Designing a HER2/neupromoter to drive α1,3galactosyltransferase expression for targeted anti-αGal antibody-mediated tumor cell killing

The enzyme α1,3galactosyltransferase (αGalT) is responsible for the synthesis of galactose-α1,3galactose-β1,4N-acetylglucosamine-R (αGal) epitopes in all mammals except Old World primates [1]. Highly expressed in nonprimate mammals, prosimians and New World monkeys, this glycosyltransferase has been mutationally inactivated during the course of evolution, starting from Old World primates [2]. We have previously shown that, in human cells, transcription of the αGalT gene is interrupted by the presence of a strong stop signal in exon 7, which leads to a chimeric mRNA comprising the first four coding exons and part of intron VII, but lacking the last two exons corresponding to the catalytic domain of the enzyme [3]. As a consequence, and given the broad circulation of αGal carbohydrate antigens, humans, apes and Old World monkeys produce large amounts of anti-αGal antibodies, which represent approximately 1% of total IgG in humans [4]. These antibodies are responsible for the hyperacute rejection of xenografts and thus prevent trials on transplantation of pig organs to humans [5, 6]. Conversely, they represent a potential constitutive tool for therapeutic applications because their highly efficient cytolytic activity could be directed against pathological cells transgenically modified to express αGal epitopes [7‐10].

Gene therapy based on the induction of cytotoxicity generally makes use of transgenes that encode prodrug activating enzymes [11]. In the case of anti-αGal-induced cytotoxicity, no chemical drug is needed to obtain the effect of αGalT because natural circulating anti-αGal antibodies are sufficient to promote cell lysis via complement activation. One common problem with gene therapy is target cell selectivity. A frequent solution to this is the use of tissue-specific or tumor-activated promoters to drive expression of the transgene [12, 13]. Human c-erbB-2 (synonyms erbB2, HER2/neu), a member of the erbB family that is overexpressed in about one third of breast tumors and in a variety of other tumors, is often correlated with a poor prognosis [14‐16]. This gene is normally expressed at a low level in a variety of human embryonic and adult epithelial and hematopoietic cells [17, 18]. The high overexpression of c-erbB-2 in tumor cells [19] results from multiple mechanisms, including gene amplification and transcription modulation [20‐22]. c-erbB-2 is a 185 kDa transmembrane tyrosine kinase receptor related to the epidermal growth factor receptor that functions as a growth factor receptor to regulate cell growth and transformation [23‐25]. Regulation of the c-erbB-2 promoter (pNeu) has been extensively investigated in a domain located within the 700 bp region upstream of its transcription start site. A -213/-87 fragment relative to the gene's transcription start site contains the minimal promoter region able to drive preferential transgene expression in breast cancer cells [26].

Efforts to develop anticancer therapies based on suicide transgenes generally focus on prodrug activating enzymes [38] combined with effective targeting of pathological cells. We have been studying αGalT gene expression and the very high efficiency of natural anti-αGal antibodies in inducing complement-mediated cell killing [39, 40] in the field of hyperacute xenograft rejection. We attempted to take advantage of this constitutive immune system to target tumor cells. Several authors have demonstrated the efficacy of natural anti-αGal antibodies for destruction of tumor cells [7‐10]. Indeed, the high numbers of circulating oligosaccharides bearing αGal epitopes are responsible for constant booster immunizations. This may explain the high plasma level (1% of IgG) of anti-αGal antibodies in humans [4] and their constant de novo synthesis in αGalT knockout mice [41, 42]. Natural anti- αGal antibodies are highly cytotoxic and cytolytic as the result of highly efficient complement activation, and this results in hyperacute rejection of xenografts [2, 40]. They have also been shown capable of protecting αGalT-deficient mice against engrafted αGal⁺ colon cancer cells [9].

The purpose of this study was the targeting of tumor cells known to overexpress c-erbB-2 using a selected form of its promoter pNeu to drive an active αGalT. This approach was designed to take advantage of the effective antibodies preexisting in all humans. In a similar study, a derived form of the human telomerase promoter was shown to render human pancreatic carcinoma cells susceptible to αGal/complement-mediated cell killing [43]. We selected pNeu because it has been well characterized and is overexpressed in a variety of tumors [16, 44]. Because definition of a precise pNeu sequence with well-restricted activation in tumor cells remains uncertain, however, we analyzed several forms of this promoter. The shortest form, pNeu209, which comprises the only PEA3 motif adjacent to the TATA box plus two SP1 sites and one AP-2 site downstream of the transcription start site, promoted very weak αGal expression. The minimal forms, pNeu392 and pNeu250, were equally capable of selectively inducing αGalT in breast tumor cells compared with HEK-293 cells. We thus concluded that the Ap-2 and SP1 motifs downstream of the transcription start site (Fig. 2) were not essential. The noticeable absence of a CCAAT box in pNeu209 probably explains its disrupted activity because in cells over-expressing c-erbB-2, the CCAAT box is up-regulated rather than the TATAA box [45]. Further studies thus focused on comparing pNeu250 with the longest form, pNeu664. This last form contains several PEA3, NF-kB, HER2 transcription factor (HTF) and SP1 sites upstream of the minimal pNeu250. The role played by the Ets family and activator protein-2 (AP-2) factors has been extensively studied in breast tumor cells. While the AP-2 binding site was present in both pNeu250 and pNeu664, the main difference between these forms was the presence of three additional PEA3 motifs in pNeu664. Activation of pNeu664 was virtually the same in MCF-7 and SK-BR-3 tumor cells, whereas a marked decrease in pNeu250 activity was observed only in SK-BR-3 cells (Fig. 3), in complete contrast to their high c-erbB-2 expression (Fig. 2c). As discussed above, the striking overexpression of c-erbB-2 in SK-BR-3 cells can be explained by their multiple gene copies. In MCF-7 cells, the differential promoting activity of pNeu250 could be explained by the relative increase in the transcription level compared with HEK-293 cells. In other aspects, in association with c-erbB-2 gene amplification, up-regulation of transcriptional factors that control endogenous pNeu remains possible. Conflicting results have been published on Ets regulation of c-erbB-2, with activation and repression of pNeu by PEA3 factors having been reported [46, 47]. The observation that Ets binding leads to a severe bend in DNA could be further support for our findings [46]. When the number of PEA3 binding sites is reduced from four in pNeu664 to one in pNeu250, over-occupation of the single remaining site in pNeu250 might hinder formation of the required DNA conformation rather than favor its reading.

The differential promoting activity of pNeu664 and pNeu250 in HEK-293 cells (Fig. 3) does not appear to be relevant to the transcriptional regulation of c-erbB-2 because this gene is only weakly expressed in these cells (Fig. 1, lane 1). In contrast, HEK-293 cells continuously express ad5 E1A, which has been shown to target pNeu [48] as a repressor of HER2/neu overexpression [49], and has been proposed for use in cancer gene therapy [50]. Like the Ets factors expressed in tumor cells, an equal level of E1A in HEK-293 cells might activate pNeu664, which contains four PEA3 motifs, and repress pNeu250, which has only one.

Our efforts to take advantage of natural cytotoxic anti-αGal antibodies as a means of destroying breast tumor cells, and to design a promoter specific for these undesirable cells, have to be considered as a preliminary contribution to the field of cancer gene therapy, given that our results have been obtained in cell line culture models. It has been shown that human primary breast tumors can be successfully engrafted into NOD/SCID mice and maintained in a growing state for more than 100 days [51]. Moreover, αGalT(-/-) KO mice have been fortunately generated by others [52, 53]. So to progress towards a gene therapy application, we are developing a two step procedure in mice. First, the distribution and expression of the transgene pNeu250/mαGalT, cloned into the retroviral vector pcPMΔU3 (see Materials and methods), will be studied in a human breast cancer xenograft model. Various types of human breast tumor differentially expressing HER2/neu will be implanted in NOD/SCID mice, and thereafter the transgene will be injected by a variety of methods. Second, αGalT-transduced tumor pieces will be transplanted into immunocompromised αGalT KO mice to evaluate the tumor destroying activity of purified human anti-αGal antibodies.

Our results show that the association pNeu250/mαGalT could be used to target tumor cells overexpressing c-erbB-2, and thus expose them to the cytolytic activity of natural anti-αGal antibodies. Development of a discriminating in vivo system capable of targeting tumor cells according to their level of c-erbB-2 expression could prove beneficial.