Sensitive In Vivo Detection of Primary T Cells Expressing Membrane-Anchored Gaussia Luciferase for the Study of Adoptive T Cell Immunotherapy in Murine Models of Malignancy.

Abstract Current approaches to in vivo bioluminescent imaging (BLI) of T cells utilizing luciferase enzymes may be compromised by poor transduction efficiencies in primary T cells, and low photon emissions. Here we describe a novel and widely applicable approach to in vivo BLI of primary T cells utilizing a membrane-anchored form of the naturally secreted Guassia luciferase (GLuc) enzyme, termed exGLuc. We constructed exGLuc by fusion of the GLuc gene to the coding region of the CD8 transmembrane domain. The resulting exGLuc enzyme is anchored to the cell surface of retrovirally transduced cells. In vitro, cells which expressed the exGLuc enzyme demonstrated a markedly (>9 fold) increased bioluminescent signal when compared to cells which expressed the native GLuc (GLuc), the related Rhenilla luciferase (RLuc), a membrane-anchored form of RLuc (exRLuc), or a green fluorescent protein (GFP)-fire fly luciferase fusion protein. Following injection into SCID-Beige mice, MHC-mismatched C57BL6 T cells transduced to express exGLuc were detected by BLI generating graft versus host disease at an earlier time point and demonstrated a >10 fold increased bioluminescent signal when compared to infused C57BL6 T cells expressing GLuc, RLuc, or exRLuc. We further demonstrate homing of OVA-specific DO11.10 exGLuc+ T cells to A20(OVA) but not A20(GFP) subcutaneous tumors in both SCID-Beige and Balb/c mice. We further successfully applied this strategy to the study of in vivo human T cell homing using a xenogeneic SCID-Beige tumor model. Specifically, we demonstrate that human T cells, retrovirally co-transduced to express both a CD19-specific chimeric antigen receptor (CAR), 19z1, and exGLuc, could be monitored over time, and that these T cells quite rapidly home to subcutaneous CD19+ acute lymphoblastic leukemia (NALM-6) tumors when compared to T cells co-expressing an irrelevant CAR with exGLuc. Furthermore, we were able to demonstrate for the first time specific homing of 19z1/exGLuc+ human T cells to systemic NALM-6 tumor, detecting exGLuc+ T cells in deep tissues including the bone marrow, spleen, and liver. We conclude that the exGLuc enzyme emits a superior bioluminescent signal when compared to other commonly utilized luciferase enzymes; that primary T cells are readily transduced to express the exGLuc enzyme; and that exGLuc+ T cells may be accurately monitored in vivo over time by BLI. Based on these data, we believe that this novel approach to primary T cell in vivo imaging will significantly enhance the study of adoptive T cell immunotherapy in murine models of leukemia and other malignancies.

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Toward Development and Production of Human T Cells in Swine for Potential Use in Adoptive T Cell Immunotherapy

Tissue Engineering Part A ◽

10.1089/ten.tea.2008.0117 ◽

2009 ◽

Vol 15 (5) ◽

pp. 1031-1040 ◽

Cited By ~ 11

Author(s):

Brenda M. Ogle ◽

Bruce E. Knudsen ◽

Ryuta Nishitai ◽

Kiyoshi Ogata ◽

Jeffrey L. Platt

Keyword(s):

T Cells ◽

T Cell ◽

Cell Immunotherapy ◽

Human T Cells ◽

Potential Use ◽

T Cell Immunotherapy

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Phase I Study of Adoptive Immunotherapy with HA-1-Specific CD8+ and CD4+ Memory T Cells for Children and Adults with Relapsed Acute Leukemia after Allogeneic Hematopoietic Stem Cell Transplantation (HCT): Trial in Progress

Blood ◽

10.1182/blood-2020-137726 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 45-46 ◽

Cited By ~ 1

Author(s):

Elizabeth F Krakow ◽

Corinne Summers ◽

Ann Dahlberg ◽

Merav Bar ◽

Melinda Ann Biernacki ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Phase I ◽

Research Funding ◽

Phase I Trial ◽

Memory T Cells ◽

Cell Immunotherapy ◽

T Cell Immunotherapy

Background: Donor T cells specific for minor histocompatibility (H) antigens can deliver potent, selective anti-leukemic effects after allogeneic HCT when the antigen is negligibly or not expressed by non-hematopoietic tissues, not present in the donor, and expressed by the recipient. We reported a new minor H antigen-directed T-cell therapy that can be deployed after HCT to manage persistent or recurrent measurable residual hematologic malignancies or overt relapse (Blood 2018;131(1):108). We developed a transgene with 4 components: 1) a high-affinity T-cell receptor (TCR) specific for the hematopoietic-restricted minor H antigen, HA-1 that is presented on HLA-A*02:01; 2) a CD8 co-receptor to enhance function of the class I-restricted TCR in CD4+ T cells so they promote cytotoxic CD8+ T cell function and survival; 3) an inducible caspase-9 safety switch, which can be triggered by the drug rimiducid in case of in vivo toxicity; and 4) a CD34-CD20 epitope to facilitate selection of the engineered product during manufacturing and track HA-1 TCR T cells in the recipient. The 21-day manufacturing process entails CD45RA+ naïve T cell depletion (minimizes the risk of GvHD), and subsequent CD4+ and CD8+ separation (provides a consistent 1:1 CD4:CD8 ratio). The separate cultures are transduced with the lentivirus construct iCasp9-HA1-TCR2-RQR-CD8, expanded, and selected using the CD34 marker to ensure removal of untransduced T cells. Study Design and Methods: The single-center phase I trial (NCT03326921) evaluates the feasibility and safety of infusion of HA-1 TCR T-cell immunotherapy. Primary end points are 1) Feasibility of manufacturing and administering HA-1 TCR CD8+ and CD4+ memory T cells and 2) Dose-limiting toxicity of HA-1 TCR T cells. Major inclusion criteria are: HLA-A*02:01-positive, HA-1-positive patients who underwent HCT for acute leukemia, myelodysplastic syndrome, BPDCN, CML, CMML or JMML from a HLA-A*-02:01+/HA-1-negative donor or HLA-A*02:01-negative haploidentical or mismatched donor (excluding umbilical cord). HA-1 genotype screening is performed on patient and donor blood, hair follicle or cheek swab samples shipped to Fred Hutchinson Cancer Research Center. To be eligible for treatment, patients must develop measurable residual disease or overt relapse after HCT but may receive other standard or investigational therapies prior to treatment with HA-1 TCR T-cell immunotherapy if clinically indicated. Some systemic immunosuppression may be continued, but prior grade IV acute GVHD and prior severe chronic GVHD are key exclusions. Two groups, <16 and ≥16 years, will be treated at dose levels ranging from 3 x 106 to30 x 106 cells/kg, in cohorts of 3-6 subjects, up to approximately 24 subjects in total. Fludarabine lymphodepletion will be used in most subjects, followed by a single T-cell infusion, with an option for a subsequent infusion(s) if the subject demonstrates an initial response without severe toxicity. Bone marrow aspirations are performed prior to T-cell infusion and at several time points following infusion. Recruitment and Patient Characteristics: To date, 3 subjects have been treated on the phase I clinical trial and received a total of 5 infusions (Table 1). HA-1 TCR T cell persistence in blood and bone marrow has been documented from >3 months to >13 months. Clear in vivo anti-leukemic activity was observed at the first dose level, including in a subject with aggressive, highly refractory T-ALL and early post-HCT relapse. Outlook: Minor H antigen-specific T-cell immunotherapy may offer effective management of post-HCT relapse while avoiding GvHD and other off-target effects. Due to population genetics of HA-1 and HLA-A*02:01, HA-1 TCR T-cell immunotherapy is applicable to 10-15% of HCT recipients with various hematological malignancies. The ongoing phase I trial is actively recruiting patients. Development of T-cell immunotherapy targeting other minor H antigen/HLA combinations is also underway to increase the broad applicability of minor H antigen-targeted T-cell immunotherapy. Disclosures Krakow: HighPass Bio: Research Funding. Cunningham:HighPass Bio: Research Funding. Vartanian:HighPass Bio: Research Funding. Bleakley:HighPass Bio: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

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Enhancement of the in vivo persistence and antitumor efficacy of CD19 chimeric antigen receptor T cells through the delivery of modified TERT mRNA

Cell Discovery ◽

10.1038/celldisc.2015.40 ◽

2015 ◽

Vol 1 (1) ◽

Cited By ~ 20

Author(s):

Yun Bai ◽

Shifeng Kan ◽

Shixin Zhou ◽

Yuting Wang ◽

Jun Xu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Reverse Transcriptase ◽

Chimeric Antigen Receptor ◽

Antigen Receptor ◽

Telomerase Reverse Transcriptase ◽

Cell Immunotherapy ◽

T Cell Immunotherapy

Abstract Chimeric antigen receptor T cell immunotherapy is a promising therapeutic strategy for treating tumors, demonstrating its efficiency in eliminating several hematological malignancies in recent years. However, a major obstacle associated with current chimeric antigen receptor T cell immunotherapy is that the limited replicative lifespan of chimeric antigen receptor T cells prohibits the long-term persistence and expansion of these cells in vivo, potentially hindering the long-term therapeutic effects of chimeric antigen receptor T cell immunotherapy. Here we showed that the transient delivery of modified mRNA encoding telomerase reverse transcriptase to human chimeric antigen receptor T cells targeting the CD19 antigen (CD19 chimeric antigen receptor T cells) would transiently elevate the telomerase activity in these cells, leading to increased proliferation and delayed replicative senescence without risk of insertion mutagenesis or immortalization. Importantly, compared to conventional CD19 chimeric antigen receptor T cells, after the transient delivery of telomerase reverse transcriptase mRNA, these CD19 chimeric antigen receptor T cells showed improved persistence and proliferation in mouse xenograft tumor models of human B-cell malignancies. Furthermore, the transfer of CD19 chimeric antigen receptor T cells after the transient delivery of telomerase reverse transcriptase mRNA enhanced long-term antitumor effects in mouse xenograft tumor models compared with conventional CD19 chimeric antigen receptor T cell transfer. The results of the present study provide an effective and safe method to improve the therapeutic potential of chimeric antigen receptor T cells, which might be beneficial for treating other types of cancer, particularly solid tumors.

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Three-Compartment Model of CAR T-cell Immunotherapy

10.1101/779793 ◽

2019 ◽

Author(s):

Brendon de Jesus Rodrigues ◽

Luciana R. Carvalho Barros ◽

Regina C. Almeida

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

Tumor Burden ◽

Long Term Memory ◽

Car T Cell ◽

Cell Immunotherapy ◽

Car T ◽

T Cell Immunotherapy

AbstractImmunotherapy has gained great momentum with chimeric antigen receptor T-cell (CAR T) therapy, in which patient’s T lymphocytes are genetically manipulated to recognize tumor-specific antigens to increase tumor elimination efficiency. Improved CAR T cell immunotherapy requires a better understanding of the interplay between CAR T cell doses and tumor burden, administration protocol, toxicity, resistance to immunotherapy, among other features. We developed a three-compartment mathematical model to describe tumor response to CAR T cell immunotherapy in immunodeficient mouse models. It encompasses interactions between tumor cells, effector and long-term memory CAR T cells such as tumor induced immunosuppression effects, conversion of memory T cells into effector T cells in the presence of tumor cells, and individual specificities considered as uncertainties in the parameters of the model. The model was able to represent two different immunotherapy scenarios with different CAR receptors and tumor targets reported in the literature. Further in silico studies considering different dosing quantities and tumor burden showed that the proposed model can represent the three possible therapy outcomes: tumor elimination, equilibrium, and escape. We found that therapy effectiveness may also depend on small variations in the parameter values, regarded as intrinsic individual specificities, as T cell proliferation capacity, as well as immunosuppressive tumor microenvironment factors. These issues may significantly reduce the chance of tumor elimination. In this way, the developed model provides potential use for assessing different CAR T cell protocols and associated efficacy without further in vivo experiments.

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Truncated Cell-Surface CD19 As a Conditional Suicide Switch For Adoptive T Cell Immunotherapy

Blood ◽

10.1182/blood.v122.21.1660.1660 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1660-1660 ◽

Cited By ~ 1

Author(s):

Lihua Elizabeth Budde ◽

Armen Mardiros ◽

Wen-Chung Chang ◽

Xiuli Wang ◽

Carolina Berger ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Surface ◽

Intracellular Signaling ◽

Flow Cytometric Analysis ◽

Dependent Manner ◽

Cell Immunotherapy ◽

T Cell Immunotherapy

Background Modification of T cells with chimeric antigen receptors (CAR) has emerged as a promising treatment modality for human malignancies. However, the potential for insertional mutagenesis and toxicities due to the infused cells have made development of safe Methods for removing transferred cells after treatment an important consideration. In addition, there is a lack of effective commercially available agents which allow for monitoring of CAR expression, tracking, isolating, and eliminating CAR- transduced cells. Therefore, adoptive T cell immunotherapy would benefit from a molecule which is stably expressed on the cell surface, of human origin, easily detected on transduced cells, lacking active biological function at baseline and capable of effectively ablating transduced cells on demand. Truncated CD19 (CD19t) harbors excellent features to be such a molecule. Its truncation shortens the intracytoplasmic domain to only 19 amino acids with removal of all conserved tyrosine residues that mediate known intracellular signaling transduction. It has been used successfully to mark transduced CAR T cells by several research groups. In this study, we set out to evaluate the activity of this truncated CD19 as a conditional suicide switch. Methods Lentiviral constructs containing a CD20 CAR and CD19t were used to transduce Jurkat T cells and primary human T cells to generate cells that express both molecules on the cell surface. CD19-mediated selection was carried out using PE conjugated anti-CD19 antibody. Internalization experiments were performed using transduced Jurkat cells that were kept at 4°C or 37°C. Surface CD19 expression was determined by flow cytometric analysis at 0 hour, 1 hour, 2 hours, and 4 hours after initial primary anti-CD19 antibody staining. NIH3T3 cells with truncated CD19 expressed on the surface (NIH3T3-19t) were generated and used for in vitro ablation experiments. Cells were left untreated or incubated withincreasing concentrations of CD19-ETA’, an anti-CD19 Pseudomonas toxin conjugate. The viability of NIH3T3-CD19t was determined by trypan blue exclusion at various time points. Results Using flow cytometry, we confirmed the expression of CAR and truncated CD19 on the transduced cell surface. Truncated CD19 was able to enrich transduced cells to more than 90% purity when used as a selectable marker. For CD19t to function as a conditional suicide switch, it needs to retain its ability to mediate antigen-antibody conjugate internalization. As expected, only up to 10% of CD19t remained on the surface of transduced cells after 4-hour period at 37°C. On the contrary, surface CD19t expression level remained largely unchanged when the cells were incubated at 4°C for 4 hours. CD19t also mediated robust ablation of transduced cells in vitro. More than 90% of transduced cells were ablated after 72-hour incubation with 10ug/ml CD19-ETA’. Mouse xenograft experiments are currently ongoing to test the in vivo tracking ability of CD19t and its ablation effect of transferred T cells upon administration of anti-CD19-drug conjugates. Conclusions These in vitro Results suggest that CD19t retains the ability to mediate antibody internalization upon its engagement. When exposed to an anti-CD19-drug conjugate, cells expressing truncated CD19 are effectively ablated in a dose dependent manner. We therefore predict that CD19t will be an excellent molecule to mark, select, track and eliminate modified T cells in vivo and it will be a useful tool for detection of engineered T cells and improvement of the safety of adoptive T cell immunotherapy. Disclosures: No relevant conflicts of interest to declare.

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Thermal Control of T-cell Immunotherapy

10.1101/2020.04.16.045146 ◽

2020 ◽

Author(s):

Mohamad H. Abedi ◽

Justin Lee ◽

Dan I. Piraner ◽

Mikhail G. Shapiro

Keyword(s):

T Cells ◽

T Cell ◽

Focused Ultrasound ◽

Thermal Control ◽

Genetically Engineered ◽

The Body ◽

External Control ◽

Cell Immunotherapy ◽

T Cell Immunotherapy

ABSTRACTGenetically engineered T-cells are being developed to perform a variety of therapeutic functions. However, no robust mechanisms exist to externally control the activity of T-cells at specific locations within the body. Such spatiotemporal control could help mitigate potential off-target toxicity due to incomplete molecular specificity in applications such as T-cell immunotherapy against solid tumors. Temperature is a versatile external control signal that can be delivered to target tissues in vivo using techniques such as focused ultrasound and magnetic hyperthermia. Here, we test the ability of heat shock promoters to mediate thermal actuation of genetic circuits in primary human T-cells in the well-tolerated temperature range of 37–42°C, and introduce genetic architectures enabling the tuning of the amplitude and duration of thermal activation. We demonstrate the use of these circuits to control the expression of chimeric antigen receptors and cytokines, and the killing of target tumor cells. This technology provides a critical tool to direct the activity of T-cells after they are deployed inside the body.

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Human Orthogonal IL-2/IL-2Rβ As a Tunable Approach to Enhance CD19-Speific CAR-T Cell Antitumor Activity

Blood ◽

10.1182/blood-2020-140463 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 48-48

Author(s):

Qian Zhang ◽

Morgan Hresko ◽

Michael Jacob Hollander ◽

Lora Picton ◽

Leon Su ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Adoptive Transfer ◽

Cell Expansion ◽

Private Company ◽

Car T Cell ◽

Cell Immunotherapy ◽

Car T ◽

T Cell Immunotherapy

Immunotherapy using T cells engineered with a chimeric antigen receptors (CAR) can produce deep and durable control of B-cell malignancies that is associated with the engraftment and persistence of the CAR T cells following adoptive transfer. Interleukin-2 (IL-2) is a central T cell cytokine that promotes T cell proliferation and effector function with a potential to enhance CAR T cell immunotherapy; however, the use of recombinant IL-2 to support T cell immunotherapy is limited by the significant toxicity associated with IL-2 infusion. Previous work has demonstrated the feasibility of engineering murine IL-2 and it's receptor to create an orthogonal (ortho) cytokine-receptor pair capable of delivering IL-2's signal without the associated toxicity (Sockolosky JT et al. Science 2018). In order to translate this novel approach to humans, we engineered a human orthogonal IL-2 (ortho-hIL-2) and human orthogonal IL-2Rβ (ortho-hIL-2Rβ) pair based upon similar mutations to those required in the murine system. We show that hoIL-2 induces JAK-STAT signaling and supports proliferation of the IL-2 dependent cutaneous T-cell lymphoma (CTCL) line, SeAx, and primary human CD8+ primary T cells when hoIL-2Rb is expressed in these cells. HoIL-2 at concentrations 1000-fold higher than wild-type IL-2 failed to induce signaling in the absence of the orthogonal receptor illustrating the high selectivity of ortho-hIL-2 for the ortho-hIL-2Rβ. Using a CD19-specific, 4-1BB-costimulated CAR T cell (CART19), we show that ortho-hIL-2 induced a dose-dependent increase in ortho-hIL-2Rβ CAR-T cell expansion and serum cytokines in vivo by as much as 1000-fold at 2 weeks following adoptive transfer into NSG mice bearing CD19+ Nalm6 leukemia xenografts and daily ortho-hIL-2 treatment. We further demonstrate that hoIL-2 can rescue the anti-leukemic effect of an otherwise suboptimal CAR T cell dose. In addition to augmenting initial CAR T cell engraftment and efficacy, we also show that ortho-hIL-2 administration initiated at the time of leukemic relapse following CAR T cell therapy can rescue an otherwise failed anti-leukemic response. In aggregate, these data demonstrate the ability of the human orthogonal IL-2 cytokine-receptor system to support T cell expansion in vivo and the potential of combining this approach with CAR T cell immunotherapy to augment the anti-tumor efficacy of engineered T cells. Disclosures Sockolosky: Synthekine Therapeutics: Current equity holder in private company. Garcia:Synthekine: Current equity holder in private company, Other: founder. Milone:Novartis: Patents & Royalties.

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627 The DLL3-targeted half-life extended bispecific T cell engager (HLE BiTE®) immune-oncology therapy AMG 757 has potent antitumor activity in neuroendocrine cancer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0627 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A663-A663

Author(s):

Keegan Cooke ◽

Juan Estrada ◽

Jinghui Zhan ◽

Jonathan Werner ◽

Fei Lee ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Growth ◽

Mouse Models ◽

T Cell Activation ◽

Phase 1 ◽

Phase 1 Study ◽

Human T Cells

BackgroundNeuroendocrine tumors (NET), including small cell lung cancer (SCLC), have poor prognosis and limited therapeutic options. AMG 757 is an HLE BiTE® immune therapy designed to redirect T cell cytotoxicity to NET cells by binding to Delta-like ligand 3 (DLL3) expressed on the tumor cell surface and CD3 on T cells.MethodsWe evaluated activity of AMG 757 in NET cells in vitro and in mouse models of neuroendocrine cancer in vivo. In vitro, co-cultures of NET cells and human T cells were treated with AMG 757 in a concentration range and T cell activation, cytokine production, and tumor cell killing were assessed. In vivo, AMG 757 antitumor efficacy was evaluated in xenograft NET and in orthotopic models designed to mimic primary and metastatic SCLC lesions. NSG mice bearing established NET were administered human T cells and then treated once weekly with AMG 757 or control HLE BiTE molecule; tumor growth inhibition was assessed. Pharmacodynamic effects of AMG 757 in tumors were also evaluated in SCLC models following a single administration of human T cells and AMG 757 or control HLE BiTE molecule.ResultsAMG 757 induced T cell activation, cytokine production, and potent T cell redirected killing of DLL3-expressing SCLC, neuroendocrine prostate cancer, and other DLL3-expressing NET cell lines in vitro. AMG 757-mediated redirected lysis was specific for DLL3-expressing cells. In patient-derived xenograft and orthotopic models of SCLC, single-dose AMG 757 effectively engaged human T cells administered systemically, leading to a significant increase in the number of human CD4+ and CD8+ T cells in primary and metastatic tumor lesions. Weekly administration of AMG 757 induced significant tumor growth inhibition of SCLC (figure 1) and other NET, including complete regression of established tumors and clearance of metastatic lesions. These findings warranted evaluation of AMG 757 (NCT03319940); the phase 1 study includes dose exploration (monotherapy and in combination with pembrolizumab) and dose expansion (monotherapy) in patients with SCLC (figure 2). A study of AMG 757 in patients with neuroendocrine prostate cancer is under development based on emerging data from the ongoing phase 1 study.Abstract 627 Figure 1AMG 757 Significantly reduced tumor growth in orthotopic SCLC mouse modelsAbstract 627 Figure 2AMG 757 Phase 1 study designConclusionsAMG 757 engages and activates T cells to kill DLL3-expressing SCLC and other NET cells in vitro and induces significant antitumor activity against established xenograft tumors in mouse models. These preclinical data support evaluation of AMG 757 in clinical studies of patients with NET.Ethics ApprovalAll in vivo work was conducted under IACUC-approved protocol #2009-00046.

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CARTmath—A Mathematical Model of CAR-T Immunotherapy in Preclinical Studies of Hematological Cancers

Cancers ◽

10.3390/cancers13122941 ◽

2021 ◽

Vol 13 (12) ◽

pp. 2941

Author(s):

Luciana R. C. Barros ◽

Emanuelle A. Paixão ◽

Andrea M. P. Valli ◽

Gustavo T. Naozuka ◽

Artur C. Fassoni ◽

...

Keyword(s):

Mathematical Model ◽

T Cells ◽

T Cell ◽

Mouse Models ◽

In Silico ◽

Car T Cells ◽

Car T Cell ◽

Cell Immunotherapy ◽

Car T ◽

T Cell Immunotherapy

Immunotherapy has gained great momentum with chimeric antigen receptor T cell (CAR-T) therapy, in which patient’s T lymphocytes are genetically manipulated to recognize tumor-specific antigens, increasing tumor elimination efficiency. In recent years, CAR-T cell immunotherapy for hematological malignancies achieved a great response rate in patients and is a very promising therapy for several other malignancies. Each new CAR design requires a preclinical proof-of-concept experiment using immunodeficient mouse models. The absence of a functional immune system in these mice makes them simple and suitable for use as mathematical models. In this work, we develop a three-population mathematical model to describe tumor response to CAR-T cell immunotherapy in immunodeficient mouse models, encompassing interactions between a non-solid tumor and CAR-T cells (effector and long-term memory). We account for several phenomena, such as tumor-induced immunosuppression, memory pool formation, and conversion of memory into effector CAR-T cells in the presence of new tumor cells. Individual donor and tumor specificities are considered uncertainties in the model parameters. Our model is able to reproduce several CAR-T cell immunotherapy scenarios, with different CAR receptors and tumor targets reported in the literature. We found that therapy effectiveness mostly depends on specific parameters such as the differentiation of effector to memory CAR-T cells, CAR-T cytotoxic capacity, tumor growth rate, and tumor-induced immunosuppression. In summary, our model can contribute to reducing and optimizing the number of in vivo experiments with in silico tests to select specific scenarios that could be tested in experimental research. Such an in silico laboratory is an easy-to-run open-source simulator, built on a Shiny R-based platform called CARTmath. It contains the results of this manuscript as examples and documentation. The developed model together with the CARTmath platform have potential use in assessing different CAR-T cell immunotherapy protocols and its associated efficacy, becoming an accessory for in silico trials.

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