Exploring the therapeutic potential of ADC combination for triple-negative breast cancer

Abstract

Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer. Currently, standard treatment options for TNBC are limited to surgery, adjuvant chemotherapy, and radiotherapy. However, these treatment methods are associated with a higher risk of intrinsic or acquired recurrence. Antibody–drug conjugates (ADCs) have emerged as a useful and promising class of cancer therapeutics. ADCs, also known as “biochemical missiles”, use a monoclonal antibody (mAb) to target tumor antigens and deliver a cytotoxic drug payload. Currently, several ADCs clinical studies are underway worldwide, including sacituzumab govitecan (SG), which was recently approved by the FDA for the treatment of TNBC. However, due to the fact that only a small portion of TNBC patients respond to ADC therapy and often develop resistance, growing evidence supports the use of ADCs in combination with other treatment strategies to treat TNBC. In this review, we described the current utilization of ADCs and discussed the prospects of ADC combination therapy for TNBC.

Introduction of antibody–drug conjugates

ADCs have emerged as a promising strategy for the targeted delivery of therapeutic molecules into cancer cells [1]. Initially, successful trastuzumab emtansine (TDM1) treatment of breast cancer has led to ongoing clinical trials evaluating additional ADCs [2]. Each ADC consists of three elements: a recombinant monoclonal antibody, a cytotoxic chemical (called a warhead), and a stable linking agent [3]. Successful ADC discovery relies heavily on the selection of the right target antigen. It is essential that the antigens are expressed exclusively in cancer cells, rather than in normal cells, in order for the antibody to bind accurately and adequately to the target antigen [4]. The linker is very important for the accurate release of the warheads from antibodies. The unstable linker has a broader toxicity profile compared with the stable one due to increased warhead release [5]. Most of the warheads being used in ADC are cytotoxic drugs, mainly microtubule targeting drugs and DNA-damaging agents. The cytotoxic drugs should exhibit a substantially greater toxic potency, the ability to trigger apoptosis, reasonable solubility, and stable in aqueous formulations that are commonly used for antibodies [6].

The use of mAbs to target warheads to cancer cells could date back to the 1960s [7]. The first ADC approved by FDA was gemtuzumab ozogamicin, an anti-CD33 antibody conjugated to a calicheamicin derivative, for the treatment of acute myeloid leukemia (AML) [8]. However, subsequent clinical data did not reveal clinical advantages, and patients treated with ADC had a high rate of fatal toxicity [9]. Therefore, the failure of first-generation ADCs led to the discovery of second-generation ones. The antibodies of ADCs have improved selective binding to cancer cells and decreased cross-reactivity with normal cells. Representative approvals of second-generation ADCs were brentuximab vedotin in 2011 [10] and trastuzumab emtansine in 2013 [11]. Nevertheless, the therapeutic window for second-generation ADCs was narrow due to off-target toxicity, competition with unconjugated antibodies, and aggregation or rapid clearance of conjugates with a drug-to-antibody ratio (DAR) of 8 [4]. Third-generation ADCs raised hope for an expanded therapeutic window. These ADCs have site-specific binding and ensure antibody conjugate drugs with a clear DAR. Third-generation ADCs exhibit a homogeneous DAR of 2 or 4 and employ fully humanized antibodies, which enhance their stability, anti-cancer activity, and reduce toxicity [12]. Targeted delivery of cytotoxic drugs to cancer cells could lower the minimum effective dose and increase the maximum tolerated dose [3]. ADCs have demonstrated greater efficacy compared to single- and double-agent chemotherapy in HER2-positive and triple-negative breast cancer, resulting in improved PFS and OS [13].

Over the past few years, there have been promising advancements in the use of ADCs in the treatment of breast cancer and various other types of malignancies. These successful trials have shown that ADCs can be highly effective in combating cancer while also minimizing toxicity. In fact, in certain cases, ADCs have shown potential in replacing conventional chemotherapy as a treatment option.

ADCs for cancer therapy

The utilization of ADCs in conjunction with other agents is on the rise, particularly as a primary treatment for cancer. With the advancement of technology in producing these intricate therapeutics, a significant number of ADCs have been granted approval or are currently undergoing late-phase clinical trials [14].

Brentuximab vedotin (also known as SGN-35 or Adcetris) is an innovative antibody–drug conjugate that combines an anti-CD30 monoclonal antibody with a tubulin inhibitor called MMAE [15]. This powerful combination enables the drug to target CD30 + lymphocytes, exerting its anti-cancer effects directly. Additionally, brentuximab vedotin utilizes other mechanisms of action, including antibody-dependent cellular phagocytosis and bystander killing, further enhancing its therapeutic capabilities [16]. In 2017, BV obtained FDA approval as a treatment option for patients with refractory primary cutaneous anaplastic large cell lymphoma (ALCL) or CD30 + mycosis fungoides.

Trastuzumab emtansine (T-DM1), also known as Kadcyla, is an antibody–drug conjugate (ADC) that combines the anti-ErbB2 (also known as anti-HER2) monoclonal antibody trastuzumab with the microtubule inhibitor DM1 [17]. Currently, there are 36 active phase I–III clinical trials registered on ClinicalTrials.gov that are investigating the use of T-DM1 in various combinations for ErbB2-positive breast cancer, lung cancer, colorectal cancer, and other solid tumors.

Inotuzumab ozogamicin, known as Besponsa, is an antibody–drug conjugate (ADC) that consists of an anti-CD22 monoclonal antibody linked to a semi-synthetic derivative of calicheamicin called Calich-DMH [18]. Calicheamicin is a potent cytotoxic agent obtained from the soil bacterium Micromonospora echinospora, known for its ability to damage DNA [19]. In 2017, the FDA granted approval to inotuzumab ozogamicin (InO) for the treatment of relapsed or refractory acute lymphoblastic leukemia (ALL) with CD22-positive expression.

Gemtuzumab ozogamicin (Mylotarg) is a medication composed of an anti-CD33 antibody linked to a calicheamicin derivative known as N-acetyl gamma-calicheamicin-dimethyl hydrazide [20]. In 2017, gemtuzumab ozogamicin regained approval from the FDA after demonstrating effectiveness at a reduced dosage and a fractionated dosing schedule for treating CD33-positive acute myeloid leukemia (AML).

Polatuzumab vedotin (Polivy) is a type of ADC that combines an anti-CD79b monoclonal antibody with a tubulin inhibitor called MMAE [18]. In 2019, polatuzumab vedotin was granted accelerated approval by the FDA for the treatment of relapsed or refractory diffuse large B-cell lymphoma (DLBCL).

Trastuzumab deruxtecan (DS-8201a, Enhertu) is an ADC consisting of an anti-ErbB2 monoclonal antibody with an identical amino acid sequence to trastuzumab. It is conjugated with DXd (a derivative of DX-8951). Notably, trastuzumab deruxtecan has a higher drug-to-antibody ratio in comparison to T-DM1, specifically 8 compared to 3 to 4 as cited in reference [18]. Trastuzumab deruxtecan is currently being investigated in a dozen active phase I–III clinical trials registered on ClinicalTrials.gov. These trials aim to explore its potential in treating various solid organ malignancies, such as ErbB2-expressing non-small-cell lung cancer, ErbB2-positive gastric cancer, and ErbB2 low breast cancer. Additionally, there are trials studying its combination with immunotherapy, specifically nivolumab, in advanced breast and urothelial cancers.

Enfortumab vedotin (ASG-22ME, Padcev) is an ADC that combines a monoclonal antibody directed toward nectin-4 with a cytotoxic payload called MMAE, which inhibits tubulin [18]. In December 2019, enfortumab vedotin (EV) was granted accelerated approval by the FDA for the treatment of patients with relapsed or refractory locally advanced or metastatic urothelial cancer.

Sacituzumab govitecan, also known as IMMU-132 or Trodelvy, is an antibody–drug conjugate (ADC) designed to specifically target tumor-associated calcium signal transducer 2 (TROP-2). This targeted approach allows for the selective delivery of SN-38, a potent anti-cancer drug [21]. Sacituzumab govitecan is currently under evaluation for its effectiveness in treating various types of cancer. Specifically, there are ongoing clinical trials investigating its potential use in endometrial cancer (NCT04251416), glioblastoma (NCT03995706), and urothelial cancer (NCT03547973).

Belantamab mafodotin, also known as GSK2857916 or Blenrep, is an antibody–drug conjugate (ADC) designed to target BCMA, a cell-surface B-cell maturation antigen expressed on multiple myeloma cells [22]. BCMA plays a crucial role in the growth and survival of plasma cells, and its serum levels are associated with therapy response and overall survival in multiple myeloma patients [23]. Recently, belantamab mafodotin has received FDA approval as a monotherapy treatment for patients with relapsed or refractory multiple myeloma, who must have previously received at least four therapies, including an anti-CD38 monoclonal antibody, a proteasome inhibitor, and an immunomodulatory agent.

ADCs for triple-negative breast cancer

Sacituzumab govitecan

Sacituzumab govitecan was recently approved by U.S. Food and Drug Administration for the treatment of patients with metastatic TNBC (mTNBC) who have received at least two prior therapies. SG is an ADC targeting anti-trophoblast cell-surface antigen 2 (Trop-2), with the antibody coupled to the topoisomerase I inhibitor SN-38 through a proprietary hydrolyzable linker (Fig. 1). The approval of topoisomerase 1 inhibitors by the FDA marked a significant breakthrough in the selection of antibody–drug conjugate payloads. The first one to gain approval was trastuzumab deruxtecan, and it was later followed by sacituzumab govitecan. Together, these agents represent the most recent family of antibody–drug conjugate payloads to be accepted by the FDA. Trop2, associated with the aggressive biological behavior of cancers, is a transmembrane calcium signal transducer. Trop2 is overexpressed in more than 85% of TNBC. Trop2 has been implicated in numerous biological processes including self-renewal, cell proliferation, invasion, and migration. Trop2 upregulated the levels of phosphorylated MAPK and cyclin D1, thus mediating cell cycle progression. Besides, Trop2 could interact with IGF-1 and attenuate IGF-1R signaling, suppressing the expression of downstream genes including AKT/ERK genes, β-catenin, and slug. Furthermore, trop2 could be transcriptionally modulated by a complex network of transcription factors. Previous experiments suggested that Trop2 expression was highly associated with tumor aggressiveness and drug resistance. Targeting Trop2 could significantly inhibit tumor growth and increase drug sensitivity both in vitro and in vivo, suggesting that Trop2 might be a promising target in cancer treatment.

Fig. 1.

Diagram showing the role of targeted ADCs in TNBC. These ADCs can effectively hinder cell proliferation, invasion, migration, mitosis, and other associated processes by specifically targeting and inhibiting key pathways including PI3K/AKT, EMT, MAPK, and microtubule reorganization

In a phase 1/2, single-group, basket, multicenter study, SG monotherapy demonstrated the preliminary single-agent benefit among patients with heavily pretreated mTNBC [21]. Furthermore, additional patients were recruited to investigate the efficacy and safety of this ADC in a single-arm trial, including 69 mTNBC patients treated with a median of 5 prior treatments. This trial demonstrated an objective response rate of 30% (19 partial responses; 2 complete responses), a median PFS of 6 months (95% CI 5.0–7.3 months), and an OS of 16.6 months. In addition, sacituzumab govitecan was well tolerated in these patients. The majority of tumor tissues were moderately to strongly positive for Trop-2 expression [24]. The trial was further expanded to enroll a total of 108 patients with mTNBC who have received at least 2 prior therapies. The response rate was 33% (95% CI 24.6–43.1), and the median PFS was 5.5 months (95% CI 4.1–6.3). Besides, the median OS was 13 months (95% CI 11.2–13.7) and the median duration of response was 7.7 months (95% CI 4.9–10.8) [25]. Grade 3 or 4 adverse events mostly included anemia and neutropenia [26]. The median treatment duration with sacituzumab govitecan was 5.1 months, which was approximately twice as long as previous cancer treatments (2.5 months). This emphasizes the clinical efficacy of sacituzumab govitecan in patients with challenging-to-treat mTNBC [26]. Based on these positive results, the FDA granted accelerated approval of sacituzumab govitecan for the treatment of heavily pretreated mTNBC patients.

The confirmatory phase 3 ASCENT trial compared sacituzumab govitecan with single-agent chemotherapy of physician’s choice (capecitabine, gemcitabine, vinorelbine, and eribulin) in patients with refractory metastatic TNBC. The trial enrolled 468 patients with 235 patients treated with sacituzumab govitecan and 233 patients receiving single-agent chemotherapy. The median PFS was 5.6 months (95% CI 4.3–6.3 months) for patients treated with sacituzumab govitecan and 1.7 months (95% CI 1.5–2.6 months) for patients treated with chemotherapy. The median OS data demonstrated a significant difference between sacituzumab govitecan and chemotherapy. With sacituzumab govitecan, the median OS was 12.1 months (95% CI 10.7–14.0 months), whereas with chemotherapy, it was 6.7 months (95% CI 5.8–7.7 months). The ORR for the sacituzumab govitecan and chemotherapy group was 35% and 5%, respectively [27]. The incidences of key treatment-related adverse events of Grade 3 or higher were neutropenia (51% with sacituzumab govitecan vs. 33% with chemotherapy), leukopenia (10% vs. 5%), diarrhea (10% vs. < 1%), anemia (8% vs. 5%), and febrile neutropenia (6% vs. 2%) [27]. In this trial, 98% of people taking sacituzumab govitecan experienced side effects, compared with 86% of patients receiving chemotherapy. The most serious and life-threatening side effects included neutropenia, diarrhea, and anemia [28]. While sacituzumab govitecan did lead to higher occurrence of side effects in comparison to chemotherapy, these adverse reactions were generally mild to moderate in severity and could be effectively managed.

Ladiratuzumab vedotin

Ladiratuzumab vedotin (SGN-LIV1A) consists of three different elements: a humanized antibody targeting the zinc transporter LIV-1, monomethyl auristatin E(MMAE), together with a proteolytically cleavable linker [29], and IgG1 antibody targets the zinc transporter LIV-1 through a MMAE payload. LIV-1 is a multi-span transmembrane protein which can act as zinc-influx transporters. LIV-1 can interact with STAT3 and Snail to activate the EMT process [30, 31]. The majority of breast cancer has a moderate to high expression of LIV-1, that has been associated with epidermal-to-mesenchymal transition (EMT) signaling. Ladiratuzumab vedotin was currently being investigated in phase 1/2 clinical trials in breast cancer. Modi et al. evaluated the efficacy and tolerability of SGN-LIV1A in patients with refractory advanced or metastatic breast cancer [32]. 51 TNBC patients were enrolled and treated with a median of three cycles of SGN-LIV1A. Most adverse events were Grade 1 or 2, which commonly included fatigue, nausea, and peripheral neuropathy. Adverse events of Grade 3 or higher mainly included neutropenia and anemia. Febrile neutropenia occurred in two patients, and one treatment-related death was included. The ORR and DCR (disease control rate) was 32% (14 PR) and 64% (14 PR, 14 SD), respectively [32]. Tsai and colleagues recently updated the results of SGNLVA-001 (NCT01969643) in patients with second-line mTNBC, in which SGNLVA-001 at a dose of 1.25 mg/kg achieved an ORR of 28%, and was associated with a manageable safety profile [33].

Trastuzumab deruxtecan (DS-8201a)

Trastuzumab deruxtecan is a next-generation HER2-targeting ADC consisting of a humanized monoclonal anti-HER2 antibody, a cleavable peptide-based linker, and a topoisomerase I inhibitor DX-8951 derivative (DXd), attached to 8 DXd payloads through a maleimide-based mc-GGFG-am protease cleavable linker [33]. DS-8201a shares the same amino acid sequence as trastuzumab and retains the HER2 binding affinity of trastuzumab. DXd, a derivative of camptothecin (CPT), is a water-soluble synthetic CPT analog with stronger inhibitory and anti-tumor activity than SN-38 and other CPT analogs. The researchers discovered that Dxd maintained its effectiveness as a potent drug, while also allowing for the successful attachment of up to 8 DXd molecules to each antibody without causing noticeable aggregation. Despite having lower passive membrane permeability compared to exatecan mesylate, the DXd payload has been utilized in various exclusive ADC programs due to its enhanced safety characteristics [33]. The tetrapeptide linker is a maleimide glycine-phenylalanine-glycine (GGFG) peptide that contributes to plasma drug stabilization and broadens the therapeutic window. The antibody attaches to the HER2 antigen of cancer cells, giving rise to the internalization of ADCs and the selective entry of cytotoxic drugs into cells. Compared to other ADCs, DS-8201a has a higher drug-to-antibody ratio of 7–8 and enables a more potent anti-tumor activity. Collectively, these physicochemical properties offer great therapeutic potential for DS-8201A. Based on the positive results from the phase 2 DESTINY-Breast01 trial (NCT03248492) demonstrating durable anti-tumor activity of DS8201a in patients with HER2-positive MBC who had more than two lines of prior anti-HER2 therapies, DS-8201a was granted accelerated approval in the United States to treat the heavily pretreated metastatic breast cancer. Apart from the efforts of DS-8201a in HER2-positive breast cancer, multiple clinical trials are evaluating its efficacy and safety in HER2-negative or HER2-low MBC. In most of the published data and ongoing clinical trials, HER2-low MBC is defined as tumors with an HER2 IHC score of 1 + or 2 + and negative ISH assay [33], based on the current scoring systems and available data. Therefore, it is estimated that more than half of breast cancers may qualify as HER2-low. It is important to note that the clinical definition of HER2-low relies on the testing technique, specifically the standard IHC/ISH approach, as clear parameters for defining HER2-low using other assays have not been formally established.

The phase 2 DESTINY-Breast01 trial demonstrated the strong efficacy of trastuzumab deruxtecan in patients with HER2-positive metastatic breast cancer who had shown resistance or refractoriness to trastuzumab emtansine. This population of patients lacks effective treatment options, making these findings particularly significant. The subsequent DESTINY-Breast02 trial (NCT03523585) was to compare the effectiveness and safety of trastuzumab deruxtecan with the treatment chosen by the physician in patients belonging to the same population. It showed the favorable benefit–risk profile of trastuzumab deruxtecan in patients with HER2 positive metastatic breast cancer, as previously reported in DESTINY-Breast01, and was the first randomized study to show that one antibody–drug conjugate can overcome resistance to a previous one. However, DS-8201a was also associated with a higher incidence of adverse events. The most frequently reported adverse events that emerged during treatment were nausea (73% with trastuzumab deruxtecan vs. 37% with the treatment of physician’s choice), vomiting (38% vs. 13%), alopecia (37% vs. 4%), fatigue (36% vs. 27%), diarrhea (27% vs. 54%), and palmar-plantar erythrodysesthesia (2% vs. 51%). Grade 3 or higher treatment emergent adverse events were observed in 213 patients (53%) who received trastuzumab deruxtecan, compared to 86 patients (44%) in the treatment of physician’s choice group. It was worth noting that drug-related interstitial lung disease occurred in 42 patients (10%), including two Grade 5 events leading to death in the trastuzumab deruxtecan group, while it was only reported in 1 patient (< 1%) in the treatment of physician’s choice group [34].

Since DS-8201a has bystander killing effects on tumor cells and neighboring cells as a result of the high membrane permeability of Dxd, DS-8201a eliminates not only HER2 + but also HER2 − cells in vitro and in vivo studies. In contrast, this bystander killing effect of DS-8201a was not detected in other HER2-targeted ADCs. The safety and activity of DS-8201a in advanced HER2-expressing (low or high) or HER2-mutated solid tumors were investigated in phase 1, non-randomized, open-label study including a dose-escalation study and dose-expansion study (NCT02564900). In the dose-escalation portion, the ORR of 41% and the disease control rate (DCR) of 88% were observed in 17 BC patients. Recently, results for DS-8201a in HER2-low (IHC1 + or IHC2 + /in situ hybridization-) breast cancer was updated. Fifty-four extensively pretreated patients were recruited and treated with ≥ 1 dose of DS-8201a. The confirmed ORR was 37% (95% CI 24.3–51.3) and the median duration of response was 10.4 months (95% CI 8.8–not evaluable). Furthermore, the authors confirmed that the ORRs of the HER2 IHC1 + and IHC2 + subgroups were similar, suggesting that HER2 expression may not be highly correlated with the clinical activity of DS-8201a. Most toxicities were gastrointestinal adverse effects and hematologic toxicities. Three fatal cases of interstitial lung disease (ILD) have been reported. In conclusion, this study provided evidence that DS-8201a has promising clinical efficacy in HER2-negative patients with manageable drug safety toxicities.

A phase 3, randomized, open-label clinical trial (DESTINY-Breast04) evaluated the clinical efficacy and toxicity of DS-8201a vs. the physician’s choice of chemotherapy (TPC) in patients with HER2-low expressed metastatic breast cancer who were previously treated with one or second lines of palliative chemotherapy [35]. Five hundred fifty-seven patients were randomly assigned with the ratio of 2:1 to receive DS-8201a or TPC. Among these patients, 494 patients displayed positive expression of hormone receptor (HR), and 63 patients showed negative HR expression. In HR-positive cohort, the median PFS was 10.1 months (95% CI 9.5–11.5) in the DS-8201a group, while the PFS was only 5.4 months (95% CI 4.4–7.1) in the TPC group. The median overall survival (OS) also differed between the two groups, with 23.9 months (95% CI 20.8–24.8) for patients receiving DS-8201a and 17.5 months (95% CI 15.2–22.4) for TPC patients. Besides, the ORR rate was 52.3% in the DS-8201a group and 16.3% in the TPC group [34]. In the HR-negative cohort, the corresponding ORR values were 50.0% (95% CI 33.8 to 66.2) and 16.7% (95% CI 3.6 to 41.4). 52.6% of patients receiving trastuzumab deruxtecan had Grade 3 or higher adverse events, while 67.4% of patients receiving chemotherapy experienced Grade 3 or higher adverse events. Patients treated with DS-8201a developed ILD in 12.1% of cases, with most cases being mild or moderate. Early detection and glucocorticoid treatment are recommended to reduce the severity of the condition. In the DESTINY-Breast04 trial, despite the limited number of HR-negative, HER2-low patients, DS-8201a showcased notable benefits in terms of objective response rate (ORR) compared to the chemotherapy selected by physicians. These findings indicate that DS-8201a could be a compelling therapeutic choice for patients belonging to this population with a poor prognosis. Considering the recent approval of TRODELVY for triple-negative breast cancer (TNBC), we anticipate future studies comparing and sequencing sacituzumab govitecan and trastuzumab deruxtecan in HR-negative and HER2-negative breast cancers.

Glembatumumab vedotin

Glembatumumab vedotin (GV) is a novel antibody–drug conjugate consisting of a gpNMB-specific IgG2 antibody conjugated to the MMAE via a proteolytic linker, and were developed using a specific marine-derived cytotoxic molecule as payload called auristatin. gpNMB (glycoprotein non-metastatic B) is an internalize transmembrane protein overexpressed in 40–60% of TNBC. Preclinical studies have demonstrated that gpNMB was involved in cancer cell invasion, metastasis, and angiogenesis. The phase 2 trial known as "EMERGE" (NCT00704158) demonstrated initial clinical activity of GV in patients with triple-negative breast cancer (TNBC) who exhibited overexpression of the tumor marker gpNMB. Furthermore, the randomized phase 2b METRIC study (NCT0199733) compared the progression-free survival (PFS) of GV to capecitabine in gpNMB-overexpressing TNBC patients who had previously received anthracycline and taxane-based chemotherapy. Out of 327 patients, 213 were assigned to the GV group and 92 to the capecitabine group. The primary endpoint of PFS was 2.9 months for GV compared to 2.8 months for capecitabine. Unfortunately, GV failed to demonstrate a significant improvement in relative risks and benefits when compared to capecitabine. As of now, GV has not proven to be therapeutically beneficial in the treatment of triple-negative breast cancer. Several factors, including tumor heterogeneity, biomarker selection, and drug resistance, may contribute to the lack of efficacy observed with GV in TNBC. Future strategies for GV studies could consider enrolling a subset of patients with gpNMB-overexpressing metastatic TNBC who have previously shown sensitivity to taxanes or have had limited exposure to taxane-based treatment regimens.

Preclinical and clinical evidence of combination therapy with ADCs in triple-negative breast cancer

Despite the potential of antibody–drug conjugates (ADCs) as a viable breast cancer treatment option, their effectiveness is often hindered by the development of drug resistance in most tumors over time, presenting a common challenge [27]. The use of ADCs in TNBC is relatively limited. However, considering the efficacy profiles of TDM1 in HER2-positive breast cancer, potential resistance mechanisms of ADCs may include reduced expression of target antigens, mutations in ADC binding sites, activation of alternative signaling pathways like RTK or PI3K, or triggering of immune responses [23]. The intricate nature of antibody–drug conjugates (ADCs) has led to the identification and exploration of various resistance mechanisms that target different components of ADCs. These mechanisms include reduced antigen expression, impaired ADC trafficking and processing, resistance to the cytotoxic payload, and enhanced efflux of the agent from the cells [36]. However, the mechanisms underlying combination therapies are not yet fully comprehended. Further molecular research is required to elucidate the mechanisms of combination therapy and explore more effective combinations. Continued research aimed at optimizing the treatment of ADCs and exploring combination therapies holds the promise of providing further insights into the most effective utilization of these agents. On the other hand, based on findings from current clinical trials for TNBC, it has become apparent that the benefits offered by a single conventional anti-cancer therapy or immunotherapy are insufficient. This is primarily due to the heterogeneity of tumors, tumor evolution, and the development of drug resistance. Consequently, combination therapy has emerged as the preferred approach for TNBC treatment [37]. Consequently, investigations have focused on combining ADCs with other strategies such as immunotherapy, chemotherapy, and targeted therapy, aiming to achieve synergistic effects and expand the therapeutic window of ADCs (Fig. 2).

Fig. 2.

The current therapeutic strategies that could be combined with ADCs in TNBC. The therapeutic strategies mainly included chemotherapy, CDK4/6 inhibitors, PARP inhibitors, PD1/PDL1 inhibitors

ADCs combined with chemotherapy

The favorable initial results of T-Dxd in HER2-low expressing and HER2-positive breast cancer have sparked interest in investigating its combination with chemotherapy in patients with HER2-low expressing triple-negative breast cancer (TNBC). In the phase 1b trial DESTINY-Breast 08, the efficacy and safety of T-Dxd were assessed in combination with capecitabine or durvalumab plus paclitaxel specifically in patients with low levels of HER2 expression. The article consists of two sections. In the first part, the focus is on determining the recommended phase 2 dose and ensuring safety. The second part primarily examines the toxicity, while secondary outcomes include ORR (overall response rate), PFS (progression-free survival), and OS (overall survival) [24, 25]. Trastuzumab duocarmazine (SYD985) is a combination of trastuzumab linked to a synthetic duocarmycin analog, seco-duocarmycin-hydroxybenzamide-azaindole, through a cleavable valine-citrulline peptide [26, 27]. An investigation of SYD985's effectiveness in inhibiting tumor growth in a mouse xenograft model of trastuzumab-sensitive BT474 breast cancer revealed its dose dependency [27]. A phase 1 clinical trial (NCT02512237) demonstrated a certain level of effectiveness in patients with heavily pretreated HER2-negative breast cancer, with an ORR rate of up to 75% in patients receiving doses over 1.2 mg/kg SYD985 [27] (Table 1). Another ongoing phase 1 trial (NCT04602117) is evaluating the safety of the HER2 antibody–drug conjugate SYD985 in combination with paclitaxel in patients with metastatic breast cancer, including TNBC. The primary outcomes being measured are ORR, clinical benefit rate (CBR) at 6 months, and Common Toxicity Criteria for Adverse Events (CTCAE). The secondary outcomes being monitored are PFS and duration of response (DOR) [28]. However, this trial was currently withdrawn as scientific interest in pursuing the SYD985 + paclitaxel combination has diminished.

Table 1.

Novel ADC-based combination therapeutics actively being evaluated in clinical trials for TNBC

Clinical trial	Starting date	Disease	Number of patients	Class of agent	Drug (s)	Phase	Primary endpoints	Status
NCT04556773	2020	TNBC	182	ADC + other anti-cancer agents (chemotherapy, immunotherapy + chemotherapy, AKT inhibitor, aromatase inhibitor, or estrogen receptor antagonist)	Trastuzumab deruxtecan (T-DXd), capecitabine, durvalumab and paclitaxel, capivasertib, anastrozole or fulvestrant	IB	AEs, SAEs	Recruiting
NCT04602117	2021	Solid malignancy (including TNBC)	27	ADC + chemotherapy	Vic-trastuzumab duocarmazine (SYD985), taxanes	I	AEs, CBR, ORR	Recruiting
NCT02161679	2016	TNBC	80	ADC + chemotherapy	Sacituzumab govitecan, platinum	II	PFS	Withdrawn
NCT04927884	2021	TNBC	79	ADC + chemotherapy	Sacituzumab govitecan, cyclophosphamide	IB/II	MTD, HTD, ORR	Active, not recruiting
NCT04230109	2020	TNBC	50	ADC + PD-1 inhibitor	Sacituzumab govitecan, pembrolizumab	II	pCR rate	Active, not recruiting
NCT04468061	2020	TNBC	110	ADC + PD-1 inhibitor	Sacituzumab govitecan, pembrolizumab	II	PFS	Recruiting
NCT05382286	2022	TNBC	440	ADC + PD-1 inhibitor/ chemotherapy	Sacituzumab govitecan, pembrolizumab, paclitaxel, nab-paclitaxel, gemcitabine, carboplatin	III	PFS	Recruiting
NCT03310957	2018	TNBC	72	ADC + PD-1 inhibitor	Ladiratuzumab vedotin (LV), pembrolizumab		ORR, AEs	Recruiting
NCT03424005	2018	TNBC	260	ADC + PD-L1 inhibitor	Ladiratuzumab vedotin (LV), atezolizumab	IB/II	ORR	Active, not recruiting
NCT04434040	2020	TNBC	40	ADC + PD-L1 inhibitor	Sacituzumab govitecan, atezolizumab	II	Rate of undetectable circulating tumor cfDNA- 6 cycles	Recruiting
NCT03971409	2019	TNBC	150	ADC + PD-L1 inhibitor	Sacituzumab govitecan, aveumab	II	BORR	Recruiting
NCT03742102	2018	TNBC	100	ADC + PD-L1 inhibitor	Datopotamab deruxtecan, durvalumab, capivasertib, oleclumab, paclitaxel, trastuzumab deruxtecan	I/II	AEs	Active, not recruiting
NCT04039230	2019	TNBC	65	ADC + PARP inhibitor	Sacituzumab govitecan, talazoparib	IB/II	Dose-limiting toxicity	Recruiting
NCT03992131	2019	Solid malignancy. (including TNBC)	329	ADC + PARP inhibitor	Sacituzumab govitecan, Rucaparib, lucitanib	IB/II	TEAEs, ORR	Terminated
NCT05113966	2021	TNBC	45	ADC + CDK 4/6 inhibitor	Sacituzumab govitecan, trilaciclib	II	PFS	Recruiting
NCT04958785	2021	TNBC	110	ADC + CCR4 inhibitor	Sacituzumab govitecan, magrolimab, paclitaxel, Nab-paclitaxel	II	PFS	Recruiting
NCT05008510	2021	TNBC	216	ADC + tubulin inhibitor	Sacituzumab govitecan, sabizabulin	II	PFS	Withdrawn
NCT03243331	2018	TNBC	18	ADC + PI3K/ mTOR inhibitor	PTK7-ADC, gedatolisib	I	Safety	Completed

Percentage of participants with adverse events (AEs), serious adverse events (SAEs), clinical benefit rate (CBR), overall response rate (ORR), treatment emergent adverse events (TEAEs), progression-free survival (PFS), maximum tolerated dose (MTD), highest tolerated dose (HTD), pathological complete response(pCR) rate, best overall response rate (BORR), recommended phase II dose (RP2D)

Combination chemotherapy for patients with TNBC heavily relies on the use of platinum-based agents, which play a vital role [29]. There was another phase 2 study (NCT02161679) conducted to explore the utilization of carboplatin in combination with SG in TNBC patients. However, it is important to note that this trial has been withdrawn because FDA asked to administratively split from IND115621 to open a new IND you need to file a protocol,while the researchers only drafted it to get the IND open and never initiated. A phase 1b/2 trial (NCT04927884) is ongoing to explore the safety and efficacy of sacituzumab govitecan-hziy in combination with chemoimmunotherapy (cyclophosphamide, N-803, and PD-L1 t-haNK) in TNBC with completion expected in October 2024. This trial may provide guidance for the subsequent use of SG in combination with chemoimmunotherapy.

ADCs combined with anti-PD1/PDL1 therapy

In comparison to non-TNBC cells, TNBC cells have a higher concentration of tumor-infiltrating lymphocytes (TILs) and higher levels of PD-L1 expression [38]. Pembrolizumab has been evaluated for its safety and efficacy in multiple tumor types, including TNBC [39, 40]. Several clinical studies are investigating the combination of ADCs and pembrolizumab for the treatment of TNBC. A phase 2 clinical trial (NCT04230109) is underway to evaluate the safety and efficacy of neoadjuvant SG therapy in patients with localized BC in combination with pembrolizumab. The estimated completion date of the trial is October 2025, and its primary outcome measure is to get a pathological complete response (pCR) rate with sacituzumab govitecan. Other secondary objectives include disease-free survival (DFS), OS, change in breast conserving surgery rate (BCS), and assessment of quality of life (QOL). Recently, the results of the phase 2 study demonstrated that the radiological response rate with SG alone was 62% (n = 31, 95% CI 48%, 77%). Further research on SG combined with pembrolizumab is worth looking forward to. Another phase 2 clinical trial (NCT04468061) is evaluating if the treatment of pembrolizumab and SG in PD-L1-negative TNBC patients with metastatic disease is safe and effective in achieving its intended outcome. The primary endpoint of the clinical trial was PFS, and secondary endpoints included OS, time to progression (TTP), and CBR. The results of the trial have shown a median PFS difference at 3 months with 80% power. The PFS in the single-SG cohort was 5.5 months and in the SG plus pembrolizumab cohort was 8.5 months. A clinical trial (NCT05382286) comparing the progression-free survival of locally advanced, inoperable, or metastatic triple-negative breast cancer (TNBC) patients receiving SG and pembrolizumab vs. pembrolizumab and other treatment options chosen by their physicians is expected to be completed in February 2027. The primary outcome of the trial is PFS, and secondary outcomes include OS, ORR, and other measures. Ladiratuzumab vedotin (LV) is a novel ADC class of humanized anti-LIV-1 IgG1 monoclonal antibodies combined with the microtubule-interfering agent monomethylorlipatin E. The LIV-1-mediated MMAE delivery which disrupts microtubules and induces cell cycle arrest and apoptosis shows encouraging efficacy for TNBC treatment [41]. Phase 1b/2 clinical trial (NCT03310957) examining the effectiveness of LV in combination with pembrolizumab in patients with unresectable locally advanced or metastatic TNBC is currently underway. It is expected that the preliminary results will be available in April 2023, with the main endpoints being objective response rate (ORR), AEs, and other measures. The secondary endpoints include DOR, PFS, and OS. In this ongoing trial, for 26 patients, a follow-up survey of no less than 3 months was found, and the confirmed objective response rate was 54% (95% CI, 33.4, 73.4). A total of 51 patients have received LV plus pembrolizumab (7 and 44 patients at LV 2.0 and 2.5 mg/kg, respectively). No DLT was observed at LV 2.0 mg/kg, but two patients enrolled at LV 2.5 mg/kg were found to have DLT of Grade 3 colitis and Grade 3 diarrhea. TEAEs most commonly reported across both LV dose levels were nausea and vomiting (53%). There were 16% of Grade 3 + AEs due to neutropenia, and 6% of SAEs due to abdominal pain and constipation. Based on preliminary results, LV plus pembrolizumab has an encouraging clinical activity when applied to TNBC [42].

Atezolizumab is a monoclonal antibody targeting PD-L1 that can be used in combination with nab-paclitaxel to treat patients with locally advanced TNBC [43–45]. Currently, several clinical trials are exploring the combination of atezolizumab with ADCs. A phase 1b/2 trial (NCT03424005) is evaluating the role of atezolizumab plus SG, and atezolizumab plus SGN-LIV1A in mTNBC patients. Primary outcomes included ORR and secondary outcomes included PFS, DCR, OS, and DOR. A phase 2 clinical trial (NCT04434040) is currently underway to evaluate the effectiveness of atezolizumab and SG combination therapy in treating patients with TNBC. The aim is to reduce the risk of recurrence and explore its therapeutic potential. Its primary endpoint is the rate of undetectable circulating tumor cfDNA after six cycles, while the secondary endpoints are OS, invasive disease, and undetectable circulating tumor DNA after one and three cycles, invasive disease-free survival (iDFS), distant metastasis-free survival (DMFS), and 3-year recurrence rate. Avelumab monotherapy has shown encouraging clinical efficacy in TNBC patients[46]. An aveumab and sacituzumab govitecan combination clinical trial (NCT03971409) has been investigated in patients with stage IV or non-surgically resectable and recurrent TNBC with the primary endpoint being the best overall response rate (BORR) and secondary endpoints being ORR, CBR, PFS, and OS. Thomas Bachelot et al. provided evidence for the monotherapy of durvalumab in patients with TNBC, but no studies have been conducted on its combination [47]. A phase 1B/2 trial (NCT03742102) investigated the efficacy and safety of durvalumab and datopotamab deruxtecan for mTNBC, with AEs and laboratory findings as the primary endpoints and secondary endpoints including ORR, PFS, OS. Based on preliminary results, 29 patients received the combination of Dato-DXd and durvalumab-D, 24 of whom remained on treatment, with a median follow-up of 3.9 months (ranging from 2 to 6 months). The confirmed ORR was 20/27 (74%; 95% CI 54–89); 2 (7%) patients had a complete response, and 18 (67%) had a partial response, all were in response at data cutoff. Common AEs were stomatitis (69%), nausea (66%), and alopecia (66%). Grade 3 + AEs occurred in eight (28%) patients. Discontinued treatment due to AE included one case of an anaphylactic reaction and one case of troponin increase. The trial may provide a new combination regimen for the clinical treatment of TNBC, the combination of Dato-DXd with durvalumab demonstrated robust response rates with a manageable safety profile in this preliminary analysis [48].

ADCs combined with targeted therapy

Combined treatment of ADCs and PARP inhibitors

The PARP poly (ADP-ribose) polymerase is essential for chromatin remodeling, DNA repair, and apoptosis [49–51]. A few phase 3 clinical trials have examined the effectiveness of PARP inhibitors in patients with gBRCA-associated breast cancer, including OlympiAD, EMBRACA, and BROCADE 3 [52–54]. As with other chemotherapy agents, PARPi also faced drug resistance and dose-dependent toxicity [55, 56]. Numerous studies are currently exploring the effectiveness of combining PARP inhibitors with ADCs to combat drug resistance and achieve a synergistic effect in the treatment of breast cancer. In a preclinical setting, the targeted antibody-based delivery of SN-38 induced stabilized TOP1 cleavage complexes (TOP1CCs). No matter if BRCA1/2 was mutated or not, SG in combination with all three PARPi increased DNA damage and inhibited tumor growth synergistically [57]. A phase 1b/2 clinical trial (NCT04039230) is investigating the efficacy of SG in combination with talazoparib in patients with metastatic TNBC. The dose-escalation phase of the clinical trial has achieved successful enrollment, determining the recommended phase-2 dose (R2PD) for the sequential administration of SG (10 mg/kg on days 1 and 8) with talazoparib (1 mg on days 15–21), every 21 days. The enrolled patients were previously treated with at least one therapeutic regimen for TNBC. The primary outcome was dose-limiting toxicity (DLT). Secondary outcomes included time-to-tumor response (TTR), DOR, PFS, and OS. Recently, the results from the phase 1b study have been reported. The staggered schedule (SG day 18, every 21 days with talazoparib) was relatively well tolerated without DLTs. Besides, the staggered schedule demonstrated encouraging evidence of clinical activity with objective responses in patients with pre-treated mTNBC [58].

Another phase 1b/2 study (NCT03992131) evaluated the efficacy and safety of rucaparib in combination with sacituzumab govitecan in patients with an advanced/metastatic solid malignancy (TNBC or urothelial cancer or ovarian cancer or solid tumor with deleterious mutation in homologous repair genes), with AE and ORR as primary endpoints, and both the PFS and DOR as secondary endpoints. Preliminary results from six patients treated with the combination of SG and rucaparib have been recently updated [59]. Among the six patients, two of whom had metastatic TNBC, two of whom had ovarian cancer, one of whom had endometrial cancer, and one of whom had urothelial cancer, all had received a median of 4 prior treatments. Three patients were previously treated with PARP inhibitors. The six patients were divided into two cohorts. Three patients in cohort 1 received rucaparib 300 mg twice daily combined with 6 mg/kg intravenous SG each cycle. Patients in cohort 2 were treated with an oral regimen of 300 mg rucaparib once daily plus 6 mg/kg of SG. There were two patients in cohort 1 with neutropenia of Grade 4. No patients in cohort 2 had DLTs. All patients displayed either SD or PR according to RECIST v1.1. Despite the small number of enrolled samples, the results suggest that ADC plus PARPi has promising anti-tumor activity in patients with advanced or metastatic solid tumors, including PARPi-exposed patients with or without HR-related gene mutations. The clinical introduction and application of PARP inhibition marked a significant milestone in the treatment of TNBC. However, the low prevalence of BRCA1 or BRCA2 germline mutations and transitory benefit greatly hindered its therapeutic performance in clinical. These ongoing clinical trials might provide a good basis for subsequent ADCs in combination with PARP inhibitors to maximize the therapeutic potential of PARPi in TNBC.

ADCs plus CDK4/6 inhibitors

The cell cycle entry process, regulated by the cyclin D-CDK4/6-INK4-Rb pathway, plays a crucial role in cancer development [60–62]. For ER-positive/HER2-negative breast cancer patients, three CDK4/6 inhibitors have received FDA approval [63]. In triple-negative breast cancer, there is limited clinical research focusing on the combination of SG and CDK4/6 inhibitors, particularly to emphasis on mitigating bone marrow toxicity. Trilaciclib, a small-molecule CDK4/6 inhibitor, has shown protective effects on hematopoietic stem cells, progenitor cells, and immune cells from chemotherapy-induced damage when administered concomitantly with cytotoxic chemotherapy [64]. A phase 2 experimental study (NCT05113966) is underway to investigate to assess the safety and efficacy of combining SG with CDK4/6 inhibitors (trilaciclib) in advanced or metastatic TNBC patients who have received at least one prior treatment in the metastatic setting. Patients were administered with trilaciclib prior to sacituzumab govitecan-hziy. The study evaluates primary outcomes such as PFS, and secondary outcomes including ORR, DOR, CBR, OS, and evaluation of the myeloprotective effects of trilaciclib. Initial data from the first 18 patients demonstrated a meaningful reduction of trilaciclib of over 50% in multiple adverse events related to the use of single-agent SG, demonstrating the targeted effect of trilaciclib [27, 65]. We await the progress of this trial and the presentation of efficacy results, expecting on-target effects of trilaciclib in reducing adverse effects.

ADC in combination with other drugs

A phase 2 trial (NCT04958785) is underway to evaluate the efficacy of a CD47 monoclonal antibody in combination with SG for the treatment of TNBC. The primary outcome of the study is ORR, while DOR and PFS are secondary outcomes. Another phase 2 trial (NCT05008510) is underway to assess the combination of the oral tubulin inhibitor sabizabulin and the antibody sacituzumab govitecan-hziy for TNBC, with the primary endpoint being radiographic progression-free survival (rPFS) and ORR as secondary outcomes. This study has been withdrawn but is hopefully to be reopened in the future. In addition, ADC drugs targeting RON have shown promising anti-tumor activity against TNBC cells in vitro, and their combined application with chemotherapy drugs, immune checkpoint antibodies, and other drugs require further investigation [66]. The RN927C drug, a targeted Trop-2 ADC, uses a site-specific transglutaminase-mediated conjugation method along with a proprietary microtubule inhibitor (MTI) linker-payload called PF-06380101.,exhibits strong lethality against TNBC tumor cells in vitro and inhibits tumor growth. Currently, there has been no exploration into combination therapy based on RN927C. [67]. Besides, PI3K/mTOR inhibitor gedatolisib in combination with PTK7-ADC is currently undergoing a preliminary safety assessment (NCT03243331) in metastatic TNBC. Gedatolisib, a pan-class I isoform PI3K/mTOR inhibitor, and PTK7-ADC, an antibody–drug conjugate targeting the cell-surface PTK7 protein (a Wnt pathway co-receptor) with an Auristatin payload [68], are two promising compounds. PTK7 is an attractive secondary target as it is upregulated after PI3K inhibition and known to be overexpressed in TNBC. Recent data have demonstrated that the combination of PTK7-ADC and gedatolisib exhibits a unique concept of "double synergy." This is attributed to gedatolisib increasing the expression of the PTK7 target, leading to one mechanism of synergy, while the Auristatin payload on PTK7-ADC acts synergistically with gedatolisib, providing a second mechanism of synergy [69]. Its results may serve the basis for future research of ADC-based combination strategies.

Conclusion

The utilization of ADCs presents a promising therapeutic strategy for enhancing conventional chemotherapy by reducing its toxicity and improving its effectiveness. Notably, ADCs have demonstrated initial success in treating cases of TNBC. Several ADCs, including SG, SGN-LIV1A, DS-8201a, and GV, have undergone evaluation. Additionally, the combination of ADC therapy with other medications is emerging as a potentially effective approach, aiming to achieve synergistic effects and target tumor heterogeneity. Numerous clinical trials are currently exploring the efficacy and tolerability of combining ADCs with chemotherapy, PD1/PDL1 inhibitors, PARP inhibitors, CDK4/6 inhibitors, and other therapeutic strategies. Promising preliminary data have been reported, and the majority of these clinical trials are expected to conclude within the next 5 years. These findings may pave the way for the future development of ADC combination therapies in TNBC. With the dedicated efforts of researchers, it is anticipated that the synergistic potential of ADC combination therapy will continue to show promising prospects in the future.

Abbreviations

TNBC

Triple-negative breast cancer

ADCs

Antibody–drug conjugates

mAb

Monoclonal antibody

SG

Sacituzumab govitecan

TDM1

Trastuzumab emtansine

AML

Acute myeloid leukemia

DAR

Drug-to-antibody ratio

mTNBC

Metastatic triple-negative breast cancer

Trop-2

Anti-trophoblast cell-surface antigen 2

SGN-LIV1A

Ladiratuzumab vedotin

MMAE

Monomethyl auristatin E

EMT

Epidermal-to-mesenchymal transition

ORR

Objective response rate

DCR

Disease control rate

DXd

DX-8951 derivative

CPT

Camptothecin

GGFG

Glycine-phenylalanine-glycine

ILD

Interstitial lung disease

TPC

The physician’s choice

HR

Hormone receptor

GV

Glembatumumab vedotin

gpNMB

Glycoprotein non-metastatic B

CBR

Clinical benefit rate

CTCAE

Common toxicity criteria for adverse events

DOR

Duration of response

TILs

Tumor infiltrating lymphocytes

pCR

Pathological complete response

DFS

Disease-free survival

BCS

Breast conserving surgery rate

QOL

Quality of life

TTP

Time to progression

iDFS

Invasive disease-free survival

DMFS

Distant metastasis-free survival

BORR

Best overall response rate

TOP1CCs

TOP1 cleavage complexes

DLT

Dose limiting toxicity

TTR

Time-to-tumor response

rPFS

Radiographic progression-free survival

MTIs

Microtubule inhibitors

Author contribution

All authors contributed to the conception of the study and the preparation and approval of the paper. LL took the lead in writing the manuscript, while ZN, ZC, and CF played vital roles in conducting the literature review, collecting and analyzing the data. KC and YS were responsible for conceptualizing the study, analyzing the data, critically reviewing the content, and providing valuable revisions to the manuscript.

Funding

This research was supported by the Natural Science Foundation of Jiangsu Province (Grant numbers BK20210096), National Natural Science Foundation of China (Grant numbers 82100109 and 82303622), Soochow University College Students Innovation and Entrepreneurship Fund (Grant numbers 5731514922).

Data availability

Not applicable.

Declarations

Conflict of interest

The authors declare that there are no conflicts of interest.

Ethics approval and consent to participate

The patients/participants provided written informed consent to participate in this study.

Consent for publication

All authors read and approved the manuscript for publication.