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1.
Proc Natl Acad Sci U S A ; 119(23): e2201562119, 2022 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-35653561

RESUMEN

The utilization of avidity to drive and tune functional responses is fundamental to antibody biology and often underlies the mechanisms of action of monoclonal antibody drugs. There is increasing evidence that antibodies leverage homotypic interactions to enhance avidity, often through weak transient interfaces whereby self-association is coupled with target binding. Here, we comprehensively map the Fab­Fab interfaces of antibodies targeting DR5 and 4-1BB that utilize homotypic interaction to promote receptor activation and demonstrate that both antibodies have similar self-association determinants primarily encoded within a germline light chain complementarity determining region 2 (CDRL2). We further show that these determinants can be grafted onto antibodies of distinct target specificity to substantially enhance their activity. An expanded characterization of all unique germline CDRL2 sequences reveals additional self-association sequence determinants encoded in the human germline repertoire. Our results suggest that this phenomenon is unique to CDRL2, and is correlated with the less frequent antigen interaction and lower somatic hypermutation associated with this loop. This work reveals a previously unknown avidity mechanism in antibody native biology that can be exploited for the engineering of biotherapeutics.


Asunto(s)
Afinidad de Anticuerpos , Regiones Determinantes de Complementariedad , Células Germinativas , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Quimioterapia , Fragmentos Fab de Inmunoglobulinas
2.
J Biol Chem ; 299(5): 104685, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-37031819

RESUMEN

The exquisite specificity, natural biological functions, and favorable development properties of antibodies make them highly effective agents as drugs. Monoclonal antibodies are particularly strong as inhibitors of systemically accessible targets where trough-level concentrations can sustain full target occupancy. Yet beyond this pharmacologic wheelhouse, antibodies perform suboptimally for targets of high abundance and those not easily accessible from circulation. Fundamentally, this restraint on broader application is due largely to the stoichiometric nature of their activity-one drug molecule is generally able to inhibit a maximum of two target molecules at a time. Enzymes in contrast are able to catalytically turnover multiple substrates, making them a natural sub-stoichiometric solution for targets of high abundance or in poorly accessible sites of action. However, enzymes have their own limitations as drugs, including, in particular, the polypharmacology and broad specificity often seen with native enzymes. In this study, we introduce antibody-guided proteolytic enzymes to enable selective sub-stoichiometric turnover of therapeutic targets. We demonstrate that antibody-mediated substrate targeting can enhance enzyme activity and specificity, with proof of concept for two challenging target proteins, amyloid-ß and immunoglobulin G. This work advances a new biotherapeutic platform that combines the favorable properties of antibodies and proteolytic enzymes to more effectively suppress high-bar therapeutic targets.


Asunto(s)
Anticuerpos Monoclonales , Terapia Biológica , Endopeptidasas , Péptido Hidrolasas , Inmunoglobulina G , Péptido Hidrolasas/metabolismo , Terapia Biológica/métodos
3.
Anal Chem ; 92(10): 6839-6843, 2020 05 19.
Artículo en Inglés | MEDLINE | ID: mdl-32309925

RESUMEN

There are many pharmacokinetic challenges associated with administering protein therapeutics, including biotransformation via clipping, deamidation, isomerization, oxidation, etc. In the case of engineered multivalent tethered antibody formats, proteolysis or deconjugation at the fusion or conjugation site present further issues. Unlike degradations associated with antibody drug conjugates, such biotransformations of tethered antibody formats usually result in degraded products with large mass differences. These large differences can result in processing or mass spectrometry response bias among the resulting product species that can lead to inaccurate stability quantitation. Herein, we describe an assay strategy for characterizing and quantitating degradations accurately for multivalent antibodies by incorporating response bias corrections. For the multivalent tethered antibody molecules selected, an ∼30-80% difference in response, compared to the cleaved product, was observed. To correct for the response bias, selected tethered multivalent antibodies and an IgG antibody (representing the stable intact and the degraded product species, respectively) were spiked in serum at known ratios for analysis. Following affinity capture, we generated calibration curves (five-parameter logistic fit p < 0.05) by plotting the measured ratios of the MS ion responses against the known spiked-in ratios (CVs < 8% for calibration standards). The qualified calibration curve (accuracy within 8% and 2% for measuring degradations of 5% and 15% product, respectively) was then used, through interpolation, to determine stability profiles for the same multivalent tethered antibody formats from both in vitro serum and pharmacokinetic study samples.


Asunto(s)
Anticuerpos/análisis , Inmunoconjugados/análisis , Cromatografía Liquida , Espectrometría de Masas
4.
Anal Chem ; 92(2): 2186-2193, 2020 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-31880920

RESUMEN

With the rapid rise of therapeutic antibodies and antibody-drug conjugates, significant investments have been made in developing workflows that utilize mass spectrometry to detect these intact molecules, the large fragments generated by their selective digestion, and the peptides generated by traditional proteomics workflows. The resultant data is used to gain insight into a wide range of parameters, including primary sequence, disulfide bonding, glycosylation patterns, biotransformation, and more. However, many of the technologies utilized to couple these workflows to mass spectrometers have significant limitations that force nonoptimal modifications to upstream sample preparation steps, limit the throughput of high-volume workflows, and prevent the harmonization of diverse experiments onto a single hardware platform. Here, we describe a new analytical platform that enables direct and high-throughput coupling to electrospray ionization mass spectrometry. The SampleStream platform is compatible with both native and denaturing electrospray, operates with a throughput of up to 15 s/sample, provides extensive concentration of dilute samples, and affords similar sensitivity to comparable liquid chromatographic methods.


Asunto(s)
Anticuerpos Monoclonales/análisis , Ensayos Analíticos de Alto Rendimiento , Inmunoconjugados/análisis , Ensayos Analíticos de Alto Rendimiento/instrumentación , Programas Informáticos , Espectrometría de Masa por Ionización de Electrospray/instrumentación
5.
Mol Pharm ; 15(10): 4529-4537, 2018 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-30118239

RESUMEN

A critical part of the clinical development path for a therapeutic antibody involves evaluating the physical and chemical stability of candidate molecules throughout the manufacturing process. In particular, the risks of chemical liabilities that can impact antigen binding, such as deamidation, oxidation, and isomerization in the antibody CDR sequences, need to be controlled through formulation development or eliminated by replacing the amino acid motif displaying the chemical instability. Commonly, the antibody CDR sequence contains multiple sequence motifs (potential hotspots) for chemical instability. However, only a subset of these motifs results in actual chemical modification, and thus, experimental assessment of the extent of instability is necessary to identify positions for potential sequence engineering. Ideally, this information should be available prior to antibody humanization at the stage of parental rodent antibody identification. Early knowledge of liabilities allows for ranking of clones or the mitigation of liabilities by concurrent engineering with the antibody humanization process instead of time-consuming sequential activities. However, concurrent engineering of chemical liabilities and humanization requires translatability of the chemical modifications from the rodent parental antibody to the humanized. We experimentally compared the stability of all sequence motifs by mass spectrometric peptide mapping between the rodent parental antibody and the final humanized antibody and observed a linear correlation. These results have enabled a streamlined developability assessment process for therapeutic antibodies from lead discovery to clinical development.


Asunto(s)
Anticuerpos/inmunología , Secuencia de Aminoácidos , Animales , Cromatografía Liquida , Desaminación , Concentración de Iones de Hidrógeno , Isomerismo , Metionina/química , Ratones , Oxidación-Reducción , Espectrometría de Masas en Tándem , Triptófano/química
6.
Bioconjug Chem ; 28(10): 2538-2548, 2017 10 18.
Artículo en Inglés | MEDLINE | ID: mdl-28885827

RESUMEN

The incorporation of cysteines into antibodies by mutagenesis allows for the direct conjugation of small molecules to specific sites on the antibody via disulfide bonds. The stability of the disulfide bond linkage between the small molecule and the antibody is highly dependent on the location of the engineered cysteine in either the heavy chain (HC) or the light chain (LC) of the antibody. Here, we explore the basis for this site-dependent stability. We evaluated the in vivo efficacy and pharmacokinetics of five different cysteine mutants of trastuzumab conjugated to a pyrrolobenzodiazepine (PBD) via disulfide bonds. A significant correlation was observed between disulfide stability and efficacy for the conjugates. We hypothesized that the observed site-dependent stability of the disulfide-linked conjugates could be due to differences in the attachment site cysteine thiol pKa. We measured the cysteine thiol pKa using isothermal titration calorimetry (ITC) and found that the variants with the highest thiol pKa (LC K149C and HC A140C) were found to yield the conjugates with the greatest in vivo stability. Guided by homology modeling, we identified several mutations adjacent to LC K149C that reduced the cysteine thiol pKa and, thus, decreased the in vivo stability of the disulfide-linked PBD conjugated to LC K149C. We also present results suggesting that the high thiol pKa of LC K149C is responsible for the sustained circulation stability of LC K149C TDCs utilizing a maleimide-based linker. Taken together, our results provide evidence that the site-dependent stability of cys-engineered antibody-drug conjugates may be explained by interactions between the engineered cysteine and the local protein environment that serves to modulate the side-chain thiol pKa. The influence of cysteine thiol pKa on stability and efficacy offers a new parameter for the optimization of ADCs that utilize cysteine engineering.


Asunto(s)
Cisteína/química , Inmunoconjugados/química , Benzodiazepinas/química , Estabilidad de Medicamentos , Inmunoconjugados/genética , Maleimidas/química , Modelos Moleculares , Mutación , Conformación Proteica , Pirroles/química
7.
Blood ; 119(9): 2074-82, 2012 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-22246035

RESUMEN

HM1.24, an immunologic target for multiple myeloma (MM) cells, has not been effectively targeted with therapeutic monoclonal antibodies (mAbs). In this study, we investigated in vitro and in vivo anti-MM activities of XmAb5592, a humanized anti-HM1.24 mAb with Fc-domain engineered to significantly enhance FcγR binding and associated immune effector functions. XmAb5592 increased antibody-dependent cellular cytotoxicity (ADCC) several fold relative to the anti-HM1.24 IgG1 analog against both MM cell lines and primary patient myeloma cells. XmAb5592 also augmented antibody dependent cellular phagocytosis (ADCP) by macrophages. Natural killer (NK) cells became more activated by XmAb5592 than the IgG1 analog, evidenced by increased cell surface expression of granzyme B-dependent CD107a and MM cell lysis, even in the presence of bone marrow stromal cells. XmAb5592 potently inhibited tumor growth in mice bearing human MM xenografts via FcγR-dependent mechanisms, and was significantly more effective than the IgG1 analog. Lenalidomide synergistically enhanced in vitro ADCC against MM cells and in vivo tumor inhibition induced by XmAb5592. A single dose of 20 mg/kg XmAb5592 effectively depleted both blood and bone marrow plasma cells in cynomolgus monkeys. These results support clinical development of XmAb5592, both as a monotherapy and in combination with lenalidomide, to improve patient outcome of MM.


Asunto(s)
Anticuerpos Monoclonales Humanizados/inmunología , Antígenos CD/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Mieloma Múltiple/terapia , Animales , Anticuerpos Monoclonales Humanizados/administración & dosificación , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Degranulación de la Célula , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Técnicas de Cocultivo , Sinergismo Farmacológico , Femenino , Proteínas Ligadas a GPI/inmunología , Humanos , Células Asesinas Naturales/inmunología , Lenalidomida , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Depleción Linfocítica , Macaca fascicularis , Ratones , Ratones SCID , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunología , Células Plasmáticas/efectos de los fármacos , Células Plasmáticas/inmunología , Talidomida/administración & dosificación , Talidomida/análogos & derivados , Talidomida/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto
8.
Cancer Immunol Res ; 12(1): 60-71, 2024 01 03.
Artículo en Inglés | MEDLINE | ID: mdl-37902604

RESUMEN

T cell-retargeting therapies have transformed the therapeutic landscape for hematologic diseases. T cell-dependent bispecific antibodies (TDB) function as conditional agonists that induce a polyclonal T-cell response, resulting in target cell destruction and cytokine release. The relationship between this response and its effects on surrounding innate immune populations has not been fully explored. Here we show that treatment with mosunetuzumab in patients results in natural killer (NK) cell activation in the peripheral blood. We modeled this phenomenon in vitro and found that TDB-mediated killing activated NK cells, increasing NK function and antibody-dependent cellular cytotoxicity (ADCC), and enhanced the capability of macrophages to perform antibody-dependent cellular phagocytosis (ADCP). This enhancement was triggered by cytokines released through TDB treatment, with IL2 and IFNγ being major drivers for increased ADCC and ADCP, respectively. Surprisingly, cytolytic ability could be further augmented through neutralization of IL10 for NK cells and TNFα for macrophages. Finally, we showed that TDB treatment enhanced the efficacy of Fc-driven killing to an orthogonal solid tumor target in vivo. These results provide rationale for novel antibody therapy combinations that take advantage of both adaptive and innate immune responses.


Asunto(s)
Anticuerpos Biespecíficos , Citocinas , Humanos , Línea Celular Tumoral , Citotoxicidad Celular Dependiente de Anticuerpos , Linfocitos T , Inmunidad Innata , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico
9.
Nat Commun ; 15(1): 642, 2024 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-38245524

RESUMEN

The ability to leverage antibodies to agonize disease relevant biological pathways has tremendous potential for clinical investigation. Yet while antibodies have been successful as antagonists, immune mediators, and targeting agents, they are not readily effective at recapitulating the biology of natural ligands. Among the important determinants of antibody agonist activity is the geometry of target receptor engagement. Here, we describe an engineering approach inspired by a naturally occurring Fab-Fab homotypic interaction that constrains IgG in a unique i-shaped conformation. i-shaped antibody (iAb) engineering enables potent intrinsic agonism of five tumor necrosis factor receptor superfamily (TNFRSF) targets. When applied to bispecific antibodies against the heterodimeric IL-2 receptor pair, constrained bispecific IgG formats recapitulate IL-2 agonist activity. iAb engineering provides a tool to tune agonist antibody function and this work provides a framework for the development of intrinsic antibody agonists with the potential for generalization across broad receptor classes.


Asunto(s)
Anticuerpos Biespecíficos , Receptores del Factor de Necrosis Tumoral , Inmunoglobulina G/genética , Ingeniería de Proteínas
10.
Exp Cell Res ; 317(9): 1278-85, 2011 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-21459085

RESUMEN

Several novel technologies have evolved over the last decade for the modification of antibodies to enhance their inherent effector functions. All focus on the constant Fc domain and utilize either amino acid substitutions or glycoform perturbations to modulate their interaction with Fc receptors and the effector cells that bear them. We review these technologies with an emphasis on their validation with animal models and human clinical data.


Asunto(s)
Anticuerpos/inmunología , Animales , Proteínas del Sistema Complemento/inmunología , Citotoxicidad Inmunológica , Diseño de Fármacos , Humanos
11.
Nat Rev Drug Discov ; 21(10): 715-735, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-35790857

RESUMEN

Antibodies are the cardinal effector molecules of the immune system and are being leveraged with enormous success as biotherapeutic drugs. A key part of the adaptive immune response is the production of an epitope-diverse, polyclonal antibody mixture that is capable of neutralizing invading pathogens or disease-causing molecules through binding interference and by mediating humoral and cellular effector functions. Avidity - the accumulated binding strength derived from the affinities of multiple individual non-covalent interactions - is fundamental to virtually all aspects of antibody biology, including antibody-antigen binding, clonal selection and effector functions. The manipulation of antibody avidity has since emerged as an important design principle for enhancing or engineering novel properties in antibody biotherapeutics. In this Review, we describe the multiple levels of avidity interactions that trigger the overall efficacy and control of functional responses in both natural antibody biology and their therapeutic applications. Within this framework, we comprehensively review therapeutic antibody mechanisms of action, with particular emphasis on engineered optimizations and platforms. Overall, we describe how affinity and avidity tuning of engineered antibody formats are enabling a new wave of differentiated antibody drugs with tailored properties and novel functions, promising improved treatment options for a wide variety of diseases.


Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Afinidad de Anticuerpos , Diseño de Fármacos , Epítopos/metabolismo , Humanos
12.
Comput Struct Biotechnol J ; 20: 4952-4968, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36147680

RESUMEN

Antibodies are fundamental effectors of humoral immunity, and have become a highly successful class of therapeutics. There is increasing evidence that antibodies utilize transient homotypic interactions to enhance function, and elucidation of such interactions can provide insights into their biology and new opportunities for their optimization as drugs. Yet the transitory nature of weak interactions makes them difficult to investigate. Capitalizing on their rich structural data and high conservation, we have characterized all the ways that antibody fragment antigen-binding (Fab) regions interact crystallographically. This approach led to the discovery of previously unrealized interfaces between antibodies. While diverse interactions exist, ß-sheet dimers and variable-constant elbow dimers are recurrent motifs. Disulfide engineering enabled interactions to be trapped and investigated structurally and functionally, providing experimental validation of the interfaces and illustrating their potential for optimization. This work provides first insight into previously undiscovered oligomeric interactions between antibodies, and enables new opportunities for their biotherapeutic optimization.

13.
Cancer Sci ; 101(5): 1080-8, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20331636

RESUMEN

Oncogenic mutations of the KRAS gene have emerged as a common mechanism of resistance against epidermal growth factor receptor (EGF-R)-directed tumor therapy. Mutated KRAS leads to ligand-independent activation of signaling pathways downstream of EGF-R. Thereby, direct effector mechanisms of EGF-R antibodies, such as blockade of ligand binding and inhibition of signaling, are bypassed. Thus, a humanized variant of the approved EGF-R antibody Cetuximab inhibited growth of wild-type KRAS-expressing A431 cells, but did not inhibit KRAS-mutated A549 tumor cells. We then investigated whether killing of tumor cells harboring mutated KRAS can be improved by enhancing antibody-dependent cellular cytotoxicity (ADCC). Protein- and glyco-engineering of antibodies' Fc region are established technologies to enhance ADCC by increasing antibodies' affinity to activating Fcgamma receptors. Thus, EGF-R antibody variants with increased affinity for the natural killer (NK) cell-expressed FcgammaRIIIa (CD16) were generated and analyzed. These variants triggered significantly enhanced mononuclear cell (MNC)-mediated killing of KRAS-mutated tumor cells compared to wild-type antibodies. Additionally, cells transfected with mutated KRAS were killed as effectively by ADCC as vector-transfected control cells. Together, these data demonstrate that KRAS mutations are not sufficient to render tumor cells resistant to ADCC. Consequently Fc-engineered EGF-R antibodies may prove effective against KRAS-mutated tumors, which are not susceptible to signaling inhibition by EGF-R antibodies.


Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos , Receptores ErbB/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Mutación , Neoplasias/genética , Ingeniería de Proteínas , Proteínas Proto-Oncogénicas/genética , Proteínas ras/genética , Línea Celular Tumoral , Proliferación Celular , Humanos , Leucocitos Mononucleares/inmunología , Neoplasias/inmunología , Proteínas Proto-Oncogénicas p21(ras) , Receptores de IgG/inmunología
14.
MAbs ; 12(1): 1818436, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32936727

RESUMEN

Treatment of ocular disease is hindered by the presence of the blood-retinal barrier, which restricts access of systemic drugs to the eye. Intravitreal injections bypass this barrier, delivering high concentrations of drug to the targeted tissue. However, the recommended dosing interval for approved biologics is typically 6-12 weeks, and frequent travel to the physician's office poses a substantial burden for elderly patients with poor vision. Real-world data suggest that many patients are under-treated. Here, we investigate IgMs as a novel platform for treating ocular disease. We show that IgMs are well-suited to ocular administration due to moderate viscosity, long ocular exposure, and rapid systemic clearance. The complement-dependent cytotoxicity of IgMs can be readily removed with a P436G mutation, reducing safety liabilities. Furthermore, dodecavalent binding of IgM hexamers can potently activate pathways implicated in the treatment of progressive blindness, including the Tie2 receptor tyrosine kinase signaling pathway for the treatment of diabetic macular edema, or the death receptor 4 tumor necrosis family receptor pathway for the treatment of wet age-related macular degeneration. Collectively, these data demonstrate the promise of IgMs as therapeutic agonists for treating progressive blindness.


Asunto(s)
Sistemas de Liberación de Medicamentos , Inmunoglobulina M/farmacología , Degeneración Macular , Cuerpo Vítreo/metabolismo , Animales , Células CHO , Cricetulus , Humanos , Inyecciones Intravítreas , Degeneración Macular/tratamiento farmacológico , Degeneración Macular/metabolismo , Ratas
15.
Mol Immunol ; 45(15): 3926-33, 2008 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-18691763

RESUMEN

The humoral immune response requires antigen-specific B cell activation and subsequent terminal differentiation into plasma cells. Engagement of B cell antigen receptor (BCR) on mature B cells activates an intracellular signaling cascade, including calcium mobilization, which leads to cell proliferation and differentiation. Coengagement by immune complex of BCR with the inhibitory Fc receptor FcgammaRIIb, the only IgG receptor expressed on B cells, inhibits B cell activation signals through a negative feedback loop. We now describe antibodies that mimic the inhibitory effects of immune complex by high-affinity coengagement of FcgammaRIIb and the BCR coreceptor complex on human B cells. We engineered the Fc domain of an anti-CD19 antibody to generate variants with up to approximately 430-fold greater affinity to FcgammaRIIb. Relative to native IgG1, the FcgammaRIIb binding-enhanced (IIbE) variants strongly inhibited BCR-induced calcium mobilization and viability in primary human B cells. Inhibitory effects involved phosphorylation of SH2-containing inositol polyphosphate 5-phosphatase (SHIP), which is known to be involved in FcgammaRIIb-induced negative feedback of B cell activation by immune complex. Coengagement of BCR and FcgammaRIIb by IIbE variants also overcame the anti-apoptotic effects of BCR activation. The use of a single antibody to suppress B cell functions by coengagement of BCR and FcgammaRIIb may represent a novel approach in the treatment of B cell-mediated autoimmune diseases.


Asunto(s)
Anticuerpos/inmunología , Antígenos CD19/inmunología , Linfocitos B/inmunología , Fragmentos Fc de Inmunoglobulinas/genética , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de IgG/inmunología , Anticuerpos/genética , Apoptosis , Linfocitos B/citología , Linfocitos B/metabolismo , Calcio/metabolismo , Línea Celular , Proliferación Celular , Humanos , Inositol Polifosfato 5-Fosfatasas , Activación de Linfocitos , Monoéster Fosfórico Hidrolasas/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de IgG/genética , Transducción de Señal , Resonancia por Plasmón de Superficie
16.
Mol Cancer Ther ; 7(8): 2517-27, 2008 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-18723496

RESUMEN

The contribution of Fc-mediated effector functions to the therapeutic efficacy of some monoclonal antibodies has motivated efforts to enhance interactions with Fcgamma receptors (FcgammaR). Although an early goal has been enhanced FcgammaRIIIa binding and natural killer (NK) cell antibody-dependent cell-mediated cytotoxicity (ADCC), other relevant cell types such as macrophages are dependent on additional activating receptors such as FcgammaRIIa. Here, we describe a set of engineered Fc variants with diverse FcgammaR affinities, including a novel substitution G236A that provides selectively enhanced binding to FcgammaRIIa relative to FcgammaRIIb. Variants containing this substitution have up to 70-fold greater FcgammaRIIa affinity and 15-fold improvement in FcgammaRIIa/FcgammaRIIb ratio and mediate enhanced phagocytosis of antibody-coated target cells by macrophages. Specific double and triple combination variants with this substitution are simultaneously capable of exhibiting high NK-mediated ADCC and high macrophage phagocytosis. In addition, we have used this unique set of variants to quantitatively probe the relative contributions of individual FcgammaR to effector functions mediated by NK cells and macrophages. These experiments show that FcgammaRIIa plays the most influential role for macrophages and, surprisingly, that the inhibitory receptor FcgammaRIIb has little effect on effector function. The enhancements in phagocytosis described here provide the potential to improve the performance of therapeutic antibodies targeting cancers.


Asunto(s)
Anticuerpos/inmunología , Macrófagos/inmunología , Fagocitosis , Receptores de IgG/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Sitios de Unión de Anticuerpos , Línea Celular , Humanos
17.
MAbs ; 11(5): 942-955, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-30982394

RESUMEN

A cell-based assay employing Madin-Darby canine kidney cells stably expressing human neonatal Fc receptor (FcRn) heavy chain and ß2-microglobulin genes was developed to measure transcytosis of monoclonal antibodies (mAbs) under conditions relevant to the FcRn-mediated immunoglobulin G (IgG) salvage pathway. The FcRn-dependent transcytosis assay is modeled to reflect combined effects of nonspecific interactions between mAbs and cells, cellular uptake via pinocytosis, pH-dependent interactions with FcRn, and dynamics of intracellular trafficking and sorting mechanisms. Evaluation of 53 mAbs, including 30 marketed mAb drugs, revealed a notable correlation between the transcytosis readouts and clearance in humans. FcRn was required to promote efficient transcytosis of mAbs and contributed directly to the observed correlation. Furthermore, the transcytosis assay correctly predicted rank order of clearance of glycosylation and Fv charge variants of Fc-containing proteins. These results strongly support the utility of this assay as a cost-effective and animal-sparing screening tool for evaluation of mAb-based drug candidates during lead selection, optimization, and process development for desired pharmacokinetic properties.


Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacocinética , Antígenos de Histocompatibilidad Clase I/inmunología , Receptores Fc/inmunología , Transcitosis/inmunología , Animales , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales Humanizados/metabolismo , Bioensayo/métodos , Perros , Glicosilación , Humanos , Inmunoglobulina G/metabolismo , Células de Riñón Canino Madin Darby , Ratones
18.
MAbs ; 11(6): 996-1011, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31156033

RESUMEN

Agonism of members of the tumor necrosis factor receptor superfamily (TNFRSF) with monoclonal antibodies is of high therapeutic interest due to their role in immune regulation and cell proliferation. A major hurdle for pharmacologic activation of this receptor class is the requirement for high-order clustering, a mechanism that imposes a reliance in vivo on Fc receptor-mediated crosslinking. This extrinsic dependence represents a potential limitation of virtually the entire pipeline of agonist TNFRSF antibody drugs, of which none have thus far been approved or reached late-stage clinical trials. We show that tetravalent biepitopic targeting enables robust intrinsic antibody agonism for two members of this family, OX40 and DR5, that is superior to extrinsically crosslinked native parental antibodies. Tetravalent biepitopic anti-OX40 engagement co-stimulated OX40low cells, obviated the requirement for CD28 co-signal for T cell activation, and enabled superior pharmacodynamic activity relative to native IgG in a murine vaccination model. This work establishes a proof of concept for an engineering approach that addresses a major gap for the therapeutic activation of this important receptor class.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Recubrimiento Inmunológico , Ligando OX40/agonistas , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/agonistas , Transducción de Señal/inmunología , Linfocitos T/inmunología , Animales , Antígenos CD28/inmunología , Células CHO , Cricetulus , Humanos , Células Jurkat , Ratones , Ratones SCID , Ratones Transgénicos , Ligando OX40/inmunología , Receptores Fc/inmunología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/inmunología , Linfocitos T/citología
19.
Mol Immunol ; 44(8): 1986-98, 2007 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-17079018

RESUMEN

We introduce a new method of humanization based on a novel and immunologically relevant metric of antibody humanness, termed human string content (HSC), that quantifies a sequence at the level of potential MHC/T-cell epitopes. Use of this quantity rather than global identity as an optimization goal enables the sampling of human diversity from distinct human germline sequences across the framework and CDR regions, and allows for the generation of multiple diverse candidate sequences. As a result engineering is carried out at finer sequence resolution relative to standard CDR grafting methods, providing for the optimization of antibody properties beyond immunogenicity such as antigen affinity and solution behavior. We have applied this method to the humanization of four antibodies with different antigen specificities. The resulting variable domains differ fundamentally from CDR-grafted antibodies in that they are immunologically more human and their humanness is derived from several discrete germline sequences. Furthermore, these antibodies bind their respective antigens better than or comparable to those of the parent antibodies without the need for affinity maturation.


Asunto(s)
Anticuerpos Monoclonales/genética , Especificidad de Anticuerpos/genética , Genes Sintéticos , Región Variable de Inmunoglobulina/genética , Ingeniería de Proteínas , Animales , Humanos , Ratones
20.
Nat Rev Drug Discov ; 17(3): 197-223, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29192287

RESUMEN

Antibodies are the most rapidly growing drug class and have a major impact on human health, particularly in oncology, autoimmunity and chronic inflammatory diseases. Many of the best understood and most tractable cell surface and secreted targets with known roles in human diseases have been extensively exploited for antibody drug development. In this Review, we focus on emerging and novel mechanisms of action of antibodies and innovative targeting strategies that could extend their therapeutic applications, including antibody-drug conjugates, bispecific antibodies and antibody engineering to facilitate more effective delivery. These strategies could enable the pursuit of difficult to hit, less well-understood or previously undruggable targets - the 'high-hanging fruit'.


Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Inmunoconjugados/uso terapéutico , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Ingeniería de Proteínas/métodos , Humanos
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