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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 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.

Isolated a 1 chains of collagen are cleaved by cyanogen bromide into discrete peptides which may be separated chromatographically. One of these peptides containing 36 amino acid residues (al-CB2) has been utilized to study the helix-coil transition characteristic of collagen. It is reversibly transformed at low temperatures to a helical product which has been characterized by optical rotation, circular dichroism, molecular sieve chromatography, and ultracentrifugation. It consists primarily of trimer but may contain small amounts of higher molecular weight aggregates. The circular dichroism S tudies of the mechanism of formation of the triple-helical structure of collagen have encountered difficulties because of the great chain length and the heterogeneity of available samples. We have attempted to simpify the problem by utilizing a relatively small peptide of known amino acid sequence isolated from cyanogen bromide digests of the a 1 chain of rat skin collagen (Bornstein and Piez, 1966). The peptide has the sequence (Bornstein, 1967) Gly-The positions shown as containing hydroxyproline contain varying amounts of proline as a result of incomplete hydroxylation (Bornstein, 1967).Since this sequence is composed of triplets of the form Gly-X-Y and contains one third proline plus hydroxyproline, it would be expected to form a collagen-like helix at sufficiently low temperatures and a random coil' at higher temperatures. Preliminary to the detailed thermodynamic and kinetic analysis of this renaturation process reported in the following paper (Piez and Sherman, 1970), it was necessary to determine the homogeneity of the product, the number of chains utilized in its formation, the nature of the helical structure, and the degree of helicity. These properties have been explored using the Archibald technique of ultracentrifugation, molecular sieve chromatography, and circular dichroism spectroscopy, The term random coil is used here to denote the denatured form of the peptide, characterized by a temperature-insensitive, low-negative optical rotation at 313 nm, and does not imply a Guassian distribution of chain elements. spectrum at 2" is characteristic of a collagen-like structure with a helical content of 90 i 10%. The incomplete helicity may indicate some misalignment of the peptide chains. Molecular sieve chromatography at 5 o reveals the presence of only two forms, monomer and trimer, in concentration-dependent equilibrium. The distribution coefficient for chromatography of the trimer on a standardized polyacrylamide column suggests an asymmetric structure consistent with calculated dimensions of 113 X 11.4 A for a rod-like molecule similar t o collagen. Experimental SectionPreparation of al-CB2. Soluble collagen was obtained from the skin of rats given /3-aminopropionitrile to inhibit collagen cross-linking. The peptide was prepared from cyanogen bromide digests of the a 1 chain of this collagen by chromatography on phosphocellulose as described by Bornstein and Piez (1966). It was further purified as follows. Fract...

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