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Content archived on 2024-06-16

Therapeutic synthetic antibodies -binding bodies- against gastrin to treat pancreatic cancer

Final Report Summary - BINDING GASTRIN (Therapeutic synthetic antibodies -binding bodies- against gastrin to treat Pancreatic Cancer)

Pancreatic cancer is diagnosed 60 000 times in the United States and the European Union each year. Almost the same number of people die of this disease each year. This illustrates that all existing therapies have little effect once the disease is diagnosed; survival time after diagnosis is 80 to 160 days. Gastrin drives the tumour growth. Recently, a vaccine against gastrin is developed able to double the survival time and to improve the quality of life of pancreatic cancer patients. Unfortunately, only a limited number of individuals respond to the vaccine and produce antibodies, and even then development of antibodies is slow which is especially problematic for this fast progressing disease.

We proposed to develop an alternative which will take effect immediately in all individuals treated. To this end, we proposed to develop high affinity and low cost synthetic antibodies -'binding bodies'- against gastrin. Binding bodies are two or more peptides which represent CDR's (complementary determining regions, which are the hypervariable and antigen-interaction parts of the binding site of an antibody) coupled covalently to a small chemical scaffold. The concept works: ample evidence shows that peptides representing single CDR's can bind antigen. Preliminary results show that combining peptides representing at least two different CDR's give much higher binding and much more specificity. We speculate that further optimisation should yield low cost synthetic antibodies - binding bodies - with activities similar to natural antibodies.

It was concluded that many of the results published previously by research groups active in the field of antibody-mimicry draw a too optimistic view for solving this highly-interesting scientific problem. Published data claiming that linear and single-loop peptides provide good mimics of antibody binding sites, with affinities only 10-100 fold lower in Kd, could not be reproduced and most likely are based upon incorrect interpretation of binding data (strong aggregation). In our hands such mimics were of low-affinity and non-specific binders. Therefore, the starting point of this project was much different than expected.

Nevertheless, we succeeded in this project to provide proof-of-principle data for the development of lowMW bb's (~2.5 kDa) for the peptide-hormone gastrin based on CDR sequence information of high-affinity anti-gastrin mAb's and anti-gastrin scFv-fragments developed through this project. Previous work has always focussed on the binding of large proteins. Performing a similar result with a low MW targets like gastrine (MW = 2.5 kDa) should be regarded as a major achievement by itself.

With the help of high-throughput screening and bioinformatics a new generation of binding bodies was developed that binds gastrin with 100 - 500 microM affinity and indeed was able to neutralise the bioactivity of gastrin in a tumour-cell bioassay. The major hurdle to be taken when it comes to increasing the binding affinity of anti-gastrin bb's is to solve the problem of self-aggregation. Most bb's suffer from this problem and 99 % of the efforts to improve affinity of bb's also increases their tendency to self-aggregate. This turned out to be the key problem in characterising lowMW bb's and optimising their binding affinity. Natural antibodies hardly suffer from this problem, since evolution likely has solved this problem already long ago. The double- and triple-CDR bb's as developed in this project may provide a solid structural basis for developing high-affinity bb's. Additional studies that reveal if these bb's adopt secondary structure in solution, and if so, which structural entities are responsible for that, are desperately needed and also provide the basis for developing a scaffold with well-defined three-dimensional (3D)-structure in solution with general applicability in the field of antibody- and in general protein-mimicry. For therapeutic and clinical applications of anti-gastrin bb's, high-affinity binding is definitely needed.

In this project, we have provided the proof-of-principle that selective recognition of gastrin with lowMW-bb's is indeed feasible. The development of high-affinity binders for gastrin is still fully realistic, However, further studies will be required to achieve this, for which the duration of this 2.5- years project was unfortunately too limited.