RESUMO
Fc engineering to increase the binding affinity of IgG antibodies to FcRn has been reported to reduce the elimination of IgG antibodies. Herein, we present a novel non-FcRn-dependent approach to reduce the elimination of IgG antibodies. Pharmacokinetic studies conducted in normal mice of various humanized IgG4 antibodies, which had identical constant regions but different variable region sequences, revealed that an antibody with a lower isoelectric point (pI) has a longer half-life. These antibodies exhibited comparable binding affinity to FcRn, and with the antibodies with lower pIs, a longer half-life was also observed in beta2-microglobulin knockout mice, suggesting that differences in the pharmacokinetics were due to a non-FcRn-dependent mechanism. On the basis of our findings, we attempted to engineer the pharmacokinetic properties of a humanized anti-IL6 receptor IgG1 antibody. Selected substitutions in the variable region, without substitution in the Fc region, lowered the pI but did not reduce the biological activity and showed a significant reduction in the clearance of the antibody in cynomolgus monkey. These results suggest that lowering the pI by engineering the variable region could reduce the elimination of IgG antibodies and could provide an alternative to Fc engineering of IgG antibodies.
Assuntos
Engenharia Genética/métodos , Imunoglobulina G/genética , Região Variável de Imunoglobulina/genética , Engenharia de Proteínas/métodos , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacocinética , Células CHO , Cricetinae , Cricetulus , Meia-Vida , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunoglobulina G/metabolismo , Ponto Isoelétrico , Camundongos , Receptores Fc/metabolismoRESUMO
In order to evaluate the targeting potential of mouse monoclonal antibody ONS-M21 recognizing a human astrocytoma- and medulloblastoma-associated antigen, the internalization ability of this antibody and the selective cytotoxicity in the toxin-conjugated form were examined. Internalization assay with 125I-labeled ONS-M21 showed that about 20% of the total radioactivities was detected in the cellular fraction of human medulloblastoma cell line ONS-76 cells and that the reaction reached a plateau level in 30 min. To examine the selective delivery capacity of a high molecular substance in place of 125I, an immunotoxin was prepared with ricin A chain and ONS-M21 via disulfide bonds. A cytotoxic effect against ONS-76 cells was found with [3H]thymidine incorporation assay using the immunotoxin, but not against antigen-negative HuH-7 and SW480 cells. These results suggest that ONS-M21 could effectively deliver toxins, chemotherapeutic agents or radionuclei to malignant glioma specifically.
Assuntos
Anticorpos Monoclonais , Antígenos de Neoplasias/análise , Glioma/imunologia , Animais , Astrocitoma/imunologia , Sobrevivência Celular , Estudos de Avaliação como Assunto , Humanos , Imunotoxinas , Meduloblastoma/imunologia , Camundongos , Ricina/farmacologia , Células Tumorais CultivadasRESUMO
A humanized ONS-M21 antibody (hM21) against human medulloblastoma and glioma cells was engineered as a single-chain Fv fragment (scFv), and its ability to internalize into tumor cells was evaluated by conjugation with ricin A. The scFv of hM21 (schM21) was easily purified from E.coli by one-step affinity column chromatography. Purified schM21 bound to a medulloblastoma ONS-76 cell with almost equal antigen-binding activity of hM21-Fab fragment. Furthermore, the schM21-ricin A conjugate inhibited the growth of ONS-76 cells, but not that of antigen-negative hepatoma HuH-7 cells, suggesting that the schM21 can be internalized after binding to antigen-positive cells. Thus, schM21 could be expected to act as a novel carrier of diagnostic and therapeutic agents for brain tumors.
Assuntos
Anticorpos Monoclonais , Fragmentos Fab das Imunoglobulinas , Região Variável de Imunoglobulina , Imunotoxinas/toxicidade , Ricina/toxicidade , Complexo Antígeno-Anticorpo , Antígenos de Neoplasias/imunologia , Sítios de Ligação de Anticorpos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Proteínas Recombinantes , Células Tumorais CultivadasRESUMO
An immunosorbent assay system to detect genetically engineered IL-6 receptor (IL-6R) was established, whereby soluble IL-6 receptor (sIL-6R) was detected in the culture medium when sIL-6R cDNA was transfected into COS1 cells. A stably transformed Chinese hamster ovary (CHO) cell line constitutively expressing sIL-6R has been established. The recombinant sIL-6R was purified to homogeneity by sequential filtration and chromatography of the culture medium. The recombinant sIL-6R augmented the sensitivity of M1 cells to IL-6 in growth inhibition assay in a dose-dependent manner. Furthermore, a radioisotope immunosorbent assay (RIA) utilizing recombinant sIL-6R was established which could detect IL-6 in a quantity as low as 10 ng/ml.
