Granted Patent B2

US 12,612,597 · App. 17/467,385 · Granted Apr 28, 2026

Inventors: Anthony Ward (Rancho Santa Fe, CA); Roberto Campos-Gonzalez (Carlsbad, CA); Alison Skelley (Riverside, CA); Khushroo Gandhi (Palo Alto, CA); Curt Civin (Baltimore, MD); James C. Sturm (Princeton, NJ); Michael Grisham (Richmond, VA)

Assignees: ZEON CORPORATION; UNIVERSITY OF MARYLAND, BALTIMORE; THE TRUSTEES OF PRINCETON UNIVERSITY

C12N5/0636A61K40/11A61K40/31A61K40/42A61K40/4254A61P35/02B01L3/502753B01L3/502761C12N5/0087B01L2200/0652B01L2300/0816B01L2300/0864B01L2400/086C12N2531/00C12N2533/54C12N2533/74

Abstract

Claims (23)

1 . A method of producing CAR T cells, comprising:

a) obtaining an apheresis sample composition, wherein said sample composition is from a patient and comprises T cells;

b) performing deterministic lateral displacement (DLD) followed by separation based on magnetic beads that specifically bind to T cells on the sample composition to reduce the total number of platelets present by at least 70%, wherein DLD is carried out on a microfluidic device comprising:

i) 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;

ii) an array of obstacles arranged in rows in the channel, each subsequent row of obstacles being shifted laterally with respect to a previous row, and wherein said obstacles are disposed in a manner such that, when the crude fluid composition is applied to an inlet of the device and fluidically passed through the channel, T cells in the composition flow to one or more collection outlets where an enriched product is collected, and wherein platelets and other materials smaller than T cells flow to one more waste outlets that are separate from the collection outlets;

and wherein the separation based on magnetic beads is based on binding of the magnetic beads to T cells;

c) genetically engineering the T cells in the enriched product obtained in step b) to produce chimeric antigen receptors (CARs) on their surface;

d) culturing the genetically engineered T cells to expand their number, wherein the number of the genetically engineered T cells obtained after 14 days in culture is at least two times higher than the number produced by the same procedure performed using centrifugation instead of DLD;

e) transferring the genetically engineered T cells into a pharmaceutical composition for administration to a patient.

2 . The method of claim 1 , wherein at least 90% of platelets are removed in step b) and wherein, prior to or during culturing, cells are exposed to a T cell activator.

3 . The method of claim 1 , wherein, in step b) and prior to step c), after performing DLD and separation based on magnetic beads, cells are transferred into a medium in which a T cell activator is present or added.

4 . The method of claim 3 , wherein after performing DLD and separation based on magnetic beads in step b) and prior to step c), the medium containing the activator is further processed by DLD to separate the activator from cells and to transfer cells into a medium in which a vector for recombinantly engineering cells is present or added.

5 . The method of claim 1 , wherein cells are not frozen during steps a) to d).

6 . The method of claim 1 , wherein, in step d), the number of the genetically engineered T cells obtained after 14 days in culture is at least four times higher than the number produced by the same procedure performed using centrifugation instead of DLD.

7 . A method of preparing CAR T cells for the treatment of a patient comprising:

a) obtaining cells collected from a patient by apheresis;

b) performing DLD followed by separation based on magnetic beads that specifically bind to T cells on the cells obtained in step a) to separate leukocytes from other cells and particles, and to transfer the leukocytes into a medium that supports their growth and that has, or is supplemented with, a T cell activator, wherein the separation based on magnetic beads is based on binding of the magnetic beads to T cells;

c) separating T cells from the medium of step b) into medium that contains a vector for genetically engineering the T cells to produce chimeric antigen receptors (CARs) on their surface;

d) performing DLD to separate T cells from reagents and to transfer the cells into a medium for administration to a patient, wherein the yield of T cells exhibiting the desired CAR T phenotype is at least 20% greater than identical cells isolated by centrifugation and not exposed to DLD.

8 . The method of claim 7 , wherein cells are not frozen during steps a) to c).

9 . The method of claim 7 , wherein in step b) a product is obtained in which the ratio of platelets to leukocytes is at least 50% lower than the ratio obtained using centrifugation or elutriation instead of DLD.

10 . The method of claim 7 , wherein the time necessary to produce a sufficient number of T cells for the treatment of a patient is at least 10% shorter than when the same method is carried out using centrifugation rather than DLD to isolate cells from apheresis starting material.

11 . The method of claim 7 , wherein, when cells prepared by said method are administered to a patient, they exhibit at least 10% less senescence than cells that have been processed from an apheresis composition using centrifugation or elutriation instead of DLD.

Assignments (7)

Continuity (8)

References Cited (304)