Chlorotoxin (EQ)-CD28-CD3zeta-CD19t-expressing CAR T-lymphocytes
chlorotoxin (eq)-cd28-cd3zeta-cd19t-expressing car t-lymphocytes
"A preparation of genetically modified T-lymphocytes transduced with a lentiviral vector expressing a chimeric antigen receptor (CAR) comprised of a CD28 co-stimulatory signaling domain fused to the zeta chain of the TCR/CD3 complex (CD3zeta), a truncated form of CD19 (CD19t), an immunoglobulin (Ig) G4-Fc (EQ) spacer, and a peptide derived from chlorotoxin (CLTX), with potential imaging and antineoplastic activities. Upon administration, chlorotoxin (EQ)-CD28-CD3zeta-CD19t-expressing CAR T-lymphocytes are re-directed to specific tumor cells in the brain inducing selective toxicity in these tumor cells. CLTX, a 36-amino acid peptide found in the venom of the deathstalker scorpion (Leiurus quinquestriatus) and a chloride channel blocker, preferentially binds to glioma (and other neuroectodermal origin) cells via membrane bound forms of the endopeptidase matrix metalloproteinase-2 (MMP-2). This may direct the T-lymphocytes to and induce selective toxicity in MMP-2-expressing tumor cells. Additionally, binding to MMP-2 on glioma cells may both interfere with transmembrane chloride exchange and inhibit proteolytic extracellular matrix remodeling by MMP-2, which may further limit the spread of these tumor cells. MMP-2 is specifically upregulated in gliomas and related cancers, but is not normally expressed in brain. The CD28 co-stimulatory molecule signaling domain enhances activation and signaling; its inclusion may increase proliferation of T-cells and antitumor activity compared to the inclusion of the CD3 zeta chain alone. IgG4-Fc (EQ) contains two point mutations in its spacer region which prevents recognition of the CAR by Fc receptors (FcRs) without altering the ability of the CAR to mediate antigen-specific lysis. CD19t, which lacks the cytoplasmic signaling tail, provides a non-immunogenic surface marker that allows for accurate measurement, efficient cell tracking and/or imaging of the therapeutic T-cells in vivo following adoptive transfer. Additionally, co-expression of CD19t functions as a \"suicide\" switch via clinically available antibodies or immunotoxins which can be used to selectively eliminate the genetically modified cells."