Human Anti-IgG1 Hinge Autoantibodies Reconstitute the Effector Functions of Proteolytically Inactivated IgGs

Abstract

A number of proteases of potential importance to human physiology possess the ability to selectively degrade and inactivate Igs. Proteolytic cleavage within and near the hinge domain of human IgG1 yielded products including Fab and F(ab′)2 possessing full Ag binding capability but absent several functions needed for immune destruction of cellular pathogens. In parallel experiments, we showed that the same proteolytically generated Fabs and F(ab′)2s become self-Ags that were widely recognized by autoantibodies in the human population. Binding analyses using various Fab and F(ab′)2, as well as single-chain peptide analogues, indicated that the autoantibodies targeted the newly exposed sequences where proteases cleave the hinge. The point of cleavage may be less of a determinant for autoantibody binding than the exposure of an otherwise cryptic stretch of hinge sequence. It was noted that the autoantibodies possessed an unusually high proportion of the IgG3 isotype in contrast to Abs induced against foreign immunogens in the same human subjects. In light of the recognized potency of IgG3 effector mechanisms, we adopted a functional approach to determine whether human anti-hinge (HAH) autoantibodies could reconstitute the (missing) Fc region effector functions to Fab and F(ab′)2. Indeed, in in vitro cellular assays, purified HAH autoantibodies restored effector functions to F(ab′)2 in both Ab-dependent cellular cytotoxicity and complement-dependent cytotoxicity assays. The results indicate that HAH autoantibodies selectively bind to proteolytically cleaved IgGs and can thereby provide a surrogate Fc domain to reconstitute cell lytic functions.

Proteinase-nicked IgGs: an unanticipated target for tumor immunotherapy

“…However, when tested over time, these reactivity levels were relatively constant at least a year (REJ, unpublished observations). 14 In any case, the results consistently revealed a large disparity between average low levels of AHA binding to the papain cleavage site (threonine 224, EU numbering) and higher reactivity to the pepsin site (leucine 234). These results suggest a connection to the previously described disparity of platelet clearance.…”

“…(a) Several alternatives have been shown to restore function to disabled scIgGs by secondary binding of intact anti-hinge antibodies. The sources of the anti-hinge Abs have been AHAs isolated from human serum, 14 a monoclonal anti-hinge mAb, 48 immunization-derived immune responses to peptide analogs of the cleaved hinge, 47 and proteinase-resistant versions of a monoclonal anti-hinge mAb. 53 The anti-hinge antibodies bind to the cleaved primary antibodies on the cell surface to provide an intact hinge and Fc that are capable of restoring ADCC and complement against the cancer cells.…”

“…The peptides each possess a respective free C-terminus that largely defines the neoepitopes exposed in the IgG1 hinge. 14 (c) The sequence of the IgG1 hinge and the sites of proteinolytic cleavage determined for selected proteinolytic enzymes. 31 …”

“…These peptides were presented outwardly as if in a cell-bound IgG fragment (Figure 1(b)). 14 Test sample sources were typically small pools of donor sera or purified intravenous gamma globulin preparations isolated from thousands of donors (Figure 1(b)). When individual donor sera were assessed, AHA ELISA reactivity levels were highly variable.…”

Engineered Protease-resistant Antibodies with Selectable Cell-killing Functions

“…ADCC-The ADCC assays were performed as described previously with several modifications (42). ADCC assays were performed with increasing anti-CD20 IgG1 antibody variant concentrations.…”