RESUMEN
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.
Asunto(s)
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
PD-1 blockade unleashes CD8 T cells1, including those specific for mutation-associated neoantigens (MANA), but factors in the tumour microenvironment can inhibit these T cell responses. Single-cell transcriptomics have revealed global T cell dysfunction programs in tumour-infiltrating lymphocytes (TIL). However, the majority of TIL do not recognize tumour antigens2, and little is known about transcriptional programs of MANA-specific TIL. Here, we identify MANA-specific T cell clones using the MANA functional expansion of specific T cells assay3 in neoadjuvant anti-PD-1-treated non-small cell lung cancers (NSCLC). We use their T cell receptors as a 'barcode' to track and analyse their transcriptional programs in the tumour microenvironment using coupled single-cell RNA sequencing and T cell receptor sequencing. We find both MANA- and virus-specific clones in TIL, regardless of response, and MANA-, influenza- and Epstein-Barr virus-specific TIL each have unique transcriptional programs. Despite exposure to cognate antigen, MANA-specific TIL express an incompletely activated cytolytic program. MANA-specific CD8 T cells have hallmark transcriptional programs of tissue-resident memory (TRM) cells, but low levels of interleukin-7 receptor (IL-7R) and are functionally less responsive to interleukin-7 (IL-7) compared with influenza-specific TRM cells. Compared with those from responding tumours, MANA-specific clones from non-responding tumours express T cell receptors with markedly lower ligand-dependent signalling, are largely confined to HOBIThigh TRM subsets, and coordinately upregulate checkpoints, killer inhibitory receptors and inhibitors of T cell activation. These findings provide important insights for overcoming resistance to PD-1 blockade.
Asunto(s)
Antígenos de Neoplasias/inmunología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Regulación de la Expresión Génica , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Antígenos de Neoplasias/genética , Linfocitos T CD8-positivos/inmunología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Células Cultivadas , Humanos , Memoria Inmunológica , Neoplasias Pulmonares/genética , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , RNA-Seq , Receptores de Interleucina-7/inmunología , Análisis de la Célula Individual , Transcriptoma/genética , Microambiente TumoralRESUMEN
Discovery of off-target CRISPR-Cas activity in patient-derived cells and animal models is crucial for genome editing applications, but currently exhibits low sensitivity. We demonstrate that inhibition of DNA-dependent protein kinase catalytic subunit accumulates the repair protein MRE11 at CRISPR-Cas-targeted sites, enabling high-sensitivity mapping of off-target sites to positions of MRE11 binding using chromatin immunoprecipitation followed by sequencing. This technique, termed DISCOVER-Seq+, discovered up to fivefold more CRISPR off-target sites in immortalized cell lines, primary human cells and mice compared with previous methods. We demonstrate applicability to ex vivo knock-in of a cancer-directed transgenic T cell receptor in primary human T cells and in vivo adenovirus knock-out of cardiovascular risk gene PCSK9 in mice. Thus, DISCOVER-Seq+ is, to our knowledge, the most sensitive method to-date for discovering off-target genome editing in vivo.
Asunto(s)
Sistemas CRISPR-Cas , Proproteína Convertasa 9 , Humanos , Animales , Ratones , Proproteína Convertasa 9/genética , Edición Génica/métodos , GenomaRESUMEN
Developing therapeutic agents with potent antitumor activity that spare normal tissues remains a significant challenge. Clonal loss of heterozygosity (LOH) is a widespread and irreversible genetic alteration that is exquisitely specific to cancer cells. We hypothesized that LOH events can be therapeutically targeted by "inverting" the loss of an allele in cancer cells into an activating signal. Here we describe a proof-of-concept approach utilizing engineered T cells approximating NOT-gate Boolean logic to target counterexpressed antigens resulting from LOH events in cancer. The NOT gate comprises a chimeric antigen receptor (CAR) targeting the allele of human leukocyte antigen (HLA) that is retained in the cancer cells and an inhibitory CAR (iCAR) targeting the HLA allele that is lost in the cancer cells. We demonstrate that engineered T cells incorporating such NOT-gate logic can be activated in a genetically predictable manner in vitro and in mice to kill relevant cancer cells. This therapeutic approach, termed NASCAR (Neoplasm-targeting Allele-Sensing CAR), could, in theory, be extended to LOH of other polymorphic genes that result in altered cell surface antigens in cancers.
Asunto(s)
Biomarcadores de Tumor , Inmunoterapia , Pérdida de Heterocigocidad , Terapia Molecular Dirigida , Neoplasias/etiología , Neoplasias/terapia , Alelos , Antígenos de Neoplasias/inmunología , Tratamiento Basado en Trasplante de Células y Tejidos , Antígenos HLA/genética , Antígenos HLA/inmunología , Humanos , Inmunoterapia/métodos , Inmunoterapia Adoptiva , Terapia Molecular Dirigida/efectos adversos , Terapia Molecular Dirigida/métodos , Anticuerpos de Cadena Única/farmacología , Anticuerpos de Cadena Única/uso terapéuticoRESUMEN
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.
Asunto(s)
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
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.
RESUMEN
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.
Asunto(s)
Anticuerpos , Proteínas Proto-Oncogénicas p21(ras) , Proteínas Proto-Oncogénicas p21(ras)/genética , Reconocimiento en Psicología , Interacciones Hidrofóbicas e Hidrofílicas , Antígenos HLA-A/genéticaRESUMEN
Identification and quantification of low-frequency mutations remain challenging despite improvements in the baseline error rate of next-generation sequencing technologies. Here, we describe a method, termed SaferSeqS, that addresses these challenges by (1) efficiently introducing identical molecular barcodes in the Watson and Crick strands of template molecules and (2) enriching target sequences with strand-specific PCR. The method achieves high sensitivity and specificity and detects variants at frequencies below 1 in 100,000 DNA template molecules with a background mutation rate of <5 × 10-7 mutants per base pair (bp). We demonstrate that it can evaluate mutations in a single amplicon or simultaneously in multiple amplicons, assess limited quantities of cell-free DNA with high recovery of both strands and reduce the error rate of existing PCR-based molecular barcoding approaches by >100-fold.
Asunto(s)
Análisis Mutacional de ADN/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , ADN de Neoplasias/sangre , ADN de Neoplasias/genética , Humanos , Mutación , Tasa de Mutación , Reacción en Cadena de la PolimerasaRESUMEN
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.
Asunto(s)
Antígenos de Neoplasias , Neoplasias , Antígenos de Neoplasias/genética , Humanos , Factores Inmunológicos/uso terapéutico , Inmunoterapia , Mutación , Neoplasias/terapia , OncogenesRESUMEN
Chimeric antigen receptor (CAR) T cells have emerged as a promising class of therapeutic agents, generating remarkable responses in the clinic for a subset of human cancers. One major challenge precluding the wider implementation of CAR therapy is the paucity of tumor-specific antigens. Here, we describe the development of a CAR targeting the tumor-specific isocitrate dehydrogenase 2 (IDH2) with R140Q mutation presented on the cell surface in complex with a common human leukocyte antigen allele, HLA-B*07:02. Engineering of the hinge domain of the CAR, as well as crystal structure-guided optimization of the IDH2R140Q-HLA-B*07:02-targeting moiety, enhances the sensitivity and specificity of CARs to enable targeting of this HLA-restricted neoantigen. This approach thus holds promise for the development and optimization of immunotherapies specific to other cancer driver mutations that are difficult to target by conventional means.
Asunto(s)
Antígeno HLA-B7/química , Isocitrato Deshidrogenasa/metabolismo , Ingeniería de Proteínas/métodos , Receptores Quiméricos de Antígenos/química , Animales , Antígenos de Neoplasias/metabolismo , Células COS , Línea Celular , Chlorocebus aethiops , Epítopos , Antígeno HLA-B7/metabolismo , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Isocitrato Deshidrogenasa/química , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/inmunología , Mutación , Biblioteca de Péptidos , Conformación Proteica , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T/fisiologíaRESUMEN
Immunotherapies such as chimeric antigen receptor (CAR) T cells and bispecific antibodies redirect healthy T cells to kill cancer cells expressing the target antigen. The pan-B cell antigen-targeting immunotherapies have been remarkably successful in treating B cell malignancies. Such therapies also result in the near-complete loss of healthy B cells, but this depletion is well tolerated by patients. Although analogous targeting of pan-T cell markers could, in theory, help control T cell cancers, the concomitant healthy T cell depletion would result in severe and unacceptable immunosuppression. Thus, therapies directed against T cell cancers require more selective targeting. Here, we describe an approach to target T cell cancers through T cell receptor (TCR) antigens. Each T cell, normal or malignant, expresses a unique TCR ß chain generated from 1 of 30 TCR ß chain variable gene families (TRBV1 to TRBV30). We hypothesized that bispecific antibodies targeting a single TRBV family member expressed in malignant T cells could promote killing of these cancer cells, while preserving healthy T cells that express any of the other 29 possible TRBV family members. We addressed this hypothesis by demonstrating that bispecific antibodies targeting TRBV5-5 (α-V5) or TRBV12 (α-V12) specifically lyse relevant malignant T cell lines and patient-derived T cell leukemias in vitro. Treatment with these antibodies also resulted in major tumor regressions in mouse models of human T cell cancers. This approach provides an off-the-shelf, T cell cancer selective targeting approach that preserves enough healthy T cells to maintain cellular immunity.
Asunto(s)
Anticuerpos Biespecíficos , Trastornos Linfoproliferativos/terapia , Linfocitos T/patología , Humanos , Receptores de Antígenos de Linfocitos T alfa-betaRESUMEN
Mutations in the RAS oncogenes occur in multiple cancers, and ways to target these mutations has been the subject of intense research for decades. Most of these efforts are focused on conventional small-molecule drugs rather than antibody-based therapies because the RAS proteins are intracellular. Peptides derived from recurrent RAS mutations, G12V and Q61H/L/R, are presented on cancer cells in the context of two common human leukocyte antigen (HLA) alleles, HLA-A3 and HLA-A1, respectively. Using phage display, we isolated single-chain variable fragments (scFvs) specific for each of these mutant peptide-HLA complexes. The scFvs did not recognize the peptides derived from the wild-type form of RAS proteins or other related peptides. We then sought to develop an immunotherapeutic agent that was capable of killing cells presenting very low levels of these RAS-derived peptide-HLA complexes. Among many variations of bispecific antibodies tested, one particular format, the single-chain diabody (scDb), exhibited superior reactivity to cells expressing low levels of neoantigens. We converted the scFvs to this scDb format and demonstrated that they were capable of inducing T cell activation and killing of target cancer cells expressing endogenous levels of the mutant RAS proteins and cognate HLA alleles. CRISPR-mediated alterations of the HLA and RAS genes provided strong genetic evidence for the specificity of the scDbs. Thus, this approach could be applied to other common oncogenic mutations that are difficult to target by conventional means, allowing for more specific anticancer therapeutics.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Antígenos de Neoplasias , Biomarcadores de Tumor/antagonistas & inhibidores , Proteínas Mutantes/antagonistas & inhibidores , Proteínas ras/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Anticuerpos Biespecíficos/inmunología , Antígenos de Neoplasias/química , Antígenos de Neoplasias/inmunología , Biomarcadores de Tumor/química , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/inmunología , Línea Celular , Reacciones Cruzadas , Antígenos HLA/inmunología , Humanos , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Proteínas Mutantes/química , Proteínas Mutantes/inmunología , Mutación , Fragmentos de Péptidos , Unión Proteica/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Proteínas ras/química , Proteínas ras/genética , Proteínas ras/inmunologíaRESUMEN
TP53 (tumor protein p53) is the most commonly mutated cancer driver gene, but drugs that target mutant tumor suppressor genes, such as TP53, are not yet available. Here, we describe the identification of an antibody highly specific to the most common TP53 mutation (R175H, in which arginine at position 175 is replaced with histidine) in complex with a common human leukocyte antigen-A (HLA-A) allele on the cell surface. We describe the structural basis of this specificity and its conversion into an immunotherapeutic agent: a bispecific single-chain diabody. Despite the extremely low p53 peptide-HLA complex density on the cancer cell surface, the bispecific antibody effectively activated T cells to lyse cancer cells that presented the neoantigen in vitro and in mice. This approach could in theory be used to target cancers containing mutations that are difficult to target in conventional ways.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Anticuerpos Antineoplásicos/inmunología , Antígenos de Neoplasias/inmunología , Antígeno HLA-A2/inmunología , Neoplasias/terapia , Proteína p53 Supresora de Tumor/inmunología , Alelos , Animales , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Antineoplásicos/química , Anticuerpos Antineoplásicos/uso terapéutico , Arginina/genética , Células COS , Chlorocebus aethiops , Femenino , Células HEK293 , Antígeno HLA-A2/química , Antígeno HLA-A2/genética , Histidina/genética , Humanos , Inmunización Pasiva , Células Jurkat , Activación de Linfocitos , Ratones Endogámicos NOD , Mutación , Linfocitos T/inmunología , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Specific targeting of inflammation remains a challenge in many pathologies, because of the necessary balance between host tolerance and efficacious inflammation resolution. Here, we discovered a short, 4-mer peptide which possesses antagonist properties towards CC chemokine receptor 2 (CCR2), but only when displayed on the surface of lipid nanoparticles. According to BLAST analysis, this peptide motif is a common repeating fragment in a number of proteins of the CC chemokine family, which are key players in the inflammatory response. In this study, self-assembled, peptide-conjugated nanoparticles (CCTV) exhibited typical properties of CCR2 antagonism, including affinity to the CCR2 receptor, inhibition of chemotactic migration of primary monocytes, and prevention from CC chemokine ligand 2 (CCL2)-induced actin polymerization. Furthermore, CCTV ameliorated NFkB activation and downregulated the secondary, but not the primary, inflammatory response in cultured macrophages. When conjugated with gadolinium or europium cryptates, CCTV enabled targeted imaging (via magnetic resonance imaging and time-resolved fluorescence) of atherosclerosis, a chronic inflammatory condition in which the CCL2/CCR2 axis is highly dysfunctional. CCTV targeted CCR2hiLy6Chi inflammatory monocytes in blood and the atherosclerotic plaque, resulting in cell-specific transcriptional downregulation of key inflammatory genes. Finally, CCTV generated pronounced inflammasome inactivation, likely mediated through reactive oxygen species scavenging and downregulation of NLRP3. In summary, our work demonstrates for the first time that a short peptide fragment presented on a nanoparticle surface exhibit potent receptor-targeted antagonist effects, which are not seen with the peptide alone. Unlike commonly used cargo-carrying, vector-directed drug delivery vehicles, CCTV nanoparticles may act as therapeutics/theranostics themselves, particularly in inflammatory conditions with CCL2/CCR2 pathogenesis, including cardiovascular disease and cancer.
Asunto(s)
Imagen por Resonancia Magnética , Nanopartículas/química , Péptidos/química , Receptores CCR2/antagonistas & inhibidores , Citoesqueleto de Actina/efectos de los fármacos , Animales , Aterosclerosis/diagnóstico por imagen , Aterosclerosis/patología , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Medios de Contraste/química , Regulación hacia Abajo/efectos de los fármacos , Humanos , Inflamasomas/metabolismo , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Péptidos/metabolismo , Péptidos/farmacología , Células RAW 264.7 , Especies Reactivas de Oxígeno/metabolismo , Receptores CCR2/metabolismoRESUMEN
Many immunotherapeutic approaches under development rely on T-cell recognition of cancer-derived peptides bound to human leukocyte antigen molecules on the cell surface. Direct experimental demonstration that such peptides are processed and bound is currently challenging. Here, we describe a method that meets this challenge. The method entailed an optimized immunoprecipitation protocol coupled with two-dimensional chromatography and mass spectrometry. The ability to detect and quantify minute amounts of predefined antigens should be useful both for basic research in tumor immunology and for the development of rationally designed cancer vaccines.