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.Bispecific antibodies are designed to target two different antigens simultaneously (1). In the context of a tumor therapy they can be applied to selectively recruit potent effector cells of the immune system such as cytotoxic T lymphocytes to tumor cells (2). This is achieved by binding on the one side to a tumorassociated antigen and on the other side to a trigger molecule on the effector cell, leading to the activation of the effector cell and tumor cell destruction. To reduce potential side effects elicited by the Fc part of antibodies (3) small recombinant bispecific antibody formats composed only of the variable regions, which define the binding unit of an antibody, have been developed (1, 4). These formats include bispecific diabodies (Db), 4 single chain diabodies (scDb), and tandem scFv (taFv) molecules, which have been applied successfully in vitro and also in vivo for the retargeting of cytotoxic T lymphocytes (via T-cell receptor molecule CD3) to tumor cells (e.g. recognizing CEA, EpCAM, or CD19) (5-8).However, these small bispecific antibody molecules with molecular masses between 50 and 60 kDa are rapidly cleared from circulation with an initial half-life of less than 30 min (9, 10). This puts some obstacles on therapeutic applications, e.g. requirement of high doses and repeated injections or infusion...

The concept of using bispecific antibodies to retarget immune effector cells for cancer therapy was conceived more than 20 years ago. However, initial clinical studies were rather disappointing mainly due to low efficacy, severe adverse effects and immunogenicity of the bispecific antibodies. A deeper understanding of effector cell biology and especially developments in the field of antibody engineering has led to the generation of new classes of bispecific antibodies capable of circumventing many of these obstacles. Furthermore, new applications were established for bispecific antibodies, such as pre-targeting strategies in radioimmunotherapy or dual targeting approaches in order to improve binding, selectivity, and efficacy. This review summarizes recent progress in the development of bispecific antibodies and describes some new concepts developed for cancer immunotherapy.

Deregulated molecular signaling pathways are responsible for the altered adhesive, migratory, and invasive properties of cancer cells. The different breast cancer subtypes are characterized by the expression of distinct miRNAs, short non-coding RNAs that posttranscriptionally modulate the expression of entire gene networks. Profiling studies have revealed downregulation of miR149 in basal breast cancer. Here, we show that miR149 expression severely impairs cell spreading, migration, and invasion of basal-like breast cancer cells. We identify signaling molecules, including the small GTPases Rap1a and Rap1b, downstream of integrin receptors as miR149 targets, providing an explanation for the defective Src and Rac activation during cell adhesion and spreading upon miR149 expression. Suppression of cell spreading by miR149 could be rescued, at least in part, by expression of constitutively active Rac. Finally, we demonstrate that increased miR149 levels block lung colonization in vivo. On the basis of our findings, we propose that miR149 downregulation in basal breast cancer facilitates the metastatic dissemination of tumor cells by supporting aberrant Rac activation. Cancer Res; 74(18); 5256-65. Ó2014 AACR.

The therapeutic application of small recombinant antibody molecules is often limited by a short serum half-life. In order to improve the pharmacokinetic properties, we have investigated a strategy utilizing fusion with an albumin-binding domain (ABD) from streptococcal protein G. This strategy was applied to a bispecific single-chain diabody (scDb CEACD3) developed for the retargeting of cytotoxic T cells to CEA-expressing tumor cells. This novel tri-functional fusion protein (scDb-ABD) was expressed in mammalian cells and recognized both antigens as well as human and mouse serum albumin. scDb-ABD was capable to retarget T cells to CEA-expressing target cells in vitro and to activate the effector cells as measured by stimulation of IL-2 release. Although activity was reduced 3-fold compared with scDb and further reduced 4-fold in the presences of human serum albumin, this assay demonstrated that scDb-ABD is active when exposed to all three antigens. Compared with scDb, the circulation time of scDb-ABD in mice was prolonged 5- to 6-fold similar to a previously described scDb-HSA fusion protein. This strategy, which adds only a small protein domain (46 amino acids) and which utilizes high-affinity, non-covalent albumin interaction, should be broadly applicable to improve serum half-lives of small recombinant antibody molecules.

Small recombinant antibody molecules such as bispecific single-chain diabodies (scDb) possessing a molecular mass of ϳ55 kDa are rapidly cleared from circulation. We have recently extended the plasma half-life of scDb applying various strategies including PEGylation, N-glycosylation and fusion to an albumin-binding domain (ABD) from streptococcal protein G. Here, we further analyzed the influence of these modifications on the biodistribution of a scDb directed against carcinoembryonic antigen (CEA) and CD3 capable of retargeting T cells to CEAexpressing tumor cells. We show that a prolonged circulation time results in an increased accumulation in CEA ؉ tumors, which was most pronounced for scDb-ABD and PEGylated scDb. Interestingly, tumor accumulation of the scDb-ABD fusion protein was ϳ2-fold higher compared with PEGylated scDb, although both molecules exhibit similar plasma half-lives and similar affinities for CEA. Comparing half-lives in neonatal Fc receptor (FcRn) wild-type and FcRn heavy chain knock-out mice the contribution of the FcRn to the long plasma half-life of scDb-ABD was confirmed. The half-life of scDb-ABD was ϳ2-fold lower in the knock-out mice, while no differences were observed for PEGylated scDb. Binding of the scDb derivatives to target and effector cells was not or only marginally affected by the modifications, although, compared with scDb, a reduced cytotoxic activity was observed for scDb-ABD, which was further reduced in the presence of albumin. In summary, these findings demonstrate that the extended half-life of a bispecific scDb translates into improved accumulation in antigen-positive tumors but that modifications might also affect scDb-mediated cytotoxicity.Bispecific single-chain diabodies (scDb) 2 are recombinant molecules composed of the variable heavy and light chain domains of two antibodies connected by three linkers in the order V H A-V L B-V H B-V L A (1). These domains assemble into molecules with a compact structure and molecular masses of ϳ55 kDa. Bispecific single-chain diabodies have been developed for various applications including the retargeting of cytotoxic T lymphocytes to tumor cells for cellular cancer therapy (2).Although scDb are capable of efficiently retargeting effector cells to tumor cells the small size leads to their rapid elimination after i.v. injection. The terminal half-life of these molecules in mice is only in the range of 5-6 h, compared with several days for whole IgG molecules (3, 4). The fast clearance of such small molecules from circulation hampers therapeutic applications, e.g. requiring infusions or repeated injections to maintain a therapeutically effective dose over a prolonged period of time (5). For example, a bispecific tandem scFv directed against CD19 and CD3 (blinatumomab) having a similar size as an scDb molecule had to be given as an 8-week infusion (maximum dose 60 g/m 2 per day) in a clinical phase I trial for the treatment of B cell lymphoma patients (6).To extend plasma half-lives of therapeutic proteins and thus to improve ph...

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The therapeutic efficacy of recombinant antibodies such as single-chain Fv fragments and small bispecific or bifunctional molecules is often limited by rapid elimination from the circulation because of their small size. Here, we have investigated the effects of N-glycosylation on the activity and pharmacokinetics of a small bispecific single-chain diabody (scDb CEACD3) developed for the retargeting of cytotoxic T cells to CEA-expressing tumor cells. We could show that the introduction of N-glycosylation sequons into the flanking linker and a C-terminal extension results in the production of N-glycosylated molecules after expression in transfected HEK293 cells. N-Glycosylated scDb variants possessing 3, 6, or 9 N-glycosylation sites, respectively, retained antigen binding activity and bispecificity for target and effector cells as shown in a target cell-dependent IL-2 release assay, although activity was reduced ϳ3-5-fold compared with the unmodified scDb. All N-glycosylated scDb variants exhibited a prolonged circulation time compared with scDb, leading to a 2-3-fold increase of the area under curve (AUC). In comparison, conjugation of a branched 40-kDa PEG chain increased AUC by a factor of 10.6, while a chimeric anti-CEA IgG1 molecule had the longest circulation time with a 17-fold increase in AUC. Thus, N-glycosylation complements the repertoire of strategies to modulate pharmacokinetics of small recombinant antibody molecules by an approach that moderately prolongs circulation time.Whole antibodies, especially chimeric, humanized or fully human IgG molecules, exhibit a long circulation time in the human body that can reach a half-life of 27 days (1, 2). In contrast, antibody fragments, e.g. Fab fragments, or recombinant formats, such as single-chain Fv fragments (scFv) 3 or bispecific derivatives thereof (tandem scFv, diabodies, single-chain diabodies), are rapidly cleared from circulation (2-4). This is mainly due to the small size leading to rapid renal clearance and the lack of recycling processes mediated by the neonatal Fc receptor (FcRn). Thus, repeated injections or infusions are required to maintain a therapeutically effective dose in the body (5). Several strategies to improve pharmacokinetic properties and thus dosing and therapeutic efficacy of recombinant antibodies have been developed in recent years. These strategies can be divided into: 1) those based on reducing renal clearance by increasing the apparent size of the therapeutic molecule, and 2) those that in addition implement FcRn-mediated recycling processes (6 -8).PEGylation of proteins is a well-established strategy to improve pharmacokinetic properties by increasing the molecular mass and the hydrodynamic radius (9 -11). Several PEGylated proteins are in clinical use, e.g. PEGylated interferon ␣-2a (PegIntron, PEGASYS) and PEGylated granulocyte-colony stimulating factor (Neulasta), or are under clinical development, e.g. a PEGylated anti-TNF Fab fragment (certolizumab pegol) (9, 12) Furthermore, direct fusion to albumin or an albumin bin...

Fusion of small recombinant antibody fragments to an albumin-binding domain (ABD) from streptococcal protein G strongly extends their plasma half-life. This ABD binds with nanomolar affinity to human (HSA) and mouse serum albumin (MSA). It was speculated that an increase in albumin-binding affinity should lead to a further increase in half-life. In the present study, we analyzed the effects of affinity and valency of the ABD on the pharmacokinetic properties of a bispecific single-chain diabody (scDb), applied previously to investigate various half-life extension strategies. The scDb is directed against carcinoembryonic antigen (CEA) and CD3 capable of mediating T cell retargeting to tumor cells. Two scDb derivatives with increased (scDb-ABD-H) and decreased (scDb-ABD-L) affinity as well as an scDb molecule fused to two ABD (scDb-ABD(2)) were generated and produced in mammalian cells. The altered binding of these constructs to HSA and MSA was confirmed by ELISA and quartz crystal microbalance measurements. All constructs bound efficiently to CEA and CD3-positive cells and were able to activate T cells in a target cell-dependent manner, although T cell activation was reduced in the presence of serum albumin. All three derivatives showed a strongly increased half-life in mice as compared with scDb. Compared with the wild-type scDb-ABD, the half-life of scDb-ABD-H exhibited a prolonged half-life and scDb-ABD-L a reduced half-life, while the half-life scDb-ABD(2) was almost identical to that of scDb-ABD. However, these changes were only moderate, indicating that the half-life-extending property of the ABD in mice is only weakly influenced by affinity for serum albumin or valency of albumin binding.