JNJ-64457107, a CD40 Agonist, Induces Cell Death in BCL6hiIRF4neg GCB Subtype of DLBCL
Authors: Rusbuldt Joshua J. , Smith Andressa A. , Balasubramanian Sriram , Borzillo Gary , Johnson Amy J. , Alvarez Arias Diana 
Published: October 2019

Abstract
CD40 is a member of the TNF receptor family that is expressed in multiple tumor types and normal cells including antigen presenting cells (APC), endothelium and platelets. CD40-CD40L signaling is one of the pivotal mechanisms essential for robust antigen presentation and adaptive immunity. JNJ-64457107 (JNJ-107) is an anti-CD40 IgG1 agonistic antibody that is currently in a Phase 1 clinical trial (NCT02829099). JNJ-107/ADC-1013 induced significant anti-tumor activity and immunological memory (Mangsbo 2015). It was also reported that a CD40 agonist, SGN-40, induced direct cytotoxicity in diffuse large B cell lymphoma (DLBCL) cell lines (Burlington 2011), and clinical responses have been noted in this indication (Advani 2009). However, not all DLBCL patients and DLBCL cell lines respond to SGN-40. Thus, a patient selection strategy in B cell malignancies may be beneficial. Here, we investigated the mechanism of action and potential biomarkers of response to JNJ-107 in DLBCL.
Here, we show that JNJ-107 induced dose-dependent APC activation measured by upregulation of co-stimulatory molecules CD86, CD54, HLA-DR and CD23 on peripheral B cells in ex vivo whole blood and in vitro generated dendritic cells by flow cytometry. The highest APC activation was at 0.1-5 µg/ml of JNJ-107. We also found that JNJ-107 induced cell death of CD40 positive DLBCL cell lines representing both, germinal center B-cell (GCB) and activated B-cell (ABC) subtypes at similar doses. Cell death was measured by the Cell Titer-Glo assay and confirmed by staining with PI/AnnexinV. JNJ-107 at 1 µg/ml induced cell death (in 30-90% of cells) in 11/20 DLBCL cell lines (called "sensitive") after 4 days of treatment, with 2 GCB lines (BJAB and SU-DHL4) showing >90% cell death. No DLBCL cell line proliferation was observed in response to JNJ-107.
CD40 signals through the NF-kB pathway in DLBCL. We investigated, if basal expression of NF-kB signaling components predicts responses to JNJ-107. First, we assessed activation of the NF-kB pathway in DLBCL lines by Western blot before and after treatment with JNJ-107. We used p65 and p52 as markers of canonical and non-canonical NF-kB activation, respectively. We found that JNJ-107 predominantly activated non-canonical NF-kB signaling in DLBCL cell lines by inducing p100 cleavage and release of p52 (Figure 1). A lower rate of p52 induction was observed in JNJ-107 "sensitive" lines that had constitutive expression of p52 (Pfeiffer, OCI-Ly7). Thus, we did not observe strong correlation between p52 induction and cell death in response to JNJ-107.
BCL6 and IRF4 are markers of cell of origin in DLBCL. BCL6 and IRF4 are constitutively expressed in GCB and ABC subtypes, respectively. Interestingly, "sensitive" GCB lines had higher BCL6 expression than resistant lines. BCL6 protein was rapidly degraded (within 16h) in "sensitive" GCB DLBCL lines stimulated with JNJ-107. BCL6 is a transcriptional repressor that is regulated by many factors including IRF4. IRF4 is induced downstream of CD40, and directly represses BCL6 expression by binding to its promoter (Saito 2007). Consistent with constitutive activation of NF-kB, ABC DLBCL lines constitutively expressed IRF4. Surprisingly, several GCB lines also constitutively expressed IRF4 and were unable to further upregulate IRF4 in response to JNJ-107. The latter lines showed a lower degree of cell death in response to JNJ-107 compared to lines negative for IRF4 expression (except Pfeiffer).
In summary, APC activation in whole blood, direct cytotoxicity and non-canonical NF-kB activation in DLBCL cell lines were seen with CD40 agonist, JNJ-107. GCB cell lines with high BCL6 expression and negative for IRF4 (BCL6hiIRF4neg) were more sensitive to JNJ-107 induced cell death. Based on these findings, GCB DLBCL patients with functional CD40 signaling in the tumor cells (BCL6hiIRF4neg phenotype) may show enhanced anti-tumor responses.
Figure 1. JNJ-107 potently induces non-canonical NF-kB signaling in DLBCL cell lines. DLBCL lines were treated with 0.1 µg/ml JNJ-107 for 15 min, then 5 µg/ml cross-linker, IgG F(ab')2 for 16h. p65 and p52 fold induction was determined by Western blot of whole lysates. Representative data of 2 independent experiments are shown.
Figure 1. Figure 1.

Disclosures
Rusbuldt: Janssen R&D: Employment. Smith:Janssen R&D: Employment. Balasubramanian:Janssen Research & Development: Employment, Equity Ownership. Borzillo:Janssen R&D: Employment. Alvarez Arias:Janssen R&D: Employment.