Periodic Reporting for period 1 - CD28 (Probing CD28 as checkpoint for T cell co-stimulation in cancer and infection)
Berichtszeitraum: 2021-10-29 bis 2023-10-28
Cancer and infections are nowadays amongst the deadliest pathologies all over the world, becoming a serious concern for public health, as they keep producing millions of deaths annually. In this regard, T lymphocytes are key immune cells in the development of effective adaptive responses against these pathologies. Understanding how the activity and proliferation of these lymphocytes is regulated at cellular and molecular level is essential to develop effective therapies. Overall, the response delivered by T cells is determined by several co-stimulatory receptors as the one addressed in this project, CD28. In particular, this receptor provides a second activation signal which definitely leads to T cell proliferation, whereas others, like CTLA-4 or PD-1, exert inhibitory effects that lead T lymphocytes to remain anergic, become tolerant to certain antigens or even die. In tumoral or chronically infected tissues, T cells become exhausted (tolerant) and do not properly neutralize the threat. We hypothesized that this effect arises from CD28 inhibition produced by aberrant, hypesialylated antigens, which are a common signature of tumoral cells for example. Therefore, in the present project, we aim to identify the molecular basis for recognition of sialic acid-containing glycans by CD28 (aim 1), and develop molecular conjugates of sialidase that target exhausted T cells and restore/enhance CD28 co-stimulation by removal of surface sialic acid moieties (sialidase) (aim 2). Revival of these exhausted T cells may have a great therapeutic potential in treatment of cancer and chronic infections.
For aim 1, structural studies were carried out to understand how the CD28-sialic acid interaction (inhibition) takes place at a molecular level, such that this knowledge might be exploited to control the ultimate T cell fate by switching on or off the CD28-driven co-stimulation. The protein of interest, CD28, was expressed in mammalian cells, and the protocol optimized to further attempt its expression in isotopically enriched medium (13C, 15N), so the 3D NMR experiments could be recorded. In parallel, a representative library of small oligosaccharides was chemoenzymatically produced to test the ligand specificity of CD28. The binding affinities were evaluated by making use of three experimental techniques: BioLayer Interferometry (BLI), MST (Thermophoresis) and Saturation Transfer Difference (STD-NMR). In each case, the reducing end of the glycans was modified to enable the proper detection of the binding event (biotin for BLI, fluorescein for MST, and free end for STD). The whole Work Package 2 was finished, but regretfully, we didn’t observe any binding between CD28 and sialylated glycans, hence the 3D-NMR experiments were not performed, and the further scheduled X-Ray studies had to be re-thought as well. The whole Work Package 1 was redesigned (see next section).
Regarding aim 2, the therapeutic potential of the anti-PD-1-sialidase conjugates to reestablish CD28 co-stimulation was evaluated making use of suitable murine models of tumor and chronic infection. Firstly, the sialidase-anti-PD-1 conjugates were prepared and the protocol optimized to get enough amounts for the corresponding in vitro and in vivo tests with exhausted T cells. The sialidase and anti-PD-1 were independently expressed and then chemically coupled using a sortase-based strategy. As a result, a homogeneous product consisting of a dimeric anti-PD-1 with two attached sialidases was isolated and its sialidase activity confirmed in vitro (Work package 4 completed). Then, the in vitro tests with exhausted T cells extracted from hosts were performed. Finally, the in vivo tests were done to compare the efficiency of anti-PD-1-sialidase conjugates with that of anti-PD-1 alone in the reduction of solid tumors. As no convincing differences were found at this point between both treatments, further scheduled studies were discontinued and the whole strategy rethought. At this point, all tasks of work package 3 were considered as done, but the experiments didn’t yield the expected results.
Regarding aim 2, the therapeutic potential of the anti-PD-1-sialidase conjugates to reestablish CD28 co-stimulation was evaluated making use of suitable murine models of tumor and chronic infection. Firstly, the sialidase-anti-PD-1 conjugates were prepared and the protocol optimized to get enough amounts for the corresponding in vitro and in vivo tests with exhausted T cells. The sialidase and anti-PD-1 were independently expressed and then chemically coupled using a sortase-based strategy. As a result, a homogeneous product consisting of a dimeric anti-PD-1 with two attached sialidases was isolated and its sialidase activity confirmed in vitro (Work package 4 completed). Then, the in vitro tests with exhausted T cells extracted from hosts were performed. Finally, the in vivo tests were done to compare the efficiency of anti-PD-1-sialidase conjugates with that of anti-PD-1 alone in the reduction of solid tumors. As no convincing differences were found at this point between both treatments, further scheduled studies were discontinued and the whole strategy rethought. At this point, all tasks of work package 3 were considered as done, but the experiments didn’t yield the expected results.
The project has included the novel synthesis of conjugates aimed at triggering two desired effects (Blocking of inhibitory PD-1 and stimulation of co-receptor CD28 via sialidase activity) at the same time, such that the expected positive joint effect might be enhanced (stimulation of exhausted T cells) and the tumor growth might be significantly reduced. At the same time, the sialic acid-CD28 interaction was a novel, totally unknown scientific area from the structural point of view. Unfortunately, this last point couldn´t be further proved for the initial receptor, CD28, so we considered a related receptor, also involved in T cell responses and communication: siglec-15. This receptor suppresses antigen-specific T cell responses in tumoral environments as a result of T cell interaction with tumor-associated macrophages (TAMs), which express this siglec in their surface. Siglec-15 blockage with high-affinity ligands is under evaluation for the treatment of several types of cancer. This membrane receptor has structural properties similar to these of CD28 and its interaction with sialylated ligands is known but poorly described at molecular level. Our structural studies with the glycan library so far provided novel data about ligand specificity of this protein. Also, our perspectives for the remaining year of the grant point toward the novel description of the crystallographic structure of siglec-15 in complex with artificially sialylated glycans. We have preliminar, promising data about the enhanced activity of these kind of ligands, open a great door to the development of similar structure-based mimetics to efficiently block this receptor, which will positively impact on the extensive quest for efficient therapies to reduce tumor progression.