Separation of antigen-specific memory b cells with a conjugated biopolymer surface

A B cell and polymer technology, applied in the field of equipment for extracting antigen-specific memory B cells, can solve problems such as high cost, speed, stability, fidelity, regulatory compliance, and product expression difficulties

Inactive Publication Date: 2012-05-02
BIOFACTURA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Currently available methods suffer from various issues such as high cost of discovery and production of antibody-based drugs and difficulties in speed, stability, fidelity, regulatory compliance and product expression

Method used

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  • Separation of antigen-specific memory b cells with a conjugated biopolymer surface
  • Separation of antigen-specific memory b cells with a conjugated biopolymer surface
  • Separation of antigen-specific memory b cells with a conjugated biopolymer surface

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Embodiment 1

[0075] Recent work has demonstrated the unique properties of chitosan, a linear b-1,4-linked polysaccharide produced by partial deacetylation of chitin, and its use in functional biomanufacturing 19-23 . Two main properties of this biopolymer include its pH-dependent solubility and the reactivity of its amine groups in enzymatic conjugation. The former property enables the spatially oriented assembly of chitosan by electrodeposition taking advantage of the distributed pH gradient on the cathode surface. Studies have shown to stabilize the assembly of functionalized conjugated chitosan in thin films on micropatterned gold electrodes in microfluidic devices 24 . The latter property allows enzymatic assembly of functionalized conjugated chitosan. In 2008, Wu et al. described the tyrosinase-mediated conjugation of tyrosine pentapeptide-labeled modified protein G on electrodeposited chitosan biopolymer surfaces, and the self-assembly of fluorescently labeled IgG antibodies to t...

Embodiment 2

[0081] Cells captured on antigen-conjugated chitosan or other polymer surfaces can be activated to induce differentiation and clonal expansion into antibody secreting cells (ASCs). Recent and previous studies have identified activators of soluble immune cells such as B cells 37-45 . B-cell activating factors of the TNF family (BAFF) have been shown to cooperate with signaling from the BCR to stimulate B-cell survival, class-switching DNA recombination, and antibody production 46 . Synergistically with IL-10, products of microbial origin including CpG-containing oligodeoxynucleotides (ODNs) and bacterial DNA can activate human B cells by opening the TLR9 pathway 47-49 . Recombinant CD40L, IL-4, IL-5, IL-6, IL-15, IL-21 or other additives can be added to increase activation, differentiation, proliferation and selection of mAb isotype (ie IgG). Once activated and expanded, B cells will form ASC colonies. Other suitable activators known to those skilled in the art can be used...

Embodiment 3

[0084] The flow chamber used to isolate, activate and colonize B cells was designed to allow visualization of colonies by microscopy and to facilitate transfer of colonies using micromanipulators in a sterile environment. These colonies can be transferred to 96-well plates for additional expansion and supernatants screened for antigen-specific mAbs by ELISA. The antigen binding / affinity of mAbs in these supernatants can be further characterized by surface plasmon resonance (Biacore) to determine the correlation of antibody avidity as a function of B cell migration distance.

[0085] The sterility of the flow chamber design, visualization and transfer of colonies can be functionally tested. These tests can be performed with antibiotic-free media and sterile maintenance steps prior to cell culture experiments. If it is not possible to activate and culture certain antigen-specific B cells in a microfluidic device, isolation of cells after capture and transfer to plates may be ne...

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Abstract

The present invention provides materials and methods for the isolation of immune cells. In one embodiment, the present invention provides a device for the isolation and separation of antigen-specific memory B cells. Immune cells will be separated by flowing the cells along a ligand-conjugated biopolymer surface. The ligand may be distributed in a concentration gradient along the z- axis of the biopolymer surface. Cells that display receptors specific for the ligands on the biopolymer will interact with the ligands and roll along the surface in the fashion of leukocyte rolling. Additive adhesive interactions will cause differential cell separation and eventual immobilization.

Description

[0001] Cross References to Related Applications [0002] This patent application claims priority to US Provisional Patent Application No. 61 / 158,649, filed March 9, 2009, which is specifically incorporated herein by reference in its entirety. Background technique [0003] The potential application of viruses (e.g., poxviruses, filoviruses) as biological weapons has received increasing attention, especially for human diseases for which there are currently no effective countermeasures. The administration of therapeutic antibodies is a suitable strategy for the treatment and / or prophylaxis of individuals exposed to or infected with viral disease agents of interest to military and counter-bioterrorism efforts. Currently, vaccinia virus (VACV) immunoglobulin (VIG), a polyclonal Ig product derived from human blood, is the "go-to" therapy for disseminated VACV infection caused by adverse smallpox vaccination events . In addition, cross-reactive mAb preparations can be used as thera...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12M1/00
CPCC12M47/04C12M1/40
Inventor 达里尔·B·桑佩
Owner BIOFACTURA INC
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