Granted Patent B2

US 12,378,526 · App. 17/746,835 · Granted Aug 5, 2025

Inventors: Myoung Ho Jang (Seoul, KR); Chun-Pyo Hong (Gyeonggi-do, KR); Dong Woo Ko (Seoul, KR); June Sub Lee (Gyeonggi-do, KR)

Assignee: GI CELL, INC.

C12N5/0646A61K35/17C12N5/0636C12N2501/2302C12N2501/51

Abstract

Claims (49)

1. A method for culturing an isolated natural killer cell, which comprises:

culturing the isolated natural killer cell without CD3+ or CD56− cells in a composition comprising a fusion protein dimer,

wherein the fusion protein comprises the following structural formula (I) or (II):

N′—X-[linker (1)]n-Fc domain-[linker (2)]m-Y—C′—  formula (I)

N′—Y-[linker (1)]n-Fc domain-[linker (2)]m-X—C′—  formula (II),

wherein, N′ is the N-terminus of the fusion protein,

C′ is the C-terminus of the fusion protein,

X is a CD80 protein or a fragment thereof,

Y is an IL-2 variant thereof,

the linkers (1) and (2) are peptide linkers, and

n and m are each independently 0 or 1,

wherein the CD80 fragment comprises the extracellular domain of CD80,

wherein the IL-2 variant is obtained by any one selected from the following substitution combinations (a) to (d) in the amino acid sequence of SEQ ID NO: 10:

(a) R38A/F42A

(b) R38A/F42A/Y45A

(c) R38A/F42A/E61R

(d) R38A/F42A/L72G.

2. The method of claim 1 , wherein the IL-2 variant consists of the amino acid sequence of SEQ ID NO: 6.

3. The method of claim 1 , wherein the fragment of CD80 consists of the 35th to 242nd amino acids in the amino acid sequence of SEQ ID NO: 11.

4. The method of claim 1 , wherein the fusion protein consists of the amino acid sequence of SEQ ID NO: 9, 26, 28, or 30.

5. A method for culturing a natural killer cell without CD3+ or CD56-cells, which comprises:

i) isolating a cell, comprising one of (A), (B), or (C):

(A) isolating a cell that does not express CD3 from peripheral blood mononuclear cells (PBMCs),

(B) isolating a cell that expresses CD56 from PBMCs, or

(C) isolating a cell that does not express CD3 from PBMCs, and isolating a cell that expresses CD56 from the cell that does not express CD3, isolated in the first isolating step; and

ii) culturing the isolated cell in the presence of a fusion protein dimer,

wherein the fusion protein comprises the following structural formula (I) or (II):

N′—X-[linker (1)]n-Fc domain-[linker (2)]m-Y—C′—  formula (I)

N′—Y-[linker (1)]n-Fc domain-[linker (2)]m-X—C′—  formula (II),

wherein, N′ is the N-terminus of the fusion protein,

C′ is the C-terminus of the fusion protein,

X is a CD80 protein or a fragment thereof,

Y is an IL-2 variant thereof,

the linkers (1) and (2) are peptide linkers, and

n and m are each independently 0 or 1,

wherein the CD80 fragment comprises the extracellular domain of CD80,

wherein the IL-2 variant has any one selected from the following substitution combinations (a) to (d) in the amino acid sequence of SEQ ID NO: 10:

(a) R38A/F42A

(b) R38A/F42A/Y45A

(c) R38A/F42A/E61R

(d) R38A/F42A/L72G.

6. The method of claim 5 , wherein the isolating of step i) comprises:

(C) isolating a cell that does not express CD3 from PBMCs, and isolating a cell that expresses CD56 from the cell that does not express CD3, isolated in the first isolating step.

7. The method of claim 6 , wherein the fusion protein dimer in said culturing ii) is at a concentration of 1 nM to 500 nM.

8. The method of claim 6 , wherein the culture period in the culturing step is 5 days to 25 days.

9. The method of claim 5 , wherein the isolating i) comprises:

(A) isolating a cell that does not express CD3 from peripheral blood mononuclear cells (PBMCs).

10. The method of claim 5 , wherein the isolating i) comprises:

(B) isolating a cell that expresses CD56 from PBMCs.

Assignments (1)

Priority Claims (2)

Continuity (2)

References Cited (68)