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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

Humanization of 1E11 by CDR grafting.Heavy chain (A) and light chain (B) amino acid sequence alignment of murine, human germline, and humanized 1E11. The CDR residues, defined according to the Kabat definition, are shown in bold. Amino acids are numbered according to the Kabat numbering scheme. Colons represent common residues between murine and human germline sequences.
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pone.0134600.g001: Humanization of 1E11 by CDR grafting.Heavy chain (A) and light chain (B) amino acid sequence alignment of murine, human germline, and humanized 1E11. The CDR residues, defined according to the Kabat definition, are shown in bold. Amino acids are numbered according to the Kabat numbering scheme. Colons represent common residues between murine and human germline sequences.

Mentions: To develop the humanized antibody, the VH and VL sequences of the murine 1E11 were compared with human germline V and J gene repertories using IMGT/V-QUEST analysis tools [24]. For the heavy chain, IGHV3-48*03 and IGHJ4*01 exhibited the highest homology to the 1E11 counterparts, sharing 85% and 87% identity, respectively. For the light chain, human IGKV1-39*01 and IGKJ1*01 genes displayed identity of 80% and 81%, respectively. These human genes were selected as acceptor sequences for the grafting of the murine CDRs. Among the Vernier zone, which consists of residues in the framework region that are involved in the presentation of CDR structures by supporting the CDR loops [18, 25], only one residue at position 49H of heavy chain differed between murine and human sequences. Consequently, humanized 1E11, hz1E11, has only one murine residue in the framework regions (Fig 1).


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)

Humanization of 1E11 by CDR grafting.Heavy chain (A) and light chain (B) amino acid sequence alignment of murine, human germline, and humanized 1E11. The CDR residues, defined according to the Kabat definition, are shown in bold. Amino acids are numbered according to the Kabat numbering scheme. Colons represent common residues between murine and human germline sequences.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134600.g001: Humanization of 1E11 by CDR grafting.Heavy chain (A) and light chain (B) amino acid sequence alignment of murine, human germline, and humanized 1E11. The CDR residues, defined according to the Kabat definition, are shown in bold. Amino acids are numbered according to the Kabat numbering scheme. Colons represent common residues between murine and human germline sequences.
Mentions: To develop the humanized antibody, the VH and VL sequences of the murine 1E11 were compared with human germline V and J gene repertories using IMGT/V-QUEST analysis tools [24]. For the heavy chain, IGHV3-48*03 and IGHJ4*01 exhibited the highest homology to the 1E11 counterparts, sharing 85% and 87% identity, respectively. For the light chain, human IGKV1-39*01 and IGKJ1*01 genes displayed identity of 80% and 81%, respectively. These human genes were selected as acceptor sequences for the grafting of the murine CDRs. Among the Vernier zone, which consists of residues in the framework region that are involved in the presentation of CDR structures by supporting the CDR loops [18, 25], only one residue at position 49H of heavy chain differed between murine and human sequences. Consequently, humanized 1E11, hz1E11, has only one murine residue in the framework regions (Fig 1).

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