Improvement In Therapeutic Index Research Articles

A CD79b Targeting ADC with Superior Anti-Tumor Activity and Safety Resulting in Significantly Improved Therapeutic Index (TI): Safe and Efficacious CD79b ADC

The Araris' site-specific and 1-step enzymatic linker conjugation technology aims at generating safe and highly potent ADCs without the need for antibody engineering prior to linker-payload conjugation. We developed a very stable anti-CD79b-MMAE ADC (ARC-02) with this technology showing a higher therapeutic index (TI) compared to the approved ADC polatuzumab-vedotin (Polivy®) in different preclinical models. Therefore, ARC-02 is considered a promising next generation treatment alternative for patients suffering from Non-Hodgkin lymphoma (NHL). Using native polatuzumab as the targeting antibody and monomethyl auristatin E (MMAE) as payload, we generated within 24 hours the highly homogeneous and pure ARC-02 development candidate with a well-defined drug-to-antibody ratio (DAR) of 1.9 and with high purity (monomer content > 98%). ARC-02 is highly stable under stressed conditions at elevated temperatures and maintains the FcyR/FcRn-binding properties of the parental mAb. In-vitro ARC-02 demonstrated potent cytotoxicity in four different NHL cell lines tested so far with similar potency (EC50 values) compared to the approved Polivy® ADC despite of the lower toxin load of ARC-02 (DAR of 1.9) compared to Polivy® (DAR of 3.5). ARC-02 clearly differentiates from Polivy® by extraordinary stability in mouse, cynomolgus monkey and human sera exemplified by the absence of payload deconjugation or linker cleavage under physiological conditions (at 37°C) and long-term incubation (>2 weeks). Exceptional stability could also be confirmed in vivo in pharmacokinetic studies in mice, rats and cynomolgus monkeys demonstrating an antibody-like exposure profile comparable to the unmodified polatuzumab parental antibody without premature toxin deconjugation in the circulation resulting in improved pharmacokinetics of ARC-02 compared to Polivy® (half-life in mice 10 days for ARC-02 vs 5 days for Polivy®). Despite the excellent stability of ARC-02 in vitro and in vivo, the MMAE toxin is still efficiently released from ARC-02 in lysosomal human Cathepsin B cleavage or human liver-lysosome release assays which is providing the foundation for the observed robust anti-tumor activity in vivo: ARC-02 was compared head-to-head to Polivy® in two different CD79b-expressing established tumor models in vivo (Granta-519 and Ramos). Single dose treatment of ARC-02 at very low payload doses induced effective and long-lasting anti-tumor responses with complete tumor eradication achieved at about half of the Polivy® payload dose in both NHL established tumor models. Based on these in vivo efficacy experiments, the minimal effective dose (MED) of ARC-02 that is still inducing eradication of established tumors is determined to be as low as 10 μg/kg based on the MMAE payload dose or 1 mg/kg based on the total ADC dose. Finally, the highest non-severely toxic dose (HNSTD) of ARC-02 in repeat dose toxicology studies was determined to be ≥30 mg/kg and ≥24 mg/kg in rats and cynomolgus monkeys, respectively, which is substantially higher than the HNSTD reported for Polivy® (i.e. 10 mg/kg in the rat and 3 mg/kg in the cynomolgus monkey as described in the Polivy® Biologic License Application). Therefore, the tolerability in rodents and non-human primates is at least 3 and 8 times higher when compared to historical safety data of Polivy®. Using the HNSTD derived from repeat dose non-human primate toxicity study (24 mg/kg) and the MED calculated from single dose rodent efficacy studies (1 mg/kg) an impressive TI of 24 can be calculated for ARC-02 which is substantially higher than the TI of Polivy®. These encouraging results obtained so far indicate that ARC-02 a) has very favorable biophysical properties, b) shows a consistent and highly defined DAR, c) is highly stable in vitro and in vivo, d) is highly potent and efficacious in multiple tumor models and e) has improved tolerability in rodents and non-human primates and f) shows an impressive improvement in TI and ultimately warrant further development of ARC-02.

Design, Synthesis, and Structure-Activity Relationship Studies of Bisamide Derivatives of Amphotericin B with Potent Efficacy and Low Toxicity.

Amphotericin B (AMB, 1) is the most powerful antibiotic in treating potentially life-threatening invasive fungal infections (IFIs), though severe toxicity derived from self-aggregation greatly limits its clinical application. Herein, we applied a bisamidation strategy at the C16-COOH and C3'-NH2 to improve the therapeutic properties by suppressing self-aggregation. It was found that basic amino groups at the residue of C16 amide were beneficial to activity, while lipophilic fragments contributed to toxicity reduction. Additionally, N-methyl-amino acetyl and amino acetyl moieties at C3' amide could help keep the fungistatic effectiveness. The modification work culminated in the discovery of 36 (ED50 = 0.21 mg/kg), which exerted a 1.5-fold stronger antifungal efficacy than amphamide, the optimal derivative theretofore, in mice, low self-aggregation propensity, and thus low acute toxicity. With the improvement in therapeutic index and good PK profile, 36 is promising for further development as a second-generation polyene antifungal agent.

Abstract 2910: A CD79b targeting ADC with superior anti-tumor activity and therapeutic index

Abstract The Araris’ site-specific and 1-step linker conjugation technology aims at generating safe and highly potent ADCs without the need for antibody engineering prior to linker-payload conjugation. We developed a very stable anti-CD79b-MMAE ADC with this technology showing a higher therapeutic index compared to polatuzumab-vedotin in preclinical models. Our ADC may represent a safe and efficacious alternative for the treatment of patients with diffuse-large B-cell lymphoma (DLBCL).Using native polatuzumab (non-engineered, same antibody sequence as present in approved polatuzumab-vedotin) as the targeting antibody and monomethyl auristatin E (MMAE) as payload, we generated within 24hours highly homogeneous and pure ADCs with a well-defined drug-to-antibody ratio (DAR) of 1.9, with a &gt 98% monomer content. The ADC is highly stable under stressed conditions at elevated temperatures and maintains the FcyR/FcRn-binding properties of the parental mAb. In in-vitro assays our ADC demonstrated potent cytotoxicity in four tested cell-lines, similar to the approved polatuzumab-vedotin (Polivy®). Moreover, our anti-CD79b ADC (ARADC) is highly stable in mouse, cynomolgus and human sera exemplified by the absence of payload deconjugation or linker cleavage. Though highly stable, the ADC is still efficiently released by lysosomal human Cathepsin B cleavage or human liver-lysosome enzymes. Most importantly, the resulting ADC is extremely stable in circulation as shown in pharmacokinetic studies in mice and rats demonstrating an antibody-like exposure profile comparable to the unmodified polatuzumab antibody and twice as long as the approved polatuzumab-vedotin (half-life 10d vs 5d).Most importantly, the in vivo efficacy of ARADC (DAR 1.9) was compared with approved polatuzumab-vedotin (DAR 3.5) in two CD79b-expressing tumor models: Granta-519 and Ramos. ARADC provided equal tumor growth inhibition and survival at about half the payload dose relative to polatuzumab-vedotin in both models. At approximately equal payload doses, ARADC treatment led to greater antitumor effects and a considerably longer survival advantage over polatuzumab-vedotin in both models. Finally, the highest non-severely toxic dose (HNSTD) of ARADC was determined at 30mg/kg in a 4-week repeat dose toxicology study in rats. This observation, together with the high anti-tumor potency at low dose - the minimal effective dose (MED), results in an overall 4-6-fold increased therapeutic index (TI).These encouraging results obtained so far indicate that ARADC a) has very favorable biophysical properties, b) shows a clearly defined drug-to-antibody ratio, c) is highly stable in vitro and in vivo, d) is highly potent and efficacious in multiple tumor models and e) showed an improvement in TI by a factor of 4-6 and ultimately warrant further development of ARADC. Citation Format: Isabella Attinger-Toller, Philipp Probst, Romain Bertrand, Ramona Stark, Roger Santimaria, Emma Renard, Rachael Fay, Dragan Grabulovski, Bernd Schlereth, Philipp René Spycher. A CD79b targeting ADC with superior anti-tumor activity and therapeutic index [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2910.

Tumor-selective activity of XTX202, a protein-engineered IL-2, in mice without peripheral toxicities in nonhuman primates.

2563 Background: High-dose recombinant human interleukin-2 (aldesleukin) elicits anti-tumor immunity and is approved for the treatment of renal cell carcinoma and melanoma based on durable complete remissions. However, use of aldesleukin is limited due to treatment-related life-threatening toxicities. Recent second-generation efforts to alleviate toxicities have largely focused on eliminating binding to IL-2Rα, often with half-life extension. We have determined that mice and non-human primates (NHPs) treated with a second generation IL-2 surrogate still experience characteristic dose-limiting toxicities, including vascular leak syndrome. To overcome these toxicities and improve the therapeutic index (TI) of IL-2 as an anti-tumor immunotherapy, we employed protein engineering to generate XTX202, a highly potent third generation masked IL-2. XTX202 is unmasked in the tumor microenvironment by proteolytic activation resulting in full restoration of binding to IL-2Rβ without binding to IL-2Rα. The current study characterizes the therapeutic index of XTX202 versus aldesleukin and a second generation IL-2 surrogate. Methods: XTX202 bioactivity was measured using STAT-5 phosphorylation in human PBMCs and reporter cell lines. Anti-tumor efficacy and peripheral immune activation were evaluated in mice bearing syngeneic tumor models. Safety was evaluated in rodents and Cynomolgus monkeys. XTX200, an unmasked half-life extended IL-2 that does not bind to IL-2Rα, was used as a surrogate second generation IL-2. Results: Masked XTX202 showed limited IL-2R-dependent STAT-5 signaling in vitro. Proteolytic activation of XTX202 resulted in CD8+ T and NK cell activation and over 1000-fold reduction in Treg activation as compared to WT IL-2. XTX202 achieved potent tumor growth inhibition in syngeneic mouse models as a single agent with no evidence of toxicity or peripheral immune activation, thus demonstrating tumor selective activity. XTX202 efficacy in mice at 2 mg/kg dose was equivalent to that achieved with the MTD dose of 0.5 mg/kg of a second generation IL-2 surrogate. XTX202 was well tolerated in NHPs in a 4-week repeat dose study at doses up to 30 mg/kg QW whereas a second generation IL-2 surrogate was not tolerated beyond 0.7 mg/kg QW. Based on these data, XTX202 has a 10 fold improvement in TI vs second generation IL-2. Based on comparative efficacy studies with aldesleukin and literature NHP tolerability data, XTX202 is projected to have a ≥150 fold greater TI than aldesleukin. Conclusions: XTX202, a third generation, tumor-selective IL-2, inhibits tumor growth and is well tolerated in repeat dose studies in NHPs at high doses. GLP toxicity studies with XTX202 are underway and first-in-human studies are expected to initiate this year. XTX202 has the potential to be a best-in-class IL-2 immunotherapy by expanding the curative anti-tumor activity of IL-2 while minimizing dose-limiting toxicities.

Improvement of Therapeutic Index by the Combination of Enhanced Peptide Cationicity and Proline Introduction.

Antimicrobial peptides (AMPs) are promising candidates for new therapeutics to combat the emergence of an increasing number of multidrug-resistant pathogens. However, a major obstacle to the systemic application of AMPs is their possible toxicity. In this study, we improved the therapeutic index of the typical AMP F5W-magainin 2 by simultaneously introducing positive charges (+9-+10) and Pro residues. The former and latter contributed to enhanced antimicrobial activity and reduced cytotoxicity, respectively. The results were sensitive to the positions of Pro substitution. The antimicrobial mechanism was considered to involve both membrane permeabilization and DNA binding. The latter was affected by the peptide charge but not the presence of Pro. The neutralization of lipopolysaccharides, another important role of AMPs, was not very sensitive to either the peptide charge or Pro introduction. This strategy using intrinsic amino acids is also promising from the viewpoints of the economic mass production of AMPs and safety of metabolized peptides.

Reducing the antigen-independent toxicity of antibody-drug conjugates by minimizing their non-specific clearance through PEGylation

Recently, we described a family of non-targeting monomethylauristatin E (MMAE) antibody-drug conjugates (ADCs) whose pharmacokinetics could be tuned through incorporation of a short polyethylene glycol (PEG) moiety of up to twelve units into a drug-linker to render the ADC surface more hydrophilic. That work demonstrated that more hydrophilic ADCs were simultaneously more effective and better tolerated in mouse models, suggesting an improvement in therapeutic index via this strategy. Here, we describe the biodistribution and toxicology assessments in Sprague-Dawley rats after intravenous dosing with the aim of elucidating the relationships between these biological outcomes and the underlying physicochemical properties of non-targeted ADCs. Dosing a non-PEGylated ADC exhibited rapid nonspecific cellular uptake, leading to ADC catabolism and rapid release of the cytotoxic payload which reached peak plasma and tissue concentrations within the first day. Introduction of a PEG chain of four, eight, or twelve units resulted in increasingly slower uptake and decreases in peak payload concentrations in all tissues. These ADCs with minimal non-specific uptake also exhibited substantially less hematologic toxicity, with reduced histologic depletion of bone marrow and less dramatic decreases and/or more rapid recovery in peripheral hematologic cell counts (neutrophils, platelets, and reticulocytes). These results support a strong correlation between ADC hydrophobicity, rate of non-specific uptake, peak tissue concentration of released payload, and resulting toxicology parameters. Should these correlations be translatable to the clinic, this would provide a more general and highly tractable strategy for reducing the antigen-independent toxicity of ADCs through drug-linker design to modulate non-specific biodistribution.

Understanding the effect of controlling phosphorothioate chirality in the DNA gap on the potency and safety of gapmer antisense oligonucleotides.

Therapeutic oligonucleotides are often modified using the phosphorothioate (PS) backbone modification which enhances stability from nuclease mediated degradation. However, substituting oxygen in the phosphodiester backbone with sulfur introduce chirality into the backbone such that a full PS 16-mer oligonucleotide is comprised of 215 distinct stereoisomers. As a result, the role of PS chirality on the performance of antisense oligonucleotides (ASOs) has been a subject of debate for over two decades. We carried out a systematic analysis to determine if controlling PS chirality in the DNA gap region can enhance the potency and safety of gapmer ASOs modified with high-affinity constrained Ethyl (cEt) nucleotides in the flanks. As part of this effort, we examined the effect of systematically controlling PS chirality on RNase H1 cleavage patterns, protein mislocalization phenotypes, activity and toxicity in cells and in mice. We found that while controlling PS chirality can dramatically modulate interactions with RNase H1 as evidenced by changes in RNA cleavage patterns, these were insufficient to improve the overall therapeutic profile. We also found that controlling PS chirality of only two PS linkages in the DNA gap was sufficient to modulate RNase H1 cleavage patterns and combining these designs with simple modifications such as 2′-OMe to the DNA gap resulted in dramatic improvements in therapeutic index. However, we were unable to demonstrate improved potency relative to the stereorandom parent ASO or improved safety over the 2′-OMe gap-modified stereorandom parent ASO. Overall, our work shows that while controlling PS chirality can modulate RNase H1 cleavage patterns, ASO sequence and design are the primary drivers which determine the pharmacological and toxicological properties of gapmer ASOs.

Baicalein enhances the antitumor efficacy of docetaxel on nonsmall cell lung cancer in a β-catenin-dependent manner.

The side effects of docetaxel have limited its antitumor performances in the treatment of nonsmall cell lung cancer (NSCLC). To address the problem, baicalein, a bioactive flavone that exhibits antitumor activity, was combined with docetaxel so as to achieve better efficacy and lower toxicity. The combination treatment enhanced the stabilization of microtubules and halted the cell-cycle progression, thus synergistically inhibiting the proliferation and inducing the apoptosis of A549 cells and Lewis lung carcinoma cells. The decreased expression of Cyclin-dependent kinase 6 and Cyclin B1 confirmed its regulation in cell cycle, with β-catenin being an important upstream effector, as evidenced by the decreased expression in the cytoplasm and nucleus as well as the attenuated aggregation in the nucleus. Furthermore, baicalein plus docetaxel evinced better antitumor efficacy by the suppressed tumor growth, increased apoptosis, and decreased tumor angiogenesis in vivo, with no increased toxicity discovered in both tumor-bearing and non-tumor-bearing mice, and an improvement in therapeutic index. This study has demonstrated that baicalein plus docetaxel is an appropriate combination simultaneously with augmented antitumor efficacy and acceptable safety, which might be a promising strategy for patients with advanced NSCLC.

Moving toward Noninvasive, Focused Ultrasound Therapeutic Delivery of Drugs in the Brain: Prolonged Opening of Blood-Brain Barrier May Not Be Needed.

Moving toward Noninvasive, Focused Ultrasound Therapeutic Delivery of Drugs in the Brain: Prolonged Opening of Blood-Brain Barrier May Not Be Needed.

Abstract 3815: Decomposition of parameters contributing to the improved therapeutic index of ADCs obtained by GlycoConnect™ and HydraSpace™ Technologies

Abstract We have found that conjugation of toxic payloads to the native glycan of a monoclonal antibody by chemoenzymatic remodeling (GlycoConnect™ technology) consistently provides antibody-drug conjugates (ADCs) with enhanced therapeutic index (TI) increases versus those of ADCs prepared by mainstream clinical technologies. For example, head-to-head comparison of GlycoConnect™ ADCs -based on the same antibody and payload components- with the marketed drugs Adcetris and Kadcyla revealed an improvement in both efficacy and safety, which is further enhanced by use of a polar HydraSpace™ technology. Importantly, a similar improvement in TI was noted for comparison of a GlycoConnect™ ADC versus or a site-specific ADC derived from a cysteine-engineered antibody, the most important emerging technology in the clinic. With the aim to better understand the superiority of ADCs based on combined GlycoConnect™ and HydraSpace™ technologies, we have performed in-depth in vitro and in vivo investigation into factors contributing to the overall performance of ADCs prepared by glycan conjugation with a polar spacer. For example, it is found that stability is significantly improved versus mainstream technologies in terms of aggregation and linker stability. Rapid aggregation was observed for both Kadcyla and Adcetris, as well as ADC decomposition due to retro-Michael reaction resulting in free payload (for Adcetris) or albumin-conjugated DM1 (for Kadcyla), as indicated by LC-MS and immunoassay analysis. The latter image is confirmed by in vivo pharmacokinetic analysis, showing a dramatic difference in total antibody versus total conjugated antibody by premature release of payload. Finally, analysis with a solid 3D tumoroid model indicates enhanced penetration of site-specific versus randomly conjugated ADC, which may also contribute to better efficacy in solid tumors. The data generated further underline the importance of conjugation and linker technology as critical quality attributes for next-generation ADCs with higher therapeutic index. Citation Format: Floris van Delft, Brian Janssen, Remon van Geel, Marloes Wijdeven, Jorge Verkade, Sander van Berkel. Decomposition of parameters contributing to the improved therapeutic index of ADCs obtained by GlycoConnect™ and HydraSpace™ Technologies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3815.

Dose evaluation of Grid Therapy using a 6 MV flattening filter-free (FFF) photon beam: A Monte Carlo study.

Spatially fractionated radiotherapy is a strategy to overcome the main limitation of radiotherapy, i.e., the restrained normal tissue tolerances. A well-known example is Grid Therapy, which is currently performed at some hospitals using megavoltage photon beams delivered by Linacs. Grid Therapy has been successfully used in the management of bulky abdominal tumors with low toxicity. The aim of this work was to evaluate whether an improvement in therapeutic index in Grid Therapy can be obtained by implementing it in a flattening filter-free (FFF) Linac. The rationale behind is that the removal of the flattening filter shifts the beam energy spectrum towards lower energies and increase the photon fluence. Lower energies result in a reduction of lateral scattering and thus, to higher peak-to-valley dose ratios (PVDR) in normal tissues. In addition, the gain in fluence might allow using smaller beams leading a more efficient exploitation of dose-volume effects, and consequently, a better normal tissue sparing. Monte Carlo simulations were used to evaluate realistic dose distributions considering a 6MV FFF photon beam from a standard medical Linac and a cerrobend mechanical collimator in different configurations: grid sizes of 0.3×0.3cm2 , 0.5×0.5cm2 , and 1×1cm2 and a corresponding center-to-center (ctc) distance of 0.6, 1, and 2cm, respectively (total field size of 10×10cm2 ). As figure of merit, peak doses in depth, PVDR, output factors (OF), and penumbra values were assessed. Dose at the entrance is slightly higher than in conventional Grid Therapy. However, it is compensated by the large PVDR obtained at the entrance, reaching a maximum of 35 for a grid size of 1×1cm2 . Indeed, this grid size leads to very high PVDR values at all depths (≥10), which are much higher than in standard Grid Therapy. This may be beneficial for normal tissues but detrimental for tumor control, where a lower PVDR might be requested. In that case, higher valley doses in the tumor could be achieved by using an interlaced approach and/or adapting the ctc distance. The smallest grid size (0.3×0.3cm2 ) leads to low PVDR at all depths, comparable to standard Grid Therapy. However, the use of very thin beams might increase the normal tissue tolerances with respect to the grid size commonly used (1×1cm2 ). The gain in fluence provided by FFF implies that the important OF reduction (0.6) will not increase treatment time. Finally, the intermediate configuration (0.5×0.5cm2 ) provides high PVDR in the first 5cm, and comparable PVDR to previous Grid Therapy works at depth. Therefore, this configuration might allow increasing the normal tissue tolerances with respect to Grid Therapy thanks to the higher PVDR and thinner beams, while a similar tumor control could be expected. The implementation of Grid Therapy in an FFF photon beam from medical Linac might lead to an improvement of the therapeutic index. Among the cases evaluated, a grid size of 0.5×0.5cm2 (1-cm-ctc) is the most advantageous configuration from the physics point of view. Radiobiological experiments are needed to fully explore this new avenue and to confirm our results.

Role of positively charged residues on the polar and non-polar faces of amphipathic α-helical antimicrobial peptides on specificity and selectivity for Gram-negative pathogens.

We have designed de novo and synthesized eight 26-residue all D-conformation amphipathic α-helical cationic antimicrobial peptides (AMPs), four with "specificity determinants" which provide specificity for prokaryotic cells over eukaryotic cells and four AMPs without specificity determinants. The eight AMPs contain six positively charged Lys residues on the polar face in four different arrangements to understand the role of these residues have on antimicrobial activity against 14 Acinetobacter baumannii strains, seven of which were resistant to polymyxin B and colistin; six diverse Pseudomonas aeruginosa strains and 17 Staphylococcus aureus strains, nine of which were methicillin-sensitive, and eight of which were methicillin-resistant. The four AMPs without specificity determinants are extremely hemolytic. In contrast, the four AMPs with specificity determinants had dramatic improvements in therapeutic indices showing the importance of specificity determinants in removing eukaryotic cell toxicity. The specificity determinants combined with the location of positively charged residues on the polar face provide Gram-negative pathogen selectivity between A. baumannii and S. aureus. Specificity determinants maintain excellent antimicrobial activity in the presence of human sera, whereas the AMPs without specificity determinants were inactive. This study clearly shows the potential of amphipathic α-helical AMPs with specificity determinants as therapeutics to replace existing antibiotics.

Abstract PR10: Significant improvements in therapeutic index for conjugated payloads using a nanoparticle-drug conjugate (NDC) platform to provide sustained drug release and potentially improved anticancer effects

Abstract Cerulean Pharma Inc.'s Dynamic Tumor Targeting™ Platform creates nanoparticle-drug conjugates (NDCs) designed to significantly mitigate a payload's limitations by providing sustained drug delivery to the tumor and superior therapeutic index through controlled release kinetics. By conjugating drug payloads to our novel β-cyclodextrin-PEG copolymer through linker strategies that allow modulation of release and pharmacokinetics (PK), we provide advantages over entrapped nanoparticle strategies, e.g., polymeric nanoparticles and liposomes. Cerulean has two NDCs in the clinic, CRLX101 and CRLX301, evidencing the translatability of our technology. CRLX101 has been dosed in over 350 patients and CRLX301 is in an ongoing Phase 1/2a trial. Our Dynamic Tumor Targeting Platform is applicable to a diverse range of drug payload possibilities, including small molecules with a host of physiochemical properties, i.e., hydrophobic and hydrophilic payloads, and functional groups with chemical handles for conjugation. To illustrate the capabilities of our platform we will present the biological impact of a series of cabazitaxel-containing NDCs with linkers encompassing a diversity of in vitro release rates. In vivo PK studies showed high and sustained levels of released drug in tumor tissues (>168 hrs), and in vivo mouse tumor model studies demonstrated vastly improved efficacy, i.e., tumor regression and significant tumor growth delay, and survival over separately dosed cabazitaxel including efficacy in a taxane-resistant tumor model. Cerulean continues to expand its platform through the development of new and emerging capabilities to treat patients living with cancer. In this regard, we also will address the future evolution of NDCs, including the conjugation of multiple payloads to a single NDC and the development of antibody-conjugated NDCs. This abstract is also being presented as Poster B43. Citation Format: Chester Metcalf, III, Derek van der Poll, Liang Zhao, Tiffany Halo, Doug Lazarus, Adam Stockmann, Christian Peters, Donna Brown, Roy Case, Ellen Rohde, Lata Jayaraman, Hongwei Wang, Tiffany Crowell, Adrian Senderowicz, Scott Eliasof. Significant improvements in therapeutic index for conjugated payloads using a nanoparticle-drug conjugate (NDC) platform to provide sustained drug release and potentially improved anticancer effects. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr PR10.

Nanoparticles for Effective Combination Therapy of Cancer

Cancer continues to remains a major healthcare problem across the world despite strong translational research efforts towards tackling the disease. Surgery, when possible, along with radiation and chemotherapy continue to remain the mainstay of cancer treatment. Novel targeted therapies or biologics and immunotherapies have recently been approved to improve treatment efficacies while reducing collateral damage to normal, non-cancerous tissues. Combination therapies have shown better results than individual monotherapies in the clinic but often the improvements in therapeutic indices remain marginal, at best. Several combinations treatments have been clinically approved for different types of cancer. Nanomedicine, the application of nanotechnology for medicine, has already made some positive impacts on the clinical care in this fight against cancer. Several nano-sized formulations of conventional chemotherapies have been clinically approved. Nanotechnology provides a novel way to deliver combination therapies with spatiotemporal control over drug release. This review explores the recent advances in nanotechnology-mediated combination treatments against cancer. Multifunctional nanomedicines for mechanism-based combination therapies are likely to deliver the right drugs to the right place at the right time for optimal treatment responses with reduced morbidity. No nanomedicine that combines two or more drugs in a single platform has been approved for clinical use yet. This is because several challenges still remain in the development of nano-combinations including but not limited to - the optimal drug ratios in these nanomedicines, control over these drug ratios over multiple batches, large scale, reproducible manufacturing of these nanomedicines and cost of these nano-combinations among others. These challenges need to be addressed soon using a multidisciplinary approach with collaborations between academia, the pharmaceutical industry and the regulatory bodies involved to ensure that nano-combination therapy delivers on its promise of better treatment outcomes while severely reducing morbidity thus improving the quality of life in cancer patients.

Design and characterization of short hybrid antimicrobial peptides from pEM‐2, mastoparan‐VT1, and mastoparan‐B

Antimicrobial peptides are considered to be excellent templates for designing novel antibiotics because of their broad-spectrum antimicrobial activity and their low prognostic to induce antibiotic resistance. In this study, for the first time, a series of short hybrid antimicrobial peptides combined by different fragments of venom-derived alpha-helical antimicrobial peptides pEM-2, mastoparan-VT1, and mastoparan-B were designed with the intent to improve the therapeutic index of the parental peptides. Short hybrid antimicrobial peptides PV, derived from pEM-2 and mastoparan-VT1, was found to possess the highest antibacterial, hemolytic, and cytotoxic activity. Short hybrid antimicrobial peptides PV3, derived from pEM-2 and three fragments of mastoparan-VT1, showed more than threefold improvement in therapeutic index compared with parental peptides pEM-2 and mastoparan-VT1. PV had the highest antimicrobial activity in stability studies. Except BVP, designed based on all three parental peptides, the other short hybrid antimicrobial peptides at their minimal inhibitory concentration and 2× minimal inhibitory concentration required less than 120 and 60min to reduce >3log10 the initial inoculum, respectively. All peptides had membrane-disrupting activity in a time-dependent manner. Collectively, this study highlights the potential for rational design of improved short hybrid antimicrobial peptides such as PV3 that was an ideal candidate for further assessment with the ultimate purpose of development of effective antimicrobial agents.

Roles for the Mitogen-activated Protein Kinase (MAPK) Phosphatase, DUSP1, in Feedback Control of Inflammatory Gene Expression and Repression by Dexamethasone

Glucocorticoids act on the glucocorticoid receptor (NR3C1) to repress inflammatory gene expression. This is central to their anti-inflammatory effectiveness and rational improvements in therapeutic index depend on understanding the mechanism. Human pulmonary epithelial A549 cells were used to study the role of the mitogen-activated protein kinase (MAPK) phosphatase, dual-specificity phosphatase 1 (DUSP1), in the dexamethasone repression of 11 inflammatory genes induced, in a MAPK-dependent manner, by interleukin-1β (IL1B). Adenoviral over-expression of DUSP1 inactivated MAPK pathways and reduced expression of all 11 inflammatory genes. IL1B rapidly induced DUSP1 expression and RNA silencing revealed a transient role in feedback inhibition of MAPKs and inflammatory gene expression. With dexamethasone, which induced DUSP1 expression, plus IL1B (co-treatment), DUSP1 expression was further enhanced. At 1 h, this was responsible for the dexamethasone inhibition of IL1B-induced MAPK activation and CXCL1 and CXCL2 mRNA expression, with a similar trend for CSF2. Whereas, CCL20 mRNA was not repressed by dexamethasone at 1 h, repression of CCL2, CXCL3, IL6, and IL8 was unaffected, and PTGS2 repression was partially affected by DUSP1 knockdown. At later times, dexamethasone repression of MAPKs was unaffected by DUSP1 silencing. Likewise, 6 h post-IL1B, dexamethasone repression of all 11 mRNAs was essentially unaffected by DUSP1 knockdown. Qualitatively similar data were obtained for CSF2, CXCL1, IL6, and IL8 release. Thus, despite general roles in feedback inhibition, DUSP1 plays a transient, often partial, role in the dexamethasone-dependent repression of certain inflammatory genes. Therefore this also illustrates key roles for DUSP1-independent effectors in mediating glucocorticoid-dependent repression.

Synthetic Aminoglycosides Efficiently Suppress Cystic Fibrosis Transmembrane Conductance Regulator Nonsense Mutations and Are Enhanced by Ivacaftor

New drugs are needed to enhance premature termination codon (PTC) suppression to treat the underlying cause of cystic fibrosis (CF) and other diseases caused by nonsense mutations. We tested new synthetic aminoglycoside derivatives expressly developed for PTC suppression in a series of complementary CF models. Using a dual-luciferase reporter system containing the four most prevalent CF transmembrane conductance regulator (CFTR) nonsense mutations (G542X, R553X, R1162X, and W1282X) within their local sequence contexts (the three codons on either side of the PTC), we found that NB124 promoted the most readthrough of G542X, R1162X, and W1282X PTCs. NB124 also restored full-length CFTR expression and chloride transport in Fischer rat thyroid cells stably transduced with a CFTR-G542XcDNA transgene, and was superior to gentamicin and other aminoglycosides tested. NB124 restored CFTR function to roughly 7% of wild-type activity in primary human bronchial epithelial (HBE) CF cells (G542X/delF508), a highly relevant preclinical model with endogenous CFTR expression. Efficacy was further enhanced by addition of the CFTR potentiator, ivacaftor (VX-770), to airway cells expressing CFTR PTCs. NB124 treatment rescued CFTR function in a CF mouse model expressing a human CFTR-G542X transgene; efficacy was superior to gentamicin and exhibited favorable pharmacokinetic properties, suggesting that in vitro results translated to clinical benefit in vivo. NB124 was also less cytotoxic than gentamicin in a tissue-based model for ototoxicity. These results provide evidence that NB124 and other synthetic aminoglycosides provide a 10-fold improvement in therapeutic index over gentamicin and other first-generation aminoglycosides, providing a promising treatment for a wide array of CFTR nonsense mutations.

Mechanism of MEK inhibition determines efficacy in mutant KRAS- versus BRAF-driven cancers

KRAS and BRAF activating mutations drive tumorigenesis through constitutive activation of the MAPK pathway. As these tumours represent an area of high unmet medical need, multiple allosteric MEK inhibitors, which inhibit MAPK signalling in both genotypes, are being tested in clinical trials. Impressive single-agent activity in BRAF-mutant melanoma has been observed; however, efficacy has been far less robust in KRAS-mutant disease. Here we show that, owing to distinct mechanisms regulating MEK activation in KRAS- versus BRAF-driven tumours, different mechanisms of inhibition are required for optimal antitumour activity in each genotype. Structural and functional analysis illustrates that MEK inhibitors with superior efficacy in KRAS-driven tumours (GDC-0623 and G-573, the former currently in phase I clinical trials) form a strong hydrogen-bond interaction with S212 in MEK that is critical for blocking MEK feedback phosphorylation by wild-type RAF. Conversely, potent inhibition of active, phosphorylated MEK is required for strong inhibition of the MAPK pathway in BRAF-mutant tumours, resulting in superior efficacy in this genotype with GDC-0973 (also known as cobimetinib), a MEK inhibitor currently in phase III clinical trials. Our study highlights that differences in the activation state of MEK in KRAS-mutant tumours versus BRAF-mutant tumours can be exploited through the design of inhibitors that uniquely target these distinct activation states of MEK. These inhibitors are currently being evaluated in clinical trials to determine whether improvements in therapeutic index within KRAS versus BRAF preclinical models translate to improved clinical responses in patients.

In Vitro Evaluation of the Efficacy of Liposomal and Pegylated Liposomal Hydroxyurea

Breast cancer is one of the most frequent cancer types within women population. Hydroxyurea (HU) is a chemotherapy compound for treatment of patients with cancer diagnosis, including breast cancer associated with several adverse effects. In this study, we applied nanotechnology to decreased drug side effects along with improvement of therapeutic index. Liposomation is widely used in modern pharmacological developments in order to enhance the effects of the drugs. To achieve this, in this study a mixture of phosphatidylcholine and cholesterol was made up and HU was added to the resultant mixture, was then pegylated using Polyethylene Glycol 2000 to increase resistance, applicability and solubility. The mean diameters of nanoliposomal and pegylated nanoliposomal HU were measured by Zeta sizer device and obtained about 402.5 and 338.2nm. The efficiency of non-pegylated and pegylated liposomal HU was 70.8 and 64.2, respectively. Releasing HU in both formulations was estimated about 25.8 and 21.7%. Also, this study investigated the cytotoxicity effect of nanoliposomal and pegylated nanoliposomal HU using MTT assay. Results of this investigation showed that the cytotoxic properties of pegylated HU was 3.6% more than those non-pegylated form, while was 38.93% more than ordinary from of HU. This study showed that the stability, releasing pattern and cytotoxicity of the pegylated nanoliposomal HU is better than that of nanoliposomal HU.

Fasting vs dietary restriction in cellular protection and cancer treatment: from model organisms to patients

The dietary recommendation for cancer patients receiving chemotherapy, as described by the American Cancer Society, is to increase calorie and protein intake. Yet, in simple organisms, mice, and humans, fasting--no calorie intake--induces a wide range of changes associated with cellular protection, which would be difficult to achieve even with a cocktail of potent drugs. In mammals, the protective effect of fasting is mediated, in part, by an over 50% reduction in glucose and insulin-like growth factor 1 (IGF-I) levels. Because proto-oncogenes function as key negative regulators of the protective changes induced by fasting, cells expressing oncogenes, and therefore the great majority of cancer cells, should not respond to the protective signals generated by fasting, promoting the differential protection (differential stress resistance) of normal and cancer cells. Preliminary reports indicate that fasting for up to 5 days followed by a normal diet, may also protect patients against chemotherapy without causing chronic weight loss. By contrast, the long-term 20 to 40% restriction in calorie intake (dietary restriction, DR), whose effects on cancer progression have been studied extensively for decades, requires weeks-months to be effective, causes much more modest changes in glucose and/or IGF-I levels, and promotes chronic weight loss in both rodents and humans. In this study, we review the basic as well as clinical studies on fasting, cellular protection and chemotherapy resistance, and compare them to those on DR and cancer treatment. Although additional pre-clinical and clinical studies are necessary, fasting has the potential to be translated into effective clinical interventions for the protection of patients and the improvement of therapeutic index.