ABSTRACT
The increasing need for mass spectrometric analysis of RNA molecules calls for a better understanding of their gas-phase fragmentation behaviors. In this study, we investigate the effect of terminal phosphate groups on the fragmentation spectra of RNA oligonucleotides (oligos) using high-resolution mass spectrometry (MS). Negative-ion mode collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) were carried out on RNA oligos containing a terminal phosphate group on either end, both ends, or neither end. We find that terminal phosphate groups affect the fragmentation behavior of RNA oligos in a way that is dependent on the precursor charge state and the oligo length. Specifically, for precursor ions of RNA oligos of the same sequence, those with 5'- or 3'-phosphate, or both, have a higher charge state distribution and lose the phosphate group(s) in the form of a neutral (H3PO4 or HPO3) or an anion ([H2PO4]- or [PO3]-) upon CID or HCD. Such a neutral or charged loss is most conspicuous for precursor ions of an intermediate charge state, e.g., 3- for 4-nt oligos or 4- and 5- for 8-nt oligos. This decreases the intensity of sequencing ions (a-, a-B, b-, c-, d-, w-, x-, y-, z-ions) and hence is unfavorable for sequencing by CID or HCD. Removal of terminal phosphate groups by calf intestinal alkaline phosphatase improved MS analysis of RNA oligos. Additionally, the intensity of a fragment ion at m/z 158.925, which we identified as a dehydrated pyrophosphate anion ([HP2O6]-), is markedly increased by the presence of a terminal phosphate group. These findings expand the knowledge base necessary for software development for MS analysis of RNA.
ABSTRACT
Four trinucleotides 5'-ATA-3' (I), 5'-ATC-3' (II), 5'-CTA-3' (III) and 5'-CTC-3' (IV) were introduced to interact with a diazido-based photoactivatable anticancer prodrug trans,trans,trans-[PtIV(N3)2(OH)2(py)2] (py = pyridine; 1) upon light irradiation. Using electrospray ionization mass spectrometry (ESI-MS), we aimed to investigate the possibility of 1,3-intrastrand crosslinks at adenine and/or cytosine in the trinucleotides via the bi-functional trans-[PtII(py)2]2+ species generated by photodecomposition of complex 1. The primary mass spectrometry results showed that although mono- and di-platinated trinucleotides bound by mono-functional trans-[PtII(N3)(py)2]+ species were the major platinated adducts, comparable amounts of bifunctional trans-[PtII(py)2]2+-bound trinucleotides were also observed. Further tandem mass spectrometry of the trans-[PtII(py)2]2+-bound trinucleotides showed the formation of 1,3-crosslinks between adenine-adenine, adenine-cytosine and cytosine-cytosine bases in the trinucleotides. The formation of such unique structures is not only distinct from the action modes of cisplatin with DNA but also an important complement to the acknowledged 1,3-GNG intrastrand crosslink by trans-Pt species, which may support the promising and distinct anticancer activities of such photoactivatable diazido Pt(IV) anticancer prodrugs and deserve further studies.
Subject(s)
Antineoplastic Agents , Prodrugs , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Organoplatinum Compounds/pharmacology , Organoplatinum Compounds/chemistry , Prodrugs/pharmacology , Prodrugs/chemistry , Adenine , CisplatinABSTRACT
Natural killer (NK) cells are promising alternatives for the production of "off-the-shelf" CAR products, posing a lower risk of cytokine release syndrome (CRS) than CAR-T cells. We synthesized four single VHH-directed anti-BCMA CARs, incorporating various intracellular regions (2B4 versus CD28) and hinge domains (CD28 versus IgG1) and ectopically producing IL-15. NK cells derived from peripheral blood (PB) were expanded ex vivo by K562-mbIL21 feeder cells. Stable CAR transduction was obtained through lentiviral transduction with the BaEV-Rless pseudotyped lentiviral vector. BCMA-CD28-IL15 CAR-NK cells with ectopic expression of IL-15 exhibited superior cytotoxicity were compared to BCMA-CD28 CAR-NK cells lacking IL-15 and BCMA-hIgG1-IL15 CAR-NK cells with an IgG1 hinge domain. We further assessed the cytotoxic capabilities of BCMA-2B4-IL15 CAR-NK cells with 2B4 intracellular domain. The BCMA-CD28-IL15 CAR-NK cells revealed stronger cytotoxicity and higher cytokine secretion against BCMA+ tumor cells than BCMA-2B4-IL15 CAR-NK cells in vitro. In the MM.1S-Luc mouse model, BCMA-CD28-IL15 CAR-NK inhibited the growth of tumor cells and prolonged mouse survival. These results show that the single VHH-directed BCMA CAR-NK cells exhibited remarkable specific killing ability, making them a potential candidate for immunotherapy in multiple myeloma treatment.
ABSTRACT
With the increasing throughput of modern sequencing instruments, the cost of storing and transmitting sequencing data has also increased dramatically. Although many tools have been developed to compress sequencing data, there is still a need to develop a compressor with a higher compression ratio. We present a two-step framework for compressing sequencing data in this paper. The first step is to repack original data into a binary stream, while the second step is to compress the stream with a LZMA encoder. We develop a new strategy to encode the original file into a LZMA highly compressed stream. In addition an FPGA-accelerated of LZMA was implemented to speedup the second step. As a demonstration, we present repaq as a lossless non-reference compressor of FASTQ format files. We introduced a multifile redundancy elimination method, which is very useful for compressing paired-end sequencing data. According to our test results, the compression ratio of repaq is much higher than other FASTQ compressors. For some deep sequencing data, the compression ratio of repaq can be higher than 25, almost four times of Gzip. The framework presented in this paper can also be applied to develop new tools for compressing other sequencing data. The open-source code of repaq is available at: https://github.com/OpenGene/repaq.
ABSTRACT
The interaction of a photoactivatable diazidodihydroxido Pt(IV) prodrug, trans,trans,trans-[Pt(N3)2(OH)2(py)2] (py = pyridine; 1), with a hexamer straight human telomeric DNA unit sequence (5'-T1T2A3G4G5G6-3', I) upon light irradiation was investigated by electrospray ionization mass spectroscopy (ESI-MS). In the primary mass spectrum, two major mono-platinated I adducts with the bound Pt moieties, trans-[PtII(N3)(py)2]+ (1') and trans-[PtII(py)2]2+ (1''), respectively, were detected. It is rare to observe such high abundance and nearly equal intensity platinated DNA adducts formed by these two PtII species because 1' is usually the only major reduced Pt(II) species produced by the photodecomposition of complex 1 in the presence of DNA while 1'' was rarely detected as the major reduced PtII species reported previously. Subsequent tandem mass spectrometric analysis by collision-induced dissociation (CID) showed that in the former adduct {I + 1'}2+, G6 and A3 were the platination sites. While in the latter adduct {I + 1''}2+, a potential intrastrand crosslink was speculated after G4 and G6 sites were identified. Additionally, other minor platinated adducts like di-platinated I adduct by 1' with platination sites at G4 and G6 and mono-platinated I adducts containing base oxidation were also detected by mass spectrometry. Due to the rich guanines and their sensitivity to oxidation, the oxidation induced by 1 most probably occurred at guanine. The oxidation adducts were proposed as 8-hydroxyl guanine, spiroiminodihydantoin (Sp), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), 5-guanidinohydantoin (Gh), and/or dehydroguanidinohydantoin (DGh) referring to previous reports. The obtained results provide useful chemical information about the photoreaction between photoactivatable Pt(IV) anticancer prodrugs and human telomeric DNA. Such special damages of Pt(IV) prodrugs on human telomeric DNA implicate its active role in the mechanism of Pt(IV) prodrugs and further support the unique sequence-dependent photointeraction profile of complex 1 reacting with DNA.
Subject(s)
Antineoplastic Agents , Prodrugs , Humans , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Prodrugs/chemistry , Organoplatinum Compounds/pharmacology , Organoplatinum Compounds/chemistry , DNA/chemistry , DNA Adducts , Guanine/chemistryABSTRACT
Mass spectrometry (MS)-based analysis of RNA oligonucleotides (oligos) plays an increasingly important role in the development of RNA therapeutics and epitranscriptomics research. However, MS fragmentation behaviors of RNA oligomers are understood insufficiently. Herein, we characterized the negative-ion-mode fragmentation behaviors of 26 synthetic RNA oligos containing four to eight nucleotides using collision-induced dissociation (CID) on a high-resolution, accurate-mass instrument. We found that in CID spectra acquired under the normalized collision energy (NCE) of 35%, approximately 70% of the total peak intensity was attributed to sequencing ions (a-B, a, b, c, d, w, x, y, z), around 25% of the peak intensity came from precursor ions that experienced complete or partial loss of a nucleobase in the form of either a neutral or an anion, and the remainder were internal ions and anionic nucleobases. The top five sequencing ions were the y, c, w, a-B, and a ions. Furthermore, we observed that CID fragmentation behaviors of RNA oligos were significantly impacted by their precursor charge. Specifically, when the precursors had a charge from 1- to 5-, the fractional intensity of sequencing ions decreased, while that of precursors that underwent either neutral or charged losses of a nucleobase increased. Additionally, we found that RNA oligos containing 3'-U tended to produce precursors with HNCO and/or NCO- losses, which presumably corresponded to isocyanic acid and cyanate anion, respectively. These findings provide valuable insights for better comprehending the mechanism behind RNA fragmentation by MS/MS, thereby facilitating the future automated identification of RNA oligos based on their CID spectra in a more efficient manner.
Subject(s)
Oligonucleotides , Tandem Mass Spectrometry , Oligonucleotides/chemistry , Tandem Mass Spectrometry/methods , RNA , Ions/chemistry , Anions , Spectrometry, Mass, Electrospray IonizationABSTRACT
CpG and its cytosine-methylated counterpart (5mCpG) are a unique reversible pair of sequences in regulating the expression of genes epigenetically. As DNA is the potential target of Pt-based anticancer metallodrugs, herein, we comparatively investigate the interactions of 5'-CpG and 5'-5mCpG with a photoactivatable anticancer Pt(IV) prodrug, trans,trans,trans-[PtIV(N3)2(OH)2(py)2] (1; py = pyridine), to explore the effects of methylation on the platination and ROS-induced oxidation of the CpG motif. Mono-platinated dinucleotides were demonstrated by ESI-MS to be the main products for both 5'-CpG and 5'-5mCpG with the bound Pt moiety as [PtII(N3)(py)2] generated by the photodecomposition of complex 1 under irradiation with blue light, accompanied by the formation of less abundant di-platinated adducts. G-N7 and C-N3/5mC-N3 were shown to be the major and minor platination sites, respectively, with G-N1 as the third and weakest platination site, in particular, in di-platinated products. Moreover, platinated dinucleotides associated with guanine and/or cytosine oxidation were also observed. Apart from 8-oxo-guanine (oxG) and N-formylamidoiminohydantoin (RedSp) reported previously, novel oxidation adducts 5-guanidinohydantoin (Gh) derived from guanine and 1-carbamoyl-4,5-dihydroxy-2-oxoimidazolidine (ImidCyt) derived from cytosine in CpG, and diimino imidazole (DIz) and 2,5-diaminoimidazol-4-one (imidazolone, Iz) derived from guanine and Imid5mCyt derived from 5mC in 5mCpG were proposed according to MS information. These results showed that methylation exerted little effects on the platination modes of CpG, but triggered distinct oxidation pathways of CpG, perhaps causing discriminated DNA damage to CpG-rich genes. This work provides novel insights into the role of the anticancer photoactivatable Pt(IV) prodrug through damaging the epigenetically modified DNA sequences.
Subject(s)
Antineoplastic Agents , Prodrugs , Antineoplastic Agents/pharmacology , Prodrugs/pharmacology , Organoplatinum Compounds/pharmacology , Guanine , Cytosine , DNA AdductsABSTRACT
Background: Consolidative allogeneic hematopoietic stem cells (allo-HSCs) after chimeric antigen receptor T cells (CAR-T) therapy is an emerging modality in hematologic malignancies. Knowledge about the success of allogeneic hematopoietic stem cell transplantation (allo-HSCT) after CAR-T therapy without a conditioning regimen is limited. Case presentation: We report a patient with relapsed/refractory (R/R) Ph+ B-cell acute lymphoblastic leukemia (ALL) who underwent anti-CD19 CAR-T immunotherapy. After 1 month of treatment, bone marrow hyperplasia remained reduced with no hematopoietic improvements. In line with this, allogeneic hematopoietic stem cells (HSCs) were extracted from an HLA-matched sibling donor and administered to the patient on day 33 after CAR-T cell therapy to support hematopoiesis. On day 40, the level of immature bone marrow lymphocytes was at 0% and minimal residual disease-negative, and the fusion gene BCR/ABL 190 was negative. Chimerism analysis showed full donor chimerism. Three months after CAR-T cells infusion, the patient was still in complete remission with full donor chimerism. However, decreased liver function with skin pigmentation and festering, indicative of acute graft versus host disease, was noted. The treatment was halted owing to financial reasons. Conclusion: We report the successful engraftment of allogeneic HSCs using CAR-T cell therapy as a conditioning regimen for R/R B-ALL patients.
Subject(s)
Hematopoietic Stem Cell Transplantation , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Receptors, Chimeric Antigen , Hematopoietic Stem Cells , Humans , T-LymphocytesABSTRACT
The bispecific T-cell engager (BiTE) blinatumomab against CD19 and CD3 has emerged as the most successful bispecific antibody (bsAb) to date; however, a significant proportion of patients do not respond to the treatments or eventually experience relapse after an initial response, and the recurrence rate increases significantly due to escape or downregulation of the CD19 antigen. To enhance antitumor efficacy and overcome potential immune escape, we developed a novel approach to design a CD19/CD22/CD3 trispecific antibody (tsAb) by site-specifically fusing anti-CD19 scFv (FMC63) and anti-CD22 nanobody (Nb25) to the defined sites of the CD3 antigen-binding fragment (Fab, SP34). This strategy allows for the optimal formation of immune synapses mediated by CD19/CD22/CD3 between target cells and T cells. Optimized tsAb can be superior for inducing T-cell-specific cytotoxicity and cytokine production against CD19+ and/or CD22+ tumor cells compared to other tsAb formats, and demonstrated significantly enhanced antitumor efficacy and the ability to overcome immune escape compared with the corresponding bsAbs alone or in combination, as well as with blinatumomab. In addition, tsAb treatment can lead to the long-term elimination of primary B-ALL patient samples in the PDX model and significantly prolong survival. This novel approach provides unique insight into the structural optimization of T-cell-redirected multispecific antibodies using site-specific recombination, and may be broadly applicable to heterogeneous and resistant tumor populations as well as solid tumors.
Subject(s)
Antibodies, Bispecific , Burkitt Lymphoma , Lymphoma, B-Cell , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Humans , Antigens, CD19 , CD3 Complex , Neoplasm Recurrence, Local/drug therapy , Antibodies, Bispecific/pharmacology , Antibodies, Bispecific/therapeutic use , Lymphoma, B-Cell/pathology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Burkitt Lymphoma/drug therapy , Cytokines , Sialic Acid Binding Ig-like Lectin 2ABSTRACT
Photoactivatable Pt(IV) anticancer prodrug trans,trans,trans-[Pt(N3)2(OH)2(pyridine)2] (1) has been shown to bind to and induce oxidation of all four DNA nucleobases. Herein, to further render the binding spectrum of complex 1 to nucleic acids, the interaction between complex 1 and uridine, an exclusive RNA component, was investigated by electrospray ionization mass spectrometry (ESI-MS) and NMR spectroscopy. The results showed that complex 1 can bind to uridine through the N3 (major) and O4 (minor) sites upon light irradiation to form the major mono-platinated uridine adduct and the minor di-platinated uridine adduct. Moreover, mono-platinated uridine associated with the oxidation of uridine to 5-hydroxyuridine and 6-hydroxyuridine was also observed. This is the first report that the photoactivatable Pt(IV) prodrug binds to and induces the oxidation of uridine, and also the last piece of the puzzle for the interactions of complex 1 with nucleobases. Combined with our previous results about the interactions between complex 1 and DNA bases, these data showed a wide interaction spectrum of this kind of photoactivatable diazido Pt(IV) prodrugs with nucleobases through such dual-action modes, strongly suggesting that RNA may be a potential target of Pt(IV) prodrugs.
Subject(s)
Antineoplastic Agents , Prodrugs , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , DNA/chemistry , Organoplatinum Compounds/chemistry , Organoplatinum Compounds/pharmacology , Prodrugs/chemistry , RNA , UridineABSTRACT
Background: Lung cancer is the leading cause of cancer-related death worldwide, of which lung adenocarcinoma (LUAD) is one of the main histological subtypes. Mitochondria are vital for maintaining the physiological function, and their dysfunction has been found to be correlated with tumorigenesis and disease progression. Although, some mitochondrial-related genes have been found to correlate with the clinical outcomes of multiple tumors solely. The integrated relationship between nuclear mitochondrial genes (NMGs) and the prognosis of LUAD remains unclear. Methods: The list of NMGs, gene expression data, and related clinical information of LUAD were downloaded from public databases. Bioinformatics methods were used and obtained 18 prognostic related NMGs to construct a risk signature. Results: There were 18 NMGs (NDUFS2, ATP8A2, SCO1, COX14, COA6, RRM2B, TFAM, DARS2, GARS, YARS2, EFG1, GFM1, MRPL3, MRPL44, ISCU, CABC1, HSPD1, and ETHE1) identified by LASSO regression analysis. The mRNA expression of these 18 genes was positively correlated with their relative linear copy number alteration (CNA). Meanwhile, the established risk signature could effectively distinguish high- and low-risk patients, and its predictive capacity was validated in three independent gene expression omnibus (GEO) cohorts. Notably, a significantly lower prevalence of actionable EGFR alterations was presented in patients with high-risk NMGs signature but accompanied with a more inflame immune tumor microenvironment. Additionally, multicomponent Cox regression analysis showed that the model was stable when risk score, tumor stage, and lymph node stage were considered, and the 1-, 3-, and 5-year AUC were 0.74, 0.75, and 0.70, respectively. Conclusion: Together, this study established a signature based on NMGs that is a prognostic biomarker for LUAD patients and has the potential to be widely applied in future clinical settings.
ABSTRACT
Tumor-infiltrating immune cells have been implicated in the tumorigenesis and progression of esophageal squamous cell carcinoma (ESCC). However, the functionalities and clinical significance of immune cells remain largely unveiled. In this study, the gene expression data from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) were extracted. The relative infiltrating levels were estimated by single-sample gene set enrichment analysis. Some cytotoxic immune cells were attenuated, and resting cytotoxic immune cells were accumulated in ESCC. Remarkably, we also observed that infiltrating levels of macrophage M2 and resting natural killer (NK) cells were increased in nonresponders of CRT, and T cells that had anticancer activities such as activated memory CD4 and T helper 2 (Th2) cells were significantly reduced in ESCC tissues of the nonresponders. Moreover, the high infiltrations of the resting natural killer (NK) and dendritic cell (DC) were observed to result in a shorter overall survival in ESCC. Consistently, high expression of immune checkpoint genes, CTLA4 and HAVCR2, was associated with poor prognosis. Furthermore, STAT5B, a key transcription factor, as well as its target genes, involved in the regulation of T cells, was significantly downregulated in ESCC, especially subgroup I, indicating that downregulation of STAT5B might be associated with reduced T cell-mediated anticancer activity. In conclusion, the present study significantly improved our understanding of the regulatory roles of immune cells in ESCC.
Subject(s)
Esophageal Neoplasms/immunology , Esophageal Squamous Cell Carcinoma/immunology , Lymphocytes, Tumor-Infiltrating/immunology , Down-Regulation/genetics , Esophageal Neoplasms/drug therapy , Esophageal Neoplasms/genetics , Esophageal Neoplasms/radiotherapy , Esophageal Squamous Cell Carcinoma/drug therapy , Esophageal Squamous Cell Carcinoma/genetics , Esophageal Squamous Cell Carcinoma/radiotherapy , Gene Expression Regulation, Neoplastic , Humans , Immune Checkpoint InhibitorsABSTRACT
SMAD4 is mutated in human lung cancer, but the underlying mechanism by which Smad4 loss-of-function (LOF) accelerates lung cancer metastasis is yet to be elucidated. Here, we generate a highly aggressive lung cancer mouse model bearing conditional KrasG12D, p53fl/fl LOF and Smad4fl/fl LOF mutations (SPK), showing a much higher incidence of tumor metastases than the KrasG12D, p53fl/fl (PK) mice. Molecularly, PAK3 is identified as a downstream effector of Smad4, mediating metastatic signal transduction via the PAK3-JNK-Jun pathway. Upregulation of PAK3 by Smad4 LOF in SPK mice is achieved by attenuating Smad4-dependent transcription of miR-495 and miR-543. These microRNAs (miRNAs) directly bind to the PAK3 3'UTR for blockade of PAK3 production, ultimately regulating lung cancer metastasis. An inverse correlation between Smad4 and PAK3 pathway components is observed in human lung cancer. Our study highlights the Smad4-PAK3 regulation as a point of potential therapy in metastatic lung cancer.
Subject(s)
Lung Neoplasms/pathology , MicroRNAs/genetics , Smad4 Protein/metabolism , p21-Activated Kinases/metabolism , 3' Untranslated Regions , Animals , Cell Movement , Gene Expression Regulation, Neoplastic , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , Loss of Function Mutation , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Mice , MicroRNAs/metabolism , Neoplasm Metastasis , Proto-Oncogene Proteins c-jun/metabolism , Signal Transduction , Smad4 Protein/genetics , Transcriptional Activation , p21-Activated Kinases/geneticsABSTRACT
OBJECTIVE: Chimeric antigen receptor-modified T (CAR-T) cells have shown impressive results against relapsed/refractory B cell malignancies. However, the traditional manufacture of CAR-T cells requires leukapheresis to isolate large amounts of peripheral blood T cells, thus making some patients ineligible for the procedure. METHODS: We developed a simple method for CAR-T cell preparation requiring small volumes of peripheral blood. First, CD3+ T cells isolated from 50 mL peripheral blood from patients (B-cell malignancies) were stimulated with immobilized anti-CD3/RetroNectin in 6-well plates and then transduced with CAR-expressing lentiviral vector. After 4 d, the T cells were transferred to culture bags for large-scale CAR-T cell expansion. In vitro and animal experiments were performed to evaluate the activity of the manufactured CAR-T cells. Finally, 29 patients with B-cell acute lymphoblastic leukemia (B-ALL) and 9 patients with B-cell lymphoma were treated with the CAR-T cells. RESULTS: The CAR-T cells were expanded to 1-3 × 108 cells in 8-10 d and successfully killed B cell-derived malignant tumor cells in vitro and in vivo. For patients with B-ALL, the complete remission rate was 93% 1 month after CAR-T cell infusion; after 12 months, the overall survival (OS) and leukemia-free survival rates were 69% and 31%, respectively. For patients with lymphoma, the objective response rate (including complete and partial remission) was 78% 2 months after CAR-T cell infusion, and after 12 months, the OS and progression-free survival rates were 71% and 43%, respectively. Cytokine-release syndrome (CRS) occurred in 65.51% and 55.56% of patients with B-ALL and B-cell lymphoma, respectively; severe CRS developed in 20.69% of patients with B-ALL and in no patients with lymphoma. CONCLUSIONS: Our novel method can generate sufficient numbers of CAR-T cells for clinical use from 50-100 mL peripheral blood, thus providing an alternative means of CAR-T cell generation for patients ineligible for leukapheresis.
Subject(s)
B-Cell Maturation Antigen/immunology , Drug Resistance, Neoplasm , Immunoglobulin Heavy Chains/metabolism , Immunotherapy, Adoptive/methods , Multiple Myeloma/therapy , Neoplasm Recurrence, Local/therapy , Single-Chain Antibodies/metabolism , Humans , Immunoglobulin Heavy Chains/genetics , Multiple Myeloma/immunology , Multiple Myeloma/pathology , Neoplasm Recurrence, Local/immunology , Neoplasm Recurrence, Local/pathology , Remission Induction , Single-Chain Antibodies/geneticsABSTRACT
Chimeric antigen receptor (CAR) T therapy represents a form of immune cellular therapy with clinical efficacy and a specific target. A typical chimeric antigen receptor (CAR) construct consists of an antigen binding domain, a transmembrane domain, and a cytoplasmic domain. Nanobodies have been widely applied as the antigen binding domain of CAR-T due to their small size, optimal stability, high affinity, and manufacturing feasibility. The nanobody-based CAR structure has shown a proven function in more than ten different tumor-specific targets. After being transduced in Jurkat cells, natural killer cells, or primary T cells, the resulting nanobody-based CAR-T or CAR-NK cells demonstrate anti-tumor effects both in vitro and in vivo. Interestingly, anti-BCMA CAR-T modulated by a single nanobody or bi-valent nanobody displays comparable clinical effects with that of single-chain variable fragment (scFv)-modulated CAR-T. The application of nanobodies in CAR-T therapy has been well demonstrated from bench to bedside and displays great potential in forming advanced CAR-T for more challenging tasks.
Subject(s)
Immunotherapy, Adoptive/methods , Single-Domain Antibodies , Humans , Jurkat Cells , Killer Cells, Natural/immunology , Single-Chain Antibodies/immunologyABSTRACT
Photoactivatable diazidodihydroxido Pt(iv) complex trans,trans,trans-[Pt(N3)2(OH)2(py)2] (1; py = pyridine) is a promising anticancer agent which can be activated by visible light to induce cancer cell death. DNA has been thought to be involved in the mechanism of action of this kind of Pt(iv) prodrug. However, the detailed photodecomposition pathways of complex 1 and its interaction modes with DNA are complex. Herein we report that upon light irradiation complex 1 can bind to all four nucleosides covalently with the reduced Pt(ii) species. Moreover, apart from the covalent coordination, various oxidation adducts of these four nucleosides induced by the reactive oxidative species (ROS) generated during the photoactivation of the complex 1 have also been identified, especially the induced oxidation of adenosine and cytidine which was firstly reported for this kind of photoactivatable Pt(iv) prodrug. Such dual-action may contribute to the highly potent photo-antiproliferativity of complex 1 towards cancer cells, which may account for the unique mechanism of action of the photoactivatable diazido Pt(iv) anticancer complexes.
Subject(s)
Antineoplastic Agents/chemistry , Azides/chemistry , Nucleosides/chemistry , Organoplatinum Compounds/chemistry , Prodrugs/chemistry , Molecular Structure , Oxidation-Reduction , Photochemical Processes , Spectrometry, Mass, Electrospray IonizationABSTRACT
Platinum based anticancer agents are widely applied in clinic and their major target is believed to be DNA. Herein, the interaction of a photoactivatable diazido Pt(iv) anticancer prodrug trans,trans,trans-[Pt(N3)2(OH)2(py)2] (py = pyridine; 1) with a 15-mer single-G-containing oligodeoxynucleotide (ODN I: 5'-CT2CTCTTG8T9CT11TCTC-3') was investigated by mass spectrometric methods. Up to penta-platinated ODN I adducts were identified from primary mass spectra while the mono- and di-platinated adducts had the highest intensity. Fragmentation of mono-, di- and tri-platinated I adducts in tandem MS revealed that T2, G8, T11 and T9 are binding sites. No cytosine sites were identified which may be due to the facile loss of Pt adducts from cytosine during CID. The intensity of {Pt(py)2}-bound adducts was comparable to that of {Pt(N3)(py)2}-bound adducts, indicating that the photo-reduction pathway of complex 1 from Pt(iv) to Pt(ii) through two one-electron donations from two azides was substantial. Moreover, no transformation of N3 to NH3 on the {Pt(N3)(py)2}-bound adducts was observed, whereas it is very popular during the reactions of complexes with short ODNs or mono-nucleotides. The oxidation on I induced by the reactive oxygen species (ROS) formed by the photodecomposition of complex 1 was significant, and the oxidation of G8 to 8-hydroxyguanine (8-OH-G), spiroiminodihydantoin (Sp) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) was discovered. These results unambiguously revealed a sequence-length-dependent photochemical reactivity of complex 1 when it interacted with different ODNs, providing deeper understanding in the reactivity of photoactivatable diazido anticancer Pt(iv) prodrugs to DNA.
Subject(s)
Antineoplastic Agents/chemistry , Azides/chemistry , Oligodeoxyribonucleotides/chemistry , Organoplatinum Compounds/chemistry , Prodrugs/chemistry , Antineoplastic Agents/chemical synthesis , Molecular Structure , Organoplatinum Compounds/chemical synthesis , Photochemical Processes , Prodrugs/chemical synthesisABSTRACT
BACKGROUND: Reactivation of hepatitis B virus (HBV) infection is a well-recognized complication in patients with chronic or resolved HBV infection undergoing anticancer therapy. There is a risk of HBV reactivation after infusion of chimeric antigen receptor (CAR) T cells for patients with refractory/relapsed (R/R) multiple myeloma (MM). METHODS: We administered B cell maturation antigen (BCMA) CAR-T cell by infusion to nine patients with R/R MM with chronic or resolved HBV infection. Patient serum was analyzed to determine the expression of five components of HBV and the copy number of HBV DNA. HBV reactivation was defined if a patient re-exhibited hepatitis B surface antigen (HBsAg) or HBV DNA regrowth after CAR-T therapy. RESULTS: In one patient who was HBsAg-positive, no HBV reactivation was observed during the follow-up period of 9.8 months after administration of anti-HBV drugs before and after CAR-T therapy. Among eight patients with MM who had resolved HBV infection, two patients administered prophylactic anti-HBV drugs did not exhibit HBV reactivation. Of the six patients who did not use prophylactic antiviral drugs, five did not exhibit HBV reactivation, while one showed recurrence of HBsAg without detection of HBV DNA or damage to liver function. The best objective response rate was 100%, and the progression-free survival (PFS) at 12 months was of 88.89% (median PFS was not observed). CONCLUSIONS: These findings showed that BCMA CAR-T cell therapy could be used in patients with R/R MM with chronic or resolved HBV infection and that antiviral drugs should be administered in these patients during CAR-T cell therapy.