Chimeric antigen receptor-modified T cells therapy in prostate cancer: A comprehensive review on the current state and prospects

Abstract

Recent immunotherapy research has focused on chimeric antigen receptor-modified T cells (CAR-Ts). CAR-T therapies have been clinically applied to manage hematologic malignancies with satisfactory effectiveness. However, the application of CAR-T immunotherapy in solid tumors remains challenging. Even so, current CAR-T immunotherapies for prostate cancer (PCa) have shown some promise, giving hope to patients with advanced metastatic PCa. This review aimed to elucidate different types of prostate tumor-associated antigen targets, such as prostate-specific membrane antigen and prostate stem cell antigen, and their effects. The current status of the corresponding targets in clinical research through their applications was also discussed. To improve the efficacy of CAR-T immunotherapy, we addressed the possible applications of multimodal immunotherapy, chemotherapy, and CAR-T combined therapies. The obstacles of solid tumors were concisely elaborated. Further studies should aim to discover novel potential targets and establish new models by overcoming the inherent barriers of solid tumors, such as tumor heterogeneity and the immunosuppressive nature of the tumor microenvironment.

Graphical abstract

1. Introduction

Prostate cancer (PCa) is a highly prevalent malignancy in humans. According to statistics, nearly 1.4 million new cases of PCa were diagnosed in 2020, with 375,000 deaths worldwide, making it the second most prevalent cancer in men, after lung cancer, and the fifth leading cause of cancer-related deaths in men [1]. Based on the risk classification provided by the European Urology Association, patients with low-risk PCa can undergo active surveillance without surgical intervention [2]. For early-stage localized PCa, radical prostatectomy (RP) and postoperative radiotherapy are the treatment of choice [3]. Androgen deprivation therapy (ADT) is the first-line therapy for advanced PCa [4]. However, the effectiveness of ADT for advanced metastatic PCa is only temporary and is associated with severe multisystemic side effects, while the 5-year survival rate is approximately 30% [[5], [6], [7], [8]]. Over time, the effects of ADT fade, and most patients progress to castration-resistant PCa (CRPC) [9]. Metastatic CRPC (mCRPC) currently has no cure and a poor prognosis; hence, the patient's quality of life is poor [10,11]. Immunotherapy, which refers to the anti-tumor effect of the body's immune system, is a novel cancer treatment strategy used as a component of multimodality therapy for advanced PCa [12,13]. Chimeric antigen receptor-modified T cell (CAR-T) therapy, a type of immunotherapy, has shown significant efficacy in treating hematologic malignancies [14] and has great potential in oncology management. Chimeric antigen receptors (CARs) are essentially cell-engineered receptors designed to enable lymphocytes (most commonly T cells) to recognize and eliminate tumor cells that express relevant target antigens after the artificial introduction of specific tumor-associated antigens (TAAs) [15]. The variety of introduced TAAs determines the heterogeneity of CAR-T functions (shown in Fig. 1). This review describes the target antigens for PCa cells and their current clinical applications. Owing to the satisfactory therapeutic effect of CAR-T therapy in hematologic malignancy, the application of CAR-Ts in the field of solid tumors is anticipated [16]. However, compared to hematologic tumors, PCa-based CAR-T immunotherapy faces numerous limitations inherent to solid tumors, such as tumor heterogeneity and the tumor microenvironment (TME) [[17], [18], [19], [20]]. Current research on CAR-T immunotherapy in solid tumors is still primarily based on second-generation CARs and has not achieved the expected outcomes [21]. Clinical studies on the efficacy and in vivo survival time of CAR-Ts still have significant room for further exploration. Breaking through the restrictions on solid tumors has become a major hotspot in current medical research. Therefore, we review the current progress of CAR-T immunotherapy in PCa, briefly discuss the current challenges of CAR-Ts in the treatment of solid tumors, and suggest possible research directions. We also propose the possible application of multi-immunotherapy regimens and combination therapies of chemotherapy and CAR-T immunotherapy, aiming to bypass the inherent limitations of solid tumors and enhance the efficacy of CAR-T immunotherapy.

Fig. 1.

Preparation of CAR-Ts.

1.1. Literature search

We searched several databases, such as PubMed, Embase, and the National Library of Medicine (NLM), for published articles. Consequently, we collected resources pertinent to the updated novel research progress on CAR-T immunotherapy for PCa.

2. Structure, function, and classification of CAR-Ts

2.1. Structure and function

CAR-Ts essentially contain fusion proteins, whose most important component is the CAR. CAR comprises four parts: extracellular antigen recognition domain (EARD), extracellular hinge region (EHR), transmembrane domain (TD), and intracellular signaling domain (ISD) [22,23]. EARD consists of a single-chain variable fragment (scFv) derived from the light and heavy chain regions of the antibody and does not require the involvement of major histocompatibility complex (MHC) molecules to interact with TAAs [23,24]. The EHR and TD link the EARD to the ISD and are essential for the stability of the entire receptor [22,23]. The hinge region provides sufficient flexibility to overcome spatial constraints and a sufficient length to facilitate access to the target antigen [22]. The TD usually comprises cluster of differentiation (CD)3ζ, CD4, CD8, or CD28. They play important roles in T-cell activation [22,24]. The ISD is mainly responsible for signal activation and transduction and thus activates T cells. T-cell activation depends on the immunoreceptor tyrosine-based activation motif (ITAM) derived from CD3ζ; therefore, the ISD includes at least one CD3ζ-containing activation domain [[22], [23], [24], [25]]. However, therapeutic responses are very limited in first-generation CAR-Ts, where only one CD3ζ-containing activation domain is present. To increase the activation, persistence, and cytotoxicity of T cells, other co-stimulatory molecules, such as CD27, CD28, and CD137, have been added to the ISD [[22], [23], [24]]. Thus, based on the structure of the ISD, CARs have evolved into the fifth generation.

2.2. Classification

• •First (I) generation: only one CD3ζ-containing activation domain is present.
• •Second (II) generation: based on the first generation, a co-stimulatory molecule is added to the ISD. Compared with the first generation, the second-generation CAR has equal antigen specificity but enhanced T-cell proliferation, as well as cytokine and anti-apoptotic protein secretion, which delays cell death [25].
• •Third (III) generation: based on the second generation, one more co-stimulatory molecule is added; the ISD consists of two co-stimulatory molecules and CD3ζ. Its cytotoxic and anti-tumor effects are stronger than those of second-generation CARs [24,26].
• •Fourth (IV) generation: based on the second generation, instead of adding additional co-stimulatory molecules, proinflammatory cytokines, such as interleukin (IL)-2, IL-15, etc., are added to help recruit and activate immune cells, thereby enhancing the immune response [27,28].
• •Fifth-generation (V) CARs or “next-generation CARs” are also based on second-generation CARs, but they contain a truncated β-chain structural domain of the cytoplasmic IL-2 receptor with a binding site for the transcription factor STAT3. After antigen-specific activation, T-cell receptors (via the CD3ζ structural domain), co-stimulatory factors (CD28 structural domain), and JAK-STAT3/5 signaling are triggered simultaneously, which effectively provides all necessary synergistic signals that are required for T-cell activation and proliferation physiologically (shown in Fig. 2 and Table 1) [29,30].

Fig. 2.

Schematic illustration of different generations of CAR-Ts.

Table 1.

Classification of CAR-Ts.

	Intracellular signaling domain
I	Only a single CD3ζ chain.
II	CD3ζ + one additional co-stimulatory molecule, such as CD28 or 4-1BB.
III	CD3ζ + two additional co-stimulatory molecules.
IV	Based on the second generation; CD3ζ + one co-stimulatory molecule + cytokine inducer to secrete cytokines (IL-2, IL-15).
V	Based on the second generation; CD3ζ + one co-stimulatory molecule (CD28) + a truncated β-chain structural domain of the cytoplasmic IL-2 receptor with a binding site for the transcription factor STAT3.

Recently, an increasing number of CAR-Ts have been developed. Several new CAR-Ts have been derived based on second-generation CAR-Ts by altering the intra- and extracellular structural domains, including T cells redirected for universal cytokine-mediated killing (TRUCK), tandem CAR, and compound CAR-T (CCAR-T), to enhance CAR-T specific recognition [31].

The second-generation CAR-T technology is relatively mature, among which CD19-CAR-Ts have been widely studied in clinical practice. The drugs Yescarta™ and Kymriah™ have been approved for marketing by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) [32,33]. Unlike the results achieved with CAR-T immunotherapies used to treat hematologic cancer, differences in the characteristics of solid tumors, such as PCa, and hematologic malignancies dictate that the essence of the cell therapy itself, its mode of action, and some other factors must be reconsidered in the case of such a therapeutic paradigm, thus limiting the utilization of CAR-T therapies in PCa (described later in this review).

3. Application of CAR-Ts in PCa

PCa is a perfect model for oncological immunotherapy because the prostate is non-essential and has numerous TAAs as potential targets. In addition, PCa is a slowly progressive disease that provides sufficient time to generate an anti-tumor immune response [13,34]. Many antigens expressed in PCa are either restricted or not expressed in other tissues. The currently known TAAs for PCa are prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), prostate-specific antigen (PSA), prostate acid phosphatase (PAP) [35], epithelial cell adhesion molecule (EpCAM), and transient receptor potential (Trp)-p8 (shown in Table 2).

Table 2.

Current clinical trials of CAR-Ts according to target PCa TAA.

Registration ID	Engaged TAA	Phase	First posted
NCT03089203	PSMA-TGFβRDN-CAR-T	I	March 24, 2017
NCT05354375	PSMA-CAR-T	I	April 29, 2022
NCT04249947	P-PSMA-101 autologous CAR-T	I	January 31, 2020
NCT05489991	TmPSMA-02 autologous CAR-T	I/II	August 5, 2022
NCT04429451	4SCAR-PSMA-T	I/II	June 12, 2020
NCT03873805	PSCA-CAR-T	I	March 13, 2019
NCT02744287	autologous PSCA-specific CAR-Ts (BPX-601)	I/II	April 20, 2016
NCT03013712	EpCAM -CAR	I/II	January 6, 2017
NCT05022849	KLK2 CAR-T	I/II	August 26, 2021
NCT04107142	NKG2DL-targeting CAR-grafted γδ T	I	September 27, 2019

3.1. PSMA

PSMA is a type II transmembrane protein containing glutamate carboxypeptidase [36]. It is mainly expressed in five sites: prostate, kidney, salivary gland, nervous system glial cells, and small intestine jejunal brush border. However, it is overexpressed in PCa cells, and its expression increases with the Gleason score (GS) and tumor malignancy, especially in CRPC [[36], [37], [38]]. Zuccolotto et al. [39] used PC3-PIP cells to construct an experimental PCa mouse model. They found that CAR-Ts targeting PSMA survived, proliferated, and recognized PSMA-positive cells in immunodeficient mice, demonstrating their ability to resist PCa. Second-generation CAR-Ts with only PSMA have already demonstrated their potential to treat PCa. To continue exploring the tumor-killing effects of the new generation of CAR-Ts with multiple co-stimulatory molecules, Wang et al. [40] designed a set of IL23 monoclonal antibody (IL23 mAb)-PSMA-CAR-Ts, showing that IL23mAb-T2A-PSMA-CAR-Ts functioned well. Tumors were gradually eradicated, and weight was immediately restored in the experimental mice starting from day 14 after T-cell infusion. Tumor eradication was further demonstrated by reverse infusion experiments using the same model. A phase I clinical trial of PSMA-targeting TGFβ-insensitive armored CAR-Ts (NCT03089203) was used to treat mCRPC and evaluate CAR-Ts with dominant-negative TGFβ receptors [41]. These results confirmed their safety and feasibility. Recently, several clinical trials of PSMA-targeted CAR-T immunotherapies have been conducted. In addition, the PSMA-targeted radioligand 68 Ga-PSMA11 has become one of the most successful radiopharmaceuticals in positron emission tomography/computed tomography for PCa diagnosis and clinical applications [38,42].

3.2. PSCA

PSCA is a small, 123 amino acid-containing glycoprotein belonging to the Thy-1/Ly-6 family of glycosylphosphatidylinositol (GPI)-anchored cell surface antigens and is one of the most prominent prostate-specific markers [43,44]. It is overexpressed in most PCas, is highly expressed in PCa bone metastases, and increases with GS and tumor staging [[43], [44], [45], [46]]. Although the exact physiological function of PSCA-directed intracellular signal transduction remains unknown, a study suggests that it may play different physiological roles in epithelial cells of different tissues; it may be oncogenic for some epithelial cells and tumor suppressive for others [47]. PSCA is of great importance in PCa immunotherapy because of its prostate-specific properties. Priceman et al. [48] constructed CD28-PSCA-CAR and 4-1BB-PSCA-CAR. Their results suggested that compared to CD28-PSCA-CAR, 4-1BB-PSCA-CAR showed equal anti-tumor capacity but better persistence with reduced T-cell depletion. Another study found that when PSMA and PSCA were introduced into CAR-Ts to treat PCa, PSMA-PSCA-CAR-Ts only destroyed tumors positive for both PSMA and PSCA while preserving tumors that expressed either of these two antigens alone. PSMA-PSCA-CAR-Ts are expected to increase the precision of mCRPC targeting and reduce the aggressiveness of tissues expressing a single antigen alone, opening up a new strategy to treat mCRPC [49,50]. Recently, a clinical trial related to PSCA-CAR-Ts was conducted (NCT03873805).

3.3. PSA

PSA is a kallikrein-like serine protease expressed almost exclusively by prostate epithelial cells and secreted into the seminal fluid, thereby inducing specific T-cell responses [35,51]. Its main function is to hydrolyze semenogelin (Sg) and thus release semen from the semen coagulum [52]. The prostate specificity of PSA makes it the target of choice for CAR-T therapy. Following the destruction of prostate tissue, regardless of the cause, PSA secretion is achieved by cytosolic emesis and diffuses from luminal cells via their epithelial basement membranes and prostatic mesenchyme to the serum, where it is detected [53]. However, PSA is not only found in the prostate but also in other organs outside the prostate, such as the breast, colon, and pancreas, and elevated PSA levels can also be seen in breast or colorectal cancer [54,55]. Therefore, PSA is not absolutely cancer-specific. Hence, the mechanism by which PSA-CAR-Ts attack only cancer cells instead of normal cells must be elucidated. It has been shown that PSA precursor (Precursor PSA, proPSA) and its derivatives can better distinguish PCa from non-PCa [56]; therefore, they have the potential to become new CAR-T targets for PCa.

3.4. PAP

PAP, a member of the acid phosphatase superfamily, is secreted by benign and malignant prostate cells and is highly expressed in prostate tumors with a GS of 6 or 7 [35,57]. Quiroz-Munoz et al. [58] found elevated PAP activity during PCa bone metastases. It has been reported that PAP is relatively more accurate in determining prostate micrometastases than PSA and could be used as a CAR-T therapy for CRPC [59]. However, in the early stage of PCa, PAP is specific to some extent, but as PCa progresses, PAP expression gradually disappears, with its specificity reduced, thus losing its ability as a PCa-specific tumor marker [[60], [61], [62]]. Some carcinoid tumors and pancreatic islet cell tumors also express small amounts of PAP [63], so the specificity of PAP as a target for CAR-T treatment of PCa is poor. However, there is still room for research on targets to treat multiple systemic metastases.

3.5. EpCAM

EpCAM (CD326) is a single transmembrane structural domain-containing component, first known as a humoral antigen expressed on colon cancer cells in 1979 [64,65]. EpCAM is highly expressed in rapidly proliferating epithelial or epithelial-derived tumor tissues. In primary PCa, higher EpCAM expression indicates a poorer prognosis [[66], [67], [68]]. Therefore, EpCAM can be an alternative target antigen for PCa, especially in advanced metastatic prostate tumors. Yang et al. [69] used EpCAM as a target antigen to construct EpCAM and ICAM-1 bispecific CAR-Ts and showed that EpCAM made cancer cells more susceptible to attack by ICAM-1-EpCAM-CAR-Ts. A clinical trial of EpCAM-CAR is currently underway (NCT03013712).

3.6. Trp-p8

Trp-p8 is a specific gene expressed mainly in prostate epithelial cells but also in other malignant tumors [70]. Zhang et al. [71] found that combining PSA density (PSA-D) with Trp-p8 could be used for the early detection of PCa. Kiessling et al. [72] studied HLA-A*0201-restricted T-cell expression of Trp-p8. Trp-p8 mRNA was expressed in all types of PCa and healthy prostate tissues, indicating that Trp-p8 might be a suitable target for PCa therapy.

4. Challenges of CAR-T therapy in the management of PCa

The highest hurdle for CAR-T immunotherapy in PCa is simultaneously achieving safety and efficacy. The occurrence of various adverse events like “on-target off-tumor effects” or “cytokine storm” greatly reduces the safety of treatment, while the efficacy of CAR-T therapies is influenced by the antigen affinity, in vivo survival time, homing ability, degree of tumor infiltration, and TME [73,74].

4.1. Tumor heterogeneity

Tumors exhibit striking heterogeneity in many morphological and physiological features, allowing them to exhibit pluripotency in cell surface receptor expression, proliferation, metabolism, and angiogenesis [75]. Precise microdissection revealed that phenotypically similar tumor lesions could have different genotypes, suggesting the heterogeneity of tumor genotypes [76]. Thus, the above-mentioned multiple reasons allow tumors to curtail immune system attacks in several ways. In addition to heterogeneity between tumor types, there is heterogeneity within the same tumor between cells [77]. The lack of a tumor-specific antigen (TSA), which is expressed only in cancer cells instead of normal cells [78], is one of the most direct manifestations of tumor heterogeneity. The TAAs used as targets for PCa immunotherapy described above are not unique to tumor tissues but are also expressed to a small extent in normal tissues, resulting in CAR-T attack regardless of malignant or healthy tissues, which leads to damage to the body, thus causing on-target off-tumor effects [79]. A strategy to improve safety is to enhance the recognition specificity of CAR-Ts so that CAR-Ts start working only after recognizing two different antigens to reduce the possibility of attacking normal tissues [50,80]. In contrast, another strategy is to reduce the attack on normal tissues with low antigen expression by adjusting the immunocidal activity, such as reducing CAR-T affinity to target cells or developing inhibitory CARs (iCARs) [80]. Theoretically, although the former is more capable of tumor clearance, the dual-target feature inevitably leads to a limited selection of target antigens; the latter has a reduced attack capability, which broadens the choice of tumor antigens but weakens the efficacy [81]. Therefore, finding a more precise, safe, and effective TSA and TAA would greatly increase the therapeutic effectiveness of CAR-Ts.

4.2. Cytokine release syndrome (CRS)

CRS is a prominent adverse event that affects nearly 67% of patients after treatment [76]. CRS marks the beginning of the immune response, resulting in the elevation of multiple cytokines, such as IL-10, IL-6, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF) [[82], [83], [84]]. The clinical manifestations of CRS vary, including high fever, nausea, headache, fatigue, cramps, myalgia, acute respiratory distress syndrome (ARDS), hypotension, capillary leak syndrome (CLS), tachycardia, liver failure, renal impairment, and disseminated intravascular coagulation (DIC) are commonly seen and the severity is highly correlated with cytokine levels [82,83]. In treating CRS, corticosteroids suppress the strong inflammatory response without diminishing CAR-T anti-tumor effectiveness, which is the main therapeutic strategy in cases of CRS caused by CAR-T therapy [[82], [83], [84], [85]]. For patients with glucocorticoid-resistant CRS, researchers have shown that combining etanercept (TNF inhibitor) and tocilizumab (IL-6 receptor blocker) can rapidly reduce clinical symptoms and has been commonly used in the treatment of CRS [85,86].

4.3. Tumor infiltration

Solid tumors, such as prostate cancer, are in a more complex environment than hematologic cancers, making it more difficult for CAR-Ts to infiltrate the tumor [87]. In addition, solid tumors lack many mature blood vessels [88], including high endothelial venules (HEV) and their cancer-associated fibroblasts (CAF), and other cells, such as vascular endothelial growth factor (VEGF), which are more sensitive to hypoxia and acidity in the TME and generate tumor blood vessels different from normal blood vessels [89]. They are irregular in structure and concentrated at the tumor margins, forming a vascular barrier that makes it difficult for CAR-Ts to enter the tumor [89,90]. The physical matrix barrier of the TME is formed by connective tissue and extracellular matrix (ECM) containing collagen, proteoglycans, fibronectin, and non-structural matrix proteins [91]. On the one hand, it restricts the entry of CAR-Ts; on the other hand, it promotes the proliferation and migration of cancer cells [91,92]. Therefore, developing CAR-Ts that can degrade ECM will sufficiently improve solid tumor infiltration.

4.4. Homing ability

Localizing CAR-Ts to cancer cells and activating their proliferation are crucial for CAR-T immunotherapy [87,93]. The majority of CAR-Ts are administered intravenously, so only migration of CAR-Ts from the peripheral blood to the tumor site is a prerequisite for their anti-tumor effect [25]. Chemokines and their receptors mediate the targeted migration of T cells, greatly contributing to immune-mediated reactions. CXCR3 is a chemokine receptor mainly expressed in CD4⁺ and CD8⁺ T cells [94,95]. However, cancer cells rarely secrete ligands for CXCR3, thus resulting in the inability of CXCR3-expressing CD8⁺-CAR-Ts to match with cancer cells [96] because cancer cells release chemokines, such as CXCLl5, T cells lack receptors for the relevant chemokines on their surface and therefore fail to recognize tumors [97]. These two reasons lead to tumors not being localized, together with the tumor ECM barrier described in Section 3.3, causing limited CAR-T migration and homing ability. Therefore, modifying CAR-Ts according to the chemokines released by different tumors to express receptors for these chemokines will substantially enhance their migration and homing abilities, thus improving tumor infiltration, and enhancing efficacy.

4.5. Immunosuppressive nature of the TME

The TME is a highly acidic, hypoxic, and glucose- and nutrient-deficient immunosuppressive environment that favors the production of helper T cell (Th) type 2 cytokine responses and is not conducive to the production of Th1 cytokines, such as IFN-γ and TNF-β [[98], [99], [100]]. Therefore, anti-tumor effector molecules, such as IFN-γ and TNF-β, are produced to a lower extent [100], limiting the anti-tumor effectiveness of CAR-T immunotherapy and fostering tumor cell growth and metastasis. In addition to the vascular and ECM barriers mentioned in Section 3.3, the main factors affecting their immunosuppressive properties include the following (shown in Fig. 3).

Fig. 3.

Immunosuppressive nature of solid tumors: Regulatory T cells (Tregs) contribute to immune homeostasis by curbing the amplified immune response, mediating immunological tolerance. Treg cells extend their affinity for IL-2 cells via their IL-2 receptors by suppressing T effector cells (CD4⁺, CD8⁺ T cells) and arresting their proliferation and release of other mediators, such as interleukins and interferons. Treg cells also express CD152 and IL-10, which deactivate T effector cells by downregulating CD80/CD60 in antigen-presenting cells. Due to this Treg cell-driven immune defense, the TME maneuvers to combat these anti-tumor immune responses. In the TME, monocytic myeloid-derived suppressor cells rapidly differentiate into tumor-associated macrophages, which in turn diminish the anti-tumor response by promoting factors such as VEGF, epidermal growth factors, transforming growth factor-β, and matrix metallopeptidase 9. In addition, CD-8 T cells induce PD-1, an immune checkpoint receptor that inhibits T-cell activation by binding to PD-L 1 and bypassing anti-tumor defense.

4.5.1. Suppressive immune cells

Tregs maintain self-tolerance and immune homeostasis via their suppressive function to prevent excessive immune activation and consequent self-tissue damage [101]. However, in the TME, Treg cells take up IL-2 from the periphery through their high-affinity IL-2 receptors, resulting in the inability of IL-2 to act on effector T cells (Teffs) [102,103]. Therefore, the Teffs cannot proliferate, differentiate, and release lymphokines, such as interleukins and interferons, leading to a decreased immune effect. In addition, they constitutively express CD152, which downregulates CD80/CD86 expression in antigen-presenting cells (APCs), thereby inhibiting Teff activation. Treg cells also produce immunosuppressive cytokines, such as IL-10, downregulating APC function [[102], [103], [104]]. Therefore, Tregs can suppress the anti-tumor response, thus promoting the development of TME, leading to cancer progression, depleting the anti-tumor immunity of CAR-Ts, and aiding tumor immune escape [101,105]. Monocytic myeloid-derived suppressor cells (M-MDSCs) flow via blood circulation from the bone marrow to solid tumors [105,106]. In the TME, due to hypoxia, M-MDSCs rapidly differentiate into tumor-associated macrophages (TAM), which maintain tumor growth by producing growth factors (e.g., VEGF, epidermal growth factor (EGF)), and promote extracellular matrix remodeling by releasing proteases and soluble mediators and matrix metallopeptidase 9 (MMP-9) or other soluble factors (e.g., VEGF, CXCL8) to provoke angiogenesis and lymphangiogenesis and releasing soluble mediators to suppress the anti-tumor immune response [[105], [106], [107]].

4.5.2. Immunosuppressive molecules

The TME promotes the expression of receptors for immunosuppressive molecules [108] such as prostaglandin E2 (PGE2), adenosine, IDO, transforming growth factor (TGF)-β, and IL-10 on T cells, resulting in the suppression of T cell activation. Tumors and macrophages promote PGE2 production by secreting cyclooxygenase-2 (COX-2), and the hypoxic environment is prone to adenosine production [105,108]. Subsequently, PGE2 and adenosine activate the protein kinase A (PKA) pathway, resulting in immunosuppression. TGF-β not only promotes tumor matrix production and tumor metastasis but also causes the immune balance of Th1/Th2 cells to shift to Th2 cells and directly inhibits the function of Teff [105,[109], [110], [111]]. PD-1 is a well-known immune checkpoint receptor often induced by activated CD8⁺ T cells and Tregs in the TME, which inhibits T-cell activation [112,113]. PD-1 expression further leads to the inhibition of activation and the proliferation of T cells by binding to programmed death ligand 1(PD-L1) on cancer cell surfaces, promoting the immune escape of tumors [113,114].

As mentioned above, the TME is one of the biggest obstacles to CAR-T treatment of solid tumors such as PCa. The modification of CAR-Ts by genetic engineering methods to express various inhibitory immune molecule receptors to cope with the inflammatory environment of the TME so that CAR-Ts can maintain long-lasting activity in the TME has become a hot research topic.

5. Combination therapy with CAR-T immunotherapy

Despite the advances that have been made in the field of cancer treatment, there are still uncertainties regarding the efficacy of single-agent therapy. The pathogenesis of PCa results from multiple etiological factors; hence, completely curing tumors using a single treatment method is unfeasible. Therefore, a combination of multimodal therapies is needed.

5.1. Immune checkpoint inhibitor (ICPI) + CAR-Ts

The immune checkpoint (ICP) is an immunosuppressive pathway of immune cells and is essential for maintaining autoimmune tolerance and regulating immune responses in the peripheral tissues [115]. The expression of ICP and its ligands allows cancer cells to escape host immune surveillance, where they further inhibit the anti-tumor properties of immune cells, thereby promoting tumorigenesis [116,117]. Therefore, the production of monoclonal antibodies targeting ICP to conduct antibody-mediated blockade against the expression of ICPs on T cells is important to improve anti-tumor efficiency. PD-1 and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) have been widely studied [118,119].

PD-1 ligand is well expressed in various cancer cells, including PCa [120], and almost 90% of prostate CD8⁺ T cells are PD-1 positive [121]. In addition to the mechanism of PD-1 action in the TME described in Section 3.5.2, PD-L1 expression is up-regulated when T cells attack cancer cells [122]. Rafiq S et al. [123] designed CAR-Ts to target the delivery of PD-1 blocking scFv and protect PD-1 signaling molecules from the immunosuppressive effect of cancer cells, thereby improving the anti-tumor activity of CAR-Ts. The results demonstrated that PD-l scFv-CAR-Ts could prolong the survival period of experimental mice and that there were long-term surviving CAR-Ts in mice, which could initiate an anti-tumor immune response during cancer recurrence. Therefore, blocking PD-1 or its receptor can prevent the activity of T cells from being suppressed and enhance their immunocidal ability. CAR-T therapy with tandem PD-1scFv may be a safer and more effective treatment option than mono-ICPI immunotherapy. Meanwhile, a related study showed that PD-L1 blockade normalized the CD4⁺/CD8⁺ cell ratio and restored the cytotoxicity of CD8⁺ T cells [124]. Therefore, the application of CD8⁺-PDL1-CAR-T therapy is highly possible. PD-1 and CTLA-4 also mediate the immune cell suppression pathway. In preclinical treatment models, combining anti-PD1 and anti-CTLA4 eliminated these PD-1/CTLA4-mediated inhibitory effects and enhanced CAR-T function [122,125]. Thus, ICPI shelters the anti-tumor capacities of T cells from cancer cell attack and enhances T-cell cytotoxicity. With ICPI and CAR-Ts, tumor-specific recognition ability is maintained, and the survival time of CAR-Ts is greatly increased, thereby substantially enhancing the anti-tumor efficacy.

5.2. Chemotherapy + CAR-Ts

Chemotherapy suppresses the activity of the immune system [126]. The core of CAR-T immunotherapy is to activate the immune system after implanting artificially-modified T cells, thereby enhancing its ability to kill cancer cells. The two mechanisms of action appear to be opposite, but chemotherapy leads to an increase in antigen presentation, which causes an immune response conducted by the immune system to attack cancer cells [127,128]. Chemotherapeutic drugs may drastically reduce the number of Tregs and activate cytotoxic T lymphocytes, thus restoring immune activity and allowing the immune system to exert its anti-tumor activity [129]. Although the exact mechanism of action is not completely understood, chemotherapy combined with CAR-T immunotherapy offers a new therapeutic strategy for advanced metastatic PCa. Currently, there are no clinical trials of CAR-T immunotherapy combined with chemotherapy, but clinical trials with PD-1 blockers combined with docetaxel are underway (NCT02861573, NCT03834506, and NCT03338790). We hope that combining ICPI-expressed CAR-T immunotherapy and chemotherapy will facilitate the treatment of advanced PCa metastasis.

5.3. Radiotherapy + CAR-Ts

Radiotherapy plays a vital role in the treatment of PCa by precisely targeting the tumor, developing the radiotherapy plan, and assessing the effectiveness of the treatment using a complex array of multimodal medical imaging techniques [130]. The fundamental principle of radiotherapy is to directly or indirectly induce intracellular DNA damage, thereby destroying tumor cells through various forms of radiation exposure [131,132]. Furthermore, this procedure stimulates the release of TAAs and energizes the immune cells, thereby creating a favorable TME that facilitates the optimal functioning of CAR-Ts [133]. Therefore, combining radiotherapy with CAR-T immunotherapy has the potential for significant benefits [134,135], as both approaches can synergistically eliminate PCa cells through both intracorporeal and extracorporeal interventions. However, the function and stability of CAR-Ts following radiation exposure remain uncertain, which may ultimately result in reduced effectiveness of CAR-T therapy [136]. Hence, achieving successful combinations of radiotherapy and CAR-T immunotherapy may necessitate the modification of standard radiotherapy regimens and target volumes to optimally maintain immune fitness and enhance antitumor immune responses for meaningful clinical benefit. Several ongoing clinical studies are currently underway to evaluate and refine meticulous radiotherapy planning and dosage delivery strategies for optimal outcomes [135,[137], [138], [139]].

6. Conclusion

CAR-T immunotherapy for PCa remains promising, with challenges and opportunities in terms of modifying CAR-Ts or combining them with other existing treatment modalities, especially for advanced metastatic cancer. Split, universal, and programmable (SUPRA) CAR-Ts were developed using a leucine zipper as a junction system, referred to as the SUPRA CAR system, which could improve the safety and efficacy of immunotherapy [140]. The BiTE (bispecific T-cell engager), which modifies T cells to produce bispecific antibodies, is a typical example of a relatively successful novel CAR-T model. BiTEs produce direct anti-tumor effects and simultaneously release antibodies to activate their immune function further and reduce immune escape, thus exerting a more potent anti-tumor effect [141]. Several nanobodies or VHHs (single variable domain on a heavy chain) have been designed against relevant PCa antigens. VHH-based CAR-Ts are believed to have the potential to target any TAA for adoptive CAR-T immunotherapy of solid tumors [142]. Moreover, recent advancements in nano-drug delivery systems have offered a novel approach to solid tumor management. Nanomedicine, with its distinctive benefits, holds the potential for targeted drug delivery and the ability to modify physicochemical, pharmacokinetic, and pharmacodynamic properties on demand which encompass aspects such as solubility, stability, and circulation time in the blood, thereby enabling precise control over drug release and transportation, ultimately enhancing efficacy [143–145]. Consequently, by utilizing nanomedicine as a carrier for CAR-T and other therapeutic agents like androgen deprivation therapy (ADT) and chemotherapeutic medicines, we can expect enhanced effectiveness. PCa is a typical epithelial adenocarcinoma; therefore, insights for developing immunotherapeutic approaches for PCa also apply to other types of epithelial cancer. The combined CAR-T immunotherapy + ICPI + chemotherapy + radiotherapy strategy will also bring unlimited possibilities for mCRPC, and better multimodality treatments will emerge.

7. Statements

Author contribution statement

All authors listed have significantly contributed to the development and the writing of this article.

Data availability statement

No data was used for the research described in the article.

Declaration of Competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article.