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Introduction:

The hormone gastrin17 acts as a trophic factor in several neoplasias such as pancreatic, gastric and colorectal cancers due to the over-expression of gastrin and its receptor. Antibodies generated in cancer patients by using a vaccine composed of the first 9 amino acids of gastrin17 coupled to diphtheria toxoid (Gastrimmune) improves the survival in vaccine responders (66% of patients). We have used different gastrin molecules combined with the diphtheria toxoid, to generate and select mouse monoclonal antibodies as well as human and murine scFvs against different regions of gastrin.

Experimental Procedures:

To generate the antibodies, we used gastrin17 and gastrin12 (the first 12 amino acids of gastrin17) coupled to DT or biotin at the N-terminal or the C-terminal end and three strategies, i) using human phage display antibody libraries, ii) immunizing Balb/c mices and generating anti-gastrin mouse monoclonal antibodies (Mab) and their cloning and expression like single chain variable fragments (scFvs) and iii) isolating murine scFv from a phage display antibody library derived from the spleen of a gastrin-immunized mouse. Their interaction with gastrin was characterized by ELISA, Surface Plasmon Resonance and Ala-scan for epitope delineation. We assessed the neutralizing ability of the different anti-gastrin antibodies with the AR42JB13 rat exocrine pancreatic tumour cell line transfected with the human c-fos luciferase reporter and TK-Renilla luciferase control reporter.

Results:

We obtained seventeen human and murine scFvs to gastrin 17. The murine scFvs presented a KD ranging between 15-500 nM, whereas the affinity of the human scFvs ranged between 2-200nM and 0.1-1.0μM, respectively. Three different epitopes were identified in the gastrin molecule; two of them located in the N and C-terminal end and the third one in the middle of the molecule (polyE region). The neutralizing activity of the different antibodies showed that antibodies against the N-terminus are the most efficient for blocking gastrin activity. However, when combined with antibodies against the C-terminus they showed a synergistic effect.

Conclusion:

We have produced and isolated 17 human and murine anti-gastrin scFvs directed against three different epitopes of the molecule that could be used with therapeutic purposes since all of them are able to neutralize the gastrin activity at different levels. A synergistic effect was observed when antibodies against different epitopes were combined. These findings should contribute to a more rational design of antibody-based anti-gastrin therapies in cancer. Moreover, the use of passive vaccination with these anti-gastrin scFvs might contribute to solve the problems associated with non-responders patients to gastrin vaccination.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA