Small Bispecific Antibody Research Articles

Functional integration of protein A binding ability to antibody fragments for convenient and tag-free purification

ABSTRACTAlthough the development of small therapeutic antibodies is important, the affinity tags used for their purification often result in heterogeneous production and immunogenicity. In this study, we integrated Staphylococcus aureus protein A (SpA) binding ability into antibody fragments for convenient and tag-free purification. SpA affinity chromatography is used as a global standard purification method for conventional antibodies owing to its high binding affinity to the Fc region. SpA also has a binding affinity for some variable heavy domains (VH) classified in the VH3 subfamily. Through mutagenesis based on alignment and structural modeling results using the SpA-VH3 cocrystal structure, we integrated the SpA-binding ability into the anti-CD3 single-chain Fv. Furthermore, we applied this mutagenesis approach to more complicated small bispecific antibodies and successfully purified the antibodies using SpA affinity chromatography. The antibodies retained their biological function after purification. Integration of SpA-binding ability into conventional antibody fragments simplifies the purification and monitoring of the production processes and, thus, is an ideal strategy for accelerating the development of small therapeutic antibodies. Furthermore, because of its immunoactivity, the anti-CD3 variable region with SpA-binding ability is an effective building block for developing engineered cancer therapeutic antibodies without the Fc region.

Complementary Design for Pairing between Two Types of Nanoparticles Mediated by a Bispecific Antibody: Bottom-Up Formation of Porous Materials from Nanoparticles.

Recent advances in biotechnology have enabled the generation of antibodies with high affinity for the surfaces of specific inorganic materials. Herein, we report the synthesis of functional materials from multiple nanomaterials by using a small bispecific antibody recombinantly constructed from gold-binding and ZnO-binding antibody fragments. The bispecific antibody-mediated spontaneous linkage of gold and ZnO nanoparticles forms a binary gold-ZnO nanoparticle composite membrane. The relatively low melting point of the gold nanoparticles and the solubility of ZnO in dilute acidic solution then allowed for the bottom-up synthesis of a nanoporous gold membrane by means of a low-energy, low-environmental-load protocol. The nanoporous gold membrane showed high catalytic activity for the reduction of p-nitrophenol to p-aminophenol by sodium borohydride. Here, we show the potential utility of nanoparticle pairing mediated by bispecific antibodies for the bottom-up construction of nanostructured materials from multiple nanomaterials.

Engineering the hinge region of human IgG1 Fc-fused bispecific antibodies to improve fragmentation resistance

Fc domain fusion can improve the therapeutic effects of relatively small biological molecules such as peptides, cytokines, and antibody fragments. Fc fusion proteins can also be used to enhance the cytotoxic effects of small bispecific antibodies (bsAbs). However, fragmentation of Fc fusion proteins, which mainly occurs around the hinge regions during production, storage, and circulation in the blood, is a major issue. In this study, we first investigated the mechanisms of fragmentation around the hinge region during storage using Fc-fused bsAbs with specificity for epidermal growth factor receptor and CD3 as a model. The fragmentation peaks generated by gel filtration analysis indicated that both contaminating proteases and dissolved active oxygen should be considered causes of fragmentation. We designed and constructed variants by introducing a point mutation into the upper hinge region, which reduced the cleavage caused by dissolved active oxygen, and shortened the hinge region to restrict access of proteases. These hinge modifications improved fragmentation resistance and did not affect the biological activity of the bsAbs in vitro. We confirmed the versatility of the hinge modifications using another Fc-fused bsAb. Our results show that hinge modifications to the Fc fusion protein, especially the introduction of a point mutation into the upper hinge region, can reduce fragmentation substantially, and these modifications can be used to improve the fragmentation resistance of other recombinant Fc fusion proteins.

High-throughput cytotoxicity and antigen-binding assay for screening small bispecific antibodies without purification

The cytotoxicity of T cell-recruiting antibodies with their potential to damage late-stage tumor masses is critically dependent on their structural and functional properties. Recently, we reported a semi-high-throughput process for screening highly cytotoxic small bispecific antibodies (i.e., diabodies). In the present study, we improved the high-throughput performance of this screening process by removing the protein purification stage and adding a stage for determining the concentrations of the diabodies in culture supernatant. The diabodies were constructed by using an Escherichia coli expression system, and each diabody contained tandemly arranged peptide tags at the C-terminus, which allowed the concentration of diabodies in the culture supernatant to be quantified by using a tag-sandwich enzyme-linked immunosorbent assay. When estimated diabody concentrations were used to determine the cytotoxicity of unpurified antibodies, results comparable to those of purified antibodies were obtained. In a surface plasmon resonance spectroscopy-based target-binding assay, contaminants in the culture supernatant prevented us from conducting a quantitative binding analysis; however, this approach did allow relative binding affinity to be determined, and the relative binding affinities of the unpurified diabodies were comparable to those of the purified antibodies. Thus, we present here an improved high-throughput process for the simultaneous screening and determination of the binding parameters of highly cytotoxic bispecific antibodies.

A semi high-throughput method for screening small bispecific antibodies with high cytotoxicity

Small bispecific antibodies that induce T-cell–mediated cytotoxicity have the potential to damage late-stage tumor masses to a clinically relevant degree, but their cytotoxicity is critically dependent on their structural and functional properties. Here, we constructed an optimized procedure for identifying highly cytotoxic antibodies from a variety of the T-cell–recruiting antibodies engineered from a series of antibodies against cancer antigens of epidermal growth factor receptor family and T-cell receptors. By developing and applying a set of rapid operations for expression vector construction and protein preparation, we screened the cytotoxicity of 104 small antibodies with diabody format and identified some with 103-times higher cytotoxicity than that of previously reported active diabody. The results demonstrate that cytotoxicity is enhanced by synergistic effects between the target, epitope, binding affinity, and the order of heavy-chain and light-chain variable domains. We demonstrate the importance of screening to determine the critical rules for highly cytotoxic antibodies.

In vitro selection of bispecific diabody fragments using covalent bicistronic DNA display

Bispecific antibodies with two different antigen-binding sites have been widely used for a variety of medical applications. The activity and stability of antibody fragments can be improved by in vitro evolution. Although the affinity and stability of small bispecific antibody fragments such as diabodies can be further optimized by in vitro display technologies, cell-free display of bispecific antibody fragments has not been reported. In this study, we applied a covalent bicistronic DNA display for the in vitro selection of heterodimeric diabodies. First, we confirmed the antigen-binding activities of a diabody synthesized by an in vitro transcription and translation system. However, when we performed DNA-display selection of a model diabody library in a proof-of-principle experiment, no enrichment of the diabody gene was observed, likely due to a low yield of the diabody heterodimer. To overcome this issue, we introduced cysteine residues at the VH-VL interface of the diabody heterodimer. Using the disulfide-stabilized diabodies, we successfully enriched the diabody gene from a model library. Our results indicate that the covalent bicistronic DNA display technique could be useful for improving the stability and affinity of bispecific diabody fragments.

Functionalization of Bispecific Therapeutic Antibodies Based on Protein Engineering

Although antibodies have been used as molecularly targeted agents for difficult-to-treat diseases such as cancers, the high production costs associated with mammalian expression systems continue to be a drawback. In addition, the clinical efficacy of conventional IgG antibodies is limited. Several types of recombinant antibody (e.g. fused with anticancer drugs, multivalent, or multispecific) have been designed in efforts to develop next-generation antibodies with higher functionality. We used protein engineering to construct several anticancer recombinant antibodies by developing bispecific antibodies that induced specific antitumor effects against cancer cells through the recruitment of lymphocytes. We found that a humanized small bispecific antibody (Ex3) that targets epidermal growth factor receptor on tumor cells and CD3 on T lymphocytes had marked anticancer activity. Furthermore, the function of Ex3 was enhanced by fusion with the human Fc region, domain rearrangement, multimerization, and affinity maturation; a combination of these modifications showed at least additive cytotoxic effects. Interestingly, merely rearranging the domain order of Ex3 induced substantial cytotoxic enhancements, even though the structural format remained the same. Here, we describe our efforts to develop highly functional bispecific antibodies as next-generation therapeutic antibodies using protein engineering.

Update on antigen-specific immunotherapy of acute myeloid leukemia.

Among the few drugs that have shown a benefit for patients with acute myeloid leukemia (AML) in randomized clinical trials over the last several decades is the CD33 antibody-drug conjugate, gemtuzumab ozogamicin (GO). Undoubtedly, this experience has highlighted the value of antigen-specific immunotherapy in AML. A wide variety of therapeutics directed against several different antigens on AML cells are currently explored in preclinical and early clinical studies. On the one hand, these include passive strategies such as unconjugated antibodies targeting one or more antigens, antibodies armed with drugs, toxic proteins, or radionuclides, or adoptive immunotherapies, in particular utilizing T cells engineered to express chimeric antigen receptors (CARs) or modified T cell receptor (TCR) genes; on the other hand, these include active strategies such as vaccinations. With the documented benefit for GO and the emerging data with several classes of therapeutics in other leukemias, in particular small bispecific antibodies and CAR T cells, the future is bright. Nevertheless, a number of important questions related to the choice of target antigen(s), patient population, exact treatment modality, and supportive care needsremain open. Addressing such questions in upcoming studies will ultimately be required to optimize the clinical use of antigen-specific immunotherapies in AML and ensure that such treatments become an effective, versatile tool for this disease for which the outcomes have remained unsatisfactory in many patients.

Cytotoxic Enhancement of a Bispecific Diabody by Format Conversion to Tandem Single-chain Variable Fragment (taFv): THE CASE OF THE hEx3 DIABODY

Diabodies (Dbs) and tandem single-chain variable fragments (taFv) are the most widely used recombinant formats for constructing small bispecific antibodies. However, only a few studies have compared these formats, and none have discussed their binding kinetics and cross-linking ability. We previously reported the usefulness for cancer immunotherapy of a humanized bispecific Db (hEx3-Db) and its single-chain format (hEx3-scDb) that target epidermal growth factor receptor and CD3. Here, we converted hEx3-Db into a taFv format to investigate how format affects the function of a small bispecific antibody; our investigation included a cytotoxicity assay, surface plasmon resonance spectroscopy, thermodynamic analysis, and flow cytometry. The prepared taFv (hEx3-taFv) showed an enhanced cytotoxicity, which may be attributable to a structural superiority to the diabody format in cross-linking target cells but not to differences in the binding affinities of the formats. Comparable cross-linking ability for soluble antigens was observed among hEx3-Db, hEx3-scDb, and hEx3-taFv with surface plasmon resonance spectroscopy. Furthermore, drastic increases in cytotoxicity were found in the dimeric form of hEx3-taFv, especially when the two hEx3-taFv were covalently linked. Our results show that converting the format of small bispecific antibodies can improve their function. In particular, for small bispecific antibodies that target tumor and immune cells, a functional orientation that avoids steric hindrance in cross-linking two target cells may be important in enhancing the growth inhibition effect.

Improved Pharmacokinetics of Recombinant Bispecific Antibody Molecules by Fusion to Human Serum Albumin

Recombinant bispecific antibodies such as tandem scFv molecules (taFv), diabodies (Db), or single chain diabodies (scDb) have shown to be able to retarget T lymphocytes to tumor cells, leading to their destruction. However, therapeutic efficacy is hampered by a short serum half-life of these small molecules having molecule masses of 50-60 kDa. Thus, improvement of the pharmacokinetic properties of small bispecific antibody formats is required to enhance efficacy in vivo. In this study, we generated several recombinant bispecific antibody-albumin fusion proteins and analyzed these molecules for biological activity and pharmacokinetic properties. Three recombinant antibody formats were produced by fusing two different scFv molecules, bispecific scDb or taFv molecules, respectively, to human serum albumin (HSA). These constructs (scFv(2)-HSA, scDb-HSA, taFv-HSA), directed against the tumor antigen carcinoembryonic antigen (CEA) and the T cell receptor complex molecule CD3, retained full binding capacity to both antigens compared with unfused scFv, scDb, and taFv molecules. Tumor antigen-specific retargeting and activation of T cells as monitored by interleukin-2 release was observed for scDb, scDb-HSA, taFv-HSA, and to a lesser extent for scFv(2)-HSA. T cell activation could be further enhanced by a target cell-specific costimulatory signal provided by a B7-DbCEA fusion protein. Furthermore, we could demonstrate that fusion to serum albumin strongly increases circulation time of recombinant bispecific antibodies. In addition, our comparative study indicates that single chain diabody-albumin fusion proteins seem to be the most promising format for further studying cytotoxic activities in vitro and in vivo.

Diabodies: small bispecific antibody fragments.

Although bispecific antibodies (bAb) are widely recognised to offer great promise in the therapy of malignancies and other diseases, significant difficulties in their production and purification have limited their progress to clinical application. Techniques employed to overcome this bottleneck in the production of clinical-grade bAb (starting from traditional bAb preparation by chemical crosslinking or hybrid-hybridoma technology) include improved purification methods for hybrid hybridomas (Allard et al. 1993), selective crosslinking chemistries for bispecific Fab2 (Brennan et al. 1985; Glennie et al. 1987; Carter et al. 1992) and the use of amphipathic helices to drive association (Kostelny et al. 1992). Recently protein engineering has been used to design new formats for bAb. These adesignero bAb are based on antibody Fv or scFv fragments as building blocks rather than whole antibodies (reviewed by Holliger and Winter 1993) and can be expressed in recombinant form as the main product and purified to homogeneity using a one-step purification procedure. One such bispecific antibody fragment is the diabody (Holliger et al. 1993). Instead of a single polypeptide chain with four domains, diabodies are dimers, each chain comprising two domains. Each chain consists of a VH domain connected to a VL domain using a linker is too short to allow pairing between domains on the same chain, thus driving the pairing between complementary domains on different chains to recreate the two antigen-binding sites. Thus, for a bispecific diabody (made from antibodies A and B), the first chain is assembled from the VH domain of antibody A and the VL domain of antibody B and vice versa to create the two chains VHA-VLB, VHB-VLA. Each chain alone is incapable of binding to antigen, but associates with the other chain to form a bispecific diabody (Fig. 1). In diabodies the antigen-binding sites are at opposite ends of the molecule, as shown by X-ray crystallography (Peresics et al. 1994). In contrast to IgG or Fab92 (and presumably bscFv), in which the two binding sites can take up a range of orientations and spacings, the diabody structure is more rigid and compact, with the two binding sites separated by 6.5 nm (less than half the distance in IgG). Molecular modelling indicates that the two Fv heads of the diabody can adopt a range of different conformations and move within 30° of the mean position (Holliger et al. 1996), so their flexibility is clearly restrained. Despite their limited flexibility, diabodies appear to be as effective at recruiting cytotoxic T cells as are bispecific bscFv and Fab92: in the BCL-1 lymphoma model a diabody directed against the idiotope and mouse CD3 was able to activate naive T cells and induce antigen-dependent cytotoxicity towards lymphoma cells at concentrations of a few nanograms per millilitre (Holliger et al. 1996). The potency of the diabody (weight/volume) in the T cell activation and the cytotoxicity assay appears to be similar to that of a bscFv (refolded from inclusion bodies) comprising the same antibody V genes B1 and 2C11 (De Jonge et al. 1995). Likewise when Zhu et al. 1996) compared the effectiveness of a diabody directed against p185 (HER2) and human CD3 with the analogous Fab92 bsAb in inducing cytotoxic T lymphocyte cytotoxicity towards HER2+ tumour cells, they found the diabody to be equally potent. Apart from cytotoxic T cells, other immune effector functions can be recruited using bispecific diabodies, e. g. the complement cascade by retargeting complement component Clq (Konterman et al. in press) on the whole gamut of antibody effector functions (including complement) by Work presented at the 5th World Conference on Bispecific Antibodies under the Auspices of EFIC; 25±28 June 1997, Volandam, The Netherlands

A small bispecific antibody construct expressed as a functional single-chain molecule with high tumor cell cytotoxicity.

Construction of a bispecific single-chain antibody derivative is described that consists of two different single-chain Fv fragments joined through a Gly-Ser linker. One specificity of the two Fv fragments is directed against the CD3 antigen of human T cells and the other is directed against the epithelial 17-1A antigen; the latter had been found in a clinical trial to be a suitable target for antibody therapy of minimal residual colorectal cancer. The construct could be expressed in CHO cells as a fully functional protein, while its periplasmic expression in Escherichia coli resulted in a nonfunctional protein only. The antigen-binding properties of the bispecific single-chain antibody are indistinguishable from those of the corresponding univalent single-chain Fv fragments. By redirecting human peripheral T lymphocytes against 17-1A-positive tumor cells, the bispecific antibody proved to be highly cytotoxic at nanomolar concentrations as demonstrated by 51Cr release assay on various cell lines. The described bispecific construct has a molecular mass of 60 kDa and can be easily purified by its C-terminal histidine tail on a Ni-NTA chromatography column. As bispecific antibodies have already been shown to be effective in vivo in experimental tumor systems as well as in phase-one clinical trials, the small CD3/17-1A-bispecific antibody may be more efficacious than intact antibodies against minimal residual cancer cells.