Hermann Oppermann

Protein engineering of single-chain Fv analogs and fusion proteins.

Publisher Summary This chapter describes the minimal antibody binding site through the protein engineering of single-chain Fv analog and fusion proteins. In this approach, the genes encoding VH and VL domains of a given monoclonal antibody are connected at the DNA level by an appropriate oligonucleotide, and on translation, this gene forms a single polypeptide chain with a linker peptide bridging the two variable domains. This offers economy of design, wherein a single-chain Fv (sFv) gene encodes a single Mr 26,000 protein that forms the entire antibody combining site. The chapter focuses on sFv analogs and fusion proteins giving insight into their design, production, and properties. The sFv is well suited to applications in immunotargeting, because a given sFv gene may be fused to a particular effector protein gene to yield a bifunctional sFv fusion protein. The fusion of effector domains to either chain terminus of the sFv appears to be practical without perturbation of the antigen combining site.

Medical Applications of Single-Chain Antibodies

A single-chain antibody or single-chain Fv (sFv) incorporates the complete antibody binding site in a single polypeptide chain of minimal size, with an approximate molecular weight of 26,000. In antibodies, the antigen combining site is part of the Fv region, which is composed of the VH and VL variable domains on separate heavy and light chains. Efforts over nearly two decades have indicated that Fv fragments can only rarely be prepared from IgG and IgA antibodies by proteolytic dissection. Beginning in 1988, single-chain analogues of Fv fragments and their fusion proteins have been reliably generated by antibody engineering methods. The first step involves obtaining the genes encoding VH and VL domains with desired binding properties; these V genes may be isolated from a specific hybridoma cell line, selected from a combinatorial V-gene library, or made by V gene synthesis. The single-chain Fv is formed by connecting the component V genes with an oligonucleotide that encodes an appropriately designed linker peptide, such as (Gly4-Ser)3. The linker bridges the C-terminus of the first V region and N-terminus of the second, ordered as either VH-linker-VL or VL-linker-VH. In principle, the sFv binding site can faithfully replicate both the affinity and specificity of its parent antibody combining site, as demonstrated in our model studies with the 26-10 anti-digoxin sFv. Furthermore, the sFv remains stable at low concentrations that promote VH and VL dissociation from the Fv heterodimer, resulting in loss of Fv binding. Intravenously administered sFv proteins exhibit accelerated biodistribution and exceptionally fast clearance compared to IgG or Fab. These pharmacokinetic properties allow rapid imaging by sFv, which therefore may be labeled with a short-lived isotope such as Tc-99m. Expression of a single gene product from fused sFv and effector genes facilitates immunotargeting of the effector protein, as shown for single-chain Fv toxin fusion proteins.

Cartilage‐Derived Morphogenetic Proteins and Osteogenic Protein‐1 Differentially Regulate Osteogenesis

Cartilage‐derived morphogenetic proteins‐1 and ‐2 (CDMP‐1 and CDMP‐2) are members of the bone morphogenetic protein (BMP) family, which play important roles in embryonic skeletal development. We studied the biological activities of recombinant CDMP‐1 and CDMP‐2 in chondrogenic and osteogenic differentiation and investigated their binding properties to type I and type II serine/threonine kinase receptors. In vivo, CDMP‐1 and CDMP‐2 were capable of inducing dose‐dependently de novo cartilage and bone formation in an ectopic implantation assay. In vitro studies using primary chondrocyte cultures showed that both CDMP‐1 and CDMP‐2 stimulated equally de novo synthesis of proteoglycan aggrecan in a concentration‐dependent manner. This activity was equipotent when compared with osteogenic protein‐1 (OP‐1). In contrast, CDMPs were less stimulatory than OP‐1 in osteogenic differentiation as evaluated by alkaline phosphatase activity and expression levels of bone markers in ATDC5, ROB‐C26, and MC3T3‐E1 cells. CDMP‐2 was the least osteogenic in these assays. Receptor binding studies of CDMP‐1 and CDMP‐2 revealed that both have affinity for the BMP receptor type IB (BMPR‐IB) and BMPR‐II, and weakly for BMPR‐IA. Moreover, using a promoter/reporter construct, transcriptional activation signal was transduced by BMPR‐IB in the presence of BMPR‐II upon CDMP‐1 and CDMP‐2 binding. Our data show that distinct members of the BMP family differentially regulate the progression in the osteogenic lineage, and this may be due to their selective affinity for specific receptor complexes.

Antigen recognition and targeted delivery by the single-chain Fv

The single-chain Fv (sFv) has proven attractive for immunotargeting, both alone and as a targeting element within sFv fusion proteins. This chapter summarizes the features of sFv proteins that have sparked this interest, starting with the conservation of Fv architecture that makes general sFv design practical. The length and composition of linkers used to bridge V domains are discussed based on the sFv literature; special emphasis is given to the (Gly4Ser)3 15-residue linker that has proven of broad utility for constructing Fv regions of antibodies and other members of the immunoglobulin superfamily. The refolding properties of sFv proteins are summarized and examples given from our laboratory. Spontaneous refolding from the fully reduced and denatured state, typified by 26-10 sFv, is contrasted with disulfide-restricted refolding, exemplified by MOPC 315 and R11D10 sFv proteins, which recover antigen binding only if their disulfides have been oxidized prior to removal of denaturant. The medical value of sFv proteins hinges on their reliability in antigen recognition and rapidity in targeted delivery. Detailed analysis of specificity and affinity of antigen binding by the 26-10 antidigoxin sFv has demonstrated very high fidelity to the binding properties of the parent 26-10 sFv. These results gave confidence to the pursuit of more complex biomedical applications of these proteins, which is indicated by our work with the R11D10 sFv for the imaging of myocardial infarctions. Diagnostic imaging and therapeutic immunotargeting by sFv present significant opportunities, particularly as a result of their pharmacokinetic properties. Intravenously administered sFv offers much faster clearance than conventional Fab fragments or intact immunoglobulin with minimal background binding.

Biosynthetic antibody binding sites: Development of a single-chain Fv model based on antidinitrophenol IgA myeloma MOPC 315

The functional antigen binding region of antidinitrophenol mouse IgA myeloma MOPC 315 has been produced as a single-chain Fv (sFv) protein inE. coli. Recombinant 315 proteins included sFv alone, a bifunctional fusion protein with amino-terminal fragment B (FB) of staphylococcal protein A, and a two-chain 315 Fv fragment. Successful refolding of the 315 sFv required formation of disulfide bonds while the polypeptide was in a denatured state, as previously observed for the parent Fv fragment. Affinity-purified recombinant 315 proteins showed full recovery of specific activity, with values forKa,app of 1.5 to 2.2×106 M−1, equivalent to the parent 315 Fv fragment. As observed for natural 315 Fv, the sFv region of active FB-sFv315 fusion protein was resistant to pepsin treatment, whereas inactive protein was readily degraded. These experiments will allow the application of protein engineering to the 315 single-chain Fv; such studies can advance structure-function studies of antibody combining sites and lead to an improved understanding of single-chain Fv proteins.

Tumor targeting in a murine tumor xenograft model with the (sFv′)2 divalent form of anti-c-erbB-2 single-chain Fv

This investigation has utilized novel forms of the single-chain Fv (sFv), wherein a cysteine-containing peptide has been fused to the sFv carboxyl terminus to facilitate disulfide bonding or specific crosslinking of this sFv′ to make divalent (sFv′)2. The 741F8 anti-c-erbB-2 monoclonal antibody was used as the basis for construction of 741F8 sFv, from which the sFv′ and (sFv′)2 derivatives were prepared. Recombinant c-erbB-2 extracellular domain (ECD) was prepared in CHO cells and the bivalency of 741F8 (sFv′)2 demonstrated by its complex formation with ECD. The tumor binding properties of125I-labeled anti-c-erbB-2 741F8 sFv, sFv′, and (sFv′)2 were compared with radiolabeled antidigoxin 26-10 sFv′ and (sFv′)2 controls. Following intravenous administration of radiolabeled species to severe combined immune-deficient (SCID) mice bearing SK-OV-3 tumors (which overexpress c-erbB-2), blood and organ samples were obtained as a function of time over 24 h. Comparative analysis of biodistribution and tumor-to-organ ratios demonstrated the 741F8 sFv, sFv′, and (sFv′)2 had excellent specificity for tumors, which improved with time after injection. This contrasted with nonspecific interstitial pooling in tumors observed with the 26-10 sFv, sFv′, and (sFv′)2, which decreased with time after administration. Tumor localization was significantly better for disulfide or peptide crosslinked 741F8 (sFv′)2 having Gly4Cys tails than for monovalent 741F8 sFv′ or Fab. The superior properties of the 741F8 (sFv′)2 in targeting SK-OV-3 tumors in SCID mice suggests the importance of further investigations of divalent sFv analogs for immunotargeting.

Preclinical studies and optimization of specific tumor delivery for 741F8 sFv, an anti-c-erbB-2 single-chain species

AbstractSingle-chain Fv fragments (sFv) that bind tumor-associated antigens exhibit highly specific tumor targeting characteristics, based on studies of intravenous sFv administration to immunodeficient mice bearing human tumor xenografts. These features suggest that sFv may be used as targeting vehicles for diverse agents such as radionuclides, toxins, chemotherapeutic agents, or genes. However, the quantitative tumor retention of sFv molecules is lowered by their fast clearance and rapid dissociation from antigen. We have found that the retention of tumor-specific 74IF8 sFv in tumor is an antigen-specific event and not the result of extravascular pooling. We also have produced divalent 741F8 (sFv)2 species and have examined their pharmacokinetics and biodistribution properties in relevant tumor-bearing scid mice. Finally, we have employed a number of strategies to optimize the tumor-specific retention of radiolabeled sFv molecules; escalations in administered doses, repetitive intravenous bolus adminis...