Introduction
Triple-negative breast cancer (TNBC) is a subtype characterized by the absence of estrogen and progesterone receptor expression, as well as HER2 overexpression or gene amplification. Clinically, TNBC is associated with rapid tumor growth, a shorter time to recurrence after initial radical treatment, and shorter progression-free survival (PFS) and overall survival (OS) in patients receiving palliative care compared to other subtypes. It accounts for approximately 15% of all diagnosed breast cancers, and in 35% of patients, it is the direct cause of death [1]. Among patients treated with curative intent, 25% experience local recurrence or disease dissemination [2]. The aggressive course of the disease and unsatisfactory outcomes of cytotoxic chemotherapy, which has little impact on OS, underscore the need for new, more effective therapies.
Structure of antibody-drug conjugates
The use of monoclonal antibodies in cancer therapy, including HER2-positive breast cancer, has significantly improved clinical outcomes due to their precise targeting of specific receptor proteins on the surface of cancer cells [3]. Antibody-drug conjugates (ADCs) enable the selective delivery of cytotoxic agents to cells with high expression of the molecular target, while minimizing toxicity to normal tissues with low or no expression of the target antigen [4].
To achieve high clinical efficacy, ADCs must meet several criteria:
antigen expression on tumor cells and absence on normal cells,
stability in serum to ensure a long biological half-life,
efficient internalization of the antibody-antigen complex into cancer cells [5].
Antibody-drug conjugates should also exhibit low immunogenicity, which determines the extent and severity of side effects in clinical practice. Immunoglobulin G1 (IgG1) antibodies are most commonly used due to their lower tendency to aggregate in serum compared to IgG2, resulting in greater stability and therapeutic efficacy. IgG3 antibodies, due to their high immunogenicity and associated side effects, are used only in limited cases.
First-generation ADCs were based on murine antibodies conjugated with classical cytotoxic agents. These had low clinical efficacy and high toxicity due to immunogenicity and unstable linkers, which led to premature drug release in the bloodstream [6].
Second-generation ADCs used humanized or fully human antibodies, improving antigen specificity and reducing immunogenicity. Linker modifications enhanced drug-antibody stability. An example is trastuzumab emtansine, used in HER2-positive breast cancer.
Third-generation ADCs aimed to further reduce toxicity by using fully human antibodies, improving specificity, and minimizing immune responses. They also featured higher drug-to-antibody ratios (DAR) and hydrophilic linkers to increase serum stability and half-life [3, 7]. Examples include enfortumab vedotin (targets nectin-4) and disitamab vedotin (targets HER2).
Fourth-generation ADCs, such as trastuzumab deruxtecan (HER2) and sacituzumab govitecan (SG; TROP-2), have high DAR values (7.8 and 7.6, respectively), ensuring high intracellular drug concentrations. They also feature enhanced stability and internalization [8].
Mechanism of action of antibody-drug conjugates
The mechanism of action of ADCs varies depending on the antibody, target receptor, and cytotoxic payload. Upon binding to specific surface antigens, the antibody-antigen complex is internalized via endocytosis. The cytotoxic agent is then released into the cytoplasm and transported to the nucleus, where it disrupts the cell cycle:
monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), mitoxantrone and maytansinoid derivatives disrupt microtubule polymerization, affecting the M phase;
calicheamicin causes DNA double-strand breaks or crosslinks;
deruxtecan and SN-38 (active metabolite of irinotecan) inhibit topoisomerase I.
The final outcome is apoptosis or necrosis of cancer cells [3, 9]. Some ADCs, such as SG and trastuzumab deruxtecan, also exhibit a bystander effect, where the cytotoxic drug is released in the vicinity of tumor cells or diffuses into neighboring cells. This is crucial in heterogeneous tumors, allowing the drug to affect antigen-negative cells, enhancing therapeutic efficacy. The bystander effect depends on the chemical properties of the payload and linker stability. For example, MMAF has limited membrane permeability, while MMAE can penetrate the tumor microenvironment [5, 10].
Additionally, some ADCs (e.g., trastuzumab deruxtecan) engage the immune system, activating immune cells to recognize and destroy antigen-expressing tumor cells via:
complement-dependent cytotoxicity (CDC),
antibody-dependent cellular cytotoxicity (ADCC),
antibody-dependent cellular phagocytosis (ADCP) [3].
Trophoblast cell surface antigen 2 receptor
The TROP-2 (trophoblast cell surface antigen 2) receptor is a protein product of the TACSTD2 gene, located on the short arm of chromosome 1 (1p32.1). Its activation leads to the stimulation of several signaling pathways involved in cancer transformation and the formation of distant metastases. TROP-2 expression is observed in 80–90% of patients with advanced TNBC and is associated with increased tumor cell proliferation and poor prognosis [11].
Activation of the TROP-2 receptor through ligand interaction leads to ERK protein phosphorylation (part of the MAPK signaling pathway), increased expression of cyclin D1, and proto-oncogene c-myc exclusively in cancer cells (via TROP-2-ICD-β-catenin complex formation) [12]. Combined with increased expression of cyclin E, CDK2, and CDK4, this promotes cell cycle progression from G1 to S phase. These processes are further enhanced by inhibition of p16 and bax proteins, and increased expression of bcl-2 and E-cadherin, leading to intensified proliferation, migration, and apoptosis inhibition of cancer cells [12].
Metastasis stimulation following TROP-2 activation results from phosphorylation of cell motility kinases (FAK, Src, PAK4), reduced cell adhesion, and increased expression of RACK1 (receptor for activated protein kinase C 1), which translocates from the cytoplasm to the cell membrane. There, it interacts with β1 integrin and talin, reducing cell-cell adhesion among cancer cells. TROP-2 overexpression also correlates with increased VEGF expression, a key factor in tumor angiogenesis. Additionally, TROP-2 induces expression of genes responsible for the barrier phenotype, which prevents immune cells from infiltrating the tumor microenvironment [12].
Sacituzumab govitecan
The clinical efficacy and safety of SG, an anti-TROP-2 antibody-drug conjugate linked to the active metabolite of irinotecan (SN-38), were initially evaluated in the IMMU-132-01 cohort study in patients with advanced TNBC. Among 108 patients, SG achieved an objective response rate (ORR) of 33%, with median PFS of 5.5 months and median OS of 13.0 months (Table 1) [13]. These results led to a multicenter, randomized phase III trial comparing SG to physician’s choice chemotherapy in patients with relapsed or refractory metastatic TNBC previously treated with taxanes. Patients with stable CNS metastases were eligible but excluded from the primary endpoint analysis, which focused on PFS in patients without CNS metastases. Secondary endpoints included OS, PFS in the overall population, ORR, duration of response, and safety profile. The study enrolled 468 patients, randomized 1 : 1 to SG or chemotherapy (capecitabine, vinorelbine, gemcitabine, or eribulin). Sacituzumab govitecan significantly improved median PFS (5.6 vs. 1.7 months) and reduced the risk of disease progression by 59% (HR = 0.41; 95% CI: 0.32–0.52; p < 0.001). Benefits were observed across all predefined subgroups, including patients ≥ 65 years (7.1 vs. 2.4 months), those previously treated with immune checkpoint inhibitors (4.2 vs. 1.6 months), and patients with liver metastases (4.2 vs. 1.5 months) [14]. Sacituzumab govitecan also improved median OS (12.1 vs. 6.7 months) and reduced the risk of death by 52% (HR = 0.48; 95% CI: 0.38–0.59; p < 0.001) [12]. Objective response rate was 35% vs. 5%, with complete response in 4% and partial response in 31% of SG-treated patients [14]. The median duration of response was 6.3 months for SG vs. 3.6 months for chemotherapy (Table 1).
Table 1
Efficacy of ADCs treatment of metastatic triple-nagative breast cancer
| Clinical trial | Phase | ADC | ORR | mPFS [months] | mOS [months] | mDOR [months] | Ref. |
|---|---|---|---|---|---|---|---|
| IMMU-132-01 | I/II | SG | 33% | 5.5 | 13.0 | 7.7 | [13] |
| ASCENT | III | SG vs. Chth | 35% vs. 5% | 5.6 vs. 1.7 HR = 0.41; p< 0.001 | 12.1 vs. 6.7 HR = 0.48; p< 0.001 | 6.3 vs. 3.6 | [14, 15] |
| TROP-2 low 2.7 vs. 1.5 TROP-2 high 6.9 vs. 2.8 | TROP-2 low 8.7 vs. 7.0 TROP-2 high 14.5 vs. 7.1 | [15] | |||||
| ASCENT-04 /KEYNOTE-D19 | III | SG + pembrolizumab vs. Chth | CPS ≥10% HR 0.65; p= 0.0009 | NR | NR | [16] | |
| TROPION-PanTumor01 | I | Dato-DXd | 31.8% | 4.4 | 13.5 | 16.8 | [17] |
| BEGONIA | Ib/II | Dato-DXd+durvalumab | 79% | 13.8 | 15.5 | [21] | |
| DESTINY-Breast04 | III | Trastuzumab-DXd vs. Chth | 52.3% vs. 16.3% HR– 50.0% vs. 16.7% | 9.9 vs. 5.1 HR 0.50; p< 0.001 HR– 8.5 vs. 2.9 | 23.4 vs. 16.8 HR = 0.64; p= 0.001 HR– 18.3 vs. 8.3 | 10.7 vs. 6.8 HR– 8.6 vs. 4.9 | [23] |
| NCT02980341 | I/II | Patritumab-DXd | 22.6% | 5.5 | 14.6 | 5.9 | [25, 26] |
In 2024, final analyses were presented evaluating SG efficacy based on TROP-2 and HER2 expression [15]. According to Bardia et al., SG benefits were observed regardless of expression level, though numerically greater PFS was seen with higher TROP-2 expression (mPFS 2.7 vs. 1.5 months in low expression; 6.9 vs. 2.8 months in high expression). Similar trends were noted for OS (mOS 8.7 vs. 7.0 months in low expression; 14.5 vs. 7.1 months in high expression) [15], though statistical significance was marginal. HER2 expression was assessed via immunohistochemistry in 78% of ASCENT trial patients, with HER2 IHC0 in 71% and HER2-low in 29%. Median OS and PFS in HER2 expression subgroups were comparable to the overall population. Importantly, high TROP-2 expression was independent of HER2 expression level [15].
In 2025, results from the ASCENT-04/KEYNOTE-D19 trial were presented. This study enrolled 443 patients receiving first-line treatment for unresectable locally advanced or metastatic TNBC with PD-L1 CPS ≥ 10%. Patients were randomized to receive SG at 10 mg/m2 on days 1 and 8, combined with pembrolizumab at 200 mg in 21-day cycles. The comparator arm included paclitaxel, nab-paclitaxel or carboplatin with gemcitabine, also in 21-day cycles. After a median follow-up of 14 months, a significant improvement in PFS was observed in the experimental arm (HR = 0.65; 95% CI: 0.51–0.84; p = 0.0009). Overall survival data had not yet reached statistical maturity (Table 1). No new safety signals were identified. This study may potentially support the use of SG in combination with pembrolizumab as a first-line treatment, though definitive conclusions await full publication of the results [16].
Datopotamab deruxtecan
Datopotamab deruxtecan (Dato-DXd) is an IgG1 antibody-drug conjugate linked to deruxtecan, a derivative of exatecan that inhibits topoisomerase I in the cell nucleus. Due to several modifications, including the linker between the antibody and cytotoxic agent, a significant reduction in systemic toxicity has been achieved [17]. Like SG, Dato-DXd can induce a bystander effect, enabling the destruction of TROP-2-negative tumor cells in the tumor microenvironment. Its clinical efficacy and safety in TNBC were initially evaluated in the phase I TROPION-PanTumor01 trial [17]. An objective response (CR or PR) was observed in 14 of 44 patients (31.8%), including one complete response. Disease control was achieved in 79.5% of patients, with a median duration of response of 16.8 months. Median PFS and OS were 4.4 and 13.5 months, respectively. Slightly better outcomes were observed in patients who had not previously received topoisomerase I inhibitors (DCR 83.3%, mPFS 7.3 months, mOS 14.3 months) (Table 1).
In 2024, early data from the TUXEDO-2 trial were presented at the ESMO Breast conference, evaluating Dato-DXd in TNBC patients with CNS metastases [18]. Intracranial objective responses were observed in 60% of patients not previously treated with SG. Median PFS in the study population was 4.2 months. These findings require confirmation in larger cohorts.
In animal models, combining Dato-DXd with PD-1/PD-L1 inhibitors resulted in enhanced clinical efficacy compared to Dato-DXd monotherapy [19]. This is attributed to immunomodulatory effects on the tumor microenvironment, including upregulation of PD-L1, dendritic cell activation, and enhanced antigen presentation. The release of the cytotoxic payload also induces immunogenic cell death and increased infiltration by cytotoxic T cells [20]. These preclinical findings were supported by the phase Ib/II BEGONIA trial (NCT03742102), which evaluated Dato-DXd in combination with durvalumab as first-line treatment for locally advanced unresectable or metastatic TNBC. The objective response rate (ORR) was 79% (95% CI: 67–88), with a median duration of response of 15.5 months (95% CI: 9.9–NC) and median PFS of 13.8 months (95% CI: 11–NC) (Table 1) [21]. The combination did not significantly increase toxicity. Currently, phase III randomized trials are underway to evaluate Dato-DXd monotherapy in first-line treatment of unresectable or metastatic TNBC (TROPION-Breast02) and its combination with durvalumab (TROPION-Breast 03, -04, -05), with preliminary results expected in 2026.
HER family receptors
As mentioned earlier, TNBC is characterized by the absence of HER2 receptor overexpression. HER2 status is initially assessed using immunohistochemistry (IHC), which evaluates the intensity of membrane staining and the percentage of tumor cells showing signal. According to CAP (College of American Pathologists) guidelines, HER2 overexpression is diagnosed when complete, intense membrane staining is observed in more than 10% of tumor cells (HER2 IHC3+) [22]. In cases of weaker, incomplete, or absent membrane staining, or when fewer than 10% of tumor cells show signal, HER2 is considered not overexpressed (IHC0, IHC1+, IHC2+). For tumors with HER2 IHC2+, ErbB2 gene amplification must be assessed. If amplification is absent (ISH–), the tumor is classified as HER2-negative.
Trastuzumab deruxtecan
The phase III DESTINY-Breast04 trial demonstrated that patients with HER2-low tumors (HER2 IHC1+, IHC2+/ISH–) benefit from trastuzumab deruxtecan, an anti-HER2 ADC linked to a topoisomerase I inhibitor [23]. The study included 557 patients previously treated with no more than two lines of therapy for advanced HER2-negative breast cancer, of whom 63 (11.3%) were hormone receptor-negative (HR–). Patients were randomized 2 : 1 to receive trastuzumab deruxtecan or physician’s choice chemotherapy (capecitabine, eribulin, nab-paclitaxel, paclitaxel, or gemcitabine). The primary endpoint was PFS in the HR+ subgroup. Secondary endpoints included PFS and OS in the entire study population, as well as OS in the HR+ subgroup. The study also evaluated response rate, duration of response, and treatment safety. Trastuzumab deruxtecan significantly prolonged median PFS compared to chemotherapy in the overall population (9.9 vs. 5.1 months), with a 50% reduction in the risk of progression or death (HR = 0.50; p < 0.001). In the HR– subgroup, median PFS was 8.5 vs. 2.9 months (HR = 0.46). OS analysis confirmed the clinical benefit of the ADC: median OS was 23.4 vs. 16.8 months in the overall population (HR = 0.64; p = 0.001), and 18.3 vs. 8.3 months in the HR– subgroup (HR = 0.48) [23]. Response rates were 52.3% in the trastuzumab deruxtecan arm vs. 16.3% in the control arm, and were similar in the HR– subgroup (50.0% vs. 16.7%) (Table 1).
Patritumab deruxtecan
Patritumab deruxtecan is an ADC targeting the extracellular domain of the HER3 receptor, preventing its interaction with neuregulin. This blocks HER3 heterodimerization with HER2 or EGFR, transphosphorylation of the HER3 intracellular domain, and ultimately inhibits activation of the PI3K-AKT signaling pathway [24]. In a phase 1/2 trial, patritumab deruxtecan was administered to patients with advanced TNBC after failure of no more than two prior lines of therapy. The objective response rate (ORR) was 22.6%, with a median response duration of 5.5 months. Median PFS and OS were 5.5 and 14.6 months, respectively (Table 1) [25]. As with other ADCs, the efficacy of patritumab deruxtecan was independent of HER3 expression levels [26]. Preliminary results from studies evaluating patritumab in TNBC have not been confirmed in trials involving seribantumab or lumretuzumab.
Summary
The development of monoclonal ADCs has marked a significant breakthrough in breast cancer treatment, including triple-negative subtypes. Targeting TROP-2, HER2, and HER3 receptors has led to improvements in PFS, OS, and response rates. These therapeutic advances are reflected in clinical guidelines from leading organizations such as ASCO and ESMO, and have become the basis for ongoing clinical trials evaluating new ADCs with different molecular targets and cytotoxic mechanisms. However, since most current data come from comparisons with traditional chemotherapy, the optimal sequencing of ADCs in TNBC treatment remains unclear.
