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Biotechnology and Applied Biochemistry Mar 2020DAB IL-2 (Denileukin diftitox) is considered an immunotoxin, and it is the first immunotoxin approved by Food and Drug Administration. It is used for the treatment of a...
DAB IL-2 (Denileukin diftitox) is considered an immunotoxin, and it is the first immunotoxin approved by Food and Drug Administration. It is used for the treatment of a cutaneous form of T-cell lymphoma. This fusion protein has two disulfide bonds in its structure that play an essential role in toxicity and functionality of the immunotoxin. Escherichia coli (E. coli) strain BL21 (DE3) is not capable of making disulfide bonds in its reductive cytoplasm, but the E. coli strain Rosetta-gami (DE3) is a proper strain for the correct expression of the protein due to mutations in glutaredoxin reductase and thioredoxin reductase. In this study, a pET21a vector with the His6-tag fused at the N-terminus of DAB IL-2 was used to express the soluble immunotoxin in E. coli Rosetta-gami (DE3). After the purification of the soluble protein by two-step column chromatographies, the structure of DAB IL-2 was analyzed using the Native-PAGE and circular dichroism methods. In the following, the nuclease activity of soluble DAB IL-2 and its cytotoxicity activity were determined. It is concluded that the soluble recombinant protein expressed in the E. coli Rosetta-gami (DE3) has an intact structure and also functional; hence, this form of immunotoxin could be competitive with its commercial counterparts.
Topics: Antineoplastic Agents; Diphtheria Toxin; Escherichia coli; Interleukin-2; Protein Domains; Recombinant Fusion Proteins; Solubility
PubMed: 31600001
DOI: 10.1002/bab.1833 -
Immunotherapy Sep 2019T regulatory cells (Tregs) are an important T cell population for immune tolerance, prevention of autoimmune diseases and inhibition of antitumor immunity. The... (Review)
Review
T regulatory cells (Tregs) are an important T cell population for immune tolerance, prevention of autoimmune diseases and inhibition of antitumor immunity. The tumor-promoting role played by Tregs in cancer has prompted numerous approaches to develop immunotherapeutics targeting Tregs. One approach to depletion of Treg cells is retargeting the highly potent cytotoxic activity of bacterial toxins. These agents capitalize on the well-characterized bacterial toxins, diphtheria toxin and exotoxin A-both of which harbor membrane translocation domains and enzymatic domains that catalytically halt protein synthesis within intoxicated eukaryotic cells and act at picomolar or subpicomolar concentrations. In this review, we summarize the preclinical and clinical development of several Treg-depleting cancer immunotherapies based on these two bacterial toxins.
Topics: ADP Ribose Transferases; Animals; Bacterial Toxins; Clinical Trials as Topic; Diphtheria Toxin; Drug Evaluation, Preclinical; Exotoxins; Humans; Immunity, Cellular; Immunotherapy; Lymphocyte Depletion; Neoplasms; T-Lymphocytes, Regulatory; Tumor Microenvironment; Virulence Factors; Pseudomonas aeruginosa Exotoxin A
PubMed: 31361167
DOI: 10.2217/imt-2019-0060 -
Frontiers in Medicine 2019Mycosis fungoides (MF) and Sézary syndrome (SS) are the most common subtypes of cutaneous T-cell lymphoma. The majority of MF cases present with only patches and... (Review)
Review
Mycosis fungoides (MF) and Sézary syndrome (SS) are the most common subtypes of cutaneous T-cell lymphoma. The majority of MF cases present with only patches and plaques and the lesions are usually limited to the skin. On the other hand, in some cases, patients show skin tumors or erythroderma followed by lymph node involvement and rarely visceral organ involvement. SS is a rare, aggressive cutaneous T-cell lymphoma marked by exfoliative erythroderma, lymphadenopathy, and leukemic blood involvement. Because patients with relapsed or refractory MF/SS display a poor prognosis and the current treatment options are characterized by high rates of relapse, there is unmet need for the efficient treatment. This review provides a discussion of the recent and future promising therapeutic approaches in the management of advanced MF/SS. These include mogamulizumab, brentuximab vedotin, alemtuzumab, immune checkpoint inhibitors, IPH4102 (anti-KIR3DL2 antibody), histone deacetylase inhibitors (vorinostat, romidepsin, panobinostat, belinostat, and resminostat), pralatrexate, forodesine, denileukin diftitox, duvelisib, lenalidomide, and everolimus.
PubMed: 31192214
DOI: 10.3389/fmed.2019.00116 -
Proceedings of the National Academy of... Feb 2019Denileukin diftitox (DAB-IL-2, Ontak) is a diphtheria-toxin-based fusion protein that depletes CD25-positive cells including regulatory T cells and has been approved for...
Denileukin diftitox (DAB-IL-2, Ontak) is a diphtheria-toxin-based fusion protein that depletes CD25-positive cells including regulatory T cells and has been approved for the treatment of persistent or recurrent cutaneous T cell lymphoma. However, the clinical use of denileukin diftitox was limited by vascular leak toxicity and production issues related to drug aggregation and purity. We found that a single amino acid substitution (V6A) in a motif associated with vascular leak induction yields a fully active, second-generation biologic, s-DAB-IL-2(V6A), which elicits 50-fold less human umbilical vein endothelial cell monolayer permeation and is 3.7-fold less lethal to mice by LD analysis than s-DAB-IL-2. Additionally, to overcome aggregation problems, we developed a production method for the fusion toxin using that secretes fully folded, biologically active, monomeric s-DAB-IL-2 into the culture medium. Using the poorly immunogenic mouse B16F10 melanoma model, we initiated treatment 7 days after tumor challenge and observed that, while both s-DAB-IL-2(V6A) and s-DAB-IL-2 are inhibitors of tumor growth, the capacity to treat with higher doses of s-DAB-IL-2(V6A) could provide a superior activity window. In a sequential dual-therapy study in tumors that have progressed for 10 days, both s-DAB-IL-2(V6A) and s-DAB-IL-2 given before checkpoint inhibition with anti-programmed cell death-1 (anti-PD-1) antibodies inhibited tumor growth, while either drug given as monotherapy had less effect. s-DAB-IL-2(V6A), a fully monomeric protein with reduced vascular leak, is a second-generation diphtheria-toxin-based fusion protein with promise as a cancer immunotherapeutic both alone and in conjunction with PD-1 blockade.
Topics: Amino Acid Substitution; Antibodies; Cell Proliferation; Corynebacterium diphtheriae; Diphtheria Toxin; Human Umbilical Vein Endothelial Cells; Humans; Immunosuppressive Agents; Immunotoxins; Interleukin-2; Interleukin-2 Receptor alpha Subunit; Melanoma, Experimental; Programmed Cell Death 1 Receptor; Receptors, Interleukin-2; Recombinant Fusion Proteins; T-Lymphocytes, Regulatory
PubMed: 30718426
DOI: 10.1073/pnas.1815087116 -
Journal of Immunotherapy (Hagerstown,... 2019Cellular immunotherapy using allogeneic natural killer (NK) cells may overcome chemotherapy-refractory acute myeloid leukemia. Our goal was to document NK cell... (Clinical Trial)
Clinical Trial
Cellular immunotherapy using allogeneic natural killer (NK) cells may overcome chemotherapy-refractory acute myeloid leukemia. Our goal was to document NK cell homing/persistence in the bone marrow following adoptive immunotherapy. Our cohort included 109 patients who received NK cell therapy for refractory acute myeloid leukemia following lymphodepleting conditioning +/- denileukin diftitox, +/- low-dose total body irradiation. We evaluated the NK cell density in bone marrow core biopsies performed an average of 14 days after NK cell transfer using a CD56 immunohistochemical stain. The NK cell density in core biopsies showed only moderate correlation with NK cell percentage in bone marrow aspirates evaluated by flow cytometry (rs=0.48) suggesting that distribution of CD56 cells in the bone marrow niche offers unique insight into NK cell homing. Better leukemia control was associated with increased NK cell density, such that patients with <5% blasts had a higher NK cell density (P=0.01). As well, NK cell density above the median of reference group was significantly associated with morphologic remission of leukemia (P=0.01). Moreover, the NK cell density varied significantly between conditioning protocols. Our findings suggest that the use of low-dose irradiation or CD25-targeting immunocytokine (denileukin diftitox, IL2DT) as part of conditioning results in increased NK cell homing/persistence in the bone marrow. These novel results will help guide future immunotherapy with NK cells.
Topics: Adolescent; Adult; Aged; Bone Marrow; Child; Child, Preschool; Female; Humans; Immunotherapy, Adoptive; Killer Cells, Natural; Leukemia, Myeloid, Acute; Male; Middle Aged; Young Adult
PubMed: 30489431
DOI: 10.1097/CJI.0000000000000250 -
Best Practice & Research. Clinical... Sep 2018Novel systemic therapies are generally prescribed to patients with advanced-stage disease or those with early-stage disease refractory to skin-directed therapies. In... (Review)
Review
Novel systemic therapies are generally prescribed to patients with advanced-stage disease or those with early-stage disease refractory to skin-directed therapies. In general, systemic chemotherapy should be reserved for patients who fail to respond to biological agents. Such biological agents include interferon alfa, bexarotene, histone deacetylase inhibitors (vorinostat, romidepsin), brentuximab vedotin and mogamulizumab. Extracorporeal photopheresis is particularly effective for patients with Sézary Syndrome. Allogeneic transplantation is becoming increasing used for younger patients. Novel agents in advanced development include the monoclonal antibody IPH4102,duvelisib,and the new modified formulation of denileukin diftitox. The choice of agents for patients is typically a balance of patient factors (age, co-morbidities, geographic location), relative efficacy and toxicity.
Topics: Allografts; Antineoplastic Agents; Hematopoietic Stem Cell Transplantation; Humans; Mycosis Fungoides; Photopheresis; Sezary Syndrome
PubMed: 30213403
DOI: 10.1016/j.beha.2018.07.007 -
Current Oncology Reports Mar 2018Cutaneous T-cell lymphoma (CTCL) is a rare form of non-Hodgkin lymphoma. Globally, the most common subtypes of CTCL are mycosis fungoides and Sézary syndrome. CTCL can... (Review)
Review
PURPOSE OF REVIEW
Cutaneous T-cell lymphoma (CTCL) is a rare form of non-Hodgkin lymphoma. Globally, the most common subtypes of CTCL are mycosis fungoides and Sézary syndrome. CTCL can confer significant morbidity and even mortality in advanced disease. Here we review the current and potential future treatments for advanced-stage CTCL.
RECENT FINDINGS
Heterogeneity of treatment choice has been demonstrated both in US and non-US centers. Systemic treatment choice is currently guided by prognostic features, incorporating stage, immunophenotypic and molecular findings, and patient-specific factors such as age and comorbidities. Randomized controlled studies are uncommon, and the literature is composed predominantly of retrospective, cohort, and early-phase studies. International consensus guidelines are available; however, the lack of comparative trials means that there is no clear algorithmic approach to treatment. This review article reports on the systemic treatment options in current use for advanced CTCL, and on the possible future therapies, acknowledging that an algorithmic approach is not yet forthcoming to guide treatment prioritization.
Topics: Combined Modality Therapy; Humans; Mycosis Fungoides; Prognosis; Sezary Syndrome; Skin Neoplasms
PubMed: 29572582
DOI: 10.1007/s11912-018-0678-x -
Cancer Science Mar 2018E7777, a recombinant cytotoxic fusion protein comprising diphtheria toxin fragments A and B and human interleukin-2, shares an amino acid sequence with denileukin...
E7777, a recombinant cytotoxic fusion protein comprising diphtheria toxin fragments A and B and human interleukin-2, shares an amino acid sequence with denileukin diftitox but has improved purity and an increased percentage of active protein monomer species. A phase I study was carried out to evaluate the tolerability, safety, pharmacokinetics, and antitumor activity of E7777 in Japanese patients with relapsed/refractory peripheral and cutaneous T-cell lymphoma. E7777 (6, 12, and expanded 9 μg/kg/day) was given to 13 patients by i.v. infusion on five consecutive days per 21-day cycle. Dose-limiting toxicities, including increased alanine aminotransferase, hyponatremia (n = 2), hypokalemia, lymphopenia, fatigue, hypoalbuminemia, rash, and increased lipase (n = 1), were observed in all three patients in the 12 μg/kg/day cohort, whereas two of six patients in the 9 μg/kg/day cohort showed decreased appetite or fatigue. The maximum tolerated and recommended dose of E7777 was 9 μg/kg/day for five consecutive days per 21-day cycle. The objective response rate was 38% (5/13) and did not appear to depend on tumor expression of CD25. E7777 was well tolerated, assuming careful management of adverse events during treatment, and preliminary but clinically meaningful antitumor activity was observed. Subsequent studies of E7777 for T-cell lymphomas are warranted. This study was registered with www.ClinicalTrials.gov (NCT1401530).
Topics: Administration, Oral; Adult; Aged; Antineoplastic Agents; Cohort Studies; Diphtheria Toxin; Drug Administration Schedule; Drug Resistance, Neoplasm; Female; Humans; Infusions, Intravenous; Interleukin-2; Lymphoma, T-Cell, Cutaneous; Male; Maximum Tolerated Dose; Middle Aged; Neoplasm Recurrence, Local; Recombinant Fusion Proteins; Skin Neoplasms; Treatment Outcome; Young Adult
PubMed: 29363235
DOI: 10.1111/cas.13513 -
Experimental Gerontology May 2018Cancer immunotherapy has shown remarkable recent progress. Immune checkpoint blocking antibodies have become the most successful anti-cancer agent class ever developed,...
Cancer immunotherapy has shown remarkable recent progress. Immune checkpoint blocking antibodies have become the most successful anti-cancer agent class ever developed, with six distinct agents approved since 2011 for a wide variety of cancers. Although age is the biggest risk factor for cancer (aside from selected early-onset pediatric cancers), these agents were tested pre-clinically in young hosts, and there is remarkably little published on the effects of host age on treatment outcomes in pre-clinical studies or human clinical trials. The three principal immune checkpoints against which blocking antibodies have been FDA-approved for human use are CTLA-4, PD-1 and PD-L1. We used a mouse model of transplantable, orthotopic B16 melanoma to test age effects of treatments with anti-CTLA-4, anti-PD-1 and anti-PD-L1 antibodies. All three agents were highly effective in treating young tumor-bearing hosts as expected. Anti-PD-L1 as a single agent had no effect on tumor growth in aged hosts, anti-CTLA-4 had detectable, modest effects and anti-PD-1 was essentially as effective in aged as in young hosts, the first single agent we have identified not to lose efficacy with age in this model. Other important differences in young versus aged hosts included lack of anti-CTLA-4-mediated depletion of intratumor regulatory T cells in aged hosts and poorer ability of all three agents to activate T cells in aged versus young hosts. Anti-CTLA-4 efficacy appeared to improve when combined with anti-PD-L1. Regulatory T cell depletion with FDA-approved denileukin diftitox did not improve treatment by any single agent. Aged mice tolerated treatments as well as young mice without obvious toxicities at equivalent doses.
Topics: Aging; Animals; B7-H1 Antigen; CTLA-4 Antigen; Diphtheria Toxin; Disease Models, Animal; Female; Immunotherapy; Interleukin-2; Male; Melanoma; Mice; Mice, Inbred C57BL; Programmed Cell Death 1 Receptor; Recombinant Fusion Proteins; T-Lymphocytes, Regulatory
PubMed: 29326088
DOI: 10.1016/j.exger.2017.12.025 -
JAAD Case Reports Nov 2017
PubMed: 29296637
DOI: 10.1016/j.jdcr.2017.06.031