Engineered drug resistant γδ T cells kill glioblastoma cell lines during a chemotherapy challenge: a strategy for combining chemo- and immunotherapy.

Classical approaches to immunotherapy that show promise in some malignancies have generally been disappointing when applied to high-grade brain tumors such as glioblastoma multiforme (GBM). We recently showed that ex vivo expanded/activated γδ T cells recognize NKG2D ligands expressed on malignant glioma and are cytotoxic to glioma cell lines and primary GBM explants. In addition, γδ T cells extend survival and slow tumor progression when administered to immunodeficient mice with intracranial human glioma xenografts. We now show that temozolomide (TMZ), a principal chemotherapeutic agent used to treat GBM, increases the expression of stress-associated NKG2D ligands on TMZ-resistant glioma cells, potentially rendering them vulnerable to γδ T cell recognition and lysis. TMZ is also highly toxic to γδ T cells, however, and to overcome this cytotoxic effect γδ T cells were genetically modified using a lentiviral vector encoding the DNA repair enzyme O(6)-alkylguanine DNA alkyltransferase (AGT) from the O(6)-methylguanine methyltransferase (MGMT) cDNA, which confers resistance to TMZ. Genetic modification of γδ T cells did not alter their phenotype or their cytotoxicity against GBM target cells. Importantly, gene modified γδ T cells showed greater cytotoxicity to two TMZ resistant GBM cell lines, U373(TMZ-R) and SNB-19(TMZ-R) cells, in the presence of TMZ than unmodified cells, suggesting that TMZ exposed more receptors for γδ T cell-targeted lysis. Therefore, TMZ resistant γδ T cells can be generated without impairing their anti-tumor functions in the presence of high concentrations of TMZ. These results provide a mechanistic basis for combining chemotherapy and γδ T cell-based drug resistant cellular immunotherapy to treat GBM.

O6-methylguanine DNA methyltransferase expression in tumor cells predicts outcome of radiotherapy plus concomitant and adjuvant temozolomide therapy in patients with primary glioblastoma.

Expression of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene has been shown to correlate with clinical outcomes in patients with glioblastoma multiforme treated with alkylating agents. We evaluated MGMT protein expression in 53 primary glioblastomas by the immunohistochemistry (IHC) and analyzed the correlation between results of immunostaining and patient outcomes. There were 28 MGMT-immunopositive and 25 negative glioblastomas. Patients with MGMT-immunonegative glioblastomas showed significantly longer progression-free survival (PFS) (P = 0.0032), but no statistically significant benefits on overall survival (OS) (P = 0.0825) were shown. In 41 glioblastomas treated with temozolomide (TMZ) therapy (MGMT-immunopositive: n = 22, negative: n = 19), both PFS and OS were significantly better in MGMT-immunonegative glioblastomas. (PFS: P = 0.0015, OS: P = 0.0384). We conclude that MGMT expression on immunohistochemistry (IHC) correlates with outcomes in patients with primary glioblastoma receiving TMZ and suggest the use of MGMT-IHC as a surrogate marker for predicting tumor chemosensitivity.

The availability of a recombinant anti-SNAP antibody in VHH format amplifies the application flexibility of SNAP-tagged proteins.

Antibodies are indispensable reagents in basic research, and those raised against tags constitute a useful tool for the evaluation of the biochemistry and biology of novel proteins. In this paper, we describe the isolation and characterization of a single-domain recombinant antibody (VHH) specific for the SNAP-tag, using Twist2 as a test-protein. The antibody was efficient in western blot, immunoprecipitation, immunopurification, and immunofluorescence. The sequence corresponding to the anti-SNAP has been subcloned for large-scale expression in vectors that allow its fusion to either a 6xHis-tag or the Fc domain of rabbit IgG2 taking advantage of a new plasmid that was specifically designed for VHH antibodies. The two different fusion antibodies were compared in immunopurification and immunofluorescence experiments, and the recombinant protein SNAP-Twist2 was accurately identified by the anti-SNAP Fc-VHH construct in the nuclear/nucleolar subcellular compartment. Furthermore, such localization was confirmed by direct Twist2 identification by means of anti-Twisit2 VHH antibodies recovered after panning of the same naïve phage display library used to isolate the anti-SNAP binders. Our successful localization of Twist2 protein using the SNAP-tag-based approach and the anti-Twist2-specific recombinant single-domain antibodies opens new research possibilities in this field.

Inducing efficient cross-priming using antigen-coated yeast particles.

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.

Relationship between expression of O6-methylguanine-DNA methyltransferase, glutathione-S-transferase pi in glioblastoma and the survival of the patients treated with nimustine hydrochloride: an immunohistochemical analysis.

Drug resistance is one of the important factors that determine tumor response to chemotherapy. Several candidates for resistance to various chemotherapeutic agents have been elucidated. O6-methylguanine-DNA methyltransferase (MGMT) removes methylation damage induced by nitrosourea from the O6 position of DNA guanines before cell injury. Glutathione-S-transferase (GST) pi is also involved in nitrosourea resistance. We examined the expression of MGMT and GST pi in 18 glioblastomas (GBM) using immunohistochemistry and compared the results with patients' survival after administration of 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU)-based chemotherapy. According to the Kaplan-Meier's method, although median progression free survival (PFS) of eight patients whose tumors retained high MGMT (3+ approximately 2+), and 10 patients whose tumors showed low MGMT expression (1+ approximately 0) were nine and 15 months, respectively (p = 0.09), median overall survival (OS) of the two groups were 12 and 22 months, respectively, which were significantly different (p = 0.01). GST pi expression in GBM was not a prognostic factor. It is suggested that GBM with strong staining of MGMT activity may show more resistance to ACNU-based chemotherapy compared to that with low MGMT. The simple immunohistochemical analysis of MGMT in GBM can be a useful method to determine whether ACNU or another treatment regimen should be recommended.

Analysis of O(6)-methylguanine-DNA methyltransferase in melanoma tumours in patients treated with dacarbazine-based chemotherapy.

In a retrospective study we analysed the levels of the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) in melanoma metastases in patients receiving dacarbazine (DTIC) either as a single drug or as part of combination chemotherapy regimens, and related the expression levels to the clinical response to treatment. Biopsies of subcutaneous and lymph node metastases obtained before chemotherapy in 65 patients with disseminated malignant melanoma were examined for MGMT protein levels by immunohistochemistry using a monoclonal anti-human MGMT antibody. All patients received chemotherapy with DTIC, given either as a single drug or in combination with vindesine and in some cases cisplatin. DTIC as single agent was given to 44 patients, while 21 received combination chemotherapy. Objective responses to chemotherapy were seen in 12 patients, while 53 patients failed to respond to treatment. The expression of MGMT was determined according to the proportion of antibody-stained tumour cells, using a cut-off level of 50%. In 12 of the patients more than one metastasis was analysed, and in seven of these cases the MGMT expression differed between tumours in the same individual. Among the responders a larger proportion (six out of 12, 50%) had tumours containing less than 50% MGMT-positive tumour cells than among the non-responders (12 out of 53, 23%). These data are consistent with the hypothesis that MGMT contributes to resistance to DTIC-based treatment, although the difference between responders and non-responders with respect to the proportion of MGMT-positive tumour cells was not statistically significant (P = 0.077).

Enhanced ultrasensitive detection of structurally diverse antigens using a single immuno-PCR assay protocol.

Our studies of DNA damage and repair in autoimmune disease, lymphomagenesis, and carcinogenesis, require identification of an immunoassay approach that is capable of ultrasensitive detection in a routine human tissue biopsy of several physicochemically diverse antigens, some of which will be present at very low level. Immuno-polymerase chain reaction (immuno-PCR) is a recently described method for ultrasensitive antigen detection that combines the amplification power of PCR with a method similar to a standard antibody capture, enzyme-linked immunosorbent assay (ELISA). As a test of the universality of immuno-PCR, and as an assessment of the suitability of this method for our studies, we used a single immuno-PCR protocol to assay purified forms of the following physicochemically diverse antigens: oligomeric pyruvate dehydrogenase complex (PDC; Mr 8.5 x 10(6)), the promutagenic DNA base adduct O(6)-methylguanosine (Mr 298) and its monomeric repair enzyme, O(6)-methylguanine-DNA methyltransferase (MGMT; Mr 22,000), and a peptide from the N-terminus of MGMT (Mr 2310). We found that all antigens could be ultrasensitively assayed using the single immuno-PCR protocol. Assay limits observed using antigen-specific (primary) antibodies at 1 microg/ml, were in the approximate range of 10(2)-10(9) molecules, with O(6)-methylguanosine being detected most sensitively. Sensitivity of the antigen assay appeared to positively correlate with primary antibody titres determined by ELISA. Furthermore, we observed a substantial increase in detection sensitivity for all antigens by the use of primary antibodies at the higher level of 10 microg/ml. The latter approach permitted antigen assay within the approximate range of 10(0)-10(7) molecules. The combination of higher titre primary antibodies and their use at higher input level, produced an increase of immuno-PCR assay sensitivity of up to four orders of magnitude greater than those previously reported through the use of this assay to measure other antigens. This represents up to a nine order of magnitude increase in immunoassay sensitivity compared to ELISA. Our findings provide compelling evidence that immuno-PCR is indeed a universal ultrasensitive antigen detection method. Using the indicated assay enhancements. immuno-PCR performed as detailed here can offer greatly increased sensitivity for antigen measurement compared to other methods. Thus, our findings suggest that parallel quantitation of several different antigens in very small samples of human tissue will be readily attainable using immuno-PCR.

Quantitative immunohistochemical estimates of O6-alkylguanine-DNA alkyltransferase expression in normal and malignant human colon.

A major mechanism of resistance to nitrosoureas is O6-alkylguanine-DNA-alkyltransferase. The alkyltransferase biochemical assay measures mean tissue activity but requires availability of fresh tissue and cannot assess tumor heterogeneity, an important component of tumor resistance to alkylating agents. We assessed the levels of alkyltransferase in human colon carcinoma and normal colon by biochemical assay, Western blot, conventional immunohistochemistry, and quantitative immunohistochemistry (using 5H7 and mT3.1 monoclonal IgGs) to correlate whole tissue levels with cell-specific expression. Alkyltransferase activity was 18.0 +/- 4.6 fmol/microgram DNA in normal colon and 15.0 +/- 6.5 fmol/microgram DNA in tumors. By Western blot estimates, alkyltransferase in normal colon was 14.8 +/- 4.2 fmol/microgram DNA and in tumors was 16.2 +/- 7.8 fmol/microgram DNA. Alkyltransferase estimates by biochemical and Western blots were correlated strongly (P < 0.0001). Conventional immunohistochemistry demonstrated that alkyltransferase was predominantly nuclear and in normal colon was concentrated in glandular epithelial mucosal cells close to the lumen, whereas in tumors, expression was heterogenous but localized to malignant epithelial cells. Two parameters of quantitative immunohistochemistry, integrated gray and mean gray, were correlated strongly with each other (P < 0.002) and with biochemical and Western blot estimates (P = 0.004-0.04). Thus, quantitative immunohistochemical estimates of alkyltransferase in fixed tissues are a reasonable alternative to biochemical analysis and have an added advantage of identifying heterogeneity of alkyltransferase expression in tumors.