Immune Checkpoint Drug Discovery In Vitro Assays

Background of Immune Checkpoint

Immune checkpoints regulate the immune system by blocking cell self-attack. Inhibitory checkpoints are targets for cancer antibody-based immunotherapies. Inhibitors of the immune checkpoints are developed to block immune checkpoints, kill cancer cells, and maintain immune homeostasis.

Distributions and Functions of Immune Checkpoint

Immune checkpoints are a normal component of immune cells, which play a crucial role in the regulation of immune tolerance and preventing self-immune attacks. Immune checkpoints bind to partner proteins, preventing the overreaction of T cell immune responses to foreign proteins. However, when immune checkpoints react to cancer cells, they prevent the immune system from destroying tumors. Immune checkpoint inhibitors have been developed as immune therapeutics for cancers, interdicting the combination of immune checkpoints and partner proteins, and thus T cells could kill tumor cells.

Fig.1 Interferon signaling in adaptive programmed cell death 1 ligand 1 expression. (Kalbasi, 2020)

Subtypes and Mechanisms of Immune Checkpoint

It has been demonstrated that immune checkpoints as antibody-based therapy targets could regulate the immune responses to autoimmune diseases, cancer cells, and other diseases. CTLA-4, PD-1, and PD-L1 are the three main target molecules of the therapeutic studies.

Immune Checkpoint	Gene	Mechanism	Inhibitors
CTLA-4	CTLA4	CTLA-4 is expressed on CD4+ and CD8+ T cells, involved in maintaining immune homeostasis CTLA-4 binds B7 proteins, inhibiting T cell and antigen presenting cells CTLA-4 fusion proteins are effective therapeutics in autoimmune diseases and organ rejection	ipilimumab tremelimumab HBM4003
PD-1	PDCD1	PD-1 binds to the ligand PD-L1, inhibiting T cells PD-1 is antibody-based therapies target for multiple tumor types, such as non-small cell lung cancer, renal cell carcinoma, melanoma, solid tumours, lymphomas etc. as well as Alzheimer's disease and asthma	AUNP-12 pembrolizumab tislelizumab spartalizumab retifanlimab dostarlimab
CD22	CD22	CD22 is a B cell I-type lectin involved in activation, adhesion, and B cell localization in lymphoid tissues CD22 inhibits immune cell activation by ITIM in the cytosolic domains CD22 is meaningful drug target for B cell malignancies	inotuzumab ozogamicin moxetumomab pasudotox
CD28	CD28	CD28 is expressed on T cells involved in T cell activation, proliferation, and survival, Th2 cell development, and stimulation of cytokine release CD28 binds B7 proteins on the antigen presenting cells, stimulating T cells. CD28 is the drug target for autoimmune diseases	lulizumab pegol FR104
CD33	CD33	CD33 is a myeloid cell I-type lectin, which inhibits immune cell activation by ITIM in the cytosolic domains CD33 inhibitors can inhibit the growth of leukemic cells in vitro and in vivo	lintuzumab vadastuximab talirine gemtuzumab ozogamicin
CD40	CD40	CD40 is expressed on multiple immune cells, such as macrophages, B cells, and DCs CD40 binds the endogenous ligand CD40L, activating immune system CD40 is the drug target for immunotherapy and immuno-oncology	CFZ533 ABBV-323

Published Data

Paper Title	Comprehensive in vitro characterization of PD-L1 small molecule inhibitors
Journal	Scientific reports
Published	2019
Abstract	A powerful strategy in cancer immunotherapy is the inhibition of the programmed cell death 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) interaction. Developing potent PD-1/PD-L1 inhibitors has emerged to be an efficient treatment strategy for cancer. The study aimed to guide the development of next-generation compounds that inhibit PD-1/PD-L1 interaction. Therefore, they evaluated and understood the structure-activity-cytotoxicity relationships of these first-generation PD-1/PD-L1 inhibitors via rigorous and systematic in vitro profiling of multiple compounds, including a macrocyclic peptide inhibitor (BMSpep-57), a peptidomimetic inhibitor (Aurigene-1), and non-peptidic small-molecule inhibitors (BMS-103, BMS-142).
Result	They performed biochemical assays. The results demonstrated that BMS-103 and BMS-142 were strongly active. Acute cytotoxicity of both compounds greatly compromised the immunological activity, while Aurigene-1 did not show any activity in either biochemical or immunological assays. In addition, they also reported the discovery of a small-molecule immune modulator, which exhibited favorable drug-like properties and strong immunological activity. The results of the study results may play an important role in guiding the development of next-generation PD-1/PD-L1 small molecule inhibitors. Fig.2 Ability of compounds to inhibit PD-1/PD-L1 binding in an ELISA competition assay. (Ganesan, 2019)

References

Kalbasi, A.; Ribas, A. Tumour-intrinsic resistance to immune checkpoint blockade. Nature Reviews Immunology. 2020, 20(1): 25-39.
Ganesan, A.; et al. Comprehensive in vitro characterization of PD-L1 small molecule inhibitors. Scientific reports. 2019, 9(1): 1-19.

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