A long-acting C-natriuretic peptide for achondroplasia.

The C-natriuretic peptide (CNP) analog vosoritide has recently been approved for treatment of achondroplasia in children. However, the regimen requires daily subcutaneous injections in pediatric patients over multiple years. The present work sought to develop a long-acting CNP that would provide efficacy equal to or greater than that of vosoritide but require less frequent injections. We used a technology for half-life extension, whereby a drug is attached to tetra-polyethylene glycol hydrogels (tetra-PEG) by ß-eliminative linkers that cleave at predetermined rates. These hydrogels-fabricated as uniform ∼60-µm microspheres-are injected subcutaneously, where they serve as a stationary depot to slowly release the drug into the systemic circulation. We prepared a highly active, stable CNP analog-[Gln6,14]CNP-38-composed of the 38 C-terminal amino acids of human CNP-53 containing Asn to Gln substitutions to preclude degradative deamidation. Two microsphere [Gln6,14]CNP-38 conjugates were prepared, with release rates designed to allow once-weekly and once-monthly administration. After subcutaneous injection of the conjugates in mice, [Gln6,14]CNP-38 was slowly released into the systemic circulation and showed biphasic elimination pharmacokinetics with terminal half-lives of ∼200 and ∼600 h. Both preparations increased growth of mice comparable to or exceeding that produced by daily vosoritide. Simulations of the pharmacokinetics in humans indicated that plasma [Gln6,14]CNP-38 levels should be maintained within a therapeutic window over weekly, biweekly, and likely, monthly dosing intervals. Compared with vosoritide, which requires ∼30 injections per month, microsphere [Gln6,14]CNP-38 conjugates-especially the biweekly and monthly dosing-could provide an alternative that would be well accepted by physicians, patients, and patient caregivers.

Long-Acting Poly(ADP-ribose) Polymerase Inhibitor Prodrug for Humans.

Poly(ADP-ribose) polymerase inhibitors (PARPi) have been approved for once or twice daily oral use in the treatment of cancers with BRCA defects. However, for some patients, oral administration of PARPi may be impractical or intolerable, and a long-acting injectable formulation is desirable. We recently developed a long-acting PEGylated PARPi prodrug, PEG∼talazoparib (TLZ), which suppressed the growth of PARPi-sensitive tumors in mice for very long periods. However, the release rate of TLZ from the conjugate was too fast to be optimal in humans. We prepared several new PEG∼TLZ prodrugs having longer half-lives of drug release and accurately measured their pharmacokinetics in the rat. Using the rates of release of TLZ from these prodrugs and the known pharmacokinetics of free TLZ in humans, we simulated the pharmacokinetics of the macromolecular prodrugs and released TLZ in humans. From several possibilities, we chose two conjugates that could be administered intravenously every 2 weeks and maintain TLZ within its known therapeutic window. We describe situations where the PEG∼TLZ conjugates would find utility in humans and suggest how the intravenously administered long-acting prodrugs could in fact be more effective than daily oral administration of free TLZ.

Attenuation of the Reaction of Michael Acceptors with Biologically Important Nucleophiles.

ß-Elimination of drugs tethered to macromolecular carbamates provides a platform for drug half-life extension. However, the macromolecular Michael acceptor products formed upon drug release can potentially react with biological amines and thiols and may raise concerns about safety. We desired to mitigate this possibility by developing linkers that have predictable rates of ß-elimination but suppressed rates of nucleophilic addition to their Michael acceptor products. We prepared Michael acceptor products of ß-eliminative linkers that contained a methyl group at the Cß carbon or a gem-dimethyl group at the Cγ carbon and studied the kinetics of their reactions with the most prevalent biological nucleophiles-amine and thiol groups. Aza-Michael reactions with glycine are slowed about 20-fold by methylation of the ß-carbon and 175-fold with a gem-dimethyl group at the γ-carbon. Likewise, addition of the glutathione thiol to γ-gem-dimethyl Michael acceptors was retarded 7-24-fold compared to parent unsubstituted linkers. It was estimated that in an in vivo environment of ∼0.5 mM macromolecular thiols or ∼20 mM macromolecular amines-as in plasma-the reaction half-life of a typical Michael acceptor with a γ-gem-dimethyl linker could exceed 3 years for thiols or 25 years for amines. We also prepared a large series of γ-gem-dimethyl ß-eliminative linkers and showed excellent structure-activity relationships of elimination rates with corresponding unsubstituted parent linkers. Finally, we compared the first-generation unsubstituted and new gem-dimethyl ß-eliminative linkers in a once-monthly drug delivery system of a 39 amino acid peptide. Both linkers provided the desired half-life extension of the peptide, but the Michael acceptor formed from the gem-dimethyl linker was much less reactive. We conclude that the γ-gem-dimethyl ß-eliminative linkers provide high flexibility and greatly reduce potential reactions of Michael acceptor products with biologically important nucleophiles.

Subcutaneously Administered Self-Cleaving Hydrogel-Octreotide Conjugates Provide Very Long-Acting Octreotide.

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 ß-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.

Hydrogel Drug Delivery System Using Self-Cleaving Covalent Linkers for Once-a-Week Administration of Exenatide.

We have developed a unique long-acting drug-delivery system for the GLP-1 agonist exenatide. The peptide was covalently attached to Tetra-PEG hydrogel microspheres by a cleavable ß-eliminative linker; upon s.c. injection, the exenatide is slowly released at a rate dictated by the linker. A second ß-eliminative linker with a slower cleavage rate was incorporated in polymer cross-links to trigger gel degradation after drug release. The uniform 40 µm microspheres were fabricated using a flow-focusing microfluidic device and in situ polymerization within droplets. The exenatide-laden microspheres were injected subcutaneously into the rat, and serum exenatide measured over a one-month period. Pharmacokinetic analysis showed a t1/2,ß of released exenatide of about 7 days which represents over a 300-fold half-life extension in the rat and exceeds the half-life of any currently approved long-acting GLP-1 agonist. Hydrogel-exenatide conjugates gave an excellent Level A in vitro-in vivo correlation of release rates of the peptide from the gel, and indicated that exenatide release was 3-fold faster in vivo than in vitro. Pharmacokinetic simulations indicate that the hydrogel-exenatide microspheres should support weekly or biweekly subcutaneous dosing in humans. The rare ability to modify in vivo pharmacokinetics by the chemical nature of the linker indicates that an even longer acting exenatide is feasible.

Approach for Half-Life Extension of Small Antibody Fragments That Does Not Affect Tissue Uptake.

The utility of antigen-binding antibody fragments is often limited by their short half-lives. Half-life extension of such fragments is usually accomplished by attachment or binding to high-molecular-weight carriers that reduce the renal elimination rate. However, the higher hydrodynamic radius results in greater confinement in the vascular compartment and, thus, lower tissue distribution. We have developed a chemically controlled drug delivery system in which the drug is covalently attached to hydrogel microspheres by a self-cleaving ß-eliminative linker; upon subcutaneous injection, the t1/2,ß of the released drug acquires the t1/2 of linker cleavage. In the present work, we compared the pharmacokinetics of an anti-TNFα scFv, the same scFv attached to 40 kDa PEG by a stable linker, and the scFv attached to hydrogel microspheres by a self-cleaving linker. We also developed a general approach for the selective attachment of ß-eliminative linkers to the N-termini of proteins. In rats, the scFv had a t1/2,ß of 4 h and a high volume of distribution at steady state (Vd,SS), suggesting extensive tissue distribution. The PEG-scFv conjugate had an increased t1/2,ß of about 2 days but showed a reduced Vd,SS that was similar to the plasma volume. In contrast, the tissue-penetrable scFv released from the hydrogel system had a t1/2,ß of about 2 weeks. Thus, the cleavable microsphere-scFv conjugate releases its protein cargo with a prolonged half-life comparable to that of most full-length mAbs and in a form that has the high tissue distribution characteristic of smaller mAb fragments. Other antigen-binding antibody fragments should be amenable to the half-life extension approach described here.

The mechanism of pseudouridine synthases from a covalent complex with RNA, and alternate specificity for U2605 versus U2604 between close homologs.

RluB catalyses the modification of U2605 to pseudouridine (Ψ) in a stem-loop at the peptidyl transferase center of Escherichia coli 23S rRNA. The homolog RluF is specific to the adjacent nucleotide in the stem, U2604. The 1.3 Å resolution crystal structure of the complex between the catalytic domain of RluB and the isolated substrate stem-loop, in which the target uridine is substituted by 5-fluorouridine (5-FU), reveals a covalent bond between the isomerized target base and tyrosine 140. The structure is compared with the catalytic domain alone determined at 2.5 Å resolution. The RluB-bound stem-loop has essentially the same secondary structure as in the ribosome, with a bulge at A2602, but with 5-FU2605 flipped into the active site. We showed earlier that RluF induced a frame-shift of the RNA, moving A2602 into the stem and translating its target, U2604, into the active site. A hydrogen-bonding network stabilizes the bulge in the RluB-RNA but is not conserved in RluF and so RluF cannot stabilize the bulge. On the basis of the covalent bond between enzyme and isomerized 5-FU we propose a Michael addition mechanism for pseudouridine formation that is consistent with all experimental data.

Hydrogel drug delivery system with predictable and tunable drug release and degradation rates.

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 ß-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 ß-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 ß-eliminative linker to tether a drug to the hydrogel, and another ß-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.

Long-term stabilization of maleimide-thiol conjugates.

Michael-addition of a thiol to a maleimide is commonly used for bioconjugation of drugs to macromolecules. Indeed, both current FDA-approved antibody-drug conjugates-Brentuximab vedotin and Trastuzumab emtansine-and one approved PEGylated conjugate-Cimzia-contain a thiol-maleimide adduct. However, the ultimate in vivo fate of such adducts is to undergo disruptive cleavage by thiol exchange or stabilizing ring opening. Therapeutic efficacy of a conjugate can be compromised by thiol exchange and the released drug may show toxicities. However, if the succinimide moiety of a maleimide-thiol conjugate is hydrolyzed, the ring-opened product is stabilized toward cleavage. We determined rates of ring-opening hydrolysis and thiol exchange of a series of N-substituted succinimide thioethers formed by maleimide-thiol conjugation. Ring-opening of conjugates prepared with commonly used maleimides were too slow to serve as prevention against thiol exchange. However, ring-opening rates are greatly accelerated by electron withdrawing N-substituents, and ring-opened products have half-lives of over two years. Thus, conjugates made with electron-withdrawing maleimides may be purposefully hydrolyzed to their ring-opened counterparts in vitro to ensure in vivo stability.

Biodegradable tetra-PEG hydrogels as carriers for a releasable drug delivery system.

We have developed an approach to prepare drug-releasing Tetra-PEG hydrogels with exactly four cross-links per monomer. The gels contain two cleavable ß-eliminative linkers: one for drug attachment that releases the drug at a predictable rate, and one with a longer half-life placed in each cross-link to control biodegradation. Thus, the system can be optimized to release the drug before significant gel degradation occurs. The synthetic approach involves placing a heterobifunctional connector at each end of a four-arm PEG prepolymer; four unique end-groups of the resultant eight-arm prepolymer are used to tether a linker-drug, and the other four are used for polymerization with a second four-arm PEG. Three different orthogonal reactions that form stable triazoles, diazines, or oximes have been used for tethering the drug to the PEG and for cross-linking the polymer. Three formats for preparing hydrogel-drug conjugates are described that either polymerize preformed PEG-drug conjugates or attach the drug postpolymerization. Degradation of drug-containing hydrogels proceeds as expected for homogeneous Tetra-PEG gels with minimal degradation occurring in early phases and sharp, predictable reverse gelation times. The minimal early degradation allows design of gels that show almost complete drug release before significant gel-drug fragments are released.

Predictable and tunable half-life extension of therapeutic agents by controlled chemical release from macromolecular conjugates.

Conjugation to macromolecular carriers is a proven strategy for improving the pharmacokinetics of drugs, with many stable polyethylene glycol conjugates having reached the market. Stable conjugates suffer several limitations: loss of drug potency due to conjugation, confining the drug to the extracellular space, and the requirement for a circulating conjugate. Current research is directed toward overcoming such limitations through releasable conjugates in which the drug is covalently linked to the carrier through a cleavable linker. Satisfactory linkers that provide predictable cleavage rates tunable over a wide time range that are useful for both circulating and noncirculating conjugates are not yet available. We describe such conjugation linkers on the basis of a nonenzymatic ß-elimination reaction with preprogrammed, highly tunable cleavage rates. A set of modular linkers is described that bears a succinimidyl carbonate group for attachment to an amine-containing drug or prodrug, an azido group for conjugation to the carrier, and a tunable modulator that controls the rate of ß-eliminative cleavage. The linkers provide predictable, tunable release rates of ligands from macromolecular conjugates both in vitro and in vivo, with half-lives spanning from a range of hours to >1 y at physiological pH. A circulating PEG conjugate achieved a 56-fold half-life extension of the 39-aa peptide exenatide in rats, and a noncirculating s.c. hydrogel conjugate achieved a 150-fold extension. Using slow-cleaving linkers, the latter may provide a generic format for once-a-month dosage forms of potent drugs. The releasable linkers provide additional benefits that include lowering C(max) and pharmacokinetic coordination of drug combinations.

Could a Long-Acting Prodrug of SN-38 be Efficacious in Sacituzumab Govitecan-Resistant Tumors?

We previously proposed that sacituzumab govitecan (SG, Trodelvy®) likely acts as a simple prodrug of systemic SN-38 as well as an antibody drug conjugate (ADC). In the present commentary, we assess whether a long-acting SN-38 prodrug, such as PLX038, might be efficacious in SG-resistant patients. We first describe possible mechanisms of action of SG, with new insights on pharmacokinetics and TROP2 receptor occupancy. We argue that SG is not an optimal conventional ADC and that the amount of systemic SN-38 spontaneously hydrolyzed from the ADC is so high it must have activity. Then, we describe the concept of time-over-target as related to the pharmacology of SG and PLX038 as SN-38 prodrugs. To be clear, we are not in any way suggesting that PLX038 or any SN-38 prodrug is superior to SG as an anticancer agent. Clearly, SG has the benefit over antigen-independent SN-38 prodrugs in that it targets cells with the TROP2 receptor. However, we surmise that PLX038 should be a more efficacious and less toxic prodrug of systemic SN-38 than SG. Finally, we suggest possible mechanisms of SG resistance and how PLX038 might perform in the context of each. Taken together, we argue that-contrary to many opinions-SG does not exclusively act as a conventional ADC, and propose that PLX038 may be efficacious in some settings of SG-resistance.

A platform technology for ultra-long acting intratumoral therapy.

Intratumoral (IT) therapy is a powerful method of controlling tumor growth, but a major unsolved problem is the rapidity that injected drugs exit tumors, limiting on-target exposure and efficacy. We have developed a generic long acting IT delivery system in which a drug is covalently tethered to hydrogel microspheres (MS) by a cleavable linker; upon injection the conjugate forms a depot that slowly releases the drug and "bathes" the tumor for long periods. We established technology to measure tissue pharmacokinetics and studied MSs attached to SN-38, a topoisomerase 1 inhibitor. When MS ~ SN-38 was injected locally, tissues showed high levels of SN-38 with a long half-life of ~ 1 week. IT MS ~ SN-38 was ~ tenfold more efficacious as an anti-tumor agent than systemic SN-38. We also propose and provide an example that long-acting IT therapy might enable safe use of two drugs with overlapping toxicities. Here, long-acting IT MS ~ SN-38 is delivered with concurrent systemic PARP inhibitor. The tumor is exposed to both drugs whereas other tissues are exposed only to the systemic drug; synergistic anti-tumor activity supported the validity of this approach. We propose use of this approach to increase efficacy and reduce toxicities of combinations of immune checkpoint inhibitors such as αCTLA-4 and αPD-1.

Synergistic Antitumor Activity of Talazoparib and Temozolomide in Malignant Rhabdoid Tumors.

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.

PLX038A, a long-acting SN-38, penetrates the blood-tumor-brain-barrier, accumulates and releases SN-38 in brain tumors to increase survival of tumor bearing mice.

Central nervous system tumors have resisted effective chemotherapy because most therapeutics do not penetrate the blood-tumor-brain-barrier. Nanomedicines between ~ 10 and 100 nm accumulate in many solid tumors by the enhanced permeability and retention effect, but it is controversial whether the effect can be exploited for treatment of brain tumors. PLX038A is a long-acting prodrug of the topoisomerase 1 inhibitor SN-38. It is composed of a 15 nm 4-arm 40 kDa PEG tethered to four SN-38 moieties by linkers that slowly cleave to release the SN-38. The prodrug was remarkably effective at suppressing growth of intracranial breast cancer and glioblastoma (GBM), significantly increasing the life span of mice harboring them. We addressed the important issue of whether the prodrug releases SN-38 systemically and then penetrates the brain to exert anti-tumor effects, or whether it directly penetrates the blood-tumor-brain-barrier and releases the SN-38 cargo within the tumor. We argue that the amount of SN-38 formed systemically is insufficient to inhibit the tumors, and show by PET imaging that a close surrogate of the 40 kDa PEG carrier in PLX038A accumulates and is retained in the GBM. We conclude that the prodrug penetrates the blood-tumor-brain-barrier, accumulates in the tumor microenvironment and releases its SN-38 cargo from within. Based on our results, we pose the provocative question as to whether the 40 kDa nanomolecule PEG carrier might serve as a "Trojan horse" to carry other drugs past the blood-tumor-brain-barrier and release them into brain tumors.

Prostate-Specific Membrane Antigen Targeted StarPEG Nanocarrier for Imaging and Therapy of Prostate Cancer.

The tumor uptake of large non-targeted nanocarriers primarily occurs through passive extravasation, known as the enhanced permeability and retention (EPR) effect. Prior studies demonstrated improved tumor uptake and retention of 4-arm 40 kDa star polyethylene glycol (StarPEG) polymers for cancer imaging by adding prostate-specific membrane antigen (PSMA) targeting small molecule ligands. To test PSMA-targeted delivery and therapeutic efficacy, StarPEG nanodrugs with/without three copies of PSMA-targeting ligands, ACUPA, are designed and synthesized. For single-photon emission computed tomography (SPECT) imaging and therapy, each nanocarrier is labeled with 177Lu using DOTA radiometal chelator. The radiolabeled nanodrugs, [177Lu]PEG-(DOTA)1 and [177Lu]PEG-(DOTA)1(ACUPA)3, are evaluated in vitro and in vivo using PSMA+ PC3-Pip and/or PSMA- PC3-Flu cell lines, subcutaneous xenografts and disseminated metastatic models. The nanocarriers are efficiently radiolabeled with 177Lu with molar activities 10.8-15.8 MBq/nmol. Besides excellent in vitro PSMA binding affinity (kD = 51.7 nM), the targeted nanocarrier, [177Lu]PEG-(DOTA)1(ACUPA)3, demonstrated excellent in vivo SPECT imaging contrast with 21.3% ID/g PC3-Pip tumors uptake at 192 h. Single doses of 18.5 MBq [177Lu]PEG-(DOTA)1(ACUPA)3 showed complete resolution of the PC3-Pip xenografts observed up to 138 days. Along with PSMA-targeted excellent imaging contrast, these results demonstrated high treatment efficacy of [177Lu]PEG-(DOTA)1(ACUPA)3 for prostate cancer, with potential for clinical translation.

ß-Eliminative releasable linkers adapted for bioconjugation of macromolecules to phenols.

We recently reported a chemical approach for half-life extension that utilizes sets of releasable linkers to attach drugs to macromolecules via a cleavable carbamate group (Santi et al., Proc. Nat. Acad. Sci. U.S.A. 2012, 109, 6211-6216). The linkers undergo a ß-elimination cleavage to release the free, native amine-containing drug. A limitation of the technology is the requirement for an amino group on the drug in order to form the carbamate bond, since most small molecules do not have an amine functional group. Here, we describe an approach to adapt these same ß-elimination carbamate linkers so they can be used to connect other acidic heteroatoms, in particular, phenolic hydroxyl groups. The approach utilizes a methylene adaptor to connect the drug to the carbamate nitrogen, and an electron-withdrawing group attached to carbamate nitrogen to stabilize the system against a pH-independent spontaneous cleavage. Carbamate cleavage is driven by ß-elimination to give a carboxylated aryl amino Mannich base which rapidly collapses to give the free drug, an aryl amine, and formaldehyde.

A Very Long-acting Exatecan and Its Synergism with DNA Damage Response Inhibitors.

Exatecan (Exa) is a very potent inhibitor of topoisomerase I and anticancer agent. It has been intensively studied as a single agent, a large macromolecular conjugate and as the payload component of antigen-dependent antibody-drug conjugates. The current work describes an antigen-independent conjugate of Exa with polyethylene glycol (PEG) that slowly releases free Exa. Exa was conjugated to a 4-arm 40 kDa PEG through a ß-eliminative cleavable linker. Pharmacokinetic studies in mice showed that the conjugate has an apparent circulating half-life of 12 hours, which reflects a composite of both the rate of renal elimination (half-life ∼18 hours) and release of Exa (half-life ∼40 hours). Remarkably, a single low dose of 10 µmol/kg PEG-Exa-only approximately 0.2 µmol/mouse-caused complete suppression of tumor growth of BRCA1-deficient MX-1 xenografts lasting over 40 days. A single low dose of 2.5 µmol/kg PEG-Exa administered with low but efficacious doses of the PARP inhibitor talazoparib showed strong synergy and caused significant tumor regression. Furthermore, the same low, single dose of PEG-Exa administered with the ATR inhibitor VX970 at doses of the DNA damage response inhibitor that do not affect tumor growth show high tumor regression, strong synergy, and synthetic lethality. Significance: A circulating conjugate that slowly releases Exa is described. It is efficacious after a single dose and synergistic with ATR and PARP inhibitors.

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

BACKGROUND: Interleukin-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 very short half-life and a single administration does not provide the sustained exposure required for optimal stimulation of target immune cells. The purpose of this work was to develop a very long-acting prodrug that would maintain IL-15 within a narrow therapeutic window for long periods-similar to a continuous infusion. METHODS: We prepared and characterized hydrogel microspheres (MS) covalently attached to IL-15 (MS~IL-15) by a releasable linker. The pharmacokinetics and pharmacodynamics of MS~IL-15 were determined in C57BL/6J mice. The antitumor activity of MS~IL-15 as a single agent, and in combination with a suitable therapeutic antibody, was tested in a CD8+ T cell-driven bilateral transgenic adenocarcinoma mouse prostate (TRAMP)-C2 model of prostatic cancer and a NK cell-driven mouse xenograft model of human ATL (MET-1) murine model of adult T-cell leukemia. RESULTS: On subcutaneous administration to mice, the cytokine released from the depot maintained a long half-life of about 168 hours over the first 5 days, followed by an abrupt decrease to about ~30 hours in accordance with the development of a cytokine sink. A single injection of MS~IL-15 caused remarkably prolonged expansions of NK and ɣδ T cells for 2 weeks, and CD44hiCD8+ T cells for 4 weeks. In the NK cell-driven MET-1 murine model of adult T-cell leukemia, single-agent MS~IL-1550 µg or anti-CCR4 provided modest increases in survival, but a combination-through antibody-depedent cellular cytotoxicity (ADCC)-significantly extended survival. In a CD8+ T cell-driven bilateral TRAMP-C2 model of prostatic cancer, single agent subcutaneous MS~IL-15 or unilateral intratumoral agonistic anti-CD40 showed modest growth inhibition, but the combination exhibited potent, prolonged bilateral antitumor activity. CONCLUSIONS: Our results show MS~IL-15 provides a very long-acting IL-15 with low Cmax that elicits prolonged expansion of target immune cells and high anticancer activity, especially when administered in combination with a suitable immuno-oncology agent.

PLX038: A Long-Acting Topoisomerase I Inhibitor With Robust Antitumor Activity in ATM-Deficient Tumors and Potent Synergy With PARP Inhibitors.

Alterations in the ATM gene are among the most common somatic and hereditary cancer mutations, and ATM-deficient tumors are hypersensitive to DNA-damaging agents. A synthetic lethal combination of DNA-damaging agents and DNA repair inhibitors could have widespread utility in ATM-deficient cancers. However, overlapping normal tissue toxicities from these drug classes have precluded their clinical translation. We investigated PLX038, a releasable polyethylene glycol-conjugate of the topoisomerase I inhibitor SN-38, in ATM wild-type and null isogenic xenografts and in a BRCA1-deficient xenograft. PLX038 monotherapy and combination with PARP inhibition potently inhibited the growth of both BRCA1- and ATM-deficient tumors. A patient with an ATM-mutated breast cancer treated with PLX038 and the PARP inhibitor rucaparib achieved rapid, symptomatic, and radiographic complete response lasting 12 months. Single-agent PLX038 or PLX038 in combination with DNA damage response inhibitors are novel therapeutic paradigms for patients with ATM-loss cancers.