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Two types of engineered T cells have been successfully used to treat patients with cancer, one with an antigen recognition domain derived from antibodies [chimeric antigen receptors (CARs)] and the other derived from T cell receptors (TCRs). CARs use high-affinity antigen-binding domains and costimulatory domains to induce T cell activation but can only react against target cells with relatively high amounts of antigen. TCRs have a much lower affinity for their antigens but can react against target cells displaying only a few antigen molecules. Here, we describe a new type of receptor, called a Co-STAR (for costimulatory synthetic TCR and antigen receptor), that combines aspects of both CARs and TCRs. In Co-STARs, the antigen-recognizing components of TCRs are replaced by high-affinity antibody fragments, and costimulation is provided by two modules that drive NF-κB signaling (MyD88 and CD40). Using a TCR-mimic antibody fragment that targets a recurrent p53 neoantigen presented in a common human leukocyte antigen (HLA) allele, we demonstrate that T cells equipped with Co-STARs can kill cancer cells bearing low densities of antigen better than T cells engineered with conventional CARs and patient-derived TCRs in vitro. In mouse models, we show that Co-STARs mediate more robust T cell expansion and more durable tumor regressions than TCRs similarly modified with MyD88 and CD40 costimulation. Our data suggest that Co-STARs may have utility for other peptide-HLA antigens in cancer and other targets where antigen density may limit the efficacy of engineered T cells.
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Neoplasias , Receptores de Antígenos de Linfocitos T , Receptores Quiméricos de Antígenos , Humanos , Animales , Receptores Quiméricos de Antígenos/metabolismo , Receptores Quiméricos de Antígenos/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Neoplasias/inmunología , Neoplasias/terapia , Ratones , Linfocitos T/inmunología , Linfocitos T/metabolismo , Línea Celular Tumoral , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Transducción de SeñalRESUMEN
Antibody and chimeric antigen receptor (CAR) T cell-mediated targeted therapies have improved survival in patients with solid and haematologic malignancies1-9. Adults with T cell leukaemias and lymphomas, collectively called T cell cancers, have short survival10,11 and lack such targeted therapies. Thus, T cell cancers particularly warrant the development of CAR T cells and antibodies to improve patient outcomes. Preclinical studies showed that targeting T cell receptor ß-chain constant region 1 (TRBC1) can kill cancerous T cells while preserving sufficient healthy T cells to maintain immunity12, making TRBC1 an attractive target to treat T cell cancers. However, the first-in-human clinical trial of anti-TRBC1 CAR T cells reported a low response rate and unexplained loss of anti-TRBC1 CAR T cells13,14. Here we demonstrate that CAR T cells are lost due to killing by the patient's normal T cells, reducing their efficacy. To circumvent this issue, we developed an antibody-drug conjugate that could kill TRBC1+ cancer cells in vitro and cure human T cell cancers in mouse models. The anti-TRBC1 antibody-drug conjugate may provide an optimal format for TRBC1 targeting and produce superior responses in patients with T cell cancers.
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Inmunoconjugados , Leucemia de Células T , Linfoma de Células T , Receptores de Antígenos de Linfocitos T alfa-beta , Linfocitos T , Animales , Femenino , Humanos , Ratones , Inmunoconjugados/inmunología , Inmunoconjugados/uso terapéutico , Inmunoterapia Adoptiva , Leucemia de Células T/tratamiento farmacológico , Leucemia de Células T/inmunología , Linfoma de Células T/tratamiento farmacológico , Linfoma de Células T/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We previously described an approach called RealSeqS to evaluate aneuploidy in plasma cell-free DNA through the amplification of ~350,000 repeated elements with a single primer. We hypothesized that an unbiased evaluation of the large amount of sequencing data obtained with RealSeqS might reveal other differences between plasma samples from patients with and without cancer. This hypothesis was tested through the development of a machine learning approach called Alu Profile Learning Using Sequencing (A-PLUS) and its application to 7615 samples from 5178 individuals, 2073 with solid cancer and the remainder without cancer. Samples from patients with cancer and controls were prespecified into four cohorts used for model training, analyte integration, and threshold determination, validation, and reproducibility. A-PLUS alone provided a sensitivity of 40.5% across 11 different cancer types in the validation cohort, at a specificity of 98.5%. Combining A-PLUS with aneuploidy and eight common protein biomarkers detected 51% of the cancers at 98.9% specificity. We found that part of the power of A-PLUS could be ascribed to a single feature-the global reduction of AluS subfamily elements in the circulating DNA of patients with solid cancer. We confirmed this reduction through the analysis of another independent dataset obtained with a different approach (whole-genome sequencing). The evaluation of Alu elements may therefore have the potential to enhance the performance of several methods designed for the earlier detection of cancer.
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Neoplasias , Humanos , Reproducibilidad de los Resultados , Neoplasias/diagnóstico , Neoplasias/genética , Elementos de Nucleótido Esparcido Corto , Aprendizaje Automático , AneuploidiaRESUMEN
Despite exciting developments in cancer immunotherapy, its broad application is limited by the paucity of targetable antigens on the tumor cell surface. As an intrinsic cellular pathway, nonsense-mediated decay (NMD) conceals neoantigens through the destruction of the RNA products from genes harboring truncating mutations. We developed and conducted a high throughput screen, based on the ratiometric analysis of transcripts, to identify critical mediators of NMD. This screen implicated disruption of kinase SMG1's phosphorylation of UPF1 as a potential disruptor of NMD. This led us to design a novel SMG1 inhibitor, KVS0001, that elevates the expression of transcripts and proteins resulting from truncating mutations in vivo and in vitro . Most importantly, KVS0001 concomitantly increased the presentation of immune-targetable HLA class I-associated peptides from NMD-downregulated proteins on the surface of cancer cells. KVS0001 provides new opportunities for studying NMD and the diseases in which NMD plays a role, including cancer and inherited diseases. One Sentence Summary: Disruption of the nonsense-mediated decay pathway with a newly developed SMG1 inhibitor with in-vivo activity increases the expression of T-cell targetable cancer neoantigens resulting from truncating mutations.
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BACKGROUND: Existing (artificial intelligence [AI]) tools in radiology are modeled without necessarily considering the expectations and experience of the end user-the radiologist. The literature is scarce on the tangible parameters that AI capabilities need to meet for radiologists to consider them useful tools. OBJECTIVE: The purpose of this study is to explore radiologists' attitudes toward AI tools in pancreatic cancer imaging and to quantitatively assess their expectations of these tools. METHODS: A link to the survey was posted on the www.ctisus.com website, advertised in the www.ctisus.com email newsletter, and publicized on LinkedIn, Facebook, and Twitter accounts. This survey asked participants about their demographics, practice, and current attitudes toward AI. They were also asked about their expectations of what constitutes a clinically useful AI tool. The survey consisted of 17 questions, which included 9 multiple choice questions, 2 Likert scale questions, 4 binary (yes/no) questions, 1 rank order question, and 1 free text question. RESULTS: A total of 161 respondents completed the survey, yielding a response rate of 46.3% of the total 348 clicks on the survey link. The minimum acceptable sensitivity of an AI program for the detection of pancreatic cancer chosen by most respondents was either 90% or 95% at a specificity of 95%. The minimum size of pancreatic cancer that most respondents would find an AI useful at detecting was 5 mm. Respondents preferred AI tools that demonstrated greater sensitivity over those with greater specificity. Over half of respondents anticipated incorporating AI tools into their clinical practice within the next 5 years. CONCLUSION: Radiologists are open to the idea of integrating AI-based tools and have high expectations regarding the performance of these tools. Consideration of radiologists' input is important to contextualize expectations and optimize clinical adoption of existing and future AI tools.
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Neoplasias Pancreáticas , Radiología , Humanos , Inteligencia Artificial , Motivación , Radiólogos , Radiología/métodos , Neoplasias Pancreáticas/diagnóstico por imagenRESUMEN
Specificity remains a major challenge to current therapeutic strategies for cancer. Mutation associated neoantigens (MANAs) are products of genetic alterations, making them highly specific therapeutic targets. MANAs are HLA-presented (pHLA) peptides derived from intracellular mutant proteins that are otherwise inaccessible to antibody-based therapeutics. Here, we describe the cryo-EM structure of an antibody-MANA pHLA complex. Specifically, we determine a TCR mimic (TCRm) antibody bound to its MANA target, the KRASG12V peptide presented by HLA-A*03:01. Hydrophobic residues appear to account for the specificity of the mutant G12V residue. We also determine the structure of the wild-type G12 peptide bound to HLA-A*03:01, using X-ray crystallography. Based on these structures, we perform screens to validate the key residues required for peptide specificity. These experiments led us to a model for discrimination between the mutant and the wild-type peptides presented on HLA-A*03:01 based exclusively on hydrophobic interactions.
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Anticuerpos , Proteínas Proto-Oncogénicas p21(ras) , Proteínas Proto-Oncogénicas p21(ras)/genética , Reconocimiento en Psicología , Interacciones Hidrofóbicas e Hidrofílicas , Antígenos HLA-A/genéticaRESUMEN
Cell-free DNA (cfDNA) concentrations from patients with cancer are often elevated compared with those of healthy controls, but the sources of this extra cfDNA have never been determined. To address this issue, we assessed cfDNA methylation patterns in 178 patients with cancers of the colon, pancreas, lung, or ovary and 64 patients without cancer. Eighty-three of these individuals had cfDNA concentrations much greater than those generally observed in healthy subjects. The major contributor of cfDNA in all samples was leukocytes, accounting for â¼76% of cfDNA, with neutrophils predominating. This was true regardless of whether the samples were derived from patients with cancer or the total plasma cfDNA concentration. High levels of cfDNA observed in patients with cancer did not come from either neoplastic cells or surrounding normal epithelial cells from the tumor's tissue of origin. These data suggest that cancers may have a systemic effect on cell turnover or DNA clearance. SIGNIFICANCE: The origin of excess cfDNA in patients with cancer is unknown. Using cfDNA methylation patterns, we determined that neither the tumor nor the surrounding normal tissue contributes this excess cfDNA-rather it comes from leukocytes. This finding suggests that cancers have a systemic impact on cell turnover or DNA clearance. See related commentary by Thierry and Pisareva, p. 2122. This article is featured in Selected Articles from This Issue, p. 2109.
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Ácidos Nucleicos Libres de Células , Neoplasias Colorrectales , Neoplasias Ováricas , Humanos , Femenino , Ácidos Nucleicos Libres de Células/genética , Metilación de ADN , ADN de Neoplasias/genética , Páncreas/patología , Neoplasias Ováricas/genética , Pulmón/patología , Neoplasias Colorrectales/genética , Biomarcadores de Tumor/genéticaRESUMEN
It is often challenging to distinguish cancerous from non-cancerous lesions in the brain using conventional diagnostic approaches. We introduce an analytic technique called Real-CSF (repetitive element aneuploidy sequencing in CSF) to detect cancers of the central nervous system from evaluation of DNA in the cerebrospinal fluid (CSF). Short interspersed nuclear elements (SINEs) are PCR amplified with a single primer pair, and the PCR products are evaluated by next-generation sequencing. Real-CSF assesses genome-wide copy-number alterations as well as focal amplifications of selected oncogenes. Real-CSF was applied to 280 CSF samples and correctly identified 67% of 184 cancerous and 96% of 96 non-cancerous brain lesions. CSF analysis was considerably more sensitive than standard-of-care cytology and plasma cell-free DNA analysis in the same patients. Real-CSF therefore has the capacity to be used in combination with other clinical, radiologic, and laboratory-based data to inform the diagnosis and management of patients with suspected cancers of the brain.
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Neoplasias del Sistema Nervioso Central , Humanos , Reacción en Cadena de la Polimerasa/métodos , Neoplasias del Sistema Nervioso Central/diagnóstico , Neoplasias del Sistema Nervioso Central/genética , Neoplasias del Sistema Nervioso Central/líquido cefalorraquídeo , Técnicas de Amplificación de Ácido Nucleico , Elementos de Nucleótido Esparcido Corto , Sistema Nervioso CentralRESUMEN
Nearly 30% of Pancreatic ductal adenocarcinoma (PDAC)s exhibit a marked overexpression of Monocarboxylate Transporter 1 (MCT1) offering a unique opportunity for therapy. However, biochemical inhibitors of MCT1 have proven unsuccessful in clinical trials. In this study we present an alternative approach using 3-Bromopyruvate (3BP) to target MCT1 overexpressing PDACs. 3BP is a cytotoxic agent that is known to be transported into cells via MCT1, but its clinical usefulness has been hampered by difficulties in delivering the drug systemically. We describe here a novel microencapsulated formulation of 3BP (ME3BP-7), that is effective against a variety of PDAC cells in vitro and remains stable in serum. Furthermore, systemically administered ME3BP-7 significantly reduces pancreatic cancer growth and metastatic spread in multiple orthotopic models of pancreatic cancer with manageable toxicity. ME3BP-7 is, therefore, a prototype of a promising new drug, in which the targeting moiety and the cytotoxic moiety are both contained within the same single small molecule. One Sentence Summary: ME3BP-7 is a novel formulation of 3BP that resists serum degradation and rapidly kills pancreatic cancer cells expressing high levels of MCT1 with tolerable toxicity in mice.
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The analysis of cell-free DNA (cfDNA) from plasma offers great promise for the earlier detection of cancer. At present, changes in DNA sequence, methylation, or copy number are the most sensitive ways to detect the presence of cancer. To further increase the sensitivity of such assays with limited amounts of sample, it would be useful to be able to evaluate the same template molecules for all these changes. Here, we report an approach, called MethylSaferSeqS, that achieves this goal, and can be applied to any standard library preparation method suitable for massively parallel sequencing. The innovative step was to copy both strands of each DNA-barcoded molecule with a primer that allows the subsequent separation of the original strands (retaining their 5-methylcytosine residues) from the copied strands (in which the 5-methylcytosine residues are replaced with unmodified cytosine residues). The epigenetic and genetic alterations present in the DNA molecules can then be obtained from the original and copied strands, respectively. We applied this approach to plasma from 265 individuals, including 198 with cancers of the pancreas, ovary, lung, and colon, and found the expected patterns of mutations, copy number alterations, and methylation. Furthermore, we could determine which original template DNA molecules were methylated and/or mutated. MethylSaferSeqS should be useful for addressing a variety of questions relating genetics and epigenetics.
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Variaciones en el Número de Copia de ADN , Neoplasias , Femenino , Humanos , Metilación , 5-Metilcitosina , ADN/genética , Mutación , Neoplasias/genética , Metilación de ADNRESUMEN
The therapeutic applications of antibodies are manifold and the emergence of SARS-CoV-2 provides a cogent example of the value of rapidly identifying biologically active antibodies. We describe an approach called SLISY (Sequencing-Linked ImmunoSorbent assaY) that in a single experiment can assess the binding specificity of millions of clones, be applied to any screen that links DNA sequence to a potential binding moiety, and requires only a single round of biopanning. We demonstrate this approach using an scFv library applied to cellular and protein targets to identify specific or broadly reacting antibodies. For a cellular target, we use paired HLA knockout cell lines to identify a panel of antibodies specific to HLA-A3. For a protein target, SLISY identifies 1279 clones that bound to the Receptor Binding Domain of the SARS-CoV-2 spike protein, with >40% of tested clones also neutralizing its interaction with ACE2 in in vitro assays. Using a multi-comparison SLISY against the Beta, Gamma, and Delta variants, we recovered clones that exhibited broad-spectrum neutralizing potential in vitro. By evaluating millions of scFvs simultaneously against multiple targets, SLISY allows the rapid identification of candidate scFvs with defined binding profiles facilitating the identification of antibodies with the desired biological activity.
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COVID-19 , SARS-CoV-2 , Humanos , Glicoproteína de la Espiga del Coronavirus/genética , Anticuerpos Neutralizantes , Anticuerpos AntiviralesRESUMEN
PURPOSE: A wide array of benign and malignant lesions of the pancreas can be cystic and these cystic lesions can have overlapping imaging appearances. The purpose of this study is to compare the diagnostic accuracy of a radiomics-based pancreatic cyst classifier to an experienced academic radiologist. METHODS: In this IRB-approved retrospective single-institution study, patients with surgically resected pancreatic cysts who underwent preoperative abdominal CT from 2003 to 2016 were identified. Pancreatic cyst(s) and background pancreas were manually segmented, and 488 radiomics features were extracted. Random forest classification based on radiomics features, age, and gender was evaluated with fourfold cross-validation. An academic radiologist blinded to the final pathologic diagnosis reviewed each case and provided the most likely diagnosis. RESULTS: 214 patients were included (64 intraductal papillary mucinous neoplasms, 33 mucinous cystic neoplasms, 60 serous cystadenomas, 24 solid pseudopapillary neoplasms, and 33 cystic neuroendocrine tumors). The radiomics-based machine learning approach showed AUC of 0.940 in pancreatic cyst classification, compared with AUC of 0.895 for the radiologist. CONCLUSION: Radiomics-based machine learning achieved equivalent performance as an experienced academic radiologist in the classification of pancreatic cysts. The high diagnostic accuracy can potentially maximize the efficiency of healthcare utilization by maximizing detection of high-risk lesions.
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Quiste Pancreático , Neoplasias Pancreáticas , Humanos , Estudios Retrospectivos , Neoplasias Pancreáticas/patología , Radiólogos , ComputadoresRESUMEN
We describe the creation of an isogenic cell line panel representing common cancer pathways, with features optimized for high-throughput screening. More than 1,800 cell lines from three normal human cell lines were generated using CRISPR technologies. Surprisingly, most of these lines did not result in complete gene inactivation despite integration of sgRNA at the desired genomic site. A subset of the lines harbored biallelic disruptions of the targeted tumor suppressor gene, yielding a final panel of 100 well-characterized lines covering 19 frequently lost cancer pathways. This panel included genetic markers optimized for sequence-based ratiometric assays for drug-based screening assays. To illustrate the potential utility of this panel, we developed a high-throughput screen that identified Wee1 inhibitor MK-1775 as a selective growth inhibitor of cells with inactivation of TP53. These cell lines and screening approach should prove useful for researchers studying a variety of cellular and biochemical phenomena.
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BACKGROUND: The role of adjuvant chemotherapy in stage II colon cancer continues to be debated. The presence of circulating tumor DNA (ctDNA) after surgery predicts very poor recurrence-free survival, whereas its absence predicts a low risk of recurrence. The benefit of adjuvant chemotherapy for ctDNA-positive patients is not well understood. METHODS: We conducted a trial to assess whether a ctDNA-guided approach could reduce the use of adjuvant chemotherapy without compromising recurrence risk. Patients with stage II colon cancer were randomly assigned in a 2:1 ratio to have treatment decisions guided by either ctDNA results or standard clinicopathological features. For ctDNA-guided management, a ctDNA-positive result at 4 or 7 weeks after surgery prompted oxaliplatin-based or fluoropyrimidine chemotherapy. Patients who were ctDNA-negative were not treated. The primary efficacy end point was recurrence-free survival at 2 years. A key secondary end point was adjuvant chemotherapy use. RESULTS: Of the 455 patients who underwent randomization, 302 were assigned to ctDNA-guided management and 153 to standard management. The median follow-up was 37 months. A lower percentage of patients in the ctDNA-guided group than in the standard-management group received adjuvant chemotherapy (15% vs. 28%; relative risk, 1.82; 95% confidence interval [CI], 1.25 to 2.65). In the evaluation of 2-year recurrence-free survival, ctDNA-guided management was noninferior to standard management (93.5% and 92.4%, respectively; absolute difference, 1.1 percentage points; 95% CI, -4.1 to 6.2 [noninferiority margin, -8.5 percentage points]). Three-year recurrence-free survival was 86.4% among ctDNA-positive patients who received adjuvant chemotherapy and 92.5% among ctDNA-negative patients who did not. CONCLUSIONS: A ctDNA-guided approach to the treatment of stage II colon cancer reduced adjuvant chemotherapy use without compromising recurrence-free survival. (Supported by the Australian National Health and Medical Research Council and others; DYNAMIC Australian New Zealand Clinical Trials Registry number, ACTRN12615000381583.).
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Antineoplásicos , Quimioterapia Adyuvante , ADN Tumoral Circulante , Neoplasias del Colon , Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Australia , Quimioterapia Adyuvante/métodos , ADN Tumoral Circulante/análisis , ADN Tumoral Circulante/sangre , Neoplasias del Colon/sangre , Neoplasias del Colon/mortalidad , Neoplasias del Colon/patología , Neoplasias del Colon/terapia , Supervivencia sin Enfermedad , Fluorouracilo/uso terapéutico , Humanos , Recurrencia Local de Neoplasia/prevención & control , Estadificación de Neoplasias , Oxaliplatino/uso terapéuticoRESUMEN
HIV-1 infection is incurable due to the persistence of the virus in a latent reservoir of resting memory CD4+ T cells. "Shock-and-kill" approaches that seek to induce HIV-1 gene expression, protein production, and subsequent targeting by the host immune system have been unsuccessful due to a lack of effective latency-reversing agents (LRAs) and kill strategies. In an effort to develop reagents that could be used to promote killing of infected cells, we constructed T cell receptor (TCR)-mimic antibodies to HIV-1 peptide-major histocompatibility complexes (pMHC). Using phage display, we panned for phages expressing antibody-like variable sequences that bound HIV-1 pMHC generated using the common HLA-A*02:01 allele. We targeted three epitopes in Gag and reverse transcriptase identified and quantified via Poisson detection mass spectrometry from cells infected in vitro with a pseudotyped HIV-1 reporter virus (NL4.3 dEnv). Sequences isolated from phages that bound these pMHC were cloned into a single-chain diabody backbone (scDb) sequence, such that one fragment is specific for an HIV-1 pMHC and the other fragment binds to CD3ε, an essential signal transduction subunit of the TCR. Thus, these antibodies utilize the sensitivity of T cell signaling as readouts for antigen processing and as agents to promote killing of infected cells. Notably, these scDbs are exquisitely sensitive and specific for the peptide portion of the pMHC. Most importantly, one scDb caused killing of infected cells presenting a naturally processed target pMHC. This work lays the foundation for a novel therapeutic killing strategy toward elimination of the HIV-1 reservoir.
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Anticuerpos Biespecíficos , Infecciones por VIH , Seropositividad para VIH , VIH-1 , Linfocitos T CD4-Positivos , Humanos , Imitación Molecular , Receptores de Antígenos de Linfocitos T , Latencia del VirusRESUMEN
Several current immunotherapy approaches target private neoantigens derived from mutations that are unique to individual patients' tumors. However, immunotherapeutic agents can also be developed against public neoantigens derived from recurrent mutations in cancer driver genes. The latter approaches target proteins that are indispensable for tumor growth, and each therapeutic agent can be applied to numerous patients. Here we review the opportunities and challenges involved in the identification of suitable public neoantigen targets and the development of therapeutic agents targeting them.