Business Risk Mitigation in the Development Process of New Monoclonal Antibody Drug Conjugates for Cancer Treatment.

Recent developments aim to extend the cytotoxic effect and therapeutic window of mAbs by constructing antibody-drug conjugates (ADCs), in which the targeting moiety is the mAb that is linked to a highly toxic drug. According to a report from mid of last year, the global ADCs market accounted for USD 1387 million in 2016 and was worth USD 7.82 billion in 2022. It is estimated to increase in value to USD 13.15 billion by 2030. One of the critical points is the linkage of any substituent to the functional group of the mAb. Increasing the efficacy against cancer cells' highly cytotoxic molecules (warheads) are connected biologically. The connections are completed by different types of linkers, or there are efforts to add biopolymer-based nanoparticles, including chemotherapeutic agents. Recently, a combination of ADC technology and nanomedicine opened a new pathway. To fulfill the scientific knowledge for this complex development, our aim is to write an overview article that provides a basic introduction to ADC which describes the current and future opportunities in therapeutic areas and markets. Through this approach, we show which development directions are relevant both in terms of therapeutic area and market potential. Opportunities to reduce business risks are presented as new development principles.

Synthesis of 68Ga-Labeled Biopolymer-based Nanoparticle Imaging Agents for Positron-emission Tomography.

AIM: The purpose of this study was to develop a folate receptor-targeted 68Ga-labeled agent for the detection of cancer cells in mouse models of ovarian cancer by dual positron-emission tomography (PET) and magnetic resonance imaging (MRI). Moreover, we aimed to develop a controlled biopolymer-based chemistry that enables linking metal-binding (here Ga-68) chelators. MATERIALS AND METHODS: The nanoparticle (NP) agent was created by self-assembling of folic acid-modified polyglutamic acid and chelator-modified chitosan followed by radiolabeling with 68Ga (III) ions (68Ga-NODAGA-FA). The structure of modified biopolymers was characterized by spectroscopy. Particle size and mobility were determined. RESULTS: Significant selective binding of NPs was established in vitro using folate receptor-positive KB and - negative MDA-MB-231 cell lines. In vivo tumor uptake of folate-targeted 68Ga3+-radiolabeled NPs was tested using subcutaneous tumor-bearing CB17 SCID mice models. PET/MR dual modalities showed high tumor uptake with 6.5 tumor-to-muscle ratio and NP localization. CONCLUSION: In vivo results supporting the preliminary in vitro tests demonstrated considerably higher 68Ga-NODAGA-FA nanoparticle accumulation in KB tumors than in MDA-MB-231 tumors, thereby confirming the folate receptor-mediated uptake of this novel potential PET imaging agent.

Biopolymer based nanosystem for doxorubicin targeted delivery.

This study describes formation of an actively and passively targeted, water-soluble drug delivery system (DDS) which contains doxorubicin (DOX). The system comprises two biocompatible and biodegradable polymers: poly-γ-glutamic acid (PGA) and chitosan (CH). Self-assembly of these biopolymers in aqueous medium results stable nanoparticles (NPs) with a hydrodynamic size of 80-150 nm and slightly negative surface charge. Folic acid (FA) was used as targeting agent bonded to the polyanion (PA) and also to the surface of the NPs. The NP's physical stability, active targeting effect, cellular toxicity, release profile and in vivo anti-tumor efficacy were investigated. It was found that the targeted, self-assembled nanoparticles are stable at 4°C for several months, cause better in vitro toxicity effect on folate receptor (FR) positive cell lines than the doxorubicin or the non-targeted nanosystem and based on its release profile it is expected, that the nanosystem will remain stable during the circulation in the body. Pharmacodynamic studies demonstrated that the DOX-loaded nanoparticles can deliver greater tumor growth inhibition than the free drug molecules and the liposomal compound, with less general toxicity. It was observed that the overall survival is the main benefit of the biopolymer based drug delivery system.

Folate receptor targeted self-assembled chitosan-based nanoparticles for SPECT/CT imaging: demonstrating a preclinical proof of concept.

A new biocompatible, biodegradable, self-assembled chitosan-based nanoparticulate product was successfully synthesized and radiolabeled with technetium-99m, and studied as a potential new SPECT or SPECT/CT imaging agent for diagnosis of folate receptor overexpressing tumors. In the present study we examined the conditions of a preclinical application of this labeled nanosystem in early diagnosis of spontaneously diseased veterinary patient using a human SPECT/CT device. The results confirmed that the nanoparticles accumulated in tumor cells overexpressing folate receptors, contrast agent revealed higher uptake in the tumor for a long time. Preclinical trials verified that the new nanoparticles are able to detect folate-receptor-overexpressing tumors in spontaneously diseased animal models with enhanced contrast.

Tumor-specific localization of self-assembled nanoparticle PET/MR modalities.

AIM: The aim of this work was to synthesize and study in vitro and in vivo nanocarriers used as magnetic resonance imaging (MRI) contrast agents that accumulate in tumor cells specifically overexpressing folate receptors. MATERIALS AND METHODS: Nanoparticles were prepared by self-assembly of poly-γ-glutamic acid and chitosan biopolymers and were complexed with gadolinium ions. Folic acid served as a targeting molecule. Rat hepatocellular carcinoma (HeDe) cells overexpressing folate receptors were used as a model system. For in vivo experiments, HeDe cells were transplanted under the renal capsule of F344 rats. RESULTS: In vitro results showed the significant internalization of nanoparticles into HeDe cells. MRI measurements revealed that targeting nanocarriers accumulated in tumors. The MRI/PET fusion images resulted in the exact localization of tumors. CONCLUSION: The nanocarrier provides a suitable means for the early diagnosis of tumors based on their overexpression of folate receptors.

(99m)Tc-labelled nanosystem as tumour imaging agent for SPECT and SPECT/CT modalities.

We report the synthesis, in vitro and in vivo investigation of folate-targeted, biocompatible, biodegradable self-assembled nanoparticles radiolabelled with (99m)Tc, as potential new SPECT or SPECT/CT imaging agent. Nanoparticles with hydrodynamic size in the range of 75-200 nm were prepared by self-assembly of chitosan and folated poly-γ-glutamic acid, and then radiolabelled with (99m)Tc. The nanoparticles target tumour cells overexpressing folate receptors and internalize specifically into them to realize early tumour diagnosis detected by SPECT and SPECT/CT modalities. Rat hepatocellular carcinoma cells were used as model system. Cell specificity and tumour targeting efficacy of these nanosystems were investigated in vitro, and in vivo using SPECT and fusion nanoSPECT/CT imaging. In vitro results showed that the radiolabeled nanosystem was efficiently internalized by tumour cells. Whole-body biodistribution of the new radiolabelled, folate-targeted nanoparticles revealed higher uptake in the tumorous kidney compared to the non-tumorous contralateral side. Uptake by the lungs and thyroids was negligible, which confirmed the stability of the nanoparticles in vivo. In vivo SPECT and SPECT/CT imaging visually reinforced the uptake results and were in accordance with the biodistribution data: the new nanoparticles as a targeted contrast agent improve tumour targeting and are able to detect folate-receptor-overexpressing tumours in animal models with enhanced contrast.

Composition and characterization of in situ usable light cured dental drug delivery hydrogel system.

Biodegradable polymers are compatible, permeable and nontoxic, thus they can provide a useful tool for drug delivery or tissue engineering. These polymers can form hydrogels, which are suitable vehicles for different types of materials e.g. drugs, bioactive molecules or cells. In the case of dentistry, photopolymerization is an obvious method to obtain in situ useable devices which can provide a more efficient way of tailoring drug release. A hydrogel system was developed based on poly-gamma-glutamic acid that was modified with methacryloyl groups to achieve this purpose. The resulting new reactive structure was proved by NMR spectroscopy. The swelling ratio of this type of hydrogel has been found remarkable, over 300 % after 24 h, and it can release 5 ng/mm(2) metronidazole. The prepared hydrogels were nontoxic as viability, cytotoxicity tests and cell morphology investigations proved it. These results render this model system an excellent candidate for use as an in situ curing local drug delivery device. The new photoactive system can be utilized in the treatment of periodontal diseases or raising the effectiveness of drugs used only in the minimal effective dose.

Cancer cell targeting and imaging with biopolymer-based nanodevices.

We report here the synthesis, in vitro and in vivo investigation of magnetic resonance imaging (MRI) active nanoparticles, which target folate receptor overexpressing tumor cells. Self-assembled nanoparticles with a hydrodynamic size of 50-200 nm were prepared from poly-γ-glutamic acid and chitosan biopolymers with Gd-ions. The nanoparticles are biocompatible, non-toxic and stable for several months in aqueous media. In vitro assays using confocal microscopy, flow cytometry and MR imaging on HeLa human cervix carcinoma tumor cells showed that folic acid targeted nanoparticles were internalized specifically in a folate receptor dependent manner. In vivo study confirmed, that, considerable accumulation of nanosystems was found compared with the control animal represented by the MR images. Relaxometry measurements demonstrated that the nanoparticle-Gd complexes drastically change the signal intensity of the tumor cells. Because of the contrast enhancement, they are attractive candidates as potential contrast agents for a variety of diagnostic applications including early diagnosis of tumors.

Nanoparticles formed by complexation of poly-gamma-glutamic acid with lead ions.

The present investigation describes the preparation and characterization of novel biodegradable nanoparticles based on complexation of poly-gamma-glutamic acid (gamma-PGA) with bivalent lead ion. The prepared nano-systems were stable in aqueous media at low pH, neutral and mild alkaline conditions. The particle size and the size of the complexes were identified by dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements. It was found that the size of the complexes depended on the pH and concentrations of gamma-PGA and lead ions. Particle sizes measured by TEM revealed that at low concentrations, nanosized particles were formed, however, at high concentrations of gamma-PGA and lead ions, the formation of large aggregates with a broad size distribution was promoted. The size of individual particles was in the range of 40-100 nm measured by TEM. The results from the DLS measurements showed that the low and high pH values in mixtures with high concentrations of gamma-PGA and Pb2+ ions favored the growth of large complexes. The gamma-PGA nanoparticles, composed of a biodegradable biomaterial with high flocculating and heavy metal binding activity, may be useful for various water treatment applications.

[Synthesis of nanoparticles for dental drug delivery systems]. / Fogászatban alkalmazható hatóanyagok leadására alkalmas nanorészecskék szintézise.

Modern drug delivery systems are designed for targeted controlled slow drug release. Up to now polymer based hydrogels have been applied in dentistry, which systems can affect the rate of the release due to their structure. Recently, intensive research for other methods is performed all over the world in order to improve the effectiveness of delivery systems. Nanotechnology is one of the most dynamically developing disciplines and is a powerful tool to increase the bioavailability of drugs. The aim of this work is to synthesise biocompatible nanoparticles by free radical initiated copolymerization of the monomers, 2-hydroxyethyl methacrylate (HEMA) and polyethyleneglycol dimethacrylate (PEGDMA) in aqueous solution, which can support the formation of nanoparticles that can be used as a drug delivery system for dental applications. The polymer-based nanoparticles were prepared via micellar polymerisation, which resulted a well dispersible white powder material. The size of particles was determined by Dynamic Laser Light Scattering (DLS) and Scanning Electron Microscopy (SEM). The size of particles is in range of 50-180 nm, measured by SEM. These values are commensurable with the results obtained by DLS experiments, where two size ranges were observed, as 40 +/- 15 nm and 180 +/- 30 nm. The nanoparticles are suitable for incorporation into a hydrogel matrix and to design new drug delivery devices for dental applications.

Direct amidation of poly(gamma-glutamic acid) with benzylamine in dimethyl sulfoxide.

Partially benzylamidated, amphipathic poly(gamma-glutamic acid) (BzPGA) was synthesized from poly(gamma-glutamic acid) (PGA) and benzylamine by direct amidation in dimethyl sulfoxide (DMSO). Benzylamine and PGA were heated in DMSO for 1 to 26 h at temperatures between 110 and 130 degrees C, producing derivatives of various degrees of benzylamidation as a function of the reaction time and temperature. Neither any carboxyl-activating agent nor catalyst is needed for the reaction to proceed. After purification by dialysis, the product was identified by 1H and 13C 1D and 2D NMR in DMSO-d(6). BzPGA prepared by the new direct amidation method was identical to that obtained with a conventional carbodiimide-mediated reaction in water. The one-pot amidation procedure described in the present article can probably be applied to the synthesis of amides from other amines and carboxylic acids.

[Synthesis and compression strength of hydrogels]. / Hidrogélek szintézise és nyomási szilárdságuk vizsgálata.

The purpose of the present investigation was the synthesis of crosslinked polymers and the assay of the effect of the monomer ratio to the compression values. Hydrogels were made by free radical photoinitiation from 2-hydroxyethyl metachrylate (HEMA) monomer, poly(ethylene glycol) dimethacrylate (PEG-DMA) crosslinker and sodium anthraquinone-2-sulphonate initiatior. The concentration of HEMA and PEG-DMA was 30 %, and their ratio was varied to obtain gels with different crosslinking density. It was found, that increasing the amount of the crosslinker in the gels, the force of the deformation increased, however the elasticity decreased. The lowest difference was found between the molar ratio of 50% HEMA: 50% PEG-DMA and the 75% HEMA: 25% PEG-DMA gels. Compression strength was strongly influenced on the ratio of the crosslinking agent. The smallest compression strength was found at the lowest amount of PEG-DMA (90% HEMA: 10% PEG-DMA) (0.0475 MPa +/- 0.0117) and the highest value was found at the highest amount of the PEG-DMA (10% HEMA: 90% PEG-DMA) (0.1366 MPa +/- 0.0546). The values of the compression strength at gels with similar composition was not significantly different. On the base of the present investigations the elasticity of the materials could be calculated from the ratio of monomers. It could be an important parameter in the further applications of the gels as a local delivery system in the parodontology.

[Modification of resin using reactive polymeric nanoparticles]. / Fogászati kompozitok gyantájának módosítása reaktiv polimer nanorészecskékkel.

Volumetric shrinkage during polymerization has always caused problems since polymer based composites are used as dental filling materials. This value has been reduced to 2-4 volume % with the improvement of dental filling materials and of suitable filling technique. The purpose of our study was to synthesize reactive polymeric nanoparticles which can be applied to modify the polymerization shrinkage of dental resin. It means that certain part of a commercial dental resin would be substituted with reactive polymeric nanoparticles as organic filler. According to our hypothesis the volume contraction would significantly be reduced by applying the modified resin with organic filler. Nanoparticles were prepared by free radical copolymerization from mono-, di- and trifunctional monomers in emulsion and organic solution. In emulsion their size distribution is narrow, but it is quite variable in solutions. The size, size distribution and reactivity of nanoparticles were examined, and these properties can be influenced by the mol ratio and concentration of the monomers. It was shown that the nanoparticles could be swollen by the matrix material and these build a unique structure that can be photo-polymerized. During polymerization the shrinkage of the copolymer can be considered zero, consequently the total contraction originates from the shrinkage of the matrix. In the modified resin the reactive groups of monomers and the pendant double bounds of nanoparticles were connected by covalent bond.

Synthesis and study of cross-linked chitosan-N-poly(ethylene glycol) nanoparticles.

The present investigation describes the synthesis and characterization of novel biodegradable nanoparticles based on chitosan. Poly(ethylene glycol) dicarboxylic acid was used for intramolecular cross-linking of the chitosan linear chains. The condensation reaction of carboxylic groups and pendant amino groups of chitosan was performed by using water-soluble carbodiimide. The prepared nanosystems were stable in aqueous media. The structure of the products was determined by nuclear magnetic resonance (NMR) spectroscopy, and the particle size was identified by dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements. It was found that biodegradable cross-linked chitosan nanoparticles experienced considerable swelling because of the length and flexibility of the cross-linking agent. The aqueous solutions or dispersions of nanoparticles were stable and clear or mildly opalescent systems depending on the ratio of cross-linking and molecular weight of chitosan, findings consistent with values of transmittance above 75%. Particle size measured by TEM varied in the range of 4-24 nm. In the swollen state, the average size of the individual particles measured by DLS was in the range of 50-120 nm depending on the molecular weight of chitosan and the ratio of cross-linking.

Prediction of folding preference of 10 kDa silk-like proteins using a Lego approach and ab initio calculations.

Because of their great flexibility and strength resistance, both spider silks and silkworm silks are of increasing scientific and commercial interest. Despite numerous spectroscopic and theoretical studies, several structural properties at the atomic level have yet to be identified. The present theoretical investigation focuses on these issues by studying three silk-like model peptides: (AG)(64), [(AG)(4)EG](16), and [(AG)(4)PEG](16), using a Lego-type approach to construct these polypeptides. On the basis of these examples it is shown that thermoneutral isodesmic reactions and ab initio calculations provide a capable method to investigate structural properties of repetitive polypeptides. The most probable overall fold schema of these molecules with respect to the type of embedded hairpin structures were determined at the ab initio level of theory (RHF/6-311++G(d,p)//RHF/3-21G). Further on, analysis is carried out on the possible hairpin and turn regions and on their effect on the global fold. In the case of the (AG)(64) model peptide, the optimal beta-sheet/turn ratio was also determined, which provided good support for experimental observations. In addition, lateral shearing of a hairpin "folding unit" was investigated at the quantum chemical level to explain the mechanical properties of spider silk. The unique mechanical characteristics of silk bio-compounds are now investigated at the atomic level.

Preparation and characterization of chitosan-based nanoparticles.

The present investigation describes the synthesis and characterization of novel biodegradable nanoparticles based on chitosan for biomedical applications. Natural di- and tricarboxylic acids were used for intramolecular cross-linking of the chitosan linear chains. The condensation reaction of carboxylic groups and pendant amino groups of chitosan was performed by using water-soluble carbodiimide. This method allows the formation of polycations, polyanions, and polyampholyte nanoparticles. The prepared nanosystems were stable in aqueous media at low pH, neutral, and mild alkaline conditions. The structure of products was determined by NMR spectroscopy, and the particle size was identified by laser light scattering (DLS) and transmission electron microscopy (TEM) measurements. It was found that particle size depends on the pH, but at a given pH, it was independent of the ratio of cross-linking and the cross-linking agent. Particle size measured by TEM varied in the range 60-280 nm. In the swollen state, the average size of the particles measured by DLS was in the range 270-370 nm depending on the pH. The biodegradable cross-linked chitosan nanoparticles, as solutions or dispersions in aqueous media, might be useful for various biomedical applications.