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The NCG triple immunodeficient mice on a NOD/Nju background lack functional/mature T, B, and NK cells, and have reduced macrophage and dendritic cell function. This study characterized the NCG mouse model for toxicity, engraftment and tumorigenicity assessments of cell therapies, using CD34+ hHSPC adult mobilized cells with two myeloablation regimens. Mice received sub-lethal irradiation or busulfan and were then injected intravenously with CD34+ hHSPCs (1.0 x 106 cells/mouse) or PBS (control), while positive control animals received 2 x 106 HL-60 cells/mouse. hCD34+ cell donors were treated with the mobilizing agent G-CSF prior to leukapheresis. Following injections, mouse blood samples were collected to assess engraftment rates by flow cytometry with body weights recorded periodically up to 20 weeks post-cell injection. No significant clinical signs or body weight changes were observed. At week 10 post-cell injection, the peripheral blood chimerism of hCD45+ cells was above 20%. While mCD45+ concentration was constant between week 10 and 17 in whole blood samples, hCD45+ concentration and chimerism slightly decreased at week 17. However, chimerism remained above 10%, with busulfan-treated mice presenting higher values. Chimerism was further assessed by quantifying human Alu sequences in blood and multiple organs using qPCR. Alu sequences were most abundant in the spleen and bone marrow, while lowest in the testes. In the positive control group, expected mortalities due to tumorigenesis were observed between days 27 and 40 post-cell injection. Overall, study results may be used to inform study design and potential toxicological endpoints relevant to non-clinical cell therapy development.

Figure S1.ABBV-744 exhibits potent anti-proliferative activity against AML cells through cell cycle arrest and induction of apoptosis.

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Dual bromodomain BET inhibitors that bind with similar affinities to the first and second bromodomains across BRD2, BRD3, BRD4, and BRDT have displayed modest activity as monotherapy in clinical trials. Thrombocytopenia, closely followed by symptoms characteristic of gastrointestinal toxicity, have presented as dose-limiting adverse events that may have prevented escalation to higher dose levels required for more robust efficacy. ABBV-744 is a highly selective inhibitor for the second bromodomain of the four BET family proteins. In contrast to the broad antiproliferative activities observed with dual bromodomain BET inhibitors, ABBV-744 displayed significant antiproliferative activities largely although not exclusively in cancer cell lines derived from acute myeloid leukemia and androgen receptor positive prostate cancer. Studies in acute myeloid leukemia xenograft models demonstrated antitumor efficacy for ABBV-744 that was comparable with the pan-BET inhibitor ABBV-075 but with an improved therapeutic index. Enhanced antitumor efficacy was also observed with the combination of ABBV-744 and the BCL-2 inhibitor, venetoclax compared with monotherapies of either agent alone. These results collectively support the clinical evaluation of ABBV-744 in AML (Clinical Trials.gov identifier: NCT03360006).

Background/Aim: The therapeutic potential of bromodomain and extra-terminal motif (BET) inhibitors in hematological cancers has been well established in preclinical and early-stage clinical trials, although as of yet, no BET-targeting agent has achieved approval. To add insight into potential response to mivebresib (ABBV-075), a broad-spectrum BET inhibitor, co-clinical modeling of individual patient biopsies was conducted in the context of a Phase I trial in acute myeloid leukemia (AML). Materials and Methods: Co-clinical modeling involves taking the patient’s biopsy and implanting it in mice with limited passage so that it closely retains the original characteristics of the malignancy and allows comparisons of response between animal model and clinical data. Procedures were developed, initially with neonate NOD/Shi-scid-IL2rγnull (NOG) mice and then optimized with juvenile NOG-EXL as host mice, eventually resulting in a robust rate of engraftment (16 out of 26, 62%). Results: Results from the co-clinical AML patient-derived xenograft (PDX) modeling (6 with >60% inhibition of bone marrow blasts) were consistent with the equivalent clinical data from patients receiving mivebresib in monotherapy, and in combination with venetoclax. The modeling system also demonstrated the activity of a novel BD2-selective BET inhibitor (ABBV-744) in the preclinical AML setting. Both agents were also highly effective in inhibiting blast counts in the spleen (10/10 and 5/6 models, respectively). Conclusion: These findings confirm the validity of the model system in the co-clinical setting, establish highly relevant in vivo models for the discovery of cancer therapy, and indicate the therapeutic value of BET inhibitors for AML and, potentially, myelofibrosis treatment.

Checkpoint inhibitors including anti-PD-1/PD-L1 are a type of immunotherapy that have become the standard of care for cancer patients. Continual focus is placed on developing new treatments for cancer, including breast, lung and colon cancer, which are the most common worldwide. Humanized mice are an important in-vivo animal model that recapitulate crucial aspects of human tumor biology and anti-tumoral immunity allowing for assessment of immunotherapies. Currently these models are primarily created using cord blood, which is in limited supply. This leads to the isolation of low numbers of hCD34+ stem cells, resulting in small cohorts of animals. This limitation represents a challenge in working with humanized mice making it important to consider alternative sources of hCD34+ stem cells. In this study, we compared NCG mice engrafted with an alternative source of adult healthy donor mobilized hCD34+ stem cells to those engrafted with cord blood derived hCD34+ stem cells.After evaluating engraftment kinetics between the two cell sources we examined the anti-tumoral effect of an anti-human PD-1 checkpoint inhibitor in mice bearing three different human tumor xenograft cell lines (CDX) (A549 lung, COLO-205 colon and MDA-MB-436 breast cancer). NCG mice were humanized using either cord blood derived or adult healthy donor mobilized hCD34+ stem cells. Levels of human immune cell engraftment were measured in peripheral blood using multi-color flow cytometry. Humanized NCG mice were implanted subcutaneously with cancer cells, and tumor bearing mice (TBM) were randomized into treatment groups when the average tumor size reached comparable volumes for each tumor type. Vehicle control mice were treated with isotype control IgG antibody, whereas TBM were dosed with anti-hPD-1 antibody. Clinical observations, body weights and tumor growth kinetics were recorded throughout the study. At the time of euthanasia whole blood, spleen and tumor tissues were collected and processed for immune profiling by multi-color flow cytometry. Adult healthy donor mobilized hCD34+ humanized mice have sustained humanization levels out to 34 weeks post-injection and can be successfully engrafted with human CDX tumors. The ability to monitor tumor growth kinetics and observe a response to an anti-hPD-1 checkpoint inhibitor across multiple tumor types indicates that NCG mice humanized with adult healthy donor mobilized CD34+ stem cells are useful when assessing immunotherapies. Future directions of this model will expand beyond monotherapies to include the evaluation of additional treatment methods such as bispecific antibodies and cell-directed therapies. Citation Format: Jenny Rowe, Steven Bronson, Christoph Eberle, Ann Fiore, Bob Mihalek, Stephen Festin. Assessment of a novel method using adult healthy donor mobilized human CD34+hematopoietic stem cells in NCG mice for use in tumor modeling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 39.

Background: Inhibitors of the PD-1/PD-L1 checkpoint pathway were among the first FDA approved immunotherapies and are now among the standard of care for cancer patients. In vivo animal models that recreate crucial aspects of human tumor biology and anti-tumor immunity are needed for evaluating new therapy developments across cancer types. In 2019, we reported an animal model using non-adult (cord blood derived) hCD34+ hematopoietic stem cells, considered the standard cell source, for humanizing the immune system of NCG mice. In this study we assessed a mouse model using adult patient mobilized hCD34+ stem cells as an alternative source and evaluated engraftment and tumor modulation using checkpoint inhibitors. Study Details: We evaluated the anti-tumor effects of an anti-human PD-1 checkpoint inhibitor on basal lung cell adenocarcinoma (A549) in NCGs humanized with adult patient mobilized hCD34+ stem cells. Humanized mice were implanted subcutaneously with A549 cells, and tumor bearing mice (TBM) were randomized into treatment groups when the average tumor size reached a volume of ~80-120 mm3. Vehicle control mice were treated with isotype control IgG antibody, whereas A549 TBM were dosed with anti-hPD-1 antibody biweekly (10 mg/kg) for three weeks. Clinical observations, body weights and tumor growth kinetics were recorded throughout the study. At study termination, whole blood, spleen and tumor tissue were collected and processed for immune profiling by multi-color flow cytometry. Results: Anti-hPD-1 monotherapy significantly inhibited A549 tumor growth. NCG mice injected with adult patient mobilized hCD34+ stem cells sustained engraftment. At study termination common human leukocytes were distributed in peripheral blood, spleen and tumors of surviving mice. Phenotyping of the cancer microenvironment revealed the presence of lymphoid (T-lymphocytes and subsets including Tregs) and myeloid immune cells (DC, TAM) with the latter lineage less detectable overall. PD-1 checkpoint blockade on average increased CD8+ T-cell infiltration and decreased Treg frequencies among the TIL population. Generally, human cytokines (IFN-γ, IL-2 and TNF-α) were secreted by viable tumor-infiltrating total T-cells (CD3+) and subsets (CD4+ and CD8+) following ex vivo PMA/Ionomycin stimulation in the presence of Brefeldin A, demonstrating the capability to produce polyfunctional responses. Conclusions: This study demonstrates that adult patient mobilized hCD34+ cells can successfully reconstitute NCG mice. An immune modulating anti-tumor response was elicited with tissue infiltration patterns of human leukocytes overall comparable to those found in cord blood derived hCD34+ humanized NCG mice. These findings make this model utilizing NCG mice engrafted with adult patient mobilized hCD34+ stem cells a viable approach for cell transfer studies when assessing targeted immuno-oncology therapies. Citation Format: Jenny Rowe, Christoph Eberle, Ann Fiore, Robert Mihalek, Brianna Schoen, Stephen Festin. Assessment of a novel tumor model using adult patient mobilized human CD34+ hematopoietic stem cells in NCG mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1647.

Dual bromodomain BET inhibitors that bind with similar affinities to the first and second bromodomains across BRD2, BRD3, BRD4, and BRDT have displayed modest activity as monotherapy in clinical trials. Thrombocytopenia, closely followed by symptoms characteristic of gastrointestinal toxicity, have presented as dose-limiting adverse events that may have prevented escalation to higher dose levels required for more robust efficacy. ABBV-744 is a highly selective inhibitor for the second bromodomain of the four BET family proteins. In contrast to the broad antiproliferative activities observed with dual bromodomain BET inhibitors, ABBV-744 displayed significant antiproliferative activities largely although not exclusively in cancer cell lines derived from acute myeloid leukemia and androgen receptor positive prostate cancer. Studies in acute myeloid leukemia xenograft models demonstrated antitumor efficacy for ABBV-744 that was comparable with the pan-BET inhibitor ABBV-075 but with an improved therapeutic index. Enhanced antitumor efficacy was also observed with the combination of ABBV-744 and the BCL-2 inhibitor, venetoclax compared with monotherapies of either agent alone. These results collectively support the clinical evaluation of ABBV-744 in AML (Clinical Trials.gov identifier: NCT03360006).

Background Breast and colon cancer rank second and third, respectively, in world-wide prevalence of malignancies and present a large unmet medical need. The correlation between lymphocyte infiltration into the tumor microenvironment and efficacy of anti-cancer immunotherapies has been established. Therefore, relevant and cost-saving pre-clinical models are needed for developing new treatment approaches to predominant human tumor types. HuCD34NCG mice facilitate studying human immune responses in vivo elicited by experimental therapeutic antibodies. We characterized growth kinetics and human immune responses to checkpoint blockade in human breast and colon tumor-bearing HuCD34NCG mice. Aging, non tumor-bearing HuCD34NCG mice were also monitored for indicators of spontaneous hematopoietic cancer formation. Methods HSC engraftment was quality controlled prior to inoculating HuCD34NCG mice with either colon adenocarcinoma (COLO 205) or triple negative breast cancer (MDA-MB-436) cells (both purchased from American Type Culture Collection, Manassas, VA). Mice were randomized into treatment groups based on tumor size, and checkpoint inhibitor antibodies were dosed twice weekly (anti-human PD-1, BioXcell clone: RMP1-14 or Keytruda; anti-human CTLA-4, BioXcell clone: BN13; and combination therapy). Body weights, general health status and survival were monitored. Peripheral blood (PB) and selected tissues were analyzed for the presence and composition of human immune cells by acoustic focusing flow cytometry. Non tumor-bearing aged HuCD34NCG mice (27 weeks post-engraftment) were sampled biweekly over ten weeks for lymphoma immunophenotyping. Results Both tumor-bearing models showed significant anti-hPD-1 and anti-hCTLA-4 responses, but combination therapy only enhanced growth reduction significantly in MDA-MB-436 tumors. Flow cytometric analysis identified viable human leukocytes in tumor and spleen at study termination. These tumor-infiltrating lymphocytes (TIL) and splenocytes from surviving COLO 205 and MDA-MB-436 mice consisted of a total T-cell phenotype (CD3+) with proliferating (Ki67+), CD4+, CD8+ and Treg subsets. Additionally, myeloid cells (CD11b+, CD11c+) and M1/M2 macrophages were detected within these infiltrates. Splenic and tumor-infiltrating T-cells readily secreted human cytokines (IFN-γ, IL-2, TNF-α) and granzyme B upon ex vivo activation exhibiting polyfunctional and cytotoxic capabilities in all treatment groups. Baseline murine and human cytokine levels were distinguished in plasma from aging, non tumor-bearing HuCD34NCGs. Their phenotypes also showed no conclusive indicators of abnormal blood cells developing or graft failure. Conclusions Breast and colon tumor cell-line derived models were established in HuCD34NCG mice. Standard checkpoint inhibitor treatment promoted human T-cell infiltration into tumor microenvironments inhibiting growth. These results demonstrate that HuCD34NCG are a robust and relevant host for various human cell xenotransplants to advance preclinical immuno-oncology drug development. Ethics Approval Animal studies were executed in compliance with local Charles River IACUC guidelines, IACUC number I-033.

Background Bladder cancer is the fifth most common cancer in the US, and the ninth most common cancer worldwide. Treatment of bladder cancer has evolved over time to encompass traditional modalities of chemotherapy and surgery, but has been particularly impacted by the recent use of immunotherapy. Modern immunotherapy has focused on checkpoint protein inhibitors that impede immune function. The inhibitors for several checkpoint targets (programmed death-ligand 1 [PD-L1], programmed cell death protein1 [PD-1], and cytotoxic T-lymphocyte-associated protein 4 [CTLA4]) were either approved or in late-stage development. In this study we examined the effect of PD-1 inhibitor pembrolizumab and cisplatin in a panel of bladder patient-derived xenografts (PDX) with distinct patterns of PD-L1 expression in CD34+ stem cell humanized NCG (HuCD34NCG) mice. Methods Three bladder PDX models PNX0428, PNX0434 and PNX1028 have been established under informed consent from the patients at the Fox Chase Cancer Center, Philadelphia. These models have been profiled for the levels of PD-L1 protein using immunohistochemical staining with SP263 antibody (Ventana) and used to establish the growth kinetics and sensitivity to the PD-1 check point inhibitor pembrolizumab and standard of care chemotherapeutic agent cisplatin in female HuCD34NCG and standard NCG mice from Charles River Laboratories. Results We have established the ability of three bladder PDX models to grow in both the HuCD34NCG and standard NCG mice. The tumor growth kinetics of these models was slightly delayed in HuCD34NCG animals compared to NCG. We observed variable responses to cisplatin and pembrolizumab treatments among the PDX models that did not correlate with the level of PD-L1 expression in these tumors. Despite the presence of ~70% PD-L1 positive cells in the PNX0428 model, these tumors produced minor responses to pembrolizumab in HuCD34NCG mice that correspond to progressive disease in patients. Interestingly, pembrolizumab treatment in the PNX1028 model and even more significantly in the PNX0434 model in HuCD34NCG mice produced strong statistically significant tumor growth inhibition that correlates with stable disease in patients despite negative staining for PD-L1 protein in these tumors. The standard of care treatment cisplatin produced significant tumor growth inhibition in all three PDX models in both HuCD34NCG and standard NCG mice. Conclusions Our data indicates that abundant expression of PD-L1 protein in tumors should not be used as the only biomarker for patient stratification for the treatment with PD-1/PD-L1 check point inhibitors. The HuCD34NCG mouse model is an effective tool for supporting tumor growth and evaluating immunotherapies. Ethics Approval Animal studies were approved by Nexus Pharma, IACUC number 08-22.Three bladder PDX models PNX0428, PNX0434 and PNX1028 have been established under informed consent from the patients at the Fox Chase Cancer Center, Philadelphia, IRB protocol 11-866.

Background: In vivo syngeneic models continue to be an important tool in cancer biology research. Charles River C57BL/6 and Balb/c mice, two frequently used inbred strains, can be implanted with different murine tumor cell lines. One aspect of syngeneic model development is to characterize the extent and composition of infiltrating leukocytes (TIL) of tumor-bearing mice. The ability to analyze TILs is a relevant factor in preclinical model selection for studying immunomodulatory responses. Study Details: We evaluated growth rates and basal infiltrates in syngeneic models of solid mouse tumors. In four separate studies 7-week-old Charles River mice were implanted subcutaneously on the flank with either MC38 or B16F10 cells (C57BL/6) or with either RENCA or EMT6 cells (Balb/c), respectively. Mice were treated with isotype control IgG1 antibody (BioXcell clone MOPC-21) when tumors reached sizes of ~50-100 mm3. Surviving mice were euthanized when tumor burden reached or exceeded 2000 mm3 in line with internal IACUC guidelines. Spleen and tumors were then processed into single cell suspensions for immunophenotyping by multicolor acoustic-assisted flow cytometry. Results: Growth kinetics of all four tumor cell lines, MC38, B16F10, RENCA and EMT6, were monitored throughout each study. At study termination murine leukocyte infiltration was found in all models. These TILs were immune profiled to determine the frequencies and composition of murine immune cells in the lymphoid and myeloid compartments. Phenotyping data confirmed the presence of viable CD45+ cells with total T-cells (CD3+), T-cell subsets (CD4+, CD8+ and Treg) and myeloid populations (CD11b+) including M1 and M2 TAMs as well as CD11c+ cells. M1/M2 TAM, CD8/Treg and CD4/CD8 ratios were also calculated, and tumor models were assessed based on their baseline TIL composition. Conclusions: Four different allograft models of solid mouse tumors could be established using Charles River C57BL/6 and Balb/c mice. Their basal immune microenvironments could be characterized and therefore can be useful as pharmacodynamic readouts in efficacy studies of potential anti-cancer therapeutics. Citation Format: Christoph S. Eberle, Jenny Rowe, Ann Fiore, Robert Mihalek, Stephen Festin. Evaluation of tumor growth and baseline leukocyte infiltration in Charles River C57BL/6 and Balb/c mice engrafted with MC38, B16F10, RENCA and EMT6 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2770.

Immunotherapy is revolutionizing cancer treatment. As a consequence, there is an urgent need for predictive preclinical models to support compound development with the aim of bringing optimal drug candidates into clinical trials. Patient-derived xenografts (PDX) are the gold-standard in cancer drug development. One major drawback of PDX is the lack of an immunological competent host. To overcome this hurdle triple immunodeficient mice are humanized with hematopoietic stem cells (HSC) to enable the interaction between human immune cells and human tumor cells in a rodent host. The current study uses HSC humanized NCG (NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl) mice, HuCD34NCG, to evaluate the sensitivity of five NSCLC PDX models towards anti CTLA-4 (Ipilimumab), anti-PD-1 (Nivolumab) and the combination thereof. We identified one predicted responder and one non-responder and in a single mouse trial approach (SMT) and confirmed the results in a conventional experimental set-up with n=5 per treatment arm. Infiltration of human immune cells was detected by flow cytometry in tumor tissue (TIL) and hematopoietic organs. Serum of tumor bearing animals was analyzed for human and mouse cytokine secretion pre- and post-treatment. The NCG mouse strain was genetically characterized by whole exome sequencing (WES) and the mutational profile compared to other common mouse strains. The mutational profile showed the highest overlap with the genome data from other triple immune compromised mice and with NOD/SCID indicating their close hereditary relationship. Non-tumor bearing HuCD34NCG mice depicted engraftment of human CD45+ cells in peripheral blood of >25% and >60% in bone marrow and spleen after 8-12 wks post injection. Thus, they fulfilled enrollment criteria for conducting a drug screen for immune modulating compounds. The five NSCLC PDX models showed a distinct sensitivity profile ranging from partial remission (tumor growth inhibition, TGI, of 87.5% compared to untreated control) to progression (no TGI). In all models the combination treatment was the most active therapy (TGI: 60%), followed by Ipilimumab (TGI: 55%) and Nivolumab (TGI: 48%). Nevertheless, in selected models anti-CTLA-4 treatment was the most efficacious therapy. The two models examined in more detail LXFE 397 (=responder, TGI 55% in both formats) and LXFA 400 (non-responder, TGI 2%SMTvs 5%, n=5/group) displayed the same sensitivity pattern in the conventional set-up as compared to the SMT screening approach. The TIL analysis revealed a pronounced rise in the CD3+ subset and increased CD4+/CD8+ ratio in the most effective treatment arms. Subsequent serum analyses will help identify possible biomarkers predicting drug response towards checkpoint inhibition. Taken together our study proves that the HuCD34NCG mouse is a fully functional drug development tool. In combination with highly predictive PDX tumor models this in vivo platform provides a further step to support the development of new drugs targeting the host immune response. Citation Format: Julia Schüler, Jenny Rowe, Eva Oswald, Stephen Festin. The single mouse trial format predicts the sensitivity towards checkpoint inhibitor treatment in NSCLC PDX models in HuCD34NCG/CRL mice, an innovative triple immune deficient mouse strain [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5634.

Background: Checkpoint blockade inhibitors targeting PD-1 and CTLA-4 pathways are clinically approved therapies for multiple cancer types. The performance of targeted interventions has been effective, but clinical response rates vary. In vivo models of human immunity in human tumor bearing mice (TBM) is an important tool for studying mechanisms of targeted therapies and developing new and effective treatments. The NCG (NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl), a recently developed triple immunodeficient mouse strain, is a stable host for both human immune cells and tumors for the study of immuno-oncology-based therapeutics. Study Details: We evaluated the anti-tumor effects of immune checkpoint inhibitors (anti-human PD-1, including Pembrolizumab, and CTLA-4) on colon epithelial carcinoma (RKO) and basal lung cell adenocarcinoma (A549) cell lines in a human donor immune cell-humanized NCG/CRL mouse model (HuCD34NCG). In separate studies Charles River humanized NCG (HuCD34NCG) mice were implanted subcutaneously on the flank with either RKO or A549 tumors. Group randomization occurred when the average tumor size reached a volume of ~100mm3 (A549) or 30-60mm3 (RKO). Control mice were treated with isotype control IgG antibodies. RKO TBM were treated with anti-PD-1 antibody alone, while A549 TBM were dosed with anti-PD1-1 and anti-CTLA-4 antibodies independently and in combination therapy. Results: Human immune cell engraftment levels were confirmed in the peripheral blood, spleen and tumor (hCD45, hCD3, hCD4, hCD8, hCD19, NK, myeloid, macrophages; markers vary based on study) of HuCD34NCG humanized mice. Tumor growth kinetics were monitored throughout the study. Inhibition of RKO and A549 tumor growth upon anti-PD-1 monotherapy was significant. Human T-cell infiltration was observed in A549 and RKO tumors with the majority of live T-cells responsive post infiltration. Human cytokines (IFN-γ, IL-2 and TNF-α) were released by tumor-infiltrating total T-cells (CD3+) and subsets (CD4+ and CD8+), as demonstrated by intracellular cytokine staining following PMA/Ionomycin stimulation. Polyfunctional T-cell responses were detected in all treatment groups at study termination. Conclusions: The results from these studies demonstrate significant immunomodulatory anti-tumor response to immune checkpoint inhibitors. The newly developed HuCD34NCG humanized mouse model showed robust and sustained engraftment of human immune cell populations and demonstrated infiltration of T-cells into tissues and tumors making this mouse model ideal for immuno-oncology studies. Citation Format: JENNY ROWE, Christoph Eberle, Elizabeth Reap, Ann Fiore, Anya Avrutskaya, Paula Miliani de Marval, Robert Mihalek, Stephen Festin. Evaluation of in vivo anti-tumor response of solid tumors in a novel immune cell-humanized NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl mouse model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5625.

The humanized mouse model has been developed as a model to identify and characterize human immune responses to human pathogens and has been used to better identify vaccine candidates. In the current studies, the humanized mouse was used to determine the ability of a vaccine to affect the immune response to infection with Mycobacterium tuberculosis. Both human CD4+ and CD8+ T cells responded to infection in humanized mice as a result of infection. In humanized mice vaccinated with either BCG or with CpG-C, a liposome-based formulation containing the M. tuberculosis antigen ESAT-6, both CD4 and CD8 T cells secreted cytokines that are known to be required for induction of protective immunity. In comparison to the C57BL/6 mouse model and Hartley guinea pig model of tuberculosis, data obtained from humanized mice complemented the data observed in the former models and provided further evidence that a vaccine can induce a human T-cell response. Humanized mice provide a crucial pre-clinical platform for evaluating human T-cell immune responses in vaccine development against M. tuberculosis.

Acute myeloid leukemia (AML) co-clinical modeling has been optimized with peripheral blood mononuclear cells (PBMCs) collected from low volume (14 mL) patient samples to establish an algorithm for efficiently co-clinically modeling AML patients. Methods: PBMCs were ficoll gradient purified and viably cryopreserved. Intrahepatic (i.h.) inoculation of AML PBMCs in neonate NOD/Shi-scid-IL2rγnull (NOG) mice and intravenous (i.v.) inoculation in both juvenile NOG mice and juvenile human transgenic IL-3/GMCSF NOD/Shi-scid-IL2rγnull mice (NOG-EXL) were evaluated. Bone marrow (BM) aspirates, splenocytes and PBMCs from mice were evaluated by fluorescence-activated cell sorting (FACS) at 12 weeks post AML inoculation for engraftment as determined by % ratio of human CD33+ cells to total CD45+ cells (human + murine cells). Humerus bones from inoculated animals were also evaluated by human CD33 immunohistochemistry (IHC). Results: Cells from 2/6 AML patient samples (CTG-2224 and CTG-2357) successfully engrafted into neonate mice. Animals were dosed with vehicle, cytarabine, ABBV-075 (clinical trial-staged BET family bromodomain (BD) inhibitor), or ABBV-744 (a preclinical BDII selective inhibitor) and evaluated for tumor burden six weeks post drug treatment initiation. ABBV-075 and ABBV-744 treated animals had lower tumor burden in the CTG-2224 model, 17% (p Citation Format: Neal C. Goodwin, Daniel H. Albert, Angela M. Davies, Jenny Rowe, Gerold Feuer, Michael Boyiadzis, Kathleen A. Dorritie, Maria Mancini, Regina Gandour-Edwards, Warren M. Kati, Mark D. McKee, Keith F. McDaniel, David J. Frost. Acute myeloid leukemia human/mouse co-clinical trial feasibility study optimized in human transgenic IL-3/GMCSF NOD/Shi-scid-IL2rγnull mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1150.

The new Supreme Court of the United Kingdom, which heard its first case in October 2009, sits at the apex of the UK's justice systems. Located in the painstakingly restored Grade II Listed Middlesex Guildhall in Parliament Square, it will be the highest appeal court in England, Wales and Northern Ireland and for civil cases in Scotland. Jenny Rowe, Chief Executive of the Court, reveals how the new Justices' Library is at the heart of this landmark building.

Varicella-zoster virus (VZV) replicates in quiescent T cells, neurons, and skin cells. In cultured fibroblasts (HFFs), VZV induces host cyclin expression and cyclin-dependent kinase (CDK) activity without causing cell cycle progression. CDK1/cyclin B1 phosphorylates the major viral transactivator, and the CDK inhibitor roscovitine prevents VZV mRNA transcription. We investigated the antiviral effects of additional compounds that target CDKs or other cell cycle enzymes in culture, ex vivo, and in vivo. Cytotoxicity and cell growth arrest doses were determined by Neutral Red assay. Antiviral effects were evaluated in HFFs by plaque assay, genome copy number, and bioluminescence. Positive controls were acyclovir (400 microM) and phosphonoacetic acid (PAA, 1 mM). Test compounds were roscovitine, aloisine A, and purvalanol A (CDK inhibitors), aphidicolin (inhibits human and herpesvirus DNA polymerase), l-mimosine (indirectly inhibits human DNA polymerase), and DRB (inhibits casein kinase 2). All had antiviral effects below the concentrations required for cell growth arrest. Compounds were tested in skin organ culture at EC(99) doses; all prevented VZV replication in skin, except for aloisine A and purvalanol A. In SCID mice with skin xenografts, roscovitine (0.7 mg/kg/day) was as effective as PAA (36 mg/kg/day). The screening systems described here are useful models for evaluating novel antiviral drugs for VZV.

To efficiently generate varicella-zoster virus (VZV) mutants, we inserted a bacterial artificial chromosome (BAC) vector in the pOka genome. We showed that the recombinant VZV (VZVBAC) strain was produced efficiently from the BAC DNA and behaved indistinguishably from wild-type virus. Moreover, VZV’s cellassociated nature makes characterizing VZV mutant growth kinetics difficult, especially when attempts are made to monitor viral replication in vivo. To overcome this problem, we then created a VZV strain carrying the luciferase gene (VZVLuc). This virus grew like the wild-type virus, and the resulting luciferase activity could be quantified both in vitro and in vivo. Using PCR-based mutagenesis, open reading frames (ORF) 0 to 4 were individually deleted from VZVLuc genomes. The deletion mutant viruses appeared after transfection into MeWo cells, except for ORF4, which was essential. Growth curve analysis using MeWo cells and SCID-hu mice indicated that ORF1, ORF2, and ORF3 were dispensable for VZV replication both in vitro and in vivo. Interestingly, the ORF0 deletion virus showed severely retarded growth both in vitro and in vivo. The growth defects of the ORF0 and ORF4 mutants could be fully rescued by introducing wild-type copies of these genes back into their native genome loci. This work has validated and justified the use of the novel luciferase VZV BAC system to efficiently generate recombinant VZV variants and ease subsequent viral growth kinetic analysis both in vitro and in vivo. Varicella-zoster virus (VZV) is a human alphaherpesvirus that is a significant pathogen in the United States. Primary infection of VZV typically occurs during childhood and leads to varicella (chickenpox). Like all herpesviruses, VZV establishes lifelong latency in the host, specifically in trigeminal ganglia and dorsal root ganglia. The end sequela of VZV