Publications by authors named "Stephen L Sazinsky"

9 Publications

  • Page 1 of 1

Engineering Aglycosylated IgG Variants with Wild-Type or Improved Binding Affinity to Human Fc Gamma RIIA and Fc Gamma RIIIAs.

J Mol Biol 2017 08 8;429(16):2528-2541. Epub 2017 Jul 8.

Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139, United States; Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139, United States; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139, United States. Electronic address:

The binding of human IgG1 to human Fc gamma receptors (hFcγRs) is highly sensitive to the presence of a single N-linked glycosylation site at asparagine 297 of the Fc, with deglycosylation resulting in a complete loss of hFcγR binding. Previously, we demonstrated that aglycosylated human IgG1 Fc variants can engage the human FcγRII class of the low-affinity hFcγRs, demonstrating that N-linked glycosylation of the Fc is not a strict requirement for hFcγR engagement. In the present study, we demonstrate that aglycosylated IgG variants can be engineered to productively engage with FcγRIIIA, as well as the human Fc gamma RII subset. We also assess the biophysical properties and serum half-life of the aglycosylated IgG variants to measure stability. Aglycosylated constructs N297D/S298T (DTT)-K326I/A327Y/L328G (IYG) and N297D/S298A-IYG optimally drove tumor cell phagocytosis. A mathematical model of phagocytosis suggests that hFcγRI and hFcγRIIIA dimers were the main drivers of phagocytosis. In vivo tumor control of B16F10 lung metastases further confirmed the variant DTT-IYG to be the best at restoring wild-type-like properties in prevention of lung metastases. While deuterium incorporation was similar across most of the protein, several peptides within the CH2 domain of DTT-IYG showed differential deuterium uptake in the peptide region of the FG loop as compared to the aglycosylated N297Q. Thus, in this study, we have found an aglycosylated variant that may effectively substitute for wild-type Fc. These aglycosylated variants have the potential to allow therapeutic antibodies to be produced in virtually any expression system and still maintain effector function.
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http://dx.doi.org/10.1016/j.jmb.2017.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5584586PMC
August 2017

Comprehensive experimental and computational analysis of binding energy hot spots at the NF-κB essential modulator/IKKβ protein-protein interface.

J Am Chem Soc 2013 Apr 10;135(16):6242-56. Epub 2013 Apr 10.

Department of Chemistry, Boston University, 590 Commonwealth Avenue, Boston, Massachusetts 02215, USA.

We report a comprehensive analysis of binding energy hot spots at the protein-protein interaction (PPI) interface between nuclear factor kappa B (NF-κB) essential modulator (NEMO) and IκB kinase subunit β (IKKβ), an interaction that is critical for NF-κB pathway signaling, using experimental alanine scanning mutagenesis and also the FTMap method for computational fragment screening. The experimental results confirm that the previously identified NEMO binding domain (NBD) region of IKKβ contains the highest concentration of hot-spot residues, the strongest of which are W739, W741, and L742 (ΔΔG = 4.3, 3.5, and 3.2 kcal/mol, respectively). The region occupied by these residues defines a potentially druggable binding site on NEMO that extends for ~16 Å to additionally include the regions that bind IKKβ L737 and F734. NBD residues D738 and S740 are also important for binding but do not make direct contact with NEMO, instead likely acting to stabilize the active conformation of surrounding residues. We additionally found two previously unknown hot-spot regions centered on IKKβ residues L708/V709 and L719/I723. The computational approach successfully identified all three hot-spot regions on IKKβ. Moreover, the method was able to accurately quantify the energetic importance of all hot-spot residues involving direct contact with NEMO. Our results provide new information to guide the discovery of small-molecule inhibitors that target the NEMO/IKKβ interaction. They additionally clarify the structural and energetic complementarity between "pocket-forming" and "pocket-occupying" hot-spot residues, and further validate computational fragment mapping as a method for identifying hot spots at PPI interfaces.
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http://dx.doi.org/10.1021/ja400914zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3680600PMC
April 2013

Rapid evolution of functional complexity in a domain family.

Sci Signal 2009 Sep 8;2(87):ra50. Epub 2009 Sep 8.

Department of Protein Engineering, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA.

Multicellular organisms rely on complex, fine-tuned protein networks to respond to environmental changes. We used in vitro evolution to explore the role of domain mutation and expansion in the evolution of network complexity. Using random mutagenesis to facilitate family expansion, we asked how versatile and robust the binding site must be to produce the rich functional diversity of the natural PDZ domain family. From a combinatorial protein library, we analyzed several hundred structured domain variants and found that one-quarter were functional for carboxyl-terminal ligand recognition and that our variant repertoire was as specific and diverse as the natural family. Our results show that ligand binding is hardwired in the PDZ fold and suggest that this flexibility may facilitate the rapid evolution of complex protein interaction networks.
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http://dx.doi.org/10.1126/scisignal.2000416DOI Listing
September 2009

Directed evolution of a secretory leader for the improved expression of heterologous proteins and full-length antibodies in Saccharomyces cerevisiae.

Biotechnol Bioeng 2009 Aug;103(6):1192-201

Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

Because of its eukaryotic nature, simple fermentation requirements, and pliable genetics, there have been many attempts at improving recombinant protein production in Saccharomyces cerevisiae. These strategies typically involve altering the expression of a native protein thought to be involved in heterologous protein trafficking. Usually, these approaches yield three- to tenfold improvements over wild-type strains and are almost always specific to one type of protein. In this study, a library of mutant alpha mating factor 1 leader peptides (MFalpha1pp) is screened for the enhanced secretion of a single-chain antibody. One of the isolated mutants is shown to enhance the secretion of the scFv up to 16-fold over wild type. These leaders also confer a secretory improvement to two other scFvs as well as two additional, structurally unrelated proteins. Moreover, the improved leader sequences, combined with strain engineering, allow for a 180-fold improvement over previous reports in the secretion of full-length, functional, glycosylated human IgG(1). The production of full-length IgG(1) at milligram per liter titers in a simple, laboratory-scale system will significantly expedite drug discovery and reagent synthesis while reducing antibody cloning, production, and characterization costs.
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http://dx.doi.org/10.1002/bit.22338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2847895PMC
August 2009

Aglycosylated immunoglobulin G1 variants productively engage activating Fc receptors.

Proc Natl Acad Sci U S A 2008 Dec 12;105(51):20167-72. Epub 2008 Dec 12.

Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.

Immunoglobulin G plays a vital role in adaptive immunity and antibody-based therapy through engagement of its Fc region by the Fc gamma receptors (Fc gammaRs) on immune cells. In addition to specific protein-protein contacts, N-linked glycosylation of the IgG Fc has been thought to be essential for the recognition of Fc by Fc gammaR. This requirement for the N-linked glycan has limited biomanufacture of therapeutic antibodies by restricting it to mammalian expression systems. We report here aglycosylated Fc domain variants that maintain engagement to Fc gammaRs, both in vitro and in vivo, demonstrating that Fc glycosylation is not strictly required for the activation of immune cells by IgG. These variants provide insight into how the N-linked glycan is used biologically in the recognition of Fc by Fc gammaRs, as well as represent a step toward the production in alternative expression systems of antibody-based therapeutics capable of eliciting immune effector functions.
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http://dx.doi.org/10.1073/pnas.0809257105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2629253PMC
December 2008

A specificity map for the PDZ domain family.

PLoS Biol 2008 Sep;6(9):e239

Terrence Donnelly Center for Cellular and Biomolecular Research, Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada.

PDZ domains are protein-protein interaction modules that recognize specific C-terminal sequences to assemble protein complexes in multicellular organisms. By scanning billions of random peptides, we accurately map binding specificity for approximately half of the over 330 PDZ domains in the human and Caenorhabditis elegans proteomes. The domains recognize features of the last seven ligand positions, and we find 16 distinct specificity classes conserved from worm to human, significantly extending the canonical two-class system based on position -2. Thus, most PDZ domains are not promiscuous, but rather are fine-tuned for specific interactions. Specificity profiling of 91 point mutants of a model PDZ domain reveals that the binding site is highly robust, as all mutants were able to recognize C-terminal peptides. However, many mutations altered specificity for ligand positions both close and far from the mutated position, suggesting that binding specificity can evolve rapidly under mutational pressure. Our specificity map enables the prediction and prioritization of natural protein interactions, which can be used to guide PDZ domain cell biology experiments. Using this approach, we predicted and validated several viral ligands for the PDZ domains of the SCRIB polarity protein. These findings indicate that many viruses produce PDZ ligands that disrupt host protein complexes for their own benefit, and that highly pathogenic strains target PDZ domains involved in cell polarity and growth.
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http://dx.doi.org/10.1371/journal.pbio.0060239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2553845PMC
September 2008

Structural and functional analysis of the PDZ domains of human HtrA1 and HtrA3.

Protein Sci 2007 Nov;16(11):2454-71

Department of Medicinal Chemistry, Genetech, Inc., South San Francisco, CA 94080, USA.

High-temperature requirement A (HtrA) and its homologs contain a serine protease domain followed by one or two PDZ domains. Bacterial HtrA proteins and the mitochondrial protein HtrA2/Omi maintain cell function by acting as both molecular chaperones and proteases to manage misfolded proteins. The biological roles of the mammalian family members HtrA1 and HtrA3 are less clear. We report a detailed structural and functional analysis of the PDZ domains of human HtrA1 and HtrA3 using peptide libraries and affinity assays to define specificity, structural studies to view the molecular details of ligand recognition, and alanine scanning mutagenesis to investigate the energetic contributions of individual residues to ligand binding. In common with HtrA2/Omi, we show that the PDZ domains of HtrA1 and HtrA3 recognize hydrophobic polypeptides, and while C-terminal sequences are preferred, internal sequences are also recognized. However, the details of the interactions differ, as different domains rely on interactions with different residues within the ligand to achieve high affinity binding. The results suggest that mammalian HtrA PDZ domains interact with a broad range of hydrophobic binding partners. This promiscuous specificity resembles that of bacterial HtrA family members and suggests a similar function for recognizing misfolded polypeptides with exposed hydrophobic sequences. Our results support a common activation mechanism for the HtrA family, whereby hydrophobic peptides bind to the PDZ domain and induce conformational changes that activate the protease. Such a mechanism is well suited to proteases evolved for the recognition and degradation of misfolded proteins.
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http://dx.doi.org/10.1110/ps.073049407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2211686PMC
November 2007

Isolating and engineering human antibodies using yeast surface display.

Nat Protoc 2006 ;1(2):755-68

Department of Chemical Engineering and Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue E19-563, Cambridge, Massachusetts 02139, USA.

This protocol describes the process of isolating and engineering antibodies or proteins for increased affinity and stability using yeast surface display. Single-chain antibody fragments (scFvs) are first isolated from an existing nonimmune human library displayed on the yeast surface using magnetic-activated cell sorting selection followed by selection using flow cytometry. This enriched population is then mutagenized, and successive rounds of random mutagenesis and flow cytometry selection are done to attain desired scFv properties through directed evolution. Labeling strategies for weakly binding scFvs are also described, as well as procedures for characterizing and 'titrating' scFv clones displayed on yeast. The ultimate result of following this protocol is a panel of scFvs with increased stability and affinity for an antigen of interest.
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http://dx.doi.org/10.1038/nprot.2006.94DOI Listing
September 2007

Stably folded de novo proteins from a designed combinatorial library.

Protein Sci 2003 Jan;12(1):92-102

Department of Chemistry, Princeton University, Princeton, NJ 08544-1009, USA.

Binary patterning of polar and nonpolar amino acids has been used as the key design feature for constructing large combinatorial libraries of de novo proteins. Each position in a binary patterned sequence is designed explicitly to be either polar or nonpolar; however, the precise identities of these amino acids are varied extensively. The combinatorial underpinnings of the "binary code" strategy preclude explicit design of particular side chains at specified positions. Therefore, packing interactions cannot be specified a priori. To assess whether the binary code strategy can nonetheless produce well-folded de novo proteins, we constructed a second-generation library based upon a new structural scaffold designed to fold into 102-residue four-helix bundles. Characterization of five proteins chosen arbitrarily from this new library revealed that (1) all are alpha-helical and quite stable; (2) four of the five contain an abundance of tertiary interactions indicative of well-ordered structures; and (3) one protein forms a well-folded structure with native-like features. The proteins from this new 102-residue library are substantially more stable and dramatically more native-like than those from an earlier binary patterned library of 74-residue sequences. These findings demonstrate that chain length is a crucial determinant of structural order in libraries of de novo four-helix bundles. Moreover, these results show that the binary code strategy--if applied to an appropriately designed structural scaffold--can generate large collections of stably folded and/or native-like proteins.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2312408PMC
http://dx.doi.org/10.1110/ps.0228003DOI Listing
January 2003