Granted Patent B2

US 12,312,595 · App. 18/894,924 · Granted May 27, 2025

Inventors: Anh Tuan Nguyen (San Francisco, CA); Jeffrey Kim (Berkeley, CA); Keunho Ahn (Pleasanton, CA); Daixuan Zhang (Fremont, CA)

Assignee: Slingshot Biosciences, Inc.

C12N5/0018A61K35/17C12M35/06C12N5/0636C12N2500/50C12N2501/51C12N2501/515

Abstract

Claims (37)

1. A method for separating a target cell from an aqueous mixture, the method comprising:

a) incubating an aqueous mixture comprising the target cell and a magnetic capture hydrogel particle, thereby producing a target cell-bound magnetic capture hydrogel particle, wherein the magnetic capture hydrogel particle comprises:

i) a hydrogel matrix comprising a magnetic material embedded and/or encapsulated therein, such that the magnetic capture hydrogel particle is capable of being attracted to or repelled by a magnetic field;

ii) disulfide crosslinks enabling the hydrogel matrix to dissolve upon addition of a reducing agent; and

iii) a ligand capable of binding the hydrogel matrix to the target cell;

b) applying a magnetic field to the incubated aqueous mixture to isolate the target cell-bound magnetic capture hydrogel particle from the aqueous mixture;

c) retrieving the isolated target cell-bound magnetic capture hydrogel particle; and

d) contacting the hydrogel matrix with a reducing agent, thereby dissolving the hydrogel matrix and releasing the target cell from the magnetic capture hydrogel particle.

2. The method of claim 1 , wherein the reducing agent comprises dithiothreitol, Tris(2-carboxyethyl) phosphine hydrochloride, 2-mercaptoethanol, or a combination thereof.

3. The method of claim 1 , wherein the hydrogel matrix comprises a polymerized monomer and a bifunctional monomer.

4. The method of claim 3 , wherein the polymerized monomer, the bifunctional monomer, or both are functionalized with acrylate or acrylamide.

5. The method of claim 1 , wherein the hydrogel matrix comprises a monomer selected from the group consisting of hydroxyethyl methacrylate, ethyl methacrylate, 2-hydroxyethyl methacrylate (HEMA), propylene glycol methacrylate, acrylamide, N-vinylpyrrolidone (NVP), methyl methacrylate, glycidyl methacrylate, glycerol methacrylate (GMA), glycol methacrylate, ethylene glycol, fumaric acid, 2-hydroxyethyl methacrylate, hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate, methoxyethyl methacrylate, methoxyethoxyethyl methacrylate, methoxydiethoxyethyl methacrylate, poly(ethylene glycol) methacrylate, methoxy-poly(ethylene glycol) methacrylate, methacrylic acid, sodium methacrylate, glycerol methacrylate, hydroxypropyl methacrylate, hydroxybutyl methacrylate, phenyl acrylate, phenyl methacrylate, benzyl acrylate, benzyl methacrylate, 2-phenylethyl acrylate, 2-phenylethyl methacrylate, 2-phenoxyethyl acrylate, 2-phenoxyethyl methacrylate, phenylthioethyl acrylate, phenylthioethyl methacrylate, 2,4,6-tribromophenyl acrylate, 2,4,6-tribromophenyl methacrylate, pentabromophenyl acrylate, pentabromophenyl methacrylate, pentachlorophenyl acrylate, pentachlorophenyl methacrylate, 2,3-dibromopropyl acrylate, 2,3-dibromopropyl methacrylate, 2-naphthyl acrylate, 2-naphthyl methacrylate, 4-methoxybenzyl acrylate, 4-methoxybenzyl methacrylate, 2-benzyloxyethyl acrylate, 2-benzyloxyethyl methacrylate, 4-chlorophenoxyethyl acrylate, 4-chlorophenoxyethyl methacrylate, 2-phenoxyethoxyethyl acrylate, 2-phenoxyethoxyethyl methacrylate, N-phenyl acrylamide, N-phenyl methacrylamide, N-benzyl acrylamide, N-benzyl methacrylamide, N,N-dibenzyl acrylamide, N,N-dibenzyl methacrylamide, N-diphenylmethyl acrylamide N-(4-methylphenyl) methyl acrylamide, N-1-naphthyl acrylamide, N-4-nitrophenyl acrylamide, N-(2-phenylethyl) acrylamide, N-triphenylmethyl acrylamide, N-(4-hydroxyphenyl) acrylamide, N,N-methylphenyl acrylamide, N,N-phenyl phenylethyl acrylamide, N-diphenylmethyl methacrylamide, N-(4-methyl phenyl) methyl methacrylamide, N-1-naphthyl methacrylamide, N-4-nitrophenyl methacrylamide, N-(2-phenylethyl) methacrylamide, N-triphenylmethyl methacrylamide, N-(4-hydroxyphenyl) methacrylamide, N,N-methylphenyl methacrylamide, N,N′-phenyl phenylethyl methacrylamide, N-vinylcarbazole, 4-vinylpyridine, 2-vinylpyridine, and a combination thereof.

6. The method of claim 1 , wherein the hydrogel matrix comprises a monomer selected from the group consisting of N,N′-methylenebisacrylamide, N,N′methylene bismethacrylamide, N,N′-ethylene bisacrylamide, N,N′-ethylene bismethacrylamide, N,N′propylenebisacrylamide, and N,N′-(1,2-dihydroxyethylene) bisacrylamide.

7. The method of claim 1 , wherein the magnetic capture hydrogel particle has a diameter of at least 5 nm.

8. The method of claim 1 , wherein the magnetic capture hydrogel particle is spherical and has a diameter of between about 2 μm and about 5 μm.

9. The method of claim 1 , wherein the target cell is selected from the group consisting of a B cell, a T cell, a natural killer cell, a lymphokine-activated killer cell, a monocyte, a macrophage, a neutrophil, a granulocyte, a mast cell, a platelet, a Langerhans cell, a stem cell, a dendritic cell, a peripheral blood mononuclear cell, a tumor infiltrating (TIL) cell, a gene modified immune cell, and a drug modified immune cell.

10. The method of claim 1 , wherein the target cell comprises a biomarker.

11. The method of claim 1 , wherein the target cell comprises a target molecule selected from the group consisting of CD2, CD3, CD4, CD8, CD19, CD20, CD25, CD27, CD28, CD30, CD40, CD40L, CD45RO, CD62L, CD69, CD62L, CD80, CD84, CD86, CD95, CD122, CD127, CD134, CD137, CD137L, CD150, CD154, CD226, CD229, CD244, CD270, CD278, CD319, CD352, CD355, CD357, membrane-bound IL-15, HLA-DR, CCR6, and FOXP3.

12. The method of claim 9 , wherein the magnetic capture hydrogel particle comprises one or more molecules that support cell growth, stimulate proliferation, or activate the target cell.

13. The method of claim 12 , wherein the one or more molecules comprises cytokines, growth factors, cytokine receptors, extracellular matrix, transcription factors, secreted polypeptides, growth factor receptors, fragments thereof, or a combination thereof,

wherein the one or more molecules are embedded or encapsulated within the hydrogel matrix, or attached to a surface of the magnetic capture hydrogel particle.

14. The method of claim 1 , wherein the magnetic capture hydrogel particle is capable of inducing or enhancing T cell growth, division, differentiation, expansion, proliferation, activity, viability, exhaustion, anergy, quiescence, apoptosis, cell death, or a combination thereof.

15. The method of claim 1 , wherein the magnetic capture hydrogel particle is capable of stimulating or expanding a T cell in the aqueous mixture, or depleting or removing a T cell from the aqueous mixture.

16. The method of claim 15 , wherein the T cell is a natural killer (NK) cell, a CD3+ T cell, a CD4+ T cell, a CD8+ T cell, a regulatory T cell (Treg), a cytotoxic T cell, a Th1 cell, a Th2 cell, or a recombinant T cell.

17. The method of claim 16 , wherein the recombinant T cell is a CAR-T cell.

18. The method of claim 1 , wherein the magnetic capture hydrogel particle comprises one or more T cell costimulatory molecules.

19. The method of claim 18 , wherein the one or more T cell costimulatory molecules comprise D28, 4.1BB (CD137), OX40 (CD134), CD27 (TNFRSF7), GITR (CD357), CD30 (TNFRSF8), HVEM (CD270), LTBR (TNFRSF3), DR3 (TNFRSF25), ICOS (CD278), CD226 (DNAM1), CRTAM (CD355), TIM1 (HAVCR1, KIM1), CD2 (LFA2, OX34), SLAM (CD150, SLAMF1), 2B4 (CD244, SLAMF4), Ly108 (NTBA, CD352, SLAMF6), CD84 (SLAMF5), Ly9 (CD229, SLAMF3), CRACC (CD319, BLAME), or a combination thereof.

20. The method of claim 1 , wherein the magnetic capture hydrogel particle comprises a cytokine, an interleukin, a member of the tumor necrosis factor superfamily, or a combination thereof.

21. The method of claim 20 , wherein the cytokine is selected from the group consisting of IL-1, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL-15, IL-17, IL-21, interferon γ, IFN alpha, IFN beta, lymphotoxin α, TNFα, and TNFβ.

22. The method of claim 1 , wherein the magnetic capture hydrogel particle comprises one or more of IL-2, IL-7, IL-15, IL-21, CD137L, and CD137.

23. The method of claim 22 , wherein the magnetic capture hydrogel particle comprises:

a) IL-2, IL-15, IL-21, CD137L, and CD137 (TNPRSF9; 4-1BB); or

b) IL-15, IL-21, CD137L, and CD137.

24. The method of claim 1 , wherein the ligand comprises an antibody or a fragment thereof.

25. The method of claim 1 , wherein the ligand comprises an anti-CD3 antibody or fragment thereof, an anti-CD28 antibody or fragment thereof, or a combination thereof.

26. The method of claim 1 , wherein the target cell released in step (d) has a purity of at least 91%.

27. The method of claim 26 , wherein the target cell released in step (d) has a yield of at least 60%.

Assignments (2)

Continuity (7)

References Cited (347)