Description

• CD20 is a marker of B cells, and it begins to express in Pre-B cells and loses expression after differentiating into plasma cells. CD20 is expressed on the surface of normal and about 95% malignant B lymphocytes, but not expressed in hematopoietic stem cells, plasma cells, or other normal tissues. CD20 is regarded as an ideal target for the treatment of B-cell lymphoma and autoimmune diseases.

• The coding region genome of mouse Cd20 gene was replaced by human CD20 coding region in B-hCD20 mice.

• Human CD20 were only detectable in homozygous B-hCD20 mice. Humanization of CD20 does not change the overall frequency or distribution of immune cell types in the spleen, blood, or lymph nodes. Humanized of CD20 does not change the blood cell composition and morphology, ALT, or AST levels or health of liver. Humanized CD20 does not affect B cell-related humoral immunity. Anti–human CD20 antibody can effectively eliminate B cells in B-hCD20 mice.

• This product is used for pharmacodynamics and safety evaluation of B-cell lymphoma and autoimmune diseases.

mRNA expression analysis

Frequency of leukocyte subpopulations in the spleen

Frequency of leukocyte subpopulations in the spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 mice and homozygous B-hCD20 mice (female, 9-week-old, n=3). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequencies of T cell subpopulations. Percentages of T cells, B cells, NK cells, DCs, monocytes, macrophages, neutrophils, CD4+ T cells, CD8+ T cells and Tregs in B-hCD20 mice were similar to those in C57BL/6 mice, demonstrating that the humanization of CD20 does not change the frequency or distribution of these cell types in the spleen. Values are expressed as mean ± SEM.

Frequency of leukocyte subpopulations in the blood

Frequency of leukocyte subpopulations in the blood by flow cytometry. Blood cells were isolated from wild-type C57BL/6 mice and homozygous B-hCD20 mice (female, 9-week-old, n=3). A. Flow cytometry analysis of the blood cells was performed to assess the frequency of leukocyte subpopulations. B. Frequencies of T cell subpopulations. Percentages of T cells, B cells, NK cells, DCs, monocytes, macrophages, neutrophils, CD4+ T cells, CD8+ T cells and Tregs in B-hCD20 mice were similar to those in C57BL/6 mice, demonstrating that the humanization of CD20 does not change the frequency or distribution of these cell types in the blood. Values are expressed as mean ± SEM.

Frequency of leukocyte subpopulations in the lymph node

Frequency of leukocyte subpopulations in the lymph node by flow cytometry. Leukocytes were isolated from wild-type C57BL/6 mice and homozygous B-hCD20 mice (female, 9-week-old, n=3). A. Flow cytometry analysis of the leukocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequencies of T cell subpopulations. Percentages of T cells, B cells, NK cells, CD4+ T cells, CD8+ T cells and Tregs in B-hCD20 mice were similar to those in C57BL/6 mice, demonstrating that the humanization of CD20 does not change the frequency or distribution of these cell types in the lymph node. Values are expressed as mean ± SEM.

Hematology analysis

Complete blood count (CBC) of B-hCD20 mice. Values are expressed as mean ± SD.

Biochemistry analysis

Biochemical test of B-hCD20 mice. Values are expressed as mean ± SD.

IgG analysis of b-hCD20 mice immunized with OVA/CFA

B-hCD20 mice (n=6) were intraperitoneally immunized with OVA/CFA or PBS on Day 0. Blood was collected on Day 0, Day 14 and Day 21 and analyzed by ELISA with mouse IgG antibody and OVA specific IgG antibody (A). Concentration of mouse IgG and mouse OVA specific IgG were significantly increased after immunization. This indicates that humanized CD20 does not affect B cell-related humoral immunity.

B cell depletion of CD20 antibody in B-hCD20 mice

Sterile anti–human CD20 antibody Obinutuzumab analog (in house), Rituximab analog (in house) and the control  hIgG1 were administered into B-hCD20 mice (female, 6-week-old, n=3) through a single tail vein injection. Blood were collected after treatment. After red blood cell lysis, the frequency of mCD19+ B cell was analyzed by flow cytometry analysis. (A) The proportion of B cells in the CD45+ cell during treatment. (B) Representative FACS images of B cells in the blood. The results indicate that Obinutuzumab analog (in house)  and Rituximab analog (in house) can effectively eliminate B cells in B-hCD20 mice.