Gene targeting strategy for B-hVEGFR2 mice. The exons 2-15 of mouse Vegfr2 gene that encodes the extracellular region coding sequences were replaced by human VEGFR2 exons 2-15 in B-hVEGFR2 mice.

Strain specific analysis of VEGFR2 gene expression in wild type and B-hVEGFR2 mice by RT-PCR. Mouse Vegfr2 mRNA was detectable only in embryo of wild-type mice (+/+). Human VEGFR2 mRNA was detectable only in homozygous B-hVEGFR2 mice (H/H) but not in wild-type mice.

Strain specific VEGFR2 expression analysis in homozygous B-hVEGFR2 mice by flow cytometry. Embryonic lung endothelial cells were collected from wild-type (+/+) and homozygous B-hVEGFR2 mice (H/H) and analyzed by flow cytometry with anti-VEGFR2 antibody. Anti-mouse VEGFR2 antibody was cross-reactive between mouse and human. Combined with mRNA expression in wild-type and homozygous B-hVEGFR2 mice, it can be proved that mouse VEGFR2 was detectable in wild-type mice. Human VEGFR2 was exclusively detectable in homozygous B-hVEGFR2 mice (H/H) but not in wild-type mice

Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hVEGFR2 mice (n=3, 6-week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for the CD45+ population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hVEGFR2 mice were similar to those in the C57BL/6 mice, demonstrating that VEGFR2 humanized does not change the overall development, differentiation or distribution of these cell types in spleen. Values are expressed as mean ± SEM.

Analysis of spleen T cell subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hVEGFR2 mice (n=3, 6-week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ cells were gated for TCRβ+ T cell population and used for further analysis as indicated here. B. Results of FACS analysis. The percent of CD4+ T cells, CD8+ T cells, and Tregs in homozygous B-hVEGFR2 mice were similar to those in the C57BL/6 mice, demonstrating that VEGFR2 humanized does not change the overall development, differentiation or distribution of these T cell subtypes in the spleen. Values are expressed as mean ± SEM.

Antitumor activity of anti-human VEGFR2 antibody in B-hVEGFR2 mice. (A) Ramucirumab (in house) inhibited MC38 tumor growth in B-hVEGFR2 mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hVEGFR2 mice (female, 8-week-old, n=6). Mice were grouped when tumor volume reached approximately 100 mm³, at which time they were treated with Ramucirumab (in house) with doses and schedules in panel A. (B) Body weight changes during treatment. (C) Proportion of CD31-positive cell area of total area analyzed in tumor tissue. As shown in panel A, anti-human VEGFR2 antibody was efficacious in controlling tumor growth in B-hVEGFR2 mice, demonstrating that the B-hVEGFR2 mice provide a powerful preclinical model for in vivo evaluation of anti-human VEGFR2 antibodies. As shown in panel C, anti-human VEGFR2 antibody Ramucirumab inhibits vascular endothelial cell proliferation. Values are expressed as mean ± SEM.