[Show abstract][Hide abstract] ABSTRACT: Members of the CD28 family play important roles in regulating T-cell functions and share a common gene structure profile. We have identified VSTM3 as a protein whose gene structure matches that of the other CD28 family members. This protein (also known as TIGIT and WUCAM) has been previously shown to affect immune responses and is expressed on NK cells, activated and memory T cells, and Tregs. The nectin-family proteins CD155 and CD112 serve as counter-structures for VSTM3, and CD155 and CD112 also bind to the activating receptor CD226 on T cells and NK cells. Hence, this group of interacting proteins forms a network of molecules similar to the well-characterized CD28-CTLA-4-CD80-CD86 network. In the same way that soluble CTLA-4 can be used to block T-cell responses, we show that soluble Vstm3 attenuates T-cell responses in vitro and in vivo. Moreover, animals deficient in Vstm3 are more sensitive to autoimmune challenges indicating that this new member of the CD28 family is an important regulator of T-cell responses.
European Journal of Immunology 04/2011; 41(4):902-15. DOI:10.1002/eji.201041136 · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Bispecific antibodies (bsAbs) present an attractive opportunity to combine the additive and potentially synergistic effects exhibited by combinations of monoclonal antibodies (mAbs). Current challenges for engineering bsAbs include retention of the binding affinity of the parent mAb or antibody fragment, the ability to bind both targets simultaneously, and matching valency with biology. Other factors to consider include structural stability and expression of the recombinant molecule, both of which may have significant impact on its development as a therapeutic. Here, we incorporate selection of stable, potent single-chain variable fragments (scFvs) early in the engineering process to assemble bsAbs for therapeutic applications targeting the cytokines IL-17A/A and IL-23. Stable scFvs directed against human cytokines IL-23p19 and IL-17A/A were isolated from a human Fab phage display library via batch conversion of panning output from Fabs to scFvs. This strategy integrated a step for shuffling V regions during the conversion and permitted the rescue of scFv molecules in both the V(H)V(L) and the V(L)V(H) orientations. Stable scFvs were identified and assembled into several bispecific formats as fusions to the Fc domain of human IgG1. The engineered bsAbs are potent neutralizers of the biological activity of both cytokines (IC(50) < 1 nM), demonstrate the ability to bind both target ligands simultaneously and display stability and productivity advantageous for successful manufacture of a therapeutic molecule. Pharmacokinetic analysis of the bsAbs in mice revealed serum half-lives similar to human mAbs. Assembly of bispecific molecules using stable antibody fragments offers an alternative to reformatting mAbs and minimizes subsequent structure-related and manufacturing concerns.
Protein Engineering Design and Selection 12/2009; 23(3):115-27. DOI:10.1093/protein/gzp073 · 2.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Binding of immune complexes to cellular FcgammaRs can promote cell activation and inflammation. In previous studies, a recombinant human (rh) soluble FcgammaR, rh-FcgammaRIA (CD64A), was shown to block inflammation in passive transfer models of immune complex-mediated disease. To assess whether rh-FcgammaRIA could block inflammation in a T cell- and B cell-dependent model of immune complex-mediated disease, the efficacy of rh-FcgammaRIA in collagen-induced arthritis was evaluated. Mice with established arthritis were treated with a single s.c. injection of rh-FcgammaRIA (0.2-2.0 mg/dose) given every other day for 11 days. Relative to mice injected with vehicle alone, mice treated with rh-FcgammaRIA exhibited lower serum concentrations of IL-6, anti-type II collagen Abs, and total IgG2a. These changes were correlated with lower levels of paw swelling and joint damage in the rh-FcgammaRIA-treated mice and occurred in the presence of a significant murine Ab response to rh-FcgammaRIA. Comparison of the serum rh-FcgammaRIA concentration vs time profiles for rh-FcgammaRIA administered at two dose levels by i.v. and s.c. injection revealed that the bioavailabilty of s.c. administered rh-FcgammaRIA was 27-37%. Taken together, these data show that rh-FcgammaRIA is an effective inhibitor of inflammation in a model of established arthritis in mice.
The Journal of Immunology 07/2009; 182(11):7272-9. DOI:10.4049/jimmunol.0803497 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Binding of Ag-Ab immune complexes to cellular FcgammaR promotes cell activation, release of inflammatory mediators, and tissue destruction characteristic of autoimmune disease. To evaluate whether a soluble FcgammaR could block the proinflammatory effects of immune complexes, recombinant human (rh) versions of FcgammaRIA, FcgammaRIIA, and FcgammaRIIIA were prepared. Binding of rh-FcgammaRIA to IgG was of high affinity (KD=1.7x10(-10) M), whereas rh-FcgammaRIIA and rh-FcgammaRIIIA bound with low affinity (KD=0.6-1.9x10(-6) M). All rh-FcgammaR reduced immune complex precipitation, blocked complement-mediated lysis of Ab-sensitized RBC, and inhibited immune complex-mediated production of IL-6, IL-13, MCP-1, and TNF-alpha by cultured mast cells. Local or systemic delivery only of rh-FcgammaRIA, however, reduced edema and neutrophil infiltration in the cutaneous Arthus reaction in mice. 125I-labeled rh-FcgammaRIA was cleared from mouse blood with a rapid distribution phase followed by a slow elimination phase with a t1/2gamma of approximately 130 h. The highest percentage of injected radioactivity accumulated in blood approximately liver approximately carcass>kidney. s.c. dosing of rh-FcgammaRIA resulted in lower serum levels of inflammatory cytokines and prevented paw swelling and joint damage in a murine model of collagen Ab-induced arthritis. These data demonstrate that rh-FcgammaRIA is an effective inhibitor of type III hypersensitivity.
The Journal of Immunology 01/2008; 180(1):580-9. DOI:10.4049/jimmunol.180.1.580 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The proinflammatory cytokines IL-17A and IL-17F have a high degree of sequence similarity and share many biological properties. Both have been implicated as factors contributing to the progression of inflammatory and autoimmune diseases. Moreover, reagents that neutralize IL-17A significantly ameliorate disease severity in several mouse models of human disease. IL-17A mediates its effects through interaction with its cognate receptor, the IL-17 receptor (IL-17RA). We report here that the IL-17RA-related molecule, IL-17RC is the receptor for IL-17F. Notably, both IL-17A and IL-17F bind to IL-17RC with high affinity, leading us to suggest that a soluble form of this molecule may serve as an effective therapeutic antagonist of IL-17A and IL-17F. We generated a soluble form of IL-17RC and demonstrate that it effectively blocks binding of both IL-17A and IL-17F, and that it inhibits signaling in response to these cytokines. Collectively, our work indicates that IL-17RC functions as a receptor for both IL-17A and IL-17F and that a soluble version of this protein should be an effective antagonist of IL-17A and IL-17F mediated inflammatory diseases.
The Journal of Immunology 11/2007; 179(8):5462-73. DOI:10.4049/jimmunol.179.8.5462 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Corticotroph-derived glycoprotein hormone (CGH), also referred to as thyrostimulin, is a noncovalent heterodimer of glycoprotein hormone alpha 2 (GPHA2) and glycoprotein hormone beta 5 (GPHB5). Here, we demonstrate that both subunits of CGH are expressed in the corticotroph cells of the human anterior pituitary, as well as in skin, retina, and testis. CGH activates the TSH receptor (TSHR); (125)I-CGH binding to cells expressing TSHR is saturable, specific, and of high affinity. In competition studies, unlabeled CGH is a potent competitor for (125)I-TSH binding, whereas unlabeled TSH does not compete for (125)I-CGH binding. Binding and competition analyses are consistent with the presence of two binding sites on the TSHR transfected baby hamster kidney cells, one that can interact with either TSH or CGH, and another that binds CGH alone. Transgenic overexpression of GPHB5 in mice produces elevations in serum T(4) levels, reductions in body weight, and proptosis. However, neither transgenic overexpression of GPHA2 nor deletion of GPHB5 produces an overt phenotype in mice. In vivo administration of CGH to mice produces a dose-dependent hyperthyroid phenotype including elevation of T(4) and hypertrophy of cells within the inner adrenal cortex. However, the distinctive expression patterns and binding characteristics of CGH suggest that it has endogenous biological roles that are discrete from those of TSH.