Kirstin A. Zettlitz

N-glycosylation as novel strategy to improve pharmacokinetic properties of bispecific single-chain diabodies.

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 ∼3–5-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.

The effects of affinity and valency of an albumin-binding domain (ABD) on the half-life of a single-chain diabody-ABD fusion protein

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.

ATROSAB, a humanized antagonistic anti-tumor necrosis factor receptor one-specific antibody

Tumor necrosis factor (TNF) signals through two membrane receptors, TNFR1 and TNFR2, and TNFR1 is known to be the major pathogenic mediator of chronic and acute inflammatory diseases. Present clinical intervention is based on neutralization of the ligand TNF. Selective inhibition of TNF receptor 1 (TNFR1) provides an alternative opportunity to neutralize the pro-inflammatory activity of TNF while maintaining the advantageous immunological responses mediated by TNFR2, including immune regulation, tissue homeostasis and neuroprotection. We recently humanized a mouse anti-human TNFR1 monoclonal antibody exhibiting TNFR1-neutralizing activity. This humanized antibody has been converted into an IgG1 molecule (ATROSAB) containing a modified Fc region previously demonstrated to have greatly reduced effector functions. Purified ATROSAB, produced in CHO cells, showed strong binding to human and rhesus TNFR1-Fc fusion protein and mouse embryonic fibroblasts transfected with a recombinant TNFR1 fusion protein with an affinity identical to the parental mouse antibody H398. Using chimeric human/mouse TNFR1 molecules, the epitope of ATROSAB was mapped to the N-terminal region (amino acid residues 1-70) comprising the first cysteine-rich domain (CRD1) and the A1 sub-domain of CRD2. In vitro, ATROSAB inhibited typical TNF-mediated responses like apoptosis induction and activation of NFκB-dependent gene expression such as IL-6 and IL-8 production. These findings open the way to further analyze the therapeutic activity of ATROSAB in relevant disease models in non-human primates.

A novel expression and purification system for the production of enzymatic and biologically active human granzyme B.

The serine protease granzyme B (grB) has previously been shown to induce perforin-independent apoptosis in membrane Hsp70 positive tumor cells in a number of in vitro experimental systems. Ongoing studies that are investigating the in vivo relevance of these findings by assessing the therapeutic potential of grB in a human xenograft tumor mouse model required the development of an expression system for the production of high yields of enzymatic and biologically active human grB. In order to maintain potentially important posttranslational modifications that occur in mammalian cells, human embryonal kidney cells (HEK293) were stably transfected with human grB. The HEK293 host cells were protected from apoptotic cell death by fusing an inactivation site coupled to a (His)(6) tag to the gene sequence of GrB. Inactive grB which was actively released from HEK293 cells by insertion of a Igκ leader sequence was purified on a nickel column utilizing the (His)(6) tag. After enterokinase digestion and heparin affinity chromatography, high yields of enzymatic and biologically active human grB were obtained. The perforin-independent interaction of grB with membrane Hsp70 positive tumor cells appeared to be associated with mammalian glycosylation and mediated by the oligosaccharide moiety of neuraminic acid (NANA).

Murine endoglin-specific single-chain Fv fragments for the analysis of vascular targeting strategies in mice.

Endoglin has been identified as a promising cell surface antigen for vascular targeting approaches in cancer therapy, e.g. employing antibody molecules as targeting moieties. However, in vivo analysis of such strategies in mouse models requires antibodies recognizing endoglin on mouse endothelial cells. Here we describe the isolation of single-chain Fv fragments (scFvs) from phage display libraries, which bind to the extracellular region of mouse endoglin. One of these clones, scFv mE12, showed strong (K(d)=11 nM) and selective binding to purified endoglin and also to the endoglin-expressing mouse endothelioma cell line eEnd.2. This antibody recognized a linear epitope located in the N-terminal region (aa 27-361) of endoglin. Cell binding was further increased by generating a bivalent scFv-Fc fusion protein composed of scFv mE12 and the human gamma1 Fc part. Moreover, scFv mE12 was endowed with an additional cysteine residue in the linker region and applied for the generation of anti-endoglin scFv immunoliposomes capable of selectively binding to endoglin-expressing cells. Thus, anti-mouse endoglin scFv mE12 should be useful to analyze vascular targeting strategies in mice.

Humanization of a Mouse Monoclonal Antibody Directed Against a Cell Surface-Exposed Epitope of Membrane-Associated Heat Shock Protein 70 (Hsp70)

The translocation of heat shock protein 70 (mHsp70) into the plasma membrane has been found to be associated with various cancers including breast cancer, head-and-neck cancer, and acute myeloid leukemia. Parts of the C-terminal substrate-binding domain (SBD) of mHsp70 are accessible to binding by monoclonal antibodies (mAb). One of these mAbs, cmHsp70.1, has been extensively studied and showed promising results as diagnostic and therapeutic antibody. Here, we describe cloning and humanization of cmHsp70.1 by complementarity determining region grafting resulting in an antibody (humex) possessing a similar affinity (3 nM) as the parental antibody and an improved production and thermal stability. Epitope mapping confirmed that the parental, chimeric, and humanized antibodies recognize the same region including amino acids 473–504 of the SBD. Hence, this humanized antibody provides a basis for further development of an anti-mHsp70 antibody therapy.

Monovalent TNF receptor 1-selective antibody with improved affinity and neutralizing activity

ABSTRACT Selective inhibition of tumor necrosis factor (TNF) signaling through the proinflammatory axis of TNF-receptor 1 (TNFR1) while leaving pro-survival and regeneration-promoting signals via TNFR2 unaffected is a promising strategy to circumvent limitations of complete inhibition of TNF action by the approved anti-TNF drugs. A previously developed humanized antagonistic TNFR1-specific antibody, ATROSAB, showed potent inhibition of TNFR1-mediated cellular responses. Because the parental mouse antibody H398 possesses even stronger inhibitory potential, we scrutinized the specific binding parameters of the two molecules and revealed a faster dissociation of ATROSAB compared to H398. Applying affinity maturation and re-engineering of humanized variable domains, we generated a monovalent Fab derivative (13.7) of ATROSAB that exhibited increased binding to TNFR1 and superior inhibition of TNF-mediated TNFR1 activation, while lacking any agonistic activity even in the presence of cross-linking antibodies. In order to improve its pharmacokinetic properties, several Fab13.7-derived molecules were generated, including a PEGylated Fab, a mouse serum albumin fusion protein, a half-IgG with a dimerization-deficient Fc, and a newly designed Fv-Fc format, employing the knobs-into-holes technology. Among these derivatives, the Fv13.7-Fc displayed the best combination of improved pharmacokinetic properties and antagonistic activity, thus representing a promising candidate for further clinical development.