RESUMEN
Chimeric antigen receptors (CARs) are immunoreceptors that redirect T cells to selectively kill tumor cells. Given their clinical successes in hematologic malignancies, there is a strong aspiration to advance this immunotherapy for solid cancers; hence, molecular CAR design and careful target choice are crucial for their function. To evaluate the functional significance of the biophysical properties of CAR binding (i.e., affinity, avidity, and antigen density), we generated an experimental system in which these properties are controllable. We constructed and characterized a series of CARs, which target the melanoma tumor-associated antigen Tyr/HLA-A2, and in which the affinity of the single-chain Fv binding domains ranged in KD from 4 to 400 nmol/L. These CARs were transduced into T cells, and each CAR T-cell population was sorted by the level of receptor expression. Finally, the various CAR T cells were encountered with target cells that present different levels of the target antigen. We detected nonmonotonic behaviors of affinity and antigen density, and an interrelation between avidity and antigen density. Antitumor activity measurements in vitro and in vivo corroborated these observations. Our study contributes to the understanding of CAR T-cell function and regulation, having the potential to improve therapies by the rational design of CAR T cells.See related article on p. 946.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Animales , Línea Celular Tumoral , Femenino , Humanos , RatonesRESUMEN
Substrates tagged with (poly)ubiquitin for degradation can be targeted directly to the 26 S proteasome where they are proteolyzed. Independently, ubiquitin conjugates may also be delivered by bivalent shuttles. The majority of shuttles attach to the proteasome through a ubiquitin-like domain (UBL) while anchoring cargo at a C-terminal polyubiquitin-binding domain(s). We found that two shuttles of this class, Rad23 and Dsk2, dock at two different receptor sites embedded within a single subunit of the 19 S proteasome regulatory particle, Rpn1. Their association/dissociation constants and affinities for Rpn1 are similar. In contrast, another UBL-containing protein, the deubiquitinase Ubp6, is also anchored by Rpn1, yet it dissociates slower, thus behaving as an occasional proteasome subunit that is distinct from the transiently associated shuttles. Two neighboring subunits, Rpn10 and Rpn13, show a marked preference for polyubiquitin over UBLs. Rpn10 attaches to the central solenoid portion of Rpn1, although this association is stabilized by the presence of a third subunit, Rpn2. Rpn13 binds directly to Rpn2. These intrinsic polyubiquitin receptors may compete with substrate shuttles for their polyubiquitin-conjugate cargos, thereby aiding release of the emptied shuttles. By binding multiple ubiquitin-processing factors simultaneously, Rpn1 is uniquely suited to coordinate substrate recruitment, deubiquitination, and movement toward the catalytic core. The broad range of affinities for ubiquitin, ubiquitin-like, and non-ubiquitin signals by adjacent yet nonoverlapping sites all within the base represents a hub of activity that coordinates the intricate relay of substrates within the proteasome, and consequently it influences substrate residency time and commitment to degradation.
Asunto(s)
Poliubiquitina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Endopeptidasas/genética , Endopeptidasas/metabolismo , Poliubiquitina/genética , Complejo de la Endopetidasa Proteasomal/genética , Unión Proteica , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Therapeutic monoclonal antibodies are becoming a significant and rapidly growing class of therapeutic pharmaceuticals. Their discovery and development requires fast and high-throughput methodologies for screening and selecting appropriate candidate antibodies having high affinity for the target as well as high specificity and low cross-reactivity. This study demonstrates the use of the ProteOn XPR36 protein interaction array system and its novel approach, termed One-Shot Kinetics, for the rapid screening and selection of high-affinity antibodies. This approach allows multiple quantitative protein binding analyses in parallel, providing association, dissociation, and affinity constants for several antibodies or supernatants simultaneously in one experiment. We show that the ProteOn XPR36 system is a valuable tool for use across multiple stages of the therapeutic antibody discovery and development process, enabling efficient and rapid screening after panning, affinity maturation, assay validation, and clone selection.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Animales , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos/inmunología , Antígenos/inmunología , Células CHO , Células Clonales , Cricetinae , Cricetulus , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Cinética , Mutación/genética , Unión Proteica , Receptores de IgG/inmunología , Reproducibilidad de los ResultadosRESUMEN
The ability of CheY, the response regulator of bacterial chemotaxis, to generate clockwise rotation is regulated by two covalent modifications - phosphorylation and acetylation. While the function and signal propagation of the former are widely understood, the mechanism and role of the latter are still obscure. To obtain information on the function of this acetylation, we non-enzymatically acetylated CheY to a level similar to that found in vivo, and examined its binding to its kinase CheA, its phosphatase CheZ and the switch protein FliM - its target at the flagellar switch complex. Acetylation repressed the binding to all three proteins. These results suggest that both phosphorylation and acetylation determine CheY's ability to bind to its target proteins, thus providing two levels of regulation, fast and slow respectively. The fast level is modulated by environmental signals (e.g. chemotactic and thermotactic stimuli). The slow one is regulated by the metabolic state of the cell and it determines, at each metabolic state, the fraction of CheY molecules that can participate in signalling.
Asunto(s)
Proteínas Bacterianas/metabolismo , Quimiotaxis , Escherichia coli/fisiología , Proteínas de la Membrana/metabolismo , Acetilación , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Histidina Quinasa , Lisina/genética , Lisina/metabolismo , Proteínas de la Membrana/genética , Proteínas Quimiotácticas Aceptoras de Metilo , Datos de Secuencia MolecularRESUMEN
As a signal for substrate targeting, polyubiquitin meets various layers of receptors upstream to the 26S proteasome. We obtained structural information on two receptors, Rpn10 and Dsk2, alone and in complex with (poly)ubiquitin or with each other. A hierarchy of affinities emerges with Dsk2 binding monoubiquitin tighter than Rpn10 does, whereas Rpn10 prefers the ubiquitin-like domain of Dsk2 to monoubiquitin, with increasing affinities for longer polyubiquitin chains. We demonstrated the formation of ternary complexes of both receptors simultaneously with (poly)ubiquitin and found that, depending on the ubiquitin chain length, the orientation of the resulting complex is entirely different, providing for alternate signals. Dynamic rearrangement provides a chain-length sensor, possibly explaining how accessibility of Dsk2 to the proteasome is limited unless it carries a properly tagged cargo. We propose a mechanism for a malleable ubiquitin signal that depends both on chain length and combination of receptors to produce tetraubiquitin as an efficient signal threshold.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Poliubiquitina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal/fisiología , Ubiquitinas/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Modelos Moleculares , Poliubiquitina/química , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/genética , Unión Proteica , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitinas/química , Ubiquitinas/genéticaRESUMEN
The production of antibodies for diagnostic and therapeutic applications is a major focus for biotechnology and pharmaceutical companies, and it requires the development of fast, high-throughput methodologies for screening and selecting appropriate candidate antibodies for development. These candidates must have very high affinity for the target as well as high specificity and low cross-reactivity. This study demonstrates the use of the ProteOn XPR 36 protein interaction array system for the rapid screening and selection of high-affinity antibodies--"one-shot" kinetics. This approach allows multiple quantitative protein binding analyses in parallel, providing association, dissociation, and affinity constants for several antibodies simultaneously in one experiment. We have used this new methodology to screen hundreds of monoclonal supernatants containing antibodies against two proteins of potential clinical interest: human interleukin 12 (IL-12) and a human hemoglobin (Hb) variant. In fact, approximately 250 supernatants raised against each antigen were screened in approximately 17 h, providing several high-affinity candidate monoclonal antibodies for each of these antigens.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Técnicas Biosensibles/métodos , Animales , Especificidad de Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Humanos , Hibridomas , Interleucina-12/inmunología , Cinética , Ratones , Ratones Endogámicos BALB C , Modelos TeóricosRESUMEN
We have characterized the molecular basis of the interaction between ASPP2 and Bcl-2, which are key proteins in the apoptotic pathway. The C-terminal ankyrin repeats and SH3 domain of ASPP2 (ASPP2(Ank-SH3)) mediate its interactions with the antiapoptotic protein Bcl-2. We used biophysical and computational methods to identify the interaction sites of Bcl-2 and its homologues with ASPP2. Using peptide array screening, we found that ASPP2(Ank-SH3) binds two homologous sites in all three Bcl proteins tested: (i) the conserved BH4 motif, and (ii) a binding site for proapoptotic regulators. Quantitative binding studies revealed that binding of ASPP2(Ank-SH3) to the Bcl-2 family members is selective at two levels: (i) interaction with Bcl-2-derived peptides is the tightest compared to peptides from the other family members, and (ii) within Bcl-2, binding of ASPP2(Ank-SH3) to the BH4 domain is tightest. Sequence alignment of the ASPP2-binding peptides combined with binding studies of mutated peptides revealed that two nonconserved positions where only Bcl-2 contains positively charged residues account for its tighter binding. The experimental binding results served as a basis for docking analysis, by which we modeled the complexes of ASPP2(Ank-SH3) with the full-length Bcl proteins. Using peptide arrays and quantitative binding studies, we found that Bcl-2 binds three loops in ASPP2(Ank-SH3) with similar affinity, in agreement with our predicted model. Based on our results, we propose a mechanism in which ASPP2 induces apoptosis by inhibiting functional sites of the antiapoptotic Bcl-2 proteins.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Sitios de Unión , Simulación por Computador , Humanos , Modelos Moleculares , Mutación , Unión ProteicaRESUMEN
A ProteOn XPR36 parallel array biosensor was used to characterize the binding kinetics of a set of small molecule/enzyme interactions. Using one injection with the ProteOn's crisscrossing flow path system, we collected response data for six different concentrations of each analyte over six different target protein surfaces. This "one-shot" approach to kinetic analysis significantly improves throughput while generating high-quality data even for low-molecular-mass analytes. We found that the affinities determined for nine sulfonamide-based inhibitors of the enzyme carbonic anhydrase II were highly correlated with the values determined using isothermal titration calorimetry. We also measured the temperature dependence (from 15 to 35 degrees C) of the kinetics for four of the inhibitor/enzyme interactions. Our results illustrate the potential of this new parallel-processing biosensor to increase the speed of kinetic analysis in drug discovery and expand the applications of real-time protein interaction arrays.