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Dive into the research topics where Brian Robert Miller is active.

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Featured researches published by Brian Robert Miller.


mAbs | 2009

Anti-tumor activity of stability-engineered IgG-like bispecific antibodies targeting TRAIL-R2 and LTβR

Jennifer S. Michaelson; Stephen J. Demarest; Brian Robert Miller; Aldo Amatucci; William B. Snyder; Xiufeng Wu; Flora Huang; Samantha Phan; Sharon X. Gao; Adam Doern; Graham K. Farrington; Alexey Lugovskoy; Ingrid Joseph; Veronique Bailly; Xin Wang; Ellen Garber; Jeffrey L. Browning; Scott Glaser

Bispecific antibodies (BsAbs) represent an emerging class of biologics that achieve dual targeting with a single agent. Recombinant DNA technologies have facilitated a variety of creative bispecific designs with many promising therapeutic applications; however, practical methods for producing high quality BsAbs that have good product stability, long serum half-life, straightforward purification, and scalable production have largely been limiting. Here we describe a protein-engineering approach for producing stable, scalable tetravalent IgG-like BsAbs. The stability-engineered IgG-like BsAb was envisioned to target and crosslink two TNF family member receptors, TRAIL-R2 (TNF-Related Apoptosis Inducing Ligand Receptor-2) and LTβR (Lymphotoxin-beta Receptor), expressed on the surface of epithelial tumor cells with the goal of triggering an enhanced anti-tumor effect. Our IgG-like BsAbs consists of a stability-engineered anti- LTβR single chain Fv (scFv) genetically fused to either the N- or C-terminus of the heavy chain of a full-length anti-TRAIL-R2 IgG1 monoclonal antibody. Both N- or C-terminal BsAbs were active in inhibiting tumor cell growth in vitro, and with some cell lines demonstrated enhanced activity relative to the combination of parental Abs. Pharmacokinetic studies in mice revealed long serum half-lives for the BsAbs. In murine tumor xenograft models, therapeutic treatment with the BsAbs resulted in reduction in tumor volume either comparable to or greater than the combination of parental antibodies, indicating that simultaneously targeting and cross-linking receptor pairs is an effective strategy for treating tumor cells. These studies support that stability-engineering is an enabling step for producing scalable IgG-like BsAbs with properties desirable for biopharmaceutical development.


Protein Engineering Design & Selection | 2010

Stability engineering of scFvs for the development of bispecific and multivalent antibodies

Brian Robert Miller; Stephen J. Demarest; Alexey Lugovskoy; Flora Huang; Xiufeng Wu; William B. Snyder; Lisa J. Croner; Norman Wang; Aldo Amatucci; Jennifer S. Michaelson; Scott Glaser

Single-chain Fvs (scFvs) are commonly used building blocks for creating engineered diagnostic and therapeutic antibody molecules. Bispecific antibodies (BsAbs) hold particular interest due to their ability to simultaneously bind and engage two distinct targets. We describe a technology for producing stable, scalable IgG-like bispecific and multivalent antibodies based on methods for rapidly engineering thermally stable scFvs. Focused libraries of mutant scFvs were designed using a combination of sequence-based statistical analyses and structure-, and knowledge-based methods. Libraries encoding these designs were expressed in E. coli and culture supernatants-containing soluble scFvs screened in a high-throughput assay incorporating a thermal challenge prior to an antigen-binding assay. Thermally stable scFvs were identified that retain full antigen-binding affinity. Single mutations were found that increased the measured T(m) of either the V(H) or V(L) domain by as much as 14 degrees C relative to the wild-type scFv. Combinations of mutations further increased the T(m) by as much as an additional 12 degrees C. Introduction of a stability-engineered scFv as part of an IgG-like BsAb enabled scalable production and purification of BsAb with favorable biophysical properties.


mAbs | 2011

A stable IgG-like bispecific antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor demonstrates superior anti-tumor activity

Jianying Dong; Arlene Sereno; Dikran Aivazian; Emma Langley; Brian Robert Miller; William Snyder; Eric Chan; Matt Cantele; Ronald Morena; Ingrid B.J.K. Joseph; Antonio Boccia; Cyrus Virata; James Gamez; Grace Yco; Michael Favis; Xiufeng Wu; Christilyn Graff; Qin Wang; Ellen Rohde; Rachel Rennard; Lisa Berquist; Flora Huang; Ying Zhang; Sharon X. Gao; Steffan N. Ho; Stephen J. Demarest; Mitchell E Reff; Kandasamy Hariharan; Scott Glaser

The epidermal growth factor receptor (EGFR) and the type I insulin-like growth factor receptor (IGF-1R) are two cell surface receptor tyrosine kinases known to cooperate to promote tumor progression and drug resistance. Combined blockade of EGFR and IGF-1R has shown improved anti-tumor activity in preclinical models. Here, we report the characterization of a stable IgG-like bispecific antibody (BsAb) dual-targeting EGFR and IGF-1R that was developed for cancer therapy. The BsAb molecule (EI-04), constructed with a stability-engineered single chain variable fragment (scFv) against IGF-1R attached to the carboxyl-terminus of an IgG against EGFR, displays favorable biophysical properties for biopharmaceutical development. Biochemically, EI-04 bound to human EGFR and IGF-1R with sub nanomolar affinity, co-engaged the two receptors simultaneously, and blocked the binding of their respective ligands with similar potency compared to the parental monoclonal antibodies (mAbs). In tumor cells, EI-04 effectively inhibited EGFR and IGF-1R phosphorylation, and concurrently blocked downstream AKT and ERK activation, resulting in greater inhibition of tumor cell growth and cell cycle progression than the single mAbs. EI-04, likely due to its tetravalent bispecific format, exhibited high avidity binding to BxPC3 tumor cells co-expressing EGFR and IGF-1R, and consequently improved potency at inhibiting IGF-driven cell growth over the mAb combination. Importantly, EI-04 demonstrated enhanced in vivo anti-tumor efficacy over the parental mAbs in two xenograft models, and even over the mAb combination in the BxPC3 model. Our data support the clinical investigation of EI-04 as a superior cancer therapeutic in treating EGFR and IGF-1R pathway responsive tumors.


Proteins | 2009

Conserved amino acid networks involved in antibody variable domain interactions

Norman Wang; William F. Smith; Brian Robert Miller; Dikran Aivazian; Alexey Lugovskoy; Mitchell Reff; Scott Glaser; Lisa J. Croner; Stephen J. Demarest

Engineered antibodies are a large and growing class of protein therapeutics comprising both marketed products and many molecules in clinical trials in various disease indications. We investigated naturally conserved networks of amino acids that support antibody VH and VL function, with the goal of generating information to assist in the engineering of robust antibody or antibody‐like therapeutics. We generated a large and diverse sequence alignment of V‐class Ig‐folds, of which VH and VL domains are family members. To identify conserved amino acid networks, covariations between residues at all possible position pairs were quantified as correlation coefficients (ϕ‐values). We provide rosters of the key conserved amino acid pairs in antibody VH and VL domains, for reference and use by the antibody research community. The majority of the most strongly conserved amino acid pairs in VH and VL are at or adjacent to the VH–VL interface suggesting that the ability to heterodimerize is a constraining feature of antibody evolution. For the VH domain, but not the VL domain, residue pairs at the variable‐constant domain interface (VH–CH1 interface) are also strongly conserved. The same network of conserved VH positions involved in interactions with both the VL and CH1 domains is found in camelid VHH domains, which have evolved to lack interactions with VL and CH1 domains in their mature structures; however, the amino acids at these positions are different, reflecting their different function. Overall, the data describe naturally occurring amino acid networks in antibody Fv regions that can be referenced when designing antibodies or antibody‐like fragments with the goal of improving their biophysical properties. Proteins 2009.


Proteins | 2009

Structural understanding of stabilization patterns in engineered bispecific Ig-like antibody molecules

Jacob L. Jordan; Joseph Arndt; Karl Hanf; Guohui Li; Janine Hall; Stephen J. Demarest; Flora Huang; Xiufeng Wu; Brian Robert Miller; Scott Glaser; Erik J. Fernandez; Deping Wang; Alexey Lugovskoy

Bispecific immunoglobulin‐like antibodies capable of engaging multiple antigens represent a promising new class of therapeutic agents. Engineering of these molecules requires optimization of the molecular properties of one of the domain components. Here, we present a detailed crystallographic and computational characterization of the stabilization patterns in the lymphotoxin‐beta receptor (LTβR) binding Fv domain of an anti‐LTβR/anti‐TNF‐related apoptosis inducing ligand receptor‐2 (TRAIL‐R2) bispecific immunoglobulin‐like antibody. We further describe a new hierarchical structure‐guided approach toward engineering of antibody‐like molecules to enhance their thermal and chemical stability. Proteins 2009.


Journal of Biological Chemistry | 2011

Stable IgG-like Bispecific Antibodies Directed toward the Type I Insulin-like Growth Factor Receptor Demonstrate Enhanced Ligand Blockade and Anti-tumor Activity

Jianying Dong; Arlene Sereno; William B. Snyder; Brian Robert Miller; Susan Tamraz; Adam Doern; Michael Favis; Xiufeng Wu; Hon Tran; Emma Langley; Ingrid Joseph; Antonio Boccia; Rebecca Kelly; Kathleen Wortham; Qin Wang; Lisa Berquist; Flora Huang; Sharon X. Gao; Ying Zhang; Alexey Lugovskoy; Shelly Martin; Heather Gouvis; Steven A. Berkowitz; Gisela G. Chiang; Mitchell Reff; Scott Glaser; Kandasamy Hariharan; Stephen J. Demarest

Bispecific antibodies (BsAbs) target multiple epitopes on the same molecular target or different targets. Although interest in BsAbs has persisted for decades, production of stable and active BsAbs has hindered their clinical evaluation. Here, we describe the production and characterization of tetravalent IgG-like BsAbs that combine the activities of allosteric and competitive inhibitors of the type-I insulin-like growth factor receptor (IGF-1R). The BsAbs, which were engineered for thermal stability, express well, demonstrate favorable biophysical properties, and recognize both epitopes on IGF-1R. Only one BsAb with a unique geometry, denoted BIIB4-5scFv, was capable of engaging all four of its binding arms simultaneously. All the BsAbs (especially BIIB4-5scFv) demonstrated enhanced ligand blocking over the single monoclonal antibodies (mAbs), particularly at high ligand concentrations. The pharmacokinetic profiles of two IgG-like BsAbs were tested in nude mice and shown to be comparable with that of the parental mAbs. The BsAbs, especially BIIB4-5scFv, demonstrated an improved ability to reduce the growth of multiple tumor cell lines and to inhibit ligand-induced IGF-1R signaling in tumor cells over the parental mAbs. BIIB4-5scFv also led to superior tumor growth inhibition over its parental mAbs in vivo. In summary, BsAbs that bridge multiple inhibitory mechanisms against a single target may generally represent a more effective strategy for intervention in oncology or other indications compared with traditional mAb therapy.


Archives of Biochemistry and Biophysics | 2012

Secretion from bacterial versus mammalian cells yields a recombinant scFv with variable folding properties

Michelle C. Vendel; Michael Favis; William B. Snyder; Flora Huang; Allan D. Capili; Jianying Dong; Scott Glaser; Brian Robert Miller; Stephen J. Demarest

Escherichia coli (E. coli) is the most commonly used organism for expressing antibody fragments such as single chain antibody Fvs (scFvs). Previously, we have utilized E. coli to express well-folded scFvs for characterization and engineering purposes with the goal of using these engineered proteins as building blocks for generating IgG-like bispecific antibodies (BsAbs). In the study, described here, we observed a significant difference in the secondary structure of an scFv produced in E. coli and the same scFv expressed and secreted from chinese hamster ovary (CHO) cells as part of a BsAb. We devised a proteolytic procedure to separate the CHO-derived scFv from its antibody-fusion partner and compared its properties with those of the E. coli-derived scFv. In comparison to the CHO-derived scFv, the E. coli-derived scFv was found trapped in a misfolded, but monomeric state that was stable for months at 4 °C. The misfolded state bound antigen in a heterogeneous fashion that included non-specific binding, which made functional characterization challenging. This odd incidence of obtaining a misfolded scFv from bacteria suggests careful characterization of the folded properties of bacterially expressed scFvs is warranted if anomalous issues with antigen-binding or non-specificity occur during an engineering campaign. Additionally, our proteolytic methodology for obtaining significant levels of intact scFvs from highly expressed IgG-like antibody proteins serves as a robust method for producing scFvs in CHO without the use of designed cleavage motifs.


Methods of Molecular Biology | 2009

Rapid Screening Platform for Stabilization of scFvs in Escherichia coli

Brian Robert Miller; Scott Glaser; Stephen J. Demarest

The poor biophysical properties of antibody fragments such as scFvs and diabodies can preclude their use as therapeutic agents. The non-ideal biophysical properties and insufficient thermal stability of antibody fragments often leads to poor expression, poor solubility, and a predisposition of the proteins to aggregate. We have developed a general platform for engineering stability into antibody fragments. By promoting Escherichia coli cultures to secrete scFvs directly into growth media, automated screening methods can be applied to empirically evaluate multiple stability design strategies including rational, sequence-based, and structure-based designs. Once stabilized, these antibody fragments demonstrate improved expression and durability during purification, handling, and storage. Stabilized antibody fragments can also be used as building blocks for multivalent or bispecific antibody-like molecules.


Archive | 2007

Stabilized polypeptide compositions

Scott Glaser; Stephen J. Demarest; Brian Robert Miller; William B. Snyder; Xiufeng Wu; Norman Wang; Lisa J. Croner; Alexey Lugovskoy


Archive | 2008

COMPOSITIONS THAT BIND MULTIPLE EPITOPES OF IGF-1R

Scott Glaser; Stephen J. Demarest; Brian Robert Miller; Kandasamy Hariharan; Steffan N. Ho; Jianying Dong; Alexey Lugovskoy

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