Patent 6,949,346

The present invention relates to an antibody for the detection quantification, or isolation of basophils, mast cells, the precursor cells of basophils or mast cells, or a surface structure of basophils or mast cells. This antibody corresponds to an antibody with the designation 97A6, produced and released by hybridoma cells that were deposited in accordance with the Budapest Treaty on Feb. 12, 1997 under accession number DSM ACC 2297 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ).

SUMMARY OF THE INVENTION

In view of the above, it is an object of the present invention to provide an antibody for the use mentioned at the outset.

According to the present invention, this object is achieved by a use of an antibody in which binding of the antibody occurs to that surface structure of the cells to which can bind the antibody with the designation 97A6, produced and released by hybridoma cells that were deposited in accordance with the Budapest Treaty on Feb. 12, 1997 under number DSM ACC 2297 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DMSZ. All restrictions upon public access to the deposit will be irrevocably removed upon the grant of a patent on this Application and the deposit will be replaced if viable samples cannot be dispensed by the depository.

The storage term of these hybridoma cells has been correspondingly extended.

Also understood as an antibody within the meaning of the invention are antibody fragments, for example F(ab), conjugates with antibodies and/or antibody fragments, and all compositions which contain antibodies, antibody fragments, and conjugates with antibodies and/or antibody fragments.

A surface structure to which an antibody binds can be, within the meaning of the invention, an individual molecule, for example a membrane protein, or also an association of two or more molecules, for example an ion channel or receptor comprising several subunits.

The object underlying the invention is completely achieved in this fashion.

Specifically, the inventors of the present Application have recognized that the surface structure to which antibody 97A6 can bind is expressed, with the exception of a few megakaryocytic cell lines, only on basophils and mast cells and on their precursor cells, and on mast cell line HMC-1 and basophilic leukemia cell line KU-812. This was not to be expected based on DE 197 08 877 C1, since the latter describes the fact that antibody 97A6 is specific for megakaryocytes.

Recent results of research by the inventor indicate, however, that antibody 97A6 recognizes a few megakaryocytic cell lines, but no native megakaryocytes.

Since the antibody used according to the present invention is well-suited for the purification of cells, the invention also concerns a substantially pure population of basophils and/or mast cells and/or of precursor cells of basophils and/or mast cells, said cells being capable of binding a reagent that specifically binds to those surface structures of said cells that are recognized by antibody 97A6. The invention moreover concerns a corresponding reagent for binding said cells.

Using antibody 97A6, it is readily possible to isolate the surface structure to which the antibody binds, and to produce both monoclonal and polyclonal antibodies against it.

Via FACS analysis it could be shown by now, that the surface structure represents the phosphodiesterase/nucleotide pyrophosphatase ectoenzyme PDNP3, which is also referred to as NPP3 or PD-Ibeta. The sequence was published by Jin-Hua et al. in Genomics 45, 421-415 (1997).

One great advantage of the use according to the present invention of an antibody lies in the fact that it is thereby reliably possible to monitor the number of basophils in the blood of patients with chronic myeloid leukemia (CML), since many of the basophils present in immature form, which hitherto were not morphologically identifiable as such, can be reliably recognized thereby. The exact number of basophils in such patients, and thus the status of their disease, can thus be ascertained.

The use according to the present invention of an antibody furthermore advantageously makes possible the isolation of precursor cells from which, depending on the cultivation conditions, mast cells or basophils can be generated. The use according to the present invention thus facilitates analysis of the differentiation of such cells. It is particularly advantageous in this context that with the antibody that is used, as compared with other antibodies hitherto known, basophils are detectable at very early development stages, and remain detectable with the antibody as they develop further.

An important advantage of the use according to the invention of an antibody in terms of routine diagnostics is the capability of detecting with a single antibody, for example, mast cells from various tissues and basophils from blood. Different antibodies and antibody combinations were hitherto necessary for this purpose, involving considerable extra cost. Basophils in blood samples can also be histochemically stained, for example using May-Grünwald-Giemsa. Experienced personnel are then needed to identify and count the basophils, and subjective fluctuations in the results are possible. Immature basophils cannot be identified in this manner. The use according to the present invention of antibodies, on the other hand, makes possible analysis using objectively analyzable standard methods, such as ELISA or FACS, which can be performed even by less-experienced personnel and allow a much greater sample throughput than the subjective evaluation of stains.

The inventors of the present Application have moreover recognized that the activation of basophils, for example in conjunction with an allergic reaction, results in an enhanced presentation of the surface structure recognized by the antibody that is used according to the present invention. The detection and/or quantification of this surface structure on basophils is therefore well suited for studying the activation of basophils.

In a preferred embodiment, the antibody used is a monoclonal antibody.

The use of a monoclonal antibody has the advantage that the antibody is reproducible in standardized fashion, and thus can potentially be produced in unlimited quantities. The binding properties of a monoclonal antibody are furthermore always constant, so that each use of a monoclonal antibody can also be standardized.

In a further preferred embodiment, the antibody used according to the present invention essentially does not interact with immunoglobulins of the IgE class.

The reason is that in studies in conjunction with the activation of basophils, the use of such an antibody offers the unexpected advantage that it does not itself activate the basophils by crosslinking cell-bound IgE immunoglobulins, and thus does not influence the study results.

In a further embodiment of the present invention, antibody 97A6 itself is used for detection and/or isolation.

The use of this antibody has the advantage that this antibody is already well-characterized and is available in large quantities.

The invention further concerns the use according to the present invention of an antibody in conjunction with the analysis of hematopoiesis.

As already mentioned previously, the use according to the present invention creates the possibility of isolating precursor cells of basophils and mast cells, and thus of analyzing hematopoiesis in terms of those cells. This analysis is, however, not only of scientific interest but can also be used in clinical diagnosis in the investigation of blood formation disorders.

In a preferred embodiment, the use according to the present invention of an antibody occurs in conjunction with the analysis of patient samples, in particular of tissue biopsies, bone marrow biopsies, and/or blood samples. In this context, the use according to the invention serves in the case of tissue biopsies to detect mast cells, in the case of bone marrow biopsies to detect precursor cells of mast cells and/or basophils, and in the case of blood samples to detect mature and immature basophils.

As already mentioned, this makes possible, with a single antibody, analyses that differ depending on the starting material and that hitherto could be performed only with antibody combinations or not at all.

The use according to the present invention of an antibody further concerns the diagnostic classification of tumors, in particular of leukemias.

The diagnosis and classification of leukemias is performed on the basis of bone marrow biopsies or blood samples. For example, in one manifestation of leukemia, in CML, the status of all myeloid cells is analyzed based on bone marrow analyses. The antibody according to the present invention has, in this context, the advantage of supplying information about the portion of precursor cells for basophils and mast cells. In a blood analysis, the use according to the present invention makes possible—as already mentioned—the detection of basophils, in particular immature basophils, that hitherto could not be recognized. But since the number of basophils is a critical parameter in the progression of CML, the use according to the present invention offers the advantage of making possible a more reliable diagnostic conclusion than was previously possible as to the stage of the CML.

In a further preferred embodiment of the invention, the antibody used is joined to a marker, in particular to a fluorescent marker.

It is advantageous in this context that the antibody can then be detected with high sensitivity, so that only small quantities of the antibody need to be used for diagnosis. It is also possible to use an antibody of this kind in an ELISA, or when flow cytometry is utilized.

In a further embodiment according to the present invention, the detection of bound antibodies is accomplished by way of a usual immunological detection method, in particular ELISA or FACS analysis.

This use has the advantage that it allows a determination of cells in patient samples that is sensitive, rapid, highly specific, and can be performed in automated fashion.

The invention further concerns the use according to the present invention of an antibody for detecting and/or quantifying activated basophils.

A use of this kind has the advantage of making it relatively easy to investigate the causes of an activation of basophils, since as compared to nonactivated basophils, activated basophils bind a greater number of antibodies used according to the present invention. For example, basophils can be incubated with potentially activating agents and with antibodies used according to the invention. Activated basophils can then be distinguished from nonactivated ones by way of the increased expression of the 97A6 antigen. This enhanced expression can be quantified in the flow cytometer.

The use according to the invention of an antibody moreover concerns the determination of the extent to which basophils are activated.

By determining the extent of antibody binding to individual basophils, it is also possible to determine the extent of antigen expression and thus of the activation of such cells.

This use according to the present invention thus offers the advantageous possibility of investigating agents which activate basophils in terms of the extent to which they contribute to the activation of such cells. Agents, for example allergens, can thus be classified in terms of their ability to activate basophils. It is thereby possible, for example, to estimate the allergy-triggering potential of an agent.

The invention further concerns a method for investigating allergies, comprising the steps:

	incubating a blood sample with an agent that is suspected of triggering an allergic reaction;
	incubating said blood sample with an antibody used according to the present invention;
	quantifying the antibodies bound to cells.

This method according to the present invention creates the possibility of performing an allergy test without having to subject the person being investigated to an annoying and unpleasant skin test. All that is necessary for the method according to the present invention is to perform a test in vitro with various allergens using a few milliliters of blood from the person being tested; 15 minutes after incubation with an agent, incubation can be performed with the antibody used according to the present invention. By quantifying the antibodies bound by the basophils, it is thereby possible to draw a conclusion not only as to whether an agent can trigger an allergic reaction, but also as to the strength of that allergic reaction.

The invention further concerns a method for providing hematopoietic precursor cells that can differentiate into mast cells or basophils, comprising the steps:

isolation and provision of bone marrow cells from an organism;

incubation of said bone marrow cells with an antibody used according to the invention;

isolation of the antibody-bound cells using usual methods, in particular FACS and MACS (magnetically activated cell sorting).

Provision of these hematopoietic precursor cells makes it possible to investigate the development of mast cells and/or basophils. This can be done for research purposes and also, in the case of improper hematopoiesis, for diagnostic purposes. It is also possible to culture from precursor cells, in vitro, cells which later can be introduced back into the donor of the precursor cells without causing immunological problems. This can be useful, for example, in patients exhibiting hematopoietic disorders.

The invention furthermore concerns a substantially pure population of basophils and/or mast cells and/or of the precursor cells of basophils and/or mast cells, said cells being capable of being bound by a reagent that binds specifically to those surface structures of said cells to which antibody 97A6 binds.

Basophils or mast cells do not represent cell lines but rather primary cells, which can only be identified and purified having knowledge of the invention.

The advantage of a population of this kind lies, for example, in the fact that the properties of the cells can thereby be explored very specifically without having the results influenced by other cells. This is important, for example, when the intention is to explore which substances are produced and given off by specific cells under specific conditions, since such substances are generally produced only by a large population of cells in a quantity sufficient that an analysis or identification of said substances can be made therewith. If the population contained different cell types, it would not be possible to draw a conclusion, from the detection of a specific substance, as to which cells were producing that substance.

The invention further concerns a reagent for binding basophils and/or mast cells, and/or the precursor cells of basophils and/or mast cells, that contains an antibody used according to the present invention, preferably antibody 97A6.

The advantage of such a reagent is that cells bound to it can be detected and selected. A further advantage of such a reagent is that it can also contain, in addition to an antibody according to the present invention, further constituents that allow the detection of bound antibodies or the selection of antibody-bound cells. These can be, for example, fluorescent-labeled antibodies or antibodies coupled to magnetic beads, which bind to the antibody according to the present invention.

Claim 1 of 9 Claims

1. A method for binding cells, comprising:

providing said cells;

contacting said cells with antibody 97A6, which is produced by e hybridoma on deposit as No. DSM ACC 2297, to produce a cell-antibody complex;

separating the cell-antibody complex from unbound cells and/or unbound antibody; and

detecting a level of the cell-antibody complex,

wherein said cells are selected from the group consisting of basophils, and mast cells.

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