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ABSTRACT: Based on their inability to express argininosuccinate synthetase (ASS), some cancer entities feature the characteristic of l-arginine (Arg) auxotrophy. This inability to intrinsically generate Arg makes them applicable for arginine deiminase (ADI) treatment, an Arg-depleting drug. Arg is also used for the synthesis of endothelial nitric oxide (NO), which mainly confers vasodilatation but is also considered to have a major influence on tumor vascularization. The purpose of this study was to define changes in tumor vasculature in an ADI-treated melanoma xenograft mouse model using the blood pool agent AngioSense 750 and fluorescence molecular tomography (FMT). We used an ASS-negative melanoma xenograft mouse model and subjected it to weekly ADI treatment. Changes in tumor size were measured, and alterations in tumor vasculature were depicted by FMT and CD31 immunohistochemistry (IHC). On ADI treatment and effective antitumor therapy, we observed a drop in NO plasma levels and visualized changes in tumor vascularization with FMT and IHC. ADI treatment in melanoma xenografts has a tumor-reducing effect, which can be noninvasively imaged by quantifying tumor vascularization with FMT and IHC.
Molecular Imaging 02/2013; 12(1):67-73. · 3.18 Impact Factor
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Anurag Gupta,
Natko Nuber,
Christoph Esslinger,
Mareike Wittenbrink,
Martin Treder,
Alexandro Landshammer,
Takuro Noguchi,
Marcus Kelly,
Sacha Gnjatic,
Erika Ritter,
Lotta von Boehmer,
Hiroyoshi Nishikawa,
Hiroshi Shiku, Lloyd Old,
Gerd Ritter,
Alexander Knuth,
Maries van den Broek
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ABSTRACT: We investigated whether antibodies against intracellular tumor-associated antigens support tumor-specific immunity when administered together with a treatment that destroys the tumor. We propose that released antigens form immune complexes with the antibodies, which are then efficiently taken up by dendritic cells. We cloned the first human monoclonal antibodies against the Cancer/Testis (CT) antigen, NY-ESO-1. We tested whether the monoclonal anti-NY-ESO-1 antibody (12D7) facilitates cross-presentation of a NY-ESO-1-derived epitope by dendritic cells to human CD8+ T cells, and whether this results in the maturation of dendritic cells in vitro. We investigated the efficacy of 12D7 in combination with chemotherapy using BALB/c mice bearing syngeneic CT26 tumors that express intracellular NY-ESO-1. Human dendritic cells that were incubated with NY-ESO-1:12D7 immune complexes efficiently stimulated NY-ESO-1(157-165)/HLA-A2-specific human CD8+ T cells to produce interferon-γ, whereas NY-ESO-1 alone did not. Furthermore, the incubation of dendritic cells with NY-ESO-1:12D7 immune complexes resulted in the maturation of dendritic cells. Treatment of BALB/c mice that bear CT26/NY-ESO-1 tumors with 5-fluorouracil (5-FU) plus 12D7 was significantly more effective than chemotherapy alone. We propose systemic injection of monoclonal antibodies (mAbs) against tumor-associated antigens plus a treatment that promotes the local release of those antigens resulting in immune complex formation as a novel therapeutic modality for cancer.
Cancer immunity: a journal of the Academy of Cancer Immunology 01/2013; 13:3.
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Paul J Sabbatini,
Takemasa Tsuji,
Luis Ferran,
Erika Ritter,
Christine Sedrak,
Kevin Tuballes,
Achim A Jungbluth,
Gerd Ritter,
Carol Aghajanian,
Katherine Bell-McGuinn, [......],
Jason Konner,
William Tew,
David R Spriggs,
Eric W Hoffman,
Ralph Venhaus,
Linda Pan,
Andres M Salazar,
Catherine Magid Diefenbach, Lloyd Old,
Sacha Gnjatic
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ABSTRACT: PURPOSE: Long peptides are efficiently presented to both CD4+ and CD8+ T-cells after intracellular processing by antigen presenting cells. To investigate the safety and in vivo immunogenicity of synthetic overlapping long peptides (OLP) from a human tumor self-antigen, we conducted a phase I clinical trial with OLP from cancer-testis antigen NY-ESO-1 in various adjuvant combinations. Experimental design: Twenty-eight advanced ovarian cancer patients in second or third remission were enrolled sequentially in 3 cohorts and received at least one vaccination. Patients in Cohort 1 (n=4) received 1.0 mg OLP, Cohort 2 (n=13) received OLP in Montanide-ISA-51, and Cohort 3 (n=11) received OLP + 1.4 mg Poly-ICLC in Montanide-ISA-51 on weeks 1, 4, 7, 10, and 13. Humoral and cellular responses were evaluated by standardized immunomonitoring techniques (ELISA, ELISPOT assay, intracellular cytokine staining, tetramer staining). RESULTS: The vaccine was generally well tolerated with injection site reactions and fatigue that resolved. NY-ESO-1-specific antibody and CD8+ T-cells were undetectable after vaccination with OLP alone, but found in 6/13 (46%) and 8/13 (62%) patients respectively after vaccination with OLP+Montanide, and in 10/11 (91%) and 10/11 (91%) patients respectively after vaccination with OLP+Montanide+Poly-ICLC. NY-ESO-1-specific CD4+ T-cells were detected in all patients, with greater frequency and polyclonality when Montanide-ISA-51 was used for vaccination. Inclusion of Poly-ICLC as adjuvant further accelerated induction of NY-ESO-1-specific immune responses. CONCLUSIONS: The current study demonstrates that NY-ESO-1 OLP vaccine is safe and rapidly induces consistent integrated immune responses (antibody, CD8+ and CD4+) in nearly all vaccinated patients when given with appropriate adjuvants.
Clinical Cancer Research 10/2012; · 7.74 Impact Factor
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ABSTRACT: Because of deficiencies in l-arginine biosynthesis, some cancers are susceptible to therapeutic intervention with arginine deiminase (ADI), an enzyme responsible for consuming the dietary supply of l-arginine to deprive the disease of an essential nutrient. ADI is currently being evaluated in several clinical trials, and fully realizing the drug's potential will depend on invoking the appropriate metrics to judge clinical response. Without a clear biologic mandate, PET/CT with (18)F-FDG is currently used to monitor patients treated with ADI. However, it is unclear if it can be expected that (18)F-FDG responses will indicate (or predict) clinical benefit.
(18)F-FDG responses to ADI therapy were studied in preclinical models of melanoma in vitro and in vivo. The molecular mechanism of response to ADI therapy was also studied, with a particular emphasis on biologic pathways known to regulate (18)F-FDG avidity.
Although proliferation of SK-MEL 28 was potently inhibited by ADI treatment in vitro and in vivo, no clear declines in (18)F-FDG uptake were observed. Further investigation showed that ADI treatment induces the posttranslational degradation of phosphatase and tensin homolog and the activation of the PI3K signaling pathway, an event known to enhance glycolysis and (18)F-FDG avidity. A more thorough mechanistic study showed that ADI triggered a complex mechanism of cell death, involving apoptosis via poly (ADP-ribose) polymerase cleavage-independent of caspase 3.
These findings suggest that some unexpected pharmacologic properties of ADI preclude using (18)F-FDG to evaluate clinical response in melanoma and, more generally, argue for further studies to explore the use of PET tracers that target apoptotic pathway activation or cell death.
Journal of Nuclear Medicine 02/2012; 53(2):281-6. · 6.38 Impact Factor
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Margarita T Murri-Plesko,
Alzbeta Hulikova,
Egbert Oosterwijk,
Andrew M Scott,
Anna Zortea,
Adrian L Harris,
Gerd Ritter, Lloyd Old,
Stefan Bauer,
Pawel Swietach,
Christoph Renner
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ABSTRACT: Carbonic anhydrase IX (CAIX) is a hypoxia-induced, membrane-tethered enzyme that is highly expressed in many cancers. It catalyses the hydration of CO(2) to HCO(3)(-) and H(+), and the reverse dehydration reaction. Recent studies have shown an important role for CAIX in pH regulation and it has been speculated that CAIX may play a role in supporting cancer progression towards more aggressive forms of the disease. Clinical correlative studies in many tumours have shown that high expression is related to poor outcome. In the present study, we have selected antigen-binding antibody fragments (Fab) against human CAIX by phage-display, and tested these for inhibitory potency on CAIX catalytic activity. Inhibition was assessed from the kinetics of the CAIX-catalysed reaction, using assays performed on intact cells over-expressing CAIX, and their CAIX-containing membrane fragments. Inhibition was also assessed in multi-cellular tissue-models (spheroids) from the kinetics of CO(2) venting. We have identified a Fab antibody, labelled MSC8, and its corresponding full-length IgG that inhibited CAIX by up to 57% and 76%, respectively, with half-maximal inhibition at 0.3μg/ml. Incubation of CAIX-expressing cells with MSC8 IgG produced a lasting inhibitory effect. The inhibitory effect was prompt and was also observed in isolated membrane-fragments, suggesting that a direct inhibitory interaction takes place between the antibody and CAIX. The inhibitory effects in spheroids argue for a physiological relevance of the antibody. Biologically-active antibodies against CAIX can be used as selective, high-affinity inhibitors in experimental studies to dissect the role of CAIX and, possibly, therapeutically by targeting a catalytically-active cancer-related protein.
European journal of pharmacology 02/2011; 657(1-3):173-83. · 2.59 Impact Factor
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ABSTRACT: Unlike chemotherapy, which acts directly on the tumor, cancer immunotherapies exert their effects on the immune system and demonstrate new kinetics that involve building a cellular immune response, followed by changes in tumor burden or patient survival. Thus, adequate design and evaluation of some immunotherapy clinical trials require a new development paradigm that includes reconsideration of established endpoints. Between 2004 and 2009, several initiatives facilitated by the Cancer Immunotherapy Consortium of the Cancer Research Institute and partner organizations systematically evaluated an immunotherapy-focused clinical development paradigm and created the principles for redefining trial endpoints. On this basis, a body of clinical and laboratory data was generated that supports three novel endpoint recommendations. First, cellular immune response assays generate highly variable results. Assay harmonization in multicenter trials may minimize variability and help to establish cellular immune response as a reproducible biomarker, thus allowing investigation of its relationship with clinical outcomes. Second, immunotherapy may induce novel patterns of antitumor response not captured by Response Evaluation Criteria in Solid Tumors or World Health Organization criteria. New immune-related response criteria were defined to more comprehensively capture all response patterns. Third, delayed separation of Kaplan-Meier curves in randomized immunotherapy trials can affect results. Altered statistical models describing hazard ratios as a function of time and recognizing differences before and after separation of curves may allow improved planning of phase III trials. These recommendations may improve our tools for cancer immunotherapy trials and may offer a more realistic and useful model for clinical investigation.
CancerSpectrum Knowledge Environment 09/2010; 102(18):1388-97. · 14.07 Impact Factor
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ABSTRACT: Medullary breast carcinoma (MBC) is a relatively rare malignancy with heavy lymphocytic infiltration that despite cytologically anaplastic features and high mitotic index has more favorable prognosis than other types of breast cancer. Lymphocytic infiltration of tumors reflects ongoing immune response against tumor antigens which could represent a great interest as potential targets for cancer immunotherapy. The search for MBC antigens by SEREX methodology has not been successful due to a very high titer of false positive clones, representing immunoglobulin genes. Here, we describe a novel approach for generating cDNA expression libraries from MBC tumor samples which are depleted of IgG cDNA clones and, therefore, are suitable for the identification of novel tumor-associated antigens (TAA) by SEREX approach. Modified methodology allowed us to isolate a panel of known and novel TAA which are currently under further investigation.
Molecular Biotechnology 05/2010; 46(2):105-12. · 2.17 Impact Factor
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ABSTRACT: The cancer-testis (CT) antigen synovial sarcoma X break point 2 (SSX2) was expressed in Pichia pastoris as a means to produce a delayed-type hypersensitivity skin test reagent for monitoring SSX2-specific anti-cancer immune responses. SSX2 was detected intracellularly in P. pastoris despite the addition of the Saccharomyces cerevisiae alpha-mating factor secretion signal. Increasing the SSX2 gene copy number did not improve its secretion but did enhance intracellular SSX2 levels. SSX2 with its C-terminal nuclear localization signal (NLS) deleted (SSX2NORD), however, was secreted. Indirect immunofluorescence indicated that SSX2 containing the NLS did not translocate to the nucleus but accumulated in the endoplasmic reticulum (ER). Experimental results further suggested that SSX2 containing the NLS was misfolded in the ER, while deletion of the NLS facilitated correct folding of SSX2 inside the ER and improved its secretion. Production of SSX2NORD was scaled-up to a 2-L fermentor using a fed-batch protocol to maintain methanol at a concentration of 1 g L(-1). Decreasing the cultivation temperature from 25 degrees C to 16 degrees C improved protein stability in the culture supernatant. In this process, after 120 h cultivation, the wet cell weight of P. pastoris reached 280 mg mL(-1), and the yield of SSX2NORD was 21.6 mg L(-1).
Applied Microbiology and Biotechnology 10/2009; 86(1):243-53. · 3.42 Impact Factor
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Yun Lin,
Humilidad Gallardo,
Geoffrey Ku,
Hao Li,
Gregor Manukian,
Teresa Rasalan,
Yinyan Xu,
Stephanie Terzulli, Lloyd Old,
James Allison,
Alan Houghton,
Jedd Wolchok,
Jianda Yuan
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ABSTRACT: Background aims Monitoring cellular immune responses is one prerequisite for the rational development of cancer vaccines. Methods We describe an extensive effort to optimize and validate quantitatively an in vitro T-cell culture method by determining the phenotype and function of both CD4(+) and CD8(+) T cells, including measurement of the phenotype markers CCR7, CD45RA, CD28 and CD27 and the functional markers interferon (IFN)-gamma, interleukin (IL)-2, macrophage inflammatory protein (MIP)-1beta, tumor necrosis factor (TNF)-alpha and CD107a. Results Autologous peripheral blood mononuclear cells (PBMC) were potent stimulators that expanded antigen (Ag)-specific CD8(+) T cells during short-term culture with the addition of IL-2 and IL-15 cytokines. Polyfunctional Ag-specific CD4(+) and CD8(+) T cells were detectable using this method. Conclusions Our culture system represents a robust human T-cell culture protocol that permits phenotypic, quantitative and qualitative evaluation of vaccine-induced CD4(+) and CD8(+) T-cell responses.
Cytotherapy 09/2009; · 3.63 Impact Factor
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Cedrik Michael Britten,
Sylvia Janetzki,
Leah Ben-Porat,
Timothy M Clay,
Michael Kalos,
Holden Maecker,
Kunle Odunsi,
Michael Pride, Lloyd Old,
Axel Hoos,
Pedro Romero
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ABSTRACT: The Cancer Vaccine Consortium of the Cancer Research Institute (CVC-CRI) conducted a multicenter HLA-peptide multimer proficiency panel (MPP) with a group of 27 laboratories to assess the performance of the assay.
Participants used commercially available HLA-peptide multimers and a well characterized common source of peripheral blood mononuclear cells (PBMC). The frequency of CD8+ T cells specific for two HLA-A2-restricted model antigens was measured by flow cytometry. The panel design allowed for participants to use their preferred staining reagents and locally established protocols for both cell labeling, data acquisition and analysis.
We observed significant differences in both the performance characteristics of the assay and the reported frequencies of specific T cells across laboratories. These results emphasize the need to identify the critical variables important for the observed variability to allow for harmonization of the technique across institutions.
Three key recommendations emerged that would likely reduce assay variability and thus move toward harmonizing of this assay. (1) Use of more than two colors for the staining (2) collect at least 100,000 CD8 T cells, and (3) use of a background control sample to appropriately set the analytical gates. We also provide more insight into the limitations of the assay and identified additional protocol steps that potentially impact the quality of data generated and therefore should serve as primary targets for systematic analysis in future panels. Finally, we propose initial guidelines for harmonizing assay performance which include the introduction of standard operating protocols to allow for adequate training of technical staff and auditing of test analysis procedures.
Cancer Immunology and Immunotherapy 04/2009; 58(10):1701-13. · 3.70 Impact Factor
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ABSTRACT: Numerous techniques are available for investigating protein-ligand interactions. The phage display technique is one such method routinely used to identify antibody-antigen interactions and has the benefit of being easily adaptable to high-throughput screening platforms. Once identified, antigen-binding domains on fragment antibodies or single-chain fragment antibodies (scFv) can be expressed and purified for further studies. In this chapter, we describe a method for high-level expression of a phage display-derived scFv in Pichia pastoris. The phage display-derived antibody A33scFv recognizes a cell surface glycoprotein (designated A33) expressed in colon cancer that serves as a target antigen for radioimmunoimaging and/or immunotherapy of human colon cancer. The expression and purification of A33scFv was optimized for the methylotrophic yeast P. pastoris. P. pastoris with a Mut(S) phenotype was selected to express A33scFv under regulation of the methanol-inducible AOX1 promoter. Here we describe a large-scale fed-batch fermentation process with an efficient online closed-loop methanol control for the production of the recombinant protein. Purification of A33scFv from clarified culture medium was done using a two-step chromatographic procedure using anion exchange and hydrophobic interaction chromatography, resulting in a final product with more than 90% purity. This chapter provides protocols that can be used as a base for process development of recombinant protein expression in P. pastoris and purification of these proteins for use in further functionality studies and in diagnostic and therapeutic applications.
Methods in molecular biology (Clifton, N.J.) 02/2009; 562:225-36.
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ABSTRACT: Urothelial carcinoma of the bladder is a solid tumor entity for which the immunomodulatory agent Bacillus-Calmette Guerin (BCG) has been shown to have substantial efficacy with approximately 90% cure rates in select patients with superficial disease. Immune-based therapies for patients with more invasive disease do not currently exist. We previously showed that invasive urothelial carcinomas express the NY-ESO-1 tumor antigen. Here we evaluated the safety and immunogenicity of a recombinant NY-ESO-1 protein vaccine, which was administered with granulocyte macrophage colony-stimulating factor and BCG as immunologic adjuvants in a cohort of urothelial carcinoma patients. Sixty-two urothelial carcinoma patients were screened for enrollment onto the vaccine clinical trial and 6 patients met all eligibility criteria to receive vaccination. Patients with localized disease underwent surgical removal of their bladders as treatment for their disease. Tumor tissues were tested for NY-ESO-1 expression and eligible patients, shown to have NY-ESO-1 tumors, were vaccinated in the postoperative setting. Peripheral blood samples were analyzed for vaccine-induced antibody and T-cell responses. The vaccine regimen was well tolerated with only mild injection site reactions. NY-ESO-1-specific antibody responses were induced in 5/6 patients whereas CD8 T-cell responses occurred in 1/6 patients and CD4 T-cell responses were found in 6/6 patients. This study demonstrates safety and feasibility of the NY-ESO-1 recombinant protein in combination with BCG and granulocyte macrophage colony-stimulating factor to induce predominantly antibody and CD4 T-cell responses in urothelial carcinoma patients. Induction of higher frequency of CD8 T-cell responses may be possible in clinical trials implementing NY-ESO-1 vaccination in combination with other immunomodulatory agents.
Journal of immunotherapy (Hagerstown, Md.: 1997) 11/2008; 31(9):849-57. · 3.20 Impact Factor
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Ramziya Kiyamova,
Vitalina Gryshkova,
Galina Ovcharenko,
Dmytro Lituyev,
Sergyy Malyuchik,
Vasiliy Usenko,
Yuliya Khozhayenko,
Vadym Gurtovyy,
Beatrice Yin,
Gerd Ritter, Lloyd Old,
Valeriy Filonenko,
Ivan Gout
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ABSTRACT: Homeostasis of inorganic phosphate in the human body is maintained by regulated absorption, metabolism, and excretion. Sodium-dependent phosphate transporters (NaPi) mediate the transport of inorganic phosphate (P(i)) in cells in response to dietary phosphate consumption, hormones, and growth factors. NaPi2b is a member of the sodium-dependent phosphate transporter family, with a distinct pattern of expression and regulation. Signaling pathways activated by mitogens, glucocorticoids, and metabolic factors have been implicated in regulating P(i) transport via NaPi2b. Inactivation of NaPi2b function by mutations has been linked to human pathologies, such as pulmonary alveolar microlithiasis. In this study, we describe the generation and characterization of monoclonal antibodies against human NaPi2b. The monoclonal antibodies were shown to recognize specifically transiently overexpressed and endogenous NaPi2b in commonly used immunoassays, including Western blotting, immunoprecipitation, and immunohistochemistry. These properties make them particularly valuable reagents for elucidating NaPi2b function in health and disease.
Hybridoma (2005) 09/2008; 27(4):277-84. · 0.42 Impact Factor
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ABSTRACT: Saccharomyces cerevisiae stimulates dendritic cells (DCs) and represents a promising candidate for cancer vaccine development. Effective cross-presentation of antigen delivered to DCs is necessary for successful induction of cellular immunity. Here, we present a yeast-based vaccine approach that is independent of yeast's ability to express the chosen antigen, which is instead produced separately and conjugated to the yeast cell wall. The conjugation method is site-specific (based on the SNAP-tag) and designed to facilitate antigen release in the DC phagosome and subsequent translocation for cross-presentation. We demonstrate that nonsite-specific chemical conjugation of the same protein hinders cross-presentation. Phagosomal antigen release was further expedited through the insertion of the invariant chain ectodomain as a linker, which is rapidly cleaved by Cathepsin S. The dose of delivered antigen was increased in several ways: by using yeast strains with higher surface amine densities, by using yeast hulls (cell wall fragments) instead of whole cells, and by conjugating multiple layers of antigen. The novel multilayer conjugation scheme takes advantage of Sfp phosphopantetheinyl transferase and remains site-specific; it enables the antigen dose to grow linearly with the number of layers. We show that whole yeast cells coated with 1 layer of the cancer-testis antigen NY-ESO-1 and yeast hulls bearing 3 layers were able to cross-prime naive CD8 T cells in vitro, with the latter resulting in higher frequencies of antigen-specific cells after 10 days. This cross-presentation-efficient antigen conjugation scheme is not limited to yeast and can readily be applied toward the development of other particulate vaccines.
Journal of immunotherapy (Hagerstown, Md.: 1997) 08/2008; 31(7):607-19. · 3.20 Impact Factor
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Sylvia Adams,
David W O'Neill,
Daisuke Nonaka,
Elizabeth Hardin,
Luis Chiriboga,
Kimberly Siu,
Crystal M Cruz,
Angelica Angiulli,
Francesca Angiulli,
Erika Ritter, [......],
Yongzhao Shao,
Olivier Manches,
Linda Pan,
Ralph R Venhaus,
Eric W Hoffman,
Achim Jungbluth,
Sacha Gnjatic, Lloyd Old,
Anna C Pavlick,
Nina Bhardwaj
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ABSTRACT: T cell-mediated immunity to microbes and to cancer can be enhanced by the activation of dendritic cells (DCs) via TLRs. In this study, we evaluated the safety and feasibility of topical imiquimod, a TLR7 agonist, in a series of vaccinations against the cancer/testis Ag NY-ESO-1 in patients with malignant melanoma. Recombinant, full-length NY-ESO-1 protein was administered intradermally into imiquimod preconditioned sites followed by additional topical applications of imiquimod. The regimen was very well tolerated with only mild and transient local reactions and constitutional symptoms. Secondarily, we examined the systemic immune response induced by the imiquimod/NY-ESO-1 combination, and show that it elicited both humoral and cellular responses in a significant fraction of patients. Skin biopsies were assessed for imiquimod's in situ immunomodulatory effects. Compared with untreated skin, topical imiquimod induced dermal mononuclear cell infiltrates in all patients composed primarily of T cells, monocytes, macrophages, myeloid DCs, NK cells, and, to a lesser extent, plasmacytoid DCs. DC activation was evident. This study demonstrates the feasibility and excellent safety profile of a topically applied TLR7 agonist used as a vaccine adjuvant in cancer patients. Imiquimod's adjuvant effects require further evaluation and likely need optimization of parameters such as formulation, dose, and timing relative to Ag exposure for maximal immunogenicity.
The Journal of Immunology 08/2008; 181(1):776-84. · 5.79 Impact Factor
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Kazuhide Tsuji,
Toshitada Hamada,
Akiko Uenaka,
Hisashi Wada,
Eiichi Sato,
Midori Isobe,
Kenji Asagoe,
Osamu Yamasaki,
Hiroshi Shiku,
Gerd Ritter,
Roger Murphy,
Eric W Hoffman, Lloyd J Old,
Eiichi Nakayama,
Keiji Iwatsuki
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ABSTRACT: NY-ESO-1 is a cancer/testis antigen highly immunogenic in cancer patients. Cholesterol-bearing hydrophobized pullulan (CHP) is a nanoparticle-forming antigen-delivery vehicle and CHP complexed with NY-ESO-1 protein (CHP-NY-ESO-1) efficiently activates CD4 and CD8 T cells in vitro.
In this study we report on a 50-year-old male melanoma patient with multiple skin and organ metastases (T4N3M1c) who was vaccinated with CHP-NY-ESO-1 at biweekly intervals and who had an unusual disease course. We characterized in this patient humoral and cellular immune responses, immune regulatory cells, and cytokine profiles in the peripheral blood and at local tumor sites.
Ten days after the second CHP-NY-ESO-1 vaccination (day 25), blisters appeared on the skin at the metastatic lesions associated with inflammatory changes. A skin biopsy showed the presence of many NY-ESO-1-expressing apoptotic melanoma cells as determined by a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) test. However, the tumors continued to grow, and the patient died of pulmonary failure due to multiple metastases on day 48. Serum antibody responses were detected after the second CHP-NY-ESO-1 vaccination and antibody titer increased with subsequent vaccinations. Th1 dependent IgG1 was the predominant immunoglobulin subtype. Both, NY-ESO-1-specific CD4 and CD8 T cell responses were detected in PBMC by IFN-gamma secretion assays. After CHP-NY-ESO-1 vaccination a slight decrease in CD4(+)CD25(+)Foxp3(+) Tregs was observed in PBMC but significantly increased numbers of CD4(+)CD25(+)Foxp3(+) Tregs and CD68(+) immunoregulatory macrophages were detected at the local tumor sites. CD4(+)CD25(+)Foxp3(+) Tregs were also increased in the blister fluid. Cytokines in the serum suggested a polarization towards a Th1 pattern in the PBMC and those in the blister fluid suggested a Th2-type response at the tumor site.
Our observations indicate induction of specific humoral and cellular immune responses against NY-ESO-1 after CHP-NY-ESO-1 vaccination in a melanoma patient. The concomitant appearance of regulatory T cells and of immune regulatory macrophages and cytokines at the local tumor sites in this patient may explain immune escape.
Cancer Immunology and Immunotherapy 04/2008; 57(10):1429-37. · 3.70 Impact Factor
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ABSTRACT: Assessing cancer margins, lymph nodes, and small cancer deposits intraoperatively can be challenging. A new device has become available that allows the detection of positron emission tomography (PET) radiotracers through both high-energy gamma and short-range beta emissions. These PET probes are handheld, allowing for real-time evaluation of cancer using a tool that provides surgeons with better intraoperative assessment of tumor sites.
Within the context of two institutional review board (IRB)-approved protocols investigating new applications of antibody-labeled PET scanning, (124)I-labeled humanized monoclonal antibodies specific for colorectal cancer (huA33) and renal tumors (cG250) were constructed. Patients underwent preoperative PET scans, approximately seven days post-tracer infusion, when tumor-to-nontumor ratios were high. Suspected tumor deposits were evaluated intraoperatively with handheld beta and gamma PET probes.
Handheld PET probes detected emissions from all tumors. Count rates from the gamma probe on tumor ranged from 48 to 306 cps, and for the beta probe ranged from 18 to 190 cps. Gamma and beta emissions exhibited a strong positive correlation. The ratio of gamma and beta counts was at least twice that of the background counts for all tumors evaluated.
This study is the first to demonstrate the utility of beta probes for the intraoperative detection of radiolabeled antibodies targeting cancer. Importantly, the recorded beta count rates from the beta probe correlate with the count rates from the high-energy gamma probe. Furthermore, the beta probe may offer superior specificity for real-time localization of small tumor deposits, compared to gamma probes. The intraoperative portable PET probe may prove a valuable bridge to combining tumor biology and PET technology to guide surgical therapy.
Surgical Endoscopy 03/2008; 22(2):386-91. · 4.01 Impact Factor
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ABSTRACT: We have been studying the native autoimmune response to cancer through the isolation of human monoclonal antibodies that are cancer specific from cancer patients. To facilitate this work we previously developed a fusion partner cell line for human lymphocytes, MFP-2, that fuses efficiently with both human lymph node lymphocytes and peripheral blood lymphocytes. Using this unique trioma fusion partner cell line we isolated a panel of autologous human monoclonal antibodies, from both peripheral blood and lymph node lymphocytes, which are representative of the native repertoire of anti-cancer specific antibodies from breast cancer patients.
The current study employs immunocytochemistry, immunohistochemistry, Western blot analysis as well as Northern blots, Scatchard binding studies and finally SEREX analysis for target antigen identification.
By application of an expression cloning technique known as SEREX, we determined that the target antigen for two monoclonal antibodies, 27.B1 and 27.F7, derived from lymph node B-cells of a breast cancer patient, is the PDZ domain-containing protein known as GIPC1. This protein is highly expressed not only in cultured human breast cancer cells, but also in primary and metastatic tumor tissues and its overexpression appears to be cancer cell specific. Confocal microscopy revealed cell membrane and cytoplasmic localization of the target protein, which is consistent with previous studies of this protein.
We have determined that GIPC1 is a novel breast cancer-associated immunogenic antigen that is overexpressed in breast cancer. Its role, however, in the initiation and/or progression of breast cancer remains unclear and needs further clarification.
BMC Cancer 02/2008; 8:248. · 3.01 Impact Factor
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Lee M Krug,
Daniel T Milton,
Achim A Jungbluth,
Lin-Chi Chen,
Emilio Quaia,
Neeta Pandit-Taskar,
Andrew Nagel,
Jessica Jones,
Mark G Kris,
Ronald Finn,
Peter Smith-Jones,
Andrew M Scott, Lloyd Old,
Chaitanya Divgi
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ABSTRACT: Lewis Y (Le(y)) is a blood group antigen with robust expression on the surface of epithelial tumors, including small cell lung cancer (SCLC), making it a potential target for antibody-based immunotherapy. 3S193, an immunoglobulin G3 monoclonal antibody, has demonstrated superior specificity, affinity, and cytotoxicity over other anti-Le(y) antibodies. A phase I trial of humanized 3S193 (hu3S193) with dosing up to 40 mg/m2 demonstrated tumor targeting without serious toxicities or the development of human anti-human antibodies.
We tested the targeting and pharmacokinetics of hu3S193 in patients with SCLC. Eligibility required progressive SCLC treated with up to three previous chemotherapy regimens, measurable disease not previously irradiated, and tumor samples positive for 3S193 by immunohistochemistry. Patients received four weekly injections of hu3S193, five patients at 10 mg/m2 and five patients at 20 mg/m2. The first and fourth injections were radiolabeled with indium-111 for gamma camera imaging.
Of 40 patients screened, 25 of 34 (74%) assessable SCLC tumor samples were 3S193 positive by immunohistochemistry. Ten patients were treated with hu3S193; nine completed all four injections. All fluorodeoxyglucose (FDG)-avid lesions >2 cm were visualized on antibody single-photon emission computed tomography. Some lesions overlying vascular structures could not be visualized. No difference was noted in imaging or pharmacokinetics between the first and fourth injections. Toxicities included grade 2 urticaria (n = 1), grade 1 vomiting (n = 2), and grade 2 hypertension (n = 1) transiently after infusion at the higher dose.
Given the strong tumor targeting, particularly at the higher dose, the favorable toxicity profile, and the potential for immunomodulatory effects, hu3S193 warrants further investigation in SCLC.
Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer 11/2007; 2(10):947-52. · 4.55 Impact Factor
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ABSTRACT: NY-ESO-1 is a highly immunogenic tumor antigen and a promising vaccine candidate in cancer immunotherapy. Access to purified protein both for vaccine formulations and for monitoring antigen-specific immune responses is vital to vaccine development. Currently available recombinant Escherichia coli-derived NY-ESO-1 is isolated from inclusion bodies as a complex protein mixture and efforts to improve the purity of this antigen are required, especially for later-stage clinical trials. Using yeast cell surface display and fluorescence activated cell sorting techniques, we have engineered an NY-ESO-1 variant (NY-ESO-L5; C(75)A C(76)A C(78)A L(153)H) with a 100x improved display level on yeast compared to the wild-type protein. This mutant can be effectively produced as an Aga2p-fusion and purified in soluble form directly from the yeast cell wall. In the process, we have identified the epitope recognized by anti-NY-ESO-1 mAb E978 (79-87, GARGPESRL). The availability of an alternative expression host for this important antigen will help avoid artifactual false positive tests of patient immune response due to reaction against expression-host-specific contaminants.
Protein Expression and Purification 09/2006; 48(2):232-42. · 1.59 Impact Factor
