Methods of making chimeric antigen receptor-expressing cells

Patent metadata

Field	Value
Publication number	US-2019269727-A1
Application number	US-201616066788-A
Country	US
Kind code	A1
Filing date	Dec 27, 2016
Priority date	Dec 28, 2015
Publication date	Sep 5, 2019
Grant date	—

Abstract

The invention provides methods of making immune effector cells (e.g., T cells, NK cells) that can be engineered to express a chimeric antigen receptor (CAR), and compositions and reaction mixtures comprising the same.

First claim

1 . A method of making a population of immune effector cells (e.g., T cells) that can be engineered to express a chimeric antigen receptor (CAR), the method comprising:
a) providing a frozen input sample comprising immune effector cells, b) thawing the frozen input sample, to produce a thawed sample, and c) performing elutriation on the thawed sample and collecting immune effector cells, thereby producing an output sample comprising immune effector cells that are suitable for expression of a CAR.

2 . The method of claim 1 , wherein:
(i) the frozen input sample is a plasma apheresis sample; (ii) the input sample comprises:
(1) at least 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 35%, or 40% monocytes;
(2) less than 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% T cells; and/or
(3) at least 1%, 2%, 5%, 10%, 15%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% B cells;
(iii) the output sample comprises:
(1) less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 2%, 1%, 0.5%, 0.2%, or 0.1% monocytes;
(2) at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% T cells;
(3) less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 2%, 1%, 0.5%, 0.2%, or 0.1% B cells;
(4) at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% CD4+CD25+ cells; and/or
(5) at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% CD8+CD25+ cells; or
(iv) the method has a T cell yield recovery of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% T cells.

3 . The method of claim 1 , wherein:
(i) the method further comprises one, two, three or all of:
d) depleting CD19+ cells under flow conditions;
e) performing density centrifugation using a medium comprising iodixanol and/or having a density greater than Ficoll;
f) performing a wash step on the thawed sample with a buffer comprising dextrose and/or sodium chloride; and
g) performing a positive selection of CD3/CD28+ cells under flow conditions;
(ii) the method comprises a step of adjusting the viscosity of the thawed sample by adding an isotonic solution to the thawed sample; or (iii) the elutriation is performed:
(1) using a flow rate of from about 30-82 mL/min or 50-80 mL/min and/or the collection volume is about 250-1250 mL or 300-1000 mL for each fraction;
(2) using a collection volume of about 250, 400, 500, 900, or 975 mL; and/or
(3) at about 2400 rpm.

4 - 8 . (canceled)

9 . The method of claim 1 , wherein the output sample is contacted with a nucleic acid encoding a CAR.

10 . The method of claim 9 , wherein, after contacting the output sample with the nucleic acid encoding a CAR,
(i) the output sample comprises:
(1) at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% CAR+CD4+ central memory cells;
(2) at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% CAR+ cells; and/or
(3) at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% CAR+CD8+ central memory cells;
(ii) the output sample produces less than 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 pg IFN-gamma per transduced cell; and/or (iii) the output sample comprises a cytotoxicity level of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30.

11 . (canceled)

12 . A method of making a population of immune effector cells that can be engineered to express a CAR, the method comprising:
a) providing an input sample comprising immune effector cells, and b) performing density centrifugation step using a medium comprising iodixanol, thereby producing an output sample comprising immune effector cells that are suitable for expression of a CAR.

13 . The method of claim 12 , wherein:
(i) the method further comprises performing one, two, three, or all of:
c) depleting CD19+ cells under flow conditions;
d) elutriation on the input sample, wherein the input sample is a thawed input sample;
e) performing a wash step with a buffer comprising dextrose and/or sodium chloride; and
f) positive selection of CD3/CD28+ cells under flow conditions;
(ii) the method does not comprise one or more of:
(1) using a solution comprising glycol;
(2) performing a wash step in a buffer comprising dextrose and/or sodium chloride; or
(3) performing a positive selection step; or
(iii) the density centrifugation is performed using a cell separation device.

14 - 15 . (canceled)

16 . The method of claim 12 , wherein:
(i) the input sample comprises:
(1) less than 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, or 15% T cells;
(2) at least 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% monocytes; and/or
(3) at least 1%, 2%, 5%, 10%, 15%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% B cells;
(ii) the output sample comprises:
(1) at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% T cells;
(2) less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 2%, 1%, 0.5%, 0.2%, or 0.1% monocytes; and/or
(3) less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.05%, or 0.01% B cells; or
(iii) the method has a T cell yield recovery of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% T cells.

17 - 18 . (canceled)

19 . A method of making a population of immune effector cells that can be engineered to express a CAR, the method comprising:
a) providing an input sample comprising immune effector cells, and b) removing CD19+ cells from the input sample under flow conditions or positively selecting for CD3+/CD28+ cells from the input sample under flow conditions, thereby producing an output sample comprising immune effector cells that are suitable for expression of a CAR.

20 . The method of claim 61 , wherein:
(i) the method further comprises performing one, two, three or all of:
c) elutriation on the input sample, wherein the input sample is a thawed input sample;
d) a density centrifugation step using a medium comprising iodixanol and/or having a density greater than Ficoll;
e) performing a wash step with a buffer comprising dextrose and/or sodium chloride; and
f) positive selection of CD3/CD28+ cells under flow conditions; or
(ii) the method does not comprise performing elutriation or density centrifugation.

21 . (canceled)

22 . The method of claim 61 , wherein:
(i) the CD19+ cells comprise B cells; (ii) the input sample comprises:
(1) at least 1%, 2%, 5%, 10%, 15%, 20%, 30%, 35%, 40%, 45%, or 50% CD19+ cells;
(2) at least 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 35%, or 40% monocytes; and/or
(3) less than 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20% T cells;
(iii) the output sample comprises:
(1) less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.05%, or 0.01% CD19+ cells;
(2) less than 50%, 45%, 40%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 2%, 2%, or 1% the percentage of CD19+ cells compared to the input sample;
(3) less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 2%, 1%, 0.5%, 0.2%, or 0.1% monocytes;
(4) at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% T cells; and/or
(5) less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 2%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.05%, or 0.01% B cells;
(iv) the method has a T cell yield recovery of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% T cells; or (

Assignees

• Fachin Fabio
• Cao Lan
• Greene Michael R
• Golovina Tatiana
• Novartis Ag
• Univ Pennsylvania

Inventors

• Fachin Fabio
• Cao Lan
• Greene Michael R
• Golovina Tatiana

Classifications

• A61P35/02

  specific for leukemia · CPC title

• A61P35/00

  Antineoplastic agents · CPC title

• C07K1/042

  characterised by the nature of the carrier · CPC title

• C07K14/7051

  T-cell receptor (TcR)-CD3 complex · CPC title

• C12N5/0636 Primary

  T lymphocytes · CPC title