Engineering of Chimeric Proteins to Enhance Immunogenicity for the Production of High-Affinity Specific Monoclonal Antibodies

Abstract

A wide range of biomedical research and diagnostic applications utilize high-affinity antibodies, the production of which depends on effective immunogens. However, some proteins are intrinsically weak immunogens incapable of eliciting a strong immune response for the production of useful antibodies. To remedy this problem, weak immunogens are conjugated to exogenous carrier proteins to increase immunogenicity and provide an adjuvant effect. In the present study, we tested an alternative strategy of using chimeric fusion protein constructs to enhance immunogenicity. We first applied the N-terminal domain of glucose-regulated protein 96 (GRP-NTD), a protein with newly identified activities in autoimmune diseases, as a carrier to present several experimentally identified weak immunogens: cardiac troponin C (cTnC), the N-terminal peptide of cardiac troponin T (N69), and the C-terminal peptide of calponin 2 (C85). Antigen peptides were engineered to the N-terminus of the carrier, expressed in E. coli, large scale purified, and injected intramuscularly into adult mice without adjuvants. The effects of immunization were assessed by ELISA titration of serum samples after three immunizations and/or the outcome of high-affinity specific monoclonal antibodies (mAb) in hybridoma development. The results demonstrate significantly enhanced immunogenicity for cTnC and N69, but not C85. Based on an observation that cloned mouse ApoE protein served as an effective carrier for the production of mAb against a small peptide, we constructed a C85-ApoE fusion protein for hybridoma development. Surprisingly, C85-ApoE immunization was not effective for C85 but yielded high-affinity mAbs against mouse ApoE, an endogenous protein abundant in host plasma. The results suggest a potentially new strategy of using chimeric immunogens to improve the production of specific antibodies.