Inhibition of immune cells by immune checkpoint conjugates

Abstract

The present invention provides cells (Immune Checkpoint Junction 'ICE' cells) having enhanced Immune Checkpoint Junction function. In some embodiments, the ICE cells have a PD-1 conjugation function (PD-1 conjugant cells). The ICE cells reduce adaptability and innate immunity when transplanted into a subject as compared to parental cells with corresponding unmodified immune checkpoint conjugation function. In some embodiments, the PD-1 zygote cells are low immune pluripotent (HIP) cells. In further embodiments, the HIP cell is type O blood (HIPO), rhesus monkey factor (Rh) negative (HIP-), or both type O and type Rh (HIPO-). In other embodiments, the PD-1 zygote cells have been derived or differentiated from HIP, HIP-, or HIPO-cells. In other embodiments, the ICE cell expresses a ligand that binds to a suppressive immune cell receptor selected from the group consisting of a LILRB1 zygote, a LILRB3 zygote, and a TIM3 zygote. In other embodiments, the ICE cell is a PD-1 zygote cell that also expresses a ligand that binds to another inhibitory immune cell receptor selected from the group consisting of CD47, truncated CD47, a LILRB1 zygote, a LILRB3 zygote, and a TIM3 zygote.

Description

[0001] [I. Cross-reference to related applications]

[0002] Pursuant to 35 U.S.C. §119(e), this application claims priority to U.S. Provisional Application No. 63 / 330,539, filed on April 13, 2022, and U.S. Provisional Application No. 63 / 448,081, filed on February 24, 2023, and is incorporated herein by reference in its entirety.

[0003] II. Field of the Invention

[0004] The present invention provides cells with enhanced immune checkpoint engagement (ICE) function. In some embodiments, the cell has a programmed cell death protein 1 (PD-1) engagement function (PD-1 engaged daughter cell) that resists an immune response when transplanted into a subject, compared to a parent cell with an unmodified PD-1 engagement function. In some embodiments, the immune checkpoint engaged daughter cell is a low immune cell. In other embodiments, the cell is a pluripotent cell. In other embodiments, the cell is an embryonic stem cell (ES) or an induced pluripotent stem cell (iPSC). In other embodiments, the immune checkpoint engaged daughter cell is a differentiated somatic cell. In other embodiments, the immune checkpoint engaged daughter cell is a low immune pluripotent cell (HIP cell). In a further embodiment, the HIP cell is blood type O (HIPO), rhesus factor (Rh) negative (HIP - ) or O and Rh - Type (HIPO - In other embodiments, the immune checkpoint engager cells are composed of HIP, HIP - or HIPO - Cells are derived or differentiated. In other embodiments, the immune checkpoint engager cells contain antibody Fc receptors to protect them from antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In other embodiments, ICE cells express a ligand that binds to an inhibitory immune cell receptor, which is selected from the group consisting of LILRB1 engagers, LILRB3 engagers, and TIM3 engagers. In other embodiments, ICE cells are PD-1 engager cells that additionally express a ligand that binds to another inhibitory immune cell receptor, which is selected from the group consisting of CD47, truncated CD47, SIRPα engagers, LILRB1 engagers, LILRB3 engagers, and TIM3 engagers.

[0005] [III. Background of the invention]

[0006] Natural Killer cells, or NK cells, are cytotoxic lymphocytes that are critical to the innate immune system. The role of NK cells is similar to that of cytotoxic T cells in the adaptive immune response of vertebrates. NK cells provide a rapid response to virus-infected cells and cancer cells. Typically, NK cells are activated by downregulating the major histocompatibility complex (MHC) of target cells, as this is the primary inhibitory NK cell signal. NK cell activation triggers cytokine release, leading to lysis or apoptosis. NK cells are unique because they can recognize stressed cells, as they upregulate other stimulatory NK cell signals, and do not require prior exposure to certain cellular epitopes. This allows them to react very rapidly. They can also respond rapidly to cells loaded with antibodies, as binding of free antibody Fc is a strong stimulatory NK cell signal. NK cells do not require primary activation to kill cells that lack MHC class I "self" markers, except for exposure to some cytokines such as IL-2 or IL-15. This role is particularly important because harmful cells with downregulated or absent MHC I markers cannot be detected and destroyed by other immune cells such as T lymphocytes.

[0007] NK cells are large granular lymphocytes that differentiate from common lymphocyte progenitor cells that give rise to B and T lymphocytes. They differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus before entering the circulation.

[0008] Programmed cell death protein 1, also known as PD-1 and CD279 (cluster of differentiation 279), is a protein present on the surface of T, B and some NK cells that regulates the immune system's response to the body's cells by downregulating the immune system and promoting self-tolerance by inhibiting the inflammatory activity of T cells. This prevents autoimmune diseases, but it can also prevent the immune system from killing cancer cells.

[0009] PD-1 often exhibits high and sustained expression levels during persistent antigen encounters, which can occur in the setting of chronic infection and cancer. PD-1 is an immune checkpoint that prevents autoimmunity through two mechanisms. First, it promotes apoptosis (programmed cell death) of antigen-specific T cells in the lymph nodes. Second, it reduces apoptosis of regulatory T cells (anti-inflammatory, suppressive T cells).

[0010] The PD-1 / PD-L1 (PD-1 ligand) pathway has important value in cancer immunotherapy and has become an important immune checkpoint. Antibody inhibitors of the PD-1 / PD-L1 interaction have shown clinical efficacy in many tumors.

[0011] The PD-1 protein is encoded by the PDCD1 gene in humans. PD-1 is a cell surface receptor that belongs to the immunoglobulin superfamily and is expressed on T cells and pre-B cells. PD-1 binds two ligands, PD-L1 and PD-L2.

[0012] Programmed death ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a protein that is encoded by the CD274 gene in humans. PD-L1 is a 40 kDa type 1 transmembrane protein that is hypothesized to play an important role in suppressing the adaptive arm of the immune system during specific events such as pregnancy, tissue allografts, autoimmune diseases, and other disease states such as hepatitis. Normally, the adaptive immune system responds to antigens associated with immune system activation via exogenous or endogenous danger signals. In turn, antigen-specific CD8 + T cells and / or CD4 + The clonal expansion of the helper cells is propagated. The binding of PD-L1 to the inhibitory checkpoint molecule PD-1 transmits an inhibitory signal based on its interaction with the phosphatase (SHP-1 or SHP-2) through the immunoreceptor tyrosine-based switch motif (ITSM). This reduces the proliferation of antigen-specific T cells in the lymph nodes and at the same time reduces the apoptosis of regulatory T cells (anti-inflammatory, suppressive T cells). This is further mediated by the lower regulation of the gene Bcl-2.

[0013] Programmed cell death ligand 1 (also known as PD-L2, B7-DC) is a protein that in humans is encoded by the PDCD1LG2 gene. PDCD1LG2 is also designated CD273 (cluster of differentiation 273). PDCD1LG2 is an immune checkpoint receptor ligand that plays a role in the negative regulation of the adaptive immune response. PD-L2 is one of the two known ligands for programmed cell death protein 1 (PD-1).

[0014] SIRPα is a member of the signal regulatory protein (SIRP) family and 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. Phosphotyrosine residues recruit SH2 domain-containing tyrosine phosphatases (PTPs) and serve as their substrates. SIRPα is involved in signal transduction mediated by various growth factor receptors.

[0015] CD47 is a ligand for SIRPα. CD47 is a "self-marker" protein that is widely overexpressed in tumor types. It is emerging as a new and effective macrophage immune checkpoint for cancer immunotherapy. CD47 in tumor cells sends a "don't eat me" signal that inhibits macrophage phagocytosis. This brings opportunities and challenges to CD47 inhibitors as monotherapy and combination therapy for blood cancers and solid tumors. Some of these drugs are currently in clinical trials.

[0016] Cytoplasmic signaling of CD47 can be mediated by its intracellular domain (ICD), although few proteins that directly interact with the CD47 cytoplasmic tail have been identified to date (Lamy L., J Biol Chem. 278: 23915-21 (2003); Wu AL, Mol. Cell. 4: 619-25 (1999)). Ubiquilin-1, one such binding partner, binds to Gβγ, thereby linking heterotrimeric G proteins to CD47 (N'Diaye EN, J Cell Biol. 163: 1157-65 (2003)). In this case, Ubiquilin-1 inhibits chemotaxis signaled by the Gi-coupled receptor CXCR4 (Sick E., Glia. 59: 308-19 (2011)). The above contents are incorporated herein by reference in their entirety.

[0017] Human primary NK cells were shown to express SIRPα and bind to CD47 upon stimulation, which reduces their killing effect on cells expressing CD47 (see PCT / US20 / 39220, incorporated herein by reference in its entirety).

[0018] Autologous induced pluripotent stem cells (iPSCs) theoretically constitute an unlimited cell source for patient-specific cell-based organ repair strategies. However, their generation presents technical and manufacturing challenges and is a lengthy process that conceptually prevents any acute therapeutic modality. From a production perspective, allogeneic iPSC-based therapies or embryonic stem cell-based therapies are easier and can produce well-screened, standardized, high-quality cell products. Since pluripotent stem cells can differentiate into any cell type in the three germ layers, the potential applications of stem cell therapies are wide-ranging. Differentiation can be performed ex vivo or in vivo by transplanting progenitor cells that continue to differentiate and mature in the organ environment of the implantation site. Ex vivo differentiation allows researchers or clinicians to closely monitor the procedure and ensure that the appropriate cell population is generated before transplantation. However, due to their allogeneic origin, such cell products may be rejected.

[0019] [IV. Summary of the invention]

[0020] The present invention provides cells with enhanced immune checkpoint engagement function (immune checkpoint engagement "ICE" cells). In some embodiments, ICE cells have PD-1 engagement function (PD-1 engagement daughter cells). Compared with parent cells with corresponding unmodified immune checkpoint engagement function, ICE cells reduce adaptive and innate immunity when transplanted into a subject. In some embodiments, PD-1 engagement daughter cells are low immune pluripotency (HIP) cells. In further embodiments, HIP cells are blood type O (HIPO), rhesus factor (Rh) negative (HIP - ) or O and Rh - Type (HIPO - In other embodiments, the PD-1 engaged daughter cells have been derived from HIP, HIP - or HIPO - Derived from or differentiated from cells.

[0021] Therefore, the present invention provides an immune checkpoint engagement (ICE) cell, which includes an engagement submolecule expressed on the cell surface, wherein the engagement submolecule engages with the immune checkpoint molecule on the immune cell, wherein the engagement submolecule is expressed at least at a level that protects the ICE cell from being killed by the immune cell. In some aspects, the engagement submolecule is a protein that does not engage with SIRPα on the immune cell. In other aspects, the engagement submolecule engages with PD-1.

[0022] In other aspects of the invention, the protein comprises an immunoglobulin domain that engages PD-1. In other aspects, the immunoglobulin domain is an antibody VH domain. In other aspects, the antibody VH domain comprises at least 90% sequence identity with amino acid numbers 146-264 of SEQ ID NO: 1. In a preferred aspect, the antibody VH domain comprises the sequence of amino acid numbers 146-264 of SEQ ID NO: 1. In other aspects, the immunoglobulin domain is an antibody VL domain. In other aspects, the antibody VL domain comprises at least 90% sequence identity with amino acid numbers 21-130 of SEQ ID NO: 1. In a preferred aspect, the antibody VH domain comprises the sequence of amino acid numbers 21-130 of SEQ ID NO: 1.

[0023] In certain aspects of the invention, the protein expressed by the ICE cell is a fusion protein. In other aspects, the fusion protein comprises at least 90% sequence identity with the IL-2 signal peptide of SEQ ID NO: 2. In preferred aspects, the fusion protein comprises the IL-2 signal peptide of SEQ ID NO: 2. In other aspects, the fusion protein comprises at least 90% sequence identity with the CD8a hinge peptide of SEQ ID NO: 3. In preferred aspects, the fusion protein comprises the CD8a hinge peptide of SEQ ID NO: 3. In certain aspects, the fusion protein comprises at least 90% sequence identity with the PDGFR transmembrane domain (TMD) of SEQ ID NO: 4. In preferred aspects, the fusion protein comprises the PDGFR TMD of SEQ ID NO: 4.

[0024] In certain aspects of the invention, the engager molecule is a protein having at least 90% sequence identity to SEQ ID NO: 1. In preferred aspects, the engager molecule has sequence identity to SEQ ID NO:1.

[0025] In certain aspects of the invention, the engager molecule comprises an antibody Fab or single chain variable fragment (scFV) that binds to PD-1. In other aspects, the Fab or scFV binds to PD-1 with an affinity measured by its dissociation constant (Kd), wherein Kd is between about 10 -7 and 10 -13 In other aspects, the engager molecule comprises one or more antibody complementarity determining regions (CDRs) that bind to PD-1. In other aspects, the one or more CDRs have at least 90% sequence identity with any one of SEQ ID NOs: 18-23. In other aspects, the one or more CDRs have the sequence of any one of SEQ ID NOs: 18-23.

[0026] In certain aspects of the invention, the engager molecule is a fusion protein comprising a heterologous transmembrane domain (TMD). In other aspects, the TMD comprises a single alpha helix, multiple alpha helices or a rolled beta sheet. In other aspects, the heterologous TMD is selected from 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, CD23 3. CD230, CD90, CD74, CD3d, CD340, CD236, CD61, CD18, CD54, CD29, CD1a, CD5, CD220, CD2, CD66e, CD51, CD141, CD115, CD42b, CD221, CD271, CD 55. 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, CD15 5. CD119, CD19, CD116, CD46, CD1e, CD1d, CD227, CD44, CD62P, CD104, CD43, CD140a, CD31, CD152, CD326, CD62E, CD36, CD127, CD49b, CD105, CD 35. CD223, CD138, CD325, CD58, CD106, CD53, CD120a, CD224, CD21, CD33, CD22, CD120b, CD11a, CD11c, CD363, CD73, CD88, CD204, CD332, CD9, C D203a, 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、In other aspects, 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).

[0027] In other aspects, the TMD comprises a sequence having at least 90% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6. In preferred aspects, the TMD comprises the sequence of SEQ ID NO: 5 or SEQ ID NO: 6. In other aspects, the TMD is from a 7 transmembrane protein (7TM) or an immunoglobulin cell surface protein.

[0028] In certain aspects of the invention, the engager molecule does not have an intracellular domain (ICD). In other aspects, the engager molecule has an intracellular domain from CD16, CD32, CD64, CD8, CD3, CD28 or CD137.

[0029] In certain aspects of the invention, the adaptor molecules have one or more linker or hinge regions connecting the ECD, TMD or ICD sequences.

[0030] In other aspects, the protein is an antibody, receptor, ligand, or adhesion protein.

[0031] In certain aspects of the invention, the ICE cells as described herein further comprise reduced or eliminated HLA-I or HLA-II expression. In other aspects, the cells are ABO blood type O. In other aspects, the cells are Rhesus factor negative (Rh - ). In other aspects, the cells have reduced or eliminated ABO blood group antigens selected from the group consisting of A1, A2 and B. In other aspects, the cells have reduced or eliminated Rh protein antigen expression 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.

[0032] In certain aspects, the ICE cells of the invention are low immunogenicity (HI) cells comprising: reduced function of endogenous major histocompatibility complex class I (HLA-I) compared to an unmodified parental cell, and reduced function of endogenous major histocompatibility complex class II (HLA-II) compared to an unmodified parental cell.

[0033] In other aspects, the conjugate cells comprise 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, 4-1B B, 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 B9, TGFβ, CD73, CD39, LAG3, IL1R2, ACKR2, TNFRSF22, TNFRSF23, TNFRS10, DAD1, PVR or IFNγR1 d39, wherein the zygote cells are ABO blood type O or rhesus factor negative (Rh - ).

[0034] In other aspects, the ICE cells of the present invention also include elevated expression of antibody Fc receptors on the cell surface, wherein the Fc receptors help to escape antibody-dependent cellular cytotoxicity (ADCC) or complement-mediated cytotoxicity (CDC). In other aspects, the Fc receptor is CD16, CD32, CD64 or truncated CD64.

[0035] In certain aspects, the ICE cells of the present invention are pluripotent. In other aspects, the cells are hypoimmune pluripotent (HIP) cells. In other aspects, the cells are hypoimmune pluripotent cells with ABO blood type O (HIPO). In other aspects, the cells are hypoimmune pluripotent cells that are Rh factor negative (HIP - In other aspects, the cell is of ABO blood type O and Rh negative (HIPO- ) of a low immune pluripotent cell. In other aspects, the cell is a pluripotent (PSC) cell, an induced PSC (iPSC) or an embryonic stem cell (ESC).

[0036] In certain aspects of the invention, the ICE cells as described herein are specific tissue types. In other aspects, the cell is a chimeric antigen receptor (CAR) cell, a T cell, a NK cell, an endothelial cell, a dopaminergic neuron, a cardiomyocyte, a pancreatic beta cell, a macrophage, a thyroid epithelial cell, a parathyroid cell, or a retinal pigment epithelial cell. In other aspects, the ICE cells of the present invention are CAR-T cells, CAR-NK cells, TCR T cells, or TCR NK cells. In other aspects, the conjugated daughter cells are differentiated from pluripotent cells.

[0037] The present invention provides a pharmaceutical composition comprising the ICE cell as described herein and a pharmaceutically acceptable carrier.

[0038] The present invention provides a medicament comprising the ICE cell as described herein and a pharmaceutically acceptable carrier.

[0039] The present invention provides a method of treating a disease in a subject, comprising transplanting an ICE cell as described herein into the subject. In other aspects, the disease is type 1 diabetes, heart disease, a neurological disease, an endocrine disease, cancer, blindness or a vascular disease.

[0040] The present invention provides the use of ICE cells as described herein for preparing a pharmaceutical composition for treating a disease in a subject. In other aspects, the disease is type 1 diabetes, heart disease, a neurological disease, an endocrine disease, cancer, blindness or a vascular disease.

[0041] In certain aspects of the invention, an ICE cell as described herein further includes a signal regulatory protein alpha (SIRPα) engager molecule on the cell surface, which engages with a SIRPα protein on an immune cell, wherein SIRPα engagement prevents the engager cell from being killed by the immune cell, wherein the SIRPα engager cell surface molecule lacks a functional intracellular domain. In other aspects, the engager molecule is a protein. In other aspects, the protein is a fusion protein. In other aspects, the fusion protein comprises a CD47 extracellular domain (ECD). In other aspects, the engager molecule comprises an immunoglobulin superfamily domain. In other aspects, the SIRPα engager molecule comprises one or more antibody complementary determining regions (CDRs) that bind to SIRPα. In other aspects, the CDR comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 18-23. In other aspects, the CDR comprises any one of SEQ ID NOs: 18-23.

[0042] The present invention provides an ICE cell as disclosed herein, wherein the engager molecule binds to one or more immune cell receptors selected from the group consisting of PD-1, TIM3, LILRB3 and LILRB1.

[0043] The present 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 to target the cell for destruction.

[0044]

V.Brief description of the attached drawings

[0045] Figure 1 Figure 1 shows an iPSC-derived endothelial cell (iEC) bioluminescence imaging (BLI) NK cell killing assay. Natural killer cells (NK cells) were stimulated with IL-2 for 72 h. NK cells were then added to FLuc + B2M - / - CIITA - / - (HLA class I and II double knockout, DKO) and B2M that additionally expresses the PD-1 agonist - / - CIITA - / - iECs (DKO PD-1 engager iECs). The decrease in BLI signal was assessed after 4 hours (mean ± SD, 3 independent experiments per group, two-way Student's t-test). PD-1 engager molecules provide iECs with protection from NK cell killing.

[0046] Figure 2 iEC BLI CD8 T cell killing assay is shown. Peripheral blood mononuclear cells (PBMCs) were primed with wt iEC in vitro and sorted with CD3 + CD8 + T cells were used as effector cells in this assay. + CD8 + T cells added to FLuc + WT and WT PD-1 agonist iECs. BLI signal decrease was assessed 4 h later (mean ± SD, 3 independent experiments per group, two-way Student’s t-test). PD-1 agonist molecules protect iECs from CD3 + CD8 + T cell killing.

[0047] Figure 3A and 3B In vitro impedance killing assay is shown. The target cells are HLA-A2 positive human pancreatic β cells. Figure 3A and 3B In the upper row, the beta cells are not modified. Figure 3A and 3B In the bottom row, β cells express the PD-1 agonist. 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 this assay. + CD8 + When T cells were added to the assay, unmodified β cells were killed, but human pancreatic β cells expressing the PD-1 engager survived (mean ± SD, 3 independent experiments per time point).

[0048] Figure 4 Binding of recombinant PD-1 to DKO PD-1 engager iECs is shown. Flow cytometry shows specific binding of PD-1 to the secondary antibody. The secondary antibody alone was used as a negative control.

[0049] Figure 5 :Produce endothelial cells (iEC) derived from human induced pluripotent stem cells (iPSC), which express firefly luciferase and further include B2M and CIITA double knockouts (DKO).Use lentiviral particles to transduce the exemplary TIM3 conjugate protein connected to red fluorescent protein (RFP) tags into these cells.By flow cytometry, they are analyzed.Histograms show the RFP signals of the iEC DKO (left peak) of non-transduced and the iEC DKO+TIM3-E (right peak) of transduction, thus confirming their expression of TIM3 conjugates.

[0050] Fig. 6A and 6B : Flow cytometry analysis of TIM3 surface expression on primary human peripheral blood NK cells and macrophages. 96.6% of NK cells were positive for TIM3 ( Fig. 6A ), 67.2% of macrophages were positive for TIM3 ( Figure 6B ).

[0051] Fig. 7A and 7B: BLI killing assay within 2 hours with NK cells. Human induced pluripotent stem cell (iPSC)-derived endothelial cells (iEC) expressing firefly luciferase and further comprising B2M and CIITA double knockout (DKO) were generated. These cells were seeded with iEC DKO+TIM3-E and adhered for 16 hours. Human peripheral blood NK cells were then added to the wells and incubated for 2 hours. The percentage of BLI signal after incubation is shown. The BLI signal of iEC DKO completely disappeared ( Fig. 7A The BLI signal of iECDKO+TIM3-E remained stable within 2 hours ( Figure 7B ).

[0052] Fig. 8A and 8B : BLI killing assay within 2 hours using macrophages. Human iEC DKO and iEC DKO+TIM3-E expressing firefly luciferase were plated and allowed to adhere for 16 hours. Then, human peripheral blood macrophages were added to the wells and incubated for 2 hours. The percentage of BLI signal after incubation is shown. The BLI signal of iEC DKO completely disappeared ( Fig. 8A The BLI signal of iEC DKO+TIM3-E remained stable within 2 hours ( Figure 8B ).

[0053] Fig. 9A and 9B : BLI killing assay with NK cells within 24 hours. Human iEC DKO and iEC DKO+TIM3-E expressing firefly luciferase were plated and adhered for 16 hours. Then, human peripheral blood NK cells were added to the wells and incubated for 24 hours. The percentage of BLI signal after 24 hours of NK incubation is shown. The BLI signal of iEC DKO completely disappeared ( Fig. 9A The BLI signal of iEC DKO+TIM3-E remained stable over 24 hours ( Fig. 9B ).

[0054] Fig. 10A and 10B : BLI killing assay with macrophages within 24 hours. Human iEC DKO and iEC DKO+TIM3-E expressing firefly luciferase were plated and allowed to adhere for 16 hours. Then, human peripheral blood macrophages were added to the wells and incubated for 24 hours. The percentage of BLI signal after 24 hours of macrophage incubation is shown. The BLI signal of iEC DKO completely disappeared ( Fig. 10A The BLI signal of iEC DKO+TIM3-E remained stable within 24 hours and even increased ( Fig. 10B ).

[0055] Fig.11:Generate human induced pluripotent stem cell (iPSC) derived endothelial cells (iEC), which express firefly luciferase and further contain B2M and CIITA double knockout (DKO). Use lentiviral particles to transduce the exemplary LILRB3 conjugate protein connected to the red fluorescent protein (RFP) tag into these cells. They are analyzed by flow cytometry. The histogram shows the RFP signal of non-transduced iEC DKO (left peak) and transduced iEC DKO+LILRB3-E (right peak), thereby confirming their expression of LILRB3 conjugate.

[0056] Fig. 12A and 12B : Flow cytometry analysis of LILRB3 surface expression on primary human peripheral blood NK cells and macrophages. 17.8% of NK cells were positive for LILRB3 ( Fig. 12A ), 95.3% of macrophages were positive for LILRB3 ( Fig. 12B ).

[0057] Fig.13A and 13B : BLI killing assay within 2 hours with NK cells. Human induced pluripotent stem cell (iPSC)-derived endothelial cells (iEC) expressing firefly luciferase and further comprising a B2M and CIITA double knockout (DKO) were generated. These cells were plated, along with iEC DKO+LILRB3-E that additionally expressed the LILRB3 conjugate. The cells were allowed to adhere for 16 hours. Human peripheral blood NK cells were then added to the wells and incubated for 2 hours. The percentage of BLI signal after incubation is shown. The BLI signal of the iECDKO was completely lost ( Fig.13A The BLI signal of iEC DKO+LILRB3-E decreased significantly less within 2 hours ( Fig. 13B ).

[0058] Fig.14A and 14B : BLI killing assay within 2 hours with macrophages. Human iEC DKO and iEC DKO+LILRB3-E expressing firefly luciferase were plated and allowed to adhere for 16 hours. Then, human peripheral blood macrophages were added to the wells and incubated for 2 hours. The percentage of BLI signal after incubation is shown. The BLI signal of iEC DKO completely disappeared ( Fig.14A The BLI signal of iEC DKO+LILRB3-E decreased significantly less within 2 hours ( Fig. 14B ).

[0059] Fig.15A and 15B: BLI killing assay with NK cells within 24 hours. Human iEC DKO and iEC DKO+LILRB3-E expressing firefly luciferase were plated and allowed to adhere for 16 hours. Then, human peripheral blood NK cells were added to the wells and incubated for 24 hours. The percentage of BLI signal after 24 hours of NK incubation is shown. The BLI signal of iEC DKO completely disappeared ( Fig.15A The BLI signal of iECDKO+LILRB3-E decreased significantly less within 24 hours ( Fig. 15B ).

[0060] Fig.16A and 16B : BLI killing assay with macrophages within 24 hours. Human iEC DKO and iEC DKO+LILRB3-E expressing firefly luciferase were plated and allowed to adhere for 16 hours. Then, human peripheral blood macrophages were added to the wells and incubated for 24 hours. The percentage of BLI signal after 24 hours of macrophage incubation is shown. The BLI signal of iEC DKO was completely lost ( Fig.16A The BLI signal of iEC DKO+LILRB3-E decreased significantly less within 24 hours, and even increased ( Fig. 16B ).

[0061] Fig.17 :Generate human induced pluripotent stem cell (iPSC) derived endothelial cells (iEC), which express firefly luciferase and further contain B2M and CIITA double knockout (DKO). Use lentiviral particles to transduce the exemplary LILRB1 conjugate protein connected to the red fluorescent protein (RFP) tag into these cells. They are analyzed by flow cytometry. The histogram shows the RFP signal of the non-transduced iEC DKO (left peak) and the transduced iEC DKO+LILRB1-E (right peak), thereby confirming their expression of the LILRB1 conjugate.

[0062] Fig.18A and 18B : Flow cytometric analysis of LILRB1 surface expression on primary human peripheral blood NK cells and macrophages. 75.8% of NK cells were positive for LILRB1 ( Fig.18A ), 99.9% of macrophages were positive for LILRB1 ( Fig.18B ).

[0063] Fig.19A and 19B: BLI killing assay within 2 hours with NK cells. Human induced pluripotent stem cell (iPSC)-derived endothelial cells (iEC) expressing firefly luciferase and further comprising a B2M and CIITA double knockout (DKO) were generated. These cells were plated, along with iEC DKO+LILRB1-E that additionally expressed the LILRB1 conjugate. The cells were allowed to adhere for 16 hours. Human peripheral blood NK cells were then added to the wells and incubated for 2 hours. The percentage of BLI signal after incubation is shown. The BLI signal of the iEC DKO was completely lost ( Fig.19A The BLI signal of iEC DKO+LILRB1-E remained stable within 2 hours ( Fig.19B ).

[0064] Fig. 20A and 20B : BLI killing assay within 2 hours with macrophages. Human iEC DKO and iEC DKO+LILRB1-E expressing firefly luciferase were plated and allowed to adhere for 16 hours. Then, human peripheral blood macrophages were added to the wells and incubated for 2 hours. The percentage of BLI signal after incubation is shown. The BLI signal of iEC DKO completely disappeared ( Fig. 20A The BLI signal of iEC DKO+LILRB1-E remained stable within 2 hours ( Fig. 20B ).

[0065] Fig.21A and 21B : BLI killing assay with NK cells over 24 hours. Human iEC DKO and iEC DKO+LILRB1-E expressing firefly luciferase were plated and allowed to adhere for 16 hours. Then, human peripheral blood NK cells were added to the wells and incubated for 24 hours. The percentage of BLI signal after 24 hours of NK incubation is shown. The BLI signal of iEC DKO completely disappeared ( Fig.21A The BLI signal of iECDKO+LILRB1-E remained stable over 24 hours ( Fig.21B ).

[0066] Fig.22A and 22B : BLI killing assay with macrophages within 24 hours. Human iEC DKO and iEC DKO+LILRB1-E expressing firefly luciferase were plated and allowed to adhere for 16 hours. Then, human peripheral blood macrophages were added to the wells and incubated for 24 hours. The percentage of BLI signal after 24 hours of macrophage incubation is shown. The BLI signal of iEC DKO completely disappeared ( Fig.22A The BLI signal of iEC DKO+LILRB1-E remained stable and even increased over 24 hours ( Fig. 22B ).

[0067] Fig.23A and 23B The expression of the PD-1 selector transgene was shown to be maintained during differentiation. iPSC DKO were transduced using lentiviral particles (iPSC DKO+PD-1-E) to express the PD-1 selector. Flow cytometry showed that robust expression could be achieved ( Fig.23A ). When iPSC DKO+PD-1-E differentiated into iEC DKO+PD-1-E, transgene expression remained stable ( Fig. 23B ).

[0068] Fig.24A and 24B The expression of the TIM3-binding transgene was shown to be maintained during differentiation. iPSC DKO were transduced with lentiviral particles (iPSC DKO+TIM3-E) to express the TIM3-binding transgene. Flow cytometry showed that robust expression could be achieved ( Fig.24A ). When iPSC DKO+TIM3-E differentiated into iEC DKO+TIM3-E, transgene expression remained stable ( Fig. 24B ).

[0069] Fig.25A and 25B The expression of the LILRB3-conjugant transgene was shown to be maintained during differentiation. iPSC DKO were transduced using lentiviral particles (iPSC DKO+LILRB3-E) to express the LILRB3-conjugant. Flow cytometry showed that robust expression could be achieved ( Fig.25A When iPSC DKO+LILRB3-E differentiated into iEC DKO+LILRB3-E, transgene expression remained stable ( Fig.25B ).

[0070] Fig.26A and 26B The expression of the LILRB1 conjugate transgene was shown to be maintained during differentiation. iPSC DKO were transduced using lentiviral particles (iPSC DKO+LILRB1-E) to express the LILRB1 conjugate. Flow cytometry showed that robust expression could be achieved ( Fig.26A When iPSC DKO+LILRB1-E differentiated into iEC DKO+LILRB1-E, transgene expression remained stable ( Fig.26B ).

[0071] Fig.27A and 27BThe threshold of TIM3-E expression required to protect cells from NK cell killing is shown. To evaluate whether there is a functional threshold for TIM3-E expression to exert its protective effect against NK cell killing, iEC DKO was transduced with lentiviral particles carrying the TIM3-E transgene. After 3 days, the transduced iEC pool was divided into three groups based on TIM3-E expression. The three groups were labeled iEC DKO+TIM3-E high, medium and low ( Fig.27A For subsequent BLI killing assays, the different populations were incubated with NK cells for 2 h ( Fig.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 completely disappeared. The BLI signals of iEC DKO+TIM3-E high and medium remained stable, but the BLI signal of iEC DKO+TIM3-E low also dropped to the background. These results indicate that the functional threshold of TIM3-E is between the low and medium expression levels.

[0072] Fig.28A and 28B The threshold of LILRB1-E expression required to protect cells from NK cell killing is shown. To evaluate whether there is a functional threshold for LILRB1-E expression to exert its protective effect against NK cell killing, iEC DKO were transduced with lentiviral particles carrying the LILRB1-E transgene. After 3 days, the transduced iEC pool was divided into three groups based on their LILRB1-E expression. The three groups were labeled iEC DKO+LILRB1-E high, medium, and low ( Fig.28A For subsequent BLI killing assays, the different populations were incubated with NK cells for 2 h ( Fig.28B ). All target cells were luciferase positive. The percentage of BLI signal after 2 h of NK incubation is shown. The BLI signal of iEC DKO completely disappeared. 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 suggest that the functional threshold of LILRB1-E is between the low and medium expression levels.

[0073] [VI. Detailed description of the invention]

[0074] The present invention provides immune checkpoint engaged (ICE) cells, which have reduced immunity when transplanted into a subject compared to parental cells. In some embodiments, ICE cells have enhanced programmed cell death protein 1 (PD-1) engagement function (PD-1 engaged daughter cells), which have reduced immunity when transplanted into a subject compared to parental cells with unmodified PD-1 engagement function. In some embodiments, PD-1 engaged daughter cells are low immune cells. In other embodiments, the cells are pluripotent cells. In other embodiments, the cells are embryonic stem cells (ES) or induced pluripotent stem cells (iPSC). In other embodiments, PD-1 engaged daughter cells are differentiated somatic cells. In other embodiments, PD-1 engaged daughter cells are low immune pluripotency (HIP) cells. In further embodiments, HIP cells are blood type O (HIPO), rhesus factor (Rh) negative (HIP - ) or O and Rh - Type (HIPO - In other embodiments, PD-1-engaged daughter cells have been derived from HIP, HIP - or HIPO - Cells are derived or differentiated. In other embodiments, the PD-1 ligand cell contains an antibody Fc receptor to protect it from antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In other embodiments, the ICE cell expresses a ligand that binds to an inhibitory immune cell receptor, which is selected from the group consisting of LILRB1 ligands, LILRB3 ligands, and TIM3 ligands. In other embodiments, the ICE cell is a PD-1 ligand cell that also expresses a ligand that binds to another inhibitory immune cell receptor, which is selected from the group consisting of CD47, truncated CD47, LILRB1 ligands, LILRB3 ligands, SIRPα ligands, and TIM3 ligands.

[0075] Surprisingly, agonists against PD-1, TIM3, LILRB3, and LILRB1 all inhibit immune effector cells to protect therapeutic cells from cell killing. Thus, the present invention recognizes that immune checkpoint engagers as a class, rather than just individual members, are valid targets for agonists to inhibit immune effector cells. Furthermore, the present invention now recognizes that such agonists must be expressed at a threshold level to protect ICE cells from immune cell killing.

[0076] The present invention provides compositions and uses for PD-1 agonists to inhibit immune effector cells from producing cell therapeutic agents in regenerative medicine or immuno-oncology to reduce host-to-graft rejection. In some embodiments, a sequence fragment is taken from a PD-1 agonist antibody (U.S. Patent No. 10,493,148, which is incorporated herein by reference in its entirety) and implemented in a newly designed fusion protein (SEQ ID NO: 1). The fusion protein of this embodiment comprises the following parts: (IL-2 or CD8) signal peptide-VL-(GGGGS) 3 linker-VH-CD8a hinge-PDGFR TMD.

[0077] In other embodiments, PD-1 engaged daughter cells have been derived or differentiated from the above cells. For example, differentiated PD-1 engaged daughter cells can be endothelial cells, cardiomyocytes, hepatocytes, dopaminergic neurons, islet cells, retinal pigment epithelial cells and other cell types for transplantation and medical therapy. These include chimeric antigen receptor (CAR) cells and T cell receptor engineered cells, such as CAR-T cells, TCR T cells, NK cells and CAR-NK cells.

[0078] As used herein, the term "subject" or "patient" refers to any animal, such as a domesticated animal, a zoo animal, or a human. A "subject" or "patient" can be a mammal, such as a dog, a cat, a bird, livestock, or a human. Specific examples of "subjects" and "patients" include, but are not limited to, individuals (especially humans) suffering from diseases or conditions related to the liver, heart, lungs, kidneys, pancreas, brain, nervous tissue, blood, bones, bone marrow, etc.

[0079] Mammalian cells can be from humans or non-human mammals. Exemplary non-human mammals include, but are not limited to, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, cows, and non-human primates (e.g., chimpanzees, macaques, and apes).

[0080] By "low immunogenicity" cells, "low immunity" cells or "HI" cells herein is meant cells that produce a reduced immune rejection response when transferred into an allogeneic host. In a preferred embodiment, HI cells do not produce an immune response. Thus, "low immunogenicity" refers to a significantly reduced or eliminated immune response compared to the immune response of the parental cell (i.e., "wt") cell prior to immunoengineering.

[0081] The "low immunogenic cells" referred to in this article are - ”, “Low immunogenicity ORh - "Cells or "HIO - "Cells are also ABO blood type O and rhesus factor Rh - HI cells. -The cells may be derived from O - Produced by cells, enzymatically modified to O - , or genetically engineered to be O - .

[0082] The "HLA" or "human leukocyte antigen" complex as used herein refers to the gene complex that encodes the major histocompatibility complex (MHC) proteins in humans. These cell surface proteins that make up the HLA complex are responsible for regulating the immune response to antigens. In humans, there are two types of MHC, class I and class II, "HLA-I" and "HLA-II". HLA-I includes HLA-A, HLA-B, HLA-C, etc. These present peptides from the inside of 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, as well as others that present extracellular antigens to T lymphocytes. This stimulates CD4 + Cells (also called helper T cells). It should be understood that the use of "MHC" or "HLA" is not meant to be limiting, as it depends on whether the gene is from a human (HLA) or a non-human (MHC). Therefore, as it relates to mammalian cells, these terms are used interchangeably herein.

[0083] As used herein, "gene knockout" refers to the process of inactivating a particular gene in the host cell in which it is located, resulting in the production of no or an inactive form of the protein of interest. As will be appreciated by those skilled in the art and described further below, this can be accomplished in a variety of different ways, including removing nucleic acid sequences from the gene, or interrupting the sequence with other sequences, changing the reading frame, or changing the regulatory components of the nucleic acid. For example, all or part of the coding region of the target gene 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, a translation initiation sequence can be removed or replaced, etc.

[0084] "Gene knock-in" as used herein refers to the process of adding a genetic function to a host cell. This results in an increase in the level of the encoded protein. As will be appreciated by those skilled in the art, this can be achieved in several ways, including adding one or more additional gene copies to the host cell or altering the regulatory components of the endogenous gene, thereby increasing the expression of the protein. This can be achieved by modifying the promoter, adding a different promoter, adding an enhancer, or modifying other gene expression sequences. "Beta-2 microglobulin" or "Beta 2M" or "B2M" protein refers to the human beta 2M protein having the amino acid and nucleic acid sequence shown below; Accession number of the human gene RefSeq NM_004048.4.

[0085] "CD47 protein" refers to the human CD47 protein having the amino acid and nucleic acid sequences shown below; the accession number of the human gene is RefSeq NM_001777.4.

[0086] After connecting to the PD-1 receptor, the natural ligand PD-L1 or PD-L2 expressed on the cell can initiate downstream signal transduction in the cell and may cause unwanted disturbances to its physiological function. Only to achieve protection against immune cell killing, certain aspects of the present invention use the PD-1 conjugate described herein to separate the extracellular PD-1 binding function from the intracellular signal transduction in the engineered cells. In other aspects, the present invention provides a PD-1 conjugate fusion protein with agonistic PD-1 binding activity, but lacks unwanted intracellular signal transduction in the engineered cells. Other aspects provide a PD-1 conjugate fusion protein comprising a PD-L1 or PD-L2 extracellular domain (ECD). In certain aspects of the present invention, this fusion protein will have a homologous or heterologous intracellular domain. In other aspects, the fusion protein will have a truncated intracellular domain or no intracellular domain.

[0087] The present invention provides a PD-1 binding fusion protein, which is expressed on engineered cells and is designed to bind to PD-1 on immune cells in an agonistic manner, thereby activating PD-1 signaling. Effector immune cells can be any immune cells expressing PD-1, and can be from myeloid lineages (e.g., monocytes, macrophages, or polymorphonuclear cells) and lymphoid lineages (e.g., T cells, B cells, or NK cells).

[0088] The PD-1 / PD-L1 pathway is an established immune checkpoint. While forced overexpression of PD-L1 on target cells can effectively induce inhibitory signals in immune effector cells, PD-L1 can induce unwanted signals in engineered cells. This invention provides a new strategy to exploit this immune checkpoint without disrupting the physiology of engineered cells. The PD-1 engager protein disclosed herein does not have any intracellular signaling domains.

[0089] Therefore, 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 generally do not have an ICD and are limited to the ECD and TMD.

[0090] In certain aspects, the ECD includes a PD-L1 or PD-L2 ECD, a complementary determining region (CDR) of an agonistic anti-PD-1 antibody, or a single chain variable fragment (scFv) of an agonistic anti-PD-1 antibody. The region of interest on the ECD includes at least one CDR sequence, wherein the CDR can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acids. Alternatively, the ECD of interest comprises a plurality of antibody variable regions. For example, the CDR of an anti-PD-1 antibody is disclosed in U.S. Patent No. 10,493,148, which is incorporated herein by reference in its entirety.

[0091] In certain aspects, one or more residues of the sequence are altered to modify binding to achieve a more favorable on-rate, a more favorable off-rate, or both, thereby achieving optimized binding.

[0092] In other aspects, the ECD comprises a linker region or hinge that connects the TMDs or to each other. In other aspects, modifications are made within one or more linker regions or hinge regions as long as these modifications do not abolish the binding affinity of the fusion protein to PD-1.

[0093] In certain aspects, the ECD has a contiguous sequence of at least about 10 amino acids, as set forth in SEQ ID NO: 1, 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 entire region provided. The ECD also includes sequences that differ by at most 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.

[0094] Generally, the transmembrane domain (TMD) of PD-1 ligand fusion protein is not limited to a specific TMD sequence. Preferably, TMD allows the fusion protein to be stably anchored in the cell membrane expressing the fusion protein (e.g., endothelial cells, cardiomyocytes, pancreatic beta cells, T cells, NK cells, or hematopoietic cells, etc.). It further allows ECD to be combined with PD-1. In some aspects, the fusion protein does include ICD, and is combined with PD-1 to allow signal transmission by ICD. If this signal transduction enhances the intrinsic function of the cell, this may be beneficial to the engineered cells. For example, enhanced adhesion can be achieved by activating integrins. In other aspects, the fusion protein does not include ICD, but is truncated after TMD. In the latter case, the combination of fusion protein and PD-1 does not result in intracellular signal transduction in engineered cells.

[0095] TMDs extend through the cell membrane lipid bilayer with a single α-helix, multiple α-helices or rolled β-sheets. Some of these "single-channel" and "multi-channel" proteins insert a covalently linked fatty acid chain in the cytoplasmic lipid monolayer. Other membrane proteins are exposed only on one side of the membrane. Some of them are anchored to the cytoplasmic surface by amphipathic α-helices, which are distributed into the cytoplasmic monolayer of lipid bilayers by the hydrophobic face of the helix. Others are only connected to the bilayer by covalently linked lipid chains-or fatty acid chains or isoprenyl-in the cytoplasmic monolayer, or are connected to phosphatidylinositols in non-cytoplasmic monolayers by 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)).

[0096] In certain aspects, exemplary TMDs of immune checkpoint engager molecules are selected from 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, CD 230, CD90, CD74, CD3d, CD340, CD236, CD61, CD18, CD54, CD29, CD1a, CD5, CD220, CD2, CD66e, CD51, CD141, CD115, CD42b, CD221, CD271, CD55, CD243, CD9 8. CD10, CD41, CD14, CD45, CD228, CD16a, CD49e, CD126, CD63, CD48, CD7, CD140b, CD3g, CD117, CD28, CD8b, CD37, CD11b, CD107a, CD331, CD222, CD20, CD 79a, 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, CD 89. 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 aspects, the TMD of the fusion protein is from CD47 (SEQ ID NO: 5), CD64 (SEQ ID NO: 6), or PDGFR (SEQ ID NO: 4).

[0097] In other aspects, the PD-1 engager fusion protein does not have an intracellular domain (ICD) to avoid signaling in engineered cells. However, if considered beneficial, the ICD of the fusion protein can be an ICD from CD16, CD32, CD64, CD8, CD3, CD28 or CD137.

[0098] "CIITA protein" refers to the human CIITA protein having the amino acid and nucleic acid sequences shown below; RefSeq accession number of the human gene is NM_000246.4.

[0099] In the context of "wild type", a cell refers to a cell found in nature. However, in the context of cell therapeutics, as used herein, this also means that the cell can contain nucleic acid changes that lead to an immortal or dedifferentiated state after reprogramming, but has not been subjected to the immunoediting procedures of the present invention to achieve low immunogenicity, antibody evasion ability, or expression of immune checkpoint engagers.

[0100] As used herein, "homologous" refers to the genetic similarity or identity of the host organism and the cell transplantation such that there is immunological compatibility; for example, no immune response will be generated.

[0101] As used herein, "allogeneic" refers to the genetic differences in the host organism that generate the immune response and the cells that are transplanted.

[0102] The “B2M - / - ” refers to a diploid cell having the B2M gene inactivated on both chromosomes. This can be accomplished in a variety of ways, as described herein.

[0103] As used herein, “PD-1tg”, “PD-1 transgenic” or “PD-1 + ” refers to host cells expressing PD-1, in some cases by having at least one additional copy of the PD-1 gene or a PD-1 fusion protein gene.

[0104] The “CIITA” mentioned in this article - / - ” refers to a diploid cell having the CIITA gene inactivated on both chromosomes. This can be accomplished in a variety of ways, as described herein.

[0105] As used herein, “PD-L1 tg”, “PD-L1 transgenic” or “PD-L1 + ” refers to host cells expressing PD-L1, in some cases by having at least one additional copy of the PD-L1 gene or a PD-L1 fusion protein gene.

[0106] As used herein, the terms “PD-L2 tg”, “PD-L2 transgenic” or “PD-L2 + ” refers to host cells expressing PD-L2, in some cases by having at least one additional copy of the PD-L2 gene or a PD-L2 fusion protein gene.

[0107] As used herein, the terms “CD47 tg”, “CD47 transgenic” or “CD47 + ” refers to host cells expressing CD47, in some cases by having at least one additional copy of the CD47 gene or a CD47 fusion protein gene.

[0108] The term "identity", in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have the same specified percentage of nucleotides or amino acid residues when compared and aligned to achieve maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms suitable for technicians) or by visual inspection. Depending on the application, the percentage "identity" can exist in the region of the compared sequences, for example, in the functional domain, or, in the entire length of the two sequences to be compared. For sequence comparison, usually one sequence serves as a reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are entered into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Then, the sequence comparison algorithm calculates the percentage sequence identity of the test sequence relative to the reference sequence according to the specified program parameters.

[0109] Optimal alignment of sequences can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology consensus algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., below).

[0110] An example of an algorithm suitable for determining 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 analysis is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0111] "Inhibitors," "activators," and "modulators" affect the function or expression of a biologically relevant molecule. The term "modulator" includes inhibitors and activators. They can be identified using in vitro and in vivo assays for the expression or activity of target molecules.

[0112] "Inhibitors" are agents that, for example, inhibit the expression and binding of a target molecule or protein. They may partially or completely block stimulation or have protease inhibitor activity. They may reduce, decrease, prevent or delay activation, including inactivating, desensitizing or down-regulating the activity of the target protein. Modulators may be antagonists of a target molecule or protein.

[0113] "Activators" are agents that, for example, induce or activate the function or expression of a target molecule or protein. They may bind to, stimulate, increase, turn on, activate or promote the activity of a target molecule. Activators can be agonists of a target molecule or protein.

[0114] "Homologs" are biologically active molecules that are similar to reference molecules at the nucleotide sequence, peptide sequence, functional or structural level. Homologs may include sequence derivatives that share a certain percentage of identity with the reference sequence. Thus, in one embodiment, homologous or derived sequences share at least 70% sequence identity. In a specific embodiment, homologous or derived sequences share at least 80% or 85% sequence identity. In a specific embodiment, homologous or derived sequences share at least 90% sequence identity. In a specific embodiment, homologous or derived sequences share at least 95% sequence identity. In a more specific embodiment, homologous or derived sequences share 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 derived nucleic acid sequences can also be defined by their ability to remain bound to a reference nucleic acid sequence under high stringency hybridization conditions. Homologues with structural or functional similarity to a reference molecule may be chemical derivatives of the reference molecule. Methods for detecting, generating and screening structural and functional homologues and derivatives are known in the art.

[0115] "Hybridization" generally depends on the ability of denatured DNA to reanneal when the complementary strand is present in an environment below its 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. Therefore, higher relative temperatures tend to make the reaction conditions more stringent, while lower relative temperatures are less stringent. For more detailed information and explanations on the 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.

[0116] The "stringency" of a hybridization reaction is easily determined by any person of ordinary skill in the art and is generally an empirical calculation dependent on probe length, washing temperature, and salt concentration. Typically, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures.

[0117] "Stringent conditions" or "high stringency conditions" as defined herein can be identified by the following conditions: (1) using low ionic strength and high temperature for washing, for example, using 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; (2) using a denaturing agent, such as formamide, during hybridization at 42°C, for example, 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% polysucrose / 0.1% polyvinyl pyrrolidone / 50 mM sodium phosphate buffer (pH 6.5) with 750 mM sodium chloride, 75 mM sodium citrate; or (3) using 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.5) at 42°C. 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50 μl / ml), 0.1% SDS and 10% dextran sulfate solution for overnight hybridization, wash with 0.2× SSC (sodium chloride / sodium citrate) at 42°C for 10 minutes, and then wash with high stringency consisting of 0.1× SSC containing EDTA at 55°C for 10 minutes.

[0118] As used herein, a "pharmaceutically acceptable carrier" or "therapeutically effective carrier" is aqueous or non-aqueous (solid), such as alcoholic or oily, or a mixture thereof, and may contain surfactants, emollients, lubricants, stabilizers, dyes, fragrances, preservatives, acids or bases for adjusting pH, solvents, emulsifiers, gelling agents, humectants, stabilizers, wetting agents, time-release agents, moisturizers, or other ingredients typically included in a particular form of a pharmaceutical composition. Pharmaceutically acceptable carriers are recognized in the art and include, for example, aqueous solutions (such as water or physiologically buffered saline) or other solvents or carriers (such as ethylene glycol, glycerol) and oils (such as olive oil). Pharmaceutically acceptable carriers may contain physiologically acceptable compounds that act to stabilize or increase the absorption of specific inhibitors, 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.

[0119] The pharmaceutical composition can be in the form of a sterile injection preparation, for example, as a sterile injection aqueous or oily suspension. The suspension can be prepared using a suitable dispersant or wetting agent (e.g., Tween 80) and a suspending agent according to techniques known in the art. The sterile injection preparation can also be a sterile injection solution or suspension, dissolved in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution of 1,3-butanediol. Acceptable carriers and solvents include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are generally used as solvents or suspension media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, can be used to prepare injections, natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially its polyoxyethylated version. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as Ph. Helv or similar alcohols.

[0120] It is intended that each maximum numerical limitation given herein include each lower numerical limitation, as if such lower numerical limitations were expressly written herein. Each minimum numerical limitation given herein will include each higher numerical limitation, as if such higher numerical limitations were expressly specified herein. Each numerical range given herein will include each narrower numerical range within such broader numerical range, as if such narrower numerical ranges were expressly written herein.

[0121] As used herein, the term "modification" refers to a change that physically distinguishes a modified molecule from a parent molecule. In some embodiments, insertions, deletions, substitutions, or other types of amino acid changes in PD-1 conjugates or SIRPα conjugate proteins. In some embodiments, the modification is prepared according to methods described herein and known in the art in PD-1, PD-L1, PD-L2, SIRPα, CD47, CD16, CD32, CD64, truncated CD64, HSVtk, EC-CD, or iCasp9 variant polypeptides. This modification distinguishes them from corresponding parents (e.g., wild-type proteins, naturally occurring mutant proteins, or other engineered proteins that do not contain such variant polypeptide modifications) that are not modified according to the methods described herein. In another embodiment, the variant polypeptide includes one or more modifications that distinguish the function of the variant polypeptide from the unmodified polypeptide. For example, amino acid changes in the variant polypeptide can affect its receptor binding spectrum. In other embodiments, the variant polypeptide includes substitution, deletion, or insertion modification, or a combination thereof. In another embodiment, a variant polypeptide includes one or more modifications that increase its affinity for a receptor compared to the affinity of the unmodified polypeptide.

[0122] In one embodiment, variant polypeptide comprises one or more displacements, insertions or deletions relative to corresponding native or parental sequences. In certain embodiments, variant 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 to 50 or 51 or more modifications.

[0123] As used herein, "episomal vector" refers to a genetic vector that can exist and replicate autonomously in the cytoplasm of a cell; for example, it is not integrated into the genomic DNA of the host cell. Many episomal vectors are known in the art, as described below.

[0124] In the context of a gene, "knockout" means that the host cell carrying the knockout does not produce a functional protein product of the gene. As described herein, knockout can be caused in a variety of ways, from removing all or part of the coding sequence, introducing a frameshift mutation to not produce a functional protein (truncated or nonsense sequence), removing or changing regulatory elements (e.g., promoters) so that the gene is not transcribed, preventing translation by binding to mRNA, etc. Typically, the knockout is performed at the genomic DNA level, so that the offspring of the cell will also permanently carry the knockout.

[0125] In the context of genes, "knock-in" means that the host cell carrying the knock-in has more functional protein that is active in the cell. As described herein, knock-in can be accomplished in a variety of ways, typically by introducing at least one copy of a transgenic (tg) encoding a protein into the cell, although this can also be accomplished by replacing regulatory components, such as by adding a constitutive promoter to an endogenous gene. In general, knock-in techniques result in the integration of additional copies of the transgene into the host cell.

[0126] [VII. Cells of the present invention]

[0127] The present invention provides ICE cells. In some embodiments, they are PD-1 engaged daughter cells. In other embodiments, they are low immune cells. Low immune cells may include HIP cells, HIPO cells, HIPO - In other embodiments, the PD-1 ligand cell is a differentiated somatic cell. In other embodiments, the PD-1 ligand cell is a hypoimmunoplastic (HIP) cell. In further embodiments, the HIP cell is a type O blood cell (HIPO), a rhesus factor (Rh) negative (HIP - ) or O and Rh - Type (HIPO - In other embodiments, PD-1-engaged daughter cells have been derived from HIP, HIP- or HIPO - In other embodiments, the PD-1 engaging daughter cells comprise an antibody Fc receptor to protect them from antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0128] The present invention provides compositions and methods for producing PD-1 engaged daughter cells. In some aspects, the cells are low immune cells. In other aspects, the cells are differentiated somatic cells. In other aspects, the cells are pluripotent cells, such as HIP cells, HIP - Cells, HIPO - Cell. In other aspects, PD-1 conjugate cells are pluripotent (PSC) cells suitable for transplantation and / or differentiation. PSC cells include induced PSC (iPSC) or embryonic stem cells (ESC). In other aspects, cells belong to a specific tissue type and have been differentiated from the above-mentioned PD-1 conjugate cells. For example, differentiated PD-1 conjugate cells can be endothelial cells, cardiomyocytes, hepatocytes, dopaminergic neurons, islet cells, retinal pigment epithelial cells, and other cell types for transplantation and drug therapy. These cells include chimeric antigen receptor (CAR) cells, such as CAR-T cells, CAR-NK cells, TCR T cells, TCR NK cells, TCR innate lymphocytes (ILC) and other engineered cell populations. See WO2018 / 132783, WO2020 / 018620, WO2020 / 018615, PCT / US2020 / 032272, and U.S. Patent Application Nos. 16 / 870,959 and 16 / 870,960, which are incorporated herein by reference in their entirety.

[0129] The present invention provides PD-1 ligand cells with PD-1 ligand proteins that interact with PD-1 on the surface of T cells, NK cells, and other immune cells. Immune checkpoint ligand molecules can prevent cell killing and reduce adaptive immunity and innate immunity. In some embodiments, the PD-1 ligand protein is an anti-PD-1 antibody tethered to the surface of a PD-1 ligand cell. In some embodiments, the anti-PD-1 antibody is tethered to the cell surface through its fragment crystallizable (Fc) portion. In other embodiments, the antigen binding portion of the anti-PD-1 antibody (scFv) is bound to the cell surface through a transmembrane domain (TMD). In a preferred embodiment, the TMD comprises one or more alpha helices. In other preferred embodiments, the TMD is from a 7-transmembrane protein (7TM). In other preferred embodiments, the TMD is from an immunoglobulin cell surface protein. In a more preferred embodiment, the immunoglobulin cell surface protein is an antibody, a receptor, a ligand, or an adhesion protein. In some embodiments, the PD-1 ligand cell is produced by a PD-L1 or PD-L2 fusion protein anchored on the cell surface.

[0130] Expression of PD-1 ligand protein can be accomplished in a variety of ways, which those skilled in the art who use "knock-in" or transgenic techniques will appreciate. In some cases, expression of PD-1 ligand protein is caused by one or more transgenes.

[0131] Thus, in some embodiments, one or more copies of a PD-1 ligand protein expression gene are added to the PD-1 ligand cells under the control of an inducible or constitutive promoter (the latter being preferred). In some embodiments, a lentiviral construct as described herein or known in the art is employed. These genes can be integrated into the genome of the host cell under the control of a suitable promoter known in the art.

[0132] In some embodiments, the expression of a gene can be increased by changing the regulatory sequence of the endogenous gene locus, for example, by exchanging the endogenous promoter for a constitutive promoter or a different inducible promoter. This can generally be accomplished using known technologies such as CRISPR.

[0133] Once changed, the presence of sufficient PD-1 ligand protein expression can be determined by using known techniques (such as those described in the Examples), such as Western blot, ELISA assay or FACS assay using appropriate antibodies. Generally speaking, in this case, "sufficient" refers to an increase in cell surface PD-1 ligand protein expression, thereby silencing NK cell killing.

[0134] 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, bispecific or multispecific antibodies, llama antibodies, nanobodies, diabodies, affibodies, Fv, Fab, F(ab')2, Fab', scFv, scFv-Fc, etc. The term also includes antibody fusion proteins, such as Ig chimeras. Preferred antibodies include humanized or fully human monoclonal antibodies or fragments thereof.

[0135] 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., as long as bispecific antibodies exhibit the desired biological activity) and may also include certain antibody fragments (as described in more detail herein). The antibody may be a chimeric antibody, a human antibody, a humanized antibody and / or an affinity matured antibody.

[0136] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to antibodies in their substantially intact form, rather than antibody fragments as defined below. These terms refer specifically to antibodies having heavy chains that include an Fc region. An "antibody fragment" comprises a portion of an intact antibody, preferably comprising its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical, except for possible mutations, for example, naturally occurring mutations that may be present in small amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.

[0137] In certain embodiments, such monoclonal antibodies generally include antibodies with polypeptide sequences that can bind to targets, wherein the target binding polypeptide sequence is obtained by including a process of selecting a polypeptide sequence that a single target binds from a plurality of polypeptide sequences. For example, the selection process can be to select a unique clone from a plurality of clones, such as a hybridoma clone, a phage clone, or a recombinant DNA clone library. It should be understood that the selected target binding sequence can be further changed, for example, to improve the affinity to the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to produce multi-specific antibodies, etc., and the antibody comprising the target binding sequence of the change is also a monoclonal antibody of the present invention. Compared with polyclonal antibody preparations that generally include different antibodies for different determinants (epitopes), each monoclonal antibody of the monoclonal antibody preparation is for a single determinant on the antigen. In addition to specificity, the advantage of monoclonal antibody preparations is that they are generally not polluted by other immunoglobulins.

[0138] An antibody that specifically binds to an antigen has a high affinity for that antigen. Antibody affinity can be measured by a dissociation constant (Kd). In certain embodiments, the antibodies provided herein have a Kd of equal to or less than about 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, or 0.001 nM (e.g., 10 -7 M or lower, 10 -7 M~10 -13 M, 10 -8 M~10 -13 M or 10 -9 M~10 -13 The dissociation constant (Kd) of the

[0139] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed with a Fab version of the target antibody and its antigen, as described in the following assay. The solution binding affinity of Fab for antigen is measured by equilibrating the Fab with a minimum concentration (125I) labeled antigen in the presence of a titration gradient of unlabeled antigen, followed by capturing the bound antigen with an anti-Fab antibody coated plate (e.g., see Chen et al., J. Mol. Biol. 293: 865-881 (1999)). To establish assay conditions, Multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) and subsequently blocked with 2% (w / v) bovine serum albumin in PBS for 2 to 5 hours at room temperature (about 23°C). In non-adsorbent plates (Nunc #269620), 100 μM or 26 μM [125I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody, Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation may be continued for a longer period of time (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to the capture plate and incubated at room temperature (e.g., 1 hour). The solution is then removed and the plate is incubated with 0.1% polysorbate 20 The plates were washed 8 times with PBS solution. When the plates were dry, 150 μl / well of scintillant (MICROSCINT-20 TM ; Packard) and in TOPCOUNT TM The plates were counted for 10 minutes in a gamma counter (Packard).The concentration of each Fab that gave less than or equal to 20% of maximal binding was selected for competitive binding assays.

[0140] According to another embodiment, Kd is calculated using or (BIAcore, Inc., Piscataway, NJ) at 25 ° C using surface plasmon resonance assays, for example, at ~ 10 response units (RU) of fixed antigen CM5 chips. Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) were activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Before injection at a flow rate of 5 μl / min, the antigen was diluted to 5 μg / ml (~0.2 μM) with 10 mM sodium acetate (pH 4.8) to obtain approximately 10 response units (RU) of coupled protein. After injection of the antigen, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) were injected into a 4% PBS solution containing 0.05% polysorbate 20 (TWEEN-20 TM ) surfactant-free PBS (PBST). The flow rate was approximately 25 μl / min. By simultaneously fitting the binding and dissociation sensorgrams, a simple one-to-one Langmuir binding model ( Evaluation software version 3.2) to calculate the binding rate (K on ) and dissociation rate (K off The equilibrium dissociation constant (Kd) was calculated as k off / k on For example, see Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate measured by surface plasmon resonance above exceeds 10 6 M -1 s -1 , this can be done by using a spectrophotometer (e.g., a stopped-flow spectrophotometer (AvivInstruments) or an 8000-series SLM-AMINCO with a stirred cuvette) TM Binding rate is determined by fluorescence quenching technique which measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2 at 25°C with increasing antigen concentrations measured in a spectrophotometer (ThermoSpectronic). Other coupling chemistries for target antigens on the chip surface (e.g., streptavidin / biotin, hydrophobic interactions, or disulfide bond chemistry) are also readily available to replace the above amine coupling method (CM5 chip), as will be appreciated by those of ordinary skill in the art.

[0141] 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 made by a variety of techniques, including, for example, the hybridoma method (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 methods (e.g., see U.S. Pat. No. 4,816,567), phage display technology (e.g., see Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Sidhu et al., J. Mol. Biol. 226:581-597 (1992)). al., J. Mol. Biol. 338(2):299-310 (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)), and techniques for producing human or human-like antibodies in animals having partial or complete human immunoglobulin loci or genes encoding human immunoglobulin sequences (e.g., see WO98 / 24893; WO96 / 34096; WO96 / 33735; WO91 / 10741; Jakobovits et al., J. Immunol. Methods 284(1-2):119-132 (2004)). 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. Patent 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 references are incorporated by reference in their entirety.

[0142] The "humanized" form of non-human (e.g., mouse) antibodies is a chimeric antibody comprising the minimum sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (receptor antibody), wherein the residues from the receptor hypervariable region are replaced by the hypervariable region residues from a non-human species (donor antibody), such as mice, rats, rabbits, or non-human primates with desired specificity, affinity, and / or capacity. In some cases, the framework region (FR) residues of human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may be included in residues not found in receptor antibodies or donor antibodies. These modifications can be made to further improve antibody performance. In general, humanized antibodies substantially comprise all of at least one (usually two) variable domains, wherein all or nearly all hypervariable loops correspond to the loops of non-human immunoglobulins, and all or substantially all FRs are FRs of human immunoglobulin sequences. Humanized antibodies optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin. For more detailed information, 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). See also 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.

[0143] A "human antibody" comprises an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human, and / or has been prepared using any of the techniques disclosed herein for making human antibodies. Such techniques include screening human-derived combinatorial libraries, such as phage display libraries (e.g., see Marks et al., J. Mol. Biol, 222: 581-597 (1991) and Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991)); using human myeloma and mouse-human heteromyeloma cell lines to produce human monoclonal antibodies (e.g., see 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., Monoclonal Antibody Production Techniques and Applications, pp. 55-93 (Marcel Dekker, Inc., New York, 1987); and al., J. Immunol, 147:86 (1991)); monoclonal antibodies are produced in transgenic animals (e.g., mice) that are capable of producing a complete human antibody repertoire in the absence of endogenous immunoglobulin production (e.g., see 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 explicitly excludes humanized antibodies that contain antigen-binding residues from non-human animals.

[0144] A. Methods of genetic alteration

[0145] The present invention includes methods for modifying nucleic acid sequences in cells or under cell-free conditions to produce PD-1 conjugated daughter cells. Example technologies 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 technologies. These technologies can achieve double-stranded DNA breaks at the desired gene locus. These controlled double-strand breaks promote homologous recombination at specific gene locus points. The process focuses on specific sequences of targeted nucleic acid molecules, such as chromosomes, and endonucleases recognize and bind sequences and induce double-strand breaks in nucleic acid molecules. Double-strand breaks are repaired by error-prone non-homologous end joining (NHEJ) or homologous recombination (HR).

[0146] As will be appreciated by those skilled in the art, a number of different techniques may be used to engineer the modified cells of the invention, and to engineer them to be less immunogenic as outlined herein.

[0147] Typically, these techniques can be used alone or in combination. For example, in the production of PD-1 ligand cells, CRISPR can be used to express PD-1 ligand proteins, such as anti-PD-1 immunoglobulins. In another example, viral technology (e.g., lentivirus) is used to express PD-1 ligand proteins.

[0148]

a.CRISPR technology

[0149] In one embodiment, the cells are manipulated using clustered regularly interspaced short palindromic repeats) / Cas ("CRISPR") technology known in the art. CRISPR can be used to generate PD-1 zygote cells. There are a number of CRISPR-based technologies, such as Doudna and Charpentier, Science doi: 10.1126 / science.1258096, which is hereby cited. CRISPR technology and kits are sold commercially.

[0150]

b.TALEN technology

[0151] In some embodiments, the cells of the invention are prepared using a transcription activator-like effector nuclease (TALEN) approach. TALEN is a combination of a restriction endonuclease and a nuclease that is engineered to bind and cleave almost any desired DNA sequence. TALEN kits are sold commercially.

[0152]

c. Zinc finger technology

[0153] In one embodiment, cells are manipulated using Zn finger nuclease technology. Zinc finger nucleases are artificial restriction endonucleases created by fusing zinc finger DNA binding domains to DNA cleavage domains. Zinc finger domains can be engineered to target specific desired DNA sequences, which enables zinc finger nucleases to target unique sequences in complex genomes. By exploiting endogenous DNA repair mechanisms, these agents can be used to precisely alter the genomes of higher organisms, similar to CRISPR and TALEN.

[0154]

d. Virus-based technology

[0155] There are a variety of viral technologies that can be used to generate some embodiments of the PD-1 engaging daughter cells of the present invention, including but not limited to the use of retroviral vectors, lentiviral vectors, adenoviral vectors and Sendai virus vectors. The free vectors used to generate the cells are described below.

[0156] For all of these techniques, well-known recombinant techniques are used to generate the recombinant nucleic acids outlined herein. In certain embodiments, a recombinant nucleic acid encoding a PD-1 ligand 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 is generally suitable for use in a host cell and is treated. Various types of appropriate expression vectors and suitable regulatory sequences for a variety of host cells are known in the art. Typically, 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, a transcription start and stop sequence, a translation start and stop sequence, and an enhancer or activator sequence. Constitutive or inducible promoters known in the art are also contemplated. The promoter may be a naturally occurring promoter or a composite promoter combining elements of more than one promoter. The expression construct may be present in a 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 to allow selection of transformed host cells. Certain embodiments include an expression vector comprising a nucleotide sequence encoding a variant polypeptide operably linked to at least one regulatory sequence. Regulatory sequences used herein include promoters, enhancers and other expression control elements. In certain embodiments, the design of the expression vector is used to select the host cell to be transformed, the specific variant polypeptide desired to be expressed, the copy number of the vector, the ability to control the copy number, or the expression of any other protein encoded by the vector, such as an antibiotic marker.

[0157] Examples of suitable mammalian promoters include, for example, promoters from the following genes: the ubiquitin / S27a promoter of hamsters (WO 97 / 15664), the simian vacuolating virus 40 (SV40) early promoter, the adenovirus major late promoter, the mouse metallothionein-I promoter, the long terminal repeat region of Rous sarcoma virus (RSV), the mouse mammary tumor virus promoter (MMTV), the long terminal repeat region of Moloney murine leukemia virus, the early promoter of human cytomegalovirus (CMV), the eukaryotic translation elongation factor 1 alpha (EF-1 alpha), and the chicken β-actin promoter coupled to the CMV early enhancer (CAG). Other examples of heterologous mammalian promoters are actin, immunoglobulin or heat shock promoters.

[0158] In additional embodiments, promoters for mammalian host cells can be obtained from the genomes of viruses, such as polyoma virus, fowl pox virus (UK 2,211,504, published on July 5, 1989), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and simian virus 40 (SV40). In further embodiments, heterologous mammalian promoters are used. Examples include actin promoters, immunoglobulin promoters and heat shock promoters. The early and late promoters of SV40 are conveniently obtained in the form of SV40 restriction fragments, which also contain the SV40 viral replication origin. Fiers et al., Nature 273: 113-120 (1978). The immediate early promoter of human cytomegalovirus is conveniently obtained in the form of HindIII E restriction fragments. Greenaway, PJ et al., Gene 18: 355-360 (1982). The above references are incorporated by reference in their entirety.

[0159] In some embodiments, the PD-1 engaging daughter cells are derived from stem cells.

[0160] The term "pluripotent cell" refers to a cell that can self-renew and proliferate while maintaining an undifferentiated state, and under appropriate conditions, can be induced to differentiate into a specialized cell type. The term "pluripotent cell" used herein includes embryonic stem cells (ESC) and other types of stem cells, including fetal stem cells, amniotic membrane stem cells or somatic stem cells. Exemplary human stem cell lines include H9 human embryonic stem cell lines. Other exemplary stem cell lines include stem cell lines available through the National Institutes of Health Human Embryonic Stem Cell Registry (National Institutes of Health Human Embryonic Stem Cell Registry) and the Howard Hughes Medical Institute HUES collection (Howard Hughes Medical Institute HUES collection) (such as Cowan, CA et.al, New England J. Med. 350: 13 (2004), incorporated herein by reference in its entirety).

[0161] "Pluripotent stem cells" as used herein have the potential to differentiate into any of the following three germ layers: endoderm (e.g., gastric junction, gastrointestinal tract, lung, etc.), mesoderm (e.g., muscle, bone, blood, urogenital tissue, etc.), or ectoderm (e.g., epidermal tissue and nervous system tissue). The term "pluripotent stem cell" as used herein also includes "induced pluripotent stem cells" or "iPSC," a pluripotent stem cell derived from a non-pluripotent cell. Examples of parental cells include somatic cells that have been reprogrammed to induce a pluripotent, undifferentiated phenotype in various ways. Such "iPS" or "iPSC" cells can be produced by inducing the expression of certain regulatory genes or by exogenous application of certain proteins. Methods for inducing iPS cells are known in the art and are further described below (see, for example, 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 herein by reference in its entirety). The generation of induced pluripotent stem cells (iPSCs) is summarized as follows. As used herein, "hiPSCs" are human induced pluripotent stem cells, while "miPSCs" are mouse induced pluripotent stem cells.

[0162] "Pluripotent stem cell characteristics" refers to the cell characteristics that distinguish pluripotent stem cells from other cells. Under appropriate conditions, it is a pluripotent stem cell characteristic to be able to produce offspring that differentiate into cell types, wherein these cell types jointly show characteristics associated with cell lineages from all three germ layers (endoderm, mesoderm, and ectoderm). The expression or non-expression of certain molecular marker combinations is also a characteristic of pluripotent stem cells. For example, human pluripotent stem cells express at least several, and in some embodiments, express all markers from the following non-limiting list: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49 / 6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Rex1, and Nanog. The cell morphology associated with pluripotent stem cells is also a pluripotent stem cell characteristic. As described herein, cells do not need to be reprogrammed into endoderm progenitor cells and / or hepatocytes through pluripotency.

[0163] B. Generation of low-immunogenic PD-1 engaged daughter cells

[0164] The production of HI cells can be completed with only three genetic changes, thereby minimizing the damage to cell activity but conferring cellular immune silence. This technology is disclosed in WO2018 / 132783, WO2020 / 018620, WO2020 / 018615, PCT / US2020 / 032272 and U.S. Patent Application Nos. 16 / 870,959 and 16 / 870,960, which are incorporated herein by reference in their entirety. These technologies will be briefly discussed below.

[0165] As described herein, one embodiment utilizes reduction or elimination of the protein activity of MHC I and II (HLA I and II when the cell is human). This can be achieved by changing the genes encoding its 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 reduce gene translation. Another embodiment is to change the gene that regulates the susceptibility of macrophage phagocytosis. This may be a "knock-in" of the gene using viral technology.

[0166] 1. HLA-I reduction

[0167] The HIPD-1 engager cells of the present invention include attenuation of MHC I (HLA I when the cells are derived from human cells) function.

[0168] As will be appreciated by those skilled in the art, reduction of function can be accomplished in a variety of ways, including removal of nucleic acid sequences from a gene, interruption of a sequence with another sequence, or alteration of regulatory components of the nucleic acid. For example, all or part of the coding region of a target gene can be removed or replaced with a "nonsense" sequence, frameshift mutations can be performed, all or part of a regulatory sequence such as a promoter can be removed or replaced, a translation initiation sequence can be removed or replaced, etc.

[0169] As will be appreciated by those skilled in the art, successful reduction of MHC I (HLA I when the cells are derived from human cells) function in PD-1 engaged cells can be measured as follows using techniques known in the art; for example, using FACS techniques with labeled antibodies that bind to the HLA complex; for example, using commercially available HLA-A, B, C antibodies that bind to the alpha chain of human major histocompatibility HLA class I antigens.

[0170]

a. β2M changes

[0171] In one embodiment, the reduction of HLA-I activity is accomplished by interfering with the expression of the beta-2 microglobulin gene in HIPD-1 zygote cells, as disclosed herein. This alteration is generally referred to herein as a gene "knockout," and in the cells of the invention, it is accomplished on both alleles in the host cell. Typically, the techniques for performing these two perturbations are the same.

[0172] One particularly useful embodiment uses CRISPR technology to interfere with the gene. Another embodiment uses programmable transcriptional memory ( JK, Cell. 184: 2503-2519 (2021), incorporated herein by reference in its entirety). In some cases, CRISPR technology is used to make small deletions / insertions into the coding region of a gene so that no functional protein is produced, which is usually the result of a frameshift mutation that results in the generation of a stop codon, thereby producing a truncated, non-functional protein.

[0173] Therefore, a useful technique is to use a designed CRISPR sequence to target the coding sequence of the B2M gene in mice or the B2M gene in humans. After gene editing, the transfected PD-1 zygote cell culture is dissociated into single cells. The single cells are expanded into full-size populations and CRISPR editing is detected by screening for the presence of abnormal sequences from the CRISPR cutting site. A clone in which both alleles are deleted is selected. This clone does not express B2M, as shown by PCR, nor does it express HLA-I, as shown by FACS analysis.

[0174] Assays for detecting whether the B2M gene has been inactivated are known and described herein. In one embodiment, the assay is a Western blot of cell lysates probed with B2M protein antibodies. In another embodiment, reverse transcriptase polymerase chain reaction (rt-PCR) confirms the presence of the inactivating change.

[0175] In addition, the cells can be tested to confirm that the HLA I complex is not expressed on the cell surface. As described above, this can be determined by FACS analysis using antibodies against one or more HLA cell surface components.

[0176] 2. HLA-II reduction

[0177] In some embodiments, in addition to the reduction of HLA I, the HIPD-1 engager cells of the present invention may also lack MHC II function (HLA II is from a human-derived cell).

[0178] As will be appreciated by those skilled in the art, reduction of function can be achieved in a variety of ways, including removal of nucleic acid sequences from a gene, addition of nucleic acid sequences to a gene, disruption of the reading frame, interruption of a sequence with another sequence, or alteration of the regulatory elements of the nucleic acid. In one embodiment, all or part of the coding region of the target gene can be removed or replaced with a "nonsense" sequence. In another embodiment, regulatory sequences such as promoters can be removed or replaced, translation initiation sequences can be removed or replaced, etc.

[0179] The successful reduction of MHC II (HLA II) function in PD-1 engaged daughter cells or their derivatives can be measured using techniques known in the art, such as Western blotting using protein antibodies, FACS technology, rt-PCR technology, and the like.

[0180]

a.CIITA changes

[0181] In one embodiment, the reduction of HLA-II activity is accomplished by disrupting the expression of the CIITA gene in PD-1 conjugated cells, as shown herein. This change is generally referred to as gene "knockout" herein, and in the PD-1 conjugated cells of the present invention, it is accomplished on both alleles of the host cell.

[0182] Assays to test whether the CIITA gene has been inactivated are known and described herein. In one embodiment, the assay is a Western blot of cell lysates probed with CIITA protein antibodies. In another embodiment, reverse transcriptase polymerase chain reaction (rt-PCR) confirms the presence of the inactivating change.

[0183] In addition, cells can be tested to confirm that the HLA II complex is not expressed on the cell surface. Again, the assay is performed in a manner known in the art. Example analyses include Western Blot or FACS analysis using commercial antibodies that bind to human HLA class II HLA-DR, DP, and most DQ antigens, as described below.

[0184] One particularly useful embodiment uses CRISPR technology to disrupt the CIITA gene. CRISPR ae is designed to target the coding sequence of the CIITA gene, which is an essential transcription factor for all MHC II molecules. After gene editing, the transfected cell cultures are dissociated into single cells. They are expanded into full-size populations and the success of the CRISPR editing is tested by screening for the presence of abnormal sequences at the CRISPR cutting site. Deletion clones that do not express CIITA are identified by PCR and may show non-expression of MHC II / HLA-II by FACS analysis. Another embodiment uses programmable transcriptional memory through CRISPR-based epigenome editing.

[0185] 【3. O blood type Rh negative cells】

[0186] Blood products can be divided into different groups (ABO blood types) based on the presence or absence of antigens on the surface of each red blood cell in a person's body. The A, B, AB, and A1 antigens are determined by the oligosaccharide sequences on the red blood cell glycoproteins. The genes in the blood group antigen group provide the instructions for making the antigen proteins. The blood group antigen proteins have a variety of functions within the cell membrane of the red blood cells. These protein functions include transporting other proteins and molecules into and out of the cell, maintaining cell structure, attaching to other cells and molecules, and participating in chemical reactions.

[0187] The Rhesus factor (Rh) blood group system is the second most important blood group system after the ABO blood group system. The Rh blood group system consists 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 with a positive or negative suffix after the ABO type. The terms "Rh factor," "Rh positive," and "Rh negative" refer only to the Rh(D) antigen. Antibodies to the Rh antigens can be involved in hemolytic transfusion reactions, and antibodies to the Rh(D) and Rh(c) antigens pose a significant risk of hemolytic disease to the fetus and newborn. ABO antibodies are formed early in everyone's life. However, Rh - Rhesus antibodies in humans are only produced when humans are sensitized. This is done by giving birth to rh + The baby may receive Rh + This is achieved through blood transfusion.

[0188] The present invention provides a method for producing a human prostate having ABO type O and / or rhesus factor negative (O - )Pluripotent (PSCO - ) cell population, suitable for transplantation and / or differentiation. - Induced pluripotent stem cells (iPSCs) - ), embryonic ESCs (ESC - ) and cells differentiated from these cells, including O - Endothelial cells, O - Myocardial cells, O - Hepatocytes, O - Dopaminergic neurons, O - Pancreatic islet cells, O - Retinal pigment epithelial cells, and other O cells for transplantation and drug therapy - cell types. These would include O - Chimeric antigen receptor (CAR) cells, such as CAR-T cells, CAR-NK cells and other engineered cell populations. In some embodiments, these cells are not hematopoietic stem cells. The present invention also provides generally accepted "off-the-shelf" ESCOs. - s and PSCO - s and their derivatives for the generation or regeneration of specific tissues and organs.

[0189] Another aspect of the present invention provides for the production of PSCO for transplantation - 、iPSCO - 、ESCO - and other O - The invention also provides methods of treating diseases, disorders and conditions that benefit from transplantation of pluripotent or differentiated cells.

[0190] In some embodiments of the invention, ABO blood type O is caused by decreased expression of ABO blood type proteins. In other aspects, the ABO blood type is endogenous type O. In certain aspects of the invention, HIPO - The cell has ABO blood type O, which is caused by the destruction of human exon 7 of the ABO gene. In some embodiments, both alleles of exon 7 of the ABO gene are destroyed. In some embodiments, the destruction of both alleles of exon 7 of the ABO gene is caused by a clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 reaction that destroys both alleles. Another embodiment uses programmable transcriptional memory to inactivate the gene through CRISPR-based epigenome editing.

[0191] In other aspects, ABO blood group O is produced by enzymatic modification of ABO gene products on the cell surface. In a preferred aspect, the enzymatic modification removes carbohydrates from the ABO gene products. In another preferred aspect, the enzymatic modification removes carbohydrates from the ABO A1 antigen, A2 antigen or B antigen.

[0192] In some embodiments of the present invention, the Rh blood type is endogenous Rh - On the other hand, Rh - Blood type is the result of reduced or eliminated Rh protein expression. - The type is caused by disrupting a gene encoding Rh C antigen, Rh E antigen, Kell K antigen (KEL), Duffy (FY) Fya antigen, Duffy Fy3 antigen, Kidd (JK) Jkb antigen, or / and or Kidd SLC14A1. In some embodiments, the disruption is caused by a CRISPR / Cas9 reaction that disrupts both alleles of a gene encoding Rh C antigen, Rh E antigen, Kell K antigen (KEL), Duffy (FY) Fya antigen, Duffy Fy3 antigen, Kidd (JK) Jkb antigen, or / and or Kidd SLC14A1.

[0193] In some embodiments of the present invention, the O - Cells (e.g. PSCO -、iPSCO - 、ESCO - and cells derived therefrom) are from a mammal, 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.

[0194] In a specific embodiment, the present invention provides a human ABO blood type O rhesus monkey factor negative (HIPO - ) conjugated cells with low immunity PD-1 conjugated cells, these cells escape the rejection reaction of the host allogeneic immune system and avoid blood antigen type rejection reaction. In some embodiments, HIPO - The cells are engineered to reduce or eliminate the expression of HLA-I and HLA-II, increase the expression of endogenous proteins, reduce the sensitivity of pluripotent cells to macrophage phagocytosis, and contain the universal blood type O Rh - (“O - ”) blood type. Universal blood typing can be achieved by eliminating ABO blood group A and B antigens and Rh factor expression, or starting with the O cell line. These new HIPO - The cells evade host immune rejection because of their impaired antigen presentation capacity and protection from innate immune clearance, and lack of blood group rejection.

[0195] 4. Suicide gene

[0196] In some embodiments, the present invention provides HIPD-1 zygote cells comprising a "suicide gene" or "suicide switch". These are incorporated to act as a "safety switch" that may cause cell death if the cell grows and divides in an unexpected manner. The "suicide gene" ablation method includes a suicide gene in a gene transfer vector that encodes a protein that causes cell killing only when activated by a specific compound. The suicide gene may encode an enzyme that selectively converts non-toxic compounds into highly toxic metabolites. The result is the specific elimination of cells expressing the enzyme. 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 entirety.

[0197] 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 sequence of human FK506 binding protein FKBP12, has an F36V mutation, and is connected to the gene encoding human caspase 9 through a series of amino acids. FKBP12-F36V binds to the small molecule dimerizer AP1903 with high affinity. Therefore, in the present invention, the suicide function of iCasp9 is triggered by the administration of a dimerization chemical inducer (CID). In some embodiments, CID is the small molecule drug AP1903. Dimerization leads to rapid induction of apoptosis (see WO2011146862; 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 herein incorporated by reference in its entirety).

[0198] In other embodiments, the binding epitope of existing CAR T cells is included in the extracellular domain of the conjugated daughter cell as a safety strategy. This binding epitope can be CD19, CD19 extracellular domain or specific anti-CD19 binding domains from, for example, CAR T cells FMC63, 4G7-2E3 or 3B10 (see Klesmith JR, et al., Biochemistry.3; 58 (48): 4869-4881 (2019), incorporated herein by reference in their entirety). In order to eliminate PD-1-conjugated daughter cells, CAR T cells with anti-CD19 binding domains incorporated by targeting are used. CAR T cells detect the incorporated binding epitopes and kill conjugated daughter cells. The binding domain 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.

[0199] 【5.Fc isolation】

[0200] If the antibody binds to unprotected cells through its Fab region, the Fc can bind to NK cells (mainly through its CD16 receptor), macrophages (mainly through their CD16, CD32 or CD64), B cells (mainly through CD32) or granulocytes (mainly through CD16, CD32 or CD64). These can mediate antibody-dependent cellular cytotoxicity (ADCC). If complement binds to Fc, complement-dependent cytotoxicity (CDC) can be induced.

[0201] In some embodiments, the PD-1 engaged daughter cells of the present invention contain elevated levels of receptors that recognize the Fc portion of IgG. Receptors that recognize the Fc portion of IgG are divided into four different categories: FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIV. This reduces the tendency of the immune system of the cell transplant recipient to reject allogeneic material. Cells expressing elevated CD16, CD32, CD64, or truncated CD64 escape ADCC or CDC. Fc isolation is disclosed in WO2021076427, which is incorporated herein by reference in its entirety.

[0202] 【6. SIRPα conjugated daughter cells】

[0203] In some embodiments, the cells of the present invention, when transplanted into a subject, also have enhanced signal regulatory protein α (SIRPα) binding function (PD-1 / SIRPα binding daughter cells) compared to parent cells with unmodified SIRPα binding function, which binding function resists innate immunity. In some embodiments, the PD-1 / SIRPα binding daughter cells are low immune cells. In other embodiments, the PD-1 / SIRPα binding daughter cells are differentiated somatic cells. In other embodiments, the PD-1 / SIRPα binding daughter cells are pluripotent cells or low immune pluripotent (HIP) cells. In a further embodiment, the HIP cells are blood type O (HIPO), rhesus factor (Rh) negative (HIP - ) or O and Rh - Type (HIPO - In other embodiments, the PD-1 / SIRPα ligand cells have been derived from or differentiated from HIP, HIP - or HIPO - Cells. In other embodiments, the PD-1 / SIRPα engaging cell comprises an antibody Fc receptor to protect the cell from antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). SIRPα engaging cells are described in PCT / US21 / 62008, which is incorporated herein by reference in its entirety.

[0204] 【7.HI phenotype determination】

[0205] Once HI cells are generated, their low immunogenicity can be assayed as generally described herein.

[0206] For example, low immunogenicity is determined using a variety of techniques. An example technique includes transplantation into an allogeneic host and monitoring HIPD-1 zygote cell survival. Cells can be transduced to express luciferase, which can then be tracked using bioluminescent imaging. Similarly, the T cells or B cells of the host animal are tested for reaction to the HIPD-1 zygote cells to confirm that they do not cause an immune response in the host animal. T cell function is assessed by Elispot, Elisa, FACS, PCR or mass cytometry (CYTOF). B cell response or antibody response is assessed using FACS or luminex. In addition, or, the ability of cells to avoid innate immune responses, such as NK cell killing, can be detected. The cytolytic activity of NK cells is assessed in vitro or in vivo using techniques known in the art.

[0207]

【C. Low immunity (HI) - Generation of PD-1 Engagement Daughter Cells

[0208] In certain aspects of the invention, the PD-1 conjugated daughter cells produced as described above will already be ABO blood type O and Rh factor negative (-) cells, since the process will be from cells with O - Blood type NK cells start.

[0209] Other aspects of the invention relate to enzymatic conversion of the A and B antigens. In a preferred aspect, the B antigen is converted to O using an enzyme. In a more preferred aspect, the enzyme is an α-galactosidase. This enzyme can eliminate the terminal galactose residue of the B antigen. Other aspects of the invention relate to enzymatic conversion of the A antigen to O. In a preferred aspect, α-N-acetylgalactosaminidase is used to convert the A antigen to O. Enzymatic conversion has been discussed, for example, Olsson et al., Transfusion Clinique et Biologique 11: 33-39 (2004); U.S. Patent Nos. 4,427,777, 5,606,042, 5,633,130, 5,731,426, 6,184,017, 4,609,627 and 5,606,042; and Int'l Pub. No. WO9923210, each of which is incorporated herein by reference in its entirety.

[0210] Other embodiments of the invention relate to genetically engineering cells by knocking out ABO gene exon 7 or silencing SLC14A1 (JK) gene. Other embodiments of the invention relate to knocking out C and E antigens of Rh blood group system (RH), K in Kell system (KEL), Fya and Fy3 in Duffy system (FY), Jkb in Kidd system (JK) or U and S in MNS blood group system. Any knockout method known in the art or described herein, such as CRISPR, talens or homologous recombination, can be used.

[0211] The production of hypoimmune ABO blood group O Rh factor ( - ) cell technology, which is incorporated herein by reference in its entirety.

[0212] [D. Embodiments of the present invention]

[0213] The PD-1 conjugate cells of the present invention or their derivatives can be used to treat, for example, type 1 diabetes, heart disease, neurological diseases, cancer, blindness, vascular diseases and other diseases / disorders that respond to regenerative medicine therapies. In particular, the invention contemplates the use of PD-1 conjugate cells to differentiate into any cell type. Thus, provided herein are iPSCs, ESCs, HIPs, iPSCOs, ESCOs, HIPOs, iPSCOs - 、ESCO - and HIPO - PD-1 ligand cells, or derivatives or differentiated cells thereof, which exhibit pluripotency, do not induce host immune response or significantly reduce the immune response when transplanted into an allogeneic host (such as a human patient).

[0214] In one aspect, the present invention provides a PD-1 engager cell or a derivative thereof, comprising a nucleic acid encoding a chimeric antigen receptor (CAR), wherein endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated, and a PD-1 engager molecule is provided on the cell surface. CAR may include an extracellular domain, a transmembrane domain, and an intracellular signal transduction 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, 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, and TnMUC1. In certain embodiments, the extracellular domain comprises a single-chain variable fragment (scFv). In some embodiments, the transmembrane domain includes CD3ζ, CD4, CD8α, CD28, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, CD64, PDGF and BTLA. In certain embodiments, the intracellular signal transduction domain includes CD3ζ, CD28, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3 and BTLA.

[0215] In certain embodiments, CAR comprises an anti-CD19 scFv domain, a CD28 transmembrane domain, and a CD3 ζ signal transduction intracellular domain. In some embodiments, CAR comprises an anti-CD19 scFv domain, a CD28 transmembrane domain, a 4-1BB signal transduction intracellular domain, and a CD3 ζ signal transduction intracellular domain.

[0216] In another aspect of the present invention, isolated PD-1 engaging CAR-T cells or low immune CAR-T cells are provided, which are produced by in vitro differentiation of any of the pluripotent cells described herein. In some embodiments, the CAR-T cells are cytotoxic HIPO - CAR-T cells.

[0217] In certain aspects, the present invention provides a PD-1 engaged NK or CAR-NK cell, or an iPSC-derived NK cell.

[0218] In various embodiments, in vitro differentiation includes culturing PD-1 engaged daughter cells or derivatives thereof, carrying CAR constructs in a culture medium containing 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 includes 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.

[0219] In a specific embodiment, the isolated PD-1 engaging CAR-T or CAR-NK cells are iPSCs, ESCs, HIPs, iPSCOs, ESCOs, HIPOs, iPSCOs carrying CAR-T constructs. - 、ESCO - or HIPO - In other embodiments, they are used to treat cancer.

[0220] In another aspect of the present invention, a method for treating a cancer patient by administering a composition comprising a therapeutically effective amount of any one of the isolated PD-1 engaging CAR-T CAR-NK cells is provided. In some embodiments, the composition further comprises a therapeutically effective carrier.

[0221] In some embodiments, the administering step comprises intravenous administration, subcutaneous administration, intranodal administration, intratumoral administration, intrathecal administration, intrapleural administration and intraperitoneal administration. In some cases, administration also comprises bolus injection or by continuous infusion.

[0222] In some embodiments, the cancer is a blood 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.

[0223] In another aspect, the present invention provides a method for preparing any of the isolated PD-1-engaged CAR-TCAR-NK cells described herein. The method comprises in vitro differentiation of iPSC, ESC, HIP, iPSCO, ESCO, HIPO, iPSCO of the present invention - 、ESCO - or HIPO -Any of the PD-1 ligands. In vitro differentiation may include culturing cells in a culture medium containing 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 includes 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.

[0224] In some embodiments, the in vitro differentiation comprises culturing iPSC, ESC, HIP, iPSCO, ESCO, HIPO, iPSCO on feeder cells. - 、ESCO - or HIPO - PD-1 engaged daughter cells. In various embodiments, the in vitro differentiation comprises culturing under simulated microgravity. In some cases, the culturing is under simulated microgravity for at least 72 hours.

[0225] In certain aspects, provided herein are isolated, engineered hypoimmunogenic cardiac cells, such as cardiomyocytes, derived from iPSC, ESC, HIP, iPSCO, ESCO, HIPO, iPSCO - 、ESCO - or HIPO - Differentiated from PD-1 engorged daughter cells.

[0226] It was previously thought that cardiomyocytes lacked ABO blood group antigens. The observation that differentiation of an ABO type B human embryonic stem cell line into cardiomyocyte-like cells results in loss of B antigens suggests that loss of these antigens may occur early in human embryogenesis. For example, see et al., Transplantation. 86(10):1407-13 (2008), incorporated herein by reference in its entirety. Other studies have also reported that differentiation of induced human pluripotent stem cells into cardiomyocyte-like cells results in a progressive loss of ABO blood group A antigens in these cells. See, for example, et al., Scientific Reports. 13072: 1-14 (2017). However, surprisingly, the inventors determined that cardiomyocytes express ABO blood group antigens, which would lead to the rejection of such cells to mismatched recipients.

[0227] Thus, in certain aspects, provided herein is a method of treating a patient with a cardiac problem or disease. The method comprises administering a composition comprising a cell derived from iPSC, ESC, HIP, iPSCO, ESCO, HIPO, iPSCO - 、ESCO - or HIPO - A therapeutically effective amount of any one of the isolated PD-1 engaging cardiomyocyte populations of PD-1 engaging daughter cells, as described herein. In some embodiments, the composition further comprises a therapeutically effective carrier.

[0228] In some embodiments, administration comprises implantation into cardiac tissue of a patient, intravenous injection, intra-arterial injection, intracoronary injection, intramuscular injection, intraperitoneal injection, intramyocardial injection, transendocardial injection, transepicardial injection, or perfusion.

[0229] In some embodiments, the cardiac disorder or disease is selected from pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, other cardiomyopathies, myocarditis, myocardial ischemia-reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, end-stage heart disease, atherosclerosis, ischemia, hypertension, restenosis, angina, rheumatic heart disease, arterial inflammation or cardiovascular disease.

[0230] In certain aspects, the present invention provides a method for generating a cardiac cell population from a PD-1 ligand cell population by in vitro differentiation, wherein endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated, and PD-1 ligand molecules are provided on the cell surface. The method comprises: (a) culturing a PD-1 ligand cell population in a culture medium containing a GSK inhibitor; (b) culturing a PD-1 ligand cell population in a culture medium containing a WNT antagonist to generate a precardiac cell population; and (c) culturing a precardiac cell population in a culture medium containing insulin to generate O - Low immune cardiomyocyte population. In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some cases, the concentration range 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 variant thereof. In some cases, the concentration range of the WNT antagonist is about 2 μM to about 10 μM.

[0231] In certain aspects, provided herein are cells derived from iPSCs, ESCs, HIPs, iPSCOs, ESCOs, HIPOs, iPSCOs - 、ESCO - or HIPO- In another aspect, the isolated, engineered PD-1 engaging endothelial cell is differentiated from a PD-1 engaging daughter cell. - or - The low-immunity 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.

[0232] In certain aspects provided herein are methods of treating a patient with a vascular problem or disease. In some embodiments, the method comprises administering a composition comprising a therapeutically effective amount of an isolated, engineered PD-1 engaging endothelial cell population.

[0233] In some embodiments, the method comprises administering a composition comprising a therapeutically effective amount of any of the isolated, engineered PD-1-engaging endothelial cell populations. In some embodiments, the composition further comprises a therapeutically effective carrier. In some embodiments, administration comprises implantation into a patient's cardiac tissue, intravenous injection, intra-arterial injection, intracoronary injection, intramuscular injection, intraperitoneal injection, intramyocardial injection, transendocardial injection, transepicardial injection, or infusion.

[0234] In some embodiments, the vascular disorder or disease is selected from vascular injury, cardiovascular disease, vascular disease, ischemic disease, myocardial infarction, congestive heart failure, hypertension, ischemic tissue damage, limb ischemia, stroke, neuropathy, and cerebrovascular disease.

[0235] In certain aspects, the present invention provides a method for producing a cell line derived from iPSC, ESC, HIP, iPSCO, ESCO, HIPO, iPSCO by in vitro differentiation. - 、ESCO - or HIPO - A method for generating a PD-1 engaged endothelial cell population in a PD-1 engaged cell population, wherein endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated, and PD-1 engager molecules are provided on the cell surface. The method comprises: (a) culturing cells in a first culture medium containing a GSK inhibitor; (b) culturing the cell population in a second culture medium containing VEGF and bFGF to generate an endothelial progenitor cell population; and (c) culturing the endothelial progenitor cell population in a third culture medium containing a ROCK inhibitor and an ALK inhibitor to generate a low immune endothelial cell population.

[0236] In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some cases, the concentration range 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 cases, the concentration range of the ROCK inhibitor is 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 cases, the concentration range of the ALK inhibitor is about 0.5 μM to about 10 μM.

[0237] In some embodiments, the first culture medium comprises 2 μM to about 10 μM CHIR-99021. In some embodiments, the second culture medium comprises 50 ng / ml VEGF and 10 ng / ml bFGF. In other embodiments, the second culture medium further comprises Y-27632 and SB-431542. In various embodiments, the third culture medium comprises 10 μM Y-27632 and 1 μM SB-431542. In certain embodiments, the third culture medium further comprises VEGF and bFGF. In special cases, the first culture medium and / or the second culture medium does not contain insulin.

[0238] In certain aspects, provided herein are isolated, engineered PD-1 engaging dopaminergic neurons (DNs) differentiated from PD-1 engaging daughter cells, wherein endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated, PD-1 engaging daughter molecules are provided on the cell surface, and the neurons are type O and Rh - Blood type.

[0239] In some embodiments, the isolated PD-1 engaging dopaminergic neuron is selected from the group consisting of a neuronal stem cell, a neuronal progenitor cell, an immature dopaminergic neuron, and a mature dopaminergic neuron.

[0240] In certain aspects, provided herein is a method for treating a patient with a neurodegenerative disease or condition. In some embodiments, the method comprises administering a composition comprising a therapeutically effective amount of any of the separated PD-1 engaged dopaminergic neuron populations. In some embodiments, the composition further comprises a therapeutically effective carrier. In some embodiments, the isolated population of low-immunity dopaminergic neurons is located on a biodegradable scaffold. In some embodiments, administration may include transplantation or injection. In some embodiments, the neurodegenerative disease or condition is selected from Parkinson's disease, Huntington's disease, and multiple sclerosis.

[0241] In certain aspects, provided herein is a method for generating a population of PD-1 engaged dopaminergic neurons from a population of PD-1 engaged cells by in vitro differentiation, wherein endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated, PD-1 engager molecules are provided on the cell surface, and blood types are O and Rh - In some embodiments, the method includes (a) culturing a cell population in a first culture medium comprising one or more of sonic hedgehog (SHH), BDNF, EGF, bFGF, FGF8, WNT1, retinoic acid, a GSK3β inhibitor, an ALK inhibitor, and a ROCK inhibitor to produce an immature dopaminergic neuron population; and (b) culturing the immature dopaminergic neuron population in a second culture medium different from the first culture medium to produce a dopaminergic neuron population.

[0242] In some embodiments, the GSKβ inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some cases, 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 cases, the concentration of the ALK inhibitor ranges from about 1 μM to about 10 μM. In some embodiments, there is no animal serum in the first culture medium and / or the second culture medium.

[0243] In some embodiments, the method further comprises isolating the hypoimmune dopaminergic neuron population from the non-dopaminergic neurons. In some embodiments, the method further comprises cryopreserving the isolated hypoimmune dopaminergic neuron population.

[0244] In certain aspects, provided herein are isolated PD-1 engaged low immune islet cells differentiated from PD-1 engaged daughter cells, wherein endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated, PD-1 engaged daughter molecules are provided on the cell surface, and blood types are O and Rh - .

[0245] In some embodiments, the isolated PD-1 engaging islet cell is selected from the group consisting of an islet progenitor cell, an immature islet cell, and a mature islet cell.

[0246] In certain aspects, provided herein is a method for treating a diabetic patient. The method comprises administering a composition comprising a therapeutically effective amount of any of the isolated PD-1-engaged islet cell populations described herein. In some embodiments, the composition further comprises a therapeutically effective carrier. In some embodiments, the isolated low-immunity islet cell population is located on a biodegradable scaffold. In some cases, administration comprises transplantation or injection.

[0247] In certain aspects, the present invention provides a method for obtaining a HIPO by in vitro differentiation. - Method for producing a PD-1 engaged islet cell population, wherein endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated, PD-1 engager molecules are provided on the cell surface, HIPO - The blood types in the cells are O and Rh - The method comprises: (a) culturing a PD-1 engaging cell population in a first culture medium, the culture medium comprising one or more factors selected from insulin-like growth factor (IGF), transforming growth factor (TGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), sonic hedgehog (SHH) and vascular endothelial growth factor (VEGF), transforming growth factor-β (TGFβ) superfamily, bone morphogenetic protein-2 (BMP2), bone morphogenetic protein-7 (BMP7), GSK3β inhibitor, ALK inhibitor, BMP type 1 receptor inhibitor and retinoic acid to produce an immature islet cell population; and (b) culturing the immature islet cell population in a second culture medium different from the first culture medium to produce a low immune islet cell population.

[0248] In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some cases, the concentration of the GSK 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 cases, the concentration of the ALK inhibitor ranges from about 1 μM to about 10 μM. In some embodiments, there is no animal serum in the first culture medium and / or the second culture medium.

[0249] In some embodiments, the method further comprises isolating the PD-1 engaging islet cell population from non-islet cells. In some embodiments, the method further comprises cryopreserving the isolated immuno-low islet cell population.

[0250] In certain aspects, provided herein are isolated, engineered PD-1-engaging retinal pigment epithelial (RPE) cells differentiated from PD-1-engaging daughter cells, wherein endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated, PD-1-engaging daughter molecules are provided on the cell surface, and blood types are O and Rh - .

[0251] In some embodiments, the isolated PD-1 engaged daughter RPE cells are selected from the group consisting of RPE progenitor cells, immature RPE cells, mature RPE cells, and functional RPE cells.

[0252] In certain aspects, provided herein is a method for treating an ocular disease patient. The method includes administering a composition comprising any PD-1 ligand RPE cell group of the separation of a therapeutically effective amount described herein. In some embodiments, the composition also includes a therapeutically effective carrier. In some embodiments, the isolated low-immunity RPE cell group is located on a biodegradable support. In some embodiments, administration includes transplanting or injecting into the patient's retina. In some embodiments, the eye problem is selected from wet macular degeneration, dry macular degeneration, juvenile macular degeneration, Leber congenital amaurosis, retinitis pigmentosa and retinal detachment.

[0253] In some aspects, there is provided herein a method for producing PD-1 from PD-1 ligand cell group by in vitro differentiation to engage retinal pigment epithelium (RPE) cell group, wherein endogenous beta-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated, and PD-1 ligand molecules are provided on cell surface. The method includes: (a) culturing PD-1 ligand cell group in a first culture medium, the culture medium comprising any factor in activin A, bFGF, BMP4 / 7, DKK1, IGF1, noggin, BMP inhibitor, ALK inhibitor, ROCK inhibitor and VEGFR inhibitor, to produce anterior RPE cell group; and (b) culturing anterior RPE cell group in a second culture medium different from the first culture medium, to produce low immune RPE cell group.

[0254] In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. In some cases, 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 cases, the concentration of the ROCK inhibitor ranges from about 1 μM to about 10 μM.

[0255] In some embodiments, animal serum is absent from the first culture medium and / or the second culture medium.

[0256] In some embodiments, the method further comprises isolating the PD-1 engaging RPE cell population from the non-RPE cells. In some embodiments, the method further comprises cryopreserving the isolated low immunogenic RPE cell population.

[0257]

E.HIPD-1 zygote cell transplantation

[0258] As will be appreciated by those skilled in the art, HIPD-1 conjugated cells are transplanted using techniques known in the art. Typically, the HIPD-1 conjugated cells of the present invention are transplanted into a specific location of a patient by intravenous injection or injection. When transplanted into a specific location, the cells may be suspended in a gel matrix to prevent them from dispersing as they take root.

[0259] In order to more fully understand the invention described herein, examples are listed below. It should be understood that these examples are for illustrative purposes only and should not be construed as limiting the present invention in any way.

[0260] [VIII. Embodiment]

[0261] [Example 1: PD-1 agonists expressed on engineered cells inhibit NK cell killing]

[0262] B2M - / - CIITA - / - iPSC-derived endothelial cells (DKO iECs) are rapidly killed by IL-2-activated NK cells. Using lentiviral transduction, PD-1 agonists were expressed in B2M - / - CIITA - / - As shown by bioluminescent imaging (BLI) killing assay, B2M - / - CIITA - / - The killing of PD-1 zygote transgenic iEC (DKO-PD-1 zygote) was significantly reduced ( Figure 1 ).

[0263] [Example 2: PD-1 agonists expressed on engineered cells inhibit CD8 T cell killing]

[0264] PBMCs from HLA-A2-negative allogeneic healthy donors were incubated with HLA-A2-positive wild-type (wt) iPSC-derived endothelial cells (iECs) for 14 days. Cells were harvested and analyzed for CD3 + CD8 + T cells were sorted. Wt iECs underwent T cell killing. PD-1 conjugates were expressed on wt iECs using lentiviral transduction. As shown in the BLI killing assay, killing of wt PD-1 conjugate transgenic iECs was significantly reduced ( Figure 2 ). [Example 3: PD-1 agonists expressed on engineered human pancreatic β cells inhibit CD8 T cell killing]

[0265] PBMCs from HLA-A2-negative allogeneic healthy donors were incubated with HLA-A2-positive wild-type (wt) iPSC-derived endothelial cells (iECs) for 14 days. Cells were harvested and analyzed for CD3 + CD8 + T cells were sorted. HLA-A2 positive human pancreatic β cells were grown on in vitro impedance assay E plates. In the top row, the β cells were unmodified. In the bottom row, the β cells expressed the PD-1 engager. The PD-1 engager was expressed using lentiviral transduction. Detection in flow cytometry was performed using the PD-1 engager sequence (SEQ ID NO: 24) with a CD8a signal peptide (SEQ ID NO: 25) and a myc tag (SEQ ID NO: 26). Unmodified β cells underwent T cell killing. PD-1 engager transgenic β cells survived ( Figure 3A and 3B ).

[0266] [Example 4: PD-1 conjugated daughter cells bind to recombinant PD-1]

[0267] B2M - / - CIITA - / - PD-1 zygote transgenic iEC (DKO-PD-1 zygote) were incubated with recombinant human PD-1 and secondary antibodies. Flow cytometry histograms show specific binding of recombinant human PD-1 to DKO-PD-1 zygote cells ( Figure 4 ).

[0268] [Example 5: Generation of TIM3 conjugated daughter cells]

[0269] Lentivirus preparation. TIM3 conjugate (SEQ ID NO: 27) is expressed under the enhanced EF1α promoter. It includes 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). RFP-blasticidin dual fusion marker is expressed under RSV promoter.

[0270] The expression lentiviral vector was co-transfected with a lentiviral packaging plasmid (Cat#: HT-pack, Gentarget, San Diego) into a lentiviral production cell line (Cat#: TLV-C, Gentarget). According to GenTarget's virus production protocol, the lentivirus was packaged in DMEM medium containing 10% serum. The lentivirus was then concentrated to obtain the desired viral titer. Viral titers were measured by ELISA P24 assay according to kit production.

[0271] Lentiviral transduction of TIM3 conjugates. Human induced pluripotent stem cell (iPSC)-derived endothelial cells (iEC) with double knockout of B2M and CIITA (iEC DKO) were transduced with the above lentivirus in pre-coated 12-well plates. They were plated at 5×10 4 The cells were plated at a density of 100 μg / mL and then incubated overnight at 37°C and 5% CO2. The next day, the cells were incubated overnight at 37°C and 5% CO2 with lentiviral particles carrying the TIM3 conjugate transgene under an enhanced EF1α promoter (Gentarget, custom product). The lentivirus also contains an RFP selection marker and is used at a multiplicity of infection of 4. Polybrene (8 μg / mL, Millipore) was added to the culture medium, the plate was centrifuged at 800 g for 30 minutes, and then incubated overnight. The cell population was sorted using the RFP tag on a FACSAria (BD Biosciences).

[0272] Flow cytometry analysis shows that TIM3 zygote protein is expressed in transduced DKO induced embryonic stem cells (iECs). TIM3 zygote transgenes linked to a red fluorescent protein (RFP) tag were quantified by flow cytometry. The histogram shows the RFP signal of non-transduced iEC DKO (left peak) and transduced iEC DKO+TIM3-E (right peak), confirming their expression of TIM3 zygote ( Figure 5 ).

[0273] [Example 6: TIM3 engagement protects cells from immune cell killing]

[0274] Flow cytometric analysis of TIM3 surface expression on primary human peripheral blood NK cells and macrophages showed that engagement of TIM3 protected cells from invasion by NK and macrophages.

[0275] Fig. 6A showed that 96.6% of NK cells were positive for TIM3, Figure 3B showed that 67.2% of macrophages were positive for TIM3 ( Figure 6B ).

[0276] BLI killing assay was performed with NK cells within 2 hours. iEC DKO expressing firefly luciferase or iEC DKO+TIM3 conjugate (TIM3-E) expressing firefly luciferase were plated and allowed to adhere for 16 hours. Then, human peripheral blood NK cells were added to the wells and incubated for 2 hours. The percentage of BLI signal after incubation is shown. The BLI signal of iEC DKO was completely lost ( Fig. 7A The BLI signal of iEC DKO+TIM3-E remained stable within 2 hours ( Figure 7B ).

[0277] Fig. 8A and 8B : BLI killing assay within 2 hours with macrophages. Human iEC DKO or iEC DKO+TIM3-E expressing firefly luciferase were plated and allowed to adhere for 16 hours. Then, human peripheral blood macrophages were added to the wells and incubated for 2 hours. The percentage of BLI signal after incubation is shown. The BLI signal of iEC DKO completely disappeared ( Fig. 8A The BLI signal of iEC DKO+TIM3-E remained stable within 2 hours ( Figure 8B ).

[0278] Fig. 9A and 9B : BLI killing assay with NK cells within 24 hours. Human iEC DKO or iEC DKO+TIM3-E expressing firefly luciferase were plated and adhered for 16 hours. Then, human peripheral blood NK cells were added to the wells and incubated for 24 hours. The percentage of BLI signal after 24 hours of NK incubation is shown. The BLI signal of iEC DKO completely disappeared ( Fig. 9A The BLI signal of iEC DKO+TIM3-E remained stable over 24 hours ( Fig. 9B ).

[0279] Fig. 10A and 10B : BLI killing assay within 24 hours with macrophages. Human iEC DKO and iEC DKO+TIM3-E expressing firefly luciferase were plated and allowed to adhere for 16 hours. Then, human peripheral blood macrophages were added to the wells and incubated for 24 hours. The percentage of BLI signal after 24 hours of macrophage incubation is shown. The BLI signal of iEC DKO completely disappeared ( Fig. 10A The BLI signal of iEC DKO+TIM3-E remained stable within 24 hours and even increased ( Fig. 10B ).

[0280] [Example 7: Generation of LILRB3 conjugated daughter cells]

[0281] Lentivirus preparation. The LILRB3 conjugate (SEQ ID NO: 32) was expressed under the enhanced EF1α promoter. It included a CD8a signal peptide (SEQ ID NO: 28), a myc tag (SEQ ID NO: 26), an anti-LILRB3 antibody heavy chain variable region (SEQ ID NO: 33), a (GGGG)3 linker (SEQ ID NO: 30), an anti-LILRB3 antibody light chain variable region (SEQ ID NO: 34), a CD8a hinge (SEQ ID NO: 3) and a platelet-derived growth factor receptor (PDGFR) transmembrane domain (SEQ ID NO: 4). The RFP-blasticidin dual fusion marker was expressed under the RSV promoter.

[0282] The expression lentiviral vector was co-transfected with a lentiviral packaging plasmid (Cat#: HT-pack, Gentarget, San Diego) into a lentiviral production cell line (Cat#: TLV-C, Gentarget). According to GenTarget's virus production protocol, the lentivirus was packaged in DMEM medium containing 10% serum. The lentivirus was then concentrated to obtain the desired viral titer. Viral titers were measured by ELISA P24 assay according to kit production.

[0283] Lentiviral transduction of LILRB3 conjugates. Human induced pluripotent stem cell (iPSC)-derived endothelial cells (iEC) with double knockout of B2M and CIITA (iEC DKO) were transduced with the above lentivirus in pre-coated 12-well plates. They were plated at 5×10 4 The cells were plated at a density of 100 μg / mL and then incubated overnight at 37°C and 5% CO2. The next day, the cells were incubated with lentiviral particles carrying the LILRB3 conjugate transgene under the enhanced EF1α promoter (Gentarget) at 37°C and 5% CO2 overnight. The lentivirus also contained an RFP selection marker and was used at an infection multiplicity of 4. Polybrene (8 μg / mL, Millipore) was added to the culture medium and the plates were centrifuged at 800 g for 30 minutes and then incubated overnight. The cell population was sorted using the RFP tag on a FACSAria (BD Biosciences).

[0284] Flow cytometric analysis shows that LILRB3-zygote protein is expressed in transduced DKO induced embryonic stem cells (iECs). The LILRB3-zygote transgene linked to a red fluorescent protein (RFP) tag was quantified by flow cytometry. The histogram shows the RFP signal of non-transduced iEC DKO (left peak) and transduced iEC DKO+LILRB3-E (right peak), confirming their expression of LILRB3-zygote ( Fig.11 ).

[0285] [Example 8: LILRB3 engagement protects cells from immune cell killing]

[0286] Flow cytometric analysis of LILRB3 surface expression on primary human peripheral blood NK cells and macrophages showed that LILRB3 was expressed on some NK cells and most macrophages.

[0287] Fig. 12A showed that 17.8% of NK cells were positive for LILRB3, Fig. 12B It showed that 95.36% of macrophages were positive for LILRB3.

[0288] BLI killing assay was performed with NK cells within 2 hours. iEC DKO expressing firefly luciferase or iEC DKO+LILRB3-E expressing firefly luciferase were plated and allowed to adhere for 16 hours. Then, human peripheral blood NK cells were added to the wells and incubated for 2 hours. The percentage of BLI signal after incubation is shown. The BLI signal of iEC DKO disappeared completely ( Fig.13A The BLI signal of iECDKO+LILRB3-E decreased significantly less within 2 hours ( Fig. 13B ).

[0289] Fig.14A and 13B : BLI killing assay within 2 hours with macrophages. Human iEC DKO or iEC DKO+LILRB3-E expressing firefly luciferase were plated and allowed to adhere for 16 hours. Then, human peripheral blood macrophages were added to the wells and incubated for 2 hours. The percentage of BLI signal after incubation is shown. The BLI signal of iEC DKO completely disappeared ( Fig.14A The BLI signal of iEC DKO+LILRB3-E decreased significantly less within 2 hours ( Figure 4 B).

[0290] Fig.15A and 15B : BLI killing assay with NK cells over 24 hours. Human iEC DKO or iEC DKO+LILRB3-E expressing firefly luciferase were plated and allowed to adhere for 16 hours. Then, human peripheral blood NK cells were added to the wells and incubated for 24 hours. The percentage of BLI signal after 24 hours of NK incubation is shown. The BLI signal of iEC DKO completely disappeared ( Fig.15A The BLI signal of iECDKO+LILRB3-E decreased significantly less within 24 hours ( Fig. 15B ).

[0291] Fig.16A and 16B: BLI killing assay with macrophages within 24 hours. Human iEC DKO and iEC DKO+LILRB3-E expressing firefly luciferase were plated and allowed to adhere for 16 hours. Then, human peripheral blood macrophages were added to the wells and incubated for 24 hours. The percentage of BLI signal after 24 hours of macrophage incubation is shown. The BLI signal of iEC DKO completely disappeared ( Fig.16A The BLI signal of iEC DKO+LILRB3-E decreased significantly less within 24 hours, and even increased ( Fig. 16B ).

[0292] [Example 9: Generation of LILRB1 zygote cells]

[0293] Lentivirus preparation. The LILRB1 conjugate (SEQ ID NO: 35) was expressed under the enhanced EF1α promoter, and the RFP-blasticidin dual fusion marker was expressed under the RSV promoter. It includes 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-blasticidin dual fusion marker was expressed under the RSV promoter.

[0294] The expression lentiviral vector was co-transfected with a lentiviral packaging plasmid (Cat#: HT-pack, Gentarget, San Diego) into a lentiviral production cell line (Cat#: TLV-C, Gentarget). According to GenTarget's virus production protocol, the lentivirus was packaged in DMEM medium containing 10% serum. The lentivirus was then concentrated to obtain the desired viral titer. Viral titers were measured by ELISA P24 assay according to kit production.

[0295] Lentiviral transduction of LILRB1 conjugates. Human induced pluripotent stem cell (iPSC)-derived endothelial cells (iEC) with double knockout of B2M and CIITA (iEC DKO) were transduced with the above lentivirus in pre-coated 12-well plates. They were plated at 5×10 4The cells were plated at a density of 100 μg / mL and then incubated overnight at 37°C and 5% CO2. The next day, the cells were incubated with lentiviral particles carrying the LILRB1 conjugate transgene under the enhanced EF1α promoter (Gentarget) at 37°C and 5% CO2 overnight. The lentivirus also contained the RFP selection marker and was used at an infection multiplicity of 4. Polybrene (8 μg / mL, Millipore) was added to the culture medium and the plates were centrifuged at 800 g for 30 minutes and then incubated overnight. The cell population was sorted using the RFP tag on a FACSAria (BD Biosciences).

[0296] Flow cytometric analysis shows that LILRB1 conjugate protein is expressed in transduced DKO induced embryonic stem cells (iECs). The LILRB1 conjugate transgene linked to a red fluorescent protein (RFP) tag was quantified by flow cytometry. The histogram shows the RFP signal of non-transduced iEC DKO (left peak) and transduced iEC DKO+LILRB1-E (right peak), confirming their expression of LILRB1 conjugate ( Fig.17 ).

[0297] [Example 10: LILRB1 binding protects cells from immune cell killing]

[0298] Flow cytometric analysis of LILRB1 surface expression on primary human peripheral blood NK cells and macrophages showed that LILRB1 was expressed on many NK cells and most macrophages.

[0299] Fig.18A showed that 75.8% of NK cells were positive for LILRB1, Fig.18B It was shown that 99.96% of macrophages were positive for LILRB1.

[0300] BLI killing assay was performed with NK cells within 2 hours. iEC DKO expressing firefly luciferase or iEC DKO+LILRB1-E expressing firefly luciferase were plated and allowed to adhere for 16 hours. Then, human peripheral blood NK cells were added to the wells and incubated for 2 hours. The percentage of BLI signal after incubation is shown. The BLI signal of iEC DKO was completely lost ( Fig.19A The BLI signal of iECDKO+LILRB1-E remained stable within 2 hours ( Fig.19B ).

[0301] Fig. 20A and 20B: BLI killing assay within 2 hours with macrophages. Human iEC DKO or iEC DKO+LILRB1-E expressing firefly luciferase were plated and allowed to adhere for 16 hours. Then, human peripheral blood macrophages were added to the wells and incubated for 2 hours. The percentage of BLI signal after incubation is shown. The BLI signal of iEC DKO completely disappeared ( Fig. 20A The BLI signal of iEC DKO+LILRB1-E remained stable within 2 hours ( Fig. 20B ).

[0302] Fig.21A and 21B : BLI killing assay with NK cells within 24 hours. Human iEC DKO or iEC DKO+LILRB1-E expressing firefly luciferase were plated and allowed to adhere for 16 hours. Then, human peripheral blood NK cells were added to the wells and incubated for 24 hours. The percentage of BLI signal after 24 hours of NK incubation is shown. The BLI signal of iEC DKO completely disappeared ( Fig.21A The BLI signal of iECDKO+LILRB1-E remained stable over 24 hours ( Fig.21B ).

[0303] Fig.22A and 22B : BLI killing assay with macrophages over 24 hours. Human iEC DKO and iEC DKO+LILRB1-E expressing firefly luciferase were plated and allowed to adhere for 16 hours. Then, human peripheral blood macrophages were added to the wells and incubated for 24 hours. The percentage of BLI signal after 24 hours of macrophage incubation is shown. The BLI signal of iEC DKO was completely lost ( Fig.22A The BLI signal of iEC DKO+LILRB1-E remained stable and even increased over 24 hours ( Fig. 22B ).

[0304] [Example 11: Stable expression of immune checkpoint agonists after differentiation]

[0305] Expression of the PD-1 conjugate transgene was maintained during differentiation. iPSC DKO were transduced using lentiviral particles (iPSC DKO+PD-1-E) to express the PD-1 conjugate. Flow cytometry showed that robust expression could be achieved ( Fig.23A When iPSCDKO+PD-1-E differentiated into iEC DKO+PD-1-E, transgene expression remained stable ( Fig. 23B ).

[0306] Expression of the TIM3-binding transgene is maintained during differentiation. iPSC DKO were transduced using lentiviral particles (iPSC DKO+TIM3-E) to express the TIM3-binding transgene. Flow cytometry showed that robust expression could be achieved ( Fig.24A When iPSCDKO+TIM3-E differentiated into iEC DKO+TIM3-E, transgene expression remained stable ( Fig. 24B ).

[0307] Maintaining expression of the LILRB3-zygote transgene during differentiation. iPSC DKO were transduced using lentiviral particles (iPSC DKO+LILRB3-E) to express the LILRB3-zygote. Flow cytometry showed that robust expression could be achieved ( Fig.25A When iPSC DKO+LILRB3-E differentiated into iEC DKO+LILRB3-E, transgene expression remained stable ( Fig.25B ).

[0308] Maintaining expression of the LILRB1 conjugate transgene during differentiation. iPSC DKO were transduced using lentiviral particles (iPSC DKO+LILRB1-E) to express the LILRB1 conjugate. Flow cytometry showed that robust expression could be achieved ( Fig.26A When iPSC DKO+LILRB1-E differentiated into iEC DKO+LILRB1-E, transgene expression remained stable ( Fig.26B ).

[0309] [Example 12: Threshold Levels of Immune Checkpoint Engagement Required for Protection]

[0310] Fig.27A and 27B The threshold of TIM3-E expression required to protect cells from NK cell killing is shown. To evaluate whether there is a functional threshold for TIM3-E expression to exert its protective effect against NK cell killing, iEC DKO was transduced with lentiviral particles carrying the TIM3-E transgene. After 3 days, the transduced iEC pool was divided into three groups based on TIM3-E expression. The three groups were labeled iEC DKO+TIM3-E high, medium and low ( Fig.27A For subsequent BLI killing assays, the different populations were incubated with NK cells for 2 h ( Fig.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 completely disappeared. The BLI signals of iEC DKO+TIM3-E high and medium remained stable, but the BLI signal of iEC DKO+TIM3-E low also dropped to the background. These results indicate that the functional threshold of TIM3-E is between the low and medium expression levels.

[0311] Fig.28A and 28B The threshold of LILRB1-E expression required to protect cells from NK cell killing is shown. To evaluate whether there is a functional threshold for LILRB1-E expression to exert its protective effect against NK cell killing, iEC DKO were transduced with lentiviral particles carrying the LILRB1-E transgene. After 3 days, the transduced iEC pool was sorted into three populations based on their LILRB1-E expression. The three populations were labeled iEC DKO+LILRB1-E high, medium, and low ( Fig.28A For subsequent BLI killing assays, the different populations were incubated with NK cells for 2 h ( Fig.28B ). All target cells were luciferase positive. The percentage of BLI signal after 2 h of NK incubation is shown. The BLI signal of iEC DKO completely disappeared. 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 suggest that the functional threshold of LILRB1-E is between the low and medium expression levels.

[0312]

method

[0313] NK cell culture Human primary NK cells were purchased from Stemcell Technologies (70036, Vancouver, Canada) and cultured in RPMI-1640 supplemented with 10% FCS hi and 1% pen / strep before assay.

[0314] Human iPSC culture and transduction to express firefly luciferase Human wt or B2M iPSCs were cultured in Essential 8Flex medium (ThermoFisher Scientific) on 10 cm dishes coated with dilute feeder-free Matrigel (hESC qualified, BD Biosciences, San Jose, CA). - / - CIITA - / - iPSCs. The medium was changed every 24 hours, and cells were passaged at a ratio of 1:6 using Versene (Gibco). For luciferase transduction, 1×10 5iPSCs were plated in 6 wells and incubated overnight at 37°C and 5% CO2. The next day, the medium was changed and a vial of Fluc lentiviral particles expressing the luciferase II gene under the re-engineered EF1α promoter (Gen Target, San Diego, CA) was added to 1.5 ml of medium. After 36 hours, 1 ml of cell culture medium was added. After 24 hours, a complete medium change was performed. After 2 days, luciferase expression was confirmed by adding D-luciferin (Promega, Madison, WI). The signal was quantified in p / s / cm2 / sr.

[0315] Differentiation of iPSC-derived endothelial cells (iEC). + wt or B2M - / - CIITA - / - iEC was obtained from FLuc as follows + B2M - / - CIITA - / -Differentiation from iPSC. The differentiation protocol was initiated when 60% iPSCs were aggregated, and the culture medium was replaced with RPMI-1640 (Gibco, Thermo Fisher Scientific brand) containing 2% B-27 without insulin (Gibco, Thermo Fisher Scientific brand) and 5 μM CHIR-99021 (Selleckchem, Munich, Germany). On the second day, the culture medium was replaced with a reducing medium: RPMI-1640 containing 2% B-27 without insulin (Gibco) and 2 μM CHIR-99021 (Selleckchem). From culture day 4 to day 7, cells were exposed to RPMI-1640EC medium containing 2% B-27 minus insulin and 50 ng / ml human vascular endothelial growth factor (VEGF; R and D Systems, Minneapolis, MN), 10 ng / ml human fibroblast growth factor basic (FGFb; R and D Systems), 10 μM Y-27632 (Sigma-Aldrich), and 1 μM SB 431542 (Sigma-Aldrich, St. Louis, MO). Endothelial cell clusters were visible from day 7 onwards, and cells were maintained in Endothelial Cell Basal Medium 2 (PromoCell, Heidelberg, Germany) plus supplements, 10% FCS hi (Gibco), 1% pen / strep, 25ng / ml VEGF, 2ng / ml FGFb, 10μM Y-27632 (Sigma-Aldrich), and 1μM SB 431542 (Sigma-Aldrich). The differentiation protocol was completed after 14 days; undifferentiated cells were separated during the differentiation process. TrypLE Express (Gibco) was used to passage cells at a 1:3 ratio every 3 to 4 days.

[0316] Differentiation of macrophages from PBMCs. PBMCs were separated from fresh blood by Ficoll and resuspended in RPMI-1640 containing 10% FCS HI and 1% penicillin-streptomycin (all Gibco). Cells were cultured at 1×10 6 Cells were plated at a concentration of 10 cells / ml in 24-well plates. 10 ng / ml human M-CSF (PeproTech) was added to the culture medium. The culture medium was changed every other day. Starting from day 6, 1 μg / ml human IL-2 (PeproTech) was added to the culture medium for 24 hours before the assay.

[0317] Lentiviral transduction of PD-1 agonists. iECs were plated at 5 × 104 The cells were plated at a density of 100 μg / mL and then incubated overnight at 37°C and 5% CO2. The next day, the cells were incubated overnight at 37°C and 5% CO2 with lentiviral particles carrying the PD-1 zygote transgene under an enhanced EF1α promoter (Gentarget, custom product). The lentivirus also contains an RFP selection marker and is used at a multiplicity of infection of 4. Polybrene (8 μg / m, Millipore) was added to the culture medium, the plate was centrifuged at 800g for 30 minutes, and then incubated overnight. The cell population was sorted using the RFP tag on a FACSAria (BD Biosciences).

[0318] iEC BLI NK cell killing assay. + B2M - / - CIITA - / - (DKO)iEC and B2M - / - CIITA - / - (DKO) PD-1 transgenic iECs were counted and 1×10 3 The concentration of cells was plated. 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 1 μg / mL concentration of human IL-2 (Life Technologies) for 72 hours. After 4 hours of BLI killing assay, luciferase expression was detected by adding D-luciferin (Promega). As a control, target cells were left untreated or treated with 2% Triton X-100 in cell-specific culture medium. The signal was quantified with Ami HT (spectral instrument imaging) at p / s / cm2 / sr, and the relative BLI intensity relative to untreated target cells was measured.

[0319] In vitro T cell sensitization. Blood from HLA-A2 negative donors was collected and PBMCs were obtained after Ficoll separation. 5 × 10 5 HLA-A2 positive wt iEC cells and 1×10 6PBMCs were sensitized with 100 PBMCs. The culture medium consisted of a 1:1 mixture of endothelial cell culture medium and PBMC culture medium and was changed every 3 days. After 14 days, cells in suspension were harvested and sorted using the following substances: APC mouse anti-human CD3 antibody (clone SP34-2, catalog no. 557597, BD Biosciences) and isotype-matched control APC mouse IgG1 antibody (clone MOPC-21, catalog no. 550854, BD Biosciences) and BV421 mouse anti-human CD8 antibody (clone SK1, catalog no. 344748, Biolegend) and isotype-matched control mouse IgG1 antibody (clone MOPC-21, catalog no. 400157, Biolegend). CD3 was quantified using a FACS Aria flow cytometer (BD Biosciences). + CD8 + Cells were sorted and used for CD8 T cell killing assays.

[0320] iEC BLI CD8 T cell killing assay. + wt iEC and wt PD-1 transgenic iEC were counted and plated at 1 × 10 per 96-well plate. 3 The target cells were plated at a concentration of 10 cells. All target cells were then mixed with ex vivo sensitized CD8 T cells at an E:T ratio of 10:1. Luciferase expression was detected by adding D-luciferin (Promega) 4 hours after the BLI killing assay. As a control, target cells were left untreated or treated with 2% Triton X-100 in cell-specific culture medium. The signal was quantified with Ami HT (spectral instrument imaging) at p / s / cm2 / sr.

[0321] Human pancreatic β cells. Human iPSC-derived pancreatic β cells were purchased from TaKaRa (ChiPSC22, catalog number Y10106) and cultured in the Cellartis hiPSβ cell culture medium kit (TaKaRa, catalog number Y10108). Cells were plated in 12-well plates according to the manufacturer's protocol. Some cells were transduced with PD-1-engaged lentiviral particles (GenTarget).

[0322] Islet cells (pancreatic beta cells) with and without PD-1-engager expression were then plated on the XCelligence platform. They attached to the plastic dish but also grew in clusters, with a cell index that increased over time. Despite the clustered growth pattern, the assay was still very sensitive in detecting target cell killing. Islet cells display the A2 type of HLA. Therefore, we used sensitized CD3 + CD8 + T cells were generated as effector cells as described above. Unedited islet cells were killed by primed T cells with typical T cell killing kinetics. In contrast, islet β cells expressing the PD-1 engager were protected from killing by primed T cells.

[0323] To show the binding of DKO PD-1-engaging iECs to PD-1, we used recombinant human PD-1 protein (FcChimera Active, ab221398, Abcam, 0.1 μg / mL) and mouse anti-human IgG Fc secondary antibody (3D3cc, Novus Biologicals, allophycocyanin, 1:200). Flow cytometry was performed on an LSRII (BD Biosciences).

[0324] [Example sequence]

[0325] [SEQ ID NO: 1: PD-1 engager with IL-2 signal peptide]

[0326]

[0327] [SEQ ID NO: 2: IL-2 signal peptide]

[0328] MYRMQLLSCI ALSLALVTNS

[0329] [SEQ ID NO: 3: CD8a hinge]

[0330] TTTPAPRPPT PAPTIASQPL SLRPEACRPA AAVHTRGLDF ACD

[0331] [SEQ ID NO: 4: Platelet-derived growth factor receptor (PDGFR) transmembrane domain (TMD)]

[0332] AAVLVLLVIV IISLIVLVVI W

[0333] [SEQ ID NO: 5: CD47 transmembrane domain (TMD)]

[0334] NILIVIFPIF AILLFWGQFG IKTLKYRSGG MDEKTIALLV AGLVITVIVI VGAILFVPGE

[0335] YSLKNATGLG LIVTSTGILI LLHYYVFSTA IGLTSFVIAI LVIQVIAYIL AVVGLSLCIA

[0336] ACIPMHGPLL ISGLSILALA QLLGLVYM

[0337] [SEQ ID NO: 6: CD64 transmembrane domain (TMD)]

[0338] VLFYLAVGIM FLVNTVLWVT I

[0339] [SEQ ID NO: 7: CD47 intracellular domain (ICD)]

[0340] KFVASNQKTIQPPRKAVEEP LNAFKESKGMMNDE

[0341] [SEQ ID NO: 8: Human SIRPα]

[0342] >NP_001317657.1 Tyrosine protein phosphatase non-receptor substrate 1 isoform 2 precursor [Homo sapiens]

[0343]

[0344]

[0345] [SEQ ID NO: 9: human CD47]

[0346] >NP_001768.1 Leukocyte surface antigen CD47 isoform 1 precursor [Homo sapiens]

[0347]

[0348] [SEQ ID NO: 10: CD47 extracellular domain (ECD)]

[0349]

[0350] [SEQ ID NO: 11: CD47 immunoglobulin superfamily domain]

[0351]

[0352] [SEQ ID NO: 12: PD-L1 (isoform 1)]

[0353]

[0354] [SEQ ID NO: 13: PD-L1 (isoform 2)]

[0355]

[0356] [SEQ ID NO: 14: PD-L1 (isoform 3)]

[0357]

[0358] [SEQ ID NO: 15: PD-L2 (isoform 1)]

[0359]

[0360] [SEQ ID NO: 16: PD-L2 (isoform 2)]

[0361]

[0362] [SEQ ID NO: 17: PD-L2 (isoform 3)]

[0363]

[0364] [SEQ ID NO: 18: Antibody 1 heavy chain CDR1]

[0365] KVSGYSLSKY DMS

[0366] [SEQ ID NO: 19: Antibody 1 heavy chain CDR2]

[0367] IIYTSGYTDY AQKFQG

[0368] [SEQ ID NO: 20: Antibody 1 heavy chain CDR3]

[0369] ATGNPYYTNG FNS

[0370] [SEQ ID NO: 21: Antibody 1 light chain CDR1]

[0371] QASQSPNNLL A

[0372] [SEQ ID NO: 22: Antibody 1 light chain CDR2]

[0373] YGASDLPS

[0374] [SEQ ID NO: 23: Antibody 1 light chain CDR3]

[0375] QNNYYVGPVS YA

[0376] [SEQ ID NO: 24: PD-1 engager with CD8a signal peptide and myc tag]

[0377]

[0378] [SEQ ID NO: 25: CD8a signal peptide]

[0379] MALPVTALLL PLALLHAAR P

[0380] [SEQ ID NO: 26: myc tag]

[0381] EQKLISEEDL

[0382] [SEQ ID NO: 27: TIM3 ligand protein]

[0383]

[0384] [SEQ ID NO: 28: CD8a signal peptide]

[0385] MALPVTALLL PLALLHAAR P

[0386] [SEQ ID NO: 29: Heavy chain variable region (V H )】

[0387]

[0388] [SEQ ID NO: 30: (GGGG) 3 linker]

[0389] GGGGSGGGGS GGGGS

[0390] [SEQ ID NO: 31: Light chain variable region (V L )】

[0391]

[0392] [SEQ ID NO: 32: LILRB3 conjugate protein]

[0393]

[0394] [SEQ ID NO: 33: Heavy chain variable region (V H )】

[0395]

[0396] [SEQ ID NO: 34: Light chain variable region (V L )】

[0397]

[0398] [SEQ ID NO: 35: LILRB1 conjugate protein]

[0399]

[0400] [SEQ ID NO: 36: Heavy chain variable region (V H )】

[0401]

[0402] [SEQ ID NO: 37: Light chain variable region (V L )】

[0403]

[0404] All publications and patent documents disclosed or mentioned herein are incorporated herein by reference in their entirety. The above description is for illustration and description purposes only. This description is not intended to limit the invention to the precise form disclosed. The scope of the invention is intended to be defined by the claims appended hereto.

Claims

1. An immune checkpoint engager (ICE) cell comprising an engager molecule expressed on the cell surface, wherein the engager molecule engages with an immune checkpoint molecule on an immune cell, wherein the engager molecule is expressed at least at a level that protects the ICE cell from being killed by the immune cell. 2 . The ICE cell according to claim 1 , wherein the engager molecule is a protein that does not bind to SIRPα on the immune cell.

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

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

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

6. The ICE cell according to claim 5, wherein the antibody VH domain comprises at least 90% sequence identity with amino acid numbers 146 to 264 of SEQ ID NO:

1. 7 . The ICE cell according to claim 6 , wherein the antibody VH domain comprises the sequence of amino acid numbers 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 according to claim 8, wherein the antibody VL domain comprises at least 90% sequence identity with amino acid numbers 21 to 130 of SEQ ID NO:

1. 10 . The ICE cell according to claim 9 , wherein the antibody VH domain comprises the sequence of amino acid numbers 21 to 130 of SEQ ID NO:

1.

11. The ICE cell according to 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 comprises at least 90% sequence identity to the IL-2 signal peptide of SEQ ID NO:

2.

13. The ICE cell according to 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 comprises at least 90% sequence identity to the CD8a hinge peptide of SEQ ID NO:

3.

15. The ICE cell according to claim 14, wherein the fusion protein comprises the CD8a hinge peptide of SEQ ID NO:

3.

16. The ICE cell of claim 11, wherein the fusion protein comprises at least 90% sequence identity to the PDGFR transmembrane domain (TMD) of SEQ ID NO:

4.

17. The ICE cell according to claim 16, wherein the fusion protein comprises PDGFRTMD of SEQ ID NO:

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 a sequence identity of SEQ ID NO:

1.

20. The ICE cell according to 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 ICE cell of claim 20, wherein the Fab or scFV binds to PD-1 with an affinity measured by its dissociation constant (Kd), wherein the Kd is about 10 -7 and 10 -13 Between M.

22. The ICE cell according to 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 the one or more CDRs have at least 90% sequence identity with any one of SEQ ID NOs: 18 to 23.

24. The PD1 engager cell according to 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 a single alpha helix, multiple alpha helices, or a coiled beta sheet.

26. The ICE cell of claim 24, wherein the heterologous TMD is selected from the group consisting of 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, CD 233. CD230, CD90, CD74, CD3d, CD340, CD236, CD61, CD18, CD54, CD29, CD1a, CD5, CD220, CD2, CD66e, CD51, CD141, CD115, CD42b, CD221, CD271, CD55, CD2 43. CD98, CD10, CD41, CD14, CD45, CD228, CD16a, CD49e, CD126, CD63, CD48, CD7, CD140b, CD3g, CD117, CD28, CD8b, CD37, CD11b, CD107a, CD331, CD222, C D20, 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, CD2 52. 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, 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 SEQ ID NO: 5 or SEQ ID NO:

6.

28. The ICE cell of claim 24, wherein the TMD comprises the sequence of SEQ ID NO: 5 or SEQ ID NO:

6.

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

30. The ICE cell according to any one of claims 1 to 29, wherein the engager molecule does not have an intracellular domain (ICD).

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

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

33. The ICE cell according to 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 eliminated HLA-I or HLA-II expression.

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

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

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

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

39. The ICE cell according to any one of claims 1 to 38, wherein the cell is a low immunogenicity (HI) cell comprising: Reduced endogenous major histocompatibility complex class I (HLA-I) function compared to unmodified parental cells, and Reduced endogenous major histocompatibility complex class II (HLA-II) function compared to said unmodified parental cell.

40. The ICE cell according to any one of claims 1 to 39, wherein the conjugate cell comprises modulated expression of one or more of the following relative to the parental cell: 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 B9, TGFβ, CD73, CD39, LAG3, IL1R2, ACKR2, TNFRSF22, TNFRSF23, TNFRS10, DAD1, PVR or IFNγR1 d39, wherein the zygote cell is ABO blood type O or rhesus factor negative (Rh - ).

41. The ICE cell according to any one of claims 1 to 40, further comprising elevated expression of an antibody Fc receptor on the cell surface, wherein the Fc receptor helps to escape antibody-dependent cellular cytotoxicity (ADCC) or complement-mediated cytotoxicity (CDC).

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

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

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

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

46. ​​The ICE cell according to claim 45, wherein the cell is Rh factor negative (HIP - ) of low immune pluripotent cells.

47. The ICE cell according to claim 46, wherein the cell has ABO blood type O and Rh factor negative (HIPO - ) of low immune pluripotent cells.

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

49. The ICE cell according to any one of claims 1 to 42, wherein the zygote cell is of a specific tissue type.

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 cardiomyocyte, a pancreatic beta cell, a thyroid epithelial cell, a parathyroid cell, or a retinal pigment epithelial cell.

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

53. A pharmaceutical composition comprising: the ICE cell according to any one of claims 49 to 52 and a pharmaceutically acceptable carrier.

54. A medicine comprising: the ICE cell according to any one of claims 49 to 52 and a pharmaceutically acceptable carrier.

55. A method of treating a disease in a subject, comprising: The ICE cell according to any one of claims 49 to 52 is transplanted into the subject.

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

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

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

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

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

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

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

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

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

65. The ICE cell according to any one of claims 60 to 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 to 23.

67. The ICE cell according to claim 66, wherein the CDR comprises any one of the sequences of SEQ ID NOs: 18 to 23.

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

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

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) having a ganciclovir-triggered herpes simplex virus thymidine kinase gene (HSV-tk), (b) having a 5-fluorocytosine (5-FC)-triggered Escherichia coli cytosine deaminase gene (EC-CD), or (c) Inducible iCasp9 protein with AP1903 triggering.

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