Cellular Implants as mini-factories for the production of therapeutic antibodies against Altzheimer s disease. rapport research week Jonas Schiffmann

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1 Cellular Implants as mini-factories for the production of therapeutic antibodies against Altzheimer s disease rapport research week Jonas Schiffmann Supported by Aurélien Lathuilière, Bernard Schneider Laboratory for the Study of Neurodegenerative Diseases

2 Introduction A lot of people suffer from Alzheimer's disease, a major health problem for our aging societies. Alzheimer s dementia is caused by a massive loss of neurons, leading to a decrease of the brain mass. At the cellular and biochemical levels, a figure 1 : healthy brain figure 1 : Alzheimer's disease brain figure 3 : plaques in the brain possible reason for neuronal loss is the accumulation of aggregated Amyloid-beta peptides in so called plaques, which build up in the brain of diseased people. The idea of the research is creating implants which are able to produce antibodies directed against the Amyloid-beta peptide. These implants will be put under the skin and will secrete antibodies in the blood circulation and penetrate the brain to eliminate disease-causing plaques. So what we need are cells that secrete antibodies against the Amyloid-beta peptide. In particular, we use a dividing cell line derived from the muscle. Normal muscle cells wouldn t do that, that s why we have to genetically modify them using a virus which changes the cellular DNA. By introducing a gene containing the figure 2 : an example of an effect by a concerned person specific sequence, cells start to produce our monoclonal antibody. 2

3 To find the amount of lentivirus leading to maximal antibody production, we used 5 different doses of the virus which were tested in duplicates for our experiments. Methods To assess the secretion level of genetically engineered cells, we performed an Enzyme Linked Immunosorbent Assay (ELISA). Briefly, the cells infected with the various virus doses were plated at a density of 100'000 cells per well in 6-well plates in figure 5 ELISA duplicates. The day after, fresh culture medium was applied for one hour and collected for the assay. The cells were then counted by using an automated cell counter. The antibody concentration measured in the ELISA assay was used to determine the rate of antibody secretion, which is then expressed in picogram per cell per day. figure 6 : western plot figure 7 : figure 8 : IgG antibody 3

4 Arbitrary Units Arbitrary Units To further characterize the expression of both heavy and light antibody chains, we performed a western blotting of cell extracts. Cells were harvested and lysed in a detergent-containing buffer. Total protein concentration was measured using the BCA assay. 40 micrograms of total proteins were loaded on either a 12% (light chain blot) or a 7.5% (heavy chain blot) acrylamide gel. After migration in an electrical field, proteins were transferred on a membrane and probed with either an antimouse IgG light chain-specific antibody or an anti-mouse IgG heavy chain-specific antibody. Results Op cal density of full IgG an body Op cal density of free light chains Mul plicity Of Infec on (MOI) Mul plicity Of Infec on (MOI) 4

5 Discussion/conclusion We found that the cellular production of antibodies directly depends on the amount of lentivirus used to infect the cells. However, if more than 1000 copies of effective virus is applied per cell, there is no more increase in the amount of antibody secreted because the saturation is reached and the cells can t produce more. The accumulation of the complete antibody showed that the cells synthetize both the light chain and the heavy chain. However, it remains unclear if free light chains are produced, which could be a problem for the therapeutic application of these cells. 5

6 Thank I want to thank Aurélien Lathuilière for all his patience and explanations. Then I thank also Bernard Schneider for his help. But also big thanks go to the Laboratory for the Study of Neurodegenerative Diseases and the EPFL which helped and supported me during my researches. Thanks also to Schweizer Jugend Forscht which made it for me possible to have such a great research week. 6

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