AXL-directed antibody–drug conjugate therapy in soft-tissue sarcoma

Abstract

AXL is a membrane-accessible receptor tyrosine kinase and a potential antibody–drug conjugate (ADC) target, but its therapeutic relevance in soft-tissue sarcoma remains incompletely defined. We evaluated the AXL-directed pyrrolobenzodiazepine ADC ADCT-601/mipasetamab uzoptirine using a sarcoma tissue microarray, patient-derived cell lines, and patient-derived xenografts. AXL expression was broad but heterogeneous, with the highest histotype-level expression in myxofibrosarcoma and undifferentiated pleomorphic sarcoma. In the patient-linked tissue-microarray cohort, AXL H-score was not associated with metastasis-free or overall survival after clinicopathologic adjustment. ADCT-601 was substantially more potent than a non-targeting anti-gp120 control ADC carrying the same SG3199-containing payload. Free-payload sensitivity varied between cell lines, indicating that intrinsic payload susceptibility contributes to activity, while the payload-matched comparison supports an additional effect of AXL-mediated delivery. ADCT-601 induced cell-cycle perturbation, apoptosis, and γ-H2AX accumulation. Combination experiments identified enhanced activity with DNA damage-response inhibition, particularly ATR inhibition, and in vivo studies supported antitumor activity in two sarcoma xenograft models. These data support biomarker-enriched development of next-generation AXL-directed ADC strategies while emphasizing antigen heterogeneity and therapeutic-index optimization.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s13045-026-01851-1.

To the editor

Soft-tissue sarcomas (STS) remain therapeutically constrained after anthracycline-based therapy. AXL, a TAM-family receptor tyrosine kinase implicated in invasion, cell-state plasticity, and therapeutic resistance [1, 2], is an attractive surface antigen because it can mediate antibody–drug conjugate (ADC) binding, internalization, and payload delivery even when it is not the dominant oncogenic driver. Clinical experience with AXL-directed ADCs also suggests that antigen expression is more likely to serve as an enrichment biomarker than as a binary predictor [3–6].

We evaluated the AXL-directed pyrrolobenzodiazepine (PBD) ADC ADCT-601/mipasetamab uzoptirine in a translational STS platform. Across 616 evaluable tissue-microarray cores, AXL expression was broad but heterogeneous. Myxofibrosarcoma and undifferentiated pleomorphic sarcoma (UPS) had the highest mean H-scores (60.3 and 43.6), whereas synovial sarcoma and adult fibrosarcoma showed low expression. Individual values and representative staining are shown in Supplementary Figs. S1–S2 and S10A and Supplementary Table S1. Among 379 patients with localized disease and valid metastasis-free follow-up (133 events), AXL H-score was not associated with metastasis-free survival in univariable analysis (hazard ratio [HR] per 10 points, 0.99; 95% confidence interval [CI], 0.96–1.02; P = 0.61) or after adjustment for age, sex, tumor size, depth, grade, and histotype (HR, 1.00; 95% CI, 0.97–1.03; P = 0.85). AXL was likewise not associated with overall survival in 409 patient-linked cases after adjustment (HR per 10 points, 1.00; 95% CI, 0.98–1.02; P = 0.86), and no signal emerged in exploratory UPS- or myxofibrosarcoma-specific analyses (Supplementary Fig. S10; Supplementary Tables S6–S8). Thus, histology may enrich for AXL prevalence, but direct testing remains necessary and AXL abundance is not a prognostic surrogate.

Across 11 AXL-positive sarcoma cell lines, the median ADCT-601 IC50 was 0.006 nM (range, 0.001–0.040), compared with 5.849 nM (range, 0.211–288.2) for B12-PL1601 (Fig. 1; Supplementary Figs. S1–S3; Supplementary Tables S2–S3). B12-PL1601 is a non-targeting ADC directed against HIV-1 gp120 and carries the same SG3199-containing PBD payload as ADCT-601 [3]. The median B12-PL1601/ADCT-601 IC50 ratio was 737-fold. Conversely, SG3199/ADCT-601 ratios ranged from 0.10 to 65.5 among cell lines with both measurements, demonstrating that intrinsic payload sensitivity varies substantially. Thus, payload susceptibility contributes in a cell-line-dependent manner, while the markedly greater potency of ADCT-601 than the payload-matched non-targeting ADC supports an additional contribution from AXL-mediated delivery.

Fig. 1.

AXL-directed PBD antibody–drug conjugate therapy in soft-tissue sarcoma. A, Translational rationale linking AXL expression, target-mediated PBD ADC delivery, DNA damage, and ATR inhibition. B, Payload-matched IC50 comparison between ADCT-601 and non-targeting B12-PL1601 across AXL-positive sarcoma cell lines. C, Fold-selectivity of ADCT-601 over B12-PL1601, calculated as B12-PL1601 IC50 divided by ADCT-601 IC50. D, Summary of in vivo survival analyses from patient-derived xenograft experiments. Hazard ratios are versus vehicle; values below 1 favor treatment. ADC, antibody–drug conjugate; ATRi, ATR inhibitor; PBD, pyrrolobenzodiazepine; STS, soft-tissue sarcoma

ADCT-601 induced cell-cycle perturbation, apoptosis, and γ-H2AX accumulation in AXL-positive sarcoma cells (Supplementary Figs. S4–S6). In IB115 cells, combination with the ATR inhibitor ceralasertib (AZD6738) produced positive synergy across ZIP, HSA, Bliss, and Loewe models; ATM inhibition with AZD0156 was more modest, and combination effects were weak or absent in AXL-negative MCF-7 cells (Supplementary Figs. S7–S8; Supplementary Table S4).

In vivo, ADCT-601 improved survival in the KN473 and JR588 UPS patient-derived xenograft models (hazard ratios versus vehicle, 0.013 and 0.090, respectively), whereas B12-PL1601 showed weaker activity (0.182 and 0.190). The magnitude of the targeting advantage differed between models, which may reflect differences in antigen density or localization, internalization, growth kinetics, and intrinsic PBD susceptibility; these variables were not individually quantified in each xenograft. Target-independent exposure to released payload may also contribute to B12-PL1601 activity. In a separate JR588 experiment, only ADCT-601 plus berzosertib significantly improved survival (hazard ratio, 0.32; P = 0.021) (Fig. 1; Supplementary Fig. S9; Supplementary Table S5).

These findings should be interpreted in the context of heterogeneous clinical results with enapotamab vedotin and mecbotamab vedotin [4–6, 7] and the discontinuation of ADCT-601 after a favorable benefit–risk profile could not be demonstrated during dose optimization/expansion [8]. Our study therefore establishes biological proof of concept rather than a case for resuming ADCT-601 unchanged. Future development should prioritize standardized membranous AXL scoring, separate analysis of AXL-null tumors, pharmacodynamic confirmation of payload delivery, and optimization of antibody, linker, payload, dose, and schedule. ATR combinations remain mechanistically attractive but require careful therapeutic-index evaluation. Overall, AXL remains a credible ADC target in selected adult pleomorphic STS when biomarker selection and modern ADC engineering are incorporated from the outset.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

ADC

Antibody–drug conjugate

ATM

Ataxia telangiectasia mutated

ATR

Ataxia telangiectasia and Rad3-related

CI

Confidence interval

HR

Hazard ratio

IC50

Half-maximal inhibitory concentration

MCF-7

Michigan Cancer Foundation-7

MFS

Myxofibrosarcoma

OS

Overall survival

PBD

Pyrrolobenzodiazepine

PDX

Patient-derived xenograft

STS

Soft-tissue sarcoma

TMA

Tissue microarray

UPS

Undifferentiated pleomorphic sarcoma

Author contributions

Conceptualization: AI, NE, TS; Methodology: TS, NE; Experimental work: TS, NE, VC, TD In vivo experiments: MD; Pathological analyses: LV; Data analysis, statistical interpretation: TS, NE, AB, CR, JPWriting original draft: NE, TS; Writing review & editing: NE, TS, AI. Supervision: AI. All authors read and approved the final manuscript.

Funding

This work received no specific funding.

Data availability

All data supporting the findings reported in this correspondence are included in the article, the supplementary data file and the source-data workbook. Additional raw experimental data are available from the corresponding author on reasonable request, subject to institutional and ethical restrictions.

Declarations

Ethics approval and consent to participate

Human tissue: Sarcoma cell lines and tissue-microarray specimens were derived from human surgical samples collected at Institut Bergonié after written informed consent and approval by the Institutional Review Board of Institut Bergonié. Animal work: All animal experiments were approved by the Structure du Bien-Être Animal, Université de Bordeaux, under protocol DAP #27630 and were performed in accordance with French and European Union regulations. The maximal tumor burden permitted by the approved protocol was 2,000 mm³, and this limit was not exceeded.

Consent for publication

Not applicable.

Authors’ information

Not applicable.

Competing interests

The authors declare no competing interests.