Antigen specifically expressed on the surface of B cells and Hodgkin's cells

The invention relates to a new antigen termed BLA-35 specifically expressed on the surface of Hodgkin's cells, Reed-Sternberg cells and B lymphocytes, and to a new monoclonal antibody (anti-BLA-36) specific thereto. The antigen is characterized by the following properties: PA1 a molecular weight of about 36,000 D; PA1 the presence of an epitope recognized by antibody to said protein; PA1 specific expression by Hodgkin's cells and Reed-Sternberg cells in all subsets of Hodgkin's disease, and by activated and early proliferating B cells; PA1 no expression by T cells; PA1 capability of reacting with its antibody in both frozen and fixed/paraffin embedded tissues; PA1 a function associated with the growth of cells capable to express said antigen protein.

FIELD OF THE INVENTION 
The present invention relates to a new antigen, termed BLA-36, specifically 
expressed on the surface of Hodgkin's cells, Reed-Sternberg cells and B 
lymphocytes, and to a new monoclonal antibody (anti-BLA-36) specific 
thereto. By detecting BLA-36 in human tissues, using anti-BLA-36 antibody, 
Hodgkin's disease and early B cell lymphomas can be diagnosed, imaged and 
treated by radio-immuno-therapy. 
BACKGROUND OF THE INVENTION 
Hodgkin's disease is a malignant lymphoma of mixed cell type which is 
usually classified according to the Lukes-Butler classification [Lukes et 
al., Cancer Res. 26, 1311 (1966)]. Cases of Hodgkin's disease of all four 
major histologic subtypes (lymphocyte predominance, mixed cellularity, 
lymphocyte depleted, and nodular sclerosis) contain variable numbers of 
cells that characterize the Hodgkin's disease process. The largest and 
most characteristic of these cells are termed Reed-Sternberg cells. The 
cell is typically binucleate, multinucleate, or has a polylobulated 
nucleus. Mononuclear variants of this cell are termed Hodgkin's cells. 
Morphological and immunohistochemical studies suggest that lymphocyte 
predominant Hodgkin's disease is a B-cell neoplasm and as such is distinct 
from other subtypes of Hodgkin's disease [see e.g. Pinkus, G. S. and Said, 
J. W., Am. J. Pathol. 133, 211-217 (1988); Burns et al., Am. J. Surg. 
Pathol. 8, 253-261 (1984); Hansmann et al., J. Cancer Res. Clin. Oncol. 
114, 405-410 (1988)]. The cellular derivation of other types of Hodgkin's 
disease remains controversial. 
The non-Hodgkin lymphomas are traditionally classified by standard 
histologic methods. However, since this classification provides little 
information about the origin or biology of these tumors, recently 
immunologic classification, emphasizing the principal immunologic cell 
type in the lymphoma, is favored. B cell-type lymphomas are at present 
classified according to the microscopic features of the cells, especially 
the nuclei. These are thought to represent different stages of 
differentiation of B cells between stem cells and plasma cells [see "Basic 
& Clinical Immunology" 3rd Edition, Fundenberg, H. H., Stites, D. P., 
Caldwell, J. L., Wells, J. V., Eds., Los Altos, Calif., 1980, Chapter 31]. 
The summary of an NCI-sponsored study of classification of non-Hodgkin's 
lypmomas was published in Cancer 49, 2112-2135 (1982). 
The development of Hodgkin's and various B cell lines has permitted the 
production of monoclonal antibodies against such cell lines, and the 
identification of antigen molecules recognized by the antibodies. 
The Fourth International Workshop of Leukocyte Differentiation Antigens 
(1989) listed no less than 78 major cluster designations (CDs1-78) for 
leukocyte antigens, with several CDs including more than one related 
antigens (e.g. CD11A, CD11B, CD11C, etc.). The molecular weights of the 
majority of these antigens have been defined. Of the 78 different CD 
antigens, approximately one dozen show some specificity for B cells (e.g. 
CDs 19-24, CD32, CD37, CD40, CDs 73-77). Only a minority of these show 
restricted specificity for B cells, and most are expressed on a limited 
number of other cell types. 
Antigens that show some preferential expression on Reed-Sternberg cells and 
their mononuclear variants, Hodgkin's cells, in Hodgkin's disease include 
CD15, CD30, and CD74. CD15 was detected by antibody LeuM1, CD30 by 
antibody Ki-1 and related antibodies, and CD74 by antibody LN2. LeuM1 is, 
for example, described in the following publications: Hsu and Jaffe, Am. 
J. Clin. Pathol. 82, 29-32 (1984); Pinkus et al., Am. J. Pathol. 119, 
244-252 (1985); and Hsu et al., JNCI 77, 363-370 (1986). Ki-1 and related 
antibodies are disclosed in numerous scientific publications, including 
Schwab et al., Nature 299, 65-67 (1982); Stein et al., Int. J. Cancer. 30, 
445-449 (1982); O'Connor et al., Histopathology 11, 733-740 (1987). 
Articles concerning antibody LN2 include Epstein et al., J. Immunol. 133, 
1028-1036 (1984) and Sherrod et al., Cancer 57, 2135-2139 (1986). 
A further anti-Reed-Sternberg cell antibody, HeFi-1 is disclosed by Hecht 
et al., J. Immunol. 134. 4231-4236 (1985). 
All of these antibodies identify only a portion of Reed-Sternberg cells in 
some of the subsets of Hodgkin's disease, and their specificity is not 
satisfactory. For example, upon more extensive study, the reactivity of 
Ki-1 appears not to be restricted to Reed-Sternberg cells, but includes 
the malignant cells of some of the non-Hodgkin's lymphomas, as well as a 
subset of apparently normal cells, the identity of which is not yet known, 
but which show some features of myeloid cells [Stein et al., Int. J. 
Cancer 29, 283-290 (1982)]. 
Monoclonal antibodies EPB-1 and EPB-2, reactive with human lymphoma, are 
described by Pawlak-Byczkowska et al., Cancer Research 49, 4568-4577 
(1989). EPB-1 is reported to be specific to normal and malignant B cells 
and to Hodgkin's disease related cells, and is identified as having 
IgG.sub.1 isotype. Its antigen has an estimated molecular weight of 
35,000. The immunogen agent used to make EPB-1 was a B cell lymphoma. 
Functional aspects of the antigen molecules recognized by any of these 
antibodies have not yet been reported. In addition, although there have 
been some sporadic reports describing the existence of inhibitory or 
cytotoxic factors in Hodgkin's disease [Taylor, C. R., "Hodgkin's Disease 
and the Lymphomas", Annual Research Reviews, D. Horrobin, Series ed. 
Churchill Livingston/Eden Press, London/New York, 1980], almost no 
information is available with respect to mechanisms of regulation of 
growth and differentiation of Hodgkin's or Reed-Sternberg cells, other 
than the general .concept that the mononuclear Hodgkin's cell is the 
proliferating element from which the Reed-Sternberg cells are derived 
[Taylor, C. R., "Upon the enigma of Hodgkin's disease and the 
Reed-Stenberg cell." In: Controversies in the Management of Lymphomas II., 
J. M. Bennet, ed. Martinus Nijhoff Publishers, Boston, pp. 91-110, 1983; 
Taylor, C. R., Recent Results Cancer Res., 64, 214-231 (1978)]. 
Immunophenotypic characterization of lymphomas by monoclonal antibodies has 
proved a valuable adjunct to histologic diagnosis and has facilitated 
understanding of the lineage of certain lymphomas. 
Monoclonal antibodies detecting various antigens have been used or proposed 
for a number of purposes in research, and for diagnostic studies of 
leukemias and lymphomas in men and animals. The techniques employed 
include: 
1. Leukocyte identification by phenotype, utilizing flow cytometry, 
immunofluorescence, immunoenzyme techniques, or immuno electron 
microscopy. 
2. Leukocyte separation techniques, including flow cytometry and panning. 
3. Identification and classification of leukemias. 
4. Radioimmunimaging of lymphomas in animals and man. 
5. Radioimmunotherapy of lymphomas in animals and man. 
6. Studies of leukocyte differentiation, maturation and function in 
experimental models and human disease. 
However, the vast majority of the antibodies described to date recognize 
epitopes that are sensitive to the process of fixation or embedment in 
paraffin wax. Such antibodies detect their corresponding antigens only in 
frozen sections exposed to minimal fixation (such as 10 seconds in 
acetone), and do not detect the corresponding antigens following formalin 
or B5 fixation and embedment in paraffin. This fact hampers the 
utilization of many of these antibodies for diagnostic purposes in which 
only fixed and paraffin embedded sections are available. Thus, particular 
diagnostic importance is attached to those antibodies that reliably detect 
their corresponding antigens in fixed tissues. Since this fact has been 
realized, the number of such antibodies available has been increased, but 
still constitutes only a small minority overall. 
Accordingly, for successful diagnosis of lymphomas, antibodies that are 
sufficiently specific to the lymphoma to be identified, and detect the 
corresponding antigens following fixation and embedment in paraffin, are 
required. 
With regard to the identification of Hodgkin's disease, of the 
above-mentioned antibodies LeuM1 and LN2 are both reactive in paraffin 
embedded tissues. Antibodies related to Ki-1 which are effective in 
paraffin embedded tissues, also have become available. However, as 
hereinabove mentioned, all three of these antibodies identify only a 
portion of Reed-Sternberg cells in some of the subsets of Hodgkin's 
disease. None of them identify Reed-Sternberg cells in all cases of 
Hodgkin's disease. EPB-1 is also reported to remain active after fixation 
and paraffin embedment, and appears to have a better specificity to B 
cells and Hodgkin's disease related cells than any of the earlier 
published antibodies. 
However, there is no disclosure of the functional aspects of the antigens 
identified by any of these antibodies on Reed-Sternberg and Hodgkin's 
cells. Whereas the literature contains many reports describing the 
existence of inhibitory or cytotoxic factors in the serum of patients with 
Hodgkin's disease, the corresponding antigens have not been identified and 
little information is available with respect to the mechanisms of 
regulation of growth and differentiation of Hodgkin's or Reed-Sternberg 
cells. 
There is a great need for monoclonal antibodies with specificity and high 
reactivity to Reed-Sternberg cells, which retain their immunoreactivity in 
tissues that have been fixed and embedded in paraffin. Such antibodies 
would have wide applications, since paraffin sections remain the standard 
in diagnostic histopathology, based upon convenience, safety, superior 
morphology preservation, and economic factors. Additionally, it would be 
desirable to identify antigen(s) specifically expressed on Hodgkin's 
disease related cells, which have regulatory functions in the growth 
and/or differentiation of such cells. Study of the function of such 
antigens would be an invaluable tool in understanding and ultimately, 
treating Hodgkin's disease. 
SUMMARY OF THE INVENTION 
The goal of the present invention is to provide a monoclonal antibody with 
specificity for Reed-Sternberg cells which retains its immunoreactivity in 
tissues that are routinely fixed and embedded in paraffin. The specificity 
of such antibody enables its use in the diagnosis of Hodgkin's disease 
and, by its pattern of reactivity, may shed light on the cellular origin 
of the malignant cells of Hodgkin's disease. Furthermore, the availability 
of an antibody specific for Reed-Sternberg cells, leads to the 
characterization and isolation of the antigens involved, as a preliminary 
to determining their functional role. 
According to the present invention, a hitherto unrecognized antigen 
specifically expressed on the surface of Hodgkin's and Reed-Sternberg 
cells and on activated and early proliferating B lymphocytes was 
identified using a newly developed monoclonal antibody produced by 
immunization of a Balb/c mouse with a Hodgkin's cell line (HDLM-3). To 
indicate its specificity and molecular weight (36,000), this antigen was 
termed "B lymphocyte antigen 36" (BLA-36); and the antibody is referred to 
as anti-BLA-36. 
In one aspect, the present invention relates to substantially pure 8 
lymphocyte antigen 36 (BLA-36). 
In another aspect, the present invention relates to a substantially pure 
antigen protein having the following characteristics: 
a molecular weight of about 36,000 Dalton; 
the presence of an epitope recognized by antibody to the protein; 
specific expression by Hodgkin's cells and Reed-Sternberg cells in all 
subsets of Hodgkin's disease, and by activated and early proliferating B 
cells; 
no expression by T cells; 
capability of reacting with its antibody in both frozen and fixed/paraffin 
embedded tissues; 
a function associated with the growth of cells capable of the expression of 
this antigen protein. 
The term "substantially pure" is used to indicate that the protein is 
substantially devoid of other proteins associated therewith in nature. 
"Substantially" means that such proteins cannot be detected by standard 
techniques conventionally used for the detection of proteins. 
In a further aspect, the present invention relates to a monoclonal antibody 
or a fragment thereof having the following properties: 
IgG.sub.3 subtype; 
reactivity with Hodgkin's cells and Reed-Sternberg cells in all subsets of 
Hodgkin's disease, and with activated and early proliferating B cells; 
lack of reactivity with T cells; 
capability of reacting with the corresponding antigen in both frozen and 
fixed/paraffin embedded tissues; 
reversible, dose-dependent growth-inhibitory effect on cells capable of 
expressing the corresponding antigen. 
In a still further aspect, the present invention relates to a method for 
the isolation of an antigen protein having the above properties from 
Reed-Sternberg cells, Hodgkin's cells or activated or early proliferating 
B cells, comprising the steps of: 
disrupting the membranes of said cells; 
preparing cell extract containing solubilized proteins; 
contacting said extract with a monoclonal antibody specific to said 
protein; 
separating the fraction containing protein reacting with said monoclonal 
antibody; and 
isolating said protein. 
In a still further aspect, the present invention relates to monoclonal 
antibody or a fragment thereof specific to B lymphocyte antigen 36. 
The present invention further relates to a hybridoma cell line producing 
antibody specific to B lymphocyte antigen 36. 
In another aspect, the present invention relates to a method for detection 
of an antigen protein having the above properties in mammalian tissues or 
cells in order to diagnose Hodgkin's disease or B cell lymphoma, 
comprising the steps of: 
obtaining monoclonal antibody that is specific to the antigen protein, 
contacting said antibody with tissue or cells obtained from a mammal to be 
diagnosed; and 
detecting the antigen protein, if present, as well as assay kit for 
performing such method. 
The invention also relates to methods for imaging lesions characteristic of 
Hodgkin's disease, comprising the steps of: 
obtaining monoclonal antibody specific to an antigen having the above 
properties, said antibody being labeled; 
labeling said antibody; 
contacting said labeled antibody with tissue obtained from a mammal; and 
imaging said label. 
In another aspect, the present invention relates to a method for inhibiting 
the growth of Reed-Sternberg cells, Hodgkin's cells or activated or early 
proliferating B cells, comprising the step of contacting such cells with a 
growth-inhibiting amount of anti-BLA-36 antibody or a (Fab').sub.2 
fragment thereof.

DETAILED DESCRIPTION OF THE INVENTION 
In the course of the experimental work that has led to the present 
invention, a developmentally related antigen expressed on the plasma 
membrane of B lymphocytes and on the surface of Reed-Sternberg cells and 
Hodgkin's cells was identified using a monoclonal antibody produced by 
immunization of a BALB/c mouse with a Hodgkin's cell line (HDLM-3F). The 
antigen with a molecule weight of 36,000 Dalton has been termed B 
lymphocyte antigen 36 (BLA-36), and its antibody was called anti-BLA-36. 
Later experiments, in which BLA-36 was isolated from various B cell lines, 
Hodgkin's lines and fresh Hodgkin's tissue, indicated that the molecular 
weight of this antigen was not dependent on its source. 
When immunoperoxidase techniques were used, anti-BLA-36 reacted strongly 
with the Hodgkin's cell line that served as immunogen, and to a lesser 
degree with pre-B and B cell lines, but showed no detectable binding 
activity with other hematopoietic cell lines. 
In normal tissues, BLA-36 was detectable predominantly on cells in the 
germinal center and mantle zone of reactive follicles in lymph nodes and 
spleen. In hematopoietic malignancy, BLA-36 antigen was detectable on the 
surface of Reed-Sternberg cells, mononuclear Hodgkin's cells, and on 
malignant cells of B-cell lineage. Under these conditions, T lymphocytes, 
histiocytes, granulocytes, macrophages, and stromal cells in lymphoid 
tissue were consistently negative for the expression of the antigen. 
BLA-36 antigen was not detectable on unactivated (resting) peripheral 
monocytes, B or T cells and on activated T cells, and was expressed on 
phorbol diester (PMA)-activated peripheral B cells. 
The findings in lymphomas exactly mirrored the patterns of staining 
observed in the cell line panels that were examined. T-cell lymphomas and 
diffuse histiocytic lymphomas were consistently nonreactive. B-cell 
lymphomas (Raji and Daudi) and the so called pre-B cell line (SUAMB-1 and 
SUAMB-2) were, by contrast, clearly reactive, as were examples of 
lymphoblastoid and undifferentiated lymphoma (BL-1 and Nu-LB-1), all of 
which show some features of B-cell differentiation. Furthermore, acute 
lymphoblastic leukemia of B-cell derivation (BALL-1 and BALM-2) clearly 
showed positivity with the antibody. 
The experimental results indicated that anti-BLA-36, unlike most other 
antileukocyte antibodies, retained its immunoreactivity in 
paraffin-embedded tissue sections, and distinguished Reed-Sternberg cells 
and B-cell lymphomas from all other malignant cells. 
BLA-36 also appears to have a role in growth regulation. When antibody to 
BLA-36 is added to BLA-36 positive cell line (B cell or Hodgkin's 
disease-derived), growth of the cell line is markedly inhibited. Cell 
proliferation is halted, and DNA synthesis is markedly reduced. This 
effect is cytostatic, rather than cytotoxic, and is reversible on removal 
of the monoclonal antibody from the culture medium. This growth regulatory 
effect, with inhibition in these conditions, further distinguishes BLA-36 
and its corresponding antibody, from other known B cell 
antigen-antibodies, which do not show inhibitory activity. 
The antibody anti-BLA-36 of the present invention may be obtained through 
conventional monoclonal antibody preparation techniques. Antigen BLA-36 
may be separated from cells (tissues) by which it is specifically 
expressed, through conventional separation techniques. First, the cell 
membranes are disrupted by freezing and thawing, by mechanical techniques, 
or by other suitable methods. The soluble cytoplasmic fraction is then 
separated from the generally insoluble structural elements and nuclei, for 
example by centrifugation. The antigen may, for example, be isolated by 
gel electrophoresis, under denaturing conditions. 
If the anti-BLA-36 is to be used for diagnostic purposes, the 
antigen-antibody reaction needs to be visualized. Antigen-antibody 
reactions can be visualized by a variety a methods known in the art, using 
markers to label either the antibody or the antigen. At present, the most 
commonly used markers are chromogens, such as fluorochromes, enzymes, 
radioactive and radiopaque compounds. 
Fluorochromes are dyes that absorb radiation, for example ultraviolet 
light, are excited by it and as a result, emit visible light. 
Fluorochromes that are useful as markers must be capable of forming 
covalent bonds with protein molecules and having a high fluorescence 
emission in the visible spectrum with a color different from that of 
tissues. At present, the most commonly used fluorochromes are fluoresce in 
isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). 
The methods that use antibodies labeled with fluorochrome markers are 
usually referred to as immunofluorescence methods. In the so called 
"direct method" fluorochrome-labeled antibody is applied to the 
preparation containing the corresponding antigen. In the "indirect method" 
the antigen is treated with its corresponding unlabeled antibody, and the 
resultant antigen-antibody complex is treated with a fluorochrome-labeled 
antibody to the immunoglobulin of the animal species that provided the 
unlabeled antibody used in the first step. In diagnostic immunology, the 
antigen-containing substrate is incubated with the patient's serum, and 
then with fluorochrome-labeled mouse, rabbit or goat antibody to human 
immunoglobulins. The indirect method is usually preferred, due to its 
higher sensitivity. 
For visualizing immunofluorescent specimens, fluorescence microscopes, that 
are simple modifications of standard transmitted light microscopes, can be 
used. If necessary, the results may be recorded by photomicrography. 
Enzymes may also be used as labels if, on interaction with their substrate, 
they form a visible well-defined, colored precipitate. Immunoenzyme 
procedures can be used to localize antigens with the aid of enzyme-labeled 
antibodies. Several enzymes have been employed as markers, but the most 
commonly used is horseradish peroxidase, and the methods based upon the 
use of this enzyme are referred to an immunoperoxidase procedures. Another 
commonly used enzyme marker is alkaline phosphatase. The most widely used 
protocol for the detection of antigens by means of enzyme-linked 
antibodies is referred to as Enzyme Linked Immunosorbent Assay (ELISA) 
that may be performed as a direct method or in sandwich format. 
As radioactive markers, any of the well-known medical radionuclides can be 
used. Suitable radionuclides include Tc-99m, I-123, In-111, In-113m, 
Ga-67, or other suitable gamma-emitters. 
The radionuclides can be conjugated to the monoclonal antibody of the 
present invention by conventional techniques. Iodination, for example, mag 
be accomplished using the chloramine-T method described by S. Mills, et 
al. .sup.123 I-Radiolabeling of Monoclonal Antibodies for In Vivo 
Procedures, Hybridoma 5, 265-275 (1986). This technique may be used to 
effect iodination to render the antibody radiopaque, or to attach a 
radionuclide, such as I-125 or I-131. 
Other radionuclides may be attached to the antibody through chelation with 
benzyl EDTA or DPTA conjugation procedures. Still other suitable 
techniques include the iodogen method disclosed by M. Pimm, et al., In 
Vivo Localization of Anti-Osteogenic Sarcoma 791T Monoclonal Antibody, 
Int. J. Cancer. 30, 75 (1982), and direct iodination with radioactive 
sodium iodide. 
Radiopaque materials suitable for labeling antibodies include iodine 
compounds, barium compounds, gallium compounds, thallium compounds, and 
the like. Specific examples of radiopaque materials include barium, 
diatrizoate, ethiodized oil, gallium citrate, iocarmic acid, iocetamic 
acid, iodamide, iodipamide, iodoxamic acid, iogulamide, iohexol, 
iopamidol, iopanoic acid, ioprocemic acid, iosefamic acid, ioseric acid, 
iosulamide meglumine, iosumetic acid, iotasul, iotetric acid, iothalamic 
acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid, ipodate, meglumine, 
metrizamide, metrizoate, propylidone, and thallous chloride. 
In another aspect, the invention relates to methods for imaging lesions 
characteristic of Hodgkin's disease. For this purpose, the anti-BLA-36 
antibody is labeled. Suitable labels include, for example, radiolabels, 
radiopaque materials, and magnetic resonance-enhancing materials. 
The radiolabels and radiopaque materials have been discussed hereinabove. 
Suitable techniques for imaging labels localized in tissues expressing the 
BLA-36 antibody are known in the art. For example, if the label is a 
gamma-emitting radionuclide, suitable imaging techniques include gamma 
cameras and single photon emission computed tomography (SPECT) techniques. 
If the antibody has been labeled with a radiopaque material, radiographic 
imaging may be applied. Other suitable techniques include computed axial 
tomography (CAT) scans, fluoroscopy and conventional X-ray imaging. 
Materials that can be detected by or that enhance the effects of magnetic 
resonance imaging equipment also may be conjugated to the antibodies. 
Suitable conventional magnetic resonance-enhancing compounds include 
gadolinium, copper, iron, and chromium. It is preferred that these metal 
atoms be prepared in the form of conventional organometallic chelates, 
which are then bound to the antibody. 
The foregoing methods along with other routine techniques of 
immunodiagnosis are disclosed in standard laboratory textbooks. See, for 
example, Rose, N. R. and Pierluigi, E. B. in Methods in Immunodiagnosis, 
Second Edition, John Wiley & Sons, Publishers, New York, Chichester, 
Brisbane, Toronto, 1980; Current Protocols in Molecular Biology, Green 
Publishing Associates and Wiley-Interscience, 1987. 
In a particularly important aspect, the present invention relates to a 
method for inhibiting the growth of Reed-Sternberg cells, Hodgkin's cells 
or activated or early proliferating B cells by contacting such cells with 
a growth-inhibiting amount of anti-BLA-36 antibody or a (Fab').sub.2 
fragment thereof. Although the amount of antibody that may be needed for 
the inhibition of cell growth is a function of the cell type, the extent 
of undesired cell proliferation, and other conditions, it generally is 
between about 0.1 and about 1.0 .mu.g/ml. 
Further details of the invention are set forth in the following 
non-limiting Examples. 
More specifically, details of the identification and characterization of 
the BLA-36 cell surface glycoprotein and the anti-BLA-36 antigen are given 
in Example 1. 
Results of a study, exploring the diagnostic utility of anti-BLA-36 by 
application to a variety of Hodgkin's and non-Hodgkin's lymphomas, in 
comparison with other antibodies which also react in B5-fixed and 
paraffin-embedded tissue, are disclosed in Example 2. 
EXAMPLE 1 
Characterization of a Cell-Surface Molecule Expressed on B Lymphocytes and 
Hodgkin's Cells 
A. Materials and Methods 
Materials. Affinity purified immunoglobulin G fraction of horse antimouse 
immunoglobulin and avidin-biotin-peroxidase complex (ABC) were purchased 
from Vector Laboratories, Burlingame, Calif. Reagents for electrophoresis 
and Western blotting were bought from Bio-Rad Laboratory, Richmond, Calif. 
Endo-B-N-acetylglucosaminidase H was obtained from Boehringer Mannheim 
Biochemicals. Pepsin and other chemical reagents were of the highest 
purity available from Sigma Chemical Co., St. Louis, Mo. 
Cell Lines. The Hodgkin's cell line HDLM-3 derived from a malignant pleural 
effusion in a patient with Hodgkin's disease was provided by Dr. George 
Moore, University of Colorado, Denver. This and related cell lines HDLM-1 
and HDLM-2 have been described elsewhere (6-9). The cell lines utilized in 
this study are listed in Table 1. 
Cell Fusion and Hybrid Selection. Three-weeks old BALB/c mice were 
immunized with 3 separate injections of 10.sup.7 HDLM-3 cells/mouse at 
three-week intervals. The spleen was removed on the 4th day after the last 
injection and the spleen cells were fused with the mouse myeloma cell line 
(SP-2) as first described by Kohler and Milstein (10). 
Screening, Selection and Cloning of Hybridoma. The initial screening of 
hybridoma supernatants was performed using cytopreparations of Hodgkin's 
(HDLM-3), B (Raji) and T (CEM) cell lines. Two hundred thousand cells in 
100 .mu.l of medium were used for making each cytopreparation. Cells were 
fixed for 30 seconds with cold acetone prior to immunocytological 
staining. Supernatants (100 .mu.l from wells exhibiting hybrid growth were 
applied and incubated with the cytospin preparation for 30 min. The slides 
were washed with PBS, and 100 .mu.l of biotinylated horse antimouse 
immunoglobulin at a predetermined dilution was added to each. After an 
incubation of 30 min, slides were again washed as above and were incubated 
with 100 .mu.l of avidin-biotin-horseradish peroxidase complex (ABC) for 
30 min. Following a wash with PBS, the bound ABC was visualized by 
addition of a mixture of the substrate H.sub.2 O.sub.2, and chromogen, 
aminoethyl carbazole (AEC). 
Supernatants containing antibodies with strong reactivity to Hodgkin's 
cells but none with the T cell lines, were subsequently screened on 
freshly frozen tissue 
TABLE 1 
__________________________________________________________________________ 
Reactivity of Antibody to BLA-36 with Hodgkin's 
and Hematopoietic Cell Lines by an Indirect 
Immunocytological Staining Method 
Reactivity with Reactivity with 
Cell Line Antibody to BLA-36 
Cell Line antibody to BLA.36 
__________________________________________________________________________ 
Hodgkin's disease: Acute lymphoblastic leukemia: 
HDLM-3 +++ T cell-CEM - 
Burkitt's lymphoma: MOLT-4 - 
RAJI + HSB-2 - 
DAUDI + HPB-ALL - 
SU-AMB-1 + JM - 
SU-AMB-2 + Null cell - NALL - 1 
- 
Undifferentiated REH - 
lymphoma:.sup.b B cell - BALL-1 
+ 
NU-DUL-1 + BALM-2 + 
U-698-M + NALM-6 (pre B) + 
Lymphoblastoid:.sup.b 
NALM-1 (pre B from CML) 
+ 
BL-1 + Myeloid leukemia: 
NU-LB-1 + Erythroid - K562 (CML) 
Large cell lymphoma: Myeloid - ML-2 - 
SU-DHL-1 - Promyelocytic - HL-60 
- 
SU-DHL-2 - Monocytic - TPC-1-0 
- 
SU-DHL-4 - Myeloma: 
U-937 - IJ-266 
ARH-77 
__________________________________________________________________________ 
NOTE: 
Sections were scored for intensity on a scale from - to +++: 
- = abscence of staining; 
+ = weak staining; 
++ = moderate staining; 
+++ = intense staining. 
.sup.b Show evidence of B cell differentiation. and on B5-fixed and 
paraffin-embedded tissue sections of lymph nodes from patients with 
Hodgkin's disease. All antibodies showing reactivity against the Hodgkin's 
cell line also reacted .with the B cell lines. Differential reactivity 
with B cells, therefore, could not be used as a selection criterion. 
Hybrid cells secreting antibodies limiting dilution. Supernatants from 
wells with single clonal growth were again subjected to the above 
screening procedures. Clones showing good production of antibody at this 
stage were subjected to two cycles of recloning before final selection of 
a clone for subsequent studies. 
Purification of Monoclonal Antibody to BLA-361 Anti-BLA-36 was purified by 
applying the spent-medium containing the antibody to a column packed with 
Protein A-Sepharose 4B conjugate. The specifically bound material was 
eluted from the column with 0.1M Glycine-HCl buffer, pH 2.7. Following 
elution, the antibody was immediately dialyzed with several changes of PBS 
at 4.degree. C., and was concentrated to yield 1 mg protein per ml in PBS. 
The purity of the antibody was determined by SDS-polyacrylamide gel 
electrophoretic analysis. The antibody was characterized as IgG.sub.3 
subtype. The labelling of the purified antibody with biotin was carried 
out at room temperature as described previously (11). Subsequently, 
F(ab').sub.2 fragments of monoclonal antibody to BLA-36 were prepared 
according to the method of Parham (12). F(ab').sub.2 fragments were then 
purified on a 2-m-long Sephacryl S-200 column and were assessed for purity 
by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). 
Both the whole and F(ab').sub.2 fragments of the antibody were sterilized 
by filtration before use. 
Comparison of BLA-36 With Other Known Antigens of B Cells. Competitive 
immunoblocking straining assays were performed using immunocytological 
techniques in order to investigate the nature of the epitopes recognized 
by anti-BLA-36 in relation to other known antigens of human hematopoietic 
cells. The cytopreparations of Hodgkin's and B cell lines were incubated 
first with the unlabelled test antibodies that recognize B cell antigens 
[(BA-1 (13), BA-2 (14), B-1 (15), B-4 (16), LN-1 and LN-2 (17), anti-mu 
chain or SC2 antibody to HLA-DR (Table 2)], followed by incubation with 
biotinylated antibody to BLA-36. The remainder of the staining procedure 
was as described above. Any change in the intensity of staining with 
reference to control preparations was recorded. 
Preparation and Staining of Tissue Sections. Normal neoplastic and fetal 
tissues were obtained from the surgical pathology files of the University 
of Southern California School of Medicine, Los Angeles County Medical 
Center. The tissues were fixed in formalin or B5, dehydrated, cleared and 
embedded in paraffin. Tissue sections were cut at five micron thickness 
for both Hematoxylin and Eosin preparations and immunostaining. 
An indirect unlabelled primary antibody method was used for localizing 
antigen with the specific antibody as described elsewhere (18). 
Biotinylated horse antimouse immunoglobulin was used as the link between 
the specific antibody and the ABC. Throughout this study, primary and 
secondary antibodies and ABC reagent were used at a constant concentration 
as determined by initial optimal titration analysis. The visual estimates 
of intensities were scored as follows: -, absence; 1+, weak; 2+, moderate; 
and 3+, intense. For each experiment, negative controls were performed to 
ensure the specificity of the reaction: these included the use of specific 
antibody following absorption with the immunogen (HDLM-3) and an 
irrelevant antibody of the same immunoglobulin class in lieu of the 
specific antibody. 
Preparation of Cell Lysate. The cell lines (10.sup.7 cell/ml) were washed 
three times in serum-free medium. The cells were lysed with 20 mM Tris HCI 
buffer, pH 7.5, 
TABLE 2 
______________________________________ 
Comparison of Epitopes Detected by Anti-BLA-36 and Other 
Known Antibodies to Human Leukocyte Antigens by an 
Immunocytochemical Steric Inference (Blocking) Assay 
Initial Second Immunoperoxidase 
Intensity 
Incubation.sup.a 
Incubation labelling of staining.sup.b 
______________________________________ 
PBS Biotinylated 
ABC +++ 
anti-BLA-36 
anti-BLA-36 
Biotinylated 
ABC - 
anti-BLA-36 
anti-BA-1 
Biotinylated 
ABC +++ 
anti-BLA-36 
anti-BA-2 
Biotinylated 
ABC +++ 
anti-BLA-36 
anti-B-1 Biotinylated 
ABC +++ 
anti-BLA-36 
anti-B-4 Biotinylated 
ABC +++ 
anti-BLA-36 
LN-1 Biotinylated 
ABC +++ 
anti-BLA-36 
LN-2 Biotinylated 
ABC +++ 
anti-BLA-36 
anti-mu chain 
Biotinylated 
ABC +++ 
anti-BLA-36 
SC-2 Biotinylated 
ABC +++ 
anti-BLA-36 
______________________________________ 
.sup.a Positive controls to demonstrate effective binding of antiBA-2, 
antiB1, antiB4, antimu chain and SC2 monoclonal antibodies were performed 
using the indirect immunoperoxidase method and the appropriate cell lines 
(HDLM3, DAUDI and RAJI) and confirm that each of these antibodies bound t 
target cells. 
.sup.b Mean value for three cell lines (HDLM3, RAJI, and DAUDI) utilized 
in this study. containing 0.5% (v/v) noidet P-40 (NP-40), 1.0 mM 
phenylmethylsulphonyl fluoride and 0.5 mM 
chloromethyl-L-(2-phenyl-1-toluenesulphoamido) ethyl ketone (lysis 
buffer). Lysates were incubated on ice for 15 min and clarified by 
centrifugation at 100,000.times.g and 4.degree. C. for 15 min. The 
supernatant was used immediately or stored at -70.degree. C. until further 
use. 
Western Blot Analysis. Following separation of the NP-40 solubilized 
material from Hodgkin's tissue, from BLA-36-positive cell lines 
(Hodgkin's, pre-B or B cell) and BLA-36-negative cell lines (T and Null 
cell acute lymphoblastic leukemia, myeloid leukemia) by sodium dodecyl 
sulphate-polyacrylamide gel electrophoresis (19), the bands were 
electrophoretically transferred to nitrocellulose filter paper as 
described by Towbin, et al. (20). Each lane was cut from the filter paper 
incubated with PBS containing 1% (w/v) gelatin for 1 hour to block the 
non-specific binding of antibody to filter paper. Each strip of filter 
paper was incubated for 30 min with 10 ml of PBS and 100 .mu.l of spent 
medium containing anti-BLA-36 (10 .mu.g/ml), anti-BLA-36 (10 .mu.g/ml) 
absorbed with the lysate of HDLM-3 cell line (1 mg/ml), or an equivalent 
amount of an irrelevant IgG.sub.3 antibody that recognizes a glycoprotein 
on the surface of human breast epithelium. Following incubation, the 
strips were washed thoroughly with PBS containing 0.05% (v/v) Tween 20 
and incubated with horseradish peroxidase conjugated goat antimouse 
immunoglobulin in an appropriate dilution for 30 min. The strips were once 
again washed as above. Finally, the color was developed by incubating the 
strip with PBS containing 1 mM diaminobenzidine and 0.01% hydrogen 
peroxide for 5 min. 
Competitive Western immunoblot analyses were performed to ascertain the 
nature of antigen recognized by anti-BLA-36 in relation to other closely 
related antigens, such as the one recognized by LN2 (CD75). Prior to 
application of the NP-40 solubilized extract of Raji cell line on 
SDS-PAGE, the samples were first preabsorbed with antibody to BLA-36 or 
CD75, immobilized individually to Sepharose 4B as described previously 
(21). Each antibody (5 mg/ml of Sepharose 4B suspension) was separately 
mixed and incubated with the Raji extract (1 mg protein/ml). Following an 
overnight incubation, the suspension was centrifuged at 10,000.times.g and 
4.degree. C. for 15 min. The supernatant containing the preabsorbed 
extract was removed and subsequently analyzed by Western immunoblot 
method. Following the application of the unabsorbed or absorbed extracts 
on SDS-PAGE, the protein bands were electrophoretically transferred to 
nitrocellulose filter-paper and then each strip of the filter-paper was 
allowed to react with the appropriate antibody. The negative control 
consisted of strip containing the extract which was preabsorbed with the 
antibody that also subsequently served as a reacting antibody. 
Determination of epitope recognized by anti-BLA-36. Studies were conducted 
to determine whether the antibody was directed to a protein and/or the 
carbohydrate antigen as described below. Protein (250 .mu.l) from 
NP-40-solubilized extract of HDLM-3 was incubated at 37.degree. C. for 18 
hours in 100 .mu.l of 0.1M sodium citrate buffer, pH 5.5, containing 50 
milliunits of endo-.beta.-N-acetylglucosaminidase H as described by 
Tarentino, et al. (22). Following incubation, the reaction mixture was 
mixed with an equal volume of cold 12.5% (w/v) trichloracetic acid (TCA) 
for 15 min at 4.degree. C. The mixture was centrifuged at 12,000.times.g 
for 15 min, and the supernatant was removed and dialyzed against several 
changes of PBS at 4.degree. C. The pellet was dissolved in 100 .mu.l of 
PBS and dialyzed. To ensure complete precipitation, a control containing 
only the protein in the absence of the enzyme was included. Protein (250 
.mu.) from NP-40-solubilized extract of HDLM- 3 was dissolved in 100 .mu.l 
of 0.07M sodium acetate buffer, pH 4.0, containing 0.05M NaCl and 15 .mu.g 
of pepsin, and the reaction mixture was incubated at 37.degree. C. in a 
water-bath for 18 hours. At the end of the enzyme-digestion period, the pH 
of the solution was adjusted to 8 with NaOH and was dialyzed against 
several changes of PBS. 
Effect of the Growth of Hodgkin's Cell Line. Hodgkin's, B, T, and 
histiocytic lymphoma cell lines were cultured in triplicate 
(1.8.times.10.sup.5 cells/ml) in serum-free defined medium (HB 101, Hana 
Biologics, Alameda, Calif.) in 24-well tissue culture plates. Cells were 
cultured in the presence of various concentrations ranging from 0.12 to 
4.0 .mu.g of intact or F(ab').sub.2 fragments of (a) specific antibody, or 
(b) equivalent amounts of specific antibody preabsorbed with the extract 
of HDLM-3 or (c) an irrelevant control antibody of the same immunoglobulin 
class, in order to assess the effect of anti-BLA-36 on cellular 
proliferation. BLA-36-negative hematopoietic cell lines also served as 
negative controls. The cells in each well were incubated for various 
lengths of time and their numbers were counted by hemacytometer on day 4 
(for the titration of antibody) or at intervals of 24 hrs. for 5 days. The 
viability of cells was determined by trypan-blue-dye exclusion limit. 
B. Results 
Generation, Cloning and Screening of Monoclonal Antibodies. Supernatants 
from wells exhibiting hybridoma growth were screened by immunocytochemical 
staining of cytopreparations of a Hodgkin's (HDLM-3), a B (Raji) and a T 
(CEM) cell line. Supernatants with no reactivity or with reactivity 
against all three cell lines were rejected. A small number of supernatants 
lacked reactivity against the T cell lines, but showed strong positivity 
with Hodgkin's cells and B cells. Supernatants with reactivity against the 
Hodgkin's cell line alone were not detected. Those hybrids producing 
antibodies with strong reactivity to Hodgkin's cell line, but not to T 
cells were repeatedly subcloned, until one clone, producing consistently 
high levels of monoclonal antibody with the above properties, was selected 
for detailed study. Double immunodiffusion studies with goat antibodies to 
subclass of mouse immunoglobulin revealed that anti-BLA-36 is an IgG.sub.3 
immunoglobulin. The primary parameter for selection of anti-BLA-36 was its 
ability to react strongly with its antigen both in a Hodgkin's related 
cell line and tissue sections. The screening process also revealed that 
the epitope recognized by anti-BLA-36 is resistant to B5- or 
formalin-fixation and paraffin-embedding procedures. 
Immunocytologic Localization of Antigen in Cell Lines. In order to test the 
specific expression of the target antigen, several cell lines of human 
hematopoietic lineage were incorporated in an indirect immunoperoxidase 
staining technique. The Hodgkin's cell line and also pre-B and B cell 
lines included in the assay showed reactivity with the antibody. 
Anti-BLA-36 showed strong binding reactivity predominantly with the 
surface membrane and to a lesser extent with the cytoplasm of the 
Hodgkin's cell line (FIG. 1). There was no detectable binding reactivity 
with non-B hematopoietic cell lines, including those of T cell, myeloid, 
and monocyte origin, suggesting that the antigen concerned is expressed on 
Hodgkin's and B cell lines only (Table 1). Absorption of specific antibody 
with the immunogen led to a complete abolition of staining of the 
Hodgkin's cells. Also, negative controls in which anti-BLA-36 was replaced 
by an irrelevant antibody of the same immunoglobulin class showed no 
reactivity. 
Comparisons of Epitope Recognized bv Anti-BLA-36 with Other Known and 
Characterized Lymphoid Antigens. The expression of antigen recognized by 
anti-BLA-36 was compared with other well characterized hematopoietic 
antigens to which commercial antibodies are available. Anti-BLA-36 showed 
strong reactivity with the Hodgkin's cell line and also with B and pre-B 
cell lines and no reactivity with large cell lymphoma or T cells lines. 
Antibody B-1 failed to react with the Hodgkin's cell line while antibodies 
BA-2 and B-4 showed weak reactivity. Conversely, antibodies to T cells 
(Leu4) or granulocytes/monocytes (OKM1 and LeuM1) showed no evidence of 
binding reactivity with Hodgkin's line. Furthermore, preadsorption of B 
cell antibodies BA-1, BA-2, B-1, B-4, LN-1, LN-2, IgM (mu chain) and 
HLA-DR (SC2) to the Hodgkin's cell line showed no reduction staining when 
the Hodgkin's cell line was subsequently treated with biotinylated 
anti-BLA-36. The result suggests that the epitope recognized by 
anti-BLA-36 is distinct from those recognized by other antibodies studied 
(Table 2). 
Immunohistological Localization of Antigen in Tissue Section. The 
specificity and pattern of anti-BLA-36 was further studied in fresh-frozen 
and B5-fixed and paraffin-embedded tissue sections using an indirect 
avidin-biotin peroxidase complex (ABC) method. 
Initially, the antibody was reacted with frozen tissue sections of normal 
lymph nodes and spleen, in addition to lymph nodes from patients with 
Hodgkin's disease. In later studies, formalin or B5-fixed and 
paraffin-embedded tissue sections were employed. The pattern of reactivity 
of anti-BLA-36 was identical in frozen and formalin or B-5-paraffin 
sections, leading to a preference for the latter based on superior 
morphology. 
Anti-BLA-36 showed strong reactivity to a subset of lymphocytes in the 
follicle and mantle zone of normal lymph nodes (FIG. 2a). The percentage 
of cells showing reactivity in mantle and within the follicle was 70 to 80 
and 100% respectively, encompassing both small and large cells. BLA-36 was 
predominantly localized on the cell-surface. The specificity and patterns 
of reactivity of the antibody in normal spleen were similar to those 
obtained with lymph node. Under these conditions, the tangible body 
macrophages, dendritic reticulum cells, interdigitating reticulum cells, 
sinus histiocytes and endothelial cells 
TABLE 3 
______________________________________ 
Reactivity of Antibody to BLA-36 with Hematopoietic Cells 
in Freshly Frozen or B5-Fixed Paraffin-Embedded Sections 
Tissue Reactivity with Antibody to BLA-36 
______________________________________ 
Lymph node: 
Germinal center 
+++ 
Mantle zone +++ 
T cell zone - 
Interdigitating histiocyte 
- 
Sinus histiocyte 
- 
Endothelium - 
Spleen: 
White pulp +++ 
Red pulp - 
______________________________________ 
were consistently negative, as were presumptive T lymphocytes in the 
paracortex (Table 3). 
Anti-BLA-36 also showed strong reactivity with Reed-Sternberg cells and 
their variants in lymph nodes of patients with Hodgkin's disease. The 
reactivity was predominantly on the plasma membrane (FIG. 2b), but with 
definite cytoplasmic staining visible in the Golgi area of larger 
Reed-Sternberg cells. A high proportion (90%) of Reed-Sternberg cells 
showed intense staining (Table 4). Scattered small lymphocytes present in 
lymph nodes involved by Hodgkin's disease also showed reactivity, 
especially where residual follicles were identifiable. The Reed-Sternberg 
cells and mononuclear Hodgkin's cells were positive in all 28 cases of 
Hodgkin's disease that were studied. 
Subsequently, 31 cases of non-Hodgkin's lymphomas were examined. The 
antibody showed strong reactivity with B cell lymphomas (Table 5), 
including follicular center cell lymphomas (large and small cell types), 
mantle zone (also known as intermediate cell) lymphomas, and immunoblastic 
sarcomas. The T cell lymphomas and chronic lymphocytic leukemia (CLL) 
consistently showed no reactivity (Table 5). 
Under identical condition, anti-BLA-36 showed no reactivity with normal 
epithelial cells, including adrenal gland, breast, colon, lung, salivary 
gland, skin and stomach and their malignant counterparts. A variable 
proportion of Kupffer cells in normal liver, however, showed weak 
reactivity with the antibody. Connective tissue elements were otherwise 
nonreactive, with the exception of scattered lymphocytes presumed to be B 
cells. 
Characterization of Epitope Recognized by Anti-BLA-36. The immunoreactivity 
of anti-BLA-36 was abolished by its absorption with TCA-soluble fraction 
obtained by eno-b-N-acetyglucosaminidase H treatment of the Hodgkin's cell 
line (Table 6). Absorption with the TCA-precipitable fractions of the 
endoglycosidase-treated or pepsin-treated 
TABLE 4 
______________________________________ 
Immunohistological Distribution of BLA-36 Detected by a 
Xenogenic Monoclonal Antibody in B5-Fixed Paraffin-Embedded 
Tissue Sections from Patients with Hodgkin's Disease 
Total No. Reed-Sternberg 
no. of Posi- cells and variants 
Intensity 
Histology cases tive (% cells staining) 
of staining 
______________________________________ 
Nodular sclerosis 
12 12 85-95 +++ 
Mixed cellularity 
9 9 75-90 +++ 
Lymphocyte 3 3 75-90 +++ 
depleted 
Lymphocyte pre- 
4 4 75-90 +++ 
dominant 
______________________________________ 
TABLE 5 
______________________________________ 
Immunohistological Distribution of BLA-36 Detected by 
a Xenogeneic Monoclonal Antibody in B5-Fixed Paraffin- 
Embedded Tissue Sections from Patients with Hematopoietic 
Malignant Diseases (Other Than Hodgkin's Disease) 
Total No. Intensity 
no. of posi- Intensity % of 
of 
Histology cases tive cells staining 
staining 
______________________________________ 
B-immunoblastic 
7 7 70-90 +++ 
sarcoma 
T-immunoblastic 
6 0 0 - 
sarcoma 
Follicular center cell 
8 8 75-85 ++ 
(large) lymphoma.sup.a 
Follicular center cell 
3 3 80 +++ 
(small cleaved) 
lymphoma.sup.b 
Follicular center cell 
3 3 80 +++ 
(small non-cleaved) 
lymphoma 
Mantle zone 2 2 80 +++ 
lymphoma 
Chronic lymphocytic 
2 0 0 - 
______________________________________ 
.sup.a Combines large cleaved and large noncleaved types. 
.sup.b Large cell component, small cells showed weak reactivity. 
TABLE 6 
______________________________________ 
Determination of the Nature of Epitope Recognized by 
Monoclonal Antibody to BLA-36 
Binding properties 
of antibody to BLA-36 
bsorbed with treated extract 
Treatment of HDLM.3 cells to cellular 
of HDLM.3 extract antigen in tissue sections 
______________________________________ 
(a) Endo-.beta.-N-acetylglucosamin- 
Reactive 
idase H (TCA-precipitable fraction) 
(b) Pepsin Non-reactive 
(TCA-soluble fraction 
Reactive 
______________________________________ 
Anti-BLA-36 antibody was absorbed with the extract of Hodgkin's cell line 
(HLDM.3) treated with (a) Trichloroacetic acid (TCA) precipitable and 
soluble fractions resulting from endoN-acetylglucosaminidase; and (b) 
pepsin as described in the text. cell extracts has no effect on the 
intensity of the immunostaining. The results are consistent with the 
thesis that the antibody recognizes an epitope expressed in the 
carbohydrate domain of a glycoprotein. 
Characterization and Comparison of BLA-36 with Other Known Lymphoid 
Antigen. Antigen specifically recognized by anti-BLA-36 was analyzed by 
Western-immunoblotting method. Western-immuno-blot analysis of non-reduced 
extracts (NP-40 solubilized) of the Hodgkin's, B and pre-B cell lines and 
lymph node from a patient with Hodgkin's disease, when reacted with the 
monoclonal antibody, yielded an antigen with an apparent molecular weight 
of 36 kilodaltons (FIG. 3, Lanes B-E). Chemical reduction of the cell 
extract before electrophoresis had no effect on the migration of the 
antigen (result not illustrated). The use of the extracts of histiocytic 
and T cell lines in the above experiment failed to yield the antigen (FIG. 
3, Lanes F, G) complementing the results obtained in the immunocytological 
staining of these cells with anti-BLA-36 (Table 1). Furthermore, 
application of anti-BLA-36 preabsorbed with an extract of BLA-36-positive 
cell line also showed no reactivity with its corresponding band on the 
strip (FIG. 3, Lane H). 
Finally, BLA-36 was compared with other known lymphoid antigens by 
Western-immunoblot method. Owing to a weak reactivity of LN2 (CD75) with 
HDLM-3 cell line, the NP-40 solubilized extract of a B cell line (Raji) 
was used as a source of target antigens for the both antibodies included 
in this experiment. The anti-BLA-36 and CD75 showed a strong reactivity 
with their corresponding antigens with the apparent molecular weights of 
36 and 35 kilodaltons respectively (FIG. 4, Lanes A and E). The 
application of anti-BLA-36 on the strip of filter-paper containing the 
extract of Raji cell line (FIG. 4, Lane B) or Hodgkin's disease tissue 
(FIG. 4, Lane C), which were preabsorbed with immobilized LN2 antibody, 
exhibited a band corresponding to BLA-36. Likewise, preabsorption of the 
extract with the immobilized anti-BLA-36 showed no effect on the antigen 
recognized by LN2 antibody (FIG. 4, Lane F). The strip containing extract 
which was preabsorbed and subsequently incubated with the same antibody 
led to the abolition of the reactivity, attesting to the specificity of 
reaction and the effectiveness of the absorption procedure (FIG. 4, Lanes 
D and G). 
Effect of Anti-BLA-36 on Cell-Growth. In order to explore the effect of 
anti-BLA-36 on cell growth, Hodgkin's B, T, and large cell lymphoma cell 
lines were grown for 4 days in the presence of various concentration 
ranging from 0.12 to 4.0 .mu.g of intact or F(ab').sub.2 fragments of the 
antibody. The antibody concentrations in the range of 1 to 2 .mu.g/ml 
completely inhibited the growth of the Hodgkin's cell line (FIG. 5) and B 
cell lines (results not shown). 
The inhibition of growth of the Hodgkin's cells by the intact antibody to 
BLA-36 gave a similar titration curve to that shown in FIG. 5. The wells 
that received either preabsorbed antibody to BLA-36 or an irrelevant 
monoclonal antibody of the same immunoglobulin class in the above 
concentrations range showed no inhibition of growth under the same 
conditions. The number of cells (approximately 0.61.times.10.sup.5 cells 
per well) in the above wells were comparable to those that received medium 
alone at the end of 4 day incubation period. 
Subsequently, Hodgkin's cell line was grown in the presence of the optimum 
concentration (2 .mu.g) of intact or F(ab').sub.2 fragments of 
anti-BLA-36. Following the incubation period, the number of cells in each 
well was counted by hemacytometer and their viability was determined by 
trypan-blue-dye exclusion limit at intervals of 24 hrs. for 5 days (FIG. 
6). The viability of cells in each well was approximately 98% as 
determined by the dye-exclusion limit, suggesting that the 
growth-inhibitory effect of the antibody was cytostatic. Furthermore, such 
growth inhibitory effect of anti-BLA-36 was reversed following the removal 
of the antibody from the culture medium (results not shown). The 
inhibition of growth was not mediated by the Fc portion of the antibody, 
since F(ab').sub.2 fragments were as effective as the whole antibody (FIG. 
6). An irrelevant monoclonal antibody of the same immunoglobulin class in 
the above concentration range exhibited no inhibition of growth under the 
same conditions (FIG. 6). In addition, anti-BLA-36 pre-absorbed with an 
extract of BLA-36-positive cell line (HDLM-3 or RAJI) exhibited no 
inhibition of growth under the same condition (results not shown). No 
growth-inhibitory effect was observed when the antibody in the above 
concentration range was incubated with antigen negative cell lines (CEM, 
MOLT-4, U-937, and SU-DUL-1) (results not shown). 
C. Discussion 
The goal of the present study was to develop monoclonal antibodies with 
specificity Reed-Sternberg cells, which retained their immunoreactivity in 
tissues that have been routinely-fixed and embedded in paraffin. It was 
anticipated that such antibodies might have value in the diagnosis of 
Hodgkin's diseases, and by their pattern of reactivity, also shed light on 
the cellular origin of the malignant cell of Hodgkin's disease. 
Furthermore, with the availability of such antibodies, attempts might be 
made to characterize and isolate the antigens involved, as a preliminary 
to determining their functional role. In order to achieve this goal, 
monoclonal antibodies generated by immunization of mice with HDLM-3, a 
presumptive Reed-Sternberg or Hodgkin cell line derived from a patient 
with Hodgkin's disease (9), were screened in a system that required 
reactivity with Reed-Sternberg cells in fixed paraffin embedded tissues as 
a selection criterion. Initial screening was performed against the 
immunogen (HDLM-3, Hodgkin's cell line) using an indirect immunoperoxidase 
method. Simultaneously, cryopreparations from known B and T cell lines 
were included in the screening procedure. The initial goal was to discover 
the presence of antibodies specific to the Hodgkin's cell line with no 
evidence of reactivity against B or T cells. In these studies, several 
supernatants containing antibody were identified that reacted with HDLM-3, 
and not with the T cell line, but all of these showed some reactivity 
against the B cell lines. These supernatants were subjected to further 
screening against a broader panel of cell lines of B, T, or histiocytic 
type. On this basis, the antibody, eventually designated anti-BLA-36, was 
selected for further studies. It showed strong reactivity with the cell 
surface membrane of Reed-Sternberg and Hodgkin's cells in 
cytopreparations, and frozen or paraffin sections. A less intense 
reactivity with pre-B and B cell lines and a subset of normal B 
lymphocytes in tissue was also observed. A variety of other hematopoietic 
cell lines were uniformly nonreactive, attesting to the specificity of 
anti-BLA-36 for Reed-Sternberg cells and a subset of B cells. 
Competitive experiments were performed to determine whether the reactivity 
of anti-BLA-36 differed significantly from other anti-leukocyte monoclonal 
antibodies which are commercially available or described in the 
literature. These findings are summarized in Table 2 and FIG. 4. Taken in 
conjunction with specificity and molecular weight data these clearly 
distinguish anti-BLA-36 from all of the following antibodies that 
recognize B cell related antigens [LN-1 and LN-2 (17), B-1 (14), B-2 (29), 
B-4 (15), BVA-1' (13), BA-2 (13), BL-2 (30), B532 (31,32), CB-2 (32), 
BB-01 (33), B-LAST-1 (34), K19 (35), FMC series (36), GB series (37), OKB 
series (38), anti Y29-55 (39), BLA (40) and PCA-1 and 2 (41)]. In 
addition, the KI-1 antibody that recognizes Reed-Sternberg cells and has 
shown reactivity with certain of the large cell non-Hodgkin's lymphomas, 
shows no reaction with normal B cells and is clearly distinct, while 
Leu-M1 differs in being reactive with histiocytes rather than B cells 
(42). Although the tissue-distribution of BLA-36 is different from B7/BB-1 
(43,44), the molecular size of BLA-36 is similar to B7-BB-1. In contrast 
to BLA-36, CD is not known to be N-glycosylated. Finally, the binding 
activity of antibody to CD20 with Reed-Sternberg cells has not been 
reported and was also not detectable in HDLM-3 cell line in this study. 
In tissue from patients with Hodgkin's disease, anti-BLA-36 showed evidence 
of reactivity with Reed-Sternberg cells and mononuclear Hodgkin's cells in 
frozen and paraffin-embedded tissue sections from all subtypes of 
Hodgkin's disease. Reactivity was predominantly associated with the plasma 
membrane, but some of the larger Reed-Sternberg cells showed focal 
reactivity in a paranuclear compartment, consistent with the location of 
the Golgi apparatus. The percentage of cells showing positive reactions 
varied from 75% to almost 100% (Table 4). Of the other cell types 
identified with lymph nodes involved by Hodgkin's disease, none showed 
evidence of reactivity, with the exception of scattered B cells in areas 
where there appeared to be residual follicles with identifiable mantle 
zones. This pattern of reactivity therefore resembled that observed in 
normal lymphoid tissue where anti-BLA-36 gave distinctive reactivity 
within the mantle zone and within the follicle proper. Presumptive T 
lymphocytes in the paracortex were nonreactive as were other cell types. 
When applied to non-Hodgkin's lymphomas, anti-BLA-36 showed reactivity with 
B cell lymphomas, particularly large cell follicular center cell lymphomas 
and immunoblastic sarcomas. Mantle zone lymphomas also showed a high 
proportion of positive cells. T cell lymphomas showed no evidence of 
reactivity and a wide variety of carcinomas and melanomas tested also were 
non-reactive. Anti-BLA-36 may, therefore, be of value in immunohistologic 
studies for distinguishing Hodgkin's disease from this latter group of 
conditions metastatic to lymph node. It is likely to be less helpful in 
distinguishing the cellular forms of Hodgkin's disease, or lymphocyte 
depleted Hodgkin's disease from the large cell lymphomas of B lymphocytic 
origin. In the present study, the intense reactivity observed with 
Reed-Sternberg and mononuclear Hodgkin's cells was clearly of value in 
recognizing these cells when they occurred in small numbers in lymphoid 
tissues. 
The findings in lymphomas exactly mirrored the patterns of staining 
observed in the cell line panels that were examined. T cell lymphomas, 
diffuse histiocytic lymphomas, and carcinomas were consistently 
nonreactive. Burkitt's lymphomas (Raji, Dandi, SU-AMB-1, SU-AMVB-2) were 
by contrast clearly reactive, as were examples of lymphoblastoid (BL-1, 
NU-LB-1), and undifferentiated lymphomas (NU-DUL-1, U-698-M), all of which 
show some features of B cell differentiation. Furthermore, acute 
lymphoblastic leukemia cell lines of B cell derivation (BALL-1 and BALM-2) 
clearly showed positivity with the antibody. The results suggest that 
anti-BLA.26, unlike most other anti-leukocyte antibodies, retains its 
immunoreactivity in paraffin-embedded tissues sections, and it 
distinguishes Reed-Sternberg cells and B cell lymphomas from all other 
malignant cells. Therefore, anti-BLA-36 appears to have diagnostic 
utility. 
Finally, the demonstration that anti-BLA-36 has growth-inhibitory effects 
in a dose-dependent manner on the antigen positive cell lines suggests a 
possible growth-regulatory function of BLA-36. The inhibition of growth 
was not mediated by the Fc portion of the antibody, since F(ab').sub.2 
fragments were as effective as the whole antibody. Preliminary studies 
suggest that the expression of BLA-36 may subserve a receptor function in 
the sense that it mediates the transmission of signals facilitating 
proliferation of cells. These aspects are the subject of continuing study. 
EXAMPLE 2 
Diagnostic Use of Anti-BLA-36 Antibody 
A. Materials and Methods 
Tissue Sections 
Cases with the diagnosis of Hodgkin's disease or non-Hodgkin's lymphoma 
were obtained from the files of the Hematopathology Division of the 
Department of Pathology at the Los Angeles County-University of Southern 
California Medical Center and from the consultation files of the 
inventors. Cases from the Medical Center were fixed in B5 solution. In the 
cases obtained from consultation files, the types of fixation could not be 
determined with certainty in every case. 
Cases of Hodgkin's disease were classified according to the Rye 
Modification of the Lukes-Butler classification (55). Non-Hodgkin's 
lymphomas were arranged according to the Working Formulation of 
Non-Hodgkin's Lymphomas (56). 
Tissue sections from B5-fixed, paraffin-embedded blocks were cut at 6 
microns for hematoxylin and eosin staining and immunohistologic studies. 
Immunohistological Staining 
Monoclonal antibodies used in this study included anti-BLA-36 (49,50), L26 
(51,52), LN-1 (CDW75) (17,47,53), LeuM1 (CD15) (14,53,23), Ber-H2 (CD30) 
(48,53), and UCHL-1 (CD45RO) (54). An indirect unlabelled primary antibody 
method (ABC) was used for localizing antigen with the specific antibody as 
described elsewhere (46,50). All primary antibodies were used at optimum 
dilutions determined in preliminary studies of cases showing positive or 
negative reactivity for the respective antigens. Antibody incubations were 
for 30 minutes at ambient temperature (25.degree.-30.degree. C.). 
Biotinylated horse anti-mouse immunoglobulin antibody was used in the 
linking reagent in an ABC conjugate method (reagents from Vector, 
Burlingame, Calif.). Amino ethyl carbazole was used as the chromogen (46). 
Visual estimates of intensity of immunostains were evaluated independently 
by three of the authors (DRD, RKB, and CRT) and were scored as: 0=absent; 
+=weak; ++=moderate; +++=strong. Table 1 reflects a positive result in 
cases where the consensus score was at least in the + category for 20% or 
more of the critical cells under evaluation. For each stain, negative 
controls were performed to insure the specificity of the reaction. For 
anti-BLA-36 this also included use of the antibody absorbed with immunogen 
(cell line HDLM-3), and an irrelevant antibody of the same immunoglobulin 
class in lieu of the specific antibody. In addition, the stains were 
performed in large batches of 42-60 slides such that the different cases 
served as positive and negative controls, each against the others. 
Finally, for most of the antibodies, individual tissue sections from most 
cases contained at least some cells reactive with the antibody under 
evaluation; these cells served as internal positive controls attesting to 
the validity of the staining procedure (e.g., residual B cells for BLA-36, 
L26; residual T cells for UCHL-1). 
Twenty-four cases of non-Hodgkin's lymphomas of B-lymphocyte origin were 
evaluated. Thirteen of these were characterized at the time of initial 
diagnosis by immunophenotyping using cryostat-sectioned frozen tissue and 
B5-fixed tissue. The remaining eleven cases were immunophenotyped prior to 
use in this study using B5-fixed tissue alone. Four of these had distinct 
follicular patterns providing further presumptive sarcomas (BIBS) with 
monoclonal intracellular immunoglobulin; two were mantle zone lymphomas; 
one was diffuse small non-cleaved, one diffuse large non-cleaved, and the 
last was a case morphologically typical of BIBS, but in which monoclonal 
immunoglobulin was not demonstrable. 
Sixteen cases of Hodgkin's disease were examined including three cases of 
lymphocyte predominant, six cases of nodular sclerosis, five cases of 
mixed cellularity, and two cases of lymphocyte depletion. Frozen section 
immunohistochemistry had been performed in five of these cases; the 
findings were consistent with the diagnosis of Hodgkin's disease. 
Frozen Tissue 
Twenty cases in this study were evaluated using both frozen and B5-fixed 
tissue. This included five cases of Hodgkin's disease, 13 cases of 
non-Hodgkin's lymphoma of B-lymphocyte origin, and two non-Hodgkin's 
lymphomas of T-lymphocyte origin. 
B. Results 
Hodgkin's Disease 
Anti-BLA-36 
The results of anti-BLA-36 reactivity in 16 cases of Hodgkin's disease are 
shown in Table 7. A case of Hodgkin's disease was judged positive when 20% 
or more of Reed-Sternberg cells and variants showed reactivity with an 
intensity of + or greater. Classical Reed-Sternberg cells and variants 
including mononuclear Hodgkin's cells (256), L&H (lymphocytic and 
histiocytic variant) cells of the lymphocyte predominant type, lacunar 
cells in nodular sclerosis, pleomorphic Reed-Sternberg cells in lymphocyte 
depletion and necrobiotic "mummy" cells showed strong staining in all of 
the cases of Hodgkin's disease that were examined. The predominant 
staining pattern was of strong membrane reactivity with weak to moderate 
diffuse cytoplasmic staining (FIGS. 7,8,9). The cytoplasmic staining was 
occasionally punctate and paranuclear. Anti-BLA-36 marked Hodgkin's cells 
more consistently and sensitively than any of the other antibodies used in 
this panel including Leu-M1 and Ber-H2 (Table 7). Findings in frozen 
sections paralleled those described for paraffin sections in terms of 
types of cells stained, numbers of cells stained, and intensity of 
staining. 
TABLE 7 
__________________________________________________________________________ 
Summary of Patterns of Reactivity in Hodgkin's Disease 
and the Non-Hodgkin's Lymphomas 
# Cases 
BLA-36 
L26 
UCHL-1 
Ber-H2 
LN-1 
LeuM1 
__________________________________________________________________________ 
HODGKIN'S DISEASE 
Subtype 
Lymphocyte predominant 
3 3 2 0 0 2 0 
Nodular sclerosing 
6 6 0 0 3 0 4 
Mixed cellularity 5 5 3 0 4 4 4 
Lymphocyte depleted 
2 2 0 0 1 0 1 
Total 16 16 5 0 8 6 9 
NON-HODGKIN'S LYMPHOMAS 
B Cell 
Small lymphocytic lymphoma 
2 2 2 0 0 2 0 
Mantle zone lymphoma 
3 1 3 0 0 2 0 
Small cleaved FCC lymphoma 
4 4 4 0 0 4 0 
Small non-cleaved FCC 
3 3 3 0 0 2 0 
lymphoma 
Large non-cleaved FCC 
6 6 6 0 0 5 0 
lymphoma 
BIBS 6 4 4 0 0 2 0 
Total 24 20 22 0 0 17 0 
T CELL 
TIBS 8 1 1 6 0 1 0 
Lymphoblastic lymphoma 
2 0 0 0 0 0 0 
Mycosis fungoides 1 0 0 1 1 0 0 
Total 11 1 1 7 1 1 0 
__________________________________________________________________________ 
L26 
L26 stained Reed-Sternberg cells and L&H cells in two of three case of 
lymphocyte predominant Hodgkin's disease. 
In the one non-reactive case, not only were Reed-Sternberg cells 
non-reactive, but B lymphocytes in residual follicles did not stain, 
suggesting that this finding may represent a false negative attributable 
to loss of antigen during processing. L26 stained Reed-Sternberg cells in 
three of five cases of mixed cellularity Hodgkin's disease but did not 
stain Hodgkin's cells in nodular sclerosis of lymphocyte depletion. 
UCHL-1 
This pan-T lymphocyte marker stained only scattered small lymphocytes in 
all cases of Hodgkin's disease. 
Ber-H2 
Ber-H2 recognizes the Ki-1 (CD30) antigen (48). This antibody stained 
Reed-Sternberg cells inconsistently in this study, but stained plasma 
cells strong and consistently. Thus these cells served as positive 
internal controls. Reed-Sternberg cells in four of five cases of the mixed 
cellularity type and three of six cases of nodular sclerosis type showed 
positive stained with Ber-H2. No lymphocyte predominant cases marked with 
this antibody. One case of lymphocyte depleted disease was positive, the 
other negative. 
LN-1 
Reed-Sternberg cells and L&H cells were stained in six cases, two cases of 
lymphocyte predominant Hodgkin's disease and four cases of mixed 
cellularity Hodgkin's disease. 
Leu-M1 
Leu-M1 stained Reed-Sternberg cells and variants in four of six cases of 
nodular sclerosis, one of two cases of lymphocyte depleted, and four of 
five cases of mixed cellularity type. Leu-M1 did not stain the 
Reed-Sternberg cells in cases of lymphocyte predominant Hodgkin's disease. 
Non-Hodgkin's Lymphomas 
Thirty-five cases were evaluated: 13 lymphomas of follicular center cell 
origin, two cases of small lymphoma, three cases of mantle zone lymphoma, 
six plasmacytoid immunoblastic sarcomas (BIBS), eight cases of T 
immunoblastic sarcoma (TIBS), two cases of lymphoblastic lymphoma, and one 
case of mycosis fungoides. Twenty of these cases had been immunophenotyped 
with fresh frozen tissue at the time of initial diagnosis. 
Anti-BLA-36 
Immunoreactivity of anti-BLA-36 in a variety of non-Hodgkin's lymphomas is 
listed in Table 7. In cases where residual normal or benign lymphoid 
tissue was present, anti-BLA-36 reacted strongly with most of the 
follicular center cells, about 30% of mantle zone lymphocytes and with 
medium and large transformed lymphocytes scattered in the interfollicular 
regions. Anti-BLA-36 had previously been shown to react strongly with 
follicular center cells and mantle zone lymphocytes in normal lymphoid 
tissue (50). 
In lymphomas of B-lymphocyte origin, the most characteristic staining 
pattern was moderate to strong membrane staining with weak to moderate 
diffuse cytoplasmic staining. Occasionally punctate paranuclear staining 
was seen, often superimposed upon diffuse cytoplasmic staining. 
Overall, larger lymphoid cells stained with anti-BLA-36, whereas small 
lymphocytes did not. Thus, the mature small lymphocytes of small 
lymphocytic lymphoma did not react with anti-BLA-36, but the transformed 
lymphocytes in the proliferation centers (pseudofollicles) of small 
lymphocytic lymphoma stained strongly (FIG. 10). Similarly in the mantle 
zone and follicular center cell lymphomas, stained was most evident in the 
larger cell components that formed a variable part of these tumors. 
In cases of large cell lymphoma derived from follicular center cells, the 
neoplastic lymphocytes marked moderately to strongly (FIG. 11). Four cases 
of plasmacytoid immunoblastic lymphoma showed strong staining of the tumor 
cells; two cases failed to show definite positivity. In one of these 
cases, all antibodies tested were non-reactive, and morphologic 
examination suggested inadequate fixation. In the remaining case, BLA-36 
was judged non-reactive, while L26 stained approximately 10% of the tumor 
cells. 
Anti-BLA-36 was judged non-reactive in 10 of the 11 T-cell lymphomas. One 
case of TIBS showed positivity of approximately 30% of cells for BLA-36 
and 15% for L26. This case was recorded as a positive case in Table 7. A 
second case showing approximately 10% of cells reactive with anti-BLA-36 
and L25 was listed as negative in accordance with the criteria set forth 
under methods. In both of these cases, UCHL-1 (anti-T cell) stained many 
small lymphocytes and scattered larger cells. Frozen section immunotyping 
was not available in the first of these cases and was inconclusive in the 
second; classification of these cases had therefore been based primarily 
on morphology. 
L26 
L26 stained the great majority of all B lymphocytes in benign and 
neoplastic conditions. This held true across the spectrum of B-lymphocyte 
neoplasms. L26 failed to stain T-lymphocyte lymphomas in all cases but 
one: as noted above, this case also showed some positivity for BLA.26 and 
previous attempts at establishing an immunophenotype has been 
inconclusive. Likewise, L26 did not stain T-lymphocyte zones in benign 
areas of any lymph node evaluated in the study. 
UCHL-1 
UCHL-1 stained only scattered small lymphocytes in the neoplasms of 
B-lymphocyte origin. Six of the eight cases designated as TIBS showed 
positivity of large apparently neoplastic cells; in the other two cases 
only small lymphocytes stained convincingly. 
LN-1 
LN-1 marked the neoplastic lymphocytes in 11 of 13 follicular center cell 
lymphomas, plus two of three mantle zone lymphomas and the "pseudo 
follicle" cells of both cases of small lymphocytic lymphoma. Two of six 
cases of BIBS were positive, while rare positive cells were observed in 
one of eight cases of TIBS. Benign follicular center cells stained 
strongly in those cases with residual follicles. 
Ber-H2 and Leu-M1 
These two antibodies contributed little to the evaluation of the 
B-lymphocyte lymphomas. Ber-H2 stained plasma cells, and Leu-M1 strongly 
stained scattered granulocytes, neither of which finding contributed to 
diagnostic evaluation. Cases of Ber-H2 (Ki-1)-positive large cell 
lymphomas were not encountered in this study. 
C. Discussion 
Morphological and immunohistochemical studies suggest that lymphocyte 
predominant Hodgkin's disease is a B-cell neoplasma and as such is 
distinct from other subtypes of Hodgkin's disease (51,57-65). This view 
derives at least in part from the observation that the Reed-Sternberg 
cells and L&H cells of this subtype of Hodgkin's disease react with 
B-lymphocyte markers such as L26 (51,52), KiB3 (60), LN-1 (47,53), B1, Leu 
14 and Dapo pan-B (64), but not with other antibodies such as Leu-M1 (24) 
or Ber-H2 (53) that do not stain B cells. Conversely, Reed-Sternberg cells 
and variants in the other subtypes of Hodgkin's disease typically do not 
stain with antibodies that mark B cells, but do stain with Leu-M1 (24) and 
Ber-H2 antibodies (53). In the present study, anti-BLA-36, a marker of 
certain B lymphocytes and B cell lymphomas (49,50), stained Reed-Sternberg 
cells and variants consistently in all four subtypes of Hodgkin's disease. 
The findings of this study are in accord with several recent lines of 
evidence accumulating from clinical (63,66), immunohistochemical 
(47,51,53,57) and gene rearrangement studies (68,69) which offer some 
support for the concept that, at least in some instances, the 
Reed-Sternberg cell may be derived from a cell in common lineage with the 
B lymphocyte. As noted above, the lymphocyte predominant subtype, possibly 
the most differentiated form of Hodgkin's disease (70), appears to be 
derived from B lymphocytes based on morphological (57,60,63,64) and 
immunohistochemical (47,51,53) characteristics. In addition, progressive 
transformation of germinal centers commonly occurs in association with, or 
may precede the development of, lymphocyte predominant Hodgkin's disease 
(57-62,71). Clonal rearrangements of immunoglobulin heavy or light chain 
genes have been found in tissue samples, rich in Reed-Sternberg cells, 
from patients with other subtypes of Hodgkin's disease (68,69), an 
observation consistent with a B-lymphocyte origin for the Reed-Sternberg 
cell. Clinicopathologic evidence for a B-cell origin of Reed-Sternberg 
cells takes the form of rare occurrences of lymphomas in which both 
Hodgkin's disease and non-Hodgkin's lymphoma (composite lymphomas) are 
found in the same lymph node group (66,72,73). In these cases, the 
non-Hodgkin's lymphoma is usually a B-cell neoplasm. Moreover, the 
co-existence of a large cell lymphoma of B-cell origin is especially 
common with lymphocyte predominant Hodgkin's disease (66). 
Finally, a recent report demonstrating Epstein-Barr virus (EBV) genomes 
within Reed-Sternberg cells gives credence to the notion that 
Reed-Sternberg cells may be of B-lymphocyte lineage (74). C3d, which is 
found on most mature B cells, is identical to the receptor for EBV; thus, 
B cells are the usual human host cell for EBV (75). 
The present work identified a monoclonal antibody raised to a Hodgkin's 
disease cell line that also detects a protein expressed on early B cells 
and on activated B cells. As far as we are aware, this is the first 
antibody raised to a Hodgkin's cell line which also reacts with benign and 
malignant B lymphocytes. Other antibodies that have been found to stain 
both Reed-Sternberg cells and B cells have been raised against B cells, 
e.g., L26 (52,76), LN-1 and LN-2 (17), EPB-1 (77). The reciprocal nature 
of this reactivity may provide evidence linking the Reed-Sternberg cell 
and the B lymphocyte. The BLA-36 is expressed on early B cells and 
activated B cells (but not resting or peripheral blood B cells). It is 
also present on the surface of Reed-Sternberg cells and their mononuclear 
variants, often referred to as Hodgkin's cells (25). However, the 
significance of BLA-36 expression in cellular function, or in terms of 
elucidating the origin of the Reed-Sternberg cell, still is not entirely 
known. We have shown (49,50) that addition of anti-BLA-36 antibody to in 
vitro cultures of B cells, B cell lines, or Hodgkin's lines produces an 
immediate, but reversible, inhibition of cell division, with a less 
immediate and less profound suppression of DNA synthesis, as measured by 
uptake of tritiated thymidine (50). It is tempting, therefore, to 
speculate that BLA-36 may subserve the function of a cell surface receptor 
for an as yet unknown growth factor. 
Apart from the implications anti-BLA-36 may have in exploring B-cell 
proliferation or in delineating the origin of the Reed-Sternberg cell, 
this study demonstrates that, on a more practical level, anti-BLA-36 is of 
value in the identification of Reed-Sternberg cells in all four major 
types of Hodgkin's disease. It is specific and sensitive, and it has the 
advantage of being effective in B5-fixed paraffin-embedded material. 
Anti-BLA-36 may be of particular value in differentiating between 
lymphocyte predominant Hodgkin's disease and other benign and malignant 
lymphoproliferative disorders, including small lymphocytic lymphoma, 
T-zone hyperplasia, sarcoidosis, progressive transformation of germinal 
centers, and giant lymph node hyperplasia. The key to the 
histopathological differential is identifying the presence of rare 
Reed-Sternberg and L&H cells. The combination of immunohistological 
staining for BLA-36 and the excellent morphology of B5- or formalin-fixed 
paraffin-embedded sections facilitates recognition of L&H cells and 
Reed-Sternberg cells and permits their distinction from any positively 
stained transformed B lymphocytes which may be encountered in conditions 
other than Hodgkin's disease. 
Anti-BLA-36 may also have a role in the evaluation of B-cell lymphomas and 
their distinction from T-cell processes. Those B-cell lymphomas with a 
predominant large cell component (large non-cleaved follicular center cell 
lymphoma and BIBS) are mostly positive (10 or 12), while large cell 
lymphomas of T-cell type (TIBS) are mostly negative (7 of 8). Furthermore, 
review of the discrepant cases raised doubts as to the validity of the 
original diagnoses that had been made in the absence of conclusive 
immunologic data. In B-cell lymphomas composed of smaller cells, 
anti-BLA-36 serves to highlight the presence of foci of larger transformed 
lymphocytes (pseudofollicles) in small lymphocytic lymphoma, and reveals 
minority populations of large follicular center cells or follicle 
structures in small cleaved cell lymphoma, thereby abetting the diagnosis 
of these conditions. 
With regard to other antibodies employed in this study, we noted that L26, 
a widely used pan-B cell marker, reacted with B cells in most cases of 
non-Hodgkin's lymphoma, thereby showing considerable overlap with 
anti-BLA-36. However, L26 stained Reed-Sternberg cells and variants in 
only 5 of 16 cases of Hodgkin's disease. The limited ability of L26 to 
stain Reed-Sternberg cells in Hodgkin's disease, other than lymphocyte 
predominant type, is in agreement with the experience of others (51). The 
failure of L26 to stain the Reed-Sternberg cells and L&H cells in one of 
our cases of lymphocyte predominant Hodgkin's disease is at variance with 
a report by Pinkus and Said (51) describing uniform reactivity of L26 in 
this condition. A possible explanation for this discrepancy may related to 
accidental destruction of antigen during tissue processing, since residual 
B cells in this case also were non-reactive for L26. 
We found other minor discrepancies with published reports. Hsu et al (23) 
described positivity for LeuM1 in Reed-Sternberg cells of all 20 cases of 
Hodgkin's disease that they studied; we observed positivity in 9 of 13, 
excluding the lymphocyte predominant subtype. Other investigators have 
reported positivity for LeuM1 of approximately 90% (24,53). There appears 
to be uniform agreement that the Reed-Sternberg cells and variants of 
lymphocyte predominant Hodgkin's disease are nonreactive for LeuM1. LN-1 
was positive in Reed-Sternberg cells of 6 of 16 cases in our series, 
approximately 30% of cases in the series described by Chittal and 
colleagues (53) and 28% of the cases in the earlier publications on LN-1 
by Sherrod et al (47). Finally, in the present study we observed Ber- H2 
positivity in Reed-Sternberg cells in 8 of 16 cases; Chittal and 
colleagues (53) reported positivity in 75% of their cases, while Stein and 
collaborators in an earlier report (67) cited positivity for Ki-1 in 100% 
of cases. The consensus appears to be that lymphocyte predominant disease 
is least often positive for Ki-1 (or Ber-H2) [Chittal et al: 3 of 18 
(53)]. In our series all of our Ber-H2 (Kil) positive cases were in the 
nodular sclerosis, mixed cellularity or lymphocyte depleted category for 
an overall positivity rate of 69%. 
These findings extend the essentials of our previous report (50) that 
revealed BLA-36 positivity in Reed-Sternberg cells of 28 of 28 cases of 
Hodgkin's disease and 23 of 25 cases of B-cell lymphoma. Furthermore, 
anti-BLA-36 was considered to be distinct from other human anti-leukocyte 
antibodies, a conclusion supported by the present study. These results 
provide further incentive for additional studies of the diagnostic utility 
of this antibody. Recognition of a cell surface antigen restricted to B 
cells and Reed-Sternberg cells also raises the possibility of the use of 
anti-BLA-36 for imaging and radio-immunotherapy of B-cell lymphomas and 
Hodgkin's disease. 
Notwithstanding that reference has been made to particular preferred 
embodiments, it will be understood that the present invention is not to be 
construed as limited to such, rather to the lawful scope of the appended 
claims. 
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