Diphtheria Toxin Fusion Protein Research Articles

Efficacy and Safety of E7777 (improved purity Denileukin diftitox [ONTAK]) in Patients with Relapsed or Refractory Cutaneous T-Cell Lymphoma: Results from Pivotal Study 302

Introduction: Denileukin diftitox (Dd) is a recombinant fusion protein of diphtheria toxin and human interleukin-2. Dd was approved and marketed as ONTAK in the US from 1999-2014 for treatment of pts with relapsed/refractory CTCL. ONTAK was voluntarily taken off the market in 2014 due to manufacturing difficulties. E7777 was developed as a result of a refined manufacturing process and has an increased percentage of protein monomer species, with a concomitant decrease in levels of misfolded protein and protein aggregates. E7777 has ~1.5-2 times greater specific bioactivity in non-clinical assays compared with ONTAK and is considered a new drug by the FDA. Methods: Objectives: Study 302 (NCT01871727) is a multicenter, open-label, single-arm registrational trial designed to assess efficacy and safety of E7777 in patients with relapsed/refractory CTCL. E7777 was given IV at 9 mcg/kg/day (established from Lead-in) over 60 min for 5 days every 21 days up to 8 cycles. Key inclusion/exclusion criteria included histopathologic diagnosis of CTCL (MF or SS); ECOG performance status 0 or 1; no prior exposure to ONTAK. The primary efficacy endpoint was objective response rate (ORR; CR+PR) per Independent Review Committee (IRC) assessment based on Global Response Score GRS (Olsen, et al., 2011). Secondary efficacy endpoints included duration of response (DOR), time-to-response (TTR), skin response (according to modified Severity Weighted Assessment Tool [mSWAT]), safety and tolerability of E7777, PK, and immunogenicity. Exploratory objectives included progression free survival (PFS), time to progression (TTP), pruritus improvement, and quality of life assessment. Results: A total of 112 patients with CTCL stage I-IV were enrolled in the study: 21 in the Lead-in and 91 in the Main Study. The primary efficacy population is comprised of 69 patients with stage I-III CTCL treated at 9 mcg/kg/day. Median number of prior anticancer therapies in Study 302 were 4.0 (range: 1 to 18). Forty-five patients (65.2%) received ≥4 prior anticancer therapies. Approximately half (34 of 69; 49.3%) of patients had prior exposure to 1 or more FDA-approved targeted therapeutics: romidepsin, brentuximab, or mogamulizumab. Demographic and baseline characteristics of patients included a median age of 64 (range 28-87); ECOG status 0 (56.5%) and 1 (43.5%); Race: 72.5% White, 18.8% Black; 9% Other. There disease stages were 43.5% IA/IB/IIA; 34.8% IIB; and 21.7% IIIA/IIIB. The median (range) number of cycles of E7777 received was 6.0 (1, 42). The primary efficacy endpoint, ORR (95% CI) by IRC, was 36.2% (95% CI: 25.0%, 48.7%), with 8.7% achieving a CR. ORR (95% CI) by Investigator was 42.3% (30.6%, 54.6%), with 8.5% achieving a CR (Table 1). DOR was at least 6 months for 13 (52.0%) responders and at least 12 months for 5 (20.0%) responders. TTR was rapid (median=1.41 months); the majority of responders (~70%) had a response after 1 to 2 cycles of treatment. Treatment with E7777 did not demonstrate any new or unexpected safety findings as compared to prior ONTAK experience. Most patients (98.6%) experienced at least 1 TEAE. Most common TEAEs were nausea (43.5%); fatigue (31.9%); and increased ALT, chills, and peripheral edema (27.5% each). Mean numbers of TEAEs per subject were higher in the first 1 to 2 treatment cycles, then decreased and remained low/stable over the remainder of therapy. Key adverse events of special interest (AESIs) associated with ONTAK in the past (i.e. capillary leak syndrome, hepatotoxicity, infusion reaction, and visual impairment) were primarily Grade 1/2 in our current study and will be discussed in detail in a separate abstract. Conclusions: In study 302, E7777 at a dose of 9 mcg/kg/day demonstrated clinical efficacy and clinically meaningful benefit in heavily pre-treated patients with relapsed/refractory CTCL. The ORR of 36.2% per IRC (42.3% by investigator assessment) showed that a substantial proportion of these heavily pretreated patients experienced clinical benefit after E7777 treatment similar to ONTAK. The observed tumor responses were rapid, durable, and deep. A BLA submission to the FDA is planned for the 2nd half of 2022. E7777 will provide a novel, efficacious, safe, and non-cross-resistant therapeutic option for CTCL patients who have failed other treatments and thus fulfill a serious unmet need. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Preclinical and clinical researches of Denileukin Diftitox (Genetical Recombination) (Remitoro®), a novel agent for T-cell lymphoma

Denileukin Diftitox (DD) is a recombinant fusion protein of diphtheria toxin (DT) fragments and human interleukin-2 (IL-2). DD binds to IL-2 receptor (IL-2R) expressed on tumor cells and is taken up into the cells. Subsequently, DT fragments with adenosine diphosphate ribosylation enzyme inhibit protein synthesis, then ultimately trigger cell death. DD binds to both high- and intermediate-affinity IL-2Rs via IL-2 domain and inhibits growth of human T-cell lymphomas cell lines. E7777, which contains DD as an active component, has improved purity and an increased percentage of active monomer compared with the approved drug E7272 (ONTAK in the US, not approved in Japan). In the phase I clinical study in Japanese patients with relapsed or refractory peripheral T-cell lymphoma (PTCL) and cutaneous T-cell lymphoma (CTCL), the maximum tolerated dose and recommended dose of E7777 were 9 μg/kg/day (administered on Days 1-5 of each cycle) based on the evaluation of dose-limiting toxicity. In the phase II clinical study, the objective response rate was 36.1%, showing comparable efficacy to existing therapies. E7777 showed anti-tumor activity observed across the range of CD25 expression. Grade 3 or higher adverse events (AE) occurred in 94.6%, and serious AE such as capillary leak syndrome and rhabdomyolysis were reported. Therefore, safety monitoring activities have been continued along with alerting related events. Based on these results, E7777 obtained a new drug approval in Japan in March 2021 for the indication of relapsed or refractory PTCL/CTCL.

Effective Host-Directed Therapy for Tuberculosis by Depletion of Myeloid-Derived Suppressor Cells and Related Cells Using a Diphtheria Toxin Fusion Protein.

Myeloid-derived suppressor cells (MDSCs) are present in elevated numbers in tuberculosis patients and have been found to be permissive for Mycobacterium tuberculosis proliferation. To determine whether depletion of MDSCs may improve host control of tuberculosis, we used a novel diphtheria toxin-based fusion protein DABIL-4 that targets and depletes interleukin 4 (IL-4) receptor-positive cells. We show that DABIL-4 depletes both polymorphonuclear MDSCs and monocytic MDSCs, increases interferon-γ + T cells, and reduces the lung bacillary burden in a mouse tuberculosis model. These results indicate that MDSC-depleting therapies targeting the IL-4 receptor are beneficial in tuberculosis and offer an avenue towards host-directed tuberculosis therapy.

Orphan designation: recombinant human interleukin-3 truncated diphtheria toxin fusion protein, Treatment of acute myeloid leukaemia

Orphan designation: recombinant human interleukin-3 truncated diphtheria toxin fusion protein, Treatment of acute myeloid leukaemia

Orphan designation: recombinant human interleukin-3 truncated diphtheria toxin fusion protein, Treatment of acute myeloid leukaemia

Orphan designation: recombinant human interleukin-3 truncated diphtheria toxin fusion protein, Treatment of acute myeloid leukaemia

Orphan designation: recombinant human interleukin-3 truncated diphtheria toxin fusion protein, Treatment of blastic plasmacytoid dendritic cell neoplasm

Orphan designation: recombinant human interleukin-3 truncated diphtheria toxin fusion protein, Treatment of blastic plasmacytoid dendritic cell neoplasm

Orphan designation: recombinant human interleukin-3 truncated diphtheria toxin fusion protein, Treatment of blastic plasmacytoid dendritic cell neoplasm

Orphan designation: recombinant human interleukin-3 truncated diphtheria toxin fusion protein, Treatment of blastic plasmacytoid dendritic cell neoplasm

Abstract LB-293: IL-2 diphtheria toxin fusion protein with anti-PD1: in mouse B16-F10 melanoma model

Abstract In tumor immunotherapy, regulatory T lymphocytes (Tregs) play an important role in the suppression of beneficial anti-tumor responses. In the non-immunogenic B16-F10 mouse melanoma model, tumors are found to be highly infiltrated by Tregs. DAB389IL-2 (Ontak, denileukin diftitox) is an IL-2 diphtheria toxin-related fusion protein that has been showed to deplete Tregs in both humans and mice. We treated mice with established B16F10 tumors with s-DAB389IL-2 (produced as a secreted protein from C. diphtheria) and observed a significant decrease in tumor growth rate in C57BL/6 mice. Also, s-DAB389IL-2 treatment increased the frequency of CD8+IFNγ+ T cells (TILs) in tumors and spleens. S-DAB389IL-2 treatment followed by PD-1 blockade resulted in greater tumor growth inhibition than either monotherapy alone. These data suggest that s-DAB389IL-2 plus anti-PD1 treatment can improve anti-tumor responses in non-immunogenic B16F10 melanoma and inhibit tumor growth. Additionally, we mutated a vascular leak inducing motif to construct s-DAB389IL-2 (V6A), which cause less vascular leak in vitro. S-DAB389IL-2 (V6A) inhibited tumor growth with similar efficacy to the original fusion toxin. As vascular leak is the major adverse effect caused by Ontak, DAB389IL-2 (V6A) shows promise as a better tolerated drug for further studies as a cancer immunotherapy. Citation Format: Juan Fu, Drew Pardoll, Laurene Cheung. IL-2 diphtheria toxin fusion protein with anti-PD1: in mouse B16-F10 melanoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-293.

Abstract B034: Preclinical immune antitumor activity of myeloid-targeting E7046 and Treg depleting E7777

Abstract Purpose: To assess the combined activity of E7046 and E7777 against multiple murine syngeneic tumor models. Background: Both regulatory T cells (Tregs)and myeloid cells are significant constituents in the tumor microenvironment in many types of cancer, where they help to maintain an immunosuppressive milieu that inhibits cytotoxic T cell function, which favors tumor persistence. Prostaglandin E2 (PGE2) engages the EP4 receptor on monocytes to direct myeloid cell activities away from antigen presentation and toward immunosuppression (wound healing). PGE2-driven myeloid responses have been reported to enhance Treg immunosuppressive activity, while Tregs with high expression of the FoxP3 transcription factor have been reported to up-regulate cyclooxygenase expression and PGE2 secretion. E7046 is a highly selective small molecule that potently competes with PGE2 binding to EP4. Single agent E7046 slows the growth of multiple syngeneic murine tumor types, in a mechanism dependent upon the presence of T cells. Denileukin diftitox (ONTAK®), an IL-2/diphtheria toxin fusion protein, was originally approved by the US FDA for treating patients with relapsed/refractory cutaneous T cell lymphoma. E7777 is a new version of denileukin diftitox developed using an improved manufacturing process. We and others have shown that denileukin diftitox selectively depletes Tregs. Neither E7046 nor E7777 affects tumor cell viability in vitro. Here we assessed their combined activity against syngeneic tumor models and the mechanisms driving their activity. Methods: Tumors were implanted subcutaneously in groups of 5-10 female BALB/c mice. When tumors were > 50 mm3, mice were treated with E7046 (150 mg/kg, QD x 21, p.o.) and/or E7777 (2.5 or 3 μg/head, Q7D x 2 or 3, i.v). Tumors, spleens, and tumor-draining lymph nodes were excised and analyzed by flow cytometry for immune cell composition and function. Results: As single agents, both E7046 and E7777 delayed the growth of established tumors. When combined, their anti-tumor activities were significantly enhanced, with up to 20% of animals rendered stably tumor-free. Activity was significantly reduced if animals were treated with antibody to deplete CD8+ T cells. In the tumors, the combination treatment dramatically increased the ratio of CD8 T cells to CD4+CD25+Foxp3+ Tregs, and also decreased the frequency of immunosuppressive myeloid cells. Importantly, the CD8+ T cells in the treated tumors, but not in the control tumors, were found to express granzyme B. Spleens from the treated animals also showed an increased ratio of CD8+ T cells vs the highly active CD4+CD25+Foxp3+ ICOS+ Tregs. Conclusions: The combination of E7046 and E7777 showed promising preclinical anti-tumor activity via an immune-mediated mechanism. Citation Format: Diana I. Albu, Kuan-Chun Huang, Jiayi Wu, Xingfeng Bao, Kenichi Nomoto, Mary Woodall-Jappe. Preclinical immune antitumor activity of myeloid-targeting E7046 and Treg depleting E7777. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B034.

Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) Patient-Derived Xenografts Are Faithful Genomic and Phenotypic Models of Primary Leukemia and Respond to the IL3 Receptor Targeting Agent SL-401 In Vivo

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive acute leukemia/lymphoma recently classified as a malignant transformation of plasmacytoid dendritic cells (pDCs) and a subtype of acute myeloid leukemia (AML). BPDCN has no standard treatment and a poor prognosis, with median survival <1 year. A significant roadblock to better understanding BPDCN is a lack of adequate model systems. We generated patient-derived xenografts (PDX) of BPDCN in NOD/Scid/IL2rgnull (NSG) mice. Bone marrow or peripheral blood cells involved by BPDCN blasts (CD45 low, CD123 high, HLA-DR high, CD3 neg) were transplanted into irradiated NSG recipients. Nine of 16 BPDCNs caused lethal leukemia involving blood, spleen, and bone marrow 2-6 months after transplantation. All nine BPDCN PDXs were serially transplantable. Flow characterization of each patient's BPDCN and corresponding xenograft revealed no major differences in BDCA2, BDCA4, FCeR1, ILT7, or cytoplasmic TCL1 staining. All samples maintained high expression of the human interleukin-3 (IL3) receptor (IL3Ralpha/CD123), a hallmark feature of BPDCN. To further characterize BPDCN pathogenesis we performed whole transcriptome sequencing (RNA-seq) on sorted blasts from 11 patients and on normal pDCs isolated from 4 healthy donors. These were compared to RNA-seq in six PDXs. The spectrum of mutations in BPDCN transcriptomes overlapped with that seen in other hematologic malignancies, particularly myeloid disorders, and was similar to reported DNA mutations in BPDCN, including in ASXL1, CTCF, IDH2, NRAS, RUNX1, STAG2, TET2, and TP53. Particularly striking was the presence of a canonical mutation in an RNA splicing factor in 7 of 11 cases (SRSF2 P95H/L/R in four, ZRSR2 R295* and gene locus deletion in two, and SF3B1 K666N in one). Known oncogenic mutations in the original disease were retained in the PDXs, including all splicing factor mutations, with the exception of an IDH2 R140Q that was lost in one PDX. BPDCN PDXs grouped together in unsupervised clustering of expression profiles, distinct from AML and ALL PDXs in an analysis of 134 models from the DFCI Public Repository of Xenografts (http://PRoXe.org). Gene set enrichment analysis (GSEA) of KEGG and REACTOME pathways associated with differentially expressed genes between primary BPDCNs and non-malignant pDCs revealed signatures related to dendritic cell activation, cell cycle, and apoptosis. In addition, 3 of the top 11 sets were genes involved in mRNA processing, mRNA splicing, and processing capped intron-containing pre-mRNAs (all FDR<1e-6). To test the efficacy of BPDCN-targeted therapy using primary human leukemias in vivo, we performed a pre-clinical trial in NSG mice using SL-401, a recombinant biologic consisting of a fusion protein of IL3 and diphtheria toxin. Three independent BPDCN xenografts were injected into 20 NSG mice each, and followed by weekly peripheral blood monitoring for human CD45 and CD123. When leukemia burden reached >0.5% in at least half of the mice in the cohort, animals were randomized to receive SL-401 at 100 ug/kg or vehicle intraperitoneally daily for 5 days. Two mice in each group were sacrificed at day 7 for response assessment, and peripheral blood was followed weekly in the remaining mice for evidence of progression (>5% human CD45/CD123-positive cells). 7 days after treatment, mice receiving SL-401 had dramatic reductions in BPDCN in the peripheral blood, spleen, and bone marrow (0.31% vs 37.6% in marrow of SL-401 vs vehicle). SL-401 prolonged progression-free survival in all BPDCNs tested (12 vs 48 days, P<0.0001 by log-rank test). At the time of progression after SL-401, relapsing mice were re-randomized to receive a 2nd and in some cases 3rd cycle of SL-401 or vehicle. Repeated treatment in mice that progressed after SL-401 resulted in second and third peripheral blood remissions. All PDXs responded to SL-401 including those with and without splicing factor and TP53 mutations. CD123 expression was maintained at high levels on all SL-401 treated BPDCNs even after repeated cycles. Primary xenografts of BPDCN are faithful models of the human disease, maintain genetic and transcriptomic characteristics of the original tumor, and respond to multiple courses of IL3-DT in vivo, suggesting that they provide a valuable resource to study disease biology and response/resistance to targeted therapy. DisclosuresChen:Stemline Therapeutics, Inc.: Employment. Brooks:Stemline Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Lane:Stemline Therapeutics, Inc.: Research Funding.

Clearance of acute myeloid leukemia by haploidentical natural killer cells is improved using IL-2 diphtheria toxin fusion protein

AbstractHaploidentical natural killer (NK) cell infusions can induce remissions in some patients with acute myeloid leukemia (AML) but regulatory T-cell (Treg) suppression may reduce efficacy. We treated 57 refractory AML patients with lymphodepleting cyclophosphamide and fludarabine followed by NK cell infusion and interleukin (IL)-2 administration. In 42 patients, donor NK cell expansion was detected in 10%, whereas in 15 patients receiving host Treg depletion with the IL-2-diphtheria fusion protein (IL2DT), the rate was 27%, with a median absolute count of 1000 NK cells/μL blood. IL2DT was associated with improved complete remission rates at day 28 (53% vs 21%; P = .02) and disease-free survival at 6 months (33% vs 5%; P < .01). In the IL2DT cohort, NK cell expansion correlated with higher postchemotherapy serum IL-15 levels (P = .002), effective peripheral blood Treg depletion (<5%) at day 7 (P < .01), and decreased IL-35 levels at day 14 (P = .02). In vitro assays demonstrated that Tregs cocultured with NK cells inhibit their proliferation by competition for IL-2 but not for IL-15. Together with our clinical observations, this supports the need to optimize the in vivo cytokine milieu where adoptively transferred NK cells compete with other lymphocytes to improve clinical efficacy in patients with refractory AML. This study is registered at clinicaltrials.gov, identifiers: NCT00274846 and NCT01106950.

Transient regulatory T-cell (Treg) depletion with IL-2 diphtheria toxin fusion protein enhances clearance of acute myeloid leukemia by haploidentical natural killer (NK) cells

Meeting abstracts Adoptive transfer of haploidentical natural killer (NK) cells can induce remissions in patients with AML. Despite lymphodepleting chemotherapy failure may result from suppression by host regulatory T cells (Treg). We report outcomes of 57 refractory AML patients treated with

DAB389IL-2 suppresses autoimmune inflammation in the CNS and inhibits T cell-mediated lysis of glial target cells

In multiple sclerosis (MS) and its rodent model, experimental autoimmune encephalomyelitis (EAE), activated CD4+ T cells with upregulated IL-2R mediate inflammation and demyelination in the central nervous system (CNS). DAB389IL-2, a chimeric fusion protein of IL-2 and diphtheria toxin, inhibits human and rodent IL-2 activated T cells that express the high affinity interleukin-2 receptor. In the present study, DAB389IL-2 was used to treat rats with EAE. We wanted to investigate the possibility that DAB389IL-2 could prevent tissue destruction within the CNS. We used a suboptimal dose of DAB389IL-2 that allowed substantial transmigration of inflammatory cells across the blood–brain barrier. DAB389IL-2 inhibited infiltration of CD4+, CD8+, CD25+ and TCR αβ+ associated mononuclear cells and inflammatory macrophages in the spinal cord on day 13 post-immunization, at the peak of disease. Gene expression study showed that DAB389IL-2 treatment suppressed TNF-α and IFN-γ as well as IL-10 cytokine gene expression in the spinal cord of rats with EAE on day 13. DAB389IL-2 in vitro treatment suppressed cytotoxicity of MBP-activated T cells from rats with EAE against oligodendrocytes in culture by 66%. Astrocytes were less targeted by MBP activated T cells in vitro. This study suggests that DAB389IL-2 directly targets CD4+ and CD25+ (IL-2R) T cells and effector T cell function and also indirectly suppresses the activation of macrophage CD169+ (ED3+) and microglia CD11b/c (OX42+) populations in the CNS.

Diphtheria Toxin–Epidermal Growth Factor Fusion Protein DAB389EGF for the Treatment of Bladder Cancer

The novel fusion protein, DAB(389)EGF, is composed of both the catalytic and the translocation domains of diphtheria toxin that are fused to the human EGF, providing a targeting and a toxicity component. We tested DAB(389)EGF for antitumor activity in both in vitro and in vivo urinary bladder cancer models. Human bladder cancer lines were treated with DAB(389)EGF and assessed for growth inhibition and clonogenic suppression. Using 6- to 8-week-old female athymic nude mice implanted orthotopically with HTB9 cells, DAB(389)EGF was administered intravesically twice weekly for 2 weeks. The response of the luciferase-expressing HTB9 cells was monitored via bioluminescence as the primary endpoint. Treatment response with DAB(389)EGF was specific and robust, with an IC(50) ranging from 0.5 to 15 ng/mL in eight tested bladder cancer cell lines, but greater than 50 ng/mL in the EGF receptor (EGFR)-negative H520 control cell line. Simulating short-duration intravesical therapy used clinically, a 2-hour treatment exposure of DAB(389)EGF (10 ng/mL) produced clonogenic suppression in three selected bladder cancer cell lines. In vivo, luciferase activity was suppressed in five of six mice treated with DAB(389)EGF [70 μL (1 ng/μL) per mouse], as compared with only one of six mice treated with a control diphtheria toxin (DT) fusion protein. Histologic assessment of tumor clearance correlated with the bioluminescent changes observed with DAB(389)EGF treatment. Immunocompetent mice treated with intravesical DAB(389)EGF did not show any nonspecific systemic toxicity. The intravesical delivery of targeted toxin fusion proteins is a novel treatment approach for non-muscle-invasive urinary bladder cancer. With appropriate targeting, the treatments are effective and well-tolerated in vivo.

DT390-triTMTP1, a Novel Fusion Protein of Diphtheria Toxin with Tandem Repeat TMTP1 Peptide, Preferentially Targets Metastatic Tumors

Peptide-based therapies have emerged as one of the most promising therapeutics strategy in cancer-targeted therapy. Using our laboratory newly identified peptide TMTP1 and diphtheria toxin, we developed a new fusion protein that showed remarkable ability to target highly metastatic tumors. Fusion protein toxins were generated by fusing the first 390 amino acids of diphtheria toxin [truncated diphtheria toxin (DT390)] to different repeats of peptide TMTP1 (DT390-TMTP1, DT390-biTMTP1, and DT390-triTMTP1). Efficacies of the recombinant fusion proteins on tumor growth and metastasis were evaluated in vitro and in vivo. DT390-triTMTP1 showed the most powerful toxicity against cancer, which led to tumor growth retardation or regression and prolonged survival of human prostate cancer PC-3M-1E8 subcutaneously bearing or gastric cancer MKN-45 orthotopic nude mice. Increased TUNEL and caspase-3 staining and reduced ki67 staining in tumor cells suggested that the anticancer effects of DT390-triTMTP1 were through selectively inducing apoptosis and inhibiting proliferation of cancer cells. In a murine model of human orthotopic gastric carcinoma, DT390-biTMTP1 significantly inhibited metastases to liver and spleen, while DT390-triTMTP1 not only totally suppressed metastasis but also reduced primary tumors by 66.6%. In the biodistribution test, DT390-triTMTP1 was observed to home to tumor tissue rapidly and lasted over 48 h, with only a transient appearance in liver and kidney immediately after injection. Thus, our present study provided a novel recombinant fusion protein DT390-triTMTP1 with preferential targeting and high cytotoxicity, which may be a promising strategy for the targeted therapy of cancer metastasis.

Diphtheria toxin/human B-Cell activating factor fusion protein kills human acute lymphoblastic leukemia BALL-1 cells: An experimental study

This study aimed to express a fusion protein of diphtheria toxin and human B cell-activating factor (DT388sBAFF) in Escherichia coli (E. coli) and investigate its activity in human B-lineage acute lymphoblastic leukemia 1 cells (BALL-1). A fragment of DT388sBAFF fusion gene was separated from plasmid pUC57-DT388sBAFF digested with Nde I and Xho I, and inserted into the expression vector pcold II digested with the same enzymes. Recombinants were screened by the colony polymerase chain reaction (PCR) and restriction map. The recombinant expression vector was transformed into BL21 and its expression was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG). The recombinant protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, and then purified by Ni(2+)-NTA affinity chromatography. The expression level of B cell-activating factor receptor (BAFF-R) on BALL-1 cells was assessed by real-time PCR. The receptor binding capacity of recombinant protein was determined by cell fluorescent assay. The specific cytotoxicity of recombinant protein on BALL-1 cells was detected by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. The expression level of recombinant protein was 50% of total bacterial proteins in E. coli, and the recombinant protein could bind to BAFF-R-positive BALL-1 cells and thereby produce a cytotoxic effect on the cells. The fusion protein expression vector DT388sBAFF was successfully constructed and the recombinant protein with selective cytotoxicity against BALL-1 cells was obtained, providing foundation for further study of the therapy of human B-lineage acute lymphoblastic leukemia.

Therapeutic Enhancement of Protective Immunity during Experimental Leishmaniasis

BackgroundLeishmaniasis remains a significant cause of morbidity and mortality in the tropics. Available therapies are problematic due to toxicity, treatment duration and emerging drug resistance. Mouse models of leishmaniasis have demonstrated that disease outcome depends critically on the balance between effector and regulatory CD4+ T cell responses, something mirrored in descriptive studies of human disease. Recombinant IL-2/diphtheria toxin fusion protein (rIL-2/DTx), a drug that is FDA-approved for the treatment of cutaneous T cell lymphoma, has been reported to deplete regulatory CD4+ T cells.Methodology/Principal FindingsWe investigated the potential efficacy of rIL-2/DTx as adjunctive therapy for experimental infection with Leishmania major. Treatment with rIL-2/DTx suppressed lesional regulatory T cell numbers and was associated with significantly increased antigen-specific IFN-γ production, enhanced lesion resolution and decreased parasite burden. Combined administration of rIL-2/DTx and sodium stibogluconate had additive biological and therapeutic effects, allowing for reduced duration or dose of sodium stibogluconate therapy.Conclusions/SignificanceThese data suggest that pharmacological suppression of immune counterregulation using a commercially available drug originally developed for cancer therapy may have practical therapeutic utility in leishmaniasis. Rational reinvestigation of the efficacy of drugs approved for other indications in experimental models of neglected tropical diseases has promise in providing new candidates to the drug discovery pipeline.

Frequency of Circulating Tregs with Demethylated FOXP3 Intron 1 in Melanoma Patients Receiving Tumor Vaccines and Potentially Treg-Depleting Agents

Regulatory T cells (Tregs) are thought to inhibit antitumor immune responses, and their depletion could therefore have a synergistic effect with therapeutic cancer vaccines. We investigated the impact of three medications on blood Treg frequency in vaccinated cancer patients. To date, the most specific marker for human Tregs is demethylation in the DNA that encodes the transcription factor FOXP3. Thus, we used a FOXP3 methylation-specific quantitative PCR assay (MS-qPCR) to measure Treg frequencies in the peripheral blood mononuclear cells (PBMCs) of melanoma patients. The patients participated in three clinical trials that combined tumor vaccines with potential Treg-depleting agents: low-dose cyclophosphamide, anti-CD25 monoclonal antibody daclizumab, and the IL-2/diphtheria toxin fusion protein denileukin diftitox. In the nine control patients, blood Treg frequencies varied over time; there was a 46% reduction in one patient. In treated patients, a more than 2-fold decrease in Tregs was observed in one out of 11 patients receiving cyclophosphamide and in four out of 13 receiving daclizumab, but there was no such Treg decrease in any of the six patients who received denileukin diftitox. As a positive control, a more than 2-fold increase in blood Tregs was detected in four out of nine patients who were treated with interleukin-2. We used a MS-qPCR method that detects Tregs but not other activated T lymphocytes; however, none of the Treg-depleting strategies that we tested led, in the majority of patients, to a conservative 50% reduction in blood Tregs.

A diphtheria toxin interleukin-3 fusion protein synergizes with tyrosine kinase inhibitors in killing leukemic progenitors from BCR/ABL positive acute leukemia

Despite initial remissions, most patients with Ph chromosome positive (Ph +) acute leukemia (AL) become refractory to tyrosine kinase inhibitors (TKIs) such as imatinib and dasatinib. This study was designed to determine if targeting the interleukin-3 receptor (IL-3R) with a diphtheria toxin fusion protein (DT 388IL3) would improve the effectiveness of TKIs against Ph + AL cells. IL-3R subunits were detected on most Ph + cells and the IC50 for killing of colony forming cell (CFC) with DT 388IL3 correlated with the level of IL-3Rα subunit by FACS. DT 388IL3 synergized with both imatinib and dasatinib for killing of malignant CFCs. Long-term suspension culture-initiating cells (SC-ICs) and quiescent leukemic cells (G 0 in cell cycle) also were studied and synergistic interactions were again demonstrated. Thus, cotreatment with TKIs and DT 388IL3 is much more effective in eliminating Ph + leukemic progenitors that express IL-3R than either agent alone.

Expression of IL-13Rα2 in liver cancer cells and its effect on targeted therapy of liver cancer

Liver cancer is the third leading cause of cancer-related deaths globally. The number of liver cancers diagnosed in the world is increasing at an alarming rate. It is of great significance to find the new targets of the tumor cells and specific medicine. This research investigated the expression of interleukin-13 receptor alpha2 (IL-13Ralpha2) in different liver cancer cell lines and liver cancer tissues, and assessed the cytotoxin DT389-hIL13-13E13K (IL-13 and diphtheria toxin fusion protein) targeted killing effect on liver cancer cells. Based on study above, we further analyzed the function of IL-13Ralpha2 on the targeted liver cancer therapy. The results will provide a novel strategy and an alternative way for liver cancer therapy. The expression of IL-13Ralpha2 in different liver cancer cell lines and tissues were analyzed by RT-PCR and immunohistochemistry. Cytotoxicity assay of DT389-hIL13-13E13K was performed in eight different concentrations in liver cancer cell lines in vitro. At the same time, siRNA-mediated knockdown was introduced to assess the role of IL-13Ralpha2 in liver cancer therapy. Two out of four tested liver cancer cell lines and 27 out of 33 (81.82%) liver tissues expressed the IL-13Ralpha2. The fusion protein DT(389)-hIL13-13E13K showed a moderate cytotoxicity to the cancer cell line BEL-7402 in vitro, which 50% inhibition (IC(50)) concentration occurred at 1.4 x 10(-5 )M. Besides, the sensitivity to fusion protein DT(389)-hIL13-13E13K was decreased in siRNA-transfected liver cells compared with control ones. These results suggest that IL-13Ralpha2 chain is a specific target for IL-13-directed fusion protein. We reported the expression of IL-13Ralpha2 in liver cancer cell lines and tissues as well as investigated the cytotoxin (DT389-hIL13-13E13K) targeted killing efficiency of liver cancer cells and potential role of IL-13Ralpha2 in the cancer treatment.