RESUMO
Interleukin 6 plays a key role in mediating inflammatory reactions in autoimmune diseases and cancer, where it is also involved in metastasis and tissue invasion. Neutralizing antibodies against IL-6 and its receptor have been approved for therapeutic intervention or are in advanced stages of clinical development. Here we describe the crystal structures of the complexes of IL-6 with two Fabs derived from conventional camelid antibodies that antagonize the interaction between the cytokine and its receptor. The x-ray structures of these complexes provide insights into the mechanism of neutralization by the two antibodies and explain the very high potency of one of the antibodies. It effectively competes for binding to the cytokine with IL-6 receptor (IL-6R) by using side chains of two CDR residues filling the site I cavities of IL-6, thus mimicking the interactions of Phe(229) and Phe(279) of IL-6R. In the first antibody, a HCDR3 tryptophan binds similarly to hot spot residue Phe(279) Mutation of this HCDR3 Trp residue into any other residue except Tyr or Phe significantly weakens binding of the antibody to IL-6, as was also observed for IL-6R mutants of Phe(279) In the second antibody, the side chain of HCDR3 valine ties into site I like IL-6R Phe(279), whereas a LCDR1 tyrosine side chain occupies a second cavity within site I and mimics the interactions of IL-6R Phe(229).
Assuntos
Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Interleucina-6/antagonistas & inibidores , Receptores de Interleucina-6/química , Receptores de Interleucina-6/imunologia , Animais , Camelus , Humanos , Interleucina-6/química , Interleucina-6/imunologia , Camundongos , Estrutura Quaternária de ProteínaRESUMO
Highly potent human antibodies are required to therapeutically neutralize cytokines such as interleukin-6 (IL-6) that is involved in many inflammatory diseases and malignancies. Although a number of mutagenesis approaches exist to perform antibody affinity maturation, these may cause antibody instability and production issues. Thus, a robust and easy antibody affinity maturation strategy to increase antibody potency remains highly desirable. By immunizing llama, cloning the 'immune' antibody repertoire and using phage display, we selected a diverse set of IL-6 antagonistic Fabs. Heavy chain shuffling was performed on the Fab with lowest off-rate, resulting in a panel of variants with even lower off-rate. Structural analysis of the Fab:IL-6 complex suggests that the increased affinity was partly due to a serine to tyrosine switch in HCDR2. This translated into neutralizing capacity in an in vivo model of IL-6 induced SAA production. Finally, a novel Fab library was designed, encoding all variations found in the natural repertoire of VH genes identified after heavy chain shuffling. High stringency selections resulted in identification of a Fab with 250-fold increased potency when re-formatted into IgG1. Compared with a heavily engineered anti-IL-6 monoclonal antibody currently in clinical development, this IgG was at least equally potent, showing the engineering process to have had led to a highly potent anti-IL-6 antibody.
Assuntos
Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Mutação/genética , Biblioteca de Peptídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos , Camelídeos Americanos/genética , Humanos , Fragmentos Fab das Imunoglobulinas/química , Interleucina-6/imunologia , Modelos Imunológicos , Modelos Moleculares , Proteínas Recombinantes/química , Alinhamento de SequênciaRESUMO
Monoclonal antibodies (mAbs) have been used for decades as tools to probe the biology and pharmacology of receptors in cells and tissues. They are also increasingly being developed for clinical purposes against a broad range of targets, albeit to a lesser extent for G-protein-coupled receptors (GPCRs) relative to other therapeutic targets. Recent pharmacological, structural and biophysical data have provided a great deal of new insight into the molecular details, complexity and regulation of GPCR function. Whereas GPCRs used to be viewed as having either "on" or "off" conformational states, it is now recognized that their structures may be finely tuned by ligands and other interacting proteins, leading to the selective activation of specific signaling pathways. This information coupled with new technologies for the selection of mAbs targeting GPCRs will be increasingly deployed for the development of highly selective mAbs that recognize conformational determinants leading to novel therapeutics.
Assuntos
Anticorpos Monoclonais/farmacologia , Desenho de Fármacos , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/química , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Humanos , Ligantes , Conformação Proteica/efeitos dos fármacos , Engenharia de Proteínas/tendências , Estabilidade Proteica/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
TRAIL is a trimeric protein that potently induces apoptosis in cancer cells by binding to the trimeric death receptors (DR4 or DR5). Death receptors are attractive therapeutic targets through both the recombinant TRAIL ligand as well as receptor agonist monoclonal antibodies. Although efficacy of the ligand is hampered by its short half-life, agonistic antibodies have a much longer half-life and have shown some clinical efficacy as antitumor agents. However, the efficacy of these antibodies may be limited by their bivalent nature that does not optimally mimic the trimeric ligand. To overcome limitations of currently used death receptor-targeting agents, we engineered trimeric proteins called Atrimer complexes that selectively bind DR4 and potently induce apoptosis in a variety of cancer cells. Atrimer complexes are based on human tetranectin, a trimeric plasma protein of approximately 60 kDa. Loop regions within the tetranectin C-type lectin domains (CTLD) were randomized to create a large phage display library that was used to select DR4-binding complexes. A panel of unique and potent agonist DR4 Atrimer complexes with subnanomolar affinity to DR4 and no detectable binding to DR5 or the decoy receptors was identified. Mechanism of action studies with a selected Atrimer complex, 1G(2), showed that Atrimer complexes induce caspase-dependent and DR4-specific apoptosis in cancer cells while sparing normal human fibroblasts and, importantly, hepatocytes. This proof-of-principle study supports the use of alternative proteins engineered to overcome limitations of therapeutically desirable molecules such as TRAIL.
Assuntos
Lectinas Tipo C/química , Lectinas Tipo C/metabolismo , Multimerização Proteica , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células HCT116 , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Multimerização Proteica/efeitos dos fármacos , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Immune evasion in cancer is increasingly recognized as a contributing factor in the failure of a natural host antitumor immune response as well as in the failure of cancer vaccine trials. Immune evasion may be the result of a number of factors, including expansion of regulatory T cells, production of immunosuppressive cytokines, downregulation of HLA class I and tumor-associated antigens and upregulation of immunosuppressive molecules on the surface of tumor cells. CD200, a cell surface ligand that plays a role in regulating the immune system, has been shown to be upregulated on the surface of some hematologic and solid tumor malignancies. This review characterizes the role of CD200 in immune suppression, and describes strategies to target this molecule in the oncology setting, thus directly modulating immune regulation and potentially altering tolerance to tumor antigens.
Assuntos
Antígenos CD/imunologia , Neoplasias Hematológicas/terapia , Terapia de Imunossupressão , Imunoterapia , Leucemia Linfocítica Crônica de Células B/terapia , Animais , Ensaios Clínicos como Assunto , Neoplasias Hematológicas/imunologia , Humanos , Leucemia Linfocítica Crônica de Células B/imunologiaRESUMO
CD200 is an immunosuppressive molecule overexpressed in multiple hematologic malignancies such as B cell chronic lymphocytic leukemia, multiple myeloma, and acute myeloid leukemia. We previously demonstrated that up-regulation of CD200 on tumor cells suppresses antitumor immune responses and that antagonistic anti-human CD200 mAbs enabled human PBMC-mediated tumor growth inhibition in xenograft NOD/SCID human (hu)-mouse models. Ab variants with effector function (IgG1 constant region (G1)) or without effector function (IgG2/G4 fusion constant region (G2G4)) exhibited high antitumor activity in a human tumor xenograft model in which CD200 was expressed. In this report, we seek to select the best candidate to move forward into the clinic and begin to decipher the mechanisms of tumor cell killing by comparing anti-CD200-G1 vs anti-CD200-G2G4 in two related animal models. In a CD200-expressing xenograft NOD/SCID hu-mouse model where CD200 ligand/receptor interactions are already established before initiating treatment, we find that anti-CD200-G1 is a less effective Ab compared with anti-CD200-G2G4. Separately, in a model that evaluates the effect of the Abs on the immune cell component of the xenograft NOD/SCID hu-mouse model distinctly from the effects of binding to CD200 on tumor cells, we find that the administration of anti-CD200-G1 Abs completely abolished human PBMC-mediated tumor growth inhibition. Along with supporting in vitro studies, our data indicate that anti-CD200-G1 Abs efficiently mediate Ab-dependent cellular cytotoxicity of activated T cells, critical cells involved in immune-mediated killing. These studies suggest important implications regarding the selection of the constant region in anti-CD200 immunotherapy of cancer patients.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígenos CD/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Animais , Anticorpos Monoclonais/farmacologia , Antígenos CD/análise , Antígenos CD/imunologia , Humanos , Camundongos , Camundongos Endogâmicos , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Induction of CTL responses by dendritic cell (DC)-based vaccines requires efficient DC-loading strategies for class I Ags. Coupling Ags to cell-penetrating peptides (CPPs) or receptor-specific Abs improves Ag loading of DCs. In contrast to CPPs, receptor-specific Abs deliver conjugated Ags to DCs with high specificity, which is advantageous for in vivo strategies. It has, however, been speculated that CPPs facilitate uptake and endosomal escape of conjugated Ags, which would potently enhance cross-presentation. In this study, we directly compare the in vitro targeting efficiency of a humanized D1 Ab directed against the human DC surface receptor DC-SIGN hD1 to that of three CPPs. The three CPPs colocalized within endosomes when targeted to human monocyte-derived DCs simultaneously, whereas hD1 was present in a different set of endosomes. However, within 75 min after uptake CPPs and hD1 colocalized extensively within the lysosomal compartment. Ab-mediated targeting of class I-restricted peptides to DC-SIGN enhanced cross-presentation of the peptides, while only one of the CPPs enhanced peptide presentation. This CPP and hD1 enhanced cross-presentation with equal efficiencies. Thus, we found no evidence of CPP specifically favoring the delivery of conjugated Ag to the DC class I presentation pathway. Given the specificity with which Abs recognize their targets, this favors the use of DC receptor-specific Abs for in vivo vaccination strategies.
Assuntos
Apresentação de Antígeno , Apresentação Cruzada , Células Dendríticas/imunologia , Peptídeos/imunologia , Anticorpos/imunologia , Moléculas de Adesão Celular/imunologia , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Endossomos/imunologia , Endossomos/metabolismo , Humanos , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Peptídeos/metabolismo , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Multiple cancer vaccine trials have been carried out using ex vivo generated autologous dendritic cells (DCs) loaded with tumor antigen before readministration into patients. Though promising, overall immunologic potency and clinical efficacy might be improved with more efficient DC-based therapies that avoid ex vivo manipulations, but are instead based on in vivo targeting of DCs. For initial in vivo proof of concept studies, we evaluated targeting of proteins or peptides to DCs through DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN). Because the biology of DC-SIGN is different between mice and humans, we assess human DC-SIGN targeting in the setting of elements of a human immune system in a mouse model. Administration of anti-DC-SIGN antibodies carrying either tetanus toxoid peptides or keyhole limpet hemocyanin (KLH) to Rag2gammaC mice reconstituted with human immune cells raised stimulatory human T-cell responses to the respective antigen without additional adjuvant requirements. Furthermore, administration of anti-DC-SIGN antibody-KLH conjugate enhanced the adjuvant properties of KLH resulting in inhibition of RAJI (Human Burkitt's Lymphoma Cell Line) cell tumor growth in Nonobese Diabetic/Severe Combined Immunodeficient mice transplanted with human immune cells. Thus, mouse models reconstituted with human immune cells seem to be suitable for evaluating DC-targeted vaccines, and furthermore, targeting to DCs in situ via DC-SIGN may provide a promising vaccine platform for inducing strong immune responses against cancer and infectious disease agents.
Assuntos
Adjuvantes Imunológicos , Células Apresentadoras de Antígenos/imunologia , Moléculas de Adesão Celular/imunologia , Células Dendríticas/imunologia , Lectinas Tipo C/imunologia , Neoplasias Experimentais/imunologia , Receptores de Superfície Celular/imunologia , Animais , Apresentação de Antígeno , Antígenos CD40/imunologia , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Células Dendríticas/metabolismo , Hemocianinas/imunologia , Humanos , Lectinas Tipo C/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Receptores de Superfície Celular/metabolismo , Linfócitos T/imunologia , Transplante HeterólogoRESUMO
In vivo targeting of antigen-presenting cells (APCs) with antigens coupled to antibodies directed against APC-specific endocytic receptors is a simple and a promising approach to induce or modulate immune responses against those antigens. In a recent in vitro study, we have shown that targeting of APCs with an antigen coupled to an antibody directed against the endocytic receptor DC-SIGN effectively induces a specific immune response against that antigen. The aim of the present study was to determine the ability of the murine antihuman DC-SIGN antibody AZN-D1 to target APCs in a cynomolgus macaque model after its administration in vivo. Immunohistochemical analysis demonstrated that macaques injected intravenously with AZN-D1 have AZN-D1-targeted APCs in all lymph nodes (LNs) tested and in the liver. DC-SIGN-positive cells were mainly located in the medullary sinuses of the LNs and in the hepatic sinusoids in the liver. No unlabeled DC-SIGN molecules were found in the LN of AZN-D1-injected macaques. Morphologic criteria and staining of sequential LN sections with a panel of antibodies indicated that the DC-SIGN-targeted cells belong to the myeloid lineage of APCs. In conclusion, this is the first study that shows specific targeting of APCs in vivo by using antibodies directed against DC-SIGN.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Moléculas de Adesão Celular/imunologia , Células Dendríticas/imunologia , Lectinas Tipo C/imunologia , Tecido Linfoide/imunologia , Macrófagos/imunologia , Receptores de Superfície Celular/imunologia , Animais , Moléculas de Adesão Celular/isolamento & purificação , Moléculas de Adesão Celular/metabolismo , Células Dendríticas/metabolismo , Células de Kupffer/imunologia , Lectinas Tipo C/isolamento & purificação , Lectinas Tipo C/metabolismo , Linfonodos/citologia , Linfonodos/imunologia , Linfonodos/metabolismo , Macaca fascicularis , Macrófagos/metabolismo , Receptores de Superfície Celular/isolamento & purificação , Receptores de Superfície Celular/metabolismoRESUMO
Although the immune system is capable of mounting a response against many cancers, that response is insufficient for tumor eradication in most patients due to factors in the tumor microenvironment that defeat tumor immunity. We previously identified the immune-suppressive molecule CD200 as up-regulated on primary B cell chronic lymphocytic leukemia (B-CLL) cells and demonstrated negative immune regulation by B-CLL and other tumor cells overexpressing CD200 in vitro. In this study we developed a novel animal model that incorporates human immune cells and human tumor cells to address the effects of CD200 overexpression on tumor cells in vivo and to assess the effect of targeting Abs in the presence of human immune cells. Although human mononuclear cells prevented tumor growth when tumor cells did not express CD200, tumor-expressed CD200 inhibited the ability of lymphocytes to eradicate tumor cells. Anti-CD200 Ab administration to mice bearing CD200-expressing tumors resulted in nearly complete tumor growth inhibition even in the context of established receptor-ligand interactions. Evaluation of an anti-CD200 Ab with abrogated effector function provided evidence that blocking of the receptor-ligand interaction was sufficient for control of CD200-mediated immune modulation and tumor growth inhibition in this model. Our data indicate that CD200 expression by tumor cells suppresses antitumor responses and suggest that anti-CD200 treatment might be therapeutically beneficial for treating CD200-expressing cancers.
Assuntos
Anticorpos Bloqueadores/uso terapêutico , Antígenos CD/efeitos dos fármacos , Imunoterapia/métodos , Neoplasias/terapia , Proteínas Recombinantes/uso terapêutico , Animais , Anticorpos Bloqueadores/genética , Anticorpos Bloqueadores/imunologia , Antígenos CD/análise , Antígenos CD/imunologia , Linhagem Celular Tumoral , Membrana Celular/química , Humanos , Leucócitos Mononucleares/imunologia , Camundongos , Camundongos Endogâmicos , Neoplasias/tratamento farmacológico , Biblioteca de Peptídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We searched for cell-surface-associated proteins overexpressed on B cell chronic lymphocytic leukemia (CLL) to use as therapeutic antibody targets. Antibodies binding the immunosuppressive molecule CD200 were identified by cell panning of an antibody phage display library derived from rabbits immunized with primary CLL cells. B cells from 87 CLL patients exhibited 1.6- to 5.4-fold cell-surface up-regulation of CD200 relative to normal B cells. An effect of increased CD200 expression by CLL cells on the immune system was evaluated in mixed lymphocyte reactions. Addition of primary CLL but not normal B cells to macrophages and T cells downregulated the Th1 response, as seen by a 50-95% reduction in secreted IL-2 and IFN-gamma. Antibodies to CD200 prevented downregulation of the Th1 response in most B cell CLL samples evaluated, indicating abrogation of the CD200/CD200R interaction can be sufficient to restore the Th1 response. A disease-progression-associated shift of the immune response from Th1 to Th2 has been observed in numerous cancers. Because this cytokine shift is also believed to promote the induction of regulatory T cells, reverting the immune response to Th1 through direct targeting of the cancer cells may provide therapeutic benefits in CLL by encouraging a cytotoxic T cell response.
Assuntos
Anticorpos/química , Antígenos de Neoplasias , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/imunologia , Biblioteca de Peptídeos , Anticorpos Monoclonais/química , Linfócitos B/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Separação Celular , Citocinas/metabolismo , Células Dendríticas/citologia , Regulação para Baixo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Fragmentos de Imunoglobulinas/química , Imunoprecipitação , Imunossupressores/farmacologia , Imunoterapia/métodos , Interleucina-2/metabolismo , Leucócitos Mononucleares/metabolismo , Teste de Cultura Mista de Linfócitos , Linfócitos/imunologia , Macrófagos/metabolismo , Espectrometria de Massas , Monócitos/metabolismo , Linfócitos T Reguladores/citologia , Células Th1 , Células Th2/imunologiaRESUMO
The C-type lectin L-SIGN is expressed on liver and lymph node endothelial cells, where it serves as a receptor for a variety of carbohydrate ligands, including ICAM-3, Ebola, and HIV. To consider targeting liver/lymph node-specific ICAM-3-grabbing nonintegrin (L-SIGN) for therapeutic purposes in autoimmunity and infectious disease, we isolated and characterized Fabs that bind strongly to L-SIGN, but to a lesser degree or not at all to dendritic cell-specific ICAM-grabbing nonintegrin (DC-SIGN). Six Fabs with distinct relative affinities and epitope specificities were characterized. The Fabs and those selected for conversion to IgG were tested for their ability to block ligand (HIV gp120, Ebola gp, and ICAM-3) binding. Receptor internalization upon Fab binding was evaluated on primary human liver sinusoidal endothelial cells by flow cytometry and confirmed by confocal microscopy. Although all six Fabs internalized, three Fabs that showed the most complete blocking of HIVgp120 and ICAM-3 binding to L-SIGN also internalized most efficiently. Differences among the Fab panel in the ability to efficiently block Ebola gp compared with HIVgp120 suggested distinct binding sites. As a first step to consider the potential of these Abs for Ab-mediated Ag delivery, we evaluated specific peptide delivery to human dendritic cells. A durable human T cell response was induced when a tetanus toxide epitope embedded into a L-SIGN/DC-SIGN-cross-reactive Ab was targeted to dendritic cells. We believe that the isolated Abs may be useful for selective delivery of Ags to DC-SIGN- or L-SIGN-bearing APCs for the modulation of immune responses and for blocking viral infections.