A Homing System Targets Therapeutic T cells to Brain Cancer

Through statistical analysis the American Cancer Society estimates that there will be over 1.7 million new cancer cases, and just over 600,000 deaths from cancer in the United States during the year of 2018.¹ Cancer results from different mutations in a person’s cells that cause uncontrollable cell replication. The set of mutations leading to cancer can be different for each patient, which makes treatment complicated.² T cell immunotherapy is a promising new type of cancer therapy in which T cells, a part of the immune system, can be engineered to target and combat cancer cells.^3,4 There is a problem implementing this therapy for brain cancer however, because the cancer prevents the T cells from entering the brain through the blood-brain barrier, and thus T cells cannot effectively fight the cancer.⁵ Samaha and associates aimed to solve this problem by engineering T cells with a molecule that allows them to pass through the blood-brain barrier, enabling them to combat brain cancer.

            In other brain diseases that illicit a T cell response, the T cells bind to a protein called Activated Leukocyte cell adhesion molecule (ALCAM) on the endothelial cells that make up the surface of the blood-brain barrier, and then the T cells are assisted in entering the brain by two other cell adhesion molecules called ICAM1 and VCAM1.⁶ Samaha and her colleagues examined samples from the common brain cancers, using immunofluorescence to find that ALCAM is overexpressed on primary tumor endothelial cells. Immunofluorescence is a technique involving the use of antibodies to certain proteins that can be visualized under a microscope. More on this technique can be found here. They created a model of the blood-brain barrier by placing endothelial cells on one side of a polycarbonate membrane and placing human brain cells called vascular pericytes on the other. T cells were not able to pass, showing that tumor endothelial cells expressing ALCAM alone do not provide a strong enough adhesion to the T cells.

            They hypothesized that enhancing the bind of CD6 ligand on the T cell to the ALCAM receptor on the epithelial cell would result in the T cell being able to cross the endothelial cells. To do this they engineered their own “homing system (HS)” to match the mapped out ALCAM binding receptor. Using Invitrogen’s, GeneArt gene synthesis, they created a gene sequence that would result in a cell expressing a molecule with a tighter bind to the ALCAM receptor then the regular CD6 protein. They referred to their construct as a “homing system,” or HS molecule. This DNA sequence was implanted into T cells through a process called transfection, resulting in T cells that express the new HS molecule, hypothesized to facilitate T cell migration through the blood-brain barrier.  They show the schematic for the binding of CD6 to ALMAC and their plan for creating various HS molecules, that would replace the CD6 ligand. 

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The part of the HS molecule that binds to the ALCAM receptor is called “domain 3.” They made two other versions, one with three repeats of "domain 3" multimerized together, and another with five repeats, to determine if more "domain 3" regions would result in a stronger interaction. They also created tailless versions of the HS molecules, resulting in six total HS molecules as shown above, to test if the signaling domain of the molecule was important for the T cells binding. Without the tail, the protein would still bind to the ALCAM receptor, however, since the signaling domain is missing, the rest of the cell would not receive any signal that the bind occurred meaning nothing inside the cell will change from the binding.

            When a T cell is pulled from the bloodstream, through the blood-brain barrier, the T cell is slowed down by binding to ALMAC, and rolls along the endothelial cells, picking up more and more interactions before it can go through. To test how their new HS molecules impacted this process, they placed T cells expressing their HS constructs, inside a microfluidics flow chamber, lined with  endothelium cells at a pressure similar to the tumor capillary vessels. They found that indeed the T cells with a HS molecule were captured quicker, rolled more slowly, and stopped before regular T cells. The more repeats of “domain 3” that the HS had, the quicker it stopped.  They went back to their original blood-brain barrier model, and tested the migration of the HS T cells, finding that T cells with the 5HS molecule containing a signaling domain had the highest trans-endothelial migration percentage.

            They then investigated why the signaling tail of the construct is important for increasing the strength of the bind. They discovered that, upon binding to the ALCAM receptor, the T cell shows increasing expression of proteins involved in creating LPA in such a confirmation that it can bind to ICAM-1, another receptor on the endothelial cell. The signaling part of the HS construct allows the T cell to bind to more receptors other than ALCAM on the endothelial cells, which adds to the strength of the total endothelial to T cell interaction. The investigators found that the signaling domain of the HS constructs also resulted in a change in cytoskeletal confirmation that assists the T cell in crossing the endothelial cells.

            With the 5HS construct containing a signaling tail determined as the best construct for getting T cells through the blood-brain barrier, they tested it on mice that had been given human glioblastoma cancer, a specific brain cancer. Their data shows that indeed the 5HS construct with the signaling domain homed to the mouse blood-brain barrier and crossed more efficiently than normal T cells. They then tested their constructs ability to deliver a therapeutic complex to the mice. They gave their T cells a specific antigen receptor that will allow the T cell to bind to a protein expressed by the tumor cells. They found that the engineered T cells were able to induce tumor remission in the mice.

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They show that with no T cell treatment, the tumor grows very large by day 19. Regular T cells armed with the antigen receptor to target the tumor cells only prolong the tumor growth, resulting in a large tumor at day 25. The 5HS T cells with the antigen receptor however, gets rid of the tumor by day 19, and the tumor resurfaces at day 30, but is quite small.

            The data of this paper suggests that they were successful in creating a construct that enables T cells to go through a cancerous blood-brain barrier to combat brain cancer. Using mice as a model, they showed that T cells expressing their 5HS molecule and an antigen receptor that targeted tumor cells, were able to cause tumor remission. This data suggests that T cells armed with other therapeutic elements to treat infected brain tissue can also be homed more efficiently to the brain with the T cells expressing the 5HS molecule. This paper shows that treatment of the mice with the engineered T cells results in tumor remission for a while, then the tumor starts to grow back at day 30. It would be useful data for them to record a second treatment and see if the tumor goes into remission again, and for how long, to make sure that successive treatments don’t become less effective. The system works because the cancer cells are expressing a particular antigen not found on normal cells, so if successive treatments winds up alerting antigen expression of the tumor cells when they resurface, successive treatments would not be effective without alterations. To further test this as a cancer treatment, they can do more experiments like this one using mice expressing other types of tumors, to make sure other therapeutic treatments don’t interfere somehow with the homing system molecule. This paper provides a promising solution to the problem faced with T cell immunotherapy of brain cancer, in which the T cells were unable to cross the blood brain barrier.  

You can find Samaha and colleague’s full paper here.

References

1-     Siegel, R. L., Miller, K. D. and Jemal, A. Cancer statistics 2018. CA: A Cancer Journal for Clinicians, 68: 7-30. (2018).

2-     Douglas Hanahan, Robert A. Weinberg. Hallmarks of Cancer: The Next Generation. Cell 144, 646-674 (2011).

3-     Rosenberg, S. A. & Restifo, N. P. Adoptive cell transfer as personalized immunotherapy for human cancer. Science 348, 62–68 (2015).

4-     Bonini, C. & Mondino, A. Adoptive T-cell therapy for cancer: The era of engineered T cells. Eur. J. Immunol. 45, 2457–2469 (2015).

5-     Sackstein, R., Schatton, T. & Barthel, S. R. T-lymphocyte homing: an underappreciated yet critical hurdle for successful cancer immunotherapy. Lab. Invest. 97, 669–697 (2017).

6-     Bullard, D. C. et al. Intercellular adhesion molecule-1 expression is required on multiple cell types for the development of experimental autoimmune encephalomyelitis. J. Immunol. 178, 851–857 (2007).