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.
Gene targeting strategy for B-hPD-1/hPD-L1/hCD40 mice. The exon 2 of mouse PD-1 gene that encodes the extracellular domain was replaced by human PD-1 exon 2 in B-hPD-1/hPD-L1/hCD40 mice. The exon 3 of mouse pdl1 gene that encode the extracellular domain was replaced by human PD-L1 exon 3 in B-hPD-1/hPD-L1/hCD40 mice. The exon 2-7 of mouse tigit gene that encodes the extracellular domain was replaced by human CD40 exon 2-7 in B-hPD-1/hPD-L1/hCD40 mice.
Protein expression analysis
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.
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 durin g 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)