Piloting a scale-up platform for high-quality human T-cells production.

Cell and gene therapies are an innovative solution to various severe diseases and unfulfilled needs. Adoptive cell therapy (ACT), a form of cellular immunotherapies, has been favored in recent years due to the approval of chimeric antigen receptor CAR-T products. Market research indicates that the industry's value is predicted to reach USD 24.4 billion by 2030, with a compound annual growth rate (CAGR) of 21.5%. More importantly, ACT is recognized as the hope and future of effective, personalized cancer treatment for healthcare practitioners and patients worldwide. The significant global momentum of this therapeutic approach underscores the urgent need to establish it as a practical and standardized method. It is essential to understand how cell culture conditions affect the expansion and differentiation of T-cells. However, there are ongoing challenges in ensuring the robustness and reproducibility of the manufacturing process. The current study evaluated various adoptive T-cell culture platforms to achieve large-scale production of several billion cells and high-quality cellular output with minimal cell death. It examined factors such as bioreactor parameters, media, supplements and stimulation. This research addresses the fundamental challenges of scalability and reproducibility in manufacturing, which are essential for making adoptive T-cell therapy an accessible and powerful new class of cancer therapeutics.

Identification of HEXIM1 as a positive regulator of p53.

Hexamethylene bisacetamide-inducible protein 1 (HEXIM1) is best known as the inhibitor of positive transcription elongation factor b (P-TEFb), which regulates the transcription elongation of RNA polymerase II and controls 60-70% of mRNA synthesis. Our previous studies show that HEXIM1 interacts with two key p53 regulators, nucleophosmin and human double minute-2 protein (HDM2), implying a possible connection between HEXIM1 and the p53 signaling pathway. Here we report the interaction between p53 and HEXIM1 in breast cancer, acute myeloid leukemia, and colorectal carcinoma cells. The C-terminal regions of p53 and HEXIM1 are required for the protein-protein interaction. Overexpression of HEXIM1 prevents the ubiquitination of p53 by HDM2 and enhances the protein stability of p53, resulting in up-regulation of p53 target genes, such as Puma and p21. Induction of p53 can be achieved by several means, such as UV radiation and treatment with anti-cancer agents (including doxorubicin, etoposide, roscovitine, flavopiridol, and nutlin-3). Under all the conditions examined, elevated protein levels of p53 are found to associate with the increased p53-HEXIM1 interaction. In addition, knockdown of HEXIM1 significantly inhibits the induction of p53 and releases the cell cycle arrest caused by p53. Finally, the transcription of the p53 target genes is regulated by HEXIM1 in a p53-dependent fashion. Our results not only identify HEXIM1 as a positive regulator of p53, but also propose a novel molecular mechanism of p53 activation caused by the anti-cancer drugs and compounds.

Specific profiles of house dust mite sensitization in children with asthma and in children with eczema.

Sensitization to house dust mites (HDM) is highly prevalent among the young atopic population in Singapore. Previously published data suggest that individuals with skin allergies show preferred sensitization to Dermatophagoides pteronyssinus while individuals with pure respiratory allergies show preferred sensitization to Blomia tropicalis. The aim of our study was to compare the sensitization profiles between children with asthma and those with eczema to D. pteronyssinus and B. tropicalis and their specific allergens. A total of 60 children, 30 with asthma and 30 with eczema were recruited. IgE levels specific for a panel of HDM allergens from the two mite species were measured using enzyme-linked immunosorbent assay. The asthma group showed highest sensitization to Blo t5 while the eczema group showed highest sensitization to Der p5. Comparison between the two disease groups showed that the eczema group had significantly higher IgE levels for Der p (p = 0.042) and its allergens Der p1 (p = 0.019) and Der p5 (p = 0.001). Generally, the eczema group was more sensitized to the panel of allergens compared to the asthma group. Individuals with asthma and those with eczema showed different sensitization profiles to HDM. These findings highlighted possible mechanisms for different manifestation of allergy.

Mite amylase from Blomia tropicalis (Blo t 4): differential allergenicity linked to geographical regions.

BACKGROUND: Blomia tropicalis is an important domestic dust mite in the tropics and subtropics. This study describes cDNA cloning of the group 4 allergen of B. tropicalis, and the evaluation of the sensitization of this allergen in atopic populations from 2 geographic regions. METHODS: cDNA cloning was carried out using the Smart RACE cDNA amplification kit. The full-length Blo t 4 cDNA was isolated by cDNA library screening, 5' and 3' rapid amplification of cDNA ends and long-distance PCR. Sequence analysis was performed with a combination of the Clustal W, CGC and Blast program packages. The cDNA was expressed in Pichia pastoris yeast. The skin prick test was used to evaluate the sensitization profile of recombinant Blo t 4, crude dust mite allergen extracts and major B. tropicalis recombinant allergen Blo t 5. RESULTS: The cloned Blo t 4 had a molecular weight of 56 kDa and had 68% amino acid homology with group 4 allergens of Dermatophagoides pteronyssinus and 65% with those of Euroglyphus maynei. A sensitization profile to the expressed recombinant Blo t 4 allergen (28%) showed an unusually higher frequency than to the major allergen Blo t 5 (22%) in allergic subjects from Chengdu, PR China. In comparison, the subjects from Singapore showed very low sensitization to Blo t 4 (4%) compared with Blo t 5 (84%). CONCLUSIONS: Group 4 allergens of B. tropicalis may be an important dust mite allergen in certain distinct populations.

Burrow characteristics of the co-existing sibling species Mus booduga and Mus terricolor and the genetic basis of adaptation to hypoxic/hypercapnic stress.

BACKGROUND: The co-existing, sibling species Mus booduga and Mus terricolor show a difference in site-preference for burrows. The former build them in flat portion of the fields while the latter make burrows in earthen mounds raised for holding water in cultivated fields. In northern India which experiences great variation in climatic condition between summer and winter, M. booduga burrows have an average depth of 41 cm, as against 30 cm in southern India with less climatic fluctuation.M. terricolor burrows are about 20 cm deep everywhere. The three chromosomal species M. terricolor I, II and III have identical burrows, including location of the nest which is situated at the highest position. In contrast, in M. booduga burrows, the nest is at the lowest position. RESULTS: The nest chamber of M. booduga is located at greater depth than the nest chamber of M. terricolor. Also, in the burrows of M. booduga the exchange of air takes place only from one side (top surface) in contrast to the burrows of M. terricolor where air exchange is through three sides. Hence, M. booduga lives in relatively more hypoxic and hypercapnic conditions than M. terricolor.We observed the fixation of alternative alleles in M. booduga and M. terricolor at Superoxide dismutase-1 (Sod-1), Transferrin (Trf) and Hemoglobin beta chain (Hbb) loci. All the three are directly or indirectly dependent on oxygen concentration for function. In addition to these, there are differences in burrow patterns and site-preference for burrows suggesting difference in probable adaptive strategy in these co-existing sibling species. CONCLUSION: The burrow structure and depth of nest of the chromosomal species M. terricolor I, II and III are same everywhere probably due to the recency of their evolutionary divergence. Moreover, there is lack of competition for the well-adapted 'microhabitats' since they are non-overlapping in distribution. However, the co-existing sibling species M. booduga and M. terricolor exhibit mutual "exclusion" of the 'microhabitats' for burrow construction. Thus, location, structure and depth of the burrows might have been the contributory factors for selection of alternative alleles at three loci Sod-1, Trf and Hbb, which reflect difference in probable adaptive strategy in M. booduga and M. terricolor.

Production of monoclonal antibody by DNA immunization with electroporation.

DNA immunization with in vivo electroporation is an efficient alternative protocol for the production of monoclonal antibodies (mAb). Generation of mAb by DNA immunization is a novel approach to circumvent the following technical hurdles associated with problematic antigens: low abundance and protein instability and use of recombinant proteins that lack posttranslational modifications. This chapter describes the use of a DNA-based immunization protocol for the production of mAb against a house dust mite allergen, designated as Blo t 11, which is a paramyosin homologue found in Blomia tropicalis mites. The Blo t 11 cDNA fused at the N terminus to the sequence of a signal peptide was cloned into the pCI mammalian expression vector. The DNA construct was injected intramuscularly with in vivo electroporation into mice, and the specific antibody production in mice was analyzed by enzyme-linked immunosorbent assay (ELISA). Hybridomas were generated by fusing mouse splenocytes with myeloma cells using the ClonaCell-HY Hybridoma Cloning Kit. Six hybridoma clones secreting Blo t 11 mAb were successfully generated, and these mAb are useful reagents for immunoaffinity purification and immunoassays.

The Blomia tropicalis allergens.

Blomia tropicalis allergens are the most important mite allergens in tropical regions. Most of them only have 30-40% sequence identity with their Dermatophagoides counterparts and they share low IgE cross reactivity and exhibit different immunobiology. Unlike the pyroglyphid counterparts, Blo t 5 is the major allergen whereas Blo t 1 only has modest allergenicity.

In vitro rejoining of double-strand breaks in genomic DNA.

Recent genetic and biochemical studies have provided important insights into the mechanism of nonhomologous end-joining (NHEJ) in higher eukaryotes, and have helped to characterize several components including DNA-PKcs, Ku, DNA ligase IV, and XRCC4. There is evidence, however, that additional factors involved in NHEJ remain to be characterized. The biochemical characterization of NHEJ in higher eukaryotes has benefited significantly from in vitro plasmid-based end-joining assays. However, because of differences in the organization and sequence of genomic and plasmid DNA, and because multiple pathways of NHEJ are operational, it is possible that different factors are preferred for the rejoining of double-strand breaks (DSBs) induced in plasmid vs genomic DNA organized in chromatin. Here, we describe an in vitro assay that allows the study of DSB rejoining in genomic DNA. The assay utilizes as a substrate DSBs induced by various means in genomic DNA prepared from agarose-embedded cells after appropriate lysis. Two extremes in terms of state of DNA organization are described: "naked" DNA and DNA organized in chromatin. Here, we describe the protocols developed to carry out and analyze these in vitro reactions, including procedures for preparation of cell extract and the preparation of the substrate DNA ("naked" DNA or nuclei).

HEXIM1, a New Player in the p53 Pathway.

Hexamethylene bisacetamide-inducible protein 1 (HEXIM1) is best known as the inhibitor of positive transcription elongation factor b (P-TEFb), which controls transcription elongation of RNA polymerase II and Tat transactivation of human immunodeficiency virus. Besides P-TEFb, several proteins have been identified as HEXIM1 binding proteins. It is noteworthy that more than half of the HEXIM1 binding partners are involved in cancers. P53 and two key regulators of the p53 pathway, nucleophosmin (NPM) and human double minute-2 protein (HDM2), are among the factors identified. This review will focus on the functional importance of the interactions between HEXIM1 and p53/NPM/HDM2. NPM and the cytoplasmic mutant of NPM, NPMc+, were found to regulate P-TEFb activity and RNA polymerase II transcription through the interaction with HEXIM1. Importantly, more than one-third of acute myeloid leukemia (AML) patients carry NPMc+, suggesting the involvement of HEXIM1 in tumorigenesis of AML. HDM2 was found to ubiquitinate HEXIM1. The HDM2-mediated ubiquitination of HEXIM1 did not lead to protein degradation of HEXIM1 but enhanced its inhibitory activity on P-TEFb. Recently, HEXIM1 was identified as a novel positive regulator of p53. HEXIM1 prevented p53 ubiquitination by competing with HDM2 in binding to p53. Taken together, the new evidence suggests a role of HEXIM1 in regulating the p53 pathway and tumorigenesis.

Regulation of PRDX1 peroxidase activity by Pin1.

Pin1 isomerizes the phosphorylated Ser/Thr-Pro peptide bonds and regulates the functions of its binding proteins by inducing conformational changes. Involvement of Pin1 in the aging process has been suggested based on the phenotype of Pin1-knockout mice and its interaction with lifespan regulator protein, p66 (Shc) . In this study, we utilize a proteomic approach and identify peroxiredoxin 1 (PRDX1), another regulator of aging, as a novel Pin1 binding protein. Pin1 binds to PRDX1 through interacting with the phospho-Thr ( 90) -Pro ( 91) motif of PRDX1, and this interaction is abolished when the Thr ( 90) of PRDX1 is mutated. The Pin1 binding motif, Thr-Pro, is conserved in the 2-Cys PRDXs, PRDX1-4 and the interactions between Pin1 and PRDX2-4 are also demonstrated. An increase in hydrogen peroxide buildup and a decrease in the peroxidase activity of 2-Cys PRDXs were observed in Pin1 (-/-) mouse embryonic fibroblasts (MEFs), with the activity of PRDXs restored when Pin1 was re-introduced into the cells. Phosphorylation of PRDX1 at Thr ( 90) has been shown to inhibit its peroxidase activity; however, how exactly the activity of PRDX1 is regulated by phosphorylation still remains unknown. Here, we demonstrate that Pin1 facilitates the protein phosphatase 2A-mediated dephosphorylation of PRDX1, which helps to explain the accumulation of the inactive phosphorylated form of PRDX1 in Pin1 (-/-) MEFs. Collectively, we identify Pin1 as a novel PRDX1 binding protein and propose a mechanism for Pin1 in regulating the metabolism of reactive oxygen species in cells.

In vitro rejoining of double strand breaks in genomic DNA.

Recent genetic and biochemical studies have provided important insights into the mechanism of nonhomologous end joining (NHEJ) pathways in higher eukaryotes, and have facilitated the functional characterization of several of its components including DNA-PKcs, Ku, DNA ligase IV, XRCC4, XLF/Cernunnos, and Artemis. Nevertheless, there is evidence that as of yet uncharacterized repair factors may contribute to the efficiency of NHEJ, for example by modulating the activity of known factors. Also, the discovery of alternative pathways of NHEJ that function as backup to the classical DNA-PK-dependent pathway of NHEJ has added yet another dimension in the set of activities involved. The biochemical characterization of NHEJ in higher eukaryotes has benefited significantly from in vitro plasmid-based end joining assays. However, because of differences in the organization and sequence of genomic and plasmid DNA, and because multiple pathways of NHEJ are operational, it is possible that different factors are preferred for the rejoining of DSBs induced in plasmid versus genomic DNA organized in chromatin. Here, we describe an in vitro assay that allows the study of DSB rejoining in genomic DNA. The assay utilizes as a substrate DSBs induced by various means in genomic DNA prepared from agarose-embedded cells after appropriate lysis. Two extremes in terms of state of DNA organization are described: "naked" DNA and DNA organized in chromatin. We describe the protocols developed to carry out and analyze these in vitro reactions, including procedures for the preparation of cell extract and the preparation of the substrate DNA ("naked" DNA or nuclei).

NPMc(+) AML cell line shows differential protein expression and lower sensitivity to DNA-damaging and p53-inducing anticancer compounds.

Nucleophosmin (NPM), an important regulator in p53 signaling pathway, is one of the most frequently mutated genes in acute myeloid leukemia (AML). In our previous study, we found that hexamethylene bisacetamide inducible protein 1 (HEXIM1) interacted with both wild-type NPM and cytoplasmic-misallocated NPMc(+) mutant, leading to an increase in RNA polymerase II transcription. Here, we examine the protein expression in wild-type NPM (AML2) and NPMc(+) mutant (AML3) AML cell lines. Significant lower levels of NPM, HEXIM1 and p53 proteins are detected in AML3 cells, and such differential protein expression is not regulated at transcriptional or post-translational stages. Effects of several anticancer compounds on cell viability of AML2 and AML3 cells are investigated. Compared to AML3 cells, AML2 cells are more sensitive to the treatment of the DNA-damaging compounds (doxorubicin and etoposide) and a specific p53-inducing compound (nutlin-3). However, no significant difference in cytotoxicity was observed when AML2 and AML3 cells were treated with cyclin-dependent kinase inhibitors, flavopiridol and CYC202. Our results provide a novel insight into the functional impact of the NPMc(+) mutation on protein expression and the potential approaches for selective therapy of AML.

Initial characterization of a low-molecular-weight factor enhancing the checkpoint response.

In higher eukaryotes, DNA double-strand breaks (DSBs) induced by ionizing radiation activate checkpoints that delay progression through the cell cycle. Compared to delays in other phases of the cell cycle, delays induced in G(2) are longer and frequently correlate with resistance to killing by radiation. Therefore, modulation of the G(2) checkpoint offers a means to modulate cellular radiosensitivity. Although compounds are known that reduce the G(2) checkpoint and act as radiosensitizers, compounds enhancing this checkpoint have not been reported. Here we summarize evidence for a factor with such properties. We show that a highly radioresistant rat embryo fibroblast (REF) cell line displays a strong G(2) checkpoint partly as a result of a factor excreted into the growth medium by nonirradiated cells. Various tests indicate that this G(2)-arrest modulating activity (GAMA) is a small molecule showing detectable retention only after passing through filters with a molecular weight cutoff limit of less than 1,000 Da. GAMA is heat stable and resistant to treatment with proteases or nucleases. Electroelution tests show that GAMA is uncharged at neutral pH, a result that is in agreement with the observed failure to bind S- or Q-Sepharose. Investigations on the mechanism of GAMA function indicate ligand-receptor interactions and allow the classification of cells as producers, responders or both. Compounds with properties such as those of GAMA bridge intercellular communication with the DNA damage response and may function as radioprotectors.

Expression of the Blomia tropicalis paramyosin Blo t 11 and its immunodominant peptide in insect cells.

Blo t 11, a dust-mite (Blomia tropicalis) paramyosin, is an allergen with significant IgE reactivity that has potential as a diagnostic/therapeutic reagent for house-dust-mite allergy. The present study describes the successful expression of Blo t 11 and its immunodominant peptide fD in insect cells using a baculovirus expression system. The Blo t 11 and fD genes were cloned into the pMelBacA vector and the resulting vectors were co-transfected into Sf9 insect cells with Bac-N-Blue DNA. Plaque assay was used to select for recombinant virus that was then used to infect High Five insect cells for protein expression. Secreted proteins were harvested by immuno-affinity purification using monoclonal antibodies to Blo t 11. Purified proteins were analysed by immunoblotting, N-terminal sequencing and ELISA. Immunoblot analyses revealed the full-length Blo t 11 cDNA expressed as a minor protein band of approx. 200 kDa and two major protein bands of approx. 60 and 70 kDa. Clones expressing fD cDNA fragment produced a protein of approx. 30 kDa that was confirmed to be fD by N-terminal sequencing. Approx. 4-7.5 mg/l of fD and 1 mg/l of Blo t 11 were obtained by affinity purification. ELISA results showed that human IgE reactivity to these recombinant allergens was lower as compared with that of the native Blo t 11, suggesting that these baculovirus-expressed allergens exhibiting reduced allergenicity could be useful for the development of immunotherapeutic reagents for mite allergy.

Molecular cloning of Blomia tropicalis allergens--a major source of dust mite allergens in the tropics and subtropics.

Allergic asthma, rhinitis, rhinoconjunctivitis and atopic dermatitis are the most common allergic disorders that are caused by the house dust mite (HDM). Beside pyroglyphid mites, the clinical importance of non-pyroglyphid mites has also been increasingly recognized in the recent years. Blomia tropicalis is the most important and ubiquitous mite species in tropical and subtropical regions of the world. Well-standardized and characterized allergens are essential for the diagnosis and therapy of house dust mite allergy as well as for the study of the pathophysiology of allergic disease. With the introduction of molecular biology, a number of house dust mite allergens were obtained by cloning the genes encoding the allergens. To date, seven allergens from Blomia tropicalis have been identified and characterized. Among these, Blo t 5, with unknown function, is the major allergen of Blomia tropicalis, with up to 92% of allergic patients sensitized to it. Native Blo t 5 has been purified and shown to consist of multiple isoforms. With advancing knowledge of these specific allergens, it is anticipated that targeted, effective, diagnostic, efficacious and safe therapeutic modalities would be developed.

An extensive study of human IgE cross-reactivity of Blo t 5 and Der p 5.

BACKGROUND: Dual sensitization by Blomia tropicalis and Dermatophagoides pteronyssinus mites is common in tropical and subtropical countries. The human IgE cross-reactivity between clinical important group 5 allergens, Blo t 5 and Der p 5, remains controversial. OBJECTIVE: This study was undertaken to assess the levels of the IgE cross-reactivity between Blo t 5 and Der p 5 by using sera from a large cohort of asthmatic children in subtropical and tropical countries. METHODS: Purified recombinant Blo t 5 and Der p 5 were produced in Pichia pastoris and tested against sera from 195 asthmatic children. The IgE cross-reactivity was examined by direct, inhibitory and competitive human IgE enzyme-linked immunosorbent assay as well as skin prick tests. RESULTS: The Blo t 5 IgE responses were 91.8% (134 of 146) and 73.5% (36 of 49) for Taiwanese and Malaysian sera, respectively. The Blo t 5 specific IgE titers were significantly higher than those of Der p 5 (P <.02). The correlation of IgE reactivity between Blo t 5 and Der p 5 was low, and only limited cross-reactivity was observed. This was further confirmed by the dose-response inhibition studies. Skin prick tests performed on asthmatic children in Thailand also showed differential IgE response to Blo t 5 and Der p 5. CONCLUSION: By using a large panel of asthmatic sera and a combination of in vitro and in vivo assays, the major allergen of B tropicalis in tropical and subtropical regions, Blo t 5, exhibits low levels of IgE cross-reactivity with homologous Der p 5. These findings suggest that highly specific clinical reagents are necessary for precise diagnosis and immunotherapeutic treatment of sensitization to group 5 mite allergens.

Identification of the major allergenic components in Blomia tropicalis and the relevance of the specific IgE in asthmatic patients.

BACKGROUND: Blomia tropicalis has been reported to be a clinically important allergen in house dust. High prevalence of sensitization to B. tropicalis has been noted in asthmatic patients in Taiwan; however, the allergenic components and its impact on asthmatic patients remain to be clarified. OBJECTIVE: To analyze the prevalence of IgE against B. tropicalis and each allergenic component in asthmatic patients. METHODS: A series of recombinant allergenic components were used for skin tests. The B. tropicalis specific IgE in the serum were measured using the Pharmacia CAP System and immunoblot analysis. RESULTS: A total of 131 patients were included in this study: 44% of these 131 patients were allergic to B. tropicalis, 43% of the 80 B. tropicalis-sensitive patients were allergic to Blo t 5, and 75% of the 65 Blo t 5-sensitive patients were allergic to Blo t 5 fragment 3 (Blo t 5 70-117). The sera IgE binding activity to B. tropicalis was repeatedly tested after Dermatophagoides pteronyssinus absorption, and results showed that most patients were concurrently sensitized to D. pteronyssinus and B. tropicalis. In addition, in 2 (18%) of 11 patients, the B. tropicalis sensitization was caused by the cross-reactivity of D. pteronyssinus. CONCLUSION: A high prevalence of B. tropicalis sensitization was detected in our asthmatic patients, and most of them were concurrently sensitized to D. pteronyssinus and B. tropicalis. The major allergenic component and its IgE binding fragments in Blo t 5 have been identified. These allergenic components can be used for the allergenic determination in B. tropicalis and for further immunotherapy.