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Supplementary Figure S1. Comparable internalization rates for 5T4_0108 and the 5T4-Tub ADC;Supplementary Figure S2. Affinity optimization improves in vitro cytotoxicity of a 5T4-ADC;Supplementary Figure S3. Induction of apoptosis by 5T4-PBD and 5T4-Tub;Supplementary Figure S4. 5T4 is expressed on CSCs of multiple cancer cell lines;Supplementary Figure S5. Only PBD is capable of inducing cytotoxicity of CSC populations in vitro;Supplementary Table S1. Improved cytotoxic potency following affinity optimization of 5T4 antibody; Supplementary Table S2. Quantification of cell surface-associated 5T4 in various cancer cell lines;Supplementary Table S3. Tumorigenicity of CSC populations derived from NCI- N87 and MDA-MB-361 xenografts; Supplementary Materials & Methods

Multiple myeloma is a hematologic cancer that disrupts normal bone marrow function and has multiple lines of therapeutic options, but is incurable as patients ultimately relapse. We developed a novel antibody–drug conjugate (ADC) targeting CS-1, a protein that is highly expressed on multiple myeloma tumor cells. The anti–CS-1 mAb specifically bound to cells expressing CS-1 and, when conjugated to a cytotoxic pyrrolobenzodiazepine payload, reduced the viability of multiple myeloma cell lines in vitro. In mouse models of multiple myeloma, a single administration of the CS-1 ADC caused durable regressions in disseminated models and complete regression in a subcutaneous model. In an exploratory study in cynomolgus monkeys, the CS-1 ADC demonstrated a half-life of 3 to 6 days; however, no highest nonseverely toxic dose was achieved, as bone marrow toxicity was dose limiting. Bone marrow from dosed monkeys showed reductions in progenitor cells as compared with normal marrow. In vitro cell killing assays demonstrated that the CS-1 ADC substantially reduced the number of progenitor cells in healthy bone marrow, leading us to identify previously unreported CS-1 expression on a small population of progenitor cells in the myeloid–erythroid lineage. This finding suggests that bone marrow toxicity is the result of both on-target and off-target killing by the ADC.

Supplemental Figure S1: Knockdown of BRCA1 or BRCA2 sensitizes Hela cells to PBD payload and PBD-based ADC in vitro. Supplemental Figure S2: Genetic deletion of BRCA1 sensitizes cells to PBD-based ADC. Supplemental Table S1: BRCA mutation(s) in patient-derived xenograft models. Supplemental Figure S3: Mean tumor volume graphs of 23 BRCA-deficient PDX tumors response to PBD-ADC treatment compared to untreated group. Supplemental Figure S4: Mean tumor volume graphs of BRCA wild-type PDX tumors response to PBD-ADC treatment compared to untreated group. Supplemental Figure S5: Representative IHC images of 5T4 staining in PDX models. Supplemental Figure S6: No in vivo efficacy was observed in tumor model DMS-114 that does not express 5T4.

Supplementary Materials and Methods, Supplementary Table S1. Sources of cell lines and cell growth media; Supplemental Table S2. Sources of antibodies for flow cytometry; Supplementary Table S3. CS-1 ADC Characteristics, Supplemental Table S4. Pharmacokinetic profile of the CS-1 ADC; Supplemental Figure S1. Characterization of the CS-1 ADC molecule; Supplemental Figure S2. CS-1 ADC effect on tumor cell growth; Figure S3. Detailed results of colony forming assays; Supplementary Figure S4. CS-1 mAb and elotuzumab bind to similar cells in bone marrow.

Supplementary Data. Supplemental Table S1: Summary of MEDI-573 IC50 on the proliferation of sarcoma cell lines with or without exogenously added IGF-1 or IGF-2. Supplemental Figure S1: Effect of MEDI-573 in combination with AZD2014 on SJSA-1 tumor growth in vivo.

MEDI-573 is a human antibody that neutralizes insulin-like growth factor (IGF) I and IGFII. IGFs are overexpressed in multiple types of cancer; their overexpression is a potential mechanism for resistance to IGFI receptor (IGFIR)-targeting therapy. Effects of IGF on cell proliferation, differentiation, and survival are mediated through its binding to and activation of IGFIR or insulin receptor A (IR-A). In this study, we measured the mRNA levels of IGFI, IGFII, and IGFIR in human pediatric sarcoma xenografts, and protein levels in sarcoma cell lines. MEDI-573 potently inhibited in vitro proliferation of sarcoma cell lines, with Ewing sarcoma cell lines being the most sensitive. In addition, MEDI-573 inhibited IGFI- and IGFII-induced sarcoma cell proliferation in vitro. The effect of MEDI-573 on IGF signaling was also examined. Treatment with MEDI-573 markedly reduced levels of pIGFIR, pIR-A, and pAKT and significantly blocked IGFI- and IGFII-induced activation of the IGFIR and AKT pathways. MEDI-573 inhibited the growth of sarcoma xenografts in vivo and inhibition correlated with neutralization of IGFI and IGFII. Combination of MEDI-573 with either rapamycin or AZD2014, another mTOR inhibitor (mTORi), significantly enhanced the antitumor activity of MEDI-573, and this response correlated with modulation of AKT and mTOR signaling. In summary, sarcoma cells respond to autocrine or paracrine growth stimulation by IGFI and IGFII, and inhibition of IGFI and IGFII by MEDI-573 results in significant slowing of tumor growth rate in sarcoma models, particularly in Ewing sarcoma. These data provide evidence for the potential benefits of MEDI-573 and mTORi combinations in patients with Ewing sarcoma. Mol Cancer Ther; 13(11); 2662–73. ©2014 AACR.

Pyrrolobenzodiazepine dimers (PBD) form cross-links within the minor groove of DNA causing double-strand breaks (DSB). DNA repair genes such as BRCA1 and BRCA2 play important roles in homologous recombination repair of DSB. We hypothesized that PBD-based antibody–drug conjugates (ADC) will have enhanced killing of cells in which homologous recombination processes are defective by inactivation of BRCA1 or BRCA2 genes. To support this hypothesis, we found 5T4–PBD, a PBD-dimer conjugated to anti-5T4 antibody, elicited more potent antitumor activity in tumor xenografts that carry defects in DNA repair due to BRCA mutations compared with BRCA wild-type xenografts. To delineate the role of BRCA1/2 mutations in determining sensitivity to PBD, we used siRNA knockdown and isogenic BRCA1/2 knockout models to demonstrate that BRCA deficiency markedly increased cell sensitivity to PBD-based ADCs. To understand the translational potential of treating patients with BRCA deficiency using PBD-based ADCs, we conducted a “mouse clinical trial” on 23 patient-derived xenograft (PDX) models bearing mutations in BRCA1 or BRCA2. Of these PDX models, 61% to 74% had tumor stasis or regression when treated with a single dose of 0.3 mg/kg or three fractionated doses of 0.1 mg/kg of a PBD-based ADC. Furthermore, a suboptimal dose of PBD-based ADC in combination with olaparib resulted in significantly improved antitumor effects, was not associated with myelotoxicity, and was well tolerated. In conclusion, PBD-based ADC alone or in combination with a PARP inhibitor may have improved therapeutic window in patients with cancer carrying BRCA mutations.

Purpose: To use preclinical models to identify a dosing schedule that improves tolerability of highly potent pyrrolobenzodiazepine dimers (PBDs) antibody drug conjugates (ADCs) without compromising antitumor activity.Experimental Design: A series of dose-fractionation studies were conducted to investigate the pharmacokinetic drivers of safety and efficacy of PBD ADCs in animal models. The exposure–activity relationship was investigated in mouse xenograft models of human prostate cancer, breast cancer, and gastric cancer by comparing antitumor activity after single and fractionated dosing with tumor-targeting ADCs conjugated to SG3249, a potent PBD dimer. The exposure–tolerability relationship was similarly investigated in rat and monkey toxicology studies by comparing tolerability, as assessed by survival, body weight, and organ-specific toxicities, after single and fractionated dosing with ADCs conjugated to SG3249 (rats) or SG3400, a structurally related PBD (monkeys).Results: Observations of similar antitumor activity in mice treated with single or fractionated dosing suggests that antitumor activity of PBD ADCs is more closely related to total exposure (AUC) than peak drug concentrations (Cmax). In contrast, improved survival and reduced toxicity in rats and monkeys treated with a fractionated dosing schedule suggests that tolerability of PBD ADCs is more closely associated with Cmax than AUC.Conclusions: We provide the first evidence that fractionated dosing can improve preclinical tolerability of at least some PBD ADCs without compromising efficacy. These findings suggest that preclinical exploration of dosing schedule could be an important clinical strategy to improve the therapeutic window of highly potent ADCs and should be investigated further. Clin Cancer Res; 23(19); 5858–68. ©2017 AACR.

Supplementary Figure 1. (a,b) Structure of the payloads SG3249 and SG3400; Supplementary Figure 2. PK concentration vs. time profile of R347-SG3400 in monkeys after either a single IV injection of 4.5mg/kg or 3 weekly IV injections of 1.5mg/kg; Supplementary Table 1. Ocular findings in rats after single or fractionated dosing with IC1-SG3249*

Elevated levels of the proinflammatory cytokine IL6 are associated with poor survival outcomes in many cancers. Antibodies targeting IL6 and its receptor have been developed for chronic inflammatory disease, but they have not yet been shown to clearly benefit cancer patients, possibly due to antibody potency or the settings in which they have been tested. In this study, we describe the development of a novel high-affinity anti-IL6 antibody, MEDI5117, which features an extended half-life and potent inhibitory effects on IL6 biologic activity. MEDI5117 inhibited IL6-mediated activation of STAT3, suppressing the growth of several tumor types driven by IL6 autocrine signaling. In the same models, MEDI5117 displayed superior preclinical activity relative to a previously developed anti-IL6 antibody. Consistent with roles for IL6 in promoting tumor angiogenesis, we found that MEDI5117 inhibited the growth of endothelial cells, which can produce IL6 and support tumorigenesis. Notably, in tumor xenograft assays in mice, we documented the ability of MEDI5117 to enhance the antitumor activities of chemotherapy or gefitinib in combination treatment regimens. MEDI5117 also displayed robust activity on its own against trastuzumab-resistant HER2+ tumor cells by targeting the CD44+CD24− cancer stem cell population. Collectively, our findings extend the evidence of important pleiotropic roles of IL6 in tumorigenesis and drug resistance, and offer a preclinical proof of concept for the use of IL6 antibodies in combination regimens to heighten therapeutic responses and overcome drug resistance. Cancer Res; 76(2); 480–90. ©2016 AACR.

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