Targeted Anticancer Agent with Original Mode of Action Prepared by Supramolecular Assembly of Antibody Oligonucleotide Conjugates and Cationic Nanoparticles.

Despite their clinical success, Antibody-Drug Conjugates (ADCs) are still limited to the delivery of a handful of cytotoxic small-molecule payloads. Adaptation of this successful format to the delivery of alternative types of cytotoxic payloads is of high interest in the search for novel anticancer treatments. Herein, we considered that the inherent toxicity of cationic nanoparticles (cNP), which limits their use as oligonucleotide delivery systems, could be turned into an opportunity to access a new family of toxic payloads. We complexed anti-HER2 antibody-oligonucleotide conjugates (AOC) with cytotoxic cationic polydiacetylenic micelles to obtain Antibody-Toxic-Nanoparticles Conjugates (ATNPs) and studied their physicochemical properties, as well as their bioactivity in both in vitro and in vivo HER2 models. After optimising their AOC/cNP ratio, the small (73 nm) HER2-targeting ATNPs were found to selectively kill antigen-positive SKBR-2 cells over antigen-negative MDA-MB-231 cells in serum-containing medium. Further in vivo anti-cancer activity was demonstrated in an SKBR-3 tumour xenograft model in BALB/c mice in which stable 60% tumour regression could be observed just after two injections of 45 pmol of ATNP. These results open interesting prospects in the use of such cationic nanoparticles as payloads for ADC-like strategies.

SURGICAL APPROACH FOR TREATMENT OF OBSTRUCTIVE JAUNDICE IN PATIENTS OF DIFFERENT AGE GROUPS.

OBJECTIVE: The aim: Assessment of the effectiveness of using minimally invasive and open methods of bile duct decompression for treatment of obstructive jaundice (OJ) by comparing complications in patients of di!erent age groups. PATIENTS AND METHODS: Materials and methods: We analyzed the results of surgical treatment of 250 patients with OJ. The patients were assigned to two groups: Group I (n = 100) consisting of young and middle-age patients, and Group II (n = 150) consisting of elderly, senile and long-living patients. The average age was 52 ± 6.0 years. RESULTS: Results: 62 (24.8%) Group I patients and 74 (29.6%) Group II patients were submitted to minimally invasive surgical interventions. 38 (15.2%) Group I patients and 76 (30.4%) Group II patients were submitted to open surgical interventions. Complications after minimally invasive surgery (n = 62) in Group I patients were observed in 2 (3.2%) cases, and in 4 (10.5%) cases after open surgeries (n = 38). Complications following minimally invasive interventions (n = 74) in Group II patients were registered in 5 (6.8%) cases, and in 9 (11.8%) cases following open operations (n = 76). 2 (2.6%) Group II patients died for transmural myocardial infarction. CONCLUSION: Conclusions: The use of minimally invasive surgical interventions for treatment of young and middle-aged OJ patients compared to patients of older age groups makes it possible to reduce the frequency of complications by 2.1 times, which is a statistically signi"cant (p <0.05). The frequency of complications after open surgical interventions of bile ducts in patients of di!erent age groups is not statistically signi"cant (p >0.05).

CHARACTERIZATION OF STRUCTURAL DISORDERS OF THE LIVER DEPENDING ON THE DURATION OF SUBHEPATIC CHOLESTASIS IN PATIENTS OF DIFFERENT AGE GROUPS.

OBJECTIVE: The aim: To study structural disorders of the liver depending on the duration of subhepatic cholestasis in patients of different age groups. PATIENTS AND METHODS: Materials and methods: 50 obstructive jaundice patients were subdivided into two groups. Group I (n = 25) consisted of young (18-44-year-old) and middle-aged patients (45-59-year-old), while the Group II (n = 25) included elderly (60-74-year-old) and senile patients (75-90-year-old). RESULTS: Results: We performed morphological and morphometric studies of 50 liver biopsy specimens taken from patients of different age groups with different duration of obstructive jaundice: less than 7 days, 7-14 days, 14-21 days, 21-28 days, and over 28 days. CONCLUSION: Conclusions: In patients of the Groups I and II, pathological hepatic changes in the early stages of mechanical jaundice were manifested in the form of he-patocyte dystrophy and hepatitis development. In the Group I patients, manifestations of steatohepatitis, fibrosis and initial signs of liver cirrhosis were noted in the late stages of subhepatic cholestasis. In addition to the above-mentioned changes, Group II patients, in the late stages of mechanical jaundice, presented signs of severe fibrosis and well-shaped liver cirrhosis. Taking into account the above morphological changes in the liver with different duration of subhepatic cholestasis, we consider reasonable to decompress bile ducts in patients of older age groups at earlier stages of mechanical jaundice compared to young and middle-aged patients, thus preventing post-decompression liver dysfunction and the subsequent development of biliary cirrhosis.

Investigating Ugi/Passerini Multicomponent Reactions for the Site-Selective Conjugation of Native Trastuzumab*.

Site-selective modification of proteins has been the object of intense studies over the past decades, especially in the therapeutic field. Prominent results have been obtained with recombinant proteins, for which site-specific conjugation is made possible by the incorporation of particular amino acid residues or peptide sequences. In parallel, methods for the site-selective and site-specific conjugation of native and natural proteins are starting to thrive, allowing the controlled functionalization of various types of amino acid residues. Pursuing the efforts in this field, we planned to develop a new type of site-selective method, aiming at the simultaneous conjugation of two amino acid residues. We reasoned that this should give higher chances of developing a site-selective strategy compared to the great majority of existing methods that solely target a single residue. We opted for the Ugi four-centre three-component reaction to implement this idea, with the aim of conjugating the side-chain amine and carboxylate groups of two neighbouring lysine and aspartate/glutamate. Herein, we show that this strategy can give access to valuable antibody conjugates bearing several different payloads; furthermore, the approach limits the potential conjugation sites to only six on the model antibody trastuzumab.

On the use of DNA as a linker in antibody-drug conjugates: synthesis, stability and in vitro potency.

Here we present the synthesis and evaluation of antibody-drug conjugates (ADCs), for which antibody and drug are non-covalently connected using complementary DNA linkers. These ADCs are composed of trastuzumab, an antibody targeting HER2 receptors overexpressed on breast cancer cells, and monomethyl auristatin E (MMAE) as a drug payload. In this new ADC format, trastuzumab conjugated to a 37-mer oligonucleotide (ON) was prepared and hybridized with its complementary ON modified at 5-end with MMAE (cON-MMAE) in order to obtain trastuzumab-DNA-MMAE. As an advantage, the cON-MMAE was completely soluble in water, which decreases overall hydrophobicity of toxic payload, an important characteristic of ADCs. The stability in the human plasma of these non-engineered ON-based linkers was investigated and showed a satisfactory half-life of 5.8 days for the trastuzumab-DNA format. Finally, we investigated the in vitro cytotoxicity profile of both the DNA-linked ADC and the ON-drug conjugates and compared them with classical covalently linked ADC. Interestingly, we found increased cytotoxicity for MMAE compared to cON-MMAE and an EC50 in the nanomolar range for trastuzumab-DNA-MMAE on HER2-positive cells. Although this proved to be less potent than classically linked ADC with picomolar range EC50, the difference in cytotoxicity between naked payload and conjugated payload was significant when an ON linker was used. We also observed an interesting increase in cytotoxicity of trastuzumab-DNA-MMAE on HER2-negative cells. This was attributed to enhanced non-specific interaction triggered by the DNA strand as it could be confirmed using ligand tracer assay.

Acyl Fluorides: Fast, Efficient, and Versatile Lysine-Based Protein Conjugation via Plug-and-Play Strategy.

We report a plug-and-play strategy for the preparation of functionally enhanced antibodies with a defined average degree of conjugation (DoC). The first stage (plug) allows the controllable and efficient installation of azide groups on lysine residues of a native antibody using 4-azidobenzoyl fluoride. The second step (play) allows for versatile antibody functionalization with a single payload or combination of payloads, such as a toxin, a fluorophore, or an oligonucleotide, via copper-free strain-promoted azide-alkyne cycloaddition (SPAAC). It is notable that in comparison to a classical N-hydroxysuccinimide ester (NHS) strategy, benzoyl fluorides show faster and more efficient acylation of lysine residues in a PBS buffer. This translates into better control of the DoC and enables the efficient and fast functionalization of delicate biomolecules at low temperature.