Yuan QA, Simmons HH, Robinson MK, Russeva M, Marasco WA, Adams GPDevelopment of engineered antibodies specific for the Mullerian inhibiting substance type II receptor: a promising candidate for targeted therapy of ovarian cancer. Mol Cancer Ther 5(8): 2096-2105
The Müllerian inhibiting substance type II receptor (MISIIR) is involved in Müllerian duct regression as part of the development of the male reproductive system. In adult females, MISIIR is present on ovarian surface epithelium and is frequently expressed on human epithelial ovarian cancer cells. Müllerian inhibiting substance has been found to be capable of inhibiting the growth of primary human ovarian cancer cells derived from ascites and ovarian cancer cell lines. This suggested to us that MISIIR could be an attractive target for antibody-based tumor targeting and growth inhibition strategies. Here, we describe the production of recombinant human MISIIR extracellular domain-human immunoglobulin Fc domain fusion proteins and their use as targets for the selection of MISIIR-specific human single-chain variable fragments (scFv) molecules from a human nonimmune scFv phage display library. The binding kinetics of the resulting anti-MISIIR scFv clones were characterized and two were employed as the basis for the construction of bivalent scFv:Fc antibody-based molecules. Both bound specifically to human ovarian carcinoma cells in flow cytometry assays and cross-reacted with mouse MISIIR. These results indicate that antibody-based constructs may provide a highly specific means of targeting MISIIR on human ovarian carcinoma cells for the purpose of diagnosing and treating this disease.
"Amhr2 expression is detectable in 2-day-old mouse ovaries, a time when there are no follicles larger than primordial, but Amhr2 has not been localized to a particular cell type (Durlinger et al., 2002a). Given that AMH has been shown to inhibit in vitro invasion and migration of epithelial ovarian cancer cell lines that express its receptor AMHR2 (Chang et al., 2011) and that AMHR2 localizes to the surface epithelium in human ovaries (Yuan et al., 2006), it is interesting to speculate that exogenous AMH could have inhibited the migration of cells from the ovarian surface epithelium in the study by Nilsson et al. (2011). These results also imply that AMH activity/ availability should be tightly restricted during primordial follicle assembly to prevent disruption of the breakdown process. "
"Thus, homodimerization but not heterotetramerization is necessary for correct folding, which could allow for more efficient expression. In general, scFv-Fcs exhibit characteristics equivalent to their parent IgG (Shu et al., 1993; Powers et al., 2001; Cao et al., 2009) and have been tested for similar applications (Li et al., 2000; Yuan et al., 2006; Mori and Kim, 2008; De Lorenzo and D'Alessio, 2009; Olafsen et al., 2009; Riccio et al., 2009). "
[Show abstract][Hide abstract] ABSTRACT: Intracellular trafficking and subcellular deposition are critical factors influencing the accumulation and posttranslational modifications of proteins. In seeds, these processes are not yet fully understood. In this study, we set out to investigate the intracellular transport, final destination, N-glycosylation status, and stability of the fusion of recombinant single-chain variable fragments to the crystallizing fragment of an antibody (scFv-Fc) of two antiviral monoclonal antibodies (2G12 and HA78). The scFv-Fcs were expressed in Arabidopsis (Arabidopsis thaliana) seeds and leaves both as secretory molecules and tagged with an endoplasmic reticulum (ER) retention signal. We demonstrate differential proteolytic degradation of scFv-Fcs in leaves versus seeds, with higher degradation in the latter organ. In seeds, we show that secretory versions of HA78 scFv-Fcs are targeted to the extracellular space but are deposited in newly formed ER-derived vesicles upon KDEL tagging. These results are in accordance with the obtained N-glycosylation profiles: complex-type and ER-typical oligomannosidic N-glycans, respectively. HA78 scFv-Fcs, expressed in seeds of an Arabidopsis glycosylation mutant lacking plant-specific N-glycans, exhibit custom-made human-type N-glycosylation. In contrast, 2G12 scFv-Fcs carry exclusively ER-typical oligomannosidic N-glycans and were deposited in newly formed ER-derived vesicles irrespective of the targeting signals. HA78 scFv-Fcs exhibited efficient virus neutralization activity, while 2G12 scFv-Fcs were inactive. We demonstrate the efficient generation of scFv-Fcs with a controlled N-glycosylation pattern. However, our results also reveal aberrant subcellular deposition and, as a consequence, unexpected N-glycosylation profiles. Our attempts to elucidate intracellular protein transport in seeds contributes to a better understanding of this basic cell biological mechanism and is a step toward the versatile use of Arabidopsis seeds as an alternative expression platform for pharmaceutically relevant proteins.
"PS11 scFv, an antibody targeting the Tat-recognition motif (TRM) of cyclin T1 , was chosen as a model for optimizing functional expression of scFv on the surface of mammalian cells. To gain bivalency and increase the sensitivity of detecting antigens bound to surface antibody, the PS11 scFv was expressed as an scFvFc fusion protein –. For anchoring to the cell membrane, PS11 scFvFc protein was fused, in frame, to a transmembrane (TM) moiety. "
[Show abstract][Hide abstract] ABSTRACT: Isolation of human antibodies using current display technologies can be limited by constraints on protein expression, folding and post-translational modifications. Here we describe a discovery platform that utilizes self-inactivating (SIN) lentiviral vectors for the surface display of high-affinity single-chain variable region (scFv) antibody fragments on human cells and lentivirus particles.
Bivalent scFvFc human antibodies were fused in frame with different transmembrane (TM) anchoring moieties to allow efficient high-level expression on human cells and the optimal TM was identified. The addition of an eight amino acid HIV-1 gp41 envelope incorporation motif further increased scFvFc expression on human cells and incorporation into lentiviral particles. Both antibody-displaying human cells and virus particles bound antigen specifically. Sulfation of CDR tyrosine residues, a property recently shown to broaden antibody binding affinity and antigen recognition was also demonstrated. High level scFvFc expression and stable integration was achieved in human cells following transduction with IRES containing bicistronic SIN lentivectors encoding ZsGreen when scFvFc fusion proteins were expressed from the first cassette. Up to 10(6)-fold enrichment of antibody expressing cells was achieved with one round of antigen coupled magnetic bead pre-selection followed by FACS sorting. Finally, the scFvFc displaying human cells could be used directly in functional biological screens with remarkable sensitivity.
This antibody display platform will complement existing technologies by virtue of providing properties unique to lentiviruses and antibody expression in human cells, which, in turn, may aid the discovery of novel therapeutic human mAbs.
PLoS ONE 02/2008; 3(9):e3181. DOI:10.1371/journal.pone.0003181 · 3.23 Impact Factor
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