PD-1 (Programmed death-1) is mainly expressed on the surface of T cells and primary B cells. The two PD-1 ligands, PD-L1 and PD-L2, are widely expressed on antigen-presenting cells (APCs). PD-1 interacts with its ligands and plays an important role in the negative regulation of the immune response. PD-L1 protein expression is detected in many human tumor tissues. PD-L1 expression in tumor cells could be induced by the microenvironment of tumor cells. PD-L1 expression is favorable for tumorigenesis and growth, for induction of anti-tumor T Cell Apoptosis, and for escaping responses by the immune system. Inhibition of PD-1 binding to its ligand can result in tumor cells that are exposed to the killing version of the immune system, and thus is a target for cancer treatments. PD-L1 (Programmed cell death ligand-1), also known as B7-H1 and CD274, is mainly expressed in antigen-presenting cells (APCs) and activated T cells, and is one of the two ligands of PD-1. The interaction between PD1 and PD-L1 plays an important role in the negative regulation of the immune response. PD-L1 is highly expressed in a variety of solid tumors. PD-1 and PD-L1 interactions can reduce T Cell Activation and promote tumor immune escape. The PD-1/PD-L1 signaling pathway can be blocked and antitumor immune response can be restored by using by anti-PD-1 or anti-PD-L1 antibodies to block the binding of PD1 to PD-L1. CD40 (CD40 molecule) is a member of the tumor necrosis factor receptor superfamily expressed on APC such as dendritic cells (DC), B cells, and monocytes as well as many non-immune cells and a wide range of tumors. Engagement with its trimeric ligand CD154 on activated T helper cells results in APC activation, which has been found to be essential in mediating a broad variety of immune and inflammatory responses including T cell-dependent immunoglobulin class switching, memory B cell development, and germinal center formation. Agonistic CD40 monoclonal antibodies (mAbs) have been shown to activate APC and promote anti-tumor T cell responses. Thus, agonistic anti-CD40 mAb may serve as a new class of antitumor therapeutics that potentiate immunity via a different mechanism than the blocking immune checkpoint inhibitors such as anti-CTLA4 or anti-PD-1.
Protein expression in splenocytes
Strain specific PD-1, PD-L1 and CD40 expression analysis in homozygous B-hPD-1/hPD-L1/hCD40 mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hPD-1/hPD-L1/hCD40 (H/H) mice analyzed by flow cytometry with species-specific anti-PD-1 antibody. Mouse PD-1, PD-L1 and CD40 were detectable in WT mice. Human PD-1, PD-L1 and CD40 were exclusively detectable in homozygous B-hPD-1/hPD-L1/hCD40 but not WT mice.
Protein expression analysis in stimulated splenocytes
Strain specific PD-1, PD-L1 and CD40 expression analysis in homozygous B-hPD-1/hPD-L1/hCD40 mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hPD-1/hPD-L1/hCD40 (H/H) mice after stimulation with anti-CD3ε in vivo (7.5 μg/mice), and without stimulation, and analyzed by flow cytometry with species-specific anti-PD-1 antibody. Mouse PD-1, PD-L1 and CD40 were detectable in WT mice. Human PD-1, PD-L1 and CD40 were exclusively detectable in homozygous B-hPD-1/hPD-L1/hCD40 but not WT mice. After anti-CD3ε stimulation, hPD-1 protein expression was significantly increased.
Combination therapy of Pembrolizumab and Selicrelumab
Antitumor activity of of Pembrolizumab and Selicrelumab in B-hPD-1/hPD-L1/hCD40 mice. (A) anti-human PD-1 antibody combined with anti-human CD40 antibody inhibited MC38-hPD-L1 tumor growth in B-hPD-1/hPD-L1/hCD40 mice. Murine colon cancer MC38-hPD-L1 cells (5×105) were subcutaneously implanted into homozygous B-hPD-1/hPD-L1/hCD40 mice (female, 7 week-old, n=5). Mice were grouped when tumor volume reached approximately 100-150 mm3, at which time they were treated with Pembrolizumab and Selicrelumab with doses and schedules indicated in panel A. (B) Body weight changes during treatment. As shown in panel A, combination of Pembrolizumab and Selicrelumab shows more inhibitory effects than individual groups, demonstrating that the B-hPD-1/hPD-L1/hCD40 mice provide a powerful preclinical model for in vivo evaluating combination therapy efficacy of PD-1 antibodies combined with anti-human CD40 antibodies. Values are expressed as mean ± SEM.(All antibodies were made in-house)