Protein expression analysis

Strain specific CD40 expression analysis in homozygous B-hCD40 mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hCD40 (H/H) mice stimulated with anti-CD3ε in vivo (7.5 μg/mice), and analyzed by flow cytometry with species-specific anti-CD40 antibody. Mouse CD40 was exclusively detectable in WT mice. Human CD40 were exclusively detectable in homozygous B-hCD40 but not WT mice.

CD40 in B cells-Spleen 

Analysis of spleen T cell subpopulations in B-hCD40 mice

In vivo efficacy of anti-human CD40 antibodies

Antitumor activity of anti-human CD40 antibodies in B-hCD40 mice. (A) Anti-human CD40 antibodies inhibited MC38 tumor growth in B-hCD40 mice. Murine colon cancer MC38 cells (5ⅹ10⁵) were subcutaneously implanted into heterozygous B-hCD40 mice (female, 4 week-old, n=5). Mice were grouped when tumor volume reached approximately 100 mm³, at which time they were treated with three anti-human CD40 antibodies with doses and schedules indicated in panel (B) Body weight changes during treatment. As shown in panel A, anti-human CD40 antibodies were efficacious in controlling tumor growth in B-hCD40 mice, demonstrating that the B-hCD40 mice provide a powerful preclinical model for in vivo evaluation of anti-human CD40 antibodies. Values are expressed as mean ± SEM

In vivo efficacy of anti-human CD40 antibodies

Antitumor activity of anti-human CD40 antibody in B-hCD40 mice. (A) Anti-hCD40 antibody Selicrelumab (in house) inhibited MC38 tumor growth in B-hCD40 mice. Murine colon cancer MC38 cells(5ⅹ10⁵) were subcutaneously implanted into homozygous B-hCD40 mice (female,8-9 week-old, n=5). Mice were grouped when tumor volume reached approximately 150±50 mm³, at which time they were treated with anti-human CD40 antibody Selicrelumab (in house) with doses and schedules indicated in panel A. (B) Body weight changes during treatment. As shown in panel A, anti-human CD40 antibody Selicrelumab (in house) was efficacious in controlling tumor growth in B-hCD40 mice, demonstrating that the B-hCD40 mice provide a powerful preclinical model for in vivo evaluation of anti-human CD40 antibodies. Values are expressed as mean ± SEM.

High does of selicrelumab (anti human CD40) moderately reduced body weight in B-hCD40 mice but not in wild type C57BL/6 mice. Homozygous B-hCD40  and C57BL/6 mice were treated with vehicle or selicrelumab (in house) on days indicated by red arrows (n=3). Selicrelumab at 20 mg/kg caused significant body weight reduction compared with vehicle control in B-hCD40 mice, while the same treatment had no effect in C57BL/6 mice. Values are expressed as mean ± SEM.

Selicrelumab  Analog Toxicity Evaluation

High-dose human CD40 antibody caused significant changes in AST and ALT in B-hCD40 mice. Homozygous B-hCD40  and C57BL/6 mice were treated with PBS or selicrelumab (in house) as indicated on the Dosing Schedule (n=3). The blood biochemical analysis was conducted on B-hCD40 mice treated with anti-human CD40 antibody 24 hours after the termination of efficacy evaluation experiment. Selicrelumab at 20 mg/kg caused significant increase of  AST and ALT compared with PBS control in B-hCD40 mice, while the same treatment had no effect in C57BL/6 mice. Values are expressed as mean ± SEM.

High-dose human CD40 antibody caused significant changes in complete blood count in B-hCD40 mice. Homozygous B-hCD40  and C57BL/6 mice were treated with PBS or selicrelumab (in house) as indicated on the graph (n=3). The complete blood count (CBC) was performed 24 hours after last dosing on day 11. Selicrelumab at 20 mg/kg caused significant reduction of WBC, RBC, Hb, HCT and LY#, and increase of RDW and MPV compared with PBS control in B-hCD40 mice, while the same treatment had no effect in C57BL/6 mice. Values are expressed as mean ± SEM.

High dose of selicrelumab therapy resulted in increased lymphocyte infiltration in liver and kidney in B-hCD40 mice but not in C57BL/6 mice