作者: Suzuki, A ; Yoshioka, H ; Tedeschini, T ; Pasut, G ; Campara, B ; Matsuno, Y ; Grigoletto, A ; Cannella, L ; Zanotto, I ; Armirotti, A ; Gabbia, D ; De Martin, S

In this work, we conceived and developed antibody-drug conjugates (ADCs) that could efficiently release the drug after enzymatic cleavage of the linker moiety by tumoral proteases. The antibody-drug linkers we used are the result of a rational optimization of a previously reported PEGylated linker, PUREBRIGHT® MA-P12-PS, which showed excellent drug loading capacities but lacked an inbuilt drug discharge mechanism, thus limiting the potency of the resulting ADCs. To address this limitation, we chose to incorporate a protease-sensitive trigger into the linker to favor the release of a "PEGless" drug inside the tumor cells and, therefore, obtain potent ADCs. Currently, most marketed ADCs are based on the Val-Cit dipeptide followed by a self-immolative spacer for releasing the drug in its unmodified form. Here, we selected two untraditional peptide sequences, a Phe-Gly dipeptide and a Val-Ala-Gly tripeptide and placed one or the other in between the drug on one side (N-terminus) and the rest of the linker, including the PEG moiety, on the other side (C-terminus), without a self-immolative group. We found that both linkers responded to cathepsin B, a reference lysosomal enzyme, and liberated a PEG-free drug catabolite, as desired. We then used the two linkers to generate ADCs based on trastuzumab (a HER2-targeting antibody) and DM1 (a microtubule-targeted cytotoxic agent) with an average drug-to-antibody ratio (DAR) of 4 or 8. The ADCs showed restored cytotoxicity in vitro, which was proportional to the DM1 loading and generally higher for the ADCs bearing Val-Ala-Gly in their structure. In an ovarian cancer mouse model, the DAR 8 ADC based on Val-Ala-Gly behaved better than Kadcyla® (an approved ADC of DAR 3.5 used as control throughout this study), leading to a higher tumor volume reduction and more prolonged median survival. Taken together, our results depict a successful linker optimization process and encourage the application of the Val-Ala-Gly tripeptide as an alternative to other existing protease-sensitive triggers for ADCs.

2024-09-01·Bulletin of Experimental Biology and Medicine

Effects of Neonatal Administration of Non-Opiate Analogues of Leu-Enkephalin on the Delayed Cardiac Consequences of Intrauterine Hypoxia

Article

作者: Malofey, Yu. B. ; Gusev, I. A. ; Sazonova, E. N. ; Malofey, Yu B ; Gusev, I A ; Sazonova, E N

Intrauterine hypoxia (gestation days 15-19, pO₂ 65 mm Hg, duration 4 h) led to an increase in the expression of p53, beclin-1, endothelial NO synthase (eNOS), and caspase-3 proteins in cardiomyocytes and reduced the number of mast cells in the heart of 60-day-old albino rats. Administration of a non-opiate analogue of leu-enkephalin (NALE peptide: Phe-D-Ala-Gly-Phe-Leu-Arg, 100 μg/kg) on days 2-6 of the neonatal period decreased the severity of delayed posthypoxic myocardial reaction. The content of eNOS⁺ cardiomyocytes and the total number of mast cells of these animals did not differ from the control parameters; the content of p53⁺ cardiomyocytes was significantly lower than in animals exposed to intrauterine hypoxia. The cardioprotective activity of NALE was partially neutralized by co-administration with the NO synthase inhibitor (L-NAME, 50 mg/kg). Correction of the delayed posthypoxic changes, similar to the effects of NALE peptide, was observed after neonatal administration of its arginine-free analogue, G peptide (Phe-D-Ala-Gly-Phe-Leu-Gly; 100 μg/kg). Non-opiate analogues of leu-enkephalin NALE and G peptides can be considered as promising substances capable of preventing long-term cardiac consequences of intrauterine hypoxia.

2024-09-01·Metabolic Engineering

Ala-Cys-Cys-Ala dipeptide dimer alleviates problematic cysteine and cystine levels in media formulations and enhances CHO cell growth and metabolism

Article

作者: Ladiwala, Pranay ; Naik, Harnish Mukesh ; Antoniewicz, Maciek R ; Schilling, Martin ; Aliyu, Lateef ; Betenbaugh, Michael J ; Cai, Xiangchen

Cysteine and cystine are essential amino acids present in mammalian cell cultures. While contributing to biomass synthesis, recombinant protein production, and antioxidant defense mechanisms, cysteine poses a major challenge in media formulations owing to its poor stability and oxidation to cystine, a cysteine dimer. Due to its poor solubility, cystine can cause precipitation of feed media, formation of undesired products, and consequently, reduce cysteine bioavailability. In this study, a highly soluble cysteine containing dipeptide dimer, Ala-Cys-Cys-Ala (ACCA), was evaluated as a suitable alternative to cysteine and cystine in CHO cell cultures. Replacing cysteine and cystine in basal medium with ACCA did not sustain cell growth. However, addition of ACCA at 4 mM and 8 mM to basal medium containing cysteine and cystine boosted cell growth up to 15% and 27% in CHO-GS and CHO-K1 batch cell cultures respectively and led to a proportionate increase in IgG titer. ¹³C-Metabolic flux analysis revealed that supplementation of ACCA reduced glycolytic fluxes by 20% leading to more efficient glucose metabolism in CHO-K1 cells. In fed-batch cultures, ACCA was able to replace cysteine and cystine in feed medium. Furthermore, supplementation of ACCA at high concentrations in basal medium eliminated the need for any cysteine equivalents in feed medium and increased cell densities and viabilities in fed-batch cultures without any significant impact on IgG charge variants. Taken together, this study demonstrates the potential of ACCA to improve CHO cell growth, productivity, and metabolism while also facilitating the formulation of cysteine- and cystine-free feed media. Such alternatives to cysteine and cystine will pave the way for enhanced biomanufacturing by increasing cell densities in culture and extending the storage of highly concentrated feed media as part of achieving intensified bioproduction processes.

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适应症	最高研发状态	国家/地区	公司	日期
视网膜色素变性	临床3期	澳大利亚	BioDiem Ltd.	-
糖尿病性黄斑水肿	临床2期	-	Biodem	-
黄斑变性	临床1期	澳大利亚	-	-
早产儿视网膜病变	临床1期	美国	University of Wisconsin-Milwaukee	-
早产儿视网膜病变	临床1期	澳大利亚	-	-