HIV-TAT Protein Transduction Domain Research Articles

TAT-MYC Recombinant Fusion Protein Enhances Hematopoietic Stem Cell Graft Performance and Immune Cell Reconstitution after Transplantation

The long interval between hematopoietic stem cell transplantation (HSCT) and hematopoietic engraftment and immune reconstitution is a dangerous time for patients. Although neutrophil counts usually rise within several weeks following stem cell transplantation, recovery of functional T and B cell repertoires can often take months; during this period patients are at risk for severe infections, and graft vs. tumor effects may be attenuated. We have examined the use of a recombinant fusion protein consisting of the N-terminal 9 amino acid segment of the HIV TAT protein transduction domain (PTD) fused to the MYC protein as a means of enhancing stem cell graft performance.Methods: Coding sequences for the 9-amino acid TAT PTD and the cDNA encoding MYC were ligated in-frame and cloned into the pET101/D-Topo vector for expression in E Coli. The fusion protein, which we designated TBX-4000, was purified to homogeneity, analyzed for contaminants, and prepared to clinical grade for administration in accordance with good manufacturing practices.Results: Following a 1-hour incubation of TBX-4000 with human cord blood cells, the chimeric protein appeared in the nucleus of target cells within 2 hours, but was rapidly metabolized, vanishing by 72 hours. Whole murine marrow and purified murine LSK+ cells that were incubated with TBX-4000 significantly out-performed control cells in competitive engraftment experiments in immune deficient recipient mice, even at ratios of 1:9 treated to control cells. Significantly higher numbers of T and B cells were recovered from both peripheral blood and splenic tissue of irradiated immune deficient mice transplanted with stem cell grafts that had been cultured for 60 minutes with TBX-4000, relative to control mice. TBX-4000 cultured cells demonstrate resistance to apoptosis induced by Granzyme B, which may account for their hardiness during engraftment in marrow niches recently exposed to the stress of pre-transplant conditioning therapy. Extensive toxicologic studies demonstrated that prolonged exposure to TBX-4000 does not cause malignant transformation of hematopoietic cells in culture, and does not lead to tumor formation in mice repeatedly injected with the recombinant protein. Mice transplanted with stem cells incubated with TBX-4000 did not develop leukemia or solid tumors during 4 months of follow-up, or following three “generations” of successive, serial transplantation. Because TBX-4000 augments immune reconstitution following hematopoietic stem cell transplantation and may stimulate graft vs. host diseases reactions, T and B cell depletion of the graft will be performed in pilot studies in humans to prevent this complication.Conclusion: Brief exposure to the TAT-MYC recombinant fusion protein enhances the performance of hematopoietic stem cells in a pre-clinical transplantation model. The brief presence of the protein protects engrafting cells from apoptotic stimuli, and does not lead to transformation of the cells following transplantation. The efficacy and safety of TBX-4000 in our pre-clinical studies serve as the basis for currently planned clinical trials of this chimeric protein in patients undergoing stem cell transplantation. DisclosuresGregory:Taiga Biotechnologies, Inc.: Employment, Equity Ownership. Turner:Taiga Biotechnologies, Inc.: Employment, Equity Ownership. Refaeli:Taiga Biotechnologies, Inc.: Employment, Equity Ownership.

Mn-TAT PTD-Ngb attenuates oxidative injury by an enhanced ROS scavenging ability and the regulation of redox signaling pathway

Neurological diseases have a close relationship to excessive reactive oxygen species (ROS). Neuroglobin (Ngb), an intrinsic protective factor, protected cells from hypoxic/ischemic injury. In the present, we reported a novel neuroprotective manganese porphyrin reconstituted metal protein, Mn-TAT PTD-Ngb, consisting of a HIV Tat protein transduction domain sequence (TAT PTD) attached to the N-terminal of apo-Ngb. Mn-TAT PTD-Ngb had a stronger ROS scavenging ability than that of TAT PTD-Ngb, and reduced intracellular ROS production and restored the function of the mitochondria and inhibited the mitochondria-dependent apoptosis. Besides, Mn-TAT PTD-Ngb activated the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway, which up-regulated the expression of nuclear factor E2-related factor 2 (Nrf2), Heme oxygenase-1 (HO-1), superoxide dismutase (SOD), catalase (CAT). The results showed that the redox chemistry of Mn-TAT PTD-Ngb and redox regulation of multiple signaling pathways attenuated the oxidative injury.

Development of Safe and Non-Self-Immunogenic Mucosal Adjuvant by Recombinant Fusion of Cholera Toxin A1 Subunit with Protein Transduction Domain.

Potential use of cholera toxin (CT) as a mucosal vaccine adjuvant has been documented in a variety of animal models. However, native CT is highly toxic to be used as a mucosal adjuvant in humans. Here, we demonstrate a new approach to generate a mucosal adjuvant by replacing the B subunit of CT with HIV-1 Tat protein transduction domain (PTD), which efficiently delivers fusion proteins into the cell cytoplasm by unspecific binding to cell surface. We compared the adjuvanticity and toxicity of Tat PTD-CTA1-Tat PTD (TCTA1T) with those of CT. Our results indicate that intranasal (i.n.) delivery of ovalbumin (OVA) with TCTA1T significantly augments the OVA-specific systemic and mucosal antibody responses to levels comparable to those seen with CT adjuvant. Moreover, in vivo cytotoxic T lymphocyte activity elicited by TCTA1T was significantly higher than that elicited by a mutant TCTA1T (TmCTA1T) lacking ADP-ribosyltransferase function. In addition, coadministration of influenza M2 protein with TCTA1T conferred near complete protection against lethal influenza virus challenge. Importantly, TCTA1T, in contrast to CT, did not induce serum IgG antibody responses to itself and was shown to be nontoxic. These results suggest that TCTA1T may be a safe and effective adjuvant when given by mucosal routes.

Enhanced anti-tuberculosis immunity by a TAT-Ag85B protein vaccine in a murine tuberculosis model

Background: The development of more effective anti-tuberculosis vaccines would contribute to the control of the global problem of infection with Mycobacterium tuberculosis (MTB). Recently, increasing evidences showed that HIV-Tat protein transduction domain is implicated in promotion of vaccines by inducing cellular immuno-response. However, it is rare known about the role of TAT in vaccines against MTB.Methods: In this study, we expressed recombinant protein-fused Ag85B with TAT (TAT-Ag85B) which was used as a vaccine to inoculate mice infected with MTB.Results: As s result, both IgG2a in serum and IFN-γ or TNFα produced by spleen cells were all increased significantly in the mice inoculated by TAT-Ag85B. Furthermore, consistently, TAT-Ag85B inoculation significantly reduced MTB loads both in lung and spleen.Conclusions: These findings demonstrate that a novel protein vaccine of TAT-Ag85B enhances immune response both in humoral and cellular immunity, and contributes to protective efficacy against MTB.

δPKC interaction with the d subunit of F1Fo ATP synthase impairs energetics and exacerbates ischemia/reperfusion injury in isolated rat hearts

Previously, we demonstrated protection against hypoxic injury in neonatal cardiac myocytes and reduced release of cardiac troponin I from perfused rat hearts by a novel peptide inhibitor [NH2-YGRKKRRQRRRMLATRALSLIGKRAISTSVCAGRKLALKTIDWVSFDYKDDDDK-] of the delta protein kinase C (δPKC) interaction with the “d” subunit of mitochondrial F1Fo ATP synthase (dF1Fo). This peptide was developed in our laboratory and contains: an HIV-Tat protein transduction domain; a mitochondrial targeting motif; the δPKC-dF1Fo inhibitor sequence; and a FLAG epitope. In the present study the δPKC-dF1Fo inhibitor attenuated co-immunoprecipitation of δPKC with dF1Fo, improved recovery of contractility, diminished levels of tissue t-carbonyls and 4-hydroxy-2-nonenal (HNE), and reduced 2,3,5-triphenyltetrazolium chloride-monitored infarct size following simulated global ischemia/reperfusion (IR) exposures. Perfusion of hearts with this peptide prior to IR enhanced ATP levels 2.1-fold, improved ADP (state 3)- and FCCP (maximal)-stimulated respiration in mitochondrial oxygen consumption assays, and attenuated Ca++-induced mitochondrial swelling following ischemic injury. Mitochondrial membrane potential (assessed by JC-1) was also improved 1.6-fold by the inhibitor in hearts subsequently exposed to IR injury. Brief IR exposures did not cause mitochondrial loss of cytochrome c in the presence or absence of the inhibitor. Additionally, the inhibitor did not modify accumulation of the autophagy marker LC3II after brief IR injury. Our results support the potential for this first-in-class peptide as a translational agent for combating cardiac IR injury.

Porcine Mx1 fused to HIV Tat protein transduction domain (PTD) inhibits classical swine fever virus infection in vitro and in vivo.

BackgroundClassical swine fever (CSF) caused by CSF virus (CSFV) is highly contagious andcauses significant economic losses in the pig industry throughout the world. Previously we demonstrated that porcine Mx1 (poMx1), when fused to HIV Tat protein transduction domain (PTD), inhibits CSFV propagation in PK-15 cells, but it is unknown whether PTD-poMx1 exhibits antiviral activity in other porcine lines and it is efficacious for controlling CSFV infection in pigs in China.MethodsTwo porcine cell lines, ST and 3D4/21, were used to investigate in vitro antiviral activity of PTD-poMx1 against CSFV using confocal microscopy, western blot, flow cytometry, and real-time RT-PCR. Furthermore, in vivo antiviral activity of PTD-poMx1 was assessed by means of rectal temperature, clinical score, pathological lesion, white blood cell count, viral load, etc.ResultsPTD-poMx1 entered both cell lines within 3 h and maintained for 16 h, but did not affect CSFV binding and uptake. Viral titers and qRT-PCR data showed that PTD-poMx1 inhibited CSFV replication in both cell lines, showing significant antiviral activity after infection. Injection of PTD-poMx1 into CSFV-challenged pigs attenuated CSFV symptoms and viremia in dose-dependent manner but did not completely block virus replication within 14 days post challenge, suggesting that PTD-poMx1 confers partial protection against a lethal challenge.ConclusionWe demonstrated the anti-CSFV activity of PTD-poMx1 in vitro and in vivo. The results have shown that treatment with PTD-poMx1 alleviated symptoms and viral load in infected pigs. The results support our previous in vitro studies and suggest that PTD-poMx1 could be promising in reducing the clinical signs caused by CSFV.

TATκ Fusion Protein of OCT-3/4 and KLF-4: Stable Mixed Population Cell Lines Capable of Delivering Fusion Proteins to Target Cells

A rapidly growing body of evidence has shown that enforced expression of a small panel of genes, including Oct-3/4, KLF4, Sox2 and c-Myc, can induce the reprogramming of previously differentiated cells, to generate induced Pluripotent Stem Cells (iPSCs). However, the generation of iPSCs by genetic modifications raises the risk of malignant transformation. Therefore, efficient in vitro reprogramming of differentiated cells, without irreversible genetic modification is highly desirable. The improved trans-activator of transcription kappa (TATκ), a synthetic TAT-HIV, confers the ability to deliver several proteins into target cells, thus, making it a potential alternative delivery mechanism for transcription factors or therapeutic agents. Using Green Fluorescence Protein (GFP) and Apoptin as model proteins, we have recently described a strategy to generate cell lines that secrete proteins carrying a modified HIV-TAT Protein Transduction Domain (PTD) for subsequent protein transduction mediated uptake by target cells. Using this strategy we have generated 293T cells secreting the pluripotent factors Oct-3/4 or KLF4 in fusion with TATκ. Oct-3/4 and KLF4 were detected in the culture medium of the transduced 293T cell and uptake of Oct-3/4 by haematopoietic cell lines JURKAT and FDCP-1 was confirmed by Western blot analysis. KLF4 was present in the culture medium of the producer 293T cell but we are still unable to demonstrate uptake by target cells. Based on results obtained, we hope that these stable mix population cell lines can play an important role in generation of iPSCs for therapeutic purposes.

HIV-TAT mediated protein transduction of Cu/Zn-superoxide dismutase-1 (SOD1) protects skin cells from ionizing radiation

BackgroundRadiation-induced skin injury remains a serious concern during radiotherapy. Cu/Zn-superoxide dismutase (Cu/Zn-SOD, SOD1) is a conserved enzyme for scavenging superoxide radical in cells. Because of the integrity of cell membranes, exogenous molecule is not able to be incorporated into cells, which limited the application of natural SOD1. The aim of this study was to evaluate the protective role of HIV-TAT protein transduction domain mediated protein transduction of SOD1 (TAT-SOD1) against ionizing radiation.MethodsThe recombinant TAT-SOD1 and SOD1 were obtained by prokaryotic–based protein expression system. The transduction effect and biological activity of TAT-SOD1 was measured by immunofluorescence and antioxidant capability assays in human keratinocyte HaCaT cells. Mito-Tracker staining, reactive oxygen species (ROS) generation assay, cell apoptosis analysis and malondialdehyde (MDA) assay were used to access the protective effect of TAT- SOD1.ResultsUptake of TAT-SOD1 by HaCaT cells retained its biological activity. Compared with natural SOD1, the application of TAT-SOD1 significantly enhanced the viability and decreased the apoptosis induced by X-ray irradiation. Moreover, TAT-SOD1 reduced ROS and preserved mitochondrial integrity after radiation exposure in HaCaT cells. Radiation-induced γH2AX foci, which are representative of DNA double strand breaks, were decreased by pretreatment with TAT-SOD1. Furthermore, subcutaneous application of TAT-SOD1 resulted in a significant decrease in 45 Gy electron beam-induced ROS and MDA concentration in the skins of rats.ConclusionsThis study provides evidences for the protective role of TAT-SOD1 in alleviating radiation-induced damage in HaCaT cells and rat skins, which suggests a new therapeutic strategy for radiation-induced skin injury.

Single Quantum Dot Tracking Reveals that an Individual Multivalent HIV-1 Tat Protein Transduction Domain Can Activate Machinery for Lateral Transport and Endocytosis

The mechanisms underlying the cellular entry of the HIV-1 Tat protein transduction domain (TatP) and the molecular information necessary to improve the transduction efficiency of TatP remain unclear due to the technical limitations for direct visualization of TatP's behavior in cells. Using confocal microscopy, total internal reflection fluorescence microscopy, and four-dimensional microscopy, we developed a single-molecule tracking assay for TatP labeled with quantum dots (QDs) to examine the kinetics of TatP initially and immediately before, at the beginning of, and immediately after entry into living cells. We report that even when the number of multivalent TatP (mTatP)-QDs bound to a cell was low, each single mTatP-QD first locally induced the cell's lateral transport machinery to move the mTatP-QD toward the center of the cell body upon cross-linking of heparan sulfate proteoglycans. The centripetal and lateral movements were linked to the integrity and flow of actomyosin and microtubules. Individual mTatP underwent lipid raft-mediated temporal confinement, followed by complete immobilization, which ultimately led to endocytotic internalization. However, bivalent TatP did not sufficiently promote either cell surface movement or internalization. Together, these findings provide clues regarding the mechanisms of TatP cell entry and indicate that increasing the valence of TatP on nanoparticles allows them to behave as cargo delivery nanomachines.

In vitro inhibition of vesicular stomatitis virus replication by purified porcine Mx1 protein fused to HIV-1 Tat protein transduction domain (PTD)

Vesicular stomatitis virus (VSV) is the causative agent of Vesicular stomatitis (VS), a highly contagious fatal disease of human and pigs. Few effective antiviral drugs are currently available against VSV infection. Mx proteins are interferon (IFN)-induced dynamin-like GTPases present in all vertebrates with a range of antiviral activities. Previous studies have shown that the transfected cell lines expressing either porcine Mx1 or human MxA acquired a high degree of resistance to VSV. To explore the feasibility of taking porcine Mx1 protein expressed in Escherichia coli as an antiviral agent, we applied the pCold system to express this fusion protein (PTD-poMx1), which consisted of an N-terminal HIV-1 Tat protein transduction domain (PTD) and the full-length porcine Mx1, and investigated its effects on the replication of VSV in Vero cells. The results demonstrated that the purified PTD-poMx1 fusion proteins could transduct into cells after incubated for 5h and had no cytotoxic. Furthermore, plaque reduction assay, determination of TCID50, real-time PCR and Western blot analyses were carried out to confirm the antiviral activity of purified fusion proteins in VSV-infected Vero cells. Altogether, these data suggested that PTD-poMx1 fusion proteins might be applicable to inhibit VSV replication as a novel antiviral therapeutic agent.

HIV Tat Domain Improves Cross-correction of Human Galactocerebrosidase in a Gene- and Flanking Sequence-dependent Manner

Krabbe disease is a devastating neurodegenerative lysosomal storage disorder caused by a deficiency of β-galactocerebrosidase (GALC). Gene therapy is a promising therapeutic approach for Krabbe disease. As the human brain is large and it is difficult to achieve global gene transduction, the efficacy of cross-correction is a critical determinant of the outcome of gene therapy for this disease. We investigated whether HIV Tat protein transduction domain (PTD) can improve the cross-correction of GALC. Tat-PTD significantly increased (~6-fold) cross-correction of GALC through enhanced secretion and uptake in a cell-culture model system. The effects of Tat-PTD were gene and flanking amino acids dependent. Tat-fusion increased the secretion of α-galactosidase A (α-gal A), but this did not improve its cross-correction. Tat-fusion did not change either secretion or uptake of β-glucocerebrosidase (GC). Tat-PTD increased GALC protein synthesis, abolished reactivity of GC to the 8E4 antibody, and likely reduced mannose phosphorylation in all these lysosomal enzymes. This study demonstrated that Tat-PTD can be useful for increasing cross-correction efficiency of lysosomal enzymes. However, Tat-PTD is not a mere adhesive motif but possesses a variety of biological functions. Therefore, the potential beneficial effect of Tat-PTD should be assessed individually on each lysosomal enzyme.

Protective effect of recombinant protein SOD-TAT on radiation-induced lung injury in mice

AimsRadiation-induced lung injury is one of the limiting factors for radiation therapy. SOD-TAT, a fusion protein of HIV-1 Tat protein transduction domain and hCuZn-superoxide dismutase (SOD), has been proved to be effective in preventing and treating the damage of the skin of guinea pigs by UVB radiation. In this study, we demonstrated SOD-TAT's radioprotective effects on lung injury in irradiated mice. Main methodsSOD-TAT was purified from yeast culture with ion exchange chromatography. Kunming mice were randomly divided into three groups: a control group, a group injected with wild SOD and a group injected with SOD-TAT. Pulmonary SOD activity of mice was determined 4.5h after injection. C57BL/6 mice were randomly divided into four groups: a control group, an irradiation group, an irradiation group treated with amifostine 0.5h before the irradiation and an irradiation group treated with SOD-TAT 4.5h before irradiation. The monthly growth rate of every mouse's weight was calculated and the level of hydroxyproline content and antioxidant activity in lung were determined 5months after irradiation. Key findingsSOD-TAT was transduced into the lung in vivo. SOD-TAT pretreatment could improve the growth rate of irradiated mice, significantly reduce the pulmonary hydroxyproline content, and maintain the SOD activity, glutathione peroxidase (GSH-Px) activity and total anti-oxidation capacity (T-AOC). Compared with amifostine, SOD-TAT was more effective in increasing the activities of pulmonary antioxidant enzymes. SignificanceCompared with amifostine, SOD-TAT treatment more effectively enhanced pulmonary antioxidant ability, reduced radiation-induced pulmonary fibrosis and improved the living quality of irradiated mice.

Cerebellar abnormalities in purine nucleoside phosphorylase deficient mice

Inherited defects in purine nucleoside phosphorylase (PNP) cause severe T cell immunodeficiency and progressive neurological dysfunction, yet little is known about the effects of PNP deficiency on the brain. PNP-KO mice display metabolic and immune anomalies similar to those observed in patients. Our objectives were to characterize brain abnormalities in PNP-KO mice and determine whether restoring PNP activity prevents these abnormalities.We analyzed structural brain defects in PNP-KO mice by magnetic resonance imaging, while assessing motor deficits using the accelerating rotarod and stationary balance beam tests. We detected morphological abnormalities and apoptosis in the cerebellum of PNP-KO mice by hematoxylin and eosin, electron microscopy, TUNEL and activated caspase 3 staining. We treated PNP-KO mice with PNP fused to the HIV-TAT protein transduction domain (TAT-PNP) from birth or from 4weeks of age.Magnetic resonance imaging revealed a smaller than normal cerebellum in PNP-KO mice. PNP-KO mice displayed motor abnormalities including rapid fall from the rotating rod and frequent slips from the balance beam. The cerebellum of PNP-KO mice contained reduced purkinje cells (PC), which were irregular in shape and had degenerated dendrites. PC from the cerebellum of PNP-KO mice, expanded ex vivo, demonstrated increased apoptosis, which could be corrected by supplementing cultures with TAT-PNP. TAT-PNP injections restored PNP activity in the cerebellum of PNP-KO mice. TAT-PNP from birth, but not treatment initiated at 4weeks of age, prevented the cerebellar PC damage and motor deficits.We conclude that PNP deficiency cause cerebellar abnormalities, including PC damage and progressive motor deficits. TAT-PNP treatment from birth can prevent the neurological abnormalities in PNP-KO mice.

Inhibition of Influenza Virus Replication by Constrained Peptides Targeting Nucleoprotein

Because of high mutation rates, new drug-resistant viruses are rapidly evolving, thus making the necessary control of influenza virus infection difficult. We screened a constrained cysteine-rich peptide library mimicking μ-conotoxins from Conus geographus and a proline-rich peptide library mimicking lebocin 1 and 2 from Bombyx mori by using influenza virus RNA polymerase (PB1, PB2 and PA) and nucleoprotein (NP) as baits. Among the 22 peptides selected from the libraries, we found that the NP-binding proline-rich peptide, PPWCCCSPMKRASPPPAQSDLPATPKCPP, inhibited influenza replicon activity to mean±sd 40.7%±15.8% when expressed as a GFP fusion peptide in replicon cells. Moreover, when the GFP fusion peptide was transduced into cells by an HIV-TAT protein transduction domain sequence, the replication of influenza virus A/WSN/33 (WSN) at a multiplicity of infection of 0.01 was inhibited to 20% and 69% at 12 and 24 h post-infection, respectively. In addition, the TAT-GFP fusion peptide was able to slightly protect Balb/c mice from WSN infection when administrated prior to the infection. These results suggest the potential of this peptide as the seed of an anti-influenza drug and reveal the usefulness of the constrained peptide strategy for generating inhibitors of influenza infection. The results also suggest that influenza NP, which is conserved among the influenza A viruses, is a good target for influenza inhibition, despite being the most abundant protein in infected cells.

A Pilot Trial of WT1 Peptide-Loaded Allogeneic Dendritic Cell Vaccine and Donor Lymphocyte Infusion for WT1-Expressing Hematologic Malignancies and Post-Transplant Relapse

Abstract 3055 Background:Treatment of relapse after allogeneic hematopoietic stem cell transplantation (alloHSCT) remains challenging. The Wilms' tumor 1 (WT1) gene product is a tumor-associated antigen that is expressed in acute leukemia and other hematologic malignancies with limited expression in normal tissues. This pilot trial incorporates antigen-specific immunotherapy and allogeneic adoptive cell transfer for pediatric and adult patients with relapsed hematologic malignancies after alloHSCT. The primary objectives are to assess safety and feasibility of a peptide-loaded donor-derived dendritic cell (DC) vaccine and donor lymphocyte infusion (DLI) designed to enhance the graft-vs -leukemia effect. Secondary objectives are to determine if immunologic and clinical responses to WT1 specific peptides can be generated by this novel vaccine strategy after alloHSCT. Design:HLA-A2+ patients with WT1-expressing hematologic malignancies that have relapsed after alloHSCT are eligible. Donor-derived DC vaccines are given every 2 weeks for 6 doses and DLI every 4 weeks for 3 doses. Peripheral blood monocyte-derived DCs are loaded with a combination of three HLA-A2 binding WT1 peptides (WT1 37–45; WT1 126–134; WT1 187–195) linked to the 11-mer HIV TAT protein transduction domain peptide (47–57) designed to enhance antigen presentation. The DCs are generated from donor peripheral blood monocytes that are separated from DLI collected by apheresis. Monocytes are incubated with GM-CSF and IL-4 followed by maturation with LPS and IFN-g. WT1 expression is assessed using immunohistochemistry and/or quantative RT-PCR. Study endpoints included toxicity, feasibility, and antigen-specific immune and clinical responses. Results:4 patients, aged 9–19 years have been treated to date, 3 with acute lymphoblastic leukemia (ALL) and one with Hodgkin lymphoma (HL). Donors were matched siblings in 3 cases and a 10/10 HLA matched father in one case. Vaccines were successfully produced from all donors. All patients tolerated vaccine and DLI administrations well. The most common adverse events were mild, reversible pain and pruritus at vaccine administration and delayed type hypersensitivity (DTH) skin test sites. One patient developed Grade I skin GVHD that did not require treatment. Immune responses were observed in all 3 patients with ALL. ELISPOT was considered positive when it was at least two times greater than and had at least 10 spots more than background. 3 of 4 (75%) patients had positive ELISPOT responses to WT1 peptides. 3 of 4 (75%) patients had positive DTH responses to the keyhole limpet hemocyanin (KLH) control and 2 of 4 (50%) to the WT1 peptides. Median overall survival was 12 months. 1 patient remains in remission 12 months after initiation of therapy and 3 have died of disease. All donors tolerated apheresis well and there were no complications from donor procedures. Conclusions:This novel allogeneic immunotherapy regimen is feasible, well-tolerated and can induce immune responses in the allogeneic setting. Accrual is ongoing.Table:Summary of DTH and ELISPOT ResponsesPatient #DiagnosisKLH DTH ResponseWT1 DTH ResponseWT1 ELISPOT Response1ALL+++2HL---3ALL+-+4ALL+++ Disclosures:Off Label Use: This is a pilot clinical trial of an investigational cancer vaccine.

B cells “transduced” with TAT-fusion proteins can induce tolerance and protect mice from diabetes and EAE

Antigen-immunoglobulin fusion protein expressing B cells have been shown as excellent tolerogenic antigen-presenting cells in multiple disease models. Using efficient protein transduction by fusion with a HIV TAT protein transduction domain, we herein tested the TAT-fusion protein transduced B cells for their effects in antigen-specific tolerance induction in two animal models, experimental autoimmune encephalomyelitis (EAE) and type 1 diabetes. We demonstrated that transfer of TAT-MOG35-55 (myelin oligodendrocyte glycoprotein)-Ig 'transduced B cells' 10 days after EAE induction significantly protected mice from disease. Similarly, the onset of disease was delayed when NOD mice received insulin specific TAT-B9-23-B cells. Surprisingly, no protection against EAE was observed in a prophylactic protocol when transduced B cells were given before disease induction. Moreover, TAT-ovalbumin transduced cells were tolerogenic in primed but not naïve mice. Our results suggest that TAT-fusion protein transduced B cells were tolerogenic in antigen primed recipients, a condition clinically relevant to autoimmune diseases.

Peptide-Mediated Disruption of Calmodulin–Cyclin E Interactions Inhibits Proliferation of Vascular Smooth Muscle Cells and Neointima Formation

Cell cycle progression in vascular smooth muscle cells (VSMCs) is a therapeutic target for restenosis. Having discovered that calmodulin (CaM)-dependent cyclin E/CDK2 activity underlies Ca(2+)-sensitive G(1)-to-S phase transitions in VSMCs, we sought to explore the physiological importance of the CaM-cyclin E interaction. A peptide based on the CaM binding sequence (CBS) of cyclin E was designed to interfere with CaM-cyclin E binding. Compared with control peptides, CBS blocked activating Thr160 phosphorylation of CDK2, decreased basal cyclin E/CDK2 activity, and eliminated Ca(2+)-sensitive cyclin E/CDK2 activity in nuclear extracts from mouse VSMCs. Nucleofection with CBS, or treatment with CBS conjugated to the HIV-1 TAT protein transduction domain to improve bioavailability, inhibited G(1)-to-S cell cycle progression in a dose-dependent manner. These effects were not observed with control peptides. TAT-CBS inhibited (3)H-thymidine incorporation in primary human aortic SMCs (HA-SMCs) in vitro, manifested greater transduction into HA-SMCs compared with endothelial cells in vitro, and limited decreased SM22α expression, neointima formation, and medial thickening without affecting collagen deposition or reendothelialization in a mouse model of carotid artery injury in vivo. The antiproliferative effects of CBS remained evident in mouse embryonic fibroblasts derived from wild-type mice but not cyclin E1/E2 double knockout mice. A synthetic peptide designed to disrupt CaM-cyclin E binding inhibits Ca(2+)/CaM-dependent CDK2 activity, cell cycle progression, and proliferation in VSMCs and limits arterial remodeling following injury. Importantly, this effect appears to be cyclin E-dependent and may form the basis of a potentially novel therapeutic approach for restenosis.

Cell Penetrating Peptides. How do they Cross Membranes?

Cell penetrating peptides (CPPs) are cationic peptides which, when linked to proteins, genes, or nanoparticles, facilitate the transport of these entities across the cell membrane. Different models have been suggested for their mode of action. One possibility is the binding of CPPs to negatively charged lipids and the formation of non-bilayer structures. We have studied the binding of two CPPs (R9, HIV-1 TAT) to model membranes with isothermal titration calorimetry and observe indeed a strong binding to the membrane. However, the bilayer remains intact and non-bilayer structures can be excluded based on 2H- and 31P-NMR spectroscopy. A second mechanism is the binding of CPPs to sulfated sugars at the membrane surface, followed by endocytosis. We have characterized the binding of a large variety of CPPs to heparin sulfate (HS), heparin, and related sulfated glycosaminoglycans (GAGs) and find binding constants in the order of 106 - 107 M−1 per binding site. An even stronger interaction is found between CPPs and DNA with binding constants in 107 - 108 M−1. We have further synthesized a fluorescent derivative of the HIV-1 TAT protein transduction domain (Fg-CPPTAT(PTD)) and have observed its uptake into non-fixated living fibroblasts with time-lapse confocal microscopy. We find that Fg-CPPTAT(PTD) enters the cytoplasm and nucleus of non-fixated fibroblasts within seconds. With differential interference contrast microscopy we furthermore detect dense aggregates on the cell surface. Several observations suggest that these aggregates consist of Fg-CPPTAT(PTD) bound to membrane-associated heparan sulfate (HS). Finally a pore-forming peptide, melittin, also binds to sulfated GAGs but not magainin 2 or nisin Z. The binding of the melittin to lipid bilayers is furthermore an excellent model system to elucidate the thermodynamic parameters of membrane-induced α-helix and β-sheet formation..

Enhanced Induction of T Cell Immunity Using Dendritic Cells Pulsed with HIV Tat and HCMV-pp65 Fusion ProteinIn Vitro

BackgroundCytotoxic T lymphocytes (CTLs) appear to play an important role in the control and prevention of human cytomegalovirus (HCMV) infection. The pp65 antigen is a structural protein, which has been defined as a potential target for effective immunity against HCMV infection. Incorporation of an 11 amino acid region of the HIV TAT protein transduction domain (Tat) into protein facilitates rapid, efficient entry into cells.MethodsTo establish a strategy for the generation of HCMV-specific CTLs in vitro, recombinant truncated N- and C-terminal pp65 protein (pp65 N&C) and N- and C-terminal pp65 protein fused with Tat (Tat/pp65 N&C) was produced in E.coli system. Peripheral blood mononuclear cells were stimulated with dendritic cells (DCs) pulsed with pp65 N&C or Tat/pp65 N&C protein and immune responses induced was examined using IFN-γ ELISPOT assay, cytotoxicity assay and tetramer staining.ResultsDCs pulsed with Tat/pp65N&C protein could induce higher T-cell responses in vitro compared with pp65N&C. Moreover, the DCs pulsed with Tat/pp65 N&C could stimulate both of CD8+ and CD4+ T-cell responses. The T cells induced by DCs pulsed with Tat/pp65 N&C showed higher cytotoxicity than that of pp65-pulsed DCs against autologous lymphoblastoid B-cell line (LCL) expressing the HCMV-pp65 antigen.ConclusionOur results suggest that DCs pulsed with Tat/pp65 N&C protein effectively induced pp65-specific CTL in vitro. Tat fusion recombinant protein may be useful for the development of adoptive T-cell immunotherapy and DC-based vaccines.

Use of Recombinant Cell-Permeable Small Peptides To Modulate Glucocorticoid Sensitivity of Acute Lymphoblastic Leukemia Cells

Glucocorticoid (GC) hormones induce apoptosis in T-cell and pre-B-cell acute lymphoblastic leukemia (ALL) cells. Steroid-mediated apoptosis requires a threshold level of the glucocorticoid receptor (GR) protein, and increasing the intracellular GR levels in ALL cells would augment their hormone sensitivity. A protein transduction domain (PTD) approach was used to accomplish this. We produced an HIV Tat PTD domain fusion protein (Tat-GR(554-777)) that potentially competes for the degradation of GR protein by the ubiquitin-proteasome system and should thus increase its intracellular levels by "stabilizing" the GR. We also designed a fusion peptide for the c-Myb DNA binding domain, Tat-c-Myb DBD, since the biological function of this peptide as a dominant negative inhibitor of the c-Myb protein was already known. Purified, bacterially expressed Tat-c-Myb DBD and Tat-GR(554-777) exhibited highly efficient transduction into cultured ALL cell lines including 697 (pre-B-ALL) and CEM-C7 (T-ALL) cells. As expected, the transduced Tat-c-Myb DBD peptide inhibited steroid-mediated stimulation of a GR promoter-luciferase reporter gene. Significantly, transduced Tat-GR(554-777) effectively increased intracellular GR levels in the GC-resistant T-ALL cell line, CEM-C1, and in the pre-B-ALL 697 cell line. Furthermore, transduction of Tat-GR(554-777) rendered GC-resistant CEM-C1 cells sensitive to steroid killing and further sensitized 697 cells to steroid. The use of Tat-fusion peptide transduction may eventually lead to innovative therapeutic modalities to improve the clinical response of patients suffering from T-cell and pre-B-cell acute lymphoblastic leukemia by increasing steroid responsiveness and perhaps converting steroid-resistant leukemia to a hormone-responsive phenotype.