Making chemistry selectable by linking it to infectivity

Abstract

The link between recognition and replication is fundamental to the operation of the immune system. In recent years, modeling this process in a format of phagedisplay combinatorial libraries has afforded a powerful tool for obtaining valuable antibodies. However, the ability to readily select and isolate rare catalysts would expand the scope of library technology. A technique in which phage infection controlled the link between recognition and replication was applied to show that chemistry is a selectable process. An antibody that operated by covalent catalysis to form an acyl intermediate restored phage infectivity and allowed selection from a library in which the catalyst constituted 1 in 10 5 members. Three different selection approaches were examined for their convenience and generality. Incorporating these protocols together with well known affinity labels and mechanism-based inactivators should allow the procurement of a wide range of novel catalytic antibodies.Much of the work of the biochemical world is accomplished as a result of protein-ligand binding. In the attempt to mimic proteins found in Nature, selection methods from large libraries of molecules have been extremely valuable. In this regard, the phage-display format is particularly attractive in that it duplicates in vitro the essence of the in vivo immune response by linking the fundamental processes of recognition and replication (1-5). Yet, in both of these in vitro and in vivo selection systems, the recognition event that drives replication is based on noncovalent interactions, wherein the outcome is selection based on binding rather than chemistry. When searching for antibody-antigen interactions, the precise utilization of binding energy is unimportant so long as it is sufficient to confer the function of immunological recognition. In contrast, the selection for function is especially relevant for procuring new enzymes, where a substrate must undergo a chemical transformation upon binding.We recently developed several paradigms for the selection of catalytic antibodies on the basis of chemical reactivity. The direct selection from combinatorial libraries (6, 7) and reactive immunization (8) both afforded a subpopulation of antibodies in which chemistry was installed in the combining site. While investigation of these methods will continue to realize their full potential, the underlying concept could be refined in a design that establishes a more intimate link between chemistry and the replication process. The utilization of phage that are selectively infective (9-11) provides a means to achieve enrichment of antibody catalysts, since selection could be strictly governed by chemistry. In this way, antibodies that carry out a chemical reaction can be identified, isolated, and replicated because the chemical event distinguishes the phage-bearing antibodies from the rest of the population. Herein, we describe the implementation of this approach to select for an antibody that operates by means of covalent catalysis. MATERIALS AND METHODSS...

Factor VIII Hydrolysis Mediated by Anti-Factor VIII Autoantibodies in Acquired Hemophilia

“…Seminal works in the field of Ab catalysis have shown that Abs with germline configuration are endowed with catalytic functions as diverse as oxy-cope rearrangement (34), Diels-Alder reaction (35,36), or peptide hydrolysis (37,38), thus demonstrating that the catalytic potential of immunoglobulins is encoded in the genome. The observation of amidase activity performed by circulating IgG, IgM, and IgA from healthy donors (39,40), and our finding of baseline levels of FVIII hydrolysis by normal immunoglobulins, indicates that the repertoire of immunoglobulins endowed with catalytic functions is expressed under physiological conditions.…”

“…IVIg exhibited a hydrolytic activity of 55.6 Ϯ 25.9 mol/min/mol toward FVIII, a value statistically indistinguishable from the mean rate of FVIII hydrolysis measured in the case of IgG from nine healthy blood donors (54.9 Ϯ 22.5 mol/min/mol, data not shown). Purified IgG from 21 of the 45 patients (46.6%) included in the study (i.e., patients 1,2,5,8,10,13,14,15,16,17,21,22,23,29,32,35,36,37,41,43, and 45) exhibited a FVIIIhydrolyzing activity that was significantly higher than that of IVIg (Table II, We have previously documented that FVIII-hydrolyzing IgG behave as serine proteases in the absence of contamination by adventitious proteases (8). In this study, the absence of contaminating proteases was confirmed by MALDI-TOF analysis of trypsin digests of the IgG preparations (data not shown).…”

Making artificial antibodies: A format for phage display of combinatorial heterodimeric arrays

“…Two murine catalytic antibodies, 21H3 and 2H6 (15)(16)(17)(18), and one murine anti-cocaine antibody, 92H2 (generated by our laboratory), were used to construct V H -(Gly 4 Ser)-pVII and V L -(Gly 4 Ser)-pIX fusion proteins. The V H sequences in each case were fused to the N terminus of pVII, and the V L sequences were fused to the N terminus of pIX.…”

“…The PCR products were digested by restriction enzymes NcoI and NheI and inserted into the phagemid vector pCGMT (15).…”

“…The microtiter plates (Corning) were coated with 25 l per well of 10 g͞ml mouse anti-Flag antibody to assay Flag constructs with pVII͞pIX, PCP-BSA to assay 21H3 and 2H6 constructs, and the cocaine conjugate GNC-BSA (21) to assay the 92H2 construct. The phage ELISA was performed as described (15,22).…”