Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-05-10
2003-06-10
Chan, Christina (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007240, C435S373000, C435S383000
Reexamination Certificate
active
06576428
ABSTRACT:
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH
Not Applicable
1. Technical Field
The invention is in the field of analysis of cell populations and cell separation and the compositions obtained thereby. More particularly, the invention concerns analysis and separation of antigen-specific T cells based on primary labeling of cells with their secreted products through capture of these products by a specific binding partner for the product anchored or bound to the cell surface.
2. Background Art
Numerous attempts have been made to analyze populations of cells and to separate cells based on the products which they produce. Such approaches to cell analysis and separation are especially useful in assessing those cells which are capable of secreting a desired product (the “product”), or which are relatively high secretors of the product. These methods include cloning in microtiter plates and analysis of the culture supernatant for product, cloning in agar and analysis by methods for identification of the product of the localized cells; the identification methods include, for example, plaque assays and western blotting. Most methods for analysis and selection of cells based upon product secretion involve physically isolating the cell, followed by incubation under conditions that allow product secretion, and screening of the cell locations to detect the cell or cell clones that produce the product. When cells are in suspension, after the cells have secreted the product, the product diffuses from the cell without leaving a marker to allow identification of the cell from which it was secreted. Thus, secretor cells cannot be separated from non-secretor cells with these types of systems.
In other cases, both secretor and non-secretor cells can associate the product with the cell membrane. An example of this type of system are B cell derived cell lines producing monoclonal antibodies. It has been reported that these types of cell lines were separated by fluorescence activated cell sorting (FACS) and other methods reliant upon the presence of antibody cell surface markers. However, procedures that analyze and separate cells by markers that are naturally associated with the cell surface can not accurately identify and/or be used in the separation of secretor cells from non-secretor cells. In addition, systems such as these are not useful in identifying quantitative differences in secretor cells (i.e., low level secretors from high level secretors).
A method that has been used to overcome the problems associated with product diffusion from the cells has been to place the cell in a medium that inhibits the rate of diffusion from the cell. A typical method has been to immobilize the cell in a gel-like medium (agar), and then to screen the agar plates for product production using a system reliant upon blotting, for example Western blots. These systems are cumbersome and expensive if large numbers of cells are to be analyzed for properties of secretion, non-secretion, or amount of secretion.
Köhler et al. have described a negative-selection system in which mutants of a hybridoma line secreting IgM with anti-trinitrophenyl (anti-TNP) specificity were enriched by coupling the hapten (i.e., TNP) to the cell surface and incubating the cells in the presence of complement. In this way, cells secreting wild-type Ig were lysed, whereas cells secreting IgM with reduced lytic activity or not binding to TNP preferentially survived. Köhler and Schulman (1980)
Eur. J. Immunol.
10:467-476.
More recently, a system has been described for labeling and separating cells based on secreted product. PCT/US93/10126. In this system, a specific binding partner for a secreted product is coupled to the surface of cells. The product is secreted, released, and bound to the cell by the specific binding partner. Cells are then separated based on the degree to which they are labeled with the bound product.
Other systems allow the cells to secrete their products in the context of microdroplets of agarose gel which contain reagents that bind the secretion products, and encapsulation of the cells. Such methods have been disclosed in publications by Nir et al. (1990)
Applied and Environ. Microbiol.
56:2870-2875; and Nir et al. (1991)
Applied and Environ. Microbiol.
56:3861-3866. These methods are unsatisfactory for a variety of reasons. In the process of microencapsulation, statistical trapping of numbers of cells in the capsules occurs, resulting in either a high number of empty capsules when encapsulation occurs at low cell concentrations, or multiple cells per capsule when encapsulation occurs at high cell concentrations. Secreted product is trapped in the agarose drop by the capture antibody and detected by a second fluorochromated antibody. This process, while allowing for the detection and isolation of cells based on secreted product, is complicated, requires special equipment, and is not suited to all types of sorting methods. In order to analyze and separate single cells or single cell clusters by this technique, large volumes must be handled to work with relatively small numbers of cells because of the numbers of empty capsules and because of the size of the microcapsules (50-100 &mgr;m). The large volume of droplets results in background problems using flow cytometry analysis and separation. In addition, the capsules do not allow separation using magnetic beads or panning for cell separation.
Various methods have been used to couple labels to cell surfaces where the label such as a fluorochrome is intended for direct detection. For example, hydrophobic linkers inserted into the cell membrane to couple fluorescent labels to cells have been described in PCT WO 90/02334, published Mar. 8, 1990. Antibodies directed to HLA have also been used to bind labels to cell surfaces. Such binding results in a smaller dimension than the encapsulated droplets described above and such cells can be conveniently used in standard separation procedures including flow cytometry and magnetic separations.
ELISpot assays and methods for intracellular cytokine staining have been used for enumeration and characterization of antigen-specific CD4
+
and CD8
+
T cells. Lalvani et al. (1997)
J. Exp. Med.
186:859-865; and Waldrop et al. (1997)
J. Clin Invest.
99:1739-1750. These methods can be quite useful for T-cell epitope mapping or for monitoring immunogenicity in vaccine trials, but they do not allow isolation of live antigen-specific T cells, e.g., for clinical trials of specific adoptive immunotherapy of cancer or infections. Kern et al. (1998)
Nat. Med.
4:975-978; E1 Ghazali et al. (1993)
Curr. Opin Immunol.
23:2740-2745; and Yee et al (1997)
Curr. Opin. Immund.
9:702-708. Soluble multivalent complexes of peptide-loaded major histocompatibility complex (MHC) molecules have been exploited recently to detect and also isolate antigen-specific T cells. Altman et al. (1996)
Science
274:94-96; Dunbar et al. (1998)
Curr. Biol.
8:413-416; Ogg et al. (1998) 279:2103-2106; Luxembourg et al. (1998)
Nat. Biotechnol.
16:281-285; Murali-Krishna et al. (1998)
Immunity
8:177-187; Gallimore et al. (1998)
J. Exp. Med.
187:1383-1393; and Flynn et al. (1998)
Immunity
8:683-691. These reagents are highly specific but the approach is limited to well defined combinations of antigenic peptides and restricting HLA alleles.
The immune system comprises two types of antigen-specific cells: B cells and T cells. T cells can be characterized phenotypically by the manner in which they recognize antigen, by their cell surface markers, and by their secreted products. Unlike B cells, which recognize soluble antigen, T cells recognize antigen only when the antigen is presented to them in the form of small fragments bound to major histocompatibility complex (MHC) molecules on the surface of another cell. Any cell expressing MHC molecules associated with antigen fragments on its surface can be regarded as an antigen-presenting cell (APC). In most situations, however, more than the mere display of an MHC-bound antigen f
Assenmacher Mario
Miltenyi Stefan
Schmitz Jurgen
Belyavskyi Michail
Chan Christina
Miltenyi Biotech GmbH
Morrison & Foerster / LLP
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