Kevin Hollevoet

In Vitro and In Vivo Activity of the Low-Immunogenic Antimesothelin Immunotoxin RG7787 in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, and new therapies are needed. RG7787 is a novel low-immunogenic antimesothelin recombinant immunotoxin (RIT), engineered to overcome the limitations of SS1P, a RIT now in clinical trials. In vitro activity was evaluated on five established PDAC cell lines (KLM-1, AsPC-1, BxPC-3, Panc 3.014, and PK-1) and on PDAC cells directly established from a patient tumor (GUMC108). RG7787 had subnanomolar IC50s in most cell lines, and was significantly more active than SS1P in GUMC108, KLM-1, and Panc 3.014 cells. GUMC108 was most sensitive, with RG7787 killing >99% of the cells. In a subcutaneous KLM-1 xenograft mouse model, two cycles of 3 × 2.5 mg/kg RG7787 QOD combined with two cycles of 1 × 50 mg/kg paclitaxel induced near-complete responses, with all tumors regressing below 5 mm3 within 30 days after therapy was initiated (>95% decrease) and no significant growth increase for at least another 3 weeks. RG7787 alone gave limited but significant regressions and paclitaxel by itself arrested tumor growth. Quantifying the uptake of Alexa Fluor 647–labeled RG7787 in tumors showed that the RIT reached only 45% of KLM-1 cells, accounting in part for the limited responses. Paclitaxel did not improve RG7787 uptake, which thus cannot explain the beneficial effect of the combination therapy. In conclusion, RG7787 has high cytotoxic activity on PDAC cell lines as well as on primary patient cells. In vivo, this novel RIT gives durable near-complete tumor responses when combined with paclitaxel. RG7787 merits further evaluation for the treatment of PDAC. Mol Cancer Ther; 13(8); 2040–9. ©2014 AACR.

Combining the antimesothelin immunotoxin SS1P with the BH3-mimetic ABT-737 induces cell death in SS1P-resistant pancreatic cancer cells.

SS1P is an antimesothelin recombinant immunotoxin (RIT). Pancreatic ductal adenocarcinoma (PDAC) cell lines are resistant to SS1P, despite high mesothelin expression. The aim of this study is to examine whether combining SS1P and BH3-mimetic ABT-737 induces cell death in a panel of PDAC cell lines. ABT-737 binds and neutralizes several antiapoptotic BCL2 family proteins, but has a low affinity for the short-lived MCL1 and BCL2A1. SS1P inhibits protein synthesis, which has shown to downregulate MCL1. PDAC cell lines KLM-1, BxPc-3, and Panc 3.014 were resistant to SS1P or ABT-737 alone. Combining both compounds led to a significant increase in cell death. After 48 hours of treatment, cell death was observed in 92% of KLM-1, 55% of BxPc-3, and 23% of Panc 3.014 cells. Panc 3.014 had the highest number of mesothelin-binding sites (92×103), followed by KLM-1 (58×103) and BxPc-3 (3×103). ABT-737 had no effect on SS1P internalization, but enhanced SS1P-induced protein synthesis inhibition significantly in KLM-1, to a lesser extent in BxPc-3, and very little in Panc 3.014. SS1P alone or in combination with ABT-737 downregulated MCL1 in KLM-1 and BxPc-3, but not in Panc 3.014. Similar observations were made for BCL2A1, which had the highest levels in Panc 3.014. Compared with KLM-1, Panc 3.014, and BxPc-3 also had lower proapoptotic BAK and a trend toward higher MCL1. Proapoptotic BAX was similar in KLM-1 and BxPc-3, but lower in Panc 3.014. In conclusion, combining SS1P with ABT-737 overcomes SS1P-resistance in PDAC, although to a variable extent. The efficacy of the combination is mainly associated with the RIT-associated inhibition of protein synthesis and the ability to downregulate MCL1 and BCL2A1, while levels of other key apoptotic proteins may also be important. Our data support the combination of an RIT and a BH3-mimetic, and identify factors that potentially limit the efficacy of such therapeutic approach.

Quantification of recombinant immunotoxin delivery to solid tumors allows for direct comparison of in vivo and in vitro results

Solid tumors present challenges for delivery of protein therapeutics; current methods cannot quantify the functional effects of these agents. RG7787 (anti-mesothelin recombinant immunotoxin) is highly cytotoxic to pancreatic cancer cell lines, but with limited activity in vivo. To investigate this discrepancy, we developed a flow cytometry method to quantify the amount of RG7787 internalized per cell in tumors and used it to analyze tumor responses by determining the number of molecules of RG7787 internalized per cell in vivo and comparing it to that needed to kill cells in vitro. At a maximum tolerated dose of 7.5 mg/kg, tumor cells in vivo internalized a wide range of RG7787 with the average amount equivalent to the amount that induced growth arrest in vitro. However, 20% of cells accumulated 20,300 ITs per cell, sufficient to kill cells in vitro. At 2.5 mg/kg the top 20% of cells internalized enough RG7787 to only induce growth arrest. These data are in agreement with tumor responses; 22% regression following a 7.5 mg/kg dose and growth stabilization following 2.5 mg/kg. Comparing amounts of RIT delivered in vivo and in vitro can explain tumor responses and should facilitate the development of more active immunotoxins and other antibody based agents.

State of play and clinical prospects of antibody gene transfer

Recombinant monoclonal antibodies (mAbs) are one of today’s most successful therapeutic classes in inflammatory diseases and oncology. A wider accessibility and implementation, however, is hampered by the high product cost and prolonged need for frequent administration. The surge in more effective mAb combination therapies further adds to the costs and risk of toxicity. To address these issues, antibody gene transfer seeks to administer to patients the mAb-encoding nucleotide sequence, rather than the mAb protein. This allows the body to produce its own medicine in a cost- and labor-effective manner, for a prolonged period of time. Expressed mAbs can be secreted systemically or locally, depending on the production site. The current review outlines the state of play and clinical prospects of antibody gene transfer, thereby highlighting recent innovations, opportunities and remaining hurdles. Different expression platforms and a multitude of administration sites have been pursued. Viral vector-mediated mAb expression thereby made the most significant strides. Therapeutic proof of concept has been demonstrated in mice and non-human primates, and intramuscular vectored mAb therapy is under clinical evaluation. However, viral vectors face limitations, particularly in terms of immunogenicity. In recent years, naked DNA has gained ground as an alternative. Attained serum mAb titers in mice, however, remain far below those obtained with viral vectors, and robust pharmacokinetic data in larger animals is limited. The broad translatability of DNA-based antibody therapy remains uncertain, despite ongoing evaluation in patients. RNA presents another emerging platform for antibody gene transfer. Early reports in mice show that mRNA may be able to rival with viral vectors in terms of generated serum mAb titers, although expression appears more short-lived. Overall, substantial progress has been made in the clinical translation of antibody gene transfer. While challenges persist, clinical prospects are amplified by ongoing innovations and the versatility of antibody gene transfer. Clinical introduction can be expedited by selecting the platform approach currently best suited for the mAb or disease of interest. Innovations in expression platform, administration and antibody technology are expected to further improve overall safety and efficacy, and unlock the vast clinical potential of antibody gene transfer.

Fibulin-3 as a biomarker for pleural mesothelioma.

n engl j med 368;2 nejm.org january 10, 2013 189 ization. Recently published results of a substudy of TRILOGY ACS on platelet function showed a lower frequency of high on-treatment platelet reactivity with prasugrel than with clopidogrel but no independent association between on-treatment platelet reactivity and ischemic outcomes.1 Dedicated pharmacogenomics analyses involving a portion of the trial population are under way. As further analyses from the trial are completed, we believe that a story will unfold that will provide insights into the mechanism underlying the main trial results and inform treatment options for patients with acute coronary syndromes who are treated without revascularization. Matthew T. Roe, M.D., M.H.S. E. Magnus Ohman, M.B., Ch.B.

Methylation-Associated Partial Down-Regulation of Mesothelin Causes Resistance to Anti-Mesothelin Immunotoxins in a Pancreatic Cancer Cell Line

Anti-mesothelin Pseudomonas exotoxin A-based recombinant immunotoxins (RITs) present a potential treatment modality for pancreatic ductal adenocarcinoma (PDAC). To study mechanisms of resistance, the sensitive PDAC cell line KLM-1 was intermittently exposed to the anti-mesothelin SS1-LR-GGS RIT. Surviving cells were resistant to various anti-mesothelin RITs (IC50s >1 μg/ml), including the novel de-immunized RG7787. These resistant KLM-1-R cells were equally sensitive to the anti-CD71 HB21(Fv)-PE40 RIT as KLM-1, indicating resistance was specific to anti-mesothelin RITs. Mesothelin gene expression was partially down-regulated in KLM-1-R, resulting in 5-fold lower surface protein levels and decreased cellular uptake of RG7787 compared to KLM-1. Bisulfite sequencing analysis found that the mesothelin promoter region was significantly more methylated in KLM-1-R (59 ± 3.6%) compared to KLM-1 (41 ± 4.8%), indicating hypermethylation as a mechanism of mesothelin downregulation. The DNA methyltransferase inhibitor 5-azacytidine restored original mesothelin surface expression to more than half in KLM-1-R and increased sensitivity to RG7787 (IC50 = 722.4 ± 232.6 ng/ml), although cells remained significantly less sensitive compared to parental KLM-1 cells (IC50 = 4.41 ± 0.38 ng/ml). Mesothelin cDNA introduction in KLM-1-R led to 5-fold higher surface protein levels and significantly higher RG7887 uptake compared to KLM-1. As a result, the original sensitivity to RG7787 was fully restored (IC50 = 4.49 ± 1.11 ng/ml). A significantly higher RG7787 uptake was thus required to reach the original cytotoxicity in resistant cells, hinting that intracellular RIT trafficking is also a limiting factor. RNA deep sequencing analysis of KLM-1 and KLM-1-R cells supported our experimental findings; compared to KLM-1, resistant cells displayed differential expression of genes linked to intracellular transport and an expression pattern that matched a more general hypermethylation status. In conclusion, resistance to anti-mesothelin RITs in KLM-1 is linked to a methylation-associated down-regulation of mesothelin, while aberrations in RIT trafficking could also play a role.

Mesothelin-targeted immunotoxin RG7787 has synergistic anti-tumor activity when combined with taxanes

Recombinant immunotoxins (RITs) are antibody-based therapeutics that carry a toxin payload. The RG7787 RIT targets the cancer antigen mesothelin to deliver a recombinantly-engineered, reduced immunogenicity variant of Pseudomonas exotoxin A (PE) to the cytosol where it inhibits protein synthesis. Here we demonstrate that maximal doses of RG7787 temporarily halt growth of pancreatic cancer tumor xenografts, similar to the approved drugs gemcitabine and nab-paclitaxel, however, combination of the RIT with nab-paclitaxel produces durable complete regressions in most mice. Synergy between taxane and anti-MSLN RITs has been previously demonstrated in mouse models, but direct interaction of the combination in cell culture was not observed. Here, we show that this favorable interaction occurs in cell culture, is dependent on the dose and duration of RG7787 exposure, requires the catalytically active PE, and still occurs with RIT targeting a non-MSLN surface antigen. Unexpectedly, the combination does not increase RG7787-mediated protein synthesis inhibition nor perturb downstream apoptotic markers of RIT-mediated killing, but does augment levels of acetylated tubulin, a marker of taxane activity. Taken together, these data suggest that PE increases cell sensitivity to taxane-mediated killing by increasing taxane-mediated microtubule stability and priming cells for apoptosis by decreasing levels of the pro-survival factor Mcl-1.

Long-term in vivo expression of trastuzumab following intramuscular electrotransfer of the encoding DNA in mice

In vivo antibody expression is at crossroads between monoclonal antibody (mAb) and gene therapy. Following a single intramuscular injection of the DNA that encodes a therapeutic mAb, the muscle is turned into a ‘bioreactor’, resulting in prolonged mAb secretion in circulation [1-3]. This innovative approach addresses several challenges associated with conventional mAb proteins. In R&D, in vivo mAb expression allows production and evaluation of leads directly into animal models. In the clinic, it can improve treatment efficacy and patient comfort by avoiding repeated high-dose injections, and provide a cost-effective answer to the increasing need for mAb combination therapies - in the field of cancer immunotherapy and beyond. This study outlines the development and delivery of a trastuzumab-encoding plasmid for in vivo mAb expression. To improve intramuscular DNA delivery in mice, we first established an optimal electrotransfer protocol using novel reporter plasmids. Following the optimized intramuscular electrotransfer of the trastuzumab-encoding plasmid, trastuzumab was detected with a commercial ELISA at therapeutically relevant concentrations (1-15 µg/ml) in the sera of athymic nude mice (n=16) for the full duration of the ongoing follow-up (>3 months). A cell viability assay demonstrated similar activity of the expressed versus commercial trastuzumab in the BT-4747 breast cancer cell line. In conclusion, we achieved proof of concept for the long-term in vivo expression of biologically active trastuzumab in mice. Ongoing work focuses on optimizing in vivo mAb expression for clinical application and evaluation for combination therapy.

Prolonged in vivo expression and anti-tumor response of DNA-based anti-HER2 antibodies

Antibody gene transfer presents an appealing alternative to conventional antibody protein therapy. This pre-clinical study evaluates the impact of various parameters on the pharmacokinetics and efficacy of in vivo expressed DNA-based anti-HER2 monoclonal antibodies (mAbs), newly engineered and delivered via intramuscular electrotransfer in mice. Plasma concentrations of trastuzumab and 4D5, its murine IgG1 equivalent, peaked on average between 1–15 µg/ml, depending on the administration and configuration of the encoding plasmid DNA (pDNA). A dual expression cassette system outperformed a single 2A-based cassette, and the CAG promoter was superior to a muscle-specific ΔUSE-based promoter. A ‘gene therapy-compatible’ Gene Transport Unit (gtGTU, FIT Biotech), a plasmid backbone that co-encodes viral elements, failed to improve in vivo reporter and mAb expression compared to a conventional plasmid. In BALB/c mice, trastuzumab detection was lost within two weeks after pDNA administration due to anti-drug antibodies. This host immune response was addressed by expressing trastuzumab in immune-compromised mice, or by gene transfer of murine 4D5 in BALB/c mice. Both approaches maintained single-digit µg/ml mAb concentrations for at least six to nine months, and allowed to boost mAb expression over time by pDNA re-dosing. In a breast cancer mouse model, prophylactic and therapeutic DNA-based trastuzumab or 4D5 led to complete tumor regressions, thereby rivalling with the administration of milligrams of mAb protein. In conclusion, our study demonstrates proof of concept for antibody gene transfer in cancer, provides critical insights in the engineering and application of DNA-based antibodies, and serves to advance this modality in oncology and beyond.

Abstract 4505: Anti-tumor activity in pancreatic cancer of a low immunogenic and clinically optimized anti-mesothelin immunotoxin RG7787

Background Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis and new therapies are needed. SS1P is a recombinant immunotoxin (RIT) containing an anti-mesothelin dsFv fused to a cytotoxic fragment of Pseudomonas Exotoxin A (PE). In clinical trials, patients developed dose-limiting neutralizing anti-drug antibodies within the first cycle of treatment. RG7787 is a next generation, clinically optimized RIT with decreased immunogenicity and increased resistance to lysosomal proteases. In this study, we evaluated the in vitro and in vivo activity of RG7787 in PDAC. Method Five established PDAC cell lines (KLM-1, AsPC-1, BxPC-1, BxpC3, Panc 3.014, and PK-1) and one primary PDAC line directly obtained from a patient tumor (GUMC108) were used. Cell viability and proliferation were calculated by measuring ATP levels with Cell Titer-Glo assays. In vivo activity was evaluated in a subcutaneous KLM-1 mouse tumor model. Delivery of fluor-labeled RG7787 to mouse tumors was assessed by flow cytometry. Results RG7787 had significant in vitro activity in the PDAC cell lines tested with most IC50 values in the subnanomolar range. It was significantly more active than SS1P in KLM-1, Panc 3.014 and GUMC108, with similar activity in the other cell lines. GUMC108 (IC50 = 18.9 pM) was the most sensitive cell line. In KLM-1 cells, SS1P and RG7787 had similar internalization rates. RG7787 could be safely administered to mice at a dose of 2.5 mg/ kg IV QOD x3, which is more than five times the maximum tolerated murine dose of SS1P. In vivo uptake studies in a KLM-1 subcutaneous mouse xenograft model demonstrated intracellular delivery of RG7787-Alexa647 to 50% of tumor cells after a single 2.5 mg/kg IV dose. Tumor volume was reduced by 13% after a single cycle of RG7787 treatment (n=6). Two cycles of combination treatment with paclitaxel (50 mg/kg IP) and RG7787 shrank tumors to Conclusions RG7787 has superior or similar activity compared to SS1P in established PDAC cell lines and a primary PDAC cell line in vitro. In a KLM-1 mouse xenograft model, the combination of paclitaxel with RG7787 cured 18.1% of mice and reduced tumor size by more than 90% in all other mice treated. RG7787 merits further development for the treatment of pancreatic cancer. Citation Format: Ira Pastan, Kevin Hollevoet, Emily Mason-Osann, Christine Alewine, Xiu-Fen Liu. Anti-tumor activity in pancreatic cancer of a low immunogenic and clinically optimized anti-mesothelin immunotoxin RG7787. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4505. doi:10.1158/1538-7445.AM2014-4505