RESUMO
Chemokines play a key role in leukocyte recruitment during inflammation and are implicated in the pathogenesis of a number of autoimmune diseases. As such, inhibiting chemokine signaling has been of keen interest for the development of therapeutic agents. This endeavor, however, has been hampered due to complexities in the chemokine system. Many chemokines have been shown to signal through multiple receptors and, conversely, most chemokine receptors bind to more than one chemokine. One approach to overcoming this complexity is to develop a single therapeutic agent that binds and inactivates multiple chemokines, similar to an immune evasion strategy utilized by a number of viruses. Here, we describe the development and characterization of a novel therapeutic antibody that targets a subset of human CC chemokines, specifically CCL3, CCL4, and CCL5, involved in chronic inflammatory diseases. Using a sequential immunization approach, followed by humanization and phage display affinity maturation, a therapeutic antibody was developed that displays high binding affinity towards the three targeted chemokines. In vitro, this antibody potently inhibits chemotaxis and chemokine-mediated signaling through CCR1 and CCR5, primary chemokine receptors for the targeted chemokines. Furthermore, we have demonstrated in vivo efficacy of the antibody in a SCID-hu mouse model of skin leukocyte migration, thus confirming its potential as a novel therapeutic chemokine antagonist. We anticipate that this antibody will have broad therapeutic utility in the treatment of a number of autoimmune diseases due to its ability to simultaneously neutralize multiple chemokines implicated in disease pathogenesis.
Assuntos
Anticorpos Neutralizantes/imunologia , Doenças Autoimunes/tratamento farmacológico , Quimiocinas CC/imunologia , Imunomodulação/imunologia , Imunoterapia/métodos , Transdução de Sinais/imunologia , Animais , Anticorpos Neutralizantes/uso terapêutico , Doenças Autoimunes/imunologia , Quimiotaxia/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Fosforilação , Ressonância de Plasmônio de SuperfícieRESUMO
Monoclonal antibodies have begun to show great clinical promise for the treatment of cancer. Antibodies that can directly affect a tumor cell's growth and/or survival are of particular interest for immunotherapy. Previously, we described monoclonal antibody DMF10.62.3 that had antiproliferative and proapoptotic effects when it bound an antigen of unknown identity on tumor cells in vitro. In this report, we determined that DMF10.62.3 and a clonally related antibody DMF10.167.4 recognize the ganglioside GM2. These antibodies react with a GM2 epitope that is expressed on a large number of tumor cell lines, including human melanoma and small cell lung carcinoma, but not on normal primary lines or most normal tissues. Interestingly, this pattern of cellular reactivity is distinct from that reported for other previously described GM2 antibodies, a difference that is presumably due to DMF10.167.4's binding to a unique GM2-associated epitope. Additional characterization of DMF10.167.4 revealed that this antibody was able to induce apoptosis and/or block cellular proliferation when cultured in vitro with the human Jurkat T lymphoma, CHL-1 melanoma, and SBC-3 small cell lung carcinoma lines. In vivo, DMF10.167.4 antibody was well tolerated in mice and did not detectably bind to or damage normal tissues. However, this antibody was able to prevent murine E710.2.3 lymphoma, human CHL-1 melanoma, and SBC-3 small cell lung carcinoma lines from establishing tumors in vivo and blocked progression of established CHL-1 and SBC-3 tumors in vivo. Therefore, monoclonal antibody DMF10.167.4 has immunotherapeutic potential.
Assuntos
Anticorpos Monoclonais/imunologia , Carcinoma de Células Pequenas/terapia , Gangliosídeo G(M2)/imunologia , Imunização Passiva/métodos , Neoplasias Pulmonares/terapia , Melanoma/terapia , Animais , Anticorpos Monoclonais/farmacologia , Especificidade de Anticorpos , Apoptose/imunologia , Carcinoma de Células Pequenas/imunologia , Carcinoma de Células Pequenas/patologia , Cricetinae , Epitopos/imunologia , Feminino , Humanos , Células Jurkat , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Melanoma/imunologia , Melanoma/patologia , Camundongos , Camundongos Endogâmicos AKR , Camundongos SCIDRESUMO
Expression of recombinant proteins is an important step towards elucidating the functions of many genes discovered through genomic sequencing projects. It is also critical for validating gene targets and for developing effective therapies for many diseases. Here we describe a novel method to express recombinant proteins that are extremely difficult to produce otherwise. The increased protein expression level is achieved by using a fusion partner, MTB32-C, which is the carboxyl terminal fragment of the Mycobacterium tuberculosis antigen, MTB32 (Rv0125). By fusing MTB32-C to the N-termini of target genes, we have demonstrated significant enhancement of recombinant protein expression level in Escherichia coli. The inclusion of a 6xHis tag and the 128-amino acid of MTB32-C will add 13.5 kDa to the fusion molecule. Comparison of the mRNA levels of the fusion and non-fusion proteins indicated that the increased fusion protein expression may be regulated at translational or post-translational steps. There are many potential applications for the generated fusion proteins. For example, MTB32-C fusion proteins have been used successfully as immunogens to generate both polyclonal and monoclonal antibodies. These antibodies have been used to characterize cellular localization of the proteins and to validate gene targets at protein level. In addition, these antibodies may be useful in diagnostic and therapeutic applications for many diseases. If desired, the MTB32-C portion in the fusion protein can be removed after protein expression, making it possible to study protein structure and function as well as to screen for potential drugs. Thus, this novel fusion expression system has become a powerful tool for many applications.
Assuntos
Biotecnologia/métodos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Sequência de Aminoácidos , Animais , Anticorpos/genética , Anticorpos/imunologia , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Sequência de Bases , Linhagem Celular , Escherichia coli , Citometria de Fluxo , Expressão Gênica , Engenharia Genética/métodos , Vetores Genéticos , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Mycobacterium tuberculosis , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
HER2/neu is a compelling cancer vaccine candidate because it is overexpressed on some cancer cells relative to normal tissues, it is known to be immunogenic in both animal models and in humans, and it is already known to be targetable by the antibody component of the immune system in the form of monoclonal antibody therapy with trastuzumab. Vaccines offer the theoretical advantage of being able to elicit T-cell responses in addition to antibody responses. HER2 vaccines have been shown to provide benefit in animal models and to be immunogenic in humans. However, the optimal vaccine formulation is not yet known and the therapeutic efficacy of the vaccines in humans has not yet been evaluated. HER2 vaccine approaches currently being tested include peptide-based, DNA plasmid-based, and protein-based vaccines. Our group has developed and started testing a protein-based vaccine composed of both the extracellular domain of HER2 and the carboxyl terminal autophosphorylation portion of the intracellular domain. The extracellular domain was retained to provide for antibody targeting. The kinase domain of the intracellular domain was excluded because of its high degree of homology to other human kinases. The carboxyl terminal autophosphorylation domain was retained because it is the most unique and possibly most immunogenic portion of the HER2 molecule with the least homology to other members of the HER family. The vaccine, termed dHER2, is immunogenic in mice and primates. In animal models it can elicit CD8 and CD4 T-cell responses as well as antibody responses that suppress the growth of HER2-positive cancer cells in vitro and in vivo. Vaccine trials are contemplated in patients with breast cancer that will determine whether the vaccine construct is similarly immunogenic in humans.