WO2023235856A1 - Cellules immunitaires résistantes à l'apoptose avec récepteur antigénique chimérique du complexe majeur d'histocompatibilité - Google Patents

Cellules immunitaires résistantes à l'apoptose avec récepteur antigénique chimérique du complexe majeur d'histocompatibilité Download PDF

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WO2023235856A1
WO2023235856A1 PCT/US2023/067853 US2023067853W WO2023235856A1 WO 2023235856 A1 WO2023235856 A1 WO 2023235856A1 US 2023067853 W US2023067853 W US 2023067853W WO 2023235856 A1 WO2023235856 A1 WO 2023235856A1
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cells
cell
car
mhc
engineered immune
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PCT/US2023/067853
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Julie NORVILLE
Elizabeth Wood
Cameron GARDNER
Kerry DOBBS
Xiao-bing CUI
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Jura Bio, Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • 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
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • MS multiple sclerosis
  • autoreactive T cells invade the blood brain barrier, initiating an inflammatory response that leads to myelin destruction and axonal loss.
  • autoreactive T cells and B cells contribute to the diseases.
  • MHC The major histocompatibility complex
  • HLA human leukocytes
  • MHC complexes bind to antigens derived from pathogens and display such to T cells, which are then activated, leading to elimination of cells displaying foreign antigens.
  • the MHC displays a self-antigen to the T cells and activates such cells, thus set forth the cascade of generating pathogenic autoreactive T cells and B cells.
  • the present disclosure is based, at least in part, on the discovery that exemplary
  • apoptosis inhibitors when expressed in engineered host cells, successfully reduced cell death caused by interaction with effector cells (e.g., NK cells) capable of degranulating or otherwise including cell death of the engineered host cells, which may co-express exogenous HLA molecules.
  • effector cells e.g., NK cells
  • Expression of such apoptosis inhibitors provided some level of protection against cell death induced by NK-like effector
  • immune cells expressing chimeric antigen receptors, including MHC-based chimeric antigen receptors) against cytotoxicity and apoptosis induced by effector immune cells, for example, pathogenic (e.g., autoreactive) T and B cells.
  • effector immune cells for example, pathogenic (e.g., autoreactive) T and B cells.
  • compositions and methods comprising engineered
  • apoptosis-resistant, chimeric antigen receptors (CAR)-expressing immune cells for use in suppressing target disease cells such as autoreactive immune cells.
  • the apoptosis-resistant, CAR-expressing immune cells as disclosed herein can be used for suppressing aberrant immune responses, such as autoimmunity.
  • the apoptosis-resistant immune cells can be engineered to express major histocompatibility complex based chimeric
  • MHC-CAR 25 antigen receptors carrying an antigenic peptide associated with n autoimmune disease.
  • the antigenic peptide loaded MHC-CARs subsequently direct the engineered immune cells expressing such to target and destroy pathogenic immune cells, such as the pathogenic T and B cells involved in autoimmune diseases.
  • the MHC-CAR expressing, apoptosis-resistant, cells can escape cell death in the cytotoxic environment arising from the
  • the engineered CAR-expressing immune cells can remain longer in vivo to execute their own cytotoxic function against the targeted disease cells such as pathogenic (e.g., autoreactive) T and B cells.
  • an engineered immune cell containing: (1) an exogenous apoptosis inhibitor; and
  • an exogenous apoptosis inhibitor refers to apoptosis inhibiting molecules that do not exist or that are not expressed from the genome of the wild-type counterparts of the engineered immune cells.
  • the exogenous apoptosis inhibitor as disclosed herein can be expressed from exogenous nucleic acids encoding such that have been introduced into immune cells to produce the engineered immune cells provided herein.
  • the exogenous nucleic acids may exist in the engineered immune cells extra-chromosomally.
  • the exogenous nucleic acids may be integrated into the genome of the immune cells.
  • the exogenous encoding nucleic acid is integrated at a genomic site that is different from the native loci of the endogenous gene.
  • the apoptosis inhibitor can be a granzyme B inhibitor, for example, a cytokine response modifier A (CRMA).
  • the CRMA may comprise the amino acid sequence of SEQ ID NO: 2.
  • the apoptosis inhibitor can be a serpin, for example, a proteinase inhibitor 9 (PI9).
  • PI9 protein may comprise the amino acid sequence of SEQ ID NO: 4.
  • the apoptosis inhibitor may be a peptidase C 14A, for example, a cellular FLICE inhibitory protein (cFLIP).
  • cFLIP cellular FLICE inhibitory protein
  • the cFLIP may comprise the amino acid sequence of SEQ ID NO: 6.
  • the engineered immune cells may express a combination of any of the apoptosis inhibitors provided herein.
  • the CAR expressed in the engineered immune cell is a major histocompatibility complex based chimeric receptor (MHC-CAR), in which the extracellular antigen binding domain of (i) comprises an extracellular domain of an MHC molecule conjugated to an antigenic peptide.
  • MHC-CAR major histocompatibility complex based chimeric receptor
  • the MHC molecule is a class I MHC molecule, for example, a human class I MHC molecule. Examples include, but are not limited to, an HLA-A, HLA-B, HLA-C, HLA-G, or HLA-E molecule.
  • the MHC molecule is a class II MHC molecule, for example, a human class II MHC.
  • a class II MHC for example, a human class II MHC. Examples include, but are not limited to, HLA-DR2, HLA-DR3, HLA- DR4, HLA-DR9, HLA-DR15, HLA-DP, or HLA-DQ.
  • Exemplary types of the engineered immune cell include a natural killer (NK) cell, a macrophage cell, and a T cell.
  • the engineered immune cell is an NK cell, which can be an NK-92 cell or an KHYG-1 cell.
  • the NK cell can be deficient in killer-cell immunoglobulin- like receptor (KIR).
  • the engineered immune cell can be a T cell.
  • the T cell is a CD8+ T regulatory (T reg ) cell.
  • the T cell can be a CD4+ T reg cell.
  • the CAR e.g. , a MHC-CAR expressed in the engineered immune cell contains at least one co-stimulatory domain and the cytoplasmic signaling domain.
  • the cytoplasmic signaling domain is from CD3 .
  • the engineered immune cell is an NK cell and the CAR expressed therein e.g., an MHC-CAR) contains a co-stimulatory domain of 2B4 (CD244).
  • the engineered immune cell is a macrophage and the CAR expressed therein (e.g., an MHC-CAR) contains a co-stimulatory domain of MegflO or FcRy.
  • the engineered immune cell can be T cells (e.g., the CD8+ or CD4+ T regulatory cell), and the CAR comprises a co-stimulatory domain of CD28 or 4- IBB.
  • the antigenic peptide in the MHC-CAR is of a protein associated with an autoimmune disease.
  • Examples of the protein associated with the autoimmune disease can be found in Tables 3-5 of the application.
  • the present disclosure provides a population of cells comprising a plurality of any of the engineered immune cells as disclosed herein. Also within the scope of the present disclosure is a pharmaceutical composition comprising any of the engineered immune cells disclosed herein or the population of cells comprising such, and a pharmaceutically acceptable carrier.
  • the present disclosure features a method for suppressing disease cells in a subject, the method comprising administering to a subject in need thereof an effective amount of any of the engineered immune cells disclosed here or the pharmaceutical composition comprising such.
  • the engineered immune cell comprises a CAR (e.g., an MHC- CAR) that targets the disease cells.
  • the engineered immune cell is allogenic to the subject.
  • the engineered immune cell is autologous to the subject.
  • the CAR is an MHC-CAR and the disease cells are autoreactive immune cells.
  • the engineered immune cell expressing the MHC-CAR can be used for treating an autoimmune disease in a human patient having such.
  • any of the engineered immune cells disclosed herein or pharmaceutical compositions comprising such for use in suppressing target disease cells (e.g., autoreactive immune cells) and treating corresponding target diseases e.g., autoimmune diseases), and uses of such engineered immune cells for manufacturing a medicament for the intended therapeutic purposes.
  • target disease cells e.g., autoreactive immune cells
  • target diseases e.g., autoimmune diseases
  • the present disclosure contemplates a method for producing a population of the engineered immune cells described herein.
  • a method may comprise: introducing into a plurality of immune cells one or more nucleic acids, which collectively encode the exogenous apoptosis inhibitor and the CAR, thereby producing the engineered immune cells expressing the exogenous apoptosis inhibitor and the MHC-CAR.
  • the one or more nucleic acids are one or more messenger RNA molecules.
  • the one or more nucleic acids are one or more expression vectors, which optionally are viral vectors.
  • FIG. 1 is a bar chart illustrating the effects of exemplary apoptosis inhibitors CRMA
  • PI9, and CFLIP in protecting HEK WT cells from cell death against KHYG-1 effector cells.
  • FIG. 2 is a bar chart illustrating the effects of exemplary apoptosis inhibitors CRMA, PI9, and CFLIP in protecting HEK WT cells from cell death.
  • FIG. 3 is a bar chart illustrating the effects of engineered MHC (eMHC) and exemplary apoptosis inhibitors CRMA, PI9, and CFLIP in protecting HEK WT cells from cell death against KHYG-1 effector cells.
  • eMHC engineered MHC
  • CRMA exemplary apoptosis inhibitors
  • PI9 exemplary apoptosis inhibitors
  • CFLIP exemplary apoptosis inhibitors
  • FIG. 4 is a bar chart illustrating the effects of engineered MHC (eMHC) and exemplary apoptosis inhibitors CRMA, PI9, and CFLIP in protecting HEK WT cells from cell death.
  • eMHC engineered MHC
  • CRMA exemplary apoptosis inhibitors
  • PI9 exemplary apoptosis inhibitors
  • CFLIP exemplary apoptosis inhibitors
  • the success of cell -based therapy depends on the health of the therapeutic cells, e.g., CAR-expressing immune cells such as MHC-CAR-expressing immune cells.
  • CAR-expressing immune cells such as MHC-CAR-expressing immune cells.
  • the MHC-CAR expressing immune cells that target pathogenic T- and/or B-cells can be susceptible to the cytotoxic effects of the pathogenic immune cells, which cause death of the MHC-CAR expressing immune cells via, e.g., induction of apoptosis.
  • the lytic granules directionally released from the activated cytotoxic T-lymphocyte (CTL) carry perforins.
  • CTL cytotoxic T-lymphocyte
  • These molecules target the cell surface of the MHC-CAR expressing immune cells and generate transmembrane pores, through which a second group of proteins, granzymes, can gain entry to the cytosol and induce an apoptotic series of events.
  • the second method occurs by apop to tic signaling via membrane-bound Fas molecules on the target cell surface and Fas ligand on the CTL surface.
  • the processes of antigen recognition, CTL activation, and delivery of apoptotic signals to the target cell can be accomplished within 10 minutes.
  • the apoptotic process in the targeted cell may take 4 hours or more and continues long after the CTL has moved on to interact with other potential targets.
  • the MHC-CAR expressing immune cells can also be vulnerable to the drugs that are used to remove undesired cells associated with diseases, e.g., rapidly growing cancer cells and pathogenic immune cells.
  • diseases e.g., rapidly growing cancer cells and pathogenic immune cells.
  • MTX methotrexate
  • DHFR dihydrofolate reductase
  • Dasatinib a second generation BCR/ABL and Src family tyrosine kinase inhibitor, is used to treat chronic myelogenous leukemia, acute lymphoblastic leukemia, and rheumatoid arthritis.
  • the Src tyrosine kinase is a critical link of multiple signal pathways that regulate proliferation, invasion, and survival. Inhibiting the pathways leads to cell death.
  • Other tyrosine kinese inhibitors include ibrutinib, alpelisib (BYL719), ruxolitinib and tofacitinib.
  • Dexamethasone is a corticosteroid used to treat inflammation.
  • the present disclosure relates to immune cells expressing CAR such as MHC-CAR that are apoptosis resistant. That is, these immune cells can resist or escape cell death that is induced by the targeted pathologic T- or B-cells. These immune cells can also resist or escape cell death that is induced by therapeutic drugs used to treat diseases or disorders, such as
  • apoptosis-resistant strategy is the expression of an apoptosis inhibitor. Accordingly, expression of an exogenous apoptosis
  • the apoptosis inhibitor may be a granzyme inhibitor, a Fas inhibitor, a methotrexate-resistant dihydrofolate reductase (mr-DHFR), a dasatinib inhibitor, a dexamethasone inhibitor, an ibrutinib inhibitor, an alpelisib inhibitor, a ruxolitinib inhibitor, and/or a tofcitibib inhibitor.
  • the apoptosis-resistant immune cell may have more than one
  • the apoptosis-resistant immune cell may have a granzyme inhibitor and a Fas inhibitor.
  • the apoptosis inhibitor would reduce the ability of the pathogenic T- or B-cells to activate the cell death pathways that can induce loss of function of the engineered immune cells.
  • Additional anti-apoptotic proteins including caspase inhibitors, granzyme inhibitors, fasL inhibitors, trail inhibitors described in
  • an apoptosis inhibitor refers to a protein that acts directly or indirectly on the pathway (s) that block programmed cell death (i.e., apoptosis), thereby halting apoptosis.
  • the apoptosis inhibitor can be a naturally occurring protein.
  • the apoptosis inhibitor can be a mutant protein of a naturally occurring protein, the mutant protein conferring resistance to a drug that inhibits the protein in its naturally occurring form.
  • the engineered immune cell described herein expresses a granzyme inhibitor, such as serine proteinase inhibitor 2 (also known as cytokine response modifier A or CRMA), serpin proteinase inhibitor 9 (PI9 or SERPINB9), Serpin Peptidase Inhibitor, Clade B (Ovalbumin), Member 4 (Serpin Family B Member 4; SERPINB4), BCL2 apoptosis regulator, and E3 ubiquitin ligase.
  • a Fas inhibitor such as cFLTP. Further examples of cell death pathways and the proteins that can provide resistance apoptosis are shown in Table 1.
  • the apoptosis inhibitor disclosed herein can be a granzyme inhibitor, for example, an inhibitor of granzyme B or granzyme M.
  • Engineered immune cells expressing such an apoptosis inhibitor would have enhanced resistance to granzyme-mediated cell death, e.g., cell-death mediated by granzyme B or granzyme M.
  • Granzymes are serine proteases released by lytic granules from activated cytotoxic T cells and natural killer (NK) cells. They induce programmed cell death (apoptosis) in the target cell, thus eliminating targeted cells that have become cancerous or are infected with viruses or bacteria. Granzyme-mediated cell death is the major pathway for cytotoxic lymphocytes to kill virus-infected and tumor cells. In humans, five different granzymes (i.e., GrA, GrB, GrH, GrK, and GrM) are known that all induce cell death.
  • Granzyme B is responsible for rapid induction of caspase-dependent apoptosis, promoting caspase activation directly and indirectly, through proteolysis of the Bcl-2 family proteins.
  • human Granzyme B cleaves BH3 interacting domain death agonist (Bid) more efficiently than it cleaves caspases.
  • Proteolysis of Bid by Granzyme B results in the translocation of the C terminus of Bid (tBid) to mitochondria.
  • tBid C terminus of Bid
  • mitochondria outer membrane Granzyme B also cleaves anti- apop to tic Bcl-2 family protein Myeloid cell leukemia sequence 1 (Mcl-1).
  • Cytochrome C mitochondrial Cytochrome c somatic
  • Smac/Diablo Diablo homolog
  • Caspase-3 is the central effector caspase within the Granzyme B-initiated caspase cascade. It completes maturation of Caspase-8 and Caspase-10 and activates Caspase-2, Caspase-6 and Caspase-9.
  • the engineered immune cells disclosed herein express a Granzyme B inhibitor, which can be a cytokine response modifier A (crmA or CRMA; also known as serine proteinase inhibitor 2).
  • CRMA was the first caspase inhibitor discovered. It was a cowpox virus-encoding protein and is a potent inhibitor of Interleukin- 1 beta converting enzyme and related proteases. CRMA inhibits caspase- 1 -dependent cytokine maturation as well as caspase-8 activity, thereby allowing viruses to evade elimination by the host's immune responses or apoptosis of infected cells.
  • an exemplary CRMA may comprise an amino acid sequence at least 80% identical to SEQ ID NO:2, for example, at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 2.
  • the CRMA for expressing in the engineered immune cells disclosed herein comprises (e.g., consisting of) the amino acid sequence of SEQ ID NO: 2.
  • the “percent identity” of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J.
  • the engineered immune cells disclosed herein express an apoptosis inhibitor, which is a serpin.
  • Serpins are a superfamily of proteins with similar structures that were first identified for their protease inhibition activity and are found in all kingdoms of life.
  • the acronym serpin was originally coined because the first serpins to be identified act on chymotrypsin-like serine proteases (serine protease inhibitors). They are notable for their unusual mechanism of action, in which they irreversibly inhibit their target protease by undergoing a large conformational change to disrupt its active site.
  • Expression of intracellular serine protease inhibitors is one of the mechanisms by which tumor cells evade cytotoxic lymphocyte-mediated killing. Intracellular expression of SERPINB9 by 5 tumor cells renders the tumor cells resistant to GrB-induced apoptosis.
  • SERPINB4 is also shown to be effective against granzyme M-induced cell death (P. J. A. de Koning, 2011, PLOS ONE, 6(8): e22645).
  • SERPINB9 is an endogenous inhibitor of interleukin 1 betaconverting enzyme (caspase- 1) activity in human vascular smooth muscle cells. These are important inhibitors of serine proteases-mediated cell death.
  • SERPINB4 is a protease
  • the serpin can be proteinase inhibitor 9 (PI9, also known as SERPINB9) belongs to the large superfamily of serine proteinase inhibitors (serpins), which bind to and inactivate serine proteinases. These interactions are involved in many cellular i processes, including coagulation, fibrinolysis, complement fixation, matrix remodeling, and apoptosis.
  • PI9 proteinase inhibitor 9
  • an exemplary P19 protein may comprise an amino acid sequence at least 80% identical to SEQ ID NO: 4, for example, at least 85%, at least 90%, or at least 95%, identical to SEQ ID NO: 4.
  • the PI9 protein may comprise (e.g., consisting of) the amino acid sequence of SEQ ID NO: 4.
  • the engineered immune cells disclosed herein express an apoptosis inhibitor, which is a FAS inhibitor.
  • the cell-surface Fas receptor also termed Apo-1 or CD95, is a member of the tumor necrosis factor (TNF) and nerve growth factor (NGF) family of receptors.
  • FasL Upon interacting with its ligand, FasL, the consequential intracellular signaling is initiated and cell death follows. The activation of the Fas pathway
  • CFLAR also known as cellular FLICE inhibitory protein or cFLIP
  • CFLAR also known as cellular FLICE inhibitory protein or cFLIP
  • cFLIP belongs 30 to the peptidase C14 family and more specifically peptidase C14A subfamily, which includes members CASP1, CASP2, CASP3, CASP4, CASP5, CASP6, CASP7, CASP8, CASP9, CASP10, CASP12, CASP14, and CFLAR/cFLIP.
  • the peptidase C14A subfamily has been known to be play a central role in regulating apoptosis.
  • cFLIP is structurally similar to caspase-8 but the protein lacks caspase activity and appears to be itself cleaved into two peptides by caspase-8.
  • CFLAR/cFLIP is a master anti- apoptotic regulator and resistance factor that suppresses tumor necrosis factor-a (TNF-a), Fas-L, and TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, as well as apoptosis triggered by chemotherapy agents in malignant cells.
  • TNF-a tumor necrosis factor-a
  • Fas-L Fas-L
  • TRAIL TNF-related apoptosis-inducing ligand
  • CFLIP is expressed as long (cFLIP(L)), short (cFLIP(S)), and cFLIP(R) splice variants in human cells.
  • cFLIP binds to FADD and/or caspase-8 or -10 and TRAIL receptor 5 (DR5) in a ligand-dependent and - independent fashion and forms an apoptosis inhibitory complex (AIC). This interaction in turn prevents death-inducing signaling complex (DISC) formation and subsequent activation of the caspase cascade.
  • cFLIP(L) and cFLIP(S) are also known to have multifunctional roles in various signaling pathways, as well as activating and/or upregulating several cytoprotective and pro-survival signaling proteins including Akt, ERK, and NF-kB. Upregulation of cFLIP has been found in various tumor types, and its silencing has been shown to restore apoptosis triggered by cytokines and various chemotherapeutic agents.
  • an exemplary cFLIP protein may comprise an amino acid sequence at least 80% identical to SEQ ID NO: 6, for example, at least 85%, at least 90%, or at least 95%, identical to SEQ ID NO: 6.
  • the cFLIP protein may comprise (e.g., consisting of) the amino acid sequence of SEQ ID NO: 6.
  • the apoptosis inhibitor provided herein may be an BCL2 protein.
  • BCL2 protein was first discovered in follicular B-cell lymphoma where a translocation of the BCL2 gene (otherwise B-cell lymphoma 2 gene, bcl-2) enhanced the BCL2 protein transcription and was found to inhibit cell death.
  • BCL2 apoptosis by the preservation of mitochondrial membrane integrity as its hydrophobic carboxyl-terminal domain is linked to the outer membrane.
  • BCL2 prevents and inactivates several apoptogenic molecules oligomerization.
  • BCL2 also regulate the activation of several initiator caspases like caspase-2 that act upstream or independently of cytochrome c release from mitochondria.
  • BCL2 directly blocks cytochrome c release and therefore prevents APAF- 1 and caspase-9 activation.
  • the apoptosis inhibitor provided herein may be an E3 ligase inhibitor.
  • the ubiquitin-proteasome system (UPS) consists mainly of E3 ligases and deubiquitinating enzymes (DUBs) are the key regulator of the apoptosis process by regulating the pro- or anti- apopto tic proteins and dictate the cell survival vs. death.
  • E3 ubiquitin ligases are the ultimate enzymes involved in the transfer of ubiquitin to substrate proteins. The addition of ubiquitin on to the substrate proteins destine the substrate proteins for degradation by the proteasome.
  • the apoptosis inhibitor may be a B cell blocker.
  • Anti-B cell antibody can inhibit B cell activation.
  • antibody encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) mutants, multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, VHH antibody (or nanobody) antigen binding fragment of heavy chain only antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity.
  • An antibody can be of any of the five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy -chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively.
  • the different classes of immunoglobulins have different and well known subunit structures and three-dimensional configurations.
  • an anti-B cell antibody examples include rituximab, ocrelizumab, ofatumumab, eculizumab, adalimumab, tocilizumab, teplizumab, and ublituximab.
  • Engineered immune cells expressing additional exemplary apoptosis inhibitors are provided below.
  • an engineered immune cell comprising a mr- DHFR and an MHC-CAR.
  • the engineered immune cell would have enhanced resistance to apoptosis sensitizers such as MTX.
  • Antifolates such as MTX, are the treatment of choice for numerous cancers and select autoimmune diseases.
  • MTX inhibits dihydrofolate reductase (DHFR), which is essential for cell growth and proliferation.
  • DHFR dihydrofolate reductase
  • Mammalian cells can acquire resistance to antifolate treatment through a variety of mechanisms but decreased antifolate titers due to changes in drug efflux or influx, or alternatively, the amplification of the DHFR gene are the most commonly acquired resistance mechanisms.
  • resistant phenotypes are associated with DHFR mutations, creating a mr-DHFR.
  • An example of a mr- DHFR is one that has reduced binding affinity to methotrexate due to a mutation of the leucine amino acid residue at position 22 (L22) and/or a mutation of the phenylalanine amino acid residue at positions 31 (F31).
  • the reference molecule is non- mutated DHFR.
  • the mutation of L22 and/or F31 of mr-DHFR may be a substitution, optionally, the amino acid substitution at L22 is L22F, L22P, or L22Y and/or the amino acid substitution at F31 is F31G or F3 IS.
  • the engineered immune cell described herein comprises a mr-DHFR having at least one of the following mutation: L22F, F31S, L31Y, F31S, L22 F, F31G, L22Y, and F31G.
  • the mr-DHFR has one of the following pairs of mutations: L22F and F31S; L22Y and F31S, L22F and F31G; L22P and F31G; L22Yand
  • the mr-DHFR is not inhibited by MTX at the dosage that would have inhibited a non-mutated wild type DHFR.
  • the binding affinity of the mutated DHFR and the consequential conferred protection from cytotoxity can be indirect measured by any method known in the art. For example, as described by Ercikan-Abali, I.R. et al., 1996, Cancer Research’ 56:4142-4145. Briefly, cells containing the vectors encoding the different i o DHFR variants can be exposed to various concentration of MTX for several days in a 96 well format at 400 cells/well.
  • the medium was replaced 24 h later with medium containing MTX at various concentrations, and then cells were cultured for 5 additional days. Cytotoxicity was measured by a colorimetric assay, for example using tetrazolium compounds such as 3- [4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and sodium 3,3'- ib [l[(phenylamino)carbonyl]-3,4- tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT).
  • MTT 3- [4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
  • XTT sodium 3,3'- ib [l[(phenylamino)carbonyl]-3,4- tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate
  • an engineered immune cell comprising a
  • an engineered immune cell comprising an inhibitor of dasatinib, nilotinib, imatinib, dexamathesome, alpelisib, ibrutinib, ruxolitinib, or tofacitinib and an MHC-CAR.
  • the engineered immune cell would have enhanced resistance to apoptosis sensitizing tyrosine kinase inhibitors such as dasatinib, nilotinib, imatinib, alpelisib ibrutinib, ruxolitinib, and tofacitinib, or increased
  • the engineered immune cell having a dasatinib inhibitor has an IC50 of at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold
  • the drug IC50 may be determined by any methods known in the art, such as those taught by S. Soverini et al., 2007, Haematologica; 92:401-404.
  • Dasatinib is a second generation tyrosine kinase inhibitor that is used for the treatment of chronic myeloid leukemia or Philadelphia chromosome-positive acute lymphoblastic leukemia. Dasatinib exhibits more durable hematological and cytogenetic effects and greater potency than the first-generation tyrosine kinase inhibitor imatinib.
  • Examples of a dasatinib inhibitor is a BCR-ABL fusion protein, ATP Binding Cassette Subfamily B Member 1 protein (ABCB1), and ATP-binding cassette super- family G member 2 protein (ABCG2), optionally, the BCR-ABL fusion protein has a threonine to isoleucine change at codon 315 (T315I) mutation, a threonine to alanine change at codon 315 (T315A) mutation and/or a phenylalanine to isoleucine change at codon 317 (F317I).
  • T315I threonine to isoleucine change at codon 315
  • T315A threonine to alanine change at codon 315
  • F317I phenylalanine to isoleucine change at codon 317
  • BCR-ABL is a mutation that is formed by the combination of two genes, known as BCR and ABL.
  • BCR and ABL nowell P, Hungerford D. 1960 Science 132: 1497; S. Salesse and C. M. Verfaillie, 2002, Oncogene 21: 8547-8559.
  • a balanced translocation occurs between chromosome 9 and 22 which leads to the formation of the chimeric gene BCR/ ABL on chromosome 22 and a reciprocal ABL/BCR on chromosome 9.
  • the ABL/BCR gene although transcriptionally active, although no ABL/BCR protein has, as yet, been identified.
  • BCR/ ABL genes can be formed and the most common is a 210 kDa cytoplasmic fusion protein, p2 io BCR7ABL (ABX82702.1), which is deregulated and is a constitutively active tyrosine kinase.
  • p2 io BCR7ABL ABX82702.1
  • Select mutations at residues 315 and 317 in the BCR-ABL kinase domain are associated with resistance to dasatinib in Philadelphia-positive leukemia patients (S. Salesse and C. M. Verfaillie, Supra).
  • the exogenous BCR/ ABL is overexpressed in the engineered immune cells described herein.
  • the level of overexpression is at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or more over the level of expression of the endogenous BCR or ABL.
  • P-glycoprotein 1 permeability glycoprotein, abbreviated as P-gp or Pgp
  • MDR1 multidrug resistance protein 1
  • ATP-binding cassette sub-family B member P-glycoprotein 1 (permeability glycoprotein, abbreviated as P-gp or Pgp) also known as multidrug resistance protein 1 (MDR1) or ATP-binding cassette sub-family B member
  • ABCB1 (NP_000918.2) or cluster of differentiation 243 (CD243) is an important protein of the cell membrane that pumps many foreign substances out of cells. More formally, it is an ATP-dependent efflux pump with broad substrate specificity. It exists in animals, fungi, and bacteria, and it likely evolved as a defense mechanism against harmful substances. Overexpression of ABCB 1 in the engineered immune cells described herein allow these cells to remove the dasatinib inhibitor before the inhibitor cause irreversible damage to the cells, and thereby facilitating the survival of the engineered immune cells in vivo during dasatinib treatment.
  • the ABCB 1 expressed has a mutation at amino acid position 5 1199.
  • the mutation is a substitution, for example, mutation G1199A.
  • the exogenous ABCB1 is overexpressed in the engineered immune cells described herein.
  • the level of overexpression is at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or more over the level of expression of the endogenous ABCB1 .
  • the exogenous ABCB 1 that is overexpressed is a mutant ABCB 1 as i o disclosed herein.
  • the ATP-binding cassette transporter G2 (ABCG2; also known as breast cancer resistance protein, BCRP) (NM_001257386.2) is similar to other ABC transporters such as ABCB1 (P-glycoprotein).
  • ABCG2 excretes a variety of endogenous and ib exogenous substrates including chemotherapeutic agents, such as mitoxantrone and several tyrosine kinase inhibitors.
  • chemotherapeutic agents such as mitoxantrone and several tyrosine kinase inhibitors.
  • ABCG2 is expressed on the apical membranes and plays a pivotal role in tissue protection against various xenobiotics.
  • the ABCG2 expressed has a mutation at amino acid position 141 or 482 or both positions 141 and 482.
  • the mutation is a substitution, for example, mutation Q141K and R482G.
  • the exogenous ABCG2 is overexpressed in the engineered immune cells described herein.
  • the level of overexpression 25 is at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or more over the level of expression of the endogenous ABCG2.
  • the exogenous ABCG2 that is overexpressed is a mutant ABCG2 as disclosed herein.
  • Dexamethasone is a corticosteroid used in a wide range of conditions for its antiinflammatory and immunosuppressant effects. In immunotherapy, CAR-T cell-mediated side 3 o effects such as cytokine release syndrome are mitigated through administration of dexamethasone.
  • An example of a dexamethasone inhibitor for the present invention is a mutant nuclear receptor subfamily 3 group C member 1 (NR3C1) (NM_000176.3) (also known as glucocorticoid receptor) where the protein that has mutations at the amino acid positions 477, 559, 676, 714, and/or 753.
  • the mutations are substitutions such as L753F, R714Q, I559N, R477H, and R679S. See A. Molnar et al., BMC Medical Genetics volume 19, Article number: 37 (2018).
  • the NR3C1 mutation is a homozygous mutation or a heterozygous mutation in the engineered immune cell, that is, the NR3C1 has mutations on both the alleles in the cell or on only one allele in the cell.
  • Ibrutinib is an inhibitor of Bruton's tyrosine kinase (BTK).
  • BTK Bruton's tyrosine kinase
  • Ibrutinib is a first- generation BTK inhibitor that is FDA approved to treat various B-cell malignancies and to prevent chronic graft-versus-host disease in stem cell transplant recipients.
  • BTK also known as tyrosine-protein kinase BTK, is a tyrosine kinase that is encoded by the BTK gene in humans.
  • BTK plays a crucial role in B cell development as it is required for transmitting signals from the pre-B cell receptor that forms after successful immunoglobulin heavy chain rearrangement. It also has a role in mast cell activation through the high-affinity IgE receptor.
  • an ibrutinib inhibitor is a mutant BTK (NM_000061.3) that has a mutation at amino acid position 481.
  • the mutation is a substitution such as C481S. See J. A Woyach et al., 2014, New England Journal of Medicine 370(24).
  • Alpelisib (BYL719) is an orally bioavailable, small-molecule, a-specific Phosphoinositide 3-kinase (PI3K) inhibitor that selectively inhibits pl 10a approximately 50 times as strongly as other isoforms.
  • PI3K is a group of plasma membrane-associated lipid kinases, consisting of three subunits: p85 regulatory subunit, p55 regulatory subunit, and pl 10 catalytic subunit.
  • An example of an alpelisib inhibitor is phosphatidylinositol-4,5- bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) (NM_006218.3) that has a mutation at amino acid position 545. In one embodiment, the mutation is a substitution such as E545K. See H. Blesinger et al., PLOS, 2018, ONE 13(7): e0200343.
  • Ruxolitinib and tofacitinib are Janus kinase (JAK) inhibitors that interfere with phosphorylation of signal transducer and activator of transcription (STAT) proteins that are involved in vital cellular functions, including signaling, growth, and survival.
  • Ruxolitinib is an oral JAK inhibitor selective for JAK1 and JAK2.
  • Tofacitinib is the prototypical JAK inhibitor, predominantly selective for JAK1 and JAK3, with modest activity against JAK2, and, as such, can block signaling from gamma-chain cytokines (e.g., IL-2, IL-4) and gp 130 proteins (e.g., IL-6, IL-11, interferons).
  • Tofacitinib is also FDA approved for the treatment of psoriatic arthritis, juvenile idiopathic arthritis, and ulcerative colitis.
  • Example of a ruxolitinib inhibitor or a tofacitinib inhibitor is a mutant Janus kinase 2 (JAK2) (NM_001322194.2) having mutations at amino acid position 931 or 935 or at both positions. In some embodiments, the mutations are substitutions such as Y931C or G935R. See N. Kopp et al., 2013, Blood (2013) 122 (21): 1429.
  • the dasatinib inhibitor, dexamethasone inhibitor, ibrutinib inhibitor, alpelisib inhibitor, ruxolitinib inhibitor or tofacitinib inhibitor described herein is expressed under the control of an inducible promoter.
  • mammalian inducible promoters examples include tetracycline responsive promoters, albumin, lymphoid specific promoters, T-cell promoters, neurofilament promoter, pancreas specific promoters, milk whey promoter; hox promoters, a-fetoprotein promoter, human LIMK2 gene promoters, FAB promoter, insulin gene promoter, transphyretin promoter, alpha.1 -antitrypsin promoter, plasminogen activator inhibitor type 1 (PAI-1) promoter, apolipoprotein myelin basic protein (MBP) promoter, GFAP promoter, OPSIN promoter, NSE promoter, tetracycline promoter, metallothionine promoter, ecdysone promoter, a mammalian virus promoter, steroidresponsive promoters, rapamycin responsive promoters, as well as mammalian virus promoter such as an adenovirus late promoter or a
  • the genes encoding these exemplary granzyme inhibitors are disclosed in Table 1.
  • the encoding genes of the granzyme inhibitors are cloned into expression vectors known in the art and transfected into the immune cells.
  • the expression vectors may integrate into the genome of the immune cell.
  • the granzyme inhibitors are placed under the control of an inducible promoter so that the expression of the granzyme inhibitors may be regulated. Examples of an inducible promoter system in mammals is the tetracycline on/off system. Examples of dihydrofolate reductase mutants that can provide resistance to methotrexate induced apoptosis sensitization are provided in Table 2.
  • the engineered immune cell described herein also comprise a chimeric antigen receptor (CAR), such as a MHC based chimeric receptor (MHR- CAR).
  • CAR chimeric antigen receptor
  • MHR- CAR MHC based chimeric receptor
  • the MHC-CAR described herein comprises an MHC moiety, which is conjugated to an antigenic peptide (e.g. , a misfolded one), and at least one cell signaling moiety, which can be a cytoplasmic signaling domain (e.g., that of CD3Q, one or more co-stimulatory domains (e.g., that of 2B4, MegflO, 41BB, CD28, or FcRy), or a combination thereof.
  • cytoplasmic signaling domain e.g., that of CD3Q
  • co-stimulatory domains e.g., that of 2B4, MegflO, 41BB, CD28, or FcRy
  • the antigenic peptide can be part of a fusion polypeptide of the CAR. In other instances, the antigenic peptide does not form a fusion polypeptide with the CAR but forms a complex with the CAR.
  • conjugated means that at least two components are physically associated, either via covalent bonds or via non-covalent interactions.
  • the CAR described herein may be a multi-chain protein complex, for example, a heterodimer, comprising one polypeptide that comprises the antigenic peptide.
  • an exogenous cytokine moiety is included as a fusion polypeptide with the antigen.
  • the antigenic peptide or polypeptide may be expressed as a separate polypeptide, which may form a complex (e.g., a trimer) with the MHC components.
  • the antigenic polypeptide can be a misfolded antigenic protein that binds to the MHC.
  • the CAR may further comprise a hinge domain, which may be adjacent to the antigenic peptide and/or the MHC moiety, a signal peptide at the N-terminus, and/or one or more tagging sites, for example, a histidine protein tag and/or an RQR domain that additionally acts as a kill-switch site.
  • a kill switch as used in this disclosure is a safety mechanism used to shut off expression of exogenous gene in an emergency, when it cannot be shut down in the usual manner.
  • MHC-CAR expressed in these immune cells are specially designed to be expressed and function in cytotoxic host cells such as natural killer (NK) cells, macrophages, monocytes or CD8 T regulatory cells for targeting autoreactive immune cells such as autoreactive T cells and B cells.
  • MHC-CAR may comprise one or more MHC polypeptides or an extracellular domain thereof and one or more cell signaling domains, for example, a cytoplasmic signaling domain (e.g., that from CD3Q, at least one co-stimulatory domain (e.g., that from 2B4, CD28, 41BB, MegflO or FcRy), or both.
  • the CAR may further comprise an antigenic peptide from an autoantigen or a foreign antigen that mimics an autoantigen in eliciting autoimmune responses.
  • cytotoxic immune cells may be modified with chimeric antigen receptor(s) targeting T cell and/or B cell surface markers, such as CD 19 or CD 20, either alone or in combination with any of the MHC-CARs disclosed herein.
  • the genetically modified cells may be used to inhibit pathogenicity at an early stage of a target disease, to control disease progression at a middle stage of the disease or to suppress pathology via, e.g., inducing cytotoxicity of pathologic CD8+ T cells at a late stage of an autoimmune disease.
  • the engineered immune cell described herein may be irradiated to limit its self-proliferation and the time window for activation of these cells expressing CAR. In some instances, these irradiated cells can still target pathogenic cells. (i) Components of CARs
  • the CAR constructs disclosed herein comprise an MHC moiety, which may comprise one or more MHC polypeptides or an extracellular domain thereof.
  • the MHC moiety may be derived from a suitable source, for example, human or a non-human mammal (e.g., monkey, mouse, rat, rabbit, pig, etc.)
  • the MHC moiety is from a human MHC molecule (also known as HLA).
  • the domains that interact with molecules from other cells are from a human MHC molecule.
  • TCR or BCR are from a human MHC molecule.
  • MHC class I molecules MHC class II molecules, both of which can be used for constructing the CARs described herein.
  • MHC class I and class II molecules of various species are available from public gene datasets, for example, the IPD-MHC database and the IMGT/HLA database provided by EMBL-EBI and the dbMHC database provided by National Center for Biotechnology Information (NCBI).
  • MHC class I molecules are heterodimers containing an alpha chain and P- microglobulin. The extracellular domain of an alpha chain includes three subdomains, al, a2, and a3.
  • the MHC moiety may include the alpha chain of a MHC class I molecule, or an extracellular domain thereof, for example, the al domain, the a2 domain, the a3 domain, or a combination thereof.
  • the MHC class I molecule may be a human HLA-A molecule, a human HLA-B molecule, or a human HLA-C molecule.
  • the alpha chain of the MHC class I molecule may be fused with -microglobulin to produce a single chain fusion protein.
  • the MHC Class I moiety is from HLA A3, which can be co-used with a PLP peptide. Honma et al., J. Neuroimmunol.
  • the MHC Class I is from HLA A2, which can be used with the same PLP peptide and display of a viral peptide such as TAX.
  • TAX is from the protein tax or p40 (Genbank accession no. BAB20130.1) that is a molecular mimic of a human neuronal protein and from the HTLV-1 virus, which is implicated in diseases such as rheumatoid arthritis, system lupus erythematosus, and Sjogren’s syndrome.
  • Garboczi et al. The Journal of Immunology, 157(12):5403-5410, 1996. Quaresma, et al., 2015. Viruses, 8(1):5 2015.
  • the class 1 protein and peptide may additionally contain modifications to enable more robust peptide loading such replacement of the invariant tyrosine at position 84 of the heavy chain with alanine; or alternatively the position 84 tyrosine can be replaced with cysteine as can the second position of the peptide-p2m linker to create a disulfide trap. Hansen et al. Trends in immunology, 31(10):363 (2010).
  • MHC class II molecules are also heterodimers consisting of two homogenous peptides, an a-chain and a P-chain.
  • the extracellular domain of each of the a-chain and the P-chain contains two subdomains al/a2, and pi/p2.
  • the MHC moiety may include two subunits, one including the a-chain or a portion thereof, for example, an extracellular domain thereof (e.g. , al, a2, or both), the other including the b-chain or a portion thereof, for example, an extracellular domain thereof (e.g., pi, P2, or both).
  • the MHC class II molecule may be a human HLA
  • the MHC class II molecule is a human HLA DR molecule, for example HLA DR*1501. Certain HLAs are associated with autoimmune disease. See Table 5 below. Hence, the HLA selected for the MHC may be the ones associated with the autoimmune disease for which the CAR is designed to treat.
  • any configuration of artificial MHCs known in the art is contemplated for constructing the MHC-CAR.
  • class I single chain trimer as disclosed US8895020, US20190201443, Kotsiou E, et al., Antioxid Redox Signal. 2011 ; 15(3):645- 655)
  • class II single chain trimer as disclosed in Zhu X, et al., Eur J Immunol. 1997 Aug;27(8):1933-41
  • disulfide trap MHC class I and MHC class II molecules as disclosed in US8992937 and US20180127481).
  • the contents of these references are incorporated herein by reference in their entireties.
  • the engineered immune cell of the invention comprises an engineered MHC (eMHC) moiety having an amino acid sequence at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 7.
  • eMHC engineered MHC
  • the antigenic peptides of the CAR described herein are an antigenic peptide that is recognizable by pathogenic immune cells (e.g., autoreactive T cells or B cells) involved in an autoimmune disease. When presented by a suitable MHC molecule, such an antigenic peptide would interact with the antigen- specific T cell receptors of pathogenic T cells, leading to downstream immune responses .
  • pathogenic immune cells e.g., autoreactive T cells or B cells
  • a specific antigenic peptide can be designed for a specific autoimmune disease patient such as an MS patient, using methods known in the art.
  • Programs like NetMHC enable personalized design of antigenic peptides that are specific to the patients MHC, and have been used to develop personalized cancer vaccines.
  • personalized CAR T and Treg therapies for autoimmune disorders are also within the scope of the present disclosure.
  • a personalized therapy can be utilized to treat a large patient class at different stages of the disease. Recent studies have also demonstrated that Class II MHCs and specifically the HLAs implicated in autoimmune disorders can display entire antigenic proteins rather than just processed peptides.
  • the antigenic peptides used herein may be fragments of autoantigens involved in autoimmune diseases, for example, myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), and proteolipid protein (PLP) involved in multiple sclerosis, insulin and glutamate decarboxylase (GAD) involved in type I diabetes,
  • MBP myelin basic protein
  • MOG myelin oligodendrocyte glycoprotein
  • PGP proteolipid protein
  • GAD glutamate decarboxylase
  • the antigenic peptide can be a fragment of a pathogen protein such as a viral or a bacterial protein that is highly homologous to a self-antigen involved in an autoimmune disease. Such an antigenic peptide also can target pathogenic T cells. If needed, the antigenic peptide can be a (typically misfolded) antigenic protein or protein fragment that can be i o expressed separately and binds directly to the MHC moiety of a CAR described herein.
  • the antigenic peptides for use in the CAR described herein may contain up to 20 amino acid residues, the extracellular domain of the antigenic protein, or the full-length antigenic protein. When co-used with a MHC class I moiety, the antigenic peptide may be 8- 25 10 amino acid-long. Such antigenic peptides would fit well into the peptide binding site of a
  • Antigenic peptides to be co-used with MHC class II moieties can be longer, for example, containing 15-24 amino acid residues or up to the full length of the antigenic protein, since the antigen-binding groove of MHC class II molecules is open at both ends, while the corresponding antigen-binding groove on class I molecules is usually closed at each end.
  • antigenic peptide may be derived from known autoantigen associated with autoimmune diseases. Exemplary of such autoantigen can be found in WO 2019/094847 and disclosed in Table 3 below.
  • the antigenic peptide may be composed of multiple peptides (with binding affinity to class I or class II MHC can also be created that are linked with glycine serine linkers, in the case where it is desirable to overstimulate the targeted cells, in our desired application using one or more autoimmune epitopes, and potentially recoding the epitopes to avoid recombination since for this implementation they will be encoded by a vector. Falk, K., Rdtzschke, O. and Strominger, J.L., 2000. Antigen-specific elimination of T cells induced by oligomerized hemagglutinin (HA) 306-318. European journal of immunology, 30(10), pp.3012-3020.
  • the antigenic peptides that bind HLA-E type MHC CAR, or HLA-E for use in suppressing receptors include peptides from HSP-60, bacterial heat shock protein HSP-65 from mycobacterial tuberculosis, peptides from HLA, peptides from TCR-VP, and peptides from CMV such as VMAPRTLIL (SEQ ID NO: 17) from UL40 that in combination with HLA-E*01:01 and HLA-E*01:03 binds to the NKG2A/CD94 receptor.
  • HLA-E also interact with HLA-E bound to the VMAPRTLFL (SEQ ID NO: 22) peptide derived from HLA-G.
  • Table 4 discloses antigenic peptide epitopes that commonly bind to HLA-E.
  • the CAR described herein comprises an antigenic peptide epitope disclosed in Table 4.
  • a HLA- E-based MHC-CAR can bind NKG2A receptors and enable their suppression. If the NK cell line is autologous, it is ideal that it has mismatches that prevents negative selection of CMV epitopes presented by HLA-E.
  • an NK cell line for treatment should not contain (HLA-A: *02, *10, *23, *24, *25, *26, *28, *34 *43, *66, *68, or *69) and should be mismatched against those alleles, as these can block NK cell recognition through the NKG2A receptor.
  • Haploidentical NK cells may help eliminate EBV, as EBV infected B cells are resistant to autologous NK cells, until lytic infection.
  • Table 5 below provides HLA and classes commonly associated with autoimmune disorders though in the exemplary case the HLA or a portion of the HLA will be patient specific and derived from a high-resolution sequence of the patient suffering from the disorder or a serological equivalent.
  • the antigenic peptides or antigenic polypeptides are patient specific and designed for the patient’ s MHC.
  • a physician can diagnose the patient with an autoimmune disorder and determine the severity of the disease.
  • the patient’s Class I (HLA-A, B, C) and II (HLA-DR, DQ, DP) regions can be typed, which can now be performed at high resolution using DNA sequencing and with comparison to a reference database (www.ebi.ac.uk/ipd/imgt/hla/). Kir regions can also be typed.
  • the patient’s Class I and II MHC with the strongest evidence of autoimmune involvement can be identified for the disorder. Those known to be associated with a particular autoimmune disorder can be used as references. See, e.g., Tables 4 and 5.
  • Personalized CARs lenti virus, transposon vectors and transposase, or mRNA can be prepared for the patient to enable targeting of pathogenic immune cells in the patient.
  • the personalized lentivirus, transposon vectors and transposes, or mRNA is used to prepare autologous or allogeneic engineered immune cells that can be combined with additional cellular modifications or cell treatments for various purposes. For example, cell treatment
  • expand refers to increasing in number, as in an increase in the number of engineered immune cells described herein or the targeted pathogenic T cells.
  • Cellular modifications to reduce the natural expression of endogenous molecules that stimulate pathogenic immune cells in the patient e.g. , endogenous MHC and co-stimulatory molecules for pathogenic cells, the endogenous MHC and co-stimulatory molecules are produced from the engineered immune cells innately) or the secretion of cytokines (to induce cell proliferation of the engineered immune cells and/or induced activation or increase cytotoxic potency).
  • the CAR described herein may comprise one or more co-stimulatory signaling domains.
  • co-stimulatory signaling domain refers to at least a portion of a protein that mediates signal transduction within a cell to induce an immune response such as an effector function.
  • the co-stimulatory signaling domain of the CAR described herein can be a cytoplasmic signaling domain from a co- stimulatory protein that transduces a signal and modulates responses mediated specifically by NK cells, CD8+ regulatory cells, dendritic cells, macrophages, or monocytes.
  • Activation of a co-stimulatory signaling domain in a host cell may induce the cell to increase or decrease the production and secretion of cytokines, phagocytic properties, proliferation, differentiation, survival, and/or cytotoxicity.
  • the co- stimulatory signaling domains compatible for use in the CAR described herein are the co- stimulatory domains of 2B4, MegflO, CD28, 41BB or FcRy. Additional cytoplasmic signaling domains contemplated for the CAR described herein are shown in Tables 7 and 8.
  • CD244 (also known as natural killer cell receptor or 2B4) is a signaling lymphocyte activation molecule (SLAM) family immunoregulatory receptor found on many immune cell types, including NK cells, a subset of T cells, DCs, and MDSCs. The interaction between NK-cell and target cells via this receptor mediates non-major histocompatibility complex (MHC) restricted killing and modulates NK-cell cytolytic activity.
  • SLAM signaling lymphocyte activation molecule
  • MHC non-major histocompatibility complex
  • the human CD244 gene is found in GENBANK Gene ID: 51744, and the protein sequence is found in UniProtKB ID: Q9BZW8.
  • MegflO is a membrane receptor involved in phagocytosis by macrophages and astrocytes of apoptotic cells.
  • the human MegflO gene is found in GENBANK Gene ID: 84466, and the protein sequence is found in UniProtKB ID: Q96KG7.
  • Fc receptor gamma is a protein of the immunoglobulin superfamily and is found on the surface of many cells - including, among others, B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, human platelets, and mast cells - that contribute to the protective functions of the immune system.
  • This family includes several members, FcyRI (CD64), FcyRIIA (CD32), FcyRIIB (CD32), FcyRIIIA (CD16a), FcyRIIIB (CD16b), which differ in their antibody affinities due to their different molecular structure.
  • the CAR may comprise a combination (e.g., 2 or 3) co-stimulatory domains, which may be from the same co-stimulatory receptor or from different costimulatory receptors.
  • the co-stimulatory domain is preceded by a short linker.
  • the short linker may be TS (i.e., a MHC internal Linker); for a class I CAR, the short linker may be PG.
  • TS i.e., a MHC internal Linker
  • the short linker may be PG.
  • Such linkers and other linkers for conjugation different types of protein sections are known in the art, e.g., as described in disclosed in the International Patent Publication No. WO 2019/094847, the content is incorporated by reference in its entirety.
  • 2B4 co-stimulatory domain for use in the CAR described herein is WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPT SQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFD VYS (SEQ ID NO: 39).
  • This 2B4 cytoplasmic domain may be modified with decreased number of lysine residue (the lysine residues underlined) to resist degradation when the MHC is recycled in the cell.
  • the cytoplasmic portion of the CAR described herein are modified to reduce or increase the number of lysine residues therein compared to the original, natural number of lysine residues in the domain. All lysine residues in the cytoplasmic portion of the CAR are substituted with other amino acid residues (e.g., alanine) thereby eliminating the intracellular lysines. Alternatively, other amino acid residues are substituted to lysines, thereby increasing the number of intracellular lysines.
  • other amino acid residues e.g., alanine
  • the lysine compositions in the cytoplasmic portion of the CAR affects the degradation rate of the CAR when the MHC is recycled in the cell. Proteins are marked for degradation by the attachment of ubiquitin to the amino group of the side chain of a lysine residue. By substituting out the lysines, the CAR may remain in the cell longer.
  • the cytoplasmic domains (ie., cytoplasmic signaling and/or co-stimulatory domains) of the CAR have a 10%, 20%, 30%, 40%, 50% or more reduction in the number of lysine residues compared to the natural number of lysine residues in the domain. Lysine substitutions may be made by any method known in the art.
  • the CAR constructs described herein may include no co- stimulatory domain.
  • it may contain a non-traditional element such as a TALEN nuclease, activators, or repressors which may now be implemented in a clinically applicable lentiviral form using a recoded or non-repeat containing TAL domain and would be linked to a single chain CAR through a membrane domain derived from Notch.
  • any cytoplasmic signaling domain comprising an immunoreceptor tyrosine-based activation motif can be used to construct the chimeric receptors described herein.
  • An “IT AM,” as used herein, is a conserved protein motif that is generally present in the tail portion of signaling molecules expressed in many immune cells. The motif may comprises two repeats of the amino acid sequence YxxL/I separated by 6-8 amino acids, wherein each x is independently any amino acid, producing the conserved motif YxxL/Ix(6-8)YxxL/I.
  • the cytoplasmic signaling domain comprising an IT AM is of CD3 .
  • the CAR does not comprise a co-stimulatory domain and the cytoplasmic signaling domain is preceded by a short linker.
  • the short linker may be TS (i.e. a MHC internal Linker).
  • the short linker may be PG.
  • the linker may be AHA or absent, such as certain instances when a costimulatory domain occurs before a signaling domain.
  • the CAR may include no cytoplasmic signaling domain, for example, that of CD3 ⁇ . Such CD3 ⁇ -free CAR would have suppressive effects against target cells or induce target cell death.
  • the CAR described herein may optionally further include one or more of the following components: a hinge domain, a transmembrane domain, a signal (leader) peptide, an exogenous cytokine and a peptide linkers.
  • a hinge domain a transmembrane domain
  • a signal (leader) peptide an exogenous cytokine and a peptide linkers.
  • the CAR constructs disclosed herein, comprising one or more components described herein, may be configured in any suitable format.
  • a CAR construct containing a MHC class I moiety as described herein may be a single fusion polypeptide that comprise the MHC class I moiety, the antigenic peptide, and a signaling domain (e.g., a co-stimulatory domain, a cytoplasmic signaling domain, or a combination thereof), and optionally one or more of the additional components described herein.
  • a MHC Class I CAR construct contains a hinge domain adjacent to the antigenic peptide.
  • a MHC class I CAR may not contain p2-microglobulin (b2m). When expressed on a cell surface, such a CAR may form a heterodimer with endogenous b2m.
  • a MHC class I CAR may also include b2m, which may be fused with the alpha chain to produce a single polypeptide.
  • a MHC class I CAR may contain two subunits, one including the alpha chain or a portion thereof (e.g., an extracellular domain), and the other including b2m or a portion thereof (e.g., an extracellular domain).
  • the antigenic peptide may be fused to the alpha chain.
  • the antigenic peptide may be fused to b2m.
  • a MHC class I CAR may contain peptide linkers between two components.
  • MHC class II CAR constructs typically contain two subunits, one including the alpha chain or a portion thereof (e.g., an extracellular domain) and the other including the beta chain or a portion thereof (e.g., an extracellular domain).
  • the antigenic peptide can be fused to either the alpha chain or the beta chain.
  • a MHC class II CAR can also be in a single fusion polypeptide format, in which the alpha and beta chains are fused to form a single polypeptide.
  • the alpha chain and beta chain of a MHC class II CAR may be derived from the same MHC class II molecule. Alternatively, they may be from different MHC class II molecules.
  • a MHC class II CAR may contain an alpha chain from HLA DRA*1010 and a beta chain from HLA DRB1*15O1, which may be fused with an antigenic peptide, such as an MBP peptide.
  • MHC class I and MHC class II constructs described herein can be further fused to one or more signaling domains and optionally one or more of the additional components (e.g., linkers, exogenous cytokines, transmembrane domains etc.).
  • the CAR constructs described herein are free of signaling domains.
  • a CAR as described herein contains matched MHC moiety and antigenic peptide, e.g. , a MHC molecule that would present the antigenic peptide or homologous analogs in natural state; however in some cases the MHC-CAR or derivative may match the immune cell line rather than the patient, such as when the MHC-CAR is used to suppress a KIR or NKG2A receptor.
  • a CAR described herein may contain an alpha chain or a beta chain from HLA DRB1*15O1 and an antigenic peptide associated with this HLA allele, e.g., those MBP peptides described herein and others as well.
  • an antigenic peptide associated with this HLA allele e.g., those MBP peptides described herein and others as well.
  • the association between antigenic peptides involved in an autoimmune disease and a specific HLA allele is well known in the art or can be identified via routine practice, for example, library screening.
  • any of the CAR constructs described herein can be prepared by a routine method, such as recombinant technology.
  • Methods for preparing the chimeric receptors herein involve generation of a nucleic acid or a nucleic acid set that encodes or collectively encodes a CAR construct (including a single polypeptide or two subunits).
  • the nucleic acid also encodes a self-cleaving peptide (e.g., P2A, T2A, or E2A peptide) between the coding sequences for the two subunits of a CAR, or between the coding sequence for a CAR and the coding sequence for other genes to be co-expressed with the CAR in a host cell (see discussions below).
  • Sequences of each of the components of the CARs may be obtained via routine technology, e.g., PCR amplification from any one of a variety of sources known in the art.
  • sequences of one or more of the components of the CARs are obtained from a human cell.
  • the sequences of one or more components of the CARs can be synthesized.
  • Sequences of each of the components e.g. , domains
  • the nucleic acid encoding the CAR may be synthesized.
  • the nucleic acid is DNA.
  • the nucleic acid is RNA.
  • any of the CAR proteins, nucleic acid encoding such, and expression vectors carrying such nucleic acid can be mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition, which is also within the scope of the present disclosure.
  • a pharmaceutically acceptable carrier to form a pharmaceutical composition, which is also within the scope of the present disclosure.
  • “Acceptable” means that the carrier is compatible with the active ingredient of the composition (e.g., the nucleic acids, vectors, cells, or therapeutic antibodies) and does not negatively affect the subject to which the composition(s) are administered.
  • Any of the pharmaceutical compositions to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formations or aqueous solutions.
  • Pharmaceutically acceptable carriers including buffers, are well known in the art, and may comprise phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; and other carbohydrates; metal complexes; and/or non-ionic surfactants. See, e.g. Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. III. Engineered Immune cells
  • Engineered immune cells expressing any of the apoptosis inhibitors provided herein and a CAR such as any of the MHC-CARs described herein provide a specific population of cells that can recognize pathogenic cells (e.g., autoreactive pathogenic T cells) involved in autoimmune diseases via MHC/peptide-TCR engagement. Such engineered immune cells are also resistant to apoptosis.
  • pathogenic cells e.g., autoreactive pathogenic T cells
  • Such engineered immune cells are also resistant to apoptosis.
  • the interaction between the MHC-peptide portion of the CAR and the cognate TCR on the pathogenic cells would activate the CAR expressing immune cells via the signaling domains (s) of the CAR (optionally by recruiting cell membrane signaling molecules of the immune cells), leading to proliferation and/or cytotoxic effector functions of the CAR-expressing immune cells, which in turn eliminate the pathogenic cells.
  • the CAR described herein can be expressed in a variety of immune cells.
  • Immune cells expressing the apoptosis inhibitor and MHC-CAR described herein provide a specific population of cells that can recognize pathogenic cells (e.g. , autoreactive T cells) involved in autoimmune diseases via MHC/peptide-TCR engagement.
  • pathogenic cells e.g. , autoreactive T cells
  • the interaction between the MHC-peptide portion of the MHC-CAR and the cognate TCR on the pathogenic cells would activate the MHC-CAR expressing immune cells via the signaling domains(s) of the MHC-CAR (optionally by recruiting cell membrane signaling molecules of the immune cells), leading to proliferation and/or effector functions of the MHC-CAR-expressing immune cells, which in turn eliminate the pathogenic cells.
  • the immune cells can be T cells, NK cells, macrophages, neutrophils, eosinophils, or any combination thereof.
  • the immune cells are T cells.
  • the immune cells are NK cells.
  • Table 8 shows some examples of host immune cells for engineering the immune cells expressing the CAR described herein.
  • the host immune cells may have additional treatment to limit uncontrolled cell proliferation and activation of the host immune cells themselves and the targeted pathogenic cells of the autoimmune disease.
  • Exemplary kill switches such as caspase 9 kill switches are described in (US20160263155, WO2011146862A1, Straathof, 2005, Blood, 105(11):4247-54; WO2011146862) and included here in their entirety. Such kill switches can be incorporated into the engineered immune cells.
  • Table 8. Exemplary Host Immune Cells for Producing the Engineering Immune Cells
  • the host immune cell may be obtained from a subject.
  • subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.
  • the subject is a human.
  • the cells can be obtained from a number of sources, including apheresis products, peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord.
  • a source suitable for obtaining the type of immune cells desired would be evident to one of skill in the art.
  • the population of immune cells can be obtained from any source, such as peripheral blood mononuclear cells (PBMCs), bone marrow, tissues such as spleen, lymph node, thymus, tumor tissue, or established cell lines.
  • PBMCs peripheral blood mononuclear cells
  • a source suitable for obtaining the type of immune cells desired would be evident to one of skill in the art.
  • the population of immune cells is derived from PBMCs.
  • the type of immune cells desired may be expanded within the population of cells obtained by co-incubating the cells with stimulatory molecules, for example, anti-CD3 and anti-CD28 antibodies may be used for expansion of T cells.
  • the immune cell is a natural killer (NK) cell or a macrophage cell or a cell line thereof.
  • NK natural killer
  • macrophage cell line a cell line or macrophage cell line.
  • Such cell lines preserve most properties of the normal immature NK or macrophage cells (monocyte).
  • the cells can be obtained from different sources: from the patient (autologous), the patient’s human leukocyte antigen (HLA)-matched siblings, or haploidentical family members or unrelated (allogeneic) donors.
  • the cells could be collected in advance, cryopreserved and thawed before infusion.
  • the cells may be expanded in culture prior to and also after transfection of the vector expression construct for the MHC-CAR, the exogenous cytokine(s), and knock-out systems for the endogenous MHCs, endogenous costimulatory molecules, and/or endogenous MHC binding receptors.
  • the expressed CAR in the host immune cell directs the immune cell towards pathogenic T cells that are causing the autoimmune diseases via the antigenic peptide in the CAR.
  • the immune cell executes its cytotoxic effects upon the targeted cell to kill the cell. For example, by releasing granules to induce cell death in the targeted pathogenic T cell or phagocytose the targeted pathogenic T cells and digesting it.
  • NK Natural killer
  • NK cells are a type of cytotoxic lymphocyte (a white blood cell) capable of inducing cell death in targeted cells.
  • NK cells are activated in response to interferons or macrophage-derived cytokines (e.g., tumor necrosis factor (TNF), IL-1, IL-6, IL-8, and IL- 12).
  • TNF tumor necrosis factor
  • IL-1 tumor necrosis factor
  • IL-6 IL-6
  • IL-8 IL-8
  • IL- 12 macrophage-derived cytokines
  • the cytoplasmic granules of NK cells contain special proteins such as perforin and proteases known as granzymes. Upon release in close proximity to a cell slated for killing, perforin forms pores in the cell membrane of the target cell through which the granzymes and associated molecules can enter, inducing apoptosis.
  • NK cells serve to contain viral infections while the
  • Natural Killer (NK) cells are an emerging cell type that is being used as a cellular chassis for CAR therapies in oncology.
  • NK cells have limited in vivo persistence, reduced risk of clonal expansion, and a smaller risk of toxicities such as cytokine release syndrome or i o neurotoxicity.
  • the reduced risk of toxicity makes NK based therapies potentially amenable to use in an outpatient setting.
  • Survival, proliferation, and/or retention of cytotoxic activity of NK cells in vivo requires stimulation by cytokines, such as 11-2, 11-7, 11-12, 11-15, 11-18, CCL5, IL-21, or IL-34.
  • cytokines such as 11-2, 11-7, 11-12, 11-15, 11-18, CCL5, IL-21, or IL-34.
  • Historically, some clinical protocols relied on 11-2 administration, to prolong NK survival in patients; however, in autoimmune diseases, 11-2 may not travel to all immune i privileged regions or sites of pathogenic cell expansion, and 11-2
  • human NK cell lines are used to express CAR described herein.
  • Human NK cell lines includes but are not limited to YTS, KHYG-1, KNK92, NK3.3, NK101, and NKL (G. Suck, 2005, Exp. Hematol.; G. Suck, Int. Immunol., 2006; M. Yagita, Leukemia, 2000; J.T. Gunesh, 2019, Mol Immunol, 115:64-75; U.S. Patent Nos.: 8,313,943; 9,181,322;
  • NKL are the three commonly used cell lines. These originate from malignant expansions of NK cell leukemia/lymphoma.
  • the NK92 cell line is derived from the peripheral blood of a male patient with large granular lymphocyte (LGL)-non-Hodgkins lymphoma and is IL-2 dependent.
  • NK92 cells are positive for cell surface receptors CD56, CD2, CD7, CDlla,
  • NK92 also have germline configuration for beta and gamma genes of the T cell receptor (TCR). While NK92 cells express few killer immunoglobulin-like receptors (KIRs), they do have a relatively diverse activating receptor repertoire including expression of NKp30, NKp46, NKG2D, CD28, and 2B4. NK92 cells also have the potential to kill through lytic granule-independent pathways as is indicated by
  • NK92 cells show high cytotoxic potential against susceptible target cells.
  • NK101 is derived from a patient with extra- nodal natural killer/T-cell lymphoma (H. G. Yang et al., Journal for ImmunoTherapy of Cancer volume 7, Article number: 138 (2019)
  • YTS cells are a sub-clone of the YT NK cell line which originates from the pericardial fluid of a male patient with acute lymphoblastic lymphoma.
  • YTS are positive for CD56, CD7, CD28, and CD45RO but negative for CD2 and CD16, with TCR genes in germline configuration. This cell line does not require exogenous IL-2 for maintenance in culture. Due to the high expression of CD28, YTS readily kill 721.221 target cells that express high levels of B7.1, but have reduced cytolytic potential for other common NK cell targets.
  • the NKL cell line is derived from the peripheral blood of a male patient with LGL- leukemia and, like NK92 cells, require IL-2 for survival. They are CD2, CD6, CDl la, CD27, CD29 and CD94 positive. Depending on their time in culture, NKL can rapidly lose expression of CD16, CD56, and CD57 resulting in cultures that are CD56 negative with minimally detectable CD 16.
  • NK3.3 The non-malignant cell line, NK3.3, was generated by in vitro NK cell cloning from the blood of a healthy donor.
  • NK3.3 originates from the peripheral blood of a normal donor expanded in mixed lymphocyte culture and are IL-2 dependent. They are positive for CD2, CD1 la, CD38, CD45, CD16 and CD56.
  • NK3.3 cells are dependent on IL-2 for prolonged survival.
  • NK3.3 have cytolytic activity against susceptible target cells (K562 and MOLT-4).
  • KHYG-1 are highly cytotoxic cells from a patient with aggressive leukemia, and require 11-2 for survival. They carry a p53 point mutation. They are CD2, CD6, CD7, and CD8positive. They have cytolytic activity against susceptible target cells (K562).
  • iPSC-derived NK cells and umbilical cord blood-derived NK cells described by F. Cinchoki (Science Translational Medicine, 2020,12 (568):eaaz5618) and B.H. Goldenson (Front. Immunol., 15 October 2020.), and in U.S. Patent No.: 9,260,696; and U.S. Patent Application No.: US20180326029).
  • Other methods of generating NK cells are also known in the art, e.g., in U.S. Pat. No. 8,926,964 and U.S. Application No.: US20150225697. These references are incorporated by reference in their entirety.
  • Macrophages and monocyte-like cell lines are also be used.
  • Macrophages are specialized, long-lived, white blood cell of the immune system that engulfs and digests cellular debris, foreign substances, microbes, cancer cells, and anything else that does not have the type of proteins specific to healthy body cells on its surface. In addition, they can also present antigens to T cells and initiate inflammation by release cytokines that activate other cells. Macrophages are differentiated from monocytes that develop from hematopoietic stem cells in the bone marrow or arise during embryonic development.
  • Monocytes are able to differentiate into macrophages and dendritic cells (DC) when stimulated by different growth factors, including granulocyte- macrophage colony stimulating factor (GM-CSF) or macrophage colony stimulating factor (M-CSF), culminating in an effective controlling and clearing of the inflamed areas.
  • differentiated cells can be further activated by various cytokines that result in their polarization, yielding further release of pro-inflammatory cytokines and chemokines including TNF-a, TL-6, TL-113 and MCP-1 (CCL2).
  • human macrophage cell lines or monocyte- like cell lines (MCLCs) are used to express the CAR described herein.
  • THP-1 and HL-60 cells are derived from patients with acute monocytic leukemia and U-937 cells are immortalized from a patient with histiocytic lymphoma. These cell lines are used routinely as surrogates for isolated CD 14+ human peripheral blood mononuclear cells (PBMCs). These cell lines have been extensively characterized based on the mRNA expression levels of a selection of inflammatory mediators, including cytokines and chemokines (P.J. Groot- Kormelink, 2012, BMC Immunology 13:57; D.M. Hohenhaus, 2013, Immunobiology 218:1345-1353; M. Daigneault, PLoS ONE 5:e8668).
  • cytokines and chemokines P.J. Groot- Kormelink, 2012, BMC Immunology 13:57; D.M. Hohenhaus, 2013, Immunobiology 218:1345-1353; M. Daigneault, PLoS ONE 5:e8668.
  • Mono Mac 1 and 6 are human monocytic cell lines with several features of mature blood monocytes such as CD 14 antigen expression, phagocytotic ability, and the functional ability to produce cytokines (P, Neustock, 1993, Immunobiology, 188(3):293-302).
  • iPSC induced pluripotent stem cells
  • iPSC-derived macrophage cells described in U.S. Patent No.: 10,724,003, the content is incorporated by reference in its entirety.
  • Methods for constructing the CAR expression vector, choices of promoters, markers, delivery into the immune cells, expansion of the resultant engineered cells and expression of the CAR are described in the International Patent Publication No. WO 2019/094847, the content is incorporated by reference in its entirety.
  • expression vectors for stable or transient expression of the apoptosis inhibitor(s) and chimeric receptor construct may be constructed via conventional methods as described herein and introduced into immune host cells.
  • nucleic acids encoding the apoptosis inhibitor(s) and MHC-CAR may be cloned into a suitable expression vector, such as a viral vector (e.g., a lenti viral vector) in operable linkage to a suitable promoter (e.g. , T7 promoter, EFlalpha promoter, or MND promotor).
  • the nucleic acids and the vector may be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined with a ligase.
  • synthetic nucleic acid linkers can be ligated to the termini of the nucleic acid encoding the apoptosis inhibitor(s) or chimeric receptors.
  • the synthetic linkers may contain nucleic acid sequences that correspond to a particular restriction site in the vector. The selection of expression vectors/plasmids/viral vectors would depend on the type of host cells for expression of the chimeric receptors, but should be suitable for integration and replication in eukaryotic cells.
  • promoters can be used for expression of the apoptosis inhibitor(s) or MHC-CAR constructs described herein, including, without limitation, cytomegalovirus (CMV) intermediate early promoter, a viral LTR such as the Rous sarcoma virus LTR, HIV- LTR, HTLV-1 LTR, the simian virus 40 (SV40) early promoter, herpes simplex tk virus promoter.
  • CMV cytomegalovirus
  • viral LTR such as the Rous sarcoma virus LTR, HIV- LTR, HTLV-1 LTR
  • SV40 simian virus 40
  • herpes simplex tk virus promoter herpes simplex tk virus promoter.
  • Additional promoters for expression of the chimeric receptors include any constitutively active promoter in an immune cell. Alternatively, any regulatable promoter may be used, such that its expression can be modulated within an immune cell.
  • the vector may contain, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in host cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColEl for proper episomal replication; internal ribosome binding sites (IRESes), versatile multiple cloning sites; T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA; a “suicide switch” or “suicide gene” which when triggered causes cells carrying the vector to die (e.g., HSV thymidine kinase, an inducible caspase such as iCasp9), and reporter gene for assessing expression of the apoptosis inhibitor(s) or MHC-CAR.
  • a selectable marker gene such as the neo
  • the marker/sorting/suicide molecules for use in the present disclosure can be used for killing with rituximab and/or for sorting with QB END.
  • Philip et al., Blood 124(8): 1277-87; 2014 One example is RQR8, which contains rituximab mimotope and QB END- 10 epitope.
  • Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art. Any of the vectors comprising a nucleic acid sequence that
  • a vector may be delivered into host immune cells by a suitable method. Methods of delivering vectors to immune cells are well known in the art and may include DNA electroporation, RNA electroporation, transfection reagents such as liposomes, or viral transduction. In some embodiments, the vectors for expression of the
  • apoptosis inhibitor(s) or MHC-CAR are delivered to host cells by viral transduction.
  • Exemplary viral methods for delivery include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S. Pat. Nos. 5,219,740 and 4,777,127; GB Patent No. 2,200,651; and EP Patent No. 0 345 242), alphavirus-based i vectors, and adeno-associated virus (AAV) vectors (see, e.g. , PCT Publication Nos.
  • retroviruses see, e.g., PCT Publication Nos.
  • the vectors for expression of the chimeric receptors are retroviruses. In some embodiments, the vectors for expression of the chimeric receptors are lentiviruses.
  • viral particles that are capable of infecting the immune cells and carry the vector may be produced by any method known in the art and can be found, for example in PCT Application No. WO 1991/002805A2, WO 1998/009271 Al, and U.S. Patent 6,194,191.
  • the viral particles are harvested from the cell culture supernatant and may be isolated and/or purified prior to contacting the viral particles
  • the cells are cultured under conditions that allow for expression of the chimeric receptor.
  • the nucleic acid encoding the apoptosis inhibitor(s) or the MHC-CAR is regulated by a regulatable promoter, the host cells are cultured in
  • regulatable promoter e.g. , Tet off/on inducible system
  • the promoter is an inducible promoter and the immune cells are cultured in the presence of the inducing molecule or in conditions in which the inducing molecule is produced. Determining whether the MHC-CAR is expressed will be evident to one of skill in the art and may be assessed by any known method, for example, detection of the chimeric receptor-encoding mRNA by quantitative reverse transcriptase PCR (qRT-PCR) or detection of the chimeric receptor protein by methods including Western blotting, fluorescence microscopy, and flow cytometry. See also Examples below. Alternatively, expression of the apoptosis inhibitors) or MHC-CAR may take place in vivo after the immune cells are administered to a subject.
  • qRT-PCR quantitative reverse transcriptase PCR
  • MHC-CAR may take place in vivo after the immune cells are administered to a subject.
  • RNA molecules encoding the apoptosis inhibitor or MHC-CAR constructs can be prepared by in vitro transcription or by chemical synthesis.
  • the RNA molecules can then introduced into suitable host cells such as immune cells (e.g., T cells, NK cells, macrophages, neutrophils, eosinophils, or any combination thereof) by, e.g., electroporation.
  • immune cells e.g., T cells, NK cells, macrophages, neutrophils, eosinophils, or any combination thereof
  • electroporation e.g., electroporation.
  • RNA molecules can be synthesized and introduced into host immune cells following the methods described in Rabinovich et al., Human Gene Therapy, 17:1027-1035 and WO WO2013/040557.
  • the methods of preparing host immune cells expressing any of the apoptosis inhibitor(s) or MHC-CARs described herein may comprise expanding the host immune cells ex vivo. Expanding host immune cells may involve any method that results in an increase in the number of cells expressing an apoptosis inhibitor and a MHC-CAR, for example, allowing the host cells to proliferate or stimulating the host cells to proliferate. Methods for stimulating expansion of host cells will depend on the type of host cell used for expression of the chimeric receptors and will be evident to one of skill in the art. In some embodiments, the host immune cells expressing any of the apoptosis inhibitor(s) and the MHC-CAR described herein can be expanded ex vivo prior to administration to a subject. c) Additional cell treatments and modifications
  • the host immune cells may be treated to increase cell proliferation thereby increasing the number of cells available for transfecting the CAR construct described herein. Additionally, one or more additional genetic modifications can be introduced into host immune cells before, concurrently with, or after the transfection of the CAR construction, e.g., to immortalize the host cells to make cell lines or incorporating an inducible kill switch so as to prevent uncontrolled cell proliferation after activation in vivo. Furthermore, the resultant engineered immune cells may be subsequently modified by irradiation treatment prior to use in treating patients. Inducible promoters are known in the art, for example, the TET on or TET off system. i) Ex vivo cell expansion
  • the host immune cells for expressing the apoptosis inhibitor and CAR described herein may be expanded ex vivo by co-incubating the cells with stimulatory molecules such as anti-CD2 and anti-CD335 antibodies and cytokines such as 11-2, 11-7, 11-12, 11-15, 11-18, IL- 21, IL-34, or a combination thereof.
  • stimulatory molecules such as anti-CD2 and anti-CD335 antibodies and cytokines such as 11-2, 11-7, 11-12, 11-15, 11-18, IL- 21, IL-34, or a combination thereof.
  • ex vivo refers to cells that have been removed from a living organism, (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).
  • NK cells e.g., NK cells, macrophages, neutrophils, eosinophils, cell lines such as Jurkat E6-1 (ATCC TIB-152) or derivatives, Jurkat with deleted TCR chains (one or both) for example J.RT3-T3.5 (ATCC TIB- 153), K562 (ATCC CCL-243), NK-92 (ATCC CRL-2407), or NK- 92 derivatives (NK-92 MI (ATCC CRL-2408), NK-92 CD16.F/F (ATCC pta-8837; ATCC PTA-8836), NK-92 (ATCC PTA-6967), KHYG-1 (ACC 725), NKL, NKG, YT (ACC 434), NK101 or any combination thereof) may be expanded prior to transfer of the CAR construct.
  • Immortalization to make host cell lines e.g., NK cells, macrophages, neutrophils, eosinophils, cell lines such as
  • primary NK cells, macrophages, or monocyte cells from autologous or allogeneic donors may be immortalized by TERT overexpression, or potentially by other modifications that would replicate the transcriptional outcome of contact with 11-21 containing feeder cells in vitro.
  • the TERT gene provides instructions for making one component of an enzyme called telomerase. Telomerase maintains structures called telomeres, which are composed of repeated segments of DNA found at the ends of chromosomes.
  • telomeres protect chromosomes from abnormally sticking together or breaking down (degrading). In most cells, telomeres become progressively shorter as the cell divides. After a certain number of cell divisions, the telomeres become so short that they trigger the cell to stop dividing or to self-destruct (undergo apoptosis). Telomerase counteracts the shortening of telomeres by adding small repeated segments of DNA to the ends of chromosomes each time the cell divides. Methods of genetically incorporating a TERT gene to immortalize a cell are described in U.S. Patent No. 7,569,385, the content is incorporated by reference in its entirety.
  • Certain mammalian viruses can also be used to immortalize immune cells, e.g., Herpesvirus saimiri and Epstein Barr Virus. Methods of immortalize immune cells with viruses are known in the art, e.g. , as described in U.S. Patent No. 8,765,470, the content is incorporated by reference in its entirety. iii) Irradiation treatment In some aspects, there may be a need to minimize the feedback back loop of uncontrolled cell activation and/or proliferation.
  • This uncontrolled cell activation and/or proliferation may occur in the engineered apoptosis-resistant, immune cell expressing the CAR described herein, in the pathogenic autoreactive immune cells of the autoimmune disease, and also in the immortalized host immune cells for use in expressing the CAR described herein, self- activation or self-proliferation.
  • the host immune cells e.g., the immortalized cell lines
  • the host immune cells are irradiated to reduce the cell proliferation capability. These cells retain their cytotoxic function for at least 24 hours after irradiation. Irradiation doses of 30 Gy, 50 Gy, 70 Gy, 100 Gy, or more (e.g., 1000 Gy) may be used.
  • the apoptosis-resistant, CAR-expressing engineered immune cell are treated and modified with irradiation.
  • This treatment blocks the proliferation of the engineered immune cell. See Table 8 above for examples of proliferation resistant host cell types that may be used to decrease pathogenic cell proliferation through lack of therapeutic cell stimulus compared to a CAR T therapy (which can radically expand in number and through time).
  • the CAR-expressing engineered immune cells are irradiated prior to administering to a patient when the CAR-expressing engineered immune cells are used for treating an autoimmune disease in a patient in need thereof.
  • the term “therapeutic,-’ as used herein, means a treatment administered to a subject who exhibits signs of pathology for the purpose of diminishing or eliminating those signs.
  • the engineered, apoptosis-resistant, CAR-expressing immune cells described herein co-express an anti-B cell antibody or anti-B cell CAR.
  • the anti-B cell antibody or anti-B cell CAR on the engineered cell redirects the cytotoxicity of immune effector cells toward B cells.
  • the anti-B cell antibody or anti-B cell CAR comprises antigen binding fragments thereof, such as camel Ig, Ig NAR, Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fv, single chain Fv proteins (“scFv”), bis-scFv, (scFv)2, minibodies, diabodies, triabodies, tetrabodies, disulfide stabilized Fv proteins (“dsFv”), and single-domain antibody (sdAb, Nanobody) and portions of full length antibodies responsible for antigen binding.
  • antigen binding fragments thereof such as camel Ig, Ig NAR, Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fv, single chain Fv proteins (“scFv”), bis-scFv, (scFv)2, minibodies, diabodies, triabodies, tetrabodies, disul
  • the anti-B cell antibody can be an anti-CD19 or anti-CD20 antibody that targets the CD19 or CD20 in B cell surface respectively.
  • the anti-B cell CAR can comprise portions of full length antibodies responsible for antigen binding in an anti-CD19, anti-CD20 or anti-CD22.
  • the anti-B cell antibody or anti-B cell CAR is a bispecific anti-B cell antibody or CAR.
  • anti-CD19 and anti-CD20 on a B cell.
  • the engineered, apoptosis-resistant, MHC-CAR-expressing immune cells described herein are expanded ex vivo before use. It is contemplated that large- scale clinical-grade expansion of engineered immune described cells are made for commercial use. Methods for enhancing cell proliferation are known, e.g., U.S. Patent Publication No.: US20130011376; and U.S. Patent Nos. 9125869 and 10,428,305. These references are incorporated by reference in their entirety.
  • the engineered, apoptosis-resistant, MHC-CAR-expressing immune cells described herein may be cryopreserved before use.
  • the cryopreservation may occur before ex vivo expansion, after ex vivo expansion, or both before and after ex vivo expansion.
  • Methods for cell cryopreserved are known, e.g., U.S. Patent Publication No.: US20180094232 and US20190037832; and U.S. Patent Nos. 8,936,905 and 10,271,543.
  • the engineered, apoptosis-resistant, MHC-CAR-expressing immune cells described herein are treated to reduce the cell proliferation capability of the engineered cells.
  • the engineered, CAR- expressing immune cells described herein are irradiated to reduce the cell proliferation capability. These cells retain their cytotoxic function for at least 24 hours after irradiation. Irradiation doses of 30 Gy, 50 Gy, 70 Gy, 100 Gy, or more (e.g., 1000 Gy) may be used.
  • cytotoxic functions and/or phagocytotic functions for any of the engineered immune cells described herein and compared with non- irradiated cells (used as control cells).
  • target cells can be labelled with a violet tag (to identify the target cell population), then mixed with a population of the engineered immune cells described herein at a variety of ratios, and assayed for viable target cells by measuring the viable cells remaining.
  • Cytotoxic function can be measured for irradiated and non-irradiated cells (control cells for comparison).
  • GFP-expressing target cells can be used for the assay, incubated with a population of the engineered immune cells described herein at a variety of ratios, and the mixture of cells are counting double positive cells (+target cell GFP, +macrophage marker CDllb+) using FACS.
  • Other known methods are disclosed in HG, Klingemann et al., 1996, Europe PMC, 2(2):68-75; H. Bergman, et al., 2020, Anticancer Research 40 (10) 5355-5359; Morrissey et al., 2018, eLife, e36688; and A.T. Pinto et al., 2016, Sci. Rep. 6: 18765.
  • the cells may be contacted with a variety of molecules or cells, such as a soluble TCR (e.g., a soluble single chain TCR), Jurkat cell that lacks TCR engineered with an exogenous TCR, and an expanded or non-expanded T cell population (either autologous or allogeneic).
  • a soluble TCR e.g., a soluble single chain TCR
  • Jurkat cell that lacks TCR engineered with an exogenous TCR
  • an expanded or non-expanded T cell population either autologous or allogeneic
  • the viability of the engineered immune cell can be assessed by 7AAD measurement, after contacting with the variety of molecules or cells, or after irradiation of the engineered immune cell.
  • endogenous cytokine released by the engineered immune cell after contact with the variety of molecules or cells can be measured and compared to a control obtained from the engineered immune cells that where not contacted with the variety of molecules or cells.
  • the engineered immune cells disclosed herein, expressing an apoptosis inhibitor(s) and a CAR (such as an MHC-CAR) described herein are useful for targeting and eliminating pathogenic cells involved in autoimmune diseases, such as those described in Tables 3-5.
  • the subject is a mammal, such as a human, monkey, mouse, rabbit, or domestic mammal.
  • the subject is a human, for example, a human patient having, suspected of having, or at risk for an autoimmune disease.
  • a population of engineered immune cells described herein for the manufacture of medicament for the treatment of an autoimmune disease in a subject in need thereof.
  • An engineered, apoptosis-resistant, CAR-expressing immune cells or compositions comprising these cells may be used to treat a patient that has or is at risk of having an autoimmune disorder, to suppress autoreactive immune cells such as pathogenic T cells and B cells associated with the autoimmune disorder.
  • the apoptosis-resistant, CAR-expressing immune cells can be mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition, which is also within the scope of the present disclosure.
  • an effective amount of the immune cells expressing any of the CAR constructs described herein can be administered into a subject in need of the treatment.
  • the immune cells may be autologous to the subject, i.e. , the immune cells are obtained from the subject in need of the treatment, genetically engineered for expression of the apoptosis inhibitor(s) and CAR constructs and optionally contains one or more of the additional genetic modifications as described herein, and then administered to the same subject.
  • Administration of autologous cells to a subject may result in reduced rejection of the immune cells as compared to administration of non- autologous cells.
  • the immune cells are allogeneic cells, i.e.
  • the cells are obtained from a first subject, genetically engineered for expression of the CAR construct, and administered to a second subject that is different from the first subject but of the same species.
  • allogeneic immune cells may be derived from a human donor and administered to a human recipient who is different from the donor.
  • the immune cells are co-used with a therapeutic agent for the target immune disease, for example, Alemtuzumab for treating MS.
  • a therapeutic agent for the target immune disease for example, Alemtuzumab for treating MS.
  • Such immunotherapy is used to treat, alleviate, or reduce the symptoms of the target immune disease for which the immunotherapy is considered useful in a subject.
  • the efficacy of the CAR immunotherapy may be assessed by any method known in the art and would be evident to a skilled medical professional.
  • the efficacy of the immunotherapy may be assessed by survival of the subject and/or reduction of disease symptoms in the subject.
  • the immune cells expressing any of the apoptosis inhibitor(s) and CAR disclosed herein are administered to a subject who has been treated or is being treated with a therapeutic agent for an autoimmune disease.
  • the immune cells expressing any one of the apoptosis inhibitor(s) and CAR disclosed herein may be co-administered with the therapeutic agent.
  • the immune cells may be administered to a human subject simultaneously with the therapeutic agent.
  • the immune cells may be administered to a human subject during the course of a treatment involving the therapeutic agent.
  • the immune cells and the therapeutic agent can be administered to a human subject at least 4 hours apart, e.g., at least 12 hours apart, at least 1 day apart, at least 3 days apart, at least one week apart, at least two weeks apart, or at least one month apart.
  • an effective amount of the apoptosisresistant, immune cells expressing CAR or compositions thereof can be administered to a subject (e.g., a human MS patient) in need of the treatment via a suitable route, such as intravenous administration. Any of the apoptosis-resistant, immune cells expressing CAR or compositions thereof may be administered to a subject in an effective amount.
  • an effective amount refers to the amount of the respective agent e.g., the immune cells expressing CAR or compositions thereof) that upon administration confers a therapeutic effect on the subject. Determination of whether an amount of the cells or compositions described herein achieved the therapeutic effect would be evident to one of skill in the art. Effective amounts vary, as recognized by those skilled in the art, depending on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner.
  • the effective amount alleviates, relieves, ameliorates, improves, reduces the symptoms, or delays the progression of any disease or disorder in the subject.
  • the subject is a human. In some embodiments, the subject is a human cancer patient.
  • the subject is a human patient suffering from an autoimmune disease, which is characterized by abnormal immune responses attacking a normal body part.
  • autoimmune diseases include multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, juvenile idiopathic arthritis (also known as juvenile idiopathic arthritis), Sjogren’s syndrome, systemic sclerosis, ankylosing spondylitis, Type 1 diabetes, autoimmune thyroid diseases (Grave’s and Hashimoto’s), multiple sclerosis myasthenia gravis, inflammatory bowel disease (Crohn’s or ulcerative colitis), Psoriasis, or a diseases mentioned in Tables 4-6.
  • the engineered apoptosis-resistant, CAR-expressing immune cells or compositions comprising these cells may be used to treat a patient at different stages of an autoimmune disease, e.g., at mild, moderate, and severe stages.
  • the engineered apoptosis-resistant, CAR-expressing immune cells or compositions comprising these cells are irradiated before being administered to a patient.
  • the engineered immune cells described herein may be used to remove pathogenic T cells or B cells in ex vivo.
  • a patient may be scheduled to undergo hematopoietic stem cell transplantation (HSCT), such as a multiple sclerosis or scleroderma patient.
  • HSCT hematopoietic stem cell transplantation
  • the patient’ s T cells are collected and combined ex vivo with the engineered immune cells described herein in order to create an autoimmune cell depleted T cell population that can be returned to the patient to reduce risk and complications of neutropenia.
  • the engineered immune cells are allogeneic or autologous to the patient’s T cells. According, provided herein an ex vivo method of removing / eliminating pathogenic immune cells (c.g.
  • T cells from a sample of immune cells
  • the method comprises mixing the sample of immune cell with a population of engineered immune cells described herein.
  • the sample of immune cells is obtain from a subject who has an autoimmune disease.
  • the immune cells of the sample and the population of engineered immune cells may be autologous or allogeneic.
  • the mixing allows the engineered immune cells to kill and remove the autoreactive pathogenic T cells.
  • the resulted cells may be separated from the engineered immune cells described herein and then infused back into the subject for the treatment of an autoimmune disease in a subject in need thereof.
  • the engineered apoptosis-resistant, immune cells carrying MHC-CAR are made from a patient’s own CD8+ regulatory T cells.
  • a patient has an autoimmune disorder and is schedule to undergo HSCT.
  • the patient’s CD8+ regulatory T cells are collected and transfected with the MHC-CAR and modified to have one or more additional features described herein.
  • CD8+ regulatory T cells are modified with a MHC-CAR (HLA-E MHC and VMAPRTVLL peptide; SEQ ID NO: 12) designed to suppress NKG2A expression or NKG2A is suppressed using another method ( ⁇ ?.g., gene knockout via CRISPR/Cas9; RNAi as disclosed in US20210046112).
  • the Treg cells may also be modified to overexpress HELIOS.
  • HELIOS expression can increase stability in CD8+ regulatory T cell in vivo (US20190192565). The contents of these references are incorporated herein by reference in their entireties.
  • the resultant autologous engineered CD8+ regulatory T cells are then infused back into the patient.
  • haploidentical NK cells are the starting cells for engineered apoptosis-resistant, immune cells carrying MHC-CAR described herein.
  • the resultant engineered immune cells may be further modified with a kill switch to allow for inducible destruction of the engineered cells as needed, e.g., after the ex vivo incubation with patient’s pathogenic T cells.
  • the resultant engineered immune cells may be irradiated to prevent cell proliferation in vivo after infusion into the patient.
  • any of the engineered apoptosis-resistant, immune cells carrying MHC- CAR described herein may also be engineered with a kill switch which may be integrated into the genome as disclosed in WO2011146862 and US9951349, and the contents of these are incorporated herein by reference in their entireties.
  • the patient being treated with any of the engineered apoptosisresistant, immune cells carrying MHC-CAR described herein may receive addition therapeutics such as CD3, CD28, and rapamycin which amplify the endogenous populations of CD8+ Tregs cell in vivo.
  • a patient having an autoimmune disorder is treated with CD3, CD28, and rapamycin.
  • CD8+ Tregs cell are collected and prepared for transfection of the CAR described herein and the described modifications to add the disclosed features described herein.
  • the patient’s CD8+ Tregs cell are collected without pre-treatment with CD3, CD28, and rapamycin.
  • the CD8+ Tregs cells are expanded ex vivo with CD3, CD28, and rapamycin, and then transfected with any CAR described herein together with the modifications to add the disclosed features described herein.
  • Other addition therapeutics include anti-CD45, CD34, and/or CD117 antibodies.
  • kits for use of the apoptosis-resistant, CAR- expressing immune cells for use in suppressing pathogenic immune cells such as autoreactive T cells in autoimmunity may include one or more containers comprising compositions comprising immune cells expressing MAR-CAR such as those described herein), and a pharmaceutically acceptable carrier.
  • the kit can comprise instructions for use in any of the methods described herein.
  • the included instructions can comprise a description of administration of the apoptosis-resistant, CAR-expressing immune cells to a subject who needs the treatment, e.g., an MS patient.
  • the kit may further comprise a description of selecting a subject suitable for treatment based on identifying whether the subject is in need of the treatment.
  • the instructions comprise a description of administering the immune cells to a subject who is in need of the treatment.
  • the instructions relating to the use of the apoptosis-resistant, immune cells expressing the CAR described herein generally include information as to dosage, dosing schedule, and route of administration for the intended treatment.
  • the containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses.
  • Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert.
  • the label or package insert indicates that the pharmaceutical compositions are used for treating, delaying the onset, and/or alleviating a disease or disorder in a subject.
  • kits provided herein are in suitable packaging.
  • suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like.
  • packages for use in combination with a specific device may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the container may also have a sterile access port.
  • At least one active agent in the pharmaceutical composition is immune cells expressing CAR as described herein.
  • Kits optionally may provide additional components such as buffers and interpretive information.
  • the kit comprises a container and a label or package insert(s) on or associated with the container.
  • the disclosure provides articles of manufacture comprising contents of the kits described above.
  • Example 1 Exemplary Methods and Assays for Producing and Analyzing Engineered Immune Cells Expressing an Apoptosis Inhibitor and an MHC-CAR
  • This example provides exemplary methods and assays for making and evaluating engineered immune cells expressing an apoptosis inhibitor and/or an engineered MHC or MHC-CAR.
  • IVTT in vitro transcription
  • DNA plasmids for IVT were designed with an upstream T7 promoter and a downstream cut site in a pcDNA3.1 IP free vector (Genscript).
  • the cut site downstream of the stop codon was EcoRI or Xbal.
  • the region between the T7 promoter and the downstream cut site contained a DNA coding for an ATG start codon, engineered MHC (eMHC) - where the eMHC in some cases may have an internal 2A sequence, an 2A sequence, an apoptosis inhibitor, a second 2A sequence, a gfp coding sequence, and a TAA stop codon.
  • eMHC engineered MHC
  • sequences encoded a ATG start, an apoptosis inhibitor, an 2A sequence, a gfp coding sequence, and a TAA stop codon In some cases, sequences encoded ATG start, eMHC (where the eMHC in some cases has an internal 2 A sequence), an 2 A sequence, a gfp coding sequence, and a TAA stop codon. In some cases, the final 2A-gfp coding sequence was not present or replaced with an alternate apoptosis inhibitor. In some cases, an RQR8 sequence (Philip, B. M. RQR8: A universal safety switch for cellular therapies. Diss.
  • A. mRNA Generation The IVT plasmid was linearized using EcoRI or Xbal and purified. mRNA was generated using the HiScribeTM T7 ARCA mRNA Kit (with tailing). One ul of the reaction was saved to run on a gel. The final reaction was purified using a Monarch® RNA Cleanup Kit.
  • APC clone L243 anti-HLA-DR antibody, 7AAD, brilliant violet 421 annexin V were from Biolegend and stained according to manufacturer’s instructions.
  • Indirect expression 2 For constructs expressing the apoptosis inhibitor and gfp, the expression was analyzed using gfp expression as a proxy. Live (annexin V- and 7AAD-) vs gfp+. Alternatively, live (7AAD-) vs (gfp+).
  • Direct expression of tagged apoptosis resistant constructs For constructs expressing an apoptosis inhibitor with an N or C terminal flag tag or eMHC with C terminal flag tag, cells were fixed with fixation buffer (Biolegend), permeabilized with intracellular staining permeabilization wash buffer (biolegend). D. Expression in NK cell lines, unstimulated NK cells (fresh or frozen), expanded NK cells, cord blood NK cells, PBMC, Macrophage/Monocyte cell lines
  • AIM V media (Thermo) with 5% CTS Immune Cell SR (Thermo), LGM-3 media (Lonza) with 5% CTS Immune Cell SR (Thermo) or Serum-free stem cell growth media (CellGenix).
  • the cells were maintained in LGM-3 with the FBS replaced with human serum albumin.
  • KHYG-1 was maintained in RPMI1640 media supplemented with 10% fetal bovine serum and antibiotics and 200 U/ml IL-2 (R&D Techne).
  • NK-92 was maintained in Alpha Minimum Essential medium without ribonucleosides and deoxyribonucleosides but with 2 mM L-glutamine and 1.5 g/L sodium bicarbonate (i.e., 0.2 mM inositol; 0.1 mM 2-mercaptoethanol ; 0.02 mM folic acid; 100-200 U/ml recombinant IL-2; adjust to a final concentration of 12.5% horse serum and 12.5% fetal bovine serum).
  • Thp-1 was maintained in RPMI1640 media supplemented with 10% fetal bovine serum and antibiotics and 0.5 mM 2-mercaptoethanol.
  • K562 expansion lines (expressing 41BB ligand and membrane bound IL-15 or 11-21) were maintained in RPMI1640 media supplemented with 10% fetal bovine serum and antibiotics.
  • Cord blood units (CB units) and peripheral blood mononuclear cells (PBMCs) from peripheral blood of donors were isolated using ficoll gradient centrifugation (Sigma Histopaque), with or without subsequent cryopreservation in LN2 (using CS10 media (Biolife solutions) or 10% DMSO and 90% FBS).
  • PBMC or CB unit derived purified unstimulated or expanded NK cells Peripheral blood mononuclear cells were isolated from the peripheral blood of donors using ficoll gradient centrifugation, with or without subsequent cry opreservation in LN2 (using CS10 media or 10% DMSO and 90% FBS). NK cells were then purified using either a MojoSort human NK cell isolation kit, or an Easysep human NK cell isolation kit with or without subsequent preservation in LN2 (using CS10 media or 10% DMSO and 90% FBS).
  • PBMC, CB units, or purified For cell expanded PBMC or CB unit derived NK cells, PBMC, CB units, or purified
  • NK cells were cocultured in the the presence of irradiated or mitomycin treated (25 mg/ml with incubation at 37 for 45 minutes, followed by three washes in media) K562 cells expressing 4-1BB ligand and membrane bound 11-15 or 11-21 and cocultured for 6-12 days in the presence of 10-500 lU/ml 11-2 and 5-15 ng/ml 11-15. Cells could be restimulated up to 3 times with new addition of K562 cells.
  • PBMC, CB units, or purified NK cells were cocultured in the the presence of the human NK cell expansion activator kit (Miltenyi) or the Cloudz human NK cell expansion kit (R&D Techne) in the presence of 10- 500 lU/ml 11-2 and 5-15 ng/ml 11-15.
  • human NK cell expansion activator kit Miltenyi
  • R&D Techne Cloudz human NK cell expansion kit
  • MaxCyte GT electroporation, PBMCs, CB units, NK cell lines, unstimulated or stimulated NK cells were electroporated (100 million cells) using EP buffer (MaxCyte) as the electroporation buffer and electroporation program ‘Expanded-NK#3’ or ‘Unstimulated- NK#1’ ; alternatively they were flow electroporated using EP buffer and the program ‘Expanded-NK#1-Flow’.
  • Lentiviral vectors were analogous to the IVT vectors above for the internal construct. Instead of the T7 promoter, they had an EFlalpha promoter and the stop codon was followed by a WPRE (SEQ ID NO: 40) sequence.
  • the base vector was a royalty free pALD-lenti vector (Aldevron).
  • Alpharetrovirus packaging system was used (Muller S, Bexte T, Gebel V, et al. Front Immunol. 2020;10:3123. Published 2020 Jan 24. doi: 10.3389/fimmu.2019.03123).
  • the lentivirus was VSV-G pseudotyped and packaged using a royalty free vector packaging vector kit (Aldevron) including PALD-Rev, PALD-GagPol, and PALD-
  • VSV-G For Baboon envelope pseudotyped vector, BAEV (wt, R, less) replaced VSV-G in the packaging vectors. Similarly, for feline endogenous retrovirus, RD114/TR replaced VSV- G in the packaging vectors. Directly prior to transduction, the cells were pretreated with low dose IL-15 (10 ng/ml) for NK cells. BAEV, RD114/TR (GenBank: X87829.1), and VSV-G pseudotyped alpharetrovirus vectors were also used. (Girard-Gagnepain, Anais, et al. Blood,
  • Viral supernatants were added to plates with (+/- retronectin coated or +/- vectronectin in solution) then cells were added +/- 10 ng/ml IL-15. In some cases spinfection (800 g 2 hours at 37) and polybrene (4-8 ug/ml) were used.
  • transposon/transposase - rAAV or mRNAA was used to deliver a normal or hyperactive piggybac, sleeping beauty, or tc buster transposase to cells, followed by electroporation or flow electroporation was used to deliver a minicircle (Kay MA, He CY, Chen ZY. A robust system for production of minicircle DNA vectors. Nat Biotechnol. 2010 Dec;28(12): 1287-9. doi: 10.1038/nbt.l708. Epub 2010 Nov 21. PMID: 21102455; PMCID: PMC4144359.) or doggybone transposon vector (Karda, Rajvinder, et al.
  • Cells modified with methotrexate resistance gene containing RNA, virus, or transposon were cultured and/or expanded with 250 nM (i.e., 50-300 nM) methotrexate or 50 nM (5-100 nM dasatinib) (for 1 day to 1 week).
  • Modified cells were cocultured with target cells (that either expand in the presence of B cells or monocytes cultured with the peptides containing the epitope of interest) in the presence of 250 nM (25-300 nM) methotrexate or 50 nM dasatinib for 4-6 hours, and then the target cells cells vere assessed for viability (target cells were stained with violet tag ii before co-culturing) and then the violet stained fraction was assessed for viability using
  • target cells were pretreated with 5-300 nM methotrexate or 5-100 nM dasatinib just before coculturing.
  • GMP compliant cell sorting after culturing with 250 nM (200-300 nM) methotrexate (1 day to 1 week), viable cells were retained (cells were sorted using viobility (live/dead) dye (Miltenyi) in the tyto, after pre-treatment with the dead cell removal kit or easy sep dead cell removal kit, and cell straining).
  • TCR dextramers (Bethune, Michael T., et al. "Preparation of peptide-MHC and T-cell receptor dextramers by biotinylated dextran doping.” Biotechniques 62.3 (2017): 123-130.), TCR bound to streptavidin, or TCR were used to label eMHC expressing cells +/- 50 nM dasatinib. Activation markers and cytokine release are assessed post TCR dextramer binding.
  • TCR dextramers fluorescently labeled TCR dextramers (Bethune, Michael T., et al. "Preparation of peptide-MHC and T-cell receptor dextramers by biotinylated dextran doping.” Biotechniques 62.3 (2017): 123-130.), TCR bound to streptavidin, or TCR were used to label eMHC expressing cells +/- 50 nM dasatinib. The TCR bound cells were then sorted using the Tyto (macs sorting). Prototype experiments were performed using a Sony SH800 (FACS sorting) of flow cytometry.
  • cells were sorted indirectly using a tag (RQR8, LNGFR) and labelled antibody targeting them (for example, a PE-labelled antibody) in the tyto.
  • a tag for example, a PE-labelled antibody
  • cells could be purified for a specific cell type after sorting (for instance with a NK selection kit).
  • monocytes and T cells are from the same donor.
  • Monocytes or B cells are purified (for example CD14+ positive isolation kit, Stemcell Technologies).
  • Untreated T cells of interest are pre purified using CD8 isolation kit or CD4 isolation kit (Miltenyi).
  • Monocytes are irradiated or treated with mitomycin as above. Afterwards they were incubated with 10 ug/ml peptide comprising the epitope of interest. The T cells of interest were then cocultured with the monocytes for 7 days with up to two additional restimulations.
  • the TCR of the expanded cells are sequenced using a 10X kit (Single cell Immune profiling, Single-cell V(D)J Immune Profiling solution, 10X Genomics).
  • This example illustrates the use of exemplary apoptosis inhibitors in protecting cells from cell death in vitro.
  • Three exemplary anti-apoptosis proteins CRMA, PI9, and cFLIP were evaluated, individually or in combination, for effects in reducing cell death from interacting with target cells that can degranulate or otherwise cause cell death of the engineered effector cells with and without addition of exogenous HLA molecules.
  • 2E5 HEK293T cells were seeded in each well of three 24 well plates (Corning) in ImL DMEM (Thermo Fisher) supplemented with 10% FBS (VWR) and lOOug/mL Primocin (InvivoGen). 24 hours later, 500ng of each plasmid was lipofected into corresponding wells using Lipofectamine 3000 (Thermo Fisher).
  • the media on the HEK cells was changed to RPMI 1640 (Thermo Fisher) + 200U/mL IL2 (Peprotech) with 25% V/V KHYG-1 culture supernatant, and 1:4000 dilution of Cytotox Red (Sartorius) before 1E5 KHYG-1 cells were added to each non-control well of lipofected HEK293T cells. Cells were then imaged every hour for ⁇ 48H on an Incucyte S3 (Sartorius) live cell imager.
  • the amino acid sequences of the exemplary apoptosis inhibitors and engineered MHC used in this example are provided below.
  • inventive embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed.
  • inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein.
  • a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified.
  • “at least one of A and B” can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

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Abstract

Des cellules immunitaires resistantes à l'apoptose qui expriment un récepteur antigénique chimérique tel qu'un récepteur chimérique basé sur le complexe majeur d'histocompatibilité sont conçues pour cibler et tuer des cellules immunitaires pathogènes telles que des lymphocytes T autoréactifs. La résistance à l'apoptose dans ces cellules immunitaires modifiées permet aux cellules de survivre aux effets cytotoxiques de lymphocytes T et B pathogènes. L'invention concerne également des compositions et des méthodes comprenant ces cellules.
PCT/US2023/067853 2022-06-03 2023-06-02 Cellules immunitaires résistantes à l'apoptose avec récepteur antigénique chimérique du complexe majeur d'histocompatibilité WO2023235856A1 (fr)

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Citations (1)

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WO2019094847A1 (fr) * 2017-11-10 2019-05-16 Jura Bio, Inc. Récepteurs chimériques à base de complexe majeur d'histocompatibilité et utilisations associées pour le traitement de maladies auto-immunes

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Publication number Priority date Publication date Assignee Title
WO2019094847A1 (fr) * 2017-11-10 2019-05-16 Jura Bio, Inc. Récepteurs chimériques à base de complexe majeur d'histocompatibilité et utilisations associées pour le traitement de maladies auto-immunes

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Title
DE KONING PIETER J. A., KUMMER J. ALAIN, DE POOT STEFANIE A. H., QUADIR RAZI, BROEKHUIZEN ROEL, MCGETTRICK ANNE F., HIGGINS WAYNE : "Intracellular Serine Protease Inhibitor SERPINB4 Inhibits Granzyme M-Induced Cell Death", PLOS ONE, vol. 6, no. 8, 1 January 2011 (2011-01-01), US , pages 1 - 9, XP093119838, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0022645 *
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