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Method for producing antibodies from plasma cells

A technology of plasma cells and antibodies, applied in the field of producing antibodies from plasma cells, can solve problems such as expensive, unsuitable for high-throughput, and inefficient acquisition

Active Publication Date: 2011-12-14
INSTITUTE FOR RESEARCH IN BIOMEDECINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is difficult, expensive, time-consuming, not amenable to high-throughput, and cannot efficiently obtain rare antibodies produced by a small fraction of the plasma cell total repertoire

Method used

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  • Method for producing antibodies from plasma cells
  • Method for producing antibodies from plasma cells
  • Method for producing antibodies from plasma cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Example 1: Plasma cells in mesenchymal stromal cell cultures

[0095] The inventors observed primary cultures of human mesenchymal stromal cells established from normal bone marrow according to standard methods (Pittenger et al, 1999, Science 284:143-147; Bieback et al, 2004 Stem cells 22:625-634; Dominici et al, 2006, Cytotherapy 8:315-317; Sotiropoulou et al 2006, Stem Cells 24:462-471) contain antibody-secreting cells. These cells were detected by ELISPOT and identified as plasma cells. Plasma cells in mesenchymal stromal cell cultures were still detectable in vitro after 3 weeks (data not shown).

Embodiment 2

[0096] Example 2: Culture of human plasma cells up to 50 days

[0097] In order to develop a culture system in which individual plasma cells can remain viable so that the antibodies produced can accumulate over time in culture, the inventors tested different sources of primary mesenchymal stromal cells prepared according to standard methods. Briefly, tissue culture flasks were pre-coated with FCS for 1 hour. Make bone marrow cells supplemented with 30% FCS and 10 -8M dexamethasone was cultured overnight in complete IMDM medium. Unattached cells were washed away, and adherent cells were cultured in complete DMEM-10% FCS. Three of the seven lines tested supported human plasma cell survival but ceased proliferation after a few passages. In subsequent experiments, fixed mesenchymal stromal cells transduced with the telomerase reverse transcriptase gene (MSC-TERT) were used. These cells are those isolated by Mihara et al. (Br J Haematol 2003, 120, 846-849).

[0098] Peripheral...

Embodiment 3

[0099] Example 3: 3-week cultures of individual plasma cells

[0100] Isolation of plasma cells from peripheral blood or bone marrow using PE-conjugated anti-CD138 antibody followed by anti-PE microbeads and cell sorting, and seeding mesenchymal matrix in 96-well plates at a density of 0.5 cells / well on a cell monolayer. IgG-containing cultures were monitored over a 22-23 day period by taking regular samples. Medium was changed on day 16. IgG productivity in monoclonal cultures was constant at 72-134 pg / cell / day throughout the culture period ( figure 2 a, Peripheral blood-derived (4 cultures); figure 2 b, Bone marrow derived (5 cultures)).

[0101] Plating efficiencies of blood and bone marrow plasma cells ranged from 30% to 65% in 5 limiting dilution experiments (data not shown). In addition, plasma cells recovered from polyclonal cultures can be replated into unicellular cultures where they maintain a constant rate of Ig secretion (data not shown). The linear accumul...

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PUM

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Abstract

The invention relates to methods of producing antibodies, including monoclonal antibodies, comprising culturing a limited number of plasma cells. It also relates to methods of identifying antibodies by performing assays on the antibodies produced by the cultured plasma cells to determine their function, binding specificity, epitope specificity, and / or their ability to neutralize a toxin or a pathogen. The invention also relates to antibodies and antibody fragments produced by the methods of the invention as well as methods of using the antibodies and antibody fragments.

Description

[0001] This application claims priority from UK Patent Application No. 0819376.5 filed 22 October 2008, US Provisional Patent Application Serial No. 61 / 181,582 filed 27 May 2009, PCT Patent Application No. PCT / US2009 / 051851 and US Patent Application No. 12 / 509,731, both filed on July 27, 2009. Background technique [0002] Plasma cells are terminally differentiated, non-proliferative cells that secrete antibodies at very high rates (thousands of molecules per second, corresponding to approximately 30-50 pg per cell per day). [0003] Isolation of antibodies, such as monoclonal antibodies, from plasma cells relies on the cloning and expression of immunoglobulin genes. This can be done using phage display libraries of promiscuous VH and VL genes isolated from plasma cells, or by isolating pairs of VH and VL genes from single plasma cells using single cell PCR. However, for screening of antibodies produced by plasma cells, immunoglobulin genes need to be cloned and expressed in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/00C12N5/0781C07K16/10C07K16/12
CPCC07K16/1018C07K2317/21C07K16/1282C12N2502/1394C12N5/0635C07K16/00A61P31/04A61P37/00A61P37/06A61P37/08A61K39/395C07K16/18
Inventor A·兰扎韦基亚D·雅罗塞
Owner INSTITUTE FOR RESEARCH IN BIOMEDECINE
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