Proteolytic cleavage and loss of function of biologic agents that neutralize tumor necrosis factor in the mucosa of patients with inflammatory bowel disease.

BACKGROUND & AIMS: Many patients with inflammatory bowel disease (IBD) fail to respond to anti-tumor necrosis factor (TNF) agents such as infliximab and adalimumab, and etanercept is not effective for treatment of Crohn's disease. Activated matrix metalloproteinase 3 (MMP3) and MMP12, which are increased in inflamed mucosa of patients with IBD, have a wide range of substrates, including IgG1. TNF-neutralizing agents act in inflamed tissues; we investigated the effects of MMP3, MMP12, and mucosal proteins from IBD patients on these drugs. METHODS: Biopsy specimens from inflamed colon of 8 patients with Crohn's disease and 8 patients with ulcerative colitis, and from normal colon of 8 healthy individuals (controls), were analyzed histologically, or homogenized and proteins were extracted. We also analyzed sera from 29 patients with active Crohn's disease and 33 patients with active ulcerative colitis who were candidates to receive infliximab treatment. Infliximab, adalimumab, and etanercept were incubated with mucosal homogenates from patients with IBD or activated recombinant human MMP3 or MMP12 and analyzed on immunoblots or in luciferase reporter assays designed to measure TNF activity. IgG cleaved by MMP3 or MMP12 and antihinge autoantibodies against neo-epitopes on cleaved IgG were measured in sera from IBD patients who subsequently responded (clinical remission and complete mucosal healing) or did not respond to infliximab. RESULTS: MMP3 and MMP12 cleaved infliximab, adalimumab, and etanercept, releasing a 32-kilodalton Fc monomer. After MMP degradation, infliximab and adalimumab functioned as F(ab')2 fragments, whereas cleaved etanercept lost its ability to neutralize TNF. Proteins from the mucosa of patients with IBD reduced the integrity and function of infliximab, adalimumab, and etanercept. TNF-neutralizing function was restored after incubation of the drugs with MMP inhibitors. Serum levels of endogenous IgG cleaved by MMP3 and MMP12, and antihinge autoantibodies against neo-epitopes of cleaved IgG, were higher in patients who did not respond to treatment vs responders. CONCLUSIONS: Proteolytic degradation may contribute to the nonresponsiveness of patients with IBD to anti-TNF agents.

Engineered protease-resistant antibodies with selectable cell-killing functions.

Molecularly engineered antibodies with fit-for-purpose properties will differentiate next generation antibody therapeutics from traditional IgG1 scaffolds. One requirement for engineering the most appropriate properties for a particular therapeutic area is an understanding of the intricacies of the target microenvironment in which the antibody is expected to function. Our group and others have demonstrated that proteases secreted by invasive tumors and pathological microorganisms are capable of cleaving human IgG1, the most commonly adopted isotype among monoclonal antibody therapeutics. Specific cleavage in the lower hinge of IgG1 results in a loss of Fc-mediated cell-killing functions without a concomitant loss of antigen binding capability or circulating antibody half-life. Proteolytic cleavage in the hinge region by tumor-associated or microbial proteases is postulated as a means of evading host immune responses, and antibodies engineered with potent cell-killing functions that are also resistant to hinge proteolysis are of interest. Mutation of the lower hinge region of an IgG1 resulted in protease resistance but also resulted in a profound loss of Fc-mediated cell-killing functions. In the present study, we demonstrate that specific mutations of the CH2 domain in conjunction with lower hinge mutations can restore and sometimes enhance cell-killing functions while still retaining protease resistance. By identifying mutations that can restore either complement- or Fcγ receptor-mediated functions on a protease-resistant scaffold, we were able to generate a novel protease-resistant platform with selective cell-killing functionality.

A monoclonal antibody against hinge-cleaved IgG restores effector function to proteolytically-inactivated IgGs in vitro and in vivo.

We report a chimeric monoclonal antibody (mAb) directed to a neo-epitope that is exposed in the IgG lower hinge following proteolytic cleavage. The mAb, designated 2095-2, displays specificity for IdeS-generated F(ab')2 fragments, but not for full-length IgG or for closely-related F(ab')2 fragments generated with other proteases. A critical component of the specificity is provided by the C-terminal amino acid of the epitope corresponding to gly-236 in the IgG1 (also IgG4) hinge. By its ability to bind to IdeS-cleaved anti-CD20 mAb, mAb 2095-2 fully restored antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against WIL2-S cells to the otherwise inactive anti-CD20 IgG1 F(ab')2 fragment. Similarly, 2095-2 reinstated ADCC against MDA-MB-231 cells to an anti-CD142 IgG1 F(ab')2 fragment. mAb 2095-2 was also capable of eliciting both CDC and ADCC to IgG4 F(ab')2 fragments, an IgG subclass that has weaker ADCC and CDC when intact relative to intact IgG1. The in vitro cell-based efficacy of 2095-2 was extended to the in vivo setting using platelets as a cell clearance surrogate. In a canine model, the co-administration of 2095-2 together with IdeS-generated, platelet-targeting anti-CD41/61 F(ab')2 fragment not only restored platelet clearance, but did so at a rate and extent of clearance that exceeded that of intact anti-CD41/61 IgG at comparable concentrations. To further explore this unexpected amplification effect, we conducted a rat study in which 2095-2 was administered at a series of doses in combination with a fixed dose of anti-CD41/61 F(ab')2 fragments. Again, the combination, at ratios as low as 1:10 (w/w) 2095-2 to F(ab')2, proved more effective than the anti-CD41/61 IgG1 alone. These findings suggest a novel mechanism for enhancing antibody-mediated cell-killing effector functions with potential applications in pathologic settings such as tumors and acute infections where protease activity is abundant.

Development of a recombinant toxin fragment vaccine for Clostridium difficile infection.

Clostridium difficile infection (CDI) is the major cause of antibiotic-associated diarrhea and pseudomembranous colitis, a disease associated with significant morbidity and mortality. The disease is mostly of nosocomial origin, with elderly patients undergoing anti-microbial therapy being particularly at risk. C. difficile produces two large toxins: Toxin A (TcdA) and Toxin B (TcdB). The two toxins act synergistically to damage and impair the colonic epithelium, and are primarily responsible for the pathogenesis associated with CDI. The feasibility of toxin-based vaccination against C. difficile is being vigorously investigated. A vaccine based on formaldehyde-inactivated Toxin A and Toxin B (toxoids) was reported to be safe and immunogenic in healthy volunteers and is now undergoing evaluation in clinical efficacy trials. In order to eliminate cytotoxic effects, a chemical inactivation step must be included in the manufacturing process of this toxin-based vaccine. In addition, the large-scale production of highly toxic antigens could be a challenging and costly process. Vaccines based on non-toxic fragments of genetically engineered versions of the toxins alleviate most of these limitations. We have evaluated a vaccine assembled from two recombinant fragments of TcdB and explored their potential as components of a novel experimental vaccine against CDI. Golden Syrian hamsters vaccinated with recombinant fragments of TcdB combined with full length TcdA (Toxoid A) developed high titer IgG responses and potent neutralizing antibody titers. We also show here that the recombinant vaccine protected animals against lethal challenge with C. difficile spores, with efficacy equivalent to the toxoid vaccine. The development of a two-segment recombinant vaccine could provide several advantages over toxoid TcdA/TcdB such as improvements in manufacturability.

Development and optimization of a novel assay to measure neutralizing antibodies against Clostridium difficile toxins.

Clostridium difficile produces two major virulence toxins, toxin A (TcdA) and toxin B (TcdB). Antitoxin antibodies, especially neutralizing antibodies, have been shown to be associated with a lower incidence of C. difficile infection (CDI) recurrence, and antibody levels are predictive of asymptomatic colonization. The development of an assay to detect the presence of neutralizing antibodies in animal and human sera for the evaluation of vaccine efficacy is highly desired. We have developed such an assay, which allows for the quantification of the effect of toxins on eukaryotic cells in an automated manner. We describe here the optimization of this assay to measure toxin potency as well as neutralizing antibody (NAb) activity against C. difficile toxins using a design-of-experiment (DOE) methodology. Toxin concentration and source, cell seeding density, and serum-toxin preincubation time were optimized in the assay using Vero cells. The assay was shown to be robust and to produce linear results across a range of antibody concentrations. It can be used to quantify neutralizing antibodies in sera of monkeys and hamsters immunized with C. difficile toxoid vaccines. This assay was shown to correlate strongly with traditional assays which rely on labor-intensive methods of determining neutralizing antibody titers by visual microscopic inspection of intoxicated-cell monolayers. This assay has utility for the selection and optimization of C. difficile vaccine candidates.