Antibody-like Affinity Research Articles

Evaluation of 18F labeled glial fibrillary acidic protein binding nanobody and its brain shuttle peptide fusion proteins using a neuroinflammation rat model.

Astrogliosis is a crucial feature of neuroinflammation and is characterized by the significant upregulation of glial fibrillary acidic protein (GFAP) expression. Hence, visualizing GFAP in the living brain of patients with damaged central nervous system using positron emission tomography (PET) is of great importance, and it is expected to depict neuroinflammation more directly than existing neuroinflammation imaging markers. However, no PET radiotracers for GFAP are currently available. Therefore, neuroimaging with antibody-like affinity proteins could be a viable strategy for visualizing imaging targets that small molecules rarely recognize, such as GFAP, while we need to overcome the challenges of slow clearance and low brain permeability. The E9 nanobody, a small-affinity protein with high affinity and selectivity for GFAP, was utilized in this study. E9 was engineered by fusing a brain shuttle peptide that facilitates blood-brain barrier permeation via two different types of linker domains: E9-GS-ApoE (EGA) and E9-EAK-ApoE (EEA). E9, EGA and EEA were radiolabeled with fluorine-18 using cell-free protein radiosynthesis. In vitro autoradiography showed that all radiolabeled proteins exhibited a significant difference in neuroinflammation in the brain sections created from a rat model constructed by injecting lipopolysaccharide (LPS) into the unilateral striatum of wildtype rats, and an excess competitor displaced their binding. However, exploratory in vivo PET imaging and ex vivo biodistribution studies in the rat model failed to distinguish neuroinflammatory lesions within 3 h of 18F-EEA intravenous injection. This study contributes to a better understanding of the characteristics of small-affinity proteins fused with a brain shuttle peptide for further research into the use of protein molecules as PET tracers for imaging neuropathology.

Abstract 5552: BT7480, a novel fully synthetic tumor-targeted immune cell agonist (TICATM) induces tumor localized 4-1BB agonism

Abstract Costimulatory molecules expressed on activated T and NK cells such as 4-1BB (CD137/TNFRSF9) can be leveraged for cancer immunotherapy. Despite compelling preclinical data, 4-1BB agonistic antibodies have been hampered by failure to delineate hepatoxicity from efficacy in the clinic [1,2]. Next generation strategies are focused on bispecific approaches aimed at promoting target-mediated clustering of 4-1BB to limit systemic and liver effects [3,4]. Bicycles® are fully synthetic, constrained bicyclic peptides that have antibody-like affinity and selectivity to their targets. Unlike traditional biologic approaches, the small size (~2 kDa) and tunable pharmacokinetic (PK) parameters of Bicycles enable superior tumor penetration and allow exploration into the relationship between pulsatile dosing and 4-1BB activation while de-risking hepatoxicity concerns due to a differentiated renal elimination mechanism combined with a tumor-localized immune response. We hypothesized that clustering and activation of 4-1BB could be achieved by conjugating a 4-1BB binding Bicycle to a tumor antigen targeting Bicycle. BT7480 is a tumor-targeted immune cell agonist (TICATM) targeting Nectin-4 and agonizing 4-1BB. Nectin-4 (PVRL4) is highly expressed on numerous tumors with unmet medical need, including bladder, pancreatic, breast, ovarian, esophageal, and lung. BT7480 exhibits highly potent 4-1BB agonism in an engineered 4-1BB reporter system that correlates with Nectin-4 surface expression on the co-cultured tumor cells. In addition, BT7480 induces robust production of interleukin-2 (IL-2) and interferon gamma (IFNγ) in primary PBMC/tumor cell co-culture assays. This activity is strictly dependent on the tumor cells expressing Nectin-4 and on the ability of the TICA to bind to both Nectin-4 and 4-1BB. Nectin-4/4-1BB TICAs are also target-specific immune cell stimulators of patient-derived lung tumors with an intact immune microenvironment. Intermittent dosing of BT7480 led to robust anti-tumor efficacy with 22 out of 24 complete responders (CRs) in a MC38 (Nectin-4-expressing) syngeneic mouse model. Importantly, a memory response was established as the CR mice were resistant to re-challenge with MC38 tumors. Additionally, BT7480 led to increased intratumoral T cell infiltration without elevation of liver enzymes in a CT26 (Nectin-4-expressing) syngeneic mouse model. In non-human primates (NHPs), BT7480 exhibits dose linear exposure and is well tolerated up to 10mpk. Further dose-range finding and safety analysis in NHPs is currently ongoing. BT7480 represents a new generation of chemically synthetic tumor antigen targeted 4-1BB agonists.

Use of Super-Resolution Optical Microscopy To Reveal Direct Virus Binding at Hybrid Core-Shell Matrixes.

Polymer particles with antibody-like affinity, i.e., molecularly imprinted polymers, offer an ideal platform for biopharmaceutical virus purification. In recent years, attempts combining molecular imprinting technology with a variety of visualization and detection techniques have been reported for directly confirming the localized presence of the template. Direct target visualization is crucial for the characterization of molecularly imprinted polymers, especially if biological templates such as viruses are used. In the present study, for the first time the viral binding behavior at virus-imprinted polymers (VIPs) via stimulated emission depletion (STED) microscopy is shown by imaging individual, fluorescently labeled virus particles. STED microscopy achieves among various other super-resolution techniques the best temporal resolution at high spatial resolution. An innovative virus purification material selective for human adenovirus type 5 (AdV5) offered highly purified virus for the subsequent fluorescent labeling procedure, thus enabling STED imaging. Excellent binding affinities (150-fold higher versus control particles) and high selectivity toward the target virus (AdV5) were observed at those VIPs, even in competitive binding experiments with minute virus of mice using dual-label STED microscopy.

Understanding Cell Penetration of Cyclic Peptides.

Approximately 75% of all disease-relevant human proteins, including those involved in intracellular protein-protein interactions (PPIs), are undruggable with the current drug modalities (i.e., small molecules and biologics). Macrocyclic peptides provide a potential solution to these undruggable targets because their larger sizes (relative to conventional small molecules) endow them the capability of binding to flat PPI interfaces with antibody-like affinity and specificity. Powerful combinatorial library technologies have been developed to routinely identify cyclic peptides as potent, specific inhibitors against proteins including PPI targets. However, with the exception of a very small set of sequences, the vast majority of cyclic peptides are impermeable to the cell membrane, preventing their application against intracellular targets. This Review examines common structural features that render most cyclic peptides membrane impermeable, as well as the unique features that allow the minority of sequences to enter the cell interior by passive diffusion, endocytosis/endosomal escape, or other mechanisms. We also present the current state of knowledge about the molecular mechanisms of cell penetration, the various strategies for designing cell-permeable, biologically active cyclic peptides against intracellular targets, and the assay methods available to quantify their cell-permeability.

Molecularly imprinted polymer based sensor directly responsive to attomole bovine serum albumin

Through years of extensive research and development, molecularly imprinted polymers (MIPs) are still inferior to their biological rivals such as antibodies, enzymes etc. In this study we report a protein-imprinted cryogel, showing antibody-like affinity and selectivity against the protein template (bovine serum albumin, BSA). The MIP was synthesized from the co-polymerization of acrylamide, N,N-methylenebisacrylamide, acrylic acid and diallylamine. Due to the participation of the ionizable monomers (acrylic acid and diallylamine), imprinted cavities with inner surface-clung charged groups were created to recognize BSA. Therefore each cavity appears like a molecular capacitor charged by carboxyl and amino groups. As the cavities are all of a molecule-size volume, a membrane made of the MIP contains a huge array of the molecular capacitors. This will produce a synergistic effect and greatly amplify the impedance signal deviations when template sorption/desorption takes place on the sensor. When the MIP was used as an artificial antibody to make an electrochemical sensor, high sensitivity and selectivity were achieved at the same time. Results indicate that BSA could be determined in a linear range of 1.5 × 10–16–10–12 mol–L−1. Meanwhile a low limit of detection was achieved at 7.2 × 10–18 mol L−1. Conclusively protein-imprinted amphoteric polyacrylamide cryogels are materials of a great potential to sense and determine charged objects like molecules, cells, microorganisms or other particles.

The substrate lends a hand.

Duramycin is a small post-translationally modified peptide with antibody-like affinity for phosphatidylethanolamine. As it turns out, the same functionality that is essential for duramycin activity helps to catalyze the formation of its conformationally constrained and compact polycyclic architecture.

Cell-Permeable Bicyclic Peptidyl Inhibitors against NEMO-IκB Kinase Interaction Directly from a Combinatorial Library.

Macrocyclic peptides are capable of binding to flat protein surfaces such as the interfaces of protein-protein interactions with antibody-like affinity and specificity, but generally lack cell permeability in order to access intracellular targets. In this work, we designed and synthesized a large combinatorial library of cell-permeable bicyclic peptides, in which the first ring consisted of randomized peptide sequences for potential binding to a target of interest, while the second ring featured a family of different cell-penetrating motifs, for both cell penetration and target binding. The library was screened against the IκB kinase α/β (IKKα/β)-binding domain of NF-κB essential modulator (NEMO), resulting in the discovery of several cell-permeable bicyclic peptides, which inhibited the NEMO-IKKβ interaction with low μM IC50 values. Further optimization of one of the hits led to a relatively potent and cell-permeable NEMO inhibitor (IC50 = 1.0 μM), which selectively inhibited canonical NF-κB signaling in mammalian cells and the proliferation of cisplatin-resistant ovarian cancer cells. The inhibitor provides a useful tool for investigating the biological functions of NEMO/NF-κB and a potential lead for further development of a novel class of anti-inflammatory and anticancer drugs.

Facile Synthesis of Dimeric Thioether-Macrocyclic Peptides with Antibody-like Affinity against Plexin-B1.

Macrocyclic peptides have gained increasing attention due to their ease of discovery through various in vitro display platforms as well as their potential in possessing favorable properties of both small molecule and antibody drug classes. It is well-known that the avidity achieved through the bivalent binding mode of antibodies gives rise to their slow dissociation rates and thus high potency as drug molecules. Here, we report the synthesis of dimeric thioether-macrocyclic peptides through a branched synthesis approach allowing for synthesis of dimeric peptides in a comparable number of steps as monomers and tunability of linker lengths from 30 to 200 Å. Applying this method to synthesize dimers of a model PlexinB1-binding macrocyclic peptide showed close to 300-fold increases in their apparent binding affinity, bringing the KD down from 8 nM to 30 pM as well as affording improved biological activities when compared to their monomeric counterparts. These enhancements demonstrate that this is a simple synthetic strategy to harness the benefits of bivalence that antibodies naturally possess.

A mini-review and perspective on multicyclic peptide mimics of antibodies

Affinity reagents are important tools in the biological sciences for understanding biological processes and for studying protein expression, localization and interactions. However, traditional affinity reagents such as antibodies (and their fragments) and non-immunoglobulin (non-Ig) scaffold binders, usually suffer from problems of poor cellular uptake efficiency, high production cost, and low structural stability. This leads to rapid development of small antibody-like affinity reagents such as scaffold-free cyclic and multicyclic peptides, which usually have 5–30 amino acid residues, thus lying between non-Ig scaffolds and small molecules in size. In this mini-review, we highlight the recent development in mono- and multi-cyclic peptide mimics of antibodies, including cyclic peptide affinity reagents that have been developed for use in antibody-like applications, novel synthetic strategies for multicyclic peptides, and promising peptide library screening platforms. We also provide a perspective on the future development in multicyclic peptide mimics of antibodies.

Bicyclic Peptides as Next-Generation Therapeutics.

Bicyclic peptides have greater conformational rigidity and metabolic stability than linear and monocyclic peptides and are capable of binding to challenging drug targets with antibody-like affinity and specificity. Powerful combinatorial library technologies have recently been developed to rapidly synthesize and screen large bicyclic peptide libraries for ligands against enzymes, receptors, and protein-protein interaction targets. Bicyclic peptides have been developed as potential therapeutics against a wide range of diseases, drug targeting agents, imaging/diagnostic probes, and research tools. In this Minireview, we provide a summary of the recent progresses on the synthesis and applications of bicyclic peptides.

Evolution of thermophilic DNA polymerases for the recognition and amplification of C2'-modified DNA.

The PCR amplification of oligonucleotides enables the evolution of sequences called aptamers that bind specific targets with antibody-like affinity. However, the use of these aptamers is limited in many applications by nuclease-mediated degradation. In contrast, oligonucleotides that are modified at their sugar C2' positions with methoxy or fluorine substituents are stable to nucleases but cannot be synthesized by natural polymerases. Here, we report the development of a polymerase evolution system and its use to evolve thermostable polymerases that efficiently interconvert C2'-OMe modified oligonucleotides and their DNA counterparts via “transcription” and “reverse transcription,” or more importantly, PCR amplify partially C2'-OMe or C2'-F modified oligonucleotides. A mechanistic analysis demonstrates that the ability to amplify the modified oligonucleotides was evolved by optimizing interdomain interactions that stabilize the catalytically competent closed conformation of the polymerase. The evolved polymerases should find practical applications and the developed evolution system should be a powerful tool for the tailoring of polymerases to have other types of novel function.

Toward a Universal Method for Preparing Molecularly Imprinted Polymer Nanoparticles with Antibody-like Affinity for Proteins.

We describe a potentially universal, simple and cheap method to prepare water-compatible molecularly imprinted polymer nanoparticles (MIP-NPs) as synthetic antibodies against proteins. The strategy is based on a solid phase synthesis approach where glass beads (GBs) are functionalized with a metal chelate, acting as a general affinity ligand to attract surface-bound histidines present on proteins. This configuration enables an oriented immobilization of the proteins, upon which thermoresponsive MIP-NPs are synthesized. The GBs play the role of both a reactor and a separation column since, after synthesis, the MIP-NPs are released from the support by a simple temperature change, resulting in protein-free polymers. The resulting MIP-NPs are endowed with improved binding site homogeneity, since the binding sites have the same orientation. Moreover, they are stable (no aggregation) in a buffer solution for prolonged storage time and exhibit apparent dissociation constants in the nanomolar range, with little or no cross-reactivity toward other proteins.

Abstract P5-01-10: In vitro and in vivo imaging of Her2 with cyclic peptides derived from directed evolution

Abstract The implementation of personalized medicine for Her2-positive breast cancer treatment (Trastuzumab and Pertuzumab) has made a dramatic effect on overall patient outcome. Unfortunately, there are currently no FDA-approved imaging agents to monitor Her2-positive breast cancer leaving physicians to rely on invasive biopsies and anatomical imaging to monitor treatment with Her2-targeted therapeutics. There is also increasing evidence that women initially diagnosed with Her2-negative breast can present with Her2-positive disease upon recurrence. Noninvasive, whole-body visualization of Her2 would identify Her2-recurrent disease and serve as a powerful tool to monitor the effectiveness of new and emerging Her2-targeted therapeutics (e.g. TDM-1). While many Her2-antibody based imaging agents have been used in preclinical applications, their long circulating half-lives, high liver uptake, and poor synthetic accessibility pose a challenges for clinical translation. Here we describe the use of highly stable cyclic peptides derived from biological display as imaging agents for Her2-positive breast cancer. These peptides have antibody-like high affinity (<10 nM) for Her2-positive tumors and their size (<2 kDa) allows for higher rates of clearance, lower background signal, and consequently higher image sensitivity. Anti-Her2 peptides were pre-optimized for affinity, protease resistance and bio-stability prior to labeling with the Cy7.5 near-infrared dye. Her2-specificity was determined by in vitro assays with Her2-postive (BT474 and SKOV3), HER2-normal (MCF7) and Her2-negative (MDA-MB-231) cell lines. Sequences that showed superior affinity and selectivity were used to acquire fluorescent images of ectopic and orthotopic murine models of Her2-positive breast cancer. Our preliminary imaging data suggests that this scaffold has excellent translational potential for targeted molecular imaging of Her2-postive breast cancer. Citation Format: Lindsay E Kelderhouse, Amanda Hardy, Yong Pan, Patrea Rhea, Argentina Ornelas, Seth Gammon, Peiying Yang, Stephen Fiacco, Steven W Millward. In vitro and in vivo imaging of Her2 with cyclic peptides derived from directed evolution [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-01-10.

Abstract 656: A bispecific HER2/CD3 targeting FynomAb with excellent tumor killing and favorable pharmacokinetic properties

Abstract There is increasing evidence that T cells are able to control tumor growth and increase survival of cancer patients. However, tumor-specific T cell responses are difficult to mount and sustain in cancer patients, and are limited by numerous immune escape mechanisms of tumor cells. A promising approach in the immunotherapy of cancer is to engage T cells to target tumor cells using bispecific therapeutics targeting a tumor-associated surface antigen and CD3e on T cells. Such therapeutics elicit T cell mediated lysis of tumor cells independent of T cell specificity. Fynomers are small 7 kDa globular proteins derived from the SH3 domain of the human Fyn kinase (Fyn SH3) that can be engineered to bind with antibody-like affinity and specificity to virtually any target of choice. Fynomers can be fused to N-terminal and/or C-terminal ends of antibodies to generate multispecific therapeutics (FynomAbs) with tailored architectures. FynomAbs can be produced using standard antibody technology (GMP production yield of 3.3 g/L at 1000 L scale achieved), and show IgG-like biophysical properties and pharmacokinetic profiles. We have generated a novel bispecific FynomAb which can simultaneously bind HER2 on tumor cells and CD3 on T cells. The bispecific HER2/CD3 targeting FynomAb COVA420 potently redirected T cells to HER2 expressing tumor cells showing picomolar tumor cell lysis activity. We present for the first time that a tailored architecture of the FynomAb leads to optimal tumor cell killing properties. The activity of COVA420 was found to be highly specific, as no lysis of cells was observed in the absence of HER2 expression. In addition, COVA420 demonstrated an antibody-like pharmacokinetic profile in mice. We anticipate that the increased half-life of T cell recruiting FynomAbs compared to other bispecific formats translates into a significant benefit for patients, since it circumvents the need for continuous infusion and prolongs the intervals between successive treatments. In summary, bispecific T cell recruiting FynomAbs represent a novel platform technology to redirect T cells to tumor cells with optimal biophysical properties, long-half lives and tailored architectures. Citation Format: Ulrich Wuellner, Fabian Buller, Kristina Klupsch, Simon Brack, Irene Zbinden, Roger Santimaria, Isabella Attinger-Toller, Susann König-Friedrich, Julian Bertschinger, Dragan Grabulovski. A bispecific HER2/CD3 targeting FynomAb with excellent tumor killing and favorable pharmacokinetic properties. [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 656. doi:10.1158/1538-7445.AM2014-656

Abstract 658: A bispecific HER2 targeting FynomAb with superior anti-tumor activity and novel mode of action

Abstract Upregulation of HER2 is a hallmark of 20% to 30% of invasive breast cancers, rendering this receptor an attractive target for cancer therapy. Although HER2 targeting agents have provided substantial clinical benefit as cancer therapeutics, there is a need for the development of new agents aiming at circumventing anti-HER2 resistance. Fynomers are small 7 kDa globular proteins derived from the SH3 domain of the human Fyn kinase (Fyn SH3) that can be engineered to bind with antibody-like affinity and specificity to virtually any target of choice. Fynomers can be fused to N-terminal and/or C-terminal ends of antibodies to generate multispecific therapeutics (FynomAbs) with tailored architectures. FynomAbs can be produced using standard antibody technology (GMP production yield of 3.3 g/L at 1000 L scale achieved), and show IgG-like biophysical properties and pharmacokinetic profiles. Based on the approved antibody pertuzumab we have created a panel of bispecific FynomAbs which target two epitopes on HER2. The activity of the HER2 targeting FynomAbs was found to depend on the FynomAb architecture, i.e. the spatial arrangement of the binding sites of antibody and the Fynomer. The most potent of these FynomAbs, termed COVA208, demonstrated superior tumor cell growth inhibition in vitro compared to pertuzumab and trastuzumab. COVA208 was characterized in detail and showed an increased ability to induce rapid HER2-internalization and apoptosis in vitro. Moreover, it elicited a stronger inhibition of downstream HER2 signaling which was accompanied by a reduction of HER2, HER3 and EGFR levels in vitro and in vivo. The therapeutic potential of COVA208 has been demonstrated in vivo in four different HER2 mouse models, where COVA208 exhibited excellent anti-tumor activity. Importantly, COVA208 demonstrated superior activity in vivo compared to trastuzumab and pertuzumab. The bispecific FynomAb COVA208 has the potential to enhance the clinical efficacy and expand the scope of HER2-directed therapies, and delineates a paradigm for designing a new class of antibody-based therapeutics for other receptor targets. Citation Format: Babette Schade, Simon Brack, Isabella Attinger-Toller, Kristina Klupsch, Richard Woods, Helen Hachemi, Ulrike von der Bey, Susann Koönig-Friedrich, Julian Bertschinger, Dragan Grabulovski. A bispecific HER2 targeting FynomAb with superior anti-tumor activity and novel mode of action. [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 658. doi:10.1158/1538-7445.AM2014-658

Synthetic Polymer Nanoparticles with Antibody-like Affinity for a Hydrophilic Peptide

Synthetic polymer nanoparticles with antibody-like affinity for a hydrophilic peptide have been prepared by inverse microemulsion polymerization. Peptide affinity was achieved in part by incorporating the target (imprint) peptide in the polymerization reaction mixture. Incorporation of the imprint peptide assists in the creation of complementary binding sites in the resulting polymer nanoparticle (NP). To orient the imprint peptide at the interface of the water and oil domains during polymerization, the peptide target was coupled with fatty acid chains of varying length. The peptide--NP binding affinities (ca. 90-900 nM) were quantitatively evaluated by a quartz crystal microbalance (QCM). The optimal chain length was established that created high affinity peptide binding sites on the surface of the nanoparticles. This method can be used for the preparation of nanosized synthetic polymers with antibody-like affinity for hydrophilic peptides and proteins ("plastic antibodies").

Design of Cyclic Peptides That Bind Protein Surfaces with Antibody-Like Affinity

There is a pressing need for new molecular tools to target protein surfaces with high affinity and specificity. Here, we describe cyclic messenger RNA display with a trillion-member covalent peptide macrocycle library. Using this library, we have designed a number of high-affinity, redox-insensitive, cyclic peptides that target the signaling protein G alpha i1. In addition to cyclization, our library construction took advantage of an expanded genetic code, utilizing nonsense suppression to insert N-methylphenylalanine as a 21st amino acid. The designed macrocycles exhibit several intriguing features. First, the core motif seen in all of the selected variants is the same and shares an identical context with respect to the macrocyclic scaffold, consistent with the idea that selection simultaneously optimizes both the cyclization chemistry and the structural placement of the binding epitope. Second, detailed characterization of one molecule, cyclic G alpha i binding peptide (cycGiBP), demonstrates substantially enhanced proteolytic stability relative to that of the parent linear molecule. Third and perhaps most important, the cycGiBP peptide binds the target with very high affinity ( K i approximately 2.1 nM), similar to those of many of the best monoclonal antibodies and higher than that of the betagamma heterodimer, an endogenous G alpha i1 ligand. Overall the work provides a general route to design novel, low-molecular-weight, high-affinity ligands that target protein surfaces.