Suppression of immune cells by immune checkpoint engagers

Abstract

The present invention provides cells with enhanced immune checkpoint engagement function (immune checkpoint engager "ICE" cells). In some embodiments, the ICE cells have PD-1 engagement function (PD-1 engager cells). The ICE cells attenuate adaptive and innate immunity when transplanted into a subject, compared to parental cells with corresponding unmodified immune checkpoint engagement function. In some embodiments, the PD-1 engager cells are hypoimmune pluripotent (HIP) cells. In further embodiments, the HIP cells are blood type O (HIPO), Rh factor (Rh) negative (HIP-), or blood type O and Rh- (HIPO-). In other embodiments, the PD-1 engager cells are derived or differentiated from HIP cells, HIP- cells, or HIPO- cells. In other embodiments, the ICE cells express a ligand that binds to an inhibitory immune cell receptor selected from the group consisting of LILRB1 engager, LILRB3 engager, and TIM3 engager. In other embodiments, the ICE cells are PD-1 engager cells that further express a ligand that binds to another inhibitory immune cell receptor selected from the group consisting of CD47, truncated CD47, LILRB1 engager, LILRB3 engager, and TIM3 engager.

Description

[Technical field]

[0001] I. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 330,539, filed April 13, 2022, and U.S. Provisional Application No. 63 / 448,081, filed February 24, 2023, which are incorporated by reference in their entireties.

[0002] II. Technology Field The present invention provides cells with enhanced immune checkpoint engagement (ICE) function. In some embodiments, the cells have programmed cell death protein 1 (PD-1) engagement function (PD-1 engager cells), and the cells resist immune responses when transplanted into a subject, compared to parental cells with unmodified PD-1 engagement function. In some embodiments, the immune checkpoint engager cells are hypoimmune cells. In other embodiments, the cells are pluripotent cells. In other embodiments, the cells are embryonic stem cells (ES cells) or induced pluripotent stem cells (iPSCs). In other embodiments, the immune checkpoint engager cells are differentiated somatic cells. In other embodiments, the immune checkpoint engager cells are hypoimmune pluripotent cells (HIP cells). In further embodiments, the HIP cells are blood type O (HIPO), Rh factor (Rh) negative (HIP-), or blood type O and Rh- (HIPO-). In other embodiments, the immune checkpoint engager cells are derived or differentiated from HIP cells, HIP-cells, or HIPO-cells. In other embodiments, the immune checkpoint engager cells comprise an antibody Fc receptor for protection from antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In other embodiments, the ICE cells express a ligand that binds to an inhibitory immune cell receptor selected from the group consisting of LILRB1 engagers, LILRB3 engagers, and TIM3 engagers. In other embodiments, the ICE cells are PD-1 engager cells that further express a ligand that binds to another inhibitory immune cell receptor selected from the group consisting of CD47, truncated CD47, SIRPα engagers, LILRB1 engagers, LILRB3 engagers, and TIM3 engagers. [Background technology]

[0003] III. Background technology Natural killer cells (NK cells) are cytotoxic lymphocytes essential to the innate immune system. The role played by NK cells is similar to that of cytotoxic T cells in the adaptive immune response of vertebrates. NK cells show rapid responses against virus-infected cells and cancer cells. Normally, NK cells are activated by target cells by downregulation of major histocompatibility complex (MHC), which is one of the major inhibitory NK cell signals. NK cell activation results in the release of cytokines, which lead to hemolysis or apoptosis. NK cells are unique in that they recognize stressed cells by upregulating other stimulatory NK cell signals, and do not require prior exposure to specific cellular epitopes. This allows them to respond very quickly. They can also respond quickly to antibody-loaded cells, because binding of free antibody Fc is a strong stimulatory NK cell signal. NK cells do not require significant activation to kill cells that lack MHC class 1 "self" markers, except for exposure to some cytokines such as IL-2 or IL-15. This role is particularly important. This is because harmful cells with downregulated or missing MHC I markers cannot be detected and destroyed by other immune cells, such as T lymphocytes.

[0004] NK cells are large granular lymphocytes that differentiate from a common lymphoid progenitor that gives rise to B and T lymphocytes. NK cells differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus, and enter the circulation.

[0005] Programmed cell death protein 1, also known as PD-1, or CD279 (cluster of differentiation 279), is a protein present on the surface of T cells, B cells, and some NK cells that plays a role in modulating the immune system's response and promoting self-tolerance by downregulating the immune system and suppressing the inflammatory activity of T cells. This prevents autoimmune diseases, but can also prevent the immune system from killing cancer cells.

[0006] PD-1 often shows high and sustained expression levels during persistent antigen encounters, as seen in chronic infections and cancer settings. PD-1 is an immune checkpoint that protects against autoimmunity by two mechanisms: first, it promotes apoptosis (programmed cell death) of antigen-specific T cells in lymph nodes; second, it reduces apoptosis of regulatory T cells (anti-inflammatory, suppressive T cells).

[0007] The PD-1 / PD-L1 (PD-1 ligand)-based pathway has great value in cancer immunotherapy and represents an important immune checkpoint. Antibody inhibitors of the PD-1 / PD-L1 interaction have shown clinical efficacy in many tumors.

[0008] The human PD-1 protein is encoded by the PDCD1 gene. PD-1 is a cell surface receptor that belongs to the immunoglobulin superfamily and is expressed on T cells and pro-B cells. PD-1 binds to two ligands, PD-L1 and PD-L2.

[0009] Programmed death-ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a protein encoded by the CD274 gene in humans. PD-L1 is a 40 kDa type 1 transmembrane protein that is believed to play a key role in suppressing the adaptive function of the immune system during certain disease states, such as pregnancy, tissue transplantation, autoimmune diseases, and hepatitis. Normally, the adaptive immune system responds to antigens associated with immune system activation by exogenous or endogenous danger signals. This results in the promotion of clonal expansion of antigen-specific CD8+ T cells and / or CD4+ helper cells. Binding of PD-L1 to the inhibitory checkpoint molecule PD-1 transmits an inhibitory signal based on its interaction with phosphorylation enzymes (SHP-1 or SHP-2) via an immunoreceptor tyrosine-based switch motif (ITSM). This results in reduced proliferation of antigen-specific T cells in lymph nodes, as well as reduced apoptosis of regulatory T cells (anti-inflammatory, suppressive T cells), which is further mediated by downregulation of the gene Bcl-2.

[0010] Programmed cell death 1 ligand 2 (PD-L2, also known as B7-DC) is a protein encoded by the PDCD1LG2 gene in humans. PDCD1LG2 is also known as CD273 (cluster of differentiation 273). PDCD1LG2 is a ligand for immune checkpoint receptors and is involved in the negative regulation of adaptive immune responses. PD-L2 is one of two known ligands for programmed cell death protein 1 (PD-1).

[0011] SIRPα is a member of the signal-regulatory protein (SIRP) family, which also belongs to the immunoglobulin superfamily. SIRP family members are receptor-type transmembrane glycoproteins known to be involved in the negative regulation of receptor tyrosine kinase-coupled signal transduction processes. SIRPα can be phosphorylated by tyrosine kinases. The phosphorylated tyrosine residues recruit and serve as substrates for SH2 domain-containing tyrosine phosphatases (PTPs). SIRPα is involved in signal transduction through various growth factor receptors.

[0012] CD47 is a ligand for SIRPα. CD47 is a "marker of self" protein that can be widely overexpressed across tumor types. It has emerged as a novel and powerful macrophage immune checkpoint for cancer immunotherapy. CD47 on tumor cells sends a "don't eat me" signal that inhibits macrophage phagocytosis. This represents an opportunity and challenge for CD47 inhibitors, both as monotherapy and combination therapy for hematological cancers and solid tumors. Several of these agents are currently in clinical trials.

[0013] Cytoplasmic signaling of CD47 may occur through its intracellular domain (ICD), but few proteins have been identified so far that directly interact with the cytoplasmic tail of CD47 (Lamy L., J Biol Chem. 278:23915-21 (2003); Wu AL, Mol Cell.4:619-25 (1999)). Ubiquilin-1 is one such binding partner, which binds to Gβγ, thereby tethering heterotrimeric G proteins to CD47 (N'Diaye EN,. J Cell Biol. 163:1157-65 (2003)). In this context, ubiquilin-1 inhibits migration signaled by the Gi-coupled receptor CXCR4 (Sick E., Glia. 59:308-19 (2011)). The aforementioned documents are incorporated herein by reference in their entirety.

[0014] Upon stimulation, primary human NK cells have been shown to express SIRPα and bind to CD47, thereby reducing the killing effect of CD47-expressing cells (see PCT / US20 / 39220, which is incorporated herein by reference in its entirety). [Prior art documents] [Non-patent literature]

[0015] [Non-Patent Document 1] Lamy L., J Biol Chem. 278:23915-21 (2003) [Non-Patent Document 2] Wu AL, Mol Cell. 4:619-25 (1999) [Non-Patent Document 3] N'Diaye EN,.J Cell Biol. 163:1157-65 (2003) [Non-Patent Document 4] Sick E., Glia.59:308-19 (2011) Summary of the Invention [Problem to be solved by the invention]

[0016] Autologous induced pluripotent stem cells (iPSCs) are theoretically an unlimited source of cells for patient-specific cell-based organ repair strategies. However, their generation poses technical and manufacturing challenges and is a lengthy process, conceptually preventing treatment in the acute phase. Therapeutics with allogeneic iPS cells or with embryonic stem cells are easier from a manufacturing standpoint, allowing the generation of well-screened and standardized high-quality cell products. The potential applications of stem cell therapy are broad, as pluripotent stem cells can differentiate into any cell type of the three germ cell layers. Differentiation can be performed ex vivo or in vivo by transplanting progenitor cells that continue to differentiate and mature in the organ environment at the transplant site. Ex vivo differentiation allows researchers and clinicians to closely monitor the procedure and ensure the generation of the appropriate cell population before transplantation. However, such cells are of allogeneic origin and may be subject to rejection. [Means for solving the problem]

[0017] IV. Overview of the Invention The present invention provides cells with enhanced immune checkpoint engagement function (immune checkpoint engager "ICE" cells). In some embodiments, the ICE cells have PD-1 engagement function (PD-1 engager cells). The ICE cells attenuate adaptive and innate immunity when transplanted into a subject, compared to parental cells with corresponding unmodified immune checkpoint engagement function. In some embodiments, the PD-1 engager cells are hypoimmune pluripotent (HIP) cells. In further embodiments, the HIP cells are blood type O (HIPO), Rh factor (Rh) negative (HIP-), or blood type O and Rh- (HIPO-). In other embodiments, the PD-1 engager cells are derived or differentiated from HIP cells, HIP- cells, or HIPO- cells.

[0018] Thus, the present invention provides immune checkpoint engager (ICE) cells, wherein an engager molecule is expressed on the cell surface, said engager molecule engages with an immune checkpoint molecule on an immune cell, said engager molecule being expressed at least at a level that protects the ICE cell from being killed by the immune cell. In some embodiments, the engager molecule is a protein that does not engage with SIRPα on an immune cell. In other embodiments, the engager molecule engages with PD-1.

[0019] In another embodiment of the invention, the protein comprises an immunoglobulin domain that binds to PD-1. In another embodiment, the immunoglobulin domain is an antibody VH domain. In another embodiment, the antibody VH domain has at least 90% sequence identity to amino acids 146-264 of SEQ ID NO:1. In a preferred embodiment, the antibody VH domain comprises the sequence of amino acids 146-264 of SEQ ID NO:1. In another embodiment, the immunoglobulin domain is an antibody VL domain. In another embodiment, the antibody VL domain has at least 90% sequence identity to amino acids 21-130 of SEQ ID NO:1. In a preferred embodiment, the antibody VH domain comprises the sequence of amino acids 21-130 of SEQ ID NO:1.

[0020] In some embodiments of the invention, the ICE cells express a protein that is a fusion protein. In other embodiments, the fusion protein has at least 90% sequence identity with the IL-2 signal peptide of SEQ ID NO:2. In a preferred embodiment, the fusion protein comprises the IL-2 signal peptide of SEQ ID NO:2. In other embodiments, the fusion protein has at least 90% sequence identity with the CD8a hinge peptide of SEQ ID NO:3. In a preferred embodiment, the fusion protein comprises the CD8a hinge peptide of SEQ ID NO:3. In some embodiments, the fusion protein has at least 90% sequence identity with the PDGFR transmembrane domain (TMD) of SEQ ID NO:4. In a preferred embodiment, the fusion protein comprises the PDGFR TMD of SEQ ID NO:4.

[0021] In some embodiments of the invention, the engager molecule is a protein having at least 90% sequence identity to SEQ ID NO: 1. In preferred embodiments, the engager molecule has sequence identity to SEQ ID NO: 1.

[0022] In some embodiments of the invention, the engager molecule comprises an antibody Fab or single chain variable fragment (scFV) that binds to PD-1. In other embodiments, the Fab or scFV binds to PD-1 with an affinity as measured by the dissociation constant (Kd), where the Kd is about 10 -7 ~10 -13 In other embodiments, the engager molecule comprises one or more antibody complementarity determining regions (CDRs) that bind to PD-1. In other embodiments, one or more CDRs have at least 90% sequence identity to any one of SEQ ID NOS: 18-23. In other embodiments, one or more CDRs comprise the sequence of any one of SEQ ID NOS: 18-23.

[0023] In some embodiments of the invention, the engager molecule is a fusion protein comprising a heterologous transmembrane domain (TMD). In other embodiments, the TMD comprises an alpha helix, multiple alpha helices, or a rolled up beta sheet. In other embodiments, the heterologous TMD comprises CD85f, CD349, CD284, CD261, CD172b, CD277, CD186, CD156c, CD304, CD254, CD263, CD267, CD337, CD170, CD283, CD133, CD327, CD205, CD232, CD282, CD16b, CD85i, CD85a, CD85c, CD275, CD108, CD358, CD335, CD218b, CD355, CD336, CD160, CD25, CD4, CD8a, CD4, CD5a, CD5c ... D235a, CD233, CD230, CD90, CD74, CD3d, CD340, CD236, CD61, CD18, CD54, CD29, CD1a, CD5, CD220, CD2, CD66e, CD51, CD141, CD115, CD42b , CD221, CD271, CD55, CD243, CD98, CD10, CD41, CD14, CD45, CD228, CD16a, CD49e, CD126, CD63, CD48, CD7, CD140b, CD3g, CD117, CD28, CD8 b, CD37, CD11b, CD107a, CD331, CD222, CD20, CD79a, CD32, CD143, CD324, CD42c, CD107b, CD56, CD102, CD49d, CD66a, CD142, CD59, CD62L , CD121a, CD122, CD13, CD155, CD119, CD19, CD116, CD46, CD1e, CD1d, CD227, CD44, CD62P, CD104, CD43, CD140a, CD31, CD152, CD326, CD6 2E, CD36, CD127, CD49b, CD105, CD35, CD223, CD138, CD325, CD58, CD106, CD53, CD120a, CD224, CD21, CD33, CD22, CD120b, CD11a, CD11c, CD363, CD73, CD88, CD204, CD332, CD9, CD203a, CD334, CD333, CD206, CD49f, CD238, CD252, CD89, CD124, CD181, CD182, CD24, CD95, CD40,CD49c、CD159a、CD159c、CD314、CD27、CD123、CD26、CD82、CD121b、CD34、CD38、CD30、CD1b、CD1c、CD154、CD6、CD52、CD132、CD32、CD66b、CD171、CD191、CD197、CD185、CD131、CD50、CD70、CD153、CD144、CD80、CD362、CD68、CD361、CD147、CD309、CD135、CD292、CD103、CD130、CD42d、CD66d、CD66c、CD96、CD110、CD79b、CD200、CD192、CD231、CD86、CD212、CD118、CD146、CD134、CD158a、CD158b1、CD158b2、CD158e、CD158k、CD158j、CD158i、CD178、CD295、CD151、CD97、CD183、CD39、CD239、CD193、CD194、CD195、CD196、CDw198、CDw199、CD296、CD298、CD49a、CD322、CD85g、CD184、CD172a、CD156a、CD339、CD156b、CD213a1、CD129、CD83、CD125、CD241、CD269、CD202b、CD87、CD164、CD136、CD137、CD249、CD69、CD91、CDw210b、CD167a、CD300c、CD157、CD317、CD148、CD161、CD215、CD150、CD11d、CD218a、CD210、CD166、CD162、CD213a2、CD242、CD158g、CD158h、CD279、CD111、CD281、CD226、CD234、CD167b、CD300e、CD276、CD305、CD300g、CD300d、CD109、CD272、CD163、CD302、CD158f1、CD85h、CD85d、CD177、CD158z、CD158f2、CD85j、CD300f、CD92、CD351、CD112、CD100、CD270、CD101、CD297、CD316、CD352、CD217、CD307b、CD307a、CD307c、CD307d、CD307e、CD114、CD180、CD158d、CD273、CD290、CD244、CD169、CD299、CD318、CD360、CD229, CD248, CD354, CD320, CD93, CD319, CD113, CD163b, CD289, CD288, CD329, CD274, CD353, CD172g, CD315, CD280, CD264, CD300a, CD312, CD84, CD344, CD350, CD246, CD201, CD338, CD208, CD257, CD328, CD286, CD357, CD294, CD321, CD265, CD278, ITGA7, ITGA8, ITGA9, ITGA10, ITGA11, CD51, CD41, CD29, CD18, CD61, and CD104. In other embodiments, the TMD of the fusion protein is derived from CD47 (SEQ ID NO:5), CD64 (SEQ ID NO:6), or PDGFR (SEQ ID NO:4).

[0024] In other embodiments, the TMD comprises a sequence having at least 90% sequence identity to SEQ ID NO:5 or SEQ ID NO:6. In preferred embodiments, the TMD comprises the sequence of SEQ ID NO:5 or SEQ ID NO:6. In other embodiments, the TMD is derived from a seven transmembrane protein (7TM) or an immunoglobulin cell surface protein.

[0025] In some embodiments of the invention, the engager molecule has no intracellular domain (ICD), hi other embodiments, the engager molecule has an intracellular domain derived from CD16, CD32, CD64, CD8, CD3, CD28, or CD137.

[0026] In some embodiments of the invention, engager molecules have one or more linker or hinge regions connecting the ECD, TMD, or ICD sequences.

[0027] In other embodiments, the protein is an antibody, a receptor, a ligand, or an adhesion protein.

[0028] In some embodiments of the present invention, the ICE cells described herein further have reduced or absent expression of HLA-I or HLA-II. In other embodiments, the cells are ABO blood group type O. In other embodiments, the cells are Rh factor negative (Rh-). In other embodiments, the cells have reduced or absent expression of an Rh protein antigen selected from the group consisting of Rh C antigen, Rh E antigen, Kell K antigen (KEL), Duffy (FY) Fya antigen, Duffy Fy3 antigen, Kidd (JK) Jkb antigen, MNS antigen U, and MNS antigen S.

[0029] In some aspects, the ICE cells of the present invention are hypoimmunogenic (HI) cells having reduced endogenous major histocompatibility complex class I (HLA-I) function compared to an unmodified parent cell and reduced endogenous major histocompatibility complex class II (HLA-II) function compared to an unmodified parent cell.

[0030] In other embodiments, the engager cells are selected from the group consisting of HLA-I human leukocyte antigen, HLA-II human leukocyte antigen, CD64, CD47, CD38, CCR5, CXCR4, NLRC5, CIITA, B2M, HLA-A, HLA-B, HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47, CI-inhibitor, IL-35, RFX-5, RFXAP, RFXANK, NFY-A, NFY-B, NFY-C, IRF-1, OX40, GITR, 4-1BB, CD28, B7-1, B7-2, ICOS, CD27, HVEM, SLAM, CD226, PD1, CTL4, LAG3, TIGIT, TIM3, CD160, BTLA, CD244, CD30, TLT, VISTA , B7-H3, PD-L2, LFA-1, CD2, CD58, ICAM-3, TCRA, TCRB, FOXP3, HELIOS, ST2, PCSK9, APOC3, CD200, FASLG, CLC21, MFGE8, SERPIN Expression of one or more of B9, TGFβ, CD73, CD39, LAG3, IL1R2, ACKR2, TNFRSF22, TNFRSF23, TNFRS10, DAD1, PVR, or IFNγR1 d39 is modulated compared to the parental cells, and the engager cells are ABO blood group O or Rh factor negative (Rh-).

[0031] In other embodiments, the ICE cells of the present invention have enhanced expression of antibody Fc receptors on the cell surface, which aid in evading antibody-dependent cellular cytotoxicity (ADCC) or complement-mediated cytotoxicity (CDC). In other embodiments, the Fc receptor is CD16, CD32, CD64, or cleaved CD64.

[0032] In some embodiments, the ICE cells of the present invention are pluripotent. In other embodiments, the cells are hypoimmune pluripotent (HIP) cells. In other embodiments, the cells are hypoimmune pluripotent cells with ABO blood group type O (HIPO). In other embodiments, the cells are hypoimmune pluripotent cells that are Rh factor negative (HIP-). In other embodiments, the cells are hypoimmune pluripotent cells that are ABO blood group type O and Rh factor negative (HIPO-). In other embodiments, the cells are pluripotent (PSC) cells, induced PSC (iPSC) cells, or embryonic stem cells (ESC).

[0033] In some embodiments of the present invention, the ICE cells described herein are of a specific tissue type. In other embodiments, the cells are chimeric antigen receptor (CAR) cells, T cells, NK cells, endothelial cells, dopaminergic neurons, cardiac cells, pancreatic islet beta cells, macrophages, thyroid epithelial cells, parathyroid cells, or retinal pigment epithelial cells. In other embodiments, the ICE cells of the present invention are CAR-T cells, CAR-NK cells, TCR T cells, or TCR NK cells. In other embodiments, the engager cells are differentiated from pluripotent cells.

[0034] The invention provides a pharmaceutical composition comprising an ICE cell as described herein and a pharma- ceutically acceptable carrier.

[0035] The invention provides a pharmaceutical preparation comprising an ICE cell as described herein and a pharma- ceutically acceptable carrier.

[0036] The present invention provides a method of treating a disease in a subject comprising transplanting ICE cells as described herein into the subject. In other embodiments, the disease is type 1 diabetes, a cardiac disease, a neurological disease, an endocrine disease, cancer, blindness, or a vascular disease.

[0037] The present invention provides the use of ICE cells as described herein for the preparation of a pharmaceutical composition for treating a disease in a subject. In other aspects, the disease is type 1 diabetes, a cardiovascular disease, a neurological disease, an endocrine disease, cancer, blindness, or a vascular disease.

[0038] In some embodiments of the invention, the ICE cells described herein further comprise a signal regulatory protein alpha (SIRPα) engager molecule on the cell surface that engages with a SIRPα protein on an immune cell, where engagement of SIRPα protects the engager cell from being killed by the immune cell, and where the surface molecule of the SIRPα engager cell lacks a functional intracellular domain. In other embodiments, the engager molecule is a protein. In other embodiments, the protein is a fusion protein. In other embodiments, the fusion protein comprises a CD47 extracellular domain (ECD). In other embodiments, the engager molecule comprises an immunoglobulin superfamily domain. In other embodiments, the SIRPα engager molecule comprises one or more antibody complementarity determining regions (CDRs) that bind to SIRPα. In other embodiments, the CDRs comprise a sequence having at least 90% sequence identity to any one of SEQ ID NOS: 18-23. In other embodiments, the CDRs comprise any one of SEQ ID NOS: 18-23.

[0039] The present invention provides an ICE cell as disclosed herein, wherein the engager molecule engages one or more immune cell receptors selected from the group consisting of PD-1, TIM3, LILRB3, and LILRB1.

[0040] The invention provides an ICE cell as disclosed herein further comprising a safety switch. In some aspects, the safety switch is a suicide gene. In other aspects, the suicide gene is a herpes simplex virus thymidine kinase gene (HSV-tk) with a ganciclovir trigger. In other aspects, the suicide gene is an Escherichia coli cytosine deaminase gene (EC-CD) with a 5-fluorocytosine (5-FC) trigger. In other embodiments, the safety switch is a protein tag expressed on the cell surface that is recognized by a clinical antibody and targets the cell for destruction. [Brief description of the drawings]

[0041] [Figure 1]NK cell killing assay by bioluminescence imaging (BLI) of iPSC-derived endothelial cells (iECs). Natural killer cells (NK cells) were stimulated with IL-2 for 72 h. The NK cells were added to FLuc+ B2M- / - CIITA- / - (double knockout for HLA class I and HLA class II, DKO) and B2M- / - CIITA- / - iECs additionally expressing PD-1 engager (DKO PD-1 engager iECs). Reduction of BLI signal was assessed after 4 h (mean ± SD, three independent experiments per group, two-way Student's t test). PD-1 engager molecule provided protection of iECs from killing by NK cells. [Diagram 2] iEC BLI CD8 T cell killing assay. Peripheral blood mononuclear cells (PBMCs) were primed in vitro with wt iECs and sorted CD3+CD8+ T cells were used as effector cells in this assay. CD3+CD8+ T cells were then added to FLuc+ wt and wt PD-1 engager iECs. Reduction in BLI signal was assessed after 4 hours (mean ± SD, three independent experiments per group, two-way Student's t test). PD-1 engager molecules protected iECs from killing by CD3+CD8+ T cells. [Diagram 3] In vitro impedance killing assay. Target cells are HLA-A2 positive human pancreatic beta cells. In the top panels of Figure 3A and Figure 3B, the beta cells are unmodified. In the bottom panels of Figure 3A and Figure 3B, the beta cells express PD-1 engager. Peripheral blood mononuclear cells (PBMCs) were primed in vitro with HLA-A2 positive wt iECs and sorted. CD3+CD8+ T cells were used as effector cells in the assay. When primed CD3+CD8+ T cells were added to the assay at time 0, unmodified beta cells were killed, whereas human pancreatic beta cells expressing PD-1 engager survived (mean ± SD, 3 independent experiments per time point). [Figure 4]Binding of recombinant PD-1 to DKO PD-1 engager iECs. Flow cytometry shows specific binding of PD-1 to secondary antibody. Secondary antibody alone was used as a negative control. [Diagram 5] Human induced pluripotent stem cell (iPSC)-derived endothelial cells (iECs) were generated that express firefly luciferase and further contain a double knockout (DKO) of B2M and CIITA. An exemplary TIM3 engager protein linked to a red fluorescent protein (RFP) tag was introduced into these cells using lentiviral particles. They were analyzed by flow cytometry. The histograms show the RFP signal of untransduced iECs DKO (left peak) and transduced iECs DKO+TIM3-E (right peak), confirming the expression of the TIM3 engager. [Figure 6] Flow cytometry analysis of TIM3 surface expression on primary human peripheral blood NK cells and macrophages. 96.6% of NK cells were TIM3 positive (Figure 6A), and 67.2% of macrophages were TIM3 positive (Figure 6B). [Figure 7] Two-hour BLI killing assay with NK cells. Human induced pluripotent stem cell (iPSC)-derived endothelial cells (iECs) were generated that express firefly luciferase and further contain double knockout (DKO) of B2M and CIITA. These cells were plated with iEC DKO+TIM3-E and allowed to adhere for 16 hours. Human peripheral blood NK cells were then added to the wells and cultured for 2 hours. The percentage of BLI signal after culture is shown. The BLI signal of iEC DKO was completely lost (Figure 7A). The BLI signal of iEC DKO+TIM3-E was stable for 2 hours (Figure 7B). [Figure 8]2-hour BLI killing assay with macrophages. Human iEC DKO and iEC DKO+TIM3-E expressing firefly luciferase were plated and allowed to attach for 16 hours. Human peripheral blood macrophages were then added to the wells and cultured for 2 hours. Percentage of BLI signal after culture is shown. The BLI signal of iEC DKO was completely lost (Figure 8A). The BLI signal of iEC DKO+TIM3-E remained stable for 2 hours (Figure 8B). [Figure 9] 24-hour BLI killing assay with NK cells. Human iEC DKO and iEC DKO+TIM3-E expressing firefly luciferase were plated and allowed to attach for 16 hours. Human peripheral blood NK cells were then added to the wells and cultured for 24 hours. Percentage of BLI signal after 24 hours of NK culture is shown. The BLI signal of iEC DKO was completely lost (Figure 9A). The BLI signal of iEC DKO+TIM3-E was stable for 24 hours (Figure 9B). [Figure 10] 24-hour BLI killing assay with macrophages. Human iEC DKO and iEC DKO+TIM3-E expressing firefly luciferase were plated and allowed to attach for 16 hours. Human peripheral blood macrophages were then added to the wells and cultured for 24 hours. The percentage of BLI signal after 24 hours of macrophage culture is shown. The BLI signal of iEC DKO was completely lost (Figure 10A). The BLI signal of iEC DKO+TIM3-E was stable for 24 hours and even increased slightly (Figure 10B). [Figure 11] Human induced pluripotent stem cell (iPSC)-derived endothelial cells (iECs) were generated that express firefly luciferase and further contain a double knockout (DKO) of B2M and CIITA. An exemplary LILRB3 engager protein linked to a red fluorescent protein (RFP) tag was introduced into these cells using lentiviral particles. They were analyzed by flow cytometry. The histograms show the RFP signal of untransduced iECs DKO (left peak) and transduced iECs DKO+LILRB3-E (right peak), confirming the expression of the LILRB3 engager. [Figure 12] Flow cytometry analysis of LILRB3 surface expression on primary human peripheral blood NK cells and macrophages. 17.8% of NK cells were LILRB3 positive (Figure 12A), and 95.3% of macrophages were LILRB3 positive (Figure 12B). [Figure 13] Two-hour BLI killing assay with NK cells. Human induced pluripotent stem cell (iPSC)-derived endothelial cells (iECs) were generated that expressed firefly luciferase and contained a double knockout (DKO) of B2M and CIITA. These cells were plated, along with iECs DKO+LILRB3-E, which expressed the LILRB3 engager. Cells were allowed to adhere for 16 hours. Human peripheral blood NK cells were then added to the wells and cultured for 2 hours. Percentage of BLI signal after culture is shown. The BLI signal of iECs DKO was completely lost (Figure 13A). The BLI signal of iECs DKO+LILRB3-E showed less significant decline at 2 hours (Figure 13B). [Figure 14] BLI killing assay with macrophages for 2 hours. Human iEC DKO and iEC DKO+LILRB3-E expressing firefly luciferase were plated and allowed to attach for 16 hours. Human peripheral blood macrophages were then added to the wells and cultured for 2 hours. Percentage of BLI signal after culture is shown. The BLI signal of iEC DKO was completely lost (Figure 14A). The BLI signal of iEC DKO+LILRB3-E showed less significant decline at 2 hours (Figure 14B). [Figure 15] 24-hour BLI killing assay with NK cells. Human iEC DKO and iEC DKO+LILRB3-E expressing firefly luciferase were plated and allowed to attach for 16 hours. Human peripheral blood NK cells were then added to the wells and cultured for 24 hours. Percentage of BLI signal after 24 hours of NK culture is shown. The BLI signal of iEC DKO was completely abolished (Figure 15A). The BLI signal of IEC DKO+LILRB3-E showed less significant decline at 24 hours (Figure 15B). [Figure 16]24-hour BLI killing assay with macrophages. Human iEC DKO and iEC DKO+LILRB3-E expressing firefly luciferase were plated and allowed to attach for 16 hours. Human peripheral blood macrophages were then added to the wells and cultured for 24 hours. Percentage of BLI signal after 24 hours of macrophage culture is shown. The BLI signal of iEC DKO was completely abolished (FIG. 16A). The BLI signal of iEC DKO+LILRB3-E showed less significant decline and even a slight increase at 24 hours (FIG. 16B). [Figure 17] Human induced pluripotent stem cell (iPSC)-derived endothelial cells (iECs) were generated that express firefly luciferase and further contain a double knockout (DKO) of B2M and CIITA. An exemplary LILRB1 engager protein linked to a red fluorescent protein (RFP) tag was introduced into these cells using lentiviral particles. They were analyzed by flow cytometry. The histograms show the RFP signal of untransduced iECs DKO (left peak) and transduced iECs DKO+LILRB1-E (right peak), confirming the expression of the LILRB1 engager. [Figure 18] Flow cytometry analysis of LILRB1 surface expression on primary human peripheral blood NK cells and macrophages. 75.8% of NK cells were LILRB1 positive (FIG. 18A), and 99.9% of macrophages were LILRB1 positive (FIG. 18B). [Figure 19] Two-hour BLI killing assay with NK cells. Human induced pluripotent stem cell (iPSC)-derived endothelial cells (iECs) were generated that expressed firefly luciferase and contained a double knockout (DKO) of B2M and CIITA. These cells were plated, along with iECs DKO+LILRB1-E, which expressed the LILRB1 engager. Cells were allowed to adhere for 16 hours. Human peripheral blood NK cells were then added to the wells and cultured for 2 hours. The percentage of BLI signal after culture is shown. The BLI signal of iECs DKO was completely lost (Figure 19A). The BLI signal of iECs DKO+LILRB1-E was stable for 2 hours. [Figure 20] BLI killing assay with macrophages for 2 hours. Human iEC DKO and iEC DKO+LILRB1-E expressing firefly luciferase were plated and allowed to attach for 16 hours. Human peripheral blood macrophages were then added to the wells and cultured for 2 hours. Percentage of BLI signal after culture is shown. The BLI signal of iEC DKO was completely lost (Figure 20A). The BLI signal of iEC DKO+LILRB1-E was stable for 2 hours (Figure 20B). [Figure 21] 24-hour BLI killing assay by NK cells. Human iEC DKO and iEC DKO+LILRB1-E expressing firefly luciferase were plated and allowed to attach for 16 hours. Human peripheral blood NK cells were then added to the wells and cultured for 24 hours. The percentage of BLI signal after 24 hours of NK culture is shown. The BLI signal of iEC DKO was completely lost (Figure 21A). The BLI signal of iEC DKO+LILRB1-E was stable for 24 hours (Figure 21B). [Figure 22] 24-hour BLI killing assay with macrophages. Human iEC DKO and iEC DKO+LILRB1-E expressing firefly luciferase were plated and allowed to attach for 16 hours. Human peripheral blood macrophages were then added to the wells and cultured for 24 hours. The percentage of BLI signal after 24 hours of macrophage culture is shown. The BLI signal of iEC DKO was completely abolished (Figure 22A). The BLI signal of iEC DKO+LILRB1-E was stable for 24 hours and even increased slightly (Figure 22B). [Diagram 23] Figure 23 shows that transgene expression of PD-1 engager was maintained during differentiation. iPSC DKO were transduced with lentiviral particles to express PD-1 engager (iPSC DKO+PD-1-E). Flow cytometry shows robust expression (Figure 23A). Transgene expression remained stable when iPSC DKO+PD-1-E were differentiated into iEC DKO + PD-1-E (Figure 23B). [Figure 24]We show that TIM3 engager transgene expression is maintained during differentiation. iPSC DKO were transduced with lentiviral particles to express TIM3 engager (iPSC DKO+TIM3-E). Flow cytometry shows robust expression (Figure 24A). When iPSC DKO + TIM3-E were differentiated into iEC DKO + TIM3-E iEC, transgene expression remained stable (Figure 24B). [Diagram 25] We show that expression of the LILRB3 engager transgene is maintained during differentiation. iPSC DKO was transduced with lentiviral particles to express the LILRB3 engager (iPSC DKO+LILRB3-E). Flow cytometry shows that robust expression was obtained (Figure 25A). When iPSC DKO + LILRB3-E was differentiated into iEC DKO + LILRB3-E, transgene expression remained stable (Figure 25B). [Figure 26] We show that the expression of the LILRB1 engager transgene is maintained during differentiation. iPSC DKO was transduced with lentiviral particles to express the LILRB1 engager (iPSC DKO+LILRB1-E). Flow cytometry shows that robust expression was obtained (Figure 26A). When iPSC DKO + LILRB1-E was differentiated into iEC DKO + LILRB1-E, the expression of the transgene remained stable (Figure 26B). [Figure 27]The threshold of TIM3-E expression required to protect cells from killing by NK cells is shown. To assess whether there is a functional threshold of TIM3-E expression to exert a protective effect against killing by NK cells, iEC DKO were transduced with lentiviral particles carrying the TIM3-E transgene. After 3 days, the pool of transduced iECs was sorted into three populations based on the expression of TIM3-E. The three populations are shown as iEC DKO + TIM3-E high, medium, and low (Figure 27A). Each of these populations was cultured with NK cells for 2 hours, followed by a BLI killing assay (Figure 27B). All target cells were luciferase positive. The percentage of BLI signal after 2 hours of NK incubation is shown. The BLI signal of iEC DKO was completely abolished. The BLI signal of iEC DKO + TIM3-E high and medium remained stable, while the BLI signal of iEC DKO + TIM3-E low also dropped to background. These results indicate that the functional threshold of TIM3-E lies between low and intermediate expression levels. [Figure 28] The threshold of LILRB1-E expression required to protect cells from killing by NK cells is shown. To assess whether there is a functional threshold of LILRB1-E expression to exert a protective effect against killing by NK cells, iEC DKO were transduced with lentiviral particles carrying the LILRB1-E transgene. After 3 days, the pool of transduced iECs was sorted into three populations based on LILRB1-E expression. The three populations are shown as iEC DKO+LILRB1-E high, medium, and low (Figure 28A). Each of these populations was cultured with NK cells for 2 hours, followed by a BLI killing assay (Figure 28B). All target cells were luciferase positive. The percentage of BLI signal after 2 hours of NK incubation is shown. The BLI signal of iEC DKO was completely abolished. The BLI signals of iEC DKO+LILRB1-E high and medium remained stable, while the BLI signal of iEC DKO+LILRB1-E low also dropped to background. These results indicate that the functional threshold of LILRB1-E lies between low and moderate expression levels. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] VI. MODE FOR CARRYING OUT THEINVENTION The present invention provides immune checkpoint-engaged (ICE) cells that are immune-reducing when transplanted into a subject, as compared to parental cells. In some embodiments, the ICE cells have enhanced programmed cell death protein 1 (PD-1)-engaging function (PD-1 engager cells) that are immune-reducing when transplanted into a subject, as compared to parental cells with unmodified PD-1-engaging function. In some embodiments, the PD-1 engager cells are hypoimmune cells. In other embodiments, the cells are pluripotent cells. In other embodiments, the cells are embryonic stem cells (ES cells) or induced pluripotent stem cells (iPSCs). In other embodiments, the PD-1 engager cells are differentiated somatic cells. In other embodiments, the PD-1 engager cells are hypoimmune pluripotent (HIP) cells. In further embodiments, the HIP cells are blood type O (HIPO), Rh factor (Rh) negative (HIP-), or blood type O and Rh- (HIPO-). In other embodiments, the PD-1 engager cells are derived or differentiated from HIP cells, HIP-cells, or HIPO-cells. In other embodiments, the PD-1 engager cells comprise an antibody Fc receptor for protection from antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In other embodiments, the ICE cells express a ligand that binds to an inhibitory immune cell receptor selected from the group consisting of LILRB1 engager, LILRB3 engager, and TIM3 engager. In other embodiments, the ICE cells are PD-1 engager cells that further express a ligand that binds to another inhibitory immune cell receptor selected from the group consisting of CD47, truncated CD47, LILRB1 engager, LILRB3 engager, SIRPα engager, and TIM3 engager.

[0043] It was unexpected that agonists of PD-1, TIM3, LILRB3, and LILRB1 all suppressed immune effector cells and protected therapeutic cells from cell death. Thus, the present invention recognizes that immune checkpoint engagers, not only as individual members but as a class, are valid targets for agonists that inhibit immune effector cells. Furthermore, the present invention recognizes that this class of agonists must be expressed at a threshold level to protect ICE cells from killing by immune cells.

[0044] The present invention provides compositions and uses of PD-1 agonists that inhibit immune effector cells to generate cell therapies in regenerative medicine and immuno-oncology to reduce host-versus-graft rejection. In some embodiments, a sequence fragment of a PD-1 agonist antibody (see U.S. Pat. No. 10,493,148, which is incorporated herein by reference in its entirety) was taken and introduced into a newly designed fusion protein (SEQ ID NO: 1). The fusion protein of this embodiment comprises a (IL-2 or CD8) signal peptide-VL-(GGGGS)3 linker-VH-CD8a hinge-PDGFR TMD portion.

[0045] In other embodiments, the PD-1 engager cells are derived or differentiated from the aforementioned cells. By way of example, the differentiated PD-1 engager cells may be endothelial cells, cardiomyocytes, hepatocytes, dopaminergic neurons, pancreatic islet cells, retinal pigment epithelial cells, and other cell types used in transplantation and medical treatment. This may include chimeric antigen receptor (CAR) cells and T cell receptor modified cells, such as CAR-T cells, TCR T cells, NK cells, and CAR-NK cells.

[0046] As used herein, the term "subject" or "patient" refers to any animal, such as a domestic animal, a zoo animal, or a human. A "subject" or "patient" is a mammal, such as a dog, a cat, a bird, a livestock animal, or a human. Specific examples of "subjects" and "patients" include, but are not limited to, individuals (particularly humans) who have a disease or disorder associated with the liver, heart, lungs, kidneys, pancreas, brain, nervous tissue, blood, bone, bone marrow, and the like.

[0047] Mammalian cells may be from humans or non-human mammals, including, but not limited to, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, and non-human primates (e.g., chimpanzees, macaques, and apes).

[0048] As used herein, "low immunogenic" cells, "low immune" cells, or "HI" cells refer to cells that, when transferred into an allogeneic host, reduce immunological rejection. In a preferred embodiment, HI cells do not elicit an immune response. Thus, "low immunogenic" refers to a significantly reduced or absent immune response compared to the immune response of the parental (i.e., "wt") cells prior to immune manipulation.

[0049] By "hypoimmunogenic O-cells," "hypoimmunogenic ORh-cells," or "HIO-" cells is meant herein HI cells that are ABO blood group O and Rh-factor. HIO-cells are generated from O-cells that have been enzymatically modified to O- or genetically engineered to be O-.

[0050] As used herein, "HLA" or "human leukocyte antigen" complex refers to the complex of genes that code for the human major histocompatibility complex (MHC) proteins. These cell surface proteins that make up the HLA complex are responsible for controlling the immune response to antigens. In humans, there are two MHCs: class I and class II, or "HLA-I" and "HLA-II." HLA-I includes HLA-A, HLA-B, and HLA-C. They present peptides from inside the cell, and the antigens presented by the HLA-I complex attract killer T cells (also known as CD8+ T cells or cytotoxic T cells). HLA-I proteins are associated with beta-2 microglobulin (B2M). HLA-II includes HLA-DP, HLA-DM, HLA-DOB, HLA-DQ, and HLA-DR, and present antigens from outside the cell to T lymphocytes, which stimulate CD4+ cells (also known as T helper cells). It should be understood that the use of either "MHC" or "HLA" is not limiting, as it depends on whether the genes are of human (HLA) or non-human (MHC) origin. Thus, insofar as mammalian cells are concerned, these terms are used interchangeably herein.

[0051] As used herein, "gene knockout" refers to the process of inactivating a particular gene in a host cell in which the gene is present, so that the protein of interest is not produced or is in an inactive form.As will be understood by those skilled in the art and as will be further described below, this can be done in many different ways, such as removing a nucleic acid sequence from the gene, interrupting the sequence with another sequence, changing the reading frame, or changing the regulatory components of the nucleic acid.For example, all or part of the coding region of the gene of interest can be removed or replaced with a "nonsense" sequence, all or part of a regulatory sequence such as a promoter can be removed or replaced, or the translation initiation sequence can be removed or replaced.

[0052] As used herein, "gene knock-in" refers to the process of adding a gene function to a host cell, thereby increasing the levels of the encoded protein. As will be appreciated by those skilled in the art, this can be done in a number of ways, including adding one or more copies of the gene to the host cell, or altering regulatory components of the endogenous gene to increase expression of the protein. This may be done by modifying the promoter, adding a different promoter, adding enhancers, or modifying other gene expression sequences. "Beta-2 microglobulin" or "β2M" or "B2M" protein refers to the human β2M protein, having the amino acid and nucleic acid sequences set forth below, and the human gene has the accession number RefSeq NM_004048.4.

[0053] "CD47 protein" refers to the human CD47 protein having the amino acid and nucleic acid sequences set forth below, the human gene having the accession number RefSeq NM_001777.4.

[0054] Upon ligation to the PD-1 receptor, the natural ligands PD-L1 or PD-L2 expressed on the cell may initiate downstream signaling within the cell, which may unfavorably affect its physiological function. To protect only the killing of immune cells, some embodiments of the invention use PD-1 engagers as described herein to separate the extracellular PD-1 binding function from the intracellular signaling in the engineered cells. In other embodiments, the invention provides PD-1 engager fusion proteins that have agonist-like PD-1 binding activity but lack undesirable intracellular signaling in the engineered cells. Other embodiments provide PD-1 engager fusion proteins that include the PD-L1 or PD-L2 extracellular domain (ECD). In some embodiments of the invention, such fusion proteins will have a homologous or heterologous intracellular domain. In other embodiments, the fusion proteins have truncated or no intracellular domain. The present invention provides PD-1 engager fusion proteins that are expressed on engineered cells and are designed to bind agonistically to PD-1 on immune cells to activate PD-1 signaling. Effector immune cells can be any immune cell that expresses PD-1, and can be myeloid (such as monocytes, macrophages, polymorphonuclear cells) or lymphoid (such as lymphocytes, T cells, B cells, or NK cells).

[0055] The PD-1 / PD-L1 pathway is a well-established immune checkpoint. Forced overexpression of PD-L1 in target cells can effectively induce inhibitory signals in immune effector cells, but PD-L1 may induce undesirable signaling in modified cells. The present invention provides a novel strategy to exploit this immune checkpoint without disrupting the physiology of modified cells. The PD-1 engager proteins disclosed herein do not have an intracellular signaling domain.

[0056] Thus, in some embodiments, the fusion proteins provided herein comprise an extracellular domain (ECD) and a transmembrane domain (TMD), and may or may not comprise an intracellular domain (ICD). Fusion proteins typically do not have an ICD, and are limited to an ECD and a TMD.

[0057] In some embodiments, the ECD comprises a PD-L1 or PD-L2 ECD, a complementarity determining region (CDR) of an agonist anti-PD-1 antibody, or a single chain variable fragment (scFv) of an agonist anti-PD-1 antibody. A region of interest on an ECD includes at least one CDR sequence, where a CDR is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acids. Alternatively, an ECD of interest comprises multiple antibody variable regions. CDRs of anti-PD-1 antibodies are disclosed, for example, in U.S. Patent No. 10,493,148, which is incorporated herein by reference in its entirety.

[0058] In some embodiments, one or more residues of the sequence are changed or modified to more favorably adjust the on-rate, off-rate, or both of the binding to optimize binding.

[0059] In other embodiments, the ECD includes a linker region or hinge that connects the provided sequences to the TMD or to each other, hi other embodiments, modifications are made to one or more of the linker or hinge regions that do not abolish the binding affinity of the fusion protein to PD-1.

[0060] In some aspects, the ECD has a contiguous sequence of at least about 10 amino acids, at least about 15 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 30 amino acids, up to the complete region provided, in the sequence set forth in SEQ ID NO: 1. The ECD also includes a sequence that differs by up to 1, 2, 3, 4, 5, 6 or more amino acids compared to the amino acid sequence set forth in SEQ ID NO: 1. In other embodiments, the ECD has at least about 80%, 85%, 90%, 95%, or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.

[0061] In general, the transmembrane domain (TMD) of the PD-1 engager fusion protein is not limited to a specific TMD sequence. Preferably, the TMD allows the fusion protein to be stably anchored to the membrane of the cell expressing the fusion protein (e.g., endothelial cells, cardiomyocytes, pancreatic beta cells, T cells, NK cells, or hematopoietic cells, etc.). The TMD further allows the ECD to bind to PD-1. In some embodiments, the fusion protein includes an ICD, and binding to PD-1 allows signaling through the ICD. Such signaling can be beneficial to the modified cell if it enhances the cell's inherent function. The improved function can be achieved, for example, by enhancing adhesion through integrin activation. In other embodiments, the fusion protein does not include an ICD and is truncated after the TMD. In the latter case, binding of the fusion protein to PD-1 does not result in intracellular signaling of the modified cell.

[0062] TMDs extend across the cell membrane lipid bilayer as single α-helices, multiple α-helices, or rolled-up β-sheets. Some of these "single-pass" and "multi-pass" proteins are inserted into the cytoplasmic lipid monolayer by covalently attached fatty acid chains. Other membrane proteins are exposed to only one side of the membrane. Some of these are anchored to the cytoplasmic surface by amphipathic α-helices and insert into the cytoplasmic monolayer of the lipid bilayer via the hydrophobic face of this helix. Others are attached to the bilayer only by lipid chains covalently attached to either fatty acid chains or prenyl groups in the cytoplasmic monolayer, or to phosphatidylinositol in the non-cytoplasmic monolayer via oligosaccharide linkers. (Alberts B, Johnson A, Lewis J, et al., Molecular Biology of the Cell.4th edition. New York: Garland Science; ISBN-10:0-8153-3218-1 (2002)).

[0063] In some embodiments, exemplary TMDs of immune checkpoint engager molecules are CD85f, CD349, CD284, CD261, CD172b, CD277, CD186, CD156c, CD304, CD254, CD263, CD267, CD337, CD170, CD283, CD133, CD327, CD205, CD232, CD282, CD16b, CD85i, CD85a, CD85c, CD275, CD108, CD358, CD335, CD218b, CD355, CD336, CD160, CD25, CD4, CD8a, CD23 5a, CD233, CD230, CD90, CD74, CD3d, CD340, CD236, CD61, CD18, CD54, CD29, CD1a, CD5, CD220, CD2, CD66e, CD51, CD141, CD115, CD42b, CD221, CD271, CD5 5, CD243, CD98, CD10, CD41, CD14, CD45, CD228, CD16a, CD49e, CD126, CD63, CD48, CD7, CD140b, CD3g, CD117, CD28, CD8b, CD37, CD11b, CD107a, CD331, CD 222, CD20, CD79a, CD32, CD143, CD324, CD42c, CD107b, CD56, CD102, CD49d, CD66a, CD142, CD59, CD62L, CD121a, CD122, CD13, CD155, CD119, CD19, CD116 , CD46, CD1e, CD1d, CD227, CD44, CD62P, CD104, CD43, CD140a, CD31, CD152, CD326, CD62E, CD36, CD127, CD49b, CD105, CD35, CD223, CD138, CD325, CD58, CD106, CD53, CD120a, CD224, CD21, CD33, CD22, CD120b, CD11a, CD11c, CD363, CD73, CD88, CD204, CD332, CD9, CD203a, CD334, CD333, CD206, CD49f, CD23 8, CD252, CD89, CD124, CD181, CD182, CD24, CD95, CD40, CD49c, CD159a, CD159c, CD314, CD27, CD123, CD26, CD82, CD121b, CD34, CD38, CD30, CD1b, CD1c,CD154、CD6、CD52、CD132、CD32、CD66b、CD171、CD191、CD197、CD185、CD131、CD50、CD70、CD153、CD144、CD80、CD362、CD68、CD361、CD147、CD309、CD135、CD292、CD103、CD130、CD42d、CD66d、CD66c、CD96、CD110、CD79b、CD200、CD192、CD231、CD86、CD212、CD118、CD146、CD134、CD158a、CD158b1、CD158b2、CD158e、CD158k、CD158j、CD158i、CD178、CD295、CD151、CD97、CD183、CD39、CD239、CD193、CD194、CD195、CD196、CDw198、CDw199、CD296、CD298、CD49a、CD322、CD85g、CD184、CD172a、CD156a、CD339、CD156b、CD213a1、CD129、CD83、CD125、CD241、CD269、CD202b、CD87、CD164、CD136、CD137、CD249、CD69、CD91、CDw210b、CD167a、CD300c、CD157、CD317、CD148、CD161、CD215、CD150、CD11d、CD218a、CD210、CD166、CD162、CD213a2、CD242、CD158g、CD158h、CD279、CD111、CD281、CD226、CD234、CD167b、CD300e、CD276、CD305、CD300g、CD300d、CD109、CD272、CD163、CD302、CD158f1、CD85h、CD85d、CD177、CD158z、CD158f2、CD85j、CD300f、CD92、CD351、CD112、CD100、CD270、CD101、CD297、CD316、CD352、CD217、CD307b、CD307a、CD307c、CD307d、CD307e、CD114、CD180、CD158d、CD273、CD290、CD244、CD169、CD299、CD318、CD360、CD229、CD248、CD354、CD320、CD93、CD319、CD113、CD163b、CD289、CD288、CD329、CD274、CD353、In other embodiments, the TMD of the fusion protein is derived from CD47 (SEQ ID NO:5), CD64 (SEQ ID NO:6), or PDGFR (SEQ ID NO:4).

[0064] In other embodiments, the PD-1 engager fusion protein lacks an intracellular domain (ICD) to avoid signaling in the engineered cell, however, if deemed beneficial, the ICD of the fusion protein can be an ICD derived from CD16, CD32, CD64, CD8, CD3, CD28, or CD137.

[0065] "CIITA protein" refers to the human CIITA protein having the amino acid and nucleic acid sequences set forth below, the human gene having RefSeq accession number NM_000246.4.

[0066] "Wild type" in the context of a cell refers to a cell that exists in nature, however, in the context of cell therapy as used herein, it also refers to a cell that may contain nucleic acid alterations that result in an immortalized or dedifferentiated state after reprogramming, and has not undergone the immunoediting techniques of the present invention to achieve low immunogenicity, acquire the ability to evade antibodies, or express immune checkpoint engagers.

[0067] As used herein, "syngeneic" refers to the genetic similarity or identity between the host organism and the cell graft, and means that there is immunological compatibility, e.g., no immune response is elicited.

[0068] As used herein, "allogeneic" refers to the genetic similarity between the host organism in which the immune response is generated and the cell graft.

[0069] As used herein, "B2M- / -" refers to a state in which the B2M gene is inactivated on both chromosomes in a diploid cell. As described herein, this can be accomplished in a variety of ways.

[0070] As used herein, "PD-1 tg," "PD-1 transgene," or "PD-1+" means that the host cell expresses PD-1, optionally by having at least one additional copy of the PD-1 gene or a PD-1 fusion protein gene.

[0071] As used herein, "CIITA- / -" means that a diploid cell has the CIITA gene inactivated on both chromosomes. As described herein, this can be accomplished in a variety of ways.

[0072] As used herein, "PD-L1 tg," "PD-L1 transgene," or "PD-L1+" means that the host cell expresses PD-L1, in some embodiments, by having at least one additional copy of the PD-L1 gene or PD-L1 fusion protein gene.

[0073] As used herein, "PD-L2 tg," "PD-L2 transgene," or "PD-L2+" means that the host cell expresses PD-L2, in some embodiments, by having at least one additional copy of the PD-L2 gene or PD-L2 fusion protein gene.

[0074] As used herein, "CD47 tg," "CD47 transgene," and "CD47+" mean that the host cell expresses CD47.

[0075] The term "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are identical in a stated percentage of nucleotides or amino acid residues, compared and aligned for maximum correspondence, using one of the sequence comparison algorithms described below (e.g., BLASTP, BLASTN, or other algorithms available to those skilled in the art), or measured visually. In some applications, the percent "identity" may be over a region of the sequences being compared, e.g., a functional domain, or over the entire length of the two sequences being compared. In sequence comparison, one sequence is usually designated as a reference sequence, to which a test sequence is compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the designated program parameters.

[0076] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), computer implementations of these algorithms (GAP, BESTFIT, FASTA, TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

[0077] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0078] "Inhibitors," "activators," and "regulators" affect the function or expression of biologically relevant molecules. The term "modulator" includes both inhibitors and activators. These can be identified using in vitro and in vivo assays for the expression or activity of the target molecule.

[0079] "Inhibitors" are agents that, for example, inhibit expression or bind to a target molecule or protein. They may partially or totally block stimulation or have protease inhibitor activity. They may suppress, reduce, prevent, or delay activation of the described target protein, including inactivation, desensitization, or downregulation of activity. Modulators may be antagonists of the target molecule or protein.

[0080] An "activator" is an agent that, for example, induces or activates the function or expression of a target molecule or protein. They bind to, stimulate, increase, open, activate, or promote the activity of the target molecule. An activator may be an agonist of the target molecule or protein.

[0081] A "homolog" is a biologically active molecule that is similar to a reference molecule at the nucleotide sequence, peptide sequence, functional or structural level. Homologs may include sequence derivatives that share a certain percent identity with a reference sequence. Thus, in one embodiment, a homologous or derivative sequence shares at least 70% sequence identity. In a specific embodiment, a homologous or derivative sequence shares at least 80% or 85% sequence identity. In a specific embodiment, a homologous or derivative sequence shares at least 90% sequence identity. In a specific embodiment, a homologous or derivative sequence shares at least 95% sequence identity. In more specific embodiments, a homologous or derivative sequence shares at least 50, 55, 60, 65, 70, 75, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity. Homologous or derivative nucleic acid sequences may be defined by their ability to remain bound to the reference nucleic acid sequence under high stringency hybridization conditions. Homologs that have structural or functional similarity with the reference molecule may be chemical derivatives of the reference molecule. Methods for detecting, producing and screening structural and functional homologs and derivatives are known in the art.

[0082] "Hybridization" generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below the melting temperature. The higher the degree of homology required between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, higher relative temperatures result in more stringent reaction conditions, while lower relative temperatures result in less stringent reaction conditions. For details and explanations of stringency of hybridization reactions, see Ausubel et al, Current Protocols in Molecular Biology, Wiley Interscience Publishers (1995), which is incorporated herein by reference in its entirety.

[0083] "Stringency" of a hybridization reaction is readily determinable by one of ordinary skill in the art and is generally an empirical calculation dependent upon probe length, washing temperature, and salt concentration. Generally, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures.

[0084] As used herein, "stringent conditions" or "high stringent conditions" can be defined by the following conditions: (1) low ionic strength and high temperature are used for washing, e.g., 0.015M sodium chloride / 0.0015M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; (2) a denaturing agent is used during hybridization, e.g., a solution containing 50% (v / v) formamide / 0.1% ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5, 750 mM sodium chloride, 75 mM sodium citrate, containing 0.1% bovine serum albumin, is used at 42°C. (3) Hybridization was performed overnight at 42°C in a solution containing 50% formamide, 5x SSC (0.75 M sodium chloride, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Dennard's solution, sonicated salmon sperm DNA (50 μl / ml), 0.1% SDS, and 10% dextran sulfate, followed by a 10-minute wash with 0.2x SSC (sodium chloride / sodium citrate) at 42°C, followed by a 10-minute high-stringency wash with 0.1x SSC containing EDTA at 55°C.

[0085] As used herein, a "pharmaceutically acceptable carrier" or a "therapeutically effective carrier" can be aqueous or non-aqueous (solid), such as alcoholic or oily, or a mixture thereof, and can include surfactants, emollients, lubricants, stabilizers, colorants, fragrances, preservatives, acids or bases for adjusting pH, solvents, emulsifiers, gelling agents, humectants, stabilizers, wetting agents, sustained release agents, humectants, or other ingredients that are typically included in pharmaceutical compositions of a particular formulation. Pharmaceutically acceptable carriers are known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline, and other solvents or vehicles such as glycols, glycerol, oils such as olive oil, etc. Pharmaceutically acceptable carriers can include physiologically acceptable compounds that act to stabilize or increase the absorption of a particular inhibitor, such as carbohydrates such as glucose, sucrose, or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, or other stabilizers or excipients.

[0086] The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oily suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, a solution in 1,3-butanediol. Acceptable carriers and solvents that can be used include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as a solvent or suspending medium. For this purpose, any grade of fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils such as olive oil or castor oil, especially those polyoxyethylated. These oil solutions or suspensions may contain long-chain alcohol diluents or dispersants, such as the Swiss Pharmacopoeia or similar alcohols.

[0087] Every maximum numerical limitation given throughout this specification is intended to include every lower numerical limitation, as if that lower numerical limitation was expressly written herein. Every minimum numerical limitation given throughout this specification is intended to include every upper numerical limitation, as if that upper numerical limitation was expressly written herein. Every numerical range given throughout this specification is intended to include every narrower numerical range that is contained within a broader numerical range, as if that narrower numerical range was expressly written herein.

[0088] The term "modification" as used herein refers to a modification that physically distinguishes the modified molecule from the parent molecule. In some embodiments, the modification includes an insertion, deletion, substitution, or other type of amino acid change in the PD-1 engager or SIRPα engager protein. In some embodiments, the modification is in a PD-1, PD-L1, PD-L2, SIRPα, CD47, CD16, CD32, CD64, truncated CD64, HSVtk, EC-CD, or iCasp9 variant polypeptide prepared according to the methods described herein and known in the art. Such modifications distinguish the corresponding parent protein that is not modified according to the methods described herein, such as a wild-type protein, a naturally occurring mutant protein, or other modified protein that does not contain such mutant polypeptide modifications. In other embodiments, the mutant polypeptide contains one or more modifications that distinguish the function of the mutant polypeptide from an unmodified polypeptide. For example, the amino acid changes in the mutant polypeptide affect its receptor binding profile. In other embodiments, the mutant polypeptide contains a substitution, deletion, insertion modification, or a combination thereof. In other embodiments, the mutant polypeptide contains one or more modifications that increase the affinity for the receptor compared to the affinity of the unmodified polypeptide.

[0089] In one embodiment, a mutant polypeptide comprises one or more substitutions, insertions, or deletions relative to the corresponding native or parent sequence, hi certain embodiments, a mutant polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31-40, 41-50, or 51 or more modifications.

[0090] As used herein, "episomal vector" refers to a gene vector that can exist and replicate autonomously in the cytoplasm of a cell.For example, it is not integrated into the genomic DNA of a host cell.Many episomal vectors are known in the art and are described below.

[0091] "Knockout" in the context of a gene means that the host cell carrying the knockout does not produce a functional protein product of that gene. As outlined herein, knockouts can be achieved in a variety of ways, including removing all or part of the coding sequence, introducing a frameshift mutation (either a truncated or nonsense sequence) such that no functional protein is produced, removing or modifying regulatory components (e.g., promoters) such that the gene is not transcribed, or blocking translation by binding to mRNA. Generally, knockouts are performed at the genomic DNA level, and the descendants of the cell will permanently inherit the knockout.

[0092] "Knock-in" in the context of a gene means that the host cell carrying the knock-in has more functional protein active in the cell. As outlined herein, knock-ins can be performed in a variety of ways, typically by introducing at least one copy of a transgene (tg) that codes for the protein into the cell, but can also be performed by replacing regulatory components, for example by adding a constitutive promoter to an endogenous gene. In general, knock-in techniques incorporate an extra copy of a transgene into the host cell.

[0093] VII. Cells of the Invention The present invention provides ICE cells. In some embodiments, they are PD-1 engager cells. In other embodiments, they are hypoimmune cells. Hypoimmune cells include HIP cells, HIPO cells, HIPO- cells, etc. In other embodiments, the PD-1 engager cells are differentiated somatic cells. In other embodiments, the PD-1 engager cells are hypoimmune pluripotent (HIP) cells. In further embodiments, the HIP cells are blood type O (HIPO), Rh factor (Rh) negative (HIP-), or blood type O and Rh- (HIPO-). In other embodiments, the PD-1 engager cells are derived or differentiated from HIP cells, HIP- cells, or HIPO- cells. In other embodiments, the PD-1 engager cells contain antibody Fc receptors for protection from antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0094] The present invention provides compositions and methods for generating PD-1 engager cells. In some embodiments, the cells are hypoimmune cells. In other embodiments, the cells are differentiated somatic cells. In other embodiments, the cells are pluripotent cells, such as HIP cells, HIP-cells, HIPO-cells, etc. In other embodiments, the PD-1 engager cells are pluripotent stem cell (PSC) cells suitable for transplantation and / or differentiation. PSC cells include induced PSC cells (iPSC) or embryonic stem cells (ESC). In other embodiments, the cells are tissue-specific and differentiated from the aforementioned PD-1 engager cells. By way of example, the differentiated PD-1 engager cells may be endothelial cells, cardiomyocytes, hepatocytes, dopaminergic neurons, pancreatic islet cells, retinal pigment epithelial cells, and other cell types used for transplantation and medical treatment. This includes chimeric antigen receptor (CAR) cells such as CAR-T cells, CAR-NK cells, TCR T cells, TCR NK cells, TCR innate lymphoid cells (ILCs), and other engineered cell populations. See WO 2018 / 132783, WO 2020 / 018620, WO 2020 / 018615, International Application No. PCT / US2020 / 032272, U.S. Application No. 16 / 870,959, and U.S. Application No. 16 / 870,960, all of which are incorporated by reference herein in their entireties.

[0095] The present invention provides PD-1 engager cells having a PD-1 engager protein that interacts with PD-1 on the surface of T cells, NK cells, and other immune cells. Immune checkpoint engager molecules prevent cell death and mitigate adaptive and innate immunity. In some embodiments, the PD-1 engager protein is an anti-PD-1 antibody tethered to the surface of the PD-1 engager cell. In some embodiments, the anti-PD-1 antibody is tethered to the cell surface via its fragment crystallizable (Fc) portion. In other embodiments, the antigen-binding portion of the anti-PD-1 antibody (scFv) binds to the cell surface via a transmembrane domain (TMD). In preferred embodiments, the TMD comprises one or more α-helices. In other preferred embodiments, the TMD is derived from a seven transmembrane protein (7TM). In other preferred embodiments, the TMD is derived from an immunoglobulin cell surface protein. In more preferred embodiments, the immunoglobulin cell surface protein is an antibody, a receptor, a ligand, or an adhesion protein. In some embodiments, PD-1 engager cells are generated from PD-L1 or PD-L2 fusion proteins anchored on the cell surface.

[0096] Expression of the PD-1 engager protein may be achieved in a number of ways, using "knock-in" or transgenic techniques, as will be appreciated by those of skill in the art. In some embodiments, expression of the PD-1 engager protein is achieved by one or more transgenes.

[0097] Thus, in some embodiments, one or more copies of a PD-1 engager protein expression gene are added to the PD-1 engager cell under the control of an inducible or constitutive promoter, with the latter being preferred. In some embodiments, a lentiviral construct is employed as described herein or known in the art. The gene is integrated into the genome of the host cell under the control of a suitable promoter, as known in the art.

[0098] In some embodiments, expression of a gene can be increased by altering the regulatory sequences of the endogenous locus, for example, by replacing the endogenous promoter with a constitutive promoter or with a different inducible promoter, which can generally be accomplished using known techniques such as CRISPR.

[0099] Once altered, the presence of sufficient PD-1 engager protein expression can be assayed using known techniques such as those described in the Examples, such as Western blot, ELISA assay, or FACS assay using an appropriate antibody. Generally, "sufficient" in this context means that expression of PD-1 engager protein on the cell surface is increased and killing by NK cells is inhibited.

[0100] Also within the scope of the present invention are polypeptides that are antibodies. The term antibody includes monoclonal antibodies, polyclonal antibodies, humanized antibodies, antibody fragments (e.g., Fc domains), Fab fragments, single chain antibodies, bi- or multispecific antibodies, llama antibodies, nanobodies, diabodies, affibodies, Fv, Fab, F(ab')2, Fab', scFv, scFv-Fc, and the like. Also included in the term are antibody fusion proteins, such as Ig chimeras. Preferred antibodies include humanized or fully human monoclonal antibodies or fragments thereof.

[0101] The terms "antibody" and "immunoglobulin" may include monoclonal antibodies (e.g., full length or intact monoclonal antibodies), polyclonal antibodies, monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), and may also include certain antibody fragments (described in more detail herein). Antibodies may be chimeric, human, humanized, and / or affinity matured.

[0102] As used herein, the terms "full length antibody", "intact antibody" and "whole antibody" are used interchangeably to refer to an antibody in a substantially intact form, rather than an antibody fragment as defined below. This term particularly refers to an antibody having a heavy chain including an Fc region. An "antibody fragment" includes a portion of an intact antibody, preferably including the antigen-binding region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are identical except for minor amounts of possible mutations, such as naturally occurring mutations. Thus, the modifier "monoclonal" indicates that the antibody is not a mixture of different antibodies.

[0103] In certain embodiments, such monoclonal antibodies typically include antibodies that include a polypeptide sequence that binds to a target, the target-binding polypeptide sequence being obtained by a process that includes selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may include selecting a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It should be noted that the selected target-binding sequence may be further modified, for example, to improve affinity to the target, to humanize the target-binding sequence, to improve production in cell culture, to reduce immunogenicity in vivo, to create a multispecific antibody, and the antibody that includes the modified target-binding sequence is also a monoclonal antibody of the present invention. Unlike polyclonal antibody preparations, which usually include different antibodies against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation targets one determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are usually not contaminated by other immunoglobulins.

[0104] An antibody that specifically binds to an antigen has a high affinity for that antigen. The affinity of an antibody can be measured by the dissociation constant (Kd). In certain embodiments, the antibodies provided herein have a dissociation constant (Kd) of about 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, or 0.001 nM or less (e.g., 10 -7 M or less, 10 -7 M~10 -13 M, 10 -8 M~10 -13 M, or 10 -9 M~10 -13 M).

[0105] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab form of the antibody of interest and its antigen, as described by the assay below. The solution binding affinity of the Fab to the antigen is measured by equilibrating the Fab with a minimal concentration of (125I)-labeled antigen with repeated titrations of unlabeled antigen and capturing the bound antigen on an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish assay conditions, MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 μg / ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate, pH 9.6, and then blocked for 2-5 hours at room temperature (approximately 23° C.) in PBS containing 2% (w / v) bovine serum albumin. In a non-adsorbent plate (Nunc#269620), 100 μM or 26 μM [125I] antigen is mixed with serial dilutions of the Fab of interest (e.g., as evaluated for anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight, although the incubation time may be extended (e.g., about 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate and incubated at room temperature (e.g., 1 hour). The solution is then removed and the plate is washed 8 times with PBS containing 0.1% polysorbate 20 (TWEEN-20®). Once the plate has dried, 150 μl of scintillant (MICROSCINT-20™; Packard) is added per well and counted for 10 minutes in a TOPCOUNT™ gamma counter (Packard). For use in competitive binding assays, concentrations of each Fab are selected that give binding less than or equal to 20% of maximal binding.

[0106] According to another embodiment, Kd is measured by surface plasmon resonance assay using BIACORE®-2000 or BIACORE®-3000 (BIAcore, Piscataway, NJ) at 25°C. For example, an immobilized antigen CM5 chip of about 10 response units (RU) is used. Briefly, a carboxymethylated dextran biosensor chip (CM5, BIACORE) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The antigen is diluted to 5 μg / ml (about 0.2 μM) with 10 mM sodium acetate (pH 4.8) before injection and injected at a flow rate of 5 μl / min to obtain a binding protein of about 10 response units (RU). After injection of the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25 °C with a flow rate of approximately 25 μl / min. A simple one-to-one Langevin binding model (BIACORE® Evaluation Software version 3.2) was used to simultaneously fit the association and dissociation sensorgrams to determine the binding rate (K on ) and dissociation rate (K off The equilibrium dissociation constant (Kd) is calculated as the ratio koff / kon (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). If the on-rate is 10 6 M -1 s -1If the on-rate exceeds 100%, the fluorescence quenching technique can be used to measure the on-rate. In this technique, the increase or decrease in the fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (in the form of Fab) in PBS (pH 7.2) at 25°C is measured with increasing concentrations of antigen. The measurements are performed using a spectrometer such as a stopped-flow spectrophotometer (Aviv Instruments) or a 8000 series SLM-AMINCO™ spectrophotometer with a stirred cell (ThermoSpectronic). As will be appreciated by those skilled in the art, instead of the amine coupling method (CM5 chip) described above, other coupling chemistries between the target antigen and the chip surface (e.g., streptavidin / biotin, hydrophobic interactions, or disulfide chemistries) can be easily used.

[0107] The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in accordance with the present invention can be produced using hybridoma techniques (e.g., Kohler et al., Nature, 256: 495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas pp. 563-681 (Elsevier, NY, 1981)), recombinant DNA techniques (e.g., U.S. Pat. No. 4,816,567), phage display techniques (e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol., 222: 581-597 (1992); Sidhu et al., J. Mol. Biol., 338(2): 299-310 (1993)), and the like. (2004); Lee et al., J. Mol. Biol., 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA, 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods, 284(1-2): 119-132 (2004)), or techniques for producing human or human-like antibodies in animals that have some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (e.g., WO 98 / 24893; WO 96 / 34096; WO 96 / 33735; WO 91 / 10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-258 (1993);Bruggemann et al., Year in Immunol., 7:33 (1993);U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016;Marks et al., Bio. Technology, 10: 779-783 (1992);Lonberg et al., Nature, 368:856-859 (1994);Morrison, Nature, 368:812-813 (1994);Fishwild et al., Nature Biotechnol., 14:845-851 (1996); Neuberger, Nature Biotechnol., 14:826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol., 13: 65-93 (1995)). The above patents, publications, and literature citations are incorporated by reference in their entirety.

[0108] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate, thereby imparting a desired specificity, affinity, or capacity. In some embodiments, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further improve antibody performance. In general, humanized antibodies contain a large portion of at least one, and usually two, variable domains, in which all or almost all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or almost all of the FRs are of human immunoglobulin sequences. A humanized antibody optionally also contains at least a portion of an immunoglobulin constant region (Fc), usually that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). Also see the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994). The above references are incorporated by reference in their entirety.

[0109] A "human antibody" is an antibody that contains an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human and / or an antibody made using any of the human antibody production techniques disclosed herein. Such techniques include screening of recombinant libraries of human origin, such as phage display libraries (see, e.g., Marks et al., J. Mol. Biol., 222: 581-597 (1991) and Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991)), production of human monoclonal antibodies using human myeloma and mouse-human heteromyeloma cell lines (see, e.g., Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 55-93 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991)), as well as the generation of monoclonal antibodies in transgenic animals (e.g., mice) capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255 (1993); Bruggermann et al., Year in Immunol., 7: 33 (1993)). This definition of a human antibody specifically excludes humanized antibodies, which contain antigen-binding residues derived from an animal other than human.

[0110] A. Methods of Genetic Modification The present invention includes methods for modifying nucleic acid sequences to generate PD-1 engager cells in cells or under cell-free conditions. Examples of techniques include homologous recombination, knock-in, ZFN (zinc finger nuclease), TALEN (transcription activator-like effector nuclease), CRISPR (clustered regularly interspaced short palindromic repeats) / Cas9, and other site-specific nuclease techniques. These techniques can be used to break double-stranded DNA at a locus of interest. This controlled double-strand break promotes homologous recombination at the specific locus. This process focuses on targeting a specific nucleic acid molecule sequence, such as a chromosome, by endonucleases that recognize and bind to specific sequences, causing a double-strand break in the nucleic acid molecule. This double-strand break is repaired by error-prone non-homologous end joining (NHEJ) or by homologous recombination (HR).

[0111] As will be appreciated by those of skill in the art, a variety of techniques can be used to generate the modified cells of the present invention, as well as to render them less immunogenic as described herein.

[0112] In general, these techniques can be used individually or in combination. For example, in the generation of PD-1 engager cells, CRISPR can be used to express a PD-1 engager protein, such as an anti-PD-1 immunoglobulin. In another example, viral techniques (e.g., lentivirus) can be used to express a PD-1 engager protein.

[0113] a.CRISPR technology In one embodiment, as known in the art, cells are engineered using clustered regularly interspaced short palindromic repeats / Cas ("CRISPR") technology. CRISPR can be used to generate PD-1 engager cells. There are many CRISPR-based technologies, see, for example, Doudna and Charpentier, Science doi:10.1126 / science.1258096, which is incorporated herein by reference. CRISPR technology and kits are commercially available.

[0114] b.TALEN technology In some embodiments, the cell of the present invention is produced using the method of transcription activator-like effector nuclease (TALEN). TALEN is a combination of restriction enzyme and nuclease, which can be designed to bind and cut almost any DNA sequence. TALEN kits are commercially available.

[0115] c. Zinc finger technology In one embodiment, cells are engineered using Zn finger nuclease technology. Zn finger nucleases are artificial restriction enzymes created by fusing a zinc finger DNA binding domain to a DNA cleavage domain. The zinc finger domain can be designed to target specific DNA sequences, allowing zinc finger nucleases to target specific sequences within complex genomes. These reagents can be used to precisely modify the genomes of higher organisms, similar to CRISPR and TALEN, by exploiting the inherent DNA repair mechanisms.

[0116] d. Virus-based technology There are a variety of viral techniques that can be used to generate some embodiments of the PD-1 engager cells of the present invention, including but not limited to the use of retroviral vectors, lentiviral vectors, adenoviral vectors, Sendai viral vectors, and the episomal vectors used to generate the cells are listed below.

[0117] In all of these techniques, known recombinant techniques are used to generate the recombinant nucleic acids outlined herein. In certain embodiments, a recombinant nucleic acid encoding a PD-1 engager protein, such as an anti-PD-1 immunoglobulin, may be operably linked to one or more regulatory nucleotide sequences in an expression construct. The regulatory nucleotide sequence will generally be appropriate for the host cell and the subject to be treated. A large variety of suitable expression vectors and suitable regulatory sequences for various host cells are known in the art. Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, a promoter sequence, a leader or signal sequence, a ribosome binding site, transcriptional start and end sequences, translational start and end sequences, and an enhancer or activator sequence. Constitutive or inducible promoters are also contemplated, as known in the art. The promoter may be a naturally occurring promoter or a hybrid promoter that combines elements of multiple promoters. The expression construct may be present in the cell on an episome, such as a plasmid, or the expression construct may be inserted into a chromosome. In a specific embodiment, the expression vector includes a selectable marker gene that allows for the selection of transformed host cells. Certain embodiments include expression vectors that comprise a nucleotide sequence that encodes a mutant polypeptide operably linked to at least one regulatory sequence.Regulatory sequence as used herein includes promoters, enhancers, and other expression control elements.In certain embodiments, expression vectors are designed for the selection of host cells to be transformed, the specific mutant polypeptide to be expressed, the copy number of the vector, the ability to control that copy number, or the expression of other proteins encoded by the vector, such as antibiotic markers.

[0118] Examples of suitable mammalian promoters include the hamster ubiquitin / S27a promoter (WO 97 / 15664), the simian vacuolar virus 40 (SV40) early promoter, the adenovirus major late promoter, the mouse metallothionein-I promoter, the long terminal repeat region of the Ruth sarcoma virus (RSV), the mouse mammary tumor virus promoter (MMTV), the long terminal repeat region of the Moloney murine leukemia virus, the early promoter of the human cytomegalovirus (CMV), the eukaryotic translation elongation factor 1 alpha (EF-1 alpha), and the promoter from the gene of the chicken β-actin promoter (CAG) combined with the CMV early enhancer. Examples of other heterologous mammalian promoters are actin, immunoglobulin, or heat shock promoters.

[0119] In further embodiments, promoters for use in mammalian host cells can be obtained from polyoma virus, fowlpox virus (GBP 2,211,504 published July 5, 1989), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, and hepatitis B virus, and simian virus 40 (SV40). In further embodiments, heterologous mammalian promoters are used. Examples include actin promoter, immunoglobulin promoter, heat shock promoter, and the like. The early and late promoters of the SV40 promoter are conveniently obtained as an SV40 restriction fragment containing the SV40 viral origin of replication. Fiers et al., Nature 273: 113-120 (1978). The immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hind III E restriction fragment. Greenaway, PJ et al., Gene 18:355-360 (1982). The above cited references are incorporated by reference in their entirety.

[0120] In some embodiments, the PD-1 engager cells are derived from stem cells.

[0121] The term "pluripotent cells" refers to cells that are capable of self-renewal and proliferation while remaining undifferentiated and can be induced to differentiate into specialized cell types under appropriate conditions. As used herein, the term "pluripotent cells" encompasses embryonic stem cells (ESCs) and other types of stem cells, including fetal stem cells, amniotic stem cells, and somatic stem cells. Exemplary human stem cell lines include the H9 human embryonic stem cell line. Further examples of stem cell lines include those available through the National Institutes of Health Human Embryonic Stem Cell Registry and the Howard Hughes Medical Institute's HUES collection (as described in Cowan, CA et al., New England J. Med. 350:13 (2004), which are incorporated by reference in their entirety).

[0122] As used herein, "pluripotent stem cells" have the potential to differentiate into any of three germ cell layers: endoderm (e.g., stomach lining, digestive tract, lungs, etc.), mesoderm (e.g., muscle, bone, blood, urogenital tissue, etc.), and ectoderm (e.g., epidermal tissue, nervous system tissue, etc.). As used herein, the term "pluripotent stem cells" also encompasses "induced pluripotent stem cells" or "iPSCs," which are a type of pluripotent stem cell derived from a non-pluripotent cell. Examples of parent cells include somatic cells that have been reprogrammed in various ways to induce a pluripotent undifferentiated phenotype. Such "iPS" or "iPSC" cells can be generated by inducing the expression of certain regulatory genes or by exogenously applying certain proteins. Methods for deriving iPS cells are known in the art and are described further below. (See, e.g., Zhou et al., Stem Cells 27 (11): 2667-74 (2009); Huangfu et al., Nature Biotechnol. 26 (7): 795 (2008); Woltjen et al., Nature 458 (7239): 766-770 (2009); and Zhou et al., Cell Stem Cell 8:381-384 (2009); each of which is incorporated by reference in its entirety.) The generation of induced pluripotent stem cells (iPSCs) is generally described below. As used herein, "hiPSCs" are human induced pluripotent stem cells, and "miPSCs" are mouse induced pluripotent stem cells.

[0123] "Pluripotent stem cell characteristics" refers to cellular characteristics that distinguish pluripotent stem cells from other cells. The ability to generate progeny that, under appropriate conditions, differentiate into cell types that exhibit a combination of characteristics associated with cell lineages derived from all three germ cell layers (endoderm, mesoderm, and ectoderm) is a characteristic of pluripotent stem cells. The expression or non-expression of a combination of specific molecular markers is also a characteristic of pluripotent stem cells. For example, human pluripotent stem cells express at least some, or in some embodiments all, of the following markers, including but not limited to SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49 / 6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Rex1, and Nanog. Cell morphology associated with pluripotent stem cells is also a characteristic of pluripotent stem cells. As described herein, it is not necessary to go through pluripotency to reprogram cells into endodermal progenitor cells and / or hepatocytes.

[0124] B. Generation of Low Immunogenic PD-1 Engager Cells The generation of HI cells is achieved with only three genetic changes, endowing the cells with immunosuppressive functions while minimizing the impact on cellular activity. This technology is disclosed in International Publication No. WO 2018 / 132783, International Publication No. WO 2020 / 018620, International Publication No. WO 2020 / 018615, International Application No. PCT / US2020 / 032272, and U.S. Application No. 16 / 870,959, and U.S. Application No. 16 / 870,960, which are incorporated herein by reference in their entireties. The methodology is briefly described below.

[0125] As discussed herein, one embodiment utilizes the reduction or inactivation of protein activity of MHC I and MHC II (HLA I and HLA II if the cell is human). This can be done by altering the genes that code for the components. In one embodiment, CRISPR is used to disrupt the coding region or regulatory sequence of the gene. In another embodiment, interfering RNA technology is used to suppress translation of the gene. Another embodiment is the alteration of a gene that controls the phagocytosis susceptibility of macrophages. This can be a "knock-in" of the gene using viral technology.

[0126] 1. Decrease in HLA-I The HI PD-1 engager cells of the present invention contain reduced MHC I function (HLA I if derived from human cells).

[0127] As will be appreciated by those skilled in the art, loss of function can be achieved in many ways, including removing nucleic acid sequences from a gene, interrupting the sequence with another sequence, altering regulatory components of the nucleic acid, etc. For example, all or part of the coding region of the gene of interest can be removed or replaced with a "nonsense" sequence, a frameshift mutation can be created, all or part of a regulatory sequence such as a promoter can be removed or replaced, or a translation initiation sequence can be removed or replaced.

[0128] As will be appreciated by those of skill in the art, appropriate reduction in MHC I function (or HLA I if the cells are of human origin) in PD-1 engager cells can be measured using techniques known in the art, as described below, such as FACS techniques using labeled antibodies that bind to HLA complexes; for example, using commercially available HLA-A, HLA-B, HLA-C antibodies that bind to the alpha chain of human major histocompatibility HLA class I antigens.

[0129] a. Modification of β2M In one embodiment, the reduction in HLA-I activity is achieved by disrupting the expression of the beta-2 microglobulin gene in HI PD-1 engager cells as disclosed herein. This modification, generally referred to herein as a "knockout" of the gene, is performed on both alleles of the host cell in the cells of the invention. Generally, the approach to disrupt both is the same.

[0130] In a particularly useful embodiment, CRISPR technology is used to disrupt genes. In another embodiment, programmable transcriptional memory is used through CRISPR-based epigenetic editing (Nunez JK, Cell. 184:2503-2519 (2021), which is incorporated by reference in its entirety). In some embodiments, CRISPR technology is used to introduce small deletions / insertions into the coding region of a gene so that no functional protein is made. In many cases, frameshift mutations result in the creation of stop codons that result in truncated, non-functional proteins.

[0131] Therefore, a useful technique is to use CRISPR sequences designed to target the coding sequence of the mouse B2M gene or the human B2M gene. After gene editing, the transfected PD-1 engager cell cultures are dissociated into single cells. The single cells are grown into full-sized colonies and CRISPR editing is tested by screening for the presence of aberrant sequences from the CRISPR cut sites. Clones carrying deletions in both alleles are selected. Such clones are confirmed by PCR to not express B2M and by FACS analysis to not express HLA-I.

[0132] Assays for testing whether the B2M gene is inactivated are known and described herein. In one embodiment, the assay is a Western blot of cell lysates probed with an antibody against the B2M protein. In another embodiment, the presence of the inactivated variant is confirmed by reverse transcriptase polymerase chain reaction (rt-PCR).

[0133] Additionally, cells can be tested to ensure that HLA I complexes are not expressed on the cell surface, which can be assayed by FACS analysis using antibodies against one or more HLA cell surface components, as described above.

[0134] 2. Decrease in HLA-II In some embodiments, HI PD-1 engager cells of the present invention, in addition to being HLA I reduced, may also lack MHC II function (HLA II from human derived cells).

[0135] As will be appreciated by those skilled in the art, the reduction in function can be achieved in many ways, such as removing a nucleic acid sequence from a gene, adding a nucleic acid sequence to a gene, disrupting the reading frame, interrupting the sequence with another sequence, or changing the regulatory components of the nucleic acid. In one embodiment, all or part of the coding region of the gene of interest can be removed or replaced with a "nonsense" sequence. In other embodiments, regulatory sequences such as promoters can be removed or replaced, and translation initiation sequences can be removed or replaced.

[0136] Appropriate reduction of MHC II (HLA II) function in PD-1 engager cells or derivatives thereof can be measured using techniques known in the art, such as Western blotting using antibodies against the protein, FACS techniques, rt-PCR techniques, and the like.

[0137] a. CIITA modification In one embodiment, the reduction in HLA-II activity is achieved by disrupting expression of the CIITA gene in PD-1 engager cells, as demonstrated herein, This modification, commonly referred to herein as a "knockout" of the gene, is achieved in both alleles of the host cell in the PD-1 engager cells of the invention.

[0138] Assays for testing whether the CIITA gene is inactivated are known and described herein.In one embodiment, the assay is a Western blot of cell lysate probed with an antibody against the CIITA protein.In another embodiment, the presence of the inactivated variant is confirmed by reverse transcriptase polymerase chain reaction (rt-PCR).

[0139] Additionally, cells can be tested to ensure that HLA II complexes are not expressed on the cell surface. Example analyses include Western blot or FACS analysis using commercially available antibodies that bind to human HLA class II HLA-DR, DP, and most DQ antigens, as outlined below.

[0140] In a particularly useful embodiment, CRISPR technology is used to disrupt the CIITA gene. CRISPR is designed to target the coding sequence of the CIITA gene, which is a transcription factor essential for all MHC II molecules. After editing the gene, the transfected cell culture is dissociated into single cells. These are grown into full-sized colonies, and the presence of abnormal sequences from the CRISPR cut site is screened to test whether the CRISPR editing is successful. Clones with deletions that do not express CIITA are determined by PCR, and are confirmed by FACS analysis that they do not express MHC II / HLA-II. In another embodiment, programmable transcriptional memory is used by CRISPR-based epigenetic editing.

[0141] 3.Blood type O, Rh negative cells Blood products can be classified into different groups by the presence or absence of antigens (ABO blood types) on the surface of all red blood cells in a person's body. The arrangement of oligosaccharides on the glycoproteins of red blood cells determines the A, B, AB and A1 antigens. Genes for blood group antigens provide the instructions for making antigen proteins. Blood group antigen proteins perform various functions within the cell membrane of red blood cells. Functions of these proteins include transporting other proteins and molecules in and out of the cell, maintaining cell structure, adhering to other cells and molecules, and participating in chemical reactions.

[0142] The Rh blood group is the second most important blood group system after the ABO blood group system. It is composed of 49 defined blood group antigens, of which five antigens, D, C, c, E, and e, are the most important. An individual's Rh(D) status is usually described by the ABO type followed by a positive or negative suffix. The terms "Rh factor", "Rh positive", and "Rh negative" refer to the Rh(D) antigen only. Antibodies to Rh antigens can be involved in hemolytic transfusion reactions, and antibodies to Rh(D) and Rh(c) antigens pose a significant risk of hemolytic disease of the fetus and newborn. ABO antibodies are expressed in all humans during early childhood. However, Rh- individuals develop Rh antibodies only after sensitization, which occurs by giving birth to an Rh+ newborn or receiving an Rh+ blood transfusion.

[0143] The present invention provides PD-1 engager cells with ABO blood group O and / or RH factor negative (O-) pluripotent (PSCO-) cell populations suitable for transplantation and / or differentiation. PSCO-cells include induced iPSCs (iPSCO-), embryonic ESCs (ESCO-), and cells differentiated therefrom, including O-endothelial cells, O-cardiomyocytes, O-hepatocytes, O-dopaminergic neurons, O-pancreatic islet cells, O-retinal pigment epithelial cells, and other O-cell types used for transplantation and medical treatment. This may include O-chimeric antigen receptor (CAR) cells, such as CAR-T cells, CAR-NK cells, and other engineered cell populations. In some embodiments, the cells are not hematopoietic stem cells. The present invention further provides universally acceptable "off the shelf" ESCO-s and PSCO-s and their derivatives for generating or regenerating specific tissues and organs.

[0144] Other aspects of the invention provide methods for generating populations of PSCO-, iPSCO-, ESCO- and other O-cells for transplantation. The invention also provides methods for treating diseases, disorders and conditions that would benefit from the transplantation of pluripotent or differentiated cells.

[0145] In some embodiments of the invention, the ABO blood group type O is due to reduced expression of ABO blood group proteins. In other aspects, the ABO blood group is endogenously type O. In some aspects of the invention, the HIPO-cell has ABO blood group type O due to disruption of human exon 7 of the ABO gene. In some embodiments, both alleles of exon 7 of the ABO gene are disrupted. In some embodiments, disruption of both alleles of exon 7 of the ABO gene is generated by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / Cas9 reaction that disrupts both alleles. In other embodiments, the gene is inactivated using programmable transcriptional memory by CRISPR-based epigenetic editing.

[0146] In other embodiments, ABO blood group O results from enzymatic modification of the cell surface ABO gene product. In a preferred embodiment, the enzymatic modification removes carbohydrate from the ABO gene product. In another preferred embodiment, the enzymatic modification removes carbohydrate from the ABO A1, A2, or B antigen.

[0147] In some embodiments of the present invention, the Rh blood group is an intrinsic Rh- type. In other aspects, the Rh- blood group is caused by the reduced or absent expression of Rh protein. In other aspects, the Rh- type is caused by the disruption of the gene coding for Rh C antigen, Rh E antigen, Kell K antigen (KEL), Duffy (FY) Fya antigen, Duffy Fy3 antigen, Kidd (JK) Jkb antigen, or / and Kidd SLC14A1. In some embodiments, the disruption is caused by a CRISPR / Cas9 reaction that disrupts both alleles of the gene coding for Rh C antigen, Rh E antigen, Kell K antigen (KEL), Duffy (FY) Fya antigen, Duffy Fy3 antigen, Kidd (JK) Jkb antigen, or / and Kidd SLC14A1.

[0148] In some embodiments of the invention, the O-cells of the invention (e.g., PSCO-, iPSCO-, ESCO- and cells derived therefrom) are of mammalian origin, e.g., human, cow, pig, chicken, turkey, horse, sheep, goat, donkey, mule, duck, goose, buffalo, camel, yak, llama, alpaca, mouse, rat, dog, cat, hamster, or guinea pig.

[0149] In a specific embodiment, the invention provides hypoimmune PD-1 engager cells with ABO blood group O Rh factor negative (HIPO-) cells that avoid rejection by the host alloimmune system and avoid blood antigen type rejection. In some embodiments, HIPO- cells are engineered to reduce or eliminate expression of HLA-I and HLA-II and increase expression of endogenous proteins that reduce the susceptibility of pluripotent cells to phagocytosis by macrophages, to comprise universal blood group O Rh- ("O-") blood type. Universal blood types are obtained by eliminating expression of ABO blood group A and B antigens and Rh factor, or by starting from an O- cell line. These novel HIPO- cells avoid host immune rejection due to impaired antigen presentation, protection from innate immune clearance, and lack of blood group rejection.

[0150] 4. Suicide Gene In some embodiments, the invention provides HI PD-1 engager cells that contain a "suicide gene" or "suicide switch." These are engineered to function as a "safety switch," allowing cells to die if they grow and divide in an undesirable way. The "suicide gene" ablation approach includes a suicide gene in a gene transfer vector that encodes a protein that will kill cells only when activated by a specific compound. The suicide gene may encode an enzyme that selectively converts a non-toxic compound into a highly toxic metabolite. As a result, cells expressing the enzyme are specifically eliminated. In some embodiments, the suicide gene is the herpes virus thymidine kinase (HSV-tk) gene and the trigger is ganciclovir. In other embodiments, the suicide gene is the Escherichia coli cytosine deaminase (EC-CD) gene and the trigger is 5-fluorocytosine (5-FC) (Barese et al., Mol. Therap. 20(10):1932-1943 (2012), Xu et al., Cell Res. 8:73-8 (1998), both of which are incorporated herein by reference in their entireties.)

[0151] In other embodiments, the suicide gene is an inducible caspase protein. The inducible caspase protein comprises at least a portion of a caspase protein capable of inducing apoptosis. In a preferred embodiment, the inducible caspase protein is iCasp9. It comprises the base sequence of human FK506-binding protein FKBP12 with the F36V mutation and is linked to a gene encoding human caspase 9 via a series of amino acids. FKBP12-F36V binds with high affinity to the small molecule dimerizer AP1903. Thus, the suicide function of iCasp9 in the present invention is triggered by administration of a chemical dimerization inducer (CID). In some embodiments, the CID is the small molecule drug AP1903. Dimerization causes rapid induction of apoptosis. (See WO 2011 / 146862; Stasi et al, N. Engl. J. Med 365;18 (2011); Tey et al., Biol. Blood Marrow Transplant. 13:913-924 (2007), each of which is incorporated by reference in its entirety.)

[0152] In other embodiments, as a safety strategy, a binding epitope for existing CAR T cells is included in the extracellular structure of the engager cells. Such a binding epitope may be CD19, CD19 ectodomain, or a specific anti-CD19 binding region, for example, from CAR T cells FMC63, 4G7-2E3, or 3B10. (See Klesmith JR, et al., Biochemistry.3;58(48):4869-4881 (2019), which is incorporated by reference in its entirety.) To eliminate PD-1-engager cells, CAR T cells are used that target the incorporated anti-CD19 binding region. The CAR T cells detect the incorporated binding epitope and kill the engager cells. The binding region can be selected from other CAR T cell targets such as CD20, CD22, CD38, CD123, CS1, CD171, BCMA, MUC16, ROR1, AXL 2, B7- H3, CD147, CD171, CD20, CD44v6, CD70, CEA, CLDN18.2, CLDN6, DLL3, DR5, EGFR, EGFRvIII, EpCAM, ErbB, FRα, GD2, gp100, GPC3, HER2, IL-13Rα2, LFA1, MMP2, MSLN, MUC1, MUC1*, MUC16ecto, NECTIN4, NKG2D, NKG2DL, PSCA, PSMA, ROR2, TM4SF1, TnMUC1, or WT1.

[0153] 5.Fc Sequestration When an antibody binds to an unprotected cell via its Fab region, the Fc may be bound by NK cells (mainly via the CD16 receptor), macrophages (mainly via CD16, CD32, or CD64), B cells (mainly via CD32), or granulocytes (mainly via CD16, CD32, or CD64). These can mediate antibody-dependent cellular cytotoxicity (ADCC). Binding of complement to the Fc leads to complement-dependent cytotoxicity (CDC).

[0154] In some embodiments, the PD-1 engager cells of the present invention have high levels of receptors that recognize the Fc portion of IgG. Receptors that recognize the Fc portion of IgG are divided into four different classes: FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIV. This reduces the tendency of the immune system of the cell graft recipient to reject the allogeneic material. Cells with high expression of CD16, CD32, CD64, or cleaved CD64 avoid ADCC or CDC. Fc sequestration is disclosed in WO2021 / 076427, which is incorporated herein by reference in its entirety.

[0155] 6. SIRPα Engager Cells In some embodiments, the cells of the invention further have increased signal regulatory protein alpha (SIRPα) engagement function that resists innate immunity upon transplantation into a subject (PD-1 / SIRPα engager cells) compared to parental cells with unmodified SIRPα engagement function. In some embodiments, the PD-1 / SIRPα engager cells are hypoimmune cells. In other embodiments, the PD-1 / SIRPα engager cells are differentiated somatic cells. In other embodiments, the PD-1 / SIRPα engager cells are pluripotent cells or hypoimmune pluripotent (HIP) cells. In further embodiments, the HIP cells are blood type O (HIPO), Rh factor (Rh) negative (HIP-), or blood type O and Rh- (HIPO-). In other embodiments, the PD-1 / SIRPα engager cells are derived or differentiated from HIP cells, HIP- cells, or HIPO- cells. In other embodiments, PD-1 / SIRPα engager cells contain antibody Fc receptors for protection from antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). SIRPα engager cells are described in International Application No. PCT / US21 / 62008, which is incorporated herein by reference in its entirety.

[0156] 7. Assay for HI Phenotype Once the HI cells are generated, they can be assayed for hypoimmunogenicity as generally described herein.

[0157] For example, low immunogenicity can be assayed using a number of techniques. One technique involves transplantation into an allogeneic host and monitoring survival of the HI PD-1 engager cells. The cells can be transduced to express luciferase and tracked using bioluminescence imaging. Similarly, the host animal's T or B cell response to the HI PD-1 engager cells is tested to ensure that they do not elicit an immune response in the host animal. T cell function is assayed by Elispot, Elisa, FACS, PCR, or mass cytometry (CYTOF). B cell or antibody responses are assayed using FACS or Luminex. Additionally or alternatively, the cells may be assayed for their ability to evade innate immune responses, such as killing by NK cells. The cytolytic activity of NK cells is assayed in vitro or in vivo using techniques known in the art.

[0158] C. Generation of Hypoimmune (HI)O-PD-1 Engager Cells In some embodiments of the present invention, the PD-1 engager cells generated as described above are already ABO blood group O and Rh factor negative (-) cells, since the procedure began with NK cells with blood type O.

[0159] Another embodiment of the invention involves enzymatic conversion of A and B antigens. In a preferred embodiment, the B antigen is converted to O using an enzyme. In a more preferred embodiment, the enzyme is α-galactosidase. This enzyme removes the terminal galactose residues of the B antigen. In another embodiment of the invention, the A and B antigens are converted enzymatically. In a preferred embodiment, the A antigen is converted to O using α-N-acetylgalactosaminidase. Enzymatic conversion is described, for example, in Olsson et al., Transfusion Clinique et Biologique 11:33-39 (2004); U.S. Pat. Nos. 4,427,777, 5,606,042, 5,633,130, 5,731,426, 6,184,017, 4,609,627, and 5,606,042; and WO 99 / 23210, each of which is incorporated by reference in its entirety.

[0160] Other embodiments of the invention include genetically engineering cells by knocking out exon 7 of the ABO gene or silencing the SLC14A1 (JK) gene. Other embodiments of the invention include knocking out C and E antigens of the Rh blood group system (RH), K of the Kell system (KEL), Fya and Fy3 of the Duffy system (FY), Jkb of the Kidd system (JK), or U and S of the MNS blood group system. Knockout methodologies known in the art or described herein, such as CRISPR, talen, and homologous recombination, can be employed. Techniques for generating hypoimmune ABO blood group O Rh factor (−) cells are described in US Provisional Application No. 62 / 846,399, which is incorporated herein by reference in its entirety.

[0161] D. Embodiments of the Invention The PD-1 engager cells of the present invention, or derivatives thereof, can be used to treat, for example, type 1 diabetes, cardiac disease, neurological disease, cancer, blindness, vascular disease, and other diseases / disorders that respond to regenerative medicine treatment. In particular, the present invention contemplates using PD-1 engager cells to differentiate into any cell type. Thus, provided herein are iPSC, ESC, HIP, iPSCO, ESCO, HIPO, iPSCO-, ESCO-, and HIPO- PD-1 engager cells, or derivatives or differentiated cells thereof, that exhibit pluripotency but do not elicit or elicit a significantly attenuated host immune response when transplanted into an allogeneic host, such as a human patient.

[0162] In one aspect, the present invention provides a PD-1 engager cell, or derivative thereof, comprising a nucleic acid encoding a chimeric antigen receptor (CAR), in which endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator of transcription (CIITA) gene activity have been inactivated, and a PD-1 engager molecule has been provided at the cell surface. The CAR may comprise an extracellular domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the extracellular domain binds to an antigen selected from the group consisting of CD19, CD20, CD22, CD38, CD123, CS1, CD171, BCMA, MUC16, ROR1, WT1, AXL2, B7-H3, CD147, CD171, CD20, CD44v6, CD70, CEA, CLDN18.2, CLDN6, DLL3, DR5, EGFR, EGFRvIII, EpCAM, ErbB, FRα, GD2, gp100, GPC3, HER2, IL-13Rα2, LFA1, MMP2, MSLN, MUC1, MUC1*, MUC16ecto, NECTIN4, NKG2D, NKG2DL, PSCA, PSMA, ROR2, TM4SF1, and TnMUC1. In certain embodiments, the extracellular domain comprises a single chain variable fragment (scFv). In some embodiments, the transmembrane domain comprises CD3zeta, CD4, CD8α, CD28, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, CD64, PDGF, and BTLA. In certain embodiments, the intracellular signaling domain comprises CD3zeta, CD28, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, and BTLA.

[0163] In certain embodiments, the CAR comprises an anti-CD19 scFv domain, a CD28 transmembrane domain, and a CD3 zeta signaling intracellular domain. In some embodiments, the CAR comprises an anti-CD19 scFv domain, a CD28 transmembrane domain, a 4-1BB signaling intracellular domain, and a CD3 zeta signaling intracellular domain.

[0164] In another aspect of the invention, there is provided an isolated PD-1 engager CAR-T cell or hypoimmune CAR-T cell produced by in vitro differentiation of any one of the pluripotent cells described herein. In some embodiments, the CAR-T cell is a cytotoxic HIPO- CAR-T cell.

[0165] In some embodiments, the present invention provides PD-1 engager NK cells or CAR-NK cells, or iPSC-derived NK cells.

[0166] In various embodiments, the in vitro differentiation comprises culturing the PD-1 engager cells or derivatives thereof bearing the CAR construct in a culture medium comprising one or more growth factors or cytokines selected from the group consisting of bFGF, EPO, Flt3L, IGF, IL-3, IL-6, IL-15, GM-CSF, SCF, and VEGF. In some embodiments, the culture medium further comprises one or more growth factors or cytokines selected from the group consisting of a BMP activator, a GSK3 inhibitor, a ROCK inhibitor, a TGFβ receptor / ALK inhibitor, and a NOTCH activator.

[0167] In certain embodiments, the isolated PD-1 engager CAR-T or CAR-NK cells are produced by in vitro differentiation of any one of iPSC, ESC, HIP, iPSC, ESC, HIP, iPSCO, ESCO, HIPO, iPSCO-, ESCO-, or HIPO- PD-1 engager cells bearing a CAR-T construct, which in other embodiments are used to treat cancer.

[0168] In another aspect of the invention, there is provided a method of treating a cancer patient comprising administering a composition comprising a therapeutically effective amount of any of the isolated PD-1 engager CAR-T CAR-NK cells described herein, hi some embodiments, the composition further comprises a therapeutically effective carrier.

[0169] In some embodiments, the administering step comprises intravenous administration, subcutaneous administration, intranodal administration, intratumoral administration, intrathecal administration, intrapleural administration, and intraperitoneal administration. In some aspects, the administering further comprises bolus administration or continuous perfusion.

[0170] In some embodiments, the cancer is a hematological cancer selected from the group consisting of leukemia, lymphoma, and myeloma. In various embodiments, the cancer is a solid tumor cancer or a liquid tumor cancer.

[0171] In another aspect, the present invention provides a method of making any one of the isolated PD-1 engager CAR-T cells, CAR-NK cells described herein. The method comprises in vitro differentiation of any one of the iPSC, ESC, HIP, iPSCO, ESCO, HIPO, iPSCO-, ESCO-, or HIPO- PD-1 engager cells of the present invention. The in vitro differentiation may comprise culturing the cells in a culture medium comprising one or more growth factors or cytokines selected from the group consisting of bFGF, EPO, Flt3L, IGF, IL-2, IL-3, IL-6, IL-7, IL-15, GM-CSF, SCF, and VEGF. In some embodiments, the culture medium further comprises one or more growth factors or cytokines selected from the group consisting of BMP activators, GSK3 inhibitors, ROCK inhibitors, TGFβ receptor / ALK inhibitors, and NOTCH activators.

[0172] In some embodiments, the in vitro differentiation comprises culturing iPSC, ESC, HIP, iPSCO, ESCO, HIPO, iPSCO-, ESCO-, or HIPO- PD-1 engager cells on feeder cells. In various embodiments, the in vitro differentiation comprises culturing under simulated microgravity. In some aspects, the culturing under simulated microgravity is for at least 72 hours.

[0173] In some aspects, provided herein are isolated and engineered hypoimmune cardiac cells (hypoimmunogenic cardiac cells), e.g., cardiomyocytes, differentiated from iPSC, ESC, HIP, iPSCO, ESCO, HIPO, iPSCO-, ESCO-, or HIPO- PD-1 engager cells.

[0174] It was previously believed that cardiomyocytes do not carry ABO blood group antigens. When ABO blood group B human embryonic stem cell lines were differentiated into cardiomyocyte-like cells, the B antigens were observed to disappear, suggesting that this loss of antigens may occur early in human embryonic development. See, e.g., Molne et al., Transplantation. 86(10):1407-13 (2008), which is incorporated by reference in its entirety. Other studies have also reported that when induced human pluripotent stem cells are differentiated into cardiomyocyte-like cells, the cells gradually lose ABO blood group A antigens. See, e.g., Saljo et al., Scientific Reports. 13072: 1-14 (2017). However, the present inventors have unexpectedly found that cardiomyocytes express ABO blood group antigens, which may cause rejection in incompatible recipients.

[0175] Thus, in some aspects, provided herein is a method of treating a patient suffering from a cardiac condition or disease, comprising administering a composition comprising a therapeutically effective amount of any one of a population of isolated PD-1 engager cardiac cells derived from iPSC, ESC, HIP, iPSCO, ESCO, HIPO, iPSCO-, ESCO-, or HIPO- PD-1 engager cells described herein. In some embodiments, the composition further comprises a therapeutically effective carrier.

[0176] In some embodiments, administration comprises implantation, intravenous injection, intra-arterial injection, intracoronary injection, intramuscular injection, intraperitoneal injection, intramyocardial injection, transendocardial injection, transepicardial injection, or infusion into the patient's cardiac tissue.

[0177] In some embodiments, the cardiac condition or disease is selected from the group consisting of pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, postpartum cardiomyopathy, inflammatory cardiomyopathy, other cardiomyopathies, myocarditis, myocardial ischemia-reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, end-stage heart disease, arteriosclerosis, ischemia, hypertension, restenosis, angina pectoris, rheumatic heart disease, arterial inflammation, or cardiovascular disease.

[0178] In some aspects, provided herein is a method of producing a population of cardiac cells from a population of PD-1 engager cells by in vitro differentiation, wherein endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity are inactivated and a PD-1 engager molecule is provided on the cell surface. The method includes (a) culturing the population of PD-1 engager cells in a medium comprising a GSK inhibitor; (b) culturing the population of PD-1 engager cells in a medium comprising a WNT antagonist to produce a precardiac cell population; and (c) culturing the precardiac cell population in a medium comprising insulin to produce a O-hypoimmune cardiomyocyte population. In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some embodiments, the concentration of the GSK inhibitor is about 2 μM to about 10 μM. In some embodiments, the WNT antagonist is IWR1, a derivative thereof, or a mutant thereof. In some aspects, the concentration of the WNT antagonist ranges from about 2 μM to about 10 μM.

[0179] In some aspects, provided herein are isolated and engineered PD-1 engager endothelial cells differentiated from iPSC, ESC, HIP, iPSCO, ESCO, HIPO, iPSCO-, ESCO-, or HIPO- PD-1 engager cells. In other aspects, the isolated and engineered O- or O-hypoimmune endothelial cells are selected from the group consisting of capillary endothelial cells, vascular endothelial cells, aortic endothelial cells, brain endothelial cells, and kidney endothelial cells.

[0180] In some aspects, provided herein are methods of treating a patient suffering from a vascular condition or disease, hi some embodiments, the methods comprise administering a composition comprising a therapeutically effective amount of a population of isolated and engineered PD-1 engager endothelial cells.

[0181] In some embodiments, the method comprises administering a composition comprising a therapeutically effective amount of any one of the populations of isolated and engineered PD-1 engager endothelial cells described herein. In some embodiments, the composition further comprises a therapeutically effective carrier. In some embodiments, the administration comprises implantation, intravenous injection, intra-arterial injection, intracoronary injection, intramuscular injection, intraperitoneal injection, intramyocardial injection, transendocardial injection, transepicardial injection, or infusion into the patient's cardiac tissue.

[0182] In some embodiments, the vascular condition or disease is selected from the group consisting of vascular injury, cardiovascular disease, vascular disease, ischemic disease, myocardial infarction, congestive heart failure, hypertension, ischemic tissue injury, limb ischemia, stroke, neuropathy, and cerebrovascular disease.

[0183] In some aspects, provided herein is a method of producing a population of PD-1 engager endothelial cells by in vitro differentiation from a population of iPSC, ESC, HIP, iPSCO, ESCO, HIPO, iPSCO-, ESCO-, or HIPO- PD-1 engager cells, in which endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been inactivated and a PD-1 engager molecule is provided on the cell surface, comprising: (a) culturing the cells in a first medium comprising a GSK inhibitor; (b) culturing the cell population in a second medium comprising VEGF and bFGF to produce a pre-endothelial cell population; and (c) culturing the pre-endothelial cell population in a third medium comprising a ROCK inhibitor and an ALK inhibitor to produce a hypoimmune endothelial cell population.

[0184] In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some embodiments, the concentration of the GSK inhibitor is about 1 μM to about 10 μM. In some embodiments, the ROCK inhibitor is Y-27632, a derivative thereof, or a variant thereof. In some embodiments, the concentration of the ROCK inhibitor is in the range of about 1 μM to about 20 μM. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. In some embodiments, the concentration of the ALK inhibitor is in the range of about 0.5 μM to about 10 μM.

[0185] In some embodiments, the first medium comprises 2 μM to about 10 μM CHIR-99021. In some embodiments, the second medium comprises 50 ng / ml VEGF and 10 ng / ml bFGF. In other embodiments, the second medium further comprises Y-27632 and SB-431542. In various embodiments, the third medium comprises 10 μM Y-27632 and 1 μM SB-431542. In certain embodiments, the third medium further comprises VEGF and bFGF. In particular, the first medium and / or the second medium does not comprise insulin.

[0186] In some aspects, provided herein are isolated and engineered PD-1 engager dopaminergic neurons (DNs) differentiated from PD-1 engager cells, in which endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator of T cell proliferation (CIITA) gene activity have been inactivated, PD-1 engager molecules are provided on the cell surface, and the neurons are blood type O and Rh-.

[0187] In some embodiments, the isolated PD-1 engager dopaminergic neuron is selected from the group consisting of neuronal stem cells, neuronal progenitor cells, immature dopaminergic neurons, and mature dopaminergic neurons.

[0188] In some aspects, provided herein is a method of treating a patient suffering from a neurodegenerative disease or condition. In some embodiments, the method comprises administering a composition comprising a therapeutically effective amount of any one of a population of isolated PD-1 engager dopaminergic neurons. In some embodiments, the composition further comprises a therapeutically effective carrier. In some embodiments, the population of isolated hypoimmune dopaminergic neurons is disposed on a biodegradable scaffold. In some embodiments, the administration comprises implantation or injection. In some embodiments, the neurodegenerative disease or condition is selected from the group consisting of Parkinson's disease, Huntington's disease, and multiple sclerosis.

[0189] In some aspects, provided herein are methods of producing a population of PD-1 engager dopaminergic neurons by in vitro differentiation from a population of PD-1 engager cells, wherein endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity are inactivated, a PD-1 engager molecule is provided on the cell surface, and the blood group is O and Rh-. In some embodiments, the method comprises: (a) culturing a population of cells in a first culture medium containing one or more factors selected from the group consisting of sonic hedgehog (SHH), BDNF, EGF, bFGF, FGF8, WNT1, retinoic acid, a GSK3β inhibitor, an ALK inhibitor, and a ROCK inhibitor to produce a population of immature dopaminergic neurons; and (b) culturing the population of immature dopaminergic neurons in a second culture medium different from the first culture medium to produce a population of dopaminergic neurons.

[0190] In some embodiments, the GSKβ inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some embodiments, the concentration of the GSKβ inhibitor ranges from about 2 μM to about 10 μM. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. In some embodiments, the concentration of the ALK inhibitor ranges from about 1 μM to about 10 μM. In some embodiments, the first culture medium and / or the second culture medium does not contain animal serum.

[0191] In some embodiments, the method also includes isolating a population of hypoimmune dopaminergic neurons from non-dopaminergic neurons, hi some embodiments, the method further includes cryopreserving the isolated population of hypoimmune dopaminergic neurons.

[0192] In some aspects, provided herein are isolated PD-1 engager hypoimmune islet cells differentiated from PD-1 engager cells, in which endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity are inactivated, a PD-1 engager molecule is presented on the cell surface, and blood type is O and Rh-.

[0193] In some embodiments, the isolated PD-1 engager islet cells are selected from the group consisting of islet progenitor cells, immature islet cells, and mature islet cells.

[0194] In some aspects, provided herein is a method of treating a patient suffering from diabetes. The method comprises administering a composition comprising a therapeutically effective amount of any one of the populations of isolated PD-1 engager islet cells described herein. In some embodiments, the composition further comprises a therapeutically effective carrier. In some embodiments, the population of isolated hypoimmune islet cells is disposed on a biodegradable scaffold. In some aspects, the administration comprises implantation or injection.

[0195] In some aspects, provided herein are methods for producing a population of PD-1 engager islet cells from a population of HIPO- cells by in vitro differentiation, wherein endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity are inactivated, a PD-1 engager molecule is provided on the cell surface, and the HIPO- cells are blood type O and Rh-. The method includes (a) culturing a population of PD-1 engager cells in a first medium containing one or more factors selected from the group consisting of insulin-like growth factor (IGF), transforming growth factor (TGF), fibroblast growth factor (EGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), sonic hedgehog (SHH), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGFβ) superfamily, bone morphogenetic protein-2 (BMP2), bone morphogenetic protein-7 (BMP7), a GSK3β inhibitor, an ALK inhibitor, a BMP type 1 receptor inhibitor, and retinoic acid, to produce a population of immature islet cells; and (b) culturing the immature islet cell population in a second medium different from the first medium to produce a population of hypoimmune islet cells.

[0196] In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some embodiments, the concentration of the GSK inhibitor is about 2 μM to about 10 μM. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. In some embodiments, the concentration of the ALK inhibitor ranges from about 1 μM to about 10 μM. In some embodiments, the first culture medium and / or the second culture medium does not contain animal serum.

[0197] In some embodiments, the method also includes separating the population of PD-1 engager islet cells from non-islet cells, hi some embodiments, the method further includes cryopreserving the isolated population of hypoimmune islet cells.

[0198] In some aspects, provided herein are isolated and engineered PD-1 engager retinal pigment epithelial (RPE) cells differentiated from PD-1 engager cells, in which endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator of T cell (CIITA) gene activity are inactivated, a PD-1 engager molecule is presented on the cell surface, and the blood type is O and Rh-.

[0199] In some embodiments, the isolated PD-1 engager RPE cells are selected from the group consisting of RPE progenitor cells, immature RPE cells, mature RPE cells, and functional RPE cells.

[0200] In some aspects, provided herein is a method of treating a patient suffering from an ocular condition. The method comprises administering a composition comprising a therapeutically effective amount of any one of the populations of isolated PD-1 engager RPE cells described herein. In some embodiments, the composition further comprises a therapeutically effective carrier. In some embodiments, the population of isolated hypoimmune RPE cells is disposed on a biodegradable scaffold. In some embodiments, the administration comprises implantation or injection into the patient's retina. In some embodiments, the ocular condition is selected from the group consisting of wet macular degeneration, dry macular degeneration, juvenile macular degeneration, Leber's congenital amaurosis, retinitis pigmentosa, and retinal detachment.

[0201] In some aspects, provided herein is a method for producing a population of PD-1 engager retinal pigment epithelial (RPE) cells by in vitro differentiation from a population of PD-1 engager cells, in which endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity are inactivated and PD-1 engager molecules are provided on the cell surface. The method includes: (a) culturing the population of PD-1 engager cells in a first medium containing any one factor selected from the group consisting of activin A, bFGF, BMP4 / 7, DKK1, IGF1, Noggin, BMP inhibitor, ALK inhibitor, ROCK inhibitor, and VEGFR inhibitor to produce a population of pre-RPE cells; and (b) culturing the population of pre-RPE cells in a second medium different from the first medium to produce a population of hypoimmune RPE cells.

[0202] In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. In some embodiments, the concentration of the ALK inhibitor ranges from about 2 μM to about 10 μM. In some embodiments, the ROCK inhibitor is Y-27632, a derivative thereof, or a variant thereof. In some embodiments, the concentration of the ROCK inhibitor ranges from about 1 μM to about 10 μM.

[0203] In some embodiments, the first medium and / or the second medium are free of animal serum.

[0204] In some embodiments, the method further comprises separating the population of PD-1 engager RPE cells from non-RPE cells, hi some embodiments, the method further comprises cryopreserving the isolated population of hypoimmune RPE cells.

[0205] E. Transplantation of HIPD-1 Engager Cells As will be appreciated by those skilled in the art, HI PD-1 engager cells are implanted using techniques known in the art. Generally, the HI PD-1 engager cells of the present invention are implanted intravenously or by injection into a specific location in a patient. When implanted into a specific location, the cells may be suspended in a gel matrix to prevent dispersion while they settle.

[0206] In order that the invention described herein may be more fully understood, the following examples are set forth below, it being understood that these examples are for illustrative purposes and are not to be construed as limiting the invention in any manner whatsoever. EXAMPLES

[0207] Example 1: PD-1 engagers expressed on engineered cells inhibit killing by NK cells B2M- / - CIITA- / - iPSC-derived endothelial cells (DKO iECs) were rapidly killed by IL-2-activated NK cells. PD-1 engager was expressed in B2M- / - CIITA- / - iECs using lentivirus. Bioluminescence immunoassay (BLI) killing assays showed that killing of B2M- / - CIITA- / - PD-1 engager transgenic iECs (DKO-PD-1 engager) was significantly attenuated (Figure 1).

[0208] Example 2: PD-1 engagers expressed on engineered cells inhibit killing by CD8 T cells PBMCs from HLA-A2-negative allogeneic healthy donors were cocultured with HLA-A2-positive wild-type (wt) iPSC-derived endothelial cells (iECs) for 14 days. Cells were harvested and CD3+CD8+ T cells were sorted by flow cytometry. Wt iECs were killed by T cells. PD-1 engager was expressed in wt iECs using lentivirus. BLI killing assays showed that killing of wt PD-1 engager transgenic iECs was significantly attenuated (Figure 2).

[0209] Example 3: PD-1 engagers expressed on artificial human pancreatic beta cells inhibit killing of CD8 T cells PBMCs from an allogeneic healthy donor negative for HLA-A2 were cocultured with HLA-A2 positive wild type (wt) iPS cell-derived endothelial cells (iECs) for 14 days. Cells were harvested and CD3+CD8+ T cells were sorted by flow cytometry. HLA-A2 positive human pancreatic beta cells were cultured on in vitro impedance assay E-plates. In the top panel, beta cells are unmodified. In the bottom panel, beta cells express PD-1 engager. PD-1 engager was expressed using lentiviral transduction. For detection by flow cytometry, PD-1 engager sequence (SEQ ID NO: 24) with CD8a signal peptide (SEQ ID NO: 25) and myc tag (SEQ ID NO: 26) was used. Unmodified beta cells were killed by T cells. PD-1 engager transgenic beta cells survived. (Figures 3A and 3B)

[0210] Example 4: PD-1 engager cells bind recombinant PD-1 B2M- / - CIITA- / - PD-1 engager transgenic iECs (DKO-PD-1 engager) were cultured with recombinant human PD-1 and secondary antibody. Flow cytometry histograms show that recombinant human PD-1 specifically bound to DKO-PD-1 engager cells (Figure 4).

[0211] Example 5: Generation of TIM3 engager cells Preparation of lentivirus. The TIM3 engager (SEQ ID NO:27) was expressed under the enhanced EF1α promoter, containing the CD8a signal peptide (SEQ ID NO:28), myc tag (SEQ ID NO:26), anti-TIM3 antibody heavy chain variable region (SEQ ID NO:29), (GGGG)3 linker (SEQ ID NO:30), anti-TIM3 antibody light chain variable region (SEQ ID NO:31), CD8a hinge (SEQ ID NO:3), and platelet-derived growth factor receptor (PDGFR) transmembrane domain (SEQ ID NO:4). The RFP-blastidine dual fusion marker was expressed under the RSV promoter.

[0212] The expression lentivector was co-transfected with lentivirus packaging plasmid (catalog number: HT-pack, Gentarget, San Diego) into a lentivirus production cell line (catalog number: TLV-C, Gentarget). Following the virus production protocol of GenTarget, the lentivirus was packaged in DMEM medium with 10% serum. The lentivirus was then concentrated to obtain the desired virus titer. The virus titer was measured by ELISA P24 assay according to the kit production.

[0213] Lentiviral transduction of TIM3 engager. Human induced pluripotent stem cell (iPSC)-derived endothelial cells (iEC) with B2M and CIITA double knockout (iEC DKO) were transduced with the above lentivirus on a precoated 12-well plate. 5 × 10 4 The cells were plated at a concentration of 0.01 mg / mL and incubated overnight at 37°C with 5% CO2. The next day, cells were incubated overnight at 37°C with lentiviral particles carrying a TIM3 engager transgene under an enhanced EF1α promoter (Gentarget, custom-made). The lentivirus further contained an RFP selection marker and was used at a multiplicity of infection of 4. Polybrene (8 μg / m, Millipore) was added to the medium and the plates were centrifuged at 800 g for 30 min and incubated overnight. Cell populations were sorted using the RFP tag on a FACSAria (BD Biosciences).

[0214] Flow cytometry analysis demonstrated expression of the TIM3 engager protein (potein) in transduced DKO-induced embryonic stem cells (iECs). The TIM3 engager transgene linked to a red fluorescent protein (RFP) tag was quantified by flow cytometry. Histograms show RFP signal in untransduced iEC DKO (left peak) and transduced iEC DKO+TIM3-E (right peak), confirming expression of the TIM3 engager. (Figure 5.)

[0215] Example 6: TIM3 engagement protects cells from killing by immune cells Analysis of TIM3 surface expression on primary human peripheral blood NK cells and macrophages by flow cytometry demonstrated that TIM3 engagement conferred protection to NK and macrophage cells.

[0216] FIG. 6A shows that 96.6% of NK cells were TIM3 positive, and FIG. 3B shows that 67.2% of macrophages were TIM3 positive (FIG. 6B).

[0217] BLI killing assay with NK cells was examined for 2 hours. Firefly luciferase-expressing iEC DKO or firefly luciferase-expressing iEC DKO+TIM3 engager (TIM3-E) were plated and allowed to attach for 16 hours. Human peripheral blood NK cells were then added to the wells and cultured for 2 hours. Percentage of BLI signal after culture is shown. The BLI signal of iEC DKO was completely lost (Figure 7A). The BLI signal of iEC DKO+TIM3-E was stable for 2 hours (Figure 7B).

[0218] Figure 8A and Figure 8B are 2-hour BLI killing assays with macrophages. Human iEC DKO or iEC DKO+TIM3-E expressing firefly luciferase were plated and allowed to attach for 16 hours. Human peripheral blood macrophages were then added to the wells and cultured for 2 hours. The percentage of BLI signal after culture is shown. The BLI signal of iEC DKO disappeared completely (Figure 8A). The BLI signal of iEC DKO+TIM3-E remained stable for 2 hours (Figure 8B).

[0219] Figures 9A and 9B are 24-hour BLI killing assays by NK cells. Human iEC DKO or iEC DKO+TIM3-E expressing firefly luciferase were plated and allowed to attach for 16 hours. Human peripheral blood NK cells were then added to the wells and cultured for 24 hours. The percentage of BLI signal after 24 hours of NK culture is shown. The BLI signal of iEC DKO was completely lost (Figure 9A). The BLI signal of iEC DKO+TIM3-E was stable for 24 hours (Figure 9B).

[0220] Figures 10A and 10B are 24-hour BLI killing assays by macrophages. Human iEC DKO and iEC DKO+TIM3-E expressing firefly luciferase were plated and allowed to attach for 16 hours. Human peripheral blood macrophages were then added to the wells and cultured for 24 hours. The percentage of BLI signal after 24 hours of macrophage culture is shown. The BLI signal of iEC DKO was completely lost (Figure 10A). The BLI signal of iEC DKO+TIM3-E was stable for 24 hours and even increased slightly (Figure 10B).

[0221] Example 7: Generation of LILRB3 engager cells Preparation of lentivirus. The LILRB3 engager (SEQ ID NO: 32) was expressed under the enhanced EF1α promoter, which contains the CD8a signal peptide (SEQ ID NO: 28), myc tag (SEQ ID NO: 26), anti-LILRB3 antibody heavy chain variable region (SEQ ID NO: 33), (GGGG)3 linker (SEQ ID NO: 30), anti-LILRB3 antibody light chain variable region (SEQ ID NO: 34), CD8a hinge (SEQ ID NO: 3), and platelet-derived growth factor receptor (PDGFR) transmembrane domain (SEQ ID NO: 4). The RFP-blastidine dual fusion marker was expressed under the RSV promoter.

[0222] The expression lentivector was co-transfected with lentivirus packaging plasmid (catalog number: HT-pack, Gentarget, San Diego) into a lentivirus production cell line (catalog number: TLV-C, Gentarget). Following the virus production protocol of GenTarget, the lentivirus was packaged in DMEM medium with 10% serum. The lentivirus was then concentrated to obtain the desired virus titer. The virus titer was measured by ELISA P24 assay according to the kit production.

[0223] Lentiviral transduction of LILRB3 engagers. Human induced pluripotent stem cell (iPSC)-derived endothelial cells (iEC) with B2M and CIITA double knockout (iEC DKO) were transduced with the above lentivirus on a precoated 12-well plate. 5 × 10 4 The cells were plated at a concentration of 0.01 mg / mL and incubated overnight at 37°C with 5% CO2. The next day, they were incubated overnight at 37°C with lentiviral particles carrying the LILRB3 engager transgene under an enhanced EF1α promoter (Gentarget). The lentivirus further contained an RFP selection marker and was used at a multiplicity of infection of 4. Polybrene (8 μg / m, Millipore) was added to the medium and the plates were centrifuged at 800 g for 30 min and incubated overnight. The cell populations were sorted using the RFP tag on a FACSAria (BD Biosciences).

[0224] Flow cytometry analysis showed that LILRB3 engager protein (potein) was expressed in transduced DKO-induced embryonic stem cells (iECs). LILRB3 engager transgene linked to a red fluorescent protein (RFP) tag was quantified by flow cytometry. Histograms show RFP signals in untransduced iEC DKO (left peak) and transduced iEC DKO+LILRB3-E (right peak), confirming expression of LILRB3 engager (Figure 11).

[0225] Example 8: Engagement of LILRB3 protects cells from killing by immune cells Flow cytometric analysis of LILRB3 surface expression on primary human peripheral blood NK cells and macrophages demonstrated that LILRB3 was expressed on a subset of NK cells and the majority of macrophages.

[0226] FIG. 12A shows that 17.8% of NK cells were LILRB3 positive, and FIG. 12B shows that 95.36% of macrophages were LILRB3 positive.

[0227] BLI killing assay with NK cells was examined for 2 hours. Firefly luciferase-expressing iEC DKO or firefly luciferase-expressing iEC DKO+LILRB3-E were plated and allowed to attach for 16 hours. Human peripheral blood NK cells were then added to the wells and cultured for 2 hours. Percentage of BLI signal after culture is shown. The BLI signal of iEC DKO was completely abolished (Figure 13A). The BLI signal of iEC DKO+LILRB3-E showed less significant decline at 2 hours (Figure 13B).

[0228] Figures 14A and 14B are 2-hour BLI killing assays with macrophages. Human iEC DKO or iEC DKO+LILRB3-E expressing firefly luciferase were plated and allowed to attach for 16 hours. Human peripheral blood macrophages were then added to the wells and cultured for 2 hours. The percentage of BLI signal after culture is shown. The BLI signal of iEC DKO was completely lost (Figure 14A). The BLI signal of iEC DKO+LILRB3-E showed less significant decline at 2 hours (Figure 4B).

[0229] Figures 15A and 15B are 24-hour BLI killing assays by NK cells. Human iEC DKO or iEC DKO+LILRB3-E expressing firefly luciferase were plated and allowed to attach for 16 hours. Human peripheral blood NK cells were then added to the wells and cultured for 24 hours. The percentage of BLI signal after 24 hours of NK culture is shown. The BLI signal of iEC DKO was completely lost (Figure 15A). The BLI signal of iEC DKO+LILRB3-E showed less significant decline at 24 hours (Figure 15B).

[0230] Figures 16A and 16B are 24-hour BLI killing assays with macrophages. Human iEC DKO and iEC DKO+LILRB3-E expressing firefly luciferase were plated and allowed to attach for 16 hours. Human peripheral blood macrophages were then added to the wells and cultured for 24 hours. The percentage of BLI signal after 24 hours of macrophage culture is shown. The BLI signal of iEC DKO was completely abolished (Figure 16A). The BLI signal of iEC DKO+LILRB3-E showed less significant decline and even a slight increase at 24 hours (Figure 16B).

[0231] Example 9: Generation of LILRB1 engager cells Preparation of lentivirus. The LILRB1 engager (SEQ ID NO: 35) was expressed under the enhanced EF1α promoter, and the RFP-blastidine double fusion marker was expressed under the RSV promoter, which contains the CD8a signal peptide (SEQ ID NO: 28), myc tag (SEQ ID NO: 26), anti-LILRB1 antibody heavy chain variable region (SEQ ID NO: 36), (GGGG)3 linker (SEQ ID NO: 30), anti-LILRB1 antibody light chain variable region (SEQ ID NO: 37), CD8a hinge (SEQ ID NO: 3), and platelet-derived growth factor receptor (PDGFR) transmembrane domain (SEQ ID NO: 4). The RFP-blastidine double fusion marker was expressed under the RSV promoter.

[0232] The expression lentivector was co-transfected with lentivirus packaging plasmid (catalog number: HT-pack, Gentarget, San Diego) into a lentivirus production cell line (catalog number: TLV-C, Gentarget). Following the virus production protocol of GenTarget, the lentivirus was packaged in DMEM medium with 10% serum. The lentivirus was then concentrated to obtain the desired virus titer. The virus titer was measured by ELISA P24 assay according to the kit production.

[0233] Lentiviral transduction of LILRB1 engager. Human induced pluripotent stem cell (iPSC)-derived endothelial cells (iEC) with B2M and CIITA double knockout (iEC DKO) were transduced with the above lentivirus on a precoated 12-well plate. 5 × 10 4The cells were plated at a concentration of 0.01 mg / mL and incubated overnight at 37°C with 5% CO2. The next day, they were incubated overnight at 37°C with lentiviral particles carrying the LILRB1 engager transgene under an enhanced EF1α promoter (Gentarget). The lentivirus further contained an RFP selection marker and was used at a multiplicity of infection of 4. Polybrene (8 μg / m, Millipore) was added to the medium and the plates were centrifuged at 800 g for 30 min and incubated overnight. The cell populations were sorted using the RFP tag on a FACSAria (BD Biosciences).

[0234] Flow cytometry analysis showed that LILRB1 engager protein (potein) was expressed in transduced DKO-induced embryonic stem cells (iECs). LILRB1 engager transgene linked to a red fluorescent protein (RFP) tag was quantified by flow cytometry. Histograms show RFP signals of untransduced iEC DKO (left peak) and transduced iEC DKO+LILRB1-E (right peak), confirming the expression of LILRB1 engager (Figure 17).

[0235] Example 10: Engagement of LILRB1 protects cells from killing by immune cells Flow cytometric analysis of LILRB1 surface expression on primary human peripheral blood NK cells and macrophages demonstrated that LILRB1 was expressed on most NK cells and the majority of macrophages.

[0236] FIG. 18A shows that 75.8% of NK cells are LILRB1 positive, and FIG. 18B shows that 99.96% of macrophages are LILRB1 positive.

[0237] BLI killing assay with NK cells was examined for 2 hours. Firefly luciferase-expressing iEC DKO or firefly luciferase-expressing iEC DKO+LILRB1-E were plated and allowed to attach for 16 hours. Human peripheral blood NK cells were then added to the wells and cultured for 2 hours. Percentage of BLI signal after culture is shown. The BLI signal of iEC DKO was completely lost (Figure 19A). The BLI signal of iEC DKO+LILRB1-E was stable for 2 hours (Figure 19B).

[0238] Figures 20A and 20B are 2-hour BLI killing assays with macrophages. Human iEC DKO or iEC DKO+LILRB1-E expressing firefly luciferase were plated and allowed to attach for 16 hours. Human peripheral blood macrophages were then added to the wells and cultured for 2 hours. The percentage of BLI signal after culture is shown. The BLI signal of iEC DKO disappeared completely (Figure 20A). The BLI signal of iEC DKO+LILRB1-E was stable for 2 hours (Figure 20B).

[0239] Figures 21A and 21B are 24-hour BLI killing assays by NK cells. Human iEC DKO or iEC DKO+LILRB1-E expressing firefly luciferase were plated and allowed to attach for 16 hours. Human peripheral blood NK cells were then added to the wells and cultured for 24 hours. The percentage of BLI signal after 24 hours of NK culture is shown. The BLI signal of iEC DKO was completely lost (Figure 21A). The BLI signal of iEC DKO+LILRB1-E was stable for 24 hours (Figure 21B).

[0240] Figures 22A and 22B are 24-hour BLI killing assays by macrophages. Human iEC DKO and iEC DKO+LILRB1-E expressing firefly luciferase were plated and allowed to attach for 16 hours. Human peripheral blood macrophages were then added to the wells and cultured for 24 hours. The percentage of BLI signal after 24 hours of macrophage culture is shown. The BLI signal of iEC DKO was completely lost (Figure 22A). The BLI signal of iEC DKO+LILRB1-E was stable for 24 hours and even increased slightly (Figure 22B).

[0241] Example 11: Immune checkpoint engagers are stably expressed after differentiation PD-1 engager transgene expression is maintained during differentiation. iPSC DKO were transduced with lentiviral particles to express PD-1 engager (iPSC DKO+PD-1-E). Flow cytometry shows robust expression (Figure 23A). Transgene expression remained stable when iPSC DKO+PD-1-E were differentiated into iEC DKO + PD-1-E (Figure 23B).

[0242] Expression of the TIM3 engager transgene is maintained during differentiation. iPSC DKO were transduced with lentiviral particles to express the TIM3 engager (iPSC DKO+TIM3-E). Flow cytometry shows robust expression (Figure 24A). When iPSC DKO + TIM3-E were differentiated into iEC DKO + TIM3-E iEC, transgene expression remained stable (Figure 24B).

[0243] Expression of the LILRB3 engager transgene is maintained during differentiation. iPSC DKO were transduced with lentiviral particles to express the LILRB3 engager (iPSC DKO+LILRB3-E). Flow cytometry shows that robust expression was obtained (Figure 25A). When iPSC DKO + LILRB3-E were differentiated into iEC DKO + LILRB3-E, transgene expression remained stable (Figure 25B).

[0244] Expression of the LILRB1 engager transgene is maintained during differentiation. iPSC DKO were transduced with lentiviral particles to express the LILRB1 engager (iPSC DKO+LILRB1-E). Flow cytometry shows that robust expression was obtained (Figure 26A). When iPSC DKO + LILRB1-E were differentiated into iEC DKO + LILRB1-E, transgene expression remained stable (Figure 26B).

[0245] Example 12: Threshold Levels of Immune Checkpoint Engagers Required for Protection Figures 27A and 27B show the threshold of TIM3-E expression required to protect cells from killing by NK cells. To assess whether there is a functional threshold of TIM3-E expression to exert a protective effect against killing by NK cells, iEC DKO were transduced with lentiviral particles carrying the TIM3-E transgene. After 3 days, the pool of transduced iECs was sorted into three populations based on the expression of TIM3-E. The three populations are shown as iEC DKO + TIM3-E high, medium, and low (Figure 27A). Each of these populations was cultured with NK cells for 2 hours, followed by a BLI killing assay (Figure 27B). All target cells were luciferase positive. The percentage of BLI signal after 2 hours of NK incubation is shown. The BLI signal of iEC DKO was completely abolished. The BLI signal of iEC DKO + TIM3-E high and medium remained stable, but the BLI signal of iEC DKO + TIM3-E low also dropped to background. These results indicate that the functional threshold of TIM3-E lies between low and intermediate expression levels.

[0246] Figures 28A and 28B show the threshold of LILRB1-E expression required to protect cells from killing by NK cells. To assess whether there is a functional threshold of LILRB1-E expression to exert a protective effect against killing by NK cells, iEC DKO were transduced with lentiviral particles carrying the LILRB1-E transgene. After 3 days, the pool of transduced iECs was sorted into three populations based on the expression of LILRB1-E. The three populations are shown as iEC DKO+LILRB1-E high, medium, and low (Figure 28A). Each of these populations was cultured with NK cells for 2 hours, followed by a BLI killing assay (Figure 28B). All target cells were luciferase positive. The percentage of BLI signal after 2 hours of NK incubation is shown. The BLI signal of iEC DKO was completely abolished. The BLI signals of iEC DKO+LILRB1-E high and medium remained stable, but the BLI signal of iEC DKO+LILRB1-E low also dropped to background. These results indicate that the functional threshold of LILRB1-E lies between low and moderate expression levels.

[0247] method: NK cell culture. Human primary NK cells were purchased from Stemcell Technologies (Vancouver, Canada, 70036) and cultured in RPMI-1640 supplemented with 10% FCS hi and 1% penicillin / streptomycin prior to assay.

[0248] Culture and transduction of human iPSCs expressing firefly luciferase. Human wt or B2M- / - CIITA- / - iPSCs were cultured in Essential 8 Flex medium (Thermo Fisher Scientific) on 10 cm dishes coated with diluted feeder-free Matrigel (hESC compatible, BD Biosciences, San Jose, CA). Medium was changed every 24 h and cells were passaged with Versene (Gibco) at a ratio of 1:6. For luciferase transduction, 1 × 10 5 iPSCs were plated in one 6-well and cultured overnight at 37°C, 5% CO2. The next day, the medium was changed and one vial of Fluc lentiviral particles expressing the luciferase II gene under a re-modified EF1α promoter (Gen Target, San Diego, CA) was added to 1.5 ml medium. After 36 hours, 1 ml of cell medium was added. After 24 hours, the medium was completely changed. After 2 days, D-luciferin (Promega, Madison, WI) was added to confirm luciferase expression. The signal was quantified in p / s / cm2 / sr.

[0249] Differentiation of iPSC-derived endothelial cells (iECs). Fluc+ wt or B2M- / - CIITA- / - iECs were differentiated from FLuc+ B2M- / - CIITA- / - iPSCs as follows. The differentiation protocol was started when iPSCs reached 60% confluency. The medium was changed to RPMI-1640 containing 2% B-27 (without insulin) (both Thermo Fisher Scientific brands, Gibco) and 5 μM CHIR-99021 (Selleckchem, Munich, Germany). On day 2, the medium was changed to reduced medium containing RPMI-1640 (Gibco) containing 2% B-27 (without insulin) and 2 μM CHIR-99021 (Selleckchem). From days 4 to 7 of culture, cells were exposed to RPMI-1640 EC medium, RPMI-1640 plus 2% B-27 (without insulin), 50 ng / ml human vascular endothelial growth factor (VEGF; R&D Systems, Minneapolis, MN), 10 ng / ml human basic fibroblast growth factor (FGFb; R&D Systems), 10 μM Y-27632 (Sigma-Aldrich), and 1 μM SB 431542 (Sigma-Aldrich, St. Louis, MO). Clusters of endothelial cells were visible by day 7. Cells were maintained in Endothelial Cell Basal Medium 2 (PromoCell, Heidelberg, Germany) supplemented with supplements, 10% FCS hi (Gibco), 1% penicillin / streptomycin, 25 ng / ml VEGF, 2 ng / ml FGFb, 10 μM Y-27632 (Sigma-Aldrich), and 1 μM SB 431542 (Sigma-Aldrich). The differentiation protocol was terminated after 14 days. Undifferentiated cells were detached during the differentiation process. Cells were passaged at a ratio of 1:3 every 3–4 days using TrypLE Express (Gibco).

[0250] Differentiation of macrophages from PBMCs. PBMCs were isolated from fresh blood by Ficoll separation and resuspended in RPMI-1640 containing 10% FCS HI and 1% penicillin-streptomycin (all from Gibco). Cells were cultured at 1 × 10 per milliliter in 24-well plates. 6 The plates were plated at a concentration of 10 ng ml -1 Human M-CSF (PeproTech) was added to the medium. The medium was changed every other day. From day 6 onwards, 1 μg ml -1 of human IL-2 (PeproTech) was added to the medium for 24 hours.

[0251] Lentiviral transduction of PD-1 engager. 5 × 10 iECs were cultured in a pre-coated 12-well plate. 4 The cells were plated at a concentration of 0.01 mg / mL and incubated overnight at 37°C with 5% CO2. The next day, cells were incubated overnight at 37°C with lentiviral particles carrying a PD-1 engager transgene under an enhanced EF1α promoter (Gentarget, custom-made). The lentivirus further contained an RFP selection marker and was used at a multiplicity of infection of 4. Polybrene (8 μg / m, Millipore) was added to the medium and the plates were centrifuged at 800 g for 30 min and incubated overnight. Cell populations were sorted using the RFP tag on a FACSAria (BD Biosciences).

[0252] iEC BLI NK cell killing assay. Fluc+ B2M- / -CIITA- / -(DKO) iECs and B2M- / -CIITA- / -(DKO)PD-1 engager transgenic iECs were counted and cultured at 1 × 10 per 96-well. 3All target cells were plated at a concentration of 10:1 cells. All target cells were then mixed with primary human NK cells at an E:T ratio of 10:1. All NK cells were pre-incubated with human IL-2 (Life Technologies) at a concentration of 1 μg / mL for 72 h. After 4 h, luciferase expression was detected by adding D-luciferin (Promega) in a BLI killing assay. As controls, target cells were left untreated or treated with 2% Triton X-100 added to the cell-specific medium. Signal was quantified using an Ami HT (Spectral Instruments Imaging) in p / s / cm2 / sr to measure the relative BLI intensity compared to untreated target cells.

[0253] Ex vivo T cell priming. Blood was collected from HLA-A2 negative donors and PBMCs were obtained after Ficoll separation. PBMCs were cultured at 5 × 10 5 1 x 10 HLA-A2 positive wt iEC cells and 1 x 10 6 PBMCs were primed by co-culture on gelatin-coated flasks. A 1:1 mixture of endothelial cell medium and PBMC medium was replaced every 3 days. After 14 days, cells in suspension were harvested and sorted using APC mouse anti-human CD3 antibody (clone SP34-2, catalog no. 557597, BD Biosciences) and its isotype control APC mouse IgG1 antibody (clone MOPC-21, catalog no. 550854, BD Biosciences), as well as BV421 mouse anti-human CD8 antibody (clone SK1, catalog no. 344748, Biolegend) and its isotype control mouse IgG1 antibody (clone MOPC-21, catalog no. 400157, Biolegend). CD3+CD8+ cells were sorted using a FACS Aria flow cytometer (BD Biosciences) and used for CD8 T cell killing assays.

[0254] iEC BLI CD8 T cell killing assay. Fluc+ wt iECs and wt PD-1 engager transgenic iECs were counted and plated at 1 × 10 per 96-well. 3 All target cells were then mixed with ex vivo primed CD8 T cells at an E:T ratio of 10:1. After 4 hours, luciferase expression was detected by adding D-luciferin (Promega) in a BLI killing assay. As controls, target cells were left untreated or treated with 2% Triton X-100 in cell-specific medium. Signal was quantified in p / s / cm2 / sr using an Ami HT (Spectral Instruments Imaging).

[0255] Human pancreatic beta cells. Human iPSC-derived pancreatic beta cells were purchased from Takara Bio Inc. (ChiPSC22, Cat. No. Y10106) and cultured with Cellartis hiPS Beta Cell Media Kit (Takara Bio Inc., Cat. No. Y10108). Cells were plated in 12-well plates according to the manufacturer's protocol. Some cells were transduced with PD-1 engagerentiviral particles (GenTarget).

[0256] Then, islet cells (pancreatic beta cells) expressing and not expressing PD-1 engagers were plated on the XCelligence platform. The cells attached to the plastic dishes but also formed clusters, and the cell index continued to increase over time. Despite the clustered growth pattern, the assay detected target cell killing with high sensitivity. The islet cells exhibited HLA A2 type. Therefore, primed CD3+CD8+ T cells generated as above were used as effector cells. Unedited islet cells were killed by primed T cells with typical T cell killing kinetics. In contrast, islet beta cells expressing PD-1 engagers were protected from killing by primed T cells.

[0257] To demonstrate binding of DKO PD-1 engagers to PD-1 on iECs, recombinant human PD-1 protein (Fc Chimera Active, ab221398, Abcam, 0.1ug / mL) was used with a mouse anti-human IgG Fc secondary antibody (3D3cc, Novus Biologicals, allophycocyanin, 1:200). Flow cytometry was performed on an LSRII (BD Biosciences).

[0258] Example array: [ka] [ka] [ka] [ka] [ka]

[0259] All publications and patent documents disclosed or cited herein are incorporated by reference in their entirety. The foregoing description has been presented for purposes of illustration and description only. It is not intended to limit the invention to the precise form disclosed. It is intended that the scope of the invention be defined by the appended claims.

Claims

1. An immune checkpoint engager (ICE) cell, wherein an engager molecule is expressed on the cell surface, said engager molecule engages with an immune checkpoint molecule on an immune cell, and said engager molecule is expressed at least at a level that protects said ICE cell from being killed by said immune cell.

2. The ICE cell of claim 1 , wherein the engager molecule is a protein that does not engage with SIRPα on the immune cell.

3. The ICE cell of claim 2, wherein the engager molecule engages with PD-1.

4. The ICE cell of claim 2, wherein the protein comprises an immunoglobulin domain that binds to PD-1.

5. The ICE cell of claim 4 , wherein the immunoglobulin domain is an antibody VH domain.

6. The ICE cell of claim 5, wherein the antibody VH domain has at least 90% sequence identity with amino acids 146-264 of SEQ ID NO:

1.

7. The ICE cell of claim 6, wherein the antibody VH domain comprises the sequence of amino acids 146 to 264 of SEQ ID NO:

1.

8. The ICE cell of claim 4 , wherein the immunoglobulin domain is an antibody VL domain.

9. The ICE cell of claim 8, wherein the antibody VL domain has at least 90% sequence identity with amino acids 21-130 of SEQ ID NO:

1.

10. The ICE cell of claim 9, wherein the antibody VH domain comprises the sequence of amino acids 21 to 130 of SEQ ID NO:

1.

11. The ICE cell of any one of claims 2 to 10, wherein the protein is a fusion protein.

12. The ICE cell of claim 11, wherein the fusion protein has at least 90% sequence identity with the IL-2 signal peptide of SEQ ID NO:

2.

13. The ICE cell of claim 12, wherein the fusion protein comprises the IL-2 signal peptide of SEQ ID NO:

2.

14. The ICE cell of claim 11, wherein the fusion protein has at least 90% sequence identity with the CD8a hinge peptide of sequence ID number 3.

15. The ICE cell of claim 14, wherein the fusion protein comprises a CD8a hinge peptide of sequence ID number 3.

16. The ICE cell of claim 11, wherein the fusion protein has at least 90% sequence identity with the PDGFR transmembrane domain (TMD) of sequence ID number 4.

17. The ICE cell of claim 16, wherein the fusion protein comprises the PDGFR TMD of sequence ID number 4.

18. The ICE cell of claim 11, wherein the engager molecule is a protein having at least 90% sequence identity to SEQ ID NO:

1.

19. The ICE cell of claim 18, wherein the engager molecule has sequence identity to sequence ID number 1.

20. The ICE cell of any one of claims 1 to 11, wherein the engager molecule comprises an antibody Fab or a single chain variable fragment (scFv) that binds to PD-1.

21. The Fab or scFv binds to PD-1 with an affinity as measured by a dissociation constant (Kd), and the Kd is about 10 －７ ~10 －１３ 21. The ICE cell of claim 20,

22. The ICE cell of any one of claims 1 to 11, wherein the engager molecule comprises one or more antibody complementarity determining regions (CDRs) that bind to PD-1.

23. The ICE cell of claim 22, wherein one or more CDRs have at least 90% sequence identity with any one of sequence ID numbers 18-23.

24. The PD1 engager cell of any one of claims 1 to 15, wherein the engager molecule is a fusion protein comprising a heterologous transmembrane domain (TMD).

25. The ICE cell of claim 24, wherein the TMD comprises an α-helix, multiple α-helices, or a rolled-up β-sheet.

26. The heterologous TMDs are CD85f, CD349, CD284, CD261, CD172b, CD277, CD186, CD156c, CD304, CD254, CD263, CD267, CD337, CD170, CD283, CD133, CD327, CD205, CD232, CD282, CD16b, CD85i, CD85a, CD85c, CD275, CD108, CD358, CD335, CD218b, CD355, CD336, CD160, CD25, CD4, CD8a, CD235a, CD233, CD230, CD90, CD74, CD3d, CD340, CD236, CD61, CD18, CD54, CD29, CD1a, CD5, CD220, CD2, CD66e, CD51, CD141, CD115, CD42b, CD221, CD271, CD55, CD243, CD98, CD10, CD41, CD14, CD45, CD228, CD16a, CD49e, CD126, CD63, CD48, CD7, CD140b, CD3g, CD117, CD28, CD8b, CD37, CD11b, CD107a, CD331, CD222, CD20, CD79a, CD32, CD143, CD324, CD42c, CD107b, CD56, CD102, CD49d, CD66a, CD142, CD59, CD62L, CD121a, CD122, CD13, CD155, CD119, CD19, CD116, CD46, CD1e, CD1d, CD227, CD44, CD62P, CD104, CD43, CD140a, CD31, CD152, CD326, CD62E, CD36, CD127, CD49b, CD105, CD35, CD223, CD138, CD325, CD58, CD106, CD53, CD120a, CD224, CD21, CD33, CD22, CD120b, CD11a, CD11c, CD363, CD73, CD88, CD204, CD332, CD9, CD203a, CD334, CD333, CD206, CD49f, CD238, CD252, CD89, CD124, CD181, CD182, CD24, CD95, CD40, CD49c, CD159a, CD159c, CD314, CD27, CD123, CD26, CD82, CD121b, CD34, CD38, CD30, CD1b, CD1c, CD154, CD6, CD52, CD132, CD32CD66b, CD171, CD191, CD197, CD185, CD131, CD50, CD70, CD153, CD144, CD80, CD362, CD68, CD361, CD147, CD309, CD135, CD292, CD103, CD130, CD42d, CD66d, CD66c, CD96, CD110, CD79b, CD200, CD192, CD231, CD86, CD212, CD118, CD146, CD134, CD158a, CD158b1, CD158b2, CD158e, CD158k, CD158j, CD158i, CD178, CD295, CD151, CD97, CD183, CD39, CD239, CD193, CD194, CD195, CD196, CDw198, CDw199, CD296, CD298, CD49a, CD322, CD85g, CD184, CD172a, CD156a, CD339, CD156b, CD213a1, CD129, CD83, CD125, CD241, CD269, CD202b, CD87, CD164, CD136, CD137, CD249, CD69, CD91, CDw210b, CD167a, CD300c, CD157, CD317, CD148, CD161, CD215, CD150, CD11d, CD218a, CD210, CD166, CD162, CD213a2, CD242, CD158g, CD158h, CD279, CD111, CD281, CD226, CD234, CD167b, CD300e, CD276, CD305, CD300g, CD300d, CD109, CD272, CD163, CD302, CD158f1, CD85h, CD85d, CD177, CD158z, CD158f2, CD85j, CD300f, CD92, CD351, CD112, CD100, CD270, CD101, CD297, CD316, CD352, CD217, CD307b, CD307a, CD307c, CD307d, CD307e, CD114, CD180, CD158d, CD273, CD290, CD244, CD169, CD299, CD318, CD360, CD229, CD248, CD354, CD320, CD93, CD319, CD113, CD163b, CD289, CD288, CD329, CD274, CD353, CD172g, CD315, CD280, CD26425. The ICE cell of claim 24, selected from the group consisting of CD300a, CD312, CD84, CD344, CD350, CD246, CD201, CD338, CD208, CD257, CD328, CD286, CD357, CD294, CD321, CD265, CD278, ITGA7, ITGA8, ITGA9, ITGA10, ITGA11, CD51, CD41, CD29, CD18, CD61, CD104, CD47 (SEQ ID NO: 5), CD64 (SEQ ID NO: 6), and PDGFR (SEQ ID NO: 4).

27. The ICE cell of claim 24, wherein the TMD comprises a sequence having at least 90% sequence identity to sequence ID number 5 or sequence ID number 6.

28. The ICE cell of claim 24, wherein the TMD comprises the sequence of sequence ID number 5 or sequence ID number 6.

29. The ICE cell of claim 24, wherein the TMD is derived from a seven-transmembrane protein (7TM) or an immunoglobulin cell surface protein.

30. 30. The ICE cell of any one of claims 1 to 29, wherein the engager molecule has no intracellular domain (ICD).

31. 30. The ICE cell of any one of claims 1 to 29, wherein the engager molecule has an intracellular domain derived from CD16, CD32, CD64, CD8, CD3, CD28, or CD137.

32. 32. The ICE cell of any one of claims 2 or 4-31, wherein the engager molecule has one or more linker or hinge regions connecting the ECD, TMD, or ICD sequences.

33. 33. The ICE cell of any one of claims 2 or 4 to 32, wherein the protein is an antibody, a receptor, a ligand, or an adhesion protein.

34. The ICE cell according to any one of claims 1 to 33, further comprising reduced or absent expression of HLA-I or HLA-II.

35. 35. The ICE cell of any one of claims 1 to 34, wherein the cell is ABO blood group type O.

36. 36. The ICE cell of any one of claims 1 to 35, wherein the cell is Rh factor negative (Rh-).

37. 37. The ICE cell of any one of claims 1 to 36, wherein the cell has reduced or absent ABO blood group antigens selected from the group consisting of A1, A2 and B.

38. The ICE cell according to any one of claims 1 to 37, wherein the cell has reduced or absent expression of a Rh protein antigen selected from the group consisting of Rh C antigen, Rh E antigen, Kell K antigen (KEL), Duffy (FY) Fya antigen, Duffy Fy3 antigen, Kidd (JK) Jkb antigen, MNS antigen U, and MNS antigen S.

39. ICE cells according to any one of claims 1 to 38, wherein the cells are hypoimmunogenic (HI) cells having reduced function of endogenous major histocompatibility complex class I (HLA-I) compared to an unmodified parent cell and reduced function of endogenous major histocompatibility complex class II (HLA-II) compared to an unmodified parent cell.

40. The engager cells are selected from the group consisting of HLA-I human leukocyte antigen, HLA-II human leukocyte antigen, CD64, CD47, CD38, CCR5, CXCR4, NLRC5, CIITA, B2M, HLA-A, HLA-B, HLA-C, HLA-E, HLA-G, PD-L1, CTLA-4-Ig, CD47, CI-inhibitor, IL-35, RFX-5, RFXAP, RFXANK, NFY-A, NFY-B, NFY-C, IR F-1, OX40, GITR, 4-1BB, CD28, B7-1, B7-2, ICOS, CD27, HVEM, SLAM, CD226, PD1, CTL4, LAG3, TIGIT, TIM3, CD160, BTL A, CD244, CD30, TLT, VISTA, B7-H3, PD-L2, LFA-1, CD2, CD58, ICAM-3, TCRA, TCRB, FOXP3, HELIOS, ST2, PCSK9, APOC3, 40. The ICE cell of any one of claims 1-39, wherein expression of one or more of CD200, FASLG, CLC21, MFGE8, SERPIN B9, TGFβ, CD73, CD39, LAG3, IL1R2, ACKR2, TNFRSF22, TNFRSF23, TNFRS10, DAD1, PVR, or IFNγR1 d39 is modulated compared to a parent cell, and the engager cell is ABO blood group O or Rh factor negative (Rh-).

41. 41. The ICE cell of any one of claims 1 to 40, wherein the expression of an antibody Fc receptor on the cell surface is enhanced, and the Fc receptor helps to evade antibody-dependent cellular cytotoxicity (ADCC) or complement-mediated cytotoxicity (CDC).

42. 42. The ICE cell of claim 41, wherein the Fc receptor is CD16, CD32, CD64, or truncated CD64.

43. The ICE cell of any one of claims 1 to 42, wherein the cell is pluripotent.

44. 44. The ICE cell of claim 43, wherein the cell is a hypoimmune pluripotent (HIP) cell.

45. 45. The ICE cell of claim 44, wherein the cell is a hypoimmune pluripotent cell having ABO blood group O (HIPO).

46. 46. ​​The ICE cell of claim 45, wherein the cell is a hypoimmune pluripotent cell that is Rh factor negative (HIP-).

47. 47. The ICE cell of claim 46, wherein the cell is a hypoimmune pluripotent cell that has ABO blood group type O and is Rh factor negative (HIPO-).

48. 48. The ICE cell of any one of claims 43 to 47, wherein the cell is a pluripotent (PSC) cell, an induced PSC (iPSC) cell, or an embryonic stem cell (ESC).

49. ICE cells according to any one of claims 1 to 42, wherein the engager cells are of a specific tissue type.

50. 50. The ICE cell of claim 49, wherein the cell is a chimeric antigen receptor (CAR) cell, a T cell, a NK cell, an endothelial cell, a dopaminergic neuron, a cardiac cell, a pancreatic islet beta cell, a thyroid epithelial cell, a parathyroid cell, or a retinal pigment epithelial cell.

51. 50. The ICE cell of claim 49, wherein the cell is a CAR-T cell, a CAR-NK cell, a TCR T cell, or a TCR NK cell.

52. 52. The ICE cell of any one of claims 1 to 42 or 49 to 51, wherein the engager cell is differentiated from a pluripotent cell.

53. A pharmaceutical composition comprising the ICE cells of any one of claims 49 to 52 and a pharma- ceutically acceptable carrier.

54. A pharmaceutical product comprising the ICE cells according to any one of claims 49 to 52 and a pharma- ceutically acceptable carrier.

55. A method for treating a disease in a subject, the method comprising transplanting an ICE cell according to any one of claims 49 to 52 into the subject.

56. 56. The method of claim 55, wherein the disease is type 1 diabetes, a cardiovascular disease, a neurological disease, an endocrine disease, cancer, blindness, or a vascular disease.

57. Use of an ICE cell according to any one of claims 49 to 52 for the preparation of a pharmaceutical composition for treating a disease in a subject.

58. Use of an ICE cell according to any one of claims 49 to 52 for treating a disease in a subject.

59. 59. The use of any one of claims 57 or 58, wherein the disease is type 1 diabetes, a cardiovascular disease, a neurological disease, an endocrine disease, cancer, blindness, or a vascular disease.

60. 53. The ICE cell of any one of claims 1 to 52, further comprising a signal regulatory protein alpha (SIRPα) engager molecule on the cell surface that engages with a SIRPα protein on the immune cell, wherein engagement of the SIRPα protects the engager cell from being killed by the immune cell, and wherein the SIRPα engager cell surface molecule lacks a functional intracellular domain.

61. 61. The ICE cell of claim 60, wherein the engager molecule is a protein.

62. 62. The ICE cell of claim 61 , wherein the protein is a fusion protein.

63. 63. The ICE cell of claim 62, wherein the fusion protein comprises the CD47 extracellular domain (ECD).

64. 64. The ICE cell of any one of claims 60 to 63, wherein the engager molecule comprises an immunoglobulin superfamily domain.

65. 65. The ICE cell of any one of claims 60-64, wherein the SIRPα engager molecule comprises one or more antibody complementarity determining regions (CDRs) that bind to SIRPα.

66. The ICE cell of claim 65, wherein the CDR comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 18-23.

67. The ICE cell of claim 66, wherein the CDR comprises any one of the sequences set forth in SEQ ID NOs: 18-23.

68. The ICE cell of claim 2, wherein the engager molecule engages with one or more immune cell receptors selected from the group consisting of PD-1, TIM3, LILRB3, and LILRB1.

69. 70. The ICE cell of any one of claims 1 to 53 or 68, further comprising a safety switch.

70. 70. The ICE cell of claim 69, wherein the safety switch is a suicide gene.

71. The ICE cell of claim 70, wherein the suicide gene is (a) a thymidine kinase gene of herpes simplex virus (HSV-tk) with a ganciclovir trigger, (b) an Escherichia coli cytosine deaminase gene (EC-CD) with a 5-fluorocytosine (5-FC) trigger, or (c) an inducible iCasp9 protein with an AP1903 trigger.

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