الفهرس 
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Abstract
Pre-existing ‘natural’ anti-PEG antibodies were extensively found in about 45% of healthy population. Due to such widespread of anti-PEG antibodies, the clinical efficacy of PEGylated therapeutic agents might be compromised. This usually occurs by the so called “accelerated blood clearance” (ABC) phenomenon. The ABC phenomenon was primarily attributed to the interaction of anti-PEG antibodies with the PEG moiety anchored on PEGylated product(s) followed by substantial complement activation. Several clinically approved PEGylated products lost their extended circulation because of high occurrence of anti-PEG antibodies. Circulating anti-PEG antibodies were accompanied with loss of PEGasparaginase and Pegloticase activity. The ABC phenomenon was extensively studied in animal models during the last two decades. PEGylated liposomes (PEG-Lip) lost longer duration when administered twice in the same mice or rat during the seroconversion time.
In addition to ABC, complement activation mediates infusion reactions which represent a major obstacle against the development and the wide use of several therapeutic agents. Since complement activation related pseudo allergy (CARPA) is characterized with immediate hypersensitivity reactions and entails broad manifestations like pulmonary distress, skin rash, hypo/hypertension, it is one of the best candidates could outline infusion reactions. 7-10% of cancer patients treated with intravenous infusion of PEGylated liposomal doxorubicin developed infusion reactions within a short period of treatment initiation and such symptoms were correlated with complement activation. CARPA symptoms were first initiated through complement activation and might be followed with mast cell activation and cytokine release. Hence, role of complement activation in initiation and exacerbation of infusion reactions was extensively reported over the last two decades. Complement activation was found to be associated with several hematological and hemodynamic changes which are considered markers for CARPA syndrome at different animal species and human. Incidence of mild to severe clinical CARPA symptoms ranged from 2% up to 30% of patients within few minutes of injection of nano PEGylated formulations and might be life-threatening or fatal at limited occasions. Furthermore, anti-PEG antibody-mediated complement activation not only initiated infusion reactions but also resulted in destabilization of drug loaded PEG-Lip membrane and potential toxicity threatening. Binding of PEG-Lip with anti-PEG antibodies induces complement activation and eventually the development of membrane attack complex (C5b-9) which accelerates the release of entrapped drug and evolves a safety hazard. The relationship between anti-PEG antibodies provoked by PEGylated therapeutics, pre-existing or induced, and complement activation is considered pivotal for safety issues. Therefore, understanding the factors affecting/controlling complement activation is of crucial importance to discover means or approaches to overcome or attenuate such factors and accordingly improve the safe use of nano PEGylated formulations particularly PEG-Lip.
Therefore, in this thesis, HIK-M11 monoclonal anti-PEG antibodies (HIK-M11 mAbs), secreted from HIK-M11 hybridoma cells, were selected as anti-PEG antibody model. HIK-M11 mAbs showed higher affinity toward PEG-Lip in vitro and in vivo. Aggregation studies of the interaction between HIK-M11 mAbs and PEG-Lip confirmed the ability of PEG-Lip to form aggregates of larger mean diameter or size in the presence of HIK-M11 mAbs. HIK-M11 mAbs were able to substantially activate the complement system upon interaction with PEG-Lip in vitro and in vivo detected using ELISA and biolayer interferometry (BLI). HIK-M11 mAbs mediated complement activation increased the uptake of PEG-Lip by naïve splenocytes by either the marginal or follicular B-cells. Also, immune complex of PEG-Lip and HIK-M11 mAbs was able to sensitize naïve splenocytes toward IL-6 secretion. In addition, presence of anti-PEG antibodies drastically increased the secretion of IL-6 from whole blood after incubation with PEG-Lip while PEG-Lip only failed to induce IL-6 release.
The effect with different injection rates on the aggregation size of PEG-Lip in the presence of anti-PEG antibodies was extensively studied in the thesis to see its effect on the different responses specially complement activation and it related immune responses. Under all conditions used, the very fast or very slow injection of PEG-Lip into anti-PEG containing solution developed smaller immune aggregates when compared with the intermediate injection rate. Moreover, the different injection rates, at which different aggregations size developed, resulted in different magnitude of complement activation in vitro and in vivo in the presence of anti-PEG antibodies. The very fast or very slow injection/infusion rates of PEG-Lip, at which the smaller aggregates developed, into anti-PEG containing solutions or mice resulted in less magnitude to activate complement system detected using quantification of the level of C3a and sC5b-9 complement split products or western blotting. While intermediate injection/infusion rates, at which the largest aggregates developed, resulted in more increase in the level of complement activation in vitro and in vivo. The intermediate infusion rate of PEG-Lip into HIK-M11 mAbs bearing mice, at which maximum complement activation occurs, resulted in maximum ABC effect, maximum IL-6 release, and maximum thrombocytopenia. While very fast or very slow injection/infusion rates, at which smaller magnitude of complement activation occurs, resulted in less ABC effect, less IL-6 release, and less thrombocytopenia.
Doxil is well known to induce infusion reactions in about 45% while l-OHP PEG-Lip in about 9% of cancer patients. The results obtained correlated the ability of the therapeutic PEGylated liposomes to form aggregates in the existence of the anti-PEG antibodies. Doxil was able to develop larger aggregates in the presence of anti-PEG antibodies than l-OHP PEG-Lip under all conditions specified. The different injection/infusion rates developed aggregates of different mean diameter or size which was identical to that showed using empty PEG-Lip.
By applying the same procedures to PEGylated cationic liposomes loaded with siRNA (PCL-siRNA) resulted in identical results to that obtained using empty PEG-Lip at the different injection/infusion rates in the presence of HIK-M11 mAbs.
In conclusion, anti-PEG antibodies possess a causal role in the development of infusion reactions to PEGylated products. The aggregation of PEG-Lip in the presence of anti-PEG antibodies is the major determinant of the magnitude of complement activation and eventually complement related immune adverse effects.