Expression and purification of monospecific and bispecific recombinant antibody fragments derived from antibodies that block the CD80/CD86-CD28 costimulatory pathway.

The development of recombinant techniques for rapid cloning, expression, and characterization of cDNAs encoding antibody (Ab) subunits has revolutionized the field of antibody engineering. By fusion to heterologous protein domains, chain shuffling, or inclusion of self-assembly motifs, novel molecules such as bispecific Abs can be generated that possess the subset of functional properties designed to fit the intended application. We describe the engineering of Ab fragments produced in bacteria for blocking the CD28-CD80/CD86 costimulatory interaction in order to induce tolerance against transplanted organs. We designed single-chain Fv antibodies, monospecific and bispecific diabodies, and a bispecific tetravalent antibody (BiTAb) molecule directed against the CD80 and/or CD86 costimulatory molecules. These recombinant Ab molecules were expressed in Escherichia coli, followed by purification and evaluation for specific interaction with their respective antigen in an enzyme-linked immunosorbent assay (ELISA). A specific sandwich ELISA confirmed the bispecificity of the bispecific diabodies and the BiTAb.

Dincq S ，Bosman F ，Buyse MA ，Degrieck R ，Celis L ，de Boer M ，Van Doorsselaere V ，Sablon E ... -  《PROTEIN EXPRESSION AND PURIFICATION》

A novel bispecific tetravalent antibody fusion protein to target costimulatory activity for T-cell activation to tumor cells overexpressing ErbB2/HER2.

Biburger M ，Weth R ，Wels WS 《JOURNAL OF MOLECULAR BIOLOGY》

Building novel binding ligands to B7.1 and B7.2 based on human antibody single variable light chain domains.

Ligands specific for B7.1 (CD80) and B7.2 (CD86) have applications in disease indications that require inhibition of T-cell activity. As we observed significant sequence and structural similarity between the B7-binding ligand, cytotoxic T-lymphocyte associated protein-4 (CTLA-4), and antibody variable light chain domains (VLs), we have explored the possibilities of making novel B7 binding molecules based on single VL domains. We first describe the "rational" design and construction of a VL/CTLA-4 hybrid molecule in which we have grafted both the CDR1 and CDR3-like loops of CTLA-4 onto a single VL light chain, at sites determined by sequence and structure-based alignment. This molecule was secreted as a soluble product from Escherichia coli, but did not show any binding to B7.1 and B7.2. In a second approach we constructed a VL library in which human VL genes derived from B-cells were spiked with the CDR3-like loop of CTLA-4 and further diversified by DNA shuffling. This library was displayed on phage, and after selection gave B7.1 binding ligands which competed with CTLA-4. In order to evaluate the possible general utility of VL domains as binding ligands, we have constructed a non-biased VL library. From this DNA-shuffled human VL library we have selected single VL domains specific for B7.1, B7.2 or human IgG. Two B7.1-specific VL ligands and one B7.2-specific VL ligand showed competition with CTLA-4. One candidate VL domain-specific for B7.1 was affinity matured by simultaneous randomisation of all CDR loops using DNA shuffling with degenerate CDR-spiking oligonucleotides. From this library, a single VL domain with affinity of 191 nM for B7.1 was obtained, which also showed binding to B7.1 in situ. This VL had mutations in CDR1 and CDR3, indicating that antigen recognition for this single VL is most likely mediated by the same regions as in the VL domain of whole antibodies. The B7.1 and B7.2-specific VL domains described in this study may form the basis of a new family of immunomodulatory recombinant molecules. Furthermore, our studies suggest that it is feasible to create specific single VL domains to diverse targets as is the case for single VH domains.

van den Beucken T ，van Neer N ，Sablon E ，Desmet J ，Celis L ，Hoogenboom HR ，Hufton SE ... -  《JOURNAL OF MOLECULAR BIOLOGY》

Construction and characterization of bispecific costimulatory molecules containing a minimized CD86 (B7-2) domain and single-chain antibody fragments for tumor targeting.

Rohrbach F ，Gerstmayer B ，Biburger M ，Wels W ... -  《CLINICAL CANCER RESEARCH》

Bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics.

To increase the valency, stability and therapeutic potential of bispecific antibodies, we designed a novel recombinant molecule that is bispecific and tetravalent. It was constructed by linking four antibody variable domains (VHand VL) with specificities for human CD3 (T cell antigen) or CD19 (B cell marker) into a single chain construct. After expression in Escherichia coli, intramolecularly folded bivalent bispecific antibodies with a mass of 57 kDa (single chain diabodies) and tetravalent bispecific dimers with a molecular mass of 114 kDa (tandem diabodies) could be isolated from the soluble periplasmic extracts. The relative amount of tandem diabodies proved to be dependent on the length of the linker in the middle of the chain and bacterial growth conditions. Compared to a previously constructed heterodimeric CD3xCD19 diabody, the tandem diabodies exhibited a higher apparent affinity and slower dissociation from both CD3(+)and CD19(+)cells. They were also more effective than diabodies in inducing T cell proliferation in the presence of tumor cells and in inducing the lysis of CD19(+)cells in the presence of activated human PBL. Incubated in human serum at 37 degrees C, the tandem diabody retained 90 % of its antigen binding activity after 24 hours and 40 % after one week. In vivo experiments indicated a higher stability and longer blood retention of tandem diabodies compared to single chain Fv fragments and diabodies, properties that are particularly important for potential clinical applications.

Kipriyanov SM ，Moldenhauer G ，Schuhmacher J ，Cochlovius B ，Von der Lieth CW ，Matys ER ，Little M ... -  《JOURNAL OF MOLECULAR BIOLOGY》