Constitutive overexpression of a novel 21 kDa protein by Hodgkin lymphoma and aggressive non-Hodgkin lymphomas

Abstract

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.

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.

Figure 2

KMH2 cells labelled with clone R24.1 mab. Both membrane and cytoplasmic staining was observed. Magnification ×400.

Figure 3

Tissue sections from clinical Biopsies labelled with clone R24.1 mab. On the left panel, a HL tissue section shows intense staining with a membrane and Golgi pattern. On the right panel, an ALCL tissue section shows intense staining of neoplastic ALCL cells. Magnification ×400.

Figure 4

PHA stimulation of T cells. T cells exposed to PHA expressed CD30 within 24 hours, peaking at 48 hours. Expression of R23.1 and R24.1 remained at control levels even up to 7 days after PHA stimulation, indicating that activation of T cells does not induce expression of R23.1 and R24.1 antigens, unlike CD30.

Figure 5

Competitive binding assay. Clone R24.1 and clone R23.1 mabs did not block the binding of anti-CD30 (BerH2 antibody) to KMH2 cells.

Figure 6

Western-blot analysis of DEL, KMH2 and L-428 cell lysates. A protein with a mass of 21 KDa was detected by clone R23.1 (A) and clone R24.1 (B) mabs in the cell lysates. Lane 1 contains molecular weights and lanes 2–4 contain cell lysates of DEL, KMH2 and L-428 respectively.

Figure 7

Immunoprecipitation and Western-blot analysis of DEL, KMH2 and L-428 cell lysates. 23.1 (Panel A and C) and R24.1 (Panel B and D). Samples were separated on 4–20% Life-gel and transferred onto nitrocellulose membranes. The membranes were then blocked in 5% non-fat dry milk and probed with monoclonal antibody clones R23.1 (Panel A and B) and R24.1 (Panel C and D) followed by goat-anti-mouse IgG-AP. The membranes were developed in BCIP/NBT. A protein band of ~21 KDa as well as IgG light chain were detected in all three lysates examined. Lane 1 contains molecular weights and lanes 2–4 contain Immunoprecipitated samples of DEL, KMH2 and L-428.