# US 2020/0399600 A1 · Methods for Preparing Therapeutically Active Cells Using Microfluidics

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- Number: US 2020/0399600 A1
- Document ID: US20200399600A1
- Status: Published application
- Kind: A1
- Application number: 17/009,797
- Filed: 2020-09-02
- Published: 2020-12-24
- Earliest claimed: 2017-09-01
- Assignees: GPB Scientific, LLC; University of Maryland, Baltimore; The Trustees of Princeton University
- Applicants: Gpb Scientific, Inc.; University of Maryland, Baltimore; The Trustees of Princeton University
- Inventors: Anthony WARD; Roberto CAMPOS-GONZALEZ; Alison SKELLEY; Khushroo GANDHI; Curt CIVIN; James C. STURM; Michael GRISHAM
- Web page: https://patentgrep.com/patent/US20200399600A1

## CPC

- C12N 5/0636: {T lymphocytes}
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/31: Chimeric antigen receptors [CAR]
- A61K 40/42: Cancer antigens
- A61K 40/4254: {Adhesion molecules, e.g. NRCAM, EpCAM or cadherins}
- A61P 35/02: specific for leukemia
- B01L 3/502753: {characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation}
- B01L 3/502761: {specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads or physically stretching molecules}
- C12N 5/0087: {Purging against subsets of blood cells, e.g. purging alloreactive T cells}
- B01L 2200/0652: Sorting or classification of particles or molecules
- B01L 2300/0816: Cards, e.g. flat sample carriers usually with flow in two horizontal directions
- B01L 2300/0864: comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
- B01L 2400/086: using baffles or other fixed flow obstructions
- C12N 2531/00: Microcarriers
- C12N 2533/54: Collagen; Gelatin
- C12N 2533/74: Alginate
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE

## Abstract

The present invention is directed to the use of microfluidics in the preparation of cells and compositions for therapeutic uses.

## Claims

### Claim at position 1

1-273. (canceled)

### Claim 274

A method for preparing T cells from a sample that has been obtained from a patient and which comprises T cells and platelets, said method comprising:
a) purifying the T cells from the sample by a combination of:
i) a size based separation on a microfluidic device, wherein the device comprises:
aa) at least one channel extending from a sample inlet to fluid outlets;
bb) obstacles that are disposed in the channel such that when the sample is applied to the sample inlet and passed through the channel, T cells are diverted from the direction of bulk fluid flow to one or more collection outlets where an enriched product is collected and platelets follow the direction of bulk fluid flow to one or more waste outlets that are separate from the one or more collection outlets;
ii) an affinity separation that is performed in addition to the size based separation; and
b) expanding the T cells purified in step a) by culturing the T cells;
wherein the T cells prepared by the method comprise a higher percentage of central memory T cells than the percentage of central memory T cells in the sample prior to performing the method.

### Claim 275 (depends on 274)

The method of claim 274, wherein the sample is an apheresis sample from a patient with cancer.

### Claim 276 (depends on 275)

The method of claim 275, wherein the apheresis sample is obtained by leukapheresis.

### Claim 277 (depends on 274)

The method of claim 274, wherein the sample is an apheresis sample from a patient with an autoimmune or infectious disease.

### Claim 278 (depends on 277)

The method of claim 277, wherein the apheresis sample is obtained by leukapheresis.

### Claim 279 (depends on 274)

The method of claim 274, wherein during the expansion of the T cells by culturing, a greater percentage of T cells express cluster of differentiation (CD) antigens characteristic of a memory phenotype than T cells obtained by affinity separation alone.

### Claim 280 (depends on 274)

The method of claim 274, wherein the affinity separation step comprises binding the T cells to magnetic beads that bind specifically to T cells.

### Claim 281 (depends on 274)

The method of claim 274, wherein after the T cells have been purified in step a), they are genetically engineered to express a therapeutically active protein.

### Claim 282 (depends on 281)

The method of claim 281, wherein the sample is obtained from a patient with cancer and the T cells are genetically engineered to produce chimeric antigen receptors on their surface.

### Claim 283 (depends on 282)

The method of claim 282, further comprising, administering the cells genetically engineered to produce chimeric antigen receptors on their surface to the same patient from which the sample was obtained.

### Claim 284 (depends on 283)

The method of claim 283, wherein the sample is an apheresis sample.

### Claim 285 (depends on 284)

The method of claim 284, wherein the apheresis sample is obtained by leukapheresis.

### Claim 286

A method for preparing T cells from a sample that has been obtained from a patient and that comprises T cells and platelets, said method comprising:
a) purifying the T cells from the sample by a combination of:
i) deterministic lateral displacement (DLD);
ii) an affinity separation that is performed in addition to said DLD; and
b) expanding the T cells purified in step a) by culturing the T cells;
wherein the T cells prepared by the method comprise a higher percentage of central memory T cells than the percentage of central memory T cells in the sample prior to performing the method.

### Claim 287 (depends on 286)

The method of claim 286, wherein said DLD is performed before said affinity separation step.

### Claim 288 (depends on 286)

The method of claim 286, wherein the affinity separation step comprises binding the T cells to magnetic beads that bind specifically to T cells.

### Claim 289 (depends on 288)

The method of claim 288, wherein after the T cells have been purified in step a), they are genetically engineered to express a therapeutically active protein.

### Claim 290 (depends on 289)

The method of claim 289, further comprising, administering the cells genetically engineered to express a therapeutically active protein to the same patient from which the sample was obtained.

### Claim 291 (depends on 288)

The method of claim 288, wherein the patient has cancer and the T cells are genetically engineered to produce chimeric antigen receptors on their surface.

### Claim 292 (depends on 291)

The method of claim 291, further comprising, administering the cells genetically engineered to produce chimeric antigen receptors on their surface to the same patient from which the sample was obtained.

### Claim 293 (depends on 292)

The method of claim 292, wherein the sample obtained from said patient is an apheresis sample.

### Claim 294 (depends on 288)

The method of claim 288, wherein during the expansion of the T cells by culturing, a greater percentage of T cells express cluster of differentiation (CD) antigens characteristic of a memory phenotype than T cells obtained by affinity separation alone.

### Claim 295 (depends on 288)

The method of claim 288, wherein the yield of T cells expressing the chimeric receptors on their surface is at least 20% greater than T cells isolated by apheresis or leukapheresis and subjected to magnetic separation but not DLD.

### Claim 296 (depends on 274)

A method of producing CAR T cells for administration to a patient, comprising:
a) obtaining an apheresis sample from a patient, wherein the apheresis sample comprises T cells;
b) purifying the T cells by the method of claim 274, step a);
c) genetically engineering the T cells purified in step b) to produce chimeric antigen receptors (CARs) on their surface;
d) expanding the T cells purified in step b) by culturing the T cells;
e) transferring the T cells into a pharmaceutical composition for administration to a patient.

### Claim 297 (depends on 296)

The method of claim 296, wherein the affinity separation step comprises binding the T cells to magnetic beads that bind specifically to T cells.

### Claim 298 (depends on 297)

The method of claim 297, wherein, in step a)i), T cells are separated from platelets by deterministic lateral displacement (DLD) performed on a microfluidic device comprising:
a) 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;
b) 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 sample obtained from said patient 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 cells, or particles that are in the sample and that are of a different size than the T cells, flow to one more waste outlets that are separate from the collection outlets.

### Claim 299 (depends on 298)

The method of claim 298, wherein the yield of T cells expressing the chimeric receptors on their surface is at least 20% greater than T cells isolated by apheresis or leukapheresis and subjected to magnetic separation but not DLD.

### Claim 300 (depends on 298)

The method of claim 298, further comprising, administering the cells genetically engineered to produce chimeric antigen receptors on their surface to the same patient from which the sample was obtained.