Adoptive cell therapy employing gene-modified T-cells expressing chimeric antigen receptors (CARs) has shown promising preclinical activity in a range of model systems and is now being tested in the ...clinical setting. The manufacture of CAR T-cells requires compliance with national and European regulations for the production of medicinal products. We established such a compliant process to produce T-cells armed with a first-generation CAR specific for carcinoembryonic antigen (CEA). CAR T-cells were successfully generated for 14 patients with advanced CEA
+
malignancy. Of note, in the majority of patients, the defined procedure generated predominantly CD4
+
CAR T-cells with the general T-cell population bearing an effector–memory phenotype and high in vitro effector function. Thus, improving the process to generate less-differentiated T-cells would be more desirable in the future for effective adoptive gene-modified T-cell therapy. However, these results confirm that CAR T-cells can be generated in a manner compliant with regulations governing medicinal products in the European Union.
Repair of oxidative damage to DNA bases is essential to prevent mutations and cell death. Endonuclease III is the major DNA glycosylase activity in Escherichia coli that catalyzes the excision of ...pyrimidines damaged by ring opening or ring saturation, and it also possesses an associated lyase activity that incises the DNA backbone adjacent to apurinic/apyrimidinic sites. During analysis of the area adjacent to the human tuberous sclerosis gene (TSC2) in chromosome region 16p13.3, we identified a gene, OCTS3, that encodes a 1-kb transcript. Analysis of OCTS3 cDNA clones revealed an open reading frame encoding a predicted protein of 34.3 kDa that shares extensive sequence similarity with E. coli endonuclease III and a related enzyme from Schizosaccharomyces pombe, including a conserved active site region and an iron/sulfur domain. The product of the OCTS3 gene was therefore designated hNTH1 (human endonuclease III homolog 1). The hNTH1 protein was overexpressed in E. coli and purified to apparent homogeneity. The recombinant protein had spectral properties indicative of the presence of an iron/sulfur cluster, and exhibited DNA glycosylase activity on double-stranded polydeoxyribonucleotides containing urea and thymine glycol residues, as well as an apurinic/apyrimidinic lyase activity. Our data indicate that hNTH1 is a structural and functional homolog of E. coli endonuclease III, and that this class of enzymes, for repair of oxidatively damaged pyrimidines in DNA, is highly conserved in evolution from microorganisms to human cells.
We previously isolated a 34-kDa nuclease (AN34) from apoptotic human leukemia cells. Here, we identify AN34 as an N-terminally truncated form of human AP endonuclease (Ape1) lacking residues 1–35 ...(Δ35-Ape1). Although Ape1 has hitherto been considered specific for damaged DNA (specific to AP site), recombinant AN34 (Δ35-Ape1) possesses significant endonuclease activity on undamaged (normal) DNA and in chromatin. AN34 also displays enhanced 3′-5′ exonuclease activity. Caspase-3 activates AN34 in a cell-free system, although caspase-3 cannot cleave Ape1 directly in vitro. We also found that Ape1 itself preferentially cleaves damaged chromatin DNA isolated from cells treated with apoptotic stimuli and that silencing of Ape1 expression decreases apoptotic DNA fragmentation in DFF40/CAD-deficient cells. Thus, we propose that AN34 and Ape1 participate in the process of chromatin fragmentation during apoptosis.
The expression of two or more genes from a single viral vector has been widely used to label or select for cells containing the transgenic element. Identification of the foot-and-mouth disease virus ...(FMDV) 2A cleavage peptide as a polycistronic linker capable of producing equivalent levels of transgene expression has greatly improved this approach in the field of gene therapy. However, as a consequence of 2A posttranslational cleavage the upstream protein is left with a residual 19 amino acids from the 2A sequence on its carboxy terminus, and the downstream protein is left with an additional 2 to 5 amino acids on its amino terminus. Here we have assessed the functional consequences of the FMDV 2A cleavage motif on two secreted proteins (interleukin IL-2 and transforming growth factor TGF-β) when expressed from a retroviral bicistronic vector. Whereas IL-2 expression and function were found to be unaffected by the 2A motif in either orientation, functional expression of secreted TGF-β was significantly abrogated when the transgene was expressed upstream of the 2A sequence. We believe this is a consequence of aberrant cleavage and intracellular trafficking of the TGF-β polyprotein. These results highlight that to achieve functional expression of secreted proteins consideration must be taken of the transgenic protein's posttranslational modification and trafficking when using 2A-based bicistronic cassettes.
The development of Adoptive Cell Therapy (ACT) based immunotherapies using either naturally-occurring or genetically engineered T cells to target tumours have reached a stage where the approach is ...delivering clinically significant results in: 1 gene engineered T cells using either Chimeric Antigen Receptors (CAR) in B cell lymphoma or tumour specific T cell receptors in metastatic melanoma or metastatic synovial cell Sarcoma; and 2 Naturally-occurring tumour specific T cells called Tumour Infiltrating Lymphocytes in metastatic melanoma. However, the current delivery of these treatments is often referred to as a “just in time” product (i.e. where cell manufacturing, the hospital and patient are synchronised for the delivery of the ACT) and hence is not sustainable to treat suitable numbers of patients for either large scale multi-centre trials or to ultimately provide the treatment as a standard therapy. In order for effective reproducible treatments to be manufactured for ACT, the most significant challenge is maintaining the stability of the starting material, from the patient, prior to processing and the final product after formulation for infusion. In essence, GMP requires the manufacturing process to routinely maintain inter-product uniformity and hence minimise the impact of the manufacturing process on the final clinical outcome. In addition, by stabilising starting materials and products this also allows manufacturing sites to schedule production runs both of these points increase the likelihood that ACTs can be manufactured routinely. Aqueous solutions have a tendency to undercool, i.e. to cool significantly below their melting point before ice nucleation occurs. In addition, control of ice nucleation is recognised as a critical step in the cryopreservation of embryos and oocytes for IVF. Therefore, we assessed the effect of spontaneous or controlled ice nucleation on the viability and function of T cells either as starting materials such as Peripheral Blood Mononuclear cells (PBMC) or after in vitro expansion to provide supportive pre-clinical data T cells for ACT in early phase trials. In this study we demonstrated controlled ice nucleation is capable of improving the recovery of T cells from either frozen PBMCs or culture enriched T cells in cryovials, using both controlled-rate freezing and passive freezing. Analysis of culture purified T cells, expanded following controlled nucleation, showed that there was a significant improvement over spontaneous nucleation at either an early (96 hours) or at a later (14 days) time point. In addition, assessment of various cryopreservatives revealed that high serum content negated the benefits of controlled nucleation. These findings could be expected to improve the reliability and efficacy of GMP manufacturing for natural or genetically engineered T cell based ACTs. Controlled ice nucleation is also likely to improve the recovery of cryopreservation starting materials for personalised medicines, clinical delivery of cell therapies, and the quality of PBMCs in biobanks.
Source of funding: University of Manchester, The Christie, UK Technology Strategy Board (Grant 130328 & MR/K500732/1), Kay Kendal Foundation & European Union FP7.
Conflict of interest: Co-founder of Cellular Therapeutics Ltd.
Ryan.Guest@manchester.ac.uk
B-cell malignancies seem to be particularly amenable to immunotherapy and as such make particularly attractive targets for adoptive T-cell therapy. Murine T cells gene-modified to express a chimeric ...immune receptor specific for CD19+ (aCD19z) efficiently kill CD19 B-cell lymphoma cells in vitro. aCD19z T cells also secrete high levels of interleukin-2 during culture with target cells in a CD86 independent manner. aCD19z T cells proved effective at eradicating established B-cell lymphoma in a syngeneic model system when combined with a lymphodepleting preconditioning regimen. In mice deficient of T, B, and natural killer cells (severe combined immunodeficient/Beige), aCD19z T cells efficiently eradicated long-term (13 d) established tumors with 100% of treated animals remaining tumor free for greater than 77 days. Although gene-modified CD4+ and CD8+ were both active in this setting, poor engraftment by CD8+ T cells coupled with the rigorous expansion of CD4+ cells in the Balb/c background suggests that CD4+ T cells may be playing a predominant role in lymphoma rejection in this model. Taken together, the therapeutic effectiveness of aCD19z T cells in this model supports a recently opened phase 1 trial of this receptor in non-Hodgkin lymphoma.
The clinical potential of chimeric antigen receptors in adoptive cellular therapy is beginning to be realized with several recent clinical trials targeting CD19 showing promising results in advanced ...B cell malignancies. This increased efficacy corresponds with improved engineering of the chimeric receptors with the latest-generation receptors eliciting greater signaling and proliferation potential. However, the antigen-binding single-chain variable fragment (scFv) domain of the receptors is critical in determining the activity of the chimeric receptor-expressing T cells, as this determines specificity and affinity to the tumor antigen. In this study, we describe a mammalian T cell line screening protocol employing a 2A-based bicistronic retroviral vector to isolate functional scFvs. This approach involves expression of the scFv library in a chimeric antigen receptor, and is based on selection of clones capable of stimulating CD69 upregulation in a T cell line and has a number of advantages over previously described methods in that the use of a 2A cassette ensures the exclusion of nonexpressing scFvs and the screening using a chimeric receptor in a mammalian T cell line ensures selection in the optimum context for therapeutic use. Proof-of-principle experiments show that the protocol was capable of a 10(5)-fold enrichment of positive clones after three rounds of selection. Furthermore, an antigen-specific clone was successfully isolated from a partially enriched scFv library, confirming the strength of the protocol. This approach has the potential to identify novel scFvs of use in adoptive T cell therapy and, potentially, wider antibody-based applications.
The early detection of relapse following primary surgery for non-small-cell lung cancer and the characterization of emerging subclones, which seed metastatic sites, might offer new therapeutic ...approaches for limiting tumour recurrence. The ability to track the evolutionary dynamics of early-stage lung cancer non-invasively in circulating tumour DNA (ctDNA) has not yet been demonstrated. Here we use a tumour-specific phylogenetic approach to profile the ctDNA of the first 100 TRACERx (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy (Rx)) study participants, including one patient who was also recruited to the PEACE (Posthumous Evaluation of Advanced Cancer Environment) post-mortem study. We identify independent predictors of ctDNA release and analyse the tumour-volume detection limit. Through blinded profiling of postoperative plasma, we observe evidence of adjuvant chemotherapy resistance and identify patients who are very likely to experience recurrence of their lung cancer. Finally, we show that phylogenetic ctDNA profiling tracks the subclonal nature of lung cancer relapse and metastasis, providing a new approach for ctDNA-driven therapeutic studies.
With the use of a mouse model expressing human Fc-gamma receptors (FcγRs), we demonstrated that antibodies with isotypes equivalent to ipilimumab and tremelimumab mediate intra-tumoral regulatory ...T (Treg) cell depletion in vivo, increasing the CD8+ to Treg cell ratio and promoting tumor rejection. Antibodies with improved FcγR binding profiles drove superior anti-tumor responses and survival. In patients with advanced melanoma, response to ipilimumab was associated with the CD16a-V158F high affinity polymorphism. Such activity only appeared relevant in the context of inflamed tumors, explaining the modest response rates observed in the clinical setting. Our data suggest that the activity of anti-CTLA-4 in inflamed tumors may be improved through enhancement of FcγR binding, whereas poorly infiltrated tumors will likely require combination approaches.
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•Anti-CTLA-4 of hIgG1 and hIgG2 isotypes promote depletion of intra-tumoral Treg cells•hIgG2 antibodies mediate in vivo depletion of intra-tumoral Treg cells via CD32a•Anti-CTLA-4 with enhanced Fc effector function improves therapeutic outcomes•The CD16-V158F SNP is associated with response to ipilimumab in inflamed tumors
Arce Vargas et al. use a mouse model expressing human FcγRs to show that antibodies with isotypes equivalent to ipilimumab increase the CD8+ to Treg ratio by depleting intra-tumoral Tregs to promote tumor rejection. In melanoma patients, response to ipilimumab is associated with a high affinity FcγR polymorphism.
Abstract
Despite the tremendous potential of liquid biopsies to revolutionise cancer care, there has been limited success translating blood-circulating proteomic and genomic biomarkers into the ...clinic. This is fundamentally due to the extremely low concentration of tumour-derived biomolecules in blood circulation, particularly at an early disease stage, which makes the discovery phase of the biomarker pipeline extremely challenging. Nanotechnology offers a promising solution, with a nanoparticle-biomolecule enrichment tool recently developed to enrich low-abundant, low molecular weight proteins in the blood of ovarian cancer patients.1 Proteomic analysis followed by immunoassay-based validation of selected proteins demonstrated the potential of the nanoparticle-platform proposed to discover novel biomarkers with greater specificity and sensitivity than the clinically used biomarkers. In addition, we recently confirmed the presence of cell-free DNA (cfDNA) captured onto the surface lipid nanoparticles incubated ex vivo with human plasma.2 A significantly higher abundance of cfDNA was detected in the nanoparticle-enriched plasma samples of late-stage ovarian cancer patients compared to age-matched female controls. Proteomic analysis of the same samples also revealed tumour-specific elevations in histone proteins, which are commonly found in circulation complexed with cfDNA. These findings have highlighted the opportunity for the development of a nano-proteogenomics platform able to simultaneously purify both proteins and cell-free nucleic acids from human plasma, an important step in the discovery of novel multi-omic biomarker panels. Utilising the above patented nanotechnology, we have compared proteomic and genomic profiles derived from nanoparticle-biomolecule samples of cancer patients with age- and sex-matched controls to uncover new potential blood-based biomarkers in a proof-of-principle study. In brief, ex-vivo plasma samples were incubated with lipid-based nanoparticles and purified using a two-step size-based purification protocol. The purified samples were then analysed by label-free proteomics (LC-MS/MS) and next-generation sequencing to uncover both proteomic and genomic tumour-specific signatures, including differentially abundant proteins, genomic copy number alterations and tumour-specific mutations. This work highlights the potential of our nanotechnology-based enrichment platform to enhance the discovery of cancer-specific proteogenomic biomarker panels, a vital step in developing sensitive and specific liquid biopsies for the early detection of cancer.
References: 1 M. Hadjidemetriou, L. Papafilippou, R. D. Unwin, J. Rogan, A. Clamp, K. Kostarelos, Nano Today 2020, 34, 100901. 2 L. Gardner, J. Warrington, J. Rogan, D. G. Rothwell, G. Brady, C. Dive, K. Kostarelos, M. Hadjidemetriou, Nanoscale Horizons 2020, 5, 1476.
Citation Format: Lois Gardner, Dominic G. Rothwell, Caroline Dive, Kostas Kostarelos, Marilena Hadjidemetriou. Nanonets for multiomics blood analysis and cancer biomarker discovery abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 568.
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