Constitutive overexpression of a novel 21 kDa protein by Hodgkin Lymphoma and Aggressive Non-Hodgkin Lymphomas

Background CD30, a 120 kDa surface phosphorylated protein is a member of tumour necrosis/nerve growth factor receptor (TNF/NGFR) family and constitutively expressed by Hodgkin and Reed-Sternberg (HRS) cells of Hodgkin lymphoma (HL) and the neoplastic cells of Anaplastic Large Cell Lymphoma (ALCL). A disease-specific protein marker is yet to be identified in Hodgkin lymphoma cells. In order to define HL-specific biomarkers, novel murine monoclonal antibodies were developed in our laboratory. Results Murine monoclonal antibodies (mabs) were raised against the B3 sub clone of HL-derived cell line KM-H2. Two of these mabs (clone R23.1 mab and clone R24.1 mab) are IgG1 class antibodies that recognize a 21 kDa protein present at the cell membrane and in the cytoplasm in HL-derived cell lines. Clone R24.1 mab recognizes a formalin-resistant epitope and labels HRS cells in tissue samples from patients with HL of the classical type, ALCL, and subsets of T and B cell aggressive Non-Hodgkin Lymphomas (NHL). The antigen recognized by the clone R23.1 mab and clone R24.1 mab does not share epitopes with CD30 cluster regions A, B, or C, and, unlike CD30, is not expressed by phytohemagglutinin (PHA) activated T cells. Conclusion The 21 kDa protein detected by clone R23.1 and clone R24.1 mabs is a novel membrane-associated protein that may be a potential marker for the diagnosis and targeted therapy of HL and aggressive T and B cell NHL.

The identification of cell surface molecules that are not activation-associated markers, and have specificity for HRS cells thus remains a desirable goal. To this end, we have developed and characterized 2 novel monoclonal antibodies, R23.1 and R24.1, that recognize a 21 kDa molecule expressed by H/RS and ALCL cells, but not by phytohemagglutinin (PHA) activated CD30+ T lymphocytes.

Reactivity of R23.1 and R24.1 against CD30+ and CD30cell lines
The two antibodies were reactive against cell surface antigens of almost all CD30+ cell lines as assessed by FACS analysis. Of 14 CD30+ cell lines, R23.1 and R24.1 labelled 12 (86%) ( Table 1). Of 14 CD30 negative cell lines, none was labelled by either R23.1 or R24.1 ( Table 2). Examples of cell surface labelling of HL cell line KMH2 by the two antibodies as well as anti-CD30 antibody BerH2 are shown in Figure 1. Relative antigen densities as indicated by the position of the fluorescence peak channel tended to vary with each antibody as well as each cell line (data not shown).

Both antibodies labelled cytoplasmic antigens in all
Hodgkin and ALCL cell lines tested (Table 3). Examples are shown in Figure 2. The pattern was generally diffuse in both mononuclear and multinucleated forms of the cells, though strong staining was observed on the cell mem-brane. The staining pattern was similar to that observed with the BerH2 anti-CD30 antibody.

Immunohistochemistry in tissue sections
When staining was performed using cryostat sections, both antibodies labelled HRS cells in classical HL cases, of both nodular sclerosis and mixed cellularity subtypes. In lymphocyte predominance (LPHD) cases, one of two cases contained L&H variants which were labelled by both antibodies (data not shown). In formalin fixed sections clone R23.1 mab was not reactive with any cells. In a series of formalin-fixed HL and NHL cases, clone R24.1 mab labelled none of LPHD cases, 100% of classical Hodgkin lymphoma cases, 1 of 4 T cell-rich B cell lymphomas (TCRBCL), 63% of diffuse large B cell lymphomas (DLBCL), 100% of Anaplastic large cell lymphomas (ALCL), and 80% of peripheral T cell lymphomas (PTCL), respectively (Table 4). A survey of non-lymphoid tissue, both benign and malignant (n = 75), including skin melanomas, lung, colorectal, urinary bladder, and mesenchymal tumours revealed no reactivity to the clone R24.1 mab. Examples of positive staining in HL and ALCL clinical biopsies are shown in Figure 3.

Activation of normal peripheral blood T lymphocytes
PHA activation of peripheral blood T cells resulted in the expression of CD30 which peaked within 48 hours. CD30 were expressed by 50% of CD3+ T cells within 1 day post simulation, peaking within 48 hours at which time point 70% of CD3+ T cells were CD30 positive, while the antigens recognized by clone R24.1 mab and clone R23.1 mab were not expressed by PHA activated CD3+ T cells even up to 7 days post-stimulation ( Figure 4). This indicates that clone R24.1 and clone R23.1 mabs recognize epitopes/ antigens unrelated to CD30.
FACS analysis of Hodgkin cell line KMH2 after labeling with clone R23.1, clone R24.1, or CD30 mabs Figure 1 FACS analysis of Hodgkin cell line KMH2 after labeling with clone R23.1, clone R24.1, or CD30 mabs. Blue lines indicate isotype control binding. Solid red curves indicate mab binding.

Competitive binding assays
Neither clone R24.1 mab nor clone anti-R23.1 mab blocked the ability of three different anti-CD30 antibodies specific for cluster regions A, B, or C of the CD30 molecule, to bind to KMH2 cells ( Figure 5). Similarly, none of the anti-CD30 antibodies blocked the binding of clone R23.1 mab or clone R24.1 mab to KMH2 cells (data not shown).
With the intention of developing additional antibodies with specificity for non-activation markers of Hodgkin lymphoma, we have characterized 2 novel antibodies that detect a protein constitutively overexpressed in Hodgkin lymphoma. One of the two antibodies, clone R24.1 mab, recognises a formalin-resistant epitope. Although the specificity of the 2 antibodies initially raised the possibility that they are directed against CD30, subsequent experiments proved otherwise. Two B cell lymphoma cell lines (OCI Ly1 and OCI Ly19,  much smaller than the 120 kDa phosphorylated glycoprotein CD30 [2]. We are in the process of determining the identity of the 21 kDa protein by mass spectrometry and its encoding gene by expression cloning.
A non-activation induced cell surface protein expressed in Hodgkin lymphoma and aggressive non-Hodgkin lymphomas may have immunotherapeutic potential as antibodies that target such a molecule are less likely to be toxic to normal activated immune cells. Anti-CD30 immunotherapy is being explored in pre-clinical models and a few human clinical trials have been conducted, but toxicity has been a problem [38][39][40][41][42][43][44][45][46][47][48][49][50][51][52] and the long-term sequelae are not yet known. Novel immunotherapy targets and antibodies still require exploration. We are currently evaluating the cytotoxic effects of the two antibodies against HL cell lines in vitro and in vivo HL xenograft models of Hodgkin lymphoma. Tissue microarrays are being assembled from large numbers of Hodgkin lymphoma, diffuse large T and B cell lymphoma, and Anaplastic Large Cell Lymphoma clinical specimens from patients with longterm follow-up in order to determine whether the 21 kDa protein expression has any prognostic significance.

Conclusion
Clone R23.1 and clone R24.1 mabs recognize a novel 21 kDa cell surface and cytoplasmic protein in HL and ALCL cells as indicated by immunoprecipitation and Western-

Methods
Animal care and experiments were carried out in accordance with the guidelines of the Canadian Council on Animal Care and all protocols were approved by the Animal Care Committee of the University of Toronto.
Research (immunohistochemistry) was carried out on archived paraffin blocks from clinical biopsies. The use of the archived paraffin tissue was approved by the University of British Columbia Clinical Research Ethics Board in compliance with the Helsinki Declaration (Approval Certificate number H06-60016).  [53]. Hybridoma supernatants were screened for IgG and IgM production by ELISA. Six IgM and 10 IgG producers were identified. Supernatants from the 10 IgG producers were screened by FACS analysis for anti-KMH2 antibodies and 5 of the 10 were found to be producing such antibodies. These were cloned twice by limiting dilution and the supernatants were re-screened by FACS for anti-KM-H2

Determination of cell surface expression of antigens on cell lines
Cells were washed (4 min.; 300 × g) two times in calcium and magnesium-free phosphate buffered saline (PBS -) and then treated as follows to block non-specific binding of mabs. Cells undergoing direct staining were blocked with 10 µg of pure mouse IgG (Sigma, Oakville, ON, Canada) for 20 minutes at 4°C. Cells undergoing indirect staining were blocked with 10% normal goat serum (NGS) for 20 minutes at 4°C. Cells were then washed in PBS -. Cell concentrations were adjusted so each tube received 1 × 10 6 cells. CD30-FITC (Dako, Mississauga, ON, Canada) was added at 1 µg per tube, the two mabs were added at 1 µg per tube, the mouse IgG1 isotype controls (IgG1-FITC and mouse IgG unconjugated) were both added at 1 µg per tube. Primary antibody staining was done for 30 minutes at 4°C in the dark. Cells were then washed in PBS -, and goat anti-mouse IgG -PE (Dako) was added and allowed to incubate for 30 minutes at 4°C in the dark. Viability was assayed by propidium iodide exclusion. Cells were washed in PBSand 300 µl of fixative (1% paraformaldehyde) was added. Cells were analyzed on a FACSort flow cytometer (Becton Dickinson) within 15 minutes.

Immunocytochemistry and immunohistochemistry
Immunocytochemistry was performed using monoclonal antibody BerH2 (Dako), and the 3 monoclonal antibodies. Cytospins were prepared using the cell lines KMH2, Immunohistochemistry was carried out on deparaffinized tissue sections (5 micron thickness). Endogenous peroxidase was quenched by treating the sections with 3% hydrogen peroxide for 10 minutes, followed by rinsing in PBS. Antigen retrieval was done by pepsin digestion (0.4% pepsin for 3 minutes, then rinsing in PBS -) prior to labelling. Non-specific binding was blocked with 10% human AB serum for 20 minutes at room temperature. R24.1 hybridoma supernatant was used at 1/4 dilution whereas purified R24.1 was used at 16 µg/ml. The slides were incubated at room temperature for 60 minutes, rinsed in PBS and then incubated with biotinylated goat anti mouse IgG (Zymed) at 1/200 at room temperature for 20 minutes, followed by rinsing in PBS. They were then incubated for 20 minutes in 1/4 dilution of streptavidinbiotin complex labelled with horseradish peroxidase for 20 min. (Ultra Streptavidin Detection System; Signet). After rinsing in PBS -, the slides were incubated with AEC for 10 to 20 minutes, rinsed in PBSand counterstained with Mayer's hematoxylin.

Chemicals and reagents
IMDM media was purchased from StemCell Technologies Inc. (Vancouver, Canada). Protein G-sepharose beads were purchased from Sigma (St. Louis, Mo, USA). Fungizone was purchased from Gibco (Grand Island, NY, USA). Proteinase inhibitor cocktail, 30% polyacrylamide (acrylamide: bis = 29:1) and substrate BCIP/NBT were purchased from Sigma (St. Louis, MO, USA). Seize Classic investigator and director of the laboratory and responsible for the design of the study and its coordination. He interpreted the results independently of the author 1, and contributed in the preparation of the manuscript.