Jochem Gokemeijer

Phase I and Pharmacokinetic Study of CT-322 (BMS-844203), a Targeted Adnectin Inhibitor of VEGFR-2 Based on a Domain of Human Fibronectin

Purpose: To determine the maximum tolerated dose (MTD), safety, pharmacokinetics, pharmacodynamics, immunogenicity, and preliminary antitumor activity of CT-322 (BMS-844203), a VEGFR-2 inhibitor and the first human fibronectin domain–based targeted biologic (Adnectin) to enter clinical studies. Experimental Design: Patients with advanced solid malignancies were treated with escalating doses of CT-322 intravenously (i.v.) weekly (qw), or biweekly (q2w). Plasma samples were assayed for CT-322 concentrations, plasma VEGF-A concentrations, and antidrug antibodies. Results: Thirty-nine patients completed 105 cycles of 0.1 to 3.0 mg/kg CT-322 i.v. either qw or q2w. The most common treatment-emergent grade 1/2 toxicities were fatigue, nausea, proteinuria, vomiting, anorexia, and hypertension. Grade 3/4 toxicities were rare. Reversible proteinuria, retinal artery, and vein thrombosis, left ventricular dysfunction, and reversible posterior leukoencephalopathy syndrome were dose limiting at 3.0 mg/kg. The MTD was 2 mg/kg qw or q2w. CT-322 plasma concentrations increased dose proportionally. Plasma VEGF-A levels increased with dose and plateaued at 2 mg/kg qw. Anti–CT-322 antibodies developed without effects on pharmacokinetics, VEGF-A levels, or safety. Minor decreases in tumor measurements occurred in 4 of 34 evaluable patients and 24 patients had stable disease. Conclusions: CT-322 can be safely administered at 2 mg/kg i.v. qw or q2w and exhibits promising antitumor activity in patients with advanced solid tumors. The absence of severe toxicities at the MTD, demonstration of plasma drug concentrations active in preclinical models, and clinical pharmacodynamic evidence of VEGFR-2 inhibition warrant further development of CT-322 and suggest strong potential for Adnectin-based targeted biologics. Cancer Res; 17(2); 363–71. ©2011 AACR. Clin Cancer Res; 17(2); 363–71. ©2011 AACR.

A fibronectin scaffold approach to bispecific inhibitors of epidermal growth factor receptor and insulin-like growth factor-I receptor.

Engineered domains of human fibronectin (Adnectins™) were used to generate a bispecific Adnectin targeting epidermal growth factor receptor (EGFR) and insulin-like growth factor-I receptor (IGF-IR), two transmembrane receptors that mediate proliferative and survival cell signaling in cancer. Single-domain Adnectins that specifically bind EGFR or IGF-IR were generated using mRNA display with a library containing as many as 1013 Adnectin variants. mRNA display was also used to optimize lead Adnectin affinities, resulting in clones that inhibited EGFR phosphorylation at 7 to 38 nM compared to 2.6 μM for the parental clone. Individual, optimized, Adnectins specific for blocking either EGFR or IGF-IR signaling were engineered into a single protein (EI-Tandem Adnectin). The EI-Tandems inhibited phosphorylation of EGFR and IGF-IR, induced receptor degradation, and inhibited down-stream cell signaling and proliferation of human cancer cell lines (A431, H292, BxPC3 and RH41) with IC50 values ranging from 0.1 to 113 nM. Although Adnectins bound to EGFR at a site distinct from those of anti-EGFR antibodies cetuximab, panitumumab and nimotuzumab, like the antibodies, the anti-EGFR Adnectins blocked the binding of EGF to EGFR. PEGylated EI-Tandem inhibited the growth of both EGFR and IGF-IR driven human tumor xenografts, induced degradation of EGFR, and reduced EGFR phosphorylation in tumors. These results demonstrate efficient engineering of bispecific Adnectins with high potency and desired specificity. The bispecificity may improve biological activity compared to monospecific biologics as tumor growth is driven by multiple growth factors. Our results illustrate a technological advancement for constructing multi-specific biologics in cancer therapy.

Anti-tumor effect of CT-322 as an Adnectin inhibitor of vascular endothelial growth factor receptor-2

CT-322 is a new anti-angiogenic therapeutic agent based on an engineered variant of the tenth type III domain of human fibronectin, i.e., an AdnectinTM, designed to inhibit vascular endothelial growth factor receptor (VEGFR)-2. This PEGylated Adnectin was developed using an mRNA display technology. CT-322 bound human VEGFR-2 with high affinity (KD, 11 nM), but did not bind VEGFR-1 or VEGFR-3 at concentrations up to 100 nM, as determined by surface plasmon resonance studies. Western blot analysis showed that CT-322 blocked VEGF-induced phosphorylation of VEGFR-2 and mitogen-activated protein kinase in human umbilical vascular endothelial cells. CT-322 significantly inhibited the growth of human tumor xenograft models of colon carcinoma and glioblastoma at doses of 15-60 mg/kg administered 3 times/week. Anti-tumor effects of CT-322 were comparable to those of sorafenib or sunitinib, which inhibit multiple kinases, in a colon carcinoma xenograft model, although CT-322 caused less overt adverse effects than the kinase inhibitors. CT-322 also enhanced the anti-tumor activity of the chemotherapeutic agent temsirolimus in the colon carcinoma model. The high affinity and specificity of CT-322 binding to VEGFR-2 and its anti-tumor activities establish CT-322 as a promising anti-angiogenic therapeutic agent. Our results further suggest that Adnectins are an important new class of targeted biologics that can be developed as potential treatments for a wide variety of diseases.

Synthesis and Biologic Evaluation of a Novel 18F-Labeled Adnectin as a PET Radioligand for Imaging PD-L1 Expression

The programmed death protein (PD-1) and its ligand (PD-L1) play critical roles in a checkpoint pathway cancer cells exploit to evade the immune system. A same-day PET imaging agent for measuring PD-L1 status in primary and metastatic lesions could be important for optimizing drug therapy. Herein, we have evaluated the tumor targeting of an anti–PD-L1 adnectin after 18F-fluorine labeling. Methods: An anti–PD-L1 adnectin was labeled with 18F in 2 steps. This synthesis featured fluorination of a novel prosthetic group, followed by a copper-free click conjugation to a modified adnectin to generate 18F-BMS-986192. 18F-BMS-986192 was evaluated in tumors using in vitro autoradiography and PET with mice bearing bilateral PD-L1–negative (PD-L1(–)) and PD-L1–positive (PD-L1(+)) subcutaneous tumors. 18F-BMS-986192 was evaluated for distribution, binding, and radiation dosimetry in a healthy cynomolgus monkey. Results: 18F-BMS-986192 bound to human and cynomolgus PD-L1 with a dissociation constant of less than 35 pM, as measured by surface plasmon resonance. This adnectin was labeled with 18F to yield a PET radioligand for assessing PD-L1 expression in vivo. 18F-BMS-986192 bound to tumor tissues as a function of PD-L1 expression determined by immunohistochemistry. Radioligand binding was blocked in a dose-dependent manner. In vivo PET imaging clearly visualized PD-L1 expression in mice implanted with PD-L1(+), L2987 xenograft tumors. Two hours after dosing, a 3.5-fold-higher uptake (2.41 ± 0.29 vs. 0.82 ± 0.11 percentage injected dose per gram, P < 0.0001) was observed in L2987 than in control HT-29 (PD-L1(–)) tumors. Coadministration of 3 mg/kg ADX_5322_A02 anti–PD-L1 adnectin reduced tumor uptake at 2 h after injection by approximately 70%, whereas HT-29 uptake remained unchanged, demonstrating PD-L1–specific binding. Biodistribution in a nonhuman primate showed binding in the PD-L1–rich spleen, with rapid blood clearance through the kidneys and bladder. Binding in the PD-L1(+) spleen was reduced by coadministration of BMS-986192. Dosimetry estimates indicate that the kidney is the dose-limiting organ, with an estimated human absorbed dose of 2.20E–01 mSv/MBq. Conclusion: 18F-BMS-986192 demonstrated the feasibility of noninvasively imaging the PD-L1 status of tumors by small-animal PET studies. Clinical studies with 18F-BMS-986192 are under way to measure PD-L1 expression in human tumors.

How Close Are We to Profiling Immunogenicity Risk Using In Silico Algorithms and In Vitro Methods?: an Industry Perspective

In silico HLA-binding algorithms and in vitro T cell-based assays as predictive tools for human immunogenicity risk have made inroads in the biotherapeutic drug discovery and development process. Currently, these tools are being used only for candidate selection or characterization and not for making a go/no-go decision for further development. A clear limitation for a broader implementation is the lack of correlation between the predicted T cell epitope content/immune reactivity potential of a biotherapeutic and the subsequent ADA-related clinical immunogenicity outcome. The current state of technologies and their pros and cons were discussed as a part of the 2016 AAPS National Biotechnology Conference in a themed session. A review of the advances in the area and the session talks along with the ensuing discussions are summarized in this commentary.

Abstract 2586: Adnectins as a platform for multi-specific targeted biologics: A novel bispecific inhibitor of EGFR and IGF-IR growth factor receptors

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor-1 (IGFR) are transmembrane receptor tyrosine kinases that mediate proliferative and invasive cell signaling in cancer. Inhibition of either receptor reduces tumor growth in both mouse models and in human clinical studies. Blocking the EGFR pathway can induce compensatory activation of the IGFR pathway to drive tumor growth and IGFR inhibition can result in activation of EGFR signaling in preclinical models. Therefore, blocking both receptors simultaneously may achieve superior efficacy to blocking either pathway alone. We developed individual optimized Adnectins™ specific for blocking either EGFR or IGFR signaling and engineered them into a single protein that linked both Adnectins together to construct a bi-specific Adnectin targeting the EGFR and IGFR (EI-tandem). The bifunctional molecule blocked activation of EGFR and IGFR, inhibited both EGF and IGF-induced down-stream cell signaling (MAPK and AKT pathways) and was antiproliferative in human cancer cell lines. Potency of the EI-tandem was comparable to anti-EGFR and anti-IGFR antibodies. The EI-tandem demonstrated a synergistic inhibition of IGFR phosphorylation and down-stream cell signaling compared to Adnectins specific for only EGFR or IGFR alone. Although Adnectins bound to the EGFR at a site distinct from the clinically approved anti-EGFR antibodies cetuximab, panitumumab and nimotuzumab, they still blocked binding of EGF to the EGFR. PEGylated EI-tandem inhibited the growth of human tumor xenografts driven by both EGFR and IGFR signaling, degraded EGFR and IGFR, and reduced phosphorylation of EGFR in tumors. Treatment of mice with EI-tandem caused increases in levels of the circulating ligands TGFα and IGF1 resulting from blockade of their respective receptors and provided convenient soluble biomarkers of target suppression. These results show that a bifunctional Adnectin can confer improved biological activity compared to monospecific biologics in tumors where growth is driven by multiple growth factors. 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 2586.

Altering drug tolerance of surface plasmon resonance assays for the detection of anti-drug antibodies

Anti-drug antibody (ADA) responses are a concern for both drug efficacy and safety, and high drug concentrations in patient samples may inhibit ADA assays. We evaluated strategies to improve drug tolerance of surface plasmon resonance (SPR) assays that detect ADAs against a bispecific Adnectin drug molecule that consists of an anti-VEGFR2 domain linked to an anti-IGF-1R domain (V-I-Adnectin). Samples containing ADAs against V-I-Adnectin and various drug concentrations were tested in the presence of 1 M guanidine hydrochloride (Gdn), at pH values ranging from 4.5 to 7.4 and temperatures of up to 37 °C. Temperature had a negligible effect in weakening the affinity of interaction of monoclonal antibodies with polyethylene glycol(PEG)-V-I-Adnectin and did not increase drug tolerance of the ADA assay. Low pH increased drug tolerance of the assay relative to pH 7.4 but caused nonspecific binding of the drug during competition experiments. The chaotropic agent Gdn lowered the affinity of interaction between an anti-V-Adnectin monoclonal antibody and the drug (from K(D)=0.93 nM to K(D)=348 nM). That decrease in the affinity of drug-ADA interaction correlated with an increase of assay drug tolerance. Conditions that lower drug-ADA interaction affinity could also be used to develop drug-tolerant SPR assays for other systems.

Abstract 871: [18F]BMS-986192 as a novel PET imaging agent for assessment of PD-L1 expressionin vivo

Objectives Inhibition of the Programmed Death Ligand-1 (PD-L1)/PD-1 interaction allows for potent anti-tumor activity and antibodies that disrupt this interaction have been approved for the treatment of multiple cancer types. PD-L1 expression has been investigated clinically as a potential biomarker to predict response to anti-PD-1/PD-L1 therapy. BMS-986192, an Adnectin with high affinity and specificity for human PD-L1, was selected in vitro from a complex library. Here we report the discovery and first preclinical evaluation of [18F]BMS-986192 as a PET imaging agent to detect PD-L1 expression in vivo. Methods [18F]BMS-986192 was radiolabeled via copper-free click chemistry and assessed for its ability to detect PD-L1 expression. Tracer binding to human L2987 (PD-L1+) and HT-29 (PD-L1-) xenografts as well as human non-small cell lung cancer (NSCLC) tissue samples was assessed by autoradiography (ARG). Tracer binding was compared to PD-L1 expression assessed independently with anti-PD-L1 immunohistochemistry (IHC). In vivo performance of the tracer was also assessed by PET imaging in mice bearing bilateral L2987 and HT-29 xenografts, and tracer biodistribution was further assayed in these animals ex vivo by gamma counter. Finally, initial in vivo biodistribution and radiation dosimetry was measured by PET in cynomolgus monkey. Results ARG studies showed increased [18F]BMS-986192 total binding to PD-L1(+) L2987 xenograft compared to PD-L1(-) HT-29 xenograft tissue. Radiotracer binding was higher in all tested human NSCLC tissue samples compared to xenografts. Dose-dependent blockade was seen in all PD-L1(+) tissues co-incubated with cold BMS-986192, and binding was unaffected by co-incubation with cold non-PD-L1 binding control. Visual comparison of tracer binding aligns closely with PD-L1 IHC both spatially as well as in intensity. Preferential accumulation of [18F]BMS-986192 was noted in PD-L1(+) L2987 compared to PD-L1(-) HT-29 xenografts in tumor-bearing mice. PET studies in cynomolgus monkeys confirmed binding to PD-L1(+) tissue (e.g. spleen) with minimal nonspecific background signal exclusive of primary clearance organs. Radiation dosimetry of [18F]BMS-986192 indicates an estimated single administration dose limit of 6.2 mCi for an average human subject. Conclusions ARG, PET studies, and ex vivo measurements in rodent and cynomolgus monkey demonstrated sensitive and specific [18F]BMS-986192 binding to PD-L1. Low background signal in cynomolgus monkey in the context of endogenous PD-L1 expression further supports the potential of this tracer for sensitive detection of PD-L1(+) lesions in vivo. Radiation dosimetry suggests that [18F]BMS-986192 can be safely administered in human trials, with estimated absorbed radiation doses well within safe parameters for human administration. [18F]BMS-986192 has potential as a sensitive PD-L1 imaging agent for same-day imaging in patients. Citation Format: Ralph A. Smith, David Donnelly, Paul E. Morin, Dasa Lipovsek, Jochem Gokemeijer, Daniel Cohen, Joonyoung Kim, Adrienne Pena, Olufemi Adelakun, Xi-Tao Wang, Patrick Chow, Samuel J. Bonacorsi, Wendy Hayes. [18F]BMS-986192 as a novel PET imaging agent for assessment of PD-L1 expression in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 871. doi:10.1158/1538-7445.AM2017-871