Gold Glyconanoparticles Combined with 91–99 Peptide of the Bacterial Toxin, Listeriolysin O, Are Efficient Immunotherapies in Experimental Bladder Tumors

Simple Summary We propose a novel type of immunotherapy for bladder cancer using gold nanoparticles bound to a peptide of a bacterial toxin with anti-tumor capacities as listeriolysin O called Listeria nanovaccines. Here, we present the pre-clinical experiments on a mice model of bladder cancer and blood cells of patients with bladder cancer using these Listeria nanovaccines that activate the immune system, block the tumor immunosuppression environment, and reduce the tumor size. The impact of Listeria nanovaccines on the field of immunotherapies for solid tumors can be extended to other solid tumors containing lymphocyte infiltration. Therefore, we propose Listeria nanovaccines as immunotherapy for tumors such as melanoma, urothelial bladder carcinoma, non-small cell lung carcinoma, and glioblastoma. Abstract This study presents proof of concept assays to validate gold nanoparticles loaded with the bacterial peptide 91–99 of the listeriolysin O toxin (GNP-LLO91–99 nanovaccines) as immunotherapy for bladder tumors. GNP-LLO91–99 nanovaccines showed adjuvant abilities as they induce maturation and activation of monocyte-derived dendritic cells (MoDCs) to functional antigen-presenting cells in healthy donors and patients with melanoma or bladder cancer (BC), promoting a Th1 cytokine pattern. GNP-LLO91–99 nanovaccines were also efficient dendritic cell inducers of immunogenic tumor death using different bladder and melanoma tumor cell lines. The establishment of a pre-clinical mice model of subcutaneous BC confirmed that a single dose of GNP-LLO91–99 nanovaccines reduced tumor burden 4.7-fold and stimulated systemic Th1-type immune responses. Proof of concept assays validated GNP-LLO91–99 nanovaccines as immunotherapy by comparison to anti-CTLA-4 or anti-PD-1 antibodies. In fact, GNP-LLO91–99 nanovaccines increased percentages of CD4+ and CD8+ T cells, B cells, and functional antigen-presenting DCs in tumor-infiltrated lymphocytes, while they reduced the levels of myeloid-derived suppressor cells (MDSC) and suppressor T cells (Treg). We conclude that GNP-LLO91–99 nanovaccines can work as monotherapies or combinatory immunotherapies with anti-CTLA-4 or anti-PD-1 antibodies for solid tumors with high T cell infiltration, such as bladder cancer or melanoma.


Introduction
In the past 10 years, nanotechnology has been widely studied for cancer treatment, as nanoparticles can play a significant role in drug delivery systems [1]. In this regard, inorganic nanoparticles such as gold nanoparticles (AuNP) are useful candidates since the gold core is inert and non-toxic and can be surface-functionalized with different compounds. Functionalization with carbohydrates might enhance their accumulation into tumors and overcome drug resistance. In addition, the AuNP combination with peptides might target them to specific cells or confer novel features [2,3]. In this regard, gold glyconanoparticles (AuGNP)-here, GNP for simplicity-present several advances such as high water solubility, ability to target antigen-presenting cells (APC), lack of toxicity [4,5], and capacity to combine in the same design with different ligands, such as carbohydrates and peptides [6,7].
Adjuvants are classical immunotherapies that activate the tumoral immune responses by acting on antigen-presenting cells (APCs) (e.g., dendritic cells, DCs); examples are inorganic nanoparticles, cancer vaccines, oligonucleotides, bacterial compounds, and antagonists of Toll-like receptors [8,9]. However, the development of immune checkpoint inhibitors (ICIs) (e.g., anti-CTLA-4 or anti-PD-1/PD-L1 antibodies) as new immunotherapies changed the focus of immune system activation to T cells, as they block the negative immune controls of T cells and boost the anti-tumoral immune responses [10]. ICIs have been approved as either a neoadjuvant or first-line treatment for tumors such as melanoma, non-small cell lung (NSCLC), renal, or bladder cancer (BC). In general, ICIs seem to benefit oncologic patients with tumors presenting high T cell infiltration and mutational variance. However, in some cases, ICI resistance or adverse effects minimize their efficiency [11], along with the failure to respond to ICI of tumors with low-medium mutational variance (e.g., hepatocarcinoma) or infiltrated T cells (e.g., glioblastoma) [12][13][14][15][16].
Bladder cancer (BC) is the ninth most common malignancy diagnosed worldwide. As a tumor, BC is highly immunogenic and T cell-infiltrated. It has been postulated that a dysregulation of the immune system within the bladder promotes BC growth. However, to date, this immunosuppression within BC has not been characterized in detail [17]. BC arises from the urothelium, the epithelium that lines the urinary bladder, and only 25% at most are muscle-invasive metastatic BCs. Metastatic BC is treated with chemotherapy regimens as first-line treatments and, recently, ICI as neoadjuvant, maintenance or secondline treatment [18]. Most BCs are superficial, low-grade, noninvasive, or superficial tumors confined to the mucosa layer. Noninvasive BC standard therapy implies tumor resection followed by adjuvant therapy with the bacterium Bacillus Calmette-Guerin (BCG) to activate the immune system [19,20]. In this regard, Listeria monocytogenes is a human bacterial pathogen also used for the treatment of several tumors such as prostate cancer, cervix carcinoma, or pancreatic cancer, using attenuated mutants that lack the C-terminal domain of the main virulence factor, listeriolysin O (LLO) [21][22][23]. Moreover, LLO is a pore-forming toxin that, along with its cytolytic activity, induces cell death of different cell types such as DCs, macrophages, or T cells [24]. LLO seems to induce different types of cell death, necrosis, necroptosis, pyroptosis, or apoptosis, causing different effects [25]. The classical assumption that necrosis induces inflammatory immune responses while apoptosis triggers anti-inflammatory immune responses does not seem applicable in all contexts of cell death by bacterial toxins. While the tuberculosis-necrotizing toxin (TNT) induces immunological silent cell death that recruits macrophages and induces poor immune responses [26], LLO action onto tumor cells appears to induce an immunogenic cell death characterized by promoting inflammatory and anti-tumor immune responses. Previous work of our group quoted LLO anti-neoplastic abilities to 91-99 N-terminal peptides and explored different vaccine vectors as therapy for experimental melanoma [5][6][7]. Here, we extend these studies using a nanocarrier vector GNP coupled to two ligands, the 91-99 LLO peptide and β-D-glucose (here called GNP-LLO 91-99 nanovaccines), to validate them as valid immunotherapies for bladder tumors and inducers of immunogenic apoptosis.

Patients
Nine patients with advanced solid tumors were included in this study: three patients with stage IV melanoma before enrollment in immunotherapy (MEL-1 and MEL-3), a stage IIIB cutaneous melanoma after surgery resection (MEL-2), a lung and bladder carcinoma treated with cisplatin-etoposide (BC-1), two urothelial bladder carcinomas without treatment (BC-2, BC-3), a hepatocellular carcinoma treated with ablation by microwaves (HEP-1), a prostate adenocarcinoma treated with taxocel (PROST-1), and a multiform glioblastoma treated with temozolomide and radiotherapy (GLIO-1). MEL-1, MEL-3, BC-1, HEP-1, PROST-1, and GLIO-1 were diagnosed at the Medical Oncology Department; BC-2 and BC-3 were diagnosed at the Urology Department; and MEL-2, the stage IIIB cutaneous melanoma, was diagnosed at the Dermatology Department (Hospital U. Marqués de Valdecilla, HUMV, Santander, Spain). The patients participated in the study voluntarily, signed an informed consent agreement at the physician consultation, and received an information document on the research study. Patients were able to revoke the informed consent at any time. Healthy donors were obtained from the blood bank at our hospital. Blood samples were obtained in heparin tubes at the Dermatology, Urology, or Medical Oncology Departments on the day of patient consultation and processed at the IDIVAL laboratory within the following 2 h.

Direct and Immunogenic Apoptosis of Melanoma and Bladder Tumor Cells
Cell toxicity was first evaluated using Trypan blue staining and incubation for 16 h with GNP-LLO 91-99 nanovaccines. Direct apoptosis was evaluated in the different tumor cell lines after incubation for 16 h with GNP-LLO 91-99 (50 µg/mL). Immunogenic apoptosis implies the incubation of tumor cell lines with 1 2 supernatants of ex vivo differentiated DCs (for murine tumors) or MoDCs from healthy donors (for human tumors), pre-treated for 16 h with 50 µg/mL of GNP-LLO 91-99 nanovaccines. Direct and immunogenic apoptosis was examined by FACS after double staining with the DNA marker, 7-AAD (7-AAD-PE), and the apoptotic marker, annexin V (annexin V-APC). The staining of cells with 7-ADD corresponded to necrotic cell death, whereas the staining of cells with annexin-V alone corresponded to early apoptotic programmed cell death (mean ± SD). Experiments involving human samples were performed three times and five times for mice assays. Results are expressed as the % of apoptotic cells ± SD of triplicate samples (p ≤ 0.5).

Bladder Tumor Auto Transplants Followed by GNP-LLO 91-99 and ICI Immunotherapies
Murine MB-49 bladder cell lines [27] or B16.F10 melanoma were transplanted into 8-12-week-old C57BL/6 male or female mice (auto transplants), respectively, with a single subcutaneous (s.c.) injection (10 6 cells) in a volume of 100 µL (n = 10/group). At 14 days, tumor transplanted mice received or not (NT) a single intravenous (i.v.) inoculation of GNP-LLO 91-99 nanovaccines (50 µg/mouse). On day 7, mice were sacrificed, and tumor sizes were measured with a caliper. Values shown for tumor volume (TV) were calculated using the following formula: (length × (width) 2 )/2, as reported [6]. Mean and SD of the tumor volumes per group were calculated. Sera were also obtained, processed, and used for cytokine measurements. Tumors were minced, homogenized, passed through a 70 µm strainer, and then centrifuged over a Ficoll gradient at a 1.077 g/mL density (Histopaque-1077, Sigma-Aldrich, St. Louis, MO, USA) to recover TILs in the interphase gradient while collecting tumor cells in pellets. For immunotherapeutic studies, treatment of MB-49 established transplants with GNP-LLO 91-99 nanovaccines were performed as above, alone or in combination with the following antibodies: anti-CTLA-4 or anti-PD-1 (50 µg/mouse). Cell populations of TILs and spleens were analyzed by FACS. All experiments were performed at least five times.

Statistical Analysis
For statistical analysis, Student's t-test was applied to mice with auto transplants; each group included 5 mice for all assays reported (p ≤ 0.05 was considered significant). Experiments in mice were performed at least five times each, and experiments with MoDCs from healthy donors or oncologic patients and tumor cell lines were performed three times each. ANOVA analysis was applied to cytokine measurements and flow cytometry analysis per the manufacturer s recommendations (p ≤ 0.05 was considered significant). For statistical purposes, each flow cytometry sample was performed in triplicate. The GraphPad software was used for the generation of all the graphs presented.

Results and Discussion
We initiated this study with the hypothesis that GNP-LLO 91-99 nanovaccines might function as immunotherapies for bladder tumors and planned the following assays: adjuvant abilities using MoDCs from human donors, the in vitro immunogenic death of murine and human tumor cell lines, the in vivo anti-neoplastic action after the establishment of a mouse bladder cancer model, and immunotherapeutic actions in comparison to anti-CTLA-4 or anti-PD-1 immunotherapies.

GNP-LLO 91-99 Nanovaccines Showed No Toxicity in Human MoDCs or Mice
Before initiating any experiment, we prepared a homogeneous batch of GNP-LLO 91-99 nanovaccines to be used in all the experiments and performed the quality and toxicity controls in vivo and in vitro. GNP-LLO 91-99 nanovaccines are formed by a gold core covalently linked to LLO 91-99 peptide and β-D glucose (GNP-LLO 91-99 nanovaccines schematic representation in Figure 1a). The gold core had a spheric shape and nanometric size with an average of 2 nm, as observed by the analysis of transmission electron microscopy ( Figure 1b). Different concentrations of GNP-LLO 91-99 nanovaccines or LLO 91-99 peptide (5-500 µM) were non-toxic for human MoDCs after incubation for 16 h, as determined by evaluating the cell viability with Trypan blue staining (Figure 1c). We also observed no toxicity in mice (C52BL/6 male and female, n = 10) when we inoculated intraperitoneally (i.p.) for 16 h with GNP-LLO 91-99 nanovaccines or LLO 91-99 peptide (5-500 µM) (Figure 1c, center and right columns) and examined mice health conditions and sera IL-1 concentrations. Results are expressed as the mean of viable cells ± SD. For in vivo analyses, we inoculated mice (12 months old male, n = 5, or female, n = 5) intravascularly (i.v.) with the same concentrations of GNP-LLO91-99 nanovaccines and LLO91-99 peptide as in (a) (5-500 µM) in C57BL/6 mice for 16 h, examined mice health conditions, and measured IL-1 concentration in mice sera. Experiments were performed five times each. Results are expressed as the mean cytokine concentrations (pg/mL) ± SD. *Sick, refers to animals with hairless and difficulties to move and feed.

GNP-LLO 91-99 Nanovaccines Served as Adjuvants for Human MoDCs of Oncologic Patients
Once we established that GNP-LLO 91-99 nanovaccines showed no toxicity in human MoDC, we next explored their abilities as adjuvants. Functional and activated MoDCs are characterized by three molecules relevant to combat tumors, the surface expression of MHC-I and MHC-II molecules necessary for antigen presentation, and CD86 co-stimulatory molecules essential for T cell activation. MoDC cytokine production is also a determinant of T cell activation or suppression. In this regard, MoDC activation corresponds with a high production of Th1 cytokines, such as IL-12p70, which stimulates cytotoxic T cells, or TNF-a, which implements innate immunity. Meanwhile, suppression is induced by classical Th2 cytokines, such as IL-6 or IL-10, which promote significant numbers of T reg [19] as well as KC or MIP-2 chemokines that also participate, as they can trigger the migration of MDSC. The ability of an adjuvant to induce both maturation and activation of ex vivo cultured DCs requires an optimal and basal functionality of these cells [30], which is not always observed in cancer patients, as reported in hepatocarcinoma or multiple myeloma [12,13,31,32]. For this reason, we evaluated GNP-LLO 91-99 nanovaccines as adjuvants after the ex vivo incubation of MoDCs from healthy donors or patients with melanoma (MEL-1, MEL-2) or bladder carcinoma (BC-1, BC-2) from our institution (Hospital U. Marqués de Valdecilla, HUMV, Santander, Spain) (Supplementary Table S1). GNP-LLO 91-99 nanovaccines showed three-fold increases in the percentages of MHC-I or MHC-II and five-fold increases in the co-stimulatory CD86 molecules after incubation of MoDCs in healthy donors (CONT), patients with melanoma (MEL-1, MEL-2), lung and bladder tumors (BC-1), or urothelial bladder tumors (BC-2) (GNP-LLO 91-99 bars in Figure 2a). Empty GNP nanovaccines or LLO 91-99 peptides (data not shown) had no effect on the surface expression of MHC-I, MHC-II, or CD86 molecules, similar to MoDCs treated with saline (CONT + GNP and CONT bars in Figure 2a), as reported previously [7].
Analysis of Th1 and Th2 cytokines indicated that GNP-LLO 91-99 nanovaccines increased the production of Th1 cytokines in MoDCs of healthy donors, especially TFN-α and IL-12p70 (CONT versus CONT + GNP-LLO 91-99 rows in Table 1). Empty GNP nanovaccines showed similar patterns as saline-treated MoDCs (CONT versus CONT + GNP rows in Table 1), indicating that empty GNP showed no adjuvant effect. MoDCs from patients with melanoma or lung and bladder carcinoma showed under non-stimulated conditions a clear Th2 cytokine pattern (e.g., IL-6 and IL-10, high levels) that reflects the systemic cytokine pattern of the patient's sera (Supplementary Table S1 Table 1). Therefore, GNP-LLO 91-99 nanovaccines seem to be valid adjuvants for patients with melanoma or BC but also for MoDCs of patients with other tumors (e.g., prostate adenocarcinoma, NSCLC lung cancer, or glioblastoma), except for those reported with a low DC functionality such as hepatocellular carcinoma [13] (Supplementary Figure S1).  Apoptosis was examined by FACS using the DNA marker, 7-AAD (7-AAD-PE), and the apoptotic marker, annexin V (annexin V-APC). Experiments were performed at least three times. Results are expressed as percentages of apoptotic cells ± SD (p ≤ 0.05).

GNP-LLO 91-99 Nanovaccines Induced Immunogenic Apoptosis in Bladder Tumors
Once we evaluated the possibility that GNP-LLO 91-99 nanovaccines served as efficient adjuvants, we examined their induction of tumor apoptosis. For this purpose, we used available tumor cell lines of melanoma (murine B16.F10 or human A-375 cell lines) or BC (murine MB-49 or human T-24 cell lines). We examined two types of apoptosis by FACS: direct apoptosis and immunogenic DC-mediated apoptosis. GNP-LLO 91-99 nanovaccines induced low percentages of direct apoptosis onto BC or melanoma tumor cell lines, barely 5% in murine B16.F10 melanoma (black bars in Figure 2b), and showed no cytotoxicity (Supplementary Figure S2). Assays of DC-mediated immunogenic apoptosis imply incubation of tumor cell lines with 1 2 of the supernatants of DCs or MoDCs, pre-incubated with GNP-LLO 91-99 nanovaccines. GNP-LLO 91-99 nanovaccines induced high percentages (35-55%) of immunogenic apoptosis in melanoma and BC (gray bars in Figure 2b). GNP-LLO 91-99 induction of immunogenic apoptosis seemed effective for tumors with significant T cell infiltration such as melanoma, BC, NSCLC lung tumors (murine TC-21 or human A-549 cell lines), or glioblastoma (murine O627 or human RG-1 cell lines), while not in tumors with low T cell infiltration, such as in hepatocellular carcinoma (murine Hepa 1-6 cell lines) [12,13], ovary tumors (hamster CHO cell lines), or SV-40-induced tumors (murine IC-21 macrophage-like cell lines) (Supplementary Figure S2). We conclude that GNP-LLO 91-99 nanovaccine induction of DC-mediated immunogenic apoptosis appears to explain their anti-neoplastic abilities.

Mechanisms of Anti-Neoplastic Abilities of GNP-LLO 91-99 Nanovaccines in Mice Models of Melanoma and BC
To validate the anti-neoplastic abilities of GNP-LLO 91-99 nanovaccines for BC, we established subcutaneous (s.c.) transplants of murine bladder MB-49 and melanoma B16.F10 tumor cell lines (protocol for all the following experiments is shown in Figure 3a Figure 3b). The sizes of control tumors were 10-fold larger than tumors at 7 days, and survival of mice was 60% and 80% reduced, respectively, presenting tumor ulcerations (Table 2, SR and U columns). To avoid unnecessary pain to mice, for further experiments, we established a protocol of 14 days transplantation and 7 days treatment with GNP-LLO 91-99 nanovaccines (Figure 3a). GNP-LLO 91-99 nanovaccines implemented the survival rates ( Table 2, SR columns of files labeled as + GNP-LLO 91-99 ) and blocked tumor growth at all times tested (TV columns).
Second, we explored the immune mechanisms able to explain the anti-neoplastic abilities of GNP-LLO 91-99 nanovaccines in BC mouse models, analyzing the cytokines in mouse sera. We confirmed a Th2 cytokine versus a Th1 pattern in mice transplanted with bladder MB-49 cell lines (light gray bars in Figure 4a), similar to the pattern detected with melanoma B16.F10 [6,7] (white bars in Figure 4a). In this regard, we detected high levels of chemokines recruiting neutrophils such as KC/CXCL1, participating in MDSC and tumor progression such as MIP-2, and high levels of cytokines such as IL-6 and TNF-a cytokines, while low levels of pro-inflammatory Th1-Th17 cytokines such as IFN-γ, IL-2, and IL-17A (CONT-B16F10 and CONT-MB-49, white and light gray bars in Figure 4a). Therefore, systemic production of chemokines/cytokines in mice transplanted with bladder MB-49 cell lines suggested an immunosuppression status [33] similar to the levels of BC or melanoma patients (Supplementary Table S1). Cancers 2022, 14, x FOR PEER REVIEW 11 of 18 First, we performed experiments to examine the effects of a single dose of GNP-LLO91-99 nanovaccines at different times, i.e., 7, 14, and 23 days post-treatment, and observed similar results ( Table 2, files labeled as + GNP-LLO91-99). Mice were intravenously (i.v.) inoculated in their tails with a single dose of GNP-LLO91-99 nanovaccines (50 µg/mouse), and 7 days post-treatment, mice were sacrificed, and sera and tumors were collected to examine the effects of nanovaccines (Figure 3b). GNP-LLO91-99 nanovaccines reduced 5-fold tumor volume of B16.F10 melanoma and 4.7-fold of MB-49 bladder cell lines (GNP-LLO91-99 bars in Figure 3b). The sizes of control tumors were 10-fold larger than tumors at 7 days, and survival of mice was 60% and 80% reduced, respectively, presenting tumor ulcerations ( Table 2, SR and U columns). To avoid unnecessary pain to

GNP-LLO 91-99 Nanovaccines Are Effective Immunotherapies That Blocked Immunosuppression
Next, we confirmed the immunosuppression status after isolation of tumor-infiltrated lymphocytes (TILs) of mice s.c. transplanted with melanoma or BC, and treated or not with GNP-LLO 91-99 nanovaccines. As shown in Figure 5a, BC basal status showed a higher immunosuppressive environment than melanoma, with higher levels of T reg and MDSC (Ly6G + CD11b + ) and lower levels of activated DCs (CD11c + MHC-II + ) and macrophages (F4/80 + ) (compare light gray versus white bars in Figure 4a). cytokines KC, MIP-2, IL-6, and TNF-α and increased the levels of IFN-γ and IL-2, confirming the shift of Th2 to Th1 cytokine pattern in both mouse models of tumor transplantation, B16.F10 melanoma and MB49 bladder cell lines, suggesting a common action (black and dark gray bars in Figure 4a,b).

GNP-LLO91-99 Nanovaccines Are Effective Immunotherapies That Blocked Immunosuppression
Next, we confirmed the immunosuppression status after isolation of tumor-infiltrated lymphocytes (TILs) of mice s.c. transplanted with melanoma or BC, and treated or not with GNP-LLO91-99 nanovaccines. As shown in Figure 5a, BC basal status showed a higher immunosuppressive environment than melanoma, with higher levels of Treg and MDSC (Ly6G + CD11b + ) and lower levels of activated DCs (CD11c + MHC-II + ) and macrophages (F4/80 + ) (compare light gray versus white bars in Figure 4a).  GNP-LLO 91-99 nanovaccine action reduced immunosuppressed cells (T reg , MDSC) in both tumor models and increased activated DCs and macrophages (compare black and dark gray bars in Figure 5a). Therefore, GNP-LLO 91-99 nanovaccines eliminated immune blockers in BC and melanoma, suggesting that they might function as immunotherapies for BC, as reported for melanoma [7].
Next, we verified that GNP-LLO 91-99 nanovaccines acted as immunotherapies in mice models of BC, exploring the immune populations in TILs of mice inoculated with a single dose of GNP-LLO 91-99 nanovaccines alone (50 µg/mouse) or in combination with anti-CTLA-4 (50 µg/mouse) or anti-PD-1 (50 µg/mouse) immunotherapies. GNP-LLO 91-99 nanovaccines, alone or in combination with anti-CTLA-4 or anti-PD-1 antibodies, increased the percentages of DCs with an activated MHC-II + CD40 + CD86 + phenotype of functional APC and the numbers of T and B cells (e.g., CD4 + , CD8 +, and CD19 + cells), while these treatments reduced the percentages of suppressor innate cells such as MDSC (Ly6G + CD11b + granulocytes) and T reg (CD25 + cells) (compare black, light gray, and dark gray bars in Figure 5b). We also included as controls mice treated with anti-CTLA-4 or anti-PD-1. As shown in Figure 5b, ICI alone cannot induce the percentages of activated DCs observed in GNP-LLO 91-99 nanovaccines combined with ICI. Anti-CTLA-4 treatment alone caused a moderate reduction in T reg but not as prominent as observed with GNP-LLO 91-99 nanovaccines combined with anti-CTLA-4.
In summary, GNP-LLO 91-99 nanovaccines appear to be valid immunotherapy for BC as well as melanoma. Moreover, the ability of GNP-LLO 91-99 nanovaccines to induce immunogenic apoptosis in solid tumors with high or low T cell infiltration, such as NSCLC lung cells or glioblastoma, and to activate MoDCs from patients with T cell-infiltrated solid tumors, such as prostate adenocarcinoma, multiform glioblastoma, or lung carcinoma, opens the possibility for future validation of GNP-LLO 91-99 nanovaccines as a neoadjuvant or immunotherapy for tumors with high or medium T cell infiltration.

Conclusions
GNP-LLO 91-99 nanovaccines appear to act as adjuvants for BC patients, as they promoted (i) the antigen presentation capacities of MoDCs of patients with BC (e.g., increasing the levels of MHC-I and MHC-II molecules as well as co-stimulatory CD86 molecules) and (ii) a Th1 cytokine pattern by releasing high levels of IL-12p70 and TNF-α with antineoplastic abilities.
In this study, GNP-LLO 91-99 nanovaccines presented anti-neoplastic abilities for BC, as they induced immunogenic apoptosis directed by dendritic cells and the cytokines released by them.
Moreover, GNP-LLO 91-99 nanovaccines showed immunotherapeutic abilities for BC, as they blocked the immunosuppression status of BC, increasing the numbers of cytotoxic T cells and DCs within the tumors and decreasing the number of immunosuppressive cells (T reg , MDSC). In fact, GNP-LLO 91-99 nanovaccines adequately combine well and potentiate the action of ICI-both anti-CTLA-4 and anti-PD-1-supporting them as a novel nano-immunotherapy for BC.