Mesothelin-targeted chimeric antigen receptors and uses thereof
Inventors
Adusumilli, Prasad S. • Sadelain, Michel • Dimitrov, Dimiter S. • Feng, Yang
Assignees
Memorial Sloan Kettering Cancer Center • US Department of Health and Human Services
Publication Number
US-10633441-B2
Publication Date
2020-04-28
Expiration Date
2035-06-05
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Abstract
The presently disclosed subject matter provides for methods and compositions for enhancing the immune response toward cancers and pathogens. It relates to chimeric antigen receptors (CARs) that specifically target human mesothelin, and immunoresponsive cells comprising such CARs. The presently disclosed mesothelin-targeted CARs have enhanced immune-activating properties, including anti-tumor activity.
Core Innovation
The presently disclosed subject matter provides mesothelin-targeted chimeric antigen receptors (CARs) comprising an extracellular antigen-binding domain specifically binding human mesothelin with an affinity from about 1 nM to about 25 nM, a transmembrane domain, and an intracellular domain including CD3ζ and optionally co-stimulatory domains such as CD28 or 4-1BB. Immunoresponsive cells expressing these CARs, including T cells and other lymphoid lineage cells, are designed to enhance immune responses against cancers and pathogens, particularly solid tumors with high mesothelin expression.
The problem being solved arises from the limitations of current CAR T cell therapies in treating solid tumors. These include difficulties in T cell trafficking and infiltration due to restrictive anatomical compartments, lack of costimulatory ligands, tumor immunosuppressive microenvironments, and selection of optimal immune targets that allow potent tumor eradication with minimal toxicity. The invention addresses needs for CARs capable of inducing effective tumor eradication with enhanced immune activation, minimal toxicity, and reduced immunogenicity, as well as methods of administration and combinations with immunomodulatory agents to overcome the hostile tumor microenvironment.
Claims Coverage
The patent discloses 40 inventive features primarily related to chimeric antigen receptors (CARs) targeting human mesothelin, their structural components, their expression in immunoresponsive cells, and their therapeutic applications.
Mesothelin-specific single-chain variable fragment (scFv)
A CAR comprising an extracellular antigen-binding domain with an scFv that specifically binds human mesothelin, comprising a heavy chain variable region at least 95% identical to amino acids 1-119 of SEQ ID NO:1 with invariable CDRs SEQ ID NOS:11-13, and a light chain variable region at least 95% identical to amino acids 1-107 of SEQ ID NO:3 with invariable CDRs SEQ ID NOS:14-16.
Human-derived scFv and fusion proteins
The scFv is a human scFv and can be part of a fusion protein with a heterologous sequence forming the extracellular antigen-binding domain.
Selective mesothelin binding threshold
The extracellular antigen-binding domain recognizes human mesothelin at expression levels of about 1,000 or more binding sites per cell.
Specific heavy and light chain variable regions
The heavy chain variable region comprises amino acids 1-119 of SEQ ID NO:1; the light chain variable region comprises amino acids 1-107 of either SEQ ID NO:3 or SEQ ID NO:5; including combinations of these sequences.
CDR sequences defining specificity
The scFv comprises specific heavy chain CDRs (SEQ ID NOS:11-13) and light chain CDRs (SEQ ID NOS:14-16) defining antigen specificity.
Linker and leader components
The scFv includes a linker connecting heavy and light chains and a leader sequence covalently joined to the N-terminus, wherein the leader comprises a CD8 polypeptide.
Transmembrane domain composition
The transmembrane domain comprises one or more polypeptides selected from CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, BTLA, or combinations thereof, with specific embodiments of CD8 or CD28 polypeptides.
Intracellular domain composition
The intracellular domain comprises a CD3ζ polypeptide and may further include co-stimulatory signaling regions comprising CD28, 4-1BB, OX40, ICOS, or combinations thereof.
Specific CAR embodiments
Exemplary CARs include Mz (transmembrane CD8, intracellular CD3ζ), M28z (transmembrane CD28, intracellular CD3ζ and CD28 co-stimulatory domain), and MBBz (transmembrane CD8, intracellular CD3ζ and 4-1BB co-stimulatory domain).
Vector expression
CARs are recombinantly expressed, preferably from γ-retroviral vectors.
Immunoresponsive cells expressing CARs
Isolated immunoresponsive cells, such as T cells, NK cells, CTLs, regulatory T cells, or pluripotent stem cell derivatives, comprising the described CARs, expressing about 1 to 4 vector copy numbers per cell.
Exogenous co-stimulatory ligand and cytokine co-expression
Immunoresponsive cells may co-express exogenous co-stimulatory ligands (e.g., 4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14) and/or cytokines (e.g., IL-2, IL-3, IL-6, IL-7, IL-11, IL-12, IL-15, IL-17, IL-21), in particular 4-1BBL or IL-12.
Co-expression of a second antigen receptor
Immunoresponsive cells may co-express a second antigen recognizing receptor binding to a tumor or pathogen antigen different from human mesothelin.
Methods of immunoresponsive cell production
Methods for producing immunoresponsive cells transduced with a nucleic acid sequence encoding the described CAR including the specific scFv and CAR structural domains.
Nucleic acids and vectors encoding CARs
Nucleic acids encoding the mesothelin-targeted CARs and vectors comprising these nucleic acids, with particular reference to γ-retroviral vectors.
Pharmaceutical compositions comprising CAR cells
Pharmaceutical compositions comprising an effective amount of the CAR expressing immunoresponsive cells with pharmaceutically acceptable excipients.
Kits for treatment or prevention
Kits comprising immunoresponsive cells expressing the mesothelin-specific CAR, nucleic acids encoding the CAR, and instructions for use in treating or preventing neoplasia, pathogen infection, autoimmune or inflammatory diseases, allogeneic transplant rejection, or graft rejection.
The claims comprehensively cover mesothelin-specific CARs with defined human scFv sequences and structural features including extracellular antigen-binding, transmembrane, and intracellular costimulatory domains. They include immunoresponsive cells expressing such CARs, methods for generating these cells, therapeutic compositions, and kits for treatment of various diseases, emphasizing their structure, function, and clinical utility.
Stated Advantages
Enhanced immune-activating properties, including potent anti-tumor activity with minimal toxicity and reduced immunogenicity.
Ability of second generation CARs to overcome tumor-mediated immunosuppressive microenvironment via costimulatory signaling, leading to sustained T cell proliferation and survival.
Regional (intrapleural) administration of CAR T cells requires lower cell doses for tumor eradication, enhances T-cell accumulation and activation in tumor sites, and promotes long-term systemic tumor immunity.
CD28 co-stimulation promotes superior cytokine secretion and proliferation especially in CD4+ T cell subset, providing multifunctional cytotoxic and helper functions.
4-1BB co-stimulation confers enhanced functional persistence and resistance to tumor-mediated inhibition compared to CD28, improving therapeutic efficacy at low T-cell doses.
Documented Applications
Treatment or prevention of cancers, including solid tumors such as malignant pleural mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer (including triple-negative breast cancer), colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial carcinoma, stomach cancer, and cholangiocarcinoma.
Use in immunotherapy to reduce tumor burden, diminish tumor size, induce tumor eradication, increase or lengthen survival of subjects having neoplasia, and increase immune-activating cytokine production.
Treatment or prevention of pathogen infections including viral infections such as cytomegalovirus (CMV), Epstein Barr Virus (EBV), Human Immunodeficiency Virus (HIV), and influenza virus infection, particularly in immunocompromised subjects.
Prevention or treatment of inflammatory diseases, e.g., pancreatitis.
Prevention of graft rejection in organ transplant recipients, including pancreas transplant recipient.
Use of regulatory T cells expressing mesothelin-specific CARs as immunoinhibitory cells to treat inflammatory diseases or prevent graft rejection.
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