Combinatorial libraries against libraries for selecting neoepitope activation-specific antibodies

Abstract

A systematic approach to the discovery of conformation-specific antibodies or those that recognize activation-induced neoepitopes in signaling molecules and enzymes will be a powerful tool in developing antibodies for basic science and therapy. Here, we report the isolation of antibody antagonists that preferentially bind activated integrin Mac-1 (α M β 2 ) and are potent in blocking neutrophil adhesion and migration. A novel strategy was developed for this task, consisting of yeast surface display of Mac-1 inserted (I) domain library, directed evolution to isolate active mutants of the I domain, and screening of phage display of human antibody library against the active I domain in yeast. Enriched antibody library was then introduced into yeast surface two-hybrid system for final quantitative selection of antibodies from monomeric antigenantibody interaction. This led to highly efficient isolation of intermediate to high affinity antibodies, which preferentially reacted with the active I domain, antagonized the I domain binding to intercellular adhesion molecule (ICAM)-1, complement C3 fragment iC3b, and fibronectin, and potently inhibited neutrophil migration on fibrinogen. The strategy demonstrated herein can be broadly applicable to developing antibodies against modular domains that switch between inactive and active conformations, particularly toward the discovery of antibody antagonists in therapeutic and diagnostic applications.antibody antagonist | mac-1 integrin | phage display | yeast display

Cross-domain inhibition of TACE ectodomain

“…As solution-phase phage display typically produces antibodies with conformational (i.e., nonlinear) epitopes, this technology is also theoretically capable of providing the intricate macromolecular cross-domain binding desirable in an ADAM inhibitor. Recent technical advances in phage display have produced antibodies recognizing multiple disparate antigens (21) and different conformations of the same antigen (22,23). These approaches use a combination of calculated selection conditions and/or antibody variable-domain chain-shuffling to produce antibodies against previously unique epitopes.…”

Tunable physiologic interactions of adhesion molecules for inflamed cell-selective drug delivery

“…A number of mutations have been isolated to increase their binding affinity to ICAM-1, mimicking activation signals transmitted to the I domain by the neighboring domains in full-length integrins [28,35]. To test the specificities of LFA-1 and Mac-1 I domains, they were expressed on yeast cell surface in their wild-type (WT) or with activating mutations (F265S/F292G for LFA-1 [28] and F302L for Mac-1 [35]). Yeast cells expressing the WT of LFA-1 or Mac-1 I domains failed to bind strongly to HMEC-1 cells even after LPS-induced ICAM-1 expression (Fig.…”

“…Some of the I domains were conjugated to Alexa Fluor 488 (AF488, Invitrogen) according to the instruction of the vendor. For GFP-Id-HA fusion protein, eGFP (Val2 to Lys239) [35] was first inserted into pET28a vector between NheI and BamHI and then Id-HA (Asn129 to Tyr307) followed by a stop codon was placed between BamHI and XhoI. GFP-Id-HA was produced from soluble fractions in BL21 cells.…”

“…For confocal microscopy (Leica TCS SP2), HeLa cells were labeled with 10 mg/ml of mAb LB-2 or GFP-Id-HA in 200 ml of labeling buffer on ice for 30 min. Mammalian cell surface binding of yeast cells displaying various I domains was performed as previously described [35]. In short, yeast cells expressing WT or activated mutants of LFA-1 and Mac-1 I domains were washed and resuspended in washing buffer (PBS (pH 7.4), 0.5% BSA, 10 mM MgCl 2 ).…”

Recombinant renewable polyclonal antibodies

“…These include antibodies recognizing one protein, but not a closely related one, 16 spliced variants, 17,18 specific epitopes, 19 or active conformations. [20][21][22] Furthermore, affinities or specificities can be improved by in vitro evolution, 23,24 and selected antibodies can be fused to additional functional elements, such as enzymes or antibody constant regions. Here, we describe the use of display methods to generate recombinant renewable polyclonal antibodies, as a valid alternative to the non-renewable polyclonal antibody products presently on the market.…”