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Testing of T cell–based cancer therapeutics often involves measuring cancer antigen–specific T-cell populations with the assumption that they arise from in vivo clonal expansion. This analysis, using peptide/MHC tetramers, is often ambiguous. From a leukemia cell line, we identified a CDK4-derived peptide epitope, UNC-CDK4-1 (ALTPVVVTL), that bound HLA-A*02:01 with high affinity and could induce CD8+ T-cell responses in vitro. We identified UNC-CDK4-1/HLA-A*02:01 tetramer+ populations in 3 of 6 patients with acute myeloid leukemia who had undergone allogeneic stem cell transplantation. Using tetramer-based, single-cell sorting and T-cell receptor β (TCRβ) sequencing, we identified recurrent UNC-CDK4-1 tetramer–associated TCRβ clonotypes in a patient with a UNC-CDK4-1 tetramer+ population, suggesting in vivo T-cell expansion to UNC-CDK4-1. In parallel, we measured the patient's TCRβ repertoire and found it to be highly restricted/oligoclonal. The UNC-CDK4-1 tetramer–associated TCRβ clonotypes represented >17% of the entire TCRβ repertoire—far in excess of the UNC-CDK4-1 tetramer+ frequency—indicating that the recurrent TCRβ clonotypes identified from UNC-CDK-4-1 tetramer+ cells were likely a consequence of the extremely constrained T-cell repertoire in the patient and not in vivo UNC-CDK4-1–driven clonal T-cell expansion. Mapping recurrent TCRβ clonotype sequences onto TCRβ repertoires can help confirm or refute antigen-specific T-cell expansion in vivo. Cancer Immunol Res; 3(3); 228–35. ©2015 AACR.

The document contains 4 supplemental tables: 1. RT-PCR Primers for TCRbeta sequencing 2. HPLC-MS identified peptide sequences 3. The amino acid sequences of recurrent TCRbeta clonotypes 4. The distribution of the CDR3 amino acid length in the analyzed patient. There are 2 supplementary figures: 1. Tetramer flow cytometry showing the generation of an oligoclonal T-cell population with a UNC-CDK4-1 high affinity T-cell population. 2. More detailed information regarding the tetramer gating strategy employed.

PDF file - 706k, Figure S1. CG is ubiquitinated in AML. Figure S2. CG1 is a naturally processed epitope that is eluted from leukemia cell surface. Figure S3. CG expression by each of the AML samples used in the cytotoxicity assays. Figure S4. Gating strategy used to determine CG1-CTL frequency in AML patient blood. Supplementary Table S1. Characteristics of cathepsin G-derived peptides with highest binding affinity to HLA-A*0201 Supplementary Table S2. AML samples used in determining cathepsin G expression

Purpose: Immunotherapy targeting aberrantly expressed leukemia-associated antigens has shown promise in the management of acute myeloid leukemia (AML). However, because of the heterogeneity and clonal evolution that is a feature of myeloid leukemia, targeting single peptide epitopes has had limited success, highlighting the need for novel antigen discovery. In this study, we characterize the role of the myeloid azurophil granule protease cathepsin G (CG) as a novel target for AML immunotherapy.Experimental Design: We used Immune Epitope Database and in vitro binding assays to identify immunogenic epitopes derived from CG. Flow cytometry, immunoblotting, and confocal microscopy were used to characterize the expression and processing of CG in AML patient samples, leukemia stem cells, and normal neutrophils. Cytotoxicity assays determined the susceptibility of AML to CG-specific cytotoxic T lymphocytes (CTL). Dextramer staining and cytokine flow cytometry were conducted to characterize the immune response to CG in patients.Results: CG was highly expressed and ubiquitinated in AML blasts, and was localized outside granules in compartments that facilitate antigen presentation. We identified five HLA-A*0201 binding nonameric peptides (CG1-CG5) derived from CG, and showed immunogenicity of the highest HLA-A*0201 binding peptide, CG1. We showed killing of primary AML by CG1-CTL, but not normal bone marrow. Blocking HLA-A*0201 abrogated CG1-CTL–mediated cytotoxicity, further confirming HLA-A*0201-dependent killing. Finally, we showed functional CG1-CTLs in peripheral blood from AML patients following allogeneic stem cell transplantation.Conclusion: CG is aberrantly expressed and processed in AML and is a novel immunotherapeutic target that warrants further development. Clin Cancer Res; 19(1); 247–57. ©2012 AACR.

Oxidation of RNA can be effected by two different techniques: a photochemical, electron-transfer method termed "flash-quench" and direct oxidation by metal oxo complexes. The flash-quench method produces selective oxidation using a metal photosensitizer, tris(bipyridyl)ruthenium(III) trichloride (Ru(bpy)(3)(3+)), and quencher, pentaamminechlorocobalt(III) chloride (Co(NH(3))(5)Cl(2+)). We have optimized the flash-quench technique for the following RNAs: tRNA(Phe), human ferritin iron-responsive element (IRE), and a mutated human ferritin IRE. We have also employed a chemical footprinting technique involving the oxoruthenium(IV) complex (Ru(tpy)(bpy)O(2+) (tpy = 2,2',2''-terpyridine; bpy = 2,2'-bipyridine)) to oxidize guanine. Comparison of the two methods shows that the flash-quench technique provides a visualization of nucleotide accessibility for a static conformation of RNA while the Ru(tpy)(bpy)O(2+) complex selectively oxidizes labile guanines and gives a visualization of a composite of multiple conformations of the RNA structure.

The interaction of particles with cells is known to be strongly influenced by particle size, but little is known about the interdependent role that size, shape, and surface chemistry have on cellular internalization and intracellular trafficking. We report on the internalization of specially designed, monodisperse hydrogel particles into HeLa cells as a function of size, shape, and surface charge. We employ a top-down particle fabrication technique called PRINT that is able to generate uniform populations of organic micro- and nanoparticles with complete control of size, shape, and surface chemistry. Evidence of particle internalization was obtained by using conventional biological techniques and transmission electron microscopy. These findings suggest that HeLa cells readily internalize nonspherical particles with dimensions as large as 3 μm by using several different mechanisms of endocytosis. Moreover, it was found that rod-like particles enjoy an appreciable advantage when it comes to internalization rates, reminiscent of the advantage that many rod-like bacteria have for internalization in nonphagocytic cells.

Testing of T cell–based cancer therapeutics often involves measuring cancer antigen–specific T-cell populations with the assumption that they arise from in vivo clonal expansion. This analysis, using peptide/MHC tetramers, is often ambiguous. From a leukemia cell line, we identified a CDK4-derived peptide epitope, UNC-CDK4-1 (ALTPVVVTL), that bound HLA-A*02:01 with high affinity and could induce CD8+ T-cell responses in vitro. We identified UNC-CDK4-1/HLA-A*02:01 tetramer+ populations in 3 of 6 patients with acute myeloid leukemia who had undergone allogeneic stem cell transplantation. Using tetramer-based, single-cell sorting and T-cell receptor β (TCRβ) sequencing, we identified recurrent UNC-CDK4-1 tetramer–associated TCRβ clonotypes in a patient with a UNC-CDK4-1 tetramer+ population, suggesting in vivo T-cell expansion to UNC-CDK4-1. In parallel, we measured the patient's TCRβ repertoire and found it to be highly restricted/oligoclonal. The UNC-CDK4-1 tetramer–associated TCRβ clonotypes represented >17% of the entire TCRβ repertoire—far in excess of the UNC-CDK4-1 tetramer+ frequency—indicating that the recurrent TCRβ clonotypes identified from UNC-CDK-4-1 tetramer+ cells were likely a consequence of the extremely constrained T-cell repertoire in the patient and not in vivo UNC-CDK4-1–driven clonal T-cell expansion. Mapping recurrent TCRβ clonotype sequences onto TCRβ repertoires can help confirm or refute antigen-specific T-cell expansion in vivo. Cancer Immunol Res; 3(3); 228–35. ©2015 AACR.

The binding of small molecules to distinctive three-dimensional structures in mRNA provides a new dimension in RNA control, previously limited to the targeting of secondary structures with antisense and RNA interference; such targeting can modulate mRNA function and rates of protein biosynthesis. Small molecules that selectively bind the iron-responsive element (IRE), a specific three-dimensional structure in the noncoding region of the ferritin mRNA model that is recognized by the iron-regulatory protein repressor, were identified by using chemical footprinting. The assay used involved an oxoruthenium(IV) complex that oxidizes guanine bases in RNA sequences. Small molecules that blocked oxidation of guanines in the internal loop region were expected to selectively increase the rate of ferritin synthesis, because the internal loop region of the ferritin IRE is distinctive from those of other IREs. The natural product yohimbine was found (based on gel mobility shifts) to block cleavage of the internal loop RNA site by >50% and seemed to inhibit protein binding. In the presence of yohimbine, the rate of biosynthesis of ferritin in a cell-free expression system (rabbit reticulocyte lysate) increased by 40%. Assignment of the IRE–yohimbine interaction as the origin of this effect was supported by a similar increase in synthesis of luciferase protein in a chimera of the IRE and luciferase gene. The identification of a small, drug-like molecule that recognizes a naturally occurring three-dimensional mRNA structure and regulates protein biosynthesis rates raises the possibility that small molecules can regulate protein biosynthesis by selectively binding to mRNA.

The electrochemical detection of nucleic acid targets at low concentrations has a number of applications in diagnostics and pharmaceutical research. Self-assembled monolayers of alkanethiol-derivatized oligonucleotides on gold electrodes provide a useful platform for such detectors, and the electrocatalytic oxidation of nucleobases included in the DNA targets is a particularly sensitive method of electrochemical detection. A strategy has been developed for combining these two aspects by substituting either 7,8-dihydro-8-oxoguanine (8G) or 5-aminouridine (5U) into DNA targets. Upon hybridization of targets containing these modified nucleobases, electrocatalytic signals at probe-modified gold electrodes are observed in the presence of Os(bpy)32+, which oxidizes both 8G and 5U upon oxidation to the Os(III) state. Self-assembled monolayers were prepared on both macro (1.6 mm) and micro (25 μm) gold electrodes using published procedures involving C6-terminated alkanethiol oligonucleotides and mercaptohexanol a...

Chromosomally mediated penicillin resistance in Neisseria gonorrhoeae occurs in part through alterations in penicillin-binding proteins (PBPs) and a decrease in outer membrane permeability. However, the genetic and molecular mechanisms of transformation of a penicillin-susceptible strain of N. gonorrhoeae to high-level penicillin resistance have not been clearly elucidated. Previous studies suggested that alterations in PBP 1 were involved in high-level penicillin resistance. In this study, we identified a single amino acid mutation in PBP 1 located 40 amino acids N terminal to the active-site serine residue that was present in all chromosomally mediated resistant N. gonorrhoeae (CMRNG) strains for which MICs of penicillin were ≥1 μg/ml. PBP 1 harboring this point mutation (PBP 1*) had a three- to fourfold lower rate of acylation ( k 2 / K' ) than wild-type PBP 1 with a variety of β-lactam antibiotics. Consistent with its involvement in high-level penicillin resistance, replacement of the altered ponA gene ( ponA1 ) in several CMRNG strains with the wild-type ponA gene resulted in a twofold decrease in the MICs of penicillin. Surprisingly, transformation of an intermediate-level penicillin-resistant strain (PR100; FA19 penA4 mtr penB5 ) with the ponA1 gene did not increase the MIC of penicillin for this strain. However, we identified an additional resistance locus, termed penC , which was required along with ponA1 to increase penicillin resistance of PR100 to a high level (MIC = 4 μg/ml). The penC locus by itself, when present in PR100, increases the MICs of penicillin and tetracycline twofold each. These data indicate that an additional locus, penC , is required along with ponA1 to achieve high-level penicillin resistance.

The 7-deaza analogues of guanine and adenine were incorporated into polymerase chain reaction (PCR) products by substitution of the appropriate nucleotide triphosphates into the reaction. These PCR products can be immobilized on ITO electrodes and detected by catalytic cyclic voltammetry with ruthenium polypyridyl complexes. Immobilization on indium tin oxide (ITO) electrodes of 330- and 1200-base pair (bp) PCR amplicons from the E. coli dacA gene containing one or both of the 7-deazapurines was effected by precipitation from a 9:1 DMF/acetate solution. Amplicons containing the 7-deazaguanine base were detected by observing current enhancement in the cyclic voltammogram of Ru(dmb)3(3)+/2+ (dmb = 4,4'-dimethyl-2,2'-bipyridine) due to the selective oxidation of the modified base by this mediator. Oxidation of incorporated 7-deazaadenine bases in addition to native guanines gives rise to a higher current enhancement in the cyclic voltammogram of Ru(bpy)3(3)+/2+ (bpy = 2,2'-bipyridine) compared to the enhancement observed in the presence of guanine only. This strategy was employed to simultaneously detect the 330-bp sequence containing 7-deazaadenine and the 1200-bp sequence containing 7-deazaguanine on the same ITO electrode. Such a strategy may provide a means for detecting multiple genes on a single microlocation and may thereby lead to more highly multiplexed gene assays.

The oxidizability of substituted nucleobases was evaluated through theoretical calculations and the ability of individual bases to induce current enhancement in the cyclic voltammograms of metal complexes. Formation of the guanine derivatives 7-deazaguanine and 8-oxoguanine is known to lower the energy for oxidation of guanine. The similar derivatives of adenine were examined and gave lower predicted redox energies as well as current enhancement with Ru(bpy)32+ (7-deazaadenine) and Fe(bpy)32+ (8-oxoadenine). Oxidizable, substituted pyrimidines were identified using a computational library that gave 5-aminocytosine and 5-aminouracil as promising electron donors. Again, these predictions were verified using catalytic electrochemistry. In addition, the computations predicted that 6-aminocytosine would be redox-active but not as easily oxidized as 5-aminocytosine, which was also confirmed experimentally. In addition to calculating the relative one-electron redox potentials, we used calculations to evaluate th...

The ponA gene encoding penicillin-binding protein 1 (PBP 1) from Neisseria gonorrhoeae was cloned by a reverse genetic approach. PBP 1 was purified from solubilized membranes of penicillin-susceptible strain FA19 by covalent ampicillin affinity chromatography and used to obtain an NH2-terminal amino acid sequence. A degenerate oligonucleotide based on this protein sequence and a highly degenerate oligonucleotide based on a conserved amino acid motif found in all class A high-molecular-mass PBPs were used to isolate the PBP 1 gene (ponA). The ponA gene encodes a protein containing all of the conserved sequence motifs found in class A PBPs, and expression of the gene in Escherichia coli resulted in the appearance of a new PBP that comigrated with PBP 1 purified from N. gonorrhoeae. A comparison of the gonococcal ponA gene to its homolog isolated from Neisseria meningitidis revealed a high degree of identity between the two gene products, with the greatest variability found at the carboxy terminus of the two deduced PBP 1 protein sequences.

Purpose: Immunotherapy targeting aberrantly expressed leukemia-associated antigens has shown promise in the management of acute myeloid leukemia (AML). However, because of the heterogeneity and clonal evolution that is a feature of myeloid leukemia, targeting single peptide epitopes has had limited success, highlighting the need for novel antigen discovery. In this study, we characterize the role of the myeloid azurophil granule protease cathepsin G (CG) as a novel target for AML immunotherapy. Experimental Design: We used Immune Epitope Database and in vitro binding assays to identify immunogenic epitopes derived from CG. Flow cytometry, immunoblotting, and confocal microscopy were used to characterize the expression and processing of CG in AML patient samples, leukemia stem cells, and normal neutrophils. Cytotoxicity assays determined the susceptibility of AML to CG-specific cytotoxic T lymphocytes (CTL). Dextramer staining and cytokine flow cytometry were conducted to characterize the immune response to CG in patients. Results: CG was highly expressed and ubiquitinated in AML blasts, and was localized outside granules in compartments that facilitate antigen presentation. We identified five HLA-A*0201 binding nonameric peptides (CG1-CG5) derived from CG, and showed immunogenicity of the highest HLA-A*0201 binding peptide, CG1. We showed killing of primary AML by CG1-CTL, but not normal bone marrow. Blocking HLA-A*0201 abrogated CG1-CTL–mediated cytotoxicity, further confirming HLA-A*0201-dependent killing. Finally, we showed functional CG1-CTLs in peripheral blood from AML patients following allogeneic stem cell transplantation. Conclusion: CG is aberrantly expressed and processed in AML and is a novel immunotherapeutic target that warrants further development. Clin Cancer Res; 19(1); 247–57. ©2012 AACR.

Uridine kinase is the rate-limiting enzyme in the pyrimidine salvage pathway of all mammalian cells. A cDNA for uridine kinase from mouse brain has been isolated, sequenced, and characterized. This is the first report of a complete nucleotide sequence for mammalian uridine kinase. The isolated cDNA is only 95% complete, missing the first 17 codons. The correct 5′-terminus sequence was obtained from high-stringency screening of a mouse liver genomic DNA library. The translated cDNA sequence encodes a protein of 277 amino acids (Mr31,068). A truncated form of the cDNA was expressed inEscherichia coli.The expressed protein displayed uridine kinase activity and readily formed a tetramer, the most active form of the wild-type enzyme. Analysis of the amino acid sequence identified the three ATP-binding site consensus motifs. The predicted secondary structure for uridine kinase and the sequence comparison with three kinases having known crystal structures are consistent with uridine kinase having an α/β core structure of the nucleotide-binding fold found in many kinases. We have also isolated and cloned a nonfunctional, processed pseudogene from mouse genomic DNA. This pseudogene sequence is 94% identical with the coding DNA.

Purine nucleoside phosphorylase (EC 2.4.2.1) from bovine spleen is allosterically regulated. With the substrate inosine the enzyme displayed complex kinetics: positive cooperativity vs inosine when this substrate was close to physiological concentrations, negative cooperativity at inosine concentrations greater than 60 microM, and substrate inhibition at inosine greater than 1 mM. No cooperativity was observed with the alternative substrate, guanosine. The activity of purine nucleoside phosphorylase toward the substrate inosine was sensitive to the presence of reducing thiols; oxidation caused a loss of cooperativity toward inosine, as well as a 10-fold decreased affinity for inosine. The enzyme also displayed negative cooperativity toward phosphate at physiological concentrations of Pi, but oxidation had no effect on either the affinity or cooperativity toward phosphate. The importance of reduced cysteines on the enzyme is thus specific for binding of the nucleoside substrate. The enzyme was modestly inhibited by the pyrimidine nucleotides CTP (Ki = 118 microM) and UTP (Ki = 164 microM), but showed greater sensitivity to 5-phosphoribosyl-1-pyrophosphate (Ki = 5.2 microM).

To investigate the cellular internalization pathways of shape- and size-specific particles as a function of zeta potential in different cell types.A top-down particle fabrication technique called PRINT was utilized to fabricate monodisperse 1 microm cylindrical particles. Cellular internalization of these PRINT particles was monitored using confocal microscopy, flow cytometry, and transmission electron microscopy. The endocytic pathway used by 1 microm cationic PRINT particles was evaluated using different inhibitory strategies. Cytotoxicity assays were used to determine the toxicity of both cationic and anionic PRINT particles in multiple cell types.Particle internalization was confirmed using confocal microscopy, flow cytometry and transmission electron microscopy. The mechanism of internalization of positively charged PRINT particles was found to be predominantly clathrin-mediated endocytosis and macropinocytosis with very few particles utilizing a caveolae-mediated endocytic pathway. The exposed charge on the surface of the particles had a significant effect on the rate of endocytosis in all cell types tested, except for the macrophage cells. No significant cytotoxicity was observed for all PRINT particles used in the present study.Cylindrical 1 microm PRINT particles were readily internalized into HeLa, NIH 3T3, OVCAR-3, MCF-7, and RAW 264.7 cells. Particles with a positive zeta potential exhibited an enhanced rate of endocytosis compared to negatively charged particles with identical sizes and shapes. It was found that PRINT particles with a positive zeta potential were endocytosed into HeLa cells using predominantely clathrin-mediated and macropinocytotic pathways.

The presence of the Zn2+ ion dramatically enhances the inhibition of trypsin and tryptase by amidine-modified benzimidazole inhibitors via coordination to both the catalytically active Ser195 hydroxyl and His57 imidazole residues of the enzyme and the nitrogens of the amidine-modified benzimidazole inhibitor (Janc, J. W.; Clark, J. M.; Warne, R. L.; Elrod, K. C.; Katz, B. A.; Moore, W. R. Biochemistry 2000, 39, 4792-4800). Some new 5-amidino-2-substituted benzimidazoles were synthesized and compared to known related molecules to explore systematically the metal-mediated inhibition of bovine trypsin as a function of coordinating groups and metal ions. These compounds take advantage of the favorable interaction between the amidine group on one side of the inhibitor and the Asp189 carboxylate in the binding pocket of the enzyme. The 5-amidino-2-substituted benzimidazoles all demonstrated similar inhibition constants (Ki) of 20-50 microM in the absence of metal ions. In the presence of Zn2+, inhibition increased to varying extents, depending upon the group substituted at the 2 position of the benzimidazole. The largest increase in inhibition in the presence of Zn2+ was seen with (5-amidino-2-benzimidazolyl)-2-benzimidazolylmethane with an apparent inhibition constant (Ki') of 0.37 +/- 0.06 nM, giving a 59,000-fold increase in inhibition when Zn2+ is present. Other metal ions, including Mn2+, Sc3+, and Hg2+, also increased the inhibition by several of the benzimidazole derivatives synthesized. The compound bis(2-benzimidazolyl)methane (BBIM) was also examined because it lacks the amidine group that provides a favorable hydrogen-bonding interaction with Asp189 in the binding pocket of trypsin. In the absence of metal ions, BBIM did not have a detectable affinity for trypsin; however, in the presence of Zn2+, a Ki' of 127 +/- 3 nM was observed. This result demonstrates that an affinity for the enzyme in the absence of metal ions is not required for potent metal-mediated inhibition, greatly expanding the possibilities for metal mediation of nonmetalloenzymes.

The electrochemical detection of nucleic acid targets at low concentrations has a number of applications in diagnostics and pharmaceutical research. Self-assembled monolayers of alkanethiol-derivatized oligonucleotides on gold electrodes provide a useful platform for such detectors, and the electrocatalytic oxidation of nucleobases included in the DNA targets is a particularly sensitive method of electrochemical detection. A strategy has been developed for combining these two aspects by substituting either 7,8-dihydro-8-oxoguanine (8G) or 5-aminouridine (5U) into DNA targets. Upon hybridization of targets containing these modified nucleobases, electrocatalytic signals at probe-modified gold electrodes are observed in the presence of Os(bpy)(3)(2+), which oxidizes both 8G and 5U upon oxidation to the Os(III) state. Self-assembled monolayers were prepared on both macro (1.6 mm) and micro (25 microm) gold electrodes using published procedures involving C6-terminated alkanethiol oligonucleotides and mercaptohexanol as the diluent. The extent of electrode modification by the modified probe was assessed using radiolabeling and a standard chronocoulometry method; both approaches gave loading levels within expected ranges ((1-6) x 10(12) molecules/cm(2)). Hybridization of the modified targets where the non-native nucleobase was incorporated by solid-phase synthesis produced electrocatalytic signals from strands that were independently detected using radiolabeling and chronocoulometry. This result was used as a basis to develop an on-electrode amplification scheme where Taq polymerase was used to extend the immobilized DNA probes from solution-phase polymeric templates using modified nucleotriphosphates. This reaction produced an electrode that was modified with extended DNA containing the appropriate modified nucleotide. Radiolabeled nucleotide triphosphates were used to confirm the desired on-electrode DNA synthesis. When these electrodes were cycled in the presence of Os(bpy)(3)(2+), electrocatalytic signals were observed when as little as 40 amol (400 fM) of the desired target was present in the hybridization solution.

A phosphoramidite containing Os(bpy)(3)(2+) (Os; bpy, 2,2'-bipyridine) with a three-carbon linker was synthesized and used to prepare oligonucleotides with the Os redox catalyst appended to the 5'-end. The electrogenerated Os(III) is capable of oxidizing 7,8-dihydro-8-oxo-guanine (8G), but 8G is not electrochemically reactive at indium tin oxide electrodes because of poor electrode kinetics for the direct reaction. The hairpin-forming oligonucleotide Os-5'-ATG TCA GAT TAG CAG GCC TGA CAT 8G was synthesized and characterized by thermal denaturation and native gel electrophoresis both in the hairpin form and when hybridized to its Watson-Crick complement. The redox potential in both forms of the appended Os(III/II) couple was 0.63 V (all potentials vs Ag/AgCl), which is identical to that for the free complex. The diffusion coefficients of the hairpin form (10.2 x 10(-)(7) cm(2)/s) and the duplex form (8.7 x 10(-)(7) cm(2)/s) were consistent with values expected from studies of noncovalently bound redox labels, which suggest that the measured diffusion coefficient should be that of the appended DNA molecule. The oligonucleotide was designed such that in the duplex form, the 8G is far from the Os(III/II) couple, but in the hairpin form, the 8G is situated close to the redox center. For the duplex form, cyclic voltammetry studies showed that mediated oxidation of the 8G nucleobase occurred only through bimolecular reaction of the electrogenerated Os(III) of one duplex with the 8G of another duplex. However, in the hairpin form, intramolecular electron transfer from 8G to Os(III) in the same molecule was apparent in both chronoamperometry and cyclic voltammetry.

A cluster of three genes, rpsF, rpsR, and rpII, encoding the ribosomal proteins S6, S18, and L9, respectively, were cloned and sequenced from Neisseria gonorrhoeae. The order of the genes within the cluster was established as rpsF-rpsR-rpII. Within this cluster an additional open reading frame of unknown identity spanning 108 bp was found between rpsF and rpsR. The putative amino acid sequences deduced from all three genes show a high degree of homology to other bacterial ribosomal proteins.

Uridine kinase is the rate-limiting enzyme in the salvage pathway for uridine or cytidine of mammalian cells. Alignment of the uridine kinase sequence with other nucleoside and nucleotide kinases supports a common ancestor for all of these. Three polypeptide segments for the ATP site and three polypeptide segments for the acceptor nucleoside site have been identified. We report here the characterization of an altered form of the enzyme with a single amino acid change, Q146R, within or near the uridine-binding site. This single amino acid change leads to a 160-fold increase in Km for uridine (Km = 6.5 mM) and a decrease in kcat by more than 99%. This variant has normal affinity for ATP (Km = 130 microM), but shows substrate inhibition at ATP concentrations >3 mM. Mouse uridine kinase is normally an active tetramer that will dissociate to inactive monomers in response to CTP. In contrast, the altered protein is monomeric, but will associate to dimers and then to tetramers with increasing ATP. The Q146R enzyme has a 100-fold loss in affinity for the allosteric inhibitor CTP; this supports a model for CTP inhibition being caused by CTP binding backward at the catalytic site, as a bisubstrate analog.

Abstract 4020 Introduction: The discovery of therapeutically relevant tumor antigens requires the demonstration of endogenous antigen processing and presentation by class I HLA molecules and evidence for the antigen's ability to elicit a specific immune response. Traditional antigen discovery methods have focused on initially generating T cell clones that recognize a target with subsequent testing of sequentially purified peptide pools to determine the chemical structure of the antigen. Generating T cell clones and the subsequent antigen discovery processes are labor-intensive, time-consuming and inefficient because antigen characteristics such as source protein, tissue restriction, HLA-restriction, and binding affinity cannot be determined prior to the generation of the T cell clone. To address these limitations, we developed an antigen discovery method based upon initial identification of HLA-epitopes with subsequent immune characterization that bypasses the need to generate a T cell clone and enabled us to identify and characterize 2 leukemia associated antigens within 1 year. Methods: Wild-type HLA-A0201 was stably transduced into the U937 myeloid leukemia cell line. Approximately 2×109 HLA-A0201+U937 were lysed and the peptide/HLA-A0201 complexes were purified by immunoaffinity column chromatography. The peptides were liberated from the HLA by weak acid elution and analyzed by reverse phase HPLC/tandem mass spectrometry (MS/MS). Collision induced dissociation (CID) MS/MS data were analyzed using the Mascot search engine. From 23 unambiguously sequenced, high-abundance peptides we analyzed 2 peptides, derived from ANKRD17 (UNC-A17 = LLIERGASL) and CDK4 (UNC-CK4 = ALTPVVVTL). These peptides were selected because ANKRD17 is a known source protein for the breast cancer antigen NY-BR-16, and CDK4 initiates entry into S-phase and is currently an investigational therapeutic target for treatment of acute myeloid leukemia (AML). Results: We confirmed CDK4 protein expression and observed variable (1 sample with high expression and 2 samples with low expression) expression of ANKRD17 in 3 of 3 human AML samples using western blots. Using an in-vitro assay, we found that both antigens bound HLA-A0201 with a dissociation t1/2 > 8 hours. Because of this slow off rate, we performed T cell expansion studies from healthy donor peripheral blood mononuclear cells (PBMC). We were able to generate antigen-specific oligoclonal T cell populations for both antigens. These T cell populations demonstrated antigen-specific immune function based upon intracellular interferon-γ flow cytometry measurements using both peptide-pulsed autologous dendritic cells and HLA-A0201+U937 cells as immune targets. We next looked for evidence of in-vivo T cell immunity towards these antigens in 5 patients (2 AML, 1 CML, 1 MDS, 1 MF) who had undergone allogeneic stem cell transplant. Using UNC-A17 and UNC-CK4 peptide loaded, fluorescently conjugated HLA-A0201 tetramers we identified UNC-CK4 specific T cells in 2 samples (0.22% and 0.39% of total CD8+ cells) and UNC-A17 specific T cells in 1 sample (0.64% of total CD8+ cells). Conclusion: Using this novel methodology we were able to identify 2 new leukemia associated antigens by directly examining the target cells for antigen expression and epitope processing. This direct methodology eliminated the need to generate a T cell clone or evaluate an entire peptide pool for evidence of immunogenicity, as the first step in antigen discovery. Refinement of our HPLC-MS techniques will enable us to sequence increasing numbers of peptide epitopes from the source cells. These technical improvements combined with the standard genomics and bioinformatics tools that are currently available will allow us to narrow our focus on the discovery of immunologically relevant tumor associated self antigens and minor histocompatibility antigens that have very narrow tissue expression and favorable HLA binding characteristics. Disclosures: No relevant conflicts of interest to declare.

Abstract 2986 Background: Immunotherapy targeting aberrantly expressed leukemia associated antigens (LAA) has shown promising results in the management of myeloid leukemia. However, because of the heterogeneity and clonal evolution that is a feature of myeloid leukemia, targeting single peptide epitopes has had limited success, highlighting the need for novel antigen discovery. PR1 is a well-characterized LAA, is derived from the azurophil granule proteases neutrophil elastase (NE) and proteinase-3 (P3), and was effectively targeted in myeloid leukemia. We have previously shown that NE and P3 are aberrantly localized outside azurophil granules in myeloid leukemia and that their aberrant expression facilitates PR1 antigen presentation. Similar to NE and P3, cathepsin G (CG) is a serine protease located within azurophil granules in neutrophils. Because azurophil granules are disrupted in myeloid leukemia and since CG was shown to be immunogenic through its association with autoimmune disease, we hypothesized that CG may be aberrantly expressed in myeloid leukemia, thereby facilitating its presentation on HLA class I molecules and rendering it a novel target for myeloid leukemia immunotherapy. Methods: SYFPEITHI and IEDB binding algorithms were used to identify CG peptides with highest binding affinities. Flow cytometry based binding assays were performed using the T2 cell line followed by HLA-A0201 staining to confirm peptide binding and determine affinities of CG-derived peptides for HLA-A0201. Peptide/HLA-A0201 complexes were released from HLA-A0201 transfected U937 cells and purified using immunoaffinity chromatography with the anti-HLA-A0201 antibody BB7.2. The peptides were dissociated from HLA-A0201 by weak acid elution and analyzed using reverse-phase HPLC-tandem mass spectrometry. Tandem mass spectra were analyzed using the Mascot sequencing algorithm. Western blots (WB) and immunoflouresence confocal microscopy were used to determine CG expression by primary leukemia. Peptide-pulsed T2 cells were used to expand CG1- and PR1-cytotoxic T lymphocytes (CTLs) for use in calcein AM cytotoxicity assays. Peptide-pulsed T2 cells and primary leukemia from patients were used as target cells. Tetramer staining and cytokine flow cytometry (CFC) assays were used to show frequency and function of CG1-CTLs, respectively. CG1- and PP65- pulsed T2 cells were used as stimulators in CFC assays. Patient samples were collected after informed consent. Results: Five CG derived nonameric peptides were identified using SYFPEITHI and IEDB binding algorithms. CG1 peptide (FLLPTGAEA) was identified as having the highest binding affinity to HLA-A0201 in comparison with other CG-derived peptides and with PR1 peptide (CG1 IC50=1 uM vs. PR1=9 uM). The dissociation t1/2 for CG1 and PR1 peptides was > 8 hours. CG1 peptide was eluted from the surface of U937-A2 cell line, and using reverse-phase HPLC-tandem mass spectrometry, we confirmed CG1 antigen presentation by leukemia. WB analysis for CG showed high CG expression in 9 of 11 AML patient samples and along with immunofluorescent cell imaging, showed localization of CG outside granules in compartments that could render CG susceptible to proteasomal degradation and antigen presentation. Killing of primary AML blasts by CG1-CTL was shown in 6 of 8 AML patient samples and directly correlated with CG expression and HLA-A0201 status. Blocking HLA-A0201 with BB7.2 antibody abrogated CG1-CTL mediated cytotoxicity, indicating HLA-A0201 dependent killing by CG1-CTLs. CG1-specific CTLs were detected in AML patient samples at frequencies ranging between 0.6% to 1.4% of total CD8+ cells. Furthermore, following stem cell transplant (SCT), 4–10 fold higher frequencies of functional CG1-CTLs were detected in patient samples when compared with pre-SCT samples from the same patient, as measured by IFN-gamma and/or TNF-alpha CFC. Conclusion: We demonstrate that CG is a novel immunotherapeutic target in myeloid leukemia. We show that CG is a LAA that is aberrantly expressed in leukemia, is presented on HLA-A0201 molecule and can be effectively targeted by CG1-CTLs. Since immunity to CG has been previously reported in autoimmune diseases, our findings show that the immunogenic potential of CG can be redirected therapeutically to target leukemia. Cumulatively, our data suggest that further development of CG-targeting therapies in myeloid leukemia is warranted. Disclosures: No relevant conflicts of interest to declare.

Abstract 1167 Poster Board I-189 Alloantibody responses to HY antigens have been well described in patients with chronic graft versus host disease (cGVHD), but specific B cell reactivity to autosomal minor histocompatibility antigens has not been described. Recently, genetic polymorphisms in hemophiliacs treated with recombinant factor VIII (F8) have been associated with an increased risk of developing F8 antibody inhibitors (Viel KR NEJM 2009), highlighting the possibility that F8 is an alloantigen target in cGVHD. From a patient with cGVHD manifesting with a clinically significant antibody inhibitor to F8, we identified a novel non-synonymous mutation resulting in a glycine (G) to arginine (R) substitution at codon 2076 (G2076R) in the patient/recipient's F8 gene not present in the donor. To visualize the G to R substitution, homology modeling was performed using the SWISS-MODEL server. The G2076R substitution was found to alter the surface of the C1 domain. The homology model is expected to be particularly reliable in this case because the side chain is on the surface, making conformational changes in peptide backbone structure unlikely. Enzyme-Linked Immunosorbent Assays (ELISA) using peptides synthesized with or without the G2076R mutation were employed to measure direct IgG binding. The donor had pre-existing alloantibodies to the mutated peptide, while the recipient, prior to hematopoietic stem cell transplantation (HSCT), did not have a pre-existing allo-IgG response to the mutated peptide. The recipient had no IgG response to native or mutated peptide or whole, unmutated F8 before HSCT or at 1,2,3,6 or 9 months after HSCT, but a specific IgG response to the G2076R containing peptide developed at 11 months post-HSCT just prior to clinically evident coagulopathy. Negative control amino acid substitutions (G2076A) did not result in IgG binding and negative control G2076R peptide (oriented on the ELISA so that R was not available for IgG binding) also did not result in IgG binding. An IgG response to whole, unmutated recombinant F8 protein as measured by ELISA and Western Blot was detected by 11 months post-HSCT. The recipient was treated with a 4 week course of rituximab and corticosteroids and had a complete clinical response with normalization of coagulation parameters. Alloreactivity in the recipient was no longer detectable 6 months later, although IgG responses to unmutated F8 protein remained, suggesting that rituximab resulted in removal of the alloantibody producing B cell. Persistence of IgG reactive to unmutated whole F8 also suggests epitope spreading occurred, resulting in the development of antibodies to other F8 epitopes that contribute to the autoreactivity found in this patient. Thus, we demonstrate ‘proof of principal’ that B cell responses to autosomal minor histocompatibility antigens occur in human cGVHD. Disclosures: Off Label Use: rituximab use in chronic graft versus host disease.

Purine nucleoside phosphorylase (PNP; EC 2.4.2.1) catalyzes the phosphorolysis of (d)guanosine or inosine to form the base (guanine or hypoxanthine) and (d)ribose-1-P; the enzyme has therefore been defined as catabolic (1,2). The enzyme may also be considered as biosynthetic, since the salvage of purines to form nucleotides is largely via a one step phosphoribosyltransferase reaction. Reports characterizing this enzyme have not been consistent. Based on the native Mr, the enzyme has been reported as a trimer, a dimer, or a monomer (references in #3). Also, some studies have reported negative cooperativity with phosphate (references in #4), or nucleosides (references in #4). Some ambiguity about the above results arose when other studies reported no cooperativity with phosphate, or with nucleosides (references in #4).

BackgroundThe one-electron oxidation of guanine nucleobases is of interest for understanding the mechanisms of mutagenesis, probing electron-transfer reactions in DNA, and developing sensing schemes for nucleic acids. The electron-transfer rates for oxidation of guanine by exogenous redox catalysts depend on the base paired to the guanine. An important goal in developing the mismatch sensitivity is to identify a means for monitoring the current resulting from electron transfer at a single base in the presence of native oligonucleotides that contain all four bases.ResultsThe nucleobase 8-oxo-guanine (8G) is selectively oxidized by the redox catalyst Os(bpy)33+/2+ (bpy = 2,2′-bipyridine) in the presence of native guanine. Cyclic voltammograms of Os(bpy)3+ show current enhancements indicative of nucleobase oxidation upon addition of oligonucleotides that contain 8G, but not in the presence of native guanine. As expected, similar experiments with Ru(bpy)3+ show enhancement with both guanine and 8G. The current enhancements for the 8G/Os(III) reaction increase in the order 8G-C ~ 8G·T < 8G·G < 8G·A < 8G, the same order as that observed for guanine/Ru(III). This site-selective mismatch sensitivity can be applied to detection of a TTT deletion, which is important in cystic fibrosis.ConclusionsThe base 8G can be effectively used in conjunction with a lowpotential redox catalyst as a probe for selective electron transfer at a single site. Because of the high selectivity for 8G, rate constants can be obtained that reflect the oxidation of only one base. The mismatch sensitivity can be used to detect biologically relevant abnormalities in DNA.