ALZET Research Application Models

The and severe combined (SCID) mouse have ALZET Pump Highlights traditionally been used as recipients for human cells or tissues because they lack host immunity and easily accept heterologous cells. The introduction of • Small size for implantation • 9 pump models for mice the non-obese diabetic (NOD)/SCID mouse led to the development of highly • Continuous and controlled immunodeficient strains, able to engraft human cells and tissues more delivery of agents efficiently, which are more appropriate for generating humanized mouse • Minimize side effects and models. experimental variables • Convenient and cost- effective dosing method The humanized mouse – a mouse carrying functional human genes, cells, • Reduced animal handling tissues, and/or organs – is now a powerful research tool for the in vivo study of and stress • Delivery rates ranging from human biology and disease. Humanized mouse models enable a better 0.11 µl/hr to 8 µl/hr understanding of disease pathways and ultimately improve the translational • Delivery durations ranging value of preclinical studies. Various humanized mouse models have been from 1 day to 6 weeks developed for the study of infectious diseases, autoimmunity, transplantation, vaccine development, immunotherapy, regenerative medicine, cell development, and more. Immunodeficient Strains*

ALZET® Osmotic Pumps are used extensively with immunodeficient mice, and Nude Mouse hundreds of publications attest to their research value in these species. These SCID Mouse implantable infusion pumps offer a convenient alternative to repetitive NOD/SCID Mouse injections for continuous dosing of unrestrained lab animals. Their automatic NSG Mouse operation, small size and simple design make them suitable for chronic dosing NOG Mouse studies in humanized mouse models. No researcher intervention is required during infusion, and animal handling is kept to a minimum to reduce the risk of NRG Mouse and stress. Read on for research summaries describing the use of *See page 4 for complete ALZET pumps in humanized mouse models. Contact us for additional descriptions information at 800.692.2990 or [email protected].

DURECT CORPORATION | 10260 BUBB ROAD | CUPERTINO CA, 95014-4166 T: 408.367.4036 F: 408.865.1406 TOLL FREE 800.692.2990 WWW.ALZET.COM | [email protected] Use of ALZET Pumps in Humanized Mouse Models

Suzuki et al. Generation of engraftable hematopoietic stem cells from induced pluripotent stem cells by way of teratoma formation. Molecular Therapy 2013;21(7):1424–1431

Purpose (1) Develop an in vivo differentiation system for the generation of engraftable hematopoietic stem cells (HSCs) from induced pluripotent stem cells (iPSCs) to facilitate hematopoiesis. (2) Investigate teratomas as a suitable microenvironment for the generation of engraftable HSCs with (BM) niche migration potential.

Mouse Model Transplantation of BM cells (hiPSC-derived HSCs) of teratoma-bearing mice into irradiated NOD/SCID or NOD/SCID/ JAK3null recipient mice.

Role of ALZET Continuous, SC administration of the hematopoietic , stem cell factor (SCF) and thrombopoietin (TPO), over 2 weeks in teratoma-bearing mice to stimulate differentiation of iPSCs into HSCs.

Key Findings • HSCs were successfully induced by injection of iPSCs into immunodeficient mice and reproducing the HSC niche by way of teratoma formation. Co-administration of OP9 stromal cells along with hematopoietic cytokines dramatically enhanced the induction efficiency. • HSCs derived from iPSCs showed self-renewal and multi-lineage differentiation capabilities. • iPSC-derived HSCs were shown to migrate from teratomas into the BM niche following IV injection into irradiated recipients, where they are capable of long-term reconstitution of the hematolymphopoietic system. • Established an in vivo differentiation system to generate fully functional, xenotransplantable HSCs from iPSCs through teratoma formation.

Gang et al. Small-molecule inhibition of CBP/catenin interactions eliminates drug-resistant clones in acute lymphoblastic . Oncogene 2013:1-10

Purpose Evaluate the efficacy of ICG-001, a novel small-molecule inhibitor of Wnt/beta catenin signaling, with or without traditional chemotherapy with nilotinib (NTB) or VDL (vincristine, dexamethasone and L-asparaginase).

Mouse Model Xenograft model of primary acute lymphoblastic leukemia (ALL) in NOD/SCID gamma (NSG) mice.

Role of ALZET Model 1004 used for subcutaneous administration of ICG-001 for 28 days to ensure stable plasma dosing levels.

Key Findings • ICG-001 treatment in combination with chemotherapy significantly prolonged survival of mice engrafted with primary ALL: a mean survival time (MST) of 100 days was seen in mice treated with ICG-001 plus chemotherapy, compared to 85 days MST for chemotherapy only-treated mice, 37.5 days MST for ICG-001 only treated mice, and 34 days MST for saline control-treated mice. • Selective inhibition of CBP/catenin signaling with ICG-001 prevents self-renewal of drug-resistant leukemia- initiating cells by forced differentiation, thereby sensitizing them to chemotherapy. • This approach represents a promising therapeutic strategy for specifically eradicating drug-resistant leukemia- initiating cells.

Klug et al. Low-dose irradiation programs differentiation to an iNOS+/M1 phenotype that orchestrates effective immunotherapy. Cancer Cell 2013;24:589–602.

Purpose Evaluate the effect of low-dose irradiation (LDI) as adjuvant for improving the efficacy of adoptive T cell therapy or tumor vaccination on various xenograft tumor models.

Mouse Model Human melanoma (MeWo) xenograft model in NOD/SCID gamma (NSG) mice and NOD/SCID mice for ; RIP1-Tag5 (RT5) pancreatic tumor xenograft model in NOD/SCID mice.

Role of ALZET Model 2002 used for subcutaneous administration of the selective iNOS inhibitor 1400W over 2 weeks to evaluate the effect of iNOS inhibition on vascular normalization, activation, and T cell recruitment into RT5 tumors.

Key Findings • iNOS inhibition by 1400W treatment eliminated NO secretion in irradiated and CD8TC-treated tumors and completely inhibited vascular cell adhesion protein-1 expression. • LDI programs the differentiation of iNOS+ M1 which induce CTL recruitment and killing within solid tumors by inducing endothelial activation and TH1 chemokine expression, as well as by suppressing the production of angiogenic, immunosuppressive, and tumor growth factors. • iNOS+ macrophages are required to mediate effector T cell recruitment into tumors for successful tumor immune rejection through an NO-dependent mechanism. • Adoptive transfer of iNOS-expressing macrophages is a promising strategy to improve the efficacy of cancer immunotherapy.

DURECT CORPORATION | 10260 BUBB ROAD | CUPERTINO CA, 95014-4166 T: 408.367.4036 F: 408.865.1406 TOLL FREE 800.692.2990 WWW.ALZET.COM | [email protected] Use of ALZET Pumps in Humanized Mouse Models

Krause et al. Differential regulation of myeloid by the bone marrow microenvironment. Nature Med 2013;19:1513–1517

Purpose Evaluate the effect of human parathyroid hormone (PTH) for modulation of the bone marrow microenvironment to eliminate persistent and drug resistant leukemia stem cells (LSC) in chronic myelogenous leukemia (CML) and (AML).

Mouse Model Xenograft model of chronic myelogenous leukemia in NOD/SCID IL-2 receptor γ deficient (NSG) mice.

Role of ALZET 14-day ALZET pumps used for SC administration of human PTH (1–34) for 18 weeks (PTH treatment started 4 weeks prior to and continued for 14 weeks thereafter, with pump replacement every two weeks).

Key Findings • PTH treatment caused a 15-fold decrease in LSCs in wild type mice with CML-like myeloproliferative neoplasia and significantly reduced engraftment of primary human CML cells in NSG mice. • The beneficial effect of PTH in CML is mediated via suppressive effects of TGF-β. • Study suggests that targeting the hematopoietic microenvironment through PTH may render it hostile for CML cells, indicating that alteration of the tumor microenvironment is a potentially complementary approach to tumor cell-specific therapy.

Chandramohan et al. Recombinant anti-podoplanin (NZ-1) immunotoxin for the treatment of malignant brain tumors. Int J Cancer. 2013;132(10)

Purpose Evaluate the intracranial efficacy of an anti-podoplanin immunotoxin (NZ-1-(scdsFv)-PE38KDEL) in the meduloblastoma tumor model.

Mouse Model Intracranial meduloblastoma xenograft model in NOD/SCID gamma (NSG) mice.

Role of ALZET Model 1003D used for administration (3 days) of an anti-podoplanin immunotoxin directly into the brain tumor site to bypass the blood-brain barrier.

Key Findings • The podoplanin tumor antigen is overexpressed in several glioblastoma and medulloblastoma xenograft cells. • Recombinant immunotoxin was highly efficacious against CNS tumors expressing podoplanin, increasing survival by 41% in tumor bearing mice compared to controls. • Podoplanin is a suitable candidate for targeted therapy of malignant brain tumors.

Examples of Humanized Mouse Models Available

BLT humanized (hBLT) mouse PBMC humanized (hPBMC) mouse

• Modification of the SCID-Hu model • NSG mice engrafted with human peripheral blood • NOD/SCID mice engrafted with human and mononuclear cells (hPBMC) tissues, along with autologous CD34+ HSCs • Perfect for short-term experiments where T cell engraftment • Robust human hemato-lymphoid system observed is of primary concern between 12-16 weeks post engraftment • Enable short-term studies requiring human T cells without the • Most functional of any current humanized 12 week T cell maturation waiting period mouse model • Average lifespan of ~8.5 months

CD34+ humanized (hCD34+) mouse GTL, or NSG-BLT, mouse

• Robust multi-lineage engraftment of human immune cells • Modification of the BLT mouse: engraftment on the NSG within the NSG background, including very good T cell instead of the NOD/SCID background maturation and function • Allows longer term experiments compared with BLT mice • hCD34+ mice have longer life spans than BLT mice, since NSG mice do not develop thymic as in the enabling execution of longer duration studies NOD/SCID

DURECT CORPORATION | 10260 BUBB ROAD | CUPERTINO CA, 95014-4166 T: 408.367.4036 F: 408.865.1406 TOLL FREE 800.692.2990 WWW.ALZET.COM | [email protected] Immunodeficient mouse strains used to generate humanized mouse models

Nude Mice SCID Mice

• Spontaneous (Foxn1) results in lack of body hair • Recessive mutation results in deficient activity of an and deteriorated or absent thymus enzyme involved in DNA repair, causing impaired cellular • Incapable of cell-mediated immunity due to lack of T cells; and humoral immune response partially defective development • Intact innate immunity; NK cell activity limits engraftment • Intact innate immunity • Some functional T and B cells develop with age (referred • Recipient of allogenic and xenogenic grafts as “leakiness”) • Model organisms for immune system, cell transplantation, infectious disease, and vaccine research • Humanized models: SCID mice engrafted with human fetal tissues (SCID-Hu), or peripheral blood mononuclear cells (Hu-PBL-SCID) NOD SCID (NOD.CB17-Prkdcscid/J) NSG mouse (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ)

• SCID mutation transferred to NOD background • a.k.a NOD/SCID gamma, or NOD/SCID IL-2 receptor • Lacks functional T and B cells gamma chain knockout • Reduced NK cell activity, impaired complement pathway, • NOD/SCID mice bearing a targeted mutation in the IL-2 and reduced “leakiness” of residual T and B cells than SCID receptor-γ chain resulting in deficient signaling of multiple strain cytokines (IL2, IL4, IL7, IL9, IL15 and IL21) • Accepts allogeneic and xenogeneic grafts very efficiently; • Severe defects in innate and adaptive immunity: lacks T used for transplantation of normal and malignant human and B cells, natural killer (NK) cells, and complement cells and tissues, including isolates from hematopoietic activity; reduced macrophage and function • No T and B cell “leakiness” associated with aging • Develops thymic lymphomas by 8-9 months; best used in • Resistant to , allowing for long-term experiments short term experiments • Improved engraftment of primary human cells, including • Adoptive transfer recipient for study of autoimmune type 1 PBMCs and HSCs diabetes • Enhanced model for human hematopoiesis, cancer (leukemia, multiple myeloma), visceral diseases, autoimmune , infectious disease (AIDS), regenerative medicine, and hematology NRG mouse (NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ) NOG mouse (NOD/Shi-scid/IL-2Rγnull)

• Also called B6 Rag1; B6 inbred background simplifies • Combination of the NOD/SCID and the IL-2 receptor-γ creation of compound immunodeficient mutants chain knockout (IL2rγKO) mouse • Similar to the NSG strain, but the SCID mutation is • Similar to the NSG mouse (The Jackson Laboratory), but substituted for the Rag1 knockout mutation, which developed by the Central Institute for Experimental provides greater tolerance for irradiation and Animals (Japan) chemotherapy • Triggering through the IL-2 γ chain receptor is disabled due • Support higher levels of human stem cell to a truncated intracytoplasmic tail (receptor completely engraftment following irradiation-conditioning knocked down in NSG mice) • Model for human lymphohematopoietic cell engraftment • Vulnerable to developing lymphomas after irradiation, but studies that require a radiation-resistant host yield similar engraftment results (compared to NSG) even when not irradiated

Resources: • Ito et al. Cellular & Molecular Immunology 2012;9:208-214 • Shultz et al. Nature Reviews 2007;7:118-130 • www.jaxmice.jax.org • www.humouse.org

DURECT CORPORATION | 10260 BUBB ROAD | CUPERTINO CA, 95014-4166 T: 408.367.4036 F: 408.865.1406 TOLL FREE 800.692.2990 WWW.ALZET.COM | [email protected]