Introduction

Breast cancer (BC) forms one of the most common culprits of invasive cancers in women. BC had the highest frequency of diagnosis in women worldwide with case counts rising to 2.26 million, compared to 0.41 million in uterine, 0.6 million in cervical and 0.3 million in ovarian cancers in the year 2020 [1]. The incidence of BC has been higher in developed countries and this finding is contributed by the fact that women in developed countries give fewer births and breastfeed for shorter durations, as compared to the developing ones [2]. Though the developed countries stood foremost in incidence rates, countries located in Asia and Africa shared 63% of the total death count in 2020 [3]. Survival was better in high-income and developed countries as compared to low income and many middle-income countries [4]. Over the last three decades incidence and death rates have remarkably increased, for which population structure, environment, genetic particulars and quality of life could be few of the responsible factors [5, 6].

BC encompasses wide phenotypical heterogeneity due to which the clinical profile of each of its subtype varies as they possess distinct demeanors towards the therapy [7]. Several molecular biomarkers are defined under the pathology of this cancer such as estrogen receptor alpha-positive (ERα+), progesterone receptor-positive (PR+), human epidermal growth factor receptor-2 (HER-2/ERBB2), epidermal growth factor receptor (EGFR), Cytokeratin 5/6 (CK5/6), vascular endothelial growth factor (VEGF) and Antigen KI67 [8]. Identification of subtypes with distinguishing prognosis and therapy targets of BC stems from gene expression studies [9]. Differing immunohistochemical properties aid classification of this carcinoma into five namely Luminal A, Luminal B, HER2, Triple negative and normal-like breast cancer. The prognosis profile ranges from best for Luminal A type to worst for triple negative breast cancer (TNBC). The prevalence rates are hereby given in a descending order with the highest seen in Luminal A(70%) followed by Triple negative (15–20%), Luminal B (10–20%), HER2 (5–15%) with least rate observed in normal-like breast cancer [10]. The above statements lead us to the fact that TNBC has the worst prognostication accompanying its higher occurrence. Ergo, embarking upon TNBC, it gained its substantiality in the mid-2000s. The poor prognosis of TNBC is owed to the lack of estrogen and progesterone receptors and under-expression of HER-2 which impedes achievement of successful treatment outcomes [11]. There exists four subtypes of TNBC based on the cellular particulars that include basal-like 1 (BL1), basal-like 2 (BL2), mesenchymal (M) and luminal androgen receptor (LAR). These four TNBC subtypes are associated with distinct expression patterns with immune-modulatory infiltrates varying within each of these subtypes [12]. For further advancements in the development of successful treatment strategies, confirmatory prognostic and predictive biomarkers are desired [13].

The Traditional therapy aiming to mitigate TNBC includes Neoadjuvant therapy, Adjuvant therapy, Surgery and Radiotherapy. Contraction of TNBC using the conventional approach faced a major shortcoming of resistance accompanied by numerous side effects and this demanded the advent of a newer perspective to treat this carcinoma. Aiming to develop even more precise intervention and eradicate the limitations mentioned formerly, immunotherapy was introduced to deal with the carcinogenesis with a novel vision [14]. Immunotherapy strengthens the resident immune machinery and prepares it to recognize and destroy cancerous cells with enhanced efficiency [15]. The immunological profile of TNBC portrays a tumor microenvironment (TME) with abundant lymphocyte infiltration and overexpression of Programmed Death-Ligand 1 (PD-L1) as compared to other subtypes [16]. In addition, TNBC presents with a higher number of somatic mutations resulting from genomic instability which escalates the frequency of neoantigen availability [17]. This depicts a higher responding capability of TNBC to immunotherapy in contrast to traditional approaches. Classes that are conventionally established on the foundation of immunotherapy include monoclonal antibodies, checkpoint inhibitors, cytokines, vaccines and Chimeric Antigen Receptor-T (CAR-T) cell therapy. Monoclonal antibodies offer target specificity with low toxicity profiles. They work in one or multiple ways depending on the antigen its targeting. Checkpoint inhibitors on the other hand target specific immune checkpoint proteins that regulate the functioning of immune system [18]. Cytokine inhibitors target cytokines which are highly inducible secretory proteins that mediate the communication between the immune cells [19]. Onco-vaccines can be preventive or therapeutic that mainly use tumor associated antigens (TAAs) and tumor specific antigens (TSAs). CAR-T cell therapy uses techniques to modify T-cells to produce chimeric antigen receptors. These receptors allow T-cells to get attached to the desired antigens [20]. This review focuses on revealing additional information regarding immunotherapy and to define the targets along with the target-specific interventions for TNBC.

Molecular targets of immunotherapy for breast cancer

The pathogenesis of BC unveils contribution of numerous molecular targets in uncontrolled proliferation and promotion of survival of the tumor. Identification of these molecular targets present a lead towards development of potential drugs that rectify the overexpression of such biomarkers and help eradicate the disease [21]. The potency of these molecular targets and the drugs aimed for achieving anti-tumor activity in TNBC are presented in Fig. 1. This list of molecular targets extends from mesothelin, CD133, CTLA4, PD-1/PD-L1, LAG-3, STAT-3, IDO to CD3 and HER2 and is summarized in Table 1.

Fig.1
figure 1

Molecular targets holding clinical potential to aim immunotherapy interventions for their immunomodulatory effect. (Tumor-infiltrating lymphocyte (TIL), antigen-presenting cell (APC), programmed death-1 (PD-1), programmed death ligand-1 (PD-L1), cytotoxic T-lymphocyte–associated antigen-4 (CTLA-4), regulatory T- cells (Treg cells), indoleamine 2,3-dioxygenase (IDO), tryptophan (Trp), kynurenine (Kyn), T-cell receptor (TCR), L-tryptophan (L-TRP))

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Table 1 Molecular biomarkers qualifying as a potential immunotherapy target for TNBC
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Mesothelin (MSLN), a 40 kDa glycosyl-phosphatidyl inositol-linked membrane glycoprotein is expressed on the mesothelial cells surfaced on peritoneum, pericardium and pleura. Overexpression of MSLN factors in progression of cancers presenting with aggressive phenotypical characteristics and poorer prognosis [22]. It is a key component in sustaining the course of progression, invasion and survival of tumor cells besides developing drug resistance. This overexpression has recently been demonstrated in TNBC thus making it a potential molecular site for targeting therapeutic interventions [23].

CD133 otherwise known as prominin-1 is a pentaspan transmembrane single chain glycoprotein residing in the protrusions present in the biological membrane with specific availability in cholesterol-based lipid domains [24]. Due to its availability on surface, it qualifies as a surface marker for the detection of cancer stem cells [25]. Mislocalization of CD133 from surface to nucleus disrupts transcriptional regulation by causing an interference in the molecular cascades directly in association with proliferation and differentiation of cancerous cells [26]. This deduces CD133 as a potential target to eradicate disease pathology.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is a glycoprotein notably expressed on the cell surface of stimulated T cells, subset of Tregs and on non-lymphoid cells of different tissues alongside being expressed on the surface of some solid tumors [27]. CTLA-4 has a potential to down-regulate T-cell responses and fade peripheral tolerance which shrinks the efficacy of generated antitumor response ultimately leading to tumor immune tolerance [28]. Upregulation of CTLA-4 is one of the important underlying causes of immune evasion and this addresses the role of anti-CTLA-4 antibodies in acquiring CTLA-4 as a putative drug target to obtain optimal therapeutic action [29]. Tremelimumab and Ipilimumab drugs are both fully humanized monoclonal antibodies capable of inducing durable responses by blocking CTLA-4 antigen [30].

PD-1/PD-L1 is a programmed death-1 receptor-ligand system, also referred to as CD279 and B7-H1 is a 33-kDa type 1 transmembrane glycoprotein usually expressed on activated T cells, natural killer (NK) cells, B lymphocytes, macrophages, dendritic cells (DCs) and monocytes with higher level of expression on tumor specific T-cells [31]. PD-1 has a negative impact on adaptive and innate immune responses down-regulating antitumor responses. It inhibits proliferation of tumor-infiltrating lymphocytes, halts cytokine production and strengthens tumor to escape immune response generated by antibodies, ultimately favouring tumor survival [32]. This suggests that it is imperative to target drugs on PD-1/PD-L1 to reverse its detrimental effect. Pembrolizumab, Avelumab, Atezolizumab and Nivolumab are monoclonal antibodies with a proposed mechanism of preventing the interaction of PD-1 receptor with PD-L1 ligand to diminish the consequences of PD-1 pathway-mediated immune responses against tumor cells [33]. As this chain of mechanistic cascades also function in TNBC, the role of these drugs can be explored in diminishing the influence of this carcinoma.

Lymphocyte activation gene-3 (LAG-3) alternatively addressed as CD223 is expressed on the surface of T cells, NK cells, NK T cells and regulatory T (Treg) cells [34]. Overexpression of LAG-3 translates into negative regulation of T cell stimulation and proliferation [35]. It further may also yield a synergistic effect with PD-1/PD-L1 to facilitate depletion of immunity [36]. This ultimately worsens the course of carcinogenesis and categorizes itself as one of the immune checkpoints to target by developing drugs to accomplish superiority over tumorigenesis. LAG-3, CTLA-4 and PD-1/PD-L1 inhibitory drugs are housed under the drug class of immune checkpoint inhibitors (ICIs).

Signal transducer and activator of transcription 3 (STAT3) are designated to be an early tumor diagnostic biomarker localized in basal-like cells of breast cancer cells CD44+ CD24– [37]. Constitutive activation and overexpression of STAT3 upregulate cyclin D-1, c-myc, and bcl-2 and suppress the apoptosis of tumor cells promoting tumor survival and progression [38]. Thus, it is worthy to note STAT3 as one of the probable biomarkers to aim for during drug development process.

Indoleamine-2,3-dioxygenase (IDO) is an immunosuppressive enzyme expressed by dendritic cells residing in tissues and draining lymph nodes of breast cancer patients. IDO catabolizes the breakdown of an essential amino acid L-tryptophan into kynurenines which causes L-tryptophan deficiency [39]. Such a deficient reserve of amino acid exhausts the cytotoxicity of T cells disabling their capability to withstand the negative influence of tumor progression [40]. Therefore, IDO enzyme inhibitors evolve as an approach to consider for immunotherapy development towards TNBC.

HER2 is 180 kDa transmembrane glycoprotein present on the surface of diverse set of T cells whose overexpression is associated with promotion of malignancy of cancer by generating anti-apoptotic signals [41]. TNBC lack expression of HER2 but a part may express circ-HER2 generated from HER2. The generation of this plasmid is a result of chemical gene synthesis of the sequence of exon3-7 of HER2 alongside addition of circulation promoter sequences to 83 bp upstream and 53 bp downstream [42]. Trastuzumab-Deruxtecan has been found to be efficacious in diminishing HER2 positive TNBC. CD3 is another surface marker inhabiting T lymphocytes which has a role of prominence in extinguishing tumor environment [43]. Therefore, simultaneous targeting of both HER2 and CD3 may present remarkable efficacy in eliminating tumor cells. Bispecific antibody like Ertumaxomab addresses this co-targeting necessity by acting on an epitope of HER2 and CD3 by involving Fc fragment to yield active macrophages and antibody dependent cellular cytotoxicity (ADCC) [44]. This achievement demands further investigations and development of similar bispecific antibodies with anti-tumor properties.

Recently, adenosine receptor antagonists are being explored for immunosuppressive myeloid cells in cancer. Two G-protein coupled receptors namely, A2B and predominantly A2A mediate the immunosuppressive action of extracellular adenosine and subsequent blocking of these adenosine receptors enhance anti-tumor immune responses. INCB106385 is a novel drug that has been entrenched as a dual antagonist which binds to both A2A and A2B receptors in the single-digit nanomolar range and antagonizes the production of cAMP in A2A and A2B expressing immune cells [45]. A diagrammatic perspective of this theoretical explanation is depicted in Fig. 2. Further discovery campaigns need to be established for evaluating the efficacy of adenosine receptor antagonists.

Fig. 2
figure 2

Immunosuppressive influence of adenosine binding with A2A and A2B receptors in the tumor micro environment and mechanism of enhancement of anti-tumor response through blocking of these receptors by adenosine receptor antagonists (INCB106385). (adenosine triphosphate (ATP), adenosine monophosphate (AMP), myeloid-derived suppressor cells (MDSC).)

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Roadmap of FDA approvals for TNBC

On March 08, 2019, Atezolizumab qualified for an accelerated approval from FDA for its efficacy in unresectable locally advanced or metastatic TNBC with positive PD-L1 expression. Atezolizumab accompanied by nanoparticle albumin-bound (nab)-Paclitaxel received a joint-approval for the formerly mentioned indication based on its prolongation effect on progression-free survival (PFS). Combining nab-Paclitaxel with Atezolizumab improved the anticancer activity [46]. The recommended dose for Atezolizumab is 840 mg administered as an IV infusion over 60 min on days 1 and 15, followed by administration of 100 mg/m2 nab-paclitaxel on days 1, 8 and 15 for each 28-day cycle. This was to be continued until resolution of disease progression or occurrence of any unacceptable toxicity [47]. The FDA assessment of changes in the therapeutic landscape of metastatic TNBC concluded in voluntary withdrawal of accelerated approval of this drug combination by Genentech where drug safety and efficacy parameters were not responsible for withdrawal [48]. The voluntary withdrawal of this accelerated approval of Atezolizumab for this indication accompanied a disappointing phase but continuing extensive research in this field presents a hope of successfully finding an efficacious treatment of TNBC in the near future [49].

Another remarkable drug was added to the armamentarium of TNBC treatment on July 26, 2021, when FDA approved Pembrolizumab for high risk, early-stage TNBC in the capacity of neoadjuvant. Pembrolizumab combined with chemotherapy regimen was found to be efficacious as neoadjuvant chemotherapy and as a sole Pembrolizumab adjuvant after surgery [50]. Combination of Pembrolizumab with chemotherapy had a positive impact on pathological complete response rate (pCR) and event free survival (EFS). The recommended dose of Pembrolizumab is 200 mg every 3 weeks or 400 mg every 6 weeks as an IV infusion over 30 min with neoadjuvant therapy continuing for 24 weeks and adjuvant therapy for 27 weeks [51].

On April 7, 2021, FDA granted approval to Sacituzumab govitecan in patients previously exposed to two or more systemic therapies for unresectable locally advanced or metastatic TNBC [52]. This approval is rooted from its affirmative potential in prolonging PFS and overall survival (OS). Dosage recommendations for Sacituzumab govitecan are 10 mg/kg once a week on days 1 and 8 of 21-day cycle continued until resolution of disease progression or occurrence of any unacceptable toxicity [53]. This timeline of drug development and subsequent FDA approvals is presented diagrammatically in Fig. 3.

Fig. 3
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Timeline of FDA approvals in the successful application of immunotherapy in TNBC

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Emerging immunotherapies and ongoing investigations have led us to a possibility of more FDA drug approvals for this indication in near future.

Evidence retrieved from clinical trials

Immunotherapy has gained a valuable designation in the interventional sphere of metastatic TNBC and this prompted the investigation of its role in the neoadjuvant setting. Neoadjuvants are a part of an interventional strategy that aim to shrink down the tumor before administration of primary treatment thus improving the efficacy of primary treatment. One trial leaning towards the affirmative potential of Pembrolizumab- a humanized IgG4 monoclonal antibody in combination with chemotherapy as neoadjuvant in TNBC treatment was conducted by enrolling 60 participants and equally dividing the number into six cohorts of Pembrolizumab plus chemotherapy regimens (NCT02622074). Specified primary endpoints were safety and recommended phase II dose (RP2D) and secondary endpoints were pCR rate, objective response rate (ORR), EFS and OS. Endpoints to be explored included defining a relationship between outcome and molecular biomarkers such as PD-L1 expression and stromal tumor-infiltrating lymphocytes. Only two out of total six cohorts met RP2D threshold with 22 patients encountering dose-limited toxicity in the form of febrile neutropenia. The most common grade ≥ 3 treatment-related adverse event was noted as neutropenia (73%). Immune-mediated and infusion reactions were observed in 18 patients with grade ≥ 3 in 6 patients. Across all cohorts, the pCR rate was 60% and 12 month event-free and OS ranged between 80 and 100%. This deduced that administering combination of chemotherapy with Pembrolizumab as neoadjuvant in high-risk, early-stage TNBC showed manageable toxicity and promising antitumor potential [54]. This draws us towards the possibility of administering Pembrolizumab as adjuvant and neoadjuvant therapy in TNBC to potentiate tumor eradication.

Another trial exemplifying role of Atezolizumab- a humanized IgG1 monoclonal antibody combined with Nab-paclitaxel in previously untreated metastatic TNBC was conducted by choosing placebo combined with Nab-paclitaxel as a comparator (NCT02425891) [55]. 902 participants were enrolled with primary end points selected as PFS and OS and secondary outcomes as Objective Response of Complete Response (CR) or Partial Response (PR), Duration of response (DOR), Time to Deterioration (TTD), Percentage of patients with at least one adverse event, percentage of participants with anti-therapeutic antibodies against Atezolizumab, maximum serum concentration of Atezolizumab, minimum serum concentration (Cmin) for Atezolizumab and plasma concentrations of total Paclitaxel. The median PFS was 7.4 months for Atezolizumab plus nab-paclitaxel as compared to 4.8 months for placebo plus nab-paclitaxel receiving patients. ORR and stratified hazard ratio also favoured the administration of combination under study over placebo [46]. This draws a deduction that Atezolizumab plus nab-paclitaxel has a potential to qualify as an interventional strategy in diluting metastatic TNBC.

Although the trials support implication of ICIs in neoadjuvant, adjuvant and metastatic settings of TNBC, these cannot be solely relied upon as a complete cure for this indication as their applicability comes at a cost of incidences of immune-related adverse events (AEs). This demands further investigations to explicate the role of ICI in PD-1/PD-L1 positive TNBC. To summarize, both ongoing and completed clinical trials of immunotherapy are present in Tables 2, 3 and 4 respectively.

Table 2 Ongoing clinical trials evaluating the safety and efficacy of immunotherapy in TNBC
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Table 3 Completed clinical trials presenting supportive evidence in application of immunotherapy in TNBC
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Table 4 Completed clinical trials presenting a supportive evidence in application of immunotherapy in TNBC
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Tackling TNBC with vaccines

Under the multitude of immunotherapies, cancer vaccines have been envisioned as a core focus since a long time. Cancer vaccines (CVs) are designed to mount an immune response by recognizing TAAs and destroy cancer cells that residents them [56]. CVs can be categorized into two based on their purpose of usage namely prophylactic and therapeutic CVs. Prophylactic vaccines aim at reducing future incidences while therapeutic CVs are developed to contract existing malignancy [57]. This strategy has also been practiced for development of CVs directed towards breast cancer. The most common targets and TAAs aimed for successful vaccination in BC include E75 peptide, glycoprotein 2 (GP2) peptide, CD4 + lymphocytes, mucin short variant S1 (MUC1) antigen, melanoma associated antigen-3 (MAGE-A3), New York Esophageal Squamous Cell Carcinoma-1 (NY-ESO-1), Carcinoembryogenic antigen (CEA), human telomerase reverse transcriptase (hTERT), Wilms’ tumor antigen (WT-1), NY-BR-1, Mammaglobin-A (Mam-A) and TRICOM. Targets to develop vaccination are summarized in Table 5.

Table 5 Possible targets for development of TNBC directed vaccines
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With emerging number of antigens specifically expressed in TNBC, huge scope of vaccine development against this carcinoma is presented. To date, numerous CVs right from peptide-based to DNA, cytokines and lymphocyte-based vaccines are paving their way to qualifying for TNBC treatment [58]. Their progress is hindered by immunologically inadequate clinical settings (large tumor burden in metastatic disease), predominance of TAAs as targets, choice of vaccine delivery platform, concomitant administration of therapies with CV and existence of influential unrestrained mechanisms favouring immune escape including antigen modification, absence of Human Leukocyte Ag class-1 (HLA-1) expression and down-regulation of TAAs which cannot be recognised as a target [59], 60, 61.

Numerous clinical trials are advancing towards the search for a safe and effective vaccine. An open-label, phase 2 study was designed to assess the enhancement in tumor-specific immune response and determine efficacy of Biological AE37 Peptide vaccine combined with Pembrolizumab in metastatic triple-negative breast cancer (mTNBC) patients (NCT04024800). The study progressed with Simon two-step design and from 29 eligible patients, 13 patients were grouped as safety cohort in Stage 1 and were subjected to the combination therapy of AE37 vaccine with Pembrolizumab. The primary outcome measures to be assessed were recommended dose within 72 h of vaccination in first 13 patients and ORR determined by RECIST 1.1. The secondary outcomes selected were PFS, OS, Clinical benefit rate (CBR) and Overall toxicity. The study aims to establish the recommended biologic dose of combined AE37 vaccine with Pembrolizumab and the results of the same are expected soon [62].

Another trial presenting a noteworthy evidence is a phase 1b/2 study that evaluated safety and efficacy of metronomic combination therapy in TNBC patients who progressed on or after standard of care (SoC) chemotherapy (NCT03387085) [63]. Treatment was administered by choosing a 3 week cycle routine in a lower dose (aldoxorubicin, cyclophosphamide, cisplatin, nab-paclitaxel, 5-FU/L), antiangiogenic therapy (bevacizumab), Stereotactic Body Radiation Therapy (SBRT), engineered allogeneic CD16 NK-92 cells (haNK), IL-15RαFc (N-803), adenoviral vector-based CEA, MUC1, brachyury, and HER2 vaccines, yeast vector-based Ras, brachyury and CEA vaccines, and an IgG1 PD-L1 inhibitor, Avelumab. Selected primary endpoints for phase 1b was incidence of treatment-related adverse effect (TRAE) and serious adverse events (SAE). Secondary endpoints assessed were ORR, PFS, OS and Disease control rate (DCR). 8 subjects received 3 treatment cycles in an outpatient setting with all having atleast 1 grade ≥ 3 TRAE being chemotherapy-induced neutropenia. 2 subjects were observed to have grade ≥ 3 haNK- related effects while 2 subjects experienced SAEs. 7 subjects remained alive, 6 subjects continued to receive ongoing treatment while 1 CR and 2 PRs were noted. This trial proved that low dose chemo-radiation combined with innate and adaptive immunotherapy has a good safety profile to be administered in outpatient setting [64]. Clinical trials both ongoing and completed evaluating safety and efficacy of cancer vaccines in TNBC are summarized in Table 6.

Table 6 Completed and ongoing clinical trials of vaccines targeting TNBC
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Achievable prospects

The edges embodying immunotherapeutic applications in the neoadjuvant, adjuvant and metastatic stages in TNBC have been widened by the preclinical and clinical evidences. Adding to this, cumulative facts obtained from other solid tumors suggest early biopsy to hold a promising potential in revealing the extent of immunotherapeutic benefits reaching to the patient. Unveiling the entire spectrum of clinical benefits of immunotherapy is possible by development of specific biomarkers to precisely predict the response and possible resistance to given immunotherapy. A rational clinical trial design assisted with strong sample collection approach could provide more reliability and accuracy to the efficacy of immunotherapy in diminishing TME. A comprehensive understanding of the fundamentals of TNBC heterogeneity, molecular biomarkers, immunotherapy mechanism cascades and development of resistance could facilitate development of better immunotherapeutic regimens with maximized benefits. Novel approaches to overcome the obstacle of narrow therapeutic index of these drugs should be developed. Identification of clearly defined indications of these drugs as monotherapy and in combination is necessary to achieve increment in drug prescription rationality.

Conclusion

Extending the therapeutic strategies to include immunotherapy in eradication of TNBC has become a necessity due to the shortcomings of conventional approaches and poor prognosis of this subtype of BC. Clinical trials included lean towards the successful use of immunotherapy in neoadjuvant, adjuvant and metastatic TNBC but this is accompanied with occurrences of alarming AEs which can be perceived as a major setback of immunotherapy. Despite the possibility of unfavourable AEs, immunotherapy still can be envisioned as a potential strategy to target tumor cells as benefits outweigh the risks. Clinical trials contribute encouraging results in favour of applicability of vaccines and concrete evidence regarding the same may be expected soon. With ongoing investigations, newly established immunotherapies may prove to qualify as first line therapies for primary TNBC diagnoses. It may also enhance survival parameters in patients presenting with metastasis or recurrence.