RESUMO
Here, we show that a NOT gated cell therapy (Tmod) can exploit antigens such as epidermal growth factor receptor (EGFR) and human leukocyte antigen-E (HLA-E) which are widely expressed on cancer cells. Noncancerous cells-despite high expression of these antigens-are protected from cytotoxicity by the action of an inhibitory receptor ("blocker") via a mechanism that involves blocker modulation of CAR surface expression. The blocker is triggered by the product of a polymorphic HLA allele (e.g., HLA-A∗02) deleted in a significant subset of solid tumors via loss of heterozygosity. Moreover, Tmod constructs that target mouse homologs of EGFR or HLA-E for activation, and a mouse-equivalent of HLA-A∗02 for inhibition, protect mice from toxicity caused by the CAR alone. The blocker also controls graft vs. host response in allogeneic T cells in vitro, consistent with the use of Tmod cells for off-the-shelf therapy without additional gene-editing.
RESUMO
Next-generation T-cell therapies will likely continue to utilize T-cell receptors (TCRs) and chimeric antigen receptors (CARs) because each receptor type has advantages. TCRs often possess exceptional properties even when tested unmodified from patients' T cells. CARs are generally less sensitive, possibly because their ligand-binding domains are grafted from antibodies selected for binding affinity or avidity and not broadly optimized for a functional response. Because of the disconnect between binding and function among these receptor types, the ultimate potential of CARs optimized for sensitivity and selectivity is not clear. Here, we focus on a thoroughly studied immuno-oncology target, the HLA-A*02/HPV-E629-38 complex, and show that CARs can be optimized by a combination of high-throughput binding screens and low-throughput functional assays to have comparable activity to clinical TCRs in acute assays in vitro. These results provide a case study for the challenges and opportunities of optimizing high-performing CARs, especially in the context of targets utilized naturally by TCRs.
Assuntos
Imunoterapia Adotiva , Neoplasias/terapia , Infecções por Papillomavirus/terapia , Receptores de Antígenos Quiméricos/imunologia , Linhagem Celular , Proteínas de Fluorescência Verde , Antígeno HLA-A2/imunologia , Humanos , Interferon gama/imunologia , Luciferases de Vaga-Lume , Neoplasias/imunologia , Proteínas Oncogênicas Virais/imunologia , Proteínas E7 de Papillomavirus/imunologia , Infecções por Papillomavirus/imunologia , Peptídeos/imunologia , Proteínas Repressoras/imunologia , Anticorpos de Cadeia Única/imunologiaRESUMO
Though TCRs have been subject to limited engineering in the context of therapeutic design and optimization, they are used largely as found in nature. On the other hand, CARs are artificial, composed of different segments of proteins that function in the immune system. This characteristic raises the possibility of altered response to immune regulatory stimuli. Here we describe a large-scale, systematic comparison of CARs and TCRs across 5 different pMHC targets, with a total of 19 constructs examined in vitro. These functional measurements include CAR- and TCR-mediated activation, proliferation, and cytotoxicity in both acute and chronic settings. Surprisingly, we find no consistent difference between CARs and TCRs as receptor classes with respect to their relative sensitivity to major regulators of T cell activation: PD-L1, CD80/86 and IL-2. Though TCRs often emerge from human blood directly as potent, selective receptors, CARs must be heavily optimized to attain these properties for pMHC targets. Nonetheless, when iteratively improved and compared head to head in functional tests, CARs appear remarkably similar to TCRs with respect to immune modulation.
Assuntos
Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , HumanosRESUMO
We designed variant human TCRs composed of the full-length TCRα/ß or extracellular and transmembrane domains of the associated CD3 subunits fused to polypeptides derived from proteins thought to either enhance or inhibit normal T cell function. First, we showed that the C termini of both the TCR α- and ß-chains can accommodate specific additional sequences, without abrogating complex formation or acute sensitivity of the receptor. Replacement of ITAMs with ITIM-containing intracellular domains inverted the TCR signal (i.e., created a ligand-dependent inhibitory receptor). The normal signaling function of the CD3 complex was transferable to the TCR by eliminating all CD3 ITAMs and grafting three to six ITAMs onto the C termini of the α/ß-chains, with no effect on acute sensitivity. The observation that TCR variants of such diverse C-terminal composition can fold and function as signaling receptors demonstrates substantial structural and functional malleability of TCRs. These results add to knowledge about TCR structure-function with regard to acute signaling and may provide a route to use TCRs in different ways for T cell therapy.
Assuntos
Complexo Receptor-CD3 de Antígeno de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Proteínas de Transporte/metabolismo , Humanos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
Chimeric antigen receptors (CARs) and their parent signaling molecule, the T cell receptor (TCR), are fascinating proteins of increasing relevance to disease therapy. Here we use a collection of 1221 pMHC-directed CAR constructs representing 10 pMHC targets to study aspects of CAR structure-activity relationships (SAR), with particular focus on the extracellular and transmembrane structural components. These experiments that involve pMHC targets whose number/cell can be manipulated by peptide dosing in vitro enable systematic analysis of the SAR of CARs in carefully controlled experimental situations (Harris and Kranz, 2016). We find that CARs tolerate a wide range of structural variation, with the ligand-binding domains (LBDs) dominating the SAR of CAR antigen sensitivity. Notwithstanding the critical role of the LBD, CAR antigen-binding on the cell surface, measured by pMHC tetramer staining, is not an effective predictor of functional sensitivity. These results have important implications for the design and testing of CARs aimed toward the clinic.
Assuntos
Antígenos HLA-A/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Sítios de Ligação/imunologia , Antígenos HLA-A/metabolismo , Humanos , Células Jurkat , Ligantes , Células MCF-7 , Domínios Proteicos/imunologia , Multimerização Proteica/imunologia , Receptores de Antígenos Quiméricos/imunologia , Relação Estrutura-Atividade , Linfócitos T/metabolismoRESUMO
Cell therapy using T cell receptors (TCRs) and chimeric antigen receptors (CARs) represents a new wave of immunotherapies garnering considerable attention and investment. Further progress in this area of medicine depends in part on improving the functional capabilities of the engineered components, while maintaining the overall size of recombinant constructs to ensure their compatibility with existing gene delivery vehicles. We describe a single-variable-domain TCR (svd TCR) that utilizes only the variable domain of the ß chain (Vß). This Vß module not only works in TCR and CAR formats, but also can be used to create single-chain bispecific CARs and TCRs. Comparison of individual ligand-binding Vß domains in different formats suggests that the lone Vß sequence controls the sensitivity and a major part of the specificity of the CAR or TCR construct, regardless of signaling format, in Jurkat and primary T cells.
Assuntos
Região Variável de Imunoglobulina/imunologia , Imunoterapia Adotiva/métodos , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/transplante , Engenharia Celular , Células HEK293 , Humanos , Região Variável de Imunoglobulina/genética , Células Jurkat , Ligantes , Neoplasias/imunologia , Cultura Primária de Células , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos Quiméricos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Linfócitos T/imunologia , Transfecção , Evasão TumoralRESUMO
DNA double-strand breaks (DSBs) are hazardous lesions that threaten genome integrity and cell survival. The DNA damage response (DDR) safeguards the genome by sensing DSBs, halting cell cycle progression and promoting repair through either non-homologous end joining (NHEJ) or homologous recombination (HR). The Mre11-Rad50-Xrs2/Nbs1 (MRX/N) complex is central to the DDR through its structural, enzymatic, and signaling roles. The complex tethers DNA ends, activates the Tel1/ATM kinase, resolves protein-bound or hairpin-capped DNA ends, and maintains telomere homeostasis. In addition to its role at DSBs, MRX/N associates with unperturbed replication forks, as well as stalled replication forks, to ensure complete DNA synthesis and to prevent chromosome rearrangements. Here, we summarize the significant progress made in characterizing the MRX/N complex and its various activities in chromosome metabolism.
RESUMO
The yeast Mre11-Rad50-Xrs2 (MRX) complex has structural, signaling, and catalytic functions in the response to DNA damage. Xrs2, the eukaryotic-specific component of the complex, is required for nuclear import of Mre11 and Rad50 and to recruit the Tel1 kinase to damage sites. We show that nuclear-localized MR complex (Mre11-NLS) catalyzes homology-dependent repair without Xrs2, but MR cannot activate Tel1, and it fails to tether DSBs, resulting in sensitivity to genotoxins, replisome instability, and increased gross chromosome rearrangements (GCRs). Fusing the Tel1 interaction domain from Xrs2 to Mre11-NLS is sufficient to restore telomere elongation and Tel1 signaling to Xrs2-deficient cells. Furthermore, Tel1 stabilizes Mre11-DNA association, and this stabilization function becomes important for DNA damage resistance in the absence of Xrs2. Enforcing Tel1 recruitment to the nuclear MR complex fully rescues end tethering and stalled replication fork stability, and suppresses GCRs, highlighting important roles for Xrs2 and Tel1 to ensure optimal MR activity.
Assuntos
DNA Fúngico/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Replicação do DNA , Rearranjo Gênico/genética , Complexos Multiproteicos/metabolismo , Mutação/genética , Ligação Proteica , Estabilidade Proteica , Recombinação Genética/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Single-stranded DNA (ssDNA) intermediates are essential for homology-dependent repair of DNA double-strand breaks (DSBs) and for the DNA damage response. Here we describe methods routinely used to identify ssDNA intermediates formed by end processing of site-specific DSBs in Saccharomyces cerevisiae. These methods have been applied in other model systems and human cell lines, and are useful tools to gain insight into the enzymes that process DSBs and how they are regulated.
Assuntos
Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Fúngico/análise , DNA de Cadeia Simples/análise , Saccharomyces cerevisiae/genética , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Nocodazol/farmacologia , Hibridização de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reparo de DNA por Recombinação/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
DNA double-strand breaks (DSBs) are cytotoxic lesions that must be accurately repaired to maintain genome stability. Replication protein A (RPA) plays an important role in homology-dependent repair of DSBs by protecting the single-stranded DNA (ssDNA) intermediates formed by end resection and by facilitating Rad51 loading. We found that hypomorphic mutants of RFA1 that support intra-chromosomal homologous recombination are profoundly defective for repair processes involving long tracts of DNA synthesis, in particular break-induced replication (BIR). The BIR defects of the rfa1 mutants could be partially suppressed by eliminating the Sgs1-Dna2 resection pathway, suggesting that Dna2 nuclease attacks the ssDNA formed during end resection when not fully protected by RPA. Overexpression of Rad51 was also found to suppress the rfa1 BIR defects. We suggest that Rad51 binding to the ssDNA formed by excessive end resection and during D-loop migration can partially compensate for dysfunctional RPA.
Assuntos
Replicação do DNA/genética , Recombinação Homóloga/genética , Rad51 Recombinase/genética , Proteína de Replicação A/genética , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , DNA Helicases/genética , Reparo do DNA/genética , DNA de Cadeia Simples , Proteínas de Ligação a DNA/genética , Instabilidade Genômica/genética , RecQ Helicases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The Mre11-Rad50-Xrs2/Nbs1 (MRX/N) complex orchestrates the cellular response to DSBs through its structural, enzymatic, and signaling roles. Xrs2/Nbs1 is essential for nuclear translocation of Mre11, but its role as a component of the complex is not well defined. Here, we demonstrate that nuclear localization of Mre11 (Mre11-NLS) is able to bypass several functions of Xrs2, including DNA end resection, meiosis, hairpin resolution, and cellular resistance to clastogens. Using purified components, we show that the MR complex has equivalent activity to MRX in cleavage of protein-blocked DNA ends. Although Xrs2 physically interacts with Sae2, we found that end resection in its absence remains Sae2 dependent in vivo and in vitro. MRE11-NLS was unable to rescue the xrs2Δ defects in Tel1/ATM kinase signaling and non-homologous end joining, consistent with the role of Xrs2 as a chaperone and adaptor protein coordinating interactions between the MR complex and other repair proteins.
Assuntos
Reparo do DNA por Junção de Extremidades , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sítios de Ligação , Camptotecina/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Endonucleases/deficiência , Endonucleases/genética , Exodesoxirribonucleases/metabolismo , Regulação Fúngica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metanossulfonato de Metila/farmacologia , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de SinaisRESUMO
The Mre11-Rad50-Xrs2/NBS1 (MRX/N) nuclease/ATPase complex plays structural and catalytic roles in the repair of DNA double-strand breaks (DSBs) and is the DNA damage sensor for Tel1/ATM kinase activation. Saccharomyces cerevisiae Sae2 can function with MRX to initiate 5'-3' end resection and also plays an important role in attenuation of DNA damage signaling. Here we describe a class of mre11 alleles that suppresses the DNA damage sensitivity of sae2Δ cells by accelerating turnover of Mre11 at DNA ends, shutting off the DNA damage checkpoint and allowing cell cycle progression. The mre11 alleles do not suppress the end resection or hairpin-opening defects of the sae2Δ mutant, indicating that these functions of Sae2 are not responsible for DNA damage resistance. The purified M(P110L)RX complex shows reduced binding to single- and double-stranded DNA in vitro relative to wild-type MRX, consistent with the increased turnover of Mre11 from damaged sites in vivo. Furthermore, overproduction of Mre11 causes DNA damage sensitivity only in the absence of Sae2. Together, these data suggest that it is the failure to remove Mre11 from DNA ends and attenuate Rad53 kinase signaling that causes hypersensitivity of sae2Δ cells to clastogens.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Endonucleases/genética , Exodesoxirribonucleases/genética , Microscopia de Fluorescência , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais/genéticaRESUMO
In flowering plants, mitochondrial and chloroplast mRNAs are edited by C-to-U base modification. In plant organelles, RNA editing appears to be generally a correcting mechanism that restores the proper function of the encoded product. Members of the Arabidopsis RNA editing-Interacting Protein (RIP) family have been recently shown to be essential components of the plant editing machinery. We report the use of a strand- and transcript-specific RNA-seq method (STS-PCRseq) to explore the effect of mutation or silencing of every RIP gene on plant organelle editing. We confirm RIP1 to be a major editing factor that controls the editing extent of 75% of the mitochondrial sites and 20% of the plastid C targets of editing. The quantitative nature of RNA sequencing allows the precise determination of overlapping effects of RIP factors on RNA editing. Over 85% of the sites under the influence of RIP3 and RIP8, two moderately important mitochondrial factors, are also controlled by RIP1. Previously uncharacterized RIP family members were found to have only a slight effect on RNA editing. The preferential location of editing sites controlled by RIP7 on some transcripts suggests an RNA metabolism function for this factor other than editing. In addition to a complete characterization of the RIP factors for their effect on RNA editing, our study highlights the potential of RNA-seq for studying plant organelle editing. Unlike previous attempts to use RNA-seq to analyze RNA editing extent, our methodology focuses on sequencing of organelle cDNAs corresponding to known transcripts. As a result, the depth of coverage of each editing site reaches unprecedented values, assuring a reliable measurement of editing extent and the detection of numerous new sites. This strategy can be applied to the study of RNA editing in any organism.