Intraventricular B7-H3 CAR T Cells for Diffuse Intrinsic Pontine Glioma: Preliminary First-in-Human Bioactivity and Safety.

Diffuse intrinsic pontine glioma (DIPG) remains a fatal brainstem tumor demanding innovative therapies. As B7-H3 (CD276) is expressed on central nervous system (CNS) tumors, we designed B7-H3-specific chimeric antigen receptor (CAR) T cells, confirmed their preclinical efficacy, and opened BrainChild-03 (NCT04185038), a first-in-human phase I trial administering repeated locoregional B7-H3 CAR T cells to children with recurrent/refractory CNS tumors and DIPG. Here, we report the results of the first three evaluable patients with DIPG (including two who enrolled after progression), who received 40 infusions with no dose-limiting toxicities. One patient had sustained clinical and radiographic improvement through 12 months on study. Patients exhibited correlative evidence of local immune activation and persistent cerebrospinal fluid (CSF) B7-H3 CAR T cells. Targeted mass spectrometry of CSF biospecimens revealed modulation of B7-H3 and critical immune analytes (CD14, CD163, CSF-1, CXCL13, and VCAM-1). Our data suggest the feasibility of repeated intracranial B7-H3 CAR T-cell dosing and that intracranial delivery may induce local immune activation. SIGNIFICANCE: This is the first report of repeatedly dosed intracranial B7-H3 CAR T cells for patients with DIPG and includes preliminary tolerability, the detection of CAR T cells in the CSF, CSF cytokine elevations supporting locoregional immune activation, and the feasibility of serial mass spectrometry from both serum and CSF. This article is highlighted in the In This Issue feature, p. 1.

Molecular details of aluminium-amyloid ß peptide interaction by nuclear magnetic resonance.

Alzheimer's disease (AD) is a devastating neurodegenerative condition that poses major challenges to human health. Both amyloid ß (Aß) and metal ions such as aluminium are implicated in the development of AD. By the means of NMR, the interactions of Al3+ with Aß1-28 peptide as well as the Aß1-28 analogues were studied, and the key binding sites of Al3+ in Aß determined. NMR data showed Al3+ interacts with Aß1-28 at the NH and αH of numerous residues by exhibiting upfield shifts. Using Aß analogues where His6, His13 and His14 were individually replaced by alanine residue(s), including Aß H6A, Aß H13A, Aß H14A, and Aß H6,13,14A, the results demonstrated that the histidine residues (His6, His13 and His14) and N-terminal Asp1 were involved in the Al3+ coordination. These findings provide, for the first time, the details of the molecular interaction between Al3+ and Aß, which points to the potential role of Al3+ in Aß aggregation, hence in AD development.

Locoregional infusion of HER2-specific CAR T cells in children and young adults with recurrent or refractory CNS tumors: an interim analysis.

Locoregional delivery of chimeric antigen receptor (CAR) T cells has resulted in objective responses in adults with glioblastoma, but the feasibility and tolerability of this approach is yet to be evaluated for pediatric central nervous system (CNS) tumors. Here we show that engineering of a medium-length CAR spacer enhances the therapeutic efficacy of human erb-b2 receptor tyrosine kinase 2 (HER2)-specific CAR T cells in an orthotopic xenograft medulloblastoma model. We translated these findings into BrainChild-01 ( NCT03500991 ), an ongoing phase 1 clinical trial at Seattle Children's evaluating repetitive locoregional dosing of these HER2-specific CAR T cells to children and young adults with recurrent/refractory CNS tumors, including diffuse midline glioma. Primary objectives are assessing feasibility, safety and tolerability; secondary objectives include assessing CAR T cell distribution and disease response. In the outpatient setting, patients receive infusions via CNS catheter into either the tumor cavity or the ventricular system. The initial three patients experienced no dose-limiting toxicity and exhibited clinical, as well as correlative laboratory, evidence of local CNS immune activation, including high concentrations of CXCL10 and CCL2 in the cerebrospinal fluid. This interim report supports the feasibility of generating HER2-specific CAR T cells for repeated dosing regimens and suggests that their repeated intra-CNS delivery might be well tolerated and activate a localized immune response in pediatric and young adult patients.

Rationally Designed Transgene-Encoded Cell-Surface Polypeptide Tag for Multiplexed Programming of CAR T-cell Synthetic Outputs.

Synthetic immunology, as exemplified by chimeric antigen receptor (CAR) T-cell immunotherapy, has transformed the treatment of relapsed/refractory B cell-lineage malignancies. However, there are substantial barriers-including limited tumor homing, lack of retention of function within a suppressive tumor microenvironment, and antigen heterogeneity/escape-to using this technology to effectively treat solid tumors. A multiplexed engineering approach is needed to equip effector T cells with synthetic countermeasures to overcome these barriers. This, in turn, necessitates combinatorial use of lentiviruses because of the limited payload size of current lentiviral vectors. Accordingly, there is a need for cell-surface human molecular constructs that mark multi-vector cotransduced T cells, to enable their purification ex vivo and their tracking in vivo. To this end, we engineered a cell surface-localizing polypeptide tag based on human HER2, designated HER2t, that was truncated in its extracellular and intracellular domains to eliminate ligand binding and signaling, respectively, and retained the membrane-proximal binding epitope of the HER2-specific mAb trastuzumab. We linked HER2t to CAR coexpression in lentivirally transduced T cells and showed that co-transduction with a second lentivirus expressing our previously described EGFRt tag linked to a second CAR efficiently generated bispecific dual-CAR T cells. Using the same approach, we generated T cells expressing a CAR and a second module, a chimeric cytokine receptor. The HER2txEGFRt multiplexing strategy is now being deployed for the manufacture of CD19xCD22 bispecific CAR T-cell products for the treatment of acute lymphoblastic leukemia (NCT03330691).

Protein kinase CK2 is involved in zinc homeostasis in breast and prostate cancer cells.

The intracellular zinc profiles of breast and prostate cancer cells are diametrically opposed, with hyper-accumulation of zinc in breast cancer, and low level in prostate cancer. This phenomenon is poorly understood. This study employs two breast and two prostate cancer cell lines to investigate the role of protein kinase CK2 in regulating zinc homeostasis. CK2 was targeted by its specific inhibitors 4,5,6,7-tetrabromobenzotriazole (TBB) and CX-4945, and by the specific siRNA against each of the three CK2 genes. The effect of zinc exposure after the above CK2 manipulation was observed by MTT [3-(4,5-dimethyliazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] cell viability assay and confocal microscopy for intracellular zinc level. The results demonstrate that CK2 is involved in regulating zinc homeostasis in breast and prostate cancer cells as both TBB and CX-4945 substantially decreased cell viability upon zinc exposure. siRNA-mediated knockdown of the three CK2 subunits (α, α' and ß) revealed their discrete roles in regulating zinc homeostasis in breast and prostate cancer cells. Knockdown of CK2α' decreased the intracellular zinc level of breast cancer cells and in turn increased the cell viability while the opposite findings were obtained for the prostate cancer cells. Knockdown of CK2ß expression substantially increased the zinc level in breast cancer cell lines whilst decreased the zinc level in prostate cancer cells. Taken together, this study shows that CK2 is involved in zinc homeostasis of breast and prostate cancer cells and opens a new avenue for research on these cancers.

Manufacture of Chimeric Antigen Receptor T Cells from Mobilized Cyropreserved Peripheral Blood Stem Cell Units Depends on Monocyte Depletion.

Cytotoxic chemotherapy and radiation can render lymphocyte repertoires qualitatively and quantitatively defective. Thus, heavily treated patients are often poor candidates for the manufacture of autologous chimeric antigen receptor (CAR)-T cell products. In the United States and Europe, children with high-risk neuroblastoma undergo apheresis early in the course of treatment to collect peripheral blood stem cells (PBSCs) for cryopreservation in preparation for high-dose chemotherapy followed by autologous stem cell rescue. Here, we investigate whether these cryopreserved chemotherapy and granulocyte colony-stimulating factor (G-CSF)-mobilized PBSCs can serve as starting material for CAR-T cell manufacturing. We evaluated T cell precursor subsets in cryopreserved PBSC units from 8 patients with neuroblastoma using fluorescent activated cell sorting-based analysis. Every cryopreserved unit collected early in treatment contained both CD4 and CD8 precursors with significant numbers of naïve and central memory precursors. Significant numbers of Ki67+/PD1+ T cells were detected, presumably the result of chemotherapy-induced lymphopenia and subsequent homeostatic proliferation. Cryopreserved PBSC units containing 56 to 112 × 106 T cells were amenable to immunomagnetic selection, CD3 × 28 bead activation, lentiviral transduction, and cytokine-driven expansion, provided that CD14 monocytes were depleted before the initiation of cultures. Second- and third-generation CD171 CAR+ CD4 and CD8 effector cells derived from cryopreserved units displayed antineuroblastoma lytic potency and cytokine secretion comparable to those derived from a healthy donor and mediated in vivo antitumor regression in NSG mice. We conclude that cryopreserved PBSCs procured via standard methods during early treatment can serve as an alternative starting source for CAR-T cell manufacturing, extending the options for heavily treated patients.

EGFR806-CAR T cells selectively target a tumor-restricted EGFR epitope in glioblastoma.

Targeting solid tumor antigens with chimeric antigen receptor (CAR) T cell therapy requires tumor specificity and tolerance toward variability in antigen expression levels. Given the relative paucity of unique cell surface proteins on tumor cells for CAR targeting, we have focused on identifying tumor-specific epitopes that arise as a consequence of target protein posttranslational modification. We designed a CAR using a mAb806-based binder, which recognizes tumor-specific untethered EGFR. The mAb806 epitope is also exposed in the EGFRvIII variant transcript. By varying spacer domain elements of the CAR, we structurally tuned the CAR to recognize low densities of EGFR representative of non-gene amplified expression levels in solid tumors. The appropriately tuned short-spacer 2nd generation EGFR806-CAR T cells showed efficient in vitro cytokine secretion and glioma cell lysis, which was competitively blocked by a short peptide encompassing the mAb806 binding site. Unlike the nonselective Erbitux-based CAR, EGFR806-CAR T cells did not target primary human fetal brain astrocytes expressing wild-type EGFR, but showed a similar level of activity compared to Erbitux-CAR when the tumor-specific EGFRvIII transcript variant was overexpressed in astrocytes. EGFR806-CAR T cells successfully treated orthotopic U87 glioma implants in NSG mice, with 50% of animals surviving to 90 days. With additional IL-2 support, all tumors were eradicate without recurrence after 90 days. In a novel human induced pluripotent stem cell (iPSC)-derived teratoma xenograft model, EGFR806-CAR T cells infiltrated but were not activated in EGFR+ epidermal cell nests as assessed by Granzyme B expression. These results indicate that EGFR806-CAR T cells effectively and selectively target EGFR-expressing tumor cells.

Proteomic response to linoleic acid hydroperoxide in Saccharomyces cerevisiae.

Yeast AP-1 transcription factor (Yap1p) and the enigmatic oxidoreductases Oye2p and Oye3p are involved in counteracting lipid oxidants and their unsaturated breakdown products. In order to uncover the response to linoleic acid hydroperoxide (LoaOOH) and the roles of Oye2p, Oye3p and Yap1p, we carried out proteomic analysis of the homozygous deletion mutants oye3Δ, oye2Δ and yap1Δ alongside the diploid parent strain BY4743. The findings demonstrate that deletion of YAP1 narrowed the response to LoaOOH, as the number of proteins differentially expressed in yap1Δ was 70% of that observed in BY4743. The role of Yap1p in regulating the major yeast peroxiredoxin Tsa1p was demonstrated by the decreased expression of Tsa1p in yap1Δ. The levels of Ahp1p and Hsp31p, previously shown to be regulated by Yap1p, were increased in LoaOOH-treated yap1Δ, indicating their expression is also regulated by another transcription factor(s). Relative to BY4743, protein expression differed in oye3Δ and oye2Δ under LoaOOH, underscored by superoxide dismutase (Sod1p), multiple heat shock proteins (Hsp60p, Ssa1p, and Sse1p), the flavodoxin-like protein Pst2p and the actin stabiliser tropomyosin (Tpm1p). Proteins associated with glycolysis were increased in all strains following treatment with LoaOOH. Together, the dataset reveals, for the first time, the yeast proteomic response to LoaOOH, highlighting the significance of carbohydrate metabolism, as well as distinction between the roles of Oye3p, Oye2p and Yap1p.

Unravelling the role of protein kinase CK2 in metal toxicity using gene deletion mutants.

Metal ions, biologically essential or toxic, are present in the surrounding environment of living organisms. Understanding their uptake, homeostasis or detoxification is critical in cell biology and human health. In this study, we investigated the role of protein kinase CK2 in metal toxicity using gene deletion strains of Saccharomyces cerevisiae against a panel of six metal ions. The deletion of CKA2, the yeast orthologue of mammalian CK2α', leads to a pronounced resistant phenotype against Zn2+ and Al3+, whilst the deletion of CKB1 or CKB2 results in tolerance to Cr6+ and As3+. The individual deletion mutants of CK2 subunits (CKA1, CKA2, CKB1 and CKB2) did not have any benefit against Co2+ and Cd2+. The metal ion content in the treated cells was then measured by inductively coupled plasma mass spectrometry. Two contrasting findings were obtained for the CKA2 deletion mutant (cka2Δ) against Al3+ or Zn2+. Upon exposure to Al3+, cka2Δ had markedly lower Al3+ content than the wild type and other CK2 mutants, congruous to the resistant phenotype of cka2Δ against Al3+, indicating that CKA2 is responsible for Al3+ uptake. Upon zinc exposure the same mutant showed similar Zn2+ content to the wild type and cka1Δ. Strikingly, the selective inhibitor of CK2 TBB (4,5,6,7-tetrabromo-1H-benzotriazole) abolished the resistant phenotype of cka2Δ against Zn2+. Hence, the CK2 subunit CKA1 plays a key role in Zn2+ sequestration of the cell. Given that both zinc and CK2 are implicated in cancer development, the findings herein are of significance to cancer research and anticancer drug development.

Preclinical Assessment of CD171-Directed CAR T-cell Adoptive Therapy for Childhood Neuroblastoma: CE7 Epitope Target Safety and Product Manufacturing Feasibility.

PURPOSE: The identification and vetting of cell surface tumor-restricted epitopes for chimeric antigen receptor (CAR)-redirected T-cell immunotherapy is the subject of intensive investigation. We have focused on CD171 (L1-CAM), an abundant cell surface molecule on neuroblastomas and, specifically, on the glycosylation-dependent tumor-specific epitope recognized by the CE7 monoclonal antibody. EXPERIMENTAL DESIGN: CD171 expression was assessed by IHC using CE7 mAb in tumor microarrays of primary, metastatic, and recurrent neuroblastoma, as well as human and rhesus macaque tissue arrays. The safety of targeting the CE7 epitope of CD171 with CE7-CAR T cells was evaluated in a preclinical rhesus macaque trial on the basis of CD171 homology and CE7 cross reactivity. The feasibility of generating bioactive CAR T cells from heavily pretreated pediatric patients with recurrent/refractory disease was assessed. RESULTS: CD171 is uniformly and abundantly expressed by neuroblastoma tumor specimens obtained at diagnoses and relapse independent of patient clinical risk group. CD171 expression in normal tissues is similar in humans and rhesus macaques. Infusion of up to 1 × 108/kg CE7-CAR+ CTLs in rhesus macaques revealed no signs of specific on-target off-tumor toxicity. Manufacturing of lentivirally transduced CD4+ and CD8+ CE7-CAR T-cell products under GMP was successful in 4 out of 5 consecutively enrolled neuroblastoma patients in a phase I study. All four CE7-CAR T-cell products demonstrated in vitro and in vivo antitumor activity. CONCLUSIONS: Our preclinical assessment of the CE7 epitope on CD171 supports its utility and safety as a CAR T-cell target for neuroblastoma immunotherapy. Clin Cancer Res; 23(2); 466-77. ©2016 AACR.

The New Role for an Old Kinase: Protein Kinase CK2 Regulates Metal Ion Transport.

The pleiotropic serine/threonine protein kinase CK2 was the first kinase discovered. It is renowned for its role in cell proliferation and anti-apoptosis. The complexity of this kinase is well reflected by the findings of past decades in terms of its heterotetrameric structure, subcellular location, constitutive activity and the extensive catalogue of substrates. With the advent of non-biased high-throughput functional genomics such as genome-wide deletion mutant screening, novel aspects of CK2 functionality have been revealed. Our recent discoveries using the model organism Saccharomyces cerevisiae and mammalian cells demonstrate that CK2 regulates metal toxicity. Extensive literature search reveals that there are few but elegant works on the role of CK2 in regulating the sodium and zinc channels. As both CK2 and metal ions are key players in cell biology and oncogenesis, understanding the details of CK2's regulation of metal ion homeostasis has a direct bearing on cancer research. In this review, we aim to garner the recent data and gain insights into the role of CK2 in metal ion transport.

Revelation of molecular basis for chromium toxicity by phenotypes of Saccharomyces cerevisiae gene deletion mutants.

Chromium toxicity is increasingly relevant to living organisms such as humans, due to the environmental contamination of chromium and the application of stainless steel-based medical devices like hip prostheses. Despite the investigations in past years, the molecular details for chromium toxicity remain to be delineated. In this study, we seek to gain insights into the molecular aspects of chromium toxicity by screening a genome-wide deletion set of individual genes in Saccharomyces cerevisiae against hexavalent chromium [Cr(vi)] using chromium trioxide. From the primary data collected in this study, two lists of deletion mutants in response to Cr(vi) exposure were obtained, one for the sensitive phenotype and the other for the resistant phenotype. The functional analysis of the genes corresponding to the sensitive mutants reveals the key features of Cr(vi) toxicity, which include genotoxicity, protein damage, disruption of energy and sulfur metabolisms. DNA repair, ubiquitination-mediated protein degradation, iron homeostasis and growth attenuation are the intrinsic facets of the cell's detoxification mechanisms. Protein kinase CK2 is, for the first time, found to be involved in regulating chromium toxicity by reducing the uptake of Cr(vi). Taken together, the findings provide meaningful details into the basic understanding of chromium toxicity in terms of its uptake, modes of action, cellular detoxification and molecular regulatory mechanisms.

Protein kinase CK2 regulates metal toxicity in neuronal cells.

Protein kinase CK2 is a pleiotropic tetrameric enzyme, regulating numerous biological processes from cell proliferation to stress response. This study demonstrates for the first time that CK2 is involved in the regulation of metal uptake and toxicity in neuronal cells. After the determination of inhibitory concentrations (IC50) for a range of metal salts (ZnSO4, Al(mal)3, CoCl2, CrO3, NaAsO2 and CaCl2) in Neuro-2a mouse neuroblastoma cells, the effect of CK2 on metal toxicity was investigated by three lines of experiments using CK2 inhibitors, metal ion specific fluorophores and siRNA-mediated knockdown of CK2 expression. The results showed that both CK2 inhibitors, 4,5,6,7-tetrabromobenzotriazole (TBB) and quinalizarin, markedly reduced the toxicity of Zn(ii), Al(iii), Co(ii), Cr(vi) and As(iii). Confocal microscopy imaging revealed that Zn(ii) uptake was accompanied by the increase of intracellular Ca(ii) in Neuro-2a cells treated with IC50 of ZnSO4 (240 µM), and such concurrent elevation of intracellular Zn(ii) and Ca(ii) was blocked by TBB and quinalizarin. The role of CK2 in metal uptake was further characterised using specific siRNA against each of the three subunits (CK2α, α' and ß) and the data demonstrate that CK2α' is the prominent subunit regulating the metal toxicity. Finally, the role of CK2 in metal toxicity was found to be conserved in the distant species-Saccharomyces cerevisiae by employing the complete deletion mutants of CK2 (cka1Δ, cka2Δ, ckb1Δ and ckb2Δ). Taken together, these findings shed light on a new facet of CK2 functionality and provide a basis for further research on the regulation of Zn(ii) and Ca(ii) homeostasis by CK2.

Molecular insight into arsenic toxicity via the genome-wide deletion mutant screening of Saccharomyces cerevisiae.

Arsenic is omnipresent in soil, air, food and water. Chronic exposure to arsenic is a serious problem to human health. In-depth understanding of this metalloid's toxicity is a fundamental step towards development of arsenic-free foods and measures for bioremediation. By screening the complete set of gene deletion mutants (4873) of Saccharomyces cerevisiae, this study uncovered 75 sensitive and 39 resistant mutants against arsenite [As(III)]. Functional analysis of the corresponding genes revealed the molecular details for its uptake, toxicity and detoxification. On the basis of the hypersensitivity of yap3Δ, the transcription factor, Yap3p, is for the first time linked to the cell's detoxification against As(III). Apart from confirming the previously described role of the mitogen-activated protein kinase (MAPK) Hog1 pathway in combating arsenic toxicity, the results show that the regulatory subunits (Ckb1p and Ckb2p) of protein kinase CK2 are also involved in the process, suggesting possible crosstalk between the two key protein kinases. The sensitivity to As(III) conferred by deletion of the genes involved in protein degradation and chromatin remodelling demonstrates protein damage is the key mode of toxicity for the metalloid. Furthermore, the resistant phenotype of fps1Δ, snf3Δ and pho81Δ against As(III) links arsenic uptake with the corresponding plasma membrane-bound transporters-aquaglyceroporin (Fps1p), hexose (Snf3p) and phosphate transporters. The molecular details obtained in this screen for As(III) uptake, detoxification and toxicity provide the basis for future investigations into arsenic-related problems in the environment, agriculture and human health.

Kv1.3 contains an alternative C-terminal ER exit motif and is recruited into COPII vesicles by Sec24a.

BACKGROUND: Potassium channels play a fundamental role in resetting the resting membrane potential of excitable cells. Determining the intracellular trafficking and localization mechanisms of potassium channels provides a platform to fully characterize their maturation and functionality. Previous investigations have discovered residues or motifs that exist in their primary structure, which directly promote anterograde trafficking of nascent potassium channels. Recently, a non-conical di-acidic motif (E483/484) has been discovered in the C-terminus of the mammalian homologue of the Shaker voltage-gated potassium channel subfamily member 3 (Kv1.3), and was shown to disrupt the anterograde trafficking of Kv1.3. RESULTS: We have further investigated the intracellular trafficking requirements of Kv1.3 both in vivo and in vitro. First, three alternative C-terminal acidic residues, E443, E445, E447 were probed for their involvement within the early secretory pathway of Kv1.3. Single point (E443A, E445A, and E447A) and double point (E443A-E445A, E445A-E447A) mutations exhibited no significant changes in their endoplasmic reticulum (ER) retention. The triple point mutant E443A-E445A-E447A displayed a modest ER retention while deletion of the C-terminus showed dramatic ER retention. Second, we demonstrate in vivo the requirement for the Sec24a isoform to confer anterograde trafficking using a siRNA knockdown assay. Third, we show in vitro the association of recombinantly expressed Kv1.3 and Sec24a proteins. CONCLUSION: These results expand upon previous studies aimed at deciphering the Kv1.3 secretory trafficking mechanisms and further show in vitro evidence of the association between Kv1.3 and the COPII cargo adaptor subunit isoform Sec24a.

Functional Tuning of CARs Reveals Signaling Threshold above Which CD8+ CTL Antitumor Potency Is Attenuated due to Cell Fas-FasL-Dependent AICD.

Chimeric antigen receptor (CAR) development is biased toward selecting constructs that elicit the highest magnitude of T-cell functional outputs. Here, we show that components of CAR extracellular spacer and cytoplasmic signaling domain modulate, in a cooperative manner, the magnitude of CD8(+)CTL activation for tumor-cell cytolysis and cytokine secretion. Unexpectedly, CAR constructs that generate the highest in vitro activity, either by extracellular spacer length tuning or by the addition of cytoplasmic signaling modules, exhibit attenuated antitumor potency in vivo, whereas CARs tuned for moderate signaling outputs mediate tumor eradication. Recursive CAR triggering renders CTLs expressing hyperactive CARs highly susceptible to activation-induced cell death (AICD) as a result of augmented FasL expression. CAR tuning using combinations of extracellular spacers and cytoplasmic signaling modules, which limit AICD of CD8(+)CTLs, may be a critical parameter for achieving clinical activity against solid tumors.

Enzymes to die for: exploiting nucleotide metabolizing enzymes for cancer gene therapy.

Suicide gene therapy is an attractive strategy to selectively destroy cancer cells while minimizing unnecessary toxicity to normal cells. Since this idea was first introduced more than two decades ago, numerous studies have been conducted and significant developments have been made to further its application for mainstream cancer therapy. Major limitations of the suicide gene therapy strategy that have hindered its clinical application include inefficient directed delivery to cancer cells and the poor prodrug activation capacity of suicide enzymes. This review is focused on efforts that have been and are currently being pursued to improve the activity of individual suicide enzymes towards their respective prodrugs with particular attention to the application of nucleotide metabolizing enzymes in suicide cancer gene therapy. A number of protein engineering strategies have been employed and our discussion here will center on the use of mutagenesis approaches to create and evaluate nucleotide metabolizing enzymes with enhanced prodrug activation capacity and increased thermostability. Several of these studies have yielded clinically important enzyme variants that are relevant for cancer gene therapy applications because their utilization can serve to maximize cancer cell killing while minimizing the prodrug dose, thereby limiting undesirable side effects.

Evaluation of a UCMK/dCK fusion enzyme for gemcitabine-mediated cytotoxicity.

While gemcitabine (2'-2'-difluoro-2'-deoxycytidine, dFdC) displays wide-ranging antineoplastic activity as a single agent, variable response rates and poor intracellular metabolism often limit its clinical efficacy. In an effort to enhance dFdC cytotoxicity and help normalize response rates, we created a bifunctional fusion enzyme that combines the enzymatic activities of deoxycytidine kinase (dCK) and uridine/cytidine monophosphate kinase (UCMK) in a single polypeptide. Our goal was to evaluate whether the created fusion could induce beneficial, functional changes toward dFdC, expedite dFdC conversion to its active antimetabolites and consequently amplify cell dFdC sensitivity. While kinetic analyses revealed the UCMK/dCK fusion enzyme to possess both native activities, the fusion rendered cells sensitive to the cytotoxic effects of dFdC at the same level as dCK expression alone. These results suggest that increased wild-type UCMK expression does not provide a significant enhancement in dFdC-mediated cytotoxicity and may warrant the implementation of studies aimed at engineering UCMK variants with improved activity toward gemcitabine monophosphate.