CD30 targeting with brentuximab vedotin: a novel therapeutic approach to primary effusion lymphoma

Abstract

Primary effusion lymphoma (PEL) is an aggressive subtype of non-Hodgkin lymphoma characterized by short survival with current therapies, emphasizing the urgent need to develop new therapeutic approaches. Brentuximab vedotin (SGN-35) is an anti-CD30 monoclonal antibody (cAC10) conjugated by a protease-cleavable linker to a microtubule-disrupting agent, monomethyl auristatin E. Brentuximab vedotin is an effective treatment of relapsed CD30-expressing Classical Hodgkin and systemic anaplastic large cell lymphomas. Herein, we demonstrated that PEL cell lines and primary tumors express CD30 and thus may serve as potential targets for brentuximab vedotin therapy. In vitro treatment with brentuximab vedotin decreased cell proliferation, induced cell cycle arrest, and triggered apoptosis of PEL cell lines. Furthermore, in vivo brentuximab vedotin promoted tumor regression and prolonged survival of mice bearing previously reported UM-PEL-1 tumors as well as UM-PEL-3 tumors derived from a newly established and characterized Kaposi's sarcoma-associated herpesvirus- and Epstein-Barr virus-positive PEL cell line. Overall, our results demonstrate for the first time that brentuximab vedotin may serve as an effective therapy for PEL and provide strong preclinical indications for evaluation of brentuximab vedotin in clinical studies of PEL patients.

Figure 1

Characterization of UM-PEL-3 cells. (A) Pathological findings for the UM-PEL-3 xenograft mouse model. (I-II) Hematoxylin and eosin-stained tissue sections of the gastrointestinal tract showing extensive infiltration of the muscularis propria by lymphoma cells. (II) Higher magnification of pleomorphic lymphoma cells with hyperchromatic nuclei. (III) Immunohistochemistry for CD138 and (IV) HHV8 latent antigen LANA. Original magnification ×400 (I) and ×600 (II-IV). (B) IFA of UM-PEL-3 cells reveals positive staining for HHV8 latent antigen LANA (red). (C) UM-PEL-3 cells were stimulated with 1 mM butyrate for 4 days and RNA expression of the indicated transcripts was measured by qRT-PCR. Fold change compared with unstimulated cells indicates induction of IE (RTA), early (ORF55), or late (gB and K8.1) lytic genes. Error bars correspond to standard error of mean. (D) Immunostaining image of late lytic protein K8.1 (red) in UM-PEL-3 cells stimulated with 0.75 µM SAHA for 24 hours (bottom) and unstimulated cells (top). For panels B and D, nuclei are stained with 4,6 diamidino-2-phenylindole (DAPI; blue). Magnification ×400. Experiments depicted in panels B-D were repeated thrice independently in triplicate. The representative data from one experiment are shown.

Figure 2

CD30 expression in PEL cell lines and primary tumors. (A) Histogram of flow cytometric analysis shows CD30 staining on the indicated PEL cell lines and control WSU-NHL cell line. Data were obtained by acquiring 1 × 10⁴ cells stained with the anti-CD30-fluorescein isothiocyanate (FITC) antibody. Blue line indicates CD30-stained cell population and black line indicates background fluorescence observed with isotype control. (B-D) CD30 expression in primary PEL cells. Malignant cells from PEL patient (case 1) are highlighted with Papanicolaou stain (I). Immunocytochemistry analysis shows positive staining for epithelial membrane antigen (EMA) (II), HHV-8 latent antigen LANA (nuclear stain) (III), and CD30 (IV). Original magnification: I, III, IV ×100 and II ×50. (C) Large and discohesive malignant cells from PEL patient (case 2) are highlighted with hematoxylin and eosin stain (I). Immunocytochemistry analysis shows positive staining for EMA (II), HHV-8 latent antigen LANA (nuclear stain) (III), and CD30 (IV). Original magnification: I-IV ×100. (D) Top shows hematoxylin and eosin staining of PEL patient (case 3) lymphoma cells and bottom shows an immunohistochemistry stain for CD30 in neoplastic cells of the same PEL patient. Original magnification ×600.

Figure 3

Brentuximab vedotin blocks proliferation of PEL cells. Human PEL cell lines BC-1 (A), BC-3 (B), UM-PEL-1c (C), and UM-PEL-3c (D) were treated with brentuximab vedotin (B.V.) at indicated doses for 0, 24, 48, and 72 hours. Proliferative response at each time point was measured by MTS assay. Results are shown as fold change of proliferation compared with time 0 hours. Experiments depicted in panels A-D were repeated thrice independently in triplicate. The representative data from one experiment are shown. Error bars correspond to standard error of mean in all graphs.

Figure 4

Brentuximab vedotin induces G2/M cell cycle arrest of PEL cells. PEL cell lines BC-1 (A), BC-3 (B), UM-PEL-1c (C), and UM-PEL-3c (D) were treated with brentuximab vedotin (B.V.) at increasing concentrations. At 24 hours after treatment, cells were stained with propidium iodide to measure DNA content and analyzed by flow cytometry for cell cycle distribution. Bar graphs indicate the percentage of cells in different phases of cell cycle (G0, G1, S, G2/M). Experiments depicted in panels A-D were repeated thrice independently in triplicate. The representative data from one experiment are shown. Error bars correspond to standard error of the mean in all graphs.

Figure 5

Brentuximab vedotin triggers apoptosis of PEL cells. Lymphoma cell lines lacking CD30 expression WSU-NHL (A) and Raji (B) and CD30-expressing PEL cell lines BC-1 (C), BC-3 (D), UM-PEL-1c (E), and UM-PEL-3c (F) were treated with increasing concentrations of brentuximab vedotin (B.V.) or Ig-VcMMAE. At 72 hours after treatment, cell viability was determined by flow cytometry following YO-PRO and propidium iodide staining. Experiments depicted in panels A-E were repeated thrice independently in triplicate. The representative data from one experiment are shown. Error bars correspond to the standard error of the mean in all graphs.

Figure 6

sCD30 levels, CD30 internalization, and brentuximab vedotin cell surface binding. (A) sCD30 levels measured by ELISA in indicated cell lines and in cells directly derived from mice ascites. Error bar represents standard error of the mean between triplicate wells. (B) Cell surface CD30 expression and (C) cell surface brentuximab vedotin binding. BC-3, UM-PEL-1c, UM-PEL-3c, and Karpas 299 cells were incubated with 15 μg/mL brentuximab vedotin for 0, 24, and 48 hours followed by incubation with anti-CD30 (fluorescein isothiocyanate [FITC]) (B) or anti-hIgG (FITC) (C) to determine antigen-binding capacity values for CD30 (B) and of bound brentuximab vedotin (C), respectively. Microbeads coated with known quantities of mouse-IgG were incubated with saturating quantities of anti-CD30 (FITC) or anti-hIgG (FITC) antibodies to determine mean fluorescence intensity assessed by flow cytometry to obtain standard curves. Data are representative of 3 independent experiments and error bars correspond to the standard error of the mean.

Figure 7

Brentuximab vedotin extends the survival of PEL xenograft mice. Kaplan-Meier survival curves of PEL xenograft mice. NOD/SCID mice (n = 5/group) were injected with 25 × 10⁶ UM-PEL-1 (A) and UM-PEL-3 (B) cells. At 3 days postinjection, mice were treated for 3 weeks with interperitoneal injections of brentuximab vedotin (B.V.), PBS, or isotype-matched irrelevant Ig-vcMMAE. Only one control group is shown, because mice in both groups exhibited identical Kaplan-Meirer survival curves. Results are representative of 2 independent experiments.