WO2023235856A1 - Apoptosis resistant immune cells with major histocompatibility complex chimeric antigen receptor - Google Patents

Abstract

Apoptosis-resistance immune cells that express a chimeric antigen receptor such as a major histocompatibility complex-based chimeric receptor are designed for targeting and kill pathogenic immune cells such as autoreactive T cells. The apoptosis-resistance in these engineered immune cells allow the cells to survive the cytotoxic effects of pathogenic T- and B-cells. Compositions and methods comprising these cells are provided.

Description

APOPTOSIS RESISTANT IMMUNE CELLS WITH MAJOR

HISTOCOMPATIBILITY COMPLEX CHIMERIC ANTIGEN RECEPTOR

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application

No. 63/348,619, filed June 3, 2022, the disclosures of which are incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING The instant application contains a Sequence Listing which has been filed electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on June 2, 2023, is named 112198-0046-70007WO00_SEQ.XML and is 38,358 bytes in size. BACKGROUND OF THE INVENTION

Autoimmune diseases are characterized by abnormal immune responses against selfantigens, leading to damage or disruption of tissues. For example, multiple sclerosis (MS) is a central nervous system autoimmune disease in which activated 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.

The major histocompatibility complex (MHC), known as human leukocytes (HLA) in humans, is a set of cell surface proteins essential for the immune system to recognize foreign agents. 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. In the autoimmune disease situation, 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.

Treatment of autoimmune diseases that involves the elimination of these pathogenic cells can improved therapeutic outcomes over available therapies. Reinhard et al., Proceedings of the National Academy of Sciences, 101(Suppl. 2): 14599-14606; 2004. For example, a cell-based method of eliminating these pathogenic cells. However, both activated and non-activated pathogenic T and B cells can target the therapeutic cells used in the cellbased therapy, thus reducing the number of therapeutic cells and also the effective function of the therapeutic cells. There is a need for therapeutic cells that can survive the cytotoxicity and apoptosis induced by pathogenic T and B cells.

SUMMARY OF THE INVENTION

The present disclosure is based, at least in part, on the discovery that exemplary

5 apoptosis inhibitors, CRMA, PI9, cFLIP, or a combination thereof, 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. Expression of such apoptosis inhibitors provided some level of protection against cell death induced by NK-like effector

1 o cells that can degranulate when coming into contact with the engineered host cells expressing the anti- apoptosis proteins in a co-culture assay. Further, CRMA, either expressed alone or in combination with an eMHC molecule, was the most effective in providing protection from degranulation induced by the NK-like effectors cells as measured by Cytotox Red signal in an Incucyte live cell imager. These experimental data indicates that expression of the apoptosis i inhibitors such as those disclosed herein could protect engineered host cells (e.g. , 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.

Accordingly, provided herein are compositions and methods comprising engineered

2 o 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. In some embodiments, the apoptosis-resistant immune cells can be engineered to express major histocompatibility complex based chimeric

25 antigen receptors (MHC-CAR) 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

30 targeted pathogenic T and B cells, and/or surrounding environment. With the added apoptosis resistance, 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.

In some aspects, provided herein is an engineered immune cell containing: (1) an exogenous apoptosis inhibitor; and

(2) a chimeric antigen receptor (CAR) comprising (i) an extracellular antigen binding domain; and (ii) a co-stimulatory domain, a cytoplasmic signaling domain, or a combination thereof. As used herein, “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. For example, 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. Alternatively, the exogenous nucleic acids may be integrated into the genome of the immune cells. In case the immune cells contain an endogenous gene of the apoptosis inhibitor disclosed herein, the exogenous encoding nucleic acid is integrated at a genomic site that is different from the native loci of the endogenous gene. In some embodiments, the apoptosis inhibitor can be a granzyme B inhibitor, for example, a cytokine response modifier A (CRMA). In some examples, the CRMA may comprise the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the apoptosis inhibitor can be a serpin, for example, a proteinase inhibitor 9 (PI9). In some examples, the PI9 protein may comprise the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the apoptosis inhibitor may be a peptidase C 14A, for example, a cellular FLICE inhibitory protein (cFLIP). In some examples, the cFLIP may comprise the amino acid sequence of SEQ ID NO: 6.

In some embodiments, the engineered immune cells may express a combination of any of the apoptosis inhibitors provided herein.

In some embodiments, 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. In some instances, 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.

In other instances, the MHC molecule is a class II MHC molecule, 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. In some examples, the engineered immune cell is an NK cell, which can be an NK-92 cell or an KHYG-1 cell. In some instances, the NK cell can be deficient in killer-cell immunoglobulin- like receptor (KIR). In other examples, the engineered immune cell can be a T cell. In one example, the T cell is a CD8+ T regulatory (T_reg) cell. In another example, the T cell can be a CD4+ T_reg cell.

In some embodiments, 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. In some examples, the cytoplasmic signaling domain is from CD3 .

In some examples, 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). In other examples, 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. In yet other examples, 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.

In some embodiments, 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.

In another aspect, 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.

In yet another aspect, 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. In some instances, the engineered immune cell is allogenic to the subject. Alternatively, the engineered immune cell is autologous to the subject.

In some embodiments, 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.

Also provided herein are 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.

Additionally, the present disclosure contemplates a method for producing a population of the engineered immune cells described herein. Such 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. In some instances, the one or more nucleic acids are one or more messenger RNA molecules. Alternatively, the one or more nucleic acids are one or more expression vectors, which optionally are viral vectors. The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to the drawing in combination with the detailed description of specific embodiments presented herein. 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.

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. DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It should be understood that this disclosure is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

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. However, 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. There are two main mechanisms by which apoptosis is induced. First, the lytic granules directionally released from the activated cytotoxic T-lymphocyte (CTL) carry perforins. 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. For example, methotrexate (MTX), an antifolate, is used to treat leukemia, psoriasis and rheumatoid arthritis. MTX inhibits dihydrofolate reductase (DHFR), which is essential for cell growth and proliferation. Consequently, the undesired cells die. 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

5 MTX, dasatinib, dexamethasone, ibrutinib, alpelisib, ruxolitinib and tofacitinib and. By conferring this added apoptosis resistant property to the MHC-CAR immune cells, the immune cells are less likely to experience a loss-of-function in vivo and this would improve the therapeutic outcomes over available therapies. One apoptosis-resistant strategy is the expression of an apoptosis inhibitor. Accordingly, expression of an exogenous apoptosis

1 o inhibitor confers the apoptosis resistant property to the engineered MHC-CAR immune cells described herein. Another apoptosis-resistant strategy is the expression of a mutant mediator protein that allows the cell to survive the toxic effects of therapeutic drugs used to treat diseases or disorders.

Accordingly, disclosed herein are genetically engineered immune cells comprising an i exogenous apoptosis inhibitor and a major histocompatibility complex (MHC)-based chimeric receptors (CAR). 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

2 o type of apoptosis inhibitor. For example, 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

25 International Patent Publication WO2018227091 Al, W02007036028A1, J. P. Medema 2001, PNAS 98 (20) 11515-11520.The engineered immune cell would have enhanced resistance to activation induced cell death from the pathogenic T- or B-cells.

I. Apoptosis Inhibitors

30 The engineered immune cells disclosed herein express one or more apoptosis inhibitors. As used herein, 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. In some instances, the apoptosis inhibitor can be a naturally occurring protein. In other instances, 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.

In some embodiments, 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. In some embodiments, the engineered immune cell described herein expresses 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.

Table 1. Cell Death Pathways and Apoptosis Inhibitor Proteins

In some embodiments, 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. In the cytoplasm 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. In mitochondria outer membrane Granzyme B also cleaves anti- apop to tic Bcl-2 family protein Myeloid cell leukemia sequence 1 (Mcl-1). Those interactions induce the release of mitochondrial Cytochrome c somatic (Cytochrome C) and Diablo homolog (Smac/Diablo) into the cytosol. Cytochrome C is involved in the apoptosome pathway together with Apoptotic peptidase activating factor 1 (Apaf-1) and activated Caspase 9. Release of mitochondrial Smac/Diablo represses the inhibitors of apoptosis in target cell, making caspases more readily activated directly by Granzyme B. On the other hand, Granzyme B directly processes Caspase-8, and Caspase- 10, and then activates Caspase-3, and Caspase-7. 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.

In some instances, 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. In some examples, 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. In one specific example, 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.

Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the invention. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs e.g., XBLAST and NBLAST) can be used. In some embodiments, 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 (serpins) 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

1 o inhibitor to modulate the host immune response from natural killer cell mediated cytotoxicity against tumor cells.

In some instances, 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. In some examples, 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. In specific examples, the PI9 protein may comprise (e.g., consisting of) the amino acid sequence of SEQ ID NO: 4.

20 In some embodiments, the engineered immune cells disclosed herein express an apoptosis inhibitor, which is a FAS inhibitor. The cell-surface Fas receptor (Fas), also termed Apo-1 or CD95, is a member of the tumor necrosis factor (TNF) and nerve growth factor (NGF) family of receptors. Upon interacting with its ligand, FasL, the consequential intracellular signaling is initiated and cell death follows. The activation of the Fas pathway

25 typically displays the features of apoptotic cell death. Key players in the induction and progression of apoptosis are a series of proteases, the caspases, which cleave substrates after an aspartate residue (Pl).

An exemplary Fas inhibitor is a CASP8 and FADD-like apoptosis regulator (CFLAR, also known as cellular FLICE inhibitory protein or cFLIP). Phylogenetically, 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. 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.

In some examples, 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. In specific examples, the cFLIP protein may comprise (e.g., consisting of) the amino acid sequence of SEQ ID NO: 6.

In other embodiments, 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.

In yet other embodiments, 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. In some instances, the apoptosis inhibitor may be a B cell blocker. Blocking the activation of B cells or the associated cell signaling pathways downstream of the activation indirectly inhibits induced apoptosis by B cells. Anti-B cell antibody can inhibit B cell activation. As used herein, the term “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.

Examples of an anti-B cell antibody are rituximab, ocrelizumab, ofatumumab, eculizumab, adalimumab, tocilizumab, teplizumab, and ublituximab.

Engineered immune cells expressing additional exemplary apoptosis inhibitors are provided below.

In some instances, provided herein is 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. 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. For example, 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. In some embodiments, 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. In other embodiments, the mr-DHFR has one of the following pairs of mutations: L22F and F31S; L22Y and F31S, L22F and F31G; L22P and F31G; L22Yand

5 F31G. In one embodiment, 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). The absorbance at 450 nm was measured using a 96- well plate reader and readings compared to that obtained for wells without drug (but with cells) as 100%, and wells without cells as 0%.

In some instances, provided herein is an engineered immune cell comprising a

20 dasatinib inhibitor and an MHC-CAR. In other aspects, provided herein is 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

25 resistance to apoptosis induced by dexamethasone. Dasatinib, nilotinib, and imatinib are tyrosine-kinase inhibitors and they work by blocking a number of tyrosine kinases such as Bcr-Abl and the Src kinase family. In some embodiments, 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

30 higher with compared with the IC50 of the cells expressing unmutated BCR-ABL when incubated in vitro with dasatinib. 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).

BCR-ABL is a mutation that is formed by the combination of two genes, known as 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. Depending on the breakpoint in the BCR gene, three main types of 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. 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). Expression of such a BCR-ABL fusion protein with select mutations at residues 315 and 317 in the engineered immune cells described herein allow these cells to circumvent the inhibition of the Src kinase pathway, avoid apoptosis, and thereby facilitating the survival of the engineered immune cells in vivo during dasatinib treatment. Point mutations of residue 315 and 317 may be carried by any methods known in the art, such as taught in US 20090306094; US20100029676; US 20120021425; and US7314712; the contents are incorporated herein by reference in their entireties. In some embodiments, 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) also known as multidrug resistance protein 1 (MDR1) or ATP-binding cassette sub-family B member

1 (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. In one embodiment, the ABCB 1 expressed has a mutation at amino acid position 5 1199. In one embodiment, the mutation is a substitution, for example, mutation G1199A. In some embodiments, 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 . In one embodiment, 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). As an efflux pump exhibiting a broad substrate specificity localized on cellular plasma membrane, ABCG2 excretes a variety of endogenous and ib exogenous substrates including chemotherapeutic agents, such as mitoxantrone and several tyrosine kinase inhibitors. In the normal tissues, ABCG2 is expressed on the apical membranes and plays a pivotal role in tissue protection against various xenobiotics. Overexpression of ABCG2 in the engineered immune cells described herein allow these cells to remove the dasatinib inhibitor before the inhibitor cause irreversible damage to the cells, 20 and thereby facilitating the survival of the engineered immune cells in vivo during dasatinib treatment. In one embodiment, the ABCG2 expressed has a mutation at amino acid position 141 or 482 or both positions 141 and 482. In one embodiment, the mutation is a substitution, for example, mutation Q141K and R482G. In some embodiments, 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. In one embodiment, 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. In some embodiments, 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). In additional embodiments, 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). 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 example of an ibrutinib inhibitor is a mutant BTK (NM_000061.3) that has a mutation at amino acid position 481. In one embodiment, 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). It is an oral agent first approved by the FDA for the treatment of rheumatoid arthritis and has been shown to decrease levels of IL-6 in patients with this disease. 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.

In one preferred embodiment, 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. Examples of mammalian inducible promoters are 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 mouse mammary tumor virus long terminal repeat (MMTV-LTR). See US9388425, the content is incorporated herein by reference in its entirety.

The genes encoding these exemplary granzyme inhibitors are disclosed in Table 1. In some embodiments, 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. In other embodiments, 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.

Table 2. Examples of Apoptosis Sensitization Drugs, Protein Targets and Mutants

II. Major Histocompatibility Complex-Based Chimeric Antigen Receptors (CAR)

In addition to an apoptosis inhibitor, the engineered immune cell described herein also comprise a chimeric antigen receptor (CAR), such as a MHC based chimeric receptor (MHR- CAR). 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. In some instances, 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. As used herein, the term “conjugated” means that at least two components are physically associated, either via covalent bonds or via non-covalent interactions.

In other examples, the CAR described herein may be a multi-chain protein complex, for example, a heterodimer, comprising one polypeptide that comprises the antigenic peptide. Optionally, an exogenous cytokine moiety is included as a fusion polypeptide with the antigen. In some instances, 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. Optionally, 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. In some instances, the 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. Such 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.

In one embodiment, 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

(a) MHC Moiety

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.) In some instances, the MHC moiety is from a human MHC molecule (also known as HLA). In some instances the domains that interact with molecules from other cells (TCR or BCR) are from a human MHC molecule. There are primarily two classes of MHC molecules, MHC class I molecules and MHC class II molecules, both of which can be used for constructing the CARs described herein. Sequences of MHC class I and class II molecules of various species (e.g., human, non-human primates, canids, fish, ovids, bovines, equids, suids, murids, and gallus) 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. In some embodiments, 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. In some instances, the alpha chain of the MHC class I molecule may be fused with -microglobulin to produce a single chain fusion protein. In some examples, the MHC Class I moiety is from HLA A3, which can be co-used with a PLP peptide. Honma et al., J. Neuroimmunol. 73:7-14 (1997). In other examples, 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).

Like MHC class I molecules, 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. When a MHC class II molecule is used for constructing a CAR, 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). In cases where only the region that interacts with other cell types is used (i.e., al and pi), specific amino acid modifications may be required to enhance the folding of the mini-MHC, see mini-sequence with shaded regions and Birnbaum et al. The MHC class II molecule may be a human HLA

DP molecule, a human HLA DM molecule, a human HLA DOA molecule, a human HLA DOB molecule, a human HLA DQ molecule, or a human HLA DR molecule. In some examples, 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. For examples, 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), and 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.

In some embodiments, 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. (b) Antigenic Peptide

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 .

In some instances, 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. Hacohen et al., Cancer immunology research, 1(1): 11- 15 (2013). Also within the scope of the present disclosure are personalized CAR T and Treg therapies for autoimmune disorders. For disorders with very strong MHC associations, 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. Jiang et al., International immunology, 25(4):235-246, (2013). These MHC-protein complexes appear to induce autoantibody production in autoimmune disorders, including antibodies that do not bind to properly folded proteins as well as autoantibodies that are specific to those with specific autoimmune disorders. In some embodiments, 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,

5 tryptase involved in rheumatoid arthritis (RA), and the proteins included in Tables 4-6 below. Alternatively, 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. In their natural state (attached to an MHC rather than an MHC moiety of a CAR), such antigenic protein/MHC complexes stimulate pathogenic B cells to produce autoantibodies. For proteins such as IgGH or rheumatoid factor in rheumatoid arthritis (Jin et al., Proceedings of the National Academy of Sciences, lll(10):3787-3792, 2014), p2-glycoprotein I in ib antiphospholipid syndrome (Tanimura et al., Blood, 125(18):2835-2844, 2015) and recurrent miscarriage (Tanimura et al., Placenta, 46:108, 2016), GM-CSF in autoimmune pulmonary alveolar proteinosis (Hamano et al., ALVEOLAR MACROPHAGE BIOLOGY B32:A3147- A3147, 2016), tyrosinase in vitiligo (Arase et al. Journal of Dermatological Science, 84(l):e87, 2016), and myeloperoxidase in microscopic polyangiitis (Hiwa et al., Arthritis &

20 Rheumatology, 69(10):2069-2080, 2017), HLA mediated surface display and in some cases autoantibody binding of misfolded variant/HLA complex can occur.

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

MHC class I molecule. 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. The

30 open antigen-binding groove of MHC class II molecules implicated in autoimmune disorders can also frequently display intact (e.g., yet misfolded) antigenic proteins or splice variants. Jiang et al., International immunology, 25(4):235-246, 2013. Additional sequence examples of antigenic peptides are disclosed in the International Patent Publication No. WO 2019/094847, the content is incorporated by reference in its entirety. In some embodiments, 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. MHC binding peptide oligomers and methods of use US20020058787A1. 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. NKG2A/CD94 and

HLA-E also interact with HLA-E bound to the VMAPRTLFL (SEQ ID NO: 22) peptide derived from HLA-G.

Table 3. Autoantigens of Various Autoimmune Disorders

Table 4 discloses antigenic peptide epitopes that commonly bind to HLA-E.

These epitopes can be used in the CAR described herein. In one embodiment, 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. For instance if the strain of CMV in an autoimmune patient has epitope VMAPRTLVL (SEQ ID NO: 9) in UL40, then preferably 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 (for instance from a family member) may help eliminate EBV, as EBV infected B cells are resistant to autologous NK cells, until lytic infection.

Table 4. Antigenic Peptide Epitopes that Commonly Bind to HLA-E.

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.

Table 5. HLA Types and Classes Commonly Associated with Autoimmune Disease

In some embodiments, the antigenic peptides or antigenic polypeptides are patient specific and designed for the patient’ s MHC. For example, 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

31 such as irradiation to reduce the capacity of the engineered immune cell described herein to expand. The term “expand” as used herein 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).

(c) Co-stimulatory signaling domains

Many immune cells require co-stimulation, in addition to stimulation of an antigenspecific signal, to promote cell proliferation, differentiation and survival, as well as to activate effector functions of the cell. The CAR described herein may comprise one or more co-stimulatory signaling domains. The term “co-stimulatory signaling domain,” as used herein, 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 (e.g., an immune 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. 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 (FcRy) 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.

Table 6. Cytoplasmic Internal Domains for CAR Function in NK cells

Table 7. Cytoplasmic Internal Domains for CAR Function in Macrophages

In some instances, 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. In some embodiments, the co-stimulatory domain is preceded by a short linker. For example, for a class II CAR, the short linker may be TS (i.e., a MHC internal Linker); for a class I CAR, 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.

An example of a 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.

In some aspects, the cytoplasmic portion of the CAR described herein (ie., the co- stimulatory domain and/or the cytoplasmic signaling domain) 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. Altering 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. In some embodiments, 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. For example, by site-directed mutation(s) disclosed in US. Patent No: US6329178, the content of which is incorporated herein by reference in its entirety. In some instances, the CAR constructs described herein may include no co- stimulatory domain. Alternatively, 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.

(d) Cytoplasmic signaling domain

Any cytoplasmic signaling domain comprising an immunoreceptor tyrosine-based activation motif (IT AM) 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. In some examples, the cytoplasmic signaling domain comprising an IT AM is of CD3 . In some examples, the CAR does not comprise a co-stimulatory domain and the cytoplasmic signaling domain is preceded by a short linker. For example, for a class II CAR, the short linker may be TS (i.e. a MHC internal Linker). For example for a class I CAR, the short linker may be PG. In some cases the linker may be AHA or absent, such as certain instances when a costimulatory domain occurs before a signaling domain. In some embodiments, 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. Moisini, et al., The Journal of Immunology, 180(5), pp.3601- 3611. The sequences for a cytoplasmic signaling domain from CD3f and nucleic acid sequences encoding a cytoplasmic signaling domain from CD3^ are described in in the International Patent Publication No. WO 2019/094847, the content is incorporated by reference in its entirety.

(e) Additional components

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. Methods of using these components to construct fusion polypeptides comprising an antigenic peptide, the MHC component (alpha chain and the beta chain), the cytoplasmic signaling domain, the co-stimulatory domain, and the exogenous cytokine are well known in the art. For example, the methods described in the International Patent Publication No. WO 2019/094847, the content is incorporated by reference in its entirety.

(ii) Configuration of CARs

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. In some examples, 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. Alternatively, a MHC class I CAR may also include b2m, which may be fused with the alpha chain to produce a single polypeptide. In some instances, 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). In some examples, the antigenic peptide may be fused to the alpha chain. In other examples, the antigenic peptide may be fused to b2m. Optionally, 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. In some instances, 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. For example, 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.

Any of 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.). In some instances, the CAR constructs described herein are free of signaling domains. Preferably, 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. In some instances, 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. 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. ( Hi ) Preparation of CARs

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). In some embodiments, 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. In some embodiments, sequences of one or more of the components of the CARs are obtained from a human cell. Alternatively, the sequences of one or more components of the CARs can be synthesized. Sequences of each of the components (e.g. , domains) can be joined directly or indirectly (e.g., using a nucleic acid sequence encoding a peptide linker) to form a nucleic acid sequence encoding the CAR, using methods such as PCR amplification or ligation. Alternatively, the nucleic acid encoding the CAR may be synthesized. In some embodiments, the nucleic acid is DNA. In other embodiments, the nucleic acid is RNA.

Any of the CAR proteins, nucleic acid encoding such, and expression vectors carrying such nucleic acid, all of which are within the scope of the present disclosure, can be mixed with 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. 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.

In one aspect, 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. 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. In some embodiments, the immune cells are T cells. In some embodiments, 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

In one embodiment, the host immune cell may be obtained from a subject. Nonlimiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Preferably, 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 (e.g., T cells, NK cells, macrophages, neutrophils, eosinophils, or any combination thereof) 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. A source suitable for obtaining the type of immune cells desired would be evident to one of skill in the art. In some embodiments, 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.

In certain embodiments, any number of cell lines or progenitor cell lines available in the art, may be used. Preferably, the immune cell is a natural killer (NK) cell or a macrophage cell or a cell line thereof. For example, a NK 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. In some embodiments, 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.

Without wishing to be bound by any particular theory, 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 then 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. a) Natural killer (NK) cell and NK cell lines

NK cells (also known as K cells, killer cells, and or large granular lymphocytes (LGL)) 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). 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

5 adaptive immune response generates antigen- specific cytotoxic T cells that can clear the infection.

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. 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 may also induce toxicity.

In one aspect, 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;

20 10,138,462, the contents are incorporated by reference in their entirety.). NK92, YTS, and

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,

25 CD28, and CD45 but are CD16 negative. 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

30 their expression of FasL, TRAIL, and TNFot. 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.

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).

Other human NK cell lines developed from induced pluripotent stem cells (iPSC) or modified NK cells can also be used. For examples, 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. b) Macrophages and monocyte-like cell lines

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. In addition, 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). In one aspect, human macrophage cell lines or monocyte- like cell lines (MCLCs) are used to express the CAR described herein. These includes but are not limited to THP-1, HL- 60, NB-40, U937, cell lines having the ATCC accession numbers: CRL 9850, CRL 9851, CRL 9852, CRL 9853, CRL 9854, CRL 9855, and CRL 9856, and the monocytic cell lines, Mono Mac 1 and 6 (CVCL_1426) cells (D.M. Riddy, 2018, PLoS ONE 13(5): e0197177; U.S. Patent No.: 5,447,861, the contents of these are incorporated herein by reference in their entireties).

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). Mono Mac 1 and 6 (from clone 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).

Other human macrophage or monocyte cell lines developed from induced pluripotent stem cells (iPSC) can also be used. For examples, the 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. To construct the immune cells that express any of the apoptosis inhibitor(s) and the MHC-CAR constructs described herein, 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. For example, 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. Alternatively, 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.

A variety of 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. 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.

Additionally, 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.

In some embodiments, 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

5 encodes an apoptosis inhibitor or a MHC-CAR construct described herein is also within the scope of the present disclosure. Such 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

1 o 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. WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655). In some embodiments, the vectors for expression of the chimeric receptors are retroviruses. In some embodiments, the vectors for expression of the chimeric receptors are lentiviruses.

In examples in which the vectors encoding apoptosis inhibitor/ s) or chimeric

2 o receptors are introduced to the host cells using a viral vector, 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

25 with the immune cells.

Following introduction into the host cells a vector encoding any of the MHC-CAR provided herein, the cells are cultured under conditions that allow for expression of the chimeric receptor. In examples in which the nucleic acid encoding the apoptosis inhibitor(s) or the MHC-CAR is regulated by a regulatable promoter, the host cells are cultured in

3 o conditions wherein the regulatable promoter is activated (e.g. , Tet off/on inducible system).

In some embodiments, 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.

Alternatively, expression of an apoptosis inhibitor or MHC-CAR construct in any of the immune cells disclosed herein can be achieved by introducing RNA molecules encoding the apoptosis inhibitor or MHC-CAR constructs. Such RNA molecules 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. For example, 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. The term “ex vivo,” as used herein, 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). The type of immune cells that can be used (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. ii) Immortalization to make host cell lines

In some cases, 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. Fujisaki, H., et al. (2009). British journal of haematology, 145(5), 606-613; Yang, Y., et al., 2020. Molecular Therapy-Methods & Clinical Development, 18, pp.428-445. 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. In one embodiment, to limit the proliferation of the CAR-expressing engineered immune cell, less proliferative cell types of host immune cells are used to express the CAR described herein. The host immune cells (e.g., the immortalized cell lines) 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.

Alternatively, 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). In one embodiment, 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. iv) Co-expression of anti-B cell antibody or anti-B cell CAR

In certain aspects, 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. In some embodiments, 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. In some embodiments, 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. In other embodiments, the anti-B cell antibody or anti-B cell CAR is a bispecific anti-B cell antibody or CAR. For example, anti-CD19 and anti-CD20 on a B cell. Methods for expression of anti-B cell antibody or anti-B cell CAR are described in the U.S. Patent Nos. 10,357,514 and 10,253,086. These references are incorporated by reference in their entirety. In one embodiment, 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.

In one embodiment, 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.

These references are incorporated by reference in their entirety.

In one embodiment, the engineered, apoptosis-resistant, MHC-CAR-expressing immune cells described herein are treated to reduce the cell proliferation capability of the engineered cells. In one embodiment, 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.

Any known methods in the art may be used to determine the 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). For example, for cytotoxic determination, 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). For phagocytosis measurements, 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.

To assay for the expression or function of the MHC-CAR in the engineered immune cell, 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). 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. Additionally, 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.

IV. Therapeutic Uses of the Engineered Immune 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. In some embodiments, the subject is a mammal, such as a human, monkey, mouse, rabbit, or domestic mammal. In some embodiments, the subject is a human, for example, a human patient having, suspected of having, or at risk for an autoimmune disease. According, provided herein are population of engineered immune cells described herein for the treatment of an autoimmune disease in a subject in need thereof. In another aspect, provided herein is the use of 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. To perform the methods described herein, 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. Alternatively, 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. For example, allogeneic immune cells may be derived from a human donor and administered to a human recipient who is different from the donor.

In some embodiments, the immune cells are co-used with 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. For example, the efficacy of the immunotherapy may be assessed by survival of the subject and/or reduction of disease symptoms in the subject.

In some embodiments, 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. For example, the immune cells may be administered to a human subject simultaneously with the therapeutic agent. Alternatively, the immune cells may be administered to a human subject during the course of a treatment involving the therapeutic agent. In some examples, 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. To practice the method disclosed herein, 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. As used herein, 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. In some embodiments, the effective amount alleviates, relieves, ameliorates, improves, reduces the symptoms, or delays the progression of any disease or disorder in the subject. In some embodiments, the subject is a human. In some embodiments, the subject is a human cancer patient.

In some embodiments, the subject is a human patient suffering from an autoimmune disease, which is characterized by abnormal immune responses attacking a normal body part. Examples of 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.

In some embodiment, 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.

In some embodiment, the engineered apoptosis-resistant, CAR-expressing immune cells or compositions comprising these cells are irradiated before being administered to a patient.

In some embodiments, the engineered immune cells described herein may be used to remove pathogenic T cells or B cells in ex vivo. For example, a patient may be scheduled to undergo hematopoietic stem cell transplantation (HSCT), such as a multiple sclerosis or scleroderma patient. 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. In some embodiments, 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.

In some embodiments, the engineered apoptosis-resistant, immune cells carrying MHC-CAR are made from a patient’s own CD8+ regulatory T cells. For example, a patient has an autoimmune disorder and is schedule to undergo HSCT. Prior to 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. For example, patient’s 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). In some embodiments, 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.

In other instances, haploidentical NK cells are the starting cells for engineered apoptosis-resistant, immune cells carrying MHC-CAR described herein. In some embodiments, when the haploidentical NK cells used are from cell lines, 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. In other embodiments, when the haploidentical NK cells used are from cell lines, the resultant engineered immune cells may be irradiated to prevent cell proliferation in vivo after infusion into the patient. Additionally, 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. In some instances, 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. In some embodiments, a patient having an autoimmune disorder is treated with CD3, CD28, and rapamycin. Then the 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. Alternatively, 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.

V. Kits for Therapeutic Uses

The present disclosure also provides 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. Such kits may include one or more containers comprising compositions comprising immune cells expressing MAR-CAR such as those described herein), and a pharmaceutically acceptable carrier.

In some embodiments, 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. In some embodiments, 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.

The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Also contemplated are packages for use in combination with a specific device. A kit 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. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiment, the disclosure provides articles of manufacture comprising contents of the kits described above.

Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein. General techniques

The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed. 1987); Introuction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.

1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds.(1985»; Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984»; Animal Cell Culture (R.I. Freshney, ed. (1986»; Immobilized Cells and Enzymes (IRL Press, ( 1986»; and B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.).

Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

EXAMPLES

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. A. Design of plasmids for in vitro transcription (IVT)

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. In some cases, 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. UCL (University College London), 2015.) which interacts with anti-CD34 antibodies or truncated LNGFR (Lauer, Ulrich M., et al. "A prototype transduction tag system (ALNGFR/NGF) for noninvasive clinical gene therapy monitoring." Cancer gene therapy 7.3 (2000): 430-437.) which interacts with anti-cd271 antibodies was used instead of gfp.

B. mRNA Generation The IVT plasmid was linearized using EcoRI or Xbal and purified. mRNA was generated using the HiScribe™ 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. C. Direct and indirect expression evaluation (DNA or mRNA) in MHC free cell line The constructs (DNA or IVT) were analyzed 24 hours after nucleofection into K562. Indirect expression 1 : For constructs expressing eMHC and the apoptosis inhibitor but not gfp, the expression was analyzed by flow cytometry using eMHC expression as a proxy. Live (annexin V- and 7AAD-) vs eMHC (HLA-DR+). Alternatively, live (7AAD-) vs (HLA-DR+). 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

For animal free media maintenance, cells or cell lines were maintained in 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).

Alternatively 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).

For 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). 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.

For bead expanded PBMC or CB unit derived NK 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. For Amaxa nucleofection (Lonza 4D Nucleofector), 1 million PBMCs, CB units, NK cell lines, unstimulated or stimulated NK cells were nucleofected using nucleocuvette strips (Lonza), Opti-MEM (Thermo) as the electroporation buffer and nucleofection program CA- 137. For Amax nucleofection Thp-1 cell line was nucleofected using nucleocuvettes strip, SG cell line solution and supplement 1, and nucleofection progrm FF-100.

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’.

E. Lentiviral and alpharetrovirus vectors and packaging

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). Alternatively an 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).

In some cases 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,

The Journal of the American Society of Hematology 124.8 (2014): 1221-1231; Zhang XY, La Russa VF, Reiser J., J Virol. 2004 Feb . 78(3): 1219-29; and Sandrin, Virginie, et al. Blood, The Journal of the American Society of Hematology 100.3 (2002): 823-832).

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.

F. Alternative delivery methods

Delivery using 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. "Production of lentiviral vectors using novel, enzymatically produced, linear DNA." Gene therapy 26.3 (2019): 86-92.) where the sequence between the ITRs (contains EFlalpha-ATG start codon, eMHC (where the eMHC in some cases has an internal 2A sequence), 2A, apoptosis inhibitor, 2A, gfp, TAA stop codon +/- WPRE (SEQ ID NO: 40)). Delivery using episomal vectors — A minicircle that retains (Flalpha- ATG start codon, eMHC (where the eMHC in some cases has an internal 2A sequence), 2 A, apoptosis inhibitor, 2A, gfp, TAA stop codon +/- WPR) Ebna-1 expression, OriP (EBV origin of replication) but removes an E. coli origin of replication. (Kamath, Anant, et al. "Efficient method to create integration-free, virus-free, Myc and Lin28-free human induced pluripotent stem cells from adherent cells." Future science OA 3.3 (2017): FSO211.) The minicircle is delivered to the cell using Amaxa or Maxey te (static or flow electroporation).

G. Ex vivo/in vivo expansion/selection/evaluation of transformed cells with the apoptosis inhibitor

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).

Patients were treated with methotrexate or dasatinib after delivery of the therapeutic cells.

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

7AAD and annexin 5). Alternatively, 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).

Alternatively, 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. Activation markers and cytokine release are assessed post TCR dextramer binding.

GMP compliant cell sorting: 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.

Alternatively cells were sorted indirectly using a tag (RQR8, LNGFR) and labelled antibody targeting them (for example, a PE-labelled antibody) in the tyto. Alternatively cells could be purified for a specific cell type after sorting (for instance with a NK selection kit).

H. Expanding T cells and sequencing TCR

In the following experiment, 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).

Example 2. Apoptosis Inhibitors CRMA, PI9, and cFLIP in Protecting Against Cell Death

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). 48 hours after lipofection, 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.

Amino Acid Sequence of CRMA

MDIFREIASSMKGENVFISPPSISSVLTILYYGANGSTAEQLSKYVEKEADKNKDDISF KSMNKVYGRYS AVFKDSFLRKIGDNFQTVDFTDCRTVDAINKCVDIFTEGKINPLLD

EPLSPDTCLLA1SAVYFKAKWLMPFEKEFTSDYPFYVSPTEMVDVSMMSMYGEAFN HASVKESFGNFSIIELPYVGDTSMVVILPDNIDGLESIEQNLTDTNFKKWCDSMDAMF IDVHIPKFKVTGSYNLVDALVKLGLTEVFGSTGDYSNMCNSDVSVDAMIHKTYIDV NEEYTEAAAATCALVADCASTVTNEFCADHPFIYVIRHVDGKILFVGRYCSPTTN (SEQ ID NO: 2)

Amino Acid Sequence of CRMA with C-terminus Flag Tag (tag sequence italicized) MDIFREIASSMKGENVFISPPSISSVLTILYYGANGSTAEQLSKYVEKEADKNKDDISF KSMNKVYGRYS AVFKDSFLRKIGDNFQTVDFTDCRTVDAINKCVDIFTEGKINPLLD EPLSPDTCLLAIS AVYFKAKWLMPFEKEFTSDYPFYVSPTEMVDVSMMSMYGEAFN

HASVKESFGNFSIIELPYVGDTSMVVILPDNIDGLESIEQNLTDTNFKKWCDSMDAMF IDVHIPKFKVTGSYNLVDALVKLGLTEVFGSTGDYSNMCNSDVSVDAMIHKTYIDV NEEYTEAAAATCALVADCASTVTNEFCADHPFIYVIRHVDGKILFVGRYCSPTTNGG SGDYKDDDDK (SEQ ID NO: 1)

Amino Acid Sequence of PI9_

MVLLGAKGNTATQMAQALSLNTEEDIHRAFQSLLTEVNKAGTQYLLRTANRLFGEK TCQFLSTFKESCLQFYHAELKELSFIRAAEESRKHINTWVSKKTEGKIEELLPGSSIDA ETRLVLVNAIYFKGKWNEPFDETYTREMPFKINQEEQRPVQMMYQEATFKLAHVGE VRAQLLELPYARKELSLLVLLPDDGVELSTVEKSLTFEKLTAWTKPDCMKSTEVEVL LPKFKLQEDYDMESVLRHLGIVDAFQQGKADLSAMSAERDLCLSKFVHKSFVEVNE EGTEAAAASSCFVVAECCMESGPRFCADHPFLFFIRHNRANSILFCGRFSSP (SEQ ID NO: 4)

Amino Acid Sequence of PI9 with C -terminus Flag Tag (tag sequence italicized) MVLLGAKGNTATQMAQALSLNTEEDIHRAFQSLLTEVNKAGTQYLLRTANRLFGEK TCQFLSTFKESCLQFYH AELKELSFTR A AEESRKHTNTWVS KKTEGKTEELLPGSSTD A ETRLVLVNAIYFKGKWNEPFDETYTREMPFKINQEEQRPVQMMYQEATFKLAHVGE VRAQLLELPYARKELSLLVLLPDDGVELSTVEKSLTFEKLTAWTKPDCMKSTEVEVL

LPKFKLQEDYDMESVLRHLGIVDAFQQGKADLSAMSAERDLCLSKFVHKSFVEVNE EGTEAAAASSCFVVAECCMESGPRFCADHPFLFFIRHNRANSILFCGRFSSPGG.S'G r KDDDDK SEQ ID NO: 3) Amino Acid Sequence of cFLIP

MSAEV1HQVEEALDTDEKEMLLFLCRDVA1DVVPPNVRDLLDILRERGKLSVGDLAE LLYRVRRFDLLKRILKMDRKAVETHLLRNPHLVSDYRVLMAEIGEDLDKSDVSSLIF LMKDYMGRGKISKEKSFLDLVVELEKLNLVAPDQLDLLEKCLKNIHRIDLKTKIQKY KQSVQGAGTSYRNVLQAAIQKSLKDPSNNFRLHNGRSKEQRLKEQLGAQQEPVKKS IQESEAFLPQSIPEERYKMKSKPLGICLIIDCIGNETELLRDTFTSLGYEVQKFLHLSMH

GISQILGQFACMPEHRDYDSFVCVLVSRGGSQSVYGVDQTHSGLPLHHIRRMFMGDS CPYLAGKPKMFFIQNYVVSEGQLEDSSLLEVDGPAMKNVEFKAQKRGLCTVHREAD FFWSLCTADMSLLEQSHSSPSLYLQCLSQKLRQERKRPLLDLHIELNGYMYDWNSR

VSAKEKYYVWLQHTLRKKLILSYT (SEQ ID NO: 6)

Amino Acid Sequence of cFLIP with C-Terminus Flag Tag (tag sequence italicized)

MSAEVIHQVEEALDTDEKEMLLFLCRDVAIDVVPPNVRDLLDILRERGKLSVGDLAE LLYRVRRFDLLKRILKMDRKAVETHLLRNPHLVSDYRVLMAEIGEDLDKSDVSSLIF LMKDYMGRGKISKEKSFLDLVVELEKLNLVAPDQLDLLEKCLKNIHRIDLKTKIQKY KQS VQGAGTS YRNVLQAAIQKSLKDPSNNFRLHNGRSKEQRLKEQLGAQQEPVKKS

IQESEAFLPQSIPEERYKMKSKPLGICLIIDCIGNETELLRDTFTSLGYEVQKFLHLSMH GISQILGQFACMPEHRDYDSFVCVLVSRGGSQSVYGVDQTHSGLPLHHIRRMFMGDS CPYLAGKPKMFFIQNYVVSEGQLEDSSLLEVDGPAMKNVEFKAQKRGLCTVHREAD FFWSLCTADMSLLEQSHSSPSLYLQCLSQKLRQERKRPLLDLHIELNGYMYDWNSR VSAKEK YYVWLQHTLRKKLILS NEGGSGDYKDDDDK (SEQ ID NO: 5)

Amino Acid Sequence of Exemplary Engineered MHC (eMElC)

MATGSRTSLLLAFGLLCLPWLQEGSAELAGIGILTVGGGGSGGGGSGGGGSIQRTPKI

5 QVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYL LYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGS GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGP EYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLR GYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTC i o VEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQR DGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRW EPSSQPTIPIVGIIAGLVLFGAVITGAVVAAVMWRRKSSGGEGVKDRKGGSYTQAAS SDSAQGSDVSLTACKV (SEQ ID NO: 7) i KHYG-1 effector cell-induced apoptosis in HEK cells expressing the exemplary apoptosis inhibitors CRMA, P19, cFLIP, or a combination of cFLIP and P19 was investigated. As shown in FIG. 1, the tested apoptosis inhibitors showed protective effects against KHYG- 1 effector cells (NK cells). CRMA exhibited the best anti- apop totic effects among all tested apoptosis inhibitors or combinations thereof.

20 The anti-apoptotic effect of CRMA, PI9, cFLIP, or a combination of cFLIP and PI9 expressed in HEK cells in the absence of effector cells was also investigated and the results are provided in FIG. 2.

Further, apoptosis of HEK cells co-expressing the eMHC molecule and the exemplary apoptosis inhibitors CRMA, PI9, cFLIP, or a combination of cFLIP and PI9, either in the

25 presence or absence of KHYG-1 effector cells, was investigated. The results are shown in FIG. 3 and FIG. 4

Overall, the results show that expression of the apoptosis inhibitors in HEK cells provided varied levels of protection against cell death induced by KHYG-1 effector cells in the co-culture assay provided herein. In particular, CRMA was the most effective in

30 providing protection from degranulation of KHYG-1 in vitro as measured by Cytotox Red signal in an Incucyte live cell imager, either alone (FIG. 1) or co-expressed with the eMHC (FIG. 3). OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

EQUIVALENTS

While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing 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. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms. All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, 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.

As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, 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. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or 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.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

This invention is further illustrated by the following example which should not be construed as limiting. The contents of all references cited throughout this application.

Claims

What Is Claimed Is:

1. An engineered immune cell comprising:

(1) an exogenous apoptosis inhibitor; and

(2) a chimeric antigen receptor (CAR) comprising (i) an extracellular antigen binding domain; and (ii) a co- stimulatory domain, a cytoplasmic signaling domain, or a combination thereof; wherein the apoptosis inhibitor is a serine proteinase inhibitor (serpin), a granzyme B inhibitor, a FS-7 associated surface antigen (Fas) inhibitor, a peptidase C14A protein, or a combination thereof.

2. The engineered immune cell of claim 1, wherein the apoptosis inhibitor is a cytokine response modifier A (CRMA), a proteinase inhibitor 9 (PI9), a cellular FLICE inhibitory protein (cFLIP), or a combination thereof.

3. The engineered immune cell of claim 1 or 2, wherein the CAR 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.

4. The engineered immune cell of claim 3, wherein the MHC molecule is a class

I MHC molecule, which optionally is a human class I MHC molecule.

5. The engineered immune cell of claim 4, wherein the human class I MHC molecule is an HLA-A, HLA-B, HLA-C, HLA-G, or HLA-E molecule.

6. The engineered immune cell of claim 3, wherein the MHC molecule is a class

II MHC molecule, which optionally is a human class II MHC.

7. The engineered immune cell of claim 6, wherein the human class II MHC is HLA-DR2, HLA-DR3, HLA-DR4, HLA-DR9, HLA-DR15, HLA-DP, or HLA-DQ.

8. The engineered immune cell of any one of claims 1-7, wherein the CAR comprises at least one co-stimulatory domain and the cytoplasmic signaling domain, which optionally is from CD3^.

9. The engineered immune cell of any one of claims 1-8, wherein the immune cell is a natural killer (NK) cell, a macrophage cell, or a T cell, which optionally is a CD8+ or CD4+ T regulatory cell.

10. The engineered immune cell of claim 9, wherein the immune cell is the NK cell, optionally wherein the NK cell is an NK-92 cell or an KHYG-1 cell or wherein the NK cell is deficient in killer-cell immunoglobulin-like receptor (KIR).

11. The engineered immune cell of claim 10, wherein the CAR comprises a costimulatory domain of 2B4 (CD244).

12. The engineered immune cell of any one of claims 1-9, where the immune cell is the macrophage, and optionally wherein the CAR comprises the co-stimulatory domain of Megfl 0 or FcRy.

13. The engineered immune cell of any one of claims 1-9, where the immune cell is the CD8+ or CD4+ T regulatory cell, and optionally wherein the CAR comprises the co- stimulatory domain of CD28 or 4- IBB.

14. The engineered immune cell of any one of claims 3-13, wherein the antigenic peptide in the MHC-CAR is of a protein associated with an autoimmune disease, optionally wherein the protein associated with the autoimmune disease is set forth in Tables 3-5.

15. The engineered immune cell of any one of claims 1-14, wherein the apoptosis inhibitor is:

(a) CRMA comprising the amino acid sequence of SEQ ID NO: 2

(b) PI9 comprising the amino acid sequence of SEQ ID NO: 4; or

(c) cFLIP comprising the amino acid sequence of SEQ ID NO: 6.

16. A population of cells, comprising a plurality of the engineered immune cells set forth in any one of claims 1-15.

17. A pharmaceutical composition comprising the engineered immune cell of any one of claims 1-15 or the population of cells of claim 16, and a pharmaceutically acceptable carrier.

18. A method for suppressing disease cells in a subject, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of claim 17, wherein the engineered immune cell in the pharmaceutical composition comprises the CAR that targets the disease cells.

19. The method of claim 18, wherein the CAR is an MHC-CAR and the disease cells are autoreactive immune cells.

20. The method of claim 19, wherein the subject is a human patient having an autoimmune disease.

21. The method of any one of claims 18-20, wherein the engineered immune cells in the pharmaceutical composition are autologous to the subject.

22. The method of any one of claims 18-20, wherein the engineered immune cells in the pharmaceutical composition are allogenic to the subject.

23. A method for producing a population of the engineered immune cells set forth in any one of claims 1-15, the method comprising: 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.

24. The method of claim 23, wherein the one or more nucleic acids are one or more messenger RNA molecules; or wherein the one or more nucleic acids are one or more expression vectors, which optionally are viral vectors.