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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

Humanized 1E11 shows equivalent binding properties and biological activity to the parental murine antibody.A, The binding activities of hz1E11 and ch1E11 to HER2-ECD protein were analyzed by ELISA. Trastuzumab (TRA) was used as a positive control antibody against HER2 protein. B, The binding activity of the antibodies was analyzed by ELISA using recombinant HER2 sub-domain proteins. C, NCI-N87 cells were treated with antibodies for 4 days in the complete growth media and the cell viability was measured in duplicates (mean ± SD) using WST-1 reagent. The 100% viability was defined as the viability of the antibody-untreated wells. D, Mice bearing NCI-N87 xenograft tumors were treated with indicated dose of control antibody, trastuzumab, hz1E11, or trastuzumab plus hz1E11. Palivizumab was used as the isotype control antibody. Tumor volume (mm3) was expressed as mean ± SD (n = 6 mice/group). For clarity, only positive error bars are shown. **, P < 0.01; n.s., not significant as determined by two-way repeated measures ANOVA followed by Bonferroni post test.
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pone.0134600.g002: Humanized 1E11 shows equivalent binding properties and biological activity to the parental murine antibody.A, The binding activities of hz1E11 and ch1E11 to HER2-ECD protein were analyzed by ELISA. Trastuzumab (TRA) was used as a positive control antibody against HER2 protein. B, The binding activity of the antibodies was analyzed by ELISA using recombinant HER2 sub-domain proteins. C, NCI-N87 cells were treated with antibodies for 4 days in the complete growth media and the cell viability was measured in duplicates (mean ± SD) using WST-1 reagent. The 100% viability was defined as the viability of the antibody-untreated wells. D, Mice bearing NCI-N87 xenograft tumors were treated with indicated dose of control antibody, trastuzumab, hz1E11, or trastuzumab plus hz1E11. Palivizumab was used as the isotype control antibody. Tumor volume (mm3) was expressed as mean ± SD (n = 6 mice/group). For clarity, only positive error bars are shown. **, P < 0.01; n.s., not significant as determined by two-way repeated measures ANOVA followed by Bonferroni post test.

Mentions: Binding activity of hz1E11 to extracellular region (ECD) of HER2 was equivalent to that of ch1E11 (Fig 2A), and the affinity of trastuzumab, ch1E11, and hz1E11 was 3 nM, 23 nM, and 23 nM, respectively. We also confirmed that hz1E11 bound to sub-domain IV like the parental ch1E11. Trastuzumab also binds to sub-domain IV [26]. The in vitro anti-proliferative activities of hz1E11 as a single agent and in combination with trastuzumab were also equivalent to those of ch1E11 (Fig 2C). In the previous study [22] it was reported that ch1E11 had in vivo antitumor activity comparable to that of trastuzumab as a single agent, and the combination of ch1E11 and trastuzumab resulted in the tumor regression index (TGI) of 95.1%. Similarly, hz1E11 as a single agent had similar in vivo antitumor activity to trastuzumab (S1 Fig) and the combination of hz1E11 with trastuzumab also showed dose-dependent anti-tumor activity in the NCI-N87 xenograft mouse model (Fig 2D). Antibody combination treatment at 1 mg/kg each of hz1E11 and trastuzumab resulted in similar anti-tumor activity with trastuzumab single treatment at 10 mg/kg, and at the 2.5 mg/kg combination and above the established tumor stabilized or started regressing (No statistically significant difference [P < 0.05] was observed among 2.5, 5, and 10 mg/kg combinations). These results show that hz1E11 has the affinity and biological activity equivalent to ch1E11, and that hz1E11 enhances the anti-tumor activity of trastuzumab when used in combination.


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)

Humanized 1E11 shows equivalent binding properties and biological activity to the parental murine antibody.A, The binding activities of hz1E11 and ch1E11 to HER2-ECD protein were analyzed by ELISA. Trastuzumab (TRA) was used as a positive control antibody against HER2 protein. B, The binding activity of the antibodies was analyzed by ELISA using recombinant HER2 sub-domain proteins. C, NCI-N87 cells were treated with antibodies for 4 days in the complete growth media and the cell viability was measured in duplicates (mean ± SD) using WST-1 reagent. The 100% viability was defined as the viability of the antibody-untreated wells. D, Mice bearing NCI-N87 xenograft tumors were treated with indicated dose of control antibody, trastuzumab, hz1E11, or trastuzumab plus hz1E11. Palivizumab was used as the isotype control antibody. Tumor volume (mm3) was expressed as mean ± SD (n = 6 mice/group). For clarity, only positive error bars are shown. **, P < 0.01; n.s., not significant as determined by two-way repeated measures ANOVA followed by Bonferroni post test.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4520604&req=5

pone.0134600.g002: Humanized 1E11 shows equivalent binding properties and biological activity to the parental murine antibody.A, The binding activities of hz1E11 and ch1E11 to HER2-ECD protein were analyzed by ELISA. Trastuzumab (TRA) was used as a positive control antibody against HER2 protein. B, The binding activity of the antibodies was analyzed by ELISA using recombinant HER2 sub-domain proteins. C, NCI-N87 cells were treated with antibodies for 4 days in the complete growth media and the cell viability was measured in duplicates (mean ± SD) using WST-1 reagent. The 100% viability was defined as the viability of the antibody-untreated wells. D, Mice bearing NCI-N87 xenograft tumors were treated with indicated dose of control antibody, trastuzumab, hz1E11, or trastuzumab plus hz1E11. Palivizumab was used as the isotype control antibody. Tumor volume (mm3) was expressed as mean ± SD (n = 6 mice/group). For clarity, only positive error bars are shown. **, P < 0.01; n.s., not significant as determined by two-way repeated measures ANOVA followed by Bonferroni post test.
Mentions: Binding activity of hz1E11 to extracellular region (ECD) of HER2 was equivalent to that of ch1E11 (Fig 2A), and the affinity of trastuzumab, ch1E11, and hz1E11 was 3 nM, 23 nM, and 23 nM, respectively. We also confirmed that hz1E11 bound to sub-domain IV like the parental ch1E11. Trastuzumab also binds to sub-domain IV [26]. The in vitro anti-proliferative activities of hz1E11 as a single agent and in combination with trastuzumab were also equivalent to those of ch1E11 (Fig 2C). In the previous study [22] it was reported that ch1E11 had in vivo antitumor activity comparable to that of trastuzumab as a single agent, and the combination of ch1E11 and trastuzumab resulted in the tumor regression index (TGI) of 95.1%. Similarly, hz1E11 as a single agent had similar in vivo antitumor activity to trastuzumab (S1 Fig) and the combination of hz1E11 with trastuzumab also showed dose-dependent anti-tumor activity in the NCI-N87 xenograft mouse model (Fig 2D). Antibody combination treatment at 1 mg/kg each of hz1E11 and trastuzumab resulted in similar anti-tumor activity with trastuzumab single treatment at 10 mg/kg, and at the 2.5 mg/kg combination and above the established tumor stabilized or started regressing (No statistically significant difference [P < 0.05] was observed among 2.5, 5, and 10 mg/kg combinations). These results show that hz1E11 has the affinity and biological activity equivalent to ch1E11, and that hz1E11 enhances the anti-tumor activity of trastuzumab when used in combination.

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