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Affinity Maturation of Monoclonal Antibody 1E11 by Targeted Randomization in CDR3 Regions Optimizes Therapeutic Antibody Targeting of HER2-Positive Gastric Cancer.

Ko BK, Choi S, Cui LG, Lee YH, Hwang IS, Kim KT, Shim H, Lee JS - PLoS ONE (2015)

Bottom Line: Milder selection pressure favored the selection of more diverse clones, whereas higher selection stringency resulted in the convergence of the panning output to a smaller number of clones with improved affinity.Clone 1A12 inhibited tumor growth of NCI-N87 xenograft model with similar efficacy to trastuzumab alone, and the combination treatment of 1A12 and trastuzumab completely removed the established tumors.These results suggest that humanized and affinity matured monoclonal antibody 1A12 is a highly optimized molecule for future therapeutic development against HER2-positive tumors.

View Article: PubMed Central - PubMed

Affiliation: Therapeutic antibody research center, AbClon Inc., Seoul, Korea.

ABSTRACT
Anti-HER2 murine monoclonal antibody 1E11 has strong and synergistic anti-tumor activity in HER2-overexpressing gastric cancer cells when used in combination with trastuzumab. We presently optimized this antibody for human therapeutics. First, the complementarity determining regions (CDRs) of the murine antibody were grafted onto human germline immunoglobulin variable genes. No difference in affinity and biological activity was observed between chimeric 1E11 (ch1E11) and humanized 1E11 (hz1E11). Next, affinity maturation of hz1E11 was performed by the randomization of CDR-L3 and H3 residues followed by stringent biopanning selection. Milder selection pressure favored the selection of more diverse clones, whereas higher selection stringency resulted in the convergence of the panning output to a smaller number of clones with improved affinity. Clone 1A12 had four amino acid substitutions in CDR-L3, and showed a 10-fold increase in affinity compared to the parental clone and increased potency in an in vitro anti-proliferative activity assay with HER2-overepxressing gastric cancer cells. Clone 1A12 inhibited tumor growth of NCI-N87 xenograft model with similar efficacy to trastuzumab alone, and the combination treatment of 1A12 and trastuzumab completely removed the established tumors. These results suggest that humanized and affinity matured monoclonal antibody 1A12 is a highly optimized molecule for future therapeutic development against HER2-positive tumors.

No MeSH data available.


Related in: MedlinePlus

Three residues in CDR-H3 are important for antigen-antibody binding.The binding activities of alanine scanning mutants of CDR-H3 (A) and CDR-L3 (B) were analyzed by ELISA. C, Quantitative dissociation kinetics of indicated mutants were analyzed by surface plasmon resonance. HER2-ECD protein was immobilized on a CM5 sensor chip followed by exposure to indicated mutant Fab antibodies. The 100% association was defined as the response unit (RU) at 200 seconds from the beginning of Fab injection. koff values were calculated using the BIAevaluation software.
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pone.0134600.g003: Three residues in CDR-H3 are important for antigen-antibody binding.The binding activities of alanine scanning mutants of CDR-H3 (A) and CDR-L3 (B) were analyzed by ELISA. C, Quantitative dissociation kinetics of indicated mutants were analyzed by surface plasmon resonance. HER2-ECD protein was immobilized on a CM5 sensor chip followed by exposure to indicated mutant Fab antibodies. The 100% association was defined as the response unit (RU) at 200 seconds from the beginning of Fab injection. koff values were calculated using the BIAevaluation software.

Mentions: To identify the critical residues for antigen-antibody interaction, alanine scanning mutagenesis was carried out in CDR3 regions of heavy and light chains (CDR-H3 and CDR-L3). The effects of the mutations were assessed by analyzing the binding activity of purified Fab proteins by indirect ELISA. In CDR-H3, alanine substitution at position Gly98H abolished the binding of the Fab to HER2, and G97HA and T99HA mutants showed reduced binding activity (Fig 3A). In CDR-L3, the alanine substitutions had much smaller effects (Fig 3B). The koff values of alanine scanning mutants were analyzed using SPR against immbolized HER2-ECD protein. We confirmed that the heavy chain G98A mutant completely lost binding activities, and the neighboring T99HA and G97HA mutants resulted in 32-fold and 17-fold increased koff values, respectively (Fig 3C). These results indicate that heavy chain Gly98H is functionally critical and its adjacent residues are also functionally important for antigen-antibody interaction.


Affinity Maturation of Monoclonal Antibody 1E11 by Targeted Randomization in CDR3 Regions Optimizes Therapeutic Antibody Targeting of HER2-Positive Gastric Cancer.

Ko BK, Choi S, Cui LG, Lee YH, Hwang IS, Kim KT, Shim H, Lee JS - PLoS ONE (2015)

Three residues in CDR-H3 are important for antigen-antibody binding.The binding activities of alanine scanning mutants of CDR-H3 (A) and CDR-L3 (B) were analyzed by ELISA. C, Quantitative dissociation kinetics of indicated mutants were analyzed by surface plasmon resonance. HER2-ECD protein was immobilized on a CM5 sensor chip followed by exposure to indicated mutant Fab antibodies. The 100% association was defined as the response unit (RU) at 200 seconds from the beginning of Fab injection. koff values were calculated using the BIAevaluation software.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520604&req=5

pone.0134600.g003: Three residues in CDR-H3 are important for antigen-antibody binding.The binding activities of alanine scanning mutants of CDR-H3 (A) and CDR-L3 (B) were analyzed by ELISA. C, Quantitative dissociation kinetics of indicated mutants were analyzed by surface plasmon resonance. HER2-ECD protein was immobilized on a CM5 sensor chip followed by exposure to indicated mutant Fab antibodies. The 100% association was defined as the response unit (RU) at 200 seconds from the beginning of Fab injection. koff values were calculated using the BIAevaluation software.
Mentions: To identify the critical residues for antigen-antibody interaction, alanine scanning mutagenesis was carried out in CDR3 regions of heavy and light chains (CDR-H3 and CDR-L3). The effects of the mutations were assessed by analyzing the binding activity of purified Fab proteins by indirect ELISA. In CDR-H3, alanine substitution at position Gly98H abolished the binding of the Fab to HER2, and G97HA and T99HA mutants showed reduced binding activity (Fig 3A). In CDR-L3, the alanine substitutions had much smaller effects (Fig 3B). The koff values of alanine scanning mutants were analyzed using SPR against immbolized HER2-ECD protein. We confirmed that the heavy chain G98A mutant completely lost binding activities, and the neighboring T99HA and G97HA mutants resulted in 32-fold and 17-fold increased koff values, respectively (Fig 3C). These results indicate that heavy chain Gly98H is functionally critical and its adjacent residues are also functionally important for antigen-antibody interaction.

Bottom Line: Milder selection pressure favored the selection of more diverse clones, whereas higher selection stringency resulted in the convergence of the panning output to a smaller number of clones with improved affinity.Clone 1A12 inhibited tumor growth of NCI-N87 xenograft model with similar efficacy to trastuzumab alone, and the combination treatment of 1A12 and trastuzumab completely removed the established tumors.These results suggest that humanized and affinity matured monoclonal antibody 1A12 is a highly optimized molecule for future therapeutic development against HER2-positive tumors.

View Article: PubMed Central - PubMed

Affiliation: Therapeutic antibody research center, AbClon Inc., Seoul, Korea.

ABSTRACT
Anti-HER2 murine monoclonal antibody 1E11 has strong and synergistic anti-tumor activity in HER2-overexpressing gastric cancer cells when used in combination with trastuzumab. We presently optimized this antibody for human therapeutics. First, the complementarity determining regions (CDRs) of the murine antibody were grafted onto human germline immunoglobulin variable genes. No difference in affinity and biological activity was observed between chimeric 1E11 (ch1E11) and humanized 1E11 (hz1E11). Next, affinity maturation of hz1E11 was performed by the randomization of CDR-L3 and H3 residues followed by stringent biopanning selection. Milder selection pressure favored the selection of more diverse clones, whereas higher selection stringency resulted in the convergence of the panning output to a smaller number of clones with improved affinity. Clone 1A12 had four amino acid substitutions in CDR-L3, and showed a 10-fold increase in affinity compared to the parental clone and increased potency in an in vitro anti-proliferative activity assay with HER2-overepxressing gastric cancer cells. Clone 1A12 inhibited tumor growth of NCI-N87 xenograft model with similar efficacy to trastuzumab alone, and the combination treatment of 1A12 and trastuzumab completely removed the established tumors. These results suggest that humanized and affinity matured monoclonal antibody 1A12 is a highly optimized molecule for future therapeutic development against HER2-positive tumors.

No MeSH data available.


Related in: MedlinePlus