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New treatments are needed to address persistent unmet clinical needs for diffuse large B-cell lymphoma (DLBCL). Overexpression of transferrin receptor 1 (TFR1) is common across cancer and permits cell-surface targeting of specific therapies in preclinical and clinical studies of various solid tumors. Here, we developed novel nanocarrier delivery of chemotherapy via TFR1-mediated endocytosis, assessing this target for the first time in DLBCL. Analysis of published datasets showed novel association of increased TFR1 expression with high-risk DLBCL cases. Carbon–nitride dots (CND) are emerging nanoparticles with excellent in vivo stability and distribution and are adaptable to covalent conjugation with multiple substrates. In vitro, linking doxorubicin (Dox) and transferrin (TF) to CND (CND–Dox–TF, CDT) was 10–100 times more potent than Dox against DLBCL cell lines. Gain- and loss-of-function studies and fluorescent confocal microscopy confirmed dependence of these effects on TFR1-mediated endocytosis. In contrast with previous therapeutics directly linking Dox and TF, cytotoxicity of CDT resulted from nuclear entry by Dox, promoting double-stranded DNA breaks and apoptosis. CDT proved safe to administer in vivo, and when incorporated into standard frontline chemoimmunotherapy in place of Dox, it improved overall survival by controlling patient-derived xenograft tumors with greatly reduced host toxicities. Nanocarrier-mediated Dox delivery to cell-surface TFR1, therefore, warrants optimization as a potential new therapeutic option in DLBCL.Significance:Targeted nanoparticle delivery of doxorubicin chemotherapy via the TRF1 receptor presents a new opportunity against high-risk DLBCL tumors using potency and precision.

Supplemental Figures 1-9. Figure S1. Carbon-Nitride Dot Synthesis. Figure S2. Characterization of CND-Dox-TF. Figure S3. CND cytotoxicity. Figure S4. Cell surface TFR1 expression and receptor recycling. Figure S5. Rapid nuclear entry by Dox after CND-Dox-TF treatment in DLBCL. Figure S6. HEK293 is a model cell line for elucidating CND-Dox-TF mechanism. Figure S7. CND-Dox-TF working dose identified. Figure S8. CND-Dox-TF has improved toxicity profile compared to Dox. Figure S9. R-nanoCHOP treatment has favorable toxicity profile in non-malignant organs.

New treatments are needed to address persistent unmet clinical needs for diffuse large B-cell lymphoma (DLBCL). Overexpression of transferrin receptor 1 (TFR1) is common across cancer and permits cell-surface targeting of specific therapies in preclinical and clinical studies of various solid tumors. Here, we developed novel nanocarrier delivery of chemotherapy via TFR1-mediated endocytosis, assessing this target for the first time in DLBCL. Analysis of published datasets showed novel association of increased TFR1 expression with high-risk DLBCL cases. Carbon–nitride dots (CND) are emerging nanoparticles with excellent in vivo stability and distribution and are adaptable to covalent conjugation with multiple substrates. In vitro, linking doxorubicin (Dox) and transferrin (TF) to CND (CND–Dox–TF, CDT) was 10–100 times more potent than Dox against DLBCL cell lines. Gain- and loss-of-function studies and fluorescent confocal microscopy confirmed dependence of these effects on TFR1-mediated endocytosis. In contrast with previous therapeutics directly linking Dox and TF, cytotoxicity of CDT resulted from nuclear entry by Dox, promoting double-stranded DNA breaks and apoptosis. CDT proved safe to administer in vivo, and when incorporated into standard frontline chemoimmunotherapy in place of Dox, it improved overall survival by controlling patient-derived xenograft tumors with greatly reduced host toxicities. Nanocarrier-mediated Dox delivery to cell-surface TFR1, therefore, warrants optimization as a potential new therapeutic option in DLBCL. Significance: Targeted nanoparticle delivery of doxorubicin chemotherapy via the TRF1 receptor presents a new opportunity against high-risk DLBCL tumors using potency and precision.

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 >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.