Background
The T-cell receptor (TCR)- CD3 complex plays a critical role in mediating T-cell activation and cellular immune response. Key signaling subunits of the CD3 co-receptor include CD3, CD3, and CD3. In recent years, an attractive therapeutic approach for cancer treatment uses bispecific antibodies that target human CD3 and a tumor-associated antigen (TAA) to ultimately redirect T cell cytotoxicity towards tumor cells. However, evaluation of therapeutics targeting human CD3 in animal models is difficult due to the relatively low sequence homology between the extracellular domain of human CD3 and mouse CD3.
Methods
To address the need for murine models suitable for directly evaluating CD3-based therapeutics, Biocytogen generated two versions of humanized CD3 mice. In B-hCD3E mice the extracellular domain of human CD3 replaced the mouse counterpart, while in the B-hCD3EDG mouse strain the coding sequences of human CD3 subunits, CD3, CD3, and CD3, replaced their mouse counterparts. We first confirmed gene and protein expression by RT-PCR and flow cytometry, respectively. To assess immune cell development and in vitro T cell activation following stimulation with an anti-CD3 antibody, flow cytometric analysis was performed using B-hCD3E and B-hCD3EDG mice. Using MC38-based tumor models, in vivo efficacy of anti-human CD3/CD19 and anti-human CD3/BCMA bispecific antibodies were evaluated in B-hCD3E and B-hCD3EDG mice, respectively.
Results
Here we present evidence that humanized B-hCD3E and B-hCD3EDG mice demonstrated normal immune cell development compared with wild-type C57BL/6 mice. When stimulated with an anti-CD3 antibody in vitro, wild-type C57BL/6 and humanized CD3-based mice displayed similar levels of T-cell activation and cytokine production. Finally, bispecific anti-human CD3/CD19 antibody blinatumomab dose-dependently inhibited MC38-hCD19 tumor growth in B-hCD3E mice, and bispecific anti-human CD3/BCMA antibody dose-dependently inhibited MC38-hBCMA tumor growth in B-hCD3EDG mice.
Conclusions
Biocytogen’s validated humanized B-hCD3E and B-hCD3EDG mice exhibited normal immune cell profiles, robust in vitro activity, and efficacious in vivo response to bispecific antibodies. These mice present a useful model for accelerated in vivo evaluation of anti-human CD3 therapeutics. Our growing list of CD3-based humanized mice includes B-hCD3E/hCD28, B-hCD3E/h4–1BB, B-hCD3E/hCD20, B-hCD3EDG/hBCMA, and B-hCD3EDG/hTROP2 which can be used for in vivo evaluation of combination therapies.
Ethics Approval
All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Biocytogen Beijing Co., Ltd.