A humanized monovalent CD3 antibody which can activate homologous complement

Abstract

The rat monoclonal antibody (mAb) YTH12.5, specific for the CD3 antigen complex on human T cells has been modified in order to improve its efficacy in human therapy. With the aim of rendering it less immunogenic, it has been humanized using the method of framework grafting. During this process sequence analysis of the YTH12.5 VL gene indicated that it was of the lambda subclass, however, it was markedly dissimilar from previously published rat and mouse V lambda gene sequences and may represent a new V lambda gene family. The humanization of this light chain represents the first successful reshaping of a lambda light chain V region. To improve the effector function of the antibody we have created a monovalent form (1 Fab, 1 Fc) using a novel method involving the introduction of an N-terminally truncated human IgG1 heavy chain gene into cells producing the humanized CD3 mAb. Comparison of the mono- and bivalent humanized mAb in a complement-mediated cell lysis assay revealed that the monovalent antibody mediated lysis of human T cell blasts whereas the bivalent form did not. The availability of a humanized, complement-fixing CD3 mAb may improve opportunities for human therapy, in the management of organ rejection, autoimmunity and the treatment of T cell lymphoma.

Temporal Pharmacokinetic/Pharmacodynamic Interaction between Human CD3εAntigen–Targeted Monoclonal Antibody Otelixizumab and CD3εBinding and Expression in Human Peripheral Blood Mononuclear Cell Static Culture

“…Otelixizumab (also known as ChAglyCD3) is an aglycosylated nonmitogenic recombinant antibody (human g1) directed against CD3« chain, a protein forming part of the CD3/T-cell receptor complex (TCR) on T lymphocytes (Routledge et al, 1991;Bolt et al, 1993). Otelixizumab reference standard at 12.1 mg/ml was used for all experiments.…”

Human T cells resistant to complement lysis by bivalent antibody can be efficiently lysed by dimers of monovalent antibody

“…15 cDNA versions of the genes were used for antibody production in this present study. A second version of the H-chain cDNA was created, in which the serine codon (TCT) at position 444 (numbering based on human myeloma protein EU) 19 was changed to cysteine (TGC).…”

“…Antibodies to such antigens are generally poor inducers of complement lysis, but the problem can be overcome to some extent using enzymatic or genetic engineering techniques to remove or inactivate one of the Fab domains of the mAb. 1,[13][14][15] This renders the mAb monovalent, preventing it from cross-linking the target antigen. This strategy has been shown to improve complement lysis activity, but the drawback is that it also reduces mAb-binding avidity by a factor of 6, 15 significantly increasing the concentration of mAb that must be used (or dose that must be given therapeutically) to achieve effective coating of the target cell.…”

Non-Fc Receptor-Binding Humanized Anti-CD3 Antibodies Induce Apoptosis of Activated Human T Cells

“…A second generation of genetically engineered anti-CD3 mAbs has been developed not only by grafting complementarity-determining regions (CDRs) 3 of murine anti-CD3 mAb into human IgG sequences (2)(3)(4)(5), but also by introducing non-FcR-binding mutations into the Fc (6,7). Humanization of the mAb results in decreased immunogenicity and improved mAb half-life.…”