In our previous blog, we discussed the two-predominant types of T cells, and some in vitro assays these cells are employed in to test the function and specificity of bi-specific antibodies. In this blog, we discuss the in vivo assays that test the efficacy of these antibodies and tie it all together by reviewing some data generated with these assays.
What are some T cell-based in vivo assays used to examine bi-specific antibody efficacy?
Most preclinical in vivo experiments that examine the efficacy of bi-specific antibodies are performed using immuno-deficient mice, such as the NOD scid gamma (NSG) mice. These mice are on the NOD genetic background which reduces the role of the innate immune system, including the function of macrophages and dendritic cells. Additionally, to block the development of T and B cells, these mice carry the scid mutation, which prevents V(D)J recombination, to inhibit T and B cell maturation. Finally, the IL-2 receptor gamma chain is knocked out to prevent the development of NK cells in these mice. Together, these NSG mice are immuno-deficient and are ideal for performing studies on potential therapeutics that utilize the immune system.
The NSG mice are ideal for studying molecules that utilize the immune system because the immune system of these mice can be reconstituted by introducing human T cells, or PBMCs, from different individuals through intravenous injection. Once introduced, these cells begin to circulate within the animal and can act as a surrogate immune system that is able to respond to cancerous cells or infections. Additionally, because the transferred cells are human, the cancer or infection model will be more indicative of a human response, as opposed to a mouse one. Therefore, NSG mice represent an ideal mouse model to study the human immune response.
Because the studies require functional immune cells, high quality and fully characterized pan T cells, enriched CD4, or enriched CD8+T cells obtained from reliable sources, such as iQ Biosciences, are very important for performing meaningful and reproducible experiments. If the T cells are of low quality, the cells may be non-functional and lead to erroneous data, including the interpretation of the test article as non-efficacious. Therefore, the source and quality of T cells are quite important. Most groups that use these T cells can also perform in vitro assays with the same donor prior to moving to the in vivo studies to fully bridge the data sets.
Typically, in vivo experiments are performed by injecting cells (usually, a cell line) that model a disease of interest into NSG mice. This is then followed by injection of immune cells and dosing with the test article, such as a bi-specific antibody, into the mice at different intervals. The difference in most experiments center on the readout of the efficacy of the molecule. Traditionally, the readout of tumor size is measured using calipers. However, more recent experiments monitor tumor size by using target cells that express a fluorescent or luminescent reporter and a compatible bio-imaging technology to visualize the tumors non-invasively. These imaging methods tend to be more high-throughput as multiple animals can be imaged simultaneously and require minimal handling of them. More importantly, the bio-imaging techniques can provide a more precise measurement of the tumor size compared to two-dimensional measurement using calipers. Additionally, test articles can be labeled and tracked for tumor penetration or bio-distribution.
Selection of the right in-vivo group to work with is critical to obtain reliable, robust, and reproducible results. These projects can be expensive over the long haul when considering the many types of studies that could potentially be conducted, including pharmacokinetics, distribution, efficacy, and exploratory safety experiments.
A review of data from in vitro and in vivo assays to support the development of a bi-specific antibody program