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Supplemental Figures S1-S8, supplemental methods

PDF file - 1.5 MB, Reversed-phase HPLC analyses for cleavage of ME-capped CL2E-SN-38 by cathepsin-B

Chemoimmunotherapy with antibody–drug conjugates (ADC) is emerging as a promising therapy for solid tumors, whereas radioimmunotherapy (RAIT) of solid tumors has been relatively ineffective because of their resistance to radiation. We developed antibody–SN-38 conjugates that have significant antitumor activity in xenograft models at nontoxic doses. The goal of this study was to determine if an ADC could be combined with RAIT to enhance efficacy without a commensurate increase in host toxicity. Nude mice bearing human pancreatic cancer xenografts (Capan-1 and BxPC-3) were treated with a single dose of 90Y-labeled antimucin antibody (hPAM4; clivatuzumab tetraxetan) alone or in combination with an anti-Trop-2–SN-38 conjugate, typically administered twice weekly over 4 weeks. The combination, even at RAIT's maximum tolerated dose, controlled tumor progression and cured established xenografts significantly better than the individual treatments without appreciable toxicity. The ADC could be started 1 week after or up to 2 weeks before RAIT with similar enhanced responses, but delaying RAIT for 2 weeks after the ADC was less effective. A nonspecific ADC provided additional benefit over using free drug (irinotecan), but the response was enhanced with the specific ADC. When targeting Capan-1 with ample mucin, hPAM4 could be used as the RAIT and the ADC agent without losing effectiveness, but in BxPC-3 with less mucin, targeting of different antigens was preferred. These studies show the feasibility of combining ADC and RAIT for improved efficacy without increased toxicity. Mol Cancer Ther; 10(6); 1072–81. ©2011 AACR.

Labetuzumab govitecan (IMMU-130), an antibody–drug conjugate (ADC) with an average of 7.6 SN-38/IgG, was evaluated for its potential to enhance delivery of SN-38 to human colonic tumor xenografts. Mice bearing LS174T or GW-39 human colonic tumor xenografts were injected with irinotecan or IMMU-130 (SN-38 equivalents ∼500 or ∼16 μg, respectively). Serum and homogenates of tumors, liver, and small intestine were extracted, and SN-38, SN-38G (glucuronidated SN-38), and irinotecan concentrations determined by reversed-phase HPLC. Irinotecan cleared quickly from serum, with only 1% to 2% injected dose/mL after 5 minutes; overall, approximately 20% was converted to SN-38 and SN-38G. At 1 hour with IMMU-130, 45% to 63% injected dose/mL of the SN-38 was in the serum, with >90% bound to the ADC over 3 days, and with low levels of SN-38G. Total SN-38 levels decreased more quickly than the IgG, confirming a gradual SN-38 release from the ADC. AUC analysis found that SN-38 levels were approximately 11- and 16-fold higher in LS174T and GW-39 tumors, respectively, in IMMU-130–treated animals. This delivery advantage is amplified >30-fold when normalized to SN-38 equivalents injected for each product. Levels of SN-38 and SN-38G were appreciably lower in the liver and small intestinal contents in animals given IMMU-130. On the basis of the SN-38 equivalents administered, IMMU-130 potentially delivers >300-fold more SN-38 to CEA-producing tumors compared with irinotecan, while also reducing levels of SN-38 and SN-38G in normal tissues. These observations are consistent with preclinical and clinical data showing efficacy and improved safety. Mol Cancer Ther; 17(1); 196–203. ©2017 AACR.

PDF file - 60 KB, Reversed-phase HPLC analyses of cathepsin B incubates and buffer control incubates of ME-CL2A-SN-38, pH 5, 37 degrees Celsius. Values represent % of applied product.

Supplementary Figure 1 from Combination Radioimmunotherapy and Chemoimmunotherapy Involving Different or the Same Targets Improves Therapy of Human Pancreatic Carcinoma Xenograft Models

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. Mol Cancer Ther; 12(6); 968–78. ©2013 AACR.

HLA-DR is a member of the MHC class II antigen family expressed on hematologic and solid tumors. Antibodies directed against HLA-DR have demonstrated some clinical success, but toxicities limited development. IMMU-140 is an anti–HLA-DR antibody–drug conjugate composed of the active metabolite of irinotecan, SN-38, conjugated to a humanized anti–HLA-DR IgG4 antibody (IMMU-114); the IgG4 naked antibody is devoid of immune functions. Our aim was to determine if SN-38, the metabolite of a drug not commonly used in hematopoietic cancers, would be effective and safe when targeted to HLA-DR–expressing tumors. IMMU-140 had dual-therapeutic mechanisms, as evidenced by its retention of nonoverlapping anti–HLA-DR nonclassical apoptotic signaling and classical apoptosis mediated by its SN-38 payload. In seven human disease models [acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), multiple myeloma (MM), acute myeloid leukemia (AML), diffuse large B-cell lymphoma (DLBCL), Hodgkin lymphoma (HL), and melanoma], IMMU-140 provided significant therapeutic efficacy compared with controls, in vitro, in 3D spheroid models, and in vivo. Except for MM and HL, IMMU-140 imparted significantly improved antitumor effects compared with parental IMMU-114. Even in intractable AML and ALL, where IMMU-114 only had modest antitumor effects, IMMU-140 therapy mediated >80% improvement in survival. Therapy was well tolerated, as demonstrated by no marked loss in body weight. Combined with doxorubicin, IMMU-140 produced significantly greater antitumor effects in HL than with monotherapy and without any added toxicity. The dual-therapeutic action of IMMU-140 resulted in promising therapeutic activity in a range of hematopoietic tumors and melanoma, and therefore warrants clinical development. Mol Cancer Ther; 17(1); 150–60. ©2017 AACR.

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Supplemental Data Table S1. Area under the curve comparisons between IMMU-140 treated A-375 melanoma-bearing mice and all other treatment groups; Fig. S1. Synthesis Scheme for CL2A-SN-38; Fig. S2. Fluorescent Images of A-375 Spheroids Stained with PI and Calcein AM; Fig. S3. Survival Curves of IMMU-140 versus IMMU-114 Treatment of Disseminated Xenograft Disease Models of Human ALL, AML, MM, and CLL; Fig. S4. Tolerability of IMMU-140 Combined with Doxorubicin or Bortezomib in SCID Mice; Fig. S5. HLA-DR a-Chain Expression on Hematological Cancer Xenografts.

Supplementary Table S1. Extraction efficiency for isolation of SN-38 in buffer with or without human serum albumin (HSA) or from the ADC with or without acid hydrolysis followed by extraction; Supplementary Figure S1. Schematic depiction of the conjugation of CL2A-SN-38 to mildly reduced IgG yielding site-specifically coupled SN-38 to 8 well-defined sites; Supplementary Figure S2. CL2A-SN38 amide capped with N-acetylcysteine; Supplementary Figure S3. SDS-Page gels comparing profiles for labetuzumab (unconjugated hMN-14 IgG) and labetuzumab govitecan (CL2A-SN-38 conjugated hMN-14 IgG; IMMU-130); Supplementary Figure S4. In vitro cell binding assay performed on LS174T cells, evaluating the binding of unconjugated hMN-14 IgG and SN-38-conjugated hMN-14 IgG (IMMU-130); Supplementary Figure S5. Stability of IMMU-130 in serum from different sources; Supplementary Figure S6. Evaluation of lactone ring stability of the CL2A-linked SN-38 compared to SN-38 at pH 7.4.

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Purpose: Sacituzumab govitecan (IMMU-132) is an antibody–drug conjugate (ADC) targeting Trop-2, a surface glycoprotein expressed on many epithelial tumors, for delivery of SN-38, the active metabolite of irinotecan. This phase I trial evaluated this ADC as a potential therapeutic for pretreated patients with a variety of metastatic solid cancers.Experimental Design: Sacituzumab govitecan was administered on days 1 and 8 of 21-day cycles, with cycles repeated until dose-limiting toxicity or progression. Dose escalation followed a standard 3 + 3 scheme with 4 planned dose levels and dose delay or reduction allowed.Results: Twenty-five patients (52–60 years old, 3 median prior chemotherapy regimens) were treated at dose levels of 8 (n = 7), 10 (n = 6), 12 (n = 9), and 18 (n = 3) mg/kg. Neutropenia was dose limiting, with 12 mg/kg the maximum tolerated dose for cycle 1, but too toxic with repeated cycles. Lower doses were acceptable for extended treatment with no treatment-related grade 4 toxicities and grade 3 toxicities limited to fatigue (n = 3), neutropenia (n = 2), diarrhea (n = 1), and leukopenia (n = 1). Using CT-based RECIST 1.1, two patients achieved partial responses (triple-negative breast cancer, colon cancer) and 16 others had stable disease as best response. Twelve patients maintained disease control with continued treatment for 16 to 36 weeks; 6 survived 15 to 20+ months. No preselection of patients based on tumor Trop-2 expression was done.Conclusions: Sacituzumab govitecan had acceptable toxicity and encouraging therapeutic activity in patients with difficult-to-treat cancers. The 8 and 10 mg/kg doses were selected for phase II studies. Clin Cancer Res; 21(17); 3870–8. ©2015 AACR.

Purpose: Evaluate the efficacy of an SN-38-anti-Trop-2 antibody–drug conjugate (ADC) against several human solid tumor types, and to assess its tolerability in mice and monkeys, the latter with tissue cross-reactivity to hRS7 similar to humans.Experimental Design: Two SN-38 derivatives, CL2-SN-38 and CL2A-SN-38, were conjugated to the anti-Trop-2–humanized antibody, hRS7. The immunoconjugates were characterized in vitro for stability, binding, and cytotoxicity. Efficacy was tested in five different human solid tumor-xenograft models that expressed Trop-2 antigen. Toxicity was assessed in mice and in Cynomolgus monkeys.Results: The hRS7 conjugates of the two SN-38 derivatives were equivalent in drug substitution (∼6), cell binding (Kd ∼ 1.2 nmol/L), cytotoxicity (IC50 ∼ 2.2 nmol/L), and serum stability in vitro (t/½ ∼ 20 hours). Exposure of cells to the ADC demonstrated signaling pathways leading to PARP cleavage, but differences versus free SN-38 in p53 and p21 upregulation were noted. Significant antitumor effects were produced by hRS7-SN-38 at nontoxic doses in mice bearing Calu-3 (P ≤ 0.05), Capan-1 (P P P Conclusions: The anti-Trop-2 hRS7-CL2A-SN-38 ADC provides significant and specific antitumor effects against a range of human solid tumor types. It is well tolerated in monkeys, with tissue Trop-2 expression similar to humans, at clinically relevant doses, and warrants clinical investigation. Clin Cancer Res; 17(10); 3157–69. ©2011 AACR.

Supplementary sections, including additional Materials and Methods, as well as Results and Tables. Table S1. SN-38 and SN-38G standard curves using triplicate dilutions of each concentration with single HPLC run. Table S2. SN-38 extraction efficiency. Table S3. Area under the curve (AUC) analysis of various products in the serum of nude mice given irinotecan or IMMU-132. Table S4. In vitro glucuronidation assay. Table S5. Analysis of products in the large intestine of animals given irinotecan or IMMU-132 (Study 2). Values represent the mean {plus minus} SD (N = 3) and are expressed in µg.

Purpose: We evaluated a Trop-2–targeting antibody conjugated with SN-38 in metastatic small cell lung cancer (mSCLC) patients.Experimental Design: Sacituzumab govitecan was studied in patients with pretreated (median, 2; range, 1–7) mSCLC who received either 8 or 10 mg/kg i.v. on days 1 and 8 of 21-day cycles. The primary endpoints were safety and objective response rate (ORR); duration of response, progression-free survival (PFS), and overall survival (OS) were secondary endpoints.Results: Sixty percent of patients showed tumor shrinkage from baseline CTs. On an intention-to-treat basis (N = 50), the ORR was 14% (17% for the 10-mg/kg group); the median response duration, 5.7 months; the clinical benefit rate (CBR ≥4 months), 34%; median PFS, 3.7 months; and median OS, 7.5 months. There was a suggested improvement in PR, CBR, and PFS with sacituzumab govitecan in second-line patients who were sensitive to first-line therapy, but no difference between first-line chemosensitive versus chemoresistant patients in the overall population. There was a statistically significant higher OS in those patients who received prior topotecan versus no topotecan therapy in a small subgroup. Grade ≥3 adverse events included neutropenia (34%), fatigue (13%), diarrhea (9%), and anemia (6%). Trop-2 tumor staining was not required for patient selection. No antibodies to the drug conjugate or its components were detected on serial blood collections.Conclusions: Sacituzumab govitecan appears to have a safe and effective therapeutic profile in heavily pretreated mSCLC patients, including those who are chemosensitive or chemoresistant to first-line chemotherapy. Additional studies as a monotherapy or combination therapy are warranted. Clin Cancer Res; 23(19); 5711–9. ©2017 AACR.

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 high-performance liquid chromatography (HPLC).Results: In serum, >98% irinotecan cleared within 5 minutes; 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 to 8 hours. 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 hours, which closely reflected the in vitro rate of SN-38 released from the conjugate in mouse serum (i.e., 17.5 hours), whereas the IgG portion of the conjugate cleared with a half-life of 67.1 hours. In vitro and in vivo studies disclosed IgG-bound SN-38 was protected from glucuronidation. Area under the curve (AUC) analysis indicated that 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.Conclusions: These studies confirm a superior SN-38 tumor delivery by IMMU-132 compared with irinotecan. Clin Cancer Res; 21(22); 5131–8. ©2015 AACR.

Serum concentration of intact sacituzumab govitecan and hRS7 IgG taken 30 min after injection for each of the 4 dose levels tested.

Evaluation of the procedure used to quantitate the constitutive products derived from irinotecan or IMMU-132.

This file contains additional methods, including eligibility criteria, study design, pharmacokinetics and immunogenicity, and immunohistology, as well as additional results related to several notable patient narratives.

Immunohistology expression of Trop-2 vs response

Time to progression in relation to the immunohistological score in 16 patients with diverse cancers enrolled in the sacituzumab govitecan trial who provided archival tissue specimens.

Trop-2 expression in archived tissue specimens from 6 patients enrolled in the clinical trial with sacituzumab govitecan.

Evaluation of IHC staining vs best overall response in SCLC treated with 8 or 10 mg/kg starting dose of sacituzumab govitecan

Supplementary Figures S1-S2; Supplementary Materials and Methods; Supplementary Tables S1-S2.

Antibody–drug conjugates (ADC) are a rapidly growing class of targeted cancer treatments, but the field has experienced significant challenges from their complex design. This study examined the multiscale distribution of sacituzumab govitecan (SG; Trodelvy), a recently clinically approved ADC, to clarify the mechanism(s) of efficacy given its unique design strategy. We employed a multiscale quantitative pharmacokinetic approach, including near-infrared fluorescence imaging, single-cell flow cytometry measurements, payload distribution via γH2AX pharmacodynamic staining, and a novel dual-labeled fluorescent technique to track the ADC and payload in a high trophoblast cell-surface antigen 2 expression xenograft model of gastric cancer (NCI-N87). We found that rapid release of the SN-38 payload from the hydrolysable linker inside cells imparts more DNA damage in vitro and in vivo than an ADC with a more stable enzyme cleavable linker. With SG, little to no extracellular payload release in the tumor was observed using a dual-labeled fluorescence technique, although bystander effects were detected. The high dosing regimen allowed the clinical dose to reach the majority of cancer cells, which has been linked to improved efficacy. In addition, the impact of multiple doses (day 1 and day 8) of a 21-day cycle was found to further improve tissue penetration despite not changing tumor uptake [percent injected dose per gram (%ID/g)] of the ADC. These results show increased ADC efficacy with SG can be attributed to efficient tumor penetration and intracellular linker cleavage after ADC internalization. This quantitative approach to study multiscale delivery can be used to inform the design of next-generation ADCs and prodrugs for other targets.

Background Uterine serous carcinoma (USC) is a biologically aggressive variant of uterine cancer. Effective treatment options for recurrent, chemotherapy-resistant USC are extremely limited. Case We describe a 74-year-old woman with recurrent and widespread treatment-resistant disease, who experienced a dramatic response to sacituzumab govitecan, a novel antibody-drug conjugate (ADC) targeting human trophoblast-cell-surface antigen (TROP-2), after failing multiple chemotherapy and immunotherapy. The impressive clinical response (66% reduction of target lesions by RECIST 1.1 with a duration response of over 10 months) was confirmed with serial CT scans in the absence of significant adverse events. Conclusion Sacituzumab govitecan may present a new treatment option for recurrent USC patients harboring Trop-2+ tumors resistant to chemotherapy. Clinical trials with sacituzumab govitecan are warranted., Highlights • Uterine serous carcinoma (USC) is a rare but aggressive variant of uterine cancer. • Sacituzumab-govitecan (IMMU-132) is an antibody-drug conjugate targeting TROP-2. • IMMU-132 may represent a new treatment option for USC overexpressing TROP-2.

This abstract was withdrawn by the authors.

Background: mTNBC has an aggressive course with limited therapeutic options. Sacituzumab govitecan (IMMU-132) is a novel antibody drug conjugate consisting of SN-38, the active metabolite of the topoisomerase I inhibitor, irinotecan, conjugated to a humanized mAb targeting Trop-2, which is highly expressed in most epithelial cancers, including TNBC. A phase I/II basket trial (NCT01631552) was conducted in patients (pts) with multiple, advanced epithelial cancers. We previously reported preliminary results in mTNBC (N=69; objective response rate [ORR] = 30%, Bardia et al., JCO 2017;35:2141-2148). In 2016, sacituzumab govitecan was granted Breakthrough Designation based on this encouraging data, and we resumed enrollment in a more defined patient population (≥3rd-line setting in mTNBC). Methods: Pts received sacituzumab govitecan on days 1 & 8 of a 21-day cycle until progression or unacceptable toxicity. Eligibility included > 2 prior lines of therapy for metastatic disease, measurable disease by CT or MRI and prior taxane. Efficacy was assessed locally by RECIST 1.1 and confirmed by independent centralized blinded review. ORR, DOR, progression-free survival (PFS) and overall survival (OS) were determined. Adverse events (CTCAE v4.0), immunogenicity, and Trop-2 expression in archived tumor samples, when available, were evaluated. Results: 110 mTNBC pts (109 female, 1 male; median age 55 yrs, range 31-81), including 53 from the previously reported cohort of 69 pts who had received ≥2 prior regimens for metastatic disease, were accrued between 7/2013 and 2/2017. As of data cutoff on 6/30/2017, 71 are deceased, 23 in long-term follow-up, and 16 still on treatment. All pts were treated at the 10 mg/kg IMMU-132 dose level, receiving 14.5 median doses (range 1-88). Treatment was well tolerated, with no treatment-related deaths, 2 treatment discontinuations for toxicity, and no anti-drug antibodies detected. Grade ≥ 3 toxicity (≥10%) included neutropenia, 39%; leukopenia, 14%; anemia, 10%; the incidence of febrile neutropenia was low (7%). By local radiologist assessment, the ORR is 34% (37/110), including 3 CRs and 34 PRs, the clinical benefit rate (CBR: CR+PR+SD>6 mo.) is 46%, the KM median DOR and PFS are 7.6 mo. (95% CI: 4.8 to 11.3) and 5.5 mo. (95% CI: 4.8 to 6.6), respectively, including 10% (11 pts) with long-term PFS (12 to 30+ mo.), and the KM median OS is 12.7 mo. (95% CI: 10.8 to 13.6). Results of the independent central blinded review along with sensitivity analyses of prior treatment regimens, including checkpoint inhibitor use, and exploratory biomarker analysis of Trop-2 expression will be presented at the meeting. Conclusions: Sacituzumab govitecan demonstrated significant clinical activity as a single agent in the ≥3rd-line setting for patients with relapsed/refractory mTNBC. Given the high unmet medical need, data from this trial is being submitted for consideration of accelerated approval, and a global confirmatory randomized Phase III trial (NCT02574455) is underway. Additional studies including rational combinations are currently being evaluated for mTNBC and other breast cancer subsets. Citation Format: Bardia A, Vahdat LT, Diamond J, Kalinsky K, O'Shaughnessy J, Moroose RL, Isakoff SJ, Tolaney SM, Santin AD, Abramson V, Shah NC, Govindan SV, Maliakal P, Sharkey RM, Wegener WA, Goldenberg DM, Mayer IA. Sacituzumab govitecan (IMMU-132), an anti-Trop-2-SN-38 antibody-drug conjugate, as ≥3rd-line therapeutic option for patients with relapsed/refractory metastatic triple-negative breast cancer (mTNBC): efficacy results [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr GS1-07.

HLA-DR is a member of the MHC class II antigen family expressed on hematologic and solid tumors. Antibodies directed against HLA-DR have demonstrated some clinical success, but toxicities limited development. IMMU-140 is an anti–HLA-DR antibody–drug conjugate composed of the active metabolite of irinotecan, SN-38, conjugated to a humanized anti–HLA-DR IgG4 antibody (IMMU-114); the IgG4 naked antibody is devoid of immune functions. Our aim was to determine if SN-38, the metabolite of a drug not commonly used in hematopoietic cancers, would be effective and safe when targeted to HLA-DR–expressing tumors. IMMU-140 had dual-therapeutic mechanisms, as evidenced by its retention of nonoverlapping anti–HLA-DR nonclassical apoptotic signaling and classical apoptosis mediated by its SN-38 payload. In seven human disease models [acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), multiple myeloma (MM), acute myeloid leukemia (AML), diffuse large B-cell lymphoma (DLBCL), Hodgkin lymphoma (HL), and melanoma], IMMU-140 provided significant therapeutic efficacy compared with controls, in vitro, in 3D spheroid models, and in vivo. Except for MM and HL, IMMU-140 imparted significantly improved antitumor effects compared with parental IMMU-114. Even in intractable AML and ALL, where IMMU-114 only had modest antitumor effects, IMMU-140 therapy mediated >80% improvement in survival. Therapy was well tolerated, as demonstrated by no marked loss in body weight. Combined with doxorubicin, IMMU-140 produced significantly greater antitumor effects in HL than with monotherapy and without any added toxicity. The dual-therapeutic action of IMMU-140 resulted in promising therapeutic activity in a range of hematopoietic tumors and melanoma, and therefore warrants clinical development. Mol Cancer Ther; 17(1); 150–60. ©2017 AACR.

Purpose: We evaluated a Trop-2–targeting antibody conjugated with SN-38 in metastatic small cell lung cancer (mSCLC) patients. Experimental Design: Sacituzumab govitecan was studied in patients with pretreated (median, 2; range, 1–7) mSCLC who received either 8 or 10 mg/kg i.v. on days 1 and 8 of 21-day cycles. The primary endpoints were safety and objective response rate (ORR); duration of response, progression-free survival (PFS), and overall survival (OS) were secondary endpoints. Results: Sixty percent of patients showed tumor shrinkage from baseline CTs. On an intention-to-treat basis (N = 50), the ORR was 14% (17% for the 10-mg/kg group); the median response duration, 5.7 months; the clinical benefit rate (CBR ≥4 months), 34%; median PFS, 3.7 months; and median OS, 7.5 months. There was a suggested improvement in PR, CBR, and PFS with sacituzumab govitecan in second-line patients who were sensitive to first-line therapy, but no difference between first-line chemosensitive versus chemoresistant patients in the overall population. There was a statistically significant higher OS in those patients who received prior topotecan versus no topotecan therapy in a small subgroup. Grade ≥3 adverse events included neutropenia (34%), fatigue (13%), diarrhea (9%), and anemia (6%). Trop-2 tumor staining was not required for patient selection. No antibodies to the drug conjugate or its components were detected on serial blood collections. Conclusions: Sacituzumab govitecan appears to have a safe and effective therapeutic profile in heavily pretreated mSCLC patients, including those who are chemosensitive or chemoresistant to first-line chemotherapy. Additional studies as a monotherapy or combination therapy are warranted. Clin Cancer Res; 23(19); 5711–9. ©2017 AACR.

Purpose Trop-2, expressed in most triple-negative breast cancers (TNBCs), may be a potential target for antibody-drug conjugates. Sacituzumab govitecan, an antibody-drug conjugate, targets Trop-2 for the selective delivery of SN-38, the active metabolite of irinotecan. Patients and Methods We evaluated sacituzumab govitecan in a single-arm, multicenter trial in patients with relapsed/refractory metastatic TNBC who received a 10 mg/kg starting dose on days 1 and 8 of 21-day repeated cycles. The primary end points were safety and objective response rate; secondary end points were progression-free survival and overall survival. Results In 69 patients who received a median of five prior therapies (range, one to 12) since diagnosis, the confirmed objective response rate was 30% (partial response, n = 19; complete response, n = 2), the median response duration was 8.9 (95% CI, 6.1 to 11.3) months, and the clinical benefit rate (complete response + partial response + stable disease ≥ 6 months) was 46%. These responses occurred early, with a median onset of 1.9 months. Median progression-free survival was 6.0 (95% CI, 5.0 to 7.3) months, and median overall survival was 16.6 (95% CI, 11.1 to 20.6) months. Grade ≥ 3 adverse events included neutropenia (39%), leukopenia (16%), anemia (14%), and diarrhea (13%); the incidence of febrile neutropenia was 7%. The majority of archival tumor specimens (88%) were moderately to strongly positive for Trop-2 by immunohistochemistry. No neutralizing antibodies to the ADC or antibody were detected, despite repeated cycles developed. Conclusion Sacituzumab govitecan was well tolerated and induced early and durable responses in heavily pretreated patients with metastatic TNBC. As a therapeutic target and predictive biomarker, Trop-2 warrants further research.

BACKGROUND Sacituzumab govitecan (IMMU-132), an antitrophoblastic cell-surface antigen (anti-Trop-2) humanized antibody-SN-38 conjugate, had encouraging efficacy in the phase 1 clinical trial. This report further examines the pharmacokinetics and safety of multiple cycles of IMMU-132 at doses of 8 or 10 mg/kg in patients with diverse advanced epithelial cancers. METHODS Patients who had multiple prior therapies received IMMU-132 on days 1 and 8 of 21-day treatment cycles. Trop-2 staining of archived tumor specimens, clearance of IMMU-132 and its constituents (ie, immunoglobulin G [IgG], SN-38 [a camptothecin, the active component of irinotecan], and glucuronidated SN-38 [SN-38G]), antibody responses, and uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) levels were determined. Safety was assessed according to Common Terminology Criteria for Adverse Events version 4.0, and responses were assessed using Response Evaluation Criteria in Solid Tumors, version 1.1. RESULTS Patients with diverse metastatic cancers who received IMMU-132 at 8 mg/kg (n = 81) and 10 mg/kg (n = 97) were examined. Trop-2 was positive in 93% of the available specimens. IMMU-132 cleared with a half-life of approximately 11 to 14 hours, reflecting the release of SN-38 from the conjugate; IgG cleared more slowly (half-life, approximately 103-114 hours). Most SN-38 in the serum (>95%) was bound to IgG. SN-38G concentrations were lower than SN-38 concentrations. Dose-limiting neutropenia after the first cycle was not correlated with SN-38 in serum or with UGT1A1 genotype. No antibody responses were detected. Objective responses were observed in several indications, including metastatic triple-negative breast cancer, confirming that 10 mg/kg produced an encouraging overall response. CONCLUSIONS Sacituzumab govitecan has a predictable pharmacokinetic profile and manageable toxicity at doses of 8 and 10 mg/kg. With objective responses and a good therapeutic index at 10 mg/kg, this dose was chosen for future development. Cancer 2017;123:3843–3854. © 2017 American Cancer Society

Topoisomerase inhibitors represent a novel class of compounds that have been used as stand-alone anticancer agents, and their incorporation into antibody conjugates adds a targeting element to the development of more selective chemotherapy. Topoisomerases are nuclear enzymes involved in relaxing DNA strands for replication and transcription, and inhibitors of these enzymes act by stabilizing DNA–topoisomerase complexes, leading to double-strand breaks. SN-38, the active drug form of the cancer drug irinotecan is a topoisomerase I (TOP1) inhibitor that has been employed in various formulations to improve bioavailability. However, antibody conjugates of SN-38 represent a new approach for improving cancer chemotherapy. This review focuses on the design of antibody–SN-38 conjugates and on the current clinical results obtained for a number of cancers. A more potent form of a TOP1-inhibiting camptothecin derivative, exatecan mesylate, is currently being examined clinically in the antibody–drug conjugate (ADC) format, and DS-8201a, an ADC targeting human epidermal growth factor receptor 2-positive (HER2+) cancers, has shown promising Phase I clinical results in breast and gastric cancers. These ADCs also lend themselves to combination therapy with poly(ADP-ribose)polymerase (PARP) inhibitors and immune checkpoint inhibitors for potentially improving clinical outcomes.

Background: In current clinical trials (ClinicalTrials.gov, NCT01631552), triple-negative breast cancer (TNBC) patients treated with IMMU-132, which is composed of the active metabolite of irinotecan, SN-38, conjugated to an anti-Trop-2 antibody, shows manageable toxicity and very encouraging responses in relapsed/refractory cases. Synthetic lethality is a concept in which a cell harboring one out of two possible gene or protein defects is viable, while a cell containing both defects is nonviable. BRCA1/2 mutations are linked to deficiencies in DNA repair and are associated with TNBC. Other repair mechanisms involve poly(adenosine diphosphoribose) polymerase (PARP), which can be used by cancer cells to overcome loss of BRACA1/2. Treatment of TNBC cells with either IMMU-132 or paclitaxel results in cleavage and deactivation of PARP, whereas the small molecule olaparib directly inhibits PARP. Therefore, the rationale of combining IMMU-132 with either paclitaxel or olaparib to effectively knock-out PARP activity was investigated in TNBC xenografts to ascertain if these combinations will result in synthetic lethality. Methods: Mice bearing human TNBC xenografts (MDA-MB-468 or HCC1806) were treated with 15 mg/kg paclitaxel weekly for 5 weeks. IMMU-132 was administered either at 10 mg/kg or 12.5 mg/kg on days 1, 8, 22, and 29. In vitro, various human TNBC cell lines were incubated with either a constant amount of IMMU-132 in combination with various amounts of olaparib or constant olaparib with varying amounts of IMMU-132. A combination index number was calculated to determine whether the interaction was synergistic, additive, or antagonistic. Mice bearing TNBC tumors were treated with olaparib (50 mg/kg, qdx5d, for 4 wks), or IMMU-132 (10 mg/kg, 2xwkly x 4 wks), or the combination of both. Results: Mice bearing MDA-MB-468 tumors treated with the combination of IMMU-132 and paclitaxel exhibited superior anti-tumor effects with >11-fold shrinkage of tumors in comparison to 1.4-fold shrinkage in the IMMU-132 group alone (P=0.0003) or 11.4-fold increase in tumor size in those mice treated with paclitaxel alone (P Conclusions: Targeting the PARP DNA repair pathway in BRCA1/2 mutant TNBC tumors by combining IMMU-132 therapy with either paclitaxel or olaparib achieved synthetic lethality in this disease model with no observable toxicity. These data provide the rationale for the clinical evaluation of IMMU-132 in combination with other chemotherapeutics that likewise target DNA-repair mechanisms in patients with TNBC. Citation Format: Goldenberg DM, Cardillo TM, Govindan SV, Zalath M, Arrojo R, Sharkey RM. Synthetic lethality in TNBC mediated by an anti-Trop-2 antibody-drug conjugate, sacituzumab govitecan (IMMU-132), when combined with paclitaxel or the PARP inhibitor, olaparib. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-15-02.

Trop-2 is a novel target for ADC therapy because of its high expression by many solid cancers. The rational development of IMMU-132 represents a paradigm shift as an ADC that binds a well-known moderately-cytotoxic drug, SN-38, to the anti-Trop-2 antibody. In vitro and in vivo studies show enhanced efficacy, while there is a gradual release of SN-38 that contributes to the overall effect. IMMU-132 is most efficacious at a high drug:antibody ratio (DAR) of 7.6:1, which does not affect binding and pharmacokinetics. It targets up to 136-fold more SN-38 to a human cancer xenograft than irinotecan, SN-38's prodrug. IMMU-132 delivers SN-38 in its most active, non-glucuronidated form, which may explain the lower frequency of severe diarrhea than with irinotecan. Thus, this ADC, carrying a moderately-toxic drug targeting Trop-2 represents a novel cancer therapeutic that is showing promising activity in patients with several metastatic cancer types, including triple-negative breast cancer, non-small-cell and small-cell lung cancers.

Purpose: Sacituzumab govitecan (IMMU-132) is an antibody–drug conjugate (ADC) targeting Trop-2, a surface glycoprotein expressed on many epithelial tumors, for delivery of SN-38, the active metabolite of irinotecan. This phase I trial evaluated this ADC as a potential therapeutic for pretreated patients with a variety of metastatic solid cancers. Experimental Design: Sacituzumab govitecan was administered on days 1 and 8 of 21-day cycles, with cycles repeated until dose-limiting toxicity or progression. Dose escalation followed a standard 3 + 3 scheme with 4 planned dose levels and dose delay or reduction allowed. Results: Twenty-five patients (52–60 years old, 3 median prior chemotherapy regimens) were treated at dose levels of 8 (n = 7), 10 (n = 6), 12 (n = 9), and 18 (n = 3) mg/kg. Neutropenia was dose limiting, with 12 mg/kg the maximum tolerated dose for cycle 1, but too toxic with repeated cycles. Lower doses were acceptable for extended treatment with no treatment-related grade 4 toxicities and grade 3 toxicities limited to fatigue (n = 3), neutropenia (n = 2), diarrhea (n = 1), and leukopenia (n = 1). Using CT-based RECIST 1.1, two patients achieved partial responses (triple-negative breast cancer, colon cancer) and 16 others had stable disease as best response. Twelve patients maintained disease control with continued treatment for 16 to 36 weeks; 6 survived 15 to 20+ months. No preselection of patients based on tumor Trop-2 expression was done. Conclusions: Sacituzumab govitecan had acceptable toxicity and encouraging therapeutic activity in patients with difficult-to-treat cancers. The 8 and 10 mg/kg doses were selected for phase II studies. Clin Cancer Res; 21(17); 3870–8. ©2015 AACR.

// David M. Goldenberg 1 , Thomas M. Cardillo 1 , Serengulam V. Govindan 1 , Edmund A. Rossi 1 , Robert M. Sharkey 1 1 Immunomedics, Inc., Morris Plains, NJ, USA * Presented in part as a lecture by DMG, “Challenging the Dogmas: Clinical Efficacy of SN-38-conjugated Antibodies in Solid Tumors,” at the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapy, Barcelona, Spain, November 20, 2014. Correspondence to: David M. Goldenberg, e-mail: dmg.gscancer@att.net Keywords: antibody-drug conjugate, Trop-2, SN-38, solid cancers, triple-negative breast cancer Received: April 28, 2015 Accepted: June 05, 2015 Published: June 18, 2015 ABSTRACT Trop-2 is a novel target for ADC therapy because of its high expression by many solid cancers. The rational development of IMMU-132 represents a paradigm shift as an ADC that binds a well-known moderately-cytotoxic drug, SN-38, to the anti-Trop-2 antibody. In vitro and in vivo studies show enhanced efficacy, while there is a gradual release of SN-38 that contributes to the overall effect. IMMU-132 is most efficacious at a high drug:antibody ratio (DAR) of 7.6:1, which does not affect binding and pharmacokinetics. It targets up to 136-fold more SN-38 to a human cancer xenograft than irinotecan, SN-38′s prodrug. IMMU-132 delivers SN-38 in its most active, non-glucuronidated form, which may explain the lower frequency of severe diarrhea than with irinotecan. Thus, this ADC, carrying a moderately-toxic drug targeting Trop-2 represents a novel cancer therapeutic that is showing promising activity in patients with several metastatic cancer types, including triple-negative breast cancer, non-small-cell and small-cell lung cancers.

Sacituzumab govitecan (IMMU-132) is an antibody-drug conjugate (ADC) made from a humanized anti-Trop-2 monoclonal antibody (hRS7) conjugated with the active metabolite of irinotecan, SN-38. In addition to its further characterization, as the clinical utility of IMMU-132 expands to an ever-widening range of Trop-2-expressing solid tumor types, its efficacy in new disease models needs to be explored in a nonclinical setting. Unlike most ADCs that use ultratoxic drugs and stable linkers, IMMU-132 uses a moderately toxic drug with a moderately stable carbonate bond between SN-38 and the linker. Flow cytometry and immunohistochemistry disclosed that Trop-2 is expressed in a wide range of tumor types, including gastric, pancreatic, triple-negative breast (TNBC), colonic, prostate, and lung. While cell-binding experiments reveal no significant differences between IMMU-132 and parental hRS7 antibody, surface plasmon resonance analysis using a Trop-2 CM5 chip shows a significant binding advantage for IMMU-132 over hRS7. The conjugate retained binding to the neonatal receptor, but it lost greater than 60% of the antibody-dependent cell-mediated cytotoxicity activity compared to that of hRS7. Exposure of tumor cells to either free SN-38 or IMMU-132 demonstrated the same signaling pathways, with pJNK1/2 and p21(WAF1/Cip1) upregulation followed by cleavage of caspases 9, 7, and 3, ultimately leading to poly-ADP-ribose polymerase cleavage and double-stranded DNA breaks. Pharmacokinetics of the intact ADC in mice reveals a mean residence time (MRT) of 15.4 h, while the carrier hRS7 antibody cleared at a similar rate as that of the unconjugated antibody (MRT ∼ 300 h). IMMU-132 treatment of mice bearing human gastric cancer xenografts (17.5 mg/kg; twice weekly × 4 weeks) resulted in significant antitumor effects compared to that of mice treated with a nonspecific control. Clinically relevant dosing schemes of IMMU-132 administered either every other week, weekly, or twice weekly in mice bearing human pancreatic or gastric cancer xenografts demonstrate similar, significant antitumor effects in both models. Current Phase I/II clinical trials ( ClinicalTrials.gov , NCT01631552) confirm anticancer activity of IMMU-132 in cancers expressing Trop-2, including gastric and pancreatic cancer patients.

Background: TNBC, comprising 15-20% of all invasive breast cancers, represents an aggressive phenotype with high risk of recurrence and mortality. Trop-2 is a cell-surface glycoprotein expressed on many human carcinomas, including TNBC. High Trop-2 expression is associated with more aggressive disease and poor prognosis in several cancers, including breast cancer. We report interim results from a Phase I/II trial evaluating a novel ADC, IMMU-132 (isactuzumab govitecan), comprising a humanized anti-Trop-2 antibody conjugated to the topoisomerase I inhibitor, SN-38 (active metabolite of irinotecan). The drug:antibody ratio of 7.6 facilitates the delivery of high-dose chemotherapy preferentially to the tumor cells. Methods: Patients (pts) with relapsed/refractory metastatic epithelial tumors were enrolled at escalating IMMU-132 doses (8 to 18 mg/kg), given on days 1 and 8 of a 21-day cycle. The Phase II dose at this schedule was 10 mg/kg. CT scans were performed every 6-8 weeks to assess response using RECIST 1.1. During the dose-escalation portion, evidence of antitumor activity, including 3 partial responses (TNBC, small-cell lung cancer and colorectal cancer) and many with durable stable disease (SD), was observed, leading to Phase II expansion. Results: As of Sept. 25, 2014, a total of 132 pts have been enrolled, including 30 with advanced/metastatic TNBC. Currently evaluable TNBC pts (N=17) had a median age of 50 (33-77), with a median of 4 prior drug regimens (range 1-8), and 67% having received prior platinum-containing regimens. In this heavily pre-treated population, there were 4 PRs (25%) and 9 SDs (56.3%) per RECIST v1.1, representing a disease control (PR+SD > 4 mos) of 53% among evaluable pts with adequate follow-up. A maximum shrinkage of target lesions of 33%, 44%, 51%, and 60% for pts with PRs, and 14%, 19%, and 27% for 3 pts with SD, was determined. Biomarker CA15.3 directional changes correlated with RECIST. All but one pathology specimen were Trop-2+ by immunohistochemistry. HPLC analysis of serum samples found Conclusions: Based on laboratory and initial clinical results, IMMU-132 is an ADC that selectively delivers a topoisomerase I inhibitor to cancer cells without the need for enrichment by a companion diagnostic. It is safe, well-tolerated, with preliminary evidence of encouraging efficacy in heavily-pretreated pts with relapsed/refractory metastatic TNBC. Randomized Phase III and combination trials are being planned. Citation Format: Aditya Bardia, Alexander Starodub, Rebecca L Moroose, Ingrid A Mayer, Jennifer R Diamond, Ellen Chuang, Serengulam V Govindan, Robert M Sharkey, Pius Maliakal, William A Wegener, Steven A Hamburger, Allyson J Ocean, David M Goldenberg, Linda T Vahdat. IMMU-132, a new antibody-drug conjugate (ADC) against Trop-2, as a novel therapeutic for patients with relapsed/refractory, metastatic, triple-negative breast cancer (TNBC): Results from Phase I/II clinical trial (NCT01631552) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-19-27.

Purpose The objectives were to evaluate dosing schedules of labetuzumab govitecan, an antibody-drug conjugate targeting carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) for tumor delivery of 7-ethyl-10-hydroxycamptothecin (SN-38), in an expanded phase II trial of patients with relapsed or refractory metastatic colorectal cancer. Patients and Methods Eligible patients with at least one prior irinotecan-containing therapy received labetuzumab govitecan once weekly at 8 and 10 mg/kg, or two times per week at 4 and 6 mg/km on weeks 1 and 2 of 3-week repeated cycles. End points were safety, response, pharmacokinetics, and immunogenicity. Results Eighty-six patients who had undergone a median of five prior therapies (range, one to 13) were each enrolled into one of the four cohorts. On the basis of Response Evaluation Criteria in Solid Tumors 1.1, 38% of these patients had a tumor as well as plasma carcinoembryonic antigen reduction from baseline after labetuzumab govitecan treatment; one patient achieved a partial response with a sustained response spanning > 2 years, whereas 42 patients had stable disease as the best overall response. Median progression-free survival and overall survival were 3.6 and 6.9 months, respectively. The major toxicities (grade ≥ 3) among all cohorts were neutropenia (16%), leukopenia (11%), anemia (9%), and diarrhea (7%). The antibody-drug conjugate’s mean half-life was 16.5 hours for the four cohorts. Anti-drug/anti-antibody antibodies were not detected. The two once-weekly dose schedules, showing comparable toxicity and efficacy, were chosen for further study. Conclusion Monotherapy with labetuzumab govitecan demonstrated a manageable safety profile and therapeutic activity in heavily pretreated patients with metastatic colorectal cancer, all with prior irinotecan therapy. Further studies of labetuzumab govitecan treatment alone or in combination with other therapies in earlier settings are indicated.

Purpose Trop-2, expressed in most solid cancers, may be a target for antibody-drug conjugates (ADCs) in non–small-cell lung cancer (NSCLC). We studied sacituzumab govitecan (IMMU-132), a Trop-2 ADC, for the targeting of SN-38. Patients and Methods We evaluated IMMU-132 in a single-arm multicenter trial in patients with pretreated metastatic NSCLC who received either 8 or 10 mg/kg on days 1 and 8 of 21-day cycles. The primary end points were safety and objective response rate (ORR). Progression-free survival and overall survival were secondary end points. Results Fifty-four patients were treated. In the response-assessable study population (n = 47), which had a median of three prior therapies (range, two to seven), the ORR was 19%; median response duration, 6.0 months (95% CI, 4.8 to 8.3 months); and clinical benefit rate (complete response + partial response + stable disease ≥ 4 months), 43%. ORR in the intention-to-treat (ITT) population was 17% (nine of 54). Responses occurred with a median onset of 3.8 months, including patients who had relapsed or progressed after immune checkpoint inhibitor therapy. Median ITT progression-free survival was 5.2 months (95% CI, 3.2 to 7.1 months) and median ITT overall survival, 9.5 months (95% CI, 5.9 to 16.7 months). Grade 3 or higher adverse events included neutropenia (28%), diarrhea (7%), nausea (7%), fatigue (6%), and febrile neutropenia (4%). One patient developed a transient immune response, despite patients receiving a median of 10 doses. More than 90% of 26 assessable archival tumor specimens were highly positive (2+, 3+) for Trop-2 by immunohistochemistry, which suggests that Trop-2 is not a predictive biomarker for response. Conclusion IMMU-132 was well-tolerated and induced durable responses in heavily pretreated patients with metastatic NSCLC. This ADC should be studied further in this disease and in other patients with Trop-2–expressing tumors.

Labetuzumab govitecan (IMMU-130), an antibody–drug conjugate (ADC) with an average of 7.6 SN-38/IgG, was evaluated for its potential to enhance delivery of SN-38 to human colonic tumor xenografts. Mice bearing LS174T or GW-39 human colonic tumor xenografts were injected with irinotecan or IMMU-130 (SN-38 equivalents ∼500 or ∼16 μg, respectively). Serum and homogenates of tumors, liver, and small intestine were extracted, and SN-38, SN-38G (glucuronidated SN-38), and irinotecan concentrations determined by reversed-phase HPLC. Irinotecan cleared quickly from serum, with only 1% to 2% injected dose/mL after 5 minutes; overall, approximately 20% was converted to SN-38 and SN-38G. At 1 hour with IMMU-130, 45% to 63% injected dose/mL of the SN-38 was in the serum, with >90% bound to the ADC over 3 days, and with low levels of SN-38G. Total SN-38 levels decreased more quickly than the IgG, confirming a gradual SN-38 release from the ADC. AUC analysis found that SN-38 levels were approximately 11- and 16-fold higher in LS174T and GW-39 tumors, respectively, in IMMU-130–treated animals. This delivery advantage is amplified >30-fold when normalized to SN-38 equivalents injected for each product. Levels of SN-38 and SN-38G were appreciably lower in the liver and small intestinal contents in animals given IMMU-130. On the basis of the SN-38 equivalents administered, IMMU-130 potentially delivers >300-fold more SN-38 to CEA-producing tumors compared with irinotecan, while also reducing levels of SN-38 and SN-38G in normal tissues. These observations are consistent with preclinical and clinical data showing efficacy and improved safety. Mol Cancer Ther; 17(1); 196–203. ©2017 AACR.

Antibody-drug conjugates (ADCs), as well as antibody conjugates of protein toxins (immunotoxins) and cytokines (immunocytokines), are showing clinical efficacy, with manageable toxicities, in cancer treatment.The utility of an ADC is governed by the antibody and the target, as well as by the drug-linker component of the conjugate. The conjugation site, conjugating group, drug/antibody ratios and site-specific conjugation for product homogeneity are all aspects to consider in optimizing the ADC and enhancing its therapeutic window. Immunotoxin and immunocytokine construction by recombinant methods can be modulated to improve efficacy and reduce toxicity. The Dock-and-Lock (DNL) platform technology provides a flexible approach to assemble mono- or bispecific constructs carrying multiple toxin or cytokine molecules for targeted therapy.Conjugation chemistry and recombinant technologies have had a significant impact on the therapeutic prospects of immunoconjugates, particularly in hematopoietic diseases. Continued concerted efforts from different scientific disciplines are needed, together with newer treatment paradigms, for greater progress in the more challenging therapy of solid tumors.

Chemoimmunotherapy with antibody–drug conjugates (ADC) is emerging as a promising therapy for solid tumors, whereas radioimmunotherapy (RAIT) of solid tumors has been relatively ineffective because of their resistance to radiation. We developed antibody–SN-38 conjugates that have significant antitumor activity in xenograft models at nontoxic doses. The goal of this study was to determine if an ADC could be combined with RAIT to enhance efficacy without a commensurate increase in host toxicity. Nude mice bearing human pancreatic cancer xenografts (Capan-1 and BxPC-3) were treated with a single dose of 90Y-labeled antimucin antibody (hPAM4; clivatuzumab tetraxetan) alone or in combination with an anti-Trop-2–SN-38 conjugate, typically administered twice weekly over 4 weeks. The combination, even at RAIT's maximum tolerated dose, controlled tumor progression and cured established xenografts significantly better than the individual treatments without appreciable toxicity. The ADC could be started 1 week after or up to 2 weeks before RAIT with similar enhanced responses, but delaying RAIT for 2 weeks after the ADC was less effective. A nonspecific ADC provided additional benefit over using free drug (irinotecan), but the response was enhanced with the specific ADC. When targeting Capan-1 with ample mucin, hPAM4 could be used as the RAIT and the ADC agent without losing effectiveness, but in BxPC-3 with less mucin, targeting of different antigens was preferred. These studies show the feasibility of combining ADC and RAIT for improved efficacy without increased toxicity. Mol Cancer Ther; 10(6); 1072–81. ©2011 AACR.

Purpose: Evaluate the efficacy of an SN-38-anti-Trop-2 antibody–drug conjugate (ADC) against several human solid tumor types, and to assess its tolerability in mice and monkeys, the latter with tissue cross-reactivity to hRS7 similar to humans. Experimental Design: Two SN-38 derivatives, CL2-SN-38 and CL2A-SN-38, were conjugated to the anti-Trop-2–humanized antibody, hRS7. The immunoconjugates were characterized in vitro for stability, binding, and cytotoxicity. Efficacy was tested in five different human solid tumor-xenograft models that expressed Trop-2 antigen. Toxicity was assessed in mice and in Cynomolgus monkeys. Results: The hRS7 conjugates of the two SN-38 derivatives were equivalent in drug substitution (∼6), cell binding (Kd ∼ 1.2 nmol/L), cytotoxicity (IC50 ∼ 2.2 nmol/L), and serum stability in vitro (t/½ ∼ 20 hours). Exposure of cells to the ADC demonstrated signaling pathways leading to PARP cleavage, but differences versus free SN-38 in p53 and p21 upregulation were noted. Significant antitumor effects were produced by hRS7-SN-38 at nontoxic doses in mice bearing Calu-3 (P ≤ 0.05), Capan-1 (P Conclusions: The anti-Trop-2 hRS7-CL2A-SN-38 ADC provides significant and specific antitumor effects against a range of human solid tumor types. It is well tolerated in monkeys, with tissue Trop-2 expression similar to humans, at clinically relevant doses, and warrants clinical investigation. Clin Cancer Res; 17(10); 3157–69. ©2011 AACR.

Overexpression, activation, and mutations of the epidermal growth factor receptor (EGFR) are commonly found in solid tumors. The aim of this study was to develop a PET-based method for detecting the constitutively active mutant de2-7 EGFR, which is associated with disease progression and resistance to chemotherapy and radiotherapy in glioma. Methods: The chimeric antibody ch806, which selectively binds an epitope of the EGFR that is exposed only on overexpressed, mutant, or ligand-activated forms of the receptor, was conjugated to the radiohalogen 124I via the residualizing ligand IMP-R4, and in vitro properties were characterized. In vivo biodistribution and small-animal PET studies were performed in BALB/c nude mice bearing U87MG.de2-7 glioma xenografts. Imaging results were correlated with measured tumor uptake of the radioconjugate. Results:124I-IMP-R4-ch806 had an immunoreactivity of 78.3% and was stable for 7 d when incubated in serum in vitro. The biodistribution analysis of 124I-IMP-R4-ch806 demonstrated a maximal uptake of 30.95 ± 6.01 percentage injected dose per gram (%ID/g) in U87MG.de2-7 xenografts at 48 h after injection, with prolonged tumor retention (6.07 ± 0.80 %ID/g at 216 h after injection). The tumor-to-blood ratio increased from 0.44 at 4 h after injection to a maximum of 4.70 at 168 h after injection. PET of 124I-IMP-R4-ch806 biodistribution was able to clearly detect the U87MG.de2-7 tumors at 24 h after injection and for at least 168 h after injection. Correlation between tumor PET image quantitation of 124I-IMP-R4-ch806 and %ID/g determined from resected tissues (r = 0.9350) was excellent. Conclusion: These results show that immuno-PET with 124I-IMP-R4-ch806 is feasible and allows noninvasive quantitation of de2-7 EGFR expression in vivo.