Harnessing the untapped potential of nucleotide‐binding oligomerization domain ligands for cancer immunotherapy

Abstract In the last decade, cancer immunotherapy has emerged as an effective alternative to traditional therapies such as chemotherapy and radiation. In contrast to the latter, cancer immunotherapy has the potential to distinguish between cancer and healthy cells, and thus to avoid severe and intolerable side‐effects, since the cancer cells are effectively eliminated by stimulated immune cells. The cytosolic nucleotide‐binding oligomerization domains 1 and 2 receptors (NOD1 and NOD2) are important components of the innate immune system and constitute interesting targets in terms of strengthening the immune response against cancer cells. Many NOD ligands have been synthesized, in particular NOD2 agonists that exhibit favorable immunostimulatory and anticancer activity. Among them, mifamurtide has already been approved in Europe by the European Medicine Agency for treating patients with osteosarcoma in combination with chemotherapy after complete surgical removal of the primary tumor. This review is focused on NOD receptors as promising targets in cancer immunotherapy as well as summarizing current knowledge of the various NOD ligands exhibiting antitumor and even antimetastatic activity in vitro and in vivo.

also at the plasma membrane. 22 Their recruitment to the cell membrane has been observed in various epithelial cells and recognized as a crucial event for activation of the NF-κB signaling pathway following bacterial PGN binding. 23,24 Interestingly, although similar in terms of cell localization, NOD1 and NOD2 are very differently expressed in cells and tissues throughout the body. NOD1 is extensively expressed in a variety of cell types, whereas NOD2 has been found mostly in professional immune cells (macrophages, 25 dendritic cells, 26 and Paneth cells 27 ), osteoblasts, 28 keratinocytes, 29 intestinal stem cells, 30 and various epithelial cells. [31][32][33] 2.2 | Structure NOD1 and NOD2 are multiple domain proteins consisting of a C-terminal, leucine-rich repeat domain (LRR) (also widely accepted as the bona fide sensor domain that is responsible for recognition of ligands), a centrally located nucleotidebinding oligomerization domain (NACHT) that mediates self-oligomerization and is crucial for NOD activation, and one (NOD1) or two (NOD2) N-terminal caspase recruitment domains (CARDs) that interact with downstream signaling molecules. 34,35 Normally, NODs are kept in a monomeric autoinhabitable state in the cell cytosol, being activated following ligand binding. 19 In addition to being able to recognize ligands in the cytosol, NODs are capable of trafficking dynamically to the cell membrane and of recognizing bacteria at the point of entry. 36

| The canonical signaling pathway (NF-κB and MAPK)
On activation by their native ligands, NODs undergo conformational changes and self-oligomerization through homophilic CARD-CARD interactions, allowing the recruitment and activation of the CARD-containing adaptor receptor-interacting serine/threonine-protein kinase 2 (RIPK2). The latter is important for downstream signal transduction. [37][38][39] In an established protein complex, RIPK2 is later polyubiquitinated by several E3 ubiquitin ligases, namely tumor necrosis factor receptor-associated factors (TRAFs), cellular inhibitor of apoptosis protein (cIAP) 1, cIAP2, and X-linked inhibitor of apoptosis protein (XIAP). [40][41][42][43][44][45] Polyubiquitin chains attached to RIPK2 then facilitate the formation and activation of a protein complex consisting of tumor growth factor β-activated kinase 1 (TAK1) and TAK1-binding proteins (TAB) 1 to 3. 46,47 TAK1 is an upstream activator of the inhibitory κB kinase (IKK) complex. 48 This activation leads to the phosphorylation and degradation of a protein inhibitor of NF-κB (IκB), resulting in translocation of NF-κB to the nucleus and transcription of NF-κB target genes. 42,46,49 On the other hand, TAK1 also activates three mitogen-activated protein kinases (MAPKs), namely p38, extracellular signalregulated kinase (ERK), and c-Jun N-terminal kinase (JNK), resulting in the activation of activator protein 1 (AP-1) transcription factor. 50 Moreover, RIPK2 also interacts with the IKKγ/NEMO subunit of IKK complex, resulting in ubiquitination of IKKγ/NEMO and activation of the IKK complex, which is important for downstream NF-κB activation ( Figure 2). 15 In addition to NF-κB and MAPK signaling pathways, NODs are involved in the activation of other innate immunity systems such as autophagy, apoptosis, inflammasome activation, and even antiviral response, as described briefly below.

Apoptosis pathway
NODs have been reported to interact indirectly with the apoptotic pathway through the inhibitor of the apoptosis (IAP) family of proteins (cIAP1, cIAP2, and XIAP) as with the proapoptotic protein BH3-interacting domain death agonist (Bid) (Figure 2). 40,43,51,52 IAP proteins participate in NOD signaling by polyubiquitinating RIPK2 and consequently stimulating NF-κB activation and stress kinases activities, 40,43,51 while Bid has been suggested to bridge the NODs to the IKK complex thereby impacting NF-κB and ERK activation. 52 Furthermore, it has been demonstrated that stimulation of NOD1 activates caspase 8, which has been linked to its underlying antitumor activity. 53,54 Autophagy Autophagy is a highly conserved degradation process in eukaryotic cells, vitally involved in the normal functioning of the innate immune system. [55][56][57][58] It has recently been discovered that NOD-mediated recognition of bacteria induces autophagy and bacterial clearance. [59][60][61] NODs have been shown to recruit the autophagy protein ATG16L1 to the cell membrane, to target bacteria at the point of entry, independently of RIPK2 ( Figure 2). 61 In addition to their role in sensing bacteria, NODs are involved in autophagosome formation. It has been demonstrated that autophagosome formation is induced in epithelial cells, fibroblasts or dendritic cells (DCs) on stimulation by NOD agonists. [59][60][61] In contrast to the cell membrane targeting function, the induction of autophagy by NOD2 is an RIPK2-dependent process leading to downstream ERK and p38 activation. 60 It should be noted that signaling through RIPK2 deactivates protein phosphatase 2A, which negatively regulates NOD-dependent autophagy. 60

Induction of an antiviral response
In addition to the previously described signaling pathways, studies have also provided evidence for PGNindependent role of NODs. 66,67 Specifically, NOD2 can act as a cytoplasmic viral PRR that activates an antiviral response, resulting in type I interferon (IFN) production. After detection of viral ssRNA, NOD2 translocates to the mitochondria where it supposedly interacts with mitochondrial antiviral signaling (MAVS) protein. This promotes the formation of a complex with serine/threonine-protein kinases TBK1 and IKKα, enabling activation interferon regulatory factor (IRF) 3 and resulting in the production of IFN-β ( Figure 2). 15,67

| THE ROLE OF NOD PROTEINS IN CANCER DEVELOPMENT
Although NODs were initially recognized as receptors for pathogen recognition within the scope of the innate immune response, recent findings have further confirmed their involvement in mechanisms underlying cancer development. On the one hand, NOD activation can prevent, inhibit, or block carcinogenesis by controlling epithelial cell regeneration while, on the other hand, it can promote carcinogenesis via the production of pro-inflammatory F I G U R E 2 Canonical and noncanonical signaling pathways of NOD1 and NOD2. A, B, NF-κB and MAPK signaling pathways. NOD1 and NOD2 recognize bacterial PGN fragments, iE-DAP and MDP, respectively. After ligand recognition, NODs undergo conformational changes and self-oligomerization through homophilic CARD-CARD interactions, allowing the recruitment and activation of the adaptor protein RIPK2. In an established protein complex, RIPK2 is polyubquitinated by TRAFs and cIAPs, allowing the recruitment of polyubiquitinated NEMO or TAK1 to the established protein complex. On one hand, NEMO triggers activation of the NF-κB pathway by phosphorylation of IκB resulting in release NF-κB transcription factor. The latter translocates to the nucleus where causes induction of pro-inflammatory genes. On the other hand, TAK1 recruits TAB1/2/3 activating both NF-κB and MAPK pathway (JNK, ERK, and p38). Activated MAPKs translocate to the nucleus and activate AP-1 transcription factor resulting transcription of genes involved in inflammatory response. C, Apoptosis pathway. NODs have been reported to interact with the apoptotic pathway indirectly through IAP family of proteins (cIAP1, cIAP2, and XIAP) and proapoptotic protein Bid. D, Inflammasome activation. NODs associate with several other NLRs (NLRP1 and NLRP3) to form inflammasome protein complexes. After detection of their cognate ligand, NLRs interact with ASC protein and procaspase-1, resulting in formation of inflammasomes. Once formed, inflammasomes activate procaspase-1, which in turn proteolytically processes pro-inflammatory cytokines IL-1β and IL-18. E, Autophagy. Activated NODs recruit the autophagy protein ATG16L1 to the cell membrane and facilitate the formation of autophagosome around invading bacteria. F, Antiviral response. Viral ssRNA activates NOD2, which translocates to the mitochondria and supposedly binds to MAVS. This promotes formation of a complex with TBK1 and IKKα, which enables activation of interferon regulatory factor (IRF) 3 resulting in production of IFN type I. ASC, apoptosis-associated speck-like protein containing caspase recruitment domain; CARD, caspase recruitment domain; cIAP, cellular inhibitor of apoptosis protein; ERK, extracellular signal-regulated kinase; IAP, inhibitor of the apoptosis; iE-DAP, D-glutamyl-meso-diaminopimelic acid; IFN, interferon; IKK, IκB kinase; IκB, protein inhibitor of NF-κB; IL, interleukin; JNK, c-Jun N-terminal kinase; NOD, nucleotide-binding oligomerization domain; NF-κB, nuclear factor κB; MAPK, mitogen-associated protein kinase; MAVS, mitochondrial antiviral signaling; MDP, muramyl dipeptide; NEMO, nuclear factor κB essential modulator; NLRs, nucleotide-binding oligomerization domain-like receptors; PGN, peptidoglycan; RIPK2, receptorinteracting serine/threonine-protein kinase 2; TAB, transforming growth factor binding protein; TAK1, transforming growth factor β-activated kinase 1; TBK1, TRAF-associated nuclear factor-κB activator-binding kinase 1; TRAFs, tumor necrosis factor receptor-associated factors; XIAP, X-linked inhibitor of apoptosis protein [Color figure can be viewed at wileyonlinelibrary.com] cytokines that contribute to chronic inflammation. 21,68 Furthermore, increased cancer risk is also associated with the presence of polymorphisms in genes CARD4 and CARD15. 21 These polymorphisms can produce altered NODs with   disrupted cytokine-producing profiles and therefore pose an increased risk, causing inflammation and cancer. Briefly,   NOD2 gene polymorphisms have been associated with increased risk of lymphoma, colorectal, gastric, breast, ovarian,   lung, and laryngeal cancers while NOD1 gene polymorphisms have been linked to increased risk of lymphoma, gastric, colorectal, ovarian, prostate, and lung cancer, as well as the cancer types whose etiology is related to Crohn's disease and sarcoidosis. 21 NODs have been studied to a greater extent in cancers of the gastrointestinal tract, such as colorectal cancer (CRC) and gastric cancer (GC), although studies in breast cancer, oral squamous cell carcinoma (OSCC), head and neck squamous cell carcinoma (HNSCC), and pancreatic cancer (PC) have also been conducted (Table 1). Stimulation of NOD1 and NOD2 was found to be protective in inflammation-induced CRC, [69][70][71]80 whereas there was no straightforward answer as to whether activation of NOD1 in the stomach promotes or prevents the development of GC. [72][73][74] Moreover, NOD1 was found to be upregulated in PC, 79 HNSCC, 77,78 OSCC, 76 and GC, 73,74 as opposed to certain studies that reported NOD1 downregulation in the cases of OSCC 75 and GC. 72 NOD2 was also found to be upregulated in GCs. 74

| TARGETING NOD RECEPTORS IN CANCER IMMUNOTHERAPY
Extensive research has shown that cancer is not just a group of malignant cells but a complex structure within a tumor microenvironment (TME). [81][82][83] Besides malignant cells, TMEs comprise a variety of immune and nonimmune cell types that, in concert with the many other factors that they secrete, create an effective environment that favors tumor growth and metastatic dissemination. 8,81 Infiltration of TME by immune cells such as macrophages, lymphocytes, natural killer (NK) cells, and DCs in the early stages of tumor development is crucial for an appropriate anticancer immune response. Unfortunately, the beneficial effect produced by these cells is often inhibited by the action of immunosuppressive cells, including regulatory T cells, type 2 (M2) macrophages, and myeloid-derived suppressor cells (MDSCs) that also infiltrate the TME of developing tumors. 83 In such an immunosuppressive environment, cancer cells are able to adapt and remain undetected by host immunosurveillance. 83 The overarching goal of cancer immunotherapy is to overcome the immunosuppression in TME, thereby enabling immune cells to effectively eliminate cancer cells without causing intolerable side-effects. 84 To achieve this, various strategies have been used, among which targeting of PRR, including NOD1 and NOD2, constitutes an interesting and novel approach that could be used as an adjunct to current cancer therapies. 8 In terms of anticancer activity, NOD agonists can act as (i) immunotherapeutics or (ii) adjuvants in cancer vaccines whereas NOD antagonists have recently proposed to mediate their antitumor activity by preventing the formation of an inflammatory TME.

| NOD agonists as immunotherapeutic agents
When NOD agonists act as immunotherapeutics, they activate the cytotoxic potential of immune cells residing in the TME and, consequently, facilitate their engagement with cancer cells. Such enhancement of anticancer immunity has been investigated in the context of NOD2 agonists which, mainly, activate monocytes and macrophages 85 although stimulation of NK cells 86,87 and DCs 88-90 has also been reported. In general, two possible mechanisms on how NOD agonists activate the antitumor activity of macrophages have been suggested. They can either induce tumoricidal macrophages that, in turn, attack cancer cells or stimulate macrophages to mediate anticancer activity indirectly by the release of pro-inflammatory molecules and other factors. Moreover, macrophages also collaborate with Th1 cells to effectively recognize and eliminate malignant cells. Specifically, type 1 (M1) macrophages and Th1 cells reinforce one another, with M1-produced IL-12 maintaining the Th1 phenotype and Th1-produced IFN-γ maintaining the M1 phenotype. 91 In this Th1-driven environment, pro-inflammatory NABERGOJ ET AL.  In addition to immunostimulation, there is evidence suggesting that NOD1 agonists also possess anticancer activity. [104][105][106] In the 1980s, researchers from Fujisawa Pharmaceutical Co synthesized a series of structurally related meso-DAP incorporating analogs that exhibited antitumor activity in vivo. 104  In subsequent studies, 4 was indeed shown to stimulate NK cells and induced tumoricidal activities of murine macrophages. 105,106 Remarkably, small amounts of 4 (more than 0.1 µg/kg) stimulated NK cell activity and inhibited experimental lung metastasis formation when administered prophylactically 2 or 3 days before inoculation of B16 melanoma cells. 106 Conversely, in the study of Inamura et al 106  were not effective in controlling pulmonary metastasis when administered 3 days after B16 tumor cell inoculation, they probably destroy only circulating but not extravascular, metastatic cells. Interestingly, repeated intravenous or subcutaneous injections of 4 given at high doses of 1 to 10 mg/kg after B16 tumor cell inoculation significant reduced the number of pulmonary metastases in an established experimental lung metastasis model. 106 In our opinion, the difference between the efficacy of prophylactic and therapeutic treatment is probably due to the different mechanisms responsible for the antimetastatic activity. Namely, it is likely that antimetastatic effect observed in the case of therapeutic treatment involves macrophage activation, and not NK cell activation since

| NOD2 agonists
MDP (9) is the minimal structural component of PGN that activates NOD2. Structurally speaking, MDP is a small molecule composed of an N-acetylmuramic acid linked to a dipeptide consisting of L-alanine (L-Ala) and D-isoglutamine (D-isoGln) ( Figure 4). Due to its low molecular weight, MDP is highly water soluble and rapidly excreted from the body, 107 resulting in weaker in vivo activity. 108 To overcome this issue and obtain NOD2 agonists with improved immunostimulatory and anticancer activities, the parent structure of MDP has been (i) incorporated into different nanocarrier delivery systems such as liposomes and nanocapsules or (ii) equipped with various classes of compounds including lipophilic molecules, biomolecules, drugs, and many others. To date, several hundred MDP analogs have been synthesized (reviewed in 109,110 ). According to the type of modifications in MDP, NOD2 agonists possessing anticancer activities can be classified into four groups namely, lipophilic derivatives, hydrophilic derivatives, conjugates, and desmuramylpeptides.

| Lipophilic MDP derivatives
As noted previously, lipophilic derivatives with improved immunostimulatory and anticancer activities have been

Mifamurtide
Muramyl tripeptide phosphatidylethanolamine or MTP-PE (10; Figure 5) is a fully synthetic lipophilic derivative of MDP that has monocyte-and macrophage-activating properties similar to those of the parent compound, with additional improvements in terms of longer half-life in plasma and lower toxicity. 108 In mifamurtide, 10 is encapsulated into multilamellar liposomes by combining the active substance with phospholipids at a ratio 1:250.
This formulation facilitated the delivery of 10 to monocytes and macrophages, especially those in the liver, lungs, and spleen. Following phagocytosis by monocytes and macrophages, liposomes incorporating 10 are degraded and release 10, resulting in activated monocytes and macrophages. 111 It has been proposed that the anticancer activity of 10 is associated with its ability to induce tumoricidal monocytes and macrophages that attack cancer cells directly, as well as with the release of pro-inflammatory molecules such as TNF-α, IL-1, IL-6, IL-8, and IL-12. [112][113][114][115] In vitro studies showed that human monocytes activated by mifamurtide selectively recognized and killed tumor | 1459 many similarities with osteosarcoma in humans. In both, dogs and humans, osteosarcoma arises from long bones and has the same pattern of metastasis, with more than 80% of metastases occurring in the lungs. Specifically, mifamurtide (2 mg/m 2 , dosed twice weekly for 8 weeks) significantly improved overall survival as compared with placebo in dogs with spontaneous osteosarcoma and splenic hemangiosarcoma when used as part of adjuvant therapy after resection of primary tumor. [121][122][123][124] In contrast, mifamurtide was not effective in mice with high tumor burden, or in cats and dogs with mammary metastatic tumors, which suggests that the impact of macrophage activation on controlling tumor growth depends on tumor burden and tumor location. 125 On the other hand, mice immunized with tumor antigens derived from unrelated P825 tumor and 10 were not able to reject a challenge with SL2 cells evidently highlighting the need to develop specific antitumor immunity. The obtained results also revealed that mouse peritoneal macrophages isolated from immunized mice 5 to 7 days after tumor challenge demonstrated high nonspecific cytotoxicity in vitro (macrophages destroyed the SL2 as well as the nonrelated P815 cells), and that no major cytotoxic lymphocyte activity or substantial cytotoxic antibody titers were detectable. These results indicate that although tumor cells can be destroyed by nonspecific macrophage cytotoxicity, T cells should be involved at least in the induction of tumor immunity due to the specificity in the tumor rejection. Although exact mechanism underlying inducing antitumor immunity due to the specificity in the tumor rejection by liposomal formulations containing tumor antigens and 10 has not been fully characterized, 10 clearly showed the potential to be used as an adjuvant in cancer vaccines.
The clinical efficacy of mifamurtide has been evaluated in a large number of phase I and II trials. 132 Additionally, one large, randomized, prospective, open-label, multicenter phase III trial (Intergroup Study 0133) has been conducted evaluating the addition of mifamurtide to a three-drug combination (DOX, CDDP, and high-dose MTX) and to a four-drug combination (DOX, CDDP, high-dose MTX, and IFO) chemotherapy for the treatment of osteosarcoma. 132,133 Briefly, after the resection of the primary tumor, three-or four-drug chemotherapy was given to patients to complete the full course of therapy. Mifamurtide treatment started simultaneously with chemotherapy following surgery. Mifamurtide was administered intravenously at doses 2 mg/m 2 twice weekly for 12 weeks, and then twice weekly for an additional 24 weeks (altogether 48 doses in 36 weeks). Importantly, the results demonstrated that addition of mifamurtide was associated with a statistically significant improvement in 6-year overall survival (but not event-free survival) in patients with newly diagnosed, high-grade, nonmetastatic, resectable osteosarcoma in comparison to the patients who did not receive mifamurtide treatment (P = 0.03). 132,133 Currently, mifamurtide holds an orphan drug status in the United States and is marketed in Europe for the treatment, in combination with other chemotherapeutics, of high-grade, nonmetastatic, resectable osteosarcoma in children, adolescents, and young adults (aged between 2 and 30 years), following complete surgical removal. The recommended regimen of mifamurtide is 2 mg/m 2 intravenously administered over 1 hour twice weekly (at least 3 days apart) for an initial 12 weeks, followed by 2 mg/m 2 once weekly for additional 24 weeks (it amounts to 48 doses in 36 weeks). In general, mifamurtide therapy is safe and well tolerated. The major adverse events are fever and chills, which are usually transient and associated with initial administration. Most patients rapidly develop tolerance leading to no adverse events with subsequent administration. 115,132 Although the exact mechanism by which mifamurtide improves overall survival in patients has not been fully elucidated, it may eliminate micrometastasis or tumor cells after surgery that are not removed by or are resistant to, chemotherapy. 115,132 In our opinion, it may be possible that mifamurtide could exhibit similar beneficial effects in the therapy of other cancers, especially those which predominantly metastasize to the lungs. Mifamurtide should be further examined to ascertain and potentially harness its potential in the treatment of other cancers.

Romurtide
Romurtide (11), also known under names MDP-Lys(L18) and muroctasin, is a synthetic stearoyl-MDP derivative and an effective immunostimulant in vitro and in vivo. 134,135 When injected subcutaneously for 10 consecutive days into healthy cynomolgus monkeys 11 (1 mg/dose) significantly increased the number of peripheral neutrophils, monocytes, and platelets. This effect may be the consequence of the ability of 11 to augment the production of several cytokines by the monocytes including colony-stimulating factors (CSFs), IL-1, and IL-6, which have central roles in the regulation of hematopoiesis. 136 Similar results, in terms of increased hematopoiesis, were also obtained in immunosuppressed mice in which multiple injections of 11 (100 µg/dose) effectively restored the white blood cell count, mainly due to an increase in neutrophil counts. 137 In both studies (healthy cynomolgus monkeys and mice), the increase in white blood count may be attributable to the augmenting effect of 11 on the production of CSFs, followed by the proliferation and differentiation of stem cells in bone marrow. 136,137 Due to the success in animal studies, 11 was entered into clinical trials in which it demonstrated a restorative effect on leukopenia in cancer patients. 138,139 In 1991, 11 (trade name Nopia) was put in the market in Japan for treating cancer patients with leukopenia induced by chemotherapy or radiotherapy. 134,135 Besides its important role in stimulating hematopoiesis and immune functions, 11 also elicits antitumor immunity against tumors and metastases in vivo. 88

MDP-GDP
Conjugation of MDP to GDP led to the formation of lipophilic MDP-GDP (19) which was recognized as a very potent immunostimulant, especially when incorporated into liposomes. Specifically, liposomal 19 was able to induce the cytotoxic activity of macrophages in in vitro and in situ studies, as well as antimetastatic activity in vivo. [153][154][155][156][157][158] Phillips et al 153

| MDP conjugates
MDP is rapidly excreted from biological systems, resulting in a weaker stimulation of immune cells in vivo. As described earlier, its activity can be enhanced by introducing lipophilic moieties or by its incorporation into liposomes. Another mode of enhancing the biological activities includes the conjugation of MDP to macromolecules, such as IgG, IgM, BSA, fibronectin, cholesterol, and 10-mer polyguanylic acid. As in the case of liposomes, biomolecules facilitate the transport of MDP and enable its phagocytosis by target cells, monocytes, and macrophages. Within these cells, molecules are released and then bind to NOD2 resulting in the activation of NOD2 signaling pathway. Alternatively, MDP has also been conjugated to small molecule drugs such as paclitaxel, batracylin, and acridine. In contrast to MDP-biomolecule conjugates which mainly induce the antitumor activity of monocytes or macrophages, these conjugates induce anticancer activity either by direct cell killing or via the modulation of TME.

Conjugates with biomolecules
The to untreated controls, respectively. In contrast, the inhibition of metastasis was not observed in the absence of pretreatment. 165 Results suggested that administration of 23 before tumor inoculation allows the circulating tumor cells to encounter activated macrophages. However, after the establishment of metastases in liver parenchyma, access by the activated liver macrophages is more restrained. In an attempt to enhance the antimetastatic activity of 23 in the liver, nanocapsulated 23 was tested in combination with nanocapsules containing indomethacin, a nonsteroidal anti-inflammatory drug, based on the finding that liposomal indomethacin demonstrated antimetastatic activity in mice bearing 3LL Lewis lung carcinoma. 166,167 Two separate injections of nanoencapsulated 23 (5 µg/dose) and nanoencapsulated indomethacin (100 µg/dose) beginning 2 days before tumor cell injection resulted in enhanced antimetastatic activity which seemed to be additive. 166 The exact mechanism of additive antimetastatic activity has yet to be elucidated but may, at least in the case of 23, include activation of monocytes and macrophages. Attempts have also been made to improve the anticancer activity of MDP by binding it to various protein carriers, namely neoglycoproteins, 168 antibodies, 169

Conjugates with small molecule drugs
Natural and synthetic acridines/acridones are known as potent cytotoxic agents but their clinical application is limited or has even been discontinued due to their severe side effects. 173  | 1467 expression and production of TNF-α and IL-12, particularly at a concentration of 5 µM or higher in a dosedependent manner. Interestingly, the ability of 31 to induce expression and production of TNF-α and IL-12 even surpassed that of PTX at a concentration of 5 µM. 177 The ensuing in vivo experiments on mice bearing metastasis of LLC, however, showed that 31 was completely devoid of antimetastatic activity. 178 To obtain analogs of MDP with antitumor and antimetastatic activities, compound 31 was further modified by replacing the muramic acid moiety by various aromatic groups, leading to the discovery of MTC-220 (32). 178  factors, and MMPs, thus leading to the increased growth and survival of tumor cells. 179 Although 32 exerts its antimetastatic activity through modulation of TME, there is still a lot unknown about the exact mechanism of this modulation. It has been speculated that 32 could also inhibit the TLR4 signaling pathway in cancer cells given the fact that the structure of 32 contains the PTX motif, which has been shown to bind to TLR4 receptors.

| Hydrophilic MDP derivatives
GMDP GMDP (33; Figure 7) is a hydrophilic MDP derivative with an N-acetylglucosamine residue attached to N-acetylmuramic acid by a β(1,4)-glycosidic bond. It is an effective immunomodulator, already marketed in Russia as Likopid, for combined treatment of various infectious diseases. 180,181 In addition to its immunomodulatory activities, 33 also mediates antitumor activity against several murine tumors including adenocarcinoma, LLC, melanoma, and sarcoma. The GMDP-mediated growth inhibition of these tumors was, however, less than 60%. 182 Furthermore, 33 also exhibits antimetastatic activity when given as prophylactic treatment. Experiments in LLC bearing mice showed that 33 reduced, 4.4-to 5.6-fold, the number of metastases as well as their size (7-10-fold), as a result of GMDP-mediated activation of NK cells. 183 Since several studies revealed the ability of muramyl peptides indicating that 33 accelerated the TNF-α-induced apoptosis. 185 Furthermore, even better results in terms of cytotoxicity were observed when 33 was used in combination with TNF-α and anticancer drugs such as actinomycin D (ActD) and CDDP in vitro. 185,186 For example, treatment of L929 cells with a TNF-α in combination with ActD (4 µg/mL) resulted in 100% dead cells at 250 U/mL concentration, whereas in the presence of 33 (1.4 µM), a similar effect was observed even when TNF-α and ActD were used at much lower 50 U/mL and 1 µg/L concentrations, respectively. 185 In addition, 33 (1 µg/mL) also significantly potentiated the cytotoxic effect of TNF-α (500 U/mL) and CDDP (3-6 µM) against several other murine and human cell lines (L929, EAT, U-93, and MCF-7). 186 Besides antitumor activity in vitro, 33 also exhibited antitumor activity in an in vivo setting. Specifically, it augmented the antitumor activity of TNF-α and CDDP against Ehrlich ascites carcinoma and against melanoma B16 bearing mice. 187 More importantly, treatment of mice with 33 at a dose as low as 0.05 µg/mouse decreased the toxicity of CDDP (40 µg/mouse)/TNF-α (500 U/mouse) combination and normalized changes in hematological parameters (decreased lymphocytes, increased monocytes, and neutrophils) attributed to CDDP/TNF-α treatment. 187 Results obtained in these studies indicated that 33 synergizes with TNF-α (and anticancer drugs) as well as augments anticancer effect of TNF-α, one of the essential cytokines involved in regulation of cancer. Despite promising anticancer activity, the clinical application of TNF-α is limited due to its high toxicity and deleterious side effects. 188 Combination of 33 with TNF-α and anticancer drugs reduced therapeutic doses of TNF-α and anticancer drugs as well as augmented their therapeutic effect. This is particularly important since chemotherapeutics used in therapeutic doses, besides cancer cells, kill also normal rapid-dividing cells such as the cells of bone marrow. The utilization of drugs characterized by synergistic effects, therefore, enables the reduction of therapeutically efficient doses, thereby decreasing the toxicity against normal cells.
F I G U R E 7 Hydrophilic muramyl dipeptide (MDP) derivatives with anticancer activity NABERGOJ ET AL.

| 1469
Murabutide Replacement of D-isoGln with a D-Gln-n-butyl-ester residue in the peptide part of MDP afforded murabutide (34), another hydrophilic MDP derivative, that has similar adjuvant activity but lacks the pyrogenicity and toxicity of MDP. [189][190][191][192] Besides its adjuvant activity, 34 has also been shown to activate the immune system to fight cancer.
Although in vast majority of studies MDP derivatives have been shown only to enhance anticancer activity of monocytes and macrophages, a study of Vidal et al 89  and IL-2 (5 × 10 6 U/kg, 5 or 3 times per week) following 2 weeks of treatment. In fact, complete tumor regression was achieved in nearly 70% tumor-bearing mice when they were treated with both compounds together. 193 Importantly, concomitant treatment with both compounds was well-tolerated since the net gain in body weight was not significantly different from that observed in the control group. The exact mechanism on how 34 potentiates antitumor activity of IL-2 has not been elucidated in this study. On the basis of results of cytokine profile induced by a combination of 34 and IL-2 in vitro, it was suggested that the antitumor effect may be due to induction of the Th1 cytokines IL-12 and IFN-y. 193 In addition, stimulation of other effector mechanisms such as activation of NK cells killing cell activities could also be involved in potentiated antitumor effect. Similarly, synergistic antitumor activity was also observed when Meth A fibrosarcoma bearing mice were treated with 34 in combination with IFNα/β. 194 Multiple injections of 34 (10 mg/kg) and IFN-α/β (1.25 × 10 6 U/kg), both given three times per week following 2 weeks of treatment into Meth A fibrosarcoma bearing mice resulted in almost 50% tumor-free mice. In sharp contrast, treatment of tumor-bearing mice with IFN-α/β or 34 by themselves did not bring about a significant regression of tumor size. 194 Collectively, the conducted studies show a limited efficacy and dose-dependent toxicity of therapeutic cytokines in anticancer therapy. In our opinion, the utilization of safe immunomodulators such as compound 34, capable of potentiating the anticancer activities of cytokines could, therefore, represent a significant advantage in the therapy of cancer. By employing this approach lower doses of therapeutic cytokines are needed for anticancer activity resulting in reduced toxicity, typically associated with high-dose and long-term administrations.

| Desmuramylpeptides
The finding that the presence of the N-acetylmuramyl moiety is not necessary for the immunomodulatory properties of MDP led to the design and synthesis of a new class of MDP derivatives, termed desmuramylpeptides. [195][196][197][198][199][200][201][202] Desmuramylpeptides are devoid of the N-acetylmuramyl moiety and have therefore more lipophilic character than MDP. This class contains compounds that are able to enhance host defense against microbial infections as well as exhibit strong adjuvant activity and, even, remarkable antitumor potency. 202 The latter was extensively studied using two nor-MDP analogs, namely LK-409 (35; Figure 8) and LK-410 (36), in which the N-acetylmuramyl moiety was replaced by the N-(7-oxooctanoyl) and N-trans-2-((2′-(acetylamino)cyclohexyl)oxy) acetyl groups, respectively. 203,204 It was demonstrated that treatment of SA-1 fibrosarcoma bearing mice with multiple intraperitoneal injections of 35 (2.5 or 25 µg/dose) or 36 (25 µg/dose) administered 5 consecutive days after tumors achieved 35 mm 3 in size resulted in a moderate but statistically significant inhibition of tumor growth measured as tumor growth delay (time required for the tumor to achieve a volume of 150 mm 3 ). An antitumor effect was substantially augmented when either 35 or 36 were administered together with TNF-α analog TNFNv3.
The most significant tumor growth delay was seen when mice with established SA-1 tumors received 2.5 µg of 36 (five injections in 5 consecutive days) and 5 × 10 5 U of TNFNv3 (three injections given every second day), and was prolonged to 9.2 days when compared to untreated controls and 3.2 days when compared to TNFNv3 (5 × 10 5 U/ dose) treated mice. Remarkably, 36 also reduced the side effects of TNFNv3 in mice, resulting in lower body weight loss and better general conditions. 202 Results indicate that desmuramylpeptides effectively potentiated the antitumor activity of TNF-α analog, which is especially desirable since lower doses of compounds, in comparison to both compounds by themselves, are needed to achieve a similar antitumor effect. The exact mechanism on how these nor-MDP analogs mediated antitumor activity has yet to be elucidated, but it most probably includes activation of macrophages. Moreover, 35 and 36 also demonstrated pronounced immunorestorative effects in vivo. 203,204 The effectiveness of 35 even surpassed that of romurtide (11) in restoring the activity of the immune response in tumor-bearing mice and in immunocompromised animals evaluated in numerous in vitro and in vivo experiments. 204 On the other hand, multiple injections of 36 at doses 10 and 100 mg/kg significantly increased the survival of mice suppressed by CTX and challenged with a suspension of Candida albicans. 203 To evaluate the mechanism underlying its immunorestorative activity, 36 was further evaluated in several immunopharmacological models. It has been shown that 36 stimulated maturation of B cells as well as increasing the activity of B cells, T cells, and macrophages, but had no effect on cell counts. 203 Since desmuramylpeptides have shown promising antitumor activity when used in combination with cytokines in addition to their ability to restore immune cell functions, in our opinion they could represent an excellent adjunct to current cancer therapy. Also, based on the observation that desmuramylpeptides can potentiate the antitumor activity of cytokines, it might be interesting to also test them in combination with other chemotherapeutics at low doses, potentially resulting in fewer side effects. Finally, desmuramylpeptides with immunorestorative activity could also restore the immune cell functions often impaired by chemotherapeutics, which is of particular importance, given the fact that the patients with the impaired immune system are more vulnerable to infection.

| SAR of MDP
The structure of MDP in correlation with its adjuvant and antitumor activities has been widely studied.
Investigations of their structure-activity relationship (SAR) are summarized in Figure 9.
Modifications of the peptide moiety L-histidine (L-His)) than that of MDP. 206-208 D-isoGln (α-amide) is an essential part of MDP, since its substitution with L-Glu (γ-amide) or D-aspartic acid (D-Asp) (side chain shortened by one methylene group) produces analogs devoid of adjuvant activity. 209 In the context of the antitumor activity, conjugation of MDP at the C-terminal end of the peptide part with lipophilic molecules such as stearic acid, dipalmitoyl phosphatidylethanolamine, GDP, cholesterol, m-nitrocinnamic acid, acridine analogs, and paclitaxel analogs resulted in analogs with antitumor activity.

Modifications of the carbohydrate moiety
Besides substitutions in the peptide portion, modifications can also be introduced into the carbohydrate moiety of MDP. The hydroxyl group at the C1 position can be eliminated, replaced by thiol, or substituted by αor β-glycosides without loss of adjuvant activity. 210  structure of MDP led to analogs with enhanced adjuvant activity. 210,214 The chiral center of the lactic acid moiety at the C3 position appears to have a minimal effect on the biological activities of MDP. Elimination of the methyl group at the chiral center, or its substitution with 3′-n-propyl group, gives MDP analogs with adjuvant activity but lower toxicity than MDP. 210 Substitution of the hydroxyl group at the C4 position is not that common, although stimulated the cytotoxic activity of NK cells derived from healthy and Ab melanoma-bearing animals. 86 Moreover, some studies also showed that replacement of the N-acetylmuramic acid in MDP with certain acyclic, cyclic, and carbohydrate moieties led to the retained immunomodulatory activity of MDP and, in some cases, even exhibited antitumor activities. [195][196][197][198][199][200][201][202] 5.3 | NOD1 and NOD2 antagonists NOD antagonists have recently been recognized as potential anticancer agents with a unique mechanism of action.
In sharp contrast to NOD agonists, which generally produce a pro-inflammatory TME and enhance the ability of immune cells to fight cancer cells, it has been proposed that NOD antagonists mainly mediate their antitumor activity by preventing the formation of an inflammatory TME. Several compounds have so far been synthesized, among which DY-16-43 (37; Figure 10), 38, MDC-405 (39) and salutaxel (40) exhibit the most effective anticancer activity.
F I G U R E 1 0 NOD1 and NOD2 antagonists with anticancer activity. NOD, nucleotide-binding oligomerization domain NABERGOJ ET AL.

| 1473
An NOD2 antagonist 37 (a noncleavable analog of 32) markedly increased the therapeutic efficacy of PTX. It has been shown that concomitant treatment of LLC tumor-bearing mice with multiple injections of PTX (12 mg/kg) and 37 (30 mg/kg) resulted, not only in the reduction of tumor weight but also in prevention of tumor metastasis.
Surprisingly, treatment with 37 alone demonstrated no significant improvement in inhibition of tumor growth and prevention of metastasis. It was therefore proposed that, in mice, NOD2 was activated mainly by DAMPs generated as a result of the treatment with PTX and, in turn, resulted in TME remodeling, chemoresistance, and metastasis. Furthermore, it was suggested that 37 prevented the establishment of an inflammatory TME by blocking DAMPs and therefore sensitized the chemotherapeutic response of PTX. 217  Under the same experimental conditions, 5.7 mg/kg of DTX inhibited tumor growth only by 19%. Moreover, compound 40 also exhibited a significant antimetastatic effect at all doses used (5, 10, or 20 mg/kg) and was also significantly superior to DTX in the prevention of tumor metastasis when administered at doses of 10 or 20 mg/kg.
In addition, compound 40 (10 mg/kg) was also tested in combination with DOX (4 mg/kg) and exhibited superior antitumor and antimetastatic effect in 4T1 carcinoma model in comparison to DTX (5.7 mg/kg)/DOX combination.
Importantly, no significant body weight loss in mice was observed under the experimental conditions indicating that compounds were well tolerated at all tested concentrations. Mechanistically, 40 suppressed the accumulation of MDSC in the spleen of tumor-bearing mice and decreased levels of several pro-inflammatory molecules. 219 As noted previously, MDSCs infiltrate the TME of developing tumors and promote their invasion and metastasis, most probably through the release of MMPs. 220 Compound 40 was reported to significantly suppress MMP9 expression in the serum, spleen, and lungs of tumor-bearing mice as well as to decrease the serum levels of tissue inhibitor of metalloproteinase (TIMP) 1 whose activity is also associated with the progression of several cancer types. 221 Moreover, lung tissue derived from 4T1 tumor-bearing mice treated with compound 40 demonstrated a significant decrease in the mRNA levels of prokineticin (PROK) 2, MMP8, S100 calcium-binding protein A8 (S100A8) and S100A9, which might be also critically involved in metastasis formation. 219 In addition, compound 40 also suppressed accumulation of neutrophils in the blood of 4T1 tumor-bearing mice. This effect could be advantageous, since several studies have shown a correlation between elevated blood neutrophil count and poor clinical outcome in many cancers. 222,223 Moreover, it has been shown that dysfunctional neutrophils express reduced levels of NOD1 and that treatments that blocked the NOD1/NF-κB pathway resulted in inhibition of neutrophil migration and their phagocytic killing capacity. 224 Due to these findings, it has been proposed that compound 40 inhibits neutrophil recruitment by blocking the NOD1 pathway.

| CONCLUSION
To date, a plethora of NOD ligands have been synthesized among which several compounds showed potent antitumor and even antimetastatic activity in numerous in vitro and in vivo studies (Supporting Information Table   S1) as well as in clinical trials. Specifically, NOD2 agonist mifamurtide certainly represents the most important compound to have been granted with marketing authorization by the European Medicine Agency for treating osteosarcoma in combination with other chemotherapeutics, following complete surgical removal of a primary tumor. For a long time, only NOD agonists were considered as promising antitumor and antimetastatic compounds.
Recently, a shift in this paradigm has occurred with the emerging knowledge that NOD antagonists could also facilitate the elimination of some cancers. Incidentally, the NOD1 antagonist salutaxel currently holds the status of an investigational new drug for cancer therapy. Furthermore, preclinical studies have also shown the great potential of NOD ligands for fighting cancer in a synergistic manner, in combination with chemotherapeutics, TLR ligands, cytokines, or when used as adjuvants in cancer vaccines.
To conclude, the innate immune receptors NOD1 and NOD2 constitute promising targets in cancer immunotherapy. It is, however, unlikely that NOD ligands alone could be sufficient for complete elimination of cancer, but their tumor-suppressing capacities could be harnessed by introducing them as adjuncts to already existing cancer immunotherapies or to traditional cancer therapies such as chemotherapy and radiation.