RESUMEN
The CD137 receptor plays a key role in mediating immune response by promoting T cell proliferation, survival, and memory. Effective agonism of CD137 has the potential to reinvigorate potent antitumor immunity either alone or in combination with other immune-checkpoint therapies. In this study, we describe the discovery and characterization of a unique CD137 agonist, 7A5, a fully human IgG1 Fc effector-null monoclonal antibody. The biological properties of 7A5 were investigated through in vitro and in vivo studies. 7A5 binds CD137, and the binding epitope overlaps with the CD137L binding site based on structure. 7A5 engages CD137 receptor and activates NF-κB cell signaling independent of cross-linking or Fc effector function. In addition, T cell activation measured by cytokine IFNγ production is induced by 7A5 in peripheral blood mononuclear cell costimulation assay. Human tumor xenograft mouse models reconstituted with human immune cells were used to determine antitumor activity in vivo. Monotherapy with 7A5 inhibits tumor growth, and this activity is enhanced in combination with a PD-L1 antagonist antibody. Furthermore, the intratumoral immune gene expression signature in response to 7A5 is highly suggestive of enhanced T cell infiltration and activation. Taken together, these results demonstrate 7A5 is a differentiated CD137 agonist antibody with biological properties that warrant its further development as a cancer immunotherapy. GRAPHICAL ABSTRACT: http://mct.aacrjournals.org/content/molcanther/19/4/988/F1.large.jpg.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Activación de Linfocitos/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Animales , Apoptosis , Linfocitos T CD8-positivos/inmunología , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/patología , Proliferación Celular , Femenino , Humanos , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , FN-kappa B/metabolismo , Transducción de Señal , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Targeted monoclonal antibody therapy is an important strategy in cancer therapeutics. Among the most promising characteristics of therapeutic targets are those that modulate the growth and survival of malignant neoplasms and their sensitivity to anticancer therapies. The insulin-like growth factor-I receptor (IGF-IR) is overexpressed in many types of solid and hematopoietic malignancies, and has been implicated as a principal cause of heightened proliferative and survival signaling. IGF-IR has also been shown to confer resistance to cytotoxic, hormonal, and targeted therapies, suggesting that therapeutics targeting IGF-IR may be effective against a broad range of malignancies. IMC-A12 (ImClone Systems Incorporated), a fully human monoclonal IgG1 antibody that binds with high affinity to the IGF-IR, inhibits ligand-dependent receptor activation and downstream signaling. IMC-A12 also mediates robust internalization and degradation of the IGF-IR. In human tumor xenograft models, IGF-IR blockade by IMC-A12 results in rapid and profound growth inhibition of cancers of the breast, lung, colon, and pancreas, and many other neoplasms. Although promising single-agent activity has been observed, the most impressive effects of targeting the IGF-IR with IMC-A12 have been noted when this agent was combined with cytotoxic agents or other targeted therapeutics. The results with IMC-A12 to date suggest that it may be an effective therapeutic in a diverse array of oncologic indications.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Inmunoglobulina G/uso terapéutico , Neoplasias/terapia , Receptor IGF Tipo 1/inmunología , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Ensayos Clínicos como Asunto , Humanos , Inmunoglobulina G/inmunología , Neoplasias/inmunología , Neoplasias/metabolismoRESUMEN
BACKGROUND: Modulation of the PD-1/PD-L1 axis through antagonist antibodies that block either receptor or ligand has been shown to reinvigorate the function of tumor-specific T cells and unleash potent anti-tumor immunity, leading to durable objective responses in a subset of patients across multiple tumor types. RESULTS: Here we describe the discovery and preclinical characterization of LY3300054, a fully human IgG1λ monoclonal antibody that binds to human PD-L1 with high affinity and inhibits interactions of PD-L1 with its two cognate receptors PD-1 and CD80. The functional activity of LY3300054 on primary human T cells is evaluated using a series of in vitro T cell functional assays and in vivo models using human-immune reconstituted mice. LY3300054 is shown to induce primary T cell activation in vitro, increase T cell activation in combination with anti-CTLA4 antibody, and to potently enhance anti-tumor alloreactivity in several xenograft mouse tumor models with reconstituted human immune cells. High-content molecular analysis of tumor and peripheral tissues from animals treated with LY3300054 reveals distinct adaptive immune activation signatures, and also previously not described modulation of innate immune pathways. CONCLUSIONS: LY3300054 is currently being evaluated in phase I clinical trials for oncology indications.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígeno B7-H1/antagonistas & inhibidores , Inmunoglobulina G/inmunología , Neoplasias/inmunología , Animales , Línea Celular , Cricetulus , Femenino , Humanos , Macaca fascicularis , Ratones , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
Unfortunately, after publication of this article [1], it was noticed that corrections to the legends of Figs. 1 and 2 were not correctly incorporated. The correct legends can be seen below.
RESUMEN
The insulin-like growth factor I receptor (IGF-IR) is overexpressed in many diverse tumor types and is a critical signaling molecule for tumor cell proliferation and survival. Therapeutic strategies targeting the IGF-IR may therefore be effective broad-spectrum anticancer agents. Through screening of a Fab phage display library, we have generated a fully human antibody (A12) that binds to the IGF-IR with high affinity (4.11 x 10(-11) M) and inhibits ligand binding with an IC(50) of 0.6-1 nM. Antibody-mediated blockade of ligand binding to the IGF-IR inhibited downstream signaling of the two major insulin-like growth factor (IGF) pathways, mitogen-activated protein kinase and phosphatidylinositol 3'-kinase/Akt, in MCF7 human breast cancer cells. As a result, the mitogenic and proliferative potential of IGF-I and IGF-II were significantly reduced. A12 did not block insulin binding to the insulin receptor but could block binding to atypical IGF-IR in MCF7 cells. In addition, A12 was shown to induce IGF-IR internalization and degradation on specific binding to tumor cells, resulting in a significant reduction in cell surface receptor density. In xenograft tumor models in vivo, IGF-IR blockade by A12 was shown to occur rapidly, resulting in significant growth inhibition of breast, renal, and pancreatic tumors. Histological analysis of tumor sections demonstrated a marked increase in apoptotic tumor cells in antibody-treated animals. These results demonstrate that A12 possesses strong antitumor activity in vitro and in vivo and may therefore be an effective therapeutic candidate for the treatment of cancers that are dependent on IGF-IR signaling for growth and survival.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Receptor IGF Tipo 1/antagonistas & inhibidores , Animales , Especificidad de Anticuerpos , Neoplasias de la Mama/terapia , División Celular/efectos de los fármacos , Femenino , Humanos , Factor I del Crecimiento Similar a la Insulina/antagonistas & inhibidores , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ligandos , Ratones , Ratones Desnudos , Biblioteca de Péptidos , Fosforilación , Receptor IGF Tipo 1/inmunología , Receptor IGF Tipo 1/metabolismo , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Bi-specific antibodies (BsAbs), which can simultaneously block 2 tumor targets, have emerged as promising therapeutic alternatives to combinations of individual monoclonal antibodies. Here, we describe the engineering and development of a novel, human bi-functional antibody-receptor domain fusion molecule with ligand capture (bi-AbCap) through the fusion of the domain 2 of human vascular endothelial growth factor receptor 1 (VEGFR1) to an antibody directed against insulin-like growth factor - type I receptor (IGF-IR). The bi-AbCap possesses excellent stability and developability, and is the result of minimal engineering. Beyond potent neutralizing activities against IGF-IR and VEGF, the bi-AbCap is capable of cross-linking VEGF to IGF-IR, leading to co-internalization and degradation of both targets by tumor cells. In multiple mouse xenograft tumor models, the bi-AbCap improves anti-tumor activity over individual monotherapies. More importantly, it exhibits superior inhibition of tumor growth, compared with the combination of anti-IGF-IR and anti-VEGF therapies, via powerful blockade of both direct tumor cell growth and tumor angiogenesis. The unique "capture-for-degradation" mechanism of the bi-AbCap is informative for the design of next-generation bi-functional anti-cancer therapies directed against independent signaling pathways. The bi-AbCap design represents an alternative approach to the creation of dual-targeting antibody fusion molecules by taking advantage of natural receptor-ligand interactions.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Anticuerpos Neutralizantes/farmacología , Receptores de Somatomedina/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/farmacología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/farmacología , Afinidad de Anticuerpos , Cromatografía Líquida de Alta Presión , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Immunoblotting , Inmunoprecipitación , Ratones , Ratones Desnudos , Microscopía Confocal , Neoplasias Experimentales/tratamiento farmacológico , Estabilidad Proteica , Receptor IGF Tipo 1 , Resonancia por Plasmón de Superficie , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Recombinant protein production in plants such as corn is a promising means to generate high product yields at low comparable production cost. The anti-EGFR monoclonal antibody C225, cetuximab, is a well-characterized receptor antagonist antibody recently approved for the treatment of refractory colorectal cancer. We initiated a study to test and compare the functional activity of glycosylated and aglycosylated C225 produced in stable transgenic corn seed. Both corn antibodies were shown to be functionally indistinguishable from mammalian-derived C225 in demonstrating high-affinity binding to the EGF receptor, blocking of ligand-dependent signaling, and inhibiting cell proliferation. In addition, consistent with cetuximab, both corn antibodies possessed strong anti-tumor activity in vivo. Acute dose primate pharmacokinetic studies, however, revealed a marked increase in clearance for the glycosylated corn antibody, while the aglycosylated antibody possessed in vivo kinetics similar to cetuximab. This experimentation established that corn-derived receptor blocking monoclonal antibodies possess comparable efficacy to mammalian cell culture-derived antibody, and offer a cost effective alternative to large-scale mammalian cell culture production.
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Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/farmacología , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/inmunología , Zea mays/genética , Zea mays/inmunología , Animales , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales Humanizados , Citotoxicidad Celular Dependiente de Anticuerpos , Antineoplásicos/farmacocinética , Cetuximab , Femenino , Humanos , Técnicas In Vitro , Cinética , Macaca fascicularis , Masculino , Ratones , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/terapia , Plantas Modificadas Genéticamente , Unión Proteica , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/farmacología , Trasplante HeterólogoRESUMEN
Stem cell factor receptor (c-Kit) exerts multiple biological effects on target cells upon binding its ligand stem cell factor (SCF). Aberrant activation of c-Kit results in dysregulated signaling and is implicated in the pathogenesis of numerous cancers. The development of more specific and effective c-Kit therapies is warranted given its essential role in tumorigenesis. In this study, we describe the biological properties of CK6, a fully human IgG1 monoclonal antibody against the extracellular region of human c-Kit. CK6 specifically binds c-Kit receptor with high affinity (EC 50 = 0.06 nM) and strongly blocks its interaction with SCF (IC 50 = 0.41 nM) in solid phase assays. Flow cytometry shows CK6 binding to c-Kit on the cell surface of human small cell lung carcinoma (SCLC), melanoma, and leukemia tumor cell lines. Furthermore, exposure to CK6 inhibits SCF stimulation of c-Kit tyrosine kinase activity and downstream signaling pathways such as mitogen-activated protein kinase (MAPK) and protein kinase B (AKT), in addition to reducing tumor cell line growth in vitro. CK6 treatment significantly decreases human xenograft tumor growth in NCI-H526 SCLC (T/C% = 57) and Malme-3M melanoma (T/C% = 58) models in vivo. The combination of CK6 with standard of care chemotherapy agents, cisplatin and etoposide for SCLC or dacarbazine for melanoma, more potently reduces tumor growth (SCLC T/C% = 24, melanoma T/C% = 38) compared with CK6 or chemotherapy alone. In summary, our results demonstrate that CK6 is a c-Kit antagonist antibody with tumor growth neutralizing properties and are highly suggestive of potential therapeutic application in treating human malignancies harboring c-Kit receptor.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Inmunoglobulina G/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Melanoma Experimental/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-kit/metabolismo , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/administración & dosificación , Dacarbazina/administración & dosificación , Etopósido/administración & dosificación , Femenino , Xenoinjertos , Humanos , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/farmacología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones Desnudos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/inmunología , Transducción de Señal , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Carcinoma Pulmonar de Células Pequeñas/patologíaRESUMEN
PURPOSE: To evaluate the antibody-dependent cellular cytotoxicity (ADCC) of cetuximab, an anti-epidermal growth factor receptor (EGFR) IgG1 antibody, in vitro. METHODS: Binding to human Fc receptors was measured by ELISA. ADCC against a panel of tumor cell lines was evaluated using peripheral blood mononuclear cells or NK cells as effectors and lactate dehydrogenase release as a marker of cell killing. Cetuximab was compared with two glycan variants of cetuximab and with panitumumab, an anti-EGFR IgG2. RESULTS: Cetuximab bound with high affinity to FcγRI (EC50 = 0.13 nM) and FcγRIIIa (EC50 = 6 nM) and effectively induced ADCC across multiple tumor cell lines. Panitumumab and aglycosylated cetuximab did not bind to FcγRI or FcγRIIIa nor have ADCC activity even at high effector-target cell ratios, even though the EGFR-binding affinity of cetuximab and panitumumab were shown to be comparable (KD = 87 pM and 83 pM, respectively). The extent of cetuximab-elicited ADCC was associated with the level of EGFR expression on tumor cells. CONCLUSIONS: Cetuximab elicits effective ADCC activity against a wide range of tumor cells in vitro. This activity is dependent on antibody glycosylation and IgG1 isotype as well as tumor-cell EGFR expression. These findings suggest that ADCC may contribute to the antitumor activity of cetuximab.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Antineoplásicos/inmunología , Receptores ErbB/metabolismo , Inmunoglobulina G/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales Humanizados , Antineoplásicos/metabolismo , Línea Celular Tumoral , Cetuximab , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Receptores ErbB/inmunología , Glicosilación , Humanos , Isotipos de Inmunoglobulinas , Células Asesinas Naturales/inmunología , L-Lactato Deshidrogenasa/metabolismo , Leucocitos Mononucleares/inmunología , PanitumumabRESUMEN
PURPOSE: Transforming growth factor beta (TGFbeta) is a pleiotropic cytokine that affects tumor growth, metastasis, stroma, and immune response. We investigated the therapeutic efficacy of anti-TGFbeta receptor II (TGFbeta RII) antibody in controlling metastasis and tumor growth as well as enhancing antitumor immunity in preclinical tumor models. EXPERIMENTAL DESIGN: We generated neutralizing antibodies to TGFbeta RII and assessed the antibody effects on cancer, stroma, and immune cells in vitro. The efficacy and mechanism of action of the antibody as monotherapy and in combination with chemotherapy in suppression of primary tumor growth and metastasis were evaluated in several tumor models. RESULTS: Anti-TGFbeta RII antibody blocked TGFbeta RII binding to TGFbeta 1, 2, and 3, and attenuated the TGFbeta-mediated activation of downstream Smad2 kinase, invasion of cancer cells, motility of endothelial and fibroblast cells, and induction of immunosuppressive cells. Treatment with the antibody significantly suppressed primary tumor growth and metastasis and enhanced natural killer and CTL activity in tumor-bearing mice. Immunohistochemistry analysis showed cancer cell apoptosis and massive necrosis, and increased tumor-infiltrating T effector cells and decreased tumor-infiltrating Gr-1+ myeloid cells in the antibody-treated tumors. Fluorescence-activated cell sorting analysis indicated the significant reduction of peripheral Gr-1+/CD11b+ myeloid cells in treated animals. Concomitant treatment with the cytotoxic agent cyclophosphamide resulted in a significantly increased antitumor efficacy against primary tumor growth and metastasis. CONCLUSIONS: These preclinical data provide a foundation to support using anti-TGFbeta RII antibody as a therapeutic agent for TGFbeta RII-dependent cancer with metastatic capacity.
Asunto(s)
Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/farmacología , Neoplasias/tratamiento farmacológico , Proteínas Serina-Treonina Quinasas/inmunología , Receptores de Factores de Crecimiento Transformadores beta/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Células Asesinas Naturales/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias/inmunología , Receptor Tipo II de Factor de Crecimiento Transformador beta , Proteína Smad2/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Although many multiple myeloma (MM) patients initially respond to cytotoxic therapy, most eventually relapse. Novel therapeutic strategies employing a combination of chemotherapy with targeted biologics may significantly enhance the response of tumor cells to treatment. We tested a fully human anti-IGF-IR antibody (A12) against MM, and showed specific inhibition of IGF-I or serum-induced IGF-IR signaling in MM cells in vitro. The A12 as a single agent was demonstrated to exert modest to significant inhibition of tumor growth in vivo in various subcutaneous xenograft MM models. The A12 was also evaluated in a disseminated xenograft MM.1S NOD/SCID model as monotherapy or in combination with other drugs (bortezomib, melphalan) currently in clinical use. The tumor burden, as determined by luciferase bioimaging, was sharply decreased, and overall survival significantly prolonged when the therapies were combined. Immunohistochemical analysis demonstrated that the A12 treated tumors had significantly decreased vascularization compared to control tumors. Furthermore, most MM lines constitutively secreted significant quantities of VEGF, and this was enhanced following IGF-I treatment. Inhibition of IGF-IR by the A12 in vitro suppressed both constitutive and IGF-I-induced secretion of VEGF, indicating that a putative anti-angiogenic mechanism associated with the A12 treatment may contribute to its anti-tumor effect.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Receptor IGF Tipo 1/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Antineoplásicos/administración & dosificación , Antineoplásicos/inmunología , Ácidos Borónicos/administración & dosificación , Bortezomib , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Humanos , Inyecciones Intravenosas , Inyecciones Subcutáneas , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mieloma Múltiple/inmunología , Mieloma Múltiple/patología , Pirazinas/administración & dosificación , Receptor IGF Tipo 1/biosíntesis , Receptor IGF Tipo 1/inmunología , Relación Estructura-Actividad , Tasa de Supervivencia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Both the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGFR) have been implicated in the tumorigenesis of a variety of human cancers. Effective tumor inhibition has been achieved both experimentally and clinically with a number of strategies that antagonize either receptor activity. Here we constructed and produced two fully human recombinant bispecific antibodies (BsAb) that target both EGFR and IGFR, using two neutralizing human antibodies originally isolated from a phage display library. The BsAb not only retained the antigen binding capacity of each of the parent antibodies, but also were capable of binding to both targets simultaneously as demonstrated by a cross-linking enzyme-linked immunosorbent assay. Furthermore, the BsAb effectively blocked both ligands, EGF and IGF, from binding to their respective receptors, and inhibited tumor cell proliferation as potently as a combination of both the parent antibodies. More importantly, the BsAb were able to completely block activation of several major signal transduction molecules, including Akt and p44/p42 MAP kinases, by both EGF and IGF, whereas each individual parent antibody was only effective in inhibiting those signal molecules activated by the relevant single growth factor. The BsAb molecules retained good antigen binding activity after incubation with mouse serum at 37 degrees C for up to 6 days. Taken together, our results underscore the benefits of simultaneous targeting multiple growth factor receptor pathways for more efficacious cancer treatment. This report describes the first time use of a recombinant BsAb for targeting two tumor-associated molecules on either a single or adjacent tumor cells for enhanced antitumor activity.