Cloning and Sequencing of the VH and YK Genes of an Anti-CD3 Monoclonal Antibody, and Construction of a Mouse/Human Chimeric Antibody

Abstract

Mouse monoclonal antibodies against CD3 on human T lymphocytes have been used for therapy in organ-transplant patients as a potent immunosuppressive agent or for treatment of cancer as a potent T cell activating agent. However, an inherent problem in their in vivo application is the human anti-mouse antibody response. In this study, we cloned and sequenced the variable region genes of the heavy and light chains (VH and V kappa) of a mouse anti-human CD3 monoclonal antibody (OKT3) using the reverse transcription-polymerase chain reaction method. Then, we constructed a mouse/human chimeric antibody, designated as Ch OKT3, by fusing the OKT3 VH and V kappa genes to the human heavy and light chain constant region genes (C gamma 1 and C kappa) derived from a human plasma cell leukemia line (ARH77), respectively. The chimeric gene constructs were sequentially co-transfected into mouse non-Ig-producing hybridoma cells (Sp2/0) by electroporation. The Ch OKT3 antibody thus prepared bound to human peripheral blood mononuclear cells and competitively inhibited the binding of the parental MAb OKT3 to the blood mononuclear cells, indicating that this chimeric antibody seems to be suitable for in vivo therapeutic approaches.

Cytotoxic Enhancement of a Bispecific Diabody by Format Conversion to Tandem Single-chain Variable Fragment (taFv)

“…The gene constructs, hEx3-taFv-3C-Fc and hEx3-taFvЈ-3C-Fc, were inserted into pcDNA3.1/Neo mammalian expression vectors (Invitrogen). The leader peptide sequences for protein secretion were derived from mouse OKT3 IgG (18).…”

T-cell activation and cytokine production via a bispecific single-chain antibody fragment targeted to blood-stage malaria parasites

“…The single-chain genes were assembled by adding a linker (Gly 4 Ser) 3 and inserted into the phage display vector pCANTAB5E (Pharmacia Biotech) to construct pCANTAB5E-5.2scFv. Binding functionality of the single-chain to PfMSP-1 19 was first analyzed by phage display system (Pharmacia Biotech). The selected single-chain genes were sequenced and the nucleotide sequence data have been deposited in GenBank database under the accession numbers AB028875 and AB028876.…”

“…Based on the sequence information of the OKT3 V H and V L genes, 19 the OKT3 V H and V L genes were amplified from the cDNAs with specific sets of primers, pOKT3V H -1/pOKT3V H -2 and pOKT3V L -1/pOKT3V L -2, respectively ( Table 1). After amplification of the V H and V L genes, overlapping PCR was performed to construct the OKT3 scFv gene under the same conditions, but with the pOKT3V H -1 and pOKT3V L -2 primers.…”

“…PfMSP-1 is a prominent antigen in malaria vaccine development and has been found to induce antibodies that bind to the merozoite surface, inhibit parasite growth in vitro, and induce a protective immune response against malaria infection in nonhuman primates in vivo. [13][14][15][16] Although portions of the PfMSP-1 gene undergo allelic variation, 19-kDa C-terminal processing product of PfMSP-1 (PfMSP-1 19 ), which appears to be an important target of the protective immune response, is highly conserved and may serve as a potential target of immunotherapy. Taken together, the bridging between blood-stage parasites and T cells via anti-CD3 biscFv with anti-PfMSP-1 19 scFv would be expected to trigger cellular activation and the release of the cytokines responsible for macrophage or neutrophil activation, resulting in phagocytosis and killing of blood-stage malaria parasites.…”

“…In this paper we describe a genetically engineered biscFv consisting of anti-CD3 scFv and anti-PfMSP-1 19 scFv. This 5.2-OKT3 biscFv was expressed in insect cells using a recombinant baculovirus expression system.…”