Mesothelin domain-specific monoclonal antibodies and use thereof
Inventors
Ho, Mitchell • Pastan, Ira H. • Phung, Yen T. • Zhang, Yifan • Gao, Wei • Hassan, Raffit
Assignees
US Department of Health and Human Services
Publication Number
US-9803022-B2
Publication Date
2017-10-31
Expiration Date
2033-08-16
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Abstract
Described herein is the use of rabbit hybridoma technology, along with a panel of truncated mesothelin domain fragments, to identify anti-mesothelin mAbs that bind specific regions of mesothelin. In one aspect of the present disclosure, the rabbit mAbs bind an epitope that is not part of Region I. In particular, the identified mAbs (YP187, YP223, YP218 and YP3) bind either Region II (391-486), Region III (487-581) or a native conformation of mesothelin with subnanomolar affinity. These antibodies do not compete for binding with the mesothelin-specific immunotoxin SS1P or mesothelin-specific antibody MORAb-009. In another aspect, disclosed is a high-affinity rabbit mAb that binds Region I of mesothelin (YP158). YP158 binds native mesothelin protein in cancer cells and tissues with high affinity and specificity.
Core Innovation
The invention disclosed concerns monoclonal antibodies specific for mesothelin, including antibodies raised against fragments of mesothelin. The antibodies disclosed bind with high affinity to full-length mesothelin and/or mesothelin fragments. The disclosure includes immunoglobulin molecules such as IgG antibodies and antibody fragments, compositions comprising these antibodies, nucleic acid molecules encoding them, expression vectors, host cells expressing the nucleic acids, and immunoconjugates comprising the antibodies linked to effector molecules like toxins.
The background identifies human mesothelin as a 40 kDa cell-surface GPI-linked glycoprotein expressed at low levels in normal mesothelial cells but highly expressed in various cancers such as mesotheliomas, stomach cancer, squamous cell carcinomas, prostate cancer, pancreatic cancer, lung cancer, cholangiocarcinoma, breast cancer, and ovarian cancer. The limited expression of mesothelin on normal cells makes it a viable target for tumor immunotherapy. Existing anti-mesothelin antibodies primarily bind Region I of the protein, but this region also binds mucin MUC16/CA125, sequestering the antibodies and limiting efficacy. In addition, soluble mesothelin detection in patients treated with certain antibodies is problematic due to epitope overlap.
The disclosed antibodies include newly identified rabbit monoclonal antibodies that bind to mesothelin epitopes outside Region I, including Region II and Region III, or recognize native conformations, with subnanomolar affinity. These antibodies, including YP187, YP223, YP218 and YP3, do not compete with existing therapeutic antibodies such as SS1P or MORAb-009 for binding to mesothelin. Another high-affinity rabbit monoclonal antibody YP158 binds Region I with high affinity and specificity. The antibodies and immunoconjugates can be used for diagnosis and treatment of a variety of cancers expressing mesothelin by detecting mesothelin levels or inhibiting tumor growth and metastasis.
Claims Coverage
The patent includes multiple independent claims directed to isolated monoclonal antibodies specific for mesothelin, their immunoconjugates, nucleic acid molecules encoding such antibodies, methods of detection and treatment, and engineered constructs such as chimeric antigen receptors and bispecific antibodies. The claims specify distinctive complementarity determining regions (CDRs) and combinations of VH and VL domain sequences.
Isolated monoclonal antibodies with specified CDR sequences binding mesothelin
Antibodies comprising variable heavy (VH) and variable light (VL) domains that include complementarity determining regions derived from specified SEQ ID NOs: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27, particularly those from groups comprising YP223, YP218, YP3, YP187 clones 1 and 2, and YP158 clones with high specificity to mesothelin epitopes.
Monoclonal antibodies as fragments or IgG isotypes
Antibodies in various forms including Fab, Fab′, F(ab)′2 fragments, single chain variable fragments (scFv), disulfide stabilized variable fragments (dsFv), or full IgG molecules, demonstrating versatility in antibody formats.
Monoclonal antibodies that are chimeric, humanized, synthetic, or labeled
Antibodies that may be engineered in chimeric or humanized forms, synthesized artificially, or conjugated with detectable labels such as fluorescent, enzymatic, or radioactive moieties.
Isolated immunoconjugates comprising the antibodies and effector molecules
Immunoconjugates formed by covalent linkage of the mesothelin-specific antibodies to effector molecules including cytotoxic agents such as Pseudomonas exotoxin or its variants (e.g., PE38), shown in specific amino acid sequences.
Nucleic acid molecules and expression vectors encoding the antibodies or immunoconjugates
Nucleotide sequences encoding the VH and VL domains of the antibodies, or their fusion to toxin sequences, operably linked to promoters, contained within vectors and expressed in host cells.
Methods of detecting mesothelin in samples using the antibodies
Diagnostic methods involving contacting samples suspected of containing mesothelin with the disclosed monoclonal antibodies and detecting antibody binding, thereby identifying mesothelin-positive cancers.
Use of antibodies in chimeric antigen receptors (CARs) and bispecific antibodies
Engineered immune effector constructs comprising mesothelin-specific antibody fragments allowing targeted immunotherapeutic applications, including CAR T cells and bispecific antibodies targeting both mesothelin and T cell receptor components.
The claims cover a panel of monoclonal antibodies and immunoconjugates with specific VH and VL domain CDRs targeting distinct mesothelin epitopes, including formats suitable for therapy and diagnosis, nucleic acid sequences encoding the antibodies, diagnostic methods using these antibodies, and engineered immunotherapeutic constructs like CARs and bispecific antibodies. The coverage emphasizes high-affinity binding outside of prior art epitopes and functional applications.
Stated Advantages
The antibodies bind mesothelin with high affinity, including subnanomolar affinity to specific subdomains and conformations not targeted by existing antibodies.
The antibodies do not compete with existing mesothelin-targeting therapeutics such as SS1P or MORAb-009, allowing detection of soluble mesothelin despite presence of these drugs.
Immunotoxins constructed with these antibodies are more potent in cytotoxicity assays against mesothelin-expressing cancer cells and patient-derived primary cells, showing improved efficacy over existing immunotoxins like SS1P.
Antibodies enable highly sensitive sandwich ELISA detection of soluble mesothelin, even in the presence of therapeutics that interfere with current detection methods.
The antibodies can be used for both diagnostic detection of mesothelin expression and therapeutic inhibition of tumor growth and metastasis.
Documented Applications
Diagnosis and confirmation of mesothelin-expressing cancers by detecting mesothelin in patient tissue or blood samples using the monoclonal antibodies in immunoassays, including ELISA and immunohistochemistry.
Therapeutic treatment of cancers expressing mesothelin, such as mesothelioma, prostate cancer, lung cancer, stomach cancer, squamous cell carcinoma, pancreatic cancer, cholangiocarcinoma, breast cancer, or ovarian cancer, by administration of the monoclonal antibodies or immunoconjugates.
Use of immunotoxins comprising the antibodies fused to Pseudomonas exotoxin for targeted killing of mesothelin-expressing tumor cells in vitro and in animal models.
Engineering of cytotoxic T lymphocytes (CTLs) expressing chimeric antigen receptors (CARs) incorporating the mesothelin-specific antibody fragments for adoptive cell immunotherapy of mesothelin-positive cancers.
Generation of bispecific antibodies combining the mesothelin-specific antibodies with T cell receptor components (e.g., CD3) to engage immune effector cells in tumor targeting.
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