Brief Report. The Science of Humanized Mouse Model Development for Testing Cancer Therapies. White Paper

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1 Brief Report The Science of Humanized Mouse Model Development for Testing Cancer Therapies White Paper Date: September 29, 2014

2 NOTE: This document is intended for educational purposes only. Victoria A. Love, Ph.D. Owner T:(760) F:(760) W: 2

3 Table of Contents Introduction*...*4! Overview*of*Mouse*Cancer*Models*...*5! Application*of*Human*Immune*System*Mouse*Oncology*Models*...*11! Summary*and*Conclusions*...*13! References*...*14! * * * 3

4 Introduction Evaluating models of human disease, particularly in mice, provides insight into fundamental biological processes. Experiments with murine models also increase understanding of the cellular and molecular pathways that regulate tumorigenesis and other pathological processes aiding discovery of molecularly targeted cancer drugs and agents for other therapeutic indications. Additionally, rodent models facilitate characterization of key events governing hematopoietic and immune system development and function 1,2. While conventional animal studies provide indispensable tools for research, limitations arise because most preclinical models lack predictive value; that is, data from rodent experiments often fail to reflect clinical response rates 3. Furthermore, conventional mouse models do not provide suitable methods for studying human-specific infectious diseases and therapies that modulate human immune system cells. In contrast, humanized mouse platforms may circumvent some challenges associated with conventional animal models of disease and have potential for translating preclinical discoveries into clinical applications 4,5. In addition to their use for evaluating drug efficacy and mechanism of action (MOA), recent guidance from the Food and Drug Administration (FDA) states that humanized mice in conjunction with in vitro and in silico analysis of protein therapeutics may be adapted for immunogenicity testing 6. Generating these specialized models requires the use of highly immunodeficient mouse strains engrafted with human hematopoietic cells or tissues. Multiple humanized immune system mouse platforms exist; and while certain populations of the engrafted human immune cells retain their normal functionality, critical components of the human immune system may fail. For example, transferred T cells readily expand in specific immunocompromised mouse strains but the same hosts may not support functional B cell responses or engraftment of dendritic and myeloid cells 7,8. Still, hundreds of reports exist that describe humanized mouse model experiments with human immunodeficiency virus (HIV) and other infectious viruses 9. Several reviews have also outlined humanized mouse models of liver disease, graft versus host disease (GVHD), and other diseases 10,11. Devising strategies for studying human immune responses against cancer in humanized mice lags behind progress with humanized mouse virology models; but with the expanding availability of novel tools and protocols, the potential for producing more robust preclinical 4

5 oncology studies increases. This review summarizes information about conventional approaches for testing drug responses in human tumors and emphasizes methods for studying tumor rejection in emerging humanized mouse model platforms. Overview of Mouse Cancer Models * Preclinical mouse models for testing small molecules, biologic therapies, or rational combination treatment modalities support oncology drug development. The models continue increasing in complexity, yet despite refinement they remain imperfect representations of human genetic diseases. In general, preclinical oncology models do not capture the vast heterogeneity, range of molecular subtypes, or the tumor microenvironment associated with human cancers 12,13. Another limitation is that tumor studies frequently rely on immunodeficient mice. Because host immune responses to cancerous cells likely slow tumor growth kinetics, conventional tumor xenografts with immunocompromised mice may provide misleading functional and efficacy data 14. Examples of platforms enabling in vivo efficacy testing of cancer agents include the following models: ectopic or orthotropic xenografts with tumor cell lines, genetically engineered mouse (GEM) models, primary tumor xenografts and humanized immune system models (with human cell lines or primary human tumors). Table 1 compares and contrasts some of the major advantages and disadvantages associated with commonly used models to study human cancers 15. Table 1: Mouse Cancer Models for Studying Human Cancer Therapies Cancer Model Major Advantages Major Disadvantages Xenograft of human tumors cell lines (nude or SCID mouse strains) Genetically engineered mice (GEM) (Balb/c, C57BL/6, other backgrounds) Xenografts using immunodeficient mice with reconstituted human immune system (various mouse strains) Provides a rapid and relatively cost effective method for testing anti-cancer agents Used to study defined mutations identified in human tumors Tumors develop in the presence of a fully functioning immune system May predict drug response in human tumors Mimics tumor microenvironment May predict drug response in human tumors Enables testing of immune modulatory drugs Immunocompromised mice provide no information about human anti-tumor immune responses Limited in predicting clinical response Costly in time and resources Targets a limited number of cancer associated genes Not useful for evaluating drugs that only target human antigens Costly in time and resources May not support engraftment of fully reconstituted human immune system Implanted tumors may not grow due to robust anti-tumor rejection 5

6 Despite some of the inherent drawbacks associated with mouse models, their strategic use accelerates the drug discovery process. The strength of these models is that they may provide valuable information about the following: pharmacodynamics/pharmacokinetic relationships, tumorigenesis, anti-tumor immune responses, potential of new drug targets, treatment regimens, tumor metastasis, drug resistance mechanisms, identification of predictive versus prognostic biomarkers, and determination of safety margins and toxicity 16,17. When devising in vivo experiments, it is imperative to understand that only well-designed clinical trials provide accurate and reliable efficacy and safety data; nevertheless, there is a crucial need for improved translational models. In supporting research and development, important considerations should include selecting models that test hypothesis-driven questions and that minimize clinical attrition rates for investigational oncology agents. Several reviews describe the history of mouse cancer model development, detailed information on the application of xenografts and GEM models, and attrition rates in oncology research 18,19. Table 2 and Table 3 provide general information about immunocompromised mouse strains available for oncology studies and other areas of research 20,21. For full details regarding the precise genetic mutations and phenotypes described for immunodeficient and humanized mice, visit The Jackson Laboratory (JAX), Charles River Laboratories (CRL), and Taconic (T) websites 22,23,24. 6

7 Table 2: Nude, NOD and SCID Mouse Platforms Adapted for Humanized Mouse Models in Oncology Research Nomenclature Common Name/ (Major Suppliers) Phenotype Strengths Limitations Foxn1 nu Nude Mice (1966) 25 (CRL, JAX, T) Lacks T lymphocytes, imparied NK cell activity; may develop B cell responses Enables xenotransplantation of human and mouse tumor lines and some primary tumors; hairless phenotype simplifies tumor measurements Intact innate immune system; does not readily support engraftment of primary cells; not suitable for testing immune modulatory drugs CB17/lcrPrkdc scid/scid SCID Mice (1983) 26 (CRL, JAX, T) Abnormal B and T lymphocyte activity; normal lymphocytes may develop over time due to leaky Scid mutation Enables xenotransplantation of human and mouse tumor lines; used extensively for cancer xenografts Intact innate immune system and high NK activity; does not readily support engraftment of primary cells; limited use for testing immune modulatory drugs CB17.B6-Prkdc scid Lyst bg SCID-beige (1993) 27 (CRL, T) Abnormal B and T lymphocyte activity; imparied NK cell function Impaired NK cell activity enables human leukocytes engraftment; supports xenografts in hosts with partially reconstituted human immune system; supports testing of immune modulatory drugs; mice do not develop GVHD Relatively low levels of engrafted human leukocytes NOD.CB17-Prkdc scid NOD/SCID Mice (1995) 28 (CRL, JAX, T) Defective adapative and innate immune cell function; has some leakiness due to SCID mutation Enables engraftment of hematopoietic cancer cell lines; supports xenotransplantation of some human tumors Mice develop thymic lymphomas; use limited to short-term studies NODShi.Cg-Prkdc scid Il2rgtm1 NOG Mice NOD/SCID-IL2Rγ null (2002) 29,30 (T) Severely immunodeficient strain; lacks functional adaptive and immate immune responses; reduced complement activity; phenotype nearly identical to NSG and NRG mice Enables xenotransplantation of human and mouse primary tumors; supports engraftment of human PBMCs and CD34 + -HSCs; tolerates radiation doses 250 cgy; strain does not develop thymic lymphomas and thus support long-term studies Tolerates radiation doses 250 cgy, may not be used in applications that requires high doses of radiation (i.e. some cancer radiation protocols); engraftment of PBMCs induces xenogeneic GVHD that may confound tumor studies NOD.Cg Prkdc scid Il2rg tm1wjl /SzJ NSG Mice NOD/SCID-IL2Rγ null (2005) 31 (JAX) Phenotype nearly identical to NOG and NRG mice Enables xenotransplantation of human and mouse primary tumors; supports engraftment of human PBMCs and CD34 + -HSCs; strain does not develop thymic lymhomas and thus support long-term studies Tolerates radiation doses 400 cgy, may not be used in applications that requires high doses of radiation (i.e. some cancer radiation protocols); engraftment of PBMCs induces xenogeneic GVHD that may confound tumor studies NOD.Cg- Rag1tm1Mom Il2rg tm1wjl /SzJ NRG Mice NOD/Rag -/- IL2Rγ null (2008) 32 (JAX) Phenotype nearly identical to NOG and NSG mice Enables xenotransplantation of human and mouse primary tumors; supports engraftment of human PBMCs and CD34 + -HSCs; compared to NSG, NRG tolerates higher radiation doses ( 650 cgy); strain does not develop thymic lymphomas and thus support long-term studies Engraftment of PBMCs induces xenogeneic GVHD that may confound tumor studies Abbreviations: CRL: Charles River Laboratories; cgy: centigray; foxn1: forkhead box n1; GVHD graft-versus-host-disease; HLA: histocompatibility leukocyte antigen; IL-2R: interleukin-2 receptor; JAX: The Jackson Laboratory; NOD: nonobese diabetic; Prkdc: DNA-dependent protein kinase catalytic subunit; SCID: severe combined immunodeficiency; T: Taconic 7

8 Table 3: BALB/c-Rag 2null Mouse Strains Adapted for Humanized Mouse Models Platforms for Oncology Research Nomenclature Common Name/ (Major Suppliers) Phenotype Strengths Limitations Rag2 null BALB/c-Rag2 null Mice (1992) 33 (T) Complete absence of mature B and T lymphocytes; functional innate immune responses May support engraftment of human and mouse tumor lines; does not have a leaky SCID mutation Intact innate immune system; not suitable for primary cell implantation Rag2 null IL2Rγ null BRG Mice (CIEA BRG) BALB/c-Rag2 null IL2Rg null (2004) 34,35 (T) Severely immunodeficient strain; lacks functional adaptive and immate immune responses; reduced complement activity; phenotype similar to NSG and NOG mice Enables xenotransplantation of human and mouse primary tumors; supports engraftment of human PBMCs and CD HSCs; compared to NSG, NRG tolerates higher radiation doses ( 650 cgy); strain does not develop thymic lymphomas and thus support long-term studies; slower GVHD onset compared to NSG/NOG strains Engraftment of PBMCs induces xenogeneic GVHD that may confound tumor studies Abbreviations: CIEA: Central Institute for Experimental Animals; cgy centigray: GVHD graft-versus-host-disease; HLA: histocompatibility leukocyte antigen; IL-2R: interleukin-2 receptor; NOD: nonobese diabetic; Prkdc: DNA-dependent protein kinase catalytic subunit; SCID: severe combined immunodeficiency; T: Taconic 8

9 Nude mice and other mouse strains used for evaluating drug responses in established tumor cell lines provide a rapid and cost effective means for screening investigational new cancer drugs and therapeutic regimens. However, these models do not sufficiently support engraftment of primary tumors; nor do they accurately reflect the complexity of human cancers. Compared to xenograft models with established cell lines, patient-derived xenografts (PDX) models may retain and more accurately reflect the molecular diversity, heterogeneity, and histology of tumors seen in patients with cancer 36. Severe combined immunodeficiency (SCID) mice and other strains such as nude mice and SCID-beige mice may be used for xenotransplantation of certain patient-derived tumors; but, these conventional mouse strains do not support the high levels of human hematopoietic cell engraftment required for reconstituting a functional human immune system presumably due to the presence of endogenous innate immune cells. Therefore, conventional tumor xenograft experiments are not suitable for testing a compound with an immune-mediated MOA that induces rejection of human tumors, or for evaluating anti-cancer therapies in the context of the tumor microenvironment. Recently, new strategies were developed that minimize rejection and increase survival of human immune cells transferred into immunocompromised mice 37. Experiments with humanized immune system mice may provide more predictive translational oncology models. Over the past decade, genetic modifications to conventional immunocompromised mice produced associated congenic strains with immune system impairments greater than those observed in SCID or nude mice (Table 2 and Table 3). For example, NSG and NOG mice are two NOD/SCID strains that lack signaling via the common interleukin-2 receptor gamma chain (IL-2Rγ). Inactivation of IL-2Rγ inhibits specific cytokine networks in certain immune system cells. An additional NOD strain, the NRG mouse, also lacks IL-2Rγ expression and has a phenotype similar to NSG and NOG mice; however, NRG mice lack functional immune responses due to deletion of recombination activation gene 1 (Rag1) gene and do not carry the SCID mutation. Deletions or truncations of IL-2Rγ bred onto recombination activating gene 2 (Rag2) deficient Balb/c background also exist collectively: the NSG, NOG, NRG and BRG mouse strains represent the most severely immunodeficient mice available 38. Multiple cytokine pathways that regulate leukocyte activity signal through the IL-2Rγ chain (Figure 1). Deleting the IL-2Rγ gene in NSG and NRG mice completely eliminates protein expression, and a mutation in NOG and BRG mice deactivates the intracytoplasmic domain of 9

10 IL2-Rγ. Both mutations cause profoundly impaired endogenous innate and adaptive immune responses in the genetically modified mice 39. The IL-2 Receptor Figure 1. This figure depicts the three transmembrane subunits that comprise the IL-2R complex. The common gamma-chain (γ c ) is shown in green 40. The common-gamma chain is a component of six different interleukin receptor complexes. Disrupted signal transduction through IL-2Rγ negatively affects the IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 pathways and results in a lack of mature T, B and functional NK cells. Additionally, IL-2Rγ deficient mice have defective dendritic cells (DCs) and macrophages (Mϕ). Because the mice lack functional immune responses, transferring human-derived tissues, hematopoetic stem cells (HSCs) or peripheral blood mononuclear cells (PBMCs) into IL-2Rγ null mice results in significantly improved human tissue or cell engraftment compared with conventional SCID or nude mouse models 41,42. Table 4 compares and contrasts properties associated with cytokine receptors that share the γ C Subunit 43. Table 4: Cytokine Receptors Containing the γ C Subunit Receptor Subunits: Receptor Expression Profile: Cytokine Produced By: IL-2Rα, IL-2Rβ; γ C T cells, B cells and NK cell T cells and DCs IL-4R, γ C T cells, B cells, NK cells mast cells and Basophils T cells, NKT cells, eosinophils and mast cells IL-7Ra; γ C Stromal cells, epithelial cells, and fibroblasts T cells, pre-b cells and DCs IL-9R; γ C T cells, mast cells, epithelial cells, and eosionophils T cells IL-15Rα, IL-2Rβ, γ C T cells and NK cells Monocytes, DCs and epithelial cells IL-21R; γ C T cells, B cells, NK cells and DCs CD4 T cells and NKT cells Presently, several congenic strains of NSG, NOG, NRG and BRG mice exist. Introducing various genetic modifications in strains with the IL-2Rγ null genotype further hindered activity in mouse leukocytes; and thus enhanced human leukocyte function in mice with a reconstituted human immune system. 10

11 Use of transgenes or targeted gene deletions produced the following modifications in IL-2Rγ null mice: insertions of human genes expressing specific cytokines hematopoietic growth factors, or human leukocyte antigen (HLA); and deletions of genes required for expression of major histocompatibility complex class I (MHC I) or associated beta-2 microglobulin (B2M) protein. Multiple studies and reviews reported findings about these novel strains and their applications in detail 44,45. Application of Human Immune System Mouse Oncology Models Several reports describe oncology experiments using immunocompromised mice engrafted with human-derived cells or tissues. Table 4 provides a representative list of humanized immune system model studies. Despite the limitations of these models, they may provide suitable methods for testing oncology drugs in the context of an intact tumor microenvironment or for evaluating the anti-tumor activity of immune modulatory drugs. Baia et al. presented one potentially powerful method for developing a humanized immune system mouse platform (Table 5). The investigators transferred HLA-A2 + -CD34 + fetal liver HSCs into 4-week old female NOG mice. Peripheral blood analysis indicated that by week 12 post HSC transfer, up to 60% of leukocytes present in the mice expressed human CD45 + a cell surface leukocyte marker. Subsequently, the investigators generated individual cohorts of humanized mice implanted with HLA-matched primary human tumors. The xenotransplanted cohorts consisted of the following types: non-small cell lung carcinoma, melanoma, colorectal cancer, and other solid tumors 46. The model has potential for testing agents that may enhance immune cell function and augment tumor rejection. Because sufficient immune system reconstitution is achieved using this particular model, ex vivo analysis to evaluate PK/PD relationships and biomarker analysis associated with immune system modulating agents may be possible. One draw back of the model is that the human T cells that develop in the mouse thymus are MHC-restricted rather than HLA-restricted; therefore, the reconstituted human immune system does not closely model conditions under which human T cells normally develop. There are recent reports that describe methods for generating HLA-restricted T cells in humanized immune system mouse to study infectious diseases, vaccines, and potentially cancer

12 Table 5: Emerging Oncology Mouse Models for Evaluating Human Immune Responses Human Immune System Model Example Major Observations Human PBL (or PBMC)-engrafted SCID Human PBMC-engrafted SCIDbeige 1. Injection of SCID xenografts with human PBL. Subsequently, mice were administered a bispecific monoclonal or control antibody and survival of the mice was tracked over time 48,49 2. Co-implantation of human prostate cancer cell line and freshly isolated PBMC into SCIDbeige mice. Mice were subsequently administered T cell modulating monoclonal antibody once established ectopic tumors were measurable in the mice 50,51 Limited engraftment of human leukocytes; polyclonal T cell responses to implanted tumors rather than tumor antigen-specific rejection; xenogeneic GVHD does not normally develop; enables testing of leukocyte-specific immune modulators in a tumor model; easy to establish Human-PBMC engrafted NSG/NOG Intravenous injection of PBMCs into NOG or NSG mice 52,53,54 Mice develop severe GVHD that may confound tumor studies; use of NOG or NSG mice expressing the beta-2-microglobulin transgene may delay GVHD onset Patient-derived xenograft (PDX) and in vivo expansion of tumorassociated leukocytes Patient derived xenograft (PDX) combined with CD34 + -engrafted NOD/SCID and NSG/NOG mice with solid tumors Patient derived xenograft (PDX) NOD/SCID and NSG/NOG mice with hematological tumors Bone marrow, liver, thymus mice (BLT) Mice implanted with tumor cell line Partial reconstitution of human immune system in NSG mice after implantation of patient-derived lung or ovarian tumors 55,56 1. Multiple different PDX models generated in CD34 + -engrafted NOG mice Non-small cell lung cancer PDX model established in CD34 + -engrafted NSG mice Two different non-small cell lung PDX experiments and a gastric cancer PDX experiment conducted in CD34 + -engrafted NOD/SCID mice Acute Myeloid Leukaemia (AML) engraft model using human cytokine/growth factor transgenic NSG mice engrafted with CD34 + cells expressing AML inducing transgene NSG mice injective with AML cells were used to establish a Paediatric AML PDX model. Mice were subsequently administered IL-27 which reduced disease burden in the model 61. NSG mice used to establish a BLT platform that supported development of functional melanoma antigen specific, HLA-A2 restricted, cytotoxic T lymphocytes 62,63,64. No drug efficacy data were reported for these studies; complex models to establish; limited sample availability which may not be suitable for extensive pre-clinical testing In general, these models are technically complex and expensive; lymphocytes are MHC-restricted unless NSG/NOG mice expressing the HLA-2A transgene are used; mice do not develop GVHD; human tumor microenvironment is partially maintained; allows for testing of drugs that modulate human immune responses; using congenic NSG/NOG expressing transgenes encoding human cytokines and growth factors enables human adaptive and innate immune system reconstitution PDX models established with CD34 + HSC support haematological tumour growth in the presence of an intact human immune system; Large-scale studies may be limited due to availability of human specimens Complex model to establish; requires use of human fetal tissues; melanoma antigen T cell receptor transgene used to produce antigen-specific CTL; this particular model makes use of a human cell line; T cells in the model are also highly manipulated; model may be useful for large-scale studies Abbreviations: CD34: cluster of differentiation 34; GVHD: graft-versus-host-disease; HLA-A2: human leukocyte antigen-a2; IL27: sinterleukin 27; PBL: peripheral blood lymphocytes; PBMC: peripheral blood mononuclear cell 12

13 Summary and Conclusions While comparing and contrasting the numerous existing and emerging humanized mouse oncology models, it is apparent that the strengths and limitations of the models differ widely according to the biology of the mouse strain used to conduct the experiments. Some humanized models may provide a more physiologically relevant method for testing cancer therapeutics compared to others. For example, while transferring human PBMCs into human tumor-bearing SCID-beige mice may provide a relatively straightforward method for evaluating immune modulatory agents, this model lacks the immunological complexity of BLT PDX platform (Tables 5). The latter model potentially allows testing of tumor-antigen specific, HLA-restricted responses to primary tumors; however, establishing and validating the BLT PDX model likely presents not-so-trivial technical challenges. While devising animal studies, experiments must address a particular mechanistic question. Ideally, the simplest model that answers the question should be used. Selecting the appropriate humanized models to support hypothesis-driven experiments is imperative for timely production of robust data while minimizing animal use and other resources. Humanized mouse models for translational research have applications for a wide range of therapeutic areas including, autoimmunity, infectious disease, cancer immunology and other areas. Research involving these models will continue gaining momentum as protocols for implementing their use become more defined. The technology continues to transition from its developmental phase towards a phase that might allow more direct applications. Advances in model development may expand scientific understanding and contribute to developing new treatment options for patients living with debilitating medical conditions. 13

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