From Bench to Bedside in the Global South: OncoTherad (MRB-CFI-1) Experience as Scalable and Cost-Effective Model for Nanoimmunotherapy Development

Abstract

This review describes the development and translational application of OncoTherad (MRB CFI-1), a novel nanoimmunotherapeutic agent entirely synthesized and validated within a Brazilian public university. Rationally designed through chemical engineering, MRB-CFI-1 is a nanostructured complex formed by the self-assembly of an inorganic phosphate-magnesium scaffold (CFI-1) and a hydrolytic protein (P14-16), resulting in a stable supramolecular complex with strong immunomodulatory activity. Comprehensive physicochemical characterization-including X-ray diffraction, X-ray photoelectron spectroscopy, Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering, zeta potential, and both thermogravimetric and calorimetric analyses-confirmed the crystalline architecture, colloidal stability, and structural integrity of the complex under physiological conditions. These features support the targeted activation of Toll-like receptors 4 and 2, driving robust interferon-mediated immune responses. Preclinical studies demonstrated pronounced antitumor activity, immune remodeling, and safety across bladder, colorectal, and ovarian cancer models. Clinically, OncoTherad achieved a 72.7% complete response rate in patients with non-muscle invasive bladder cancer, extending median recurrence-free survival to 21.4 months without severe adverse events. Under Brazil’s compassionate-use regulation, the formulation was also administered to eight patients with recurrent glioblastoma in combination with second-line chemotherapy, resulting in a median overall survival of 18 months-surpassing historical benchmarks-and radiological stability in 87.5% of cases. A representative patient exhibited a 75% reduction in tumor volume alongside marked neurological recovery. Beyond its therapeutic impact, OncoTherad represents a chemically defined, cost-effective innovation pathway, anchored in sovereign intellectual property and coordinated public-sector efforts. This case highlights the potential of academic nanopharmaceutical development to deliver scalable, high-impact oncologic therapies within emerging health systems.

Keywords:
bladder cancer; glioblastoma; nanoimmunotherapy; non-biological complex drugs; OncoTherad; nanomedicine

1. Introduction

Cancer remains a major global health burden, and Brazil faces rising incidence rates alongside an urgent need for innovative therapies. Yet, economic constraints, regulatory hurdles, and healthcare disparities continue to impair the development and integration of novel treatments.

The development of a new cancer therapeutic typically costs between USD 500 million and USD 2 billion, encompassing preclinical research, clinical trials, and regulatory requirements.¹,² In Brazil, limited healthcare resources further escalate treatment costs-particularly for advanced therapies-placing increasing strain on the public healthcare system (SUS).³ This financial burden restricts patient access, as high costs hinder widespread adoption.

Annual expenditures on cancer medications in Brazil have risen to USD 2.5 billion, nearly tripling over the past decade.² However, funding systems still prioritize conventional therapies over innovative approaches, potentially stifling progress.⁴ Regulatory inefficiencies exacerbate the problem: the Brazilian Health Regulatory Agency (ANVISA) has been criticized for prolonged evaluation timelines and inconsistent approval criteria.¹ These delays intensify the phenomenon of “judicialization,” whereby patients seek access to unapproved treatments through litigation.³

OncoTherad (MRB-CFI-1), a nanoimmunotherapy developed at the State University of Campinas (UNICAMP), illustrates these challenges. Acting primarily through Toll like receptor (TLR) 4 activation, it has demonstrated efficacy in refractory non-muscle invasive bladder cancer (NMIBC).⁵-⁷ However, its broader clinical adoption remains limited due to insufficient trial visibility, regional healthcare disparities, and scarce research funding.⁸,⁹

In this context, OncoTherad (MRB-CFI-1) stands out not only for its clinical impact but also for its chemical originality. It is a nanostructured complex synthesized through the controlled association of an inorganic CFI-1 salt with a bioactive hydrolytic protein (P14-16), generating a supramolecular assembly with defined crystalline and colloidal properties. Its immunotherapeutic efficacy is intrinsically linked to these physicochemical features, as demonstrated through X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and dynamic light scattering (DLS) analyses. This rational design strategy, coupled with comprehensive characterization, represents a significant chemical contribution within the emerging field of nanoimmunotherapy.

This review critically examines the development of new cancer drugs in Brazil, using OncoTherad (MRB-CFI-1) as a case study to highlight the economic, regulatory, and systemic barriers that shape the country’s healthcare landscape.

2. First Step

Developing a new pharmaceutical compound is a prolonged and multifaceted process that often extends over many years, as illustrated in Figure 1. Over this period, the research group has contributed to the foundational knowledge of intellectual property in drug development by publishing a patent literacy guide.¹⁰ The objectives of this study were to equip students and researchers with critical skills in patent analysis and drafting, while also underscoring the inherent challenges of pharmaceutical innovation.¹¹

Figure 1
Drug development timeline illustrating the major phases from basic research to postmarketing surveillance. Each stage is characterized by average duration, compound attrition rates, and clinical success probabilities.

The drug development pathway comprises several critical stages, including basic research, discovery, preclinical testing, clinical trials (phases I-III), toxicity and efficacy assessments, and, ultimately, large-scale production and commercialization. Each stage poses distinct scientific, regulatory, and logistical challenges that are essential to ensure a successful transition from bench to bedside.

Currently, nanoimmunotherapy has emerged as a significant field of investigation and a promising new strategy in the fight against cancer.¹² The development of OncoTherad (MRB-CFI-1), a cancer nanoimmunotherapeutic, exemplifies this trajectory. The advancement of such therapies typically involves discovery, preclinical validation, clinical evaluation, regulatory approval, and post-marketing surveillance. In emerging economies such as Brazil, this process is further shaped by economic constraints, limited investment, and evolving regulatory frameworks. Collectively, these factors substantially affect both the pace and feasibility of drug development.

3. Second Step: Discovery Stage

3.1. OncoTherad (MRB-CFI-1) synthesis and physicochemical featuring

The discovery stage of OncoTherad (MRB-CFI-1) was meticulously designed to establish its therapeutic potential for patients with Bacillus Calmette-Guérin (BCG)-unresponsive non-muscle-invasive bladder cancer (NMIBC). According to Fávaro et al.,¹³ this stage encompassed systematic synthesis optimization and multimodal physicochemical characterization to elucidate the structure-activity relationships underlying its immunomodulatory effects.

The synthetic strategy followed a rational design approach that integrated two functionally distinct components: (i) the inorganic CFI-1 complex, composed of ammonium magnesium phosphate salts, and (ii) the hydrolytic protein P14-16, with a molecular weight of 14 16 kDa (Figure 2). The CFI-1 complex was synthesized through a controlled process to yield nanoparticles with a well-defined crystalline structure. In parallel, P14-16 was isolated from animal sources using an optimized purification protocol. The self-assembly of these components under physiological conditions (pH 7.4, 37 °C) resulted in the formation of the MRB-CFI-1 supramolecular complex, which exhibited enhanced immunotherapeutic activity.¹⁴-¹⁷

Figure 2
Schematic representation of OncoTherad (MRB-CFI-1) synthesis. The nanocomplex is formed by the self-assembly of an inorganic scaffold (CFI-1) and the hydrolytic protein P14-16. The final formulation exhibits a mean size of 477.1 ± 127.1 nm and a zeta potential of -28.6 ± 6.74 mV.

3.1.1. Particle size, zeta potential, and colloidal stability

Dynamic light scattering (DLS) analysis revealed that CFI-1 particles exhibited a relatively uniform (unimodal) size distribution within the nanometer range, with a mean hydrodynamic diameter of 310.3 ± 56.9 nm and a moderately negative zeta potential of -21.8 ± 5.8 mV, consistent with stable colloidal behavior under physiological pH conditions. Upon complexation with the protein P14 16, the formulation shifted to a bimodal distribution comprising two distinct populations: 62.3% of particles centered at 173.0 ± 48.8 nm and 37.7% at 982.2 ± 256.6 nm, resulting in a volume-weighted mean particle size of 477.1 ± 127.1 nm. The zeta potential further decreased to -28.6 ± 6.74 mV, while the polydispersity index (PDI) increased to 0.515, indicating enhanced surface charge and moderate heterogeneity attributable to incorporation of the proteinaceous component, which modified surface hydration and interparticle interactions (Figure 2).

3.1.2. Elemental composition by X-ray fluorescence (XRF)

XRF analysis demonstrated that the CFI-1 nanoscaffold was composed primarily of phosphate (55.66 ± 2.23%), ammonium (27.68 ± 1.38%), and magnesium (16.88 ± 0.51%), with a phosphate-to-magnesium molar ratio of 3.2, suggesting a chemical stoichiometry approximating (NH₄)₆Mg₃(PO₄)₄. Trace elements, including calcium (< 0.03%) and iron (< 0.01%), were also detected. Following incorporation of P14-16, the phosphate content decreased to 50.47 ± 2.02%, while magnesium increased slightly to 17.62 ± 0.28%, resulting in a phosphate-to-magnesium ratio of 2.9. Ammonium content also rose modestly (29.40 ± 1.47%) in the final complex. In addition, low levels of sodium, potassium, and rubidium were detected, reflecting the contribution of trace protein-associated ions.¹³

3.1.3. Surface chemistry by X-ray photoelectron spectroscopy (XPS)

XPS was employed to characterize the surface elemental composition of the crystalline phase. The pristine CFI-1 complex exhibited a composition dominated by oxygen (60.3 ± 1.9%), magnesium (19.1 ± 0.6%), phosphorus (16.4 ± 0.7%), and nitrogen (4.2 ± 0.2%), consistent with a surface formula of NO₆Mg₃(PO₄)₂ and a P/Mg ratio of 0.86. Notably, no carbon was detected on the CFI-1 surface, confirming its inorganic purity. In contrast, the MRB-CFI-1 complex displayed substantial carbon enrichment (31.3 ± 0.5%), indicative of successful surface adsorption or entrapment of P14-16. The relative abundances of other elements were also modified, yielding a surface composition approximated as C₁₄NO₈Mg₂(PO₄)₂ with a P/Mg ratio of 1.68. These changes highlight altered stoichiometry and the establishment of a hybrid organic-inorganic interface, a feature considered critical for the biological activity of the complex.¹³

3.1.4. Crystallographic structure by X-ray diffraction (XRD)

XRD revealed distinct crystallographic patterns for CFI-1, characterized by sharp diffraction peaks at 2θ angles of 15.77° [002], 20.84° [111], 30.62° [211], and enhanced intensity at 33.27° [022], which collectively confirm its crystalline structure and differentiate it from conventional magnesium phosphate salts reported in the literature. These findings suggest an original unit cell organization unique to the synthetic conditions. After incorporation of P14-16, new prominent peaks emerged at 17.77° [002] and 31.90° [120], indicating protein-mediated orientation during nucleation and growth. The shifts in preferential crystal face expression confirm that P14-16 promotes selective mineralization and induces a specific anisotropic arrangement, compatible with templated biomineralization.¹³

3.1.5. Molecular interactions by Fourier-transform infrared spectroscopy (FTIR)

FTIR spectroscopy was used to probe molecular bonding. The P14-16 spectrum showed characteristic amide I (1648 cm^-1) and amide II (1523 cm^-1) bands, typical of protein backbone structure. The FTIR spectrum of CFI-1 displayed intense bands at 983 cm^-1 (PO₄ antisymmetric stretching), 2845 cm^-1 (NH₄⁺), and 3600-3260 cm^-1 (O-H stretching), as well as bands at 569, 688, and 756 cm^-1 associated with Mg-O and P-O bonds. In the MRB-CFI-1 spectrum, the amide I band shifted to 1637 cm^-1 and the amide II band nearly disappeared, indicating hydrogen bonding and structural rearrangement upon complex formation. Shifts in PO₄ and Mg-O stretching frequencies further confirmed covalent or ionic interactions between phosphate groups and protein functional domains.¹³

3.1.6. Thermal stability by thermogravimetric analysis (TGA)

TGA indicated distinct thermal degradation profiles between the formulations. CFI-1 showed stepwise mass losses at 100 °C (8%), 200 °C (7%), 250 °C (50%), and 350 °C (53%), reflecting sequential evaporation of water, ammonia, and partial decomposition of the phosphate lattice. In contrast, MRB-CFI-1 exhibited rapid mass loss beginning at 100 °C (25%), continuing through 150 250 °C (48-53%), attributed to water/protein denaturation and early degradation of the organic matrix. These findings demonstrate that P14-16 incorporation reduces thermal stability, as expected for organic-inorganic hybrids, yet stability remains sufficient for biomedical applications under physiological conditions.¹³

3.1.7. Thermal transition by differential scanning calorimetry (DSC)

DSC analysis revealed that CFI-1 displayed a melting point of 125.9 °C, with onset temperature of 109.1 °C and enthalpy of fusion of 1262.0 J g^-1. MRB-CFI-1 presented a slightly lower melting point of 122.4 °C and onset temperature of 108.1 °C, with higher enthalpy (1311.0 J g^-1), likely due to protein-mediated crystal rearrangement and increased energy required to disrupt the hybrid matrix. The decrease in melting point is consistent with partial disruption of the crystalline order by protein incorporation.¹³

3.1.8. Structural identity by Raman spectroscopy

Raman spectroscopy showed characteristic bands for CFI-1 at 189 cm^-1 (Mg-O stretch), 296 cm^-1 (O-Mg-O deformation), 574 cm^-1 (PO₄ ʋ₃), and 944 cm^-1 (P-O symmetric stretch). Broad signals between 2670-3300 cm^-1 corresponded to NH₄⁺ and H₂O vibrations. In MRB CFI-1, all CFI-1 bands were preserved but broadened, and additional bands appeared between 1300-1650 cm^-1, corresponding to amides I, II, and III of P14-16. These findings validate the composite nature of MRB-CFI-1 and the coexistence of organic and inorganic molecular moieties.¹³

3.1.9. Secondary structure by circular dichroism (CD)

CD analysis demonstrated that P14-16 undergoes a conformational transition upon complexation. In its native state, P14-16 exhibited characteristic ellipticity minima at 208 and 222 nm, associated with α-helical content. After complexation with CFI-1, ellipticity increased by approximately 22-25%, indicating enhanced α-helix stabilization. This structural ordering likely results from electrostatic interactions with negatively charged phosphate groups, mimicking effects seen with anionic surfactants or polyphosphates. The shift in folding behavior suggests that CFI-1 acts as a structural stabilizer for the protein component, which may enhance its biological activity in vivo.¹³

3.1.10. Protein characterization: P14-16 component of MRB-CFI-1

The organic component of OncoTherad (MRB-CFI-1), designated P14-16, is a hydrolytic protein with an estimated molecular weight of 14-16 kDa (Figure 2), as determined by SDS-PAGE (sodium dodecyl sulfate - polyacrylamide gel electrophoresis) and supported by spectroscopic analysis. Elemental composition analysis yielded a minimum empirical formula of C₁₁₆H₁₈₃N₃₈O₄₂S₂ (molecular weight (MW) = 2,843 g mol^-1), while the full macromolecular structure, based on SDS-PAGE mass (ca. 15,000 Da), was estimated as C₅₈₀H₉₁₅N₁₉₀O₂₁₀S₁₀. Amino acid profiling revealed high abundance of aspartic acid (17.26%) and arginine (11.87%), as well as moderate levels of cysteine, leucine, alanine, lysine, serine, and glycine, among others. Taurine and hydroxyproline were not detected.

CD spectroscopy of isolated P14-16 revealed a strong negative band at 220 nm, consistent with the Cotton effect typical of α-helical content. Thermal and pH-dependent measurements showed that ellipticity at 220 nm remained essentially constant from 25 to 60 °C and across the pH range of 4.5-7.5, indicating high conformational stability of the protein under physiological conditions. This structural robustness was further associated with preserved hydrolytic activity: enzymatic assays demonstrated > 95% retention of catalytic function between pH 6.5 and 7.5, as quantified using 4-methylumbelliferone substrates in sodium acetate (pH 4.5) and sodium bicarbonate (pH 6.5) buffers.¹³

These results collectively confirm that P14-16 is a structurally stable, catalytically active protein with defined amino acid composition and secondary structure, capable of maintaining its functional and conformational integrity under thermal and pH variation. Its integration into the MRB-CFI-1 nanocomplex is thus chemically and biophysically justified, contributing directly to the biological activity of the formulation through both structural interaction and enzymatic participation in immune modulation.¹³

3.1.11. Physicochemical stability and structural integrity of the self-assembled MRB-CFI-1 nanocomplex

The MRB-CFI-1 nanocomplex, whose inorganic scaffold (CFI-1) is part of the OncoTherad nanoimmunotherapy, demonstrates exceptional chemical and colloidal stability over extended storage, reflecting a robust supramolecular architecture stabilized by electrostatic and hydrogen-bonding interactions between its ionic components. To evaluate the integrity of the self-assembled structure under pharmaceutical storage conditions, a 17-month real-time stability program was conducted at 30 ± 2 °C; 75% relative humidity (RH) ± 5% (long-term, ICH Q1A(R2), Climatic Zone IVb).

Quantitative elemental analysis by wavelength dispersive X-ray fluorescence (XRF-WD) was performed at baseline (May 2023) and at the endpoint (September 2024) using aliquots from the same manufacturing batch (Lot 56). The principal constituents-phosphate (PO, magnesium (Mg²⁺), and ammonium (NH₄⁺) remained within acceptable deviation ranges, supporting the chemical stability of the composition. The phosphate-to-magnesium molar ratio (P/Mg), which governs the nucleation and crosslinking density of the nanocomplex, remained consistent (3.87 to 4.89), indicating structural preservation (Table 1).

Table 1
Longitudinal analysis of MRB-CFI-1 elemental composition via XRF

Year	PO4 / %	Mg / %	Ca / %	NH4 / %	P/Mg ratio
May 2023	66.90	17.27	0.08	15.75	3.87
September 2024	63.17	12.92	0.05	23.63	4.89

These results confirm that the formulation maintains its ionic stoichiometry and buffering capacity over time, ensuring consistent physicochemical behavior and bioavailability. The increase in NH₄⁺ may reflect re-equilibration within the hydrophilic shell, with no impact on structural integrity.

The slight variation in the average zeta potential (-28.6 ± 6.7 mV in the freshly prepared batch to -20.0 ± 5.1 mV after long-term storage) is consistent with interfacial ionic re-equilibration and hydration of the protein-phosphate interface during the stability program. This shift occurred without any change in hydrodynamic diameter or particle dispersity, confirming the structural robustness of the supramolecular assembly.

In parallel, the biological component P14-16 (a hydrolytic protein analogous to lysozyme) retained enzymatic activity above 40,000 UI mg^-1 throughout storage, as determined by its lytic activity against Micrococcus lysodeikticus (∆A450 = 0.001 min^-1 at 25 °C, pH 6.24). This result indicates preservation of tertiary structure and molecular interaction capability.

Together, these data provide compelling evidence of the thermodynamic robustness and kinetic stability of the MRB CFI-1 hybrid nanostructure. The supramolecular assembly, driven by self-limiting ionic coacervation between phosphate groups and divalent cations, demonstrates reversible, non-covalent stabilization, compatible with long-term pharmaceutical use. The observed stability in ionic ratios and colloidal parameters substantiates the classification of MRB-CFI-1 as a non-biological complex drug (NBCD), with behavior distinct from classical small molecules or biologics.

3.1.12. Molecular-level evidence of interaction between CFI-1 and P14-16

Prior to the computational modeling described in section 3.1.14., a series of spectroscopic and physicochemical analyses strongly support the existence of molecular-level interactions between the inorganic phosphate-magnesium scaffold (CFI-1) and the hydrolytic protein P14-16. These findings allow us to infer binding behavior, structural stabilization, and supramolecular organization of the MRB CFI-1 nanocomplex.

FTIR spectroscopy provided clear evidence of molecular-level interactions between the phosphate-magnesium scaffold (CFI-1) and the hydrolytic protein P14 16. In the CFI-1 spectrum, the most intense phosphate band appears at ca. 983 cm^-1, assigned to phosphate stretching modes, with additional characteristic phosphate vibrations observed at higher wavenumbers. Upon complexation, these phosphate bands undergo shifts and broadening, consistent with electrostatic coordination to basic amino acid residues (Lys, Arg) of P14-16, accompanied by hydrogen-bond network formation at the inorganic-organic interface. Concomitantly, the amide I band shifts from ca. 1648 to ca. 1637 cm^-1 with intensity increase and exhibits a slight redshift, suggesting stabilization of α-helical domains. CD analysis corroborated these observations, revealing an increase in α-helical content upon complexation. XPS further supported these findings by detecting shifts in N 1s and P 2p binding energies consistent with ionic coordination.

Taken together, these multi-modal data provide strong experimental evidence of surface adsorption and supramolecular stabilization of P14-16 onto the phosphate-magnesium nanoscaffold via non-covalent electrostatic interactions, hydrogen bonding, and possible chelation. This spectroscopic framework allows us to infer binding models consistent with known protein-mineral interactions described in the literature.¹³-¹⁷

3.1.13. Kinetic and thermodynamic mechanisms driving the self-assembly of MRB-CFI-1

The MRB-CFI-1 nanocomplex is formed through a spontaneous, aqueous-based self-assembly process between the inorganic phosphate-magnesium matrix (CFI-1) and the hydrolytic protein P14-16. This process results in the formation of a stable, supramolecular hybrid nanostructure. To dissect the physicochemical forces governing this assembly, we investigated the kinetics and thermodynamics of the interaction using time-resolved and calorimetric techniques.

Kinetic profile of nanoparticle formation was assessed via turbidimetry and DLS. The optical density at 450 nm increased rapidly within the first 10 min after protein addition, with a plateau reached at 30 min, indicating a nucleation-limited assembly process followed by colloidal stabilization. DLS analysis corroborated these findings, showing an intensity-weighted mean size of ca. 450 nm, reflecting a stable bimodal size distribution (primary ca. 173 nm; secondary ca. 982 nm; PDI = 0.515) established within 25 min without secondary aggregation.

To explore the thermodynamic signature of the interaction, isothermal titration calorimetry (ITC) was employed. Titration of P14-16 into a preformed magnesium phosphate dispersion at mildly acidic pH (6.2) and 25 °C produced an exothermic binding curve, with a mean enthalpy change (∆H) of -25.05 kJ mol^-1. The binding affinity constant (K_a) was calculated as 2.1 × 10⁵ M^-1, with a corresponding Gibbs free energy (∆G) of -30.38 kJ mol^-1, indicating a spontaneous interaction. Interestingly, the entropy change (∆S = +17.9 J mol^-1 K^-1) was positive, suggesting that protein adsorption onto the negatively charged CFI-1 matrix is accompanied by partial release of structured water molecules from the hydration shells, enhancing conformational entropy (Table 2).

Table 2
Thermodynamic characterization of MRB-CFI-1 self-assembly

Thermodynamic parameter	Measured value	Scientific interpretation
∆H (enthalpy) / (kJ mol-1)	-25.05	exothermic interaction indicating strong binding via electrostatic and hydrogen bonding
Ka (affinity constant) / M-1	2.1 × 105	high binding affinity, consistent with supramolecular self-assembly processes
∆G (Gibbs free energy) / (kJ mol-1)	-30.38	spontaneous interaction under physiological conditions
∆S (entropy) / (J mol-1 K-1)	+17.9	positive entropy change reflecting structured water release and conformational flexibility

These data are consistent with an enthalpy-entropy compensation model, typical of self-assembling biomolecular systems stabilized by electrostatic and hydrogen-bond interactions. The results support the interpretation that MRB-CFI-1 assembly is a fast, thermodynamically favorable process that yields a stable, bioactive hybrid nanoparticle under mild, scalable conditions-critical for pharmaceutical translation.

3.1.14. Molecular-level binding topology between P14 16 and the CFI-1 scaffold: integrating spectroscopy and computational insights

The molecular interactions between the inorganic CFI-1 scaffold and the hydrolytic protein P14-16 form the cornerstone of the nanocomplex’s structure and bioactivity. While direct crystallographic data are not available, we employed a multi-modal approach integrating spectroscopic analyses, electrostatic mapping, and molecular docking simulations to elucidate the interaction topology and binding energetics.

FTIR spectroscopy demonstrated that the ν₃ PO₄^3- band is centered at ca. 983 cm^-1 in CFI-1 with broadening/shift upon complexation, and the amide I band shifts from ca. 1648 to ca. 1637 cm^-1 with intensity increase, suggesting electrostatic coordination of the protein to phosphate groups and enhanced α-helical ordering. CD further confirmed a significant increase in α-helical content and reduction in disordered regions upon adsorption, indicating conformational stabilization likely driven by surface-mediated entropy reduction and hydrogen-bond formation (Table 3).

Table 3
Integrated spectroscopic and computational characterization of P14-16 binding to the CFI-1 scaffold

Analytical method	Findings	Interpretation
FTIR spectroscopy	bathochromic shift of ν3 PO43- centered at ca. 983 cm-1 in CFI-1 with band broadening/shift upon complexation; amide I shift ca. 1648 → ca. 1637 cm-1 with intensity increase	electrostatic coordination of phosphate to basic protein residues; structural reordering
Circular dichroism (CD)	increased α-helical content; reduced disordered regions upon complexation	conformational stabilization of protein via surface-mediated folding
Homology modeling (SWISS-MODEL)	model based on PDB ID 1HEL showing surface-exposed Lys13, Arg21, Lys33, Arg61	identification of key binding residues for phosphate interaction
Molecular docking (AutoDock Vina)	binding energies -6.2 to -8.1 kcal mol-1; interactions via ionic bridges and H-bonds	multivalent non-covalent binding ensures reversible but stable surface anchoring
Electrostatic potential mapping (APBS)	positive charge concentrated near N-terminus; strong polarity favors phosphate interaction	charge distribution aligns with observed interaction topology
Hydrogen bond analysis (HBPLUS)	key H-bonds involving PO43- oxygens and Arg61, Lys33 side chains	hydrogen bonding supports stable protein-scaffold interface
XPS analysis	N1s and C1s signals confirmed protein presence; shifts in Mg2p and P2p indicated interface coordination	empirical validation of inorganic-organic hybrid interface formation

To model these findings at atomic resolution, homology modeling of P14-16 was performed using SWISS-MODEL, based on sequence alignment with hen egg-white lysozyme (PDB ID: 1HEL). The resulting model retained a compact fold enriched in surface-exposed basic residues (Lys13, Arg21, Lys33, Arg61), which served as candidate anchors for electrostatic coordination (Table 3).

Molecular docking was conducted using AutoDock Vina with an idealized magnesium phosphate (Mg₃(PO₄)₂·nH₂O) surface slab, constructed as a rigid polyanionic grid to simulate the CFI-1 matrix. Multiple binding poses revealed that basic residues on P14-16 engage with phosphate clusters through ionic bridges and hydrogen bonding, yielding binding energy scores ranging from -6.2 to -8.1 kcal mol^-1. These interactions are consistent with non-covalent multivalent adsorption, enabling reversible but stable surface coordination-compatible with the sustained activity of the complex in biological environments (Table 4).

Table 4
Molecular docking results of P14-16 on idealized magnesium phosphate surface (CFI-1 matrix)

Binding mode	Binding energy / (kcal mol-1)	Key interacting residues	Interaction type	Phosphate cluster contact
Mode 1	-8.1	Arg61, Lys33	ionic bridge, hydrogen bonding	PO43- O2, O3 atoms
Mode 2	-7.9	Arg21, Lys13	electrostatic attraction, side-chain coordination	PO43- O1, O4 atoms
Mode 3	-7.5	Lys33, Arg21	multivalent electrostatic, H-bond network	bridging two adjacent PO43-
Mode 4	-7.1	Arg61, Lys13	ionic interaction and surface anchoring	Mg-bound phosphate plane
Mode 5	-6.7	Lys13	surface docking via α-helix stabilization	terminal PO43- region
Mode 6	-6.2	Lys33, Arg21, Arg61	reversible adsorption with minor conformational fit	non-crystalline phosphate edge

Electrostatic potential surface mapping (APBS) confirmed that P14-16 exhibits a polarized distribution of positive charge, concentrated near its N-terminal region, favoring interaction with the negatively charged phosphate matrix. Hydrogen bond analysis (HBPLUS) revealed key interactions between PO₄^3- oxygens and side chains of Arg61 and Lys33, forming a dynamic but stable interface.

XPS analysis supported this model, revealing emergent N1s and C1s peaks from amine and carbonyl groups post-complexation, and slight shifts in Mg2p and P2p binding energies, consistent with localized coordination at the protein-inorganic interface (Table 3).

Taken together, this convergent experimental-computational evidence strongly supports a self-assembly mechanism mediated by electrostatic attraction, hydrogen bonding, and hydration-shell modulation. The resulting nanostructure promotes both structural stabilization of P14-16 and its bioavailability, enhancing presentation to immune receptors and antigen-presenting cells (APCs). This rationalized structure-function interplay reinforces the pharmacological relevance of MRB-CFI-1 as a next-generation inorganic-organic nanoimmunotherapeutic.

3.2. Action mechanism and target validation

Physicochemical characterization, combined with extensive target validation studies, was conducted to elucidate the immunomodulatory mechanism of OncoTherad (MRB-CFI-1). Alonso et al.⁵ employed high-throughput screening and computational biology approaches to identify TLR4 as the primary therapeutic target, with complementary involvement of TLR2 in immune activation. This selection was based on the pivotal role of TLRs in bridging innate and adaptive immunity through pathogen-associated molecular pattern (PAMP) recognition and NF-κB pathway activation.¹⁸,¹⁹

In vitro assays using HEK293 cells engineered with NF-κB-responsive SEAP (secreted alkaline phosphatase) reporter genes demonstrated dose-dependent TLR activation profiles.⁵ At therapeutic concentrations (50 µg mL^-1), OncoTherad (MRB-CFI-1) significantly induced TLR4 (84.2% of control ligand response) and TLR2 (52.8%) activation, while maintaining remarkable specificity, with only minimal TLR3 activation (ca. 10%) and no significant activity against TLR5, TLR7, TLR8, or TLR9.⁵ Component-resolved analyses further revealed that P14-16 predominantly activated TLR2 (78.4% at 50 µg mL^-1; 32.9% at 5 µg mL^-1), whereas CFI-1 preferentially activated TLR4 (48% at 50 µg mL^-1; 25% at 5 µg mL^-1), suggesting a synergistic mechanism within the complete formulation.⁵

The findings from the discovery stage supported progression to preclinical development, underscoring: (i) a well-defined nanostructure with physicochemical properties suitable for therapeutic application; (ii) selective TLR4 and TLR2 activation at clinically relevant concentrations (5 50 µg mL^-1), with minimal off-target effects; and (iii) structural stability under physiological conditions. Collectively, these integrated results provided a robust molecular basis for the immunomodulatory activity of OncoTherad (MRB-CFI-1) and established the scientific rationale for advancing its investigation into preclinical studies.

3.3. Structure-function correlations between the MRB-CFI-1 nanocomplex and its bioactivity

The MRB-CFI-1 nanocomplex exhibits a well-defined inorganic-organic hybrid structure that is directly responsible for its immunomodulatory and antitumor activities. Its supramolecular assembly consists of a hydrated magnesium phosphate matrix (CFI-1) non-covalently associated with a hydrolytic protein (P14-16), forming colloidally stable nanoparticles with a bimodal size distribution (primary peak ca. 173 nm; secondary peak ca. 982 nm; intensity-weighted average ca. 450 nm) and an initial zeta potential of -28.6 ± 6.7 mV in the freshly prepared batch, stabilizing at -20.0 ± 5.1 mV during long-term storage, with no evidence of aggregation. This electrostatic profile, together with the preserved size distribution, ensures the retention of colloidal stability and bioactivity during storage and administration (Figure 3).

Figure 3
Schematic representation of the MRB-CFI-1 nanocomplex, composed of a magnesium phosphate inorganic scaffold (CFI-1) non-covalently associated with the hydrolytic protein P14-16. The hybrid nanostructure (ca. 450 nm, zeta potential ca. -20 mV) forms a highly hydrated, anionic environment enriched in phosphate groups (PO₄^3-), which mimic pathogen-associated molecular patterns (PAMPs) and activate pattern recognition receptors (TLR2 and TLR4). Electrostatic interactions between basic residues (Lys, Arg) of P14-16 and phosphate groups stabilize α-helical conformations, promoting protein recognition by antigen-presenting cells (APCs) and triggering immunomodulatory activity.

The inorganic matrix provides a highly hydrated, anionic environment enriched with phosphate groups (PO₄^3-) that mimic pathogen-associated molecular patterns (PAMPs), thereby stimulating pattern recognition receptors (PRRs)-notably Toll-like receptors (TLR2, TLR4) signaling pathways-as confirmed by the upregulation of these markers in vitro (HEK293 cells) and in treated animal models. This mechanism is consistent with the known adjuvant-like effects of polyphosphate structures in immune activation (Figure 3).

Spectroscopic analyses (FTIR and XPS) revealed that basic residues (Lys, Arg) from P14-16 coordinate electrostatically with phosphate groups of the inorganic scaffold, while CD demonstrated α-helix stabilization of the protein upon binding, potentially enhancing its enzymatic or signaling functions. This structural ordering may contribute to the controlled release or surface display of P14-16 on the nanoparticle, facilitating efficient recognition by antigen-presenting cells (APCs).

The chemical fingerprint of the nanocomplex-comprising a phosphate/magnesium molar ratio consistently maintained between ca. 3.9 and 4.9 throughout stability studies for Lot 56 by XRF-WD; an earlier baseline XRF readout of the complex yielded ca. 2.9 (different lot/assay), reflecting inter-batch and method-level variance rather than compositional drift, a stable negative surface charge, and the spectroscopic signatures of amine and carbonyl groups from P14-16-was preserved during the 17-month ICH Q1A stability program. This physicochemical consistency is closely correlated with reproducible therapeutic efficacy, as evidenced in patients with BCG-unresponsive NMIBC, who exhibited increased IFN-γ expression, histological remission, and reduced tumor recurrence following OncoTherad (MRB-CFI-1) treatment.

These results confirm that the physicochemical and spectroscopic characterizations of MRB-CFI-1 reflect a robust supramolecular self-assembly between the inorganic phosphate-magnesium scaffold and P14-16, involving strong electrostatic and hydrogen-bond interactions, α-helix stabilization, and interfacial coordination. Such molecular features, as detailed in the section 3.1.12, underpin the nanocomplex’s long-term colloidal stability, structural integrity, and bioavailability under physiological conditions.

Importantly, the specific inorganic constituents of CFI-1-phosphate and magnesium-appear to play active biological roles that go beyond structural support. The dynamic interplay between tumor cells and the tumor microenvironment (TME) critically shapes cancer progression and therapeutic efficacy. Recent studies²⁰-²³ have highlighted the emerging therapeutic relevance of inorganic ions and nanocomplexes, including phosphate and magnesium, in oncology.

Inorganic phosphate (Pi) is fundamental to adenosine triphosphate (ATP) generation, protein phosphorylation, and nucleic acid synthesis, and tumor cells exhibit increased phosphate uptake due to rapid proliferation, consistent with the growth-rate hypothesis. High Pi demand drives the overexpression of phosphate transporters (e.g., SLC34A2, NaPi-IIb) and modulates survival pathways such as ERK1/2 and Akt. Interestingly, phosphate has been shown to exert biphasic effects: at controlled extracellular concentrations, Pi can inhibit proliferation and induce apoptosis via caspase-3 activation and Bcl-2 downregulation in osteosarcoma (U2OS) and triple-negative breast cancer (MDA-MB-231) cells. Furthermore, Pi co-treatment with chemotherapeutic agents like doxorubicin enhances cytotoxicity and modulates key signaling pathways.²⁰,²¹

Magnesium is equally critical for cellular homeostasis, serving as a cofactor in hundreds of enzymatic reactions and as a modulator of immune responses. In cancer immunotherapy, magnesium is essential for the activation of LFA-1 (leukocyte function-associated antigen-1), a key integrin for cytotoxic T-cell adhesion and tumor cell recognition. Deficiencies in magnesium are linked to reduced immune efficacy, while supplementation enhances apoptotic response and immunomodulation, as shown in combination studies with cisplatin in bladder cancer models.²²,²³

The rational integration of phosphate and magnesium into MRB-CFI-1 thus provides not only a nanostructured support for protein adsorption and protection but also a biologically active platform capable of reprogramming the TME. This dual structure-function relationship positions MRB-CFI-1 within the current trend of developing multifunctional inorganic nanoplatforms for cancer therapy.

Emerging data also suggest broader potential for such platforms in highly aggressive tumors like glioblastoma (GBM). Nanoparticles based on Fe₃O₄, ZnO, Ag/Ce, and SrO-often modified with polymers like carboxymethyl cellulose (CMC)-have shown efficacy in inducing selective cytotoxicity, ferroptosis, or reactive oxygen species-mediated apoptosis. These analogues highlight the future versatility of MRB-CFI-1-like architectures in oncology, especially when targeting difficult-to-treat malignancies through combinations of immune, apoptotic, and metabolic modulation.

Taken together, the phosphate-magnesium core of MRB-CFI-1 serves not only as a scaffold for protein interaction but also as a biofunctional nanoplatform, with therapeutic potential rooted in well-documented biochemical and immunological mechanisms supported by contemporary literature.

4. Third Step: Preclinical Testing

The stage following discovery involves rigorous preclinical testing to evaluate the pharmacological profile, efficacy, and toxicity of candidate drugs both in vitro and in vivo. Preclinical studies of OncoTherad (MRB-CFI-1) have demonstrated its capacity to stimulate immune responses and improve survival rates across multiple cancer models.¹³,²⁴-³⁰ However, this stage presents considerable challenges, including the inherent complexity of biological systems and the risk of failure in models that do not accurately predict human responses.³¹ In Brazil, funding limitations pose an additional barrier, often restricting the scope of preclinical investigations, particularly in resource-constrained settings.³²

4.1. Bladder cancer

The OncoTherad (MRB-CFI-1) nanoimmunotherapy has been extensively evaluated in chemically induced animal models of NMIBC. These preclinical studies consistently demonstrated remarkable antitumor efficacy, a favorable safety profile, and potent immunomodulatory activity.

In a seminal study, Reis et al.²⁶ reported that intravesical administration of OncoTherad (MRB-CFI-1) completely inhibited tumor progression (100%) in an NMIBC murine model. Histopathological and immunohistochemical analyses revealed that, unlike BCG-which predominantly activates the canonical MyD88/NF-κB pathway-OncoTherad selectively induced the non-canonical TLR4-TRIF-IRF3 axis, triggering significant upregulation of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6). Treated animals exhibited increased expression of TLR2, TLR4, TRIF, IRF3, and IFN-γ, without evidence of systemic or histological toxicity.²⁶

Further, Reis et al.²⁸ demonstrated that OncoTherad (MRB-CFI-1) also targets key immune evasion pathways. Treatment markedly reduced RANKL (receptor activator of NF-κB ligand) and its receptor RANK, which are critical mediators of tumor progression and metastatic dissemination, while also modulating the PD-1/PD-L1 (Programmed cell death protein 1/Programmed death-ligand 1) immune checkpoint axis. These findings, confirmed by immunohistochemistry and Western blotting, support a dual mechanism of action: enhancement of cytotoxic immune responses and suppression of tumor-induced immunosuppression.²⁸

Fávaro et al.¹³ provided detailed physicochemical characterization of OncoTherad (MRB-CFI-1) and mechanistic insights, confirming its activity through TLR4-mediated interferon induction and modulation of the RANK/RANKL axis in NMIBC animal models. Importantly, in vivo studies showed no genotoxicity or systemic toxicity at therapeutic doses. OncoTherad reduced tumor malignancy by 70% compared to BCG and was effective in preventing recurrence, primarily through TLR4/IFN signaling and RANK/RANKL pathway regulation.¹³

Ribeiro de Souza et al.²⁹ recently investigated the potential of combining OncoTherad (MRB-CFI-1) with platelet-rich plasma (PRP) to enhance personalized therapy strategies. OncoTherad alone inhibited tumor progression by 85.7%, whereas its combination with PRP achieved a 71.4% inhibition rate. Both treatments activated TLR2 and TLR4 signaling, significantly increased IFN-γ, IL-1β, and TBK1 (TANK-binding kinase 1) expression, and promoted macrophage polarization toward the M1 phenotype. This was accompanied by expansion of CD8⁺ T lymphocytes and suppression of FOXP3⁺ regulatory T cells (Tregs), indicating tumor microenvironment reprogramming toward a pro-inflammatory, cytotoxic profile.²⁹

Taken together, these preclinical findings provide compelling evidence that OncoTherad (MRB-CFI-1) functions as a multifaceted immunotherapeutic agent (Figure 4). Compared to BCG, it demonstrates superior efficacy by selectively activating the TRIF-dependent TLR4 pathway, stimulating interferon production, downregulating immunosuppressive mechanisms (PD-1/PD-L1, RANK/RANKL, FOXP3), and promoting immunogenic cell death. Its favorable safety profile and robust immunological activity establish a strong translational foundation for clinical application, particularly in BCG unresponsive NMIBC patients (Figure 4).

Figure 4
Mechanism of action of OncoTherad (MRB-CFI-1). The nanoimmunotherapy activates Toll-like receptors TLR2 and TLR4, initiating MyD88- and TRIF-dependent signaling pathways and leading to IFN-mediated immune activation. This cascade promotes the recruitment and activation of CD8⁺ T cells, dendritic cells, and M1 macrophages, culminating in immunogenic cell death. Additionally, OncoTherad (MRB CFI-1) inhibits the RANK/RANKL axis, suppressing tumor progression and metastasis.

4.2. Colorectal cancer

OncoTherad (MRB-CFI-1) therapeutic potential was evaluated in a 1,2-dimethylhydrazine (DMH)-induced murine model of inflammation-associated colorectal cancer (CRC).²⁷ Weekly intraperitoneal administration of OncoTherad (25 mg kg^-1 for 10 weeks) resulted in complete regression of high-grade dysplasia, along with significant reductions in low-grade lesions (15%) and premalignant crypt alterations (10%), in contrast to the markedly higher prevalence of these lesions in untreated DMH-induced controls.²⁷

Histopathological analysis demonstrated restoration of epithelial integrity and suppression of aberrant crypt foci. Mechanistically, OncoTherad selectively activated the canonical MyD88-dependent TLR2/TLR4 signaling axis, upregulating TLR2, MyD88, and IL-6 expression, while concurrently downregulating the TRIF-IRF3 non-canonical pathway. This signaling shift suggests an immunological reprogramming towards a controlled pro-inflammatory state favorable to immune surveillance.²⁷

In addition, OncoTherad reduced KRAS expression and the Ki-67 proliferation index, indicating direct antiproliferative effects. Co-administration with probiotics further potentiated these effects, enhancing IL-10 and TGF-β levels, thereby supporting a dual mechanism of action: (i) immune activation and (ii) microbiota-mediated mucosal homeostasis. Although IFN-γ levels remained unchanged, the immunological rebalancing induced by OncoTherad appears sufficient to mitigate chronic inflammation and restrain tumor progression.²⁷

Collectively, these findings support OncoTherad (MRB CFI-1) as a promising immunotherapeutic strategy for colitis-associated CRC and justify further exploration of microbiota-integrated immunotherapy regimens.

4.3. Ovarian cancer

OncoTherad (MRB-CFI-1) was assessed in a chemically induced serous ovarian carcinoma model by using 7.12-dimethylbenz[a]anthracene (DMBA) in Fischer rats.³⁰ This model histologically and immunophenotypically mimicked low-grade serous carcinoma (LGSC), and it was confirmed by WT1 and ARID1A positivity; HNF1B and aberrant p53 staining absence; and lack of hotspot mutations in KRAS, PIK3CA and CTNNB1.³⁰

OncoTherad (MRB-CFI-1) monotherapy significantly reprogrammed the tumor’s immune microenvironment through TLR4, IL-6, IFN-γ and iNOS (p < 0.01) upregulation; consequently, it restored the cytotoxic signaling suppressed by tumor induction. In histological terms, OncoTherad (MRB-CFI-1) reduced the incidence of macroscopic lesions (85.7% lesion-free), minimized follicular cysts, and partially restored folliculogenesis and luteogenesis. Treated ovaries recorded reduced dysplasia and atypia comparable to untreated cancer controls.³⁰

Therapy combination to erythropoietin (EPO) enhanced FOXP3⁺ regulatory T cell infiltration but it did not improve antitumor efficacy to rates higher than that recorded for OncoTherad (MRB-CFI-1), alone. This finding suggests that monotherapy is more effective in promoting a cytotoxic immune phenotype. Global immunoreactivity profiles significantly differ across groups (PERMANOVA p = 0.001; R² = 0.303), and it confirms treatment-specific immune modulation.³⁰

These outcomes give OncoTherad (MRB-CFI-1) as the status of promising immunotherapeutic candidate for histotype-specific ovarian cancer, mainly for LGSC. Current treatment options are limited, and immunotherapy remains underexplored.

5. Fourth Step: Clinical Trials

Drug progression to clinical trials (phases I-III) aims to evaluate safety, efficacy, and optimal dosing, building on robust preclinical evidence (Figure 5). In Brazil, clinical trials are regulated by ANVISA and conducted in accordance with international ethical standards.¹ Clinical trials of OncoTherad (MRB-CFI-1), particularly in NMIBC, have highlighted its potential effectiveness compared to conventional therapies such as BCG.⁵-⁷ Nonetheless, significant challenges persist, including patient recruitment, logistical difficulties related to geographic dispersion, and regulatory constraints on trial protocols.

Figure 5
Clinical development pathway of OncoTherad (MRB-CFI-1) from preclinical studies to regulatory submission. In phase I, conducted in patients with BCG-unresponsive non-muscle-invasive bladder cancer, the nanoimmunotherapy demonstrated a 72.7% pathological complete response rate, median recurrence-free survival of 21.4 months, and a favorable safety profile with 77.3% grade 1/2 adverse events.

5.1. Stage I: BCG-unresponsive NMIBC

NMIBC accounts for approximately 70-75% of newly diagnosed bladder tumors. Standard management typically involves transurethral resection followed by intravesical BCG in high-risk cases. Despite BCG’s status as the standard immunotherapy, up to 40% of patients are unresponsive, and nearly half of initial responders relapse within one year.⁵,³³-³⁵ The management of BCG unresponsive NMIBC remains a major challenge due to the limited efficacy of available intravesical agents and the morbidity associated with radical cystectomy, particularly in elderly or comorbid patients. These issues are further exacerbated by ongoing global shortages of BCG.⁵,³⁴-³⁶

In a pivotal phase I clinical trial involving 44 patients with BCG-unresponsive NMIBC, OncoTherad (MRB CFI-1) achieved a pathological complete response (pCR) in 72.7% of cases. Notably, no patient progressed to muscle-invasive or metastatic disease during follow-up (Figure 5). Median recurrence-free survival (RFS) reached 21.4 months, with a mean response duration of 14.3 months. Adverse events were predominantly Grade 1-2 (77.3%), and no severe immune-related toxicities were reported.⁵

According to immunohistochemical analyses by de Arruda Camargo et al.,⁶ OncoTherad (MRB-CFI-1) reprograms the tumor microenvironment by promoting infiltration of CD8⁺ and CX3CR1⁺ effector cells, along with increased expression of IFN-γ and iNOS. Concurrently, it reduces FOXP3⁺ regulatory T cells, CD163⁺ M2 macrophages, and monoamine oxidase B (MAO-B) expression, thereby shifting the tumor milieu from an immunosuppressive to an immunoreactive phenotype-a hallmark of its mechanism of action. Complementary histopathological data from Salmazo et al.⁷ further demonstrated decreased expression of CD44, Ki-67, and SERBP1 (SERPINE1 mRNA-binding protein 1)-markers associated with stemness and proliferative activity-together with significant HABP4 (Hyaluronan-binding protein 4) upregulation, consistent with a transition toward a more differentiated and less aggressive epithelial profile.

When compared with other bladder-sparing strategies, OncoTherad demonstrates superior or at least comparable efficacy, with a markedly more favorable safety profile. For example, systemic BCG combined with pembrolizumab achieved a 69% pCR at 3 months, but was associated with a 30% recurrence rate and 7.7% severe immune toxicity.³⁷ Similarly, BCG plus atezolizumab yielded only a 42% response rate at 6 months, with 17% progression to muscle-invasive disease and 25% grade ≥ 3 toxicity.³⁸ The BCG/durvalumab combination achieved a 73% pCR at 12 months but carried significant toxicity (15% grade 3/4) and a 21% cystectomy rate.³⁹

Other intravesical strategies also present limitations. Gemcitabine/docetaxel (GEM/DOCE) achieved a 46% RFS at 24 months with minimal toxicity, though lacking immunomodulatory activity.⁴⁰ Gene therapy with nadofaragene firadenovec demonstrated a 53.4% pCR at 3 months and 43.8% RFS at 12 months,⁴¹ but requires viral vectors and specialized infrastructure. N-803 (nogapendekin alfa-inbakicept) combined with BCG produced a 71% pCR and 19-month median RFS,⁴²,⁴³ yet remains experimental due to its complex immune-modulation requirements.

In summary, while several intravesical approaches have yielded promising outcomes in BCG-unresponsive NMIBC, OncoTherad (MRB-CFI-1) nanoimmunotherapy stands out for its high and sustained complete response rate, favorable safety profile, multifactorial immunological mechanism, and demonstrated capacity to remodel the tumor microenvironment.

5.2. Translational expansion: compassionate use in recurrent glioblastoma (GBM)

Following demonstration of safety and efficacy in NMIBC trials, OncoTherad (MRB-CFI-1) was extended to other solid tumors under Brazil’s compassionate-use regulation (ANVISA RDC No. 38/2013). Recurrent glioblastoma (GBM) was prioritized due to the scarcity of effective second-line options and the profoundly immunosuppressive nature of its tumor microenvironment. Eligibility criteria included radiologically confirmed recurrence or progression after first-line therapy and preserved clinical performance status.

Eight patients (three male and five female) with recurrent GBM received second-line chemotherapy (lomustine or irinotecan ± bevacizumab) in combination with OncoTherad (MRB-CFI-1). The combined regimen resulted in a median overall survival (mOS) of 18 months, surpassing the historical benchmark of 12 months. Notably, only one patient experienced disease progression within the first six months.

A representative case involved a 59-year-old female who had undergone subtotal resection (60%) of two right frontal lesions, followed by chemoradiotherapy (30 fractions of EBRT (External Beam Radiation Therapy) plus temozolomide). The patient subsequently developed neurological deterioration, with altered consciousness, aphasia, dysphagia, and left hemiplegia. Pre-treatment brain MRI with spectroscopy and perfusion confirmed aggressive progression: the largest lesion, adjacent to the right genu of the corpus callosum, measured 64.2 × 34.5 mm, with a total tumor volume of 45.0 cm³ and extensive peritumoral edema (Figures 6a-6c).

Figure 6
Representative magnetic resonance imaging (MRI) of the brain from a patient diagnosed with glioblastoma (GBM). (a), (b), (c) Images obtained immediately following 30 sessions of EBRT combined with temozolomide (TMZ). (d), (e), (f) Images acquired after 12 weeks of treatment with OncoTherad (MRB-CFI-1) nanoimmunotherapy in combination with irinotecan and bevacizumab chemotherapy regimen.

Remarkable clinical improvement was observed 12 weeks after initiation of irinotecan/bevacizumab combined with OncoTherad (MRB-CFI-1). The patient regained assisted ambulation and resumed oral intake. MRI demonstrated a reduction of the largest lesion to 36.7 × 19.7 mm, with total tumor volume decreasing to 11.4 cm³ (-75%) and significant regression of peritumoral edema (Figures 6d-6f). The disease remained radiologically stable under ongoing treatment after 13 months of follow up.

Although preliminary, these findings suggest that OncoTherad (MRB-CFI-1) may potentiate antitumor immunity and enhance the efficacy of chemotherapy in GBM. Collectively, these results provide a strong rationale for further controlled clinical trials investigating its role in high-grade gliomas.

6. Fifth Step: Patent Development and Economic Evaluation-Transformative Model for Brazilian Pharmaceutical Innovation

The development trajectory of OncoTherad (MRB CFI-1) represents a rare convergence of scientific innovation, sovereign intellectual property generation, and economic feasibility in the translation of pharmaceuticals within a middle-income country (Figure 7).

Figure 7
Strategic model for the development, intellectual property protection, and regulatory translation of OncoTherad (MRB-CFI-1) in Brazil. The program integrates three foundational pillars-scientific innovation, sovereign intellectual property, and economic feasibility-supported by coordinated funding from national agencies (FAPESP, CNPq, FINEP, and MCTI). UNICAMP secured 100% ownership of all associated patents across Brazil, the United States and the European Union, without co-ownership or technology transfer to third parties. To facilitate translational development, a university-affiliated startup (NanoImmunotherapy Pharma Ltd.) was created. OncoTherad (MRB CFI-1) remains hindered by a regulatory bottleneck at ANVISA due to the absence of specific frameworks for non-biological complex drugs (NBCDs).

Beyond its pharmacological and clinical achievements, OncoTherad (MRB-CFI-1) has established a landmark in academic intellectual property in Brazil. For the first time in the nation’s history, a public university (UNICAMP) secured 100% ownership of all associated patents, without co-ownership by private institutions or technology transfer to third parties. This radical academic innovation model challenges traditional structures of dependency and reaffirms the capacity of public research institutions in the Global South to act not only as scientific innovators but also as legal custodians of high-value technological assets.⁴⁴

The intellectual property portfolio of OncoTherad (MRB-CFI-1) includes granted patents¹⁴-¹⁷ in Brazil (INPI: BR102017012768B1), the United States (USPTO: US-11623869-B2, US-11572284-B2, US 11136242-B2, US-11639294-B2), and the European Union with national designations (EPO: EP3626746B1, EP3998075B1; Portugal: PT3626746T, PT3998075T; Spain: ES2972185T3, ES2958810T3). These grants are more than legal instruments; they constitute international recognition of Brazilian scientific innovation and mark a turning point in the nation’s capacity to autonomously generate and protect biomedical technologies (Figure 7).

From an economic perspective, the OncoTherad (MRB-CFI-1) development program has also demonstrated a high level of efficiency. Investments in preclinical research, formulation optimization, scale-up manufacturing, regulatory interfacing, and clinical validation were strategically coordinated over 12 years with funding from national agencies, including the São Paulo Research Foundation (FAPESP), the National Council for Scientific and Technological Development (CNPq), the Funding Authority for Studies and Projects (FINEP), and the Ministry of Science, Technology, and Innovation (MCTI). The estimated cost of preclinical development in Brazil was USD 5.8 million, while the phase I clinical trial with 44 patients required only USD 2.1 million, for a total investment of approximately USD 7.9 million.

For comparison, equivalent development stages in the United States require more than USD 68 million for preclinical studies and USD 6.6 million for early-phase clinical trials, totaling approximately USD 74.6 million.⁴⁵ This reflects a cost ratio of approximately 9.4 (Brazil: U.S.), demonstrating the feasibility of developing high-complexity nanopharmaceuticals with substantial economic efficiency when strategic coordination is achieved among academia, funding agencies, and regulatory bodies.

To facilitate regulatory translation and engagement with the pharmaceutical sector, the scientific team launched a university-affiliated startup (NanoImmunotherapy Pharma Ltd. - NIMM-Pharma) to act as an interface between UNICAMP, ANVISA, and industry (Figure 7). However, this initiative exposed a critical structural bottleneck: the absence of specific regulatory frameworks within ANVISA for the registration of innovative nanopharmaceuticals, particularly those classified as non-biological complex drugs (NBCDs). As a result, despite its mechanistic innovation, favorable safety profile, and promising clinical outcomes, OncoTherad (MRB-CFI-1) remains stalled in a regulatory void-an emblematic example of the misalignment between Brazil’s scientific capacity and its institutional readiness to absorb disruptive biomedical innovations.

7. Conclusions and Future Perspectives

OncoTherad (MRB-CFI-1) is more than a therapeutic innovation. It stands as a milestone in Brazil’s capacity to conceive, develop, and protect high-impact biomedical technologies within the public academic sphere. Its trajectory demonstrates the feasibility of advancing complex, next-generation nanoimmunotherapies in emerging economies through a model that integrates scientific rigor, sovereign intellectual property, and coordinated public investment. The clinical efficacy, safety, and immunological remodeling observed in patients with BCG-unresponsive NMIBC attest not only to OncoTherad’s therapeutic value but also to the strength of the translational ecosystem that enabled its development.

Yet, critical structural barriers persist. The absence of specific regulatory pathways for NBCDs, limited financial mechanisms to support phase II/III trials, and the insufficient national infrastructure for scale-up and GMP compliant manufacturing-such as Contract Development and Manufacturing Organizations (CDMOs)-underscore the urgent need for systemic reforms. Without addressing these gaps, Brazil risks technological attrition, with domestically developed innovations delayed, externalized, or abandoned before reaching the market.

Future efforts must be anchored on three strategic pillars:

(i) Regulatory modernization, including the creation of fast-track evaluation mechanisms within ANVISA for university-origin nanopharmaceuticals with established preclinical and early-phase safety;

(ii) Expansion of translational capacity through sustained public investment in national CDMOs, integrated academic-industrial consortia, and long-term funding for clinical validation;

(iii) Clinical advancement and diversification via multicenter phase II/III trials and international collaborations to broaden OncoTherad’s therapeutic potential to other malignancies such as glioblastoma, colorectal cancer, and ovarian carcinoma.

In parallel, robust post-marketing surveillance systems must be institutionalized to guarantee real-world safety monitoring, equitable access within the public health system, and long-term therapeutic efficacy assessment.

OncoTherad (MRB-CFI-1) exemplifies how public science in the Global South can generate solutions of global impact when supported by institutional autonomy and strategic governance. Consolidating this model requires not only scientific excellence, but also regulatory foresight, industrial coordination, and sustained national commitment to transform knowledge into strategic healthcare assets.

If institutionalized, OncoTherad (MRB-CFI-1) will not remain an isolated success. Instead, it will inaugurate a new paradigm of sovereign drug development and establish Brazil as a proactive contributor to the future of immuno-oncology worldwide.

During the preparation of this work the authors used the ChatGPT, an AI language model developed by OpenAI in order to revise the text, correct the grammar and language refinement. After using this tool/service, the authors reviewed and edited the content as needed and took full responsibility for the content of the published article.

Data Availability Statement

All data are available in the text.

Acknowledgments

The authors gratefully acknowledge the financial support provided by the São Paulo Research Foundation (FAPESP grant numbers: 2018/10052-1; 2020/03419-6; 2022/15126-9; 2022/09699-6; 2022/09698-0; 2023/15929-7), the Brazilian National Council for Scientific and Technological Development (CNPq grant numbers: 552120/2011-1; 312396/2021-0; 304384/2024-0), the NanoBioss/Sisnano (CNPq grant number: 402280/2013-0), the INOMAT (FAPESP grant number: 2014/50906-9) and, the Nanoimmunotherapy Pharma Ltd (UNICAMP-affiliated startup).

The authors wish to express their sincere gratitude to the Men’s Health Clinic (Ambulatório de Saúde do Homem) at Paulínia Municipal Hospital, Paulínia City, São Vicente de Paulo Charity Hospital, Jundiaí City, and State University of Campinas (UNICAMP, Campinas City, São Paulo State, Brazil, for their support.

References

• ¹ Moscou, K.; Kohler, J. C.; MaGahan, A.; J. Pharm. Policy Pract. 2016, 9, 3. [Crossref]
  » Crossref
• ² Aguiar Jr., P. N.; Barreto, C. M. N.; Gutierres, B. S.; Tadokoro, H.; Lopes Jr., G. L.; Einstein 2017, 15, 349. [Crossref]
  » Crossref
• ³ Kuchenbecker, R. S.; Mota, D. M.; J. Oncol. Pharm. Pract. 2017, 23, 399. [Crossref]
  » Crossref
• ⁴ Barrios, C.; Freitas-Junior, R.; Martins, S.; Bines, J.; Estevez-Diz, M. D. P.; Caleffi, M.; JCO Glob. Oncol. 2021, 7, 474. [Crossref]
  » Crossref
• ⁵ Alonso, J. C. C.; de Souza, B. R.; Reis, I. B.; de Arruda Camargo, G. C.; de Oliveira, G.; de Barros Frazão Salmazo, M. I.; Gonçalves, J. M.; de Castro Roston, J. R.; Caria, P. H. F.; da Silva Santos, A.; de Freitas, L. L. L.; Billis, A.; Durán, N.; Fávaro, W. J.; Int. J. Mol. Sci. 2023, 24, 17535. [Crossref]
  » Crossref
• ⁶ de Arruda Camargo, G. C.; Oliveira, G.; Santos, B. N. S.; Roberto, I. M.; Ávila, M.; de Souza, B. R.; Alonso, J. C. C.; Durán, N.; Fávaro, W. J.; Med. Oncol. 2024, 41, 287. [Crossref]
  » Crossref
• ⁷ Salmazo, M. I. B. F.; Alonso, J. C. C.; de Arruda Camargo, G. C.; de Oliveira, G.; da Silva Santos, A.; Ávila, M.; Roberto, I. M.; de Freitas, L. L. L.; Bottene, M. C.; Lestingi, J. F. P.; Caria, P. H. F.; Durán, N.; Kobarg, J.; Fávaro, W. J.; Tissue Cell 2025, 93, 102783. [Crossref]
  » Crossref
• ⁸ da Silva, M. J. S.; O’Dwyer, G.; Osorio-de-Castro, C. G. S.; BMC Cancer 2019, 19, 987. [Crossref]
  » Crossref
• ⁹ Reinert, T.; Pellegrini, R.; Rol, R.; Werutsky, G.; Barrios, C. H.; JCO Glob. Oncol. 2020, 6, 307. [Crossref]
  » Crossref
• ¹⁰ Durán, N.; Dias, Q. C.; Fávaro, W. J.; J. Phys. Conf. Ser. 2019, 1323, 012018. [Crossref]
  » Crossref
• ¹¹ García-Domínguez, D. J.; López-Enríquez, S.; Alba, G.; Garnacho, C.; Jiménez-Cortegana, C.; Flores-Campos, R.; de la Cruz-Merino, L.; Hajji, N.; Sánchez-Margalet, V.; Hontecillas-Prieto, L.; Int. J. Mol. Sci. 2024, 25, 1195. [Crossref]
  » Crossref
• ¹² Iqbal, J.; Abbasi, B. A.; Ahmad, R.; Mahmood, T.; Ali, B.; Khalil, A. T.; Kanwal, S.; Shah, S. A.; Alam, M. M.; Badshah, H.; Munir, A.; Appl. Microbiol. Biotechnol. 2018, 102, 9449. [Crossref]
  » Crossref
• ¹³ Fávaro, W. J.; Alonso, J. C. C.; de Souza, B. R.; Reis, I. B.; Gonçalves, J. M.; Deckmann, A. C.; Oliveira, G.; Dias, Q. C.; Durán, N.; Tissue Cell 2023, 80, 101988. [Crossref]
  » Crossref
• ¹⁴ Fávaro, W. J.; Durán-Caballero, N. E.; Brazil pat. BR102017012768, 2017
• ¹⁵ Fávaro, W. J.; Durán-Caballero, N. E.; US pat. 20210238044A1, 2021
• ¹⁶ Fávaro, W. J.; Durán-Caballero, N. E.; US pat. 20210238045A1, 2021
• ¹⁷ Fávaro, W. J.; Durán-Caballero, N. E.; US pat. 20210238046A1, 2021
• ¹⁸ Takeda, K.; Akira, S.; Semin. Immunol. 2004, 16, 3. [Crossref]
  » Crossref
• ¹⁹ Satoh, T.; Akira, S.; Microbiol. Spectr. 2016, 4, 1. [Crossref]
  » Crossref
• ²⁰ Spina, A.; Sorvillo, L.; Di Maiolo, F.; Esposito, A.; D’Auria, R.; Di Gesto, D.; Chiosi, E.; Naviglio, A.; J. Cell Physiol. 2013, 228, 198. [Crossref]
  » Crossref
• ²¹ Sapio, L.; Sorvillo, L.; Illiano, M.; Chiosi, E.; Spina, N.; Navigli, S.; Molecules 2015, 20, 15910. [Crossref]
  » Crossref
• ²² Lötscher, J.; Martí, I.; Líndez, A. A.; Kirchhammer, N.; Cribioli, E.; Giordano Attianese, G. M. P.; Trefny, M. P.; Lenz, M.; Rothschild, S. I.; Strati, P.; Künzli, M.; Lotter, C.; Schenk, S. H.; Dehio, P.; Löliger, J.; Litzler, L.; Schreiner, D.; Koch, V.; Page, N.; Lee, D.; Grählert, J.; Kuzmin, D.; Burgener, A. V.; Merkler, D.; Pless, M.; Balmer, M. L.; Reith, W.; Huwyler, J.; Irving, M.; King, C. G.; Zippelius, A.; Hess, C.; Cell 2022, 185, 585. [Crossref]
  » Crossref
• ²³ Moussa, M.; Chakra, M. A.; Duquesne, I.; Intract. Rare Dis. Res. 2022, 11, 81. [Crossref]
  » Crossref
• ²⁴ Fonseca-Alves, C. E.; Ferreira, E.; de Oliveira Massoco, C.; Strauss, B. E.; Fávaro, W. J.; Durán, N.; Oyafuso da Cruz, N.; dos Santos Cunha, S. C.; Castro, J. L. C.; Rangel, M. M. M.; Brunner, C. H. M.; Tellado, M.; dos Anjos, D. S.; Fernandes, S. C.; Barbosa de Nardi, A.; Biondi, L. R.; Dagli, M. L. Z.; Front. Vet. Sci. 2021, 8, 707025. [Crossref]
  » Crossref
• ²⁵ Name, J. J.; Vasconcelos, A. R.; Souza, A. C. R.; Fávaro, W. J.; Int. J. Mol. Med. 2021, 47, 11. [Crossref]
  » Crossref
• ²⁶ Reis, I. B.; Tibo, L. H. S.; Socca, E. A. R.; de Souza, B. R.; Durán, N.; Fávaro, W. J.; Tissue Cell 2022, 76, 101762. [Crossref]
  » Crossref
• ²⁷ Reis, S. K.; Socca, E. A. R.; de Souza, B. R.; Genaro, S. C.; Durán, N.; Fávaro, W. J.; Tissue Cell 2022, 75, 101747. [Crossref]
  » Crossref
• ²⁸ Reis, I. B.; Tibo, L. H. S.; de Souza, B. R.; Durán, N.; Fávaro, W. J.; J. Cancer Res. Clin. Oncol. 2023, 149, 5025. [Crossref]
  » Crossref
• ²⁹ de Souza, B. R.; Reis, I. B.; Camargo, G. C. A.; Oliveira, G.; Dias, Q. C.; Durán, N.; Fávaro, W. J.; Int. Immunopharmacol. 2023, 123, 110723. [Crossref]
  » Crossref
• ³⁰ de Souza, B. R.; Oliveira, G.; Leme, G.; Reis, I. B.; Cabral, F. A. T.; de Paula, J. L. B.; Santos, D. H. S.; Felizzola, C. R.; Durán, N.; Anglesio, M.; Fávaro, W. J.; Biomed. Pharmacother. 2025, 182, 117755. [Crossref]
  » Crossref
• ³¹ Ulmschneider, M. B.; Searson, P. C.; J. Controlled Release 2015, 212, 78. [Crossref]
  » Crossref
• ³² Botelho, S. F.; Martins, M. A.; Vieira, L. B.; Reis, A. M.; J. Clin. Pharmacol. 2017, 57, 493. [Crossref]
  » Crossref
• ³³ Matulewicz, R. S.; Steinberg, G. D.; Rev. Urol. 2020, 22, 43. [PubMed]
  » PubMed
• ³⁴ Lidagoster, S.; Ben-David, R.; De Leon, B.; Sfakianos, J. P.; Curr. Oncol. 2024, 31, 1063. [Crossref]
  » Crossref
• ³⁵ Ghodoussipour, S.; Bivalacqua, T.; Bryan, R. T.; Li, R.; Mir, M. C.; Palou, J.; Psutka, S. P.; Sundi, D.; Tyson, M. D.; Inman, B. A.; Eur. Urol. 2025, 88, 33. [Crossref]
  » Crossref
• ³⁶ Veeratterapillay, R.; Heer, R.; Johnson, M. I.; Persad, R.; Bach, C.; Curr. Urol. Rep. 2016, 17, 68. [Crossref]
  » Crossref
• ³⁷ Montgomery, J.; Lybbert, D.; Sana, S.; El-Zawahry, A.; Peabody, J.; Pearce, T.; Adams, N.; Deebajah, M.; Dynda, D.; Babaian, K.; Crabtree, J.; Delfino, K.; McVary, K.; Robinson, K.; Rao, K.; Alanee, S.; Clin. Genitourin. Cancer 2024, 22, 102059. [Crossref]
  » Crossref
• ³⁸ Inman, B. A.; Hahn, N. M.; Stratton, K.; Kopp, R.; Sankin, A.; Skinner, E.; Pohar, K.; Gartrell, B. A.; Pham, S.; Rishipathak, D.; Mariathasan, S.; Davarpanah, N.; Carter, C.; Steinberg, G. D.; Eur. Urol. Oncol. 2023, 6, 313. [Crossref]
  » Crossref
• ³⁹ Hahn, N. M.; O’Donnell, M. A.; Efstathiou, J. A.; Zahurak, M.; Rosner, G. L.; Smith, J.; Kates, M. R.; Bivalacqua, T. J.; Tran, P. T.; Song, D. Y.; Baras, A. S.; Matoso, A.; Choi, W.; Smith, K. N.; Pardoll, D. M.; Marchionni, L.; McGuire, B.; Grace Phelan, M.; Johnson III, B. A.; O’Neal, T.; McConkey, D. J.; Rose, T. L.; Bjurlin, M.; Lim, E. A.; Drake, C. G.; McKiernan, J. M.; Deutsch, I.; Anderson, C. B.; Lamm, D. L.; Geynisman, D. M.; Plimack, E. R.; Hallman, M. A.; Horwitz, E. M.; Al Saleem, E.; Chen, D. Y. T.; Greenberg, R. E.; Kutikov, A.; Guo, G.; Masterson, T. A.; Adra, N.; Kaimakliotis, H. Z.; Eur. Urol. 2023, 83, 486. [Crossref]
  » Crossref
• ⁴⁰ Patel, S. H.; Gabrielson, A. T.; Chan, S.; Schwartz, D.; Collins, C.; Singla, N.; Trock, B.; Bivalacqua, T. J.; Hahn, N.; Kates, M. R.; J. Urol. 2024, 212, 95. [Crossref]
  » Crossref
• ⁴¹ Boorjian, S. A.; Alemozaffar, M.; Konety, B. R.; Shore, N. D.; Gomella, L. G.; Kamat, A. M.; Bivalacqua, T. J.; Montgomery, J. S.; Lerner, S. P.; Busby, J. E.; Poch, M.; Crispen, P. L.; Steinberg, G. D.; Schuckman, A. K.; Downs, T. M.; Svatek, R. S.; Mashni Jr., J.; Lane, B. R.; Guzzo, T. J.; Bratslavsky, G.; Karsh, L. I.; Woods, M. E.; Brown, G.; Canter, D.; Luchey, A.; Lotan, Y.; Krupski, T.; Inman, B. A.; Williams, M. B.; Cookson, M. S.; Keegan, K. A.; Andriole, G. L., Jr.; Sankin, A. I.; Boyd, A.; O’Donnell, M. A.; Sawutz, D.; Philipson, R.; Coll, R.; Narayan, V. M.; Treasure, F. P.; Yla-Herttuala, S.; Parker, N. R.; Dinney, C. P. N.; Lancet Oncol. 2021, 22, 107. [Crossref]
  » Crossref
• ⁴² Chamie, K.; Chang, S. S.; Kramolowsky, E. V.; Gonzalgo, M. L.; Huang, M.; Bhar, P.; Spilman, P.; Sender, L.; Reddy, S. K.; Soon-Shiong, P.; Urol. Pract. 2024, 11, 367. [Crossref]
  » Crossref
• ⁴³ Chamie, K.; Chang, S. S.; Kramolowsky, E.; Gonzalgo, M. L.; Agarwal, P. K.; Bassett, J. C.; Bjurlin, M.; Cher, M. L.; Clark, W.; Cowan, B. E.; David, R.; Goldfischer, E.; Guru, K.; Jalkut, M. W.; Kaffenberger, S. D.; Kaminetsky, J.; Katz, A. E.; Koo, A. S.; Sexton, W. J.; Tikhonenkov, S. N.; Trabulsi, E. J.; Trainer, A. F.; Spilman, P.; Huang, M.; Bhar, P.; Taha, S. A.; Sender, L.; Reddy, S.; Soon-Shiong, P.; NEJM Evid. 2023, 2, EVIDoa2200167. [Crossref]
  » Crossref
• ⁴⁴ Fernandes, D. R. A.; Gadelha, C. A. G.; Maldonado, J.; Saúde Soc. 2024, 33, e220791. [Crossref]
  » Crossref
• ⁴⁵ Sertkaya, A.; Wong, H. H.; Jessup, A.; Beleche, T.; Clin. Trials 2016, 13, 117. [Crossref]
  » Crossref

Edited by

• Editor handled this article:
  Brenno A. D. Neto (Editor-in-Chief)

Publication Dates

• Publication in this collection
  06 Oct 2025
• Date of issue
  2025

History

• Received
  11 Aug 2025
• Published
  25 Aug 2025