Krista Kinneer

A Human Antibody–Drug Conjugate Targeting EphA2 Inhibits Tumor Growth In vivo

The EphA2 receptor tyrosine kinase is selectively expressed on the surface of many different human tumors. We have previously shown that tumor cells can be targeted by EphA2 monoclonal antibodies and that these antibodies function, in part, by inducing EphA2 internalization and degradation. In this report, we describe the isolation and characterization of a fully human monoclonal antibody (1C1) that selectively binds both the human and rodent EphA2 receptor. After cell binding, the antibody induces rapid tyrosine phosphorylation, internalization, and degradation of the EphA2 receptor. Because monoclonal antibodies that selectively bind tumor cells and internalize provide a vehicle for targeted delivery of cytotoxics, 1C1 was conjugated to the microtubule inhibitor monomethylauristatin phenylalanine using a stable maleimidocaproyl linker. The anti-EphA2 antibody-drug conjugate [1C1-maleimidocaproyl-MMAF (mcMMAF)] stimulated the activation of caspase-3/caspase-7 and the death of EphA2-expressing cells with IC(50) values as low as 3 ng/mL. Similarly, the conjugate induced degradation of the EphA2 receptor and inhibited tumor growth in vivo. Administration of 1C1-mcMMAF at doses as low as 1 mg/kg once weekly resulted in significant growth inhibition of EphA2-expressing tumors without any observable adverse effects in mouse xenograft and rat syngeneic tumor models. Our data support the use of an antibody-drug conjugate approach to selectively target and inhibit the growth of EphA2-expressing tumors.

Direct targeting of αvβ3 integrin on tumor cells with a monoclonal antibody, Abegrin™

The humanized monoclonal antibody Abegrin™, currently in phase II trials for treatment of solid tumors, specifically recognizes the integrin αvβ3. Due to its high expression on mature osteoclasts, angiogenic endothelial cells, and tumor cells, integrin αvβ3 functions in several pathologic processes important to tumor growth and metastasis. Targeting of this integrin with Abegrin™ results in antitumor, antiangiogenic, and antiosteolytic activities. Here, we exploit the species specificity of Abegrin™ to evaluate the effects of direct targeting of tumor cells (independent of targeting of endothelia or osteoclasts). Flow cytometry analysis of human tumor cell lines shows high levels of αvβ3 on many solid tumors, including cancers of the prostate, skin, ovary, kidney, lung, and breast. We also show that tumor growth of αvβ3-expressing tumor cells is inhibited by Abegrin™ in a dose-dependent manner. We present a novel finding that high-dose administration can actively impair the antitumor activity of Abegrin™. We also provide evidence that antibody-dependent cellular cytotoxicity contributes to in vitro and in vivo antitumor activity. Finally, it was observed that peak biological activity of Abegrin™ arises at serum levels that are consistent with those achieved in clinical trials. These results support a concept that Abegrin™ can be used to achieve selective targeting of the many tumor cells that express αvβ3 integrin. In combination with the well-established concept that αvβ3 plays a key role in cancer-associated angiogenesis and osteolytic activities, this triad of activity could provide new opportunities for therapeutic targeting of cancer. [Mol Cancer Ther 2006;5(12):3122–9]

EphB4 promotes or suppresses Ras/MEK/ERK pathway in a context-dependent manner: Implications for EphB4 as a cancer target

EphB4 is a member of the Eph receptor tyrosine kinase family shown to act in neuronal guidance and mediate venal/arterial separation. In contrast to these more established roles, EphB4’s function in cancer is much less clear. Here we illustrate both tumor promoting as well as suppressing roles of EphB4, by showing that its activation resulted in inhibition of the Ras/ERK pathway in endothelial cells but activation of the same pathway in MCF-7 breast cancer cells. This was true if EphB4 was stimulated with EphrinB2, its natural ligand, or an agonistic monoclonal antibody for EphB4. Correspondingly, EphB4 activation stimulated MCF7 growth while inhibiting HUVEC cell proliferation. The reason for these dramatic differences is due to functional coupling of EphB4 to different downstream effectors. Reduction of p120 RasGAP in HUVEC cells attenuated the inhibitory effect of EphB4 activation on the ERK pathway, whereas knockdown of PP2A in MCF7 cells attenuated EphB4 activation of the ERK pathway. This represents the first time a functional coupling between Eph receptor and PP2A has been demonstrated leading to activation of an oncogenic pathway. Our study illustrates the caveats and potential challenges of targeting EphB4 for cancer therapy due to the conflicting effects on cancer cell and endothelial cell compartments.

Efficient Preparation of Site-Specific Antibody–Drug Conjugates Using Cysteine Insertion

Antibody-drug conjugates (ADCs) are a class of biopharmaceuticals that combine the specificity of antibodies with the high-potency of cytotoxic drugs. Engineering cysteine residues in the antibodies using mutagenesis is a common method to prepare site-specific ADCs. With this approach, solvent accessible amino acids in the antibody have been selected for substitution with cysteine for conjugating maleimide-bearing cytotoxic drugs, resulting in homogeneous and stable site-specific ADCs. Here we describe a cysteine engineering approach based on the insertion of cysteines before and after selected sites in the antibody, which can be used for site-specific preparation of ADCs. Cysteine-inserted antibodies have expression level and monomeric content similar to the native antibodies. Conjugation to a pyrrolobenzodiazepine dimer (SG3249) resulted in comparable efficiency of site-specific conjugation between cysteine-inserted and cysteine-substituted antibodies. Cysteine-inserted ADCs were shown to have biophysical properties, FcRn, and antigen binding affinity similar to the cysteine-substituted ADCs. These ADCs were comparable for serum stability to the ADCs prepared using cysteine-mutagenesis and had selective and potent cytotoxicity against human prostate cancer cells. Two of the cysteine-inserted variants abolish binding of the resulting ADCs to FcγRs in vitro, thereby potentially preventing non-target mediated uptake of the ADCs by cells of the innate immune system that express FcγRs, which may result in mitigating off-target toxicities. A selected cysteine-inserted ADC demonstrated potent dose-dependent anti-tumor activity in a xenograph tumor mouse model of human breast adenocarcinoma expressing the oncofetal antigen 5T4.

A Potent HER3 Monoclonal Antibody That Blocks Both Ligand-Dependent and -Independent Activities: Differential Impacts of PTEN Status on Tumor Response

HER3/ERBB3 is a kinase-deficient member of the EGFR family receptor tyrosine kinases (RTK) that is broadly expressed and activated in human cancers. HER3 is a compelling cancer target due to its important role in activation of the oncogenic PI3K/AKT pathway. It has also been demonstrated to confer tumor resistance to a variety of cancer therapies, especially targeted drugs against EGFR and HER2. HER3 can be activated by its ligand (heregulin/HRG), which induces HER3 heterodimerization with EGFR, HER2, or other RTKs. Alternatively, HER3 can be activated in a ligand-independent manner through heterodimerization with HER2 in HER2-amplified cells. We developed a fully human mAb against HER3 (KTN3379) that efficiently suppressed HER3 activity in both ligand-dependent and independent settings. Correspondingly, KTN3379 inhibited tumor growth in divergent tumor models driven by either ligand-dependent or independent mechanisms in vitro and in vivo. Most intriguingly, while investigating the mechanistic underpinnings of tumor response to KTN3379, we discovered an interesting dichotomy in that PTEN loss, a frequently occurring oncogenic lesion in a broad range of cancer types, substantially blunted the tumor response in HER2-amplified cancer, but not in the ligand-driven cancer. To our knowledge, this represents the first study ascertaining the impact of PTEN loss on the antitumor efficacy of a HER3 mAb. KTN3379 is currently undergoing a phase Ib clinical trial in patients with advanced solid tumors. Our current study may help us optimize patient selection schemes for KTN3379 to maximize its clinical benefits. Mol Cancer Ther; 15(4); 689–701. ©2016 AACR.

Preclinical assessment of an antibody–PBD conjugate that targets BCMA on multiple myeloma and myeloma progenitor cells

MP, Ries RE, et al. Expression and functional characterization of CD33 transcript variants in human acute myeloid leukemia. Oncotarget. 2016;7:43281–94. 12. Humbert O, Peterson CW, Norgaard ZK, Radtke S, Kiem HP. A nonhuman primate transplantation model to evaluate hematopoietic stem cell gene editing strategies for betahemoglobinopathies. Mol Ther Methods Clin Dev. 2018;8:75–86. 13. Correnti CE, Laszlo GS, de van der Schueren WJ, Godwin CD, Bandaranayake A, Busch MA, et al. Simultaneous multiple interaction T-cell engaging (SMITE) bispecific antibodies overcome bispecific T-cell engager (BiTE) resistance via CD28 costimulation. Leukemia. 2018;32:1239–43. 14. Haworth KG, Ironside C, Norgaard ZK, Obenza WM, Adair JE, Kiem HP. In vivo murine-matured human CD3(+ ) cells as a preclinical model for T cell-based immunotherapies. Mol Ther Methods Clin Dev. 2017;6:17–30. 15. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9:671–5.

Synthesis of a heterotrifunctional linker for the site-specific preparation of antibody-drug conjugates with two distinct warheads

Codelivery of multiple therapeutic agents with different anticancer mechanisms can overcome drug resistance as well as generate additive or synergistic anticancer effects that may enhance the antitumor efficacy. Antibody-drug conjugates (ADCs) can be used for highly specific delivery of multiple therapeutic agents with different anticancer mechanisms, though more research is required towards designing flexible platforms on which dual drug ADCs could be prepared. Herein, we describe the synthesis of a heterotrifunctional linker that could be used to construct flexible platforms for preparing dual-cytotoxic drug conjugates in a site-specific manner. As a proof of concept, we synthesized dual drug ADCs carrying monomethyl auristain E (MMAE, tubulin polymerization inhibitor) and pyrrolobenzodiazepine dimer (PBD, DNA minor groove alkylator). We then evaluated the dual drug ADCs for in vitro efficacy and confirmed the dual mechanism of action.

Abstract P4-07-05: MEDI3379, an antibody against HER3, is active in HER2-driven human breast tumor models

HER3 (ERBB3) is a member of the EGFR/HER family of receptor tyrosine kinases (RTK), consisting of EGFR, HER2, HER3 and HER4. Unlike the other HER family members, HER3 lacks intrinsic tyrosine kinase activity and therefore needs to form heterodimers with EGFR, HER2 or other kinase-proficient RTKs to be functionally active. Dimerization is induced by overexpression of EGFR or HER2 in a ligand-independent (LI) manner. In this process HER3 acts as a driver in divergent cancer types including HER2-positive breast cancer (BC) via induction of the PI3K-AKT pathway. Alternatively, heregulin (NRG1/HRG), the major HER3 ligand, induces a conformational shift in HER3 which leads to dimer formation with a partner RTK in a ligand-dependent (LD) manner. We have developed an antagonistic human monoclonal antibody against HER3, termed MEDI3379, and tested it in multiple breast cancer cell lines. We observed effective suppression of constitutive pHER3 and pAKT with MEDI3379, leading to anti-proliferation effects in cell culture models. Preclinical evaluation of MEDI3379 demonstrated antitumor activity in several orthotopic BC xenografts in nude mice which did not express HRG. For example, the BC xenograft model BT474 with amplified HER2 responded to MEDI3379 (65% dTGI). In conclusion, our findings with targeting of HER3 in mouse models support continued development of MEDI3379 for cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-07-05.

Abstract 307: EphB4 promotes or suppresses Ras/ERK pathway depending on cellular contexts: Implications for EphB4 as a cancer target

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC EphB4 is a member of the Eph RTK family. Its classical role has been shown to act in neuronal guidance and mediate venal/arterial separation. In contrast to these more established roles, EphB4s function in cancer is controversial since both tumor promoting as well as tumor suppressing functions have been ascribed to it. Here we provide a vivid illustration of this intriguing dichotomy by showing that EphB4 activation resulted in inhibition of the Ras/ERK pathway in endothelial cells but induction of the same pathway in MCF-7 breast cancer cells. This was true if EphB4 was stimulated with ephrinB2, its natural ligand, or an EphB4-specific fully human monoclonal antibody. The reason for these dramatic differences is due to potential functional coupling of EphB4 to different downstream effectors. In HUVEC cells, the RNAi knockdown of p120 RasGAP, a Ras inhibitor, abrogated the inhibitory effect on Ras/ERK pathway. However, in MCF7 cells, RNAi knockdown of PP2A, which has been shown to activate the Ras/ERK pathway through removal of the inhibitory phosphorylation on c-Raf, negated EphB4-driven activation of the Ras/ERK pathway. This represents the first time a functional coupling between Eph receptor and PP2A has been established, and this interaction appears to confer activation but not suppression of an oncogenic pathway. Our study also demonstrates the caveats and potential challenges of targeting EphB4 for cancer therapy due to the presumed conflicting effects on cancer cell and endothelial cell compartments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 307.