Targeting B-Cell Malignancies With Anti-CD20-Interleukin-21 Fusokine
Authors: Bhatt Shruti , Zhu Daxing , Jiang Xiaoyu , Shin Seung-uon , Timmerman John M , Rosenblatt Joseph D. , Lossos Izidore S 
Published: October 2019

Abstract
The anti-CD20 antibody rituximab has revolutionized the treatment for B cell non-Hodgkin lymphomas (NHLs). However, rituximab has limited effectiveness as a single agent in some NHL subtypes and its clinical efficacy is compromised by acquired drug resistance. As a result, many patients still succumb to NHLs. Hence, strategies that enhance the activity of anti-CD20 antibody may improve patient outcome.
Interleukin-21 (IL21), a member of the IL2 cytokine family, exerts diverse regulatory effects on natural killer (NK), T and B cells. IL21 has been reported to possess potent anti-tumor activity against a variety of cancers not expressing IL21 receptor (IL21R) through activation of the immune system and is in clinical trials for renal cell carcinoma and metastatic melanoma. We have recently reported that apart from immuno-stimulatory effects, IL21 exerts direct cytotoxicity on IL21R expressing diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) cell lines and primary tumors both in vitro as well in vivo (Sarosiek et al Blood 2010; Bhatt et al AACR 2013).
Herein we designed a fusion protein comprising IL21 linked to the N-terminus of anti-CD20 antibody (αCD20-IL21 fusokine) to improve efficacy of its individual components and prolong IL21 half-life. We have verified the expression of full length fusion protein and demonstrated that αCD20-IL21 fusokine retained binding ability to its individual components; CD20 and IL21R, as analyzed by immunofluorescence and flow-cytometry analyses. Similar to our previous study of IL21 in DLBCL, treatment of B cell lymphoma cell lines with fusokine lead to phosphorylation of STAT1 and STAT3, upregulation of cMYC and BAX and downregulation of BCL-2 and BCL-XL, implying the activation of IL21R dependent signaling to trigger cytotoxic effects. In vitro, direct cell death induced by αCD20-IL21 fusokine in DLBCL (RCK8, WSU and Farage) and MCL (Mino, HBL2 and SP53) cell lines was markedly increased compared to its individual components (IL21 and parent αCD20-IgG1 antibody). More importantly, fusokine treatment resulted in cell death of MCL cell lines (L128, G519 and UPN1) that were found to be resistant to IL21 alone treatment. Furthermore, treatment of freshly isolated primary NHL cells with the αCD20-IL21 fusokine also exhibited a 40-50% increase in direct cell death compared to its individual components.
Previous studies reported that IL21 enhances antibody-dependent cellular cytotoxicity (ADCC) of therapeutic antibodies by activation of NK cells. ADCC assays using chromium release with purified human NK cells demonstrated that ADCC induced by the parent antibody was enhanced in the presence of IL21 while IL21 alone had minimal effect on the lysis of Raji, Daudi, and Jeko1 target cells. Notably, αCD20-IL21 fusokine demonstrated increased ADCC activity in comparison to parent antibody plus IL21 in Raji, Daudi and Jeko-1 cells (p<0.001, p<0.005 and p<0.001, respectively). Similar results were obtained in primary MCL tumor cells. Consistent with this finding, fusokine treatment resulted in enhanced activation of the NK cells as assessed by CD69 upregulation and CD16 downregulation using flow-cytometry. Complement dependent cytotoxicity (CDC) of the fusokine was similar to the parent antibody and rituximab in Raji cells. Studies analyzing in vivo effects of the fusokine are in progress and will be presented at the meeting.
These data strongly suggest that together with direct apoptotic potential, an anti-CD20 IL21 fusokine retains the ability to trigger indirect cell killing mediated via activation of immune effector cells. These dual effects may give remarkable advantage to the fusokine over existing anti-CD20 antibodies for the treatment of NHL tumors. Collectively, our study demonstrates that anti-tumor effects of IL21 and anti-CD20 antibodies can be enhanced by conjugation of IL21 with anti-CD20 antibody that may serve as a novel anti-lymphoma therapy.

Disclosures:
Rosenblatt: Seattle Genetics, Inc.: Research Funding.