Methods of producing patient-specific anti-cancer therapeutics and methods of treatment therefor
Inventors
Chapman, Kevin T. • White, Mark P. • Wang, Xiaohua • PARK, MINHA • Stadler, Guido K. • Lowe, JR., Randall D. • Radstrom, Xiao Guan • McEwen, Jason M. • Wang, Gang F. • Fox, George L. • Radel, Peggy A.
Assignees
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Publication Number
US-11971409-B2
Publication Date
2024-04-30
Expiration Date
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.
Core Innovation
The invention provides a method of identifying an isolated B cell that produces antibodies capable of binding to a cancer cell-associated antigen. A dissociated cell sample obtained from at least one solid tumor sample from a patient is loaded into a microfluidic device having at least one flow region and at least one sequestration chamber with an isolation region fluidically connected to the flow region.
Within the microfluidic device, at least one B cell is moved from the dissociated cell sample into at least one isolation region to obtain at least one isolated B cell. Antibody binding capability is assessed by introducing the cancer cell-associated antigen into the microfluidic device, allowing identification of isolated B cells that produce antibodies capable of binding the cancer cell-associated antigen.
The disclosed approach further includes refining how B cells are moved into the isolation region, including using DEP force and/or gravity and localized fluid flow. Additional refinements include contacting the B cell with a stimulating agent, including CD40L+ feeder cells and/or a TLR agonist such as CpG2006, and introducing the antigen via antigen-carrying micro-objects selected from cells, liposomes, lipid nanorafts, and beads.
Claims Coverage
The independent claim broadly covers one inventive workflow: microfluidic isolation of B cells from a patient solid tumor sample and in-device identification of B cells that produce antibodies capable of binding to a cancer cell-associated antigen. The dependent claims further refine B-cell movement, stimulation, antigen presentation, and dissociation.
Microfluidic isolation of B cells in a sequestration chamber
Loading a dissociated cell sample from at least one solid tumor sample obtained from a patient into a microfluidic device having at least one flow region and at least one sequestration chamber comprising an isolation region, wherein the isolation region is fluidically connected to the flow region, and 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.
In-device identification via cancer cell-associated antigen introduction
Identifying at least one isolated B cell that produces antibodies capable of binding to a cancer cell-associated antigen by introducing the cancer cell-associated antigen into the microfluidic device.
B cell relocation using DEP force, gravity, and localized fluid flow
Moving at least one B cell from the dissociated cell sample into at least one isolation region in the microfluidic device using DEP force and/or gravity and localized fluid flow.
B cell activation using CD40L+ feeder cells and a TLR agonist
Contacting the B cell with a stimulating agent, including CD40L+ feeder cells and/or a TLR agonist such as CpG2006, with stimulation that may be substantially continuous over a defined period.
Antigen introduction using antigen-carrying micro-objects
Introducing the cancer cell-associated antigen into the microfluidic device using micro-objects selected from cells, liposomes, lipid nanorafts, and beads.
Tumor cell dissociation using collagenase and DNase or a cell dissociator instrument
Dissociating individual cells from a dissociated cell sample using collagenase plus DNase digestion and/or a cell dissociator instrument.
Overall, the inventive coverage centers on isolating B cells in a microfluidic sequestration chamber connected to a flow region and identifying antibody-producing B cells by introducing a cancer cell-associated antigen into the same microfluidic device, with additional refinements for B-cell movement, stimulation, antigen presentation, and dissociation.
Stated Advantages
Parallel rapid testing for patient-derived antibodies.
Documented Applications
Identifying patient-derived isolated B cells that produce antibodies capable of binding a cancer cell-associated antigen, including downstream antibody generation and characterization activities described in the document (e.g., sequencing heavy/light chain variable domains, hybridoma generation, and generating antibody or antibody fragments for therapy and/or detection).
Using B-cell outputs to generate engineered antibody constructs and engineered T/NK/CAR-T constructs as described in the document.
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