Tregs have the potential to establish long-term immune tolerance in patients recently diagnosed with type 1 diabetes (T1D) by preserving β cell function. Adoptive transfer of autologous thymic Tregs, although safe, exhibited limited efficacy in previous T1D clinical trials, likely reflecting a lack of tissue specificity, limited IL-2 signaling support, and in vivo plasticity of Tregs. Here, we report a cell engineering strategy using bulk CD4+ T cells to generate a Treg cell therapy (GNTI-122) that stably expresses FOXP3, targets the pancreas and draining lymph nodes, and incorporates a chemically inducible signaling complex (CISC). GNTI-122 cells maintained an expression profile consistent with Treg phenotype and function. Activation of CISC using rapamycin mediated concentration-dependent STAT5 phosphorylation and, in concert with T cell receptor engagement, promoted cell proliferation. In response to the cognate antigen, GNTI-122 exhibited direct and bystander suppression of polyclonal, islet-specific effector T cells from patients with T1D. In an adoptive transfer mouse model of T1D, a mouse engineered-Treg analog of GNTI-122 trafficked to the pancreas, decreased the severity of insulitis, and prevented progression to diabetes. Taken together, these findings demonstrate in vitro and in vivo activity and support further development of GNTI-122 as a potential treatment for T1D.

Supplementary Tables S1-S3. Supplementary Table S1: Changes in tumor deposition in the control and cyclophosphamide-treated groups as measured by PET/CT. Supplementary Table S2: Effects of cyclophosphamide on HER2 expression, collagen type I deposition and vascular parameters in BT474-M3 tumors. Supplementary Table S3. Reagents for immunofluorescence on FFPE and frozen tumor sections

Supplementary Figures S1-S6. Supplementary Fig. S1. The effect of cyclophosphamide on liposome delivery is dose dependent, requires a predose rather than a co-injection, and is not mediated by changes in blood clearance. Supplementary Fig. S2. Cyclophosphamide induces DNA-damage and tumor cell apoptosis. Supplementary Fig. S3. Effects of cyclophosphamide, ifosfamide, paclitaxel and eribulin on HER2-tPLD delivery, stromal cell density, total cell density, and interstitial space area. Supplementary Fig. S4. Pretreatment of tumors with cyclophosphamide significantly enhances nuclear delivery of doxorubicin following HER2-tPLD injection. Supplementary Fig. S5. Pretreatment of tumors with cyclophosphamide enhances the delivery of HER2-tPLD in multiple tumor models. Supplementary Fig. S6. The combination of a cyclophosphamide predose with HER2-tPLD results in synergistic anti-tumor activity.

Supplementary Materials and Methods. This file contains information on the reagents, methods for the evaluation of cell viability, preparation of liposomes and details on the image analysis.

Supp. Figure 1 MM-302 and trastuzumab co-localize in BT474-M3 xenograft tumors. Supp. Figure 2 In vivo deposition of MM-302 is specifically increased by trastuzumab or T-DM1. Supp. Figure 3 Pharmacokinetics (PK) of MM-302 remains the same with or without co-administration of trastuzumab. Supp. Figure 4 Combination of MM-302 and trastuzumab demonstrates synergistic anti-tumor activity in models of HER2-positive breast and gastric cancer.

Fig. S1. MM-302 tumor deposition in preclinical xenografts as a function of HER2 expression. Fig. S2. Preclinical assessment of minimum 64Cu-MM-302 tumor delivery for anti-tumor activity. Fig. S3. Maximum intensity projection images of selected patients with average and rapid clearance of 64Cu-MM-302. Fig. S4. Stability of 64Cu-MM-302 in patients. Fig. S5. Hepatic tumor lesion with similar uptake as normal liver tissue. Fig. S6. Estimated parameter values (k1, k-1, VVF) for lesions from 3 patients who underwent 3 scans. Table S1. Model parameters for tracer kinetic modeling of 64Cu-MM-302 liposome transport into and out of tumors. Table S2. Patient Demographic (n=19). Table S3. Radiation Dosimetry of 64Cu-MM-302 (n=11).

The high-risk human papillomavirus (HPV) strain 16 accounts for ~ 70% of all cervical cancers and 80% of head and neck cancers associated with HPV infection., Despite available therapies, there remains a critical need for new treatment options for advanced HPV 16-associated cancers. To address this, we have genetically engineered red blood cells to create an allogeneic artificial antigen presenting cell (APC), RTX-321, that expresses an HPV E7 peptide bound to MHC I (HLA-A*02:01), 4-1BBL and IL-12 on the cell surface to mimic the biology of T cell APC interactions. RTX-321 is designed to enhance both the quantity and quality of endogenous tumor-specific T cells. In this study, we evaluated the mechanisms through which RTX-321 promotes anti-tumor immune responses, particularly its ability to engage and activate HPV 16-specific CD8+ T cells in the context of mixed immune populations in peripheral blood mononuclear cells (PBMCs) and to modulate other key immune cell types in PBMCs. Primary CD8+ T cells were engineered to express the HPV E7 TCR, resulting in HPV E7-specific CD8+ T cells (E7 TCR-T). To determine whether RTX-321’s immune modulatory activities are dependent on the presence of antigen-specific T cells, we examined the effect of RTX-321 on PMBCs admixed with E7 TCR-T as compared to PBMCs alone. RTX-321 selectively expanded HPV E7-specific CD8+ T cells from the PBMC mixture in a dose-dependent manner. RTX-321 preferentially upregulated activation markers and effector molecules, and promoted effector memory on HPV E7-specific CD8+ T cells, compared to HPV independent CD8+ T cells. In addition to these HPV antigen-specific responses, modulations on other immune cells were observed with RTX-321 treatment. RTX-321 induced activation marker upregulation on general CD8+ T cells, as well as NK cell expansion, activation and effector molecule upregulation, independent of the presence of E7 TCR-T. In PBMCs, RTX-321 increased proinflammatory cytokine and chemokine secretion, including IFNγ, TNFα and CXCL10. The addition of E7 TCR-T cells further increased the production of IFNγ and TNFα in PBMCs, suggesting its potential role in further promoting the immune response by secreting effector molecules. Overall, our data indicate that RTX-321 not only engages HPV 16 antigen-specific CD8+ T cells, but also other key immune cell populations of the adaptive and innate immune systems to promote a broad and robust anti-tumor response. An Investigational New Drug application for RTX-321 for the treatment of patients with HPV 16-positive solid tumors is planned by the end of 2020. Citation Format: Mengyao Luo, Shamael S. Dastagir, Xuqing Zhang, Andrea Schmidt, Beatriz Marques, Timothy J. Lyford, Billy Blanco, Laurence A. Turka, Thomas J. Wickham, Tiffany F. Chen. RTX-321, an allogeneic red blood cell-based artificial antigen presenting cell, expressing MHC I-peptide, 4-1BBL and IL-12, engages primary human HPV-specific T cells and boosts other general immune responses [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO044.

Checkpoint inhibitors and T-cell therapies have highlighted the critical role of T cells in anti-cancer immunity. However, limitations associated with these treatments drive the need for alternative approaches. Here, we engineer red blood cells into artificial antigen-presenting cells (aAPCs) presenting a peptide bound to the major histocompatibility complex I, the costimulatory ligand 4-1BBL, and interleukin (IL)-12. This leads to robust, antigen-specific T-cell expansion, memory formation, additional immune activation, tumor control, and antigen spreading in tumor models in vivo. The presence of 4-1BBL and IL-12 induces minimal toxicities due to restriction to the vasculature and spleen. The allogeneic aAPC, RTX-321, comprised of human leukocyte antigen-A*02:01 presenting the human papilloma virus (HPV) peptide HPV16 E7 11-19 , 4-1BBL, and IL-12 on the surface, activates HPV-specific T cells and promotes effector function in vitro. Thus, RTX-321 is a potential ‘off-the-shelf’ in vivo cellular immunotherapy for treating HPV + cancers, including cervical and head/neck cancers.

Recombinant agonists that activate co-stimulatory and cytokine receptors have shown limited clinical anticancer utility, potentially due to narrow therapeutic windows, the need for coordinated activation of co-stimulatory and cytokine pathways and the failure of agonistic antibodies to recapitulate signaling by endogenous ligands. RTX-240 is a genetically engineered red blood cell expressing 4-1BBL and IL-15/IL-15Rα fusion (IL-15TP). RTX-240 is designed to potently and simultaneously stimulate the 4-1BB and IL-15 pathways, thereby activating and expanding T cells and NK cells, while potentially offering an improved safety profile through restricted biodistribution. We assessed the ability of RTX-240 to expand and activate T cells and NK cells and evaluated the in vivo efficacy, pharmacodynamics and tolerability using murine models. Treatment of PBMCs with RTX-240 induced T cell and NK cell activation and proliferation. In vivo studies using mRBC-240, a mouse surrogate for RTX-240, revealed biodistribution predominantly to the red pulp of the spleen, leading to CD8 + T cell and NK cell expansion. mRBC-240 was efficacious in a B16-F10 melanoma model and led to increased NK cell infiltration into the lungs. mRBC-240 significantly inhibited CT26 tumor growth, in association with an increase in tumor-infiltrating proliferating and cytotoxic CD8 + T cells. mRBC-240 was tolerated and showed no evidence of hepatic injury at the highest feasible dose, compared with a 4-1BB agonistic antibody. RTX-240 promotes T cell and NK cell activity in preclinical models and shows efficacy and an improved safety profile. Based on these data, RTX-240 is now being evaluated in a clinical trial.

MM-302 is an anti-HER2 antibody-targeted pegylated liposomal doxorubicin designed to deliver doxorubicin specifically to HER2-expressing solid tumors. The delivery and activity of MM-302 were evaluated in orthotopic, transgenic, and intravenous breast cancer models expressing varying levels of HER2 that metastasize to some of the most common sites of dissemination for breast cancer, namely, lung, liver, and brain. Metastatic burden was quantified by gross evaluation, immunohistochemistry (IHC), and bioluminescent imaging. Liposome delivery was quantified by IHC and ex vivo fluorescent imaging. Unlike its non-targeted counterpart, pegylated liposomal doxorubicin (PLD), MM-302 showed activity at controlling both primary and metastatic tumor burden in all models tested. The effect of HER2-targeting was greatest in the lung where lymphatic vessel density and MM-302 delivery were highest. Our data indicate that the therapeutic advantage of actively targeting a nanoliposome with an antibody is influenced by both target expression and the tumor microenvironment.

T cell-based therapies have demonstrated efficacy in a small subset of cancers; however, they have the potential to proliferate uncontrollably and manufacturing these therapies at scale has proven difficult. To address this limitation, Rubius Therapeutics has genetically engineered red cells to create allogeneic artificial antigen presenting cells (RCT-aAPCs) that express MHC class I loaded with a tumor specific antigen, together with costimulatory molecules that recapitulate normal APC-T cell interactions. These RCT-aAPC cells are designed to expand and activate tumor-specific T cells already present within the patient, thus eliminating the need to individually manufacture patient-derived T cells. As a proof of principle, red cells were engineered to express mouse MHC class I H-2Kb loaded with OVA 257-264 peptide and murine 4-1BBL. These cells induced in vitroT cell proliferation of OVA antigen-specific OT1 cells, whereas red cells expressing only MHC I or 4-1BBL did not induce proliferation. The RCT-aAPC expanded OT1 cells demonstrated an activated phenotype with increased CD44 expression, secretion of both IFNγ and IL2, as well as antigen-specific tumor killing of EG7.OVA tumor cells. To test in vivo efficacy, a mouse surrogate RCT-aAPC was created using murine red blood cells chemically conjugated with H-2Kb OVA and the m4-1BBL molecule. CellTrace Violet (CTV)-labeled OT1 cells were adoptively transferred into B6 Cd45.1 mice followed by intravenous dosing of the RCT-aAPC several hours later. Significant OT1 proliferation was observed 3-4 days post-dosing as measured by CTV dilution. Administration of a second RCT-aAPC dose at this time drove >200-fold expansion of OT1 cells with a memory-like phenotype in the peripheral blood and secondary lymphoid organs. Using a similar dosing strategy, administration of RCT-aAPC to mice bearing EG7.OVA tumors caused 60% tumor growth inhibition by Day 7 after dosing, which corresponded with the increased expansion of the OT1s. Treatment with RCT-aAPC significantly prolonged survival compared to the control group (p-val = 0.0024). After interacting with RCT-aAPC, antigen-specific T cells, traffic to the lymph nodes and tumor as demonstrated by OT1 presence at these sites. Based on the proof of concept using a murine system, human RCT-aAPCs expressing [human] 4-1BBL and [human] HLA-A2 loaded with an HPV E7 peptide were developed to expand and activate HPV E7-specific T cells. These RCT-aAPC cells activated TCR signaling in primary HPV E7-specific T cells as measured by upregulation of Nur77 expression and in engineered HPV E7-specific TCR Jurkat lines, measured using an NFAT luciferase reporter assay. Further validation of RCT-aAPC is ongoing and will be the focus for future clinical development in patients with HPV-positive cancers. Citation Format: Xuqing Zhang, Shamael R. Dastagir, Naren Subbiah, Mengyao Luo, Vikram Soman, Sneha Pawar, Douglas C. McLaughlin, Nicholas Bayhi, Viral Amin, Torben Straight Nissen, Christopher L. Carpenter, Thomas J. Wickham, Tiffany F. Chen. Engineered red-cell therapeutics (RCT) as artificial antigen presenting cells promotein vivoexpansion and anti-tumor activity of antigen specific T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3260.

Recombinant IL-12 is a potent cytokine that has held significant promise as an immunotherapeutic. In preclinical studies, recombinant IL-12 has impressive anti-tumor activity; however toxicity and a narrow therapeutic window halted development in humans. To address these limitations, Rubius Therapeutics has developed genetically engineered red cell therapeutics (RCTs) with cell surface expression of IL-12 alone (RTX-IL-12) or in combination with co-stimulatory or other cytokine molecules. These cells present ligands in their native form that potently activate and expand both T and NK cells through potent cell-cell interactions. On-target, off-tissue toxicity of RCTs may be limited due to their biodistribution, which is restricted to the vascular system. Based on its reported role in promoting a TH1 response and proliferation of cytotoxic NK and CD8 T cells, RTX-IL-12 activity was evaluated in vitro and demonstrated proliferation of human CD8 T cells (2-3 fold), Th1 differentiation of naïve CD4 T cells, IFNγproduction (6-11 fold increase over control), as well as NK cell activation and cytotoxicity against K562 targets (2-5 fold increase over control). To evaluate in vivoimmune response, anti-tumor activity and safety, a mouse surrogate red cell therapeutic, mRBC-IL-12, was developed by chemically conjugating recombinant Fc-IL-12 to mouse red blood cells. This surrogate overcame the rapid clearance of human red cells in mice. Administration of mRBC-IL-12 (1X109cells) to C57Bl6 mice that received B16F10 melanoma cells IV, showed a 52% decrease in the number of lung metastases and was associated with increased proliferating and cytotoxic CD8 and NK cells in the lungs (p=0.0002). Administration of mRBC-IL-12 (1X109cells) to mice bearing B16F10 and MC38 subcutaneous models exhibited 82% and 80% tumor growth inhibition, respectively. When combined with anti-PD1 treatment, mRBC-IL-12 (1X109cells) efficacy was improved in these two models and showed 86% and 85% tumor growth inhibition, respectively. Efficacy was accompanied by an increase in survival (p=0.0004 B16F10, p=0.0003 MC38 ) and the infiltration of M1 macrophages (p=0.007) into the tumor. Mice treated with the highest feasible dose of mRBC-IL-12 displayed no significant body weight loss and 3-10-fold lower serum IFNg levels compared to soluble rec. Fc-IL-12. By combining IL-12 with IL-15TP or 4-1BBL on the same red cell, tumor growth was strongly inhibited and in some cases improved over mRBC-IL-12 alone (TGI 46% and 63% in B16F10 SC, TGI 83%, 77% in MC38 and 78%, 70% in B16F10 IV). In summary, the data demonstrate that sequestering IL-12 in the vasculature through expression on red cells drives significant reductions in tumor growth, while improving the tolerability profile of the cytokine,supporting further testing in cancer patients. Citation Format: Anne-Sophie Dugast, Enping Hong, Maegan Hoover, Arjun Bollampalli, Douglas C. McLaughlin, Omkar Bhate, Timothy J. Lyford, Torben Straight Nissen, Christopher L. Carpenter, Thomas J. Wickham, Sivan Elloul. RTX-IL-12, an allogeneic red cell therapeutic expressing IL-12, exhibits potent in vitro and in vivo activity and favorable safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3256.

Abstract Next generation sequencing technologies, coupled with personal neoantigen identification approaches, have significantly improved the capability to develop patient-specific T-cell-based therapies targeting tumors. Current peptide neoantigen vaccine approaches are promising, but do not adequately stimulate and expand patient T cells to the levels required to achieve robust efficacy. To address these limitations, Rubius Therapeutics has developed allogeneic artificial antigen presenting cells (aAPCs), which express the required signals for complete T cell activation: antigenic peptide-MHC complex, costimulatory ligand and cytokine. By engineering red cells to express immunomodulatory signals 1, 2 and 3, these aAPCs dramatically expand antigen-specific T cells in vivo and promote T cell memory and effector function. To use the aAPC approach with personal neoantigens, Rubius Therapeutics has developed a loadable MHC system that enables the rapid generation of aAPCs. Unloaded MHC class I molecules typically do not express robustly on the cell surface, as innate biophysical instability and internal cellular quality control mechanisms prevent MHC molecules that lack a loaded peptide from displaying on the cell surface. We demonstrate here that the empty MHC class I complex can be stably presented on the red cell surface. This was achieved by fusing wild-type MHC class I [human] HLA-A2 and β2 microglobulin to the glycophorin A transmembrane domain. Of note, disulfide engineering of HLA-A2 did not change expression when compared to wild-type constructs on the red cells. Fluorescently labelled peptides were used to measure the kinetics of peptide loading, which demonstrated that disulfide-engineering dramatically increased binding rates. Further, peptide competition experiments indicated slightly increased affinity for peptide with disulfide engineered HLA-A2. Functional testing revealed that addition of exogenous HPV E7 peptide and co-incubation of loaded cells with Jurkat-Lucia NFAT cells expressing HPV E7-specific TCR cells demonstrated TCR-specific activity. Additional TCR activity assays showed that peptide loaded onto empty wild-type HLA-A2 was stable up to 3 days, while disulfide-engineered HLA-A2 activity was abrogated after 1 day. Finally, this concept was extended to other HLA genes, demonstrating that our approach could achieve expression of a variety of HLA alleles on the red cell surface, including MHC class II alleles in the HLA-DR and HLA-DP gene families. Collectively, these results demonstrate that Rubius’ loadable aAPC system is highly generalizable and can be applied to produce aAPC populations presenting multiple antigenic peptides across a range of functional alleles on the red cell surface. Rubius’ allogeneic aAPC system represents a novel approach to generate effective personal neoantigen-specific therapies in a wide patient population with requisite ease of manufacturability Citation Format: Christopher L Moore, Sneha Pawar, Mellissa Nixon, Timothy J Lyford, Douglas C McLaughlin, Shamael R Dastagir, Abigail Bracha, Lori Melancon, Christopher L Carpenter, Thomas J Wickham, Tiffany F Chen. Enabling the rapid generation of allogeneic artificial antigen presenting cell (aAPC) Red Cell Therapeutics with a loadable MHC system [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B062. doi:10.1158/1535-7163.TARG-19-B062

Recombinant agonists that activate co-stimulatory and cytokine receptors have shown limited clinical activity perhaps due to 1) toxicity; 2) a need for coordinated activation of co-stimulatory and cytokine pathways; or 3) the failure of agonist antibodies to recapitulate signaling by endogenous ligands. To address these limitations, Rubius Therapeutics developed genetically engineered red cells with cell surface expression of both co-stimulatory and cytokine ligands, which present ligands in their native form through cell-cell contact to potently activate and expand both T and NK cells. On-target, off-tissue toxicity may be limited due their biodistribution, which is restricted to the vascular system. IL-15 is known to promote NK survival and CD8 T cell memory and 4-1BB agonists are known to promote T cell prolioferation and survival. Rubius Therapeutics has developed RTX-212, an allogeneic red cell therapeutic genetically engineered to co-express 4-1BBL and an IL-15/IL-15Rαfusion (IL-15TP). In vitro assays demonstrated that the combination of both ligands on RTX-212 expanded both memory CD8 T cells (2.5-fold) and NK cells (10-15-fold), in the absence of TCR stimulation. RTX-212 further induced dramatic proliferation of CD8 T cells and CD4 T cells in the presence of TCR stimulation with increased IFNγsecretion. In addition to the synergistic effects of 4-1BBL and IL-15TP, each molecule provided complementary functions that expanded the activity of RTX-212 beyond RTX-4-1BBL or RTX-IL-15TP alone. IL-15TP uniquely activated NK cytotoxicity and ADCC, while 4-1BBL uniquely stimulated CD4 and CD8 T cell proliferation and production of IFNγ. To evaluate in vivo immune responses, anti-tumor activity and safety, a mouse surrogate therapeutic, mRBC-212, was developed where recombinant Fc-IL-15-sushi and m4-1BBL were chemically conjugated to mouse red blood cells. This surrogate overcame the rapid clearance of human red cells in mice. Intravanous administration of mRBC-212 to C57Bl6 mice that received B16F10 melanoma cells IV, showed a 66% decrease in the number of lung metastases compared to control mice (p=0.0001) and was associated with a significant increase in NK cell infiltration into the lungs (p=0.02). In a CT26 tumor model, mRBC-212 treated mice exhibited 55% tumor growth inhibition, which was accompanied by a 1.7-fold increase in the tumor infiltration of proliferating and cytotoxic CD8 T cells. Mice treated with the highest feasible dose of mRBC-212 showed no change in serum transaminases, infiltration of CD8 T cells and macrophages to the liver or liver inflammation score compared to agonistic 4-1BB antibodies treated mice. Taken together, these data indicate that RTX-212 has the potential to be an effective therapy with an improved safety profile compared to 4-1BB agonist antibodies and IL-15 agonists, supporting its clinical development. Citation Format: Anne-Sophie Dugast, Shannon McArdel, Maegan Hoover, Enping Hong, Shannon Curtis Leonard, Arjun Bollampalli, Douglas C. McLaughlin, Jennifer Mellen, Torben Straight Nissen, Christopher L. Carpenter, Thomas J. Wickham, Sivan Elloul. RTX-212, an allogeneic red cell therapeutic expressing 4-1BBL and IL-15TP, exhibits potent in vitro and in vivo activity and a favorable safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3272.

This phase 1 dose-escalation trial studied MM-302, a novel HER2-targeted PEGylated antibody–liposomal doxorubicin conjugate, in HER2-positive locally advanced/metastatic breast cancer. Patients were enrolled in four cohorts: MM-302 monotherapy (8, 16, 30, 40, and 50 mg/m2 every 4 weeks [q4w]); MM-302 (30 or 40 mg/m2 q4w) plus trastuzumab (4 mg/kg q2w); MM-302 (30 mg/m2) plus trastuzumab (6 mg/kg) q3w; MM-302 (30 mg/m2) plus trastuzumab (6 mg/kg) and cyclophosphamide (450 mg/m2) q3w. Sixty-nine patients were treated. The most common adverse events (AEs) were fatigue and nausea. Grade 3/4 AEs of special interest included neutropenia, fatigue, mucosal inflammation, anemia, thrombocytopenia, febrile neutropenia, and palmar-plantar erythrodysesthesia. The MTD was not reached. With MM-302 ≥ 30 mg/m2, overall response rate (ORR) was 13% and median progression-free survival (mPFS) 7.4 months (95% CI: 3·5–10·9) in all arms. In 25 anthracycline-naïve patients, ORR was 28·0% and mPFS 10·9 months (95% CI: 1·8–15·3). Imaging with 64Cu-labeled MM-302 visualized tumor-drug penetrance in tumors throughout the body, including the brain. MM-302 monotherapy, in combination with trastuzumab, or trastuzumab plus cyclophosphamide, was well tolerated and showed promising efficacy. The selected phase 2 MM-302 dose was 30 mg/m2 plus 6 mg/kg trastuzumab q3w.

Abstract Purpose: Therapeutic nanoparticles are designed to deliver their drug payloads through enhanced permeability and retention (EPR) in solid tumors. The extent of EPR and its variability in human tumors is highly debated and has been proposed as an explanation for variable responses to therapeutic nanoparticles in clinical studies. Experimental Design: We assessed the EPR effect in patients using a 64Cu-labeled nanoparticle, 64Cu-MM-302 (64Cu-labeled HER2-targeted PEGylated liposomal doxorubicin), and imaging by PET/CT. Nineteen patients with HER2-positive metastatic breast cancer underwent 2 to 3 PET/CT scans postadministration of 64Cu-MM-302 as part of a clinical trial of MM-302 plus trastuzumab with and without cyclophosphamide (NCT01304797). Results: Significant background uptake of 64Cu-MM-302 was observed in liver and spleen. Tumor accumulation of 64Cu-MM-302 at 24 to 48 hours varied 35-fold (0.52–18.5 %ID/kg), including deposition in bone and brain lesions, and was independent of systemic plasma exposure. Computational analysis quantified rates of deposition and washout, indicating peak liposome deposition at 24 to 48 hours. Patients were classified on the basis of 64Cu-MM-302 lesion deposition using a cut-off point that is comparable with a response threshold in preclinical studies. In a retrospective exploratory analysis of patient outcomes relating to drug levels in tumor lesions, high 64Cu-MM-302 deposition was associated with more favorable treatment outcomes (HR = 0.42). Conclusions: These findings provide important evidence and quantification of the EPR effect in human metastatic tumors and support imaging nanoparticle deposition in tumors as a potential means to identify patients well suited for treatment with therapeutic nanoparticles. Clin Cancer Res; 23(15); 4190–202. ©2017 AACR.

Although HER2-targeted therapies such as pertuzumab and ado-trastuzumab emtansine (T-DM1) have improved patient outcomes, treatment resistance typically occurs. MM-302 is a HER2-targeted antibody-liposomal doxorubicin conjugate in development by Merrimack Pharmaceuticals. In a Phase 1 study, patients with HER2-positive metastatic breast cancer (MBC) were treated with MM-302 alone and in combination with trastuzumab with or without cyclophosphamide. MM-302 had an acceptable safety profile, and promising efficacy was observed in patients not previously exposed to an anthracycline. Trial design: HERMIONE is a randomized Phase 2, two-arm, open-label trial in patients with anthracycline naïve, trastuzumab-, pertuzumab- and T-DM1-pretreated HER2-positive locally advanced breast cancer (LABC)/MBC. Patients are randomized 1:1 to receive MM-302 (30mg/m2, Q3W) plus trastuzumab (6mg/kg, Q3W) or chemotherapy of physician's choice (vinorelbine, capecitabine, or gemcitabine) plus trastuzumab (6mg/kg, Q3W). Eligibility criteria: Centrally confirmed HER2-positive LABC/MBC, no prior anthracycline exposure, prior trastuzumab, prior T-DM1 in the LABC/MBC setting, prior pertuzumab in LABC/MBC setting or disease recurrence within 12 months of neoadjuvant/adjuvant treatment, unlimited prior lines, ECOG 0-1 and LVEF ≥50%. CNS metastases are permitted if stable and without symptoms or steroids for 4 weeks. Specific aims: The primary endpoint is progression free survival (PFS) assessed by an independent blinded review. Secondary endpoints include investigator assessed PFS, overall survival, response rate, safety and patient related outcomes. Statistics: 250 patients will be enrolled to observe 191 PFS events for 90% power to detect a HR of 0.625. The MM-302 arm will be compared to the control arm on the primary endpoint of PFS using a stratified log-rank test at one-sided 0.025 level. Status: First patient treated was in December 2014 and enrollment is expected to be complete in 2017. Sites are open in the US, Canada and Western Europe. Clinical trial identification: NCT02213744 Legal entity responsible for the study: Merrimack Pharmaceuticals Merrimack Pharmaceuticals