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Shortened engineered human antibody CH2 domains: increased stability and binding to the human neonatal Fc receptor.

ABSTRACT: The immunoglobulin (Ig) constant CH2 domain is critical for antibody effector functions. Isolated CH2 domains are promising scaffolds for construction of libraries containing diverse binders that could also confer some effector functions. We have shown previously that an isolated human CH2 domain is relatively unstable to thermally induced unfolding, but its stability can be improved by engineering an additional disulfide bond (Gong, R., Vu, B. K., Feng, Y., Prieto, D. A., Dyba, M. A., Walsh, J. D., Prabakaran, P., Veenstra, T. D., Tarasov, S. G., Ishima, R., and Dimitrov, D. S. (2009) J. Biol. Chem. 284, 14203-14210). We have hypothesized that the stability of this engineered antibody domain could be further increased by removing unstructured residues. To test our hypothesis, we removed the seven N-terminal residues that are in a random coil as suggested by our analysis of the isolated CH2 crystal structure and NMR data. The resulting shortened engineered CH2 (m01s) was highly soluble, monomeric, and remarkably stable, with a melting temperature (T(m)) of 82.6 °C, which is about 10 and 30 °C higher than those of the original stabilized CH2 (m01) and CH2, respectively. m01s and m01 were more resistant to protease digestion than CH2. A newly identified anti-CH2 antibody that recognizes a conformational epitope bound to m01s significantly better (>10-fold higher affinity) than to CH2 and slightly better than to m01. m01s bound to a recombinant soluble human neonatal Fc receptor at pH 6.0 more strongly than CH2. These data suggest that shortening the m01 N terminus significantly increases stability without disrupting its conformation and that our approach for increasing stability and decreasing size by removing unstructured regions may also apply to other proteins.

SUBMITTER: Gong R

PROVIDER: S-EPMC3149322 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Shortened engineered human antibody CH2 domains: increased stability and binding to the human neonatal Fc receptor.

Gong Rui R Wang Yanping Y Feng Yang Y Zhao Qi Q Dimitrov Dimiter S DS

The Journal of biological chemistry 20110613 31

The immunoglobulin (Ig) constant CH2 domain is critical for antibody effector functions. Isolated CH2 domains are promising scaffolds for construction of libraries containing diverse binders that could also confer some effector functions. We have shown previously that an isolated human CH2 domain is relatively unstable to thermally induced unfolding, but its stability can be improved by engineering an additional disulfide bond (Gong, R., Vu, B. K., Feng, Y., Prieto, D. A., Dyba, M. A., Walsh, J. ...[more]

PMID: 21669873

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Project description:BackgroundDuring infection with human cytomegalovirus (HCMV) several viral proteins occur on cell surfaces in high quantity. We thus pursue an HLA-independent approach for immunotherapy of HCMV using chimeric antigen receptors (CARs) and bispecific BiTE® antibody constructs. In this context, HCMV-encoded proteins that mediate viral immune evasion and bind human IgG might represent particularly attractive target antigens. Unlike in observations of similar approaches for HIV and hepatitis B and C viruses, however, HCMV-infected cells develop a striking resistance to cytotoxic effector functions at later stages of the replication cycle. In our study we therefore wanted to test two hypotheses: (1) CAR T cells can efficiently inhibit HCMV replication independently from cytotoxic effector functions, and (2) HCMV can be targeted by CH2-CH3 IgG spacer domains that contain mutations previously reported to prevent exhaustion and to rescue CAR T cell function in vivo.MethodsReplication of GFP-encoding recombinant HCMV in fibroblasts in the presence and absence of supernatants from T cell co-cultures plus/minus cytokine neutralizing antibodies was analyzed by flow cytometry. CARs with wild type and mutated CH2-CH3 domains were expressed in human T cells by mRNA electroporation, and the function of the CARs was assessed by quantifying T cell cytokine secretion.ResultsWe confirm and extend previous evidence of antiviral cytokine effects and demonstrate that CAR T cells strongly block HCMV replication in fibroblasts mainly by combined secretion of IFN-γ and TNF. Furthermore, we show that fibroblasts infected with HCMV strains AD169 and Towne starting from day 3 have a high capacity for binding of human IgG1 and also strongly activate T cells expressing a CAR with CH2-CH3 domain. Importantly, we further show that mutations in the CH2-CH3 domain of IgG1 and IgG4, which were previously reported to rescue CAR T cell function by abrogating interaction with endogenous Fc receptors (FcRs), still enable recognition of FcRs encoded by HCMV.ConclusionsOur findings identify HCMV-encoded FcRs as an attractive additional target for HCMV immunotherapy by CARs and possibly bispecific antibodies. The use of specifically mutated IgG domains that bind to HCMV-FcRs without recognizing endogenous FcRs may supersede screening for novel binders directed against individual HCMV-FcRs.

Dataset Information

Shortened engineered human antibody CH2 domains: increased stability and binding to the human neonatal Fc receptor.

Publications

Shortened engineered human antibody CH2 domains: increased stability and binding to the human neonatal Fc receptor.

Similar Datasets

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