DOHH-2

EBV activates NKL homeobox genes in DOHH-2 Cells

NKL homeobox genes encode developmental transcription factors regulating basic processes in cell differentiation. EBV infects B-cells and influences the activity of signaling pathways. Therefore, EBV infection impacts the pathogenesis and the outcome of B-cell malignancies including Hodgkin lymphoma and diffuse large B-cell lymphoma (DLBCL).

Limited dilution of DLBCL-derived cell line DOHH-2 was performed to end up with pure EBV-positive and EBV-negative subclones. Subsequent FISH and PCR analyses confirmed the presence and absence of EBV in all cells of the isolated subclones (Fig. 1A and 1B). To determine the latency program operating in EBV-positive DOHH-2 subclones, an RT-PCR analysis of selected EBV-encoded genes (Fig. 1C) was performed. Quantification of CD21 expression by RQ-PCR demonstrated high levels in EBV-positive DOHH-2 cells while expression in EBV-negative DOHH-2 cells was barely detectable (Fig 1D). In contrast, the expression levels of the B-cell receptor cofactor CD19 were similar in both DOHH-2 subclones (Fig. 1D).

Expression profiling data generated from EBV-positive and EBV-negative DOHH-2 subclones showing the top-1000 differentially expressed genes reflected their deregulation (Fig. 2A). In addition, the differing expression levels of these genes were confirmed by RQ-PCR. The EBV-positive subclone expressed significantly decreased levels of BCL6, BACH2, and IL4R (Fig. 2B) while the genes for IRF4, MIR155HG, and PRDM1 showed elevated transcript levels (Fig. 2C). However, RQ-PCR and Western blot analyses showed no significant difference between EBV-positive and EBV-negative subclones (Fig. 2C). RQ-PCR analysis of these genes in EBV-positive/negative DOHH-2 cells demonstrated significant differences for HLX, NKX6-3 and MSX1 while the levels of HHEX were similar in both subclones (Fig. 2D). As compared to primary hematopoietic cells/tissues, EBV-positive DOHH-2 cells expressed enhanced levels of HLX, resembling those in bone marrow and exceeding the levels in lymph nodes and spleen (Fig. 2E).

Fig. 1 Characterization of EBV-positive/negative DOHH-2 subclones. (Nagel S, et al., 2019)

Fig. 2 Gene expression analyses in EBV-positive/negative DOHH-2 subclones. (Nagel S, et al., 2019)

Targeting B-cell malignancies with anti-CD37 radioimmunoconjugate ¹⁷⁷Lu-NNV003

CD37 has been used as a target for naked antibodies and two antibody-drug conjugates. Furthermore, the next generation RIC is the murine version of ¹⁷⁷Lu-NNV003 and is currently in clinical trials for relapsed/refractory lymphomas.

¹⁷⁷Lu-NNV003 showed an antiproliferative effect against the cell lines tested (Fig. 3). The biodistribution of ¹⁷⁷Lu-NNV003 was performed in NSG mice with s.c. MEC-2 xenografts and in CB17 SCID mice with s.c. DOHH-2 xenografts (Fig. 4). ¹⁷⁷Lu-NNV003 reached a maximum uptake of 45% of injected activity/g in MEC-2 tumors after three days and 15% in DOHH-2 tumors after two days. The injected activity/g was approximately 12% for DOHH-2 and 10% for MEC-2 7 days after injection. After initial distribution to normal organs, no redistribution of the radionuclide was observed. The absorbed radiation dose after injection of 100 MBq/kg ¹⁷⁷Lu-NNV003 in DOHH-2 tumors was 4.6 Gy and lower than 1.2 Gy in all of the normal tissues, except in blood (Fig. 5).

Fig. 3 Anti-tumor effect of ¹⁷⁷Lu-NNV003 in REC-1 and DOHH-2 cells. (Lin JT, et al., 2022)

Fig. 4 Biodistribution of ¹⁷⁷Lu-NNV003. (Lin JT, et al., 2022)

Fig. 5 Absorbed radiation dose (Gy) to organs and tumor for CB17 SCID mice with DOHH-2 s.c. xenografts injected with ¹⁷⁷Lu-NNV003. (Lin JT, et al., 2022)