Development of Chimeric Antigen Receptor T cells to Target Development of Cancer
Faculty Mentor: Dr. Scott Weber, Department of Microbiology and Molecular Biology
Cancer negatively affects the lives of millions of individuals, and remains a difficult ailment to treat. In 2014, approximately 585,720 deaths and 1,665,540 new cases of cancer were reported in the USA alone.1 Treatment of cancer is challenging due to cancer mutating to combat the body’s immune system. An example is shown in major histocompatibility complexes (MHCs) located on the surface of nearly every cell. These are patrolled by T cells to ensure that cells are properly functioning and healthy. With cancer however, these become downregulated allowing the cancer to grow and proliferate since T cells are unable to detect them. Chimeric antigen receptor T cells (CAR T cells) overcome this by bypassing the use of MHCs and allowing specific targeting and destruction of cancer cells based on a surface epitope. Recently, CAR T cells targeting B cell cancers have resulted in cancer recession or removal, but overall have had limitations to the extent of cancers treatable.2 This is due to the same epitopes being expressed on both cancer cells and normal, healthy cells, resulting in both cell’s destruction. However, an epitope has been found by the Dr. Kim O’Neill which is highly overexpressed on many types of cancers, allowing T cells to target the malignant cells only. In collaboration with the O’Neill lab, we have begun generating a CAR T cell that specifically targets this epitope. This process has involved sequencing of antibodies that target the epitope; creating a single chain fragment variable (scFv), which links the heavy and light chains of an antibody; and placing this into a T cell. For this project, the sequencing and creation of the scFv was performed.
In order to create the scFv for the CAR T cell, B cells (CB1) were generated to target the epitope with an IgG1 antibody. The heavy and light chains of the B cell receptor or antibody were then sequenced. This was done by searching databases, such as IMGT, for previously sequenced light and heavy chains of IgG1 antibodies and developing degenerate primers, which could amplify those regions in a polymerase chain reaction (PCR). The primers used focused on regions of high homology within the variable region of the light and heavy chains.
After PCR of the antibody chains, the products underwent gel electrophoresis to ensure the proper target was being amplified and to aid in DNA extraction. The light and heavy chains of CB1 were sequenced at the BYU sequencing center. Analysis through Geneious compared the homology of the CB1 antibody sequences to other similar sequences of the whole antibody and the highly variable CDR regions where antibodies bind to epitopes. Once the sequences were confirmed, restriction sites and a Gly-Ser linker were added to both the heavy and light chain through the use of tailed primers. The products were then spliced with overlap extension PCR using the overlapping regions of the Gly-Ser linker. The restriction sites were added for insertion into a vector specific for CAR T cells.
Initial amplification of antibody sequence came from degenerate primers loosely based off of the IgG1 antibody. Once some product was obtained, that was sequenced to develop stronger primers capable of accurately amplifying the entire light and heavy chains of CB1. Through this we determined that the heavy chain belonged to the IgG1 class family 14 and the light chain belonged to the IgG1 class family 4. Our results were compared to several complete IgG1 antibody sequences using the program Geneious. On average there was a 51.8% homology to other IgG1 antibodies with only a 22.7% homology within the highly variable CDR regions.
After ensuring that we had a unique antibody specific to the epitope, development of the scFv was done using tailed primers. These added two different restriction sites to opposite ends of the heavy and light chains, while adding a partial Gly-Ser linker sequence to the other end of the amplified sequence. Using primers specific for the restriction sites and linker, the heavy and light chains were linked together to form the scFv product. This was confirmed using gel electrophoresis and sequencing.
The homology between the CB1 antibody variable regions and other IgG1 antibodies suggests that there is significant difference in the binding of epitopes. This allows us to presume that the CB1 antibody binds specifically the targeted epitope and not to other epitopes which could be present throughout the body. After performing the overlap extension PCR, gel electrophoresis and sequencing showed that the scFv was properly made. This would then be placed into a vector which could be introduced into T cells for expression. For a CAR T cell to function properly, the vector also contains the sequences for a signal peptide and costimulatory domains, which allows the receptor to activate the T cell against its target. Once the CAR T cell has been generated, tests will follow to ensure proper targeting of cancer cells ranging across multiple cell lines.
The sequencing of the B cells initially made against the epitope was necessary for the creation of the CAR T cell. This allows the CAR T cell to target multiple cancer cells even when they’ve undergone mutations preventing regular T cells from destroying them. Although my time in research ended with the creation of the scFv, others have continued on with the process. The vector containing the scFv of the CB1 antibody variable regions and costimulatory domains have been inserted into a T cell line and tested against cancer cells. A video was created showing targeting of the T cells to the cancer cells and in the case of one cell, apoptosis ensued. There is still more experimentation and development needed for the project, but progress is continually being made by members of both the O’Neill and Weber labs.