Phage antibodies able to internalize rapidly into the SKBR-3 carcinoma breast cancer cell line were obtained using a protocol based on a 15 min incubation at 37°C of the phage antibody library with the cells. Within the 55 SKBR-3 binding antibodies analyzed, approximately 50% were specific of ErbB2 (with two unique antibodies) (Poul et al., 2000
), and approximately 40% bound TfR (with six unique antibodies) a receptor known to be rapidly internalized after binding of its natural ligand (transferrin) (Dautry-Varsat, 1986
). The six fully human anti-TfR scFvs are currently being characterized with respect to their affinity for cells, yield of production in E. coli
, and their efficiency in inhibition of SKBR-3 cell proliferation, to identify the best candidate for transferrin receptor-mediated drug delivery strategies (Daniels et al., 2006
; Qian et al., 2002
). Five additional binders (non ErbB2 and non TfR binding) specific for cancer cells and showing differential binding pattern on a panel of breast cancer and colon cancer cell lines were further characterized to identify the antigen recognized by using immunoprecipitation.
Only two antibodies (3GA5 and 3GA9) immunoprecipitated a cell surface protein detectable by Western Blot. Immunoprecipitation using Ni-NTA agarose, which captures scFvs via their (His)6 tag, led to immunoprecipitation of many intracellular proteins, complicating antigen identification by in gel digestion and LC-MS-MS. In contrast, immunoprecipitation with Protein A agarose gave much cleaner immunoprecipitates. Since approximately 25% of the scFvs in the parental phage antibody library have human VH3 VH domains and bind protein-A, the results suggest that when possible Protein A-sepharose be used for immunoprecipitation.
Mass spectrometry analysis of the 130 kD antigen immunoprecipitated by scFv 3GA5 led to the identification of CD9P-1, which was initially described as a major partner for the CD9 and CD81 tetraspanins (Charrin et al., 2001
; Stipp et al., 2001b
). Tetraspanins are a large super family of evolutionarily conserved cell-surface proteins characterized by four transmembrane domains and two extracellular loops. At the molecular level, tetraspanins have been shown to interact with each other and also with other transmembrane proteins like integrins (Stipp et al., 2003
) forming a molecular network known as the “Tetraspanin Web”. The Tetraspanin Web function is related to spatial and temporal organisation of membrane complexes/domains (Hemler, 2003
). At the cellular level, tetraspanins have been implicated in various physiological processes including cell motility, metastasis, cell proliferation and differentiation. CD9P-1 has been shown to interact directly with the large extracellular loop and/or the fourth transmembrane domain of CD9 tetraspanin through a stoichiometric association (Charrin et al., 2001
). Considering the putative implication of CD9P-1 in the acquisition of a transformed phenotype (Charrin et al., 2001
) and the lack of information on its role within the tetraspanin web, we explored using scFv-3GA5 to obtain more information on CD9P-1 function.
Single chain Fv-3GA5 targeting led to rapid internalization of immunoliposomes into SK-BR-3 cells as demonstrated by both fluorescence microscopy and quantitative flow cytometry analysis. We therefore evaluated the ability of 3GA5 to downregulate cell surface CD9P-1. Despite the fact that 3GA5 was rapidly endocytosed, no variation of cell surface levels of CD9P-1 or of partner CD9 and CD81 were observed following 2 days incubation with saturation conditions of soluble scFv-3GA5. This might be due to rapid recycling of CD9P-1 to the cell surface. Sustained incubation of soluble scFv-3GA5 with SKBR-3 cells, or with HS-578T cells which express higher levels of CD9P-1, also had no effect on cell proliferation nor on cell adhesion. These indicate that targeting CD9P-1 with the 3GA5 scFv does not down regulate the receptor nor cause associated anti-tumor effects. However, 3GA5 can be used to delivery nanoparticles specifically to CD9P-1 expressing tumor cells. Such an approach merits further study with respect to the ability to achieve a tumor specific cytotoxic effect in vitro
and in vivo
(Nielsen et al., 2002
; Park et al., 2002
; Noble et al., 2004
; Mamot et al., 2005
Single chain Fv-3GA5 mass was evaluated at around 28 kD by size exclusion chromatography (not shown) indicating that it does not spontaneously form dimers (also called “diabodies”) which have been observed with other scFvs, which would enhance its affinity for cells and perhaps favor more efficient binding and/or internalization. Internalisation via CD9P-1 might be more efficient in the immunoliposome format due to increased avidity due to multivalent binding, since each liposome bears 30 molecules of scFv/liposome. In conclusion, the ability of 3GA5 to internalize into target cells via the tumor marker CD9P-1 makes 3GA5 a highly suitable candidate for targeted immunotherapy via liposomal drug format.
Since soluble scFv 3GA5 did not downregulate CD9P-1 when added to culture media, intracellular expression of single chain Fv antibodies, so called ‘intrabodies’, was explored as a means to knock down CD9P-1. Intracellular expression of antibody fragments like scFvs can result in correct folding and conserved antigen-binding properties in certain examples (Cardinale et al., 2004
). The intracellular scFv trafficking can also be directed by fusion to a specific intracellular localization sequence (Persic et al., 1997
). As a result of these properties, the interaction of intrabodies with Ag can achieve a “phenotypic knockout” that may give insights into Ag function (Visintin et al., 2004
) and, in the case of tumor associated antigens, revert a transformed phenotype (Tanaka and Rabbitts, 2003
). Intracellular expression of scFv 3GA5 was tested for the ability to knock down the surface display of CD9P-1. The ER retention signal KDEL sequestered scFv 3GA5 in the ER and this sequestration resulted in a significant decrease of the surface display of CD9P-1, possibly because 3GA5 bound to the newly synthesized CD9P-1 polypeptide. Other evidence for this phenotypic knock out of surface CD9P-1 is that nearly 50% of CD9P-1 from the total cell extracts showed smaller apparent molecular weights of 105 kD, which indicated the glycosylation of CD9P-1 during post-translational modifications along the secretory pathway was hampered. The remaining approximately 50% of CD9P-1 with molecular weight of 130 kD, could be due to the incomplete knock-down effect resulting from transient expression of scFv 3GA5 and/or to low CD9P-1 cell surface recycling rate.
In summary, we have demonstrated that direct selection of phage antibody libraries on tumor cell lines can be used to generate panels of antibodies with differential tumor cell binding. Antibody specificity can be identified using immunoprecipitation and Western blotting using mAbs to known antigens, or by immunoprecipitation with the novel scFv and antigen identification by LC-MS-MS. Resulting scFv can be used to study whether the antibody-antigen pair have associated anti-tumor biology amenable to blockade with a naked antibody approach. In the case of the 3GA5 scFv and CD9P-1 antigen studied here, this was not the case. Antibodies generated using this approach, however, are also rapidly internalized by cells expressing antigen, opening up targeted therapeutic approaches using immunotoxins, immunoliposomes, or antibody-drug conjugates.