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International Journal of Molecular Sciences 24 (24): 17535 (2023)
Ovarian Cancer Research Centre (Vancouver, B.C.)
Alonso, João Carlos Cardoso
de Souza, Bianca Ribeiro
Reis, Ianny Brum
de Arruda Camargo, Gabriela Cardoso
de Oliveira, Gabriela
de Barros Frazão Salmazo, Maria Izabel
Gonçalves, Juliana Mattoso
de Castro Roston, José Ronaldo
Caria, Paulo Henrique Ferreira
da Silva Santos, André
de Freitas, Leandro Luiz Lopes
Billis, Athanase
Durán, Nelson
Fávaro, Wagner José
This study assessed the safety and efficacy of OncoTherad® (MRB-CFI-1) nanoimmunotherapy for non-muscle invasive bladder cancer (NMIBC) patients unresponsive to Bacillus Calmette-Guérin (BCG) and explored its mechanisms of action in a bladder cancer microenvironment. A single-arm phase I/II study was conducted with 44 patients with NMIBC who were unresponsive to BCG treatment. Primary outcomes were pathological complete response (pCR) and relapse-free survival (RFS). Secondary outcomes comprised response duration and therapy safety. Patients’ mean age was 65 years; 59.1% of them were refractory, 31.8% relapsed, and 9.1% were intolerant to BCG. Moreover, the pCR rate after 24 months reached 72.7% (95% CI), whereas the mean RFS reached 21.4 months. Mean response duration in the pCR group was 14.3 months. No patient developed muscle-invasive or metastatic disease during treatment. Treatment-related adverse events occurred in 77.3% of patients, mostly grade 1–2 events. OncoTherad® activated the innate immune system through toll-like receptor 4, leading to increased interferon signaling. This activation played a crucial role in activating CX3CR1+ CD8 T cells, decreasing immune checkpoint molecules, and reversing immunosuppression in the bladder microenvironment. OncoTherad® has proved to be a safe and effective therapeutic option for patients with BCG-unresponsive NMIBC, besides showing likely advantages in tumor relapse prevention processes.
Citation: Alonso, J.C.C.; de Souza,B.R.; Reis, I.B.; de Arruda Camargo,G.C.; de Oliveira, G.; de BarrosFrazão Salmazo, M.I.; Gonçalves,J.M.; de Castro Roston, J.R.; Caria,P.H.F.; da Silva Santos, A.; et al.OncoTherad® (MRB-CFI-1)Nanoimmunotherapy: A PromisingStrategy to Treat Bacillus Calmette–Guérin-Unresponsive Non-Muscle-Invasive Bladder Cancer: Crosstalkamong T-Cell CX3CR1, ImmuneCheckpoints, and the Toll-LikeReceptor 4 Signaling Pathway. Int. J.Mol. Sci. 2023, 24, 17535. https://doi.org/10.3390/ijms242417535Academic Editor: Hideki FuruyaReceived: 8 November 2023Revised: 9 December 2023Accepted: 13 December 2023Published: 15 December 2023Copyright: © 2023 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). International Journal of Molecular SciencesArticleOncoTherad® (MRB-CFI-1) Nanoimmunotherapy: A PromisingStrategy to Treat Bacillus Calmette–Guérin-UnresponsiveNon-Muscle-Invasive Bladder Cancer: Crosstalk among T-CellCX3CR1, Immune Checkpoints, and the Toll-Like Receptor 4Signaling PathwayJoão Carlos Cardoso Alonso 1,2,*, Bianca Ribeiro de Souza 3 , Ianny Brum Reis 4 ,Gabriela Cardoso de Arruda Camargo 1, Gabriela de Oliveira 1, Maria Izabel de Barros Frazão Salmazo 1,Juliana Mattoso Gonçalves 1, José Ronaldo de Castro Roston 1, Paulo Henrique Ferreira Caria 1 ,André da Silva Santos 1, Leandro Luiz Lopes de Freitas 5, Athanase Billis 5, Nelson Durán 1and Wagner José Fávaro 1,*1 Laboratory of Urogenital Carcinogenesis and Immunotherapy (LCURGIN), Universidade Estadual deCampinas (UNICAMP), Campinas 13083-865, São Paulo, Brazil; gcacamargo0@gmail.com (G.C.d.A.C.);gabriela.ufscar@gmail.com (G.d.O.); mabelfrazao@hotmail.com (M.I.d.B.F.S.);jumattosogg@gmail.com (J.M.G.); rroston@uol.com.br (J.R.d.C.R.); phcaria@unicamp.br (P.H.F.C.);andre.s.s@me.com (A.d.S.S.); nelsonduran1942@gmail.com (N.D.)2 Paulínia Municipal Hospital, Paulínia 13140-000, São Paulo, Brazil3 Obstetrics & Gynecology Department, Ovarian Cancer Research Group University of British Columbia,Vancouver, BC V6Z 2K8, Canada; bianca.ribeiro@ubc.ca4 Diagnosis and Surgery Department, Dentistry School, São Paulo State University (UNESP),Araraquara 14801-903, São Paulo, Brazil; iannybrumreis@yahoo.com.br5 Pathology Department, Medical School, Universidade Estadual de Campinas (UNICAMP),Campinas 13083-888, São Paulo, Brazil; leandrollfreitas@gmail.com (L.L.L.d.F.); athanase@unicamp.br (A.B.)* Correspondence: jocaalonso1@gmail.com (J.C.C.A.); favarowj@unicamp.br (W.J.F.)Abstract: This study assessed the safety and efficacy of OncoTherad® (MRB-CFI-1) nanoimmunother-apy for non-muscle invasive bladder cancer (NMIBC) patients unresponsive to Bacillus Calmette-Guérin (BCG) and explored its mechanisms of action in a bladder cancer microenvironment. Asingle-arm phase I/II study was conducted with 44 patients with NMIBC who were unresponsiveto BCG treatment. Primary outcomes were pathological complete response (pCR) and relapse-freesurvival (RFS). Secondary outcomes comprised response duration and therapy safety. Patients’ meanage was 65 years; 59.1% of them were refractory, 31.8% relapsed, and 9.1% were intolerant to BCG.Moreover, the pCR rate after 24 months reached 72.7% (95% CI), whereas the mean RFS reached21.4 months. Mean response duration in the pCR group was 14.3 months. No patient developedmuscle-invasive or metastatic disease during treatment. Treatment-related adverse events occurredin 77.3% of patients, mostly grade 1–2 events. OncoTherad® activated the innate immune systemthrough toll-like receptor 4, leading to increased interferon signaling. This activation played a crucialrole in activating CX3CR1+ CD8 T cells, decreasing immune checkpoint molecules, and reversingimmunosuppression in the bladder microenvironment. OncoTherad® has proved to be a safe andeffective therapeutic option for patients with BCG-unresponsive NMIBC, besides showing likelyadvantages in tumor relapse prevention processes.Keywords: bladder cancer; immunotherapy; Oncotherad; bacillus Calmette–Guerin; nanotechnology1. IntroductionBladder cancer is a significant global public health issue with high morbidity andmortality rates [1,2]. It is known for its tendency to recur and progress, even in localizedInt. J. Mol. Sci. 2023, 24, 17535. https://doi.org/10.3390/ijms242417535 https://www.mdpi.com/journal/ijmsInt. J. Mol. Sci. 2023, 24, 17535 2 of 30disease stages [2,3]. Patients diagnosed with bladder cancer often require several intravesi-cal treatments, whereas advanced and metastatic cases need complex surgical and systemictherapies. Consequently, bladder cancer poses a substantial burden on healthcare resourcesand it results in significant economic costs [2,4].Non-muscle-invasive bladder cancer (NMIBC) accounts for most bladder tumors(70–75%). NMIBC is confined to the urothelium as a papillary tumor (pTa) or carcinoma insitu (pTis or CIS) without stromal invasion or with limited invasion into the lamina propria(pT1) [5–7]. This cancer type encompasses a heterogeneous group of tumors whose rates ofprogression to the muscle-invasive phenotype range from 0.8% to 50% within 5 years [8,9].According to previous studies, significant risk factors for NMIBC progression comprisethe combined presence of CIS, a high tumor grade, and the T1 stage [8,9]. Moreover,multiplicity, tumor size ≥3 cm, and history of relapse are also considered risk factors forthis disease [8,9].The therapeutic approach established to manage high-risk non-muscle-invasive blad-der cancer (HR-NMIBC) typically involves initial transurethral resection of the bladdertumor (TURBT), which is followed by intravesical instillations of Bacillus Calmette–Guérin(BCG) [7,10,11]. BCG represents the most efficacious intravesical therapy for individuals di-agnosed with HR-NMIBC [7,10–14]. Despite an initial complete response to BCG observedin approximately 80% of patients, more than half of these individuals experience diseaserecurrence and progression within the initial year of treatment. Consequently, a substan-tial proportion of patients eventually develop what is referred to as BCG-unresponsivedisease [7,11,12]. Disease progression can increase mortality rates and delay the timelyadministration of appropriate radical interventions. Consequently, this process affectspatients’ overall survival [7,13,14]. Hence, the early identification of patients prone to treat-ment failure before starting BCG therapy is of paramount importance, since it can enablethese individuals to significantly benefit from early radical cystectomy, notwithstandingits potential impact on their quality of life [14]. Moreover, it is essential emphasizing theglobal shortage of BCG, as well as the pressing need of finding alternative therapeuticmodalities [11,15].Radical cystectomy stands as the most definitive treatment for NMIBC in this par-ticular context [13–15]. However, this procedure is linked to a significant perioperativemorbidity rate, and a considerable number of patients either lack the willingness or thecapability to undergo such a comprehensive intervention [13–15]. Numerous agents haveundergone assessment as potential intravesical therapies following BCG treatment [11,15].Regrettably, none of these agents has demonstrated a consistently robust and enduring re-sponse. Consequently, there remains a pressing unmet clinical need for the development ofan intravesical treatment that is both effective and durable, while also being safe. This needis particularly critical for patients who wish to avoid the extensive surgical intervention ofradical cystectomy.The therapeutic approach established to manage high-risk non-muscle-invasive blad-der cancer (HR-NMIBC) typically involves initial transurethral resection of the bladdertumor (TURBT), which is followed by intravesical instillations of Bacillus Calmette–Guérin(BCG) [7,10,11]. Although the initial response to BCG treatment is often favorable, itslong-term effectiveness is only moderately effective, and it leads to a substantial risk ofdisease relapse and progression [7,11,12]. Disease progression can increase mortality ratesand delay the timely administration of appropriate radical interventions. Consequently,this process affects patients’ overall survival [7,13,14]. Hence, the early identification ofpatients prone to treatment failure before starting BCG therapy is of paramount importance,since it can enable these individuals to significantly benefit from early radical cystectomy,notwithstanding its potential impact on their quality of life [14]. Moreover, it is essential toemphasize the global shortage of BCG, as well as the pressing need of finding alternativetherapeutic modalities [11,15].Modulating the tumor microenvironment (TME) and enhancing T cell infiltration intotumors are pivotal strategies to assist in managing different tumor types [16]. One of theInt. J. Mol. Sci. 2023, 24, 17535 3 of 30effective approaches involves using innate immunity modulators, more specifically, toll-likereceptor (TLR) agonists [16,17]. These agonists aid in increasing the activity of anti-tumoreffector cells, such as cytotoxic T cells and natural killer (NK) cells, while simultaneouslysuppressing immunosuppressive cell populations, such as regulatory T cells (Tregs) andmyeloid suppressor cells. Furthermore, they play a significant role in influencing immunecells’ recruitment within the tumor microenvironment [16,17]. Checkpoint inhibitor ther-apies’ primarily function is to enhance antitumor responses mediated by CD8+ T cells.Chemokine receptor 1, CX3C (CX3CR1), was recently identified as a T cell differentiationmarker [18]. Notably, CX3CR1 receptors are consistently expressed in CD8+ T cells through-out their differentiation process from CX3CR1−CD8+ T cells during the effector phase [18].This feature turns CX3CR1 into a valuable biomarker, mainly in comparison to moleculespresenting transient expression patterns in T cells.TLRs’ activation triggers a complex signaling cascade that activates both innate andadaptive immune responses and releases pro-inflammatory cytokines [16,17]. Upon ac-tivation, TLRs recruit adapter proteins presenting toll/interleukin-1 receptor (TIR) do-mains. These critical TLR signaling adapters encompass myeloid differentiation factor 88(MyD88), toll/interleukin-1 receptor (TIR) domain-containing adapter protein (TIRAP),toll/interleukin-1 receptor (TIR) domain-containing adapter-inducing interferon-β (TRIF)and translocating chain-associating membrane protein (TRAM) [16,17,19]. MyD88 worksas common adapter for all TLRs, except for TLR3. MyD88 activation triggers nuclearfactor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways, andultimately produces inflammatory cytokines [16,17,19,20]. TLR4 recruits TRIF throughTRAM, whereas TLRs 2 and 4 use TIRAP to recruit MyD88 [16,17,20].TLR signaling can be classified into two different pathways, namely, the MyD88-dependent (canonical) pathway, which promotes the production of inflammatory cytokines,such as tumor necrosis factor-alpha (TNF-α), as well as interleukins such as IL-6, IL-1β,and IL-10; and the TRIF-dependent (non-canonical) pathway, which activates interferonregulatory factors (IRF-3, IRF-5, and IRF-7)—this process results in interferon produc-tion [16,17,19,20]. More specifically, TLR4 uses all four adapter molecules (MyD88, TRIF,TIRAP, and TRAM) to trigger its signaling cascade [16,17,19,20].The emergence of novel immunotherapeutic agents has revolutionized the treatmentapplied to different tumor types, such as urothelial carcinoma [10]. These immunother-apies have shown promising outcomes in bladder cancer cases. Accordingly, our re-search team has developed the OncoTherad® (MRB-CFI-1) nanoimmunotherapy, whichrecorded favorable outcomes in cancer therapy, mainly in bladder cancer cases [21–26].OncoTherad® (MRB-CFI-1) comprises nanometric components formed by phosphate andmetal salts (CFI-1) linked to glycosidic proteins (P14 and P16 proteins) whose size rangesfrom 420 nm to 530 nm [21,27]. It holds granted patents both in the United States (USPTO:US-11623869-B2; US-11572284-B2; US-11136242-B2; US-11639294-B2) and in Brazil (INPI:BR102017012768B1) [21,28–32].OncoTherad® (MRB-CFI-1) triggers the human innate immune system by activatingTLR2 and TLR4. This process enhances the IFN signaling pathway activation process,which involves TLR4, TRIF, IRF-3, IFN-α, and IFN-γ [26–28,33–36]. More specifically,OncoTherad® (MRB-CFI-1) induces immune stimulation through TLR2. TLR4 takes placeby phosphorylating hydroxylated amino acids such as tyrosine, threonine, and serineusing compounds containing phosphate salts. This activation process stimulates thestimulator of interferon genes (STING) and leads to increased IFN-α and IFN-γ produc-tion [21,26–32,35,36]. Moreover, OncoTherad® (MRB-CFI-1) downregulates the expressionof both the receptor activator of nuclear factor-κB (RANK) and the receptor activator ofthe nuclear factor-κB ligand (RANK-L) system. Therefore, it aids in preventing metastasisformation and inhibiting disease progression [24,26–28,37].The aims of the current study were to assess both the safety and the effectiveness ofOncoTherad® (MRB-CFI-1) nanoimmunotherapy applied to patients with NMIBC whowere unresponsive to BCG, as well as to investigate the underlying action mechanismsInt. J. Mol. Sci. 2023, 24, 17535 4 of 30of this nanoimmunotherapy in the intricate microenvironment of bladder cancer cases byfocusing on interrelated factors and signaling pathways.2. Results2.1. OncoTherad® (MRB-CFI-1) Nanoimmunotherapy Primarily Activated Toll-Like Receptor 4 (TLR4)OncoTherad® (MRB-CFI-1) samples used at the concentration of 50 µg/mL led tosignificant expression of both TLR4 and TLR2, which accounted for approximately 84.2%and 52.8% of control ligands, respectively (Figure 1). Furthermore, OncoTherad® (MRB-CFI-1) samples used at the concentration of 5 µg/mL presented a remarkable expression effecton TLR4 and accounted for approximately 49.5% of control ligand responses (Figure 1). Onthe other hand, OncoTherad® (MRB-CFI-1) samples did not trigger significant TLR5, TLR7,TLR8, or TLR9 expression (Figure 1). However, they had a minor expression effect on TLR3,at both 50 µg/mL and 5 µg/mL concentrations, and it corresponded to approximately10.7% and 9.3% of control ligands, respectively (Figure 1).Figure 1. Human TLR ligand screening—OncoTherad® (MRB-CFI-1).On the other hand, P14-16 protein samples had a significant expression effect on humanTLR2 at the concentration of 5 µg/mL and accounted for approximately 32.9% of controlligand responses (Figure S1). Furthermore, P14-16 protein samples led to significant TLR2and TLR4 expression at the concentration of 50 µg/mL. They accounted for approximately78.4% and 38.2% of control ligand responses, respectively (Figure S1). However, P14-16protein did not induce significant human TLR3, TLR5, TLR7, TLR8, or TLR9 expression(Figure S1).Inorganic component (CFI-1) samples of OncoTherad® (MRB-CFI-1) had a significantexpression effect on human TLR4 at the concentration of 5 µg/mL and accounted forapproximately 25.0% of control ligand responses (Figure S2). Moreover, CFI-1 samples ledto significant TLR4 and TLR2 expression at the concentration of 50 µg/mL. They accountedfor approximately 48.0% and 29.1% of control ligand responses, respectively (Figure S2).However, CFI-1 samples did not induce significant human TLR5, TLR7, TLR8, or TLR9Int. J. Mol. Sci. 2023, 24, 17535 5 of 30expression (Figure S2). Nonetheless, they had a minor expression effect on TLR3 at theconcentration of 50 µg/mL and corresponded to approximately 9.4% of control ligandresponses (Figure S2).2.2. Patients’ Baseline Demographics and General FeaturesIn total, 44 patients who met the inclusion criteria were subjected to treatment withOncoTherad® (MRB-CFI-1). Among them, 30 (68.2%) were men and 14 (31.8%) werewomen, with a mean age of 65 years (Table 1). With respect to bladder cancer risk factors,31.8% of patients were smokers, 52.3% were former smokers, and 13.6% were non-smokers(Table 1). In addition, one patient reported to have been continuously exposed to aniline ina paint factory (Table 1).Table 1. Patients’ baseline demographics and general features (n = 44).Characteristics No. (%)Age, median (years) (Minimum–Maximum) 65 (34–96)SexMale 30 (68.2%)Female 14 (31.8%)RaceWhite 41 (93.2%)Black or African-American 01 (2.3%)Asian 02 (4.5%)SmokingNever 06 (13.6%)Former 23 (52.3%)Actual 14 (31.8%)Chronic exposure to aniline 01 (2.3%)Prior BCG cycles1 07 (15.9%)2 37 (84.1%)Intravesical chemotherapy 09 (20.4%)BCG failureBCG refractory 26 (59.1%)BCG relapsing 14 (31.8%)BCG intolerant 04 (9.1%)Histological grade after BCG and/or intravesical chemotherapyHigh-grade pT1 14 (32.4%)High-grade pTa 25 (56.8%)pTis 05 (10.8%)Tumor Focality after BCG and/or intravesical chemotherapyMultifocal 28 (63.6%)Single 16 (36.4%)Tumor size after BCG and/or intravesical chemotherapy<3 cm 08 (18.2%)>3 cm 36 (81.8%)Number of Relapses after BCG and/or intravesical chemotherapy1 04 (9.1%)2 29 (65.9%)3 09 (20.5%)4 02 (4.5%)Mean (Standard deviation) 2.2 (0.67)Most patients (84.1%) had previously undergone two courses of intravesical BCGtherapy, as shown in Table 1. Nine patients (20.4%) had previously undergone intravesicalchemotherapy with gemcitabine either after BCG therapy failure or without it (Table 1).When it comes to BCG responses, 59.1% of patients presented a refractory response type,31.8% experienced disease relapse, and 9.1% were deemed intolerant to it (Table 1).Int. J. Mol. Sci. 2023, 24, 17535 6 of 30With respect to initial disease staging and histological grading after BCG and/orintravesical chemotherapy treatment, tumors’ prevalence was categorized as high-gradepTa (56.8%, Figure 2E,F), which was followed by high-grade pT1 (32.4%, Figure 2G,H) andpTis (10.8%, Figure 2C,D) (Table 1). A multifocal tumor was observed in 63.6% of patients,whereas single lesions were found in 36.4% of cases (Table 1). Furthermore, tumors largerthan 3 cm were identified in 81.8% of patients, whereas tumors smaller than 3 cm weredetected in 18.2% of cases (Table 1).Figure 2. Representative ultrasonography, cystoscopy, and photomicrographs of the urinary bladderbefore OncoTherad® (MRB-CFI-1) treatment: (A) Ultrasonography image revealing an oval-shapedhyperechoic lesion, measuring 3.0 × 1.0 × 0.9 cm in the left lateral wall; (B) Cystoscopy imageconfirming a vegetative lesion >3 cm in the left lateral wall; (C,D) Flat carcinoma in situ (pTis);(E,F) Non-invasive papillary urothelial carcinoma (pTa) 2 + 2 (Unicamp) or high-grade (WHO/ISUP);(G,H) Urothelial papillary carcinoma 2 + 2 (Unicamp) or high-grade (WHO/ISUP) with focal invasion(arrows) of the lamina propria (pT1).The mean number of relapses after prior BCG and/or intravesical chemotherapy withgemcitabine was 2.2 relapses per patient (Table 1).2.3. OncoTherad® (MRB-CFI-1) Nanoimmunotherapy Has Shown High Pathological CompleteResponse (pCR) Rates, as Well as Increased Relapse-Free Survival (RFS) and Response Duration(RD), within a 24-Month Follow-UpNone of the investigated patients experienced disease relapse during initial follow-up(from 1 to 6 months) after OncoTherad® (MRB-CFI-1) treatment application, as indicated inTable 2. However, a subset of patients (27.3%) experienced relapses during the 24 monthfollow-up duration: 8.3% of relapse cases were observed at the 7-to-9-month follow-up;41.7% of cases were observed during the 10-to-12-month follow-up; 33.3% were observedduring the 13-to-18-month follow-up; and 16.7% were observed during the 19-to-24-monthfollow-up (Table 2).Int. J. Mol. Sci. 2023, 24, 17535 7 of 30Table 2. Descriptive measures of quantitative variables (number of relapses, relapse-free survival, andresponse duration) and categorical variables (histological grade and pathological complete response)within the 24-month follow-up of patients treated with OncoTherad® (MRB-CFI-1).Parameters N % Mean StandardDeviationNumber of Relapses1-to-6-month follow-up 0 0 0 07-to-9-month follow-up 01 8.3 0.02 0.1510-to-12-month follow-up 05 41.7 0.11 0.3213-to-18-month follow-up 04 33.3 0.09 0.2919-to-24-month follow-up 02 16.7 0.04 0.21Absence Frequency = 32Total 12 27.3 0.27 0.46Relapse-Free Survival (RFS) 44 100 21.4 4.8Response Duration (RD) 12 27.3 14.3 4.1Histological gradepTis 02 16.7High-grade pT1 02 16.7High-grade pTa 01 8.3Low-grade pTa 07 58.3Absence Frequency = 32Pathological CompleteResponse (pCR) 32 72.7Furthermore, based on the comparison between outcomes observed for OncoTherad®(MRB-CFI-1) nanoimmunotherapy and those observed for prior treatments involving BCGand/or intravesical chemotherapy, it was possible to see statistically significant differ-ences (p < 0.0055) * in relapse incidence reductions in patients treated with OncoTherad®(Tables 2 and 3). Most importantly, no statistically significant disparity in relapse rates wasdetected during different follow-up periods after OncoTherad® (MRB-CFI-1) treatmentapplication (p > 0.05) * (Table 2).Table 3. Histological grade, tumor size, and tumor focality assessment before (BCG and/or intravesi-cal chemotherapy) and after OncoTherad® (MRB-CFI-1) treatment application.BCG and/or Intravesical Chemotherapy OncoTherad®(MRB-CFI-1) p-ValueParameters N % N %Histological GradeHigh-grade 44 100 04 33.3 *0.0055 (Fisher)Low-grade 0 0 08 66.7 *Absence Frequency = 32Tumor Size0.0055 (Fisher)<3 cm 08 18.2 08 66.7 *>3 cm 36 81.8 04 33.3 *Absence Frequency = 32Tumor Focality0.0055 (Fisher)Multifocal 28 63.6 04 33.3 *Single 16 36.4 08 66.7 *Absence Frequency = 32Mean Number ofRelapses 2.2 100 0.27 27.3 * 0.0055 (Fisher)Absence Frequency = 32* Statistical significance.With respect to histological staging and grading after OncoTherad® (MRB-CFI-1) treat-ment application, relapses were observed across different categories, namely, 66.7% of caseswere low-grade pTa, 16.7% were high-grade pT1, 8.3% were high-grade pTa, and 8.3% werepTis tumors (Table 2). Furthermore, OncoTherad® (MRB-CFI-1) nanoimmunotherapysignificantly reduced histological tumor grading in comparison to prior treatments involv-Int. J. Mol. Sci. 2023, 24, 17535 8 of 30ing BCG and/or intravesical chemotherapy (Table 3). More specifically, 100% of patientstreated with BCG and/or intravesical chemotherapy presented high-grade tumors, whereas66.7% and 33.3% of patients subjected to OncoTherad® (MRB-CFI-1) treatment presentedlow-grade and high-grade tumors, respectively (Table 3).Likewise, OncoTherad® (MRB-CFI-1) nanoimmunotherapy enabled a substantialreduction in lesion size and focality in comparison to outcomes observed for previoustreatments with BCG and/or intravesical chemotherapy (Table 3). In most cases (66.7%),patients experiencing relapse after OncoTherad® (MRB-CFI-1) treatment presented lesionssmaller than 3 cm, and these relapses were mostly single lesions.In total, 32 patients (72.7%) subjected to OncoTherad® (MRB-CFI-1) treatment achievedpCR after the 24-month follow-up (Table 2). The Kaplan–Meier curve plotted for RFSindicated a mean RFS of 21.4 months (equivalent to 642 days) during the 24-month follow-up of patients treated with OncoTherad® (MRB-CFI-1) (Table 2 and Figure S3). Moreover,the mean RD was 14.3 months (Table 2).2.4. OncoTherad® (MRB-CFI-1) Nanoimmunotherapy Promoted Non-Neoplastic Cystoscopic andTissue Changes in Patients’ Urinary Bladder at the End of the 24-Month Follow-UpInitial ultrasound and/or cystoscopic examinations evidenced neoplastic lesions >3 cmin the urinary bladder of most patients (81.8%; Tables 1 and 3) who had undergone previoustreatment with BCG and/or intravesical chemotherapy (Figure 2A–H).At the end of the 24-month follow-up of patients treated with OncoTherad® (MRB-CFI-1), 6.8% (Table S1) of them showed normal ultrasound (Figure 3A), cystoscopy (Figure 3B),and histology (Figure 3C) results. Non-neoplastic tissue alterations, such as follicularcystitis (Figure 3J–L), discrete chronic cystitis (Figure 3D,E), erosive cystitis with granulationtissue (Figure 3G–I), and flat urothelial hyperplasia with reactive atypia (Figure 3F) wereobserved in 79.6%, 6.8%, 4.5%, and 2.3% of patients, respectively (Table S1).Figure 3. Representative ultrasonography, cystoscopy, and photomicrographs of the urinary bladderafter OncoTherad® (MRB-CFI-1) treatment: (A) bladder with regular walls and mucosa free ofvegetative lesions, showing only the prior (B) transurethral resection (TUR); (C) normal urotheliumand normal lamina propria; (D,E) mild chronic cystitis; (F) flat urothelial hyperplasia with reactive atypia;Int. J. Mol. Sci. 2023, 24, 17535 9 of 30(G) bladder mucosa with lesions exhibiting short, scattered projections, histologically characterized in(H,I) as granulation tissue (erosive cystitis + granulation tissue); (J) cystoscopic image revealing a flatlesion (circle) <3 cm on the bladder floor, characterized as follicular cystitis; (K,L) photomicrographsof the same flat lesion found during cystoscopy: secondary lymphoid follicle.Chronic cystitis (Figure 3D,E) was histologically featured by the presence of lympho-plasmacytic infiltrate and scarce reactive urothelial atypia in response to inflammation.Flat urothelial hyperplasia with reactive atypia (Figure 3F) was histologically featured bythickening and reactive urothelial atypia, focal chronic non-specific inflammatory infiltrate,edema, and mucosal hyperemia.Erosive cystitis with granulation tissue was featured in cystoscopy by the presence ofshort and scattered projections (Figure 3G) and histologically featured by the presence oflymphoplasmacytic inflammatory infiltrate with granulation tissue fragments (Figure 3H,I).Follicular cystitis was identifiable based on the observation of multiple scatteredflat lesions on the bladder lining surface during cystoscopy, as shown in Figure 3J. Inhistological terms, bladder tissues presented several primary and secondary lymphoidfollicles. Secondary follicles stood out due to the presence of germinal center-presentinglymphocytes, as well as centrocytes (small and cleaved cells), centroblasts (large cells withprominent nucleoli), and macrophages, as shown in Figure 3K,L.Therefore, one of the primary outcomes of OncoTherad® (MRB-CFI-1) nanoimmunother-apy relied on stimulating lymphoid follicle formation, a fact that may be linked to itsimmune-protective effects.2.5. OncoTherad® (MRB-CFI-1) Nanoimmunotherapy Induced Mild-to-Moderate Side Effectsduring the 24-Month Follow-UpThirty-four (n = 34; 77.3%) of the herein assessed 44-patient cohort experienced adversereactions attributable to OncoTherad® treatment, whereas 10 patients (22.7%) remainedfree from any discernible adverse effects, as shown in Figure 4.Figure 4. Occurrence of some type of adverse reaction according to the degree of intensity.Grade 1–2 adverse reactions were significantly more prevalent than the grade 3–4 ones,as shown in Figure 4 and outlined, in detail, in Table 4. More specifically, 28 patients (63.7%)exclusively presented grade 1–2 adverse reactions, whereas six patients (13.6%) experiencedboth grade 1–2 and 3–4 adverse reactions (Figure 4). Dysuria, itching, cystitis, arthralgia,fatigue, skin rash, and fever were the grade 1–2 adverse reactions (≥20% incidence) mostreported by patients (Table 4). Conversely, the most common grade 3–4 adverse reactions(≥4% incidence) comprised skin rash, diarrhea, cough, and shortness of breath (Table 4).Furthermore, comprehensive biochemical serological analyses did not show significantdifferences in parameters such as glucose, hemoglobin, leukocyte count, and platelet count,as well as AST, ALT, GGT, urea, and creatinine levels, before and after OncoTherad® (MRB-CFI-1) treatment application (Table S2). Consequently, these findings provided evidenceInt. J. Mol. Sci. 2023, 24, 17535 10 of 30that OncoTherad® (MRB-CFI-1) administration at the prescribed therapeutic dosage didnot show systemic toxicity indications.Table 4. Adverse reactions in patients with NMIBC who were subjected to OncoTherad® (MRB-CFI-1)intravesical and intramuscular treatment.Adverse ReactionsOncoTherad® (MRB-CFI-1)n = 44Grade 1–2N (%)Grade 3–4N (%)General ReactionsFatigue 14 (31.8%) 0Peripheral edema 05 (11.4%) 0Pyrexia 09 (20.4%) 0Gastrointestinal ReactionsDiarrhea 03 (6.8%) 02 (4.5%)Nausea 06 (13.6%) 0Vomiting 03 (6.8%) 01 (2.3%)Constipation 04 (9.1%) 0Abdominal pain 07 (15.9%) 01 (2.3%)Urinary Tract ReactionsDysuria 23 (52.3%) 0Cystitis 19 (43.2%) 0Musculoskeletal and Connective Tissue ReactionsArthralgia 15 (34.1%) 0Skin and Subcutaneous Tissue ReactionsPruritus 22 (50.0%) 0Rash 12 (27.3%) 03 (6.8%)Respiratory, Thoracic, and Mediastinal ReactionsCough 07 (15.9%) 02 (4.5%)Dyspnea 03 (6.8%) 02 (4.5%)2.6. OncoTherad® (MRB-CFI-1) Nanoimmunotherapy Triggered Innate Immune SystemActivation through TLR4-Mediated Mechanisms and Led to Interferon Signaling PathwayAugmentation, which Was Followed by CD8+ T Cell ActivationOncoTherad® (MRB-CFI-1) induced lymphoid follicles’ formation (Figure 3J–L) inmost patients (79.6%—Table S1) after the 24-month follow-up. Therefore, immunoreactionsand immunohistochemical analyses conducted after the 24-month follow-up of patientstreated with OncoTherad® (MRB-CFI-1) have prioritized regions showing lymphoid folli-cles (LFs), given the significance of LF formation in bladder tissue.Immunoreactivity observed for TLR4 (Figure 5A,B) was significantly higher in bladderbiopsies conducted after OncoTherad® (MRB-CFI-1) treatment application than in pre-treatment biopsies (Figure 5A,B; Table 5).Similarly, immunoreactivity levels observed for antigens TRIF (Figure 5C,D), TBK1(Figure 5E,F), and IRF3 (Figure 5G,H), which are integral components of the TLR4-mediatedinterferon signaling pathway, recorded statistically significant increases in bladder biopsiesconducted after OncoTherad® (MRB-CFI-1) treatment application in comparison to pre-treatment biopsies (Table 5). Moreover, immunoreactivity observed for IFN-γ (Figure 5I,J)recorded significant increases in post-OncoTherad® treatment bladder biopsies in compari-son to pre-treatment biopsies (Table 5).Int. J. Mol. Sci. 2023, 24, 17535 11 of 30Figure 5. Representative immunolabeling of antigens in the urinary bladder before (A,C,E,G,I) andafter (B,D,F,H,J) OncoTherad® (MRB-CFI-1) treatment. TLR4 (A,B); TRIF (C,D); TBK1 (E,F); IRF3(G,H); IFN-γ (I,J). Scale bars = 50 µm.Table 5. Mean ± standard deviation of total immunoreactivity (%) observed for different antigens,both before and after OncoTherad® (MRB-CFI-1) treatment application.Groups (n = 10 Fields/Group)Antigens Before OncoTherad®TreatmentAfter OncoTherad®TreatmentTLR4 50.33 ± 5.0 a 95.83 ± 6.6 bTRIF 47.96 ± 6.9 a 91.24 ± 4.0 bTBK1 53.92 ± 5.8 a 91.01 ± 3.4 bIRF3 42.36 ± 6.2 a 90.32 ± 4.5 bIFN-γ 33.32 ± 3.2 a 82.99 ± 3.7 bCX3CR1 20.00 ± 5.7 a 93.20 ± 3.3 biNOS 31.49 ± 3.7 a 94.94 ± 4.3 bFOXP3 91.28 ± 6.1 a 34.86 ± 9.8 bPD-L1 56.78 ± 7.3 a 56.43 ± 4.7 aCTLA4 92.10 ± 5.0 a 31.98 ± 2.8 bValues followed by different letters on the same line point out significant differences between groups (p < 0.05).Int. J. Mol. Sci. 2023, 24, 17535 12 of 30A significantly augmented interferon signaling pathway was observed after OncoTherad®(MRB-CFI-1) treatment application, and it notably increased CX3CR1 (Figure 6A,B) andiNOS (Figure 6I,J) immunoreactivity in opposition to values recorded for pre-treatmentbiopsies (Table 5). These findings have indicated that this nanoimmunotherapy typeinduced a cytotoxic immune response.Figure 6. Representative immunolabeling of antigens in the urinary bladder before (A,C,E,G,I) andafter (B,D,F,H,J) OncoTherad® (MRB-CFI-1) treatment. CX3CR1 (A,B); FOXP3 (C,D); PD-L1 (E,F);CTLA4 (G,H); iNOS (I,J). Scale bars = 50 µm.In order to investigate the potential correlation between CX3CR1 protein levels andindividuals’ responses to intramuscular administration of OncoTherad® (MRB-CFI-1)nanoimmunotherapy, these levels were assessed in the peripheral blood of 20 patientsbefore and after treatment application. CX3CR1 protein levels were significantly higher afterOncoTherad® (MRB-CFI-1) treatment application (Figure 7 and Figure S4). These findingssuggest that this immunotherapy was effective in promoting CD8+ T cells’ effector phase.Int. J. Mol. Sci. 2023, 24, 17535 13 of 30Figure 7. Representative immunoblots and semiquantitative determination of CX3CR1 protein levels.Representative protein profiles grouped from five patients per sample pool: before (pools 1, 2, 3, 4)and after (pools 1′, 2′, 3′, 4′) OncoTherad® (MRB-CFI-1) treatment. Immunoblots were performed induplicate. The graphs represent the relative integrated optical density (IOD) for CX3CR1 protein,normalized to β-actin and expressed as mean ± standard deviation. *, **, #, + Statistical significance(p < 0.01): * Pool 1′ vs. Pool 1; ** Pool 2′ vs. Pool 2; # Pool 3′ vs. Pool 3; + Pool 4′ vs. Pool 4.2.7. OncoTherad® (MRB-CFI-1) Nanoimmunotherapy Reduced the Immunosuppression State, asWell as the Immune Checkpoint Immunoreactivity, within the Tumor MicroenvironmentImmunoreactivity observed for the immune checkpoint molecules PD-L1 (Figure 6E,F)and CTLA4 (Figure 6G,H) significantly decreased in bladder biopsies conducted afterOncoTherad® (MRB-CFI-1) treatment application in comparison to values recorded forpre-treatment biopsies (Table 5).Similarly, immunoreactivity observed for FOXP3 (Figure 6C,D) significantly decreasedin bladder biopsies conducted after OncoTherad® (MRB-CFI-1) treatment application incomparison to values recorded for pre-treatment biopsies (Table 5). Based on this finding,Int. J. Mol. Sci. 2023, 24, 17535 14 of 30this nanoimmunotherapy type was capable of reversing the immunosuppression statewithin tumor microenvironment.3. DiscussionNMIBC is a diverse condition featuring varying progression and relapse rates whichare influenced by several clinical and pathological factors [10,38–40]. Intravesical BCGtherapy is widely acknowledged as effective against this condition, and this is the reasonwhy it was the initial treatment adopted to reduce the risk of relapse and progression inhigh-grade NMIBC cases [10,38–40]. Approximately 70% of patients can achieve completeremission depending on their risk profile. However, up to 60% of them may experiencerelapse within a year [8,40]. Approximately 20% of high-risk cases may progress to muscle-invasive disease within four years despite the BCG treatment application [40,41].A critical aspect to be taken into consideration in cases of NMIBC relapse, despiteadequate BCG therapy application, lies in whether there is a large enough window of timeto explore alternative treatment options before contemplating radical cystectomy. Evidenceavailable in the literature has suggested that most patients have approximately one yearbefore experiencing prognosis decline [42]. Moreover, a study did not find significant dif-ferences in five-year overall or cancer-specific survival rates between patients participatingin clinical trials and those who chose the option for cystectomy right away [43]. However,it is noteworthy that patients presenting any lymphovascular invasion or prostatic urethralinvolvement degree tend to have poorer prognosis and may not be suitable candidates forsecond-line therapy.In cases where intravesical treatment options prove ineffective, the recommendedcourse of action is radical cystectomy with intestinal diversion. While radical cystectomyhas the potential to halt local disease progression, it is accompanied by a substantialrisk of postoperative complications, a diminished quality of life for patients, and thepotential for overtreatment of individuals whose disease may not be significantly advanced.Furthermore, not all patients are suitable candidates for cystectomy due to factors suchas advanced age or the presence of severe comorbidities. Considering the life-alteringconsequences and the adverse impact on quality of life associated with radical cystectomy,a significant number of patients opt to decline this surgical procedure. Consequently,there exists an urgent need for the development of innovative therapies that can offer anon-surgical second-line treatment option.The BCG non-response point is a crucial time for patients. It is well-establishedthat a third BCG therapy course provides minimal benefit and means the option forundergoing aggressive second-line therapy over cystectomy can potentially lead to tumorprogression, among other unfavorable outcomes. On the other hand, radical cystectomy isassociated with increased comorbidity rates and may represent over-treatment. Therefore,it is imperative to engage in comprehensive and careful discussions with patients beforemaking these life-changing decisions. Regrettably, guidelines provided by the AmericanUrological Association (AUA) provide limited guidance in this context. Given the scarcityof robust data, many experts in the NMIBC field see cystectomy as the safest option forpatients with BCG-non-responsive high-grade NMIBC and for those with high-gradeNMIBC who are unable to tolerate full-dose BCG induction and maintenance therapy.In addition to challenges related to BCG’s toxicity and therapeutic ineffectivenessin high-grade NMIBC patients, the ongoing global shortage in BCG production, as wellas its distribution disruptions, contribute to significantly worsening patients’ prognoses.The current BCG shortage has forced clinics to implement rationing and prioritizationstrategies, and it may have contributed to increase disease relapse and progression ratesamong NMIBC patients [44–46]. Given the aforementioned BCG shortage, patients havebeen subjected to reduced BCG therapy courses, a fact that may increase the number ofpatients undergoing cystectomy [44]. Moreover, the BCG shortage has also affected patients’access and eligibility to participate in clinical trials [44].Int. J. Mol. Sci. 2023, 24, 17535 15 of 30Only two NMIBC treatments were approved by the United States Food and DrugAdministration (FDA) in the past three decades. Valrubicin, which is classified as anthracy-cline, was granted FDA approval in 1998. It was followed by pembrolizumab, which is animmune checkpoint inhibitor immunotherapy approved in 2020 [47,48]. NMIBC researchprogress has been hampered by a lack of consensus concerning clinical trial endpoints andsuitable control groups, as well as by challenges associated with enrolling patients in early-stage clinical trials [49]. However, in 2016, the International Bladder Cancer Group (IBCG)introduced guidelines to guide single-arm trials focused on assessing new therapies incases lacking well-accepted control groups. Subsequently, these guidelines were endorsedby the FDA [10,50]. This governmental agency recommends complete response (CR) asthe primary efficacy endpoint for treatment, as well as a follow-up schedule comprisingassessments conducted every three months in the first year after treatment, semi-annuallyin the second year, and on a yearly basis from the third year onwards [51]. On the otherhand, the IBCG recommends using CR for carcinoma in situ (CIS) tumors and relapse-freesurvival (RFS) for high-grade papillary tumors as primary efficacy endpoints [50]. Theseguidelines have encouraged the development of new studies focused on investigating theuse of immunotherapies to treat BCG-unresponsive NMIBC patients. The current studyinvolving OncoTherad® (MRB-CFI-1) immunotherapy adheres to recommendations madeby both the FDA and IBCG. It used CR and RFS as primary endpoints, along with a follow-up schedule involving quarterly clinical assessments in the first year and semi-annualassessments in the second year.Nowadays, second-line treatment options available for BCG-unresponsive NMIBCcases are less than ideal. Although several conventional chemotherapeutic agents, such asgemcitabine, mitomycin, gemcitabine combined with mitomycin, docetaxel, and valrubicin,have been explored, none of them was superior to BCG and remains under investigation.Studies focusing on bladder preservation therapies after BCG relapse reported RFS rateslower than 25% after two years based on single-agent intravesical instillations of chemother-apy and biological preparations. Chemotherapy combinations, most often gemcitabinecombination with mitomycin C or docetaxel, recorded RFS rates equal to or lower than 35%after two years [52]. Valrubicin, which is an intravesical analog of doxorubicin and the soledrug approved by FDA for this purpose, recorded a one-year disease-free rate in only 8% ofpatients [53]. Off-label second-line options comprise interferon-alpha (one-year disease-freerate of 12%) [54,55], gemcitabine (21%) [56], and mitomycin C (23%) [57]. However, if onetakes into consideration that several studies reported five-year disease-free rates rangingfrom 20% to 40% in patients subjected to radical cystectomy, it is quite clear that cystectomyremains the safest option in these cases.The bladder cancer treatment scenario has significantly evolved over time, since itnot only encompasses traditional approaches, such as surgery and chemotherapy, but alsoimmunotherapy integration. BCG immunotherapy has been applied to prevent NMIBCrelapse and progression for more than four decades. However, recent improvementsin scientists’ understanding about this topic have opened new room to enhance its anti-tumor effectiveness. Alongside BCG, the introduction of immune checkpoint inhibitors hasrevolutionized the standard care provided to patients with metastatic bladder cancer, aswell as expanded their potential use in BCG-unresponsive NMIBC cases. These immunecheckpoint inhibitors, such as anti-PD1 or anti-PD-L1 agents, have garnered increasinginterest within the bladder cancer-related scientific community. Critical considerationscomprise whether using immune checkpoint inhibitors can delay or change patients’ needof undergoing surgery and potentially affect their outcome, as well as whether toxicity isproportional to NMIBC severity.Pembrolizumab (an anti-PD1 agent) has recently received FDA approval to be appliedto high-risk BCG-unresponsive NMIBC patients with and without concomitant CIS who arenot suitable candidates for cystectomy. This approval was based on findings resulting fromthe ongoing phase 2 KEYNOTE-057 study, which achieved a three-month CR rate of 40.6%and a median RD of 16.2 months after more than two years of follow-up with 96 patients.Int. J. Mol. Sci. 2023, 24, 17535 16 of 30In total, 46.2% of all 39 patients who achieved CR presented RD equal to or longer than12 months [40,58]. Nine (9) of 57 non-responders experienced disease progression at threemonths, whereas three out of 36 patients who underwent cystectomy showed diseaseprogression to T2 bladder cancer at least [58].According to the phase 2 SWOG S1605 monotherapy study conducted with 74 CISpatients treated with atezolizumab (anti-PD-L1 agent), CR rates reached 41.1% at threemonths and 26.0% at six months [59]. However, this trial was terminated because it didnot meet its primary endpoint. Preliminary results of the ongoing phase-2 QUILT-3.032study enabled the FDA to grant innovative therapy designation to the ALT-803 + BCGIL-15 receptor superagonist in BCG-unresponsive NMIBC patients [60]. In total, 90% ofthe herein assessed 20 CIS patients achieved CR, whereas 16 high-grade pTa/pT1 patientsrecorded a 75% survival rate at six months and 54% at nine months [60].Vicinium (oportuzumab monatox) is a fusion protein composed of an epithelial celladhesion molecule-specific antibody fragment and Pseudomonas exotoxin. It exerts itsantitumor effects by inhibiting protein synthesis [61]. Phase 1 and 2 studies involvingintravesical vicinium application reported CR rates ranging from 29% to 40% at threemonths in patients with CIS [61]. The recently concluded phase 3 VISTA study enabledvicinium fast-track designation by the FDA. Initial phase results have indicated a 40% CRrate at three months, while 52% of patients maintained CR for 12 months or longer [62,63].Intravesical gene therapy is a promising alternative to treat NMIBC, given the blad-der’s suitability for such a treatment. Nadofaragene firadenovec, which is a gene-mediatedintravesical therapy based on using human IFN-α2b, recorded a CR rate of 53.4% at threemonths in a phase 3 study conducted with CIS patients—24.3% of them remained free fromhigh-grade relapse after one year [64]. In total, 72.9% and 43.8% of patients with singlehigh-grade pTa/pT1 tumors maintained relapse-free status at three and twelve months,respectively—patients’ responses to the adopted treatment extended up to one year [64].OncoTherad® (MRB-CFI-1) nanoimmunotherapy, which provides innovative thera-peutic advantages in comparison to conventional treatments, has emerged as promisingcandidate to help managing high-risk NMIBC, mainly in cases of unsuccessful BCG therapy.Extensive prior research efforts made by our group have evidenced a range of beneficialtherapeutic properties and effects associated with OncoTherad® (MRB-CFI-1) [21–28,36].OncoTherad® (MRB-CFI-1) used in preclinical trials has shown approximately 80% an-titumor effectiveness, as well as presented clear superiority over BCG in comparativeeffectiveness and safety studies [21–28,36].These promising preclinical effectiveness findings have encouraged our research teamto perform a clinical-veterinary trial with dogs diagnosed with naturally occurring bladdercancer. Dogs participating in this trial were treated with intravesical (44 mg/mL) andintramuscular (22 mg/mL) OncoTherad® (MRB-CFI-1) applications on a weekly basis forsix consecutive weeks. Subsequently, they underwent intravesical and intramuscular appli-cations of it twice a week for six months, then intravesical and intramuscular applicationson a monthly basis for an additional six months (24 applications in total). It is noteworthythat 83.3% of patients presented stabilized disease, whereas 16.7% of them presented partialdisease remission after the initial three OncoTherad® (MRB-CFI-1) applications. All (100%)patients achieved partial disease remission after the 24th OncoTherad® (MRB-CFI-1) appli-cation. The median relapse-free/progression-free survival time was 402 days. Moreover,OncoTherad® (MRB-CFI-1) administration did not show indications of systemic toxicity atthe prescribed therapeutic doses [21,22,29–32,65].Another prospective multicenter clinical-veterinary study was conducted with 19 dogsdiagnosed with recurrent oral melanoma who had previously undergone at least onecourse of first-line chemotherapy [33]. OncoTherad® (MRB-CFI-1) treatment based onintramuscular applications (22 mg/mL) administered twice a week for three months wasapplied to Group G1, which comprised 10 dogs. Subsequently, treatment frequency wasreduced to applications every 15 days until the end of the 12-month treatment. Group G2(nine dogs) was treated with OncoTherad® (MRB-CFI-1) in combination with chemother-Int. J. Mol. Sci. 2023, 24, 17535 17 of 30apy (Carboplatin: 250–300 mg/m2—intravenous) or electrochemotherapy (Bleomycin:15,000 IU/m2—intravenous). Based on treatment outcomes assessed through iRECISTstudy criteria, complete response occurred in 31.6% of cases in total (Group G1—40.0%;Group G2—22.2%), whereas partial response was observed in 42.1% of cases (Group G1—30.0%; Group G2—55.6%), and stabilized disease was observed in 10.5% of patients (GroupG1—20.0%; Group G2—0.0%). Only 15.8% of all patients presented disease progression(Group G1—10.0%; Group G2—22.2%). The median overall progression-free survival timewas 640 days (Group G1—676.5 days, Group G2—599.4 days). Therefore, OncoTherad®(MRB-CFI-1) immunotherapy was effective in managing chemotherapy-resistant diseaseand presented potential advantages in preventing canine oral melanoma progression [33].The current study recorded a pCR rate of 72.7% (95% CI) and a mean RFS of 21.4 months.The mean response duration among all 32 patients with pCR was 14.3 months. No patientprogressed to muscle-invasive or metastatic disease during treatment. Altogether, thesefindings have emphasized the highly promising antitumor effects of OncoTherad® (MRB-CFI-1) immunotherapy, which surpassed outcomes observed for standard BCG therapyand for other therapeutic regimens used for BCG and/or intravesical chemotherapy non-responsive NMIBC cases. This factor turns OncoTherad® (MRB-CFI-1) immunotherapyinto a valuable therapeutic option in NMIBC management.BCG toxicity can be classified into two categories: local and systemic side effects.Local side effects are more prevalent but, oftentimes, less severe. Sometimes, they canlead to treatment discontinuation [66]. It is noteworthy that most local side effects have atemporary nature [67]. Local side effects’ incidence remains relatively consistent, whetherpatients are on maintenance therapy or not. These effects often include lower urinary tractsymptoms (observed in 57–71% of patients undergoing maintenance therapy and in 38–59%of those who are not undergoing it) and hematuria (observed in 20% of patients undergoingmaintenance therapy and 29% of those who are not undergoing it) [68].Similarly, studies did not identify significant differences in systemic side effects be-tween non-maintenance and maintenance therapy groups [69]. Some of them have evenreported reduced toxicity after the initial six-month treatment [66]. Systemic side effects canbe categorized into infectious (e.g., bacterial cystitis, epididymitis, prostatitis, and systemicinfection) and non-infectious (i.e., arthralgias, skin reactions, and anaphylaxis). Among themost often reported systemic toxicities, one finds fever, chills and flu-like symptoms thataffect 22% to 30% of patients undergoing maintenance therapy, as well as 19% to 26% ofnon-maintenance therapy patients. Epididymitis, prostatitis, and urethral infections werereported in 4% of patients undergoing maintenance therapy and in 4% of non-maintenancetherapy patients, whereas systemic infection was observed in 7% of patients undergoingmaintenance therapy and in 1% of non-maintenance therapy patients. Less severe systemictoxicity symptoms, such as fever higher than 39.5 ◦C, rash, arthralgias, and BCGitis (es-tablished BCG infection with manifestations in another organ) recorded incidence ratesranging from 2% to 6% [67,68].Adverse effects observed for pembrolizumab used as a monotherapy were assessedin a cohort of 102 patients. Results have shown that 97.1% of patients experienced at leastone adverse event, 65.7% of them had treatment-related adverse events, and 29.4% of theinvestigated population experienced grade 3–5 adverse events [70].With respect to OncoTherad® (MRB-CFI-1) nanoimmunotherapy’s safety profile, thecurrent findings have evidenced that treatment-related adverse events (TRAEs) werereported by 77.3% of patients, whereas 22.7% of them did not experience any adverseevent. Grade 1–2 TRAEs were the most prevalent types; they were reported by 63.7% ofpatients in opposition to grade 3–4 events. The most often reported reactions comprisedcystitis, dysuria, arthralgia, itching, skin rash, fatigue, and fever. Furthermore, serumlevels of several biochemical parameters remained within the normal reference values afterOncoTherad® (MRB-CFI-1) treatment application.Previous studies conducted by our research team have evidenced this nanocom-pound’s low toxicity in vitro in grade II urinary bladder carcinoma cells (5637 cell line), asInt. J. Mol. Sci. 2023, 24, 17535 18 of 30well as 75% cell viability, and suggested that neoplastic cell death induced by OncoTherad®(MRB-CFI-1) likely depends on immune system activation [27]. Moreover, the afore-mentioned studies have shown that OncoTherad® (MRB-CFI-1) immunotherapy did notinduce either local or systemic adverse effects on rats, mice, and rabbits at a therapeuticdose of 20 mg/kg [21,23,27,29]. However, OncoTherad® (MRB-CFI-1) doses of 50 mg/kgand 100 mg/kg resulted in moderate and intense inflammatory reactions in the bladder,ureter, and kidneys of the tested animal models. Furthermore, OncoTherad® (MRB-CFI-1) treatment at doses of 20 mg/kg, 50 mg/kg, and 100 mg/kg did not induce hepato-toxicity and nephrotoxicity in rats, mice, or rabbits—the maximum tolerated dose was100 mg/kg [21,23,27,29].Briefly, the current study has shown that OncoTherad® (MRB-CFI-1) nanoimmunother-apy induced lower intensity and frequency adverse effects in comparison to those associatedwith standard BCG therapy and with immune checkpoint inhibitor use. This finding high-lights both the safety and the effectiveness of OncoTherad® (MRB-CFI-1) immunotherapyas a viable option to aid in managing patients with carcinoma of the high-risk NMIBC typein situ.The immune microenvironment of the urinary bladder has unique features. On theone hand, the bladder uses several non-specific defense mechanisms to remain free frominfections [71]. These strategies involve the presence of a thick mucin barrier, regular uri-nation, and the secretion of antibacterial agents, such as β-defensins and cathelicidin [71].Conversely, the bladder maintains an immunosuppressive environment to avoid unde-sired immune responses [71,72]. Furthermore, bladder tumors’ microenvironments areimmunosuppressive, besides leading to tumor-infiltrating lymphocytes’ (TILs) deficiencyin effectively ruling out tumor cells [73]. Bladder tumors are often associated with increasedregulatory T cell (Tregs) levels, as well as with the expression of inhibitory Th1 responsecytokines, such as transforming growth factor-beta (TGF-β) and IL-10. Consequently,understanding the strategies used by bladder tumors to evade the immune system is ofparamount importance to assist in developing immunotherapies aimed at fighting bladdercancer [74].Nowadays, TLR agonists are acknowledged as effective immune stimulants withsignificant potential to be used in immunotherapy applied to different tumor types, in-cluding bladder cancer. One of the immune mechanisms accounting for both detectingand controlling tumor cell proliferation lies in TLR4-mediated production of inflammatorycytokines in macrophages [75,76]. There is downregulation of TLR4 expression in tumortissues in comparison to surrounding or normal tissues. Most importantly, TLR4 expressionis positively associated with overall survival (hazard ratio [HR] = 0.38) and cancer-specificsurvival (HR = 0.15) rates in bladder cancer patients [77]. In addition, low TLR4 expressionis mainly observed in high-grade tumors [77].The non-canonical TLR4 signaling pathway plays a pivotal role in triggering inter-feron (IFN) production. IFNs have strong antitumor effects since they enable TNF-relatedapoptosis-inducing ligand (TRAIL) generation. TRAIL is known for its ability to inducecell death in tumor cells [76]. Tumor-suppressive actions performed by the immune systemmostly rely on the activities of IFN-γ, which stimulates several antitumor and antipro-liferative pathways in both macrophages and neoplastic cell lines [75]. Moreover, IFNsplay a key role in early protection against [75] metastasis, as well as in integral parts ofmechanisms involving the innate immune system activation for tumor detection and T cellresponse modulation purposes [78–81]. CD8+ T lymphocytes’ activation against tumorswas compromised in mice lacking STING. Studies conducted in vitro by Woo et al. [81]have evidenced that DNA deriving from neoplastic cells triggered IFN production andactivated dendritic cells (DCs) through both the STING pathway and IRF3 and indicatedthe involvement of a cytosolic DNA detection mechanism. DNA extracted from malignantneoplastic cells from models in vivo was detected within host antigen-presenting cells(APCs), which were correlated to IFN production and STING activation [81].Int. J. Mol. Sci. 2023, 24, 17535 19 of 30The current study has shown that OncoTherad® (MRB-CFI-1) immunotherapy wascapable of activating the innate immune system mediated by TLR4. This finding wasconsistent with data available in the literature and with our pre-clinical and veterinaryclinical findings on bladder cancer. The aforementioned activation led to a substantialincrease in the interferon signaling pathway involving TRIF, TBK1, IRF3, and IFN-γ. Thisactivation plays a critical role in stimulating CD8+ CX3CR1+ T cells, as well as in reducingthe number of immune checkpoint molecules (PD-1/PD-L1 and CTLA4) and in reversingindividuals’ immunosuppressive state, with emphasis on decreased FOXP3+ Tregs withinthe urinary bladder microenvironment.The TLR4-mediated interferon signaling pathway activation induced by OncoTherad®(MRB-CFI-1) also has a significant influence on both the formation and organization oflymphoid follicles (LFs) within the bladder tissue. LFs work as the architectural basis forthe development of follicular dendritic cell networks, which play a pivotal role in germinalcenter reactions. These reactions are essential for affinity maturation processes accountingfor optimizing antibodies during adaptive immune responses [82]. TLR expression bymyeloid lineage cells establishes a critical link between the innate and adaptive immuneresponses, mainly in dendritic cells and monocyte–macrophage lineage cells accounting forexpressing TLRs. This interaction enhances the function of antigen-presenting cells andenables the release of cytokines that play essential roles in activating CD8+ and CD4+ Tcells [82,83]. These findings emphasize the pivotal role played by TLRs in driving processesthat ultimately lead to the development of a fully functional cellular immune response.More specifically, TLR4 is implicated in the maturation of follicular dendritic cells, as wellas in the establishment of germinal centers within lymphoid follicles [82].The role played by TLRs in enhancing humoral immune responses, as well as in affect-ing B cells both directly and indirectly through their interactions with other immune cells,has been increasingly acknowledged. B cells themselves express TLRs. Moreover, they un-dergo polyclonal stimulation when they are exposed to TLR ligands such as OncoTherad®(MRB-CFI-1). This stimulation leads to B cells’ differentiation and proliferation into cells ac-counting for secreting immunoglobulins. TLR signaling also plays a crucial role in enablingB lymphocytes’ migration to and clustering within lymphoid follicles. This process pro-motes sustained B cell proliferation, besides generating memory B and plasma cells [82]. Inaddition to its direct effects on B cells, TLR signaling indirectly supports T cell-dependentB cell response induction through its cooperative interactions with CD4+ T cells. Thissynergy between TLRs and CD4+ T cells contributes to enhanced B cell responses withinthe immune system.Although TLRs’ influence on triggering humoral immune responses is well-documentedfor helper T cells and for the intrinsic activation of B cells, the general understandingconcerning the potential consequences of TLR ligands in LFs and follicular dendritic cellsremains limited. Consequently, the current study has emphasized the notable influenceof the TLR4 signaling pathway activation induced by (MRB-CFI-1) on LFs’ developmentand arrangement processes. This OncoTherad® phenomenon may be associated withOncoTherad’s immunoprotective effects on bladder tissue.Identifying blood-based biomarkers capable of reflecting dynamic changes in thetumor microenvironment and of predicting tumor responses to immunotherapies is apromising path for enhancing current treatment strategies [18]. The current study focusedon investigating CX3CR1 as a potential predictive biomarker to measure bladder cancerresponses to OncoTherad® (MRB-CFI-1) immunotherapy. According to Yamauchi et al. [18],the administration of immune checkpoint inhibitor immunotherapies to tumor-bearingmice has increased both T-cell receptor frequency and clonality within the peripheralCX3CR1+CD8+ T-cell subset. Furthermore, their study evidenced that the early surge inCX3CR1+ subset frequency among circulating CD8+ T cells was correlated to both responsesby and prolonged survival of non-small cell lung cancer patients undergoing anti-PD-1therapy. The current findings have indicated that increased CX3CR1 levels, both in bladdertissue and peripheral blood samples, mirrored dynamic fluctuations observed in CD8+Int. J. Mol. Sci. 2023, 24, 17535 20 of 30TILs within the tumor microenvironment after OncoTherad® (MRB-CFI-1) treatment appli-cation. This increase in CX3CR1 levels is associated with anti-tumor responses triggered byOncoTherad® (MRB-CFI-1) therapy applied to BCG-unresponsive patients with carcinomain situ. Consequently, the current results support CX3CR1 adoption as a biomarker to helpin prognosticating clinical responses to OncoTherad® (MRB-CFI-1) therapy.Our study has certain limitations that should be acknowledged. Firstly, the rela-tively small sample size may not provide a precise reflection of the safety and efficacy ofOncoTherad® (MRB-CFI-1) immunotherapy. It is important to note that our results arederived from a single institution with expertise in NMIBC treatment protocols. However, itis conceivable that these findings may not be readily applicable to other institutions withlower case volumes of NMIBC.Moreover, our single-arm study was constrained by the absence of a direct comparatorgroup. Nevertheless, it’s worth emphasizing that there are limited effective treatmentalternatives available beyond radical cystectomy for this patient population. Additionally,comparing our findings with off-label treatments is a complex endeavor due to historicalinconsistencies in defining adequate BCG therapy, variations in BCG dosing regimens, theinherent heterogeneity of patient populations, and differences in monitoring or surveil-lance protocols.In recognition of these limitations, our research group is actively pursuing furtherinvestigations to substantiate the safety and efficacy of OncoTherad® (MRB-CFI-1) im-munotherapy. Our future research initiatives will encompass expansions to multipleclinical research centers and a substantial augmentation of the sample size.4. Materials and Methods4.1. Assessing Toll-like Receptor (TLR) Ligands in Human Cells: OncoTherad® (MRB-CFI-1) andIts Components (CFI-1 and P14-16 Protein)A series of experiments involving NF-κB activation in HEK293 cells genetically engi-neered to express specific TLRs was conducted to assess the impact of both OncoTherad®(MRB-CFI-1) and its constituent components (CFI-1 and P14-16 protein) on toll-like recep-tors (TLRs). An NF-κB activation-responsive promoter was used to control the expressionof a secreted alkaline phosphatase (SEAP) reporter gene, and it enabled monitoring of theTLR signaling through NF-κB activation.A screening test was applied to assess the effects of both OncoTherad® (MRB-CFI-1)and its constituents (CFI-1 and P14-16 protein) on seven different human TLRs, namely,TLR2, TLR3, TLR4, TLR5, TLR7, TLR8 and TLR9. Ligands acknowledged for these TLRswere used for control purposes, as follows:• TLR2: Heat-killed Listeria monocytogenes (HKLM). Concentration: 108 cells/mL.• TLR3: Poly(I:C). Concentration: 1 µg/mL.• TLR4: Escherichia coli K12 lipopolysaccharide (E. coli K12 LPS). Concentration: 100 ng/mL.• TLR5: Salmonella Typhimurium flagellin (S. Typhimurium flagellin). Concentration:100 ng/mL.• TLR7: CL097. Concentration: 1 µg/mL.• TLR8: CL075. Concentration: µg/mL.• TLR9: CpG ODN 2006. Concentration: 1 µg/mL.• NF-κB control cells: TNF-α. Concentration: 100 ng/mL.Samples of both OncoTherad® (MRB-CFI-1) and its constituents (CFI-1 and P14-16 protein) were prepared by diluting them in 1 mL of dimethyl sulfoxide (DMSO) toachieve a stock concentration of 5 mg/mL. Subsequently, these samples were tested at finalconcentrations of 0.5, 5, and 50 µg/mL—results were compared to control ligands. Eachstep of this process was carried out in duplicate. In total, 20 µL of OncoTherad® (MRB-CFI-1) samples or of its constituents (CFI-1 and P14-16 protein), along with positive controlligands, were introduced in 96-well plates—each well contained the total volume of 200 µLand 50,000 cells. Culture medium was added to the wells used to detect NF-κB-inducedInt. J. Mol. Sci. 2023, 24, 17535 21 of 30SEAP expression. Optical density (OD) was measured in a Beckman Coulter AD 340CAbsorbance Detector at 650 nm after 16–20 h of incubation.4.2. Pharmacological Treatment Delivered to NMIBC Patients: OncoTherad® (MRB-CFI-1)NanoimmunotherapyIn total, 58 patients originating at Paulínia Municipal Hospital (HMP), Paulínia City,São Paulo State, Brazil, who had been diagnosed with NMIBC classified as refractory, recur-rent, or intolerant to Bacillus Calmette–Guérin (BCG) and/or to intravesical chemotherapy,were included in the current study. Forty-four (44) of them met the predefined inclusioncriteria and started being treated with OncoTherad® (MRB-CFI-1). The recruitment phasespanned from August 2018 to June 2021. It was followed by a subsequent treatment and bya 24-month clinical follow-up.Eligible participants were patients aged 18 years or older, presenting with histologicallyconfirmed high-risk NMIBC, who were either not suitable candidates for or declined radicalcystectomy. Hematological and biochemical laboratory assessments were mandatory14 days prior to the commencement of the study. To meet eligibility criteria, patientsunderwent transurethral resection of the bladder tumor (TURBT) four weeks prior tothe initiation of the study. This procedure was conducted at HMP and was performedunder either general or epidural anesthesia, with the choice being determined throughconsultation between anesthetists and patients. Confirmation of diagnosis through bladderbiopsies was carried out by the Pathology Department of HMP (Figure 8).BCG-unresponsive disease was defined as: (i) BCG refractory: failure to achieve adisease-free state by six months after initial BCG therapy because of persistent or rapidlyrecurrent disease. This category also includes patients with stage or grade progression atthree months despite BCG therapy; (ii) persistent high-risk NMIBC within six months afteradequate BCG therapy; (iii) recurrent high-risk NMIBC within nine months after the lastBCG instillation, despite adequate BCG therapy; (iv) BCG intolerant: disease recurrenceafter the administration of a less than adequate course of BCG therapy because of a seriousadverse event or symptomatic intolerance. Adequate BCG therapy was defined as a mini-mum of five induction instillations (adequate induction) and at least seven instillations ofBCG within nine months after the first instillation, consistent with the FDA-recommendeddefinition (≥5 out of six doses of an initial induction course plus ≥2 out of three doses ofmaintenance therapy or ≥2 out of six doses of a second induction course). Eligible patientshad not received intervening intravesical chemotherapy or immunotherapy from the timeof the most recent cystoscopy or TURBT until study entry.Patients with histologically confirmed muscle-invasive carcinoma, locally advancedunresectable or metastatic urothelial carcinoma, or concurrent extravesical urothelial car-cinoma (in the prostatic urethra, distal urethra, ureter, or renal pelvis) were excluded(Figure 8).Upon diagnosis, patients provided informed consent and subsequently started treat-ment with OncoTherad® (MRB-CFI-1). The OncoTherad® treatment regimen comprisedtwo phases (Figure 8):(a) Induction Phase: Intravesical (at concentration of 120 mg/mL, intravesically retainedfor 1 h, while patients were asked to rotate positions to maximise bladder surfaceexposure) and intramuscular (at concentration of 25 mg/mL) OncoTherad® (MRB-CFI-1) administrations on a weekly basis for six consecutive weeks.(b) Maintenance Phase: Biweekly OncoTherad® (MRB-CFI-1) administrations (both in-travesical and intramuscular) for three months, followed by monthly administrations(both intravesical and intramuscular) for additional nine months, totaling one year oftreatment. Subsequently, quarterly OncoTherad® (MRB-CFI-1) administrations (bothintravesical and intramuscular) were maintained for another year.The current study was approved by the Research Ethics Committee/UNICAMP(CAAE: 93619718.7.0000.5404) and registered in the Brazilian Clinical Trials Registry(RBR-6swqd2).Int. J. Mol. Sci. 2023, 24, 17535 22 of 30Figure 8. OncoTherad® (MRB-CFI-1) study design. BCG: Bacillus Calmette–Guérin; NMIBC: Non-muscle-invasive bladder cancer.4.3. Assessing the Therapeutic Effectiveness of OncoTherad® (MRB-CFI-1) NanoimmunotherapyThe primary criteria used to assess treatment effectiveness comprised pathologicalcomplete response (pCR), relapse-free survival (RFS), and response duration (RD). pCRwas defined as the number of patients who did not show any evidence of disease or diseaseInt. J. Mol. Sci. 2023, 24, 17535 23 of 30progression. RFS refers to the time interval from the beginning of OncoTherad® (MRB-CFI-1) treatment to either disease relapse or death as an initial event. Relapse was featured as anyhistologically confirmed tumor recurrence, regardless of its grade. Response duration wasdefined as the time interval between achieving pCR and experiencing therapeutic failure.All patients were subjected to regular follow-up consultations at the outpatient clinic ofHMP to assess therapeutic effectiveness. This follow-up process encompassed histopatho-logical assessments, urinary tract ultrasounds, cystoscopies, and laboratory examinationsevery three months during the first follow-up year and, subsequently, every six monthsduring the second year (Figure 8).4.4. Inclusion and Exclusion Criteria4.4.1. Inclusion CriteriaPatients meeting the following criteria were eligible to be included in the current study:• Aged ≥18 years• Histologically confirmed diagnosis of NMIBC (Ta, T1, and/or carcinoma in situ—CIS)with urothelial carcinoma—predominant histology.• Presence of multiple tumors (>2 lesions) and/or recurrent and/or large tumors (>3 cm)classified as TaG1-2.• Diagnosis of grade 3 urothelial bladder carcinoma.• Completed ≥1 adequate course of BCG induction therapy for the treatment of NMIBC.• Persistent or recurrent high-risk NMIBC after adequate induction therapy.• Patients who were refractory or intolerant to BCG treatment.• Patients who were recurrent or refractory to first and second-line chemotherapy sincethey are at higher risk of disease progression to invasive and/or metastatic cancer.• Willingness to provide informed consent.• Clinical ineligibility for radical cystectomy.• Patients who declined radical cystectomy.• Adequate organ function.4.4.2. Exclusion CriteriaPatients meeting any of the following criteria were excluded from the current study:• Diagnosis of muscle-invasive bladder tumors.• Metastatic bladder tumors.• Histological types other than urothelial carcinoma, including adenocarcinoma andsquamous cell carcinoma.• Patients with cardiac function classified as higher than class III since it indicates theneed of cardiac procedures or treatments.• Presence of comorbidities, such as demyelinating diseases of the central nervoussystem (e.g., multiple sclerosis, optic neuritis), congestive heart failure class II orhigher, chronic obstructive pulmonary disease, interstitial lung diseases (includingpulmonary fibrosis), tuberculosis (any form), chronic kidney failure stage II or higher,liver cirrhosis of any cause, chronic viral hepatitis, HIV infection (with or withoutAIDS), leprosy, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, anyglomerular kidney disease, bullous skin diseases, chronic fungal infections, chronicparasitic diseases, or being a transplant recipient on immunosuppressants.• Current or prior diagnosis of any other malignant neoplasm (except for non-melanomaskin cancer) unrelated to the inclusion tumor.• Having access to any antineoplastic immunotherapy approved to treat their respec-tive tumor.4.5. Histopathological AnalysisBladder samples collected from all patients through cystoscopy or transurethral re-sections of bladder tumors (TURBT) were fixed in 10% formaldehyde for 24 h and thenembedded in paraffin. Subsequently, they were sliced into 5 µm-thick sections with aInt. J. Mol. Sci. 2023, 24, 17535 24 of 30semi-automatic microtome (CUT5062; Slee Mainz, Munich, Germany). These sectionswere stained with hematoxylin–eosin and examined under a Leica DM2500 microscopeequipped with a DFC295 camera (Leica, Munich, Germany) in order to capture images.Diagnosis based on bladder biopsies was initially performed by the Pathology De-partment of HMP and further reviewed by a senior pathologist from the Medical Schoolat the University of Campinas (UNICAMP). Urothelial lesions were categorized based onthe staging criteria proposed by the World Health Organization/International Society ofUrological Pathology consensus [84].4.6. Toxicological Analyses of OncoTherad® (MRB-CFI-1) NanoimmunotherapyAll patients underwent clinical assessments using the Toxicity Scale, as outlined in the4th Common Terminology Criteria for Adverse Events [85], to investigate potential localand systemic toxicities associated with OncoTherad® (MRB-CFI-1) nanoimmunotherapy.Blood and urine samples were collected every three months during the first follow-up year,as well as every six months during the second year, to perform the following assessments:• Complete blood count, including hemoglobin and white blood cell counts.• Thrombogram.• Serum glucose, aspartate transaminase (AST), alanine transaminase (ALT), gamma-glutamyl transferase (GGT), urea, and creatinine levels.These assessments were conducted at the Clinical Analysis Laboratory of HMP. Localtoxic effects were investigated based on cystoscopy findings, on the incidence of hematuria,as well as on the assessment of lower urinary tract symptoms.4.7. Immunohistochemical Analyses Applied to TLR4, TRIF, TBK1, IRF3, IFN-γ, CX3CR1,FOXP3, PD-L1, CTLA4, and iNOS in Bladder Biopsies, Both before and after OncoTherad®(MRB-CFI-1) Treatment ApplicationBoth pre- and post-treatment bladder biopsy samples (n = 44) collected from patientstreated with OncoTherad® (MRB-CFI-1) were subjected to immunohistochemical analysesafter histopathological diagnosis. In order to do so, bladder tissue samples were sliced into5-µm thick sections with a semi-automatic microtome, Slee CUT5062 RM 2165 (Slee Mainz,Mainz, Germany), and placed on silanized slides. Then, these sections were subjected todeparaffinization with xylene, rehydrated with graded alcohols, and rinsed with deionizedwater. Antigen retrieval was carried out using citrate buffer (pH 6.0) in a microwaveat 100 ◦C and at 800 W power for 20 min. This procedure was followed by blockingendogenous peroxidase activity using 0.3% H2O2 for 15 min. Non-specific protein bindingwas blocked based on using a 5% goat serum solution in TBS-T buffer at room temperaturefor 30 min.Subsequently, the immunolocalization of antigens such as TLR4, TRIF, TBK1, IRF3,IFN-γ, CX3CR1, FOXP3, PD-L1, CTLA4 and iNOS was performed using specific primaryantibodies (Table S3), which were diluted in 1% goat serum and incubated at 4 ◦C overnight.Antigen detection was achieved using the EasyLink One Polymer HRP IHC kit (EP-12-20504, EasyPath) according to the manufacturer’s guidelines. Bladder tissue sectionswere exposed to horseradish peroxidase (HRP)-conjugated secondary antibody from theEasyLink One Polymer HRP IHC kit for 40 min, after which they were rinsed with TBS-T buffer. Subsequently, they were visualized using chromogen 3-3′-diaminobenzidinetetrahydrochloride (DAB), counterstained with Harris hematoxylin, and assessed under aLeica DM2500 microscope equipped with a DFC295 camera (Leica, Munich, Germany).Ten (10) fields (at 400×magnification) were selected for each patient and antibody toassess antigen immunoreactivity intensity in bladder tissue. Immunostaining results wereanalyzed in Image J software version 1.54f. Macro profile analysis was used to quantifypositive cells [28,36]. Total immunoreactivity was calculated as the rate of negative cells fora specific antibody subtracted from 100%. It represented the total number of cells in thefield that were immunoreactive to the assessed antibody [28,36].Int. J. Mol. Sci. 2023, 24, 17535 25 of 304.8. Western Blotting Analysis Applied to CX3CR1 in Patients’ Peripheral Blood, Both before andafter OncoTherad® (MRB-CFI-1) Treatment ApplicationPeripheral blood samples were collected from 20 patients, both before and afterOncoTherad® (MRB-CFI-1) immunotherapy application. Patients refrained from takingmedication 72 h before sample collection in order to minimize potential drug effects on theirimmune system. Blood drawn from each patient was placed in 3.5 mL tubes (Vacuette—REF 454327, Greiner Bio-One GmbH, Kremsmünster, Austria) using 0.5 mL of 3.2% (w/v)sodium citrate as an anticoagulant; the blood/sodium citrate ratio was kept at 9:1. Bloodsamples were centrifuged in a Rotina 380R centrifuge (Hettich Zentrifugen, Munich, Ger-many) at 3500 rpm at 25 ◦C for 10 min right after collection. After the centrifugation processwas over, plasma was separated from each patient’s sample with a 200 µL micropipetteand transferred to 2.0 mL microtubes. Then, 10 µL/mL of protease inhibitor cocktail(MilliporeSigma, Burlington, MA, USA) was added to each microtube filled with plasma.A portion of each sample was used to determine the protein concentration based on theBradford microplate assay method. Subsequently, 50 micrograms of protein were loadedonto an SDS-polyacrylamide gel for electrophoresis purposes, then they were electricallytransferred to nitrocellulose membranes [27].Nitrocellulose membranes were blocked with 3% bovine serum albumin (BSA) solutiondiluted in TBS-T buffer to avoid non-specific protein binding. These membranes wereincubated with primary antibodies, such as anti-CX3CR1 (dilution: 1:1500 in 1% BSA) andanti-β-actin (dilution: 1:1500 in 1% BSA), at 4 ◦C overnight (Table S3). Subsequently, theywere incubated with HRP-conjugated secondary antibodies (diluted 1:3000 in 1% BSA;MilliporeSigma, Burlington, MA, USA) for 2 h.Bands’ immunoreactivity was visualized by incubating them with DAB (diaminoben-zidine) chromogen (Sigma Chemical Co., St. Louis, MO, USA). Samples collected from all20 patients, both before and after OncoTherad® (MRB-CFI-1) treatment application, weregrouped into four different pools (each pool comprised n = 5 samples). Immunoblots wereconducted in duplicate, and semiquantitative densitometric analysis (IOD—IntegratedOptical Density) applied to the bands was performed in Image J software version 1.54f.This analysis was followed by statistical assessment, whose results were expressed as themean ± standard deviation in comparison to β-actin labeling intensity, which was used asan endogenous positive control [27,28].4.9. Statistical AnalysesCategorical variables were summarized in frequency tables, which comprised abso-lute frequencies (n) and percentages (%), whereas numerical variables were summarizedthrough descriptive statistics (mean, standard deviation, minimum, median, and maximum)to provide an overview of sample features in comparison to the investigated variables.Tumor size, focus, and frequencies (both absolute and percentage) were tabulated toassess tumor response to OncoTherad® (MRB-CFI-1) treatment with respect to histologicalgrade. Fisher’s exact test was used for statistical analysis purposes.Quantitative variables were introduced based on central tendency and dispersionmeasures for the analysis applied to relapse and biochemical serological parameters, bothbefore and after OncoTherad® (MRB-CFI-1) treatment application. Data distribution wasassessed based on parameters such as homogeneity of variance and adherence to thenormal curve through the Kolmogorov–Smirnov test. Non-parametric tests (specifically theKruskal–Wallis test) were applied to variables that did not conform to normal distribution.Descriptive statistics were computed (mean, standard deviation, minimum, median,and maximum) based on the time taken to achieve relapse-free status, more specifically,pathological complete response. This was done in order to assess relapse-free survival ratesover time. A relapse-free survival curve was plotted based on the Kaplan–Meier method.A McNemar test was used to assess the incidence of adverse reactions (categorized asNo or Yes) based on their intensity grade (1–2 or 3–4). Descriptive statistics were used tocompare the median values recorded for the total number of grade 1–2 reactions to those ofInt. J. Mol. Sci. 2023, 24, 17535 26 of 30grade 3–4 reactions based on the Wilcoxon test. Moreover, each reaction type was assessedseparately, while reaction frequencies (both absolute and percentage) were recorded.Quantitative data resulting from immunohistochemistry and Western blotting testswere expressed as mean ± standard deviation. Parametric analysis of variance (ANOVA)was carried out. It was followed by Tukey testing whenever normality and homoscedasticityassumptions were met. Non-parametric analysis of variance (Kruskal–Wallis test) was usedin cases whose data did not follow normal distribution. Student–Newman–Keuls testingwas used for post-hoc comparisons.All statistical analyses were conducted by the Biostatistics Service of the Medical Schoolat UNICAMP in the SAS System for Windows (Statistical Analysis System) version 9.4,as well as in R software version 3.4.2, which was developed by The R Foundation forStatistical Computing.5. ConclusionsOncoTherad® (MRB-CFI-1) treatment effectively activated patients’ innate immunesystem through TLR4, and it significantly upregulated the interferon signaling pathway.This activation played a crucial role in stimulating CD8+ CX3CR1+ T cells, while reduc-ing the number of immune checkpoint markers and reversing the immunosuppressiveconditions within the urinary bladder microenvironment. Moreover, the TLR4-mediatedinterferon signaling pathway induced by OncoTherad® (MRB-CFI-1) contributed to boththe formation and organization of lymphoid follicles in the bladder tissue. This process haspotentially contributed to its antitumor and immune-protective effects.Therefore, OncoTherad® (MRB-CFI-1) nanoimmunotherapy emerges as a safe andeffective treatment option for patients with BCG-unresponsive NMIBC and for those whohave undergone intravesical chemotherapy. This therapy has the potential to help reducetumor relapse rates and potentially delay or rule out the need for conducting radicalsurgical interventions in these patients.6. PatentsOncoTherad® (MRB-CFI-1) nanoimmunotherapy holds patents granted in Brazil(INPI: BR102017012768B1), in the United States (USPTO: US-11623869-B2; US-11572284-B2;US-11136242-B2; US-11639294-B2), and in Europe (EPO: EP3626746B1; EP3998075B1).Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms242417535/s1.Author Contributions: Conceptualization, J.C.C.A., N.D. and W.J.F.; Methodology, J.C.C.A., P.H.F.C.,A.d.S.S., L.L.L.d.F., A.B. and W.J.F.; Software, B.R.d.S., I.B.R., G.C.d.A.C., G.d.O., M.I.d.B.F.S. andJ.M.G.; Validation, A.d.S.S., L.L.L.d.F., A.B., N.D. and W.J.F.; Formal Analysis, J.C.C.A., B.R.d.S.,I.B.R., G.C.d.A.C., G.d.O., M.I.d.B.F.S., J.M.G., P.H.F.C., L.L.L.d.F., A.B., N.D. and W.J.F.; Investigation,J.C.C.A., J.R.d.C.R., A.d.S.S., L.L.L.d.F., A.B. and W.J.F.; Resources, J.C.C.A., L.L.L.d.F., A.B., N.D.and W.J.F.; Data Curation, J.C.C.A., N.D. and W.J.F.; Writing—Original Draft Preparation, J.C.C.A.and W.J.F.; Writing—Review and Editing, J.C.C.A., J.R.d.C.R., P.H.F.C., A.d.S.S., L.L.L.d.F., A.B.,N.D. and W.J.F.; Visualization, J.C.C.A., B.R.d.S., I.B.R., G.C.d.A.C., G.d.O., M.I.d.B.F.S., J.M.G.,J.R.d.C.R., P.H.F.C., A.d.S.S., L.L.L.d.F., A.B., N.D. and W.J.F.; Supervision, J.C.C.A. and W.J.F.; ProjectAdministration, J.C.C.A. and W.J.F.; Funding Acquisition, W.J.F. All authors have read and agreed tothe published version of the manuscript.Funding: This research was funded by the São Paulo Research Council [FAPESP grant numbers:2018/10052-1; 2020/03419-6], and the Brazilian National Council for Scientific and TechnologicalDevelopment [CNPq grant numbers: 552120/2011-1; 312396/2021-0].Institutional Review Board Statement: The study was conducted according to the guidelines of theDeclaration of Helsinki, approved by the Institutional Ethics Committee of Universidade Estadualde Campinas (UNICAMP) (CAAE number: 69189017.5.0000.5404), and registered in the BrazilianClinical Trials Registry (RBR-6swqd2).Int. J. Mol. Sci. 2023, 24, 17535 27 of 30Informed Consent Statement: Informed consent was obtained from all subjects involved in thestudy. Written informed consent has been obtained from the patients to publish this paper.Data Availability Statement: The data presented in this study are available on request from thecorresponding author. The data are not publicly available due to patent issues.Acknowledgments: The authors wish to express their gratitude for the assistance provided by theAmbulatório de Saúde do Homem (Men’s Health Clinic) at Paulínia Municipal Hospital (HMP) inPaulínia, São Paulo, Brazil. Special thanks are extended to Magali Codogno Maciel and HumbertoAlves Ferrari for their invaluable support to the patients involved in this study.Conflicts of Interest: The authors declare no conflict of interest.References1. Faguet, G.B. A brief history of cancer: Age-old milestones underlying our current knowledge database. Int. J. Cancer 2015, 136,2022–2036. [CrossRef]2. Celada Luis, G.; Albers Acosta, E.; de la Fuente, H.; Velasco Balanza, C.; Arroyo Correas, M.; Romero-Laorden, N.; Alfranca,A.; Olivier Gómez, C. A Comprehensive Analysis of Immune Response in Patients with Non-Muscle-Invasive Bladder Cancer.Cancers 2023, 15, 1364. [CrossRef]3. 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Available online: https://ctep.cancer.gov/protocoldevelopment/electronic_applications/docs/CTCAE_v5_Quick_Reference_8.5%C3%9711.pdf (accessed on 27 October 2020).Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individualauthor(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury topeople or property resulting from any ideas, methods, instructions or products referred to in the content.
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OncoTherad® (MRB-CFI-1) Nanoimmunotherapy: A Promising Strategy to Treat Bacillus Calmette–Guérin-Unresponsive Non-Muscle-Invasive Bladder Cancer: Crosstalk among T-Cell CX3CR1, Immune Checkpoints, and the Toll-Like Receptor 4 Signaling Pathway
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