Humanization Of Murine Antibodies Research Articles

Isolation of A Human GM-CSF Antibody with Potent Neutralizing Activity from a Subject with Idiopathic Pulmonary Alveolar Proteinosis (96.24)

Abstract Background. GM-CSF has been implicated in the development of autoimmune disorders. The efficacy of neutralizing anti-GM-CSF has been demonstrated in several animal disease models rheumatoid arthritis, asthma and lung inflammation. While humanization of murine antibodies (Abs) and phage-display technology have generated potential therapeutic leads currently in development, these engineered leads face potential challenge of serum stability and immunoreactivity. We have isolated a fully human anti-GM-CSF from a patient of idiopathic pulmonary alveolar proteinosis (iPAP) who produces anti-GM-CSF as autoantibody. Method. To isolate natural human Abs, we used a short term memory B cell culture system to generate Ab-containing supernatants and screened for anti-GM-CSF that inhibited the proliferation of TF-1 cells in response to human GM-CSF. The specificity for GM-CSF was then confirmed via secondary binding screening in Alphascreen assays. Recombinant human Abs were isolated from the corresponding memory B cells using RT-PCR and subcloning. Results. By analyzing the serum from several iPAP patients for the neutralizing activity against TF-1 proliferation, we selected one peripheral blood sample for Ab isolation. TCN-350 was isolated with IC50 of 27 pM for TF-1 inhibition. It binds human GM-CSF with an affinity of 39 pM and cross-reacts with rabbit GM-CSF with an affinity of 1 nM. The high neutralization potency of TCN-350 warrants further evaluation as a therapeutic lead.

Transgenèse animale et humanisation des anticorps

The properties of monoclonal antibodies explain why they are such a successful class of therapeutic molecules. However, pionneered initial antibodies were of murine origin and triggered an immune response which limited the therapeutic potential of the antibody and generated deleterious effects. Consequently, tremendous efforts have been developped to engineer these murine Ig by introducing human sequences in vitro, or in vivo by humanization of murine antibodies, leading to chimeric immunoglobulins, and more recently generation of fully human antibodies in transgenic mice with a more or less diversified V repertoire. These approaches have led to the development of an increasing number of these chimeric or humanized monoclonal antibodies entering pharmaceutical pipelines.

Thermodynamic Consequences of Mutations in Vernier Zone Residues of a Humanized Anti-human Epidermal Growth Factor Receptor Murine Antibody, 528

To investigate the role of Vernier zone residues, which are comprised in the framework regions and underlie the complementarity-determining regions (CDRs) of antibodies, in the specific, high affinity interactions of antibodies with their targets, we focused on the variable domain fragment of murine anti-human epidermal growth factor receptor antibody 528 (m528Fv). Grafting of the CDRs of m528Fv onto a selected framework region of human antibodies, referred to as humanization, reduced the antibody's affinity for its target by a factor of 1/40. The reduction in affinity was due to a substantial reduction in the negative enthalpy change associated with binding. Crystal structures of the ligand-free antibody fragments showed no noteworthy conformational changes due to humanization, and the loop structures of the CDRs of the humanized antibodies were identical to those of the parent antibodies. Several mutants of the CDR-grafted (humanized) variable domain fragment (h528Fv), in which some of the Vernier zone residues in the heavy chain were replaced with the parental murine residues, were constructed and prepared using a bacterial expression system. Thermodynamic analyses of the interactions between the mutants and the soluble extracellular domain of epidermal growth factor receptor showed that several single mutations and a double mutation increased the negative enthalpy and heat capacity changes. Combination of these mutations, however, led to somewhat reduced negative enthalpy and heat capacity changes. The affinity of each mutant for the target was within the range for the wild-type h528Fv, and this similarity was due to enthalpy-entropy compensation. These results suggest that Vernier zone residues make enthalpic contributions to antigen binding and that the regulation of conformational entropy changes upon humanization of murine antibodies must be carefully considered and optimized.

Humanization of a mouse monoclonal antibody neutralizing TNF-α by guided selection

With the advent of phage display antibody libraries, humanization of murine antibodies can be achieved by epitope guided selection. In present study, guided selection was applied to the humanization of the mouse mAb Z8 that is directed to human TNF-α and can neutralize the cytotoxicity of TNF-α. First, the Z8 Fd gene was paired as a template with a repertoire of human κ chains, and displayed on the filamentous phage, forming a hybrid phage antibody library. Selected by four rounds of panning against TNF-α, hybrid antibody fragments that bound to TNF-α and contained human κ chains were obtained. Meanwhile human Fd genes were selected by pairing the human Fd repertoire with the Z8 κ chain and performing the same procedure of panning. One of the isolated human Fd genes (huFd2), which showed the strongest reactivity, was chosen to pair with 12 of selected human κ chains. Two of the resulting human Fabs (huFd2-huκ1 and huFd2-huκ2), with same Fd and different κ chains, bound to TNF-α specifically. Their human origin was proved by ELISA and sequencing analysis. The human Fabs competitive ELISA and in vitro TNF-α neutralization assay demonstrated that the human Fabs resembled its parental mouse mAb Z8 in that they both recognized the same epitope and neutralized the cytotoxicity of TNF-α. These results suggest that guided selection is a promising strategy in murine mAb humanization.