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.
TNFRSF9 (Tumor necrosis factor receptor super family, member 9), also called CD137 and 4-1BB, is a co-stimulatory molecule and is mainly expressed on the surface of T, NK and mononuclear cells. CD137 is activated by its ligand CD137L or activating anti-CD137 antibodies, resulting in T Cell activation, proliferation and cytokine production. CD137L is expressed on antigen presenting cells such as dendritic cells and macrophages and activated B cells. In vivo studies show that CD137 activation increases anti-tumor immune responses, providing a new target for immunotherapy.
Gene targeting strategy for B-hPD-L1/h4-1BB mice. The exon 3 of mouse Pdl1 gene that encode the IgV domain were replaced by human PD-L1 exon 3 in B-hPD-L1/h4-1BB mice. The exon 2-7 of mouse 4-1BB gene that encode the extracellular domain were replaced by human 4-1BB exon 2-7 in B-hPD-L1/h4-1BB mice.
Protein Expression Analysis
Strain specific PD-L1 and 4-1BB expression analysis in homozygous B-hPD-L1/h4-1BB mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hPD-L1/h4-1BB (H/H) mice stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-hPD-L1 and anti-h4-1BB antibody. Mouse Pdl1 and 4-1BB was detectable in WT mice but not in homozygous B-hPD-L1/h4-1BB mice. Human PD-L1 and 4-1BB was exclusively detectable in homozygous B-hPD-L1/h4-1BB mice but not in WT mice.
In vivo efficacy of combination of anti-human PD-L1 antibody and anti-human 4-1BB antibody
Antitumor activity of combination of anti-human PD-L1 antibody (atezolizumab，in house）and anti-human 4-1BB (urelumab, in house) in B-hPD-L1/h4-1BB mice. (A) combination of anti-human PD-L1 antibody and anti-human 4-1BB antibody inhibited MC38 tumor growth in B-hPD-L1/h4-1BB mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hPD-L1/h4-1BB mice (female, 6-week-old, n=6). Mice were grouped when tumor volume reached approximately 100 mm3, at which time they were treated with anti-human PD-L1 antibody and anti-human 4-1BB antibody with doses and schedules indicated in panel. (B) Body weight changes during treatment. As shown in panel A, combination of anti-human PD-L1 antibody and anti-human 4-1BB antibody were efficacious in controlling tumor growth in B-hPD-L1/h4-1BB mice, demonstrating that the B-hPD-L1/h4-1BB mice provide a powerful preclinical model for in vivo evaluation of combination of anti-human PD-L1 antibody and anti-human 4-1BB antibody. Values are expressed as mean ± SEM.
4-1BB-PD-L1 bispecific antibody Efficacy Evaluation
Antitumor activity of anti-4-1BB antibody and anti-4-1BB-PD-L1 bispecific antibody in B-hPD-L1/h4-1BB mice. (A) Anti-4-1BB antibody and anti-4-1BB-PD-L1 bispecific antibody inhibited B16F10-hPD-L1 tumor growth in B-hPD-L1/h4-1BB mice. B16F10-hPD-L1 cells were subcutaneously implanted into homozygous B-hPD-L1/h4-1BB mice (female, 7-week-old, n=6). Mice were grouped when tumor volume reached approximately 100 mm3, at which time they were treated with anti-4-1BB antibody and bispecific antibody with doses and schedules indicated in panel. (B) Body weight changes during treatment. As shown in panel A, the bispecific antibody were more efficacious in controlling tumor growth in B-hPD-L1/h4-1BB mice compared with PBS and the anti-4-1BB antibody group, demonstrating that the B-hPD-L1/h4-1BB mice provide a powerful preclinical model for in vivo evaluation of anti-human PD-L1 antibody, anti-human 4-1BB antibody or related bispecific antibody. Values are expressed as mean ± SEM.
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