In healthy adults, approximately 90% of NK cells in peripheral blood belong to the cytotoxic CD56^dimCD16⁺ subpopulation capable of developing ADCC responses. A second major NK cell subpopulation, defined by a CD56^bright phenotype and the absence of the CD16A receptor, accounts for 10% of circulating NK cells, prevails in secondary lymphoid organs and lacks ADCC potential. Among CD56^dimCD16⁺ NK cells, several subsets displaying different NK cell receptor combinations are found at variable frequencies. Interindividual variability on the NK cell receptor repertoire is dictated by genetic and environmental factors. Major genetic factors include KIR and HLA-I genotypes. The KIR locus contains a variable number of genes, which together with their allelic diversity, determine the existence of a substantial number of distinct KIR haplotypes distributed in the world population (34, 35). KIR genes are stochastically expressed along NK cell differentiation, generating NK cell clones with discrete KIR combinations (34, 67). Only NK cell clones expressing at least one inhibitory receptor specific for self-HLA-I achieve functional maturity. Thus, KIR–HLA-I interactions contribute to set the overall functional potential in the patient NK cell repertoire (68–70). Whether certain KIR–HLA-I gene combinations can modulate the efficacy of anti-HER2 mAbs in breast cancer patients remains unaddressed, yet associations between distinct paired KIR/KIR-ligands and clinical responses to other tumor antigen-specific mAbs, such as anti-GD2 dinutuximab, anti-CD20 rituximab, or anti-EGFR cetuximab have been reported (71, 72).

Another genetic factor known to modulate antibody-dependent NK cell activation is the CD16A (FcγRIIIA) 158V/F allelic dimorphism encoding for two receptor variants harboring either a phenylalanine (F) or valine (V) at amino acid position 158 in the receptor IgG-binding domain (73). Presence of a V residue defines receptors with high affinity for IgG1 (73). An initial association between the high affinity CD16A 158V/V genotype and complete clinical responses to trastuzumab-based treatment was described in a retrospective analysis of a small cohort of metastatic breast cancer patients (74). Nonetheless, the association of CD16A 158V/F dimorphism with time to relapse and overall survival in larger patient cohorts receiving trastuzumab in adjuvancy remains controversial (75–77). Possible caveats accounting for the different results in these studies have been discussed in critical reviews (9, 78).

Environmental factors challenging the immune system, such as autoimmune or chronic inflammatory diseases and infections, can also shape the configuration of the NK cell compartment. In this regard, infection by human cytomegalovirus (HCMV) promotes, in some individuals, a persistent adaptive expansion of long-lived NK cells hallmarked by the elevated expression of the CD94/NKG2C activating receptor (79–81). Adaptive NKG2C⁺ NK cells are functionally mature and have been associated with the control of HCMV infection in kidney transplant recipients (82, 83) as well as with protection from leukemia relapse upon hematopoietic stem cell transplantation (84). Remarkably, NKG2C⁺ NK cells display enhanced effector function upon antibody-driven recognition of virus-infected targets and rituximab-coated B lymphoblastoid cell lines in vitro (85–87).

Hormone receptor status differentiates two HER2⁺ breast tumor subgroups with distinct pathological response rate and overall survival upon anti-HER2 mAb treatment (88). The benefit of anti-HER2 therapy is highest in estrogen receptor (ER)-negative tumors and progressively decreases in tumors with increased ER expression (89). Globally, many immune parameters in HER2⁺ breast tumors (i.e., TILs, CD8⁺ infiltrate) are inversely correlated with ER or progesterone receptor expression (90), and it is tempting to propose a possible relationship between decreased clinical benefit of ER⁺ tumors to anti-HER2 mAbs and their increased resistance to NK cell-mediated ADCC. E-cadherin expression associated to ER⁺ breast carcinomas (91, 92) dampens trastuzumab-dependent ADCC through its specific interaction with the inhibitory killer cell lectin-like receptor G1 (KLRG1) on NK cells in preclinical in vitro and in vivo models (93, 94). Remarkably, resistance to trastuzumab-based treatment has been associated to E-cadherin expression in tumors from patients with HER2⁺ metastatic breast cancer (94). In addition, estrogens regulate the transcription of SerpinB9/proteinase inhibitor 9, a granzyme B inhibitor shown to decrease the susceptibility of ER⁺ breast cancer cells to NK and CD8⁺ T cell cytotoxicity in vitro (95, 96). Estrogens also upregulate HLA-I transcription through a cis-regulatory element in breast cancer cell lines (97–99), potentially modulating their susceptibility to NK cell-mediated ADCC. The relationship between ER and HLA-I expression has been confirmed by HLA-I immunohistochemical score in ER⁺/HER2⁺ as compared to ER⁻/HER2⁺ tumors (90). Whether other molecular features underlying breast carcinoma heterogeneity (e.g., mutations in PI3K, PTEN, p53, or p95HER2) (8) may modulate the susceptibility to NK cell-mediated ADCC remains uncertain.

HER2 dual targeting with trastuzumab in combination with pertuzumab is nowadays the gold standard therapeutic approach for HER2⁺ breast cancer in the neoadjuvant setting and in the first-line treatment of metastatic disease. Patients that have progressed to prior trastuzumab, pertuzumab, and T-DM1 are treated with lapatinib. Both therapeutic strategies augment the coating of HER2⁺ tumors with IgG1 increasing the possibilities for NK cell-mediated ADCC antitumor responses. Simultaneous binding of pertuzumab and trastuzumab to HER2 increases the density of FcγR binding sites on HER2⁺ tumors; lapatinib does so, by preventing HER2 phosphorylation and internalization, hence increasing HER2 availability for trastuzumab (100–103).

Genetic engineering of the antibody Fc domain for optimizing FcγR engagement is one of the current strategies explored for enhancing the clinical success of several tumor antigen-specific mAbs (104). Margetuximab, an Fc-optimized HER2-specific mAb in clinical development, displayed increased binding to CD16A and elicited enhanced ADCC in breast cancer preclinical models (105). Promising single-agent activity of margetuximab has been recently reported for HER2⁺ breast and gastric cancer patients with advanced disease (106). Results of an ongoing two-arm open-label Phase 3 clinical trial in front of trastuzumab (NCT02492711) will reveal whether margetuximab displays superior efficacy, particularly for patients homozygous for the CD16A 158F/F low affinity genotype, in whom margetuximab showed the highest enhancement of NK cell-mediated ADCC in preclinical studies (105).

In addition to anti-HER2 mAbs, concomitant chemotherapy regimens may significantly impact on NK cell ADCC responses. Several chemotherapeutic agents currently combined or sequentially administered with anti-HER2 mAbs (i.e., anthracyclines, cyclophosphamide, taxanes) elicit a particular type of apoptosis, known as immunogenic cell death (ICD), that is accompanied by the coordinated release of DAMPs (e.g., ATP, and HMGB1) (107). DAMPs released along ICD activate a panel of pattern-recognition receptors (e.g., TLRs, P2RX7) and promote type I IFN release from cancer cells and the secretion of pro-inflammatory cytokines by immune cells (107, 108). Among DAMPs released along ICD, HMGB1 has been shown to enhance NK cell activation and recruitment to the tumor in a TLR2/4-dependent manner in preclinical models (109, 110) whereas type I IFNs have been shown to be necessary for the therapeutic efficacy of anti-HER2 mAb in MMTV-ErbB-2 transgenic mouse model (58). Indeed, a type I IFN signature predicted clinical responses to anthracycline-based chemotherapy in several independent cohorts of patients with breast cancer (108). In addition, in vitro treatment with anthracyclines and taxanes enhanced anti-HER2 mAb-induced ADCC by promoting endoplasmic reticulum-stress and the upregulation of NKG2D-ligands in breast carcinoma cells (111–113). Contravening the traditional view that chemotherapeutic drugs suppress patient immunity, anthracyclines- and taxanes-based treatments associated to enhanced NK cell function in breast cancer patients (60, 113–116).

On the whole, studies integrating information on the patient NK cell repertoire, NK cell receptor ligands on tumor cells and concomitant treatments might shed light on putative resistance mechanisms to anti-HER2 mAbs in HER2⁺ breast cancer patients.

Several observations provide the rationale for combinatorial approaches including anti-HER2 mAbs and antibodies targeting surface NK cell receptors or co-receptors with activating and inhibitory function, termed immune checkpoints modulators. Nonetheless and despite promising results in preclinical models, clinical trials combining anti-HER2 mAbs and immune checkpoint-targeting antibodies are currently lacking.

IFNγ secretion by NK cells has been shown to contribute to the tumor adaptive immune resistance response (154) by upregulating the expression of HLA-I and PD-L1 in HER2⁺ breast cancer cells in vitro and in vivo (58, 155, 156). HLA-I and PD-L1 can be, respectively, recognized by KIR, CD94/NKG2A, LILRB1, and PD-1 inhibitory receptors, modulating the subsequent recognition of transformed cells by NK and T lymphocytes.

Blocking mAbs targeting HLA-I-specific inhibitory receptors with constitutive expression in NK cells include an anti-NKG2A (monalizumab, IPH2201) and an anti-KIR (lirilumab, IPH2101, BMS-986015) (45). Both antibodies are currently in early clinical development being tested for safety and efficacy mostly for the treatment of hematological malignancies.² No clinical trials combining anti-HER2 mAbs and blocking agents targeting KIR or CD94/NKG2A are being developed, yet the safety and early efficacy of monalizumab and cetuximab (anti-EGFR) combination is being tested for the treatment of head and neck cancer (NCT02643550). Of note, an unexpected NK cell unresponsiveness consequent to treatment with lirilumab associated with treatment limited clinical efficacy in multiple myeloma patients (157, 158) warned about the undesired consequences of chronic targeting of HLA-I-specific NK cell receptors.

An alternative strategy, with unprecedented success as standalone treatment for several cancer types, is the blockade of the immune cell inhibitory PD-1/PD-L1 axis. Though generally considered a T cell co-receptor, PD-1 is also expressed by human exhausted NK cells (159) and circulating PD-1⁺ NK cell subpopulations were reported to be enriched in individuals with chronic viral infections as well as in cancer patients (159–161). PD-1⁺ expression is restricted to mature CD56^dimCD16⁺ NK cells and interferes with activation via NKp30, NKp46, or CD16 receptors (159). PD-L1 expression was preferentially detected in HER2⁺ breast tumors showing a strong cytotoxic local immune response (162) and the numbers of PD-1⁺ tumor-infiltrating lymphocytes were associated with poor prognosis in HER2⁺ breast cancer (163, 164). Remarkably, combination of HER2-specific mAbs with blocking antibodies targeting the PD-1/PD-L1 showed greater efficacy in preclinical models (58, 62). These observations support the suitability of combining anti-HER2 mAbs with immunotherapy targeting the PD1/PD-L1 axis. Several clinical trials assessing the benefit of mAbs targeting the PD1–PD1-L axis as monotherapy or in combination with chemotherapy, radiotherapy or hormone therapy are currently being developed for ER⁺ or triple-negative breast tumors (see text footnote 2); likewise, combinatorial approaches with anti-HER2 mAbs are warranted.

TIGIT, a nectin-binding inhibitory co-receptor showing overlapping ligand specificity with the activating DNAM-1, is another inducible receptor with the capacity to modulate NK cell ADCC responses (165, 166). Both receptors recognize CD155 (also known as PVR) and CD112 (also known as Nectin-2), ubiquitous cell-adhesion molecules (167) overexpressed in HER2⁺ breast cancer cell lines (51). Besides CD8⁺ T cells, TIGIT is preferentially expressed on CD16⁺ NK cells and upregulated upon activation via ADCC (168, 169). TIGIT blockade has been shown to enhance trastuzumab-triggered antitumor response by human NK cells in vitro (169). Currently, an anti-TIGIT blocking mAb (OMP-313M32) is in early clinical development being tested for safety as standalone treatment in patients with locally advanced or metastatic solid tumors (NCT03119428).

Another immune checkpoint shown to synergize with anti-HER2 mAb in xenotransplant models of breast cancer is CD137 (58, 170). CD137 (4-1BB; TNFRSF9) is a co-stimulatory receptor induced in activated leukocytes, originally described for its capacity to enhance antitumor T cell responses (171, 172). CD137 expression following CD16 ligation has been shown in murine and human NK cells (173) and CD137 upregulation has been well documented on ex vivo circulating NK cells from breast and head and neck cancer patients upon tumor antigen-specific mAb infusion (170, 174). Two agonistic anti-CD137 mAb are currently in clinical development (urelumab and utomilumab), being tested alone or in combination with anti-PD-1 mAbs in advanced solid and hematologic tumors (45).

Of note, since NK and some T lymphocyte subsets share many receptor/ligand pairs involved in their functional regulation (e.g., PD-1, TIGIT, 4-1BB/CD137, and CD94/NKG2A), combinations between anti-HER2 therapeutic mAbs and distinct immune checkpoint modulators would promote antitumor immunity by dual targeting T and NK cell functional exhaustion.

Several attempts to potentiate NK cell antitumor function by systemic treatment with recombinant cytokines have also been carried out. Besides their effects on T cells, IL-2, and IL-15 signaling through STAT 5 enhance NK cell antitumor function (41, 175, 176).

IL-2 enhanced NK cell-mediated ADCC triggered by anti-HER2 mAb against breast cancer cell lines in vitro and in vivo (177, 178). However, clinical trials including combined administration of IL-2 with trastuzumab did not show improved disease outcome in metastatic HER2⁺ breast cancer patients (179, 180). Caveats of systemic IL-2 administration include treatment-associated toxicity, its rapid clearance in vivo and IL-2 pro-tumor effects through the concurrent activation of CD4⁺ regulatory T cells. Nonetheless, low-dose IL-2 is currently included in a number of clinical trials to support cellular adoptive approaches with combined infusions of NK cells and trastuzumab in HER2⁺ breast cancer patients (NCT02030561, NCT02843126).

IL-15 is an essential cytokine for human NK cell homeostasis; nonetheless, early clinical assays including systemic IL-15 were withdrawn due to concurrent adverse events and dose-limiting toxicities (181). Similarly, IL-15 enhanced the antitumor activity of trastuzumab, yet causing fatal side effects in a humanized tumor mice model (182). Current research efforts include the development of cytokine variants with extended in vivo half-life and targeted action on precise lymphocyte subsets and tumor sites (i.e., engineered IL-2 “superkine,” IL-15Rα Sushi-Fc fusion protein; IL-15 tri and tetraspecific killer engagers) (183–186).

IL-12 has been shown to enhance the antitumor actions of trastuzumab via the enhancement of NK cell IFN-γ production in mouse models (56, 57). In a clinical trial in which IL-12 was combined with trastuzumab and paclitaxel, increased levels of IFN-γ and several chemokines were detected in sera from patients with clinical benefit, but not in patients with progressive disease (187). Currently, two clinical trials are ongoing including IL-12 and trastuzumab combined treatment (NCT00004074, NCT00028535). Preclinical studies are focused on the development of approaches for targeting cytokine expression in the tumor site to avoid toxicities associated to systemic treatment (i.e., tumor-targeting immunocytokines, gene therapy with loco-regional injections of cytokine-encoding plasmid) (188).

Toll-like receptor TLR ligands have been shown to improve both the quality and the magnitude of host antitumor innate and adaptive immune responses (189). TLR2, TLR3, TLR8, and TLR9 agonists have been shown to prime NK cell effector function (39, 40) and to synergize with anti-HER2 mAb therapy in a type I and II IFNs-, NK-, and CD8⁺ T cell-dependent manner in preclinical models (190–192). In the context of breast cancer, TLR ligands are being tested as adjuvants in diverse HER2-peptide vaccination strategies (i.e., TLR9-ligand CpG ODN in NCT00640861; TLR7 agonist imiquimod in NCT02276300; AS15 mixture in NCT02364492, NCT00058526, NCT00140738; TLR3 agonist Hiltonol in NCT01532960), including trastuzumab in some instances (i.e., the TLR9-ligand PF03512676-CpG 7909 or agatolimod-: NCT03512676, NCT00043394, NCT00031278). Strategies for delivering TLR agonists into the tumor site would likely potentiate NK cell-mediated ADCC synergizing with anti-HER2 mAbs antitumor function.

One of the consequences of CD16-mediated NK cell activation is the shedding of CD16 extracellular domain by the induced action of the A disintegrin and metalloproteinase 17 (ADAM17), thus limiting subsequent CD16A receptor engagement and NK cell activation (193). Intriguingly, ADAM10 (with constitutive activity) and ADAM17 (inducible) also control the release of ligands for EGFR/HER receptors (194) and promote the shedding of B7-H6 and MICA/B ectodomains, amplified and overexpressed in breast tumors (195, 196) limiting NKp30- and NKG2D-mediated NK cell activation (140). In fact, ADAM10 and ADAM17 levels have been associated with poor responses and shorter relapse-free survival after trastuzumab treatment (197, 198). In this scenario, inhibition of ADAM17/10 could improve NK cell-mediated ADCC triggered by anti-HER2 mAb, preventing CD16 and B7-H6 shedding as well as enhancing HER2 surface availability. ADAM17 specific inhibitor prevented CD16 shedding and improved NK cell-mediated ADCC responses in vitro (199). Two clinical trials tested the combination of an ADAM17 inhibitor (INCB7839) with trastuzumab (NCT01254136, NCT00864175) yet the development of the compound was suspended by the sponsor corporation and no results were published. Currently, the possibility of enhancing NK cell-mediated ADCC by combining ADAM17 inhibitor (INCB7839) and tumor antigen-specific antibodies is being tested in combination with rituximab (NCT02141451).