hCD3EDG/hCD19/hCD20(2)

Fig.1 Detection of hCD3/hCD19/hCD20 expression on T cells and B cells in spleen of WT C57BL/6 mice and HO hCD3EDG/hCD19/hCD20 mice by FACS. Leukocytes were collected from wild-type C57BL/6 mice (female, 11 weeks old) and homozygous hCD3EDG/hCD19/hCD20 humanized mice (female, 11 weeks old), and analyzed by flow cytometry with anti-mCD3,  anti-mCD19, anti-mCD20, anti-hCD3, anti-hCD19, and anti-hCD20. hCD3/hCD19/hCD20 was detectable in hCD3EDG/hCD19/hCD20 humanized mice.

Fig.2 Frequency of leukocyte subpopulations in bone marrow by flow cytometry. In the bone marrow of HO hCD3/hCD19/hCD20 mice, the percentages of T cells, B cells, dendritic cells (DCs), neutrophils, macrophages, and monocytes were similar to those in WT C57BL/6 mice. However, HO mice exhibited a lower percentage of NK cells compared to WT mice in both female and male groups. Bone marrow were collected from WT C57BL/6 mice and HO hCD3/hCD19/hCD20 mice (female and male, 8-week-old, n=5). Flow cytometry analysis of frequencies of lymphocyte and myeloid subpopulations in the bone marrow. Bar indicated as mean ± SEM.

Abbr.  HO, homozygous; WT, wild type.

Fig.3 Frequency of leukocyte subpopulations in spleen by flow cytometry. In the spleen of HO hCD3/hCD19/hCD20 mice, the percentages of T cells, B cells, dendritic cells (DCs), neutrophils, macrophages, and monocytes were similar to those in WT C57BL/6 mice. However, HO mice exhibited a lower percentage of NK cells compared to WT mice, especially in female groups. Spleen were collected from WT C57BL/6 mice and HO hCD3/hCD19/hCD20 mice (female and male, 8-week-old, n=5). Flow cytometry analysis of frequencies of lymphocyte and myeloid subpopulations in the spleen. Bar indicated as mean ± SEM.

Abbr.  HO, homozygous; WT, wild type.

Fig.4 Frequency of leukocyte subpopulations in blood by flow cytometry. In the blood of HO hCD3/hCD19/hCD20 mice, the percentages of T cells, B cells, dendritic cells (DCs), neutrophils, macrophages, and monocytes were similar to those in WT C57BL/6 mice. However, HO mice exhibited a lower percentage of NK cells compared to WT mice, especially in female groups. Blood were collected from WT C57BL/6 mice and HO hCD3/hCD19/hCD20 mice (female and male, 8-week-old, n=5). Flow cytometry analysis of frequencies of lymphocyte and myeloid subpopulations in the blood. Bar indicated as mean ± SEM.

Abbr.  HO, homozygous; WT, wild type.

Fig.5 In vivo B cell depletion of Bispecific Antibodies in hCD3EDG/hCD19/hCD20 Mice. B cell depletion by anti-human CD3/CD20 or CD3/CD19 bispecific antibodies in humanized mice. (A) Representative flow cytometric analysis showing B cell depletion in peripheral blood of HO-hCD3E/hCD19/hCD20 mice. (B) Quantitative summary of B cell depletion in peripheral blood of hCD3E/hCD19/hCD20 mice (female, 8 weeks old, n=3-5 per group) after a single administration. Mice received Vehicle, Glofitamab (anti-CD3/CD20), or Surovatamig (anti-CD3/CD19). The percentage of human CD45⁺cells that are B cells is plotted over time (Day-1 [baseline], Day 3, and Day 5 post-administration).