Dasa Lipovsek

Adnectins: engineered target-binding protein therapeutics

AdnectinsTM are a new family of therapeutic proteins based on the 10th fibronectin type III domain, and designed to bind with high affinity and specificity to therapeutically relevant targets. Adnectins share with antibody variable domains a beta-sheet sandwich fold with diversified loops, but differ from antibodies in primary sequence and have a simpler, single-domain structure without disulfide bonds. As a consequence, Adnectins bind targets with affinity and specificity as high as those of antibodies, but are easier to manipulate genetically and compatible with bacterial expression systems. Adnectins that bind macromolecular targets with nanomolar and picomolar affinity have been selected using in vitro evolution methods, including mRNA display, phage display and yeast display. CT-322, a PEGylated, anti-angiogenic Adnectin that binds vascular endothelial growth factor (VEGF) receptor 2 and blocks its interaction with VEGF A, C and D, is being evaluated in Phase II clinical trials for efficacy in several oncology indications.

Chapter six – Target-Binding Proteins Based on the 10th Human Fibronectin Type III Domain (10Fn3)

We describe concepts and methods for generating a family of engineered target-binding proteins designed on the scaffold of the 10th human fibronectin type III domain ((10)Fn3), an extremely stable, single-domain protein with an immunoglobulin-like fold but lacking disulfide bonds. Large libraries of possible target-binding proteins can be constructed on the (10)Fn3 scaffold by diversifying the sequence and length of its surface loops, which are structurally analogous to antibody complementarity-determining regions. Target-binding proteins with high affinity and specificity are selected from (10)Fn3-based libraries using in vitro evolution technologies such as phage display, mRNA display, or yeast-surface display. (10)Fn3-based target-binding proteins have binding properties comparable to those of antibodies, but they are smaller, simpler in architecture, and more user-friendly; as a consequence, these proteins are excellent building blocks for the construction of multidomain, multifunctional chains. The ease of engineering and robust properties of (10)Fn3-based target-binding proteins have been validated by multiple independent academic and industrial groups. In addition to performing well as specific in vitro detection reagents and research tools, (10)Fn3-based binding proteins are being developed as therapeutics, with the most advanced candidate currently in Phase II clinical trials.

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.

Ensemble Modeling and Intracellular Aggregation of an Engineered Immunoglobulin-Like Domain.

The production of recombinant proteins in Escherichia coli frequently results in the formation of insoluble protein aggregates called inclusion bodies (IBs). The determinants of IB formation remain poorly understood and are of much interest for biotechnological and research applications, as well as offering insight into disease-related in vivo protein aggregation. Here we investigate a set of engineered target-binding proteins based upon the fibronectin type III domain, and we find that variations in sequence at just three positions in a solvent-exposed loop greatly alter the extent of IB formation. The loop is analogous to the third complementarity-determining region of immunoglobulin variable domains and has been shown to be conformationally mobile. In contrast to studies of other proteins, the extent of IB formation is not explained by differences in thermal stability measured by differential scanning calorimetry. Instead, IB formation is correlated with the average local stability of the FG loop, as modeled by an ensemble of structures generated using Rosettas kinematic closure loop reconstruction method. This correlation suggests that loop instability may promote local unfolding, exposing aggregation-prone surfaces. Consistent with this mechanism, sequence-based predictions of aggregation propensity produced by Zyggregator are also correlated with IB formation, though not with modeled loop stability. The combination of average model energy scores with sequence-based aggregation predictions accounts for the variation in IB formation remarkably well (R(2)=0.8). The comparison with experimental data validates the ensemble modeling approach, which may be applicable to dynamic protein loops involved in a wide range of phenomena.

Adnectin–drug conjugates for Glypican-3-specific delivery of a cytotoxic payload to tumors

Abstract Tumor-specific delivery of cytotoxic agents remains a challenge in cancer therapy. Antibody–drug conjugates (ADC) deliver their payloads to tumor cells that overexpress specific tumor-associated antigens—but the multi-day half-life of ADC leads to high exposure even of normal, antigen-free, tissues and thus contributes to dose-limiting toxicity. Here, we present Adnectin–drug conjugates, an alternative platform for tumor-specific delivery of cytotoxic payloads. Due to their small size (10 kDa), renal filtration eliminates Adnectins from the bloodstream within minutes to hours, ensuring low exposure to normal tissues. We used an engineered cysteine to conjugate an Adnectin that binds Glypican-3, a membrane protein overexpressed in hepatocellular carcinoma, to a cytotoxic derivative of tubulysin, with the drug-to-Adnectin ratio of 1. We demonstrate specific, nanomolar binding of this Adnectin–drug conjugate to human and murine Glypican-3; its high thermostability; its localization to target-expressing tumor cells in vitro and in vivo, its fast clearance from normal tissues and its efficacy against Glypican-3-positive mouse xenograft models.

Conformational Assessment of Adnectin and Adnectin-Drug Conjugate by Hydrogen/Deuterium Exchange Mass Spectrometry

AbstractHigher-order structure (HOS) characterization of therapeutic protein-drug conjugates for comprehensive assessment of conjugation-induced protein conformational changes is an important consideration in the biopharmaceutical industry to ensure proper behavior of protein therapeutics. In this study, conformational dynamics of a small therapeutic protein, adnectin 1, together with its drug conjugate were characterized by hydrogen/deuterium exchange mass spectrometry (HDX-MS) with different spatial resolutions. Top-down HDX allows detailed assessment of the residue-level deuterium content in the payload conjugation region. HDX-MS dataset revealed the ability of peptide-based payload/linker to retain deuterium in HDX experiments. Combined results from intact, top-down, and bottom-up HDX indicated no significant conformational changes of adnectin 1 upon payload conjugation. Graphical Abstractᅟ

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