Blood–brain barrier permeable nano immunoconjugates induce local immune responses for glioma therapy

Brain glioma treatment with checkpoint inhibitor antibodies to cytotoxic T-lymphocyte-associated antigen 4 (a-CTLA-4) and programmed cell death-1 (a-PD-1) was largely unsuccessful due to their inability to cross blood–brain barrier (BBB). Here we describe targeted nanoscale immunoconjugates (NICs) on natural biopolymer scaffold, poly(β-L-malic acid), with covalently attached a-CTLA-4 or a-PD-1 for systemic delivery across the BBB and activation of local brain anti-tumor immune response. NIC treatment of mice bearing intracranial GL261 glioblastoma (GBM) results in an increase of CD8+ T cells, NK cells and macrophages with a decrease of regulatory T cells (Tregs) in the brain tumor area. Survival of GBM-bearing mice treated with NIC combination is significantly longer compared to animals treated with single checkpoint inhibitor-bearing NICs or free a-CTLA-4 and a-PD-1. Our study demonstrates trans-BBB delivery of tumor-targeted polymer-conjugated checkpoint inhibitors as an effective GBM treatment via activation of both systemic and local privileged brain tumor immune response.

G lioblastoma multiforme (GBM) is the most common and most aggressive form of primary brain tumor in adults, with an incidence of 3.2 per 100,000 population. Comprehensive advances in molecular profiling including our work under The Cancer Genome Atlas (TCGA) project for GBM consortium have led to the identification of key prognostic factors 1,2 , but this has not translated into change in therapy or survival 3,4 . Immunotherapy is one of the fastest developing approaches in clinical oncology with successful treatment of different cancers 5 . However, the unique immune environment of the central nervous system (CNS) needs consideration when pursuing immunotherapeutic approaches for gliomas. Treatment options are limited, in part because of inefficient drug delivery across the blood-brain barrier (BBB) [6][7][8] . A recently published review 9 summarized the results of immunotherapy clinical trials in glioma: 28 clinical trials for vaccines (e.g., a peptide vaccine that targets EGFRvIII or IDH1); 13 clinical trials completed for oncolytic viruses; 15 phase III clinical trials for checkpoint inhibitor antibodies (e.g., CheckMate 143 trial); and genetically modified T cells expressing chimeric antigen receptors (CAR-T cells). Unfortunately, no treatment so far has been superior to the standard-of-care for GBM, represented by temozolomide/ radiation therapy with 14.6 months average survival 9 .
Blockade of cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) using the antagonistic monoclonal antibody (mAb) ipilimumab was the first strategy to achieve a significant clinical benefit for stage IV melanoma patients 10,11 . Humanized mAbs against immune system response modulators CTLA-4 (ipilimumab), and programmed cell death-1 (PD-1) (pembrolizumab and nivolumab), received FDA approval. Their effect is due to the suppression of regulatory T cells (Tregs) and activation of antitumor immune response by cytotoxic T lymphocytes (CTLs). Systemic administration of CTLA-4 or PD-1 and programmed cell death ligand 1 (PD-L1) mAbs can suppress some tumors, but has low efficacy against brain tumors 9,12,13 .
Despite growing evidence to support an interaction between the CNS and general immune system 14 , clinical trials using nivolumab and ipilimumab in GBM showed serious safety issues, but not a significant anti-tumor treatment effect 15 . Although CTLA-4, PD-1 and other antibodies do not cross the BBB [16][17][18] , some efficacy has been demonstrated against GBM, possibly due to general immune system activation.
Recent studies highlighted significant roles of the tumor microenvironment in tumor development and progression. Tumor-associated macrophages/microglia (TAMs) are a major stromal cell component in GBM. It was shown that both mouse and human TAMs express PD-1. TAM PD-1 expression increases over time in mouse cancer models and with higher disease stage in human cancers. PD-1 expression by TAMs inhibits phagocytosis and tumor immunity [19][20][21] . Macrophage polarization into M1 and M2 phenotypes with distinct functional consequences is well established, and M1 anti-tumor macrophages have shown promise to function as a cancer immunotherapy 22,23 . M1 macrophages, in response to IFNγ or TNFα, convert arginine into nitric oxide (NO) through inducible nitric oxide synthase (iNOS) to promote anti-tumor activity. M2 macrophages, polarized by TGFβ and IL-10, accumulate in tumors and can induce Treg cells that suppress CTLs. They can also inhibit activation of NK cells through immunosuppressive TGFβ. The immune response to tumors appears to be the result of interactions between T cells (Tregs and effector cells), NK cells, and TAMs 22,23 .
In order to achieve successful immunotherapeutic effects in gliomas, the corresponding drugs should be able to cross the BBB and reach the tumor. We combine nanotechnology and immunotherapy advances 23,24 to deliver nanoscale immunoconjugate (NIC) drugs across the BBB and treat GBM. A versatile drug carrier, poly(β-L-malic acid) (PMLA), a natural polymer obtained from the slime mold Physarum polycephalum 25,26 , is used to deliver covalently conjugated CTLA-4 and PD-1 antibodies (a-CTLA-4 and a-PD-1) to brain tumor cells, which results in local immune system activation and prolonged survival of intracranial GBM GL261-bearing mice. PMLA-based nanotherapeutics target brain tumors by crossing the BBB using transferrin receptor (TfR)-mediated transcytosis 25 . We also use an alternative mechanism of delivery through BBB with PMLA-conjugated Angiopep-2 (AP-2) peptide, which is a synthetic low-density lipoprotein receptor-related protein 1 (LRP-1) ligand 27 . To our knowledge, this is the first successful use of polymer-based carriers with covalently attached immunotherapeutics to activate local immune response and treat brain tumors.
Free a-CTLA-4, a-PD-1, and a-msTfR showed somewhat higher binding affinity toward their respective antigens on a plate surface compared with polymer-bound antibodies, due to the bulkier size of the NICs. In addition, we proved the presence of pairs of two antibodies (i.e. a-CTLA-4 and a-msTfR or a-PD-1 and a-msTfR) within one single polymer chain using pull-down ELISA (Fig. 1f, g, Supplementary Fig. 1C, D). The ELISA signal intensity of a-msTfR on P/a-CTLA-4 conjugate was comparable to that of a-CTLA-4 on the same conjugate, confirming the presence of both a-msTfR and a-CTLA-4 on the conjugate (Fig. 1d). Similar results were obtained for P/a-PD-1, when the surface was coated with PD-1 (Fig. 1e), and for P/a-CTLA-4 and P/a-PD-1, when the surface was coated with msTfR (Supplementary Fig. 1A, B). ELISA results confirmed the reactivity and the presence of each antibody in the conjugates. FTIR analysis for P/a-CTLA-4 shows peaks at the O-H stretching frequencies of 2880 cm −1 (carboxylic acid O-H) that can be seen in both preand final NIC, as well as peaks that are present at the a-CTLA-4 spectrum, which are also present in the final NIC (mainly peaks at 3270 and 2953 cm −1 , which are N-H and O-H stretching frequencies, and lower frequencies peaks at 1120 and 1630 cm −1 ) (Supplementary Fig. 2A). A similar trend could also be seen for P/ a-PD-1, with significant specific peaks at 2875, 1664, 1031, and 942 cm −1 (Supplementary Fig. 2B). The analysis of the FTIR spectra, together with SE-HPLC and ELISA results, suggested that the antibodies were conjugated successfully to the pre-conjugate.
ζ-Potentials of NICs were in the range of −9.9 to −11.0 mV, reflecting the design and the intrinsic antibody charges. Hydrodynamic size was in the range of 28.0-28.5 nm by intensity, in agreement with design and molecular masses of constituents and the absence of aggregates, and endotoxin level was reduced below 0.1 EU/mL by phase separation method 31 (Supplementary Table 1).

NICs cross the BBB and reach the tumor interstitium.
Rhodamine-labeled a-CTLA-4 and a-PD-1 or their combination could hardly be detected outside of the blood vessels positive for lectins (tomato and RCA120) in the intracranial tumors at 4 and 6 h after intravenous (I.V.) injection, indicating their inability to cross the BBB and reach the tumor parenchyma (Fig. 2a top; 2b top row). In contrast, rhodamine-labeled checkpoint inhibitor antibodies covalently attached to the polymer, P/a-CTLA-4 and P/a-PD-1 (schematics on Fig. 2a bottom) or their combination, were readily detected in the tumor parenchyma 4 h after I.V. injection (Fig. 2a, b bottom row). NICs were distributed through the tumor area (but not in the healthy brain), mostly outside the blood vessels, with only occasional presence in the vessels (Fig. 2a,  b). Very similar results were obtained after vessel labeling for von Willebrand factor (Fig. 2c). Thus, NICs cross the BBB, allowing conjugated mAbs to bind to CTLA-4 and PD-1 and modulate the immune response in the tumor area. Optical imaging data analysis performed with ImageJ Fiji 6,32 revealed significant differences (F = 46.52, DF = 5, p < 0.0001 for lectins; and F = 11.36, DF = 6, p < 0.0001 for von Willebrand factor by ANOVA) between fluorescent areas outside the vessels for NICs vs. free mAbs (Fig. 2c, e). Multiple pairwise comparisons by ANOVA with Sidak's posttest also showed significantly more labeled NICattached checkpoint inhibitors or their combination than the free ones or their combination in the tumor parenchyma using both vessel labeling methods (Fig. 2c, e).

Tregs in tumor
To further study the local immune response, ex vivo brain tumors were collected near the end point of mice after treatment with NICs. CD8+ and CD4+FoxP3+ T cells were revealed by immunofluorescent staining of brain tumor cryosections and quantitated using Fiji software. For CD8+ staining, we analyzed 8499 cells in tumors from 24 mice representing different treatment groups, four mice per group (Fig. 4a, b). On average, each analyzed image contained 116 ± 25 cells, and cell densities were uniform for all data included in our analysis. We observed a significant increase in the number of CD8+ T cells in tumor tissue after NIC treatment (F = 5.383, DF = 5, by ANOVA with Sidak's posttest; p = 0.0003; Fig. 4b). The percentage of CD8+ T cells was significantly increased vs. PBS group following treatments with P/a-PD-1 (p < 0.007) and P/a-CTLA-4+P/a-PD-1 (p < 0.0001). CD8+ T cell percentage was also significantly increased after P/a-CTLA-4+P/a-PD-1 compared to treatment with a-CTLA-4 (p = 0.001) and a-PD-1 (p < 0.006) (Fig. 4b). The NIC combination showed a tendency for increase in CD8+ T cells vs. NICs with single antibodies but it did not reach significance. Overall, the combined NIC treatment was the most effective at recruiting CD8+ T cells into the tumor tissue.
To determine the effect of our drug treatments on Treg in tumors, we also measured the percentage of CD4 and FoxP3 double immunolabeled T cells. Cells that were exclusively FoxP3+ were not counted. We analyzed 9898 cells in tumor tissues from 24 mice (four mice per group) (Fig. 4c, d). On average, each analyzed image contained 127 ± 26 cells; cell densities were uniform. We observed a significant decrease in the incidence of CD4+FoxP3+T cells in treated tumor tissue (F = 7.567, DF = 5, by ANOVA with Sidak's posttest; p < 0.0001; Fig. 4d). Specifically, a significantly lower percentage of CD4+FoxP3+ T cells were seen following treatment with P/a-CTLA-4 (p < 0.009), P/a-PD-1 (p < 0.003) and P/a-CTLA-4+P/a-PD-1 (p = 0.0004) compared to PBS (Fig. 4d). These results were almost exactly inverse to CD8+ T cell data. A significant reduction of Tregs was also observed when comparing the a-CTLA-4 with the P/a-CTLA-4 (p < 0.0001) and P/a-CTLA-4+P/a-PD-1 (p < 0.0001) treatments (Fig. 4d). These data suggested that combination treatment was the most effective at inhibiting CD4+FoxP3+Tregs in the tumor tissue. The differences in Treg abundance between flow cytometry and immunohistochemistry could be due to tissue harvest for the latter analysis at the end of animals' life when NIC treatment led to marked reduction in Ki67+ proliferating tumor cells and the formation of multiple tumor necroses ( Supplementary Fig. 5). It is possible that Tregs were affected by developing hypoxia/necrosis that caused their reduction after NIC treatment. Overall, flow cytometry and histology results confirmed that NIC treatment stimulated local brain immune system allowing CTLs to attack the tumor (summarized in Supplementary Movie 1).
Delivery of checkpoint inhibitors as part of NICs and activation of brain immunity resulted in significant increase of tumor macrophages (MΦ). Both total and in particular, M1 MΦ increased that are responsible for tumoricidal effect by production of iNOS to promote anti-tumor activity. Their numbers by flow cytometry were significantly higher in all NIC groups vs. PBS or free mAbs (F = 10.51, DF = 6, p = 0.0001; F = 8.745, DF = 6, p < 0.0001, respectively, by ANOVA with Sidak's posttest; Fig. 5a, b). M2 MΦ numbers were also higher in NIC-treated groups vs. PBS (F = 9.125, DF = 6, by ANOVA with Sidak's posttest, p < 0.0001; Fig. 5c), possibly triggered by massive hypoxic/necrotic areas in the tumor ( Supplementary Fig. 5) as a result of treatment. However, the differences between NICs and free antibodies were non-significant. NK cells 10,33 known as "tumor killers" were also increased in all NIC groups vs. PBS or free antibodies (F = 10.53, DF = 6, by ANOVA with Sidak's posttest, p < 0.0001; Fig. 5d), as well as interferon γ producing anti-tumor IFNγ+ NK and to a lesser extent, NKT cells 34 (F = 3.766, DF = 6, p = 0.0038; F = 2.447, DF = 6, p = 0.0385, respectively, by ANOVA with Sidak's posttest; Fig. 5d-f).
Immunohistochemical analysis for anti-tumor M1 MΦ marker iNOS was also performed on tumor sections after various treatments (Fig. 6b). The staining was nonuniform and showed evidence of "hot spots", which were analyzed quantitatively. In accordance with flow cytometry data, immunostaining also showed significant increase of M1 MΦ after combined NIC treatment as compared with combination of free antibodies (F = 53.98, DF = 2, by ANOVA with Sidak's posttest, p < 0.01) or PBS (p < 0.0001).

NICs increase survival of GL261 glioblastoma-bearing mice.
Most preclinical studies with checkpoint inhibitors used intraperitoneal (I.P.) administration 35,36 , to avoid anaphylactic shock after I.V. injection. However, systemic I.V. drug administration is widely considered as the clinically accepted method in brain cancer treatment. In animal models, we observed a rapid and fatal hypersensitivity reaction with repeated I.V. injections of a-CTLA-4, a-PD-1, and NICs during our initial experiments. Therefore, to avoid acute immune-mediated anaphylaxis-like side effects, premedication must be used [37][38][39] .
Although the first two I.V. treatments did not cause noticeable side effects, mice experienced a severe drop in body temperature, piloerection, loss of spontaneous activity, dyspnea, and lethargy 15-20 min after the following injections. About 20% of mice died after the third treatment and up to 100% after the fifth treatment. Different regimens to reduce treatment dosage and frequency, however, did not eliminate toxicity. To counteract these side effects, 10 mg/kg anti-histamine Triprolidine and/or 5 mg/kg platelet activating factor (PAF) antagonist CV6209 were administered prior to each drug injection following the first one. Although both Triprolidine and CV6209 reduced side effects to some level, they prevented side effects in 100% of cases only when administered as a combination regimen. Premedication allowed five repeated I.V. treatments using a 10 mg/kg antibody dose, fully comparable to the current clinical dosage of checkpoint inhibitor mAbs.
Survival of mice bearing intracranial GBM GL261 and treated with free mAbs or NICs alone or in combination was investigated. Both a-CTLA-4 and a-PD-1 free mAbs and their combination failed to increase survival compared to PBS treatment (Fig. 7f, g), in line with their inability to cross the BBB (Fig. 2) and increase mouse survival, as well as with the failure of a clinical trial with such mAbs as GBM treatment 9 . Crucially, either P/a-CTLA-4 or P/a-PD-1 (Fig. 7a, b) significantly improved animal survival compared to PBS (p < 0.008 and p < 0.002, respectively, by log-rank test) or the respective free antibody treatment (p < 0.04 and p < 0.004, respectively) (Fig. 7f,  g). A combination of P/a-CTLA-4+P/a-PD-1 further improved survival (by 40% median) in GL261 tumor-bearing mice compared to PBS (p < 0.0001), a-CTLA-4 (p < 0.0001), a-PD-1 (p < 0.0001), P/a-CTLA-4 (p < 0.0001), and P/a-PD-1 (p < 0.004) (Fig. 7f, g). When checkpoint inhibitor mAb was attached to NIC with tumor vasculature targeting AP-2 peptide, mouse survival was also significantly improved, similar to a-TfR-mediated BBB delivery (p = 0.003, Fig. 7h).

Discussion
Cancer immunotherapy with checkpoint inhibitor mAbs represents a major advance in cancer treatment in the last decade 9 and was recently recognized by a Nobel prize. A number of "hot" immunogenic tumors, such as melanoma and other cancers with high rate of lymphocyte infiltration, have been successfully treated with immunotherapy by activating general anti-tumor immune response. However, many brain tumor clinical trials yielded mediocre results 9 partly because gliomas are considered "cold" and poorly infiltrated with lymphocytes 9,40 . The most significant obstacles in the treatment with checkpoint inhibitors are tumor resistance and toxicity 41 , and in the case of brain tumors, their inability to cross biological barriers 16,17 . GBM, a very aggressive tumor with short survival and with limited and poorly effective treatment options, presents a particular challenge for drug delivery because of its location in the CNS and the necessity for the drugs to cross the BBB 7 . A recent study has described survival improvement and immune system activation in glioma-bearing mice treated with free checkpoint inhibitor mAbs (a-PD1 and a-CTLA-4) 42 . Although such antibodies do not bind known receptors capable of BBB transcytosis and are unlikely to function effectively in the brain, they may still provide some activation of the brain immune system, possibly working through systemic immune stimulation 17,42 .
The glioma immune microenvironment is very complex, but, with proper checkpoint inhibitor delivery through the BBB, the basic concept for immune treatment still holds true: disrupting the PD-1/PD-L1 and CTLA-4/B7-1 complex formation in the tumor is key to the improved survival of glioma-bearing mice. We thus hypothesized that effective treatment of brain cancer with immunotherapy should involve the activation of brain local immune system by drugs able to reach the brain tumor by crossing the BBB, which cannot be readily achieved by free checkpoint inhibitor They were able to cross the BBB (Fig. 2) via the proven transcytosis delivery system using polymer-conjugated a-msTfR 28,29 , or AP-2 peptide 27 , as a vehicle for nanodrugs to cross the BBB 27,43,44 and elicit anti-tumor immune responses in brain glioma in a mouse model. NICs were immunochemically verified for their biological ability to bind their substrates, mimicking the natural conditions for naked a-CTLA-4 and a-PD-1. The satisfactory analytical results allowed us to move to the systemic drug delivery and glioma therapy. We demonstrated the efficacy of NICs carrying a-PD-1 and a-CTLA-4 in treating GBM in a murine model vs. free a-CTLA-4 and a-PD-1. We established that NICs, as single nano agent therapies and as a combination of P/a-PD-1+P/a-CTLA-4, crossed the BBB and significantly increased survival of GL261 tumor-bearing mice activating the brain resident immune system compared to treatment with free antibodies (Supplementary Animation Video 1). To look into mechanisms behind increased animal survival, various subsets of immune cells were examined in the treated tumors. After NIC treatment compared to free mAbs, both flow cytometry and immunohistochemistry showed an increase in effector T cells (CD4+ and CD8+) that can mediate tumor attack (Figs. 3 and 4). Additionally, NK cells, another effector cell population, were increased after NIC treatment (Fig. 5). These data agreed well with increased animal survival after NIC treatment, especially with drug combination.
We also found an increase of proinflammatory M1 MΦ that are active against tumors 45 (Figs. 5 and 6). At the same time, M2 MΦ were not reduced (Fig. 5). It has been shown that in hypoxic/ necrotic tumor areas MΦ become polarized towards the M2 phenotype 45 . This could explain a persistence of M2 MΦ after NIC treatment, as pronounced tissue necrosis was found in treated tumors. This necrosis could also explain lack of tumor size reduction on MRI images after NIC treatment ( Supplementary  Fig. 5). GL261 tumor apparently showed typical pseudoprogression 46 rather than real progression as was also evidenced by a marked decrease in proliferating Ki67+ cells in tumors treated with NIC but not with free antibodies (Supplementary Fig. 5).
The situation with Tregs appears to be more complicated. Immunostaining for CD4+FoxP3+ Tregs clearly showed their decrease after NIC treatment vs. free mAbs, especially after NIC combination (Fig. 4b). However, flow cytometry revealed a small Treg increase after treatment (combined NIC actually showed the smallest increase). Immunohistochemical data may be more accurate than flow cytometry quantitation because we can assess more precisely the tumor area (nearly 100%), whereas flow results would comprise tumor and adjacent non-cancerous area even with accurate tumor harvesting and only detect fully viable cells. Additionally, NIC treatment led to the formation of multiple tumor necroses, more evident at the end of animals' life when tissue was harvested for immunostaining ( Supplementary Fig. 5). Marked hypoxia/necrosis could contribute to Treg reduction after NIC treatment, similar to severe drop in Ki67+ proliferating tumor cells.
As a consequence of immune system activation, serum levels of cytokines produced by T cells to regulate immune response 47 including IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, and TNFα were significantly increased after combination treatment with P/ a-CTLA-4+P/a-PD-1 compared to other NICs or PBS. In general, IL-1β, IL-2, IL-12, IFNγ, and TNFα are part of T helper 1 (Th1) response, whereas IL-4, IL-5, IL-6, and IL-10 are part of T helper 2 (Th2) response. IL-2 secretion promotes T and B lymphocyte activity, enhances anti-tumor immunity, stimulates microglia, and regulates Tregs [48][49][50] . IFNγ is mainly produced by CD4+ and CD8+ T cells, NK cells, and microglia to boost the cytotoxic immune response 50,51 . In a self-stimulating loop, the microglia can also secrete IL-12, activating NK and stimulating T cells 52 . IL-10 is a multifunctional immune cytokine with antiangiogenic properties 53,54 . Finally, cytokines like IL-1β and TNFα can suppress tumors via stimulation of cell-mediated humoral immune reaction 55 . Some of these cytokines have been used in anti-cancer treatment: IL-4 for GBM 56,57 IL-12 for breast cancer brain metastasis 58 , IL-2 for melanoma brain metastasis 59,60 , and IFNα and IL-2 for renal cell carcinoma 61 . CTLA-4 and PD-1 suppression also increased IL-4, IL-5, IL-6, and IL-10, which is a known effect of such treatments in other cancer types [62][63][64] . In our study, all these cytokines were elevated in serum of animals treated with NIC combination (Supplementary Fig. 6), supporting increased cytotoxic activity of the immune system. Previous animal work mostly used intraperitoneal (I.P.) administration for multiple injections. It was tolerated by mice, but is not effective for the drug delivery, in particular for brain tumors shielded by BBB. Using previously undescribed premedication, we were able to safely deliver the same drugs in mice multiple times by I.V. route. Two pathways of anaphylaxis are known in mice: one is caused by antigen crosslinking of IgE on mast cells followed by release of histamine 65,66 , and the second one is mediated by IgG1 and basophil or neutrophil release of PAF 38 . Immunotherapy with a-CTLA-4 and a-PD-1 caused anaphylaxis-like side effects after repeated administrations. However, we observed absence of adverse effects after premedication consisting of anti-histamine, Triprolidine, and PAF antagonist, CV6209, previously shown to prevent anaphylaxis in other contexts 22 . In our study, premedication safely allowed for five repeated I.V. administrations at a therapeutic dosage comparable with the clinical settings. PAF inhibitor drugs are currently available for clinical use in cardiac rehabilitation and could be adopted in future clinical trials to mitigate adverse immunemediated events related to checkpoint inhibitor therapy 38 .
Overall, our results show that BBB-crossing NICs stimulate the brain resident immune system, prompting the proliferation of CD8+ T-cells and triggering the release of several cytokines, increasing production of M1 MΦ, and thus orchestrating immune response against GBM. These NICs provide a useful tool h Treatment with PBS, and P/a-CTLA-4+P/a-PD-1. This experiment was performed with AP-2 peptide to cross BBB as an alternative to a-TfR antibody. NIC treatment significantly improves survival compared to PBS. Data are mean ± SEM, N = 6 per group. p-Values were obtained using a log-rank test where **p < 0.01; ****p < 0.0001; N.S. = non-significant for the delivery of immunotherapy and targeted therapies to brain tumors. They may be used for treatment of primary brain tumors and brain metastases, where current treatment is very inefficient 67 , due to the inability to deliver therapeutics through the BBB and activate brain privileged immune system. Our regimen of premedication appears to alleviate adverse immune-mediated effects upon repeated I.V. injections of NICs, allowing the use of this clinically relevant route of administration in animal studies of checkpoint inhibitors.
In conclusion, we may highlight several important steps in our work. The presence of two antibodies in the active form on the same PMLA polymer was confirmed by state-of-the-art ELISA. We provided clear experimental evidence that nanoscale immunodrugs are efficient against GBM using several methods: flow cytometry, immunohistochemistry and the golden efficacy standard, animal survival. We compared our treatment with checkpoint inhibitor antibody delivery to brain tumors using not only anti-TfR Ab, but also AP-2 peptide. The anti-TfR Ab was used for the sake of prospective clinical trials and ELISA confirmation method for antibody, not peptides, and thus anti-TfR Ab was selected as a priority delivery vehicle. After five I.V. treatments with Triprolidine+CV6209 premedication, no animals died from toxicity, which plagues clinical trials. These data were obtained with our covalent NICs, which are different from nanoparticles. Here, the key innovative point is brain delivery of a-PD-1 and a-CTLA-4, which were never before used with "old" or "unknown" delivery vehicles, and their action in the brain, which may be used for other checkpoint inhibitors and treatments of not only untreatable GBM or brain metastases, but also of Alzheimer's and other neurodegenerative diseases.
Pull-down ELISA. NUNC MaxiSorp plates (Thermo Fisher Scientific) were coated with PD-1, CTLA-4 proteins (Acrobiosystems), or mouse TfR (500 ng/well) (recombinant protein made by California Institute of Technology) in coating buffer (Protein Detector™ HRP Microwell Kit; SeraCare) at 4°C overnight. The plates were blocked with 4% skim milk for 1 h at room temperature and washed once. The samples (a-CTLA-4, a-PD-1, a-msTfR, and nanoconjugates P/a-CTLA-4 or P/ a-PD-1) were incubated in binding buffer containing 0.5% milk for 1 h followed by washing four times. Secondary HRP-labeled antibodies (goat anti-rat from Abcam; goat anti-mouse and goat anti-hamster antibodies from SeraCare) were used for the detection of free and conjugated a-msTfR and conjugated a-CTLA-4 or a-PD-1. The conjugated a-msTfR was detected with anti-rat/HRP secondary antibody when the other antibody a-CTLA-4 or a-PD-1 was attached to its plate-adsorbed antigen, to confirm the presence of both antibodies on one polymer chain (pull-down ELISA). Pull-down ELISA was also performed for the detection of a-CTLA-4 or a-PD-1 when the other antibody a-msTfR was attached to its plate-adsorbed antigen similarly.
Cell line. Mouse glioblastoma cell line GL261 was a gift from B. Badie's lab (City of Hope Beckman Research Institute) and was cultured in Dulbecco's modified Eagle medium (DMEM; ATCC) containing 10% fetal bovine serum with 1% mixture of penicillin (100 U/mL), streptomycin (100 μg/mL), and amphotericin B (0.25 μg/mL) at 37°C with 5% CO 2 . This cell line is not in the database of ICLAC's commonly misidentified cell lines. Cells were routinely checked for mycoplasma (a kit from Lonza) with negative results. Twenty thousand GL261 cells in 2 μL PBS were implanted intracranially into the right basal ganglia of immunocompetent 8 weeks old female C57BL/6J mice (The Jackson Laboratory). All treatments were started on the 6th day after tumor cell inoculation. Free antibodies and NICs were administered at a dose of~10 mg/kg via tail vein injections, twice per week for a total of five injections. The tumor-bearing mice were randomized into different groups for various drug treatments a day before the treatment started. Because of the use of several experimental and control drugs plus standard control group, there was no possibility to perform blinded treatment study in order to not mix the groups. However, imaging of BBB permeation was performed using animal numbers only by researchers blinded to a specific treatment group.
To prevent anaphylactic-like adverse effects, starting with the second treatment, all mice (including the control group) received 200 μg anti-histamine Triprolidine (Sigma-Aldrich) and 100 μg platelet-activating factor (PAF) antagonist CV6209 (Santa Cruz Biotechnology) via intraperitoneal injection, respectively, 30 and 45 min prior to NIC injection. Six to ten mice per treatment group were used (flow cytometry and drug treatment), and mean value of cell counts as well as the standard error from each group were used for further analysis. Three mice were used for BBB permeation imaging and immunostaining experiments. Two or more independent experiments were performed for each assay. The number of samples per group was set to yield statistically significant data.
Optical imaging data were analyzed using ImageJ Fiji software 32 . Images acquired at ×40 or ×63 magnification were first normalized for size. This was done by cropping the ×40 images (85% of data) to a dimension that equaled the ×63 image. The position of the crop was randomized so that cells included in the final analysis were objectively selected. Total cell numbers were calculated from manual counts of DAPI-labeled nuclei; counts were performed with the Fiji cell counter tool.
Cells that were labeled with anti-CD8, anti-CD4, and anti-FoxP3 antibodies were counted separately by two investigators, and ratios were calculated against the number of DAPI-labeled cells. We analyzed 3-5 images from four mice for each drug condition. In total, 74 images were analyzed to produce CD8+ data, and 79 images were analyzed to produce FoxP3+ cell counts. Statistical analysis and plots were made using Prism 7 (GraphPad). Cell counts were compared by one-way ANOVA combined with pairwise post-hoc comparisons. Quantitative analyses demonstrated significant amount of labeled NICs in all nanoconjugate fluorescence measurements in brain tumors and presented as relative fluorescence intensities (Fig. 2, Supplementary Fig. 3; a total of four mice).
Spectral flow cytometry analysis. Mouse intracranial tumors were harvested and dissociated with 1.5 U/mL Liberase TL (Roche Diagnostics GmbH) in RPMI medium at 37°C for 30 min. The dissociated GL261 tumor cells were filtered through 70 µm cell strainers and loaded into a 96-well plate (~10 6 /well) for flow cytometry staining. After blocking with rat anti-mouse CD16/32 (Mouse BD Fc Block, clone 2.4G2, BD Biosciences), the cells from each tumor were split into two 96-well plates and stained with a panel of T-cell antibodies and a panel of NK/ macrophage antibodies in parallel (Supplementary Table 2). Flow cytometry was performed using the spectral flow cytometer SONY SA3800 (SONY Biotechnology). From each well, 100,000 events were recorded and analyzed with the SA3800 Software (SONY Biotechnology). The gating strategy for each specific population of immune cells is shown in Supplementary Figs. 3 and 4.
Cytokine analysis. Blood was drawn from tumor-bearing mice injected with NICs 24 h after the third treatment, and serum was separated for cytokine analysis. The multiplex cytokine assay was performed using a custom Bio-Plex Pro Assay kit (Bio-Rad) including antibodies targeting IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 (p70), IFNγ, and TNFα, according to the manufacturer's instructions. Results were obtained using a Bio-Plex 200 System (Bio-Rad) equipped with Bio-Plex Manager Software (Bio-Rad) and data were processed using the same software.
Tumor volume measurement by MRI. To determine the tumor volume, mice were anesthetized by inhalation of isoflurane (3.5% to effect) inside an induction chamber. MultiHance, a contrast agent, was used for tumor demarcation and was dissolved in PBS at a dose of 0.4 mmol/kg, and administered via the tail vein using a 1 mL syringe and a 30-gauge needle. Anesthesia was maintained during measurements by nose cone administration of 1-1.5% isoflurane. The mouse bed was heated to prevent animal cooling during anesthesia. Breathing was regulated in the range of 45-65 breaths/min during the measurements by slightly increasing or decreasing the percentage of isoflurane. Images were recorded immediately after contrast agent injection. Spin-echo images of the entire brain were acquired. Axial slices were positioned to cover the whole brain. A multislice multiecho sequence (8 spin_echos) was used with a TR = 450 ms. Slices were acquired with a 1.0 mm thickness for a 1.8-1.8 cm field of view with a 256-196 matrix size providing an inplane resolution of 70-92 μm/pixel. Total scan time was around 6 min. Animals underwent MRI scanning on a 9.4 T small-animal scanner (BioSpec 94/20USR, Bruker Biospin MRI GmbH). Each animal was placed inside a transmission whole body coil (T10325 V3, Bruker Biospin) with a four-channel surface array coil (T11071 V3, Bruker Biospin) positioned over the brain. The transmission body coil was used for all radio frequency transmission; the surface coil was used for detection. Representative images of MRI brain scan for a mouse from each cohort (n = 5) are shown ( Supplementary Fig. 5). A region of interest (ROI) was drawn along the perimeter of tumors (marked by pink dotted line) and the total volume was determined for each mouse by combining all ROIs of induvial slices.
Statistical analysis. Statistical analysis of survival data was carried out using Kaplan-Meier curves and log-rank test by GraphPad Prism 7 software. All data are presented as mean ± standard error. One-way ANOVA with Sidak's posttest was used in Prism 7 for multiple treatment group pairwise comparisons. P < 0.05 was considered statistically significant.
Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability
The original flow cytometry, drug distribution morphometry, and survival data that support the findings of this study are available from the corresponding author upon reasonable request. The authors declare that other data supporting the findings of this study including details of the nanodrug synthesis and characterization are available within the paper and its supplementary information file. The manuscript has been deposited to BioRxiv: https://doi.org/10.1101/466508. A reporting summary for this article is available as a Supplementary Information file.