All Publications


  • Engineering a Single-Agent Cytokine/Antibody Fusion That Selectively Expands Regulatory T Cells for Autoimmune Disease Therapy JOURNAL OF IMMUNOLOGY Spangler, J. B., Trotta, E., Tomala, J., Peck, A., Young, T. A., Savvides, C. S., Silveria, S., Votavova, P., Salafsky, J., Pande, V. S., Kovar, M., Bluestone, J. A., Garcia, K. 2018; 201 (7): 2094–2106
  • Engineering a Single-Agent Cytokine/Antibody Fusion That Selectively Expands Regulatory T Cells for Autoimmune Disease Therapy. Journal of immunology (Baltimore, Md. : 1950) Spangler, J. B., Trotta, E., Tomala, J., Peck, A., Young, T. A., Savvides, C. S., Silveria, S., Votavova, P., Salafsky, J., Pande, V. S., Kovar, M., Bluestone, J. A., Garcia, K. C. 2018

    Abstract

    IL-2 has been used to treat diseases ranging from cancer to autoimmune disorders, but its concurrent immunostimulatory and immunosuppressive effects hinder efficacy. IL-2 orchestrates immune cell function through activation of a high-affinity heterotrimeric receptor (composed of IL-2Ralpha, IL-2Rbeta, and common gamma [gammac]). IL-2Ralpha, which is highly expressed on regulatory T (TReg) cells, regulates IL-2 sensitivity. Previous studies have shown that complexation of IL-2 with the JES6-1 Ab preferentially biases cytokine activity toward TReg cells through a unique mechanism whereby IL-2 is exchanged from the Ab to IL-2Ralpha. However, clinical adoption of a mixed Ab/cytokine complex regimen is limited by stoichiometry and stability concerns. In this study, through structure-guided design, we engineered a single agent fusion of the IL-2 cytokine and JES6-1 Ab that, despite being covalently linked, preserves IL-2 exchange, selectively stimulating TReg expansion and exhibiting superior disease control to the mixed IL-2/JES6-1 complex in a mouse colitis model. These studies provide an engineering blueprint for resolving a major barrier to the implementation of functionally similar IL-2/Ab complexes for treatment of human disease.

    View details for PubMedID 30104245

  • Intermolecular correlations are necessary to explain diffuse scattering from protein crystals IUCRJ Peck, A., Poitevin, F., Lane, T. J. 2018; 5: 211–22

    Abstract

    Conformational changes drive protein function, including catalysis, allostery and signaling. X-ray diffuse scattering from protein crystals has frequently been cited as a probe of these correlated motions, with significant potential to advance our understanding of biological dynamics. However, recent work has challenged this prevailing view, suggesting instead that diffuse scattering primarily originates from rigid-body motions and could therefore be applied to improve structure determination. To investigate the nature of the disorder giving rise to diffuse scattering, and thus the potential applications of this signal, a diverse repertoire of disorder models was assessed for its ability to reproduce the diffuse signal reconstructed from three protein crystals. This comparison revealed that multiple models of intramolecular conformational dynamics, including ensemble models inferred from the Bragg data, could not explain the signal. Models of rigid-body or short-range liquid-like motions, in which dynamics are confined to the biological unit, showed modest agreement with the diffuse maps, but were unable to reproduce experimental features indicative of long-range correlations. Extending a model of liquid-like motions to include disorder across neighboring proteins in the crystal significantly improved agreement with all three systems and highlighted the contribution of intermolecular correlations to the observed signal. These findings anticipate a need to account for intermolecular disorder in order to advance the interpretation of diffuse scattering to either extract biological motions or aid structural inference.

    View details for PubMedID 29765611

  • Tungstate as a Transition State Analog for Catalysis by Alkaline Phosphatase JOURNAL OF MOLECULAR BIOLOGY Peck, A., Sunden, F., Andrews, L. D., Pande, V. S., Herschlag, D. 2016; 428 (13): 2758-2768

    Abstract

    The catalytic mechanisms underlying Escherichia coli alkaline phosphatase's (AP) remarkable rate enhancement have been probed extensively. Past work indicated that whereas the serine nucleophile (Ser102) electrostatically repels the product phosphate, another oxyanion, tungstate, binds more strongly in the presence of Ser102. These results predict a covalent bond between the serine nucleophile and tungstate, a model that we test herein. The crystal structure of tungstate-bound alkaline phosphatase provides evidence for a covalent adduct model and further shows that the ligand adopts trigonal bipyramidal geometry, which is infrequently observed for tungstate in small molecules and other active sites but mirrors the geometry of the presumed phosphoryl transfer transition state. The AP active site is known to stabilize another oxyanion, vanadate, in trigonal bipyramidal geometry, but the extent to which binding of either ligand reproduces the energetics of the transition state cannot be deduced from structural inspection alone. To test for transition state analog behavior, we determined the relationship between catalytic activity and affinity for tungstate and vanadate for a series of 20 AP variants. Affinity and activity were highly correlated for tungstate (r(2) = 0.89) but not vanadate (r(2) = 0.23), indicating that the tungstate•AP complex may better mimic this enzyme's transition state properties. The results herein suggest that tungstate will be a valuable tool for further dissecting AP catalysis and may prove helpful in mechanistic studies of other phosphoryl transfer enzymes.

    View details for DOI 10.1016/j.jmb.2016.05.007

    View details for Web of Science ID 000378958500007

    View details for PubMedID 27189921

  • Conformational heterogeneity of the calmodulin binding interface NATURE COMMUNICATIONS Shukla, D., Peck, A., Pande, V. S. 2016; 7

    Abstract

    Calmodulin (CaM) is a ubiquitous Ca(2+) sensor and a crucial signalling hub in many pathways aberrantly activated in disease. However, the mechanistic basis of its ability to bind diverse signalling molecules including G-protein-coupled receptors, ion channels and kinases remains poorly understood. Here we harness the high resolution of molecular dynamics simulations and the analytical power of Markov state models to dissect the molecular underpinnings of CaM binding diversity. Our computational model indicates that in the absence of Ca(2+), sub-states in the folded ensemble of CaM's C-terminal domain present chemically and sterically distinct topologies that may facilitate conformational selection. Furthermore, we find that local unfolding is off-pathway for the exchange process relevant for peptide binding, in contrast to prior hypotheses that unfolding might account for binding diversity. Finally, our model predicts a novel binding interface that is well-populated in the Ca(2+)-bound regime and, thus, a candidate for pharmacological intervention.

    View details for DOI 10.1038/ncomms10910

    View details for Web of Science ID 000373529600001

    View details for PubMedID 27040077

    View details for PubMedCentralID PMC4822001

  • Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site ELIFE Sunden, F., Peck, A., Salzman, J., Ressl, S., Herschlag, D. 2015; 4

    Abstract

    Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called 'catalytic residues' are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modes of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes.

    View details for DOI 10.7554/eLife.06181

    View details for Web of Science ID 000372713000001

    View details for PubMedCentralID PMC4438272

  • Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site. eLife Sunden, F., Peck, A., Salzman, J., Ressl, S., Herschlag, D. 2015; 4

    Abstract

    Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called 'catalytic residues' are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modes of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes.

    View details for DOI 10.7554/eLife.06181

    View details for PubMedID 25902402

    View details for PubMedCentralID PMC4438272

  • Alternative activation in systemic juvenile idiopathic arthritis monocytes CLINICAL IMMUNOLOGY Macaubas, C., Nguyen, K. D., Peck, A., Buckingham, J., Deshpande, C., Wong, E., Alexander, H. C., Chang, S., Begovich, A., Sun, Y., Park, J. L., Pan, K., Lin, R., Lih, C., Augustine, E. M., Phillips, C., Hadjinicolaou, A. V., Lee, T., Mellins, E. D. 2012; 142 (3): 362-372

    Abstract

    Systemic juvenile idiopathic arthritis (SJIA) is a chronic autoinflammatory condition. The association with macrophage activation syndrome, and the therapeutic efficacy of inhibiting monocyte-derived cytokines, has implicated these cells in SJIA pathogenesis. To characterize the activation state (classical/M1 vs. alternative/M2) of SJIA monocytes, we immunophenotyped monocytes using several approaches. Monocyte transcripts were analyzed by microarray and quantitative PCR. Surface proteins were measured at the single cell level using flow cytometry. Cytokine production was evaluated by intracellular staining and ELISA. CD14(++)CD16(-) and CD14(+)CD16(+) monocyte subsets are activated in SJIA. A mixed M1/M2 activation phenotype is apparent at the single cell level, especially during flare. Consistent with an M2 phenotype, SJIA monocytes produce IL-1β after LPS exposure, but do not secrete it. Despite the inflammatory nature of active SJIA, circulating monocytes demonstrate significant anti-inflammatory features. The persistence of some of these phenotypes during clinically inactive disease argues that this state reflects compensated inflammation.

    View details for DOI 10.1016/j.clim.2011.12.008

    View details for PubMedID 22281427

  • Monocyte phenotypes in systemic juvenile idiopathic arthritis Macaubas, C., Khoa Nguyen, Peck, A., Wong, E., Buckingham, J., Goertz, Y., Deshpande, C., Alexander, H., Chang, S., Sun, Y., Park, J., Lee, T., Begovich, A., Mellins, E. AMER ASSOC IMMUNOLOGISTS. 2011
  • Distribution of circulating cells in systemic juvenile idiopathic arthritis across disease activity states CLINICAL IMMUNOLOGY Macaubas, C., Nguyen, K., Deshpande, C., Phillips, C., Peck, A., Lee, T., Park, J. L., Sandborg, C., Mellins, E. D. 2010; 134 (2): 206-216

    Abstract

    Juvenile idiopathic arthritis (JIA) encompasses a group of chronic childhood arthritides of unknown etiology. One subtype, systemic JIA (SJIA), is characterized by a combination of arthritis and systemic inflammation. Its systemic nature suggests that clues to SJIA pathogenesis may be found in examination of peripheral blood cells. To determine the immunophenotypic profiles of circulating mononuclear cells in SJIA patients with different degrees of disease activity, we studied PBMC from 31 SJIA patients, 20 polyarticular JIA patients (similar to adult rheumatoid arthritis), and 31 age-matched controls. During SJIA disease flare, blood monocyte numbers were increased, whereas levels of myeloid dendritic cells (DC) and gammadelta T cells were reduced. At both flare and quiescence, increased levels of CD14 and CD16 were found on SJIA monocytes. Levels of CD16-DC were elevated at SJIA quiescence compared both to healthy controls and to SJIA subjects with active disease. Overall, our findings suggest dysregulation of innate immunity in SJIA and raise the possibility that quiescence represents a state of compensated inflammation.

    View details for DOI 10.1016/j.clim.2009.09.010

    View details for PubMedID 19879195

  • Plasticity of T-cell phenotype and function: the T helper type 17 example IMMUNOLOGY Peck, A., Mellins, E. D. 2010; 129 (2): 147-153

    Abstract

    Mature T helper type 1 (Th1) and Th2 cells antagonize the development of the opposing subset to sustain lineage-specific responses. However, the recent identification of a third distinct subset of helper T cells - the Th17 lineage - collapses the established Th1/Th2 dichotomy and raises intriguing questions about T-cell fate. In this review, we discuss the Th17 subset in the context of the effector and regulatory T-cell lineages. Initial studies suggested reciprocal developmental pathways between Th17/Th1 subsets and between Th17/regulatory T-cell subsets, and identified multiple mechanisms by which Th1 and Th2 cells antagonize the generation of Th17 cells. However, recent observations reveal the susceptibility of differentiated Th17 cells to Th1 polarization and the enhancement of Th17 memory cells by the Th1 factors interferon-gamma and T-bet. In addition, new data indicate late-stage plasticity of a subpopulation of regulatory T cells, which can be selectively induced to adopt a Th17 phenotype. Elucidating the mechanisms that undermine cross-lineage suppression and facilitate these phenotype shifts will not only clarify the flexibility of T-cell differentiation, but may also shed insight into the pathogenesis of autoimmunity and cancer. Furthermore, understanding these phenomena will be critical for the design of immunotherapy that seeks to disrupt lineage-specific T-cell responses and may suggest ways to manipulate the balance between pathogenic and regulatory lymphocytes for the restoration of homeostasis.

    View details for DOI 10.1111/j.1365-2567.2009.03189.x

    View details for Web of Science ID 000273458000001

    View details for PubMedID 19922424

    View details for PubMedCentralID PMC2814457

  • Precarious Balance: Th17 Cells in Host Defense INFECTION AND IMMUNITY Peck, A., Mellins, E. D. 2010; 78 (1): 32-38

    Abstract

    Lineage-specific responses from the effector T-cell repertoire form a critical component of adaptive immunity. The recent identification of Th17 cells-a third, distinct lineage of helper T cells-collapses the long-accepted paradigm in which Th1 and Th2 cells distinctly mediate cellular and humoral immunity, respectively. In this minireview, we discuss the involvement of the Th17 lineage during infection by extracellular bacteria, intracellular bacteria, and fungi. Emerging trends suggest that the Th17 population bridges innate and adaptive immunity to produce a robust antimicrobial inflammatory response. However, because Th17 cells mediate both host defense and pathological inflammation, elucidation of mechanisms that attenuate but do not completely abolish the Th17 response may have powerful implications for therapy.

    View details for DOI 10.1128/IAI.00929-09

    View details for Web of Science ID 000272984300002

    View details for PubMedID 19901061

    View details for PubMedCentralID PMC2798221

  • Breaking old paradigms: Th17 cells in autoimmune arthritis CLINICAL IMMUNOLOGY Peck, A., Mellins, E. D. 2009; 132 (3): 295-304

    Abstract

    Aberrant helper T cell activation has been implicated in the pathogenesis of an array of autoimmune diseases. In this review, we summarize evidence that suggests the involvement of a novel T cell subset, the Th17 lineage, in rheumatoid arthritis. In particular, we focus on the role of Th17 cells in inducing and perpetuating the chronic inflammation, cartilage damage, and bone erosion that are hallmark phases of joint destruction and consider current and emerging therapies that seek to disrupt the inflammatory Th17 network and shift the immune system back towards homeostasis.

    View details for DOI 10.1016/j.clim.2009.03.522

    View details for Web of Science ID 000268783900001

    View details for PubMedID 19403336

    View details for PubMedCentralID PMC2720426