Breast cancer is the most commonly diagnosed cancer in women and is a leading cause of cancer death in women worldwide. Despite the available treatment options, such as surgery, chemotherapy, radiotherapy, endocrine therapy and molecular targeted therapy, breast cancer treatment remains a challenge. The advent of immunotherapy has revolutionized the treatment of breast cancer as it utilizes the host’s immune system to directly target tumor cells.
Immunotherapy refers to harnessing the patient’s own immune system to eradicate cancer. The first immunotherapy attempt was introduced in the late 19th century by two German physicians, Fehleisen and Busch, who noticed significant tumor regression after erysipelas infection 
. The following years, the role of immunotherapy was extensively studied by William B. Coley, also known as the “Father of Immunotherapy”, who conducted experiments which involved injections of live bacteria S. pyogenes
and S. marcescens
(referred to as Coley’s toxin) into patients with inoperable bone cancers. In recent years, accumulating data support a key role for the immune system in determining both the response to standard and adjuvant therapy in patients with breast cancer 
The immune system consists of monitoring mechanisms that detect and respond to the presence of cancer cells, a process called “immunosurveillance” 
. Specifically, immunosurveillance consists of three phases: elimination, equilibrium and escape 
. During the elimination phase, the immune system cells recognize tumor-specific antigens and respond by destroying tumor cells. The latter is achieved due to tumor immunogenicity as well as the interaction of tumor-specific antigens with immune system cells. Immunogenicity refers to the ability of a tumor to induce immune responses which inhibit its survival and proliferation. Next, tumor cells that survive the elimination phase, enter a state of equilibrium, which is a transition period from the elimination phase to the onset of the disease 
. Lastly, tumor cells manage to escape immune surveillance and proliferate after establishing an immune suppressive tumor microenvironment (TME) 
Breast cancer (BC) is the most commonly diagnosed cancer among women worldwide with 2.26 million new cases in 2020 and 684,996 deaths 
. According to the World Health Organization, malignancies account for 107.8 million disability-adjusted life years (DALYs), of which 19.6 million DALYs are attributed to breast cancer 
. The significant prognostic differences in patient outcomes have led to the classification of the disease based on estrogen receptor (ER) and progesterone receptor (PR) status, as well as the expression of human epidermal growth factor receptor 2 (HER2) into luminal A (defined as ER+ and/or PR+, HER2−), luminal B (defined as ER+ and/or PR+, HER2+), HER2 overexpressing (defined as EGFR+, ER−, PR−) and triple negative breast cancer (TNBC) which is defined by the absence of ER, PR and HER2. TNBCs represent 10–20% of all BCs with 71–91% of them having a basal-like phenotype and approximately 20% expressing ER or HER2 to a certain extent 
. TNBC is diagnosed based on immunohistochemistry (IHC) in clinical practice. However, guidelines recommend additional analysis in IHC samples with ambiguous HER2 status in order to avoid false positive or negative results 
The number of risk factors of breast cancer is significant and includes both modifiable factors and non-modifiable factors. Non-modifiable factors include age, sex, ethnicity, genetic mutations, family and reproductive history and previous radiation therapy, while modifiable factors include body mass index (BMI), physical activity, alcohol and processed food intake, smoking, exposure to certain chemicals, vitamin supplementation and chosen drugs. It is estimated that about 80% of patients with BC are white non-Hispanic female individuals aged >50 with mutations in the BRCA1, BRCA2, TP53, CDH1, PTEN and STK11 genes and less often in the ATM, PALB2, BRIP1, CHEK2 and XRCC2 genes.
2. Monoclonal Antibodies against HER-2 Receptor Protein
According to the National Institute of Health, monoclonal antibodies are artificially made proteins that can recognize specific targets and are widely used as a targeted cancer therapy 
. Most often, they are categorized into those targeting immune molecules, otherwise known as immune checkpoint inhibitors, or into those targeting oncogenic membrane receptors 
. Figure 1
demonstrates the two types of monoclonal antibodies.
Monoclonal antibody immunotherapies. (A
) Demonstrates the mechanism of action of anti-PD-1 and anti-CTLA-4 immune checkpoint inhibitors. T cells require a total of 3 signals for proper activation. Antigen-specific recognition through T cell receptor (TCR): major histocompatibility complex (MHC) interaction between costimulatory molecules and cytokine signals 
. Cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4) and programmed cell death-1 (PD-1) are negative checkpoint molecules that drive the inactivation of T cells upon interaction with their corresponding ligands, CD80/86 on antigen-presenting cells (APCs) and PD-L1 on tumor cells. Immune checkpoint inhibitors block that interaction and allow positive checkpoint molecules (e.g., CD28) to interact with their corresponding ligands (i.e., CD80/86) and deliver the 2nd signal for T cell activation. (B
) Illustrates the mechanism of action of an anti-HER-2 monoclonal antibody by preventing dimerization of HER subunits. This ultimately leads to the perturbation of downstream signaling events and consequently promotes cell death, apoptosis and cell cycle arrest (created using BioRender.com
, URL accessed on 16 May 2023).
Approximately one third of breast cancer patients exhibit upregulation of HER2 receptor which has led to the development of monoclonal antibodies against this particular receptor such as trastuzumab or pertuzumab 
. Trastuzumab, commercially known as Herceptin, was the first humanized monoclonal antibody to be approved by the Food and Drug Administration (FDA) in 1998 for metastatic HER2+ breast cancer. It is known to function through multiple mechanisms, from mediating HER2 receptor internalization and subsequent degradation via recruitment of ubiquitin ligase to dampen HER-2-dependent signaling pathways such as the RAS/MAPK or PI3K/Akt pathways 
Antibody-drug conjugates (ADCs) are made up of antibodies fused together to cytotoxic agents that allows the efficient delivery of these agents within cancer cells. Antibodies allow for specific recognition of malignant cells followed by ADC internalization and the release of cytotoxic effects mediated by the drug within the cell 
. Trastuzumab emtansine (T-DM1) is an example of an ADC that is composed of trastuzumab as the monoclonal antibody linked to DM1, a cytotoxic agent that targets microtubule assembly and consequently leading to cell death 
This has led to the development of alternative ADCs such as Trastuzumab deruxtecan. The latter contains the same antibody as T-DM1 but with a topoisomerase I inhibitor instead and was first approved in the USA in 2019 
. Using patients previously treated with T-DM1 and having progressed with it, Trastuzumab deruxtecan showed significant clinical efficacy; however, some noticeable adverse effects related to lung disease were observed in a substantial percentage of those patients 
3. Immune Checkpoint Inhibitors (ICIs)
Immune checkpoint inhibitors (ICIs) are monoclonal antibodies that specifically target immune checkpoint molecules on the surface of T cells or tumor cells to promote the activation of immune cells and consequently elicit an immune response against the cancer cells 
. Several studies have detected significant levels of the immunosuppressive PD-L1 checkpoint molecule in HER-2+ breast cancer 
which would suggest that PD-L1 blockade with ICIs would be beneficial.
Several clinical studies are evaluating the use of such antibodies in patients with TNBC—a subtype negative for progesterone, estrogen and HER-2 receptors 
. Atezolizumab is an anti-PD-L1 monoclonal antibody that managed to increase the ORR in patients with metastatic TNBC by approximately 20% compared to non-treated patients 
. Other PD-L1 checkpoint inhibitors such as avelumab have also been shown to augment the ORR in TNBC patients 
. In both cases, the efficacy of the antibodies seemed to be dictated by the level of PD-L1 expression in patients. Those with higher expression seemed to benefit more compared to those with lower to no expression 
. Due to the ability of chemotherapy agents to promote antigen presentation and PD-L1 expression, several studies have turned their attention to combining these agents with ICIs 
. The anti-PD-L1 monoclonal antibody, pembrolizumab, was combined with nab-paclitaxel, paclitaxel and gemcitabine/carboplatin chemotherapy in metastatic TNBC patients and led to augmented PFS 
. This again was restricted to PD-L1-positive patients.
The KEYNOTE trial was the first clinical trial ever to use an anti-PD-1 agent, pembrolizumab, in patients diagnosed with PD-L1+ metastatic TNBC. Overall, the different trial phases suggest that pembrolizumab exhibits a low toxicity profile; however, response rates including ORR and OS remained relatively low. Furthermore, the study failed to show that pembrolizumab was superior to chemotherapy as a monotherapy treatment for metastatic TNBC patients 
. The PANACEA trial enrolled patients diagnosed with HER2+ breast cancer previously progressed on trastuzumab monotherapy to assess the effect of pembrolizumab plus trastuzumab combination therapy.
4. Breast Cancer Vaccines
Cancer vaccines is another type of immunotherapy that uses tumorigenic antigens as a way to train the immune system to recognize those antigens and initiate an immune response against them 
. There are several types of cancer vaccines including those who utilize a patient’s own cancer antigens in order for the vaccine to be specific for the patient’s tumor or those who make use of a patient’s own immune or tumor cells 
. Protein-based vaccines are considered to be the conventional method of vaccination and Figure 2
briefly summarizes how they function.
Mechanism of action of protein-based cancer vaccines. Protein-based cancer vaccines are used to train T cells to recognize specific tumorigenic antigens. Cancer antigens are taken up by antigen presenting cells (APCs) such as dendritic cells through MHC Class II molecules. Active APCs migrate to lymph nodes and present antigens to T cells through MHC: TCR interactions which lead to T cell activation. Active T cells are then able to recognize those same antigens on the surface of cancer cells and initiate an immune response against those tumor cells (created using BioRender.com
, URL accessed on 16 May 2023).
HER-2 positive breast cancer has been reported as a subtype with a strong association between its tumor immune landscape and disease progression 
. More specifically, the NeoALTTO trial has observed a reduction in disease progression in patients with this particular breast cancer subtype following tumorigenic T cell infiltration 
. Moreover, studies have also provided evidence that immune responses against HER-2 positive tumors are initiated by both CD8+ and CD4+ T lymphocytes 
. The evidence for the link between disease recurrence and immune cell infiltration for this cancer subtype is overwhelming; thus, several clinical trials have been targeting this receptor using either protein-based or cell-based vaccination.
The field of DNA vaccine development is rapidly evolving, especially having gone through the COVID-19 pandemic. The AST-301 (pNGVL3-hICD) is a DNA-based vaccine that contains the pNGVL3-hICD plasmid which encodes the intracellular domain of HER-2 receptor protein 
. A clinical trial evaluating the clinical efficacy of the following vaccine plus GM-CSF therapy is still underway.
5. Cytokine Therapies
Cytokines consist of small proteins that are released upon specific stimuli to promote numerous cellular processes. Cytokines can have both anti- and pro-inflammatory effects and can promote cell growth or death 
. For example, interleukin-1β (IL-1β) is a cytokine often found within the TME and can have both anti- and pro-tumorigenic effects. In vivo studies using breast cancer models reported that IL-1β deficiency results in tumor regression due to the intratumoral recruitment and differentiation of inflammatory monocytes. IL-1β deficiency caused low levels of macrophages and recruitment of CD11b+ dendritic cells which consequently led to an interleukin-12 (IL-12) upregulation and interleukin-10 (IL-10) downregulation.
Interleukin-2 (IL-2) is a cytokine often used in cancer immunotherapy mostly due to its ability to activate and expand immune cells, including T lymphocytes and natural killer (NKs) cells. For that reason, studies are looking into IL-2 intervention as a treatment course for cancer in general. For example, a recently commenced clinical trial is aiming to recruit TNBC patients to assess the efficacy of IL-2 administration 
. In addition, the combination of IL-2 with chemotherapy has also been proven to improve the response rate and survival of patients with advanced breast cancer 
. TNBC patients are often reported to be positive for epidermal growth factor receptor (EGFR) which has given enough reason for anti-EGFR antibodies such as cetuximab to be considered as a treatment option 
Interleukin-12 (IL-12) is a cytokine that plays a crucial role in the immune response against cancer. Similar to IL-2, it has been shown to activate T cells, NKs and dendritic cells which together can lead to tumor regression. For that reason, several studies have explored the approach of using this cytokine as another immunotherapy. For example, an in vitro study using SCK murine mammary carcinoma cells, described that IL-12 and IL-18 synergistically inhibit tumor angiogenesis and tumor development 
. Furthermore, clinical studies have also demonstrated a favorable safety profile and high efficacy of IL-12 therapy in patients diagnosed with metastatic breast cancer 
Tumor necrosis factor-alpha (TNF-α) is another type of cytokine and there is a lot of debate in terms of its potential in cancer therapeutics mostly because it has been shown to exert both pro- and anti-tumorigenic effects. For example, studies have reported TNFα to recruit immunosuppressive immune cells to the tumor microenvironment whilst other have shown that similar to other ILs, TNFα can activate the immune system as well 
. However, its use in breast cancer treatment has been limited by its toxic side effects. For that reason, a modified form of TNF-α called PEGylated TNF-α (PEG-TNF-α) has been developed that has a longer half-life and reduced toxicity. Clinical trials have shown that PEG-TNF-α can have antitumor effects in breast cancer, but further studies are needed to determine its optimal use 
Chemokines are small cytokines often found to be involved in cancer progression, Particularly, the C-C chemokine motif ligand 2/C-C chemokine receptor 2 (CCL2/CCR2) pathway has been shown to drive a pro-inflammatory microenvironment which favors tumor progression 
. The following study evaluated the safety profile and potential efficacy of a CCL2 inhibitor, propagermanium (PG), as an antimetastatic drug in perioperative patients with primary breast cancer. Overall, a low-grade adverse event profile was reported. IL-6 levels were reduced in a dose-dependent manner which, given the fact that IL-6 is implicated in the metastatic dissemination of breast cancer 
, suggests that PG may have the ability to hinder metastasis in human BC.
6. Adoptive Cell Therapies (ACTs)
6.1. Tumor-Infiltrating Lymphocyte (TIL) Therapy
TIL therapy often involves the ex vivo expansion of either CD8+ or CD4+ T cells and their infusion back into the patient 
. Both allogeneic and autologous transplants have been attempted for breast cancer patients in the past with each approach having its own harms and benefits. For example, allogeneic transplants exhibited significant toxicity whereas autologous transplants were less efficacious compared to allogeneic 
. TIL therapy has been found to be most effective in patients with a high somatic mutational burden as it allows for the expansion of TILs specifically against tumor-specific neoantigens. For example, a case study for a 49-year-old patient diagnosed with ER-positive metastatic breast cancer reported disease regression following a combination of TIL therapy, specifically against proteins mutated in that patient, IL-2 cytokine therapy and PD-1 immune checkpoint inhibition 
6.2. Engineered TCR Therapy
Engineered TCR therapy describes the genetic engineering of the TCR of a patient’s own T cells to specifically recognize tumor antigens 
. Most αβTCR-engineered cells can only be used against antigens presented by MHC molecules expressed on immune cells such as dendritic cells 
. For instance, Qiongshu et al. (2018) successfully engineered CD8+ T cells to specifically recognize PLAC1 in different breast cancer cell lines. PLAC1 is an example of an antigen known to be often expressed in breast cancer 
6.3. Chimeric Antigen Receptor (CAR) T Cell Therapy
Chimeric antigen receptor (CAR) therapy includes the genetic modification of a patient’s own T cells that involves the addition of a CAR gene. The latter encodes for a receptor that specifically recognizes certain cancer antigens. This allows for a robust and potent anti-tumor response 
. For example, the success of the anti-CD19 CAR T cell therapy against B cell lymphoma leading to its FDA approval is well known 
On the contrary, CAR T cell therapy has not been as successful in solid malignancies such as breast cancer mainly because of the presence of an immunosuppressive tumor microenvironment as opposed to haematological malignancies where there is none. An important factor to consider is choosing an appropriate target antigen that is highly expressed in tumor cells but not so much in healthy tissue 
Overall, several other antigens have been identified so far that appear to be promising targets for the generation of such CAR T cells such as 
. For example, receptor tyrosine kinases (RTKs) drive the activation of several signaling pathways such as the PI3K, MAPK or JAK/STAT pathways, leading to numerous cellular processes such as proliferation, differentiation and apoptosis 
. RTKs identified in breast cancer as promising candidates for CAR cells include HER-2, EGFR and ROR1 
. Cell surface antigens present on tumor cells are another possible candidate for CAR cells. For instance, MUC1 upregulation was detected in the majority of breast cancer subtypes and promotes the metastatic dissemination of tumor cells through the activation of ERK1/2 and NF-kB pathways 
7.1. Immune Checkpoint Inhibitors (ICIs)
Despite the worldwide implementation of ICIs in clinical practice, there are multiple challenges which must be taken into consideration. Firstly, predictors of response are considered the most important challenge among physicians, especially in the case of non-small-cell lung cancer (NSCLC), since only 30–40% of patients seem to benefit from ICIs 
. Up to date, there are gaps in clinical evidence for concurrent administration of steroids and ICIs. Retrospective studies showed that patients taking steroids had a lower progression-free survival or overall survival 
but were not negatively affected while taking steroids for managing side effects 
. It remains unclear if steroids reduce the efficacy of ICIs or if there is an eventual difference driven by a more aggressive form of disease. ICI-induced adverse effects, also known as immune-related adverse effects (irAEs), are more prevalent in organs such as the skin, lungs, liver, kidneys, gastrointestinal and nervous system. ICIs have been associated with gastrointestinal and hepatic adverse events, with colitis being the most common side effect. According to comprehensive systematic review by Wang et al., the majority of deaths were attributed to colitis (70%) with anti-CTLA-4 therapy and pneumonitis and hepatitis (22%) with anti-PD-1/PD-L1-therapy. With combination PD-1/CTLA-4 therapy, deaths were frequently due to colitis (37%) and myocarditis (25%) 
. The median time of onset of neurological adverse events (nAEs) was 6 weeks after ICIs treatment initiation. The incidence of severe nAEs (grade 3 and 4) with anti-CTLA4 therapy was slightly higher (0.7%) than with anti-PD1 therapy (0.4%) 
. The incidence of ICI-induced liver dysfunction is much lower compared to diarrhoea and is reported in about 1–6% of patients, mostly at grades 1 and 2 
. The greater incidence and severity of irAEs with anti-CTLA-4 therapy compared to ani-PD-1/PD-L1 therapies reflects the potentially more dominant role of CTLA-4 compared to PD-1 as a negative T-cell regulator. It is worth mentioning that irAEs of combination ICIs are more severe than those of monotherapy 
. Combination ICIs have been associated with endocrinopathies such as thyroid disorders and hypophysitis, which can be life-threatening if not recognized early 
To tackle the aforementioned challenges, microsatellite instability-high (MSI-H) or deficient mismatch repair (dMMR) could be implemented, as it correlates with stronger responses with ICIs. In fact, it has already found application in as many as 24 cancer types with the highest percentage of MSI-H in endometrial cancer (17%), gastric adenocarcinoma (9%), small intestinal malignancies (8%), colorectal adenocarcinoma (6%) and lower for breast carcinoma (0.6%) 
. The management of grade 1–2 hepatic dysfunction generally involves close monitoring with liver tests to avoid grade 3–4 liver toxicity. The management of grade 3–4 hepatic dysfunction requires high dose intravenous glucocorticoids for 24–48 h, followed by an oral steroid over at least a period of 30 days 
. Regarding neurological AEs, no standard treatment has yet been defined. Clinical improvements have only been reported after the ICI treatment discontinuation 
7.2. Breast Cancer Vaccines
BC vaccines present with good tolerance but without significant clinical benefit. Particularly, the Theratope®
(STn) vaccine for metastatic BC and the NeuVax™ [Nelipepimut-S (NPS), or E75] vaccine for adjuvant BC both failed to display clinical benefit during phase 3 studies 
7.3. Cytokine Therapies
Just like with every treatment, cytokine therapy has its own limitation—not particularly specific for breast cancer. The major challenges presented here originate from the core principles of cytokines. First of all, being pleiotropic makes their targeting not specific to the TME; thus, off-target effects often occur. Additionally, due to the fact that different cytokines can regulate the same pathways, blocking one of them may not be enough as the others can compensate for that loss. Finally, due to their ability to regulate the immune system of the host, a blockade of any cytokines can lead to impairment of the immune system and can lead to autoimmunity or tissue damage 
. The introduction of point mutations or fusion with their receptor counterparts is a way of overcoming the broad specificity of cytokines 
. For example, IL-2 can activate both T cells and NKs but regulatory T cells (Tregs) as well. The latter lead to immunosuppression whereas the former to activation of the immune system 
. Several studies have used site-directed mutagenesis to reduce the affinity of IL-2 for the receptor on Tregs. Overall, these mutants have shown comparable activity to their wild-type forms and enhanced efficacy in in vivo settings 
7.4. Adoptive Cellular Therapies (ACTs)
Each type of ACT from TIL to CAR T cell therapy has its own disadvantages as well. Some patients have non-functional T cells or even no T cells at all as they have what is known as a “cold” tumor (i.e., not immunogenic) 
and therefore must be further modified to be applied in those patients. For example, they must either be co-cultured with IL-2 to further expand and activate the cells or select T cells that are tumor-specific 
Immunotherapy has revolutionized the treatment of breast cancer, significantly improving the curative effect when added to standard therapies. Despite challenges, immunotherapy remains a promising therapeutic strategy and ongoing trials will confirm its clinical benefit in combination with conventional therapies. Further knowledge about the immunosuppressive tumor microenvironment could aid the selection of patients who are most likely to respond to the aforementioned regimens.