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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.

Supplemental Figures

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.

Despite recent advances in treatment, breast cancer remains the second-most common cause of cancer death among American women. A greater understanding of the molecular characteristics of breast tumors could ultimately lead to improved tumor-targeted treatment options, particularly for subsets of breast cancer patients with unmet needs. Using an unbiased genomics approach to uncover membrane-localized tumor-associated antigens (TAAs), we have identified glial cell line derived neurotrophic factor (GDNF) family receptor α 1 (GFRA1) as a breast cancer TAA. Immunohistochemistry (IHC) revealed that GFRA1 displays a limited normal tissue expression profile coupled with overexpression in specific breast cancer subsets. The cell surface localization as determined by fluorescence-activated cell sorting (FACS) and the rapid internalization kinetics of GFRA1 makes it an ideal target for therapeutic exploitation as an antibody-drug conjugate (ADC). Here, we describe the development of a pyrrolobenzodiazepine (PBD)-armed, GFRA1-targeted ADC that demonstrates cytotoxicity in GFRA1-positive cell lines and patient-derived xenograft (PDX) models. The safety profile of the rat cross-reactive GFRA1-PBD was assessed in a rat toxicology study to find transient cellularity reductions in the bone marrow and peripheral blood, consistent with known off-target effects of PBD ADC's. These studies reveal no evidence of on-target toxicity and support further evaluation of GFRA1-PBD in GFRA1-positive tumors.

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.

BACKGROUND: The goal study of this was to explore attitudes, health knowledge, and experiences with healthcare setting and providers among gay, lesbian, bisexual, transgender, queer/questioning (GLBTQ) individuals and to identify areas for improvement. METHODS: Members of Equality Florida™ residing in the five counties of the Tampa Bay region were recruited through email invitation to complete a 60-item questionnaire assessing demographics, attitudes, and experiences with healthcare providers (HCPs). Additional open-ended questions focused on experiences with HCPs and suggestions for ways to improve HCPs’ cultural competency. RESULTS: 632 respondents completed the survey of which 41% were gay men and 29% were lesbian. The majority of participants was White, non-Hispanic (93%), married/partnered (78%), and had health insurance (88%). The majority (67%) reported they always or often disclosed their sexual orientation/identity to an HCP and few had negative reactions in the healthcare setting (

Pyrrolobenzodiazepine dimers (PBD) form cross-links within the minor groove of DNA causing double-strand breaks (DSB). DNA repair genes such as

Thiosuccinimide-linked antibody-drug conjugates (ADCs) are susceptible to drug loss over time due to a retro-Michael reaction, which can be prevented by selecting stable conjugation positions or hydrolysis of the thiosuccinimide. Here, we investigate pyrrolobenzodiazepine (PBD) ADC drug-linkers equipped with N-phenyl maleimide functionality for stable thiol conjugation via thiosuccinimide hydrolysis. Two PBD drug-linker formats (enzyme-cleavable and non-cleavable) were evaluated following site-specific conjugation to an engineered cysteine incorporated at position T289, which is known to be unstable for N-alkyl maleimide conjugates. N-phenyl maleimide PBDs conjugated to antibodies with similar efficiencies as N-alkyl maleimide PBDs and enhanced thiosuccinimide hydrolysis for N-phenyl maleimide PBDs was confirmed by mass spectrometry, capillary isoelectric focusing, and a SYPRO Orange dye binding assay. All of the PBD ADCs were highly potent in vitro regardless of maleimide- or linker-type, exhibiting low pM EC50 values. Thiol conjugation to N-phenyl maleimide PBD minimized the retro-Michael reaction in both rat and mouse serum. However, cleavage of the valine-alanine dipeptide in mouse serum for ADCs containing cleavable drug-linker led to drug loss regardless of maleimide type, which impacted ADC potency in tumor growth inhibition studies that were conducted in mouse models. Therapeutic improvement in mouse tumor models was realized for ADCs prepared with non-cleavable PBD drug-linkers that were conjugated through N-phenyl maleimide, where a stronger tumor growth inhibition (TGI) response was achieved when compared to the analogous N-alkyl maleimide drug-linker ADC. Altogether, our findings highlight the stability and efficacy benefits of N-phenyl maleimide functionality for ADCs that are produced with thiol-maleimide conjugation chemistry.

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.

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.

Antibody–drug conjugates (ADC) are used to selectively deliver cytotoxic agents to tumors and have the potential for increased clinical benefit to cancer patients. 5T4 is an oncofetal antigen overexpressed on the cell surface in many carcinomas on both bulk tumor cells as well as cancer stem cells (CSC), has very limited normal tissue expression, and can internalize when bound by an antibody. An anti-5T4 antibody was identified and optimized for efficient binding and internalization in a target-specific manner, and engineered cysteines were incorporated into the molecule for site-specific conjugation. ADCs targeting 5T4 were constructed by site-specifically conjugating the antibody with payloads that possess different mechanisms of action, either a DNA cross-linking pyrrolobenzodiazepine (PBD) dimer or a microtubule-destabilizing tubulysin, so that each ADC had a drug:antibody ratio of 2. The resulting ADCs demonstrated significant target-dependent activity in vitro and in vivo; however, the ADC conjugated with a PBD payload (5T4-PBD) elicited more durable antitumor responses in vivo than the tubulysin conjugate in xenograft models. Likewise, the 5T4-PBD more potently inhibited the growth of 5T4-positive CSCs in vivo, which likely contributed to its superior antitumor activity. Given that the 5T4-PBD possessed both potent antitumor activity as well as anti-CSC activity, and thus could potentially target bulk tumor cells and CSCs in target-positive indications, it was further evaluated in non-GLP rat toxicology studies that demonstrated excellent in vivo stability with an acceptable safety profile. Taken together, these preclinical data support further development of 5T4-PBD, also known as MEDI0641, against 5T4+ cancer indications. Mol Cancer Ther; 16(8); 1576–87. ©2017 AACR.

Despite recent advances in treatment, breast cancer remains the second-most common cause of cancer death among American women. A greater understanding of the molecular characteristics of individual tumors could ultimately lead to improved treatment options, particularly for subsets of breast cancer patients with unmet needs. Using an unbiased genomics approach to identify membrane-localized tumor associated antigens (TAAs), we have identified a novel breast cancer TAA that can potentially be exploited through antibody-drug conjugate (ADC) therapy. Here, we describe the development of a GFRA1-targeted ADC that demonstrates activity in GFRA1-positive cell lines and PDX tumor models. Work in non-Good Laboratory Practice (GLP) rat toxicology models revealed acceptable toxicity profiles and supports further evaluation of GFRA1-PBD in GFRA1-positive tumors. Citation Format: Emily E. Bosco, Ronald J. Christie, Rosa Carrasco, Darrin Sabol, Jiping Zha, Karma DaCosta, Lee Brown, Sandrina Phipps, Qun Du, Binyam Bezabeh, Shannon Breen, Cui Chen, Molly Reed, MaryJane Hinrichs, Haihong Zhong, Zhan Xiao, Rakesh Dixit, Ronald Herbst, David A. Tice. Preclinical evaluation of a GFRA1 targeted antibody-drug conjugate in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 759.

Colorectal cancer (CRC) is a heterogeneous disease with a broad spectrum of genetic and epigenetic changes. A comprehensive molecular characterization of CRC by The Cancer Genome Atlas Network detected the overexpression of the insulin-like growth factor 2 (IGF2) gene, encoding a ligand for the insulin-like growth factor 1 receptor (IGF-1R), in a subset of CRC tumors. In this study, we investigated the oncogenic potential of IGF-2 in IGF2-overexpressing CRC models and the efficacy of MEDI-573, an IGF-1/2-neutralizing antibody. We found that a subset of CRC cell lines express high IGF-2 levels owing to an increased DNA copy number and hypermethylation in the H19 promoter of the IGF2 gene. MEDI-573 efficiently neutralized IGF-2 and induced apoptosis, which resulted in significant tumor growth inhibition in CRC mouse models that express high levels of IGF-2. These effects were specific to CRCs overexpressing IGF-2, as MEDI-573 did not affect the growth CRC cell lines with normal levels. Moreover, blockade of IGF-2 by MEDI-573 modulated other signaling pathways, suggesting combination therapies with inhibitors of these pathways. Indeed, in vivo efficacy was significantly enhanced when MEDI-573 was used in combination with trastuzumab, AZD2014 (dual mTORC1/2i), AZD5363 (AKTi) and selumetinib (AZD6244/ARRY-142886, MEK1/2i) or cetuximab. These results demonstrate that overexpressed IGF-2 is the major tumorigenic driver in a subset of CRCs and encourage testing of MEDI-573, alone and in combinations, in IGF2-overexpressing CRC patients.

A drawback of targeting soluble antigens such as cytokines or toxins with long-lived antibodies is that such antibodies can prolong the half-life of the target antigen by a "buffering" effect. This has motivated the design of antibodies that bind to target with higher affinity at near neutral pH relative to acidic endosomal pH (∼pH 6.0). Such antibodies are expected to release antigen within endosomes following uptake into cells, whereas antibody will be recycled and exocytosed in FcRn-expressing cells. To understand how the pH dependence of antibody-antigen interactions affects intracellular trafficking, we generated three antibodies that bind IL-6 with different pH dependencies in the range pH 6.0-7.4. The behavior of antigen in the presence of these antibodies has been characterized using a combination of fixed and live cell fluorescence microscopy. As the affinity of the antibody:IL-6 interaction at pH 6.0 decreases, an increasing amount of antigen dissociates from FcRn-bound antibody in early and late endosomes, and then enters lysosomes. Segregation of antibody and FcRn from endosomes in tubulovesicular transport carriers (TCs) into the recycling pathway can also be observed in live cells, and the extent of IL-6 association with TCs correlates with increasing affinity of the antibody:IL-6 interaction at acidic pH. These analyses result in an understanding, in spatiotemporal terms, of the effect of pH dependence of antibody-antigen interactions on subcellular trafficking and inform the design of antibodies with optimized binding properties for antigen elimination.

Angiopoietin 2 (Ang2) is an important regulator of angiogenesis, blood vessel maturation and integrity of the vascular endothelium. The correlation between the dynamic expression of Ang2 in tumors with regions of high angiogenic activity and a poor prognosis in many tumor types makes Ang2 an ideal drug target. We have generated MEDI3617, a human anti-Ang2 monoclonal antibody that neutralizes Ang2 by preventing its binding to the Tie2 receptor in vitro, and inhibits angiogenesis and tumor growth in vivo. Treatment of mice with MEDI3617 resulted in inhibition of angiogenesis in several mouse models including: FGF2-induced angiogenesis in a basement extract plug model, tumor and retinal angiogenesis. In xenograft tumor models, treatment with MEDI3617 resulted in a reduction in tumor angiogenesis and an increase in tumor hypoxia. The administration of MEDI3617 as a single agent to mice bearing human tumor xenografts resulted in tumor growth inhibition against a broad spectrum of tumor types. Combining MEDI3617 with chemotherapy or bevacizumab resulted in a delay in tumor growth and no body weight loss was observed in the combination groups. These results, combined with pharmacodynamic studies, demonstrate that treatment of tumor-bearing mice with MEDI3617 significantly inhibited tumor growth as a single agent by blocking tumor angiogenesis. Together, these data show that MEDI3617 is a robust antiangiogenic agent and support the clinical evaluation and biomarker development of MEDI3617 in cancer patients.

MEDI-573 is a human antibody that neutralizes the insulin-like growth factors, IGF-1 and IGF-2. IGFs are over-expressed in multiple types of cancer; their over-expression is a potential mechanism for resistance to IGF-1 receptor (IGF-1R)-targeting therapy. Effects of IGFs on cell proliferation, differentiation, and survival are mediated through their binding to and activation of IGF-1R or insulin receptor A (IR-A). In this study, we studied the anti-tumor activity and mechanism of action of MEDI-573 in models of sarcoma. MEDI-573 potently inhibited in vitro proliferation of several sarcoma cell lines, with Ewing's sarcoma cell lines being the most sensitive. This inhibition also occurred after growth stimulation with added IGF-1- and IGF-2. The effect of MEDI-573 on IGF signaling was also examined. Treatment with MEDI-573 markedly reduced levels of pIGF-1R, pIR, and pAKT, and significantly blocked IGF-1- and IGF-2-induced activation of the IGF-1R and AKT pathways. MEDI-573 inhibited the growth of sarcoma xenografts in vivo, and inhibition correlated with neutralization of IGF-1 and IGF-2. Combination of MEDI-573 with either rapamycin or another mTOR inhibitor, AZD2014, significantly enhanced the anti-tumor 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 secreted IGF-1 and IGF-2, and inhibition of IGF-1 and IGF-2 by MEDI-573 results in potent anti-tumor activity in several sarcoma models. Our data provide evidence for evaluation of MEDI-573 and mTORi combinations in clinical studies of sarcoma patients. Note: This abstract was not presented at the meeting. Citation Format: Haihong Zhong, Christine Fazenbaker, Shannon Breen, Cui Chen, Jiaqi Huang, Morehouse Chris, Yihong Yao, Robert Hollingsworth. Medi-573 alone or in combination with mammalian target of rapamycin inhibitors, targets the insulin-like growth factor pathway in sarcomas. [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 4493. doi:10.1158/1538-7445.AM2014-4493

Bi-specific antibodies are suggested to possess unique advantages when compared with mono-specific antibodies as anti-cancer therapeutics. These advantages include greater efficacy, superior combination with standard of care agents, and evasion of resistance mechanisms. Data are presented supporting the hypothesis that the increased avidity of a tetravalent (non BiTE-like) bi-specific antibody mediates such advantages when compared with conventional bi-valent mono-specific antibodies. Experiments include functional comparisons of antibodies binding the Epidermal Growth Factor (EGFR) and the Insulin-like Growth Factor 1 Receptor (IGF1R) at the biochemical, cellular and tumor xenograft levels. These data support the ongoing use of multi-specific antibodies in oncology research and describe a mechanism that may contribute to superior activity compared to mono-specific antibodies and drug combinations. It is noted that advantages of bi-specific antibody formats remain to be seen clinically in terms of both efficacy and safety. Citation Format: Kris F. Sachsenmeier, Nazzareno Dimasi, Qihui Huang, Erin Sult, Binyam Bezabeh, Ryan Fleming, Carl Hay, Helen Zhong, Shannon Breen, Changshou Gao, Balaji Agoram, Lutz Jermutus, Robert Hollingsworth. The avidity hypothesis: comparing bispecific and monospecific antibodies in preclinical oncology models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4635. doi:10.1158/1538-7445.AM2013-4635

Delta-like ligand 4 (DLL4) is a membrane bound ligand for Notch receptors essential for functional vessel formation during development and angiogenesis. We have shown previously that MEDI0639, a human anti-DLL4 antibody, has potent in vitro and in vivo activity in blocking DLL4-Notch interaction. However, because MEDI0639 has low binding affinity to mouse DLL4, to enable in vivo pharmacology studies using xenograft tumor models in mice, MEDI0639 was engineered to bind more strongly to mouse DLL4. The resulting affinity-optimized variant antibody (anti-mDLL4 mAb) bound with high affinity to both human and mouse DLL4 and maintained binding specificity to DLL4. In a number of human cancer xenograft models in nude mice, the MEDI0639 variant antibody markedly inhibited the growth of tumors. qRT-PCR analysis using the Fluidigm platform showed decreases in mouse genes downstream of the Notch signaling pathway at efficacious doses, indicating on-target antibody effect on tumor vasculature. In addition, IHC and imaging analysis of xenograft tumors from the anti-DLL4 mAb dosed animals showed a dose-dependent increase in microvessel density and decrease in tumor perfusion compared to the control groups. In summary, we describe here the generation of mouse and human DLL4 cross-reactive antibody and the utilization of this antibody in demonstrating a positive correlation between the anticipated mechanism of action of DLL4 blockade on vasculature and inhibition of tumor growth. Citation Format: Song H. Cho, David W. Jenkins, Patrick Strout, Martin Korade, Haihong Zhong, Ching Ching Leow, Shannon Breen, Jon Chesebrough, Nicholas Holoweckyj, Ivan Inigo, Gennady Gololobov, Ping Tsui, Kimberly Cook, Melissa Damschroder, Meggan Czapiga, Philip Brohawn, Jiaqi Huang, Sally-Ann Ricketts, Juliana Maynard, Adeela Kamal. A mouse DLL4 cross-reactive variant of MEDI0639 disrupts functional vessel formation and inhibits tumor growth in preclinical models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5098. doi:10.1158/1538-7445.AM2013-5098