing step was introduced, which subsequently showed the expected amount of SN-38 associated with IMMU-132. Furthermore, triplicate samples of serum spiked with SN38G that were first processed by acid hydrolysis and then extracted found only 3.0 ± 0.2% of the SN-38G was converted to SN-38, indicating there was a negligible impact on the determination of SN-38G in the acid-hydrolyzed samples. Concentrations of Products in Serum Although animals given irinotecan in Study 1 examined serum taken over 24 h, none of the products was detected at this time, and therefore subsequent studies focused on intervals over 6-8 h. Measurements of IMMU-132 and its SN-38 products were assayed over 72 h, with detection by both ELISAs and SN-38 [TOTAL] over this entire period of time, but unbound SN-38 and SN-38G were within detectable levels only through 48 and 6 h, respectively (Fig. 1A-D). In Study 1, irinotecan concentrations at 5 min averaged 1.7 ± 0.2% injected dose per mL (% ID/mL), indicating that >98% of the irinotecan already had cleared from the blood. Both SN-38 and SN38G were detected at 5 min, illustrating rapid conversion and glucuronidation (Fig. 1A). Concentrations of SN-38 and SN-38G were similar over 6 h (AUC5 min-6 h of SN-38 and SN-38G were 2.52 and 2.84 µg/mL·h, respectively) (Supplementary Table S3). The combined AUCs of SN-38 and SN-38G divided by the AUC of irinotecan estimated the conversion rate to be ~25%, confirming the more efficient conversion in mice compared to humans (6, 15, 16). In the IMMU-132-treated animals, at 1 h SN-38 [TOTAL] concentrations accounted for 58.7 ± 3.3% ID/mL of the estimated amount of SN-38 equivalents. At each interval examined, free SN-38 levels 8
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were 95% of the total SN-38 in the serum is bound to the IgG, (c) the amount of free SN-38G in the serum is only a fraction of the free SN-38, and (d) there is no evidence of glucuronidation of SN-38 bound to IgG. Analysis of serum samples taken from patients given 8 to 10 mg/kg of IMMU-132 on days 1 and 8 of 21-day cycles has found free SN-38 concentrations at levels of ~100 ng/mL 30 minutes after 17
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the end of a 2-3 h infusion, while the total SN-38 levels average ~4000 ng/mL (34). These levels decrease steadily, being undetectable within 2-3 days; SN-38G levels in sera never exceeded SN-38. Thus, we suspect the levels of free SN-38 in the serum of patients receiving IMMU-132 therapy, which are similar to the peak levels of SN-38 seen in patients given irinotecan therapy (18), contribute to the dose-limiting neutropenia in patients given IMMU-132, whereas the significantly lower levels of SN-38G with IMMU-132 reduces the incidence of severe diarrhea. In conclusion, this ADC platform departs from the current practice using stably-linked ultra-toxic agents (subnanomolar), employing instead a moderately-toxic agent (low nanomolar) that is released in serum. IMMU-132 was found to deliver much higher levels of SN-38 to tumors than irinotecan and, importantly, all of the SN-38 delivered to the tumor by IMMU-132 is released in its most potent form. In relationship to gastrointestinal toxicity, the much lower amounts of SN-38G in the serum and significantly lower amounts of SN-38/SN38G in the intestine with IMMU-132 are expected to reduce the risk for severe diarrhea in patients, which is confirmed in clinical studies (34-38). Thus, IMMU-132 is an ADC that appears to have unique properties that combine to make an effective therapeutic agent in the solid cancers tested.
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Acknowledgements We thank Jennifer Inghrim, Nalini Sathyanarayan, Jayson Jebson, Roberto Arrojo, and Preeti Trisal, Maria Zalath for their technical assistance.
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Table 1. Examination of SN-38 concentrations in tissues of nude mice bearing 2 human cancer xenografts. IMMU-132 Irinotecan (SN-38 (SN-38 Intervals equivalents)a equivalents)a Tissues examined Study Tumor (N = 3 animals/interval)
1
b
2
3
Capan-1
Irinotecan 5 min, 1, 2, 6, and 24 h IMMU-132 1, 6, 24, 48, 72 h
Tumor, serum, liver, small intestine contents
1.0 mg (16 µg)
773 µg (448 µg)
Capan-1
Irinotecan 1 and 6 h IMMU-132 1, 6, and 24 h
Tumor, serum, liver, small and large intestine contents
1.0 mg (16 µg)
808 µg (468 µg)
NCI-N87
Irinotecan 5 min, 1, 2, 6, and 8 h IMMU-132 1, 6, 24, 48, 72 h
Tumor, serum
1.0 mg (16 µg)
840 µg (486 µg)
a
SN-38 equivalents for IMMU-132 based on spectrophotometric determinations of protein and SN-38 concentrations. SN-38 equivalents for irinotecan based on mass, with SN-38 representing approximately 58% of irinotecan’s mass. .
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Table 2. Concentrations of products over time (AUC) in mice bearing Capan-1 or NCI-N87 tumors administered irinotecan or IMMU-132a. Tissue Capan-1 Study 1 only [Study 1+2] NCI-N87
Irinotecan-treated SN-38 SN-38G Irinotecan 0.40 [1.09] 2.11
1.08 [1.04] 0.31
48.37 [46.73] 45.69
IMMU-132-treated SN-38 [TOTAL]
SN-38 deliver ratio IMMU-132/Irinotecan
54.25 [49.02] 43.88
135.6 [45.0] 20.8
a
AUC are expressed as (µg/g·h). In Study 1, SN-38 AUC derived from 5 min – 2 h; SN-38G and irinotecan from 5 min to 6 h. AUC from 5 min to 6 h for irinotecan-treated mice combining Studies 1 and 2. For IMMU-132-treated animals SN-38 [TOTAL] AUC are derived from 1 h to 72 h.
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Table 3. Determination of tumor/serum AUC ratiosa. Capan-1 [Study 1+2] Serum Irinotecan-treated SN-38 3.27 1.09 SN-38G 4.02 Irinotecan 27.48 IMMU-132-treated Free SN-38 Free SN-38G SN-38 [TOTAL]
3.87 1.00 157.21
49.02
NCI-N87
Tumor/serum AUC ratio Capan-1 NCI-N87
2.11
0.33
0.65
43.88
12.7
11.3
a
AUCs, expressed as µg/g·h were averaged from all 3 studies, with values derived from 5 min to 8 h for irinotecan-treated animals or from 1 h to 72 h for IMMU-132-treated animals.
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Figure Legends
Figure 1. Clearance of constitutive products in tumor-bearing nude mice given irinotecan or IMMU132. (A) Study 1, Capan-1-beaing mice; irinotecan. (B) Study 1; IMMU-132. (C) Study 3, NCI-N87-bearing mice; irinotecan. (D) Study 3; IMMU-132. Area-under-the-curve (AUC) estimates are provided in the table for products with 3 or more samples. Figure 2. Concentrations of products extracted from Capan-1 and NCI-N87 tumors isolated from animals given irinotecan or IMMU-132. (A) Concentrations of SN-38, SN-38G and irinotecan in Capan-1 tumors from mice given irinotecan in Study 1 and Study 2. In Study 1, SN-38 was below the threshold of detection in the 6-h sample, whereas SN-38G and irinotecan was detected in all samples. Study 2 only examined samples taken at 1 and 6 h. (B) Concentrations of products in NCI-N87 tumors from animals given irinotecan (Study 3). (C) Uptake and clearance of SN-38 [TOTAL] in mice bearing Capan-1 (Studies 1 and 2 combined) and NCI-N87 (Study 3). No other products were detected in the tumors. (D) Comparison of SN-38 [TOTAL] concentrations in Capan-1 xenografts from mice in Study 1 and Study 2. Figure 3. Contents of the upper small intestine analyzed for the various products in mice treated with irinotecan or IMMU-132 (Studies 1 and 2 combined, N = 3 to 6). Irinotecan animals were examined from 5 min to 24 h, while IMMU-132-treated animals were examined 1 to 72 h, but no products were found at detectable levels after 24 h.
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Enhanced Delivery of SN-38 to Human Tumor Xenografts with an Anti-Trop-2-SN-38 Antibody Conjugate (Sacituzumab Govitecan) Robert M Sharkey, William J McBride, Thomas M. Cardillo, et al. Clin Cancer Res Published OnlineFirst June 23, 2015.
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