RESUMO
B and T lymphocyte attenuator (BTLA) is an attractive target for a new class of therapeutics that attempt to rebalance the immune system by agonizing checkpoint inhibitory receptors (CIRs). Herpesvirus entry mediator (HVEM) binds BTLA in both trans- and cis-orientations. We report here the development and structural characterization of three humanized BTLA agonist antibodies, 22B3, 25F7, and 23C8. We determined the crystal structures of the antibody-BTLA complexes, showing that these antibodies bind distinct and non-overlapping epitopes of BTLA. While all three antibodies activate BTLA, 22B3 mimics HVEM binding to BTLA and shows the strongest agonistic activity in functional cell assays and in an imiquimod-induced mouse model of psoriasis. 22B3 is also capable of modulating HVEM signaling through the BTLA-HVEM cis-interaction. The data obtained from crystal structures, biochemical assays, and functional studies provide a mechanistic model of HVEM and BTLA organization on the cell surface and informed the discovery of a highly active BTLA agonist.
Assuntos
Receptores Imunológicos , Linfócitos T , Camundongos , Animais , Linfócitos T/metabolismo , Receptores Imunológicos/metabolismo , Anticorpos/metabolismoRESUMO
Biologics have the potential to induce an immune response when used therapeutically. A number of in vitro assays are currently used preclinically to predict the risk of immunogenicity, but the validation of these preclinical tools suffers from the relatively small number of accessible immunogenic molecules and the limited understanding of the mechanisms underlying the immunogenicity of biologics. Here, we present the post-hoc analysis of three monoclonal antibodies with high immunogenicity in the clinic. Two of the three antibodies elicited a CD4 T cell proliferative response in multiple donors in a peripheral blood mononuclear cell assay, but required different experimental conditions to induce these responses. The third antibody did not trigger any T cell response in this assay. These distinct capacities to promote CD4 T cell responses in vitro were mirrored by different capacities to stimulate innate immune cells. Only one of the three antibodies was capable of inducing human dendritic cell (DC) maturation; the second antibody promoted monocyte activation while the third one did not induce any innate cell activation in vitro. All three antibodies exhibited a moderate to high internalization by human DCs and MHC-associated peptide proteomics analysis revealed the presence of potential T cell epitopes that were confirmed by a T-cell proliferation assay. Collectively, these findings highlight the existence of distinct immune stimulatory mechanisms for immunogenic antibodies. These findings have implications for the preclinical immunogenicity risk assessment of biologics.
Assuntos
Anticorpos Monoclonais/imunologia , Formação de Anticorpos/imunologia , Apresentação de Antígeno/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Ativação Linfocitária/imunologia , Anticorpos Monoclonais/farmacologia , Formação de Anticorpos/efeitos dos fármacos , Apresentação de Antígeno/efeitos dos fármacos , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/efeitos dos fármacosRESUMO
Fas ligand (FasL) is a 40-kDa type II transmembrane protein belonging to the tumor necrosis factor (TNF) family of proteins and binds to its specific receptor, Fas, a member of the TNF receptor family. Membrane-bound FasL can be processed into a soluble form by a metalloprotease similar to that which cleaves TNFalpha. Elevated levels of FasL have been implicated in a wide variety of diseases ranging from cancer to inflammatory abnormalities, which could be targeted by antibody therapy. We generated a fully human high-affinity antibody against FasL that binds to and neutralizes the activity of both soluble and membrane-associated human FasL. In order to elucidate the mechanism of function of this antibody, we have mapped the region and critical residues involved in the recognition of FasL using a combination of homology modeling, immunoprecipitation, hydrogen-deuterium exchange mass spectrometry (H/DXMS), and alanine scanning site-directed mutagenesis. These studies have revealed the antibody binding site on human FasL. Furthermore, through molecular homology modeling, we have proposed a mechanism for the neutralizing activity of this antibody that involves interference with the docking of the ligand to its receptor by the antibody.
Assuntos
Anticorpos , Epitopos/química , Proteína Ligante Fas/química , Proteína Ligante Fas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos/química , Anticorpos/imunologia , Apoptose/imunologia , Sítios de Ligação , Mapeamento de Epitopos , Epitopos/imunologia , Proteína Ligante Fas/genética , Humanos , Células Jurkat , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/metabolismoRESUMO
Menin is a 70-kDa protein encoded by MEN1, the tumor suppressor gene disrupted in multiple endocrine neoplasia type 1. In a yeast two-hybrid system based on reconstitution of Ras signaling, menin was found to interact with the 32-kDa subunit (RPA2) of replication protein A (RPA), a heterotrimeric protein required for DNA replication, recombination, and repair. The menin-RPA2 interaction was confirmed in a conventional yeast two-hybrid system and by direct interaction between purified proteins. Menin-RPA2 binding was inhibited by a number of menin missense mutations found in individuals with multiple endocrine neoplasia type 1, and the interacting regions were mapped to the N-terminal portion of menin and amino acids 43 to 171 of RPA2. This region of RPA2 contains a weak single-stranded DNA-binding domain, but menin had no detectable effect on RPA-DNA binding in vitro. Menin bound preferentially in vitro to free RPA2 rather than the RPA heterotrimer or a subcomplex consisting of RPA2 bound to the 14-kDa subunit (RPA3). However, the 70-kDa subunit (RPA1) was coprecipitated from HeLa cell extracts along with RPA2 by menin-specific antibodies, suggesting that menin binds to the RPA heterotrimer or a novel RPA1-RPA2-containing complex in vivo. This finding was consistent with the extensive overlap in the nuclear localization patterns of endogenous menin, RPA2, and RPA1 observed by immunofluorescence.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas , Animais , Proteínas de Bactérias/metabolismo , Western Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Cromatografia em Gel , DNA/metabolismo , Dano ao DNA , DNA Complementar/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Fase G1 , Glutationa Transferase/metabolismo , Células HeLa , Humanos , Camundongos , Microscopia de Fluorescência , Neoplasia Endócrina Múltipla/genética , Mutação de Sentido Incorreto , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Proteína de Replicação A , Fase S , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
Stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 play a critical role in mobilization and redistribution of immune cells and hematopoietic stem cells (HSCs). We evaluated effects of two CXCR4-targeting agents, peptide antagonist LY2510924 and monoclonal antibody LY2624587, on mobilizing HSCs and white blood cells (WBCs) in humans, monkeys, and mice. Biochemical analysis showed LY2510924 peptide blocked SDF-1/CXCR4 binding in all three species; LY2624587 antibody blocked binding in human and monkey, with minimal activity in mouse. Cellular analysis showed LY2624587 antibody, but not LY2510924 peptide, down-regulated cell surface CXCR4 and induced hematological tumor cell death; both agents have been shown to inhibit SDF-1/CXCR4 interaction and downstream signaling. In animal models, LY2510924 peptide induced robust, prolonged, dose- and time-dependent WBC and HSC increases in mice and monkeys, whereas LY2624587 antibody induced only moderate, transient increases in monkeys. In clinical trials, similar pharmacodynamic effects were observed in patients with advanced cancer: LY2510924 peptide induced sustained WBC and HSC increases, while LY2624587 antibody induced only minimal, transient WBC changes. These distinct pharmacodynamic effects in two different classes of CXCR4 inhibitors are clinically important and should be carefully considered when designing combination studies with immune checkpoint inhibitors or other agents for cancer therapy.
RESUMO
Sclerostin antibodies increase bone mass by stimulating bone formation. However, human and animal studies show that bone formation increases transiently and returns to pre-treatment level despite ongoing antibody treatment. To understand its mechanism of action, we studied the time course of bone formation, correlating the rate and extent of accrual of bone mass and strength after sclerostin antibody treatment. Ovariectomized (OVX) rats were treated with a sclerostin-antibody (Scle-ab) at 20mg/kg sc once weekly and sacrificed at baseline and 2, 3, 4, 6, and 8weeks post-treatment. In Scle-ab treated rats, serum PINP and OCN rapidly increased at week 1, peaked around week 3, and returned to OVX control levels by week 6. Transcript analyses from the distal femur revealed an early increase in bone formation followed by a sustained decrease in bone resorption genes. Lumbar vertebral (LV) osteoblast surface increased 88% by week 2, and bone formation rate (BFR/BS) increased 138% by week 4. Both parameters were below OVX control by week 8. Bone formation was primarily a result of modeling based formation. Endocortical and periosteal BFR/BS peaked around week 4 at 313% and 585% of OVX control, respectively. BFR/BS then declined but remained higher than OVX control on both surfaces through week 8. Histomorphometric analyses showed LV-BV/TV did not further increase after week 4, while BMD continued to increase at LV, mid femur (MF), and femoral neck (FN) through week 8. Biomechanical tests showed a similar improvement in bone strength through 8weeks in MF and FN, but bone strength plateaued between weeks 6 and 8 for LV. Our data suggest that bone formation with Scle-ab treatment is rapid and modeling formation dominated in OVX rats. Although transient, the bone formation response persists longer in cortical than trabecular bone.
Assuntos
Anticorpos/farmacologia , Proteínas Morfogenéticas Ósseas/imunologia , Osso e Ossos/patologia , Osso e Ossos/fisiopatologia , Marcadores Genéticos/imunologia , Osteogênese/efeitos dos fármacos , Ovariectomia , Animais , Biomarcadores/sangue , Fenômenos Biomecânicos , Reabsorção Óssea/sangue , Reabsorção Óssea/patologia , Osso e Ossos/efeitos dos fármacos , Osso Esponjoso/efeitos dos fármacos , Osso Esponjoso/patologia , Densitometria , Feminino , Fêmur/efeitos dos fármacos , Fêmur/patologia , Fêmur/fisiopatologia , Vértebras Lombares/efeitos dos fármacos , Vértebras Lombares/patologia , Vértebras Lombares/fisiopatologia , Tamanho do Órgão/efeitos dos fármacos , Ratos Sprague-Dawley , Fatores de Tempo , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
SDF-1 and CXCR4 are a chemokine and chemokine receptor pair playing critical roles in tumorigenesis. Overexpression of CXCR4 is a hallmark of many hematological malignancies including acute myeloid leukemia, chronic lymphocytic leukemia and non-Hodgkin's lymphoma, and generally correlates with a poor prognosis. In this study, we developed a humanized anti-CXCR4 monoclonal antibody, LY2624587 as a potent CXCR4 antagonist that was advanced into clinical study for cancer. LY2624587 blocked SDF-1 binding to CXCR4 with an IC50 of 0.26 nM, and inhibited SDF-1-induced GTP binding with a Kb of 0.66 nM. In human lymphoma U937 and leukemia CCRF-CEM cells expressing endogenous CXCR4, LY2624587 inhibited SDF-1-induced cell migration with IC50 values of 3.7 and 0.26 nM, respectively. This antibody also inhibited CXCR4 and SDF-1 mediated cell signaling including activation of MAPK and AKT in tumor cells expressing CXCR4. Bifocal microscopic and flow cytometry analyses revealed that LY2624587 mediated receptor internalization and caused CXCR4 down-regulation on the cell surface. In human hematologic cancer cells, LY2624587 caused dose dependent apoptosis in vitro and in vivo. In mouse xenograft models developed with human leukemia and lymphoma cells expressing high levels of CXCR4, LY2624587 exhibited dose-dependent tumor growth inhibition and provided significant survival benefit in a disseminated lymphoma model. Collectively, we have demonstrated that CXCR4 inhibition by LY2624587 has the potential for the treatment of human hematological malignancies.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Hematológicas/metabolismo , Receptores CXCR4/antagonistas & inibidores , Animais , Anexina A5/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Quimiocina CXCL12/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Regulação para Baixo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/mortalidade , Neoplasias Hematológicas/patologia , Humanos , Camundongos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores CXCR4/metabolismo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
MEN1 is a likely tumor suppressor gene that encodes a novel protein, menin. Menin is a 610 amino-acid residue protein with as yet unknown function(s). We have used tandem affinity purification and mass spectroscopy to isolate and identify proteins associating with menin from cultured HeLa cell extracts. This strategy has resulted in the isolation and identification of nonmuscle myosin type II-A heavy chain (NMHC II-A) as a menin interacting protein. This interaction was confirmed by glutathione-S-transferase pulldown assays, by coimmunoprecipitation, and by actin selection of myosin. We have further identified the amino-terminal region of menin and the head domain of NMHC II-A to be regions required for this interaction. Moreover menin was seen to colocalize with this myosin isoform in the cleavage furrow of dividing cells by indirect immunofluoresence. These data indicate that menin through binding to NMHC II-A could participate in cell division and in other processes that involve NMHC II-A.
Assuntos
Cadeias Pesadas de Miosina/metabolismo , Proteínas de Neoplasias/metabolismo , Miosina não Muscular Tipo IIA/metabolismo , Proteínas Proto-Oncogênicas , Núcleo Celular/metabolismo , Imunofluorescência , Humanos , Mutação , Cadeias Pesadas de Miosina/isolamento & purificação , Proteínas de Neoplasias/genética , Miosina não Muscular Tipo IIA/isolamento & purificação , Testes de PrecipitinaRESUMO
Multiple endocrine neoplasia type 1 (MEN1), among all syndromes, causes tumors in the highest number of tissue types. Most of the tumors are hormone producing (e.g., parathyroid, enteropancreatic endocrine, anterior pituitary) but some are not (e.g., angiofibroma). MEN1 tumors are multiple for organ type, for regions of a discontinuous organ, and for subregions of a continuous organ. Cancer contributes to late mortality; there is no effective prevention or cure for MEN1 cancers. Morbidities are more frequent from benign than malignant tumor, and both are indicators for screening. Onset age is usually earlier in a tumor type of MEN1 than of nonhereditary cases. Broad trends contrast with those in nonneoplastic excess of hormones (e.g., persistent hyperinsulinemic hypoglycemia of infancy). Most germline or somatic mutations in the MEN1 gene predict truncation or absence of encoded menin. Similarly, 11q13 loss of heterozygosity in tumors predicts inactivation of the other MEN1 copy. MEN1 somatic mutation is prevalent in nonhereditary, MEN1-like tumor types. Compiled germline and somatic mutations show almost no genotype/phenotype relation. Normal menin is 67 kDa, widespread, and mainly nuclear. It may partner with junD, NF-kB, PEM, SMAD3, RPA2, FANCD2, NM23beta, nonmuscle myosin heavy chain II-A, GFAP, and/or vimentin. These partners have not clarified menin's pathways in normal or tumor tissues. Animal models have opened approaches to menin pathways. Local overexpression of menin in Drosophila reveals its interaction with the jun-kinase pathway. The Men1+/- mouse has robust MEN1; its most important difference from human MEN1 is marked hyperplasia of pancreatic islets, a tumor precursor stage.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasia Endócrina Múltipla/genética , Neoplasia Endócrina Múltipla/fisiopatologia , Proteínas Proto-Oncogênicas/genética , Animais , Humanos , Neoplasia Endócrina Múltipla/patologiaRESUMO
Epitope mapping of antibodies is the identification and characterization of binding sites of monoclonal antibodies (mAbs) on target antigens. This knowledge can be useful in generating novel antibodies to a particular target as well as elucidating an antibody mechanism of action. Several techniques are available to identify antibody epitopes among which are preliminary and simple ones like sequence homology analysis ELISA and Western blotting. However, the more widely used robust methods typically involve the use of mass spectrometry to fully analyze and interpret the data and accurately identify the binding site. Such methods include epitope extortion/excision, hydrogen deuterium exchange.