Mouse Model For Preclinical Testing Research Articles

EPEN-27. GAINING A CLUE FROM 1Q: A HIGH-RISK CHROMOSOMAL GAIN IN PEDIATRIC POSTERIOR FOSSA EPENDYMOMA

Abstract BACKGROUND Ependymomas are neuroepithelial tumours that can be classified into distinct molecular subgroups, each exhibiting a unique clinical outcome. Therapeutic efforts against the most aggressive and abundant subgroup of pediatric ependymoma (posterior fossa group A, PFA) have traditionally been hampered by a lack of known recurrent driver mutations. However, approximately 25% of PFA tumours exhibit gains of the chromosome arm 1q which is associated with an extremely poor prognosis despite aggressive treatment. By interrogating the multi-omic landscape of PFAs harbouring this copy number variation, we aimed to uncover genetic dependencies from which actionable therapeutics could be derived. METHODS Transcriptomic (bulk/single-cell RNA sequencing), proteomic (LC-MS/MS) and phosphoproteomic analyses were conducted on patient tissues and primary patient-derived cell lines to gain insight into the pathways that drive 1q gain pathogenesis. Chromatin immunoprecipitation sequencing of H3K27me3 and H3K27ac marked histones was carried out to profile the epigenetic landscape of these tumours, and whole genome CRISPR knockout screens were performed to reveal the contribution of essential genes unique to tumours harboring 1q gains. RESULTS Through the comparison of 1q gain and balanced PFA samples, our multi-omic analyses identified the up-regulation and essentiality of known glioma oncogenic drivers previously uncharacterized in PFA. The investigation of essential candidate genes showed a convergence on pathways related to ciliogenesis, with genetic vulnerabilities informing in vitro drug screening. Candidate genes were further validated by in vivo knockdown using established xenograft mouse models for pre-clinical testing. CONCLUSIONS This combined approach has allowed for the derivation of a functional cancer signature underlying 1q gains in PFA and presents novel therapeutic targets for this high-risk subgroup. PFA ependymoma is a deadly malignancy whose complex biology has so far eluded therapy, and new insights from this project will help to identify the first effective therapies for this vulnerable patient population.

Eμ-TCL1 adoptive transfer mouse model of chronic lymphocytic leukemia.

Despite being the most common adult leukemia in the western world, Chronic Lymphocytic Leukemia (CLL) remains a life-threatening and incurable disease. Efforts to develop new treatments are highly dependent on the availability of appropriate mouse models for pre-clinical testing. The Eμ-TCL1 mouse model is the most established pre-clinical approach to study CLL pathobiology and response to treatment, backed by numerous studies highlighting its resemblance to the most aggressive form of this malignancy. In contrast to the transgenic Eμ-TCL1 model, employing the adoptive transfer of Eμ-TCL1-derived splenocytes in immunocompetent C57BL/6 mice results in a comparably rapid (e.g., leukemic development within weeks compared to months in the transgenic model) and reliable model mimicking CLL. In this chapter, we would like to provide readers with a thoroughly optimized, detailed, and comprehensive protocol to use the adoptive transfer Eμ-TCL1 model in their research.

A humanized CD3ε-knock-in mouse model for pre-clinical testing of anti-human CD3 therapy.

Pre-clinical murine models are critical for translating drug candidates from the bench to the bedside. There is interest in better understanding how anti-human CD3 therapy works based on recent longitudinal studies of short-term administration. Although several models have been created in this pursuit, each have their own advantages and disadvantages in Type-1 diabetes. In this study, we report a murine genetic knock-in model which expresses both a murine and a humanized-CD3ε-exon, rendering it sensitive to manipulation with anti-human CD3. These huCD3εHET mice are viable and display no gross abnormalities. Specifically, thymocyte development and T cell peripheral homeostasis is unaffected. We tested immune functionality of these mice by immunizing them with T cell-dependent antigens and no differences in antibody titers compared to wild type mice were recorded. Finally, we performed a graft-vs-host disease model that is driven by effector T cell responses and observed a wasting disease upon transfer of huCD3εHET T cells. Our results show a viable humanized CD3 murine model that develops normally, is functionally engaged by anti-human CD3 and can instruct on pre-clinical tests of anti-human CD3 antibodies.

Fidelity of human ovarian cancer patient-derived xenografts in a partially humanized mouse model for preclinical testing of immunotherapies

BackgroundImmune checkpoint blockers (ICBs) have been approved by the Food and Drug Administration to be used alone in front-line therapies or in combination with other regimens for certain advanced cancers....

Editorial: Current Concepts of Cellular and Biological Drugs to Modulate Regulatory T Cell Activity in the Clinic.

The Editorial on the Research Topic Current Concepts of Cellular and Biological Drugs to Modulate Regulatory T Cell Activity in the Clinic Regulatory T (Treg) cells are essential for the maintenance of peripheral tolerance and prevent the development of autoimmunity and allergy. While on the one hand being indispensable for the perpetuation of tolerance to harmless antigens or self-antigens, Treg cells contribute to cancer pathogenesis and progression (1). Hence, the potential to treat a multitude of different human diseases by pharmacological modulation of Treg cells is enormous. Consequently, this T cell population is in the focus of biomedical research and development. Currently, isolated and in vitro expanded Treg cells as cellular therapeutics are intensively tested in clinical trials for their ability to prevent and ameliorate graft-versus-host disease (GvHD) and first biologicals to modulate Treg cell activity directly in man are being developed and tested in preclinical and clinical trials. This Research Topic comprised seven review articles; the authors discuss selected developments on the way from basic research on manipulation of peripheral tolerance mechanisms toward clinical application. Mahnke et al. present an updated view on the critical role of dendritic cells (DC) in control and maintenance of peripheral tolerance under non-inflammatory conditions based on findings obtained in preclinical mouse models showing that factors of local microenvironment and extrinsic inflammatory signals dictate the function of these “sentinels of immunity” (2): inducers of tolerance versus immunity. Analogous to this overview of basic DC research in the mouse, Raker et al. provide an overview of translational research on human tolerogenic DC, the important role of IL-10 for stability of their phenotype and their potential as cellular therapeutics for reestablishment of peripheral tolerance in patients with autoimmune or allergic diseases (Raker et al.). Hahn et al. discuss current developments in establishment of humanized mouse models for preclinical testing of novel biologicals to manipulate Treg cell suppressive function in vivo. Additionally, the risk and benefit of cellular therapies using in vitro expanded Treg cells or pharmacological modulators of Treg cell function like rapamycin or IL-2 is comparatively discussed by Perdigoto et al. Complementary, Pham et al. provide an updated view on the use of low dose IL-2 in combination with tissue-specific antigens as a therapeutic approach to expand and activate antigen-specific Treg cells in patients with type I diabetes (Pham et al.), an approach that will be currently used by different groups for treatment of T cell-mediated autoimmunity. A well-known strategy for tolerance induction first described by Hermann Waldman’s group in 1990 is the application of non-depleting anti-CD4 antibodies that induces a long lasting antigen-specific tolerance in mouse models of solid organ transplantation (3). In this Research Topic, Konig et al. report on their clinical experiences by using a humanized anti-CD4 antibody (BT-061, Tregalizumab) for treatment of rheumatoid arthritis in patients. This anti-CD4 mAb, first tested in patients with psoriasis, induces suppressive function of human Treg without blocking T helper cell activities per se. Additionally, Hui et al. discuss recent advances of management of advanced non-small cell lung cancer by PD-1-blocking antibodies and their impact on cytokine-induced killer cell activity. In summary, this Research Topic of Frontiers in Immunology provides a snapshot of our recent understanding of Treg cell biology and function with a special focus on current concepts of therapeutic strategies to modulate peripheral tolerance mechanisms and Treg cell activity in humans. Given the recent advances in our understanding of the molecular mechanisms underlying cell type- and ­tissue-specific Treg cell function, it seems promising that Treg cell-based cellular therapeutics as well as novel biologicals to directly and cell-specifically modulate Treg cell function are entering the clinic, and first results of their potential as tolerance modifiers will be available in near future.

009.2 Establishment of the gonorrhoea mouse model for pre-clinical testing of antimicrobial agents againstneisseria gonorrhoeae

Introduction New antibiotics for gonorrhoea are needed due to the emergence of resistance to the extended-spectrum cephalosporins (ESCs) in Neisseria gonorrhoeae . Here we established the 17β-estradiol mouse model of gonococcal genital tract infection for testing antibiotics against gonorrhoea by defining the in vivo efficacy of cefixime (CFX) and ceftriaxone (CRO) against strain FA1090 (ESC S ) and the multi-drug resistant strain H041 (ESC R ). Methods Estradiol-treated female BALB/c mice were inoculated vaginally with FA1090 or H041 bacteria. PBS or different doses of CFX or CRO were administered two days later (n = 9 mice/group) and vaginal swabs were quantitatively cultured for N. gonorrhoeae for 8 consecutive days. The percentage of mice colonised over time was compared among groups using the Log-rank test. Results A single oral dose of 60, 12, 6 or 3 mg/kg CFX showed significant activity against strain FA1090 with the two highest doses clearing infection within 48 hr. One or two mice in the groups that received 6 or 3 mg/kg CFX did not clear infection. None of four higher concentrations (120, 60, 12, and 6 mg/kg) of CFX cleared H041 infection, but gentamycin (48 mg/kg, i.p. injection, 5 days, q24h) was effective compared to PBS. Five concentrations (30, 15, 5, 1.5, and 0.5 mg/kg) of a single i.p. dose of CRO had significant activity against FA1090, while 60, 30, 15, or 1.5 mg/kg had no effect against H041. Conclusion The gonorrhoea mouse model shows a dose-dependent response for CRO and CFX against an ESC S strain with in vivo break-points less than 0.5 and 3 mg/kg, respectively. Higher doses of these antibiotics were not effective against an ESC R strain. We are currently correlating in vivo efficacy with pharmacokinetic analyses to further strengthen the usefulness of this model to test antimicrobial compounds against gonorrhoea. Disclosure of interest statement This work was supported by NIH/NIAID (Interagency Agreement AAI14024–001) and USUHS (USU-DOD MIC73–2493).

ITOC2 – 005. Humanized mouse models for preclinical testing of antitumour immune therapy

The identification and validation of new targets for antitumour immune therapy is still a challenge for the preclinical research as the classical syngeneic tumour models are of limited translational value and the patient derived human tumour xenograft models (PDX) are growing on immunodeficient animals. Our aim is the development of PDX models on mice with a functional human immune system to improve predictability of drug efficacy and safety. We reconstituted a human immune system by engrafting human hematopoietic stem cells in immunodeficient mice. Twelve weeks later stable expression of lymphoid cell lineages in peripheral blood and secondary lymphoid tissues could be detected by flow cytometry and immunohistochemistry analysis. We investigated the co-application of cytokine treatment (IL-3, IL-2 and IL-15) on the differentiation of immune cells. Treatment with IL-3 increased the proliferation rate, however not the differentiation pattern. At the time when the human immune system is developed, established melanoma PDXs were transplanted on these humanized mice. Human tumours developed on humanized mice without evidence of rejection. Drug-sensitivity testing with various immunoconjugates in these tumour-bearing humanized mice will follow. Our humanized mouse models will enable a more appropriate preclinical assessment of immune-based therapeutic antitumour strategies especially when combining the humanized mouse with patient-derived xenografts.

195 A humanized mouse model for preclinical testing of molecules targeting immune checkpoints

195 A humanized mouse model for preclinical testing of molecules targeting immune checkpoints

Challenges in pre-clinical testing of anti-cancer drugs in cell culture and in animal models

Experiments with cultures of human tumor cell lines, xenografts of human tumors into immunodeficient mice, and mouse models of human cancer are important tools in the development and testing of anti-cancer drugs. Tumors are complex structures composed of genetically and phenotypically heterogeneous cancer cells that interact in a reciprocal manner with the stromal microenvironment and the immune system. Modeling the complexity of human cancers in cell culture and in mouse models for preclinical testing is a challenge that has not yet been met although tremendous advances have been made. A combined approach of cell culture and mouse models of human cancer is most likely to predict the efficacy of novel anti-cancer treatments in human clinical trials.

Unlocking the power of cross-species genomic analyses: identification of evolutionarily conserved breast cancer networks and validation of preclinical models

The application of high-throughput genomic technologies has revealed that individual breast tumors display a variety of molecular features that require more personalized approaches to treatment. Several recent studies have demonstrated that a cross-species analytic approach provides a powerful means to filter through genetic complexity by identifying evolutionarily conserved genetic networks that are fundamental to the oncogenic process. Mouse-human tumor comparisons will provide insights into cellular origins of tumor subtypes, define interactive oncogenetic networks, identify potential novel therapeutic targets, and further validate as well as guide the selection of genetically engineered mouse models for preclinical testing.

Harnessing genetically engineered mouse models for preclinical testing

Recent studies cast doubt on the value of traditionally used models as tools for testing therapies for human cancer. Although the standard practice of xenografting tumors into immunocompromised mice generates reproducible tumors, drug testing in these models has low predictive power when compared to the clinical responses in Phase II trials. The use of tumor-bearing genetically engineered mouse models holds promise for improving preclinical testing. These models recapitulate specific molecular pathways in tumor initiation or progression and provide a biological system in which to study the disease process for assessing efficacy of new therapies and proof-of-principle for testing molecularly targeted drugs. In this review, we discuss the advantages and limitations of genetically engineered mice and plausible solutions for adapting these valuable tumors for wider use in preclinical testing by transplantation into naïve recipients. We also provide examples of comparative molecular analysis of mammary tumors from MMTV-Polyoma Middle-T antigen and MMTV-wnt1 models as tools for finding clinical correlates, validating existing models and guiding the development of new genetically engineered mouse models for cancer.

The Use of Targeted Mouse Models for Preclinical Testing of Novel Cancer Therapeutics

The use of genetically engineered cancer-prone mice as relevant surrogates for patients during the development of pertinent clinical applications is an unproven expectation that awaits direct demonstration. Despite the generally disappointing findings using tumor xenografts and certain early transgenic cancer models to predict therapeutic efficacy in patients, the dramatic progress of mouse models in recent years engenders optimism that the newest generation of mouse models will provide a higher standard of predictive utility in the process of drug development.

Mouse models for pretesting of immunotherapeutic strategies for cancer patients

There are two major reasons for using mouse models for preclinical testing of immunotherapetic strategies before proceeding to clinical trial. First, the requirements of regulatory authorities for toxicity testing and, second, the need of the investigator to convince himself/herself and the grant-giving bodies that proceeding with a clinical trial is scientifically justified. In both cases, the mouse model presents problems specific to a therapy depending on immune effector cells and their products. These are particularly evident in evaluating therapies for cancer patients, where analysis of cellular responses can often be better evaluated in in vitro studies with human peripheral blood leukocytes. However, where mouse models can show an effect on tumour growth, they can be extremely useful for evaluating the mechanisms underlying the effect, as we have found in evaluating tumour rejection of MUC1 expressing tumours. Moreover, strains carrying transgenes of human target antigens allow testing for auto-immunity.

Potential mouse tumor model for pre-clinical testing of mage-specific breast cancer vaccines.

Currently, there is a lack of suitable pre-clinical mouse models for testing and optimization of experimental cancer vaccines. Here, in situ developed mammary tumors of MMTV-v-Ha-ras and MMTV-c-myc transgenic mice and normal mammary, liver, spleen, and testis were screened for expression of tumor-associated antigens (TAA) Mage-b1/2/3 by reverse-transcriptase polymerase chain reaction (RT-PCR) and Southern blot hybridization. Mage-b1/2/3 are homologues of the human TAA MAGE-B1/2/3. Expression of these human MAGE genes has been found in tumors of various histological types, including breast cancer. Mage-specific RT-PCR products (using primers that amplify all three Mage-b1/2/3) were detected in mammary tumors of the MMTV-v-Ha-ras and MMTV-c-myc transgenic mice and in testis, but not in other normal tissues. RT-PCR products obtained from the mammary tumors (using primers that amplify the complete protein-encoding region of Mage-b1/2/3) were cloned and sequenced, and appeared to be most homologous with Mage-b3. Comparison of the Mage-b3 gene in mammary tumors and normal tissues suggest that somatic mutations did not occur in the Mage-b3 gene of the ras- and myc-induced mammary tumors. In addition, no differences were found between the Mage-b3 cDNA of testis, the only normal tissue that expresses Mage-b3, and Mage-b3 in genomic DNA of normal kidney, where Mage-b3 is silent. The MMTV-v-Ha-ras and MMTV-c-myc transgenic mice of this study are the first immune competent mouse models with in situ developed mammary tumors in which the expression of Mage-b3 TAA has been demonstrated. This makes them potentially suitable as a mouse model for pre-clinical testing of Mage-specific cancer vaccines in vivo.

Tailoring cancer vaccines to the elderly: the importance of suitable mouse models

The incidence of cancer has increased over the last decade, mainly due to an increase in the elderly population. Vaccine therapy for cancer is potentially less toxic than chemotherapy or radiation and could, therefore, be especially effective in older, more frail cancer patients. However, it has been shown that older individuals do not respond to vaccine therapy as well as younger adults. This has been attributed to T cell unresponsiveness, a phenomenon also observed in cancer patients per se. Activation of tumor-specific T cells by cancer vaccines might be an approach, especially suitable for elderly patients, to eradicate or to prevent recurrence of tumors after primary treatment. To tailor pre-clinical testing of vaccine therapies to the elderly, it is important to have mouse models in which tumors develop at equivalent time points in their life span, as in humans. Such models are currently not available. This progress report first summarizes the current knowledge of tumor-immunological parameters potentially involved in T cell unresponsiveness in relation to aging in mice and humans. Secondly, it reviews those cancer vaccines that are known for their potential to induce tumor-specific T cell responses. Thirdly, it discusses the usefulness of currently available mouse models for pre-clinical testing of cancer vaccines applicable to the elderly population. Finally, experimental approaches are proposed, as to how to develop mouse models that allow the induction of specific tumors at will at different ages, expressing tumor-specific antigens in an ‘immune competent’ environment. These mouse models may teach us how to overcome immune deficits in the elderly, thereby facilitating the development of effective and safe cancer vaccines.