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

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

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

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

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

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

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

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

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

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.

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).

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

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 373.4 and 270.8 mm3 for PDX 626 and 635, respectively, on day 84 (Figures 1 and 2). Data are represented as individual tumors

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 930.9 mm3 (Figure 3).

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 1585.4 mm3 (Figure 4).

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

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

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

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