Application of humanized immunodeficient mice for precision medicine 擬人化免疫缺陷小鼠在精準醫學的應用 Shu-Wha Lin, Ph.D. 林淑華 台大醫技系 Professor, Department of Clinical Laboratory Science and Medical Biotechnology, College of Medicine, National Taiwan University Principal Investigator of Transgenic Mouse Model Core (TMMC) Facility Ministry of Science and Technology, Taiwan

Design a rational combination therapy Apply treatment on patient Precision oncology Determine tumor-driving networks that function in a particular patient’s tumor Design a rational combination therapy Test in vitro/in vivo Apply treatment on patient Panomic data from tissue samples, obtained from tumours and surrounding healthy tissue, are analysed to produce a list of hypothetical aberrant driver networks. Drug candidates that target specific molecular pathways are selected and validated in models if possible; for example, in vitro (in tumour-derived cell lines) or in vivo (in mouse models). If the decision is made to move forward with that treatment, the patient is treated and monitored using rapid measures, such as imaging and serum biomarkers. Failure to respond, or disease recurrence, might lead one to choose a different drug or combination based upon a fresh analysis. Nat Rev Clin Oncol. 2014 Feb;11(2):109-18.

PDX (patient derived xenograft) an ideal in-vivo model for preclinical testing Utility of humanized mouse models for predictive in vivo preclinical testing of physiology, pathology, and therapy. With the generation of predictive humanized mouse model systems, knowledge of primary human-specific physiology and pathology can be accelerated and utilized for medical improvements. 3

因應人類組織/細胞異種移植發展出各式免疫缺陷小鼠 Blood. 2015 Apr 23;125(17):2630-40.

NOD-scid IL2rγ null (NSG) mice JAX Lab NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ JAX Lab Lack of mature T, B, and functional NK cells Deficient in signaling of multiple cytokines (IL2, IL4, IL7, IL9, IL15 and IL21) Engraftment of human tissues, HSC, and PBMC: Significantly improved Resistant to lymphoma, allowing for long-term experiments Application: Reconstitution of human immune systems (hu-PBL/HSC mice) Patient-derived xenograft (PDX) (Ito, Takahashi et al. 2012 Cell Mol Immunol. 208-14)

Patient Tumor Characteristics NSG™ vs scid WHY NSG? Patient Tumor Characteristics NSG™ vs scid Cancer Patient’s Lung Tumor Fragment NSG™ C.B-17 scid H&E H&E Human cells (anti-HLA) Mouse blood cells (anti-mCD45) Simpson-Abelson MR et al. 2008.J Immunol 180(10):7009-18. PMID:18453623 JAX® Mice | 18

Humanized tumor-bearing NSGTM Engraftment of human tumor in mice with Hu-PDX Humanized tumor-bearing NSGTM Engraftment of human tumor in mice with human immune system The next step in cancer modeling NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSGTM) (005557) In Vivo Pharmacology Services | 48

Tumor in humanized PDX NSG v.s Tumor in PDX NSG WHY Hu-PDX ? Tumor in humanized PDX NSG v.s Tumor in PDX NSG Genetic drift in tumor cells PDX Hu-PDX Disease Models & Mechanisms 1, 78-82 (2008) doi:10.1242/dmm.000976

WHY Hu-PDX ? 免疫療法仰賴病人自身免疫細胞攻擊癌細胞  以 humanized PDX mouse model 測試療效 More faster and robust reconstitution of human immune system (CD34+ HSC) 4 weeks, majority CD33+ myeloid cells, Tregs Human PDX breast tumor Human PDX lung tumor Testing the response of patient’s tumor to immune check point inhibitors can be possible 以anti-PD-1抗體治療 以anti-PD-1抗體治療 以anti-CTLA4抗體治療 Pembrolizumab (Keytruda): anti-PD-1 antibody Ipilimumab (Yervoy): anti-CTLA4 antibidy 9 JAX Lab

改善人類腫瘤小鼠模式缺少人類腫瘤微環境的缺點 擬人化免疫缺陷小鼠在精準醫學的應用 改善人類腫瘤小鼠模式缺少人類腫瘤微環境的缺點 移植病人造血幹細胞 HM: humanized mice (擬人化小鼠) Cancer Res. 2016 Nov 1;76(21):6153-6158.

Our efforts on humanized mouse models

建立測試移植人類免疫系統的擬人化小鼠平台 周邊血 移植成功定義 hCD45+ cells > 15% T, B cells patient Human PBMC Humanized mice are immunodeficient mice engrafted with human HSC, PBMC or tumor tissues 12 12

建立測試移植人類免疫系統的擬人化小鼠平台 周邊血 移植成功定義 hCD45+ cells > 15% T, B cells patient 功能測試 Human PBMC Humanized mice are immunodeficient mice engrafted with human HSC, PBMC or tumor tissues Humanized Hemophilia NSG mice 功能測試 13 13

建立測試移植人類免疫系統的擬人化小鼠平台 利用病人周邊血移植入經CRISPR改造的NSG血友病小鼠 移植成功定義 hCD45+ cells > 15% T, B cells patient Inhibitor(+) 功能測試 IgG titer (與病人相當) Human PBMC Humanized mice are immunodeficient mice engrafted with human HSC, PBMC or tumor tissues Humanized Hemophilia NSG mice 功能測試 Presence of Treg細胞 (Antigen challenge) 14 14

Who we are and our achievement Prof. Shu-Wha Lin P.I. ---National Transgenic Mouse Model Core facility Ministry of Science and Technology, TAIWAN http://140.112.133.74/ The core facility has generated > 400 KO/KI mice; helped our user to win a technology transfer to Novo Nordisk for USD$13 millions

Hemophilia A-NSG mice deletions in exon 1 of the FVIII gene gender # of Mice Genotyping available results WT Indel Male 12 9 4 5 Female 8 Total 20 17 16

3rd generation Hemophilia A NSG mice Hemophilia A NSG mice were crossed with NSG mice to more than three generation to eliminate potential off-targets Genotyping 17

Liver FVIII mRNA level (quantitative RT-PCR)：> 50% decrease hemophilia A-NSG mice showed reduced mRNA levels and clotting activities Liver FVIII mRNA level (quantitative RT-PCR)：> 50% decrease FVIII activity (APTT): greatly reduced, comparable to that detected in the widely-used HemoA mice (B6;129S-F8tm1Kaz/J) (B6/129) 18

模擬血友病病人的NSG小鼠平台

Setup hu-NSG mouse model Engraftment of PBMC from healthy donor into NSG mice (1): Injection route hPBMC engraftment Weekly Flow cytometry for proportion of hCD45+ cell Successful engraftment rates intravenous injection (I.V.) 58% (14/24) intrafemoral (I.F.) 100% (n=8) intrasplenic injection (I.Sp.) 90~100% (n=10) Successful engraftment hCD45+/mCD45+ > 15% in mice  Humanized mice

Engraftment of PBMC from healthy donor into NSG mice (2): Time for reconstitution of human immune system I.V. (A) & I.F. (B) 4th~5th weeks I.Sp. (C) 2nd ~3rd weeks

Engraftment of PBMC from healthy donor into NSG mice (3): Proportion of human T and B cells in mouse PB B cells detected in mice by Intra-splenic & intra-femoral injection

Engraftment of PBMC from healthy donor into NSG mice (4): Detection of human total IgG

Study with patients’ samples

hu-NSG model: PBMC from Hemo A patients into NSG mice Intra-splenic injection Data provided by Dong-Yan Tsai, in Dr. 林國儀’s Lab #4 patient Density gradient separation Anti-CD19 beads plasmablasts Rest of the cells CD45+ Intra-femoral 2.1x106 Viable cells 25

B cells and plasma cells in Hu-PBL NSG mice Mouse #1

Hu-NSG hemophilia mice human T & B cells in the bone marrow and spleen Human B cell : hCD45+hCD19+ 27

Immunization of hu-PBL mice with antigen Intra-splenic injection Spleen B cells Human cytokine AAV: Blys, IL-4, GM-CSF Hemo A NSG Treg Immunization Treg B cells Treg

High titer Ag-specific antibody in Hu-PBL NSG mice hHA-NSG#1 總抗體效價 (anti-FVIII IgG) 中和性抗體效價 病例: 4號病人 (50歲, 未曾接受ITI) 抗體效價 123 BU (2016.06) hHA-NSG#2 總抗體效價 (anti-FVIII IgG) 中和性抗體效價 29

Re-engraftment (繼代移植) hPBMC engraftment hPBMC engraftment Inhibitor+ patient Primary recipient HemoA-NSG mice GVHD (weight loss >15%) 2nd recipient HemoA-NSG mice 3rd recipient HemoA-NSG mice 30

Stable engraftment of human Immune system into NSG mice hHA-NSG#5 病例: 4號病人 (50歲, 未曾接受ITI) 抗體效價 123 BU (2016.06) Primary recipient mice 每天輸注高劑量第八因子蛋白 繼代移植 2nd recipient mice 每天輸注高劑量第八因子蛋白 31 31

Conclusion Humanized NSG mice Platform has been established rate of successful engraftment ~100% (> 30 mice tested) route of transplantation is important Majority T cells with detectable B cells and Treg cells Antigen specific IgG (inhibitors) is detectable, i.e., functional immune response Will set up hu-NSG by repopulating mice with peripheral CD34+ cells 32

The End Acknowledgement 檢醫部/小兒部 (NTUH) SWL lab 楊永立醫師 (Dr. Y-L Yang) 顏靜慈博士 (Dr. CT Yen) 范夢倪 (M-N Fan) 徐玉真博士 (Dr. Y-C Hsu) 白振學博士 (Dr. J-H Pai) NARL 國家實驗動物中心 莊博士 (Dr. Chuang) 中榮 VG Hospital 王建得醫師 (Dr. J-T Wang) 雙和醫院 (SH Hospital) 陳淑惠醫師 (Dr. S-H Chen) 中研院 陶秘華研究員(Dr. M-H Tao) 林國儀研究員(Dr. K-I Lin) 檢醫部/小兒部 (NTUH) 楊永立醫師 (Dr. Y-L Yang) 動物中心 (LAC-NTUCM) 陳惠文主任 (Dr. S Chen) 游益興助理研究員(Dr. I-S Yu) 張家宜獸醫師 (Dr. C-Y Chang) 醫技系 (CLSMB, NTUCM) 莊雅惠老師 (Dr. Y-H Chuang) 胡忠怡老師 (Dr. CY Hu) 內科 (NTUH) 周聖傑醫師 (Dr. S-J Chou) 基蛋所 (NTUCM) 陳佑宗老師 (Dr. T-Z Chen) The End

Challenges limiting the utility of HM model Cancer Res. 2016 Nov 1;76(21):6153-6158.