Vitamin C is an antioxidant vitamin that has been hypothesized to antagonize the effects of reactive oxygen species-generating antineoplastic drugs.
The therapeutic efficacy of the widely-used antineoplastic drugs, doxorubicin, cisplatin, vincristine, methotrexate and imatinib were compared in leukemia (K562) and lymphoma (RL) cell lines with and without pre-treatment with dehydroascorbic acid, the commonly transported form of vitamin C. The impact of vitamin C on viability, clonogenicity, apoptosis, P-glycoprotein, reactive oxygen species (ROS) and mitochondrial membrane potential was determined.
Pre-treatment with vitamin C caused a dose-dependent attenuation of cytotoxicity as measured by trypan blue exclusion and colony formation after treatment with all anti-neoplastic agents tested. Vitamin C administered prior to doxorubicin treatment led to a substantial reduction of therapeutic efficacy in mice with RL cell-derived xenogeneic tumors. Vitamin C treatment led to a dose-dependent decrease in apoptosis in cells treated with the antineoplastic agents that was not due to up-regulation of P-glycoprotein or vitamin C retention modulated by anti-neoplastics. Vitamin C had only modest effects on intracellular ROS and a more general cytoprotective profile than N-acetylcysteine; suggesting a mechanism of action that is not mediated by ROS. All antineoplastic agents tested caused mitochondrial membrane depolarization that was inhibited by vitamin C.
These findings indicate that vitamin C administered prior to mechanistically dissimilar antineoplastic agents antagonizes therapeutic efficacy in a model of human hematopoietic cancers by preserving mitochondrial membrane potential. These results support the hypothesis that vitamin C supplementation during cancer treatment may detrimentally affect therapeutic response.
cancer; reactive oxygen species; mitochondria; apoptosis
Ionizing radiation (IR) and certain chemotherapeutic drugs are designed to generate cytotoxic DNA double-strand breaks (DSBs) in cancer cells. Inhibition of the major DSB repair pathway, nonhomologous end joining (NHEJ), will enhance the cytotoxicity of these agents. Screening for inhibitors of the DNA ligase IV (Lig4), which mediates the final ligation step in NHEJ, offers a novel target-based drug discovery opportunity. For this purpose, we have developed an enzymatic assay to identify chemicals that block the transfer of [α-33P]-AMP from the complex Lig4-[α-33P]-AMP onto the 5′ end of a double-stranded DNA substrate and adapted it to a scintillation proximity assay (SPA). A screen was performed against a collection of 5,280 compounds. Assay statistics show an average Z′ value of 0.73, indicative of a robust assay in this SPA format. Using a threshold of >20% inhibition, 10 compounds were initially scored as positive hits. A follow-up screen confirmed four compounds with IC50 values ranging from 1 to 30 μM. Rabeprazole and U73122 were found to specifically block the adenylate transfer step and DNA rejoining; in whole live cell assays, these compounds were found to inhibit the repair of DSBs generated by IR. The ability to screen and identify Lig4 inhibitors suggests that they may have utility as chemo- and radio-sensitizers in combination therapy and provides a rationale for using this screening strategy to identify additional inhibitors.
Approved therapeutic antineoplastic antibodies have targeted extracellular or cell-surface molecules. ESK1 is the first fully human T-cell receptor-like antibody targeting an intracellular tumor-associated antigen, Wilm’s tumor 1 (WT1). In murine xenograft models, ESK1 exhibits a high specificity and exert robust antineoplastic effects against human cancers that express WT1 epitopes on HLA-A201 molecules.
WT1; TCR-like; monoclonal antibodies; cancer; MHC
Alpha particle-emitting isotopes have been proposed as novel cytotoxic agents for augmenting targeted therapy. Properties of alpha particle radiation such as their limited range in tissue of a few cell diameters and their high linear energy transfer leading to dense radiation damage along each alpha track are promising in the treatment of cancer, especially when single cells or clusters of tumor cells are targeted. Actinium-225 (225Ac) is an alpha particle-emitting radionuclide that generates 4 net alpha particle isotopes in a short decay chain to stable 209Bi, and as such can be described as an alpha particle nanogenerator. This article reviews the literature pertaining to the research, development, and utilization of targeted 225Ac to potently and specifically affect cancer.
Actinium-225; 225Ac; alpha particle-emitter; nano-device; targeted therapy; monoclonal antibody; DOTA; radioimmunotherapy
Iron is required for nearly all organisms, playing important roles in oxygen transport and many enzymatic reactions. Excess iron, however, can be cytotoxic. Emerging evidence suggests that radioresistance can be achieved in lower organisms by the protection of proteins, but not DNA, immediately following ionizing radiation (IR) exposure, allowing for improved DNA repair. One potential mechanism for protein protection is controlling and limiting the amount of free iron in cells, as has been demonstrated in the extremophile Deinococcus Radiodurans, reducing the potential for oxidative damage to proteins during exposure to IR. We found that iron regulatory protein 1 (IRP1) expression was markedly reduced in human myeloid leukemia HL60 cells resistant to low linear energy transfer (LET) gamma rays, but not to high LET alpha particles. Stable knockdown of IRP1 by short-hairpin RNA (shRNA) interference in radiosensitive parental cells led to radioresistance to low LET IR, reduced intracellular Fenton chemistry, reduced protein oxidation, and more rapid DNA double-strand break (DSB) repair. The mechanism of radioresistance appeared to be related to attenuated free radical-mediated cell death. Control of intracellular iron by IRPs may be a novel radioresistance mechanism in mammalian cells.
The sphingomyelin pathway involves the enzymatic cleavage of sphingomyelin to produce ceramide, a second messenger that serves as a key mediator in the rapid apoptotic response to various cell stressors. Low-linear energy transfer (LET) γ radiation can initiate this pathway, independent of DNA damage, via the cell membrane. Whether short-ranged, high-LET a particles, which are of interest as potent environmental carcinogens, radiotherapies and potential components of dirty bombs, can act through this mechanism to signal apoptosis is unknown. Here we show that irradiation of Jurkat cells with a particles emitted by the 225Ac-DOTA-anti-CD3 IgG antibody construct results in dose-dependent apoptosis. This apoptosis was significantly reduced by pretreating cells with cholesterol-depleting nystatin, a reagent known to inhibit ceramide signaling by interfering with membrane raft coalescence and ceramide-rich platform generation. The effects of nystatin on α-particle-induced apoptosis were related to disruption of the ceramide pathway and not to microdosimetry alterations, because similar results were obtained after external irradiation of the cells with a broad beam of collimated a particles using a planar 241Am source. External irradiation allowed for more precise control of the dosimetry and geometry of the irradiation, independent of antibody binding or cell internalization kinetics. Mechanistically consistent with these findings, Jurkat cells rapidly increased membrane concentrations of ceramide after external irradiation with an average of five α-particle traversals per cell. These data indicate that a particles can activate the sphingomyelin pathway to induce apoptosis.
Selective inhibitors of human peptide deformylase (HsPDF) are predicted to constitute a new class of antitumor agents. We report the identification of benzofuran-4,5-diones as the first known selective HsPDF inhibitors and we describe their selectivity profile in a panel of metalloproteases. We characterize their struture activity relationships for antitumor activity in a panel of cancer cell lines, and we assess their in vivo efficacy in a mouse xenograft model. Our results demonstrate that selective HsPDF inhibitors based on the benzofuran-4,5-dione scaffold constitute a novel class of antitumor agents that are potent in vitro and in vivo.
Human peptide deformylase; Benzofuran-4,5-diones; Structure activity relationships; Fluorescence polarization; Antiproliferative agents
Humanized A33 (huA33) is a promising monoclonal antibody that recognizes A33 antigen, which is present in more than 95% of colorectal cancers and in normal bowel. In this study, we took advantage of quantitative PET to evaluate 124I huA33 targeting, biodistribution, and safety in patients with colorectal cancer. We also determined the biodistribution of 124I-huA33 when a large dose of human intravenous IgG (IVIG) was administered to manipulate the Fc receptor or when 124I-huA33 was given via hepatic arterial infusion (HAI).
We studied 25 patients with primary or metastatic colorectal cancer; 19 patients had surgical exploration or resection. Patients received a median of 343 MBq (44.4–396 MBq) and 10 mg of 124I-huA33. Nineteen patients received the antibody intravenously and 6 patients via HAI, and 5 patients also received IVIG.
Ten of 12 primary tumors were visualized in 11 patients. The median concentration in primary colon tumors was 0.016% injected dose per gram, compared with 0.004% in normal colon. The PET-based median ratio of hepatic tumor uptake to normal-liver uptake was 3.9 (range, 1.8–22.2). Quantitation using PET, compared with well counting of serum and tissue, showed little difference. Prominent uptake in bowel hindered tumor identification in some patients. Pharmacokinetics showed that patients receiving IVIG had a significantly shorter serum half-time (41.6 ± 14.0 h) than those without (65.2 ± 9.8 h). There were no differences in clearance rates among the intravenous group, IVIG group, and HAI group, nor was there any difference in serum area under the curve, maximum serum concentration, or volume of distribution. Weak titers of human–anti-human antibodies were observed in 6 of 25 patients. No acute side effects or significant toxicities were associated with huA33.
Good localization of 124I-huA33 in colorectal cancer with no significant toxicity has been observed. PET-derived 124I concentrations agreed well with those obtained by well counting of surgically resected tissue and blood, confirming the quantitative accuracy of 124I-huA33 PET. The HAI route had no advantage over the intravenous route. No clinically significant changes in blood clearance were induced by IVIG.
A33; 124I; antibody; arterial; positron; colon
The rapid development of techniques that enable synthesis (and manipulation) of matter on the nanometer scale, as well as the development of new nano-materials, will play a large role in disease diagnosis and treatment, specifically in targeted cancer therapy. Targeted nanocarriers are an intriguing means to selectively deliver high concentrations of cytotoxic agents or imaging labels directly to the cancer site. Often solubility issues and an unfavorable biodistribution can result in a suboptimal response of novel agents even though they are very potent. New nanoparticulate formulations allow simultaneous imaging and therapy (“theranostics”), which can provide a realistic means for the clinical implementation of such otherwise suboptimal formulations. In this review we will not attempt to provide a complete overview of the rapidly enlarging field of nanotechnology in cancer; rather, we will present properties specific to nanoparticles, and examples of their uses, which demonstrate their importance for targeted cancer therapy.
Anti-angiogenic therapies are frequently used with concomitantly administered cancer chemotherapy to improve outcomes, but the mechanism for the benefit of the combination is uncertain. We describe a mechanism by which a specific, cytotoxic anti-vascular agent causes vascular remodeling and improved chemotherapy results. By selectively killing tumor neovasculature using short-ranged alpha particles targeted to VE-cadherin on vascular endothelial cells (by use of 225Ac-labeled E4G10 antibody) we were able to both reduce tumor growth and to increase the efficacy of chemotherapy, an effect seen only when the chemotherapy was administered several days after the vascular targeting agent, but not if the order of administration was reversed. Immunohistochemical and immunofluorescence studies showed that the vasculature of 225Ac-E4G10 treated tumors was substantially depleted; the remaining vessels appeared more mature morphologically and displayed increased pericyte density and coverage. Tumor uptake and micro-distribution studies with radioactive and fluorescent small molecule drugs showed better accumulation and more homogenous distribution of the drugs within 225Ac-E4G10 treated tumors. These results show that 225Ac-E4G10 treatment leads to ablation and improvement of the tumor vascular architecture, and also demonstrate that the resulting vascular remodeling can increase tumor delivery of small molecules, thus providing a process for the improved outcomes observed after combining anti-vascular and chemotherapy. This study directly shows evidence for what has long been a speculated mechanism for anti-angiogenic therapies. Moreover, targeting the vessel for killing provides an alternative mode of improving chemotherapy delivery and efficacy, potentially avoiding some of the drawbacks of targeting a highly redundant angiogenic pathway.
cancer; radiotherapy; anti-vascular therapy; angiogenesis
Lintuzumab (HuM195), a humanized anti-CD33 antibody, targets myeloid leukemia cells and has modest single-agent activity against acute myeloid leukemia (AML). To increase the antibody’s potency without the nonspecific cytotoxicity associated with β-emitters, the α particle-emitting radionuclide bismuth-213 (213Bi) was conjugated to lintuzumab. This phase I/II trial was conducted to determine the maximum tolerated dose (MTD) and antileukemic effects of 213Bi-lintuzumab, the first targeted α-emitter, after partially cytoreductive chemotherapy.
Thirty-one patients with newly diagnosed (n = 13) or relapsed/refractory (n = 18) AML (median age, 67 years; range, 37–80) were treated with cytarabine 200 mg/m2/day for 5 days followed by 213Bi-lintuzumab 18.5–46.25 MBq/kg.
The MTD of 213Bi-lintuzumab was 37 MB/kg; myelosuppression lasting > 35 days was dose-limiting. Extramedullary toxicities were primarily limited to ≤ grade 2 events, including infusion-related reactions. Transient grade 3/4 liver function abnormalities were seen in 5 patients (16%). Treatment-related deaths occurred in 2 of 21 patients (10%) who received the MTD. Significant reductions in marrow blasts were seen at all dose levels. The median response duration was 6 months (range, 2–12). Biodistribution and pharmacokinetic studies suggested that saturation of available CD33 sites by 213Bi-lintuzumab was achieved after partial cytoreduction with cytarabine.
Sequential administration of cytarabine and 213Bi-lintuzumab is tolerable and can produce remissions in patients with AML.
Radioimmunotherapy; Alpha-particles; Acute myeloid leukemia; Monoclonal Antibodies; Bismuth-213
Alpha-particle-emitting elements are of increasing importance as environmental and occupational carcinogens, toxic components of radiation dispersal devices and accidents, and potent therapeutics in oncology. Alpha particle radiation differs from radiations of lower linear energy transfer in that it predominantly damages DNA via direct action. Because of this, radical scavengers effective for other radiations have had only limited effect in mitigating alpha particle toxicity. We describe here a simple assay and a pilot screen of 3,119 compounds in a high-throughput screen (HTS), using the alpha-particle-emitting isotope, 225Ac, for the discovery of compounds that might protect mammalian cells from alpha particles through novel mechanisms. The assay, which monitored the viability of a myeloid leukemic cell line upon alpha particle exposure, was robust and reproducible, yielding a Z' factor of 0.66 and a signal-to-noise ratio of nearly 10 to 1. Surprisingly, 1 compound emerged from this screen, epoxy-4,5-α-dihydroxysantonin (EDHS), that showed considerable protective activity. While the value of EDHS remains to be determined, its discovery is a proof of concept and validation of the utility of this HTS methodology. Further application of the described assay could yield compounds useful in minimizing the toxicity and carcinogenesis associated with alpha particle exposure.
Effective targeting and killing of intraperitoneally disseminated micrometastases remains a challenge.
In this work, we evaluated the potential of antibody-labeled PEGylated large liposomes as vehicles for direct intraperitoneal (i.p.) drug delivery with the aim to enhance the tumor-to-normal organ ratio and to improve the bioexposure of cancer cells to the delivered therapeutics while shifting the toxicities toward the spleen. These targeted liposomes are designed to combine: (1) specific targeting to and internalization by cancer cells mediated by liposome-conjugated tumor-specific antibodies, (2) slow clearance from the peritoneal cavity, and (3) shift of normal organ toxicities from the liver to the spleen due to their relatively large size.
Conjugation of anti-HER2/neu antibodies to the surface of large (approximately 600 nm in diameter) PEGylated liposomes results in fast, specific binding of targeted liposomes to cancer cells in vitro, followed by considerable cellular internalization. In vivo, after i.p. administration, these liposomes exhibit fast, specific binding to i.p. cancerous tumors. Large liposomes are slowly cleared from the peritoneal cavity, and they exhibit increased uptake by the spleen relative to the liver, while targeted large liposomes demonstrate specific tumor uptake at early times. Although tissue and tumor uptake are greater for cationic liposomes, the tumor-to-liver and spleen-to-liver ratios are similar for both membrane compositions, suggesting a primary role for the liposome’s size, compared to the liposome’s surface charge.
The findings of this study suggest that large targeted liposomes administered i.p. could be a potent drug-delivery strategy for locoregional therapy of i.p. micrometastatic tumors.
Large liposomes; targeted liposomes; trastuzumab; intraperitoneal therapy; ovarian cancer
Blood vessels are formed either by sprouting of resident tissue endothelial cells (angiogenesis) or by recruitment of bone marrow (BM)–derived circulating endothelial progenitor cells (EPCs, vasculogenesis). Neovascularization has been implicated in tumor growth and inflammation, but its roles in graft-vs-host disease (GVHD) and in tumors after allogeneic BM transplantation (allo-BMT) were not known.
We analyzed neovascularization, the contribution of endothelial cells and EPCs, and the ability of anti–vascular endothelial-cadherin antibody, E4G10, to inhibit neovascularization in mice with GVHD after allo-BMT using immunofluorescence microscopy and flow cytometry. We examined survival and clinical and histopathologic GVHD in mice (n = 10–25 per group) in which GVHD was treated with the E4G10 antibody using immunohistochemistry, flow cytometry, and cytokine immunoassay. We also assessed survival, the contribution of green fluorescent protein-marked EPCs to the tumor vasculature, and the ability of E4G10 to inhibit tumor growth in tumor-bearing mice (n = 20–33 per group) after allo-BMT using histopathology and bioluminescence imaging. All statistical tests were two-sided.
We found increased neovascularization mediated by vasculogenesis, as opposed to angiogenesis, in GVHD target tissues, such as liver and intestines. Administration of E4G10 inhibited neovascularization by donor BM-derived cells without affecting host vascularization, inhibited both GVHD and tumor growth, and increased survival (at 60 days post-BMT and tumor challenge with A20 lymphoma, the probability of survival was 0.29 for control antibody-treated allo-BMT recipients vs 0.7 for E4G10-treated allo-BMT recipients, 95% confidence interval = 0.180 to 0.640, P < .001).
Therapeutic targeting of neovascularization in allo-BMT recipients is a novel strategy to simultaneously ameliorate GVHD and inhibit posttransplant tumor growth, providing a new approach to improve the overall outcome of allogeneic hematopoietic stem cell transplantation.
Deformylases are metalloproteases in bacteria, plants, and humans that remove the N-formyl-methionine off peptides in vitro. The human homolog of peptide deformylase (HsPDF) resides in the mitochondria, along with its putative formylated substrates; however, the cellular function of HsPDF remains elusive. Here we report on the function of HsPDF in mitochondrial translation and oxidative phosphorylation complex biogenesis. Functional HsPDF appears to be necessary for the accumulation of mitochondrial DNA-encoded proteins and assembly of new respiratory complexes containing these proteins. Consequently, inhibition of HsPDF reduces respiratory function and cellular ATP levels, causing dependence on aerobic glycolysis for cell survival. A series of structurally different HsPDF inhibitors and control peptidase inhibitors confirmed that inhibition of HsPDF decreases mtDNA-encoded protein accumulation. Therefore, HsPDF appears to have a role in maintenance of mitochondrial respiratory function, and this function is analogous to that of chloroplast PDF.
Long-term survival still eludes most patients with leukemia and non-Hodgkin’s lymphoma. No approved therapies target the hallmark of the B cell, its mIgM, also known as the B-cell receptor (BCR). Aptamers are small oligonucleotides that can specifically bind to a wide range of target molecules and offer some advantages over antibodies as therapeutic agents. Here, we report the rational engineering of aptamer TD05 into multimeric forms reactive with the BCR that may be useful in biomedical applications. Systematic truncation of TD05 coupled with modification with locked nucleic acids (LNA) increased conformational stability and nuclease resistance. Trimeric and tetrameric versions with optimized polyethyleneglycol (PEG) linker lengths exhibited high avidity at physiological temperatures both in vitro and in vivo. Competition and protease studies showed that the multimeric, optimized aptamer bound to membrane-associated human mIgM, but not with soluble IgM in plasma, allowing the possibility of targeting leukemias and lymphomas in vivo. The B-cell specificity of the multivalent aptamer was confirmed on lymphoma cell lines and fresh clinical leukemia samples. The chemically engineered aptamers, with significantly improved kinetic and biochemical features, unique specificity and desirable pharmacological properties, may be useful in biomedical applications.
Single wall carbon nanotube (SWCNT) constructs were covalently appended with radiometal-ion chelates (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid [DOTA] or desferrioxamine B [DFO]) and the tumor neovascular-targeting antibody E4G10. The E4G10 antibody specifically targeted the monomeric vascular endothelial-cadherin (VE-cad) epitope expressed in the tumor angiogenic vessels. The construct specific activity and blood compartment clearance kinetics were significantly improved relative to corresponding antibodyalone constructs. We performed targeted radioimmunotherapy with a SWCNT-([225Ac]DOTA) (E4G10) construct directed at the tumor vasculature in a murine xenograft model of human colon adenocarcinoma (LS174T). The specific construct reduced tumor volume and improved median survival relative to controls. We also performed positron emission tomographic (PET) radioimmunoimaging of the tumor vessels with a SWCNT-([89Zr]DFO)(E4G10) construct in the same murine LS174T xenograft model and compared the results to appropriate controls. Dynamic and longitudinal PET imaging of LS174T tumor-bearing mice demonstrated rapid blood clearance (<1 hour) and specific tumor accumulation of the specific construct. Incorporation of the SWCNT scaffold into the construct design permitted us to amplify the specific activity to improve the signal-to-noise ratio without detrimentally impacting the immunoreactivity of the targeting antibody moiety. Furthermore, we were able to exploit the SWCNT pharmacokinetic (PK) profile to favorably alter the blood clearance and provide an advantage for rapid imaging. Near-infrared three-dimensional fluorescent-mediated tomography was used to image the LS174T tumor model, collect antibody-alone PK data, and calculate the number of copies of VE-cad epitope per cell. All of these studies were performed as a single administration of construct and were found to be safe and well tolerated by the murine model. These data have implications that support further imaging and radiotherapy studies using a SWCNT-based platform and focusing on the tumor vessels as the target.
actinium-225 (225Ac); zirconium-89 (89Zr); angiogenesis; vascular endothelialcadherin; radioimmunotherapy (RIT); radioimmunoPET
Peptide deformylase proteins (PDFs) participate in the N-terminal methionine excision pathway of newly synthesized peptides. We show that the human PDF (HsPDF) can deformylate its putative substrates derived from mitochondrial DNA-encoded proteins. The first structural model of a mammalian PDF (1.7 Å), HsPDF, shows a dimer with conserved topology of the catalytic residues and fold as non-mammalian PDFs. The HsPDF C-terminus topology and the presence of a helical loop (H2 and H3), however, shape a characteristic active site entrance. The structure of HsPDF bound to the peptidomimetic inhibitor actinonin (1.7 Å) identified the substrate-binding site. A defined S1′ pocket, but no S2′ or S3′ substrate-binding pockets, exists. A conservation of PDF–actinonin interaction across PDFs was observed. Despite the lack of true S2′ and S3′ binding pockets, confirmed through peptide binding modeling, enzyme kinetics suggest a combined contribution from P2′ and P3′ positions of a formylated peptide substrate to turnover.
human deformylase; peptide deformylase; crystal structure
Modern combination strategies are active in chronic lymphocytic leukemia (CLL) but can have significant myelosuppression and immunosuppression that may require dose attenuation for safety. We explored a sequential treatment strategy to allow safe delivery of active agents at full doses. Previously, we studied sequential therapy with fludarabine followed by cyclophosphamide (F→C). In that study, cyclophosphamide consolidation improved the frequency of complete response (CR) four-fold. Subsequently, rituximab was added to this regimen (F→C→R).
Patients and Methods
Thirty-six previously untreated CLL patients received therapy with fludarabine 25 mg/m2 on days 1 through 5 every 4 weeks for six cycles, followed by consolidation with cyclophosphamide 3,000 mg/m2 administered every 3 weeks for three cycles, followed by consolidation with weekly rituximab 375 mg/m2 for four cycles. Evaluation for minimal residual disease included flow cytometry and a highly sensitive clonotypic polymerase chain reaction (PCR). The median age was 59 years (range, 37 to 71 years), 61% of patients had high-risk disease, and 58% had unmutated IgVH genes.
There were 32 responses (89%), including 22 CRs (61%). Consolidation with cyclophosphamide improved responses in 13 patients (36%); nine patients (25%) further improved their response with rituximab. Twenty patients (56%) achieved flow cytometric CRs, and 12 patients (33%) achieved a molecular CR (PCR negative). Patients achieving molecular CRs had an excellent prognosis with a plateau in the response duration curve, and 90% remain in clinical CR at 5 years. For the entire group, 5-year survival rate is 71% compared with a rate of 48% with our prior F→C regimen (P = .10).
Sequential therapy with F→C→R yields improvement in quality of response, with many patients achieving a PCR-negative state.
Cyclin D1 is over-expressed in various human tumors and therefore can be a potential oncogenic target antigen. However, only a limited number of T cell epitopes has been characterized. We aimed at identifying human cyclin D1-derived peptides that include both CD4 and CD8 T cell epitopes and to test if such multi-epitope peptides could yield improved cytotoxic CD8 T cell responses as well as cytotoxic CD4 T cells. Five HLA-DR.B1-binding peptides containing multiple overlapping CD4 epitopes and HLA-A0201-restricted CD8 T cell epitopes were predicted by computer algorithms. Immunogenicity of the synthetic peptides was assessed by stimulating T cells from healthy donors in vitro and the epitope recognition was measured by IFN-γ ELISPOT and 51Chromium release assays. A HLA-DR.B1 peptide, designed “DR-1”, in which a HLA-A0201-binding epitopes (D1-1) was imbedded, induced CD3 T cell responses against both DR-1 and D1-1 peptides in IFN-γ ELISPOT assay. This suggested processing of the shorter D1-1 epitope from the DR-1 sequence. However, only DR-1-stimulated CD4 or CD3 T cells possessed cytotoxicity against peptide-pulsed autologous DCs and a cancer cell line, that expresses a high level of cyclin D1. Monoclonal antibody to HLA-DR abrogated the epitope-specific responses of both CD3 and CD4 T cells, demonstrating class II-mediated killing. Our studies suggest a possible role of CD4 T cells in anti-tumor immunity as cytotoxic effectors against HLA-DR expressing cancers and provide a rationale for designing peptide vaccines that include CD4 epitopes.
Prostate specific antigen (PSA) molecules secreted by cancerous and normal prostate cells differ in their N-linked glycan composition, while the peptide backbone appears to be conserved. Antibodies selectively recognizing such differentially glycosylated PSA structures could form a basis for a new diagnostic assay for prostate cancer. Twenty-amino acid PSA fragments carrying di-, tri-, and tetrabranched complex-type glycans were prepared by total synthesis and conjugated to maleimide-modified keyhole limpet hemocyanin (KLH) carrier protein through backbone Cys residues. These glycopeptide/KLH conjugates were then used for antibody generation.
Wilms tumor protein (WT1) is a transcription factor selectively overexpressed in leukemias and cancers; clinical trials are underway that use altered WT1 peptide sequences as vaccines. Here we report a strategy to study peptide-MHC interactions by incorporating non-natural and photo-reactive amino acids into the sequence of WT1 peptides. Thirteen WT1 peptides sequences were synthesized with chemically modified amino acids (via fluorination and photo-reactive group additions) at MHC and T cell receptor binding positions. Certain new non-natural peptide analogs could stabilize MHC class I molecules better than the native sequences and were also able to elicit specific T-cell responses and sometimes cytotoxicity to leukemia cells. Two photo-reactive peptides, also modified with a biotin handle for pull-down studies, formed covalent interactions with MHC molecules on live cells and provided kinetic data showing the rapid clearance of the peptide-MHC complex. Despite “infinite affinity” provided by the covalent peptide bonding to the MHC, immunogenicity was not enhanced by these peptides because the peptide presentation on the surface was dominated by catabolism of the complex and only a small percentage of peptide molecules covalently bound to the MHC molecules. This study shows that non-natural amino acids can be successfully incorporated into T cell epitopes to provide novel immunological, biochemical and kinetic information.
Targeted α-particle emitters hold great promise as therapeutics for micrometastatic disease. Because of their high energy deposition and short range, tumor targeted α-particles can result in high cancer-cell killing with minimal normal-tissue irradiation. Actinium-225 is a potential generator for α-particle therapy: it decays with a 10-day half-life and generates three α-particle emitting daughters. Retention of 225Ac daughters at the target increases efficacy; escape and distribution throughout the body increases toxicity. During circulation, molecular carriers conjugated to 225Ac cannot retain any of the daughters. We previously proposed liposomal encapsulation of 225Ac to retain the daughters, whose retention was shown to be liposome-size dependent. However, daughter retention was lower than expected: 22% of theoretical maximum decreasing to 14%, partially due to binding of 225Ac to the phospholipid membrane. In this study, MUltiVEsicular Liposomes (MUVELs) composed of different phospholipids were developed to increase daughter retention. MUVELs are large liposomes with entrapped smaller lipid-vesicles containing 225Ac. PEGylated MUVELs stably retained over time 98% of encapsulated 225Ac. Retention of 213Bi, the last daughter, was 31% of the theoretical maximum retention of 213Bi for the liposome sizes studied. MUVELs were conjugated to an anti-HER2/neu antibody (immunolabeled MUVELs), and were evaluated in vitro with SKOV3-NMP2 ovarian cancer cells, exhibiting significant cellular internalization (83%). This work demonstrates that immunolabeled MUVELs could be able to deliver higher fractions of generated α-particles per targeted 225Ac compared to the relative fractions of α-particles delivered by 225Ac-labeled molecular carriers.
actinium-225; bismuth-213; multivesicular liposomes; intraperitoneal alpha therapy; ovarian cancer micrometastases