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Anti-NKG2D (KYK-1.0) h(4-1BB-CD3ζ) CAR-T Cells [Humanized Mouse (CD34+ HSC)] (MCAR-P192)

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All products and services are For Research Use Only and CANNOT be used in the treatment or diagnosis of disease.

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 humanized mouse (hu-mouse) model with a functional human immune system and genetically-matched (autologous) Colorectal cancer that permits modeling of NKG2D-targeted CAR T cell therapy in immunocompetent hosts without allogeneic or xenogeneic immune responses. Our anti-NKG2D CAR T cells are prepared from freshly isolated T lymphocytes in a syngeneic mouse model, followed by ex vivo transduction with anti-NKG2D CAR lentiviral particles and amplification process to generate CAR T cells. These humanized CD34+ mice-derived CAR-T cells offer many advantages including permitting the evaluation of antitumor responses in immunocompetent NSG 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
  • NKG2D
  • Applications
  • The humanized NKG2D CAR-T cells can be used for evaluation of the safety, toxicity, persistence and exhaustion of CAR T cells in a humanized CD34+ mouse model.
  • T Cell Source
  • Humanized CD34+ HSC Mouse Model
  • Donor Mouse Strain
  • BALB/c
  • Donor Tumor Cell Line
  • Colorectal cancer Cell Line
  • Mouse Modeling
  • Humanized CD34+ mice is a study-ready mouse model with a human-like immune system, created by adoptive transfer of CD34+ stem cells. Humanized CD34+ mice are supreme in vivo models to study immunology and immuno-oncology.

    Our Hu-CD34+ mice produced by injecting CD34+ hematopoietic stem cells (hu-CD34) demonstrate robust multi-lineage engraftment of human immune cell populations including very good T cell maturation and function for long-term studies. Successful humanization of each mouse is quantified from mouse peripheral blood via flow cytometry using anti- hu-CD45+ and anti-murine CD45+. The presence of human immune cells are verified in the peripheral blood as early as 12-15 weeks post-engraftment, as verified by multi-color flow cytometry. These immune cells are detectable in mouse tissues, including bone marrow, spleen, liver, and lung, and have been shown to infiltrate implanted tumors.
  • T Cell Isolation
  • The human T cells were isolated from hu-mouse spleens by MACS using anti-human CD3 micro-beads.

CAR Design

  • CAR Introduction
  • The vector of anti-NKG2D chimeric antigen receptor (CAR) is constructed for the engineering of T cells to target Human NKG2D. The T cells are genetically modified through transduction with a lentiviral vector expressing scFv of anti-NKG2D antibody linked to 4-1BB and CD3ζ signaling domains. And the vector product was designed for the treatment of Colorectal cancer.
  • scFv Clone
  • KYK-1.0
  • Target Species
  • Human
  • scFv Host Species
  • Human
  • CAR Construction
  • scFv-41BB-CD3ζ
  • CAR Signaling Cassetes
  • 41BB
    CD137 (also known as 4-1BB) is a surface co-stimulatory glycoprotein originally described as present on activated T lymphocytes, which belongs to the tumor necrosis factor (TNF) receptor superfamily. It is expressed mainly on activated CD4+ and CD8+ T cells, and binds to a high-affinity ligand (4-1BBL) expressed on several antigen-presenting cells such as macrophages and activated B cells. On the basis of preclinical observation, this molecule can promote the persistence of antigen-specific and antigennonspecific chimeric antigen receptor T-cells to significantly increases antitumor activity.
    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 4-1BB 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
  • Colorectal cancer

CAR-T Cell

  • CAR-T Cell Preparation
  • Freshly human T cells were isolated from hu-mouse spleens by MACS using anti-human CD3 micro-beads, and then transduced with anti-PD-L1 CAR-expressing lentiviral particles.
  • CAR-T Cell Expansion
  • The CAR virus transduced T cells were then expanded for about 193 weeks using our in-house protocol. The expanded CAR-T cells should be used immediately in hu-mice.
  • CAR-T Cell Characterization
  • The expression of human PD-L1 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 - 8°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.

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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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