Single-chain Antibody Fusion Protein Research Articles

Improved expression of a soluble single chain antibody fusion protein containing tumor necrosis factor in Escherichia coli

The immunocytokine scFvMEL/TNF is a fusion protein composed of tumor necrosis factor (TNF-alpha) and a single-chain Fv antibody scFvMEL targeting the melanoma-associated gp240 antigen. The fusion protein containing thioredoxin and a hexa-histidine tag was expressed in two Escherichia coli host cells, AD494 (DE3) pLysS and T7 Express I (q). The cell growth and expression level of target protein, His-tagged scFvMEL/TNF, were highly dependent on the induction temperature, inducer types and host strains. The ratio of insoluble to soluble target proteins was found to be controllable and could be minimized using cold shock conditions at less than 18 degrees C. The total productivity of soluble target protein was further improved by high cell density cultivation using a DO-STAT feeding strategy. The scFvMEL/TNF purified under their conditions was specifically cytotoxic to gp240-antigen positive melanoma A375-M cells as previously described.

An immunoglobulin C kappa-reactive single chain antibody fusion protein induces tolerance through receptor editing in a normal polyclonal immune system.

Understanding immune tolerance mechanisms is a major goal of immunology research, but mechanistic studies have generally required the use of mouse models carrying untargeted or targeted antigen receptor transgenes, which distort lymphocyte development and therefore preclude analysis of a truly normal immune system. Here we demonstrate an advance in in vivo analysis of immune tolerance that overcomes these shortcomings. We show that custom superantigens generated by single chain antibody technology permit the study of tolerance in a normal, polyclonal immune system. In the present study we generated a membrane-tethered anti-Igκ–reactive single chain antibody chimeric gene and expressed it as a transgene in mice. B cell tolerance was directly characterized in the transgenic mice and in radiation bone marrow chimeras in which ligand-bearing mice served as recipients of nontransgenic cells. We find that the ubiquitously expressed, Igκ-reactive ligand induces efficient B cell tolerance primarily or exclusively by receptor editing. We also demonstrate the unique advantages of our model in the genetic and cellular analysis of immune tolerance.

A bispecific single-chain antibody fusion protein for targeted depletion of autoreactive B cells via unstimulated human T lymphocytes

Autoantigen-specific B cells are culprits in the pathogenesis of many autoimmune diseases either through the production of autoreactive antibodies or as very effective antigen-presenting cells. A general depletion of B cells by a CD20-specific monoclonal IgG1 antibody has recently been validated as an effective strategy for treating rheumatoid arthritis. However, general elimination of B cells can lead to immunosuppression and increased risk of infection. In search for a more specific approach, we have generated a fusion protein for the antigen-specific targeting of autoreactive B cells for re-directed lysis by resting human T lymphocytes. We describe the design, purification and characterization of MOGxanti-CD3, a single-chain bispecific antibody fusion protein recognizing B cell receptors specific for the human myelin oligodendrocyte glycoprotein (MOG) and to CD3 on human T cells. MOGxAnti-CD3 induced selective and very efficient redirected lysis of MOG-reactive B cells through freshly isolated, unstimulated human T cells. Fusions between autoantigens and an anti-CD3 single-chain antibody may be suitable to develop very specific therapeutic approaches for the selective depletion of autoreactive B cells in autoimmune diseases.

A bispecific single-chain antibody fusion protein for targeted depletion of autoreactive B cells via unstimulated human T lymphocytes

Autoantigen-specific B cells are culprits in the pathogenesis of many autoimmune diseases either through the production of autoreactive antibodies or as very effective antigen-presenting cells. A general depletion of B cells by a CD20-specific monoclonal IgG1 antibody has recently been validated as an effective strategy for treating rheumatoid arthritis. However, general elimination of B cells can lead to immunosuppression and increased risk of infection. In search for a more specific approach, we have generated a fusion protein for the antigen-specific targeting of autoreactive B cells for re-directed lysis by resting human T lymphocytes. We describe the design, purification and characterization of MOGxanti-CD3, a single-chain bispecific antibody fusion protein recognizing B cell receptors specific for the human myelin oligodendrocyte glycoprotein (MOG) and to CD3 on human T cells. MOGxAnti-CD3 induced selective and very efficient redirected lysis of MOG-reactive B cells through freshly isolated, unstimulated human T cells. Fusions between autoantigens and an anti-CD3 single-chain antibody may be suitable to develop very specific therapeutic approaches for the selective depletion of autoreactive B cells in autoimmune diseases.

Improved yield and stability of L49-sFv-beta-lactamase, a single-chain antibody fusion protein for anticancer prodrug activation, by protein engineering.

The L49 single-chain Fv fused to beta-lactamase (L49-sFv-bL) combined with the prodrug C-Mel is an effective anticancer agent against tumor cells expressing the p97 antigen. However, large-scale production of L49-sFv-bL from refolded E. coli inclusion bodies has been problematic due to inefficient refolding and instability of the fusion protein. Sequence analysis of the L49-sFv framework regions revealed three residues in the framework regions at positions L2, H82B, and H91, which are not conserved for their position, occurring in <1% of sequences in Fv sequence databases. One further unusual residue, found in <3% of variable sequences, was observed at position H39. Each unusual residue was mutated to a conserved residue for its position and tested for refolding yield from inclusion bodies following expression in E. coli. The three V(H) single mutants showed improvement in the yield of active protein and were combined to form double and triple mutants resulting in a 7-8-fold increased yield compared to the parental protein. In an attempt to further improve yield, the orientation of the triple mutant was reversed to create a bL-L49-sFv fusion protein resulting in a 3-fold increase in expressed inclusion body protein and producing a 20-fold increase in the yield of purified protein compared to the parental protein. The triple mutants in both orientations displayed increased stability in murine plasma and binding affinity was not affected by the introduced mutations. Both triple mutants also displayed potent in vitro cytotoxicity and in vivo antitumor activity against p97 expressing melanoma cells and tumor xenografts, respectively. These results show that a rational protein-engineering approach improved the yield, stability, and refolding characteristics of L49-sFv-bL while maintaining binding affinity and therapeutic efficacy.

Construction and Functional Evaluation of a Single-Chain Antibody Fusion Protein With Fibrin Targeting and Thrombin Inhibition After Activation by Factor Xa

Recombinant technology was used to produce a new anticoagulant that is preferentially localized and active at the site of the clot. The variable regions of the heavy and light chains of a fibrin-specific antibody were amplified by polymerase chain reaction (PCR) with hybridoma cDNA. To obtain a functional single-chain antibody (scFv), a linker region consisting of (Gly(4)Ser)(3) was introduced by overlap PCR. After the scFv clones were ligated with DNA encoding the pIII protein of the M13 phage, high-affinity clones were selected by 10 rounds of panning on the Bbeta15-22 peptide of fibrin (beta-peptide). Hirudin was genetically fused to the C-terminus of the variable region of the light chain. To release the functionally essential N-terminus of hirudin at the site of a blood clot, a factor Xa recognition site was introduced between scFv(59D8) and hirudin. The fusion protein was characterized by its size on SDS-PAGE (36 kDa), by Western blotting, by its cleavage into a 29-kDa (single chain alone) and 7-kDa (hirudin) fragment, by its binding to beta-peptide, and by thrombin inhibition after Xa cleavage. Finally, the fusion protein inhibited appositional growth of whole blood clots in vitro more efficiently than native hirudin. A fusion protein was constructed that binds to a fibrin-specific epitope and inhibits thrombin after its activation by factor Xa. This recombinant anticoagulant effectively inhibits appositional clot growth in vitro. Its efficient and fast production at low cost should facilitate a large-scale evaluation to determine whether an effective localized antithrombin activity can be achieved without systemic bleeding complications.

Construction, expression, and activities of L49-sFv-beta-lactamase, a single-chain antibody fusion protein for anticancer prodrug activation.

The L49 (IgG1) monoclonal antibody binds to p97 (melanotransferrin), a tumor-selective antigen that is expressed on human melanomas and carcinomas. A recombinant fusion protein, L49-sFv-bL, that contains the antibody binding regions of L49 fused to the Enterobacter cloacae r2-1 beta-lactamase (bL) was constructed, expressed, and purified to homogeneity in an Escherichia coli soluble expression system. The variable regions of L49 were cloned by reverse transcription-polymerase chain reaction from L49 hybridoma mRNA using signal sequence and constant region primers. Construction of the gene encoding L49-sFv-bL was accomplished by hybridization insertion of VH, VL, and sFv linker sequences onto a pET phagemid template containing the bL gene fused to the pelB leader sequence. Optimal soluble expression of L49-sFv-bL in E. coli was found to take place at 23 degrees C with 50 microM isopropyl beta-D-thiogalactopyranoside induction and the use of the nonionic detergent Nonidet P-40 for isolation from the bacteria. Construction and expression of a soluble form of the p97 antigen in Chinese hamster ovary cells allowed affinity-based methods for analysis and purification of the fusion protein. Surface plasmon resonance, fluorescent activated cell sorting, and Michaelis-Menten kinetic analyses showed that L49-sFv-bL retained the antigen binding capability of monovalent L49 as well as the enzymatic activity of bL. In vitro experiments demonstrated that L49-sFv-bL bound to 3677 melanoma cells expressing the p97 antigen and effected the activation of 7-(4-carboxybutanamido)cephalosporin mustard (CCM), a cephalosporin nitrogen mustard prodrug. On the basis of these results, L49-sFv-bL was injected into nude mice with subcutaneous 3677 tumors, and localization was determined by measuring bL activity. Tumor to blood conjugate ratios of 13 and 150 were obtained 4 and 48 h post conjugate administration, respectively, and the tumor to liver, spleen, and kidney ratios were even higher. A chemically produced L49-Fab'-bL conjugate yielded a much lower tumor to blood ratio (5.6 at 72 h post administration) than L49-sFv-bL. Therapy experiments established that well-tolerated doses of L49-sFv-bL/CCM combinations resulted in cures of 3677 tumors in nude mice. The favorable pharmacokinetic properties of L49-sFv-bL allowed prodrug treatment to be initiated 12 h after the conjugate was administered. Thus, L49-sFv-bL appears to have promising characteristics for site-selective anticancer prodrug activation.

New recombinant multimer of two fusion proteins, each including part of C4 binding protein; recombinant complement-C4 binding protein and CD4, CD8, CD16, CD35, Rhesus-D, antigen, enzyme or single chain antibody fusion protein multimer for use as a recombinant vaccine or diagnostic : Univ. Paris Pierre Marie Curie and Univ. Rheims Champagne Ardennes World 9704 109; 6 February 1997

New recombinant multimer of two fusion proteins, each including part of C4 binding protein; recombinant complement-C4 binding protein and CD4, CD8, CD16, CD35, Rhesus-D, antigen, enzyme or single chain antibody fusion protein multimer for use as a recombinant vaccine or diagnostic : Univ. Paris Pierre Marie Curie and Univ. Rheims Champagne Ardennes World 9704 109; 6 February 1997