“So You Think You Have a Drug You Want to Develop”

Mark Klempner, MD, Executive Vice Chancellor, MassBiologics of UMass Medical School and Professor of Medicine, describes MassBiologics and their process for developing a new medicine. MassBiologics is the only non-profit, FDA-licensed manufacturer of vaccines and biologics in the United States

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

Background. Enterotoxigenic Escherichia coli (ETEC) infections are the major cause of diarrheal morbidity among children living in developing countries. ETEC mediates small intestine adherence through bacterial adhesion followed by production of enterotoxins that induce diarrhea. Currently there is no vaccine available for ETEC. One of the most predominant adhesin of pathogenic ETEC strains is colonization factor antigen I (CFA/I). The CFA/I adhesion tip, CfaE, is required for ETEC binding to human intestinal cells and colonization. Human antibodies against CfaE have potential to block colonization of ETEC and serve as a potent immunoprophylactic against ETEC-related diarrhea. Methods. A panel of human IgG1 monoclonal antibodies (HuMAbs) were generated against CfaE. The antibodies were tested in vitro for blockage of bacterial adhesion to intestinal cells and in vivo for inhibition of bacterial colonization in the ileum. Antibody epitope analysis were performed using BioLuminate software (Schrodinger, Inc.), followed by mutagenesis of the predicted residues located in the antibody/CfaE interface and in-vitro binding assays. Results. The lead IgG1 anti-CfaE HuMAbs blocked 50% of adhesion of ETEC bacterial cells to human intestinal cells at concentrations ranging from 0.3 to 1.3 ug/ml. In vivo studies revealed 2 to 4 log decrease in colony forming units in the small intestine when the bacteria were pre-incubated with anti-CfaE MAbs as compared to an irrelevant isotype control. In silico epitope analysis revealed critical residues involved in the MAbs interaction with CfaE. Two of the leads HuMabs recognize epitopes sequence conserved across other 6 major adhesins. Conclusions. We have identified a panel of fully human IgG1 monoclonal antibodies against CfaE protein of ETEC. These antibodies are capable of blocking in vitro and in vivo ETEC adhesion to intestinal cells at low concentrations. Two lead antibodies recognizing sequence conserved epitopes have the potential for cross-protection against multiple ETEC strains

Identification of GDF-6 blocking antibodies as anti-melanoma therapeutics

Through comparative oncogenomic studies and functional analyses, we have identified the bone morphogenetic protein (BMP) factor GDF6 as a new melanoma oncogene. The secreted, carboxy-terminal portion of GDF6 is the active form that binds to cell-surface receptors to initiate BMP signaling. Targeted antibodies directed against secreted proteins are a proven therapeutic modality in several diseases. To develop therapeutic antibodies against the active form of GDF6, we generated a panel of monoclonal antibodies. Due to the high similarity of human and mouse GDF6 proteins, the C-terminal GDF6 protein was expressed as bacterial recombinant protein with fusion tags to enhance immunogenicity. The Expresso Screening System (Lucigen) was used to select fusion tags, and MBP and SlyD were chosen for optimal protein solubility and purification recovery. Ten CD1 mice were immunized with GDF6-MBP fusion protein and robust immune responses were observed in all animals after 5 immunizations. Animals were sacrificed for hybridoma fusion, and hybridoma clones were screened by ELISA using GDF6-SlyD fusion protein to select clones with specific binding activity to GDF6. Over 70 monoclonal antibodies were identified with strong reactivity to GDF6, and a subset has been shown to recognize the endogenous, secreted form of GDF6 via western blot. These antibodies will be screened for their activity to block GDF6 binding to melanoma cells and ability to inhibit downstream signaling using both in vitro assays and in vivo xenograft models

Identification of fully human monoclonal antibodies against the adhesin domain of colonizing factor antigen I of Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) causes significant diarrheal illness in infants in the developing world and travelers to endemic countries including military personnel. Infection of the host involves bacterial colonization of the small intestinal epithelium and toxin secretion leading to watery diarrhea. CFA/I is the most common colonizing factor antigens expressed on the surface of ETEC isolates. The CFA/I adhesin, CfaE, appears to be required for ETEC binding to human intestinal cells for colonization. Human antibodies against the binding domain of CfaE have potential to block colonization of ETEC and serve as a potent immunoprophylactic therapeutic for ETEC-related diarrhea. In the current study, we generated a panel of fully human monoclonal antibodies (HuMabs) against the adhesin domain of CfaE using mice transgenic for human immunoglobulin genes and identified lead antibodies utilizing a series of in vitro assays. Mice were immunized with the N-terminal binding domain of CfaE fused to maltose binding protein. Over thirty unique IgG1 HuMabs were identified with binding activity to recombinant CfaE. These antibodies were tested for inhibition of hemagglutination of type A human erythrocytes by ETEC. Two lead HuMabs, 837-6 and 840-53, inhibited hemagglutination at low concentrations (\u3c 1 nM). Both antibodies also blocked the binding of ETEC with intestinal epithelial cells. Biacore analysis revealed an affinity of less than 2 nM with distinct epitopes of CfaE. Our analysis suggests that CfaE specific HuMabs 837-6 and 840-53, as the first isolated fully human monoclonal antibodies against CfaE adhesion domain, could potentially be used in combination with heat labile toxin neutralizing antibodies to prevent traveler’s diarrhea

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

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

Pre-exposure Immunoprophylaxis by Genetically Encoded DMAb anti-OspA Human Monoclonal Antibody to Prevent Lyme Disease

Tick transmission of Borrelia spirochetes to humans results in significant morbidity from Lyme disease. Animal studies have demonstrated that transmission of Borrelia from tick vector to the mammalian host can be blocked by antibodies against outer surface protein A (OspA). We have recently developed borreliacidal human IgG1 monoclonal antibodies (HuMabs) directed against OspA. HuMab 319-44 was borreliacidal against B. burgdorferi (IC50Borreliatransmission after a single dose of 2 mg/kg administered on the day of tick challenge. Since passively administered IgG1 antibodies do not have a sufficient half-life to provide protection for the 6-7 month peak risk period, we investigated a novel approach of vector-mediated gene transfer of HuMabs that could potentially provide protection against Lyme disease during the seasonal risk period. A modified HuMab, 319-44 mod, expressed by a synthetic DNA plasmid (DMAb) was optimized and characterized in in vitro OspA binding and bactericidal assays. To assess in vivo protection, mice were administered a single DMAb injection into the quadriceps followed by electroporation. The mice were then challenged by B. burgdorferi-infected nymphs. Tissue samples were monitored by dark-field microscopy for spirochete growth. Serum samples were analyzed by ELISA to determine antibody concentrations. The modified 319-44 DMAb maintained in vitro biological activity comparable to the un-modified wild type antibody, and formulation-based delivery of DMAb resulted in long-term expression. This led to effective pre-exposure prophylaxis preventing transmission of spirochetes in 80% of mice in the murine model of tick-transmitted Lyme disease. These studies represent the first demonstration of employing DNA transfer as a rapid, novel delivery system for biologically relevant functional full-length HuMAbs in an in vivo animal model and provide support for such an approach for pre-exposure immunoprophylaxis to prevent Lyme disease

Structural and Molecular Analysis of a Protective Epitope of Lyme Disease Antigen OspA and Antibody Interactions

The murine monoclonal antibody LA-2 recognizes a clinically protective epitope on outer surface protein (OspA) of Borrelia burgdorferi, the causative agent of Lyme disease in North America. Human antibody equivalence to LA-2 is the best serologic correlate of protective antibody responses following OspA vaccination. Understanding the structural and functional basis of the LA-2 protective epitope is important for developing OspA-based vaccines and discovering prophylactic antibodies against Lyme disease. Here, we present a detailed structure-based analysis of the LA-2/OspA interaction interface and identification of residues mediating antibody recognition. Mutations were introduced into both OspA and LA-2 based on computational predictions on the crystal structure of the complex, and experimentally tested for in-vitro binding and borreliacidal activity. We find that Y32 and H49 on the LA-2 light chain, N52 on the LA-2 heavy chain and residues A208, N228 and N251 on OspA were the key constituents of OspA/LA-2 interface. These results reveal specific residues that may be exploited to modulate recognition of the protective epitope of OspA and have implications for design of vaccines against Lyme disease

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