Targeted systemic dendrimer delivery of CSF‐1R inhibitor to tumor‐associated macrophages improves outcomes in orthotopic glioblastoma

Abstract Glioblastoma is the most common and aggressive form of primary brain cancer, with median survival of 16–20 months and a 5‐year survival rates of <5%. Recent advances in immunotherapies have shown that addressing the tumor immune profile by targeting the colony‐stimulating factor 1 (CSF‐1) signaling pathway of tumor‐associated macrophages (TAMs) has the potential to improve glioblastoma therapy. However, such therapies have shown limited successes in clinical translation partially due to lack of specific cell targeting in solid tumors and systemic toxicity. In this study, we present a novel hydroxyl dendrimer‐mediated immunotherapy to deliver CSF‐1R inhibitor BLZ945 (D‐BLZ) from systemic administration selectively to TAMs in glioblastoma brain tumors to repolarize the tumor immune environment in a localized manner. We show that conjugation of BLZ945 to dendrimers enables sustained release in intracellular and intratumor conditions. We demonstrate that a single systemic dose of D‐BLZ targeted to TAMs decreases pro‐tumor expression in TAMs and promotes cytotoxic T cell infiltration, resulting in prolonged survival and ameliorated disease burden compared to free BLZ945. Our results demonstrate that dendrimer‐drug conjugates can facilitate specific, localized manipulation of tumor immune responses from systemic administration by delivering immunotherapies selectively to TAMs, thereby improving therapeutic efficacy while reducing off‐target effects.

is less than 5%. 2 In addition, patients face significant impacts to their cognitive functions and qualities of life. 3 These prognoses have improved negligibly despite advancements in anticancer treatments, 4,5 thereby requiring the development of novel delivery strategies to address the significant technological gap between clinical needs and effective therapies.
Recent developments in cancer immunotherapies have revealed macrophage receptor signaling as a promising target due to their roles in regulating inflammatory responses. 6 The activity of macrophages is governed by their cytokine expression profile, which can be broadly categorized into the classical M1 and alternative M2 phenotypes. 7 To facilitate tumor progression, resident microglia and infiltrating macrophages are repolarized into an M2-dominant phenotype through signals secreted by tumor cells. 8 These tumor-associated macrophages (TAMs) have been implicated in tumor initiation, progression, metastasis, and drug resistance in a variety of cancers. 9,10 Targeting TAMs to shift their polarization away from the tumor-supporting state and toward an antitumor phenotype has become an attractive strategy in immunotherapies.
The colony-stimulating factor 1 (CSF-1) pathway has sparked interest to block tumor recruitment of macrophages into TAMs, 11 and multiple clinical trials are underway for agents targeting the CSF-1 pathway for cancer treatment (NCT02829723, NCT02452424, NCT01349049). CSF-1 phosphorylation facilitates macrophage proliferation and conversion to TAMs, as well as tumor cell invasion and metastasis. 8,12 CSF-1 receptor inhibitors such as BLZ945 has shown some promise for suppressing tumor progression in clinical studies. However, low response rates, poor brain and tumor penetration, and systemic safety concerns have been major obstacles limiting their clinical translation. 13,14 Therefore, targeted delivery vehicles that can bring immunotherapies from systemic circulation into brain tumors and specifically to TAMs while limiting off-target activity may achieve significant improvements to glioblastoma treatment.
Hydroxyl-terminated polyamidoamine (PAMAM) dendrimers are promising nanocarriers for delivering therapies specifically to activated microglia/macrophages from systemic circulation. 15,16 Their small size, neutral surface charge, and high density of surface hydroxyls enable them to cross the blood brain barrier and localize within activated microglia/macrophages without targeting moieties.
This intrinsic targeting has been shown to significantly enhance the efficacy of antioxidants by targeting activated glia in neuroinflammation. 15 In orthotopic brain tumor models, we have shown previously that systemically administered hydroxyl PAMAM dendrimers penetrate throughout the solid tumor and localize selectively in TAMs in an orthotopic model of malignant glioma. 17,18 These dendrimers are also well-positioned for clinical translation due to their safe profile in vivo and scalability. 19,20 Here, we present a novel nanoparticle formulation of BLZ945 conjugated to the generation 4 hydroxyl PAMAM dendrimer via a triggered release linker that can locally repolarize the tumor immune profile for improved efficacy in glioblastoma treatment.

| Preparation and characterization of dendrimer-BLZ945 conjugate
To improve formulation and delivery, we conjugated BLZ945 (BLZ) (1) to G4-OH dendrimers. The hydroxyl group on the cyclohexane ring in BLZ945(1) was modified by reacting with succinic anhydride (2) (5). 1 H-NMR spectra of compounds indicate successful formation of (3) and successful chemical conjugation of (3) to (5). Key identifying protons are denoted ml reduced phosphorylation by 50% compared to unstimulated controls. This knockdown in receptor activation translated to dosedependent decrease in expression of arginase-1 (Arg-1), an antiinflammatory cytokine that mediates tumor progression and metastasis and correlates with cancer grade and prognosis (Figure 4b). 27 Notably, during the timeframe of in vitro experiments ( TAMs in the tumor exhibited highly ameboid morphology, consistent with previous reports that TAMs exhibit loss of ramified structure correlating with immunosuppressive activation (Figure 5a). 17 TAMs in mice treated with DBLZ exhibit partial recovery of ramified structure, while TAMs in free BLZ treated mice have ameboid morphology similar to TAMs in untreated mice. Surface area to volume ratio, a measure of sphericity, indicates that DBLZ treatment significantly increased this ratio 2-fold more (+18.5% vs. control; p = .07 BLZ vs. DBLZ) compared to free BLZ (+8.5% vs. control). We have observed previously that resting macrophages exhibit 2-fold greater surface area to volume ratio than TAMs, so DBLZ treatment induced a partial recovery away from TAMs activation and toward resting phenotype. 17 Consistent with this, DBLZ significantly decreased the expression of

| DISCUSSION
Despite nanomolar binding affinity to the CSF-1 receptor, BLZ requires high doses when administered systemically to achieve therapeutic efficacy (e.g., 200 mg/kg oral daily dose in orthotopic brain tumor models). 24,32 In addition, its low aqueous solubility and potential toxicity due to ablation of systemic macrophages and microglia in healthy brain tissue are major obstacles for its clinical translation. 13,24 To overcome these challenges, we conjugated BLZ to nontoxic hydroxyl PAMAM dendrimers undergoing clinical translation to achieve specific targeting to TAMs and improve aqueous solubility. 17 We have previously demonstrated that these dendrimers penetrate into orthotopic brain tumors and localize specifically within TAMs, as well as in activated glia in central nervous system (CNS) disease models, with negligible uptake in other cell types. 16 Preclinical and clinical studies of anticancer medicines have long focused on survival and have used progression-free survival as a proxy for QoL. 43 However, recent patient surveys have revealed that survival and patient QoL are not well correlated, and studies of cancer therapies often underreport or underemphasize adverse impacts to patient wellbeing. 44 Impacts of treatments on patient QoL is therefore an important, often overlooked parameter that should be taken into consideration, particularly in glioblastoma, which is associated with significant cognitive and behavioral burden. 3 As proxies for QoL we assessed brain tumor bearing mice with parameters associated with mouse health and anxiety, including kyphosis, rearing behavior, and motor function. Kyphosis is a measure of hunched posture that is used as a marker for mouse health and in the context of neurodegeneration and glioblastoma results from spinal cord muscular atrophying. 45,46 Rearing behavior an exploratory instinct used in behavioral neuroscience as a marker for fear/anxiety associated with disease burden. 47 Mobility in open field experiments as a proxy for anxiety and fatigue has been shown to correlate with tumor burden, with mice exhibiting decreased mobility when inoculated with tumors and partial recovery upon tumor resection. 48

| BLZ945 release from dendrimer conjugate
The drug release of BLZ945 from the DBLZ were measured in PBS (pH 7.4) and citrate buffer (pH 5.5) to simulate plasma and intracellular/intratumor conditions, respectively, based on previously published procedure. 51    tumor bearing mice were used for these analyses with three images analyzed and averaged per animal. Image analyses were performed blinded.

| Survival study and evaluation of markers of disease progression
For longitudinal studies to evaluate survival and disease progression, brain tumor bearing mice were assigned randomly to a treatment group: control (n = 11), BLZ (n = 11), and DBLZ (n = 13). An intrave-

CONFLICT OF INTEREST
The authors have awarded and pending patents relating to TAMs targeting ability of high surface hydroxyl dendrimers. R. M. K. and S. K. are cofounders and have financial interests in Ashvattha Therapeutics, Inc., Orpheris, Inc., and RiniSight, three startups undertaking clinical translation of the dendrimer drug delivery platform. RS currently works with Ashvattha Therapeutics and has share ownership.
This work was performed before RS joined Ashvattha.

DATA AVAILABILITY STATEMENT
Data are available on request from the authors.