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.
T-cell immunoglobulin domain and mucin domain-3 (TIM3) is an activation-induced inhibitory molecule involved in immune tolerance and T-cell exhaustion in chronic viral infection and cancers. TIM3 maturation and cell surface expression is facilitated by forming a heterodimeric interaction with CD66a. Co-blockade of CD66a and TIM3 enhanced anti-tumor immune responses, and eliminated tumors in mouse colorectal cancer models.
Gene targeting strategy of B-hPD-1/hTIM3 mice. The targeting strategy of B-hTIM3 mice is to the exon 2 of mouse Tim3 gene that encode the extracellular domain were replaced by human TIM3 exon 2 . The targeting strategy of B-hPD-1 mice is to the exon 2 of mouse PD-1 gene that encode the extracellular domain were replaced by human PD-1 exon 2. The B-hPD-1/hTIM3 double knock-in model, was developed by breeding the B-hPD-1 mice and the B-hTIM3 mice, has a functional mouse immune system.
mRNA Expression Analysis
RT-PCR analysis of the B-hPD-1/hTIM3 humanized mice. The splenocytes of the B-hPD-1/hTIM3 homozygous mice showed expression of human PD-1 and TIM3 mRNA but not mouse PD-1 and TIM3 mRNA.
Protein Expression Analysis
Strain specific TIM3 expression analysis in homozygous B-hPD-1/hTIM3 mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hPD-1/hTIM3 (H/H) mice stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-TIM3 antibody. Mouse TIM3 was detectable in WT mice. Human TIM3 was exclusively detectable in homozygous B-hPD-1/hTIM3 but not WT mice.
Splenocytes from both wild type (WT) C57BL/6 and homozygous B-hPD-1/hTIM3 mice were analyzed by flow cytometry. Mouse TIM3+ and PD-1+ T cells were detectable in the WT C57BL/6 mice, while human TIM3+ and PD-1+ T cells were detectable in the homozygous B-hPD-1/hTIM3 mice.
Combination Therapy of PD-1 mAb (Keytruda) and TIM3 mAb
Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hPD-1/hTIM3 mice. Mice were grouped when the tumor size was approximately 150±50 mm3 (n=6). Anti-TIM3 antibody and the anti-hPD-1 antibody Keytruda shows obviously inhibitory effects respectively. Combination of anti-hTIM3 antibody and the anti-hPD-1 antibody Keytruda shows better inhibitory effects than single treatment, suggesting that B-hPD-1/hTIM3 mouse is a powerful tool for in vivo evaluating combination therapy efficacy of hPD-1 antibody and hTIM3 antibody.
- Nat Commun. 2017 Feb 6;8:14369. doi: 10.1038/ncomms14369.
- EMBO J. 1992 Nov;11(11):3887-95.
- Int. J. Mol. Sci. 2017, 18, 645; doi:10.3390/ijms18030645