Current Role of Topoisomerase I Inhibitors for the Treatment of Mesenchymal Malignancies and Their Potential Future Use as Payload of Sarcoma-Specific Antibody-Drug Conjugates Open Access

Deoxyribonucleic acid (DNA) topoisomerases are found ubiquitously expressed in all organisms as they are enzymes involved in essential cellular processes [1‒3]. DNA topoisomerases are involved in a broad range of functions related to DNA, including the packaging of DNA in the nucleus, preventing and resolving DNA entanglements, and organizing DNA supercoiling during transcription and replication [1‒3]. In eukaryotes, there are two main subfamilies of DNA topoisomerases, namely, type I and type II [1, 2]. There are 4 genes in human cells encoding for type I topoisomerase: type IB consisting of nuclear topoisomerase I [TOP1] and mitochondrial DNA topoisomerase [TOP1MT], and type IA consisting of TOP3A, and TOP3B [2, 4].

The TOP1 gene encodes for the enzyme topoisomerase I [1, 2], which is essential to genomic stability as it removes positive and negative DNA supercoils that might otherwise lead to DNA breaks [5]. Topoisomerase I catalyzes the relaxation of supercoiled double-stranded DNA (dsDNA) [1, 2, 4, 6, 7]. During vital cell processes, such as DNA replication, transcription, and recombination, topoisomerase I reversibly cleaves with the supercoiled dsDNA and cuts one DNA strand on the dsDNA helix [1, 2, 4, 6, 7]. Topoisomerase I forms a covalent bond between the cut 3’-phosphotyrosyl end (3’-end) of the DNA, called the topoisomerase I cleavage complex (TOP1CC) [2, 4]. The torsional strain in the supercoiled DNA allows the rotation of the 5’-hydroxyl end (5’-end) of the cut DNA, around the intact DNA strand [2, 4, 6, 7]. The 5’-end DNA is then realigned with the 3’-end DNA allowing for the religation, or reattachment, of the DNA and the dissociation of topoisomerase I [2, 4]. This relieves the DNA’s torsional strain allowing relaxation of the supercoiled DNA, enabling the dsDNA to separate to form the templates needed during replication, transcription, recombination, and repair [1, 2, 4, 6, 7].

Human topoisomerase I expression levels are lower in healthy cells, but higher in rapidly dividing cancer cells [8]. Rapid, uncontrolled cell proliferation is a characteristic of aggressive malignancies, and these tumor cells would be reliant on topoisomerases to allow the high replication and transcription rates of DNA [9]. High TOP1 gene copy number, mRNA, and protein levels, as well as enzyme activity, are associated with unfavorable prognosis in some epithelial cancers [10, 11]. Inhibition of topoisomerases is an important principle of anticancer treatment [1, 9], and inhibitors of topoisomerases I and II are broadly used in the clinic. A correlation has been observed between the level of human topoisomerase I expression in cells and the sensitivity of cells to topoisomerase I inhibitors [8]. During the topoisomerase I cleavage reaction and the formation of TOP1CC, topoisomerase I is vulnerable to topoisomerase I inhibitors [4]. Topoisomerase I inhibitors reversibly bind to TOP1CC at the topoisomerase I-DNA interface, trapping the TOP1CC, and this stabilization of TOP1CC inhibits the religation of the 5’-end DNA with the 3’-end DNA [2, 4, 7, 8]. Topoisomerase I inhibitors thereby cause disruption of the DNA strand, leading to DNA damage, cell cycle arrest, and apoptosis of malignant cells [2, 4, 7, 8].

The efficacy of topoisomerase inhibitors is positively correlated with the expression level of topoisomerase [12]. Human cancer cells depleted of topoisomerase I become resistant to the topoisomerase I inhibitor camptothecin [13]. However, it is not known whether the correlation between topoisomerase levels and drug response is a simple linear relationship and which assay would be optimal for quantifying the level of topoisomerase I. The TOP1 locus (20q12) is often amplified in colon carcinoma [14], and the increased expression of topoisomerase I in this tumor type may in part explain why its inhibitors are a mainstay therapy for this disease [15]. The expression level of topoisomerase I and its relevance as a prognostic and/or predictive biomarker in sarcoma are unknown. Similarly, in vitro and in vivo studies have shown that levels of expression of Top2A determine the response to doxorubicin (a topoisomerase II poison), with resistance to doxorubicin being produced with the suppression of Top2A [16]. Furthermore, in patients with high-risk soft tissue sarcoma (STS) treated with neoadjuvant chemotherapy, Top2A levels have been found to be a predictive indicator of survival [17].

Natural sources of topoisomerase I inhibitors include camptothecin, which was discovered in plant extracts over 60 years ago and was first isolated in the wooden stem of the native Chinese tree Camptotheca acuminata (the bark of which is used in traditional Chinese medicine). Camptothecin has subsequently been chemically synthesized [18]. Topoisomerase I inhibitors have been approved for the treatment of various epithelial malignancies [18, 19]. The registered topoisomerase I inhibitors are related to camptothecin: irinotecan is approved for metastatic colorectal cancer [20]; topotecan is approved for metastatic ovarian cancer, small cell lung cancer (SCLC), and cervical cancer [19, 21, 22]; and the irinotecan liposomal injection MM-398 is approved for metastatic pancreatic cancer [19, 23, 24]. Several other camptothecin derivatives have been explored in the preclinical setting or in early clinical trials, without achieving regulatory approval, such as rubitecan, exatecan, lurtotecan, and gimatecan [4, 8]. No new topoisomerase I inhibitor has been approved as a free drug for any oncology indication since 2007, while the liposomal injection MM-398 obtained FDA approval in pancreatic cancer in 2015 [1, 25].

There are characteristic adverse events (AEs) associated with the systemic use of free drug and prodrug topoisomerase I inhibitors. The short plasma half-life (t_1/2) and chemical instability may reduce the efficacy of camptothecin-based topoisomerase I inhibitors and contribute to AEs [7]. Dose-limiting toxicities (DLTs) with topoisomerase I inhibitors can occur relatively frequently and can limit their usage, with the main DLTs being diarrhea, other gastrointestinal AEs, and myelosuppression [5, 20, 21, 24, 26]. Early- and late-onset diarrhea can occur with irinotecan; diarrhea can happen shortly after administration, while late-onset can develop 24 h after irinotecan administration and can be life-threatening [5, 20, 26]. The early-onset diarrhea seen is part of a cholinergic syndrome and can be prevented by the use of atropine [20]. Irinotecan and topotecan cause myelosuppression, severe neutropenia can be associated with infection, and sepsis can occur which can be fatal [5, 20, 21]. The toxicity of topoisomerase I inhibitors can be enhanced in patients with hereditary enzyme deficiencies such as genetic variations in UDP-glucuronosyltransferase 1A1 gene (UGT1A1) [5, 27]. The UGT1A1 enzyme catalyzes the glucuronidation of SN-38 (the active metabolite of irinotecan) to the detoxified SN-38 glucuronide which is excreted in the bile, and patients with inherited UGT1A1 deficiency have impaired clearance of SN-38 and increased risk of severe irinotecan toxicity [5, 27‒29].

Sarcomas are a heterogenous group of solid, often very aggressive tumors with very limited systemic treatment options, and there is an unmet clinical need for effective treatments to improve outcomes especially for those with relapsed, locally advanced/inoperable, and/or metastatic disease [30]. An overview of key preclinical and clinical studies with conventional and experimental topoiso-merase I inhibitors for the treatment of sarcoma is the focus of this review.

The camptothecin analog irinotecan is a water-soluble prodrug inhibitor which is converted endogenously by carboxylesterase-converting enzyme to SN-38, the active metabolite [26, 31]. In vitro studies using purified calf thymus topoisomerase I and DNA demonstrated that SN-38 (1 nmol/L) had 1,000-fold greater potency than irinotecan (1 µmol/L) in inhibiting the religation reaction of the TOP1CC; similar results were obtained from studies with P388 leukemia and Ehrlich carcinoma cells [32]. Studies in P388 cells showed that irinotecan required 1,000-fold higher concentrations than SN-38 to achieve the same single-strand DNA breaks [32]. Irinotecan and SN-38 demonstrated activity in vitro, in various tumors including breast, colorectal, ovarian, and non-small cell lung cancer (NSCLC) [32]. Synergistic or additive activity was observed with the combination of irinotecan/SN-38 and cisplatin or 5-fluorouracil (5-FU) in human cell lines of CRC (C4), leukemia (MOLT-3), lymphoma (Dauji), and in 4 NSCLC cell lines [32].

In vivo, SN-38 acts through the stabilization of the topoisomerase I-DNA complex, which prevents religation of DNA, causing S-phase-specific cytotoxicity [2, 4, 26, 31]. In vivo studies have shown that irinotecan has a broad spectrum of activity in murine tumor models when administered intravenously, orally, and intraperitoneally [32]. Irinotecan was more active than doxorubicin in L1210 leukemia and had notable efficacy in Ehrlich carcinoma, Lewis lung carcinoma, mammary carcinoma of C3H/HeN, Meth A fibrosarcoma, MH134 hepatoma, and S180 sarcoma [32]. While investigations into sarcoma models were not the focus of early preclinical studies, notable antitumor activity was observed with irinotecan in S180 sarcoma with curative activity observed in 4/10 and 6/10 mice when administered orally or intravenously, respectively [32]. In human tumor xenografts implanted in nude mice, significant tumor inhibition was observed with irinotecan in several solid tumors including childhood rhabdomyosarcomas, indicating its potential as a therapeutic agent for this type of cancer [32]. In vivo, synergistic activity was observed with irinotecan and 5-FU in L1210 leukemia, and superior efficacy was observed with the irinotecan and cisplatin versus cisplatin alone in human lung tumor xenografts in nude mice [32].

In studies in rodents and dogs, the main toxic effects of irinotecan were myelosuppression and lymphoid organ depletion, with dose-related diarrhea observed in dogs [32], and the DLTs of myelosuppression and diarrhea observed in early clinical studies reflected these findings [33]. In vitro and in vivo studies with irinotecan and cisplatin combinations showed drug ratio-dependent antitumor activity [34]. Irinotecan and radiation combinations showed greater tumor growth delays than any single treatment [35].

Topotecan is a water-soluble semi-synthetic analog of camptothecin with a broad spectrum of antitumor activity [36‒39], developed to improve the bioavailability and to decrease the toxicity compared with the parent drug [36, 37]. In vitro, topotecan demonstrated efficacy against a range of human tumor models, including breast, colorectal, NSCLC, ovarian, and renal cell cancer [38, 40]. Topotecan has shown activity in tumor cells with resistance to other chemotherapies including cyclophosphamide, doxorubicin, etoposide, and 5-FU [38, 40].

In vivo, promising activity was observed in xenografts derived from ovarian cancer, osteosarcoma, rhabdomyosarcoma, and brain tumors and neuroblastomas [36, 37]. Objective responses were seen in Ewing and rhabdomyosarcoma, including 1/5 rhabdomyosarcoma xenografts and 1/5 Ewing sarcoma xenografts obtaining maintained complete responses [37]. In rhabdomyosarcoma xenografts, topotecan significantly reduced tumor volumes compared with the control [37]. Maximal activity appeared to be obtained with sustained exposure of topotecan at lower doses compared with an intermittent schedule at higher doses [36, 37]. Sustained, daily lower doses of topotecan (i.e., daily doses for 5 consecutive days, every 3 weeks, for 20 weeks) in rhabdomyosarcoma xenografts were found to have greater activity and minor toxicity compared with higher single doses [36]. In xenografts, antitumor activity was observed with various schedules and routes of administration of topotecan [36, 38]. When comparing oral and intravenous routes, in A549 NSCLC tumor, COCF colon tumor, POVD SCLC tumor, SKOV-3 ovarian tumor, and U87 glioblastoma tumor xenografts, tumor growth inhibition was observed regardless of the route of administration [36]. In vivo, the DLT of intermittent schedules was neutropenia, while the DLT of sustained schedules, such as prolonged infusions or continuous intravenous infusion, was thrombocytopenia [39].

Exatecan (Daiichi Pharmaceutical Co Ltd, Tokyo, Japan) is a water-soluble analog of camptothecin that was developed to have an improved therapeutic activity compared to other camptothecin analogs, with decreased AEs [41]. Exatecan is not a substrate of P-glycoprotein and thus may overcome P-glycoprotein-mediated drug resistance [7, 18]. In preclinical studies, exatecan was more potent than SN-38, topotecan, and camptothecin [42]. The exatecan inhibitory effect is 3, 10, and 20 times greater than SN-38, topotecan, and camptothecin, respectively [41, 43]. Stronger topoiso-merase I trapping, higher levels of DNA damage, and apoptosis have been observed with exatecan compared with topotecan, irinotecan, or SN-38 [44].

In vitro, exatecan demonstrated high potency against a series of 32 cell lines which included breast, colon, stomach, lung, ovarian cancers, and leukemias [45]. Exatecan was also active in irinotecan-, SN-38-, and topotecan-resistant cell lines and in Pgp-mediated multidrug-resistant cell lines [41, 43, 45].

In vivo, exatecan was active against a range of human tumor xenografts in nude mice, including breast, lung, ovarian, gastric, colon, pancreatic cancers, and myelo-genous leukemia [41, 43, 45]. Exatecan also had antitumor activity in an intracranial xenograft of human RH30 rhabdomyosarcoma [41]. While sarcoma was not the focus of earlier preclinical studies, investigations that did involve sarcoma models showed that cyclical exatecan dosing patterns at lower doses were found to be more effective than single dosing schedules in the homologous Meth A mouse fibrosarcoma model [41]. In various human tumor xenografts, exatecan had greater activity than irinotecan, topotecan, and lurtotecan (GG-211) [43, 46].

In rodents and dogs, the DLTs of exatecan were non-cumulative myelosuppression, in particular neutropenia, which was observed in single and divided dosing regimens [41]. Exatecan is currently being developed as tumor-targeted delivery payload in several formulations, including antibody-drug conjugates (ADCs) [44]. A human epidermal growth factor receptor 2 (HER2)-targeting ADC with exatecan was tested in vitro and in vivo in breast and gastric cancer models, and results with the exatecan-based ADC showed potent antitumor activity and an improved pharmacokinetic profile [47].

Rubitecan is a camptothecin derivative [48], and 9-nitrocamptothecin (9-NC), the prodrug of rubitecan, is metabolically converted into 9-aminocamptothecin (9-AC) [48]. Both 9-NC and 9-AC are active, although 9-AC did not show meaningful activity in clinical trials in NSCLC and CRC and had modest responses in SCLC, ovarian cancer, and lymphoma [49, 50].

In vitro models showed time- and dose-dependent cell cycle arrest with apoptosis at nanomolar concentrations [51]. 9-NC had a high inhibitory effect in human tumors in tissue cultures [52]. Rubitecan showed greater potency than irinotecan and topotecan in a number of tumor cell lines [51].

In vivo, rubitecan showed broad anticancer activity in human tumor xenograft studies, with activity observed in various tumors including breast, colorectal, lung, melanomas, ovarian, pancreatic, prostate, stomach, and leukemia [48]. In mice and dogs, toxicology studies showed intestinal toxicity to be the primary toxicity, while myelosuppression and gastrointestinal effects were the main DLTs in phase 1 studies [52].

The clinical trials summarized in this section are studies with either the free drug, prodrugs, or liposomal formulations.

Irinotecan has been evaluated in many clinical trials in different tumor types [26, 53‒55]. We provide an overview of sarcoma trials in Table 1. The most frequent AEs associated with irinotecan are nausea, vomiting, diarrhea, and myelosuppression [5, 20]; the main toxicities with liposomal irinotecan are diarrhea and myelosuppression [5, 24].

Phase 1 studies have been conducted with single agent irinotecan in solid tumors, which included numerous different tumor types. The studies presented here are phase 1 studies with irinotecan in combination regimens in sarcoma only.

In a phase 1b-2 study, the vincristine + irinotecan (VI) + anlotinib combination showed promising activity in advanced Ewing sarcoma, the objective response rates (ORRs) were 63% and 83%, respectively, and the recommended dose in cohort A (≥16 years) was the 15 mg/m² with the DLT being diarrhea [56]. In cohort B (<16 years), there were no DLTs and irinotecan in the combination was continued at 20 mg/m² [56].

The regimen irinotecan + temsirolimus was evaluated in adult patients with metastatic sarcoma, with disease stabilization observed in some patients (11%) [57]. DLTs included neutropenia, muscle weakness, platelet count decrease, and the recommended phase 2 dose (RP2D) was irinotecan 80 mg/m² + temsirolimus 20 mg on a weekly basis for 3 out of 4 weeks [57].

Irinotecan + niraparib or temozolomide + niraparib were evaluated in advanced Ewing sarcoma, ORR was 8.3%, and DLTs included neutropenia, thrombocytopenia, colitis, anorexia, and alanine aminotransferase elevation [58]. The maximum tolerated dose in arm 1 was temozolomide 10–30 mg/m²/day (D2–6) + niraparib 200 mg/day (D1–7), and in arm 2 was irinotecan 20 mg/m²/day (D2–6) + niraparib 100 mg/day (D1–7) [58]. While the combination of irinotecan and niraparib was tolerable, the doses were lower than conventional combinations. A triple combination study with irinotecan + niraparib + temozolomide is ongoing [58].

In a phase 2 trial, single agent irinotecan had activity (ORR of 23%) in refractory or recurrent STS (N = 30) [59]. There were also several phase 2 and retrospective studies that have assessed irinotecan in combination with various agents in patients with sarcoma (Table 1).

The VI combination had activity (ORR 26–37%) in patients with relapsed rhabdomyosarcoma (N = 92) [60]. In patients with metastatic rhabdomyosarcoma, when comparing irinotecan alone to VI or to vincristine + dactinomycin + cyclophosphamide (VDC), the VI combination achieved a higher ORR (70%) compared with VDC (64%) and irinotecan alone (42%) [61]. In this trial, a high rate of primary progressive disease (32%) in patients receiving irinotecan alone led to closure of the study [61]. However, the VI combination was highly active with non-overlapping safety profiles [61], and this combination has been evaluated further in other studies. In a randomized phase 2 trial, VI has been compared with vincristine + irinotecan + temozolomide (VIT) in patients with relapsed or refractory rhabdomyosarcoma (N = 120), the ORR was 31% with VI and 44% with VIT, and the median overall survival (mOS) was 10.3 and 15.0 months [62].

The schedule irinotecan + vincristine + pazopanib has been evaluated in patients with resistant or relapsed sarcomas in a retrospective chart review (N = 166), and the ORR was 47% with a mOS of 15 months [63]. Irinotecan + carboplatin + radiotherapy has been evaluated in patients with intermediate and high-risk rhabdomyosarcoma (N = 60), showing a 2.5-year local control of 89% [64].

Irinotecan + temozolomide (IT) has been evaluated in a retrospective chart review in patients with recurrent sarcoma (N = 24) which showed modest activity (ORR 17%, with a clinical benefit rate [CBR] of 54%) [65]. IT has also been evaluated in several retrospective studies in patients with advanced, relapsed, or recurrent Ewing sarcoma, and the ORRs in the studies have ranged from 29% to 63%, showing that IT is effective and feasible for this patient population [66‒70]. In a phase 2 study with front-line IT in patients with primary disseminated Ewing sarcoma (N = 34), while two courses of IT showed promising ORR (59%) and CBR (91%), there was no impact on event-free survival [71].

The VIT combination with a shorter 5-day regimen has been evaluated in a retrospective study in patients with relapsed and refractory Ewing sarcoma (N = 22), and this regimen was effective with an ORR of 68% [72]. In patients with relapsed alveolar rhabdomyosarcoma, a single institution’s experience in 4 patients observed a 25% (1/4) ORR [73]. Other combinations with irinotecan in sarcoma patients include a phase 2 study with irinotecan + docetaxel in recurrent or refractory Ewing sarcoma (N = 9) with an ORR of 33% [74]; a pharmacokinetic study with irinotecan + ifosfamide in osteosarcoma showing that fractionated ifosfamide altered irinotecan/SN38 pharmacokinetics [75]; and a compassionate use study with trabectedin followed by irinotecan in advanced translocation-positive sarcomas (N = 12) with an ORR of 8% and CBR of 50% [76].

A phase 3 study comparing VDC to VDC/VI with radiotherapy starting at week 4 in patients with intermediate-risk rhabdomyosarcoma (N = 448) showed that the addition of VI to VDC did not improve outcomes (4-year overall survival [OS] rate of 73% with VDC vs. 72% with VDC/VI [Table 1]) [77, 78]. In the rEECur randomized trial, patients with recurrent and primary refractory Ewing sarcoma received IT, or topotecan + cyclophosphamide (TC), or gemcitabine + docetaxel, or high-dose ifosfamide [79]. At the first interim analysis of the study, the gemcitabine-docetaxel arm was halted as it showed worse efficacy outcomes than the other arms [79]. At the second interim analysis, at a median follow-up of 9.2 months, the irinotecan-temozolomide arm showed a response rate of 20%, a mPFS of 4.7 months, and a mOS of 13.9 months [79]. In a pairwise comparison of the irinotecan-temozolomide arm with the other ongoing arms of the study, the comparisons for objective response, PFS, and OS favored the other ongoing arms of the study [79].

Based on the current experience summarized here, irinotecan remains a drug that is sporadically used in selected sarcomas, mainly in pediatric patients with relapsed/refractory mesenchymal tumors and most commonly in Ewing sarcoma. Of note, irinotecan has no regulatory approval for the treatment of sarcoma.

An overview of topotecan in the treatment of sarcoma is presented in Table 2. The most frequent AE associated with topotecan is myelosuppression [5, 21].

Phase 1 studies with single agent topotecan have been performed in various solid tumors and have not focused on patients with sarcomas. However, there are phase 1 studies with topotecan in combination regimens that have enrolled only sarcoma patients, and these are mentioned below.

A study enrolled 12 patients with relapsed or refractory Ewing sarcoma or osteosarcoma, these patients were treated with topotecan + cyclophosphamide + cabozantinib, the study was completed in October 2022, and results are awaited (NCT04661852). A phase 1 open-label expansions study started in June 2018 evaluating topo-tecan + cyclophosphamide + seclidemstat (SP-2577, LSD1 inhibitor) or single agent seclidemstat in relapsed or refractory Ewing or Ewing-related sarcomas, and the estimated study completion date is December 2023 (NCT03600649). A rollover study is also ongoing to allow continued access to seclidemstat to patients benefiting on treatment in the prior study, where patients will continue to receive either single agent seclidemstat or the topo-tecan + cyclophosphamide + seclidemstat combination as per the parent protocol, and the estimated study completion date is December 2025 (NCT05266196).

Topotecan has been evaluated as a single agent or in combination in phase 2 studies and retrospective evaluations (Table 2). As a single agent, topotecan had a low ORR (10%) in adult patients with recurrent or metastatic STS (N = 32) [80]. Single agent topotecan as salvage therapy for pretreated STS did not achieve objective responses, but 38% of patients had stable disease (N = 16) [81]. Continuous infusion of single agent topotecan was not effective in adults with advanced STS (0% ORR, 24% CBR [N = 22]) [82]. Single agent topotecan had limited activity (11% ORR, 44% CBR) in women with advanced, persistent, or recurrent uterine leiomyosarcomas (N = 36) [83].

In combination regimens, topotecan + cyclophosphamide (TC) in patients with relapsed Ewing sarcoma had an ORR of 23% (54% CBR) and 4 patients had sustained responses (N = 14) [84]. TC was active (ORR: 33%; CBR: 59%) in children and adults with refractory or relapsed Ewing tumors (N = 54) [85]. TC was tolerable with modest activity (20% ORR, 47% CBR) in adult patients with relapsed sarcoma (N = 15) [86]. However, limited activity (11% ORR, 37% CBR) was observed in adult patients with TC in relapsed or refractory pediatric-type sarcoma (N = 39) [87].

Vincristine + topotecan + cyclophosphamide added to standard chemotherapy at a compressed interval of 2 weeks was tolerable compared with historical standard therapy at a 3-week interval in children and young adults with newly diagnosed localized Ewing sarcoma, with a 5-year OS rate of 88% (N = 35) [88]. Vincristine + topotecan + cyclophosphamide has also shown efficacy (50% ORR, 64% CBR) in children and adolescents with recurrent/progressive Ewing sarcoma family tumors (N = 14) [89].

While topotecan + carboplatin alternating with carboplatin + etoposide was tolerable, it had limited efficacy (ORR 28%) in children with resistant or refractory rhabdomyosarcoma (N = 38) [90]. However, topotecan + carboplatin had modest activity (38% ORR; 71% CBR) in patients ≤21 years with metastatic sarcoma (N = 34) [91].

In a phase 2 study, pazopanib + topotecan had an ORR/CBR of 8%/71% in non-adipocytic STS, 4%/79% in osteosarcoma, and 0%/44% in liposarcoma [92]. The combination did not meet the primary objective and was associated with a high frequency of AEs (N = 153) [92].

In the phase 2–3 rEECur trial (N = 451), the interim efficacy assessment in children and adults with recurrent and primary refractory Ewing sarcoma showed high-dose ifosfamide was more effective than TC (Table 2), IT, and gemcitabine + docetaxel; McCabe et al. [79, 93] concluded that high-dose ifosfamide should be considered as the control arm in future phase 2–3 trials or could be considered in a combination regimen in this patient population. The topotecan- and irinotecan-based combinations remain to be used in highly refractory patients, given the lack of good treatment alternatives. In a randomized phase 3 trial (N = 629), adding vincristine + topotecan + cyclophosphamide to five-drug interval-compressed chemotherapy did not improve 5-year event-free survival versus standard five-drug interval-compressed chemotherapy (79% vs. 78%) nor did it improve 5-year OS (88% vs. 86%) in children and adults with newly diagnosed non-metastatic Ewing sarcoma [94].

Exatecan is a synthetic analog of camptothecin that does not require enzymatic activation [43, 95], and has been evaluated in several phase 1 trials in solid tumors, and there have been phase 2 trials in patients with sarcoma. The clinical trials summarized in this section are trials with free drug and do not include other formulations of exatecan derivatives.

There are no phase 1 studies with exatecan enrolling only patients with sarcomas, and most of the phase 1 studies are in solid tumors that may have included a few patients with sarcomas. Phase 1 studies evaluated exa-tecan using various administration schedules and durations in solid tumors, and the recommended schedule for phase 2 studies was a 30-min infusion (daily × 5) every 3 weeks [42]. The pharmacokinetic evaluations showed AUC and C_max to be proportionate to dose, while clearance and distribution were dose-independent [41].

Across all administration schedules, the toxicity profile was similar and the principal DLT of exatecan was neutropenia in minimally pretreated patients; and neutropenia and thrombocytopenia in heavily pretreated patients [41, 42]. Hematological AEs were dose-dependent and reversible, and the main non-hematological AEs were nausea, vomiting, diarrhea, ele-vated hepatic transaminases, asthenia, and alopecia [42].

Exatecan showed some antitumor activity in solid tumors including sarcoma, and partial responses (PRs) were reported in previously treated patients with solid tumors, including in a patient with sarcoma [41, 42]. In one phase 1 study, a 75-year-old man with sarcoma (previously untreated with chemotherapy) had a PR with exatecan (3.13 mg/m² administered as a 30-min infusion for 3 out of every 4 weeks) [96].

An overview of the phase 2 sarcoma trials with exa-tecan is shown in Table 3. Three trials investigating exatecan in patients with sarcoma are recorded in the NIH US National Library of Medicine ClinicalTrials.gov, of which one study has published results.

In a multicenter phase 2 (NCT00041236) trial in pretreated adult patients with advanced STS (N = 39), exatecan had a manageable tolerability, and while no objective responses were achieved, exatecan did have some activity in leiomyosarcomas patients who had previously failed one or two lines of chemotherapy (60% CBR, 3-month progression-free survival rate of 56%) [95, 97]. The other two phase 2 studies evaluating exatecan in patients with sarcoma included one study in children with relapsed or refractory rhabdomyosarcoma (NCT00055939) and the other study in children and adult patients who had relapsed or refractory Ewing sarcoma, peripheral primitive neuroectodermal tumor, or desmoplastic small round cell tumor (NCT00055952). For both of these studies, the planned number of patients was 13–27, the study’s start date was January 2003, and the study’s completion date was April 2006, although no results appear to have been published for either of these studies.

No phase 3 trials have been conducted with free exa-tecan in patients with sarcomas. The free drug is not approved for clinical use in any indication.

Most of the phase 1 and 2 studies with rubitecan or 9-AC reported disappointing antitumor results in various cancers [18]. A summary of the few rubitecan studies involving patients with sarcoma is shown in Table 4.

Rubitecan has been evaluated in numerous phase 1 studies, with different formulations, in solid tumors and leukemia [18]. In a study in solid tumors, in patients treated with 1, 1.5, or 2 mg/m²/day for 5 days followed by 2 days of rest, the DLT was hematologic, gastrointestinal AEs were the next most significant AEs, and the RP2D was 1.5 mg/m²/day for 5 days each week [52].

Ewing sarcoma and other sarcomas have a high incidence of pulmonary metastasis [99], and a phase 1–2 study in patients over the age of 10 years with Ewing sarcoma evaluated the liposomal 9-nitro-20-(S)-camptothecin aerosol alone or in combination with temozolomide (NCT00492141). Ten participants were enrolled, and the study was completed in September 2009, but no results appear to have been reported.

There are three phase 2 studies with rubitecan in patients with sarcomas: the phase 1–2 study mentioned above with the study arm with 10 patients with Ewing sarcoma, a study in patients with advanced STS, and a study in patients with advanced chordoma, STS, and gastrointestinal stromal tumor (GIST). In a study with advanced STS, 9-NC was administered in adult patients with advanced STS (N = 56) and showed minimal activity in previously treated STS (PR of 8%), and was inactive in gastrointestinal leiomyosarcomas [52].

In a study with advanced chordoma, STS, and GIST (N = 51), 9-NC had modest activity in chordoma (ORR 7%), with little to no benefit in patients with STS (ORR 4%) and GIST (ORR 0%) [98]. The main AEs with rubitecan included fatigue, gastrointestinal toxicity, and myelosuppression [51, 52, 98].

There are no phase 3 studies in patients with rubitecan and in patients with sarcoma, and the drug is not used in clinical routine.

While topoisomerase I inhibitors are active anticancer agents, their rapid elimination may limit the therapeutic concentrations needed at the tumor for sustained exposure to the agent and anticancer effects [5, 7]. Furthermore, the gastro- and myelotoxicities such as diarrhea and neutropenia limit the use with free topoisomerase I inhibitors in clinical routine [5, 20, 21, 24]. To overcome some of the limitations of these inhibitors, different delivery strategies have been investigated [5].

Liposomal nanoparticle topoisomerase I inhibitors have the potential to leverage the enhanced permeability and retention (EPR) effects which may occur in tumors (due to increased permeability of blood vessels and limited lymphatic drainage), allowing for the accumulation of the liposomal nanoparticles in the tumor, protection from degradation, and sustained drug release within the tumor [5]. However, while nano-liposomal irinotecan does have a longer t_1/2 compared to free drug, severe or life-threatening neutropenia or diarrhea can still occur [5, 24].

Another more promising and clinically validated approach is using topoisomerase I inhibitors as “payloads” for ADCs, where a monoclonal antibody (mAb) can deliver the active drug to targeted tumor cells that express specific antigens [5]. Sarcomas encompass a wide range of tumor subtypes (>80 different sarcoma subtypes), and this heterogeneity may pose a challenge to the development of ADCs due to the general lack of universally applicable tumor-specific antigens across the sarcoma subtypes [100]. However, some sarcoma subtypes do exhibit specific antigens that can be exploited for ADC targeting (such as the CD99-targeted ADCs for Ewing sarcoma, or targeting Wilms tumor 1 protein for desmoplastic small round cell tumor, etc.), and there are antigens that are expressed on multiple sarcoma subtypes that could be targets for applicable mAbs in ADCs (such as anti-CD13 mAbs in ADCs for targeting fibrosarcoma, STS, liposarcoma; or anti-CD133 [or prominin-1] mAbs for targeting osteosarcoma and Ewing sarcoma; or targeting anti-insulin-like growth factor 1 in osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, etc.) which provide a practical approach to address the heterogeneity of sarcomas [100‒107].

The use of a topoisomerase I inhibitor as a payload for ADCs can enhance delivery of the topoisomerase I inhibitor to the tumor cell, limit systemic exposure, and potentially reduce off-target toxicity [5]. Cytotoxic drugs such as topoisomerase I inhibitors, when given as free (pro)drug systemically, are non-specific in terms of the cells they target, and while they will select rapidly dividing cells (such as cancer cells), they can have off-target effects and they can also be rapidly eliminated, leading to the need for higher doses of topoisomerase I inhibitors to reach therapeutic concentrations in the tumor to achieve the desired effect [7]. Topoisomerase I-ADCs allow for targeted delivery of the inhibitor to the tumor, thereby limiting exposure of healthy cells, minimizing off-target effects, with the potential to reduce associated toxicity [7].

For a successful topoisomerase I-ADC (shown in Fig. 1a), four aspects need to be optimized: the selection of the targeted antibody, the linker connection between the mAb and the payload (choice of topoisomerase I inhibitor), and the drug-to-antibody ratio (DAR) [7, 108]. The mAb can offer high selectivity based on the targeted antigen expressed on the tumor cell [7]. An immunoglobulin G subclass is frequently used as the backbone for the ADC due to its stability and long serum t_1/2 [7, 108]. The choice of the linker (cleavable or non-cleavable) is critical to ensure that the ADC is stable during systemic circulation and that the payload is optimally released inside the tumor [7, 108]. An effective cytotoxic agent is needed for the payload, and the DAR is also important, because if the DAR is too low the desired clinical effect might not be achieved and if the DAR is too high there may be off-target toxicity due to increased circulating plasma concentrations of the cytotoxic drug [7, 108]. There has been a rapid development of ADCs to target topoisomerase I inhibitors to cancer cells [5, 7, 109], and the schematic mechanism of action of topoisomerase I-ADCs is shown in Figure 1b. In clinical development, topoisomerase I-ADCs are competing with monomethyl auristatin E (MMAE)-based ADCs for the treatment of patients with sarcoma, where preclinical studies targeting the Endo180 (also known as urokinase plasminogen activator receptor-associated protein [uPARAP]) receptor with the MMAE-ADC showed to be effective in Endo180-expressing sarcoma cell lines and in sarcoma xenografts [110].

The irinotecan derivative SN-38 and the exatecan derivative deruxtecan (Dxd) have successfully been used as payloads in ADCs in various epithelial tumor types, two of which have been approved by FDA [7, 109]. Enhertu^® (DsS8201a/DS-8201a), containing a Dxd payload joined to the HER2-directed antibody trastuzumab, is approved for the treatment of HER2+ metastatic breast cancer, HER2-low metastatic breast cancer, HER2+ advanced stomach cancer, and HER2-mutant metastatic lung cancer [7, 109]. Trodelvy^® (IMMU-132), containing the govitecan payload joined to the antibody sacituzumab (hRS7) with a trophoblast cell surface antigen 2 target, is approved for the treatment of HR+/HER2− metastatic breast cancer, metastatic triple-negative breast cancer, and advanced bladder cancer [7, 111, 113].

Numerous topoisomerase I-ADCs are in phase 1–3 clinical trials in a range of tumor types that include Dxd payloads (U3-1402 [patritumab-Dxd], Ds-1062a [datopotamab-Dxd], Ds-7300a, and Ds-6157a), SN-38 payloads (IMMU-130 [labetuzumab-govitecan], IMMU-140), payloads with AZ’0132/AZ-0132, an exatecan derivative (AZD8205), or KL610023, a belotecan derivative (SKB-264), exatecan mesylate payloads (PRO1184, PRO1102, PRO1160), AMDCPT payload (SGN-CD30c), and 7-n-butyl-10-amino-CPT or 7-n-butyl-9-amino-10,11-MDO-CPT or CPT payloads [5, 7, 109]. However, while most of these studies are evaluating topoisomerase I-ADCs in solid tumors, there are no sarcoma-specific clinical trials at the time of preparation of this manuscript.

However, “non-topoisomerase I” ADCs have been studied and are currently under preclinical and clinical investigation in sarcomas [100, 114], and potentially some of these mAb targets could also be explored with a topoisomerase I-based ADC. Several non-topoisomerase I payloads have been considered for the treatment of sarcomas and include tubulin-targeting drugs (such as MMAE or MMAF), or DNA-targeting drugs (with DNA alkylating agents such as duocarmycins, pyrrolobenzodiazepines, calicheamicins, or anthracycline), or microtubule destabilizing agents (such as maytansine) [100, 115, 116]. The advantages of these ADCs include the selective targeting of the sarcoma tumor and the potential for an improved therapeutic index, while one of the disadvantages may be the potential for resistance to develop, as with conventional chemotherapy [117, 118]. In sarcoma, mAb targets that are also under development include endosialin, glycoprotein non-metastatic b, leucine-rich repeat containing 15, neural cell adhesion molecule, endoglin, receptor tyrosine kinase-like orphan receptor, and uPARAP [100].

uPARAP (product of MRC2 gene, also known as Endo180 or CD280) is a highly interesting target. It is an endocytic receptor that collagen binds to, and uPARAP plays an important role in tissue collagen turnover [110, 119, 120]. On healthy cells, uPARAP expression is limited, but there is a high level of expression on tumor cells including glioblastoma, mesothelioma, osteosarcoma, STS, bone sarcoma, and acute myeloid leukemia [110, 119, 120]. In vitro studies with the tubulin inhibitor MMAE-ADC directed against uPARAP showed strong cytotoxicity in uPARAP-positive sarcoma cancer cell lines [110, 119]. uPARAP-targeted ADCs containing microtubulin and alkylating agent payloads showed antitumor activity in patient-derived xenograft models of osteosarcoma [121]. In tissue samples from more than 400 individual donors, uPARAP expression was found to be widely expressed at high levels in multiple sarcoma subtypes [120]. These findings together with the low expression levels of uPARAP in healthy tissue make a topoisomerase I-ADC directed against uPARAP an attractive strategy for treatment of a broad range of mesenchymal malignancies and support further investigation [120].

There is a high unmet need for novel, active, and safe treatments for patients with advanced, inoperable, or metastatic sarcoma. Progress in the complex field of mesenchymal malignancies has been slow, though the biology of many of these diseases is relatively well understood and a number of potentially relevant drug targets have been identified. Topoisomerase I inhibitors are potent cytotoxic agents with limited use as free drug in this heterogeneous family of tumors, though activity seen in preclinical models and responses observed in the few published trials highlight the potential relevance of topoisomerase I as a relevant enzyme involved in the proliferation of sarcoma cells. By repurposing topoisomerase I inhibitors as cytotoxic payload of ADCs, these drugs can be selectively delivered to tumor cells expressing specific surface antigens, thereby minimizing exposure of healthy cells to these drugs, with the potential to reduce systemic toxicity and enhance efficacy. Investigations evaluating topoisomerase I-ADCs against a variety of novel cellular targets may pave the way for more effective and tailored therapies for sarcoma patients in the future.

J. O’Regan (Bingham Mayne and Smith) provided editing/medical writing support.

P.S. involved in consulting or advisory activities, or research funding, which include consulting or advisory role – Adcendo; Adaptimmune; Amryt Pharma; Avacta Life Sciences; Biolumina; Blueprint Medicines; Boehringer Ingelheim; Boxer Capital; Cogent Biosciences; Curio Science; Deciphera; Eisai; Ellipses Pharma; Exelixis; Genmab; Intellisphere; Lilly; LLX Solutions; Loxo; Medpace; Merck Healthcare KGaA; Moleculin Biotech; PharmaMar; Plexxikon; Regeneron; Sanofi; Servier; SQZ Biotechnology; Studiecentrum voor Kernenergie (SCK CEN); Transgene; UCB; and research funding – Adcendo; Blueprint Medicines; Boehringer Ingelheim; CoBioRes NV; Eisai; Exelixis; G1 Therapeutics; Genmab; Lilly; Merck; Novartis; ONA Therapeutics; PharmaMar; Plexxikon; Sartar Therapeutics. C.-C.W., M.P.S., and A.W. have no conflicts of interest to declare.

This manuscript received no funding, and there is no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. P. Schöffski funded editorial/medical writing support.

Patrick Schöffski proposed and developed the manuscript, approved the final version of this manuscript, and was responsible for the decision to submit the manuscript. Patrick Schöffski, Chao-Chi Wang, Morris Patrick Schöffski, and Agnieszka Wozniak made substantial contributions to the manuscript, were involved in the preparation and revision of the manuscript, and reviewed and approved this manuscript.