CTLA4 (cytotoxic T-lymphocyte-associated protein 4), also known as CD152, competitively binds to B7-1 (CD80) and B7-2 (CD86) on Antigen-Presenting Cells (APCs) to block the T cell activating signal by B7 and CD28 (on T cells) interaction. The inhibition of CTLA4 by its inhibitory antibodies enhances T cell activity. The CTLA4 antibody is the first FDA-approved antibody to treat advanced melanoma. TNFRSF18 (TNF Receptor Superfamily Member 18) is also known as Glucocorticoid-induced TNFR-related protein (GITR), which is expressed on many immune cells including T cells. As a co-stimulatory signal of T cells, TNFRSF18 is upregulated upon the activation of T cells, and in turn promotes T Cell Proliferation. CD25+/CD4+ regulatory T cell is known to mediate immune tolerance, and GITR agonist antibodies can reverse this immune tolerance, and show anti-tumor effect in multiple tumor models.
Gene targeting strategy for B-hCTLA4/hGITR mice. The exon 2 of mouse Ctla4 gene that encode the extracellular domain was replaced by human CTLA4 exon 2 in B-hCTLA/hGITR mice. The exons 1-4 of mouse Tnfrsf18 gene that encode the extracellular domain were replaced by human TNFRSF18 exons 1-4 in B-hCTLA4/hGITR mice.
mRNA expression analysis
Strain specific analysis of CTLA4 and GITR gene expression in WT and B-hCTLA/hGITR mice by RT-PCR. Mouse Ctla4 and Tnfrsf18 mRNA was detectable only in splenocytes of wild-type (+/+) mice. Human CTLA4 and TNFRSF18 mRNA was detectable only in H/H, but not in +/+ mice.
Protein expression analysis
Strain specific CTLA4 and GITR expression analysis in homozygous B-hCTLA4/hGITR mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hCTLA4/hGITR (H/H) mice analyzed by flow cytometry with species-specific anti-CTLA4 antibody or anti-GITR antibody. Mouse CTLA4 and GITR were exclusively detectable in WT mice. Human CTLA4 and GITR were exclusively detectable in homozygous B-hCTLA4/hGITR but not WT mice.
Protein expression analysis
Strain specific CTLA4 and GITR expression analysis in homozygous B-hCTLA4/hGITR mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hCTLA4/hGITR (H/H) mice stimulated with anti-CD3ε in vivo (7.5 μg/mice), and analyzed by flow cytometry with species-specific anti-CTLA4 antibody or anti-GITR antibody. Mouse CTLA4 and GITR were exclusively detectable in WT mice. Human CTLA4 and GITR were exclusively detectable in homozygous B-hCTLA4/hGITR but not WT mice.
Combination therapy of anti-human CTLA4 antibody and anti-human GITR antibody
Antitumor activity of anti-human CTLA4 antibody combined with anti-human GITR antibody in B-hCTLA4/hGITR mice. (A) Anti-human CTLA4 antibody combined with anti-human GITR antibody inhibited MC38-hPD-L1 tumor growth in B-hCTLA4/hGITR mice. Murine colon cancer MC38-hPD-L1 cells were subcutaneously implanted into homozygous B-hCTLA4/hGITR mice (female, 6-7 week-old, n=6). Mice were grouped when tumor volume reached approximately 100 mm3, at which time they were treated with BMS986156 (in house) and Yervoy (in house) with doses and schedules indicated in panel A. (B) Body weight changes during treatment. As shown in panel A, combination of BMS986156 and Yervoy shows more inhibitory effects than BMS986156 individual group but not obviously in Yervoy individual group, maybe the doses and schedules should be optimization. The B-hCTLA4/hGITR mice provide a preclinical model for in vivo evaluating combination therapy efficacy of hCTLA4 antibodies and hGITR antibodies. Values are expressed as mean ± SEM.