Targeted Delivery of Diphtheria Toxin into VEGFR1/VEGFR2 Overexpressing Cells Induces Anti-angiogenesis Activity.

BACKGROUND: Vascular Endothelial Growth Factor Receptors (VEGFR1 and VEGFR2) are tyrosine kinase receptors expressed on endothelial cells and tumor vessels and play an important role in angiogenesis. In this study, three repeats of VEGFR1 and VEGFR2 binding peptide (VGB3) were genetically fused to the truncated diphtheria toxin (TDT), and its in vitro activity was evaluated. METHODS: The recombinant construct (TDT-triVGB3) was expressed in bacteria cells and purified with nickel affinity chromatography. The binding capacity and affinity of TDT-triVGB3 were evaluated using the enzyme-linked immunosorbent assay. The inhibitory activity of TDT-triVGB3 on viability, migration, and tube formation of human endothelial cells was evaluated using MTT, migration, and tube formation assays. RESULTS: TDT-triVGB3 selectively detected VEGFR1 and VEGFR2 with high affinity in an enzyme- linked immunosorbent assay and significantly inhibited viability, migration, and tube formation of human endothelial cells. CONCLUSION: The developed TDT-triVGB3 is potentially a novel agent for targeting VEGFR1/ VEGFR2 over-expressing cancer cells.

The Evaluation of the N-cadherin Promoter's ability to Block EMT by Specific Expression of Diphtheria Toxin in EMT-induced A549 Cell Lines.

During epithelial to mesenchymal transition, the ability of cancer cells to transform and metastasize is primarily determined by N-cadherin-mediated migration and invasion. This study aimed to evaluate whether the N-cadherin promoter can induce diphtheria toxin expression as a suicide gene in epithelial to mesenchymal transition (EMT)-induced cancer cells and whether this can be used as potential gene therapy. To investigate the expression of diphtheria toxin under the N-cadherin promoter, the promoter was synthesized, and was cloned upstream of diphtheria toxin in a pGL3-Basic vector. The A-549 cells was transfected by electroporation. After induction of EMT by TGF-ß and hypoxia treatment, the relative expression of diphtheria toxin, mesenchymal genes such as N-cadherin and Vimentin, and epithelial genes such as E-cadherin and ß-catenin were measured by real-time PCR. MTT assay was also performed to measure cytotoxicity. Finally, cell motility was assessed by the Scratch test. After induction of EMT in transfected cells, the expression of mesenchymal markers such as Vimentin and N-cadherin significantly decreased, and the expression of ß-catenin increased. In addition, the MTT assay showed promising toxicity results after induction of EMT with TGF-ß in transfected cells, but toxicity was less effective in hypoxia. The scratch test results also showed that cell movement was successfully prevented in EMT-transfected cells and thus confirmed EMT occlusion. Our findings indicate that by using structures containing diphtheria toxin downstream of a specific EMT promoter such as the N-cadherin promoter, the introduced toxin can kill specifically and block EMT in cancer cells.

Challenges of Diphtheria Toxin Detection.

Diphtheria toxin (DT) is the main virulence factor of Corynebacterium diphtheriae, C. ulcerans and C. pseudotuberculosis. Moreover, new Corynebacterium species with the potential to produce diphtheria toxin have also been described. Therefore, the detection of the toxin is the most important test in the microbiological diagnosis of diphtheria and other corynebacteria infections. Since the first demonstration in 1888 that DT is a major virulence factor of C. diphtheriae, responsible for the systemic manifestation of the disease, various methods for DT detection have been developed, but the diagnostic usefulness of most of them has not been confirmed on a sufficiently large group of samples. Despite substantial progress in the science and diagnostics of infectious diseases, the Elek test is still the basic recommended diagnostic test for DT detection. The challenge here is the poor availability of an antitoxin and declining experience even in reference laboratories due to the low prevalence of diphtheria in developed countries. However, recent and very promising assays have been developed with the potential for use as rapid point-of-care testing (POCT), such as ICS and LFIA for toxin detection, LAMP for tox gene detection, and biosensors for both.

Astrogliosis in the GFAP-CreERT2:Rosa26iDTR Mouse Model Does Not Exacerbate Retinal Microglia Activation or Müller Cell Gliosis under Hypoxic Conditions.

Diabetic retinopathy (DR) affects over 140 million people globally. The mechanisms that lead to blindness are still enigmatic but there is evidence that sustained inflammation and hypoxia contribute to vascular damage. Despite efforts to understand the role of inflammation and microglia in DR's pathology, the contribution of astrocytes to hypoxic responses is less clear. To investigate the role of astrocytes in hypoxia-induced retinopathy, we utilized a 7-day systemic hypoxia model using the GFAP-CreERT2:Rosa26iDTR transgenic mouse line. This allows for the induction of inflammatory reactive astrogliosis following tamoxifen and diphtheria toxin administration. We hypothesize that DTx-induced astrogliosis is neuroprotective during hypoxia-induced retinopathy. Glial, neuronal, and vascular responses were quantified using immunostaining, with antibodies against GFAP, vimentin, IBA-1, NeuN, fibrinogen, and CD31. Cytokine responses were measured in both the brain and serum. We report that while both DTx and hypoxia induced a phenotype of reduced microglia morphological activation, DTx, but not hypoxia, induced an increase in the Müller glia marker vimentin. We did not observe that the combination of DTx and hypoxic treatments exacerbated the signs of reactive glial cells, nor did we observe a significant change in the expression immunomodulatory mediators IL-1ß, IL2, IL-4, IL-5, IL-6, IL-10, IL-18, CCL17, TGF-ß1, GM-CSF, TNF-α, and IFN-γ. Overall, our results suggest that, in this hypoxia model, reactive astrogliosis does not alter the inflammatory responses or cause vascular damage in the retina.

Tat-dependent conditionally replicating adenoviruses expressing diphtheria toxin A for specifically killing HIV-1-infected cells.

HIV-1 infection remains a public health problem with no cure. Although antiretroviral therapy (ART) is effective for suppressing HIV-1 replication, it requires lifelong drug administration due to a stable reservoir of latent proviruses and may cause serious side effects and drive the emergence of drug-resistant HIV-1 variants. Gene therapy represents an alternative approach to overcome the limitations of conventional treatments against HIV-1 infection. In this study, we constructed and investigated the antiviral effects of an HIV-1 Tat-dependent conditionally replicating adenovirus, which selectively replicates and expresses the diphtheria toxin A chain (Tat-CRAds-DTA) in HIV-1-infected cells both in vitro and in vivo. We found that Tat-CRAds-DTA could specifically induce cell death and inhibit virus replication in HIV-1-infected cells mediated by adenovirus proliferation and DTA expression. A low titer of progeny Tat-CRAds-DTA was also detected in HIV-1-infected cells. In addition, Tat-CRAds-DTA showed no apparent cytotoxicity to HIV-1-negative cells and demonstrated significant therapeutic efficacy against HIV-1 infection in a humanized mouse model. The findings in this study highlight the potential of Tat-CRAds-DTA as a new gene therapy for the treatment of HIV-1 infection.

Neutralizing anti-diphtheria toxin scFv produced by phage display.

BACKGROUND: Diphtheria can be prevented by vaccination, but some epidemics occur in several places, and diphtheria's threat is considerable. Administration of diphtheria antitoxin (DAT) produced from hyperimmunized animals is the most common treatment. Recombinant human antibody fragments such as single-chain variable fragments (scFv) produced by phage display library may introduce an interesting approach to overcome the limitations of the traditional antibody therapy. In the present study, B cells of immunized volunteers were used to construct a human single-chain fragment (HuscFv) library. MATERIALS AND METHODS: The library was constructed with the maximum combination of heavy and light chains. As an antigen, Diphtheria toxoid (DTd) was used in four-round phage bio-panning to select phage clones that display DTd bound HuscFv from the library. After panning, individual scFv clones were selected. Clones that were able to detect DTd in an initial screening assay were transferred to Escherichia coli HB2151 to express the scFvs and purification was followed by Ni metal ion affinity chromatography. Toxin neutralization test was performed on Vero cells. The reactivity of the soluble scFv with diphtheria toxin were done and affinity calculation based on Beatty method was calculated. RESULTS: The size of the constructed scFv library was calculated to be 1.3 × 106 members. Following four rounds of selection, 40 antibody clones were isolated which showed positive reactivity with DTd in an ELISA assay. Five clones were able to neutralize DTd in Vero cell assay. These neutralizing clones were used for soluble expression and purification of scFv fragments. Some of these soluble scFv fragments show neutralizing activity ranging from 0.6 to 1.2 µg against twofold cytotoxic dose of diphtheria toxin. The affinity constant of the selected scFv antibody was determined almost 107 M-1. CONCLUSION: This study describes the prosperous construction and isolation of scFv from the immune library, which specifically neutralizes diphtheria toxin. The HuscFv produced in this study can be a potential candidate to substitute the animal antibody for treating diphtheria and detecting toxins.

Immunogenicity and safety of adsorbed diphtheria-purified pertussis-tetanus-inactivated polio (Sabin strain)-Haemophilus type b conjugate combined vaccine (DPT-IPV-Hib) in healthy Japanese Infants ≥ 2 and < 43 months of Age: A phase III, multicenter, active controlled, assessor-blinded, randomized, parallel-group study.

OBJECTIVE: This study investigated the immunogenicity and safety of a pentavalent vaccine Gobik (DPT-IPV-Haemophilus influenzae type b [Hib]) in healthy Japanese infants aged ≥ 2 and < 43 months using a concomitant vaccination with ActHIB® (Hib) and Tetrabik (DPT-IPV) as a comparator. METHODS: This study was conducted as a phase 3, multicenter, active controlled, assessor-blinded, randomized, parallel-group study. Participants received a total of 4 subcutaneous doses (3 primary immunization doses and a booster dose) of either the experimental drug (DPT-IPV-Hib) or the active comparator (Hib + DPT-IPV). The primary endpoints were the anti-PRP antibody prevalence rate with ≥ 1 µg/mL, and the antibody prevalence rates against pertussis, diphtheria toxin, tetanus toxin, and attenuated poliovirus after the primary immunization. RESULTS: In 267 randomized participants (133 in the DPT-IPV-Hib group and 134 in the Hib + DPT-IPV group), the antibody prevalence rates after the primary immunization in both groups were 100.0 % and 88.7 % for anti-PRP antibody with ≥ 1 µg/mL, 99.2 % and 98.5 % against diphtheria toxin, and 100.0 % and 99.2 % against tetanus toxin, respectively. The antibody prevalence rates against pertussis and attenuated poliovirus were 100.0 % in both groups. The non-inferiority of the DPT-IPV-Hib group to the Hib + DPT-IPV group was verified for all measured antibodies. In both groups, all the GMTs of antibodies after the primary immunization were higher than those before the first dose, and those after the booster dose were higher than those after the primary immunization. No safety issues were identified. CONCLUSION: A single-agent Gobik, the first DPT-IPV-Hib pentavalent vaccine approved in Japan, was confirmed to simultaneously provide primary and booster immunizations against Hib infection, pertussis, diphtheria, tetanus, and poliomyelitis and to have a preventive effect and safety comparable to concomitant vaccination with Hib (ActHIB®) and DPT-IPV quadrivalent vaccine (Tetrabik).

Neuropilin-1 Binding Peptide as Fusion to Diphtheria Toxin Induces Apoptosis in Non-small Cell Lung Cancer Cell Line.

BACKGROUND: Targeted cancer therapy can be considered as a new strategy to overcome the side effects of current cancer treatments. Neuropilin-1 (NRP-1) is a transmembrane glycoprotein that is expressed in endothelial cells and tumor vessels to stimulate angiogenesis progression. Targeted diphtheria toxin (DT)- based therapeutics are promising tools for cancer treatment. This study aimed to construct a novel NRP-1 binding peptide (as three repeats) (CRGDK) as a fusion to truncated DT (DTA) (DTA-triCRGDK) for targeted delivery of DT into NRP-1 expressing cells. METHODS: The concept of DTA-triCRGDK was designed, synthesized and cloned into the bacterial host. Expression of DTA-triCRGDK was induced by Isopropyl ß-D-1-thiogalactopyranoside (IPTG) and purification was performed using Ni-NTA chromatography. Biological activity of DTA-triCRGDK was evaluated using MTT, apoptosis, and wound healing assays. In addition, expression levels of apoptotic Bax, Bcl2, and Casp3 genes were determined by Real-time PCR. RESULTS: Cytotoxicity analysis showed the IC50 values of DTA-triCRGDK for A549 and MRC5 were 0.43 nM and 4.12 nM after 24 h, respectively. Bcl2 expression levels decreased 0.4 and 0.72 fold in A549 and MRC5, respectively. However, Bax and Casp3 expression level increased by 6.75 and 8.19 in A549 and 2.51 and 3.6 in MRC5 cells. CONCLUSION: Taken together, DTA-triCRGDK is a promising tool for targeted therapy of NRP-1 overexpressing cancer cells.

Single-cell division tracing and transcriptomics reveal cell types and differentiation paths in the regenerating lung.

Understanding the molecular and cellular processes involved in lung epithelial regeneration may fuel the development of therapeutic approaches for lung diseases. We combine mouse models allowing diphtheria toxin-mediated damage of specific epithelial cell types and parallel GFP-labeling of functionally dividing cells with single-cell transcriptomics to characterize the regeneration of the distal lung. We uncover cell types, including Krt13+ basal and Krt15+ club cells, detect an intermediate cell state between basal and goblet cells, reveal goblet cells as actively dividing progenitor cells, and provide evidence that adventitial fibroblasts act as supporting cells in epithelial regeneration. We also show that diphtheria toxin-expressing cells can persist in the lung, express specific inflammatory factors, and transcriptionally resemble a previously undescribed population in the lungs of COVID-19 patients. Our study provides a comprehensive single-cell atlas of the distal lung that characterizes early transcriptional and cellular responses to concise epithelial injury, encompassing proliferation, differentiation, and cell-to-cell interactions.

Zoonotic transmission of diphtheria toxin-producing Corynebacterium ulcerans.

BACKGROUND: Diphtheria caused by toxin-producing Corynebacterium ulcerans is a re-emerging human disease that can cause local and systemic sequelae. In Australia, toxigenic diphtheria is a rare notifiable communicable disease, due to high-vaccination coverage. The public health management of cutaneous cases of toxigenic C. ulcerans varies between jurisdictions, as opposed to the more uniform public health response to toxigenic Corynebacterium diphtheriae presenting as respiratory or laryngeal diphtheria. AIM: To report a case of zoonotically acquired C. ulcerans, review evidence on the zoonotic reservoir and reported transmission events, and examine public health guidelines for the management of human and animal contacts. METHODS AND RESULTS: In this case report, we detail our case investigation, treatment and public health management, including contact tracing and an approach to animal testing. We successfully identified companion canines as probable sources for the human case, with WGS confirming the link. The zoonotic disease link of C. ulcerans to domestic and agricultural animals is established in the literature; however, the management of animal contacts in human cases is inconsistent with jurisdictional or national guidelines. CONCLUSIONS: While a rare disease, a consistent approach to public health management is warranted to systematically elucidate the disease source and improve understanding of transmission.

Diphthamide - a conserved modification of eEF2 with clinical relevance.

Diphthamide, a complex modification on eukaryotic translation elongation factor 2 (eEF2), assures reading-frame fidelity during translation. Diphthamide and enzymes for its synthesis are conserved in eukaryotes and archaea. Originally identified as target for diphtheria toxin (DT) in humans, its clinical relevance now proves to be broader than the link to pathogenic bacteria. Diphthamide synthesis enzymes (DPH1 and DPH3) are associated with cancer, and DPH gene mutations can cause diphthamide deficiency syndrome (DDS). Finally, new analyses provide evidence that diphthamide may restrict propagation of viruses including SARS-CoV-2 and HIV-1, and that DPH enzymes are targeted by viruses for degradation to overcome this restriction. This review describes how diphthamide is synthesized and functions in translation, and covers its clinical relevance in human development, cancer, and infectious diseases.

Generation of a human embryonic stem cell line (SMUDHe010-A-1B) carrying inducible DTA expression cassette in the AAVS1 locus by CRISPR/Cas9-mediated homologous recombination.

Diphtheria toxin A (DTA) is an exotoxin secreted by Corynebacterium diphtheriae. After entering the cell through receptor-mediated manner, DTA can trigger the programmed cell death mechanism and lead to cell death. In 2001, Michiko Saito established a Diphtheria toxin receptor-mediated cell knockout system, which can conditional deplete specific cell type in transgenic mice. This system is not only very useful in the pathogenesis study of human diseases, but also has a wide application prospect in the study of organ development and regeneration. In 2008, David Voehringer described a newly generated mouse strain that encodes DTA under control of a loxP-flanked stop cassette in the ubiquitously expressed ROSA26 locus. Thereby, it can be used in combination with tissue-specific and/or inducible Cre-expressing mouse strains to achieve toxin-mediated cell ablation in vivo. The application of DTA-mediated cell knockout system in mice has been widely reported, but it has rarely been used in human cells. Accordingly, we generated a human embryonic stem cell line (SMUDHe010-A-1B) carrying inducible DTA expression cassette (loxp-stop-loxp-DTA, LSL-DTA) using CRISPR/Cas9-mediated homologous recombination. The cell line preserves normal karyotype, pluripotency and the ability to differentiate into all three germ layers. Moreover, the cell line can be used to prepare human organoid, which may provide a model for achieving conditional cell ablation in human tissues and organs.

Hosts and Heterologous Expression Strategies of Recombinant Toxins for Therapeutic Purposes.

The production of therapeutic recombinant toxins requires careful host cell selection. Bacteria, yeast, and mammalian cells are common choices, but no universal solution exists. Achieving the delicate balance in toxin production is crucial due to potential self-intoxication. Recombinant toxins from various sources find applications in antimicrobials, biotechnology, cancer drugs, and vaccines. "Toxin-based therapy" targets diseased cells using three strategies. Targeted cancer therapy, like antibody-toxin conjugates, fusion toxins, or "suicide gene therapy", can selectively eliminate cancer cells, leaving healthy cells unharmed. Notable toxins from various biological sources may be used as full-length toxins, as plant (saporin) or animal (melittin) toxins, or as isolated domains that are typical of bacterial toxins, including Pseudomonas Exotoxin A (PE) and diphtheria toxin (DT). This paper outlines toxin expression methods and system advantages and disadvantages, emphasizing host cell selection's critical role.

Targeting regulatory T cells by E7777 enhances CD8 T-cell-mediated anti-tumor activity and extends survival benefit of anti-PD-1 in solid tumor models.

Introduction: Regulatory T cell (Treg)-targeting cancer immunotherapy aims to transiently deplete Treg cells in the tumor microenvironment, without affecting effector T cells (Teff), thus both enhancing anti-tumor activity and avoiding autoimmunity. This study evaluated whether adding E7777 (a new formulation of denileukin diftitox [DD]) improved the efficacy of anti-PD-1 antibody therapy. DD is a recombinant protein containing the hydrophobic and catalytic portions of diphtheria toxin fused to full-length human IL-2. E7777 has the same amino acid sequence and brief circulatory half-life as DD, but with greater purity and potency. Methods: Subcutaneous syngeneic murine solid tumor models (colon cancer CT-26 and liver cancer H22) were used to evaluate safety, efficacy, and overall survival with E7777 and anti-PD-1 antibodies, each administered as monotherapy or in concurrent or sequential combination. In Experiment 1, treatments were compared to assess anti-tumor activity at various time points, with tumors excised and dissociated and tumor leukocytes characterized. In Experiment 2, tumor growth, response, and overall survival were characterized for 100 days following a 3-week treatment. Results: E7777 administered in combination with anti-PD-1 led to significantly increased anti-tumor activity and durable, extended overall survival compared to either treatment alone. In both tumor models, the Treg cell infiltration induced by anti-PD-1 treatment was counterbalanced by co-treatment with E7777, suggesting potential synergistic activity. Combination therapy showed the most favorable results. Treatment with E7777 was safe and well-tolerated. Discussion: Combined E7777 and anti-PD-1 therapy was well tolerated and more effective than monotherapy with either drug.

Intravenous Administration of Human Muse Cells Ameliorates Deficits in a Rat Model of Subacute Spinal Cord Injury.

Multilineage-differentiating stress-enduring (Muse) cells are newly established pluripotent stem cells. The aim of the present study was to examine the potential of the systemic administration of Muse cells as an effective treatment for subacute SCI. We intravenously administered the clinical product "CL2020" containing Muse cells to a rat model two weeks after mid-thoracic spinal cord contusion. Eight experimental animals received CL2020, and twelve received the vehicle. Behavioral analyses were conducted over 20 weeks. Histological evaluations were performed. After 20 weeks of observation, diphtheria toxin was administered to three CL2020-treated animals to selectively ablate human cell functions. Hindlimb motor functions significantly improved from 6 to 20 weeks after the administration of CL2020. The cystic cavity was smaller in the CL2020 group. Furthermore, larger numbers of descending 5-HT fibers were preserved in the distal spinal cord. Muse cells in CL2020 were considered to have differentiated into neuronal and neural cells in the injured spinal cord. Neuronal and neural cells were identified in the gray and white matter, respectively. Importantly, these effects were reversed by the selective ablation of human cells by diphtheria toxin. Intravenously administered Muse cells facilitated the therapeutic potential of CL2020 for severe subacute spinal cord injury.

Diphtheria toxin activates ribotoxic stress and NLRP1 inflammasome-driven pyroptosis.

The ZAKα-driven ribotoxic stress response (RSR) is activated by ribosome stalling and/or collisions. Recent work demonstrates that RSR also plays a role in innate immunity by activating the human NLRP1 inflammasome. Here, we report that ZAKα and NLRP1 sense bacterial exotoxins that target ribosome elongation factors. One such toxin, diphtheria toxin (DT), the causative agent for human diphtheria, triggers RSR-dependent inflammasome activation in primary human keratinocytes. This process requires iron-mediated DT production in the bacteria, as well as diphthamide synthesis and ZAKα/p38-driven NLRP1 phosphorylation in host cells. NLRP1 deletion abrogates IL-1ß and IL-18 secretion by DT-intoxicated keratinocytes, while ZAKα deletion or inhibition additionally limits both pyroptotic and inflammasome-independent non-pyroptotic cell death. Consequently, pharmacologic inhibition of ZAKα is more effective than caspase-1 inhibition at protecting the epidermal barrier in a 3D skin model of cutaneous diphtheria. In summary, these findings implicate ZAKα-driven RSR and the NLRP1 inflammasome in antibacterial immunity and might explain certain aspects of diphtheria pathogenesis.

Emerging Corynebacterium diphtheriae Species Complex Infections, Réunion Island, France, 2015-2020.

Clinical, epidemiologic, and microbiologic analyses revealed emergence of 26 cases of Corynebacterium diphtheriae species complex infections on Réunion Island, France, during 2015-2020. Isolates were genetically diverse, indicating circulation and local transmission of several diphtheria sublineages. Clinicians should remain aware of the risk for diphtheria and improve diagnostic methods and patient management.

Clinical Characteristics of Corynebacterium ulcerans Infection, Japan.

Corynebacterium ulcerans is a closely related bacterium to the diphtheria bacterium C. diphtheriae, and some C. ulcerans strains produce toxins that are similar to diphtheria toxin. C. ulcerans is widely distributed in the environment and is considered one of the most harmful pathogens to livestock and wildlife. Infection with C. ulcerans can cause respiratory or nonrespiratory symptoms in patients. Recently, the microorganism has been increasingly recognized as an emerging zoonotic agent of diphtheria-like illness in Japan. To clarify the overall clinical characteristics, treatment-related factors, and outcomes of C. ulcerans infection, we analyzed 34 cases of C. ulcerans that occurred in Japan during 2001-2020. During 2010-2020, the incidence rate of C. ulcerans infection increased markedly, and the overall mortality rate was 5.9%. It is recommended that adults be vaccinated with diphtheria toxoid vaccine to prevent the spread of this infection.

Histidine Protonation and Conformational Switching in Diphtheria Toxin Translocation Domain.

Protonation of key histidine residues has been long implicated in the acid-mediated cellular action of the diphtheria toxin translocation (T-) domain, responsible for the delivery of the catalytic domain into the cell. Here, we use a combination of computational (constant-pH Molecular Dynamics simulations) and experimental (NMR, circular dichroism, and fluorescence spectroscopy along with the X-ray crystallography) approaches to characterize the initial stages of conformational change happening in solution in the wild-type T-domain and in the H223Q/H257Q double mutant. This replacement suppresses the acid-induced transition, resulting in the retention of a more stable protein structure in solutions at pH 5.5 and, consequently, in reduced membrane-disrupting activity. Here, for the first time, we report the pKa values of the histidine residues of the T-domain, measured by NMR-monitored pH titrations. Most peaks in the histidine side chain spectral region are titrated with pKas ranging from 6.2 to 6.8. However, the two most up-field peaks display little change down to pH 6, which is a limiting pH for this protein in solution at concentrations required for NMR. These peaks are absent in the double mutant, suggesting they belong to H223 and H257. The constant-pH simulations indicate that for the T-domain in solution, the pKa values for histidine residues range from 3.0 to 6.5, with those most difficult to protonate being H251 and H257. Taken together, our experimental and computational data demonstrate that previously suggested cooperative protonation of all six histidines in the T-domain does not occur.

Diphtheria toxin-derived, anti-PD-1 immunotoxin, a potent and practical tool to selectively deplete PD-1+ cells.

Programmed death-1 (PD-1), an immune checkpoint receptor, is expressed on activated lymphocytes, macrophages, and some types of tumor cells. While PD-1+ cells have been implicated in outcomes of cancer immunity, autoimmunity, and chronic infections, the exact roles of these cells in various physiological and pathological processes remain elusive. Molecules that target and deplete PD-1+ cells would be instrumental in defining the roles unambiguously. Previously, an immunotoxin has been generated for the depletion of PD-1+ cells though its usage is impeded by its low production yield. Thus, a more practical molecular tool is desired to deplete PD-1+ cells and to examine functions of these cells. We designed and generated a novel anti-PD1 diphtheria immunotoxin, termed PD-1 DIT, targeting PD-1+ cells. PD-1 DIT is comprised of two single chain variable fragments (scFv) derived from an anti-PD-1 antibody, coupled with the catalytic and translocation domains of the diphtheria toxin. PD-1 DIT was produced using a yeast expression system that has been engineered to efficiently produce protein toxins. The yield of PD-1 DIT reached 1-2 mg/L culture, which is 10 times higher than the previously reported immunotoxin. Flow cytometry and confocal microscopy analyses confirmed that PD-1 DIT specifically binds to and enters PD-1+ cells. The binding avidities between PD-1 DIT and two PD-1+ cell lines are approximately 25 nM. Moreover, PD-1 DIT demonstrated potent cytotoxicity toward PD-1+ cells, with a half maximal effective concentration (EC50 ) value of 1 nM. In vivo experiments further showed that PD-1 DIT effectively depleted PD-1+ cells and enabled mice inoculated with PD-1+ tumor cells to survive throughout the study. Our findings using PD-1 DIT revealed the critical role of pancreatic PD-1+ T cells in the development of type-1 diabetes (T1D). Additionally, we observed that PD-1 DIT treatment ameliorated relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE), a mouse model of relapsing-remitting multiple sclerosis (RR-MS). Lastly, we did not observe significant hepatotoxicity in mice treated with PD-1 DIT, which had been reported for other immunotoxins derived from the diphtheria toxin. With its remarkable selective and potent cytotoxicity toward PD-1+ cells, coupled with its high production yield, PD-1 DIT emerges as a powerful biotechnological tool for elucidating the physiological roles of PD-1+ cells. Furthermore, the potential of PD-1 DIT to be developed into a novel therapeutic agent becomes evident.