scholarly journals 908 Humanized model for assessment of therapies targeting either lymphoid or myeloid compartment: enhanced evaluation of clinical relevancy & translatability

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A953-A953
Author(s):  
Florence Renart-Depontieu ◽  
Gaëlle Martin ◽  
Chloé Beuraud ◽  
Poonam Yakkundi ◽  
Angus Sinclair ◽  
...  
Keyword(s):  
T Cells ◽  
Mouse Model ◽  
Temperature Drop ◽  
Myeloid Cells ◽  
Body Weight Loss ◽  
Preclinical Model ◽  
Human Immune System ◽  
Short Life Span ◽  
Human Immune

BackgroundThe breakthrough of immunotherapies has unleashed new hope and new success for cancer therapy. However, the choice of a preclinical model is one of the main challenges as they are important for evaluation of translatability to help support testing in clinical studies, including potential efficacy and tolerability of immunotherapies during preclinical development.The development of mouse models featuring a human immune system (HIS) provides new paths for the investigation of the efficacy of immunotherapies in preclinical models engrafted with human tumors. Although these models provided a breakthrough in the assessment of immune targeting agents, they also come with a few significant caveats. These include: a lack of a mature human myeloid compartment in the mouse, and a short life span of the model when this compartment is promoted at non-physiological levels via the over-expression of human cytokines. Here, we report a novel mouse model (BRGSF-HIS), featuring functional human lymphoid and myeloid compartments. The human immune response of this model was assessed through examination of the immune cells composition in the tumor microenvironment (TME), and its ability to respond to biologics known to trigger cytokine release syndrome (CRS).MethodsBRGSF (balb/C Rag2-/-, IL2Rg-/-, SIRPaNOD and Flt3-/-) is a highly immunodeficient mouse, with reduced murine myeloid cells, which allows long term CD34+ HSC-engraftment and development of human lymphoid and myeloid compartments (human CD141+ and CD1c+ DC subsets, CD123+ pDC, CD14+ monocytes), in blood, spleen and bone marrow. The engraftment is stable for over twelve months with no side effects. The effect of exogenous human Flt3 ligand (Flt3L) on the composition of TME in A549 model, and an anti-CD3 antibody (OKT3)-induced CRS, were assessed.ResultsExogenous human Flt3L significantly and transiently increased the proportion of human myeloid cells. This can be recalled by continuous dosing of Flt3L. Assessment of tumor immunobiology in A549 model, showed increased tumor-infiltrating T-cells (mainly CD8+ T-cells) and myeloid cells, while tumor-infiltrating NK cells were decreased. The presence of myeloid cells provides a new opportunity for assessment of myeloid targeted therapies, as proven using pDC-depleting antibodies. OKT3 administration resulted in CRS symptoms including a temperature drop, body weight loss and a change in serum cytokine levels. Symptoms were mitigated upon administration of tocilizumab, suggesting the contribution of the myeloid compartment in the response observed.ConclusionsThese data demonstrate that BRGSF-HIS mice support development of functional human myeloid cells and that this mouse model enables preclinical evaluation of cancer immunotherapy in vivo.

scholarly journals P06.07 In vivo studies of immunomodulatory a-CTLA-4 antibody in a humanized mouse model

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A22.1-A22
Author(s):  
C Reitinger ◽  
F Nimmerjahn
Keyword(s):  
Immune System ◽  
Mouse Model ◽  
Cancer Immunotherapy ◽  
Model Systems ◽  
Checkpoint Control ◽  
Human Immune System ◽  
Humanized Mouse ◽  
Humanized Mouse Model ◽  
Human Immune

BackgroundRecent findings in cancer immunotherapy have reinforced the hypothesis that the immune system is able to control most cancers. Immunomodulatory antibodies can enhance immune responses, having the potential to generate anti-cancer immunity.1–4Materials and MethodsMost current studies addressing this question are performed in murine mouse model systems or use in vitro culture systems, which do not reflect the human in vivo situation, potentially leading to results that cannot be fully translated into human cancer therapy. Therefore, it is necessary to establish a new mouse model, which allows the study of cancer immunotherapy in the context of a human immune system. We focused on the establishment of a humanized mouse model, in which different immunomodulatory antibodies can be tested in the presence of a human immune system.ResultsFirst experiments concerning the suitability to test immunomodulatory antibodies in the humanized mouse model, revealed that effects of checkpoint-control antibody a-CTLA-4 were similar to the effects seen in patients of clinical studies. To analyse the anti-tumor activities of immunomodulatory antibodies in vivo we are establishing a human melanoma-like tumor model in humanized mice.ConclusionsThis enables us to test the efficacy of immunomodulatory agonistic antibodies (such as CP-870,893) and checkpoint control antibodies (such as anti-CTLA-4) in eliminating a melanoma-like tumor. Furthermore, parameters like tumor infiltrating human cells und cytokine/chemokine production can be analysed.ReferencesSchuster M, Nechansky A, Loibner H. Cancer immunotherapy. Biotechnol J 2006;1:138–147.Mellman I, Coukos G, Dranoff G. Cancer immunotherapy comes of age. Nature rev 2011;480:480–489.Finn OJ. Immuno-oncology: understanding the function and dysfunction of the immune system in cancer. Annals of Oncology 2012;23:vii6–vii9.Langer LF, Clay TM, Morse MA. Update on anti-CTLA-4 in clinical trials. Expert Opin Biol Ther 2007;8:1245–1256.Disclosure InformationC. Reitinger: None. F. Nimmerjahn: None.

scholarly journals Diffuse Large B Cell Pdtx in Humanized Mice Are Valuable Models to Study Host-Lymphoma Interactions and Immune-Modulating Agents

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2406-2406
Author(s):  
Giorgia Zanetti ◽  
Giuseppina Astone ◽  
Luca Cappelli ◽  
William Chiu ◽  
Maria Teresa Cacciapuoti ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
T Cell ◽  
B Cell ◽  
Research Funding ◽  
Ex Vivo ◽  
Human Immune System ◽  
Human T Cells ◽  
Human Immune

Abstract Introduction: Immunotherapy is a promising therapeutic intervention for cancer treatment. Activation of the immune system via checkpoint blockade has been shown to produce antitumor responses in patients with both solid and hematological tumors. However, many patients do not respond to checkpoint inhibitors, and additional therapies are needed to treat these patients. Testing immunotherapies requires a functional human immune system; thus, it is difficult to evaluate their effectiveness using conventional experimental models. For this reason, establishing in vivo models that closely reproduce not only human tumors, but also their interactions with the human immune system, has become mandatory. Methods: We developed a humanized mouse model and combined it with a patient-derived tumor xenograft (PDTX). Humanized mice (HuMice) were generated by transplantation of cord blood or mobilized peripheral blood CD34+ hematopoietic stem and progenitor cells into preconditioned immunodeficient mice. We compared human engraftment in 3 different mouse strains: NSG (NOD.Cg-Prkdc scidIl2rg tm1Wjl/SzJ), NSGS (NOD.Cg-Prkdc scidIl2rg tm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ) and NBSGW (NOD.Cg-Kit W-41J Tyr + Prkdc scid Il2rg tm1Wjl/ThomJ). Immune cell profiling and distribution was performed using flow cytometry and immunohistochemistry. The B cell receptor (BCR) repertoire was evaluated using an RNA-based NGS assay. To evaluate the maturation and functionality of T cells developing in HuMice we performed proliferation, degranulation and intracellular cytokine staining. Results: Two months after CD34+ cell transplantation, we observed high levels of human hematopoietic chimerism in all the 3 strains. NSGS mice supported high-level chimerism as early as 1 month after transplantation, with more than 25% of human CD45+ cells in the blood. In all mice the majority of human circulating leukocytes were CD19+ B cells. An early appearance of CD3+ human T cells was detected in NSGS mice as compared to the other strains. Notably, the T cell expansion correlated with a decrease in relative B cell abundance while the myeloid cell contribution to the graft remained steady. We documented the differentiation of CD4+ and CD8+ human T cells at a 2:1 ratio. The characterization of the T cell subsets revealed that the majority was represented by CD45RA-CCR7- effector memory cells in both the spleen and the blood of HuMice. Nevertheless, recipient mice did not exhibit overt signs of graft-versus-host disease. We also evaluated the cytotoxic potential of T cells isolated from the spleen of HuMice: ex vivo peptide antigen (i.e. EBV) presentation let to generation of effective and specific cytotoxic T-cells. After assessing a functional human immune system reconstitution in HuMice, we challenged them in vivo with low-passage tumor fragments from a diffuse large B cell lymphoma (DLBCL) PDTX. All tumor implants were successfully engrafted in both HuMice and non-humanized controls. Remarkably, all the 3 HuMice strains showed a significant reduction in the tumor volume and/or eradication compared to matched non-humanized controls. Flow cytometry analysis of the peripheral blood of humanized PDTX revealed that the tumor engraftment elicited a significant expansion of CD3+ T cells and cytotoxic CD8+ lymphocytes. Moreover, tumors developing in HuMice exhibited intermediate to high levels of tumor infiltrating T lymphocytes commingling with the neoplastic B cells, as determined by immunohistochemistry. Large areas of necrosis were often observed in PDTX of HuMice. Infiltrating CD3+ cells were TIGIT, PD-1 and Lag-3 positive, and did not efficiently proliferate ex vivo: all features consistent with an exhaustion phenotype. PDTX of HuMice often displayed larger areas of necrosis. Conclusions: Collectively, our data demonstrate that a robust reconstitution can be achieved in different strains of immunocompromised mice and that HuMice elicit effective anti-lymphoma responses. PDTX HuMice represent a powerful platform to study host-tumor interactions, and to test novel immune-based strategies (CAR-T, bifunctional Abs) and new pharmacological approaches to counteract T-cell exhaustion. Figure 1 Figure 1. Disclosures Scandura: Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Constellation: Research Funding; MPN-RF (Foundation): Research Funding; CR&T (Foudation): Research Funding; European Leukemia net: Honoraria, Other: travel fees . Roth: Janssen: Consultancy; Merck: Consultancy.

scholarly journals A Human-Immune-System (HIS) humanized mouse model for COVID-19 research (DRAGA: HLA-A2.HLA-DR4.Rag1KO.IL-2Rγc KO.NOD)

2020 ◽  
Author(s):  
Teodor-Doru Brumeanu ◽  
Pooja Vir ◽  
Ahmad Faisal Karim ◽  
Swagata Kar ◽  
Kevin K. Chung ◽  
...  
Keyword(s):  
Immune System ◽  
T Cell ◽  
Mouse Model ◽  
Human Model ◽  
Human Immune System ◽  
Humanized Mouse ◽  
Humanized Mouse Model ◽  
Hematopoietic Stem ◽  
Human Immune

We report the first Human Immune System (HIS)-humanized mouse model (DRAGA: HLA-A2.HLA-DR4.Rag1KO.IL-2RgammacKO.NOD) for SARS-CoV-2 infection and COVID-19 research. This mouse is reconstituted with HLA-matched human hematopoietic stem cells from cord blood, thereby avoiding use of fetal tissue. It engrafts human epi/endothelial cells expressing the human ACE2 receptor for SARS-CoV-2 and the TMPRSS2 serine protease, which co-localize on lung epithelia. HIS-DRAGA mice sustained SARS-CoV-2 infection, showing abrupt weight loss, ruffed fur, hunched back and reduced mobility. Infected mice developed human-like lung immunopathology including T-cell infiltrates, microthrombi, hemorrhage, and pulmonary sequelae. Among T-cell infiltrates, lung-resident (CD103+)CD8+ T cells were sequestered in epithelial (CD326+) lung niches and secreted granzyme B and perforin, indicating cytotoxic potential. Infected mice developed antibodies against the SARS-CoV-2 S protein. Hence, HIS-DRAGA mice show unique advantages as a surrogate in vivo human model for studying SARS-CoV-2 immunopathology and for testing the safety and efficacy of candidate vaccines and therapeutics.

scholarly journals Modeling human tumor-immune environments in vivo for the preclinical assessment of immunotherapies

2021 ◽  
Author(s):  
Bethany Bareham ◽  
Nikitas Georgakopoulos ◽  
Alba Matas-Céspedes ◽  
Michelle Curran ◽  
Kourosh Saeb-Parsy
Keyword(s):  
Immune Cells ◽  
Human Tumor ◽  
Preclinical Model ◽  
Murine Models ◽  
Immunocompetent Mouse ◽  
Human Immune System ◽  
Human Immune ◽  
Immunocompromised Mice ◽  
Cancer Immunotherapies

AbstractDespite the significant contributions of immunocompetent mouse models to the development and assessment of cancer immunotherapies, they inadequately represent the genetic and biological complexity of corresponding human cancers. Immunocompromised mice reconstituted with a human immune system (HIS) and engrafted with patient-derived tumor xenografts are a promising novel preclinical model for the study of human tumor-immune interactions. Whilst overcoming limitations of immunocompetent models, HIS-tumor models often rely on reconstitution with allogeneic immune cells, making it difficult to distinguish between anti-tumor and alloantigen responses. Models that comprise of autologous human tumor and human immune cells provide a platform that is more representative of the patient immune-tumor interaction. However, limited access to autologous tissues, short experimental windows, and poor retention of tumor microenvironment and tumor infiltrating lymphocyte components are major challenges affecting the establishment and application of autologous models. This review outlines existing preclinical murine models for the study of immuno-oncology, and highlights innovations that can be applied to improve the feasibility and efficacy of autologous models.

Transient accumulation of human mature thymocytes and regulatory T cells with CD28 superagonist in “human immune system” Rag2-/-γc-/- mice

Blood ◽  
2006 ◽  
Vol 108 (1) ◽  
pp. 238-245 ◽  
Author(s):  
Nicolas Legrand ◽  
Tom Cupedo ◽  
Anja U. van Lent ◽  
Menno J. Ebeli ◽  
Kees Weijer ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
T Cell ◽  
Opportunistic Infections ◽  
Human Immune System ◽  
Independent Manner ◽  
Human T Cell ◽  
Human Immune

Efficient and quick reconstitution of T-cell compartments in lymphopenic patients is of great importance to prevent opportunistic infections, but remains difficult to achieve. Human T-cell proliferation in a T-cell-receptor (TCR)-independent manner is possible in vitro with superagonist anti-CD28 antibodies, and such molecules are therefore promising therapeutic tools. Here, we investigated the in vivo effects of superagonist anti-CD28 treatment on human developing and mature T cells, in the recently developed model of “human immune system” BALB/c Rag2-/-γc-/- mice. Our results show that superagonist anti-CD28 treatment transiently induces a 7-fold increase in thymocyte numbers and up to 18-fold accumulation of mature thymocytes. The increased thymic production lead to transient accumulation of mature T cells in the periphery at the peak of treatment effect (day 6). In addition, long-term peripheral T-cell depletion was induced. Furthermore, the concomitant selective expansion and accumulation of suppressive CD4+CD25+FoxP3+ T cells was induced in a transient manner. Superagonist anti-CD28 therapy could therefore be of clinical interest in humans, both for beneficial effect on thymic T-cell production as well as regulatory T-cell accumulation. (Blood. 2006;108:238-245)

Neonatal NOD/SCID/IL2rg-Null Mice Support the Functional Development of Human Hematopoietic and Immune Systems.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2781-2781 ◽  
Author(s):  
Fumihiko Ishikawa ◽  
Masaki Yasukawa ◽  
Bonnie Lyons ◽  
Shuro Yoshida ◽  
Leonard D. Shultz ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
B Cells ◽  
Peripheral Blood ◽  
Human Immune System ◽  
Immune Systems ◽  
New Model ◽  
Cd34 Cells ◽  
Human Immune

Abstract (Purpose) We aimed to develop a new model for studying the development of a human hematopoietic and immune systems in vivo. (Methods) In order to establish a new model of xenogeneic transplantation, we establish an immune-compromised strain, NOD.Cg-PrkdcscidIL2rgtmlWjl/Sz (NOD/SCID/IL2rg-null) mice by backcrossing a complete null mutation of the IL2 receptor common gamma chain (IL2rg) onto the NOD/SCID background. 1 x 105 human CB-derived lineage antigen negative (Lin−) CD34+ cells were intravenously transplanted into newborn NOD/SCID/IL2rg-null mice following 100cGy irradiation. At 3 months post-transplantation, the engraftment of human cells was evaluated by flow cytometric analysis, immunostaining, and functional assays for production of human immunoglobulin and T-cell cytotoxicity against allogeneic cells. (Results) NOD/SCID/IL2rg-null mice showed extremely low activity of NK cells along with the complete lack of mature B cells and T cells. During post-natal development of the NOD/SCID/IL2rg-null mice, a human hematopoietic system was developed following injection of human CB-derived Lin-CD34+ cells. In BM of the recipient mice, human glycophorin A+ erythroid cells were present at 9.5 +/− 6.2% (n=5), and human CD41+ megakaryocytes were present at 1.64 +/− 0.42% (n=5). Human CB-derived Lin−CD34+ cells generated multi-lineage leukocytes, CD33+ myeloid cells, CD19+ B cells, and CD3+ T cells. The engraftment level of human CD45+ cells in peripheral blood was significantly higher (68.9 +/− 11.6%, n=5) in NOD/SCID/IL2rg-null mice than that in NOD/SCID/b2mnull mice (12.4 +/− 5.9%, n=4). Mature erythrocytes and platelets were identified in peripheral blood. The xenogeneic environment supported the systemic development of a human immune system, containing each stage of B cells and T cells in primary and secondary lymphoid tissues. CD34+CD19+ pro-B cells, CD10+CD19+ B cells, and CD19+CD20hi mature B cells were identified in the BM and spleen. Immature CD4+CD8+ double positive T cells were the major cell populations in the thymus, while spleen contained abundant single positive T cells at 1.39 +/− 0.61 (n=5) CD4/CD8 ratio, suggesting that human CB stem/progenitor-derived T cells underwent the maturation and proliferation similarly as identified in human body. Transplanted human stem cells reconstituted mucosal immunity in intestinal tracts as evidenced by human IgA+ B cells and CD3+ T cells. Adaptive human immune system cooperatively functioned in xenogeneic environment to produce antigen-specific human IgM and IgG antibodies, when engrafted mice were immunized with ovalbumin. Furthermore, human CD4+ T cells as well as CD8+ T cells generated in the xenogeneic host exerted cytotoxicity against allogeneic target cells. (Conclusion) The neonatal NOD/SCID/IL2rg-null model will facilitate studying post-natal development of the human hematopoietic and immune systems and for studying of human immune surveillance in vivo against exogenous antigens.

327 B7-H3 Chimeric Antigen Receptor Modified T Cells Show Potent Anti-Tumor Activity in a Preclinical Model of Glioblastoma

Neurosurgery ◽  
2017 ◽  
Vol 64 (CN_suppl_1) ◽  
pp. 272-272
Author(s):  
Kwong-Hon (Kevin) Chow ◽  
Sabine Heitzeneder ◽  
Peng Xu ◽  
Johanna Theruvath ◽  
Siddhartha S Mitra ◽  
...  
Keyword(s):  
T Cells ◽  
Mouse Model ◽  
Glioma Cell ◽  
Preclinical Model ◽  
Car T Cells ◽  
Car T ◽  
Glioma Cell Lines ◽  
Anti Tumor Activity

Abstract INTRODUCTION While initial phase I data suggest efficacy of local delivery of chimeric antigen receptor (CAR) modified T cells against glioblastoma (GBM), their activity remains limited in part by the intensity of antigen expression. Targeting more robust tumor associated antigens (TAAs) may help to improve anti-tumor responses. B7-H3 (CD276), a transmembrane glycoprotein which is overexpressed on many solid cancers including GBM, is a promising target. Here we generate CAR T cells specific for B7-H3 and characterize their function in a preclinical model of glioblastoma. METHODS B7-H3 CAR T cells were generated by retroviral transduction of healthy donor peripheral blood mononuclear cells (PBMCs) using a vector designed by our lab. The CAR modified T cells were tested in vitro for their ability to produce proinflammatory cytokines and kill B7-H3 positive glioma cell lines. In vivo activity of B7-H3 CAR T cells was tested using an orthotopic GBM xenograft mouse model. RESULTS >B7-H3 CAR T cells produced the proinflammatory cytokines interferon-gamma (IFN-g), interleukin-2 (IL-2), and tumor necrosis factor-alpha (TNF-a) when cocultured with B7-H3 positive glioma cell lines. B7-H3 CAR T cells also killed B7-H3 positive glioma cells in an in vitro cytotoxicity assay. Finally, B7-H3 CAR T cells demonstrated potent anti-tumor activity in vivo, producing tumor regression in our mouse model of GBM and significantly improving survival. CONCLUSION B7-H3 CAR T cells effectively target GBM and demonstrate significant anti-tumor activity in our preclinical studies. Efforts to translate this CAR for clinical use are warranted and will add to the armamentarium for treating patients with GBM and other solid cancers.

721 AT1412, a patient-derived CD9 antibody in preclinical development promoting tumor immune infiltration and inducing tumor rejection

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A763-A763
Author(s):  
Remko Schotte ◽  
Julien Villaudy ◽  
Martijn Kedde ◽  
Wouter Pos ◽  
Daniel Go ◽  
...  
Keyword(s):  
T Cells ◽  
Immune System ◽  
T Cell ◽  
Tumor Cells ◽  
Tumor Rejection ◽  
Human Immune System ◽  
Preclinical Development ◽  
High Affinity ◽  
Human Immune ◽  
A375 Melanoma

BackgroundAdaptive immunity to cancer cells forms a crucial part of cancer immunotherapy. Recently, the importance of tumor B-cell signatures were shown to correlate with melanoma survival. We investigated whether tumor-targeting antibodies could be isolated from a patient that cured (now 13 years tumor-free) metastatic melanoma following adoptive transfer of ex vivo expanded autologous T cells.MethodsPatient‘s peripheral blood B cells were isolated and tested for the presence of tumor-reactive B cells using AIMM’s immmortalisation technology. Antibody AT1412 was identified by virtue of its differential binding to melanoma cells as compared to healthy melanocytes. AT1412 binds the tetraspanin CD9, a broadly expressed protein involved in multiple cellular activities in cancer and induces ADCC and ADCP by effector cells.ResultsSpontaneous immune rejection of tumors was observed in human immune system (HIS) mouse models implanted with CD9 genetically-disrupted A375 melanoma (A375-CD9KO) tumor cells, while A375wt cells were not cleared. Most notably, no tumor rejection of A375-CD9KO tumors was observed in NSG mice, indicating that blockade of CD9 makes tumor cells susceptible to immune rejection.CD9 has been described to regulate integrin signaling, e.g. LFA-1, VLA-4, VCAM-1 and ICAM-1. AT1412 was shown to modulate CD9 function by enhancing adhesion and transmigration of T cells to endothelial (HUVEC) cells. AT1412 was most potently enhancing transendothelial T-cell migration, in contrast to a high affinity version of AT1412 or other high affinity anti-CD9 reference antibodies (e.g. ALB6). Enhanced immune cell infiltration is also observed in immunodeficient mice harbouring a human immune system (HIS). AT1412 strongly enhanced CD8 T-cell and macrophage infiltration resulting in tumor rejection (A375 melanoma). PD-1 checkpoint blockade is further sustaining this effect. In a second melanoma model carrying a PD-1 resistant and highly aggressive tumor (SK-MEL5) AT1412 together with nivolumab was inducing full tumor rejection, while either one of the antibodies alone did not.ConclusionsThe safety of AT1412 has been assessed in preclinical development and is well tolerated up to 10 mg/kg (highest dose tested) by non human primates. AT1412 demonstrated a half-life of 8.5 days, supporting 2–3 weekly administration in humans. Besides transient thrombocytopenia no other pathological deviations were observed. No effect on coagulation parameters, bruising or bleeding were observed macro- or microscopically. The thrombocytopenia is reversible, and its recovery accelerated in those animals developing anti-drug antibodies. First in Human clinical study is planned to start early 2021.Ethics ApprovalStudy protocols were approved by the Medical Ethical Committee of the Leiden University Medical Center (Leiden, Netherlands).ConsentBlood was obtained after written informed consent by the patient.

scholarly journals A Preclinical Embryonic Zebrafish Xenograft Model to Investigate CAR T Cells in Vivo

Cancers ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 567 ◽  
Author(s):  
Susana Pascoal ◽  
Benjamin Salzer ◽  
Eva Scheuringer ◽  
Andrea Wenninger-Weinzierl ◽  
Caterina Sturtzel ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cellular Therapy ◽  
Xenograft Model ◽  
Model Systems ◽  
Preclinical Model ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Chimeric antigen receptor (CAR) T cells have proven to be a powerful cellular therapy for B cell malignancies. Massive efforts are now being undertaken to reproduce the high efficacy of CAR T cells in the treatment of other malignancies. Here, predictive preclinical model systems are important, and the current gold standard for preclinical evaluation of CAR T cells are mouse xenografts. However, mouse xenograft assays are expensive and slow. Therefore, an additional vertebrate in vivo assay would be beneficial to bridge the gap from in vitro to mouse xenografts. Here, we present a novel assay based on embryonic zebrafish xenografts to investigate CAR T cell-mediated killing of human cancer cells. Using a CD19-specific CAR and Nalm-6 leukemia cells, we show that live observation of killing of Nalm-6 cells by CAR T cells is possible in zebrafish embryos. Furthermore, we applied Fiji macros enabling automated quantification of Nalm-6 cells and CAR T cells over time. In conclusion, we provide a proof-of-principle study that embryonic zebrafish xenografts can be used to investigate CAR T cell-mediated killing of tumor cells. This assay is cost-effective, fast, and offers live imaging possibilities to directly investigate CAR T cell migration, engagement, and killing of effector cells.

scholarly journals Immunoinformatic design of a COVID-19 subunit vaccine using entire structural immunogenic epitopes of SARS-CoV-2

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Esmaeil Behmard ◽  
Bijan Soleymani ◽  
Ali Najafi ◽  
Ebrahim Barzegari
Keyword(s):  
A Priori ◽  
3D Structure ◽  
Free Energy Calculations ◽  
Structural Proteins ◽  
Toll Like Receptor ◽  
Cell Binding ◽  
Human Immune System ◽  
Human Immune ◽  
Dynamics Simulations

AbstractCoronavirus disease 2019 (COVID-19) is an acute pneumonic disease, with no prophylactic or specific therapeutical solution. Effective and rapid countermeasure against the spread of the disease’s associated virus, SARS-CoV-2, requires to incorporate the computational approach. In this study, we employed various immunoinformatics tools to design a multi-epitope vaccine polypeptide with the highest potential for activating the human immune system against SARS-CoV-2. The initial epitope set was extracted from the whole set of viral structural proteins. Potential non-toxic and non-allergenic T-cell and B-cell binding and cytokine inducing epitopes were then identified through a priori prediction. Selected epitopes were bound to each other with appropriate linkers, followed by appending a suitable adjuvant to increase the immunogenicity of the vaccine polypeptide. Molecular modelling of the 3D structure of the vaccine construct, docking, molecular dynamics simulations and free energy calculations confirmed that the vaccine peptide had high affinity for Toll-like receptor 3 binding, and that the vaccine-receptor complex was highly stable. As our vaccine polypeptide design captures the advantages of structural epitopes and simultaneously integrates precautions to avoid relevant side effects, it is suggested to be promising for elicitation of an effective and safe immune response against SARS-CoV-2 in vivo.

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