Clostridium difficile infection still represents an important clinical problem which causes very substantial health care costs. Current approved treatments tackle only one aspect of primary infections: reduction of bacterial burden using antibiotics. Another need is to neutralize and clear the toxins that cause the symptoms. Tackling both bacteria and toxin might bring improvements in clinical outcomes, such as a reduction in the duration and severity of diarrhea, along with reduced death and recurrence rates.
We made a mixture of three antibodies which neutralized TcdA and TcdB in vitro
and in vivo
. These MAbs are “functionally oligoclonal,” as measured by the high valency of binding to the toxin, and hence might combine the manufacturing benefits of MAbs along with the functional benefits of a hyperimmune polyclonal serum. In order to best determine their clinical potential, we compared them against the MAbs which are the most advanced in the clinic, CDA1 and MDX-1388. These MAbs are in two phase III clinical trials (MODIFY I [identifier NCT01241552] and MODIFY II [identifier NCT01513239] [see www.clinicaltrials.gov
]) as a mixture designated MK-3415A and alone as MAbs designated MK-3415 and MK-6072, respectively, for reduction in recurrence rates and as a global cure.
The UCB MAb mixture gave 100% (11/11) protection of hamsters until day 11 and ~82% (9/11) protection at the end of the study on day 28. These were higher and more durable levels of protection than those demonstrated with CDA1 and MDX-1388 by Babcock et al. (35
) and herein. One-hundred-percent protection was observed until only day 5 with the CDA1 and MDX-1388 mixture; thereafter, protection decreased until day 20, when only 2/11 (18%) of animals survived. The higher protection levels of the UCB MAbs were supported by observations made using cecum pathology scores and animal weight changes. Animals treated with the UCB MAbs had fewer moderate and severe cecum pathology scores (2/11) than MDX MAb-treated animals (9/11). A greater number of UCB MAb-treated hamsters showed rebound from weight loss than MDX MAb-treated hamsters. It is possible that weight rebound was caused directly by the toxin-neutralizing properties of the UCB MAbs or that it was a result of increased well-being of the animals due to an earlier restoration of normal diet, digestion, and, hence, normal gut microbes (45
). Others have shown (42
) several-log reductions in the numbers of C. difficile
CFU after treatment with neutralizing antitoxin MAbs, perhaps supporting the latter suggestion. The different levels of protection between the UCB and MDX MAb treatment groups were not due to differences in antibody PK. The subtle differences between the Babcock et al. study and the one reported here, including doses and routes of administration of clindamycin (10 mg/kg i.p. versus 5 mg/kg s.c., respectively), infectious strains, and doses (140 spores of B1 versus 3.4 × 106
CFU of ATCC 43596 vegetative cells, respectively), were observed to have little or no effect on the protection test in comparison with published data (35
The incomplete and waning protection of the lower dosing groups (5 mg/kg, total dose of 20 mg/kg), even when dosed prophylactically, might reflect at least in part complex dynamics in the model between antibody dose (biodistribution and clearance), toxin burden, and relative sensitivity of hamsters to TcdA and TcdB. Human clinical studies using therapeutic dosing suggest that a total dose of 10 mg/kg can offer significant clinical benefit (38
). One other hamster infection study of neutralizing MAbs (42
) used MAb doses lower than the 50 mg/kg × 4 used here, but these data are not instructive when considering the question of minimum protective dose. The dosing regimen used in our study and by Babcock et al. (35
) involved MAb dosing on days −3, −2, −1, and 0, whereas Marozsan et al. (42
) utilized “top-up” multidosing during the infection period, with MAb dosing on days −1, 1, 3, and 5. It is probable that the serum hIgG concentrations will differ considerably between the two regimens given the ~6-day serum half-life of hIgG1 in (uninfected) hamsters and the potential for hIgG1 loss into the gut of infected hamsters. Perhaps in the hamster model, sufficiently high concentrations of antibody must be present during the critical first ~5 days of infection in order for the intestine to be protected from high levels of toxin. Lower concentrations of neutralizing IgG1 might suffice thereafter for keeping the gut healthy when challenged with lower levels of toxin, such as during a recurrence. The dynamics will be quite different in human disease, not the least in that the substantially longer serum half-life of IgG1 in humans versus hamsters (~21 versus 6 days) would ensure high drug levels during the first critical week of infection and treatment.
We have demonstrated that some hamsters which survived to day 28 were capable of mounting an anti-TcdB IgG response and, perhaps surprisingly, that there was an inverse correlation between this and hamster weight loss. It is possible that animals with persistent weight loss might have been subjected to a more persistent environmental reinfection challenge. The demonstration that surviving hamsters can mount an antitoxin response is of general interest. The mounting of an effective host antitoxin response following clearance of parenterally administered MAbs would be an excellent clinical outcome for patients in that it might confer a long-lived protection against recurrent infection.
Can we rationalize mechanisms for the higher levels of protection provided by the UCB MAbs than by the MDX sequence MAbs? Our data show that the UCB MAbs had higher affinities, higher potencies (lower EC50
s), and higher valencies of toxin binding. Collectively, these were likely to result in more effective toxin neutralization and clearance from the circulation. The data also show that UCB MAbs were more stable (higher Fab Tm
), which might confer some benefit for retention of MAb functionality in the gut lumen. Of special interest, however, is the observation that the CDA1 MAb was poorly neutralizing at high TcdA concentrations. In the hamster model (and unlike potential human clinical application), the MAbs were given as sole therapy rather than in addition to antibiotic therapy. The hamster intestine is likely to encounter very high levels (total amounts and local concentrations) of both TcdA and TcdB in the first few days after infection. CDA1 might have been largely ineffective in terms of TcdA activity neutralization in these circumstances, whereas CA997 will likely have retained substantial levels of neutralization. Of additional interest is the difference in protection against TEER loss in response to TcdA. A loss of tight junctions is a known first step toward fluid loss and toxin penetration and, hence, diarrhea and local inflammation (17
). The ability to prevent TEER loss in vitro
might be considered a marker for protection against early gut damage events in vivo
. CDA1 conferred low levels of protection (percent protection and EC50
) against TEER loss, whereas CA997 offered a maximum protection level of ~80% and an EC50
of ~80 ng/ml. One might imagine that animals treated with CA997 might have suffered less fluid loss and less gut damage than those treated with CDA1. Wet tail was found to be an inaccurate disease indicator in these experiments, but the lower prevalence of cecum damage in the UCB MAb 50-mg/kg group was at least consistent with a benefit from protection against TEER loss.
The higher valencies of binding of these (human IgG1) MAbs might have more relevance in human studies than in hamster studies because of the potential for more effective engagement of Fcγ receptors (FcγRs). The clearance of toxin from the circulation is much faster (minutes rather than hours) (51
) when an antigen is decorated with three or more Fc fragments (53
). The presence of low levels of toxin in the circulation of infected humans has been a point of some conjecture (54
), but toxin was detected using a very sensitive assay in a piglet model of infection and was shown to be the cause of systemic symptoms in mice and piglets (47
). Neutralized MAb:antigen complexes have been demonstrated to both educate (56
) and vaccinate (57
) host immune systems, and antigen:Fc fusion vaccines have been shown to offer mucosal protection (58
). In addition, oligoclonal MAb binding has been shown to confer neutralization advantages through altered binding kinetics (59
). CA997 was estimated to bind TcdA approximately 12 times, while CDA1 was shown to bind TcdA twice. Hence, there are likely to be considerably different dynamics of toxin decoration and, hence, possibly also of toxin clearance. The anti-TcdB mixture of CA1125 plus CA1151 offers a more defined set of toxin decoration/cross-linking outcomes. CA1125 was shown to only bind to TcdB once and hence is most likely to catalyze TcdB cross-linking of two TcdB molecules. In contrast, CA1151 and MDX-1388 both appear to bind to TcdB twice and thus might be able to bind bivalently to one TcdB molecule or might cross-link two TcdB molecules. Therefore, the UCB MAb mixture is perhaps more capable of both decorating and cross-linking the Clostridium difficile
toxins A and B than MDX-1388. In a piglet model of systemic disease, only anti-TcdB antibodies (including MDX-1388) were protective, whereas anti-TcdA antibodies (including CDA1) were not (23
Of interest regarding ongoing human clinical studies of CDA1 and MDX-1388 (MK-3415A) is the observation that the CDA1 produced by us showed negligible neutralization activity against TcdA of ribotypes 027 and 078, a finding confirmed by others (42
). CDA1 also showed incremental reductions of neutralization potential at the higher concentrations of all three TcdAs. These observations might have important implications in the clinic, where 15 to 50% of patients can be found to be infected with ribotype 027 strains. There also remains a need to reduce the impact on patients of the acute effects of infection (diarrhea and gut tissue damage) during the first few days of infection. Effective neutralization of TcdA might have a secondary benefit in that in vitro
experiments suggest that the initial damage caused by TcdA (loss of tight junctions) can provide deeper access of the more potent TcdB to underlying tissues and hence enhance its overall inflammatory potential (18
). UCB MAbs used proteins of ribotype 027 sequence origin as immunogens and/or screening agents, while assays were performed using that of VPI10463 (ribotype 003). Hence, UCB MAbs were likely to retain good neutralizing activity against ribotype 003 and 027. All together, the UCB MAbs have been shown to be effective against toxins produced by four different strain ribotypes: 003, 027, and 078 in vitro
and 012 in vivo
In a phase II study of CDA1 alone, there was no difference in the recurrence rate versus that of placebo (61
), and yet a substantial reduction in the recurrence rate was observed when both CDA1 and MDX-1388 were used together in another phase II study (38
). One conclusion from these studies is that TcdB is the only virulence factor which determines the risk of recurrence in humans. Indeed, the Leav et al. study (61
) highlighted that the concentration of (host) anti-TcdB-neutralizing antibodies was significantly lower in subjects whose disease recurred than in those whose disease did not recur. An alternative or additional conclusion in light of the data presented here is that the anti-TcdA component of MK-3415A (CDA1) was insufficiently protective to elicit a measurable clinical outcome in humans. It is still not clear at this stage what the relative importance and specific roles of TcdA and TcdB are in human disease or what therapeutic benefits might be observed after their complete neutralization.
In summary, we show data here for humanized IgG1, which as a mixture of three MAbs provided excellent levels of protection in a hamster model of infection. This high level of protection is likely a composite of potent toxin neutralization, high affinity/avidity toxin binding, protection from loss of tight junctions, and antibody stability. Functional oligoclonality was demonstrated through high-level molar binding equivalents during surface plasmon resonance assays. Along with high-affinity toxin binding, this property might also make this MAb mixture a good agent of toxin clearance in humans. The UCB anti-TcdA MAb was capable of effective neutralization of a range of clinically relevant toxin ribotypes at high toxin concentrations and conferred significant protection from TEER loss. Finally, we show that some infected hamsters were capable of mounting their own antitoxin response after MAb-induced survival. The characteristics of these MAbs might offer advantages to patients and health care providers in terms of reducing the duration and severity of diarrhea and the incidence rates of death and recurrence.