Anti-human pre-B cell receptor antibody

Details Anti-human pre-B cell receptor antibody Anti-human pre-B cell receptor antibody

2000-01-31

2002-01-01

Eyler, Yvonne (Department: 1646)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C424S138100, C424S130100, C530S388100, C530S389100

Reexamination Certificate

active

06335175

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to an antibody which recognizes a human pre-B cell receptor. The invention also relates to an antibody which reacts with a VpreB molecule of a surrogate light chain of a human pre-B cell receptor expressed on the surface of a human pre-B cell, VpreB in the cytoplasm of the pre-B cell, and a VpreB molecule expressed in the cytoplasm a human pro-B cell; or an active fragment of this antibody. The invention further relates to a method of detecting a human pro-B cell and a human pre-B cell by the above-mentioned antibody or its active fragment with the use of a saponin buffer. The antibody of the present invention can be applied to the diagnosis of acute lymphoblastic leukemia.
2. Related Background Art
B cell is known to differentiate from a hematopoietic stem cell of bone marrow into a plasma cell through the stages of pro-B cell, pre-B cell, immature B cell, and mature B cell.
On the surface of a B cell, immunoglobulin is expressed (may be referred to hereinafter as Ig) as an antigen-recognizing receptor. The antigen-recognizing receptor is composed of two types of polypeptides, i.e., a heavy chain and a light chain. Its composition varies as the B cell is differentiated.
Differentiation of B cell and its antigen-recognizing receptor will be described with a mouse taken as an example.
In pro-B cell, it is known that a surrogate light chain (SL chain), which is composed of two associated polypeptides called &lgr;5 and vpreB, is associated with a heavy chain (H chain) called a surrogate heavy chain (SH chain), and the associated pair is expressed on the cell surface. The surrogate light chain is so called, because the &lgr;5 molecule (may be referred to hereinafter as &lgr;5) and the VpreB molecule (may be referred to hereinafter as VpreB) are associated together to substitute for an ordinary light chain (L chain). &lgr;5 corresponds to the constant region of a light chain, while VpreB corresponds to the variable region of the light chain.
With regard to pre-B cell, a pre-B cell receptor is known to be expressed on the surface of the cell. In the pre-B cell receptor, a surrogate light chain (SL chain), which is composed of two associated polypeptides called &lgr;5 and VpreB, is associated with a heavy chain (H chain) called &mgr; chain.
In an immature B cell and a mature B cell, a B cell receptor composed of an ordinary L chain bound to &mgr; chain is expressed as an antigen-recognizing receptor.
As will be seen from the foregoing description, &lgr;5 and VpreB are expressed only in pro-B cell and pre-B cell.
With a human pre-B cell receptor, similar findings have been reported.
The following are known as antibodies which react with the human pre-B cell receptor. Their reactivity, etc. are shown in Table 1.
{circle around (1)} Anti-human pre-B cell receptor antibodies: SLC1, SLC2, SLC3, SLC4 (Cell, Vol. 73, 73-86, 1993)
{circle around (2)} Anti-human pre-B cell receptor antibody: 9C2 (Eur. J. Immunolo., Vol. 24, 257-264, 1994)
{circle around (3)} Anti-human VpreB antibodies: 3C7, 6F6 (Eur. J. Immunolo., Vol. 26, 2172-2180, 1996)
{circle around (4)} Anti-human VpreB antibodies: B-MAD176, B-MAD688, B-MAD792, B-MAD1112 (J. Exp. Med., Vol. 183, 2693-2698, 1996)
TABLE 1
Cell surface staining (FACS)
Antibody
Immunogen
Pro-B cell
Pre-B cell
Reaction site
Immunoprecipitation
Isotype
SLC1
Pre-B cell receptor
Negative
Positive
&lgr;5
No data
IgG
SLC2
Pre-B cell receptor
Negative
Positive
&lgr;5
No data
IgM
SLC3
Pre-B cell receptor
Negative
Positive
&lgr;5
No data
IgM
SLC4
Pre-B cell receptor
Negative
Positive
&lgr;5
No data
IgG
9C2
Part of synthetic
Very weakly
Positive
VpreB
No data
IgM
VpreB molecule
positive
3C7
Recombinant VpreB
Positive
Very weakly
VpreB
No data
IgM
positive
6F6
Recombinant VpreB
Positive
Very weakly
VpreB
No data
IgM
positive
B-MAD176
Recombinant VpreB
Negative
Positive
No data
No data
IgM
B-MAD688
Recombinant VpreB
Positive
Positive
VpreB
SH chain (only when
IgM
formed as complex
with SL chain)
B-MAD792
Recombinant VpreB
Negative
Positive
No data
No data
IgM
B-MAD1112
Recombinant VpreB
Positive
Positive
VpreB
No data
IgM
depending on
cell strain
SUMMARY OF THE INVENTION
An abnormality in B cell differentiation causes leukemia. Knowing at which stage of differentiation B cell multiplied abnormally is important in treating leukemia. In acute lymphoblastic leukemia in childhood, in particular, abnormal proliferation of pro-B cell or pre-B cell may be a frequent cause. Currently, B cell surface markers generally used in diagnosis are CD10, CD19, CD20, and CD21. Attempts have been made to react any of these antibodies with B cell and to determine by the results which stage the B cell is at. However, CD19, CD20, and CD21 also react with an immature B cell or a mature B cell, while CD10 also reacts with a T cell or an epithelial cell. These antibodies, therefore, were unable to detect only a human pro-B cell or a human pre-B cell specifically.
The present invention aims to provide an antibody which recognizes a human pre-B cell receptor, and which does not recognize a human pro-B cell, thereby making it possible to distinguish between human pre-B cell and human pro-B cell and detect human pre-B cell or human pro-B cell specifically.
In the field of clinical diagnosis, microscopic observation of a cell is made. Compared with other methods, this is the most convenient and fastest method that requires the smallest space for installation of equipment. If staining of the cell is very weak, however, it cannot be observed under a microscope, although it can be detected by a highly accurate detection method such as FACS. All the antibodies against a human pre-B cell receptor enumerated earlier as above-mentioned {circle around (1)} to {circle around (4)}, especially the antibodies against VpreB, only gave the results with FACS. That is, it has not been shown whether they have reactivity enough to stain the antibody-reacted cell so as to be observable microscopically. Also, it has been shown that reactivity with a human pro-B cell is only reactivity on the surface of the cell, and intracellular staining has not been shown.
It is another object of the present invention to provide an antibody which reacts specifically with a human pre-B cell and a human pro-B cell, and which can immunostain a human pre-B cell expressing a human pre-B cell receptor on the surface and a human pro-B cell expressing a VpreB molecule intracellularly so that only the immunostained cell can be observed microscopically.
To attain this object, the present invention provides an antibody which recognizes a human pre-B cell receptor and does not recognize a human pro-B cell. The antibodies of this invention are an antibody which recognizes a stereostructure formed by the &mgr; chain, &lgr;5 and VpreB of a human pre-B cell, and an antibody which reacts with a VpreB molecule, one of the constituents of the SL chain of a human pre-B cell receptor expressed on the surface of a human pre-B cell, or reacts with a VpreB molecule expressed in the cytoplasm of a human pro-B cell, but does not react with a &lgr;5 molecule, and whose reactivity is such that the human pre-B cell or human pro-B cell reacted with the antibody can be detected microscopically.
The invention also provides an anti-human pre-B cell receptor antibody as claimed in claim
1
which is a monoclonal antibody produced by hybridoma HSL2 (originally deposited on Oct. 16, 1997 under acceptance No. FERM-P16476 with National Institute of Bioscience and Human Technology (NIBHT), Agency of Industrial Science and Technology, Ministry of Industrial Trade and Industry, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan; and transferred on May 25, 1998 under acceptance No. FERM-BP6378 to NIBHT, the international deposition organization), or an active fragment of the monoclonal antibody.
The invention also provides an anti-human pre-B cell antibody or its active fragment which is a monoclonal antibody produced by hybridoma HSL96 (originally deposited with NIBHT on May 30, 1997 under acc

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