Abstract

Breast Cancer arises to be the most diagnosed cancer type in recent decades. It ranks to be the most vulnerable among women in terms of incidence and mortality. In 2020, 2.3 million women were diagnosed with breast cancer, and 6.85 lacs of death were reported globally. Here, we will focus mainly on TNBC, the most complicated breast cancer subtype. Therefore, novel treatment modalities are urgently required. Treatments like chemotherapy and radiotherapy limit the efficacy of therapeutic outcomes. Thus, new specific ideas are coming up to find a way out. For triple-negative breast cancer (TNBC), which is currently the most complex and challenging breast cancer subtype to treat, chemotherapy is still the standard of care. There has been a lot of study into novel treatments for people with TNBC because of its poor prognosis and the high chance of clinical recurrence. Chimeric antigen receptor (CAR) T cell-based immunotherapy directs the patient’s immune system to recognize and eradicate tumor cells that express tumor-associated antigens (TAAs). It opens up a new area of research. Chimeric Antigen Receptor (CAR-T) cell therapy is an immunotherapy type derived from adoptive T cell relocation. CAR-T cells are well equipped with specific antibodies to identify antigens in self-tumor cells, thus bringing out cytotoxic outcomes. CARs are the modified receptors with improved specificity and responsiveness to intensify the recognition of cancer cells. The therapeutic effects of CAR-T cell treatment, including breast cancer, have not lived up to expectations in solid tumors despite recent triumphs in treating hematologic malignancies. In this review, we will discuss some recent developments in the field of breast cancer-specific immunotherapy using CAR-T.

Keywords

CAR-T cell therapy

Breast cancer

Chimeric antigen receptor

Immunotherapy

Genetic engineering

Triple-negative breast cancer

Abbreviations

1. Introduction

In spite of the spectacular steps forward in cancer research, breast cancer continues to be a major health issue and is currently a priority for biomedical research. Over the next several years, it is anticipated that both its incidence and mortality would considerably rise globally. Recently, breast cancer has captured the attention of researchers. According to recent data, breast cancer is the most common type of cancer that kills women under the age of 45. TNBC has intricate biological characteristics and appears to be very diverse. However, age is not a factor in management, advice, alternatives, or tactics. The “complex” biology of this cancer remains unclear and unexplored [62]. Women with breast cancer are known to be treated with a combination of surgery, chemotherapy, and radiation therapy. In order to reduce side effects and increase overall survival, ongoing research initiatives started involving personalized treatment [107]. Despite recent advances in cancer treatment, current evidence-based medicine shows that advances in breast cancer have been slow for the past decade. This corresponds to a several months extension of survival in the metastatic area. This is not surprising given the significant limitations of targeted therapies currently available. Reasons for high intrinsic and acquired resistance to available target drugs include their transient antitumor activity and the inability to explain inter-individual and intra-tumor heterogeneity. Understanding this highly complex heterogeneity is crucial in the “war” against the development and metastasis of breast tumors [94].

There are 4 extensive types of breast cancer that include ductal carcinoma in situ (non-invasive cancer where abnormal cells are found in the lining of the breast milk duct), invasive ductal carcinoma (invasive cancer where abnormal cancer cells forming in the milk ducts have spread beyond the ducts into other parts of the breast tissue), inflammatory breast cancer (aggressive and fast-growing breast cancer in which cancer cells infiltrate the skin and lymph vessels of the breast), and metastatic breast cancer (classified as Stage 4 breast cancer, where cancer spreads to other parts of the body faster, usually includes the lungs, liver, bones or brain) [1]. Some more specified breast cancer types are HER2 breast cancer and TNBC.

The diagnosis of TNBC is the absence of the three most common types of receptors (oestrogen, progesterone, and HER-2 neu genes) known to promote breast cancer growth in cancerous tumors. It means that breast cancer cells have been tested negative for epidermal growth factor receptor 2 (HER-2), oestrogen receptor (ER), and progesterone receptor (PR) [1]. Because tumor cells lack the necessary receptors, conventional treatments such as hormone therapy and drugs that target oestrogen, progesterone, and HER-2 are ineffective. Using chemotherapy to treat triple-negative breast cancer remains one of the valid options. TNBC may respond better to chemotherapy in its early stages than many other cancers [1]. The majority of TNBCs have a “basal-like” molecular profile in their gene expression array. The majority of BRCA1-related breast cancers are triple-negative and basal cell-like. The extent to which BRCA1 signaling contributes to the behavior of sporadic basal breast cancer is an active area of research. Epidemiological studies have shown a high prevalence of TNBC in young African women. Accumulated evidence suggests that risk factor profiles differ between this subtype and the more common luminal subtypes. Although sensitive to chemotherapy, early recurrence is common, and a tendency for visceral metastases, including brain metastases, is observed. Targeted drugs such as epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), and poly (ADP-ribose) polymerase (PARP) inhibitors are currently in clinical trials for the treatment of this invasive disease [6].

HER2-positive breast cancer tests are positive for a protein called HER2. This protein promotes the growth of cancer cells. In about 1 of every 5 breast cancers, the cancer cells have extra copies of the gene that makes the HER2 protein [44]. Importantly, significant advances have been made in HER2-positive breast cancer, which accounts for 20% of all breast cancer patients. Identification of the HER2 signaling pathway and its dysfunction when the HER2 gene is amplified has led to the development of the well-known anti-HER2 monoclonal antibody (mAb) trastuzumab [42]. A randomized, controlled phase III trial reported that the addition of trastuzumab to chemotherapy for HER2-positive breast cancer significantly improved overall survival in metastatic and adjuvant therapies. For these reasons, trastuzumab was the standard first-line treatment for these patients [7,42,57]. However, recurrence rates and disease progression rates remain dramatically higher. The single-agent trastuzumab emtansine (T-DM1) provides a potentially improved clinical outcome. The safety and efficacy of this new drug in breast cancer and its limitations in the treatment of metastatic HER2-positive breast cancer have recently been evaluated [7,26,42]. Phase III studies are underway comparing this single drug with both different regimens and a significant variety of novel combinations of mAbs and TKIs. A great hope for the future in the fight against this aggressive and mysterious type of cancer is the discovery of new “drug” agents [7,42].

2. Threats in breast cancer

The most important risk factors for breast cancer are being a woman and aging. More than 70% of women with the disease have only these two risk factors. The risk of breast cancer increases as a woman ages. It even becomes more critical after the age of 50. Most breast cancers occur in women 55 years of age and older. There are other risk factors as well that govern the increase in breast cancer. They have different levels of risk. Having one or more influences doesn’t guarantee that the disease will occur [2].

2.1. Stronger threats

2.1.1. Genetics

BRCA1 and BRCA2 are called breast cancer genes. Changes in these genes lead to a higher risk of breast cancer and other cancers. These changes can lead to an 80% lifetime risk of developing breast cancer. Changes in other genes, such as p53, PTEN, and CHEK2, also increase breast cancer risk [2].

2.1.2. High-dose radiation exposure

Radiation therapies used to treat various conditions can inculcate risks of breast cancer. Exposure to a large amount of radiation before the age of 30 can lead to tuberculosis [2].

2.2. Moderate threats

2.2.1. Previous breast cancer diagnosis

A woman has a higher chance of recurrent cancer if she has had breast cancer in the past. The risk is around three to four times greater than that of any other woman without a history.

2.2.2. Direct family history

A woman has an increased chance of developing breast cancer if a “first-degree relative,” such as her mother, sister, or daughter, has the disease. 87% of women who are diagnosed with breast cancer do not directly have a family history of the disease. As a result, everyone should be vigilant about other risk factors and screening recommendations. A simple family history cannot guarantee that you will get breast cancer [2].

2.2.3. High-risk breast lesions

A woman is 4–5 times more likely to develop breast cancer if she has a history of benign diseases. The disorders include lobular carcinoma in situ and alterations in breast cells (breast atypia). Consequently, a woman with one of the aforementioned diseases is given the option of more regular breast cancer screening. Along with regular mammograms, this also involves breast examinations (twice a year) and MRIs. By using oral medications like tamoxifen and raloxifene, one might reduce the risk by lowering the body’s Oestrogen levels [2].

2.2.4. Weight

Breast and other cancers are more likely to develop in people who are overweight. After menopause, women should pay particular attention to this. In order to lower the risk of breast cancer, losing weight is crucial.

3. Available treatments

3.1. Immunotherapy using pd1/pdl1 inhibitors

The programmed death receptor 1 (PD-1) pathway performs a major task in calibrating the immune reaction. PD-1, an immune checkpoint inhibitor receptor intimated on activated T cells, B cells, natural killer cells, activated monocytes, dendritic cells, myeloid cells, and a subset of thymocytes, restricts autoimmunity by manipulating the activity of effector T cells within the range against feedback to an inflammatory stimulus. PD-L1, a PD-1 ligand, functions as an immunosuppressive signal and is stimulated in reprisal to pro-inflammatory signals, like interferon-γ [25,71,83,87].

3.2. Associated drugs

3.2.1. Pembrolizumab

Pembrolizumab (MK-3475) is a monoclonal antibody that specifically binds to PD-1, a transmembrane protein in T cells. The adherence of the antibody to PD-1 restricts the interaction between PD-1 in T cells and the binding of ligand PD-L1 in tumor cells thus, the immune response is elevated to eliminate abnormal consequences in tumor cells [32,36,38,117]. The KEYNOTE-01231 study was a non-randomized, 1b phase, multi-cohort study that included a subset of patients with metastatic TNBC. Tumors were screened for PD-L1 activity of a minimum of 1% expression in tumor cells or stroma, using the anti-human 22C3 antibody PD-1 (Merck & Co., Kenilworth, NJ, USA) and were identified in almost 60% of the examined patients. Of the 111 patients with TNBC, 58.6% had positive PD-L1 tumors.

Pembrolizumab has also been studied in association with eribulin in a multi-center, single-arm, open (I/II) (KEYNOTE-150) study with the aim of examining the security and activity of the combination in patients with metastatic TNBC. Eribulin may be a chemotherapeutic drug, with anti-microtubule action registered in previously treated metastatic breast cancer. The patients during this study could have been previously treated with 0–2 lines of chemotherapy for metastatic disease. Among 107 patients, 106 were evaluated and included without considering their PD-L1 status. The ORR was 26.4% (3 patients with CR and 25 with PR) and CBR was 32.8% [77,102]. It’s important to note that the ORRs were not significantly different concerning the PD-L1 status (30% in positive PD-L1 vs. 22% in negative PD-L1; of the three patients who had an entire response, one patient was PD-L1 negative) or previous exposure to chemotherapy (29% in untreated patients vs 22% in patients with 1–2 previous lines) [100]. The mixture of immunotherapy with chemotherapeutic agents such as eribulin can induce immunomodulatory changes within the tumor, like the positive regulation of PD-L1 and hyperexpression of immunogenic markers on the cell surface, where these changes within the tumor environment together can positively influence the effectiveness of immunotherapy [31,54].

3.2.2. Avelumab

Avelumab (MSB0010718C) may be a monoclonal antibody that targets the programmed cell death ligand 1 receptor, a transmembrane protein in tumor cells. Avelumab is a fully human IgG1 mAb that binds to PD-L1 [40]. Avelumab was previously the primary approved drug against metastasis by the FDA in metastatic Merkel cells and advanced urothelial carcinoma [92]. Since its approval, new studies changing the dosage supported by body weight to a flat dose of Avelumab in Metastatic Merkel Cell and Advanced Urothelial Carcinoma and its applicability in other types of tumors have been studied [40,117].

The JAVELIN study, a phase I study, evaluated the action of avelumab on solid tumors through a cohort of 168 patients, during which 58 patients had metastatic breast cancer. The dose of Avelumab was 10 mg/kg IV in every 2 weeks until progression. To be eligible, patients had to possess a biopsy-proven locally advanced or metastatic breast cancer, have received ≤3 previous lines of chemotherapy and have previously received anthracycline and taxane, unless contraindicated [40].

The ORR in those patients with metastatic carcinoma was 3.0%, including 1 CR, 4PRs, and 42 patients with SD. Of the 5 patients who had a response, 3 had TNBC. within the cohort of patients with metastatic breast cancer, 58 (34.5%) patients had TNBC. The ORR within the TNBC cohort was 5.2%, with 0 patients with complete response, 3 patients with partial response, and 15 with stable disease. PD-L1 expression of a minimum of >1% was seen in 48 of 58 patients with TNBC. The ORR for those with TNBC-supported PDL1 status was 6.1% [PDL1≥1% (n = 33)], 7.7% [PDL1≥5% (n = 13)] and 0% [PDL1≥25% (n = 2)]. In tumor-associated immune cells with PDL1≥10%, the ORR was 22.2%.30 The results of this study show that the security profile of Avelumab is tolerable and that those who have TNBC with PDL1-positive immunohistochemistry appear to have a clinical benefit with avelumab [40].

3.2.3. Atezolizumab

Atezolizumab (MPDL3280A), like avelumab, may be a monoclonal antibody that targets the programmed cell death ligand 1 receptor, a transmembrane protein in tumor cells. Unlike Avelumab, atezolizumab may be a humanized mAb of the IgG1 isotype that selectively binds to PD-L1 [76,108].

In the IMpassion130 study, the study that showed more benefits within the use of immunotherapy to treat TNBC, a PD-L1 expression above 1% in immune cells was wont to define the PD-L1+ group [36]. Interestingly, most of the patients that tested positive for PD-L1+ in tumor-infiltrating immune cells also had a positive expression of PD-L1 in tumor cells. within the IMpassion13041 biomarker subgroup analysis, the expression of PD-L1 in immune cells was positively correlated with the amount of CD8 + T cells, and both factors were associated with the increase in PFS and OS. The association with nab-paclitaxel was chosen a priori within the IMpassion130 study because it facilitates the reduced use of corticosteroids[67, B cell lym80, [85]. Although reducing the utilization of corticosteroids in oncology is very relevant, other studies have shown that better agents could also be available to increase the immunogenicity of breast cancer, citing anthracyclines, platinum salts, and other taxanes [35].

In the phase III randomized study IMpassion-130 [36,70] patients with metastatic TNBC treated with first-line and with good performance status (0–1) were randomized to weekly receive nab-paclitaxel (100 mg/m2 D1, D8, D15) plus atezolizumab (800 mg D1, D15) or placebo, in every 28-day cycle.40 Previous treatments like radiotherapy and chemotherapy, including taxanes, were allowed if they were performed a minimum of 12 months before randomization. Patients with treated asymptomatic CNS metastases were eligible. Patients were stratified consistent with the presence of liver metastases, previous taxanes within the adjuvant and/or neoadjuvant setting, and the PD-L1 expression in the immune cells infiltrated in the tumor [36,70].

The use of an anti-PD-1 or anti-PD-L1 agent, or the utilization of both of them seems to be relevant for the survival rate even in patients that did not receive immunotherapy as the first-line therapy. New studies with patients without a positive immune infiltrate must be conducted, so as to verify the efficiency in this setting, also as new clinical trials that associate immunotherapy with other agents in the first-line therapy. New predictive biomarkers also have to be developed. To date, IHC is the only FDA-approved test for measuring PD-L1 expression.

Rather than conventional immunotherapy approaches, CAR-T therapy has a better advantage over the precedent techniques because of its short treatment time and rare chances of disease relapse once treated. Now, CAR T-cell therapy is available for the medical interventions of patients in whom transplant is not likely to be curative or in patients who relapse after transplant. But depending upon the success rate of this treatment in the coming future, this therapy will be readily accessible to everyone.

4. CAR-T therapy

CAR-T treatment is an immunotherapy method that alters T cells to pinpoint cancer cells and more efficiently attack and eradicate them. Human T cells are first extracted, genetically manipulated, and then administered to patients to target tumors. The T cells usually are engineered on CAR or chimeric antigen receptors. Receptors are chimeric because they are a single receptor that combines both antigen-binding and T cell activation functions refer to Fig. 1. These receptor proteins that have undergone modifications give T cells a new ability to target specific proteins. The T cells generally used are taken from the patient’s blood (autologous) itself or from another healthy donor (allogeneic) [105]. The US Food and Drug Administration (FDA) approved CD19-CAR-T-cell therapy in 2017 for the treatment of a subset of pediatric and young adult patients with acute lymphoblastic leukemia, and since then, it has overcome several obstacles [4,59]. The CAR construct is made up of a flexible extracellular hinge and a transmembrane domain that help to bind the receptor to the cell surface and connects it to the signaling domain of the T cell receptor [134]. CAR combines the tumor recognition capacity of monoclonal antibodies (mAbs) with the potent antitumor and proliferative capacity of T cells. CAR is introduced into T cells using viral or non-viral vectors (such as retroviruses) and integrates genes such as synthetic DNA transposon systems and transient mRNA transfection. After expression on T cells, CAR-T cells can function as “living drugs” exhibiting antigen-specific recognition, activation, proliferation, and cytotoxic functions independent of MHC presentation refer to Fig. 2 [75,134].

T cell activation depends on the signal intensity received by the T cell receptor (signal 1) and the co-stimulator molecule (signal 2). Choosing the proper co-stimulation signal can increase the ability of CAR-T cells to proliferate, persist, and lyse target cells. Second-generation CARs representing the co-stimulation domain of 4-1BB have been clinically shown to promote T cell persistence and long-term control over leukemia due to fatty acid oxidation, increased central memory formation, and reduced fatigue [69]. CAR-T cell therapy was effective in treating B-cell malignancies but less efficient against solid tumors. Optimal target selection, multi-specific or other next-generation CAR-T cell development, T cell depletion, and inhibitory TME eradication, improved efficacy of T cell response, and tumor limitation Key efforts, including the development of new approaches to overcome T cell transport, have been invented to improve the efficiency of CAR-T cell therapy in solid tumors [74,101]. The recent treatments for solid tumors in the context of TNBC also consider the context of CAR components (scFv, flexible hinges, trans-membrane, and costimulatory domains) [49,109] and manufacturing processes (activation, transfection, and extended protocols) [127] that will help for better therapeutic results [79,103].

5. Potential targets of TNBC for CAR-T therapy

Over a dozen targets of TNBC have been identified both in vitro and in vivo for CAR-T therapy for proof of concept (POC) studies [28]. Some of these tumor antigens are tumor-specific, tumor-associated, or cancer germline antigens. Those antigens that are expressed on malignant tissues are referred to as tumor-specific while those which are exclusively expressed on lineage-restricted malignant cells as compared to healthy cells are called tumor-associated antigens [28]. On the other hand, cancer germline antigens are expressed only on adult somatic tissues such as ovaries in females and testis in males [3,72,89]. While most of these are actually proteins expressed on the surface of malignant cells, some of them also include some post-translational modifications of proteins like abnormal glycosylation patterns or some changes on cell surface proteins [72,111].

5.1. Receptor tyrosine kinase AXL

AXL is a receptor tyrosine kinase that has evolved to be a potential therapeutic target for TNBCs because it is involved in activating some downstream signaling cascades including PI3K, MAPK, and NF-Ƙβ pathways [132]. Also, this type of immunotherapy has the potential to outwit the immunosuppressive TME by inhibiting the release of cytokines and chemokines from TAMs [28,76,121]. On the other hand, it has been noticed that AXL is expressed on myeloid-derived suppressor cells (MDSC), which are immature myeloid cells capable of suppressing immune responses and letting the cancer cells proliferate [14]. Therefore, targeting AXL by CAR-T cells can alter the TME to a proinflammatory state by altering the TME. Invitro experimentation with AXL-specific CAR-T cells, constitutively expressing IL-7 has shown enhanced antitumor activity in xenograft mice models [130].

5.2. F_cγ receptors (F_cγR)

CAR-T cells can be specifically designed with these receptors containing signaling and co-stimulatory domains [20,21]. F_cγR expressing T cells can be used to redirect therapeutic antibodies to target antigens and thereby result in the complete elimination of solid tumors by antibody-dependent cellular cytotoxicity (ADCC) [20,28]. The potential F_cγRs that are of therapeutic interest include CD16A (expressed by Natural Killer (NK) cells) and CD32A (expressed by monocytes, macrophages, dendritic cells, and NK cells). Transmission of activation signals is brought about by these receptors through immunoreceptor-based activation motifs (ITAM) adaptor molecules [11,13]. Engagement of CD16A and CD32A with the Fc region of therapeutic antibodies results in target cell depletion by ADCC which can be a possible therapeutic strategy for CAR-T cells for TNBC shown in Fig. 3. One such therapeutically approved antibody is cetuximab which has demonstrated ADCC against EFGR-positive tumor cells. AS EFGR is expressed by TNBCs, such therapeutic antibodies along with F_cγR expressing CAR-T cells can be considered to be novel strategies against TNBCs [21,73].

5.3. Chondroitin sulfate proteoglycan 4 (CSPG4)

Expressed in TNBC, CSPG4 is a cell-surface proteoglycan that stabilizes cell-surface interactions in melanoma [28]. Invitro experiments with CSPG4-CAR-T cells on MDA-MB-231, Hs578T, and MDA-MB-468 TNBC cells have revealed controlled tumor growth in mice models along with the release of proinflammatory cytokines [9]. The major advantage is using CSPG4-specific CAR-T cells is that they can target both primary TNBC cells as well as CAFs as CSPG4 is highly expressed on stromal cells in the TNBC TME [16]. To reduce unwanted toxicity and because CSPG4 is also expressed on normal cells, several proposals for incorporation of suicide genes like caspase 9 have been proposed as safety switches that can trigger cell death upon activation [9].

5.4. Receptor tyrosine kinase epidermal growth factor receptor (EFGR)

The growth, survival, and metastatic invasion of some cancer cells are mediated by this type of receptor [84]. As more than 60% of TNBC patients have been shown to overexpress EFGR, considerable efforts can be made to target this receptor [73]. The limitations of the traditional inhibitors of EFGR can be improved by using CAR-T cells against EFGR in TNBC. Invitro experiments on some breast cancer cell lines with epithelial morphology, have demonstrated increased cytokine secretion and cytotoxic activity by third-generation CAT cells against EFGR. However, one of the problems encountered while designing such CAR-T cells is that EFGR is also expressed by normal cells [22]. EFGR variant III (EFGRvIII) is an attractive target in this case as it is only expressed by tumor cells and CAR-T cells directed against such variant receptors can reduce the chances of immune exhaustion [52]. Clinical studies are also being conducted to design novel EFGR targeting CAR-T cells containing checkpoint-blocking antibiotics anti-PD-1 and anti-cytotoxic T lymphocyte-associated protein 4 (CTLA4) to negate the immunosuppressive TME by secreting these antibodies which can prove to be a potential combinatorial therapy [127].

5.5. Folate receptor alpha (FRα)

It is a glycosylphosphatidylinositol (GPI)-lined membrane protein involved in the intracellular transport of the water-soluble vitamin folate, which is necessary for DNA biosynthesis and facilitating metabolic reactions for proliferating cells [28]. Overexpression in breast cancers of epithelial origin makes it an attractive anti-cancer therapeutic target [24]. It has been observed that expression of FRα increases in ER-negative, stage IV metastatic TNBC by approximately 70% while its expression decreases to 30% in other breast cancer subtypes thus showing an inverse correlation exists between FRα and ER (estrogen receptor) expression. Moreover, FRα-CAR-T cells have demonstrated potent in vitro killing of TNBC cells and significant tumor regression in an MDA-MB-231 xenograft mouse model. This anti-tumor activity of FRα-CAR-T cells strongly correlated with FRα surface antigen expression levels on tumor cells, suggesting that FRα could serve as a selection biomarker to improve clinical response rates. To reduce unwanted targeting of normal cells by FRα-CAR-T cells, POC for bispecific CAR-T cells simultaneously co-targeting two different tumor-associated antigens, FRα, and mesothelin has been demonstrated [67,110]. Moreover, a folate-fluorescein isothiocyanate (FITC) bispecific adaptor molecule has been shown in Fig. 4 to redirect universal anti-FITC-CAR-T cells to target tumor cells expressing the folate receptor [64].

5.6. Disialoganglioside GD2

This type of glycosphingolipid is involved in the tethering of tumor cells to ECM proteins. GD2 expression is highly upregulated in cancerous tissue and is highly restricted in normal tissue [28]. This tumor-selective expression of GD2 makes this molecule an attractive target for antibody-based immunotherapies like the recent development of the anti-GD2 monoclonal antibody Dinutuximab β for the treatment of neuroblastoma [107,116]. Recent clinical investigations have revealed that subpopulations of CD44^high and CD24^low human breast cancer highly express GD2 that phenotypically express cancer stem cells. Further attempts involve the fabrication of the third generation of CAR-T cells with an scFv derived from Dinutuximab β to target GD2 on cancer stem cells in TNBC to eliminate tumor cell formation and metastasis prevention. In vitro experimentation on orthotopic xenograft mice models revealed an effective anti-tumor response that arrested tumor growth and prevented the formation of metastases [107].

5.7. Intracellular adhesion Molecule-1 (ICAM-1)

Facilitation of endothelial transmigration of leukocytes and stabilization of cell-cell interactions are brought about by this cell surface glycoprotein, which is upregulated in many breast cancer cell lines like MDA-MB-231 TNBC cells [10]. Invitro experimentation revealed that 85% of such TNBC cells could be killed by ICAM-1 specific CAR-T cells [49].

5.8. Integrin α_vβ₃

Receptors that are usually expressed on the surface of all nucleated cells and aid in cell adhesion are called integrins. Although these types of receptors are involved in the transmission of signals between the cells and their microenvironment, recent reports indicate that they also modulate oncogenic processes including proliferation, migration, invasion, and survival [11]. One such most characterized integrins involved in oncology is integrin α_vβ₃, whose expression becomes elevated in TNBC as well as in other TMEs like TAMs, CAFs, and angiogenic epithelial cells. Invitro experimentation revealed that CAR-T cells targeting integrin α_vβ₃ showed potent antitumor activity by inhibiting tumor growth and stimulating the secretion of pro-inflammatory cytokines such as IL-2 and IFNγ [120]. Moreover, equipment of these CAR-T cells with an EFGR safety switch renders them susceptible to destruction by endogenous ADCC upon administration of anti-EFGR cetuximab to prevent unwanted toxicity which makes these receptors an important target antigen in TNBC for CAR-T cell-based immunotherapy [118].

5.9. Mesothelin

This is a cell adhesion glycoprotein that is expressed on mesothelial cells and promotes oncogenesis by activation of NF-Ƙβ, PI3K, and MAPK signaling pathways [82]. Immunohistochemical analysis revealed that this glycoprotein is highly expressed in 67% of TNBCs and lowly expressed in normal cells [114]. Thus, it has become an attractive target for anti-cancer therapy in TNBC. Invitro experimentation using engineered mesothelin-specific CAR-T has revealed significant tumor regression along with induction of invitro cytotoxicity and production of cytokine. While using mesothelin-specific CAR-T cells in these in-vitro experiments, expression of PD-1 had been knocked down by CRISPR (clustered regularly interspersed short palindromic repeat) technology. PD-1 is an immune checkpoint receptor on T cells that engages with programmed death-ligand 1 (PD-L1) expressed on tumor cells to suppress the T cell response and allow tumor cells to escape immune surveillance. Accordingly, PD-1 knockout mesothelin CAR-T cells showed superior effector function in an orthotopic xenograft mouse model of TNBC due to the downregulation of this inhibitory receptor [60,61].

5.10. Receptor tyrosine kinase c-met

Although this tyrosine kinase receptor mainly mediates wound healing and organogenesis, its main function in the context of cancer, is the activation of PI3K, Stat3, beta-catenin, and RAS signaling cascades [42, EGFR65]. One of the recent approaches in anti-cancer therapy involves engineering small interfering RNAs (si-RNAs) to silence c-Met in TNBC cell lines to stop cancer cell proliferation and migration [43,65,129]. Overexpression of c-Met occurs in 50% of TNBC patients and is lowly expressed in healthy cells. Therefore, to reduce unwanted targeting, mRNA electroporation was utilized to induce transient c-Met CAR expression in autologous T cells in TNBC xenograft animal models. This was done because mRNA-based CAR-T cell therapy provides a safer approach due to transient expression and limited persistence of cells as compared to stably transduced CAR-T cells [129].

5.11. Mucin 1 glycoprotein (MUC1)

This transmembrane protein lies on the outer surface of epithelial cells and protects host cells from infection by acting as a protective mucosal barrier. Serine and threonine residues present in the variable number tandem repeats (VNTR) region of the MUC1 extracellular domain serve as attachment sites for O-glycans [86,133]. Thus, post-translational modifications are readily observed in the MUC1 protein. TNBC cells overexpress an aberrantly glycosylated tumor form of MUC1 (tMUC1) in greater than 95% of all TNBC, and no significant tMUC1 expression is detected on normal breast tissues [131]. Second-generation tMUC1-CAR-T cells recently demonstrated potent tumor cytolytic activity and Th1 cytokine and chemokine production in vitro. Tumor cell growth was significantly inhibited in a TNBC xenograft mouse model while sparing normal breast epithelial cells. tMUC1 represents an important target for CAR-T cell therapy in TNBC given its high tumor antigen specificity. Similarly, another aberrant glycoform of MUC1 (TnMUC1) is also abundantly expressed on TNBC. CAR-T cells 440 recognizing this glycoform are being considered as bonafide CAR-T cell targets for TNBC [99,131].

5.12. Natural-killer group 2, member D (NKG2D)

It is a receptor that activates natural killer cells and functions in recognizing stress ligands on infected or transformed cells followed by subsequent elimination of these unwanted cells through cytotoxic mechanisms [122]. Some NKG2D ligands like MHC class I related chain (MIC) and UL16 binding protein (ULBP) are highly expressed on TNBC cells as compared to their minimal expression on normal cells. Not only this but NKG2D cells are also expressed on immunosuppressive cells of the TME like MDSCs and Tregs. Recent clinical studies on xenograft mice models suggested that TNBC cells expressing NKG2D ligand were susceptible to invitro cytolytic activity and release of proinflammatory cytokines along with the simultaneous regression in tumor progression. Moreover, second-generation NKG2D-CAR-T cells engineered with 4-1BB or CD27 co-stimulatory domains demonstrated superior in vivo anti-tumor activity in terms of increased T cell persistence and tumor regression compared to first-generation NKG2D-CAR-T cells [50,51] [43].

5.13. Receptor tyrosine kinase-like orphan receptor 1 (ROR1)

Mostly facilitating neuronal growth in the CNS, this protein is highly expressed in TNBCs and has limited expression in healthy cells. Moreover, in-vitro toxicology studies with ROR1-based immunotherapies against TNBC in vascularized 3D complex, revealed no significant levels of toxicity to vital organs which makes it an attractive anticancer therapeutic target [117]. ROR1-CAR-T cells were shown to infiltrate and migrate through TNBC 3D cultures and elicit potent anti-tumor immune responses. This study provides POC for the development of novel 3D micro-physiological systems to test the anti-tumor activity of CAR-T cell therapies in TNBC, compared to conventional two-dimensional culture systems and xenograft animal models [117,123].

5.14. Stage-specific embryonic antigen-4 (SSEA-4)

Mainly used to identify embryonic stem cells, this glycoprotein promotes invasion and metastasis of tumor cells along with association with poor prognosis and chemoresistance in solid tumors in TNBCs. Although SSEA-4 is expressed in normal cells, it is upregulated in TNBC tumor cells by approximately 30% [97]. The anti-tumor activity of second-generation SSEA-4-CAR-T cells was investigated in TNBC cells, where in vitro T cell degranulation, cytokine secretion, and tumor cell killing were achieved [97].

5.15. Tumor endothelial marker 8 (TEM8)

This collagen-binding cell surface glycoprotein primarily promotes the migration of endothelial cells [17]. TEM8 is overexpressed on the surface of TNBC cells. Clinical investigations are being conducted on TNBC animal models to engineer TEM8-specific CAR-T cells to use a combinatorial approach to achieve antitumor activity along with the reversal of the immunosuppressive TME [18,19]. However, unwanted targeting of CAR-T cells is a major limitation of this procedure as normal cells also slightly express TEM8 [5,96,98].

5.16. Trophoblast cell-surface antigen 2 (TROP2)

TROP2 is overexpressed in approximately 90% of TNBC tumors and correlates with poor prognosis in breast cancer due to the induction of pro-oncogenic signaling [28]. Recently, an antibody-drug conjugate targeting TROP2 called sacituzumab govitecan received FDA approval for the treatment of relapsed or refractory metastatic TNBC [8]. The antibody-drug conjugate consists of the active metabolite of the topoisomerase I inhibitor irinotecan (SN-38) conjugated to a humanized IgG antibody against the cell surface glycoprotein TROP2. Clinical trials on TNBC patients demonstrated a minimal level of toxicity in normal tissues. Thus, TROP2-specific CAR-T cells have become a promising therapeutic strategy for treating TNBC [8,44].

6. Persistence, homing, and tumor microenvironment

6.1. Persistence

To ensure there are enough CAR-T cells available to infiltrate tumor locations, it is crucial to maintain high numbers of CAR-T cells in patients’ peripheral circulation. Early studies using the first generation of CAR-T cells that targeted ovarian [62] and renal cell antigens [15] suggested that a lack of patient preconditioning or anti-CAR immune responses may be the cause of the lack of persistence. Afterward, costimulatory signals were included in CARs to boost their in vivo persistence, particularly when given to hosts who have low levels of lymphocytes [91,95,104]. To promote T cell growth in vitro, IL-2 has been chosen as a crucial cytokine. Other cytokines, such as IL-15, IL-7, and IL-21, may cause cultured T cells to become more prevalent than IL-2-expanded T cells. According to research, IL-15 can stimulate long-lasting memory cells that have experienced antigens [101], override Treg-mediated inhibition [58,58,71,83], boost T lymphocyte development, prevent apoptosis and exhaustion [93,128], and reverse anergy [13]. While avoiding systemic toxicity, CAR-T cells can be genetically altered to generate cytokines to enhance their multiplication and persistence in vivo [35,56,103,119]. Positive clinical outcomes have been seen with antibodies that block the programmed death-1 (PD-1) receptor, the PD-L1 ligand, or the cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) [54,55]. They are advantageous for triple-negative breast cancer, according to convincing evidence [47]. Before genetic alteration, selecting T cells that express naive markers like CD62L may result in CAR-T cells that are more persistent than effector or more differentiated T cells [23]. A different option is the lifelong persistence of virus-specific cytotoxic T lymphocytes (CTLs), which contain both CD4⁺ and CD8⁺ subsets, with the latter being a crucial compartment for the former’s long-term persistence [29,63]. By their ability to travel to and remain in the designated lymphoid or non-lymphoid tissues, virus-specific CTLs also exhibit the expression of homing/chemokine receptors [27]. A continuous supply of CAR-T cells could result from memory T-stem cells differentiating into memory T cells. On the other hand, CARs might be engineered into hematopoietic stem cells to create CAR-T cells over time [16,48].

6.2. Homing

The target B cells are easily accessible to CAR-T cells and express a range of costimulatory receptor ligands that can enhance CAR-T cell function, which is likely what leads to the effectiveness of CAR-T cell therapy for B-cell malignancies [40]. Recent investigations [12,38,109] have shown that chemokines are crucial and complex for lymphocyte movement [113]. To effectively target CAR-T cells, it is crucial to devise a plan that makes use of the crucial homing chemokines while avoiding the potential regulatory effects of other tumor-expressed chemokines [30,36].

6.3. Tumor microenvironment

Numerous elements, including immunosuppressive cytokines, regulatory modulators, and coinhibitory receptors, are present in the tumor microenvironment [32]. Regulatory T cells, populations of immature myeloid cells, and tumor-associated macrophages are among the immunosuppressive cell types [33,88,112,124]. It is possible to protect CAR-T cells from the tumor immunosuppressive microenvironment by genetically modifying the CAR vector to include dominant-negative TGF- receptors to counteract the negative effects of tumor-derived TGF [37,80], adopt knockdown strategies to avoid apoptosis mediated by Fas/Fas ligand [45], or the expression of survival genes like BCL-XL [34,46]. Additionally, the immunosuppressive tumor environment can be reversed by the transgenic production of cytokines like IL-15 or IL-12. A different approach could involve the transgenic production of constitutively active signaling molecules or the silencing of genes that block T-cell activity in the tumor microenvironment [87]. Finally, it may be possible for CAR-T cells to overcome the tumor microenvironment through a combination therapy that combines medicines that promote cell-based immunotherapies with medications that work around antitumor mechanisms [115].

7. Present state of CAR-T therapy

There are a number of benefits when comparing CAR-T cell treatment to other cellular immunotherapies. Gross and colleagues first proposed the idea of the CAR in 1989 [47]. They combined the T-cell receptor (TCR) signaling domain CD3ζ with the antibody-binding domain Fab to create and name it the T body. Nowadays, the most popular technique for creating T cells that are specific for a given Tumor uses CARs to genetically edit T cells [[66], [68], [81]]. CAR-engineered T cells combine the homing and killing ability of T cells with the specificity of mAbs. A single-chain antibody’s extracellular ligand-binding domain (scFv), a cytoplasmic signaling chain, a transmembrane domain, a hinge, and the probable presence of costimulatory molecules make up the majority of CARs. Initially, polyclonal T cells that have been nonspecifically stimulated are used to create CAR-T cells. As a result, they circumvent the challenge of isolating and amplifying naturally occurring CD4⁺ and CD8⁺ T cells specific for Tumors. Secondly, a major histocompatibility complex (MHC) is not required for CAR-T cells to detect the target antigens. This characteristic makes it possible for CAR-T cells to identify target cells with decreased human leukocyte antigen (HLA) expression or antigen processing and is believed to be crucial in Tumor immunological escape. Thirdly, CAR-T cells have the ability to aggressively and specifically target Tumor locations, as well as the capacity to grow and last for prolonged periods after Tumor detection in vivo. In order to produce long-lasting Tumor responses, CAR-T cells directed against tumor-associated antigens (TAAs) may therefore be more efficient than mAbs. The ability of CAR-T cells to traverse the blood-brain barrier is another distinct benefit. Although unfavorable effects specific to the central nervous system must also be taken into account, this trait is very helpful for treating malignant Tumors that include or have spread to the central nervous system [104].

Based on the three signals—TCR, costimulation, and cytokine—that are necessary for T-cell activation, CAR-T cells might be split into three generations refer to Fig. 5.

First-generation CARs	included scFv and just one CD3ζ-derived signaling domain. The results of the first-generation CAR studies, however, were underwhelming. A costimulatory signal was necessary for both full T-cell activation and preventing apoptosis.
Second-generation CARs	contained two or three CD28, 4-1BB, or other costimulatory molecule signal domains, to complete the CAR-T cell activation signal [106].
Third-generation CARs	were included into a cytokine cassette that provided the CAR-T cells with a better environment for activity or survival.

8. Clinical trials in TNBC using CAR-T therapy

Many target antigens for CAR-T in TNBCs, have been evaluated in both preclinical in vitro and in vivo clinical studies. Recently, a phase I clinical study was conducted to test the safety and efficacy of mesothelin-specific CAR-T therapy for treating TNBC, although the current status of this clinical study remains unknown [NCT02580747]. Another study has started recruiting metastatic HER2-negative breast cancer patients for a phase I clinical study to evaluate the safety and efficacy of mesothelin-specific CAR-T therapy [NCT02792114]. Another phase I study was conducted by the University of Pennsylvania to evaluate the efficacy of intratumorally injected c-Met-CAR-T cells into TNBC patients [NCT01837602]. This study demonstrated that this immunotherapy had been well tolerated by patients and elicited an inflammatory response within TNBC tumors, with no evidence of drug-related adverse effects. The most explored cell therapy was evaluating the efficacy of autologous MUC-specific CAR-T cells in TNBC patients [NCT02587689]. Of late, Minerva Biotechnologies Corporation started recruiting individuals with metastatic breast cancer to participate in a Phase I trial investigating the safety and maximum tolerated dose of autologous CAR-T cells targeting a cleaved form of MUC1 antigen [NCT04020575]. Other target antigens under clinical trials include NKG2D ligand and ROR1. Efforts are being made to evaluate the safety and efficacy of allogeneic gamma delta (γδ) T cells transduced with CARs targeting NKG2D ligands on TNBC cells in a phase I trial [NCT04107142]. Moreover, TNBC patients are also being recruited to study the efficacy of ROR1-specific CAR-T cells. Altogether, the growing number of clinical trials being conducted on different CAR-T antigens suggests that these efforts will pave the way for a stirring era in cancer immunotherapy [NCT02706392] [29].

In the following Table 1 different antigen-specific CAR-T-cell clinical trial results for TNBC and solid tumors are enumerated.

Table 1. A Summary of different antigen-specific CAR-T-cell clinical trials for TNBC and other solid tumors.

Target	CAR Design	CAR Delivery	Phase	Route of CAR-T cells administration	References
Mesothelin	4-1BB/CD3ζ	mRNA	1	Regional(lntratumoral)	[125]
	iCasp9CD28/CD3ζ	Retrovirus	1	Intravenous	[125]
	4-1BB/CD3ζ	mRNA	0	Regional(lntratumoral)	[125]
	4-1BB/CD3ζ	mRNA	1	Intravenous	[125]
TnMUC1	4-1BB/CD3ζ	Lentivirus	1	Intravenous	[125]
Folate receptor-α	4-1BB/CD3ζ	Lentivirus	1	Regional(lntrapleural)	[125]
RORI	4-1BB/CD3ζ	Lentivirus	1	Intravenous	[125]
NKG2D ligands	Full-length NKG2D/CD3ζ	Retrovirus	1	Intravenous	[125]

9. Combinatorial therapy of breast cancer using EFGR CAR-T cells and CDK7 inhibition

Targeting TNBC with a Combination Therapy of EGFR CAR-T Cells and CDK7 Inhibition has been studied both in vitro and in vivo in research by Xia et al., 2021. According to the study, EGFR CAR-T-cell-induced immunosuppressive genes were particularly susceptible to the CDK7 inhibitor THZ1, which they chose from a panel of small drugs targeting epigenetic modulators [126]. It was also linked to EGFR CAR-T-cell-activated enhancers. Third-generation EGFR CAR-T-cell treatment was shown to be resistant in one-third of the mice with TNBC tumors in this study. Transcriptomic study of EGFR CAR-T-cell resistant tumors showed that a large number of immune suppressive genes were activated. IFNγ produced by CAR-T cells most likely activated these genes. These CAR-T-cell-induced immunosuppressive genes contain CAR-T-cell-activated enhancers that are especially susceptible to the CDK7 inhibitor THZ1. As a consequence, the combination therapy using THZ1 and EGFR CAR-T cells decreased immune resistance, Tumor growth, and TNBC metastasis in mice. Because THZ1 has the ability to reduce the immunosuppressive genes that are generated by EGFR CAR-T cells, they looked into whether co-treatment with THZ1 would boost the effectiveness of EGFR CAR-T cells in eradicating TNBC cells in vitro. Combining THZ1 with EGFR CAR-T cells resulted in greater cellular toxicity than either drug used alone. Additional models used to study the effects of the combo therapy included immune-competent animals, PDX models, and EGFR CAR-T-cell-resistant MDA-MB-231 cell-derived Xenograft models. The co-treatment of CAR-T cells with THZ1 and EGFR had the most notable effects on stifling cell line proliferation in each scenario [53,127].

10. Olaparib and trastuzumab in CAR-T therapy

A study was conducted to prove that the PARPi olaparib when coupled with CAR-T cells, causes an immune response that is anti-tumor and improves CD8⁺ T cell recruitment in mice breast cancer models. It was thus hypothesized that olaparib may inhibit the release of SDF1α in CAFs via HIF1α and further reduce the recruitment of myeloid-derived suppressor cells (MDSCs) into Tumor tissue, which may enhance CD8⁺ T cell survival [39].

MDSCs have become significant immune evasion factors in solid tumors. Their immunosuppressive functions as well as their impacts on Tumor cell invasion and angiogenesis have been implicated in this detrimental effect. The immunosuppressive solid Tumor microenvironment is implicated as a modulator of CAR-T cell efficacy due to the association between tumor-induced MDSCs and poor CAR-T cell efficacy against xenografts in vivo [41].

In the study conducted, it was found that there were more CD8⁺ T cells and less MDSC recruitment in the Tumor tissue of the group receiving combination therapy. Further findings showed that in vitro co-culture with MDSCs could increase CD8⁺ T cell death and decrease CAR-T cell lytic activity. Consequently, it is possible to at least in part explain why mice treated with olaparib have higher CD8⁺ T cell residency in Tumor tissue. When compared to the control group, the CAR-T cell therapy group’s Tumor tissue had significantly more MDSCs, and this finding was significantly connected with the quantity of CAR-T cells used. These findings suggested that administering CAR-T cells may increase the recruitment of MDSCs into Tumor tissue and that the buildup of intra-tumoral MDSCs may be a factor in the immunosuppression of tumors. Complicating factors may have an impact on the recruitment of MDSCs to tumors that are produced by CAR-T cells.

Thus, olaparib may considerably enhance the anti-tumor efficaciousness of CAR-T cells in mouse carcinoma xenografts, and also the reduced enlisting of MDSCs within the Tumor microenvironment was the crucial determinant of therapeutic efficacy [28].

In pre-clinical models and clinical trials, HER2-redirected CAR-T cells have demonstrated great promise in the elimination of HER2+ trastuzumab-sensitive Tumor cell lines (SKBR3 and BT474) and the treatment of trastuzumab-naive patients.

Previous research suggests that ECM elements contribute to trastuzumab therapeutic resistance by interfering with antigen recognition, which may also harm the effectiveness of newly developed antibody-drug conjugate (ADC) therapies. Trastuzumab’s ability to bind to HER2 in JIMT-1 spheroids was decreased as a result of the overexpression of CD44/hyaluronan, which further limited the penetration of CD16.176V.NK-92 cells. As measured by the incorporation of propidium iodide, HER2-specific CAR-T cells on the other hand were able to enter the spheroid cores and elicit a robust anti-tumor response.

In a study, trastuzumab-redirected CD16.176V.NK-92 and HER2-CAR-T cells were employed to demonstrate in vivo the anti-tumor effects of JIMT-1 and MDA-HER2 tumors growing in NSG mice. Beginning on the day of Tumor cell inoculation, cells were given concurrently with trastuzumab twice a week in order to investigate the in vivo cytolytic activity of CD16.176V.NK-92 cells in the presence or absence of the immunotherapy drug. Trastuzumab-resistant MDAHER2.ffLUC and JIMT-1 tumors were still able to grow in preclinical models, despite the fact that the same treatment was previously proven to be able to completely cause the regression of existing BT474 xenografts that were trastuzumab sensitive. Although the MDA-HER2.ffLUC and JIMT-1 tumors were 250 mm3 in size and had a well-established ECM at the time of treatment, HER2-CAR-T cells administered on day 14 post Tumor inoculation caused complete and long-lasting Tumor regression in contrast to the repetitive treatment with trastuzumab and CD16.176V.NK-92 cells. HER2-specific CAR-T cells also penetrated trastuzumab-resistant xenografts in vivo, which is similar to the case with spheroids generated in vitro. But in HER2-negative MDA.ffLUC control xenografts, there was little penetration and no Tumor lysis, which shows that active invasion of an accumulation of HER2-CAR-T cells in the Tumor is triggered by particular antigen recognition. Thus, therapy with trastuzumab with CD16.176V.NK-92 cells do not effectively eradicate existing clinic-derived and in vitro-created trastuzumab-resistant tumors, despite the fact that both cells detect the identical HER2 epitope. Trastuzumab-resistant spheroids and well-established xenograft tumors are eliminated by CAR-T cells, demonstrating their ability to cross the ECM barrier. Thus, it is believed that the therapeutic use of HER2-specific CAR-T cells produced by trastuzumab may be a viable approach for the treatment of trastuzumab-resistant malignancies [63].

11. Challenges in CAR-T therapy

Identification of acceptable Tumor target antigens that are missing or expressed at very low levels on normal tissues, particularly in essential organs, is the first significant challenge associated with CAR-T cell treatment in solid Tumors. The fact that a specific CAR-T cell only has to detect a small number of receptors on the target cell in order to fully activate has made this problem more serious. An optimum target antigen should be chosen to prevent off-target effects and related toxicity (i.e., overexpression on cancer cells, with minimal to no detectable expression observed on healthy tissue). In the setting of B-cell-specific antigens, Tumor antigen expression on normal tissues is tolerable in hematologic malignancies. Due to its upregulation in more than 95% of B-cell malignancies, CD19, the antigenic target for both Kymriah and Yescarta, is a typical example of an ideal target antigen for CAR-T cells. Immunoglobulin replacement treatment can be used to lessen patient difficulties brought on by B-cell aplasia, despite the fact that CD19 is expressed on nonmalignant (normal) B cells as well. Intratumor heterogeneity in terms of antigen expression, which may result in Tumor escape, is a problem with CAR-T cell therapy in solid Tumors [90]. The immunosuppressive Tumor microenvironment is another difficulty (TME). The TME is made up of a variety of interacting parts, such as an extracellular matrix, immune cells, stromal cells, chemokines, and cytokines. Recruitment of regulatory T cells (Treg), myeloid-derived suppressor cells (MDSC), cancer-associated fibroblasts (CAF), tumor-associated macrophages (TAM), and myeloid-derived suppressor cells (MDSC), as well as the production of immunosuppressive cytokines and soluble factors (e.g., IL-10, VEGF, TGF, indoleamine 2,3-dioxygenase, and adenosine), encourages high immunosuppressive nature of TME in Solid Tumors. T cells that express immune checkpoint molecules inhibit anticancer immune responses. Research on changing the solid TME from an immunosuppressive state to a proinflammatory one, which can improve anticancer immunity, is still ongoing. Inadequate Tumor trafficking and invasion as well as a lack of durability are additional challenges with CAR-T cell treatment in solid Tumors. Together, the lack of target antigen specificity, intrinsic target antigen heterogeneity, an immunosuppressive TME, expression of immune checkpoint molecules, ineffective intra-tumoral trafficking/infiltration, and poor persistence are the main problems with CAR-T cell therapy for the treatment of solid malignancies [29,135].

12. Future prospects

For patients with TNBC who have a higher risk of metastasis and lower survival rates, there is an unfulfilled need to create effective therapeutic methods. Immunotherapeutic approaches based on CAR-T cells have recently been developed to reroute T cells to attack solid immunogenic cancers like TNBCs. B-cell malignancies have responded well to CAR-T cell therapy, as seen with Kymriah® and Yescarta®. It is being thought of using CAR-T cells that release inhibitory antibodies against immunological checkpoints like anti-CTLA-4 and anti-PD-1 instead of combo therapy. Different suicide gene “safety switches” have been incorporated into TNBC CAR-T cell designs to lessen the risk of on-tumor/off-target damage caused by low target antigen expression on healthy cells. In addition to doing away with switch-based control mechanisms, “on switches” like split CARs, which separate the antigen-binding domain from the signaling domain, have been created to treat solid cancers. Split CARs allow for precise control of CAR-T cell activation. Additionally, in preclinical investigations, autologous T cells with bispecific CARs that target various TNBC Tumor antigens showed POC. Inhibitory CARs (iCARs) that use NOT-gating logic or dual or synthetic notch CARs that use AND-gating logic have demonstrated success in lowering on-tumor/off-target toxicity in different solid cancers. Ineffective trafficking to the Tumor site is another significant issue with CAR-T cell therapy in solid malignancies; this issue has not yet been addressed in TNBC. Since TNBCs overexpress the associated chemokine ligands, engineering TNBC CAR-T cells to express chemokine receptors like CCR-2 and CCR-4 is one technique that has the potential to enhance Tumor trafficking and infiltration. In fact, different cancers may benefit from using modified anti-FITC CAR-T cells that also express CCL19 and IL-7 to promote Tumor invasion. Constitutively activated interleukin-15 (IL-15) or IL-7 receptors are being included in CAR-T cell designs as part of ongoing efforts to increase the durability of CAR-T cells in solid Tumors. Combination therapy has been shown to be a successful method of treating cancer because it can concurrently target many mechanisms of action. In the near future, chemotherapy for TNBC is unlikely to be replaced by CAR-T cell treatment, but it may be helpful in combination therapy. For the treatment of metastatic TNBC, the FDA-approved anti-PD-L1 immunotherapy atezolizumab is sold in combination with the chemotherapeutic drug nab-paclitaxel. The inclusion of specific therapeutic modalities to CAR-T cell therapy has potential, in addition to researching CAR-T cell therapy and chemotherapy combination regimens. Olaparib, a PARP inhibitor, was recently shown to promote CD8⁺ T cell infiltration via STING pathway activation in an in vivo model of BRCA1-deficient TNBC, which supports the use of CAR-T cell therapy in combination with 616 PARP inhibitors for the treatment of TNBC. Additionally, it has been demonstrated that PI3K suppression during ex vivo CAR-T cell growth causes a memory phenotype, which improves in vivo durability and anti-tumor effectiveness in leukemia. In addition, a clinical trial testing an oncolytic adenovirus in combination with CAR-T cells that target HER2 positive Tumors is now underway. Combining TNBC-targeting CAR-T cell treatment with other targeted medicines including oncolytic viruses, PARP inhibitors, and PI3K inhibitors should be assessed. TNBC treatment should also be investigated using promising next-generation strategies, such as γδ-CAR-T cells and CAR-NK cell immunotherapy. Together, the multiple therapies being tested in preclinical research and clinical trials are proof that significant progress has been made in the CAR-T cell therapy field with regard to the creation of new treatments for patients with TNBC. Clinical studies for CAR-T cell treatment in liquid Tumors and other solid cancers have much to teach us, and these cutting-edge ideas may be applicable to TNBC. Engineering TNBC CAR-T cells to achieve specificity, safety, and efficacy will be essential for the field’s continued growth, as will choosing the best possible combination therapy to enhance clinical outcomes in TNBC patients [29,78].

13. Conclusion

After lung cancer, breast cancer remains the second most prevalent cause of cancer-related death in women in the United States, and its causes include growing older, being obese, abusing alcohol, having a family history of the disease, and radiation exposure in the past. In an era of immunological advancements, the prospective effectiveness of CAR-T therapy is encouraging. The solid Tumor may be significantly eradicated by using CAR-T cells to battle the immunosuppressive effects of the Tumor microenvironment. The specificity and safety of CAR-T-cell treatment in adoptive cell transfers for breast cancer are discussed in this review.