• 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 two novel hIL12RB1/hIL12RB2 knock-in mouse strains to support related drugs development. For IL12RB1 humanized model, both in IL12RB2 plus and ad versions, the extracellular region sequences of mouse Il12rb1 gene were replaced by human IL12RB1 counterpart gene sequences. For IL12RB2 humanized model, the extracellular and transmembrane region sequences of mouse Il12rb2 were replaced by human IL12RB2 counterpart gene sequences in B-hIL12RB1 plus/hIL12RB2 plus mice. 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 plus mice and 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 plus/hIL12RB2 plus mice (112518). IL12RB1: the extracellular region sequences of mouse Il12rb1 were replaced by human IL12RB1 counterpart gene sequences. IL12RB2: the extracellular and transmembrane region sequences of mouse Il12rb2 were replaced by human IL12RB2 counterpart gene sequences.

Strain specific analysis of IL12RB1 mRNA expression by RT-PCR. Spleen RNA were isolated from wild-type C57BL/6 mice (WT), homozygous B-hIL12RB1 plus/hIL12RB2 plus mice (plus) and homozygous B-hIL12RB1 plus/hIL12RB2 ad mice (ad), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse and human strain specific IL12RB1 primers. Human IL12RB1 mRNA exclusively detectable in homozygous B-hIL12RB1 plus/hIL12RB2 plus mice and B-hIL12RB1 plus/hIL12RB2 ad mice but not in wild-type C57BL/6 mice. Mouse Il12rb1 was only detectable in wild-type C57BL/6 mice but not in humanized mice.

Strain specific analysis of IL12RB2 mRNA expression by RT-PCR. 

Spleen RNA were isolated from wild-type C57BL/6 mice (WT), homozygous B-hIL12RB1 plus/hIL12RB2 plus mice (plus) and homozygous B-hIL12RB1 plus/hIL12RB2 ad mice (ad), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse and human strain specific IL12RB2 primers. 

• Human IL12RB2 mRNA exclusively detectable in homozygous B-hIL12RB1 plus/hIL12RB2 plus mice and homozygous B-hIL12RB1 plus/hIL12RB2 ad mice but not in wild-type C57BL/6 mice. 
• Mouse Il12rb2 mRNA that PCR with mF3/mR3 primers was not detected in B-hIL12RB1 plus/hIL12RB2 ad mice and B-hIL12RB1 plus/hIL12RB2 plus mice. Whereas, mouse Il12rb2 mRNA that PCR with mF1/mR2 primers was both detectable in wild-type C57BL/6 mice and B-hIL12RB1 plus/hIL12RB2 ad mice, but not in B-hIL12RB1 plus/hIL12RB2 plus mice. It’s demonstrating that there’s an un-complete mouse Il12rb2 mRNA in B-hIL12RB1 plus/hIL12RB2 ad mice.

IL12RB1 and IL12RB2 gene expression in wild-type C57BL/6 mice and homozygous B-hIL12RB1/hIL12RB2 mice by RT-qPCR. Splenocytes were collected from wild-type C57BL/6 mice (2 female, 1 male, n=3, 14-week-old), homozygous B-hIL12RB1 plus/hIL12RB2 plus mice (male, n=3, 14-week-old) and homozygous B-hIL12RB1 plus/hIL12RB2 ad mice (male, n=3, 14-week-old). Then cDNA libraries were synthesized by reverse transcription, followed by PCR with human-specific IL12RB1 and IL12RB2 primers. Values are expressed as mean ± SEM. Significance was determined by unpaired t-test.  *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, homozygous B-hIL12A/hIL12B/hIL12RB1/hIL12RB2 mice, homozygous B-hIL12RB1 plus/hIL12RB2 plus 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) humanized mice, male, 3 mice/group, 14-week-old. 2) wild-type C57BL/6 mice, 2 female and 1 male mice, 14-week-old.

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 mice (female, n=3, 6-week-old), homozygous B-hIL12RB1 plus/hIL12RB2 plus mice (female, n=3, 9-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, dendritic cells, neutrophils, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Tregs in B-hIL12RB1 plus/hIL12RB2 plus mice and 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), homozygous B-hIL12RB1 plus/hIL12RB2 plus mice (female, n=3, 9-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, dendritic cells, neutrophils, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Tregs in B-hIL12RB1 plus/hIL12RB2 plus mice and 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), homozygous B-hIL12RB1 plus/hIL12RB2 plus mice (female, n=3, 9-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 plus mice and 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.

Values are expressed as mean ± SEM. Significance was determined by One-way or Two-way ANOVA test.  *P < 0.05, **P < 0.01, ***P < 0.001.  

Values are expressed as mean ± SEM. Significance was determined by One-way or Two-way ANOVA test.  *P < 0.05, **P < 0.01, ***P < 0.001.  

Values are expressed as mean ± SEM. Significance was determined by One-way or Two-way ANOVA test.  *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.