SN-38 Conjugate Research Articles

A New Antibody-Drug Conjugate Composed of an Anti-HLA-DR IgG4 Antibody, IMMU-114, and SN-38, Is Active in Experimental Acute Myelocytic Leukemia (AML), Acute Lymphocytic Leukemia (ALL), and Multiple Myeloma (MM)

Introduction: Relapsed AML, ALL, and MM continue to be a therapy challenge. IMMU-114 (hL243) is a humanized anti-HLA-DR IgG4 monoclonal antibody engineered to lack effector-cell functions, but retains binding and a broad range of antitumor effects in diverse hematological neoplasms (Stein et al., Blood. 2010;115:5180-90). When given subcutaneously, it has encouraging efficacy in an initial Phase I clinical trial in relapsed or refractory NHL and CLL, with a good safety profile (ClinicalTrials.gov, NCT01728207).In vitro, AML has proven to be resistant to the antitumor effects of IMMU-114, despite high expression levels of HLA-DR. Likewise, in several different human ALL and MM cell lines, IMMU-114 has demonstrated a range of antitumor effects from a low of 9% to a high of 69%. In an effort to improve the antitumor activity of IMMU-114, an antibody-drug conjugate (ADC) was made in which IMMU-114 was conjugated with the active metabolite of irinotecan, SN-38. Another ADC utilizing SN-38 (sacituzumab govitecan) being studied in solid tumors has been well tolerated, with clinically significant objective responses in patients given multiple cycles over >6 months, with manageable neutropenia being the major toxicity. Thus, our goal was to determine if SN-38, a drug not commonly used in hematopoietic cancers, would prove to be an effective and safe therapeutic when targeted with the IMMU-114 antibody.In this current work, the in vivo activity of hL243-SN-38 versus parental IMMU-114 is examined in human AML, ALL, and MM xenografts.Methods : Conjugation of SN-38 to hL243 IgG4 resulted in a drug-to-antibody-ratio range of 6.1 to 6.6. For AML and MM disease models, NSG/SCID and C.B.-17 SCID mice received 2 Gy irradiation 24 h prior to an i.v. injection of MOLM-14 (2x106) or CAG cells (1x107), respectively. ALL was established in C.B-17 SCID mice injected with MN-60 cells (1x107). All therapies began 5 days post-tumor-cell injection. Test agents, including a non-targeting anti-CEA-SN-38 ADC, were administered as 500-mg injections twice-weekly for 4 wks. Animals were sacrificed at disease progression, characterized by the onset of hind-limb paralysis or loss of more than 15% body weight.Results: In experimental AML, saline control and IMMU-114 treated mice succumbed to disease progression quickly, with a median survival time (MST) of only 14 and 15 days, respectively. Conversely, mice treated with hL243-SN-38 had a greater than 1.5-fold increase in survival (MST=37 d, P=0.0031). Further, hL243-SN-38 therapy provided a significant survival benefit when compared to anti-CEA-SN-38 control (MST=21 d, P=0.0031). In mice bearing ALL xenografts, IMMU-114 provided a >60% improvement in survival compared to saline control (MST = 37 d vs. 22.5 d, respectively; P<0.0001), whereas hL243-SN-38 increased this by another 80% (MST=66.5 d) which was significantly better than all other treatments, including IMMU-114 (P<0.0001). Finally, MM mice had a greater than 123-d MST when treated with hL243-SN-38 compared to 32 d for saline control (P<0.0001). This survival benefit also was significantly higher than for mice treated with bortezomib (0.89 mg/kg) or control ADC (MST=32.5 d for both; P<0.0001). While not significant, hL243-SN-38 does provide a >30% improvement in survival when compared to IMMU-114 therapy (MST=94.5 d, P=0.1313). In all three experiments, therapy with hL243-SN-38 was well tolerated, as evidenced by no significant loss in body weight.Conclusions: Therapy with the hL243-SN-38 ADC proved to be superior to IMMU-114 (which is active clinically in NHL and CLL) in both AML and ALL xenografts and beneficial in MM. Most importantly, in IMMU-114-refractive AML, hL243-SN-38 demonstrated a significant antitumor effect without any undue toxicity. This new ADC is a candidate for clinical evaluation in these intractable malignancies. DisclosuresGoldenberg:Immunomedics: Employment, Equity Ownership, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Sharkey:Immunomedics, Inc.: Consultancy, Employment, Equity Ownership. Govindan:Immunomedics, Inc.: Employment, Equity Ownership, Patents & Royalties. Zalath:Immunomedics, Inc.: Employment, Equity Ownership. Mostafa:Immunomedics, Inc.: Employment, Equity Ownership. Cardillo:Immunomedics, Inc.: Employment, Equity Ownership.

Self-assembled nanoparticles comprising aptide–SN38 conjugates for use in targeted cancer therapy

Self-assembled nanoparticles (NPs) have been intensively utilized as cancer drug delivery carriers because hydrophobic anticancer drugs may be efficiently loaded into the particle cores. In this study, we synthesized and evaluated the therapeutic index of self-assembled NPs chemically conjugated to a fibronectin extra domain B-specific peptide (APTEDB) and an anticancer agent SN38. The APTEDB–SN38 formed self-assembled structures with a diameter of 58 ± 3 nm in an aqueous solution and displayed excellent drug loading, solubility, and stability properties. A pharmacokinetic study revealed that the blood circulation half-life of SN38 following injection of the APTEDB–SN38 NPs was markedly higher than that of the small molecule CPT-11. The APTEDB–SN38 NPs delivered SN38 to tumor sites by both passive and active targeting. Finally, the APTEDB–SN38 NPs exhibited potent antitumor activities and low toxicities against EDB-expressing tumors (LLC, U87MG) in mice. This system merits further preclinical and clinical investigations for SN38 delivery.

Combining ABCG2 Inhibitors with IMMU-132, an Anti-Trop-2 Antibody Conjugate of SN-38, Overcomes Resistance to SN-38 in Breast and Gastric Cancers.

Sacituzumab govitecan (IMMU-132), an SN-38-conjugated antibody-drug conjugate, is showing promising therapeutic results in a phase I/II trial of patients with advanced Trop-2-expressing, metastatic, solid cancers. As members of the ATP-binding cassette (ABC) transporters confer chemotherapy resistance by active drug efflux, which is a frequent cause of treatment failure, we explored the use of known inhibitors of ABC transporters for improving the therapeutic efficacy of IMMU-132 by overcoming SN-38 resistance. Two human tumor cell lines made resistant to SN-38, MDA-MB-231-S120 (human breast cancer) and NCI-N87-S120 (human gastric cancer), were established by continuous exposure of the parental cells to stepwise increased concentrations of SN-38 and analyzed by flow cytometry for functional activities of ABCG2 and ABCB1, immunoblotting and qRT-PCR for the expression of ABCG2 at both protein and mRNA levels, and MTS assays for the potency of SN-38 alone or in combination with a modulator of ABC transporters. MDA-MB-231-S120 and NCI-N87-S120 displayed reduced sensitivity to SN-38 in vitro, with IC50 values approximately 50-fold higher than parental MDA-MB-231 and NCI-N87 cells. The increase in drug resistance of both S120 cell populations is associated with the expression of functional ABCG2, but not ABCB1. Importantly, treatment of both S120 sublines with known ABCG2 inhibitors (fumitremorgin C, Ko143, and YHO-13351) restored toxicity of SN-38, and the combination of YHO-13351 with IMMU-132 increased the median survival of mice bearing NCI-N87-S120 xenografts. These results provide a rationale for combination therapy of IMMU-132 and inhibitors of ABC transporters, such as YHO-13351. Mol Cancer Ther; 15(8); 1910-9. ©2016 AACR.

Synthesis, characterization, and evaluation of mPEG-SN38 and mPEG-PLA-SN38 micelles for cancer therapy.

7-Ethyl-10-hydroxy camptothecin (SN38) is a potent topoisomerase inhibitor and a metabolite of irinotecan. Its clinical development has been hampered by its poor solubility. To address this problem, methoxy poly(ethylene glycol)-2000 (mPEG2K)–SN38 and mPEG2K–poly(lactide) (PLA1.5K)–SN38 conjugates were prepared and then dispersed into an aqueous medium to form micelles. Physicochemical characteristics of SN38–polymer conjugate micelles, for example, micelle diameter, zeta potential, morphology, and drug content, were then evaluated. The results showed that the mean diameters of mPEG2K–SN38 and mPEG2K–PLA1.5K–SN38 micelles were ~130 and 20 nm, respectively. These two micelles had similar drug contents. mPEG2K–PLA1.5K–SN38 micelles were more homogeneous than mPEG2K–SN38 micelles. Moreover, in vitro drug release behavior of the micelles was studied by high performance liquid chromatography. SN38 release from mPEG2K–SN38 micelles was much faster than from mPEG2K–PLA1.5K–SN38 micelles. In vitro cytotoxicity, cellular uptake, and apoptosis assays of the SN38–polymer conjugate micelles were carried out on BEL-7402 human liver cancer cells. In vivo biodistribution and antitumor tumor efficacy studies were carried out in a nude mouse xenograft model derived from BEL-7402 cells. The results showed that mPEG2K–PLA1.5K–SN38 micelles were significantly more effective than mPEG2K–SN38 micelles in tumor inhibition, and the inhibitory effect of mPEG2K–PLA1.5K–SN38 micelles on tumor growth was significantly greater than that of mPEG2K–SN38 micelles (1,042 vs 1,837 mm) at 30 days. In conclusion, mPEG–PLA–SN38 is a promising anticancer agent that warrants further investigation.

HSP90 Inhibitor-SN-38 Conjugate Strategy for Targeted Delivery of Topoisomerase I Inhibitor to Tumors.

The clinical benefits of chemotherapy are commonly offset by insufficient drug exposures, narrow safety margins, and/or systemic toxicities. Over recent decades, a number of conjugate-based targeting approaches designed to overcome these limitations have been explored. Here, we report on an innovative strategy that utilizes HSP90 inhibitor-drug conjugates (HDC) for directed tumor targeting of chemotherapeutic agents. STA-12-8666 is an HDC that comprises an HSP90 inhibitor fused to SN-38, the active metabolite of irinotecan. Mechanistic analyses in vitro established that high-affinity HSP90 binding conferred by the inhibitor backbone could be exploited for conjugate accumulation within tumor cells. In vivo modeling showed that the HSP90 inhibitor moiety was required for selective retention of STA-12-8666, and this enrichment promoted extended release of active SN-38 within the tumor compartment. Indeed, controlled intratumoral payload release by STA-12-8666 contributed to a broad therapeutic window, sustained biomarker activity, and remarkable degree of efficacy and durability of response in multiple cell line and patient-derived xenograft models. Overall, STA-12-8666 has been developed as a unique HDC agent that employs a distinct mechanism of targeted drug delivery to achieve potent and sustained antitumor effects. These findings identify STA-12-8666 as a promising new candidate for evaluation as novel anticancer therapeutic.

Abstract 5537: Nanoparticle delivery of an SN38 conjugate is more effective than Irinotecan in a mouse model of Neuroblastoma

Abstract Purpose: Neuroblastoma (NB) is the most common and deadly solid tumor in childhood. The majority of NB patients have advanced stage disease and a poor prognosis, so more effective, less toxic therapy is needed. We wanted to determine if more targeted delivery of chemotherapeutic agents would improve efficacy and reduce systemic toxicity. We developed a novel nanocarrier-based strategy for tumor-targeted delivery of a prodrug of SN38, the active metabolite of irinotecan. Experimental Design: We formulated ultrasmall-sized (&amp;lt;100 nm) biodegradable poly(lactide)-poly(ethylene glycol) based nanoparticles (NPs) containing SN38 conjugated to tocopherol succinate (SN38-TS). We assessed alternative dosing schedules of SN38-TS NPs compared to irinotecan. An equivalent dose of SN38-TS NPs given 1x/2wk x 4 weeks (2 doses) had identical efficacy to irinotecan given 5x/wk x 4 weeks (20 doses), but none were cured. We then compared irinotecan 5x/wk x 8 weeks (40 doses) to SN38-TS NPs given 2x/wk x 4 weeks, 1x/wk x 8 weeks, and 2x/wk x 8 weeks (8, 8, and 16 doses, respectively). Results: All SN38-TS NP regimens were far superior to irinotecan, and “cures” were obtained in all NP arms (no cures with irinotecan). SN38-TS NP delivery resulted in 200x the amount of SN38 in NB tumors at 4 hr post-treatment, and 25% of the 4-hr SN38 level remained at 72 hr post-treatment, compared to no SN38 detected at 24 hr post-treatment for the irinotecan arm; no toxicity was seen with NPs. Conclusions: We conclude that this SN38-TS NP formulation improved delivery, retention, and efficacy, without causing systemic toxicity. Citation Format: Radhika Iyer, Jamie L. Croucher, Michael Chorny, Jennifer L. Mangino, Ivan S. Alferiev, Robert J. Levy, Venkatadri Kolla, Garrett M. Brodeur. Nanoparticle delivery of an SN38 conjugate is more effective than Irinotecan in a mouse model of Neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5537. doi:10.1158/1538-7445.AM2015-5537

Preclinical efficacy of STA-12-8666, an HSP90 inhibitor-targeted SN-38 conjugate, in small cell lung cancer (SCLC).

e18560 Background: Small cell lung cancer (SCLC) is a lethal disease, with 5 year survival of only 6%. While patients initially respond to cytotoxic chemotherapies, resistance rapidly emerges and r...

Drug conjugation to hyaluronan widens therapeutic indications for ovarian cancer.

Management of ovarian cancer still requires improvements in therapeutic options. A drug delivery strategy was tested that allows specific targeting of tumor cells in combination with a controlled release of a cytotoxic molecule. To this aim, the efficacy of a loco-regional intraperitoneal treatment with a bioconjugate (ONCOFID-S) derived by chemical linking of SN-38, the active metabolite of irinotecan (CPT-11), to hyaluronan was assessed in a mouse model of ovarian carcinomatosis. In vitro, the bioconjugate selectively interacted with ovarian cancer cells through the CD44 receptor, disclosed a dose-dependent tumor growth inhibition efficacy comparable to that of free SN-38 drug, and inhibited Topoisomerase I function leading to apoptosis by a mechanism involving caspase-3 and -7 activation and PARP cleavage. In vivo, the intraperitoneal administration of ONCOFID-S in tumor-bearing mice did not induce inflammation, and evidenced an improved therapeutic efficacy compared with CPT-11. In conclusion, SN-38 conjugation to hyaluronan significantly improved the profile of in vivo tolerability and widened the field of application of irinotecan. Therefore, this approach can be envisaged as a promising therapeutic strategy for loco-regional treatment of ovarian cancer.

Nanoparticle delivery of an SN38 conjugate is more effective than irinotecan in a mouse model of neuroblastoma

Neuroblastoma (NB) is the most common and deadly solid tumor in children. The majority of NB patients have advanced stage disease with poor prognosis, so more effective, less toxic therapy is needed. We developed a novel nanocarrier-based strategy for tumor-targeted delivery of a prodrug of SN38, the active metabolite of irinotecan. We formulated ultrasmall-sized (<100 nm) biodegradable poly(lactide)-poly(ethylene glycol) based nanoparticles (NPs) containing SN38 conjugated to tocopherol succinate (SN38-TS). Alternative dosing schedules of SN38-TS NPs were compared to irinotecan. Comparison of SN38-TS NPs (2 doses) with irinotecan (20 doses) showed equivalent efficacy but no cures. Comparison of SN38-TS NPs (8, 8, and 16 doses, respectively) to irinotecan (40 doses) showed that all SN38-TS NP regimens were far superior to irinotecan, and “cures” were obtained in all NP arms. SN38-TS NP delivery resulted in 200× the amount of SN38 in NB tumors at 4 hr post-treatment, compared to SN38 detected for the irinotecan arm; no toxicity was seen with NPs. We conclude that this SN38-TS NP formulation improved delivery, retention, and efficacy, without causing systemic toxicity.

Preclinical Testing Demonstrates Striking Efficacy of STA-12-8666, an Hsp90 Inhibitor–Targeted SN-38 Conjugate, in Small Cell Lung Cancer: Small Cell Lung Cancer

Preclinical Testing Demonstrates Striking Efficacy of STA-12-8666, an Hsp90 Inhibitor–Targeted SN-38 Conjugate, in Small Cell Lung Cancer: Small Cell Lung Cancer

Abstract 4471: AD-O64.4 - a novel bioconjugate for tumor-targeted drug delivery

Abstract Although standard chemotherapy is still one of the most effective treatment for many types of cancer - often causes side effects. Chemotherapeutic agents primarily damage cancer cells with a rapidly dividing and growing profile, thereby also destroy healthy cells with such characteristics, which leads to side effects. Scientists are continually working to identify new, safer drugs, methods of administering chemotherapy, and combinations of existing treatments that have fewer side effects. In recent years, drug delivery systems (DDS) based on a concept of conjugation anticancer agents to carrier protein have been developed, achieving a better clinical response and tolerability. Protein-drug conjugates represent a whole new concept for cancer treatment which, although highly effective, cause much fewer sides effects than traditional chemotherapy. The present work is aimed to create a new conjugate molecule which essentially demonstrates greater effect than the summarized effects of its components due to specific targeting to cancer cells. We generated AD-O64.4 complex molecule, consisting of fusion protein INF- γ - TRAIL/Apo2L conjugated with the anti-mitotic agent SN-38 via the thioether linker. Carrier protein is a fusion molecule which is composed of soluble fragment of TRAIL (acting as a targeting carrier and effector) fused with artificial dimer of IFN-γ (acting as effector). SN-38, a topoisomerase I inhibitor with low nanomolar potency, the active metabolite of irinotecan, cannot be given directly to patients because of its toxicity and poor solubility. Linking SN-38 to the carrier fusion protein results in selective drug delivery to tumors which consequently leads to increased amount of the drug reaching the tumors and minimized damage of healthy cells. Analysis of the conjugate was performed by LC-MS and resulted in determination of drug-protein ratio. Agregation studies of the molecule complex were conducted by DLS and fluorescent protein aggregation assay. Cytotoxicity was evaluated with MTT assay and efficacy was performed on female SCID mice xenograft model bearing Human Uterine Sarcoma (MES-SA/Dx5). Our results show an effective process of conjugation of anticancer compound SN-38 to the carrier protein which leads to formation of an active complex with enhanced efficacy. AD-O64.4 exhibited cytotoxic effect on various cancer cell lines, (IC50 about 10 ng/ml), but showed no toxic effect on normal cells. This conjugate was also highly cytotoxic against primary cancer cells. AD-O64.4 was highly active (up to 75% tumor remission) against subcutaneous multiple drug resistant, MES-SA/Dx5, tumor xenografts in severe combined immunodeficient mice with better activity compared to the carrier protein alone, as well as to combination of the carrier protein with free-drug. Overall, these results represent a novel and a valuable system for drug delivery to the tumor and its microenvironment. Citation Format: Wojciech Strozek, Anna Pieczykolan, Bartlomiej Zerek, Michal Szymanik, Albert Jaworski, Marlena Galazka, Katarzyna Bukato, Piotr Rozga, Sebastian Pawlak, Katarzyna Poleszak, Malgorzata Teska-Kaminska, Jerzy Pieczykolan. AD-O64.4 - a novel bioconjugate for tumor-targeted drug delivery. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4471. doi:10.1158/1538-7445.AM2014-4471

MUC1 aptamer conjugated to chitosan nanoparticles, an efficient targeted carrier designed for anticancer SN38 delivery

Molecularly targeted therapy is of great interest for diagnosis and treatment of cancerous cells due to its low toxicity for normal cells. In this study, chitosan was utilized as a promising carrier for delivery, and aptamer (Apt) was employed for active targeting of SN38 to colon cancer. SN38 cannot be used clinically due to its poor solubility and high toxicity. Developing nanoparticles (NPs) of drug–polymer conjugates can be a good candidate for overcoming such problems. N-Carboxyethyl chitosan ester (CS-EA) was synthesized as an intermediate for conjugation of SN38 to chitosan. MUC1 DNA aptamer with 5′-NH2 functional group was conjugated to the self-assembled conjugate as a targeting agent. Prepared NPs had smooth and spherical morphology with 200nm particle size. Conjugation of aptamer was confirmed by gel electrophoresis. In vitro cytotoxicity of NPs was assessed by HT-29 as MUC1 positive cell line through MTT assay. Aptamer conjugated NPs (Apt NPs) were more toxic than non-targeted NPs, however they were as toxic as free drug. Cellular uptake and targeting ability of prepared NPs were also confirmed via confocal microscopy. As a conclusion, prepared CS-SN38–Apt NPs can increase efficacy of drug SN38 through increasing solubility and specific delivery to the target tissue.

In vitro cytotoxicity, pharmacokinetics and tissue distribution in rats of MXN-004, a novel conjugate of polyethylene glycol and SN38

1. MXN-004 is a water-soluble PEGylated 7-ethyl-10-hydroxy-camptothecin (SN38). The aim of this study was to evaluate the in vitro cytotoxicity of MXN-004 and investigate pharmacokinetics and tissue distribution of MXN-004 and its active metabolite SN38 in rats.2. In vitro cytotoxicity of MXN-004 was tested in A549, HepG2 and Caco-2 cancer cell lines by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and compared with irinotecan. The pharmacokinetics and tissue distribution of MXN-004, irinotecan and their identical active metabolite SN38 were investigated after intravenous administration of MXN-004 and irinotecan at a same dose level of 16 μmol/kg in rats.3. In vitro cytotoxicity study showed that MXN-004 was more potent in comparison with irinotecan. In rats, MXN-004 exhibited a longer half-life (sixfold) and much greater Vss as compared with irinotecan. The AUC0–∞, T1/2 and Cmax of SN38 after intravenous administration of MXN-004 were higher than those of irinotecan (3.5-, 1.92- and 10.6-fold, respectively). In addition, the concentrations of SN38 released from MXN-004 were significantly higher in all tissues than those from irinotecan, especially in the lung.4. These results suggested that MXN-004 might be a more potential water-soluble antitumor agent with prolonged half-life of SN38 compared to irinotecan.

Milatuzumab–SN-38 Conjugates for the Treatment of CD74+ Cancers

CD74 is an attractive target for antibody-drug conjugates (ADC), because it internalizes and recycles after antibody binding. CD74 mostly is associated with hematologic tumors but is expressed also in solid cancers. Therefore, ADCs of the humanized anti-CD74 antibody, milatuzumab, were examined for the therapy of CD74-expressing solid tumors. Milatuzumab-doxorubicin and two milatuzumab-SN-38 conjugates with cleavable linkers, differing in their stability in serum and how they release SN-38 in the lysosome, were prepared. CD74 expression was determined by flow cytometry and immunohistology. In vitro cytotoxicity and in vivo therapeutic studies were conducted in the human cancer cell lines A-375 (melanoma), HuH-7 and Hep-G2 (hepatoma), Capan-1 (pancreatic), NCI-N87 (gastric), and Raji Burkitt lymphoma. The milatuzumab-SN-38 ADC was compared with SN-38 ADCs prepared with anti-Trop-2 and anti-CEACAM6 antibodies in xenografts expressing their target antigens. Milatuzumab-doxorubicin was most effective in the lymphoma model, whereas in A-375 and Capan-1 solid tumors, only milatuzumab-SN-38 showed a therapeutic benefit. Despite much lower surface expression of CD74 than Trop-2 or CEACAM6, milatuzumab-SN-38 had similar efficacy in Capan-1 as anti-Trop-2-SN-38, but in NCI-N87, anti-CEACAM6 and anti-Trop-2 conjugates were superior. Studies in two hepatoma lines at a single dose level showed significant benefit over saline controls but not against an irrelevant immunoglobulin G conjugate. CD74 is a suitable target for ADCs in some solid tumor xenografts, with efficacy largely influenced by uniformity of CD74 expression and with SN-38 conjugates providing the best therapeutic responses; SN-38 conjugates were preferable in solid cancers, whereas doxorubicin ADC was better in lymphoma tested.

Phase I dose-escalation study of EZN-2208 (PEG-SN38), a novel conjugate of poly(ethylene) glycol and SN38, administered weekly in patients with advanced cancer

This study evaluated the tolerability, pharmacokinetics, and preliminary antitumor activity of EZN-2208, a water-soluble poly(ethylene) glycol conjugate of SN38. Patients with advanced malignancies were enrolled in dose-escalating cohorts (3 + 3 design). EZN-2208 was administered as a 1-h intravenous infusion given weekly for 3 weeks per each 4-week cycle. Doses ranged from 1 to 12 mg/m(2). Forty-one patients received EZN-2208. All patients had received prior cancer therapy (median = 2, range = 1-11). Twenty patients (49 %) had received prior irinotecan, and one patient had received prior topotecan. One patient in the 9-mg/m(2) cohort had dose-limiting toxicity (grade 3 febrile neutropenia), and one patient in the 12-mg/m(2) cohort had grade 3 neutropenia that resulted in the inability to deliver the third dose of EZN-2208. The most commonly reported drug-related adverse events were nausea (51 %), diarrhea (46 %), fatigue (41 %), alopecia (29 %), neutropenia (24 %), and vomiting (22 %). Administration of EZN-2208 results in prolonged exposure to SN38. Stable disease, sometimes prolonged, was observed as best response. EZN-2208 has an acceptable safety profile in previously treated patients with advanced malignancies. The recommended phase II dose of EZN-2208 administered according to this schedule was 9 mg/m(2).

Synthesis, characterization, and antitumor evaluation of the albumin–SN38 conjugate

7-Ethyl-10-hydroxycamptothecin (SN38), the active metabolite of irinotecan, exerts a 100-fold to 1000-fold higher effect than irinotecan itself against several tumor cell lines. However, the water insolubility of SN38 has prevented its direct use as an antitumor drug in the clinic. To improve the water solubility and antitumor efficacy, SN38 was covalently attached to the only free sulfhydryl at cysteine-34 on the BSA site specifically through a thiol-binding linker to form a prodrug BSA-SN38 conjugate (BSA : SN38=1 : 1). The water solubility of this conjugate was similar to albumin using the current method. Also, SN38 loading in this conjugate became controllable. Size-exclusion chromatography purification and UV characterization of the SDS-PAGE electrophoresis product were carried out. Then, an MTT assay was carried out to test the antitumor effect of this conjugate on five colon cancer cell lines in vitro. The 72 h IC50 values of the BSA-SN38 conjugate ranged from 1.5 to 6.1 μmol/l. A colorectal peritoneal carcinomatosis model in mice was established to determine the intraperitoneal chemotherapy effect of the BSA-SN38 conjugate. The BSA-SN38 conjugate at an SN38 equivalent dose of 10 mg/kg/day was administrated every 4 days. Eighteen days after manipulation, the mice were euthanized and the tumors in the abdominal cavity were collected and weighed. Tumors in the BSA-SN38 conjugate treatment group (m=0.21 ± 0.15 g) were found to be significantly (P=5) lighter than those in the NS control group (m=4.74±0.73 g). The results indicated that this water-soluble BSA-SN38 conjugate exerted a strong antitumor effect on colorectal carcinoma.

Safety, pharmacokinetics, and activity of EZN‐2208, a novel conjugate of polyethylene glycol and SN38, in patients with advanced malignancies

EZN-2208 is a water-soluble, polyethylene glycol drug conjugate of SN38, which is the active moiety of irinotecan. In this study, the authors evaluated the tolerability, pharmacokinetics (PK), and activity of EZN-2208 in adult patients with advanced solid tumors. Patients in sequential cohorts (3 + 3 design) received intravenous EZN-2208 at doses between 1.25 mg/m(2) and 25 mg/m(2) once every 21 days. Thirty-nine patients received EZN-2208. The median number of prior therapies was 2 (range, 0-10 prior therapies). Seventeen patients received prior irinotecan. Two maximum tolerated doses (MTDs) were defined: EZN-2208 with (16.5 mg/m(2)) and without (10 mg/m(2)) granulocyte-colony-stimulating factor (G-CSF). The dose-limiting toxicity (DLT) was febrile neutropenia. Two of 19 patients who were heterozygous for a polymorphism in the uridine diphosphate glucuronosyltransferase 1 family, polypeptide A1 (UGT1A1) gene (UGT1A1*28) developed DLTs (dose, 25 mg/m(2) with G-CSF), and 2 patients who were homozygous for UGT1A1*28 were treated without DLTs (dose, 5 mg/m(2)). PK analysis indicated a mean terminal half-life of 19.4 ± 3.4 hours. Sixteen patients (41%) achieved stable disease, including 6 of 39 patients (15%) who had stable disease that lasted ≥ 4 months. One patient with cholangiocarcinoma (no prior irinotecan) achieved a short-lived 32% tumor regression. Among 6 patients who had stable disease that lasted for ≥ 4 months, 3 had received prior irinotecan, and 1 had KRAS-positive colorectal cancer. EZN-2208 was well tolerated and produced stable disease that lasted for ≥ 4 months/unconfirmed partial responses in 7 of 39 heavily pretreated patients (18%) with advanced solid tumors, including those who had failed prior irinotecan therapy.

Final analysis of phase II study of EZN-2208 (PEG-SN38) in metastatic breast cancer (MBC).

1017 Background: EZN-2208 is a water-soluble PEGylated conjugate of SN38. EZN-2208 results in prolonged exposure of tumors to SN38 via preferential accumulation of EZN-2208 in the tumor and prolonged release of SN38. These data represent the final analysis of our study evaluating EZN-2208 in MBC. Methods: EZN-2208 9 mg/m2 (SN38 equivalents) was delivered as a 60-minute IV infusion, weekly for 3 wks in 4‑wk cycles. The primary objective was to determine the overall response rate (RR) in female patients with metastatic breast cancer (MBC) who had received prior adjuvant or metastatic therapy with either 1) anthracycline and taxane (AT) or 2) anthracycline, taxane, and capecitabine (ATX). Secondary objectives included evaluation of RR based on tumor receptor status, duration of response, progression-free survival (PFS), overall survival (OS), and safety. Results: Patients with MBC (n=164) were treated with a median (range) of 3.3 (0.3-22) cycles of EZN-2208. The objective response rate (RR) was 20% for AT and 9% for ATX. The clinical benefit rate (CBR=%CR + %PR + %SD ≥6 months) was 41% and 27% in patients in the AT and ATX cohorts, respectively. The RR and CBR among ER+ patients were 11% (10/91 pts) and 41.8% (38/91 pts). In patients who progressed during or within 30 days of prior platinum-containing regimens (Platinum Progressors), the CBR was 20% (8/40 pts). Among triple negative breast cancer (TNBC) patients, the RR and CBR were 22.5% (11/49 pts) and 36.7% (18/49 pts). For TNBC, Platinum Progressors, the CBR was 26.1% (6/23 pts). Overall, most common reported drug-related adverse events were diarrhea, nausea and neutropenia. Conclusions: EZN-2208 has notable activity in patients with previously treated MBC and appears to be an active agent for treatment of TNBC. Patients with TNBC, who had been previously treated with a platinum-based regimen, also derive clinical benefit from EZN-2208. The safety profile of EZN-2208 is acceptable with good tolerability in most patients. Further evaluation of EZN-2208 in MBC in general and TNBC in particular is warranted.

Therapy of human solid tumor xenografts with CD74-targeted milatuzumab-SN-38 immunoconjugates.

3091 Background: CD74 is commonly considered to be an antigen present in hematopoietic cancers, but it is also expressed in a number of solid tumors. The humanized anti-CD74 antibody, milatuzumab (hLL1), was previously shown to have exquisite internalization properties, making it an attractive carrier for drug delivery. In this study, we evaluated hLL1 conjugates of a potent topo I inhibitor, SN-38, for treating solid tumors. Methods: Two hLL1-SN-38 conjugates, possessing either a pH-sensitive linker (‘CL2A’ form) or a cathepsin-B-cleavable linker ("CL2E" form), were examined. In vitro analyses were carried out in the A-375 human melanoma cell line, and therapy experiments were performed in female athymic nude mice bearing established s.c. A-375 or s.c. human pancreatic adenocarcinoma (Capan-1) xenografts using specified doses (i.p.) at a twice weekly × 4 wks schedule; animals were sacrificed when tumor volumes (TVs) reached 1 cm3. Results: Both A-375 and Capan-1 cell lines tested positive for CD74 expression by IHC. In vitro, both SN-38 derivatives liberated free drug at the same rate when exposed to cathepsin-B at pH 5, while serum stabilities for CL2A and CL2E forms of the conjugates were ~ 1 d and &gt;10 d, respectively. In the A-375 melanoma cell line, IC50 for the hLL1-SN-38 conjugates of CL2A and CL2E forms were 5 nM and 34 nM, respectively. In the aggressive A-375 s.c. model of melanoma, in nude mice (n =8; TV: 0.23 ± 0.06 cm3) treated with 12.5 mg/kg protein dose (total 1.7 mg/kg SN-38 eq.) of specific or non-specific SN-38 conjugates of CL2A and CL2E forms, only hLL1-CL2A-SN-38 conjugate was efficacious (Log-rank: P&lt;0.009 vs. all controls), with a median survival time of 28 d vs. 10.5 d for untreated. Likewise, in mice bearing Capan-1 xenografts (n = 8-10; TV=0.27 ± 0.05 cm3), two low doses of hLL1-CL2A-SN-38 (12.5 or 5 mg/kg) significantly improved survival in comparison to saline control mice (p&lt;0.035), whereas the CL2E form and a non-specific control demonstrated no efficacy. Conclusions: These results indicate the therapeutic potential of hLL1-CL2A-SN-38 conjugate and the importance of linker chemistry, and encourage additional testing in other CD74-positive solid cancers.

Abstract 2526: Optimal cleavable linker for antibody-SN-38 conjugates for cancer therapy: Impact of linker's stability on efficacy

Abstract We reported therapeutic results of antibody (MAb) conjugates of the topo I inhibitor, SN-38, in human tumor xenografts in nude mice. These involved SN-38 derivatives, CL2-SN-38 (maleimido-[x]-Phe-Lys-PABOCO-20-O-SN-38) and its ‘Phe’-deleted variant, CL2A-SN-38. Conjugates of these derivatives showed similar serum stabilities and equivalent therapeutic efficacies. A more stably linked, cathepsin-B sensitive, derivative, CL2E-SN-38 (maleimido-[x]-Phe-Lys-PABOCO-N(Me)-(CH2)2-N(Me)-CO-10-O-SN-38) was prepared to determine the relative advantage of CL2E vs. CL2A linkers for MAb-SN-38 conjugates using MAbs with a spectrum of internalizing rates in hematological and solid tumor cell lines. Initial studies found these linkers differed in the drug-release rate from the MAb conjugates in human serum, with half-lives (t½) of ∼ 1 day for CL2A- and &amp;gt;10 days for CL2E-conjugates. The t½ for cathepsin-B cleavage at the lysosomal pH (pH 5) for CL2A- and CL2E-SN-38 derivatives were 10 h and 0.5 h, respectively; the CL2A linker cleavage rate at this pH was the same with or without the enzyme, indicating a pH-mediated, but cathepsin-B insensitive, drug release. The release of SN-38 from CL2E-SN-38, requiring both the cathepsin-B cleavage and an intramolecular cyclization, proceeded with an overall t½ of 10 h at pH 5, very similar to that for CL2A-SN-38, suggesting that the corresponding MAb conjugates would liberate free SN-38 at a similar rate in the lysosome. For a slowly internalizing anti-CEACAM5 MAb, hMN-14 (labetuzumab), SN-38 conjugate with the CL2A linker was superior to that with CL2E in the LS174T colonic tumor cell line and xenograft model. With a more rapidly internalizing anti-TROP-2 MAb, hRS7, IC50s for CL2A- and CL2E-SN-38 conjugates were 9 nM and 132 nM, and 20 nM and 242 nM, in the pancreatic tumor cell line, Capan-1, and the lung adenocarcinoma cell line, Calu-3, respectively. CL2A-SN-38 and CL2E-SN-38 conjugates of the fast internalizing anti-CD22 MAb, hLL2 (epratuzumab), exhibited EC50 values of 3.2 nM and 135.8 nM, respectively, in the Raji lymphoma cell line. With an anti-CD74 MAb, hLL1 (milatuzumab), which internalizes exceptionally quickly with rapid CD74 re-cycling, CL2A- and CL2E-SN-38 conjugates showed IC50 values of 5 nM and 34 nM, respectively, in the CD74-positive melanoma cell line, A-375. The IC50 for free SN-38 ranged from 0.5 nM to 7 nM in these cell lines. While specific antitumor effects against human tumor xenografts were found using CL2A-SN-38 conjugates, specificity could not be shown in a 4-day in vitro MTS assay, most likely because of the 1-day half-life for drug release. However, with hLL1-CL2E-SN-38 conjugate in the A-375 cell line, specificity was established vs. non-targeting anti-CD22 conjugate, hLL2-CL2E-SN-38. The significance of these findings will be presented, together with the data from ongoing therapy studies in xenograft models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2526. doi:1538-7445.AM2012-2526