Enhanced chimeric antigen receptors and use thereof
a technology of chimeric antigen receptor and enhanced chimeric antigen, applied in the field of immunology and molecular biology, can solve the problems of affecting the full use of this approach in cancer treatment, poor prognosis, and many patients' inability to cur
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nt of Truncated EGFRvIII (Ev3) CAR
[0231]An Ev3-CAR was designed in which the Ev3 stalk serves as an integrative structure to transduce signals from antigen binding, such as by scFv, as well as an “off” switch, meaning it can be recognized by clinically accessible antibodies such as cetuximab to ablate CAR-positive immune cells (FIG. 1A). The Ev3-CAR may further comprise DAP12 signaling domain and is designated Ev3-DAP12-CAR. A schematic depicting Ev3 as compared to EGFR which comprise the endodomain and the EGF binding domain is shown in FIG. 2.
[0232]Retroviral vectors encoding the Ev3-CAR or Ev3-DAP12-CAR with various antigen binding domains were generated as depicted in FIGS. 3A-3D. The antigen-binding domain was both the scFv derived from the mouse anti-human antibody as well as a humanized scFv. The vectors comprise CD28 and CD3ζ signaling domains and, optionally, DAP12. The vectors also comprise a suicide gene, inducible caspase 9.
[0233]The CD19-specific CARs including CAR19.Ev...
example 2
nt of Humanized CARs
[0234]A platform was developed to generate humanized scFvs for use in producing humanized CARs. Due to structural constraints, some residues contribute more to antigen binding, thus 3-D modeling was used to identify potentially critical interactions to generate the humanized CARs. The stepwise process detailed below may be repeated iteratively to improve on previous cycles.
[0235]The CAR humanization workflow provided herein comprises 5 basic process modules as detailed below and depicted in FIG. 5A.
[0236]Module I comprises monoclonal antibody (mAb) sequence analysis. The BLAST program was harnessed in search of immunoglobulin (IG) sequences for the best match against the starting queries, such as the murine mAb sequences (FIG. 5B). BLAST can analyze nucleotide and amino acid sequences, either in series (one-by-one) or in parallel (in batches), as well as search against germline gene databases including curated sequence databases simultaneously thus maximizing the...
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