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HN125: A Novel Immunoadhesin Targeting MUC16 with Potential for Cancer Therapy.

Xiang X, Feng M, Felder M, Connor JP, Man YG, Patankar MS, Ho M - J Cancer (2011)

Bottom Line: Because of its lower immunogenicity in patients, a fully human protein is the most desirable format for clinical applications.We believe that the methods developed here may apply to the generation of other tumor-targeting immunoadhesins when it is difficult to obtain a human monoclonal antibody to a given antigen for clinical applications.The resultant immunoadhesins can have advantages usually found in monoclonal antibodies such as ease of purification, high binding affinity and effector functions.

View Article: PubMed Central - PubMed

Affiliation: 1. Antibody Therapy Unit, Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA;

ABSTRACT

Background: The mucin MUC16 expresses the repeating peptide epitope CA125 that has been known for decades to be a well-validated cancer marker that is overexpressed on the cell surface of ovarian cancers and other malignant tumors. In spite of recent efforts to make mouse monoclonal antibodies to MUC16 to treat ovarian cancer, a human monoclonal antibody against this mucin has not been described. MUC16 interacts with mesothelin, a protein that mediates heterotypic cancer cell adhesion, indicating that MUC16 and mesothelin play an important role in the peritoneal implantation and metastasis of ovarian tumors. Therefore, a suitable candidate for therapeutic targeting of MUC16 would functionally block the interaction of MUC16 and mesothelin.

Methodology/principal findings: Here we report the generation of a novel immunoadhesin, HN125, against MUC16 that consists of a functional MUC16 binding domain of mesothelin (IAB) and the Fc portion of a human antibody IgG1. The yield for purified HN125 proteins is over 100 µg/mL of HEK-293 culture supernatant. We show that HN125 has high and specific affinity for MUC16-expressing cancer cells by flow cytometry and immunohistochemistry. HN125 has the ability to disrupt the heterotypic cancer cell adhesion mediated by the MUC16-mesothelin interaction. Moreover, it elicits strong antibody-dependent cell mediated cytotoxicity against MUC16-positive cancer cells in vitro.

Conclusion/significance: This report describes a novel human immunotherapeutic agent highly specific for MUC16 with potential for treating ovarian cancer and other MUC16-expressing tumors. Because of its lower immunogenicity in patients, a fully human protein is the most desirable format for clinical applications. We believe that the methods developed here may apply to the generation of other tumor-targeting immunoadhesins when it is difficult to obtain a human monoclonal antibody to a given antigen for clinical applications. The resultant immunoadhesins can have advantages usually found in monoclonal antibodies such as ease of purification, high binding affinity and effector functions.

No MeSH data available.


Related in: MedlinePlus

Generation of immunoadhesin HN125. A. Diagram of expression plasmid pMH142 for HN125 expression in human HEK-293F cells as NH2-terminal fusions to human IgG1 Fc (hFc) (Hinge-CH2-CH3). CMV IE Enhancer/Promoter: human cytomegalovirus immediate-early enhancer/promoter; IL2 Sig: IL2 signal peptide; Tbgh: poly A bovine growth hormone tail; ori: origin of replication; Kan.: kanamycin resistance. B. The structural model of HN125. IAB is the functional binding domain in mesothelin for MUC16. CH2 and CH3: second and third constant domains of a human IgG1, respectively. C. SDS-PAGE analysis of HN125. Molecular weights: non-reduced dimers (~75 kDa) and reduced monomers (~37 kDa).
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Figure 1: Generation of immunoadhesin HN125. A. Diagram of expression plasmid pMH142 for HN125 expression in human HEK-293F cells as NH2-terminal fusions to human IgG1 Fc (hFc) (Hinge-CH2-CH3). CMV IE Enhancer/Promoter: human cytomegalovirus immediate-early enhancer/promoter; IL2 Sig: IL2 signal peptide; Tbgh: poly A bovine growth hormone tail; ori: origin of replication; Kan.: kanamycin resistance. B. The structural model of HN125. IAB is the functional binding domain in mesothelin for MUC16. CH2 and CH3: second and third constant domains of a human IgG1, respectively. C. SDS-PAGE analysis of HN125. Molecular weights: non-reduced dimers (~75 kDa) and reduced monomers (~37 kDa).

Mentions: PCR amplification of IAB from pMH118 30 was performed using primers IAB-F(EcoRI): 5'-GAATTCGATATCGGAAGTGGAGAAGACAGCCTGTCCT-3' and IAB-R(BglII): 5'-AGATCTGAGCTCATCCAGTTTATGCTTTAGG-3'. The PCR product was purified and digested with EcoRI and BglII (New England Biolabs, Ipswich, MA), then ligated with pFUSE-hIgG1-Fc2 (Invivogen, San Diego, CA) linearized with EcoRI and BglII. The resultant vector was reamplified for the IAB-hFc (Fc portion of a human antibody IgG1) part using primers IL2-F: 5'-TCGACACGTGTGATCAGATAGGGCCACCATGTACAGGATGCAACTCCTGTCT-3' and hFc-R: 5'- TCTAGAGCGGCCGCGATACTCATTTACCCGGAGACAGGGAGAG-3', The IAB-hFc PCR fragment was then digested with PmI and NotI, and inserted into the vector pVRC8400 37, 38. The final plasmid is named pMH142 (Figure 1A).


HN125: A Novel Immunoadhesin Targeting MUC16 with Potential for Cancer Therapy.

Xiang X, Feng M, Felder M, Connor JP, Man YG, Patankar MS, Ho M - J Cancer (2011)

Generation of immunoadhesin HN125. A. Diagram of expression plasmid pMH142 for HN125 expression in human HEK-293F cells as NH2-terminal fusions to human IgG1 Fc (hFc) (Hinge-CH2-CH3). CMV IE Enhancer/Promoter: human cytomegalovirus immediate-early enhancer/promoter; IL2 Sig: IL2 signal peptide; Tbgh: poly A bovine growth hormone tail; ori: origin of replication; Kan.: kanamycin resistance. B. The structural model of HN125. IAB is the functional binding domain in mesothelin for MUC16. CH2 and CH3: second and third constant domains of a human IgG1, respectively. C. SDS-PAGE analysis of HN125. Molecular weights: non-reduced dimers (~75 kDa) and reduced monomers (~37 kDa).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3100680&req=5

Figure 1: Generation of immunoadhesin HN125. A. Diagram of expression plasmid pMH142 for HN125 expression in human HEK-293F cells as NH2-terminal fusions to human IgG1 Fc (hFc) (Hinge-CH2-CH3). CMV IE Enhancer/Promoter: human cytomegalovirus immediate-early enhancer/promoter; IL2 Sig: IL2 signal peptide; Tbgh: poly A bovine growth hormone tail; ori: origin of replication; Kan.: kanamycin resistance. B. The structural model of HN125. IAB is the functional binding domain in mesothelin for MUC16. CH2 and CH3: second and third constant domains of a human IgG1, respectively. C. SDS-PAGE analysis of HN125. Molecular weights: non-reduced dimers (~75 kDa) and reduced monomers (~37 kDa).
Mentions: PCR amplification of IAB from pMH118 30 was performed using primers IAB-F(EcoRI): 5'-GAATTCGATATCGGAAGTGGAGAAGACAGCCTGTCCT-3' and IAB-R(BglII): 5'-AGATCTGAGCTCATCCAGTTTATGCTTTAGG-3'. The PCR product was purified and digested with EcoRI and BglII (New England Biolabs, Ipswich, MA), then ligated with pFUSE-hIgG1-Fc2 (Invivogen, San Diego, CA) linearized with EcoRI and BglII. The resultant vector was reamplified for the IAB-hFc (Fc portion of a human antibody IgG1) part using primers IL2-F: 5'-TCGACACGTGTGATCAGATAGGGCCACCATGTACAGGATGCAACTCCTGTCT-3' and hFc-R: 5'- TCTAGAGCGGCCGCGATACTCATTTACCCGGAGACAGGGAGAG-3', The IAB-hFc PCR fragment was then digested with PmI and NotI, and inserted into the vector pVRC8400 37, 38. The final plasmid is named pMH142 (Figure 1A).

Bottom Line: Because of its lower immunogenicity in patients, a fully human protein is the most desirable format for clinical applications.We believe that the methods developed here may apply to the generation of other tumor-targeting immunoadhesins when it is difficult to obtain a human monoclonal antibody to a given antigen for clinical applications.The resultant immunoadhesins can have advantages usually found in monoclonal antibodies such as ease of purification, high binding affinity and effector functions.

View Article: PubMed Central - PubMed

Affiliation: 1. Antibody Therapy Unit, Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA;

ABSTRACT

Background: The mucin MUC16 expresses the repeating peptide epitope CA125 that has been known for decades to be a well-validated cancer marker that is overexpressed on the cell surface of ovarian cancers and other malignant tumors. In spite of recent efforts to make mouse monoclonal antibodies to MUC16 to treat ovarian cancer, a human monoclonal antibody against this mucin has not been described. MUC16 interacts with mesothelin, a protein that mediates heterotypic cancer cell adhesion, indicating that MUC16 and mesothelin play an important role in the peritoneal implantation and metastasis of ovarian tumors. Therefore, a suitable candidate for therapeutic targeting of MUC16 would functionally block the interaction of MUC16 and mesothelin.

Methodology/principal findings: Here we report the generation of a novel immunoadhesin, HN125, against MUC16 that consists of a functional MUC16 binding domain of mesothelin (IAB) and the Fc portion of a human antibody IgG1. The yield for purified HN125 proteins is over 100 µg/mL of HEK-293 culture supernatant. We show that HN125 has high and specific affinity for MUC16-expressing cancer cells by flow cytometry and immunohistochemistry. HN125 has the ability to disrupt the heterotypic cancer cell adhesion mediated by the MUC16-mesothelin interaction. Moreover, it elicits strong antibody-dependent cell mediated cytotoxicity against MUC16-positive cancer cells in vitro.

Conclusion/significance: This report describes a novel human immunotherapeutic agent highly specific for MUC16 with potential for treating ovarian cancer and other MUC16-expressing tumors. Because of its lower immunogenicity in patients, a fully human protein is the most desirable format for clinical applications. We believe that the methods developed here may apply to the generation of other tumor-targeting immunoadhesins when it is difficult to obtain a human monoclonal antibody to a given antigen for clinical applications. The resultant immunoadhesins can have advantages usually found in monoclonal antibodies such as ease of purification, high binding affinity and effector functions.

No MeSH data available.


Related in: MedlinePlus