Durable and efficient gene silencing in vivo by hit-and-run epigenome editing.

Permanent epigenetic silencing using programmable editors equipped with transcriptional repressors holds great promise for the treatment of human diseases1-3. However, to unlock its full therapeutic potential, an experimental confirmation of durable epigenetic silencing after the delivery of transient delivery of editors in vivo is needed. To this end, here we targeted Pcsk9, a gene expressed in hepatocytes that is involved in cholesterol homeostasis. In vitro screening of different editor designs indicated that zinc-finger proteins were the best-performing DNA-binding platform for efficient silencing of mouse Pcsk9. A single administration of lipid nanoparticles loaded with the editors' mRNAs almost halved the circulating levels of PCSK9 for nearly one year in mice. Notably, Pcsk9 silencing and accompanying epigenetic repressive marks also persisted after forced liver regeneration, further corroborating the heritability of the newly installed epigenetic state. Improvements in construct design resulted in the development of an all-in-one configuration that we term evolved engineered transcriptional repressor (EvoETR). This design, which is characterized by a high specificity profile, further reduced the circulating levels of PCSK9 in mice with an efficiency comparable with that obtained through conventional gene editing, but without causing DNA breaks. Our study lays the foundation for the development of in vivo therapeutics that are based on epigenetic silencing.

Recombinant Cellular Repressor of E1A-Stimulated Genes Protects against Renal Fibrosis in Dahl Salt-Sensitive Rats.

BACKGROUND: Human cellular repressor of E1A-stimulated genes (CREG) is a secreted glycoprotein that attenuates angiotensin II-induced hypertension, alleviates myocardial fibrosis, and improves heart function. However, the role of CREG in high-salt (HS) diet-induced hypertensive nephropathy is unclear. METHODS: To determine the effects and molecular mechanisms of CREG in HS diet-induced hypertensive nephropathy, we established a hypertensive nephropathy animal model in Dahl salt-sensitive (SS) rats fed a HS diet (8% NaCl, n = 20) for 8 weeks. At week 4 of HS loading, these rats were administered recombinant CREG (reCREG; 35 µg/kg·day, n = 5) and saline (n = 5) via subcutaneously implanted pumps and were also administered the vasodilator hydralazine (20 mg/kg·day, n = 5) in drinking water. We used hematoxylin and eosin staining, Masson's trichrome staining, immunohistochemical labeling, western blotting, RT-PCR, and Tunel staining to determine the signaling pathways of CREG in HS diet-induced hypertensive nephropathy. RESULTS: After 8 weeks of HS intake, the Dahl SS rats developed renal dysfunction and severe renal fibrosis associated with reductions of 78 and 67% in CREG expression, respectively, at both mRNA and protein levels in the kidney. Administration of reCREG improved renal function and relieved renal fibrosis. Administration of CREG also inhibited monocyte infiltration and reduced apoptosis in the kidney cells. CREG overexpression upregulated forkhead box P1 expression and inhibited the transforming growth factor-ß1 signaling pathway. CONCLUSION: Our study shows that CREG protected the kidney against HS-diet-induced renal damage and provides new insights into the mechanisms underlying kidney injury.

Mucosal HPV E6/E7 Peptide Vaccination in Combination with Immune Checkpoint Modulation Induces Regression of HPV+ Oral Cancers.

High-risk human papillomavirus (HPV)-associated squamous cell carcinomas of the oropharynx (SCCOP) are among the fastest growing cancers. After standard-of-care treatment, however, patients with HPV+ SCCOP have better overall and disease-specific survival than patients with HPV- SCCOP, suggesting the importance of HPV-specific immunity. We reasoned that therapeutic vaccination targeting the HPV-16 E6 and E7 oncogenes could elicit high-affinity, high-frequency tumor antigen-specific T-cell responses, which could then be augmented and shielded from suppression in the tumor microenvironment by immune checkpoint modulation. In this study, we used a preclinical syngeneic mouse model of oral cancer comprised of mouse tonsil-derived epithelial cells stably expressing HPV-16 E6 and E7 genes along with H-ras oncogene (mEER) to identify combinations of vaccination and checkpoint antibodies capable of promoting tumor regression. Intranasal HPV E6/E7 peptide vaccination and single checkpoint antibodies failed to elicit responses in more than half of animals; however, 4-1BB agonist antibody along with either CD40 agonist antibody or CTLA-4 blockade eliminated the majority of established mEER tumors. The combination of intranasal HPV peptide vaccine and α4-1BB and αCTLA-4 antibodies produced curative efficacy and a better safety profile against orally implanted mEER tumors. Correlates of protective immunity included enhanced intratumoral levels of CD8 T cells relative to immunosuppressive regulatory T cells and myeloid-derived suppressor cells. Overall, our results demonstrate combination vaccine-immunotherapy modalities as novel treatment options for HPV+ SCCOP.Significance: Combinations of vaccine and checkpoint modulation are effective and safe treatment options for HPV+ oral cancers. Cancer Res; 78(18); 5327-39. ©2018 AACR.

Peptide-functionalized nanoparticles for the selective induction of apoptosis in target cells.

AIM: The study developed a prohibitin (PHB) targeted nanotherapy for selective induction of apoptosis in target cells. METHODS: Gold nanoparticles (AuNPs) were bifunctionalized with adipose homing and proapoptotic peptides. The efficacy and mode of cell death induced by the AuNPs were investigated in vitro on three cancer cell lines. RESULTS: The antiproliferative activity of PHB-targeted bifunctionalized AuNPs was more pronounced on cells that express the PHB receptor, and demonstrated receptor-mediated targeting and selectivity. The bifunctionalized AuNPs induced cell death by apoptosis. CONCLUSION: The PHB-targeted nanotherapy under study could potentially be used for treatment of diseases that are characterized by overexpression of PHB. As such, further investigations will be conducted in vivo.

Targeting Insulin Receptor in Breast Cancer Using Small Engineered Protein Scaffolds.

Insulin receptor (InsR) and the type I insulin-like growth factor (IGF1R) are homologous receptors necessary for signal transduction by their cognate ligands insulin, IGF-I and IGF-II. IGF1R mAbs, intended to inhibit malignant phenotypic signaling, failed to show benefit in patients with endocrine-resistant tumors in phase III clinical trials. Our previous work showed that in tamoxifen-resistant cells, IGF1R expression was lacking, but InsR inhibition effectively blocked growth. In endocrine-sensitive breast cancer cells, insulin was not growth stimulatory, likely due to the presence of hybrid InsR/IGF1R, which has high affinity for IGF-I, but not insulin. Combination inhibition of InsR and IGF1R showed complete suppression of the system in endocrine-sensitive breast cancer cells. To develop InsR-binding agents, we employed a small protein scaffold, T7 phage gene 2 protein (Gp2) with the long-term goal of creating effective InsR inhibitors and diagnostics. Using yeast display and directed evolution, we identified three Gp2 variants (Gp2 #1, #5, and #10) with low nanomolar affinity and specific binding to cell surface InsR. These Gp2 variants inhibited insulin-mediated monolayer proliferation in both endocrine-sensitive and resistant breast cancer, but did not downregulate InsR expression. Gp2 #5 and Gp2 #10 disrupted InsR function by inhibiting ligand-induced receptor activation. In contrast, Gp2 #1 did not block InsR phosphorylation. Notably, Gp2 #1 binding was enhanced by pretreatment of cells with insulin, suggesting a unique receptor-ligand-binding mode. These Gp2 variants are the first nonimmunoglobulin protein scaffolds to target insulin receptor and present compelling opportunity for modulation of InsR signaling. Mol Cancer Ther; 16(7); 1324-34. ©2017 AACR.

Cellular Repressor of E1A-Stimulated Genes Is a Critical Determinant of Vascular Remodeling in Response to Angiotensin II.

OBJECTIVE: Cellular repressor of E1A-stimulated genes (CREG) is a lysosomal glycoprotein implicated in maintaining vascular homeostasis. Here, we have hypothesized that CREG is a critical target of intervention for the prevention of hypertensive vascular remodeling. APPROACH AND RESULTS: CREG gene expression was significantly decreased accompanied by an upregulated expression of angiotensin II (Ang II) in remodeled vascular tissues of high salt-induced Dahl salt-sensitive rats and Ang II-induced mice. In particular, the downregulation of CREG gene was Ang II specific and independent from blood pressure. Prominent medial hypertrophy and vascular fibrosis in both thoracic aortas and mesenteric arteries were observed in CREG+/- mice infused with Ang II than in CREG+/+ mice, but blunted response in CREG+/+ mice received recombinant human CREG protein, suggesting that changes in CREG expression account for the different phenotype between genotypes. Within a tiled promoter array, E26 transformation-specific-1 binds to CREG promoter at high stringency with the stimulation of Ang II. Moreover, the Ang II-induced E26 transformation-specific-1 directly interacted with the CREG promoter (-1179 and -271 bp) and inhibited its transcription in vascular smooth muscle cells. Selective, pharmacological inhibition of E26 transformation-specific-1 led to restoration of CREG expression in aortas and rescue of experimental vascular remodeling by systemic administration of dominant negative E26 transformation-specific-1 membrane-permeable peptides. CONCLUSIONS: CREG is a novel mediator of vascular remodeling in response to Ang II and may be an attractive therapeutic target for prevention of vascular diseases.

Pre-immature dendritic cells (PIDC) pulsed with HPV16 E6 or E7 peptide are capable of eliciting specific immune response in patients with advanced cervical cancer.

BACKGROUND: The protein products of the early genes E6 and E7 in high-risk HPV types 16 and 18 have been implicated in the oncogenic capability of these viruses. Therefore, these peptides represent attractive vaccine therapy targets. METHODS: Thirty-two patients with advanced cervical cancer (HPV16 or 18 positive) were treated with HPV16 E6 (18-26) (Arm A) or HPV16 E7 (12-20) peptide (Arm B) pulsed on PBMCs in order to illicit immune response against the relevant peptide on both arms. These PBMCs were cultured for a short time (48 hours only) and in the presence of GM- CSF, accordingly, they were identified as "Pre-Immature Dentritic Cells". RESULTS: 51Cr release assay and ELISPOT demonstrated evidence of specific immune response against the relevant peptide in 10/16 (63%) evaluable patients in arm A and 7/12 (58%) in arm B. HPV16 E6 was found to be homologous to HPV18 E6 in both vivo and vitro. The median overall survival (OS) and progression free survival (PFS) for the full cohort was 10.0 and 3.5 months, respectively. There were no RECIST responses in any patient. The majority of toxicities were grade I and II. CONCLUSIONS: We demonstrated the feasibility and ability of Pre-Immature Dentritic Cells pulsed with HPV16 E6 (18-26) or HPV16 E7 (12-20) to induce a specific immune response against the relevant peptide despite the advanced disease of the cervical cancer patients treated on this trial. We believe that this observation deserves further investigations.

Nanoporous CREG-eluting stent attenuates in-stent neointimal formation in porcine coronary arteries.

BACKGROUND: The goal of this study was to evaluate the efficacy of a nanoporous CREG-eluting stent (CREGES) in inhibiting neointimal formation in a porcine coronary model. METHODS: In vitro proliferation assays were performed using isolated human endothelial and smooth muscle cells to investigate the cell-specific pharmacokinetic effects of CREG and sirolimus. We implanted CREGES, control sirolimus-eluting stents (SES) or bare metal stents (BMS) into pig coronary arteries. Histology and immunohistochemistry were performed to assess the efficacy of CREGES in inhibiting neointimal formation. RESULTS: CREG and sirolimus inhibited in vitro vascular smooth muscle cell proliferation to a similar degree. Interestingly, human endothelial cell proliferation was only significantly inhibited by sirolimus and was increased by CREG. CREGES attenuated neointimal formation after 4 weeks in porcine coronary model compared with BMS. No differences were found in the injury and inflammation scores among the groups. Scanning electron microscopy and CD31 staining by immunohistochemistry demonstrated an accelerated reendothelialization in the CREGES group compared with the SES or BMS control groups. CONCLUSIONS: The current study suggests that CREGES reduces neointimal formation, promotes reendothelialization in porcine coronary stent model.

Neural stem cells modified by a hypoxia-inducible VEGF gene expression system improve cell viability under hypoxic conditions and spinal cord injury.

STUDY DESIGN: An in vitro neural hypoxia model and rat spinal cord injury (SCI) model were used to assess the regulation of therapeutic vascular endothelial growth factor (VEGF) gene expression in mouse neural stem cells (mNSCs) by the EPO (erythropoietin) enhancer or RTP801 promoter. OBJECTIVE: To increase VEGF gene expression in mNSCs under hypoxic conditions in SCI lesions but avoid unwanted overexpression of VEGF in normal sites, we developed a hypoxia-inducible gene expression system consisting of the EPO enhancer and RTP801 promoter fused to VEGF or the luciferase gene, then transfected into mNSCs. SUMMARY OF BACKGROUND DATA: On the basis of the ischemic response in the injured area, poor cell survival at the transplantation site is a consistent problem with NSC transplantation after SCI. Although VEGF directly protects neurons and enhances neurite outgrowth, uncontrolled overexpression of VEGF in uninjured tissue may cause serious adverse effects. To effectively improve NSC survival in ischemic sites after transplantation, we evaluated mNSCs modified by a hypoxia-inducible VEGF gene expression system in an SCI model. METHODS: Hypoxia-inducible luciferase or VEGF plasmids were constructed using the EPO enhancer or RTP801 promoter. The effect of these systems on targeted gene expression and cell viability was evaluated in mNSCs in both hypoxic in vitro injury and a rat SCI model in vivo. RESULTS: The gene expression system containing the EPO enhancer or RTP801 promoter significantly increased the expression of the luciferase reporter gene and therapeutic VEGF gene under hypoxic conditions. The Epo-SV-VEGF plasmid transfection group had significantly fewer apoptotic cells in vitro. This system also augmented cell viability in the in vivo SCI model. CONCLUSION: These results strongly suggest the potential utility of mNSCs modified by a hypoxia-inducible VEGF gene expression system in the development of effective stem cell transplantation protocols in SCI.

An HPV 16 L1-based chimeric human papilloma virus-like particles containing a string of epitopes produced in plants is able to elicit humoral and cytotoxic T-cell activity in mice.

BACKGROUND: Even though two prophylactic vaccines against HPV are currently licensed, infections by the virus continue to be a major health problem mainly in developing countries. The cost of the vaccines limits wide-scale application in poor countries. A promising strategy for producing affordable and efficient vaccines involves the expression of recombinant immunogens in plants. Several HPV genes have been expressed in plants, including L1, which can self-assemble into virus-like particles. A plant-based, dual prophylactic/therapeutic vaccine remains an attractive possibility. RESULTS: We sought to express in tomato plants chimeric HPV 16 VLPs containing L1 fused to a string of epitopes from HPV 16 E6 and E7 proteins. The L1 employed had been modified to eliminate a strong inhibitory region at the 5' end of the molecule to increase expression levels. Several tomato lines were obtained expressing either L1 alone or L1-E6/E7 from 0.05% to 0.1% of total soluble protein. Stable integration of the transgenes was verified by Southern blot. Northern and western blot revealed successful expression of the transgenes at the mRNA and protein level. The chimeric VLPs were able to assemble adequately in tomato cells. Intraperitoneal administration in mice was able to elicit both neutralizing antibodies against the viral particle and cytotoxic T-lymphocytes activity against the epitopes. CONCLUSION: In this work, we report for the first time the expression in plants of a chimeric particle containing the HPV 16 L1 sequence and a string of T-cell epitopes from HPV 16 E6 and E7 fused to the C-terminus. The particles were able to induce a significant antibody and cytotoxic T-lymphocytes response. Experiments in vivo are in progress to determine whether the chimeric particles are able to induce regression of disease and resolution of viral infection in mice. Chimeric particles of the type described in this work may potentially be the basis for developing prophylactic/therapeutic vaccines. The fact that they are produced in plants, may lower production costs considerably.

Adenovirus-mediated intra-arterial delivery of cellular repressor of E1A-stimulated genes inhibits neointima formation in rabbits after balloon injury.

OBJECTIVE: This study examined the effect on neointimal hyperplasia of adenovirus-mediated delivery of cellular repressor of E1A-stimulated genes (CREG) to the artery after balloon injury. METHODS: Sixty rabbits were randomized into three groups and underwent balloon injury in the left common carotid arteries. The injured arterial segment was isolated by two inflated balloon catheters. Saline or recombinant adenovirus expressing CREG or green fluorescent protein was injected into the lumen of the isolated arterial segments and incubated for 30 minutes. The rabbits were euthanized for immunohistochemistry, Western blotting, and morphometric analysis at 3, 7, 14, and 28 days after balloon injury and in vivo gene transfer (5 rabbits for each time point). Common carotid artery angiography was performed before euthanasia. RESULTS: Immunohistochemistry and Western blot analysis demonstrated that CREG expression was significantly down-regulated in the acute phase of vascular injury and was gradually restored in the resolution phase. The changes of CREG expression were in parallel with those of the smooth muscle cell (SMC) differentiation markers SM alpha-actin and SM myosin heavy chain in the injured arteries. Adenovirus-mediated CREG transfer markedly increased CREG expression in the injured artery. Consequently, morphometric analysis revealed an approximate 50% reduction in the neointima and the intima/media ratio in CREG-transferred arteries compared with the saline and green fluorescent protein controls. Assay with 5-bromo-2-deoxyuridine showed that CREG transfer significantly inhibited SMC proliferation. In contrast, endothelialization of the injured artery was not affected by CREG transduction as assessed by CD31 immunostaining. CONCLUSION: These data suggest that forced expression of CREG in the artery wall after acute vascular injury inhibits SMC proliferation, induces cellular differentiation, and attenuates neointimal hyperplasia. CREG delivery may have therapeutic potential for the prevention of restenosis after vascular angioplasty.

Skin reactions to human papillomavirus (HPV) 16 specific antigens intradermally injected in healthy subjects and patients with cervical neoplasia.

We have tested the safety and feasibility of a synthetic long peptide-based HPV16-specific skin test to detect cellular immune responses to HPV16 E2, E6 and E7 in vivo. Women with cervical neoplasia (n = 11) and healthy individuals (n = 19) were intradermally challenged with 8 different pools of HPV16 E2, E6 and E7 peptides. The skin test was safe as the injections were perceived as mildly painful and no adverse events were observed. The majority of skin reactions appeared significantly earlier in HPV16+ patients (<8 days) than in healthy subjects (8-25 days). The development of late skin reactions in healthy subjects was associated with the appearance of circulating HPV16-specific T cells and the infiltration of both HPV16-specific CD4+ Th1/Th2 and CD8+ T cells into the skin. These data show that the intradermal injection of pools of HPV16 synthetic long peptides is safe and results in the migration of HPV16-specific T cells into the skin as well as in an increase in the number of circulating HPV16-specific T cells. The use of this test to measure HPV16-specific immunity is currently tested in a low resource setting for the measurement of spontaneously induced T-cell responses as well as in our HPV16 vaccination trials for the detection of vaccine-induced immunity.

IFN-gamma protects cerulein-induced acute pancreatitis by repressing NF-kappa B activation.

We explored the pathophysiological roles of IFN-gamma in cerulein-induced acute pancreatitis. In wild-type (WT) mice, cerulein injection caused acute pancreatitis as evidenced by increased serum amylase levels and pathological changes such as interstitial edema, vacuolization, acinar cell necrosis, and neutrophil infiltration in pancreas. Concomitantly, cerulein treatment augmented intrapancreatic gene expression of TNF-alpha, KC/CXCL1, MIP-2/CXCL2, cyclooxygenase-2 (COX-2), and IFN-gamma in WT mice. In situ hybridization combined with immunofluorescence analyses demonstrated that infiltrating neutrophils expressed IFN-gamma mRNA. Unexpectedly, IFN-gamma(-/-) mice exhibited exacerbated cerulein-induced pancreatic injury, with enhanced neutrophil recruitment. Moreover, intrapancreatic gene expression of TNF-alpha, KC/CXCL1, MIP-2/CXCL2, and COX-2 were significantly exaggerated in IFN-gamma(-/-) mice, compared with WT mice. Cerulein activated NF-kappaB, an indispensable transcription factor for gene transcription of TNF-alpha, KC/CXCL1, MIP-2/CXCL2, and COX-2, in pancreas of cerulein-treated WT mice as evidenced by the increases in nuclear amount and DNA-binding activity of NF-kappaB p65. In comparison with WT mice, IFN-gamma(-/-) mice exhibited exaggerated and prolonged NF-kappaB activation, probably due to reduced acetylation of Stat1, a main signal transducer of IFN-gamma, because acetylated Stat1 can inhibit NF-kappaB activation. Indeed, IFN-gamma acetylated Stat1 and reciprocally reduced NF-kappaB activation and COX-2 expression in neutrophils. Finally, even when administered 4 h after the first cerulein injection, IFN-gamma remarkably attenuated acute pancreatitis in both WT and IFN-gamma(-/-) mice, with reduced NF-kappaB activation and COX-2 expression. Thus, IFN-gamma can have anti-inflammatory effects on acute pancreatitis by depressing the proinflammatory consequences of NF-kappaB activation.

T-cell-based vaccination for morphological and functional neuroprotection in a rat model of chronically elevated intraocular pressure.

Acute or chronic glaucoma is often associated with an increase in intraocular pressure (IOP). In many patients, however, therapeutic pressure reduction does not halt disease progression. Neuroprotection has been proposed as a complementary therapeutic approach. We previously demonstrated effective T-cell-based neuroprotection in experimental animals vaccinated with the synthetic copolymer glatiramer acetate (copolymer-1, Cop-1), a weak agonist of self-antigens. This study was undertaken to test different routes and modes of vaccination with Cop-1 as treatment modalities for protection against retinal ganglion cell (RGC) death caused by chronic elevation of IOP in rats, and to determine whether anatomical neuroprotection is accompanied by functional neuroprotection. In a chronic model of unilaterally high IOP, Cop-1 vaccination, with or without an adjuvant, protected rats against IOP-induced loss of RGCs by eliciting a systemic T-cell-mediated response capable of cross-reacting with self-antigens residing in the eye. In rats deprived of T cells, Cop-1 (unlike treatment with alpha2-adrenoreceptor agonists) was not protective of RGCs, substantiating the contention that its beneficial effect is not conferred directly but is T-cell-mediated. Pattern electroretinography provided evidence of functional protection. Thus, vaccination with adjuvant-free Cop-1 can protect RGCs from the consequences of elevated IOP in rats. This protection is manifested both morphologically and functionally. These findings can be readily implemented for the development of a therapeutic vaccination to arrest the progression of glaucoma.

Combined immunization with DNA and transduced tumor cells expressing mouse GM-CSF or IL-2.

A combination of different types of vaccines usually induces enhanced immune responses in comparison to immunization with single vaccines. The highest efficacy of a heterologous prime-boost strategy is mostly achieved after priming with a DNA vaccine and boosting with a recombinant virus or a protein vaccine. The aim of this study was to determine whether the combination of a DNA and cellular vaccine elicits stronger antitumor immune responses than vaccines used alone and to find out whether the efficacy of this combined immunization depends on the sequence in which the vaccines were applied. We utilized experimental vaccines that proved to be partially effective in protection against mouse tumor cells representing models of human papillomavirus-induced malignancies. The fusion gene Sig/E7GGG/LAMP-1, inoculated via a gene gun, was used for DNA immunization. As cellular vaccines, HPV16 E6/E7 and H-ras transformed B9 or 181 cells transduced with the gene coding for GM-CSF or IL-2, respectively, were applied. In both preventive and therapeutic immunizations, inoculation first with the DNA vaccine followed by application of a cellular vaccine induced the best protection from tumor growth. These results were confirmed by detection of immune reactions with in vitro tests. We failed to enhance immune reactions by utilization of an equivalent mix of B9 and 181 cells, but the addition of the second DNA-vaccine dose applied simultaneously with a cellular-vaccine boost moderately increased antitumor response. Our findings suggest the benefit of the heterologous prime-boost strategy based on combination of a DNA vaccine with a cellular vaccine and importance of sequence in which the vaccines are administered.

Human autologous tumor-specific T-cell responses induced by liposomal delivery of a lymphoma antigen.

PURPOSE: The idiotype (Id) of the immunoglobulin on a given B-cell malignancy is a clonal marker that can serve as a tumor-specific antigen. We developed a novel vaccine formulation by incorporating Id protein with liposomal lymphokine that was more potent than a prototype, carrier-conjugated Id protein vaccine in preclinical studies. In the present study, we evaluated the safety and immunogenicity of this vaccine in follicular lymphoma patients. EXPERIMENTAL DESIGN: Ten patients with advanced-stage follicular lymphoma were treated with five doses of this second generation vaccine after chemotherapy-induced clinical remission. All patients were evaluated for cellular and humoral immune responses. RESULTS: Autologous tumor and Id-specific type I cytokine responses were induced by vaccination in 10 and 9 patients, respectively. Antitumor immune responses were mediated by both CD4+ and CD8+ T cells, were human lymphocyte antigen class I and II associated, and persisted 18 months beyond the completion of vaccination. Specific anti-Id antibody responses were detected in four patients. After a median follow-up of 50 months, 6 of the 10 patients remain in continuous first complete remission. CONCLUSIONS: This first clinical report of a liposomal cancer vaccine demonstrates that liposomal delivery is safe, induces sustained tumor-specific CD4+ and CD8+ T-cell responses in lymphoma patients, and may serve as a model for vaccine development against other human cancers and infectious pathogens.

The VIM3-AraC regimen followed by autologous stem cell transplantation in refractory or relapsing aggressive non-Hodgkin's lymphoma. A prospective study of 71 consecutive cases.

We evaluated with an intent-to-treat analysis the response rate, the disease-free survival (DFS), and the overall survival after a multidrug salvage regimen (VIM3ARAC), followed by stem-cell transplantation (SCT) in case of response, in patients with aggressive non-Hodgkin's lymphoma (NHL) who progressed on or after the first-line therapy. Seventy-one patients (refractory: 15; relapse 'on therapy': 36; and relapse 'off therapy': 20) received two courses of VIM3ARAC (teniposide, ifosfamide, mitoxantrone, mitoguazone, high-dose methotrexate, high-dose cytarabine, prednisolone). SCT was performed only in patients with minimal disease after the second course. The response rate was 72%. It was not influenced by response to first-line therapy. Forty-eight patients (68%), including 32 complete responders, fulfilled response criteria for SCT. Thirty-six patients underwent SCT (allogeneic: 3; autologous: 33). The 4-year DFS rate of the 48 responding patients was 39%. The actuarial survival at 4 years was 34% for all patients. Relapse off therapy and a performance status <2 at relapse were the only two independent favorable prognostic factors for survival. In conclusion, VIM3AraC is associated with a high response rate in relapsing and refractory aggressive NHL. Up to half of the patients could receive SCT. This chemotherapy, followed by SCT could durably salvage 34% of these patients.

VIM3-ARA C: an effective salvage regimen in refractory or recurrent aggressive non Hodgkin's lymphoma. A report on 18 cases.

Based on encouraging results of previous combination regimens, we used a combination of VM26, ifosfamide, methyl GAG, mitoxantrone (or adriamycin), high-dose (HD) methotrexate and HD Ara C to treat 18 patients with relapsed or refractory NHL. Front-line therapy had been in most of them a reinforced CHOP regimen. Twelve patients (67 per cent) responded: there were nine (50 per cent) partial responses (PR) and three (17 per cent) complete remissions (CR). Nine of these 12 responders were grafted (eight autologous, one allogeneic transplants), one relapsed before autograft could be performed and the two remaining patients were excluded from autograft because of positive bone marrow. Five of nine patients remained free of disease after 11+ to 27+ months. Response rate was higher in patients who relapsed 'off' therapy (2/3), but CR was also obtained in two refractory NHL and persisted for 11+ and 26+ months, suggesting that VIM3-ARA C was, at least partially, non-cross-resistant with front-line adriamycin-containing regimens.