Serum starved Caco-2 cells were incubated with negative control (C) or anti-hTGF (T) polyclonal antibodies (at different dilutions) for 12h and subsequently lysed with lysis buffer

Serum starved Caco-2 cells were incubated with negative control (C) or anti-hTGF (T) polyclonal antibodies (at different dilutions) for 12h and subsequently lysed with lysis buffer. a mouse model using different adjuvants. All immunogens were effective for the generation of specific humoral responses against hTGF. The inmunodominant epitope of hTGF when immunizing mice with the fusion protein involved the C-loop/C-terminal region. This region includes key residues for hTGF binding to EGFR. The anti-hTGF immune mice sera recognized the natural hTGF precursor in A431 cells and hTGF-transfected 3T3 fibroblasts as revealed by flow cytometry analysis and immunoblotting. They inhibited the binding of125I-TGF to the EGFR, EGFR-autophosphorylation, and downstream activation of MAP kinases as well Flavopiridol (Alvocidib) as proliferation of two EGFR-expressing human carcinoma cell lines. These data Flavopiridol (Alvocidib) suggest that EGFR signaling activation by the hTGF autocrine loop may be inhibited in vivo by induction of specifically blocking antibodies. The fusion protein reported in this paper could be a potential immunogen for the development of a new cancer vaccine. Keywords:hTGF, Active specific immunotherapy, Growth factor immunodeprivation, Cancer vaccine == Introduction == The epidermal growth factor receptor (EGFR) family and the corresponding peptide ligands are involved in normal and neoplastic development, and many of them are over-expressed in human carcinomas as compared with their normal counterpart [1]. The simultaneous presence of the EGFR and its ligand transforming growth factor alpha (TGF) in human tumor tissues suggests that the TGF autocrine loop drives tumor growth. In fact, TGF/EGFR co-expression is considered to be an indicator of bad prognosis for many epithelial tumors [29]. Besides, TGF is a more potent angiogenic factor than EGF [10] and cooperates with other oncogenes (e.g., c-myc and ras) or chemical carcinogens to promote tumorigenesis [11]. In this context, TGF might represent a suitable target for novel therapeutic approaches in cancer. Passive immunotherapy with specific monoclonal antibodies (MAbs) against EGFR (e.g., Imclone C225 and Theracim hR3), combined or not with chemotherapy or radiotherapy [1,12,13], and receptor tyrosine kinase inhibitor drugs [14] are currently in clinical trials. Following an active specific immunotherapy approach, our group has developed a cancer vaccine based on EGF-immune deprivation, aiming to stop EGF-dependent tumor growth. This vaccine has revealed promising preclinical [15,16] and clinical results [17,18]. Obviously, an EGF vaccine can only block the action of EGF but will have no effect on TGF. Therefore, many epithelial tumors which depend in growth on EGFR signaling would escape from EGF-immunodeprivation therapy by up-regulation of the TGF autocrine loop. Thus, an active specific immunotherapy approach targeting TGF seems highly warranted. To our best knowledge, such an approach has not yet been attempted. The main goal of this work was to assess the Flavopiridol (Alvocidib) potential of an active specific immunotherapy approach to block the TGF/EGFR autocrine loop. For the proof of the concept a fusion protein between human TGF (hTGF) and a highly immunogenic carrier protein was designed and expressed inEscherichia coli. The immunogenicity of this recombinant protein was characterized in a mouse model using different adjuvants and the induction of TGF-blocking antibodies was analyzed. Sera from immunized mice inhibited in vitro TGF-mediated EGFR signaling activation. == Materials and methods == == Cell culture == A431 human epidermoid carcinoma, H125 human lung adenocarcinoma, and Caco-2 colon LDH-A antibody cancer cell lines were obtained from the ATCC (USA). Balb-3T3 murine fibroblast cell line and Balb-3T3 cells transfected with hTGF cDNA were kindly provided by Dr. Diana del Barco (CIGB, Havana, Cuba). All cell lines were maintained in DMEM medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal calf serum (FCS, Hyclone). == Design of pMhisTGF expression vector == The gene coding for hTGF (150 bp long) was amplified by PCR using the PSK-TGF plasmid (kindly provided by CIGB, Havana, Cuba) coding for the complete hTGF gene (500 bp long) as template and oligonucleotides 4292 (sense, 5 GCT.