Developing anti-GDF6 therapeutics for treatment of advanced melanoma

Melanoma, the leading cause of skin cancer death in the U.S., is increasing in incidence. Targeted therapies have been approved for treatment of advanced melanoma, but few patients experience extended survival benefit. In order to combat poor outcomes, new therapeutic targets are needed. Using cross-species oncogenomic analyses, our lab has identified a novel melanoma driver, Growth differentiation factor 6 (GDF6), a secreted bone morphogenetic protein (BMP) ligand that is amplified and overexpressed in human melanomas. Functional analyses show GDF6 acts via the BMP-SMAD1 pathway as a pro-survival factor in melanomas. Inhibiting GDF6 or the BMP pathway using shRNAs or the small molecule inhibitor, DMH1, induces melanoma cell death thereby abrogating melanoma growth in mouse xenografts. These results suggest GDF6 is an optimal target melanoma therapy. In order to better understand the dynamics of GDF6 signaling in melanoma cells, we are currently investigating the effect of exogenous GDF6 on cells with inhibited GDF6 expression to determine the required concentration to activate SMAD1 signaling and rescue viability. As GDF6 is a secreted ligand, we proposed developing antibodies to block the GDF6 interaction at its receptor, thereby inhibiting signaling. In collaboration with MassBiologics, we have generated a panel of monoclonal antibodies targeting GDF6. To identify antibodies capable of blocking GDF6 activity, we have devised a series of assays to eliminate antibodies from the panel. First, candidates are screened for affinity to GDF6. Second, candidates are screened for ability to block interaction between GDF6 and its receptor. Third, candidates are evaluated for ability to inhibit downstream signaling via SMAD1 pathway. After selection of final candidates, we will use a xenograft model to determine ability to inhibit melanoma growth in vivo. Currently, we have identified antibodies that are able to recognize GDF6 via western blot, and are proceeding to screen these antibodies for anti-GDF6 activity

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

Rational Design of an Epitope-Based Hepatitis C Virus Vaccine

Despite improving treatment methods and therapeutic options, hepatitis C virus (HCV) remains a major global disease burden, and a vaccine would help greatly in reducing its incidence. Due to its extremely high sequence variability, HCV can readily escape the immune response, thus a vaccine must elicit an immune response toward conserved, functionally important epitopes. Using structural data of the broadly neutralizing antibody HCV1 in complex with a conserved linear epitope from the HCV E2 protein (aa 412-423, referred to as epitope I or domain E), we performed structure-based design to generate vaccine immunogens to induce antibody responses to this epitope. Designs selected for immunological characterization included a stabilized minimal epitope structure based on a defensin protein, as well as a bivalent vaccine featuring two copies of epitope I on the E2 surface. In vivo studies confirmed that these designs successfully generated robust antibody responses to this epitope, and sera from vaccinated mice neutralized HCV. In addition to presenting several effective HCV vaccine immunogens, this study demonstrates that induction of neutralizing anti-HCV antibodies is possible using an epitope-based vaccine, providing the basis for further efforts in structure-based vaccine design to target this and other critical epitopes of HCV

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

Conformational dynamics and allosteric modulation of the SARS-CoV-2 spike

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects cells through binding to angiotensin-converting enzyme 2 (ACE2). This interaction is mediated by the receptor-binding domain (RBD) of the viral spike (S) glycoprotein. Structural and dynamic data have shown that S can adopt multiple conformations, which controls the exposure of the ACE2-binding site in the RBD. Here, using single-molecule Forster resonance energy transfer (smFRET) imaging, we report the effects of ACE2 and antibody binding on the conformational dynamics of S from the Wuhan-1 strain and in the presence of the D614G mutation. We find that D614G modulates the energetics of the RBD position in a manner similar to ACE2 binding. We also find that antibodies that target diverse epitopes, including those distal to the RBD, stabilize the RBD in a position competent for ACE2 binding. Parallel solution-based binding experiments using fluorescence correlation spectroscopy (FCS) indicate antibody-mediated enhancement of ACE2 binding. These findings inform on novel strategies for therapeutic antibody cocktails

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

Pre-exposure prophylaxis with OspA-specific human monoclonal antibodies protects mice against tick transmission of Lyme disease spirochetes

Background. Tick transmission of Borrelia spirochetes to humans results in significant morbidity from Lyme disease worldwide. Serum concentrations of antibodies against outer surface protein A (OspA) were shown to correlate with protection from infection with Borrelia burgdorferi, the primary cause of Lyme disease in the United States. Methods. Mice transgenic for human immunoglobulin genes were immunized with OspA protein of B. burgdorferi to generate human monoclonal antibodies (HuMabs) against OspA. HuMabs were generated and tested in in vitro borreliacidal assays and animal protection assays. Results. Nearly 100 unique OspA specific HuMabs were generated and four HuMabs (221-7, 857-2, 319-44, and 212-55) were selected as lead candidates based on borreliacidal activity. HuMab 319-44, 857-2 and 212-55 were borreliacidal against one or two Borrelia genospecies, whereas 221-7 was borreliacidal (IC50 \u3c 1nM) against B. burgdorferi, B. afzelii and B. garinii, the three main genospecies endemic in the US, Europe and Asia. All four HuMabs completely protected mice from infection at 10 mg/kg in a murine model of tick-mediated transmission of B. burgdorferi. Conclusions. Our study indicates that OspA-specific HuMabs can prevent the transmission of Borrelia and administration of these antibodies could be employed as pre-exposure prophylaxis for 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

Structure-Based Design of Hepatitis C Virus Vaccines That Elicit Neutralizing Antibody Responses to a Conserved Epitope

Despite recent advances in therapeutic options, hepatitis C virus (HCV) remains a severe global disease burden, and a vaccine can substantially reduce its incidence. Due to its extremely high sequence variability, HCV can readily escape the immune response; thus, an effective vaccine must target conserved, functionally important epitopes. Using the structure of a broadly neutralizing antibody in complex with a conserved linear epitope from the HCV E2 envelope glycoprotein (residues 412 to 423; epitope I), we performed structure-based design of immunogens to induce antibody responses to this epitope. This resulted in epitope-based immunogens based on a cyclic defensin protein, as well as a bivalent immunogen with two copies of the epitope on the E2 surface. We solved the X-ray structure of a cyclic immunogen in complex with the HCV1 antibody and confirmed preservation of the epitope conformation and the HCV1 interface. Mice vaccinated with our designed immunogens produced robust antibody responses to epitope I, and their serum could neutralize HCV. Notably, the cyclic designs induced greater epitope-specific responses and neutralization than the native peptide epitope. Beyond successfully designing several novel HCV immunogens, this study demonstrates the principle that neutralizing anti-HCV antibodies can be induced by epitope-based, engineered vaccines and provides the basis for further efforts in structure-based design of HCV vaccines. IMPORTANCE: Hepatitis C virus is a leading cause of liver disease and liver cancer, with approximately 3% of the world\u27s population infected. To combat this virus, an effective vaccine would have distinct advantages over current therapeutic options, yet experimental vaccines have not been successful to date, due in part to the virus\u27s high sequence variability leading to immune escape. In this study, we rationally designed several vaccine immunogens based on the structure of a conserved epitope that is the target of broadly neutralizing antibodies. In vivo results in mice indicated that these antigens elicited epitope-specific neutralizing antibodies, with various degrees of potency and breadth. These promising results suggest that a rational design approach can be used to generate an effective vaccine for this virus

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