Humanized CD3e Based Bispecific Antibody Efficacy Evaluation

Many antibodies that target human CD3 do not cross-react to mouse CD3. Biocytogen’s B-hCD3ε mice provide an excellent solution where the human extracellular domain of CD3ε replaces the mouse counterpart, thus allowing direct testing of anti-human CD3 antibodies (including bispecific anti-human CD3 antibodies) in mice for in vivo efficacy assessment. These B-hCD3ε mice have the following features:

  • Human CD3ε are expressed in the homozygous B-hCD3ε mice whereas mouse CD3ε cells are absent.
  • T cell development is normal in B-hCD3ε homozygous mice.
  • T cell subtypes in the spleen, thymus, lymph nodes, and PBMC in B-hCD3ε mice are comparable to those in wildtype C57BL/6 mice.
  • Anti-mouse PD-1 antibody significantly represses the growth of MC38 tumor cells engrafted on B-hCD3ε mice, suggesting that T cells in B-hCD3ε mice are functional.
  • Treatment of B-hCD3ε mice with anti-human CD3ε antibody OKT3 leads to T cell depletion, suggesting functional human CD3ε.
  • Normal in vitro T cell proliferation and cytokine production are found in anti-hCD3ε antibody treated humanized B-hCD3ε mice.

Most importantly, B-hCD3ε mice respond to blinatumomab in a dose-dependent manner in inhibiting growth of murine colon cancer MC38-hCD19 cells.

CD3ε Mouse Model

In B-hCD3ε model, the mouse CD3ε extracellular domain was replaced by human CD3ε. We used conventional ES/HR technology to knockin human CD3ε genomic DNA from 3’ part of exon 2 to 5’ of exon7 to replace mouse corresponding exon 2-exon 6.

Case 1: Bispecific Antibody in vivo Efficacy Evaluation

Murine colon cancer MC38-hCD19 cells were subcutaneously implanted into B-hCD3ε mice. Mice were divided into control and treatment group dose(n=6) when the tumor size was approximately 150±50 mm3. High doses of hCD3ε antibody (Blinatumomab) significantly inhibited tumor growth, confirming that the B-hCD3ε mouse model is a powerful tool for in vivo anti-hCD3ε based bi-specific antibody efficacy evaluation. (A) Tumor average volume ± SEM, (B) Mice average weight ±SEM.

Case 2

Strains of Mice Group Treatment
B-hCD3ε G1 hCD3 Antibody(OKT3)
B-hCD3ε G2 CD3/CD19 Bispecific Antibody
B-hCD3ε G3 Blinatuomomab
B-hCD3ε G4 PBS
C57BL/6 G5 mCD3 antibody (145-2C11)
C57BL/6 G6 PBS

The ratio of T cells in spleen and blood were analyzed at 24h, 72h and 168h by flow cytometry.

Case 3

Murine colon cancer MC38 cells were subcutaneously implanted into C57BL/6(A) and B-hCD3e (B) mice. Mice were grouped when the tumor size was approximately 150±50mm3 (n=5). In the humanized mouse model, mPD-1 antibody significantly inhibited tumor growth, indicating normal T cell function. More aggressive tumor growth after anti-hCD3 antibody treatment was observed. This may result from activation induced cell death (AICD). As a result, the B-hCD3e mouse model is a powerful tool for in vivo CD3 antibody pharmacological efficacy studies.

Humanized Tumor Cell Lines

Humanized Tumor Cell Lines

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