Mesothelin antibodies and methods for eliciting potent antitumor activity
Inventors
Ho, Mitchell • Pastan, Ira H. • Dimitrov, Dimiter S. • Tang, Zhewei • Feng, Mingqian • Gao, Wei
Assignees
US Department of Health and Human Services
Publication Number
US-9416190-B2
Publication Date
2016-08-16
Expiration Date
2033-09-16
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Abstract
Described herein is the use of phage display antibody engineering technology and synthetic peptide screening to identify SD1 and SD2, human single-domain antibodies to mesothelin. SD1 recognizes a conformational epitope at the C-terminal end (residues 539-588) of human mesothelin close to the cell surface. SD2 binds full-length mesothelin. To investigate SD1 as a potential therapeutic agent, a recombinant human Fc (SD1-hFc) fusion protein was generated. The SD1-hFc protein exhibits strong complement-dependent cytotoxicity (CDC), in addition to antibody-dependent cellular cytotoxicity (ADCC), against mesothelin-expressing tumor cells. Furthermore, the SD1-hFc protein causes significant tumor growth inhibition of tumor xenografts in nude mice. SD1 and SD2 are the first human single-domain antibodies targeting mesothelin-expressing tumors.
Core Innovation
This invention relates to human single-domain monoclonal antibodies specific for mesothelin, particularly SD1 and SD2, identified using phage display antibody engineering technology and synthetic peptide screening. SD1 recognizes a conformational epitope at the C-terminal end of human mesothelin near the cell surface, while SD2 binds full-length mesothelin but not the C-terminal peptide. SD1 was developed into a recombinant human Fc fusion protein (SD1-hFc) which exhibits strong complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and significant tumor growth inhibition in mouse xenograft models.
The problem being addressed is the lack of effective mesothelin-targeting antibodies capable of inducing complement-dependent cytotoxicity against mesothelin-expressing tumors. Existing anti-mesothelin antibodies, including SS1P, MORAb-009, and HN1, show ADCC but do not exhibit CDC, partially because they target an epitope far from the cell membrane, limiting CDC effectiveness. Since CDC is an important mechanism for therapeutic antibodies, its absence hampers the efficacy of mesothelin-targeted therapies.
The invention provides novel human single-domain antibodies that bind mesothelin near the cell surface, enabling CDC and ADCC. SD1-hFc fusion protein induces complement recruitment to cancer cells, effectively triggering CDC in vitro, alongside ADCC activity. The antibody shows high specificity and affinity for mesothelin, binds native mesothelin on tumor cells, and demonstrates potent anti-tumor activity in animal models, suggesting a new direction for antibody-based cancer immunotherapy targeting mesothelin.
Claims Coverage
The patent includes multiple independent claims directed to human single-domain antibodies specific for human mesothelin and methods employing these antibodies. The main inventive features cover the antibody structure, immunoconjugates, fusion proteins, therapeutic and diagnostic methods, and vectors.
Human single-domain antibody with defined CDRs specific for mesothelin
An isolated human variable heavy (VH) single domain monoclonal antibody that specifically binds human mesothelin, comprising specific CDR1, CDR2, and CDR3 sequences as set forth by precise amino acid residues from SEQ ID NO: 2 or SEQ ID NO: 15, enabling specific high-affinity binding.
Immunoconjugates comprising the single-domain antibody and an effector molecule
Immunoconjugates that include the single-domain antibody linked to an effector molecule such as a toxin, specifically Pseudomonas exotoxin or its variants (SEQ ID NOs: 3-8), enabling targeted cytotoxicity against mesothelin-expressing cells.
Fusion proteins combining the single-domain antibody with heterologous proteins
Fusion proteins consisting of the single-domain antibody fused to a heterologous protein, particularly human Fc proteins such as IgGγ1 Fc, to enhance therapeutic properties like stability and effector function.
Therapeutic methods using the single-domain antibody and its derivatives
Methods of treating mesothelin-expressing cancers by administering therapeutically effective amounts of the single-domain antibody, fused to human Fc or a toxin, to inhibit tumor growth or metastasis.
Diagnostic methods for detecting mesothelin expression
Methods of detecting mesothelin in biological samples by contacting the sample with the single-domain antibody and detecting antibody binding, with increased binding compared to control indicating presence of mesothelin, thus detecting cancer.
Nucleic acid and vector constructs encoding the antibody
Isolated nucleic acid molecules encoding the single-domain antibody (e.g., comprising SEQ ID NO:1 or SEQ ID NO:14), operably linked to promoters, expression vectors comprising these nucleic acids, and host cells transformed therewith for antibody expression.
Chimeric antigen receptors (CARs) and bispecific antibodies
Chimeric antigen receptors incorporating the single-domain antibody for engineered T cells targeting mesothelin-expressing tumors, and bispecific antibodies combining the single-domain antibody with specificity to T cell receptor components such as CD3 for immunotherapy.
The independent claims cover human variable heavy single-domain antibodies with defined CDR sequences that specifically bind human mesothelin, immunoconjugates and fusion proteins comprising these antibodies, methods of treating and detecting mesothelin-expressing cancers, as well as nucleic acid constructs and engineered immune effectors comprising these antibodies.
Stated Advantages
SD1-hFc exhibits strong CDC, an important antibody-mediated tumor cell killing mechanism not shown by previous anti-mesothelin antibodies.
The antibody binds a novel epitope close to the cancer cell surface, enabling efficient complement activation and increased therapeutic efficacy.
SD1-hFc also mediates ADCC, combining two potent immune mechanisms against mesothelin-expressing tumors.
The antibody shows high specificity and affinity for human mesothelin, binding native protein on cancer cells and not cross-reacting with mouse mesothelin.
SD1-hFc causes significant tumor growth inhibition in vivo in xenograft mouse models as a single agent.
Documented Applications
Use of antibodies to confirm the diagnosis of mesothelin-expressing cancers including mesothelioma, prostate cancer, lung cancer, stomach cancer, squamous cell carcinoma, pancreatic cancer, cholangiocarcinoma, breast cancer (such as triple negative breast cancer), and ovarian cancer by detecting mesothelin in patient samples.
Method of treating mesothelin-expressing cancers in subjects by administering therapeutically effective amounts of the antibodies, immunoconjugates, or fusion proteins to inhibit tumor growth and metastasis.
Use of the antibodies in diagnostic immunoassays such as ELISA, flow cytometry, immunohistochemistry, and immunoprecipitation to detect mesothelin expression.
Generation of engineered cytotoxic T lymphocytes expressing chimeric antigen receptors (CARs) containing the mesothelin-specific antibody for immunotherapy of mesothelin-positive cancers.
Production of bispecific antibodies combining mesothelin-targeting antibodies with T cell receptor components to target T cells to tumor cells.
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