• Interleukin 12 (IL12) is a powerful cytokine encoded by two separate genes, IL12A (p35) and IL12B (p40), which exist in the form of an active heterodimer referred to as p70. The main producers of IL12 are dendritic cells and macrophages in response to antigenic stimulation. IL12 receptor (IL12R) is a complex consisting of interleukin 12 receptor beta 1 (IL12Rβ1) and interleukin 12 receptor beta 2 (IL12Rβ2) chains. IL12 is a major regulator of innate resistance and adaptive immunity, which can induce proliferation, activates cytotoxic lymphocytes and natural killer (NK) cells, increases interferon (IFN)-γ production, as well as favoring differentiation, augments the production of TH1-associated immunoglobulin. Because of its ability in pro-inflammatory and immunoregulatory, it can turn the tumor from “cold” to “hot”. IL12 is a promising anti-tumor drug. However, the administration of unmodified IL-12 may insufficiently accumulate in the tumor microenvironment specifically. In turn, it's hard to be effective and may cause severe immune-related adverse effects. So, modified IL12 or combined with other therapy agents may enhance its efficacy and overcome safety challenges.
• Mouse PD-1 was only detectable in wild-type C57BL/6 mice. Human PD-1 was exclusively detectable in homozygous B-hPD-1 plus/hIL12RB1 /hIL12RB2 mice, but not in wild-type C57BL/6 mice. Human IL12 induced the IFN-γ production in CD4+ T cells sorted from splenocytes of homozygous B-hPD-1 plus/hIL12RB1 plus/hIL12RB2 ad mice. Anti-human PD-1/IL12 bispecific antibody were efficacious in controlling tumor growth in B-hPD-1 plus/hIL12RB1 plus/hIL12RB2 ad mice. 
• This product is used for the pharmacodynamics and safety evaluation of bispecific antibodies against human PD-1 and IL12.

Gene targeting strategy for B-hPD-1 plus/hIL12RB1 plus/hIL12RB2 ad mice. 

• The extracellular region sequences of mouse Il12rb1 were replaced by human IL12RB1 counterpart gene sequences in B-hPD-1 plus/hIL12RB1 plus/hIL12RB2 ad mice. 
• Chimeric CDS, composed of human IL12RB2 extracellular region plus mouse Il12rb2 transmembrane and cytoplasmic region, was inserted into the mouse Il12rb2 gene exon2 in B-hPD-1 plus/hIL12RB1 plus/hIL12RB2 ad mice. 
• Human PD-1 gene encoding the extracellular region and mouse Pd-1 gene encoding the transmembrane and cytoplasmic region were inserted after the initiation codon ATG of mouse Pd-1 gene in B-hPD-1 plus/hIL12RB1 plus/hIL12RB2 ad mice.

Strain specific PD-1 expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hPD-1 plus/hIL12RB1 plus/hIL12RB2 ad mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice and homozygous B-hPD-1 plus/hIL12RB1 plus/hIL12RB2 ad mice stimulated with anti-mouse CD3ε antibody (7.5 μg, i.p.) in vivo for 24 hrs. Protein expression was analyzed with anti-mouse PD-1 antibody (Biolegend, 109104) and anti-human PD-1 antibody (Biolegend, 329904) by flow cytometry. Mouse PD-1 was only detectable in wild-type C57BL/6 mice. Human PD-1 was exclusively detectable in homozygous B-hPD-1 plus/hIL12RB1 plus/hIL12RB2 ad mice, but not in wild-type C57BL/6 mice.

IL12 induced the IFN-γ production in CD4+ T cells sorted from splenocytes. CD4+ T cells were sorted from the splenocytes in the wild-type C57BL/6 mice and homozygous B-hPD-1 plus/hIL12RB1 plus/hIL12RB2 ad mice, then the production of IFN-γ in supernatants were assessed after 48 hous of incubation with mouse IL-12 (mIL12) and human IL12 (hIL12) in combination with bead-associated CD3 and CD28 antibodies, under the condition in the panel. Mouse IFN-γ were both increased after responsiveness to mIL12 in wild-type C57BL/6 mice and homozygous B-hPD-1 plus/hIL12RB1 plus/hIL12RB2 ad mice. Mouse IFN-γ were increased after responsiveness to hIL12 in homozygous B-hPD-1 plus/hIL12RB1 plus/hIL12RB2 ad mice but not in wild-type C57BL/6 mice.

Antitumor activity of anti-human PD-1/IL12 bispecific antibody in B-hPD-1 plus/hIL12RB1 plus/hIL12RB2 ad mice. (A) anti-human PD-1/IL12 bispecific antibody inhibited MC38 tumor growth in B-hPD-1 plus/hIL12RB1 plus/hIL12RB2 ad mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hPD-1 plus/hIL12RB1 plus/hIL12RB2 ad mice (female, 8-week-old, n=6). Mice were grouped when tumor volume reached approximately 80-120 mm³, at which time they were intraperitoneally injected with anti-human PD-1/IL12 bispecific antibody (provided by the client) indicated in panel. (B) Body weight changes during treatment. As shown in panel A, anti-human PD-1/IL12 bispecific antibody (provided by the client) was efficacious in controlling tumor growth in B-hPD-1 plus/hIL12RB1 plus/hIL12RB2 ad mice, demonstrating that the B-hPD-1 plus/hIL12RB1 plus/hIL12RB2 ad mice provide a powerful preclinical model for in vivo evaluation of anti-human PD-1/IL12 bispecific antibody. Values are expressed as mean ± SEM.