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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.


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The binding of HN125 to MUC16 on cancer cells. A431/H9 (A), OVCAR3 (B), NCI-H226 (C) and YOU (D) cells were probed with HN125 (solid dark line). Dark gray shading: secondary antibody control. Each cell line and its GeoMean value: A431/H9 (6), OVCAR3 (1229), NCI-H226 (6) and YOU (137). GeoMean of the secondary antibody control was about 6. The binding saturation curve (E) followed by Scatchard analysis (F) showed that the KD of HN125 to OVCAR3 cells was 13 nM.
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Figure 2: The binding of HN125 to MUC16 on cancer cells. A431/H9 (A), OVCAR3 (B), NCI-H226 (C) and YOU (D) cells were probed with HN125 (solid dark line). Dark gray shading: secondary antibody control. Each cell line and its GeoMean value: A431/H9 (6), OVCAR3 (1229), NCI-H226 (6) and YOU (137). GeoMean of the secondary antibody control was about 6. The binding saturation curve (E) followed by Scatchard analysis (F) showed that the KD of HN125 to OVCAR3 cells was 13 nM.

Mentions: The binding of HN125 to membrane-bound MUC16 on cancer cells was examined by flow cytometry. All the cancer cell lines (A431/H9, OVACR3, NCI-H226 and YOU) express mesothelin on the cell surface, while only OVCAR3 and YOU cells express MUC16 30, 36, 39. As shown in Figure 2 (A-D), HN125 specifically bound to the MUC16-positive ovarian cancer cells (OVCAR3) and mesothelioma cells (YOU), but not to the MUC16-negative A431/H9 and NCI-H226 cancer cells, indicating excellent specificity of HN125 for cancer cell-associated MUC16 molecules. We have also tested the binding of HN125 on additional four MUC16-negative cancer cell lines, no signal was found in any of these lines (data not shown). OVCAR3 cells showed a 600-fold increase in MUC16 detection. YOU cells had around ten-fold less MUC16 expression on the cell surface. Interestingly, HN125 showed clear and strong staining on YOU cells, indicating high binding affinity of HN125 for MUC16-positive cancer cells including those with low MUC16 expression. To measure the binding affinity of HN125 on cancer cells, we made the binding saturation curve and Scatchard Plot (Figure 2E and 2F). The Kd of HN125 on OVCAR3 cells was 13 nM.


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)

The binding of HN125 to MUC16 on cancer cells. A431/H9 (A), OVCAR3 (B), NCI-H226 (C) and YOU (D) cells were probed with HN125 (solid dark line). Dark gray shading: secondary antibody control. Each cell line and its GeoMean value: A431/H9 (6), OVCAR3 (1229), NCI-H226 (6) and YOU (137). GeoMean of the secondary antibody control was about 6. The binding saturation curve (E) followed by Scatchard analysis (F) showed that the KD of HN125 to OVCAR3 cells was 13 nM.
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Related In: Results  -  Collection

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Figure 2: The binding of HN125 to MUC16 on cancer cells. A431/H9 (A), OVCAR3 (B), NCI-H226 (C) and YOU (D) cells were probed with HN125 (solid dark line). Dark gray shading: secondary antibody control. Each cell line and its GeoMean value: A431/H9 (6), OVCAR3 (1229), NCI-H226 (6) and YOU (137). GeoMean of the secondary antibody control was about 6. The binding saturation curve (E) followed by Scatchard analysis (F) showed that the KD of HN125 to OVCAR3 cells was 13 nM.
Mentions: The binding of HN125 to membrane-bound MUC16 on cancer cells was examined by flow cytometry. All the cancer cell lines (A431/H9, OVACR3, NCI-H226 and YOU) express mesothelin on the cell surface, while only OVCAR3 and YOU cells express MUC16 30, 36, 39. As shown in Figure 2 (A-D), HN125 specifically bound to the MUC16-positive ovarian cancer cells (OVCAR3) and mesothelioma cells (YOU), but not to the MUC16-negative A431/H9 and NCI-H226 cancer cells, indicating excellent specificity of HN125 for cancer cell-associated MUC16 molecules. We have also tested the binding of HN125 on additional four MUC16-negative cancer cell lines, no signal was found in any of these lines (data not shown). OVCAR3 cells showed a 600-fold increase in MUC16 detection. YOU cells had around ten-fold less MUC16 expression on the cell surface. Interestingly, HN125 showed clear and strong staining on YOU cells, indicating high binding affinity of HN125 for MUC16-positive cancer cells including those with low MUC16 expression. To measure the binding affinity of HN125 on cancer cells, we made the binding saturation curve and Scatchard Plot (Figure 2E and 2F). The Kd of HN125 on OVCAR3 cells was 13 nM.

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