Releasable Linker Research Articles

Leveraging long-acting IL-15 agonists for intratumoral delivery and enhanced antimetastatic activity.

IL-15 agonists hold promise as immunotherapeutics due to their ability to induce the proliferation and expansion of cytotoxic immune cells including natural killer (NK) and CD8+ T cells. However, they generally have short half-lives that necessitate frequent administration to achieve efficacy. To address this limitation, we have developed a half-life extension technology using hydrogel microspheres (MS). Here, the therapeutic is tethered to MSs by a releasable linker with pre-programed cleavage rates. We previously showed the MS conjugate of single-chain IL-15, MS~IL-15, effectively increased the half-life of IL-15 to approximately 1 week and enhanced the pharmacodynamics. We sought to determine whether the same would be true with a MS conjugate of the IL-15 agonist, receptor-linker IL-15 (RLI). We prepared a long acting MS conjugate of RLI, MS~RLI. The pharmacokinetics and pharmacodynamics of MS~RLI were measured in C57BL/6J mice and compared to MS~IL-15. The antitumor efficacy of MS~RLI was measured when delivered subcutaneously or intratumorally in the CT26 tumor model and intratumorally in the orthotopic EO771 tumor model. MS~RLI exhibited a half-life of 30 h, longer than most IL-15 agonists but shorter than MS~IL-15. The shorter than expected half-life of MS~RLI was shown to be due to target-mediated-disposition caused by an IL-15 induced cytokine sink. MS~RLI resulted in very potent stimulation of NK and CD44hiCD8+ T cells, but also caused significant injection-site toxicity that may preclude subcutaneous administration. We thus pivoted our efforts toward studying the MS~RLI for long-acting intra-tumoral therapy, where some degree of necrosis might be beneficial. When delivered intra- tumorally, both MS~IL-15 and MS~RLI had modest anti-tumor efficacy, but high anti- metastatic activity. Intra-tumoral MS~RLI and MS~RLI combined with systemic treatment with other agents could provide beneficial antitumor and anti-metastatic effects without the toxic effects of systemic IL-15 agonists. Our findings demonstrate that intra-tumorally administered long-acting IL-15 agonists counter two criticisms of loco-regional therapy: the necessity for frequent injections and the challenge of managing metastases.

Abstract 7214: A click chemistry-based HER2 targeting agent activates a decoupled MMAE payload making it highly differentiated in efficacy and safety than ADCs

Abstract Shasqi is advancing the Click Activated Protodrugs Against Cancer (CAPAC®) platform based on click chemistry, a Nobel Prize winning technology. The platform is modular and comprises of 1) an activator that targets specific antigens, and 2) inert cancer drugs, protodrugs, which are selectively activated at tumors via click chemistry. The CAPAC technology separates the tumor targeting function from the payload and reunites them at the tumor creating the flexibility to optimize activity while limiting toxicity during preclinical and clinical development. The modularity of the platform enables the rapid development of new therapies as well as unlocking unique treatment benefits such as tunable combinations and payload cycling. We envision that CAPAC will expand the scope of potential targets, widen the activity window of antibody drug conjugates (ADCs). Here we compare a HER2-targeting activator + a monomethyl auristatin E (MMAE) payload with an ADC that binds the same target and carries the same payload, as well as other targeting formats. The activator SQT01 is composed of a HER2-binding Fab conjugated with tetrazines (Tz). The protodrug SQP22 is formed by coupling trans-cyclooctene (TCO, with a releasable linker) to MMAE, a cytotoxic spindle poison. An efficient covalent reaction between tetrazine and TCO moieties releases active MMAE at the tumor. Mice bearing human NCI-N87 HER2+ tumor were treated with 1) SQT01, 2) a non-binding isotype Fab-Tz, or 3) saline. After 18 h, all mice received either vehicle (10% HPCD), SQP22 protodrug, a non-releasable MMAE TCO-protodrug analog of SQP22, or a di-peptide-containing TCO-protodrug analog. We also tested CAPAC’s approach with SQT06 (a full-length IgG trastuzumab-Tz conjugate) that was followed by an SQP22 dose after 4 days. The dosing of SQP22 with the Fab-Tz and IgG-Tz is timed to allow for systemic clearance of the targeting activator. Fabs have a shorter half-life than IgG, so we dosed SQP22 at 18 h after SQT01 instead of the 4-day timepoint with SQT06. To allow comparisons with ADCs, additional groups received a single dose of commercially available disitamab vedotin (DV) or a HER2-Fab-vedotin. SQT01+SQP22 induced a strong tumor growth inhibition comparable to SQT06+SQP22, but significantly better than DV, and SQT01+non-releasable protodrug analog. Dosing with SQT01 with a protease cleavable dipeptide-TCO protodrug analog or a molar equivalent dose of HER2-Fab-vedotin were highly toxic and intolerable. Based on preclinical observations, SQT01+SQP22 was safe and tolerated, and there was no observed body weight loss. These data demonstrate that the CAPAC platform, which separates the targeting agent from the payload, provides a clear benefit in terms of safety and efficacy compared with small or large format proteins with the classic fixed construct of ADCs (e.g. a fixed drug to antibody ratio). Citation Format: Sangeetha Srinivasan, Stefanie Wagner, George Coricor, Jesse M. McFarland, Tri-Hung Nguyen, José M. Mejía Oneto. A click chemistry-based HER2 targeting agent activates a decoupled MMAE payload making it highly differentiated in efficacy and safety than ADCs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7214.

Abstract 2700: ORM-6151: A first-in-class CD33-antibody enabled GSPT1 degrader for AML

Abstract Targeted protein degraders (TPDs) have expanded the breadth of therapeutic options through both their catalytic mechanism of action and ability to degrade previously “undruggable” target proteins. Prior reports of small-molecule GSPT1 degraders such as CC-90009 in AML demonstrate potent anti-tumor cytotoxicity, but with a potentially narrow therapeutic index. To increase the efficacy vs. tolerability window of TPDs and improve drug delivery, we introduce TPD-Squared (TPD2 TM), a dual-targeted protein degradation approach of combining the catalytic mechanism of targeted protein degradation with the precision of tumor-targeting therapeutic antibodies. We have previously shown in vitro and in vivo efficacy with a HER2-targeted TPD2 conjugate: ORM-5029. Following on that success, we generated conjugates using a CD33-targeting antibody (OR000283) produced by engineering the FAb (H&L) sequences from gemtuzumab onto an IgG1 Fc with N297A variant to inhibit Fc-γR binding. Medicinal chemistry optimization of linker-payloads led to the identification of ORM-6151, which is composed of SMol006, a highly potent GSPT1 degrader conjugated to OR000283 via a novel β-glucuronide releasable linker. ORM-6151 treatment in CD33-expressing cell lines showed picomolar activity with 10-1000-fold greater potency compared to several GSPT1 degrader molecules, including CC-90009 or Mylotarg, and had robust activity in Mylotarg-resistant lines (AML193 and Kasumi6). ORM-6151 also exhibited picomolar potency in in vitro cytotoxicity to primary relapsed/refractory AML patient blasts, with better potency than CC-90009 and Mylotarg. We evaluated ORM-6151 in several in vivo disseminated xenograft models and observed robust efficacy following a single treatment at doses as low as 0.1 mg/kg. Tumor growth inhibition correlated with the degree and duration of GSPT1 depletion and changes in the expression of previously described integrated stress response biomarker genes. In summary, ORM-6151 is a promising, potential therapy for AML and currently in preclinical development as a first-in-class targeted protein degrader therapy with CD33-targeted delivery. Citation Format: James Palacino, Pedro Lee, Hangyeol Jeong, Yeonjoon Kim, Yoojin Song, Uttapol Permpoon, Wesley Wong, Chen Bai, Nathan Fishkin, Khuloud Takouri, Eunjun Yu, Yong Yi, Anna Skaletskaya, Min-Soo Kim, Da-Yeong Kim, Dong-Ki Choi, Peter U. Park. ORM-6151: A first-in-class CD33-antibody enabled GSPT1 degrader for AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2700.

Facile preparation, closed-loop recycling of multifunctional carbon fiber reinforced polymer composites

Recyclable carbon fiber reinforced polymer (CFRP) composites have attracted considerable attention in recent years due to the combined merits of excellent mechanical and thermal properties, recyclability and reprocessability. This paper reports on the successful preparation of closed-loop recyclable CFRP by in-situ polymerization and dynamic cross-linking (ISPDC) technology. The SEM, XPS and monofilament tensile curves were measured to investigate the morphology, chemical details, and mechanical properties of the recycled carbon fiber (CF); the chemical structure of the degraded resin was further characterized and analyzed according to the NMR and FTIR spectra. The obtained results show that under mild acidic conditions, the CFRP was degraded to high-quality recycled CF and linear polymer with methacrylic acid units, which can be used to prepare regenerated CFRP. In addition, due to the dynamic feature of the hemiacetal ester structure in the CFRP matrix, the CFRP also exhibited multiple functions, such as rapid welding, reprocessing, shape shifting, and self-healing, as well as the extremely long shelf life of composite prepregs. Our analysis of the recycling and dynamic exchange processes indicates that the strategy of embedding a releasable linker is dominant for the preparation of closed-loop recyclable and multifunctional CFRP.

ORM-6151: A First-in-Class, Anti-CD33 Antibody-Enabled GSPT1 Degrader for AML

Targeted protein degradation (TPD) molecules have expanded the breadth of therapeutic options through both their catalytic mechanism of action and ability to degrade previously "undruggable" target proteins. Prior reports of small-molecule GSPT1 degraders such as CC-90009 in AML demonstrate potent anti-tumor cytotoxicity, but with a potentially narrow therapeutic index. To increase the efficacy vs. tolerability window of TPDs and improve drug delivery, we introduce TPD-Squared (TPD2TM), a dual-targeted protein degradation approach of combining the catalytic mechanism of targeted protein degradation with the precision of tumor-targeting therapeutic antibodies. We generated conjugates using a CD33-targeting antibody (OR000283) produced by engineering the FAb (H&L) sequences from gemtuzumab onto an IgG1 Fc with N297A variant to inhibit Fc-γR binding. Medicinal chemistry optimization of linker-payloads led to the identification of ORM-6151, which is composed of SMol006, a highly potent GSPT1 degrader conjugated to OR000283 via a novel β-glucoronide releasable linker. ORM-6151 treatment in CD33-expressing cell lines showed picomolar activity with 10-1000-fold greater potency compared to several GSPT1 degrader molecules including CC-90009 or Mylotarg, and had robust activity in Mylotarg-resistant lines (AML193 and Kasumi6). ORM-6151 also exhibited picomolar potency in in vitro cytotoxicity to primary relapsed/refractory AML patient blasts, with better potency than CC-90009 and Mylotarg. Moreover, ORM-6151 showed minimal cytotoxic activity to healthy hematopoietic progenitor cells, with 10-10,000 fold less toxicity than CC-90009 or Mylotarg. We evaluated ORM-6151 in several in vivo xenograft models and observed robust efficacy, following a single treatment at doses as low as 1 mg/kg. In the MV4-11 xenograft model, treatment with ORM-6151 demonstrated superior activity than CC-90009. The tumor growth inhibition correlated with the degree and duration of GSPT1 depletion and changes in expression of previously described integrated stress response biomarker genes. In summary, ORM-6151 is a promising, potential therapy for AML and currently in preclinical development as a first-in-class targeted protein degrader therapy with CD33-targeted delivery. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A very long-acting IL-15: implications for the immunotherapy of cancer

BackgroundInterleukin-15 (IL-15) is an important cytokine necessary for proliferation and maintenance of natural killer (NK) and CD8+ T cells, and with great promise as an immuno-oncology therapeutic. However, IL-15 has...

A Novel Antibody-Toxin Congujate to Treat Mantle Cell Lymphoma

IntroductionMantle cell lymphoma (MCL), while sensitive to several chemotherapeutic regimens including rituximab, cure rates remain low and new treatments are needed.We have identified a membrane bound “activated” matriptase, as an attractive antigen target for the highly selective delivery of toxins to epithelial tumors and MCL. Matriptase, a member of type II transmembrane serine proteases, is a glycoprotein (80-90 kDa) synthesized as a latent single-chain structure. Activation generates a disulfide-linked-two-chain fully active enzyme through an auto-activation step. Reactive oxygen species (ROS), as well as acidic environments present in tumors can activate matriptase. In addition to roles in initiating carcinogenesis, the enzyme also plays important roles in tumor progression, including invasiveness and metastasis.Importantly, while matriptase is present in a latent form on epithelial cells and B-cells, activated matriptase expression is restricted to the membranes of epithelial tumors and some B-cell malignancies .MethodsMaterials: The M-69 antibody was generated by CY. Bortezomib was from the CINJ pharmacy.Cell Culture: The MCL cells (JeKo-1, Mino, Maver and Z138) were cultured in 1X RPMI Media 1640 (Life Technologies) containing 10% fetal bovine serum (FBS) at 37ºC.Western blotting: The M-69 antibody was used for western analysis and Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) purchased from Millipore was used as a control.Cytotoxicity assay: Cell viability was measured by the trypan blue dye exclusion method using a Vi-Cell XR© cell viability analyzer (Beckman Coulter). The cytotoxicity data were analyzed using GraphPad Prism 4 software (GraphPad Software Inc., CA).Animal studies: NOD/SCID/IL2 receptor gamma chain null (NOD/SCID/IL2gnull, NSG) mice were obtained from the Jackson Laboratory. JeKo-1 MCL cells were used for the xenograft studies. Once tumors were palpable, the mice were treated i.p. with anti-matriptase antibody (M-69) conjugated with MMAE (ADC). Controls used were M-69 antibody alone and saline. In a second experiment mice were treated with the ADC alone, bortezomib alone or the combination. Tumor size and body weights were measured twice a week.ResultsThe linkage of monomethyl auristatin-E (MMAE), a potent tubulin-inhibitor, to M69 used the releasable linker technology which is based on Seattle Genetics' valine-citrulline-PABA linker that is cleavable by cathepsin B in the lysosome, while showing stability in circulation. MALDI-TOF mass spectrometry of the ADC showed a 7000 Da increase of the average M.W. of the antibody that corresponds to an average of 3.5 drug molecules linked to each mAb molecule.MCL (JeKo-1, Mino, Maver and Z138) cell lines were found to overexpress activated matriptase using the M-69 antibody. These cell lines were sensitive to the ADC with IC50s at single digit µg/ml of the conjugate.We elected to use the JeKo-1 cell line for the initial xenograft studies. We found that the 5 mg/kg dose of the ADC weekly produced a better anti-tumor effect (90% growth inhibition) than a 1 mg/kg dose, without any body weight loss. In combination with bortezomib, treatment with the ADC led to some complete responses.ConclusionsThe mouse antibody to human matriptase, M-69, conjugated to MMAE potently inhibited growth of four MCL cell lines and inhibited xenografts of the JeKo-1 MCL in NSG mice alone and produced enhanced effects in combination with bortezomib without significant side effects. DisclosuresRather:Phosplatin Therapeutics: Research Funding. Bertino:Phosplatin Therapeutics: Research Funding.

A Very Long-Acting PARP Inhibitor Suppresses Cancer Cell Growth in DNA Repair-Deficient Tumor Models.

PARP inhibitors are approved for treatment of cancers with BRCA1 or BRCA2 defects. In this study, we prepared and characterized a very long-acting PARP inhibitor. Synthesis of a macromolecular prodrug of talazoparib (TLZ) was achieved by covalent conjugation to a PEG40kDa carrier via a β-eliminative releasable linker. A single injection of the PEG∼TLZ conjugate was as effective as ∼30 daily oral doses of TLZ in growth suppression of homologous recombination-defective tumors in mouse xenografts. These included the KT-10 Wilms' tumor with a PALB2 mutation, the BRCA1-deficient MX-1 triple-negative breast cancer, and the BRCA2-deficient DLD-1 colon cancer; the prodrug did not inhibit an isogenic DLD-1 tumor with wild-type BRCA2. Although the half-life of PEG∼TLZ and released TLZ in the mouse was only ∼1 day, the exposure of released TLZ from a single safe, effective dose of the prodrug exceeded that of oral TLZ given daily over one month. μPET/CT imaging showed high uptake and prolonged retention of an 89Zr-labeled surrogate of PEG∼TLZ in the MX-1 BRCA1-deficient tumor. These data suggest that the long-lasting antitumor effect of the prodrug is due to a combination of its long t 1/2, the high exposure of TLZ released from the prodrug, increased tumor sensitivity upon continued exposure, and tumor accumulation. Using pharmacokinetic parameters of TLZ in humans, we designed a long-acting PEG∼TLZ for humans that may be superior in efficacy to daily oral TLZ and would be useful for treatment of PARP inhibitor-sensitive cancers in which oral medications are not tolerated. SIGNIFICANCE: These findings demonstrate that a single injection of a long-acting prodrug of the PARP inhibitor talazoparib in murine xenografts provides tumor suppression equivalent to a month of daily dosing of talazoparib.

Abstract LB-060: A very long-acting poly(ADP-ribose) polymerase inhibitor

Abstract Purpose: The purpose of this work was to prepare and characterize the anti-tumor properties of a prodrug for a very long-acting of poly(ADP-ribose) polymerase (PARP) inhibitor. Background: Four PARP inhibitors (PARPi) have been approved for QD oral use in treatment of human cancers. It is believed that PARP requires continuous inhibition for optimal anti-tumor effects, but, as with any drug with a short t1/2, daily administered PARPi exhibit high Cmax values and peak-to-trough excursions. We speculated that the prolonged exposure and lower Cmax and Cmax/Cmin of a long-acting PARPi might provide a more effective, less toxic therapeutic. In choosing which PARPi to target, a major consideration was whether the carrier has capacity to deliver sufficient levels of the drug over a long dosing interval. Talazoparib (TLZ) was chosen for this study because it is the most potent of the PARP inhibitors, requiring only 1 mg/day in adults compared to hundreds of mg/day for other PARPi's. Experimental procedures: We prepared a long-acting prodrug of TLZ by attaching it to a PEG40kDa carrier by a β-eliminative releasable linker. The chemistry was achieved by a novel alkylation of TLZ at the poorly acidic 2-NH of the phthalazinone moiety with an O-azidoalkyl-N-alkyl-N-chloromethyl carbamate, followed by coupling to PEG-cyclooctyne. Daily PO doses of TLZ or a single IP injection of the PEG-TLZ conjugate were administered to xenografts in mice possessing defects in homologous recombination - either a PALB2 mutation in the KT-10 Wilms tumor, or a BRCA1-deficient MX-1 triple-negative breast cancer. New data: PEG~TLZ was highly effective in treating both KT-10 and MX-1 xenografts. Although the t1/2 of TLZ in the mouse is only ~3 hr, tumor growth in animals treated with PEG~TLZ was suppressed for about one month. The EFS T/C values - the ratio of the median time to event between treated and control groups - of single injections of ~5 mg TLZ/kg as PEG~TLZ in either tumor was more than 4, indicating the drug is a highly active agent at low doses. The amount of TLZ in a single efficacious dose of the PEG~TLZ conjugate was equivalent to the same amount of free TLZ administered in divided daily doses for 4 or more weeks. Although we did not investigate scheduling, dosing PEG~TLZ once every 3 to 4 weeks should be sufficient to suppress tumor growth for extended periods. Conclusion: We developed a novel method of conjugating linkers to the 2N of the phthalazinone moiety of PARPi. We prepared a cleavable PEG~TLZ that releases TLZ with a t1/2 of 160 hr at pH 7.4. In mouse xenografts of tumors with defective HR, single non-toxic doses of PEG~TLZ suppresses tumor growth for ~1 month, and are equi-effective to QD administration of TLZ over that period. We posit that the long lasting effect is due to the long t1/2 of the prodrug, increased sensitivity of the tumor upon continued exposure to TLZ, tumor accumulation of the 15 nm nanomolecule, and counteracting drug resistance by efflux pumps. Citation Format: Shaun D. Fontaine, Gary W. Ashley, Peter J. Houghton, Raushan Kurmasheva, Morgan Diolati, Alan Ashworth, Daniel V. Santi. A very long-acting poly(ADP-ribose) polymerase inhibitor [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-060.

Bone-Fracture-Targeted Dasatinib-Oligoaspartic Acid Conjugate Potently Accelerates Fracture Repair.

Approximately 6.3 million bone fractures occur annually in the United States, resulting in considerable morbidity, deterioration in quality of life, loss of productivity and wages, and sometimes death (e.g., hip fractures). Although anabolic and antiresorptive agents have been introduced for treatment of osteoporosis, no systemically administered drug has been developed to accelerate the fracture-healing process. To address this need, we have undertaken to target a bone anabolic agent selectively to fracture surfaces in order to concentrate the drug's healing power directly on the fracture site. We report here that conjugation of dasatinib to a bone fracture-homing oligopeptide via a releasable linker reduces fractured femur healing times in mice by ∼60% without causing overt off-target toxicity or remodeling of nontraumatized bones. Thus, achievement of healthy bone density, normal bone volume, and healthy bone mechanical properties at the fracture site is realized after only 3-4 weeks in dasatinib-targeted mice, but it requires ∼8 weeks in PBS-treated controls. We conclude that targeting of dasatinib to bone fracture surfaces can significantly accelerate the healing process at dasatinib concentrations that are known to be safe in oncological applications.

Quantitative and Comparative Profiling of Protease Substrates through a Genetically Encoded Multifunctional Photocrosslinker

AbstractA genetically encoded, multifunctional photocrosslinker was developed for quantitative and comparative proteomics. By bearing a bioorthogonal handle and a releasable linker in addition to its photoaffinity warhead, this probe enables the enrichment of transient and low‐abundance prey proteins after intracellular photocrosslinking and prey–bait separation, which can be subject to stable isotope dimethyl labeling and mass spectrometry analysis. This quantitative strategy (termed isoCAPP) allowed a comparative proteomic approach to be adopted to identify the proteolytic substrates of an E. coli protease–chaperone dual machinery DegP. Two newly identified substrates were subsequently confirmed by proteolysis experiments.

Quantitative and Comparative Profiling of Protease Substrates through a Genetically Encoded Multifunctional Photocrosslinker.

A genetically encoded, multifunctional photocrosslinker was developed for quantitative and comparative proteomics. By bearing a bioorthogonal handle and a releasable linker in addition to its photoaffinity warhead, this probe enables the enrichment of transient and low-abundance prey proteins after intracellular photocrosslinking and prey-bait separation, which can be subject to stable isotope dimethyl labeling and mass spectrometry analysis. This quantitative strategy (termed isoCAPP) allowed a comparative proteomic approach to be adopted to identify the proteolytic substrates of an E. coli protease-chaperone dual machinery DegP. Two newly identified substrates were subsequently confirmed by proteolysis experiments.

Abstract 4615: Development of an antibody-drug conjugate with broad anticancer activity

Abstract Treatment for patients with advanced solid tumors that include triple negative breast cancer (TNBC), non-small cell lung cancer (NSCLC), and castrate resistant prostate cancer (CRPC), as well as Mantle Cell Lymphoma (MCL), while increasing survival is not curative. We have identified a membrane bound protease, “activated” matriptase, as an attractive target antigen for highly selective antibody delivery of cytotoxins as activated matriptase expression is restricted to epithelial tumors and some B-cell lymphomas. We generated a novel ADC by linking M69, a mouse antibody specific to activated matriptase, to monomethyl auristatin E (MMAE) via a PEGylated, releasable di-peptide linker as a proof-of-principle prototype. Both in cell lines and in human xenograft models of TNBC, NSCLC, CRPC and MCL, the conjugate was found to exhibit potent anticancer activity against all of these tumor types without toxicity. Encouraged by these results, we are also exploring the use of this ADC against gastric and pancreatic cancer, and in combination with chemotherapy and immunotherapy. As this is a mouse antibody, we are also generating a chimeric antibody for toxicity studies in primates. Citation Format: Siang-Yo Lin, Zoltan Szekely, Chen-Yong Lin, Joseph R. Bertino, Gulam Mohmad Rather. Development of an antibody-drug conjugate with broad anticancer activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4615. doi:10.1158/1538-7445.AM2017-4615

Abstract P1-12-05: Etirinotecan pegol: Survival advantage over standard of care drugs in a model of brain metastases of breast cancer

Abstract BACKGROUND: Brain metastasis of breast cancer is a significant cause of death among women with disseminated breast cancer. Chemotherapy, radiation, and surgery for these metastases provide only minimal benefit with considerable toxicity. The long-acting topoisomerase I inhibitor etirinotecan pegol (EP) is a novel treatment for disseminated breast cancer. EP consists of irinotecan attached to a 20kDa branched polymer via a releasable linker. The large molecular weight of EP results in tumor accumulation, including brain metastases, providing increased and sustained exposure to cytotoxic SN38. In the Phase 3 BEACON study, EP showed a significant survival benefit (HR 0.51; 95% CI 0.30-0.86) in a prespecified subgroup of women with history of treated, stable brain metastases compared to treatment of physicians choice (TPC) consisting of one of seven approved chemotherapy drugs (median OS 10.0 vs 4.8 mo). Nektar has filed a conditional marketing authorization in Europe for EP in patients with advanced breast cancer and brain metastases. A Phase 3 trial in this patient population is being initiated to support a potential U.S. filing. To further support the biological rationale for EP in these patients, an experimental preclinical model of brain metastases of breast cancer was conducted to compare the efficacy of EP to TPC in BEACON and the planned subsequent study. METHODS: Female athymic nude mice were inoculated with 1.75x105 MDA-MB-231-BrLuc cells via intracardiac injection. Starting on day 21, gemcitabine (60 mg/kg) and eribulin (1.5 mg/kg) were dosed IP every 4 days; EP (50 mg/kg), irinotecan (50 mg/kg), paclitaxel (6 mg/kg), vinorelbine (10 mg/kg), docetaxel (10 mg/kg), and vehicle (saline or dextrose) were dosed weekly via tail vein. Efficacy was measured by tumor burden and survival. Tumor burden was determined based on twice weekly bioluminescence measurements. Animals were euthanized according to international animal care guidelines. RESULTS: Median survival for all control arm groups (docetaxel, vinorelbine, eribulin, gemcitabine, irinotecan, and paclitaxel) were 39, 43, 41, 48, 35, and 42 days, respectively, none of which were significantly different from vehicle control median survival of 40 days (p>0.05). No animals receiving these drugs survived until the end of the 90-day trial. Median survival for the EP treatment group was 86 days (p<0.05) with a 40% survival rate at 90 days, the trial endpoint. Tumor burden increased nearly 100-fold in all TPC and vehicle groups, whereas EP significantly inhibited tumor growth. Quantitative autoradiography showed that EP accumulated ~10-fold in brain metastases, while accumulation in non-tumor brain tissue was only ~2-fold compared to irinotecan. EP at 50 mg/kg achieved plasma trough concentrations comparable to those observed in patients receiving the recommended dose of 145 mg/m2 every 21 days. CONCLUSIONS: In this model of brain metastases, EP preferentially accumulates in brain metastases, significantly reduces tumor burden progression and significantly improves survival in brain metastases of breast cancer compared to the most active chemotherapeutic agents available for advanced breast cancer and those used in the BEACON trial and planned subsequent Phase 3 study. Citation Format: Shah N, Mohammad AS, Adkins CE, Dolan EL, Griffith J, Jagannathan R, Hoch U, Lockman PR. Etirinotecan pegol: Survival advantage over standard of care drugs in a model of brain metastases of breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-12-05.

Re-programming pullulan for targeting and controlled release of doxorubicin to the hepatocellular carcinoma cells

A novel bioconjugate for hepatocellular carcinoma (HCC) targeting was obtained by pullulan re-programming, which involves the backbone oxidation and conjugation of targeting peptide and doxorubicin (Doxo) through a releasable linker. Preliminary in vivo studies showed that the oxidation of 40 glucopyranose units (GPU) out of 100 remarkably reduced the pullulan unspecific liver tropism. This oxidized polymer was functionalized with PreS1 to selectively target the HCC and with rhodamine (Rhod) as label to perform in vitro cell up-take investigations. PreS1 and Rhod were conjugated to the aldehydes present along the oxidized pullulan backbone through a 3.4 and 2kDa PEG spacer, respectively, and by reductive amination. The resulting PreS1-Pull-Rhod contained a mean of 8 PreS1 per oxidized pullulan chain. Cell culture studies were performed by using HepG2/SERPINB3 cells that overexpress the serpine B3 receptor and control HepG2/EMPTY cells that do not overexpress the receptor. A comparative study by cytofluorimetry and confocal microscopy performed using PreS1-Pull-Rhod and Pull-Rhod (control polymer) showed that PreS1 conveys to the conjugate high cell selectivity. Afterwards, the oxidized pullulan was exploited to generate a targeted drug delivery system by conjugation of Doxo to the polymer backbone through a hydrazone pH-sensitive bond and NH2-PEG3.4 kDa-PreS1. The PreS1-Pull-Doxo conjugate showed a two-fold increase of anticancer activity with respect to the control Pull-Doxo towards HepG2/SERPINB3 cells.

P3.02c-027 Phase I and PK Study of the Folate Receptor-Targeted Small Molecule Drug Conjugate (SMDC) EC1456 in Advanced Cancer: Lung Cancer Subset: Topic: IT

EC1456 is composed of folic acid conjugated through a releasable linker system to a potent microtubule inhibitor, tubulysin B hydrazide. EC1456 targets folate receptor (FR)-expressing cancer cells, which occur in approximately 60% of NSCLC cases and 14% of SCLC. The objectives of the ongoing Phase 1 are to determine the safety, PK, and optimal dosing schedule of EC1456 in advanced cancer pts. FR expression (not required for enrollment) is characterized in all pts using 99mTc-etarfolatide. 63 pts were dosed weekly (QW) or twice-weekly (BIW), for 2 consecutive weeks of a 3-week cycle. 8 NSCLC and 3 SCLC pts received doses ranging from 1.0-12.5 mg/m2. Toxicities are primarily Grade (Gr) 1 and 2. Common adverse events (AE) are GI, fatigue, and metabolic changes. Gr 3 infusion reaction (4.5 mg/m2) and Gr 3 headache (10.0 mg/m2) were seen in the QW cohort. The safety profile in lung cancer pts was similar to the overall population.Tabled 1BIW (N=32)QW (N=31)BIW Lung (N=4)QW Lung (N=7)AllDrug RelatedAllDrug RelatedAllDrug RelatedAllDrug Related≥ 1 AE32 (100%)25 (78%)29 (94%)23 (74%)4 (100%)4 (100%)7 (100%)4 (57%)≥ 1 grade 3 or 4 AE19 (59%)6 (19%)14 (45%)4 (13%)2 (50%)0 (0%)1 (14%)0 (0%)≥ 1 serious AE12 (38%)2 (6%)14 (45%)5 (16%)1 (25%)0 (0%)2 (29%)0 (0%)Serious drug related AEsConstipation (2/63 pts); Abd pain, Anemia, Headache, Infusion reactions, and SVT(1/63 pts each) Open table in a new tab Response and durability of response is demonstrated in the figure: EC1456 is well tolerated, with early indications of efficacy suggested by durable stable disease, and responses in this refractory population. Updated safety, PK, and efficacy data will be presented at the meeting.

Abstract 1929: Antifolates as folate receptor targeting ligands: Design and synthesis of their conjugates with cytotoxic agents

Abstract Antifolates are interfering specifically with DNA synthesis in the S-phase of the cell cycle. Thus, they are cytotoxic to rapidly dividing cancer cells, which replicate their DNA more frequently. Some antifolates bind with high affinity to the cell-membrane-associated folate receptor (FR). FR is frequently over-expressed in some of the most prevalent tumors. FR-binding antifolates can also be used as ligands for FR-targeted delivery of therapeutic agents. With this presentation we are reporting an improved synthesis of the FR-binding antifolate CB3717. Furthermore, to demonstrate its dual utility as FR-ligand and therapeutic agent, we designed and synthesized a conjugate of CB3717 with the highly cytotoxic agent Desacetylvinblastine Monohydrazide. A hydrophilic spacer unit and two biologically releasable disulfide-based linker systems were used to construct the conjugate. This conjugate exhibited high binding affinity for FR and excellent anti-tumor activity in vivo (subcutaneous FR+ KB tumors). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1929. doi:1538-7445.AM2012-1929

Abstract 4640: Development of antibody-drug conjugate technology based on novel DNA-interacting agents and unique linker technology

Abstract Antibody-Drug Conjugation (ADC) is gaining momentum as a next generation antibody therapeutics approach in oncology. Tumor specific delivery of the cell-killing agent maximizes the agent's potency while avoiding damage to healthy tissue resulting in a high therapeutic window. Synthon's Potent Payload Technology comprises the combination of highly potent DNA-alkylating duocarmycin derivatives and unique releasable linker technology. Duocarmycin analogs and the CC-1065 derivatives represent a class of highly potent, cell-killing, DNA-alkylating, minor groove binding agents. This class is suitable to target solid tumors and has shown activity in a variety of Multi-Drug Resistant (MDR) models.Synthon SpaceLink technology is the only releasable linker technology for ADC that can couple the toxin via its hydroxyl group to the antibody, validated in vivo. Synthon has combined its linkers with duocarmycin derivatives that have a hydroxyl group crucial for biological activity. The data package to be presented supports that SpaceLink and duocarmycins form an excellent chemical match for ADC therapy, possessing an intrinsic complementarity. Strong in vivo Proof of Concept with Potent Payload ADCs has been obtained against multiple targets. In human tumor xenograft models, substantial efficacies have been obtained based on low, single dose treatments, with minimal or absent side effects. We discuss applicability and several other aspects of multiple, validated Potent Payload-based Antibody-Drug Conjugates (ADCs). Latest results of preclinical development progress (in vivo efficacy, safety, etc.) will be presented, including in vitro drug and ADC potencies, plasma stabilities, cathepsin cleavage kinetics, linker cyclization kinetics and in vivo therapeutic window aspects for ADCs directed against HER2 and potentially other (collaborator) targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4640. doi:1538-7445.AM2012-4640

Synthesis of a novel cellulose nanowhisker-based drug delivery system

Recently, cellulose nanowhiskers have attracted attention as a promising material in the biomedical field because of their outstanding properties such as hydrophilicity, biocompatibility, biodegradability, and high surface area. In this work, we prepared a novel nanometric carrier molecule for amine-containing biologically active molecules and drugs employing functionalized cellulose nanowhiskers. The nanowhiskers were grafted with a spacer molecule, gamma aminobutyric acid, using a periodate oxidation and Schiff's base condensation reaction sequence. To achieve controlled and rapid delivery of the targeting moiety, syringyl alcohol, a releasable linker, was then attached to it. All the reactions were carried out in aqueous media and the resulting products were characterized by FT-IR, NMR, XPS, and TEM experiments to evaluate the functional groups, structure, and morphology respectively.

Releasable Luciferin−Transporter Conjugates: Tools for the Real-Time Analysis of Cellular Uptake and Release

The design, synthesis, and evaluation of conjugates of arginine-rich transporters and luciferin are described that release luciferin only after entry into cells that are stably transfected with luciferase. Each molecule of free luciferin that is released after entry generates a photon that can be measured allowing for real-time quantification of uptake and release in cells. The process provides a method to assay uptake and release of free luciferin as a function of variations in the releasable linker and in the transporter.