George Ehrlich

Identification of peptides that bind to the constant region of a humanized IgG1 monoclonal antibody using phage display

The pFc′ fragments of a humanized IgG1 monoclonal antibody were generated by digestion with immobilized pepsin. These pFc′ fragments were separated from F(ab′)2 fragments by affinity chromatography. The pFc′ fragments corresponding to the constant region of the humanized IgG1 monoclonal antibody were used as targets for phage display using variable‐length peptide libraries. Interacting phage‐displayed peptides were selected by repetitious cycles of target screening and phage amplification. Peptide sequences, deduced by sequencing DNA from isolated phage, were aligned and analyzed for amino acid motifs against each other and protein A. These results indicated that an amino acid motif has been identified using phage display technology that is sufficient for pFc′ binding. Furthermore, the peptides derived from this study may prove useful in the development of peptidomimetic alternatives to protein A for use in affinity chromatography. Copyright

Preparation and characterization of albumin conjugates of a truncated peptide YY analogue for half-life extension.

Recombinant human serum albumin (HSA) conjugates of a 15-amino-acid truncated peptide YY (PYY) analogue were prepared using three heterobifunctional linkers [succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC), 6-maleimidohexanoic acid N-hydroxysuccinimide ester (MHS), and N-[γ-maleimidobutyryloxy]sulfosuccinimide ester (GMBS)] in 2 synthetic steps involving (1) reaction of succinimidyl ester on linker with ε-amine of Lys2 on the peptide and (2) reaction of maleimide on peptide linker with free thiol of Cysteine 34 (Cys34) on albumin. In-process controls using ESI LC-MS were used to follow reactions and identify reaction products. Proteolytic digests of the conjugate revealed that peptide conjugation occurs at Cys34 on HSA. Conjugates were assayed in cell-based assays to determine potency at the human Y2-receptor, and selectivity at the human Y1-, Y4-, and Y5-receptors using a calcium flux assay. All three conjugates assayed were selective agonists of the Y2-receptor, and displayed nanomolar potencies. MCC and MH conjugates were selected for acute PK/PD studies in DIO mice. Significant reduction in food intake was observed with the MH conjugate, which lasted for 24 h at the 10 mg (or 4 μmol)/kg dose. While the MCC conjugate exhibited greater potency in vitro, it was slightly less effective than the MH conjugate in vivo with respect to reduction in food intake. Both conjugates were significantly less active than the peptide coupled to a 30 kDa PEG. The observed T1/2 (8-9 h) for both conjugates was significantly lower than that observed for the PEGylated peptide (∼25 h). These results suggest that, as compared with the unmodified and PEGylated peptide, the extended circulation half-life of albumin conjugates is mediated through uptake and recirculation by FcRn, and allometric scaling methods are necessary to account for interspecies variation in pharmacokinetic properties.

Affinity purification and characterization of an anti‐PEG IgM

Anti‐PEG IgM was purified by affinity chromatography using variable length PEG chains (5, 10, 20 and 30 kDa) as affinity ligands. Maximal binding of anti‐PEG IgM was observed using the 30 kDa PEG‐derivatized NuGel (single passage). Purified anti‐PEG IgM was characterized for binding to PEG functionalized proteins/peptides by surface plasmon resonance, western blotting and ELISA. Anti‐PEG IgM, in solution and adsorbed on 20 kDa PEG‐derivatized NuGel, was subjected to pepsin digestion followed by affinity chromatography. SDS–PAGE analysis of eluates in both preparations yielded one fragment that was similar in size. However, an additional lower molecular weight band was observed in solution‐digested affinity purified material that was not present in the eluate from the material subjected to pepsin digestion on the affinity matrix. The lower MW fragment could be eluted under milder conditions, suggesting loss of binding multiplicity. Analysis by mass spectrometry yielded molecular weights of 132 kDa (both) and 82 kDa (solution) for the respective fragments. N‐terminal sequencing of both fragments resulted in primary sequences (heavy and light chains) that were not only identical to each other but also to those of native IgM. The anti‐PEG IgM fragments were characterized for binding to pegylated interferon alfa‐2a by ELISA. The results from these studies suggest that affinity purified anti‐PEG IgM and fragments can be used as probes in detection assays for PEG functionalized biotherapeutics in pre‐clinical and clinical studies. Copyright

Abstract A156: Preclinical activity of MDM2 antagonist RO6839921, a pegylated prodrug for intravenous administration

The p53 tumor suppressor is a transcription factor that inhibits tumorigenesis by inducing cell cycle arrest or apoptosis in response to diverse stresses. In normal cells, p53 levels are tightly controlled by MDM2 which binds p53 and negatively regulates its activity and stability. MDM2 is overproduced in many human cancers, thereby impairing p53 function. Antagonists of p53-MDM2 interaction can enhance p53 activity and offer a novel approach to cancer therapy. The first potent and selective small-molecule inhibitors of p53-MDM2 binding, the nutlins, provided preclinical proof-of-concept for MDM2 antagonists as therapeutics for patients with tumors expressing wild-type p53. The nutlin family member idasanutlin (RG7388, RO5503781) is an oral small molecule inhibitor of MDM2 currently in clinical testing. Here we describe RO6839921, a pegylated prodrug formulated for intravenous administration. This IV MDM2 antagonist has been developed in order to improve variability in exposure seen with the oral compound, and to allow expansion into indications where patients cannot swallow or absorb the oral idasanutlin. RO6839921 is rapidly metabolized to the active principle (AP) idasanutlin which then binds selectively to the p53 site on the surface of the MDM2 molecule. In vitro testing with the AP shows high affinity with effective displacement of p53 from MDM2, leading to stabilization and accumulation of p53 protein and activation of the p53 pathway. Studies focused on in vivo investigation of activity of the prodrug RO6839921 since esterase cleavage is required to release the AP. The anti-tumor activity of RO6839921 was investigated in several sarcoma xenograft models including highly responsive wild-type (WT) p53, MDM2 overexpressing osteosarcoma models SJSA-1 and MHM. Sustained survival was seen in the WT p53 HT1080 fibrosarcoma model when combined with Doxil. Activity was also seen in the WT p53 MOLM-13 disseminated AML model alone and in combination with cytarabine, in the CRPC model 22rv1, and in the ER+ BCa model MCF-7. In these studies we see an increase in dose commensurate with exposure and activity (prodrug vs oral), less variability, and potentiated activity in combination with relevant therapeutics. Clinical studies with the oral AP (idasanutlin) have shown that p53 may be activated by this novel therapeutic strategy that releases p53 from MDM2 inhibition. In particular, patients with AML exhibit significant clinical activity (ASH 2014). In view of the existing unmet medical need in advanced cancers, and the promising activity seen with idasanutlin, RO6839921 is believed to be a promising agent that may offer new therapeutic options, and is therefore currently in clinical testing in both solid and hematologic malignancies. Citation Format: Brian Higgins, Christian Tovar, Kelli Glen, Aruna Railkar, Zoran Filipovic, Farooq Qureshi, Binh Vu, George Ehrlich, Dan Fishlock, Lin-Chi Chen, Steven Middleton, Gwen Nichols, Kathryn Packman, Lyubomir Vassilev. Preclinical activity of MDM2 antagonist RO6839921, a pegylated prodrug for intravenous administration. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A156.

Affinity 2011 – The 19th biennial meeting of the International Society for Molecular Recognition

The 19th biennial meeting of the International Society for Molecular Recognition (ISMR), Affinity 2011, was hosted by Professor Guilherme N. M. Ferreira (IBB/UALG) in Tavira, Portugal from June 16th to 19th, 2011. There were 110 registered participants, including 44 students, from 25 countries, in attendance. As stated by the organizers, the main theme of Affinity 2011 centered on “highlights of the scientific advances in molecular binding and recognition through life sciences, bioengineering and nanotechnologies,” with particular focus on aspects of molecular biorecognition related to cell signaling and differentiation, development and application of devices and affinity-based technologies. Despite an economic climate, similar to that experienced at Affinity 2009 in Iceland, Affinity 2011 was received with great success, thanks to the unwavering commitment of Professor Ferreira and his local organizing committee [co-chair Raquel Aires-Barros (IBB/IST), Ana Azevedo (IBB/IST), Cecilia Roque (Requimte/UNL), Joao Goncalves (IMM/FFUL) and DECHEMA (secretariat)]. In the tradition of past Affinity meetings, Affinity 2011 was host to a welcoming reception, the ISMR/Pierce Affinity Award, the Younger Investigator’s Award and a gala dinner featuring traditional Portuguese music, Fado, at the Tavira castle. In addition, two “Travel Awards” were kindly sponsored by the Journal of Molecular Recognition (JMR). The scientific program at Affinity 2011 was packed with 41 speakers in nine sessions covering the following topics over 3days: (1) kinetics and thermodynamics of biomolecular interactions, (2) evolutionary engineering and combinatorial design for affinity and drug discovery, (3) affinity interactions in cell biology – signaling pathways and networks, (4) affinity and protein–protein interactions in health and disease, (5) computational modeling and biomimetic design and materials, (6) affinity-based bioprocessing, (7) nanotechnology, nanomaterials, micro and nanosystems, (8) single-molecule detection: devices/sensors and in vivo tracking and (9) self-assembly and mechanisms of protein machines. Set in Algarve, the southern region of Portugal known for “sunshine breaks and relaxing holidays,” Klaus Mosbach opened Affinity 2011 with a retrospective on “The history of ISMR”. Alois Jungbauer from the Department of Biotechnology at the University of Natural Resources and Life Science in Vienna received the 2011 ISMR/Pierce Affinity Award for his outstanding contributions to the field of “downstream processing.” His lecture entitled “Staphylococcal protein A and camelid antibody affinity chromatography: engineering principles and surface characterization” described the introduction of specific camelid antibodies as an alternative to protein A-based immunoaffinity purification. Two Travel Awards, kindly donated by the Journal of Molecular Recognition, were given to the student, Jennifer D. Knoop from the University of Houston, TX, and the Post-Doc Graziella El Khoury from the University of Cambridge, UK. The Younger Investigators Award sponsored by Hoffmann-La Roche was organized by A. Cecilia Roque and George Ehrlich. Awards were given to nine younger investigators in recognition of their outstanding presentations and active participation at Affinity 2011: Nima Matias Jokilaakso and Johan Nilvebrant (Sweden), Dorota Smolarek (Poland), Matthias Meininger (Germany), Takafumi Honjo (Japan), Alessandro Cumbo (Switzerland) and Luís de Matos Borlido, João Rodrigo Cardoso Trabuco and Telma Barroso (Portugal). The meeting concluded on June 19th with an invitation to the 20th biennial meeting of the International Society for Molecular Recognition in Vienna, Austria, by Affinity 2013 host and organizer, Alois Jungbauer. The 12 peer-reviewed articles published in this special issue span the spectrum of topics presented at Affinity 2011 and cover many profound aspects of affinity-based science and technology. The first section of this volume is devoted to affinity-based technologies and openswith a review article byMaria Raquel Aires-Barrosa’s group summarizing strategies for lectin purification. The subsequent article, written by Ranjini and Vijayalakshmi, describes the adsorption of catalase on two mixed mode ligands and discusses the mechanism involved. The next paper by Tekiner et al. describes the use of cryogels for metal-based affinity chromatography for urease purification from jack beans. A similar cryogel was used by Andaç et al., who prepared a new composite protein-imprinted macroporous cryogel for depletion of albumin from human serum prior to use in proteomic applications. Following is a paper by Czarnecka et al., describes the Engineering of Candida albicans glucosamine6-phosphate synthase by insertion of His6 sequences, a commonly used affinity tag, for efficient enzyme purification. This section concludes with a paper by Sandoval et al., who discuss the use of general rate model to describe elution relationships in affinity chromatography. Two articles by the group of Maria H. L. Ribeiro are related to enzyme immobilization. The first article by Nunesa et al. is entitled “High-affinity water soluble system for efficient naringinase immobilization in polyvinyl alcohol–dimethyl sulfoxide lens-shaped particles.” The, second, by Furtado et al., is entitled “Hesperidinase encapsulation towards hesperitin production targeting improved bioavailability.” The third section deals with the topic of protein–protein interactions. The first article by Kysilka and Vondra sek discusses the analysis of protein–protein interactions in dimeric structures at the molecular level utilizing chemical composition, binding preferences and residue interaction energies, using AMBER empirical force field, in an attempt to reach a better understanding Editorial