Several publications and patent documents are referenced in this application in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications is incorporated by reference herein.
Several research groups have reported that immunization with negatively charged transition state analogs (TSAs) results in the synthesis of antibodies (Abs) with esterase activity (1,2). These attempts to prepare transacylase Abs relied on creating catalytic sites de novo over the course of somatic diversification of antibody (Ab) genes. In this strategy, induction of noncovalent shape complementary between Ab combining sites and a negatively charged oxygen atom in tetrahedral TSAs was proposed to be sufficient to achieve catalytic activity (Table 1). However, there are no examples of proteolytic Abs identified by this strategy in the peer-reviewed literature, although patents claiming peptide bond hydrolysis by Abs raised to negatively charged TSAs have been granted (e.g., U.S. Pat. No. 5,952,462). One report describes a side-by-side examination of esterase and proteolytic activities in antibodies raised to a TSA (3). The former activity was readily detected, but no peptide bond cleaving activity was observed. The failure to prepare proteolytic Abs by this approach is generally attributed to the greater energetic demands of peptide bond hydrolysis and the more complex reaction pathways for this reaction, including formation of multiple transition states in which the catalyst must form transient covalent complexes with the peptide substrates for the reaction to proceed to completion.
A breakthrough has emerged from observations that naturally occurring Abs can express proteolytic activities. Observations that vasoactive intestinal peptide (VIP) is cleaved by Abs from asthma patients provided early evidence that Abs may possess peptidase activity (4). The generality of this observation is supported by additional reports showing cleavage of thyroglobulin by auto antibodies in Hashimoto's thyroiditis (5). Further evidence for the bias towards catalytic Ab synthesis in autoimmune disease is supported by observations of DNase activity in Abs from lupus patients (6) and mouse strains with a genetic predisposition to autoimmune disease (7). More recently antibodies isolated from certain hemophilia patients were observed to hydrolyze Factor VIII, a cofactor in blood coagulation (8). Certain antibody fragments to the HIV protein gp41 are also described to hydrolyze this protein (9).
Disclosed in the present invention are data indicating that the potential for cleaving peptide bonds by a covalent catalytic mechanisms is distributed broadly in most naturally occurring Abs. Covalent catalytic mechanisms reminiscent of those utilized by non-Ab serine proteases are a distinguishing feature of the naturally occurring proteolytic Abs (10). In comparison, Abs raised to TSAs utilize noncovalent binding of the transition state, and the emergence of covalent catalytic pathways is not predicted, expect by accident.
One aspect of the present invention is to strengthen the covalent reactivity of naturally occurring Abs. This results in two outcomes: (a) the increased covalent reactivity allows emergence of Abs that can form stable bonds with polypeptides, due to the covalent character of the bonding reaction; and (b) When a water molecule is properly accommodated in the Ab active sites, the covalent Ab-polypeptide complexes can be hydrolyzed to complete the reaction cycle. To favor the latter outcome, immunization is done using polypeptide analogs that contain a bound water molecule, allowing induction of Ab active sites with sufficient room to accommodate the desired water molecule.
Proteolytic Abs can not be identified using traditional binding assays, as the catalytic cleavage of polypeptides does not allow formation of stable Ab-antigen complexes. Analogs of antigens employed previously to identify catalytic Abs have assumed that the chemical reaction center in the analogs must simulate precisely the location of the bond in polypeptide antigens that is cleaved by catalytic Abs. Disclosed in the present invention are data that the covalently reactive groups in proteolytic Abs, the serine protease-like nucleophiles, enjoys considerable conformational flexibility relative to the noncovalent binding forces responsible for the specificity of Abs for individual polypeptide epitopes.
This discovery has resulted in another major aspect of the present invention, that is, the development of polypeptide analogs in which a covalently reactive electrophile can readily be located in side chains of the amino acids instead of the polypeptide backbone. Disclosed in this invention are methods using these analogs for coordination of the Ab nucleophilic reactivity with specificity for the linear and discontinuous epitopes expressed by polypeptides, allowing the occurrence of epitope-specific nucleophilic reactions between Abs and antigens. These methods remove an important bottle-neck in development of covalent and catalytic Abs, because preparation of such antibodies to large polypeptides is presently not possible by conventional methods. Synthesis of large polypeptides with electrophiles incorporated with the backbone is outside the scope of current chemical synthesis technology, whereas the electrophiles can readily be placed on the amino acid side chains by chemical conjugation without unduly disturbing the native antigenic structure of proteins. An alternative approach to preparing electrophilic polypeptides within the backbone is the utilization of unnatural electrophilic amino acid analogs for protein synthesis by natural synthetic procedures, for example by correct recognition of the electrophilic amino acid analog by the appropriate tRNA species during the translation of mRNA in the polyribosome complex.
The proteolytic activity of naturally occurring Abs is reported to derive heritable germline lines encoding serine protease-like nucleophilic sites (11). The first Abs made by B cells over the course of their differentiation into cells that synthesize specific Abs to individual antigenic epitopes belong to the IgM class, with class switching to IgG Abs occurring at a later stage, concomitant with ongoing somatic diversification of the Ab variable domains. Disclosed in the present invention are observations indicating that IgM Abs are superior catalysts compared to IgG Abs. Also disclosed are methods to identify and induce the synthesis of antigen-specific Abs of the IgM with proteolytic activity.