Depending on the most advanced techniques and extensive experience in Chimeric Antigen Receptor (CAR) construction field, Creative Biolabs now provides customers with one-stop solution for scFv generation, CAR construction and virus packaging services via various modern molecular cloning and immunological techniques, in order to meet our customers' needs.
The basic CAR structure consists of an extracellular antigen-recognition domain attached to an extracellular spacer domain, a trans-membrane region that anchors the receptor in the plasma, and a signaling endodomain. The single-chain antibody fragment (scFv) consisting of a variable heavy (VH) and a variable light (VL) chain, both of which are derived from the same parental antibody and linked by a flexible linker. Due to its small size which can facilitate both the genetic manipulation and the CAR expression, scFv has been used extensively as the antigen-recognition domain in many CARs. The scFv determines the CAR antigen specificity and makes the CAR-T cells bind target cells in a MHC-independent, non-restricted manner with the specificity and affinity similar to the antibody which it is derived from.
Figure 1. Structure of a CAR.
We have built up three one-stop solution platforms to support our CAR development services:
Phase I. scFv generation
One-stop solution CAR development: Hybridoma cells;
We can generate the antibody by constructing hybridoma cell line and cloning the antibody gene out from it.
One-stop solution CAR development: Phage display scFv library construction and screening;
One-stop solution CAR development: Antibody sequencing.
Phase II. CAR construction
Once we obtain the scFvs, we will move on to constructing CAR plasmids and then prepare lentivirus particles (>107 virus particles).
Phase III. CAR-T cells expression
We will perform T-cell transduction, activation, CAR expression and detection in T cells.
(1) We can construct a Flag or Myc tag at the N-terminal of the scFv, and subsequently perform the anti-Flag or anti-Myc assays to detect the expression of CARs.
(2) We can use fusion Fc or Fluorescent labels to perform the detection assays, which can analyze the binding activity of CAR simultaneously.
Phase IV. Cell lysis Assays
• We guarantee any Chimeric Antigen Receptor (CAR) construction with excellent homogeneity, stability and efficacy, such as custom lentivirus particles.
• We can offer different scFvs for antibody construction, or start from your own scFv or hybridoma; then we'll synthesize the TM-Intracytoplasmic gene and insert it into the scFv vector according to your requirement.
Quality Controls Measures
The tumor cells killing efficiency is employed for evaluating the quality of CAR constructions.
Lipovska-Bhalla, e tal. (2012). "Target immunotherapy of cancer with CAR T cell: achievements and challenges". Cancer Immunol Immunother 61 (7): 953–962. doi:10.1007/s00262-012-1254-0. PMID 22527245. Retrieved 25 March 2012.
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