Periodic Reporting for period 2 - CD28 (Probing CD28 as checkpoint for T cell co-stimulation in cancer and infection)
Periodo di rendicontazione: 2023-10-29 al 2024-10-28
Cancer and autoimmune diseases 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 membrane receptors that might trigger either stimulatory/co-stimulatory effects, leading to T cell proliferation, or instead, inhibitory effects that make T cells remain anergic and become tolerant to the inspected antigen. In particular, siglecs are membrane receptors that prime inhibitory effects on T cells upon engaging by sialic-acid containing antigens (self signature). This inhibitory behavior has been widely described to be exploited by hypersialylated tumoral cells to evade T cell responses. Over the last two decades, siglecs have progressively gained interest as therapeutic targets, given that their efficient blockage in tumors might help to restore T cell function. While some advancements have been achieved for few human siglecs like CD22 and CD33 so far, others remain pretty unknown, especially from a molecular point of view. Siglec-15 is a representative example of this unbalanced knowledge about the siglec family, as this lectin has been barely documented in peer-reviewed publications over the last 15 years. This receptor suppresses antigen-specific T cell responses in tumoral environments as a result of T cell interaction with tumor-associated macrophages (TAMs), which overexpress sialylated glycans in their surface. Therefore, in the present project, we aim to characterize in detail the molecular basis for recognition of sialic acid-containing glycans by siglec-15 and find improved hits to efficiently target it (aim 1). Also, a second aim is to develop molecular conjugates containing siglec antigens (using these hits) to neutralize the inhibitory effect of both siglec receptors and PD-1 receptors, helping T cells to exert an effective immune response in tumoral environments (aim 2).
For aim 1, a soluble dimeric Siglec-15 chimera construct was successfully produced in either mammalian CHO cells and HEK293F cells, to be used for most of NMR-based experiments. In parallel, a small oligosaccharide library (natural & modified) was prepared (chemoenzymatically and/or by means of classic synthetic strategies) and subsequently tested in the presence of the protein by STD-NMR. This technique allowed to screen the ligand selectivity of the lectin and further analyze in detail the ligand epitope (binding map) of the binding sugars. Also, one of the modified glycans was identified as potential hit with improved affinity above the rest natural binders. To further understand how binding takes place, STD-NMR was used along with relaxation measurements (19F-T2) to test and compare this hit between the wild type and a mutant version of the protein (glycan binding disrupted), as well as the glycan binding in the presence of a blocking antibody for the protein (competition experiments). Unexpected results found for the ligand hit allowed to expand the scope of interest for this ligand, as its behavior with siglec-15 intriguingly differed from the original counterpart of this mimetic, the CD22 receptor. The Kds will be directly estimated through the STD data for publication, using the recently developed software RedDat. Considering that NMR provided a lot of information about the siglec-15 binders from the ligand point of view, and taking advantage of the binding results obtained for the hit in the simultaneous presence of the antibody, we decided to put the focus on the crystallography to get a detailed description of the hit ligand bound to the protein-antibody complex.
Regarding aim 2, the original work (focused on CD28) was already finished and detailed in the previous report. The glycosylated anti-PD-1 conjugates bearing improved glycan ligands for siglec-15/CD22 are being still evaluated at Scripps as part of in vivo experiments. Given that this work recently started last year, after discontinuing the previous work, several experiments are still ongoing and results will determine if more experiments will be needed. Results are not expected to be ready for the publication that is already in preparation, but for a subsequent manuscript scheduled for next year.
Regarding aim 2, the original work (focused on CD28) was already finished and detailed in the previous report. The glycosylated anti-PD-1 conjugates bearing improved glycan ligands for siglec-15/CD22 are being still evaluated at Scripps as part of in vivo experiments. Given that this work recently started last year, after discontinuing the previous work, several experiments are still ongoing and results will determine if more experiments will be needed. Results are not expected to be ready for the publication that is already in preparation, but for a subsequent manuscript scheduled for next year.
The project provided a novel, unexpected hit for siglec-15 whose structure is based on (or shared) a previous hit for murine CD22. Moreover, this ligand seemed not only to target siglec-15 with an improved affinity, but also to target the binding site in the presence of a blocking antibody. The enhanced affinity, considering it as a single epitope, turns out to be promising for the subsequent development of multivalent scaffolds with very high inhibitory potency and/or conjugates exerting independent inhibitory effects by/or targeting multiple receptors. Indeed, this strategy (conjugates) is already under testing and we expect to have some results in few months. Also, the further description of the crystallographic structure with the hit will surely 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.