Anti-IgG1 monoclonal antibody, clone L48 3H7

Therapeutic monoclonal antibodies (mAbs) have high efficacy in treating TNF alpha-related immunological diseases. Other than neutralizing TNF alpha, these IgG1 antibodies exert Fc receptor-mediated effector functions such as the complement-dependent cytotoxicity (CDC) and antibody-dependent cell cytotoxicity (ADCC). The crystallizable fragment (Fc) of these IgG1 contains a single glycosylation site at Asn 297/300 that is essential for the CDC and ADCC. Glycosylated antibodies lacking core fucosylation showed an improved ADCC. However, no structural data are available concerning the ligand-binding interaction of these mAbs used in TNF alpha-related diseases and the role of the fucosylation. We therefore used comparative modeling for generating complete 3D mAb models that include the antigen-binding fragment (Fab) portions of infliximab, complexed with TNFa (4G3Y. pdb), the Fc region of the human IGHG1 fucosylated (3SGJ) and afucosylated (3SGK) complexed with the Fc receptor subtype Fc gamma RIIIA, and the Fc region of a murine immunoglobulin (1IGT). After few thousand steps of energy minimization on the resulting 3D mAb models, minimized final models were used to quantify interactions occurring between FccRIIIA and the fucosylated/afucosylated Fc fragments. While fucosylation does not affect Fab-TNF alpha interactions, we found that in the absence of fucosylation the Fc-mAb domain and Fc gamma RIIIA are closer and new strong interactions are established between G129 of the receptor and S301 of the Chimera 2 Fc-mAb; new polar interactions are also established between the Chimera 2 Fc residues Y299, N300, and S301 and the Fc gamma RIIIA residues K128, G129, R130, and R155. These data help to explain the reduced ADCC observed in the fucosylated mAbs suggesting the specific AA residues involved in binding interactions.

Background B-cell chronic lymphocytic leukemia (CLL) is a heterogeneous disease and the most common adult leukemia in western countries. IgVH mutational status distinguishes two major types of CLL, each associated with a different prognosis and survival. Sequencing identified NOTCH1 and SF3B1 as the two main recurrent mutations. We described a novel method to clarify how these mutations affect gene expression by finding small-scale signatures that predict the IgVH, NOTCH1 and SF3B1 mutations. We subsequently defined the biological pathways and correlation networks involved in disease development, with the potential goal of identifying new drugable targets. Methods We modeled a microarray dataset consisting of 48807 probes derived from 163 samples. The use of Fisher's ratio and fold change combined with feature elimination allowed us to identify the minimum number of genes with the highest predictive mutation power and, subsequently, we applied network and pathway analyses of these genes to identify their biological roles. Results The mutational status of the patients was accurately predicted (94-99%) using small-scale gene signatures: 13 genes for IgVH, 60 for NOTCH1 and 22 for SF3B1. LPL plays an important role in the case of the IgVH mutation, whereas MSI2, LTK, TFEC and CNTAP2 are involved in the NOTCH1 mutation, and RPL32 and PLAGL1 are involved in the SF3B1 mutation. Four high discriminatory genes (IGHG1, MYBL1, NRIP1 and RGS1) are common to these three mutations. The IL-4-mediated signaling events pathway appears to be involved as a common mechanism and suggests an important role of the immune response mechanisms and antigen presentation. Conclusions This retrospective analysis served to provide a deeper understanding of the effects of the different mutations in CLL disease progression, with the expectation that these findings will be clinically applied in the near future to the development of new drugs.