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Biomarkers change on treatment

TRC105 is an anti-endoglin antibody currently being tested in combination with VEGF inhibitors. In the phase Ib trial, 38 patients were treated with both TRC105 and bevacizumab (BEV), and improved clinical outcomes were observed, despite the fact that 30 patients (79%) were refractory to prior anti-VEGF therapy. Plasma samples were tested for angiogenic and inflammatory biomarkers at baseline and on-treatment. To provide broader context of this combination biomarker study, direct cross-study comparisons were made to biomarker studies previously conducted in patients treated with either BEV or TRC105 monotherapy. Upon treatment with BEV and TRC105, pharmacodynamic changes in response to both BEV (PlGF increase) and TRC105 (soluble endoglin increase) were noted. In addition, distinct patterns of change were identified (similar, opposing, neutralizing). Similar patterns were observed when the combination elicited similar effects to those observed with monotherapy treatment (i.e., decreases of Ang-2, increases of IL6 and VCAM-1). Opposing patterns were observed when the combination led to opposing effects compared with monotherapy treatment (i.e., TGFβ1, PDGF-AA and PDGF-BB, PAI-1). Lastly, neutralizing patterns were observed when one drug led to increase, whereas the other drug led to decrease, and the combination elicited no overall effect on the marker (i.e., VEGF-A, VEGF-D, and IGFBP-3). Patients achieving partial responses or stable disease from the combination exhibited significantly lower expression of E-Cadherin, HGF, ICAM-1, and TSP-2 at baseline. Taken together, the novel biomarker modulations identified may deepen our understanding of the underlying biology in patients treated with BEV and TRC105 compared with either drug alone. Mol Cancer Ther; 17(10); 2248–56. ©2018 AACR.

Movie S3 from Dual Targeting of Tissue Factor and CD105 for Preclinical PET Imaging of Pancreatic Cancer

Supplementary Tables

Endoglin is expressed by CAFs, does not correlate to stage-III CRC patient survival and TCGA database analysis

Characterization of patient-derived NFs and CAFs

Permission letter regarding inclusion of information of CHLA-255 C-Myc over-expression

Purpose:We determined whether elimination of CD105+ cells in the tumor microenvironment (TME) with anti-CD105 antibodies enhanced anti-disialoganglioside (GD2) antibody dinutuximab therapy of neuroblastoma when combined with activated natural killer (aNK) cells.Experimental Design:The effect of MSCs and monocytes on antibody-dependent cellular cytotoxicity (ADCC) mediated by dinutuximab with aNK cells against neuroblastoma cells was determined in vitro. ADCC with anti-CD105 mAb TRC105 and aNK cells against MSCs, monocytes, and endothelial cells, which express CD105, was evaluated. Anti-neuroblastoma activity in immunodeficient NSG mice of dinutuximab with aNK cells without or with anti-CD105 mAbs was determined using neuroblastoma cell lines and a patient-derived xenograft.Results:ADCC mediated by dinutuximab with aNK cells against neuroblastoma cells in vitro was suppressed by addition of MSCs and monocytes, and dinutuximab with aNK cells was less effective against neuroblastomas formed with coinjected MSCs and monocytes in NSG mice than against those formed by tumor cells alone. Anti-CD105 antibody TRC105 with aNK cells mediated ADCC against MSCs, monocytes, and endothelial cells. Neuroblastomas formed in NSG mice by two neuroblastoma cell lines or a patient-derived xenograft coinjected with MSCs and monocytes were most effectively treated with dinutuximab and aNK cells when anti-human (TRC105) and anti-mouse (M1043) CD105 antibodies were added, which depleted human MSCs and murine endothelial cells and macrophages from the TME.Conclusions:Immunotherapy of neuroblastoma with anti-GD2 antibody dinutuximab and aNK cells is suppressed by CD105+ cells in the TME, but suppression is overcome by adding anti-CD105 antibodies to eliminate CD105+ cells.

Purpose:Endoglin is a coreceptor for TGFβ ligands that is highly expressed on proliferating endothelial cells and other cells in the tumor microenvironment. Clinical studies have noted increased programmed cell death (PD)-1 expression on cytotoxic T cells in the peripheral blood of patients with cancer treated with TRC105, an endoglin-targeting antibody. In this study, we investigated the combination of endoglin antibodies (TRC105 and M1043) with an anti-PD1 antibody.Experimental Design:The combination anti-endoglin/anti-PD1 antibodies was tested in four preclinical mouse models representing different stages of cancer development. To investigate the underlying mechanism, Fc-receptor–knockout mice were used complemented with depletion of multiple immune subsets in mice. Tumor growth and the composition of immune infiltrate were analyzed by flow cytometry. Finally, human colorectal cancer specimens were analyzed for presence of endoglin-expressing regulatory T cells (Treg).Results:In all models, the combination of endoglin antibody and PD1 inhibition produced durable tumor responses, leading to complete regressions in 30% to 40% of the mice. These effects were dependent on the presence of Fcγ receptors, indicating the involvement of antibody-dependent cytotoxic responses and the presence of CD8+ cytotoxic T cells and CD4+ Th cells. Interestingly, treatment with the endoglin antibody, TRC105, significantly decreased the number of intratumoral Tregs. Endoglin-expressing Tregs were also detected in human colorectal cancer specimens.Conclusions:Taken together, these data provide a rationale for combining TRC105 and anti-PD1 therapy and provide additional evidence of endoglin's immunomodulatory role.

Determination of NOTA to Fab Fragment Ratio

Targeting CD105+ Microenvironment cells enhances immunotherpy of Dinutuxmab with aNK cells against neuroblastoma

Purpose:Cancer-associated fibroblasts (CAF) are a major component of the colorectal cancer tumor microenvironment. CAFs play an important role in tumor progression and metastasis, partly through TGF-β signaling pathway. We investigated whether the TGF-β family coreceptor endoglin is involved in CAF-mediated invasion and metastasis.Experimental Design:CAF-specific endoglin expression was studied in colorectal cancer resection specimens using IHC and related to metastases-free survival. Endoglin-mediated invasion was assessed in vitro by transwell invasion, using primary colorectal cancer–derived CAFs. Effects of CAF-specific endoglin expression on tumor cell invasion were investigated in a colorectal cancer zebrafish model, whereas liver metastases were assessed in a mouse model.Results:CAFs specifically at invasive borders of colorectal cancer express endoglin and increased expression intensity correlated with increased disease stage. Endoglin-expressing CAFs were also detected in lymph node and liver metastases, suggesting a role in colorectal cancer metastasis formation. In stage II colorectal cancer, CAF-specific endoglin expression at invasive borders correlated with poor metastasis-free survival. In vitro experiments revealed that endoglin is indispensable for bone morphogenetic protein (BMP)-9–induced signaling and CAF survival. Targeting endoglin using the neutralizing antibody TRC105 inhibited CAF invasion in vitro. In zebrafish, endoglin-expressing fibroblasts enhanced colorectal tumor cell infiltration into the liver and decreased survival. Finally, CAF-specific endoglin targeting with TRC105 decreased metastatic spread of colorectal cancer cells to the mouse liver.Conclusions:Endoglin-expressing CAFs contribute to colorectal cancer progression and metastasis. TRC105 treatment inhibits CAF invasion and tumor metastasis, indicating an additional target beyond the angiogenic endothelium, possibly contributing to beneficial effects reported during clinical evaluations.See related commentary by Becker and LeBleu, p. 6110.

Supplementary Figure 1. Purification of heterodimer after click chemistry conjugation of ALT-836-Fab and TRC105-Fab Supplementary Figure 2. MALDI-TOF mass spectra of ALT-836-Fab: [M+H]+ = 47266.0, TRC105-Fab: [M+H]+ = 47454.7, and heterodimer: exact [M+H]+ = 103504.0. Supplementary Figure 3. Flow cytometry analysis in BXPC-3 after 1 h incubation of FITC-Fab conjugates for the comparison of their in vitro targeting Supplementary Figure 4. Radiolabeling efficiency at different copper: immunoconjugate ratios were used to determine the number of NOTA per antibody fragment molecule Supplementary Figure 5. PET imaging and biodistribution of 64CuCl2 in BXPC-3 tumor-bearing mice. Supplementary Figure 6. PET imaging and quantitative ROI analysis of 64Cu-NOTA-heterodimer with TF and CD105 dual-blocking in BXPC-3 tumor-bearing mice. Supplementary Figure 7. Coronal images and transverse PET/CT images of normal nude mice at 3, 15, 24 and 30 h following injection of 64Cu-NOTA-heterodimer Supplementary Figure 8. PET ROI-derived quantification of 64Cu-NOTA-heterodimer uptake in the tumor, normal pancreas, blood pool, liver, kidney and muscle at 3 h, 15 h, 24 h and 30 h p.i. of the tracer

Supplementary figures S1-S5

Purpose: Pancreatic adenocarcinoma is a highly aggressive cancer, currently treated with limited success and dismal outcomes. New diagnostic and treatment strategies offer the potential to reduce cancer mortality. Developing highly specific noninvasive imaging probes for pancreatic cancer is essential to improving diagnostic accuracy and monitoring therapeutic intervention.Experimental Design: A bispecific heterodimer was synthesized by conjugating an anti-tissue factor (TF) Fab with an anti-CD105 Fab, via the bio-orthogonal “click” reaction between tetrazine (Tz) and trans-cyclooctene (TCO). The heterodimer was labeled with 64Cu for PET imaging of nude mice bearing BXPC-3 xenograft and orthotopic pancreatic tumors.Results: PET imaging of BXPC-3 (TF/CD105+/+) xenograft tumors with 64Cu-labeled heterodimer displayed significantly enhanced tumor uptake (28.8 ± 3.2 %ID/g; n = 4; SD) at 30 hours postinjection, as compared with each of their monospecific Fab tracers (12.5 ± 1.4 and 7.1 ± 2.6 %ID/g; n = 3; SD). In addition, the activity–concentration ratio allowed for effective tumor visualization (tumor/muscle ratio 75.2 ± 9.4 at 30 hours postinjection.; n = 4; SD). Furthermore, 64Cu-NOTA-heterodimer enabled sensitive detection of orthotopic pancreatic tumor lesions with an uptake of 17.1 ± 4.9 %ID/g at 30 hours postinjection and tumor/muscle ratio of 72.3 ± 46.7.Conclusions: This study demonstrates that dual targeting of TF and CD105 provided synergistic improvements in binding affinity and tumor localization of the heterodimer. Dual-targeted imaging agents of pancreatic and other cancers may assist in diagnosing pancreatic malignancies as well as reliable monitoring of therapeutic response. Clin Cancer Res; 22(15); 3821–30. ©2016 AACR.

Supplementary table 1 and 2

Purpose In nonmetastatic hormone receptor-positive and Her2-negative breast cancer, preoperative endocrine therapies can yield outcomes similar with chemotherapy. We evaluated the tolerability and preliminary antitumor activity of preoperative letrozole, everolimus, and carotuximab, a monoclonal antibody targeting endoglin, in nonmetastatic breast cancer. Methods Eligible patients had newly diagnosed, stage 2 or 3, hormone receptor-positive and Her2/neu-negative breast cancer. Patients received escalating doses of everolimus; the dose of letrozole and carotuximab were fixed at 2.5 mg PO daily and 15 mg/kg intravenously every 2 weeks, respectively. The primary objective was to determine the safety and tolerability of the combination. Secondary objectives included pharmacokinetic and pharmacodynamic studies and assessments of antitumor activity. Results Fifteen patients enrolled. The recommended phase 2 dose of everolimus in combination with letrozole and carotuximab was 10 mg PO daily. The most frequent adverse events were headache (67%), fatigue (47%), facial flushing and swelling (47%), gingival hemorrhage (40%), epistaxis (33%), nausea and vomiting (27%). Headache constituted a dose-limiting toxicity. At least two signs of mucocutaneous telangiectasia developed in 92% of patients. Carotuximab accumulated in the extravascular space and accelerated the biodistribution and clearance of everolimus. All patients had residual disease. Gene expression analyses were consistent with downregulation of genes involved in proliferation and DNA repair. Among 6 patients with luminal B breast cancer, 5 converted to luminal A after one cycle of therapy. Conclusion Letrozole, everolimus, and carotuximab were tolerated in combination at their single-agent doses. Pharmacokinetic studies revealed an interaction between everolimus and carotuximab. Trial registration This trial is registered with ClinicalTrials.gov (Identifier: NCT02520063), first posted on August 11, 2015, and is active, not recruiting.

PurposeMalignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive softtissue sarcomas that lack effective treatments, underscoring the urgent need to uncover novel mediators of MPNST pathogenesis that may serve as potential therapeutic targets. Tumor angiogenesis is considered a critical event in MPNST transformation and progression. Here, we have investigated whether endoglin (ENG), a TGF-β coreceptor with a crucial role in angiogenesis, could be a novel therapeutic target in MPNSTs.Experimental DesignENG expression was evaluated in human peripheral nerve sheath tumor tissues and plasma samples. Effects of tumor cell-specific ENG expression on gene expression, signaling pathways and in vivo MPNST growth and metastasis were investigated. The efficacy of ENG targeting in monotherapy or in combination with MEK inhibition was analyzed in xenograft models.ResultsENG expression was found to be upregulated in both human MPNST tumor tissues and plasma circulating small extracellular vesicles. We demonstrated that ENG modulates Smad1/5 and MAPK/ERK pathway activation and pro-angiogenic and pro-metastatic gene expression in MPNST cells and plays an active role in tumor growth and metastasis in vivo. Targeting with ENG-neutralizing antibodies (TRC105/M1043) decreased MPNST tumor growth and metastasis in xenograft models by reducing tumor cell proliferation and angiogenesis. Importantly, combination of anti-ENG therapy with MEK inhibition reduced more effectively tumor cell growth and angiogenesis.ConclusionsOur data unveil a tumor-promoting function of ENG in MPNSTs and support the combined use of anti-ENG antibodies and MEK inhibitors as a novel potential combination to control MPNSTs growth and metastasis.Statement of translational relevanceMalignant peripheral nerve sheath tumors (MPNSTs) present a poor clinical outcome due to tumor aggressiveness and the absence of effective treatments that underline the need for identifying novel therapeutic approaches. Here, we show that ENG is upregulated in human MPNSTs and we uncover an important role for this coreceptor in MPNST. ENG-neutralizing antibodies (TRC105/M1043) decreased MPNST tumor growth and metastasis in xenograft models, supporting a novel use of anti-ENG therapies for MPNST treatment. Currently, MEK inhibitors are used in the clinic to shrink peripheral nerve sheath tumors (e.g. plexiform neurofibromas). Importantly, we demonstrate that combination of anti ENG antibodies with MEK inhibitors efficiently blocked MPNST growth and metastasis. Our findings provide a rationale for combining anti-ENG and MEK inhibitors as a new strategy for MPNST management.

Purpose: Endoglin is a coreceptor for TGFβ ligands that is highly expressed on proliferating endothelial cells and other cells in the tumor microenvironment. Clinical studies have noted increased programmed cell death (PD)-1 expression on cytotoxic T cells in the peripheral blood of patients with cancer treated with TRC105, an endoglin-targeting antibody. In this study, we investigated the combination of endoglin antibodies (TRC105 and M1043) with an anti-PD1 antibody. Experimental Design: The combination anti-endoglin/anti-PD1 antibodies was tested in four preclinical mouse models representing different stages of cancer development. To investigate the underlying mechanism, Fc-receptor–knockout mice were used complemented with depletion of multiple immune subsets in mice. Tumor growth and the composition of immune infiltrate were analyzed by flow cytometry. Finally, human colorectal cancer specimens were analyzed for presence of endoglin-expressing regulatory T cells (Treg). Results: In all models, the combination of endoglin antibody and PD1 inhibition produced durable tumor responses, leading to complete regressions in 30% to 40% of the mice. These effects were dependent on the presence of Fcγ receptors, indicating the involvement of antibody-dependent cytotoxic responses and the presence of CD8+ cytotoxic T cells and CD4+ Th cells. Interestingly, treatment with the endoglin antibody, TRC105, significantly decreased the number of intratumoral Tregs. Endoglin-expressing Tregs were also detected in human colorectal cancer specimens. Conclusions: Taken together, these data provide a rationale for combining TRC105 and anti-PD1 therapy and provide additional evidence of endoglin's immunomodulatory role.

Lessons Learned Background Endoglin is an angiogenic receptor expressed on proliferating tumor vessels and renal cell carcinoma (RCC) stem cells that is implicated as a mechanism of resistance to vascular endothelial growth factor receptor (VEGFR) inhibitors. This study evaluated an antiendoglin monoclonal antibody (carotuximab, TRC105) combined with axitinib in patients with advanced or metastatic clear cell renal cell carcinoma (mccRCC) who had progressed following one or more prior VEGF inhibitors. Methods TRAXAR was a multicenter, international randomized 1:1 (stratified by ECOG, 0 vs. 1), phase II study of carotuximab combined with axitinib versus axitinib alone in mccRCC patients who had progressed following one or more prior VEGF inhibitors. The primary endpoint was progression-free survival (PFS) assessed by independent central review (ICR) per RECIST 1.1 Results A total of 150 patients were randomized. The combination therapy resulted in shorter median PFS by RECIST 1.1 than axitinib monotherapy (6.7 vs. 11.4 months). The combination was tolerated similarly to axitinib monotherapy, and there were no treatment related deaths. Exploratory evaluation of pretreatment circulating biomarkers suggested the combination might benefit patients who have low baseline VEGF levels. Conclusion The combination of carotuximab with axitinib did not demonstrate additional efficacy over single agent axitinib in patients with mccRCC who progressed following one or more prior VEGF inhibitor treatment.

Background TRC105 is an IgG1 endoglin monoclonal antibody that potentiates VEGF inhibitors in preclinical models. We assessed safety, pharmacokinetics, and antitumor activity of TRC105 in combination with axitinib in patients with metastatic renal cell carcinoma (mRCC). Subjects, Materials, and Methods Heavily pretreated mRCC patients were treated with TRC105 weekly (8 mg/kg and then 10 mg/kg) in combination with axitinib (initially at 5 mg b.i.d. and then escalated per patient tolerance to a maximum of 10 mg b.i.d.) until disease progression or unacceptable toxicity using a standard 3 + 3 phase I design. Results Eighteen patients (median number of prior therapies = 3) were treated. TRC105 dose escalation proceeded to 10 mg/kg weekly without dose-limiting toxicity. Adverse event characteristics of each drug were not increased in frequency or severity when the two drugs were administered concurrently. TRC105 and axitinib demonstrated preliminary evidence of activity, including partial responses (PR) by RECIST in 29% of patients, and median progression-free survival (11.3 months). None of the patients with PR had PR to prior first-line treatment. Lower baseline levels of osteopontin and higher baseline levels of TGF-β receptor 3 correlated with overall response rate. Conclusion TRC105 at 8 and 10 mg/kg weekly was well tolerated in combination with axitinib, with encouraging evidence of activity in patients with mRCC. A multicenter, randomized phase II trial of TRC105 and axitinib has recently completed enrollment (NCT01806064). Implications for Practice TRC105 is a monoclonal antibody to endoglin (CD105), a receptor densely expressed on proliferating endothelial cells and also on renal cancer stem cells that is implicated as a mediator of resistance to inhibitors of the VEGF pathway. In this Phase I trial, TRC105 combined safely with axitinib at the recommended single agent doses of each drug in patients with renal cell carcinoma. The combination demonstrated durable activity in a VEGF inhibitor-refractory population and modulated several angiogenic biomarkers. A randomized Phase II trial testing TRC105 in combination with axitinib in clear cell renal cell carcinoma has completed accrual.

Silica nanoparticles have been one of the most promising nanosystems for biomedical applications due to their facile surface chemistry and non-toxic nature. However, it is still challenging to effectively deliver them into tumor sites and noninvasively visualize their in vivo biodistribution with excellent sensitivity and accuracy for effective cancer diagnosis. In this study, we design a yolk/shell-structured silica nanosystem 64Cu-NOTA-QD@HMSN-PEG-TRC105, which can be employed for tumor vasculature targeting and dual-modality PET/optical imaging, leading to superior targeting specificity, excellent imaging capability and more reliable diagnostic outcomes. By combining vasculature targeting, pH-sensitive drug delivery, and dual-modality imaging into a single platform, as-designed yolk/shell-structured silica nanosystems may be employed for the future image-guided tumor-targeted drug delivery, to further enable cancer theranostics.

This work presents the production with a cyclotron of the positron emitter 55Co via the 54Fe(d,n) and 58Ni(p,α) reactions and the Auger electron emitter 58mCo via the 57Fe(d,n) reaction after high current (40μA p and 60μA d) irradiation on electroplated targets. High specific activity radionuclides (up to 55.6 GBq/μmol 55Co and 31.8GBq/μmol 58mCo) with high radionuclidic purity (99.995% 55Co from 54Fe, 98.8% 55Co from 58Ni, and 98.7% 58mCo from 57Fe at end of bombardment, EoB), in high activity concentration (final separated radionuclide in 92%) were obtained after processing of the irradiated targets with novel radiochemical separation methods based on HCl dissolution and the resin N,N,N',N'-tetrakis-2-ethylhexyldiglycolamide (DGA, branched). One hour long irradiations using 38-65, 110-214 and 59-78mg of enriched 54Fe (99.93%), 58Ni (99.48%) and 57Fe (95.06%), respectively, electroplated over a 1.0cm2 surface, yielded 582 ± 66MBq 55Co, 372 ± 14MBq 55Co and 810 ± 186MBq 58mCo, respectively, decay corrected to EoB. The separation methods allow for the recovery of the costly enriched target materials, which were reconstituted into metallic targets after novel electroplating methods, with an overall recycling efficiency of 93 ± 4% for iron. The produced radionuclides were used to radiolabel the angiogenesis marker antibody TRC105 conjugated to the chelator NOTA as a demonstration of their quality.

Background: Metastatic undifferentiated pleomorphic sarcoma (UPS) and the genetically related myxofibrosarcoma (MFS) are soft tissue sarcoma (STS) subtypes with poor prognoses. While responses to front line chemotherapy can approach 20%, in the 2nd line setting and beyond, efficacy remains limited. Pazopanib, the only approved treatment in the refractory setting has demonstrated an objective response rate (ORR) of 4%. Envafolimab is a single domain PD-L1 antibody administered rapidly by subcutaneous (SQ) injection that is being studied in two additional pivotal trials: MSI-H cancer and biliary tract cancer. The activity of envafolimab is similar to other PD-1 antibodies administered intra-venously. Envafolimab demonstrated a 32% ORR in MSI-H colorectal cancer that failed three approved chemotherapeutics, similar to the ORR of 28% and 33%, with nivolumab and pembrolizumab, respectively. The rationale for the ENVASARC trial is based on the previously reported efficacy of checkpoint inhibition in UPS/MFS. Single agent pembrolizumab demonstrated a 23% ORR, while the combination of nivolumab and ipilimumab demonstrated a 29% ORR in refractory UPS/MFS. Methods: ENVASARC (NCT 04480502) is a pivotal multicenter (at ~25 U.S. centers) open-label, randomized, non-comparative, parallel cohort study of treatment with envafolimab 300 mg every 3 weeks by SQ injection (cohort A; n=80) or envafolimab 300 mg every 3 weeks by SQ injection combined with ipilimumab 1 mg/kg every 3 weeks intra-venously for four doses (cohort B; n=80) in patients with locally advanced, unresectable or metastatic UPS/MFS who have progressed on one or two lines of prior therapy. The primary objective of each of parallel cohort is to demonstrate an ORR with a lower limit of the 95% confidence interval that excludes 5.0% in each cohort. If ≥ 9 responders are observed of the 80 patients enrolled in each cohort, then the lower bound of the 95% confidence interval will exclude 5.0%. Secondary endpoints include duration of response (DOR), PFS and OS. Key inclusion criteria: ≤ 2 prior lines of therapy (neoadjuvant and adjuvant therapy excluded), ECOG ≤ 1. Citation Format: Sandra D'Angelo, Steven Robinson, Joelle Lam, Bonne Adams, James Freddo, Charles P. Theuer, Robert Maki. ENVASARC: A Pivotal Trial of Envafolimab, and Envafolimab in Combination with Ipilimumab, in Patients with Advanced or Metastatic Undifferentiated Pleomorphic Sarcoma or Myxofibrosarcoma who have Progressed on Prior Chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT239.

2511 Background: CD73 is implicated in tumor resistance to checkpoint immunotherapy (CPI) and plays a critical role in adenosine-mediated immune suppression. Uliledlimab, a differentiated CD73 antibody, inhibits the adenosine pathway in a non-competitive and unique intra-dimer binding mode. Uliledlimab suppresses tumor growth when combined with a PD-(L)1 inhibitor in multiple pre-clinical models. Methods: This 3+3 dose-escalation phase 1 study (NCT03835949) evaluated safety, tolerability, PK, PD and preliminary efficacy in cancer patients. Uliledlimab was administered intravenously at doses of 5, 10 or 15 mg/kg weekly (QW) or 15 or 20 mg/kg every 3 weeks (Q3W) alone in the first cycle and in combination with atezolizumab (1,200 mg Q3W) starting on week 4. Soluble CD73 in serum and CD73 receptor occupancy (RO) in circulating CD19+ B cells were measured. Expression of PD-L1, CD73 and A2A receptor was analyzed in baseline tumor specimens (n = 14). Tumor responses were assessed by RECIST/iRECIST. Results: As of 17 January 2021, 20 patients with advanced solid tumors were enrolled (M:F 8:12; mean age = 64; median prior regimens = 3 (range 1-9)). Uliledlimab was well-tolerated with no dose limiting toxicity. The most common treatment-related adverse events were first dose infusion related reactions (65%, n = 13) most commonly comprising chills/rigors, nausea, and vomiting (Grade 1 or 2) that resolved in subsequent infusions. PK appears linear at doses ≥ 10 mg/kg and modelling indicated a mean derived effective half-life of ̃19 days. Soluble CD73 was undetectable and complete RO was achieved in all patients after the first dose at ≥ 10 mg/kg. Anti-drug antibody was detected in 3/20 patients (15%). Among 13 efficacy-evaluable patients dosed at ≥ 10 mg/kg, complete response (CR = 1) and partial response (PR = 2) were observed in 3 patients (ORR = 23%) together with 3 stable disease (SD) patients (DCR = 46%). One PD-(L)1 inhibitor naïve patient with clear cell ovarian cancer achieved CR at 10 mg/kg QW and remains on study after 12 months. Two patients with NSCLC dosed at 15 mg/kg QW and 20 mg/kg Q3W, respectively, achieved PR. One patient failed nivolumab and the other received no prior PD-(L)1 inhibitor treatment. CD73 was expressed on 78% (mean) of malignant cells from archival tumor specimens in responders compared to 23% in non-responders. Conclusions: Uliledlimab is safe and well tolerated up to 20 mg/kg Q3W and 15 mg/kg QW. Full saturation of circulating and cell-bound CD73 was achieved at doses ≥ 10 mg/kg. Uliledlimab exhibited evidence of clinical activity in both PD-(L)1 treatment naïve and refractory cancer patients with high archival tumor expression of CD73. The results of this phase 1 study encourage further clinical investigation to evaluate the efficacy of uliledlimab in the treatment of solid tumors. Clinical trial information: NCT03835949.

TPS11581 Background: Metastatic undifferentiated Pleomorphic Sarcoma (UPS) and the genetically related myxofibrosarcoma (MFS) are soft tissue sarcoma (STS) subtypes with poor prognoses. While responses to front line chemotherapy can approach 20%, efficacy remains limited in the 2nd line setting and beyond. Pazopanib, the only approved treatment in the refractory setting, has demonstrated an objective response rate (ORR) of 4%. Envafolimab is a single domain PD-L1 antibody administered rapidly by subcutaneous (SQ) injection that is being studied in two additional pivotal trials: microsatellite instability-high (MSI-H) cancer and biliary tract cancer. The activity of envafolimab appears to be similar to other PD-1 antibodies administered i.v. Envafolimab demonstrated a 32% objective response rate (ORR) in MSI-H colorectal cancer patients who failed three approved chemotherapeutics, similar to the ORR of 28% and 33% with nivolumab and pembrolizumab in these patient populations, respectively. The rationale for the ENVASARC trial is based on the previously reported activity of checkpoint inhibition in UPS/MFS. Single agent pembrolizumab demonstrated a 23% ORR, while the combination of nivolumab and ipilimumab demonstrated a 29% ORR in refractory UPS/MFS. Methods: ENVASARC (NCT 04480502) is a pivotal multicenter (at ̃25 U.S. centers) open-label, randomized, non-comparative, parallel cohort study of treatment with envafolimab 300 mg every 3 weeks by SQ injection (cohort A; n = 80) or envafolimab 300 mg every 3 weeks by SQ injection combined with ipilimumab 1 mg/kg every 3 weeks i.v. for four doses (cohort B; n = 80) in patients with locally advanced, unresectable or metastatic UPS/MFS who have progressed on one or two lines of prior therapy. The primary objective of each of parallel cohort is to demonstrate an ORR with a lower limit of the 95% confidence interval that excludes 5.0% in each cohort. If ≥ 9 responders are observed of the 80 patients enrolled in each cohort, then the lower bound of the 95% confidence interval will exclude 5.0%. Secondary endpoints include duration of response (DOR), PFS and OS. Key inclusion criteria: ≤ 2 prior lines of therapy (neoadjuvant and adjuvant therapy excluded), ECOG ≤ 1. Clinical trial information: NCT 04480502.

Increased angiogenesis is a marker of aggressiveness in many cancers. Targeted radionuclide therapy of these cancers with angiogenesis-targeting agents may curtail this increased blood vessel formation and slow the growth of tumors, both primary and metastatic. CD105, or endoglin, has a primary role in angiogenesis in a number of cancers, making this a widely applicable target for targeted radioimmunotherapy. The anti-CD105 antibody, TRC105 (TRACON Pharmaceuticals), was conjugated with DTPA for radiolabeling with 177Lu (t 1/2 6.65 days). Balb/c mice were implanted with 4T1 mammary carcinoma cells, and five study groups were used: 177Lu only, TRC105 only, 177Lu-DTPA-IgG (a nonspecific antibody), 177Lu-DTPA-TRC105 low-dose, and 177Lu-DTPA-TRC105 high-dose. Toxicity of the agent was monitored by body weight measurements and analysis of blood markers. Biodistribution studies of 177Lu-DTPA-TRC105 were also performed at 1 and 7 days after injection. Ex vivo histology studies of various tissues were conducted at 1, 7, and 30 days after injection of high-dose 177Lu-DTPA-TRC105. Biodistribution studies indicated steady uptake of 177Lu-DTPA-TRC105 in 4T1 tumors between 1 and 7 days after injection (14.3 ± 2.3%ID/g and 11.6 ± 6.1%ID/g, respectively; n = 3) and gradual clearance from other organs. Significant inhibition of tumor growth was observed in the high-dose group, with a corresponding significant increase in survival (p

Purpose: We determined whether elimination of CD105+ cells in the tumor microenvironment (TME) with anti-CD105 antibodies enhanced anti-disialoganglioside (GD2) antibody dinutuximab therapy of neuroblastoma when combined with activated natural killer (aNK) cells. Experimental Design: The effect of MSCs and monocytes on antibody-dependent cellular cytotoxicity (ADCC) mediated by dinutuximab with aNK cells against neuroblastoma cells was determined in vitro. ADCC with anti-CD105 mAb TRC105 and aNK cells against MSCs, monocytes, and endothelial cells, which express CD105, was evaluated. Anti-neuroblastoma activity in immunodeficient NSG mice of dinutuximab with aNK cells without or with anti-CD105 mAbs was determined using neuroblastoma cell lines and a patient-derived xenograft. Results: ADCC mediated by dinutuximab with aNK cells against neuroblastoma cells in vitro was suppressed by addition of MSCs and monocytes, and dinutuximab with aNK cells was less effective against neuroblastomas formed with coinjected MSCs and monocytes in NSG mice than against those formed by tumor cells alone. Anti-CD105 antibody TRC105 with aNK cells mediated ADCC against MSCs, monocytes, and endothelial cells. Neuroblastomas formed in NSG mice by two neuroblastoma cell lines or a patient-derived xenograft coinjected with MSCs and monocytes were most effectively treated with dinutuximab and aNK cells when anti-human (TRC105) and anti-mouse (M1043) CD105 antibodies were added, which depleted human MSCs and murine endothelial cells and macrophages from the TME. Conclusions: Immunotherapy of neuroblastoma with anti-GD2 antibody dinutuximab and aNK cells is suppressed by CD105+ cells in the TME, but suppression is overcome by adding anti-CD105 antibodies to eliminate CD105+ cells.

Purpose: Cancer-associated fibroblasts (CAF) are a major component of the colorectal cancer tumor microenvironment. CAFs play an important role in tumor progression and metastasis, partly through TGF-β signaling pathway. We investigated whether the TGF-β family coreceptor endoglin is involved in CAF-mediated invasion and metastasis. Experimental Design: CAF-specific endoglin expression was studied in colorectal cancer resection specimens using IHC and related to metastases-free survival. Endoglin-mediated invasion was assessed in vitro by transwell invasion, using primary colorectal cancer–derived CAFs. Effects of CAF-specific endoglin expression on tumor cell invasion were investigated in a colorectal cancer zebrafish model, whereas liver metastases were assessed in a mouse model. Results: CAFs specifically at invasive borders of colorectal cancer express endoglin and increased expression intensity correlated with increased disease stage. Endoglin-expressing CAFs were also detected in lymph node and liver metastases, suggesting a role in colorectal cancer metastasis formation. In stage II colorectal cancer, CAF-specific endoglin expression at invasive borders correlated with poor metastasis-free survival. In vitro experiments revealed that endoglin is indispensable for bone morphogenetic protein (BMP)-9–induced signaling and CAF survival. Targeting endoglin using the neutralizing antibody TRC105 inhibited CAF invasion in vitro. In zebrafish, endoglin-expressing fibroblasts enhanced colorectal tumor cell infiltration into the liver and decreased survival. Finally, CAF-specific endoglin targeting with TRC105 decreased metastatic spread of colorectal cancer cells to the mouse liver. Conclusions: Endoglin-expressing CAFs contribute to colorectal cancer progression and metastasis. TRC105 treatment inhibits CAF invasion and tumor metastasis, indicating an additional target beyond the angiogenic endothelium, possibly contributing to beneficial effects reported during clinical evaluations. See related commentary by Becker and LeBleu, p. 6110.

Purpose: Pancreatic adenocarcinoma is a highly aggressive cancer, currently treated with limited success and dismal outcomes. New diagnostic and treatment strategies offer the potential to reduce cancer mortality. Developing highly specific noninvasive imaging probes for pancreatic cancer is essential to improving diagnostic accuracy and monitoring therapeutic intervention. Experimental Design: A bispecific heterodimer was synthesized by conjugating an anti-tissue factor (TF) Fab with an anti-CD105 Fab, via the bio-orthogonal “click” reaction between tetrazine (Tz) and trans-cyclooctene (TCO). The heterodimer was labeled with 64Cu for PET imaging of nude mice bearing BXPC-3 xenograft and orthotopic pancreatic tumors. Results: PET imaging of BXPC-3 (TF/CD105+/+) xenograft tumors with 64Cu-labeled heterodimer displayed significantly enhanced tumor uptake (28.8 ± 3.2 %ID/g; n = 4; SD) at 30 hours postinjection, as compared with each of their monospecific Fab tracers (12.5 ± 1.4 and 7.1 ± 2.6 %ID/g; n = 3; SD). In addition, the activity–concentration ratio allowed for effective tumor visualization (tumor/muscle ratio 75.2 ± 9.4 at 30 hours postinjection.; n = 4; SD). Furthermore, 64Cu-NOTA-heterodimer enabled sensitive detection of orthotopic pancreatic tumor lesions with an uptake of 17.1 ± 4.9 %ID/g at 30 hours postinjection and tumor/muscle ratio of 72.3 ± 46.7. Conclusions: This study demonstrates that dual targeting of TF and CD105 provided synergistic improvements in binding affinity and tumor localization of the heterodimer. Dual-targeted imaging agents of pancreatic and other cancers may assist in diagnosing pancreatic malignancies as well as reliable monitoring of therapeutic response. Clin Cancer Res; 22(15); 3821–30. ©2016 AACR.

Radioisotopes of arsenic are of considerable interest to the field of nuclear medicine with unique nuclear and chemical properties making them well-suited for use in novel theranostic radiopharmaceuticals. However, progress must still be made in the production of isotopically pure radioarsenic and in its stable conjugation to biological targeting vectors. This work presents the production and irradiation of isotopically enriched (72)Ge(m) discs in an irrigation-cooled target system allowing for the production of isotopically pure (72)As with capability on the order of 10 GBq. A radiochemical separation procedure isolated the reactive trivalent radioarsenic in a small volume buffered aqueous solution, while reclaiming (72)Ge target material. The direct thiol-labeling of a monoclonal antibody resulted in a conjugate exhibiting exceptionally poor in vivo stability in a mouse model. This prompted further investigations to alternative radioarsenic labeling strategies, including the labeling of the dithiol-containing chelator dihydrolipoic acid, and thiol-modified mesoporous silica nanoparticles (MSN-SH). Radioarsenic-labeled MSN-SH showed exceptional in vivo stability toward dearsenylation.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy, often characterized by poor prognosis following standard induction therapy. The hypomethylating agent decitabine (DAC) is an alternative treatment for elderly and relapsed/refractory AML patients, yet responses following DAC monotherapy are still modest. The transforming growth factor-β (TGF-β) receptor CD105 (endoglin) is expressed in various hematopoietic malignancies, and high CD105 expression correlates with poor prognosis in AML patients. Using a xenograft model, we have recently demonstrated that targeting CD105+ AML blasts with the TRC105 monoclonal antibody inhibits leukemia progression. Here we investigated whether administration of TRC105 along with DAC could represent a novel therapeutic option for relapsed/refractory AML. Our data show that the DAC/TRC105 combination results in a more durable anti-leukemic effect in AML xenografts compared to DAC monotherapy. Moreover, the DAC/TRC105 combination enhanced reactive oxygen species (ROS) activity, which correlated with reduced leukemia burden. RNA-sequencing studies suggest that TRC105 may alter TGF-β activity in AML blasts. Taken together, these findings provide rationale for the clinical evaluation of TRC105 in combination with DAC in AML patients., AML; TRC105; Decitabine; Xenografts; Cell biology; Antibody; Molecular biology; Cancer research; Chemotherapy; Hematology

Angiogenesis is widely recognized as one of the hallmarks of cancer. Therefore, imaging and therapeutic agents targeted to angiogenic vessels may be widely applicable in many types of cancer. To this end, the theranostic isotope pair, (86)Y and (90)Y, were used to create a pair of agents for targeted imaging and therapy of neovasculature in murine breast cancer models using a chimeric anti-CD105 antibody, TRC105. Serial positron emission tomography imaging with (86)Y-DTPA-TRC105 demonstrated high uptake in 4T1 tumors, peaking at 9.6 ± 0.3%ID/g, verified through ex vivo studies. Additionally, promising results were obtained in therapeutic studies with (90)Y-DTPA-TRC105, wherein significantly (p < 0.05) decreased tumor volumes were observed for the targeted treatment group over all control groups near the end of the study. Dosimetric extrapolation and tissue histological analysis corroborated trends found in vivo. Overall, this study demonstrated the potential of the pair (86/90)Y for theranostics, enabling personalized treatments for cancer.

Background Endoglin is an essential angiogenic receptor expressed on proliferating tumor vessels and RCC stem cells that is implicated as a mechanism of VEGF resistance. TRC105 is an endoglin monoclonal antibody that potentiates the anti-tumor activity of VEGF inhibitors in preclinical models and demonstrated a 29% RECIST response rate when combined with AX in patients with mRCC in a Phase 1b trial. Methods TRAXAR was a multicenter, randomized 1:1 (stratified by ECOG, 0 vs. 1), Phase 2 study of TRAX vs AX in patients with mRCC who had progressed following one prior VEGF inhibitor at 33 centers in the US and EU. The primary endpoint was progression-free survival (PFS) assessed by RECIST by independent review committee (IRC). Secondary endpoints included overall response rate (ORR), and safety. PFS and ORR were also assessed by Investigator review (INV) and according to Choi criteria (CC). Results Of 150 pts (TRAX, 75; AX, 75), 106 (71%) were male, 142 (95%) were white; median age was 64 years (range, 38-82). Treatment with TRAX did not prolong PFS compared to AX. ORR was not different based on IRC, INV or CC (Table). Most all-grade common adverse events (AEs) in TRAX vs AX: headache (65.8% vs. 16.2%), epistaxis (63.0% vs. 8.1%), and diarrhea (60.3% vs. 59.5%); most common serious AEs included: anemia (6.9% vs. 1.4%) and dehydration (4.1% vs. 0%). Table . 912PD IRC INV CC TRAX AX TRAX AX TRAX AX Median PFS, mo (95% CI) 6.7 (5.6-13.1) 11.4 (5.8-NE) 7.2 (5.5-9.1) 7.4 (5.5-12.8) 7.2 (5.6-9.4) 7.5 (5.6-20.3) HR (95% CI) p-value 1.42 (0.88-2.30) 0.15 1.41 (0.95-2.10) 0.09 1.30 (0.83-2.03) 0.26 ORR, % 33.8 32.9 34.8 30.6 66.2 67.1 Odds ratio (95% CI) p-value 1.04 (0.50-2.15) 0.92 1.19 (0.58-2.44) 0.63 0.96 (0.47-1.96) 0.91 NE: not estimable; CI: confidence interval; mo: months Conclusions TRC105 did not demonstrate activity when combined with AX in patients with mRCC who had received prior VEGF inhibitor treatment, whether assessed using RECIST or Choi criteria. TRAX was generally well tolerated in pts with advanced or metastatic RCC. Clinical trial identification NCT01806064. Legal entity responsible for the study TRACON Pharmaceuticals. Funding TRACON Pharmaceuticals. Disclosure T.K. Choueiri: Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: TRACON; Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca; Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution): Bayer; Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution): BMS; Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution): Cerulean; Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution): Eisai; Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution): Foundation Medicine; Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution): Exelixis; Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution): Ipsen; Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution): Genentech; Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution): Roche; Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution): Roche Products Limited; Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution): GSM; Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution): Merck; Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution): Novartis; Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution): Peloton; Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution): Pfizer; Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution): Prometheus; Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution): Corvus; Honoraria (institution): Calithera. R.K. Pachynski: Advisory / Consultancy: EMD Serono; Advisory / Consultancy: Bristol-Myers Squibb; Advisory / Consultancy: Pfizer; Advisory / Consultancy, Speaker Bureau / Expert testimony: Sanofi; Advisory / Consultancy: Jounce Therapeutics; Speaker Bureau / Expert testimony: Dendreon; Speaker Bureau / Expert testimony: Merck; Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Genentech/Roche; Speaker Bureau / Expert testimony: Genomic Health; Advisory / Consultancy: Bayer. Y. Zakharia: Advisory / Consultancy: Amgen; Advisory / Consultancy: Roche; Advisory / Consultancy: Novartis; Advisory / Consultancy: Jansen; Advisory / Consultancy: Eisai; Advisory / Consultancy: Exelixis; Advisory / Consultancy: Castle Bioscience; Advisory / Consultancy: Pfizer; Advisory / Consultancy: Array. T.H. Ho: Advisory / Consultancy: Roche; Advisory / Consultancy: Pfizer; Advisory / Consultancy: ipensenlivemeetings; Advisory / Consultancy: cardinal health; Advisory / Consultancy: Exelixis. B.E. Simpson: Shareholder / Stockholder / Stock options, Full / Part-time employment: TRACON Pharmaceuticals. B. Adams: Shareholder / Stockholder / Stock options, Full / Part-time employment: TRACON. L. Robertson: Shareholder / Stockholder / Stock options, Full / Part-time employment: TRACON. M. Darif: Advisory / Consultancy: TRACON. C. Theuer: Shareholder / Stockholder / Stock options, Full / Part-time employment, Officer / Board of Directors: TRACON. N. Agarwal: Advisory / Consultancy: Astellas; Advisory / Consultancy, Research grant / Funding (self): AstraZeneca; Advisory / Consultancy: Argos; Advisory / Consultancy: Bayer; Advisory / Consultancy: Clovis; Advisory / Consultancy, Research grant / Funding (self): Eisai; Advisory / Consultancy, Research grant / Funding (self): Exelixis; Advisory / Consultancy: EMD Serono; Advisory / Consultancy, Research grant / Funding (self): Ely Lilly; Advisory / Consultancy: Foundation One; Advisory / Consultancy: Genentech; Advisory / Consultancy, Research grant / Funding (self): Janssen; Advisory / Consultancy, Research grant / Funding (self): Merck; Advisory / Consultancy: Medivation; Advisory / Consultancy, Research grant / Funding (self): Novartis; Advisory / Consultancy: Nektar; Advisory / Consultancy, Research grant / Funding (self): Pfizer; Advisory / Consultancy: Pharmacyclics; Research grant / Funding (institution): Bavarian Nordic; Research grant / Funding (self): TRACON. All other authors have declared no conflicts of interest.

UNLABELLED The critical challenge in abdominal aortic aneurysm (AAA) research is the accurate diagnosis and assessment of AAA progression. Angiogenesis is a pathologic hallmark of AAA, and CD105 is highly expressed on newly formed vessels. Our goal was to use (64)Cu-labeled anti-CD105 antibody Fab fragment for noninvasive assessment of angiogenesis in the aortic wall in a murine model of AAA. METHODS Fab fragment of TRC105, a mAb that specifically binds to CD105, was generated by enzymatic papain digestion and conjugated to NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) for (64)Cu labeling. The binding affinity/specificity of NOTA-TRC105-Fab was evaluated by flow cytometry and various ex vivo studies. BALB/c mice were anesthetized and treated with calcium phosphate to induce AAA and underwent weekly PET scans using (64)Cu-NOTA-TRC105-Fab. Biodistribution and autoradiography studies were also performed to confirm the accuracy of PET results. RESULTS NOTA-TRC105-Fab exhibited high purity and specifically bound to CD105 in vitro. Uptake of (64)Cu-NOTA-TRC105-Fab increased from a control level of 3.4 ± 0.1 to 9.5 ± 0.4 percentage injected dose per gram (%ID/g) at 6 h after injection on day 5 and decreased to 7.2 ± 1.4 %ID/g on day 12, which correlated well with biodistribution and autoradiography studies (i.e., much higher tracer uptake in AAA than normal aorta). Of note, enhanced AAA contrast was achieved, due to the minimal background in the abdominal area of mice. Degradation of elastic fibers and highly expressed CD105 were observed in ex vivo studies. CONCLUSION (64)Cu-NOTA-TRC105-Fab cleared rapidly through the kidneys, which enabled noninvasive PET imaging of the aorta with enhanced contrast and showed increased angiogenesis (CD105 expression) during AAA. (64)Cu-NOTA-TRC105-Fab PET may potentially be used for future diagnosis and prognosis of AAA.

Actively targeted theranostic nanomedicine may be the key for future personalized cancer management. Although numerous types of theranostic nanoparticles have been developed in the past decade for cancer treatment, challenges still exist in the engineering of biocompatible theranostic nanoparticles with highly specific in vivo tumor targeting capabilities. Here, we report the design, synthesis, surface engineering, and in vivo active vasculature targeting of a new category of theranostic nanoparticle for future cancer management. Water-soluble photothermally sensitive copper sulfide nanoparticles were encapsulated in biocompatible mesoporous silica shells, followed by multistep surface engineering to form the final theranostic nanoparticles. Systematic in vitro targeting, an in vivo long-term toxicity study, photothermal ablation evaluation, in vivo vasculature targeted imaging, biodistribution and histology studies were performed to fully explore the potential of as-developed new theranostic nanoparticles.

TRC105 is an anti-endoglin antibody currently being tested in combination with VEGF inhibitors. In the phase Ib trial, 38 patients were treated with both TRC105 and bevacizumab (BEV), and improved clinical outcomes were observed, despite the fact that 30 patients (79%) were refractory to prior anti-VEGF therapy. Plasma samples were tested for angiogenic and inflammatory biomarkers at baseline and on-treatment. To provide broader context of this combination biomarker study, direct cross-study comparisons were made to biomarker studies previously conducted in patients treated with either BEV or TRC105 monotherapy. Upon treatment with BEV and TRC105, pharmacodynamic changes in response to both BEV (PlGF increase) and TRC105 (soluble endoglin increase) were noted. In addition, distinct patterns of change were identified (similar, opposing, neutralizing). Similar patterns were observed when the combination elicited similar effects to those observed with monotherapy treatment (i.e., decreases of Ang-2, increases of IL6 and VCAM-1). Opposing patterns were observed when the combination led to opposing effects compared with monotherapy treatment (i.e., TGFβ1, PDGF-AA and PDGF-BB, PAI-1). Lastly, neutralizing patterns were observed when one drug led to increase, whereas the other drug led to decrease, and the combination elicited no overall effect on the marker (i.e., VEGF-A, VEGF-D, and IGFBP-3). Patients achieving partial responses or stable disease from the combination exhibited significantly lower expression of E-Cadherin, HGF, ICAM-1, and TSP-2 at baseline. Taken together, the novel biomarker modulations identified may deepen our understanding of the underlying biology in patients treated with BEV and TRC105 compared with either drug alone. Mol Cancer Ther; 17(10); 2248–56. ©2018 AACR.

Purpose: Our goal was to explore nano‐graphene for in vivo tumor targeting and quantitatively evaluate the pharmacokinetics and tumor targeting efficacy through PET imaging, using 6 4Cu and 66Ga as the radiolabel. Methods: Nano‐graphene oxide (GO) sheets, with amino group‐ terminated PEG chains (10 kDa) covalently attached, were conjugated to NOTA (l,4,7‐triazacyclononane‐l,4,7‐triacetic acid, a suitable chelator for 6 4Cu and 6 6 Ga) and TRC105 (a mAb that binds to CD 105, overexpressed on neovasculature). FACS analyses, size measurements, and serum stability studies were performed to characterize the GO conjugates before in vivo investigation (PET, bio distribution, blocking studies, etc.) in 4T1 murine breast tumor‐bearing mice. Findings from imaging studies were then validated by histology. Results: TRC105‐conjugated GO, 20–30 nm in diameter, was specific for CD105 with little non‐specific binding. Both 64 Cu‐ and 66Ga‐ labled GO conjugates had excellent stability in mouse serum. Clearance of the GO conjugates in mice was via the hepatobiliary pathway. vCu/6 6 Ga‐NOTA‐GO‐TRC105 accumulated rapidly in the 4T1 tumor and tumor uptake remained stable over time (3.8±0.4, 4.5±0.4, 5.8±0.3, and 4.5±0.4 %ID/g at 0.5, 3, 7, and 24 h p.i. for66 Ga; 5.8±0.6, 5.3±0.6, 4.0±0.4, and3.4±0.1 %ID/g at 0.5, 3,24, and 48 h p.i. for 6 4Cu; n = 4). Blocking studies confirmed CD105 specificity of 6 4 Cu/6 6 Ga‐NOTA‐ GOTRC105, which was corroborated by bio distribution studies. Furthermore, microscopy examination of GO in light view mode and immunofluorescence staining revealed that targeting of NOTA‐GO‐TRC105 is tumor vasculature CD105 specific with little extravasation. Conclusions: For the first time, we demonstrated that GO can be specifically directed to the tumor neovasculature in vivo through targeting of CD105, a marker of tumor angiogenesis. The versatile chemistry of graphene‐based nanomaterials makes them suitable nanoplatforms for future biomedical research, such as cancer theranostics.

Dual-targeted imaging agents have shown improved targeting efficiencies in comparison to single-targeted entities. The purpose of this study was to quantitatively assess the tumor accumulation of a dual-labeled heterobifunctional imaging agent, targeting two overexpressed biomarkers in pancreatic cancer, using positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging modalities. A bispecific immunoconjugate (heterodimer) of CD105 and tissue factor (TF) Fab′ antibody fragments was developed using click chemistry. The heterodimer was dual-labeled with a radionuclide (64Cu) and fluorescent dye. PET/NIRF imaging and biodistribution studies were performed in four-to-five week old nude athymic mice bearing BxPC-3 (CD105/TF+/+) or PANC-1 (CD105/TF−/−) tumors xenografts. A blocking study was conducted to investigate the specificity of the tracer. Ex vivo tissue staining was performed to compare TF/CD105 expression in tissues with PET tracer uptake to validate in vivo results. PET imaging of 64Cu-NOTA-heterodimer-ZW800 in BxPC-3 tumor xenografts revealed enhanced tumor uptake (21.0 ± 3.4 %ID/g; n = 4) compared to the homodimer of TRC-105 (9.6 ± 2.0 %ID/g; n=4; p < 0.01) and ALT-836 (7.6 ± 3.7 %ID/g; n=4; p < 0.01) at 24 h post-injection. Blocking studies revealed that tracer uptake in BxPC-3 tumors could be decreased by four-fold with TF blocking and two-fold with CD105 blocking. In the negative model (PANC-1), heterodimer uptake was significantly lower than that found in the BxPC-3 model (3.5 ± 1.1 %ID/g; n=4; p < 0.01). The specificity was confirmed by the successful blocking of CD105 or TF, which demonstrated the dual targeting with 64Cu-NOTA-heterodimer-ZW800 provided an improvement in overall tumor accumulation. Also, fluorescence imaging validated the PET imaging, allowing for clear delineation of the xenograft tumors. Dual-labeled heterodimeric imaging agents, like 64Cu-NOTA-heterodimer-ZW800, may increase the overall tumor accumulation in comparison to single-targeted homodimers, leading to improved imaging of cancer and other related diseases.

Endoglin (CD105), a receptor of the transforming growth factor-β superfamily, has been reported to identify functional long-term repopulating hematopoietic stem cells, and has been detected in certain subtypes of acute leukemias. Whether this receptor plays a functional role in leukemogenesis remains unknown. We identified endoglin expression on the majority of blasts from patients with acute myeloid leukemia (AML) and acute B-lymphoblastic leukemia (B-ALL). Using a xenograft model, we find that CD105+ blasts are endowed with superior leukemogenic activity compared with the CD105- population. We test the effect of targeting this receptor using the monoclonal antibody TRC105, and find that in AML, TRC105 prevented the engraftment of primary AML blasts and inhibited leukemia progression following disease establishment, but in B-ALL, TRC105 alone was ineffective due to the shedding of soluble CD105. However, in both B-ALL and AML, TRC105 synergized with reduced intensity myeloablation to inhibit leukemogenesis, indicating that TRC105 may represent a novel therapeutic option for B-ALL and AML.

Brush-shaped amphiphilic block copolymers were conjugated with a monoclonal antibody against CD105 (i.e., TRC105) and a macrocyclic chelator for (64)Cu-labeling to generate multifunctional theranostic unimolecular micelles. The backbone of the brush-shaped amphiphilic block copolymer was poly(2-hydroxyethyl methacrylate) (PHEMA) and the side chains were poly(L-lactide)-poly(ethylene glycol) (PLLA-PEG). The doxorubicin (DOX)-loaded unimolecular micelles showed a pH-dependent drug release profile and a uniform size distribution. A significantly higher cellular uptake of TRC105-conjugated micelles was observed in CD105-positive human umbilical vein endothelial cells (HUVEC) than nontargeted micelles due to CD105-mediated endocytosis. In contrast, similar and extremely low cellular uptake of both targeted and nontargeted micelles was observed in MCF-7 human breast cancer cells (CD105-negative). The difference between the in vivo tumor accumulation of (64)Cu-labeled TRC105-conjugated micelles and that of nontargeted micelles was studied in 4T1 murine breast tumor-bearing mice, by serial positron emission tomography (PET) imaging and validated by biodistribution studies. These multifunctional unimolecular micelles offer pH-responsive drug release, noninvasive PET imaging capability, together with both passive and active tumor-targeting abilities, thus making them a desirable nanoplatform for cancer theranostics.

TRC105 is an endoglin-targeting drug that possesses anti-angiogenic and antitumor potential. Analysis of the initial phase I trial of TRC105 demonstrated good tolerability and efficacy in cancer patients. In this report, we analyzed multiple circulating biomarkers at baseline, cycle 2 day 1 (C2D1), and end of study (EOS) for each patient. The baseline level and the fold change from baseline to both C2D1 and EOS for each marker were statistically analyzed. At C2D1, seven markers were significantly downregulated (angiopoietin-2 [Ang-2], insulin-like growth factor-binding protein-3 [IGFBP-3], plasminogen activator inhibitor-1 [PAI-1] total, platelet-derived growth factor [PDGF]-AA, PDGF-BB, thrombospondin-1 [TSP-1], and vascular endothelial growth factor [VEGF]-D). Meanwhile, seven markers were upregulated by C2D1 (E-Cadherin, soluble Endoglin [sEnd], E-Selectin, interleukin-6 [IL-6], osteopontin [OPN], TSP-2, and von Willebrand factor [vWF]). At EOS, seven markers were upregulated including Ang-2, C-reactive protein (CRP), intercellular adhesion molecule-1 (ICAM-1), IGFBP-1, IL-6, TSP-2, and vascular cell adhesion molecule-1 (VCAM-1). A statistical trend was also seen for increases of VEGF-A and placenta growth factor (PlGF) at EOS. Throughout treatment, sEnd levels significantly increased, an observation that was recapitulated in cultured endothelial cells. This is the first report of plasma-based biomarkers in patients receiving TRC105. TRC105 treatment by C2D1 was associated with decreases in several angiogenic factors, including Ang-2, PDGF isoforms, and VEGF isoforms, offering insight into the mechanisms underlying TRC105's anti-angiogenic, antitumor function. Increases in sEnd were the most significant of all observed biomarker changes and may reflect direct drug effects. Additionally, biomarker changes in response to TRC105 are distinct from those seen in patients treated with VEGF-targeting drugs, suggesting the possible utility of combining these two classes of angiogenesis inhibitors in patients.

Background Pazopanib (P) is approved for refractory advanced STS refractory. A retrospective study of 40 AS patients treated with P, reported a median progression-free survival (PFS) of 3.1 months and median overall survival (OS) 9.9 months (Kollar et al, Acta Oncol, 2017). Endoglin is an essential angiogenic receptor expressed on AS cells that is upregulated following VEGF inhibition. TRC105, an endoglin antibody, combined with P produced a median PFS of 7.8 months in chemotherapy-refractory and P-naive patients enrolled on a phase I-II trial. Methods TAPPAS was a randomized multicenter trial of TRC105/P vs P that enrolled cutaneous and non-cutaneous AS patients and incorporated an adaptive enrichment design. Key inclusion criteria: 0-2 prior lines of therapy, ECOG ≤ 1. The primary endpoint was PFS by RECIST 1.1 by independent radiographic review (+ cutaneous lesions by photography) (IRR). Secondary endpoints included PFS by investigator review (INV) and OS. An interim analysis to determine the final sample size was conducted following enrollment of 123 patients. Results Of 123 pts (TRC105/P, 62; P, 61), 60% were female, 89% were white; median age was 68 years (range: 24-82); 46% were ECOG PS 0; 50% had cutaneous disease; and 28% had no prior treatment. TRC105/P did not prolong median PFS or OS compared to P (Table). Most common all-grade adverse events (AEs) in TRC105/P vs P: fatigue (61% vs 55%); headache (64% vs 23%), diarrhea (57% vs 51%), nausea (48% vs 49%), vomiting (38% vs 23%), anemia (44% vs 9.4%), epistaxis (56% vs 3.8%) & hypertension (36% vs.55%). Table . 1667O P TRC105/P Median PFS, mo (95% CI), IRR 4.3 (2.9-NE) 4.2 (2.8-8.3) HR (95% CI) 0.98 (0.52-1.8) p-value 0.95 Median PFS, mo (95% CI), Investigator 2.9 (2.6-4.1) 3.9 (2.6-5.5) HR (95% CI) 0.77 (0.46-1.78) p-value 0.31 Median PFS, cutaneous, mo (95% CI), IRR 5.6 (2.6-5.6) 4.2 (2.8-8.3) HR (95% CI) 1.07 (0.43, 2.7) p-value 0.89 Overall Survival, mo (95% CI) 8.7 (7.4, NE) NE (6.8, NE) Odds ratio (95% CI) 0.90 (0.46, 1.74) p-value 0.74 Overall Survival, cutaneous, mo (95% CI) 8.0 (6.7, NE) NE (6.8, NE) Odds ratio (95% CI) 0.68 (0.25, 1.84) p-value 0.45 NE: not estimable; CI: confidence interval; mo: months Conclusions TRC105 did not demonstrate activity when combined with P in angiosarcoma. This was the 1st randomized phase III trial in angiosarcoma. These data provide a benchmark of the activity of P in 1st + 2nd-line setting in AS. A number of patients derived durable benefit from the combination schedule, indicating the heterogeneity of angiosarcomas. Clinical trial identification NCT 02979899. Legal entity responsible for the study Tracon Pharmaceuticals, Inc. Funding Tracon Pharmaceuticals, Inc. Disclosure R.L. Jones: Advisory / Consultancy: Tracon; Advisory / Consultancy: Adaptimmune; Advisory / Consultancy: Blueprint; Advisory / Consultancy: Clinigen; Advisory / Consultancy: Deciphera; Advisory / Consultancy: Daichii; Advisory / Consultancy: Eisai; Advisory / Consultancy: Epizyme; Advisory / Consultancy: PharmaMar; Advisory / Consultancy: Lilly; Advisory / Consultancy: Merck. L. Liu: Full / Part-time employment: Tracon. C. Theuer: Full / Part-time employment: Tracon. All other authors have declared no conflicts of interest.

Endoglin is a transforming growth factor β (TGF-β) coreceptor that serves as a prognostic, diagnostic and therapeutic vascular target in human cancer. A number of endoglin ectodomain-targeting antibodies (Abs) can effectively suppress both normal and tumor-associated angiogenesis, but their molecular actions remain poorly characterized. Here we define a key mechanism for TRACON105 (TRC105), a humanized monoclonal Ab in clinical trials for treatment of advanced or metastatic tumors. TRC105, along with several other endoglin Abs tested, enhance endoglin shedding through direct coupling of endoglin and the membrane-type 1 matrix metalloproteinase (MMP)-14 at the cell surface to release the antiangiogenic factor, soluble endoglin (sEng). In addition to this coupling process, endoglin shedding is further amplified by increased MMP-14 expression that requires TRC105 concentration-dependent c-Jun N-terminal kinase (JNK) activation. There were also notable counterbalancing effects on canonical Smad signaling in which TRC105 abrogated both the steady-state and TGF-β-induced Smad1/5/8 activation while augmenting Smad2/3 activation. Interestingly, TRC105-induced sEng and aberrant Smad signaling resulted in an excessive migratory response through enhanced stress fiber formation and disruption of endothelial cell–cell junctions. Collectively, our study defines endoglin shedding and deregulated TGF-β signaling during migration as major mechanisms by which TRC105 inhibits angiogenesis.

Endoglin, a transforming growth factor-beta co-receptor, is highly expressed on angiogenic endothelial cells in solid tumors. Therefore, targeting endoglin is currently being explored in clinical trials for anti-angiogenic therapy. In this project, the redundancy between endoglin and vascular endothelial growth factor (VEGF) signaling in angiogenesis and the effects of targeting both pathways on breast cancer metastasis were explored. In patient samples, increased endoglin signaling after VEGF inhibition was observed. In vitro TRC105, an endoglin-neutralizing antibody, increased VEGF signaling in endothelial cells. Moreover, combined targeting of the endoglin and VEGF pathway, with the VEGF receptor kinase inhibitor SU5416, increased antiangiogenic effects in vitro and in a zebrafish angiogenesis model. Next, in a mouse model for invasive lobular breast cancer, the effects of TRC105 and SU5416 on tumor growth and metastasis were explored. Although TRC105 and SU5416 decreased tumor vascular density, tumor volume was unaffected. Strikingly, in mice treated with TRC105, or TRC105 and SU5416 combined, a strong inhibition in the number of metastases was seen. Moreover, upon resection of the primary tumor, strong inhibition of metastatic spread by TRC105 was observed in an adjuvant setting. To confirm these data, we assessed the effects of endoglin-Fc (an endoglin ligand trap) on metastasis formation. Similar to treatment with TRC105 in the resection model, endoglin-Fc-expressing tumors showed strong inhibition of distant metastases. These results show, for the first time, that targeting endoglin, either with neutralizing antibodies or a ligand trap, strongly inhibits metastatic spread of breast cancer in vivo.

A systematic study of in vitro and in vivo behavior of biodegradable mesoporous silica nanoparticles (bMSNs), designed to carry multiple cargos (both small and macromolecular drugs) and subsequently self‐destruct following release of their payloads, is presented. Complete degradation of bMSNs is seen within 21 d of incubation in simulated body fluid. The as‐synthesized bMSNs are intrinsically radiolabeled with oxophilic zirconium‐89 (89Zr, t 1/2 = 78.4 h) radionuclide to track their in vivo pharmacokinetics via positron emission tomography imaging. Rapid and persistent CD105 specific tumor vasculature targeting is successfully demonstrated in murine model of metastatic breast cancer by using TRC105 (an anti‐CD105 antibody)‐conjugated bMSNs. This study serves to illustrate a simple, versatile, and readily tunable approach to potentially overcome the current challenges facing nanomedicine and further the goals of personalized nanotheranostics.