IgA as a potential candidate for enteric monoclonal antibody therapeutics with improved gastrointestinal stability

Mucosal surfaces of the gastrointestinal tract play an important role in immune homeostasis and defense and may be compromised by enteric disorders or infection. Therapeutic intervention using monoclonal antibody (mAb) offers the potential for treatment with minimal off-target effects as well as the possibility of limited systemic exposure when administered orally. Critically, to achieve efficacy at luminal surfaces, mAb must remain stable and functionally active in the gastrointestinal environment. To better understand the impact of isotype, class, and molecular structure on the intestinal stability of recombinant antibodies, we used an in vitro simulated intestinal fluid (SIF) assay to evaluate a panel of antibody candidates for enteric mAb-based therapeutics. Recombinant IgG1 was the least stable following SIF incubation, while the stability of IgA generally increased upon polymerization, with subtle differences between subclasses. Notably, patterns of variability within and between mAbs suggest that variable regions contribute to mAb stability and potentially mediate mAb susceptibility to proteases. Despite relatively rapid degradation in SIF, mAbs targeting Enterotoxigenic Escherichia coli (ETEC) displayed functional activity following SIF treatment, with SIgA1 showing improved function compared to SIgA2. The results of this study have implications for the design of enteric therapeutics and subsequent selection of lead candidates based upon in vitro intestinal stability assessments

Analytical characterization and formulation assessment of model secretory- immunoglobulin-A (sIgAs) for their potential use as low cost, orally delivered sIgAs

Enterotoxigenic Escherichia coli (ETEC) is a major cause of bacterial diarrheal disease in developing countries, especially among children and infants. ETEC is estimated to cause 280-400 million diarrheal episodes per year in children \u3c5 years of age, resulting in 300,000 to 500,000 deaths.1 Despite the need for a vaccine, there are currently no licensed vaccines against ETEC. Alternatively, passive immunization by oral delivery of pathogen-specific immunoglobulins is another promising approach to provide “instant” protection against ETEC. The potential advantages of oral delivery are reduced cost, simplicity of administration and localized treatment within the GI tract. Secretory IgA (sIgA) is of particular interest because it is naturally found in the mucosal surfaces within the GI tract, relatively more resistant to proteolysis by digestive enzymes (vs. IgGs), and can protect against enteric bacteria by directly neutralizing virulence factors.2 One major challenge of this approach is the instability of protein molecules during oral delivery (in the digestive tract) as well as during long-term storage (in various formulations). In this study, two proteins, sIgA1 and sIgA2 against heat labile toxin (LT, one of the major virulence factors of ETEC), were used as model sIgA molecules for developing analytical techniques and assessing stability (physicochemical as well as in vitro binding) under various conditions. A combination of biochemical and biophysical methods were employed to comprehensively characterize the sIgA1 and sIgA2 model proteins including primary structure, post translational modifications (i.e., N-linked glycans), size, apparent solubility, higher order structure and conformational stability as well as in vitro antigen binding. Using these characterization and stability indicating methods, we are monitoring the stability of these two model sIgAs both in an in vitro digestion model (to mimic in vivo degradation conditions), and during accelerated stability studies (to assess storage stability). Our goal is to use the information gained by these aforementioned methods and stability studies to design stable, low-cost liquid formulations for oral delivery of sIgAs in the developing world. Please click Additional Files below to see the full abstract

Preformulation Characterization and Stability Assessments of Secretory IgA Monoclonal Antibodies as Potential Candidates for Passive Immunization by Oral Administration

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrheal disease in children in developing countries, and there are no licensed vaccines to protect against ETEC. Passive immunization by oral delivery of ETEC-specific secretory IgAs (sIgAs) could potentially provide an alternative approach for protection in targeted populations. In this study, a series of physiochemical techniques and an in vitro gastric digestion model were used to characterize and compare key structural attributes and stability profiles of three anti-heat labile enterotoxin monoclonal antibodies (sIgA1, sIgA2 and IgG1 produced in CHO cells). The mAbs were evaluated in terms of primary structure, N-linked glycan profiles, size and aggregate content, relative apparent solubility, conformational stability, and in vitro antigen binding. Compared to IgG1 mAb, sIgA1 and sIgA2 mAbs showed increased sample heterogeneity, especially in terms of N-glycan composition and the presence of higher molecular weight species. The sIgA mAbs showed overall better physical stability and were more resistant to loss of antigen binding activity during incubation at low pH, 37 degrees C with pepsin. These results are discussed in terms of future challenges to design stable, low-cost formulations of sIgA mAbs as an oral supplement for passive immunization to protect against enteric diseases in the developing world

Immune features that afford protection from clinical disease versus sterilizing immunity to Bordetella pertussis infection in a nonhuman primate model of whooping cough

The respiratory bacterial infection caused by Bordetella pertussis (whooping cough) is the only vaccine-preventable disease whose incidence has been increasing over the last 3 decades. To better understand the resurgence of this infection, a baboon animal model of pertussis infection has been developed. Naïve baboons that recover from experimental pertussis infection are resistant both to clinical disease and to airway colonization when re-challenged. In contrast, animals vaccinated with acellular pertussis vaccine and experimentally challenged do not develop disease, but airways remain colonized for 4-6 weeks. We explored the possibility that the IgG antibody response to pertussis infection is qualitatively different from antibodies induced by acellular pertussis vaccination. IgG was purified from pertussis-convalescent baboons shown to be resistant to pertussis disease and airway colonization. Purified IgG contained high titers to pertussis toxin, pertactin, and filamentous hemagglutinin. This pertussis-immune IgG or control IgG was passively transferred to naïve, juvenile baboons before experimental airway pertussis inoculation. The control animal that received normal IgG developed a typical symptomatic infection including leukocytosis, cough and airway colonization for 4 weeks. In contrast, baboons that received convalescent IgG maintained normal WBC counts and were asymptomatic. However, despite remaining asymptomatic, their airways were colonized for 4-6 weeks with B. pertussis. All animals developed IgG and IgA anti-pertussis antibody responses. Interestingly, the clearance of B. pertussis from airways coincided with the emergence of a serum anti-pertussis IgA response. These studies demonstrate that passive administration of pertussis-specific IgG from previously infected animals can prevent clinical disease but does not affect prolonged airway colonization with B. pertussis. This outcome is similar to that observed following acellular pertussis vaccination. Understanding immune mechanisms—other than IgG—that are capable of preventing airway colonization with B. pertussis will be critical for developing more effective vaccines to prevent whooping cough

Identification and Characterization of Human Monoclonal Antibodies for Immunoprophylaxis Against Enterotoxigenic Escherichia coli Infection

Background. Enterotoxigenic Escherichia coli (ETEC) cause diarrheal illness in infants in the developing world and travelers to endemic countries including military personnel. ETEC infection of the host involves colonization of the small intestinal epithelium and toxin secretion leading to watery diarrhea. There is currently no vaccine licensed to prevent ETEC. CFA/I is one of the most common colonization factor antigens (CFAs). The CFA/I adhesin subunit, CfaE, is required for ETEC adhesion to host intestinal cells. Human antibodies against CfaE have potential to block colonization of ETEC and serve as an immunoprophylactic against ETEC-related diarrhea. Methods. Mice transgenic for human immunoglobulin genes were immunized with CfaE to generate a panel of human monoclonal IgG1 antibodies (HuMAbs). The most potent IgG1 identified in the in vitro functional assays were selected and isotype switched to secretory IgA (sIgA) and tested in animal colonization assays via oral administration. Results. Over 300 unique anti-CfaE IgG1 HuMabs were identified. The lead IgG1 anti-CfaE HuMAbs completely inhibited hemagglutination and blocked adhesion of ETEC to Caco-2 cells. Epitope mapping studies revealed that HuMAbs recognized epitopes in the N-terminal domain of CfaE near the putative receptor binding site. Oral administration of anti-CfaE antibodies in either IgG or secretory IgA isotypes inhibited intestinal colonization in mice challenged with ETEC. A two to four log decrease of colony forming units was observed as compared to irrelevant isotype controls. Conclusions. We identified fully human monoclonal antibodies against CfaE adhesion domain that can be potentially employed as an immunoprophylaxis to prevent ETEC-related diarrhea

Anti-CfaE nanobodies provide broad cross-protection against major pathogenic enterotoxigenic Escherichia coli strains, with implications for vaccine design

Enterotoxigenic Escherichia coli (ETEC) is estimated to cause approximately 380,000 deaths annually during sporadic or epidemic outbreaks worldwide. Development of vaccines against ETEC is very challenging due to the vast heterogeneity of the ETEC strains. An effective vaccines would have to be multicomponent to provide coverage of over ten ETEC strains with genetic variabilities. There is currently no vaccine licensed to prevent ETEC. Nanobodies are successful new biologics in treating mucosal infectious disease as they recognize conserved epitopes on hypervariable pathogens. Cocktails consisting of multiple nanobodies could provide even broader epitope coverage at a lower cost compared to monoclonal antibodies. Identification of conserved epitopes by nanobodies can also assist reverse engineering of an effective vaccine against ETEC. By screening nanobodies from immunized llamas and a naive yeast display library against adhesins of colonization factors, we identified single nanobodies that show cross-protective potency against eleven major pathogenic ETEC strains in vitro. Oral administration of nanobodies led to a significant reduction of bacterial colonization in animals. Moreover, nanobody-IgA fusion showed extended inhibitory activity in mouse colonization compared to commercial hyperimmune bovine colostrum product used for prevention of ETEC-induced diarrhea. Structural analysis revealed that nanobodies recognized a highly-conserved epitope within the putative receptor binding region of ETEC adhesins. Our findings support further rational design of a pan-ETEC vaccine to elicit robust immune responses targeting this conserved epitope

A cross-reactive human IgA monoclonal antibody blocks SARS-CoV-2 spike-ACE2 interaction

COVID-19 caused by SARS-CoV-2 has become a global pandemic requiring the development of interventions for the prevention or treatment to curtail mortality and morbidity. No vaccine to boost mucosal immunity, or as a therapeutic, has yet been developed to SARS-CoV-2. In this study, we discover and characterize a cross-reactive human IgA monoclonal antibody, MAb362. MAb362 binds to both SARS-CoV and SARS-CoV-2 spike proteins and competitively blocks ACE2 receptor binding, by overlapping the ACE2 structural binding epitope. Furthermore, MAb362 IgA neutralizes both pseudotyped SARS-CoV and SARS-CoV-2 in 293 cells expressing ACE2. When converted to secretory IgA, MAb326 also neutralizes authentic SARS-CoV-2 virus while the IgG isotype shows no neutralization. Our results suggest that SARS-CoV-2 specific IgA antibodies, such as MAb362, may provide effective immunity against SARS-CoV-2 by inducing mucosal immunity within the respiratory system, a potentially critical feature of an effective vaccine

Opioid Misuse: An Organizational Response While Managing Cancer-Related Pain.

BACKGROUND: Opioids are commonly prescribed to manage cancer pain. Similar to the general population, patients with cancer are not excluded from the risk for opioid misuse and dependence. This situation can contribute to clinician reluctance to prescribe and manage pain using opioids. OBJECTIVES: The purpose of this article is to provide an overview of how opioid misuse may affect pain management in patients with cancer and to describe a comprehensive cancer center\u27s approach to safely managing cancer pain. METHODS: Based on a literature review, the project team developed a stewardship program. Project components included selecting a validated screening tool for risk of opioid misuse, determining if a history of addiction affects pain management in patients with cancer, and establishing a task force to focus on the opioid crisis and to follow the Joint Commission\u27s revised pain assessment and management standards. FINDINGS: The project established a hospitalwide opioid stewardship program. Through the use of a multidisciplinary, universal precautions approach to assessing misuse in all patients with cancer who are prescribed opioids, healthcare teams can potentially reduce risks associated with misuse while safely managing cancer pain

IgA MAb blocks SARS-CoV-2 Spike-ACE2 interaction providing mucosal immunity [preprint]

COVID-19 caused by SARS-CoV-2 has become a global pandemic requiring the development of interventions for the prevention or treatment to curtail mortality and morbidity. No vaccine to boost mucosal immunity or as a therapeutic has yet been developed to SARS-CoV-2. In this study we discover and characterize a cross-reactive human IgA monoclonal antibody, MAb362. MAb362 binds to both SARS-CoV and SARS-CoV-2 spike proteins and competitively blocks hACE2 receptor binding, by completely overlapping the hACE2 structural binding epitope. Furthermore, MAb362 IgA neutralizes both pseudotyped SARS-CoV and SARS-CoV-2 in human epithelial cells expressing hACE2. SARS-CoV-2 specific IgA antibodies, such as MAb362, may provide effective immunity against SARS-CoV-2 by inducing mucosal immunity within the respiratory system, a potentially critical feature of an effective vaccine

Oral administration of an anti-CfaE secretory IgA antibody protects against Enterotoxigenic Escherichia coli diarrheal disease in a non-human primate model [preprint]

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of diarrhea-associated illness in developing countries. There is currently no vaccine licensed to prevent ETEC and the development of an efficacious prophylaxis would provide an intervention with significant impact. Recent studies suggested that effective protection could be achieved by inducing immunity to block colonization of ETEC. Here, we evaluated the efficacy of secretory (s) IgA2 and dimeric (d) IgA2 of an anti-colonization factor antigen antibody, 68-61, in the Aotus nancymaae non-human primate (NHP) ETEC challenge model via oral and parental delivery. Thirty-nine animals were distributed across 3 groups of 13, and challenged with 5.0×1011 cfu of H10407 on Day 0. Group 1 received a dIgA2 68-61 subcutaneously on day 0. Group 2 received a SIgA2 68-61 orally on days −1, 0, and +1, and Group 3 received an irrelevant SIgA2 antibody orally on days −1, 0, and +1. All animals were observed for symptoms of diarrhea, and stools were collected for ETEC colony counts. SIgA2 treatment significantly lowered the attack rate, resulting in a protective efficacy of 71.4% (p=0.025) in Group 2 as compared to Group 3. Anti-CfaE dIgA2 treatment group reduced the diarrheal attack rate, although the reduction did not reach significance (57.1%; P=0.072) as compared to the irrelevant SIgA2 Group 3. Our results demonstrated the feasibility of oral administration of SIgA as a potential immunoprophylaxis against enteric infections. To our knowledge, this is the first study to demonstrate the efficacy of administrated SIgA in a non-human primate model