Immunotherapy has made revolutionary progress in cancer treatments and has become mainstream in clinical trials. Specifically, the adoptive transfer of immune cells immunotherapy, for instance, chimeric antigen receptor (CAR)-T cells has brought a new dawn for patients who suffered hematological malignancies. Different from other therapeutics, CAR-T activates in advance to recognize the specific tumor cells in vivo, avoiding the complex immune activation pathway, which makes this approach more efficient (Chen and Mellman, 2013). Hence, genetically modified T-cells expressing CARs present one of the most prominent ways of cellular immunotherapy, which activate immune cells to exert anti-tumor effects through the specific recognition of tumor-specific antigens by antibodies as well as the downstream signaling of T-cells (June et al., 2018). Due to their prominent therapeutic effects in many hematological malignancies, the US Food and Drug Administration (FDA) has approved six kinds of drugs for B-cell maturation antigen (BCMA) hitherto (Schuster et al., 2017; Abramson et al., 2020; Raychaudhuri et al., 2020; Khurana et al., 2021; Si Lim et al., 2021; Ying et al., 2021). In stark contrast, in patients with solid tumors, CAR-T therapy has not been shown to be effective (Narayan et al., 2022; Patel et al., 2022). Plenty of research has explored the mechanism, in which tumor microenvironment (TME) immunosuppressive properties and poor infiltration of T lymphocytes into the tumor joint harmed the effectiveness of treatment for solid tumors (Kumar Vodnala and Restifo, 2019; Elia and Haigis, 2021; Huang et al., 2021; Kim et al., 2022). Additionally, the limited durability of CAR-T, inefficient response rate caused by antigen escape, cytokine release syndrome (CRS), and neurotoxicity, as well as tedious and complex preparation processes, limit their clinical applications in a variety of malignant tumors (Hou et al., 2021; Marofi et al., 2021; The Lancet Oncology, 2021; Kyte, 2022). To overcome the diminished efficacy against solid tumors, introduction CARs into other innate immune cells provides revolutionary innovation in cancer immunotherapy. Among them, macrophages provide an ideal alternative and exhibited promising potency for CAR-modified strategy against solid tumors.

Macrophages are highly plasticity and polarization, and they play a double-edged sword in cancer therapy (Locati et al., 2020). First, macrophages can infiltrate tumors more efficiently than other immune cells, resulting in their highest proportion (more than 50%) in TME (Wagner et al., 2019). In addition, the growing evidence indicates that the maintenance of the treatment-resistant microenvironment to support the development and metastasis of tumors are closely related to macrophages, particularly TAM, in many malignancies tumor-bearing mice models and clinical trials, such as breast cancer, lung cancer, ovarian cancer, colorectal cancer, glioblastoma, and other cancers (Wenes et al., 2016; Halbrook et al., 2019; Lin et al., 2019; Singhal et al., 2019; Yin et al., 2019; Buonfiglioli and Hambardzumyan, 2021; Lu et al., 2021; Huang et al., 2022). More importantly, as part of the innate immune system, macrophages play a central role as effectors and regulators, and the activated macrophages present phagocytosis, cytotoxicity, as well as the function of secreting pro-inflammatory factors and presenting antigen to T-cells, which have great potential in activating anti-tumor innate immune and adaptive immune responses via immune cascade response (DeNardo and Ruffell, 2019; Mantovani et al., 2022). Therefore, these above-mentioned characteristics and inherent advantages of macrophages seem to be better suited for genetic modification to circumvent the barriers faced by CAR-T. Recently, the CAR-M strategy has also attracted extensive attention both in the laboratory and in the industry, and research reports on the feasibility of the CAR-M technology, safety, and therapeutic effect were preliminarily explored. In these existing explorations, researchers have placed significant emphasis on the design of CAR structure, the selection of donor cell sources, and the vector delivery of CAR genes into macrophages. Simultaneously, the CAR-M treatment strategy has evolved from in vitro modification of macrophages to the direct in vivo administration of gene reagents for in situ macrophage modification. In the development of CAR-M therapy, the gene delivery vector has emerged as a critical concern that restricts the effectiveness and applicability of this strategy. Due to outstanding advantages in transduction efficiency, viral vectors have become the most widely used in the field of cell therapy (Bin Umair et al., 2022). Various viral vectors, including adenoviral vectors and lentiviral vectors, have been applied to CAR-M therapy. However, the high cost and safety problems of viral vectors interfered with the development of CAR-M therapy. Differently, nonviral vectors represented by nanobiomaterials become an attractive alternative due to the advantages in the simplicity of the production process, ease of large-scale production, and lack of specific immune response (Ramamoorth and Narvekar, 2015; Zu and Gao, 2021). At present, with the above unique characteristics and advantages, a variety of nanobiomaterials including LNP formulations, cationic polymers, and biocompatible hydrogels have been employed in the development of CAR-M therapy. These materials serve as alternatives to viral vectors for delivering CAR genes and exhibit great potential in the context of in vivo CAR-M therapy strategies (Figure 1). In this review, we elaborate the CAR-M therapy’s research progress and clinical attempts and discuss ongoing innovations in this approach to enhance clinical efficacy in solid tumors, including the CAR structures design, the cellular origin of macrophages, and the use of novel nanometer biomaterials, as well as the challenges and opportunities of CAR-M-based immune cell therapy that have emerged in clinical and preclinical studies.

Macrophages are a diverse and intricate group of cells infiltration the tumor microenvironment (Wagner et al., 2019; Christofides et al., 2022). Based on the classical paradigms of M1 and M2 phenotypes with pro-inflammatory and anti-inflammatory properties, the mainstream anti-tumor therapy for TAM mainly focuses on the strategies for depletion (Mazzieri et al., 2011; Germano et al., 2013), remodeling TAM including polarization or reactivation (Hoves et al., 2018; Eisinger et al., 2020; Yang et al., 2020; Anfray et al., 2021; Sun et al., 2021; Nie et al., 2022), and inhibition of monocyte or macrophage recruitment into tumors (Qian et al., 2011; Mantovani et al., 2017; Cassetta and Pollard, 2018). Recently, a large number of clinically developed drugs mainly focus on the targets CSF-1R, CD47, CD40, and chemokines (Beatty et al., 2011; Yanagita et al., 2017; Sikic et al., 2019; Sylvestre et al., 2020). However, restricted by the high plasticity of macrophages and the lack of specificity of tumor-associated antigens, these single therapies for solid tumors have only achieved limited therapeutic effects with substantial off-target toxicity. Hence, various combination therapy strategies based on macrophages have been extensively developed. Wang et al. designed a sonoimmunity-engineered nanoplatform to reverse the physical microenvironment of the tumor by producing a substantial amount of reactive oxygen species (ROS) through the sonodynamic processes (Wang et al., 2022a). The substantial accumulation of ROS within tumors induces the polarization of TAMs towards the pro-inflammatory M1 subtype, thereby amplifying the therapeutic efficacy of immune checkpoint blockade and CAR-T therapy against solid tumors with metastasis. A similar strategy was reported by Powell Jr.’s Group (Rodriguez-Garcia et al., 2021), and they proved the selective elimination of FRβ+ TAMs by CAR-T cells enriched pro-inflammatory monocytes and stimulated tumor-specific CD8⁺ T-cells to infiltrate in the TME, eventually syngeneic tumor-bearing mice have delayed tumor progression and prolonged survival rate. These paradigms that change one point yet benefit the whole provide a rationale for using CAR-M in patients with solid cancers. Different from the traditional TAM-targeted treatments, using chimeric antigen receptor expression on the macrophage surface is a whole new attempt. This method hypothesized that macrophages expressing the chimeric antigen receptor could simultaneously boost phagocytosis, change M2-to-M1 phenotypes, and activate adaptive immunity (Figure 2) (Morrissey et al., 2018; Klichinsky et al., 2020; Kang et al., 2021; Niu et al., 2021). Since Morrissey et al. verified the phagocytosis of macrophages affected by various CAR structures (Morrissey et al., 2018), more and more studies confirmed this innovative concept. Interestingly, CAR-T which acts directly on macrophages rather than cancer cells also achieved prominent therapeutic effects on breast cancer and colorectal cancer models simultaneously. For instance, Chen et al. delivered the CD47 blocker SIRPα-Fc by CAR-T cells enhancement antitumor efficacy (Chen et al., 2022b), while Achkova et al. (2022) designed CAR-T-cells that contain M-CSF and IL-34 to mediate selective remodeling of macrophage cells that express enforced or endogenous M-CSFR. Also the synchronous secretion of cytokines including IL-2 and interferon (IFN)-γ suppressed tumor cell progression. Comprehensively, CAR-M technology might represent a novel therapeutic approach for manipulating the TAM to become an M1 phenotype, enhance the activity of phagocytosis or antigen presentation to further induce the immune response, and sensitize other common treatments, which hold potential against solid tumors.

Clinical translations of CAR-M therapies are still in the initial exploratory stages (Table 1), and until now, only two clinical trials have been disclosed on CAR-M therapy (NCT05007379 and NCT04660929), but yet still lack therapeutic outcomes. For instance, MaxCyte has developed an MCY-M11 that utilizes mRNA-transfected peripheral blood monocytes (PBMCs), the precursors of macrophages, for the production of CAR-M cells that target mesothelin-expressing cancer cells. This CAR therapy provides transient expression without the necessity of viral vectors or cell amplification via the utilization of MaxCyte GT System™, a cGMP-compliant electroporation system. This system enables rapid manufacturing and timely administration to the patient (Hung et al., 2018). Currently, a phase I clinical trial (NCT03608618) is underway to evaluate the efficacy of this treatment for advanced ovarian cancer and peritoneal mesothelioma. The drug candidate CT-0508, from CARISMA Therapeutics, is designed for the treatment of patients with relapsed/refractory HER2 overexpression (NCT04660929). This study recruited 18 patients with HER2-positive solid tumors for the first time to investigate the effects of CAR-M transduced by adenovirus. Recently, Carisma Therapeutics presented data from the phase I clinical trial of CT-0508 in patients with HER2-positive solid tumors at the 2023 American Society of Gene and Cell Therapy (ASGCT). The results highlighted the preliminary outcomes of CT-0508 in 18 patients with locally advanced (unresectable) or metastatic HER2 overexpressing solid tumors, focusing on aspects such as safety, tolerability, feasibility of cell manufacturing, transportation, and activation of the TME. However, in this clinical trial, patients need to be injected with hundreds of millions of engineered macrophages at a time, and considering the difficulty of in vitro expansion of macrophages discussed earlier, the application prospects of CT-0508 are undoubtedly challenging. It is worth noting that several companies have designed CAR-M pipelines in advance according to different molecular targets, cell sources, and transduction vectors. These ongoing results present the possibility of the advancement of CAR-M immunotherapeutic modalities for the treatment of solid tumors.

As an essential function in the innate immune system and adaptive immune system, macrophages have shown great potential for synergistic therapy in solid tumors. The emerging cellular immunotherapy approach, CAR-M, provides a novel and promising solution in cancer immunotherapy. Importantly, the nanobiomaterials-based gene transfer vector solved the technical barriers to achieving CAR-M therapy in situ, in addition to cost and safety. Although this paradigm has been validated in several tumor-bearing mice models, many challenges still need to be overcome before clinical applications. First, the efficiency of gene reprogramming determines the therapeutic effect of CAR-M, so the nanobiomaterials maybe need to provide efficient target cell transfection. Taking in vitro viral infection as an example, compared with T-cell infection, the use of viral vectors to infect macrophages is extremely inefficient, which means that a larger dose of virus is required to achieve the expected therapeutic effect, which greatly increases the cost of treatment. Second, the pharmacokinetics behavior of biomaterials gene vector in vivo is also worth considering, especially in vivo CAR-M protocol, after systemic administration, most of the exogenous vehicles will be distributed in the liver, failing to reach the tumor lesion and affecting the treatment effect. Third, solid tumor-specific targets determine the safety of CAR-cell immunotherapy in vivo. Just as in CAR-T therapy, if the specificity of the selected target is not high enough, then it may bring very serious toxic side effects, and the tumor cells with lower target antigen expression may also evade the pursuit of CAR-M and cause tumor recurrence. Therefore, the rational design of delivery vehicles for efficient targeting of special cells poses a great challenge in this field again. Last, the complex microenvironment of solid tumors may impair the therapeutic efficacy of CAR-M. Although macrophages have a stronger tumor infiltration capacity than T-cells, and CAR-M has shown promising results in mice tumor models in preclinical studies, considering that human tumor microenvironments are much more complex than those of animal models, so how to improve the infiltration of CAR-M in tumors will also be a problem to achieve therapeutic effects. The in situ reprogramming of macrophages avoids complex isolation and reprogramming operations in vitro to a certain extent, is not affected by the migration distribution of CAR-M, and has a certain degree of advanced application, but still faces the problem of in vivo delivery of reprogrammed genes. For a virus-depend delivery protocol to be adopted, the viral vector must have excellent in vivo safety and be able to accurately target macrophages to reprogram them when entering the complex physiological environment. For non-viral vector delivery, under the premise of solving the same challenges as viral vectors, the transfection efficiency and the potential cross-reaction between target cells and tumor microenvironment triggered by nanobiomaterials remain must be improved to meet the requirement of treatment.

After solving the above-mentioned challenges, the optimized CAR-M technology platform is expected to truly become a “sharp blade” for solid tumor treatment. Significantly, the development of the future CAR-M technology platform cannot simply follow the old route of CAR-T technology iteration. The structure of the CAR is originally designed to activate the function of T-cells, theoretically, the novel and efficient CAR molecules should be explored to activate the function of macrophages. Because the function of macrophages in maintaining homeostasis is more complex than that of T-cells and NK cells, the future application of the CAR-M technology platform may not be limited to a simple cell or reprogramming factor infusion. These special functions provide a new focus for applying a new generation of CAR-M technology. In addition, CAR-M has great potential in modifying the solid TME, which provides the possibility of combining CAR-M therapy with other therapies. Recently, benefiting from the rapid development of nanotechnology in cancer theranostics, the possibility of using nanobiomaterials vehicles to achieve CAR-M in situ in an efficient, economical, and specific manner against solid tumors is desired.