RESUMEN
Immunotherapy for cancer using antibodies to enhance T-cell function has been successful in recent clinical trials. Many molecules that improve activation and effector function of T cells have been investigated as potential new targets for immunomodulatory antibodies, including the tumor necrosis factor receptor superfamily members GITR and OX40. Antibodies engaging GITR or OX40 result in significant tumor protection in preclinical models. In this study, we observed that the GITR agonist antibody DTA-1 causes anaphylaxis in mice upon repeated intraperitoneal dosing. DTA-1-induced anaphylaxis requires GITR, CD4(+) T cells, B cells, and interleukin-4. Transfer of serum antibodies from DTA-1-treated mice, which contain high levels of DTA-1-specific immunoglobulin G1 (IgG1), can induce anaphylaxis in naive mice upon administration of an additional dose of DTA-1, suggesting that anaphylaxis results from anti-DTA-1 antibodies. Depletion of basophils and blockade of platelet-activating factor, the key components of the IgG1 pathway of anaphylaxis, rescues the mice from DTA-1-induced anaphylaxis. These results demonstrate a previously undescribed lethal side effect of repetitive doses of an agonist immunomodulatory antibody as well as insight into the mechanism of toxicity, which may offer a means of preventing adverse effects in future clinical trials using anti-GITR or other agonist antibodies as immunotherapies.
Asunto(s)
Anafilaxia/inmunología , Anticuerpos Monoclonales/efectos adversos , Proteína Relacionada con TNFR Inducida por Glucocorticoide/agonistas , Animales , Anticuerpos Monoclonales/administración & dosificación , Antígenos de Diferenciación/administración & dosificación , Linfocitos B/fisiología , Linfocitos T CD4-Positivos/fisiología , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Proteína Relacionada con TNFR Inducida por Glucocorticoide/genética , Proteína Relacionada con TNFR Inducida por Glucocorticoide/inmunología , Inyecciones Intraperitoneales , Interleucina-4/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Tumorales CultivadasRESUMEN
Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that plays an oncogenic role in breast, gastric and other solid tumors. However, anti-HER2 therapies are only currently approved for the treatment of breast and gastric/gastric esophageal junction cancers and treatment resistance remains a problem. Here, we engineer an anti-HER2 IgG1 bispecific, biparatopic antibody (Ab), zanidatamab, with unique and enhanced functionalities compared to both trastuzumab and the combination of trastuzumab plus pertuzumab (tras + pert). Zanidatamab binds adjacent HER2 molecules in trans and initiates distinct HER2 reorganization, as shown by polarized cell surface HER2 caps and large HER2 clusters, not observed with trastuzumab or tras + pert. Moreover, zanidatamab, but not trastuzumab nor tras + pert, elicit potent complement-dependent cytotoxicity (CDC) against high HER2-expressing tumor cells in vitro. Zanidatamab also mediates HER2 internalization and downregulation, inhibition of both cell signaling and tumor growth, antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP), and also shows superior in vivo antitumor activity compared to tras + pert in a HER2-expressing xenograft model. Collectively, we show that zanidatamab has multiple and distinct mechanisms of action derived from the structural effects of biparatopic HER2 engagement.
Asunto(s)
Anticuerpos Biespecíficos , Antineoplásicos , Neoplasias de la Mama , Humanos , Femenino , Ensayos Antitumor por Modelo de Xenoinjerto , Línea Celular Tumoral , Trastuzumab/farmacología , Trastuzumab/uso terapéutico , Receptor ErbB-2/metabolismo , Citotoxicidad Celular Dependiente de Anticuerpos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológicoRESUMEN
pHLA complexes represent the largest class of cell surface markers on cancer cells, making them attractive for targeted cancer therapies. Adoptive cell therapies expressing TCRs that recognize tumor specific pHLAs take advantage of the unique selectivity and avidity of TCR: pHLA interactions. More recently, additional protein binding domains binding to pHLAs, known as TCR mimics (TCRm), were developed for tumor targeting of high potency therapeutic modalities, including bispecifics, ADCs, CAR T and -NK cells. TCRm compounds take advantage of the exquisite tumor specificity of certain pHLA targets, including cell lineage commitment markers and cancer testis antigens (CTAs). To achieve meaningful anti-tumor responses, it is critical that TCRm compounds integrate both, high target binding affinities and a high degree of target specificity. In this review, we describe the most advanced approaches to achieve both criteria, including affinity- and specificity engineering of TCRs, antibodies and alternative protein scaffolds. We also discuss the status of current TCRm based therapeutics developed in the clinic, key challenges, and emerging trends to improve treatment options for cancer patients treated with TCRm based therapeutics in Oncology.
RESUMEN
Since the first murine monoclonal antibody was approved for human therapeutic use over a decade ago, the realization that monoclonal antibody therapeutics could be engineered to improve their efficacy has inspired an astonishing array of novel antibody constructs. Early focus was on reducing the immunogenicity of rodent antibodies via humanization and generation of antibodies in transgenic mice; as those techniques were being established and then provided marketed therapeutic antibodies, the focus expanded to include engineering for enhanced effector functions, control of half-life, tumor and tissue accessibility, augmented biophysical characteristics such as stability, and more efficient (and less costly) production. Over the past two years significant progress in designing antibodies with improved pharmacokinetic properties, via modified interaction with the neonatal Fc receptor (FcRn), has been achieved. Likewise, the ability to alter the communication of a therapeutic antibody with the immune system has been advanced, using both manipulation of the immunoglobulin protein sequence and its glycosylation. Although clinical evaluation of these engineered modifications has yet to be reported, results in primates are encouraging.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Complemento C1q/inmunología , Diseño de Fármacos , Isotipos de Inmunoglobulinas/inmunología , Ingeniería de Proteínas , Receptores Fc/inmunología , Animales , Anticuerpos Monoclonales/metabolismo , Afinidad de Anticuerpos , Citotoxicidad Celular Dependiente de Anticuerpos , Complemento C1q/metabolismo , Semivida , Humanos , Isotipos de Inmunoglobulinas/metabolismo , Receptores Fc/metabolismoRESUMEN
One of the first difficulties in developing monoclonal antibody therapeutics was the recognition that human anti-mouse antibody (HAMA) response limited the administration of murine antibodies. Creative science has lead to a number of ways to counter the immunogenicity of non-human antibodies, primarily through chimeric, humanized, de-immunized, and most recently, human-sequence therapeutic antibodies. Once therapeutic antibodies of low or no immunogenicity were available, the creativity then turned to engineering both the antigen-binding domains (e.g., affinity maturation, stability) and altering the effector functions (e.g. antibody-dependent cellular cytotoxicity, complement-dependent cellular cytotoxicity, and clearance rate).
Asunto(s)
Anticuerpos/inmunología , Anticuerpos/uso terapéutico , Ingeniería de Proteínas , Animales , Anticuerpos/química , Anticuerpos Antiidiotipos/biosíntesis , Anticuerpos Antiidiotipos/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Complemento C1q/inmunología , Humanos , Ratones , Ratones Transgénicos , Especificidad de la EspecieRESUMEN
Shotgun scanning combinatorial mutagenesis was used to study the antigen-binding site of Fab2C4, a humanized monoclonal antibody fragment that binds to the extracellular domain of the human oncogene product ErbB2. Essentially all the residues in the Fab2C4 complementarity determining regions (CDRs) were alanine-scanned using phage-displayed libraries that preferentially allowed side-chains to vary as the wild-type or alanine. A separate homolog-scan was performed using libraries that allowed side-chains to vary only as the wild-type or a similar amino acid residue. Following binding selections to isolate functional clones, DNA sequencing was used to determine the wild-type/mutant ratios at each varied position, and these ratios were used to assess the contributions of each side-chain to antigen binding. The alanine-scan revealed that most of the side-chains that contribute to antigen binding are located in the heavy chain, and the Fab2C4 three-dimensional structure revealed that these residues fall into two groups. The first group consists of solvent-exposed residues which likely make energetically favorable contacts with the antigen and thus comprise the functional-binding epitope. The second group consists of buried residues with side-chains that pack against other CDR residues and apparently act as scaffolding to maintain the functional epitope in a binding-competent conformation. The homolog-scan involved subtle mutations, and as a result, only a subset of the side-chains that were intolerant to alanine substitutions were also intolerant to homologous substitutions. In particular, the 610 A2 functional epitope surface revealed by alanine-scanning shrunk to only 369 A2 when mapped with homologous substitutions, suggesting that this smaller subset of side-chains may be involved in more precise contacts with the antigen. The results validate shotgun scanning as a rapid and accurate method for determining the functional contributions of individual side-chains involved in protein-protein interactions.
Asunto(s)
Anticuerpos/química , Receptor ErbB-2/química , Receptor ErbB-2/inmunología , Alanina/química , Secuencia de Aminoácidos , Reacciones Antígeno-Anticuerpo , Sitios de Unión , Cristalografía por Rayos X , Ensayo de Inmunoadsorción Enzimática , Epítopos , Biblioteca de Genes , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Biblioteca de Péptidos , Mutación Puntual , Unión ProteicaRESUMEN
Rituxan, a chimeric anti-CD20 antibody, has been used for treating non-Hodgkin's lymphoma and some autoimmune diseases. However, a humanized anti-CD20 antibody is desirable for long-term treatment of autoimmune diseases. CD20 is an integral membrane protein with a small intervening extracellular loop. Lacking a native soluble CD20 protein, we developed a simple cell-based enzyme-linked immunosorbent assay (ELISA) using live WIL2 cells in a 96-well format to measure relative binding affinity to support the humanization process. Although WIL2 cells grow in suspension and require centrifugation during the wash steps, the assay was quantitative and reproducible. We also demonstrated that cloned adherent transfected Chinese hamster ovary (CHO) cells could be used to improve assay throughput. For clinical studies requiring quantification of the humanized antibody in serum, we used an alternate approach and developed a high throughput ELISA using an anti-idiotypic antibody as a surrogate antigen for capture and an anti-idiotypic antibody for detection to overcome serum effects. These assay strategies may be applied for characterization of other antibodies directed to multitransmembrane proteins.
Asunto(s)
Anticuerpos Antiidiotipos/química , Anticuerpos Monoclonales/análisis , Antígenos CD20/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas Recombinantes de Fusión/análisis , Animales , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales de Origen Murino , Antígenos CD20/genética , Antígenos CD20/metabolismo , Enfermedades Autoinmunes/sangre , Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Células CHO , Células Clonales , Cricetinae , Expresión Génica , Humanos , Linfoma no Hodgkin/sangre , Linfoma no Hodgkin/tratamiento farmacológico , Linfoma no Hodgkin/inmunología , Linfoma no Hodgkin/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/uso terapéutico , Rituximab , Suero/química , Transducción GenéticaRESUMEN
With eleven therapeutic antibodies approved worldwide and many more in clinical trials, research on antibody engineering has continued to escalate and expand. This review covers recent progress in generation of antibodies by ex vivo methods, systems for screening these, and the quest for higher affinity, more stable, optimally biodistributed antibody fragments, especially for solid tumors. The latest developments in engineering antibodies for removal or enhancement of effector functions (antibody-dependent cellular cytotoxicity (ADCC), phagosytosis, complement fixation (CDC) and half-life) through protein alteration or carbohydrate optimization may now enable generation of superior antibody therapeutics. Antibody conjugates, including immunotoxins and immunocytokines, as well as multivalent and multispecific antibodies confer expanded utility of therapeutic antibodies. Finally, research into the IgA/FcalphaRI system has now provided an additional route to therapeutic antibodies.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/uso terapéutico , Animales , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/farmacología , Fragmentos Fab de Inmunoglobulinas/uso terapéutico , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunología , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/farmacologíaAsunto(s)
Anticuerpos Monoclonales/genética , Linfoma de Burkitt/genética , Linfoma de Burkitt/inmunología , Evolución Molecular Dirigida/métodos , Inmunoglobulinas Intravenosas/genética , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos/genética , Afinidad de Anticuerpos/inmunología , Formación de Anticuerpos/efectos de los fármacos , Formación de Anticuerpos/genética , Formación de Anticuerpos/inmunología , Sitios de Unión de Anticuerpos/efectos de los fármacos , Sitios de Unión de Anticuerpos/genética , Linfoma de Burkitt/metabolismo , Senescencia Celular/inmunología , Pollos , Clonación Molecular , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunoglobulinas Intravenosas/biosíntesis , Inmunoglobulinas Intravenosas/inmunología , Imitación Molecular , Mutagénesis , Ratas , Valores de Referencia , Selección Genética , Sensibilidad y Especificidad , Especificidad de la Especie , Proteína Estafilocócica A/inmunología , Proteína Estafilocócica A/metabolismo , Estreptavidina/administración & dosificación , Estreptavidina/inmunología , Células Tumorales CultivadasRESUMEN
Clinical response to the anti-CD20 antibody rituximab has been demonstrated to correlate with the polymorphism in the FcγRIIIa receptor where patients homozygous for the higher affinity V158 allotype showed a better response rate. This finding suggests that engineering of anti-CD20 for increased FcγRIIIa affinity could result in improved clinical outcome. To identify variants with increased affinity to FcγRIIIa, we developed quantitative assays using soluble receptors as well as engineered cell lines expressing FcγRI or FcγRIIIa on the cell surface. We assayed a set of anti-CD20 IgG(1) variants that had identical Fab regions, but alterations in the Fc regions, in both the soluble receptor-based and cell-based FcγRIIIa binding assays. We obtained similar relative binding affinity increases and assay precisions. The increase in affinity for FcγRIIIa correlated with the increase in activity in the antibody-dependent cellular cytotoxicity assay. These variants had unaltered FcγRI binding. In addition to Fcγ receptors, IgG also binds to FcRn, the receptor responsible for the long circulating half-life of IgG. The mutations in the anti-CD20 variants were previously found not to affect FcRn binding in the soluble receptor-based assays; consequently, we used anti-Her2 variants with different binding affinities to FcRn to study FcRn binding assays. We generated a cell line expressing FcRn on the cell surface to measure IgG binding and obtained similar ranking of these anti-Her2 variants in the cell-based and the soluble receptor-based FcRn binding assays. In conclusion, both the soluble receptor-based and cell-based binding assays can be used to identify IgG(1) variants with increased affinity to FcγRIIIa and unaltered affinity to FcγRI and FcRn.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Receptores Fc/metabolismo , Receptores de IgG/metabolismo , Sustitución de Aminoácidos , Animales , Anticuerpos Monoclonales de Origen Murino/genética , Anticuerpos Monoclonales de Origen Murino/metabolismo , Afinidad de Anticuerpos , Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos CD20/inmunología , Linfocitos B/inmunología , Células CHO , Membrana Celular/inmunología , Cricetinae , Cricetulus , Ensayo de Inmunoadsorción Enzimática , Variación Genética , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Técnicas In Vitro , Células Asesinas Naturales/inmunología , Cinética , Ratones , Ratones Transgénicos , Unión Proteica , Ingeniería de Proteínas , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/inmunología , Receptores Fc/genética , Receptores de IgG/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rituximab , SolubilidadRESUMEN
The MHC class I-like Fc receptor FcRn plays an essential role in extending the half-life (t(1/2)) of IgG antibodies and IgG-Fc-based therapeutics in the circulation. The goal of this study was to analyze the effect of human IgG1 (hIgG1) antibodies with enhanced in vitro binding to FcRn on their in vivo t(1/2) in mice expressing human FcRn (hFcRn). Mutants of the humanized monoclonal Herceptin antibody (Hu4D5-IgG1), directed against human epidermal growth factor receptor 2 (p185 (HER2)), show altered pH-dependent binding to hFcRn in vitro. Two engineered IgG1 mutants (N434A and T307A/E380A/N434A) showed a considerably extended t(1/2) in vivo compared with wild-type antibody in mice expressing an hFcRn transgene (Tg) but not in mice expressing the endogenous mouse FcRn. The efficiency of hFcRn-mediated protection was dependent on hFcRn Tg copy number. Moreover, when injected into FcRn-humanized mice at a concentration sufficient to partially saturate hFcRn, the engineered IgG1 mutants with an extended serum t(1/2) were most effective in reducing the t(1/2) of a tracer hIgG1 antibody. Finally, administration of mutant with high binding to hFcRn ameliorated arthritis induced by passive transfer with human pathogenic plasma. These results indicate that Fc regions modified for high binding affinity to hFcRn increases serum persistence of therapeutic antibodies, that the same approach can be exploited as an anti-autoimmune therapy to promote the clearance of endogenous pathogenic IgG and that FcRn-humanized mice are a promising surrogate for hIgG therapeutic development.
Asunto(s)
Anticuerpos/uso terapéutico , Artritis/terapia , Enfermedades Autoinmunes/terapia , Inmunoglobulina G/uso terapéutico , Receptores Fc/metabolismo , Animales , Anticuerpos/administración & dosificación , Anticuerpos/sangre , Anticuerpos/metabolismo , Artritis/inmunología , Enfermedades Autoinmunes/inmunología , Relación Dosis-Respuesta Inmunológica , Ingeniería Genética , Semivida , Humanos , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/sangre , Inmunoglobulina G/metabolismo , Ratones , Resultado del TratamientoRESUMEN
mAbs account for an increasing portion of marketed human biological therapeutics. As a consequence, the importance of optimal selection, design, and engineering of these not only has expanded in the past 2 decades but also is now coming into play as a competitive factor. This review delineates the 4 basic areas for optimal therapeutic antibody selection and provides examples of the increasing number of considerations necessary for, and options available for, antibody design. Though some of the advances in antibody technology (eg, antibodies derived from phage-display libraries) have already made it to market, other more recent advances, such as engineering antibodies for enhanced effector functions, may not be far behind, especially given the increasing competition for therapeutic antibodies to the same target (eg, anti-CD20 and anti-TNF-alpha).
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Citotoxicidad Celular Dependiente de Anticuerpos , Regiones Determinantes de Complementariedad , Diseño de Fármacos , Epítopos , Humanos , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/uso terapéutico , Ratones , Ingeniería de ProteínasRESUMEN
Lec13 cells, a variant Chinese hamster ovary cell line, were used to produce human IgG1 that were deficient in fucose attached to the Asn(297)-linked carbohydrate but were otherwise similar to that found in IgG1 produced in normal Chinese hamster ovary cell lines and from human serum. Lack of fucose on the IgG1 had no effect on binding to human FcgammaRI, C1q, or the neonatal Fc receptor. Although no change in affinity was found for the His(131) polymorphic form of human FcgammaRIIA, a slight improvement in binding was evident for FcgammaRIIB and the Arg(131) FcgammaRIIA polymorphic form. In contrast, binding of the fucose-deficient IgG1 to human FcgammaRIIIA was improved up to 50-fold. Antibody-dependent cellular cytotoxicity assays using purified peripheral blood monocytes or natural killer cells from several donors showed enhanced cytotoxicity, especially evident at lower antibody concentrations. When combined with an IgG1 Fc protein variant that exhibited enhanced antibody-dependent cellular cytotoxicity, the lack of fucose was synergistic.