Role of the methoxy group in immune responses to mPEG-protein conjugates.

Anti-PEG antibodies have been reported to mediate the accelerated clearance of PEG-conjugated proteins and liposomes, all of which contain methoxyPEG (mPEG). The goal of this research was to assess the role of the methoxy group in the immune responses to mPEG conjugates and the potential advantages of replacing mPEG with hydroxyPEG (HO-PEG). Rabbits were immunized with mPEG, HO-PEG, or t-butoxyPEG (t-BuO-PEG) conjugates of human serum albumin, human interferon-α, or porcine uricase as adjuvant emulsions. Assay plates for enzyme-linked immunosorbent assays (ELISAs) were coated with mPEG, HO-PEG, or t-BuO-PEG conjugates of the non-cross-reacting protein, porcine superoxide dismutase (SOD). In sera from rabbits immunized with HO-PEG conjugates of interferon-α or uricase, the ratio of titers of anti-PEG antibodies detected on mPEG-SOD over HO-PEG-SOD ("relative titer") had a median of 1.1 (range 0.9-1.5). In contrast, sera from rabbits immunized with mPEG conjugates of three proteins had relative titers with a median of 3.0 (range 1.1-20). Analyses of sera from rabbits immunized with t-BuO-PEG-albumin showed that t-butoxy groups are more immunogenic than methoxy groups. Adding Tween 20 or Tween 80 to buffers used to wash the assay plates, as is often done in ELISAs, greatly reduced the sensitivity of detection of anti-PEG antibodies. Competitive ELISAs revealed that the affinities of antibodies raised against mPEG-uricase were c. 70 times higher for 10 kDa mPEG than for 10 kDa PEG diol and that anti-PEG antibodies raised against mPEG conjugates of three proteins had >1000 times higher affinities for albumin conjugates with c. 20 mPEGs than for analogous HO-PEG-albumin conjugates. Overall, these results are consistent with the hypothesis that antibodies with high affinity for methoxy groups contribute to the loss of efficacy of mPEG conjugates, especially if multiply-PEGylated. Using monofunctionally activated HO-PEG instead of mPEG in preparing conjugates for clinical use might decrease this undesirable effect.

The role of excited Rydberg States in electron transfer dissociation.

Ab initio electronic structure methods are used to estimate the cross sections for electron transfer from donor anions having electron binding energies ranging from 0.001 to 0.6 eV to each of three sites in a model disulfide-linked molecular cation. The three sites are (1) the S-S sigma(*) orbital to which electron attachment is rendered exothermic by Coulomb stabilization from the nearby positive site, (2) the ground Rydberg orbital of the -NH(3)(+) site, and (3) excited Rydberg orbitals of the same -NH(3)(+) site. It is found that attachment to the ground Rydberg orbital has a somewhat higher cross section than attachment to either the sigma orbital or the excited Rydberg orbital. However, it is through attachment either to the sigma(*) orbital or to certain excited Rydberg orbitals that cleavage of the S-S bond is most likely to occur. Attachment to the sigma(*) orbital causes prompt cleavage because the sigma energy surface is repulsive (except at very long range). Attachment to the ground or excited Rydberg state causes the S-S bond to rupture only once a through-bond electron transfer from the Rydberg orbital to the S-S sigma(*) orbital takes place. For the ground Rydberg state, this transfer requires surmounting an approximately 0.4 eV barrier that renders the S-S bond cleavage rate slow. However, for the excited Rydberg state, the intramolecular electron transfer has a much smaller barrier and is prompt.

Selectivity of binding of PEGs and PEG-like oligomers to anti-PEG antibodies induced by methoxyPEG-proteins.

The use of methoxypoly(ethylene glycol) (mPEG) in PEG conjugates of proteins and non-protein therapeutic agents has led to the recognition that the polymer components of such conjugates can induce anti-PEG antibodies (anti-PEGs) that may accelerate the clearance and reduce the efficacy of the conjugates. Others have classified anti-PEGs as "methoxy-specific" or "backbone-specific". The results of our previous research on anti-PEGs in the sera of rabbits immunized with mPEG or hydroxyPEG (HO-PEG) conjugates of three unrelated proteins were consistent with that classification (Sherman, M.R., et al., 2012. Bioconjug. Chem. 23, 485-499). Enzyme-linked immunosorbent assays (ELISAs) were performed on rabbit antisera and rabbit monoclonal anti-PEGs with competitors including 10 kDa mPEG, 10 kDa PEG diol and six linear or cyclic oligomers of oxyethylene (CH2CH2O), with molecular weights of ca. 150-264 Da. Our results demonstrate that (1) the binding affinities of anti-mPEGs depend more on the backbone lengths of the polymers and the hydrophobicities of their end-groups than on their resemblance to the methoxy terminus of the immunogenic polymer; (2) anti-PEGs raised against HO-PEG-proteins are not directed against the terminal hydroxy group, but against the backbone; (3) rabbit anti-PEGs bind to and distinguish among PEG-like oligomers with as few as three oxyethylene groups; and (4) none of the monoclonal or polyclonal anti-PEGs was absolutely "methoxy-specific" or "backbone-specific", but displayed distinct relative selectivities. If these results are relevant to human immune responses, the clinical use of stable conjugates of HO-PEG with proteins and non-protein therapeutic agents would be expected to produce fewer and less intense immune responses than those induced by conjugates with mPEG or PEGs with larger alkoxy groups.

A theoretical model for indirect dissociative electron attachment.

In this paper, we describe a computational model that allows us to avoid having to perform a very large number of tedious calculations on electronically metastable anions when studying indirect DEA processes. By indirect, we mean that the electron attaches to an orbital in one region of the molecule but a bond is subsequently broken in another region. For such events, one must describe the coupling between two diabatic anion states, corresponding to the occupation of orbitals in the two regions of the molecule, to achieve a correct description. We introduce a simple 2 x 2 matrix model as well as physically reasonable and computationally efficient approximations to the diabatic states in regions where they are metastable. We show this model to be highly effective when applied to several indirect DEA processes that we studied earlier with brute-force methods. The main advantage of using this model is that one can avoid having to carry out a large number of calculations on metastable anion states; only one or two such calculations are required.

Coulomb-assisted dissociative electron attachment: application to a model peptide.

The fragmentation of positively charged gas-phase samples of peptides is used to infer the primary structure of such molecules. In electron capture dissociation (ECD) experiments, very low-energy electrons attach to the sample and rupture bonds to effect the fragmentation. It turns out that ECD fragmentation tends to produce cleavage of very specific types of bonds. In earlier works by this group, it has been suggested that the presence of positive charges produces stabilizing Coulomb potentials that allow low-energy electrons to exothermically attach to sigma orbitals of certain bonds and thus to cleave those bonds. In the present effort, the stabilizing effects of Coulomb potentials due to proximal positive charges are examined for a small model peptide molecule that contains a wide range of bond types. Direct attachment of an electron to the sigma orbitals of eight different bonds as well as indirect sigma bond cleavage, in which an electron first binds to a carbonyl C=O pi orbital, are examined using ab initio methods. It is found that direct attachment to and subsequent cleavage of any of the eight sigma bonds is not likely except for highly positively charged samples. It is also found that attachment to a C=O pi orbital followed by cleavage of the nitrogen-to-alpha-carbon bond is the most likely outcome. Interestingly, this bond cleavage is the one that is seen most commonly in ECD experiments. So, the results presented here seem to offer good insight into one aspect of the ECD process, and they provide a means by which one can estimate (on the basis of a simple Coulomb energy formula) which bonds may be susceptible to cleavage by low-energy electron attachment.

Charge loss in gas-phase multiply negatively charged oligonucleotides.

In an attempt to shed light on the mechanism by which gaseous samples of negatively charged oligonucleotides undergo extremely slow (i.e., over 1-1000 s) charge loss, we have carried out molecular dynamics simulations on an oligonucleotide anion, T(5)(3-), containing five thymine, deoxyribose, and phosphate units in which the first, third, and fifth phosphates are negatively charged. The study is aimed at determining the rate at which an electron is detached from such a trianion by way of an internal Coulomb repulsion induced event. In this process, the intrinsic 5.0-5.1 eV electron binding strength of each phosphate site is reduced by the repulsive Coulomb potentials of the other two negative sites. As geometrical fluctuations cause the distances among the three negative phosphate sites to change, this causes the Coulomb repulsion energies at these sites to fluctuate. Once the Coulomb potential at any phosphate site exceeds ca. 5 eV, the electron on that site is able to undergo autodetachment. Although such an electron must tunnel through a barrier to escape, it is shown that the tunneling rate is not the rate-limiting step in electron loss; instead, it is the rate at which geometrical fluctuations cause the Coulomb potentials to exceed 5 eV that determines the rate of electron loss. Because these rates are extremely slow, special techniques had to be introduced to allow results of dynamics simulations on more flexible models of T(5)(3-) to be extrapolated to predict the behavior of the actual T(5)(3-).