# US 10,479,976 B2 · Buoyancy enabled separation method and system

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,479,976 B2
- Document ID: US10479976B2
- Status: Granted
- Kind: B2
- Application number: 15/842,803
- Filed: 2017-12-14
- Published: 2018-06-21
- Granted: 2019-11-19
- Earliest claimed: 2016-12-16
- Assignees: Diagnologix, LLC
- Applicants: Diagnologix, LLC
- Inventors: Guixin Shi; Kimberly Liu; Ying-Ting Wang
- Web page: https://patentgrep.com/patent/US10479976B2

## CPC

- C12N 5/0636: {T lymphocytes}
- A61K 39/0011: {Cancer antigens}
- C12M 47/02: {Separating microorganisms from the culture medium; Concentration of biomass (separating microorganisms from their culture media C12N1/02)}
- C12N 5/0081: {Purging biological preparations of unwanted cells}
- C12N 5/0087: {Purging against subsets of blood cells, e.g. purging alloreactive T cells}
- C12N 5/0093: {Purging against cancer cells}
- G01N 33/536: with immune complex formed in liquid phase
- G01N 33/538: by sorbent column, particles or resin strip {, i.e. sorbent materials}
- G01N 33/5432: {Liposomes or microcapsules}
- G01N 33/544: the carrier being organic
- G01N 33/56972: {White blood cells}
- A61K 2039/5156: {expressing foreign proteins}
- A61K 2039/5158: {Antigen-pulsed cells, e.g. T-cells}
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 2319/03: containing a transmembrane segment
- C12N 2501/50: Cell markers; Cell surface determinants
- C12N 2501/515: CD3, T-cell receptor complex
- C12N 2501/599: with CD designations not provided for elsewhere
- 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)
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

A buoyancy enabled separation method for isolation from a sample including a variety of different cells a sparse subset of cells that is differentiated by a plurality of different cell surface markers. Microbubbles conjugated to antibodies are applied sequentially in a container with the previously used microbubbles disrupted prior to applying the next microbubbles conjugated to a different antibody. A system that includes a syringe-like container with a plunger and closeable opening. Further, a method for activating and expanding isolated T cells by applying antigen presenting microbubbbles having a flexible lipid shell mimicking an antigen presenting cell for generating immunological synapses.

## Claims

### Claim 1

A buoyancy enabled separation method for isolation from a sample including a variety of different cells a sparse subset of cells that is differentiated by a plurality of different cell surface markers, comprising:
a) placing the sample in a container;
b) adding first flexible shell microbubbles having a flexible shell with an inner bubble wall enclosing a gas and an outer bubble wall conjugated to a first antibody capable of binding to a first cell surface marker of a first subset of cells encompassed in the sample;
c) incubating over a time span sufficient to allow an interaction between the first antibody and the first cell surface marker binding the first antibody to the first cell surface marker;
d) allowing the first flexible shell microbubbles with the first subset of cells bound to the first flexible shell microbubbles to separate by flotation from a remainder of the sample;
e) removing waste from the container, including cells within the sample other than the first subset of cells;
f) disrupting the first flexible shell microbubbles such that the first set of isolated cells are no longer buoyant;
g) adding second flexible shell microbubbles having a flexible shell with an inner bubble wall enclosing a gas and an outer bubble wall conjugated to a second antibody capable of binding to a second cell surface marker of a second subset of cells encompassed within the first subset of cells;
h) incubating over a time span sufficient to allow an interaction between the second antibody and the second cell surface marker binding the second antibody to the at least one second cell surface marker;
i) allowing the second flexible shell microbubbles with the second subset of cells bound to the second flexible shell microbubbles to separate by flotation from a remainder of the sample; and
j) removing waste from the container, including cells within the first subset of cells other than the second subset of cells.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the first and second cell surface markers is one of CD8, CD62L, CD45RA, CD3 and CD28.

### Claim 3 (depends on 1)

The method of claim 1, further comprising:
k) disrupting the second flexible shell microbubbles such that the isolated first sot second subset of cells are no longer buoyant; and
l) repeating steps g) through k) for a third subset of cells by applying additional flexible shell microbubbles conjugated to not previously used antibodies capable of binding to targeted cell surface markers that have not been previously targeted.

### Claim 4 (depends on 3)

The method of claim 3, wherein the cell surface markers are CD8+, CD62L+ and CD45RA+, and the sparse subset of cells are naïve T cells.

### Claim 5 (depends on 4)

The method of claim 4, further comprising repeating step I) for the nth subset of cells by applying additional flexible shell microbubbles conjugated to not previously used antibodies capable of binding to targeted cell surface markers that have not been previously targeted.

### Claim 6 (depends on 1)

The method of one of claim 1, further comprising that the method step of removing waste from the container includes removing the waste from the bottom of the container.

### Claim 7 (depends on 6)

The method of claim 6, further comprising that the method step of removing waste from the container includes moving a plunger within the container.

### Claim 8 (depends on 1)

The method of one of claim 1, further comprising the method step of removing waste from the container by a negative selection by removing waste in form of undesired cells from the container by binding these undesired cells to respective microbubbles to be buoyant to the top of the container and removing these undesired cells bound to respective microbubbles from that top of the container.

### Claim 9 (depends on 8)

The method of claim 8, further comprising that the method step of removing waste from the container includes moving a plunger within the container.

### Claim 10 (depends on 1)

The method of claim 1, further comprising that the method step of disrupting the first, second and subsequent flexible shell microbubbles includes increasing the pressure to which the flexible shell microbubbles are subjected.

### Claim 11 (depends on 10)

The method of claim 10, further comprising that increasing the pressure to which the microbubbles are subjected includes moving a plunger within the container.

### Claim 12 (depends on 1)

The method of claim 1, wherein the sparse subset of cells includes T-cells and the method further comprises:
m) adding to the sparse subset of cells ligands presenting flexible shell microbubbles having a flexible shell with an inner bubble wall enclosing a gas and an outer bubble wall conjugated to ligands capable of forming an immunological synapse with the T-cells;
o) incubating the T cells with the ligands presenting flexible shell microbubbles over a time span that is sufficient for activating and expanding the sparse subset of T cells;
p) achieving specific T-cell activation through combining with a unique peptide bound to a recombinant MHC, and anti-CD28 or with co-stimulating molecules; and
q) achieving nonspecific T-cell activation through combining anti-CD3, and anti-CD28 or with co-stimulating molecules.

### Claim 13 (depends on 12)

The method of claim 12, further comprising r) achieving specific T-cell activation through combining with co-stimulating molecules recombinant CD80 and CD86.

### Claim 14 (depends on 12)

The method of claim 12, further comprising r) achieving nonspecific T-cell activation through combining with co-stimulating molecules recombinant CD80 and CD86.

### Claim 15 (depends on 1)

The method of claim 1, wherein the sample includes one of or a combination of T cells, B cells, tumor-infiltrating lymphocytes, dendritic cells, natural killer cells, endothelial cells, stem cells and cancer cells from human or animal blood, other human or animal body fluids or artificial buffer solutions.

### Claim 16 (depends on 1)

The method of claim 1, wherein the isolation of the sparse subset of cells from the sample including a variety of different cells is made by a positive selection, negative selection or a sequential combination of both.