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Adoptive immunotherapy using T cells expressing chimeric antigen receptors (CARs) has produced remarkable clinical outcomes. However, much of the mechanisms of action, such as the development of memory responses and sources of immune cytokines, remain elusive largely due to the challenge of characterizing human CAR T cell function in vivo. Creative Biolabs has established a syngeneic mouse model with a functional mouse immune system and genetically-matched (autologous) Acute myeloid leukemia (AML) that permits modeling of CLL1-targeted CAR T cell therapy in immunocompetent hosts without allogeneic immune responses. Our anti-CLL1 CAR T cells are prepared from freshly isolated T lymphocytes in a syngeneic mouse model, followed by ex vivo transduction with anti-CLL1 CAR lentiviral particles and amplification process to generate CAR T cells. These syngeneic mouse model-derived CAR-T cells offer many advantages including permitting the evaluation of antitumor responses in immunocompetent murine hosts, testing human CAR-T constructs against human primary malignance, and preclinical evaluation of CAR T cell therapies.
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T Cell Background
Target
CLL1
Applications
The mouse CLL1 CAR-T cells can be used for evaluation of the safety, toxicity, persistence and exhaustion of CAR T cells in syngeneic mouse model.
T Cell Source
Syngeneic Mouse Model
Donor Mouse Strain
BALB/c
Donor Tumor Cell Line
Acute myeloid leukemia (AML) Cell Line
Mouse Modeling
Syngeneic mouse models can be a powerful tool for testing immunotherapies. They consist of tumor tissues from the same genetic background as the given immuno-competent mouse strain. A syngeneic mouse model provides an effective approach for studying how cancer therapies perform in the presence of a functional immune system.
At Creative Biolabs, we have established syngeneic models by injecting a recipient of a specific genetic background with cell lines previously established through isolation of tumor cells from a mouse of the same genetic background. Our syngeneic models that have been fully characterized with known checkpoint inhibitors (e.g., anti-PDL-1, anti-PD-1, anti-CTLA-4), whole exome sequencing, and immunologic profiling data, backed by experienced scientists who can ensure the model runs smoothly.
T Cell Isolation
The mouse T cells were isolated from syngeneic mouse spleens by MACS using anti-mouse CD3 micro-beads.
CAR Design
CAR Introduction
The vector of anti-CLL1 chimeric antigen receptor (CAR) is constructed for the engineering of T cells to target Mouse CLL1. The T cells are genetically modified through transduction with a lentiviral vector expressing scFv of anti-CLL1 antibody linked to CD28 and CD3ζ signaling domains. And the vector product was designed for the treatment of Acute myeloid leukemia (AML).
scFv Clone
5D3
Target Species
Mouse
scFv Host Species
Rat
CAR Construction
scFv-CD28-CD3ζ
CAR Signaling Cassetes
CD28 CD28 (Cluster of Differentiation 28) is one of the proteins expressed on T cells that provide costimulatory signals required for T cell activation and survival. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2) proteins which are expressed on antigen-presenting cells (APC). CD28 modulates the primary TCR/CD3ζ signal in a different fashion than the late costimulatory elements OX40 and CD28. CD28 enhances the expression of downstream regulators that impact on T-cell proliferation, death, differentiation, and effector functions. CAR+ T cells containing the CD28 endodomain showed strikingly enhanced sustained T cell activation, growth, survival. And CD28 results in a brightly expressed, stable receptor as the transmembrane domain. Including CD28 costimulatory domains in CARs led to enhanced anti-malignancy efficacy. CD3ζ CD3ζ, also known as T-cell receptor zeta, which together with T-cell receptor and CD3γ, δ , ε chain, forms the TCR-CD3 complex. ζ was expressed independently from the complex. The zeta chain plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. CD3-zeta, which contains 3 ITAMs, is the most commonly used endodomain component of CARs. It transmits an activation signal to the T cell after antigen is bound. CD3-zeta may not provide a fully competent activation signal and additional co-stimulatory signaling is needed. For example, chimeric CD28 and OX40 can be used with CD3-zeta to transmit a proliferative/survival signal, or all three can be used together.
CAR Vector Name
pCDCAR1
CAR Vector Length
~8kb
CAR Vector Type
Lentiviral vector
CAR Generation
Second
Targeting Diseases
Acute myeloid leukemia (AML)
CAR-T Cell
CAR-T Cell Preparation
Freshly mouse T cells were isolated from mouse spleens by MACS using anti-mouse CD3 micro-beads, and then transduced with anti-CLL1 CAR-expressing lentiviral particles.
CAR-T Cell Expansion
The CAR virus transduced T cells were then expanded for about 2 weeks using our in-house protocol. The expanded CAR-T cells should be used immediately in mice.
CAR-T Cell Characterization
The expression of mouse CLL1 is characterized by flow cytometry.
Quality Control
Cell Purity
>95%
Cell Viability
>90%
Mycoplasma Testing
The cell line has been screened using the luciferase based mycoplasma detection kit to confirm the absence of mycoplasma species.
Sterility Testing
Creative Biolabs provides sterility testing in accordance with USP and EP regulations. All of our sterility testing is performed in an isolator or clean room environments. The cell line has been screened using the membrane filtration testing methods to confirm the absence of aerobic, anaerobic and fungi microorganisms.
Shipping and Handling
Shipping
Dry Ice
Storage
Lyophilized cells should be stored in a liquid nitrogen tank (-150°C~-190°C). Once reconstituted, the cells may be used for up to five days if properly stored at 2°C - 24°C in the buffer provided.
Handling Notes
Frozen cells should be thawed immediately upon receipt and grown according to handling procedure to ensure cell viability and proper assay performance. Note: Do not freeze the cells upon receipt as it may result in irreversible damage to the cell line. Disclaimer: We cannot guarantee cell viability if the cells are not thawed immediately upon receipt and grown according to handling procedure.
Warnings
Avoid multiple freeze/thaw cycles
Research Use Only
For research use only, not for diagnostic or therapeutic use.
Customer Reviews and Q&As
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For research use only. Not intended for any clinical use. No products from Creative Biolabs may be resold, modified for resale or used to manufacture commercial products without prior written approval from Creative Biolabs.
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