Methods for preparing therapeutically active cells using microfluidics

1. A method for preparing cells for treating a patient with cancer, comprising the steps:

a) purifying T cells from an apheresis sample from said patient, wherein the sample comprises leukocytes and platelets, wherein the leukocytes comprise T cells and wherein the T cells are purified by:

i) performing a size based separation using a microfluidic device to produce an enriched product in which, compared to the sample, the ratio of platelets to T cells has been reduced by at least 70%; and wherein the microfluidic device comprises:

at least one channel extending from a sample inlet to one or more fluid outlets, wherein the channel is bounded by a first wall and a second wall opposite from the first wall;

an array of obstacles disposed in the channel in a manner such that, when the sample is applied to an inlet of the device and fluidically passed through the channel, T cells flow to one or more collection outlets to form an enriched product, and platelets flow to one more waste outlets that are separate from the collection outlets;

ii) in addition to the size based separation, performing an affinity based separation by binding T cells to a carrier that binds to T cells with specificity, and then separating the carrier-bound T cells from cells not bound to carrier;

b) after the purification of step a), activating and expanding the T cells to produce a composition in which the percentage of T cells that are central memory T cells has increased compared to the percentage of T cells that are central memory T cells in the sample; and in which the percentage of T cells that are central memory T-cells is greater than in a method in which the sample is not purified by a size based separation using a microfluidic device to reduce the ratio of platelets to T-cells prior to activation and expansion;

c) after the purification of step a), genetically engineering activated T cells to comprise modified cell surface receptors of therapeutic benefit in the treatment of said patient's cancer;

wherein the T cells in the sample are not centrifuged during the method.

2. The method of claim 1 , wherein the carrier of paragraph a)ii) is magnetized.

3. The method of claim 1 , wherein, after the cells are prepared, they are administered to said patient.

4. The method of claim 1 , wherein in paragraph c), the modified cell surface receptors of therapeutic benefit are chimeric antigen receptors (CARs).

5. The method of claim 1 , wherein the percentage of central memory T cells relative to the total number of T cells produced in the method is at least 20% higher than in a method in which centrifugation is used in step a)i) instead of a size based separation on said microfluidic device.

6. The method of claim 1 , wherein the sample is a leukapheresis sample.

7. The method of claim 6 , wherein the carrier of paragraph a)ii) is magnetized.

8. The method of claim 6 , wherein, in paragraph c), the modified cell surface receptors of therapeutic benefit are chimeric antigen receptors (CARs).

9. The method of claim 6 , wherein the percentage of central memory T cells relative to the total number of T cells produced in the method is at least 20% higher than in a method in which centrifugation is used in step a)i) instead of a size based separation on said microfluidic device.

10. The method of claim 6 , wherein the platelets in the enriched product of paragraph a)ii) are depleted by at least 80% compared to the sample and/or there are no more than 5 platelets per leukocyte in the enriched product.

11. A method for treating a patient with cancer, comprising administering to said patient a therapeutic composition comprising genetically engineered T cells, wherein the genetically engineered T cells have been prepared by a method comprising:

a) purifying T cells from an apheresis or leukapheresis sample prepared from said patient, wherein the sample comprises leukocytes and platelets, wherein the leukocytes comprise T cells and the T cells are purified by:

i) performing a size based separation using a microfluidic device to produce an enriched product in which, compared to the apheresis or leukapheresis sample, the ratio of platelets to T cells has been reduced by at least 50%; and wherein the microfluidic device comprises:

at least one channel extending from a sample inlet to one or more fluid outlets, wherein the channel is bounded by a first wall and a second wall opposite from the first wall;

an array of obstacles disposed in the channel in a manner such that, when the sample is applied to an inlet of the device and fluidically passed through the channel, T cells flow to one or more collection outlets to form an enriched product, and platelets flow to one more waste outlets that are separate from the collection outlets;

ii) in addition to the size based separation, performing an affinity based separation by binding T cells to a carrier that binds to T cells with specificity, and then separating the carrier-bound T cells from cells not bound to carrier;

b) after the purification of step a), activating and expanding the T cells to produce a composition in which the percentage of T cells that are central memory T cells has increased compared to the percentage of T cells that are central memory T cells in the sample; and in which the percentage of T cells that are central memory T-cells is greater than in a method in which the sample is not purified by a size based separation using a microfluidic device to reduce the ratio of platelets to T-cells prior to activation and expansion;

c) after the purification of step a), genetically engineering activated T cells to comprise modified cell surface receptors of therapeutic benefit in the treatment of said patient's cancer;

wherein the T cells are not centrifuged during the method.

12. The method of claim 11 , wherein the carrier of paragraph a)ii) is magnetized.

13. The method of claim 11 , wherein in paragraph c), the modified cell surface receptors of therapeutic benefit are chimeric antigen receptors (CARs).

14. The method of claim 11 , wherein the enriched product produced in paragraph a)i) has a ratio of platelets to leukocytes that is at least 70% lower than in said apheresis or leukapheresis sample.

15. The method of claim 11 , wherein the platelets in the enriched product of paragraph a)i) are depleted by at least 80% compared to the sample and/or there are no more than 5 platelets per leukocyte in the enriched product.

16. The method of claim 11 , wherein the percentage of central memory T cells relative to the total number of T cells produced in the method is at least 20% higher than in a method in which centrifugation is used in step a)i) instead of a size based separation on said microfluidic device.

17. The method of claim 11 , wherein the T cells are not frozen prior to being genetically engineered.

18. The method of claim 11 , wherein the T cells are not activated or bound to a carrier prior to size based separation of paragraph a)i).

19. The method of claim 18 , wherein the T cells are not frozen prior to being genetically engineered.

20. The method of claim 18 , wherein:

aa) the platelets in the enriched product of paragraph ai) are depleted by at least 80% compared to the sample and/or there are no more than 5 platelets per leukocyte in the enriched product;

bb) in paragraph c) of the method, the modified cell surface receptors of therapeutic benefit are chimeric antigen receptors (CARs).

21. The method of claim 20 , wherein the percentage of central memory T cells relative to the total number of T cells produced in the method is at least 20% higher than in a method in which centrifugation is used in step a)i) instead of a size based separation on said microfluidic device.

22. A method for preparing a therapeutic composition comprising genetically engineered T cells, comprising:

a) purifying T cells from an apheresis sample prepared from a patient, wherein the sample comprises leukocytes and platelets, wherein the leukocytes comprise T cells and the T cells are purified by:

i) performing a size based separation using a microfluidic device configured to separate cells by deterministic lateral displacement to produce an enriched product in which, compared to the apheresis sample, the ratio of platelets to T cells has been reduced by at least 70%;

ii) in addition to the size based separation, performing an affinity based separation by binding T cells to a carrier that binds to T cells with specificity, and then separating the carrier-bound T cells from cells not bound to carrier;

b) after the purification of step a), activating and expanding the T cells to produce a composition in which the percentage of T cells that are central memory T cells has increased compared to the percentage of T cells that are central memory T cells in the sample;

c) genetically engineering the activated T cells to comprise modified cell surface receptors;

wherein the percentage of central memory T cells relative to the total number of T cells produced in the method is at least 20% higher than in a method in which centrifugation is used in step a)i) instead of a size based separation on said microfluidic device.

23. The method of claim 22 , wherein the sample is a leukapheresis sample.

24. The method of claim 22 , wherein the carrier of paragraph a)ii) is magnetized.

25. The method of claim 22 , wherein, after the cells are prepared, they are administered to said patient.

26. The method of claim 22 , wherein in paragraph c), the modified cell surface receptors of therapeutic benefit are chimeric antigen receptors (CARs).

27. The method of claim 22 , wherein the platelets in the enriched product of paragraph a)i) are depleted by at least 80% compared to the sample and/or there are no more than 5 platelets per leukocyte in the enriched product.