RESUMEN
Cluster of differentiation 47 (CD47) is a transmembrane protein ubiquitously expressed on human cells but overexpressed on many different tumor cells. The interaction of CD47 with signal-regulatory protein alpha (SIRPα) triggers a "don't eat me" signal to the macrophage, inhibiting phagocytosis. Thus, overexpression of CD47 enables tumor cells to escape from immune surveillance via the blockade of phagocytic mechanisms. We report here the development and characterization of CC-90002, a humanized anti-CD47 antibody. CC-90002 is unique among previously reported anti-CD47 bivalent antibodies that it does not promote hemagglutination while maintaining high-affinity binding to CD47 and inhibition of the CD47-SIRPα interaction. Studies in a panel of hematological cancer cell lines showed concentration-dependent CC-90002-mediated phagocytosis in acute lymphoblastic leukemia, acute myeloid leukemia (AML), lenalidomide-resistant multiple myeloma (MM) cell lines and AML cells from patients. In vivo studies with MM cell line-derived xenograft models established in immunodeficient mice demonstrated significant dose-dependent antitumor activity of CC-90002. Treatment with CC-90002 significantly prolonged survival in an HL-60-disseminated AML model. Mechanistic studies confirmed the binding of CC-90002 to tumor cells and concomitant recruitment of F4-80 positive macrophages into the tumor and an increase in expression of select chemokines and cytokines of murine origin. Furthermore, the role of macrophages in the CC-90002-mediated antitumor activity was demonstrated by transient depletion of macrophages with liposome-clodronate treatment. In non-human primates, CC-90002 displayed acceptable pharmacokinetic properties and a favorable toxicity profile. These data demonstrate the potential activity of CC-90002 across hematological malignancies and provided basis for clinical studies CC-90002-ST-001 (NCT02367196) and CC-90002-AML-001 (NCT02641002).
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígenos de Diferenciación/inmunología , Antígeno CD47/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Leucemia Promielocítica Aguda/tratamiento farmacológico , Macrófagos/inmunología , Receptores Inmunológicos/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antígenos de Diferenciación/metabolismo , Antineoplásicos Inmunológicos/inmunología , Antineoplásicos Inmunológicos/farmacología , Apoptosis , Antígeno CD47/metabolismo , Diferenciación Celular , Proliferación Celular , Femenino , Humanos , Leucemia Promielocítica Aguda/inmunología , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Fagocitosis , Pronóstico , Receptores Inmunológicos/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cluster of differentiation 47 (CD47) is a transmembrane protein highly expressed in tumor cells that interacts with signal regulatory protein alpha (SIRPα) and triggers a "don't eat me" signal to the macrophage, inhibiting phagocytosis and enabling tumor escape from immunosurveillance. The CD47-SIRPα axis has become an important target for cancer immunotherapy. To date, the advancement of CD47-targeted modalities is hindered by the ubiquitous expression of the target, often leading to rapid drug elimination and hematologic toxicity including anemia. To overcome those challenges a bispecific approach was taken. CC-96673, a humanized IgG1 bispecific antibody co-targeting CD47 and CD20, is designed to bind CD20 with high affinity and CD47 with optimally lowered affinity. As a result of the detuned CD47 affinity, CC-96673 selectively binds to CD20-expressing cells, blocking the interaction of CD47 with SIRPα. This increased selectivity of CC-96673 over monospecific anti-CD47 approaches allows for the use of wild-type IgG1 Fc, which engages activating crystallizable fragment gamma receptors (FcγRs) to fully potentiate macrophages to engulf and destroy CD20+ cells, while sparing CD47+CD20- normal cells. The combined targeting of anti-CD20 and anti-CD47 results in enhanced anti- tumor activity compared to anti-CD20 targeting antibodies alone. Furthermore, preclinical studies have demonstrated that CC-96673 exhibits acceptable pharmacokinetic properties with a favorable toxicity profile in non-human primates. Collectively, these findings define CC-96673 as a promising CD47 × CD20 bispecific antibody that selectively destroys CD20+ cancer cells via enhanced phagocytosis and other effector functions.
Asunto(s)
Anticuerpos Biespecíficos , Linfoma no Hodgkin , Neoplasias , Animales , Antígeno CD47 , Neoplasias/tratamiento farmacológico , Fagocitosis , Inmunoterapia , Inmunoglobulina G/uso terapéuticoRESUMEN
In normal cells, binding of the transmembrane protein CD47 to signal regulatory protein-α (SIRPα) on macrophages induces an antiphagocytic signal. Tumor cells hijack this pathway and overexpress CD47 to evade immune destruction. Macrophage antitumor activity can be restored by simultaneously blocking the CD47-SIRPα signaling axis and inducing a prophagocytic signal via tumor-opsonizing antibodies. We identified a novel, fully human mAb (BMS-986351) that binds SIRPα with high affinity. BMS-986351 demonstrated broad binding coverage across SIRPα polymorphisms and potently blocked CD47-SIRPα binding at the CD47 binding site in a dose-dependent manner. In vitro, BMS-986351 increased phagocytic activity against cell lines from solid tumors and hematologic malignancies, and this effect was markedly enhanced when BMS-986351 was combined with the opsonizing antibodies cetuximab and rituximab. A phase I dose-escalation/-expansion study of BMS-986351 for the treatment of advanced solid and hematologic malignancies is underway (NCT03783403). SIGNIFICANCE: Increasing the phagocytotic capabilities of tumor-associated macrophages by modulating macrophage-tumor cell surface signaling via the CD47-SIRPα axis is a novel strategy. Molecules targeting CD47 have potential but its ubiquitous expression necessitates higher therapeutic doses to overcome potential antigen sink effects. The restricted expression pattern of SIRPα may limit toxicities and lower doses of the SIRPα antibody BMS-986351 may overcome target mediated drug disposition while maintaining the desired pharmacology.
Asunto(s)
Neoplasias Hematológicas , Neoplasias , Humanos , Antígeno CD47/genética , Receptores Inmunológicos/genética , Fagocitosis , Macrófagos , Neoplasias/tratamiento farmacológico , Anticuerpos Antineoplásicos/metabolismo , Proteínas Opsoninas/metabolismo , Neoplasias Hematológicas/metabolismoRESUMEN
Bispecific antibodies (BsAbs) target multiple epitopes on the same molecular target or different targets. Although interest in BsAbs has persisted for decades, production of stable and active BsAbs has hindered their clinical evaluation. Here, we describe the production and characterization of tetravalent IgG-like BsAbs that combine the activities of allosteric and competitive inhibitors of the type-I insulin-like growth factor receptor (IGF-1R). The BsAbs, which were engineered for thermal stability, express well, demonstrate favorable biophysical properties, and recognize both epitopes on IGF-1R. Only one BsAb with a unique geometry, denoted BIIB4-5scFv, was capable of engaging all four of its binding arms simultaneously. All the BsAbs (especially BIIB4-5scFv) demonstrated enhanced ligand blocking over the single monoclonal antibodies (mAbs), particularly at high ligand concentrations. The pharmacokinetic profiles of two IgG-like BsAbs were tested in nude mice and shown to be comparable with that of the parental mAbs. The BsAbs, especially BIIB4-5scFv, demonstrated an improved ability to reduce the growth of multiple tumor cell lines and to inhibit ligand-induced IGF-1R signaling in tumor cells over the parental mAbs. BIIB4-5scFv also led to superior tumor growth inhibition over its parental mAbs in vivo. In summary, BsAbs that bridge multiple inhibitory mechanisms against a single target may generally represent a more effective strategy for intervention in oncology or other indications compared with traditional mAb therapy.
Asunto(s)
Anticuerpos Biespecíficos/farmacocinética , Anticuerpos Monoclonales de Origen Murino/farmacocinética , Antineoplásicos/farmacocinética , Inmunoglobulina G , Neoplasias Experimentales/tratamiento farmacológico , Receptor IGF Tipo 1/antagonistas & inhibidores , Animales , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacología , Anticuerpos Monoclonales de Origen Murino/inmunología , Anticuerpos Monoclonales de Origen Murino/farmacología , Antineoplásicos/inmunología , Antineoplásicos/farmacología , Línea Celular Tumoral , Estabilidad de Medicamentos , Humanos , Ligandos , Ratones , Ratones Desnudos , Neoplasias Experimentales/inmunología , Estabilidad Proteica , Receptor IGF Tipo 1/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
CD47 is the only 5-transmembrane (5-TM) spanning receptor of the immune system. Its extracellular domain (ECD) is a cell surface marker of self that binds SIRPα and inhibits macrophage phagocytosis, and cancer immuno-therapy approaches in clinical trials are focused on blocking CD47/SIRPα interaction. We present the crystal structure of full length CD47 bound to the function-blocking antibody B6H12. CD47 ECD is tethered to the TM domain via a six-residue peptide linker (114RVVSWF119) that forms an extended loop (SWF loop), with the fundamental role of inserting the side chains of W118 and F119 into the core of CD47 extracellular loop region (ECLR). Using hydrogen-deuterium exchange and molecular dynamics simulations we show that CD47's ECLR architecture, comprised of two extracellular loops and the SWF loop, creates a molecular environment stabilizing the ECD for presentation on the cell surface. These findings provide insights into CD47 immune recognition, signaling and therapeutic intervention.
Asunto(s)
Biomarcadores , Antígeno CD47/química , Antígeno CD47/metabolismo , Proteínas Portadoras/metabolismo , Receptores Inmunológicos/metabolismo , Anticuerpos Bloqueadores/química , Anticuerpos Bloqueadores/farmacología , Antígenos de Diferenciación/inmunología , Sitios de Unión , Antígeno CD47/efectos de los fármacos , Antígeno CD47/genética , Humanos , Macrófagos/metabolismo , Modelos Moleculares , Fagocitosis/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
The Wnt/ß-catenin signaling pathway has been implicated in human proliferative diseases such as cancer and fibrosis. The functions of ß-catenin and several other components of this pathway have been investigated in fibrosis. However, the potential role of R-spondin proteins (RSPOs), enhancers of the Wnt/ß-catenin signaling, has not been described. A specific interventional strategy targeting this pathway for fibrosis remains to be defined. We developed monoclonal antibodies against members of the RSPO family (RSPO1, 2, and 3) and probed their potential function in fibrosis in vivo. We demonstrated that RSPO3 plays a critical role in the development of fibrosis in multiple organs. Specifically, an anti-RSPO3 antibody, OMP-131R10, when dosed therapeutically, attenuated fibrosis in carbon tetrachloride (CCl4)-induced liver fibrosis, bleomycin-induced pulmonary and skin fibrosis models. Mechanistically, we showed that RSPO3 induces multiple pro-fibrotic chemokines and cytokines in Kupffer cells and hepatocytes. We found that the anti-fibrotic activity of OMP-131R10 is associated with its inhibition of ß-catenin activation in vivo. Finally, RSPO3 was found to be highly elevated in the active lesions of fibrotic tissues in mouse models of fibrosis and in patients with idiopathic pulmonary fibrosis (IPF) and nonalcoholic steatohepatitis (NASH). Together these data provide an anti-fibrotic strategy for targeting the Wnt/ß-catenin pathway through RSPO3 blockade and support that OMP-131R10 could be an important therapeutic agent for fibrosis.
Asunto(s)
Anticuerpos/uso terapéutico , Fibrosis Pulmonar Idiopática , Enfermedad del Hígado Graso no Alcohólico , Trombospondinas/fisiología , Animales , Células Cultivadas , Humanos , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Fibrosis Pulmonar Idiopática/metabolismo , Masculino , Ratones , Ratones Endogámicos DBA , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
Galiximab is a primatized monoclonal antibody that targets CD80 expressed on malignant B cells and is being studied in the clinic as a potential treatment for follicular NHL. We have recently reported that galiximab signals B-NHL cells in vitro and inhibits cell growth and sensitizes resistant tumor cells to apoptosis by chemotherapeutic drugs. This study was designed to validate the in vitro findings in in vivo in mice. Thus, we examined in vivo the antitumor activity of galiximab used alone and in combination with chemotherapeutic agents in SCID mice bearing human lymphoma xenografts. The in vivo antitumor effects of galiximab used alone and in combination with fludarabine or doxorubicin were determined in solid and disseminated human B-lymphoma tumors grown in SCID mice. Galiximab monotherapy in vivo demonstrated significant antitumor activity in a Raji lymphoma solid tumor model and in an SKW disseminated lymphoma tumor model. There was significant inhibition in tumor growth and prolongation of survival. In vitro, galiximab sensitized Raji cells to apoptosis by both fludarabine and doxorubicin. Tumor growth inhibition was significantly enhanced when the mice were treated with the combination of galiximab and fludarabine. These findings support the potential clinical application of galiximab in combination with chemotherapeutic drugs for the treatment of CD80-expressing hematological malignancies.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica , Antígeno B7-1/antagonistas & inhibidores , Linfoma no Hodgkin/tratamiento farmacológico , Vidarabina/análogos & derivados , Animales , Apoptosis/efectos de los fármacos , Línea Celular Transformada , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/uso terapéutico , Sinergismo Farmacológico , Humanos , Ratones , Ratones SCID , Vidarabina/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Galiximab (anti-CD80 monoclonal antibody) is a primatized (human IgG1 constant regions and cynomologus macaque variable regions) monoclonal antibody that is currently in clinical trials. Galiximab inhibits tumor cell proliferation through possibly cell signaling-mediated effects. Thus, we hypothesized that galiximab may signal the tumor cells and modify intracellular survival/antiapoptotic pathways such as the NF-κB pathway. This hypothesis was tested using various CD80(+) Burkitt B-NHL (non-Hodgkin lymphomas) cell lines as models. Treatment of B-NHL cells with galiximab (25-100 µg/mL) resulted in significant inhibition of NF-κB activity and its target resistant factors such as YY1, Snail, and Bcl-2/Bcl-XL. Treatment of B-NHL cells with galiximab sensitized the tumor cells to both cis-diamminedichloroplatinum(II) (CDDP)- and TRAIL-induced apoptosis. The important roles of YY1- and Snail-induced inhibition by galiximab in the sensitization to CCDP and TRAIL were corroborated following transfection of Raji cells with YY1 or Snail short interfering RNA. The transfected cells were shown to become sensitive to both CCDP- and TRAIL-induced apoptosis in the absence of galiximab. Furthermore, knockdown of YY1 or Snail inhibited Bcl-XL. The involvement of Bcl-XL inhibition in sensitization was corroborated by the use of the pan-Bcl-2 inhibitor 2MAM-3 whereby the treated cells were sensitive to both CDDP- and TRAIL-induced apoptosis. These findings show that galiximab inhibits the NF-κB/Snail/YY1/Bcl-XL circuit that regulates drug resistance in B-NHL and in combination with cytotoxic drugs results in apoptosis. The findings also support the therapeutic application of the combination of galiximab and cytotoxic drugs in the treatment of drug-resistant CD80-positive B-cell malignancies.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Apoptosis/efectos de los fármacos , FN-kappa B/metabolismo , Factores de Transcripción/metabolismo , Factor de Transcripción YY1/metabolismo , Proteína bcl-X/metabolismo , Anticuerpos Monoclonales/inmunología , Antígenos CD20/inmunología , Antígenos CD20/metabolismo , Antineoplásicos/farmacología , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cisplatino/farmacología , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , Linfoma de Células B/patología , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Factores de Transcripción de la Familia Snail , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Factores de Transcripción/genética , Factor de Transcripción YY1/genética , Proteína bcl-X/antagonistas & inhibidoresRESUMEN
The use of monoclonal antibodies (mAbs) has become a general approach for specifically targeting and treating human disease. In oncology, the therapeutic utility of mAbs is usually evaluated in the context of treatment with standard of care, as well as other small molecule targeted therapies. Many anti-cancer antibody modalities have achieved validation, including the targeting of growth factor and angiogenesis pathways, the induction of tumor cell killing or apoptosis, and the blocking of immune inhibitory mechanisms to stimulate anti-tumor responses. But, as with other targeted therapies, few antibodies are curative because of biological complexities that underlie tumor formation and redundancies in molecular pathways that enable tumors to adapt and show resistance to treatment. This review discusses the combinations of antibody therapeutics that are emerging to improve efficacy and durability within a specific biological mechanism (e.g., immunomodulation or the inhibition of angiogenesis) and across multiple biological pathways (e.g., inhibition of tumor growth and induction of tumor cell apoptosis).
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Inhibidores de la Angiogénesis/uso terapéutico , Anticuerpos Monoclonales/inmunología , Antineoplásicos/inmunología , Protocolos de Quimioterapia Combinada Antineoplásica/inmunología , Apoptosis/efectos de los fármacos , Ensayos Clínicos como Asunto , Humanos , Neoplasias/irrigación sanguínea , Neoplasias/inmunologíaRESUMEN
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
The insulin-like growth factor-I receptor (IGF-IR) is a cell surface receptor tyrosine kinase that mediates cell survival signaling and supports tumor progression in multiple tumor types. We identified a spectrum of inhibitory IGF-IR antibodies with diverse binding epitopes and ligand-blocking properties. By binding distinct inhibitory epitopes, two of these antibodies, BIIB4 and BIIB5, block both IGF-I and IGF-II binding to IGF-IR using competitive and allosteric mechanisms, respectively. Here, we explored the inhibitory effects of combining BIIB4 and BIIB5. In biochemical assays, the combination of BIIB4 and BIIB5 improved both the potency and extent of IGF-I and IGF-II blockade compared with either antibody alone. In tumor cells, the combination of BIIB4 and BIIB5 accelerated IGF-IR downregulation and more efficiently inhibited IGF-IR activation as well as downstream signaling, particularly AKT phosphorylation. In several carcinoma cell lines, the antibody combination more effectively inhibited ligand-driven cell growth than either BIIB4 or BIIB5 alone. Notably, the enhanced tumor growth-inhibitory activity of the BIIB4 and BIIB5 combination was much more pronounced at high ligand concentrations, where the individual antibodies exhibited substantially reduced activity. Compared with single antibodies, the BIIB4 and BIIB5 combination also significantly further enhanced the antitumor activity of the epidermal growth factor receptor inhibitor erlotinib and the mTOR inhibitor rapamycin. Moreover, in osteosarcoma and hepatocellular carcinoma xenograft models, the BIIB4 and BIIB5 combination significantly reduced tumor growth to a greater degree than each single antibody. Taken together, our results suggest that targeting multiple distinct inhibitory epitopes on IGF-IR may be a more effective strategy of affecting the IGF-IR pathway in cancer.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Receptor IGF Tipo 1/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/inmunología , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/inmunología , Neoplasias Óseas/patología , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/patología , Progresión de la Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Epítopos/inmunología , Femenino , Células Hep G2 , Humanos , Factor I del Crecimiento Similar a la Insulina/antagonistas & inhibidores , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/antagonistas & inhibidores , Factor II del Crecimiento Similar a la Insulina/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/patología , Ratones , Ratones Desnudos , Terapia Molecular Dirigida/métodos , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/inmunología , Osteosarcoma/patología , Receptor IGF Tipo 1/inmunología , Receptor IGF Tipo 1/metabolismo , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoAsunto(s)
Anticuerpos Monoclonales/administración & dosificación , Antineoplásicos/administración & dosificación , Linfoma Folicular/terapia , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales de Origen Murino , Ensayos Clínicos Fase I como Asunto , Ensayos Clínicos Fase II como Asunto , Evaluación de Medicamentos , Humanos , Linfoma Folicular/patología , Estudios Multicéntricos como Asunto , Proyectos Piloto , RituximabRESUMEN
PURPOSE: Rituximab [chimeric anti-CD20 monoclonal antibody], alone or combined with chemotherapy, is used in the treatment of non-Hodgkin's lymphoma (NHL). Rituximab binds to CD20 and inhibits intracellular survival/growth pathways leading to chemo/immunosensitization of tumor cells in vitro. The contribution of rituximab Fc-FcR interaction in signaling is not known. This study examined the role of Fc-FcR interactions in rituximab-induced signaling using rituximab (Fab')(2) fragments as well as rituximab devoid of the CH2 Fc-binding domain (CH2(-)). EXPERIMENTAL DESIGN: Rituximab (CH2(-)) and rituximab (Fab')(2) were tested for their activity on B-NHL cell lines. Cell signaling and sensitization to chemotherapy and immunotherapy were examined. The in vitro studies were validated in mice bearing tumor xenografts. RESULTS: Although the modified antibodies were defective in antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity functions, they retained all other biological activities such as inhibition of cell proliferation, induction of cell aggregation, and apoptosis induction. In addition, similar to rituximab, the modified antibodies inhibited the activity of cell survival/growth pathways and their associated transcription factors (e.g., NF-kappaB, YY1, SP-1), and signal transducers and activators of transcription 3 (STAT-3), and downregulated the expression of antiapoptotic gene products, such as Bcl-2/Bcl(xl), which regulate drug resistance. The modified antibodies, similar to rituximab, sensitized resistant B-NHL cells to both CDDP and Fas ligand-induced apoptosis. Furthermore, treatment of nude mice bearing Raji tumor cell xenografts with the combination of rituximab (Fab')(2) or rituximab and CDDP resulted in similar and significant inhibition of tumor growth. CONCLUSION: These findings reveal that rituximab-mediated inhibition of intracellular signaling pathways and leading to chemo/immuno-sensitization of resistant B-NHL is Fc independent.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Fragmentos Fab de Inmunoglobulinas/uso terapéutico , Fragmentos Fc de Inmunoglobulinas/fisiología , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/inmunología , Transducción de Señal , Animales , Anticuerpos Monoclonales de Origen Murino , Apoptosis , Línea Celular Tumoral , Supervivencia Celular , Resistencia a Antineoplásicos , Inmunoterapia , Linfoma de Células B/metabolismo , Ratones , Ratones Desnudos , Rituximab , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Epítopos/química , Receptores de Somatomedina/química , Sitio Alostérico , Animales , Células CHO , Rastreo Diferencial de Calorimetría , Cricetinae , Cricetulus , Mapeo Epitopo , Humanos , Factor II del Crecimiento Similar a la Insulina/química , Cinética , Ligandos , Conformación Molecular , Receptor IGF Tipo 1/metabolismoRESUMEN
Lumiliximab is a chimeric macaque-human monoclonal antibody to CD23, a protein expressed on virtually all chronic lymphocytic leukemia (CLL) cells. We examined the ability of lumiliximab to mediate apoptosis, antibody-dependent cellular cytotoxicity, and complement-dependent cytotoxicity against primary CLL cells and CD23-expressing B-cell lines. Our data suggest that lumiliximab kills CLL cells and CD23-expressing B cells predominantly by apoptosis, which occurs through the intrinsic pathway. Lumiliximab-induced apoptosis was accompanied by the down-regulation of antiapoptotic proteins Bcl-2, Bcl-X(L), and XIAP, activation of Bax, and release of cytochrome c from the mitochondria. We also found that the addition of lumiliximab to rituximab or fludarabine results in synergistic cytotoxicity of primary CLL cells and CD23-expressing B-cell lines. We investigated the in vivo activity of lumiliximab in a human disseminated CD23(+) B-cell lymphoma SCID mouse model and found greater antitumor activity with it than with control antibody. We also found that paralysis-free survival was greater with lumiliximab plus rituximab or fludarabine than with any of those agents alone. These results suggest that lumiliximab may be an effective treatment alone or in combination with rituximab or chemotherapy agents in CLL or other CD23-overexpressing B-cell malignancies.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Apoptosis/efectos de los fármacos , Leucemia Linfocítica Crónica de Células B/patología , Linfoma/metabolismo , Linfoma/patología , Receptores de IgE/metabolismo , Animales , Antineoplásicos/farmacología , Inhibidores de Caspasas , Caspasas/metabolismo , Humanos , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Inhibidores de Proteasas/farmacología , Transducción de Señal/efectos de los fármacos , Tasa de Supervivencia , Células Tumorales Cultivadas , Vidarabina/análogos & derivados , Vidarabina/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína X Asociada a bcl-2/metabolismoRESUMEN
BACKGROUND: The approval of monoclonal antibodies (MAbs) as antibody-targeted therapy in the management of patients with hematologic malignancies has led to new treatment options for this group of patients. The ability to target antibodies to novel functional receptors can increase their therapeutic efficacy. METHODS: The authors reviewed improvements in MAb design to enhance their effectiveness over the existing therapeutic MAb currently approved for treating hematologic malignancies. RESULTS: Three classes of therapeutic MAbs showing promise in human clinical trials for treatment of hematologic malignancies include unconjugated MAb, drug conjugates in which the antibody preferentially delivers a potent cytotoxic drug to the tumor, and radioactive immunotherapy in which the antibody delivers a sterilizing dose of radiation to the tumor. CONCLUSIONS: A better appreciation of how MAbs are metabolized in the body and localized to tumors is resulting in the development of new antibody constructs with improved biodistribution profiles.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Neoplasias Hematológicas/terapia , Animales , Ensayos Clínicos Fase III como Asunto , Terapia Combinada , Quimioterapia , Predicción , Neoplasias Hematológicas/inmunología , Reacción Huésped-Injerto/inmunología , Humanos , Sistema Inmunológico/efectos de los fármacos , Sistema Inmunológico/inmunología , Estudios Multicéntricos como Asunto , Radioinmunoterapia/normas , Resultado del Tratamiento , Estados Unidos/epidemiología , United States Food and Drug AdministrationRESUMEN
A major drawback of subunit vaccines is their inability to generate cytolytic T lymphocytes (CTL), a deficit attributed to segregation of the class I and class II antigen-processing pathways. We sought to understand processes involved in CTL induction by three proprietary adjuvants: Tomatine, PROVAX, and a synthesized glycolipid (Glc-N-(8/16), Glycolipid). We used in vivo models to investigate antigen uptake, macrophage involvement, TAP-independent processing, and costimulatory molecule dependencies. Glycolipid required splenic and lymph node macrophages, whereas Tomatine generated CTL independently of either macrophage population. In contrast, PROVAX showed partial macrophage requirements. Immunized TAP knockout mice revealed that ovalbumin (OVA)-Tomatine and OVA-PROVAX, but not OVA-Glycolipid, generate class I-peptide complexes. All three immunostimulants also elicited CD86-dependent TH1 cytokine responses.