RESUMEN

The objective of this study was to determine the trends in surgical approach to hysterectomy over the last decade and compare perioperative outcomes and complications. This retrospective cohort study used clinical registry data from the Michigan Hospitals that participated in Michigan Surgical Quality Collaborative (MSQC) from January 1st, 2010 through December 30th, 2020. A multigroup time series analysis was performed to determine how surgical approach to hysterectomy [open/TAH, laparoscopic (TLH/LAVH), and robotic-assisted (RA)] has changed over the last decade. Abnormal uterine bleeding, uterine fibroids, chronic pelvic pain, pelvic organ prolapse, endometriosis, pelvic mass, and endometrial cancer were the most common indications for hysterectomy. The open approach to hysterectomy declined from 32.6 to 16.9%, a 1.9-fold decrease, with an average decline of 1.6% per year (95% CI - 2.3 to - 0.9%). Laparoscopic-assisted hysterectomies decreased from 27.2 to 23.8%, a 1.5-fold decrease, with an average decrease of 0.1% per year (95% CI - 0.7 to 0.6%). Finally, the robotic-assisted approach increased from 38.3 to 49.3%, a 1.25-fold increase, with an average of 1.1% per year (95% CI 0.5 to 1.7%). For malignant cases, open procedures decreased from 71.4 to 26.6%, a 2.7-fold decrease, while RA-hysterectomy increased from 19.0 to 58.7%, a 3.1-fold increase. After controlling for the confounding variables age, race, and gynecologic malignancy, RA hysterectomy was found to have the lowest rate of complications when compared to the vaginal, laparoscopic and open approaches. Finally, after controlling for uterine weight, black patients were twice as likely to undergo an open hysterectomy compared to white patients.

Asunto(s)

Laparoscopía , Procedimientos Quirúrgicos Robotizados , Humanos , Femenino , Michigan/epidemiología , Estudios Retrospectivos , Procedimientos Quirúrgicos Robotizados/métodos , Histerectomía/métodos , Laparoscopía/métodos , Hospitales , Histerectomía Vaginal

RESUMEN

This study has employed mammalian transient expression systems to generate afucosylated antibodies and antibody Fc mutants for rapid candidate screening in discovery and early development. While chemical treatment with the fucose analogue 2-fluoro-peracetyl-fucose during transient expression only partially produced antibodies with afucosylated N-glycans, the genetic inactivation of the FUT8 gene in ExpiCHO-S™ by CRISPR/Cas9 enabled the transient production of fully afucosylated antibodies. Human IgG1 and murine IgG2a generated by the ExpiCHOfut8KO cell line possessed a 8-to-11-fold enhanced FcγRIIIa binding activity in comparison with those produced by ExpiCHO-S™. The Fc mutant S239D/S298A/I332E produced by ExpiCHO-S™ had an approximate 2-fold higher FcγRIIIa affinity than that of the afucosylated wildtype molecule, although it displayed significantly lower thermal-stability. When the Fc mutant was produced in the ExpiCHOfut8KO cell line, the resulting afucosylated Fc mutant antibody had an additional approximate 6-fold increase in FcγRIIIa binding affinity. This synergistic effect between afucosylation and the Fc mutations was further verified by a natural killer (NK) cell activation assay. Together, these results have not only established an efficient large-scale transient CHO system for rapid production of afucosylated antibodies, but also confirmed a cooperative impact between afucosylation and Fc mutations on FcγRIIIa binding and NK cell activation.

Asunto(s)

Inmunoglobulina G , Células Asesinas Naturales , Humanos , Animales , Ratones , Inmunoglobulina G/genética , Mamíferos

RESUMEN

Background: Interleukin (IL)25 has been implicated in tissue homeostasis at barrier surfaces and the initiation of type two inflammatory signaling in response to infection and cell injury across multiple organs. We sought to discover and engineer a high affinity neutralizing antibody and evaluate the antibody functional activity in vitro and in vivo. Methods: In this study, we generated a novel anti-IL25 antibody (22C7) and investigated the antibody's therapeutic potential for targeting IL25 in inflammation. Results: A novel anti-IL25 antibody (22C7) was generated with equivalent in vitro affinity and potency against the human and mouse orthologs of the cytokine. This translated into in vivo potency in an IL25-induced air pouch model where 22C7 inhibited the recruitment of monocytes, macrophages, neutrophils and eosinophils. Furthermore, 22C7 significantly reduced ear swelling, acanthosis and disease severity in the Aldara mouse model of psoriasiform skin inflammation. Given the therapeutic potential of IL25 targeting in inflammatory conditions, 22C7 was further engineered to generate a highly developable, fully human antibody while maintaining the affinity and potency of the parental molecule. Conclusions: The generation of 22C7, an anti-IL25 antibody with efficacy in a preclinical model of skin inflammation, raises the therapeutic potential for 22C7 use in the spectrum of IL25-mediated diseases.

RESUMEN

Background: Supervision of behavior analysts seeking certification and supervision of service delivery are key processes in the provision of quality behaviour analytic services to individuals with developmental disabilities. Our study is the first to examine international supervisory practices within the field of applied behaviour analysis. Method: An online survey was distributed to 92 professionals internationally, assessing supervisory practice, supervisor support, work demands, job satisfaction, and burnout. Results: Findings indicate high satisfaction with the supervisor and supervisory experience. Excessive work demands positively correlate with high burnout and low job satisfaction. Half of all professionals only worked with one or two clients before certification. Supervisor and collegial support seem to decrease the likelihood of suffering burnout and increase job satisfaction, although relationships were not statistically significant. Conclusions: Supervisor and collegial support warrant further research as protective factors. Implications for an evidence-based supervisory practice that produces ethical and competent supervisees are discussed.

Asunto(s)

Análisis Aplicado de la Conducta , Agotamiento Profesional , Satisfacción en el Trabajo , Apoyo Social , Adolescente , Adulto , Certificación , Femenino , Humanos , Masculino , Persona de Mediana Edad , Encuestas y Cuestionarios , Trabajo , Adulto Joven

RESUMEN

Naïve antibody libraries provide a rich resource for the identification of binding domains against targets of therapeutic interest. Being naïve in nature means a lack in antigen bias, resulting in a breadth of diversity with respect to epitopes that can be successfully targeted. In combination with display-based technology platforms, selection strategies allow for the generation of ortholog cross-reactive binding domains which enable critical preclinical proof-of-concept studies. However, naïve binding domains often suffer from low target affinity. In addition, construction of large naïve libraries results in non-native pairing of heavy and light v-domains which can present a challenge to molecular stability. Here we describe effective methods for the parallel evolution of antibody affinity and thermal stability which couple mutant antibody library phage display with carefully designed selection strategies.

Asunto(s)

Anticuerpos/uso terapéutico , Afinidad de Anticuerpos/inmunología , Evolución Molecular Dirigida/métodos , Temperatura , Regiones Determinantes de Complementariedad , Ensayo de Inmunoadsorción Enzimática , Fluorescencia , Biblioteca de Péptidos , Ingeniería de Proteínas , Estabilidad Proteica , Anticuerpos de Cadena Única/genética

RESUMEN

Implementation of in vitro assays that correlate with in vivo human pharmacokinetics (PK) would provide desirable preclinical tools for the early selection of therapeutic monoclonal antibody (mAb) candidates with minimal non-target-related PK risk. Use of these tools minimizes the likelihood that mAbs with unfavorable PK would be advanced into costly preclinical and clinical development. In total, 42 mAbs varying in isotype and soluble versus membrane targets were tested in in vitro and in vivo studies. MAb physicochemical properties were assessed by measuring non-specific interactions (DNA- and insulin-binding ELISA), self-association (affinity-capture self-interaction nanoparticle spectroscopy) and binding to matrix-immobilized human FcRn (surface plasmon resonance and column chromatography). The range of scores obtained from each in vitro assay trended well with in vivo clearance (CL) using both human FcRn transgenic (Tg32) mouse allometrically projected human CL and observed human CL, where mAbs with high in vitro scores resulted in rapid CL in vivo. Establishing a threshold value for mAb CL in human of 0.32 mL/hr/kg enabled refinement of thresholds for each in vitro assay parameter, and using a combinatorial triage approach enabled the successful differentiation of mAbs at high risk for rapid CL (unfavorable PK) from those with low risk (favorable PK), which allowed mAbs requiring further characterization to be identified. Correlating in vitro parameters with in vivo human CL resulted in a set of in vitro tools for use in early testing that would enable selection of mAbs with the greatest likelihood of success in the clinic, allowing costly late-stage failures related to an inadequate exposure profile, toxicity or lack of efficacy to be avoided.

Asunto(s)

Anticuerpos Monoclonales/farmacocinética , Descubrimiento de Drogas/métodos , Técnicas In Vitro , Modelos Animales , Animales , Humanos , Ratones , Ratones Transgénicos

RESUMEN

Fully-human single-chain Fv (scFv) proteins are key potential building blocks of bispecific therapeutic antibodies, but they often suffer from manufacturability and clinical development limitations such as instability and aggregation. The causes of these scFv instability problems, in proteins that should be theoretically stable, remains poorly understood. To inform the future development of such molecules, we carried out a comprehensive structural analysis of the highly stabilized anti-CXCL13 scFv E10. E10 was derived from the parental 3B4 using complementarity-determining region (CDR)-restricted mutagenesis and tailored selection and screening strategies, and carries four mutations in VL-CDR3. High-resolution crystal structures of parental 3B4 and optimized E10 scFvs were solved in the presence and absence of human CXCL13. In parallel, a series of scFv mutants was generated to interrogate the individual contribution of each of the four mutations to stability and affinity improvements. In combination, these analyses demonstrated that the optimization of E10 was primarily mediated by removing clashes between both the VL and the VH, and between the VL and CXCL13. Importantly, a single, germline-encoded VL-CDR3 residue mediated the key difference between the stable and unstable forms of the scFv. This work demonstrates that, aside from being the critical mediators of specificity and affinity, CDRs may also be the primary drivers of biotherapeutic developability.

Asunto(s)

Productos Biológicos/química , Quimiocina CXCL13/antagonistas & inhibidores , Modelos Moleculares , Anticuerpos de Cadena Única/química , Sustitución de Aminoácidos , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Complejo Antígeno-Anticuerpo/química , Complejo Antígeno-Anticuerpo/metabolismo , Sitios de Unión de Anticuerpos , Productos Biológicos/metabolismo , Quimiocina CXCL13/química , Quimiocina CXCL13/metabolismo , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/metabolismo , Humanos , Cinética , Mutación , Agregado de Proteínas , Conformación Proteica , Estabilidad Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/metabolismo , Solubilidad , Difracción de Rayos X

RESUMEN

Although humanized antibodies have been highly successful in the clinic, all current humanization techniques have potential limitations, such as: reliance on rodent hosts, immunogenicity due to high non-germ-line amino acid content, v-domain destabilization, expression and formulation issues. This study presents a technology that generates stable, soluble, ultrahumanized antibodies via single-step complementarity-determining region (CDR) germ-lining. For three antibodies from three separate key immune host species, binary substitution CDR cassettes were inserted into preferred human frameworks to form libraries in which only the parental or human germ-line destination residue was encoded at each position. The CDR-H3 in each case was also augmented with 1 ± 1 random substitution per clone. Each library was then screened for clones with restored antigen binding capacity. Lead ultrahumanized clones demonstrated high stability, with affinity and specificity equivalent to, or better than, the parental IgG. Critically, this was mainly achieved on germ-line frameworks by simultaneously subtracting up to 19 redundant non-germ-line residues in the CDRs. This process significantly lowered non-germ-line sequence content, minimized immunogenicity risk in the final molecules and provided a heat map for the essential non-germ-line CDR residue content of each antibody. The ABS technology therefore fully optimizes the clinical potential of antibodies from rodents and alternative immune hosts, rendering them indistinguishable from fully human in a simple, single-pass process.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Regiones Determinantes de Complementariedad/inmunología , Células Germinativas/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Especificidad de Anticuerpos/inmunología , Células Clonales , Regiones Determinantes de Complementariedad/química , Simulación por Computador , Ensayo de Inmunoadsorción Enzimática , Epítopos de Linfocito T/inmunología , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/inmunología , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Biblioteca de Péptidos , Estabilidad Proteica , Estructura Terciaria de Proteína , Ratas , Alineación de Secuencia , Análisis de Secuencia de Proteína , Proteínas tau/química , Proteínas tau/inmunología

RESUMEN

While myriad molecular formats for bispecific antibodies have been examined to date, the simplest structures are often based on the scFv. Issues with stability and manufacturability in scFv-based bispecific molecules, however, have been a significant hindrance to their development, particularly for high-concentration, stable formulations that allow subcutaneous delivery. Our aim was to generate a tetravalent bispecific molecule targeting two inflammatory mediators for synergistic immune modulation. We focused on an scFv-Fc-scFv format, with a flexible (A4T)3 linker coupling an additional scFv to the C-terminus of an scFv-Fc. While one of the lead scFvs isolated directly from a naïve library was well-behaved and sufficiently potent, the parental anti-CXCL13 scFv 3B4 required optimization for affinity, stability, and cynomolgus ortholog cross-reactivity. To achieve this, we eschewed framework-based stabilizing mutations in favor of complementarity-determining region (CDR) mutagenesis and re-selection for simultaneous improvements in both affinity and thermal stability. Phage-displayed 3B4 CDR-mutant libraries were used in an aggressive "hammer-hug" selection strategy that incorporated thermal challenge, functional, and biophysical screening. This approach identified leads with improved stability and>18-fold, and 4,100-fold higher affinity for both human and cynomolgus CXCL13, respectively. Improvements were exclusively mediated through only 4 mutations in VL-CDR3. Lead scFvs were reformatted into scFv-Fc-scFvs and their biophysical properties ranked. Our final candidate could be formulated in a standard biopharmaceutical platform buffer at 100 mg/ml with<2% high molecular weight species present after 7 weeks at 4 °C and viscosity<15 cP. This workflow has facilitated the identification of a truly manufacturable scFv-based bispecific therapeutic suitable for subcutaneous administration.

Asunto(s)

Anticuerpos Biespecíficos/genética , Regiones Determinantes de Complementariedad/genética , Ingeniería de Proteínas , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/metabolismo , Animales , Bacteriófagos/genética , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inyecciones Subcutáneas , Biblioteca de Péptidos , Estabilidad Proteica , Ratas , Anticuerpos de Cadena Única/genética , Temperatura

RESUMEN

Malarial parasites are exquisitely susceptible to a number of microtubule inhibitors but most of these compounds also affect human microtubules. Herbicides of the dinitroaniline and phosphorothioamidate classes however affect some plant and protozoal cells but not mammalian ones. We have previously shown that these herbicides block schizogony in erythrocytic parasites of the most lethal human malaria, Plasmodium falciparum, disrupt their mitotic spindles, and bind selectively to parasite tubulin. Here we show for the first time that the antimitotic herbicides also block the development of malarial parasites in the liver stage. Structure-based design of novel antimalarial agents binding to tubulin at the herbicide site, which presumably exists on (some) parasite and plant tubulins but not mammalian ones, can therefore constitute an important transmission blocking approach. The nature of this binding site is controversial, with three overlapping but non-identical locations on α-tubulin proposed in the literature. We tested the validity of the three sites by (i) using site-directed mutagenesis to introduce six amino acid changes designed to occlude them, (ii) producing the resulting tubulins recombinantly in Escherichia coli and (iii) measuring the affinity of the herbicides amiprophosmethyl and oryzalin for these proteins in comparison with wild-type tubulins by fluorescence quenching. The changes had little or no effect, with dissociation constants (Kd) no more than 1.3-fold (amiprophosmethyl) or 1.6-fold (oryzalin) higher than wild-type. We conclude that the herbicides impair Plasmodium liver stage as well as blood stage development but that the location of their binding site on malarial parasite tubulin remains to be proven.

Asunto(s)

Antiprotozoarios/metabolismo , Hepatocitos/parasitología , Herbicidas/metabolismo , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/crecimiento & desarrollo , Tubulina (Proteína)/metabolismo , Sitios de Unión , Línea Celular , Análisis Mutacional de ADN , Humanos , Mutagénesis Sitio-Dirigida , Unión Proteica , Tubulina (Proteína)/genética

RESUMEN

We have generated large libraries of single-chain Fv antibody fragments (>10(10) transformants) containing unbiased amino acid diversity that is restricted to the central combining site of the stable, well-expressed DP47 and DPK22 germline V-genes. Library WySH2A was constructed to examine the potential for synthetic complementarity-determining region (CDR)-H3 diversity to act as the lone source of binding specificity. Library WySH2B was constructed to assess the necessity for diversification in both the H3 and L3. Both libraries provided diverse, specific antibodies, yielding a total of 243 unique hits against 7 different targets, but WySH2B produced fewer hits than WySH2A when selected in parallel. WySH2A also consistently produced hits of similar quality to WySH2B, demonstrating that the diversification of the CDR-L3 reduces library fitness. Despite the absence of deliberate bias in the library design, CDR length was strongly associated with the number of hits produced, leading to a functional loop length distribution profile that mimics the biases observed in the natural repertoire. A similar trend was also observed for the CDR-L3. After target selections, several key amino acids were enriched in the CDR-H3 (e.g., small and aromatic residues) while others were reduced (e.g., strongly charged residues) in a manner that was specific to position, preferentially occurred in CDR-H3 stem positions, and tended towards residues associated with loop stabilization. As proof of principle for the WySH2 libraries to produce viable lead candidate antibodies, 114 unique hits were produced against Delta-like ligand 4 (DLL4). Leads exhibited nanomolar binding affinities, highly specific staining of DLL4+ cells, and biochemical neutralization of DLL4-NOTCH1 interaction.

Asunto(s)

Especificidad de Anticuerpos , Regiones Determinantes de Complementariedad/inmunología , Regiones Determinantes de Complementariedad/uso terapéutico , Biblioteca de Péptidos , Anticuerpos de Cadena Única/biosíntesis , Anticuerpos de Cadena Única/uso terapéutico , Proteínas Adaptadoras Transductoras de Señales , Animales , Especificidad de Anticuerpos/genética , Proteínas de Unión al Calcio , Clonación Molecular , Regiones Determinantes de Complementariedad/genética , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/inmunología , Ratones , Modelos Moleculares , Mutación , Receptor Notch1/antagonistas & inhibidores , Receptor Notch1/genética , Receptor Notch1/inmunología , Anticuerpos de Cadena Única/genética

RESUMEN

Highly specific antibodies to phosphoepitopes are valuable tools to study phosphorylation in disease states, but their discovery is largely empirical, and the molecular mechanisms mediating phosphospecific binding are poorly understood. Here, we report the generation and characterization of extremely specific recombinant chicken antibodies to three phosphoepitopes on the Alzheimer disease-associated protein tau. Each antibody shows full specificity for a single phosphopeptide. The chimeric IgG pT231/pS235_1 exhibits a K(D) of 0.35 nm in 1:1 binding to its cognate phosphopeptide. This IgG is murine ortholog-cross-reactive, specifically recognizing the pathological form of tau in brain samples from Alzheimer patients and a mouse model of tauopathy. To better understand the underlying binding mechanisms allowing such remarkable specificity, we determined the structure of pT231/pS235_1 Fab in complex with its cognate phosphopeptide at 1.9 Å resolution. The Fab fragment exhibits novel complementarity determining region (CDR) structures with a "bowl-like" conformation in CDR-H2 that tightly and specifically interacts with the phospho-Thr-231 phosphate group, as well as a long, disulfide-constrained CDR-H3 that mediates peptide recognition. This binding mechanism differs distinctly from either peptide- or hapten-specific antibodies described to date. Surface plasmon resonance analyses showed that pT231/pS235_1 binds a truly compound epitope, as neither phosphorylated Ser-235 nor free peptide shows any measurable binding affinity.

Asunto(s)

Enfermedad de Alzheimer/metabolismo , Anticuerpos/inmunología , Epítopos/inmunología , Proteínas tau/inmunología , Enfermedad de Alzheimer/genética , Secuencia de Aminoácidos , Animales , Anticuerpos/química , Anticuerpos/genética , Encéfalo/metabolismo , Pollos , Epítopos/química , Epítopos/genética , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Fosforilación , Proteínas tau/química , Proteínas tau/genética , Proteínas tau/metabolismo

RESUMEN

Protein engineering techniques can facilitate the direct de-convolution of specific domains, regions, and particular amino acids that contribute to protein function. Many tools are available to aid this enterprise and herein we describe one such tool, a technique we term "Molecular Scanning" (MS). MS is analogous to previously described alanine scanning in that it samples potentially functional sequence space, but differs in that it uses Error-Prone polymerase chain reaction to randomly introduce all amino acids across the sequence space, as opposed to simply introducing alanine at each desired position. We commonly use MS in conjunction with ribosome-display, selecting for specific character traits (e.g., improved affinity) which allows us to sample functionally relevant diversity on a reasonably large scale. This approach is amenable to a variety of different mutational techniques and display technologies as dictated by user requirements or needs. In this chapter we present a general outline of the process as we have previously successfully applied it.

Asunto(s)

Biología Computacional/métodos , Mutagénesis/genética , Biblioteca de Péptidos , Ingeniería de Proteínas/métodos , Ribosomas/metabolismo , Tampones (Química) , Humanos , Biosíntesis de Proteínas , ARN/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Reversa/genética , Soluciones , Estadística como Asunto , Transcripción Genética

RESUMEN

Examination of 1269 unique naive chicken V(H) sequences showed that the majority of positions in the framework (FW) regions were maintained as germline, with high mutation rates observed in the CDRs. Many FW mutations could be clearly related to the modulation of CDR structure or the V(H)-V(L) interface. CDRs 1 and 2 of the V(H) exhibited frequent mutation in solvent-exposed positions, but conservation of common structural residues also found in human CDRs at the same positions. In comparison with humans and mice, the chicken CDR3 repertoire was skewed toward longer sequences, was dominated by small amino acids (G/S/A/C/T), and had higher cysteine (chicken, 9.4%; human, 1.6%; and mouse, 0.25%) but lower tyrosine content (chicken, 9.2%; human, 16.8%; and mouse 26.4%). A strong correlation (R(2) = 0.97) was observed between increasing CDR3 length and higher cysteine content. This suggests that noncanonical disulfides are strongly favored in chickens, potentially increasing CDR stability and complexity in the topology of the combining site. The probable formation of disulfide bonds between CDR3 and CDR1, FW2, or CDR2 was also observed, as described in camelids. All features of the naive repertoire were fully replicated in the target-selected, phage-displayed repertoire. The isolation of a chicken Fab with four noncanonical cysteines in the V(H) that exhibits 64 nM (K(D)) binding affinity for its target proved these constituents to be part of the humoral response, not artifacts. This study supports the hypothesis that disulfide bond-constrained CDR3s are a structural diversification strategy in the restricted germline v-gene repertoire of chickens.

Asunto(s)

Sustitución de Aminoácidos , Pollos/genética , Regiones Determinantes de Complementariedad/genética , Cadenas Pesadas de Inmunoglobulina/genética , Mutación , Animales , Afinidad de Anticuerpos/genética , Camelus/genética , Camelus/inmunología , Pollos/inmunología , Regiones Determinantes de Complementariedad/inmunología , Disulfuros/inmunología , Humanos , Cadenas Pesadas de Inmunoglobulina/inmunología , Ratones , Estabilidad Proteica , Especificidad de la Especie

RESUMEN

Antibodies that neutralize RAGE (receptor for advanced glycation end products)-ligand interactions have potential therapeutic applications in both acute and chronic diseases. We generated XT-M4, a rat anti-RAGE monoclonal antibody that has in vivo efficacy in an acute sepsis model. This antibody was subsequently humanized. To improve the affinity of this antibody for the treatment of chronic indications, we used random and targeted mutagenesis strategies in combination with ribosome and phage-display technologies, respectively, to generate libraries of XT-M4 variants. We identified a panel of single-chain Fv antibody fragments (scFv's) that was improved up to 110-fold in a homogeneous time-resolved fluorescence competition assay against parental XT-M4 immunoglobulin G (IgG). After reformatting to bivalent scFv-Fc fusions and IgGs, we observed similar gains in potency in the same assay. Further analysis of binding kinetics as IgG revealed multiple variants with subnanomolar apparent affinity that was dictated primarily by improvements in the off-rate. All variants also had improved binding to cell surface-expressed human RAGE, and all retained, or had improved, apparent affinity for mouse RAGE. F100bL in V(H) (variable region of the heavy chain) complementarity-determining region 3 (CDR3) was one of a number of key mutations that correlated with affinity improvements and was independently identified by both mutagenesis strategies. Random mutagenesis coupled with ribosome display and high-throughput screening revealed an unexpectedly high level of mutational plasticity across the whole length of the humanized scFv, suggesting greater scope for structural optimization outside of the primary antigen-combining site defined by V(H) CDR3 and V(kappa) CDR3. In summary, our comprehensive mutagenesis approach not only achieved the desired affinity maturation of XT-M4 but also defined multiple mutational hotspots across the antibody sequence, provided an insight into the specificity-determining residues of the antibody paratope, and identified additional sites within the CDR loops where human germ-line amino acids may be introduced without affecting function.

Asunto(s)

Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/metabolismo , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/inmunología , Secuencia de Aminoácidos , Animales , Análisis Mutacional de ADN , Fluorometría , Humanos , Cinética , Ratones , Datos de Secuencia Molecular , Pruebas de Neutralización , Ratas , Receptor para Productos Finales de Glicación Avanzada

RESUMEN

Phage and ribosome display technologies have emerged as important tools in the high-throughput screening of protein pharmaceuticals. However, a challenge created by the implementation of such tools is the need to purify large numbers of proteins for screening. While some assays may be compatible with crude bacterial lysates or periplasmic extracts, many functional assays, particularly cell-based assays, require protein of high purity and concentration. Here we evaluate several methods for small-scale, high-throughput protein purification. From our initial assessment we identified the HIS-Select 96-well filter plate system as the method of choice for further evaluation. This method was optimized and used to produce scFvs that were tested in cell-based functional assays. The behavior of HIS-Select purified scFvs in these assays was found to be similar to scFvs purified using a traditional large-scale 2-step purification method. The HIS-Select method allows high-throughput purification of hundreds of scFvs with yields in the 50-100 microg range, and of sufficient purity to allow evaluation in a cell-based proliferation assay. In addition, the use of a similar 96-well-based method facilitates the purification and subsequent screening of large numbers of IgGs and Fc fusion proteins generated through reformatting of scFv fragments.

Asunto(s)

Anticuerpos Monoclonales/aislamiento & purificación , Región Variable de Inmunoglobulina/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Escherichia coli/genética , Escherichia coli/inmunología , Femenino , Humanos , Regiones Constantes de Inmunoglobulina/genética , Regiones Constantes de Inmunoglobulina/inmunología , Regiones Constantes de Inmunoglobulina/aislamiento & purificación , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Inmunoglobulina G/aislamiento & purificación , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Masculino , Periplasma/genética , Periplasma/inmunología , Proteínas Periplasmáticas/genética , Proteínas Periplasmáticas/inmunología , Proteínas Periplasmáticas/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología

RESUMEN

Microtubules play important roles in cell division, motility and structural integrity of malarial parasites. Some microtubule inhibitors disrupt parasite development at very low concentrations, but most of them also kill mammalian cells. However, the dinitroaniline family of herbicides, which bind specifically to plant tubulin, have inhibitory activity on plant cells but are relatively non-toxic to human cells. Certain dinitroanilines are also inhibitory to various protozoal parasites including Plasmodium. Here we demonstrate that the dinitroanilines trifluralin and oryzalin inhibited progression of erythrocytic Plasmodium falciparum through schizogony, blocked mitotic division, and caused accumulation of abnormal microtubular structures. Moreover, radiolabelled trifluralin interacted with purified, recombinant parasite tubulins but to a much lesser extent with bovine tubulins. The phosphorothioamidate herbicide amiprophos-methyl, which has the same herbicidal mechanism as dinitroanilines, also had antimalarial activity and a similar action on schizogony. These data suggest that P. falciparum tubulin contains a dinitroaniline/phosphorothioamidate-binding site that is not conserved in humans and might be a target for new antimalarial drugs.

Asunto(s)

Antimaláricos/farmacología , Herbicidas/farmacología , Plasmodium falciparum/efectos de los fármacos , Moduladores de Tubulina/farmacología , Tubulina (Proteína)/efectos de los fármacos , Compuestos de Anilina/química , Compuestos de Anilina/farmacología , Animales , Antimaláricos/química , Dinitrobencenos/química , Dinitrobencenos/farmacología , Herbicidas/química , Inmunohistoquímica , L-Lactato Deshidrogenasa/análisis , Microscopía Fluorescente , Estructura Molecular , Nitrobencenos/química , Nitrobencenos/farmacología , Compuestos Organotiofosforados/química , Compuestos Organotiofosforados/farmacología , Pruebas de Sensibilidad Parasitaria , Plasmodium falciparum/crecimiento & desarrollo , Ensayo de Unión Radioligante , Sulfanilamidas/química , Sulfanilamidas/farmacología , Trifluralina/química , Trifluralina/farmacología , Tubulina (Proteína)/análisis , Moduladores de Tubulina/química