• CD3 consists of four protein chains (CD3E, CD3D, CD3G and CD3Z), which are important biological markers on the T cell membrane. CD3 can form a TCR/CD3 complex with the T cell receptor, participating in the regulation of T cell antigen recognition, signal transduction and T cell development. CD19 is a biomarker for normal and neoplastic B cells, as well as follicular dendritic cells. 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.
• B-hCD3EDG/hCD20 mice were obtained by mating B-hCD3EDG mice(110039) and B-hCD20 mice(111231). In B-hCD3EDG/hCD20 mice, chimeric human CD3EDG was expressed, while mouse Cd3edg were knocked out. The exons 2-7 of mouse Cd20 gene that encode the whole molecule (ATG to STOP codon) are replaced by human counterparts in B-hCD20 mice. The promoter,5’UTR, and 3’UTR region of the mouse gene are retained. The human CD20 expression is driven by endogenous mouse Cd20 promoter, while mouse Cd20 gene transcription and translation will be disrupted.
• Human CD3E and CD20 can be detected respectively on T cells and B cells from spleen of homozygous B-hCD3EDG/hCD20 mice, but not in wild-type mice. In in vivo B cells depletion experiment, Glofitamab analog can effectively eliminate B cells in B-hCD3EDG/hCD20 mice.
• This product is used for the pharmacological and safety evaluation of TCE in B-cell lymphoma and autoimmune diseases.

Strain specific analysis of CD3DG gene expression in wild type (WT) mice and B-hCD3EDG/hCD20 mice by RT-PCR. Mouse Cd3d and Cd3g mRNA were detectable only in thymocytes of WT mice (+/+). Human CD3D and CD3G mRNA were detectable only in homozygous B-hCD3EDG/hCD20 mice (H/H;H/H) but not in WT mice (+/+).

Strain specific CD3E expression analysis in homozygous B-hCD3EDG/hCD20 mice by flow cytometry. Splenocytes were collected from wild type (WT) mice (+/+) and homozygous B-hCD3EDG/hCD20 mice (H/H;H/H), and analyzed by flow cytometry with species-specific anti-CD3E antibody. Mouse CD3E was detectable in WT mice (+/+). Human CD3E was exclusively detectable in homozygous B-hCD3EDG/hCD20 mice (H/H;H/H) but not in WT mice (+/+).

Strain specific CD20 expression analysis in homozygous B-hCD3EDG/hCD20 mice by flow cytometry. Splenocytes were collected from wild type (WT) mice (+/+) and homozygous B-hCD3EDG/hCD20 mice (H/H;H/H), and analyzed by flow cytometry with species-specific anti-CD20 antibody. Mouse CD20 was detectable in WT mice (+/+). Human CD20 was exclusively detectable in homozygous B-hCD3EDG/hCD20 mice (H/H;H/H) but not in WT mice (+/+).

In vivo B cell depletion of anti-human CD3/CD20 bispecific antibody(BsAb) in B-hCD3EDG/hCD20 mice. Anti–human CD3/CD20 bispecific antibody Glofitamab analog (Commercialized) and PBS were administered into B-hCD3EDG/hCD20 mice (female, 8-week-old, n=3) through a single dose injection. Blood  were collected at Day-1, Day2, Day4 and Day7 after treatment. The numbers and the frequency of mCD45+ cells, B cells (mCD19+) and T cells (mTCRβ+) were determined by flow cytometry. (A) The number of CD45+ cells, B cells and T cells in blood. (B) The proportion of T and B cells in the CD45+ cells. (C) B cell depletion ratio. The results indicate that Glofitamab analog can effectively eliminate B cells in B-hCD3EDG/hCD20 mice.

Anti-CD3/CD20 BsAb Induce Cytokine Storm (CRS) in B-hCD3EDG/hCD20 mice. B-hCD3EDG/hCD20 mice (female, 6-week-old, n=5) were treated once intravenously on day0 with antibodies (Commercialized) indicated in the panel. Multiplex analysis of cytokines in serum at 4, 24 and 72 h after administration. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***P < 0.001.

Antitumor Activity of Anti-Human CD3/CD20 Bispecific Antibodies (Glofitamab, Commercialized) in B-hCD3EDG/hCD20 Mice.​​ B-hCD20 MC38 mouse colon carcinoma cells were subcutaneously implanted into homozygous B-hCD3EDG/hCD20 mice (female, 9-week-old, n=6). Mice were grouped once tumor volume reached approximately 100 mm³, at which time they were intravenously injected with anti-human CD3/CD20 bispecific antibodies (indicated in the panel). (A) Anti-human CD3/CD20 bispecific antibodies inhibited B-hCD20 MC38 tumor growth in B-hCD3EDG/hCD20 mice. (B) Body weight changes during treatment. Values are expressed as mean ± SEM.

(C) Anti-CD3/CD20 BsAb Glofitamab depletes B cells in blood of B-hCD3EDG/hCD20 mice bearing B-hCD20 MC38 tumor. During in vivo efficacy study described in previous page, blood  were collected at Day 0, Day7, Day14, Day21 and Day 26 after grouping. The numbers and the frequency of mCD45+ cells, B cells (mCD19+) and T cells (mTCRβ+) were determined by flow cytometry. The results indicate that Glofitamab can effectively eliminate B cells and induced T cells death in B-hCD3EDG/hCD20 mice. Values are expressed as mean ± SEM.

(D) At the end of the experiment, spleen cells were collected and the frequency of B cells (mCD19+) and T cells (mTCRβ+) in mCD45+ cells were determined by flow cytometry. The results indicate that Glofitamab can effectively induced T cells death but not eliminate B cells in B-hCD3EDG/hCD20 mice. Values are expressed as mean ± SEM. Significance was determined by Ordinary one-way ANOVA.  *P < 0.05, **P < 0.01, ***P < 0.001.

(F) Anti-CD3/CD20 BsAb Induce Cytokine Storm (CRS) in B-hCD3EDG/hCD20 mice Bearing B-hCD20 MC38 cells. During in vivo efficacy study described in previous page, blood were collected at 4, 12, 24 hours after first dosing. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***P < 0.001.

Effects of anti-human CD3/CD20 bispecific antibody (BsAb) Glofitamab Analog on MOG₁₋₁₂₅-Induced EAE.​​ Mice received MOG₁₋₁₂₅ on day 0 and day 1. Body weight (A), clinical score (B), and EAE incidence (C) were recorded. Glofitamab analog effectively alleviated EAE symptoms. Statistical analysis was performed using two-way ANOVA with Dunnett’s test (n = 3–5; *P < 0.05, ​**​P < 0.01 vs. G2).  

FACS analysis of T and B cell depletion in blood and spleen.​​ (A) B cells in blood. (B) T cells in blood. (C) B cells in spleen. (D) T cells in spleen. Efficient depletion of B and T cells was observed in both blood and spleen following Glofitamab analog treatment. Statistical analysis was performed using two-way ANOVA with Dunnett’s test (n = 3–5; *P < 0.05, ​**​P < 0.01 vs. G2).