Stephen Burke

Macrogenics, Роквилл, Maryland, United States

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Publications (8)73.98 Total impact

  • European Journal of Cancer 11/2014; 50. DOI:10.1016/S0959-8049(14)70264-3 · 5.42 Impact Factor
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    ABSTRACT: To exploit the physiologic Fcgamma receptor IIb (CD32B) inhibitory coupling mechanism to control B cell activation by constructing a novel bispecific diabody scaffold, termed a dual-affinity retargeting (DART) molecule, for therapeutic applications. DART molecules were constructed by pairing an Fv region from a monoclonal antibody (mAb) directed against CD32B with an Fv region from a mAb directed against CD79B, the beta-chain of the invariant signal-transducing dimer of the B cell receptor complex. DART molecules were characterized physicochemically and for their ability to simultaneously bind the target receptors in vitro and in intact cells. The ability of the DART molecules to negatively control B cell activation was determined by calcium mobilization, by tyrosine phosphorylation of signaling molecules, and by proliferation and Ig secretion assays. A DART molecule specific for the mouse ortholog of CD32B and CD79B was also constructed and tested for its ability to inhibit B cell proliferation in vitro and to control disease severity in a collagen-induced arthritis (CIA) model. DART molecules were able to specifically bind and coligate their target molecules on the surface of B cells and demonstrated a preferential simultaneous binding to both receptors on the same cell. DART molecules triggered the CD32B-mediated inhibitory signaling pathway in activated B cells, which translated into inhibition of B cell proliferation and Ig secretion. A DART molecule directed against the mouse orthologs was effective in inhibiting the development of CIA in DBA/1 mice. This innovative bispecific antibody scaffold that simultaneously engages activating and inhibitory receptors enables novel therapeutic approaches for the treatment of rheumatoid arthritis and potentially other autoimmune and inflammatory diseases in humans.
    Arthritis & Rheumatology 07/2010; 62(7):1933-43. DOI:10.1002/art.27477 · 7.76 Impact Factor
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    ABSTRACT: Bispecific antibodies capable of redirecting the lytic potential of immune effector cells to kill tumor targets have long been recognized as a potentially potent biological therapeutic intervention. Unfortunately, efforts to produce such molecules have been limited owing to inefficient production and poor stability properties. Here, we describe a novel Fv-derived strategy based on a covalently linked bispecific diabody structure that we term dual-affinity re-targeting (DART). As a model system, we linked an Fv specific for human CD16 (FcgammaRIII) on effector cells to an Fv specific for mouse or human CD32B (FcgammaRIIB), a normal B-cell and tumor target antigen. DART proteins were produced at high levels in mammalian cells, retained the binding activity of the respective parental Fv domains as well as bispecific binding, and showed extended storage and serum stability. Functionally, the DART molecules demonstrated extremely potent, dose-dependent cytotoxicity in retargeting human PBMC against B-lymphoma cell lines as well as in mediating autologous B-cell depletion in culture. In vivo studies in mice demonstrated effective B-cell depletion that was dependent on the transgenic expression of both CD16A on the effector cells and CD32B on the B-cell targets. Furthermore, DART proteins showed potent in vivo protective activity in a human Burkitt's lymphoma cell xenograft model. Thus, DART represents a biologically potent format that provides a versatile platform for generating bispecific antibody fragments for redirected killing and, with the selection of appropriate binding partners, applications outside of tumor cell cytotoxicity.
    Journal of Molecular Biology 04/2010; 399(3):436-49. DOI:10.1016/j.jmb.2010.04.001 · 4.33 Impact Factor

  • Molecular Cancer Therapeutics 12/2009; 8(Supplement 1):C180-C180. DOI:10.1158/1535-7163.TARG-09-C180 · 5.68 Impact Factor
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    ABSTRACT: Background: To develop an immunotherapeutic product that neutralizes variola or other orthopoxviruses in case of bioterrorism, we evaluated the prophylactic and therapeutic activity of monoclonal antibodies (mAbs) directed against vaccinia virus antigens. Ch7D11 is a chimeric (mouse/human) version of 7D11 mAb [from A. Schmaljohn, USAMRIID] that targets the L1 protein of MV particles. Ch8A and Ch12F are chimeric (chimp/human) mAbs that target the B5 and A33 proteins, respectively, of EV particles [PNAS 103:1882; J Virol 81:8989]. Methods: Balb/c mice were infected intratracheally with vaccinia virus. mAbs were administered IP. Animals were monitored for morbidity (weight loss) and mortalilty. Results: When administered prophylactically one day prior to infection at a dose of 10 mg/kg, Ch8A or Ch12F was associated with partial survival, whereas Ch7D11 was associated with full survival, although significant weight loss was observed. Only a combination of anti-MV and anti-EV mAbs (Ch7D11:Ch8A or Ch7D11:Ch12F) at a total dose of 10 mg/kg was associated with reduced morbidity. When administered therapeutically at 2 or 3 days post-infection, none of the individual mAbs prevented death, but a Ch7D11:Ch8A or Ch7D11:Ch12F combination rescued ≥90% of the animals. Each anti-MV:anti-EV mAb combination had a half-maximal effective therapeutic dose of ~5 mg/kg and an optimal ratio of 1:1, while a mixture of two anti-EV mAbs (Ch8A:Ch12F) was ineffective. VIG (vaccinia immune globulin) at 500 mg/kg also was ineffective. Conclusions: Combinations of anti-MV and anti-EV monoclonal antibodies exhibit synergistic prophylactic and therapeutic activity in mice and are potent orthopoxvirus immunotherapeutic agents. [Supported by NIAID grant 5-U01-AI070504 and funds from DIR, NIAID]
    Infectious Diseases Society of America 2008 Annual Meeting; 10/2008
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    ABSTRACT: Monoclonal antibodies (mAbs) and IgG-Fc fusion proteins are widely used in the treatment of cancer, lymphoma, and autoimmune diseases. These therapeutic molecules act by a variety of different mechanisms, ranging from blockade of receptor oligomerization, to ligand competition or the recruitment of leukocytes effector cells leading to antibody-dependent cell cytotoxicity (ADCC). A common denominator in the latter case is the binding of the Fcγ regions of the molecule to low-affinity Fcγ receptors (FcγRs) expressed on the effector cells. As a consequence, the optimization of the Fc region of therapeutic mAb offers a means to obtain molecules with enhanced activity and a customizable binding profile suitable for different therapeutic applications. We have constructed a portfolio of novel IgG1-Fc domains by using a functional genetic screen via yeast display technology aimed at selecting variants with selective FcγR binding properties. All Fc mutants were characterized for their FcγR binding profile by ELISA and surface plasmon resonance analysis; selected Fc were then cloned into model mAbs and subjected to functional analysis. These Fc-optimized mAbs outperformed the parent antibody in vitro in ADCC assays and in xenograft tumor models in the mouse. Fcγ-optimization via molecular engineering is an effective option for designing new generations of antibodies with improved therapeutic properties.
    Advances in enzyme regulation 01/2008; 48(1):152-64. DOI:10.1016/j.advenzreg.2007.11.011
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    ABSTRACT: Monoclonal antibodies (mAb) are widely used in the treatment of non-Hodgkin's lymphoma and autoimmune diseases. Although the mechanism of action in vivo is not always known, the therapeutic activity of several approved mAbs depends on the binding of the Fcgamma regions to low-affinity Fcgamma receptors (FcgammaR) expressed on effector cells. We did functional genetic screens to identify IgG1 Fc domains with improved binding to the low-affinity activating Fc receptor CD16A (FcgammaRIIIA) and reduced binding to the low-affinity inhibitory Fc receptor, CD32B (FcgammaRIIB). Identification of new amino acid residues important for FcgammaR binding guided the construction of an Fc domain that showed a dramatically enhanced CD16A binding and greater than a 100-fold improvement in antibody-dependent cell-mediated cytotoxicity. In a xenograft murine model of B-cell malignancy, the greatest enhancement of an Fc-optimized anti-human B-cell mAb was accounted for by improved binding to FcgammaRIV, a unique mouse activating FcgammaR that is expressed by monocytes and macrophages but not natural killer (NK) cells, consistent with experimental and clinical data suggesting that mononuclear phagocytes, effector cells expressing both activating and inhibitory FcgammaR, are critical mediators of B-cell depletion in vivo. By using mice transgenic for human CD16A, enhanced survival was observed due to expression of CD16A-158(phe) on monocytes and macrophages as well as on NK cells in these mice. The design of new generations of improved antibodies for immunotherapy should aim at Fc optimization to increase the engagement of activating FcgammaR present on the surface of tumor-infiltrating effector cell populations.
    Cancer Research 10/2007; 67(18):8882-90. DOI:10.1158/0008-5472.CAN-07-0696 · 9.33 Impact Factor
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    ABSTRACT: West Nile virus is a mosquito-borne flavivirus closely related to the human epidemic-causing dengue, yellow fever and Japanese encephalitis viruses. In establishing infection these icosahedral viruses undergo endosomal membrane fusion catalysed by envelope glycoprotein rearrangement of the putative receptor-binding domain III (DIII) and exposure of the hydrophobic fusion loop. Humoral immunity has an essential protective function early in the course of West Nile virus infection. Here, we investigate the mechanism of neutralization by the E16 monoclonal antibody that specifically binds DIII. Structurally, the E16 antibody Fab fragment engages 16 residues positioned on four loops of DIII, a consensus neutralizing epitope sequence conserved in West Nile virus and distinct in other flaviviruses. The E16 epitope protrudes from the surface of mature virions in three distinct environments, and docking studies predict Fab binding will leave five-fold clustered epitopes exposed. We also show that E16 inhibits infection primarily at a step after viral attachment, potentially by blocking envelope glycoprotein conformational changes. Collectively, our results suggest that a vaccine strategy targeting the dominant DIII epitope may elicit safe and effective immune responses against flaviviral diseases.
    Nature 10/2005; 437(7059):764-9. DOI:10.1038/nature03956 · 41.46 Impact Factor