Effect of digestion on stability of palivizumab IgG1 in the infant gastrointestinal tract.

BACKGROUND: Potentially, orally administered antibodies specific to enteric pathogens could be administered to infants to prevent diarrheal infections, particularly in developing countries where diarrhea is a major problem. However, to prevent infection, such antibodies would need to resist degradation within the gastrointestinal tract. METHODS: Palivizumab, a recombinant antibody specific to respiratory syncytial virus (RSV), was used in this study as a model for examining the digestion of neutralizing antibodies to enteric pathogens in infants. The survival of this recombinant IgG1 across digestion in 11 infants was assayed via an anti-idiotype ELISA and RSV F protein-specific ELISA. Concentrations were controlled for any dilution or concentration that occurred in the digestive system using mass spectrometry-based quantification of co-administered, orally supplemented, indigestible polyethylene glycol (PEG-28). RESULTS: Binding activity of Palivizumab IgG1 decreased (26-99%) across each phase of in vivo digestion as measured by both anti-idiotype and RSV F protein-specific ELISAs. CONCLUSION: Antibodies generated for passive protection of the infant gastrointestinal tract from pathogens will need to be more resistant to digestion than the model antibody fed to infants in this study, or provided in higher doses to be most effective. IMPACT: Binding activity of palivizumab IgG1 decreased (26-99%) across each phase of in vivo infant digestion as measured by both anti-idiotype and RSV F protein-specific ELISAs. Palivizumab was likely degraded by proteases and changes in pH introduced in the gut. Antibodies generated for passive protection of the infant gastrointestinal tract from pathogens will need to be more resistant to digestion than the model antibody fed to infants in this study, or provided in higher doses to be most effective. The monoclonal antibody IgG1 tested was not stable across the infant gastrointestinal tract. The observation of palivizumab reduction was unlikely due to dilution in the gastrointestinal tract. The results of this work hint that provision of antibody could be effective in preventing enteric pathogen infection in infants. Orally delivered recombinant antibodies will need to either be dosed at high levels to compensate for digestive losses or be engineered to better resist digestion. Provision of enteric pathogen-specific recombinant antibodies to at-risk infants could provide a new and previously unexplored pathway to reducing the infection in infants. The strategy of enteric recombinant antibodies deserves more investigation throughout medicine as a novel means for treatment of enteric disease targets.

Survival of recombinant monoclonal and naturally-occurring human milk immunoglobulins A and G specific to respiratory syncytial virus F protein across simulated human infant gastrointestinal digestion.

To help rationally design an antibody for oral administration, we examined how different isotypes (IgG, IgA and sIgA) with the same variable sequence affect antibody stability across digestion. We compared the degradation of recombinant palivizumab (IgG1), and recombinant IgA and sIgA versions of palivizumab spiked in human milk to the degradation of naturally-occurring anti-respiratory syncytial virus (RSV) sIgA/IgA and IgG in human milk from four donors across gastric and intestinal phases of an in vitro model of infant digestion via a validated RSV F protein ELISA. Palivizumab IgG and IgA formats were less stable than the sIgA version after complete simulated gastrointestinal digestion: palivizumab IgG, IgA and sIgA decreased across complete simulated gastrointestinal digestion by 55%, 48% and 28%, respectively. Naturally-occurring RSV F protein-specific IgG was stable across digestion, whereas naturally-occurring sIgA/IgA was stable in the gastric phase but decreased 33% in the intestinal phase of simulated digestion.

Purification of Antibodies From Human Milk and Infant Digestates for Viral Inhibition Assays.

Oral administration of enteric pathogen-specific immunoglobulins may be an ideal approach for preventing infectious diarrhea in infants and children. For oral administration to be effective, antibodies must survive functionally intact within the highly proteolytic digestive tract. As an initial step toward assessing the viability of this approach, we examined the survival of palivizumab, a recombinant monoclonal antibody (IgG1κ), across infant digestion and its ability to neutralize respiratory syncytial virus (RSV). Human milk and infant digestive samples contain substances known to interfere with the RSV neutralization assay (our selected functional test for antibody survival through digestion), therefore, antibody extraction from the matrix was required prior to performing the assay. The efficacy of various approaches for palivizumab purification from human milk, infant's gastric and intestinal digestates, including casein precipitation, salting out, molecular weight cut-off, and affinity chromatography (protein A and G) were compared. Affinity chromatography using protein G with high-salt elution followed by 30-kDa molecular weight cut-off centrifugal filtration was the most effective technique for purifying palivizumab from human milk and infant digestates with a high yield and reduced background interference for the viral neutralization assay. This work is broadly applicable to the optimal isolation of antibodies from human milk and infant digesta for viral neutralization assays, enables the examination of how digestion affects the viral neutralization capacity of antibodies within milk and digestive samples, and paves the way for assessment of the viability of oral administration of recombinant antibodies as a therapeutic approach to prevent enteric pathogen-induced infectious diarrhea in infants.

Partial Degradation of Recombinant Antibody Functional Activity During Infant Gastrointestinal Digestion: Implications for Oral Antibody Supplementation.

Oral administration of engineered immunoglobulins has the potential to prevent enteric pathogen-induced diarrhea in infants. To prevent infection, these antibodies need to survive functionally intact in the proteolytic environment of the gastrointestinal tract. This research examined both ex vivo and in vivo the functional survival across infant digestion of palivizumab, a model FDA-approved recombinant antibody against respiratory syncytial virus (RSV) F protein. Palivizumab-fortified feed (formula or human milk), infant gastric, and intestinal samples were incubated to simulate in vivo digestion (ex vivo digestion). Palivizumab-fortified human milk was also fed to infants, followed by collection of gastric and intestinal samples (in vivo digestion). Palivizumab was purified from the samples of digestate using protein G spin columns followed by filtration through molecular weight cut-off membranes (30 kDa). Palivizumab functional survival across ex vivo and in vivo digestion was determined via an anti-idiotype ELISA and an RSV plaque reduction neutralization test. Palivizumab concentration and RSV neutralization capacity both decreased when incubated in intestinal samples (ex vivo study). The concentration and neutralization activity of orally-supplemented palivizumab also decreased across infant digestion (in vivo study). These results indicate that if recombinant IgGs were selected for oral supplementation to prevent enteric infections, appropriate dosing would need to account for degradation occurring in the digestive system. Other antibody formats, structural changes, or encapsulation could enhance survival in the infant gastrointestinal tract.

Quantitative Analysis of Antibody Survival across the Infant Digestive Tract Using Mass Spectrometry with Parallel Reaction Monitoring.

Orally delivered antibodies may be useful for the prevention of enteric pathogen infection, but to be effective they need to survive intact across digestion through the gastrointestinal tract. As a test case, we fed a recombinant human antibody, palivizumab, spiked into human milk to four infants and collected gastric, intestinal and stool samples. We identified a tryptic peptide from palivizumab (LLIYDTSK) that differs from all endogenous human antibodies and used this for quantitation of the intact palivizumab. To account for dilution by digestive fluids, we co-fed a non-digestible, non-absorbable molecule-polyethylene glycol 28-quantified it in each sample and used this value to normalize the observed palivizumab concentration. The palivizumab peptide, a stable isotope-labeled synthetic peptide and polyethylene glycol 28 were quantified via a highly sensitive and selective parallel-reaction monitoring approach using nano-liquid chromatography/Orbitrap mass spectrometry. On average, the survival of intact palivizumab from the feed to the stomach, upper small intestine and stool were 88.4%, 30.0% and 5.2%, respectively. This approach allowed clear determination of the extent to which palivizumab was degraded within the infant digestive tract. This method can be applied with some modifications to study the digestion of any protein.

Survival of Recombinant Monoclonal Antibodies (IgG, IgA and sIgA) Versus Naturally-Occurring Antibodies (IgG and sIgA/IgA) in an Ex Vivo Infant Digestion Model.

To prevent infectious diarrhea in infants, orally-supplemented enteric pathogen-specific recombinant antibodies would need to resist degradation in the gastrointestinal tract. Palivizumab, a recombinant antibody specific to respiratory syncytial virus (RSV), was used as a model to assess the digestion of neutralizing antibodies in infant digestion. The aim was to determine the remaining binding activity of RSV F protein-specific monoclonal and naturally-occurring immunoglobulins (Ig) in different isoforms (IgG, IgA, and sIgA) across an ex vivo model of infant digestion. RSV F protein-specific monoclonal immunoglobulins (IgG, IgA, and sIgA) and milk-derived naturally-occurring Ig (IgG and sIgA/IgA) were exposed to an ex vivo model of digestion using digestive samples from infants (gastric and intestinal samples). The survival of each antibody was tested via an RSV F protein-specific ELISA. Ex vivo gastric and intestinal digestion degraded palivizumab IgG, IgA, and sIgA (p < 0.05). However, the naturally-occurring RSV F protein-specific IgG and sIgA/IgA found in human milk were stable across gastric and intestinal ex vivo digestion. The structural differences between recombinant and naturally-occurring antibodies need to be closely examined to guide future design of recombinant antibodies with increased stability for use in the gastrointestinal tract.