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Abstract Background: Malignant rhabdoid tumors (MRTs) are among the most aggressive and treatment-resistant malignancies affecting infants, originating in the kidney, brain, liver, and soft tissues. The 5-year event-free survival rate for these cancers is a mere 20%. In nearly all cases of MRT, the SMARCB1 gene (occasionally SMARCA4) - a pivotal component of the SWI/SNF chromatin remodeling complex - is homozygously deleted, although the precise etiology of these tumors remains unknown. While young patients with localized MRT generally show improved outcomes, especially those who are older and have early-stage disease, the overall prognosis remains poor despite optimal standard treatments. This highlights the urgent need for more effective treatment strategies. PARP1 is a widely targeted molecule in cancer treatment and, beyond its role in DNA repair, it participates in transcriptional regulation by recruiting chromatin remodeling complexes to modulate DNA accessibility for RNA polymerases. Methods: We evaluated the antitumor activity of the PARP1 inhibitor talazoparib (TLZ) in combination with the DNA alkylating agent temozolomide (TMZ) across six MRT xenograft models in CB-17 scid mice. The dosing schedule for this combination followed a previously reported protocol (iScience 2021; 25(2):103725). Time-to-event was defined as 4-fold increase in tumor xenograft volume from the treatment initiation date. Event-free survival (EFS) between treated and control groups was analyzed using Kaplan-Meier method. Objective response categories including partial (PR), complete (CR), and maintained complete responses (MCR), were determined based on previously established criteria (Ped Blood Cancer 2007;49:928). To widen the therapeutic window of the drug combination, we employed PEGylated TLZ (PEG-TLZ), which has been reported to reduce systemic toxicity through slow drug release. Results: Remarkably, our findings indicate that five out of six MRT xenografts exhibited objective response to PEG-TLZ+TMZ therapy. Notably, SMARCB1 loss was found to confer a protective effect, correlating with elevated expression of DNA damage and repair proteins in SMARCB1-deficient MRT cells. Additionally, MGMT emerged as a potential biomarker predictive of in vivo MRT response to PEG-TLZ+TMZ treatment. In addition, our analysis revealed alterations in signaling pathways associated with the observed antitumor activity. Conclusions: This study presents a novel and efficacious therapeutic approach for MRT, along with a promising candidate biomarker for predicting tumor response. Citation Format: Elena Mironova, Sebastian Molinas, Vanessa Del Pozo, Abhik Bandyopadhyay, Zhao Lai, Dias Kurmashev, Eric L. Schneider, Daniel V. Santi, Yidong Chen, Raushan Kurmasheva. Synergistic antitumor activity of the combined PARP1 inhibition and alkylating DNA damage in malignant rhabdoid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7039.

The objective of this work was to develop a long-acting form of the lipidated peptide semaglutide that can be administered to humans once-monthly. Semaglutide was attached to hydrogel microspheres by a cleavable linker with an expected in vivo release half-life of about 1 mo. After a single subcutaneous dose, the pharmacokinetic parameters of released semaglutide and bodyweight loss were determined in mice, and results were used to estimate the dosing and pharmacokinetics of the released semaglutide in humans. The in vivo half-life of released semaglutide was ~36 d, and a single dose in diet-induced obese mice resulted in a lean-sparing body weight loss of 20% over 1 mo, statistically the same as semaglutide dosed twice daily. Simulations indicated the microsphere-semaglutide would permit once-monthly administration in humans; moreover, it could maintain the therapeutic minimum concentration (Cmin) of once-weekly semaglutide with only 75% of the once-weekly maximum concentration (Cmax), a feature that could reduce adverse side effects or allow higher dosing. The same approach should enable the conversion of other lipidated peptides from once-weekly to once-monthly administration.

Abstract The objective of this work was to develop a long-acting form of the lipidated peptide semaglutide that can be administered to humans once-monthly. Semaglutide was attached to 50 μ diameter hydrogel microspheres by a cleavable linker with an expected in vivo release half-life of about one-month. After a single subcutaneous dose, the pharmacokinetic parameters of released semaglutide were determined in normal mice and the bodyweight loss was determined in diet induced obese mice. The results were used to simulate the pharmacokinetics of semaglutide released from the microspheres in humans.Semaglutide tethered to microspheres by a cleavable linker could be completely released with an in vitro half-life of ∼55 days at pH 7.4. The in vivo half-life of released semaglutide was ∼30 days, and a single dose in diet-induced obese mice resulted in a lean-sparing body weight loss of 20% over 1 month, statistically the same as semaglutide dosed twice daily. Simulations indicated the microsphere-semaglutide would permit once-monthly administration in humans. The microsphere-semaglutide conjugate described here should be suitable for once-monthly dosing in humans, and the same approach should enable conversion of other lipidated peptides from once-weekly to once-monthly administration.

Malignant rhabdoid tumors (MRTs) are among the most aggressive and treatment-resistant malignancies affecting infants, originating in the kidney, brain, liver, and soft tissues. The 5-year event-free survival rate for these cancers is a mere 20%. In nearly all cases of MRT, the SMARCB1 gene (occasionally SMARCA4)-a pivotal component of the SWI/SNF chromatin remodeling complex-is homozygously deleted, although the precise etiology of these tumors remains unknown. While young patients with localized MRT generally show improved outcomes, especially those who are older and have early-stage disease, the overall prognosis remains poor despite optimal standard treatments. This highlights the urgent need for more effective treatment strategies. We investigated the antitumor activity of a PARP1 inhibitor (talazoparib, TLZ) combined with a DNA alkylating agent (temozolomide, TMZ) in MRT xenograft models. PARP1 is a widely targeted molecule in cancer treatment and, beyond its role in DNA repair, it participates in transcriptional regulation by recruiting chromatin remodeling complexes to modulate DNA accessibility for RNA polymerases. To widen the therapeutic window of the drug combination, we employed PEGylated TLZ (PEG~TLZ), which has been reported to reduce systemic toxicity through slow drug release. Remarkably, our findings indicate that five out of six MRT xenografts exhibited an objective response to PEG~TLZ+TMZ therapy. Significantly, the loss of SMARCB1 was found to confer a protective effect, correlating with higher expression levels of DNA damage and repair proteins in SMARCB1-deficient MRT cells. Additionally, we identified MGMT as a potential biomarker indicative of in vivo MRT response to PEG~TLZ+TMZ therapy. Moreover, our analysis revealed alterations in signaling pathways associated with the observed antitumor efficacy. This study presents a novel and efficacious therapeutic approach for MRT, along with a promising candidate biomarker for predicting tumor response.

Abstract Background. Intra-tumoral (IT) injections of therapeutics hold promise for the treatment of solid tumors. However, drugs administered directly in the tumor have short IT residence times potentially limiting their exposure and therefore efficacy. We are developing a platform of novel hydrogel microsphere (MS) conjugates that enables long-acting tumor residence after a single injection of the conjugate. We demonstrate this approach not only solves the problem of short acting intra-tumoral therapy, but also allows for the safe administration of two therapies with overlapping toxicities such as SN-38 and a PARP inhibitor (PARPi). Methods. Hydrogel microsphere (MS) conjugates were prepared and characterized. Using MS conjugates labeled with rhodamine and fluorescein, we established methods to directly measure the IT concentration and half-life of the released drug after IT administration into CT26 tumor bearing mice. We next measured the tissue pharmacokinetics and efficacy of MSs attached to SN-38, a potent topoisomerase 1 inhibitor and immunomodulator, using NSG mice bearing 22Rv1 ATM-/- xenografts. Finally, we measured the anti-tumor efficacy of MS~SN-38 in combination with an oral PARPi, talazoparib, in 22Rv1 ATM-/- xenografts. Results. We successfully established methods to quantify the concentration of released drug and MS bound drug from tumor biopsies using fluorescent surrogates. When applied to MS~SN-38 conjugates, locally injected tissues showed high levels of SN-38 with a long half-life of about one week. In contrast, the systemic half-life of free SN-38 is ~30 minutes. IT MS~SN-38 was ~10-fold more efficacious as an anti- tumor agent than systemic SN-38 from subcutaneous administration of MS~SN-38. Finally, we show high anti-tumor synergy from the SN-38 localized in a tumor by IT MS~SN-38 in combination with systemic administration of the PARPi talazoparib. Conclusions. This microsphere technology allows for sustained delivery of high local concentrations of drugs administered intratumorally. Here, IT MS~SN-38 resulted in an intra-tumoral half-life of ~1 week and marked anti-tumor efficacy in 22Rv1 ATM-/- xenografts. We also demonstrated that long-acting IT injections enables safe use of drug combinations with well-described overlapping toxicities such as SN-38 and PARPi. The microsphere technology may offer an alternative strategy for localizing and sustaining drugs in tumors and may mitigate toxicities from combination therapy where two agents have a similar toxicological profile. Other combinations, including small molecules and immuno-oncology agents, are being explored. Citation Format: John A. Hangasky, Jeff Henise, Deborah Charych, Christopher W. Carreras, Gary Ashley, Daniel V. Santi. Intra-tumoral administration of releasable hydrogel microsphere drug conjugates for long acting therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB439.

Tumoral uptake of large-size nanoparticles is mediated by the enhanced permeability and retention (EPR) effect, with variable accumulation and heterogenous tumor tissue penetration depending on the tumor phenotype. The performance of nanocarriers via specific targeting has the potential to improve imaging contrast and therapeutic efficacy in vivo with increased deep tissue penetration. To address this hypothesis, we designed and synthesized prostate cancer-targeting starPEG nanocarriers (40 kDa, 15 nm), [89Zr]PEG-(DFB)3(ACUPA)1 and [89Zr]PEG-(DFB)1(ACUPA)3, with one or three prostate-specific membrane antigen (PSMA)-targeting ACUPA ligands. The in vitro PSMA binding affinity and in vivo pharmacokinetics of the targeted nanocarriers were compared with a nontargeted starPEG, [89Zr]PEG-(DFB)4, in PSMA+ PC3-Pip and PSMA- PC3-Flu cells, and xenografts. Increasing the number of ACUPA ligands improved the in vitro binding affinity of PEG-derived polymers to PC3-Pip cells. While both PSMA-targeted nanocarriers significantly improved tissue penetration in PC3-Pip tumors, the multivalent [89Zr]PEG-(DFB)1(ACUPA)3 showed a remarkably higher PC3-Pip/blood ratio and background clearance. In contrast, the nontargeted [89Zr]PEG-(DFB)4 showed low EPR-mediated accumulation with poor tumor tissue penetration. Overall, ACUPA conjugated targeted starPEGs significantly improve tumor retention with deep tumor tissue penetration in low EPR PC3-Pip xenografts. These data suggest that PSMA targeting with multivalent ACUPA ligands may be a generally applicable strategy to increase nanocarrier delivery to prostate cancer. These targeted multivalent nanocarriers with high tumor binding and low healthy tissue retention could be employed in imaging and therapeutic applications.

The goal was to develop and characterize a companion diagnostic for the releasable PEG40kDa∼SN-38 oncology drug, PLX038, that would identify tumors susceptible to high accumulation of PLX038. PEG conjugates of the zirconium ligand desferroxamine B (DFB) of similar size and charge to PLX038 were prepared that contained one or four DFB, as well as one that contained three SN-38 moieties and one DFB. Uptake and associated kinetic parameters of the 89Zr-labeled nanocarriers were determined in tumor and normal tissues in mice using μPET/CT imaging. The data were fit to physiologically based pharmacokinetic models to simulate the mass-time profiles of distribution of conjugates in the tissues of interest. The time-activity curves for normal tissues showed high levels at the earliest time of measurement due to vascularization, followed by a monophasic loss. In tumors, levels were initially lower than in normal tissues but increased to 9% to 14% of injected dose over several days. The efflux half-life in tumors was very long, approximately 400 hours, and tumor levels remained at about 10% injected dose 9 days after injection. Compared with diagnostic liposomes, the PEG nanocarriers have a longer serum half-life, are retained in tumors at higher levels, remain there longer, and afford higher tumor exposure. The small PEG40kDa nanocarriers studied here show properties for passive targeting of tumors that are superior than most nanoparticles and might be effective probes to identify tumors susceptible to similar size therapeutic nanocarriers such as PLX038.

We developed a long-acting drug-delivery system that supports subcutaneous administration of the peptidic somatostatin agonist octreotide-a blockbuster drug used to treat acromegaly and neuroendocrine tumors. The current once-a-month polymer-encapsulated octreotide, Sandostatin LAR, requires a painful intragluteal injection through a large needle by a health-care professional. To overcome such shortcomings, Tetra-PEG hydrogel microspheres were covalently attached to the α-amine of d-Phe(1) or the ε-amine of Lys(5) of octreotide by a self-cleaving β-eliminative linker; upon subcutaneous injection in the rat using a small-bore needle, octreotide was slowly released. The released drug from the ε-octreotide conjugate showed a remarkably long serum half-life that exceeded two months. The α-octreotide conjugate had a half-life of ∼2 weeks, and showed an excellent correlation of in vitro and in vivo drug release. Pharmacokinetic models indicate these microspheres should support once-weekly to once-monthly self-administered subcutaneous dosing in humans. The hydrogel-octreotide conjugate shows the favorable pharmacokinetics of Sandostatin LAR without its drawbacks.

Many drugs and drug candidates are suboptimal because of short duration of action. For example, peptides and proteins often have serum half-lives of only minutes to hours. One solution to this problem involves conjugation to circulating carriers, such as PEG, that retard kidney filtration and hence increase plasma half-life of the attached drug. We recently reported an approach to half-life extension that uses sets of self-cleaving linkers to attach drugs to macromolecular carriers. The linkers undergo β-eliminative cleavage to release the native drug with predictable half-lives ranging from a few hours to over 1 y; however, half-life extension becomes limited by the renal elimination rate of the circulating carrier. An approach to overcoming this constraint is to use noncirculating, biodegradable s.c. implants as drug carriers that are stable throughout the duration of drug release. Here, we use β-eliminative linkers to both tether drugs to and cross-link PEG hydrogels, and demonstrate tunable drug release and hydrogel erosion rates over a very wide range. By using one β-eliminative linker to tether a drug to the hydrogel, and another β-eliminative linker with a longer half-life to control polymer degradation, the system can be coordinated to release the drug before the gel undergoes complete erosion. The practical utility is illustrated by a PEG hydrogel-exenatide conjugate that should allow once-a-month administration, and results indicate that the technology may serve as a generic platform for tunable ultralong half-life extension of potent therapeutics.