1. A new tool for oncology researchers: The RAG2-/-IL2Rg-/- double knockout (R2G2) mouse
Jamie Naden, PhD
Scientist
Veterinary Science, Research and Support
Envigo

2. Presentation overview
Overview of the of the R2G2 (B6;129-Rag2tm1FwaII2rgtm1Rsky/DwlHsd ) model characteristics
Comparative analysis of the R2G2 and NSG immune cell subsets in spleen
Comparative analysis of the R2G2 and NSG radiosensitivity
Estrogen supplementation tolerance
Chemotherapy tolerance
Tumor growth
Summary

3. R2G2 Model: Background R2G2 = “Rag2” “IL2Rg” double knockout mouse
Model licensed from Fox Chase Cancer Center (Temple)
Created by backcrossing the IL2Rg (common gamma chain) knockout mutation on to a Rag2 knockout mouse
Background is a mixture of C57-129
Nomenclature:
B6;129-Rag2tm1FwaII2rgtm1Rsky/DwlHsd

4. R2G2 Model: Characteristics
Ultra (triple) immunodeficient mouse model
Deficient in T
Deficient in B cells
Deficient in NK cells
No leakiness, compared to other SCID mice (varies with background)
Less radiosensitive
Less sensitive to radiation as compared to traditional SCID models

5. Flow results: B and T lymphocytes diminished
Wild type
Diminished B cells and T cells in NSG and R2G2
NSG
R2G2- F
R2G2- M
B220
CD3
Gating set up based on C57bl/6 spleen

6. Flow results: Th and Tc lymphocytes diminished
Wild type
Diminished helper T cells and cytotoxic T cells in NSG and R2G2
NSG
RAG2-/-IL2Rg-/-- F
RAG2-/-IL2Rg-/-- M
CD8
CD4
Gating set up based on C57bl/6 spleen

7. Flow results: NK lymphocytes diminished
Wild type
Diminished helper natural killer cells in NSG and R2G2
NSG
RAG2-/-IL2Rg-/-- F
RAG2-/-IL2Rg-/-- M
CD49b
CD335
Gating set up based on C57bl/6 spleen

8. R2G2 Model: Growth curve R2G2 has a more robust growth curve than NSG
R2G2 Growth curve. Growth curve was completed on a cohort of R2G2 mice (N=20 males and 20 females). Data represented are average ± 2 SD.
= NOG/NSG Male
= NOG/NSG Female

9. Survival analysis of R2G2 and NSG mice in response to a single dose of radiation at 4 Gy (n=10/group)
At 4 Gy, NSG survivability began to drop at 6 days post radiation and all NSG died by day 8
At 4 Gy, all R2G2 survived
At 2 Gray, there was 100% survival of both groups
At 6 gray, there was 100% mortality

10. Survival analysis of R2G2 mice in response to differing doses of estrogen (n=10/group)
Dose response as anticipated
Survival (Figure 3) was lowest at 70% (by day 26) in the group given 1.7 mg estrogen. Eighty percent of animals given the 0.36 mg and 0.72 mg pellets survived through day 60 and 90% of the animals given 0.18 mg of estrogen survived through day 60, as anticipated.

11. Survival analysis of R2G2 mice in response to chemotherapy treatment (n=10/group)
Dox at 5mg/kg was detrimental
Doxorubicin was tolerated well at the 2 mg/kg dose and body weights remained stable. At 5 mg/kg, mice continually lost weight. At 50% survival, body weights were no longer reported (Figure 6).
As mentioned earlier, 5-FU-30, CTX-100 and DOX-2 were well tolerated in the R2G2 mouse and 100% of these animals survived. Only 10% of animals in the DOX-5 group survived by day 40 (Figure 6).

12. Growth of PNX0225 Burkitt Lymphoma PDX
R2G2 had double the tumor volume vs. C.B-17 SCID at 19 days post implantation

13. Results – head and neck PDX
Head and neck PDX 626 was transplanted (2.2 mm2 tissues) into 4 sections of each of two R2G2 mice each (n=2/PDX).
Head and neck PDX 635 was transplanted (2.2 mm2 tissues) into 4 sections of each of two R2G2 mice each (n=2/PDX).
Head and neck PDX 626 and 635
Of four tissue pieces implanted in two R2G2 mice, three 626 and three 635 tumors developed. Average tumor size was and mm3 for PDX 626 and 635, respectively, on day 84 (Figures 1 and 2).
Data are represented as individual tumors

14. Results – human esophageal adenocarcinoma (OE33)
Human esophageal adenocarcinoma OE33 cells (5x106 cells per mouse) were injected into the left and right flanks of SCID (n=3), Athymic Nude (n=3) and R2G2 (n=2) mice.
Human esophageal adenocarcinoma OE33
Take rate of OE33 cells was 100% in R2G2 mice, 0% in SCID mice and 17% in Athymic Nude mice. Only one small tumor developed in the Athymic Nude mouse (data not shown). Average tumor growth on day 70 was mm3 (Figure 3).

15. Results – human esophageal adenocarcinoma (FLO1)
FLO1 cells (5x106 cells per mouse) were injected into both flanks of three each of Athymic Nude and SCID mice, and two R2G2 mice.
No tumor growth was seen in Athymic Nude or SCID mice in Study A with FLO-1 cells whereas take rate was 100% in R2G2 mice. Average growth rate at day 60 was mm3 (Figure 4).

16. R2G2 Model: Summary Flow cytometry Growth Curve Radiosensitivity
Comparable in Cytotoxic T, Th cells and NK cells to NSG
Growth Curve
Adult M/F ~ 5-10g larger than NOG/NSG at similar age
Radiosensitivity
R2G2 can tolerate double the single radiation dose than NSG mice
Burkitt PDX xenograft
R2G2 had approximately double the tumor volume at Day 20 compared to C.B-17-Prkdcscid mice
Head and Neck PDX
Difficult to grow in Athymic Nude and SCID mice; 100% take rate in R2G2 mice
Esophageal Adenocarcinoma
100% take rate was seen in R2G2 mice, whereas there was no growth in Athymic Nude or SCID mice
Estrogen supplementation tolerance
Estrogen supplementation tolerance

17. Gating Strategy: Lymphocyte panel
Single cells
Live
CD45+ (WBC)
SSC-A
FSC-A
Dead
FSC-W
CD45
Gating set up based on C57bl/6 spleen

18. Gating strategy: Lymphocyte panel
Lymphocytes
B220+ (B)
CD8+ (CTL)
CD19 sligthly better but B220 in mice good.
CD3+ (T)
CD4+ (Th)
FSC-A
B220
CD8
FSC-W
CD3
CD4
Gating set up based on C57bl/6 spleen

19. Gating strategy: Lymphocyte panel
Lymphocytes
CD335+/CD49b+ (NK)
weak Cd335 definitely +CD49b+
CD49b
FSC-A
FSC-W
CD335
Gating set up based on C57bl/6 spleen