Camelidae heavy-chain-only antibodies  have advantages over conventional antibodies in terms of their small size and stability to pH and temperature extremes, their ability to express well in microbial hosts, and to be functionally multimerized for enhanced properties. For these reasons, VHHs are showing promise as enteric disease therapeutics, yet little is known as to their pharmacokinetics (PK) within the digestive tract. To improve understanding of enteric VHH PK, we investigated the functional and structural stability of monomeric and multimeric camelid VHH-agents following in vitro incubation with intestinal extracts (chyme) from rabbits and pigs or fecal extracts from human sources, and in vivo in rabbits. The results showed that unstructured domains such as epitopic tags and flexible spacers composed of different amino acid sequences were rapidly degraded by enteric proteases while the functional core VHHs were much more stable to these treatments. Individual VHHs were widely variable in their functional stability to GI tract proteases. Some VHH-based agents which neutralize enteric Shiga toxin Stx2 displayed a functional stability to chyme incubations comparable to that of Stx2-neutralizing IgG and IgA mAbs, thus indicating that selected nanobodies can approach the functional stability of conventional immunoglobulins. Enteric PK data obtained from in vitro incubation studies were consistent with similar incubations performed in vivo in rabbit surgical gut loops. These findings have broad implications for enteric use of VHH-based agents, particularly VHH fusion proteins.Single-domain antibodies (sdAbs) derived from  Enteric diseases substantially impact health in both human and veterinary medicine. While modern sanitation methods largely control enteric disease in the developed world, food-borne pathogens remain a major cause of disease and death in the developing world, particularly among children. Vaccines and drugs such as antibiotics and anthelmintics are available to prevent and/or treat many enteric diseases but are underutilized in the developing world due to factors such as cost, appearance of drug-resistant pathogens and inadequate medical infrastructure. Antibody therapeutics could also provide enteric disease benefits but are currently costlier and more challenging to distribute and administer than vaccines and small molecule drugs.H  domains offer the potential to overcome some of the obstacles to enteric immunotherapies. VHHs can be identified that bind to pathogen targets with similar affinities to conventional antibodies, yet possess advantages such as reduced production costs, improved stability to temperature and pH extremes, expression as multimers with enhanced properties, and amenability to gene therapies (recently reviewed by [Clostridium difficile infection [Lactobacillus and Lactococcus [Single-domain antibodies (sdAbs) such as Camelid heavy-chain-only Viewed by ). A numbiewed by –4, ETEC iewed by , inflammiewed by ,7 and panfection . To bypanfection  or exprenfection  and probtococcus ,8,11.Despite the successes reported using oral therapeutic VHHs to treat enteric pathology, studies to evaluate the PK of VHHs within the GI tract are very limited. A major limitation to performing these studies is the inability to reliably detect functional VHHs that survive exposures to enteric proteases using routine immunological methods. This is because antibodies may falsely detect epitopes on partially degraded, non-functional VHHs or fail to detect surviving functional VHHs due to selective degradation of the epitopic tag used by the assay. For this reason, enteric studies on VHH PK are best performed by monitoring the survival of a specific VHH function in place of immunological methods. For example, Maffey et al  monitorein vitro studies that characterize the stability of VHH-based agents to enteric proteases. The studies were done primarily employing functional assays to assess VHH monomer and heterodimer survival to enteric protease exposure. We developed and characterized a rabbit anti-VHH serum that we demonstrate to be capable of improved core VHH recognition in ELISA and western blot assays, thus permitting the acquisition of immunological data in support of the functional data. Finally, we performed rabbit enteric PK studies in live animals to seek in vivo validation of the in vitro approaches. The results clearly show that the enteric proteases rapidly degrade unstructured regions on the VHH-based agents, such as epitopic tags and different spacers sequences, while the core VHH function is far more resistant to the proteases. Different VHHs appear to have highly variable susceptibility to enteric protease inactivation. The results should provide guidance to researchers seeking to develop effective VHH-based therapeutics for treatments of enteric diseases.In this manuscript, we employ extracts prepared from the upper intestine of rabbits and piglets (chyme), and human fecal extracts, to perform N spacers [Fig 1, western blots that recognize the epitopic E-tags which flank the six linked VHHs produced a similar partial digestion pattern when incubated with either human or porcine enteric proteases. The size of the products, multiples of ∼15 kDa, suggested that both extracts cleaved the heteromultimer within the spacers separating the 15 kDa VHH components.Early western blot studies incubating intestinal and fecal extracts with VHH multimers had suggested that the proteins were preferentially cleaved within the spacer regions separating the VHH monomer components. A particularly clear example occurred when we incubated a heterohexameric agent consisting of VHHs linked by conventional (GGGGS) spacers  with dilFig 2A, were incubated at 37°C for 10 or 60 minutes with PBS or with extracts of human feces or porcine intestinal chyme (1:10 each). One of the heterodimers (Trx/E/ALcH7/JPUA5/E) consisted of two VHHs that potently inhibit the activity of Botulinum neurotoxin A (BoNT/A) light chain protease (LC/A) [Fig 2B, the E-tags were rapidly lost after 10 minutes of incubation and became undetectable after 60’ incubation. Chyme proteases were inhibited with α1-antitrypsin (α1AT), and then the incubation products were assayed for LC/A inhibition . As a zinc protease, LC/A is not inhibited by α1AT. Surprisingly, all samples of the LC/A inhibitory VHH heterodimer retained substantial potency  even after 60’ incubation with human fecal or pig intestinal extracts, and despite being virtually undetectable with anti-E-tag reagent on the western blot. Clearly, the flanking E-tag epitopes were becoming degraded or excised from the VHH heterodimers during enteric extract incubation long before the VHH agents lost their binding and inhibition activity. In other studies, we employed VHH agents fused to other epitope tags such as myc-tag, S-tag and hexahistidine tags, however all were rapidly degraded following similar incubations with GI extracts and thus poorly useful for PK studies (not shown). Our results strongly suggested that the highly-structured functional core VHHs are much more resistant to enteric proteases than the flanking epitopic tags. The poor correlation between VHH immunological detection and VHH function when monitoring enteric VHHs with conventional anti-tag antibody reagents highlights the substantial challenge of performing simple and quantitative enteric PK studies on therapeutic proteins.To further characterize the degradation of VHH heteromultimers by enteric proteases, two different purified VHH heterodimers, each flanked by E-tag peptides and fused to thioredoxin (Trx) as shown in e (LC/A) . The sece (LC/A) . AliquotFig 3 and derive from data shown in . All cleavage sites were found to occur very close to the amino terminus of one or both core VHHs. Additional efforts to perform Edmon sequencing on ambiguous mixtures of enteric protease degradation products, identified the known enterokinase cleavage site located in the vector derived sequences upstream of the VHHs. In summary, no clear cleavage sequence specificity was apparent, although all identified sites contained a small amino acid  at the amino end. The results suggested that preferred intestinal protease cleavage sites are located in the likely unstructured spacer regions that immediately precede the component core VHHs. This specificity is suggestive of cleavage by a gastrointestinal serine protease such as neutrophil elastase [We next sought to identify some specific sites in VHH agents which are preferentially cleaved by human and porcine enteric proteases. A series of studies were performed employing different VHH heterodimers. The purified VHH heterodimers were digested for various times with human fecal or porcine intestinal extracts to identify time points at which distinct digestion products became apparent. Sufficient protein was employed to permit identification of the amino terminal sequences of digestion products by Edman degradation methods. A summary of four cleavage sites identified after digestion is shown in elastase . PrefereFig 2), we sought to identify an immunological reagent capable of detecting the functional VHH cores to facilitate more robust enteric PK studies. We initially immunized rabbits with a mixture of 13 different native VHH proteins in a non-denaturing aqueous alum adjuvant. Each of the VHHs in the pool also contained ∼50 pET32 vector-encoded, amino-terminal amino acids consisting of several purification epitopic tags and a short C-terminal myc tag (S1 Table). Due to early unsatisfactory results  with the resulting immune sera , we immunized a second pair of rabbits employing the same VHHs as before, but 50% of the VHH pool was removed, partially denatured (reduction and boiling) and recombined. The partially denatured pool was then formulated in an oil-based Freund’s adjuvant for all immunizations. The resulting immune sera was called ‘rabbit anti-denatured VHH sera’.After determining that immunologic detection of VHHs using conventional anti-epitopic tag reagents was unreliable following VHH exposures to intestinal proteases  sampled after various times of incubation  with porcine intestinal chyme (1:20). In one analysis, samples were resolved by SDS-PAGE and detected by western blotting. As shown in Fig 4B, sera derived from rabbits immunized with anti-denatured VHHs contained antibodies that were capable of recognizing distinct peptides that preferentially survived long term intestinal protease incubations, such as those observed by Coomassie staining in . The smallest band recognized was ∼14 kDa, the predicted size of core VHHs. In contrast, rabbit anti-native VHH sera poorly recognized the VHH heterodimer or the VHH monomers after only 30 minutes exposure to intestinal extract, thus displaying similar sensitivity to intestinal proteases as previously found with anti-E-tag reagent . This result suggests that the rabbit anti-native VHH sera almost exclusively recognizes the short unstructured amino and carboxy terminal peptides, such as the epitopic tags, common to all of the recombinant VHHs used for rabbit immunization.The two different rabbit anti-VHH sera preparations were tested for recognition of an Stx2-neutralizing VHH heterodimer , even in samples incubated overnight in chyme that had been reduced to a single visible band of ∼14 kDa on western blot. Note that the higher peak signal for untreated control in this and other similar ELISAs likely reflects recognition of additional flanking epitopes that are lost rapidly during chyme incubations as well as some reduction in agent affinities resulting from dimers being cleaved into monomer components. Based on the shift in the EC50 values of the treated samples compared to untreated protein, it appears that at least 10% of the original Stx2 binding activity  remained detectable by rabbit anti-denatured VHH sera following the overnight incubation with intestinal proteases under the in vitro conditions employed.While anti-VHH sera western blots proved very useful as a tool to monitor the Fig 5A). MTT assays assessed the amount of HeLa cell metabolic activity that remains following exposure to Stx2 toxin. Increased cell viability indicated the level of toxin neutralization that remained in each antibody sample dilution. The ability of antibody samples to protect cells from toxin despite greater amounts of sample dilution reflects increased antibody potency. The results demonstrated a wide range of functional chyme susceptibility in the four VHHs. The JEN-D10 VHH appeared the most susceptible to inactivation as no detectable Stx2-neutralizing activity was found after only 20 minutes of incubation. In contrast, JSY-F12 VHH appeared to be the most protease resistant VHH as the Stx-2 neutralizing potency was strong after 3 hours incubation and remained easily detected even after overnight incubation. The remaining two VHHs, JFG-H6 and JGH-G1 remained active after 1 hour or 3 hours incubation respectively, but both had lost function after overnight incubation.Studies described in the prior sections found that exposure to enteric proteases rapidly removes epitopic tags of VHH multimers and cleaves spacers resulting in monomeric core VHHs which, on longer incubations, generally lose their function and integrity. We next sought to test whether different VHH monomers have similar or widely variable susceptibility to functional inactivation by more harsh exposures to intestinal proteases. For this, we took advantage of our prior discovery of four unrelated VHHs that all recognize the same small target of Stx2 B-subunit, and all potently neutralize Stx2-induced toxicity in HeLa cells. These VHHs were each subjected to varying times of exposure to a more concentrated rabbit intestinal chyme extract (1:5 dilution). At each time point, VHHs were quenched with a protease inhibitor cocktail and then later assayed for Stx2-neutralization potency in a cell-based assay . At 0 time, some degradation is apparent in all VHHs within the ∼5 kDa vector-derived amino terminal fusion partner which contains a hexahistidine for purification and two protease-sensitive sites. The variable degradation profile between the four VHHs is consistent with the cell-based assay findings, confirming that VHHs with intact ∼14 kDa core display Stx2-neutralizing activity. These results seem to confirm the interpretation that the 14 kDa bands, which display high resistance to intestinal protease digestion as described above, represent the highly structured core VHH.The same samples were also assayed for their functional integrity by western blot using the rabbit anti-denatured VHH sera for detection .Functional assays such as the enzyme inhibition or toxin neutralizing assays shown in H and VL domains and tested both with or without the polymeric immunoglobulin receptor (pIgR). Parallel incubations with intestinal chyme at 1:5 and 1:3 chyme dilution were performed on the four different Stx2-neutralizing antibody types. All the samples were then assayed in a 1:5 dilution series for their Stx2-neutralizing potencies using the HeLa cell-based viability assay. The results showed substantial chyme protease resistance by IgG1 and IgA2 (+/- pIgR) in 1:5 chyme dilution even after overnight incubations . Under the same conditions, the Stx2-neutralizing VHH heterodimer (JFG-H6/JGH-G1) was substantially less resistant to enteric proteases than the conventional antibodies, remaining active after 3 hours incubation and losing most activity during overnight incubations. Under the harsher 1:3 chyme incubation conditions, the Stx2-neutralizing activity of the IgG and IgA was somewhat lower after overnight incubation while the VHH heterodimer lost most of its potency after only 3 hours incubation . The functional PK of the heterodimer thus approximately matched that of the JGH-G1 monomer, from Fig 5A. The results suggest that if we had selected more protease resistant component VHHs, such as JSY-F12 , the VHH-based agents would have a functional chyme stability that is more similar to that of conventional antibodies.It is not possible to exactly compare the functional stability to enteric proteases of conventional vs VHH-based antibodies due to the fundamental differences in structure of the two antibody types and the wide variation in chyme sensitivity of different VHHs. In a first effort to generally compare the protease stability of conventional and VHH antibodies, we tested the functional protease stability of an Stx2-neutralizing VHH heterodimer with that of an Stx2-neutralizing IgG1 (5C12) and a recombinant IgA2 that was engineered with the 5C12 VFig 6A) were also assessed for structural integrity by western blots . The results revealed that the incubation time-dependent loss of function in Fig 6A showed similar kinetics to the loss of integrity of these antibody types. The IgG Hc appeared to be much more sensitive to degradation compared to the IgA2 Hc, which was not reflected in a loss of neutralizing potency, suggesting that loss of IgG1 Hc integrity did not significantly impact antibody potency to neutralize Stx2. This is not surprising as long as the Fab region remained intact as suggested by the western blot. As expected, the VHH heterodimer was rapidly degraded to VHH monomers by enteric proteases while the monomer integrity appeared to correlate well with the functional potency of the different incubation time points.The same antibody samples assayed for Stx2-neutralization  heterodimers containing TcdB-inhibiting VHH components 5D and E3 . Two variables were introduced into the three heterodimer constructions, specifically the different spacers and/or flanking sequence modifications as detailed in S1 Table. In this study, two of the heterodimers completely lacked a spacer between the two VHHs. To engineer this, the carboxyl end of the 5D core VHH (…VSS) was joined directly to the amino end of E3 core VHH (QVQLVE …). One of the two heterodimers lacking a spacer was flanked by two copies of a 10 amino acid peptide PEPEPEPEPE (PE5). The PE5 peptides were included to test whether flanking proline-rich peptides might protect the amino and carboxyl ends of the functional VHH heterodimer from degradation by amino peptidases and carboxypeptidases likely present in the chyme [Fig 7B, there were no apparent differences in the rate at which the three constructs lost their TcdB-neutralizing potency with increasing chyme incubation times. The same incubations were also analyzed by western blot employing the rabbit anti-denatured VHH sera and the results  revealed no apparent differences in the rates at which the VHH heterodimers were degraded to monomers.he chyme . In addihe chyme ,20 whichS8A Fig). We employed the same VHH heterodimer in which the only difference in the three proteins was in the sequence of spacers separating the two VHHs as detailed in S2 Table. One spacer consisted of a conventional 5 amino acid polyglycine flexible spacer (5G) while the other spacers were either the proline-rich amino acid rigid spacer, PEPEPE (PE3) or a proline/glycine hybrid spacer, PGPGPG (PG3). The three heterodimers were incubated at various times in rabbit chyme (1:3) and analyzed by western blot with rabbit anti-denatured VHH sera . Again, we were unable to detect any meaningful difference in the rates at which the heterodimers were reduced to monomers.In another study, we compared the intestinal protease resistance of three different VHH spacers expected to differ in their flexibility  while a control loop located between them was injected with PBS to control for potential leakage (n = 1). One sample was removed and quenched immediately after injection and massaging to mix the sample within each loop. Additional samples were removed at 15 min, 45 min and 2 hrs following loop injections. Functional in vivo persistence of the VNA-TcdB function during intestinal gut loop chyme incubation was assessed using a cell-based TcdB-neutralization cell rounding assay of the samples removed at different time points. The control gut loop sample displayed no significant TcdB neutralization  showing that the chyme itself had negligible effect on the Vero cell rounding assay and that no detectable VNA-TcdB had passed through the surgical ties separating it from the adjacent gut loops injected with VNA-TcdB. As shown in , the gut loop samples obtained from the two loops injected with VNA-TcdB displayed clear TcdB neutralization at the earliest timepoint tested (1 min), preventing TcdB-elicited cell rounding even when diluted more than 1000-fold. The 1 min timepoint would have experienced some exposure to proteases prior to removal and quenching but provided an approximation of the initial potency of the VNA-TcdB diluted into the gut loop chyme. During the 2 hr in vivo gut loop incubation, the VHH heterodimer potency dropped about 5 and 20-fold in the replicate loops based on their estimated IC50s as compared to the 1 min timepoint. As a final measure for comparison, ELISAs were performed on the samples removed from VNA-TcdB injected loop (n = 1)  using rabbit anti-denatured VHH sera. As observed with the TcdB-neutralization assay, ELISA EC50 shifts indicated that the TcdB-binding potency of the samples dropped about 20-fold during the 2 hr in vivo gut loop residence. The results are within the range expected based on similar studies performed in vitro with chyme and support the value of using much simpler and more robust in vitro assays to obtain accurate estimates of the in vivo enteric functional PK of VHH-based agent samples. In addition, they indicate that target-binding ELISAs using rabbit anti-denatured VHH sera can provide a simpler and useful measure of the enteric functional stability of VHH-based agents.In an effort to test the Single-domain antibodies, such as VHHs, are particularly promising as components of enteric therapeutics because they can be produced at low cost in microbes and delivered orally or expressed in patients by probiotics or, possibly by engineering their own microbiome. Such products have already been reported to show some therapeutic benefits in numerous animal models of enteric diseases –11. BecaFig 2 above, showed that conventional immunologic detection tools that employ epitopic tags fused to the VHH-based agents are of limited value as intestinal protease incubations quickly degraded the tags well before the VHH biological functions were lost. Clearly the functional stability of the VHH agents is a much more relevant property to monitor when performing enteric PK studies, yet these assays can also be difficult to perform quantitatively when the samples are contaminated with the highly variable, complex mix of components, including microbes and proteases, found within intestinal chyme.Unsurprisingly, this hostile proteolytic environment of the GI tract produces substantial challenges to the performance of enteric PK studies. Initial PK studies, such as we report in in vitro enteric PK studies that mimic those in extracts of intestinal fluid. But we lack complete knowledge as to the identity and concentrations of each protease found in chyme and which proteases elicit the greatest influence on VHH-agent stability. It is known that pancreatic enzymes are stimulated by feeding [in vitro PK studies is clearly imperfect as it does not mimic the dynamic, flowing nature of the GI tract. Another complication of using crude enteric extracts is that incubations cannot be unambiguously terminated by addition of protease inhibitor cocktails due to the possible activity of unknown proteases that are not completely inhibited. If some proteases remain active, further degradation may occur during later functional assays leading to underestimates of the inherent functional stability of some agents. Despite these challenges, we were able to show that the PK results obtained from in vitro time course incubations of a VHH heterodimer with intestinal extracts compared favorably to similar time courses obtained in vivo by injecting the same VHH heterodimer into surgically extruded gut loops . In addition, when qualitatively comparing enteric PK between different samples  the findings were supported in multiple independent assays . In summary, we feel that the results reported in these studies are qualitatively significant, though quantitative outcomes must be considered as approximations.For ideal PK studies, the intestinal protease components in chyme, and their concentrations, would be consistent and well known so as to permit the use of defined mixtures for  feeding , so largFig 4). When testing the anti-native VHH sera, we found that virtually all of the VHH immunodetection signal was rapidly lost at early times of chyme incubations, well before significant drops in VHH function were detected , and similar to our observations of the epitopic tag sensitivity to chyme . It was only when using rabbit anti-denatured VHH sera that we observed significant VHH recognition of samples incubated for longer times with high concentrations of chyme . The presence of a 14 kDa product on western blots at later times of VHH agent chyme incubations appeared to correlate well with the retention of VHH function. We conclude that this band represents functional ‘core’ VHHs. Note that some VHH samples extensively degraded by chyme, such that VHH monomers were not detected by rabbit anti-denatured VHH sera, still retained detectable function in some cases , indicating that there are limits to the immunodetection of bioactive VHHs even with this reagent. The full set of data reported here highlights the substantial challenges in performing quantitative enteric PK studies of VHH-based agents when employing immunodetection tools.Because the use of epitopic tags proved to be effective only when very brief or diluted chyme incubations were employed, we sought to identify or create an anti-VHH reagent better able to detect VHH agents that had been exposed to chyme for longer time periods. Preliminary studies with some commercial anti-VHH agents proved unproductive, so we created custom sera by immunizing two pairs of rabbits with a pool of VHH monomers. In one pair of rabbits, we employed native VHH immunogens in aqueous alum adjuvant to produce ‘anti-native VHH sera’. In the second pair of rabbits, half of the VHH pool was denatured by boiling under reducing conditions prior to immunization and formulated in Freund’s oil-based adjuvant resulting in ‘anti-denatured VHH sera’. The same VHH monomer immunogens were included in all immunized rabbits, and shared small vector-derived peptides flanking the VHHs consisting mostly of epitopic and purification tags. Both rabbit sera pools strongly recognized intact VHH monomers on ELISAs and western blots  and each occurred a few residues to the amino end of a core VHH. It is difficult to know whether the small amino acids  surrounding the cleavage sites reflect a preferred protease specificity or simply occurred at sterically available sites immediately flanking the structural core VHH. We thus sought to proactively engineer spacers and flanking sequences into VHH-based agents in efforts to slow the intestinal protease activity. Numerous different spacers and flanking sequences were tested yet all remained highly susceptible to cleavage by intestinal extract incubations at times before the VHH functions themselves were significantly damaged. In one approach we tested the use of rigid or flexible spacers and exoprotease-resistant flanking peptides . We also tested core VHHs that were linked together without any intervening spacer, hypothesizing that removal of this small region of protease-susceptible unstructured peptide would resist cleavage by the enteric proteases, however even this minimal linkage remained protease susceptible . Additional spacer sequences were less rigorously tested in other studies not reported here, and again none displayed measurably improved protease resistance. In total, the data seems to suggest that the intestinal proteases rapidly degrade unstructured peptides such as epitopic tags and spacers and are much slower to degrade highly structured proteins such as the core VHHs. Since most proteins from food entering through the stomach are denatured by acidic conditions, it makes sense that the intestinal proteases would have their highest specificity for unstructured protein.Our results show that there are significant challenges to the use of VHH multimers or VHH fusion proteins as enteric therapeutics for applications requiring the components to remain connected while in the GI tract. The most sensitive sites of intestinal protease cleavage of VHH multimers were determined for several heterodimers . The results revealed dramatically different enteric PK properties of the four VHH monomers assessed under the same treatment conditions. Since the success of VHH-based products as enteric therapeutics is likely to depend on the use of VHHs that are highly resistant to destruction by enteric proteases, we developed and validated a rapid and general method to assess enteric PK properties of monomeric VHHs by sandwich ELISA . Anecdotally, our lab has performed similar rapid assays on dozens of other VHHs having a wide variety of target specificities as a down-selection screen for VHHs that show high functional resistance to enteric proteases. To detect VHH target binding in these assays, we typically use the immune alpaca antisera generated previously for the original VHH discovery. These studies have continued to reveal wide variations between different VHHs in their functional stability to intestinal chyme. To date we have not observed any obvious correlation between functional stability and the presence of specific amino acids or amino acid types  within CDRs nor CDR lengths, or the presence of unusual framework amino acids. Future studies, involving significantly more VHHs, or systematically sequence-modified VHHs, or detailed physical chemistry comparisons, will likely be required before this issue can be fully addressed.Despite the substantial resistance of core VHH monomers to proteolytic inactivation by intestinal extracts, the functional activity of all tested monomer VHHs was eventually destroyed with prolonged chyme exposure. We performed a series of studies comparing the functional stability of individual monomer VHHs to incubations with intestinal extracts. In one study, we compared the functional stability of a panel of four unrelated Stx2-neutralizing VHH monomers . Because the target binding domains of mAbs and VHHs are inherently different, the comparisons were at best approximations. The results showed that the toxin-neutralizing functions of the IgG and IgA2 were more stable to incubations with intestinal extracts than the VHH-based agent we tested. Both components of the tested VHH dimer, though, were only moderately resistant to enteric proteases  and our results suggest that use of VHHs that are pre-selected for high protease resistance will permit development of VHH-based products with enteric PK properties more similar to that of conventional antibodies.Finally, we performed preliminary studies seeking to generally compare the enteric PK of VHH agents with conventional IgG and IgA antibodies  under Protocols No. G2017-18, No. G2017-55 and No. G2017-119. Human infant fecal solids were obtained in 2018 from a deidentified donor under informed parental consent in accordance with Tufts Health Sciences Campus Institutional Review Board guidelines and in compliance with certification of exempt IRB status No. 9893.TM protease inhibitor cocktail (Thermo Fisher), using the concentrations and conditions indicated. Rabbits and piglets were sacrificed by intracardiac or intravenous administration of sodium pentobarbital at 87 mg/kg.Variable segments of the rabbit or porcine GI tract representing sections of duodenum, jejunum, ileum, cecum, colon and rectum were removed by dissection and the contents were squeezed out into 50 ml tubes and diluted approximately 1:4 (v:v) in PBS. Human infant fecal solids were similarly diluted approximately 1:4 (v:v) in PBS. Tubes were then vigorously vortexed and centrifuged . The resulting chyme or fecal extract supernatants were stored in aliquots at -80°C as 1x chyme or 1x fecal extract. Different antibody preparations and controls were incubated at 37°C with extracts of human feces or intestinal chyme of porcine or rabbit origin in PBS, and reactions terminated with either α1-antitrypsin (α1AT) (Sigma) or 1x HALT2; %), and end tidal CO2 were monitored and normocapnia (35–45mmHg) was maintained. Rabbits were kept warm using a heating pad and lamp during the experiment, IV crystalloids were administered for maintenance fluid support, and rectal temperature and blood pressure (indirect oscillometric measurement from the forelimb) were continuously monitored. The ventral abdomen was clipped and prepped aseptically. Using sterile surgical technique, an 8 cm midline abdominal incision was made, and a segment of jejunum exteriorized. Adjacent jejunal segments (5–6 cm each) were isolated by circumferential ligation of the jejunum and the terminal arcade of the jejunal artery and vein, as described previously [Specific pathogen-free New Zealand White rabbits (Covance) were anesthetized with ketamine (35mg/kg IM ) and xyleviously . AdjacenE. coli as previously reported [VHHs and VHH heterodimers were expressed in reported . In somereported .The Stx2-neutralizing IgG mAb, 5C12, was produced as previously described . The IgA3 , and NaCl . Protein was then captured using Pierce Protein L Plus Agarose (Thermo Fisher), washed with HBS-E-hs buffer , and eluted in 0.1 mM glycine, pH 2.7 . Protein-containing fractions were pooled and buffer-exchanged against HBS-E  by ultrafiltration using Amicon Ultra devices (Millipore).For the dimeric IgA production, FreeStyle 293-F (Thermo Fisher) cells were transiently co-transfected using PEI MAX (Polysciences) with the 5C12 hIgA2/hIgK/hIgJ plasmids, using the following ratio of constructs 0.25/0.25/0.5. Following 5 days of culturing, conditioned medium was harvested by centrifugation, supplemented by the addition of NaN3  and NaCl . Protein was then purified by immobilized metal-affinity chromatography using HisPur Ni-NTA resin (Thermo Fisher) followed by gel-filtration over a HiLoad 16/600 Superdex 200 pg column (Thermo Fisher) in the HBS-E buffer .Recombinant human pIgR (hpIgR) was produced by transient transfection with the hpIgR.HisAvi construct. Following 5 days of culturing, conditioned medium was harvested by centrifugation, and supplemented by the addition of NaNTwo New Zealand white rabbits (Covance) were immunized with a pool containing equal amounts of thirteen random, unrelated VHH proteins, expressed as above with only hexahistidine and myc tags, and administered under native, aqueous conditions to minimize VHH denaturation. Each rabbit was injected with 200 μg of the VHH pool followed by four subcutaneous immunizations with 100 μg of the same VHH pool, all using an aqueous alum adjuvant. The resulting serum pool was called ‘rabbit anti-native VHH sera’.A second pair of New Zealand white rabbits were immunized with the same pool of VHHs as above except that, prior to immunization, 50% of the pool was subjected to reduction and denaturation by adjusting the solution to 1 mM DTT and boiling for 30 minutes. The remaining 50% of the pool contained native VHHs. For the second pair of rabbits, the first three immunizations employed complete Freund’s adjuvant (CFA) while the last two employed alum adjuvant. This serum pool was called ‘rabbit anti-denatured VHH sera’. Serum was obtained from rabbits several days after the final immunizations from a terminal bleed performed under anesthesia with ketamine (35 mg/kg) and xylazine (5 mg/kg) administered IM.H+VL) (Jackson) and of IgA2 anti-human IgA (Hc only) (Sigma). Commercial antibodies were used at dilutions recommended by manufacturer. Signals were detected using peroxidase reagent and colorimetric or chemiluminescent substrate, and measured as described elsewhere [ELISAs and western blots were performed as previously described  with spelsewhere . Raw imaThe four Stx2-neutralizing VHHs were coated overnight at 10 μg/ml concentration onto individual columns of a 96-well Nunc Maxisorp ELISA plates, then washed but not blocked. A 2-fold dilution series of porcine intestinal chyme was applied starting at 1:5 in PBS in the top well and down the plate. The plates were then incubated at 37°C for either 1 hour or 3 hours before extensive washing and blocking of the plate overnight in 1xPBS/4% milk/0.1% Tween. An Stx2 binding ELISA was performed on each plate by incubating the plate in 0.5 μg/ml Stx2, washing and detecting bound Stx2 with an anti-Stx2 (A subunit) mAb (5C12 ).VHH proteins were incubated with enteric proteases for indicated times and resolved on a 4–20% Novex™ Tris/Glycine SDS/PAGE gel (Invitrogen). The gel was then electroblotted onto PVDF membrane and visualized with 0.02% Coomassie Brilliant blue in 40% methanol, 5% acetic acid for 20–30 seconds. Bands of interest were excised and subjected to N-terminal sequence analysis on an ABI 494 Protein Sequencer by the Tufts University Core Facility. The resulting mixture of sequences was analyzed blind to the samples by experienced staff in the facility. Raw images of all gels produced in this study are available in in vitro and in vivo incubations with enteric protease extracts were stored at -80°C until use. Prior to assays, samples were thawed on ice and diluted 30-fold in ice cold PBS containing 1x HALTTM. Diluted samples were then sterile filtered by syringe keeping all samples on ice prior to use in cell-based assays.Assay test samples from Stx2 neutralization was assayed in a cell-based assay as described previously . In summTcdB neutralization was assayed in a cell-based assay as described previously . SpecifiThis assay was performed as previously described  using thS1 Fig™ A/E, BioSentinel Pharmaceuticals) [A 20 μl aliquot of each sample from uticals)  in a 50 (PDF)Click here for additional data file.S2 FigS3A and S3B Fig).(A) Coomassie-stained SDS-PAGE (5 μg of VHH agent/lane) and (B) Coomassie-stained PVDF membrane obtained following transfer of the same samples from a preparative SDS-PAGE gel (10 μg of VHH agent/lane). All samples derive from chyme incubations of VHH heterodimer Trx/E/AH3/AA6/E (500 μg/ml)  (diagram(PDF)Click here for additional data file.S3 FigS2 Fig resulting from incubation with (A) porcine intestinal chyme or (B) human fecal extract. The five traces corresponding to the first five residues are shown with the top candidates for amino acids 1 to 5 indicated with arrows. The top amino acid calls (identified blind) are shown to the right, with secondary calls in parentheses. The sequencing results of both bands indicated an amino terminus of AQGVQ which could be unambiguously identified at the amino end of the submitted peptide shown in Figs 3 and S2C.Edman degradation traces of VHH heterodimer Trx/E/AH3/AA6/E digestion products from (PDF)Click here for additional data file.S4 FigS5 Fig).(A) Coomassie-stained SDS- PAGE (2 μg/lane) and (B) Coomassie-stained PVDF membrane obtained following transfer of the same samples from a preparative SDS-PAGE gel (4 μg of VHH agent/lane). Samples derive from chyme incubation of VHH heterodimer Trx/E/JDQF12/JDQD12/E (40 μg/ml)  (diagram(PDF)Click here for additional data file.S5 FigS4 Fig. The five traces corresponding to the first five residues are shown with the top candidates for amino acids 1 to 5 indicated with arrows. The top amino acid calls (identified blind) are shown to the left, with secondary calls in parentheses. The sequencing results indicated an amino terminus of AQVQL which could be unambiguously identified at the amino end of the submitted peptide shown in S4C Fig.Edman degradation traces of VHH heterodimer Trx/E/JDQF12/JDQD12/E digestion products from (PDF)Click here for additional data file.S6 FigS7 Fig).(A) Coomassie-stained PVDF membrane of samples that derive from chyme incubation of VHH heterodimer 6H/E/StxA5/Stx2G1/E  (diagram(PDF)Click here for additional data file.S7 FigS6 Fig. The seven traces corresponding to the first seven residues are shown with the top candidates for amino acids 1 to 7 indicated with arrows. The top amino acid calls (identified blind) are shown at the top, with secondary calls in parentheses. We conclude that the correct amino end call for residue 1 must be a glycine as no similar sequence existed in this protein in which alanine could be the correct call. The sequencing results of the degradation product(s) indicated an amino terminus of GVQAQLQ, a sequence found immediately upstream of both core VHHs as shown in S6B Fig. We suggest that the submitted product was most likely a pool of two degradation products resulting from cleavages at these two sites in both VHHs. We cannot rule out that the band resulted from cleavage at just one of the two identified sites while the second VHH was further degraded to smaller fragments.Edman degradation traces of VHH heterodimer 6H/E/StxA5/Stx2G1/E digestion products from (PDF)Click here for additional data file.S8 FigS2 Table. Each consisted of two Stx2-neutralizing core VHHs, JGH-G1 (G1) and JFG-H6 (H6) [(A) Cartoons of the three VHH heterodimer proteins tested. Each heterodimer was identical except for the sequence of spacer region as indicated in -H6 (H6) , an amin(PDF)Click here for additional data file.S1 TableFig 7A.Colored sequences correspond to VHH components diagramed in (PDF)Click here for additional data file.S2 TableS8A Fig.Colored sequences correspond to VHH components diagramed in (PDF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.