Allogeneic t-cells and methods for production thereof

Patent metadata

Field	Value
Publication number	US-2021171908-A1
Application number	US-202017088759-A
Country	US
Kind code	A1
Filing date	Nov 4, 2020
Priority date	Nov 5, 2019
Publication date	Jun 10, 2021
Grant date	—

Abstract

The present invention provides methods for producing allogeneic T-cells, including the use of an engineered nuclease under the control of a controllable promoter. By preparing T-cells with an inducible nuclease, large volumes of cells can be prepared, each of which contains the ability to individually produce the desired nuclease. These cells can then be modified as desired through the introduction of a gene of interest, or an undesired gene can be knocked-out. Also provided herein are allogeneic T-cells for use in various therapeutic applications.

First claim

What is claimed is:

1 . A method of producing a T-cell line for use in an allogeneic application, comprising:
a. introducing a nucleic acid molecule encoding an engineered nuclease under the control of a controllable promoter into a T-cell line; b. integrating the nucleic acid molecule into the genome of the T-cell line; and c. expanding the T-cell line.

2 . The method of claim 1 , wherein the engineered nuclease is selected from the group consisting of a meganuclease, a zinc finger nuclease, a transcription activator-like effector-based nuclease, and a CRISPR-associated nuclease.

3 . The method of claim 2 , wherein the CRISPR-associated nuclease is a Cas9 nuclease or a Cas12 nuclease.

4 . The method of claim 1 , wherein the controllable promoter is an inducible promoter, selected from the group consisting of a 4HT inducible promoter, a rapamycin inducible promoter, a hormone response element, and a glutamate inducible promoter.

5 . (canceled)

6 . The method of claim 1 , wherein the controllable promoter comprises a Tet-on system and is a derepressible promoter, and wherein the derepressible promoter includes one or more tetracycline operator sequences (TetO).

7 . (canceled)

8 . The method of claim 6 , wherein the nucleic acid molecule further includes a tetracycline repressor protein.

9 . (canceled)

10 . The method of claim 1 , wherein the T-cell line comprises at least about 10 9 T-cells and following the expanding, the method further comprises freezing the expanded T-cell line.

11 . (canceled)

12 . (canceled)

13 . A method of producing a genetically modified T-cell line, comprising:
a. introducing a nucleic acid molecule encoding a CRISPR-associated nuclease under the control of a controllable promoter into a T-cell line; b. integrating the nucleic acid molecule into the genome of the T-cell line; c. expanding the T-cell line; d. inducing expression of the CRISPR-associated nuclease by activating the controllable promoter; e. introducing a guide-RNA and a gene of interest into the expanded T-cell line; f. knocking out expression of a T-cell receptor and introducing the gene of interest into the genome of the T-cell line; and g. recovering the genetically modified T-cell line.

14 . The method of claim 13 , wherein the CRISPR-associated nuclease is a Cas9 nuclease or a Cas12 nuclease.

15 . The method of claim 13 , wherein the controllable promoter is an inducible promoter selected from the group consisting of a 4HT inducible promoter and a glutamate inducible promoter.

16 . (canceled)

17 . The method of claim 13 , wherein the controllable promoter is a derepressible promoter, and wherein the derepressible promoter includes one or more tetracycline operator sequences (TetO) and activating the derepressible promoter comprises adding doxycycline to the T-cell line.

18 . (canceled)

19 . The method of claim 17 , wherein the nucleic acid molecule further includes a tetracycline repressor protein.

20 . (canceled)

21 . The method of claim 13 , wherein the controllable promoter comprises a Tet-on system, and wherein the activating the Tet-on system comprises adding doxycycline to the T-cell line.

22 . (canceled)

23 . The method of claim 13 , wherein the gene of interest encodes a chimeric antigen receptor (CAR).

24 . The method of claim 13 , wherein the genetically modified T-cell line comprises at least about 10 9 T-cells and following expanding of the T-cells, and wherein the method further comprises freezing the T-cell line and thawing prior to the inducing.

25 . (canceled)

26 . A method of producing a chimeric antigen receptor (CAR) T-cell line, comprising:
a. introducing a nucleic acid molecule encoding a Cas9 nuclease or a Cas12 nuclease under the control of a controllable promoter into a T-cell line; b. integrating the nucleic acid molecule into the genome of the T-cell line; c. expanding the T-cell line; d. inducing expression of the Cas9 nuclease or Cas12 nuclease by activating the controllable promoter; e. introducing a guide-RNA and a nucleic acid encoding a chimeric antigen receptor (CAR) into the expanded T-cell line; f. knocking out expression of a T-cell receptor and introducing the nucleic acid encoding the CAR into the genome of the T-cell line; and g. recovering the CAR T-cell line.

27 . The method of claim 26 , wherein the controllable promoter is an inducible promoter selected from the group consisting of a 4HT inducible promoter and a glutamate inducible promoter.

28 . (canceled)

29 . The method of claim 26 , wherein the controllable promoter is a derepressible promoter, and wherein the derepressible promoter includes one or more tetracycline operator sequences (TetO) and activating the derepressible promoter comprises adding doxycycline to the T-cell line.

30 . (canceled)

31 . The method of claim 29 , wherein the nucleic acid molecule further includes a tetracycline repressor protein.

32 . (canceled)

33 . The method of claim 26 , wherein the controllable promoter comprises a Tet-on system, and wherein the activating the Tet-on system comprises adding doxycycline to the T-cell line.

34 . (canceled)

35 . The method of claim 26 , further comprising freezing the T-cell line following the expanding in c, and thawing prior to the inducing in d.

36 . The method of claim 26 , wherein the CAR T-cell line comprises at least about 10 9 T-cells.

37 . An allogeneic T-cell line, comprising a CRISPR-associated (Cas) nuclease under the control of a controllable promoter integrated into the genome of the T-cell line.

38 . The allogeneic T-cell line of claim 37 , wherein the CRISPR-associated nuclease is a Cas9 nuclease or a Cas12 nuclease.

39 . The allogeneic T-cell line of claim 37 , wherein the controllable promoter is an inducible promoter selected from the group consisting of a 4HT inducible promoter and a glutamate inducible promoter.

40 . (canceled)

41 . The allogeneic T-cell line of claim 37 , wherein the controllable promoter is a derepressible promoter, and wherein the derepressible promoter includes one or more tetracycline operator sequences (TetO).

42 . (canceled)

43 . The allogeneic T-cell line of claim 41 , wherein T-cell further includes a nucleic acid encoding a tetracycline repressor protein.

44 . The allogeneic T-cell line of claim 37 , comprising at least about 10 9 T-cells and, wherein the controllable promoter comprises a Tet-on system.

45 . (canceled)

46 . The allogeneic T-cell line of claim 44 , comprising at least about 10 10 T-cells.

47 . The allogeneic T-cell line of claim 44 , further comprising a transposon.

48 . The method of claim 1 , wherein the integrating the nucleic acid molecule into the genome of the T-cell line includes the use of a transposon.

49 . The method of claim 13 , wherein the integrating the nucleic acid molecule into the genome of the T-cell line includes the use of a transposon.

Assignees

• Lonza Walkersville Inc
• Lonza Israel Ltd

Inventors

• Levinson Yonatan
• Ramaswamy Senthil
• Sargent Alex
• Gilert Ariel
• Feldman Alona
• Gotlieb Frida

Classifications

• C12N2740/16043

  viral genome or elements thereof as genetic vector · CPC title

• C12N2800/80

  Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• A61P35/00

  Antineoplastic agents · CPC title

• A61K40/50

  characterised by the use of allogeneic cells · CPC title