Vaccination of mice with hybrid protein containing Exotoxin S and PcrV with adjuvants alum and MPL protects Pseudomonas aeruginosa infections.

Pseudomonas aeruginosa as a common pathogen causing urinary tract infections (UTIs) has been resistant to different antibiotics and developing an effective vaccine can be an alternative strategy. In the present study, the immunogenicity and protection efficacy of formulations composed of a hybrid protein composed of P. aeruginosa V-antigen (PcrV) and exoenzyme S (ExoS) with alum and MPL were evaluated. The hybrid protein could increase the specific systemic and mucosal immune responses, as well as cellular responses as compared with control groups. Combining of alum or MPL adjuvant with the hybrid protein significantly improved the levels of IgG1, serum IgA, mucosal IgG, and IL-17 as compared to the ExoS.PcrV alone. After bladder challenge with a P. aeruginosa strain, the bacterial loads of bladder and kidneys were significantly decreased in mice received ExoS.PcrV admixed with alum and ExoS.PcrV admixed with MPL than controls. The present study indicated that immunization of mice with a hybrid protein composed of ExoS and PcrV could induce multifactorial immune responses and opsonize the bacteria and decrease the viable bacterial cells. Because P. aeruginosa have caused therapeutic challenges worldwide, our study proposed ExoS.PcrV + alum and ExoS.PcrV + MPL as promising candidates for the prevention of infections caused by P. aeruginosa.

Ribosylation of the CD8αß heterodimer permits binding of the nonclassical major histocompatibility molecule, H2-Q10.

The CD8αß heterodimer plays a crucial role in the stabilization between major histocompatibility complex class I molecules (MHC-I) and the T cell receptor (TCR). The interaction between CD8 and MHC-I can be regulated by posttranslational modifications, which are proposed to play an important role in the development of CD8 T cells. One modification that has been proposed to control CD8 coreceptor function is ribosylation. Utilizing NAD+, the ecto-enzyme adenosine diphosphate (ADP) ribosyl transferase 2.2 (ART2.2) catalyzes the addition of ADP-ribosyl groups onto arginine residues of CD8α or ß chains and alters the interaction between the MHC and TCR complexes. To date, only interactions between modified CD8 and classical MHC-I (MHC-Ia), have been investigated and the interaction with non-classical MHC (MHC-Ib) has not been explored. Here, we show that ADP-ribosylation of CD8 facilitates the binding of the liver-restricted nonclassical MHC, H2-Q10, independent of the associated TCR or presented peptide, and propose that this highly regulated binding imposes an additional inhibitory leash on the activation of CD8-expressing cells in the presence of NAD+. These findings highlight additional important roles for nonclassical MHC-I in the regulation of immune responses.

A Methodological Approach Using rAAV Vectors Encoding Nanobody-Based Biologics to Evaluate ARTC2.2 and P2X7 In Vivo.

On murine T cells, mono-ADP ribosyltransferase ARTC2.2 catalyzes ADP-ribosylation of various surface proteins when nicotinamide adenine dinucleotide (NAD+) is released into the extracellular compartment. Covalent ADP-ribosylation of the P2X7 receptor by ARTC2.2 thereby represents an additional mechanism of activation, complementary to its triggering by extracellular ATP. P2X7 is a multifaceted receptor that may represents a potential target in inflammatory, and neurodegenerative diseases, as well as in cancer. We present herein an experimental approach using intramuscular injection of recombinant AAV vectors (rAAV) encoding nanobody-based biologics targeting ARTC2.2 or P2X7. We demonstrate the ability of these in vivo generated biologics to potently and durably block P2X7 or ARTC2.2 activities in vivo, or in contrast, to potentiate NAD+- or ATP-induced activation of P2X7. We additionally demonstrate the ability of rAAV-encoded functional heavy chain antibodies to elicit long-term depletion of T cells expressing high levels of ARTC2.2 or P2X7. Our approach of using rAAV to generate functional nanobody-based biologics in vivo appears promising to evaluate the role of ARTC2.2 and P2X7 in murine acute as well as chronic disease models.

Identification of the Mouse T Cell ADP-Ribosylome Uncovers ARTC2.2 Mediated Regulation of CD73 by ADP-Ribosylation.

Mouse T cells express the ecto-ADP-ribosyltransferase ARTC2.2, which can transfer the ADP-ribose group of extracellular nicotinamide adenine dinucleotide (NAD+) to arginine residues of various cell surface proteins thereby influencing their function. Several targets of ARTC2.2, such as P2X7, CD8a and CD25 have been identified, however a comprehensive mouse T cell surface ADP-ribosylome analysis is currently missing. Using the Af1521 macrodomain-based enrichment of ADP-ribosylated peptides and mass spectrometry, we identified 93 ADP-ribsoylated peptides corresponding to 67 distinct T cell proteins, including known targets such as CD8a and CD25 but also previously unknown targets such as CD73. We evaluated the impact of ADP-ribosylation on the capability of CD73 to generate adenosine from adenosine monophosphate. Our results show that extracellular NAD+ reduces the enzymatic activity of CD73 HEK cells co-transfected with CD73/ARTC2.2. Importantly, NAD+ significantly reduced CD73 activity on WT CD8 T cells compared to ARTC2ko CD8 T cells or WT CD8 T cells treated with an ARTC2.2-blocking nanobody. Our study provides a comprehensive list of T cell membrane proteins that serve as targets for ADP-ribosylation by ARTC2.2 and whose function may be therefore affected by ADP-ribosylation.

ADP-Ribosylation Regulates the Signaling Function of IFN-γ.

Murine T cells express the GPI-anchored ADP-ribosyltransferase 2.2 (ARTC2.2) on the cell surface. In response to T cell activation or extracellular NAD+ or ATP-mediated gating of the P2X7 ion channel ARTC2.2 is shed from the cell surface as a soluble enzyme. Shedding alters the target specificity of ARTC2.2 from cell surface proteins to secreted proteins. Here we demonstrate that shed ARTC2.2 potently ADP-ribosylates IFN-γ in addition to other cytokines. Using mass spectrometry, we identify arginine 128 as the target site of ADP-ribosylation. This residue has been implicated to play a key role in binding of IFN-γ to the interferon receptor 1 (IFNR1). Indeed, binding of IFN-γ to IFNR1 blocks ADP-ribosylation of IFN-γ. Moreover, ADP-ribosylation of IFN-γ inhibits the capacity of IFN-γ to induce STAT1 phosphorylation in macrophages and upregulation of the proteasomal subunit ß5i and the proteasomal activator PA28-α in podocytes. Our results show that ADP-ribosylation inhibits the signaling functions of IFN-γ and point to a new regulatory mechanism for controlling signaling by IFN-γ.

Isolation and characterizations of a novel recombinant scFv antibody against exotoxin A of Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is the leading cause of nosocomial infections, especially in people with a compromised immune system. Targeting virulence factors by neutralizing antibodies is a novel paradigm for the treatment of antibiotic-resistant pseudomonas infections. In this respect, exotoxin A is one of the most potent virulence factors in P. aeruginosa. The present study was carried out to identify a novel human scFv antibody against the P. aeruginosa exotoxin A domain I (ExoA-DI) from a human scFv phage library. METHODS: The recombinant ExoA-DI of P. aeruginosa was expressed in E. coli, purified by Ni-NTA column, and used for screening of human antibody phage library. A novel screening procedure was conducted to prevent the elimination of rare specific clones. The phage clone with high reactivity was evaluated by ELISA and western blot. RESULTS: Based on the results of polyclonal phage ELISA, the fifth round of biopanning leads to the isolation of several ExoA-DI reactive clones. One positive clone with high affinity was selected by monoclonal phage ELISA and used for antibody expression. The purified scFv showed high reactivity with the recombinant domain I and full-length native exotoxin A. CONCLUSIONS: The purified anti-exotoxin A scFv displayed high specificity against exotoxin A. The human scFv identified in this study could be the groundwork for developing a novel therapeutic agent to control P. aeruginosa infections.

Immunogenicity of Immunotoxins Containing Pseudomonas Exotoxin A: Causes, Consequences, and Mitigation.

Immunotoxins are cytolytic fusion proteins developed for cancer therapy, composed of an antibody fragment that binds to a cancer cell and a protein toxin fragment that kills the cell. Pseudomonas exotoxin A (PE) is a potent toxin that is used for the killing moiety in many immunotoxins. Moxetumomab Pasudotox (Lumoxiti) contains an anti-CD22 Fv and a 38 kDa portion of PE. Lumoxiti was discovered in the Laboratory of Molecular Biology at the U.S. National Cancer Institute and co-developed with Medimmune/AstraZeneca to treat hairy cell leukemia. In 2018 Lumoxiti was approved by the US Food and Drug Administration for the treatment of drug-resistant Hairy Cell Leukemia. Due to the bacterial origin of the killing moiety, immunotoxins containing PE are highly immunogenic in patients with normal immune systems, but less immunogenic in patients with hematologic malignancies, whose immune systems are often compromised. LMB-100 is a de-immunized variant of the toxin with a humanized antibody that targets mesothelin and a PE toxin that was rationally designed for diminished reactivity with antibodies and B cell receptors. It is now being evaluated in clinical trials for the treatment of mesothelioma and pancreatic cancer and is showing somewhat diminished immunogenicity compared to its un modified parental counterpart. Here we review the immunogenicity of the original and de-immunized PE immunotoxins in mice and patients, the development of anti-drug antibodies (ADAs), their impact on drug availability and their effect on clinical efficacy. Efforts to mitigate the immunogenicity of immunotoxins and its impact on immunogenicity will be described including rational design to identify, remove, or suppress B cell or T cell epitopes, and combination of immunotoxins with immune modulating drugs.

Mechanisms of Resistance to Immunotoxins Containing Pseudomonas Exotoxin A in Cancer Therapy.

Immunotoxins are a class of targeted cancer therapeutics in which a toxin such as Pseudomonas exotoxin A (PE) is linked to an antibody or cytokine to direct the toxin to a target on cancer cells. While a variety of PE-based immunotoxins have been developed and a few have demonstrated promising clinical and preclinical results, cancer cells frequently have or develop resistance to these immunotoxins. This review presents our current understanding of the mechanism of action of PE-based immunotoxins and discusses cellular mechanisms of resistance that interfere with various steps of the pathway. These steps include binding of the immunotoxin to the target antigen, internalization, intracellular processing and trafficking to reach the cytosol, inhibition of protein synthesis through ADP-ribosylation of elongation factor 2 (EF2), and induction of apoptosis. Combination therapies that increase immunotoxin action and overcome specific mechanisms of resistance are also reviewed.

A novel approach to assess domain specificity of anti-drug antibodies to moxetumomab pasudotox, an immunotoxin with two functional domains.

Biologics are potentially immunogenic and can elicit immune response. Complex biologics, such as bispecific antibodies or multi-domain molecules can induce anti-drug antibodies (ADA) with specificity to different domains. Domain specific ADAs may differently affect drug efficacy and safety, and thus, characterization of ADA domain specificity has become a regulatory expectation for multi-domain biologics. Unlike well-established methods for screening, confirmation, titer and neutralizing ADA detection, characterization of ADA domain specificity is an emerging field. The conventional approach for determination of ADA domain specificity is a competitive inhibition with domain-containing molecules. When developing a conventional domain specificity assay for moxetumomab pasudotox, a recombinant anti-CD22 immunotoxin, comprised of two functional domains (CD22-binding fragment and truncated Pseudomonas exotoxin A (PE38), we encountered a bioanalytical challenge. The method was able to detect immunodominant anti-PE38 (ADA-PE) but generated false negative results for low abundant CD22-binding domain ADA (ADA-BD) in a polyclonal sample. Troubleshooting experiments using control samples with varying levels of each ADA subtype demonstrated that a major factor for successful ADA identification was the ratio of the ADA signals contributed by each ADA subtype. To overcome this unique bioanalytical challenge, we developed a novel approach, which ensures detection of a domain-specific ADA subtype regardless of its relative level in a polyclonal ADA sample by evaluating signal inhibition by a respective domain-containing molecule at the condition when signals from all other ADAs are fully blocked. The method has been used for characterization of ADA domain specificity in moxetumomab pasudotox clinical trials, including study 1053, the pivotal Phase III study in hairy cell leukemia patients. It allowed for successful detection of ADA-BD in the presence of immunodominant ADA-PE, enabling accurate determination of domain specificity for moxetumomab pasudotox. The results demonstrated that the method was superior than the conventional approach. The method could be applied broadly to other biologics with two or more domains when there is a need to detect a minor ADA subtype in polyclonal samples.

Human single-chain antibodies that neutralize Pseudomonas aeruginosa-exotoxin A-mediated cellular apoptosis.

Targeting bacterial virulence factors directly provides a new paradigm for the intervention and treatment of bacterial diseases. Pseudomonas aeruginosa produces a myriad of virulence factors to cause fatal diseases in humans. In this study, human single-chain antibodies (HuscFvs) that bound to P. aeruginosa exotoxin A (ETA) were generated by phage display technology using recombinant ETA, ETA-subdomains and the synthetic peptide of the ETA-catalytic site as baits for selecting ETA-bound-phages from the human-scFv phage display library. ETA-bound HuscFvs derived from three phage-transfected E. coli clones neutralized the ETA-induced mammalian cell apoptosis. Computerized simulation demonstrated that these HuscFvs used several residues in their complementarity-determining regions (CDRs) to form contact interfaces with the critical residues in ETA-catalytic domain essential for ADP-ribosylation of eukaryotic elongation factor 2, which should consequently rescue ETA-exposed-cells from apoptosis. The HuscFv-treated ETA-exposed cells also showed decremented apoptosis-related genes, i.e., cas3 and p53. The effective HuscFvs have high potential for future evaluation in animal models and clinical trials as a safe, novel remedy for the amelioration of exotoxin A-mediated pathogenesis. HuscFvs may be used either singly or in combination with the HuscFv cognates that target other P. aeruginosa virulence factors as an alternative therapeutic regime for difficult-to-treat infections.

Anti-Mesothelin Recombinant Immunotoxin Therapy for Colorectal Cancer.

BACKGROUND: Mesothelin (MSLN) is a cell surface glycoprotein expressed at a high level on many malignancies, including pancreatic adenocarcinoma, serous ovarian cancer, and epithelioid mesothelioma. MSLN-targeted recombinant immunotoxins (RITs) consist of an anti-MSLN Fv fused to the catalytic domain of Pseudomonas exotoxin A. Recent data has also shown that MSLN is expressed at clinically relevant levels on the surface of colorectal cancer (CRC). In this study, CRC cell lines were tested for MSLN expression and susceptibility to MSLN-targeted RITs. MATERIALS AND METHODS: CRC cell lines were tested for membranous MSLN expression via flow cytometry. Cell lines expressing MSLN were tested by WST-8 cell viability assay for sensitivity to various RITs and chemotherapeutic agents. CRC cell line SW-48 was tested in a mouse model for response to RIT as a single agent or in combination with actinomycin D and oxaliplatin. RESULTS: CRC cell lines were susceptible to anti-MSLN RITs at half maximal inhibitory concentration levels comparable with those previously described in pancreatic cancer cell lines. In a nude mouse model, MSLN-targeted RIT treatment of SW48 CRC tumors resulted in a significant decrease in tumor volume. Although combination therapy with standard of care chemotherapeutic oxaliplatin did not improve tumor regressions, combination therapy with actinomycin D resulted in > 90% tumor volume reduction with 50% complete regressions. CONCLUSIONS: These data support the development of anti-MSLN RITs as well as other MSLN-targeted therapies for CRC.

Cloning and expression of immunogenic Clostridium botulinum C2I mutant proteins designed from their evolutionary imprints.

C2 toxin produced from Clostridium botulinum serotypes C and D has a potential role in many pathophysiological mechanisms in birds and animals. It has encompassed an ADP ribosyltransferase subunit (C2I) and a translocation/binding subunit (C2II). In the present study, we intended to produce C2I mutant proteins as recombinant subunit vaccines by using glutathione-S-transferase-gene fusion system. The mutants of this study were previously evaluated from their evolutionary imprints and suggested as suitable candidates for subunit vaccines. A synthetic C2 gene was inserted in a pGEX-2T vector, cloned and expressed in Escherichia coli BL21 host. The expressed mutant proteins were purified by using glutathione-agarose column and then examined for their ADP ribosyltransferase activity and vaccinogenic characteristics. The pGEX-2T-C2I constructs with Y298F, S347A and S350A substitutions have shown effective transformation efficiencies in E. coli XL10 competent cells but their mutagenesis efficiency was relatively low. Gene expression analysis indicated the rate of gene expression was depended on the fused mutant genes. A high-level expression was achieved for Y298F, S347A and S350A mutant proteins. All purified protein exhibited a molecular mass of 49 kDa. C2I mutant proteins exhibited a reduced ADP ribosyltransferase activity with retained immunogenic and vaccinogenic characteristics compared to the wild-type C2I subunit. The overall analysis of our study suggested the recombinant C2I proteins (S197A and Y298F) are the most promising candidates for the development of subunit vaccine or immunogen for C2 mutants mediated diseases in birds and animals.

Exotoxin A-PLGA nanoconjugate vaccine against Pseudomonas aeruginosa infection: protectivity in murine model.

Pseudomonas aeruginosa is the major infectious agent of concern for cystic fibrosis (CF) patients. Therefore, it is necessary to develop appropriate strategies for preventing colonization by this bacterium and/or neutralizing virulence factors. In this study, we formulated the encapsulation of exotoxin A into PLGA nanoparticles. The biological activities of the nanovaccine candidate were also characterized. Based on the results, ETA-PLGA can act as a suitable immunogen to stimulate the humoral and cellular immune response. The antibodies raised against ETA-PLGA significantly decreased bacterial titer in the spleens of the immunized mice after challenge with PAO1 strain, compared to the control groups. The encapsulation of PLGA into ETA led to a significantly higher production of INF-γ, TNF-α, IL-4, and IL-17A cytokine responses compared to the ETA group. ETA-PLGA enhanced IgG responses in immunized mice compared to ETA antigen. We concluded that encapsulation of Pseudomonas aeruginosa ETA to PLGA nanoparticles can increase its functional activity by decreasing the bacterial dissemination.

Prophylactic and therapeutic protection of human IgG purified from sera containing anti-exotoxin A titers against pneumonia caused by Pseudomonas aeruginosa.

Antibodies are effective alternative tools to combat infections caused by Pseudomonas aeruginosa (PA), especially multi-drug-resistant PA. Thus, to solve the urgent need for an anti-PA antibody drug, we hypothesized that anti-PA intravenous immunoglobulins could be a practical attempt. Exotoxin A (ETA) is one of the most important factors for PA infection and is also a critical target for the development of immune interventions. In this study, a total of 320 sera were collected from healthy volunteers. The concentration of ETA-specific antibodies was determined by a Luminex-based assay and then purified by affinity chromatography. The purified IgGs were able to neutralize the cytotoxicity of ETA in vitro. We showed they had a prophylactic and therapeutic protective effect in PA pneumonia and ETA toxemia models. In addition, administration of nonspecific IgGs also provided partial protection. Collectively, our results provide additional evidence for IVIG-based treatment of infections caused by multi-drug-resistant PA and suggest that patients at high risk of PA pneumonia could be prophylactically treated with anti-ETA IgGs or even with nonspecific IgGs.

HER2-Specific Targeted Toxin DARPin-LoPE: Immunogenicity and Antitumor Effect on Intraperitoneal Ovarian Cancer Xenograft Model.

High immunogenicity and systemic toxicity are the main obstacles limiting the clinical use of the therapeutic agents based on Pseudomonas aeruginosa exotoxin A. In this work, we studied the immunogenicity, general toxicity and antitumor effect of the targeted toxin DARPin-LoPE composed of HER2-specific DARPin and a low immunogenic exotoxin A fragment lacking immunodominant human B lymphocyte epitopes. The targeted toxin has been shown to effectively inhibit the growth of HER2-positive human ovarian carcinoma xenografts, while exhibiting low non-specific toxicity and side effects, such as vascular leak syndrome and liver tissue degradation, as well as low immunogenicity, as was shown by specific antibody titer. This represents prospects for its use as an agent for targeted therapy of HER2-positive tumors.

Depletion of regulatory T cells in tumors with an anti-CD25 immunotoxin induces CD8 T cell-mediated systemic antitumor immunity.

The tumor microenvironment plays a critical role in controlling tumor progression and immune surveillance. We produced an immunotoxin (2E4-PE38) that kills mouse cells expressing CD25 by attaching the Fv portion of monoclonal antibody 2E4 (anti-mouse CD25) to a 38-kDa portion of Pseudomonas exotoxin A. We employed three mouse cancer tumor models (AB1 mesothelioma, 66c14 breast cancer, and CT26M colon cancer). Tumors were implanted at two sites on BALB/c mice. On days 5 and 9, one tumor was directly injected with 2E4-PE38, and the other was not treated; 2E4-PE38 produced complete regressions of 85% of injected AB1 tumors, 100% of 66c14 tumors, and 100% of CT26M tumors. It also produced complete regressions of 77% of uninjected AB1 tumors, 47% of 66c14 tumors, and 92% of CT26M tumors. Mice with complete regressions of 66c14 tumors were immune to rechallenge with 66c14 cells. Mice with complete regressions of AB1 or CT26M tumors developed cross-tumor immunity rejecting both tumor types. Injection of anti-CD25 antibody or a mutant inactive immunotoxin were generally ineffective. Tumors were analyzed 3 days after 2E4-PE38 injection. The number of regulatory T cells (Tregs) was significantly reduced in the injected tumor but not in the spleen. Injected tumors contained an increase in CD8 T cells expressing IFN-γ, the activation markers CD69 and CD25, and macrophages and conventional dendritic cells. Treatment with antibodies to CD8 abolished the antitumor effect. Selective depletion of Tregs in tumors facilitates the development of a CD8 T cell-dependent antitumor effect in three mouse models.

Assessment of the impact of manufacturing changes on the physicochemical properties of the recombinant vaccine carrier ExoProtein A.

Efforts to develop a vaccine for the elimination of malaria include the use of carrier proteins to assemble monomeric antigens into nanoparticles to maximize immunogenicity. Recombinant ExoProtein A (EPA) is a detoxified form of Pseudomonas aeruginosa Exotoxin A which has been used as a carrier in the conjugate vaccine field. A pilot-scale process developed for purification of EPA yielded product that consistently approached a preset upper limit for host cell protein (HCP) content per human dose. To minimize the risk of bulk material exceeding the specification, the purification process was redeveloped using mixed-mode chromatography resins. Purified EPA derived from the primary and redeveloped processes were comparable following full biochemical and biophysical characterization. However, using a process specific immunoassay, the HCP content was shown to decrease from a range of 0.14-0.24% w/w of total protein to below the level of detection with the revised process. The improved process reproducibly yields EPA with highly similar quality characteristics as the original process but with an improved profile for the HCP content.

EGF ligand fused to truncated Pseudomonas aeruginosa exotoxin A specifically targets and inhibits EGFR­positive cancer cells.

Cancer cells have been known to overexpress the epidermal growth factor receptor (EGFR) and hence relevant multiple­targeted therapies have been developed, with a recent clinical application of the antibody­mediated inhibition of the EGFR. However, this strategy is not useful in cancer cells with mutations in KRAS; a GTPase downstream of EGFR which constitutively activates the pathway without EGF stimulation. Furthermore, mutations in EGFR also reduce the binding of monoclonal antibodies and thereby render them ineffective. In the present study, we designed a chimeric EGF protein fused to the truncated N­terminal domain fragment of Pseudomonas aeruginosa exotoxin A (EGF­ETA), which has ADP­ribosylation activity and induces apoptosis. The EGF­ETA protein was expressed in E. coli as a His­tagged fusion. Our results showed that EGF­ETA significantly inhibited the proliferation of EGFR­positive A431 epidermoid carcinoma (IC50 27 ng/ml) and HN5 head and neck squamous cell carcinoma (IC50 36 ng/ml) cells. However, its effect on cancer cells with little or no EGFR expression was limited (A549­IC50 1,000 ng/ml; MCF­7­IC50 >10,000 ng/ml). Compared to cetuximab, EGF­ETA was highly potent in its killing capacity of HN5 cancer cells at 1,000 ng/ml, while cetuximab had little effect at 1,000 ng/ml. Furthermore, EGF­ETA was just as potent in HCT116 (KRAS G13D) and SW480 (KRAS G12V) colon cancer cell lines harbouring KRAS hyperactivating mutations when compared to KRAS wild­type HT29 colon cancer cells. Finally, co­incubation of EGF­ETA with an anti­EGF antibody abrogated its effect on the EGFR­positive A431 cells. Our results show that the chimeric EGF­ETA toxin is extremely effective against EGFR­positive cancers and raises the potential to further develop this chimera for use in targeting EGFR­positive tumours resistant to monoclonal antibodies.