Bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics¹

Abstract

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 (V_H and V_L) 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 CD3 × CD19 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 °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.

Introduction

Bispecific antibodies (BsAb) provide an effective means of retargeting cytotoxic effector cells against tumor cells (Fanger et al., 1992). They have mainly been produced using murine hybrid hybridomas (Bohlen et al., 1993) or by chemical cross-linking Brennan et al 1985, Glennie et al 1987. However, the immunogenicity of BsAb derived from rodent monoclonal antibodies is a major drawback for clinical use (Khazaeli et al., 1994). They are also difficult to produce and purify in large quantities. Recent advances in recombinant antibody technology have provided several alternative methods for constructing and producing BsAb molecules Carter et al 1995, Pluckthun and Pack 1997. For example, single chain Fv (scFv) fragments have been genetically fused with adhesive polypeptides (de Kruif & Logtenberg, 1996) or protein domains (Müller et al., 1998c) to facilitate the formation of heterodimers. The genetic engineering of scFv-scFv tandems linked with a third polypeptide linker has also been carried out in several laboratories Gruber et al 1994, Kurucz et al 1995. A bispecific diabody was obtained by the non-covalent association of two single chain fusion products consisting of the V_H domain from one antibody connected by a short linker to the V_L domain of another antibody Holliger et al 1993, Holliger et al 1996. The two antigen binding domains have been shown by crystallographic analysis to be on opposite sides of the diabody such that they are able to cross-link two cells (Perisic et al., 1994). In contrast to native antibodies, all of the above mentioned bispecific molecules have only one binding domain for each specificity. However, bivalent binding is an important means of increasing the functional affinity and possibly the selectivity for particular cell types carrying densely clustered antigens.

We recently described the construction and characterization of a bispecific diabody with dual specificity for both the human B cell antigen CD19 and ϵ chain of the CD3/T cell receptor (TCR) complex designed for the treatment of minimal residual disease in patients with leukemias and malignant lymphomas (Kipriyanov et al., 1998). The CD19 antigen is expressed on virtually all B-lineage malignancies from acute lymphoblastic leukemia (ALL) to non-Hodgkin’s lymphoma (NHL) (Uckun & Ledbetter, 1988). Moreover, it is not shed and is absent from hemopoietic stem cells, plasma cells, T cells and other tissues. Bispecific diabodies appear to be quite effective in mediating T cell cytotoxicity Holliger et al 1996, Kipriyanov et al 1998, Zhu et al 1996. However, the co-secretion of two hybrid scFv fragments can give rise to two types of dimer: active heterodimers and inactive homodimers. A second problem is that the two chains of diabodies are held together by non-covalent associations of the V_H and V_L domains and can diffuse away from one another. Moreover, to ensure the assembly of a functional diabody, both hybrid scFv fragments must be expressed in the same cell in similar amounts. This latter requirement is difficult to uphold in eukaryotic expression systems such as yeast, which are often preferred because high yields of enriched product can be obtained Ridder et al 1995, Shusta et al 1998. Finally, the small size of bispecific diabodies (50–60 kDa) leads to their rapid clearance from the blood stream through the kidneys, thus requiring the application of relatively high doses for therapy.

To circumvent the drawbacks of diabodies and to increase the valency, stability and therapeutic potential of recombinant bispecific antibodies, we have now constructed single chain molecules comprising four antibody variable domains (V_H and V_L) of two different specificities in an orientation preventing Fv formation. They can either form bivalent bispecific antibodies by diabody-like folding (sc-diabodies) or dimerize with the formation of tetravalent bispecific antibodies (tandem diabodies). The efficacy of tandem diabody (Tandab) formation is dependent on the length of the linker between two halves of the molecule. Here we show that Tandabs are bispecific and have higher avidity resulting from the bivalency for each specificity. CD3 × CD19 Tandabs were more potent than the diabody for inducing human T cell proliferation in the presence of irradiated CD19⁺ B cells. In cytotoxic assays, Tandabs were able to retarget human T lymphocytes to malignant B cells. The efficiency of Tandab-mediated cell lysis also compared favorably to that obtained with a diabody of the same dual specificity. In vivo studies demonstrated that tetravalent Tandabs were more stable and were retained longer in the blood of normal mice compared to scFv and diabodies. This bispecific antibody format could therefore prove to be particularly advantageous for cancer immunotherapy.