• 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.
• KLK2 is an androgen-regulated kallikrein expressed in the prostate. It specifically cleaves the N-terminal peptide chain of pro-PSA to activate its function, and serves as a key regulator of PSA bioactivity. For example, KLK2 is highly expressed in prostate cancer compared to benign prostatic hyperplasia (BPH). Beyond activating pro-PSA, KLK2 also activates its own precursor (pro-KLK2) and the zymogen form of urokinase-type plasminogen activator (uPA). Increased KLK2 expression in prostate cancer tissues leads to uPA activation and concurrently inactivates its primary inhibitor, plasminogen activator inhibitor-1 (PAI-1). KLK2 also promotes angiogenesis and tumor microenvironment remodeling, enhancing tumor aggressiveness and playing a significant role in prostate cancer progression.
• The exons 2-6 of mouse Cd3e gene that encode the extracellular domain were replaced by human CD3E exons 2-7 in B-hCD3E/hKLK2/hKLK3 mice. The full coding sequences of human KLK2 and KLK3 gene, including the promoter, 5’UTR and 3’UTR are inserted into mouse Hipp11 (H11) locus in  B-hCD3E/hKLK2/hKLK3 mice.
• Mouse CD3E was detectable on T cells of wild-type C57BL/6JNifdc mice. Human CD3E was exclusively detectable on T cells of  homozygous B-hCD3E/hKLK2/hKLK3 mice but not on wild-type mice.
• Human KLK2 was detected in prostate of homozygous B-hCD3E/hKLK2/hKLK3 mice.
• Application: This product is used to evaluate the pharmacodynamics and toxicity of bispecific antibody-drug conjugates (BsADCs) for treating prostate cancer in preclinical models.

Gene targeting strategy for B-hCD3E/hKLK2/hKLK3 mice. The exons 2-6 of mouse Cd3e gene that encode the extracellular domain were replaced by human CD3E exons 2-7 in B-hCD3E/hKLK2/hKLK3 mice. The full coding sequences of human KLK2 and KLK3 gene, including the promoter, 5’UTR and 3’UTR are inserted into mouse Hipp11 (H11) locus in B-hCD3E/hKLK2/hKLK3 mice.

Strain specific CD3E expression analysis in homozygous B-hCD3E/hKLK2/hKLK3 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCD3E/hKLK2/hKLK3 mice(H/H), and analyzed by flow cytometry with species-specific anti-mouse CD3E antibody (Biolegend, 100312) and anti-human CD3E antibody (BD Horizon™, 562426). Mouse CD3E was detectable in wild-type mice. Human CD3E was  exclusively detectable on T cells of  homozygous B-hCD3E/hKLK2/hKLK3 mice but not in wild-type mice.

Western blot analysis of KLK2 protein expression in homozygous B-hCD3E/hKLK2/hKLK3 mice. Various tissue lysates were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hCD3E/hKLK2/hKLK3 mice (H/H), and then analyzed by western blot with anti-KLK2 antibody(LSbio, LS-C336461). 40 μg total proteins were loaded for western blotting analysis. KLK2 was detected in prostate of homozygous B-hCD3E/hKLK2/hKLK3 mice.

Immunohistochemical (IHC) analysis of KLK3 expression in wild-type C57BL/6JNifdc mice and homozygous B-hCD3E/hKLK2/hKLK3 mice. Various tissue were collected from wild-type mice (+/+) and homozygous B-hCD3E/hKLK2/hKLK3 mice (H/H; H/H; H/H) and analyzed by IHC with anti-KLK3 antibody (abcam, ab76113). KLK3 was exclusively detectable in prostate of B-hCD3E/hKLK2/hKLK3 mice, but not in wild-type mice.

Establishment of a B-Tg(hKLK2) MC38 model and in vivo efficacy study of the Pasritamig-analog. B-Tg(hKLK2) MC38 cells were implanted subcutaneously into homozygous B-hCD3E/hKLK2/hKLK3 mice (male, 12-weeks-old, n=6). Mice were grouped once tumor volume reached approximately 88 mm³, at which time they were intravenously injected with anti-human CD3/KLK2 bispecific antibody Pasritamig-analog (made in house).

Antitumor activity of Pasritamig-analog against syngeneic tumors. (A) Tumor growth curves. (B) Body weight changes during treatment. (C) Tumor cells growth of individual mouse. As shown in panel A, CD3/KLK3 bispecific antibodies (made in house) was efficacious in controlling tumor growth in B-hCD3E/hKLK2/hKLK3 mice. These results demonstrate that B-hCD3E/hKLK2/hKLK3 mice provide a powerful preclinical model for in vivo evaluation of anti-human CD3E/KLK2 bispecific antibodies. Values are expressed as mean ± SEM. The overage of this tumor model is 40%.