Granted Patent B2

US 12,338,459 · App. 16/966,264 · Granted Jun 24, 2025

Inventors: Hao Yan (Chandler, AZ); Bo Ning (Tempe, AZ)

Assignee: ARIZONA BOARD OF REGENTS ON BEHALF OF ARIZONA STATE UNIVERSITY

C12N5/0636A61K40/11A61K40/31A61K40/4204A61K40/4205A61K40/421A61K40/4211A61K40/4255A61K40/4257A61K40/4258C07K14/70517C12N15/115A61K2239/31A61K2239/38A61K2239/48A61K2239/49A61K2239/55B82Y5/00C07K2317/70C07K2319/03C07K2319/50C12N2310/16C12N2310/3513C12N2510/00

Abstract

Claims (38)

1. An engineered T cell comprising:

(a) an expressed engineered chimeric antigen receptor (CAR) which comprises an extracellular adaptor protein selected from O 2 -alkylcytosine DNA alkyltransferase which reacts with O 2 -benzylcytosine moiety, or O 6 -alkylguanine DNA alkyltransferase which reacts with O 2 -benzylcytosine moiety;

(b) a protein tag bound to said adaptor protein;

(c) a first oligonucleotide connected to said protein tag;

(d) a second oligonucleotide wherein a portion of the second oligonucleotide sequence is complementary to a portion of the first oligonucleotide sequence; and

(e) a targeting agent connected to the second oligonucleotide, wherein the targeting agent comprises one or a plurality of targeting molecules,

wherein the targeting agent comprises

(i) a DNA origami nanostructure comprising a central polynucleotide strand and a first staple strand which comprises the second oligonucleotide sequence and a plurality of second staple strands which comprises one or a plurality of third distinct oligonucleotide sequences; and

(ii) one or more targeting molecules connected to one or a plurality of fourth distinct oligonucleotide sequence(s),

wherein a portion of the third distinct oligonucleotide sequence is complementary to a portion of the fourth oligonucleotide sequence.

2. The engineered T cell of claim 1 , wherein the expressed engineered chimeric antigen receptor comprises:

(i) a signaling polypeptide domain;

(ii) a transmembrane polypeptide domain;

(iii) a spacer polypeptide domain configured to be between the transmembrane polypeptide domain and the adaptor protein tag domain;

(iv) a costimulatory polypeptide domain; and

(v) an adaptor protein tag domain.

3. The engineered T cell of claim 1 , further comprising a fluorescent protein domain.

4. The engineered T cell of claim 3 , wherein the fluorescent protein domain is green fluorescent protein (GFP).

5. The engineered T cell of claim 2 , further comprising an antibiotic resistant gene.

6. The engineered T cell of claim 2 , wherein the signaling polypeptide domain is CD3ζ, the transmembrane polypeptide domain is CD8, the costimulatory domain is 4-1BB, the spacer polypeptide domain is (Gly-Gly-Gly-Gly-Ser) 4 (SEQ ID NO: 2), and the adaptor protein tag domain is O 6 -alkylguanine-DNA alkyltransferase which reacts with O 6 -benzylguanine moiety.

7. The engineered T cell of claim 1 , wherein the targeting agent comprises one or a plurality of targeting molecules selected from a group of targeting molecules consisting of: an aptamer, a synbody, an antibody, and a fragment of an antibody thereof.

8. The engineered T cell of claim 7 , wherein the targeting agent is a fragment of an antibody fragment, and further wherein the fragment of an antibody is a ScFv.

9. The engineered T cell of claim 1 , wherein one of the one or a plurality of targeting molecules comprises a matrix metalloproteinase.

10. The engineered T cell of claim 1 , wherein one of the one or a plurality of targeting molecules comprises a cytokine or chemokine.

11. The engineered T cell of claim 1 , wherein one of the one or a plurality of targeting molecules comprises an inhibitory pathway overcoming agent which is an anti-PD-1L aptamer.

12. The engineered T cell of claim 7 , wherein the aptamer is scg8 having SEQ ID NO: 10.

13. The engineered T cell of claim 1 , wherein the T cell is selected from a group of T cells consisting of: a natural killer T cell, a regulatory T cell, a helper T cell, a cytotoxic T cell, a memory T cell, a gamma delta T cell and a mucosal invariant T cell.

14. A method of preparing the engineered T cell of claim 1 comprising:

(a) inserting a DNA sequence which encodes for the CAR polypeptide into a virus;

(b) contacting the virus with a T cell to form a viral-infused T cell;

(c) growing the viral-infused T cells to produce an adaptor T cell expressing the CAR polypeptide comprising an extracellular adaptor protein;

(d) isolating the adaptor T cell;

(e) contacting the isolated adaptor T cells with a first oligonucleotide functionalized with a cognate protein tag;

(f) forming a complex between the extracellular adaptor protein of the adaptor T cells with the cognate protein tag to form a first oligonucleotide-functionalized adaptor T cell; and

(g) contacting the first oligonucleotide-functionalized adaptor T cell with a second oligonucleotide comprising a targeting agent under appropriate conditions to form a hybridization complex between a portion of the first linker oligonucleotide and a portion of the second linker oligonucleotide,

wherein said virus is selected from a lentivirus, retrovirus or adeno-associated virus.

15. A method of activating an engineered T cell, the method comprising contacting a cancer cell with the engineered T cell of claim 1 .

16. The engineered T cell of claim 11 , wherein the anti-PDL1 aptamer comprises a sequence having SEQ ID NO: 5.

Assignments (1)

Continuity (2)

References Cited (230)