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-9409992-B2
Publication Date
2016-08-09
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 employs rabbit hybridoma technology and a collection of truncated mesothelin domain fragments to identify anti-mesothelin monoclonal antibodies (mAbs) that bind to specific regions of mesothelin. Notably, the identified rabbit mAbs bind epitopes outside of the commonly targeted Region I, specifically to Region II (residues 391-486), Region III (residues 487-581), or depend on the native conformation of mesothelin, with subnanomolar affinity. These antibodies do not compete for binding with existing mesothelin-specific immunotoxins such as SS1P or antibodies such as MORAb-009, both of which recognize Region I. Additionally, the disclosure includes a high-affinity rabbit mAb (YP158) that binds Region I with high affinity and specificity to native mesothelin on cancer cells and tissues.
The problem addressed relates to limitations of current anti-mesothelin therapeutic antibodies that target Region I. Region I-binding antibodies, including MORAb-009 and SS1P, recognize the highly immunogenic N-terminal domain involved in binding mucin MUC16/CA125. In patients, autoantibodies or soluble MUC16/CA125 can sequester these antibodies, limiting therapeutic efficacy. Furthermore, diagnostic detection of soluble mesothelin using approved kits is impaired due to complex formation with MORAb-009 which overlaps the diagnostic antibody's epitope. Thus, there is a need for mAbs targeting mesothelin regions other than Region I to improve therapeutic and diagnostic capabilities.
The disclosure further provides that immunotoxins comprising rabbit single chain variable fragments (scFv) of the identified domain-specific mAbs fused to Pseudomonas exotoxin (PE38) exhibit potent and selective cytotoxicity against mesothelin-expressing cancer cells. In particular, YP218scFv-PE38 demonstrated superior binding affinity and cytotoxic activity compared to SS1P in primary mesothelioma cell lines and an in vivo mouse model, inhibiting tumor relapse more effectively. Collectively, these findings extend the therapeutic and diagnostic targeting of mesothelin beyond Region I to offer alternative, potentially more efficacious options.
Claims Coverage
The patent claims encompass one independent claim directed to isolated monoclonal antibodies that bind mesothelin, defined by specific complementarity determining regions (CDRs) in the variable heavy (VH) and light (VL) domains corresponding to identified sequences. The main inventive features relate to unique antibody compositions and their functional characteristics, as well as related immunoconjugates, nucleic acids, and methods of use.
Monoclonal antibodies defined by specific CDR sequences
An isolated monoclonal antibody that binds mesothelin, wherein the VH and VL domains comprise specific CDR1, CDR2 and CDR3 sequences as set forth by amino acid residues from any of SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27, defining five different permissible combinations of VH and VL CDR sets.
Monoclonal antibodies with specified sequence identity to defined VH and VL domains
Monoclonal antibodies wherein the VH and VL domains have at least 90% or 95% amino acid sequence identity to any of SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, 23, 25, or 27, further characterizing and allowing variations of the antibodies within these identity thresholds.
Monoclonal antibodies comprising specific VH and VL domain sequences
Monoclonal antibodies comprising the full VH and VL domain amino acid sequences as set forth in any of SEQ ID NOs: 9 and 11, 13 and 15, 17 and 19, 21 or 29 and 23, or 25 and 27.
Forms of the monoclonal antibody
The monoclonal antibody may be in the form of Fab, Fab′, F(ab)′2, single chain variable fragment (scFv), or disulfide stabilized variable fragment (dsFv).
Isotypes and modifications of the antibody
The antibody can be an IgG and may be chimeric, synthetic, or humanized, and optionally labeled with a fluorescent, enzymatic, or radioactive label.
Immunoconjugates comprising the monoclonal antibody and effector molecules
Isolated immunoconjugates comprising the monoclonal antibody linked to an effector molecule, such as a toxin, in particular Pseudomonas exotoxin or its variants including those with amino acid sequences of SEQ ID NOs: 1-6, exemplified by amino acid sequences set forth in SEQ ID NOs: 31, 33, 35, 37, or 39.
Therapeutic compositions containing the antibody
Compositions comprising a therapeutically effective amount of the monoclonal antibody in a pharmaceutically acceptable carrier.
Methods of detecting mesothelin using the monoclonal antibody
A method of detecting mesothelin in a sample by contacting the sample with the monoclonal antibody, detecting binding, and determining mesothelin presence based on increased binding relative to a control sample, including application to tumor biopsies from various cancers.
Nucleotide molecules encoding the antibodies and immunoconjugates
Isolated nucleic acid molecules encoding the monoclonal antibodies or immunoconjugates, comprising specified VH and VL domain nucleotide sequences operably linked to promoters, vectors, and host cells transformed therewith.
Use of the antibodies in chimeric antigen receptors and bispecific antibodies
Chimeric antigen receptors (CARs) incorporating the monoclonal antibody or scFv fragments thereof, and bispecific antibodies incorporating the monoclonal antibody, for therapeutic use against mesothelin-expressing cancers.
The claims cover isolated monoclonal antibodies with defined VH and VL CDRs and sequences that specifically bind mesothelin, including various antibody fragments, labeling, immunoconjugates with toxins such as Pseudomonas exotoxin, nucleic acid constructs, expression systems, compositions, diagnostic and therapeutic methods, as well as engineered CARs and bispecific antibodies incorporating these monoclonal antibodies.
Stated Advantages
The antibodies bind mesothelin with high affinity and specificity, including epitopes outside Region I, thereby overcoming limitations of existing Region I-targeting antibodies.
The antibodies and immunotoxins demonstrate potent cytotoxicity against mesothelin-expressing cancer cells, with certain immunotoxins showing greater efficacy than known agents such as SS1P.
The antibodies enable sensitive and specific detection of soluble mesothelin even in the presence of competing therapeutic antibodies, improving diagnostic assays.
Alternative epitopes enable improved diagnostic and therapeutic targeting, potentially reducing antibody sequestration and enhancing cancer treatment efficacy.
Documented Applications
Diagnostic detection and confirmation of mesothelin-positive cancers by detecting antibody binding to biological samples, including tumor biopsies and blood samples.
Therapeutic treatment of cancers expressing mesothelin, including mesothelioma, prostate cancer, lung cancer, stomach cancer, squamous cell carcinoma, pancreatic cancer, cholangiocarcinoma, breast cancer and ovarian cancer, by administering therapeutically effective amounts of the antibodies or immunoconjugates.
Use of the antibodies to generate recombinant immunotoxins targeting mesothelin-expressing tumors.
Engineering of cytotoxic T lymphocytes expressing chimeric antigen receptors incorporating the antibodies for adoptive immunotherapy against mesothelin-expressing tumors.
Creation of bispecific antibodies combining the mesothelin-specific antibodies with antibodies targeting T cell receptors, for cancer immunotherapy.
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