RESUMEN
The interplay among antibody/antigen binding kinetics, antibody diffusion, and antigen metabolic turnover together determines the depth of penetration of antitumor antibodies into prevascular tumor spheroid cell clumps. A sharp boundary between an outer shell of bound high-affinity antibody and an inner antibody-free core has been previously observed and mathematically modeled and was termed the "binding site barrier." We show here that this process is well described by a simplified shrinking core model wherein binding equilibration is much more rapid than diffusion. This analysis provides the following experimentally testable predictions: (a) the binding site barrier is a moving boundary whose velocity is proportional to the time integral of antibody concentration at the spheroid surface (i.e. plasma antibody AUC); (b) the velocity of this moving boundary is independent of binding affinity, if the affinity is sufficiently high to strongly favor antibody/antigen complex formation at prevailing antibody concentrations; and (c) maximum tumor retention is achieved when the antibody/antigen dissociation rate approaches the rate of antigen metabolic turnover. The consistency of these predictions with published experimental results is demonstrated. The shrinking core model provides a simple analytic relationship predicting the effects of altered antibody pharmacokinetics, antibody molecular weight, antigen turnover rate, antigen expression level, and micrometastasis size on antibody penetration and retention. For example, a formula is provided for predicting the bolus dose necessary to accomplish tumor saturation as a function of antibody and tumor properties. Furthermore, this analysis indicates certain attributes necessary for an optimal tumor targeting agent.
Asunto(s)
Anticuerpos/metabolismo , Modelos Inmunológicos , Neoplasias/metabolismo , Animales , Anticuerpos/inmunología , Complejo Antígeno-Anticuerpo/biosíntesis , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Humanos , Fragmentos de Inmunoglobulinas/metabolismo , Cinética , Melanoma/inmunología , Melanoma/metabolismo , Ratones , Neoplasias/inmunología , Esferoides Celulares/inmunología , Esferoides Celulares/metabolismo , Distribución Tisular , Trasplante HeterólogoRESUMEN
An scFv has been engineered to bind carcinoembryonic antigen (CEA) with a dissociation half-time >4 days at 37 degrees C. Two mutations responsible for this affinity increase were isolated by screening yeast surface-displayed mutant libraries by flow cytometry. Soluble expression of the mutant scFv in a yeast secretion system was increased 100-fold by screening mutant libraries for improved yeast surface display level. This scFv will be useful as a limiting case for evaluating the significance of affinity in tumor targeting to non-internalizing antigens.
Asunto(s)
Antígeno Carcinoembrionario/inmunología , Evolución Molecular Dirigida , Calor , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/inmunología , PlásmidosRESUMEN
The epidermal growth factor receptor (EGFR) and the type I insulin-like growth factor receptor (IGF-1R) are two cell surface receptor tyrosine kinases known to cooperate to promote tumor progression and drug resistance. Combined blockade of EGFR and IGF-1R has shown improved anti-tumor activity in preclinical models. Here, we report the characterization of a stable IgG-like bispecific antibody (BsAb) dual-targeting EGFR and IGF-1R that was developed for cancer therapy. The BsAb molecule (EI-04), constructed with a stability-engineered single chain variable fragment (scFv) against IGF-1R attached to the carboxyl-terminus of an IgG against EGFR, displays favorable biophysical properties for biopharmaceutical development. Biochemically, EI-04 bound to human EGFR and IGF-1R with sub nanomolar affinity, co-engaged the two receptors simultaneously, and blocked the binding of their respective ligands with similar potency compared to the parental monoclonal antibodies (mAbs). In tumor cells, EI-04 effectively inhibited EGFR and IGF-1R phosphorylation, and concurrently blocked downstream AKT and ERK activation, resulting in greater inhibition of tumor cell growth and cell cycle progression than the single mAbs. EI-04, likely due to its tetravalent bispecific format, exhibited high avidity binding to BxPC3 tumor cells co-expressing EGFR and IGF-1R, and consequently improved potency at inhibiting IGF-driven cell growth over the mAb combination. Importantly, EI-04 demonstrated enhanced in vivo anti-tumor efficacy over the parental mAbs in two xenograft models, and even over the mAb combination in the BxPC3 model. Our data support the clinical investigation of EI-04 as a superior cancer therapeutic in treating EGFR and IGF-1R pathway responsive tumors.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Anticuerpos Monoclonales/inmunología , Receptores ErbB/inmunología , Neoplasias/inmunología , Receptor IGF Tipo 1/inmunología , Animales , Anticuerpos Biespecíficos/farmacología , Anticuerpos Monoclonales/farmacología , Afinidad de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Western Blotting , Células CHO , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/inmunología , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Receptores ErbB/metabolismo , Humanos , Inmunoglobulina G/inmunología , Ratones , Ratones Desnudos , Ratones SCID , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Fosforilación/efectos de los fármacos , Receptor IGF Tipo 1/metabolismo , Transducción de Señal/efectos de los fármacos , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/farmacología , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Integrin alpha6beta4 signaling interactions have been implicated in tumor progression, and beta4 expression has been linked to poor prognosis in certain breast cancer subtypes. We generated human antibodies to alpha6beta4 to further evaluate its role in tumor cell signaling. Biochemical characterization indicated these antibodies are specific for alpha6beta4, recognize distinct epitopes and have low nanomolar affinities for both human and murine protein. The antibodies demonstrated differing effects on alpha6beta4-mediated cellular adhesion, highlighting the existence of different functional epitopes on alpha6beta4. Interestingly however both antibodies blocked adhesion-independent growth in a panel of breast cancer cell lines. Antibody induced apoptosis and inhibition of phosphoinositide 3-kinase (PI3K) signaling were also observed within the context of matrix adhesion. Enhanced inhibitory effects were observed when the alpha6beta4 antibodies were used in combination with antibodies to epidermal growth factor receptor (EGFR) or erythoblastic leukemia viral oncogene homolog 2 (ErbB2). These findings illustrate a role for both the adhesive and signaling functions of alpha6beta4 in breast cancer cell survival. The antibodies and data generated herein advance our understanding of alpha6beta4 in regulating tumorigenic processes, and suggest that combination therapies involving alpha6beta4 may be therapeutically effective in breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Integrina alfa6beta4/metabolismo , Integrina alfa6beta4/fisiología , Anticuerpos/metabolismo , Apoptosis/efectos de los fármacos , Adhesión Celular/fisiología , Línea Celular , Receptores ErbB/metabolismo , Femenino , Humanos , Integrinas/metabolismo , Neoplasias/metabolismo , Transducción de SeñalAsunto(s)
Anticuerpos/química , Anticuerpos/genética , Afinidad de Anticuerpos , Ingeniería de Proteínas/métodos , Saccharomyces cerevisiae/genética , Vectores Genéticos , Reacción en Cadena de la Polimerasa/métodos , Reproducibilidad de los Resultados , Mapeo Restrictivo , Sensibilidad y EspecificidadRESUMEN
Therapeutic antibodies directed against the type 1 insulin-like growth factor receptor (IGF-1R) have recently gained significant momentum in the clinic because of preliminary data generated in human patients with cancer. These antibodies inhibit ligand-mediated activation of IGF-1R and the resulting down-stream signaling cascade. Here we generated a panel of antibodies against IGF-1R and screened them for their ability to block the binding of both IGF-1 and IGF-2 at escalating ligand concentrations (>1 microm) to investigate allosteric versus competitive blocking mechanisms. Four distinct inhibitory classes were found as follows: 1) allosteric IGF-1 blockers, 2) allosteric IGF-2 blockers, 3) allosteric IGF-1 and IGF-2 blockers, and 4) competitive IGF-1 and IGF-2 blockers. The epitopes of representative antibodies from each of these classes were mapped using a purified IGF-1R library containing 64 mutations. Most of these antibodies bound overlapping surfaces on the cysteine-rich repeat and L2 domains. One class of allosteric IGF-1 and IGF-2 blocker was identified that bound a separate epitope on the outer surface of the FnIII-1 domain. Using various biophysical techniques, we show that the dual IGF blockers inhibit ligand binding using a spectrum of mechanisms ranging from highly allosteric to purely competitive. Binding of IGF-1 or the inhibitory antibodies was associated with conformational changes in IGF-1R, linked to the ordering of dynamic or unstructured regions of the receptor. These results suggest IGF-1R uses disorder/order within its polypeptide sequence to regulate its activity. Interestingly, the activity of representative allosteric and competitive inhibitors on H322M tumor cell growth in vitro was reflective of their individual ligand-blocking properties. Many of the antibodies in the clinic likely adopt one of the inhibitory mechanisms described here, and the outcome of future clinical studies may reveal whether a particular inhibitory mechanism leads to optimal clinical efficacy.