• 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 IL12 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. 
• To evaluate the efficacy of IL12 analogs in preclinical studies, Biocytogen has successfully generated a novel hIL12RB1/hIL12RB2 knock-in mouse strain to support related drugs development. In this mouse model, the extracellular region sequences of mouse Il12rb1 gene were replaced by human IL12RB1 counterpart gene sequences. Chimeric CDS, composed of human IL12RB2 extracellular region plus mouse Il12rb2 cytoplasmic region, was inserted into the mouse endogenous gene locus in B-hIL12RB1 plus/hIL12RB2 ad mice. It has been verified that human IL12 induced the IFNγ production in CD4+ T cells sorted from splenocytes of homozygous B-hIL12RB1 plus/hIL12RB2 ad mice. The gene humanization was successful. During the in vivo toxicity study, besides induced the IFNγ production, human IL12 induced an increase in the percentage of liver and spleen relative to body weight and showed a tendency of AST activity increase in humanized mice. B-hIL12RB1 plus/hIL12RB2 ad mice can be used to evaluate the efficacy and toxicity of human IL12 analogs.

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

The extracellular region sequences of mouse Il12rb1 were replaced by human IL12RB1 counterpart gene sequences in B-hIL12RB1/hIL12RB2 mice ad.

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-hIL12RB1/hIL12RB2 mice ad

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 mice (female, n=3, 6-week-old) and homozygous B-hIL12RB1 plus/hIL12RB2 ad mice (female, n=3, 6-week-old). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Percentages of T cells, B cells, NK cells, DCs, neutrophils, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Tregs in B-hIL12RB1 plus/hIL12RB2 ad mice were similar to those in C57BL/6 mice. Values are expressed as mean ± SEM. Significance was determined by Multiple t tests. *P < 0.05, **P < 0.01, ***p < 0.001.

Frequency of leukocyte subpopulations in blood by flow cytometry. Blood cells were isolated from wild-type C57BL/6 mice (female, n=3, 6-week-old) and homozygous B-hIL12RB1 plus/hIL12RB2 ad mice (female, n=3, 6-week-old). A. Flow cytometry analysis of the blood cells was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Percentages of T cells, B cells, NK cells, DCs, neutrophils, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Tregs in B-hIL12RB1 plus/hIL12RB2 ad mice were similar to those in C57BL/6 mice. Values are expressed as mean ± SEM. Significance was determined by Multiple t tests. *P < 0.05, **P < 0.01, ***p < 0.001.

Frequency of leukocyte subpopulations in lymph nodes by flow cytometry. Lymph nodes cells were isolated from wild-type C57BL/6 mice (female, n=3, 6-week-old) and homozygous B-hIL12RB1 plus/hIL12RB2 ad mice (female, n=3, 6-week-old). A. Flow cytometry analysis of the lymph nodes cells was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Percentages of T cells, B cells, NK cells, CD4+ T cells, CD8+ T cells and Tregs in B-hIL12RB1 plus/hIL12RB2 ad mice were similar to those in C57BL/6 mice. Values are expressed as mean ± SEM. Significance was determined by Multiple t tests. *P < 0.05, **P < 0.01, ***p < 0.001.

IL12 induced the IFNγ production in CD4+ T cells sorted from splenocytes. CD4+ T cells were sorted from the splenocytes of wild-type C57BL/6 mice and homozygous B-hIL12RB1 plus/hIL12RB2 ad mice. The production of IFNγ in supernatants were assessed after incubation with mouse IL12 (mIL12) or human IL12 (hIL12) in combination with bead-associated 0.4 μg/mL anti-mCD3e and 0.8 μg/mL anti-mCD28 antibodies for 48 hours. Mouse IFNγ were increased after responsiveness to mIL12 in humanized mice and wild-type mice. While, only hIL12 induced mouse IFNγ increase in humanized mice. 

Mice information: 1) B-hIL12RB1 plus/hIL12RB2 ad mice, male, 3 mice/group, 14-week-old. 2) wild-type C57BL/6 mice, 2 female and 1 male mice, 14-week-old.

Antitumor activity of  human IL-12 in B-hIL12RB1 plus/hIL12RB2 ad mice. (A) Human IL12 inhibited MC38 tumor growth in B-hIL12RB1 plus/hIL12RB2 ad mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hIL12RB1 plus/hIL12RB2 ad mice (female, 6-8 weeks-old, n=8). Mice were grouped when tumor volume reached approximately 100 mm³, at which time they were intravenous injection with hIL-12 in the panel. (B) Body weight changes during treatment. As shown in panel A, the human IL-12 showed inhibitory effects in a dose-dependent manner. Values are expressed as mean ± SEM.