Abstract
Molecularly engineered antibodies with fit-for-purpose properties will differentiate next generation antibody therapeutics from traditional IgG1 scaffolds. One requirement for engineering the most appropriate properties for a particular therapeutic area is an understanding of the intricacies of the target microenvironment in which the antibody is expected to function. Our group and others have demonstrated that proteases secreted by invasive tumors and pathological microorganisms are capable of cleaving human IgG1, the most commonly adopted isotype among monoclonal antibody therapeutics. Specific cleavage in the lower hinge of IgG1 results in a loss of Fc-mediated cell-killing functions without a concomitant loss of antigen binding capability or circulating antibody half-life. Proteolytic cleavage in the hinge region by tumor-associated or microbial proteases is postulated as a means of evading host immune responses, and antibodies engineered with potent cell-killing functions that are also resistant to hinge proteolysis are of interest. Mutation of the lower hinge region of an IgG1 resulted in protease resistance but also resulted in a profound loss of Fc-mediated cell-killing functions. In the present study, we demonstrate that specific mutations of the CH2 domain in conjunction with lower hinge mutations can restore and sometimes enhance cell-killing functions while still retaining protease resistance. By identifying mutations that can restore either complement- or Fcγ receptor-mediated functions on a protease-resistant scaffold, we were able to generate a novel protease-resistant platform with selective cell-killing functionality.
Highlights
Proteases can cleave human IgG1 antibodies, resulting in loss of cell-killing functions
Proteolytic cleavage in the hinge region by tumor-associated or microbial proteases is postulated as a means of evading host immune responses, and antibodies engineered with potent cell-killing functions that are resistant to hinge proteolysis are of interest
The anti-hinge antibodies were used for immunohistochemical detection of cleaved IgGs in human head and neck squamous cell carcinoma (HNSCC)
Summary
Proteases can cleave human IgG1 antibodies, resulting in loss of cell-killing functions. Mutation of the lower hinge region of an IgG1 resulted in protease resistance and resulted in a profound loss of Fc-mediated cell-killing functions. The singly cleaved intermediate retains antigen binding capabilities as well as the long circulating half-life of the intact IgG1 counterpart [8] For these reasons, our group and others have hypothesized that antibody cleavage by tumor-associated and microbial proteases can potentially function as an immune evasion mechanism We demonstrate that mutation of the lower hinge of IgG1 confers protease resistance and results in the loss of Fc-mediated cell-killing functions. We show that specific mutations incorporated into the CH2 region of engineered mAbs with a protease-resistant lower hinge cannot only restore functional activities to IgG1 but in some cases substantially enhance Fc effector functions. We show that cleaved IgGs are detected within the tumor microenvironment of human head and neck squamous cell carcinoma, highlighting the need for a protease-resistant platform for diseases characterized by the presence of IgG-cleaving proteases
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