WO2021061648A1 - Méthodes et compositions pour la stimulation de réponses de lymphocytes t endogènes - Google Patents

Méthodes et compositions pour la stimulation de réponses de lymphocytes t endogènes Download PDF

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WO2021061648A1
WO2021061648A1 PCT/US2020/052005 US2020052005W WO2021061648A1 WO 2021061648 A1 WO2021061648 A1 WO 2021061648A1 US 2020052005 W US2020052005 W US 2020052005W WO 2021061648 A1 WO2021061648 A1 WO 2021061648A1
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cell
antigen
binding
cells
amino acids
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PCT/US2020/052005
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Darrell J. Irvine
Eric Dane
Leyuan MA
Angela ZHANG
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Massachusetts Institute Of Technology
President And Fellows Of Harvard College
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0006—Modification of the membrane of cells, e.g. cell decoration
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6006—Cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6018—Lipids, e.g. in lipopeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • ACT T cells One way to enhance the function of ACT T cells is via genetic engineering of the cells themselves, for example by introducing chimeric receptors or costimulatory molecules (see e.g., Stephan et al. (2007) Nat Med 13(12): 1440; Morgan et al. (2006) Science 314(5796): 126; Gade et al. (2005) Cancer Res 65(19): 9080). Chimeric antigen receptor (CAR) T cell therapy has emerged as a promising strategy for the treatment of cancer.
  • CAR Chimeric antigen receptor
  • Chimeric antigen receptors are genetically- engineered, artificial transmembrane receptors that confer a defined specificity for an antigen (e.g., ligand) onto an immune effector cell (e.g., a T cell, natural killer cell or other immune cell), which results in activation of the effector cell upon recognition and binding to the antigen.
  • an antigen e.g., ligand
  • an immune effector cell e.g., a T cell, natural killer cell or other immune cell
  • these chimeric receptors are used to impart the antigen specificity of a monoclonal antibody onto a T cell, referred to in the art as CAR T cells.
  • Expression of the engineered chimeric antigen receptor on the surface of CAR T cells confers on the T cells the ability to lyse any target cell with surface expression of the particular antigen recognized by the chimeric receptor.
  • CAR T cell therapy has shown encouraging results for the treatment of hematological malignancies, its application to solid tumors has thus far been limited. Its limitations in the treatment of solid tumors are believed to be several-fold: (1) lack of tumor-specific antigens, (2) poor CAR T cell expansion and persistence, (3) limited CAR T cell trafficking to solid tumors, and (4) inhibition of CAR T cell activity by the immunosuppressive tumor microenvironment. Accordingly, approaches for improving adoptive cell therapy, such as CAR T cell therapy, in the treatment of diseases such as cancer are still needed.
  • This disclosure provides methods and compositions for improving adoptive T cell therapy, such as CAR T cell therapy, through stimulating an endogenous T cell response in the microenvironment in which the adoptively transferred T cells are located as a means to thereby amplify target cell lysis in this microenvironment (e.g., solid tumor cell lysis).
  • An endogenous T cell response is stimulated via delivery of MHC-binding antigens (e.g., MHC class I-binding peptides) to the microenvironment.
  • MHC-binding antigens e.g., MHC class I-binding peptides
  • MHC- binding antigens are delivered to the microenvironment by tethering to the surface of the adoptively transferred T cells (referred to herein as exogenous T cells) in an inactive form (e.g., no or limited capability for MHC-binding), wherein activation of the exogenous T cells results in release of the antigens in active form (e.g., fully-functional capability for MHC-binding) into the microenvironment.
  • the released antigens e.g., peptides
  • MHC molecules e.g., MHC class I molecules
  • the approach of the disclosure for stimulating an endogenous T cell response can be used, for example, to recruit a synergistic endogenous T cell response (e.g., CD8+ T cell response) for amplification of target cell killing (e.g., tumor cell killing, such as within solid tumors), such as to enhance CAR T cell therapy in cancer treatment.
  • a synergistic endogenous T cell response e.g., CD8+ T cell response
  • target cell killing e.g., tumor cell killing, such as within solid tumors
  • the invention pertains to a method for stimulating an endogenous T cell response in a subject.
  • the method comprises administering to a subject an exogenous T cell having tethered to its surface an MHC- binding antigen in an inactive form, wherein activation of the exogenous T cell in the subject releases the MHC-binding antigen in active form such that the antigen binds endogenous MHC molecules and stimulates an endogenous T cell response.
  • the subject is a tumor-bearing subject and the exogenous T cell homes to the tumor.
  • the exogenous T cell is a CAR T cell that recognizes an antigen on the tumor in the subject.
  • activation of the CAR T cell in the subject and release of the MHC-binding antigen in active form stimulates an endogenous T cell response (e.g., CD8+ cytotoxic T cell response) in the environment of the tumor.
  • the disclosure provides a method for stimulating an endogenous cytolytic T cell response in a tumor environment in a tumor-bearing subject, comprising administering to the subject an exogenous T cell that homes to the tumor and that has tethered to its surface an MHC-binding antigen in an inactive form, wherein activation of the exogenous T cell in the subject releases the MHC-binding antigen in active form such that the antigen binds endogenous MHC molecules on cells in the tumor environment and stimulates an endogenous T cell response (e.g., cytolytic T cell response) in the tumor environment.
  • an endogenous T cell response e.g., CD8+ cytotoxic T cell response
  • the exogenous T cell is a CAR T cell that recognizes an antigen on the tumor in the subject.
  • the MHC-binding antigen is a peptide, such as an MHC class I-binding peptide.
  • the antigen e.g., peptide
  • the lipid vehicle is a lipid nanoparticle.
  • the lipid nanoparticle comprises maleimide- reactive groups on its surface.
  • the lipid vehicle is a liposome.
  • secretion of perforin by the exogenous T cell upon activation in the subject releases the antigen from the lipid vehicle (e.g., lipid nanoparticle) in active form such that it binds endogenous MHC molecules.
  • the antigen e.g., peptide
  • the exogenous T cell in inactive form by covalent linkage to a lipophilic tail that inserts into the exogenous T cell membrane, wherein a protease sensitive-cleavable linker is positioned between the antigen and the lipophilic tail.
  • the protease sensitive-cleavable linker is sensitive to cleavage by granzyme B.
  • the MHC-binding antigen e.g., MHC class I-binding peptide
  • the MHC-binding antigen is from a pathogen that the subject has been previously infected with prior to administration of the exogenous T cell.
  • the MHC-binding antigen is a cytomegalovirus antigen (e.g., a CMV peptide).
  • the MHC-binding antigen is an Epstein-Barr virus antigen (e.g., an EBV peptide).
  • the MHC-binding antigen is from a pathogen that the subject has been vaccinated against prior to administration of the exogenous T cell.
  • the MHC- binding antigen e.g., peptide
  • the invention pertains to a composition for stimulating an endogenous T cell response.
  • the composition comprises a T cell having tethered to its surface an MHC-binding antigen (e.g., MHC class I-binding peptide) in an inactive form, wherein activation of the T cell releases the antigen in active form such that the antigen can bind MHC molecules (e.g., MHC class I molecules).
  • MHC-binding antigen e.g., MHC class I-binding peptide
  • the T cell homes to a tumor.
  • the T cell is a CAR T cell that recognizes an antigen on a tumor.
  • the composition comprises a CAR T cell having tethered to its surface an MHC-binding peptide in an inactive form, wherein activation of the CAR T cell releases the peptide in active form such that the peptide can bind MHC molecules.
  • the T cell is a CAR T cell that recognizes an antigen in or on a tumor.
  • the MHC-binding antigen is a peptide (e.g., an MHC class I-binding peptide).
  • the antigen e.g., peptide
  • the lipid vehicle is a lipid nanoparticle.
  • the lipid nanoparticle comprises maleimide-reactive groups on its surface.
  • the lipid nanoparticle comprises maleimide- reactive groups on its surface that react with thiol groups on the T cell surface, thereby allowing conjugation of the lipid nanoparticle to the T cell surface.
  • the lipid vehicle is a liposome.
  • secretion of perforin by the T cell upon activation releases the antigen from the lipid vehicle (e.g., lipid nanoparticle) in active form such that the antigen (e.g., peptide) can bind MHC molecules (e.g., MHC class I molecules).
  • the perforin disrupts the lipid membrane of the lipid vehicle, thereby resulting in release of the antigen.
  • the antigen e.g., peptide
  • the T cell in inactive form by covalent linkage to a lipophilic tail that inserts into the T cell membrane, wherein a protease sensitive-cleavable linker is positioned between the antigen and the lipophilic tail.
  • the protease sensitive-cleavable linker is sensitive to cleavage by granzyme B.
  • secretion of granzyme B by the T cell upon activation releases the antigen from the lipophilic tail in active form such that the antigen (e.g., peptide) can bind MHC molecules.
  • the MHC-binding antigen (e.g., MHC class I-binding peptide) is from a pathogen.
  • the MHC-binding antigen is a cytomegalovirus antigen (e.g., a CMV peptide).
  • the MHC-binding antigen is an Epstein-Barr virus antigen (e.g., an EBV peptide).
  • the MHC-binding antigen (e.g., peptide) is from a pathogen selected from the group consisting of tuberculosis, measles, mumps, rubella, rotavirus, varicella, yellow fever, human papilloma virus (HPV), hepatitis A, hepatitis B and smallpox.
  • the composition comprises a population of the T cells.
  • FIG.1 is a schematic diagram of an embodiment of the disclosure.
  • MHC class I- binding peptides are loaded into lipid nanoparticles, which are then conjugated to the surface of CAR T cells.
  • the CAR T cells When infused into a tumor-bearing individual, the CAR T cells home to the tumor and become activated upon recognition of a tumor-specific target antigen (1), thereby secreting perforin and lysing the surface-conjugated lipid nanoparticle to release the encapsulated peptide (2).
  • FIGs.2A-2C are schematic diagrams of an embodiment of the invention.
  • Amphiphile peptide conjugates are prepared, composed of an MHC class I-binding peptide and a lipophilic tail, linked by a granzyme B-cleavable linker positioned between the peptide and the tail (FIG.2A).
  • the amphiphile peptide conjugates are inserted into the surface membrane of a CAR T cell via association of the lipophilic tail with membrane phospholipids (FIG.2B).
  • FIG.2C When infused into a tumor-bearing individual (FIG.2C), the CAR T cells home to the tumor and become activated upon recognition of a tumor-specific target antigen (1), thereby secreting granzyme B and cleaving the granzyme B-cleavable linker (2) to release the peptide from the amphiphile peptide conjugate (3).
  • the released peptide is loaded onto MHC class I molecules on the surface of bystander cells (4).
  • FIGs.3A-3C are graphs showing peptide encapsulation in lipid nanoparticles and antigen-specific lysis of lipid nanoparticles conjugated to the surface of CAR T cells.
  • FIG.3A is a bar graph showing BCA protein assay results quantifying the amount of SIYRYYGL (SEQ ID NO: 1) peptide encapsulated into lipid nanoparticles that were prepared as either empty (‘No peptide’) or loaded with the SIYRYYGL (SEQ ID NO: 1) peptide.
  • FIG.3B is graph showing flow cytometry results of CAR T cells having lipid nanoparticles encapsulating Alexa Fluor 647-labeled dextran conjugated to their cell surface.
  • the flow cytometry results show the proportion of CAR T cells labeled with a live/dead viability marker and with AF647-dextran.
  • FIG.3C are graphs showing flow cytometry results of anti-GFP-specific CAR T cells conjugated with AF647-dextran liposomes co-cultured for 16 hours with K562 cells expressing either intracellular GFP (iGFP) or surface (membrane-bound) GFP (sGFP).
  • iGFP intracellular GFP
  • sGFP surface (membrane-bound) GFP
  • FIGs.4A-4B are graphs showing upregulation of expression of MHC class I molecules and peptide loading onto MHC class I molecules.
  • FIG.4A shows flow cytometry results of B16F10 cells incubated with various doses of IFN- ⁇ , showing dose- dependent upregulation of H-2Kb on the cell surface.
  • FIG.4B shows flow cytometry results of IFN- ⁇ -pretreated B16F10 cells incubated with varying concentrations of the MHC class I binding peptide SIYRYYGL (SEQ ID NO: 1), showing loading of the peptide onto H-2Kb on the cell surface.
  • FIGs.5A-5B are graphs showing flow cytometry results of Jurkat cells incubated with an amphiphile conjugate of DSPE linked to FITC by a PEG2k linker (DSPE-PEG2k- FITC) (FIG.5A) or DSPE linked to a MHC class I binding peptide by a PEG2k linker with FITC at the peptide terminus (DSPE-PEG2k-CFTKKVSYYHT (SEQ ID NO: 208)- FITC) (FIG.5B) at the indicated concentrations.
  • the flow cytometry results provide the proportion of Jurkat cells labeled with FITC relative to negative control Jurkat cells incubated in PBS-only.
  • FIGs.6A-6B are graphs showing flow cytometry results of Jurkat cells incubated with DSPE-PEG 2k -FITC for the indicated duration (FIG.6A) or incubated with DSPE- PEG2k-FITC for 30 minutes followed by resuspension in media without DSPE-PEG-2k FITC for the indicated duration (FIG.6B).
  • FIGs.7A-7B are molecular structures of amphiphile-peptide conjugates.
  • FIG. 7A shows the molecular structure of Amph-IEPD-AMC, a conjugate of DSPE-PEG2k, the granzyme-cleavable linker IEPD (SEQ ID NO: 209) and the fluorogenic substrate 7- amino-4-methylcoumarin (AMC).
  • FIG.7B shows the molecular structure of Amph- GPGD-AMC, a control conjugate containing the uncleavable linker GPGD (SEQ ID NO: 210) not susceptible to granzyme-mediated cleavage.
  • FIGs.8A-8B are graphs showing fluorescence released by Amph-IEPD-AMC (FIG.8A) or Amph-GPGD-AMC (FIG.8B) upon incubation with the indicated concentrations of granzyme B.
  • T cells such as chimeric antigen receptor (CAR) T cells, having tethered to their surface MHC-binding antigens (e.g., MHC class I-binding peptides) such that the T cells can be used to recruit a synergistic endogenous T cell response (e.g., CD8+ cytolytic T cell response) in a subject upon administration to the subject, e.g., for amplification of tumor cell killing.
  • CAR chimeric antigen receptor
  • the MHC-binding antigens are tethered to the T cells’ surface in inactive form, wherein subsequent activation of the T cells (e.g., in an antigen-specific manner in the subject) releases the MHC-binding antigens in active form such that the MHC-binding antigens (e.g., MHC class I-binding antigens) can then bind MHC molecules (e.g., MHC class I molecules on endogenous bystander cells in the subject) and stimulate an endogenous T cell response in the subject.
  • MHC-binding antigens e.g., MHC class I molecules on endogenous bystander cells in the subject
  • the particular MHC-binding antigens (e.g., MHC class I-binding peptides) tethered to the T cells of the disclosure are selected based on the expected presence in the subject to whom the T cells are to be administered of endogenous T cells that are capable of recognizing and responding to the exogenously-supplied MHC-binding antigens.
  • an antigenic peptide(s) from an infectious agent that is known to be commonly encountered in the human population can be tethered to the T cells of the disclosure.
  • antigenic peptides from that infectious agent can be tethered to the T cells of the disclosure.
  • a subject can be vaccinated against a particular infectious agent (e.g., using a peptide vaccine or a live attenuated vaccine) prior to administration of the T cells of the disclosure such that the subject is expected to have T cells that are specific for the infectious agent of the vaccine. Then, antigenic peptides from the infectious agent of the vaccine can be tethered to the T cells of the disclosure.
  • the methods of the disclosure are particularly useful in amplifying an anti-tumor response in subject, e.g., within a localized microenvironment of a solid tumor.
  • the methods of the disclosure take advantage of T cell (e.g., CAR T cell) specificity for a target antigen for local, noninvasive, tumor-specific delivery of MHC-binding antigen (e.g., MHC class I-binding peptide).
  • T cell e.g., CAR T cell
  • MHC-binding antigen e.g., MHC class I-binding peptide
  • Previous studies have demonstrated the ability to increase tumor antigenicity via intratumoral injection of peptide (see e.g., Nobuoka et al. (2013) Canc. Immunol. Immunother.62(4):639-652); however, this is not only invasive, but also limits treatable tumors to those that are easily accessible.
  • MHC-binding antigens e.g., peptides
  • T cells e.g., CAR T cells
  • the T cell specificity is coupled to release of the MHC-binding antigens (e.g., peptides) in active form.
  • MHC-binding antigens e.g., peptides
  • the methods of the disclosure have the advantage of repurposing vaccination- induced anti-pathogen immune response to target tumor cells. Prior studies have demonstrated that intratumor injection of peptides can enhance tumor cell antigenicity recognized by CTLs (Nobuoka et al. (2013) Canc.
  • the methods of the disclosure provide for synergizing T cell cytotoxicity resulting from adoptive cell transfer (e.g., CAR T cells) with endogenous CD8 + T cell- mediated cytotoxicity to enable amplification of cell-mediated immunotherapy. Accordingly, the methods of the disclosure provide for amplification of tumor cell killing, potentially overcoming current limitations in the trafficking, expansion, persistence, and functional activity of adoptively transferred T cells (e.g., CAR T cells), particularly in solid tumors.
  • adoptive cell transfer e.g., CAR T cells
  • the methods of the disclosure can be used to enhance the potency of adoptive T cell therapy (e.g., CAR T cell therapy), expand the range of efficacious tumor-specific target antigens, and improve adoptive T cell (e.g., CAR T cell) therapy efficacy in the face of tumor heterogeneity, reducing the likelihood of resistance and relapse.
  • adoptive T cell therapy e.g., CAR T cell therapy
  • adoptive T cell e.g., CAR T cell therapy
  • the strategy of the disclosure greatly enhances adoptive T cell (e.g., CAR T cell) efficacy by enabling the use of T cells (e.g., CAR T cells) that target highly tumor-specific antigens that may be expressed by only a small subset of tumor cells, thereby broadening the repertoire of potential adoptive T cell (e.g., CAR T cell) target antigens.
  • adoptive T cell e.g., CAR T cell
  • the ability to recruit an effective endogenous T cell response to thereby synergize with the T cell response provided by adoptive T cell thereby could abrogate the need to substantially optimize adoptive T cells (e.g., CAR T cells) for expansion, persistence, trafficking, and functional activity by harnessing a powerful endogenous memory response
  • adoptive T cell e.g., CAR T cell
  • the invention pertains to a method for stimulating an endogenous T cell response in a subject.
  • the term “endogenous T cell response” refers to a response by T cells that are naturally-occurring (already present) in the subject, i.e., T cells that have not been administered to the subject.
  • the endogenous T cell response typically is a CD8+ T cell response, although other types of T cell responses (e.g., CD4+ T cell responses) also are encompassed.
  • the endogenous T cell response typically is a cytolytic response (i.e., a cytotoxic T cell response against a target cell resulting in target cell lysis), although other types of T cell responses (e.g., production of one or more cytokines) also are encompassed.
  • the term “stimulating” a T cell response is intended to encompass initiating, generating, increasing, enhancing and/or promoting a T cell response in the subject.
  • the term “subject” refers to any organism to which a composition in accordance with the disclosure may be administered. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). In some embodiments, a subject may be a patient, e.g., a human patient.
  • the methods of the disclosure involve administering to a subject an exogenous T cell.
  • an “exogenous T cell” refers to a T cell that is present outside of the subject prior to being administered to the subject.
  • the exogenous T cell may have originated from the subject (i.e., the exogenous T cell can be an autologous T cell that has previously been isolated from the subject).
  • the exogenous T cell may have originated from an individual other than the subject (i.e., the exogenous T cell can be an allogeneic T cell that has been isolated from an individual other than the subject).
  • the exogenous T cell is a chimeric antigen receptor (CAR) T cell.
  • CAR T cells and other suitable types of exogenous T cells for use in the methods are described in further detail in subsection II below.
  • exogenous T cells used in the methods of the disclosure have tethered to their surface (i.e., cell surface membrane) an MHC-binding antigen (e.g., MHC class I-binding antigen) in an inactive form, wherein activation of the exogenous T cell in the subject releases the MHC-binding antigen in active form such that the antigen binds endogenous MHC molecules and stimulates an endogenous T cell response.
  • an MHC-binding antigen e.g., MHC class I-binding antigen
  • an MHC-binding antigen in “an inactive form” is a form of the antigen that is incapable of binding to MHC molecules.
  • an MHC- binding antigen in “active form” is a form of the antigen that is capable of binding to MHC molecules.
  • activation of the exogenous T cell is intended to encompass recognition by the exogenous T cell of its target antigen, as well as downstream effector functions of the T cell subsequent to target antigen recognition (e.g., perforin secretion and/or granzyme B secretion by the T cell).
  • target antigen recognition e.g., perforin secretion and/or granzyme B secretion by the T cell.
  • MHC-binding antigens e.g., MHC class I-binding peptides
  • the subject is a tumor-bearing subject and the exogenous T cell homes to the tumor.
  • the exogenous T cell is a CAR T cell that recognizes an antigen on the tumor in the subject.
  • activation of the CAR T cell in the subject and release of the MHC-binding antigen in active form stimulates an endogenous CD8+ cytotoxic T cell response in the environment of the tumor.
  • the disclosure provides a method for stimulating an endogenous cytolytic T cell response in a tumor environment in a tumor-bearing subject, comprising administering to the subject an exogenous T cell that homes to the tumor and that has tethered to its surface an MHC-binding antigen in an inactive form, wherein activation of the exogenous T cell in the subject releases the MHC-binding antigen (e.g., MHC class I-binding peptide) in active form such that the antigen binds endogenous MHC molecules (e.g., MHC class I molecules) on cells in the tumor environment and stimulates an endogenous cytolytic T cell response in the tumor environment.
  • MHC-binding antigen e.g., MHC class I-binding peptide
  • the exogenous T cell is a CAR T cell that recognizes an antigen on the tumor in the subject.
  • CAR T cell recognizes an antigen on the tumor in the subject.
  • FIG.1 MHC class I-binding peptide-loaded lipid nanoparticles are synthesized and conjugated to CAR T cells specific for a target tumor antigen. This conjugation occurs via reaction of a maleimide-containing lipid within the lipid nanoparticles with free thiols on the cell surface of CAR T cells.
  • the CAR T cells Upon recognition of the target antigen on a target tumor cell (e.g., upon infusion into a tumor-bearing subject and homing of the CAR T cells to the tumor), the CAR T cells secrete perforin, which lyses the nanoparticles, thereby releasing peptide into the local tumor milieu. Peptide is then loaded onto class I MHC of bystander cells within the milieu, and stimulates trafficking of endogenous CD8 + T cells to the tumor to lyse the peptide-loaded bystander cells.
  • the peptide-MHC class I complex is recognized in a subject by endogenous CD8 + T cells generated prior to CAR T cell infusion via pre-vaccination or prior infection, resulting in bystander cell lysis (e.g., bystander tumor cells within a solid tumor).
  • bystander cell lysis e.g., bystander tumor cells within a solid tumor.
  • lipid nanoparticles have been conjugated to the surface of CAR T cells and coculture of CAR T cells with CAR target antigen-expressing tumor cells was demonstrated to trigger nanoparticle lysis and local release of peptide antigen.
  • peptide antigen in the culture medium was shown to be readily loaded onto class I MHC on bystander cells, resulting in a peptide-MHC complex.
  • the MHC class I-binding antigen e.g., peptide
  • the MHC class I-binding antigen is tethered to the exogenous T cell in inactive form by encapsulation in a lipid vehicle conjugated to the exogenous T cell surface.
  • the liposome is a lipid nanoparticle.
  • the lipid nanoparticle comprises maleimide-reactive groups on its surface.
  • the lipid vehicle is a liposome.
  • secretion of perforin by the exogenous T cell upon activation in the subject releases the antigen from the lipid vehicle (e.g., lipid nanoparticle) in active form such that it binds endogenous MHC class I.
  • lipid vehicle e.g., lipid nanoparticle
  • Lipid vehicles such as lipid nanoparticles and liposomes, preparation and peptide-loading thereof and conjugation thereof to T cells is described in further detail in subsection IV below.
  • An alternative method for activation-induced delivery of MHC class I-binding antigens (e.g., peptides) by exogenous T cells uses amphiphile antigen conjugates with protease (e.g., granzyme B)-responsive linkers.
  • the conjugates used in this approach are composed of a lipophilic tail and an antigen of interest, linked by a protease (e.g., granzyme B)-cleavable linker that is positioned between the lipophilic tail and the antigen.
  • protease e.g., granzyme B
  • amphiphile-peptide conjugates are able to insert into cell membranes via association of the lipophilic tail with membrane phospholipids, and enable local release of the peptide upon granzyme B-mediated cleavage.
  • a CAR T cell with such an amphiphile- peptide conjugate inserted into its surface cell membrane homes to the tumor and becomes activated upon recognition of a tumor-specific target antigen. This results in secretion of granzyme B by the CAR T cell and cleavage of the amphiphile-peptide conjugate via the granzyme B-cleavable linker, thereby releasing the peptide.
  • the released peptide is loaded onto class I MHC molecules on the surface of bystander cells, and the peptide-MHC complex is recognized by endogenous CD8+ T cells, resulting in bystander cell lysis.
  • the disclosure is based, at least in part, on the discovery that an amphiphile-peptide conjugate inserted in a T cell (e.g., CAR T cell) membrane remains at the cell-surface for a substantial period of time (e.g., at least 4h, 8h, 12h, 24h, 36h, 48h), providing, for example, a sufficient period for labeled T cells to be administered to a tumor-bearing invidual, home to the tumor, become activated upon target antigen recongnition, and release active peptide (e.g., prior to internalization of membrane- inserted amphiphile-peptide conjugate).
  • a substantial period of time e.g., at least 4h, 8h, 12h, 24h, 36h, 48h
  • the antigen e.g., peptide
  • the exogenous T cell in inactive form by covalent linkage to a lipophilic tail that inserts into the exogenous T cell membrane, wherein a protease (e.g., granzyme B)-cleavable linker is positioned between the antigen and the lipophilic tail.
  • a protease e.g., granzyme B
  • secretion of protease by the exogenous T cell e.g., secretion of granzyme B
  • tumor cells in the tumor microenvironment upon activation of the T cell in the subject releases the antigen from the lipophilic tail in active form such that it binds endogenous MHC class I.
  • Amphiphile-antigen conjugates, preparation thereof and insertion thereof into T cell membranes is described in further detail in subsection V below.
  • Exogenous T Cells utilize an exogenous T cell (i.e., a T cell that is present outside of a subject prior to being administered to a subject) that has tethered to its surface (e.g., cell surface membrane) an MHC-binding antigen.
  • T cells are suitable for use in the methods and compositions of the disclosure.
  • any T cell that is suitable for use in adoptive cell therapy can be utilized as an exogenous T cell of the disclosure.
  • the T cell is a CD8+ T cell, although CD4+ T cells are also encompassed.
  • the exogenous T cells typically are mammalian cells (e.g., human cells).
  • the exogenous T cell is autologous to a subject to whom the exogenous T cell is to be administered.
  • the T cell is isolated from the subject, modified as described herein to tether an MHC class I-binding antigen to its surface (optionally along with other modification(s) that may be performed on the T cell ex vivo, including those described further below) and then re-infused into the subject.
  • the T cell is allogeneic to a subject to whom the exogenous T cell is to be administered.
  • the T cell is obtained from an individual other than the subject, modified as described herein to tether an MHC class I-binding antigen to its surface (optionally along with other modification(s) that may be performed on the T cell ex vivo, including those described further below) and then administered into the subject.
  • the allogeneic T cell can be selected for optimal receptivity by the subject (e.g., MHC matching).
  • Some embodiments of the invention refer to “isolated” T cells (e.g., isolated exogenous T cells). Isolated T cells are T cells that have been separated from the environment in which they naturally occur (e.g., they have been removed from their natural environment, such as removed from a subject). T cells in vitro are an example of isolated T cells. When administered in vivo, the exogenous T cells preferably home to target site(s).
  • Suitable T cells are chosen based on their homing potential, their cell surface phenotype (for tethering of MHC class I-binding antigens), and their ability to carry the tethered antigens in inactive form and release them in active form. For example, substantial levels of free thiol (-SH) groups exist on the surfaces of T cells, thereby facilitating conjugation of liposomes, e.g., lipid nanoparticles, to such cells.
  • the exogenous T cells preferably have half-lives in vivo, following administration (or re-infusion, in some instances), of at least 48 hours, more preferably at least, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, or more.
  • the exogenous T cells can be genetically engineered to express one or more factors including without limitation costimulatory molecules or receptors including chimeric receptors (discussed further below).
  • the T cells are not genetically engineered.
  • the T cells are isolated and naturally occurring (i.e., they have not been genetically or otherwise engineered).
  • the T cells may be manipulated prior to tethering of the MHC-binding antigens (e.g., conjugation with peptide-encapsulated lipid nanoparticles).
  • the T cells however need not be surface- modified in order to facilitate tethering, e.g., conjugation of lipid nanoparticles.
  • the invention in some of its embodiments instead takes advantage of reactive groups that normally exist on the cell surface without having to incorporate reactive groups or other entities onto the cell surface.
  • reactive groups or other entities such cells do not require the presence of exogenous entities such as antibodies or antibody fragments, among others, on their surface in order to tether the MHC-binding antigens, e.g., conjugation of lipid nanoparticles.
  • Ex vivo manipulation of the exogenous T cells may also involve activation of the cells, as is routinely performed for T cells.
  • the cells may be expanded and/or activated (or stimulated, as the terms are used interchangeably herein) in vitro prior to tethering of the MHC-binding antigens (e.g., mixing with peptide-encapsulated liposomes or lipid nanoparticles).
  • Expansion and activation protocols will vary depending on the particular circumstances of use, but can include incubation with one or more cytokines, incubation with one or more cell types, incubation with one or more antigens, etc.
  • T cells activation may be performed, for example, by incubating the cells with IL-2, IL-15, IL-15 superagonist, costimulatory molecules such as B7, B7.2, CD40, antibodies to various T cell surface molecules including antibodies to cell surface receptors, anti-CD3 antibodies, anti-CD28 antibodies, anti-CTLA-4 antibodies, anti-CD40L antibodies, and the like.
  • the T cells are not coated with exogenous antibodies on their cell surface (i.e., the cells have not been contacted with antibodies or antibody fragments in vitro prior to administration). Expansion may be measured by proliferation assays involving incorporation of radiolabeled nucleotides such as tritiated thymidine.
  • Exogenous T cells may be selected prior to administration to a subject in order to enrich and thus administer higher numbers of such cells in smaller volumes and/or to remove other, potentially unwanted, cells from the administered composition. Selection may involve positive or negative selection, including for example column or plate based enrichment protocols that are known in the art, as well as cell sorting methods known in the art, such as flow cytometry. Exogenous T cells may be harvested from the peripheral blood of a subject.
  • exogenous T cells may be harvested from lymph nodes and/or spleen. Still further, exogenous T cells may be harvested from tumor-infiltrating lymphocytes (TILs) isolated from a tumor site. T cells can be harvested using standard procedures known in the art for cell harvesting, such as affinity columns, magnetic beads, fluorescence activated cell sorting (FACS), some combination thereof, and the like. In certain embodiments of the methods and compositions of the disclosures, the exogenous T cells comprise further modifications in addition to the tethering of the MHC class I-binding antigens to the surface of the T cells. For example, T cells can be further modified to redirect the antigen recognition capabilities of the T cells.
  • TILs tumor-infiltrating lymphocytes
  • the exogenous T cell is further modified to express a receptor (e.g., TCR or CAR), such as a receptor that recognizes a cancer antigen (also referred to as a tumor antigen).
  • a cancer antigen typically is an antigen that is expressed preferentially by cancer cells (i.e., it is expressed at higher levels in cancer cells than on non-cancer cells) and in some instances is expressed solely by cancer cells.
  • the cancer antigen may be expressed within a cancer cell or on the surface of the cancer cell.
  • cancer antigens include those listed in the subsection below.
  • Chimeric Antigen Receptors the disclosure provides compositions and methods to be used or performed in conjunction with chimeric antigen receptor (CAR) T cells.
  • CAR chimeric antigen receptor
  • the term "chimeric antigen receptor (CAR)" refers to an artificial transmembrane protein receptor comprising an extracellular domain capable of binding to a predetermined CAR ligand or antigen, an intracellular segment comprising one or more cytoplasmic domains derived from signal transducing proteins different from the polypeptide from which the extracellular domain is derived, and a transmembrane domain.
  • CAR chimeric antigen receptor
  • T-body chimeric immune receptor
  • CIR chimeric immune receptor
  • CAR ligand used interchangeably with “CAR antigen” means any natural or synthetic molecule (e.g. small molecule, protein, peptide, lipid, carbohydrate, nucleic acid) or part or fragment thereof that can specifically bind to the CAR.
  • intracellular signaling domain means any oligopeptide or polypeptide domain known to function to transmit a signal causing activation or inhibition of a biological process in a cell, for example, activation of an immune cell such as a T cell or a NK cell. Examples include ILR chain, CD28 and/or CD3 ⁇ .
  • cancer antigen refers to (i) tumor- specific antigens, (ii) tumor- associated antigens, (iii) cells that express tumor- specific antigens, (iv) cells that express tumor- associated antigens, (v) embryonic antigens on tumors, (vi) autologous tumor cells, (vii) tumor- specific membrane antigens, (viii) tumor- associated membrane antigens, (ix) growth factor receptors, (x) growth factor ligands, and (xi) any other type of antigen or antigen-presenting cell or material that is associated with a cancer.
  • Chimeric antigen receptors are genetically-engineered, artificial transmembrane receptors, which confer an arbitrary specificity for a ligand onto an immune effector cell (e.g. a T cell, natural killer cell or other immune cell) and which results in activation of the effector cell upon recognition and binding to the ligand.
  • an immune effector cell e.g. a T cell, natural killer cell or other immune cell
  • these receptors are used to impart the antigen specificity of a monoclonal antibody onto a T cell.
  • CARs contain three domains: 1) an ectodomain typically comprising a signal peptide, a ligand or antigen recognition region (e.g.
  • scFv scFv
  • a flexible spacer 2) a transmembrane (TM) domain; 3) an endodomain (alternatively known as an “activation domain”) typically comprising one or more intracellular signaling domains.
  • the ectodomain of the CAR resides outside of the cell and exposed to the extracellular space, whereby it is accessible for interaction with its cognate ligand.
  • the TM domain allows the CAR to be anchored into the cell membrane of the effector cell.
  • the third endodomain also known as the “activation domain” aids in effector cell activation upon binding of the CAR to its specific ligand.
  • effector cell activation comprises induction of cytokine and chemokine production, as well as activation of the cytolytic activity of the cells.
  • the CARs redirect cytotoxicity toward tumor cells.
  • chimeric antigen receptors comprise a ligand- or antigen- specific recognition domain that binds to a specific target ligand or antigen (also referred to as a binding domain).
  • the binding domain is a single- chain antibody variable fragment (scFv), a tethered ligand or the extracellular domain of a co-receptor, fused to a transmembrane domain, which is linked, in turn, to a signaling domain.
  • the signaling domain is derived from CD3 ⁇ or FcR ⁇ .
  • the CAR comprises one or more co-stimulatory domains derived from a protein such as CD28, CD137 (also known as 4-lBB), CD134 (also known as OX40) and CD278 (also known as ICOS).
  • the CAR does not comprise a co-stimulatory domain derived from CD137. Engagement of the antigen binding domain of the CAR with its target antigen on the surface of a target cell results in clustering of the CAR and delivers an activation stimulus to the CAR-containing cell.
  • the main characteristic of CARs are their ability to redirect immune effector cell specificity, thereby triggering proliferation, cytokine production, phagocytosis or production of molecules that can mediate cell death of the target antigen expressing cell in a major histocompatibility (MHC) independent manner, exploiting the cell specific targeting abilities of monoclonal antibodies, soluble ligands or cell specific co-receptors.
  • MHC major histocompatibility
  • a new generation of CARs containing a binding domain, a hinge, a transmembrane and the signaling domain derived from CD3 ⁇ or FcRy together with one or more co-stimulatory signaling domains has been shown to more effectively direct antitumor activity as well as increased cytokine secretion, lytic activity, survival and proliferation of CAR expressing T cells in vitro, in animal models and cancer patients (Milone et al., Molecular Therapy, 2009; 17: 1453-1464; Zhong et al., Molecular Therapy, 2010; 18: 413-420; Carpenito et al., PNAS, 2009; 106:3360-3365).
  • co-stimulatory signaling domains e.g., intracellular co-stimulatory domains derived from CD28, CD137, CD134 and CD278
  • chimeric antigen receptor-expressing T cells are cells that are derived from a patient with a disease or condition and genetically modified in vitro to express at least one CAR with specificity for a ligand.
  • the cells perform at least one effector function (e.g. induction of cytokines) that is stimulated or induced by the specific binding of the ligand to the CAR and that is useful for treatment of the same patient’s disease or condition.
  • the effector cell is a T cell (e.g. a cytotoxic T cell) that exerts its effector function (e.g.
  • a cytotoxic T cell response on a target cell when brought in contact or in proximity to the target or target cell (e.g. a cancer cell)
  • target or target cell e.g. a cancer cell
  • Prolonged exposure of T cells to their cognate antigen can result in exhaustion of effector functions, enabling the persistence of infected or transformed cells.
  • Recently developed strategies to stimulate or rejuvenate host effector function using agents that induce an immune checkpoint blockade have resulted in success towards the treatment of several cancers. Emerging evidence suggests that T cell exhaustion may also represent a significant impediment in sustaining long-lived antitumor activity by chimeric antigen receptor-expressing T cells (CAR T cells).
  • CAR T cells chimeric antigen receptor-expressing T cells
  • the differentiation status of the patient-harvested T cells prior to CAR transduction and the conditioning regimen a patient undergoes before reintroducing the CAR T cells can profoundly affect the persistence and cytotoxic potential of CAR T cells.
  • CAR T cells e.g., addition or exclusion of alkylating agents, fludarabine, total-body irradiation
  • cytokines such as IL- 2
  • T cell populations can also alter the differentiation status and effector function of CAR T cells (Ghoneim et al., (2016) Trends in Molecular Medicine 22(12):1000-1011).
  • T cells modified to express a CAR which binds to a universal immune receptor, a tag, a switch or an Fc region on an immunoglobulin are useful in the methods and compositions described herein.
  • T cells are modified to express a universal immune receptor or UnivIR.
  • UnivIR is a biotin-binding immune receptor (BBIR) (see e.g., US Patent Publication US20140234348 A1 incorporated herein by reference in its entirety).
  • T cells are modified to express a universal, modular, anti- tag chimeric antigen receptor (UniCAR).
  • UniCAR universal, modular, anti- tag chimeric antigen receptor
  • T cells are modified to express a switchable chimeric antigen receptor and chimeric antigen receptor effector cell (CAR-EC) switches.
  • CAR-EC switchable chimeric antigen receptor and chimeric antigen receptor effector cell
  • the CAR-EC switches have a first region that is bound by a chimeric antigen receptor on the CAR-EC and a second region that binds a cell surface molecule on a target cell, thereby stimulating an immune response from the CAR-EC that is cytotoxic to the bound target cell.
  • the CAR-EC is a T cell, wherein the CAR- EC switch may act as an “on-switch” for CAR-EC activity. Activity may be “turned off” by reducing or ceasing administration of the switch.
  • CAR-EC switches may be used with CAR-ECs disclosed herein, as well as existing CAR T-cells, for the treatment of a disease or condition, such as cancer, wherein the target cell is a malignant cell.
  • a disease or condition such as cancer
  • Such treatment may be referred to herein as switchable immunotherapy (US Patent Publication US9624276 B2 incorporated herein by reference in its entirety).
  • T cells are modified to express a receptor that binds the Fc portion of human immunoglobulins (e.g., CD16V-BB- ⁇ ) (Kudo et al., (2014) Cancer Res 74(1):93-103 incorporated herein by reference in its entirety).
  • T cells are modified to express a universal immune receptor (e.g., switchable CAR, sCAR) that binds a peptide neo-epitope (PNE).
  • a universal immune receptor e.g., switchable CAR, sCAR
  • PNE peptide neo-epitope
  • the peptide neo-epitope (PNE) has been incorporated at defined different locations within an antibody targeting an antigen (antibody switch). Therefore, sCAR-T- cell specificity is redirected only against PNE, not occurring in the human proteome, thus allowing an orthogonal interaction between the sCAR-T-cell and the antibody switch.
  • sCAR-T cells are strictly dependent on the presence of the antibody switch to become fully activated, thus excluding CAR T-cell off-target recognition of endogenous tissues or antigens in the absence of the antibody switch (Arcangeli et al., (2016) Transl Cancer Res 5(Suppl 2):S174-S177 incorporated herein by reference in its entirety).
  • switchable CARs is provided by US Patent Application US20160272718A1 incorporated herein by reference in its entirety.
  • the term “tag” encompasses a universal immune receptor, a tag, a switch, or an Fc region of an immunoglobulin as described supra.
  • an effector cell is modified to express a CAR comprising a tag binding domain.
  • the CAR binds fluorescein isothiocyanate (FITC), streptavidin, biotin, dinitrophenol, peridinin chlorophyll protein complex, green fluorescent protein, phycoerythrin (PE), horse radish peroxidase, palmitoylation, nitrosylation, alkalanine phosphatase, glucose oxidase, or maltose binding protein.
  • FITC fluorescein isothiocyanate
  • streptavidin biotin
  • biotin dinitrophenol
  • peridinin chlorophyll protein complex green fluorescent protein
  • PE phycoerythrin
  • horse radish peroxidase palmitoylation
  • nitrosylation alkalanine phosphatase
  • glucose oxidase or maltose binding protein.
  • a subject is genetically modified with a chimeric antigen receptor (Sadelain et al., Cancer Discov.3:388-398, 2013).
  • a T cell is provided and recombinant nucleic acid encoding a chimeric antigen receptor is introduced into the patient-derived T cell to generate a CAR cell.
  • T cells not derived from the subject are genetically modified with a chimeric antigen receptor.
  • T cells are allogeneic cells that have been engineered to be used as an “off the shelf” adoptive cell therapy, such as Universal Chimeric Antigen Receptor T cells (UCARTs), as developed by Cellectis.
  • UCARTs are allogeneic CAR T cells that have been engineered to be used for treating the largest number of patients with a particular cancer type.
  • Non-limiting examples of UCARTs under development by Cellectis include those that target the following tumor antigens: and C D38
  • a variety of different methods known in the art can be used to introduce any of the nucleic acids or expression vectors disclosed herein into a T cell.
  • Non-limiting examples of methods for introducing nucleic acid into a T cell include: lipofection, transfection (e.g., calcium phosphate transfection, transfection using highly branched organic compounds, transfection using cationic polymers, dendrimer-based transfection, optical transfection, particle-based transfection (e.g., nanoparticle transfection), or transfection using liposomes (e.g., cationic liposomes)), microinjection, electroporation, cell squeezing, sonoporation, protoplast fusion, impalefection, hydrodynamic delivery, gene gun, magnetofection, viral transfection, and nucleofection.
  • transfection e.g., calcium phosphate transfection, transfection using highly branched organic compounds, transfection using cationic polymers, dendrimer-based transfection, optical transfection, particle-based transfection (e.g., nanoparticle transfection), or transfection using liposomes (e.g., cationic liposomes)
  • CRISPR/Cas9 genome editing technology can be used to introduce CAR nucleic acids into T cells and/or to introduce other genetic modifications (e.g., as described below) into T cells to enhance CAR T cell activity (for use of CRISPR/Cas9 technology in connection with CAR T cells, see e.g., US 9,890,393; US 9,855,297; US 2017/0175128; US 2016/0184362; US 2016/0272999; WO 2015/161276; WO 2014/191128; CN 106755088; CN 106591363; CN 106480097; CN 106399375; CN 104894068).
  • Chimeric antigen receptors include an antigen-binding domain, a transmembrane domain, and an cytoplasmic signaling domain that includes a cytoplasmic sequence of CD3 ⁇ sequence sufficient to stimulate a T cell when the antigen-binding domain binds to the antigen, and optionally, a cytoplasmic sequence of one or more (e.g., two, three, or four) co-stimulatory proteins (e.g., a cytoplasmic sequence of one or more of B , , , , , , , , , , , , and a ligand that specifically binds to CD83) that provides for co-stimulation of the T cell when the antigen-binding domain binds to the antigen.
  • co-stimulatory proteins e.g., a cytoplasmic sequence of one or more of B , , , , , , , , , , , and a ligand that specifically binds to CD83
  • a CAR can further include a linker.
  • a linker Non-limiting aspects and features of CARs are described below. Additional aspects of CARs and CAR cells, including exemplary antigen-binding domains, linkers, transmembrane domains, and cytoplasmic signaling domains, are described in, e.g., Kakarla et al., Cancer J.20:151-155, 2014; Srivastava et al., Trends Immunol.36:494-502, 2015; Nishio et al., Oncoimmunology 4(2): e988098, 2015; Ghorashian et al., Br. J. Haematol.169:463-478, 2015; Levine, Cancer Gene Ther.
  • CARs and CAR cells including exemplary antigen-binding domains, linkers, transmembrane domains, and cytoplasmic signaling domains, are described in WO 2016/168595; WO 12/079000; 2015/0141347; 2015/0031624; 2015/0030597; 2014/0378389; 2014/0219978; 2014/0206620; 2014/0037628; 2013/0274203; 2013/0225668; 2013/0116167; 2012/0230962; 2012/0213783; 2012/0093842; 2012/0071420; 2012/0015888; 2011/0268754; 2010/0297093; 2010/0158881; 2010/0034834; 2010/0015113; 2009/0304657; 2004/0043401; 2014/0322253; 2015/0118208; 2015/0038684; 2014/0024601; 2012/0148552; 2011/0223129; 2009/0257994; 2008/0160607
  • Antigen binding domains included in the chimeric antigen receptor can specifically bind to an antigen (e.g., a tumor associated antigen (TAA) or an antigen that is not expressed on an non-cancerous cell).
  • an antigen e.g., a tumor associated antigen (TAA) or an antigen that is not expressed on an non-cancerous cell.
  • TAA tumor associated antigen
  • an antigen binding domain include: a monoclonal antibody (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgE, and IgD) (e.g., a fully human or a chimeric (e.g., a humanized) antibody), an antigen binding fragment of an antibody (e.g., Fab, Fab', or F(ab') 2 fragments) (e.g., a fragment of a fully human or a chimeric (e.g., humanized) antibody), a diabody, a triabody, a tetrabody, a minibody, a scFv, scFv-Fc, (scFv)2, scFab, bis-scFv, hc-IgG, a BiTE, a single domain antibody (e.g., a V-NAR domain or a VhH domain), IgNAR, and a multivalent antibody (e
  • an antigen binding domain includes at least one (e.g., one, two, three, four, five, or six) CDR (e.g., any of the three CDRs from an immunoglobulin light chain variable domain or any of the three CDRs from an immunoglobulin heavy chain variable domain) of an antibody that is capable of specifically binding to the target antigen, such as immunoglobulin molecules (e.g., light or heavy chain immunoglobulin molecules) and immunologically-active (antigen-binding) fragments of immunoglobulin molecules.
  • CDR e.g., one, two, three, four, five, or six
  • an antibody that is capable of specifically binding to the target antigen, such as immunoglobulin molecules (e.g., light or heavy chain immunoglobulin molecules) and immunologically-active (antigen-binding) fragments of immunoglobulin molecules.
  • an antigen binding domain is a single-chain antibody (e.g., a V-NAR domain or a V H H domain, or any of the single-chain antibodies as described herein).
  • an antigen binding domain is a whole antibody molecule (e.g., a human, humanized, or chimeric antibody) or a multimeric antibody (e.g., a bi-specific antibody).
  • antigen-binding domains include antibody fragments and multi-specific (e.g., bi-specific) antibodies or antibody fragments.
  • antibodies and antigen-binding fragments thereof include, but are not limited to: single- chain Fvs (scFvs), Fab fragments, Fab’ fragments, F(ab’) 2 , disulfide-linked Fvs (sdFvs), Fvs, and fragments containing either a VL or a VH domain.
  • Additional antigen binding domains provided herein are polyclonal, monoclonal, multi-specific (multimeric, e.g., bi-specific), human antibodies, chimeric antibodies (e.g., human-mouse chimera), single-chain antibodies, intracellularly-made antibodies (i.e., intrabodies), and antigen-binding fragments thereof.
  • the antibodies or antigen-binding fragments thereof can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.
  • the antigen binding domain is an IgG1 antibody or antigen-binding fragment thereof.
  • the antigen binding domain is an IgG 4 antibody or antigen-binding fragment thereof.
  • the antigen binding domain is an immunoglobulin comprising a heavy and light chain.
  • antigen binding domains are antigen-binding fragments of an IgG (e.g., an antigen-binding fragment of IgG1, IgG2, IgG3, or IgG4) (e.g., an antigen-binding fragment of a human or humanized IgG, e.g., human or humanized IgG1, IgG2, IgG3, or IgG4), an antigen-binding fragment of an IgA (e.g., an antigen-binding fragment of IgA1 or IgA2) (e.g., an antigen-binding fragment of a human or humanized IgA, e.g., a human or humanized IgA1 or IgA2), an antigen-binding fragment of an IgD (e.g., an antigen-binding fragment of a human or humanized IgD), an antigen-binding fragment of an IgE (e.g., an antigen-binding fragment of a human or humanized Ig
  • an antigen binding domain can bind to a particular antigen (e.g., a tumor-associated antigen) with an affinity (K D ) about or less than 1 x 10 -7 M (e.g., about or less than 1 x 10 -8 M, about or less than 5 x 10 -9 M, about or less than 2 x 10 -9 M, or about or less than 1 x 10 -9 M), e.g., in saline or in phosphate buffered saline.
  • a particular antigen e.g., a tumor-associated antigen
  • K D affinity
  • the choice of the antigen binding domain to include in the CAR depends upon the type and number of ligands that define the surface of a cell (e.g., cancer cell or tumor) to be targeted in a subject in need thereof.
  • the antigen binding domain may be chosen to recognize a ligand that acts as a cell surface marker on cancer cells, or is a tumor-associated antigen (e.g., CD19, CD30, Her2/neu, EGFR or BCMA) or a tumor-specific antigen (TSA).
  • CAR effector cells e.g., CAR T cells
  • comprise a CAR molecule that binds to a tumor antigen e.g., comprises a tumor antigen binding domain).
  • the CAR molecule comprises an antigen binding domain that recognizes a tumor antigen of a solid tumor (e.g., breast cancer, colon cancer, etc.).
  • the CAR molecule comprises an antigen binding domain that recognizes a tumor antigen of a hematologic malignancy (e.g., leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute promyelocytic leukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt’s lymphoma and marginal zone B cell lymphoma, Polycythemia vera, Hodgkin's disease, non-Hodgkin' s disease, multiple myeloma, etc.).
  • a hematologic malignancy e.g., leukemia, acute lymphocytic leukemia, acute myelocy
  • the tumor antigen is a tumor-specific antigen (TSA).
  • TSA tumor-specific antigen
  • a TSA is unique to tumor cells and does not occur on other cells in the body.
  • the tumor antigen is a tumor-associated antigen (TAA).
  • TAA tumor-associated antigen
  • a TAA is not unique to a tumor cell and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen.
  • the expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen.
  • a TAA is expressed on normal cells during fetal development when the immune system is immature and unable to respond or is normally present at extremely low levels on normal cells but which are expressed at much higher levels on tumor cells.
  • the tumor-associated antigen is determined by sequencing a patient's tumor cells and identifying mutated proteins only found in the tumor. These antigens are referred to as "neoantigens.” Once a neoantigen has been identified, therapeutic antibodies can be produced against it and used in the methods described herein.
  • the tumor antigen is an epithelial cancer antigen, (e.g., breast, gastrointestinal, lung), a prostate specific cancer antigen (PSA) or prostate specific membrane antigen (PSMA), a bladder cancer antigen, a lung (e.g., small cell lung) cancer antigen, a colon cancer antigen, an ovarian cancer antigen, a brain cancer antigen, a gastric cancer antigen, a renal cell carcinoma antigen, a pancreatic cancer antigen, a liver cancer antigen, an esophageal cancer antigen, a head and neck cancer antigen, or a colorectal cancer antigen.
  • epithelial cancer antigen e.g., breast, gastrointestinal, lung
  • PSA prostate specific cancer antigen
  • PSMA prostate specific membrane antigen
  • bladder cancer antigen e.g., a lung (e.g., small cell lung) cancer antigen
  • a colon cancer antigen e.g., an ovarian cancer antigen
  • a brain cancer antigen
  • the tumor antigen is a lymphoma antigen (e.g., non-Hodgkin's lymphoma or Hodgkin's lymphoma), a B-cell lymphoma cancer antigen, a leukemia antigen, a myeloma (e.g.., multiple myeloma or plasma cell myeloma) antigen, an acute lymphoblastic leukemia antigen, a chronic myeloid leukemia antigen, or an acute myelogenous leukemia antigen.
  • Tumor antigens e.g., tumor antigens, (e.g.
  • tumor-associated antigens TAAs
  • TSAs tumor-specific antigens
  • CAR effector cells include, but are not limited to, 1GH-IGK, 43-9F, 5T4, 791Tgp72, acyclophilin C-associated protein, alpha-fetoprotein (AFP), ⁇ -actinin-4, A3, antigen specific for A33 antibody, ART-4, B7, Ba 733, BAGE, BCR-ABL, beta-catenin, beta-HCG, BrE3-antigen, B CA225 BTAA CA125 CA 153 ⁇ CA 2729 ⁇ BCAA CA195 CA242 CA 50 CAM43, CAMEL CAP 1 carbonic anhydrase IX, c-Met, CA19-9, CA72-4, CAM 17.1, CASP- 8/m, , , , , , , , , , , , , , , , , , , , , , , , , , , ,
  • the tumor antigen is a viral antigen derived from a virus associated with a human chronic disease or cancer (such as cervical cancer).
  • the viral antigen is derived from Epstein-Barr virus (EBV), HPV antigens E6 and/or E7, hepatitis C virus (HCV), hepatitis B virus (HBV), or cytomegalovirus (CMV).
  • Exemplary cancers or tumors and specific tumor antigens associated with such tumors include acute lymphoblastic leukemia (etv6, aml1, cyclophilin b), B cell lymphoma (Ig-idiotype), glioma (E-cadherin, ⁇ -catenin, ⁇ -catenin, ⁇ -catenin, p120ctn), bladder cancer (p21ras), biliary cancer (p21ras), breast cancer (MUC family, HER2/neu, c-erbB-2), cervical carcinoma (p53, p21ras), colon carcinoma (p21ras, HER2/neu, c-erbB-2, MUC family), colorectal cancer (Colorectal associated antigen ( C C) CO 7 /G 733, C), choriocarcinoma (CEA), epithelial cell cancer (cyclophilin b), gastric cancer (HER2/neu, c-erbB-2, ga733 glycoprotein),
  • the immune effector cell comprising a CAR molecule useful in the methods disclosed herein expresses a CAR comprising a mesothelin binding domain (i.e., the CAR T cell specifically recognizes mesothelin).
  • Mesothelin is a tumor antigen that is overexpressed in a variety of cancers including ovarian, lung and pancreatic cancers.
  • the immune effector cell comprising a CAR molecule (e.g., CAR T cell) useful in the methods disclosed herein expresses a CAR comprising a CD19 binding domain.
  • the immune effector cell comprising a CAR molecule e.g., CAR T cell
  • the immune effector cell comprising a CAR molecule e.g., CAR T cell
  • the CAR effector cell expressing a CAR comprising a CD19 targeting or binding domain is Kymriah TM (tisagenlecleucel; Novartis; see WO 2016109410, herein incorporated by reference in its entirety) or YescartaTM (axicabtagene ciloleucel; Kite; see US 20160346326, herein incorporated by reference in its entirety).
  • B. Linker Provided herein are CARs that can optionally include a linker (1) between the antigen binding domain and the transmembrane domain, and/or (2) between the transmembrane domain and the cytoplasmic signaling domain.
  • the linker can be a polypeptide linker.
  • the linker can have a length of between about 1 amino acid and about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, about 14 amino acids, about 12 amino acids, about 10 amino acids, about 8 amino acids, about 6 amino acids, about 4 amino acids, or about 2 amino acids; about 2 amino acids to about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 90 amino acids, about 80 amino acids, about 70 amino acids, about 60 amino acids, about 50 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 18 amino acids, about 16 amino acids, about 14 amino acids, about 12 amino acids, about 10 amino acids, about 8 amino acids, about 6 amino acids, about 4 amino
  • the CARs described herein also include a transmembrane domain.
  • the transmembrane domain is naturally associated with a sequence in the cytoplasmic domain.
  • the transmembrane domain can be modified by one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) amino acid substitutions to avoid the binding of the domain to other transmembrane domains (e.g., the transmembrane domains of the same or different surface membrane proteins) to minimize interactions with other members of the receptor complex.
  • the transmembrane domain may be derived from a natural source. In some embodiments, the transmembrane domain may be derived from any membrane-bound or transmembrane protein. Non-limiting examples of transmembrane domains that may be used herein may be derived from (e.g., comprise at least the transmembrane sequence or a part of the transmembrane sequence of) the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, , , , , C , , , , , , , , or CD154. In some embodiments, the transmembrane domain may be synthetic.
  • the transmembrane domain may include (e.g., predominantly include) hydrophobic residues (e.g., leucine and valine).
  • the synthetic transmembrane domain will include at least one (e.g., at least two, at least three, at least four, at least five, or at least six) triplet of phenylalanine, tryptophan, and valine at the end of a synthetic transmembrane domain.
  • the transmembrane domain of a CAR can include a CD8 hinge domain. Additional specific examples of transmembrane domains are described in the references cited herein. D.
  • CAR molecules that comprise, e.g., a cytoplasmic signaling domain that includes a cytoplasmic sequence of CD3 ⁇ sufficient to stimulate a T cell when the antigen binding domain binds to the antigen, and optionally, a cytoplasmic sequence of one or more of co-stimulatory proteins (e.g., a cytoplasmic sequence of one or more of CD27, CD28, 4-1BB, OX40, CD30, CD40L, CD40, PD-1, PD-L1, ICOS, LFA-1, CD2, CD7, CD160, LIGHT, BTLA, TIM3, CD244, CD80, LAG3, NKG2C, B7-H3, a ligand that specifically binds to CD83, and any of the ITAM sequences described herein or known in the art) that provides for co-stimulation of the T cell.
  • co-stimulatory proteins e.g., a cytoplasmic sequence of one or more of CD27, CD28, 4-1BB, O
  • the stimulation of a CAR immune effector cell can result in the activation of one or more anti-cancer activities of the CAR immune effector cell.
  • stimulation of a CAR immune effector cell can result in an increase in the cytolytic activity or helper activity of the CAR immune effector cell, including the secretion of cytokines.
  • the entire intracellular signaling domain of a co-stimulatory protein is included in the cytoplasmic signaling domain.
  • the cytoplasmic signaling domain includes a truncated portion of an intracellular signaling domain of a co-stimulatory protein (e.g., a truncated portion of the intracellular signaling domain that transduces an effector function signal in the CAR immune effector cell).
  • a co-stimulatory protein e.g., a truncated portion of the intracellular signaling domain that transduces an effector function signal in the CAR immune effector cell.
  • Non-limiting examples of intracellular signaling domains that can be included in a cytoplasmic signaling domain include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any variant of these sequences including at least one (e.g., one, two, three, four, five, six, seven, eight, nine, or ten) substitution and have the same or about the same functional capability.
  • TCR T cell receptor
  • a cytoplasmic signaling domain can include two distinct classes of cytoplasmic signaling sequences: signaling sequences that initiate antigen- dependent activation through the TCR (primary cytoplasmic signaling sequences) (e.g., a CD3 ⁇ cytoplasmic signaling sequence) and a cytoplasmic sequence of one or more of co- stimulatory proteins that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences).
  • primary cytoplasmic signaling sequences e.g., a CD3 ⁇ cytoplasmic signaling sequence
  • secondary cytoplasmic signaling sequences co- stimulatory proteins that act in an antigen-independent manner to provide a secondary or co-stimulatory signal.
  • the cytoplasmic domain of a CAR can be designed to include the CD3 ⁇ signaling domain by itself or combined with any other desired cytoplasmic signaling sequence(s) useful in the context of a CAR.
  • the cytoplasmic domain of a CAR can include a CD3 ⁇ chain portion and a costimulatory cytoplasmic signaling sequence.
  • the costimulatory cytoplasmic signaling sequence refers to a portion of a CAR including a cytoplasmic signaling sequence of a costimulatory protein (e.g., CD27, CD28, 4-IBB (CD 137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83).
  • a costimulatory protein e.g., CD27, CD28, 4-IBB (CD 137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with
  • the cytoplasmic signaling sequences within the cytoplasmic signaling domain of a CAR are positioned in a random order. In some embodiments, the cytoplasmic signaling sequences within the cytoplasmic signaling domain of a CAR are linked to each other in a specific order. In some embodiments, a linker (e.g., any of the linkers described herein) can be used to form a linkage between different cytoplasmic signaling sequences. In some embodiments, the cytoplasmic signaling domain is designed to include the cytoplasmic signaling sequence of CD3 ⁇ and the cytoplasmic signaling sequence of the costimulatory protein CD28.
  • the cytoplasmic signaling domain is designed to include the cytoplasmic signaling sequence of CD3 ⁇ and the cytoplasmic signaling sequence of costimulatory protein 4-IBB. In some embodiments, the cytoplasmic signaling domain is designed to include the cytoplasmic signaling sequence of CD3 ⁇ and the cytoplasmic signaling sequences of costimulatory proteins CD28 and 4-1BB. In some embodiments, the cytoplasmic signaling domain does not include the cytoplasmic signaling sequences of 4-1BB.
  • CAR T cells are administered to the subject.
  • a methylcytosine dioxygenase gene e.g., Tetl, Tet2, Tet3
  • Tetl methylcytosine dioxygenase gene
  • Tet2 Tet2
  • Tet3 methylcytosine dioxygenase gene
  • a T cell can be engineered to express a CAR and wherein expression and/or function of Tetl, Tet2 and/or Tet3 in said T cell has been reduced or eliminated.
  • the therapeutic efficacy of CAR T cells is enhanced by using a T cell that constitutively expresses a CAR (referred to as a nonconditional CAR) and conditionally expresses another agent useful for treating cancer, as described in PCT Publication WO 2016/126608 and US Publication No.2018/0044424.
  • the conditionally expressed agent is expressed upon activation of the T cell, e.g., the binding of the nonconditional CAR to its target.
  • the conditionally expressed agent is a CAR (referred to herein as a conditional CAR).
  • the conditionally expressed agent inhibits a checkpoint inhibitor of the immune response.
  • conditionally expressed agent improves or enhances the efficacy of a CAR, and can include a cytokine.
  • the therapeutic efficacy of CAR T cells is enhanced by modifying the CAR T cell with a nucleic acid that is capable of altering (e.g., downmodulating) expression of an endogenous gene selected from the group consisting of TCR ⁇ chain, TCR ⁇ chain, beta-2 microglobulin, a HLA molecule, CTLA-4, PD1, and FAS, as described in PCT Publication WO 2016/069282 and US Publication No. 2017/0335331.
  • the therapeutic efficacy of CAR T cells is enhanced by co-expressing in the T cells the CAR and one or more enhancers of T cell priming (“ETPs”), as described in PCT Publication WO 2015/112626 and US Publication No. 2016/0340406.
  • ETPs enhancers of T cell priming
  • APC enhanced “professional” antigen-presenting cell
  • the CAR and one or more ETPs are transiently co-expressed in the T cell.
  • the engineered T cells are safe (given the transient nature of the CAR/ETP expression), and induce prolonged immunity via APC function.
  • the therapeutic efficacy of CAR T cells is enhanced by co-expressing in the T cells a CAR and an inhibitory membrane protein (IMP) comprising a binding (or dimerization) domain, as described in PCT Publication WO 2016/055551 and US Publication No.2017/0292118.
  • the CAR and the IMP are made both reactive to a soluble compound, especially through a second binding domain comprised within the CAR, thereby allowing the co-localization, by dimerization or ligand recognition, of the inhibitory signaling domain borne by the IMP and of the signal transducing domain borne by the CAR, having the effect of turning down the CAR activation.
  • the inhibitory signaling domain is preferably the programmed death-1 (PD-1), which attenuates T-cell receptor (TCR)-mediated activation of IL-2 production and T-cell proliferation.
  • PD-1 programmed death-1
  • TCR T-cell receptor
  • the therapeutic efficacy of CAR T cells is enhanced using a system where controlled variations in the conformation of the extracellular portion of a CAR containing the antigen-binding domain is obtained upon addition of small molecules, as described in PCT Publication WO 2017/032777. This integrated system switches the interaction between the antigen and the antigen binding domain between on/off states. By being able to control the conformation of the extracellular portion of a CAR, downstream functions of the CAR T cell, such as cytotoxicity, can be directly modulated.
  • a CAR can be characterized in that it comprises: a) at least one ectodomain which comprises: i) an extracellular antigen binding domain; and ii) a switch domain comprising at least a first multimerizing ligand-binding domain and a second multimerizing ligand-binding domain which are capable of binding to a predetermined multivalent ligand to form a multimer comprising said two binding domains and the multivalent ligand to which they are capable of binding; b) at least one transmembrane domain; and c) at least one endodomain comprising a signal transducing domain and optionally a co-stimulatory domain; wherein the switch domain is located between the extracellular antigen binding domain and the transmembrane domain.
  • an MHC-binding antigen is tethered to the surface of an exogenous T cell.
  • the antigen is tethered to the T cell surface such that it is inactive, i.e., unable to bind MHC molecules, prior to T cell activation but becomes active, i.e., able to bind MHC molecules, upon T cell activation.
  • the MHC-binding antigen is an MHC class I- binding antigen (e.g., peptide).
  • the MHC-binding antigen is an MHC class II-binding antigen (e.g., peptide).
  • MHC class I-binding antigens e.g., peptides
  • MHC class II-binding antigens e.g., peptides
  • the MHC-binding antigen includes at least one MHC classs I-binding antigen (e.g., peptide) such that activation of the exogenous T cell to which the antigen is tethered leads to release of the antigen in active form such that the antigen can bind to MHC class I molecules in the microenvironment to thereby stimulate MHC class I-mediated responses.
  • MHC class II-binding antigen e.g., peptide
  • MHC class I-binding or “binds to MHC class I molecules” refers to association of the antigen with an MHC class I molecule (e.g., HLA- A, HLA-B or HLA-C molecule in a human subject) such that the MHC class I/antigen complex is presented to and recognizable by immune cells (e.g., cytotoxic T cells).
  • MHC class II-binding or “binds to MHC class II molecules” refers to association of the antigen with an MHC class II molecule (e.g., HLA-D molecules in a human subject) such that the MHC class II/antigen complex is presented to and recognizable by immune cells (e.g., helper T cells).
  • MHC-binding antigen is a peptide, although other antigens capable of associating with and being presented by MHC molecules, such as peptidomimetics, glycopeptides, phosphopeptides and the like are also encompassed.
  • peptides (or peptidomimetics, glycopeptides, phosphopeptides or the like) all having the same amino acid sequence serve as the MHC- binding antigens that are tethered to the exogenous T cell surface.
  • a peptide having the amino acid sequence of a dominant T cell epitope can be used as the MHC- binding antigen.
  • a group (i.e., plurality) of peptides (or peptidomimetics, glycopeptides, phosphopeptides or the like) having different amino acid sequences serves as the MHC-binding antigens that are tethered to the exogenous T cell surface.
  • a plurality of peptides of different sequences each having the amino acid sequence of a T cell epitope can be used as the MHC-binding antigen.
  • that sequence can be used as a monomer or as a multimer (e.g., containing two, three, four, five, six, seven, eight, nine, ten or more copies of the sequence) in the MHC-binding antigen preparation.
  • the MHC-binding antigen functions to stimulate an endogenous T cell response in a subject to which a composition of the disclosure is administered.
  • the particular antigen(s) that selected for use in a particular patient are selected based on the expected presence in the subject of endogenous T cells that will recognize those particular MHC-binding antigens.
  • MHC class I- binding antigens from pathogens e.g., viruses
  • pathogens e.g., viruses
  • CMV cytomegalovirus
  • EBV Epstein-Barr virus
  • adenoviruses adenoviruses
  • rhinoviruses adenoviruses
  • enteroviruses e.g., enteroviruses
  • influenza virus e.g., varicella virus
  • the MHC-binding antigen is an MHC class I-binding antigen (e.g., peptide) from a pathogen that the subject has been previously infected with prior to administration of the exogenous T cell.
  • MHC class I-binding antigen e.g., peptide
  • Methods for determining whether a subject has previously been infected with a particular pathogen are known in the art, such as measurement of antibody titers, PCR detection of pathogen DNA and/or detection of immune cells from the subject that are specific for the pathogen.
  • the MHC-binding antigens are MHC class I-binding antigens from a pathogen that the subject has been vaccinated against prior to administration of the exogenous T cell.
  • Pathogens that are commonly vaccinated against, and thus from with the MHC class I-binding antigens can be derived include tuberculosis, measles, mumps, rubella, rotavirus, varicella, yellow fever, human papilloma virus (HPV), hepatitis A, hepatitis B and smallpox.
  • the subject is vaccinated against the pathogen in preparation for subsequent treatment with the exogenous T cell (i.e., the subject had not previously been vaccinated against the pathogen before contemplation of administering exogenous T cells to stimulate an endogenous T cell response).
  • the subject is vaccinated against the pathogen at a sufficient time prior to administration of the exogenous T cells such that an immune response against the vaccine has been developed.
  • the subject has previously been vaccinated against the pathogen before contemplation of administering the exogenous T cells (i.e., the subject is expected to have immunity against the pathogen due to prior vaccination against the pathogen at the time when administration of exogenous T cells is contemplated).
  • Methods for determining whether a subject has mounted an immune response against a particular vaccine are known in the art, such as measurement of antibody titers and/or detection of immune cells from the subject that are specific for the vaccine.
  • a subject may be administered a vaccine booster prior to administration of the exogenous T cells to thereby boost the anti-vaccine response in the subject.
  • MHC class I-binding antigens including peptides, peptidomimetics, glycopeptides, phosphopeptides and the like can be prepared using standard methods known in the art, including standard chemical synthesis and biological synthesis using recombinant DNA technology.
  • Non-limiting examples of MHC class-I binding antigens derived from pathogens that a subject may have been previously infected with or vaccinated against are described further below.
  • Cytomegalovirus Human cytomegalovirus is a ⁇ -herpesvirus with a seroprevalence of 60- 90% depending on geographic location (Hosie et al. (2017) Front. Immunol.8:1776). Although primary infection is typically asymptomatic, the virus establishes a state of persistent infection, undergoing periodic episodes of reactivation. CD8+ T cells play a critical role in controlling viral reactivation (see e.g., Elkington et al. (2003) J. Virol.
  • CMV infection has been shown to trigger one of the largest virus-specific cellular immune responses yet determined (Gillespie et al. (2000) J. Virol.74:8140-8150).
  • the dominant CTL response in CMV infection is directed against the CMV tegument protein pp65 (Wills et al. (1996) J. Virol.70:7569-7579).
  • HLA-A2 Individuals expressing the widely distributed MHC class I molecule HLA-A*0201 (HLA-A2) produce CMV-specific CTLs bearing T cell receptors that mainly recognize an epitope corresponding to residues 495-503 of pp65, having the amino acid sequence NLVPMVATV (SEQ ID NO: 2) (Wills et al. (1996) supra; Peggs et al. (2002) Blood 99:213-223). Accordingly, the CMV pp65495-503 peptide can be used as an MHC class I- binding antigen of the disclosure.
  • CMVPepVax a CMV vaccine has been developed (known in the art as CMVPepVax) that is comprised of the HLA-A*0201-restricted CD8+ T cell epitope CMV pp65 495-503 peptide having the sequence NLVPMVATV (SEQ ID NO: 2) fused with a Tetanus toxin P2 epitope (TT830-843) having the sequence QYIKANSKFIGITE (SEQ ID NO: 3) (see e.g., Nakamura et al. (2014) Blood 124:183; Nakamura et al. (2016) Lancet Haematol.3:e87-e98).
  • such a peptide fusion construct can be as an MHC class I-binding antigen of the disclosure.
  • a panel of CMV epitopes restricted by HLA-C have been described in the art (Hosie et al. (2017) Front. Immunol.8:1776) and is shown below in Table 1.
  • HLA-C*07:02 is less susceptible to viral immune evasion by CMV, thereby suggesting that HLA-C*07:02-restricted viral epitopes are promising viral epitopes for stimulating an effective anti-CMV T cell response.
  • Peptide multimers (streptamers) of HLA-C*07:02-restricted CMV epitopes have been described in the art, including HLA-C_ 07:02/IE- 1309 ⁇ 317 (CRVLCCYVL) (SEQ ID NO: 9) and HLA-C_ 07:02/MAGE-A12170-178 (VRIGHLYIL) (SEQ ID NO: 14) (Schlott et al.
  • Epstein Barr Virus Human Epstein Barr Virus is a ⁇ -herpesvirus that infects over 90% of the human population (Rickinson and Kieff (1996) in Virology, 3rd Ed. B. N. Fields et al., eds. Lippincott-Raven, Philadelphia, p.2397). EBV can establish both nonproductive (latent) and productive (lytic) infections within the cells of its host. T cells specific for EBV lytic protein epitopes are readily detectable in hosts and are usually more abundant than those specific for latent epitopes.
  • a preferred EBV-specific MHC class I-binding antigen for HLA-B8 subjects is a peptide comprising the sequence RAKFKQLL (SEQ ID NO: 16).
  • a panel of EBV epitopes restricted by HLA-A*03:01 have been described in the art (Bieling et al. (2016) Oncotarget 9:4737-4757) and is shown below in Table 3.
  • Table 3 EBV Epitopes Restricted by HLA-A*03:01 Accordingly, any or all of these epitopes shown in Table 3, in any combination, can be used as MHC class I-binding antigens of the disclosure. Furthermore, conserved EBV-specific CD8 T cell epitopes from early antigens have been described in Alonso-Padilla et al. (2017) J. Immunol. Res.2017:9363750, which are shown below in Table 4. Table 4: HLA-Restricted EBV Epitopes Accordingly, any or all of these epitopes shown in Table 4, in any combination, can be used as MHC class I-binding antigens of the disclosure.
  • any or all of these epitopes shown in Table 5, in any combination, can be used as MHC class I-binding antigens of the disclosure.
  • T cell epitopes from the EBV immediate early 1 (IE1) protein, along with their HLA restriction, have been described (Iampietro et al. (2014) Eur. J. Immunol. 44:3573-3584).
  • T cell epitopes include amino acid residues 109-117, having the amino acid sequence CIQSIGASV (SEQ ID NO: 64), which is restricted to HLA-A*02, amino acid residues 9-17, having the amino acid sequence STSMFILGK (SEQ ID NO: 65), which is restricted to HLA-A*03, amino acid residues 173-181, having the amino acid sequence CYAKMLSGK (SEQ ID NO: 66), which is also restricted to HLA-A*03 and amino acid residues 109-117, having the amino acid sequence NPEISNKEF (SEQ ID NO: 67), which is restricted to HLA-B*07.
  • any or all of these epitopes can be used as MHC class I-binding antigens of the disclosure.
  • a single epitope and a tandem epitope i.e., triplet multimer from the gp350/220 capsid protein have been described for development into an EBV vaccine.
  • the single epitope has the amino acid sequence: Q NPVYLIPETVPYIKWDNC (SEQ ID NO: 68) and the tandem epitope has the sequence Q Q Q ( Q : 69) (Widodo et al. (2016) Heliyon 4:e00564). Accordingly, these gp350/220 epitopes can be used as an MHC class I-binding antigen of the disclosure.
  • VZV Varicella Varicella-zoster virus infects about 95% of the population, persists throughout life, and may lead to herpes zoster (HZ) when the virus reactivates.
  • Primary infection elicits both humoral and cellular responses, but cellular immunity is essential for preventing herpes zoster.
  • the live attenuated zoster vaccine available in the art has been shown to generate cellular immunity (i.e., T cell responses) (see e.g., Diaz et al. (1989) J. Immunol.142:636-641; Levin et al. (1992) J. Infect. Dis.166:253-259).
  • a peptide having the sequence ALWALPHAA (SEQ ID NO: 70) (used as a multimer) was shown to stimulate a positive signal from T cells (van der Heiden et al. (2009) supra.). Accordingly, this VZV T cell epitope can be used as an MHC class-I binding antigen of the disclosure.
  • the peptide can be used as a monomer or as a multimer (e.g., tetramer, heptamer).
  • VZV ribonucleotide reductase subunit 2 a dominant HLA-A*0201-restricted epitope in the VZV ribonucleotide reductase subunit 2 has been identified and used a tetramer to analyze the phenotype and function of epitope-specific CD8 T cells (Chiu et al. (2014) PLoS Pathog. 10(3):e1004008).
  • CD8 T cells responding to this VZV epitope also recognized homologous epitopes, not only in the other ⁇ -herpesviruses (HSV-1 and HSV-2) but also the ⁇ -herpesvirus EBV.
  • This immunodominant HLA- A*0201-restricted class I epitope is a peptide having the amino acid sequence ILIEGIFFV (SEQ ID NO: 71). Accordingly, this VZV T cell epitope (that is also crossreactive with HSV-1, HSV-2 and EBV epitopes) can be used as an MHC class-I binding antigen of the disclosure.
  • the peptide can be used as a monomer or as a multimer (e.g., tetramer, heptamer).
  • Measles Measles virus can cause acute infection in early childhood, which is normally followed by life-long immunity due to the presence of neutralizing antibodies against virus surface glycoproteins H and F (see e.g., Norby, E. (1985) Ann. Inst. Pasteur/Virol.136E:561-570). Induction of cell-mediated immunity has been found to be imperative for rapid clearance of infection and for protection from subsequent disease (see e.g., Kernahan et al. (1987) Br. Med. J.295:15-18; Markowitz et al. (1988) J. Infect. Dis.158:480-483). An effective live attenuated viral vaccine against MV exists (MMR vaccine).
  • MHC class I-binding antigens of the disclosure Mycobacterium Tuberculosis (TB) is one of the most prevalent diseases in developing countries and is caused by infection with Mycobacterium tuberculosis and other members of the Mycobacterium tuberculosis complex (MTBC). The current vaccine against TB is the M.
  • MTBC Mycobacterium tuberculosis
  • bovis bacille Calmette-Guerin (BCG) live vector vaccine which has been shown to stimulate both cellular and antibody responses and has the possibility to stimulate prolonged memory T cell responses (Rapeah et al. (2006) Vaccine 24:3646-3653).
  • BCG bovis bacille Calmette-Guerin
  • HLA-A*11:01 binding motifs
  • Peptides that bound with high affinity to purified HLA molecules were then evaluated in functional assays, which led to the identification of six epitopes, each derived from unique MTB antigens, which were recognized by CD8+ T cells from active pulmonary TB patients (Liu et al.
  • Table 7 CD8+ T Cell Epitopes from MTB Antigens Accordingly, any or all of these epitopes shown in Table 7, in any combination, can be used as MHC class I-binding antigens of the disclosure. Furthermore, integrated computational and proteomic approaches were used to screen 10% of the M. tuberculosis (MTB) proteome for CD8 MTB antigens (Lewinsohn et al. (2013) PLoS One 8(6):e67016). Synthetic peptide libraries were used to screen MTB-specific CD8+ T cell clones, restricted by MHC class I molecules, which were isolated from MTB-infected humans.
  • MTB tuberculosis
  • Yellow Fever Yellow fever virus is a member of the family Flaviviridae, which includes at least 68 single-stranded RNA viruses transmitted by mosquitoes or ticks.
  • a live attenuated vaccine (strain 17D) is available, derived from the human isolate Asibi.
  • the vaccine has been effective in preventing smallpox infection in 95% of those vaccinated.
  • the vaccine was proven to prevent or substantially lessen infection when given within a few days after a person was exposed to the variola virus.
  • Cell-mediated immune responses play an important role in protection (see e.g., Freed et al. (1972) Am J. Med.52:411-420; Karupiah et al. (1996) J. Virol.70:8301-8309).
  • Virus-specific CTLs have been detected even decades after primary vaccination, demonstrating long-lasting cellular immunity memory (Demkowicz et al. (1996) J. Virol.70:2627-2631).
  • HLA-A*0201-restricted epitope has been identified that is recognized by CD8+ T cells and conserved among Orthopox virus species including variola virus and vaccinia virus (Drexler et al. (2002) Proc. Natl. Acad. Sci. USA 100:217-222).
  • the epitope which is part of a 35 kDa vaccinia virus envelope protein encoded by ORF H3L, has the amino acid sequence SLSAYIIRV (SEQ ID NO: 141) (Drexler et al. (2002) supra.). Accordingly, this epitope sequence can be used as an MHC class I-binding antigen of the disclosure.
  • a vaccinia virus epitope has been identified that binds HLA-A2.1 and confers protection against lethal vaccinia virus challenge in HLA-A2 transgenic mice (Snyder et al. (2004) J. Virol.78:7052-7060). This epitope is conserved in vaccinia viruses and in variola viruses, and thus may provide cross-protection against smallpox in HLA-A2.1 individuals, which represents almost half of the US population.
  • the epitope which is from host range protein 2 (HRP2 74-82 ), has the amino acid sequence KVDDTFYYV (SEQ ID NO: 142) (Snyder et al. (2004) supra.).
  • this epitope sequence can be used as an MHC class I-binding antigen of the disclosure.
  • a panel of vaccinia epitopes has been identified that participate in the memory response of immune donors immunized with modified vaccinia virus Ankara (MVA), an attenuated replication-deficient strain of vaccinia virus (Meyer et al. (2008) J. Immunol.181:6371-6383).
  • MAA modified vaccinia virus Ankara
  • Table 9 Vaccinia Epitopes Accordingly, any or all of these epitopes shown in Table 9, in any combination, can be used as MHC class I-binding antigens of the disclosure.
  • Human Papilloma Virus Cervical cancer accounts for about two-thirds of all cancer cases linked etiologically to Human Papilloma Virus (HPV). Fifteen oncogenic HPV types can cause cervical cancer, of which HPV16 and HPV18 account for about 70% of cases.
  • HPV 16/17 L1 virus-like particle (VLP) vaccines are employed for the prevention of HPV infection.
  • VLP virus-like particle
  • a T cell epitope within HPV16 LI has been identified that binds well to both HLA-A*0201 and HLA-A*2402 and that stimulates antigen-specific T cell responses after HPV vaccination as compared to responses prior to vaccination (Yokomine et al. (2017) Exp. Therap.
  • This epitope (L1305-313) has the amino acid sequence QIFNKPYWL (SEQ ID NO: 157)(Yokomine et al. (2017) supra.). Accordingly, this epitope sequence can be used as an MHC class I-binding antigen of the disclosure. Furthermore, bioinformatics approaches have been used to predict MHC class I- restricted (HLA-A*0201) T cell epitopes against HPV types 16 and 18 by identifying evolutionarily conserved regions of the major capsid protein (L1) (Baidya et al. (2017) Bioinformation 13:86-93).
  • MHC class I-restricted T cell epitope within the L1 protein of HPV16: (SEQ ID NO: 158). This study also identified the following MHC class I-restricted T cell epitopes with the L1 protein of HPV18: Q ( Q : 159), YNPETQRLVWAC (SEQ ID NO: 160), D TGYGAMD (SEQ ID NO: 161), P (SEQ ID NO: 162), R DNVSVDYKQTQLCI (SEQ ID NO: 163) and YSRHVEEYDLQFIF (SEQ ID NO: 164).
  • any of these epitope sequences in any combination, can be used as an MHC class I-binding antigen of the disclosure.
  • an epitope highly conserved among HPV16 strains has been identified that is recognized by CTLs and induces cytolysis (Riemer et al. (2010) J. Biol. Chem.285:29608-29622). This epitope is part of the viral E7 protein (E711-19), is HLA- A*0201-restricted and has the amino acid sequence YMLDLQPET (SEQ ID NO: 165) (Riemer et al. (2010) supra.).
  • an epitope with the HPV16 E5 protein (E5 63- 71 ), having the amino acid sequence YIIFVYIPL (SEQ ID NO: 166) has been demonstrated to be an HLA-A*0201-restricted CTL epitope (Liu et al. (2006) J. Virol. 81:2869-2879).
  • an E7 peptide (E782-90), which is conserved between HPV6b and HPV11 strains and has the amino acid sequence LLLGTLNIV (SEQ ID NO: 167), has been demonstrated to be an HLA-A*0201-restricted CTL epitope (Peng et al. (2016) Cancer Immunol. Immunotherap.65:261-271).
  • an MHC class I-binding antigen e.g., peptide
  • a lipid vehicle which vehicle is then conjugated to the surface of the T cells.
  • the lipid vehicle used is sensitive to perforin, such that release of perforin upon activation of the T cell lyses the lipid vehicle and releases the MHC class I-binding antigen (e.g., peptide) in active form such that it can bind MHC class I molecules on bystander cells.
  • the term “lipid vehicle” refers to a chemical composition that can carry and/or encapsulate an MHC class I-binding antigen and that comprises at least one lipid in its structure.
  • the lipid vehicle is a lipid nanoparticle.
  • the lipid vehicle is a liposome.
  • the lipid vesicle is a multilamellar vesicle or a solid lipid nanoparticle.
  • Conjugation of drug-loaded lipid nanoparticles to the surface of cytotoxic T- lymphocytes (CTLs) and lysis of the nanoparticles by local secretion of perforin by CTLs upon target cell recognition has been demonstrated in the art (see e.g., Jones et al. (2017) Biomaterials 117:44-53; Zheng et al. (2017) ACS Nano 11:3089-3100).
  • CTLs cytotoxic T- lymphocytes
  • Suitable lipid vehicles, including lipid nanoparticles and liposomes, preparation thereof and conjugation thereof to T cells has been described in the art (see e.g., U.S.
  • Nanoparticles are solid colloidal particles used to deliver MHC class I-binding antigens (e.g., peptides) in accordance with the disclosure. Nanoparticles are not liposomes, as used herein. The nanoparticles are not viruses or particles thereof. The nanoparticles are also to be distinguished from films or other structurally layered polymers matrices, since the nanoparticles are comprised of one or more solidified polymer(s) that is arranged in a random manner. The nanoparticles are preferably biodegradable and thus typically are not magnetic.
  • Biodegradable nanoparticles may be synthesized using methods known in the art including without limitation solvent evaporation, hot melt microencapsulation, solvent removal, and spray drying. Exemplary methods for synthesizing nanoparticles are described herein in Examples 4 and 5, as well as by Bershteyn et al., Soft Matter 4:1787-1787, 2008 and in US 2008/0014144 A1, the specific teachings of which relating to nanoparticle synthesis are incorporated herein by reference.
  • the nanoparticles are comprised of a nucleic acid internal core.
  • Such “DNA nanoparticles” (or DNA-gel nanoparticles) are described in greater detail in published U.S. application no. US 20070148246.
  • the nucleic acid core of such particles may act as a scaffold for the agents being delivered in vivo and/or it may act as the agent itself.
  • An exemplary protocol for synthesizing DNA nanoparticles is provided in Example 4.
  • the nanoparticles release their MHC class I-binding antigen “payload” over a number of days as a function of their degradation profile in vivo.
  • the nanoparticles are biodegradable in nature and thus they gradually degrade in an aqueous environment such as occurs in vivo. If the antigens are dispersed throughout the nanoparticles then their release will occur as the outermost layers of the nanoparticle degrade or as the pores within the nanoparticle enlarge.
  • the nanoparticles are preferably not engulfed by either their carrier exogenous T cells or other cells at the target site. They function rather by gradually releasing their payload into the environment of the target site(s).
  • the nanoparticles’ diameter ranges from 1-1000 nanometers (nm). In some embodiments, their diameter ranges in size from 20-750 nm, or from 20-500 nm, or from 20-250 nm.
  • their diameter ranges in size from 50-750 nm, or from 50-500 nm, or from 50-250 nm, or from about 100-300 nm. In some embodiments, their diameter is about 100, about 150, about 200 nm, about 250 nm, or about 300 nm. As used in the context of nanoparticle diameters, the term “about” means +/- 5% of the absolute value stated. Thus, it is to be understood that although these particles are referred to herein as nanoparticles, the invention intends to embrace microparticles as well. As discussed herein, the nanoparticles may be synthesized to comprise one or more reactive groups on their exterior surface for reaction with reactive groups on cell carriers (e.g., exogenous T cells).
  • cell carriers e.g., exogenous T cells
  • nanoparticle reactive groups include without limitation thiol-reactive maleimide head groups, haloacetyl (e.g., iodoacetyl) groups, imidoester groups, N-hydroxysuccinimide esters, pyridyl disulfide groups, and the like. These reactive groups react with groups on the carrier cell surface and thus the nanoparticles are bound to the cell surface. It will be understood that when surface modified in this manner, the nanoparticles are intended for use with specific carrier cells having “complementary” reactive groups (i.e., reactive groups that react with those of the nanoparticles). In some embodiments, the nanoparticles will not integrate into the lipid bilayer that comprises the cell surface.
  • the nanoparticles will not be phagocytosed (or internalized) by the carrier cells.
  • the nanoparticles do not comprise antibodies or antibody fragments on their surface, while in other embodiments they do.
  • the nanoparticles do not comprise antibodies or antibody fragments that are specific to T cell surface moieties (or exogenous moieties coated onto a T cell surface such other antibodies or antibody fragments), while in other embodiments they do.
  • the nanoparticles themselves do not stimulate carrier T cell activation simply by binding to the carrier T cell.
  • the nanoparticles do stimulate carrier T cell activation by binding to the carrier T cell (e.g., binding of the nanoparticle results in crosslinking of T cell surface moieties and this activates the carrier T cell).
  • the nanoparticles may be covalently conjugated (or attached or bound, as the terms are used interchangeably herein), or they may be non-covalently conjugated to the carrier T cells.
  • Covalent conjugation typically provides a more stable (and thus longer) association between the nanoparticles and the carrier T cells.
  • Covalent conjugation in some embodiments also can provide stability and thus more sustained localized delivery of agents in vivo.
  • Non-covalent conjugation includes without limitation absorption onto the cell surface and/or lipid bilayer of the cell membrane.
  • covalent attachment can be achieved in a two-step process in which carrier T cells are first incubated with maleimide-bearing nanoparticles to allow conjugation to the cell surface, followed by in situ PEGylation with thiol-terminated poly(ethylene glycol) (PEG) to cap remaining maleimide groups of the particles and avoid particle-mediated crosslinking of cells.
  • PEG poly(ethylene glycol)
  • This strategy allows particles ranging from simple liposomes (e.g., with an aqueous drug-loaded core) to more complex lipid-coated polymer or DNA-based nanoparticles to be stably attached to live cells.
  • the linkage chemistry is benign and non-toxic as evidenced in part by the conjugation of up to 139 ( ⁇ 29) ⁇ 200 nm-diameter lipid-coated nanoparticles to the surface of cells without any deleterious effect (data not shown).
  • liposomes and lipid-coated polymer particles are able to spontaneously adsorb to cell surfaces, in some instances covalent conjugation is preferred due to the increased stability it achieves.
  • Nanoparticles bound to carrier T cells remain localized at the cell surface as revealed by optical sectioning with confocal microscopy, scanning electron microscopy and by flow cytometry internalization assays, even following extended in vitro stimulation (data not shown).
  • Exemplary synthetic polymers which can be used to form the biodegradable nanoparticles include without limitation aliphatic polyesters, poly (lactic acid) (PLA), poly (glycolic acid) (PGA), co-polymers of lactic acid and glycolic acid (PLGA), polycarprolactone (PCL), polyanhydrides, poly(ortho)esters, polyurethanes, poly(butyric acid), poly(valeric acid), and poly(lactide-co-caprolactone), and natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof, including substitutions, additions of chemical groups such as for example alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by
  • the nanoparticles also preferably comprise a lipid bilayer on their outermost surface.
  • This bilayer may be comprised of one or more lipids of the same or different type. Examples include without limitation phospholipids such as phosphocholines and phosphoinositols. Specific examples include without limitation DMPC, DOPC, DSPC, and various other lipids such as those recited below for liposomes. Liposomes The invention also contemplates the use of liposomes in place of nanoparticles in the various embodiments described herein.
  • Liposomes are small closed vesicles comprising at least one lipid bilayer and an internal aqueous compartment. As used herein, liposomes are not nanoparticles. Liposomes may be anionic, neutral or cationic. They may be unilamellar or multilamellar. Liposome may comprise without limitation unilamellar vesicle lipids, multilamellar vesicle lipids and extruded lipids including , , , , alone or together with cholesterol to yield D and cholesterol, DOTAP and cholesterol, DOTIM and cholesterol, and DDAB and cholesterol.
  • liposomes may be synthesized to include maleimide conjugated phospholipids such as without limitation DSPE-MaL-PEG2000.
  • An exemplary synthesis protocol for liposomes is provided in Example 6.
  • V. Amphiphile-Antigen Conjugates An amphiphile vaccine technology has been developed that involves linking adjuvants or antigens (e.g., peptides) to lipophilic polymeric tails, which promotes localization of vaccines to lymph node (Liu et al. (2014) Nature 507:519-522).
  • Such amphiphile-antigens e.g., amph-peptides
  • this amphiphile technology can be used to tether an MHC-binding antigen (e.g., MHC class I-binding peptide) to the surface of an exogenous T cell for use in the methods and compositions of the disclosure.
  • an MHC-binding antigen e.g., MHC class I-binding peptide
  • the amphiphile used for tethering the antigen to the exogenous T cell is a PEG lipid.
  • the PEG-lipid includes, but not limited to 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG- DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG- distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-l,2-dimyristyloxlpropyl-3-amine (PEG- c-DMA).
  • PEG-DMG 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol
  • PEG- DSPE 1,2- distearoyl-sn-
  • the PEG lipid is DSPE-PEG2000 (DSPE-PEG2k).
  • a protease-sensitive linker is positioned between the amphiphile and the antigen (e.g., peptide) such that the antigen is kept in inactive form (i.e., tethered to the exogenous T cell and unable to bind MHC molecules on bystander cells) in the absence of the protease.
  • the exogenous T cell to which the conjugate is tethered results in cleavage of the protease-sensitive linker and release of the MHC-binding antigen (e.g., MHC class I-binding peptide) in active form (i.e., untethered from the exogenous T cell and able to bind MHC molecules on bystander cells).
  • the protease is release by the T cell upon activation.
  • proteases within T cells e.g., CTLs
  • serine proteases such as granzymes A, B and C.
  • the protease-sensitive linker is sensitive to cleavage by Granzyme B (discussed further below).
  • the protease is expressed in tumors and present in the tumor microenvironment.
  • proteases expressed in tumors and that can be expressed in the tumor microenvironment include matrix metalloproteinases (MMPs) (e.g., MT1-MMP), cathepsins (e.g., cathepsin B, cathepsin D), gelatinases (e.g., gelatinase-A, gelatinase-B), stromelysin I, interstitial collagenase, uPA and uPAR (see e.g., Koblinski et al.
  • MMPs matrix metalloproteinases
  • cathepsins e.g., cathepsin B, cathepsin D
  • gelatinases e.g., gelatinase-A, gelatinase-B
  • the protease-sensitive linker is sensitive to cleavage by granzyme B.
  • Granzyme B (also referred to herein simply as granzyme) is a serine protease found in the cytotoxic T cells (CTLs) and natural killer cells. It is secreted by these cells, along with the pore-forming protein perforin, to mediate apoptosis in target cells.
  • Granzyme B is active at a neutral pH and therefore is inactive in the acidic granules of CTLs, but becomes active upon release following antigen-specific activation of the CTLs.
  • Granzyme B contains the catalytic triad His-Asp-Ser in its active site and preferentially cleaves a tetrapeptide target sequence having an aspartic acid residue situated in the last position (the P1 position).
  • the granzyme B-sensitive cleavage site within the linker comprises the amino acid sequence Xaa1-Xaa2-Xaa3-Asp (D) (SEQ ID NO: 168), wherein Xaa1, Xaa2 and Xaa3 are any amino acid.
  • the aspartic acid residue to be cleaved associates with an arginine residue in the enzyme’s binding pocket (Bolvin et al.
  • Non-limiting examples of amino acid sequences capable of being cleaved by granzyme B include those listed in Table 10 below. Accordingly, an appropriate granzyme B-sensitive linker can be designed to incorporate any of these sequences.
  • the granzyme B-sensitive cleavage site within the linker comprises the amino acid sequence Xaa1-Xaa2-Xaa3-Asp (D), wherein Xaa1 is selected from the group consisting of Asp (D), Ile (I), Leu (L), Met (M), Pro (P) and Val (V), Xaa2 is selected from the group consisting of Ala (A), Arg (R), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), Ser (S) and Thr (T) and Xaa3 is selected from the group consisting of Ala (A), Asn (N), Gln (Q), Glu (E), Gly (G), Ile (I), Lys (K), Pro (P), Ser (S), Thr (T) and Val (V) (SEQ ID NO: 169).
  • the granzyme B-sensitive cleavage site within the linker comprises the amino acid sequence Xaa1-Xaa2-Xaa3-Asp (D), wherein Xaa1 is Ile (I) or Val (V), Xaa2 is Glu (E) and Xaa3 is Pro (P) or Thr (T) (SEQ ID NO: 203).
  • the granzyme B-sensitive cleavage site within the linker comprises the amino acid sequence I-E-P-D (SEQ ID NO: 209).
  • Granzyme B is known to have many natural substrates, including proteins in the extracellular matrix, proteins in the nucleus of target cells, viral proteins and plasma proteins.
  • an appropriate granzyme B-sensitive linker can be designed based on the sequence of a granzyme-sensitive site within a natural substrate of the enzyme.
  • natural substrates for granzyme B include caspase 3, caspase 7, caspase 8, caspase 10, BID (BH3 interacting-domain death agonist), ICAD (inhibitor of caspase-activated DNase), Mcl-1, HAX1 (Hs-1 associated protein X-1), PARP (poly ADP ribose polymerase), DNA PK (DNA protein kinase), nucleophosmin, topoisomerase 1, nucleolin, HSV 1 ICP4, NUMA (nuclear mitototic apparatus protein), DBP (DNA binding protein), fibronectin, vitronectin, laminin, aggrecan, IL-1 ⁇ , IL18, IL-1 ⁇ , PAR1 (protease activated receptor 1), von Willebrand factor and plasminogen (see e.g., Af
  • Non-limiting examples of peptide sequences shown to be granzyme B cleavage sites from various substrates include those shown below in Table 10: Table 10: Granzyme B Cleavage Sites Accordingly, an appropriate granzyme B-sensitive linker can be designed to incorporate any of the sequences shown in Table 10.
  • bioinformatics tools also have been described in the art that can predict granzyme B cleavage sites within protein sequences. Accordingly, such bioinformatics tools can be used to design an appropriate granzyme B-sensitive linker.
  • Non-limiting examples of such bioinformatics tools include GraBCas (described in Backes et al. (2005) Nucl.
  • protease-sensitive cleavage site within the linker has been described in detail with respect to granzyme B, one of ordinary skill in the art will appreciate that other protease-sensitive linkers can be prepared and used in the amphiphile-antigen conjugate using information readily available in the art on the cleavage sites for other proteases that are present in T cells (e.g., granzyme A, C) or released by tumors into the tumor microenvironment (e.g., MMPs, cathepsins, gelatinases, collagenases and the like, as described above). Such alternative protease-sensitive linkers are also encompassed by the invention.
  • amphiphile-antigen (e.g., peptide) conjugate comprising a protease-sensitive cleavage site positioned between the amphiphilic tail and the antigen (e.g., peptide) can be inserted into the cell surface membrane of an exogenous T cell to thereby tether the conjugate to the T cell.
  • the amphiphile conjugate typically is prepared via a maleimide- thiol reaction, for example between DSPE-PEG2k-maleimide and a peptide with a terminal cysteine (wherein the peptide comprises the MHC-binding antigen and the protease-sensitive linker).
  • Insertion of the amphiphile-antigen conjugate into the cell membrane of the exogenous T cell is accomplished simply by incubation of the amphiphile-antigen conjugate with the cell of interest in PBS over a period of about 30 minutes.
  • Preparation of amphiphile conjugates and insertion thereof into cell membranes is described in further detail in Example 7.
  • Protease cleavage of an amphiphile conjugate containing a linker sequence sensitive to the protease is described in further detail in Example 8. VI.
  • compositions comprising a T cell having tethered to its surface an MHC class I-binding antigen in an inactive form, wherein activation of the T cell releases the antigen in active form such that the antigen can bind MHC class I molecules.
  • the composition comprises a CAR T cell having tethered to its surface an MHC class I-binding peptide in an inactive form, wherein activation of the CAR T cell releases the peptide in active form such that the peptide can bind MHC class I molecules.
  • compositions e.g., T cells, MHC class I-binding antigens, means of tethering the antigens to the T cells
  • the compositions can be formulated such that they are suitable for administration to a subject. Effective Amounts, Regimens, Formulations
  • the compositions are administered in effective amounts.
  • An effective amount is a dosage of the composition sufficient to provide a medically desirable result.
  • the effective amount will vary with the particular condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the concurrent or combination therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner.
  • an effective amount may be that amount that reduces the tumor volume or load (as for example determined by imaging the tumor). Effective amounts may also be assessed by the presence and/or frequency of cancer cells in the blood or other body fluid or tissue (e.g., a biopsy). If the tumor is impacting the normal functioning of a tissue or organ, then the effective amount may be assessed by measuring the normal functioning of the tissue or organ.
  • the invention provides pharmaceutical compositions.
  • compositions are sterile compositions that comprise cells, tethering means (e.g., lipid nanoparticles) and/or antigen(s) (e.g., peptides), preferably in a pharmaceutically- acceptable carrier.
  • pharmaceutically-acceptable carrier means one or more compatible solid or liquid filler, diluents or encapsulating substances that are suitable for administration to a human or other subject contemplated by the invention.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the cells, tethering means (e.g., lipid nanoparticles) and antigen(s) (e.g., peptides) are combined to facilitate administration.
  • compositions when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers.
  • Pharmaceutical parenteral formulations include aqueous solutions of the ingredients.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of ingredients may be prepared as oil-based suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • fatty oils such as sesame oil
  • synthetic fatty acid esters such as ethyl oleate or triglycerides
  • liposomes Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety).
  • any conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.
  • Excipients may include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, and waters of hydration.
  • antiadherents antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, and waters of hydration.
  • excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C,
  • the formulations described herein may include at least one pharmaceutically acceptable salt.
  • pharmaceutically acceptable salts that may be included in a formulation of the disclosure include, but are not limited to, acid addition salts, alkali or alkaline earth metal salts, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
  • Representative acid addition salts include acetate, acetic acid, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzene sulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.
  • the composition is administered to the patient parenterally.
  • suitable routes of parenteral administration include intravenously, intratumorally, intramuscularly, subcutaneously and intraperitoneally. VII.
  • Subjects can be practiced in virtually any subject type that is likely to benefit from localized stimulation of an endogenous T cell response as contemplated herein.
  • Human subjects are preferred subjects in some embodiments of the invention.
  • Subjects also include animals such as household pets (e.g., dogs, cats, rabbits, ferrets, etc.), livestock or farm animals (e.g., cows, pigs, sheep, chickens and other poultry), horses such as thoroughbred horses, laboratory animals (e.g., mice, rats, rabbits, etc.), and the like.
  • Subjects also include fish and other aquatic species.
  • the subjects to whom the compositions of the disclosure are delivered may be normal subjects.
  • Such conditions include cancer (e.g., solid tumor cancers) and infections (particularly infections localized to particular regions or tissues in the body).
  • Tests for diagnosing various of the conditions embraced by the invention are known in the art and will be familiar to the ordinary medical practitioner. These laboratory tests include without limitation microscopic analyses, cultivation dependent tests (such as cultures), and nucleic acid detection tests. These include wet mounts, stain- enhanced microscopy, immune microscopy (e.g., FISH), hybridization microscopy, particle agglutination, enzyme-linked immunosorbent assays, urine screening tests, DNA probe hybridization, serologic tests, etc.
  • a subject having a cancer is a subject that has detectable cancer cells.
  • a subject at risk of developing a cancer is a subject that has a higher than normal probability of developing cancer. These subjects include, for instance, subjects having a genetic abnormality that has been demonstrated to be associated with a higher likelihood of developing a cancer, subjects having a familial disposition to cancer, subjects exposed to cancer causing agents (i.e., carcinogens) such as tobacco, asbestos, or other chemical toxins, and subjects previously treated for cancer and in apparent remission.
  • cancer causing agents i.e., carcinogens
  • Subjects having an infection are those that exhibit symptoms thereof including without limitation fever, chills, myalgia, photophobia, pharyngitis, acute lymphadenopathy, splenomegaly, gastrointestinal upset, leukocytosis or leukopenia, and/or those in whom infectious pathogens or byproducts thereof can be detected.
  • a subject at risk of developing an infection is one that is at risk of exposure to an infectious pathogen.
  • Such subjects include those that live in an area where such pathogens are known to exist and where such infections are common.
  • the subjects also include those that engage in high risk activities such as sharing of needles, engaging in unprotected sexual activity, routine contact with infected samples of subjects (e.g., medical practitioners), people who have undergone surgery, including but not limited to abdominal surgery, etc.
  • the subject may have or may be at risk of developing an infection such as a bacterial infection, a viral infection, a fungal infection, a parasitic infection or a mycobacterial infection.
  • Cancer contemplates administration of the compositions of the disclosure to subjects having or at risk of developing a cancer including for example a solid tumor cancer.
  • the cancer may be carcinoma, sarcoma or melanoma.
  • Carcinomas include without limitation to basal cell carcinoma, biliary tract cancer, bladder cancer, breast cancer, cervical cancer, choriocarcinoma, CNS cancer, colon and rectum cancer, kidney or renal cell cancer, larynx cancer, liver cancer, small cell lung cancer, non-small cell lung cancer (NSCLC, including adenocarcinoma, giant (or oat) cell carcinoma, and squamous cell carcinoma), oral cavity cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer (including basal cell cancer and squamous cell cancer), stomach cancer, testicular cancer, thyroid cancer, uterine cancer, rectal cancer, cancer of the respiratory system, and cancer of the urinary system.
  • NSCLC non-small cell lung cancer
  • Sarcomas are rare mesenchymal neoplasms that arise in bone (osteosarcomas) and soft tissues (fibrosarcomas).
  • Sarcomas include without limitation liposarcomas (including myxoid liposarcomas and pleiomorphic liposarcomas), leiomyosarcomas, rhabdomyosarcomas, malignant peripheral nerve sheath tumors (also called malignant schwannomas, neurofibrosarcomas, or neurogenic sarcomas), Ewing’s tumors (including Ewing's sarcoma of bone, extraskeletal (i.e., not bone) Ewing's sarcoma, and primitive neuroectodermal tumor), synovial sarcoma, angiosarcomas, hemangiosarcomas, lymphangiosarcomas, Kaposi's sarcoma, hemangioendothelioma
  • Melanomas are tumors arising from the melanocytic system of the skin and other organs. Examples of melanoma include without limitation lentigo maligna melanoma, superficial spreading melanoma, nodular melanoma, and acral lentiginous melanoma.
  • the cancer may be a solid tumor lymphoma. Examples include Hodgkin’s lymphoma, Non-Hodgkin’s lymphoma, and B cell lymphoma.
  • the cancer may be without limitation bone cancer, brain cancer, breast cancer, colorectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, cancer of the head and neck, gastric cancer, intra- epithelial neoplasm, melanoma neuroblastoma, Non-Hodgkin’s lymphoma, non-small cell lung cancer, prostate cancer, retinoblastoma, or rhabdomyosarcoma.
  • Infections contemplates administration of the compositions of the invention to subjects having or at risk of developing an infection such as a bacterial infection, a viral infection, a fungal infection, a parasitic infection or a mycobacterial infection.
  • the bacterial infection may be without limitation an E. coli infection, a Staphylococcal infection, a Streptococcal infection, a Pseudomonas infection, Clostridium difficile infection, Legionella infection, Pneumococcus infection, Haemophilus infection, Klebsiella infection, Enterobacter infection, Citrobacter infection, Neisseria infection, Shigella infection, Salmonella infection, Listeria infection, Pasteurella infection, Streptobacillus infection, Spirillum infection, Treponema infection, Actinomyces infection, Borrelia infection, Corynebacterium infection, Nocardia infection, Gardnerella infection, Campylobacter infection, Spirochaeta infection, Proteus infection, Bacteriodes infection, H.
  • the mycobacterial infection may be without limitation tuberculosis or leprosy respectively caused by the M. tuberculosis and M. leprae species.
  • the viral infection may be without limitation a Herpes simplex virus 1 infection, a Herpes simplex virus 2 infection, cytomegalovirus infection, hepatitis A virus infection, hepatitis B virus infection, hepatitis C virus infection, human papilloma virus infection, Epstein Barr virus infection, rotavirus infection, adenovirus infection, influenza A virus infection, H1N1 (swine flu) infection, respiratory syncytial virus infection, varicella- zoster virus infections, small pox infection, monkey pox infection, SARS infection or avian flu infection.
  • the fungal infection may be without limitation candidiasis, ringworm, histoplasmosis, blastomycosis, paracoccidioidomycosis, crytococcosis, aspergillosis, chromomycosis, mycetoma infections, pseudallescheriasis, or tinea versicolor infection.
  • the parasite infection may be without limitation amebiasis, Trypanosoma cruzi infection, Fascioliasis, Leishmaniasis, Plasmodium infections, Onchocerciasis, Paragonimiasis, Trypanosoma brucei infection, Pneumocystis infection, Trichomonas vaginalis infection, Taenia infection, Hymenolepsis infection, Echinococcus infections, Schistosomiasis, neurocysticercosis, Necator americanus infection, or Trichuris trichuria infection.
  • Combination Therapies can further include treatment of the subject with additional agents that enhance therapeutic responses, such as enhance an anti-tumor response in the subject and/or that are cytotoxic to the tumor (e.g., chemotherapeutic agents).
  • additional agents that enhance therapeutic responses such as enhance an anti-tumor response in the subject and/or that are cytotoxic to the tumor (e.g., chemotherapeutic agents).
  • Suitable therapeutic agents for use in combination therapy include small molecule chemotherapeutic agents, including protein tyrosine kinase inhibitors, as well as biological anti-cancer agents, such as anti-cancer antibodies, including but not limited to those discussed further below.
  • Combination therapy can include administering to the subject an immune checkpoint inhibitor to enhance anti- tumor immunity, such as PD-1 inhibitors, PD-L1 inhibitors and CTLA-4 inhibitors.
  • an agent that modulates an immune checkpoint is an antibody.
  • an agent that modulates an immune checkpoint is a protein or small molecule modulator.
  • the agent (such as an mRNA) encodes an antibody modulator of an immune checkpoint.
  • Non-limiting examples of immune checkpoint inhibitors that can be used in combination therapy include pembrolizumab, alemtuzumab, nivolumab, pidilizumab, ofatumumab, rituximab, MEDI0680 and PDR001, AMP-224, PF-06801591, BGB-A317, REGN2810, SHR-1210, TSR-042, affimer, avelumab (MSB0010718C), atezolizumab (MPDL3280A), durvalumab (MEDI4736), BMS936559, ipilimumab, tremelimumab, AGEN1884, MEDI6469 and MOXR0916.
  • Example 1 Peptide Encapsulation in Lipid Nanoparticles
  • a model MHC class I-binding peptide was encapsulated into a lipid nanoparticle and the amount of peptide encapsulated was quantitated.
  • a peptide having the amino acid sequence SIYRYYGL (SEQ ID NO: 1) was used as a model MHC class I-binding peptide.
  • Lipid nanoparticles encapsulating the peptide were synthesized using a 4:3:3 molar ratio of 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide] (18:1 MCC PE):HSPC:cholesterol by extrusion. More specifically, 500 nmol 18:1 MCC PE, 375 nmol HSPC, and 375 nmol cholesterol were combined in chloroform and dried to lipid films.
  • the malemide headgroup of MCC PE is incorporated in the liposomes to enable conjugation to free thiols present on a T cell surface.
  • These films were resuspended in a buffer of 10 mM HEPES, 75 mM NaCl, at pH 6.5 containing 2 mg/mL of the SIYRYYGL peptide (SEQ ID NO: 1), by multiple rounds of vortexing. Mixtures were subjected to six cycles of freezing in liquid N2 and thawing to form unilamellar nanoparticles, then extruded through a 200 nm pore size polycarbonate filter membrane.
  • Example 2 Antigen-Specific Lysis of Lipid Nanoparticles Conjugated to the Surface of CAR T Cells
  • antigen-specific lysis of lipid nanoparticles conjugated to the surface of CAR T cells was demonstrated using lipid nanoparticles loaded with a labeled substance.
  • liposomes encapsulating Alexa Fluor 647-labeled dextran were incubated in suspension with anti- green fluorescent protein (GFP) CAR T cells to allow conjugation via maleimide-thiol reaction (i.e., liposomal malemide groups and free thiols on the CAR T cell surface) prior to neutralization with PEG-SH.
  • GFP green fluorescent protein
  • CAR T cells were incubated in 1 mg/mL liposomes in X-VIVO media for 45 min at 37°C with multiple rounds of vortexing. Cells were resuspended in 1 mg/mL PEG-SH for 20 min at 37°C to neutralize remaining liposomal maleimide groups. Flow cytometry was performed to detect coupling of the AF647-dextran liposomes to the surface of the CAR T cells. Cells were labeled with a live/dead aqua stain to distinguish viable cells. The results are shown in FIG.3B, and indicate a substantial fraction (93.6%) of live cells were positive for AF647-dextran.
  • the AF647-dextran liposome-conjugated GFP-specific CAR T cells were co-cultured for 16 hours with K562 cells expressing either surface (membrane- bound) GFP (sGFP) or intracellular GFP (iGFP).
  • Untransduced T cells i.e., non-GFP- specific T cells conjugated with AF647-dextran liposome were used as a negative control.
  • the fluorophore signal was measured by flow cytometry.
  • FIG.3C The results show that the AF647-dextran signal was diminished after the CAR T cells were co-cultured with cells expressing on their surface the antigen (sGFP) specifically recognized by the CAR T cells, whereas cells expressing the antigen intracellularly (iGFP) did not cause diminution of the AF647-dextran signal.
  • sGFP antigen specifically recognized by the CAR T cells
  • iGFP antigen intracellularly
  • Example 3 Loading of MHC Class-I Binding Peptides onto MHC Class I
  • a model MHC class I-binding peptide was demonstrated to load onto MHC class I molecules expressed on the surface of cells
  • IFN- ⁇ at doses from 1 to 100 Units/ml
  • H-2Kb levels on the cell surface were measured by flow cytometry, using an APC-labeled anti-H-2Kb antibody.
  • the results are shown in FIG.4A, which confirmed upregulation of MHC class I expression by IFN- ⁇ in a dose-dependent manner. Secretion of IFN- ⁇ by CAR T cells in vivo upon antigen-specific activation is expected to act as an in vivo correlate of this in vitro preincubation step.
  • B16F10 cells were first preincubated with 50U/ml of IFN- ⁇ for 16 hours, followed by incubation at 37° C with varying concentrations of the SIYRYYGL (SEQ ID NO: 1) peptide in the culture medium for 1 hour.
  • These particles have a biodegradable poly(lactide-co-glycolide) core and a surface coating of a phospholipid bilayer. These nanoparticles can encapsulate agents in their core and/or incorporate agents in the surface lipid bilayer, enabling sustained release of proteins, peptides, or small-molecule compounds.
  • Nanoparticles were synthesized by a double emulsion/solvent evaporation process: 200 ⁇ L water was emulsified in 1 mL chloroform containing 2 mg of a lipid mixture (4:1 mole:mole DOPC:DOPG with varying quantities of dioleoyl maleimidophenyl phosphoethanolamine (MPB PE), with or without 25 ⁇ g 1,1’-dioctacdecyl-3,3,3’,3’-tetramethylindodicarbocyanine (DiD) or DiR lipid-like fluorescent dye (Invitrogen)) and 30 mg poly(lactide-co-glycolide) (PLGA, 50:50 wt:wt lactide:glycolide, 13 KDa, Lakeshore biopolymers).
  • PLGA poly(lactide-co-glycolide)
  • the lipids in the organic phase self-assemble at the oil-water interface and form a bilayer coating around the nascent PLGA-core particles; excess lipid is also present in the particle bulk.
  • the particles were purified from free lipid by centrifugation through a 60 wt% sucrose cushion, dialyzed to remove sucrose, and stored at 4°C (short term storage) or lyophilized in the presence of trehalose and stored at 4°C until used.
  • Simple variations in the processing conditions e.g., use of homogenization instead of sonication allowed particles of different size to be prepared, as determined by dynamic light scattering (DLS, data not shown).
  • DNA-gel nanoparticles To synthesize DNA-gel nanoparticles, we first generated four-armed DNA junctions, X-DNA monomers, by annealing the following oligonucleotides (Integrated DNA Technology, IDT): 1) 2) 3) 4) These oligos self-assemble into three-dimensional “X” nanostructures with complementary overhangs at the end or each arm. As recently described (Um et al. (2006) Nat. Mater., 5:797), addition of ligase to a solution of these DNA macromers leads to covalent crosslinking and the formation of DNA-base hydrogels.
  • IDT Integrated DNA Technology, IDT
  • X-DNA monomer was then admixed to 6.7 ⁇ l T4 DNA ligase (3 Weiss units/ ⁇ l, Promega), 20 ⁇ l T4 ligase buffer (Promega) and nuclease-free water (IDT) to a total volume of 200 ⁇ l, which was subsequently vortexed with a dry lipid film containing 0.396 mg DOPC, 0.101 mg DOPG, 0.63 mg MPB and 0.04 mg DiD.
  • T4 DNA ligase 3 Weiss units/ ⁇ l, Promega
  • 20 ⁇ l T4 ligase buffer Promega
  • IDT nuclease-free water
  • the resulting DNA gel-lipid mixture was sonicated on ice (5 min total, alternating power cycles of 1 W and 5 Watts every 30s with a Misonix Microson XL probe tip sonicator), and extruded 21 times through a polycarbonate filter (200 nm pore size, Whatman). Following a 3 hour incubation at 25°C and overnight incubation at 4°C to allow ligase- mediated X-DNA crosslinking, 4 ⁇ l Exonuclease III (New England Biolabs), 20 ⁇ l Buffer 1 (New England Biolabs) and nuclease-free water to a total volume of 200 ⁇ l was were added and incubated at 37°C for 90 minutes.
  • DNA-gel nanoparticles were purified from free lipids and DNA by centrifugation through a 10 wt% sucrose cushion, and washed three times with nuclease-free water. A typical yield of 10 10 DNA gel nanoparticles in the 200-250 nm diameter range was measured using a 90Plus Particles Size Analyzer (Brookhaven Instruments).
  • IL-15Sa/IL-21 encapsulation in DNA-gel nanoparticles
  • 30 ⁇ g recombinant mouse IL-15R ⁇ /Fc chimera was precomplexed with 10 ⁇ g mouse IL-15 (Peprotech) in nuclease-free water for 1 hour at room temperature to generate superagonist IL-15 (IL-15Sa), combined with 10 ⁇ g mouse IL-21 (Peprotech) and blended with the X-DNA/T4 ligase mixture for DNA-gel particle synthesis, following the procedure described above.
  • nanoparticles could be simply adsorbed to T cell surfaces stably, by incubating cells with nanoparticles at varying particle:cell ratios for different durations at 4°C or 37°C.
  • PLGA-core nanoparticles could be adsorbed to cells (in varying quantities, depending on the surface charge of the nanoparticles used)
  • T cells were found to have high levels of free thiols at the cell surface.
  • Nanoparticle carriers were prepared which included lipids with maleimide- terminated headgroups.
  • CD8 + T cells were isolated from spleens of pmel-1 TCR- transgenic using magnetic bead negative selection (Miltenyi Biotec) and expanded for 4 days in vitro using anti-CD3/anti-CD28-coated beads in the presence of 200 IU/mL human IL-2, mimicking the preparation of tumor-specific T cells for adoptive cell therapy.
  • T cells were washed and incubated (60 x 10 6 cells/mL) with maleimide- functionalized-nanoparticles (at varying concentrations) at 37°C for 45 min at varying particle:cell ratios.
  • T cells do not internalize lipid-coated PLGA nanoparticles, even during extended culture or following proliferation (discussed further below). This is in stark contrast to what we observed with dendritic cells, which phagocytosed the attached nanoparticles within minutes.
  • Example 5 Cytokine/Drug Loading in Lipid-Coated PLGA Nanoparticles
  • proteins e.g., IL-15 superagonist, TLR ligands
  • PLGA nanoparticles have been explored in numerous prior studies as vehicles for encapsulation and delivery of proteins, peptides, and small molecule drug compounds, and notably vaccine antigens/adjuvants.
  • Measurement of the amount of protein encapsulated was performed by lysing the nanoparticles for 4 hrs in 0.02 M NaOH/2% SDS, neutralizing the solution with 0.2 M HCl, and measuring released ova fluorescence calibrated against ova solution standards exposed to the same base treatment conditions. By these measurements, we found that ⁇ 1 ⁇ g of ova per mg nanoparticles was encapsulated ( ⁇ 25% encapsulation efficiency).
  • Ova is a model globular protein and as such it was chosen to illustrate the behavior of other proteins such as interleukin-15 (IL-15) superagonist molecules which can be used to support ACT T cells.
  • IL-15 interleukin-15
  • IL-15 cytokine alone
  • lipid-coated PLGA lipid-coated PLGA
  • IL-15 5 ⁇ g
  • PBS PBS
  • the kinetics of IL-15 release from the particles was determined by incubating the particles in complete RPMI medium containing 10% FCS at 37°C with gentle agitation and taking aliquots of the supernatant at staggered timepoints for ELISA analysis of cytokine content. The results showed that ⁇ 80% of the encapsulated cytokine was released by the end of this incubation period.
  • the lipid-coated particles can be loaded with protein and release encapsulated material over a ⁇ 1 week period.
  • the release kinetics can be modulated to faster or slower rates by altering the MW of the PLGA used in the particles.
  • Nanoparticles were also loaded with the TLR4 ligand MPLA and/or the TLR7 ligand, imiquimod, as potent clinically-relevant ligands for driving DC activation during T cell adoptive therapy. Due to its lipid-like structure, MPLA is quantitatively incorporated into the particles by simply co-dissolving this ligand with the other phospholipids in the chloroform phase of the particle synthesis.
  • Gardiquimod and resiquimod are imidazoquinoline derivatives that, similar to imiquimod, are selective ligands for TLR7/8. Encapsulation of gardiquimod and resiquimod and detection of their release from PLGA nanoparticles were carried out with minor modifications. For encapsulation of gardiquimod in nanoparticles, 200 ⁇ L water in the synthesis protocol described in section 4.1 was replaced with 1.8 mg of gardiquimod dissolved in 200 ⁇ L of water, and for encapsulation of resiquimod, 0.83 mg of resiquimod was dissolved along with 30 mg of PLGA in organic solvent; the rest of nanoparticle synthesis protocol outlined in section 4.1 was followed thereafter.
  • Example 6 Conjugation of Liposomes to Cells
  • a DOPC/DOPG/MPB PE/DiD lipid film (lipid ratios as in polymer nanoparticles) was hydrated with 185 ⁇ l PBS for a one-hour period with vigorous vortexing every 10 minutes.
  • Both conjugates were fluorescently labeled using fluorescein (FITC); the latter conjugate also contains the peptide C (SEQ ID NO: 208), a peptide derived from the melanoma antigen Trp1.
  • the amphiphile conjugates were prepared via standard maleimide-thiol reaction. To examine the effect of conjugate concentration on membrane insertion, Jurkat cells were incubated with various concentrations (10 nM, 100 nM, 1 ⁇ M) of conjugate in PBS for 30 minutes at 37°C, followed by flow cytometry to detect cell-associated fluorescence. The results are shown in FIG.5A (for D SPE PEG2k FITC) and FIG.5B (for D S G -C VS C).
  • Example 8 Protease Cleavage of Amphiphile Conjugates
  • Amph-IEPD-AMC a conjugate of DSPE-PEG 2k
  • the granzyme-cleavable linker IEPD SEQ ID NO: 209
  • the fluorogenic substrate 7-amino-4-methylcoumarin AMC
  • Amph-GPGD-AMC a control conjugate of DSPE-PEG2k
  • the uncleavable linker GPGD SEQ ID NO: 210) and AMC.
  • Amph-IEPD-AMC and Amph-GPGD-AMC are shown in FIGs.7A and 7B, respectively.
  • 1mM of either Amph-IEPD-AMC or Amph-GPGD-AMC was incubated with various concentrations of recombinant human granzyme B (100 nM, 50 nM, 25 nM, 12.5 nM or 6.25 nM) at 37°C for 8 hours. Fluorescence was measured with excitation 380 nm, emission 460 nm. Fluorescence is quenched for the conjugated substrate, but becomes detectable upon cleavage to release the substrate.
  • FIG.8A for Amph-IEPD- AMC
  • FIG.8B for Amph-GPGD-AMC

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Abstract

L'invention concerne des méthodes et des compositions pour stimuler une réponse de lymphocytes T endogènes chez un sujet, par exemple dans un microenvironnement tumoral pour améliorer la lyse de cellules tumorales. Dans les méthodes et les compositions de l'invention, un antigène de liaison au CMH (par exemple, un peptide de liaison au CMH de classe I) est attaché à une surface de lymphocyte T sous une forme inactive, lors de l'administration et de l'activation du lymphocyte T l'antigène est libéré et se lie à des molécules CMH sur des cellules bystander chez le sujet pour ainsi stimuler une réponse de lymphocyte T endogène.
PCT/US2020/052005 2019-09-23 2020-09-22 Méthodes et compositions pour la stimulation de réponses de lymphocytes t endogènes WO2021061648A1 (fr)

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