As research deepens, some potential immune checkpoints have emerged, such as lymphocyte activation gene 3 (LAG-3) (anti-LAG-3 monoclonal antibody or in combination with anti-PD-1 therapy), killer inhibitory receptor (KIR) (alone or in combination with anti-PD-1 or anti-CTLA-4 therapy), B7-H3, T cell immunoglobulin and mucin-3 (TIM-3), T cell immunoglobulin and immune tyrosine inhibitory motif (ITIM) domain (TIGIT), and other immune checkpoint related drugs are also being studied, broadening the path for cancer treatment.

The cytotoxic T cell, also known as CD8+ T cell, is a T cell receptor (TCR) that can kill cancer cells, cells infected by viruses, bacteria, or other forms of damage. It recognizes specific antigens. The antigens inside the cell bind to MHCI molecules and are carried to the cell surface for recognition by T cells. If TCR is specific to the antigen, it will bind to the complex, and T cells will destroy the cell. If the antigen inside the cell binds to MHC class II molecules, it is a CD4+ T cell.

Fig 1: Antigen presentation stimulates T cells to become either "cytotoxic" CD8+ cells or "helper" CD4+ cells.

Programmed cell death protein 1 (PD-1), encoded by the PDCD1 gene, is a cell surface receptor on T and B cells. PD-1 is an immune checkpoint that regulates T cell activity by promoting antigen-specific T cell apoptosis in lymph nodes and inhibiting regulatory T cell apoptosis. The expression of PD-L1 on tumor cells inhibits anti-tumor activity by binding to PD-1 on effector T cells, inducing cell apoptosis, and weakening the host's immune response to tumor cells, thereby promoting tumor cell growth and causing immune evasion of tumor cells. Antigen-presenting cells (APCs) present tumor antigens to T cell receptors (TCRs) through the major histocompatibility complex (MHC). When the MHC antigen complex specifically binds to the TCR, it triggers a series of signal transduction pathways, including phosphatidylinositol signaling, mitogen-activated protein kinase signaling pathways, etc., thereby activating the immune response of effector T cells. After PD-L1 binds to PD-1, the cytoplasmic region of PD-1 undergoes phosphorylation, thereby activating SHP2. Subsequently, SHP-2 mediated dephosphorylation of TCR-associated CD3 and ZAP70 signaling bodies while inhibiting CD28 stimulation signals, weakening downstream TCR signal intensity and cytokine secretion (such as IL-2), ultimately inhibiting T cell function.

Fig 2: The immune regulation mechanism of the PD-1/PD-L1. (Reference source: Lin, X., Kang, K., Chen, P. et al. Regulatory mechanisms of PD-1/PD-L1 in cancers. Mol Cancer 23, 108 (2024). https://doi.org/10.1186/s12943-024-02023-w)

CD47 (CD47 differentiation cluster 47), also known as integrin-associated protein (IAP), is involved in a series of cellular processes including apoptosis, proliferation, adhesion, and migration. Activation and axonal development of T cells and dendritic cells (DCs), binding of CD47 to its receptor signaling regulatory protein alpha (SIRP alpha), and downregulation of tumor cell phagocytosis, anti-CD47 antibodies can promote tumor cell death through various mechanisms. Firstly, anti-CD47 monoclonal antibodies can inhibit the interaction between CD47 and SIRP alpha, which can lead to macrophage phagocytosis of tumor cells (anti-SIRP alpha antibodies can also inhibit this pathway), reducing immune escape. Secondly, they can eliminate tumor cells through NK cell-mediated antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Thirdly, they can directly induce tumor cell apoptosis and finally present antigens to CD4+ and CD8+ T cells, thereby stimulating an anti-tumor adaptive immune response.

Fig 3: Therapeutic targeting of the CD47-SIRPα pathway can cause elimination of cancer cells through multiple mechanisms. (Reference source: Chao MP, Weissman IL, Majeti R. The CD47-SIRPα pathway in cancer immune evasion and potential therapeutic implications. )

The author inoculated tumor cell lines (selected AT3-OVA can express high levels of CD47 and PD-L1) into mice, injected a certain amount of bispecific antibodies (CD47 × PD-L1) at certain intervals, and added corresponding control groups. For CD8+ T cell experiments, anti-CD8a was used to observe. After some time, T cells were isolated from mouse tissues and bodies, and single-cell suspensions were prepared for cell flow cytometry analysis. The data was statistically analyzed, and the tumor growth inhibition rate was estimated by calculating the volume average.

Fig. 4a shows the experimental group and control group: CD47 × PD-L1 BisAb and isotype control were injected into mice receiving AT3-OVA tumors. Fig. 4b (process changes) and c (result presentation) show that the tumor volume was significantly reduced compared to receiving isotype control antibodies. Figs. 4d and e represent macrophages, monocytes, and neutrophils in the spleen and tumor, respectively, whose numbers increased after the experiment. Fig. 4f and g represent CD44 hi CD4+ T cells in the spleen and tumor. The number of CD44 hi CD8+ T cells and SIINFEKL+ CD8+ T cells showed significant increases in some cases and no significant changes in others.

Fig 4: CD47 × PD‐L1 BisAb therapy reduces tumor burden and induces CD8+ T cell expansion.

Fig. 5a Inoculate mice in the experimental group and the control group with anti-CD8 depletion antibodies. Fig. 5b shows the number of CD44 hi CD8+ T cells in the spleen and tumor. It can be seen that the number of CD44 hi CD8+ T cells decreases sharply or even does not exist. The tumor volume in Fig. 5c increases with time. The final tumor volume in Fig. 5d increases compared with other control groups. It can be seen that CD8+ T cells play a direct role in tumor regression of breast cancer, and the tumor control mechanism mediated by CD47 × PD-L1 BisAb is regulated by CD8+ T cells.

Fig 5: CD47 × PD‐L1 BisAb therapy reduces tumor burden and induces CD8+ T cell expansion.

Alpha Lifetech can provide biosimilar antibodies targeting CD47 and PD-L1, respectively, as shown in the table below. We can also provide biosimilar antibody development and bispecific antibody development services to accelerate the progress of our client's project research and provide customized services for our clients.

Tab 2: Biosmilar Antibody Products List.