Antigen experience history directs distinct functional states of CD8+ CAR T cells during the anti-leukemia response.

Chimeric antigen receptor T cells are an effective therapy for B-lineage malignancies. However, many patients relapse and this therapeutic has yet to show strong efficacy in other hematologic or solid tumors. One opportunity for improvement lies in the ability to generate T cells with desirable functional characteristics. Here, we dissect the biology of CD8+ CAR T cells (CAR8) by controlling whether the T cell has encountered cognate TCR antigen prior to CAR generation. We find that prior antigen experience influences multiple aspects of in vitro and in vivo CAR8 functionality, resulting in superior effector function and leukemia clearance in the setting of limiting target antigen density compared to antigen-inexperienced T cells. However, this comes at the expense of inferior proliferative capacity, susceptibility to phenotypic exhaustion and dysfunction, and inability to clear wildtype leukemia in the setting of limiting CAR+ cell dose. Epigenomic and transcriptomic comparisons of these cell populations identified overexpression of the Runx2 transcription factor as a novel strategy to enhance CAR8 function, with a differential impact depending on prior cell state. Collectively, our data demonstrate that prior antigen experience determines functional attributes of a CAR T cell, as well as amenability to functional enhancement by transcription factor modulation.

Genetically engineered macrophages persist in solid tumors and locally deliver therapeutic proteins to activate immune responses.

BACKGROUND: Though currently approved immunotherapies, including chimeric antigen receptor T cells and checkpoint blockade antibodies, have been successfully used to treat hematological and some solid tumor cancers, many solid tumors remain resistant to these modes of treatment. In solid tumors, the development of effective antitumor immune responses is hampered by restricted immune cell infiltration and an immunosuppressive tumor microenvironment (TME). An immunotherapy that infiltrates and persists in the solid TME, while providing local, stable levels of therapeutic to activate or reinvigorate antitumor immunity could overcome these challenges faced by current immunotherapies. METHODS: Using lentivirus-driven engineering, we programmed human and murine macrophages to express therapeutic payloads, including Interleukin (IL)-12. In vitro coculture studies were used to evaluate the effect of genetically engineered macrophages (GEMs) secreting IL-12 on T cells and on the GEMs themselves. The effects of IL-12 GEMs on gene expression profiles within the TME and tumor burden were evaluated in syngeneic mouse models of glioblastoma and melanoma and in human tumor slices isolated from patients with advanced gastrointestinal malignancies. RESULTS: Here, we present a cellular immunotherapy platform using lentivirus-driven genetic engineering of human and mouse macrophages to constitutively express proteins, including secreted cytokines and full-length checkpoint antibodies, as well as cytoplasmic and surface proteins that overcomes these barriers. GEMs traffic to, persist in, and express lentiviral payloads in xenograft mouse models of glioblastoma, and express a non-signaling truncated CD19 surface protein for elimination. IL-12-secreting GEMs activated T cells and induced interferon-gamma (IFNγ) in vitro and slowed tumor growth resulting in extended survival in vivo. In a syngeneic glioblastoma model, IFNγ signaling cascades were also observed in mice treated with mouse bone-marrow-derived GEMs secreting murine IL-12. These findings were reproduced in ex vivo tumor slices comprised of intact MEs. In this setting, IL-12 GEMs induced tumor cell death, chemokines and IFNγ-stimulated genes and proteins. CONCLUSIONS: Our data demonstrate that GEMs can precisely deliver titratable doses of therapeutic proteins to the TME to improve safety, tissue penetrance, targeted delivery and pharmacokinetics.

Human macrophages engineered to secrete a bispecific T cell engager support antigen-dependent T cell responses to glioblastoma.

BACKGROUND: Targeted and effective treatment options are needed for solid tumors, including glioblastoma (GBM), where survival rates with standard treatments are typically less than 2 years from diagnosis. Solid tumors pose many barriers to immunotherapies, including therapy half-life and persistence, tumor penetrance, and targeting. Therapeutics delivered systemically may not traffic to the tumor site. If cellular therapies or drugs are able to access the tumor site, or can be delivered directly within the tumor, treatments may not persist for the duration necessary to reduce or eliminate tumor burden. An approach that allows durable and titratable local therapeutic protein delivery could improve antitumor efficacy while minimizing toxicities or unwanted on-target, off-tissue effects. METHODS: In this study, human monocyte-derived macrophages were genetically engineered to secrete a bispecific T cell engager (BiTE) specific to the mutated epidermal growth factor variant III (EGFRvIII) expressed by some GBM tumors. We investigated the ability of lentivirally modified macrophages to secrete a functional BiTE that can bind target tumor antigen and activate T cells. Secreted BiTE protein was assayed in a range of T cell functional assays in vitro and in subcutaneous and intracranial GBM xenograft models. Finally, we tested genetically engineered macrophages (GEMs) secreting BiTE and the proinflammatory cytokine interleukin (IL)-12 to amplify T cell responses in vitro and in vivo. RESULTS: Transduced human macrophages secreted a lentivirally encoded functional EGFRvIII-targeted BiTE protein capable of inducing T cell activation, proliferation, degranulation, and killing of antigen-specific tumor cells. Furthermore, BiTE secreting macrophages reduced early tumor burden in both subcutaneous and intracranial mouse models of GBM, a response which was enhanced using macrophages that were dual transduced to secrete both the BiTE protein and single chain IL-12, preventing tumor growth in an aggressive GBM model. CONCLUSIONS: The ability of macrophages to infiltrate and persist in solid tumor tissue could overcome many of the obstacles associated with systemic delivery of immunotherapies. We have found that human GEMs can locally and constitutively express one or more therapeutic proteins, which may help recruit T cells and transform the immunosuppressive tumor microenvironment to better support antitumor immunity.

Formulation and chemical characterization of Clerodendrum infortunatum leaf extract in relation to anti-fungal activity.

The study explored to develop an eco-friendly herbal fungicide from chloroform extract of Clerodendrum infortunatum leaves. The extract upon activity guided purification using flash chromatography yielded eight fractions F3 through F10. Total phenol and flavonoid contents in the fractions ranged from 0.12-48.25 mg GAE/g and 0.03-25.29 mg QE/g. LC-MS/MS analysis confirmed the identification of seven phenolic acids across different fractions, the total of which varied between 0 and 2.17 mg/g. Emulsifiable Concentrate (20%) formulation was made with the extract and fractions and tested against Phomopsis vexans causing fruit rot disease in brinjal. Of the various fractions, F8 displayed highest antifungal activity (ED50 = 46.8 µg/ml). Antifungal activity of leaf extract/fractions was correlated with total phenol, total flavonoid and total phenolic acids (r = -0.60 to -0.69). Among the phenolic acids, benzoic acid showed maximum antifungal activity followed by t-cinnamic acid. The relationship between phenolic composition and activity is also reported.

Antisense-mediated redirection of mRNA splicing.

Antisense technology has been used to study basic biological processes, and to block these processes when they deleteriously lead to human disease. A separate, equally important application of antisense technology is to upregulate the gene expression lost in the diseased state by shifting alternative splicing of pre-messenger RNA. This strategy has commonly relied upon the use of antisense oligonucleotides; however, another approach is to use a plasmid construct to generate antisense RNA inside the cell. Antisense therapeutics based on expression vectors and viral vectors offers a gene therapy approach, whereas those based on oligonucleotides offers a more drug like approach.

Spontaneous and MNNG-induced reversion of an EGFP construct in HeLa cells: an assay for observing mutations in living cells by fluorescent microscopy.

A HeLa cell line stably expressing the enhanced green fluorescence protein (EGFP) gene, interrupted by the HBB IVS2-654 intron, was studied without treatment and after treatment with a single standard dose of 15 microM of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). This assay was done in order to prove that such a construct can revert by a variety of mechanisms and that it produces a visible phenotype, i.e., green fluorescence. The system permits visual detection of living mutant cells among a background of non-mutant cells and does not require a selective medium. The results show that the construct reverts by large deletions (-62, -100, and -162 bp), small insertions (+4 bp), small rearrangements (19 bp duplication), base substitutions at purines (G652, G653, A655, G579), and a pyrimidine (T654) between nucleotide positions 579 and 837. Splice-site mutations were recovered, and some of the mechanisms underlying these mutations are discussed. Because of the ease of detection of revertant cells under fluorescent light and the wide variety of mutations that can be recovered, further development of this system could make it a useful new mammalian cell mutagenicity assay.

Nuclear antisense effects of neutral, anionic and cationic oligonucleotide analogs.

The antisense activity of oligomers with 2'-O-methyl (2'-O-Me) phosphorothioate, 2'-O-methoxyethyl (2'-O-MOE) phosphorothioate, morpholino and peptide nucleic acid (PNA) backbones was investigated using a splicing assay in which the modified oligonucleotides blocked aberrant and restored correct splicing of modified enhanced green fluorescent protein (EGFP) precursor to mRNA (pre-mRNA), generating properly translated EGFP. In this approach, antisense activity of each oligomer was directly proportional to up-regulation of the EGFP reporter. This provided a positive, quantitative readout for sequence-specific antisense effects of the oligomers in the nuclei of individual cells. Nuclear localization of fluorescent labeled oligomers confirmed validity of the functional assay. The results showed that the free uptake and the antisense efficacy of neutral morpholino derivatives and cationic PNA were much higher than that of negatively charged 2'-O-Me and 2'-O-MOE congeners. The effects of the PNA oligomers were observed to be dependent on the number of L-lysine (Lys) residues at the C-terminus. The experiments suggest that the PNA containing Lys was taken up by a mechanism similar to that of cell-penetrating homeodomain proteins and that the Lys tail enhanced intracellular accumulation of PNA oligomer without affecting its ability to reach and hybridize to the target sequence.

Antisense effects in the cell nucleus: modification of splicing.

The surprisingly small number of human genes, which has recently been estimated to be approximately 30,000, suggests that RNA processing, and in particular alternative RNA splicing, is in large part responsible for the diversity of gene products in human and mammalian cells. The ability to manipulate alternative splicing using antisense oligonucleotides, as demonstrated in several studies during the past year, makes it an important and attractive approach to altering gene expression. A review of these studies leads to the conclusion that antisense oligonucleotides, whether designed to affect the cytoplasmic mRNA or nuclear pre-mRNA, function predominantly in the nucleus and not the cytoplasm.

Strong RNA splicing enhancers identified by a modified method of cycled selection interact with SR protein.

A modified method of cycled selection was used to characterize splicing enhancers for exon inclusion from a pool of beta-globin-based three exon/two intron pre-mRNAs with a variable number of random nucleotides incorporated in the internal exon. The pre-mRNAs generated by this method contained random sequences ranging from 0 to 18 nucleotides in length. This method was used to isolate particular splicing enhancer motifs from a previously enriched pool of extremely diverse enhancers. After four cycles of selection for mRNA containing the internal exon, a distinct enhancer motif (GACGAC...CAGCAG) was highly enriched. This motif served as strong splicing enhancers in a heterogeneous exon. We have shown here that the selected enhancer motif promotes exon inclusion through specific interaction with SRp30. We have also shown that although present in many of our selected splicing enhancers conforming to this motif, a typical purine-rich enhancer sequence is dispensable for either enhancer activity or binding with SRp30.

Persistence and metabolism of imidacloprid in different soils of West Bengal.

A laboratory experiment was performed to study the persistence of imidacloprid from two formulations (Confidor 200 g litre-1 SL and Gaucho 700 g kg-1 WS), and its metabolism in three different soils (Gangetic alluvial soil of Kalyani, lateritic soil of Jhargram and coastal alkaline soil of Canning) of West Bengal following application at 0.5 kg and 1.0 kg AIha-1. Dissipation of imidacloprid in soil followed first-order kinetics and DT50 values ranged from 28.7 to 47.8 days. The shortest half-lives (28.7 and 35.8 days) were observed in the lateritic soil of Jhargram for both liquid and powder formulations. The formation of two metabolites of imidacloprid, imidacloprid-urea and imidacloprid-olefin, was first detected on day 30 of degradation at 28 (+/- 1) degrees C in all three soils.

Modification of alternative splicing of Bcl-x pre-mRNA in prostate and breast cancer cells. analysis of apoptosis and cell death.

There is ample evidence that deregulation of apoptosis results in the development, progression, and/or maintenance of cancer. Since many apoptotic regulatory genes (e.g. bcl-x) code for alternatively spliced protein variants with opposing functions, the manipulation of alternative splicing presents a unique way of regulating the apoptotic response. Here we have targeted oligonucleotides antisense to the 5'-splice site of bcl-x(L), an anti-apoptotic gene that is overexpressed in various cancers, and shifted the splicing pattern of Bcl-x pre-mRNA from Bcl-x(L) to Bcl-x(S), a pro-apoptotic splice variant. This approach induced significant apoptosis in PC-3 prostate cancer cells. In contrast, the same oligonucleotide treatment elicited a much weaker apoptotic response in MCF-7 breast cancer cells. Moreover, although the shift in Bcl-x pre-mRNA splicing inhibited colony formation in both cell lines, this effect was much less pronounced in MCF-7 cells. These differences in responses to oligonucleotide treatment were analyzed in the context of expression of Bcl-x(L), Bcl-x(S), and Bcl-2 proteins. The results indicate that despite the presence of Bcl-x pre-mRNA in a number of cell types, the effects of modification of its splicing by antisense oligonucleotides vary depending on the expression profile of the treated cells.

Modification of alternative splicing by antisense oligonucleotides as a potential chemotherapy for cancer and other diseases.

It has been estimated that greater than 35% of all human genes undergo alternative splicing. The process of alternative splicing is highly regulated and disruption of a splicing pattern can produce splice variants that have different functions. Certain splice variants that are associated with induction of cell death, regulation of cellular proliferation and differentiation, cell signaling, and angiogenesis are present in a variety of cancers. Several of these cancer-related alternatively spliced genes will be discussed in this review. In addition, alternative splicing is associated with several genetic disorders such as beta-thalassemia, cystic fibrosis, and muscular dystrophy. Control of pre-mRNA splicing patterns with antisense oligonucleotides presents an attractive way to potentially treat and manage a variety of diseases. This review will discuss potential gene targets for antisense oligonucleotide induced modification of alternative splicing patterns. Furthermore, the chemistries and delivery strategies of antisense oligonucleotides will be discussed.

Restoration of correct splicing of thalassemic beta-globin pre-mRNA by modified U1 snRNAs.

The T-->G mutation at nucleotide 705 in the second intron of the beta-globin gene creates an aberrant 5' splice site and activates a 3' cryptic splice site upstream from the mutation. As a result, the IVS2-705 pre-mRNA is spliced via the aberrant splice sites leading to a deficiency of beta-globin mRNA and protein and to the genetic blood disorder thalassemia. We have shown previously that in cell culture models of thalassemia, aberrant splicing of beta-thalassemic IVS2-705 pre-mRNA was permanently corrected by a modified murine U7 snRNA that incorporated sequences antisense to the splice sites activated by the mutation. To explore the possibility of using other snRNAs as vectors for antisense sequences, U1 snRNA was modified in a similar manner. Replacement of the U1 9-nucleotide 5' splice site recognition sequence with nucleotides complementary to the aberrant 5' splice site failed to correct splicing of IVS2-705 pre-mRNA. In contrast, U1 snRNA targeted to the cryptic 3' splice site was effective. A hybrid with a modified U7 snRNA gene under the control of the U1 promoter and terminator sequences resulted in the highest levels of correction (up to 70%) in transiently and stably transfected target cells.

Restoration of hemoglobin A synthesis in erythroid cells from peripheral blood of thalassemic patients.

Mononuclear cells from peripheral blood of thalassemic patients were treated with morpholino oligonucleotides antisense to aberrant splice sites in mutant beta-globin precursor mRNAs (pre-mRNAs). The oligonucleotides restored correct splicing and translation of beta-globin mRNA, increasing the hemoglobin (Hb) A synthesis in erythroid cells from patients with IVS2-654/beta(E), IVS2-745/IVS2-745, and IVS2-745/IVS2-1 genotypes. The maximal Hb A level for repaired IVS2-745 mutation was approximately 30% of normal; Hb A was still detectable 9 days after a single treatment with oligonucleotide. Thus, expression of defective beta-globin genes was repaired and significant level of Hb A was restored in a cell population that would be targeted in clinical applications of this approach.

Modification of alternative splicing pathways as a potential approach to chemotherapy.

Many cancer-associated genes are alternatively spliced; their expression leads to the production of multiple splice variants. Although the functions of most of these variants are not well-defined, some have antagonistic activities related to regulated cell death mechanisms. In a number of cancers and cancer cell lines, the ratio of the splice variants is frequently shifted so that the anti-apoptotic splice variant predominates. This observation suggests that modification of splicing, which restores the proper ratio of alternatively spliced gene products, may reverse the malignant phenotype of the cells and offer a gene-specific form of anticancer chemotherapy. Our laboratory has extensively investigated the use of antisense oligonucleotides for shifting the splicing patterns of several genes. Potential application of this method for treatment of cancers, as well as of certain genetic disorders, is discussed.