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Mesothelin antibodies and methods of eliciting potent antitumor activity

A mesothelin and antibody technology, applied in the direction of anti-tumor drugs, chemical instruments and methods, antibodies, etc., can solve problems such as obstacles and lack of targeted mesothelin therapy

Active Publication Date: 2018-11-16
UNITED STATES OF AMERICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although many known mesothelin monoclonal antibodies are available, none exhibit complement-dependent cytotoxicity (CDC) against tumor cells
Therefore, current mesothelin-targeted therapies are hampered by the lack of anti-mesothelin monoclonal antibodies with potent CDC

Method used

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  • Mesothelin antibodies and methods of eliciting potent antitumor activity
  • Mesothelin antibodies and methods of eliciting potent antitumor activity
  • Mesothelin antibodies and methods of eliciting potent antitumor activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0270] Example 1: Materials and methods

[0271] This example describes the experimental procedures used in the study described in Example 2.

[0272] Cell culture

[0273] As described in the literature (Ho et al., Int J Cancer 128: 2020-2030, 2011; Yu et al., J Cancer 1: 141-149, 2010), the human cholangiocarcinoma (CCA) cell line (KMBC, Mz-ChA- 1 and HuCCT-1), as well as A431 (epidermal cancer), NCI-H226 (mesothelioma), EKVX (human non-small cell lung cancer or NSCLC), OVCAR-8 (ovarian cancer), and L55 (NSCLC) cell growth. A431 / H9 is a transfected A431 cell line that stably expresses human mesothelin (Ho et al., Clin Cancer Res 11: 3814-3820, 2005). Make HEK-293F cell line (Invitrogen, Carlsbad, CA) in FreeStyle TM Grow in serum-free medium (Invitrogen). All cell lines can only be passaged a few times (less than 1 month) after being thawed in the first freezing to reduce the total number of passages to less than 15. The first freezing occurs immediately after the cell line is ...

Embodiment 2

[0293] Example 2: Human single domain antibodies cause effective anti-tumor activity by targeting epitopes in mesothelin near the surface of cancer cells

[0294] This example describes the identification and characterization of human single-domain antibodies specific for the C-terminal epitope of human mesothelin.

[0295] Discovery and preparation of SD1 human antibody

[0296] In order to find new anti-mesothelin monoclonal antibodies that target sites close to the cell surface, the C-terminal mesothelin peptide is designed to screen small-size conjugate (VH) libraries. The human mesothelin (MSLN) gene encodes a precursor protein containing 622 amino acids (GenBank accession number: AY743922). When transported into the endoplasmic reticulum, the N-terminal signal peptide (residues: 1-33) and the C-terminal GPI anchor addition signal peptide (predicted cleavage site: Ser598) are removed, and then Those are replaced by GPI anchors. Using big-PI prediction software, the GPI cleava...

Embodiment 3

[0324] Example 3: Mesothelin-specific monoclonal antibody is used to detect cancer of a subject or determine the cancer diagnosis of a subject

[0325] This example describes the use of a mesothelin-specific monoclonal antibody (such as SD1 or SD2, or a monoclonal antibody including the CDR sequence of SD1 or SD2) such as the monoclonal antibody disclosed in the present invention to detect cancer in a subject . This example further introduces the use of these antibodies to determine the cancer diagnosis of the subject.

[0326] Blood samples come from cancers diagnosed or suspected of having mesothelin-positive cancers (ie, cancers that overexpress mesothelin, such as mesothelioma, prostate cancer, lung cancer, gastric cancer, squamous cell carcinoma, pancreatic cancer, cholangiocarcinoma, triple negative Breast cancer or ovarian cancer). A blood sample obtained from a patient who has never had cancer can be used as a control. An ELISA test is performed to detect the presence of...

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Abstract

The present invention describes the use of phage display antibody engineering and synthetic peptide screening to identify human single domain antibodies SD1 and SD2 against mesothelin. SD1 recognizes a conformational epitope near the C‑terminus (residues 539‑588) of human mesothelin near the cell surface. SD2 binds full-length mesothelin. To study SD1 as a potential therapeutic agent, a recombinant human Fc (SD1‑hFc) fusion protein was generated. SD1-hFc protein exhibits strong complement-dependent cytotoxicity (CDC) in addition to antibody-dependent cytotoxicity (ADCC) against mesothelin-expressing tumor cells. Furthermore, SD1‑hFc protein had a significant tumor growth inhibitory effect on xenograft tumors in nude mice. SD1 and SD2 are the first human single domain antibodies targeting mesothelin-expressing tumors.

Description

[0001] Cross-reference related applications [0002] This application claims the benefits of U.S. Provisional Application No. 61 / 706,396 filed on September 27, 2012, and the content of the application is hereby incorporated in its entirety. Technical field [0003] The present disclosure relates to monoclonal antibodies, such as single-domain monoclonal antibodies specific for mesothelin. The present disclosure further relates to the use of the antibody, such as for the diagnosis and treatment of cancer. Background technique [0004] Because mesothelin is highly expressed in malignant mesothelioma (Chang et al., Cancer Res 52:181-186, 1992; Chang and Pastan, proc Natl Acad Sci USA 93:136-140, 1996) and in other solid tumors, such as Gastric cancer, squamous cell carcinoma, prostate cancer, pancreatic cancer, lung cancer, cholangiocarcinoma, breast cancer and ovarian cancer are highly expressed, so mesothelin is considered a therapeutic target. (Hassan et al., Clin. Cancer Res. 10:...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/30A61K39/395A61P35/00
CPCA61P35/00A61P35/04C07K16/30C07K2317/21C07K2317/30C07K2317/52C07K2317/569C07K2317/732C07K2317/734C07K2319/00A61K2039/505C07K2317/56C07K2317/73G01N33/57492G01N2333/705
Inventor 何苗壮I·H·帕斯坦D·S·迪米特洛夫唐喆伟丰明乾高威
Owner UNITED STATES OF AMERICA