Combinatorial libraries of rearranged hypervariable V(H) and V(L) sequences from nonimmunized human donors contain antigen specificities, including anti-self reactivities, created by random pairing of V(H)s and V(L)s. Somatic hypermutation of immunoglobulin genes, however, is critical in the generation of high-affinity antibodies in vivo and occurs only after immunization. Thus, in combinatorial phage display libraries from nonimmunized donors, high-affinity antibodies are rarely found. Lengthy in vitro affinity maturation is often needed to improve antibodies from such libraries. We report the construction of human Fab libraries having a unique combination of immunoglobulin sequences captured from human donors and synthetic diversity in key antigen contact sites in heavy-chain complementarity-determining regions 1 and 2. The success of this strategy is demonstrated by identifying many monovalent Fabs against multiple therapeutic targets that show higher affinities than approved therapeutic antibodies. This very often circumvents the need for affinity maturation, accelerating discovery of antibody drug candidates.

A human anti-IL-2 antibody that potentiates regulatory T cells by a structure-based mechanism . Previous studies showed that IL-2 is highly flexible 8,9 and exists in different conformations that favor either the high-affinity trimeric IL-2R or intermediate-affinity dimeric IL-2R, resulting in the activation of different immune cells 9 . This plasticity has complicated the use of the approved drug Proleukin at high doses to treat metastatic melanoma and renal cell carcinoma 10 , due to the role of IL-2 as an essential growth factor for T regs [11][12][13] . Moreover, adverse effects of high-dose IL-2 therapy have greatly limited its use 14,15 . Several studies have shown that low-dose IL-2 therapy preferentially activates T regs due to the constitutive high expression of IL-2Rα 16 and other cell-intrinsic factors that increase signal transduction sensitivity 17 . Treatment of mice and humans with low doses of IL-2 has been shown to ameliorate autoimmune diseases and graft-versus-host disease (GvHD) as well as delaying organ allograft rejection [18][19][20][21][22] . However, IL-2 therapy has some limitations, including difficulty in predicting the efficacious dose, off-target effects on different cell populations and a short in vivo half-life 23,24 . Thus, attempts have been made to engineer or modify the IL-2 structure to improve its therapeutic potential by modulating its ability to selectively target either T effs or T regs [25][26][27][28][29][30][31] . Selective antibodies against IL-2 can alter its conformation by binding a number of potential epitopes, thereby modifying the binding interaction of IL-2 to any of the IL-2R subunits and resulting in selective expansion of T regs or T eff cell subsets 32,33 . For example, it has been demonstrated that a rat anti-mouse IL-2 monoclonal antibody (JES6-1) can be administered in complex with wild-type mouse IL-2 and used to preferentially enhance T reg populations 26 . Binding of JES6-1 to IL-2 alters its conformation to lower the affinity of mIL-2 for CD25, such that CD25 high T regs compete favorably for IL-2 binding and expansion against T effs 33 . The therapeutic potential of IL-2 to selectively activate the tolerogenic immune response, combined with the imperative to develop a human T reg -selective IL-2 compound, led us to develop a mechanism-based screening strategy to identify human antibodies against human IL-2 that exhibit an in vivo T reg potentiation profile when complexed with hIL-2. This class of monoclonal antibody, exemplified by F5111.2, blocked IL-2Rβ binding and reduced IL-2Rα binding to IL-2, and, when administered in complex with hIL-2, preferentially promoted T reg expansion and was effective in models of autoimmune disease including type 1 diabetes and experimental autoimmune encephalomyelitis (EAE) in addition to GvHD. ResultsSelective IL-2 stimulation in T regs . To directly compare T reg and T eff sensitivity to IL-2, the pSTAT5 signaling response of T regs was analyzed in a mixed population of peripheral blood mononuclear cells (PBMCs).

Intracellular antibodies (intrabodies) represent a new class of neutralizing molecules with a potential use in gene therapy. Intrabodies are engineered single-chain antibodies in which the variable domain of the heavy chain is joined to the variable domain of the light chain through a peptide linker, preserving the affinity of the parent antibody. Intrabodies are expressed inside cells and directed to different subcellular compartments where they can exert their function more effectively. The effects of intrabodies have been investigated using structural, regulatory, and enzymatic proteins of the human immunodeficiency virus (HIV-1) as targets. These intrabodies have demonstrated their versatility by controlling early as well as late events of the viral life cycle. In this article, we review studies of the use of intrabodies as research tools and therapeutic agents against HIV-1.

Thrombosis is a major cause of morbidity and mortality. Current antithrombotic drugs are not ideal in that they must balance prevention of thrombosis against bleeding risk. Inhibition of coagulation factor XI (FXI) may offer an improvement over existing antithrombotic strategies by preventing some forms of thrombosis with lower bleeding risk. To permit exploration of this hypothesis in humans, we generated and characterized a series of human immunoglobulin Gs (IgGs) that blocked FXIa active-site function but did not bind FXI zymogen or other coagulation proteases. The most potent of these IgGs, C24 and DEF, inhibited clotting in whole human blood and prevented FeCl3-induced carotid artery occlusion in FXI-deficient mice reconstituted with human FXI and in thread-induced venous thrombosis in rabbits at clinically relevant doses. At doses substantially higher than those required for inhibition of intravascular thrombus formation in these models, DEF did not increase cuticle bleeding in rabbits or cause spontaneous bleeding in macaques over a 2-week study. Anticipating the desirability of a reversal agent, we also generated a human IgG that rapidly reversed DEF activity ex vivo in human plasma and in vivo in rabbits. Thus, an active site-directed FXIa-specific antibody can block thrombosis in animal models and, together with the reversal agent, may facilitate exploration of the roles of FXIa in human disease.