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 (VZV BAC ) 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 (VZV Luc ). 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 VZV Luc 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) 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 μM) 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 EC99 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.
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).
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.
BackgroundBreast 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.MethodsHSC 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.ResultsBoth 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.ConclusionsBreast 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 ApprovalAnimal studies were executed in compliance with local Charles River IACUC guidelines, IACUC number I-033.
BackgroundBladder 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.MethodsThree 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.ResultsWe 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.ConclusionsOur 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 ApprovalAnimal 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: 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.
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