CAR-T cells with humanized CD19 ScFv with mutation in CDR 1 region
Provided is a humanized CD19 single-chain variable fragment (scFv) having a mutation of valine to glycine in CDR1, comprising VH having the amino acid sequence of SEQ ID NO: 6 and VL having the amino acid sequence of SEQ ID NO: 5. Also provided is a CD19 chimeric antigen receptor fusion protein comprising from N-terminus to C-terminus: (i) a single-chain variable fragment (scFv), (ii) a transmembrane domain, (iii) at least one co-stimulatory domains, and (iv) an activating domain. This humanized CD19-CAR-T cells have specific killing activity with secretion of cytokine IFN-gamma in CAR-T cells in vitro and in vivo.
1. A humanized single-chain variable fragment (scFv) of CD19 comprising VH having the amino acid sequence of SEQ ID NO: 6 and VL having the amino acid sequence of SEQ ID NO: 5.
2. The scFv of claim 1 , further comprises a linker in between VH and VL.
3. The scFv of claim 2 , which has the amino acid sequence of SEQ ID NO: 4.
4. A chimeric antigen receptor (CAR) fusion protein comprising from N-terminus to C-terminus:
(i) the scFv of claim 1 ,
(ii) a transmembrane domain,
(iii) at least one co-stimulatory domain, and
(iv) an activating domain;
wherein the CAR has the amino acid sequence of SEQ ID NO: 14.
5. An antibody against human CD19 comprising VH having the amino acid sequence of SEQ ID NO: 6 and VL having the amino acid sequence of SEQ ID NO: 5.
6. A nucleic acid molecule encoding the CAR fusion protein of claim 4 .
7. A vector comprising the nucleic acid molecule of claim 6 .
8. A host cell expressing the CAR fusion protein of claim 4 .
9. A formulation comprising the cell of claim 8 , and a pharmaceutically acceptable carrier, diluent or excipient.
10. The cell of claim 8 , wherein the cell is T cell or NK cell.
11. A method for treating cancer or tumor comprising administering the cell of claim 8 to a subject in need of treatment.