Methods of producing patient-specific anti-cancer therapeutics and methods of treatment therefor

Patent metadata

Field	Value
Publication number	US-10712344-B2
Application number	US-201715406289-A
Country	US
Kind code	B2
Filing date	Jan 13, 2017
Priority date	Jan 15, 2016
Publication date	Jul 14, 2020
Grant date	Jul 14, 2020

Abstract

A method of preparing an antibody therapeutic is provided comprising: (a) providing a dissociated cell sample from at least one solid tumor sample obtained from a patient; (b) loading the dissociated cell sample into a microfluidic device having a flow region and at least one isolation region fluidically connected to the flow region; (c) moving at least one B cell from the dissociated cell sample into at least one isolation region in the microfluidic device, thereby obtaining at least one isolated B cell; and (d) using the microfluidic device to identify at least one B cell that produces antibodies capable of binding to cancer cells. The cancer cells can be the patient's own cancer cells. Also provided are methods of treating patients, methods of labeling or detecting cancer, engineered T or NK cells comprising antibodies or fragments thereof, and engineered antibody constructs.

First claim

What is claimed is:

1. A method of identifying an isolated B cell that produces antibodies capable of binding to a cancer cell-associated antigen, comprising:
a. providing a dissociated cell sample from at least one solid tumor sample obtained from a patient;
b. loading the dissociated cell sample into a microfluidic device having at least one flow region and at least one isolation region fluidically connected to the flow region;
c. moving at least one B cell from the dissociated cell sample into at least one isolation region in the microfluidic device, thereby obtaining at least one isolated B cell; and
d. identifying at least one isolated B cell that produces antibodies capable of binding to a cancer cell-associated antigen,
wherein (i) the flow region of the microfluidic device comprises a microfluidic channel and each of the at least one isolation regions is part of a corresponding sequestration chamber, and wherein each sequestration chamber further comprises a connection region fluidically connecting the corresponding isolation region to the microfluidic channel, and/or
(ii) the microfluidic device comprises a substrate having a DEP configuration, and wherein moving the at least one B cell into the at least one isolation region comprises using DEP force to move the at least one B cell.

2. The method of claim 1 , wherein said isolation region comprises at least one conditioned surface that promotes B cell lymphocyte viability, said conditioned surface comprising covalently linked molecules, wherein said covalently linked molecules comprise alkylene ether moieties, amino acid moieties, saccharide moieties, or any combination thereof.

3. The method of claim 2 , wherein said at least one conditioned surface comprises a layer of covalently linked hydrophilic molecules.

4. The method of claim 1 , wherein said isolation region comprises at least one surface coated with a coating material that promotes B cell viability, wherein said coating material comprises a polymer comprising alkylene ether moieties, amino acid moieties, saccharide moieties, or any combination thereof.

5. The method of claim 1 , wherein each of the at least one isolation region(s) forms a dead-end in the microfluidic device, and wherein, when the flow region is substantially filled with a flowing first fluidic medium and the isolation region(s) are substantially filled with a second fluidic medium:
a. components of the second medium are able to diffuse into the first medium and components of the first medium are able to diffuse into the second medium; and
b. there is substantially no flow of the first medium from the flow region into the isolation region.

6. The method of claim 5 , wherein the connection region has a width W con of about 20 microns to about 100 microns.

7. The method of claim 6 , wherein each of the at least one isolation region(s) has a width W iso that is greater than the width W con of the corresponding connection region.

8. The method of claim 7 , wherein each of the at least one isolation region(s) has a width W iso that is about 50 microns to about 250 microns.

9. The method of claim 8 , wherein each of the sequestration chambers comprises a volume of about 0.5 nl to about 2.5 nl.

10. The method of claim 1 , wherein the microfluidic device comprises a substrate having a DEP configuration, and wherein moving the at least one B cell into the at least one isolation region comprises using DEP force to move the at least one B cell.

11. The method of claim 1 , wherein prior to loading the dissociated cell sample, the method further comprises labeling B cells in the dissociated cell sample with a detectable marker, and wherein moving the at least one B cell into the at least one isolation region comprises selecting at least one B cell for movement based on detection of the detectable marker.

12. The method of claim 1 , further comprising:
contacting the at least one B cell with a stimulating agent that stimulates B cell activation, wherein the stimulating agent comprises a CD40 agonist and/or one or more CD40L+ feeder cells, and wherein the at least one B cell is contacted with the stimulating agent for a period of one to ten days.

13. The method of claim 12 , wherein the stimulating agent further comprises a toll-like receptor (TLR) agonist.

14. The method of claim 12 , further comprising:
providing culture medium to the at least one B cell, wherein the culture medium comprises one or more growth-inducing agents that promote B cell expansion, and wherein the one or more growth-inducing agents include at least one agent selected from the group of IL-2, IL-4, IL-6, IL-10, IL-21, and BAFF.

15. The method of claim 14 , wherein each B cell of the at least one B cell(s) is cultured in the isolation region of the microfluidic device to a cell count of about 8 to 20 cells.

16. The method of claim 1 , wherein identifying the at least one isolated B cell that produces antibodies capable of binding to the cancer cell-associated antigen comprises introducing a fluidic medium comprising the cancer cell-associated antigen into the microfluidic device, wherein the fluidic medium comprises soluble cancer cell-associated antigen or micro-objects that comprise the cancer cell-associated antigen.

17. The method of claim 16 , wherein the micro-objects are beads and the cancer cell-associated antigen is conjugated to the beads, or wherein the micro-objects are cells and the cancer cell-associated antigen is a membrane-associated antigen present on the cell surface of cancer cells present in the at least one tumor sample.

18. The method of claim 17 , wherein the micro-objects are beads and the cancer cell-associated antigen is from a cell membrane preparation obtained from cancer cells isolated from the at least one tumor sample, or wherein the micro-objects are cancer cells isolated from the at least one tumor sample.

19. The method of claim 16 , wherein introducing the cancer cell-associated antigen into the microfluidic device comprises:
flowing a fluidic medium that contains micro-objects that comprise the cancer cell-associated antigen through the flow region of the microfluidic device;
stopping the flow of the fluidic medium when at least some of the micro-objects in the medium are located in a portion of the flow region that is proximal to the at least one isolation region; and
moving at least one of the micro-objects into at least one of the isolation regions in the microfluidic device that contains at least one B cell, thereby producing at least one isolation region having at least one micro-object and at least one B cell.

20. The method of claim 16 , wherein identifying the at least one isolated B cell that produces antibodies capable of binding to a cancer cell-associated antigen further comprises:
flowing a fluidic medium that contains a labeled antibody-binding agent through the flow region of the microfluidic device; and
stopping the flow of the fluidic medium when at least some of the labeled antibody-binding agent in the fluidic medium is located in a portion of the flow region that is proximal to the at least one isolation region; and
monitoring binding of the labeled antibody-binding agent to the cancer cell-associated antigen.

21. The method of claim 20 , wherein the labeled antibody-binding agent is provided in a mixture with the cancer cell-associated antigen.

22. The method of claim 1 , wherein providing a dissociated cell sample comprises obtaining the at least one solid tum

Assignees

• Berkeley Lights Inc

Inventors

• Chapman Kevin T
• Fox George L
• Radel Peggy A
• White Mark P
• Wang Xiaohua
• Park Minha
• Stadler Guido K
• Lowe Jr Randall D
• Radstrom Xiao Guan
• Mcewen Jason M
• Wang Gang F

Classifications

• G01N33/575 Primary

  for cancer · CPC title

• A61K40/414

  Nervous system antigens · CPC title

• A61K40/31

  Chimeric antigen receptors [CAR] · CPC title

• A61K40/11

  T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells · CPC title

• A61K2239/13

  Antibody-based · CPC title