The new frontier of immunotherapy: chimeric antigen receptor T-cell (CAR-T) and macrophage (CAR-M) therapy for breast cancer. (2023)

Author(s): Giuseppe Schepisi (corresponding author) [*]; Catherine Gianni; Michela Palleschi; Sara Bléve; Clara Casadei; Christian Loli; Laura Ridolfi; Juan Martinelli; ugo de giorgi

1. Introduction

Breast cancer (BC) is the most common cancer in women worldwide. According to Globocan, with an estimated 2.3 million new cases (11.7%) worldwide in 2020, it is the most commonly diagnosed cancer and the fifth leading cause of cancer death [1]. The incidence of MC is increasing, especially in highly developed countries where screening strategies help to reduce cancer mortality, while in poor developing countries the incidence of MC is still low but the mortality rate is still higher [1] . However, in advanced countries, the diagnosis of de novo metastatic CM still accounts for about 3% to 6% of new CM diagnoses and has not declined despite the widespread use of mammography [2].

BC is a very heterogeneous disease that is clinically differentiated into several subtypes based on hormone receptor (HR) expression and human epidermal growth factor receptor 2 (HER-2) status: BC luminal, BC HER-2 positive and triple negative BC (TNBC). HR and HER2 are the targets of several specific early- and late-stage treatments. TNBC is defined by the lack of expression of HR and HER-2 and represents about 15% of all BC [3].

TNBC has been considered a significant unmet need due to its aggressive behavior and poor prognosis due to lack of specific therapeutic targets. These tumors tend to recur early and metastasize rapidly to the lungs, liver and central nervous system, leading to poorer survival [4]. For these reasons, chemotherapy remains a mainstay in the management of this CD subtype.

Immunotherapy with checkpoint inhibitors has been shown to be effective in TNBC, particularly in tumor tissue expression of programmed death 1 (PD-1) [5]. Furthermore, TNBC has the highest tumor mutation burden (TMB) among all BC subtypes [6]. A high level of mutations can result in the production of tumor “neoantigens” that can be recognized by antigen-presenting cells in the tumor microenvironment (TME), thus enhancing the antitumor immune response [6]. Although CB has always been considered a poorly immunogenic tumor, the TN and HER2+ subtypes show considerable immunological infiltration. To demonstrate, tumor infiltrating lymphocytes (TILs) are commonly present in TN and HER2+ tumor specimens and are associated with a good prognosis and prediction of immunotherapy efficacy [7,8,9]. Otherwise, high immune infiltration has a completely different effect on luminal and lobular BC subtypes, indicating a poor prognosis [5,10,11,12]. Especially in TNBC, intrinsic molecular features (determined by mRNA profiling, gene expression and proteomics) can distinguish different intrinsic subtypes of TNBC defined as basal 1 or 2, luminal androgen receptor and mesenchymal tumors [13]. Each intrinsic subtype is associated with an individual TME composed of the molecular properties and genomic signatures of cancer cells [14]. The quality of immune cells and their distribution in tumor tissue is also important in TNBC, distinguishing between “cold tumors” and “inflamed tumors” and foci of inflammation in stromal tissue, borders or completely inflamed tumor tissue [15,16] . In general, immune-rich early TNBCs show less clonal heterogeneity, somatic mutation, and less expression of neoantigens, but high expression of TIL, CD8+ T cells, or memory T cells [17]. On the other hand, metastatic sites appear more heterogeneous and immunocompromised with fewer TILs, CD8+ T cells or dendritic cells, a low BMR and greater clonal diversity [18,19]. In contrast, in the metastatic scenario, there is a greater presence of metastasis-associated macrophages (MAM) with a pro-tumor phenotype capable of enhancing immune escape strategies and cancer dissemination [20]. Tumor-mediated immunosuppression is a real problem responsible for acquired resistance to active immunotherapy (ICI) [21].

Targeting the immune system with a combination of different targets, especially in advanced CM, becomes a valuable therapeutic strategy to achieve the best survival outcomes. This strategy aims to convert nonresponders into responders, maintain an achievable sustained response, and overcome acquired immune resistance. Interrupting immune evasion, promoting the antitumor phenotype of immune cells or enhancing antitumor immunity are expected goals [22]. Many early-stage clinical trials in various solid tumors (including BC patients) with new agents that target macrophages or neutrophils are ongoing [23]. Following the incredible results achieved in the field of onco-hematology, emerging treatments for solid tumors are being used in immunotherapy. This includes adoptive cell therapy (ACT), which uses TILs or genetically engineered T cells to express modified T cell receptors (TCRs) or chimeric antigen receptors (CARs).

CAR-based therapies using T cells or natural killer (NK) cells show promise as potential practice switch effectors in BC, particularly in tumors with low-target antigens (such as TNBC), although significant limitations remain, depending on the resistance of unfavorable MSDs and side effects [24,25]. The presence of an extracellular matrix (ECM) in the tumor stroma creates a physical barrier to the transmission of CAR-T cells. CAR-modified macrophages (CAR-M) can overcome this barrier through the production of metalloproteinases and enhance the antitumor effect through antigen-specific phagocytosis [26,27]. In our review, we provide an overview of the potential of CAR-based therapies in BC.

2. Structure of the CAR molecule

A CAR is an artificial fusion protein composed of an extracellular antigen-binding domain, including an antigen-recognition domain, such as a mAb-derived single-chain variable fragment (scFv), involved in binding between the T cell and a tumor . -associated antigen (TAA) [28]. A hinge region is attached to the scFv, providing flexibility to the CAR; its length can be modified to optimize the distance between CAR-T cells and specific cancer cells and improve the signal transduction process [29]. In addition, a transmembrane domain is involved in intracellular signal transduction pathways. To this end, this region includes signaling and costimulatory domains (e.g. CD3α, also called CD247) responsible for activating CAR-T cells [28].

3. Generations of CAR-T cells

Improvement of CAR vectors can improve the safety and efficacy of CAR T cell therapy [30]. Several generations of CARs have been developed for this purpose and the fifth generation is currently being tested [31,32]. The main differences between the CAR generations are in specific costimulatory molecules. Does the first generation only contain CD3? last signaling domain whose binding to extracellular scFv modifies and activates T cells [33]. However, due to their short survival time and incomplete T cell activation, it was necessary to develop second and third generation CARs containing (each) one or two additional costimulatory molecules such as CD27, CD28, 41BB, ICOS and OX-40. These molecules increase the persistence and cytolytic capacity of cells [34,35,36]. T cells redirected to universal cytokine-mediated killing (TRUCK) or shielded CAR-T represent the fourth generation of CAR containing a nuclear factor activated T cell domain (NFAT) [37]. The domain promotes the secretion of cytokines, mainly interleukin (IL)-12, IL-15 and granulocyte-macrophage colony-stimulating factor (GM-CSF), which aim to modulate the antitumor microenvironment. Indeed, shielded CAR-Ts exert simultaneous antitumor activity, targeting both tumor cells that express the CAR-targeted antigen and those that do not. The other advantage of this strategy is determined by the local release of IL-12 with lower risk of systemic toxicity related to cytokine secretion [38]. Such CAR-Ts can be tested for TNBC-related antigens.

A fifth generation of CARs has been developed in recent years. It contains a fragment of the IL-2 receptor β (IL-2Rβ) that induces the secretion of Janus kinases (JAKs) and transcription signal transducer and activator (STAT)-3/5 [31,39]. This new generation of CARs aims to prevent terminal phenotypic differentiation of effector cells; Consequently, fifth-generation CAR is able to promote its in vitro expansion and its sustained in vivo cytotoxicity [40].

4. Targets for CAR-T cell therapy in BC

The development of CAR led to the search for new targets for cancer therapy, particularly for histologies lacking expression of ERBB2 and HR [24], such as TNBC (Figure 1). All studies testing potential targets for CAR-T cell therapy in BC tumors are listed in Table 1.

Abbreviations: CAR: chimeric antigen receptor; CEA: carcinoembryonic antigen; EpCAM: epithelial cell adhesion molecule; FR: folate receptor; HER2: human epidermal growth factor receptor 2; MUC1: mucin1; PRLR: prolactin receptor; ROR1: orphan receptor-type tyrosine kinase; TEM8: tumor endothelial marker 8; TNBC: triple negative breast cancer; VEGFR1: Vascular endothelial growth factor receptor 1.

4.1. Integrina

In our research, we looked for membrane receptors to identify a specific target for CAR molecules. In this context, integrins represent a potential target for CARs due to their proven involvement in cell proliferation and metastasis and due to their high expression in BC [41]. In particular, avβ3 integrin-targeted CAR molecules were designed and tested, which demonstrated their cytolytic activity against various tumors in vitro, including MDA-MB-231 TNBC cell lines. After in vivo testing of these molecules, some complete responses were reported in mice affected by metastatic melanoma [43]. Furthermore, as reported in some studies, this therapy has shown selective cytotoxicity against cell lines expressing avβ3 without involving normal cells [42,43,44]. Therefore, as reported, avβ3CAR T-cell therapy looks promising and deserves further studies to confirm its efficacy in CM.

4.2. Mesotelina

Another potential target for CAR molecule development is mesothelin, a tumor differentiation glycoprotein involved in cell adhesion that is normally expressed in mesothelial cells but overexpressed in several solid neoplasms, including TNBC [64,65]. Its activity in oncogenesis has been reported through various cell signaling pathways such as MAPK, PI3K and NF-kB [83]. As its overexpression has been reported in 67% of TNBCs, with limited expression in normal mammary cells [84], mesothelin represents an attractive target for the development of CAR molecules. In this regard, Hu et al. when evaluating the expression of mesothelin in three TNBC cell lines, such as MDA-MB-231, BT-549 and Hs578T, he found that only BT-549 cells expressed the molecule. The authors then manufactured CAR-Ts with second-generation mesothelin and tested them in vitro and in vivo. It should be noted that the researchers destroyed the PD-1 gene locus in the T cells before inserting the CAR transgene. Their CAR-Ts showed an interesting increase in antitumor activity and cytokine secretion against tumor cells expressing PD-L1 in culture [85]. This is likely due to the high expression of PD-L1 in TNBC cells [86], suggesting a potential use of this CAR-T to overcome the silencing effects of the PD-1/PD-L1 axis in BC [85] . . Thanks to these interesting data, some clinical studies have been developed and are ongoing with the aim of evaluating the activity of CAR molecules in TNBC. Specifically, a phase I clinical trial (NCT02792114) is evaluating the safety and tolerability of CAR-T redirected to mesothelin in metastatic/advanced BCs that express mesothelin, including TNBCs. Another phase I/II clinical trial (NCT02414269) is testing second-generation CAR-T retargeted with mesothelin in a variety of tumors, including BC. In addition, two other clinical studies (NCT02580747 and NCT01355965, the latter only in mesothelioma) have been completed, but no official data has yet been published.

4.3. TEM8

Some studies have shown that the endothelium of various neoplasms frequently overexpresses an integrin-like protein called tumor endothelial marker 8 (TEM8), also known as ANTXR1, and is usually expressed during endothelial cell development, but rarely in adults [47,48] . As evidence, increased TEM8 expression was found both in invasive/relapsing BC [49,50] and in several BC cell lines [51], making the upregulation of this molecule a potential target for CAR-T cell development . [87.88]. In this regard, a single dose of L2CAR-T cell-specific therapy derived from the L2 monoclonal antibody (Mab) against TEM8 showed a complete response against TNBC in vitro and a significant reduction in cancer in vivo and TNBC xenografts [51]. ]. For these reasons, TEM8 represents a promising target for CAR-T cell therapy against TNBC.

4.4. MUC1

In TNBC, MUC1 represents a highly selective overexpressed target [66]. It is a glycosylated transmembrane molecule with altered epithelium [66]. It produces mucin that protects cells from pathogens [67,68,69]. Tumor cells overexpress MUC1 and activate intracellular signaling pathways involved in cancer proliferation [66,69]. The recent cohort study by Jiang et al., involving more than 5800 BC patients, demonstrated the predictive role of MUC1 and its correlation with poor prognosis [89]. In particular, neoplasms overexpress a hypoglycosylated variant of MUC1, also known as tumor-specific MUC1 (tMUC1), which exposes new epitopes to the immune system [90]. To that end, antibodies that specifically bind to tMUC1 have been developed and tested [91]. One of these molecules, designated TAB004, served as a reference point for creating a CAR molecule containing its extracellular scFv. The CAR T-derived cells known as MUC28? CAR-T cells have been shown to potently increase the expression of markers of leukocyte activation and cytokines in vitro. These effects caused significant cell lysis in vitro and reduced cancer cell growth in vivo [66]. Recently, a new CAR molecule targeting tMUC1, known as huMNC2-CAR44, was activated in a clinical study involving 69 patients with BC (HER2 positive, HER2 negative, triple negative); the estimated study completion date is January 15, 2035 (NCT04020575). Another phase I study is currently testing the safety, tolerability, feasibility, and preliminary efficacy of tMUC1-targeted CAR T cell delivery in 112 patients with advanced tMUC1-positive solid tumors (including BC) and multiple myeloma. The estimated study completion date is October 31, 2036 (NCT04025216).

4.5. ROR1

The orphan receptor tyrosine kinase (ROR)1 is a highly expressed molecule during embryogenesis, but not in adults. BC cells strongly express ROR1, especially in cases of poor prognosis; Overexpression of ROR1 has been found in some TNBC cell lines (eg in MDA-MB-231) but not in others [70]. ROR1-based CAR-T cell therapy has also been shown to induce MDA-MB-231 apoptosis in tumor models through significant levels of IL-2 and IFN? production [92]. A phase I study is testing the effectiveness of ROR1-specific CAR-T cells in 60 subjects with hematologic and solid tumors, including triple-negative DC. Patients will be followed for approximately 15 years after completion of the study. The estimated study completion date is December 1, 2023 (NCT02706392). The first results of the study were recently published and indicate an improved efficacy of CAR-T cell therapy by adding oxaliplatin to the lymphocyte depletion regimen [93]. Another Chinese phase I study is currently recruiting 40 patients with advanced solid tumors (including BC) to investigate the efficacy of TIL and CAR-TIL against various molecular targets including ROR1, MUC1, HER-2, mesothelin, PSCA, EGFR, GD1 , GPC3, Lewis Y, AXL, Claudin18.2/6 and B7-H3. The estimated study completion date is January 1, 2035 (NCT04842812).

4.6. Group 2 natural killer member D-linker (NKG2DL)

Under certain pathological conditions, innate and adaptive immune cells (including CD8+ and some CD4+ T cells, NK cells, and βd T cells) express a type II transmembrane protein termed group 2 natural killer, D-Member (NKG2D) [74], which in turn help to increase cytotoxicity and cytokine production by effector cells and promote their proliferation and survival. Furthermore, NKG2D can cooperate with other receptors (including TCR on T cells or NKp46 on NK cells) acting as a co-stimulator of their responses [94]. Stress-induced upregulation of ligands has been frequently reported in tumor cells, including TNBC cells; NKG2D can naturally recognize these ligands [95], which is why it has been considered a potential target for immunotherapy in several studies. CAR molecules obtained by fusing full-length NKG2D to the cytoplasmic CD3z region, together with endogenous costimulation of DAP10, have been shown to react with tumor cells expressing NKG2DL through the production of cytokines and chemokines, thereby increasing cytotoxicity [96] . These results were also confirmed by in vivo studies [97,98]. Recently, Han et al. tested. in TNBC cell lines and TNBC mouse models [99]. In this case, CAR-Ts redirected to NKG2DL were obtained by fusion between the extracellular domain of human NKG2D and the TCR CD3z alone or costimulatory domains such as 4-1BB or CD27. The authors showed that the increase in CD25 expression and the presence of IL-2 were necessary to promote the expansion of CAR-T in vitro in the absence of costimulatory domains. Furthermore, CAR-Ts redirected to NKG2DL were able to recognize and kill MDA-MB-231 and MDA-MB-468 cell lines expressing TNBC NKG2DL [99]. Based on these results, a phase I clinical trial (NCT04107142) evaluated the safety and tolerability of CAR-T cells redirected to NKG2DL in patients with various solid malignancies, including TNBC, but no results have been published to date.

4.7. Chondroitin sulfate proteoglycan 4 (CSPG4)

CSPG4 is a hyperglycosylated transmembrane protein with low expression in normal tissues and overexpression in several types of tumors, including TNBC. It has been suggested that CSPG4 is involved in neural network regulation and epidermal stem cell homeostasis [55]. Second-generation CSPG4-redirected CAR-Ts have been tested on several CSPG4-expressing cell lines (including SENMA, UACC-812, CLB, MDA-MB-231, MILL, PHI, and PCI-30) and demonstrated significant suppression of cell growth capacity [56]. The same results were obtained in preclinical mouse models of various human tumors (including BC). In another study, second-generation CSPG4-targeted CAR-Ts using murine scFvs reported target antigen-dependent cytotoxicity and cytokine secretion against various tumor cell lines (including BC) [100].

4.8. EpCAM

Epithelial cell adhesion molecule (EpCAM) is a well-known molecule whose expression has been associated with poor prognosis and tumor metastasis [71]. Different treatment strategies targeting EpCAM have shown benefits for different tumor types. Currently, a Chinese clinical trial is recruiting patients with BC and nasopharyngeal neoplasms to evaluate the safety of genetically modified CAR-T cells that recognize EpCAM. These molecules were developed by lentiviral transduction of third-generation CAR genes. Different cohorts of patients are receiving the experimental treatment in increasing doses; the expected completion date of the study was set in July 2022 (NCT02915445).

4.9. Intercellular Adhesion Molecule-1 (ICAM-1)

ICAM-1 is a transmembrane protein involved in white blood cell diapedesis. It is overexpressed on the surface of many cancer cells, including TNBC cells [60]. ICAM-1 plays a role in tumor growth, invasion and metastasis [61]. To prevent CAR-T-related cytotoxicity in normal cells, Park et al. generated CAR-Ts with micromolar (rather than nanomolar) affinity and showed that these CAR-Ts redirected to ICAM-1 were more efficient and safer than their higher affinity counterparts [62]. More recently, the same results were confirmed in preclinical models [63].

4.10. she-2

HER2 overexpressing BC represents the subset of tumors for which CAR-T cells were designed [101]. Several clinical trials are underway to test CAR molecules that target HER-2. One is a phase I study evaluating the safety and preliminary therapeutic efficacy of CCT303-406 cells in 15 patients with stage IV HER-2 positive solid tumors (which have failed standard care for recurrence or are difficult to treat), including BC. The estimated study completion date is April 1, 2023 (NCT04511871). A phase I/II multicenter study is underway in the US in 220 patients with HER-2 positive tumors, including BC, to evaluate the safety, tolerability, and clinical activity of HER2-derived CAR-T cells evaluating specific dual switching. BPX-603 administered with Rimididuc. The estimated study completion date is January 2, 2025 (NCT04650451). Another US dose escalation study is underway at City of Hope Medical Center (Duarte, CA) to evaluate side effects and the best dose of HER2-CAR T cells in the treatment of patients with metastatic breast, brain, or leptomeningeal cancer. . 39 patients will receive HER2-CAR T cells intraventricularly for five minutes once a week in three doses to be administered at the discretion of the investigator. Patients will be followed at the end of treatment at 4 weeks, 3, 6, 8, 10 and 12 months and then up to 15 years. The estimated study completion date is August 31, 2023 (NCT03696030). A third US phase I study is evaluating the safety and efficacy of combining HER2-specific CAR-T cells with an intratumoral injection of CAdVEC, an oncolytic adenovirus believed to activate the immune system against cancer. 45 patients with HER-2-positive tumors were included in our study. The estimated study completion date is December 30, 2038 (NCT03740256). However, most patients did not have significant complications. In some cases, the lack of specificity of HER-2 expression between tumor cells and healthy cells can lead to serious side effects; a case of cardiopulmonary failure due to excessive activation of T cells has been reported [102].

Therefore, to avoid inconveniences related to the marker's non-tumor specificity, the researchers sought to deepen their knowledge of the receptor to assess whether it is possible to find a more specific variant in CM. In this sense, p95HER2 is a potential antigen, a truncated version of HER2 was found in 40% of HER2-positive BCs. This variant is more tumor specific than the constitutive form, as it is not found in normal breast cells. p95HER2 has previously been evaluated as a target for a bispecific antibody against cancer cells in vitro and in vivo without significant side effects. Based on the encouraging results reported, this variant may therefore be a future target for the development of CAR-based therapies [103,104].

4.11. VEGF

Vascular endothelial growth factor receptor (VEGFR)1 is a tyrosine kinase receptor involved in the migration and survival of hematopoietic stem cells, and its overexpression is associated with the process of BC metastasis [105,106]. Therefore, VEGFR1 represents a potential candidate for immunotherapy. To date, VEGFR1 has been tested as part of a bispecific antibody VEGFR1-CD3 and has shown promising results against the TNBC cell lines MDA-MB-231 and MDA-MB-435. These results warrant further studies on VEGFR1 activity, eg. B. as a target for CAR-based therapies [107]. Furthermore, as the main functions of normal endothelial cells depend on VEGFR2 [105], inhibition of VEGFR1 may prevent endothelial toxicity. At the moment, this is just a hypothesis, so more research is needed.

4.12. c-MET

Hepatocyte growth factor receptor, also called c-Met, is a cell membrane protein tyrosine kinase expressed in several types of solid neoplasms, including BC [108]. Onartuzumab, an anti-c-Met monoclonal antibody, has been administered to patients with metastatic solid tumors [109,110,111,112]. Chou et al. c-Met tested as a potential target for CAR-T cell therapy; For this purpose, the scFv of the CD19 binding domain of a CD19-CAR molecule was replaced by that of onartuzumab and its efficacy against BC cells was confirmed in vitro and in vivo [113]. Subsequently, the new c-Met CAR-T cells were introduced into a cohort of patients with BC (NCT01837602) via a single intratumoral injection of mRNA. The injections were well tolerated and no significant drug-related adverse events were reported. Furthermore, after analysis of the tumor samples (four TNBC and two ER+ HER2-negative BC) injected with the CAR-T cells, extensive tumor necrosis at the injection site and macrophage infiltrates in the necrotic areas were observed [113].

4.13. AXL

AXL, a receptor in the TAM family of receptor tyrosine kinase, and its high-affinity ligand, specific growth arrest protein 6 (GAS6), are involved in the expansion, metastasis, and survival of cancer cells; Furthermore, the low expression of AXL in normal adult cells and its overexpression in several types of tumors (including BC) and some cell lines (including MDA-MB-231 [114]) make AXL a potential target for the development of molecules CAR. [52,115,116].

Wei and others. AXL-targeted CAR-Ts designed using an AXL-specific scFv; these CAR-Ts were tested on the TNBC cell line expressing AXL MDA-MB-231 and showed antigen-dependent cytotoxicity and cytokine production; these results were confirmed in an in vivo evaluation in xenograft models established with MDA-MB-231 [53]. Other researchers have generated CAR-Ts redirected to AXL with a constitutively activated IL-7 receptor (C7R); demonstrated a significant ability to kill tumor cells in the TNBC cell lines MDA-MB-231 and MDA-MB-468, which was more effective than the use of conventional CAR-T with AXL redirection. This improvement was likely due to co-expression of C7R, which helped to prolong survival and reduce tumor recurrence rates [54]. However, more research is needed to confirm these results.

4.14. Disialoganglyosid GD2

GD2 is a surface protein normally expressed only on peripheral nociceptors, neurons and melanocytes; Consequently, GD2 expression has been detected in neuroectoderm-derived tumors such as melanoma and neuroblastoma [117]. Its cell-type restriction makes GD2 a potential target for the development of CAR molecules. Indeed, GD2 has been studied primarily as a target for neuroblastoma treatments [118]. However, Seitz et al. used the dinutuximab-derived anti-GD2 mAb scFv to produce GD2 redirected CAR-Ts. The researchers evaluated GD2 expression in different TNBC cell lines and showed very low expression in MDA-MB-231, while Hs578T and BT-549 uniformly expressed GD2. However, in an in vitro test, these CAR-Ts did not show specific tumor cell killing activity for MDA-MB-231, while induced specific cytotoxicity and cytokine production when co-cultured with Hs578T and BT-549 cell lines . 59]. To date, one study is testing the feasibility, safety, and efficacy of several fourth-generation CAR-T cells targeting Her2, GD2, and CD44v6 surface antigen in BC (NCT04430595), but no results have yet been published.

4.15. PRLR

In mammals, prolactin is an important hormone for milk secretion and breast tissue growth, binding to the prolactin receptor (PRLR) in the mammary glands [119]. PRLR is overexpressed in some BC histotypes, particularly in the TNBC cell line MDA-MB-231 and even more so in the T47DHER2+ and SKBR-3 cell lines [120]. This correlation between PRLR and HER2 expression could lead to the development of a CAR-based therapy targeting PRLR against BC. However, when these two targets are combined, bispecific antibody cytotoxicity has been reported, caused primarily by combinatorial inhibition of the two rather than the effect of T-mediated cytotoxicity [121]. Although the results look promising, future studies of CAR-based therapies targeting PRLR should avoid cross-toxicity to other organs expressing the same receptor, such as prostate, liver, etc. [122].

4.16. CEA

CEA is a well-known tumor marker expressed in several solid neoplasms [75]. In normal cells, under physiological conditions, only a small amount of CEA is expressed on the cell membrane, especially towards the cell cavity to avoid recognition by CEA-targeted CAR-T cells. A phase I-II study is currently enrolling 40 patients with various solid tumors to test the efficacy and safety, recommended dose, and infusion schedule of CEA-targeted CAR-T cell therapy. The estimated study completion date is April 30, 2023 (NCT04348643).

4.17. CD44v6

CD44 domain variant 6 (CD44v6), a member of the CD44 family, has been shown to play a role in tumorigenesis, tumor cell invasion and metastasis. In normal tissues, its presence is reported only in subsets of epithelial and hematopoietic cells, particularly during embryogenesis and hematopoiesis [76]. Instead, it is expressed in multiple squamous cell carcinomas, partly in adenocarcinomas of different origins, partly in lymphomas and melanomas, and therefore represents an attractive target for cancer therapy. Currently, a phase I-II clinical trial is investigating the feasibility, safety, and efficacy of CD44v6 CAR T-cell therapy in 100 patients with various tumors, including BC. Another objective of this study is to learn more about the functions of 4SCAR-CD44v6 T cells. The estimated study completion date is December 31, 2023 (NCT04427449).

4.18. Trophoblastic cell surface antigen 2 (TROP2)

TROP2 is a transmembrane protein expressed on the cell surface of human trophoblasts and is commonly present in several types of epithelial tumors (including TNBC), where it is associated with poor prognosis [57]. Zhao et al. developed and tested in vitro (against gastric cancer cell lines) and bispecific CAR-T redirected by TROP2 and PD-L1 in vivo [58]. The authors reported greater antitumor activity with bispecific CAR-T compared with monospecific CAR-T. Although CAR-T-mediated TROP2 targeting in TNBC has not been extensively studied, the results obtained in gastric cancer cell lines warrant further investigation in other tumor types, including TNBC.

4.19. Epidermal growth factor receptor (EGFR)

EGFR is a transmembrane glycoprotein belonging to the ERBB family of receptor tyrosine kinase and is involved in tumor growth and metastasis [72]. Its overexpression has been reported in several types of tumors, including TNBC. Read al. produced CAR-Ts redirected to EGFR using the non-viral Bac piggy transposon system and demonstrated their antitumor activity first in human lung tumor xenografts and later in a phase I clinical trial (NCT03182816) against non-small cell lung tumors [123,124 ]. Regarding TNBC, Liu et al. tested the antitumor activity of CAR-T redirected to EGFR in vitro and in vivo and reported the overexpression of EGFR in several tumor cell lines, such as Hs578T, MDA-MB-468 and MDA-MB-231, respectively. These CAR-T showed antitumor activity and cytokine secretion in these cell lines [125]. Recently, Xia et al. generated third-generation EGFR-redirected CAR-Ts that showed antitumor activity and specific cytokine production. Furthermore, the authors found that markers of T cell activation (such as CD25 and CD69) were up-regulated when co-cultured with EGFR-positive TNBC cell lines [126]. However, more preclinical and clinical studies are needed to confirm these results. Several clinical studies (NCT03182816, NCT02873390, NCT02862028 and NCT03170141) test the effect of EGFR-redirected CAR-Ts on the production of anti-CTLA-4, anti-PD-1 or anti-PD-L1 antibodies. in EGFR-positive solid tumors [127]. Regarding TNBC, there are two ongoing studies. The first (NCT05341492) evaluates the safety and efficacy of EGFR/B7H3-CAR T-cell therapy in EGFR/B7H3-positive advanced solid cancers (including TNBC), and the second evaluates the antitumor activities and safety profiles of CAR-EGFR- TGFßR -KO -T cell therapy in previously treated EGFR-positive solid tumors (including TNBC) However, no official results in TNBC patients have been reported yet

4.20. Prostate Specific Membrane Antigen (PSMA)

Although it does not appear to be related to MC at first glance, this molecule has recently been shown to be present in circulating breast cancer cells and associated with a worse prognosis [77]. Although PSMA is expressed in normal prostate and is upregulated in prostate tumors, it is not limited to prostate cancer. At TNBC, PSMA is being evaluated as a target for the development of CAR molecules. In an ongoing phase I open label study, bispecific anti-PSMA/CD70 CAR T-cell therapy was tested in several cancers (including TNBC) that express PSMA or CD70, another potential tumor target that is overexpressed and weak in many cancers is expressed in normal tissue (NCT05437341). A second phase I study is underway to test the feasibility, safety and efficacy of anti-GD2/PSMA bispecific CAR T cell therapy in patients with GD2 and PSMA positive tumors (including TNBC) (NCT05437315). Additionally, a third phase I study in China is evaluating the feasibility, safety, and efficacy of PSMA-specific CAR-T cell therapy in patients with PSMA-positive malignancies, including TNBC (NCT04429451). No results have been published yet.

4.21. Alpha Folate Receptor

In normal cells, the DNA synthesis pathway is efficient in the presence of folate, which is directed into the cell through an appropriate receptor called the folate receptor (FRa). FRa is often overexpressed in BC, especially TNBC, correlating with poor clinical outcomes [95,109]. FRa-CAR T cells have been shown to target FRa+ TNBCs and reduce tumor growth in the MDA-MB-231 tumor xenograft [110]. To limit toxicity, Lanitis et al. constructed a trans signaling CAR with two different signaling domains (CD3β and CD28) located on two different CARs and a T cell to bind mesothelin and FRa in tumor cells. Under these conditions, activation occurs only when antigen binding occurs at the same time, which in turn can activate T cell activity [111]. Therefore, FRa could become a potential target for immunotherapy in BC.

5. Overcoming CAR-T-related problems in solid tumors: macrophage-based cellular therapeutics

So far, CAR-T cell therapy has shown efficacy against hematological malignancies, but in contrast its results against solid tumors have been disappointing. The reason for this difference probably lies in the presence of a TME around solid tumors that modulates the immune response against malignant cells and prevents CAR-T cell invasion [128,129,130]. To overcome the MSE obstacle, CAR molecules were developed, inspired by gamma-delta (?d) and natural killer (NK) T cells due to their specific biological properties. Indeed, these cells can identify a broad spectrum of tumor-associated antigens (TAAs) independently of the major histocompatibility complex (MHC) [131,132], with little impact in terms of immune-mediated toxicity [133]. However, problems in the expansion process of these immune cells limit its application in clinical practice [134]. On the other hand, the phenomenon of "T-cell depletion" is a well-known and still not completely solved problem [135,136,137]. Therefore, it was necessary to look for other strategies, such as exploring other immune cells [138]. In this context, macrophages seem to be an interesting option for the development of immunotherapies. Indeed, they exhibit a wide range of immune activities, including their role as antigen-presenting cells that can modulate adaptive immune responses, phagocytosis, and secretion of pro-inflammatory cytokines [139]. Furthermore, they represent a significant percentage of immune cells within the TME of solid tumors that recruit peripheral blood monocytes and then promote their differentiation into tumor-associated macrophages (TAMs) [140,141].

TAMs are often reported as M1 (pro-inflammatory) and M2 (anti-inflammatory) phenotype, but a higher concentration of M2 is more often associated with a poor prognosis [142]. Its presence is considered crucial for the regulation of the TME, especially with regard to the stimulation of tumor growth, angiogenesis and metastasis [143,144]. Furthermore, TAMs have been reported to play a role in the recruitment of cytotoxic lymphocytes into the TME [145].

In this context, the targeting of TAMs has become the goal of numerous immunological approaches, e.g. B. TAM depletion, repolarization or inhibition of suppressor molecules secreted by TAM [146]. Rather than targeting TAMs directly, several studies have examined the role of macrophages in cancer therapy. For example, antibody-dependent cellular phagocytosis is an interesting strategy (ADCP) [146]. It utilizes antibodies against specific tumor-associated antigens via the Fab region, which are internalized by the binding of Fc receptors (such as CD16a or CD32a) to macrophages. In addition, macrophages can stimulate phagocytosis through these receptors and other surface molecules, such as Mac1 or LRP1, whose intracellular mechanism of action is similar to that of CD16a and CD32a. Indeed, its cytoplasmic region is rich in tyrosine-based activation motifs that can activate MAPK and PI3K/AKT signaling pathways, with the consequent process of phagocytosis against cancer cells [147]. Bispecific antibodies directed against different TAAs or macrophage receptors are another option [148]. The involvement of phagocytosis checkpoint inhibitors such as CD47 may enhance phagocytosis mechanisms by blocking macrophage inactivation signals [138]. However, using antibodies is not that simple; To date, several challenges need to be overcome before they can advance clinically. First, macrophages display the inhibitory FC receptor (Fc?RIIb) on their extracellular membrane, which counteracts cell activation. Second, Mab therapy cannot distinguish between antitumor and/or protumor TAMs [149].

For these reasons, another option is adoptive cell therapy based on ex vivo genetically modified CAR macrophages (CAR-Ms). Among different types of CAR molecules, second- and third-generation CARs are preferred for their ability to amplify phagocytic activation signals [150,151]. CAR-Ms offer several advantages over T cells: (1) lower risk of GVHD by allowing the production of CARs in advance for "on demand" use; and (2) significant MMP production, allowing macrophages to degrade ECMs and thus approach tumor cells [152]. However, several issues remain to be resolved: (1) although its efficacy and safety profile have been reported in animal studies, it is still unclear in humans; and (2) the use of viral transfection in CAR gene transfer can promote insertions with unpredictable implications for treatment. In this context, the CRISPR/Cas9 genome targeting system may be a valuable option to overcome this problem [153].

Furthermore, regenerative medicine may represent a potential strategy to limit the high cost of CAR therapy by providing a sustainable source of CAR-M. CAR delivery to induced pluripotent stem cell (iPSC)-derived macrophages may extend CAR-M cell therapy to a larger population of patients. In a recent study, iPSC-derived CAR-Ms reduced tumor growth by activating phagocytosis in leukemia, ovarian cancer, and pancreatic cancer cell lines. Furthermore, the same results were reported in vivo in a mouse model of ovarian cancer [154].

6. CAR-M in solid tumors and BC

To date, several investigators have attempted to use CAR-M against BC and solid tumors. Several CAR phagocytes (CAR-P) have been designed to direct macrophages against specific targets. In particular, CAR-P expressing the intracellular region of FcRv or Megf10 has been shown to stimulate TAA phagocytosis through TCR-CD3γ-mediated recruitment of SYK kinase. Complete phagocytosis is usually uncommon, suggesting that CAR-P macrophage binding to target cells is insufficient to achieve this. In this regard, it should be remembered that the PI3K signaling pathway has been shown to be involved in target internalization and increased phagocytosis in macrophages [152]. For this reason, a "tandem" CAR (tandem CAR-P) was developed by connecting the PI3K p85 subunit to the CAR-P-FcRv. This molecule showed an increase in the phagocytic activity of CAR-P, particularly with regard to whole cell phagocytosis [150].

CAR-147 is a CAR molecule consisting of a single-chain antibody fragment that targets HER2, a murine IghG1 hinge region, and a mouse-derived CD147 intracellular and transmembrane domain. Co-culture of CD147 with HER2+ human BC cells resulted in intense MMP expression, demonstrating the ability of CAR-147 to target HER2 and effectively promote MMP production in macrophages. Indeed, CAR-147 macrophages have been shown to increase the number of T cells in the vicinity of tumor cells compared to controlled macrophage treated tumors, demonstrating their potential to disrupt the extracellular matrix in tumors. Furthermore, CAR-147 macrophages demonstrated antitumor activity by secreting IL-12 and IFN? levels in tumor tissue [155]. Intravenous injection of CAR-147 significantly inhibited cancer growth in 4T1 BC mouse models, but this has not been demonstrated in vitro. Recently, an adenovirus-induced CAR-M consisting of an anti-HER2 CAR and CD3? An intracellular domain was designed that demonstrated its specificity for antigen-specific phagocytosis against HER2-positive tumor cells in vitro. A single injection of anti-HER2-CAR-M has been shown to reduce tumor burden and prolong survival in mice. It was also able to convert M2 macrophages into M1 macrophages, stimulate inflammatory TME and promote antitumor cytotoxicity. Furthermore, HER2 CAR-M can generate epitope diffusion, which could be another solution to prevent tumor immune system escape [151,156].

Another study combined an anti-HER2 CAR with peripheral blood macrophages derived from transduced primary human CD14+ monocytes. These CAR-Ms promoted dose-dependent phagocytosis of the ovarian cancer cell line HER2+-SKOV3. The authors also showed that the transduction of macrophages is not affected by the anticancer effect, since their transduction with a control CAR did not have antitumor activity [151].

Furthermore, the in vivo tumor burden of SKOV3 in NOD-SCID mice was significantly lower in cases treated with primary human anti-HER2 CAR-M. The authors also showed that CAR-Ms survived and resisted the immunosuppressive cytokines secreted by the TME. In contrast, CAR-Ms secreted pro-inflammatory cytokines, triggering a macrophage conversion from an M2 to M1 phenotype and consequently converting TME into a pro-inflammatory environment. Furthermore, a combination of T cells derived from CAR-M donors enhanced the antitumor response in vivo [151].

Pierini et al. showed that infusion of mouse-derived anti-HER2-CAR-M resulted in inhibition of tumor growth, prolongation of overall survival, and increase in CD4+ and CD8+ T cells, NK cells, and dendritic cells in TME. The authors also reported that CAR-Ms play a critical role in the regulation of TME by up-regulating MHC I/II expression in cancer cells [157].

7. Clinical applications of the CAR-M strategy against CD and other solid tumors

As of December 2022, three clinical trials evaluated a CAR-M-based approach in solid tumors, two of which received FDA approval [152] (Table 2).

The first (a Phase I clinical trial) tested CT-0508 (CARISMA Therapeutics Inc., Philadelphia, PA, USA), a therapy consisting of infused anti-HER2 CAR macrophages in 18 patients with HER2-overexpressing tumors. The study evaluated the effects of CAR-M adenovirus transduction. The estimated study completion date is February 2023 (NCT04660929). The second study tested MCY-M11 (MaxCyte Inc., Gaithersburg, MD, USA), consisting of mRNA-targeted PBMCs (not just CAR-M) expressing mesothelin-CAR, in patients with relapsed/refractory and peritoneal mesothelioma (NCT03608618). A third study (CARMA-2101), not yet recruited, is being carried out at the Oscar Lambret Center (Lille, France). This observational study aims to determine the antitumor activity of the new CAR-M in organoids derived from 100 BC patients. In particular, the researchers will test the activity of CAR-M against organoids derived from HER2-negative, HER2-low and HER2-positive BCs and then compare the activity of CAR-M and unmodified macrophages. The estimated study completion date is September 1, 2023 (NCT05007379).

8. Conclusions

Recent remarkable advances in the field of immunotherapy have fundamentally changed the way we approach many types of cancer. With a better understanding of the role that immune cells play in the mechanisms of tumor progression, it was possible to design drugs that target specific immune targets and forms of immune cell-based therapy, such as B. CAR technologies that led to the development of CAR-T. Cells that are also effective in the clinical setting (particularly in the field of hematologic malignancies).

To date, several problems severely limit the application of CAR-T cells, particularly in solid tumors. For this reason, further studies of CAR-M in tumor therapy are interesting due to the known adaptability of these immune cells to solid tumors.

In fact, early results have shown that CAR-M holds promise in the fight against cancer; preclinical data have confirmed its efficacy (in terms of tumor phagocytosis and growth inhibition in vitro and in vivo) and also in several solid tumors, including TNBC. The latter represents a heterogeneous subtype of BC that is generally resistant to standard therapies. However, its immunogenic nature has resulted in favorable clinical benefits of newer immune checkpoint inhibitors such as the recently FDA-approved atezolizumab in combination with nab-paclitaxel against metastatic TNBC [158].

CAR-based therapy in TNBC is an emerging field whose advancement depends on the discovery of appropriate and targeted AATs, particularly in preclinical and early clinical stages. In our article, we discuss several new CAR-based target antigens that have been tested against BC. Many of them have only been tested in “in vitro” and “in vivo” studies, and a small part of them have also been evaluated in humans, as summarized in Table 1 and Table 2. Furthermore, scientific research is uncovering other potential targets. , such as embryo-specific antigen-4, which has been studied in “in vitro” and “in vivo” studies in BC patients, but so far with less data compared to the above targets [159].

Therefore, we still have little clinical data to assess the effective validity of these new therapeutic approaches against CM (and not only), which require further studies. Due to this lack of relevant data on small series, many questions remain unanswered. For example, is CAR-based therapy (M or T cells) more effective than standard treatments for TNBC? Is it sufficient to consider monoimmunotherapy or should it be combined with other strategies, perhaps even other forms of immunotherapy (eg immune checkpoint inhibitors)? Regarding the last question, it appears that combination therapies may lead to better efficacy, especially in terms of overcoming MSD. Indeed, a recent study performed in immunocompetent mouse models of HER2+ solid tumors (including BC) showed a combination of anti-PD-1 with HER2-CAR-M cell therapy better operating system and better tumor control than monotherapy strategies [ 78]. In this sense, other strategies were evaluated. For example, drugs that target ECM or cancer-associated fibroblasts (CAF) and macrophage or monocyte killing have been tested with the aim of enhancing the antitumor effects of CAR-T in TNBC as well [160].

It seems clear that the success of CAR-based therapy in BC depends on the ability to select the best antigens to use as the basis for designing more effective CAR molecules that are also more manageable and less toxic. It is expected that more data from ongoing studies will be available in the near future.

Author Contributions

conceptualization, G.S.; Methodology, S.G.; Editing – creation of the original draft, S.G. and C.G.; Revision and Edition, G.S., C.G., M.P., S.B., C.C., C.L., L.R., G.M. and U.D.G.; Supervision, UDG; All authors read and accepted the published version of the manuscript.

interest conflicts

The authors declare no conflict of interest.

Disclaimer/Editor's Note: The statements, opinions and data contained in all publications are the sole responsibility of the individual authors and contributors and not of MDPI or the publishers. MDPI and/or the publisher(s) disclaim all liability for any damage to persons or property resulting from any ideas, methods, instructions or products mentioned in the content.

expression of gratitude

This work was partially funded by the Italian Ministry of Health, thanks to the contribution of Ricerca Corrente, within the scope of the research line L2 (Innovative therapies, clinical trials phases I-III). Figure 1 was created with Biorender.com (https://biorender.com/ (accessed Feb 28, 2023)).

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figure and tables

Figure 1: The image summarizes all molecules currently being evaluated as potential targets for CAR development in BC. [Download the PDF to view the image]

Table 1: Studies testing potential targets for CAR-T cell therapy in BC.

Abbreviations: AXL = UFO receptor tyrosine protein kinase; CAR-T = chimeric antigen receptor T cells; CD44v6 = CD44 domain 6 variant; CEA = carcinoembryonic antigen; c-MET = protein tyrosine kinase Met; CSPG4 = chondroitin sulfate proteoglycan 4; EGFR = epidermal growth factor receptor; EpCAM = epithelial cell adhesion molecule; FRa=folate receptor alpha; GD2 = disialoganglioside; ICAM-1 = Intercellular Adhesion Molecule-1; MUC1 = mucin1; NKG2D = natural killer group 2, linker member D; PSMA = prostate-specific membrane antigen; ROR1 = orphan receptor tyrosine kinase; TEM8 = endothelial tumor marker; TIL = tumor infiltrating lymphocytes; TROP-2 = Trophoblast Cell Surface Antigen 2. *TIL and CAR-TIL target HER2, Mesothelin, PSCA, MUC1, Lewis-Y, GPC3, AXL, EGFR, Claudin18.2/6, ROR1, GD1 or B7-H3. **CAdVEC is an oncolytic adenovirus designed to help the immune system, including HER2-specific CAR-T cells, respond against tumors.

Table 2: Assays for CAR-M in BC and other solid tumors.

links do autor):

IRCCS Romagna Institute for the Study of Tumors (IRST) „Dino Amadori“, 47014 Meldola, Italy

Note(s) of the author(s):

[*] Correspondence: giuseppe.schepisi@irst.emr.it

DOI: 10.3390/Krebs15051597