Targeting macrophages in hematological malignancies: recent advances and future directions

Sydney R. Gordon et al.’s 2017 study documented a new role of the PD-1/PD-L1 signal pathway in modulating the phagocytic capacity of TAMs [43]. This study suggests that the PD-1/PD-L1 signaling pathway in macrophages could also function as the “don’t eat me” signal. They exhibited that PD-1 was expressed in mouse and human TAMs. Furthermore, PD-1 expression levels increased through cancer progression in mice models and high TNM staging in primary human malignancies [43]. PD-1⁺ TAMs exhibit an M2-like macrophage phenotype and display a considerably reduced phagocytic potency against cancer cells. However, inhibition of the cross talk between PD-1 and PD-L1, either via PD-1 or PD-L1 blockade, results in an anticancer immune response in mice that lack T cells, B cells, and NK cells but have functional macrophages [43]. The phagocytic function of PD-1⁺ TAMs is rescued in PD-L1^−/− mice in vivo. However, there is no significant difference in phagocytosis of PD-1⁻ TAMs between PD-L1-expressing and knockout tumor cells [43]. These findings strongly suggest that PD-L1-expressing cancer cells can evade TAM-mediated phagocytosis. Previous investigations have shown that PD-1 expression inhibits a wide variety of immune cells in the TME, consisting of T cells [128], B cells [129], NK cells [130], and DCs [131]. Sydney R Gordon et al. extended their study to include macrophages, further emphasizing the essential function of PD-1 expression in immune system balance across the innate and adaptive immune systems [43].

Anti-PD-1/PD-L1 treatments have been explored in various hematological cancers, but there is still a need to establish why blocking the PD-1/PD-L1 axis demonstrates clinical efficacy only in a minority of cancer types [1, 5, 132]. The influences of PD-1 blockade on TAMs in human cancer should not be ignored as it may aid the search for new disease biomarkers or therapeutic combinations. For example, PD-1 blockade exhibited high clinical responses in HL patients despite the heterogeneous expression of PD-1 on tumor-infiltrating lymphocytes (TILs). Frequently, PD-1 blockade compromised class I major histocompatibility complex (MHC-I) surface expression on tumor cells, which potentially precludes the T cell-mediated anti-tumor immune response mechanism [132]. This inconsistency in the PD-1/PD-L1 blockade could partially be explained by the TAMs-mediated anti-tumor immune effect on HL [132]. These findings indicate that adaptive and innate immune checkpoints need further investigation. The findings also suggest that the study of innate and adaptive immunity can provide a novel understanding of the heterogeneity of clinical responses to immune checkpoint inhibitors.

MHC-I expressed by tumor cells also inhibits macrophage-mediated phagocytosis [44]. MHC-I comprises HLA α-chains and β₂-microglobulin (β₂-M) chains [133]. Some tumor cells highly express the MHC-I complex to inhibit macrophage-mediated phagocytosis [44]. To better understand the process of MHC-I-induced suppression of phagocytosis, researchers have identified the receptors utilized in detecting MHC-I. The study has reported that two leukocyte immunoglobulin-like receptor (LILR) family members, LILRB1 and LILRB2, couple with MHC-I and harbor immunoreceptor tyrosine-based inhibitory motifs (ITIMs), aiding in the inhibition of intracellular signal transduction [44]. LILRB1 and LILRB2 are considered candidates for the MHC-I-modulated inhibition of phagocytosis. An analysis of human monocytes showed that LILRB1 was expressed in most monocytes, while only a small subpopulation of LILRB2⁺ was detected [44]. TAMs in several tumors highly express LILRB1 but not LILRB2. Treatment with LILRB1 mAb has been shown to significantly increase the anti-CD47-triggered phagocytosis of MHC-I⁺ cells but not MHC-I⁻ cells [44]. In contrast, anti-LILRB2 mAb have no considerable impact on phagocytosis of either MHC-I⁺ cells or MHC-I⁻ cells [44]. The MHC-I/LILRB1 signaling axis is a “don’t eat me” signaling pathway, and inhibition of either LILRB1 or MHC-I may greatly enhance tumor cell phagocytosis. Although MHC-I or LILRB1 blockade promotes macrophage-mediated phagocytosis, it does not extensively inhibit tumor growth in immunocompetent mice, indicating some of the limitations of this therapy.

Previous studies have shown that blocking LILRB2 using therapeutic antibodies can boost macrophages’ maturation and strengthen their pro-inflammatory phenotype [46]. Additionally, LILRB2 blockade combined with anti-PD-L1 mAb enhances the phagocytosis of TAMs and increases their anti-tumor effects in transgenic mice, which can express human LILRB2 on myeloid cells [46]. However, it remains unclear whether blocking LILRB2 promotes direct or indirect phagocytosis of TAMs. Although LILRB1 and LILRB2 dock to MHC-I, it is unknown whether LILRB2 ligand cross talk also works as a phagocytosis checkpoint [134, 135]. Anti-LILRB2 mAbs have been examined in a phase I clinical trial of malignancies (JTX-8064, INNATE) [136]. However, further investigations are needed to examine MHC-I expression patterns in hematological malignancies and LILRB1/2 expression patterns in associated TAMs. Based on phase I clinical data, this phagocytosis regulator is a promising target for enhancing ADCP against hematological malignancies [1]. Clinical research studies are also needed to examine the clinical efficacy of blocking the MHC-I/LILRB1/2 axis in hematological malignancies. Finally, LILRB1 is also expressed on several other distinct types of human immune cells, including B cells, DCs, NK cells and T cells [137]. Significantly, Chen et al. found that LILRB1 mAb enhanced anti-tumor activity of NK cells in multiple myeloma, leukemia and lymphoma [137]. Therefore, the study of LILRB1-mediated innate and adaptive immunity should be considered in the future.

CD24 is also called the heat-stable antigen or small-cell lung cancer cluster-4 antigen. It belongs to a glycosylphosphatidylinositol-anchored surface protein with a high glycosylation level [138, 139]. The expression level of CD24 is upregulated across almost all tumor types but not normal tissues [140‐144]. Furthermore, in tumor tissues, the expression of CD24, another known innate immune checkpoint, is notably strong compared to the expression of CD47 [47, 140]. The highest upregulation of CD24 expression has been reported in triple-negative breast cancer (TNBC) and ovarian cancer patients, who showed decreased recurrence-free survival (RFS) time and OS, respectively [47, 140]. Therefore, CD24 constitutes a “don’t eat me” signal and has been defined as an immune checkpoint via cross talk with the inhibitory receptor SIGLEC-10 on macrophages in the TME [47, 140]. These mechanisms have also been confirmed by single-cell RNA-seq results and fluorescence-activated cell-sorting assays using primary ovarian and breast cancer tissues [47, 140]. CD24 is especially upregulated in tumor cells, while SIGLEC-10 expression occurs in a subpopulation of macrophages in the TME, suggesting a potential cross talk between CD24 and SIGLEC-10 [47, 140].

Amira A Barkal et al. investigated the role of the CD24/SIGLEC-10 axis in modulating macrophage-triggered phagocytosis [47]. Studies have shown that macrophages in a co-culture system more readily phagocytose CD24^−/− MCF-7 cells than wild-type MCF-7 cells in vitro [47]. Additionally, SIGLEC-10^−/− macrophages also show significantly enhanced phagocytosis of the wild-type MCF-7 cells, further confirming that the CD24/SIGLEC-10 axis exerts a “don’t eat me” effect [47]. Anti-CD24 antibody therapy is also reported to substantially increase phagocytosis of wild-type MCF-7 cells in macrophages [47]. The pro-phagocytic effects of inhibiting the CD24/SIGLEC-10 axis are higher than the CD47 blockade treatment [47]. Another significant experimental result indicates that SIGLEC-10^−/− macrophages inhibit the phagocytosis effects of the anti-CD24 antibody, suggesting that CD24 blockade functions by disrupting the cross talk between CD24 and SIGLEC-10 [47]. An in vivo xenograft model experiment indicated that CD24-knockout MCF-7 cells had a markedly lower tumor burden than the wild-type group [47]. Moreover, macrophage depletion considerably inhibited the reduced tumor burden in CD24-knockout mice but not in wild-type mice, indicating that the anti-tumor effect may be attributed to macrophage-mediated phagocytosis [47]. These findings suggest that the CD24/SIGLEC-10 axis is a phagocytosis-related innate immune checkpoint that modulates anti-tumor immunity by regulating macrophage-mediated phagocytosis, demonstrating that CD24 blockade is a potential immunotherapy approach.

Previous preclinical investigations have shown that blocking CD24 has abundant potential in anti-tumor applications. Given that AML has a low expression of CD24, and acute lymphoblastic leukemia (ALL) and DLBCL have a moderate/high expression of CD24, it stands to reason that ALL and DLBCL may effectively respond to anti-CD24 treatment [47]. Significantly, Andrea Aroldi et al. reported on a functional study that has shown an improvement of phagocytosis through CD24/SIGLEC-10 axis inhibition in MCL [145]. Several MCL cell lines (e.g., NALM-6, Jeko-1, Granta-519, and Mino) express surface CD24, and immunosuppressive-induced M2-like macrophages demonstrated increased SIGLEC-10 expression [145]. In addition, Andrea Aroldi et al. performed a phagocytic assay via M2-like macrophages co-cultured with MCL cell lines. CD24 mAb was found to increase macrophage phagocytosis [145], which suggests a potential immunotherapeutic target in MCL with the aim of improving innate immunity via disruption of the CD24/SIGLEC-10 axis. Another in vitro study found that CD24 mAb removed more than 90% of MCL cell lines [146]. Furthermore, CD24 mAb triggered phagocytosis in primary patient-derived MCL cells by autologous macrophages [146]. Treatment for in vitro MCL cell lines with CD24 mAb was superior to CD47 mAb, which suggests that CD24 mAb may be more effective in treating MCL than CD47 mAb [146]. Despite these studies, in vivo studies are also required to confirm these in vitro efficacies documented for MCL. However, no clinical investigations have exhibited the efficacy of anti-CD24 in hematological malignancies; therefore, further studies are needed.

In conclusion, there are four major phagocytosis checkpoint blockades (Fig. 4). In hematological malignancies, those that block the CD47/SIRPα axis and PD-1/PD-L1 axis have demonstrated promising clinical responses. However, larger clinical trials with longer follow-up times are required to confirm these findings. And the TEAEs of distinct CD47 products should also be considered in future. In addition, preclinical studies have shown potential therapeutic effects for another two phagocytosis checkpoints (MHC-I/LILRB1/2, CD24/SIGLEC10) in tumors. Still, more clinical studies are needed to examine the effects of these phagocytosis checkpoints on distinct types of hematological malignancies. Identifying heterogeneity in “don’t eat me” signal expression in different hematological malignancies indicates this area of research requires further investigation to select appropriate therapeutic strategies precisely. Because the anti-tumor influences of phagocytosis checkpoint blockades depend on the presence of macrophages [22], it is necessary to detect the number of macrophages infiltrated into the microenvironment of hematological malignancies and identify biomarkers. These steps can help identify patients who may benefit the most while avoiding substantial toxicities. Furthermore, preclinical studies have demonstrated that TTI-621 triggers phagocytosis of blood cancer cells through all macrophage subsets [104]. Therefore, macrophage-mediated phagocytosis checkpoint blockades in combination with therapeutic strategies to increase macrophage numbers may enhance the efficacy and utility of these therapies.

CD47/CD20 BsAbs bind CD20 and CD47 monovalently. They have a lower affinity for CD47 than the parental antibody but maintain robust docking to CD20 [53, 54]. These properties enable BsAbs to attach specifically to CD47⁺CD20⁺ tumor cells, resulting in phagocytosis. In vitro studies reveal that CD47/CD20 BsAbs stimulate tumor cell phagocytosis in a CD20-dependent approach [53, 54]. Essentially, these in vivo studies have also indicated that the treatment of human NHL-inserted mice with CD47/CD20 BsAbs reduces lymphoma burden and prolongs the OS of mice [53, 54]. Therefore, BsAbs can produce synergistic treatment efficacy in therapies that combine anti-CD47 and anti-CD20 [53, 54].

In addition, Shanghai ImmuneOnco Biopharmaceuticals Co. (ImmuneOnco) developed a bispecific recombinant antibody trap named IMM0306. IMM0306 is a recombinant human SIRPα and anti-CD20 antibody fusion protein [174]. It can simultaneously target CD47 and CD20 on B cells but avoids docking to human RBCs in vitro. IMM0306 exhibits a strong pro-phagocytosis influence compared to CD47⁺CD20⁺ target cells and an even stronger ADCC influence than rituximab. Treating lymphoma-transplanted SCID mice with IMM0306 significantly hampered tumor growth and resulted in the complete remission of tumor cells in 62.5% of mice. This therapy was remarkably effective compared to rituximab alone or the co-administration of rituximab and SIRPα-Fc (IMM01) [174]. In conclusion, the research revealed that IMM0306 may be a promising approach for developing CD47-targeted immunotherapy. It allows for the targeted avoidance of RBC-modulated antigen drops and the onset of anemia while still exhibiting substantial anti-tumor efficacy. IMM0306 is now undergoing a phase I clinical investigation (NCT04746131). JMT601, designed by the China Shijiazhuang Pharmaceutical Company (CSPC), is another recombinant human SIRPα and anti-CD20 antibody fusion protein. A phase I study of JMT601 (NCT04853329) is ongoing. However, no clinical data have been published for CD47/CD20 BsAb.

TG-1801 (also known as NI-1701) is a fully humanized IgG1 BsAb designed to target and deplete B cells through various mechanisms [57]. In an in vitro study, TG-1801 bound to B cells in whole blood and specifically blocked CD47/SIRPα cross talk on CD19-expressing cells [57]. TG-1801 mediated the successful killing of primary and hematological cancer cell lines (Raji, Ramos, MEC-2, NALM-6, and SUDHL-4) through ADCP and ADCC [57]. Essentially, the in vitro anti-tumor activity extends in vivo killing efficacy. This study used a Raji B cell–transplanted NOD/SCID mouse model. TG-1801 led to the inhibition of tumor proliferation and a considerable increase in median OS compared to isotype control-treated mice [57]. The anti-tumor effects of TG-1801 were also demonstrated in a patient-derived xenograft model of B-ALL, where TG-1801 reduced tumors across the various organs tested [57]. The study suggests that TG-1801 offers an alternative treatment for patients who are resistant, refractory, or both to anti-CD20 therapy. A clinical investigation is currently being performed to validate the safety and efficacy of this BsAb (NCT04806035). Furthermore, Emmanuel Normant et al. explored potential synergies between TG-1801 and ublituximab alone, or thresholdisib alone, or ublituximab plus thresholdisib in B-NHL via in vitro and in vivo experiments [175]. Significantly, the tumor growth inhibition (TGI) for TG-1801 in combination with umbralisib alone, ublituximab alone, and ublituximab plus thresholdisib were 85%, 93%, and 93%, respectively [175]. Intriguingly, the anti-tumor effect of the different combinations of TG-1801 was associated with higher levels of mouse macrophage infiltration within tumors, as well as the upregulation of G-protein coupled receptor EBI2/GRP183. Additionally, the EBI2 small-molecule inhibitor NIBR189 inhibits the ADCP, B cell cytoskeleton remodeling, and inflammatory cytokine production induced by TG-1801 [175]. Taken together, these data suggest a combination strategy of TG-1801 with other anti-B-cell mechanisms, such as umbralisib and ublituximab, for the treatment of B-NHL [175]. Further clinical studies are also required to evaluate the combination strategy of TG-1801 with other anti-B-cell drugs.

Targeting CD47 stimulates the innate immune system, in particular. Thus, therapy with CD47 mAbs enhances antigen presentation in the presence of MHC via macrophages, and DCs, thereby activating T cell cross-priming in mouse models [176]. Based on this rationale, a novel BsAb (DSP107) that inhibits the CD47 signaling pathway while simultaneously stimulating anti-tumor T cell immune response has been designed [177, 178]. DSP107 exhibited a high binding affinity for both human CD47 and 4-1BB [177]. DSP107 also blocked the cross talk of SIRPα with CD47 and triggered phagocytosis in several lymphomas, leukemia, and carcinoma cell lines in vitro [177]. By binding CD47 on cancer cells, DSP107 blocks the CD47/SIRPα cross talk, thereby inducing phagocytosis of cancer cells [178, 179]. Additionally, DSP107 binds to 4-1BB, a co-stimulatory receptor that is increased in response to T cell receptor (TCR)/MHC cross talk and a proven surrogate biomarker for the tumor-reactive T cell subset in the TME [178, 179]. Furthermore, DSP107 triggers 4-1BB signaling only after binding to CD47 [178, 179]. Taken together, DSP107 unleashes innate and adaptive immune responses to target the tumor site. In vitro, DSP107 alone or combined with rituximab triggers substantial phagocytosis of numerous DLBCL cancer cell lines and primary patient-derived DLBCL cells through macrophages [178, 179]. Additionally, 4-1BB activation is only observed following DSP107 binding to human CD47, and the induction of 4-1BB co-stimulatory signaling triggers prominent T cell proliferation to augment T cell cytotoxicity in vitro [178, 179]. Injecting PBMCs into mice with generated SUDHL6 xenografts accompanied by DSP107 treatment triggers a considerable decrement in tumor size compared to PBMC-only treatment [178, 179]. Thus, DSP107 induces innate and adaptive anti-tumor immunity, potentially making it useful in treating DLBCL, leukemia, and other diseases. Despite the lack of clinical results for DSP107 in hematological malignancies, the safety and efficacy of DSP107 in advanced solid tumors have been confirmed at the 2022 ASCO meeting [180]. A clinical trial is ongoing to assess DSP107’s safety and efficacy in the treatment of MDS, AML, and T cell lymphoproliferative disease (NCT04937166).

CD33 is over-expressed in 90% of patients with AML [181]. Commonly, CD47 is also over-expressed in patients with AML [33]. Therefore, Jerome Boyd-Kirkup et al. constructed a bispecific anti-CD47xCD33 antibody (HMBD004) [60]. HMBD004 exhibited preferential binding of CD47⁺CD33⁺ cells. HMBD004 significantly inhibits CD47/SIRPα cross talk and the induction of phagocytosis in vitro [60]. However, HMBD004 does not bind to RBCs in vitro, suggesting a lower incidence of hemagglutination [60]. Additionally, in vivo cell line-derived xenograft mouse models of AML were created by subcutaneously implanting NCr nude mice with the CD47⁺CD33⁺ AML cell line HL-60. The treatment of these animals with HMBD004 resulted in a reduction in tumor burden and an increase in PFS. In conclusion, these findings indicate that HMBD004 enhances the specificity, efficacy, and safety of CD47 mAb therapy in AML. Therefore, clinical studies on HMB004 are worthwhile.

Tumor cells express both PD-L1 and CD47, whereas most normal cells have limited or undetectable expression of PD-L1. The distinct co-expression pattern of PD-L1 and CD47 in cancers compared to normal tissues provides a rationale for designing BsAbs that can selectively recognize PD-L1⁺CD47⁺ tumor cells and block their CD47 signaling to trigger the engulfment of double-positive cancer cells. While the monovalent CD47 antibody arm binds CD47 and triggers strong tumor cell phagocytic activity, it has little effect on normal human CD47 single positive cells [61]. IBI322, a CD47/PD-L1 BsAb, was generated by blocking the PD-L1 signaling pathway with the bivalent single-domain PD-L1 antibody arm [61]. Numerous preclinical studies have examined the safety and efficacy of IBI322, both in vitro and in vivo [61, 182, 183]. IBI322 selectively binds CD47⁺PD-L1⁺ tumor cells, effectively inhibits CD47/SIRPα signaling, and instigates intense phagocytosis of CD47⁺PD-L1⁺ tumor cells by macrophages in vitro [61]. However, IBI322 has minimal effects on CD47 single positive cells, such as human RBCs. Additionally, IBI322 accumulates in PD-L1⁺ Raji tumors to produce a synergistic effect, resulting in complete tumor regression in vivo [61]. Moreover, IBI322 demonstrates minimal destruction of RBCs, and IBI322 is well tolerated in repeated weekly injections, supporting the sufficient therapeutic window in the future clinical use of IBI322 for cancer therapy [61]. Further investigations are required to validate the safety and efficacy of IBI322 for the treatment of hematological malignancies.

CD47/PD-1 BsAb (HX009) is a humanized antibody fusion protein that binds to CD47 and PD-1 concurrently [62]. HX009 considerably diminished solid tumor proliferation in mouse xenograft models [62]. The first-in-human phase I dosage escalation study on HX009 was completed in individuals with advanced solid tumors. HX009 was well tolerated without DLT and exhibited enhanced anticancer efficacy in multiple tumor types [62]. A phase I/II clinical trial is ongoing to evaluate the safety and efficacy of HX009 in the treatment of R/R-lymphoma (NCT05189093). Further investigations of HX009 in hematological malignancies are warranted.

CD47 mAb or CD38 mAb is effective for the treatment of MM [92, 113, 184]. Significantly, blocking the CD47/SIRPα signaling pathway enhanced the killing effect of CD38 mAb on MM cells [112]. Based on this, the design of BsAbs targeted both CD47 and CD38 may have a synergistic effect in the treatment of MM. ISB 1442, a fully human BsAb with anti-CD38 and CD47 binding arms, was then developed for the treatment of R/R MM [185]. The CD38 Fab arm of ISB 1442 preferentially drives the binding to CD38⁺ MM cells and achieves a blockade of the proximal CD47 receptor on the same cells through induced binding [185]. The Fc portion of ISB 1442 was engineered to enhance ADCP, ADCC, and CDC. ISB 1442 was highly effective in killing both CD38^hi MM cells via CDC and CD38^low MM cells via ADCC and ADCP, in comparison to daratumumab in vitro. Significantly, the tumor-killing effect of ADCP, ADCC, and CDC induced by ISB 1442 is more powerful than magrolimab in vitro [185]. Furthermore, there was a twofold increase in MM cell killing with ISB 1442 compared to daratumumab in co-culture assays (macrophages and PBMCs from healthy donors were incubated with MM cells and human serum) [185]. To evaluate the off-target tumor specificity of ISB 1442 in vitro through binding to human RBCs, hemagglutination, and RBC depletion, hemolysis and platelet aggregation were measured in the present study. Compared to magrolimab, ISB 1442 showed no evidence of hemolysis, RBC depletion, or platelet aggregation and significantly reduced human RBC hemagglutination in vitro. In addition, the potency of ISB 1442 was evaluated in vivo using a Raji tumor xenograft model in CB17/SCID mice (which have functional complement, macrophages, and NK cells). Compared to daratumumab, there was a greater inhibition of tumor growth with ISB 1442, and, compared to magrolimab, there was a comparable reduction in tumor size. Collectively, ISB 1442 is anticipated to be a safe and effective approach for the treatment of MM. Therefore, further clinical studies on ISB 1442 are required.

CD70 is expressed by various hematopoietic and epithelial-derived cancer cells and plays a role in promoting tumor cell survival/proliferation [186]. Phase I clinical trial results have shown that the combination of CD70 mAb (ARGX-110) and azacitidine in patients with AML resulted in an ORR of 92% [187], while single-agent ARGX-110 in advanced cutaneous T-cell lymphoma showed an ORR of 23% [188]. Thereafter, researchers developed a bispecific anti-CD70/SIRPα antibody [189]. In comparison to CD70 mAb + SIRPα mAb treatment, CD70/SIRPα BsAb had a greater ability to phagocytose CD70-expressing NHL and renal cell carcinomas in vitro, but no apparent differences were observed in vivo [189]. Further studies are required to investigate the efficacies of CD70/SIRPα BsAb in CD70^hi hematological malignancies.

CD123 is highly expressed on AML blasts and leukemic stem cells (LSCs) and demonstrates only a moderate expression on normal HSCs, suggesting that CD123 is a promising target antigen [190]. SIRPα receptors on macrophages interact with CD47 to inhibit phagocytosis. SIRPα/CD123 BsAb was designed by Siret Tahk et al. based on these interactions [191]. Their results demonstrate that SIRPα/CD123 BsAb yields strong anticancer activity against AML in vitro and in vivo via enhanced NK cell-dependent ADCC and macrophage-mediated ADCP effects. SIRPα/CD123 BsAb also established its safety by demonstrating low CD47-related on-target off-leukemia toxicity. Preclinical and clinical testing of the SIRPα/CD123 BsAb is required.

MARCO constitutes a pattern recognition receptor that is a member of the scavenger receptor family of class A receptors [198]. MARCO is mostly expressed by TAMs and has been correlated with a poor prognosis in many cancers [199, 200]. In preclinical studies, anti-MARCO antibodies have been reported to inhibit tumor growth and metastasis in 4T1 mammary carcinoma and B16 melanoma mouse models [201]. The anti-tumor activity of anti-MARCO is dependent on the binding of the Fc subunit with its inhibitory Fc receptor, FcγRIIB, similar to anti-CD40 antibody-mediated reprogramming [202]. In 2020, Silke Eisinger et al. demonstrated a novel mechanism targeting MARCO on tumor macrophages and altering their polarization. In turn, NK cells were activated to kill tumor cells [200]. Anti-MARCO treatment often works in combination with T cell-targeted checkpoint therapies. This finding was confirmed in human-based experiments, wherein a new specific antibody targeting human macrophages could initiate NK cell killing and thus support the use of combinatory treatments in cancer therapy.

In summary, these studies highlight the potential of antibody-mediated macrophage reprogramming using macrophage-associated targets. These studies also stress the significance of the correct antibody designs, especially in the Fc region, for future clinical interventions. A recent study from our group exhibited that a subpopulation of macrophages in the AML microenvironment expressed high levels of MARCO, and MARCO^high macrophages shared the M2 phenotype [203]. These results suggest that AML patients with high MARCO expression may benefit from anti-MARCO therapy. Furthermore, MARCO expression in hematological malignancies should be examined in order to demonstrate how anti-MARCO treatment might be effective.

TLRs are a pathogen sensor family that recognizes bacterial and viral ligands and activates innate immune sensing [204]. TLR activation polarizes macrophages toward a pro-inflammatory phenotype [204]. Recently, Holly M. Akilesh et al. reported that activation of TLR7 and TLR9 increases the phagocytosis of monocyte-derived macrophages and causes anemia of inflammation [205]. Researchers have used different TLR ligands in various cancer models to analyze their activities during the transformation of TAMs into tumor-killing macrophages [204].

As early as the 1960s, Vassal et al. reported improved OS in pediatric leukemia patients using the TLR agonist Bacille Calmette-Guérin vaccine, which is currently used for treating bladder cancer patients [206]. The majority of clinical trials using TLR agonists to treat hematological malignancies have focused on TLR3, TLR7/8, and TLR9. Recent investigations have shown that the synchronous application of different TLR agonists may be useful for patients with various TLRs expressed in tumors. Brenda J. Weigel et al. performed a phase II clinical study of 852A in R/R hematological cancer patients [207]. The study reported that six patients had AML, five had ALL, four had NHL, one had HL, and one had MM. The mean age of the patients was 41 years (range: 12–71 years), and the median cycle of prior chemotherapy regimens was five (range: 1–14). Of the 17 cases, 13 patients completed all 24 cycles of 852A injections. Grade 3/4 toxicities included dyspnea, myalgia, nausea, malaise, fever, and cough. Patients with clinical responses included one ALL and one AML. However, nine patients showed progressive disease [207].

TACL T2009-008, a phase I clinical study, indicated that GNKG168 treatment was correlated with immunological changes in 23 patients with pediatric leukemia [208]. However, other studies with large sample sizes are required to investigate the effect of changes in disease therapy and the persistence of leukemia remission. Presently, three phase I/II studies are recruiting patients to assess the clinical efficacy of TLR3, TLR4, and TLR9 agonists combined with standard therapies for the treatment of R/R lymphomas, including low-grade B cell lymphomas, T cell lymphomas, and FL (Clinical Trial Identifiers: NCT01976585, NCT03410901, and NCT02927964).

In conclusion, TLR agonists activate macrophages toward the M1 phenotype, resulting in satisfactory preclinical therapeutic effects for hematological malignancies. Future studies are required to gain a comprehensive mechanistic understanding of the role of TLR agonists in hematological malignancies. Additionally, there is a need to examine the influence of TLR signaling on the pathogenesis of hematological malignancies and determine the appropriate clinical utility by conducting extensive cohort studies.

Under normal conditions, Tim-4⁺ macrophages recognize phosphatidylserine (PS) receptors exposed on the apoptotic cell surface or on the nuclei extruded from matured erythroid cells. Tim-4⁺ macrophages then remove these cells, thereby maintaining homeostasis and preventing the development of autoimmune diseases [72, 209]. In 2020, Houjun Xia et al. reported that Tim-4⁺ TAMs in the peritoneal cavity of ovarian cancer are embryonic in origin and locally sustained through self-renewal, while Tim-4⁻ TAMs are replenished from circulating monocytes [210]. In the ID8 ovarian cancer mouse model, Houjun Xia et al. found that Tim-4⁺ TAMs, but not Tim-4⁻ TAMs, promote the proliferation of cancer in vivo [210]. RNA-sequencing analysis revealed that Tim-4⁺ and Tim-4⁻ TAMs have different functions and phenotypes. The Tim-4⁺ TAMs exhibit higher levels of arginase-1 (ARG1), inhibiting mTORC1 activation and thus promoting mitophagy [210]. Moreover, deficiency of the autophagy element FAK family-interacting protein 200 kDa (FIP200) led to Tim-4⁺ TAM loss, enhanced T cell immune responses, and inhibited the proliferation of the ID8 tumor in vivo [210]. The complement receptor of the immunoglobulin superfamily (CRIg)⁺ TAMs in human ovarian cancer is similar to murine Tim-4⁺ TAMs in terms of transcriptional levels, metabolic effects, and functional roles. Accordingly, targeting CRIg⁺ (Tim-4⁺) TAMs may effectively treat patients with peritoneal metastases from ovarian cancer [210]. Anders Etzerodt et al. reported similar results in 2020 [211]. Their findings revealed the metastatic spread of ovarian cancer cells via a distinct population of omental macrophages with CD163⁺ Tim4⁺ on their surface [211]. Using genetic and pharmacological tools, selectively depleting of CD163⁺ Tim4⁺ macrophages could prevent tumor progression and metastatic spread by in the omentum [211]. María Casanova-Acebes et al. also reported that the peritoneal TAMs of ovarian cancer significantly promote tumor progression [212]. Additionally, the researchers demonstrated that RXR signaling controls the maintenance of these TAMs and that the deletion of RXR diminishes the accumulation of TAMs in tumors and strongly reduces ovarian tumor progression in mice [212]. In 2021, Andrew Chow et al. reported a poor prognosis after PD-1 therapy for metastatic cancer of the pleural and peritoneal cavities [213]. First, they discovered that Tim4⁺ TAMs are positively related to decreased numbers of CD8⁺ T lymphocytes with tumor-reactive characteristics in cancer patients’ pleural effusions and peritoneal ascites. Additionally, they observed that Tim-4⁺ macrophages trap and inhibit CD8⁺ T cell growth. In contrast, Tim-4 inhibition abolishes this sequestration and growth suppression and significantly improves anti-tumor effectiveness in mouse models of anti-PD-1 treatment and adoptive T cell therapy. Thus, these investigations recommend Tim-4 as a TAM target, having demonstrated that blocking Tim-4 enhances anti-PD-1 treatment in preclinical tests.

Tim-4 is highly expressed on the surface of macrophages in hematopoietic-related organs, bone marrow, spleen, lymph node, fetal liver, and so on [72, 209]. Additional study of the functional roles of Tim-4⁺ macrophages in hematological malignancies is required.

Meghan A. Morrissey et al. developed a family of chimeric antigen receptors that can guide macrophages to phagocytose antigen-specific targets, including Raji B cells, CD19, or CD22 positive cells [215]. In their study, three engineering strategies were tested. Macrophages expressing the CAR-P^Megf10 or CAR-P^FcRɣ were insufficient to trigger whole-cell engulfment [215]. Moreover, in conjunction with CD47 blockade led to a 2.5-fold increase of phagocytosis of Raji cells, suggesting CAR-P expression in combination with CD47 or SIRPα blockades has an additive effect [215]. In contrast, CAR-P^tandem enhanced the capability of macrophages to take up cancer cells, suggesting that assembling a motif of phagocytic effectors could increase the cancer cell phagocytic activity of CAR-P [66, 216]. This engineered macrophage could be identified as the first reported “CAR-M” in preclinical studies. Furthermore, the macrophages showed antigen-specific killing effects among tumor cells. In 2020, Michael Klichinsky et al. established a novel cellular therapy, CAR macrophages (CAR-Ms) [66]. These researchers hypothesized that human macrophages expressing CARs could redirect their phagocytosis function, resulting in an antigen-specific, anticancer therapeutic effect with the potential to induce an adaptive immune response. CAR-Ms led to a sustained pro-inflammatory phenotype and overcame the inherent phagocytosis-correlated resistance of primary human macrophages. In addition, CAR-Ms demonstrated antigen-specific phagocytosis and clearance of tumor cells in vitro [66]. A single transfusion of reprogrammed human CAR-Ms led to a remarkable inhibition of tumor growth and prolonged OS of mice using xenograft mouse models of solid tumor in vivo.

Transduced CARs were constructed to define the mechanism of CAR-modulated redirection of macrophage phagocytosis: CAR19ζ, which contains a CD3ζ intracellular domain, and CAR19Δζ, which lacks the CD3ζ intracellular domain. The CD3ζ molecule is homologous with the Fc common γ-chain, FcεRI-γ molecule, and ADCP signaling molecule in macrophages [66]. Therefore, only CAR19ζ was found to phagocytose CD19⁺ cancer cells in an antigen-specific manner, unlike CAR19Δζ and control untransduced (UTD) macrophages (Fig. 6) [66]. In addition, the engulfment of CAR-Ms is considered an active process requiring the polymerization of Syk, actin, and non-muscle myosin-IIA. The phagocytic process is similar to the Fc receptor-mediated ADCP. Transcriptome sequencing data demonstrate that CAR-Ms secrete pro-inflammatory cytokines and chemokines, convert M2 macrophages to M1, upregulate antigen presentation, recruit and present antigens to T cells, and resist the effects of immunosuppressive cytokines [66]. CAR-Ms yield substantially increased antigen-specific anti-tumor effects for hematological malignancies.

However, sourcing and the high cost of CAR-Ms remain a challenge. Immortalized macrophage cell lines are not applicable for clinical use, and bone marrow (BM) and peripheral blood mononuclear cells (PBMC)-derived macrophages cannot be efficiently modified or engineered. There is a need for novel and efficiently engineered macrophages [65]. Recently, induced pluripotent stem cells (iPSCs)-derived macrophages (CAR-iMac) have emerged as a promising source of cellular immunotherapy following the work of Li Zhang et al. [65]. They demonstrated that CAR-iMac cells result in antigen-specific functions for macrophages, such as the secretion of pro-inflammatory cytokines, polarization toward the anti-tumor phenotype, engulfment of antigen-specific tumor cells, and in vivo anti-tumor cell activity for both hematological malignancies and solid tumors [65]. Hence, this study presents a new source of CAR-M with satisfactory anti-tumor cell activity.

Preclinical findings of CAR-M (CT-0508) suggest that this therapy may have the potential to overcome the challenges encountered with T cell therapies in solid tumors [66]. On December 9, 2020, Carisma Therapeutics Inc. began the first clinical study of CAR-M (CT-0508): “A phase I, the first human study of adenoviral transduced autologous macrophages engineered to contain an anti-HER2 chimeric antigen receptor in subjects with HER2-overexpressing solid tumors” (ClinicalTrials.gov Identifier: NCT04660929). This clinical trial represents an important milestone in the development of gene-based therapies because it marks the first reported use of CAR-Ms in a clinical study. The study focused on solid tumors in patients with recurrent or metastatic human epidermal growth factor receptor 2 (HER2)-overexpression for cancers without any approved HER2-targeted therapies or patients who did not respond to treatment. On March 18, 2021, Carisma Therapeutics Inc. announced that the first patient had received the first dose of CT-0508, HER2-targeted CAR-M, in the phase I multicenter clinical trial. However, more time is necessary to investigate the clinical efficacy of this CAR-M.

The success of CAR-T cells in hematological malignancies suggests that CAR-M cellular therapy may also yield promising results in hematological malignancies. Indeed, IMAGINE (ClinicalTrials.gov Identifier: NCT05138458) is a multicenter, open-label, dose escalation and dose cohort expansion phase I/II clinical trial for evaluating CD5-targeted CAR-M (MT-101) treatment in patients with R/R-PTCL. This study was sponsored by Myeloid Therapeutics on December 15, 2021. On May 11, 2022, they completed the first patient dosing of MT-101 in R/R-PTCL. This patient did not exhibit cytokine release syndrome (CRS) and no apparent cytotoxicity. However, more time is needed to investigate the safety and clinical efficacy of MT-101.

Several diverse CAR-Ms have been evaluated for treating both hematological malignancies and solid tumors in preclinical studies and clinical studies [217, 218] (Table 4). The target antigen included CD5, CD19, CD22, HER2, CCR7, and ALK. More target antigens must be developed in the future to explore their therapeutic role in hematological malignancies.

In conclusion, CAR-Ms display antigen-specific anti-tumor activities in preclinical studies both in vitro and in vivo [219]. This novel cellular immunotherapy has clear potential for treating both hematological malignancies and solid tumors [220]. The strengths of CAR-M include the following points: 1) CAR-M can infiltrate tumor tissues considerably, reduce the proportion of TAMs, affect the phenotype of TAMs, and produce a positive effect on tumor treatment. 2) In addition to its role in tumor cell phagocytosis, CAR-M can promote antigen presentation and enhance T cell killing. 3) CAR-M shows little “On-Target, Off-Tumor Toxicity.”

The challenges associated with CAR-M’s wider applications in various cancer types should also be considered in future studies. The challenges include the following points: 1) The safety and efficacy of CAR-M for humans remain to be proved via clinical trials. 2) Reliable cell sources and expansion are necessary for the clinical application of CAR-M. 3) Currently, CAR-M mostly uses viral transfection, which may induce insertional mutations; however, CRISPR/Cas9 offers a solution. 4) The underlying mechanisms of CAR-M resistance need to be investigated. 5) The complex immune microenvironment should also be considered when applying CAR-M therapy. Thus, rational selection of existing therapy in combination with CAR-Ms may have a synergistic effect on anti-tumor responses.