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Principal Investigator: Samuel Brill
Organization: COLORADO STATE UNIVERSITY
Fiscal Year: 2024
Award: $48,974
Funding agency: National Cancer Institute
Project Summary
This proposal aims to train a dual-degree, DVM-PhD student for a career as a lab animal veterinarian and independent
scientist. The research outlined in this proposal will develop a pre-clinical platform to evaluate CAR-T therapy in a canine
model. Chimeric antigen receptor (CAR)-T cells have induced up to 90% remission rates for treatment
relapsed/refractory B cell malignancies. While mice have been instrumental to CAR-T progress, CAR-T therapy for solid
tumors have been hampered by this inbred, immunodeficient model. Pet dogs are a higher fidelity translational model
due to their outbred genetics, intact immune system, high incidence of cancer, and similar cancer biology.
A CAR is a fusion protein comprised of a T cell receptor signaling domain, costimulatory domain, and an antibody based
binding domain. CARs are introduced to patients’ T cells ex vivo, enabling the T cells to directly recognize tumor antigen.
CAR-T cells are a “living therapy” wherein the efficacy of the treatment relies not only on the design of the CAR, but also
how the CAR-T cells are able to home to the tumor and elicit anti-tumor effects. The CAR helps T cells to “recognize” the
tumor, but the trafficking, persistence, and effector function of these cells relies heavily on intrinsic T cell biology. To
adequately assess CAR-T cell function in vivo, design of the CAR (Aim 1) and patient T cell biology (Aim 2) will be
evaluated.
Aim 1 – Determine optimal CAR design for targeting tumor associated antigens GD2, FolR1, and CD20. CAR constructs
will be designed for the tumor associated antigens GD2, FolR1, and CD20. These CARs will be introduced to primary
canine T cells via a lentiviral vector. The CAR-T cells will be evaluated for efficacy against antigen positive tumors by IFNγ
ELISA, IL-2 ELISA, and Incucyte live cell videomicroscopy. Each of these constructs will be tested with CAR costimulatory
domains 4-1BB and CD28. The CAR constructs with the strongest reactivity will be further evaluated with an NOD scid
gamma (NSG) mouse xenograft model, measuring tumor growth inhibition and CAR-T expansion in vivo.
Aim 2 – Determine which subset of CAR-T cells preferentially traffic and persist in the tumor in vivo. To evaluate the
respective contribution of CAR-T cell subsets to anti-tumor efficacy in vivo, semi-random nucleotide barcodes will be
added to the CAR constructs allowing for the tracking of clonal lineage during CAR-T production, infusion, and post-
engraftment in mice. Using single cell sequencing, clonal diversity of the CAR-T infusion product will be compared to
clonal diversity intratumorally. Subsets of CAR-T that preferentially home to and expand in the tumor will be identified.
Together, these aims will set the basis for future studies of CAR-T therapy in a canine model. Aim 1 will provide a
candidate CAR construct to be evaluated in a canine model. Aim 2 will provide a method for understanding how CAR-T
cells traffic to and persist within a tumor in vivo. This platform will be used to screen and refine novel approaches to
CAR-T therapy in a high-fidelity, high-throughput animal model.
Terms: <Animal Model><Animal Models and Related Studies><Animals><Antibodies><Antigens><B cell malignancy><B lymphoid malignancy><B lymphoma><B-Cell Lymphomas><Back><Bar Codes><Binding><Biology><Biopsy><Bone Sarcoma><Bp35><Breast Carcinoma><CAR T cell therapy><CAR T cells><CAR T therapy><CAR modified T cells><CAR-T><CAR-Ts><CD20><CD28><CD28 gene><Cancer Biology><Cancers><Canine Species><Canis familiaris><Cell Body><Cell Function><Cell Physiology><Cell Process><Cells><Cellular Function><Cellular Physiology><Cellular Process><Cellular biology><Cellular immunotherapy><Chemicals><Chimera Protein><Chimeric Proteins><Co-Stimulator><Co-culture><Cocultivation><Coculture><Coculture Techniques><Complement><Complement Proteins><Contracting Opportunities><Contracts><Costimulator><Disease remission><Dogs><Dogs Mammals><Dorsum><Dysfunction><ELISA><Engraftment><Enzyme-Linked Immunosorbent Assay><Epidermal Thymocyte Activating Factor><Ethics><Evolution><Fc Receptor><Functional disorder><Fusion Protein><Future><Generations><Genetic><Health><Hematologic Cancer><Hematologic Malignancies><Hematologic Neoplasms><Hematological Malignancies><Hematological Neoplasms><Hematological Tumor><Hematopoietic Cancer><Home><Human><IFN-Gamma><IFN-g><IFN-γ><IFNG><IFNγ><IL-2><IL2 Protein><IVIS SpectrumCT><IVIS imaging><IVIS optical imaging><IVIS spectral imaging><IVIS spectrum><IVIS system><Immune><Immune Interferon><Immune system><Immunes><Immunocompetent><Immunodeficient Mouse><In Vitro><Inbreeding><Incidence><Individual><Infusion><Infusion procedures><Interferon Gamma><Interferon Type II><Interleukin 2><Interleukin 2 Precursor><Interleukin II><Interleukin-2><Interleukine 2><Interleukine 2 Precursor><Interleukine II><Kinetics><Lentiviral Vector><Lentivirus Vector><Leu-16><Lymphocyte Mitogenic Factor><MHC Receptor><MS4A1><MS4A1 gene><MS4A2><Major Histocompatibility Complex Receptor><Malignant Hematologic Neoplasm><Malignant Melanoma><Malignant Neoplasms><Malignant Tumor><Mammary Carcinoma><Measurement><Measures><Melanoma><Methods><Mice><Mice Mammals><Mitogenic Factor><Modeling><Modern Man><Molecular><Molecular Interaction><Murine><Mus><Neuroblastoma><Nucleotides><Osseous Sarcoma><Osteogenic Sarcoma><Osteosarcoma><Patients><Ph D student><Ph D. student><Ph. D. student><Ph.D student><Ph.D. student><PhD student><PhD. student><Phenotype><Physiopathology><Procedures><Production><RNA Seq><RNA sequencing><RNAseq><Receptor Signaling><Refractory><Relapse><Remission><Research><Sampling><Scientist><Single cell seq><Skeletal Sarcoma><Solid Neoplasm><Solid Tumor><Subcellular Process><T cell growth factor><T cell infiltration><T cells for CAR><T-Cell Antigen Receptors><T-Cell Growth Factor><T-Cell Homing Receptors><T-Cell Receptor><T-Cell Stimulating Factor><T-Cell Subsets><T-Cells><T-Lymphocyte><T-Lymphocyte Subsets><T44><Testing><Thymocyte Stimulating Factor><Training><Translating><Treatment Efficacy><Tumor Antigens><Tumor Cell Line><Tumor-Associated Antigen><Veterinarians><Video Microscopy><Videomicrography><Videomicroscopy><Xenograft Model><anti-tumor effect><antibody receptor><antitumor effect><barcode><cancer antigens><canine><canine animal model><canine model><career><cell biology><cell transduction><cell-based immunotherapy><cellular transduction><chimeric antigen T cell receptor><chimeric antigen receptor><chimeric antigen receptor (CAR) T cell therapy><chimeric antigen receptor (CAR) T cells><chimeric antigen receptor T cell therapy><chimeric antigen receptor T cells><chimeric antigen receptor T therapy><chimeric antigen receptor fusion protein T-cells><chimeric antigen receptor modified T cells><complement system><complementation><cost><design><designing><determine efficacy><doctoral student><dog model><domestic dog><driving force><efficacy analysis><efficacy assessment><efficacy determination><efficacy evaluation><efficacy examination><enzyme linked immunoassay><ethical><evaluate efficacy><examine efficacy><homes><hypoimmunity><immune cell therapy><immune competent><immune deficiency><immunodeficiency><immunogen><in vivo><in vivo imaging system><infusions><intervention efficacy><lFN-Gamma><malignancy><model of animal><mouse model><murine model><neoplasm/cancer><new approaches><novel><novel approaches><novel strategies><novel strategy><osteochondrosarcoma><osteoid sarcoma><pathophysiology><pre-clinical><pre-clinical efficacy><preclinical><preclinical efficacy><restraint><single cell next generation sequencing><single cell sequencing><success><therapeutic efficacy><therapy efficacy><thymus derived lymphocyte><timeline><trafficking><transcriptome sequencing><transcriptomic sequencing><transduced cells><translational model><tumor><tumor growth><tumor-specific antigen><xenograft transplant model><xenotransplant model>