Recombinant immunotoxin targeting mesothelin
Inventors
Pastan, Ira H. • Weldon, John • Beers, Richard
Assignees
US Department of Health and Human Services
Publication Number
US-10683362-B2
Publication Date
2020-06-16
Expiration Date
2032-05-04
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Abstract
Mesothelin is a differentiation antigen present on the surface of ovarian cancers, mesotheliomas and several other types of human cancers. Because among normal tissues, mesothelin is only present on mesothelial cells, it represents a good target for antibody mediated delivery of cytotoxic agents. The present invention is directed to improved recombinant immunotoxins comprising anti-mesothelin antibodies, including Fv molecules with particularly high affinity for mesothelin, and a Pseudomonas Exotoxin moiety which has been modified to reduce its immunogenicity and protease sensitivity and providing a better cytotoxicity for cells which express mesothelin. The RITs are well-suited for the treatment of cancers of the ovary, stomach, squamous cells, mesotheliomas and other malignant cells expressing mesothelin.
Core Innovation
The invention relates to recombinant immunotoxins (RITs) that target mesothelin, a differentiation antigen found on the surface of ovarian cancers, mesotheliomas, and other human cancers, while being limited in normal tissues to mesothelial cells, thus representing an effective target for antibody-mediated delivery of cytotoxic agents.
The invention addresses the limitations of prior RITs based on Pseudomonas exotoxin A (PE), specifically problems including poor solid tumor penetration, high immunogenicity, nonspecific toxicities, and inefficient intracellular processing, particularly poor furin cleavage in truncated variants such as SS1-LR, which reduce cytotoxic efficacy against mesothelin-expressing cells.
The core innovation is the generation of improved PE variants and chimeric molecules comprising an anti-mesothelin antibody or fragment fused via a furin cleavage site and a short flexible peptide linker composed of glycine and serine residues to a modified PE functional domain III containing specific amino acid substitutions that reduce immunogenicity and improve cytotoxicity. This configuration enhances cytotoxicity, including on patient-derived mesothelioma cells, without substantially altering furin cleavage efficiency, decreases nonspecific toxicity such as capillary leak syndrome, and allows administration at higher doses.
Claims Coverage
The patent claims focus on chimeric molecules comprising an anti-mesothelin antibody fused to a modified Pseudomonas exotoxin A fragment via specific linkers and cleavage sites, nucleic acids encoding these molecules, pharmaceutical compositions containing them, and methods for treating mesothelin-expressing cancers.
Construction of a chimeric molecule with improved cytotoxicity
A fusion polypeptide comprising an anti-mesothelin antibody or fragment (M) linked via a short linker (L1, 1-10 glycine/serine residues) to a furin cleavage site (FCS) selected from specified sequences including SEQ ID NOs: 17, 29, truncated versions, and others, followed by a flexible linker (FL) of 3-8 glycine/serine residues, and a functional PE domain III (residues 395-613 of SEQ ID NO:1) with optional substitutions at specified positions that reduce immunogenicity and enhance cytotoxicity, wherein this arrangement increases cytotoxicity relative to molecules with cysteine residues flanking the FCS.
Specific antibody formats and structure
The antibody or fragment can be a minibody, diabody, triabody, Fab′, F(ab)′2, single chain Fv (scFv), or disulfide stabilized Fv (dsFv) fragment, and may comprise disulfide stabilized light and heavy chain variable regions fused through the heavy chain to L1.
Defined CDR sequences in variable regions
The antibody includes VH and VL chains with specific CDR amino acid sequences as detailed in SEQ ID NOs: 51-53 for VH and SEQ ID NOs: 54-67 for VL CDRs, with several VL CDR3 variants enumerated.
Linker and domain substitutions preserving function
The L1 linker length is 3 to 5 amino acids; PE domain III includes substitutions (alanine, glycine or serine) at residues corresponding to specified positions for reduction of immunogenic epitopes; flexible linker FL includes specific glycine and serine concatamers such as GGS and variants.
Pharmaceutical formulations and genetic constructs
Pharmaceutical compositions containing the chimeric molecule, nucleic acids encoding the molecule, vectors comprising such nucleic acids, and host cells transformed therewith are also claimed.
Therapeutic methods
Methods of treating mesothelin-overexpressing cancers such as lung adenocarcinoma, ovarian carcinoma, mesothelioma, and epidermoid carcinoma by administration of the chimeric molecule are claimed.
The claims cover chimeric molecules combining anti-mesothelin antibodies with improved PE domain III via specified linkers and furin cleavage sites that enhance cytotoxicity against mesothelin-expressing cells, reduce immunogenicity and nonspecific toxicity, as well as related nucleic acids, compositions, and therapeutic methods for treating relevant cancers.
Stated Advantages
Reduced immunogenicity due to elimination of B cell epitopes in PE domain III.
Improved cytotoxicity on mesothelin-expressing cells including patient-derived cancer cells compared to previous RIT variants.
Decreased nonspecific toxicity such as capillary leak syndrome demonstrated in rodent models.
Capability of administration at higher doses than prior RITs due to lower toxicity.
Documented Applications
Treatment of cancers expressing or overexpressing mesothelin, including ovarian cancer, mesothelioma, non-small cell lung cancer, lung adenocarcinoma, fallopian tube cancer, head and neck cancer, cervical cancer, and pancreatic cancer.
In vitro or ex vivo elimination or purging of mesothelin-expressing malignant cells from biological samples.
Use in diagnostic and therapeutic methods involving targeting mesothelin-positive cells to inhibit growth, induce apoptosis, or monitor tumor burden in cancer patients.
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