Oxazaphosphorines (Oxaza; that is, cyclophosphamide (CPA), ifosfamide (IFO)) are antineoplasic agents widely used to treat various cancers from soft-tissue tumors to lymphomas and are still the corner stone of several polychemotherapy protocols. They are major alkylating agents of polychemotherapy protocols but limiting their toxicity and increasing their efficacy could be of major interest. For IFO, dose regimens range from 1 to 12 g/m²/day in humans1 2 and 200 to 300 mg/kg in tumor-bearing mice3 when used as cytotoxic agent; for CPA dose range from 200 to 1000 mg/m²/day in humans4–6 and from 100 to 250 mg/kg in tumor-bearing mice.7–9 IFO is an isomeric form of CPA and differs chemically from CPA in the localization of one of the chloroethyl groups leading to a different metabolization. Oxaza are prodrugs that require a metabolic activation by a specific liver cytochrome P450 (CYP). CPA is mainly metabolized by CYP2B6 leading to 4-HO-CPA, the key intermediate for alkylating mustard which reacts with DNA. The bioactivation of IFO by CYP3A4 produces 4-hydroxy derivative (4-HO-IFO), a key intermediate for isophosphoramide mustard release displaying cytotoxicity by DNA crosslinks. However, only 10%–50% of IFO is transformed into this key entity, and 50%–90% of IFO releases chloroacetaldehyde (CAA), a nephrotoxic and neurotoxic metabolite.10 11 These toxicities are increased in a high-dose setting; indeed, there is a dose–response relationship for IFO, that is, the higher the dose, the higher the IFO plasma exposure.12 Several high-dose IFO protocols (HD IFO) were studied. Le Cesne et al have studied efficacy and safety profile of HD IFO administered as a rescue medication (cumulative dose of 12 g/m²) in patients with advanced refractory soft-tissue sarcomas which have not responded at standard doses of IFO. This study has demonstrated the efficacy (20 patients out of 36 showed partial or stable disease response), but with major toxicities.13 In pediatrics, the incidence of toxicities is high mostly in HD protocols.14 A clinical study in children with osteosarcoma receiving after HD IFO (cumulative dose of 14 000 mg/m²) has shown an increase of 30% of progression-free survival, but with severe nephrotoxicity in a quarter of patients.15 The need to improve the IFO therapeutic index is an important clinical issue. Preactivated oxazaphosphorines (X-Oxaza) based on the link to terpene chains have been designed in order to bypass the metabolic pathways.16 As shown in figure 1, geranyloxy-IFO (G-IFO) leads to release 4-HO-IFO without releasing CAA. Skarbek et al 17 have shown the cytotoxicity of G-IFO on a large panel of tumor models in vitro, validating the proof of concept of preactivation. As CPA and G-IFO give high levels (66%–80%) of the alkylating entity compared with IFO (10%–50%), and CPA is known for its immunomodulatory properties, we therefore decided to explore the immune properties of IFO and G-IFO compared with CPA.

Tumor microenvironment plays an important role in tumor development. Indeed, microenvironment cells such as endothelial cells and stroma, immune cells may contribute to both tumor progression or tumor regression.18 Tumor cells may also provide mediators of immune escape through the secretion of immunosuppressive factors such as transforming growth factor-beta and through the expression of ligands for immune checkpoints establishing immune tolerance. Targeting the tumor cell environment seems to be a promising strategy to increase the efficiency of conventional chemotherapeutic agents. Immune checkpoint blockers (ICB) such as antiprogrammed cell death 1 (PD-1), antiprogrammed cell death ligand 1 (PD-L1) and anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) monoclonal antibodies (mAbs) have also proved their efficacy in many tumors such as melanoma19 and non-small-cell lung cancer treatment.20 Today, the combination of immunotherapy and conventional cancer therapy provides new and innovative antitumor strategies.21 22

Conventional antitumor chemotherapy is described as a direct cytotoxic agent used for targeting the DNA integrity (alkylating agents), the DNA and RNA synthesis (antimetabolites and cytotoxic antibiotics), the DNA replication (topoisomerase inhibitors) or cytosolic compounds (antimicrotubule agents). In addition to this direct cytotoxicity, several studies also revealed that conventional antitumor chemotherapies could modulate antitumor immune response.22 Indeed, some conventional chemotherapies can stimulate the immune system by depleting regulatory T cells (Treg), by subverting immunosuppressive mechanisms or exerting immunostimulatory activities.23 CPA has a direct cytotoxicity through DNA binding at conventional doses and an indirect antitumor effect at low dose through the activation of antitumor immunity. Many reports have demonstrated the immunomodulatory effects of CPA through different mechanisms such as Treg cell depletion or inactivation,24–29 the polarization of T cells toward Th1 and Th17,30 the modulation of myeloid cells toward inflammatory cells.31 More recently, it has been suggested that induction of pathogenic Th17 cells after CPA treatment was the consequence of the modulation of intestinal microbiota composition in tumor-bearing mice.32 Studies have described the enhancement of antitumor efficacy for CPA combined with immunotherapy through induction of immunogenic tumor death.33 34 Moreover, clinical studies have suggested that low myelosuppression in IFO-treated patients may have advantages over CPA in combination therapy.35–37 Very few publications have focused on with the influence of IFO on the immune system compared with CPA and most in vitro studies have reported a negative impact of IFO on immunity. Lind et al 38 showed that IFO and its metabolites (4-HO-IFO and CAA) decrease the intracellular-glutathione (GSH) amount in the lymphocytes of patients treated with IFO at 5 g/m². GSH is the major intracellular thiol reductant that plays a protective role against oxidative injury; thus, GSH decrease affects survival, proliferation and functions of lymphocytes. Issels et al 39 confirmed that IFO could diminish GSH in lymphocyte in vitro and Multhoff et al 40 showed that IFO could decrease the cytotoxic activity of T cells. More recently, Kuppner et al 41 suggested that IFO could decrease IFNγ production by T cells. These in vitro results seem in contradiction with data accumulated with CPA demonstrating an activation of T cell immunity. To our knowledge, no study to date has explored the immunomodulatory activity of IFO in vivo.

In the current study, we studied the capacity of IFO and G-IFO to modulate T cell polarization in tumor-bearing mice. We demonstrated that low-dose IFO and G-IFO (1) favor Th1 polarization, (2) induce the T cell-dependent antitumor effect in mice and (3) increase the efficacy of anti-PD-1 antibodies in tumor-bearing mice. At high dose, IFO and G-IFO induced lymphopenia and synergy with anti-PD-1 was completely lost. Altogether, low doses of IFO and G-IFO could represent an efficient strategy to increase the response rate in patients treated with immune checkpoint inhibitors.

IFO and CPA share a common metabolic pathway; they are prodrugs requiring bioactivation by hepatic enzymes leading to their respective cytotoxic metabolites, isophosphoramide mustard and phosphoramide mustard. Since its introduction in 1958, CPA has been widely used as an antitumor agent, but myelosuppression gives the dose-limiting toxicity of CPA. Thereby, CPA is widely used as myeloablative chemotherapy. Myeloablative activity is due to the nornitrogen mustard released from CPA after hepatic metabolization into aldophosphoramide mustard and then enzymatic oxidation into carboxyphosphoramide mustard.45 46 Note that nornitrogen mustard is also a metabolite of melphalan similarly used as a myeloablative agent. IFO, an isomeric form of CPA, was developed in the early 1970s in order to overcome this myeloablative propriety. Unlike CPA and melphalan, IFO does not release nornitrogen as one of the chloroethyl group is linked to the endocyclic nitrogen while for CPA, both chloroethyl groups are bonded to the exocyclic nitrogen, possibly explaining the absence of myeloablative activity of IFO.35 Consequently, for treatment of cancers such as soft-tissue sarcomas, the use of IFO is preferred to CPA.35 Difference in toxicity between IFO and CPA may also rely on a different metabolism rate generating threefold to fivefold less of alkylating phosphoramide mustard compared with CPA,46 47 and releasing a majority of CAA which is a nephrotoxic and neurotoxic metabolite. However, the development of preactivated analogs of IFO allowed bypassing hepatic pathway and avoiding CAA release.16 Pharmacokinetic study has confirmed the proof of concept of preactivation of IFO; the toxic pathway is bypassed and a higher level of 4-HO-IFO is released.17 We confirmed the pharmacokinetic profile of G-IFO (data not shown). As CAA is an unstable metabolite and is released at equimolar quantity with DCE-IFO, we quantified N ³-DCE-IFO in order to quantify the toxic metabolites encountered on this pathway. As expected, IFO released N ³-DCE-IFO metabolite in plasma whereas G-IFO did not; as N ³-DCE-IFO is released concomitantly with CAA, we concluded that G-IFO did not release this nephrotoxic and neurotoxic metabolite.

In the present study, we scrutinized immune modifications following i.p. injection of IFO and G-IFO in mice. The B cell population seemed very affected by oxazaphosphorines even at low dose of IFO (100 mg/kg) and G-IFO (eq. 100 mg/kg; data not shown) underlining high sensitivity of B cells to the direct killing by these cytotoxic agents as previously reported.48 The presence or absence of tumor does not affect the fate of B cells after IFO or CPA injection. Several studies have previously showed that B cells were preferentially decreased compared with T cells after CPA treatment; our results brought evidence that IFO is also highly cytotoxic for B cells. B cell sensitivity may rely on the toxic activity of CPA on differentiating B cells compared with thymocytes resulting in a rapid reduction of B cell numbers.49 The B cell decrease could be an advantage in the perspective of combination of an oxazaphosphorine with immunotherapy. Immune checkpoint inhibitors such as anti-PD-1, anti-PD-L1 and anti-CTLA-4 antibodies have frequent immune-related adverse events (irAEs). Some of these irAEs are the consequence of autoantibody induction and/or increase.50 Nowadays, corticoid administration is the main treatment for serious irAEs with interruption of immunotherapy in most cases. Thus, combining ICBs with oxazaphosphorines could lead to less frequent reactivation of autoreactive B cells and less autoimmune-related adverse events.

We demonstrated the dose-dependent antitumor effect of IFO with an immunomodulatory effect at low doses (≤150 mg/kg) and a cytotoxic effect at higher doses (>150 mg/kg). Indeed, IFO at low doses did not affect numbers or proportions of splenic CD4⁺ and CD8⁺ T cells in naive and tumor-bearing mice. This immune-pharmacodynamic study has shown that IFO at low doses could upregulate the production of IFNγ, IL-17 and IL-6 after TCR stimulation particularly with IFO 150 mg/kg. At higher doses, no significant or high levels of IFNγ and IL-17 were observed consistent with the decrease of splenic T cells. Interestingly, G-IFO at high dose (150 mg/kg) seems to trigger a non-specific splenic and intratumoral T cells decrease; however, higher secretions of IFNγ and IL-6 compared with vehicle were observed. These data highlight a difference of interaction for IFO and G-IFO with T cells and deserve further investigation.

Interestingly compared with CPA at 100 mg/kg, IFO 150 mg/kg induced a stronger secretion of IFNγ, IL-17 and IL-6. IL-6 is known to skew Treg differentiation into non-suppressive proinflammatory Th17 cells51 52; at 150 mg/kg, the significant correlation between IL-17A and IL-6 secretions (not observed for CPA) suggests that IL-6 could play a role toward Th17 differentiation after low dose-IFO treatment (data not shown). For CPA at 100 mg/kg, Treg differentiation into non-suppressive proinflammatory Th17 cells was not observed30 but rather the modification of gut microbiota composition was demonstrated as mandatory for CPA-induced Th17 differentiation.32 In our study, no correlation between IL-6 and IL-17A was found after CPA treatment (data not shown), suggesting that Th17 induction might not rely on IL-6-driven Th17 polarization. Although TCR-dependent induction of cytokines was investigated in our study, the precise origin of IL-6 was not clearly determined since we stimulated splenocytes and not purified T cells. IL-6 is a proinflammatory cytokine produced by several cell types including myeloid cells. As already stated, IFO is less myelotoxic than CPA35; thus, we cannot exclude that higher number of myeloid cells recovered after IFO treatment compared with CPA even at low dose. Myeloid cells are able to secrete large amounts of proinflammatory cytokine such as IL-6 after IFNγ triggering53 and could explain the higher IL-6 concentrations after IFO treatment compared with CPA, but this is yet to be clearly demonstrated. Regarding preactivated analog of IFO, the IL-6 secretion is proportional to G-IFO’s dose with similar secretion between high dose of G-IFO (eq. 150 mg/kg) and CPA 100 mg/kg. Interestingly, levels of IL-6 could be of importance since previous observation has suggested that low levels of secreted IL-6 could favor Treg cells while higher amounts of IL-6 could favor Th17 cell differentiation54 as observed with 150 mg/kg IFO treatment in tumor-bearing mice.

However, one should bear in mind that opposite roles for IFNγ and IL-6 have been described particularly in tumor cell proliferation and tumor growth through phosphorylation of STAT1 and STAT3, respectively. The role of the balance between activation of STAT1, considered as antitumoral, and STAT3, considered as protumoral, remains not totally clear.55 Consequently, we cannot rule out that higher secretion of IL-6 after IFO 150 mg/kg could be deleterious compared with CPA 100 mg/kg. Regarding IL-17A, the roles of Th17 cells depend strongly on the context of the ongoing cytokine environment.56 IL-17A associated with IFNγ production (ie, Th17.1 polarization) might have a better antitumor activity compared with a pure Th17 polarization as suggested after a single injection of low dose of CPA.30 32

Many murine syngeneic tumor models could be used to test immune-based therapy and previous data have shown that like in patients with cancer an immune diversity between these tumor models exists and dictates their capacity to respond to immune checkpoints blockers.57 MCA205 is known to be poorly sensitive to immunotherapy as tumor cells express low levels of PD-L1 (42–44); we confirmed in this study that MCA205 poorly responds to anti-PD-1 mAbs as a standalone treatment . As shown in figure 4, adding a single injection of G-IFO at eq. 100 mg/kg or CPA at 100 mg/kg to anti-PD-1 mAbs treatment improved the antitumor efficacy. The difference in synergy between the immunomodulatory doses of IFO, CPM and G-IFO showed that the synergy with anti-PD-1 is not only due to an increase in the level of studied cytokines, that is, INFγ, IL-17A and IL-6 (non-exhaustive selection). Besides, the treatments causing DNA damage lead to the release of DNA fragments in the cytosol. The interaction of cytosolic DNA with the cGAS protein leads to its activation and the production of cGAMP which will bind to its STING (stimulator of interferon genes) receptor. The CGAS/STING pathway is essential for the antitumor effect of ICB58; therefore, different STING agonists are currently undergoing preclinical evaluation and the rationale for use in combination with ICB for a synergistic antitumour effect has recently been described.59 We could hypothesize that phosphoramide or isophosphoramide mustard, released in greater quantities from CPA and G-IFO compared with IFO, is responsible for cGAS/STING activation. This would favor Th1 adaptive immune response as shown with IFNγ secretion where CPA and G-IFO induced systematically more IFNγ than IFO. Interestingly, a potent synergy was observed with anti-PD-1 mAbs associated with G-IFO eq. 100 mg/kg. Thus, G-IFO at low dose seemed pertinent to leverage anti-PD-1 mAbs activity. Previous studies have shown that some chemotherapy such as gemcitabine and paclitaxel increase the expression of PD-L1 on the surface of tumor cells.60 IFNγ and IL-6 cytokines upregulate PD-L1 expression on solid tumor cells through JAK-signal transcription pathway61 62; thus, G-IFO through adaptive T cell immunity modulation could also increase PD-L1 expression on tumor cells. This hypothesis remains to be explored. This study provided proof of concept on the synergy between anti-PD-1 and a new alkylating agent. As reported by Wu and Waxman reviewing works dealing with immunogenic chemotherapy, both dose and schedule are major parameters for a significant efficacy of the synergy.63 The authors show the huge number of combination strategies dealing with the dose of the chemotherapy (low, medium or high dose the number of repeated injections), the number of injections of the immunotherapy and the number of days between chemotherapy and immunotherapy. Indeed, there are multiple strategies that give multiple events, such as changes in the tumor-tolerant immune cells, chemotherapy treatment-induced drug cytotoxicity, lymphopenia or immune-stimulatory signals. We think that finding the optimal combination between IFO/G-IFO and anti-PD-1 could have taken many months of experimentation. Further studies are needed to find the optimal combination between IFO/G-IFO and anti-PD-1 including the dose, the frequency of the anti-PD-1 administrations, in the context of future clinical trials, knowing that transpose the results from murine models to human is not obvious.

The aim of the design of novel oxazaphosphorine was to optimize the metabolic balance while improving biodistribution through the terpenyl modification. In addition, as reported for others chemotherapeutic agents, immunogenic death or immunomodulation is linked to the dose. Gemcitabine used at 120 mg/kg in mouse model (a dose similar to the equivalent dose used in patients) selectively reduces the numbers of myeloid suppressor cells while preserving CD4⁺ T cells, CD8⁺ T cells and B cells64 whereas CPA is used at low doses for their immunomodulatory effects. G-IFO was investigated for its capability to deliver 4-HO-IFO in the same proportion as CPA. To generate immunomodulation effects and not lymphopenic effets, G-IFO, as others alkylating agents need to be used at low doses, especially in combination with ICB. Numerous clinical trials are currently studying CPA for its immunomodulatory properties, in combination with antitumor immunotherapy (vaccination and ICBs): 172 ongoing studies including 25 that study the association of low-dose CPA with anti-PD-1 (clinicaltrials.gov). G-IFO was investigated for its capability to deliver 4-HO-IFO in the same proportion as CPA. To generate immunomodulation effects and not lymphopenic effets, G-IFO as others alkylating agents need to be used at low doses, especially in combination with ICB.

Altogether, this study showed the importance of oxazaphosphorine’s metabolism and the well-balanced administered doses that favor T cell activation rather than lymphopenia and that G-IFO could be a promising chemotherapeutic agent candidate for combination with immunotherapy.