R-Nanochop Incorporating a TFR1-Targeted Doxorubicin Nanocarrier Is Superior to R-CHOP in a PDX Model of Diffuse Large B-Cell Lymphoma
Authors: Arumov Artavazd , Liyanage Piumi Y. , Trabolsi Asaad , Roberts Evan R. , Ferreira Braulio C. L. B. , Newsam Austin D. , Bilbao Daniel , Leblanc Roger M. , Schatz Jonathan H. 
Published: November 2020

Diffuse large B-cell lymphoma (DLBCL) comprises a third of non-Hodgkin lymphoma (NHL), with incidence steadily rising in what is already the most common hematologic malignancy. The backbone of potentially curative frontline chemoimmunotherapy combinations like R-CHOP is doxorubicin (Dox), still considered the most active anti-lymphoma agent. Relapsed or refractory (rel/ref) disease after frontline treatment carries persistently poor prognosis, even with recent advances in immunotherapy. Development and implementation of new therapeutic strategies remain urgent priorities to address unmet clinical needs in both high-risk previously untreated and rel/ref patients.
We have studied transferrin receptor (TFR1) expression as a marker of high-risk DLBCL revealing significantly worse outcomes following frontline therapy associated with high expression in diagnostic samples. TFR1 is therefore a rational target for treatments aimed specifically at high-risk DLBCL. We used third-generation carbon-nitride dot (CND) nanocarriers we have developed conjugated to Dox and holo-transferrin (TF) to develop a chemotherapeutic-nanocarrier (NanoDox) designed to deliver Dox to TFR1-expressing tumors while sparing non-malignant tissues. Dox's mechanism is primarily through nuclear DNA damage or induction of reactive oxygen species (ROS) H202. We treated DLBCL cell lines with NanoDox or Dox for 24-hours and observed induction of nuclear DNA damage marker γ-H2Ax at drastically lower doses (10 nM NanoDox vs. 100 nM Dox). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay results yielded similar findings in BJAB cells, as observed under γ-H2Ax analysis, with TUNEL induction at significantly lower NanoDox concentrations. Treatment with NanoDox did not generate significant changes in ROS H202 when compared to Dox at 24-hours. Dox is known for a strong cytotoxic profile that extends past its terminal half-life of 24-36 hours. Therefore, we treated BJAB, Farage and DHL4 cells for 24-hours with a range of NanoDox and Dox doses, followed by washout and tracked cell viability for 6 additional days. We found as low as 10 nM NanoDox induced rapid and irreversible cytotoxicity, an effect only seen at much higher doses of Dox. These results combined with fluorescent confocal microscopy studies confirmed NanoDox works primarily through rapid nuclear Dox accumulation, causing DNA damage and apoptosis.
Building on initial NanoDox dosing studies in NOD Scid Gamma (NSG) mice, we constructed R-nanoCHOP as an alternate to R-CHOP by replacing Dox with NanoDox at a working dose (WD) of 33.0 mg/kg. We next engrafted patient-derived xenograft (PDX) tumors derived from a previously untreated patient with germinal center B-cell (GCB) subtype DLBCL. We randomized NSG animals to two groups at tumor engraftment and treated with 21-day cycles of R-nanoCHOP (n=10) or R-CHOP (n=11). The treatments controlled tumor volume (TV) similarly, but R-nanoCHOP significantly improved overall survival associated with greatly reduced toxicities to host animals, which tolerated an average of 2 additional cycles of R-nanoCHOP. Studies with murine cells in vitro, including A20 B-cell lymphoma cells, confirmed these effects were not due to species differences, with human holo-TF alone and as part of NanoDox binding mouse and human TFR1 with equal affinity.
This work establishes proof of principle in DLBCL for targeting TFR1, a specific marker of clinically high-risk disease. CND nanocarriers provide a flexible platform to exploit this, with our initial studies enhancing the safety profile of Dox, which remains the most active frontline DLBCL agent more than 50 years after it entered use. We are now optimizing NanoDox by substituting the holo-TF with an anti-TFR1 single chain variable fragment (scFv), decreasing size of the overall nanoconjugate by &gt;50%. We believe dosing optimization from this change and additional optimizations to Dox delivery intracellularly will permit investigational new drug (IND) studies of absorption, distribution, metabolism and excretion (ADME). In sum, we provide compelling clinically relevant evidence for targeting TFR1 in DLBCL as a new therapeutic approach.

Disclosures
No relevant conflicts of interest to declare.