Identification of Potential Sites for Tryptophan Oxidation in Recombinant Antibodies Using tert-Butylhydroperoxide and Quantitative LC-MS

Amino acid oxidation is known to affect the structure, activity, and rate of degradation of proteins. Methionine oxidation is one of the several chemical degradation pathways for recombinant antibodies. In this study, we have identified for the first time a solvent accessible tryptophan residue (Trp-32) in the complementary-determining region (CDR) of a recombinant IgG1 antibody susceptible to oxidation under real-time storage and elevated temperature conditions. The degree of light chain Trp-32 oxidation was found to be higher than the oxidation level of the conserved heavy chain Met-429 and the heavy chain Met-107 of the recombinant IgG1 antibody HER2, which have already been identified as being solvent accessible and sensitive to chemical oxidation. In order to reduce the time for simultaneous identification and functional evaluation of potential methionine and tryptophan oxidation sites, a test system employing tert-butylhydroperoxide (TBHP) and quantitative LC-MS was developed. The optimized oxidizing conditions allowed us to specifically oxidize the solvent accessible methionine and tryptophan residues that displayed significant oxidation in the real-time stability and elevated temperature study. The achieved degree of tryptophan oxidation was adequate to identify the functional consequence of the tryptophan oxidation by binding studies. In summary, the here presented approach of employing TBHP as oxidizing reagent combined with quantitative LC-MS and binding studies greatly facilitates the efficient identification and functional evaluation of methionine and tryptophan oxidation sites in the CDR of recombinant antibodies

LC-separation UV-profiles of the real-time stability sample (9 months at 4°C) and the corresponding reference material (RS, stored at −70°C) after tryptic cleavage.

<p>The <i>m/z</i>-values of Met-253 and Trp-32 containing tryptic antibody peptides, obtained by LC-ESI-MS, are listed in the inset along with their corresponding LC fraction number. Peak identification and quantification performed by LC-MS. Chromatographic conditions are described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017708#s4" target="_blank">materials and methods</a>.</p

Low-energy CID mass spectra and resulting amino acid sequence of the triply protonated quasi molecular ions of the non-oxidized (upper panel) and oxidized (medium and lower panel) tryptic light chain fragment 25–42 at <i>m/z</i> 703.05, 704.39, and 708.38.

<p>The oxidized Trp-32 residue is highlighted in bold-face.</p

Analysis of antibody target binding by surface plasmon resonance.

<p>Biacore sensorgrams showing the pH 7.0 target binding (100 nM) following different TBHP exposures.</p

Relative quantification of HER2 methionine oxidation (Met-83, Met-107, and Met-255) by ion current chromatogram analysis of the oxidized product and its parent peptide using the quantification software GRAMS/32™.

<p>Relative quantification of HER2 methionine oxidation (Met-83, Met-107, and Met-255) by ion current chromatogram analysis of the oxidized product and its parent peptide using the quantification software GRAMS/32™.</p

Extracted ion chromatograms (EIC) of <i>m/z</i> 703.05, 704.39, and 708.38 from the reference material and the stability sample.

<p>Extracted ion chromatograms (EIC) of <i>m/z</i> 703.05, 704.39, and 708.38 from the reference material and the stability sample.</p

Relative quantification of antibody methionine and tryptophan oxidation (Met-83, Met-253, and Trp-32) by ion current chromatogram analysis of the oxidized product and its parent peptide using the quantification software GRAMS/32TM (columns 1–4).

<p>Formation of fragments and aggregates was monitored by size-exclusion chromatography (Column 5) and target binding was assessed by SPR-analysis (Column 6). Data is represented as mean ± S.D; n.d. not determined.</p

Tumor-targeted 4-1BB agonists for combination with T cell bispecific antibodies as off-the-shelf therapy

Endogenous costimulatory molecules on T cells such as 4-1BB (CD137) can be leveraged for cancer immunotherapy. Systemic administration of agonistic anti-4-1BB antibodies, although effective preclinically, has not advanced to phase 3 trials because they have been hampered by both dependency on Fc gamma receptor-mediated hyperclustering and hepatotoxicity. To overcome these issues, we engineered proteins simultaneously targeting 4-1BB and a tumor stroma or tumor antigen: FAP-4-1BBL (RG7826) and CD19-4-1BBL. In the presence of a T cell receptor signal, they provide potent T cell costimulation strictly dependent on tumor antigen-mediated hyperclustering without systemic activation by Fc gamma R binding. We could show targeting of FAP-4-1BBL to FAP-expressing tumor stroma and lymph nodes in a colorectal cancer-bearing rhesus monkey. Combination of FAP-4-1BBL with tumor antigen-targeted T cell bispecific (TCB) molecules in human tumor samples led to increased IFN-gamma and granzyme B secretion. Further, combination of FAP- or CD19-4-1BBL with CEA-TCB (RG7802) or CD20-TCB (RG6026), respectively, resulted in tumor remission in mouse models, accompanied by intratumoral accumulation of activated effector CD8(+) T cells. FAP- and CD19-4-1BBL thus represent an off-the-shelf combination immunotherapy without requiring genetic modification of effector cells for the treatment of solid and hematological malignancies