Introduction

Avastin® (bevacizumab) is an antibody widely used in antitumor therapy with the underlying principle of neutralizing vascular endothelial growth factor (VEGF) thus moderating tumor growth. Although no final assessment of the effectiveness of bevacizumab in large cohorts of cancer patients has been published, it can be considered a therapeutic antibody with high potential [1]-[5].

Fatal adverse effects, however, have been reported in tumor patients treated with this protein drug and in according to a meta-analysis, bevacizumab therapy was associated with increased treatment-related mortality [6].

More specifically, bevacizumab treatment may significantly increase the risk of serious hemorrhage, hypertension, proteinuria, cardiac toxicity, vascular thromboembolism, gastrointestinal, dermatological and endocrine toxicity in cancer patients [7]-[15]. According to the statement by Stone et al. [10], toxicity management in treatment with anti-angiogenic agents has not been an endpoint in most studies carried out up to now and there is therefore a fundamental need for investigations that will generate more evidence-based practice guidelines.

Vascular side effects may be readily assigned to biological activity of VEGF inhibition directly whereas others may be due to other mechanisms, including probable modifications of this humanized antibody. No information on Avastin® protein modifications is available so far and the only systematic mass spectrometrical approach analysing Avastin® was not designed to characterise protein modifications but rather to determine stability [16].

The absence of this information formed the rationale for the current study with the aims to identify and characterise Avastin protein and protein modifications in order to form the basis for studies linking protein modifications to adverse side effects. And indeed, heavy tyrosine nitration, known to modify protein properties and functions, was observed in the commercially available medical product.

Materials and Methods

Avastin® was purchased from Roche, Basel, Switzerland, as manufactured by Genentech, Inc., San Francisco, USA (Batch number H0102B01).

Samples of 100 μg protein were applied on immobilized pH 3–10 nonlinear gradient strips. Focusing started at 200 V and the voltage was gradually increased to 8,000 V at 4 V/min and kept constant for a further 3 h (approximately 150,000 Vh totally). Prior to the second dimensional run, strips were equilibrated twice for 15 min with gentle shaking in 10 mL of SDS equilibration buffer (50 mM, pH 8.8, Tris-HCl, 6 M urea, 30% v/v glycerol, 2% w/v SDS, trace of bromophenol blue). DTT (1% w/v) was added at the first incubation for 15 min and 4% (w/v) iodoacetamide instead of DTT at the second incubation step for 15 min. The second-dimensional separation was performed on 10–16% gradient SDS-PAGE. After protein fixation for 12 h in 50% methanol and 10% acetic acid, gels were stained with colloidal Coomassie blue (Novex, San Diego, CA, USA) for 8 h and excess of dye was washed out from the gels with distilled water. Apparent molecular weights were determined by running precision protein standard markers (Bio-Rad Laboratories, Hercules, CA, USA), covering the range of 10–250 kDa and isoelectric points of the immobilized pH gradient strips were from 3-10 [17].

In-gel digestion

Selected gel spots were picked for the investigation (Fig. 1). Gel pieces were put into a 1.5 mL tube and washed with 10 mM ammonium bicarbonate and 50% acetonitrile (ACN) in 10 mM ammonium bicarbonate repeatedly. Addition of ACN resulted in gel shrinking and the shrunk gel plugs were then dried in a Speedvac Concentrator (Eppendorf, Germany). Dried gel pieces were re-swollen and in-gel digested with 40 ng/μL trypsin (Promega, Madison, WI, USA) in digestion buffer (consisting of 5 mM octyl β-D-glucopyranoside (OGP) and 10 mM ammonium bicarbonate, pH 7.8) and incubated overnight at 37°C. Digestion with chymotrypsin (Roche Diagnostics), 25 ng/μL was done in 25 mM NH₄HCO₃ with 5 mM OGP (pH 7.8) at 30°C for 4 h. Digestion with 40 ng/μL Pepsin (Roche Diagnostics) was done in 0.1M HCL (pH 1) at 37°C for 4 h.

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Figure 1. 2-DE image of Avastin® is shown providing assignments of identified protein spots.

https://doi.org/10.1371/journal.pone.0034511.g001

Peptide extractions were performed with 15 μL of 1% formic acid (FA) in 5 mM OGP for 30 min, 15 μL 0.1% FA for 30 min and 15 μL 0.1% FA in 20% ACN for 30 min. The extracted peptides were pooled for nano-LC-ESI-CID/ETD-MS/MS or LTQ orbitrap MS/MS analyses.

Results and Discussion

As shown in Fig. 1 the commercially available protein drug Avastin® was presenting with several spots. Heavy and light chain were clearly separated and heavy chain protein showed tracking while the light chain showed three major spots. Seven spots were picked and sequence coverages, enzyme that generated matching peptides, peptide sequences, ion scores and mass errors that were less than 0.2 Da are provided in Table S1 and Figure S1. Only spots 2,3,6 and 7 were presenting with tyrosine nitrations. The proposed multiple protein modifications (PMs) rather than sequence conflicts or amino acid exchanges observed in the individual spots resulting from HCT analysis respecting CID and ETD fragmentations may have been responsible for horizontal and vertical electrophoretic shifts as given in Table S2A.

LTQ-orbitrap analyses were used to find additional PMs in some Avastin® spots, and showed the presence of PMs and the results are demonstrated in Table S2B. PMs observed by both methods are presented in Table 1. Results on different modifications of avastin® expression forms are assigned to spot numbers.

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Table 1. The modifications verified by HCT and Orbitrap.

https://doi.org/10.1371/journal.pone.0034511.t001

Table 2 shows tyrosine nitrations shown by ion trap using CID but not by ETD fragmentation as well as using LTQ orbitrap.

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Table 2. Identified nitrotyrosine or aminotyrosine modifications in Avastin by HCT and Orbitrap: the software proposes possible modifications on the peptide and subsequently assigns them to “measured m/z”. The theoretical m/z indicates the peptide mass with expected modifications.

https://doi.org/10.1371/journal.pone.0034511.t002

On the light chain as well as on the heavy chain of the antibody (Avastin®) LTQ identified this PM. HCT analysis demonstrated two tyrosine nitrations on the light chain and three at the heavy chain. LTQ orbitrap detected five tyrosine nitrations on the light chain and four on the heavy chain. Nitration was observed as nitration or as aminotyrosine [20]. Both MS/MS methods detected nitration of Y192 expressed by nitrotyrosine and aminotyrosine identification.

To support the identification of nitrotyrosine, Avastin® was reduced by sodium hydrosulfite and indeed, aminotyrosine on Y192 was detected (Figure S2), which does not rule out that the other modifications are not valid: results from different methodologies depend on fragmentation and formation of corresponding ions generated. Misidentifications of tyrosine nitration by a series of factors was ruled out [21].

Multiple nitrations of the humanised antibody Avastin® may have chemical and biological consequences: potential changes of Avastin immunochemistry may include antigenic changes and nitration has been repeatedly shown to modify this immunological property and was even leading to autoimmune phenomena [22]. The underlying cause of nitrations cannot be addressed but chemical modification by manufacturing is as plausible as nitration that occurred in vivo during primary generation of the antibody as a posttranslational modification [23]. Details from manufacturing and production are not available from the supplier or the corresponding patent (6,054,297 US patent, 2000).

Antibodies against nitrotyrosine have been observed [24] and it is known that nitrated peptides and proteins are eliciting antibody formation [25]-[27]. Moreover, antibodies against nitrated alpha synuclein produced an inflammatory response in mice that led to degeneration of dopaminergic neurons [28]. One may also suggest that nitration not only leads to autoantibody formation but also to conformational changes of the nitrated protein [29] that in turn may change antigenic properties and also protein-protein interactions with unknown functional consequences. Apart from nitration-induced antigenic changes of epitopes, nitration is known to impair function of proteins including Mn superoxide dismutase [30], [31].

A major finding indicating pathogenetic or toxic properties of nitrated proteins was presented recently: assembly of alpha-synuclein and fibrinogen were deteriorated by nitration of only a small fraction of proteins [25], [32]. As for fibrinogen, nitration is accelerating the rate of fibrin clot formation [33]. It cannot be ruled out, therefore, that nitration of Avastin® may lead to side effects based upon probable disturbed protein-protein interactions as aggregations that in turn may lead to a series of complications as listed in the drug information sheet.

As to the underlying cause of tyrosine nitration it remains open, whether it can be considered as a post-translational modification of the immunoglobulin Avastin® or technical in nature, or both [34]-[38].

Taken together, it remains unclear if the observed nitrations and other PMs detected on Avastin® may be responsible for different biological or pharmacotoxicological properties and effects. Quantification of nitrations and their presence in several batches of the product has to be taken into considerations on probable toxicity as well, but corresponding in vivo analyses based upon these findings should be carried out by the manufacturers to probably save this protein drug that may have strong potential in tumor therapy.

Supporting Information